Association Between Regulation Of Cell Death And Chemosensitivity

Anticancer drug induce tumor cell death. Cell death has been subdivided into the distinct processes of apoptosis, autophage and necrosis according to cellular morphology. An increasing number of research papers shows that the disorder of apoptosis contributes to play essential role in tumorgenesis, development of malignancy and chemosensitivity. However, the regulation mechanisms of cell death which determine the chemosensitivity of esophageal squamous cell carcinoma still remain unknown and need to be further elucidated.In the first part of the dissertation, we show that Smac, a mitochondrial apoptogenic protein, was significantly down-regulated in esophageal cancer (36.8%,25/68, P=0.001), and the Smac's expression was significantly different (P<0.05) between chemosensitive (with CR or PR responses) and chemoresistant (with SD or PD) tumors among malignant esophageal cancer patients. And consequentially the c-IAP1, an apoptotic inhibitor, was up-regulated in cytoplasm of esophageal cancer tissue (46%, 24/52). According to the information obtained from esphageal cancer tissue, two cell lines, which are high expression of c-IAP1 and Smac, were knocked down of Smac with RNA interference. Smac knock-down significantly increased the chemoresistance of esophageal cancer cells response to anticancer drug. The molecular mechanistic studies revealed that the knockdown of Smac abrogated cisplatin-induced apoptosis, mitochondrial dysfunction, cytochrome C release, caspase and apoptotic intrinsic pathway activation. Smac deficiency also reduced the effect of cellular chemosensitivity, and led to increase cisplatin resistance in vitro and vivo experiments. Meanwhile, in the cells with high expression of c-IAP1, Smac mediated degradation of c-IAP1 and contributed to apoptosis after CDDP treatment. Furthermore, LBW242, a small-molecule Smac mimetic, enhanced cisplatin-induced apoptosis and caspase activation, and restored cisplatin sensitivity in Smac-deficient cells. Those findings defined that low Smac expression is associated with resistance to cisplatin treatment.In the second part of the dissertation, we show that RIP3, a molecular switch between apoptosis and necrosis, was significantly up-regulated in esophageal cancer (67.9%,36/53, P=0.001). In KYSE140 esophageal cancer cell line which blocks apoptotic pathway, CDDP can trigger necrosis mediated by RIP3, lead to maintain the chemosensitivity. The KYSE140, which expresses high levels of RIP3, was knocked down of RIP3 with RNA interference. So the RIP3 knock-down significantly increased the chemoresistance of esophageal cancer cells response to anticancer drug. The molecular mechanistic studies revealed that CDDP induced degradation of caspase-9, blocked apoptosis intrinsic pathway, and increased the expression of RIP3. The RIP3 can regulate cellular metabolism via interacting with ENO1 which is a key enzyme of glucose metabolic pathway, and increase reactive oxygen species (ROS) production for RIP3's ability to contribute to necrosis. At the same time, we found a novel link that RIP3 could locate in the nucleus of KYSE140 and accumulate after CDDP treatment. Those findings define that RIP3-mediated regulation of necrosis is associated with chemosensitivity to cisplatin treatment.In the third part of the dissertation, we show that quantum dots (QDs) can specifically label fluorescent imaging of esophageal cancer cell stained by the QDs conjugated with antibody AIF. Based on the principle of fluorescence quenching, we found that QDs coupled with antibody revealed higher photostability of fluorescent light than traditional organic fluorescent dye. The free QDs with 5 nm diameter, can move rapidly from cytoplasm into nuclei in the presence of DAPI after UV excitation. Once in the nuclei, they are trapped and show bright green emission with high photostability.In summary, the results have revealed that apotosis associated genes including Smac and c-IAP1, and necrosis associated gene RIP3 are involved in regulating chemosensitivity of esophageal cancer cells. Quantum dots, a kind of nano fluoresent materials, can be effectively used in cellular label.