ABSTRACT
The effect of Smac gene on the TRAIL-induced apoptosis of the prostate cancer cell line PC-3 and the molecular mechanism were investigated. The Smac gene was transfected into PC-3 cells under the induction of liposome. The intrinsic Smac gene expression was detected by Western blotting. After treatment with TRAIL as an apoptosis inducer, in vitro cell growth activity was assayed by MTT colorimetry. The apoptosis rate of PC-3 cells was determined by annexin V-FITC and propidium iodide staining flow cytometry. The expression of cellular XIAP and caspase-3 genes was examined by Western blotting. Smac-transfected cells (PC-3/Smac group) had significantly increased Smac protein level as compared with PC-3 controls (P<0.01). After induction with 100-200 ng/mL TRAIL for 12-36 h, cellular proliferation rate in PC-3/Smac group was significantly lower than in PC-3 controls (P<0.05). After induction with 100 ng/mL TRAIL for 24 h, the apoptosis rate in PC-3/Smac group was significantly enhanced as compared with that of PC-3 controls (P<0.05). Accordingly, the XIAP expression level was down-regulated significantly (P<0.05) and caspase-3 subunit P20 was up-regulated significantly (P<0.05). It is suggested that the over-expression of cellular Smac can inhibit inhibitor of apoptosis proteins (IAPs), enhance caspases activity and the apoptosis rate of PC-3 cells induced by TRAIL, which may provide a useful experimental basis for prostate cancer therapy.
ABSTRACT
In our previous study, we identified a novel testis-specific expressed gene 2 (TSEG-2) from mouse testis. To further investigate its functions, 35 male Balb/c mice (8 weeks old) were divided into cryptorchidism group (n=20), sham group (n=10), and control group (n=5). In cryptorchidism group, the right testes were anchored to the inner lateral abdominal wall. In situ hybridization (ISH) was applied to measure the localization of TSEG-2 in mouse testis. Real-time quantitative PCR was performed to detect the expression of TSEG-2 gene. Meanwhile, under the mediation of polyethylenimine (PEI), the recombinant vector pEGFP-TSEG-2 (n=5) or empty vector (mock, n=5) was transfected into the testis of male mice. The transfection efficiencies were measured under a fluorescence microscope. The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling (TUNEL). The results showed that TSEG-2 was expressed in convoluted seminiferous tubules, more precisely, in spermatogonia and spermatocytes. As compared with sham and control groups, the TSEG-2 transcription was significantly enhanced (P<0.05) and was correlated with apoptosis of spermatogenic cells in cryptorchid testes (P<0.05). PEI was efficient in mediating transfection of TSEG-2 into seminiferous tubules of testis. One week post-transfection, intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo (P<0.05). These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.
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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 mitomycin 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 carcinoma 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.
ABSTRACT
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.
ABSTRACT
The 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.
ABSTRACT
The 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.
ABSTRACT
To 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 between Xba Ⅰ and HindⅢ 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-UPⅡ 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.
