Review Article
TMZ Resistance in Glioblastoma: Role of P-Glycoprotein
Veronica De Gregorio, Anna Lisa Iorio, Martina da Ros, Laura Giunti, Lorenzo Genitori, Maurizio de Martino and Iacopo Sardi*
Corresponding Author: Iacopo Sardi, MD, PhD, Neuro Oncology Unit, Department of Pediatric Oncology, Meyer Childrens University Hospital, Viale G.Pieraccini 24, 50139 Florence, Italy.
Received: November 14, 2017; Revised: January 26, 2018; Accepted: December 21, 2017
Citation: Gregorio V D, Iorio A L, Ros M D, Giunti L, Genitori L, et al. (2018) TMZ Resistance in Glioblastoma: Role of P-Glycoprotein. J Neurosurg Imaging Techniques, 3(1): 158-171.
Copyrights: ©2018 Gregorio V D, Iorio A L, Ros M D, Giunti L, Genitori L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Temozolomide is the most common antineoplastic agent used for glioblastoma therapy. Some patients can develop early resistance to this compound. Overcoming chemoresistance could be an important challenge to improve the prognosis and increase the survival of these patients. The action of some efflux transporters localized on the blood-brain barrier seems to be the main mechanism involved in the resistance. An intriguing member of this resistance mechanism is the P-glycoprotein, an ABC transporter.

In this review, we focus on discussing the role of P-glycoprotein in temozolomide resistance of glioblastoma multiforme. We summarize the current literature on structure, localization and activity of this protein, highlighting its role on the distribution of different drugs used for the treatment of brain tumors and other cancers.

 

Keywords: P-gp, Temozolomide, Chemoresistance, Glioblastoma, Blood-brain barrier, Multidrug resistance

 

Abbreviations: P-gp/ABCB1/MDR1: Permeability glycoprotein; GBM: Glioblastoma Multiforme; TMZ: Temozolomide; BBB: Blood-brain Barrier; CNS: Central Nervous System; ABC Transporter: ATP-binding Cassette Transporter; MDR: Multidrug Resistance; BCRP/ABCG2: Breast Cancer Resistance Protein; MRPs: Multidrug Resistance-Associated Proteins; OS: Overall Survival; MGMT: O6-methlyguanine-DNA-methyltransferase; MMR: Mismatch Repair; TMDs: Transmembrane Domains; THMs: Transmembrane α-Helices; NBDs: Nucleotide Binding Domains; CPT-11: Irinotecan; TKIs: Tyrosine kinase Inhibitors; EGFR: Epidermal Growth Factor Receptor; ECF: Extracellular Fluid; siRNA: Small Interfering RNA; RNAi: RNA Interference; lncRNAs: Long Non-Coding RNAs; NPs: Nanoparticles; EGF: Epidermal Growth Factor

INTRODUCTION

 

GBM is the most common and aggressive primary malignant brain tumor [1,2] with an incidence of 3 cases per 100,000 individuals each year and a median OS less than one year [1,3].

Current standard of care for GBM consists of surgical resection, radiation therapy and chemotherapy. TMZ represents the frontline chemotherapy treatment for GBM [4-6], TMZ together with surgical resection and radiotherapy has improved the prognosis for GBM patients [1,7-10]; however, despite improvements in therapeutic treatment, quality of life and prognosis remain very poor. Moreover the management of GBM patients is complicated by the presence of drug resistance mechanisms that are a common cause for therapeutic failure of several drugs, including TMZ.

TMZ is an oral alkylating chemotherapeutic compound that is able to cross the BBB and acts generating O6-methylguanine adducts which introduce mis-pairs with thymine; it is not possible to repair these adducts which thus create DNA damage resulting in cell cycle arrest, cell death  and senescence [4,11-13].

An understanding of the molecular processes associated to resistance is critical to find and develop mechanisms to sensitize GBM cells to TMZ. Several studies try to explain TMZ resistance; MGMT and the MMR system appear to be involved in the failure of TMZ treatment [4,13-17].

A possible candidate responsible of resistance to antineoplastic agents, including TMZ, in GBM patients is the P-gp which belongs to the ABC transporter family [18].

P-gp (ABCB1 or MDR1) is an ATP-driven efflux pump which utilizes ATP hydrolysis to transport various substrates across the plasma mem­brane of several tissues [4]; protein expression has been reported not only in healthy tissues but also in many tumors, including brain tumors [18-23]. Concerning cancer, P-gp is a potent efflux-pump, which through its mechanism of action, is involved in the expulsion of several drugs out of the tumor cells: this mechanism provides an explanation for the resistance of tumor cells to multiple antineoplastic agents known as MDR [4,18,24].

The present review is focused on the role played by P-gp in TMZ resistance of GBM analyzing the molecular and biological mechanisms through which this efflux pump could represent a limiting factor for success of TMZ treatment.

Structure and Function of P-gp

BBB is the greatest challenge in the treatment of CNS tumors, representing the primary obstacle to drug delivery into CNS. It is a dynamic interface that separates the brain from the blood ensuring CNS homeostasis and protecting the brain from potentially harmful substances [18,25,26].

BBB consists of capillary endothelial cells not fenestrated, joined together by tight junctions limiting the passage of solutes [18,27-29]. Moreover brain endothelial cells are characterized by the presence of specific transport systems that regulate the entry of compounds [30]. The major transport system is represented by the ABC transporter family. These efflux transporters are responsible of MDR phenotype, binding and hydrolyzing ATP; BBB is therefore strongly protective for the CNS, but at the same time becomes a limiting factor to treatment of CNS diseases regulating the entry of drugs into the brain.

ABC transporter family is an evolutionarily conserved family of proteins suggesting its paramount role in survival of the species [19,21,31-33]; so far, 49 ABC transporters have been identified and classified in different human tissues [33-36]. The prominent members of this family are P-gp, BCRP (or ABCG2) and MRPs.

P-gp was the first of these transporters to be identified and analyzed [37,38]; it is currently the best known efflux-pump in humans, probably for its significant role in cancer cells chemoresistance. P-gp is coded by the multiple drug resistance MDR1 gene localized on chromosome 7, it is a single 170 kDa polypeptide [33]. The protein is made of two TMDs, each consisting of six highly hydrophobic TMHs, and two NBDs involved in the binding and hydrolysis of ATP [33,39-41].

P-gp is expressed preferentially in organs with excretory role and in tissues with barrier function, particularly in the apical membrane of epithelial cells including liver, kidney, intestine and apical membrane of endothelial cells of the capillaries of the brain [33]: protein localization suggests a role in the defense of susceptible organs, such as the brain, from toxic compounds and in the secretion of metabolites or xenobiotics [33,42]. In addition P-gp expression was also found in pancreas, adrenal gland, placenta, testis and on the surface of hematopoietic cells [33,43].

P-gp is a plasma membrane protein able to interact with several compounds including chemotherapeutic drugs, immunosuppressive agents, calcium channel blockers and natural products, among many others, pumping them out of the cells [18,33,39,41,44-46].  P-gp substrates differ in size, structure and function, even if most of them are weakly amphipathic and relatively hydrophobic [33,41]. This broad substrate specificity is in agree with P-gp role as efflux pump involved in removing substrates from the inner to the outer side of cell plasma membrane or directly into the extracellular space, preventing the accumulation inside tissues of a variety of compounds, such as drugs, xenobiotics, toxins and metabolites [19,47].

P-gp Influences CNS Distribution of Chemical Agents

Several mechanisms seem to be involved in the poor response of brain tumors to chemotherapy, drugs delivery to the CNS continues to be a clinical challenge.

