Withaferin A: A Dietary Supplement with Promising Potential as an Anti-Tumor Therapeutic for Cancer Treatment - Pharmacology and Mechanisms

Zhichao Xing; Anping Su; Li Mi; Yujie Zhang; Ting He; Yuxuan Qiu; Tao Wei; Zhihui Li; Jingqiang Zhu; Wenshuang Wu

doi:10.2147/DDDT.S422512

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Review

Withaferin A: A Dietary Supplement with Promising Potential as an Anti-Tumor Therapeutic for Cancer Treatment - Pharmacology and Mechanisms

Authors Xing Z, Su A, Mi L, Zhang Y, He T, Qiu Y , Wei T, Li Z, Zhu J, Wu W

Received 23 May 2023

Accepted for publication 18 August 2023

Published 21 September 2023 Volume 2023:17 Pages 2909—2929

DOI https://doi.org/10.2147/DDDT.S422512

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor Anastasios Lymperopoulos

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Zhichao Xing,^1,^* Anping Su,^1,^* Li Mi,¹ Yujie Zhang,¹ Ting He,¹ Yuxuan Qiu,² Tao Wei,¹ Zhihui Li,¹ Jingqiang Zhu,¹ Wenshuang Wu¹

¹Division of Thyroid Surgery, Department of General Surgery and Laboratory of Thyroid and Parathyroid Disease, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu, People’s Republic of China; ²Department of Ultrasound, West China Hospital, Sichuan University, Chengdu, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Jingqiang Zhu; Wenshuang Wu, Division of Thyroid Surgery, Department of General Surgery and Laboratory of Thyroid and Parathyroid Disease, Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, People’s Republic of China, Email [email protected]; [email protected]

Abstract: Cancer, as the leading cause of death worldwide, poses a serious threat to human health, making the development of effective tumor treatments a significant challenge. Natural products continue to serve as crucial resources for drug discovery. Among them, Withaferin A (WA), the most active phytocompound extracted from the renowned dietary supplement Withania somnifera (L.) Dunal, exhibits remarkable anti-tumor efficacy. In this manuscript, we aim to comprehensively summarize the pharmacological characteristics of WA as a potential anti-tumor drug candidate, with the objective of contributing to its further development and the discovery of prospective drugs. Through an extensive review of literature from PubMed, Science Direct, and Web of Science, we have gathered substantial evidence showcasing WA’s significant anti-tumor effects against a wide range of cancers in both in vitro and in vivo studies. Mechanistically, WA exerts its anti-tumor influence by inducing cell cycle arrest, apoptosis, autophagy, and ferroptosis. Additionally, it inhibits cell proliferation, cancer stem cells, tumor metastasis, and also suppresses epithelial-mesenchymal transition (EMT) and angiogenesis. Several studies have identified direct target proteins of WA, such as vimentin, Hsp90, annexin II and mFAM72A, while BCR-ABL, Mortalin (mtHsp70), Nrf2, and c-MYB are potential targets of WA. Notwithstanding its remarkable anti-tumor efficacy, there are some limitations associated with WA, including potential toxicity and poor oral bioavailability, which need to be addressed when considering it as an anti-tumor candidate agent. Nevertheless, I given its promising anti-tumor attributes, WA remains an encouraging candidate for future drug development. Unveiling the exact target and comprehensive mechanism of WA’s action represents a crucial research direction to pursue in the future.

Keywords: Withaferin A, Withania somnifera, dietary supplement, anti-cancer activity, pharmacological mechanism, direct target

Graphical Abstract:

Introduction

Cancer poses a severe threat to human health and has emerged as a leading cause of death in many countries worldwide, particularly those experiencing rapid population growth and aging. According to the 2020 global cancer statistics, there were 19.3 million new cases and almost 10.0 million cancer-related deaths. It is projected that the number of cancer cases will rise to 28.4 million by the year 2040.¹ Consequently, there is an urgent need for novel anti-cancer drugs and treatment approaches.

Natural products have long been vital in drug discovery due to their unique biocompatibility, novel structural backbones, and diverse pharmacological activities. Many anti-cancer agents are either natural products or direct synthetic derivatives of natural products,² such as paclitaxel, colchicine, irinotecan (a derivative of camptothecin). However, chemotherapeutic agents often come with undesirable side effects. As a result, dietary compounds derived from food sources are being explored as potential alternatives for anti-cancer drug discovery.

