Skip participates in formation and lipid metabolism of beige adipose and might mediate the effects of SIRT1 activator BTM-0512 on beige remodeling.

Dissipating energy by activating thermogenic adipose to combating obesity attracts many interests. Ski-interacting protein (Skip) has been known to play an important role in cell proliferation and differentiation, but whether it participates in energy metabolism is not known. Our previous study revealed that BTM-0512 could induce beige adipose formation, accompanying with up-regulation of Skip, but the role of Skip in metabolism was unknown. In this study, we mainly investigated whether Skip was involved in beige remodeling of subcutaneous white preadipocytes as well as in lipid metabolism of differentiated beige adipocytes. The results showed that in high fat diet-induced obesity mice, the protein levels of Skip in subcutaneous and visceral white adipose as well as in brown adipose were all down-regulated, especially in subcutaneous white adipose. Then we cultured subcutaneous adipose derived-stem cells (ADSCs) and found knock-down of Skip (siSkip) inhibited the expressions of thermogenic adipose specific genes including PRDM16 and UCP1 in both undifferentiated ADSCs and differentiated beige adipocytes, which could abolish the effects of BTM-0512 on beige remodeling. We further observed that siSkip affected multiple rate-limiting enzymes in lipid metabolism. The expressions of ACC, GPAT-1, HSL and ATGL were down-regulated, while CPT1α expression was up-regulated by siSkip. The expression of AMPK was also decreased by siSkip. In conclusion, our study demonstrated that Skip might play an important role in the beige remodeling of white adipocytes as well as lipid metabolism of beige adipose.

[Evaluation of treatment technology of odor pollution source in petrochemical industry].

Using an environmental technology assessment system, we put forward the evaluation index system for treatment technology of the typical odor pollution sources in the petroleum refining process, which has been applied in the assessment of the industrial technology. And then the best available techniques are selected for emissions of gas refinery sewage treatment plant, headspace gas of acidic water jars, headspace gas of cold coke jugs/intermediate oil tank/dirty oil tank, exhaust of oxidative sweetening, and vapors of loading and unloading oil.

The relevance and role of vascular endothelial growth factor C, matrix metalloproteinase-2 and E-cadherin in epithelial ovarian cancer.

We investigated the correlation of vascular endothelial growth factor C, matrix metalloproteinase-2, E-cadherin to explore mechanisms of vascular endothelial growth factor C in the metastasis of ovarian cancer and the relationship of prognosis. We applied immunohistochemistry to investigate the expression of vascular endothelial growth factor C, matrix metalloproteinase-2 and E-cadherin in ovarian tissues of 227 patients. We adopted Pearson chi-square test, Spearman correlation coefficient, univariate analysis, multivariate analysis, and Kaplan-Meier method. The positive rate of vascular endothelial growth factor C, matrix metalloproteinase-2 and E-cadherin in ovarian cancer was higher than that in borderline and benign tumor (P < 0.01). Vascular endothelial growth factor C was positively correlated with matrix metalloproteinase-2 (r = 0.665, P < 0.01) while negatively with E-cadherin(r = -0.185, P < 0.05). Univariate analysis showed clinical stage, pathologic grade, lymphatic metastasis, residual disease, chemotherapy, ascites, vascular endothelial growth factor C, and matrix metalloproteinase-2-influenced survival time (P < 0.05). In Cox multivariate analysis, all the aforementioned factors were found to be independent prognostic factors except pathologic grade. Vascular endothelial growth factor C was a new target to assess the prognosis of ovarian cancer. The expression of vascular endothelial growth factor C in ovarian cancer was related to matrix metalloproteinase-2 and E-cadherin.

[Mechanisms of resveratrol bovine serum albumin nanoparticle-induced cell death in human ovarian cancer SKOV3 cells].

