siRNA-MEDIATED BCL-2 AND BCL-XL GENE SILENCING SENSITIZES HUMAN HEPATOBLASTOMA CELLS TO CHEMOTHERAPEUTIC DRUGS

SUMMARY

• 1. 

  The aim of the present study was to investigate the changes in chemotherapeutic drug sensitivity of HepG2 cells transfected with Bcl-2 and Bcl-xl siRNA expression vectors.
• 2. 

  Bcl-2 and Bcl-xl siRNA and negative siRNA expression vectors were constructed and stably transfected into HepG2 cells. Reverse transcriptase–polymerase chain reaction was used to detect the target gene expression, and the Bcl-2, Bcl-xl, Bax and caspase-3 protein levels were measured using western blots and immunofluorescence. The sensitivity of the cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) was analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and flow cytometry.
• 3. 

  The Bcl-2 and Bcl-xl gene expression and corresponding protein levels in Bcl-2 siRNA, Bcl-xl siRNA and Bcl-2/Bcl-xl siRNA transfected cells were reduced compared with negative siRNA transfected or untreated cells. The Bax protein level remained unaltered but the caspase-3 level was enhanced when Bcl-2 and Bcl-xl protein levels were reduced. The MTT results demonstrated that Bcl-2 and Bcl-xl transfected cells exhibited increased sensitivity to 5-FU or HCPT. Flow cytometry demonstrated that the sub G1 cell population increased in Bcl-2/Bcl-xl siRNA co-transfected and Bcl-xl siRNA and Bcl-2 siRNA transfected cells when compared with negative siRNA or untreated cells. The latter trend was strengthened further in the presence of 5-FU or HCPT.
• 4. 

  Thus, Bcl-2 and Bcl-xl siRNA-mediated gene silencing, in combination with chemotherapy, may be a potential therapeutic strategy against human hepatoblastoma.