One such traditional medicine is Withania somnifera (L.) Dunal (WS), also kwon as Indian ginseng (Ashwagandha) and considered the king of Ayurvedic herbs, which has been utilized for 6, 000 years in Indian.³ WS finds wide application in treating various conditions, including cancers, epilepsy, depression, arthritis, diabetes, Parkinson’s disease, schizophrenia insomnia, and hypothyroidism, and palliative effects, such as analgesic, rejuvenating, regenerating, and growth-promoting effects, as well as improvement in sexual function.^4–9 Numerous research groups have investigated the chemical constituents of Ashwagandha to identify its bioactive entities. Withaferin A (WA) is a major bioactive lactone of WS (Figure 1). WA displays a wide range of activities, including anti-inflammatory, anticancer, anticoagulant, neuroprotective, hypoglycemic, hepatoprotective, and antiarthritic effects. Furthermore, recent study have reported that WA can potentially treat or prevent the COVID-19 transmission by inhibiting the virus’s S protein from binding to the host receptor.¹⁰

Figure 1 Role for different cancer types of WA isolated from Withania somnifera.

Of particular note is WA’s highly sensitive and broadly applicable anticancer efficacy. Studies have indicated that WA is a promising candidate and may become a potential treatment option for various cancers. In this review, we provide a comprehensive examination of the pharmacology and mechanism of WA as a potential drug candidate for cancer therapy. Additionally, we discuss viable strategies to overcome limitations and enhance the feasibility of WA as a novel anti-tumor agent.

The Anti-Tumor Efficacy of WA Against Various Cancers

Despite WA’s popularity as a small molecule compound with diverse bioactivities, its antitumor activity has raised significant concerns. Nevertheless, WA shows promise in the treatment of multisystem tumors (Figure 1). In this study, we demonstrated the remarkable antitumor activity of WA in cancer cells (Table 1) and animal models (Table 2). Furthermore, we provided a comprehensive summary of the progress of WA in clinical trials.

Table 1 Anti-Cancerous Activities of WA in vitro

Table 2 Anti-Cancerous Activities of WA in vivo

In vitro Anti-Tumor Effects of WA

WA exhibits significant anti-tumor efficacy against almost all type cancers, Notably, it demonstrates promising results in reproductive system tumors, such as breast cancer,^11–13 cervical cancer,³³ ovarian cancer,^38,46 and endometrial cancer,¹¹⁹ as well as urinary system tumors, including prostate cancer^48,49,125 and renal carcinoma.⁵⁹ Additionally, WA shows potential in combating digestive system tumors, such as oral cancer,^{88,89,131,132} colorectal cancer (CRC),^61,62 pancreatic cancer,^68,69 hepatocellular carcinoma (HCC)^74,128 and gastric cancer,¹¹⁸ as well as respiratory system tumors, including lung cancer.^76,77,141 Furthermore, it displays anti-tumor effects in endocrine system tumors like adrenocortical carcinoma,¹¹⁶ and thyroid cancers,^87,130 circulatory system tumors like lymphoma⁹⁶ and leukemia^97,134 nervous system tumors including glioblastoma (GBM)¹⁰³ and neuroblastoma,¹⁰¹ and motor system tumors like osteosarcoma.^109,110,142 Moreover, WA exhibits significant tumor inhibition in other types of tumors, including in uveal melanoma,⁹³ melanoma,¹¹² mesothelioma,¹¹¹ head and neck squamous carcinoma cells¹¹⁷ and Ehrlich ascites.¹³⁸ The anti-tumor efficacy of WA is attributed to its ability to modulate multiple signaling pathways. It inhibits cell proliferation, migration, angiogenesis, and cancer stem cells (CSCs), while also inducing cell cycle arrest, apoptosis, autophagy, ferroptosis (Table 1).

In vivo Anti-Tumor Effects of WA

Various animal models have been utilized to evaluate the in vivo anti-tumor efficacy of WA (Table 2). The results consistently demonstrate that WA exhibits potent inhibitory effect on tumor growth and metastasis across multiple cancers when administered intraperitoneal (i.p.) or per os (p.o.) at doses ranging from 1–20 mg/kg. Moreover, WA has been showed to have synergistic effects when combinations with chemotherapeutics. In in vivo experiments, observations of mouse mortality and body weight suggest that WA is well-tolerated and safe, further indicating its potential for future development.

