Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Deltapsi(mt)) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases -9, -3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-kappaB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.

A triterpenediol (TPD) comprising of isomeric mixture of 3alpha, 24-dihydroxyurs-12-ene and 3alpha, 24-dihydroxyolean-12-ene from Boswellia serrata induces apoptosis in cancer cells. An attempt was made in this study to investigate the mechanism of cell death by TPD in human leukemia HL-60 cells. It inhibited cell proliferation with IC50 approximately 12 microg/ml and produced apoptosis as measured by various biological end points e.g. increased sub-G0 DNA fraction, DNA ladder formation, enhanced AnnexinV-FITC binding of the cells. Further, initial events involved massive reactive oxygen species (ROS) and nitric oxide (NO) formation, which were significantly inhibited by their respective inhibitors. Persistent high levels of NO and ROS caused Bcl-2 cleavage and translocation of Bax to mitochondria, which lead to loss of mitochondrial membrane potential (Deltapsim) and release of cytochrome c, AIF, Smac/DIABLO to the cytosol. These events were associated with decreased expression of survivin and ICAD with attendant activation of caspases leading to PARP cleavage. Furthermore, TPD up regulated the expression of cell death receptors DR4 and TNF-R1 level, leading to caspase-8 activation. These studies thus demonstrate that TPD produces oxidative stress in cancer cells that triggers self-demise by ROS and NO regulated activation of both the intrinsic and extrinsic signaling cascades.

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Tumor angiogenesis is a validated target for therapeutic intervention, but agents that are more disease selective are needed. Here, we report the isolation of secalonic acid-D (SAD), a mycotoxin from a novel source that exhibits potent antiangiogenic antitumor activity. SAD inhibited multiple HIF1a/VEGF-arbitrated angiogenesis dynamics as scored in human umbilical vascular endothelial cells and human MCF-7 breast tumor xenografts. Similarly, SAD suppressed VEGF-induced microvessel sprouting from rat aortic ring and blood vessel formation in the Matrigel plug assay in C57/BL6J mice. Under normoxic or hypoxic conditions, SAD inhibited cell survival through the Akt/mTOR/p70S6K pathway, with attendant effects on key proangiogenesis factors, including HIF1a, VEGFR, and MMP-2/MMP-9. These effects were reversed by cotreatment with the Akt inhibitors perifosine and GSK69069 or by the addition of neutralizing VEGF antibodies. The apoptotic properties of SAD were determined to be both extrinsic and intrinsic in nature, whereas the cell-cycle inhibitory effects were mediated by altering the level of key G 1 -S transitionphase proteins. In experimental mouse models of breast cancer, SAD dosing produced no apparent toxicities (either orally or intraperitoneal) at levels that yielded antitumor effects. Taken together, our findings offered a preclinical validation and mechanistic definition of the antiangiogenic activity of a novel mycotoxin, with potential application as a cancer-selective therapeutic agent. Cancer Res; 75(14); 2886-96. Ó2015 AACR.

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.

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