Study of Drug Targets Associated With Oncogenesis and Cancer Cell Survival and the Therapeutic Activity of Engineered Ashwagandha Extract Having Differential Withanolide Constitutions

Abstract

Ashwagandha (Withania somnifera) has gained worldwide popularity for a multitude of health benefits inclusive of cancer-preventive and curative effects. Despite numerous research data supporting the benefits of this wonder herb, the actual use of ashwagandha for cancer treatment in clinics is limited. The primary reason for this is the inconsistent therapeutic outcome due to highly variable composition and constitution of active ingredients in the plant extract impacting ashwagandha’s pharmacology. We investigate here an engineered yield: an ashwagandha extract (Oncowithanib) that has a unique and fixed portion of active ingredients to achieve consistent and effective therapeutic activity. Using the MCF7 cell line, Oncowithanib was studied for its anti-neoplastic efficacy and drug targets associated with cell cycle regulation, translation machinery, and cell survival and apoptosis. Results demonstrate a dose-dependent decline in Oncowithanib-treated MCF7 cell viability and reduced colony-forming ability. Treated cells showed increased cell death as evidenced by enhancement of Caspase 3 enzyme activity and decreased expressions of cell proliferation markers such as Ki67 and Aurora Kinase A. Oncowithanib treatment was also found to be associated with expressional suppression of key cellular kinases such as RSK1, Akt1, and mTOR in MCF7 cells. Our findings indicate that Oncowithanib decreases MCF7 cell survival and propagation, and sheds light on common drug targets that might be good candidates for the development of cancer therapeutics. Further in-depth investigations are required to fully explore the potency and pharmacology of this novel extract. This study also highlights the importance of the standardization of herbal extracts to get consistent therapeutic activity for the disease indication.

Introduction

Cancer is a complex group of diseases driven by multiple factors that need to align in order for the disease to advance. The complex underpinnings of carcinogenesis clearly reveal that family history, environment, viral infection, mental state, and lifestyle can all play dynamic roles in this multigenic disease.^{1 -3} However, cancer mutations in oncogenes or suppressor genes are not black and white determinants of phenotypic expression of cancer^{4 -7} since multiple genes and the result of their expression need to be corrupted or mutated in order for cancer to prevail. In addition, the genetic and metabolic features of cancer pathology that contribute to oncogenesis in its early stages may look very different from that of cancer that has evolved resulting in treatment protocols needing to evolve as well.^8,9 In addition, it has become clear that tumors harbor stem cells that coexist in an ecosystem of different cell types that support the fitness and survival of the tumor in the context of treatment with therapeutic strategies.^10,11 The genetic variation of cells within the tumor itself and the divergence of novel mutations in metastatically disseminated tumors from the primary tumor demonstrate the complexity and ever-changing environment that cancer treatments need to contend with. ¹²

Cancer investigation today focusses more on genetic expression versus genetic constitution and the state of the gene; and is evolving quickly to hone in on stem cell activity to circumvent epithelial transition to mesenchymal state. ¹³ This transition instills plasticity and tumor progression. Successful treatment requires consideration of the downstream proteome in this very context because a mutation that may “turn on” a cancer gene must still contend with tumor suppressor genes, of which more than 100 candidates were isolated ¹⁴ even as far back as 1997.^15,16 Even with how advanced medical sciences are today the treatment of most cancers comes with unavoidable cytotoxicity on healthy cells and tissues.^{17 -19} While we treat with an objective to eradicate cancer and its predecessor stem cells, we do our best to limit irreversible injury to host tissues or death resulting from treatment. Common is the development of therapy resistance through innate and acquired forms of resistance ²⁰ ; or the cancer evolving genetically to behave differently than the disease that was first diagnosed. ²¹

The degree of host toxicity associated with most treatments is a fundamental challenge.^22,23 We might be able to overcome this cytotoxicity challenge if we are able to utilize strategies that are co-administered to make cancer cells more vulnerable to the current treatments we have come to know more about and rely on. Withaferin A, a constituent of ashwagandha (Withania somnifera) has been shown to induce apoptosis in human breast cancers ²⁴ ; in pancreatic cancers^25,26; lymphoma ²⁷ and other cancers as we have also shown in the work herein. Nevertheless, Withaferin A is also highly toxic to healthy cells, like many other allopathic chemotherapeutic agents are. We have studied and reported on the various constituents of the ashwagandha plant in previous publications, an alternative medicinal agent central to the Ayurveda. It boasts as many as 35 bio-active withanolides and countless other phyto-actives. ²⁸

Here we study and report on Oncowithanib, an engineered yield of an ashwagandha extract that includes all the withanolides in the presence of an amplified Withaferin A constitution that exceeds the amount found in a typical extract. Using a standardized extraction method, Oncowithanib has been extracted to achieve consistent and effective therapeutic outcome.

