Resistance to cancer chemotherapy: failure in drug response from ADME to P-gp

Cancer chemotherapy resistance (MDR) is the innate and/or acquired ability of cancer cells to evade the effects of chemotherapeutics and is one of the most pressing major dilemmas in cancer therapy. Chemotherapy resistance can arise due to several host or tumor-related factors. However, most current research is focused on tumor-specific factors and specifically genes that handle expression of pumps that efflux accumulated drugs inside malignantly transformed types of cells. In this work, we suggest a wider and alternative perspective that sets the stage for a future platform in modifying drug resistance with respect to the treatment of cancer.


Background
In US only, the newly diagnosed cancer patient is 1,665,540 every year and the estimated death is 585,720 [1] which are increasing as countries become more developed and more people reach advanced ages. Therefore, many efforts are being done in the war against cancer [2]. One of these efforts is the continuous development of new drugs and forms of chemotherapy.
In cancer, chemotherapy represents the backbone of treatment for many cancers at different stages of the disease. Therefore, chemotherapeutic resistance results in therapeutic failure and usually, (eventually) death. To address these limitations, many researchers focus on how cancer cells manipulate their genomes and metabolism to prevent drug influx and/or facilitate efflux of accumulated drugs, the so called: "the neostrategy of cancer cells and tissues" [3]. In this work, we show that drug resistance is a multifactorial phenomenon that requires attention to the host as well as the tumor and that such factors are organized at different levels ( Figure 1).

P-gp
Permeability glycoprotein (P-gp), also known as multidrug drug resistance protein (MDR) is found along the gastrointestinal tract (GIT) [10], including the small intestine as primary site for the epithelial absorption of many orally administered drugs [11]. It has been shown that P-gp reduces the oral bioavailability of some anticancer drugs [12]. Concomitant administration of some of the antineoplastic agents leads to the overexpression of P-gp that results in bioavailability reduction of several these agents, e.g. Imatinib [13]. P-gp expression, in the gut, is a subject of interindividual variation due to either genetic polymorphism or pathologic condition [14,15] and so fluctuates the bioavailability of several antineoplastic agents e.g. paclitaxel [12].

Food
The effects of food on drug absorption and bioavailability have been attracting attention very much and stimulated a long debate of whether fasting improved or worsened a drug's bioavailability [16][17][18]. It has been shown that the half-life period of orally applied Topotecan is longer than that of intravenous administration. The administration of Topotecan with a high-fat breakfast shows a small decrease in the absorption rate but does not affect the extent of the absolute absorption [19]. St John's wort, induces the expression of Pregnane X receptor, a xenobiotic or detoxification sensor, which reduces the efficacy of some antineoplastic agents, e.g. Irinotecan [4]. CYP3A4 is a metabolising enzyme (see below) which has been found in the intestine presumably as a defense strategy against xenobiotics [20]. It is well known that grapefruit juice abates the presence of CYP3A4, which is beneficial for the application of antineoplastic oral agents with low bioavailability [21]. Thus, it becomes apparent that the interaction between food and antineoplastic agents should be cautiously monitored to maintain a sufficient bioavailability [22][23][24].

Distribution
The "volume of distribution" (V d ) of the drug is a hypothetical volume that outlines drug distribution into tissues [25,26]. A higher V d means that more of a drug penetrates into a tissue while it is more diluted (present at lower concentrations) in the plasma. The distribution of the drug between plasma and tissues relies on several factors. Some of them include: 1. Gender Metronidazole, a bactericide and protozoocide, also displays a potential activity as an anticancer drug, especially as a radiosensitizer in hypoxic regions [27], and shows a lower V d in women [ Figure 1 Diagrammatic representation describes that cancer chemotherapy resistance is a multi-factorial phenomenon that could be organized as multilevel structure.
fore, it is critical to consider the gender factor also to age if the concentration of the drug in the plasma is to be determined or needs to be defined. 2. Weight Weight is a paramount factor that determines dose adjustment [30]. Cancer patients often lose weight during tumor progression [31][32][33]. Therefore, dosing based on body weight should be routinely readjusted to the current weight [34, 35]. 3. Plasma proteins when a drug is available in the blood (plasma), the drug will be found either in free or bound form to plasma proteins (see Figure 2). Albumin and Alpha 1-acid glycoprotein are an example of drug-binding plasma proteins. While albumin carries acidic drugs, the alpha 1-acid glycoprotein will carry basic and neutral lipophilic drugs [36-38].  A drug is active while in unbound form. Therefore, the ability of the drug to bind plasma proteins and tissue reduces its activity [221], The drug's activity is modulated by ifferences in the amount of plasma proteins [222][223][224].