Clinical Research of WA

Given the significant antitumor activity of WA in cancers, several clinical trials have been conducted to investigate its safety and pharmacokinetics in the clinical treatment of cancer patients. Notably, a Phase I trial conducted by Pires N et al found that WA was generally well-tolerated in patients with advanced stage high-grade osteosarcoma at doses of 72, 108, 144 and 216 mg.¹⁴² Furthermore, a recent clinical study titled “Combination therapy with liposomal doxorubicin and WA in recurrent ovarian cancer” aims to assess the feasibility and tolerance of WA in phase I and evaluate the treatment response (complete response (CR), partial response (PR), and stable disease (SD)) in recurrent ovarian cancer patients in Phase II (ClinicalTrials.gov Identifier: NCT05610735, 2022).

The Reported Direct Binding Target of WA in Cancers

Identifying the specific targets of compounds plays a crucial role in elucidating their mechanism of action and in drug development. Active natural compounds often act on multiple targets. Several studies have identified candidate targets of WA through computer calculations and molecular simulations (Table 3). BCR-ABL,¹⁴³ ACE2,¹⁴⁴ Mortalin (mtHsp70) and Nrf2¹⁴⁵ were proposed as potential targets of WA based on these approaches. Furthermore, Clesham et al utilized the Connectivity Map (CMAP) database and identified c-MYB as a potential target of WA in acute myeloid leukemia.⁹⁷

Table 3 The Direct Target Proteins of WA in Cancers

Additionally, a few articles have demonstrated that vimentin, Hsp90, and annexin II proteins directly bind to WA using WA-biotin analogs (Table 3). Paola BM et al revealed a covalent bonding between the C3 or C6 electrophilic carbon centers of WA and Cys328 of the vimentin A-helix. Moreover, hydrogen bonding was observed between the C1 position oxygen atom and Gln324 of the vimentin A-helix, as well as between the C4 hydroxyl group and Asp331 of the vimentin A’-helix.¹⁴⁶ Yanke et al identified that WA-biotin binds to the C-terminus of Hsp90.⁶⁹ Additionally, both Falsey RR et al and Gabriel Ozorowski et al demonstrated that WA directly binds to annexin II protein.^147,148 However, Falsey RR et al found that WA binds covalently to the N-terminal domain of annexin A2, not Cys133. Furthermore, Jessica et al reported WA binds to mFAM72A with micromolar affinity in HeLa and HEK293T cells using biolayer interferometry.¹⁴⁹

Molecular Mechanism of the Antitumor Effect of WA in Various Cancers

The complete understanding of the mechanisms underlying WA’s antitumor activity remains elusive; nevertheless, it appears to involve multiple effects, such as inducting cell cycle arrest, apoptosis, autophagy, ferroptosis, and suppressing invasion, metastasis, angiogenesis, and cancer stem cells.^150–152 In light of this, we conducted a comprehensive review of the molecular mechanism of the antitumor activity of WA in various cancers (Figure 2).

Figure 2 Mechanisms and signaling pathway of WA on cancer inhibition.

Inducing Cell Cycle Arrest

In various cancers, including human breast cancer cells, ovarian cancer cells, cervical cancer cells,^34,46 uveal melanoma cells,⁹³ human head and neck cancer cells,¹¹⁷ and GBM cells,^104,105 WA has been reported to induce G2/M phase arrest. The process of WA-induced G2/M arrest involves multiple cell cycle-related proteins, such as cyclin-dependent kinase 1 (Cdk1), cyclin B1, cell division cycle 25C (Cdc25C) and Cdc25B. In MCF-7 and MDA-MB-231 cells, WA treatment led to a concentration-dependent and time-dependent decrease in the expression levels of Cdk1, Cdc25C and Cdc25B, ultimately inducing G2/M arrest.¹⁵³ Moreover, overexpression of Cdc25C in breast cancer cells partially prevented the G2/M arrest induced by WA.¹⁵³ Similarly, WA treatment downregulated Cdc25C and induced cyclin B1 in GBM and ovarian cancer cells CaOV3 and SKOV3.^34,105 Consistent with Stan’s findings,¹⁵³ WA-treated CaSki cells showed an accumulation of cyclin B1, downregulation of Cdk1, decreased complex formation between cyclin B1 and Cdk1,³⁴ and induction of the Cdk inhibitor p21.¹²⁵ Additionally, clues for G2/M arrest induced by WA emerged from the observation of mitotic spindle disruption microtubules.¹⁰⁹

Inducing Apoptosis

Apoptosis, a form of programmed cell death, is a common mechanism employed by antitumor drugs. In human cells, there are two well-characterized pathways that trigger apoptosis. One is the intrinsic pathway, which is induced by mitochondrial changes. The activation of caspases is regulated by mitochondrial Bcl-2 family proteins.^154,155 Another is the extrinsic pathway, activated by death domain-containing receptors, such as CD95, TNF receptors and TNF-related apoptosis-inducing ligands.

