Former Medical Director of the Hospital del Centro Gallego de Buenos Aires, Buenos Aires 1006, Argentina.
Correspondence to: Dr. Tomas Koltai, Former Medical Director of the Hospital del Centro Gallego de Buenos Aires, Buenos Aires 1006, Argentina; Maipu 712 dept 7 B, Buenos Aires 1006, Argentina; E-mail: email@example.com
© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Multiple drug resistance (MDR) is the tumor’s way of escaping the cytotoxic effects of various unrelated chemotherapeutic drugs. It can be either innate or acquired. MDR represents the end of the therapeutic pathway, and it practically leaves no treatment alternatives. Reversing MDR is an unfulfilled goal, despite the important recent advances in cancer research. MDR, the main cause of death in cancer patients, is a multi-factorial development, and most of its known causes have been thoroughly discussed in the literature. However, there is one aspect that has not received adequate consideration - intracellular alkalosis - which is part of wider pH deregulation where the pH gradient is inverted, meaning that extracellular pH is decreased and intracellular pH increased. This situation interacts with MDR and with the proteins involved, such as P-gp, breast cancer resistance protein, and multidrug associated resistance protein 1. However, there are also situations in which these proteins play no role at all, and where pH takes the lead. This is the case in ion trapping. Reversing the pH gradient to normal can be an important contribution to managing MDR. The drugs to manipulate pH exist, and most of them are FDA approved and in clinical use for other purposes. Furthermore, they have low or no toxicity and are inexpensive compared with any chemotherapeutic treatment. Repurposing these drugs and combining them in a reasonable fashion is one of the points proposed in this paper, which discusses the relationship between cancer’s peculiar pH and MDR.
Multidrug resistance, pH gradient inversion, reversion of the pH gradient, P-gp, pH centered treatment.
For more than 3000 years, trepanations (for unclear reasons) have been performed, since 2000 BC (and a breast cancer tumor was excised approximately at the same date); up to the 20th century, surgery was the only available treatment for solid tumors. During that time, there was nothing to be done for non-solid tumors. Things started to change in the middle of the last century.
Chemotherapy started in earnest in the 1940s, a decade in which two important advances achieved clinical status. In the first of these events, Louis Goodman and Alfred Gilman created the first alkylating agent as a derivative of the poisonous nitrogen mustard gas, a sibling of the sulfur mustard gas used in WWI. The second event had Sydney Farber as the protagonist. He introduced the first anti-metabolite (aminopterin) for acute leukemia treatment in children. Interestingly, the title of the first publication by Farber and his associates starts with “Temporary relief…”, thus, incorporating from the first moment one of the main problems of chemotherapy, the limited duration of its benefits. In both cases, the first patients treated by Goodman and Gilman on the one hand and the children with acute leukemia treated by Farber et al. on the other hand, the effects of chemotherapy were not long lived and repeat treatments were usually unsuccessful. Chemotherapy came up against its main adversary, resistance. To prolong the beneficial effects and at the same time reduce toxicity, multidrug chemotherapy protocols were introduced - successfully in many cases. Remissions lasted longer and toxicity was reduced. However, resistance and relapse still persisted at the end of the therapeutic path. Patients receiving chemotherapy can in many cases become resistant to previously effective drugs. Unfortunately, resistance is the proof of concept that cancer cells are the most adaptive cells in eukaryotes.
Resistance is the product of two different sources:
(a) The malignant cell itself; and
(b) The stroma and the vascular system.
The most important mechanism is the one in which the cancer cell develops the ability to prevent the drug from entering it or reduces the amount that can enter, simply expels the drug, or can inhibit apoptosis despite the treatment.
The stroma may contribute to resistance through its dense composition (e.g., pancreatic cancer with desmoplastic reaction) or its low vascular supply that decreases drug distribution in the tumor [Figure 1].
Figure 1. An overview of the main mechanisms of drug resistance. Drug extrusion is represented by P-gp (P-glycoprotein).
A distinction must be made between primary (or intrinsic) and acquired (or secondary) resistance. In the first case, cells are resistant to the drug before they first encounter it. In the latter, acquired resistance, the tumor develops resistance during the course of chemotherapy. This difference implies that, in the second case, tumors evolve from their initial responsive status towards an unresponsive one. The main evolution consists in upregulating the expression of some specific proteins of the ATP binding cassette family (ABC), namely P-glycoprotein (P-gp, also known as MDR1 or ABCB1), multidrug associated resistance protein 1 (MRP1 or ABCC1), and breast cancer resistance protein (BCRP or ABCG2).
The introduction of novel targeted therapies in the last twenty years was expected to dramatically change the resistance problem. That did not happen. Targeted treatments improved survival and progression-free periods in many different cancers - in some, a cure was even achieved - but resistance to treatment remained little changed in most cases.
In 1958, Burchenal and Holmberg were the first to study the short-lived remission achieved with anti-metabolites in the treatment of leukemia at a cell level. They expressed the prevailing concept at that moment - a random mutation induced by the drug - but they also established that there were many other biochemical mechanisms leading to resistance.
There is a special form of resistance consisting of invulnerability to many unrelated drugs that were never administered to the patient. This is known as MDR, a situation that severely limits the therapeutic options and consists of increased efflux of drugs. This mechanism was first proposed by Keld Danø, in 1973, as probably the main mechanism . This drug extrusion hypothesis was followed some years later by the discovery of P-glycoprotein  and other MDR proteins such as multidrug- associated resistance protein 1 (MRP1)  and BCRP, proteins that were not known when Danø proposed his theory. This meant that the extrusion culprits were finally identified.
Resistance to a drug must be differentiated from multidrug resistance. In the first case, a mutation or clonal evolution can be the cause, supposing that the malignant cells are responsive to other chemotherapeutic drugs. It is the lack of response to many different drugs that signals towards MDR, especially if these drugs were never administered before. In this last case, the most probable cause is the increased expression of one or more of the MDR proteins. Therefore, it is cancer’s resistance to many different and unrelated chemotherapeutic drugs that defines MDR, which may be intrinsic (primary) or acquired after chemotherapy. The lack of specificity is still one of the issues that is hard to explain. The causes of MDR are multiple, from poorly drug vulnerable stem cells to clonal evolution, stromal barriers, and mutations. It is not the aim of this paper to discuss MDR mechanisms but to focus exclusively on those related to pH alterations. When chemotherapy initially works, it is able to induce apoptosis in many of the malignant cells. However, tumors are very heterogeneous [8, 9] and not all malignant cells will be eliminated with the treatment. Those that survive repeated cycles of cytotoxic medication are either intrinsically resistant, or they are located in inaccessible parts of the tumor, such as very hypoxic niches without blood supply or in the middle of dense connective tissue with high interstitial pressure impeding circulation [Figure 1]. The non-resistant cells are gradually killed, while the surviving resistant cells will thrive and progress and, ultimately, fully replace the “weaker” cells, thus heralding the relapse. This is a typically Darwinian evolution where chemotherapy is the selective force for the survival of the fittest.
This paper does not analyze MDR causes in depth, which is beyond the scope of the review, but rather focuses on the relationship between the tumor dysregulated pH and MDR and explores new therapeutic avenues in this regard.
It has been known since the work of Otto Warburg  in the 1920s that tumors are acidic due to excessive production of lactic acid as a consequence of high glycolytic flux and downregulation of mitochondrial oxidative activity. Between the 1920s and the beginning of the 1970s, it was believed that there was no difference between intra- and extracellular pH. Thus, if the tumor was acidic, this concept included both sides of the cell membrane. Only in the late 1970s did it become evident that acidity was limited to the extracellular matrix, while in the intracellular milieu, pH was either unchanged or increased, compared with normal cells. The long time it took to discover the pH differences on the two sides of the cell membrane was due to the lack of adequate instruments that could accurately gauge intracellular pH [Table 1]. The awareness of different intra- and extracellular pH levels also led to the discovery of the channels, exchangers, transporters, and enzymes located on the membrane, which are in charge of maintaining this pH differential (gradient).
pH in different compartments in normal and cancer cells
|Table 1||Normal cell||Cancer cell|
|Extracellular (EC) pH (pHe)||7.30-7.35||6.4-7.0|
|Intracellular (IC) pH (pHi)||7.2||7.25-7.50|
In Table 1, the direction of the arrow indicates the pH gradient, and we can easily see that in cancer, the gradient follows exactly the opposite direction compared with non-malignant counterparts. This is the inversion of the pH gradient, a process found in all tumors, which is fundamental for cancer cell survival and progression. This means that in cancer, extracellular pH becomes acidic and intracellular pH becomes more alkaline.
Extracellular acidity, known for a long time, and the more recently discovered intracellular alkalinity were merely considered as a consequence of cancer metabolism and, to a certain extent, innocent bystanders. This notion started to change in the mid-1980s, when researchers found more and more evidence showing that the inverted pH gradient represented an important advantage for growth, proliferation, migration, and invasion. Furthermore, in 2000, Reshkin et al. showed that the first step in cellular transformation consisted in an increase of intracellular pH as a consequence of the enhanced activity of a specialized membrane channel, sodium bicarbonate exchanger 1 (NHE1). This exchanger, one of the main players in intracellular pH homeostasis, has the ability to export hydrogen ions (H+, protons) while importing Na+ from the matrix into the cells.
Reshkin et al. went one step further; they inhibited NHE1 with a drug that has been in clinical use for more than fifty years, amiloride. Interestingly, NHE1 inhibition impeded malignization. This was clear proof of the importance of pH in cancerization.
Intracellular and extracellular pH are discussed separately below, even though they are part of the same process of pH deregulation. The membrane between both compartments is the tool that keeps this different pH alive. The membrane is not a simple and passive boundary, but rather it is an active player that maintains a different environment inside and outside the cell. This is in part achieved by channels, exchangers, transporters, and enzymes spanning through it.
Normal extracellular pH, which is very close to the blood pH, decreases by around 8–10% in cancer tissues. It goes from 7.35 (normal tissues) to roughly 6.8 (malignant tissues). In some cases, it becomes even more acid. This is the result of different events:
(a) Increased CO2 production that produces carbonic acid on the cell surface and immediately ionizes, generating a bicarbonate ion that is reintroduced into the cell and a proton that remains in the extracellular matrix [Figure 2]. Two proteins located in the cell membrane participate in this process, namely membrane carbonic anhydrases (isoforms CAIX and CAXII) and sodium-bicarbonate cotransporter (NBC). CAIX is usually overexpressed in many hypoxic tumors[19-22]. CAIX is so closely associated with hypoxia that many authors consider its overexpression as a hypoxia marker[23-29].
Figure 2. CO2 of metabolic origin diffuses to the cell surface where it is hydrated to carbonic acid, which spontaneously ionizes to form a proton and a bicarbonate molecule. While the bicarbonate is reintroduced into the cell by NBC, the proton remains in the extracellular matrix, contributing to its acidification. NBC: Sodium-bicarbonate cotransporter.
CO2 abandons the cell simply by diffusion. When it reaches the cell surface, there is a tandem activity on CO2 carried out first by membrane carbonic anhydrase IX or XII and then by NBC [Figure 2].
Since Warburg’s work and until 1999, lactic acid was considered the main culprit of extracellular acidosis in cancer. Seminal research by Newell et al. showed that eliminating lactic acid production in malignant cells only minimally modified extracellular acidosis; thus, lactate is not the main and sole origin of a low pHe. It seems that CO2 production is equally important in pHe descent. Malignant cells produce large amounts of CO2 through the very active pentose phosphate pathway and fatty acid beta-oxidation.
