The incidence of GC is high in China. As many GC patients are already in the intermediate and late stages at the time of diagnosis, chemotherapy often becomes the mainstay of treatment for these patients. However, the severe adverse effects from the currently available chemotherapy drugs were often intolerable and prevent many chemotherapy patients from enduring to the end. It is therefore an urgent task to find new therapeutic targets for the treatment of GC patients. ω-3 PUFA, an eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) either alone or their combination, are reported to have an anti-tumor effect by inducing apoptosis of cancer cells[11, 12]. Study showed that ω-3 PUFAs inhibited the proliferation of colorectal cancer cells and induce their apoptosis in vitro. The administration of ω-3 PUFAs prevented the occurrence of colorectal cancer induced by AOM/DSS in mice. PUFAs may also improve the effectiveness of tumor therapy by increasing the sensitivity of tumor cells to conventional treatment. However, the specific mechanism was still unknown. Therefore, BALB/C nude mice were selected as experimental animals in this study, and the GC SGC7901 cell line was used to establish a GC subcutaneous xenograft tumor model. Using ω-3 PUFAs lipid emulsion and chemotherapy drug 5-FU for drug intervention in nude mice, the impact of ω-3 PUFAs lipid emulsion on tumor tissue growth and its anti-tumor effect combinated with 5-Fu were observed to explore the possible mechanism.
Compared with the NC control group, ω-3 PUFAs fish oil emulsion or 5-FU infusion alone showed no obvious inhibitory effect on the growth of the transplanted tumors in the nude mouse model, but the volume and weight of the transplanted tumors were significantly decreased in the combinated treatment group. The HE staining result showed that the tumor tissues had varying degrees of cell necrosis accompanied with the decreased number of small blood vessels and tissue structural destruction in the three intervention groups, especially these in the combinated group, suggesting that ω-3 PUFAs fish oil emulsion could increase the inhibitory effect of 5-fu on the growth of GC.
In recent years, the relationship between ω-3 PUFAs and tumor proliferation, invasion and metastasis has aroused increasing attention. Tumor proliferation invasion and metastasis are affected by various factors. RhoA and RhoC in the Rho family not only play a key regulatory role in tumor cell proliferation, invasion and metastasis but also changed the morphology of vascular endothelial cells and promoted tumor angiogenesis. Meanwhile, ROCK, the downstream molecule of RhoA and RhoC, is regulated by the interaction between Rho GTP enzymes, and promotes the generation of actin-mediated contractile force through phosphorylation of a variety of downstream target proteins to control cell movement and metastasis[15, 16]. It was found in our study that combination ω-3 PUFAs with 5-FU reduced the levels of RhoA both in the mRNA or protein level. Some studies also found that the levels of RhoA, RhoC and ROCK1 was closely related to the proliferation, invasion and metastasis of breast, prostate and liver cancer cells[17, 18], Therefore, in vivo, we considered ω-3 PUFAs combined with 5-FU could inhibited the level of RhoA and ROCK1 proteins, which may probably further affect the proliferation,invasion and apoptosis of GC cells. Howerver, we did not observe the variation of RhoC which suggested Rhoc was not probably related to the proliferation, invasion and metastasis of the GC cells at least in this study.
Induction of tumor cell apoptosis plays an important role in tumor therapy and it is an important target of many therapeutic strategies. In previous studies, Marnie found DHA DOX-treated mice had 50% smaller tumors than control mice and the level of Bcl-2 decreased by 24%, thought the supplementation of DHA may amplificate the effect of DOX on pro-apoptosis in MDA-MB-231 Breast Cancer Cells. It was also reported that the combination of ω-3 PUFAs fish oil emulsion and 5-FU significantly reduced the expression level of anti-apoptotic protein Bcl-2 in tumor tissues, and increased the expression level of Bax protein. And our research found that treatment with 5-FU alone or combined with ω-3 PUFAs decreased the level of Bcl-2, and the latter has a more pronounced effect, which indicated the addition of ω-3 PUFAs may enhance the killing effect of 5-FU on tumor cells. Related studies showed that Bcl-2 family promoted and inhibited apoptosis by regulating the release of mitochondrial downstream effector molecules. In the colon cancer rat model, apoptosis of the animals fed with fish oil was enhanced and the anti-apoptotic protein Bcl-2 was expressed at a lower level. Bcl-2 is located in the mitochondrial endoplasmic reticulum, and overexpression can inhibit the activity of caspase[17, 23]. By increasing the permeability of cell membrane, Bax induced apoptosis, released cytochrome C from mitochondria, and activated caspase-3 and the caspase activation pathway, leading to the shearing of Parp protein[12, 24]. Most caspases normally exists in inactive precursor form when sites in the pro-enzyme molecules are cleaved by aspartate proteases to form polymers that are activated and then play a role in promoting apoptosis. Among that, Casepase-9 is located upstream of the apoptosis cascade reaction and is an important starting factor in the apoptosis pathway of the cell-dependent mitochondrial apoptosis. In this study, we also found treatment with 5-FU alone or combined with ω-3 PUFAs activated the protein of caspase-9 and result in the cleaving of Parp, which is considered to be an important indicator of apoptosis and also commonly considered as a substrate for the action of caspases. However, adding ω-3 PUFAs alone has no significant effect on apoptosis, which probably means ω-3 PUFAs is only a role in promoting the chemotherapy effect of 5-FU or its dose is too low to play the role of apoptosis alone in this study. In addition, changes in some apoptosis proteins such as Caspase-3,Caspase-8, Bax were not observed in all intervent groups,which also should be suggested for a further study. Above that, It suggested ω-3 PUFAs fish oil emulsion combined with 5-FU may affect the mitochondrial apoptosis pathway to inhibit the growth of mouse xenografts.
In summary, ω-3 PUFAs combined with 5-FU may inhibit proliferation, invasion by reducing the levels of RhoA and ROCK1, ultimately affecting the growth of GC and promoting apoptosis of tumor cells by down-regulating the expression of bcl-2, caspase-9 and up-regulating cleaved Parp. This study may provide in vivo experimental data for clinical treatment of GC.