Epigallocatechin-3-gallate induces growth inhibition and apoptosis of human anaplastic thyroid carcinoma cells through suppression of EGFR/ERK pathway and cyclin B1/CDK1 complex.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is one of the most lethal cancers because of its aggressiveness and the lack of efficacious therapy. Epigallocatechin-3-gallate (EGCG), a major catechin in green tea, was shown to possess remarkable therapeutic potential against various types of human cancer cells in in vitro and in vivo models. The aim of this study was to investigate the effect of EGCG on the proliferation and apoptosis of ARO cells--human ATC cells. STUDY DESIGN: Experimental study. METHODS: Human ATC cell line, ARO, was treated with EGCG. Cell viability was assessed by MTT assay. Inhibition of EGFR/MAPK pathway and cell cycle-related proteins by EGCG were measured by Western blot analysis. In addition, cell cycle analysis was measured by flow cytometry. RESULTS: EGCG treatment inhibited the growth of ARO cells in a dose-dependent manner. Furthermore, EGCG suppressed phosphorylation of EGFR, ERK1/2, JNK, and p38. These changes were associated with increased p21 and reduced cyclin B1/CDK1 expression. In addition, EGCG treatment increased the accumulation of sub-G1 cell, activated caspase-3 and cleaved PARP. CONCLUSIONS: Taken together, EGCG inhibits cell proliferation and induces apoptosis via suppression of the EGFR/ERK pathway and cyclin B1/CDK1 complex in ATC cells.

Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas.

A subpopulation of cancer stem cells (CSCs), but not the majority of non-tumorigenic cancer cells, in a variety of human malignancies plays a critical role in cancer cell proliferation, invasion, metastasis, and tumor recurrence post-therapies. We report the isolation of sphere-forming cells (squamospheres) from primary head and neck squamous cell carcinomas (HNSCCs), and characterization of their CSC properties. Squamospheres appeared within 2 weeks after seeding as single-dissociated cells obtained from primary HNSCC specimens in serum-free culture conditions. Real-time RT-PCR and immunocytochemistry assays revealed that a number of stem cell markers, including CK5, OCT4, SOX2, and nestin, were up-regulated in HNSCC-driven squamospheres. Fluorescence-activated cell sorting (FACS) analysis showed that squamospheres contain enriched side population cells compared to serum-induced differentiated squamosphere cells. Furthermore, HNSCC-driven squamospheres appeared to be chemoresistant to cisplatin, 5-fluorouracil (FU), paclitaxel and doxetaxel, and showed increased levels of ABCG2, one of the ATP-binding cassette (ABC) transporters. Of particular interest, in sharp contrast to subcutaneous injection of 1×10(6) differentiated squamosphere cells, as few as 100 squamosphere cells were able to give rise to tumors in nude mice. Altogether, we assert that primary HNSCC-driven squamospheres possess CSC properties, and its functional analysis may provide a novel tool for investigating the tumorigenic process of HNSCC.

Diallyl sulfide induces growth inhibition and apoptosis of anaplastic thyroid cancer cells by mitochondrial signaling pathway.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal solid tumors arising thyroid gland with dismal prognosis. One of the constituents of garlic, diallyl sulfide (DAS) was shown to inhibit chemically induced carcinogenesis in many animal models. This study examined whether DAS could induce growth inhibition and apoptosis in ATC cells. In MTT assay, DAS treatment inhibited the proliferation of ARO cells in a dose-dependent manner. Flow cytometric analysis showed that DAS treatment increased the accumulation of sub-G1 DNA and concomitant accumulation of cells in the G2/M phase in a dose-dependent manner. In addition, DAS-induced apoptosis was associated with a decrease in the level of Bcl-2 expression and an increase in the level of Bax expression, and cytochrome c was remarkably released from mitochondrial into the cytosol by DAS. Furthermore, caspase-9 and caspase-3 were activated by DAS, and DAS cleaved PARP. Taken together, DAS decreased cell proliferation and induced apoptosis via mitochondrial signaling pathway in ATC cells.

H2O2-dependent hyperoxidation of peroxiredoxin 6 (Prdx6) plays a role in cellular toxicity via up-regulation of iPLA2 activity.

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase activity and Ca2+-independent phospholipase A2 (iPLA2) activity. Here, we report that H2O2-induced cellular toxicity acts through Prdx6 hyperoxidation. Under high concentrations of H2O2 (> 100 microm), Prdx6, and 2-Cys Prdxs were hyperoxidized. Contrary to hyperoxidation of 2-Cys Prdxs, hyperoxidation of Prdx6 was irreversible in vivo. Surprisingly, H2O2-induced cell cycle arrest at the G2/M transition correlated with hyperoxidation and increased iPLA2 activity of Prdx6. This arrest was also associated with up-regulation of p53 and p21 and with down-regulation of cyclin B1. Furthermore, the H2O2-mediated increase in iPLA2 activity was dramatically abolished in a hyperoxidation mutant (C47A), an iPLA2 mutant (S32A), and a double mutant (C47A/S32A) of Prdx6, demonstrating the essential requirement of Prdx6 C47 hyperoxidation for its iPLA2 activity. Together, our results demonstrate that H2O2-mediated hyperoxidation of Prdx6 induces cell cycle arrest at the G2/M transition through up-regulation of iPLA2 activity.

Methylene chloride fraction of Scutellaria barbata induces apoptosis in human U937 leukemia cells via the mitochondrial signaling pathway.

BACKGROUND: Scutellaria barbata D.Don has been applied to treat cancers, inflammation and urinary disease. However, its antitumor mechanism still remains unclear. METHODS: With methylene chloride fraction of Herba Scutellariae barbatae (MCSB), apoptosis-related experiments were carried out on human U937 leukemia cells by (a) 2,3-bis[2-4-nitro-5-sulphophenyl]2H-tetrazolium-5-carboxanilide (XTT) assay for cytotoxicity; (b) terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay for morphological changes; (c) cell cycle analysis; (d) Western blot analysis of poly(ADP-ribose) polymerase (PARP), caspase-8, caspase-9, caspase-3 and Bax, Bcl-2 and cytochrome c expressions for apoptosis signaling pathway. RESULTS: MCSB inhibited the proliferation of human U937 leukemia cells in a dose-dependent manner (IC50 = approximately 10 microg/ml). MCSB dose-dependently increased the sub-G1 DNA contents by cell cycle analysis. DNA fragments indicating induction of apoptosis were observed in MCSB-treated U937 cells by TUNEL assay. Caspase-9 and caspase-3 were activated while caspase-8 was intact by MCSB. Similarly, MCSB effectively cleaved PARP, increased the ratio of Bax/Bcl-2 and released the cytochrome c from mitochondria during apoptosis in U937 cells. CONCLUSIONS: Our results suggest that MCSB can induce apoptosis via the mitochondria-mediated signaling pathway.