(b) Increased lactic acid production is extruded from the cell by specialized membrane transporters such as monocarboxylate transporter 1 (MCT1) and monocarboxylate transporter 4 (MCT4). Figure 3 shows the origin of lactic acid from the glucose metabolism, which is strongly deviated towards the glycolytic pathway instead of the oxidative pathway of the Krebs cycle. MCTs can carry monocarboxylates in general; they are not exclusively dedicated to lactate transport. In cancer, these transporters are overexpressed on the cell membrane[35-38]. Hao et al. found that there was an association of CD44, CD147, MDR1, and MCTs expressions with prostate cancer progression and drug resistance.
Figure 3. Origin of lactate production. Normal cells do not use the glycolytic pathway beyond pyruvate that goes into the Krebs cycle associated with coenzyme A and converted into acetyl-CoA. Malignant cells follow the glycolytic pathway ending in lactate that is extruded by the activity of monocarboxylate transporters. Lactate cannot stay inside the cell because it would decrease intracellular pH to life-threatening levels, thus it must be swiftly exported. This soft-spot of cancer metabolism transforms MCTs into valid targets[31-34].
(c)There is the extrusion of intracellular protons through NHE1, vacuolar ATPase proton pumps.
(d)There is the extrusion of protons through endosomes that become exosomes or simply release their acidic cargo into the extracellular matrix. Endosomes can follow two pathways: (a) transformation into lysosomes; and (b) becoming carriers of compounds that will be extruded from the cell, such as protons, proteolytic enzymes, etc.
(e)Debris of stromal and tumoral cell death are produced, whether by hypoxia, invasion, chemotherapy, or radiotherapy. Necrotic cells are able to release acidic intracellular compounds, while apoptotic cells are engulfed by macrophages and there is no intracellular content released into the matrix.
Reducing extracellular pH simultaneously increases intracellular pH because the mechanism is essentially based on protons released from inside the cell and into the matrix.
Matrix acidification has advantages for malignant cells because acidity:
• Decreases and inhibits immunological attacks on the tumor;
• Activates proteolytic enzymes needed for invasion; and
• Stimulates migration at the invadopodium level,
As mentioned above, intracellular pH increases through the loss of protons towards the matrix and the import of bicarbonate. This situation is also advantageous for the malignant cell because it:
• Increases the activity of glycolytic enzymes, thus enhancing glycolytic flux that allows a higher biosynthetic activity;
• Increases proliferation; and
• Decreases the possibility of apoptosis.
The relationship between pH and MDR has major participation in the resistance process, as is discussed below. We do not have the evidence to maintain that MDR would not be possible without deregulated pH; however, there are many findings that hint towards this hypothesis. At this point, we are convinced that restoring the normal pH gradient should be an integral part of MDR targeting.
The relationship between extracellular pH and MDR has been extensively studied and is represented by a phenomenon called ion trapping.
Weakly basic chemotherapeutic drugs, such as doxorubicin, cannot enter the hydrophobic cell membrane because they undergo ionization in the acidic tumor extracellular environment. The lipid bilayer cell membrane is semipermeable, meaning that, while it allows fat-soluble non-ionized moieties to enter, it is poorly permeable to ionized water-soluble molecules. Anthracyclines and vinca alkaloids are weak basic drugs that are ionized by the microenvironmental acidity, thus substantially reducing their access to the cell[40-42].
There are many reports that serve as proof of concept regarding the relation between ion trapping and extracellular acidity:
• Reducing extracellular acidity with sodium bicarbonate, mitoxantrome cellular penetration was increase.
• Gu et al. developed a fluorescent probe based on dihydroberberine that showed that ionized berberine had a substantially lower cell penetration than the non-ionized form.
• Proton pump inhibitors, which increase extracellular pH, decrease drug resistance.
Martinez-Zaguilán et al. described the trapping of chemotherapeutic drugs in cellular endosomes. A new membrane is rapidly formed around the drug molecules, and these endosomes have a high V/ATPase proton activity, meaning that their interior is highly acidic. According to our criteria, drug release from the endosomes is inhibited by a mechanism similar to ion trapping. That is, the acidic interior ionizes weak basic drugs, impeding their transit through the membrane.
This mechanism is independent of ion trapping and consists of the increased P-gp expression when extracellular pH becomes acidic[47-50]. The mechanism that leads from extracellular acidity to increased P-gp proteins has not been fully clarified as yet.
The intracellular milieu and its sub-compartments, such as mitochondria, Golgi, endoplasmic reticulum, and endosomes, including autophagosomes and lysosomes, all have different pH, which is set according to the needs of the metabolic processes taking place in them:
• Cytoplasm is the site of glycolysis and fatty acid synthesis.
• Mitochondria are the site of the Krebs cycle, the electron transport chain, and lipid beta-oxidation.
• Golgi and endoplasmic reticulum mature proteins and secretions.
• Autophagosomes recycle organelles and other nutritional agents.
• Lysosomes and endosomes are very acid, degrade biological products, and intervene in the maturation of proteolytic enzymes.
Each of these activities requires a different optimum pH, and the cell’s homeostatic machinery maintains these different pHs (gradients) through membranes, e.g., cytoplasmic pH around 7.2, mitochondrial pH around 8, and lysosomal pH below 5.5.
Cell proliferation requires an alkaline cytoplasm, slightly above that of the resting cell.
In an extensive phylogenetic review, Busa and Nucitelli showed that almost all species increase their intracellular pH before replication. There were also some minor exceptions. This led the authors to consider intracellular pH as a signaling mechanism. High intracellular pH does not seem to be an indispensable mechanism but rather a facilitator for cell replication.
Drug-resistant cells were found to over-express or increase the activity of some of the membrane proteins discussed above:
• A subunit of a vacuolar H+-ATPase proton pump; and
Proton pumps and NHE1 [see Figure 4] are directly related to the pH gradient inversion: increasing intracellular alkalinization and decreasing extracellular pH. P-gp has a direct connection with intracellular and extracellular pH because:
Figure 4. Increase of protons in the extracellular matrix originates mainly from the extrusion of intracellular protons by NHE1 and the proton pump.
• Intracellular acidification downregulates P-gp; and
• Extracellular acidity increases P-gp expression by up to 5-10-fold in vitro .
This particular P-gp/pH relationship may explain some cases of intrinsic resistance, where there was no previous contact with any chemotherapeutic drug.
The main objective of chemotherapy consists in inducing the programmed death (apoptosis) of malignant cells[59, 60]. If this objective is not achieved, the tumor is resistant. Tumors have the ability to suppress apoptosis to a certain extent. There are many different modes for achieving resistance to apoptosis. One of them is increased signaling through the pro-survival PI3K/AKT pathway. This is usually associated with, or may produce a high expression of, the anti-apoptotic Bcl2 proteins.
For example, high expression of the anti-apoptotic Bcl2 protein induces an MDR phenotype in lymphoma cells. Apoptosis is favored by low intracellular pH and impeded when it is high. There is abundant evidence showing that the intracellular decrease of pH is favorable for apoptosis:
• Deoxyribonuclease II is an essential enzyme in programmed cell death and requires an acidic environment for its action.
• The first step in apoptosis consists of cytoplasmic acidification mainly due to proton export from mitochondria, thus alkalinizing this organelle and releasing cytochrome C.
• Maximal activation of caspases is only achieved with acidic cytoplasm.
• Pharmaceuticals that decrease intracellular pH induce apoptosis. This is the case of lansoprazole (inhibits the proton pump), salinomycin, and lonidamine. The antibiotic salinomycin has shown the ability to decrease the efflux of doxorubicin as well as other chemotherapeutic drugs in MDR[69-72] through P-gp inhibition. In addition to these effects, it also has anti-tumor activity independently of MDR inhibition[74-82]. However, we presume that many of the salinomycin’s anticancer effects are directly related to intracellular acidification. Conversely, P-gp has antiapoptotic effects, and prolonged intracellular acidification decreases P-gp expression.
It has been known since 1990 that multidrug-resistant cells have a higher pH than their non-resistant counterparts. This pH is higher on average by 0.1-0.2 and has been found in many different malignant cell lines. Although this is not a universal finding, it is quite frequent. There is also evidence that the increased intracellular pH is partly due to increased NHE1 activity.
MDR1 expressing cells were resistant to complement-mediated cytotoxicity; however, interestingly, malignant cells not expressing MDR1 that were manipulated to increase their pHi also showed similar resistance to complement-mediated cytotoxicity. Complement deposition in the cell membrane was reduced and delayed in both types of cells to a similar magnitude.
Belhoussine et al. confirmed the higher intracellular pH in MDR cells. They also found that these cells had more acidified vesicles, which were more acidic than their sensitive counterparts. According to the authors, these findings suggest that non-MDR cells have a lesser ability to remove protons.
Hamilton et al. showed that verapamil, a classic P-gp inhibitor, lowered pHi, but they considered these two factors unrelated.
In 1997, Robinson et al. found that the expression of MDR1 had the ability to delay apoptosis in fibroblasts transfected with the MDR1 gene and exposed to cytotoxic substances, colchicine in particular. The sole increase of pHi in non-transfected cells was able to induce a similar apoptotic delay. This finding suggests at least two things:
(1) Apoptosis requires low pHi; and
(2) One of the mechanisms used by MDR to oppose apoptosis is to maintain the increased pHi.
What remains to be seen is whether high pHi is a facilitating mechanism for resistance to apoptosis or a causal one. This report is in line with that by Weisburg and supports the idea that increased pHi by itself can induce an MDR phenotype.
MDR has a “protective” effect against caspase-dependent apoptosis. Inhibition of P-gp with specific antibodies increased drug- or Fas-mediated apoptosis through caspase activation in drug-resistant cells, and activating caspases requires an acidic cytoplasm. Thus, protection from apoptosis seems to be a synergic activity of MDR proteins plus intracellular alkalinity.
The pH gradient inversion, with its intracellular alkalinity and without MDR proteins, has shown that it can prevent intracellular accumulation of chemotherapeutic agents. In this regard, Simon et al. showed that intracellular alkalinity can substantially decrease accumulation and modify the intracellular distribution of weak alkaline chemotherapeutic drugs without any efflux mechanism in place. Intracellular alkalinity can prevent drugs from binding to their targets. Therefore, it is not only the extracellular ion-trapping effect that impedes the activity of weak alkaline drugs. In the experiments performed by Simon et al., non-resistant myeloma cells had a significantly lower intracellular pH compared with myeloma chemo-resistant cells (7.1 vs. 7.45).
Proton pump inhibitors have shown the ability to decrease or inhibit the MDR phenotype. Cisplatin treatment increased V-ATPase proton pump expression, and pHi was significantly increased in cisplatin-resistant cells. The DNA-binding ability of cisplatin was significantly enhanced in a more acidic pHi, suggesting that cisplatin’s cytotoxicity was modulated by pHi. Proton pump inhibitors such as bafilomycin could synergistically increase cisplatin’s cytotoxicity. These findings show that pHi is a key player in cisplatin’s effects. Unfortunately, bafilomycin cannot be used in clinical practice due to its toxicity.
Thiebaut et al. confirmed the higher pHi in resistant cells compared with their non-resistant counterparts. They performed an experiment in which they raised the extracellular pH and found that non-resistant cells maintained a pHi around 7, but the resistant cells markedly increased their pHi. pHi did not increase when they treated resistant cells with P-gp inhibitors, and they suggested that P-gp also behaves as a proton exporter.