Some studies have shown the role of P-gp and other ABC transporters, especially BCRP, in preventing therapeutic agents penetration into the brain, including conventional antitumor compounds such as vinca alkaloids, anthracyclines and taxanes [19,48-52]; indeed the expression of these transporters is associated with inherent or acquired MDR [53]. Moreover, recent literature suggests that P-gp and BCRP work together and cooperate at the BBB with a synergistic effect, reducing significantly brain penetration of drugs and consequently their effectiveness [18,54-58].

CPT-11 is an antineoplastic agent which acts inhibiting DNA topoisomerase I, a nuclear enzyme involved in DNA replication, repair and transcription [59, 60]. This compound is strongly used for colorectal cancer treatment and shows an interesting activity against other type of tumors, including GBM [59, 61]. In vivo and in vitro studies suggested that CPT-11 is able to cross the BBB, but its activity is strongly limited by P-gp action that reduces brain penetration not only of CPT-11, but also of its active metabolite [59].

de Vries et al. demonstrated that BCRP and P-gp work together to reduce plasma exposure and brain penetration of topotecan in a knockout mice model [57]. Topotecan, inhibitor of topoisomerase I, is a derivative of camptothecin; its efficacy is confirmed in the treatment of ovarian, lung, and cervical cancer and seems to have a moderate effect also in adults with primary malignant glioma [57,62].

Lapatinib, antineoplastic agent, is a member of the 4-anilinoquinazoline class of TKIs; different studies on lapatinib and other TKIs reported the involvement of the efflux transporters of the BBB, among which P-gp, as responsible of low brain concentration of these compounds [63-66]. Regarding TKIs, in another study Agarwal et al. suggested the involvement of P-gp and BCRP on the distribution of an EGFR inhibitor, gefitinib, to the CNS [54]. In vivo and in vitro experiments showed that both transporters are able to reduce the intracellular accumulation of gefitinib favoring its outflow. As confirmation of these results, brain distribution of gefitinib improved and increased when it was co-administered with a dual P-gp and BCRP inhibitor, suggesting a combined therapy of gefitinib and this type of inhibitors as a novel opportunity for cancer treatment [54].

Another inhibitor of EGFR, erlotinib, is a known substrate of P-gp which prevents its brain penetration; unfortunately erlotinib does not seem to be successful in different clinical trials for GBM [67].

In vivo preclinical studies showed that P-gp reduces the brain penetration of alkaloid compounds too and especially of vinblastine, an antineoplastic agent able to bind to tubulin and inhibit microtubule formation, resulting in disruption of mitotic spindle assembly and arrest of the cell cycle [68]. In particular, the authors compared the pharmacokinetics of vinblastine in P-gp knockout and wild type mice. They observed an increase of drug accumulation in tissues, especially in the brain, and a reduction in drug excretion in P-gp knockout mice, suggesting consequently an involvement of P-gp in the efficacy of vinblastine.

Finally by in vivo and in vitro studies, P-gp appeared to interfere with novel anticancer molecules too, such as vemurafenib, a BRAF inhibitor approved for the treatment of patients with metastatic melanoma; active efflux of vemurafenib by P-gp and BCRP strongly reduces its brain distribution [69]. These observations could be very useful for the assessment of vemurafenib in the treatment of brain metastasis.

P-gp and Anticancer Strategies

MDR phenomenon in cancer cells is associated to their resistance to a wide range of anticancer compounds, even if structurally and functionally different [33,70,71]. Intrinsic or acquired resistance could be associated to several mechanisms and biological processes such as action of efflux systems, including P-gp and other ABC transporters, enhanced DNA repair, alteration in apoptosis and metabolic modifications [72,73].

Several research studies are conducted with the purpose to find strategies to sensitize cancer cells to chemotherapeutic agents overcoming drug resistance, in particular P-gp inhibitors/modulators have been developed representing a significant opportunity in clinical setting for P-gp-mediated drug resistance.

Different P-gp inhibitors have been identified and some of these compounds may be efficient in cancer treatment in association with other antineoplastic drugs, such as vincristine and daunorubicin [31, 33]. The P-gp inhibitors are classified in different groups depending on potency, selectivity and drug-drug interaction potential; currently it is possible to distinguish four generations of P-gp inhibitors.

About first-generation inhibitors, many compounds belong to this group among which calcium channel blockers, immunosuppressants, anti-hypertensives, antiarrhythmics and antiestrogens [33]. In 1981, Tsuruo et al. using leukemia cells observed that verapamil, a calcium channel blocker, could reverse drug resistance [74]. Verapamil and cyclosporine A are two important examples of early discovery of P-gp inhibitors/modulators, both are P-gp substrates and act competing with other P-gp substrates for efflux by a mechanism of competitive inhibition [33,45]. Unfortunately, the combination of first-generation MDR inhibitors with anticancer drugs leads to toxic side effects, especially serious cardiovascular toxicity; this aspect makes their use clinically difficult, therefore they were replaced by second generation inhibitors [33,45].

Second generation inhibitors were developed in order to increase inhibitory effects and reduce toxicity at the same time. These compounds are analogues of the first generation inhibitors and they were synthesized by structurally modifications of first generation inhibitors. Valspodar is the best known second generation inhibitor, it has been evaluated in association with anticancer drugs in several clinical trials [33,75-90]. Other examples of second-generation inhibitors are non-immunosuppressive analogues of cyclosporin A, R-enantiomer of verapamil and dexverapamil [33]. Despite second-generation P-gp inhibitors have a better pharmacological profile compared to the first-generation ones, unfortunately these compounds show some disadvantages, indeed they may be responsible of unexpected drug-drug interactions and they are able to inhibit cytochrome P450 resulting in an increase of drug toxicity [74,91-93].

In order to improve the features of P-gp modulators/inhibitors, a third-generation of inhibitors with high affinity for ABC transporters, high specificity and potency has been developed. Third generation includes compounds such as tariquidar and elacridar [33]; tariquidar binds to P-gp through a non-competitive mechanism, while elacridar acts by binding to the allosteric site of P-gp [33,94].

Some studies show that the inhibition of P-gp improves brain drug delivery of some anticancer compounds and consequently the treatment of CNS tumors; Fellner et al. demonstrated that P-gp inhibition by valspodar increases paclitaxel brain levels in nude mice with intracerebrally implanted human U-118 MG glioblastoma [18,95]. Even if in vitro and in vivo studies report that these drugs are associated to an enhancement of chemosensitivity, unfortunately they do not appear associated with an improvement of OS in cancer patients [72,96,97], moreover they continue to show unexpected toxic effects [33,71].

Probably side effects and drug-drug interactions may be responsible of ABC transporter inhibitors failure, limiting their clinical application and their translation from animal model to patient. In addition, inhibiting a specific transporter, the remaining ABC transporters, that are coexpressed in the tumor, could compensate by their biological activity, interfering with inhibition and reducing the effectiveness of the drug inhibitor.

Some research studies tried to develop novel P-gp inhibitors which constitute the fourth generation [33,71]; substances belonging to this group are natural agents and their derivatives, surfactants and lipids, peptidomimetics. Surfactants are responsible of alteration of membrane lipids integrity and are able to modify P-gp structure [33,98] determining loss of P-gp function [33,98]; other substances act through other mechanisms such as limitation of P-gp ATPase activity [33,99]. Finally other agents associate transporters inhibition with another favorable biological function (dual ligands), for example some aminated thioxanthones were able to inhibit cell growth and P-gp activity at the same time [33,100].

