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Melatonin Sensitizes Human Hepatoma Cells to Endoplasmic Reticulum Stress-induced Apoptosis

Author: GaoJinSuo
Tutor: SunGuoPing
School: Anhui Medical University,
Course: Oncology
Keywords: Endoplasmic reticulum stress Melatonin HCC Tunicamycin Apoptosis
CLC: R735.7
Type: Master's thesis
Year: 2011
Downloads: 71
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Abstract


Background: HCC (hepatocellular carcinoma, HCC) is one of common malignant tumors in all human tumors account for about 6%, its incidence is rising worldwide. The overall survival of patients with hepatocellular carcinoma is very short, 5-year survival rate of less than 10%. Apoptosis resistance is a major cause of liver cancer treatment failure, hepatocellular carcinoma apoptosis resistance (Hepatocellular carcinoma, HCC) is one of the characteristics in the development of HCC development occupies an important position. Apoptosis resistance causes very complex. Endoplasmic reticulum stress (endoplasmic reticulum stress, ERS) can lead to apoptosis, but under the ERS is not obvious tumor cell apoptosis, suggesting that tumor cells may produce adapt and evade apoptosis ERS. Studies have found that the use of ERS inducer tunicamycin (Tunicamycin, TM) and brefeldin (Brefeldin A) can lead to breast cancer MCF-7 cells, COX-2 upregulation. The study found that cyclooxygenase -2 (cycloxygenase-2, COX-2) significantly inhibit apoptosis, has attracted wide attention. In this study, ERS inducer TM apoptosis of human hepatoma cell line HepG2 and human normal liver cells HL-7702 occurred ERS, observe the proliferation inhibition rate and apoptosis rate changes. After the detection of liver cancer cells ERS COX-2 expression, and to explore reactivity hepatoma cells resist apoptosis ERS possible mechanisms. COX-2 transcriptional activity, expression level and continue over time, mainly due to internal procedures and endogenous transcriptional factors, such as melatonin (MT) and interferon (interferon, INF) and other controls. Wherein, MT COX-2 as the target molecule of the control of transcription and expression, a specific COX-2 blockers. So we use block COX-2-specific inhibition of melatonin suppression of COX-2 expression in hepatoma cells was observed after ERS proliferation inhibition rate and apoptosis rate changes. For the treatment of HCC to find more ways. Objective: ERS-induced apoptosis of hepatoma cells and its mechanism, and explore melatonin in hepatoma cells resist ERS-induced cell apoptosis. Methods: MTT assay of drug action in the human hepatoma cell line HepG2 and human normal liver cell line HL-7702 inhibition of proliferation by flow cytometry with PI staining of cell cycle analysis of DNA content in apoptosis, TUNEL drug effects were observed cells were apoptotic morphology and the apoptosis rate differences, Wertern Blot Detection TM induced COX-2 protein expression and GRP 78. Results: 1. TM and TM added role of melatonin in human HepG2 cells and normal human cell line HL-7702 1.1 TM inhibited the proliferation of HepG2 cell proliferation by MTT assay, was observed on HepG2 cell proliferation TM inhibition. The results showed that different concentrations (1.5,3,6,9,12 umol / L) TM effect on HepG2 cells after the inhibition of cell proliferation is not obvious, 12umol / L TM after 48h growth inhibition rate reached 27.2%. 1.2 TM for HL-7702 cell proliferation by MTT assay, was observed for the HL-7702 TM cell proliferation inhibition. The results show different concentrations (1.5,3,6,9,12 umol / L) TM effect on HL-7702 cells was significantly inhibited cell proliferation. 1.5umol / L TM TM after 48h proliferation inhibition rate of 50.9%. 1.3 when the TM application melatonin inhibited the proliferation of HepG2 cells by MTT assay, TM was observed when melatonin inhibited the proliferation of HepG2 cells. The results showed that application of melatonin, HepG2 the TM-induced endoplasmic reticulum stress-induced apoptosis increased sensitivity, 12umol / L TM for 48h proliferation inhibition rate increased to 64.9%. 1.4 Applications of melatonin on the HL-7702 when the TM cell proliferation inhibition by MTT assay was observed when melatonin TM for HL-7702 cell proliferation inhibition. The results showed that application of melatonin and the TM group HL-7702 cells with a single group with TM proliferation inhibition rate was no significant difference. 2. TM and TM-induced joined ERS when melatonin on apoptosis of HepG2 and HL-7702 and adding effects 2.1 TM TM induced ERS when melatonin on apoptosis of HepG2 and HL-7702 2.1.1 Influence TUNEL assay TM and TM when adding melatonin-induced apoptosis of HepG2 ERS impact TUNEL assay using terminal dideoxynucleotide transferase-mediated dUTP nick labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, TUNEL) observed HepG2 and HL-7702 cell apoptosis morphological changes. Control cells and found that melatonin alone exuberant cell proliferation, apoptosis only see a little; alone TM after 48h, slight improvement in cell density decreased, showing little apoptosis; adding melatonin after 48h when TM , showing that the cell density was significantly reduced number of apoptotic cells increased significantly. 2.1.2 TM and TM when adding melatonin-induced apoptosis in HL-7702 ERS impact on the TUNEL assay findings alone control cells and cell proliferative melatonin group, only to see a few apoptotic cells; solely with TM cell group and adding melatonin and TM cell group after 48h, cell density were seen significantly reduced number of apoptotic cells increased significantly. 2.2 TM and TM-induced joined ERS when melatonin on apoptosis of HepG2 and HL-7702 affected by FCM 2.2.1 TM and TM when adding melatonin-induced apoptosis of HepG2 ERS FCM detection applications of flow cytometry instrument (Flow Cytometry, FCM) to detect cell apoptosis. In the DNA profiles G 0 / G 1 period before the emergence of a representative of apoptosis Asia G 1 peak that apoptotic peak. FCM results showed that the normal control group showed low apoptotic peak, alone and single-use melatonin TM cell group after 48h, no significant change in its apoptotic peak. When adding melatonin, the induction of the TM after 48h, the apoptotic peak increases. 2.2.2 TM and TM-induced joined ERS when melatonin on apoptosis of HL-7702 FCM FCM analysis showed that the normal control group showed low apoptotic peak melatonin alone cell group after 48h, no significant change in its apoptotic peak. When the TM and TM-induced melatonin added after 48h, the apoptotic peak increases. 3 ERS-induced liver cancer cells to resist the possible molecular mechanisms of apoptosis using Wertern Blot Detection TM induced COX-2 protein expression and GRP 78. The results show that, ERS inducer TM can induce increased expression of GRP78, ERS can induce the production of COX-2 expression increased. ERS melatonin suppression hepatoma cells induced COX-2 expression. Conclusion Melatonin improve hepatoma cell endoplasmic reticulum stress-induced apoptosis pathway sensitivity

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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