Hoffman et al. showed that the level of resistance in MDR cells was correlated with two other parameters, namely pHi and low membrane electrical potential. Resistance was induced when they increased pHi in non-resistant cells. Furthermore, membrane depolarization also conferred a mild chemoresistance without any P-gp participation.
NHE1 is the main proton exporter, albeit not the only one, and it is overexpressed/over-active in cancer cells. This is particularly so in resistant cells. Cariporide is a powerful experimental NHE1 inhibitor. Interestingly, cariporide, which acidifies cytoplasm, is also able to sensitize resistant-breast cancer cells to doxorubicin.
NHE1 inhibition with cariporide reversed imatinib resistance in BCR-ABL-expressing leukemia cells[98-100], and NHE1 knockdown sensitized malignant cells to cisplatin-induced apoptosis. Inhibiting proton extruders, among them NHE1, increased doxorubicin’s cytotoxic effects on breast cancer cell lines.
pH regulation implies the participation of many players, in addition to NHE1. Membrane carbonic anhydrases are among these participants. Interestingly, by inhibiting membrane CAs, MDR can be reversed[103-108].
The inverted pH gradient has roles at both ends of the gradient: while cytoplasmic acidification downregulated P-gp, extracellular acidity upregulated it. This finding explains why it is not enough to act pharmacologically on one of the components of pH deregulation; both need to be addressed.
Despite all the evidence supporting the idea that high pHi is an indispensable condition for the MDR phenotype, Young et al. showed that increased intracellular pH is not a necessary condition for P-gp drug extrusion activity. However, we consider that this research has a conclusion bias because it only proves acid extrusion but not drug extrusion. Actually, this research showed that P-gp seems to have proton extruding abilities.
Coley et al. found that drug resistance was related to high electric conductivity. Conductivity is not directly related to pH because it depends on the total ions (including hydrogen ions) in a solution, while pH depends only on hydrogen ions.
Mulhall et al. showed a strong inverse relationship between conductivity and initiation of apoptosis. Thus, increased cytoplasmic conductivity seems to increase the apoptosis resistance found in resistant cells.
From these two last publications, we can deduce that the “ideal” drug-resistant cells seem to be those with:
(1) high electrical conductivity; and
(2) markedly elevated cytoplasmic pH.
These “ideal” resistant cells are refractory to apoptosis induction. We can also speculate, at this point, that increased intracellular pH is not mandatory for P-gp drug extruding activity, but it is a valuable resource for resistance to apoptosis.
Confirming the importance of conductivity, it was found that the cystic fibrosis transmembrane conductance regulator (CFTR), another member of the ABC family, decreases plasma membrane electrical potential when it is over-expressed and at the same time generates an MDR phenotype, but with a low intracellular pH. There are some similarities between this regulator and P-gp and a striking difference regarding pH.
NHE1 activity has been found to be increased in many tumors, and its downregulation re-sensitized cells to chemotherapy drugs[115, 116]. Drug-induced cellular surface tension modifications can impact P-gp activity.
Surface tension (interfacial tension) causes membrane rigidity; thus, pH plays a fundamental role in this phenomenon. Furthermore, high pHi increases membrane lipid electric charges. Phosphatidylethanolamine, a normal component of the cell membrane, is involved in acid-base equilibrium with the medium.
There is abundant evidence showing that modifying cell membrane fluidity (rigidity) can reduce P-gp activity, thus reversing MDR[119-124]. Surfactants that reduce surface tension, such as Tween 80 (polysorbate 80), are able to reduce P-gp activity and improve drug delivery into the cell .
Based on the evidence discussed above, a triple approach against MDR is proposed here. This consists of a known P-gp inhibitor such as verapamil associated with a surfactant and a pH gradient reversal scheme.
Verapamil, a calcium channel blocker, was first found to be an inhibitor of MDR in 1981. It is now a well-known P-gp inhibitor that impedes P-gp protein expression at the transcriptional level  and increases ATP consumption in MDR cells. Direct binding of verapamil to P-gp has also been described. There is abundant evidence about this drug’s impact against MDR[131-137].
As mentioned above, surfactants reduce cell membrane rigidity, thus counteracting one of the tools employed by MDR proteins to reject chemotherapeutic drugs. In this regard, surfactants reduce chemoresistance. There is also abundant evidence concerning Tween 80’s anti-MDR properties[139-142].
This scheme is based on five drugs that target different cell membrane proteins involved in pH homeostasis and in the inverted pH gradient. Its objective is to partially downregulate all the participants in the pH gradient inversion. Full blown inhibition of all of them would be impossible without serious undesired consequences for normal cells. However, partial inhibition is possible with no toxicity. These pH modulators are:
(d) quercetin; and
The appropriate combination of these drugs creates an important decrease of the intracellular pH and at the same time increases extracellular pH. Targeting pH alterations in cancer is becoming a valid strategy in complementary treatments.
• Amiloride is an FDA-approved potassium-saving diuretic in clinical use for the treatment of cardiovascular diseases and is usually associated with other diuretics such as hydrochlorotiazide. Amiloride’s main objective in the scheme is the inhibition of NHE1. Although it is a weak NHE1 blocker, at clinical doses, it is the only available approved drug. There are more potent NHE1 inhibitors; however, they are neither on the market nor FDA-approved. Evidence supporting amiloride’s anticancer effects is abundant[144-150] and involves actions derived from its intracellular acidifying properties as well as its ability to inhibit urokinase-type plasminogen activator (uPA)[151-154]. In addition, amiloride decreases the release of tumor exosomes[155-158]. This exosome inhibition also reduces proton discharge and blocks an important pathway of cancer cell communication. Specifically, amiloride and its derivatives reversed MDR in different types of tumors[159-162].
• Acetazolamide is a nonspecific carbonic anhydrase inhibitor. Cytoplasmic pH lowering is a known effect of this diuretic[163-165] that has been in medical practice for over sixty years and is FDA approved for uses not related to cancer. There is also evidence of its ability to slow cancer growth[166,167] and inhibit MDR. Furthermore, Zheng et al. found that MDR in some tongue cancers was not produced by the three known MDR proteins of the ABC family, but rather by over-expression of CAIX. When CAIX was downregulated by antisense oligonucleotides or acetazolamide, the tumor was re-sensitized. Kopecka et al. showed that the other membrane carbonic anhydrase, CAXII, physically interacted with P-gp on the cell surface. Silencing CAXII or inhibiting it with acetazolamide created a low intracellular pH that altered P-gp’s ATPase activity and promoted chemosensitization in MDR cells. There is active ongoing research for specific CAIX and CAXII inhibitors that would make it possible to circumvent the side effects of acetazolamide. For the time being, and until these new molecules are approved, we can only count on acetazolamide as a CA inhibitor.
• Lansoprazole is a vacuolar ATPase proton pump inhibitor approved by the FDA for the treatment of diseases related to excessive gastroduodenal acid production. At the cellular level, lansoprazole has the ability to inhibit proton extrusion from the cell, thus acidifying the intracellular milieu. Proton pumps can be found in intracellular membranes and the cell membrane. Those located in lysosomes keep the intra-lysosomal space acid while removing protons from the cytoplasm [Figure 5].
Figure 5. Different arrangements of proton pumps in the cell membrane and lysosomes. While in the cell, membrane the pump extrudes protons towards the extracellular space, in the lysosome, it pumps the protons into it. In a further step, the lysosome releases protons into the matrix. The functional end result is the same in both cases: inversion of the pH gradient.
Lansoprazole was able to induce apoptosis in breast cancer cells. Regarding MDR, lansoprazole reversed it in pets. Other proton pump inhibitors, such as omeprazole, pantoprazole, and esomeprazole, showed incremental effects on different chemotherapeutic drugs[174-176]. Unfortunately, there are also negative findings, e.g., pantoprazole in a clinical trial for docetaxel in metastatic castration-resistant prostate cancer showed no effects, despite the favorable results in laboratory level cell tests[178-179]. Pantoprazole even increased tumor growth and decreased chemotherapeutic cytotoxicity in mice. The benefits or disadvantages of pantoprazole remain controversial. According to Wang et al., proton pump inhibitors increased chemosensitivity and improved overall survival and progression-free survival in patients with advanced colorectal cancer. It is possible that proton pump inhibitors are not all the same regarding their anticancer effects. This is the reason we choose lansoprazole, which is less controversial than pantoprazole. Proton pump inhibitors decreased cisplatin sequestration in endosomes that were finally released from the cell in a melanoma model. Luciani et al. treated malignant cells with proton pump inhibitors, improving the accumulation of intracellular cytotoxic drugs. Intermittent proton pump inhibitors associated with standard chemotherapy administration improved the clinical outcome in metastatic breast cancer patients.
• Quercetin is a natural flavonoid that is not approved as a drug by the FDA but is available as an over-the-counter nutritional supplement. However, it is the only compound that can be found on the market with a strong ability to inhibit monocarboxylate transporters[185, 186]. MCT inhibition by quercetin induces important intracellular acidification. Significantly, there is considerable evidence of its capacity to reverse the MDR phenotype[188-218]. Despite this large body of evidence, absolute lack of toxicity, and low cost, we could not find clinical trials exploring the substance’s ability for MDR reversal. Adverse events using high levels of quercetin (1 g daily) as a nutritional supplement have been rarely reported. Quercetin also has additional beneficial effects in cancer:
(c) Decrease of ROS that diminishes PKC activity;
(e) Downregulation of heat shock protein 90;
(f) Inhibition of β-catenin signaling;
(g) Inhibition of pleiotropic kinases; and
(h) Induction of apoptosis.
• Topiramate is an FDA-approved drug for the treatment of seizures and epilepsy. It has four pharmacological effects that can benefit MDR reversal: (1) carbonic anhydrase inhibition; (2) intracellular acidification; (3) inhibition of voltage-gated sodium channels; and (4) inhibition of aquaporin 1[231-235]. There are no publications on topiramate having direct effects on MDR; however, refractory epilepsy in rats has been found to be associated with increased expression of P-gp[236, 237]. Topiramate and other anticonvulsants are substrates for P-gp. Although there is no empirical proof, we suspect that topiramate may saturate P-gp extrusion capacity. The reason for including topiramate in the scheme is mainly for two of its effects: cytoplasmic acidification and voltage-gated sodium channel inhibition.
• Statins are inhibitors of the de novo synthesis of cholesterol by blocking hydromethyl glutaryl coenzyme A reductase, an enzyme that is a rate-limiting factor for mevalonate synthesis and the mevalonate pathway. Therefore, statins decrease endogenous cholesterol production. Cell membrane rigidity depends on the amount of cholesterol, among other factors.
pH homeostasis is a complex mechanism in which different transporters, exchangers, channels, and enzymes are involved in overlapping proton and ionic trafficking between different cellular compartments. The results of these ionic movements lead to the best possible pH balance for cellular functions. Tumor pH homeostasis is different from that found in normal tissues, and this difference involves a proliferative and progressive advantage for the malignant phenotype. Each enzyme in a complex organism has a specific pH in which it works at the optimum speed and capacity. This is the pK. Tumors, by creating a different pH homeostasis, are signaling which enzymes should be more active and when, thus regulating tumor metabolism.
