Gastrin In Combination With Trastuzumab Shows Enhanced Antitumor Activity In Human Gastric Cancer Cell Lines And Xenograft Model

Background:In this manuscript we reported the synergistic antitumor effect of gastrin in combination with trastuzumab on gastric cancer cell lines and xenograft model, and revealed underlying mechanism of Cholecystokinin B receptor (CCKBR) and its downstream factors during the synergistic treatment of cancer cells.Methods:1. Cell counting and colony formation assay were used to estimate the actitumor effect of gastrin and trastuzumab on HER2-positive (NCI-N87) and HER2-negative (SGC7901) gastric cancer cells, and flow cytometry was used to determine the cell cycle arrest.2. The protein level of CCKBR in SGC7901and NCI-N87cells after treated with gastrin and trastuzumab were detected by Western-blot. The mRNA level of CCKBR is each cell lines was determined by RT-PCR The CHX and MG132experiment were employed to estimate the CCKBR protein stability. Over-expression of pEGFP-CCKBR in SGC7901cells was also used to investigate the membrane localization of GFP tagged CCKBR under the gastrin and trastuzumab treatment.3. A set of proteins, CCKBR, AE1, AE2, P16, CyclinD1and β-catenin were analyzed by western blot to understand the inhibition effect of CCKBR and its downstream factors on cell proliferation. Subcellular fractionation assay was used to determine the location of the p16protein in SGC7901cells after treated with gastrin and trastuzumab.4. The xenograft animal model was developed to study the synergistic anti-tumor effects in vivo. Tumors was subject to immunohistochemical staining and western-blot. CCKBR, AE1AE2, P16, CyclinD1and etc. were measured to understand the impact of gastrin and trastuzumab on the expression of CCKBR and its downstream signaling pathway.Results1. HER2expressed only in NCI-N87cells among7gastric cancer cell lines and a nonmalignant gastric cell line GES-1cells while CCKBR expression was observed in all the above gastric cell lines.2. Trastuzumab significantly inhibited the proliferation of HER2-positive NCI-N87cells but had no inhibitory effect on HER2-negative SGC7901cells. Conversely, gastrin suppressed the growth of both cell lines. Cell counting and colony formation assay indicated the synergistic tumor growth inhibition effect of combined treatment of gastrin/trastuzumab on the proliferation of SGC7901and NCI-N87, compared to the monotherapy. Moreover, flow cytometry results showed that gastrin/trastuzumab treatment synergistically arrested SGC7901cells at G0/G1phase.3. Gastrin/trastuzumab treatment synergistically up-regulated the expression of CCKBR. Conventional PCR and Real-time PCR showed that gastrin or trastuzumab treatment did not affect CCKBR mRNA level in SGC7901cells but reduced the CCKBR protein stability and ubiquitination.4. Western-blot results showed gastrin/trastuzumab treatment could down-regulate p16, AE1, CyclinD1and β-catenin but up-regulate AE2and in the meantime facilitated cytoplasmic p16to transport to the nucleus.5. In vivo study found the similar synergistic inhibition effect as in vitro. In addition to the western blot results, immunohistochemistry showed the significant increase of membrane CCKBR after gastrin/treatment.Conclusions:1. Gastrin and trastuzumab have shown synergistic anti-tumor activity in vivo and in vitro;2. Gastrin and trastuzumab synergistically increased the abundance of CCKBR protein and promoted its membrane localization. The up-regulation of CCKBR thence magnified the gastrin signal and activated the downstream anti-tumor pathways.3. The up-regulated CCKBR facilitated the gastrin binding and inhibited the interaction of p16with AE1and AE2via down-regulating the expression of cytoplasmic p16, resulting in the AE1degradation, AE2release and membrane localization of AE2protein. The up-regulated funtional AE2protein led to the acidification of tumor cells, Wnt/β-catenin pathway inactivation, Cyclin D1decrease, and thereby inhibited gastric cancer cell proliferation.