Despite all the efforts to develop P-gp inhibitors, only few compounds have been evaluated for their capacity to enhance drug delivery into the CNS; currently researchers are trying to identify new approaches and to find new P-gp inhibitors or novel mechanisms of action, among which natural compounds, small molecule inhibitors, RNA interference and epigenetic regulation [72].

For instance, several TKIs are ABC transporters modulators and have been examined to allow drugs to overcome the BBB increasing their bioavailability; probably many TKIs are very functional for their capacity to inhibit both P-gp and BCRP at the same time. Intriguingly, gefitinib, a TKI, increased topotecan penetration into the brain ECF likely via inhibition of BCRP and P-gp [101-105]. Some in vivo or in vitro studies described other TKIs, such as nilotinib and icotinib, which appeared able to inhibit ABC transporters enhancing chemotherapeutic drugs efficacy [72,106-108].

Flavonoids, alkaloids, coumarins and terpenoids are natural compounds that in vitro or in vivo studies seem to be associated with P-gp downregulation and decrease of proteins expression; their application is promoted by low cost, low toxicity and action extended to other ABC transporters too [72,109-114]. Among these compounds curcumin, active principle of Curcuma longa, was examined in some in vitro models of breast, colon and prostate cancer and appeared to be associated to an increase of sensitivity to some drugs [72,115-118].

Another very interesting approach to fight multidrug resistance is given by the possibility to block and silence ABC transporters expression through siRNA or RNAi mechanisms, approaches which involve RNA molecules to inhibit gene expression or translation.  RNAi technology was used for the first time in 2003 in human cancer cells to knockdown the P-gp encoding mRNA reversing chemoresistance [72]. Since then several in vitro studies, especially on gastric, pancreatic, lung and ovarian cancer, designed stable vectors to overcome chemoresistance decreasing P-gp and other ABC transporter expression, sensitizing cells to antineoplastic compounds and promoting drug accumulation [72,119-120]. LncRNAs is a novel class of transcripts which includes important regulators of transcriptional processes. The lncRNAs have a wide range of functions in cellular and developmental processes and are able to regulate several genes including genes associated to anticancer resistance.

Another strategy to fight MDR is represented by the use of microRNAs, which are endogenous, nonprotein-coding, short RNAs of 20–22 nucleotides, involved in gene expression regulation by the ability to bind mRNA and silence genes. The genes inhibited by microRNAs are involved in different biological processes such as embryogenesis, cell development, proliferation and apoptosis [121,122]. Interestingly, microRNAs are dysregulated in cancer. Researchers are investigating about the possibility of using these molecules as diagnostic or predictive or prognostic biomarkers in different tumors. Their alterations are involved at various levels of chemoresistance mechanism. Some studies identified a pattern of microRNAs responsible of the inhibition of P-gp expression in MCF-7 breast cancer cells and esophageal squamous carcinoma cells [74,123-125]. Other microRNAs are involved in ABC transporters inhibition improving sensitivity to antineoplastic agents, for example miR-122 in hepatocellular carcinoma cells [72,126], miR-19a and miR-19b in gastric cancer cells [72,127], miR-145 in ovarian cancer cells [72,128], miR-137 in neuroblastoma cells in which for instance it regulates the response to doxorubicin treatment [72,129]; intriguingly, miR-21 represents another molecule involved in the response, in particular it appears to promote doxorubic in resistance in GBM T98G cells [130].

Recently some studies identified a role of epigenetic modifications, such as DNA methylation and histone modifications, in the regulation of gene expression associated to chemosensitivity and resistance [72,131].

Finally, nanotechnology-based approach is being developed to overcome multidrug resistance, this approach consisting in constructs with the function to deliver chemical compounds, including drugs, such P-gp inhibitors, or miRNA or RNAi, to specific target cells. These constructs include liposomes, polymer and peptide/protein conjugates, polymeric micelles, polymeric, lipid and inorganic NPs; they show differences in structure, for instance liposomes are small artificial vesicles consisting of lipid bilayers, while NPs are carriers with natural or synthetic polimeric matrix. Concerning these constructs in general, even if they have different conformation, they all function as vehicle of chemical compounds without giving problems associated to high dosage, cell toxicity, low specificity and uptake. Delivery of antitumor molecules to cancer cells through a nanotechnology-based approach could be an interesting novel opportunity to inhibit P-gp expression enhancing intracellular drug concentration [72,74].

P-gp Contributes to Chemoresistance in Glioblastoma

Resistance to antineoplastic agents is the main reason for treatment failure of brain tumors, in particular resistance to TMZ is often quickly acquired by GBM cells; for this reason different studies try to understand molecular mechanisms underlying TMZ resistance with the aim of developing strategies to sensitize GBM cells to TMZ.

The first predictive and prognostic molecular biomarker linked to TMZ resistance is the enzyme MGMT implicated in DNA damage repair associated to TMZ action. According to Hegi et al., hypermethylation of MGMT promoter gene, and consequently its silencing, is observed in about 50% of GBM cases and is associated to a better prognosis and a longer survival regardless of the treatment [132]. Moreover, a major survival benefit was observed in patients with methylatated MGMT promoter treated with a combination of TMZ and radiotherapy compared to patients treated with radiotherapy only, suggesting an association between MGMT methylation and response to TMZ treatment in adult GBM patients.

Unfortunately, chemoresistance is a very complex mechanism linked to different biological processes; therefore MGMT promoter methylation status is not the only marker in GBM resistance. Sardi et al. studying methylation status of MGMT promoter and analyzing the expression of MGMT in pediatric brain tumors treated with TMZ, showed that MGMT was nearly always unmethylated in contrast to what is observed in adult brain tumors [133]; moreover the expression level of MGMT appeared variable. The unmethylated status of MGMT along with the involvement of other DNA repair mechanisms could justify the reduced efficacy of TMZ in pediatric brain tumors.

Another factor associated to chemoresistance in CNS tumors is the expression and the activity of efflux pump proteins, among these P-gp is the first identified xenobiotic drugs ATP-dependent efflux pump and the most examined.

Some studies investigated about the role of P-gp in TMZ resistance. Schaich et al. in GBM patients treated with TMZ, in order to investigate the possible involvement of MDR1 gene variants in patient’s survival, discovered that the exon 12 C1236T polymorphism is predictive of the outcome independently from MGMT status [134]. This observation suggests that this polymorphism could play a role in patient response to TMZ; according to some hypotheses this effect could be associated to the genetic mechanism of linkage disequilibrium or to an altered affinity of P-gp for TMZ. In addition, in vitro analysis showed an increase of cytotoxicity and cell death in MDR1 negative cells after exposure to TMZ compared to MDR1-expressing cells. In the same study, as confirmation of the involvement of MDR1 in the resistance to TMZ, the authors observed a trend to restoration of chemosensitivity to the drug in MDR1-expressing cells when treated with a combination of TMZ and MDR1-inhibitor/modulator, especially cyclosporine A. Finally Schaich et al., reported a significant P-gp expression not only in parenchymal tissue but also in GBM vessels suggesting that drug delivery to the brain and drug resistance are influenced by the activity of P-gp of endothelial cells too [134].

In another study, using in vivo and in vitro models, authors tried to investigate the mechanisms underlying P-gp-mediated resistance to TMZ in GBM [135]. The authors distinguished an active and an inactive form of P-gp, both expressed in GBM cells; TMZ induced the active form of P-gp, indeed an increase of active P-gp was observed when GBM cells were treated with TMZ even if for a short time, the increase was greater for chronical treatment of GBM cells with TMZ. In the study was observed that the increase of active P-gp was induced by TMZ through a bifasic mechanism accomplished by two steps. Firstly, TMZ treatment promoted the traffic of active intracellular P-gp to the cell membrane directly; in a second phase the increase of P-gp was associated to an increase of transcription of MDR1 gene, induced by TMZ-mediated production of EGF, and subsequently to an increase of P-gp protein synthesis.

