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Withania somnifera Extract Protects Model Neurons from In Vitro Traumatic Injury.

ABSTRACT: Withania somnifera has been used in traditional medicine for a variety of neural disorders. Recently, chronic neurodegenerative conditions have been shown to benefit from treatment with this extract. To evaluate the action of this extract on traumatically injured neurons, the efficacy of W. somnifera root extract as a neuroprotective agent was examined in cultured model neurons exposed to an in vitro injury system designed to mimic mild traumatic brain injury (TBI). Neuronal health was evaluated by staining with annexin V (an early, apoptotic feature) and monitoring released lactate dehydrogenase activity (a terminal cell loss parameter). Potential mechanisms underlying the observed neuroprotection were examined. Additionally, morphological changes were monitored following injury and treatment. Although no differences were found in the expression of the antioxidant transcription factor nuclear factor erythroid 2-like 2 (Nrf2) or other Nrf2-related downstream components, significant changes were seen in apoptotic signaling. Treatment with the extract resulted in an increased length of neurites projecting from the neuronal cell body after injury. W. somnifera extract treatment also resulted in reduced cell death in the model neuron TBI system. The cell death factor Bax was involved (its expression was reduced 2-fold by the treatment) and injury-induced reduction in neurite lengths and numbers was reversed by the treatment. This all indicates that W. somnifera root extract was neuroprotective and could have therapeutic potential to target factors involved in secondary injury and long-term sequelae of mild TBI.

SUBMITTER: Saykally JN

PROVIDER: S-EPMC5657733 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Withania somnifera Extract Protects Model Neurons from In Vitro Traumatic Injury.

Saykally Jessica N JN Hatic Haris H Keeley Kristen L KL Jain Subhash C SC Ravindranath Vijayalakshmi V Citron Bruce A BA

Cell transplantation 20170701 7

Withania somnifera has been used in traditional medicine for a variety of neural disorders. Recently, chronic neurodegenerative conditions have been shown to benefit from treatment with this extract. To evaluate the action of this extract on traumatically injured neurons, the efficacy of W. somnifera root extract as a neuroprotective agent was examined in cultured model neurons exposed to an in vitro injury system designed to mimic mild traumatic brain injury (TBI). Neuronal health was evaluated ...[more]

PMID: 28933215

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Project description:Background:Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. Objective:In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. Materials and Methods:The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. Results:The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 ?g/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 ?g/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. Conclusion:We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. SUMMARY:The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 ?g/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance.

Project description:Though clinicians can predict which patients are at risk for developing metastases, traditional therapies often prove ineffective and metastatic disease is the primary cause of cancer patient death; therefore, there is a need to develop anti-metastatic therapies that can be administered over long durations to specifically inhibit the motility of cancer cells. Withaniasomnifera root extracts (WRE) have anti-proliferative activity and the active component, Withaferin A, inhibits the pro-metastatic protein, vimentin. Vimentin is an intermediate filament protein and is part of the epithelial to mesenchymal transition (EMT) program to promote metastasis. Here, we determined whether WRE standardized to Withaferin A (sWRE) possesses anti-metastatic activity and whether it inhibits cancer motility via inhibition of vimentin and the EMT program. Several formulations of sWRE were created to enrich for Withaferin A and a stock solution of sWRE in EtOH could recover over 90% of the Withaferin A found in the original extract powder. This sWRE formulation inhibited breast cancer cell motility and invasion at concentrations less than 1µM while having negligible cytotoxicity at this dose. sWRE treatment disrupted vimentin morphology in cell lines, confirming its vimentin inhibitory activity. To determine if sWRE inhibited EMT, TGF-β was used to induce EMT in MCF10A human mammary epithelial cells. In this case, sWRE prevented EMT induction and inhibited 3-D spheroid invasion. These studies were taken into a human xenograft and mouse mammary carcinoma model. In both models, sWRE and Withaferin A showed dose-dependent inhibition of tumor growth and metastatic lung nodule formation with minimal systemic toxicity. Taken together, these data support the hypothesis that low concentrations of sWRE inhibit cancer metastasis potentially through EMT inhibition. Moreover, these doses of sWRE have nearly no toxicity in normal mouse organs, suggesting the potential for clinical use of orally administered WRE capsules.

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Withania somnifera Extract Protects Model Neurons from In Vitro Traumatic Injury.

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Withania somnifera Extract Protects Model Neurons from In Vitro Traumatic Injury.

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