**2. The drug efflux transporter: P-glycoprotein**

Drug transporters can be grouped, according to their transport direction, into uptake and efflux pumps. Most of the known efflux transporters particularly P-glycoprotein (P-gp or MDR1; encoded by *ABCB1*), multidrug resistance protein 1 (MRP1, encoded by *ABCC1*), multidrug resistance protein 2 (MRP2, encoded by *ABCC2*) and breast cancer resistance protein (BCRP; encoded by *ABCG2*) are members of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. The ABC transporters require ATP hydrolysis for their transport activity across plasma membrane in the secretive direction. These efflux transporters share similar structural assembly across plasma membrane, composing of a membrane-spanning α-helix structure as a transmembrane domain (TMD) and a relatively hydrophilic ATP-binding site in nucleotide binding domain (NBD). High activity and expression of these ABC drug efflux pumps is a major contributing factor for development of MDR phenomenon in cancer cells [1, 4].

Among the ABC efflux transporters, P-gp is the first and most studied transporter for MDR development in chemotherapy and drug-transporter-related interaction issues. This transporter was first identified from its involvement with multidrug-resistance in cancer cells. Particularly, overexpression of P-gp in cancer cells, either intrinsic or acquired, has been strongly associated with MDR occurrence, thereby P-gp becomes a promising target for development of chemosensitizers.

#### **2.1 Overview of P-gp (structure, function, location, expression, and MDR)**

P-gp (MW approximately 170 kDa) is a single polypeptide with 1280 amino acids arranging in two duplicated units of a 6 α-helix structure hydrophobic TMD with linkage to a hydrophilic NBD (**Figure 1**) [1, 2, 7]. These two TMD with the total of 12 helices forms together into one channel as the membrane crossing passage. A substrate binds to the drug-binding site in the TMD whereas an ATP binds to the NBD. After ATP binding, ATP undergoes hydrolysis into ADP for energy to activate P-gp action through protein conformational alteration [7, 8]. This transporter, then, is able to move its substrates across lipid bilayer structure of plasma membrane to extracellular environment.

#### *2.1.1 P-gp and its normal physiological functions*

P-gp is constitutively located in the apical surface of either epithelial or endothelial linings of various normal tissues/organs such as adrenal glands, intestine, liver, kidney, pancreas, placenta, capillary vessels in the brain and testes [2, 7–10]. Some organs such as prostate, skin, heart and skeletal muscle have low constitutive expression of P-gp. It should be noting that expression level of P-gp varies in each organ. For example, the numbers of P-gp in colon and ileum are higher than those in jejunum, duodenum and stomach [11, 12]. The tissue distribution of P-gp indicates that this transporter normally serves as an intrinsic determinant of oral drug bioavailability and drug disposition [13–18]. Intestinal P-gp can influence the absorptive amount of its drug substrates, except those in BCS class I (i.e., high permeability and high solubility drugs such as verapamil), into the body after orally taken [13, 19–21]. The constitutive expression of P-gp at the mucosal surface in the lower gastrointestinal (GI) tract (i.e. jejunum, ileum, and colon) may prevent an uptake of its substrate, and perhaps also facilitate GI excretion. Moreover, the interplay between P-gp and the major phase I drug metabolizing enzymes (e.g. cytochrome P450, CYP450) can be anticipated due to their substrate similarity [22].

**191**

**Figure 1.**

*Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance*

As such, P-gp and CYP3A4 act in concert to affect metabolic biotransformation of their substrates such as paclitaxel in intestine and liver, influencing the oral drug bioavailability [22–24]. Localization of P-gp in the blood-organ barriers such as brain or testis prevents drug penetration into such organ systems such as brain, testes [13, 23, 25, 26]. The presence of P-gp on the brush border of nephron proximal tubule and hepatocytes involve with excretion of drugs and endogenous substrates into the urine and bile [13, 27]. To this end, P-gp can be considered as the protective mechanism against xenobiotics as well as pharmacokinetic influencer

Expression of P-gp at plasma membrane involves several cellular processes that linking to P-gp mRNA and protein expression. The regulatory mechanisms have been largely associated with (1) activation or inactivation of oncogenes (e.g., Ras, c-Raf) and transcriptional process, (2) MDR1 translation into P-gp and post translational modification, protein trafficking, and (3) P-gp turn over. It has been reported that the dysregulated microRNA levels (e.g., miR-21, -27a, -451, -130a, -298) could cause MDR development in various cancer cells [28–34]. For example, miR-130 was correlated to MDR1/P-gp overexpression, and cisplatin resistance in SKOV3/CIS cells [32]. Overexpression of miR-27a and miR-451 was linked to increased MDR1 expression and MDR phenotype in resistant cancer cells

