Bevacizumab + cisplatin treatment inhibited tumor growth, compared with that of cisplatin at 1 week after treatment. (D) SGC-CBP30 in vitro Quantification of bioluminescence showed no significant difference in tumor growth between bevacizumab and PBS groups 4 weeks after treatment. Bevacizumab + cisplatin treatment inhibited tumor growth compared with that of cisplatin at 4 weeks after

treatment. *P < 0.05, **P < 0.01. Hypoxia is implicated in the adaptive response To gain an insight into possible molecular mechanisms of the increased metastasis, we determined whether hypoxia development was concomitant with metastasis. Mice were assigned into four groups (PBS, bevacizumab, cisplatin and bevacizumab + cisplatin) and received bevacizumab and/or cisplatin treatments for 3 weeks. Four weeks after initial treatment, five mice from each group were sacrificed for examination. Expression of HIF-1α in pulmonary tumor nodules was analyzed by western blotting. In PBS and cisplatin groups, most tumors showed little hypoxia. In contrast, mice that received bevacizumab and bevacizumab + cisplatin therapy showed a markedly increased level of HIF-1α expression (Figure 2). Differences in HIF-1α protein levels in each group were considered statistically significant. Figure 2 Hypoxia is implicated in the adaptive

response Cilengitide mw after short-term bevacizumab treatment. Expression of HIF-1α in pulmonary tumor nodules of the four groups. (A) A representative western blot is shown. β-actin was used as a loading control. (B) While most tumors showed little expression of HIF-1α protein in PBS and cisplatin groups, mice that received bevacizumab and bevacizumab + cisplatin therapy showed a markedly increased level of HIF-1α expression.. *P < 0.05, **P < 0.01. Anti-VEGF treatment also induces increased VM The definition of VM is that tumor cells mimic endothelial cells and form vasculogenic networks. Y-27632 manufacturer CD34-PAS double staining was used to distinguish VM and endothelial-dependent

vessels. CD34 is a marker of endothelial cells, and the basement membrane is positive for PAS. Therefore, we counted PAS-positive and CD34-negative vessels for indicate. Mice were assigned into four groups (PBS, bevacizumab, cisplatin and bevacizumab + cisplatin) that received bevacizumab and/or cisplatin treatments for 3 weeks. Four weeks after initial treatment, five mice from each group were sacrificed for examination. Tumors in the bevacizumab group formed more VM channels than those of PBS and cisplatin, and bevacizumab + cisplatin groups (Figure 3). Figure 3 Anti-VEGF treatment induces increased VM. Comparison of VM channels in mice with various treatments. VM channels were positive for PAS staining and negative for CD34 staining in sections (arrow, ×400). (A) PBS (B) bevacizumab (C) cisplatinp and(D) bevacizumab + cisplatin groups. (E) Comparison of VM channels in A, B, C and D.