In order to evaluate the effect of mitofusin-2 gene (mfn2) on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-V/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively (P<0.05). The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin (CAMP) followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively (P<0.05). It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.
Antineoplastic Agents, Phytogenic/pharmacology ; Apoptosis ; Camptothecin/pharmacology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Drug Screening Assays, Antitumor ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Green Fluorescent Proteins/metabolism ; Membrane Proteins/*biosynthesis ; Membrane Proteins/*genetics ; Mitochondrial Proteins/*biosynthesis ; Mitochondrial Proteins/*genetics ; Transfection

In order to evaluate the effect of mitofusin-2 gene (mfn2) on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-V/PI double labeling method was em- ployed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 eDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the per- centage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively (P<0.05). The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin (CAMP) followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively (P<0.05). It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and pro- mote their sensitivity to CAMP with a synergic effect.

The protective effects of in vitro cultivated calculus bovis (ICCB) on the cerebral and myocardial cells in hypoxic mice and the mechanism were examined. In one group, mice were intra-gastrically (i.g.) given ICCB for 15 days and then they were subjected to acute cerebral ischemia by decapitation, and then the panting time was recorded. In the other group, 12 min after exposure to hypoxia, mice was administered the ICCB i.g. for 5 days, and then the blood serum and tissues of brain,heart, liver were harvested and examined for SOD, GSH-px and T-AOC activity and content of MDA. The tissues of brain and heart were observed electron-microscopically for ultrastructural changes. The corpus striatum and hippocampus of brain were collected and examined for content of dopamine (DA) and norepinephrine (NE). The ultrastrural examination showed that the pathological change in brain and heart in the ICCB group was very slight, while abnormal changes in the control group were obviously more serious. ICCB significantly prolonged the panting time of the hypoxic mice (P<0.001), increased the activity of SOD, GSH-px, T-AOC in serum and tissues of brain, liver,heart and elevated the content of DA and NE. ICCB also pronouncedly reduced content of MDA in serum and tissues of brain, heart and liver. Significant differences in these parameters were noted between ICCB group and controls. It is concluded that ICCB can exert protective effect on the cells of brain and myocardium by enhancing the tolerance of the tissues to hypoxia and the body's ability to remove free radicals and regulating the neurotransmitters.

The effects of hepatic ischemia/reperfusion (1/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on store-operated calcium current (Isoc) in isolated rat hepatocytes after hepatic ischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 micromol/L,) produced a concentration-dependent decrease of Isoc with IC50 value of 64. 63 +/- 10.56 micromol/L, (n = 8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.
Boron Compounds/*pharmacology ; Calcium Channel Blockers/*pharmacology ; Calcium Channels/drug effects ; Cell Separation ; Hepatocytes/metabolism ; Liver/*blood supply ; Liver/metabolism ; Rats, Sprague-Dawley ; Reperfusion Injury/*metabolism

The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on storeoperated calcium current (Isoc) in isolated rat hepatocytes after hepaticischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC50 value of 64.63±10.56 μmol/L (n= 8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.

Antineoplastic Agents ; pharmacology ; Bile ; physiology ; Bile Duct Neoplasms ; metabolism ; pathology ; Bile Ducts, Intrahepatic ; Celecoxib ; Cell Division ; drug effects ; Cell Line, Tumor ; Cholangiocarcinoma ; metabolism ; pathology ; Cyclooxygenase 2 ; Dinoprostone ; metabolism ; Humans ; Isoenzymes ; biosynthesis ; genetics ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; Pyrazoles ; RNA, Messenger ; genetics ; Sphincterotomy, Transduodenal ; adverse effects ; Sulfonamides ; pharmacology ; Up-Regulation

To investigate the expression of KLF6mRNA in primary hepatocellular carcinoma (HCC), nomal liver tissues and the tissues adjacent to the cancers, reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of the KLF6 gene in HCC, the corresponding adjacent non-cancerous tissues and normal liver tissue. Our results showed that an amplified fragment of 427 bp DNA was detected in 18 of 19 (94.7%) adjacent non-cancerous tissues and normal liver tissue, and in 12 (85.7%) of 14 HCC. There were no significant differences in the levels of KLF6 mRNA between normal liver and liver tumors (P>0.05). It is concluded that KLF6 mRNA is generally expressed in HCC.
Carcinoma, Hepatocellular ; metabolism ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; biosynthesis ; genetics ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

To investigate the effects of dendritic cells (DCs) transfected with full length wild type p53 and modified by gastric cancer lysates on immune response, the wild type P53 was transducted to DCs with adenovirus, and the DCs were modified by gastric cancer lysates (Lywt-P53DC). The concentration of the surface molecules (B7-1, B7-2, MHC-I , MHC-II) of all DCs was determined by FACS, and the ability of the DCs to induce efficient and specific immunological response in anti-51Cr-labeled target cells studied. BALB/c mice model infected with DCs and Mk28 was established. CTL response in mice immunized with Lywt-p53DC and the effectiveness of Lywt-p53DC in the treatment of tumor-bearing mice was assayed. FACS revealed that the surface molecules of Ly-wt-P53 DC had a high expression: for B7-1 86.70% +/- 0.07%, B7-2 18.77% +/- 0.08%, MHC-I 87.20% +/- 0.05%, MHC-II 56.70%+/-0.07%; The T lymphocytes had a specific CTL lysing ability induced by Lywt-P53DC with the CTL lysis rate being 81%. The immune protective effect of Lywt-p53DC group was more obvious than any other groups (P<0.05). The tumor diameter in Lywt-p53DC group was 3.10+/-0.31 mm, 2.73+/-0.23 mm, 3.70+/-0.07 mm on the day 13, 16 and 19, smaller than DC, wtp53DC and LyDC groups (P<0.05). On the other hand, the growth rate of tumor in Lywt-p53DC group was slower than any other groups (P<0.05). It was suggested that DCs transfected with wild type P53 and modified by gastric cancer lysates had specific CTL killing capability.
Adenoviridae ; genetics ; metabolism ; Animals ; Cell Line, Tumor ; Dendritic Cells ; immunology ; metabolism ; Female ; Genetic Vectors ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

To investigate the expression of KLF6mRNA in primary hepatocellular carcinoma (HCC), nomal liver tissues and the tissues adjacent to the cancers, reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of the KLF6 gene in HCC, the corresponding adjacent non-cancerous tissues and normal liver tissue. Our results showed that an amplified fragment of 427 bp DNA was detected in 18 of 19 (94.7%) adjacent non-cancerous tissues and normal liver tissue, and in 12 (85.7%) of 14 HCC. There were no significant differences in the levels of KLF6 mRNA between normal liver and liver tumors (P>0.05). It is concluded that KLF6 mRNA is generally expressed in HCC.
Carcinoma, Hepatocellular/*metabolism ; Kruppel-Like Transcription Factors/*biosynthesis ; Kruppel-Like Transcription Factors/genetics ; Liver/metabolism ; Liver Neoplasms/*metabolism ; Proto-Oncogene Proteins/*biosynthesis ; Proto-Oncogene Proteins/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction

To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cells in vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINE 2000 (LF2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by Western-blot, the growth activity was measured by MTT, and apoptosis was detected by Flow Cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.
Antigens, Neoplasm ; biosynthesis ; genetics ; Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; Fluorouracil ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; metabolism ; pathology ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; Plasmids ; RNA, Messenger ; biosynthesis ; genetics ; Transfection