South Asian Medicinal Compounds as Modulators of Resistance to Chemotherapy and Radiotherapy

Cancer is a hyperproliferative disorder that involves transformation, dysregulation of apoptosis, proliferation, invasion, angiogenesis and metastasis. During the last 30 years, extensive research has revealed much about the biology of cancer. Chemotherapy and radiotherapy are the mainstays of cancer treatment, particularly for patients who do not respond to surgical resection. However, cancer treatment with drugs or radiation is seriously limited by chemoresistance and radioresistance. Various approaches and strategies are employed to overcome resistance to chemotherapy and radiation treatment. Many plant-derived phytochemicals have been investigated for their chemo- and radio-sensitizing properties. The peoples of South Asian countries such as India, Pakistan, Sri Lanka, Nepal, Bangladesh and Bhutan have a large number of medicinal plants from which they produce various pharmacologically potent secondary metabolites. The medicinal properties of these compounds have been extensively investigated and many of them have been found to sensitize cancer cells to chemo- and radio-therapy. This review focuses on the role of South Asian medicinal compounds in chemo- and radio-sensitizing properties in drug- and radio-resistant cancer cells. Also discussed is the role of South Asian medicinal plants in protecting normal cells from radiation, which may be useful during radiotherapy of tumors to spare surrounding normal cells.


Introduction
The plant kingdom plays an essential role in the life of humans and animals. South Asia is one of the largest producers of medicinal plants and natural products for human health and disease [1]. South Asian countries such as India, Pakistan, Sri Lanka, Nepal, Bangladesh and Bhutan have a large number of plants that are used to produce medicinal agents. In particular, India is one of the largest producers of medicinal plants and can rightly be called the "Botanical Garden of the World". In India, there are about 400 families and 8000 species of medicinal plants, which include approximately 50% of all the higher flowering plant species [2].
South Asian medicinal plants and their compounds have a very long history of use for cancer treatment. The climate, environment and geography of South Asian countries enable the growth of diverse medicinal plants with novel secondary active compounds, which has attracted the attention of the scientific community. In this review, we focus on the chemosensitizing and radiosensitizing potential of bioactive compounds from South Asian medicinal plants commonly found in Bangladesh, India, Nepal, Pakistan and Sri Lanka, and discuss their mechanisms of action. The chemoprotective

South Asian Medicinal Compounds as Chemosensitizers and Radiosensitizers
Chemotherapy works by targeting cells within the body that divide rapidly, which is one of the prominent characteristics of cancer cells. Radiation treatment damages the DNA of the cancer cells, leading to cell death. One of the primary causes for therapeutic failure in cancer is resistance to radiation and chemotherapeutic drugs. Cancer cells employ several mechanisms to resist chemotherapy and radiotherapy ( Table 2). Some tumors develop resistance to structurally and chemically unrelated anticancer drugs, termed multidrug resistance (MDR). This is one of the most dreadful challenges to cancer chemotherapy [22]. Tumors usually consist of a mixed population of malignant cells, some drug-sensitive, and some drug-resistant. Cancer cells that show resistance to chemotherapy when first exposed to an anticancer drug have intrinsic MDR [23]. Chemotherapy usually destroys drug-sensitive cells, and favors the survival of drug-resistant cells. After an initial treatment with chemotherapy, the tumor begins to grow again and develop resistance to chemotherapeutic agents [24]. Furthermore, drug resistance involves altered drug transport across the plasma membrane, genetic responses, enhanced DNA repair, modification of target molecules and access to target cells, metabolic effects, and various growth factors. Some of the mechanisms used by cancer cells to resist cytotoxic drugs are also observed in normal cells. They are part of the defense mechanisms that protect cells from environmental carcinogens. Table 2. Mechanisms by which tumor cells resist radiation and chemotherapeutic drugs.

