TP, TS And VEGF Expression Regulated By Interferon-α On Chemosensitivity Of 5-Fluorouracil In Renal Cell Carcinoma

Despite significant progress in understanding the biology of renal cell carcinoma (RCC), there are an estimated 190,000 new renal cancer cases, and 91,000 deaths from renal cancer in the world each year. The mortality rate and incidence of RCC is diverse in China, there is a upward trend on the incidence and mortality rate of RCC.Treatment for RCC is limited. In contrast to many other malignancies, RCC is generally resistant to both chemotherapy and hormone therapy. RCC can often be successfully treated by radical nephrectomy with nephron-sparing surgery, if it is localized. However approximately 20～30% of patients present with metastatic disease at the time of diagnosis, and 20～40% of patients undergoing nephrectomy for clinically localized RCC are expected to relapse at distant sites. Once metastatic disease develops, the survival is poor, with a 5-yr survival of approximately 20%.In view of the biological characteristics of RCC, a single mode of treatment is not suitable for the treatment of metastatic renal cell carcinoma. In order to further improve the clinical response rate, a combination of immunotherapy and chemotherapy is carried out in domestic and abroad clinic to explore the efficacy. Combination of interferon-αand 5-fluorouracil (5-FU) is one of immunochemotherapies. Some studies indicate combination of interferon-αand 5-fluorouracil synergize efficacy, and improve the overall response survival rate in patients with metastatic RCC. But its mechanism remains to be unclear. There are still some patients with poor response rate to this combination.Interferon-αis aâ… type of interferon, and the mechanism of its treatment includes inhibiting tumor cell proliferation, controlling cell apoptosis, regulating immunoreactions, stimulating proliferation of immune cells and enhancing the effect of cytotoxic lymphocytes. Multifunctions which interferon-αshowed in the process of therapy inform other mechanisms are involved in. Some studies interferon-αhas an negative impact on angiogenesis, and can inhibit proliferation of endothelial cells. It is unknown that the function of anti-angiogenesis is realized by activation of immune cells or by other mechanism. It remains to be elucidated whether this function has involved in the synergy of anti-tumor effect with 5-FU.5-FU is widely used in the treatment of various malignancies. But its efficiency of a single drug is only 5%～20% in treatment of metastasic RCC. Thymidine phosphorylase and thymidylate synthase are two key enzymes for activation and function of 5-FU in vivo and are closely related with effects of chemotherapy of 5-FU. TP can catalyze reversible transformation of the thymidine to deoxyribose-1-phosphate and thymine, thus stabilize the level of thymidine, which is the essential material for DNA synthesis. The drug 5-FU cannot exert its antitumor function until it is activated by TP in human body. In addition, TP, known as a platelet-derived endothelial cell growth factor (PD-ECDF), have an angiogenic activity and play an important role in stimulating tumor angiogenesis. TP exerts angiogenic activity in vivo while its enzymatic activity is indispensable for its angiogenic effect。TS is a rate-limiting enzyme in the synthesis of the thymidine and converts dUMP into dTMP, the only source of thymidine for DNA synthesis and repair. Thus, TS plays an important role in the cell division and proliferation. TS is also the target enzyme of 5-FU, which exerts its antitumor activity through its inhibition of TS.Considering TP and TS are closely linked with the efficacy of chemotherapy of 5-FU, and furthermore angiogenic activity of VEGF and TP is possible regulated by interferon-α, expressions of TP, TS and VEGF have been speculated to exert an impact on the efficacy of combination of interferon-αand 5-FU. It is necessary to exactly understand the regulation of interferon-αon the expressions of TP, TS and VEGF to clarify the mechanism involved in the synergy of combination therapy mentioned above. The studies concerned about this mechanism are rare so far. There are no relevant reports on comparative analysis of