Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.     

S100A6 gene have a positive influence on the growth and proliferation of gastric cancer cell MKN45

Objective： S100A6 （a.k.a., calcyclin） is over-expressed in several human tumors, including gastric carcinoma, human melanoma, pancreatic carcinoma, squamous cell carcinoma, malignant fibrous histiocytoma （MFH）, and carcinomas of the thyroid, breast, and colon. However, little is known about the role S100A6 plays in gastric adenocarcinoma. In the present study, we intended to investigate the influence of S100A6 on the growth, proliferation, apoptosis, invasion and cell cycle of the gastric cancer cell MKN45. Methods： As an important member of S100 family, S100A6 cDNAwas subcloned into a constitutive vector pcDNA3.1 followed by transfection in gastric cancer cell line MKN45 by using liposome. Then stable transfectants were selected and appraised. The apoptosis and cell cycles of these clones were analyzed by using flow cytometric assay. The growth and proliferation were analyzed by cell growth curves and colony-forming assay respectively. The S100A6 stable expression clones （MKN-S100A6） were detected and compared with their control groups respectively. Results： MKN- S100A6 grew faster than MKN45 and MKN-PC （MKN45 transfected with pcDNA3.1 vector）. The cell counts of MKN-SI00A6 in the fifth, sixth and seventh days were significantly more than those of control groups （P 〈 0.05）. Cell cycle analysis showed that proportions of MKN-S100A6 in G0-G1 and G2-M were different significantly with those of its control groups respectively （P 〈 0.05）. The apoptosis rate of MKN-S100A6 was significantly lower than those of control groups （P 〈 0.05）. Results of colony-forming assay showed that the colon formation rate of MKN-S100A6 was higher than those of control groups （P 〈 0.05）. Conclusion： S100A6 can promote the growth and proliferation of gastric cancer cells. It can help tumor cell maintain malignant phenotype. In gastric cancer, S100A6 could be thought as a tumor-enhancing gene in some distance, but its role could be complicated.     

Effects of monoclonal antibodies against human stathmin I combined paclitaxel on proliferation of human hepatocellular carcinoma cell lines

Objective： We investigated the effects of monoclonal antibodies against stathmin 1 combined paclitaxel on the proliferation of HCC cells. Methods： HepG2 cells were treated with monoclonal antibodies against stathmin 1, paclitaxel alone or their combination, with the untreated cells used as the control, 24, 48, 72, 96 h later, the cell growth condition was observed by invert microscope and inhabitation rate was studied by MTT assay; The apoptosis was analyzed by flow cytometry with Annexin V/PI. Results： The population decreased and shape, size changed after treating with different concentration of experimental groups. Monoclonal antibodies against stathmin 1 and paclitaxel used alone or in combination both inhibited the proliferation of HepG2 cells, the inhibition ratio of their combination was more higher （P 〈 0.05）, and a synergistic effect of the two agents was noted in their combined action （P 〈 0.05）. Combined treatment of the cells resulted in significantly higher apoptosis rate than that in the other groups （P 〈 0.05）. Conclusion： Monoclonal antibodies against stathmin 1 and paclitaxel used alone or in combination both can inhibit proliferation of HepG2 cells and induce apoptosis. A synergistic effect is obsewed between the monoclonal antibodies against stathmin 1 and paclitaxel in their inhibition of HepG2 cell proliferation.     

The effects of acetaminophen combined with radiation on the radiosensitivity of irradiated human glioma cell progeny

Objective： To study the effects of acetaminophen （ACE） combined with radiation on the progeny of the human glioma cell line SHG-44, and to investigate if ACE may be an useful therapeutic radiosensitivity agent in the treatment of recurrent human glioma. Methods： A randomized, controlled experiment, was performed at the Department of Radiology Laboratory, the First Hospital Affiliated to Soochow University, between September 2004 and January 2006. Brain glioma SHG-44 cells were divided into three groups： SHG-44, SHG-44-10, and SHG-44-10 ＋ ACE cells groups. The SHG-44-10 cells group was irradiated with dose of 10 Gy by a linear accelerator （6 MVX）. It was passaged for 15 generations and cultured in RPMI-1640 culture media. Then SHG-44-10 ＋ ACE cells group was treated with ACE. Measures： Community re-double time, mean lethal dose （DO）, extrapolation number （N）, fraction surviving fraction irradiated by 2 Gy dose （SF2）, quasi-threshold dose （Dq）, and cell cycle. Results： The SF2 of the SHG-44, SHG-44-10, and SHG-44-10 ＋ ACE cells groups were 70.8%, 80.6% and 45.2%, respectively, with significance （P = 0.040）. The SHG-44-10 and SHG-44-10 ＋ ACE cells groups were irradiated with 8 Gy. After 12 hours, the G2/M ratio of the SHG-44-10 and SHG-44-10 ＋ ACE cells groups were indicating significantly higher ratio compared to pre-irradiated groups （P 〈 0.01）. After 24 hours, the G2/M ratio of the SHG-44-10 cells group decreased rapidly, while the ratio of the SHG-44-10 ＋ ACE cells group still maintained in high level. Conclusion： In the present study, Subtoxic dose of ACE increased the radiosensitivity of the progeny of irradiated human glioma cell. ACE may be an useful radiosensitivity agent in the treatment of recrudescent human malignant glioma.     