OBJECTIVE: To study the effect of resveratrol bovine serum albumin nanoparticles on SKOV3 cell line and its mechanisms. METHODS: The morphological changes of the cells exposed to the nanoparticles were observed by apoptotic body/cell nucleus DNA staining under inverted microscope and fluorescence microscope, and the pathway of cell death was determined by phosphatidylserine translocation. Western blotting was performed to detect the activation of cyto.c, caspase-3 and caspase-9. RESULTS: DNA ladder was detected with gel electrophoresis and the cell death was partially inhibited by the pan-caspase inhibitor Z-VAD-FMK. Gel electrophoresis displayed both DNA ladder and smear in RES-BSANP exposed groups, while DNA ladder disappeared in Z-VAD-FMK group and only the smear was left. Cyto.c in the cytoplasm was released at 2 h, while the expression of caspase-9 protein reached the peak level at 4 h and caspase-3 expression was obvious enhanced at 8 h. At 4 h, caspase-9 expression in the cells exposed to 100 µmol/L RES-BSANP was decreased significantly as compared to the cells treated with 50 µmol/L RES-BSANP (P>0.05). CONCLUSION: RES-BSANP can induce the necrosis and apoptosis of SKOV3 cells via either caspase-dependent or caspase-independent pathways.

[Exogenous DNA influencing fertilization of goat sperm cells and expression in early embryos].

Our early study found that goat spermatozoa could spontaneously take up foreign DNA and vary in capabilities of spermatozoa from different donors to bind and internalize exogenous DNA. In this study, three goats with considerable differences of capability were used to investigate the effect of exogenous DNA on goat spermatozoa, and feasibility and efficiency of transgenic embryo production by sperm-mediated gene transfer method. The viability, acrosomal reaction frequencies and cleavages were decreased in the groups co-cultured with exogenous DNA, compared with the control groups, and the range of decrease was correlated with the capability of sperm cells up-take foreign DNA. After fertilizing with co-cultured spermatozoa, GFP gene was introduced into oocytes and expressed in early embryos. However, different efficiencies of transgenic embryos appeared in sperm donors (P<0.05). GFP gene was detected in 16.2% (25/154), 5.3% (4/76), and 0% (0/36) embryos, respectively, when high, middle and low capability of sperm donors were used. But only 6.5% (10/154) embryos from high capability sperm donor expressed GFP. Our results demonstrate that selecting high capability of sperm donor is a key step for improving efficiency of sperm mediated-gene transfer method. However, the adverse influence of foreign DNA on spermatozoa needs to be further studied.

[Inhibitory effect of recombinant anti-angiogenic peptide of tumstatin on growth and metastasis of human ovarian cancer transplanted in nude mice].

OBJECTIVE: To evaluate the anti-angiogenic activity of peptide 21 obtained by modification of tumstatin, and its inhibitory effect on the growth and metastasis of human ovarian cancer transplanted in nude mice. METHODS: The peptide 21 was purified by affinity chromatography. Human ovarian cancer SKOV3 cells were inoculated in nude mice and the transplanted tumor was treated with the peptide 21 to observe the tumor growth and metastasis. The microvessel density (MVD) and immunohistochemical staining index of PCNA, VEGF and MMP-2 and TIMP-2 were performed to assess the inhibitory effect of the peptide 21. RESULTS: In the nude mice at 21 days after peptide 21 treatment, the inhibition rate of tumor growth was 53.17%, the tumor microvessel density was significantly reduced (P <0.05), the expression of PCNA, VEGF and MMP-2 were significantly lower (P <0.01), and TIMP-2 expression was significantly higher (P <0.01) in comparison with that of control group. CONCLUSION: The peptide 21 generated in this study has a significant anti-angiogenetic activity, showing significant inhibitory effect on the growth of human ovarian cancer transplanted in nude mice. The mechanism of its inhibitory action on ovarian cancer growth may be mediated by reduction of neovascularization and reduction of expression of angiogenetic factors.

[Effects of interleukin-12 transfection on proliferation of ovarian cancer SKOV3 cells in vitro and in vivo].