expression of these genes in combination of interferon-αand 5-FU. So in this study RT-PCR, immunohistochemistry and MTT method were adopted to examine relationship between expression of TP and TS with clinic-pathologic factors and their relevancy to chemosensitivity to 5-FU. Then regulation of interferon on expression of TP, TS and VEGF at RCC in vitro and vivo and followed effect of this regulation on apoptosis and chemosensitivity to 5-FU are investigated by RT-PCR, Western-blot, immunohistochemistry, Flow-cytometry and TUNEL to evaluate the status that expressions of TP, TS and VEGF in synergy of combination. Guidences are expected to be provided to individualized treatment On clinical oncology.Part oneResearch of the expressions of TP, TS and their correlation with the clinic pathological factors and chemosensitivity of 5-fluorouracil in renal cell carcinomaMethods1. Semi-quantitative RT-PCR was used to quantitate mRNA level of TP and TS in 43 carcinomas from renal cell carcinoma patients and 7 normal renal tissues.2. Expressions of TP, TS, and CD34 protein were examined with IHC.3. The relativity between the microvascular density reflected by protein of CD34 and TP protein was analysed by HMIAS-2000 pathological image analysis system.4. Carcinoma cells from 26 renal cell carcinoma patients were isolated by primary cell culture technology to examine their chemosensitivity to 5-FU by methyl thiazolyl tetrazolium (MTT) method. Expression of TP, TS mRNA and protein were compared with the chemosensitivity of carcinoma cells from 26 RCC to 5-FU to get the relativity between expression and chemosensitivity to 5-FU.5. Statistical analyses, including T test, multivariant analysis, and related test were performed with the software package SPSS for windows version 13.0. The accepted level of significance was P＜0.05. All p-values presented are two-sided.Results1. In both renal cell carcinoma and normal renal tissue, mRNA expressions of TP and TS were observed and mRNA expression of TP and TS varied considerably in the examined specimens. In renal cell carcinoma, the relative value of TP mRNA expression ranged from 0.31 to 0.88, and their average value is 0.7193±0.1282. The relative value of TS mRNA expression ranged from 0.48 to 0.91, and their average value is 0.7400±0.0954. In normal renal tissue, the relative value of TP mRNA expression ranged from 0.21 to 0.43, and their average value is 0.3359±0.0714. The relative value of TS mRNA expression ranged from 0.36 to 0.61, and their average value is 0.4917±0.0886. mRNA levels of TP and TS were higher in tumor than in normal tissues, differences were statistically significant(P＜0.05).2. Expressions of TP mRNA was not correlated with tumor size, stage, lymph node metastasis and distant metastasis(P＞0.05). Expression of TS mRNA was negatively correlated with tumor size, lymph node metastasis and distant metastasis(P＞0.05), but was correlated to tumor stage.3. Expression of TP and TS protein were observed in all 43 cases. TP protein was commonly expressed in cytoplasm, and expressed in nuclear in the condition of its strong expression. TS protein was only expressed in cytoplasm. In normal renal tissue, the positive rate of TP protein expression is 28.57%(2/7), and the positive rate of TS protein expression is 57.14%(4/7). TP and TS protein were expressed in renal tubular epithelial cell cytoplasm.4. Expression of TP protein was not correlated with tumor size, stage, and lymph node metastasis (P＞0.05),but was correlated with distant metastasis(P＜0.05). Expression of TS protein was not correlated with tumor size, stage, lymph node metastasis and distant metastasis(P＞0.05).5. No correlation was identified between expressions of TP mRNA and its proteins in renal cell carcinoma(P＞0.05).expressions of TS mRNA were correlated with its protein(P＜0.05).6. There was a correlation between the microvascular density and TP protein expression(P＜0.05). Accompanying evaluation of MVD degree, expressions of TP protein were increased.7. No correlation was identified between expressions of TP mRNA and chemosensitivity of carcinoma cells to 5-FU(r=0.142, P＞0.05). Expression of TS mRNA was negatively correlated with chemosensitivity of carcinoma cells