Harmine Inducing Apoptosis In Human Gastric Adenocarcinoma SGC-7901 Cells

[Background]Peganum hannala L is defined as one of the perennial herbs in tribulus division, which is redundant in the west and north-west plateau of China. It was first reported by Chinese researchers that the harmala had the anti-cancer activity in 1970's. The crude solution of the harmala was used to treat the patients of some kinds of malignant tumor (such as esophageal carcinoma;carcinoma of gastric cardia;gastric carcinoma;colon carcinoma;lymphadenosarcoma) who were not operated or not suited to operation in cilinical, and such therapy had some good effect. This indicated the finely future of exploiting the harmala as an anti-cancer agent.The recent pharmacological research found that harmine and harmaline played the mainly role in anti cancer activity among the harmala compounds. And the harmine be the most important. The experiments indicated that these two compounds could inhibit some malignant tumor cell line significantly in vivo, such as the S-180 cell, hepatoma H22 cell, leukemic cell K562, hunman cervical carcinoma Hela cell. But the firmly mechanism of the cyto -inhibition activity of harmine and harmaline was rarely reported, thus the really mechanism on how the harmala take the anti cancer affection is not clear now .Gastric cancer is the most frequent malignant tumor in the digestive system,and its mortality rate is top two. Operation treatment was preferable to gastric cancer patients, but there lacked effective treatment to the cases who were not suit orrejected to operation for some reasons. There were just several case reports on the managing of gastric cancer patients with harmala agent, but lacks of researches in vivo or vitro about the activity on gastric cancer cell by the effective compounds of harmala in literature. In this research, in order to identify the course and the pharmacal mechanism of the anti cancer activity of harmine , and eventually provide a fundamental data for the clinlical usage of it, we extracted purified harmine, and studied its anti gastric cancer cell activity in vitro, exploring the induced inhibition and apoptosis biological behaviour of gastric adenocarcinoma SGC-7901 cell by different concentration of harmine.(Purpose]To investigate the function and the possible mechanisms of the apoptosis induced in human gastric adenocarcinoma SGC-7901 cells by Harmine.[Methods]The cell line survival inhibition ratio was detected by MTT assay;apoptosis cell was observed by cell morphology examination;apoptosis ratio was determined by flow cytometry;genomic DNA agarose gel electrophoresis detect the apoptosis ladder, RT-PCR and Western Blot wre used to assay the mRNA and protein expression of the apoptosis related gene: Fas and Bcl-2.[Results](1) The survival of SGC-7901 cells was decreased. Harmine could effectively inhibit the growth of SGC-7901 cells with 1.25,2.5,5,10,20 ug/mL for incubating 48h,and the inhibiting rates were 18.8%, 6.1%, 41.2%, 54.0% 90.5% respectively (p<0.01), the IC50 is 8.34ug/ml;and after incubating 72h the inhibitory rate were 18.6%, 28.1%, 44.7%, 59.9%, 94.7% respectively (p<0.01), the IC50is 7.31ug/ml. (2) Apoptotic cells were observed by light microscope. After exposure to Harmine, the SGC-7901 cells presented some morphologic features of apoptosis,including cell shrinkage,nuclear condensation,DNA fragmentation and apoptotic bodies formation.(3)FCM analysis showed that the typical subdiploid peak of apoptosis before Gi phase increased. After SGC-7901 cells were treated with Harmine of 0,10,15,20 ug/mL for 48h,the apoptotic cell rates were 1.20 % ,2.19 % ,6.98 % ,34.94% respectively;and for 72h the apoptotic cell rate was 5.55%,7.33%, 12.88%,52.73% respectively. (4)Typical DNA Ladder were detected in DNA agarose gel electrophoresis. (5) After harmine disposaling of SGC-7901 cell for 24h, the RT-PCR detected mRNA expression strap gray scale ratio of 0, 5,10ug/ml fas were 0.8685,1.1576,1.3201 respectively;0,5,10ug/ml bcl-2 mRNA expression strap gray scale ratio were 1.6194, 1.0224, 1.0052 respectively. (6) After harmine desposaled of SGC-7901 cell for 24h, the 0,5,10ug/ml Fas protein expression strap gray scale ratio were 0.40,0.52,0.69 respectively;0,5,10ug/ml Bcl-2 protein expression strap gray scale ratio were 0.86,0.78,0.64 respectively.[Conclusions](1) Harmine could be able to inhibit the growth of human gastric adenocarcinoma SGC-7901 cell significantly, and inhibit ability was concentra- tion dependent within the concerntration from 0 to 20ug/ml. (2) Harmine could be able to induce apoptosis in human gastric adenocarcinoma SGC-7901 cells.(3)During Harmine inducing apoptosis in human gastric adenocarcinoma SGC-7901 cells, the expression of Fas was up- regulated, and the expression of Bcl-2 was down-regulated. (4)Beside inducing apoptosis in human gastric adenocarcinoma SGC-7901 cells, Harmine produced toxic effectin human gastric adenocarcinoma SGC-7901 cells, it was a multiple mechanism co- effective process.