| BackgroundGastric cancer is a common malignant tumor of the digestive system,and its mortality rate ranks second in all cancers.Surgery is the only effective treatment at present.However,more than half of patients had local recurrence or distant metastasis after radical resection of gastric cancer.Peritoneal metastasis is the main form of recurrence after radical resection of gastric cancer.This is the primary cause of death in patients with gastric cancer.Gastric cancer cells which fall off from the original cancer site into peritoneal cavity,play an important role in peritoneal metastasis of gastric cancer.These fall-off gastric cancer cells usually exist as two forms,one is isolated,single free cancer cell,the other are aggregates or multicellular spheroids(multicellular aggregates,MCAs).Some research found that dispersed intraperitoneal free cancer cells usually cause anoikis.However,the MCAs has the capability to resist anoikis,grow and proliferate in hypoxia environment in peritoneal cavity.The MCAs in peritoneal cavity may be the true "seeds" cells in peritoneal metastasis of gastric cancer.Therefore,it is of great significance to reveal the anoikis resistant molecular mechanism of gastric cancer MCAs for the treatment and prevention of peritoneal metastasis.The proto-oncogene C-src is the first cell cancer gene discovered in 1976,and its expression product,src protein,is a member of the Src family kinase(SFKs).It is also a kind of non-receptor-dependent Tyrosine protein kinase which was first found closely related to human disease.It plays a key role in the regulation of cancer cell growth,differentiation,development and other biological functions.In cell carcinogenesis,its expression product is highly activated or overexpressed in a variety of tumor cells.And the src gene isolated from cancer cells has a strong ability to transform normal cells.The current study found that c-Src protein is involved in many important intracellular signaling pathways,and is closely related with gastric cancer occurrence,development and prognosis.Such as c-Src protein can promote gastric cancer cell proliferate,promote tumor angiogenesis,participate in gastric cancer cell adhesion and invasion,and regulate tumor growth through growth factor receptor and growth factor interaction and so on.Previous studies have shown that the expression of Src in gastric cancer was significantly up-regulated.However,at home and abroad,there is no idea whether src plays an important role in the anti-anoikis of multicellular aggregates in the peritoneal metastasis of gastric cancer or not.Purpose1.Isolate and identify the native gastric cancer MCAs from ascites of gastric cancer patients with peritoneal metastasis.2.Test the expression of src protein in native gastric cancer MCAs and dispersed intraperitoneal free cancer cells3.Detect The expression of src protein in native gastric cancer MCAs and dispersed intraperitoneal free cancer cells4.Study the role of src on proliferation and biological function of 3D cultured gastric cancer MCAs in vitro through inhibiting the expression of src.5.Preliminarily reveal the role and mechanism of src in anoikis resistant of gastric cancer MCAs for the treatment and prevention of peritoneal metastasis.Methods1.Ascites was collected from chemotherapy-naive patients with peritoneal metastasis of gastric cancer.Native gastric cancer multicellular spheroids(MCAs)were isolated by filtration through a 40?m cell strainer(Becton Dickinson)and washed with phosphate-buffered saline(PBS)into a collection tube containing a 10% formalin solution for immediate fixation to make paraffin sections.HE staining was performed to confirm the existence of native gastric MCAs.2.The expression of src m RNA in native gastric cancer MCAs and dispersed intraperitoneal free cancer cells were detected by qRT-PCR.3.Three-dimensional(3D)culture of BGC823 and MKN45 is provided to simulate native gastric cancer MCAs in peritoneal cavity of patients with peritoneal metastasis.4.The expression of src protein and apoptosis related proteins in 3D cultured gastric cancer MCAs and monolayer cultured gastric cancer cells were detected by western blot.5.Lentiviral siRNA vectors of human Src protein were constructed and then transfected into gastric cell line BGC-823 and MKN45.The morphology and cell viability of 3D cultured gastric cancer MCAs were investigated by MTT.6.The effects of Src on apoptosis of BGC823 MCAs and MKN45 MCAs were detected by flow cytometry,and the effect of Src on the expression of apoptosis related proteins in BGC823 MCAs and MKN45 MCAs was detected by western blot.Results1.We first isolated the native gastric cancer MCAs from ascites of gastric cancer patients with peritoneal metastasis.The existence of native gastric MCAs was confirmed by the result of HE staining.2.The results of Real-time PCR showed that the expression level of Src in native gastric cancer MCAs cells was significantly higher than that in dispersed intraperitoneal free cancer cells(P<0.05).3.We successfully established the 3D culture pattern of gastric cancer cells in vitro to simulate native gastric cancer MCAs in peritoneal cavity.4.The results of western blot showed that the expression level of Src in 3D cultured gastric cancer MCAs cells was significantly higher than that in monolayer cultured gastric cancer cells(P<0.05).5.Human src Lentiviral siRNA vectors were successfully constructed.Inhibiting the expression of Src in gastric cancer cells BGC823 and MKN45 could significantly inhibit their ability of forming MCAs,and obviously affect the activity of the MCAs.6.The results of flow cytometry showed that the inhibition of src could significantly induce the apoptosis of BGC823 MCAs and MKN45 MCAs.Western blot revealed that inhibiting the expression of Src could default the anoikis-resistant ability of BGC823 MCAs and MKN45 MCAs by inhibiting the expression of anti-apoptotic protein Bcl-2 and promoting the expression of pro-apoptotic protein Caspase-3.Conclusion1.In this study,we successfully isolated native gastric cancer MCAs from ascites of gastric cancer patients with peritoneal metastasis.For the first time we confirmed that gastric cancer MCAs in peritoneal cavity,as a separate functional unit with anoikis resistant ability,were the true "seeds" cells in peritoneal metastasis of gastric cancer.2.Src protein,the express product of oncogene c-src,was significantly higher expressed in the gastric cancer MCAs than in monolayer cultured gastric cancer cells.Inhibiting the expression of Src could increase the apoptosis rate of gastric cancer MCAs by upregulating the expression of pro-apoptotic protein Caspase-3 and downregulating the expression of anti-apoptotic protein Bcl-2.Our study confirmed that Src protein plays an important role in anoikis resistant of gastric cancer MCAs for the first time. |
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