Nutraceuticals have played an important role in the overall well-being of humans for many years, with or without rigorous evidence backing their health claims. Traditional medicine systems around the world have utilized plants for millennia that have medicinal properties, providing an opportunity for modern day researchers to assess their efficacies against ailments such as cancer. Withania somnifera (WS) is a plant that has been used in Ayurveda (an ancient form of medicine in Asia) and in the recent past, has been demonstrated to have anti-tumorigenic properties in experimental models. While scientific research performed on WS has exploded in the past decade, much regarding the mode of action and molecular targets involved remains unknown. In this review, we discuss the traditional uses of the plant, the experimental evidence supporting its chemopreventive potential as well as roadblocks that need to be overcome in order for WS to be evaluated as a chemopreventive agent in humans.

 Withania somnifera (WS): an introduction

WS (Ashwagandha; Indian winter cherry, Indian ginseng) is a medicinal plant that has been utilized in traditional medicine in many parts of South Asia for millennia. It belongs to the diverse Solanaceae family of flowering plants. Withania species show a particularly wide distribution throughout drier climates of the world. Although there are 23 known Withania species, only W. somnifera and W. coagulans (Rishyagandha) are believed to have medicinal benefits . While they have several similarities, the WS plant is much more branched and has larger leaves compared to W. coagulans. WS is more commonly used in traditional medicine but some specific preparations also utilize W. coagulans. A few studies have identified that W. coagulans may also have important implications as a therapeutic to type II diabetes .

WS roots used in over 200 formulations in Ayurveda, Siddha and Unani medicine. Ashwagandha churna, powdered root of the WS plant is frequently used to treat a variety of ailments . Further, it is also used with other ingredients. WS is used as the major component in Saraswati churna, which is a herbal powder mixture utilized to treat neurological conditions. Ashwagandhadhi lehyam is another preparation that includes WS, primarily utilized as a rejuvenation supplement, a treatment for male impotence and as an energy enhancer . While these uses may seem highly divergent, it is likely that specific proportions and interactions with the other ingredients used in the preparations could result in highly differential outcomes. Interestingly, only the root of the plant is used for traditional medicine preparations. The use of WS in Ayurvedic concoctions has recently been evaluated by alternative medicine researchers where it has been shown that utilizing standardization, phytochemical screenings and testing for pathogen/heavy metal contamination can significantly improve the actions of Ashwagandadhi lehyam

Potential to target cancer

Mounting evidence from cell culture and animal studies suggest that WS possesses anti-tumorigenic properties. In 1967, it was first demonstrated experimentally that the root extract resulted in lowered cancer incidence in vivo . Ever since, research interest in WS as an anti-tumorigenic agent has grown. This is apparent from the increase in the number of publications citing Withaferin A (WA; a withanolide from the WS plant) over the past decade from less than 5 in 2002 to more than 50 in 2015. Researchers are just starting to scratch the surface of molecular pathways modulated by WS and its withanolides in order to counter the carcinogenic process. Not only has WS and its withanolides been shown to have therapeutic potential against cancer, some of them have also been shown to possess cancer preventive properties . These studies are discussed in detail in later sections. The cancer fighting properties have been seen not only with root extracts, but also with leaf extracts which is a relatively underused part of the Ashwagandha plant . In addition to directly protecting against carcinogenesis, WS and especially WA has been shown to be hepatoprotective . From the perspective of Ayurvedic medicine, there are several important implications of Ashwagandha for the treatment and prevention of cancer. As mentioned previously, the role of Ashwagandha in regeneration and rejuvenation can potentially be pivotal to improve longevity and quality of human life. Thus, this idea of overall health promotion may lead towards prevention of chronic disease like cancer. However, the dosage of Ashwagandha administered as treatment for cancer is presumably quite different to what is given as a general supplement that promotes good health. Careful research needs to be conducted to determine these parameters so that the factors pertaining to the use of Ashwagandha as a chemopreventive agent can be accurately established.