We have set out to target various subcellular proteins in MCF7 cancer model including Akt1 which is recognized as a central node affecting multiple signaling pathways relating to proliferation, angiogenesis, and other key metabolic features of cancer cell survival.^{29 -32} While successfully targeting Akt with our engineered drug, we also show inhibitory activity of downstream mTOR. Research indicates Akt and mTOR dysregulation is one of the most frequent abnormalities advancing malignancy.^{33 -36} The over-activated form of Ribosomal Protein S6, the translational machinery for the over-activated RSK pathway^{37 -40} is also a good target we have dedicated investigative resources to. Here we show direct downward modulation of RSK1 by our candidate drug and the same for RSK’s phosphorylation target, Ribosomal Protein S6. Cancer treatment may be successful but fail to address stem cells or other surviving forms that arise out of the silenced disease in due time with a resilience to the treatment that was successful on the first round of treatment.^{41 -44} Our results are presented alongside those of commonly used chemotherapeutic drugs revealing significantly functional results.

Recurrence of disease after successful chemotherapeutic treatment might be better addressed if we can utilize mechanisms that restore vulnerability of cancer to the original treatment; and prevent stem cell differentiation and proliferation. With this objective we also investigate cancer cell proliferative activity by way of Ki67, the expression of which strongly correlates with tumor cell proliferation and metastasis.^{45 -47} Aurora kinase A is also a critical mitotic regulator.^{48 -50} Our candidate drug is further applied to target these and other markers of proliferation and stem cell differentiation with pleasantly surprising outcomes.

A better understanding of how our candidate drug modulates these selected targets will allow us to utilize the same treatment approach to target multiple factors (such as stem cells or other stages that survive) that allow cancer cells and tumor ecosystems to survive.^20,51,52 Nevertheless, the positive implication of this strategic co-treatment might be bigger than treatment of recurring disease. If that anti-resistance strategy could be one of natural origin, we may be able to perform reliable and predictable treatment the second time around without the unpredictability of renewed host toxicity. These strategies could also prove to be functional preventive approaches circumventing recurrence altogether and the mechanism for this treatment may have been at our fingertips all along.

Materials and Methods

Cell Line

The MCF7 cancer cell line was used in the study and was kindly provided by Dr. Steven Pelech (Kinexus Bioinformatics, Canada). Cells were cultured in RPMI-1640, supplemented with 5% fetal bovine serum (FBS) and 1% antibiotics (Sigma, USA) at 37°C in a humidified cell culture incubator (Forma Scientific) with 5% CO₂.

Crystal Violet Assay

Crystal violet assay was performed to determine the cytotoxicity of Oncowithanib on MCF7 cells. The cytotoxic activity of Oncowithanib was also compared to other natural anti-neoplastic compounds such as Withaferin A, Paclitaxel, Vinblastine, and commercial ashwagandha (ASHWITH) extract. Withaferin A, Paclitaxel, and Vinblastine were obtained from Sigma, USA. Oncowithanib and ASHWITH ashwagandha were obtained from Biologic Pharmamedical, Canada. Briefly, MCF7 cells were seeded at a density of 10 × 10³ cells per well in 96-well plates and allowed to settle overnight. On the next day, cells were treated with (a) varying concentrations (0.63, 1.25, 2.5, 5, 10, and 20 µg/ml) of Oncowithanib and Withaferin A and (b) 1 and 10 µg/ml of Oncowithanib, Withaferin A, ASHWITH, Paclitaxel, and Vinblastine. After 18 hours of incubation, plates were washed to remove non-viable cells (viable cells remain attached to the plate) and incubated with 0.2% crystal violet (Sigma, USA). Stained cells were photographed using Nikon Eclipse TS100 microscope. Finally, 1% SDS was added to solubilize the stain, and the absorbance was measured at 570 nm using Versamax microplate reader (Molecular Devices). The cell viability of treated cells was calculated against the solvent control and presented as the percentage survival (compared to DSMO).

