Investigation Of The Role Of Heme Oxygenase-1 On Peritoneal Metastasis Of Gastric Cancer And Its Involved Mechanisms

【Background】Heme Oxygenase-1 is abbreviated as HO-1(also called Heat-shock protein32 ), of which relative molecular weight is 32 kDa.HO-1 gene localized on human chromosome 22q13.It is a rate-limiting enzyme catalyzing degradation of heme and plays an important role in recycling of iron. Previous studies have focused on the role of HO-1 in cardiovascular diseases, which participates in cellular homeostasis maintaining and oxidative injury reduction, inflammatory response restriction, and cellular modulation of proliferation/apoptosis. Recently, some evidences in vivo and in vitro indicate HO-1 is closely related to tumor cell proliferation, apoptosis and angiogenesis. Highly expressed HO-1 activates growth and metastasis of tumors; however, HO-1 is upregulated in tumor tissues, especially in high malignant tumor and tumor metastasis. HO-1 siRNA transfected tumor cell repressed proliferation and metastasis. As far as gastric cancer was concerned, overexpressed HO-1 promoted neoplasia and cancer cell proliferation, and its carcinogenesis was related with activation of anti-apoptosis factor p21.But the role of HO-1 on peritoneal metastasis of gastric cancer was unknown. We have established gastric cancer cells GC9811-P with potent metastasis to the peritoneum in previous study, which developed peritoneal metastasis in 13 of 13 (100%) in mice, compared with only 20% of those implanted with parental GC9811. Tumor cell growth of GC9811-P in vitro was vigorous and its ability of proliferation was fast and the time of incubation period of GC9811 was short. Motility assays demonstrated higher motility of GC9811-P than of GC9811. After GC9811-P cells were planted to abdominal cavity in athymic mouse, the metastatic foci in the peritoneum developed extensively, which is similar with the procedures of human cancer cells shedding and adhering to abdominal cavity and invasion as well as angiogenesis of cancer. We have previously found that HO-1 protein is overexpressed in the GC9811-P cell line compared with its maternal line GC9811 cell line. Therefore, we hypothesized that HO-1 protein may play an important role in the peritoneal metastasis of human gastric carcinoma.【Objectives】To investigate the expression and significance of HO-1 protein in peritoneal metastasis of gastric cancer and examine the effect of HO-1 on proliferation , angiogenesis as well as apoptosis of high potential peritoneal metastasis of gastric cancer cell lines.【Methods and Results】Compared with matched adjacent nonneoplastic tissues, the expression of HO-1 in human peritoneal metastasis of gastric cancer tissue will be studied to illuminate the relationship between HO-1 and pathology parameter. The low or absent HO-1 protein expressed gastric cancer cell was obtained by HO-1 specific inhibitor incubated with high peritoneal metastasis of gastric cancer cell GC9811-P. Proliferation and angiogenesis as well as apoptosis of the low or absent HO-1 expressed cell were studied to illuminate the role of HO-1 on peritoneal metastasis of gastric cancer and its involved mechanisms. It was found HO-1 protein was expressed at a higher level in peritoneal metastasis of gastric cancer tissues than that in matched adjacent nonneoplastic tissues. HO-1 protein expression was correlated with differentiation of tumor tissues, especially low-differentiated tissue by immunohistochemistry staining. Also, the expression of HO-1 protein was found to be at higher levels in almost all peritoneal metastasis of gastric cancer tissues compared to adjacent tissues by Western blotting analysis. After high potential peritoneal metastasis of gastric cancer cell line GC9811-P were incubated with HO-1 inhibitor ZnPPIX, proliferration of GC9811-P was inhibited at the concentrations of 1×10^-4 to 1×10^-9 mol/L, and maximal inhibitory rate was 62.04±6.13%, which was produced by 1×10^-4 mol/L ZnPPIX, with IC50 was1.2×10^-5 mol/L. Flow cytometry was used to measure the apoptosis index of GC9811-P cells, and Western blot was used to observe the expression of HO-1 protien, Bcl-2 protein and Bax protein at appointed time. After GC9811-P cells were treated with 1×10-5 mol/L ZnPPIX for12, 18, 24, 48 hours, apoptosis index of GC9811-P cells was significantly increased compared with controls. The expression of HO-1 protein was decreased after 3 hours incubation with 1×10^-5 mol/L ZnPPIX. After HO-1 inhibitor zinc protoporphyrin IX and GC9811-P cells were incubated, proliferation of GC9811-P cells was found reduced with negative correlate with concentration in the range of 10^-9-10^-4mol/L. It showed that inhibiting the expression of HO-1 protein followed with proliferation stagnation of GC9811-P cells, so HO-1 protein may play an important role in proliferation of peritoneal metastasis of gastric cancer.After HO-1 inhibitor were incubated with the GC9811-P cells, apoptosis index of GC9811-P cell significantly increased, and apoptosis index was(61.1±5.8)% after incubated 48h; Cell cycle was arrested in G1/S phase, and G1/S phase was 88.2±6.7% after 48h. HO-1, Bcl-2, Bax protein contents were detected by Western blot. The results showed that inhibiting expression of HO-1 protein followed with Bcl-2/Bax protein expression decreased, especially after incubated with 24h. Also, the time point of HO-1 reduction was much earlier than that of Bcl-2/Bax, suggesting HO-1 protein may be upstream elements that regulate apoptosis factors Bcl-2/BaxChorioallantoic membrane test: HO-1 inhibitor zinc protoporphyrin IX and GC9811-P cells were incubated on the surface of chick embryo chorioallantoic membrane for 4-5d. The embryonic blood vessel branch points in chicken were significantly reducedcompared with controls (P <0.05), which indicated HO-1 play an important role in angiogenesis of peritoneal metastasis of gastric cancer cells.【Conclusions】HO-1 protein was highly expressed in peritoneal metastasis of gastric cancer and gastric cancer cell lines with high potential of peritoneal metastasis. High level of HO-1 expression might be correlated to the malignant potential of peritoneal metastasis of gastric cancer. Inhibition of HO-1 expression may surpress proliferation, angiogenesis of high potential peritoneal metastasis of gastric cancer cell lines and induce their apoptosis. HO-1 protein may play an important role in peritoneal metastasis of gastric cancer.