Preliminary Mechanism Study On Different Sensitivity Between Human Liposarcoma SW872and SW872-S Cell Lines Induced By Bortezomib

According to previous study, tumor formation of human liposarcoma cell line SW872in nude mice can not occur in two months [1]. And it happened in SCID mice minimally. As a result, to find a gradual malignant liposarcoma model in vivo is required. Our laboratory has firstly established a highly malignant sub-cell line SW872-S which has single morphology and stable multiplication from human liposarcoma cell line SW872by re-transplantation in nude mice [2].In the first chapter, we compared human liposarcoma cell line SW872with its sub-cell line SW872-S, and found that bortezomib induced much more severe mitochondria-dependent apoptosis in SW872-S than in SW872. It is not P38MAPK phosphorylation but JNK MAPK phosphorylation occurring in SW872and SW872-S incubated with bortezomib. Phosphorylated JNK MAPK separated its downstream Bax-14-4-3complex into Bax and14-4-3and the free Bax then translocated from endochylema to mitochondria and consequently leaded to the release of cytochrome c, triggered mitochondria-dependent apoptosis in the human liposarcoma cell line SW872and the sub-cell line SW872-S. In the investigation, we found that cycloheximide (CHX) almost completely blocked the apoptosis effect induced by bortezomib. The detected result of proteasome activity indicated there is no obvious discrimination of proteasome chymolase activity between SW872and SW872-S. As a hypothesis, we supposed the potential reason for SW872-S proceeding much more severe apoptosis than SW872incubated with the same concentration of bortezomib may be SW872-S owned much more vigorous genetic transcription and protein expression system than SW872, and in the condition of the same inhibited capacity of degradation, SW872-S incubated with bortezomib accumulated more ubiquited protein which can not be degradated in time. As a result, gravis accumulation of endocellular stress in SW872-S leaded to violent apoptosis. And our hypothesis was proved correct by detecting the key protein of translation regulation pathway.In the second chapter, we investigated the role and potential mechanism of P-glycoprotein played in the tolerance of bortezomib in SW872and SW872-S cells. We found that incubated with high concentration of bortezomib, the MDR-1(P-glycoprotein, P-gp) protein in SW872and SW872-S cells were both down-regulated; While incubated with lower concentration of bortezomib, MDR-1expression was slightly up-regulated in SW872contrary to down-regulation of MDR-1in SW872-S, indicating that bortezomib may dose-dependently up-regulate the expression of MDR-1in SW872cell under a certain concentration yet it down-regulated MDR-1expression, promoted apoptosis consequently, in the dose-dependent manner as soon as the bortezomib concentration surpassed the certain range. Bortezomib has been reported to trigger apoptosis in cancer cells via down-regulation of phosphor-Akt or phosphor-IκBα. However, there was no obvious variation of PI3K/AKT signal pathway investigated in SW872-S cell incubated with bortezomib, instead, bortezomib triggered the activation of NF-κB signal pathway. JNK inhibitor SP600125could partially reverse the down-regulation of MDR-1protein and the activation of NF-κB signal pathway induced by bortezomib in the SW872-S cell, as a consequence, the pro-apoptosis activity of bortezomib had been reversed. The analogous phenomenon had been observed in human colon cancer cell lines SW480and SW620. Furthermore, we indicated bortezomib triggered the NF-κB signal pathway in SW620cell line by down-regulating the expression of IkBα protein in the dose-dependent manner. In the third chapter, antitumor activity of bortezomib in vivo was evaluated. SW872and SW872-S cell line were inoculated into armpit of nude mice. Results showed that, compared with PBS groups, bortezomib groups significantly inhibit cell growth both in SW872and SW872-S xenografts. By HE staining and Tunnel straining, we found that high-level tumor necrosis areas in xenografts of SW872and SW872-S treated by bortezomib groups were higher than the PBS control groups, and there were obvious apoptosis in tumor tissues. Western blot results confirmed that bortezomib induced apoptosis and the activation of NF-κB signal pathway in xenografts of SW872and SW872-S.In conclusion, this study has made a series of comparison between human liposarcoma cell line SW872and its sub-cell line SW872-S induced by bortezomib, and found that bortezomib induces much more obvious mitochondria-dependent apoptosis. SW872-S cell possesses more vigorous protein synthesis system compared to SW872cell. Bortezomib particularly improved the intracellular ROS level of SW872-S via inhibition of protease activity, promoting the activation of JNK MAPK signal pathway, leading to mitochondria-dependent apoptosis ultimately. Meanwhile, activated JNK could phosphorylates IκBα, down-regulated IκBα protein expression, and subsequently separate NF-κB from IκBα to trigger the activation of NF-κB signal pathway.