Background & objective: Human papillomavirus (HPV) are the most important etiologic factor for cervical carcinoma. E6 virus gene is one of the most important oncogene and expression levels of E6 transforming oncoproteins of high risk HPV genotypes, such as HPV16, appear to be necessary for maintaining the malignant phenotype. Radiation treatment represents a standardized and effective modality for contemporary cervical carcinoma therapy. The goal of this study was to investigate the radiation sensitizing effect of anti-HPV16 E6-ribozyme on cervical carcinoma cell line.
Methods: With the method of lipofectin transfection, the anti-HPV16 E6 ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell line, which named as CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in the three kinds of cells were detected by Northern blot. The growth rates of the CaSKi and transfected cells were examined by cell count and their sensitivity to radiotherapy were examined by colony formation test. The apoptosis rates of each cell was determined by PI/Annexin V stained methods. Expressions of p53, bcl-2, and bax were determined by Flow cytometry analysis.
Results: Anti-HPV16 E6-ribozyme can be expressed stably in transfected CaSKi-R cells. Northern blot showed that E6 mRNA was less in CaSKi-R than in CaSKi and CaSKi-P. The growth rate of CaSKi-R was much slower than that of CaSKi and CaSKi-P. The sensitivity of CaSKi-R cells to radiotherapy increased more than that of CaSKi and CaSKi-P cells. The ability of colony formation decreased (P < 0.05), while the apoptosis rates of CaSKi-R cells increased more than that of CaSKi and CaSKi-P cells(P < 0.01). Anti-HPV16 E6-ribozyme did significantly upregulate expression of p53, bax protein, and downregulate the expression of bcl-2 protein before and after radiotherapy (P < 0.01).
Conclusion: CaSKi-R cells transfected with Anti-HPVE 6-rivozyme showed growth inhibition and increased sensitivity to radiotherapy.