BACKGROUND & OBJECTIVE: The incidence, recurrence rate, and metastasis rate of malignant ovarian tumors are high. Interleukin-12 (IL-12) has antitumor effect. This study was to investigate the effects of IL-12 on the proliferation of human ovarian carcinoma SKOV3 cells in vitro and in vivo. METHODS: The plasmid containing mouse IL-12 gene was transfected into SKOV3 cells (SKOV3/IL-12). Blank plasmid-transfected SKOV3 cells (SKOV3/neo) and untransfected SKOV3 cells were used as controls. The expression of IL-12 protein was detected by ELISA. The proliferation inhibition rate and population doubling time (PDT) of SKOV3 cells were evaluated by MTT assay. SKOV3/IL-12, SKOV3/neo and SKOV3 cells were inoculated in nude mice subcutaneously. Tumor growth was recorded. The serum levels of IL-12 and interferon-gamma (IFN-gamma) in mice was detected by ELISA. The expression of vascular endothelial growth factor (VEGF) and IFN-gamma-inducible protein 10 (IP-10), and microvessel density (MVD) in tumor tissues was detected by immunohistochemistry. RESULTS: After transfection, the protein level of IL-12 was significantly higher in SKOV3/IL-12 cells than in SKOV3/neo and SKOV3 cells [(473.0+/-38.0) pg/ml vs. (16.0+/-1.3) pg/ml and (16.0+/-1.2) pg/ml at 48 h, (522.0+/-32.0) pg/ml vs. (18.0+/-1.6) pg/ml and (17.0+/-1.4) pg/ml at 60 h, P<0.01]. The proliferation inhibition rate of SKOV3/IL-12 cells was significantly higher than those of SKOV3/neo and SKOV3 cells (63.7% vs. 0% and 0%, P<0.01). The PDT was significantly longer in SKOV3/IL-12 cells than in SKOV3/neo and SKOV3 cells [(45.8+/-2.7) h vs. (27.6+/-2.2) h and (28.2+/-2.1) h, P<0.01]. In nude mice, the tumor formation rate of SKOV3/IL-12 cells was lower than those of control cells (5/8 vs. 8/8 and 8/8)û the tumor formation time was significantly longer in SKOV3/IL-12 group than in SKOV3/neo group and SKOV3 group [(15.0+/-5.0) days vs. (7.9+/-3.2) days and (7.8+/-2.4) days, P<0.01]. The volume and weight of tumors and the body weight of mice were significantly lower in SKOV3/IL-12 group than in SKOV3/neo group and SKOV3 group (P<0.01). The serum levels of IL-12 and IFN-gamma were significantly higher in SKOV3/IL-12 group than in SKOV3/neo group and SKOV3 group (P<0.01). The positive rate of VEGF and MVD were significantly lower and the positive rate of IP-10 was higher in SKOV3/IL-12 group than in SKOV3/neo group and SKOV3 group (P<0.01). CONCLUSIONS: Transfection of IL-12 can inhibit the proliferation and suppress the tumorigenic ability of SKOV3 cells. IL-12 can promote IFN-gamma to induce IP-10 production, thereafter, inhibit neovascularization in tumors.

[Survivin antisense RNA induces apoptosis and sensitizes ovarian cancer cell line SKOV3 to docetaxel].

BACKGROUND & OBJECTIVE: Survivin gene overexpresses in a variety of human tumors, and plays an important role in cell apoptosis and drug resistance of tumors. This study was designed to establish Survivin antisense RNA, and explore its effects on apoptosis of ovarian cancer cell line SKOV3 and sensitivity to docetaxel. METHODS: Survivin antisense eukaryotic expression vector anti-pcDNA3-svv was established, and transfected into SKOV3 cells by electroperforation. Positive clones (SKOV3-SVVanti) were screened out. Survivin mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR); Survivin protein was detected by Western blot. The effect of Survivin antisense RNA on apoptosis of SKOV3 cells was measured by flow cytometry and observed under electron microscope; its effect on sensitivity of SKOV3 cells to docetaxel was examined by MTT assay. RESULTS: The mRNA and protein levels of Survivin were obviously lower in SKOV3-SVVanti cells than in control cells. Terminal apoptosis changes were observed under electron microscope after transfection. The apoptosis rate was 19%. The 50% inhibitory concentration (IC50) of docetaxel was significantly lower for SKOV3-SVVanti cells than for control cells [(13.3+/-2.2) ng/ml vs. (53.2+/-2.4) ng/ml, P<0.05]. CONCLUSION: Surivivin antisense RNA can induce apoptosis of SKOV3 cells, and sensitize SKOV3 cells to docetaxel.

