Effects of PHA665752 and vemurafenib combination treatment on in vitro and murine xenograft growth of human colorectal cancer cells with BRAFV600E mutations

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 BRAFV600E 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.

Effects of <em>PHA</em>-<em>665752</em> and cetuximab combination treatment on in vitro and murine xenograft growth of human colorectal cancer cells with KRAS or BRAF mutations.

BACKGROUND
It remains unknown whether blockade of c-Met signaling and epidermal growth factor receptor signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. In this study, we investigated the effects of the c-Met inhibitor PHA-665752 alone and in combination with cetuximab on the growth of human CRC cells in vitro and in mouse xenografts.
METHODS
Human CRC cell lines (Caco2, HCT-116, and HT-29) and mice bearing HCT-116 xenografts were treated with cetuximab in the absence or presence of PHA-665752. Cell viability and apoptosis were examined using the MTT and TUNEL assays, respectively. Vimentin was measured by immunohistochemistry as a marker for epithelial-to-mesenchymal transition. Western blotting was used to determine signaling protein expression levels.
RESULTS
The MTT assay showed that the growth of Caco2, HCT-116, and HT-29 cells was inhibited by PHA-665752 in a dose-dependent manner, but only Caco2 cell growth was suppressed by cetuximab. Combination treatment with PHA-665752 and cetuximab inhibited the proliferation of Caco2 cells and RAS mutant CRC cell lines. However, relative to the PHA-665752-alone treatment group, HT-29 cells with a BRAF mutation showed no noticeable effect. The mean tumor volume in mice treated with cetuximab in combination with PHA-665752 was significantly smaller than that in the mice treated with only cetuximab (P = 0.033) or PHA-665752 (P < 0.01). Similarly, the expression of vimentin in the mice treated with PHA-665752 in combination with cetuximab was significantly lower than that in the mice treated with cetuximab or PHA-665752 alone (P < 0.05 in each case). TUNEL assays revealed that treatment with PHA-665752 in combination with cetuximab markedly increased CRC cell apoptosis. Western blotting analysis of signaling protein expression showed that PHA- 665752 inhibited Met phosphorylation (P < 0.05). In addition, treatment with cetuximab alone or in combination with PHA-665752 effectively inhibited EGFR phosphorylation (P < 0.05).
CONCLUSIONS
Combination treatment with PHA-665752 and cetuximab suppressed in vitro and in vivo CRC cell growth more than treatment with either agent alone did.

MET inhibitor <em>PHA</em>-<em>665752</em> suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells

Although ionizing radiation (IR) has provided considerable improvements in nasopharyngeal carcinoma (NPC), in subsets of patients, radioresistance is still a major problem in the treatment. In this study, we demonstrated that irradiation induced MET overexpression and activation, and the aberrant MET signal mediated by hepatocyte growth factor (HGF) induced radioresistance.
We also found that MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. Further investigation indicated that PHA-665752 suppressed the phosphorylation of the Akt, ERK1/2, and STAT3 proteins in a dose-dependent manner. Our data indicated that the combination of IR with a MET inhibitor, such as PHA-665752, might be a promising therapeutic strategy for NPC.

Celastrol exerts synergistic effects with <em>PHA</em>-<em>665752</em> and inhibits tumor growth of c-Met-deficient hepatocellular carcinoma in vivo.

PHA665752 (PHA), a selective small molecule c-Met Inhibitor, potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes of a variety of tumor cells including hepatocellular carcinoma cells. However, these effects were impaired in c-Met-deficient cancer cells. In the present study, we investigated the potential anti-human c-Met-deficient hepatocellular carcinoma effects of Celastrol, a novel triterpene, and its combination with PHA. Human hepatocellular carcinoma cells BEL-7402 (c-Met-positive) and Huh7 (c-Met-deficient) were treated with different dose of PHA with or without equal dose of Celastrol, and cell growth, cell cycle and apoptosis were evaluated, respectively, by MTT assay, flow cytometry and Caspase3/7 activity.
Nude mice bearing Huh7 xenografts were used to assess the in vivo anti-tumor activity. Our results showed that Celastrol at high concentration (>1.0 μM) induced G2/M arrest and apoptosis with the activation of Caspase3/7 in Huh7 cells whereas at low concentration (<1.0 μM) had no obvious effects. Low concentration Celastrol presented significant combined effects with PHA on Huh7 cells and Huh7 xenografts in terms of growth inhibition, migration inhibition and apoptosis induction. These results suggest that Celastrol and its combination with PHA present the therapeutic potential on c-Met-deficient hepatocellular carcinoma, and deserve further preclinical and clinical studies.