This essentially means that pH is a signaling molecule. If anyone doubted that pH is a molecule, he or she would be right. It is not a molecule but many molecules, or, even better, many protons. The cell behavior is thus conditioned by the number of protons present. The MDR phenotype shows a slight difference with the drug-sensitive one: a higher intracellular pH. This difference allows for two characteristics of the MDR cell:
(1) A more rigid cell membrane that plays a role in impeding cytotoxic drug access inside the cell; and
(2) A higher resistance to apoptotic signals.
A third characteristic must be added to this: the ion trapping produced by the strongly acidic extracellular matrix.
Multidrug resistance is not a one-protein job. At a certain point, P-gp and its sister molecules of the ABC family require adequate cell membrane rigidity, higher apoptosis resistance, and more ion trapping, whether in the matrix or inside lysosomes. This means the appropriate pH. MDR seems to function better with a high intracellular pH. This does not mean that one is the cause of the other. An MDR phenotype can be achieved even with low intracellular pH; this is the case of CFTR. Conversely, a high pHi can generate an MDR phenotype without over-expressing the MDR proteins.
This evidence hints towards the idea that high pHi and the MDR proteins complement each other, rather than there being a causal relationship. Both come together in one characteristic of the resistant cell: increased cell membrane rigidity. High pHi induces membrane rigidity, which in turn cooperates with MDR.
All this said, it becomes evident that for a successful fight against MDR, it is not enough to downregulate P-gp, etc., but pH and cell membrane rigidity must be tackled as well. The scheme proposed here confronts the three issues:
• P-gp with calcium channel blockers such as verapamil or others;
• pH gradient inversion with the pH-centered treatment; and
• membrane rigidity with surfactants such as Tween 80 and others.
The MDR problem has even further complexities. Balza et al., working with two different breast cancer cells, a triple-negative one and a hormone-sensitive one, found that:
(1) Associating cisplatin with an amiloride derivative was significantly more effective than treatments with cisplatin plus esomeprazole in triple-negative cells; and
(2) Esomeprazole alone was more effective in hormone-sensitive cells.
This shows that pH-centered treatments as complementary therapy may differ according to the type of cell.
Importantly, persistent intracellular acidification was able to downregulate the MDR phenotype.
Figure 7. Site of actions of the anti-MDR scheme. Reversion of the deregulated pH gradient is able to act against the MDR phenotype in two ways, by decreasing extracellular acidity and reducing intracellular pH. MDR: Multiple drug resistance.
It is important to note that extracellular acidity per se can induce P-gp expression. Figure 6 shows that the ABC family of drug extruders, intracellular alkalosis, and extracellular acidosis can all generate an MDR phenotype in an independent manner. However, there is evidence supporting the relationships among these three factors. Increased extracellular acidity induces P-gp expression. MDR cells with increased P-gp expression usually show increased intracellular alkalinity[87, 95]. This, in turn, prevents apoptosis and increases cell membrane rigidity[112, 113], creating the ideal environment for drug resistance.
MDR represents the last chapter of chemotherapeutic cancer treatment. It leaves the oncologist on a very narrow path to continue patient care. Unfortunately, there is no accepted treatment protocol. In this review, we propose a multidrug approach that simultaneously targets three important MDR characteristics, namely the MDR proteins, dysregulated pH, and cell membrane rigidity, with a rationally constructed approach.
This scheme has not been tested on clinical grounds. However, each of its components has separately provided successful experimental results, with the exception of topiramate, which has not been tested in the MDR context. This justifies their combination, as each of them targets different aspects of the MDR conundrum. Well-planned clinical trials are needed to evaluate this proposal. Furthermore, the scheme has almost no toxicity for normal cells, and there is ample clinical experience with the use of all these drugs.
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1. Goodman LS, Wintrobe MM, Dameshek W, et al. Nitrogen mustard therapy; use of methyl-bis (beta-chloroethyl) amine hydrochloride and tris (beta-chloroethyl) amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. JAMA 1946;132:126-32.DOIPubMed
2. Farber S, Diamond LK. Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-aminopteroyl-glutamic acid. N Engl J Med 1948;238:787-93.DOIPubMed
3. Burchenal JH, Holmberg EA. The utility of resistant leukemias in screening for chemotherapeutic activity. Ann N Y Acad Sci 1958;76:826-31; discussion 832.DOIPubMed
4. Danø K. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochimica et Biophysica Acta (BBA) - Biomembranes 1973;323:466-83.DOIPubMed
5. Chen YN, Mickley LA, Schwartz AM, et al. Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein. J Biol Chem 1990;265:10073-80.PubMed
6. Cole SP, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992;258:1650-4.DOIPubMed
7. Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 1998;95:15665-70.DOIPubMed PMC
8. Marusyk A, Polyak K. Tumor heterogeneity: causes and consequences. Biochim Biophys Acta 2010;1805:105-17.DOIPubMed PMC
9. Dexter DL, Leith JT. Tumor heterogeneity and drug resistance. J Clin Oncol 1986;4:244-57.DOIPubMed
10. Gillet JP, Gottesman MM. Mechanisms of multidrug resistance in cancer. Methods Mol Biol 2010;596:47-76.DOIPubMed
11. Rascio F, Spadaccino F, Rocchetti MT, et al. The pathogenic role of PI3K/AKT pathway in cancer onset and drug resistance: an updated review. Cancers (Basel) 2021;13:3949.DOIPubMed PMC
12. Mansoori B, Mohammadi A, Davudian S, Shirjang S, Baradaran B. The different mechanisms of cancer drug resistance: a brief review. Adv Pharm Bull 2017;7:339-48.DOIPubMed PMC
13. Jayaraj R, Nayagam SG, Kar A, et al. Clinical theragnostic relationship between drug-resistance specific miRNA expressions, chemotherapeutic resistance, and sensitivity in breast cancer: a systematic review and meta-analysis. Cells 2019;8:1250.DOIPubMed PMC
14. Ruan T, Liu W, Tao K, Wu C. A Review of research progress in multidrug-resistance mechanisms in gastric cancer. Onco Targets Ther 2020;13:1797-807.DOIPubMed PMC
15. Aleksakhina SN, Kashyap A, Imyanitov EN. Mechanisms of acquired tumor drug resistance. Biochim Biophys Acta Rev Cancer 2019;1872:188310.DOIPubMed
16. Vasan N, Baselga J, Hyman DM. A view on drug resistance in cancer. Nature 2019;575:299-309.DOIPubMed PMC
17. . Warburg, O. H. The metabolism of tumours: investigations from the Kaiser Wilhelm Institute for Biology; Berlin-Dahlem: Constable & Company Limited; 1930.DOI
18. Reshkin SJ, Bellizzi A, Caldeira S, et al. Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes. FASEB J 2000;14:2185-97.DOIPubMed
19. Hussain SA, Ganesan R, Reynolds G, et al. Hypoxia-regulated carbonic anhydrase IX expression is associated with poor survival in patients with invasive breast cancer. Br J Cancer 2007;96:104-9.DOIPubMed PMC
20. Benej M, Pastorekova S, Pastorek J. Carbonic anhydrase IX: regulation and role in cancer. Subcell Biochem 2014;75:199-219.DOIPubMed
21. Luong-Player A, Liu H, Wang HL, Lin F. Immunohistochemical reevaluation of carbonic anhydrase IX (CA IX) expression in tumors and normal tissues. Am J Clin Pathol 2014;141:219-25.DOIPubMed
22. Schmidt J, Oppermann E, Blaheta RA, et al. Carbonic-anhydrase IX expression is increased in thyroid cancer tissue and represents a potential therapeutic target to eradicate thyroid tumor-initiating cells. Mol Cell Endocrinol 2021;535:111382.DOIPubMed
23. Kaluz S, Kaluzová M, Liao SY, Lerman M, Stanbridge EJ. Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: a one transcription factor (HIF-1) show? Biochim Biophys Acta 2009;1795:162-72.DOIPubMed PMC
24. Koukourakis MI, Bentzen SM, Giatromanolaki A, et al. Endogenous markers of two separate hypoxia response pathways (hypoxia inducible factor 2 alpha and carbonic anhydrase 9) are associated with radiotherapy failure in head and neck cancer patients recruited in the CHART randomized trial. J Clin Oncol 2006;24:727-35.DOIPubMed
25. Olive PL, Aquino-Parsons C, MacPhail SH, et al. Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer. Cancer Res 2001;61:8924-9.PubMed
26. Chia SK, Wykoff CC, Watson PH, et al. Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma. J Clin Oncol 2001;19:3660-8.DOIPubMed
27. Yoo H, Baia GS, Smith JS, et al. Expression of the hypoxia marker carbonic anhydrase 9 is associated with anaplastic phenotypes in meningiomas. Clin Cancer Res 2007;13:68-75.DOIPubMed
28. Pastorekova S, Gillies RJ. The role of carbonic anhydrase IX in cancer development: links to hypoxia, acidosis, and beyond. Cancer Metastasis Rev 2019;38:65-77.DOIPubMed PMC
29. Pastorekova S, Ratcliffe PJ, Pastorek J. Molecular mechanisms of carbonic anhydrase IX-mediated pH regulation under hypoxia. BJU Int 2008;101 Suppl 4:8-15.DOIPubMed
30. Newell K, Franchi A, Pouysségur J, Tannock I. Studies with glycolysis-deficient cells suggest that production of lactic acid is not the only cause of tumor acidity. Proc Natl Acad Sci ;90:1127-31.DOI
31. Pinheiro C, Albergaria A, Paredes J, et al. Monocarboxylate transporter 1 is up-regulated in basal-like breast carcinoma. Histopathology 2010;56:860-7.DOIPubMed
32. Pértega-Gomes N, Vizcaíno JR, Miranda-Gonçalves V, et al. Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer. BMC Cancer 2011;11:312.DOIPubMed PMC
33. Pinheiro C, Longatto-Filho A, Azevedo-Silva J, Casal M, Schmitt FC, Baltazar F. Role of monocarboxylate transporters in human cancers: state of the art. J Bioenerg Biomembr 2012;44:127-39.DOIPubMed
34. Puri S, Juvale K. Monocarboxylate transporter 1 and 4 inhibitors as potential therapeutics for treating solid tumours: a review with structure-activity relationship insights. Eur J Med Chem 2020;199:112393.DOIPubMed
