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Molecular mechanisms of exopolysaccharide from Aphanothece halaphytica (EPSAH) induced apoptosis in HeLa cells.

ABSTRACT: The present study aims to investigate the pharmacological effect of the exopolysaccharides from Aphanothece halophytica GR02 (EPSAH) on the HeLa human cervical cancer cell line. HeLa cells were cultured in RPMI-1640-10% FBS medium containing with or without different concentrations of EPSAH. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was elevated with Wright-Giemsa staining, AO/EB double staining, and DNA fragmentation assay. Apoptosis-associated molecules from cultured HeLa cells were quantified using Western blot analysis. Our results suggest that EPASH induces apoptosis in HeLa cells by targeting a master unfolded protein response (UPR) regulator Grp78. Grp78 further promotes the expression of CHOP and downregulates expression of survivin, which leads to activate mitochondria-mediated downstream molecules and p53-survivin pathway, resulting in caspase-3 activation and causing apoptosis. These findings provide important clues for further evaluating the potential potency of EPSAH for use in cancer therapy.

SUBMITTER: Ou Y

PROVIDER: S-EPMC3900761 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Molecular mechanisms of exopolysaccharide from Aphanothece halaphytica (EPSAH) induced apoptosis in HeLa cells.

Ou Yu Y Xu Shuya S Zhu Dandan D Yang Xuegan X

PloS one 20140123 1

The present study aims to investigate the pharmacological effect of the exopolysaccharides from Aphanothece halophytica GR02 (EPSAH) on the HeLa human cervical cancer cell line. HeLa cells were cultured in RPMI-1640-10% FBS medium containing with or without different concentrations of EPSAH. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was elevated with Wright-Giemsa staining, AO/EB double staining, and DNA fragmentation assay. Apoptosis-associated molecul ...[more]

PMID: 24466342

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Project description:BackgroundAutophagy 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.ResultsThe 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-X(L), 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.ConclusionsA 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.

Project description:ObjectivesThe legume lectin family, one of the most extensively studied plant lectin families, has received increasing attention for the remarkable anti-tumor activities of its members for binding specific cancer cell surface glycoconjugates. MicroRNAs, a class of small, non-coding RNAs, control translation and stability of mRNAs at post-transcriptional and translational levels. To date, accumulating evidence has revealed that microRNAs are involved in progression of a number of human diseases, especially cancers. However, the molecular manners of microRNA-modulated apoptosis in legume lectin-treated cancer cells are still under investigation.Materials and methodsWe performed in silico analyses to study the interactions between three typical legume lectins (ConA, SFL and SAL) and some specific sugar-containing receptors (for example, EGFR, TNFR1, HSP70 and HSP90). Additionally, we predicted some relevant microRNAs which could significantly regulate these aforementioned targetreceptors and thus inhibiting down-stream cancer-related signaling pathways.ResultsThe results showed that these three legume lectins could competitively bind sugar-containing receptors such as EGFR, TNFR1, HSP70 and HSP90 in two ways, via anti-apoptotic or survival pathways. On the one hand, the legume lectins could induce cancer cell death through triggering receptor-mediated signaling pathways, which resulted from indirect binding between legume lectins and mannoses resided in receptors. On the other hand, direct binding between legume lectins and receptors could lead to steric hindrance, which would disturb efficient interactions between them, and thus, the legume lectins would induce cancer cell death by triggering receptor-mediated signaling pathways. In addition, we identified several relevant microRNAs that regulated these targeted receptors, thereby ultimately causing cancer cell apoptosis.ConclusionsThese findings provide new perspectives for exploring microRNA-modulated cell death in legume lectin-treated cancer cells, which could be utilized in combination therapy for future cancer drug development.

Dataset Information

Molecular mechanisms of exopolysaccharide from Aphanothece halaphytica (EPSAH) induced apoptosis in HeLa cells.

Publications

Molecular mechanisms of exopolysaccharide from Aphanothece halaphytica (EPSAH) induced apoptosis in HeLa cells.

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