[Study on the relationship between genesis and development of cervical cancer and the infection of human papillomavirus type 16/18, human herpesvirus II and cytomegalovirus].

OBJECTIVE: To investigate the correlation between genesis and the development of cervical cancer and infection of human papillomavirus (HPV) type 16/18, human herpesvirus II (HSV- II) and cytomegalovirus(CMV). METHODS: Different viruses were determined by polymerase chain reaction in 156 specimens of uterine including cervix 43 cervical cancer specimens,47 cervical intraepithelial neoplasia (CIN) specimens, 56 cervicitis specimens and 10 normal cervix specimens. RESULTS: (1) Positive rates on different viruses: the positive rates of HSV- II, HPV16/18 and CMV were declining in the cervical cancer specimens, CIN specimens or CIN III specimens and CIN I - II specimens, with significant differences. (2)Positive rate and grading, staging and histogenesis of cervical cancer on different viruses as well as positive rates of HPV16/18 in II staging cervical cancer specimens were significantly higher than that in I staging cervical cancer specimens while positive rates of HPV16/18 and HSV- II in high differentiation of cervical cancer specimens were significantly higher than those with medium differentiation from cervical cancer specimens. Positive rates of CMV did not seem to correlate with positive rate of HSV- II and CMV was not correlated to grading, staging or histogenesis of cervical cancer. (3)Copies of infected virus, HSV-II and HPV16/18 showing cervical cancer>CIN> cervicitis while with CMV:cervical cancer>CIN. (4) There were mixed infections of different viruses as HPV16/18 + HSV- II > HPV16/18 + CMV seen in the study. CONCLUSION: HPV 16/18, HSV- II and CMV infection were closely related to the genesis of cervical cancer and quantity of viruses which might have played an important role in carcinogenesis of cervical lesions.

[Functional study on TGF-beta/Smads signaling pathway in human ovarian cancer cells].

Resistance to the growth inhibitory effects of transforming growth factor-beta (TGF-beta) is a characteristic of many transformed cells. The purpose of this study was to determine the response of ovarian cancer cells to TGF-beta1 and to investigate the roles of components of the TGF-beta/Smads signaling pathway in carcinogenesis of ovarian cancer. Three ovarian cancer cell lines, HO-8910, HO-8910PM and SKOV3, were treated with TGF-beta1 and assayed for growth response by MTT assay. Furthermore, expression and subcellular localization of the components of TGF-beta/Smads signaling pathway in these cell lines in the absence or presence of TGF-beta1 were determined by RT-PCR and immunofluorescence analysis. We found that proliferation of SKOV3 cell was not significantly inhibited by TGF-beta1 while it expressed all components of the TGF-beta/Smads signaling pathway. After exposure to TGF-beta1, Smad7 protein in SKOV3 increased transiently and translocated to cytoplasm from nucleus while P-Smad2 translocated into nucleus from cytoplasm. Taken together, the results suggested that the TGF-beta/Smads signaling pathway remained functional in human ovarian cancer cells, HO-8910, HO-8910PM and SKOV3, and the abnormalities of the downstream effectors of Smads proteins might contribute to the resistance of SKOV3 cell to TGF-beta1.

[Experimental therapy of survivin antisense oligonucleotide for human ovarian cancer cell SKOV3].