Radioresistance Chemoresistance
Tumor hypoxic condition Increased drug efflux and decreased drug uptake Increased cellular production of cellular antioxidants Inactivation of apoptosis Activation of certain proto-oncogenes, and stromal interactions Increased drug metabolism and drug compartmentalization Amplification of DNA repair genes Increase in the repair of DNA damage Cancer stem cells as contributors to radioresistance Increased or altered the drug targets Survival signals favoured by transcription factors Survival signals favoured by transcription factors Multidrug resistance in cancer cells mainly develops by the overexpression of ATP-binding cassette (ABC) drug transporters. This is considered to be a major impediment to clinical cancer chemotherapy [25]. This condition occurs when cancer cells spontaneously become insensitive to structurally and functionally unrelated drugs. ABC transporters utilize energy derived from ATP hydrolysis to actively transport anticancer drugs across biological membranes, preventing drugs from reaching their targets within a cancer cell [26]. The ABC transporters belong to a superfamily of proteins that are classified into seven subfamilies (ABCA-ABCG) based on their sequence. However, only three major ABC drug transporters, including P-glycoprotein (P-gp; ABCB1), multidrug resistance protein 1 (MRP1; ABCC1) and ABCG2 (BCRP; MXR), are believed to seriously affect cancer chemotherapy [27].
Tumor cells acquire changes through many possible mechanisms and have increased resistance to apoptosis. They include inactivation of the p53 tumor-suppressor gene, activating mutations of the gene for PI3K, and activating mutations of the genes for the RAS/RAF pathway. Bcl-2 phosphorylation followed by activation of the NF-κB transcription factor and its downstream targets can lead to resistance to apoptosis. Tumor cell cytokinetics also can contribute to MDR [28], as well as other mechanisms, such as microsatellite instability and defective DNA repair, whereby tumour cells develop genetic changes.
Inhibiting the function of ABC drug transporters by inhibitors or modulators is one method to ameliorate drug sensitivity in multidrug-resistant cancer cells. The inhibition of ABC drug transporters during multidrug-resistant cancer chemotherapy allows elevated drug penetration, distribution and accumulation, and restores drug sensitivity. ABC drug transporter inhibitors, also called MDR modulators, chemosensitizers, or MDR reversal agents, are able to reverse resistance against anticancer drugs [29]. Inhibitors influence ABC transporters by specific interactions with proteins, changing the intracellular ATP level, which is the source of energy, or affecting membrane profiles to increase permeability. Inhibitors can affect other biological targets by nonspecific binding. However, the use of inhibitor drugs at the high concentrations necessary to reach a sufficient inhibitory effect may cause toxicity. Some of the south Asian medicinal compounds commonly used as radiosensitizers and chemosensitizers are listed in Figure 1.

Phytochemicals as Chemosensitizers
Currently three generations of chemosensitizers have been developed. Unfortunately, they are all toxic and cause side effects in recipients. Phytochemicals from medicinal plants are considered as fourth generation chemosensitizers to overcome tumor resistance. Examples include withaferin-A (from Withania somnifera), apigenin, kaempferol, anthocyanidin (from Solanum nigrum L); curcumin (from Curcuma longa), and berberine (from Tinospora cordifolia),glycyrrhizin (from Glycyrrhiza glabra) diallyldisulphide, diallyltrisulphide (from Allium sativum), and capsaicin (from Capsicum annuum). These have been shown to accomplish chemosensitizing activities both in vitro and in vivo [30]. Curcumin increases intracellular chemotherapeutic drug concentrations of drugs