Regarding P-gp cellular traffiking, TMZ treatment reduced subcellular P-gp level and at the same time increased the active P-gp in the cell membrane, the activation of P-gp requiring a conformational change; moreover the increase of P-gp protein expression appeared to be time-dependent.

As far as the transcription of MDR1 gene is concerned, TMZ promoted this molecular mechanism increasing firstly production and release of EGF, thus promoting enhancement of EGFR signaling, resulting in the activation of the heterodimeric transcription factor AP-1 which in the end promotes the transcription of MDR1 gene. An increase of transcription MDR1 gene protected GBM cells from TMZ treatment with consequent increase of cell survival; as confirmation of these results MDR1 knockdown cells showed an increase of cytotoxicity with a decrease of survival when treated with TMZ. Therefore through an autocrine mechanism TMZ-resistant GBM cells expressed EGFR and in the presence of TMZ produced EGF at the same time, in this way EGF stimulated the same cells inducing MDR1 gene expression through AP-1 activation.

In the same study, the authors observed also that on the other hand the use of kinase inhibitors, such as erlotinib, to block EGFR signaling in combination with TMZ reduced P-gp expression and promoted the action of TMZ on GBM cells, confirming the role and the involvement of EGFR signaling in the induction of MDR1 expression [135].Therefore concerning in vivo models they reported that combined therapy of erlotinib with TMZ promoted a decrease of tumor volumes, confirming the results obtained in vitro.

To summarize, TMZ activates cell surface P-gp, promotes protein expression and the enhancement of the function; combined therapy of TMZ with EGFR inhibitors prevents P-gp activation sensitizing GBM cells to TMZ treatment (Figure 1). In another report Munoz et al., using in vitro model, investigated about the interaction between P-gp and TMZ, in particular they co-administered P-gp fluorescent target with TMZ observing a competitive mechanism between TMZ and the other P-gp substrates [5]. According to the study, competitive mechanism was useful for combined treatment of GBM cells especially of TMZ with P-gp inhibitors, this association resulted in an increase of Caspase 3 activity reducing cell survival. Finally, using a computer modeling system, authors identified a specific region of interaction between TMZ and P-gp localized in the same area of interaction of other P-gp targets, and especially near ATP binding site.

Another study reported the involvement of miRNA-9 in TMZ resistance in GBM cancer stem cells CD133+ (prominin-1), this miRNA appeared connected to MDR1 gene [5,136]. In particular the authors observed that TMZ chemoresistance was associated to an increased level of miRNA-9 which promoted upregulation of MDR1 expression through activation of the SHH/PTCH1/MDR1 axis. Experiments carried out by targeted siRNA confirmed the involvement of SHH pathway in MDR1 upregulation. In general in brain tumors, some microRNAs appear involved in drug sensitivity/resistance. Giunti et al., using in vitro models, showed that miR-21, an oncogenic miRNA overexpressed in human breast cancer, was associated with resistance to doxorubicin in GBM T98G cells [130]; the authors observed a greater sensitivity of GBM T98G cells to doxorubicin, with an increase of apoptosis, when they were transfected with anti-miR-21 inhibitor compared to not trasfected control cells.

However, Zhang et al. showed in glioma cells and especially in P-gp overexpressed cells, an increase of sensitivity to P-gp substrates under the action of TMZ [137]. They reported that when TMZ was co-administered with doxorubicin, TMZ affecting P-gp activity promoted an increase of doxorubicin accumulation with a synergistic mechanism, suggesting that TMZ could reverse doxorubicin resistance improving the treatment efficacy. Doxorubicin is able to promote P-gp expression and in general the expression of the ATP-binding superfamily transporter proteins. In vitro models therefore confirmed synergistic effect between doxorubicin and TMZ showing an increase of doxorubicin accumulation in presence of TMZ, accumulation was significantly higher in presence of high dosage of TMZ. Analyzing the effect of TMZ on drug efflux pump, the authors showed that TMZ does not change P-gp protein expression, but acts inhibiting P-gp directly and especially decreasing P-gp ATPase activity. This mechanism of action could explain synergistic effect of combination between TMZ and doxorubicin; the mechanisms which promote accumulation of doxorubicin may represent a promising novel strategy against malignant gliomas (Figure 2) [137].

 

TMZ affecting P-gp sensitizes GBM cells to different drugs that are all substrates of P-glycoprotein; therefore in general drugs that synergized with TMZ are substrates of P-gp.

According to Riganti et al., also in GBM cancer stem cells TMZ promoted the accumulation of other drugs affecting P-gp activity, in particular TMZ appeared to methylate Wnt3a gene promoter reducing its expression [138]; Wnt3a is an important factor involved in cell growth, tumorigenesis and stemness maintenance. The diminished expression of Wnt3a was associated with a decrease of transcriptional activation of ABCB1 resulting in reduced P-gp protein expression and efflux pump activity. Therefore, through this mechanism, TMZ may sensitize GBM cancer stem cells to P-gp substrate promoting their accumulation in tumor cells and consequently their cytotoxic and antiproliferative effects.

Several P-gp inhibitors are not successful in clinical trials because they show low specificity and high toxicity, for all these reasons TMZ may represent an alternative strategy able to act as chemotherapeutic drug and chemosensitizer agent at the same time [138].

Concluding Remarks and Future Perspective

GBM is the most common and malignant primary brain tumor in adults with a dismal prognosis, a survival of up to 12-18 months and a very low possibility to survive longer than 5 years [137,139].

The main cause of the frequent relapse of this disease is due to chemoresistance of GBM stem cell. Generally GBM stem cell resistance is associated to alterations in different biological processes such as cell cycle, apoptosis, DNA repair; moreover, in brain tumors the BBB is the main responsible of the difficulty for some chemotherapy agent to reach CNS, promoting drug resistance.

Chemoresistance is mainly associated to the activity of efflux pumps; in particular P-gp, BCRP and MRPs proteins work together on the BBB and on the plasma membrane of brain tumors cooperating and playing a relevant role in the MDR phenomenon.

TMZ represents the frontline treatment for GBM, so it is very important to understand the mechanisms underlying TMZ-resistance and find novel approaches to overcome it.

Recently some studies analyzed the key role of P-gp in drug resistance mechanism observed in GBM and especially its implication in TMZ resistance. These reports showed the involvement of genetic variant of MDR1 gene in response to TMZ and the implication of TMZ in P-gp activation; in particular TMZ seems to promote P-gp expression and function. However, targeting P-gp increases sensitivity to TMZ resulting in an increase of apoptosis and therefore reversing the resistance to TMZ. In addition TMZ competes with P-gp substrates, including some antineoplastic agents, representing a promising opportunity for combined targeted therapies.

Further preclinical and clinical investigations are necessary to better understand and overcome resistance mechanism of GBM to TMZ associated to P-gp and other MDR mechanisms, in order to develop novel therapeutic strategies and new molecules or optimize combined therapies for the treatment of CNS tumors.

