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Inhibition of beclin1 affects the chemotherapeutic sensitivity of osteosarcoma.

ABSTRACT: This study was conducted to explore the role of autophagy in cisplatin-resistant osteosarcoma. Cisplatin-resistant osteosarcoma cell line (MG63/DDP) was obtained from parental MG63 by treating cisplatin with an intermittent stepwise selection protocol. The autophagy in MG63/DDP and MG63 was fully analyzed by immunofluorescence and western blot analysis. Meanwhile, the autophagy and the sensitivity to cisplatin for MG63/DDP and MG63 after inhibition of beclin1 were analyzed in vitro and in vivo. Increased autophagy was observed in cisplatin resistant MG63/DDP cells and in the cisplatin-treated MG63 and MG63/DDP cells. Meanwhile, inhibition the beclin1 significantly inhibited the formation of autophagosome and resulted in the increase in the sensitivity to cisplatin for both MG63 and MG63/DDP cells in vitro and in vivo. In conclusion, autophagy is implicated in the cisplatin resistant osteosarcoma, and inhibition of beclin1 could be a target for improving osteosarcoma therapy.

SUBMITTER: Wu W

PROVIDER: S-EPMC4230152 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Inhibition of beclin1 affects the chemotherapeutic sensitivity of osteosarcoma.

Wu Wenxin W Li Wei W Zhou Yong Y Zhang Chaoyue C

International journal of clinical and experimental pathology 20140915 10

This study was conducted to explore the role of autophagy in cisplatin-resistant osteosarcoma. Cisplatin-resistant osteosarcoma cell line (MG63/DDP) was obtained from parental MG63 by treating cisplatin with an intermittent stepwise selection protocol. The autophagy in MG63/DDP and MG63 was fully analyzed by immunofluorescence and western blot analysis. Meanwhile, the autophagy and the sensitivity to cisplatin for MG63/DDP and MG63 after inhibition of beclin1 were analyzed in vitro and in vivo. ...[more]

PMID: 25400807

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Project description:Heme oxygenase-1 (HO-1) and its major product carbon monoxide (CO) are known to be involved in the development and progression of many tumors. The present study was to elucidate the expression and function of HO-1 in colorectal cancer (CRC), specially focusing on the circulation CO levels in CRC patients and the possible roles of HO-1 in chemoresistance of colon cancer cells.One hundred and eighteen patients received resection for colorectal cancer and polyps at China Medical University Sheng Jing Hospital, were collected in this study. HO-1 expression in CRC tissues was analyzed by immnuohistochemical staining; circulation CO levels as carboxyhemoglobin (COHb) in CRC patients were analyzed by an ABL800 FLEX blood gas analyzer. HO-1 expression in murine colon cells C26 and human colon cancer cells HT29 and DLD1 under HO-1 inducer hemin and anticancer drug pirarubicin (THP) treatment was examined by RT-PCR, and the cell viability after each treatment was investigated by MTT assay. Data were analyzed by student's t-test or one-way ANOVA followed by Bonferroni t-test or Fisher's exact test.HO-1 expression in tumor tissues of CRC (61.0%) was significantly higher than in normal colorectal tissues and polyps tissues (29.7%, P < 0.01); well-differentiated CRC seemed to express more HO-1 (81.5%) than moderately/poorly-differentiated cancers (59.5%, P < 0.05). However, the nuclear HO-1 expression is apparently higher in moderately/poorly differentiated CRC than well-differentiated CRC probably suggesting a new mechanism of function involved in HO-1 in cancer. In parallel with HO-1 expression, circulation CO levels in CRC patients also significantly accelerated. Moreover, HO-1 expression/induction also related to the chemosensitivity of colon cells; HO inhibitor zinc protoporphyrin significantly increased cytotoxicities of THP (i.e., 2.6 - 5.3 folds compared to cells without zinc protoporphyrin treatment).These findings strongly suggested HO-1/COHb is a useful diagnostic and prognostic indicator for CRC, and inhibition of HO-1 may be a option to enhance the chemotherapeutic effects of conventional anticancer drugs toward CRC.

Project description:Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of -8.4 and -6.4?Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10?mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

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Inhibition of beclin1 affects the chemotherapeutic sensitivity of osteosarcoma.

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Inhibition of beclin1 affects the chemotherapeutic sensitivity of osteosarcoma.

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