| Background5-Fluorouracil (5-Fu) and cisplatin(DDP) have been important chemotherapeutic agents for advanced gastric cancer, and FP(5-Fu/DDP)are basic chemotherapeutic scheme. The clinical effect of chemotherapy alone is limited as gastric cancer is naturally resistant to chemotherapy. Recent researches demonstrated combinations of targeted agents and chemotherapeutic agents could improve effect. Trastuzumab, a recombinant humanized monoclonal antibody that selectively targets the human epidermal growth factor receptor 2 protein (HER-2/neu, HER2), has proved to have good curative effect for patients with HER2 positive breast cancer both alone and in combination with chemotherapeutic agents, but the conclusions of several basic studies about trastuzumab treating gastric cancer were not consistent, and the result of related clinical trial has not been obtained. In this study, we firstly assessed the effect of trastuzumab in combination with 5-Fu and / or DDP on gastric cancer cell lines and explored interaction mechanism of trastuzumab and chemotherapeutic agents, then deduced the best scheme of trastuzumab in combination with 5-Fu and DDP, so provided theoretical basis for gastric cancer therapy.MethodFirstly, six gastric cancer cell lines were evaluated for HER2 protein expression by flow cytometry, and a breast cancer cell SK-BR-3 was used for positive control. Secondly, the growth inhibitory effects of 5-Fu or DDP in combination with trastuzumab, both alone and in combinations, were assayed by MTT. Then the differences of drug effects between HER2 positive and HER2 negative cells were compared and the best sequence of drug combination was determined. After that, molecular mechanisms of drug interaction on HER2 positive gastric cancer cell were explored including: 1) analyze the effects of drug combinations on HER2-PI3K-AKT signaling transduction by flow cytometry (HER2 protein) and Western Blot (AKT,pAKT andNFκB proteins); 2) analyze the effects of drug combinations on mRNA expression of targeted enzymes such as thymidylate synthase (TS) and nucleotide excision repair cross-complementation 1 (ERCC1) by RT-PCR; 3)analyze the influences of drug combinations on cell cycle changes by flow cytometry.Results1. Among the 6 gastric cancer cells, NCI-N87 and YCC-2 showed positivity of HER2 expression, and SGC7901,MGC803,BGC823 and AGS showed negativity.2. Trastuzumab alone had no growth inhibitory effects on gastric cancer cell lines, however, it augmented the cytotoxicity of chemotherapeutic agents in HER2 positivity cells. Sequential 5-Fu followed by trastuzumab and trastuzumab plus DDP followed by trastuzumab produced the marked inhibition effects, but trastuzumab followed by trastuzumab plus 5-Fu antagonized the cytotoxicity of 5-Fu.3. Trastuzumab down regulated HER2 protein and inhibited AKT phosphorylation, but did not alter nuclear NFκB expressin. 5-Fu and DDP did not influence the expression of pAKT and NFκB, but down regulated pAKT when in combination with trastuzumab. Concurrent of trastuzumab and DDP markedly inhibited AKT phosphorylation and inhibited NFκB from entering into nucleus.4. Trastuzumab inhibited mRNA expression of ERCC1, but did not influence mRNA expression of HER2 and TS. 5-Fu alone decreased TS mRNA, but combinations of trastuzumab and 5-Fu did not show any enhanced inhibition effects on TS mRNA expression. DDP alone up regulated ERCC1 mRNA, and combination trastuzumab and DDP made ERCC1 mRNA comeback to normal level.5. Trastuzumab resulted in G0/G1 phase arrest and a decrease in S phase. G0/G1 phase arrest is also induced by 5-Fu, and S phase declined when simultaneous exposure to trastuzumab and 5-Fu. A marked S-phase block is induced by DDP, and G2/M-depletion was most pronounced when simultaneous combination of trastuzumab and DDP.Conclusion1. Trastuzumab enhanced the cytotoxicity of chemotherapeutic agents in HER2 positivity cells with a schedule-dependent character. That HER2-PI3K-AKT signaling transduction was inhibited by trastuzumab may explain the synergic effects.2. Trastuzumab in combination with DDP is more applicable to HER2 positivity gastric cancer. Trastuzumab plus DDP followed by trastuzumab may be a favorable combination schedule. The mechanism as follows may interpret synergy between Trastuzumab and DDP: 1) Trastuzumab inhibited AKT phosphorylation and NFκB from entering into nucleus, so promoted cell apoptosis. 2) Trastuzumab down regulated mRNA expression of ERCC1 and inhibited DNA repair. 3) Trastuzumab induced a block in G0/G1 phase and a decrease in G2/M phase, so inhibited cell mitosis.3. Appropriate sequence should be emphasized when trastuzumab in combination with 5-Fu because trastuzumab followed by trastuzumab plus 5-Fu antagonized the cytotoxicity of 5-Fu. That S-phase decrease induced by trastuzumab may weaken the effect of 5-Fu for 5-Fu is a cell cycle specific agent (CCSA) mainly impacting on S-phase cell.4.The best schedule of trastuzumab in combination with FP treating HER2 positive gastric cancer is several cycles of trastuzumab plus FP followed by trastuzumab monotherapy until disease progress.Significance1. Trastuzumab has ten-year history for breast cancer treatment. This study compared therapeutic effect of different sequences of trastuzumab in combination with chemotherapeutic agents, and analyzed interaction mechanism. It is the first study to aim at gastric cancer cell. The conclusions of best sequence had practical significance for gastric cancer therapy.2. This study observed the therapeutic effect of trastuzumab in combination with 5-Fu, the key chemotherapeutic agent for gastric cancer for the first time, and deduced the appropriate sequence was important for combination of these two drugs. It denied the conclusion at some degree that antagonistic interaction between these two drugs in breast cancer, so provided effective support for combination of these two drugs on treating gastric cancer.
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