[Mechanism Research of Gambogenic Acid on Proliferation,Apoptosis and Invasion of Cervical Carcinoma He La Cells].

Objective: To investigate the effects of gambogenic acid on proliferation,apoptosis and invasion of human cervical carcinoma HeLa cells. Methods: HeLa cells were given different concentrations of gambogenic acid( 0. 00,0. 63,1. 25,2. 50,5. 00 and 10. 00 mg / L) for 72 h,MTT assay was used to measure the cells proliferation. Flow cytometry was used to measure cell cycle. Fluorescence microscopy and DNA ladder analysis were used to measure the apoptosis of HeLa cells. Transwell chambers was adopted to detect the cells invasion. The expression levels of mRNA and protein of BCL-2,BAX,NF-κB and E-cadherin were detected by RT-PCR and Western blot. Results: After treatment with different concetrations of gambogenic acid for 72 h, the proliferation of HeLa cells was significantly inhibited, and in a concentration dependent manner. The ability of invasion was decreased with the concentration of gambogenic acid increased, which was detected by Transwell chamber assays in vitro. RT PCR and Western blot demonstrated that gambogenic acid up-regulated the expressions of BAX and E-cadherin and down-regulated the expression of mRNA and protein of BCL-2 and NF-κB. Conclusion: Gambogenic acid can inhibite the proliferation and invasion of HeLa cells,and promote the cells apoptosis in a concentration dependent manner.

[Expression of Micro-RNA 218 in Cervical Cancer and Its Effect on Proliferation, Apoptosis and Invasion of HeLa Cells].

OBJECTIVES: To investigate the expression and clinical significance of microRNA-218(miR-218)in human cervical cancer and the effects ofmiR-218 on proliferation, cell apoptosis and invasion of HeLa cells. METHODS: QRT-PCR was used to detect the expression ofmiR-218 in 23 cases of normal cervical tissues and 114 cases of cervical cancer, and the relationship between the expression and the clinicopathological features was analyzed; HeLa cells were devided into three groups: non transfection (control group), transfected with empty liposomes negative control goup (NC group), transfected with miR-218 mimic (miR-218M group). The cell growth inhibiting ratio of HeLa cells was assessed by MTT assay. Fluorescence activated cell sorting was used to measure cell apoptosis. Changes of cell migration ability were detected by wound healing test and Transwell assay. QRT-PCR and Western blot were used to detect the expression of Bcl-2, Bax, NF-κB and E-cadherin, respectively. RESULTS: The expression ofmiR-218 in cervical cancer was down regulated, and there were significant differences in the different pathological types, stages, lymph node metastasis and interstitial infiltration in cervical cancer tissues ( P<0.01); After being transfected with miR-218 mimic, the proliferation of HeLa cells was significantly inhibited. The ability of invasion was decreased. QRT-PCR and Western blot showed that after being transfected with miR-218 mimic, the expression levels of Bcl-2 mRNA and protein were down-regulated and Bax mRNA and protein expression levels were increased, E-cadherin mRNA and protein expression were up-regulated, but NF -κB mRNA and protein expression were down-regulated. CONCLUSIONS: he low-expression of miR-218 is correlated with the poor clinicopathological features in human cervical cancer. MiR-218 overexpression reduces cancer cell proliferation and induces apoptosis and inhibits cell migration, suggesting that miR-218 may play a key role in the progression of human cervical cancer.

[In vitro interaction of human pancreatic cancer cells and rat dorsal root ganglia: a co-culture model].