Soft-Agar Colony Formation Assay

The assay was performed as described in the protocol of Borowicz et al ⁵³ with slight modifications, to measure the clonogenic activity of MCF7 cells treated with Oncowithanib and Withaferin A. The bottom agar layer was prepared by mixing an equal volume of 1% agar (Sigma, USA) and 2× RPMI complete growth media. About 0.5 ml of this mixture was added per well in a 24-well plate and allowed to settle for 30 minutes at room temperature. The top agar layer was prepared by mixing an equal volume of 0.6% agar and MCF7 single-cell suspension required to achieve 5 × 10³ cells per well. About 0.5 ml of this mixture was added on top of the solidified bottom agar layer and allowed to settle further for 30 minutes. About 100 µl of treatment media with varying concentrations (2.5, 5, and 10 µg/ml) of Oncowithanib and Withaferin A was added to the respective wells every 3 day interval. The plate was placed into a 37°C humidified incubator and colonies were manually counted on day 26 using an inverted microscope (Nikon Eclipse TS100).

Caspase 3 Assay

Caspase 3 enzyme activity assay was performed as per the manufacturer’s instruction (#ab39401, Abcam, Canada). Oncowithanib (10 µg/ml) treated MCF7 cells were harvested, lysed using cell lysis buffer, and cell supernatant after total protein quantification was mixed with the reaction buffer and DEVD-p-NA substrate. The output was measured at 400 nm using Versamax microplate reader (Molecular Devices).

Western Blot

MCF7 cells were seeded at a density of 20 × 10⁴ cells per well in 6-well cell culture plates. Cells at 80% confluency were treated with various concentrations (5 and 10 µg/ml) of Oncowithanib for 18 hours. Afterward, cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested using RIPA lysis buffer with a protease inhibitor cocktail (Sigma, USA). Crude cell lysates were sonicated for 30 seconds followed by centrifugation at 10 000g for 10 minutes at 4°C. The cell supernatant was collected and used for total protein quantification (DC Protein assay kit, BIO-RAD, Canada). An equal amount of protein (30 µg) of each sample was mixed with SDS sample buffer (EMD Millipore Corp. USA), heated at 95°C for 5 minutes for protein denaturation, and finally separated using 7.5% polyacrylamide gel electrophoresis (BIO-RAD). After separation, proteins were transferred onto 0.2 µM nitrocellulose membrane (Amersham, Germany) using the wet transfer method. The membranes were blocked in 2.5% bovine serum albumin (BSA) blocking buffer for 1 hour and then probed with primary antibodies such as Ki67 (2:1000) (Abcam, Canada), Aurora Kinase A (2:1000), RSK1 (2:1000), Ribosomal Protein S6 (RPS6 − pS235 + pS236) (2:1000), Pan-Akt1 (1:1000), mTOR (2:1000) (Kinexus Bioinformatics, Canada) and GAPDH (1:1000) (Abcam, Canada) for overnight at 4°C. Membranes were washed twice with Tris-buffered saline with Tween (TTBS) and incubated with goat anti-rabbit secondary antibody (1:10,000) (Sigma, USA) for 1 hour at room temperature. The membranes were developed using Immobilon Forte (Western HRP substrate; EMD Millipore Corp. USA), scanned using Fluor-S MultiImager scanner, and quantified by Quantity One software (BIO-RAD). Target protein expressions were normalized to the sample’s loading control GAPDH.

Statistical Analysis

All experiments were repeated thrice, and quantitative values were presented as mean ± SD of N = 3. Unpaired Student’s t-test was applied for statistical analysis, and P < .05 was considered statistically significant.

Results

Dose-Dependent Decline of MCF7 Cell Viability and Reduction of Colony-Forming Ability After Treatment With Oncowithanib