PHA-665752

B2057-25 Biovision 631 EUR

PHA-665752

B2057-5 Biovision 196 EUR

PHA 665752

20-abx076781 Abbexa
  • 857.00 EUR
  • 342.00 EUR
  • 25 mg
  • 5 mg

PHA-665752

E1KS1070 EnoGene 5 mg 325 EUR

PHA-665752

A2307-10 ApexBio 10 mg 142 EUR

PHA-665752

A2307-100 ApexBio 100 mg 711 EUR

PHA-665752

A2307-5.1 ApexBio 10 mM (in 1mL DMSO) 154 EUR

PHA-665752

A2307-50 ApexBio 50 mg 432 EUR

PHA-665752

A2307-S ApexBio Evaluation Sample 81 EUR

PHA-665752

HY-11107 MedChemExpress 50mg 505 EUR

PHA 665752 (MET kinase inhibitor)

SIH-421-25MG Stressmarq 25 mg 580 EUR

PHA 665752 (MET kinase inhibitor)

SIH-421-5MG Stressmarq 5 mg 193 EUR

PHA-680632

B1944-25 Biovision 838 EUR

PHA-680632

B1944-5 Biovision 251 EUR

PHA-680632

E1KS1454 EnoGene 5mg 874 EUR

PHA-793887

E1KS1487 EnoGene 5mg 874 EUR

PHA 568487

B7282-10 ApexBio 10 mg 308 EUR

PHA 568487

B7282-50 ApexBio 50 mg 1136 EUR

PHA-680632

A4122-10 ApexBio 10 mg 270 EUR

PHA-680632

A4122-200 ApexBio 200 mg 1778 EUR

PHA-680632

A4122-5 ApexBio 5 mg 177 EUR

PHA-680632

A4122-50 ApexBio 50 mg 734 EUR

PHA-848125

A8501-10 ApexBio 10 mg 351 EUR

PHA-848125

A8501-5 ApexBio 5 mg 270 EUR

PHA-848125

A8501-5.1 ApexBio 10 mM (in 1mL DMSO) 305 EUR

PHA-767491

A3721-10 ApexBio 10 mg 125 EUR

PHA-767491

A3721-5.1 ApexBio 10 mM (in 1mL DMSO) 148 EUR

PHA-767491

A3721-50 ApexBio 50 mg 350 EUR

A selective small molecule inhibitor of c-Met, <em>PHA</em>-<em>665752</em>, reverses lung premalignancy induced by mutant K-ras.

The c-Met receptor tyrosine kinase has been implicated in cellular transformation induced by mutant Ras, a commonly activated proto-oncogene in non-small cell lung cancer (NSCLC). However, the role of c-Met has not been defined in K-ras-mutant NSCLC, a disease for which no effective targeted therapeutic options currently exist. To acquire a greater understanding of its role, we used genetic and pharmacologic approaches to inhibit c-Met in mice and cultured cells. In Kras(LA1) mice, which develop premalignant lung lesions that progress to multifocal lung adenocarcinomas owing to somatic mutations in K-ras, c-Met was expressed in multiple cell types within premalignant lung lesions, and high concentrations of HGF were detected in bronchoalveolar lavage samples.
Short-term treatment with PHA-665752, a c-Met inhibitor, decreased the numbers of premalignant lung lesions and induced apoptosis in tumor cells and vascular endothelial cells within lesions. In cell culture, PHA-665752 induced apoptosis of a lung adenocarcinoma cell line derived from Kras(LA1) mice (LKR-13) and a murine lung endothelial cell line (MEC). c-Met depletion by siRNA transfection induced apoptosis of MECs but not LKR-13 cells. Collectively, these findings suggest that apoptosis was an on-target effect of PHA-665752 in MECs but not in LKR-13 cells. We conclude that PHA-665752 inhibited lung tumorigenesis in Kras(LA1) mice and may provide a novel therapeutic approach to the prevention of K-ras-mutant NSCLC.

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