Enhancement of the Response of B16F1 Melanoma to Fractionated Radiotherapy and Prolongation of Survival by Withaferin A and/or Hyperthermia Guruprasad Kalthur, PhD1, and Uma Devi Pathirissery, PhD2
Abstract Withaferin A (WA), isolated from Indian medicinal plant Withania somnifera has weak antitumor and radiosensitizing property. The present investigation was planned to evaluate the tumor sensitizing effect of WA with or without local hyperthermia on the response of B16F1 melanoma to fractionated and acute radiotherapy. C57BL mice bearing tumors of 100 ± 10mm3 were treated with fractionated radiotherapy (RT, 2Gy x 5 days/week, 4 weeks), withaferin A (15mg/kg, i.p., 5 days/week, 3 weeks), local hyperthermia (HT, 43°C once a week, 3 weeks) and their combinations, or acute RT (40Gy), WA (40mg/kg), HT (43°C, 30min) and their combinations. Treatment response was studied by tumor regression, growth delay and animal survival. Acute RT+HT produced 50% partial response which increased to 62.5% with combination of WA. In fractionated regimen, trimodality combination resulted in 100% PR. Acute RT+HT and WA+RT produced similar increase in growth delay (GD) compared to RT alone which further increased in trimodality treatment. Fractionated WA+RT+HT for 3 weeks produced a higher GD and survival than all other treatments. In conclusion, WA is a better radiosensitizer than HT in fractionated regimen and the response of radioresistant tumors like melanoma can be significantly enhanced by combining nontoxic doses of WA with fractionated RT, with or without HT, allowing decrease in radiation dose.
Genomic Analysis Highlights the Role of the JAK-STAT Signaling in the Anti-proliferative Effects of Dietary Flavonoid—‘Ashwagandha’ in Prostate Cancer Cells Ravikumar Aalinkeel1, Zihua Hu2, Bindukumar B. Nair1, Donald E. Sykes1, Jessica L. Reynolds1, Supriya D. Mahajan1 and Stanley A. Schwartz1
1Department of Medicine, Division of Allergy, Immunology, and Rheumatology, Buffalo General Hospital, Kaleida Health System and 2Center for Computational Research, New York State Center of Excellence in Bioinformatics and Life Sciences and Department of Biostatistics, University at Buffalo, State University of New York (SUNY), New York State Center of Excellence, Buffalo, NY 14203, USA
aBSTRACT Phytochemicals are dietary phytoestrogens that may play a rolein prostate cancer prevention. Forty percent of Americans usecomplementary and alternative medicines (CAM) for disease preventionand therapy. Ashwagandha (Withania somnifera) contains flavonoidsand active ingredients like alkaloids and steroidal lactoneswhich are called ‘Withanolides’. We hypothesizethat the immunomodulatory and anti-inflammatory properties ofAshwagandha might contribute to its overall effectiveness asan anti-carcinogenic agent. The goal of our study was gain insightinto the general biological and molecular functions and immunomodulatoryprocesses that are altered as a result of Ashwagandha treatmentin prostate cancer cells, and to identify the key signalingmechanisms that are involved in the regulation of these physiologicaleffects using genomic microarray analysis in conjunction withquantitative real-time PCR and western blot analysis. Ashwagandhatreatment significantly downregulated the gene and protein expressionof proinflammatory cytokines IL-6, IL-1β, chemokine IL-8,Hsp70 and STAT-2, while a reciprocal upregulation was observedin gene and protein expression of p38 MAPK, PI3K, caspase 6,Cyclin D and c-myc. Furthermore, Ashwagandha treatment significantlymodulated the JAK-STAT pathway which regulates both the apoptosisprocess as well as the MAP kinase signaling. These studies outlineseveral functionally important classes of genes, which are associatedwith immune response, signal transduction, cell signaling, transcriptionalregulation, apoptosis and cell cycle regulation and provideinsight into the molecular signaling mechanisms that are modulatedby Ashwagandha, thereby highlighting the use of this bioflavanoidas effective chemopreventive agent relevant to prostate cancerprogression.
Abstract Withaferin A (WA) is derived from the medicinal plant Withania somnifera, which has been safely used for centuries in Indian Ayurvedic medicine for treatment of different ailments. We now show, for the first time, that WA exhibits significant activity against human breast cancer cells in culture and in vivo. The WA treatment decreased viability of MCF-7 (estrogen-responsive) and MDA-MB-231 (estrogen-independent) human breast cancer cells in a concentration-dependent manner. The WA-mediated suppression of breast cancer cell viability correlated with apoptosis induction characterized by DNA condensation, cytoplasmic histone–associated DNA fragmentation, and cleavage of poly-(ADP-ribose)-polymerase. On the other hand, a spontaneously immortalized normal mammary epithelial cell line (MCF-10A) was relatively more resistant to WA-induced apoptosis compared with breast cancer cells. The WA-mediated apoptosis was accompanied by induction of Bim-s and Bim-L in MCF-7 cells and induction of Bim-s and Bim-EL isoforms in MDA-MB-231 cells. The cytoplasmic histone–associated DNA fragmentation resulting from WA exposure was significantly attenuated by knockdown of protein levels of Bim and its transcriptional regulator FOXO3a in both cell lines. Moreover, FOXO3a knockdown conferred marked protection against WA-mediated induction of Bim-s expression. The growth of MDA-MB-231 cells implanted in female nude mice was significantly retarded by 5 weekly i.p. injections of 4 mg WA/kg body weight. The tumors from WA-treated mice exhibited reduced cell proliferation and increased apoptosis compared with tumors from control mice. These results point toward an important role of FOXO3a and Bim in regulation of WA-mediated apoptosis in human breast cancer cells.
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.