Inducing apoptosis is the primary anti-tumor effect of WA, observed in breast cancer,¹³ prostate cancer, leukemia,¹⁵⁶ melanoma,¹¹³ and head and neck cancer. WA induces the generation of the reactive oxygen species (ROS) in many cancer cells, leading to increased expression of Bak and Bax, which in turn induces mitochondrial apoptosis. Additionally, WA triggers Bak and Bax protein activation by reducing laminin and integrin gene expression.¹⁴ In CRC cells, WA induces apoptosis through ROS-mediated mitochondrial dysfunction and the JNK pathway.⁶¹ Tang’s study revealed that WA triggered intrinsic apoptosis in GBM cells via the ATF4-ATF3-CHOP axis.¹⁰³ Another study demonstrated that WA induced apoptosis by inhibiting the AKT/mTOR pathway. Silvia’s research indicated that by knocking down Bim and FOXO3a levels in breast cancer cell lines, WA-induced apoptosis was significantly attenuated in vivo.¹³

Moreover, it has been documented that WA enhances TNFα-related apoptosis-inducing ligand-induced apoptosis (TRAIL) by decreasing the expression of c-FLIPL and c-FLIPs (cellular FLICE-like inhibitory protein), the negative regulators of apoptosis.^157–160 In Seon’s study, WA induced cell apoptosis by inhibiting the phosphorylation of Axl and STAT3 induced by the growth inhibition specific protein 6 (rhGas6).¹⁶¹ The inhibition of STAT3 (on Tyr705) phosphorylation also results in inhibition of Janus-activated kinase 2 (JAK2) activity. Furthermore, WA causes apoptosis by downregulating STAT3-regulated genes, including Bcl-2, Bcl-xL, survivin and cyclin D1, in human renal carcinoma Caki cells.⁵⁹

Inhibiting Proliferation

Additionally, WA has demonstrated the ability to inhibit tumor cell proliferation through various mechanisms. For instance, in human endometrial carcinoma, WA inhibits tumor cell proliferation by blocking the phosphorylation of TGF-β-dependent Smad2 and the expression of other TGF-β-related proteins.¹¹⁹ In the case of myeloma cells, WA inhibits proliferation through ROS-mediated intrinsic apoptosis.¹⁶² Moreover, WA has been reported to inhibit proliferation in chronic myeloid leukemia by targeting BCR-ABL oncogenic signaling.¹⁴³ Furthermore, WA exerts its inhibitory effects on proliferation by regulating c-MYB target gene expression. This occurs through the induction of c-MYB protein ablation, resulting in reduced viability, impaired colony formation, and hindered progression of acute myeloid leukemia cells.⁹⁷ Additionally, combined with blocking SGs by targeting G3BP1, WA-induced oxidative stress combined with blocking SGs by targeting G3BP1 results in reduced survival of prostate cancer.⁵⁰

Inducing Autophagy

Physiologically, autophagy is a cellular process responsible for degrading macromolecules and organelles and has been shown to contribute to cell death.^163,164 Inducing autophagy is an important anti-tumor mechanism of WA. In three WA-treated prostate cancer cell lines (22Rv1, PC-3 and LNCaP), autophagy induction was confirmed through the results of transmission electron microscopy and Western blot analysis. The key autophagy markers, including LC3BII, SQSTM1, Atg7 and Beclin-1, were robustly increased in WA-induced cancer cells. Moreover, GABARAPL1 was found to be involved in the cytoprotective autophagy induced by WA in prostate cancer cells.⁴⁸

Additionally, WA inhibits the expression of β-catenin and p-GSK-3β proteins, leading to autophagy.¹⁶⁵ Recent studies have shown that WA induces autophagy in both a spontaneously immortalized and nontumorigenic normal human mammary epithelial cell line (MCF-10A) and human breast cancer cells (MCF-7 and MDA-MB-231). Furthermore, WA induces autophagy in MDA-MB-231 xenograft mice.¹⁵