Phytochemicals as Chemosensitizers
Currently three generations of chemosensitizers have been developed. Unfortunately, they are all toxic and cause side effects in recipients. Phytochemicals from medicinal plants are considered as fourth generation chemosensitizers to overcome tumor resistance. Examples include withaferin-A (from Withania somnifera), apigenin, kaempferol, anthocyanidin (from Solanum nigrum L); curcumin (from Curcuma longa), and berberine (from Tinospora cordifolia), glycyrrhizin (from Glycyrrhiza glabra) diallyldisulphide, diallyltrisulphide (from Allium sativum), and capsaicin (from Capsicum annuum). These have been shown to accomplish chemosensitizing activities both in vitro and in vivo [30].
Curcumin increases intracellular chemotherapeutic drug concentrations of drugs such as vinblastine or vincristine and can inhibit ABC transporters. Curcumin also enhances the cytotoxic effect of 5-FU and sensitizes prostate cancer cells through p53-independent cell-cycle arrest, consequently down-regulating NF-κB activation [31]. Curcumin has been shown to increase the cytotoxic effect of 5-FU and oxaliplatin in colon cancer cells via down-regulation of COX-2 [32,33]. (-)-Epigallocatechin-3-gallate (EGCG) can saturate drug efflux pumps, increasing the accumulation of chemotherapeutic drug within the cell. The substrates in effect sensitize cancer cells to chemotherapeutic agents. Moreover, curcumin is capable of interfering with the function of ABC transporters such as MRP, which needs a balanced supply of reduced glutathione GSH. This kind of inhibition might enhance chemotherapeutic agents by sensitizing cancer cells that overexpress MRP [34]. Targeting c-JUN expression is another clinical strategy to reduce GSH levels to overcome tumor resistance. C-JUN expressions are related with an AP-1-mediated increase in GSH synthetase levels. Curcumin is a strong inhibitor, reducing intracellular GSH at the transcriptional level [35]. Glutathione S-transferase Pi (GST-Pi) expression is linked with resistance to chemotherapeutic agents. Genistein, an isoflavone, is a promising chemosensitizer. Genistein potentiates the efficacy of gemcitabine through the down-regulation of NF-κB and Akt [36,37]; down-regulation of Akt is another mechanism to sensitize cancer cells to the apoptotic effects of TRAIL [38]. Genistein executes its chemosensitizing efficacy by modulating the pathways that cross-talk with TRAIL-induced apoptosis [39]. A number of studies have explored how quercetin enhances the efficiency of anticancer drugs and sensitizes cancer cells to chemotherapy. The efficiency of quercetin to chemosensitize cancer cells to doxorubicin depends on its ability to down-regulate HIF1-α, the downregulation of which makes them sensitive to doxorubicin [40]. Emodin is an anthraquinone obtained from Cassia obtusifolia. It is a tyrosine kinase inhibitor that inhibits HER2/neu tyrosine kinase activity, leading to a suppression of tumor growth [41]. Emodin sensitizes chemoresistant cells to paclitaxel-induced apoptosis and increases the intracellular concentration of paclitaxel. It also down-regulates the expression of antiapoptotic molecules and suppresses P-gp expression through ROS generation [42][43][44].
Resveratrol, a polyphenol, has been shown to sensitize cancer cells to chemotherapeutic drugs by modulating the molecular players of chemoresistance. Resveratrol also increases drug sensitivity through the inhibition of STAT3, NF-κB, c-FLIP, and Bcl-xL [45][46][47]. The major mechanism behind the chemosensitization of resveratrol to paclitaxel chemotherapy is through the downregulation of Bcl-2 family members and P-gp and inhibition of ERK1/2 and AP-1 pathways, leading to reduced Bcl-xL expression in resistant cells [48]. Resveratrol can be effectively used in combination with several chemotherapeutic agents that show adverse effects such as paclitaxel, vincristine, and daunorubicin in some tumor cells [49][50][51][52]. Glycyrrhetinic acid (GA) exhibits cytotoxic activities against HL-60 cells by induction of apoptosis. GA-induced apoptosis is at least partially initiated by interaction of the CD95 and CD178 signaling pathways [53]. Betulinic acid can overcome resistance or cross-resistance effectively. Combination of betulinic acid with irinotecan (IRT) and oxaliplatin (OXT) was effective against SNU-C5/5FU-R cells. Betulinic acid induced cancer cell death by apoptosis through the mitochondrial pathway [54]. The active components of south asian medicinal plants, which have interact with cell surface ABC transporters such as P-gp, ABCG2, and receptor tyrosine kinases including TRIAL-R, FLT3, KIT, VEGFR and EGFR. Some of the medicinal compounds also interact with intracellular tyrosine kinases (BCR-ABL kinase) resulting in chemosensitization ( Figure 2). The ATPase activity of P-gp can be stimulated by number of phytochemicals such as caffeic acid phenyl ester [CAPE], licochalcone A, anacardic acid, celastrol, and xanthohumol. Tumor necrosis factor-α (TNF-α)-mediated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were found to be sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, and xanthohumol. Natural NF-κB inhibitors reverse multidrug resistance. Some natural compounds, such as CAPE and licochalcone A, have dual inhibitory effects on the anticancer drug efflux transporter P-gp and NF-κB activation [55]. Natural compounds including withanolide, withaferin A and polyphenol quercetin were shown to inhibit NF-κB target genes in doxorubicin-sensitive K562 and -resistant K562/Adr cells involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance. Withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation decreases intracellular protein levels of Bcl2, Bim and P-Bad [56]. The chemosensitizing potential of certain South Asian medicinal compounds are summarized in Table 3. The ATPase activity of P-gp can be stimulated by number of phytochemicals such as caffeic acid phenyl ester [CAPE], licochalcone A, anacardic acid, celastrol, and xanthohumol. Tumor necrosis factor-α (TNF-α)-mediated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were found to be sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, and xanthohumol. Natural NF-κB inhibitors reverse multidrug resistance. Some natural compounds, such as CAPE and licochalcone A, have dual inhibitory effects on the anticancer drug efflux transporter P-gp and NF-κB activation [55]. Natural compounds including withanolide, withaferin A and polyphenol quercetin were shown to inhibit NF-κB target genes in doxorubicin-sensitive K562 and -resistant K562/Adr cells involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance. Withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation decreases intracellular protein levels of Bcl2, Bim and P-Bad [56]. The chemosensitizing potential of certain South Asian medicinal compounds are summarized in Table 3.  Down-regulation of Bcl-2 family members and MDR1/P-gp. Down-regulation of Bcl-2 family members and MDR1/P-gp. [48]

