WT1 silencing by RNAi synergizes with chemotherapeutic agents and induces chemosensitization to doxorubicin and cisplatin in B16F10 murine melanoma cells

Abstract

Abstract. The Wilm's tumor gene (WT1), encoding a transcription factor that modulates the expression of certain genes that are involved in proliferation and apoptosis, is overexpressed in numerous solid tumors. WT1 is important for cell proliferation and in the diagnosis of melanoma. The objectives of this study were to investigate whether WT1 silencing is capable of synergizing with chemotherapeutic agents and whether this silencing is capable of sensitizing cancer cells to doxorubicin and cisplatin in the B16F10 murine melanoma cell line. In the present study, B16F10 cells were simultaneously treated with median lethal doses (LD50s) of WT1-1 or WT1-2 small hairpin RNAs (shRNAs) and chemotherapeutic agents. A total of 24 h posttransfection, a [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay] MTT assay was performed. To determine whether shRNA interference (shRNAi) is capable of sensitizing B16F10 cells to chemotherapeutic agents, cells were transfected with an LD50 of each of the recombinant plasmids, treated with varying concentrations of doxorubicin or cisplatin 24 h post-transfection, and analyzed 48 h later for inhibition of cell proliferation using the MTT assay. We observed that WT1-RNAi and the two chemotherapeutic agents acted synergistically to inhibit B16F10 cell proliferation. The greatest inhibition of cell proliferation was observed with the WT1-2/cisplatin (91%) and WT1-1/cisplatin combinations (85%). WT1 silencing using shRNAi induced the chemosensitization of cells to doxorubicin and cisplatin, with the greatest inhibition (85%) of cell proliferation being observed in the cells treated with the WT1-2/cisplatin 6 ng/µl combination. Our results provide direct evidence that WT1 gene silencing has a synergistic effect with chemotherapeutic drugs and sensitizes B16F10 melanoma cells to doxorubicin and cisplatin. This suggests that these combination strategies are potentially utilized in melanoma therapy.

WT1 Enhances Proliferation and Impedes Apoptosis in KRAS Mutant NSCLC via Targeting cMyc

“…Wagner et al showed that inhibition of WT1 reduces cell proliferation, migration and vascular formation through transcriptional activation of the proto-oncogene ETS-1 [36]. A combination strategy of WT1 gene silencing and chemotherapeutic agents cisplatin and doxorubicin was found to have a synergistic effect on inhibiting cell proliferation in B16F10 murine melanoma cells [37]. In human melanoma, WT1 protein expression was found to be associated with shorter overall survival[38].…”

WT1 expression increases with malignancy and indicates unfavourable outcome in astrocytoma

“…WT1 is supposed to act as a modulator of proliferation, a transcriptional activator and as a tumour suppressor 3 4. By regulating the antiapoptotic protein Bcl-xL, WT1 contributes to the resistance against apoptotic therapeutic strategies such as tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL),5 and silencing of WT1 leads to increased tumour sensitivity to cisplatin chemotherapy 6…”

shRNA-WT1 Potentiates Anticancer Effects of Gemcitabine and Cisplatin Against B16F10 Lung MetastasesIn VitroandIn Vivo

“…Down-regulation of WT1 protein expression by antisense oligodeoxynucleotides and RNA interference in different types of cell lines (1,(19)(20)(21) was found to result in cell growth inhibition, as well as modified expression of proteins involved in the cell cycle, such as cyclin D1, and those involved in apoptosis, such as caspase-3 and poly-ADPribose polymerase (1,19). In vivo, Zamora et al observed that delivery of complexes of small hairpin RNA plasmid against WT1 (shRNA-WT1) with polyethyleneimine (PEI) by an aerosol system to lungs of mice with B16F10 lung metastases resulted in a reduction in the number and size of lung tumor foci and the number and size of tumor blood vessels, suggesting reduced angiogenesis (22).…”