Pharmacological Strategies for Overcoming Multidrug Resistance to Chemotherapy

Abstract

Actual mechanisms of multidrug resistance (MDR) to chemotherapy in oncology are considered. ABC-transporters such as P-glycoprotein, BCRP protein, and MRP proteins take part in the development of resistance. The review presents the main classes of chemosensitizers, i.e., inhibitors of ABC transporters of the 1^st-4^th generations. Plant polyphenols, i.e., flavonoids, are commonly referred to as the last (4^th) generation of MDR inhibitors. Chemosensitizers of different classes should be chosen with allowance for the patient mutation-expression profile and the receptor status of a particular tumor. The appropriate dosage of the chemosensitizer and the administration schedule can enhance the process of counteracting MDR.

Multidrug resistance (MDR) is one of the major factors limiting the efficacy of antitumor agents, including for repeated treatment regimes. MDR arises from the use of any therapeutic medicine, including for more personalized and focused treatment strategies such as immunotherapy, hormonal therapy, and targeted therapy [1]. Enhanced elimination from cells due to hyperexpression of transmembrane transporter proteins belonging to the ATP-binding cassette (ABC) transporter family [P-glycoprotein (P-gp), multidrug resistance proteins (MRP), breast cancer resistance protein (BCRP)], including the solute carrier (SLC) family of dissolved substance transporters, play the main role in MDR development to first-line therapeutic classical cytostatics, e.g., doxorubicin, cisplatin, methotrexate, 5-fluorouracil, etc. Processes such as the appearance of new mutations, a change of tumor microenvironment, deregulation of apoptosis, and up-regulation of molecular-signaling transmission enzymes also are important in the development of resistance to immunotherapy, hormonal therapy, and targeted therapy. As a result, the cytotoxic action of the drugs decreases and the tumor progresses [2]. Resistance is also associated with increased enzyme metabolic (detoxification) activity, primarily activation of CYP3A4, glutathione transferase P1 – 1, and glutathione peroxidase 4 (GPX4) [3]. Activation of these enzymes leads to changes in the pharmacokinetics of antitumor drugs and undesired drug–drug interactions. The amounts of glutathione, a co-substrate of glutathione-S-transferase, were observed to increase in cell lines resistant to alkylating compounds (embikhine, chlorbutin, melfalan, cyclophosphamide, etc.). Chemical interactions between glutathione and alkylating compounds were catalyzed by glutathione-Stransferase enzymes [4].

The search for strategies for overcoming MDR is proceeding in several directions [5]. The search for ABC-transporter inhibitors is the most rapidly developing direction and is the focus of the present review.

ATP-binding cassette transporters

ATP-binding cassette transporters (ABC-transporters) are translocase transporter systems. ABC-transporters consist of several subunits, one or two of which are transmembrane proteins; others, membrane-bound ATPases. The ATPase subunits utilize adenosine triphosphate (ATP) binding and hydrolysis energy to supply the energy necessary for the transport of substrates through membranes.

ABC-transporters promote the resistance of cells to antibiotics and antitumor drugs, actively eliminating drugs from cells [6].

Hyperexpression of ABC-transporters such as P-gp (ABCB1), MRP-1 (ABCC1), and BCRP (ABCG2) is a common mechanism of MDR development [7].

Such resistance to pharmacotherapy is called MDR because patients develop resistance to not only the drug being taken at the moment but also other drugs [8].

It could be supposed that these proteins can transfer such a large variety of drugs because of the presence of hydrophobic cavities that non-specifically bind xenobiotics.

ABC-transporters were discovered during research on cultivated tumor cells that exhibited resistance to several drugs with different chemical structures. These cells were shown to express elevated levels of an MDR transporter protein that was initially called P-glycoprotein (P-gp) [8]. It is now called MDR1 or ABCB1 [9]. The MDR1 gene is often amplified in cells with MDR. This leads to significantly increased synthesis of MDR1, P-gp itself. Substrates of mammalian ABCB1 are mainly planar lipid-soluble molecules with one or several positive charges. All substrates compete with each other for transport by analogy to serum albumin. Many drugs transported by ABCB1 are small nonpolar drugs that diffuse through the extracellular milieu into the cytosol where they block various cellular functions. Such drugs, e.g., colchicine and vinblastine, which block microtubule assembly, permeate freely through the membrane into the cytosol. However, transport of these drugs by ABCB1 reduces their intracellular concentration. Therefore, higher drug concentrations are required to destroy cells expressing ABCB1 than cells that do not have hyperexpression this transporter.

