The synergistic effect of withaferin A combined with cisplatin on cervical cancer and its mechanism / 中国药理学通报

Aim To study the synergistic effect of withaferin A (WA) combined with cisplatin (DDP) on cervical cancer and its mechanism. Methods MTT assay was employed to detect the synergistic effect of WA on DDP in cervical cancer cell lines. Annexin V-FITC/PI staining, TUNEL assay and immunoblotting were used to investigate the effect of WA combined with DDP on apoptosis of cervical cancer cells. Immunofluorescence and immunoblotting were used to detect NF-kB/MDR1 pathway related proteins. DCFH-DA and MitoSOX were applied to determine the intracellular reactive oxygen species (ROS) levels. A xenograft model was also used to evaluate the synergistic effect of WA on DDP. Results The combination of WA and DDP could inhibit the survival of cervical cancer cells, promote apoptosis, and inhibit the growth of tumor in mice. WA could inhibit DDP-induced NF-kB/MDR1 signaling pathway and promote ROS production. Conclusions WA plays a synergistic role in anti-cervical cancer by inhibiting DDP-induced NF-kB/MDR1 pathway activation and enhancing DDP induced ROS production.

Safety and pharmacokinetics of Withaferin-A in advanced stage high grade osteosarcoma: A phase I trial

Background: Withaferin-A (WA), an active principle obtained from a traditional Indian herb known asAshwagandha or the Indian ginseng, has been shown to prevent and cure urethane-induced lung tumorsin mice, and also inhibit the growth of transplanted sarcoma in mice.Objectives: In this study, we evaluated the safety and pharmacokinetics of WA in patients with advancedstage high-grade osteosarcoma.Methods: A phase I dose escalation study was planned using the classical 3 þ 3 design (C33D). Doseescalation cohorts comprised of 72, 108, 144 and 216 mg of WA administered in two to four divided dosesper day. Three patients were enrolled in each cohort and the last patient was observed for at least 30 daysfor any dose-limiting toxicity before progressing to a higher cohort. Pharmacokinetic studies wereperformed using high performance liquid chromatography (HPLC) technique with sensitivity up to 50 ng/ml. Safety evaluation including clinical examination, detailed history of adverse events, Liver FunctionTests , Renal Function Tests and complete blood counts were performed at each visit. WA was administered daily till progression. Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 wasused for grading adverse events.Results: The formulation used was generally well tolerated. Eleven adverse events of grade 1 or grade 2severity were observed. No grade 3 or grade 4 adverse events were observed. Elevation of liver enzymes(5/11) and skin rash (2/11) was the most common adverse events. Other adverse effects include fatigue,fever, edema, and diarrhea (one each). None of the patients had detectable levels of WA in circulation.Conclusion: The formulation was well tolerated. However, WA appears to have low oral bioavailability.Further studies with improved formulations are warranted.© 2019 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services byElsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Withaferin A protecting against high fat diet induced obesity by inducing white adipose tissue browning / 解剖学报

[ Abstract] Objective To investigate the protective effect of withaferin A (WA) against high fat diet induced obesity and its associated mechanism. Methods C57BL / 6 J mice at 8-week of age were divided into two groups. The mice were fed with high fat diet (HFD) and were given an intraperitoneal injection of WA or DMSO (solvent control) . The body weight and food intake of the mice were monitored. One week later, inguinal white adipose tissue (iWAT), interscapular brown adipose tissue (BAT), epididymal white adipose tissue (eWAT) and retroperitoneal white adipose tissue (rWAT) were collected and weighed. Expression levels of the genes associated with white adipose browning were detected in iWAT. HE staining was applied to observe the morphological changes of iWAT. Results The data showed that body weight and fat weight in WA group were significantly lower than those in the control group, and the food intake was not changed significantly. Real-time PCR analysis showed that the expression level of browning related genes in iWAT of the WA group was significantly increased. The result from Western blotting analysis showed that the protein levels of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) increased significantly in iWAT of the WA group. The typical morphological change of adipose browning, such as the multilocular adipocytes was observed in inguinal white adipose tissue of the mice treated with WA by using HE staining and mmunofluorescence assay. Conclusion Taken together, these observations indicate that withaferin A can protect the mice from high fat diet induced obesity by promoting white adipose tissue browning.

Roles of withaferin compounds in nervous system disorders / 中国药理学通报

Withaferin compounds have attracted increasing attention in nervous system disorders in reecnt years, which has been reported to have excellent neuroprotective role in stroke, spinal cord injury, ncuroinflammation and so on. The effects of withaferin compounds for nervous system disorders are mainly summa rized in this research, including relevant reports and our recent works.