OBJECTIVE: To establish an in vitro model of perineural invasion (PNI) with co-culture of human pancreatic cancer cells and rat root ganglion, to observe the neurite outgrowth and pancreatic cancer cell proliferation and migration, and to explore the molecular basis of perineural invasion (PNI) of pancreatic cancer. METHODS: Human pancreatic cancer cell line (MIA PaCa-2) and rat dorsal root ganglion (DRG) were co-cultured in Matrigel matrix to generate the PNI model. The neurite outgrowth, pancreatic cancer cell colony formation, neurite-colony contact and retrograde migration were observed under an inverted microscope. The data were analyzed with the Image-Pro Plus 5.0 system. The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody. In order to determine the absorbance (A) of the pancreatic cancer cells, MTT assay was used. The apoptotic index (AI) was evaluated by flow cytometry. RESULTS: Neurite outgrowth was stimulated in the presence of pancreatic cancer cells. After 72 hours of the co-culture, MIA PaCa colonies co-cultured with DRG exhibited a significantly larger colony area (242.83 ± 4.92) than that of the control (182.50 ± 5.39, P < 0.001). In the MIA PaCa-2/DRG co-culture system, the neurites exhibited a trend of growing towards the pancreatic cancer cell colony. However, the pancreatic cancer cells showed a trend of retrogradely migrating to the DRG along the neurite outgrowth, when MIA PaCa-2 colonies touched the DRG. The positive rate of Ki-67 nuclear antigen was significantly higher than in the co-culture group. The PI value was higher in the experimental group (12.80%) than that in the control group (6.81%, P < 0.01). The MTT assay showed that proliferation of the pancreatic cancer cells was more active than that in the control group. Flow cytometry analysis showed that the apoptosis rate of the pancreatic cancer cell was 2.46%, significantly lower than that of the control group (4.89%, P < 0.001). CONCLUSIONS: An in vitro co-culture model of rat dorsal root ganglion and human pancreatic cancer cell line is successfully established in this study. This MIA PaCa-2/DRG co-culture system demonstrates that the neural-pancreatic carcinoma cell interaction is a mutually beneficial process for the growth of neurites and pancreatic carcinoma cells. The pancreatic cancer cells show a trend of migrating to the DRG along the neurite outgrowth.

[Relationship between mutation of BRCA1 and susceptibility to early onset of breast cancer].

OBJECTIVE: To detect the prevalence of Breast Cancer Susceptibility Gene 1 (BRCA1) mutations and single nucleotide polymorphism (SNP) among young patients with breast cancer and to study the relationship between BRCA1 gene mutation and susceptibility to breast cancer. METHODS: 30 samples of breast cancer tissue were collected from female patients with breast cancer diagnosed when they were aged < or = 35 5 of which had at least one first-degree relative affected with breast cancer. Genomic DNA was extracted from the breast cancer tissues. The PCR products were amplified in the coding sequence of exon 2, 11C, 11F, 11L, 11I, 16, and 20 by using polymerase chain reaction. Then the PCR products were analyzed using DNA direct sequencing. The sequence was compared with the DNA Star-MagAlign software. RESULTS: A total of 14 sequence variations in BRCA1 gene were identified, including 3 frameshift mutations (cDNA2639, 2640delTA, 3343 delG, and 3398delT) and 11 spot mutations (cDNA 2570 C > T, cDNA 2620 A > T, 1473 A > G, 1561 C > T, 1594G > A, 2206 A > G, 2227 T > C, 2659 C > A, 2806T > C, 3307 A > G, and 3375 G > A). Three patients with these mutations had a family history of breast cancer. The mutation frequency of BRCA1 was 10% (3/30). No mutation was found in the exons 16 and 20. CONCLUSION: Patients with breast cancer aged < or = 35 have BRCA1 mutations located in the exon 11 mainly. The mutation frequency of the breast cancer patients aged < or = 35 with breast cancer family history is higher than those without family history. Three frameshift mutation sites may be related to early onset of breast cancer.

[Study on gene control region of mitochondrial DNA in familial breast cancer].

OBJECTIVE: To study on mutations in D-loop region which is gene control region of mitochondrial genome in patients with familial breast cancer. METHODS: Twenty-three breast cancer patients came from twenty-one families of breast cancer, and eighteen healthy controls participated in the study. PCR amplification of D-loop region in mitochondrial DNA was performed and then the product was sequenced to analyze mutations. RESULTS: One hundred and twenty-six mutations in D-loop region were found in twenty-three patients with familial breast cancer, and four mutations were new. In all of twenty-three patients, thirty-seven mutations were found in D310 which was hot spot of D-loop region in mitochondrial DNA. In these mutations, T>C in 310, TC insert in 311-312, CA deletion in 522-523 and C>G in 527 were multi-presentation mutations in patients with familial breast cancer. Mutations had no difference in the same family member of breast cancer family except that occurrence in the region of D310. In the same family, mutations in D310 of patients were different from controls. CONCLUSION: Mutations in D310 of familial breast cancer patients may enhance their susceptibility to breast cancer.