Bioactivity of Withania somnifera: Withaferin A and other withanolides

Extraction and isolation

Multiple methods are utilized to extract Ashwagandha from whole roots or leaves of the plant. Conventional methods usually involve extensive drying followed by grinding into a fine powder. Next, aqueous or organic solvent-based extraction procedures are performed where research suggests several ways in which extraction yields could be improved . For example, microwave-assisted extraction can be optimized by modifying extraction time, temperature and solvent ratio. It has been identified that the major compounds isolated through alcoholic extraction of WS are alkaloids and withanolides . Ultimately the best determinant of the success of the extraction or isolation methodology is how well the extract itself performs against a given disease process. A study showed that water extraction is just as viable as organic solvent extraction of Ashwagandha in affecting cancer cell progression . More sophisticated methods such as high performance liquid chromatography (HPLC) coupled with mass spectrometric quantification have allowed more extensive and consistent isolation of bioactives from Ashwagandha . On the other hand, non-extraction based isolation methods are also used, albeit infrequently, especially within the realm of Ayurveda where whole plant parts are dried and used directly as a powder. While this method may preserve the integrity of the plant parts, using whole plant products increases chances of contamination with pathogens and heavy metals and may also reduce the potency due to the presence of chemicals other than the bioactive components in the plant. Conversely, elements of the plant matrix could enhance the bioactivity of WA. Nonetheless, given the high variability of withanolide concentrations in different plant parts and the existence of chemotypes of WS , standardization techniques need to be incorporated into these isolation practices. Furthermore, it is very important that preparations are made in accordance with guidelines published by the World Health Organization to minimize pathogens, aflatoxins, pesticide residues and heavy metals .

Pharmacology

Characterization studies of WS have identified that the bioactive compounds present in the root, leaf and stem extract includes alkaloids and steroidal lactones. The bioactive compounds of WS have been further identified as withanolides, a type of steroidal lactone. So far, 12 alkaloids, 35 withanolides and several other sitoindosides (a withanolide containing a glucose molecule at carbon 27) have been identified  suggesting the diverse chemical makeup of the plant. Studies have shown that there is differential distribution of withanolides in different parts of the WS plant where WA is most abundant in the leaves as opposed to 12-deoxywithastramonolide and Withanolide A which is more profuse in the plant root . In an in vitro model system that closely mimicked cellular absorption using Madin-Darby canine kidney cells, WA had much lower absorption compared to other withanolides . WA has also been demonstrated to have higher bioavailability compared to Withanolide A when WS root extract was administered to Swiss Albino female mice orally . The half-life of withanolides was evaluated in the same study where t_1/2 of WA was shown to be approximately 60 minutes whereas Withanolide A had a shorter t_1/2 of 45 minutes . Given this rapid half-life, it may be worth considering twice daily (BID) or three times daily (TID) of WS in dosing regimens. While withanolides as a whole possess several properties that could potentially be utilized against a variety of diseases, the majority of research work that has been conducted on withanolides involves WA. This in part, is due to the notion that WA is the most potent withanolide identified thus far from the Ashwagandha plant and was one of the first withanolides to ever be isolated .

The pathways for the metabolism and biotransformation of the withanolides of WS are poorly understood. In vitro microbial transformation of WA to 14 alpha-hydroxywithaferin A has been shown . Given the structure of WA, it is likely that it undergoes hydrolysis (by epoxide hydrolase) and other reduction/oxidation reactions followed by conjugation to glutathione, glucuronides or sulfates. However, experimental evidence to support this claim is limited and is therefore an area that needs to be considered especially when studying the pharmacokinetics of withanolides.

Reports of major side effects of Ashwagandha are relatively scarce making it an attractive agent for cancer chemoprevention in humans. To assess acute toxicity, Wistar rats were administered a very large dose of 2000 mg/kg WS root extract for 14 days where no mortality or signs of toxicity were observed . However, in another study where Sprague-Dawley rats were fed WS (dose not noted) for 14 days changes in liver and kidney histopathology was observed . Understandably, purified withanolides have been associated with some minor side effects, likely due to the fact that biological effects are enhanced with a purified compound as compared to a crude plant extract. Administering 16 mg/kg WA intraperitoneally for 30 days to C57BL/6J mice resulted in loss of body weight and changes in serum enzymes . Some sedation, ptosis and ataxia were observed in Sprague-Dawley rats 15–20 minutes of administering a herbal concoction that contained WS at a large dose of 1–2 g/kg body weight . From a structural standpoint, it has been hypothesized that observed cytotoxicity of WA against cancer cells is attributable to its epoxide group . Further research is required to determine if the aforementioned toxic side effects can be alleviated by using structural analogs that have the epoxide group or any other potentially important chemical group modified. These studies suggest that an in vivo safe dosage range is available for WS but need to be established in pre-clinical studies using appropriate models.