Radioresistance
When irradiation is unable to reduce tumor volume or cancer recurs after a regression, then a cell is considered to be radioresistant. Chemotherapy, radiotherapy and surgery are the major therapeutic options for treatment of human malignancies. Nearly 50% of newly diagnosed cancer patients receive radiotherapy (alone or in combination with chemotherapy or surgery) for their treatment [101]. Nevertheless, the curative potential of radiotherapy is limited by intrinsic radioresistance of cancer cells and normal tissue toxicity [102]. The degree of radiosensitivity is recognized by both intrinsic and extrinsic properties. Intrinsic properties include DNA repair, cell cycle status, and survival pathways, while extrinsic properties include cues from the extracellular environment [103]. Radioresistance has an effect on various biological factors such as DNA repair, gene amplification, increases in cellular production of free radical scavengers (eg, glutathione), proto-oncogene activation, and stromal interactions. DNA repair, apoptosis, growth factors, signal transduction, the cell cycle and cell adhesion are associated with genes related to radiosensitivity. The expression of p53 [104], ras [105], raf-1 [106], Bcl-2 [107] and survivin [108] genes are associated with radioresistance. In addition, cancer stem cells have emerged as a contributor to radioresistance through the activation of the DNA damage checkpoint response and an increase in DNA repair capacity [109]. Consequently, there is an increase in interest in enhancing the radiosensitivity of cancer cells for development of effective therapies [110].

Radiosensitization
A radiosensitizer is a drug that makes tumor cells more sensitive to radiotherapy. Radiosensitization is useful in cancer therapy, as it can enhance the effect of therapeutic radiation. An incredible amount of research and clinical trials have been performed on chemical agents that mimic the radiosensitizing effects of oxygen. Metronidazole, misonidazole, etanidazole and nimorazole are some of the electron-affinity agents used for radiosensitizing effects [111]. Radiosensitizing and radioenhancing agents increase the toxicity of radiation to cancerous tissues, and cause less damage to adjacent normal parenchyma cells. In addition to normal and hypoxia-based radiosensitizers, various other types of sensitizing agents have also been tried on malignant brain tumors. Preclinical research often shows significant promise, but unfortunately, the clinical efficacy of such agents has generally been modest at best [112]. For example, grade III gliomas when treated with conventional radiotherapy and estramustine, an estradiol-based antimicrotubule effector showed a positive improved response. [113].