to 5-FU(r=-0.545, P＜0.05). No correlation was identified between expressions of TP protein and chemosensitivity of carcinoma cells to 5-FU(P＞0.05). There was a correlation between expressions of TS protein and chemosensitivity of carcinoma cells to 5-FU (P＜0.05). The ratio of chemosensitivity to 5-FU is higher in group whose carcinoma cells with weak expression of TS protein than in group whose carcinoma cells with strong expression of TS protein (P＜0.05).Part twoTP, TS and VEGF expression regulated by interferon-αon chemosensitivity of 5-Fluorouracil in renal carcinoma cell lineMethods1. Semi-quantitative RT-PCR was used to quantitate mRNA level of TP in 786-0 line treated by 0, 1000, 3000, 6000, 12000 IU/ml interferon-α2b.2. Semi-quantitative RT-PCR was used to quantitate mRNA level of TP in 786-0 cell line by interferon-α2b treated for 0, 12, 24, 36, 48, 60, 72h.3. Semi-quantitative RT-PCR was used to quantitate mRNA level of TS in 786-0 cell line treated by 0, 1000, 3000, 6000, 12000 IU/ml interferon-α2b for 72h.4. Semi-quantitative RT-PCR was used to quantitate mRNA level of VEGF in 786-0 cell linetreated by 0, 1000, 3000, 6000, 12000 IU/ml interferon-α2b for 72h.5. Effects of treatment of interferon-α2b on protein expression of TP and VEGF in 786-0 cell line were examined by western-blot method.6. Changes in TP and TS protein expression were examined in 786-0 cell line treated by interferon-α2b by cyto-immunohistochemistry.7. Flow-cytometry (FLC) was used to examine effect of combination of interferon-α2b and 5-FU on apoptosis in 786-0 cell line.8. 50% inhibitory concentration of growth (IC₅₀) of 5-FU in treated 786-0 cell line by interferon-α2b was evaluated by MTT assay.Results1. The greatest increase on TP mRNA expression was achieved by 6000 IU/ml IFN-α2b after 72 h treatment in 786-0 cell line. IFN-α2b increased TP mRNA expression in a dose-dependent manner (r=0.901, P＜0. 05). IFN-α2b also increased TP mRNA expression in a time-dependent manner (r=0.886, P＜0.01).2. IFN-α2b has no effect on TS mRNA expression under the dosage of 12000 IU/ml(P＞0.05).3. The greast inhibition on VEGF mRNA expression was achieved by 12000 IU/ ml IFN-α2b after 72h treatment in 786-0 cell line. IFN-α2b inhibited VEGF mRNA expression in a negative dose-depe, ndent manner (r=-0.934, P＜0.05).4. In control, 3000 IU/mlIFN-α2b treated and 6000 IU/mlIFN-α2b treated group. TP protein expression are gradually increased, and VEGF protein expression are gradually decreased. In the dosage of 6000 IU/ml, IFN-α2b can increase TP protein expression and decrease VEGF protein expression.5. In 786-0 cells treated by IFN-α2b, TP protein was expressed in cytoplasmand nuclear. In untreated 786-0 cells, TP protein was expressed only in cytoplasm. In treated group, TP protein expression are strong than that in control group. There are no difference on TS protein expression between treated group and control group, and TS protein only expressed in cytoplasm.6. IFN-α2b has no significant effect on apoptosis of 786-0 cells. Combination of IFN-α2b and 5-FU can increase the chemosensitivity of 786-0 cells to 5-FU. Apoptosis and necrosis were significantly increased 786-0 cells.Part threeTP, TS and VEGF expression regulated by interferon-αon chemosensitivity of 5-Fluorouracil in vivoMethods1. 786-0 cells at exponential phase (2×10⁶ cells per mouse) were implanted S.C. into the right flank of BALB/C mice, established xenograft tumor model of 786-0 cell line. Fifteen mice with xenograft tumor were randomly divided into three groups and named as control, 5-FU and combination group. Saline injection was used in control group, and intraperitoneal injection of 25 mg/kg 5-FU was used in 5-FU group. 