Nashi Widodo

Selective Killing of Cancer Cells by Leaf Extract of Ashwagandha: Identification of a Tumor-Inhibitory Factor and the First Molecular Insights to Its Effect

Purpose: Ashwagandha is regarded as a wonder shrub of India and is commonly used in Ayurvedic medicine and health tonics that claim its variety of health-promoting effects. Surprisingly, these claims are not well supported by adequate studies, and the molecular mechanisms of its action remain largely unexplored to date. We undertook a study to identify and characterize the antitumor activity of the leaf extract of ashwagandha. Experimental Design: Selective tumor-inhibitory activity of the leaf extract (i-Extract) was identified by in vivo tumor formation assays in nude mice and by in vitro growth assays of normal and human transformed cells. To investigate the cellular targets of i-Extract, we adopted a gene silencing approach using a selected small hairpin RNA library and found that p53 is required for the killing activity of i-Extract. Results: By molecular analysis of p53 function in normal and a variety of tumor cells, we found that it is selectively activated in tumor cells, causing either their growth arrest or apoptosis. By fractionation, purification, and structural analysis of the i-Extract constituents, we have identified its p53-activating tumor-inhibiting factor as withanone. Conclusion: We provide the first molecular evidence that the leaf extract of ashwagandha selectively kills tumor cells and, thus, is a natural source for safe anticancer medicine.

Selective killing of cancer cells by Ashwagandha leaf extract and its component Withanone involves ROS signaling.

Background and Purpose Ashwagandha is a popular Ayurvedic herb used in Indian traditional home medicine. It has been assigned a variety of health-promoting effects of which the mechanisms remain unknown. We previously reported the selective killing of cancer cells by leaf extract of Ashwagandha (i-Extract) and its purified component Withanone. In the present study, we investigated its mechanism by loss-of-function screening (abrogation of i-Extract induced cancer cell killing) of the cellular targets and gene pathways. Methodology/Principal Findings Randomized ribozyme library was introduced into cancer cells prior to the treatment with i-Extract. Ribozymes were recovered from cells that survived the i-Extract treatment. Gene targets of the selected ribozymes (as predicted by database search) were analyzed by bioinformatics and pathway analyses. The targets were validated for their role in i-Extract induced selective killing of cancer cells by biochemical and molecular assays. Fifteen gene-targets were identified and were investigated for their role in specific cancer cell killing activity of i-Extract and its two major components (Withaferin A and Withanone) by undertaking the shRNA-mediated gene silencing approach. Bioinformatics on the selected gene-targets revealed the involvement of p53, apoptosis and insulin/IGF signaling pathways linked to the ROS signaling. We examined the involvement of ROS-signaling components (ROS levels, DNA damage, mitochondrial structure and membrane potential) and demonstrate that the selective killing of cancer cells is mediated by induction of oxidative stress. Conclusion Ashwagandha leaf extract and Withanone cause selective killing of cancer cells by induction of ROS-signaling and hence are potential reagents that could be recruited for ROS-mediated cancer chemotherapy.

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) promotes tumor metastasis by induction of genes involved in extracellular matrix, cell movement, and angiogenesis.

Background: Cancer metastasis is a major hurdle in cancer therapy and needs identification of novel targets for drug designing. Results: hnRNP-K is highly expressed in cancer cells and regulates extracellular matrix, cell motility, and angiogenesis pathways. Conclusion: hnRNP-K is a potential target for metastasis therapy. Significance: hnRNP-K expression level may serve as a marker of metastatic cancers, and hnRNP-K-inhibiting drugs could be candidate anti-cancer and anti-metastasis reagents. Cancer is a leading cause of death and still awaits effective therapies. Rapid industrialization has contributed to increase in incidence of cancer. One of the reasons why most of the cancers fail therapy is due to their metastatic property. Hence identification of factors leading to metastasis is highly important to design effective and novel anti-cancer therapeutics. In our earlier study (Inoue, A., Sawata, S. Y., Taira, K., and Wadhwa, R. (2007) Loss-of-function screening by randomized intracellular antibodies: identification of hnRNP-K as a potential target for metastasis. Proc. Natl. Acad. Sci. U.S.A. 104, 8983–8988), we had reported that the involvement of heterogeneous nuclear ribonucleoprotein K (hnRNP-K) in metastasis. Here, we established hnRNP-K-overexpressing and -underexpressing derivative cell lines and examined their proliferation and metastatic properties in vitro and in vivo. Whereas hnRNP-K compromised cells showed delayed tumor growth, its overexpression resulted in enhanced malignancy and metastasis. Molecular basis of the hnRNP-K induced malignant and metastatic phenotypes was dissected by cDNA microarray and pathway analyses. We found that the hnRNP-K regulates extracellular matrix, cell motility, and angiogenesis pathways. Involvement of the selected genes (Cck, Mmp-3, Ptgs2, and Ctgf) and pathways was validated by gene-specific expression analysis. Our results demonstrated that the hnRNP-K is a potential target for metastasis therapy.