Structures and mechanisms of action

Novel withanolides are still being identified by researchers . As mentioned previously, extensive work has been performed with WA where several of its structural properties have been identified. The cysteine-reactive nature of the α,β- unsaturated carbonyl group of WA is well-established . WA has further been shown to directly bind to key cysteine residues of proteins such as Vimentin , GFAP , IKKβ  and β-Tubulin . WA has also been shown to modulate important cellular signaling processes such as autophagy , proteasomal degradation  and the heat shock response . Whether modulation of these processes originates from direct binding has not been elucidated. A study that evaluated the heat shock inducing activity of WA and several structural analogs showed that undesired cytotoxicity from WA could be minimized while enhancing cytoprotective activity by modifying WA structurally . This study also suggested that there are key chemical moieties of the WA molecule that might be responsible for specific biological activities.

Cancer pathways modulated by Withania somnifera and its withanolides

Cell survival/ apoptosis

Most discussions on anti-tumorigenic properties of WS pertain to its ability to activate apoptotic pathways in cancer cells. Even within the realm of cancer chemoprevention, cell survival and the activation of pro-apoptotic pathways holds important implications where successful reversal of the carcinogenesis process essentially requires the early clearance or destruction of impaired cells. Several currently known chemopreventive agents such as the isothiocyanate, sulforaphane  and the triterpenoid, CDDO-Im  exhibit this property. A plethora of in vitro evidence exists about the induction of apoptosis by WS , WA  as well as other withanolides . Some of the earliest hints of tumor suppression by WS came from a study that evaluated the potential of leaf extract to inhibit tumor formation in nude mice subcutaneously injected with fibrosarcoma HT1080 cells . It was observed that treating mice with the leaf extract (0.3 mL of 24 µg/mL extract in cell growth medium, s.c.) resulted in reduced tumor size and was in part mediated via upregulation of p53. Interestingly, the authors of the paper used NMR to identify the component responsible for this action to be withanone. Induction of apoptosis by WA has been noted in some in vivo models where treatment with 4 mg/kg WA, i.p. 5 times for 2 weeks markedly reduced MDA-MB-231 tumor weights in nude mice as well as increased apoptosis compared to tumors in control mice .

While the exact mechanisms for induction of apoptosis by WS and its withanolides are yet to be established, data from several publications suggest that enhanced expression of pro-apoptotic genes as well as the suppression of proliferative pathways are possible targets. In a study conducted on a xenograft mouse model of cervical cancer, it was shown that 8 mg/kg WA, i.p. treatment for 6 weeks resulted in 70% reduction in tumor volume compared to controls as well as heightened expression of p53 and lowered expression of pro-caspase 3/ Bcl2 . The ability of WA to downregulate oncogenic proteins that have anti-apoptotic function such as Bcl2 has been reported by others as well . Whether this phenomena occurs in vivo within the tumor micro-environment to the extent that WA can selectively slow the growth of tumor cells via the aforementioned mechanisms while stabilizing the apoptotic function of normal cells has not been clearly determined. Ultimately, to utilize WS as a chemopreventive agent, the pharmacological conditions under which normal cells will survive while pre-cancerous/ cancerous cells will undergo death need to be assessed. Selective killing of cancer cells by WA is an idea that has been put forward by many. By comparing cell lines that are cancerous and non-cancerous, WA has been shown to be cytotoxic to only cancerous cell lines . A point to note is that, these cell lines have inherent differences that can result in differential drug uptake, retention and toxicity. Therefore, mechanistic explorations of how tumor cells vs. non-tumor cells respond to WS and its withanolides require further investigation.