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The 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 micromol/L - 40 micromol/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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Curcumin/pharmacology , Dose-Response Relationship, Drug , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Tumor Cells, Cultured , Urinary Bladder Neoplasms/pathologyABSTRACT
Objective To explore the feasibility of laparoscopic-assisted partial and subtotal colectomy and per anum intrarectal pull-through procedures for Hirschsprung's disease-allied disorder(HAD).Methods From October 1999 to May 2006,38 infants and children with HAD or Hirschsprung's disease(HD) associated with HAD,to whom conservative treatments proved ineffective or relapse occurred,were treated by laparoscopic-assisted partial or subtotal colectomy and per anum intrarectal pull-through procedures.Four 5 mm trocars were inserted at the edge of umbilicus,right upper,right lower and left center quadarant of abdomen when subtotal colectomy was carried out.Two trocars were placed in the left side,lied in left upper and mid lower quadarant of abdomen respectively.Colon descendens,colon transversum,colon ascendens and ileocecal junction lateral peritoneum were mobilized under laparoscope.The operation on anus was referred to modified Soave procedures.Results 38 patients underwent the operations successfully.Left hemicolon resection were performed in 9 cases,with operation time being 110-180 min(mean,135 min).Subtotal colectomy were performed in 29 cases and colon ascendens were rotated reversal clockwise 270? and pulled down using Deloyers procedures,lengths of remaining colon ascendens being 7-13 cm(mean,11.5 cm),operation time being 140-220 min(mean,175 min),intraoperative blood loss being 15-70 ml(mean,35 ml).Postoperative pathological diagnosis showed 10 cases of intestine neuron developmental anormaly(IND),3 cases of hypoganglionosis(HG),4 cases of immature gangliocyte(IGC),9 cases were not classified,6 cases of HD complicated with IND,2 cases of HD complicated with HG,4 cases of HD complicated with ICG.38 cases were followed up for a mean of 3 years and 5 months(range,6 months to 7 years).9 cases of left hemicolon resection had 1-2 stools per day at 6 months postoperatively without stoma stenosis and constipation recurrence.Conclusions Laparoscopic-assisted partial or subtotal colectomy and radical per anum pull-through procedures for HAD are safe,effect,feasible,with minimal invasion,but some laparoscopic procedure experiences are required.
ABSTRACT
The 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 tumortargeted chemotherapeutic drugs.
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To study the growth-inhibitory effects of curcumin on human ovary cancer A2780 cells in vitro and its molecular mechanisms, the growth inhibition rates of A2780 cancer cells, after being treated with 10 micromol/L-50 micromol/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.
Subject(s)
Female , Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Cell Division , Curcuma , Chemistry , Curcumin , Pharmacology , Drugs, Chinese Herbal , Pharmacology , Nuclear Proteins , Metabolism , Ovarian Neoplasms , Pathology , Proto-Oncogene Proteins , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Proto-Oncogene Proteins c-mdm2 , Tumor Cells, Cultured , Tumor Suppressor Protein p53 , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the possibility of gene therapy for prostate cancer by blocking androgen receptor (AR) gene expression using a specific hammerhead ribozyme (RZ).</p><p><b>METHODS</b>The hammerhead ribozyme expression vector pcDNA-hAR-RZ, specific to AR mRNA, was constructed and transfected into the prostate cancer cell line LNCaP by using lipofectamine. Androgen receptor expression was measured by RT-PCR and immunohistochemical methods. Cellular proliferation activities were assayed using the tetrazolium bromide colorimetry method; cell cycle changes were observed by flow cytometry; and cell apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling method.</p><p><b>RESULTS</b>One to seven days after transfection with the ribozyme expression vector, AR mRNA expression at molecular and protein levels in LNCaP cells decreased by 32.6% - 40.7% (P < 0.05) and 21.0% - 87.64% (P < 0.05) respectively, and cell proliferation was inhibited by 18.28% - 35.34% (P < 0.05). Meanwhile, the cell cycle was arrested at the G2/M stage, and apoptotic morphological changes occurred with an apoptosis rate of 25.17% (P < 0.01).</p><p><b>CONCLUSION</b>Ribozyme specific against AR mRNA is capable of inhibiting the expression AR and inducing the apoptosis in prostate cancer cells.</p>
