| [Objective] To explore the predictive values of platinum-related genes in gastric cancer treated with oxaliplatin-based chemotherapy and to explore the potentially synergistic effects of "Chong Lou Fu Fang" (CLFF) with chemotherapeutic agents commonly used in gastric cancer therapy in vitro.[Methods] Expression of chemotherapeutic agents associated genes was measured by real time quantitative PCR. SNPs in ERCC1 Asp118Asp, XRCC1 Arg399Gln, XPD Lys751Gln and GSTP1 Ile105Val were assessed by 5'nuclease allelic discrimination assay (TaqMan) using real-time PCR and direct sequencing, The expression levels of ERCC1 and the genotypes of ERCC1 Asp118Asp, XRCC1 Arg399Gln, XPD Lys751Gln and GSTP1 Ile105Val were tested for an association with survivals in gastric cancer patients treated with oxaliplatin-based adjuvant chemotherapy regimen.The cell proliferation capacity was determined by the MTT assay. Interaction between CLFF and chemotherapeutic agents in human gastric cancer SGC-7901 and BGC-823 cell lines was evaluated using the combination index (CI) method based on the median-effect principle. Apoptosis rates of cancer cells were evaluated by means of flow cytometry.[Results]1. The ERCC1 expression analysis in all 75 patients indicated that the median value for ERCC1 was 7.32(range 0.50-147.03).Among 75 patients,52 cases received surgery followed by at least 6 cycles modified FOLFOX4 adjuvant chemotherapy, and 23 cases received surgery alone. Univariate analysis suggested that low ERCC1 levels had longer RFS and OS than those with high ERCC1 levels (median RFS,18 versus 7 months, P=0.001; MST,27 versus 11 months, P =0.001) in group received adjuvant chemotherapy, and high ERCC1 levels had longer RFS and OS than those with low ERCC1 levels (median RFS,33 versus 12 months, P=0.038; MST,43 versus 21 months, P=0.004) in group received surgery alone. Cox proportional hazards regression model multivariable analysis suggested that high ERCC1 expression is an independent prognostic marker of poor RFS (hazard ratio 2.16,95% confidence interval 1.09-4.25, P=0.026) and OS (hazard ratio 2.21,95% confidence interval 1.07-4.55, P=0.031) in oxaliplatin-based adjuvant settings, but remained significantly positive prognostic marker for OS (hazard ratio 0.17,95% confidence interval 0.04-0.71, P=0.015) in patients receiving surgery alone.2.The genotype analysis in 126 gaseric cancer patients who received at least 6 cycles modified FOLFOX4 adjuvant chemotherapy indicated the frequencies for the homozygous wildtype allele, heterozygous and homozygous polymorphic variant:64.29%,28.57%, and 7.14% for ERCC1-118; 56.35%,38.89%, and 4.76% for XRCC1-399; 84.92%,15.08%,and 0 for XPD-751; and 68.25%,30.60%,and 3.97% for GSTP1-105. Univariate analysis indicated that the ERCC1-118, the XRCC1-399 and the GSTP1-105 SNPs showed the predictive values for RFS and OS. With regard to RFS, for ERCC1-118, the median RFS was 5 months for T/T and C/T cases and 45 months for C/C patients (P<0.001). For XRCC1-399, the median RFS was 47 months for A/A and A/G cases and 8 months for G/G patients (P=0.001), and For GSTP1-105, the median RFS was 47 months for G/G and A/G cases and 12 months for A/A patients (P<0.001). As for OS, the ERCC1-118, the XRCC1-399 and the GSTP1-105 SNPs also remained significant predictive value. For ERCC1-118, the MST was 15 months for T/T and C/T patients and no defined for C/C cases (P<0.001). For the XRCC1-399, the MST was 18 months for A/A patients and no defined for A/A and A/G patients