  1. Su J, Cai M, Li W, Hou B, He H, et al. (2016) Molecularly Targeted Drugs Plus Radiotherapy and Temozolomide Treatment for Newly Diagnosed Glioblastoma: A Meta-Analysis and Systematic Review. Oncol Res 24: 117-128.
  2. Kawano H, Hirano H, Yonezawa H, Yunoue S, Yatsushiro K, et al. (2015) Improvement in treatment results of glioblastoma over the last three decades and beneficial factors. Br J Neurosurg 29: 206-212.
  3. Pope WB, Lai A, Mehta R, Kim HJ, Qiao J, et al. (2011) Apparent diffusion coefficient histogram analysis stratifies progression-free survival in newly diagnosed bevacizumab-treated glioblastoma. Am J Neuroradiol 32: 882-889.
  4. Perazzoli G, Prados J, Ortiz R, Caba O, Cabeza L, et al. (2015) Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression. PLoS One 10: e0140131.
  5. Munoz JL, Walker ND, Scotto KW, Rameshwar P (2015) Temozolomide competes for P-glycoprotein and contributes to chemoresistance in glioblastoma cells. Cancer Lett 367: 69-75.
  6. Friedman HS, Kerby T, Calvert H (2000) Temozolomide and treatment of malignant glioma. Clin Cancer Res 6: 2585-2597.
  7. Rizzo D, Scalzone M, Ruggiero A, Maurizi P, Attina G, et al. (2015) Temozolomide in the treatment of newly diagnosed diffuse brainstem glioma in children: A broken promise? J Chemother 27: 106-110.
  8. Narayana A, Gruber D, Kunnakkat S, Golfinos JG, Parker E, et al. (2012) A clinical trial of bevacizumab, temozolomide, and radiation for newly diagnosed glioblastoma. J Neurosurg 116(2): 341-345.
  9. Stupp R, Hegi ME, Neyns B, Goldbrunner R, Schlegel U, et al. (2010) Phase I/IIa study of cilengitide and temozolomide with concomitant radiotherapy followed by cilengitide and temozolomide maintenance therapy in patients with newly diagnosed glioblastoma. J Clin Oncol 28: 2712-2718.
  10. Vredenburgh JJ, Desjardins A, Kirkpatrick JP, Reardon DA, Peters KB, et al. (2012) Addition of bevacizumab to standard radiation therapy and daily temozolomide is associated with minimal toxicity in newly diagnosed glioblastoma multiforme. Int J Radiat Oncol Biol Phys 82: 58-66.
  11. Günther W, Pawlak E, Damasceno R, Arnold H, Terzis AJ (2003) Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids. Br J Cancer 88: 463-469.
  12. Kheirelseid EAH, Miller N, Chang KH, Curran C, Hennessey E, et al. (2013) Mismatch repair protein expression in colorectal cancer. J Gastrointest Oncol 4(4): 397-408.
  13. Yoshimoto K, Mizoguchi M, Hata N, Murata H, Hatae R, et al. (2012) Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma. Front Oncol 2: 186.
  14. Marchesi F, Turriziani M, Tortorelli G, Avvisati G, Torino F, et al. (2007) Triazene compounds: mechanism of action and related DNA repair systems. Pharmacol Res 56: 275-287.
  15. Melguizo C, Prados J, Gonzalez B, Ortiz R, Concha A, et al. (2012) MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy. J Transl Med 10: 250.
  16. Hsieh P, Yamane K (2008) DNA mismatch repair: molecular mechanism, cancer, and ageing. Mech Ageing Dev 129: 391-407.
  17. Goellner EM, Grimme B, Brown AR, Lin YC, Wang XH, et al. (2011) Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair. Cancer Res 71: 2308-2317.
  18. Agarwal S, Hartz AM, Elmquist WF, Bauer B (2011) Breast cancer resistance protein and P-glycoprotein in brain cancer: two gatekeepers team up. Curr Pharm Des 17: 2793-2802.
  19. Iorio AL, da Ros M, Fantappiè O, Lucchesi M, Facchini L, et al. (2016) Blood-Brain Barrier and Breast Cancer Resistance Protein: A Limit to the Therapy of CNS Tumors and Neurodegenerative Diseases. Anticancer Agents Med Chem 16: 810-815.
  20. Salphati L, Plise EG, Li G (2009) Expression and activity of the efflux transporters ABCB1, ABCC2 and ABCG2 in the human colorectal carcinoma cell line LS513. Eur J Pharm Sci 37: 463-468.
  21. Chung FS, Santiago JS, Jesus MF, Trinidad CV, See MF (2016) Disrupting P-glycoprotein function in clinical settings: what can we learn from the fundamental aspects of this transporter? Am J Cancer Res 6: 1583-1598.
  22. Burger HFJ, Look MP, Meijer-van Gelder ME, Klijn JG, Wiemer EA, et al. (2003) RNA expression of breast cancer resistance protein, lung resistance-related protein, multidrug resistance-associated proteins 1 and 2, and multidrug resistance gene 1 in breast cancer: correlation with chemotherapeutic response. Clin Cancer Res 9: 827-836.
  23. Fattori S, Becherini F, Cianfriglia M, Parenti G, Romanini A, et al. (2007) Human brain tumors: multidrug-resistance P-glycoprotein expression in tumor cells and intratumoral capillary endothelial cells. Virchows Arch 451: 81-87.
  24. Fodale V, Pierobon M, Liotta L, Petricoin E (2011) Mechanism of cell adaptation: when and how do cancer cells develop chemoresistance? Cancer J Sudbury Mass 17: 89-95.
  25. Azad TD, Pan J, Connolly ID, Remington A, Wilson CM, et al. (2015) Therapeutic strategies to improve drug delivery across the blood-brain barrier. Neurosurg Focus 38: E9.               
  26. Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57: 173-185.
  27. Begley DJ, Brightman MW (2003) Structural and functional aspects of the blood-brain barrier. Prog Drug Res 61: 39-78.
  28. Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38: 323-337.
  29. Yu AS, McCarthy KM, Francis SA, McCormack JM, Lai J, et al. (2005) Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells. Am J Physiol Cell Physiol 288: C1231-C1241.
  30. Jones PM, George AM (2004) The ABC transporter structure and mechanism: perspectives on recent research. Cell Mol Life Sci 61: 682-699.
  31. Amin ML (2013) P-glycoprotein Inhibition for Optimal Drug Delivery. Drug Target Insight 7: 27-34.
  32. Sheps JA, Ralph S, Zhao Z, Baillie DL, Ling V (2004) The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes. Genome Biol 5: R15.
  33. Silva R, Vilas-Boas V, Carmo H, Dinis-Oliveira RJ, Carvalho F, et al. (2015) Modulation of P-glycoprotein efflux pump: induction and activation as a therapeutic strategy. Pharmacol Ther 149: 1-123.
  34. Dean M, Hamon Y, Chimini G (2001) The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res 42: 1007-1017.
  35. Couture L, Nash JA, Turgeon J (2006) The ATP-binding cassette transporters and their implication in drug disposition: a special look at the heart. Pharmacol Rev 58: 244-258.
  36. Vasiliou V, Vasiliou K, Nebert DW (2009) Human ATP-binding cassette (ABC) transporter family. Hum Genomics 3: 281-290.
  37. Chen CJ, Chin JE, Ueda K, Clark DP, Pastan I, et al. (1986) Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47: 381-389.