Inducing Ferroptosis

Ferroptosis is characterized by uncontrolled lipid peroxidation of cell membranes, resulting in membrane damage and cell death.^166,167 Recent studies have reported that WA can induce a form of nonapoptotic cell death known as ferroptosis in cancer cells. This induction is attributed to the excessive activation of heme oxygenase-1 (HMOX1) by WA, which elevates intracellular labile ferrous iron (Fe²⁺) levels, consequently leading to the accumulation of toxic lipid radicals and triggering ferroptosis.¹⁰¹ Additionally, WA has been found to induce ferroptosis through directly targeting and inhibiting glutathione peroxidase 4 (GPX4), which plays a critical role in detoxifying membrane hydrogen peroxide.¹⁶⁸ Notably, according to Emilie, due to considerable overlap in ferroptosis and apoptosis kinome activity, WA induces mixed ferroptosis and apoptosis in multiple myeloma cells.¹⁶⁹

Suppressing Cancer Stem Cells

Cancer stem cells (CSCs) are a subpopulation of cells within tumors that possess self-renewal and differentiation capabilities, and they are believed to play a significant role in tumor initiation, growth, metastasis, and therapy resistance.³⁹ WA has demonstrated properties in suppressing CSC as well. Kakar et al reported the role of WA in suppressing putative CSCs in ovarian cancer.³⁸ They observed a remarkable 70–80% reduction in tumor metastasis and growth of cancerous cells. WA significantly inhibited the expressing of CSC markers, including CD24, CD44, CD117, CD34 and Oct 4, and downregulated the Hey 1, Notch 1, and Hes 1 genes. Similarly, Jade et al demonstrated that WA effectively inhibits the growth of lung CSCs, the formation of lung cancer spheroids, and decreases side population cells.⁷⁶ Additionally, Kim and Singh reported that FoxQ1 is a target of WA for inhibiting breast CSCs in vivo.¹¹ Moreover, Mayuko et al found that WA is a potent inhibitor of CSC stemness, leading to cellular senescence primarily via the induction of p21Cip1 expression.¹⁷⁰ Therefore, WA holds promise for providing potential therapeutic benefit in various cancers by suppressing CSCs through diverse pathways, making it a promising candidate for further investigation as a potential therapeutic agent for different types of cancer.

Inhibiting Cancer Metastasis and Angiogenesis

Epithelial mesenchymal transition (EMT) is a biological process in which cells lose their epithelial characteristics and acquire mesenchymal properties, facilitating the migration and invasion of tumor cells. Studies have demonstrated that WA decreases cellular mobility by countering EMT, such as decreasing Slug (SNAI2) and Twist expression, while increasing the adhesion molecule E-cadherin expression.^63,170,171 In human non-small cell lung cancer (NSCLC) cells, WA suppresses TGFβ1- and TNFα-induced EMT and inhibited cell adhesion, invasion and migration of H1299 and A549 cells. Moreover, WA inhibits EMT by preventing the nuclear translocation and phosphorylation of Smad2/3 and nuclear factor kappa B (NF-κB) in H1299 and A549 cells.⁷⁷ Additionally, Chen et al reported that WA inhibits the migration of lung cancer cells by downregulating miR-27a and miR-10b, which regulate the expression of Bax and E-cadherin.⁷⁸ Furthermore, WA suppresses the AK4-HIF-1α signaling axis and acts as a potent antimetastatic agent in lung cancer.⁷⁹ In MMTV-neu mouse and breast cancer xenografts, WA increases E-cadherin expression and reduces vimentin expression.¹⁶ Moreover, at a concentration of 700 nM, WA suppresses breast cancer metastasis and relapse by inhibiting the urokinase-like plasminogen activator (uPA) protease, which promotes cell migration and proliferation by remodeling the extracellular matrix.^17,18 Another study demonstrated that 3-azido-WA upregulates TIMP-1 and E-cadherin expression in prostate cancer cells.¹³⁵ As a vimentin inhibitor, WA suppresses the migration and invasion activity of glioblastoma cells by inhibiting vimentin.¹⁷² Additionally, WA interacts with vimentin and heterogeneous nuclear ribonucleoprotein hnRNP-K to downregulate the expression of proteins involved in tumor cell metastasis, such as MMPs, VEGF, N-cadherin, and u-PA.¹⁷³