Overexpression of P-gp particularly in MDR phenomenon has been evidently connected to up-regulation of MDR1 gene through alteration of various signaling pathways and transcription factors. Example of the transcriptional factors involving in MDR1 transcription are nuclear factor-κB (NF-ᴋB) [35, 36], Y-box binding

particularly on absorption, distribution and disposition.

*The key ABC drug efflux transporters and their selected anticancer drug substrates.*

*2.1.2 P-gp expression and signaling pathways*

A2780DX5 and KB-V1 [28].

*DOI: http://dx.doi.org/10.5772/intechopen.95553*

*Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance DOI: http://dx.doi.org/10.5772/intechopen.95553*

**Figure 1.**

*Advances in Precision Medicine Oncology*

**2. The drug efflux transporter: P-glycoprotein**

of MDR phenomenon in cancer cells [1, 4].

membrane to extracellular environment.

*2.1.1 P-gp and its normal physiological functions*

chemosensitizers.

Drug transporters can be grouped, according to their transport direction, into

Among the ABC efflux transporters, P-gp is the first and most studied transporter for MDR development in chemotherapy and drug-transporter-related interaction issues. This transporter was first identified from its involvement with multidrug-resistance in cancer cells. Particularly, overexpression of P-gp in cancer cells, either intrinsic or acquired, has been strongly associated with MDR occurrence, thereby P-gp becomes a promising target for development of

**2.1 Overview of P-gp (structure, function, location, expression, and MDR)**

P-gp (MW approximately 170 kDa) is a single polypeptide with 1280 amino acids arranging in two duplicated units of a 6 α-helix structure hydrophobic TMD with linkage to a hydrophilic NBD (**Figure 1**) [1, 2, 7]. These two TMD with the total of 12 helices forms together into one channel as the membrane crossing passage. A substrate binds to the drug-binding site in the TMD whereas an ATP binds to the NBD. After ATP binding, ATP undergoes hydrolysis into ADP for energy to activate P-gp action through protein conformational alteration [7, 8]. This transporter, then, is able to move its substrates across lipid bilayer structure of plasma

P-gp is constitutively located in the apical surface of either epithelial or endothelial linings of various normal tissues/organs such as adrenal glands, intestine, liver, kidney, pancreas, placenta, capillary vessels in the brain and testes [2, 7–10]. Some organs such as prostate, skin, heart and skeletal muscle have low constitutive expression of P-gp. It should be noting that expression level of P-gp varies in each organ. For example, the numbers of P-gp in colon and ileum are higher than those in jejunum, duodenum and stomach [11, 12]. The tissue distribution of P-gp indicates that this transporter normally serves as an intrinsic determinant of oral drug bioavailability and drug disposition [13–18]. Intestinal P-gp can influence the absorptive amount of its drug substrates, except those in BCS class I (i.e., high permeability and high solubility drugs such as verapamil), into the body after orally taken [13, 19–21]. The constitutive expression of P-gp at the mucosal surface in the lower gastrointestinal (GI) tract (i.e. jejunum, ileum, and colon) may prevent an uptake of its substrate, and perhaps also facilitate GI excretion. Moreover, the interplay between P-gp and the major phase I drug metabolizing enzymes (e.g. cytochrome P450, CYP450) can be anticipated due to their substrate similarity [22].

uptake and efflux pumps. Most of the known efflux transporters particularly P-glycoprotein (P-gp or MDR1; encoded by *ABCB1*), multidrug resistance protein 1 (MRP1, encoded by *ABCC1*), multidrug resistance protein 2 (MRP2, encoded by *ABCC2*) and breast cancer resistance protein (BCRP; encoded by *ABCG2*) are members of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. The ABC transporters require ATP hydrolysis for their transport activity across plasma membrane in the secretive direction. These efflux transporters share similar structural assembly across plasma membrane, composing of a membrane-spanning α-helix structure as a transmembrane domain (TMD) and a relatively hydrophilic ATP-binding site in nucleotide binding domain (NBD). High activity and expression of these ABC drug efflux pumps is a major contributing factor for development

