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<p><b>OBJECTIVE</b>To observe the effect of p27 gene recombinant adenovirus combined with Chinese medicine Pientzehuang ([characters: see text]) on the growth of xenografted human osteosarcoma in nude mice.</p><p><b>METHODS</b>Tissue transplantation was used to construct the orthotopic model of human osteosarcoma Saos-2 cell in nude mice. Thirty tumor-bearing nude mice were randomly divided into 5 groups with 6 mice in each group: blank control group (model of osteosarcoma), empty vector group (recombinant adeno-associated virus-multiple cloning site), Pientzehuang group, p27 gene group and combined treatment group (p27 gene combined with Pientzehuang). The effect of combined treatment on human osteosarcoma was analyzed through the tumor formation, tumor volume and inhibition rate of tumor growth. The expression of p27 was measured by immunohistochemical staining and Western blot.</p><p><b>RESULTS</b>The orthotopic model of osteosarcoma in nude mice was successfully constructed. The general appearance of tumor-bearing nude mice in Pientzehuang and p27 gene groups was markedly improved compared with the blank control group; and in the combined treatment group it was significantly improved compared with the Pientzehuang and p27 gene groups. The tumor growth in the Pientzehuang and p27 gene groups was significantly inhibited compared with the blank control group P<0.05); while in the combined treatment group it was markedly inhibited compared with the Pientzehuang and p27 gene groups (P<0.05). The rates of tumor growth inhibition were 34.1%, 56.5% and 63.8% in the Pientzehuang, p27 gene and combined treatment groups, respectively. Meanwhile, the protein expression of p27 gene in the p27 gene group was significantly increased compared with the blank control group (P<0.05); and it was significantly increased in the combined treatment group compared with the p27 gene and Pientzehuang groups (P<0.05).</p><p><b>CONCLUSION</b>p27 gene introduced by adenovirus combined with Pientzehuang can inhibit the growth of human osteosarcoma cell Saos-2 in nude mice.</p>
Subject(s)
Animals , Humans , Mice , Adenoviridae , Blotting, Western , Bone Neoplasms , Pathology , Cell Line , Cyclin-Dependent Kinase Inhibitor p27 , Genetics , Drugs, Chinese Herbal , Pharmacology , Immunohistochemistry , Mice, Inbred BALB C , Mice, Nude , Osteosarcoma , Pathology , Sarcoma, Experimental , Pathology , Transplantation, HeterologousABSTRACT
Objective To investigate the relationship between p27 gene methylation and pathology of colorectal carcinoma. Methods p27 gene methylation promotor region and p27 protein expression were detected respectively by methylation specificity polymerase chain reaction and immunohistochemical staining SP in 106 cases of colorectal carcinoma and each adjacent normal mucous membrane tissue and 22 cases of colorectal adenoma tissue. Results The positive expression rate of p27 gene methylation was statistically different in colorectal carcinoma tissue compared with normal mucous membrane and colorectal adenoma tissue (P＜0.05). Their positive expression rate were 59.4% (63/106), 18.2% (4/22) and 3.8%(4/106) respectively in colorectal carcinoma tissue,colorectal adenoma and normal mucous membrane tissue (P ＜ 0. 05). p27 gene methylation in poorly differentiated group was significantly higher than that in welldifferentiated group (48.0% vs. 24. 7%, P ＜0. 05), in Dukes-A + B stage group was significantly lower than that in Dukes C + D stage group(20. 0% vs. 41.2%, P ＜ 0. 05 ), and it was higher in lymph nodes metastases group than that in lymph nodes negative group(41.5% vs. 23. 1%, P ＜0. 05), that in positive serosa infiltration group was higher than negative serosa infiltration group(32. 5% vs. 24. 1%, P ＞ 0. 05 ).Conclusions Methylated p27 gene protein expression in colorectal carcinoma was significantly higher than normal mucous membrane and colorectal adenoma tissue. The methylation rate of p27 gene in colorectal carcinoma was significantly associated with tumor differentiation, invasive depth, Dukes stage, lymph node metastasis.
