Effect of Cilengitide and Vismodegib on Primary Cell Cultures and Cell Lines of Head and Neck Squamous Cell Carcinoma (HNSCC) after Targeting Integrin and Hedgehog Pathways

Due to the low 5-year survival rate and the high recurrence rate of HNSCC, new chemotherapeutic strategies are of high clinical interest. Integrins are important adhesion molecules and play a regulatory role in many critical biological processes such as cell proliferation, invasion, apoptosis, metastasis, and angiogenesis in squamous cell carcinoma. Cilengitide is a cyclized RGD containing pentapeptide that selectively blocks activation of the integrins. Recently, growing evidence has suggested aberrant activation of the HH pathway in squamous cell carcinoma plays a critical role during cancer development. Vismodegib (GDC-0449) is an orally administered agent that selectively suppresses SMO activity. Most previous studies used cell lines purchased from commercial companies. These cell lines may already have differentiated and lost some of their potential characteristics as tumor cells. Accordingly, we isolated and identified primary HNSCC cell cultures, and evaluated the effects of cilengitide and vismodegib on both cell lines and primary cell cultures. The application of drugs to the cell lines or primary cell cultures produced different results. Four tongue squamous cell carcinoma cell lines (SCC4, SCC9, SCC15 and SCC25) were obtained from the American Type Culture Collection. Three primary cell cultures (TU53, TU57 and TU63) were obtained, respectively, from three patients with HNSCC, who underwent surgery. DNA profiling using STRs showed that K53 and TU53, K57 and TU57, K63 and TU 63 are all homologous. To identify the biological characteristics of the primary cell cultures and cell lines, four kinds of typical immunocytochemical staining were performed. All cells expressed negative CD90, excluding a fibroblast source. Both cell lines and primary cell cultures strongly expressed cytokeratin, which is characteristic of epithelial cells. All cells showed moderate expression of vimentin, demonstrating a mesenchymally derived sarcoma tumor. All cells expressed SHH, indicating activation of the HH pathway in HNSCC. MTT and BrdU tests showed proliferation of cell lines and primary cell cultures were suppressed by cilengitide or vismodegib in a dose dependent manner without exception. Primary cell cultures expressed more significantly depressed growth than cell lines. 6.25 μM of cilengitide had a significant inhibitory effect on primary cell cultures, but a concentration as high as 100 μM was needed to produce a similar effect on cell lines. 50 μM vismodegib significantly inhibited the proliferation of primary cell cultures, but for cell lines the concentration needed to be as high as 100 μM. Primary cell cultures may retain more of their internal biological characteristics. Therefore, individualized treatment may be necessary with regard to the different stages of tumor progression. RT-PCR results showed that 50 μM cilengitide significantly reduced ITGAV, ITGB3, and ITGB5 gene expression. But incubation with 100 μM cilengitide significantly increased expression of ITGAV, ITGB3, and ITGB5 in all cells. Cilengitide can inhibit tumor cell proliferation and integrin gene expression at low concentrations, holding tremendous promise in tumor chemotherapy. However, the research on the role of RGD peptides in cancer therapy is still in its early stages and further investigations are required. RT-PCR results also showed that GLI1 gene expression was reduced significantly by incubation with 50 μM or 100 μM vismodegib. The results demonstrated that vismodegib could effectively block activation of HH signal pathway. Findings from current study have potentially important clinical implications. We found that the integrin inhibitor cilengitide and HH signal inhibitor vismodegib can inhibit HNSCC cell proliferation and reduce target gene expression at certain concentrations. Patients with HNSCC tumors may benefit from an individualized therapeutic strategy.


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