Tissue factor pathway inhibitor-2 (TFPI-2) that inhibits plasmin,trypsin and matrix metalloproteinases is a broad-spectrum inhibitor of serine proteinase.TFPI-2 can accommodate the invasion and metastasis of human non-small-cell lung cancer by maintaining the integrity of extracellular matrix and regulating the angiogenesis and apoptosis of tumor cells.It has been a new target for gene therapy of cancers.

OBJECTIVETo investigate the inhibitory effect of classic demethylating drug 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human lung adenocarcinoma cells in nude mouse xenograft models, and to observe its effect on methylation status and expression of TFPI-2 gene in the nude mouse xenograft tissues.

METHODSThe nude mouse xenograft model was established by subcutaneous inoculation of human lung adenocarcinoma A549 cells. According to different doses of 5-Aza-CdR, the tumor-bearing nude mice were randomly divided into experimental groups (0.5 mg/kg group, 1 mg/kg group, 2 mg/kg group) and control group (0 mg/kg group). The tumor growth in the nude mice was observed. The methylation status and the expression of TFPI-2 gene mRNA and protein were detected by methylation specific polymerase chain reaction, real-time fluorescent quantitative polymerase chain reaction and Western blot assay.

RESULTSThe nude mice were euthanized at 28 days after intraperitoneal injection of 5-Aza-CdR. The body weight of tumor-bearing nude mice was (27.12 ± 0.38) g in the 0 mg/kg group, (26.80 ± 0.18) g in the 0.5 mg/kg group, (26.67 ± 0.28) g in the 1 mg/kg group, and (26.50 ± 0.26) g in the 2 mg/kg group, showing no significant difference among them (P > 0.05). The volume of xenograft tumors in the 0 mg/kg group was (709.22 ± 2.87)mm³, (400.67 ± 2.68)mm³ in the 0.5 mg/kg group, (285.71 ± 2.91)mm³ in the 1 mg/kg group, and (230.44 ± 3.15)mm³ in the 2 mg/kg group, showing a significant difference (P < 0.05). There were complete methylation of TFPI-2 gene in the 0 mg/kg group, incomplete methylation in the 0.5 and 1 mg/kg groups, and unmethylation in the 2 mg/kg group. The relative mRNA level in the 0, 0.5, 1, 2 mg/kg groups were 1.00 ± 0.00, 1.67 ± 0.07, 3.40 ± 0.24, and 5.55 ± 0.61, respectively (P < 0.05). The relative expression level of TFPI-2 protein in the 0, 0.5, 1, 2 mg/kg groups was 0.18 ± 0.02, 0.36 ± 0.01, 0.64 ± 0.02, and 0.81 ± 0.20, respectively (P < 0.05).

CONCLUSIONS5-Aza-CdR suppresses the tumor growth of human lung adenocarcinoma cells in nude mouse xenograft models, and induces expression of TFPI-2 gene in the xenograft tumor cells. The mechanism might be that 5-Aza-CdR induces re-expression of demethylated TFPI-2 gene by demethylation, and thus inhibits the growth and proliferation of human lung adenocarcinoma cells.

Adenocarcinoma ; drug therapy ; pathology ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; DNA Methylation ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; genetics ; Heterografts ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Mice ; Mice, Nude ; RNA, Messenger ; metabolism ; Random Allocation

