Inhibition of cyclin D1 expression associated with increased cisplatin chemosensitivity

Objective: Cyclin D1 overexpression is associated with tumor initiation and proliferation.Its prosurvival function has been noticed in some kind of tumors. Since increasing expression of cyclin D1 is common event in oral squamous cell carcinoma and has been correlated with cisplatin resistance.We investigated if inhibition of cyclin D1 expression could increase cisplatin chemosensitivity of oral squamous cell carcinoma.

Methods: Five cyclin D1 shRNA lentiviral particles were prepared and transfected cisplatin resistant oral squamous cell carcinoma cell line Tca/cisplatin. Cyclin D1 silencing efficacy was investigated with Real-time PCR and Western Blot. Two most effective lentiviral particles were selected for following in vivo and in vitro experiments. IC50 value for cisplatin was calculated with modified MTT assay. Cisplatin induced apoptosis and cell cycle block were investigated with Flow cytometry. Tumor transplanted model was prepared in nude mice to examine the cisplatin sensitivity of Tca/cisplatin after cyclin D1 silencing treatment in vivo.

Results: Stable transfection of these five shRNAs led to significantly decreasing expression of cyclin D1 mRNA and protein. The most effective shRNA decrease 77% cyclin D1 mRNA level and 80% protein level. Two most effective shRNAs lentivirus were selected in the studies. After transfected with two cyclin D1 shRNAs, IC50 of cisplatin resistant oral squamous cell carcinoma decreased from 5.01mg/ml to 1.24mg/ml and 2.39mg/ml. More apoptosis rate was observed in cyclin D1 silencing cells. In vivo transplanted tumor models confirmed cisplatin sensitizing effect of cyclin D1 shRNA in oral squamous cell carcinoma. Tunnel assay validated an increasing apoptosis induced by cisplatin in cyclin D1 shRNA treatment groups, accompanied with decreasing expression of PCNA and CDKN1A.

Conclusion: Reduction of cyclin D1 expression in cisplatin resistant oral squamous cell carcinoma cell line inhibits cell proliferation and enhance apoptosis inducing by cisplatin. Cyclin D1 is target for future therapy with oral squamous cell carcinoma.