35. Yoshida GJ. The Harmonious interplay of amino acid and Monocarboxylate transporters induces the robustness of cancer cells. Metabolites 2021;11:27.DOIPubMed PMC
36. Payen VL, Mina E, Van Hée VF, Porporato PE, Sonveaux P. Monocarboxylate transporters in cancer. Mol Metab 2020;33:48-66.DOIPubMed PMC
37. Sun X, Wang M, Wang M, et al. Role of proton-coupled Monocarboxylate transporters in cancer: from metabolic crosstalk to therapeutic potential. Front Cell Dev Biol 2020;8:651.DOIPubMed PMC
38. Baltazar F, Pinheiro C, Morais-Santos F, et al. Monocarboxylate transporters as targets and mediators in cancer therapy response. Histol Histopathol 2014;29:1511-24. Available from:
39. Hao J, Chen H, Madigan MC, et al. Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression. Br J Cancer 2010;103:1008-18.DOIPubMed PMC
40. Roos A, Boron WF. Intracellular pH. Physiol Rev 1981;61:296-434.DOIPubMed
41. Mahoney BP, Raghunand N, Baggett B, Gillies RJ. Tumor acidity, ion trapping and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in vitro. Biochem Pharmacol 2003;66:1207-18.DOIPubMed
42. Raghunand N, Altbach MI, van Sluis R, et al. Plasmalemmal pH-gradients in drug-sensitive and drug-resistant MCF-7 human breast carcinoma xenografts measured by 31P magnetic resonance spectroscopy. Biochem Pharmacol 1999;57:309-12.DOIPubMed
43. Raghunand N, Mahoney BP, Gillies RJ. Tumor acidity, ion trapping and chemotherapeutics. Biochem Pharmacol 2003;66:1219-29.DOIPubMed
44. Gu Y, Zhao Z, Niu G, et al. Visualizing semipermeability of the cell membrane using a pH-responsive ratiometric AIEgen. Chem Sci 2020;11:5753-8.DOIPubMed PMC
45. Milito A, Fais S. Proton pump inhibitors may reduce tumour resistance. Expert Opin Pharmacother 2005;6:1049-54.DOIPubMed
46. Martı́nez-zaguilán R, Raghunand N, Lynch RM, et al. pH and drug resistance. I. functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines. Biochem Pharmacol 1999;57:1037-46.DOIPubMed
47. Thews O, Gassner B, Kelleher DK, Schwerdt G, Gekle M. Impact of extracellular acidity on the activity of P-glycoprotein and the cytotoxicity of chemotherapeutic drugs. Neoplasia 2006;8:143-52.DOIPubMed PMC
48. Sauvant C, Nowak M, Wirth C, et al. Acidosis induces multi-drug resistance in rat prostate cancer cells (AT1) in vitro and in vivo by increasing the activity of the p-glycoprotein via activation of p38. Int J Cance ;123:2532-42.DOIPubMed
49. Thews O, Dillenburg W, Fellner M, et al. Activation of P-glycoprotein (Pgp)-mediated drug efflux by extracellular acidosis: in vivo imaging with 68Ga-labelled PET tracer. Eur J Nucl Med Mol Imaging 2010;37:1935-42.DOIPubMed
50. Thews O, Nowak M, Sauvant C, Gekle M. Hypoxia-induced extracellular acidosis increases p-glycoprotein activity and chemoresistance in tumors in vivo via p38 signaling pathway. Adv Exp Med Biol 2011;701:115-22.DOIPubMed
51. Busa WB, Nuccitelli R. Metabolic regulation via intracellular pH. Am J Physiol 1984;246:R409-38.DOIPubMed
52. Simon SM, Schindler M. .DOIPubMed PMC
53. Ma L, Center MS. The gene encoding vacuolar H+-ATPase subunit C is overexpressed in multidrug resistant HL60 cells. Biochem Biophys Res Commun 1992;182:675-81.DOIPubMed
54. Chen Q, Liu Y, Zhu XL, et al. Increased NHE1 expression is targeted by specific inhibitor cariporide to sensitize resistant breast cancer cells to doxorubicin in vitro and in vivo. BMC cancer 20196;19:1-13.DOIPubMed PMC
55. Amith SR, Wilkinson JM, Baksh S, Fliegel L. The Na+/H+ exchanger (NHE1) as a novel co-adjuvant target in paclitaxel therapy of triple-negative breast cancer cells. Oncotarget 2015;6:1262-75.DOIPubMed PMC
56. Hoffmann EK, Lambert IH. Ion channels and transporters in the development of drug resistance in cancer cells. Philos Trans R Soc Lond B Biol Sci 2014;369:20130109.DOIPubMed PMC
57. Jin W, Lu Y, Li Q, et al. Down-regulation of the P-glycoprotein relevant for multidrug resistance by intracellular acidification through the crosstalk of MAPK signaling pathways. Int J Biochem Cell Biol 2014;54:111-21.DOIPubMed
58. Wei LY, Roepe PD. Low external pH and osmotic shock increase the expression of human MDR protein. Biochemistry 1994;33:7229-38.DOIPubMed
59. Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancer chemotherapy. Exp Cell Res 2000;256:42-9.DOIPubMed
60. Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 2006;25:4798-811.DOIPubMed
61. Hickman J. Apoptosis and chemotherapy resistance. Eur J Cancer 1996;32:921-6.DOIPubMed
62. Lyons SK, Clarke AR. Apoptosis and carcinogenesis. Br Med Bull 1997;53:554-69.DOIPubMed
63. Schmitt CA, Lowe SW. Apoptosis and chemoresistance in transgenic cancer models. J Mol Med (Berl) 2002;80:137-46.DOIPubMed
64. Sergeeva TF, Shirmanova MV, Zlobovskaya OA, et al. Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis. Biochim Biophys Acta Mol Cell Res 2017;1864:604-11.DOIPubMed
65. Morana S, Li J, Springer E, Eastman A. THe inhibition of etoposide-induced apoptosis by zinc is associated with modulation of intracellular pH. Int J Oncol 1994.PubMed
66. Matsuyama S, Llopis J, Deveraux QL, Tsien RY, Reed JC. Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2000;2:318-25.DOIPubMed
67. Zhang S, Wang Y, Li SJ. Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion. Biochem Biophys Res Commun 2014;448:424-9.DOIPubMed
68. Harguindey S, Stanciu D, Devesa J, et al. Cellular acidification as a new approach to cancer treatment and to the understanding and therapeutics of neurodegenerative diseases. Semin Cancer Biol 2017;43:157-79.DOIPubMed
69. Kim KY, Kim SH, Yu SN, et al. Salinomycin enhances doxorubicin-induced cytotoxicity in multidrug resistant MCF-7/MDR human breast cancer cells via decreased efflux of doxorubicin. Mol Med Rep 2015;12:1898-904.DOIPubMed PMC
70. Hermawan A, Wagner E, Roidl A. Consecutive salinomycin treatment reduces doxorubicin resistance of breast tumor cells by diminishing drug efflux pump expression and activity. Oncol Rep 2016;35:1732-40.DOIPubMed
71. Dewangan J, Srivastava S, Rath SK. Salinomycin: a new paradigm in cancer therapy. Tumour Biol 2017;39:1010428317695035.DOIPubMed
72. Dewangan J, Srivastava S, Rath SK. Salinomycin: a new paradigm in cancer therapy. Tumour Biol 2017;39:1010428317695035.DOIPubMed
73. Riccioni R, Dupuis ML, Bernabei M, et al. The cancer stem cell selective inhibitor salinomycin is a p-glycoprotein inhibitor. Blood Cells Mol Dis 2010;45:86-92.DOIPubMed
74. Koo KH, Kim H, Bae YK, et al. Salinomycin induces cell death via inactivation of Stat3 and downregulation of Skp2. Cell Death Dis 2013;4:e693.DOIPubMed PMC
75. Huczyński A, Janczak J, Antoszczak M, Wietrzyk J, Maj E, Brzezinski B. Antiproliferative activity of salinomycin and its derivatives. Bioorg Med Chem Lett 2012;22:7146-50.DOIPubMed
76. Sommer AK, Hermawan A, Mickler FM, et al. Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases. Oncotarget 2016;7:50461-76.DOIPubMed PMC
77. Lu D, Choi MY, Yu J, Castro JE, Kipps TJ, Carson DA. Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells. Proc Natl Acad Sci ;108:13253-7.DOIPubMed PMC
78. Pellegrini P, Dyczynski M, Sbrana FV, et al. Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells. Oncotarget 2016;7:35703-23.DOIPubMed PMC
79. Jiang J, Li H, Qaed E, et al. Salinomycin, as an autophagy modulator-- a new avenue to anticancer: a review. J Exp Clin Cancer Res 2018;37:26.DOIPubMed PMC
80. Yue W, Hamaï A, Tonelli G, et al. Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance. Autophagy 2013;9:714-29.DOIPubMed PMC
81. Antoszczak M. A medicinal chemistry perspective on salinomycin as a potent anticancer and anti-CSCs agent. Eur J Med Chem 2019;164:366-77.DOIPubMed
82. Kaushik V, Yakisich JS, Kumar A, Azad N, Iyer AKV. Ionophores: potential use as anticancer drugs and chemosensitizers. Cancers (Basel) 2018;10:360.DOIPubMed PMC
83. Pallis M, Russell N. P-glycoprotein plays a drug-efflux-independent role in augmenting cell survival in acute myeloblastic leukemia and is associated with modulation of a sphingomyelin-ceramide apoptotic pathway. Blood 2000;95:2897-904.PubMed
84. Lu Y, Li QH, Ma L, et al. Effect of intracellular acidification on P-glycoprotein in drug-resistant K562/A02 cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2009;17:568-73.PubMed
85. Boscoboinik D, Gupta RS, Epand RM. Investigation of the relationship between altered intracellular pH and multidrug resistance in mammalian cells. Br J Cancer 1990;61:568-72.DOIPubMed PMC
86. Weisburg JH, Roepe PD, Dzekunov S, Scheinberg DA. Intracellular pH and multidrug resistance regulate complement-mediated cytotoxicity of nucleated human cells. J Biol Chem 1999;274:10877-88.DOIPubMed
87. Belhoussine R, Morjani H, Sharonov S, Ploton D, Manfait M. Characterization of intracellular pH gradients in human multidrug-resistant tumor cells by means of scanning microspectrofluorometry and dual-emission-ratio probes. Int J Cancer 1999;81:81-9.DOIPubMed
88. Epand RF, Epand RM, Gupta RS, Cragoe EJ Jr. Reversal of intrinsic multidrug resistance in Chinese hamster ovary cells by amiloride analogs. Br J Cancer 1991;63:247-51.DOIPubMed PMC
89. Pannocchia A, Revelli S, Tamponi G, et al. Reversal of doxorubicin resistance by the amiloride analogue EIPA in multidrug resistant human colon carcinoma cells. Cell Biochem Funct 1996;14:11-8.DOIPubMed
90. Hamilton G, Cosentini EP, Teleky B, et al. The multidrug-resistance modifiers verapamil, cyclosporine A and tamoxifen induce an intracellular acidification in colon carcinoma cell lines in vitro. Anticancer Res 1993;13:2059-63.PubMed
91. Robinson LJ, Roberts WK, Ling TT, et al. Human MDR 1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts. Biochemistry 1997;36:11169-78.DOIPubMed
92. Smyth MJ, Krasovskis E, Sutton VR, Johnstone RW. The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis. Proc Natl Acad Sci U S A 1998;95:7024-9.DOIPubMed PMC
93. Taylor S, Spugnini EP, Assaraf YG, et al. Microenvironment acidity as a major determinant of tumor chemoresistance: Proton pump inhibitors (PPIs) as a novel therapeutic approach. Drug Resist Updat 2015;23:69-78.DOIPubMed