P<0.001), and For GSTP1-105, the MST was no defined for G/G and A/G cases and 21 months for A/A patients (P=0.019). Cox proportional hazards regression model multivariable analysis suggested ERCC1-118 remained significant predictive value for RFS (P<0.001, HR=2.362; CI 95%:1.458-3.827) and OS (P=0.001; HR=2.388;CI 95%:1.448-3.937), and XRCC1-399 only remained significant predictive value for RFS, and XRCC1-399 (A/A+A/G) genotype could significantly decrease the recurrence hazard of the patients (P<0.001, HR=0.569; CI 95%:0.341-0.949).3.The synergistic analysis indicated that CLFF had a synergistic effect on the cytotoxicity of 5-fluorouracil (5-FU) in a relative broad dose inhibition range (20-95% fraction affected in SGC-7901 cell lines and 5-65% fraction affected in BGC-823 cell lines), while the synergistic interaction between CLFF and oxaliplatin or docetaxel only existed in a low dose inhibition range (<50% fraction affected in both cell lines), CIs<1.4. The apoptosis analysis indicated the combination of CLFF and 5-fluorouracil, oxaliplatin or docetaxel could also induce apoptosis in a synergistic manner. The percentages of the apoptotic cells (early apoptosis plus late apoptosis) induced by CLFF and 5-fluorouracil,oxaliplatin and docetaxel were (8.4+6.8)%, (24.6+4.6)%(9.7+3.7)%and (29.5+5.9)%in SGC-7901 cell lines and (6.7+1.4)%, (12.6+3.8)%(11.6+1.3)%, (6.6+1.2)%in BGC-823 cell lines, while the percentage of them induced by drug combination in both cell lines were increased to (80.5+12.9)%, (32.5+5.7)%and (63.2+6.2)%in SGC-7901 cells, respectively, and (66.1+6.4)%, (48.6+3.1)%and (33.5+9.6)%in BGC-823 cells, respectively.5.The expression levels of ERCC1, TS, b-tubulinⅢand tau were significantly down-regulated after treatment with CLFF alone or CLFF combined with chemotherapeutic agents in SGC-7901 cell lines and BGC-823 cell lines. However, any of chemotherapeutic agents alone did not down-regulate their respective drug resistance-associated genes. TS,ERCC1,β-tublinⅢand Tau mRNA levels were markedly suppressed at (0.32±0.02),(0.30±0.03),(0.31±0.03)and (0.28±0.02) fold in SGC-7901 cell lines, respectively, and 0.46±0.03),(0.46±0.02),(0.44±0.04) and (0.56±0.07)fold in BGC-823 cell lines, respectively, by the presentation of CLFF for 24 h.At the same time, we found that the combination of CLFF and 5-FU, oxaliplatin and docetaxel for 24 h could downregulat the TS,ERCC1,β-tublinⅢand Tau mRNA levels to (0.55±0.02),(0.48±0.03),(0.59±0.04) and (0.43±0.02) fold in SGC-7901 cell lines, respectively, and (0.64±0.04),(0.55±0.03),(0.51±0.04) and (0.62±0.06) fold in BGC-823 cell lines, respectively.[Conclusion] It seemed that gastric cancer patients with low levels of ERCC1 expression showed a great benefit from oxaliplatin-based adjuvant chemotherapy, while high levels of ERCC1 expression might be a positive prognostic marker for patients receiving surgery alone.On the other hand, gastric cancer patients harboring ERCC1-118C/C genotype and XRCC1-399A/G or G/G genotypes may better receive oxaliplatin-based adjuvant chemotherapy. XRCC1-399 and GSTP1-105 polymorph-isms may be associated with the toxicities of the oxaliplatin-based chemotherapy regimen.The combination of CLFF and 5-fluorouracil, oxaliplatin or docetaxel showed significant synergistic antitumor activity in both two gastric cancer cell lines. The possible mechanisms might be the synergistic apoptotic effect and the down-regulation of chemotherapeutic agents associated genes.
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