Honokiol Induces Programmed Necrotic Cell Death,Synergizes Etoposide In Breast Cancer Cells

Honokiol (HNK), a small-molecule pharmacologically active component, exhibits competent cytotoxicity in a variety of human cancer cells. Taking its anti-tumor effect into consideration, mechanisms such as tumor-cell death induction, anti-angiogenesis, anti-migration and anti-multiple drug resistance are all taking participation. Among all the anti-tumor effects, honokiol-induced cell death is the most fundamental one.Previous study first reported honokiol induced a distinct programmed cell death modality, programmed necrotic death, which was modulated by Cyclophilin D (CyP D). And this initiates new insights and greatly expands the cognition of its antitumor activity. In the current study, we demonstrated necrotic-cell proportion (PI positive) gradually increased and early-stage apoptotic cell proportion(PI negative and AV positive) decreased in dose-and time-dependent manner after honokiol treatment. We revealed these PI positive cells were under necrotic cell death, as no late-apoptosis characteristics including conspicuous chromatin condensation or DNA ladder patterns were detected. These results revealed cells suffered death modes transition from early-stage apoptosis to programmed necrosis with the increase of honokiol dose or treatment time. Protein expressions of RIP3markedly increased parallelling with HNK-triggered death modes transition, while expressions of RIP1decreased. Cyclophilin D expression increased during cell death modes transition, and inhibited cyclophilin D by cyclosporin A apparently blocked HNK-triggered programmed necrosis. Such results indicated honokiol-induced programmed necrosis and death modes transition are potential RIP3-dependent, cyclophilinD-regulated. Further results showed blocked cyclophilinD by cyclosporin A did not affect HNK-induced RIP3overexpression, which indicated cyclophilinD was a modulator at downstream of RIP3.On the other hand, honokiol also demonstrated a prominent antitumor effects in primary cultured breast cancer cells as both mean IC50and cytotoxicity of honokiol at40μg/ml approximated those in breast cancer cell lines.Previously, we systematically and quantitatively assess its combinational effect with different chemotherapeutic agents using combination index (CI) equation. We found honokiol synergized chemotherapeutic agents both in sensitive and resistant, solid and none-solid (MCF-7, HL-60, MCF-7/ADR and HL-60/ADR) cell lines. In the current study, we further estimated mechanisms, through which honokiol synergized chemotherapeutic agents and reversed multidrug resiestance. Honokiol (40μg/ml) induced a necrotic cell death in MCF-7/ADR cells with characterized morphological and biochemical features. Co-incubation with honokiol and etoposide (VP-16) activated a complex death modality, which was composed of necrotic cell death and apoptosis. This dual-death pathway was shut down when pretreated with pan-caspase inhibitor (z-VAD-fmk) and cyclophilin D inhibitor (Cyclosporin A). Western blot results proved that honokiol also enhanced VP-16-induced apoptosis potentially via blocking NF-κB activation.In conclusion, honokiol triggered a potential RIP3-denpendent cell death modes transition from early-stage apoptosis to programmed necrosis, which is highly-regulated by CyclophilinD. And honokiol synergizes chemotherapy drugs in multidrug resistant breast cancer cells through enhanced apoptosis and additional programmed necrotic death. All these findings arise a promising way to circumvent MDR and apoptosis tolerance.