Drug-drug interactions
Cancer chemotherapy is administered in the form of a cocktail, the combined application of several chemotherapeutics. Such a combination is designed to reduce toxicity and to decrease the likelihood of resistance. One drug alone would require a higher concentration and, so the side effects caused would be increased. Using a combination reduces the side-effects of each single drug as it can be applied at a significantly lower concentration [106].
A tumor consists of a heterogeneous population and it is commonly thought that using a "cocktail" of several agents will target different populations and thus reduce the selective pressure by using single agents (the use of only one single agent might kill one defined population and positively select a pre-adapted one that will remain and grow). Therefore, a combination therapy is useful in treating cancer. Conversely, it needs to be pointed out that co-administration of drugs might result in antagonism such that one drug may counteract or neutralize another one: -Agents that target tumor vascularization: tumors require a blood supply for the provision of oxygen and nutrients [107], removal of metabolites [108] and to support metastasis [109]. It is widely assumed that administration of agents that target tumor vasculature (antiangiogenic therapy) eventually interrupts tumor progression. There are two classes of these agents (1) Angiogenesis inhibitors; they inhibit the tumor that has initiated the angiogenic process and (2) vascular disrupting agents that destruct the existing tumor vessels. Those agents might limit perfusion of cytotoxic drugs especially upon chronic administration [110]. Moreover, it is postulated that antiangiogenic therapy is useful in the management of resistance to chemotherapy [111], however, diminishing tumor vascularization may accelerate the adaptation to hypoxia while increasing the necrotic zone by accumulation of metabolites and so worsen tumor prognosis [112] -NaHCO 3 has been recently used systemically in the treatment of cancer [113]. It induces systemic alkalosis. By elevation of urine pH, methotrexate excretion is greatly enhanced. Therefore, NaHCO 3 modulates the pharmacokinetics of methotrexate [114]. -Tamoxifen is a prodrug that needs to be metabolized to its active form by CYP2D6, CYP3A, CYP2B6 and CYP2C19 [115]. Some drugs, particularly from the group of selective serotonin reuptake inhibitors, inhibit CYP2D6 and so reduce the efficacy of Tamoxifen by decreasing amount of its active metabolites [116][117][118]. -Pravastatin, an HMG-CoA reductase inhibitor, is correlated with biliary excretion [119,120] as a substrate of P-gp [121]. Also, it induces P-gp expression as well [122] and so may promote resistance to other compounds.

Microscopic (local) resistance [tumor related factors]
The loss of ability of drugs to kill cancer cells could also be due to failure at the tumor site. Such disability could occur via several mechanisms. Some of them are:

Evolutionary resistance
Also termed biochemical resistance [123,124], acquired resistance [66, 125], active resistance [126], or extrinsic resistance [127]. Evolutionary resistance is an ancient type of resistance that can be found in bacteria even prior exposure to antibiotics [128][129][130][131][132]. Evolutionary resistance could occur either through interfering with drug resident time intracellularly and/or altering its site of action.

Alteration of drug residency in cancer cells
There are several proteins that alter drug residency in cancer cells. Some of these include:

P-gp
P-glycoprotein 1 (permeability glycoprotein, abbreviated as P-gp or Pgp) also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1) or cluster of differentiation 243 (CD243). It is a glycoprotein that in humans is encoded by the ABCB1 gene [133,134]. Commonly, P-gp is localized at the plasma membrane [135] of colon, jejunum, bile canaliculi, renal tubular cells, placenta, the luminal surface of capillary endothelial cells, testes, pancreas and blood-brain barriers (BBB) [135][136][137][138]. P-gp might have a role in the normal secretion of metabolites. P-gp also induces expression of CYP3A4 [139] that in turn may deactivate some anticancer drugs (see Table 1 that shows the pharmacological modulators of P-gp).
In resistant cancer cell lines, P-gp is localized in the Golgi apparatus and the rough endoplasmic reticulum [140,141]. Also, it is expressed in mitochondrial cristae [142,143] to protect the accumulation of mitochondria [144] or prevent nuclear accumulation by expression of P-gp at the nuclear envelope [141,145].
Expression of P-gp fluctuates with elevated expression level in untreated cancer into higher level upon relapse after chemotherapy and undetectable or low level in the expression in drug sensitive tumors [134,146] which means there is no unifying theorem correlating expression of P-gp and cancer treatment.
MRPs Multidrug resistance-associated protein MRP1 (ABCC1) was the first of the xenobiotic-transporting MRP-related proteins to be cloned and was identified based on its overexpression in a multidrug-resistant lung cancer cell line [147]. The MRP family consists of the four isoforms MRP1-4 [148,149]. MRPs are similar to P-gp in that they are (I) capable of decreasing intracellular drug levels and (II) ATP-dependent [150]. Also, MRPs require glutathione GSH to extrude xenobiotics [151][152][153][154].
MXR The Mitoxantrone resistance protein MXR or the Multixenobiotic resistance protein, also known as BCRP, ABCP and ABCG2, is one member of the ABC-superfamily that plays a role in trafficking biological molecules across cell membranes [155]. Expression of MXR might be an alternative strategy of resistance if cancer cells lack p-gp and MRP [156]. MXR preferentially extrudes large hydrophobic, positively charged molecules while others members of the MRP family can eject both hydrophobic uncharged molecules and water-soluble anionic compounds [157].

Alteration of drug target
When the drug reaches its target, another mechanism of resistance could be evolved somatically. Examples, which explain this mechanism of resistance, is: -Methotrexate is a drug of choice for the treatment of rheumatoid arthritis [158][159][160]. Moreover, its activity against several types of tumors has been shown. It inhibits tumor cells via inhibition of the Dihydrofolate reductase enzyme (DHFR) which is a co-enzyme in DNA-methylation. Both, in vitro and in vivo studies show that the genomic amplification of the DHFR gene is reflected by extra copies of DHFR [161][162][163].
-5-fluorouracil is a thymidylate synthetase inhibitor that is widely used in several types of tumors. Thymidylate Table 1 Shows the pharmacological modulators of P-gp

Microenvironmental resistance
Cancer cells maintain a unique pH gradient; it is more acidic extracellularly and more alkaline intracellularly about normal tissues [167][168][169]. Such a pH gradient creates a unique environment around cancer cells. The tumor microenvironment becomes one of the cancer's hallmarks [109]. Tumor microenvironment increases tumor's fitness by blunting the immune system [167,170], activating endogenous immunosuppressive strategies [171] and inhibiting the growth of the normal cell population. Moreover, the tumor microenvironment disables the activities of several chemotherapeutic agents resulting in resistance and failure in drug response [172] either through disturbing drug partitioning, sequestering it intracellularly [173,174] or through induction of MDR expression [146].
There are several components of the tumor microenvironment that contribute to drug disability. Some of them include: pH Most anticancer agents are either weak bases or weak acids. Some of them are Zwitterions (see Table 2). Weakly basic anticancer drugs are ionizable at the interstitial fluid that decreases their partitioning, and if they cross the plasma membrane, they are sequestered into acidic vesicles (lysosomes). While weakly acid drugs increase their partitioning into the interstitial fluid, they will be rendered at cytosol due to intracellular alkalinity, and so they are slightly prevented from reaching their targets. This phenomenon is well known as "ion trapping mechanism" [123,124,[175][176][177]. While basic drugs have reduced efficacy in an acidic microenvironment, Chlorambucil is a weakly acidic compound, and its cytotoxicity is enhanced by acidic microenvironments [178]. So, any attempt to induce intracellular acidification could be an avenue to both breaking through MDR and as an anticancer therapeutic approach it own [179].
Hypoxia somehow handles drug resistance via the following factors: 1. Most anticancer agents act to activate the apoptosis pathway, and the presence of free radicals are essential to promote this process [188]. Therefore, the absence of oxygen will diminish the activity of these drugs. Hypoxia does not only confer resistance to chemotherapy [189][190][191][192] but also to radiation [27]. 2. Hypoxia induces genes expression that code for ABC-transporters [193] and so favors the developing of resistance of some of the anticancer drugs e.g. 5fluorouracil [194]. 3. Hypoxia also alters activity of some of metabolizing enzymes that are responsible for the activation and/or inactivation of some of the anticancer drugs e.g. conversion of paclitaxel metabolism into 6-α-hydroxypaclitaxel is reduced upon hypoxic conditions compared to normoxic conditions in HepaRG cells [195]. Therefore, hypoxia might alter therapeutic effectiveness. 4. Hypoxia is an important evolutionary determinant factor that shapes part of the tumor population to become hypoxia-adapted [108]. Hypoxia is associated with cellular senescence [196]. Chemotherapeutic agents which are designed to target cells that have high proliferation rates will fail even if they reach their site of action in sufficient amounts in poorly vascularized regions [197,198]. Conversely, recent data suggests that hypoxia suppresses geroconversion (the conversion of arrested cells to senescence) [199] which make the role of hypoxia in tumorigenesis and tumor resistance still unclear.

