HichPage 9 of(page number not for citation purposes)BMC Cancer 2009, 9:http
HichPage 9 of(page number not for citation purposes)BMC Cancer 2009, 9:http://www.biomedcentral.com/1471-2407/9/ControlErlotinib (80mg/kg)MP470 (50mg/kg)Erlotinib+MP470 (80mg/kg+50mg/kg)Negative control, primary antibody is replaced by blocking solutionPI staining show nucleusFigure 8 tion in of MP470-Erlotinib Effects xenograft tissues combination on Akt phosphorylaEffects of MP470-Erlotinib combination on Akt phosphorylation in xenograft tissues. IHC analysis for Akt activity was performed on the tumors harvested at the end of the treatment of an LNCaP xenograft mouse model. Paraffin embedded sections were immuno-stained for expression of phosphorylated Akt (Ser473). Tumors from mice treated with the MP470-Erlotinib combination had a near complete Aprotinin web inhibition of Akt phosphorylation compared to control or individual therapies (200 ?.may contribute to the tumor suppression seen in an LNCaP xenograft mouse model. In addition, hormonerefractory prostate cancer is a major clinical obstacle as there are no drugs to halt its progression [3]. Previous studies have shown that PI3K/Akt activation is associated with prostate cancer progression from an androgendependent to an androgen-independent state [30]. In androgen-ablated LNCaP cells, PI3K/Akt activity is elevated and required for growth and survival [31] and inhibition can restore sensitivity to apoptosis induction [32]. In a mouse xenograft model of LNCaP, conditional Akt activation promotes tumor growth in castrated animals by enhanced cell proliferation and inhibition of apoptosis [28]. Thus, blockage of Akt activity should prove beneficial for hormone-refractory prostate cancer. Our experiments showed that the MP470-Erlotinib combination efficiently inhibited Akt activity in androgen-ablated LNCaP cells (Fig. 3B), suggesting that this combination may be a viable treatment modality in patients failing androgen blockade or can be administered with androgens in front-line therapy to prevent hormone refractory status. Except for the loss of PTEN function, PI3K/Akt signaling is often dysregulated in human cancer due to constitutive activation of receptor tyrosine kinases (RTKs) [41]. Of the known RTKs, activation of the HER family (HER1, 2 and 3) and the PDGFR family has been demonstrated to associate with prostate cancer progression [16,19]. In prostate cancer cell lines, HER family receptors are over-expressed [42,43] and inhibition with specific TKIs has shown antitumor effects in vitro and in vivo [44,45]. HER familymembers are also widely expressed in cancerous tissues of the prostate and significant over-expression is found in hormone-refractory prostate cancer and metastatic tissue compared to localized prostate cancer [46]. Hence, HER family receptors have become potential therapeutic targets in prostate cancer [47]. MP470, designed as an ATPcompetitive TKI was very effective in inhibiting tyrosine phosphorylation in LNCaP and NIH3T3 cells after pervanadate stimulation (Fig. 4A and 4B). Further, the MP470-Erlotinib combination completely inhibited tyrosine phosphorylation and p85 binding (Fig. 4C) as well as Akt activity. The RTK phospho-antibody assay identified the HER family (HER1, HER2 and HER3) in LNCaP cells as targeted by MP470 (Fig. 5A). Erlotinib or MP470 alone did not totally inhibit phosphorylation of the HER family. However, MP470-Erlotinib combination completely inhibited the phosphorylation of HER1, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26104484 HER2 and HER3, the binding of PI3K regulatory subunit p85 to HER3 and d.