BACKGROUND & OBJECTIVE: Survivin abnormally overexpresses in a variety of human tumors, it may play an important role in the development of tumor. This study was designed to investigate the inhibitory effects of survivin antisense oligonucleotide (ASODN) on human ovarian carcinoma cell SKOV3 in vivo and its mechanism. METHODS: SKOV3 cells were transfected with survivin ASODN mediated by DOTAP liposome reagent, MTT method was used to observe the inhibitory rate, Western blot analysis was used to determine relating gene expression. Flow cytometry, DNA ladder analysis, and DAPI staining were used to examine cell apoptosis. Kinase activity test was used to examine the changes of caspase-3 activity. RESULTS: The expression of survivin in SKOV3 cells decreased after transfected with survivin ASODN. Survivin ASODN has obviously inhibited the growth of SKOV3 cells after transfection. The inhibitory rate of 1000 ng/ml ASODN transfection group was (60.30+/-2.95)%, which is obviously higher than control group (P< 0.05). Survivin ASODN transfection induced cell cycle arrest in G1 phrase, while the apoptotic rate increased from 0.65% to 32.10%. Caspase-3 activity (0.998+/-0.001) increased significantly after transfected with survivin ASODN (P< 0.01). CONCLUSION: Survivin ASODN,which can inhibit human ovarian carcinoma SKOV3 cells proliferation and induce apoptosis, is a prospective anti-cancer drug.

Role of ERK1/2 kinase in cisplatin-induced apoptosis in human ovarian carcinoma cells.

OBJECTIVE: To investigate the role of extracellular regulated kinase (ERK1/2) pathway in cisplatin-induced apoptosis in human ovarian carcinoma cells. METHODS: Cisplatin-induced apoptosis were stained with DAPI and was assessed microscopically in human epithelial adenocarcinoma ovarian cell line SKOV3 cells. ERK activation was determined by Western blotting using an anti-phospho-ERK antibody to detect ERK activity. The effect of PD98059 on ERK activity induced by cisplatin was detected by MTT assay. RESULTS: Marked apoptosis of SKOV3 cells resulted from 48 hours treatment with 20 microg/mL cisplatin. Strong activation of ERK was led to by 15 microg/mL cisplatin. Dose response and time course of cisplatin induced apoptosis in SKOV3 cells. Cisplatin-induced ERK activation occurred at 12 hours and increased to highest induction at 24 hours by Western blotting. The effect of PD 98059 on ERK activity induced by cisplatin at the concentration of 100 micromol/L PD 98059. Statistically significant decreased in cell survival were observed with 100 micromol/L PD 98059 at 15 and 20 microg/mL cisplatin (P < 0.05). CONCLUSIONS: Cisplatin activates the ERK signaling pathway in ovarian cancer cell line SKOV3. Inhibition of ERK activity enhances sensitivity to cisplatin cytotoxity in ovarian cancer cell line SKOV3. Evaluation of ERK activity could be useful in predicting which ovarian cancer will response most favorably to cisplatin therapy.

[Study on the correlation between the different papillomavirus type and telomerase in cervical cancer].

OBJECTIVE: To define a correlation between different human papillomavirus (HPV) types and telomerase activity in cervical cancer. METHODS: Telomerase activity was detected by TRAP-PCR, and different HPV type was determined by PCR in 83 cervical cancer, 47 cervical intraepithelial neoplasia (CIN) and 10 normal cervix cases. RESULTS: With regard to positive rates of telomerase and HPV 16/18: the results were cervical cancer > CIN > normal cervix, CIN III > CIN I, II; with regard to HPV 6/11 positive rate: the results showed CIN I, II > CIN III. Positive rates of telomerase cervical cancer and HPV were bearing on grading and staging, but they did not correlate with histologic subtypes. Positive rate of HPV 6/11 had nothing to do with grading, staging and histologic patterns. On expression strength of telomerase and HPV 16/18: the results showed cervical cancer > CIN, CIN III > CIN I, II. Regard to HPV 6/11'expression strength: the results showed CIN I, II > CIN III, CIN > cervical carcinoma. CONCLUSION: HPV 16/18 infection seemed to have played an important role in carcinogenesis of cervical lesions by activation of telomerase.