ABCC1 (MRP1) and ABCG2 (BCRP) are other ABC-transporters that promote MDR.

ABC-transporters are also expressed in membranes of healthy cells, where they facilitate transport of various endogenous and exogenous compounds. For example, ABC-transporters such as P-gp, MRP, and BCRP limit the absorption of many drugs from the intestines and transfer drugs and their metabolites from liver cells into bile [10].

Because P-gp participates in the development of resistance to not only antibiotics and cytostatics but also many drugs that are either its substrates or even inductors, the analysis of polymorphism in the genes of P-gp and other transporters involved in MDR, like the search for overcoming resistance, is assuming a larger role. For this, the use of computational technology to design inhibitors for targeted chemical synthesis was also proposed [11].

The fact that P-gp inhibitors include not only synthetic drugs but also natural polyphenols and alkaloids such as quercetin, curcumin, and tetrandine has important practical significance [12]. A tendency for combined or alternating drug therapy methods, e.g., chemotherapy and immunotherapy or hormonal and radiation therapy, was recently noted as a means to avoid MDR. Modern aspects of the battle with MDR to increase the efficacy of antitumor therapy include the search for ferroptosis inductors (e.g., using erastin) as an alternative cell-death mechanism, the search for stabilizers of epigenetic changes and chromatin confirmational changes, inhibitors of the epithelial–mesenchymal transition, and signaling pathway inhibitors [13].

Pharmacological inhibition of P-gp and other ABCtransporters

Drugs with the indication “MDR reduction” do not currently exist. However, the search for inhibitors of P-gp and other ABC-transporters is continuing. Four generations of MDR modulators/inhibitors, mainly affecting P-gp, have been identified (Table 1).

Table 1.

Typical 1^st-4^th Generation P-Glycoprotein Inhibitors [14 – 16]

The 1^st generation includes calcium-channel blockers (verapamil, diltiazem, and azidopine), quinidine derivatives, calmodulin inhibitors (trifluoroperazine and chlorpromazine), and the immunodepressants cyclosporin A and tacrolimus. Second generation modulators include derivatives of 1^st-generation compounds, e.g., R-verapamil and PSC-833 (a cyclosporin derivative). The 3^rd generation of MDR modifiers was developed for specific interaction with a certain MRP transporter, primarily 3^rd generation modulators with high affinity for P-gp, and were developed just for this purpose, in contrast to 1^st- and 2^nd-generation drugs with their own pharmacological activity.

The 3^rd generation of P-gp inhibitors comprises zosuquidar (LY335979), mitotane (NSC-38721), laniquidar (R101933), tariquidar (XR9576), ONT-093, elacridar (F12091), annamycin, HM30181, R10933, and biricodar [17]. Tariquidar is the most well studied.

Tariquidar is a 3^rd-generation specific P-gp inhibitor. It was approved by the FDA for clinical trials as a chemosensitizer and belongs to the benzanilide class of aromatic compounds containing an aniline group in which a carboxamide group is substituted by a benzene ring. They have the common structure RNC(=O)R′, where R and R′ = benzene.

It was proposed that tariquidar and its derivatives could bind to the H-binding part of P-gp through several binding mechanisms.

Phase 1 and 2 clinical trials in humans of a combination of tariquidar and antitumor drugs were halted because of insufficient efficacy and side effects. However, data from clinical trials of tariquidar in healthy volunteers regarding its ability to inhibit P-gp in blood–brain barrier cells and facilitate penetration into the brain of P-gp substrates were reported [18].

Compounds of plant origin, e.g., curcumin, quercetin, ellagic and caffeic acids, piperine, capsaicin, and limonin, did not escape the attention of researchers in the search for P-gp inhibitors.

