Study on Expression Condition Optimization of Two Recombinant Methioninases and Their Inhibitory Effects on Human Lung Adenocarcinoma Cells GLC / 中国药房

OBJECTIVE： To optimize the expression induction condition of two recombinant methioninases， and to investigate their inhibitory effects on the proliferation of human lung adenocarcinoma cells GLC. METHODS： Recombinant methioninases expression plasmid PGEX-4T1-4A1-MGL and PGEX-4T1-3B8-MGL were transfected into competent Escherichia coli Dh5α， and induced by isopropyl-β-D-thiogalactoside. Using the expression level of target protein as index， the initial OD600 nm value before induction， culture temperature and induction time were optimized by single factor test. The recombinant methioninase 4A1-MGL and 3B8-MGL were purified by affinity chromatography. The concentration of recombinant methioninase was detected by Coomassie blue method. The purity of the product was detected by sodium lauryl benzene sulfonate-polyacrylamide gel electrophoresis； its activity was detected by spectrophotometry. The proliferation of cells was detected by MTT assay after treated with low-dose， medium-dose and high-dose of recombinant methioninases （4A1-MGL or 3B8-MGL was 0.1， 0.2， 0.4 U/mL） for 24， 48， 74 h. Inhibitory rate of cells were calculated. RESULTS： The optimal induction condition of two recombinant methioninases included that initial OD600 nm of 0.9， culture temperature of 37 ℃， induction time of 5 h. The results of validation test showed that protein expression level of 4A1-MGL was 1.52±0.04， that of 3B8-MGL was 1.28±0.03 （RSD＜3％，n＝3）. After purification， the concentration， purity and activity of 4A1-MGL were （0.70±0.02）mg/mL， （96.42±3.15）％ and （0.45±0.02）    U/mg； and those of 3B8-MGL were （0.56±0.02）mg/mL， （97.43±2.96）％ and （0.91±0.03）U/mg. After treated with low-dose and medium-dose of 4A1-MGL and 3B8-MGL for 48 and 72 h， treated with high-dose of 4A1-MGL and 3B8-MGL for 24， 48 and 72 h， inhibitory rate of GLC cell was increased significantly， and high-dose group for 72 h was significantly higher than low-dose and medium-dose groups at same time point （P＜0.05）. CONCLUSIONS： The induction conditions of recombinant methioninase expression are successfully optimized in this study. The obtained 4A1-MGL and 3B8-MGL could inhibit the proliferation of GLC cells in a dose-dependent manner.

Synergetic Inhibition Effects of Methioninase and Cisplatin against the Growth of Human Lung Adenocarcinoma Cell Line GLC / 昆明医科大学学报

Objective To purify recombinant methioninase and investigate the synergetic inhibitory effect of methioninase and cisplatin on the proliferation of human lung adenocarcinoma cell line GLC. Methods Recombinant methioninase was purified with GST-column from supernatant after ultrasonic disruption of cultured Escherichia coli in the prokaryotic expression system pGEX-4T-1-Met/Dh5a. MTT assay was used to determine the inhibition rate of methioninase in combined with cisplatin on cell proliferation,and their synergistic effect was evaluated by using the q value judge method. Results The concentration of recombinant methionine cleaving enzyme was 0.22 mg/mL, the purity was 95%, and the activity was 0.568 IU/mg. After 72 h culturing, the inhibition rate of cisplatin methionine was 24.80%and 27.49%respectively,while the inhibition rate of the combined drugs was 66.80% ( =1.47>1.15) which showed a significant synergistic effect. Conclusion Both methioninase and cisplatin have the inhibition effect,and the combined drugs display a significant synergistic effect on the proliferation of GLC.

Research development of tumor treatment with methionine gamma-lyase / 生物医学工程学杂志

Methionine-dependent increase in tumor cells is a specific metabolic defect. This metabolic defect is also a target for selective treatment of cancer. Studies found that the methionine gamma-lyase (methioninase, L-methionine gamma-lyase) can specificly split the methionine of extracellular and intracellular, so it can strongly inhibit the growth of tumor cells and induce apoptosis of tumor cells. However, no effect on normal cells has been found, so the methionine gamma-lyase may play the anti-tumor role. We also explored in the present study another effect of methionine gamma-lyase as a single agent on DNA methylation levels and DNA synthesis, which may change as a result of deprivation of methionine, and thus may enhance anti-tumor effects. Animal studies and clinical trials showed that a variety of methionine dependent methionine gamma-lyase can eliminate the tumor cells. Therefore, methionine restriction is an effective anticancer strategy. Methionine gamma-lyase has shown great prospects as a new type of gene therapy. This article made a review of it.