94. Murakami T, Shibuya I, Ise T, et al. Elevated expression of vacuolar proton pump genes and cellular PH in cisplatin resistance. Int J Cancer 2001;93:869-74.DOIPubMed
95. Thiebaut F, Currier SJ, Whitaker J, et al. Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye. J Histochem Cytochem 1990;38:685-90.DOIPubMed
96. Hoffman MM, Wei LY, Roepe PD. Are altered pHi and membrane potential in hu MDR 1 transfectants sufficient to cause MDR protein-mediated multidrug resistance? J Gen Physiol 1996;108:295-313.DOIPubMed PMC
97. Stock C, Pedersen SF. Roles of pH and the Na+/H+ exchanger NHE1 in cancer: From cell biology and animal models to an emerging translational perspective? Semin Cancer Biol 2017;43:5-16.DOIPubMed
98. Harguindey S, Arranz JL, Polo Orozco JD, et al. Cariporide and other new and powerful NHE1 inhibitors as potentially selective anticancer drugs--an integral molecular/biochemical/metabolic/clinical approach after one hundred years of cancer research. J Transl Med 2013;11:282.DOIPubMed PMC
99. Jin W, Li Q, Lin Y, et al. Reversal of Imatinib resistance in BCR-ABL-positive leukemia after inhibition of the Na+/H+ exchanger. Cancer Lett 2011;308:81-90.DOIPubMed
100. Hu RH, Jin WN, Chang GQ, et al. Increasing sensitivity of leukemia cells to imatinib by inhibiting NHE1 and p38MAPK signaling pathway. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2012; 20:1341-5.PubMed
101. Lauritzen G, Jensen MB, Boedtkjer E, et al. NBCn1 and NHE1 expression and activity in DeltaNErbB2 receptor-expressing MCF-7 breast cancer cells: contributions to pHi regulation and chemotherapy resistance. Exp Cell Res 2010;316:2538-53.DOIPubMed
102. Tavares-Valente D, Sousa B, Schmitt F, Baltazar F, Queirós O. Disruption of pH Dynamics Suppresses Proliferation and Potentiates Doxorubicin Cytotoxicity in Breast Cancer Cells. Pharmaceutics 2021;13:242.DOIPubMed PMC
103. Tonissen KF, Poulsen S. Carbonic anhydrase XII inhibition overcomes P-glycoprotein-mediated drug resistance: a potential new combination therapy in cancer. Cancer Drug Resist 2021;4:343-55.DOI
104. von Neubeck B, Gondi G, Riganti C, et al. An inhibitory antibody targeting carbonic anhydrase XII abrogates chemoresistance and significantly reduces lung metastases in an orthotopic breast cancer model in vivo. Int J Cancer 2018;143:2065-75.DOIPubMed
105. Kopecka J, Rankin GM, Salaroglio IC, Poulsen SA, Riganti C. P-glycoprotein-mediated chemoresistance is reversed by carbonic anhydrase XII inhibitors. Oncotarget 2016;7:85861-75.DOIPubMed PMC
106. Zheng G, Peng C, Jia X, et al. ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH. Mol Cancer 2015;14:84.DOIPubMed PMC
107. Podolski-Renić A, Dinić J, Stanković T, et al. Sulfocoumarins, specific carbonic anhydrase IX and XII inhibitors, interact with cancer multidrug resistant phenotype through pH regulation and reverse P-glycoprotein mediated resistance. Eur J Pharm Sci 2019;138:105012.DOIPubMed
108. Ilardi G, Zambrano N, Merolla F, et al. Histopathological determinants of tumor resistance: a special look to the immunohistochemical expression of carbonic anhydrase IX in human cancers. Curr Med Chem 2014;21:1569-82.DOIPubMed PMC
109. Lu Y, Pang T, Wang J, et al. Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells. Biochem Biophys Res Commun 2008;377:441-6.DOIPubMed
110. Li Y, Xiang J, Zhang SS, et al. Analysis of the impact of extracellular acidity on the expression and activity of P-glycoprotein and on the P-glycoprotein-mediated cytotoxicity of daunorubicin in cancer cell by microfluidic chip technology. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2015;37:75-81.DOIPubMed
111. Young G, Reuss L, Altenberg GA. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression. I. nt J Biochem Mol Biol 2011;2:219-27.PubMed PMC
112. Coley HM, Labeed FH, Thomas H, Hughes MP. Biophysical characterization of MDR breast cancer cell lines reveals the cytoplasm is critical in determining drug sensitivity. Biochim Biophys Acta 2007;1770:601-8.DOIPubMed
113. Mulhall HJ, Cardnell A, Hoettges KF, Labeed FH, Hughes MP. Apoptosis progression studied using parallel dielectrophoresis electrophysiological analysis and flow cytometry. Integr Biol (Camb) 2015;7:1396-401.DOIPubMed
114. Wei L, Stutts M, Hoffman M, Roepe P. Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype. Biophys J 1995;69:883-95.DOIPubMed PMC
115. Miraglia E, Viarisio D, Riganti C, Costamagna C, Ghigo D, Bosia A. Na+/H+ exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin. Int J Cancer 2005;115:924-9.DOIPubMed
116. Li S, Bao P, Li Z, Ouyang H, Wu C, Qian G. Inhibition of proliferation and apoptosis induced by a Na+/H+ exchanger-1 (NHE-1) antisense gene on drug-resistant human small cell lung cancer cells. Oncol Rep 2009;21:1243-9.DOIPubMed
117. Omran Z, Whitehouse C, Halwani M, et al. Pinocytosis as the biological mechanism that protects PGP function in multidrug resistant cancer cells and in blood-brain barrier endothelial cells. Symmetry 2020;12:1221.DOI
118. Petelska AD, Figaszewski ZA. Interfacial tension of bilayer lipid membrane formed from phosphatidylethanolamine. Biochim Biophys Acta 2002;1567:79-86.DOIPubMed
119. Schuldes H, Dolderer J, Zimmer G, et al. Reversal of multidrug resistance and increase in plasma membrane fluidity in CHO cells with R-verapamil and bile salts. Eur J Cancer 2001;37:660-7.DOIPubMed
120. Drori S, Eytan GD, Assaraf YG. Potentiation of anticancer-drug cytotoxicity by multidrug-resistance chemosensitizers involves alterationsin membrane fluidity leading to increased membrane permeability. Eur J Biochem 1995;228:1020-9.DOIPubMed
121. Regev R, Katzir H, Yeheskely-Hayon D, Eytan GD. Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane. FEBS J 2007;274:6204-14.DOIPubMed
122. Ferté J. Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane. Eur J Biochem 2000;267:277-94.DOIPubMed
123. Zong L, Pi Z, Liu S, Xing J, Liu Z, Song F. Liquid extraction surface analysis nanospray electrospray ionization based lipidomics for in situ analysis of tumor cells with multidrug resistance. Rapid Commun Mass Spectrom 2018;32:1683-92.DOIPubMed
124. Leibovici J, Klein O, Wollman Y, et al. Cell membrane fluidity and adriamycin retention in a tumor progression movel of AKR lymphoma. Biochim Biophys Acta 1996;1281:182-8.DOIPubMed
125. Zhang H, Yao M, Morrison RA, Chong S. Commonly used surfactant, Tween 80, improves absorption of P-glycoprotein substrate, digoxin, in rats. Arch Pharm Res 2003;26:768-72.DOIPubMed
126. Kaur P, Garg T, Rath G, Murthy RS, Goyal AK. Surfactant-based drug delivery systems for treating drug-resistant lung cancer. Drug Deliv 2016;23:727-38.DOIPubMed
127. Tsuruo T, Iida H, Tsukagoshi S, Sakurai Y. Overcoming of vincristine resistance in P388 leukemia, in vivo and in vitro through enhanced cytoloxicity of vincristine and vinblastine by verapamil. Cancer Res 1981;41:1967-72.PubMed
128. Muller C, Goubin F, Ferrandis E, et al. Evidence for transcriptional control of human mdr1 gene expression by verapamil in multidrug-resistant leukemic cells. Mol Pharmacol 1995;47:51-6.PubMed
129. Broxterman HJ, Pinedo HM, Kuiper CM, et al. Induction by verapamil of a rapid increase in ATP consumption in multidrug-resistant tumor cells. FASEB J 1988;2:2278-82.DOIPubMed
130. Yusa K, Tsuruo T. Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and transport of verapamil outward across the plasma membrane of K562/ADM cells. Cancer Res 1989;49:5002-6. Available from:
131. Salmon S, Dalton W, Grogan T, et al. Multidrug-resistant myeloma: laboratory and clinical effects of verapamil as a chemosensitizer. Blood 1991;78:44-50.PubMed
132. Karwatsky J, Lincoln MC, Georges E. A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity. Biochemistry 2003;42:12163-73.DOIPubMed
133. Warr JR, Anderson, M, Fergusson J. Properties of verapamil-hypersensitive multidrug-resistant Chinese hamster ovary cells. Cancer Res 1988;48:4477-83.PubMed
134. Dönmez Y, Akhmetova L, İşeri ÖD, Kars MD, Gündüz U. Effect of MDR modulators verapamil and promethazine on gene expression levels of MDR1 and MRP1 in doxorubicin-resistant MCF-7 cells. Cancer Chemother Pharmacol 2011;67:823-8.DOIPubMed
135. Afrooz H, Ahmadi F, Fallahzadeh F, Mousavi-fard SH, Alipour S. Design and characterization of paclitaxel-verapamil co-encapsulated PLGA nanoparticles: Potential system for overcoming P-glycoprotein mediated MDR. Journal of Drug Delivery Science and Technology 2017;41:174-81.DOI
136. Williams JB, Buchanan CM, Pitt WG. Codelivery of doxorubicin and verapamil for treating multidrug resistant cancer cells. Pharm Nanotechnol 2018;6:116-23.DOIPubMed
137. Wang L, Sun Y. Efflux mechanism and pathway of verapamil pumping by human P-glycoprotein. Arch Biochem Biophys 2020;696:108675.DOIPubMed
138. Woodcock DM, Linsenmeyer ME, Chojnowski G, et al. Reversal of multidrug resistance by surfactants. Br J Cancer 1992;66:62-8.DOIPubMed PMC
139. Yuan X, Ji W, Chen S, et al. A novel paclitaxel-loaded poly(d,l-lactide-co-glycolide)-Tween 80 copolymer nanoparticle overcoming multidrug resistance for lung cancer treatment. Int J Nanomedicine 2016;11:2119-31.DOIPubMed PMC
140. Kaur H, Ghosh S, Kumar P, Basu B, Nagpal K. Ellagic acid-loaded, tween 80-coated, chitosan nanoparticles as a promising therapeutic approach against breast cancer: In-vitro and in-vivo study. Life Sci 2021;284:119927.DOIPubMed
141. Bhattacharjee J, Verma G, Aswal VK, et al. Tween 80-sodium deoxycholate mixed micelles: structural characterization and application in doxorubicin delivery. J Phys Chem B 2010;114:16414-21.DOIPubMed
142. Tsujino I, Yamazaki T, Masutani M, Sawada U, Horie T. Effect of Tween-80 on cell killing by etoposide in human lung adenocarcinoma cells. Cancer Chemother Pharmacol 1999;43:29-34.DOIPubMed
143. Neri D, Supuran CT. Interfering with pH regulation in tumours as a therapeutic strategy. Nat Rev Drug Discov 2011;10:767-77.DOIPubMed
144. Diego J P, Trilla C, Cañero RG. Potentiation by amiloride of doxorubicin effect on normal and tumor liver cells in vitro. International Hepatology Communications 1995;3:S165. Available from:
