MDRA-6 And Thermotherapy To Liver Cancer SMMC-7721 Cell Synergism Research

1 BackgroundHyperthermia of tumor is an effective and brand-new therapy following the surgery, radiotherapy and chemotherapy. Nevertheless, the mechanism is still elusive. Recently, it has been a research hot point owing to the continuous renovation of equipments and the rapid progress of technology for hyperthermia. Hyperthermia is one of the powerful methods for combined therapy of tumor as well as surgery, chemotherapy and biological therapy, approved by U S Food and Drug Administration (FDA) in 1985. The Chinese Ministry of Public Health issued "Standard Regulations of Technology on the Management of Hyperthermia for Deeper and Whole Body Tumor" in Nov.2009. mDRA-6 is a functional monoclonal antibody against death receptor 5(DR5), developed by inoculating the DR5 protein into BALB/c mouse in the Laboratory of Cellular and Molecular Immunology, Henan University. Fewer reports were on the relationship of apoptosis with DR5 after tumor hyperthermia in recent years. Much attention was paid to the p53, HSP-70 and c-Myc, et al. The combined therapy of tumor based on the apoptosis induced by hyperthermia was primarily concentrated on hyperthermia along with chemotherapy and radiotherapy. There was no report about the synergistic effect of hyperthermia with mDRA-6, or hyperthermia with mDRA-6 and radiotherapy on apoptosis in tumor cells. The current study will investigate the synergistic effect of mDRA-6 and hyperthermia or mDRA-6, radiotherapy and hyperthermia on tumor cells. We also addressed the mechanism of apoptosis induced by hyperthermia, the expression of DR5 on tumor cell surface and the effect on Ca2+ level in tumor cells after hyperthermia.2 ObjectiveTo investigate the effect of hyperthermia on the heptocarcinoma cell line SMMC-7721, the possible mechanism of apoptosis induced by hyperthermia and the synergistic effect of mDRA-6 and hyperthermia on apoptosis.3 MethodsHeptocarcinoma cell line SMMC-7721 was first heated in water bath at 42.5±0.5℃for lh and then was grown in 5% CO2 incubator with at 37℃. The morphological change was observed under inverted microscope(X400) at 0,6,12 and 24h after hyperthermia. MTT method was used to study the cellular proliferation. Annexin V/PI staining was performed for the detection of apoptosis with flow cytometry. The DR5 expression was measured with flow cytometry after treatment of the cells with antibody against DR5(366EC), and goat anti-mouse IgG-FITC. The intracellular Ca2+ concentration was also analysed by flow cytometry after loaded with Fluo-3. The MTT method was employed to test the inhibition of hepatocarcinoma cell line SMMC-7721 caused by DR5 antibody alone or combined with cisplatin or hyperthermia. The effect of varied drugs on the apoptotic rate of SMMC-7721 was also checked with flow cytometry.4 ResultsHyperthermia could lead to morphological alteration of cells, such as smaller size, rounder shape, exfoliation and vacuole formation. It also inhibited the cellular proliferation with inhibition rates 22.18%,26.76%,31.30% and 36.62% at 0h,6h,12h and 24h respectively after one hour's hyperthermia. The cellular rate of apoptosis was calculated by double staining of Annexin V/PI after one hour's hyperthermia. It was 15% at 0h(including earlier rate 2% and later rate 13%),65.45% at 6h(earlier 42.23% and later 22.22%),12.32% at 12h(earlier 4.6% and later 7.72%), and 22.58% at 24h(earlier 7.15% and 15.43%) respectively. The intracellular Ca2+ concentration (MFI) was 18.13±0.20,13.44±0.84,7.87±3.35,13.01±2.38,37.24±1.31 at 0,4, 8,12 and 24h respectively after one hour's hyperthermia with 14.28±3.65 as control. The expression rate of DR5 on cell surface was 79.74,83.22,91.28 and 92.52% at 0,6, 12 and 24h respectively after one hour's hyperthermia with 83.02% as control.The inhibition rate of proliferation without hyperthermia is 32% for cisplatin,22% for m-DRA-6 and 37% for cisplatin and m-DRA-6 combined vs 58.06% for cisplatin, 14.52% for m-DRA-6 and 91.13% for cisplatin and m-DRA-6 combined respectively in group with hyperthermia. The apoptotic rate is 13.53% 32% for cisplatin,13.66% for m-DRA-6 and 84.24% for cisplatin and m-DRA-6 respectively in group without hyperthermia vs 9.41% for hyperthermia only group,30.27% for cisplatin,57.52% for m-DRA-6 and 91.33% for cisplatin and m-DRA-6 combined respectively with hyperthermia. 5. ConclusionHyperthermia could increase the expression of DR5 on hepatocarcinoma cell line SMMC-7721. The apoptosis of SMMC-7721 was related to the higher expression of DR5 and intracellular Ca2+. The increase of intracellular Ca2+ was probably the key factor for the later apoptosis of hepatocarcinoma cells. Monoclonal antibody against DR5, m-DRA-6, could strengthen the anti-tumor functions of drugs in chemotherapy. The antibody was synergistic with drugs of chemotherapy. Hyperthermia could not only raised the apoptosis of carcinoma cells, but also stratified the anti-tumor role of anti-DR5 antibody and chemotherapical drugs.