Natural compounds such as curcumin and flavonoids (e.g., kaempferol and quercetin), which can significantly inhibit P-gp and suppress MDR [19], are considered 4th generation inhibitors. Many plant compounds are inhibitors, substrates, inductors, and/or activators of transporter-protein drugs [20].

The multi-target polyphenol quercetin and its derivatives were investigated in 2010 for treating atherosclerosis, hepatitis C, various malignant diseases, and viral infections including SARS-CoV-2 [21]. Studies of isoquercetin, among others, as an antidepressant began in 2017 [22, 23].

Also, isoquercetin was a Wnt-signaling pathway inhibitor [24]. Because activation of certain signaling pathways of epidermal growth factor receptor (EGFR), renin-angiotensin system (Ras), phosphatidylinositol-3-kinase/protein-kinase B (PI3K/Akt), Wnt, Notch, and transforming growth factor beta (TGF-β) is an important factor in the development of resistance, many natural P-gp inhibitors inhibiting these pathways correspondingly possess dual chemosensitizing mechanisms of action [25].

Several dietary polyphenols contained in grapefruit, cherries, and other red berries are human P-gp inhibitors. Singh, et al. first demonstrated that cyanidin-3-O-sophoroside from cherries was a specific P-gp inhibitor [26]. Other polyphenols tested in the work (quercetin, quercetin-3-glucoside, naringenin, ellagic acid, cynidin-3-sophoroside, oenin, malvin, kuromanin, keracyanin, caffeic acid, chlorogenic acid, trans-ferulic acid, catechin, epicatechin and narcissoside, and ellagic acid) also inhibited P-gp [26]. However, the polyphenols did not totally inhibit P-gp. Various polyphenol–verapamil combinations were tested in this research and demonstrated very strong inhibitory effects that could be used in chemotherapy protocols to treat polyresistant tumors.

Although the exact binding mode of an inhibitor to P-gp and the mechanism of action of a potential inhibitor are unknown, it is highly probable that many potent P-gp inhibitors stabilize the internally directed conformational state of the pump and; therefore, prevent formation of a sandwich-dimer of the nucleoside-binding domain and thereby inhibit ATP hydrolysis.

The effects of several P-gp inhibitors may be related to a change of fluidity (membrane viscosity). For example, quercetin alters the anisotropy of the fluorescent dyes 6-phenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH), indicating that the membrane fluidity changes. P-gp is sensitive to membrane fluidity changes. Therefore, the above specific membrane effects of quercetin could also promote its inhibitory activity for P-gp.

According to Singh, et al., quercetin increases the membrane fluidity in P-gp-positive cells but, on the other hand, reduces it in P-gp-negative cells [26]. The fluidizing effect of quercetin in P-gp-positive cells could be explained by P-gp-dependent sequestration of quercetin in the membrane acyl region, which was traced using the DPH anisotropy change [26].

Different inhibition mechanisms could occur for a single inhibitor. Dong, et al. proposed the following mechanisms [27]:

1) competitive inhibition of P-gp-mediated efflux by directly binding to the drug-binding sites on P-gp;

2) non-competitive inhibition of P-gp-mediated efflux by interacting with an allosteric residue of P-gp and modulating its active conformation;

3) non-competitive inhibition of P-gp-mediated efflux by blocking the binding of ATP to the ATP-binding site on P-gp;

4) disturbance of the membrane environment of cancer cells by interacting with the membrane lipid bilayer;

5) direct down-regulation of the expression of P-gp by targeting its upstream regulators.

The lipophilicity of a potential inhibitor was the main structural attribute for P-gp inhibitory activity. A phenol ring carrying several methoxyls (especially o-dimethoxy) increased the lipophilicity of P-gp inhibitors according to computer modeling and could participate in the formation of H-bonds with P-gp.

Several P-gp inhibitors combine several inhibition mechanisms, e.g., inhibit both the P-gp transport function and its expression (quercetin, curcumin). It is noteworthy that most of these P-gp inhibitors were studied in vitro on tumor cells.