Angiogenesis

It is widely accepted that angiogenesis is a vital process exploited by tumors to facilitate their own growth. In addition to tumor masses, early stage carcinogenic events may also utilize angiogenesis suggesting that it could be attenuated in a cancer preventive context. Angiogenesis has been categorized as a marker of cancer progression given the differences that occur in new blood vessel formation during early and late stages of carcinogenesis . The role of WS and its withanolides on angiogenesis has been studied. The first report related to anti-angiogenic effect was published in 2004, where WA was shown to be a potent inhibitor of angiogenesis both in vitro and in vivo . In another study, WS was shown to inhibit angiogenesis in a VEGF-induced neovascularization model in vivo . An in silico study along with molecular docking analyses corroborated the mechanism of this finding by showing that WA may directly bind to VEGF and thereby hamper angiogenesis . Further in vitro and in vivo experimentation is required to validate the physiological relevance of this finding.

Stress response

In recent years, the role of stress response pathways in cancer chemoprevention has been closely evaluated . WS and some of its withanolides have been shown to be mediators of the heat shock response. The heat shock response is essential to cellular homeostasis given its function in facilitating the degradation of misfolded proteins. Transcriptional regulation of multiple classes of genes by Heat shock transcription factor 1 (HSF1) is considered to be an important regulatory step of this mechanism. WA has been shown to bind HSP90 to inhibit its chaperone activity through an ATP-dependent mechanism in pancreatic cancer cells .This has been proposed to be one of the mechanisms by which WA exerts its anti-tumorigenic activity. A multiple compound screening study that utilized heat shock response induction as an endpoint identified WA as one of the potent mediators of the heat shock response wherein 1–4 µM WA was shown to be thiol-reactive and also shown to induce protein expression of HSP72 and 27 . In a subsequent analysis, Wijeratne et al.  demonstrated that modulation of heat shock inducing activity of WA is feasible by structural modifications. It is important to point out that the effect of leaf or root extracts of WS on heat shock response has not been determined.

In addition to the heat shock response, several other stress response pathways have also been shown to be affected by WS and some of its withanolides. Several reports note that WA is a strong inducer of oxidative stress, mediated primarily via the generation of reactive oxygen species . Interestingly, a report by Kaur et al.  suggested that WS extract did not provide any protection against oxidative damage caused by high glucose and hydrogen peroxide in human cancer cells, possibly suggesting that the pro-oxidant characteristics of WA would not render useful in protecting against oxidative damage. The exact percentages of withanolides in this leaf extract were not revealed, making it difficult to understand the exact mechanism underlying the observation. Furthermore, whether oxidative stress induction by WA is a very early molecular event that facilitates downstream cytoprotective pathways in order to ultimately guard cells and organisms is also currently unknown. WS and its withanolides have also been shown to up regulate the expression of several phase II enzymes suggesting that other cytoprotective pathways, such as Nrf2 directly or indirectly, may be mediated by the action of withanolides.

Inflammation and immune regulation

Researchers are on the brink of identifying the pivotal roles played by inflammation and immune function in cancer. Reducing chronic inflammation to prevent certain types of cancers (e.g., hepatitis virus-induced inflammation and liver cancer) as well as utilizing immunotherapy as a successful treatment strategy for cancer are two key widely sought after areas of current cancer research. It is indeed desirable that some future chemopreventive drugs possess anti-inflammatory properties and also exhibit the ability to induce a robust immune response against early stage malignancies. Whether certain compounds that activate the immune system could potentially be utilized to prevent cancer has not been studied in detail, perhaps due to the fact that hyperactivation of the immune system could lead to several undesired challenges. Nevertheless, controlled activation of the immune system by WS is well-documented. In fact, two human studies with WS have looked at immunological end points. These studies suggest that the mechanism of action is driven by lymphocyte and NK cell activation. Anti-inflammatory properties of WA are attributable to directly targeting cysteine 179 of IKK-β leading to the inhibition of NF-kB activity. WA has also shown COX-2 inhibitory activity in some experimental models. The anti-inflammatory and immune effects of WS and withanolides warrants further investigation, especially given the role of Ashwagandha as an adaptogen in traditional medicine.

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