3×10⁵ IFN-αSubcutaneously injection 3×10⁵ IU/d IFN-α2b and intraperitoneal injection of 25 mg/kg 5-FU was used in combination group.2. After treatment for 28 days, mice with xenograft tumor were sacrificed and transplanted tumor were weighted and measured volume.3. Expressi6n of TP, TS and VEGF mRNA were examined by RT-PCR in transplanted tumor.4. Expression and location of TP, TS and VEGF protein were examined by immunohistochemistry in transplanted tumor.5. Expression of TP and VEGF protein were examined by Western-blot.6. Cellular apoptosis were detected by TUNEL.Result1. After treatment, weight of transplanted tumor in control, 5-FU and combination group were 1.1820±0.4874g, 0.6900±0.1517g and 0.2500±0.1490g respectively, and volume in each group were 1.0648±0.5336 cm³,0.5424±0.1591 cm³ and 0.0940±0.0492 cm³ respectively. The weight and volume of transplanted tumor in 5-FU and combination group were significant smaller than that in control group.2. Expression of TP mRNA is the strongest in combination group (P＜0.05), there is no significant difference on TP mRNA expression between 5-FU and combination group (P＞0.05).3. Expression ofTS mRNA is the strongest in 5-FU group (P＜0.05). Expression of TS mRNA is the weakest in combination group. All of these differences were statistically significant (P＜0.01).4. All of differences on VEGF mRNA expression were no statistically significant in control, 5-FU and combination groups.5. TP proteins were expressed in all fifteen transplanted tumors, and the expressions are weak or modest positive in control and 5-FU group. Expressions of TP protein are strong positive in combination group.6. TS proteins were expressed in twelve transplanted tumors. The number of negative expression of TS protein is one in control group and two in combination group. Expression of TS protein is strong positive in 5-FU group and weak positive in control and 5-FU group.7. VEGF proteins were expressed in all fifteen transplanted tumors and expression is strong positive in all groups.8. Examination of Western-blot showed TP protein expressed stronger in combination group than in other groups, and difference was statistically significant (P＜0.05).9. Examination of Western-blot showed difference on expressions of VEGF protein was no statistically significant (P＞0.05).10. Apoptotic index are 5±3, 23±5 and 34±10 in control, 5-FU and combination group respectively. Compared with the control group, 5-FU group and combination group were statistically significant differences in apoptotic index. Difference in apoptotic index was no statistically significant between 5-FU and combination group (P＞0.05).Conclusion1. There are differences on expression level of TP and TS mRNA between renal cell carcinoma and corresponding normal renal tissue. The mRNA level of TP in renal cell carcinoma is significantly higher than normal renal tissuea. TP protein expression is correlated with distant metastasis and microvascular density. These three characteristic indicate TP has involved in the progression of RCC and promoted its invasion.2. The mRNA levels of TP show no association with its protein expressions, indicating post-transcriptional regulation.3. Either mRNA or protein levels, TS are related with chemosensitivity of RCC patients to 5-FU. This indicates RCC patients with weak expression of TS in carcinoma are fit for the chemotherapy of 5-FU. Expression of TP can predict the efficacy of chemotherapy.4. IFN-α2b can increase the expression of TP mRNA and protein in 786-0 cells in a dose-dependant manner without decreasing apoptotic rate. This indicates IFN-α2b inhibits anti-apoptosis function of TP and increase the chemosensitivity of 786-0 cells to 5-FU by upregulation of TP.5. The epression of TS protein are increased after chemotherapy of 5-FU in transplanted tumor. Although IFN-α2b can not influence the expression of TS mRNA in 786-0, it can inhibit the increase of expression of TS protein which caused by chemotherapy of 5-FU in transplanted tumor in vivo. This suggests there must be a mechanism to make IFN-α2b indirectly interfere expression of TS in vivo. Indirect intervention of IFN-α2b increase chemosensitivity of cells to 5-FU.6. IFN-α2b can decrease the expression ofTP mRNA and protein in 786-0 cells, but this function disappears in vivo. This indicates the regulation of IFN-α2b on VEGF is influenced by other cytokines in microenvironment of carcinoma. The relationship between expression of VEGF and synergy of combination of IFN-αand 5-FU should be studied in various aspects in vivo.7. Discrepancy of expression has been shown between TP and VEGF when these two genes were regulated by IFN-α2b in 786-0 cells. This discrepancy proves TP and VEGF are regulated by different signal passway.