MCF7 cells were treated with varying concentrations of Oncowithanib (0.63, 1.25, 2.5, 5, 10, and 20 µg/ml) for 18 hours followed by a crystal violet cell cytotoxicity assay. A dose-dependent decline in cell viability was observed with increasing concentrations of Oncowithanib treatment in MCF7 cells, and IC50 concentration was found to be 10 µg/ml (effective dose to reduce the number of a viable cell population by half). MCF7 cells were also treated with Withaferin A for comparison, which showed a similar pattern in reduction of cell viability after treatment (Figure 1A). Crystal violet-stained cells were photographed for visual observation, which further confirmed a dose-dependent reduction in viable cell population after drug treatment (Figure 1B). On the other hand, the soft-agar colony assay also showed a dose-dependent decline in the colony-forming ability of MCF7 cells treated with Oncowithanib (Figure 1C). Next, we sought to compare the potency of Oncowithanib with other natural anti-neoplastic compounds in addition to Withaferin A, such as Paclitaxel and Vinblastine, and regular commercial ashwagandha (ASHWITH) extract. MCF7 cells were treated with 1 and 10 µg/ml concentrations of Oncowithanib, Withaferin A, ASHWITH, Paclitaxel and Vinblastine for 18 hours, and cell viability was determined. The percentage cell viability was found to be 84.23 ± 8.18%, 67.79 ± 12.40%, 80.02 ± 4.91%, 72.70 ± 9.37%, and 65.09 ± 8.49% at 1 µg/ml, respectively and 45.21 ± 6.26%, 40.57 ± 8.18%, 64.42 ± 3.90% 67.05 ± 12.05%, and 68.33 ± 9.10% at 10 µg/ml, respectively. Taken together, Oncowithanib significantly reduces MCF7 survivability and has a similar potency as Withaferin A and a higher potency than regular ashwagandha extract, Paclitaxel and Vinblastine (Figure 1D).

Figure 1.

Cytotoxicity of Oncowithanib on MCF7 cells. (A) MCF7 cells were treated with Oncowithanib at 0.63, 1.25, 2.5, 5, 10, and 20 µg/ml concentrations for 18 hours, and cytotoxicity was determined by crystal violet assay. Cells were also treated with Withaferin A for a comparison. Data shows a dose-dependent decline in MCF7 cell viability and IC50 was found to be approximately 10 µg/ml for Oncowithanib treatment. (B) Crystal violet stained MCF7 cells (Magnification 200×). A dose-dependent decline in viable cell population after treatment with Oncowithanib and Withaferin A. (C) A significant reduction of MCF7 colonies cultivated in soft-agar plates after treatment with Oncowithanib and Withaferin A at 2.5, 5, and 10 µg/ml concentrations. (D) MCF7 cells were treated with Oncowithanib and other natural anti-neoplastic compounds (Withaferin A, ASHWITH, Paclitaxel, and Vinblastine) at 1 and 10 µg/ml concentrations for 18 hours, and cytotoxicity was compared by crystal violet assay. Data shows Oncowithanib has a higher cytotoxicity than regular commercial ashwagandha extract (ASHWITH), Paclitaxel and Vinblastine whereas similar cytotoxicity as Withaferin A. Quantitative data is presented as mean ± SD (standard deviation) of 3 samples (N = 3). *P indicates a statistically significant (*P < .05) difference in comparison to the control.

Induction of Apoptosis and Decline of Mitotic Activity in MCF7 Cells After Treatment With Oncowithanib

Mitotic deregulation and suppression of apoptosis create the platform for abnormal cell proliferation and survival of mutant clones in cancerous tissues. Here, we aimed to identify the underlying mechanism of Oncowithanib-mediated MCF7 cell death and inhibition of cell proliferation by investigating caspase 3 enzyme activity, and the expressions of Aurora Kinase A and Ki67. Caspases are a family of cysteine-aspartic protease enzymes that play crucial roles in apoptosis. Caspase 3 is known to act as an executioner caspase which has proteolytic functions essential to carry out the final stages of apoptosis.^{54 -56} Oncowithanib treatment at IC50 concentration (10 µg/ml) was found to increase caspase 3 enzyme activity in MCF7 cells (control 0.23 ± 0.01 versus treated 0.33 ± 0.05; *P < .05) (Figure 2A). Simultaneously, Oncowithanib treatment was found to diminish Ki67 (control 1.44 ± 0.27 versus treated 0.55 ± 0.21; *P < .05) and Aurora Kinase A (control 2.04 ± 0.69 versus treated 0.52 ± 0.20; *P < .5) expressions in MCF7 cells (Figure 2B and C). Ki67 protein level increases during mitosis and is one of the important proliferation markers often used for the assessment of chemotherapy response and cancer prognosis. ⁵⁷ On the other hand, Aurora Kinase A is a mitotic serine/threonine kinase required for cell transition from the G2 to the M phase. Overexpression of Aurora Kinase A is associated with cell cycle deregulation and hence cancer progression. ⁵⁸ Taken together, results suggest that Oncowithanib treatment diminishes the expression levels of mitotic regulators as well as activates apoptosis machinery via enhancing caspase 3 enzyme activity in MCF7 cells.

