BI-33, a novel and potent pan-Bcl-2 inhibitor, induces apoptosis in cancer cells and shows combinational effect with chemo drugs

C229 Introduction: BI-33 is a novel flavonoid compound which mimics the Bim BH3 peptide. Our study is to determine the biological effect of BI-33 and its therapeutic potential used as a single agent or in combinational treatment with chemo drugs.
 Experimental Procesures : BI-33 binds to Bcl-2, Bcl-xL and Mcl-1 with Ki value at 17nM, 1.2uM and 18nM, respectively, as shown in fluorescence-polarization-based binding assay. BI-33 inhibits cell growth in 2LMP breast cancer cell line and PC3 prostate cancer cell line as shown by 4 days WST assay, with IC50 at 50nM and 40nM, respectively. BI-33 used at 1uM for 4 days induces ~50% of cell death in these two cell lines as demonstrated by trypan blue assay and confirmed by AnnexinV-PI apoptotic assay. BI-112, served as compound control which has low binding affinity to Bcl-2, Bcl-xL and Mcl-1, does not induce cell death even at 10uM. To determine if mitochondrial apoptotic pathway is activated by BI-33, cytochrome C release and caspase3 activation is investigated. BI-33 at 10uM for 2 days induces cytochrome C release in cytosolic fraction and PARP cleavage in nuclear fraction from 2LMP cells. Caspase3 activity was increased 6 fold by BI-33 in 2LMP cells. To further determine if BI-33 targets Bcl-2 or Bcl-xL for cell killing, stable clones of Bcl-2 or Bcl-xL in MDA-MB-435 cells were established and confirmed the increased resistance to VP16. BI-33 counters overexpression of Bcl-2 or Bcl-xL as shown by efficient killing of these stable clones. To investigate if BI-33 synergizes with chemo drugs to induce cell death, Paclitaxel is tested in 2LMP cells and IC50 of Paclitaxel is decreased 10 fold by co-treatment with BI-33. We further tested Jurkat cells with overexpression of Bcl-2 or Bcl-xL, which show increased resistance to VP-16. BI-33 can also sensitize these stable clones to VP-16 treatment.
 Conclusion: Taken together, our study shows that BI-33, as a pan-Bcl-2 inhibitor, induces cancer cell death through activation of mitochondrial apoptotic pathway, and has potential to be used as single agent or in combinational treatment with chemotherapy, to kill cancer cells which have high level of Bcl-2, Bcl-xL and Mcl-1.