Rottlerin-induced autophagy leads to the apoptosis in breast cancer stem cells: molecular mechanisms

Abstract Background Autophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles. Autophagy is associated with the several pathological processes, including cancer. Cancer stem cells (CSCs) play significant roles in cancer initiation, progression and drug resistance. Recent studies have demonstrated the antitumor activities of plant-derived chemopreventive agent rottlerin (Rott). However, the molecular mechanism by which Rott induces autophagy in breast CSCs has not been investigated. Results The objectives of this study were to examine the molecular mechanism by which Rott induces autophagy which leads to apoptosis in breast CSCs. Treatment of breast CSCs with Rott for 24 h resulted in a concentration dependent induction of autophagy, followed by apoptosis as measured by flow cytometry. Electron microscopy confirmed the presence of autophagosomes in Rott treated breast CSCs. Western blot analysis showed that Rott treatment increased the expression of LC3, Beclin-1 and Atg12 that are accumulated during autophagy. Prolonged exposure of breast CSCs to Rott caused apoptosis which was associated with the suppression of phosphorylated Akt and mTOR, upregulation of phosphorylated AMPK, and downregulation of anti-apoptosis Bcl-2, Bcl-XL, XIAP and cIAP-1. Knock-down of Atg7 or Beclin-1 by shRNA inhibited Rott-induced autophagy at 24 h. Our study also demonstrates that pre-treatment of breast CSCs with autophagosome inhibitors 3-methyladenine and Bafilomycin, as well as protein synthesis inhibitor cycloheximide inhibited Rott-induced autophagy and apoptosis. Rott induces autophagy via extensive cytoplasmic vacuolization in breast CSCs. Molecular docking results between C2-domain of protein kinase C-delta and Rott indicated that both hydrogen bonding and hydrophobic interactions contributed significantly for ligand binding with minimum binding affinity of ≈ 7.5 Kcal/mol. Although, autophagy inhibitors suppress the formation of cytoplasmic vacuolization and autophagy in breast CSCs, the potency of Rott to induce autophagy and apoptosis might be based on its capability to activate several pathways such as AMPK and proteasome inhibition. Conclusions A better understanding of the relationship between autophagy and apoptosis would eventually allow us to discover novel drugs for the treatment of breast cancer by eliminating CSCs.Peer Reviewe

Curcumin sensitizes TRAIL-resistant xenografts: molecular mechanisms of apoptosis, metastasis and angiogenesis

<p>Abstract</p> <p>Background</p> <p>We have recently shown that curcumin (a diferuloylmethane, the yellow pigment in turmeric) enhances apoptosis-inducing potential of TRAIL in prostate cancer PC-3 cells, and sensitizes TRAIL-resistant LNCaP cells <it>in vitro </it>through multiple mechanisms. The objectives of this study were to investigate the molecular mechanisms by which curcumin sensitized TRAIL-resistant LNCaP xenografts <it>in vivo</it>.</p> <p>Methods</p> <p>Prostate cancer TRAIL-resistant LNCaP cells were implanted in Balb c nude mice to examine the effects of curcumin and/or TRAIL on tumor growth and genes related to apoptosis, metastasis and angiogenesis.</p> <p>Results</p> <p>Curcumin inhibited growth of LNCaP xenografts in nude mice by inducing apoptosis (TUNEL staining) and inhibiting proliferation (PCNA and Ki67 staining), and sensitized these tumors to undergo apoptosis by TRAIL. In xenogrfated tumors, curcumin upregulated the expression of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax, Bak, p21^/WAF1, and p27^/KIP1, and inhibited the activation of NFκB and its gene products such as cyclin D1, VEGF, uPA, MMP-2, MMP-9, Bcl-2 and Bcl-X_L. The regulation of death receptors and members of Bcl-2 family, and inactivation of NFκB may sensitize TRAIL-resistant LNCaP xenografts. Curcumin also inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells in mice.</p> <p>Conclusion</p> <p>The ability of curcumin to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that curcumin alone or in combination with TRAIL can be used for prostate cancer prevention and/or therapy.</p

X-ray diffraction studies on Mycobacterium smegmatis DNA

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A functionally conserved boundary element from the mouse HoxD locus requires GAGA factor in Drosophila