Subject(s)
Humans , Male , Apoptosis , Cell Line, Tumor , Gene Transfer Techniques , Genetic Therapy , Methods , Genetic Vectors , Immunohistochemistry , Prostatic Neoplasms , Therapeutics , RNA, Catalytic , Physiology , Receptors, Androgen , Genetics , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To explore the growth inhibiting effects on human bladder cancer by antisense RNA targeting the proliferating cell nuclear antigen (PCNA) gene.</p><p><b>METHODS</b>The eukaryotic expression vector for antisense PCNA cDNA was constructed and transferred into a bladder cancer EJ cell line. The PCNA expression in the cancer cells was detected by RT-PCR and Western blotting assays. The in vitro proliferation activities of the transferred cells were observed by growth curve, tetrazolium bromide (MTT) colorimetry, tritiated thymidine ((3)H-TdR) incorporation, flow cytometry and clone formation testing, while its in vivo anti-tumor effects were detected on nude mice allograft models.</p><p><b>RESULTS</b>After the antisense vector, pLAPSN, was transferred, cellular PCNA expression was inhibited at both protein and mRNA levels. The growth rates of EJ cells were reduced from 27.91% to 62.07% (P < 0.01), with an inhibition of DNA synthesis rate by 52.31% (P < 0.01). Transferred cells were blocked at G(0)/G(1) phases in cell-cycle assay, with the clone formation ability decreased by 50.81% (P < 0.01). The in vivo carcinogenic abilities of the transferred cancer cells were decreased by 54.23% (P < 0.05).</p><p><b>CONCLUSIONS</b>Antisense PCNA gene transfer could inhibit the growth of bladder cancer cells in vitro and in vivo, which provided an ideal strategy for gene therapy of human cancers.</p>
Subject(s)
Animals , Humans , Mice , Cell Division , Genetics , Gene Transfer Techniques , Genetic Therapy , Methods , Genetic Vectors , Mice, Nude , Proliferating Cell Nuclear Antigen , Genetics , RNA, Antisense , Genetics , Tumor Cells, Cultured , Urinary Bladder Neoplasms , PathologyABSTRACT
To explore a novel strategy for antisense gene therapy of cancer, the coding sequence of human proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryotic vector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with liposome. The PCNA expression in transferred cells was dynamically detected by immunofluorescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryotic vector was successfully constructed and named as pLAPSN. After transfection with it for 1-7 days, PCNA protein and mRNA levels in cancer cells were blocked by 16.74%-84.21% (P < 0.05) and 23.27%-86.15% (P < 0.05) respectively. The proliferation activities of transferred cells were inhibited by 27.91%-62.07% (P < 0.01), with cloning formation abilities being decreased by 50.81% (P < 0.01). It was concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique, which could serve as an ideal strategy for gene therapy of bladder cancer.
Subject(s)
Humans , Cell Division , Cell Line, Tumor , Cloning, Molecular , DNA, Complementary , DNA, Neoplasm , Metabolism , Eukaryotic Cells , Metabolism , Gene Expression , Genetic Vectors , Proliferating Cell Nuclear Antigen , Genetics , RNA, Antisense , Genetics , Transfection , Urinary Bladder Neoplasms , Genetics , MetabolismABSTRACT
The resistin-like molecules(RELMs)are a novel protein family with tissue-specific distribution.Recent evidences suggest their important roles in type II diabetes mellitus,inflammation,immunological reactions and cell proliferation,which provoked many interests for the researchers.This overview summarizes the structure,tissue distribution,biological functions,regulatory pathways,and their relationship with diseases of these family members.
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Aim To explore the growth inhibition effects of jasmonates on human neuroblastoma SH-SY5Y cell line,and to investigate its mechanisms.Methods After administration of 0.5~2.5 mmol?L-1 jasmonates for 6~24 hrs, the growth inhibition rates of SH-SY5Y cells were studied by MTT colorimetry.Cell cycle phases were assayed by propidium iodide staining flow cytometry. Cellular apoptosis was inspected by Hoechst 33258 fluorescent staining and Annexin V-FITC and propidium iodide staining flow cytometry.Gene expressions of PCNA, cyclin D1 and N-myc were determined by reverse transcription polymerase chain reaction.Results Jasmonates inhibited the growth of SH-SY5Y cells in a dose-and time-dependent manner,while the methyl jasmonate was the most efficient. After administration of 0.5 to 2.5 mmol?L-1 of methyl jasmonate for 24 hrs,the growth inhibition rates of cells reached 5.75%~88.7%(P