The effects of interfering in COX-2 gene expression on malignant proliferation of the human lung adenocarcinoma A2 cell in vitro

Objective： We explored the interfering effect of COX-2 gene expression and the influence on the malignant proliferation of A2 cells by RNAi after quenching COX-2 in vitro. Methods： COX-2 was selected as the subject. Three COX-2 siRNA expression vectors with human U6 promoter were constructed and three vectors and the vacant vector （pEGFP） were transfected into A2 cells with lipofectamine respectively. The cell strains transfected were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A2 cells after silencing COX- 2 were studied by cell growth curve and clonogenic assay in vitro. Results： The three siRNA and U6 promoter cloned into pEGFP plasmid were validated by PCR, restriction endonucleases identification, DNA sequencing and BLAST alignment. The cell strains transfected were named as A2-3, A2-7, A2-10 and A2-p respectively. Green fluorescence was seen in A2-p cells and not in A2-3, A2-7 and A2-10 cells in 24, 48 and 72 h after transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors produced effects and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, the levels of COX-2 mRNA of A2-3, A2-7 and A2-10 cells reduced 15.6%, 20.4% and 64.2% respectively; the levels of COX-2 protein of A2-3, A2-7 and A2-10 cells reduced 23.7%, 36.7% and 60.2% respectively. The results of cell growth curve and clonogenic assay showed the growth of A2-10 cell slowed and the colonial formation rate reduced but the growth of A2-3 and A2-7 cells had not obvious changes in contrast to the controls （A2 and A2-p）. Conclusion： Silencing the COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2 cells.     

Influence of CDK1 and CDK2 siRNA interference on tumor cell cycle and cell apoptosis

Objective： We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods： The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results： CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotransfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate ceJls among cotransfected cells were observed under the microscope. Conclusion： The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.     

The effects of CIK cells on the treatment of renal cell carcinoma

Objective： To observe the effects of cytokine-induced killer （CIK） cells on the treatment of renal cell carcinoma. Methods： Twenty-eight postoperative cases with stage Ⅰ or stage Ⅱrenal cell carcinoma were admitted in our hospital from January 2002 to June 2006, all cases were pathologically confirmed, and were divided into group A （18 cases） and group B （10 cases）. Group A was administrated 3-12 periods of CIK cells treatment combined with 5-7 cycles of IL-2 and INFα-2b, together with 4 cycles of chemotherapy （5-Fu ＋ CF）. Group B was given 4 cycles of chemotherapy （5-Fu ＋ CF） and 5-7 cycles of IL-2 and INFa-2b. Results： Three cases in group A had metastatic masses in two lungs within 1 year and died within 2 years postoperatively. The other 15 cases are still alive and in good health. Six cases in group B had metastatic masses in two lungs or/and in abdominal cavity within 1 year, and 4 of them died within 2 years. All the 6 cases died within 3 years. The other 4 cases are still alive and in good health. Conclusion： CIK cells are safe and effective for the treatment of stage I or stage II renal cell carcinoma, which should be further widely used.     

Impact of resveratrol on the expression of apoptosis related gene survivin and bax in human cancer cells

Objective： We explored the mechanism of apoptosis in human esophageal cancer Ecal09 cells by resveratrol. Methods： The suppressive ratio of resveratrol on Ecal09 cells proliferation was evaluated by MTT colorimetric assay and morphology was observed by transmission electron microscope. The expression of survivin and bax was analyzed by RT-PCR and Flow Cytometry （FCM）. Results： Resveratrol inhibited the growth of Ecal09 calls in a dose-and time-dependent man- ner, and the suppressive ratio arrived at 76.42%. Morphological apoptosis could be observed after treated with resveratrol.The bulk of some drug-treated cells turned small and the nuclear chromatin became condensed and rnarginated. The results determined by RT-PCR and FCM showed that resveratrol could down-regulate surviving, while up-regulate bax. Conclusion： Resveratrol could induce the apoptosis of human esophageal cancer Ecal09 cells, and its possible molecular mechanisms might be related to modulation the expression of survivin and bax.     

Effect of IL-12 on the proliferation and cytotoxicity of CIK cells to gastric adenocarcinoma cell BGC-823

Objective： The aim of the study was to evaluate the effect of interleukin-12 （IL-12） on the proliferation and cytotoxicity of cytokine-induced killer （CIK） cells in vitro. Methods： Three different combinations of cytokines, IL-2, IL-12 ＋ IL-2, and IL-12, were used to proliferate CIK cells, adding IFN-γ, IL-1 and CD3 McAb in each one. Phenotype of the cells was analyzed by flow cytometry. The cellular proliferation and cytotoxic activity were determined by cytometry and MTT assay. Results： CIK cells generated by the three methods showed high reproductive activity, and no obviously difference in inducing CD3＋CD56＋ cells was found among the three groups, The group of IL-2 ＋ IL-12 evidently enhanced both the proliferation and the cytotoxicity of the CIK cells compared with the other two groups （P 〈 0.05）. Conclusion： IL-12 could be used to induce the CIK cells as well as IL-2. CIK cells induced by combining IL-12 with IL-2 had stronger proliferative ability and higher cytotoxicity to tumor cells in vitro, which could be used as a potential anti-tumor adoptive immunotherapy in clinic.