Figure 2.

Caspase 3 enzyme activity, and ki67 and Aurora kinase A expressions in Oncowithanib-treated MCF7 cells. (A) Treatment with Oncowithanib shows an induction in caspase 3 enzyme activity in MCF7 cells. (B and C) Cells were treated with the vehicle (DMSO) or Oncowithanib (10 µg/ml) for 18 hours. Western blot was performed for investigating the expression level of mitotic regulators. Target protein expression was normalized to the sample’s loading control GAPDH and presented as fold change with respect to the control. The bands are representative of 3 repeat experiments. Data shows a significant reduction of Ki67 and Aurora Kinase A expressions in Oncowithanib-treated cells as compared to DMSO-treated control cells. The data is presented as the mean ± SD (standard deviation) of 3 samples (N = 3). *P indicates a statistically significant (*P < .05) difference in comparison to the DMSO-treated control.

Oncowithanib Exerts Anti-Cancer Activity by Suppressing the RSK1/P-RPS6 Axis in MCF7 Cells

Ribosomal S6 kinase (RSK) is often overexpressed in various types of human malignancies and small molecule inhibitors blocking RSK1 activity have been shown to inhibit the survival and growth of cancer cells. RSK1 regulates a plethora of cellular activities via the modulation of different substrates such as Ribosomal protein S6 (RPS6). RPS6 is an indispensable component of the 40S smaller subunit of the eukaryotic ribosome and hence contributes to protein synthesis. RPS6 activity is strictly dependent on phosphorylation by RSK1 at its conserved serine residues (S235 and S236).^52,53 Here, we sought to determine the role of the RSK1/P-RPS6 axis in MCF7 cells treated with Oncowithanib. Oncowithanib-treated MCF7 cells were analyzed for pan-RSK1 and phospho-RPS6 (pS235 + pS236) expressions by western blotting. Data showed a dose-dependent decline in the phospho-RPS6 [control 1.24 ± 0.03 versus treated (5 µg/ml 0.80 ± 0.22; *P < .05 and 10 µg/ml 0.22 ± 0.02; *P < .001)] as well as RSK1 expressions [control 1.05 ± 0.05 versus treated (5 µg/ml 0.83 ± 0.16 and 10 µg/ml 0.51 ± 0.08; *P < .001)] (Figure 3A-C). Results indicate that downregulation of the RSK1/P-RPS6 axis could be one of the mechanisms of Oncowithanib-mediated inhibition of MCF7 cell survival most likely due to the impairment of protein synthesis which is crucial for rapidly growing cancer cells.

Figure 3.

RSK1 expression and RPS6 phosphorylation in Oncowithanib-treated MCF7 cells. (A-C) Cells were treated with Oncowithanib at 5 and 10 µg/ml concentrations for 18 hours. Western blot was performed to analyze protein expression levels. Target protein expression was normalized to the sample’s loading control GAPDH and presented as fold change with respect to the control. The bands are representative of 3 repeat experiments. Data shows a dose-dependent decline in total RSK1 and phosphorylated form of RPS6 (pS235 + pS236) levels in MCF7 cells after Oncowithanib treatment. The data is presented as the mean ± SD (standard deviation) of 3 samples (N = 3). *P indicates a statistically significant (*P < .05) difference in comparison to the DMSO-treated control.

Akt and mTOR, the Other Subcellular Targets of Oncowithanib in MCF7 Cells

Akt and mTOR are the 2 important components of the cellular signaling pathways that control fundamental cellular mechanisms such as cell proliferation, survival, and apoptosis. Overexpression of Akt and mTOR have often been found in human cancers, either due to genetic mutations or post-translational modifications. ⁵⁹ Here, we studied Akt and mTOR expression levels in Oncowithanib-treated MCF7 cells. A dose-dependent decline in pan-Akt1 [control 1.37 ± 0.16 versus treated (5 µg/ml 0.5 ± 0.24; *P < .01 and 10 µg/ml 0.14 ± 0.08; *P < .001)] (Figure 4A and B) and pan-mTOR [control 2.05 ± 0.41 versus treated (5 µg/ml 0.58 ± 0.19; *P < .01 and 10 µg/ml 0.42 ± 0.24; *P < .01)] (Figure 4C and D) protein levels was observed in Oncowithanib-treated MCF7 cells in comparison to the DMSO-treated control cells.

