BackgroundAshwagandha, a traditional Indian herb, has been known for its variety of therapeutic activities. We earlier demonstrated anticancer activities in the alcoholic and water extracts of the leaves that were mediated by activation of tumor suppressor functions and oxidative stress in cancer cells. Low doses of these extracts were shown to possess neuroprotective activities in vitro and in vivo assays.Methodology/Principal FindingsWe used cultured glioblastoma and neuroblastoma cells to examine the effect of extracts (alcoholic and water) as well as their bioactive components for neuroprotective activities against oxidative stress. Various biochemical and imaging assays on the marker proteins of glial and neuronal cells were performed along with their survival profiles in control, stressed and recovered conditions. We found that the extracts and one of the purified components, withanone, when used at a low dose, protected the glial and neuronal cells from oxidative as well as glutamate insult, and induced their differentiation per se. Furthermore, the combinations of extracts and active component were highly potent endorsing the therapeutic merit of the combinational approach.ConclusionAshwagandha leaf derived bioactive compounds have neuroprotective potential and may serve as supplement for brain health.

Mitochondrial functions play a central role in energy metabolism and provide survival fitness to both normal and tumor cells. Mitochondrial chaperonin Hsp60 is involved in both pro- and anti-apoptotic functions, but how Hsp60 senses the mitochondria selective oxidative stress response is unknown. In this study, by using rotenone, an irreversible inhibitor of oxidative phosphorylation against IMR-32 and BC-8 tumor cells containing differential heat shock transcriptional machinery, we studied whether the oxidative stress response is related to Hsp60. The accelerated cytotoxicity in response to rotenone has been correlated with enhanced production of O2•−, H2O2, reactive oxygen species, and Hsp60 translocation from the mitochondria to the cytoplasm. The inability of cells to resist oxidative stress mediated Hsp60 translocation appeared to depend on mitochondrial oxyradical scavenging system and Bax translocation. A delayed oxidative stress response in hsp60 shRNA-treated cells was found to be due to increased mitochondrial translocation of Hsp60 on shRNA pre-sensitization. Overexpression of Hsp60 failed to protect cells from oxidative stress due to a lack of its mitochondrial retention upon post-rotenone treatment. These results also revealed that Hsp60 mitochondrial localization is indispensable for decreasing O2•− levels, but not H2O2 and ROS levels. However, cycloheximide treatment alone induced Hsp60 translocation, while rotenone combination delayed this translocation. In contrast to oxidative stress, MG132 and 17AAG treatments showed mitochondrial retention of Hsp60; however, MG132 combination either with hsp60 shRNA or 17AAG induced its translocation. Additionally, overexpression of Huntingtin gene also resulted in Hsp60 mitochondrial accumulation. We suggest that Hsp60 may act as a barrier to pharmacological targeting of mitochondria.

Hsp90 chaperone has been identified as an attractive pharmacological target to combat cancer. However, some metastatic tumors either fail to respond to Hsp90 inhibition or show recovery necessitating irreversible therapeutic strategies. In response to this enforced senescence has been proposed as an alternate strategy. Here, we demonstrate that inhibiting Hsp90 with 17AAG sensitizes human neuroblastoma to DNA damage response mediated cellular senescence. Among individual and combination drug treatments, 17AAG pre-treatment followed by doxorubicin treatment exhibited senescence-like characteristics such as increased nucleus to cytoplasm ratio, cell cycle arrest, SA-β-gal staining and the perpetual increase in SAHF. Doxorubicin induced senescence signaling was mediated by p53-p21CIP/WAF-1 and was accelerated in the absence of functional Hsp90. Sustained p16INK4a and H3K4me3 expressions correlating with unaffected telomerase activation annulled replicative senescence and appraised stress induced senescence. Despite increases in [(ROS)i] and [(Ca2+)i], a concomitant increase in cellular antioxidant defense system suggested oxidation independent senescence activation. Sustained activation of survival (Akt) and proliferative (ERK1/2) kinases fosters robustness of cells. Invigorating senescent cells with growth factor or snooping with mTOR or PI3 kinase inhibitors compromised cell survival but not senescence. Intriguingly, senescence-associated secretory factors from the senescence cells manifested established senescence in neuroblastoma, which offers clinical advantage to our approach. Our study discusses tumor selective functions of Hsp90 and discusses irrefutable strategies of Hsp90 inhibition in anticancer treatments.

Mitosis-targeted anti-cancer therapies gained much attention in recent years. However, lack of tumor selectivity poses limitations to the current anti-mitotic drugs to be used as broad-spectrum anti-cancer agents. In this study, we show that combination treatment of colcemid, an inhibitor of microtubule polymerization with geldanamycin, an inhibitor of cancer chaperone, Hsp90 irreversibly targets mitosis through mitotic kinase bubR1 stabilization. When the individual and combination drugs treatments were tested against tumor cells (IMR-32 and HeLa) and non-tumor cells (SRA01), the combination treatment showed significant increase in cytotoxicity only in tumor cells followed by G2/M cell cycle block. The IMR-32 cells showed enhanced cytotoxicity in response to combination treatments, compared to HeLa cells. Further studies revealed that the G2/M arrest in IMR-32 correlates with both increased bubR1 nuclear localization and metaphase arrest. The siRNA knockdown of bubR1 has decreased tumor cell response to geldanamycin suggesting Hsp90-dependent regulation of bubR1. The combination treatment also showed inactivation of non-canonical β-catenin signaling suggesting inhibition of cancer growth. In addition, the combination treatment has significantly affected the distribution and functions of bubR1 downstream mitotic kinases such as aurks and plk1 indicating the combinatorial attack of combination treatment. In conclusion, we demonstrate that colcemid and GA combination treatment compromises the division potential of tumor cells interfering with the mitosis through bubR1 kinase.

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