Heavy Iron Radiation Impacted On Tumor Angiogenesis In Renal Cell Carcinoma By The VEGF-DLL4/Notch Signaling Pathway

Section1Study on the relationship between DLL4expression and Prognosis for Patients with Renal cell CarcinomaBackground:Renal cell carcinoma (RCC) is one of the most destructive tumors, for which surgical resection is the only potentially curative therapeutic option. However, the5-year survival of operated cases is not optimistic. It’s beneficial for clinical management to explore the factors associated with early recurrence and metastasis after curative resection, which may lead to the prognostic improvement for renal cancer patients. Stromal remodeling and angiogenesis have a significant role in the process of recurrence and metastasis of renal cell carcinoma for operated cases. Vascular endothelial growth factor (VEGF) signal is one of the most important pathways for cancer cells growth and vessels reconstruction in RCC. It has been demonstrated that one of the Notch ligands, delta-like ligand4(DLL4), which is induced by VEGF and acts downstream of VEGF as a "brake" on VEGF receptor2(VEGFR-2) expression, forming an autoregulatory negative feedback loop inactivating VEGF-induced vessel growth. Consistent with the role, blockade of DLL4results in excessive, nonproductive angiogenesis and then results in decreased tumor growth, even for tumors that are resistant to anti-VEGF therapy. Recent evidence has shown that DLL4was expressed highly in several human malignancies, such as lung cancer and so on, which was correlated with poor prognosis. Therefore, DLL4-Notch signaling may be involved in renal cell carcinoma development and predict poor prognosis for patients.Objective:To determine whether high DLL4expression is correlated with poor prognosis after curative resection for clear cell renal cell carcinoma (ccRCC) and assess the relationship between DLL4expression and VEGF receptor2(VEGFR-2) expression for patients with ccRCC.Methods:From January,2001, to December,2011, the consecutive patients with a confirmed histologic diagnosis of primary ccRCC, who underwent macroscopically curative resection in second Hospital, Lan Zhou University were recruited in this study. All members had no history of adjuvant therapy before operation. We reviewed all valuable medical information during their hospitalization, which consisted of demology characteristics, private habits, medical history, clinical manifestation, laboratory results, surgical records, postoperative complications, and so on. After being discharged from the hospital, all patients received regular follow-up by phone or clinic recheck which was closed on Dec30,2011. The content included overall survival (OS), progression-free survival (PFS), adjuvant therapy and so on. On the other, surgical specimens obtained from121patients with ccRCC were immunohistochemically assessed for DLL4and vascular endothelial growth factor receptor2(VEGFR-2) levels. Prognostic significance of DLL4expression was evaluated by Kaplan-Meier method and Cox proportional hazards models with backward step wise selection. The correlations of DLL4expression with VEGFR-2expression, tumor stage, and metastasis were examined by chi-square test.Results:121ccRCC patients (male57,47.1%; female64,52.9%) aged from38to84(mean=57) were included in this study. According to pathologic classification of tumor grading,22was considered as poorly differentiated,31as moderately differentiated and68as well differentiated. As for T-staging of tumor,54was considered as T1,28 as T2,29as T3and10as T4. The mean follow-up duration at the time of analysis was48.6months (range from6to120months). There were29cases of metastasis ccRCC and92cases of non-metastasis. High DLL4expression and high VEGFR-2expression were observed in52(43.8%) and75(61.9%) patients, respectively.1, Prognostic Significance of DLL4ExpressionIn univariate survival analyses, both OS and PFS were associated with DLL4expression, VEGFR-2expression, tumor stage, tumor differentiation. Kaplan-Meier curves showed that patients with low DLL4expression had a significantly better survival than those with high DLL4expression (P<0.05).In multivariate survival analyses, high DLL4expression was shown to be poor independent prognostic factors for both OS and PFS (P<0.05).2, Correlations between DLL4expression and clinicopathological featuresDLL4expression was significantly correlated with clinicopathological factors, including VEGFR-2expression, tumor stage and metastasis (P<0.05).Conclusions:High DLL4expression is significantly associated with poor prognosis for surgically resected ccRCC, advanced tumor stage and metastasis, which is an independent poor predictor for ccRCC. Section2Heavy iron radiation impacted on Tumor angiogenesis in renal cell carcinoma by the VEGF-DLL4/Notch signaling pathwayBackground:Heavy ions are high LET (Linear energy transfer) radiation, release their highest dose near the end of their path at the Bragg Peak which can kill tumor cells furthest and maximize relative biological effectiveness. Compared with conventional radiation, heavy ions cause more serious damage to the tumor cells, especially for hypoxic cancer cells which are insensitive to radiation. Heavy ions have been recognized as the most advantageous use of radiation rays in21st Century. In this study we use heavy ion irradiation to explore the role of DLL4and VEGF in VEGF-DLL4/Notch signaling path way of tumor angiogenesis for renal cell carcinoma, to provide molecular biology evidence for the heavy ion therapy of renal cell carcinoma.Objective:To clear relationship between DLL4and VEGF and explore the role of DLL4and VEGF in VEGF-DLL4/Notch signaling pathway of tumor angiogenesis for clear cell renal cell carcinoma.Methods:DLL4and VEGF in786-0cells were assayed by Western blot after heavy ions irradiation; nude mouse model of renal cell carcinoma was established and the volume of tumor and weigh of nude mouse were measure; Renal tissues were immunohistochemically assessed for DLL4, VEGF and CD31after heavy ions irradiation. CD31was recognized as a tumor microvessel density (MVD) indicator.Results:After heavy ion irradiation12h,24h,48h, VEGF protein and DLL4were upregulated, DLL4was increasing with the rise of VEGF; In the study of1Gy、2Gy、3Gy heavy ions irradiation, we found that DLL4and VEGF expression were higher than that in control group. Similarly, DLL4was increasing with the rise of VEGF; Tumor growth in heavy ion irradiation group is slower than that in the control group, especially after the second3Gy heavy ion irradiation, the tumor volume increased slowly in the experimental group. However, there was no significant difference in weight between two groups. Immunohistochemistry showed both VEGF and DLL4were overexpressed after heavy ion irradiation, the MVD of heavy ion irradiation group was42.2±0.6, the control group was26.8±0.3, with significant difference.Conclusions:Heavy ion irradiation can promote expression of VEGF and DLL4in renal cancer cells and the expression of DLL4was induced by VEGF; Heavy ion irradiation can inhibit growth speed of transplanted kidney tumors, the possibility of mechanism is that, the increasing of DLL4expression functions as a negative regulator of angiogenesis downstream of VEGF after Heavy ion irradiation, which inhibits the functions of VEGF-DLL4/Notch signaling path way of tumor angiogenesis for renal cell carcinoma, enlarges microvascular density, promote excessive and non-productive angiogenesis to inhibit tumor growth.