Figure 4.

Akt1 and mTOR expression levels in MCF7 cells treated with Oncowithanib. Cells were treated with Oncowithanib at 5 and 10 µg/ml concentrations for 18 hours. After treatment, the expression levels of Akt1 and mTOR proteins were analyzed by western blot. Target protein expression was normalized to the sample’s loading control GAPDH and presented as fold change with respect to the control. The bands are representative of 3 repeat experiments. Data shows a dose-dependent decline in (A and B) pan-Akt1 and (C and D) pan-mTOR levels in treated MCF7 cells. The data is presented as the mean ± SD (standard deviation) of 3 samples (N = 3). *P indicates a statistically significant (*P < .05) difference in comparison to the DMSO-treated control.

Discussion

The medicinal herb ashwagandha is known for centuries for its plethora of therapeutic benefits including cancer cure. However, ashwagandha based treatment solutions are often not the first choice of therapy because of highly inconsistent outcome. Here we have investigated the pharmacology of a novel engineered ashwagandha extract, Oncowithanib that has been extracted and standardized to achieve consistent therapeutic activity. The present study investigates the efficacy and drug targets of Oncowithanib using MCF7 cancer cell line. Furthermore, we wish to better understand the interactions of multiple drug targets in the context of our polypharmacology. The notion of polypharmacology treatment protocols is a highly controversial one. Over the last 2 decades drug research has pointed its microscope at generating highly selective drugs that are supposed to elicit fewer side-effects and improved efficacy. ⁶⁰

The problem we have faced in recent years is a monumental attrition rate due to the adoption of this selective concept. ⁶¹ The concept of selectivity is a difficult one to manage for selectivity is a relative concept in that we may not see beyond the scope of the selective target unless we are looking. In addition, the likelihood that drugs even as pointed as those that are antibody modeled are, in fact, laser selective, is a difficult one to maintain since we’d expect the epitope to NOT be repeated in any tissue. ⁶² Additionally, the selective drug concept voids the viability of natural medicines that are ultimately naturally designed with inherent polypharmacology.^63,64

However, the principle of drug cocktails to treat complex diseases is an acceptable strategy in mainstream medicine as is a more recent trend toward acceptance of network pharmacology in the context of natural medicines with polypharmacology.^64,65 Polypharmacology fits into the concept of network pharmacology or systems biology where multi-node, multi-target pharmacology can address treatment demands for complex diseases like cancer. When we consider the evolutionary development of phytomedicine polypharmacology like the ones studied herein, it should not come as a complete surprise that a “controlled” polypharmacology with a strategic outcome might very well be naturally built into these plant-based medicines. This may be especially so in the context of highly conserved signaling pathways that have themselves coevolved with animals including insects grazing on these plants for thousands of years.

Most of these phytochemicals are ultimately natural pest control designed to regulate transcriptional events in grazers at multiple pathway nodes as defensive strategies.^{66 -68} Could a plant generate metabolites of these chemicals that serve as escalations of protective pharmacology over thousands of years as a natural survival mechanism—natural selection? Many of the ancient regulatory pathways like the NF-kappa-B Signaling, MAPK Signaling, and Nrf2 Signaling are highly conserved and still managing key cellular processes in a wide range of developed species.^69,70

With a better understanding of the pharmacology for key constituents in natural medicines in the context of these conserved signaling pathways we will be better equipped to engineer a targeted treatment that may consider the naturally inherent polypharmacology as a functional “program.” This requires a mechanistic understanding of the key constituents in isolation and what that isolated pharmacology means when they are aggregated again in a group or as a whole.

This knowledge also allows us an opportunity to re-engineer the multi-constituent natural agent with more potency toward a specific node by excluding some of the plant’s constituents unless, of course, the natural wholistic approach is desired. Our goal is to be able to understand natural constituents that have been studied as expansively as allopathic chemotherapeutics and studied using new-age drug research protocols so they can be engineered, if needed, and administered with strategic potency that confers low to no host risk.