Targeted antitumor drugs include compounds that can inhibit P-gp. These are primarily tyrosine kinase inhibitors (TKIs). The TKIs lapatinib and poziotinib [28] and Janus kinase 2 (JAK2) inhibitors such as fedratinib and pacritinib suppress P-gp activity and increase the sensitivity of tumor cells to vincristine [29, 30].

TKIs interact with the catalytic site of the tyrosine kinase domain and compete with ATP, which blocks the kinase activity by preventing the initiation of low-lying signaling pathways and consequently reducing cell proliferation and survival. TKIs were shown to be capable of affecting the ATPase activity of ABC-transporters. For example, nilotinib (Tasigna), a Bcr-Abl inhibitor, was approved for therapy of chronic myelogenous leukemia and inhibits the functioning of transporters ABCB1, ABCG2, and ABCC10 and eliminates drug resistance. Rivoceranib, a vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitor, eliminates ABCB1- and ABCG2-mediated resistance of tumor cells; lapatinib (Tykerb), a dual tyrosine kinase inhibitor (blocks the HER2/neu and EGFR pathways), inhibits P-gp and MRP-proteins; infigratinib (BGJ 398) is an effective chemosensitizer of tumor cells resistant to paclitaxel and doxorubicin, inhibiting P-gp and MRP-proteins of the ABCB1 family [13].

Specific TKIs targeted at tumor cells were developed because genomic aberrations of anaplastic lymphoma kinase (ALK) promote their proliferation and survival. Several studies reported that ALK inhibitors could inhibit ABC-transporters and eliminate MDR in addition to their initially found action on ALK. They included crizotinib, ceritinib, alectinib, and lorlatinib in combination with cytostatic antitumor drugs that could be used to overcome MDR in clinical practice [31].

The so-called “repurposing” of drugs approved long ago and actively used in clinical practice for new applications as chemosensitizers is a modern strategy for discovering routes to overcome MDR. For example, Lai, et al. analyzed FDA-approved drugs with inhibitory activity against P-gp and identified several candidates such as clarithromycin, sildenafil, and metformin, which are already being studied in combination with antitumor agents and could have potential for further use in oncological practice as chemosensitizers for enhancing the activity of cytostatics [32]. Experiments on animals and in cell cultures found chemosensitizing activity for the pregnane progestins progesterone and its derivatives medroxyprogesterone acetate (MPA), megestrol acetate (MA), and gestobutanoil [7]. The progestins MPA and MA could also be used as chemosensitizers in treatment of various tumors because they are indicated for treating hormone-dependent tumors.

Conclusion

The mechanisms of MDR development are varied and related not only to hyperexpression of transporter proteins such as P-gp, BCRP, and MRP-proteins but also newer evolutionally determined factors. Tumor cells probably devise more finely tuned and unique resistance mechanisms particular to each newly developed antitumor agent incorporated into clinical practice. Obviously, newer and newer resistance mechanisms will emerge.

The search for drugs inhibiting the elimination of an antitumor drug from the cell and its accelerated inactivation, i.e., the search for ABC-transporter and MRP-protein inhibitors, like the search for inhibitors of detoxification enzymes in clinical trials, has not shown specificity and efficacy. This may be related to the unfortunate design of the clinical trials where all factors were not considered in selecting volunteers, e.g., expression of MDR-proteins, receptor hormones, and growth factor and epigenetic characteristics of the tumor cell such as the degree of SUMOylation, acetylation, ubiquitination, and phosphorylation of the target proteins of the antitumor therapy. These epigenetic factors prevent effective suppression of tumor cell growth.

Also, brief inhibition of the elimination of antitumor agents could be considered a key strategy for overcoming MDR, especially in the initial chemotherapy stages. An incontrovertibly simple and universal route is blockage of ABC-transporters. The search for ways to overcome resistance from a pharmaco-economic viewpoint should involve not the selection of a new chemical structure for a chemosensitizer but an adequate regime and dosage for using it.

A personalized determination of biochemical markers and gene polymorphism, e.g., P-gp expression, CYP3A4 polymorphism, glutathione transferase P1 – 1 expression, GPX4 expression, and glutathione level in tumor, must be introduced into routine practice for timely utilization of an antitumor drug with one mechanism of action or another. Monitoring and consideration of its dynamics during treatment are even more important.