Hox genes are necessary for proper morphogenesis and organization of various body structures along the anterior-posterior body axis. These genes exist in clusters and their expression pattern follows spatial and temporal co-linearity with respect to their genomic organization. This colinearity is conserved during evolution and is thought to be constrained by the regulatory mechanisms that involve higher order chromatin structure. Earlier studies, primarily in Drosophila, have illustrated the role of chromatin-mediated regulatory processes, which include chromatin domain boundaries that separate the domains of distinct regulatory features. In the mouse HoxD complex, Evx2 and Hoxd13 are located ~9 kb apart but have clearly distinguishable temporal and spatial expression patterns. Here, we report the characterization of a chromatin domain boundary element from the Evx2-Hoxd13 region that functions in Drosophila as well as in mammalian cells. We show that the Evx2-Hoxd13 region has sequences conserved across vertebrate species including a GA repeat motif and that the Evx2-Hoxd13 boundary activity in Drosophila is dependent on GAGA factor that binds to the GA repeat motif. These results show that Hox genes are regulated by chromatin mediated mechanisms and highlight the early origin and functional conservation of such chromatin elements

ANTIHYPERGLYCEMIC AND ANTIDYSLIPIDEMIC POTENTIAL OF IPOMOEA BATATAS LEAVES IN VALIDATED DIABETIC ANIMAL MODELS

Objective: The present study was undertaken to investigate the antidiabetic potential of the leaves of Ipomoea batatas.Methods: The crude powder, 95% ethanolic, 50% ethanolic and aqueous extracts of Ipomoea batatas leaves were administered to normoglycemic and streptozotocin (STZ)-induced diabetic rats in a single dose study. The chloroform, butanol and aqueous fractions of aqueous extract were investigated for their antihyperglycemic on STZ-induced diabetic rats. Multiple dose study of an aqueous fraction was also done in STZ and neonatal STZ-induced diabetic rats. Further, the aqueous fraction was measured against the alpha glucosidase and aldose reductase enzymes, and glucose uptake in L6 myotubes.Results: The aqueous extract showed significant lowering of postprandial hyperglycemia of post sucrose loaded normal rats and significantly declined the blood glucose level of STZ-induced diabetic rats. The aqueous fraction at a single dose of 100 mg/kg b. w in comparison with chloroform and butanol fractions significantly lowered the blood glucose level of STZ-induced diabetic rats. The aqueous fraction in a multiple dose study were found to significantly improved the percent glycated hemoglobin (%HbA1c), fasting blood glucose, oral glucose tolerance (OGTT), serum insulin, lipid profile, liver and kidney parameters in STZ-induced diabetic rats. Marked improvement in OGTT and serum insulin levels was also found in neonatal STZ-induced diabetic rats. In vitro study, the aqueous fraction of I. batatas increased glucose uptake in L6 myotubes and inhibits the α-glucosidase and aldose reductase enzymes.Conclusion: The present study demonstrated the significant antidiabetic activity of the I. batatas leaves by promoting insulin secretion, alpha glucosidase and aldose reductase enzyme inhibition.Â

Hedgehog Signaling Antagonist GDC-0449 (Vismodegib) Inhibits Pancreatic Cancer Stem Cell Characteristics: Molecular Mechanisms

Recent evidence from in vitro and in vivo studies has demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in various human cancer stem cells (CSCs). Therefore, the chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. GDC-0449 (Vismodegib), orally administrable molecule belonging to the 2-arylpyridine class, inhibits SHH signaling pathway by blocking the activities of Smoothened. The objectives of this study were to examine the molecular mechanisms by which GDC-0449 regulates human pancreatic CSC characteristics in vitro.GDC-0499 inhibited cell viability and induced apoptosis in three pancreatic cancer cell lines and pancreatic CSCs. This inhibitor also suppressed cell viability, Gli-DNA binding and transcriptional activities, and induced apoptosis through caspase-3 activation and PARP cleavage in pancreatic CSCs. GDC-0449-induced apoptosis in CSCs showed increased Fas expression and decreased expression of PDGFRα. Furthermore, Bcl-2 was down-regulated whereas TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was increased following the treatment of CSCs with GDC-0449. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GDC-0449-treated pancreatic CSCs. Thus, activated Gli genes repress DRs and Fas expressions, up-regulate the expressions of Bcl-2 and PDGFRα and facilitate cell survival.These data suggest that GDC-0499 can be used for the management of pancreatic cancer by targeting pancreatic CSCs