[Clonality of the peripheral papilloma and cancerous cells of breast].

OBJECTIVE: To study the clonality status of peripheral papilloma (peri-MP), ductal carcinoma in situ (DCIS), and normal tissue of the breast using an assay based on inactivation mosaicism of the length-polymorphic X-chromosomes at the androgen receptor (AR) locus and to explore a reliable way to distinguish the benign and malignant (or pre-malignant) cases judged morphologically. METHODS: Specimens of breast tissues were obtained from 26 cases of peri-PM, 25 cases of peri-PM with atypical ductal hyperplasia (ADH), and 27 cases pf DCIS, 16 cases of developed canceration, and 20 normal women. DNA was extracted and amplified via nested-PCR with or without previous digestion by the methylation-sensitive restriction endonuclease Hha I. The products were resolved on denaturing polyacrylamide gels and visualized through silver staining. The clonality of these samples was analyzed by showing the lanes. RESULTS: Loss of polymorphism at the AR locus was found in all the cases with DCIS and in 10 cases (10/25, 40.0%) of peri-PM with ADH, indicating the monoclonality of the tumor. Twenty-four cases (92.3%) of the 26 cases with peri-PM and the 20 specimens of normal tissue were shown to be polyclonal. In the 16 cases of developed canceration identical X chromosome inactivation (monoclonal alterations) was observed in the cancer focus, parts of peri-PM with ADH, and the part of DCIS. CONCLUSION: Normal breast tissue and peri-PM show polyclonality and the peri-PM with ADH shows monoclonality. Clonality analysis may be a useful modality to screen high-risk cases from precancerous lesions or to distinguish between the benign hyperplasia and early carcinoma.

[Study on the potential mechanisms of leukemia cell resistance to TRAIL-induced apoptosis].

OBJECTIVE: To explore the potential mechanisms of leukemia cell resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis. METHODS: Cells apoptosis, changes of mitochondrial membrane potential, activity of NF-kappaB, activity of caspase-8 and expressions of apoptosis-related proteins in TRAIL treated K562 and CEM cells, were detected by flowcytometry, ELISA and Western blotting methods, respectively. RESULTS: After treated with TRAIL, the apoptosis indexes were 29.98% and 14.1%, and mitochondrial membrane potential were decreased to 73.25% and 25.4% in K562 and CEM cells respectively. Constitutive level of caspase-8 expression in CEM was lower than that in K562 cells. Both cells became over-expressed Bcl-xL and down-regulated Bax. The ratio of Bcl-xL/Bax in CEM cells was higher than that in K562 cells. Compared with that in K562, the NF-kappaB activity increased significantly in CEM after treatment with TRAIL in early stage. CONCLUSION: CEM cells were more resistant to TRAIL-induced apoptosis than K562 cells did. The potential mechanisms associated with CEM drug resistance might be the lower expression of the constitutive level of caspase-8, lower sensitivity of mitochondrial inner membrane, early increase in NF-KB activity and altered expression of Bcl-2 proteins family.

[Study on the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by tumor necrosis factor related apoptosis inducing ligand].

OBJECTIVE: To explore the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by Tumor necrosis factor related apoptosis inducing ligand and offer evidences for TRAIL application in clinic. METHODS: Apoptosis, integration of mitochondria (including DeltaPsim, cardiolipin), activity of Caspase-9 and release of cytochrome c in colon carcinoma cells SW1116 treated with TRAIL, were detected by means of flowcytometry, fluorometer method and western-blot at the different time point. RESULTS: After treated with TRAIL for 4 hours, the apoptosis index was 32.98%, and the damage of mitochondria occurred with DeltaPsim, cardiolipin decreased, and the activity of Caspase-9 and cytochrome c increased. The Caspase-9 activity at 24 hour was (48.12+/-2.21)micromol.L(-1).h(-1).mg(-1) protein. Mitochondrial damage induced by TRAIL could be inhibited by Caspase inhibitor Z-VAD. fmk. CONCLUSION: Mitochondrial pathways involved in the apoptosis of colon carcinoma cell induced by TRAIL. Cytochrome c was released and Caspase-9 was activated in the Caspase-dependent manner after the damage of mitochondrial.

Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo.

