Figure 9. ER maleate anticancer potential in tumor xenograft mice model and IHC analysis of Syk and PLK1 in human patient OSCC.

A. ER maleate inhibits growth of tumor xenografts in mice. Cal33 cells were injected in the right flank of 6 weeks old immunocompromised mice (NOD/SCID/crl). ER maleate treatment was started after 3 weeks when tumor xenografts volume was about 250 mm³ with doses ranging from 0.1-3.0 mg/kg bwt for 10 weeks. Analysis of xenograft tumors from mice treated with ER maleate showed a dose dependent suppression of tumor growth within initial 6 weeks with an efficacious pharmacodynamic effect of complete inhibition of tumor growth at 1mg/kg bwt and 3mg/kg bwt by the 10^th week. From the 7^th week, the group of mice pre-treated with 0.1mg/kg bwt ER maleate was treated with CBP at 75mg/kg bwt and the group with ER maleate at 0.3mg/kg bwt within first 6 weeks received a combination treatment of ER maleate (1mg/kg bwt) and CBP (75mg/kg bwt) shown in the grey box. The combination treatment with ER maleate and CBP inhibited tumor growth in vivo from the 8^th week; in comparison, inhibition of tumor growth by CBP alone was lesser than in combination with ER maleate. B. Effect of ER maleate treatment on body weight of mice. Weekly measurements of mice body weight after Cal33 cell injection among different groups. C–H. Histogram of apoptotic markers in the frozen tumor tissues by western blot analysis showed the levels of PARP (C), cleaved PARP (D), caspase3 (E) and its two cleaved forms at 19 kDa (F) and 17 kDa (G) in xenograft tumor from groups receiving ER maleate of 1 mg/kg bwt or 3 mg/kg bwt compared to vechile control group. (H) Representative western blot showed the expression of PARP, cleaved PARP, caspase3 and its two cleaved forms at 19 kDa and 17 kDa. b-actin served as a loading control. I. Immunohistochemical analysis of Cyclin D1, Syk and PLK1 in tumor xenografts in immunocompromised mice. Panel I shows H&E stained tumor tissue sections in untreated control mice (a) and the treatment groups (b-e). Panels II, III and IV show nuclear Cyclin D1, Syk and PLK1 expression in untreated control mice (a); reduced Cyclin D1, Syk and PLK1 expression in CBP (75mg/kg bwt) treated tumors (b); combination of CBP (75mg/kg bwt) and ER maleate (1mg/kg bwt) shows further reduction in Cyclin D1, Syk and PLK1 (c); ER maleate treatment at 1mg/kg bwt and 3mg/kg bwt show reduced Cyclin D1, Syk and PLK1 expression in comparison with untreated controls (d and e), respectively. Original magnification is x400.