  38. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM (2003) P-glycoprotein: from genomics to mechanism. Oncogene 22: 7468-7485.
  39. Hennessy M, Spiers JP (2007) A primer on themechanics of P-glycoprotein the multidrug transporter. Pharmacol Res 55: 1-15.
  40. Sharom FJ (2008) ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics 9: 105-127.
  41. Sharom FJ (2011) The P-glycoprotein multidrug transporter. Essays Biochem 50: 161-178.
  42. Döring B, Petzinger E (2014) Phase 0 and phase III transport in various organs: Combined concept of phases in xenobiotic transport and metabolism. Drug Metab Rev 46: 261-282.
  43. Eckford PDW, Sharom FJ (2009) ABC efflux pump-based resistance to chemotherapy drugs. Chem Rev 109: 2989-3011.
  44. Kim RB (2002) Drugs as P-glycoprotein substrates, inhibitors, and inducers. Drug Metab Rev 34: 47-54.
  45. Varma MV, Ashokraj Y, Dey CS, Panchagnula R (2003) P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement. Pharmacol Res 48: 347-359.
  46. Zhou SF (2008) Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition. Xenobiotica 38: 802-832.
  47. Litman T, Skovsgaard T, Stein WD (2003) Pumping of drugs by Pglycoprotein: a two-step process? J Pharmacol Exp Ther 307: 846-853.
  48. Shi Z, Tiwari AK, Shukla S, Robey RW, Singh S, et al. (2011) Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance. Cancer Res 71: 3029-3041.
  49. Black KL, Yin D, Ong JM, Hu J, Konda BM, et al. (2008) PDE5 inhibitors enhance tumor permeability and efficacy of chemotherapy in a rat brain tumor model. Brain Res 1230: 290-302.
  50. Lin F, Hoogendijk L, Buil L, Beijnen JH, Van Tellingen O (2013) Sildenafil is not a useful modulator of ABCB1 and ABCG2 mediated drug resistance in vivo. Eur J Cancer 49: 2059-2064.
  51. Kuo T, Recht L (2006) Optimizing therapy for patients with brain metastases. Semin Oncol 33: 299-306.
  52. Dai H, Chen Y, Elmquist WF (2005) Distribution of the Novel Antifolate Pemetrexed to the Brain. J Pharmacol Exp Ther 315: 222-229.
  53. Demeule M, Régina A, Jodoin J, Laplante A, Dagenais C, et al. (2002) Drug transport to the brain: key roles for the efflux pump P-glycoprotein in the blood-brain barrier. Vascul Pharmacol 38: 339-348.
  54. Agarwal S, Sane R, Gallardo JL, Ohlfest JR, Elmquist WF (2010) Distribution of gefitinib to the brain is limited by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2)-mediated active efflux. J Pharmacol Exp Ther 334: 147-155.
  55. Agarwal S, Sane R, Ohlfest JR, Elmquist WF (2011) The role of Breast Cancer Resistance Protein (ABCG2) in the Distribution of Sorafenib to the Brain. J Pharmacol Exp Ther 336: 223-233.
  56. Chen Y, Agarwal S, Shaik NM, Chen C, Yang Z, et al. (2009) P-glycoprotein and breast cancer resistance protein influence brain distribution of dasatinib. J Pharmacol Exp Ther 330: 956-963.
  57. de Vries NA, Zhao J, Kroon E, Buckle T, Beijnen JH, et al. (2007) P-glycoprotein and breast cancer resistance protein: two dominant transporters working together in limiting the brain penetration of topotecan. Clin Cancer Res 13: 6440-6449.
  58. Polli JW, Olson KL, Chism JP, John-Williams LS, Yeager RL, et al. (2009) An unexpected synergist role of P-glycoprotein and breast cancer resistance protein on the central nervous system penetration of the tyrosine kinase inhibitor lapatinib (N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine; GW572016). Drug Metab Dispos 37: 439-442.
  59. Goldwirt L, Beccaria K, Carpentier A, Farinotti R, Fernandez C (2014) Irinotecan and temozolomide brain distribution: a focus on ABCB1. Cancer Chemother Pharmacol 74: 185-193.
  60. Basili S, Moro S (2009) Novel camptothecin derivatives as topoisomerase I inhibitors. Expert Opin Ther Pat 19: 555-574.
  61. Prados MD, Lamborn K, Yung WKA, Jaeckle K, Robins HI, et al. (2006) A phase 2 trial of irinotecan (CPT-11) in patients with recurrent malignant glioma: a North American Brain Tumor Consortium study. Neuro Oncol 8: 189-193.
  62. Pipas JM, Meyer LP, Rhodes CH, Cromwell LD, McDonnell CE, et al. (2005) A phase II trial of paclitaxel and topotecan with filgrastim in patients with recurrent or refractory glioblastoma multiforme or anaplastic astrocytoma. J Neurooncol 71: 301-305.
  63. Polli JW, Humphreys  JE, Harmon KA, Castellino S, O'Mara MJ, et al. (2008) The role of efflux and uptake transporters in [N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) disposition and drug interactions. Drug Metab Dispos 36: 695-701.
  64. Heimberger AB, Learn CA, Archer GE, McLendon RE, Chewning TA, et al. (2002) Brain tumors in mice are susceptible to blockade of epidermal growth factor receptor (EGFR) with the oral, specific, EGFR-tyrosine kinase inhibitor ZD1839 (Iressa). Clin Cancer Res 8: 3496-3502.
  65. Breedveld P, Pluim D, Cipriani G, Wielinga P, van Tellingen O, et al. (2005) The effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics and brain penetration of imatinib mesylate (Gleevec): implications for the use of breast cancer resistance protein and P-glycoprotein inhibitors to enable the brain penetration of imatinib in patients. Cancer Res 65: 2577-2582.
  66. Kil KE, Ding YS, Lin KS, Alexoff D, Kim SW, et al. (2007) Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec). Nucl Med Biol 34: 153-163.
  67. de Vries NA, Buckle T, Zhao J, Beijnen JH, Schellens JH, et al. (2012) Restricted brain penetration of the tyrosine kinase inhibitor erlotinib due to the drug transporters P-gp and BCRP. Invest New Drugs 30: 443-449.
  68. Gallo JM, Li S, Guo P, Reed K, Ma J (2003) The effect of P-glycoprotein on paclitaxel brain and brain tumor distribution in mice. Cancer Res 63: 5114-5117.
  69. Mittapalli RK, Vaidhyanathan S, Sane R, Elmquist WF (2012) Impact of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on the brain distribution of a novel BRAF inhibitor: vemurafenib (PLX4032). J Pharmacol Exp Ther 342: 33-40.
  70. Pauwels EK, Erba P, Mariani G, Gomes CM (2007) Multidrug resistance in cancer: its mechanism and its modulation. Drug News Perspect 20: 371-377.
  71. Palmeira A, Sousa E, Vasconcelos MH, Pinto MM (2012) Three decades of P-gp inhibitors: skimming through several generations and scaffolds. Curr Med Chem 19: 1946-2025.
  72. Chen Z, Shi T, Zhang L, Zhu P, Deng M; et al. (2016) Mammalian drug efflux transporters of the ATP binding cassette (ABC) family in multidrug resistance: A review of the past decade. Cancer Lett 370: 153-164.
  73. Alakhova DY, Kabanov AV (2014) Pluronics and MDR reversal: an update. Mol Pharm 11: 2566-2578.
  74. Li W, Zhang H, Assaraf YG, Zhao K, Xu X, et al. (2016) Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies. Drug Resist Updat 27: 14-29.