AIM: To evaluate the effect of antisense vascular endothelial growth factor (VEGF) RNA (PCMV-FGEV) transfection on the profile of hepatocellular carcinoma (HCC) SMMC-7721 cells in vitro and in vivo. METHODS: SMMC-7721 cells were transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine as antisense group, sense group and control group respectively. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the cells were determined by RT-PCR and immunohistochemistry. Cell proliferation was observed by MTT assay. FACS analysis was used to determine the effect of PCMV-FGEV transfection on cell apoptosis. The growth of transfected cells in vivo was also observed in nude mice. RESULTS: VEGF expression was reduced in SMMC-7721 transfected with PCMV-FGEV, which was confirmed by RT-PCR and immunohistochemistry. No effect of PCMV-FGEV transfection was found on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cells transfected with PCMV-FGEV was slow in nude mice and accompanied with obvious apoptosis. The latent time of tumors in the antisense group was 25.0 +/- 1.8 d, which was longer than that in sense and control groups (F = 19.455, P < 0.01). The average tumor weight in antisense group (0.96 g +/- 0.28 g) was the smallest among the three groups (F = 21.501, P < 0.01). CONCLUSION: The expression of VEGF can be inhibited by antisense PCMV-FGEV. Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721 in vitro but can inhibit tumor growth and induce cell apoptosis in vivo.

[Reversing multidrug resistance in breast cancer cell line MCF-7/ADR by small interfering RNA].

BACKGROUND & OBJECTIVE: Multidrug resistance of tumor cells often leads to failure of chemotherapy. The over-expression of P-glycoprotein (P-gp), encoded by multidrug resistance 1 (mdr1) gene, plays an important role in multidrug resistance of breast cancer. This study was to explore the feasibility of silencing mdr1 gene by small interfering RNA (siRNA) in drug resistant breast cancer cell line MCF-7/ADR. METHODS: The siRNA oligonucleotides strand designed previously was inserted into pSilencer3.1-H1 Hygro vector, the plasmid was transformed into E.coli. After amplification, the plasmid was purified, and sequenced to determine whether the ligation between siRNA insert and the vector was correct, then transfected into MCF-7/ADR cells, and relevant sensitive MCF-7 cells. MCF-7/ADR cells were screened by hygromycin, surviving cells were cultured. The positive rate of P-gp was detected by flow cytometry, and positive rate of mdr1 gene was detected by real-time relatively quantitative polymerase chain reaction (PCR). Adriamycin (ADM) resistant experiment was performed on MCF-7/ADR cells with siRNA. RESULTS: Positive rate of P-gp in MCF-7/ADR cells was decreased from 99.8% (before siRNA transfection) to 12.3% (after siRNA transfection). Real- time PCR revealed that the threshold cycle value of MCF-7/ADR cells increased from 25.22 to 30.64 after transfected with siRNA. The IC(50) of ADM for MCF-7/ADR cells transfected with siRNA was 0.51 micromol/L, while that for MCF-7/ADR cells without transfection was 17.88 micromol/L. CONCLUSION: siRNA can silence mdr1 gene in MCF-7/ADR cells, may become a new, effective medical technique.

[Change in E-cadherin, alpha-, beta- and gamma-catenin expression after hyperthermia of a human colon carcinoma cell line in vitro].

OBJECTIVE: To investigate changes in E-cadherin, alpha-, beta-, and gamma-catenin expression after hyperthermia of a human colon cancer cell line in vitro. METHODS: E-cadherin and alpha-, beta-, and gamma-catenin expression on a human colon carcinoma cell line HT29 at 7 successive times after hyperthermia at 43 degrees C for 60 min were investigated in vitro by RT-PCR and immunochemistry. RESULTS: In vitro studies revealed that E-cadherin expression had increased significantly on HT29 cells at 24 hours after hyperthermia at 43 degrees C for 60 min. In a time-course analysis, the expression of E-cadherin had major increase at 24 to 72 hours after hyperthermia compared with an unheated control cell. gamma-catenin expression had increased significantly at 8 to 72 hours. After hyperthermia at 43 degrees C for 60 min, beta-catenin expression had decreased gradually at 4 hours to 72 hours. alpha-catenin expression kept unchanged after hyperthermia. CONCLUSION: Hyperthermia at 43C for 60 min upregulated E-cadherin and gamma-catenin expressions but downregulated beta-catenin expression on colon carcinoma cells in vitro. alpha-catenin expression was not regulated by hyperthermia.

