Background Cancer is a leading cause of death accounting for 15-20%

Background Cancer is a leading cause of death accounting for 15-20% of global mortality. ASH-WEX is cytotoxic to cancer cells selectively, and causes tumor suppression anti-tumor test in nude mice subcutaneous xenograft and tail vein metastasis models. Figure 1 Identification of cancer cell specific cytotoxicity in the water extract of Ashwagandha leaves by and assays. As shown in Figure 1D, mice fed with 500 mg of ASH-WEX/Kg body weight on every alternate day for 60 days did not show toxicity in terms of change in body weight and physical activity. After implanting HT1080 subcutaneous xenograft and tail vein injections, the nude mice were fed with ASH-WEX 250 mg/Kg body weight every alternate day and the tumor formation was monitored. In about 20 days, out of 24 control mice, 21 showed big tumor (Figures 1E and 1F), one showed small tumor and two showed no tumors. ASH-WEX fed mice, on the 196808-24-9 IC50 other hand, showed strong tumor suppression. In this group, out of 24 mice, 12 showed no tumors, 3 showed tumors buds, 5 showed small tumors and only 4 showed big tumors (Figures 1E and 1F). In lung metastasis assays, ASH-WEX fed mice showed strong suppression of metastasis. There were less than 5 tumors in ASH-WEX fed mice as compared to the control mice that had more than 50 tumors (Figure 1G). These data suggested that the ASH-WEX has considerable anticancer activity and (Figure 2E). These features (HPLC profile and proteins) were used as ASH-WEX signature to avoid batch variation during the course of the study. Figure 2 Chemical analysis of WEX (ASH-WEX). In order to characterize the anticancer activity in ASH-WEX in cell-based assays, we first inactivated the protein components by heat denaturation and proteinase-degradation (Figure 3A). The assays revealed that the cytotoxic activity of ASH-WEX was independent of its protein 196808-24-9 IC50 components (Figure 3B). Furthermore, it was size fractionated as described in the materials and methods. The active fraction (ASH-WEX-F2, as examined by cytotoxic assays) was heat denatured, dried and subjected to NMR analysis. 1H and 13C-NMR spectrums (Figure 3C, a-b) predicted the presence of triethylene glycol (TEG) as a major component in ASH-WEX-F2. Indeed, the 1H and 13C-NMR spectrums of TEG were identical 196808-24-9 IC50 to the ones obtained for ASH-WEX-F2 (Figure 3C, c-d). We performed HPLC of ASH-WEX under conditions as described in the materials and methods (HPLC-3) and using TEG as a standard. These data confirmed the presence of triethylene glycol (TEG) in ASH-WEX (Figure 3D). Figure 3 Activity-based chemical identification of anticancer component. We next investigated whether TEG is the main cytotoxic component of ASH-WEX in and nude mice assays. As described above, 196808-24-9 IC50 in assays, heat inactivation did not cause the loss of cytotoxicity in ASH-WEX. Based on this observation and in order to determine the anticancer component in ASH-WEX, we tested the effect of heat inactivation on cytotoxicity of withaferin A, withanone and TEG. As shown in Figure 4A, cytotoxicity of withaferin A and withanone to U2OS cells was compromised by heat treatment (Figure 4A, compare bar 6 with 7, P value <0.01). However, there was no significant difference in the cytotoxic effect of TEG and heat-inactivated TEG (Figure 4A, compare bar 8 with 9). Furthermore, the cytotoxicity, predominantly seen in cancer cells, was also supported by these assays (Figures 4A and 4B). Erg Normal human fibroblasts treated with serial doses of TEG showed no/minor growth arrest as shown in Figures 4B and 4C. Cell cycle analysis revealed that the ASH-WEX and TEG caused G1 arrest (Figure 4D). Figure 4 Cancer cell toxicity analysis of TEG. We next investigated an tumor suppression activity in TEG by examining the nude mice tumor formation and metastasis assays. HT1080 cells that form aggressive tumors 196808-24-9 IC50 with high lung metastasis were used. Tumor volume in control cells showed sharp increase in 28 days. Oral feeding of TEG, similar to ASH-WEX (as described above), to the mice implanted with HT1080 subcutaneous xenograft and tail vein injections showed slow tumor growth as compared to the control mice suggesting tumor suppressive activity in TEG (Figures 5A and 5B). Intraperitoneal injections (100 l of 5% TEG on every alternate day).

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