Furthermore, WA exerts potent anti-angiogenic activity in vivo.¹⁷⁴ In the Ehrlich ascites tumor model, WA exerts its anti-angiogenic activity by reducing the binding of the transcription factor specificity protein 1 (Sp1) to VEGF.¹⁷⁵ S Saha et al demonstrated that WA has a favorable binding affinity with vascular endothelial growth factor (VEGF), leading to decreased angiogenesis.¹⁷⁶ In another study, WA reduces macrophage infiltration and inhibits the expression of protein tyrosine kinase-2 (Pyk2), rho-associated kinase 1 (ROCK1), and VEGF in a hepatocellular carcinoma xenograft model, thereby suppressing tumor invasion and angiogenesis.¹⁷⁷ WA can also directly interact with the hnRNP residue domain through hydrogen bonding and hydrophobic interactions, disrupting the binding between RNA-binding protein hnRNP-k (hnRNP-k) and single-stranded DNA (ssDNA). This inhibits hnRNP-k from binding to ssDNA and subsequently lowers the downstream effects of hnRNP-k, including the expression of VEGF, PERK, and MMP2, thus suppressing the migration and invasion of HT1080 fibrosarcoma cells.¹⁷⁸ Moreover, a recent study found that WA treatment downregulates the secretion of many angiogenesis proteins in HCC.⁷⁴ According to Bilal’s findings, 3-azido WA dose-dependently suppresses the expression of p-ERK and p-AKT, which may play a significant role in inhibiting angiogenesis in mouse.¹⁷⁹ By reducing Akt signaling and MMP-9 expression, WA also decreases the invasion and migration capabilities of CasKi cells.³⁵

Synergistic Combinations

Since the first approval of synergistic combination drugs in the 1940s, the number of approved synergistic combination has experienced significant growth. The ideal synergistic combination can improve clinical efficacy, reduce drug toxicity, and delay or prevent the development of drug resistance. As a highly effective antitumor agent, WA has been studied in combination with several other drugs.

In one combination of WA and cisplatin, WA produced ROS, while cisplatin caused DNA damage, suggesting that lower doses of cisplatin combined with suboptimal doses of WA could achieve the same effect.⁴⁰ Kendra’s study demonstrated the combinatorial of sulforaphane and WA showed synergistic effects on epigenetic modifiers and cell proliferation in breast cancer cells.¹⁸⁰ Cohen et al reported that a synergistic combination of WA and sorafenib caused G2/M arrest in anaplastic and papillary thyroid cancer cells.⁸⁷ Abdullah et al reported that the combination of WA and 5-FU executed PERK axis-mediated endoplasmic reticulum (ER) stress-induced autophagy and apoptosis by upregulating the expression of ER stress sensors, such as PERK, BiP, CHOP, eIF2α, and ATF-4.⁶⁴ The combination also modulated ER stress and significantly induced antiproliferative effect and cell death in CRC cells.⁶⁴ Additionally, the combination of cisplatin and pemetrexed with WA synergistically inhibited wild-type epidermal growth factor receptor (EGFR) lung cancer cell viability. Moreover, WA further enhanced the cytotoxic effect of cisplatin in lung CSCs.⁷⁶ The combination of WA with tumor treating fields (TTFields) showed obvious synergistic effects by significantly inhibiting tumor growth in glioma cells (GBM2, GBM39, U87-MG).¹⁰⁶ WA and carnosol also exhibit a synergistic effect on pancreatic cancer cells through targeting pancreatic cancer stem cells by phosphorylating c-met and downregulating pluripotency maintenance genes (Oct-4 and Nanog).⁷⁰

Reversing Drug Resistance

Over the past two decades, drug development in the field of oncology has predominantly focused on molecular targeted drugs. However, the rapid emergence of drug resistance due to target mutations has significantly reduced drug efficacy, making overcoming drug resistance a major challenge for current antitumor drugs. Multiple studies have found that WA has the ability to reverse drug resistance in various cancers.

Kunimasa et al demonstrated that the combination of WA and phloretin (a glucose transport inhibitor) led to a significant reduction in tumor size in gefitinib-induced drug- tolerant lung cancer, indicating that EGFR-resistant lung cancer could be effectively treated with a combination of WA and metabolism-targeting therapies.¹²⁹ In sorafenib-resistant HCC cells (HepG2 and SNU449 cells), WA enhanced ferroptosis and increased Keap1 expression to counteract the effects of Nrf2 signaling activation on the ferroptosis-related protein xCT and EMT. Moreover, blockade of Keap1/Nrf2 signaling facilitated sorafenib resistance and reversed WA-induced ferroptosis. Consequently, WA attenuated sorafenib resistance and metastatic potential by regulating Keap1/Nrf2-associated ferroptosis and EMT.¹⁸¹ In sorafenib-resistant HepG2 cells, WA induced a dose-dependent reduction in vimentin expression, followed by a reduction in ABCG2 expression and a decrease in cell viability, induced by the inhibition of vimentin in both parental and sorafenib-resistant HepG2 cells.¹⁸² WA also repressed the invasiveness of cyclophosphamide, vincristine, doxorubicin, and prednisone (CHOP) chemotherapeutic regimen-resistant DLBCL cells in collagen matrices.¹⁸³ In GBM, WA could resensitize temozolomide-resistant GBM cells by depleting O6-methylguanine-DNA methyltransferase (MGMT) and inducing apoptosis through the AKT/mTOR pathway.¹⁰⁷