145. Matthews H, Ranson M, Kelso MJ. Anti-tumour/metastasis effects of the potassium-sparing diuretic amiloride: an orally active anti-cancer drug waiting for its call-of-duty? Int J Cancer 2011;129:2051-61.DOIPubMed
146. Xu S, Liu C, Ma Y, Ji HL, Li X. Potential roles of amiloride-sensitive sodium channels in cancer development. Biomed Res Int 2016;2016:2190216.DOIPubMed PMC
147. Zheng YT, Yang HY, Li T, et al. Amiloride sensitizes human pancreatic cancer cells to erlotinib in vitro through inhibition of the PI3K/AKT signaling pathway. Acta Pharmacol Sin 2015;36:614-26.DOIPubMed PMC
148. Rojas EA, Corchete LA, San-Segundo L, et al. Amiloride, an old diuretic drug, is a potential therapeutic agent for multiple myeloma. Clin Cancer Res 2017;23:6602-15.DOIPubMed
149. Cho YL, Lee KS, Lee SJ, et al. Amiloride potentiates TRAIL-induced tumor cell apoptosis by intracellular acidification-dependent Akt inactivation. Biochem Biophys Res Commun 2005;326:752-8.DOIPubMed
150. Chang WH, Liu TC, Yang WK, et al. Amiloride modulates alternative splicing in leukemic cells and resensitizes Bcr-AblT315I mutant cells to imatinib. Cancer Res 2011;71:383-92.DOIPubMed
151. Matthews H, Ranson M, Tyndall JD, Kelso MJ. Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA). Bioorg Med Chem Lett 2011;21:6760-6.DOIPubMed
152. Wang Y, Dang J, Liang X, Doe WF. Amiloride modulates urokinase gene expression at both transcription and post-transcription levels in human colon cancer cells. Clin Exp Metastasis 1995;13:196-202.DOIPubMed
153. Jankun J, Skrzypczak-Jankun E. Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride. Cancer biochem biophys 1999;17:109-23.PubMed
154. Buckley BJ, Kumar A, Aboelela A, et al. Screening of 5- and 6-substituted amiloride libraries identifies dual-uPA/NHE1 active and single target-selective inhibitors. Int J Mol Sci 2021;22:2999.DOIPubMed PMC
155. Zhou L, Zhang T, Shao W, et al. Amiloride ameliorates muscle wasting in cancer cachexia through inhibiting tumor-derived exosome release. Skelet Muscle 2021;11:17.DOIPubMed PMC
156. Chalmin F, Ladoire S, Mignot G, et al. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest 2010;120:457-71.DOIPubMed PMC
157. Dorayappan KDP, Wanner R, Wallbillich JJ, et al. Hypoxia-induced exosomes contribute to a more aggressive and chemoresistant ovarian cancer phenotype: a novel mechanism linking STAT3/Rab proteins. Oncogene 2018;37:3806-21.DOIPubMed PMC
158. Escrevente C, Keller S, Altevogt P, Costa J. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer 2011;11:108.DOIPubMed PMC
159. Diego JP, Trilla C, Cañero RG. Involvement of the activity of the MDR gene product, GP170, with PH I regulation in rat hepatoma cells in vitro. I. nternational Hepatology Communications 1995;3:S166. Available from:
160. Radvakóva I, Mirossay A, Mojzis J, Mirossay L. The effect of 5’-(N, N-dimethyl)-amiloride on cytotoxic activity of doxorubicin and vincristine in CEM cell lines. Physiol Res 2001;50:283-8.PubMed
161. Raghunand N, Gillies RJ. pH and drug resistance in tumors. Drug Resist Updat 2000;3:39-47.DOIPubMed
162. Garcãa-caã±ero R. Transport activity of the multidrug resistance protein is accompanied by amiloride-sensitive intracellular pH changes in rat hepatoma cells. Hepatology Research 1998;10:27-40.DOI
163. Geers C, Gros G. Effects of carbonic anhydrase inhibitors on contraction, intracellular pH and energy-rich phosphates of rat skeletal muscle. J Physiol 1990;423:279-97.DOIPubMed PMC
164. Swietach P, Patiar S, Supuran CT, Harris AL, Vaughan-Jones RD. The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths. J Biol Chem 2009;284:20299-310.DOIPubMed PMC
165. Chiche J, Ilc K, Laferrière J, et al. Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH. Cancer Res 2009;69:358-68.DOIPubMed
166. Bin K, Shi-Peng Z. Acetazolamide inhibits aquaporin-1 expression and colon cancer xenograft tumor growth. Hepatogastroenterology 2011;58:1502-6.DOIPubMed
167. Parkkila S, Rajaniemi H, Parkkila AK, et al. Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro. Proc Natl Acad Sci ;97:2220-4.DOIPubMed PMC
168. Duan L, Di Q. Acetazolamide suppresses multi-drug resistance-related protein 1 and p-glycoprotein expression by inhibiting aquaporins expression in a mesial temporal epilepsy rat model. Med Sci Monit 2017;23:5818-25.DOIPubMed PMC
169. Zheng G, Zhou M, Ou X, et al. Identification of carbonic anhydrase 9 as a contributor to pingyangmycin-induced drug resistance in human tongue cancer cells. FEBS J 2010;277:4506-18.DOIPubMed
170. Kopecka J, Campia I, Jacobs A, et al. Carbonic anhydrase XII is a new therapeutic target to overcome chemoresistance in cancer cells. Oncotarget 2015;6:6776-93.DOIPubMed PMC
171. Teodori E, Braconi L, Bua S, et al. Dual P-glycoprotein and CA XII inhibitors: a new strategy to reverse the P-gp mediated multidrug resistance (MDR) in cancer cells. Molecules 2020;25:1748.DOIPubMed PMC
172. Zhao X, Zhang N, Huang Y, et al. Lansoprazole alone or in combination with gefitinib shows antitumor activity against non-small cell lung cancer a549 cells in vitro and in vivo. Front Cell Dev Biol 2021;9:655559.DOIPubMed PMC
173. Huntington KE, Louie A, Zhou L, et al. Colorectal cancer extracellular acidosis decreases immune cell killing and is partially ameliorated by pH-modulating agents that modify tumor cell cytokine profiles. Am J Cancer Res 2022;12:138-51.PubMed PMC
174. Azzarito T, Venturi G, Cesolini A, Fais S. Lansoprazole induces sensitivity to suboptimal doses of paclitaxel in human melanoma. Cancer Lett 2015;356:697-703.DOIPubMed
175. Yu M, Lee C, Wang M, Tannock IF. Influence of the proton pump inhibitor lansoprazole on distribution and activity of doxorubicin in solid tumors. Cancer Sci 2015;106:1438-47.DOIPubMed PMC
176. Goh W, Sleptsova-Freidrich I, Petrovic N. Use of proton pump inhibitors as adjunct treatment for triple-negative breast cancers. An introductory study. J Pharm Pharm Sci 2014;17:439-46.DOIPubMed
177. Hansen AR, Tannock IF, Templeton A, et al. Pantoprazole Affecting Docetaxel Resistance Pathways Via Autophagy (PANDORA): phase II trial of high dose pantoprazole (autophagy inhibitor) with docetaxel in metastatic castration-resistant prostate cancer (mCRPC). Oncologist 2019;24:1188-94.DOIPubMed PMC
178. Li Z, He P, Long Y, et al. Drug repurposing of pantoprazole and vitamin c targeting tumor microenvironment conditions improves anticancer effect in metastatic castration-resistant prostate cancer. Front Oncol 2021;11:660320.DOIPubMed PMC
179. Lu ZN, Shi ZY, Dang YF, et al. Pantoprazole pretreatment elevates sensitivity to vincristine in drug-resistant oral epidermoid carcinoma in vitro and in vivo. Biomed Pharmacother 2019;120:109478.DOIPubMed
180. Tvingsholm SA, Dehlendorff C, Østerlind K, Friis S, Jäättelä M. Proton pump inhibitor use and cancer mortality. Int J Cancer 2018;143:1315-26.DOIPubMed PMC
181. Wang X, Liu C, Wang J, Fan Y, Wang Z, Wang Y. Proton pump inhibitors increase the chemosensitivity of patients with advanced colorectal cancer. Oncotarget 2017;8:58801-8.DOIPubMed PMC
182. Federici C, Petrucci F, Caimi S, et al. Exosome release and low pH belong to a framework of resistance of human melanoma cells to cisplatin. PLoS One 2014;9:e88193.DOIPubMed PMC
183. Luciani F, Spada M, De Milito A, et al. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs. J Natl Cancer Inst 2004;96:1702-13.DOIPubMed
184. Wang BY, Zhang J, Wang JL, et al. Intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer. J Exp Clin Cancer Res 2015;34:85.DOIPubMed PMC
185. Tavana E, Mollazadeh H, Mohtashami E, et al. Quercetin: a promising phytochemical for the treatment of glioblastoma multiforme. Biofactors 2020;46:356-66.DOIPubMed
186. Shim CK, Cheon EP, Kang KW, Seo KS, Han HK. Inhibition effect of flavonoids on monocarboxylate transporter 1 (MCT1) in Caco-2 cells. J Pharm Pharmacol 2007;59:1515-9.DOIPubMed
187. Albatany M, Meakin S, Bartha R. The monocarboxylate transporter inhibitor quercetin induces intracellular acidification in a mouse model of glioblastoma multiforme: in-vivo detection using magnetic resonance imaging. Invest New Drugs 2019;37:595-601.DOIPubMed
188. Borska S, Chmielewska M, Wysocka T, Drag-Zalesinska M, Zabel M, Dziegiel P. In vitro effect of quercetin on human gastric carcinoma: targeting cancer cells death and MDR. Food Chem Toxicol 2012;50:3375-83.DOIPubMed
189. Li S, Zhao Q, Wang B, et al. Quercetin reversed MDR in breast cancer cells through down-regulating P-gp expression and eliminating cancer stem cells mediated by YB-1 nuclear translocation. Phytother Res 2018;32:1530-6.DOIPubMed
190. Kumar M, Sharma G, Misra C, et al. N-desmethyl tamoxifen and quercetin-loaded multiwalled CNTs: a synergistic approach to overcome MDR in cancer cells. Mater Sci Eng C Mater Biol Appl 2018;89:274-82.DOIPubMed
191. Liu M, Fu M, Yang X, et al. Paclitaxel and quercetin co-loaded functional mesoporous silica nanoparticles overcoming multidrug resistance in breast cancer. Colloids Surf B Biointerfaces 2020;196:111284.DOIPubMed
192. Zhou Y, Zhang J, Wang K, et al. Quercetin overcomes colon cancer cells resistance to chemotherapy by inhibiting solute carrier family 1, member 5 transporter. Eur J Pharmacol 2020;881:173185.DOIPubMed
193. Marques MB, Machado AP, Santos PA, et al. Anti-MDR effects of quercetin and its nanoemulsion in multidrug-resistant human leukemia cells. Anticancer Agents Med Chem 2021;21:1911-20.DOIPubMed
194. Liu S, Li R, Qian J, et al. Combination therapy of doxorubicin and quercetin on multidrug-resistant breast cancer and their sequential delivery by reduction-sensitive hyaluronic acid-based conjugate/d-α-tocopheryl poly(ethylene glycol) 1000 succinate mixed micelles. Mol Pharm 2020;17:1415-27.DOIPubMed
195. Liu Z, Balasubramanian V, Bhat C, et al. Quercetin-based modified porous silicon nanoparticles for enhanced inhibition of doxorubicin-resistant cancer cells. Adv Healthc Mater 2017;6:1601009.DOIPubMed
196. Lv L, Liu C, Chen C, et al. Quercetin and doxorubicin co-encapsulated biotin receptor-targeting nanoparticles for minimizing drug resistance in breast cancer. Oncotarget 2016;7:32184-99.DOIPubMed PMC
197. Chen C, Zhou J, Ji C. Quercetin: a potential drug to reverse multidrug resistance. Life Sci 2010;87:333-8.DOIPubMed