Effect of Withaferin A on epithelial-mesenchymal transition and apoptosis in cells of lung squamous cell carcinoma / 肿瘤研究与临床

Objective To study the regulation of Withaferin A on apoptosis-related proteins in lung squamous cell carcinoma and its effect on cell epithelial-mesenchymal transition.Methods Lung squamous cell carcinoma cell line SK-MES-1 were cultured in vitro.The SK-MES-1 cells were treated with the final mass concentration of 0 (control),5,10,20,and 40 μg/ml for 28 h,and the general morphology and shedding of the cells were observed under phase contrast microscope;MTT assay was used for detection of cell viability;flow cytometry was used for detection of apoptosis;immunofluorescence and real-time fluorescent polymerase chain reaction (RT-PCR) was used for detection of apoptosis-related proteins and genes bcl-2 and bax,and expression of the epithelial marker E-cadherin and the interstitial marker Vimentin.Results Different concentrations (0,5,10,20,40 μg/ml) of Withaferin A inhibited the activity of SK-MES-1 cells with cell viability of 0.62±0.05,0.42±0.04,and 0.33±0.06,0.21±0.03,0.17±0.04,respectively,which suggesting that this inhibition was related to the concentration of Withaferin A (F =386.505,P =0.005).After treatment with SK-MES-1 cells for 24 h at different concentrations (0,5,10,20,40 μg/ml) of Withaferin A,the apoptosis rates of each group were (0.180±0.011) ％,(0.310±0.013) ％,(0.500±0.021) ％,(0.540±0.018) ％,and (0.410± 0.027) ％,which suggesting that Withaferin A rarely caused apoptosis,mostly necrotic cells (F =1 065.78,P =0.124).In SK-MES-1 cells treated with different concentrations of Withaferin A for 24 h,the results of immunofluorescence showed that the expression of Vimentin was decreased in the experimental group at a concentration of 20 μg/ml compared with the control group,and the fluorescence intensity was lower than that of the control group,but the fluorescence intensity of E-cadherin was higher than that of the control group;the intensity did not change significantly in the experimental group with the other concentrations.While the expression levels of bax and bcl-2 proteins in the control group and the experimental group did not change significantly.RT-PCR results showed that the mRNA expression of E-cadherin (6.7±0.6 and 6.4±0.9) in the experimental group at a concentration of 20 and 40 μg/ml was significandy higher than that in the control group (4.2±1.0),and the difference was statistically significant (both P ＜ 0.05),and the mRNA expression of Vimentin (4.7±0.5 and 4.7±0.5) was significantly lower than that in the control group (7.2±0.7),and the difference was statistically significant (both P ＜ 0.05).Conclusion Withaferin A can inhibit the growth of lung squamous cell carcinoma cells and inhibit the process of epithelial-mesenchymal transition,but it has no obvious relationship with apoptosis and apoptosis-related proteins.

Withaferin A exerts anti-tumor effects in gliobIastoma cells by arresting cell cycle at G2/M phase / 中国药理学与毒理学杂志

Glioblastoma (GBM) is the most common, malignant, and lethal primary brain tumor in adults.Up to now,there is no effective drug for GBM.Withaferin A(WFA)is mainly derived from Indian Winter cherry.It has been traditionally used in ayurvedic medicine.WFA has wide range of pharmaco-logical activities including cardioprotective,anti-inflammatory,immuno-modulatory properties.Recently, WFA was reported to inhibit the growth of many cancer cells; however, the precise molecular mecha-nisms of its anti-cancer activities in GBM remain unclear. Here, we found that treatment of WFA in U251 and U87-MG glioma cells inhibited the cell proliferation,released the cellular LDH,decreased the DNA synthesis, and inhibited the migration, invasion, and colony formation of cells. WFA also in-creased the apoptotic rate of cells, decreased the mitochondrial membrane potential, arrested cell cy-cle at G2/M,inhibited the activity of caspase 3/7,and increased the protein expression of cleaved-cas-pase 3,cleaved PARP in U251 and U87-MG cells.In addition,cell apoptosis induced by WFA was as-sociated with increasing level of Bim,Bad,P21,P53 and decreasing the level of p-CDK1,cyclin A and B. It was also shown that cell apoptosis induced by WFA was associated with P38 signal pathway. These results demonstrated that WFA induced mitochondrial dependent apoptosis in glioblastoma cells which was associated with arresting the cell cycle at G2/M phase by P38 pathway.Taken together,our findings suggest that WFA might be a promising chemotherapy drug in the treatment of GBM.

Simultaneous Estimation of Stigmasterol and Withaferin A in Union Total Herbal Formulation Using Validated HPTLC Method.

Union Total is herbal formulation made in the form of capsule which contains two standardized plant extracts Cissus quadrangularis (CQ) and Withania somnifera (WS). The present work describes development and validation of High Performance Thin Layer Chromatographic method for simultaneous analysis of Stigmasterol (STG) in Cissus quadrangularis (CQ) and Withaferin A (WFA) in Withania somnifera (WS). Stigmasterol and Withaferin A were identified on silica G60 F254 HPTLC plates by post derivatization technique and robustness study was performed by applying a central composite design (CCD) with k factor having 2k factorial runs, 2k axial experiments and five center points. In HPTLC good separation was obtained with chloroform: methanol: toluene: formic acid (6.5: 0.5: 3: 0.25 v/v/v/v) as mobile phase and anisaldehyde sulphuric acid as a derivatizing reagent at detection wavelength 530 nm. Linearity was obtained in the concentration range of 100-200 ng/band for WFA and 200-700 ng/band for STG and the % recoveries were found in the range of 100.06 % to 100.46 % for WFA and 99.97 % to 100.94 % for STG respectively. HPTLC method was found to be sensitive, precise, accurate and reproducible, which would be of use in quality control of these tablets.