Limitations of WA as a Potential Anti-Cancer Candidate and Corresponding Solutions

Despite the broad antitumor effects of WA, there were also some limitations, including potential toxicity, poor oral bioavailability, and low production.

Potential Toxicity

The toxicity of natural active compounds is often unavoidable, and WA is no exception. However, the toxicity of WA has been a subject of controversial and potential concern. As the main bioactive component of WS, there have been articles demonstrating the safety of WS extract in all tested groups.^184,185 An acute and sub-acute toxicity study of oral WA also yielded similar results, showing that the LD₅₀ of WA in mice was above 2000 mg/kg body weight.¹⁸⁶ On the other hand, other studies found that WA exhibited certain toxic side effects in mice, with an LD₅₀ of 54 mg/kg body weight.^187,188 To address these toxicity concerns, researchers have explored structural modifications to obtain derivatives of WA with comparable but safer activity. For instance, the 2-thiophene ester-linked derivative of WA, ASR488, selectively inhibited bladder cancer cells while showing no toxicity to normal cells.¹⁸⁹ Furthermore, a range of IC₅₀ values for withanolides, as reported by Zhang et al, suggests the existence of various compounds within this class with potential anti-cancer activity, warranting further exploration and development.¹⁹⁰ Notably, the ability of another WA derivate, ASR490, to inhibit the growth of colon cancer cell lines and xenotransplanted tumors without causing systemic toxicity is encouraging.¹⁹¹

Overall, these studies highlight the potential of WA as a source of lead compounds for the development of new anti-cancer drugs. However, further research in this area is essential to fully understand and address the potential toxicity concerns associated with WA and its derivatives.

Poor Oral Bioavailability

In addition to the potential toxicity issues, another limitation of WA is its poor oral bioavailability. Saurabh by Saurabh et al and Tianming et al reported oral bioavailability values 1.8% and 32.4 ± 4.8%, respectively, in male rats.^186,192 The poor oral bioavailability of WA limits its effectiveness as a drug for cancer prevention and treatment.

To address this challenge, Farrukh et al proposed an improvised implant formulation known as “coated” implants to enhance the bioavailability of WA.¹⁹³ The method involves coating polycaprolactone implants with 20–30 layers of polycaprolactone solution containing 0.5–2% of WA dissolved in dichloromethane. When compared with the ineffective intraperitoneal administration of the same total dose of WA, the drug-eluting implant significantly inhibited the growth of human lung cancer A549 xenografts in athymic nude mice. Another study by Ramesh et al also explored the use of polycaprolactone implants embedded with WA for controlled systemic delivery, resulting in nearly 60% inhibition of lung cancer A549 cell xenografts in mice.¹⁴¹

Low Production

Although WA can be isolated from the leaves, berry (winter cherry) and root of WS, its content is relatively low. The data showed that the concentration of major active compounds known as withanolides, represented by WA, in the leaves typically ranges from 0.001% to 0.5% of the dry weight.¹⁹⁴ Considering the broad pharmacological effects of WA and WS, the demand for the plant in the market continues to increase. According to data, approximately 9127 tons of dried plant material are required for the production of WA in India, while the annual yield is around 5905 tons.¹⁹⁵ Clearly, the current cultivation of the plant cannot meet the market demand. Therefore, increasing the production of withanolides in the plant has become a significant focus of attention.