Objective To investigate the inhibitory effect of classic demethylating drug 5?aza?2′?deoxycytidine( 5?Aza?CdR ) on the growth of human lung adenocarcinoma cells in nude mouse xenograft models, and to observe its effect on methylation status and expression of TFPI?2 gene in the nude mouse xenograft tissues. Methods The nude mouse xenograft model was established by subcutaneous inoculation of human lung adenocarcinoma A549 cells.According to different doses of 5?Aza?CdR, the tumor?bearing nude mice were randomly divided into experimental groups (0.5 mg/kg group, 1 mg/kg group, 2 mg/kg group) and control group (0 mg/kg group). The tumor growth in the nude mice was observed. The methylation status and the expression of TFPI?2 gene mRNA and protein were detected by methylation specific polymerase chain reaction, real?time fluorescent quantitative polymerase chain reaction and Western blot assay. Results The nude mice were euthanized at 28 days after intraperitoneal injection of 5?Aza?CdR. The body weight of tumor?bearing nude mice was (27.12±0.38)g in the 0 mg/kg group, (26.80±0.18)g in the 0.5 mg/kg group, (26.67±0.28)g in the 1 mg/kg group, and (26.50±0.26)g in the 2 mg/kg group, showing no significant difference among them (P>0.05). The volume of xenograft tumors in the 0 mg/kg group was (709.22±2.87)mm3, (400.67±2.68)mm3 in the 0.5 mg/kg group, (285.71±2.91)mm3 in the 1 mg/kg group, and (230.44 ±3.15)mm3 in the 2 mg/kg group, showing a significant difference (P<0.05). There were complete methylation of TFPI?2 gene in the 0 mg/kg group, incomplete methylation in the 0.5 and 1 mg/kg groups, and unmethylation in the 2 mg/kg group. The relative mRNA level in the 0, 0.5, 1, 2 mg/kg groups were 1.00±0.00, 1.67±0.07, 3.40±0.24, and 5.55±0.61, respectively (P<0.05). The relative expression level of TFPI?2 protein in the 0, 0.5, 1, 2 mg/kg groups was 0.18±0.02, 0.36±0.01, 0.64±0.02, and 0.81±0.20, respectively (P<0.05). Conclusions 5?Aza?CdR suppresses the tumor growth of human lung adenocarcinoma cells in nude mouse xenograft models, and induces expression of TFPI?2 gene in the xenograft tumor cells. The mechanism might be that 5?Aza?CdR induces re?expression of demethylated TFPI?2 gene by demethylation, and thus inhibits the growth and proliferation of human lung adenocarcinoma cells.

Objective To investigate the inhibitory effect of classic demethylating drug 5?aza?2′?deoxycytidine( 5?Aza?CdR ) on the growth of human lung adenocarcinoma cells in nude mouse xenograft models, and to observe its effect on methylation status and expression of TFPI?2 gene in the nude mouse xenograft tissues. Methods The nude mouse xenograft model was established by subcutaneous inoculation of human lung adenocarcinoma A549 cells.According to different doses of 5?Aza?CdR, the tumor?bearing nude mice were randomly divided into experimental groups (0.5 mg/kg group, 1 mg/kg group, 2 mg/kg group) and control group (0 mg/kg group). The tumor growth in the nude mice was observed. The methylation status and the expression of TFPI?2 gene mRNA and protein were detected by methylation specific polymerase chain reaction, real?time fluorescent quantitative polymerase chain reaction and Western blot assay. Results The nude mice were euthanized at 28 days after intraperitoneal injection of 5?Aza?CdR. The body weight of tumor?bearing nude mice was (27.12±0.38)g in the 0 mg/kg group, (26.80±0.18)g in the 0.5 mg/kg group, (26.67±0.28)g in the 1 mg/kg group, and (26.50±0.26)g in the 2 mg/kg group, showing no significant difference among them (P>0.05). The volume of xenograft tumors in the 0 mg/kg group was (709.22±2.87)mm3, (400.67±2.68)mm3 in the 0.5 mg/kg group, (285.71±2.91)mm3 in the 1 mg/kg group, and (230.44 ±3.15)mm3 in the 2 mg/kg group, showing a significant difference (P<0.05). There were complete methylation of TFPI?2 gene in the 0 mg/kg group, incomplete methylation in the 0.5 and 1 mg/kg groups, and unmethylation in the 2 mg/kg group. The relative mRNA level in the 0, 0.5, 1, 2 mg/kg groups were 1.00±0.00, 1.67±0.07, 3.40±0.24, and 5.55±0.61, respectively (P<0.05). The relative expression level of TFPI?2 protein in the 0, 0.5, 1, 2 mg/kg groups was 0.18±0.02, 0.36±0.01, 0.64±0.02, and 0.81±0.20, respectively (P<0.05). Conclusions 5?Aza?CdR suppresses the tumor growth of human lung adenocarcinoma cells in nude mouse xenograft models, and induces expression of TFPI?2 gene in the xenograft tumor cells. The mechanism might be that 5?Aza?CdR induces re?expression of demethylated TFPI?2 gene by demethylation, and thus inhibits the growth and proliferation of human lung adenocarcinoma cells.