Effect of carbon and nitrogen sources on in vitro flower and fruit formation and withanolides production in Withania somnifera (L.) Dunal.

We studied the influence of sucrose and nitrogen concentration on in vitro flowering and fruit setting in elongated shoots of Withania somnifera. BA (1.5 mg/l) and IAA (0.3 mg/l) on MS medium supplemented with 4% sucrose showed 67% of in vitro flower induction frequency, 9 flowers/shoot, 4 fruits/shoot and 11 seeds/fruit in elongated-shoots. Different concentrations of nitrogen sources (L-glutamine, adenine sulphate, ammonium nitrate, potassium nitrate and sodium nitrate 5-25 mg/l) were tested in combination with 4% sucrose and BA at 1.5 mg/l and IAA at 0.3 mg/l. Highest number of flowers (20 flowers/shoot; 2.2-fold) and fruits (16 fruits/shoot; 3.39-fold), fruit setting (12 seeds/fruit; 1.08-fold) at a higher frequency (88 %) were achieved on MS medium augmented with 15 mg/l adenine sulphate with same PGRs and sucrose concentration. The maximum production of withanolide A (0.68 mg/g DW) and withanolide B (0.77 mg/g DW) was recorded in in vitro fruits. Highest accumulation of withaferin A (2 mg/g DW) was quantified from in vitro flowers, whereas, it was low in in vitro fruits (0.49 mg/g DW withaferin A). However, withanone (0.23 mg/g DW) was found accumulated uniformly in both in vitro flowers and fruits compared to control.

Withaferin-A Inhibits Colon Cancer Cell Growth by Blocking STAT3 Transcriptional Activity

BACKGROUND: Withania somnifera (known as Ashwagandha) is a medicinal plant used in the ayurvedic medicines in India. Withaferin-A, a withanolide derived from the leaf extract of W. somnifera, has been reported to exhibit anti-tumor activity against various cancer cells, such as leukemia, breast cancer and colon cancer cells. METHODS: We investigated the anti-cancer effects of withaferin-A on the proliferation and migration of human colorectal cancer (HCT116) cells. And we evaluated the effects of withaferin-A on the transcriptional activity of STAT3 and the growth of HCT116 cells in xenograft mouse tumor model. RESULTS: In the present study, we found that withaferin-A inhibited the proliferation and migration of HCT116 cells in a concentration-dependent manner. Treatment of HCT116 cells with withaferin-A attenuated interleukin-6-induced activation of STAT3, which has been implicated in the development and progression of colon cancer. To examine the effect of withaferin-A on HCT116 cells proliferation in vivo, we generated HCT116 cells xenograft tumors in Balb/c nude mice and treated the tumor bearing mice with or without withaferin-A intraperitoneally. Treatment with withaferin-A exhibited significant decrease in the volume and weight of tumors as compared to untreated controls. CONCLUSIONS: The present study suggests that withaferin-A holds the potential to be developed as a small molecule inhibitor of STAT3 for the treatment of HCT116.

Withaferin A Inhibits Helicobacter pylori-induced Production of IL-1beta in Dendritic Cells by Regulating NF-kappaB and NLRP3 Inflammasome Activation

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, and gastric cancer. There is evidence that IL-1beta is associated with the development of gastric cancer. Therefore, downregulation of H. pylori-mediated IL-1beta production may be a way to prevent gastric cancer. Withaferin A (WA), a withanolide purified from Withania somnifera, is known to exert anti-inflammatory and anti-tumor effects. In the present study, we explored the inhibitory activity of WA on H. pylori-induced production of IL-1beta in murine bone marrow-derived dendritic cells (BMDCs) and the underlying cellular mechanism. Co-treatment with WA decreased IL-1beta production by H. pylori in BMDCs in a dose-dependent manner. H. pylori-induced gene expression of IL-1beta and NLRP3 (NOD-like receptor family, pyrin domain containing 3) were also suppressed by WA treatment. Moreover, IkappaB-alpha phosphorylation by H. pylori infection was suppressed by WA in BMDCs. Western blot analysis revealed that H. pylori induced cleavage of caspase-1 and IL-1beta, as well as increased procaspase-1 and pro IL-1beta protein levels, and that both were suppressed by co-treatment with WA. Finally, we determined whether WA can directly inhibit ac tivation of the NLRP3 inflammasome. NLRP3 activators induced IL-1beta secretion in LPS-primed macrophages, which was inhibited by WA in a dose-dependent manner, whereas IL-6 production was not affected by WA. Moreover, cleavage of IL-1beta and caspase-1 by NLRP3 activators was also dose-dependently inhibited by WA. These findings suggest that WA can inhibit IL-1beta production by H. pylori in dendritic cells and can be used as a new preventive and therapeutic agent for gastric cancer.