  75. Chauncey TR, Rankin C, Anderson JE, Chen I, Kopecky KJ, et al. (2000) A phase I study of induction chemotherapy for older patients with newly diagnosed acute myeloid leukemia (AML) using mitoxantrone, etoposide, and the MDR modulator PSC 833: a southwest oncology group study 9617. Leuk Res 24: 567-574.
  76. Sonneveld P, Burnett A, Vossebeld P, Ben-Am M, Rosenkranz G, et al. (2000) Dose-finding study of valspodar (PSC 833) with daunorubicin and cytarabine to reverse multidrug resistance in elderly patients with previously untreated acute myeloid leukemia. Hematol J 1: 411-421.
  77. Advani R, Fisher GA, Lum BL, Hausdorff J, Halsey J, et al. (2001) A phase I trial of doxorubicin, paclitaxel, and valspodar (PSC 833), a modulator of multidrug resistance. Clin Cancer Res 7: 1221-1229.
  78. Baekelandt M, Lehne G, Trope CG, Szanto I, Pfeiffer P, et al. (2001) Phase I/II trial of the multidrug-resistance modulator valspodar combined with cisplatin and doxorubicin in refractory ovarian cancer. J Clin Oncol 19: 2983-2993.
  79. Bates S, Kang M, Meadows B, Bakke S, Choyke P, et al. (2001) A Phase I study of infusional vinblastine in combination with the P-glycoprotein antagonist PSC 833 (valspodar). Cancer 92: 1577-1590.
  80. Chico I, Kang MH, Bergan R, Abraham J, Bakke S, et al. (2001) Phase I study of infusional paclitaxel in combination with the P-glycoprotein antagonist PSC 833. J Clin Oncol 19: 832-842.    
  81. Dorr R, Karanes C, Spier C, Grogan T, Greer J, et al. (2001) Phase I/II study of the P-glycoprotein modulator PSC 833 in patients with acute myeloid leukemia. J Clin Oncol 19: 1589-1599.      
  82. Fracasso PM, Brady MF, Moore DH, Walker JL, Rose PG, et al. (2001) Phase II study of paclitaxel and valspodar (PSC 833) in refractory ovarian carcinoma: a gynecologic oncology group study. J Clin Oncol 19: 2975-2982.
  83. Kang MH, Figg WD, Ando Y, Blagosklonny MV, Liewehr D, et al. (2001) The P-glycoprotein antagonist PSC 833 increases the plasma concentrations of 6alphahydroxypaclitaxel, a major metabolite of paclitaxel. Clin Cancer Res 7: 1610-1617.
  84. Baer MR, George SL, Dodge RK, O'Loughlin KL, Minderman H, et al. (2002) Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B Study 9720. Blood 100: 1224-1232.
  85. Gruber A, Bjorkholm M, Brinch L, Evensen S, Gustavsson B, et al. (2003) A phase I/II study of the MDR modulator Valspodar (PSC 833) combined with daunorubicin and cytarabine in patients with relapsed and primary refractory acute myeloid leukemia. Leuk Res 27: 323-328.
  86. Bates SE, Bakke S, Kang M, Robey RW, Zhai S, et al. (2004) A phase I/II study of infusional vinblastine with the P-glycoprotein antagonist valspodar (PSC 833) in renal cell carcinoma. Clin Cancer Res 10: 4724-4733.
  87. Bauer KS, Karp JE, Garimella TS, Wu S, Tan M, et al. (2005) A phase I and pharmacologic study of idarubicin, cytarabine, etoposide, and the multidrug resistance protein (MDR1/Pgp) inhibitor PSC-833 in patients with refractory leukemia. Leuk Res 29: 263-271.
  88. Fracasso PM, Blum KA, Ma MK, Tan BR, Wright LP, et al. (2005) Phase I study of pegylated liposomal doxorubicin and the multidrug-resistance modulator, valspodar. Br J Cancer 93: 46-53.
  89. Carlson RW, O'Neill AM, Goldstein LJ, Sikic BI, Abramson N, et al. (2006) A pilot phase II trial of valspodar modulation of multidrug resistance to paclitaxel in the treatment of metastatic carcinoma of the breast (E1195): a trial of the Eastern Cooperative Oncology Group. Cancer Invest 24: 677-681.
  90. O'Brien MM, Lacayo NJ, Lum BL, Kshirsagar S, Buck S, et al. (2010) Phase I study of valspodar (PSC-833) with mitoxantrone and etoposide in refractory and relapsed pediatric acute leukemia: a report from the Children's Oncology Group. Pediatr Blood Cancer 54: 694-702.
  91. Gottesman MM, Ludwig J, Xia D, Szakacs G (2006) Defeating drug resistance in cancer. Discov Med 6: 18-23.
  92. Kathawala RJ, Gupta P, Ashby CR Jr, Chen ZS (2015) The modulation of ABC transporter-mediated multidrug resistance in cancer: a review of the past decade. Drug Resist Updat 18: 1-17.
  93. Leonard GD, Fojo T, Bates SE (2003) The role of ABC transporters in clinical practice. Oncologist 8: 411-424.
  94. Akhtar N, Ahad A, Khar RK, Jaggi M, Aqil M, et al. (2011) The emerging role of P-glycoprotein inhibitors in drug delivery: a patent review. Expert Opin Ther Pat 21: 561-576.
  95. Fellner S, Bauer B, Miller DS, Schaffrik M, Fankhanel M, et al. (2002) Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo. J Clin Invest 110: 1309-18.
  96. Cripe LD, Uno H, Paietta EM, Litzow MR, Ketterling RP, et al. (2010) Zosuquidar, a novel modulator of P-glycoprotein, does not improve the outcome of older patients with newly diagnosed acute myeloid leukemia: a randomized, placebo-controlled trial of the Eastern Cooperative Oncology Group 3999. Blood 116: 4077-4085.
  97. Kelly RJ, Draper D, Chen CC, Robey RW, Figg WD, et al. (2011) A pharmacodynamic study of docetaxel in combination with the P-glycoprotein antagonist tariquidar (XR9576) in patients with lung, ovarian, and cervical cancer. Clin Cancer Res 17: 569-580.
  98. Hugger ED, Novak BL, Burton PS, Audus KL, Borchardt RT (2002) A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro. J Pharm Sci 91: 1991-2002.
  99. Vilas-Boas V, Silva R, Nunes C, Reis S, Ferreira L, et al. (2013) Mechanisms of P-gp inhibition and effects on membrane fluidity of a new rifampicin derivative, 1,8-dibenzoyl-rifampicin. Toxicol Lett 220: 259-266.
  100. Palmeira A, Vasconcelos MH, Paiva A, Fernandes MX, Pinto M, et al. (2012) Dual inhibitors of P-glycoprotein and tumor cell growth: (re)discovering thioxanthones. Biochem Pharmacol 83: 57-68.
  101. Carcaboso AM, Elmeliegy MA, Shen J, Juel SJ, Zhang ZM, et al. (2010) Tyrosine kinase inhibitor gefitinib enhances topotecan penetration of gliomas. Cancer Res 70: 4499-508.
  102. Shen J, Carcaboso AM, Hubbard KE, Tagen M, Wynn HG, et al. (2009) Compartment-specific roles of ATP-binding cassette transporters define differential topotecan distribution in brain parenchyma and cerebrospinal fluid. Cancer Res 69: 5885-92.
  103. Zhuang Y, Fraga CH, Hubbard KE, Hagedorn N, Panetta JC, et al. (2006) Topotecan central nervous system penetration is altered by a tyrosine kinase inhibitor. Cancer Res 66: 11305-11313.
  104. Leggas M, Panetta JC, Zhuang Y, Schuetz JD, Johnston B, et al. (2006) Gefitinib modulates the function of multiple ATP-binding cassette transporters in vivo. Cancer Res 66: 4802-4807.