Radiosensitizing Properties of South Asian Medicinal Compounds
Radiosensitization has generally been limited to the combination of radiation therapy with traditional cytotoxic agents such as hydroxyurea, 5-fluorouracil and cisplatin. Thus, radiotherapy has been combined with molecular targeted agents, which blocks specific pro-survival signaling pathways [114]. Two brain tumor radiosensitizers have received a great degree of interest of late: motexafin gadolinium (MGd) and efaproxiral [115]. Most patients with cancer receive radiotherapy at some point during the course of their treatment. The efficacy of treatment depends on the total dose of radiation, the fractionation scheme, the degree of oxygenation of tumors and the normal tissue response to radiation. The heterogeneity of tumors and their response to treatment, in early experimental and theoretical studies in radiobiology, were identified. DNA strand-break repair, repopulation of cancer cells between radiation fractions, reoxygenation of tumors after a radiation fraction and redistribution of cells into a radioresistant phase of the cell cycle were considered to be the four classical mechanisms of radioresistance [116]. Understanding the molecular basis of these processes can permit selective targeting of the molecules and pathways that regulate cellular radioresponse. Radiation activates pro-survival signaling pathways, to target normal tissue responses such as angiogenesis and also exploit radiation-induced gene expression for other therapeutic modalities [117].
Many natural or naturally derived compounds have shown cytotoxic effects in cancer cells. Compounds such as vinblastine, vincristine, taxol, and camptothecin have been approved for clinical practice [118]. These anticancer drugs act through several mechanisms to kill tumors: Microtubule polymerization, mitotic inhibition [119], and inhibition of DNA topoisomerase I enzyme [120]. Genotoxicity is the main tumoricidal effect of anticancer agents. The effectiveness of radiation therapy on cancer cells is enhanced when the anticancer products are co-administered with IR during radiotherapy. Treating cells with radiosensitizers before irradiation probably sensitizes the cell cycle phase to radiotherapy [121,122]. The presence of natural compounds during irradiation enhances their effects by increasing the toxic reactions of free radicals and inhibiting the repair of the radiation induced lethal and sub-lethal damage by misregulation of signal pathways [123]. The active components of south asian medicinal plants, which have rediosensitizing properties, and their molecular targets are described in Figure 3.
Many natural or naturally derived compounds have shown cytotoxic effects in cancer cells. Compounds such as vinblastine, vincristine, taxol, and camptothecin have been approved for clinical practice [118]. These anticancer drugs act through several mechanisms to kill tumors: Microtubule polymerization, mitotic inhibition [119], and inhibition of DNA topoisomerase I enzyme [120]. Genotoxicity is the main tumoricidal effect of anticancer agents. The effectiveness of radiation therapy on cancer cells is enhanced when the anticancer products are co-administered with IR during radiotherapy. Treating cells with radiosensitizers before irradiation probably sensitizes the cell cycle phase to radiotherapy [121,122]. The presence of natural compounds during irradiation enhances their effects by increasing the toxic reactions of free radicals and inhibiting the repair of the radiation induced lethal and sub-lethal damage by misregulation of signal pathways [123]. The active components of south asian medicinal plants, which have rediosensitizing properties, and their molecular targets are described in Figure 3.

Radioprotective Effects of Natural Compounds
Radioprotectors are agents that protect normal cells during radiotherapy. Radioprotective agents protect macromolecules, cells, and tissues from radiation-induced toxicity. These agents are administered before exposure to radiation. Several mechanisms are proposed for radioprotective agents: Direct scavenging of ROS, hydrogen donation to reactive free radicals, inducing/altering the levels of endogenous enzymes for detoxifying ROS, anti-inflammatory action, immune-stimulant activity, increasing DNA stability, reducing the production of ROS by inducing hypoxia, and

Radioprotective Effects of Natural Compounds
Radioprotectors are agents that protect normal cells during radiotherapy. Radioprotective agents protect macromolecules, cells, and tissues from radiation-induced toxicity. These agents are administered before exposure to radiation. Several mechanisms are proposed for radioprotective agents: Direct scavenging of ROS, hydrogen donation to reactive free radicals, inducing/altering the levels of endogenous enzymes for detoxifying ROS, anti-inflammatory action, immune-stimulant activity, increasing DNA stability, reducing the production of ROS by inducing hypoxia, and enhancing DNA damage repair pathways [124][125][126]. Pretreatment with natural compounds with antioxidant activity protects biological systems against oxidative stress. Because natural products have fewer side effects, they are increasingly being used as radioprotective agents. Polyphenols from herbal plants have protective effects against genotoxicity induced by γ-irradiation. Natural compounds reduce DNA damage and genomic instability induced by ROS in vitro and in vivo. Flavonoids have excellent antioxidant activity through various mechanisms, and flavonoids with more antioxidant activity have higher radioprotective effects [127]. Table 4 summarizes some of these natural products that act as radioprotectors in normal cells and radiosensitizers in cancer cells through different mechanisms. Table 4. South Asian medicinal compounds protect normal cells from radiation effects and sensitize cancer cells to radiation effects. South Asian medicinal compounds at the concentration studied protects the normal human cells and experimental animals against indicated radiation dose and sensitizes radiation effects in the cancer cells through different mechanism.

Conclusions
Cancer is a multifactorial disease that requires multichannel therapy, including radiotherapy and chemotherapy. Cancer cells acquire resistance to radiotherapy and chemotherapy through different mechanisms. Number of synthetic and semisynthetic radiosensitizers and chemosensitizers were investigated; unfortunately these compounds exhibit adverse reactions in non-target organs. Naturally occurring medicinal compounds are considered to be non-toxic fourth generation chemosensitizers and a majority of these compounds also exhibit radiosensitizing properties. This present review reveals the role and importance of South Asian medicinal compounds as radiosensitizers and chemosensitizers. South Asian medicinal compounds exhibit radiosensitizing and chemsensitizing property through modulating/inhibiting different molecular targets. Thus, South Asian medicinal compounds may be considered as an adjuvant to radio-and chemotherapy for cancer patients.