Glucose
In the 1920s, Otto Warburg discovered that cancer have high aerobic glycolysis even in the presence of oxygen [200][201][202]. Recently, metabolic reprogramming of cancer has been adopted as one of the hallmarks of cancer [109]. Glucose, especially in high loads, induces over-expression of Sodium Hydrogen Exchanger 1 (NHE-1) resulting in the alkalinization of pHi [203] and in the induction of the metabolic transformation that aggravates the tumor microenvironment [27]. Moreover, glucose uptake is associated with tumor progression [204] as well as chemotherapeutic resistance [205]. Therefore, alteration of glucose transport [206], glucose deprivation, and fasting may enhance tumor sensitivity to chemotherapy [207]. However, targeting glucose also leads to interconversion of sensitive populations into more resistant populations, which may handle relapse [208]. So, targeting glucose as a potential strategy for overcoming resistance may be a dead end.

Mesoscopic (physical, mechanical) or (regional) resistance [tumor-host interacting factors]
The physics of the tumor site has a great impact on drug activity and results in drug resistance as follows: 1. Geometric, or vascular, resistance is a complex function of vascular morphology, i.e. the number of vessels of various types, their branching pattern, their diameter, and length [209]. It has been showing that upon clonal tumor expansion, tumor perfusion, and, therefore, the amount of drug to reaching its target is decreased [198,210,211]. Vascular modulating agents may alter vascular resistance and diminish the drugs reaching their targets [212] while by reducing the geometric resistance will enhance the activity of chemotherapeutic agents [213]. 2. Blood viscosity is very significant especially for intratumoral blood flow [209]. Highly blood viscosity is an indicator for blood stasis and thus for the stagnation of drugs at certain sites. Moreover, inflammatory mediators at tumor sites might alter erythrocyte sedimentation rate and lead to greatly enhanced blood viscosity [214], at least at the tumor. Because blood flow has been correlated with oxygen diffusion kinetics [215], increasing blood viscosity may induces blood flow retardation and hypoxia becomes an adaptive strategy of survival especially for xeric phenotypes (cancer cells that grow distal from blood vessels) [108]. This also contributes to drug penetration into intratumor regions (see Figure 3). Anemia also might occur due reduction in hematocrit. In this regard, bone marrow suppression by chemotherapeutic agents should be taken into consideration.

Co-resistance
The presence of the extracellular matrix (ECM) and a stroma are crucial for carcinogenesis. Both of this play a significant role in cancer progression, metabolism and the metastatic cascade [216]. In this context, the ECM plays a critical role in mediating drug resistance [217], either by acting as a physical barrier that impairs drug diffusion [218] and/or by cooperating with tumor cells to generate chemotherapy resistance [219,220]. In this regard, this strategy of resistance could be called ECMdependent resistance [220,221].

Conclusions
From bacteria to cancer, (multi) drug resistance is becoming a central issue and a significant challenge for medicine today. Although drug resistance is often studied at the single cell level, it is important to realize that the ability of a drug to interact with its target is more complex involving many body compartments. Also, over the past decade, there have been significant changes in our understanding of some fields from biology. Whereas, before, the keylock model was supposed to uncover biology helping us to understand life, it is clear today that evolution theory needs to be introduced at the single cell level to clarify our understanding of some diseases including cancer. The famous key-lock model, as well as the long-awaited magic bullet to kill cancer, has to be revised accordingly. Therefore, we suggest reframing the concepts used in drug resistance in a more general context thereby dismantling the monolithic tone that the resistance is only a matter of genes. We propose that drug ineffectiveness results from tumor-host interactions and that a clear understanding of