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

April 2007
Nashi Widodo; Kamaljit Kaur; Bhupal G. Shrestha; Yasuomi Takagi; Tetsuro Ishii; Renu Wadhwa; Sunil C. Kaul

Abstract

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.

Ashwagandha (Withania somnifera, an evergreen shrub commonly found in the drier parts of the Indian subcontinent) is widely used in Indian natural medicine, Ayurveda. Extracts from different parts of ashwagandha have been claimed to promote physical and mental health due to its effects, ranging from antistress, antiinflammatory, antioxidant, antipyretic, analgesic, antiarthritic, antidepressant, anticoagulant, immunomodulatory, adaptogenic, cardioprotective, rejuvenating, and regenerating properties (1–13).

Few reports have characterized the activities of the root extract of ashwagandha and include an induction of nitric oxide synthase–inducible protein expression (4, 14), down-regulation of p34cdc2 expression (15), and its antioxidant, free radical–scavenging, and detoxifying properties (16–19). However, the mechanistic aspects of its effects, including tumor suppression and isolation of active components, have largely remained unexplored. Hence, the use of ashwagandha has not been developed to a systemic medicine.

Although ashwagandha roots are most commonly used in Indian Ayurvedic medicine, we undertook a study to examine the effects of its leaf extract because of the easy accessibility and abundant availability. We earlier reported that the leaf extract obtained by a series of extractions (20) has an antimutagenic effect (21).

In the present study, we examined the effect of the leaf extract on human normal and cancer cells and found that it selectively kills tumor cells. Fractionation of the tumor-inhibitory extract (i-Extract) and characterization of its constituents led to the identification of a tumor-inhibitory factor (i-Factor).

Nuclear magnetic resonance (NMR) spectra revealed its identity as withanone. By employing small hairpin RNA (shRNA) library and molecular analysis, we report for the first time that the selective killing of tumor cells by i-Extract and i-Factor involves an activation of the wild-type p53 function.

Materials and Methods

Preparation of leaf extract from ashwagandha from field-raised plants. Ashwagandha (W. somnifera) leaf extracts were prepared as described earlier (20–22). The leaves were air dried, ground to a fine powder, and subjected to extraction with methanol (60°C) in Soxhlet apparatus for 4 to 5 days. The methanol extracts were further extracted with hexane to remove chlorophyll and other pigments and then with diethyl ether that was evaporated to obtain the ether extract. Ether extract solubilized in DMSO was used for the present studies.

Nude mice assay. BALB/c nude mice (4 weeks old, female) were bought from Nihon Clea (Japan). Mice were fed on standard food pellet and water ad libitum, acclimatized to our laboratory condition at a temperature of 24 ± 2°C, relative humidity of 55% to 65%, and 12-h light/dark cycle, for 3 days. Fibrosarcoma (HT1080) cells (1 × 106 suspended in 0.5 mL of growth medium) were injected s.c. into the flank of nude mice (one site per mouse). i-Extract injections were commenced at three different times, i.e., (a) mixed with cells at the time of injection, (b) injection before the formation of tumor buds, and (c) injection when small tumor buds (5 mm) were formed.

In each case, s.c. injection (0.3 mL of 24 μg/mL i-Extract in the cell growth medium) was given. Tumor formation was monitored during the next 15 to 20 days, with local injection of i-Extract to the tumor site every third day. For oral feeding, i-Extract or its components were suspended in 2% sterile carboxymethyl cellulose (a vehicle) and injected into the digestive tract of mice using a flexible Teflon needle on alternate days.

Characterization of i-Extract by column chromatography. Ether extract of the leaves was subjected to reversed-phase high-performance liquid chromatography (HPLC) analysis on a C-18 column (5 mm, 150 × 4.6 mm internal diameter; Waters, Milford, MA or YMC, Kyoto, Japan) at 40°C or 50°C using 1% methanol/H2O (solution A) and methanol/ethanol/isopropanol (52.25:45.30:2.45; solution B) for elution. Elution was done with a gradient of 35% to 45% solution B in 25 min at a flow rate of 1 mL/min. The detection was done at 220 nm. Withaferin A, 12-deoxywithastramonolide, and withanolide D were used as standards for comparison.

Human cell culture and treatments. Normal diploid fibroblasts (TIG-1 and WI-38), osteogenic sarcoma (U2OS and Saos-2), breast carcinoma (MCF7, HS578T, and SK-BR3), fibrosarcoma (HT1080), colon carcinoma (HCT116), and lung carcinoma (PC14) cells were cultured in DMEM (Life Technologies, Gaithersburg, MD), supplemented with 10% fetal bovine serum in a humidified incubator (37°C and 5% CO2). Cells (∼50-60% confluency) were treated with i-Extract (6-36 μg/mL) for time periods as indicated.

Growth assays. Equal number of cells, counted by Neubauer hemocytometer, was plated in six-well dishes for control and treatment wells. Cells were harvested every 24 h up to 96 h, counted, and plotted as growth curves. Viability was monitored by WST-based cell proliferation kit.

Cell Proliferation Reagent WST-1, a colorimetric assay for the quantification of cell proliferation and cell viability, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dihydrogenases in viable cells.(Roche, Mannheim, Germany).

Preparation and use of shRNAs. shRNAs for the genes listed in Table 1 were cloned in a U6-driven expression vector as described earlier (23). Two target sites per gene were used; the sequences for each of the target site are listed in Table 1. Cells were plated in 96-well plates and were transfected at ∼70% confluency with 50 ng of the plasmid DNA. At 24 h posttransfection, cells were selected in puromycin (2 μg/mL) supplemented medium for 48 to 72 h, expanded to 70% confluency, and were then treated with i-Extract (24 μg/mL). shRNAs that resulted in the survival of cells (in the presence of i-Extract) were selected and taken through the second round of transfections and confirmation. Cell viability was measured by staining with Crystal Violet and AlamarBlue assay (BioSource International, Camarilo, CA).