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Role of PKC-δ in Induction of Apoptosis by TRAIL and Adriamycin in Osteosarcoma Cell Line MG-63

Author: LiGuangZhang
Tutor: TangXin
School: Dalian Medical University
Course: Surgery
Keywords: TRAIL Adriamycin PKC-δ Osteosarcoma Apoptosis
CLC: R738
Type: Master's thesis
Year: 2010
Downloads: 37
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Abstract

Objective:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of tumor necrosis factor(TNF)family. TRAIL could specifically induce apoptosis in various tumor cells, and is nontoxic to normal tissues and cells. Because not all tumor cells are sensitive to TRAIL, more and more researches are focusing on the method of combination application of TRAIL and radiation or chemotherapeutic agents recently. Recent studies have shown that synergistic induction of apoptosis by the combination of TRAIL and low dose adriamycin (ADM) in human osteosarcoma cell line in vitro. Protein kinase C-δ(protein kinase C-δ, PKC-δ) is a member of PKC isoforms, which play an important role in cell cycle regulation and apoptosis in many types of cells. PKC-δis activated when the start in DNA damage factor, tumor necrosis factor, anti-Fas antibodies induced apoptosis. To observe synergistic effect of TRAIL and low dose ADM’s effect on apoptosis of osteosarcoma cell line MG-63 in vitro. To investigate the role of PKC-8 in induction of apoptosis by TRAIL and ADM in osteosarcoma cell line MG-63 and the role of PKC-8 in this process by addition of Rottlerin, a kind of PKC inhibitor and PMA, a kind of PKC-8 specific activator.Method:The osteosarcoma cell line MG-63 were cultured in 37℃,5%CO2 incubation box of conventional training with 10% calf serum DMEM (containing non-essential amino acids) medium. The morphologic changes and characters of apoptosis were observed in the TRAIL group、the control group、ADM group and TRAIL+ADM group by inverted phase contrast microscope. The mRNA expression of PKC-8 was examined in the TRAIL group、the control group、ADM group and TRAIL+ADM group by RT-PCR. Flow Cytometry (FCM) analyzed the MG-63 apoptosis rate of Rottlerin+TRAIL+ADM group and TRAIL+ADM group, and the MG-63 apoptosis rate of PMA+TRAIL+ADM group and TRAIL+ADM group. Statistical treatment:the experimental data was analyzed by t test. Result:Under inverted phase contrast microscope, comparing with administration of TRAⅡalone、ADM alone and the control group, some cells became more rounder and began to float after 24 hours’treating with 500ng/ml TRAIL and 5μg/ml ADM, cavitation phenomenon became clear, cellular fragments of varying sizes can be seen in the cytoplasm. The result of RT-PCR showed that, comparing with administration of TRAII alone、ADM alone and the control group, the level of PKC-δ’mRNA expression in MG-63 cells in TRAIL+ADM group increased obviously, and the difference was statistically significant (P<0.01). The result of FCM showed that the MG-63 apoptosis rate was 11.88%±0.30% after 12 hours’treating with 500ng/ml TRAIL and 5μg/ml ADM, rottlerin could decrease apoptosis rate to 3.33%±0.31% in TRAIL and low dose ADM’s effect on apoptosis of osteosarcoma cell line MG-63 in vitro, and PMA could increase the apoptosis rate to 18.85%±0.41%, the difference was statistica-lly significant (P<0.01).Conclusion:TRAIL and low dose ADM can effectively induce apoptosis of osteosarcoma cell line MG-63. Rottlerin may inhibit the apoptosis in Induction of apoptosis by TRAIL and low dose ADM in osteosarcoma cell line MG-63, PMA may increase the apoptosis. PKC-8 may participate in the process in induction of apoptosis by TRAIL and low dose ADM in osteosarcoma cell line MG-63 and play a promoting role.

Role of PKC-δ in Induction of Apoptosis by TRAIL and Adriamycin in Osteosarcoma Cell Line MG-63

Abstract

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