Zheng Liduan

Growth-inhibitory effects of curcumin on ovary cancer cells and its mechanisms

SummaryTo study the growth-inhibitory effects of curcumin on human ovary cancer A2780 cellsin vitro and its molecular mechanisms, the growth inhibition rates of A2780 cancer cells, after being treated with 10 μmol/L–50 μmol/L curcumin for 6–24 h, were examined by MTT method. The morphological changes of cancer cells were observed under inversion microscopy. Cellular apoptotic rates were determined by using TUNEL. The protein expression levels of bcl-2, p53 and MDM2 in cancer cells were examined by SP immunohistochemistry. After being treated by various concentrations of curcumin, the growth of cancer cells was inhibited significantly. Some cancer cells presented characteristic morphological changes of apoptosis. The rates of apoptosis were 6.41%–28.48% (P<0.01). The expression of bcl-2 and p53 was decreased, which depended on the action time (P<0.01). There were no obvious changes in MDM2 expression. It was concluded that curcumin could significantly inhibit the growth of ovary cancer cells. The induction of apoptosis by down-regulating the expression of bcl-2 and p53 was probably one of its molecular mechanisms.

Growth inhibition and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780

Objective: To explore the growth inhibition effects and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780.Methods: After treatment with 10–50 μmol/L curcumin for 6–24 h, the growth activity of A2780 cancer cells were studied by [4,5-dimethylthiazol-2-yl]-2, 5-dipheny-Itetrazolium bromide (MTT) colorimetry. Cellular apoptosis was inspected by flow cytometery and acridine orange-ethidium bromide fluorescent staining methods. The fragmentation of cellular chromosome DNA was detected by DNA ladder, the ultrastructural change was observed under a transmission electron microscope, and the protein levels of nuclear factor-Kappa B (NF-κB, P65) and cysteinyl aspartate specific protease-3 (Caspase-3) in ovarian cancer cells were measured by immunohistochemistry.Results: After treatment with various concentrations of curcumin, the growth inhibition rates of cancer cells reached 62.05% —89. 24%, with sub-G1 peaks appearing on histogram. Part of the cancer cells showed characteristic morphological changes of apoptosis under fluorescence and electron microscopes, and the rate of apoptosis was 21.5%–33.5%. The protein expression of NF-κB was decreased, while that of Caspase-3 was increased in a time-dependent manner.Conclusion: Curcumin could significantly inhibit the growth of human ovarian cancer cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating gene expression of NF-κB is probably one of its molecular mechanisms.

Preparation of curcumin prodrugs and theirin vitro anti-tumor activities

SummaryThe curcumin prodrugs, which could be selectively activated in tumor cells, were prepared to establish a basis for the targeted chemotherapy for cancer. On the basis of the molecular structure of curcumin, the N-maleoyl-L-valine-curcumin (NVC), N-maleoyl- glycine-curcumin (NGC) were chemically synthesized and identified by IR and NMR spectroscopy. After treatment with these two prodrugs for 6–24 h, the rates of growth inhibition on human bladder cancer EJ cells and renal tubular epithelial (HKC) cells were detected by MTT colorimetry. Our results showed that after the treatment with 20 μmol/L–40 μmol/L NVC and NGC for 6–24 h, the growth inhibitory effects on EJ cells were 6.71%–65.13% (P<0.05), 10.96%–73.01% (P<0.05), respectively, in both dose- and time-dependent manners. When compared with the curcumin of same concentrations, the growth inhibitory effects of these two prodrugs on HKC cells were significantly decreased (P<0.01). It is concluded that activation of curcumin prodrugs via hydrolysis functions of cellular esterase could inhibit the growth activities of tumor cells, and reduce the side effects on normal diploid cells. This provided a novel strategy for further exploration of tumor-targeted chemotherapeutic drugs.

Smac/DIABLO promotes mitomycin C-induced apoptosis of bladder cancer T24 cells

SummaryThe enhancing effects of Smac gene on the mitomycin C-induced apoptosis of the bladder cancer cell line T24 were investigated. The Smac gene was transfected into bladder cancer cell line T24 under the induction of liposome. The intrinsic Smac level was detected by using immunohistochemistry and RT-PCR. The in vitro cellular growth activities were assayed by MTT colorimetry. Apoptosis was assayed by the flow cytometry. The results showed that as compared with the control cells, the apoptosis rate of T24 cells induced by mitomycin C was enhanced by transfected Smac gene. Flow cytometry revealed that, the apoptosis rate was 18.84% and 33.52%, and 10.72% and 26.24% respectively in blank and transfected cells treated with 0.05 or 0.005 mg/mL mitomycin C (P<0.05). It was concluded that Smac could enhance the apoptosis of T24 by mitomycin C, which could provide a useful experimental evidence for bladder cancer therapy.

Expression of X-linked inhibitor of apoptosis protein and its effect on chemotherapeutic sensitivity of bladder carcinoma

The expression of X-linked inhibitor of apoptosis protein (XIAP) gene and its effect on chemotherapeutic sensitivity in bladder carcinoma was explored. By using immunohistochemistry, the expression of XIAP was detected in 47 bladder carcinomas and 5 normal bladder tissues. The XIAP gene was transfected into bladder cancer cell line T24 by liposome and the positive clone was screened by G418. Cellular XIAP mRNA level was detected by RT-PCR. Low-dose mitocycin C was administered to induce the apoptosis of T24 cells. The in vitro growth of bladder carcinoma cells was analyzed by MTT colorimetry, and the apoptosis rate was assayed by TUNEL methods. It was found XIAP was moderately expressed in bladder carcinomas with the the positive rate being 78.73% (37/47), but the positive rate was not correlated with carcinoma stages and grades (P<0.05). XIAP mRNA level in transfected T24 cells was significantly increased by 3.8 times as compared with that in the cells not transfected with XIAP. After treatment with low-dose mitomycin C (0.005 and 0.05 mg/mL), the growth rate in XIAP no-transfected control group was increased by (11.60±0.25)% and (16.51±0.87)% (P<0.05), and the apoptosis rate was decreased by (10.1±0.2)% and (11.9±0.2%) (P<0.05) respectively as compared with XIAP transfected group. It was concluded that XIAP was expressed in most of bladder carcimoma samples. Overexpression of XIAP in T24 could significantly reduce the MMC-induced apoptosis of bladder carcinoma, suggesting its effect on the chemotherapeutic sensitivity of T24 cells.

Cloning of human Uroplakin II gene from Chinese transitional cell carcinoma of bladder and construction of its eukaryotic expression vector

SummaryTo clone Uroplakin II gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GIII/T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0, software. Full length cDNA of Uroplakin II gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted betweenXba I andHindIII restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin II negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin II were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin II cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100% homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP II for Uroplakin II, was successfully constructed. After being transferred with pcDNA-UP II for 72 h, cellular Uroplakin II mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin II gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin II gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.