Water extract of Ashwagandha leaves has anticancer activity: identification of an active component and its mechanism of action.

Background Cancer is a leading cause of death accounting for 15-20% of global mortality. Although advancements in diagnostic and therapeutic technologies have improved cancer survival statistics, 75% of the world population live in underdeveloped regions and have poor access to the advanced medical remedies. Natural therapies hence become an alternative choice of treatment. Ashwagandha, a tropical herb used in Indian Ayurvedic medicine, has a long history of its health promoting and therapeutic effects. In the present study, we have investigated an anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX). Methodology/Principal Findings Anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX) was detected by in vitro and in vivo assays. Bioactivity-based size fractionation and NMR analysis were performed to identify the active anticancer component(s). Mechanism of anticancer activity in the extract and its purified component was investigated by biochemical assays. We report that the ASH-WEX is cytotoxic to cancer cells selectively, and causes tumor suppression in vivo. Its active anticancer component was identified as triethylene glycol (TEG). Molecular analysis revealed activation of tumor suppressor proteins p53 and pRB by ASH-WEX and TEG in cancer cells. In contrast to the hypophosphorylation of pRB, decrease in cyclin B1 and increase in cyclin D1 in ASH-WEX and TEG-treated cancer cells (undergoing growth arrest), normal cells showed increase in pRB phosphorylation and cyclin B1, and decrease in cyclin D1 (signifying their cell cycle progression). We also found that the MMP-3 and MMP-9 that regulate metastasis were down regulated in ASH-WEX and TEG-treated cancer cells; normal cells remained unaffected. Conclusion We provide the first molecular evidence that the ASH-WEX and TEG have selective cancer cell growth arrest activity and hence may offer natural and economic resources for anticancer medicine.

Withanone binds to mortalin and abrogates mortalin-p53 complex: computational and experimental evidence.

Mortalin binds to p53 tumor suppressor protein and sequesters it in the cytoplasm. This results in an inhibition of the transcriptional activation and control of centrosome duplication functions of p53, thus contributing to human carcinogenesis. Abrogation of mortalin-p53 interaction and reactivation of p53 function could be a valid proposition for cancer therapy. In the present study, we first investigated in silico the interaction of withanone, a withanolide with anticancer activity, with mortalin. We found that withanone could bind to mortalin in a region, earlier predicted critical for binding to p53. Cationic rhodacyanine dye, MKT-077 has also shown to bind the same region and kill cancer cells selectively. We report the molecular dynamic simulations revealing the thermodynamic and structural stability of the withanone-mortalin complexes. We also demonstrate the experimental evidence of abrogation of mortalin-p53 complex by withanone resulting in nuclear translocation and functional reactivation of p53 in human cancer cells. The present study establishes a molecular interaction basis that could be used for screening and development of anticancer drugs with low toxicity to normal cells. Accurate knowledge of the 3D structure of mortalin would further enhance the potential of such analyses to understand the molecular basis of mortalin biology and mortalin based cancer therapy.

Deceleration of Senescence in Normal Human Fibroblasts by Withanone Extracted From Ashwagandha Leaves

Ashwagandha is an Ayurvedic shrub that forms a common ingredient of health supplements, tonics, and Indian home remedies designed to promote health and quality of life. Though sustained through experience and history, there are only a limited laboratory studies and experimental evidence to its effects. In our efforts to characterize Ashwagandha activities and their molecular mechanisms, we initially prepared leaf extract of Ashwagandha (i-Extract) that showed tumor-inhibitory activity. In the present study, we demonstrate that a major component of i-Extract and withanone (i-Factor) protected the normal human fibroblasts against the toxicity caused by withaferin A. It increased the in vitro division potential of normal human cells that appeared to be mediated by decreased accumulation of molecular damage, downregulation of the senescence-specific beta-galactosidase activity and the senescence marker protein, p21(WAF-1), protection against oxidative damage, and induction of proteasomal activity. To the best of our knowledge, we provide the first example of phytochemical(s) (i-Extract and withanone) that have both anticancer and antiaging activities and point to the molecular link between aging and cancer.