Our results show a dose-dependent decline in MCF7 cancer cell viability with Oncowithanib treatment that is as effective as purified Withaferin A and more effective than Paclitaxel and Vinblastine. This presents promising insight into the possibility that Oncowithanib could perform effectively in vivo with the possibility of conveying less toxicity than isolated Withaferin A. Clinical trials will need to be designed to further evaluate this potential. In their expansive review, Dutta et al show that multiple researchers using different breast cancer models including MCF7 demonstrate antimetastatic activity by Withaferin A as well as ROS-mediated apoptosis and Surviving protein suppression. Other research indicates direct modulation of RSK, mTOR, AKT by Withaferin A to provide corroborative support for what we are seeing in our work. ⁷¹ Nevertheless, we are showing comparable results with the Withaferin A-reduced (25% Withaferin A) Ashwagandha extract, Oncowithanib.

Knowledge and validation of the functional network polypharmacology facilitates confident prescription and application of traditional medicines as reliable treatments for disease in mainstream medical settings. In order for this confidence to be instilled these treatments must be studied in the context of allopathic drug research models that consider and justify polypharmacology in the context of an extended view of the targeted disease pathology. In particular, the multiple drug targets that might infer better treatment than the selective treatment will need to be reliably and credibly reported. This knowledge is essential for us to be able to bring acceptance to mainstream medicine for treatments that have successfully served humanity for thousands of years already as alternative or complimentary treatments.

The “medicine of the future” will be delivered by medical practitioners whose curriculum will have included the study of naturopathic, allopathic, and nutrigenomic sciences with equal attention. Treatment of disease will involve the use of the least invasive therapy followed by more biologically intrusive therapies or treatments only if required and only after all others have been exhausted. Although our conventional allopathic medicine has been driven by this directive to limit adverse events in the course of treatment, it has yet to fully embrace naturopathic and nutrigenomic tools to fully execute based on this crucial philosophy. The type of research we see executed here will be needed in abundance in order to establish this “medicine of the future.”

Study Limitations

Although this work established the foundation for a better understanding of how we can design the next steps to study these unique ashwagandha extracts we recognize design limitations that can be addressed in subsequent work. We were unable to move upstream to relevant targets as desired that would have shed more light on mechanisms due to failure of PCR equipment; equipment that will be available for subsequent work in this series. We also need to better understand the dynamic of the network in the context of cortisol secretion and the influence of prolonged exposure of elevated cortisol on proximate cells and how these extracts can modulate or circumvent deleterious effects of the dysregulated hormone. We will need to design co-cultures to gain insight on this and lead to clinical trials on human subjects or animal models to gain this systemic understanding. For now, this preliminary outcome will suffice as a platform from which to design future experimental models

Conclusion

Withaferin A, a constituent of ashwagandha (Withania somnifera) has been shown to induce apoptosis in many different cancer types.^{24 -27} Nevertheless, Withaferin A is also toxic to healthy cells, like many other allopathic chemotherapeutic agents are. We have studied and reported on the various constituents of the ashwagandha plant in previous publications, an alternative medicinal agent central to the Ayurveda. It has as many as 35 withanolide- and countless other phyto- actives. With a cautious belief in and understanding of network pharmacology and plant-based polypharmacology we consider and studied the potential of different ashwagandha extractions, the extraction yields of which can vary significantly. Our approach is to find the strategy that gets us to an effective natural cancer treatment that can be controllably repeated. Natural agents like these are classed as “privileged structures” presenting multiple advantages over the synthetic drugs commonly used as chemotherapeutic. ⁷² Natural compounds are showing promise as they have molecular features that can result in lower toxicity to host cells; in addition the long historic use of these compounds by humanity also serves as a favorable safety consideration. ⁷³ Historically, in previous papers, we have shown and reported on how various Withaferin A-voided (low to zero Withaferin A content) extractions support health-, vigor- and longevity-promoting mechanisms. With this current exercise here, we investigated an ashwagandha extract (Oncowithanib) that includes key withanolides in the presence of a largely amplified Withaferin A constitution. Using MCF7 cancer cell line, this controlled polypharmacology by Oncowithanib demonstrates chemotherapeutic activity, which reduces survivability and promotes apoptosis by targeting key cellular kinases—AKT, mTOR, RSK, and caspase related apoptotic machinery. Multiple cancer targets effectively modulated by a multiphytochemical agent that would otherwise require a drug cocktail to achieve. We have shown the potential of the engineered natural medicines that may be useful in developing novel therapeutic solutions for the dreadful diseases that require complex treatment programs.