  105. Yang CH, Huang CJ, Yang CS, Chu YC, Cheng AL, et al. (2005) Gefitinib reverses chemotherapy resistance in gefitinib-insensitive multidrug resistant cancer cells expressing ATP-binding cassette family protein. Cancer Res 65: 6943-6949.
  106. Tiwari AK, Sodani K, Wang SR, Kuang YH, Ashby CR Jr, et al. (2009) Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters. Biochem Pharmacol 78: 153-161.
  107. Tiwari AK, Sodani K, Dai CL, Abuznait AH, Singh S, et al. (2013) Nilotinib potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, andABCC10-multidrug resistance xenograft models. Cancer Lett 328: 307-317.
  108. Wang DS, Patel A, Shukla S, Zhang YK, Wang YJ, et al. (2014) Icotinib antagonizes ABCG2-mediated multidrug resistance, but not the pemetrexed resistance mediated by thymidylate synthase and ABCG2. Oncotarget 5: 4529-4542.
  109. Nabekura T, Yamaki T, Kitagawa S (2008) Effects of chemopreventive citrus phytochemicals on human P-glycoprotein and multidrug resistance protein 1. Eur J Pharmacol 600: 45-49.
  110. Endres CJ, Hsiao P, Chung FS, Unadkat JD (2006) The role of transporters in drug interactions. Eur J Pharm Sci 27: 501-517.
  111. Xu D, Lu Q, Hu X (2006) Down-regulation of P-glycoprotein expression in MDR breast cancer cell MCF-7/ADR by honokiol. Cancer Lett 243: 274-280.
  112. Battle TE, Arbiser J, Frank DA (2005) The natural product honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Blood 106: 690-697.
  113. Zhang MW, Xu XJ, Fan JX, Hung YK, Ye YB, et al. (2014) Honokiol combined with Gemcitabine synergistically inhibits the proliferation of human Burkitt lymphoma cells and induces their apoptosis. Zhongguo Shi Yan Xue Ye Xue Za Zhi 22: 93-98.
  114. Li Y, Huang L, Zeng X, Zhong G, Ying M, et al. (2014) Down-regulation of P-gp expression and function after Mulberroside A treatment: potential role of protein kinase C and NF-kappa B. Chem Biol Interact 213: 44-50.
  115. Li Y, Li S, Han Y, Liu J, Zhang J, et al. (2008) Calebin-A induces apoptosis and modulates MAPK family activity in drug resistant human gastric cancer cells. Eur J Pharmacol 591: 252-258.
  116. Collett GP, Robson CN, Mathers JC, Campbell FC (2001) Curcumin modifies Apc(min) apoptosis resistance and inhibits 2-amino 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced tumour formation in Apc(min) mice. Carcinogenesis 22: 821-825.
  117. Chearwae W, Shukla S, Limtrakul P, Ambudkar SV (2006) Modulation of the function of the multidrug resistance-linked ATP-binding cassette transporter ABCG2 by the cancer chemopreventive agent curcumin. Mol Cancer Ther 5: 1995-2006.
  118. Kunnumakkara AB, Anand P, Aggarwal BB (2008) Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer Lett 269: 199-225.
  119. Zhu CY, Lv YP, Yan DF, Gao FL (2013) Knockdown of MDR1 increases the sensitivity to adriamycin in drug resistant gastric cancer cells. Asian Pac J Cancer Prev 14: 6757-6760.
  120. Horiguchi S, Shiraha H, Nagahara T, Kataoka J, Iwamuro M, et al. (2013) Loss of runt-related transcription factor 3 induces gemcitabine resistance in pancreatic cancer. Mol. Oncol 7: 840-849.
  121. Croce CM, Calin GA (2005) miRNAs, cancer, and stem cell division. Cell 122: 6-7.
  122. Negrini M, Ferracin M, Sabbioni S, Croce CM (2007) MicroRNAs in human cancer: from research to therapy. J Cell Sci 120: 1833-1840.
  123. Bao L, Hazari S, Mehra S, Kaushal D, Moroz K, et al. (2012) Increased expression of P-glycoprotein and doxorubicin chemoresistance of metastatic breast cancer is regulated by miR-298. Am J Pathol 180: 2490-2503.
  124. Hong L, Han Y, Zhang H, Li M, Gong T, et al. (2010) The prognostic and chemotherapeutic value of miR-296 in esophageal squamous cell carcinoma. Ann Surg 251: 1056-1063.
  125. Zhu H, Wu H, Liu X, Evans BR, Medina DJ, et al. (2008) Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells. Biochem Pharmacol 76: 582-588.
  126. Xu Y, Xia F, Ma L, Shan J, Shen J, et al. (2011) MicroRNA-122 sensitizes HCC cancer cells to adriamycin and vincristine through modulating expression of MDR and inducing cell cycle arrest. Cancer Lett 310: 160-169.
  127. Wang F, Li T, Zhang B, Li H, Wu Q, et al. (2013) MicroRNA-19a/b regulates multidrug resistance in human gastric cancer cells by targeting PTEN. Biochem Biophys Res Commun 434: 688-694.
  128. Zhu X, Li Y, Xie C, Yin X, Liu Y, et al. (2014) miR-145 sensitizes ovarian cancer cells to paclitaxel by targeting Sp1 and Cdk6. Int J Cancer 135: 1286-1296.
  129. Takwi AA, Wang YM, Wu J, Michaelis M, Cinatl J, et al. (2014) miR-137 regulates the constitutive androstane receptor and modulates doxorubicin sensitivity in parental and doxorubicin-resistant neuroblastoma cells. Oncogene 33: 3717-3729.
  130. Giunti L, da Ros M, Vinci S, Gelmini S, Iorio AL, et al. (2014) Anti-miR21 oligonucleotide enhances chemosensitivity of T98G cell line to doxorubicin by inducing apoptosis. Am J Cancer Res 5(1): 231-242.
  131. Wang H, Wang X, Hu R, Yang W, Liao A, et al. (2014) Methylation of SFRP5 is related to multidrug resistance in leukemia cells. Cancer Gene Ther 21: 83-89.
  132. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, et al. (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352: 997-1003.
  133. Sardi I, Cetica V, Massimino M, Buccoliero AM, Giunti L, et al. (2009) Promoter methylation and expression analysis of MGMT in advanced pediatric brain tumors. Oncol Rep 22: 773-779.
  134. Schaich M, Kestel L, Pfirrmann M, Robel K, Illmer T, et al. (2009) A MDR1 (ABCB1) gene single nucleotide polymorphism predicts outcome of temozolomide treatment in glioblastoma patients. Ann Oncol 20: 175-181.
  135. Munoz JL, Rodriguez-Cruz V, Greco SJ, Nagula V, Scotto KW, et al. (2014) Temozolomide induces the production of epidermal growth factor to regulate MDR1 expression in glioblastoma cells. Mol Cancer Ther 13: 2399-2411.
  136. Munoz JL, Rodriguez-Cruz V, Rameshwar P (2015) High expression of miR-9 in CD133+ glioblastoma cells in chemoresistance to temozolomide. J Cancer Stem Cell Res 3.
  137. Zhang R, Saito R, Shibahara I, Sugiyama S, Kanamori M, et al. (2016) Temozolomide reverses doxorubicin resistance by inhibiting P-glycoprotein in malignant glioma cells. J Neurooncol 126: 235-242.
  138. Riganti C, Salaroglio IC, Caldera V, Campia I, Kopecka J, et al. (2013) Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/β-catenin pathway. Neuro Oncol 15: 1502-1517.
  139. Grossman SA, Ye X, Piantadosi S, Desideri S, Nabors LB, et al. (2010) Survival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States. Clin Cancer Res 16: 2443-2449.