Combined Effects Of Bcl-xl And Mcl-1 Antisense Oligonucleotides In Melanoma Drug Resistance

Objective: Malignant melanoma is the most aggressive form of skin cancer. Multidrug resistance is the vital factor of ineffective treatment in malignant melanoma. Thus, one of the main goals for melanoma treatment is to reverse chemoresistance of tumor cells.A number of studies have suggested that apoptosis and its regulation plays an important pathogenic role in the chemoresistance of human melanoma. Over expression of the antiapoptotic gene can protect cells from apoptosis induced by chemotherapeutic agents, which may be relevant to the poor prognosis and high death rate of melanoma.Bcl-2-related proteins , key regulators of apoptosis, are the most frequently studied.The delicate balance between antiapoptotic and proapoptotic Bcl-2 family members determines whether the cell survives or undergoes apoptosis. Bcl-xl and Mcl-1 are antiapoptotic members of Bcl-2 family. In recent years, it is found that downregulated Bcl-xl or Mcl-1 gene expression by using Bcl-xl AS-ODN or Mcl-1 AS-ODN separately can enhance chemosensitivity of tumor cells. Our research has showed that Mcl-1 AS-ODN can partially reverse the chemoresistance of melanoma. But tumorigenesis is a multistep process with many genes being involved in, and the chemoresistance of human tumor may be related to alteration of many genes. It is reasonable to speculate that combined utility of antisense oligonucleotides against antiapoptotic Bcl-2 family members ,such as Bcl-xl and Mcl-1, may prove even more promising. In this experiment we transfected Bcl-xl AS -ODN in combination with Mcl-1 AS-ODN into WM451 cells with lipofectamine 2000 to observe the combined effects of Bcl-xl AS-ODN and Mcl-1 AS-ODN on the chemoresistance of melanoma. The aim is to get the more convinced imformation about the function of Mcl-1 AS-ODN and Bcl-xlAS-ODN as a potential therapeutic target in melanoma, and to provide new targets for the antisense therapy of melanoma.Methods: Bcl-xl ASODN and Mcl-1 ASODN were transfected into cultured melanoma cell line WM451 with lipofectamine 2000.The expression of Bcl-xl and Mcl-1 nucleotide and protein was detected by Reverse transcription polymerase chain reaction(RT-PCR),Western blot and Immunocytochemistry. Apoptosis was assayed by Elecrton microscope and FAC.The sensitivity of WM451 cells to chemotherapeutic agents was assayed by MTT method.Results1.Transfection of Bcl-x1 AS-ODN and Mcl-1 AS-ODN in combination , could downregulate the mRNA level of Bcl-xl and Mcl-1 gene at the same time.2. After transfection with Bcl-xl and Mcl-1 AS-ODN, the protein expression level of Bcl-xl and Mcl-1was downregulated at the same time.3.Electron microscopy showed that WM451 cells exhibited characteristic morphologic changes of apoptosis after transfected with Bcl-xl AS-ODN and Mcl-1 AS-ODN. Flow cytometry demonstrated that transfection with both Bcl-xl and Mcl-1 AS-ODN resulted in significantly more cells undergoing apoptosis compared with either AS-ODN alone.4.Downregulation of Bcl-xl and Mcl-1 separately or simultaneously can enhance the chemosensitivity of cells to DTIC, with IC50 from 152.74ug/ml(Control) to 83.33ug/ml(Bcl-xl AS-ODN), 92.63 ug/ml (Mcl-1 AS-ODN), and 64.34 ug/ml(Bcl-xl AS-ODN +Mcl-1 AS-ODN), respectively. Combined utility of Bcl-xl AS-ODN and Mcl-1 AS-ODN appears to be more effective in restoring the sensitivity of cells to DTIC than either AS-ODN alone.5.Treatment with Bcl-xl and Mcl-1 AS-ODN separately as well as in combination did not result a significant reduction of IC50 value of DDP compared with S-ODN or NS-ODN treatment.Conclusions:1.Transfection of Bcl-xl or/and Mcl-1AS-ODN can induce apoptosis of WM451 by downregulating of Bcl-xl or Mcl-1expression .2.Bcl-xl and Mcl-1AS-ODN used separately or in combination, can enhance the chemosensitivity of WM451 to DTIC, and partially reverse thechemoresistance of melanoma. Our data suggest that Bcl-xl and Mcl-1 are promising targets for antisense therapy of melanoma.3.Combined effects of Bcl-xl AS-ODN and Mcl-1AS-ODN are better than that of Bcl-xl AS-ODN or Mcl-1AS-ODN alone in reversing chemo -resistance of melanoma. The results provide a useful experimental basis for the clinical combination of targeted therapies with antisense oligonucleotides.