**190**

*The key ABC drug efflux transporters and their selected anticancer drug substrates.*

As such, P-gp and CYP3A4 act in concert to affect metabolic biotransformation of their substrates such as paclitaxel in intestine and liver, influencing the oral drug bioavailability [22–24]. Localization of P-gp in the blood-organ barriers such as brain or testis prevents drug penetration into such organ systems such as brain, testes [13, 23, 25, 26]. The presence of P-gp on the brush border of nephron proximal tubule and hepatocytes involve with excretion of drugs and endogenous substrates into the urine and bile [13, 27]. To this end, P-gp can be considered as the protective mechanism against xenobiotics as well as pharmacokinetic influencer particularly on absorption, distribution and disposition.

#### *2.1.2 P-gp expression and signaling pathways*

Expression of P-gp at plasma membrane involves several cellular processes that linking to P-gp mRNA and protein expression. The regulatory mechanisms have been largely associated with (1) activation or inactivation of oncogenes (e.g., Ras, c-Raf) and transcriptional process, (2) MDR1 translation into P-gp and post translational modification, protein trafficking, and (3) P-gp turn over. It has been reported that the dysregulated microRNA levels (e.g., miR-21, -27a, -451, -130a, -298) could cause MDR development in various cancer cells [28–34]. For example, miR-130 was correlated to MDR1/P-gp overexpression, and cisplatin resistance in SKOV3/CIS cells [32]. Overexpression of miR-27a and miR-451 was linked to increased MDR1 expression and MDR phenotype in resistant cancer cells A2780DX5 and KB-V1 [28].

Overexpression of P-gp particularly in MDR phenomenon has been evidently connected to up-regulation of MDR1 gene through alteration of various signaling pathways and transcription factors. Example of the transcriptional factors involving in MDR1 transcription are nuclear factor-κB (NF-ᴋB) [35, 36], Y-box binding

protein-1 (YB-1) [37, 38], activator protein-1 (AP-1) [39], and hypoxia-inducible factor-1 (HIF-1) [38, 40]. The activities of these transcription factors have been linked to various signal transduction pathways, particularly the two major cell survival signaling cascades i.e. (1) the mitogen-activated protein kinase (MAPK) [37, 41], and (2) the phosphatidylinositol 3-kinase (PI3K) pathways [42, 43]. It has been shown that hyperactivation of either MAPK/ERK1/2 or PI3K/Akt/NF-ᴋB signaling pathways results in overexpression of P-gp in doxorubicin-resistant cells such as lung, breast and ovarian cancer cells [44–49]. An up-regulation of P-gp expression in vincristine-resistant human gastric cancer cells was associated with activation of the p-38/MAPK pathway [50].

After activation and translocation into nucleus, transcription factors such as NF-kB and YB-1 (Y-box binding protein) bind to MDR1 promoter region, leading to initiation of MDR1 transcription. Increase in YB-1 nuclear activity is related to P-gp-mediated development of MDR in several cancers including breast cancer, lung cancer, ovarian cancer, colorectal cancer, prostate cancer and osteosarcoma [38]. In response to cell stress such as hyperthermia, viral infection and chemical assault, the survival Akt and MAPKs signaling would be activated, and subsequently increase YB-1 expression and translocation into nucleus for its MDR1-transcription activity [51]. Doxorubicin is a known P-gp inducer in various cancer cells. Doxorubicin up-regulates MDR1 gene expression via the MAPK/ERK1/2 signaling that linked to activation of YB-1 in B-cell lymphoma [37]. Moreover, upregulation of P-gp has been reported after prolonged exposure to various functional unrelated compounds, leading to the loss of drug efficacy and safety [52]. Examples of the known P-gp inducers include anticancer (e.g., cisplatin, doxorubicin, etoposide vinblastine), antidepressants (e.g., trazodone, St. John's Wort), anticonvulsants (e.g., carbamazepine, phenytoin), anti-HIV (e.g., saquinavir, indinarvir, tenofovir), immunosuppressants (e.g., cyclosporine, tacrolimus), steroids (e.g., dexamethasone) [52–54]. It is worth noting that certain CYP450 inducers such as rifampin and St. John's Wort are able to up-regulate P-gp expression, possible sharing through the PXR regulation [55, 56]. Prolonged exposure to rifampin and St. John's Wort in human led to increased intestinal P-gp level, and increased digoxin absorption [57, 58]. Since, P-gp-mediated MDR in cancer is largely due to up-regulation of P-gp expression, better understanding of the signaling proteins and transcription factors will provide a promising targets in overcoming MDR for anticancer chemotherapy.

