Apoptotic effect of Epigallocatechin-3-gallate on the human gastric cancer cell line MKN45 via activation of the mitochondrial pathway

To investigate whether Epigallocatechin-3-gallate （EGCG） can induce apoptosis of the gastric cancer cell line MKN45 and its apoptotic pathway.METHODS： To determine this, apoptotic rates of MKN45 cells after EGCG treatment with or without caspase-3 inhibitor were evaluated by Annexin V-FITC ＋ PI staining The influence of EGCG on the activity of caspase-3 in the MKN45 cells was determined by ELISA. By Rhodamine123 staining, the membrane potential change of the mitochondrion was also investigated, and mRNAs and protein expression of the bcl-2 family were analyzed by RT-PCR and Western blot.RESULTS： EGCG can induce apoptosis of MKN45 cells in time- and dose-dependent manner. Eight hours after EGCG treatment, the activity of caspase-3 in the MKN45 increased, especially 12 h after treatment. The mitochondrial membrane potential was significantly weakened 4 h after EGCG insult. The mRNA and protein expression levels of pro-apoptotic members, such as Bax, Bid and Bad, were upregulated gradually as treated time increased. Moreover, the mRNA and protein expression levels of anti-apoptotic members, such as Bcl-xL and Bcl-2, were inhibited. CONCLUSION： These data support that EGCG can induce apoptosis of the human gastric cancer cell line MKN45, and the effect is in a time- and dose-dependent manner. The apoptotic pathway triggered by EGCG in MKN45 is mitochondrial-dependent.

Methylation of PTCH1a gene in a subset of gastric cancers

AIM： To establish if PTCHla transcriptional regulation region （TRR） is methylated in gastric cancer and its influence in gastric tumorigenesis.METHODS： The CpG islands in PTCHla TRR were analyzed by Methyl Primer Express v1.0 software. The region from -643 to -355 bp （the transcription initiation site of PTCHla was designated as 0） that contained 19 CpG sites was chosen for bisulfitesequencing PCR （BSP） and methylation-specific PCR （MSP） detection. The gastric cancer cell line AGS was treated with 5-aza-2′-deoxycytidine （5-Aza-dC; 1 μmol/L） for 3 d. Alterations in PTCHla TRR methylation in treated AGS cells was measured through BSP clone sequences, and their PTCH1 expression was measured by quantitative RT-PCR. The cell cycle and apoptosis were observed with flow cytometry through propidium iodide （PI） staining or annexin V/PI double staining. The prevalence of PTCHla TRR methylation was investigated in 170 gastric cancer tissue samples and the adjacent normal tissues by MSP. The correlation of PTCH1a TRR methylation with PTCH1 expression or with patients＇ clinical features was analyzed.RESULTS： Methylation of PTCHla TRR was observed in AGS ceils and a subset of gastric cancer tissues （32%, 55/170）, while no methylation amplification products were observed in any normal tissues by MSP. The methylation of PTCH1α TRR was correlated negatively with PTCH1 expression （Spearman＇s r = -0.380, P = 0.000）. However, methylation of PTCHla TRR was not related to the gastric cancer patients＇ clinical features, such as sex, age of onset, clinical stage, lymph node metastasis or histological grade. The methylation of PTCHla TRR in AGS cells was almost converted to non-methylation after 5-Aza-dC treatment, which increased PTCH1 expression （5.3 ± 2.5 times; n = 3） and apoptosis rate （3.0 ± 0.26 times; P 〈 0.05; n = 3）.CONCLUSION： Methylation of PTCH1α TRR is present in a subset of gastric cancers and correlated negatively with PTCH1 expression. This may be an early event in gastric tumorigenesis and a new treatment target.

Astragalus extract inhibits destruction of gastric cancer cells to mesothelial cells by anti-apoptosis

AIM： To determine the inhibitory effect of Astragalus memebranaceushas on gastric cancer cell supernatant- induced apoptosis of human peritoneal mesothelial cells. METHODS： Human peritoneal mesothelial cell （HPMC） line HMrSV5 was co-incubated with gastric cancer cell supernatant （MKN45） and/or Astragalus memebranaceushas. Morphological changes in gastric cancer cells were observed under phase-contrast microscope. Quantitative cell damage was determined by MTT assay. Apoptosis was determined under transmission electron microscope and quantified by detectinr, acridine orange/ethidium bromide-stained （AO/EB） condensed nuclei under fluorescent microscope or by flow cytometry. Expressions of Bcl-2 and Bax were evaluated with immunostaining.

Relationship between preoperative staging by endoscopic ultrasonography and MMP-9 expression in gastric carcinoma

AIM: To investigate the relationship between the staging by endoscopic ultrasonography (EUS) and the expression of carcinoma metastasis associated gene in the patients with gastric carcinoma. METHODS: Sixty-three patients with gastric cancer were diagnosed by electric gastroscopy and EUS. The preoperative staging of gastric cancer was measured by EUS and compared with pathologic staging and MMP-9 expression. Peripheral serum level of MMP-9 was measured with enzyme-linked immunosorbent assay (ELISA), while the expression of MMP-9 protein was tested with immunohistochemistry and hybridization in situ in the gastric carcinoma tissues. RESULTS: The total accuracy of EUS in estimating invasive depth of gastric cancer was 80.95%, while that in estimating lymphatic metastasis was 73.02%. Serum MMP-9 levels were consistent with the expression of MMP-9 protein and MMP-9 mRNA in tissue, a result closely correlated with invasive degree, staging with EUS and lymphatic metastasis in gastric cancer (P < 0.05). The total accuracy of estimating invasive depth in gastric cancer was 95.22% using both EUS and MMP-9. CONCLUSION: The MMP-9 level of preoperative serum presents the reference value for preoperative staging by EUS in the patients with gastric cancer. When serum MMP-9 level in gastric cancer is significantly high, physicians should pay closer attention to the metastasis which reaches the serosa or beyond. Combining EUS and MMP-9 improves the accuracy in deciding the invasion and metastasis in the patients with gastric carcinoma.