Currently, there are various methods to increase withanolides production in plants. Firstly, concerning plant cultivation, it has been reported that compared to Kunapa jala, farmyard manure, and inorganic fertilizer, the application of Pancha gavya can increase withanolides production in plants.¹⁹⁶ There also are some new technologies to be developed to increase the production of withanolides in plants by optimizing conditions to enhance the accumulation of effective metabolites or secondary metabolites. Studies have shown that inducers such as 100 ppm salicylic acid, 50 ppm jasmonic acid, and 100 ppm chitosan can be sprayed on the leaf surface,¹⁹⁷ short-term UV-B radiation (less than 3 hours),¹⁹⁸ or low concentrations of WcAgNPs to improve root organogenesis,¹⁹⁹ all of which promote the synthesis of withanolides compounds in plants. Endophytes can also be used to regulate the expression of withanolides biosynthetic genes in plant leaves and roots,²⁰⁰ overexpress squalene synthase (SQS)²⁰¹ or cycloartenol synthase,²⁰² or use treatments involving ultrasound, vacuum infiltration, and thiol compounds (l-cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l) to promote the integration and expression of the gusA gene in transgenic plants,²⁰³ thereby increasing withanolides content in the transgenic plants. In addition, researchers have established a multiple shoot culture system of WS using single shoot apices as explants,²⁰⁴ and examined the withanolides production from adventitious root cultures,²⁰⁵ hairy roots,²⁰⁶ and cell suspension of WS,^207,208 all of which aim to provide theoretical basis for efficient withanolides production in the industry.

Overall, the use of polycaprolactone implants embedded with WA shows promise as a potential therapeutic approach for the treatment of cancers. However, further research is needed to determine its safety, effectiveness, and production in humans before it can be used as a clinical treatment.

Conclusions and Outlook

The exploration of various plant extracts for their potential anti-tumor properties has been extensive. Among these extracts, WA, the primary bioactive component of the Ayurvedic herb WS, is a promising anti-tumor agent. While some studies have identified potential target proteins of WA, such as vimentin and heat shock proteins, the exact mechanism of action of WA and its comprehensive effects on cancer cells remain active areas of research. Understanding the systematic effects of WA on cancer cells is crucial for its development as an anti-tumor agent. A critical aspect in the development of WA as an anti-tumor agent is assessing its toxicity and safety. While animal studies have shown that WA is well-tolerated, further research is required to thoroughly evaluate its toxicity and potential side effects in humans. Safety is paramount in the development of any therapeutic agent. Moreover, to enhance its therapeutic effectiveness, innovative approaches to improve the bioavailability of WA are needed. Methods like drug-eluting implants and other delivery systems show promise in enhancing the delivery and targeting of WA to cancer cells.

In conclusion, WA holds immense potential as an anti-tumor agent, and its pharmacological properties have shown significant antitumor efficacy in various cancers. As research in this field continues to progress, we expect a better understanding of the precise mechanisms of WA’s action, its toxicity profile, and advancements in delivery strategies. These efforts will contribute to establishing WA as a potent and safe candidate for cancer treatment, opening new possibilities for clinical applications and improving the overall outlook in the fight against cancer.

Abbreviations

ACC, Adrenocortical carcinoma; AKT, Protein kinase B; Cdc25C, Cell division cycle 25C; Cdk1, Cyclin-dependent kinase 1; CRC, Colorectal cancer; CSCs, Cancer stem cells; DLBCL, Diffuse large B-cell lymphoma; DMBA, Dimethyl butanoic acid; EMT, Epithelial mesenchymal transition; ER, Endoplasmic reticulum; EGFR, Epidermal growth factor receptor;GBM, Glioblastoma; GPX4, Glutathione peroxidase 4; HCC, Hepatocellular carcinoma; HMOX1, Heme oxygenase-1; IC₅₀, Half maximal inhibitory concentration; IKK, IκB kinase; IL-6, Interleukin-6; i.p., Intraperitoneal injection; JAK2, Janus-activated kinase 2; LXR-α, Liver X receptor-α; MAPK, Mitogen-activated protein kinase; MGMT, O6-methylguanine-DNA methyltransferase; NF-κB, Nuclear factor kappa B; NSCLC, Non-small cell lung cancer; rhGas6, Recombinant human growth inhibition specific protein 6; ROS, Reactive oxygen species; SO, Sorafenib; T-ALL, Lymphoblastic leukemia; TTFields, Tumor treating fields; uPA, Urokinase-like plasminogen activator; VEGF, Vascular endothelial growth factor; WA, Withaferin A; WS, Withania somnifera (L.) Dunal.

Data Sharing Statement

Data will be made available from the corresponding author on request.

Funding

This work was financially supported by the Support Program for Science and Technology Department of Sichuan Province (2021YFS0230), the Projects of Sichuan University (2018SCUH0067) and West China Hospital (19HXFH101).

Disclosure

Zhichao Xing and Anping Su are co-first authors for this study. The authors declare no conflicts of interest in this work.

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