| Aim:To establish an orthotopic implanted model of human gastric cancer in SCID mice with a gastric cancer cell line SGC 7901, to explore the effects of NM-3 alone or combined with paclitaxel on the growth and metastasis of gastric cancer in vivo, to observe the inhibitory effects of NM-3 on the angiogenesis and apoptosis, to study the changes of angiogenesis factor such as VEGF, bFGF and TGF-β1 etc; NM-3 combined with Paclitaxel was designed to clarify whether this protocol had synergistic effects and the mechanisms involved would be investigated, we hope it will provide novel theoretical and experimental evidences for human gastric cancer treatment in the future.Methods: Moderately differentiated gastric cancer cell line SGC 7901 was adopted, with tissue mass transplantation, we established orthotopic implanted model of human gastric cancer in the stomach of SCID mice. 24 SCID mice were randomly divided into four groups: normal saline group (control group, C group), NM-3 group (NM-3 at a 10mg/kg dose was injected into abdominal cavity for 8 weeks in different time point, N group), paclitaxel group (paclitaxel at a 5mg/kg dose was injected into abdominal cavity for 3 weeks in different time point, in 2 courses, T group) and the combined group (using the same dose above, NT group). At the end of 10th weeks after orthtopic implantation, all drugs were stopped using, these mice were sacrificed 10 days later. The growth and metastasis of xenografts were observed, the apoptosis of gastric cancer cell was checked by TUNEL, the positive expression of CD34 was measured by immunohistochemistry and the microvessel density (MVD) was calculated accordingly. VEGF, bFGF, TGF-β1, bcl-2, p53, caspase3, survivin, smad3 and smad4 were measured by real time fluorescence RT-PCR and / or Western blot assay.Results: NM-3, Paclitaxel and the combined group had inhibitory effects on the growth of orthotopic gastric cancer implantation in SCID mice, the inhibitory ratio of implanted tumor was 29.55%, 25.12%and 46.18% respectively(P<0.01), there was no significantly differences between N group and T group; but the inhibitory ratio of implanted tumor in combined group was significantly increased in contrast with N group and T group(P<0.05);there was no statistically significant in animal weight before and after experimental treatment in all group(sP>0.05). The apoptotic index (AI) of group N, T, NT was 25.62±6.46%, 28.90±5.38% and 38.51±5.14% respectively, which was significantly higher than that of control group(13.24±7.75%, P<0.05), and the AI of NT group was significantly increased compared with both N and T group. The tumor metastasis ratio in group C, N, T, and NT group was 100%, 33.33%, 83.33% and 16.67% respectively, it was significantly lower among group N and NT than that of group C(P<0.05). The MVD of implanted tumor in N, T and NT group (6.47±0.92, 7.67±1.02, 4.07±0.77 respectively) were significantly lower than that in control group (11.07±0.92, P<0.01). Immunohistochemistry assay showed that the positive ratios of VEGF and bFGF in group N and NT were reduced compared with group C ( VEGF: 50%, 33.3% vs 100%; bFGF: 50%, 33.3% vs 83.3%). The mRNA expression of VEGF, bFGF and TGF-β1 was detected by the real time fluorescence RT-PCR, the results showed that three proangiogenesis factors were all reduced in group N and group NT compared with that in group C(VEGF CT value,15.04±1.12 and 16.88±1.37 vs 10.59±0.82;bFGF CT value, 12.89±0.78 and 15.57±2.34 vs 10.59±0.79;TGF-β1 CT value, 17.31±1.04 and 18.44±1.06 vs 13.92±1.07);Paclitaxel had no effect on mRNA expression of VEGF, bFGF and TGF-β1. With GAPDH as a contrast, the relative quantity of mRNA expression of p53 and bcl-2 were detected by real time RT-PCR, the results showed that NM-3, Paclitaxel and combined treatment protocol could significantly reduce the mRNA expression of p53 and bcl-2 (P<0.01)(p53 mRNA 1.28±0.48%, 2.11±0.57% and 1.07±0.82% vs 6.07±0.94%; bcl-2 mRNA 0.92±0.23%, 2.35±0.84% and 0.22±0.16% vs 5.23±1.96%), but there was no significant difference between group N and NT. The expression of caspase3 protein among group N, T, and NT was significantly higher than that of control group (88.64±11.91%, 71.87±5.68 % and 119.44±9.78 % vs 49.54±11.25 % , P<0.01), and the differences between group NT and N/T were statistically significant (P<0.01). On the contrary, the expressions of survivin protein among group N, T and NT were significantly decreased compared with control group ( 76.31±9.14 % , 86.45±6.10 % and 57.87±3.86 % vs 102.03±12.27%,P<0.05), and the differences between group NT and N/T were statistically significant (P<0.01). The protein expression of smad3 and smad4 was also done by Western blot, the results showed that the levels of smad3 and smad4 in N group and NT group were significantly enhanced compared with control group (smad3 63.64±4.22%, 81.91±12.11% vs 40.81±7.70% ; smad4 73.78±12.82%, 81.96±10.17% vs 46.16±12.59%, P<0.01), while Paclitaxel had no effect on the expression of smad3 and smad4. Conclusions: 1. NM-3 and Paclitaxel extracted from a special plant (Taxus) have inhibitory effects on the growth of orthotopic implanted tumor in the stomach of SCID mice, the combination protocol has synergetic effect and can significantly reduce the metastasis ratio of the tumor. 2. The tumor angiogenesis in SCID mice was significantly inhibited by NM-3, its inhibitory effects can be attributed to the reduction of VEGF, bFGF, TGF-β1 on the mRNA and protein level, and to the augmentation of smad3 and smad4, which may be related with the metastasis of human gastric cancer. 3. Paclitaxel has certain inhibitory effects on the angiogenesis of gastric cancer in SCID mice, but it has no effects on the expression of VEGF, bFGF and TGF-β1. 4. The combination protocol of NM-3 and Paclitaxel had synergistic inhibitory effects on the angiogenesis of orthotopic implantation of human gastric cancer in SCID mice, which may be due to the different the mechanism of each drug. 5. NM-3 and Paclitaxel could obviously induce apoptosis of gastric cancer cells by reduction the expression of p53, bcl-2 and survivin with a sequent elevated activation of caspase-3.
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