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Objective To detect p27 expression in rectal carcinoma and serum transforming growth factor ? 1 (TGF ? 1) level in these patients, and to elucidate the modulatory effect of serum TGF ?1 on p27 expression in rectal carcinoma. Methods Expression of p27 was measured in 37 cases of rectal carcinoma, 22 of rectal adenoma and 19 of normal control specimens by immunohistochemical staining using antibodies to p27. Serum level of TGF ?1 was measured in these patients by enzyme linked immunosorbent assay (ELISA) method. Results p27 protein was expressed in normal rectal tissue, rectal adenoma and rectal carcinoma, and the positive rate was 89.47%, 90.91% and 64.87%, respectively. The positive rate of p27 in rectal carcinoma was significantly lower than that of normal rectal tissue and rectal adenoma ( P =0.025). p27 was mainly located in nucleolus of normal rectal tissue and rectal adenoma, and the positive rate of p27 in cytoplasm of rectal carcinoma was higher than that of normal and rectal adenoma. The positives rates of serum TGF ?1 in normal group, rectal adenoma group and rectal carcinoma group were 21.05%, 27.27% and 51.35%( P =0.045),respectively. The expression of p27 related to histological differentiation, lymph node metastasis and infiltration depth. Serum level of TGF ?1 related to lymph node metastasis, infiltrated depth and CEA level. The positive rate of p27 in TGF ?1 negative group and positive group was 88.89% and 42.11%(Mantel Haenszel ? 2=6.755, P =0.009), respectively. Conclusion TGF ?1 may be useful in assessment of malignance and prognosis of rectal carcinoma. TGF ?1 can down regulate p27 expression in rectal carcinoma.
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Objective To investigate the expression of tumor growth tactor ?1 (TGF-?1) and p27 in gallbladder carcinoma and their relation to the development of the carcinoma. Methods The expression of TGF-?1 and p27 in 36 cases of gallbladder carcinoma was detected by SP immunohistochemical staining. Twenty cases of chronic cholecystitis were collected as control. Results The positive rate of TGF-?1 (63.9%) was higher than that of the control (10.0%), P
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Objective To study the effects of p27kip1 gene on the apoptosis in esophageal carcinoma cell lines EC 9706. Methods A replication deficient adenovirus vector encoding p27kip1(ad p27kip1) was constructed and human esophageal carcinoma cell lines EC 9706 were transduced in vitro. The cell apoptosis were determined by flow cytometry, TUNEL technique and DNA fragmentation analysis. Results Ad p27kip1 containing the p27kip1 sequence was successfully constructed and the virus titer was 1.24?10 12 CFU/ml. The transduction efficiency was 100% when multplicity of infection≥50. FCM analysis revealed a sub G 1 cell peak in ad p27kip1 transduced esophageal carcinoma cell lines. Agarose electrophoresis showed marked ladder. The difference of apoptotic index between the ad p27kip1 group and the control group was statistically significant(37.3?3.4 vs. 1.3?0.2, P
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Objective To explore the inhibition effect of recombinant mutant type(mt) p27 gene adenovirus (Ad-mt p27) transfection on the proliferation of colorectal cancer cell line SW480. Methods The recombinant mt p27 adenovirus vector was transfected into SW480 cells, western blotting analysis was applied to determine the p27 expression, and cell cycle distribution was analyzed using flow cytometry, and growth curve was drawn to observe the cell growth. Results There was high expression of p27 protein in the transfected SW480 cells. 77 9% cells were blocked in G 1/G 0 phase in the transfected group, which the percentage of the G 1/G 0 phase of Ad-LacZ transfected group and normal control group were 27 5% and 25 29% respectively. Ad-mt p27 transfection markedly inhibited SW480 cell growth in 24h and the inhibition effects was maintained for 7 days. Conclusion Recombinant adenovirus could efficaciously trasfect mt p27 gene into SW480 cells, and obtain high level mt p27 exprerssion. mt p27 markedly bolcked SW480 cell cycle progression and prominently inhibited SW480 cell growth. The mutant p27 gene transfection may be a new way of gene therapy for colorectal cancer.
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Objective To investigate the effects of recombinant trichosanthin(rTCS) on methylation status and expression level of p27 gene in HeLa cells.Methods HeLa cells was treated by different concentration(20 ?g/mL,40 ?g/mL,and 80 ?g/mL) of rTCS for 48 h and then methylation-specific polymerase chain reaction(MSP) was used to detect the promoter methylation status of the p27 gene,real-time PCR was used to detect levels of p27 and DNMT1 mRNA,and Western blotting assay was used to detect expression level of p27 protein before and after treatment with rTCS.Results Low expression level and promoter methylation status of the p27 gene were detected in HeLa cells.Treatment with 40 ?g/mL rTCS totally demethylated p27 promoter.Treatment with 20 ?g/mL,40 ?g/mL or 80 ?g/mL rTCS resulted in a 2.22-,4.00-or 6.03-folds increase in p27 mRNA level,respectively,and also a great increase in p27 protein level.A high DNMT1 expression level was observed in HeLa cells and treatment with 40 ?g/mL rTCS resulted in a 78% decrease at the DNMT1 mRNA expression.Conclusion rTCS could reverse promoter hypermethylation and re-activate the expression of p27 gene by inhibiting DNMT1 expression in HeLa cells,which indicates its potential use in cancer therapy.