Protective effect of Abutilon indicum against lead-induced reproductive toxicity in male Wistar rats

Despite ample literature on the toxic impact of lead on the environment and health, the exact mechanism of pathogenesis/toxicity is not clearly known. Because it is well established that lead induces oxidative stress, it is assumed that exposure to antioxidants may reduce the toxic impact of lead. In this study, we evaluated the impact of coadministration of the methanolic root extract of a plant Abutilon indicum (50, 100, 200 mg kg ⁻¹b.wt.) in mitigating the toxic impact of lead on the reproductive system of rats. In brief, Wistar rats were exposed to lead acetate in drinking water with or without coadministration of plant root extract and compared with that of control animals. After 45 days of exposure as outlined above, the animals were killed and the reproductive toxicity was assessed by sperm parameters, hormone and antioxidant enzyme assays, and testis histopathology. Significant reduction in testis weight, sperm count, testosterone levels, and antioxidant enzymes levels such as Superoxide Dismutase, Catalase, and Glutathione peroxidase was seen in lead-treated animals, confirming the toxic impact. The coadministration of A. Indicum (100 and 200 mg kg ⁻¹b.wt.) was found to bring the studied parameters close to the levels seen in untreated (control) animals. Our findings are indicative of the protective nature of A. Indicum against lead-induced reproductive toxicity in a dose-dependent manner. However, further characterization of the root extract is required to elucidate the probable mechanism of protection.     

Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators.     

AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.     

Caspase-2 triggers Bax-Bak-dependent and -independent cell death in colon cancer cells treated with resveratrol

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.     

Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity

Rohitukine is a chromane alkaloid possessing anti-inflammatory, anti-cancer and immuno-modulatory properties. The compound was first reported from Amoora rohituka (Meliaceae) and later from Dysoxylum binectariferum (Meliaceae) and Schumanniophyton problematicum (Rubiaceae). Flavopiridol, a semi-synthetic derivative of rohitukine is a potent CDK inhibitor and is currently in Phase III clinical trials. In this study, the isolation of an endophytic fungus, Fusarium proliferatum (MTCC 9690) from the inner bark tissue of Dysoxylum binectariferum Hook.f (Meliaceae) is reported. The endophytic fungus produces rohitukine when cultured in shake flasks containing potato dextrose broth. The yield of rohitukine was 186 μg/100 g dry mycelial weight, substantially lower than that produced by the host tissue. The compound from the fungus was authenticated by comparing the LC–HRMS and LC–HRMS/MS spectra with those of the reference standard and that produced by the host plant. Methanolic extract of the fungus was cytotoxic against HCT-116 and MCF-7 human cancer cell lines (IC₅₀ = 10 μg/ml for both cancer cell lines).