The Biological Characteristics in vitro and Anti-Tumor Effect in vivo of Lung Cancer Cells Infected with Recombinant Adenovirus Expressing hTNF-? / 中国肿瘤生物治疗杂志

Objective: To study the biological characteristics of tumor cells infected with recombinant adenovirus expressing hTNF-?, investigate the antitumor effect of recombinant adenovirus. Methods: Human lung adenocarcinoma cell line Anip973 was infected with recombinant adenovirus expressing hTNF-?. Cell growth assay, colone formation test, flowcytometry assay and morphology were used to observe the effects on tumor cells. The hTNF-a gene, which was transduced into cancer cells mediated by recombinant adenovirus, was detected by PCR and agarose gel electrophoresis and its products were detected by ELISA assay. The intratumoral injection of rAd-LacZ and rAd-hTNF-? was carried out to evaluate their antitumor effects. Results: The liter of rAd reached 1010 PFU/ml and more than 90% Anip973 cells could be infected by 30MOI rAd. Except the surface structure and ultrastructure of tumor cells infected with rAd had a light change, cell growth abillity assay, colone formation test, flow cytometry assay showed no significant difference compared with that of the control cells. The TNF-? gene expression at 24 h increased greatly. Antitumor study indicated that on the tumor-bearing mice treated with rAd the tumor grew slowly. Tumor volume was significantly smaller and survive time was prolonged than that of controls. Conclusion: There was no significantly changes occurred on tumoral cells after infected with recombinant adenovirus expressing hTNF-?. The intratumoral injection of rAd-LacZ and rAd-hTNF-? could inhibit the growth of solid tumor.

in vitro the Biological Characteristicsof Adenovirus-CD80-Transduced Human Lung Cancer Cells / 中国肿瘤生物治疗杂志

Objective: To investigate in vitro the biological characteristics of AdCD80-infected human lung cancer cells on the basis of generation of replication-deficient hB7-1(CD80) recombinant adenovirus. Methods: Human CD80 gene was transduced into lung cancer cells mediated by recombinant adenovirus and then the expression of the gene was detected by PCR and agarose gel electrophoresis. The biological characteristics of the above cells were analysed with electron microscope, FACS and etc. Results: The titers of rAd reached to 10 10 PFU/ml and more than 90% Anip973 lung cancer cells could be infected by 30 MOI rAd. The growth curve and cloning efficiency of rAdCD80-infected Anip973 cells showed no significant difference compared with that of the control cells. The cell proliferation cycle of rAdCD80-infected 973 cells showed no change through FACS test. Having been infected by rAdCD80, the surface structure and ultrastructure of 973 cells had a little change. Conclusion: These results will lay foundation for tumor vaccines.

The biological characteristics in vitro and anti-tumor effect in vivo of lung cancer cells infected with recombinant adenovirus expressing hIL-2 / 中国免疫学杂志

Objective:To study the biological characteristics of tumor cells infected with recombinant adenovirus expressing hIL 2, and investigate the antitumor effect of the rAd Methods:Human lung adenocarcinoma cell line Anip973 was infected with recombinant adenovirus expressing hIL 2(rAd hIL 2) Cell growth assay, colony formation were used to observe the effects on tumor cells hIL 2 gene was transduced into cancer cells mediated by recombinant adenovirus, then detected by PCR and agarose gel electrophoresis and its products were detected by ELISA assay The intratumor injection of rAd LacZ, rAd hIL 2 was carried out to evaluate its antitumor effects Results:The titer of rAd reached 10 10 PFU/ml and more than 90% Anip973 cells could be infected by 30MOI rAd Growth inhibition assay, colony formation assay showed no significant difference compared with that of the control cells The 24 h IL 2 expression increased greatly Antitumor study indicated that the tumor grew slowly in the bearing tumor mice treated with rAd Tumor volume was significantly smaller and survive time was prolonged than that of controls Conclusion:There were no significantly changes of tumor cells infected with recombinant adenovirus expressing hIL 2 The intratumor injection of rAd LacZ?rAd hIL 2 could inhibit the growth of solid tumor