#### *2.1.3 P-gp and multi-drug resistance in cancer*

Overexpression of P-gp has been strongly correlated with chemo-resistance and cancer relapses in several cancer patients such as breast cancer, adult acute myeloid leukemia, pheochromocytoma patients, leading to poor prognosis from therapeutic failure in patients receiving chemotherapy [1, 59–62]. Accordingly, P-gp is intrinsically expressed in various cancer types, particularly those derived from tissues with high basal MDR1 expression levels such as colon, kidney and liver tissues. Being a transmembrane efflux pump, P-gp serves as a cellular defense mechanism against drug assault by limiting intracellular drug accumulation up to toxic threshold level. Regarding this, the susceptibility of cancer to anticancer drugs being P-gp substrate varies, depending on intrinsic expressed P-gp levels. Certain types of cancers may be classified as poor responder showing their unresponsiveness to chemotherapy regimens containing P-gp substrates. For example, prostate cancer appears to be better responder to chemotherapy, as compared to colorectal or renal cancers [63, 64]. Moreover, some cancers such as leukemia, lymphoma and breast cancer having low levels of intrinsic P-gp expression, and thus initially respond well to

**193**

**Table 1.**

*Selected substrates, inducers and modulators of P-gp.*

*Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance*

chemotherapy. Later, after repeated treatment, the expression level of P-gp markedly increases, and those cancers display multi-drug resistance (MDR) phenotype [1, 65, 66]. This acquired MDR phenomenon can be viewed as cellular adaptive

Examples of substrates and modulators of P-gp are listed in **Table 1**.

Human ABC efflux transporters demonstrate their broad substrate specificity toward structurally diverse lipophilic compounds. Most of their substrates are weakly amphipathic and hydrophobic planar structure with aromatic ring and positively charged nitrogen atom [52, 54, 67, 68]. Examples of P-gp substrates are anticancer drugs (vinca alkaloids, anthracyclines, and epipodophyllotoxins), cardiovascular drugs (e.g., digoxin, quinidine, talinolol, diltiazem, losartan, verapamil), anti-microbial agents (e.g., doxycycline, erythromycin, itraconazole, rifampin), anti-viral drugs (e.g., indinavir), anticonvulsants (e.g., phenytoin), acid blockers (e.g., cimetidine), immunosuppressants (e.g., cyclosporine, tacrolimus), steroids (e.g., aldosterone, cortisol,

**P-gp modulators**

Small molecule inhibitors *First generation* Cyclosporin A Verapamil *Second generation* VX-710 (Biricodar) Dexverapamil PSC833 (Valspodar) *Third generation* GF120918 (Elacridar) XR9576 (Tariquidar) LY335979 (Zosuquidar) *Fourth generation* Capsaisin Curcumin Limonin Piperine Quercetin Monoclonal antibodies MRK 16 MRK 17 UIC 2

**Direct inhibitors Suppressors of expression**

Curcumin Dasatinib Dexverapamil Reserpine Imatinib Nilotinib Sorafenib Trifluoperazine Toremifene PSC833 (Valspodar) RNA interference

(siRNA)

Small molecule inhibitors

*MDR1* small-interfering RNA

Antisense oligonucleotides *MDR1* antisense oligonucleotides delivered via lysosomes

*DOI: http://dx.doi.org/10.5772/intechopen.95553*

survival response to cytotoxic challenge.

dexamethasone), opioids (loperamide, morphine).

**Inducers (Anti-cancer drugs)**

Daunorubicin Docetaxel Doxorubicin Flutamide Paclitaxel Vinblastine Vincristine

**2.2 Substrates and modulators**

*2.2.1 Substrates*

**Substrates (Anti-cancer drugs)**

Actinomycin D Colchicine Docetaxel Doxorubicin Daunorubicin Epirubicin Etoposide Idarubicin Imatinib Methotrexate Paclitaxel Teniposide Topotecan Vinblastine Vincristine

chemotherapy. Later, after repeated treatment, the expression level of P-gp markedly increases, and those cancers display multi-drug resistance (MDR) phenotype [1, 65, 66]. This acquired MDR phenomenon can be viewed as cellular adaptive survival response to cytotoxic challenge.