198. Scambia G, Ranelletti FO, Panici PB, et al. Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target. Cancer Chemother Pharmacol 1994;34:459-64.DOIPubMed
199. Iriti M, Kubina R, Cochis A, et al. Rutin, a quercetin glycoside, restores chemosensitivity in human breast cancer cells. Phytother Res 2017;31:1529-38.DOIPubMed
200. Limtrakul P, Khantamat O, Pintha K. Inhibition of P-glycoprotein function and expression by kaempferol and quercetin. J Chemother 2005;17:86-95.DOIPubMed
201. Chen Z, Huang C, Ma T, et al. Reversal effect of quercetin on multidrug resistance via FZD7/β-catenin pathway in hepatocellular carcinoma cells. Phytomedicine 2018;43:37-45.DOIPubMed
202. Yuan Z, Wang H, Hu Z, et al. Quercetin inhibits proliferation and drug resistance in KB/VCR oral cancer cells and enhances its sensitivity to vincristine. Nutr Cancer 2015;67:126-36.DOIPubMed
203. Daglioglu C. Enhancing tumor cell response to multidrug resistance with PH-sensitive quercetin and doxorubicin conjugated multifunctional nanoparticles. Colloids Surf B Biointerfaces 2017;156:175-85.DOIPubMed
204. Kim SH, Yeo GS, Lim YS, et al. Suppression of multidrug resistance via inhibition of heat shock factor by quercetin in MDR cells. Exp Mol Med 1998;30:87-92.DOIPubMed
205. Zhang J, Luo Y, Zhao X, et al. Co-delivery of doxorubicin and the traditional Chinese medicine quercetin using biotin-PEG2000-DSPE modified liposomes for the treatment of multidrug resistant breast cancer. RSC Adv 2016;6:113173-84.DOI
206. Singh A, Patel SK, Kumar P, et al. Quercetin acts as a P-gp modulator via impeding signal transduction from nucleotide-binding domain to transmembrane domain. J Biomol Struct Dyn 2020:1-9.DOIPubMed
207. Chen FY, Cao LF, Wan HX, et al. Quercetin enhances adriamycin cytotoxicity through induction of apoptosis and regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling in multidrug-resistant leukemia K562 cells. Mol Med Rep 2015;11:341-8.DOIPubMed
208. Czepas J, Gwoździński K. The flavonoid quercetin: possible solution for anthracycline-induced cardiotoxicity and multidrug resistance. Biomed Pharmacother 2014;68:1149-59.DOIPubMed
209. Maruszewska A, Tarasiuk J. Quercetin triggers induction of apoptotic and lysosomal death of sensitive and multidrug resistant leukaemia HL60 cells. Nutr Cancer 2021;73:484-501.DOIPubMed
210. Xu W, Xie S, Chen X, Pan S, Qian H, Zhu X. Effects of quercetin on the efficacy of various chemotherapeutic drugs in cervical cancer cells. Drug Des Devel Ther 2021;15:577-88.DOIPubMed PMC
211. Kim MK, Choo H, Chong Y. Water-soluble and cleavable quercetin-amino acid conjugates as safe modulators for P-glycoprotein-based multidrug resistance. J Med Chem 2014;57:7216-33.DOIPubMed
212. Yuan J, Wong IL, Jiang T, et al. Synthesis of methylated quercetin derivatives and their reversal activities on P-gp- and BCRP-mediated multidrug resistance tumour cells. Eur J Med Chem 2012;54:413-22.DOIPubMed
213. Choiprasert W, Dechsupa N, Kothan S, Garrigos M, Mankhetkorn S. Quercetin, quercetrin except rutin potentially increased pirarubicin cytotoxicity by non-competitively inhibiting the P-glycoprotein-and MRP1 function in living K562/adr and GLC4/adr cells. American Journal of Pharmacology and Toxicology 2010;5:24-33. Available from:
214. Kim MK, Park KS, Choo H, Chong Y. Quercetin-POM (pivaloxymethyl) conjugates: Modulatory activity for P-glycoprotein-based multidrug resistance. Phytomedicine 2015;22:778-85.DOIPubMed
215. Hyun HB, Moon JY, Cho SK. Quercetin suppresses CYR61-mediated multidrug resistance in human gastric adenocarcinoma AGS cells. Molecules 2018;23:209.DOIPubMed PMC
216. Cho CJ, Yu CP, Wu CL, et al. Decreased drug resistance of bladder cancer using phytochemicals treatment. Kaohsiung J Med Sci 2021;37:128-35.DOIPubMed
217. Lu X, Yang F, Chen D, et al. Quercetin reverses docetaxel resistance in prostate cancer via androgen receptor and PI3K/Akt signaling pathways. Int J Biol Sci 2020;16:1121-34.DOIPubMed PMC
218. Amorim R, Pinheiro C, Miranda-Gonçalves V, et al. Monocarboxylate transport inhibition potentiates the cytotoxic effect of 5-fluorouracil in colorectal cancer cells. Cancer Lett 2015;365:68-78.DOIPubMed
219. Andres S, Pevny S, Ziegenhagen R, et al. Safety aspects of the use of quercetin as a dietary supplement. Mol Nutr Food Res 2018;62:1700447.DOIPubMed
220. Gulati N, Laudet B, Zohrabian VM, Murali R, Jhanwar-Uniyal M. The antiproliferative effect of quercetin in cancer cells is mediated via inhibition of the PI3K-Akt/PKB pathway. Anticancer Res 2006;26:1177-81. Available from:
221. Bruning A. Inhibition of mTOR signaling by quercetin in cancer treatment and prevention. Anticancer Agents Med Chem 2013;13:1025-31.DOIPubMed
222. Klappan AK, Hones S, Mylonas I, Brüning A. Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity. Histochem Cell Biol 2012;137:25-36.DOIPubMed
223. Wang K, Liu R, Li J, et al. Quercetin induces protective autophagy in gastric cancer cells: involvement of Akt-mTOR- and hypoxia-induced factor 1α-mediated signaling. Autophagy 2011;7:966-78.DOIPubMed
224. Maurya AK, Vinayak M. Modulation of PKC signaling and induction of apoptosis through suppression of reactive oxygen species and tumor necrosis factor receptor 1 (TNFR1): key role of quercetin in cancer prevention. Tumour Biol 2015;36:8913-24.DOIPubMed
225. Jeong JH, An JY, Kwon YT, Rhee JG, Lee YJ. Effects of low dose quercetin: cancer cell-specific inhibition of cell cycle progression. J Cell Biochem 2009;106:73-82.DOIPubMed PMC
226. Yoshida M, Sakai T, Hosokawa N, et al. The effect of quercetin on cell cycle progression and growth of human gastric cancer cells. FEBS Letters 1990;260:10-3.DOIPubMed
227. Aalinkeel R, Bindukumar B, Reynolds JL, et al. The dietary bioflavonoid, quercetin, selectively induces apoptosis of prostate cancer cells by down - regulating the expression of heat shock protein 90. Prostate 2008;68:1773-89.DOIPubMed PMC
228. Park CH, Chang JY, Hahm ER, et al. Quercetin, a potent inhibitor against beta-catenin/Tcf signaling in SW480 colon cancer cells. Biochem Biophys Res Commun 2005;328:227-34.DOIPubMed
229. Russo GL, Russo M, Spagnuolo C, et al. Quercetin: a Pleiotropic Kinase Inhibitor Against Cancer. In: Zappia V, Panico S, Russo GL, Budillon A, Della Ragione F, editors. Advances in Nutrition and Cancer. Berlin: Springer Berlin Heidelberg; 2014. p. 185-205.DOIPubMed
230. Choi JA, Kim JY, Lee JY, et al. Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int J Oncol 2001;19:837-44.DOIPubMed
231. Marathe K, McVicar N, Li A, et al. Topiramate induces acute intracellular acidification in glioblastoma. J Neurooncol 2016;130:465-72.DOIPubMed
232. Bonnet U, Wiemann M. Topiramate decelerates bicarbonate-driven acid-elimination of human neocortical neurons: strategic significance for its antiepileptic, antimigraine and neuroprotective properties. CNS Neurol Disord Drug Targets 2020;19:264-75.DOIPubMed
233. K. Y. Intracellular Acidification in brain tumors induced by topiramate: in-vivo detection using chemical exchange saturation transfer magnetic resonance imaging. Electronic Thesis and Dissertation Repository ;Available from:
234. Albatany M, Ostapchenko VG, Meakin S, Bartha R. Brain tumor acidification using drugs simultaneously targeting multiple pH regulatory mechanisms. J Neurooncol 2019;144:453-62.DOIPubMed
235. Leniger T, Thöne J, Wiemann M. Topiramate modulates pH of hippocampal CA3 neurons by combined effects on carbonic anhydrase and Cl-/HCO3- exchange. Br J Pharmacol 2004;142:831-42.DOIPubMed PMC
236. Feldmann M, Asselin M, Liu J, et al. P-glycoprotein expression and function in patients with temporal lobe epilepsy: a case-control study. Lancet Neurol 2013;12:777-85.DOIPubMed
237. Stępień KM, Tomaszewski M, Tomaszewska J, Czuczwar SJ. The multidrug transporter P-glycoprotein in pharmacoresistance to antiepileptic drugs. Pharmacoll Rep 2012;64:1011-9.DOIPubMed
238. Balza E, Carlone S, Carta S, et al. Therapeutic efficacy of proton transport inhibitors alone or in combination with cisplatin in triple negative and hormone sensitive breast cancer models. Cancer Med 2022;11:183-93.DOIPubMed PMC
239. Heilos D, Röhrl C, Pirker C, et al. Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins. Oncotarget 2018;9:25661-80.DOIPubMed PMC
240. Inci F, Celik U, Turken B, Özer HÖ, Kok FN. Construction of P-glycoprotein incorporated tethered lipid bilayer membranes. Biochem Biophys Rep 2015;2:115-22.DOIPubMed PMC
241. Belli S, Elsener PM, Wunderli-Allenspach H, Krämer SD. Cholesterol-mediated activation of P-glycoprotein: distinct effects on basal and drug-induced ATPase activities. J Pharm Sci 2009;98:1905-18.DOIPubMed
Koltai T. The complex relationship between multiple drug resistance and the tumor pH gradient: a review. Cancer Drug Resist 2022;5:277-303. http://dx.doi.org/10.20517/cdr.2021.134
Koltai T. The complex relationship between multiple drug resistance and the tumor pH gradient: a review. Cancer Drug Resistance. 2022; 5(2):277-303. http://dx.doi.org/10.20517/cdr.2021.134
Koltai, Tomas. 2022. "The complex relationship between multiple drug resistance and the tumor pH gradient: a review" Cancer Drug Resistance. 5, no.2: 277-303. http://dx.doi.org/10.20517/cdr.2021.134
Koltai, T. The complex relationship between multiple drug resistance and the tumor pH gradient: a review. Cancer Drug Resist. 2022, 5, 277-303. http://dx.doi.org/10.20517/cdr.2021.134
Koltai T. The complex relationship between multiple drug resistance and the tumor pH gradient: a review. Cancer Drug Resist 2022;5:277-303. http://dx.doi.org/10.20517/cdr.2021.134
Koltai T. The complex relationship between multiple drug resistance and the tumor pH gradient: a review. Cancer Drug Resistance. 2022; 5(2):277-303. http://dx.doi.org/10.20517/cdr.2021.134
Koltai, Tomas. 2022. "The complex relationship between multiple drug resistance and the tumor pH gradient: a review" Cancer Drug Resistance. 5, no.2: 277-303. http://dx.doi.org/10.20517/cdr.2021.134
Koltai, T. The complex relationship between multiple drug resistance and the tumor pH gradient: a review. Cancer Drug Resist. 2022, 5, 277-303. http://dx.doi.org/10.20517/cdr.2021.134