Effects of PHA-665752 and vemurafenib combination treatment on in�vitro and murine xenograft growth of human colorectal cancer cells with BRAFV600E mutations

Abstract

Abstract. It remains unknown whether blockade of B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E signaling and MET proto-oncogene, receptor tyrosine kinase (c-Met) signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. The present study investigated the effects of the vemurafenib alone and in combination with c-Met inhibitor PHA-665752 on the growth of human CRC cells in vitro and in mouse xenografts. HT-29 and RKO CRC cell lines with BRAF V600E mutations and mice bearing HT-29 xenografts were treated with vemurafenib in the absence or presence of PHA-665752. Cell viability and cycle phase were respectively examined by using the MTT and flow cytometry assay. Immunohistochemistry was conducted to detect the protein expression levels of hepatocyte growth factor (HGF), phosphorylated (p)-c-Met, p-AKT serine/threonine kinase (AKT) and p-extracellular signal-regulated kinase (p-ERK). The MTT assay demonstrated that the growth of RKO and HT-29 cells was inhibited by PHA-665752 in a time-and dose-dependent manner (P<0.05), however no significant suppressive effects were observed with vemurafenib. Relative to the PHA-665752 or vemurafenib stand-alone treatment groups, the combination of PHA-665752 and vemurafenib had a significant inhibitory effect on the proliferation of CRC cell lines (P<0.05). The mean tumor volume in mice treated with vemurafenib in combination with PHA-665752 was significantly smaller compared with those treated with only vemurafenib or PHA-665752 (P<0.05). Flow cytometry assay revealed that the G0/G1 phase frequency was significantly increased in the combination group compared with any other treatment groups (P<0.05). Immunohistochemistry demonstrated that vemurafenib in combination with PHA-665752 effectively induced the expression of p-c-Met, p-AKT and p-ERK, however had no effect on HGF. IntroductionColorectal cancer (CRC) is one of the principal causes of cancer worldwide (1). Unfortunately, even if a 5-year survival prognosis can be given to 90% of patients in the early stages, disappointing treatment outcomes are recorded in subjects with extensive local invasion or distant metastases. Generally, their 5-year survival rate is less than 15% (2) and, even if suitable for receiving adjuvant chemotherapy, disease-related deaths remain stubbornly high.A recent steady stream of important breakthroughs has dramatically improved our understanding of CRC. One such discovery identified the BRAF mutation as common in metastatic CRC patients, especially those with a right-side colon cancer and a poorly differentiated tumor (3). Vemurafenib is a potent and selective inhibitor of mutated BRAF. Chapman et al (4) revealed that vemurafenib achieved 40% response rates in melanoma with a BRAF mutation. However, unlike melanoma, the effect of vemurafenib in CRC patients with a BRAF mutation is often negligible, resulting in a clinical response in only 5% of patients (5). This discrepancy of outcomes suggests that different cancer types may present important variat...

Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention

“…For an initial assessment of the in vivo efficacy of the c-MET inhibitor PHA-665752, we treated mice infected with the rodent malaria parasite P. berghei . A treatment regime and formulation that was previously used to control tumour growth in mice with the same inhibitor 51 led to a 3.3-fold reduction in parasite load after two doses of treatment in vivo (Fig. 6e , day 4).…”

“…The drug was prepared fresh by diluting a stock of 50 mg PHA-665752 in 0.5 ml DMSO (w/v) in water (1 : 40, v/v). The same drug formulation and treatment regime had proved effective in controlling carcinoma growth 51 .…”

Signalome-wide assessment of erythrocyte response toPlasmodiumreveals novel targets for host-directed antimalarial intervention

“…For an initial assessment of the in vivo efficacy of the c-MET inhibitor PHA-665752, we treated mice infected with the rodent malaria parasite Plasmodium berghei . A treatment regime and formulation that was previously used to control tumour growth in mice with the same inhibitor 56 led to a 3.3-fold reduction in parasite load after two doses of treatment in vivo (Fig. 6e, day 4).…”

“…The drug was prepared fresh by diluting a stock of 50 mg PHA-665752 in 0.5 ml DMSO (w/v) in water (1:40, v/v). The same drug formulation and treatment regime had proved effective in controlling carcinoma growth 56 .…”

RePhine: An Integrative Method for Identification of Drug Response-Related Transcriptional Regulators

“…In cluster 2, there were two subgroups. Cluster 2.1 contained two ALK inhibitors (PF-2341066 and TAE684), an ABL inhibitor (nilotinib), a CD4/6 inhibitor (PD-0332991), an IGF-1R inhibitor (AEW541), and two multi-kinase inhibitors (sorafenib and TKI258) [28] ; in cluster 2.2, two MEK inhibitors (PD-0325901 and AZD6244), two RAF inhibitors (PLX4720 and RAF265), an MET inhibitor (PHA-665752), which were related to MAPK signaling [31] , were clustered. All the drugs in cluster 2 were targeted therapy drugs [28] .…”