[Clinicopathological significance of homeobox gene BP1 mRNA expression in human breast cancer].

BACKGROUND & OBJECTIVE: Beta protein 1 (BP1) gene, a novel member of DLX homeobox gene family, is located in 17q21-22 region and overexpressed in both acute myeloid leukemia and acute T cell lymphocytic leukemia. However, the reports on the function of BP1 in solid tumors are rare. The study was designed to determine the expression of BP1 gene in breast cancer and to analyze its relationship with various clinicopathological factors. METHODS: With beta-actin gene as a reference, BP1 mRNA expression was detected in 82 breast cancer tissues and 12 near adjacent tissues and 10 far adjacent tissues using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression rates of BP1 in near and far adjacent tissues were 16.67% (2/12) and 0% (0/10), respectively; while the expression rate in breast cancer group was 64.63% (53/82). There were statistically significant differences among the three groups (P< 0.05). Overexpression of BP1 in breast cancer was correlated with histological grade (P< 0.05). Nevertheless, no correlation was found between BP1 expression and other clinicopathological factors, including tumor size, lymph nodal metastasis, family history, pathological type, menarcheal age, primiparous age, number of pregnancy, menopausal status, ER status and PR status (P >0.05). CONCLUSION: BP1 gene is of upregulated expression in breast tumors and could be regarded as a new and vital biomarker in breast cancer research.

[Study on inhibitory effect of antisense VEGF RNA on the growth of hepatocellular in vitro in vivo].

OBJECTIVE: To determine the effects of PCMV-FGEV transfection on the profile of SMMC-7721 hepatocellular in vitro in vivo. METHODS: SMMC-7721 hepatocellular was transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the SMMC-7721 hepatocellular were determined by in situ hybridization and immunochemical analysis. The effect of PCMV-FGEV transfection on SMMC-7721 hepatocellular proliferation was observed by MTT colorimetric assay. Flow cytometry was used to determine the effects of PCMV-FGEV transfection on cell apoptosis of SMMC-7721. The growth of transfected cells was also observed in nude mice. RESULTS: There was reduced VEGF expression in SMMC-7721 transfected with PCMV-FGEV confirmed by in situ hybridization and immunohistochemical analysis. There was no effect of PCMV-FGEV transfection on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cell with PCMV-FGEV transfected was slow in nude mice (vivo) and accompanied with obvious apoptosis. The latent time of tumor in the antisense mice group was 25.0+/-1.8 days, which was longer than that in sense and control group significantly (F=19.455, P<0.01). On the other hand, the average tumor weight in antisense group (0.96 g+/-0.28 g) was the smallest among the three groups (F=21.501, P<0.01). CONCLUSIONS: The expression of VEGF was inhibited by PCMV-FGEV. There was no effect on cell proliferation and cell apoptosis of SMMC-7721 by transferring PCMV-FGEV gene into SMMC-7721 cells in vitro. But in vivo it can inhibit tumor growth and induce cell apoptosis.

BRCA1 and BRCA2 sequence variants in Chinese breast cancer families.

A series of 45 high-risk breast cancer patients, consisting of 25 affected individuals from 16 families in China with at least two cases of breast cancer and 20 cases of breast cancer diagnosed under age 35 without reported family history, were studied for germline mutations of the BRCA1 and BRCA2 genes. Thirteen of the 16 families contained at least one case diagnosed under age 50. Three distinct protein truncating sequence variants, likely to be disease-associated, were identified: two novel mutations in BRCA1 (1584G>T and 5028delC), and a previously reported mutation in BRCA2 (7883delTTAA). Additional sequence variants identified included common polymorphisms, and several variants of unknown clinical significance, including a novel BRCA1 alteration. Based on models for predictive testing using allele frequencies and risks estimated in Western populations, our results suggest that BRCA1/2 mutations account for a somewhat smaller fraction of breast cancer cases in Tianjin than in the Caucasian populations studied. This difference could be the result of a lower penetrance of BRCA1/2 mutations due to the surrounding environmental and hormonal milieu, or a lower frequency of mutations in this population. Larger, more detailed, studies will be necessary to determine which factors underlie this difference.