Functional Significance of Minor Structural and Expression Changes in Stress Chaperone Mortalin

Abstract:  Mortalin is one of the highly conserved heat‐shock chaperones. Some of the established features of mortalin include its various subcellular localizations, multiple binding partners, and differential subcellular distribution in normal and immortal cells. It inhibits nuclear translocation, transcriptional activation, and control of centrosome‐duplication functions of p53. It also functions as an adaptive protein in a variety of stress–response mechanisms and contributes to human carcinogenesis. Interestingly, minor alterations in its structure and level of expression may lead to drastic biological consequences (for example, Myelodysplastic syndrome and old age pathologies, such as Alzheimers and Parkinsons disease). Besides being validated as a reliable target for cancer therapy, mortalin also warrants attention from the perspectives of management of old‐age diseases and healthy aging.

Stress chaperone mortalin contributes to epithelial-mesenchymal transition and cancer metastasis.

Mortalin/mthsp70 (HSPA9) is a stress chaperone enriched in many cancers that has been implicated in carcinogenesis by promoting cell proliferation and survival. In the present study, we examined the clinical relevance of mortalin upregulation in carcinogenesis. Consistent with high mortalin expression in various human tumors and cell lines, we found that mortalin overexpression increased the migration and invasiveness of breast cancer cells. Expression analyses revealed that proteins involved in focal adhesion, PI3K-Akt and JAK-STAT signaling, all known to play key roles in cell migration and epithelial-to-mesenchymal transition (EMT), were upregulated in mortalin-expressing cancer cells. We further determined that expression levels of the mesenchymal markers vimentin (VIM), fibronectin (FN1), β-catenin (CTNNB1), CK14 (KRT14) and hnRNP-K were also increased upon mortalin overexpression, whereas the epithelial markers E-cadherin (CDH1), CK8 (KRT8), and CK18 (KRT18) were downregulated. Furthermore, shRNA-mediated and pharmacological inhibition of mortalin suppressed the migration and invasive capacity of cancer cells and was associated with a diminished EMT gene signature. Taken together, these findings support a role for mortalin in the induction of EMT, prompting further investigation of its therapeutic value in metastatic disease models.

CARF: An emerging regulator of p53 tumor suppressor and senescence pathway

Replicative senescence, a major outcome of normal cells with finite lifespan, is a widely accepted in vitro model for ageing studies. Limited repair and defense mechanisms of normal cells, in addition to DNA alterations and oncogene inductions under stress, are believed to result in senescence as a protective mechanism to prevent undesirable proliferation of cells. The ARF/p53/p21(cip1/waf1) tumor suppression pathway acts as a molecular sensor and regulator of cellular stress, senescence, and immortalization. Understanding the molecular regulation of this pathway by intrinsic and extrinsic signals is extremely important to address unsolved questions in senescence and cancer. CARF was first discovered as a binding partner of ARF and has since been shown to have both ARF-dependent and -independent functions that converge to regulate p53 pathway. CARF directly binds to p53 and HDM2, and functions in a negative feedback pathway. Whereas CARF transcriptionally represses HDM2 to increase p53 activity, HDM2 in return degrades CARF. Thus, CARF may act as a novel key regulator of the p53 pathway at multiple checkpoints. The aim of this article is to discuss the current knowledge about functions of CARF and its impact on p53 pathway in regulation of senescence and carcinogenesis.

Imminent approaches towards molecular interventions in ageing

Ageing is an innate feature of living organisms. Sensational progress in its molecular understanding in the last decade has culminated into a highly complex picture. Emerging from this complexity are the distinctive roles of some of the tumor suppressor pathways including p53 and pRB in maintenance of senescence phenotype, and telomere maintaining pathways in its escape. We discuss here the current scenario of molecular ageing and the use of modern approaches for its intervention in culture system, at least. Many of the tools we describe here are the newly emergent functional RNA tools that are proved to be fruitful in decoding the human genome. These post-genomic technologies will help us in the discovery of gene targets for interventions aiming to improve the quality at later years of life beyond their mere algebraic extension.