The Radiosensitization Effect And Its Mechanism Of Smac-mimetic Compound SM-164in Breast Cancer Cells

Breast cancer is the most frequently diagnosed cancer and the leading cause ofcancer death among females. Radiotherapy is a treatment choice for local control ofbreast cancer, particularly after the removal of tumor tissues by surgery. However,intrinsic radioresistance of cancer cells limits therapeutic efficacy. The main functionof IAPs is to suppress apoptosis via binding to and thus inhibiting active caspase-3,-7and-9. XIAP, cIAP-1and cIAP-2are the important members in IAP familyproteins. SM-164is a small molecule compound that mimics SMAC, amitochondrial protein released during apoptosis to activate caspases by inhibitingcellular inhibitor of apoptosis proteins, cIAP-1and XIAP. BCL-2family membersplay an on-off role in the apoptosis of cancer cells. BM-771, a novel inhibitor ofBCL-2, can block the function of BCL-2family for suppressing apoptosis.Objective: To determine in breast cancer cells the potential radiosensitizingactivity of SM-164and explore the relating mechanism of radiosensitization. Toevaluate in breast cancer cells the radiosensitizing activity of BM-771.Methods:(1) Western blotting assay were used to examine their protein levelsof IAPs and BCL-2family, and to evaluate the effect of cIAP-1degradation inducedby SM-164. The viability of the cells was measured using one-step ATPlite cellproliferation assay. Radiosensitization of SM-164and BM-771were determined bystandard clonogenic assay.(2) Breast cancer cells (SK-BR-3and MDA-MB-468)were assigned to the following four groups: Control, SM-164200nM, Radiation6Gyand SM-164+Radiation. The nature of SM-164radiosensitization was evaluated bymorphological observation, FACS analysis, DNA fragmentation assay and westernblotting analyses determining protein levels of cleaved PARP and cleaved caspase-3.Caspase activation on SM-164radiosensitization was detected by caspase activityassays based ELISA. Caspase inhibitors and siRNA silencing were used to blockSM-164radiosensitization.Results:(1) There were many high protein levels of IAPs and BCL-2family in breast cancer cells. cIAP-1was rapidly degraded by SM-164in all four highexpression cIAP-1breast cancer cells. All four breast cancer lines with highexpression of cIAP-1are sensitive to BM-771as a single agent, but BM-771can'tinduced a radiosensitzation in breast cancer cells. We found that three of four breastcancer lines with high expression of cIAP-1are resistant to SM-164as a single agent,but SM-164at nanomolar concentrations sensitized in breast cancer cells, includingSM-164resistant SK-BR-3cells and SM-164partially sensitive MDA-MB-468cells,to radiation with a sensitization enhancement ratio (SER) of1.7~1.8. Human breastepithelia MCF10A cells, with undetectable levels of cIAP-1, were resistant toSM-164, indicating a low potential toxicity of SM-164on normal cells.(2) Asignificant increase in the number of shrunken/detached cells was seen incombinational treatment with radiation in MDA-MB-468and SK-BR-3. In FACSanalysis, had significantly enhanced radiation-induced apoptosis cells when used incombinational treatment of radiation with SM-164(P＜0.05). The assay for DNAfragmentation, revealed that in SM-164resistant SK-BR3cells, the180-bp DNAladders were only obviously seen in combinational treatment group, whereas inSM-164sensitive MDA-MB-468cells, single treatment with SM-164caused thesubstantial DNA fragmentation, which was further enhanced by radiation. PARPcleavage and caspase-3activation/cleavage also demonstrated a maximal inductionof cleaved PARP and caspase-3by combinational treatment in both cell lines. Theseresults strongly suggested that SM-164radiosensitization is associated withenhanced apoptosis with potential involvement of caspase activation. Aboutcaspase-3,-8and-9activation assay, in SK-BR-3cells, combinational treatmentsignificantly activated both caspase-9and-3up to3-fold (P<0.01), but no effect oncaspase-8activation; in MDA-MB-468cells, combinational treatment caused asignificant increase of active caspases with an up to4-,5-and8-fold activation ofcaspases-8,-9and-3, respectively (P<0.05or P<0.01). SM-164radiosensitization inSK-BR-3cells resistant to SM-164is mediated by intrinsic mitochondrial apoptosispathway through activation of caspases-9and-3, whereas in MDA-MB-468cellssensitive to SM-164, it is mediated by both intrinsic mitochondrial and extrinsicdeath receptor apoptosis pathways via activation of caspases-9,-8, and-3. z-VAD-fmk, a pan-caspase inhibitor, treatment completely blocked apoptotic DNAfragmentation induced by combination of SM-164and radiation, and largelyabrogated SM-164radiosensitization in both cell lines. In SK-BR-3cells caspase-3knockdown completely inhibited apoptotic DNA fragmentation. siRNA knockdownof caspase-9and caspase-3significantly abrogated SM-164radiosensitization withSER reduction from1.57to1.27or1.23, respectively, whereas caspase-8knockdown had no effect with SER change from1.57to1.55.Conclusions: We report here that SM-164is a potent and novel class ofradiosensitizer for breast cancer cells, and low cell toxicity in normal cells. SM-164radiosensitization is mediated by caspase activation. BM-771, a novel inhibitor ofBCL-2, is a potent cytotoxic compound in breast cancer cells, but isn't an effectiveradiosensitizer. Removal of negative blockers, such as cIAP-1and XIAP by SM-164leads to activation of caspase-9and-3via the DNA damage pathway for enhancedapoptosis induction.