| Honokiol (HNK), an active component isolated and purified from magnolia, has antioxidant, antithrombosis, antibacterial, xanthine oxidase inhibitory, and anxiolytic effect. Previous reports have demonstrated that HNK induces apoptosis in many tumor cell lines, but little is known about the nonapoptotic cell death induced by HNK. Here in our study, we investigated the phenomenon and the possible mechanism of the HNK induced nonapoptotic programmed cell death in human leukemia HL60 cell line. The main contents are divided into some sections as follows:1. HNK inhibits growth of HL60 cells in vitro in a concentration- and time-dependent mannerMTT assay shows concentration-dependent growth inhibition effect of HNK in HL60 cells, and the IC50 is about 13.47μg/ml. Time-dependent inhibition of HL60 cells exposed to 10, 15 and 20μg/ml of honokiol for different time length were examined by Lactate Dehydrogenase assay.2. HNK induces caspase-independent cell death in HL60 cell lineHL60 cells were treated with 15μgfml HNK with or without 50μM zVAD-FMK for 12 hours, and cell death ratio was determined by PI staining via FCM. The death ratio in combination group is 44.95 % ± 3.63 %, which has no significant difference with the HNK group (53.01%±3.52%)(p>0.05). In addition, after treatment with 15μg/ml HNK for 6,12,24 hours, HL60 cells shows no significant increased activity of caspase-3 by the caspase-3 detection kit (p>0.05).3. HNK does not induce the AIF translocation in HL60 cell lineAIF translocations in HL60 cells were determined by immunofluorescence after treatment with 15μg/ml HNK for 12 hours. The confocal imaging shows no AIF translocation in the HNK group.4. HNK has no effect on the cell cycle of HL60 cells, and does not induce DNA-ladderingCell cycles of HL60 cells were determined by FCM after treatment with 15μg/ml HNK for 8 hours. There is no significant change of cell cycle and no apoptotic peak in HNK group compared with control. In addition, HL60 cells were treated with 15μg/ml HNK for 6,12,18 hours and DNA was extracted by DNA-laddering Kit, but no DNA ladderingwas observed in HNK group.5. HNK-treated HL60 cells have no ultrastructural feature of apoptosisHL60 cells were observed by transmission electron microscope after treatment with 15//g/ml honokiol for 8hrs. Honokiol induces no ultrastructural features of apoptosis, while vacuoles containing membranous whorls, autophagosome-like vacuoles, and rounded mitochondria with disrupted internal structures were observed in HNK group.6. HNK induced cell death could be recovered in a time-dependent mannerAfter treatment with HNK for different time length, all the samples were washed, replaced with fresh media and incubated for another 24hrs. Cell death rates were examined with trypan blue. The cytotoxic effect can be recovered when cells were treated with HNK for relatively short time. And when cells were treated with HNK for longer time, the death ration increased even after drug wash out, but still below the death ratio without wash out.7. Cyclosporin A could inhibit HNK induced cell death in HL60 cell lineHL60 cells were treated with 15jug/m\ HNK with or without Cyclosporin A> Bongkrekic Acid and 3-Methyladenine, and cells were examined by PI staining via FCM. The cell death ratio of HNK group is 11.56% ±0.34% for 6 hours, 51.06% ±1.34% for 12 hours, and 80.07%±2.11% for 18 hours. CsA could significantly decrease the cell death ratio to 4.14% ±0.50%, 24.93% ± 1.85%, and 68.8% ± 1.20%(p<0.05). BAand 3-MA have no significant inhibition of this cell death.8. HNK induces mitochondrial PT pore opening, loss of mitochondrial membrane potential(MMP) and increased ROS productionThe opening of PT pore was examined by Calcein-AM staining and observed by confocal microscope after treatment 15/zg/ml HNK for 6 hours. The green fluorescence in HNK group is weak compared to control, indicating the opening of PT pore, while CsA could partially inhibit this opening. The MMP was examined by JC-1 staining via FCM detection after treatment with 15fig/ml HNK for 6,12,18 hours. The ratio of JC-1 Low cells increases in HNK group, indicating the loss of MMP, while CsA could partially inhibit this loss. The ROS level was examined by H2DCFDA staining via FCM detection after treatment with 15^g/ml HNK for 1,3,6,12 hours. The HNK treated HL60 cells shows increased ROS production in all the time points, while CsA could partially inhibit ROS generation.9. HNK up-regulates the mRNA level of Cyclophilin D in HL60 cell lineReal-time PCR shows the increased mRNA level of Cyclophilin D in HL60 cell after treatment with 15/jg/ml HNK for 1.5, 3, 6, 9 hours (p<0.05). The relative levels (compared to control) of Cyclophilin D mRNA at these time point are 2.42+0.42, 3.10 ±0.21, 1.71+0.11, 1.38 + 0.31. But there is no significant increase of Cyclophilin D mRNA level in HNK treated K562 cells (p>0.05).10. HNK induces similar cell death in HEK293 and MCF-7 cell linesHEK293 cells were treated with 4Qag/ml HNK for 8 hours, and the dead cells were determined by trypan blue staining. The death ration of HNK group is 49.60% + 1.59%, while CsA could significantly decrease the ratio to 23.96% ±5.32% (p<0.05). MCF-7 cells were treated with 4Qwg/ml HNK for 8 hours, and the dead cells were determined by PI staining. The death ration of HNK group is 37.16%, while CsA could decrease the ratio to 13.07%. The DNA analysis via FCM shows no apoptotic peak in both cell lines after treatment with HNK for 8 hours. Besides, there is no apoptotic feature observed by transmission electron microscope in HNK treated cell lines. Both the HEK293 and MCF-7 cells show the similar autophagosome-like vacuoles, and rounded mitochondria with disrupted internal structures with HL60 cells under electron microscope.ConclusionHNK-induced cell death in HL60 cell line is independent of caspase activation and nuclear translocation of apoptosis-inducing factor, and shows no apoptotic feature in DNA analysis and electron microscope images. HNK treated HL60 cells exhibit mitochondrial damage and extensive cytoplasmic vacuolation under electron microscope. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, loss of MMP, and increased ROS production. The PT pore inhibitors CsA could partially block the mitochondrial dysregulation and cell death. In addition, HNK could upregulate the mRNA level of Cyclophilin D in HL60 cell line, indicating the potential regulation effect of Cyclophilin D in this kind of cell death. Besides, HNK induces the similar cell death in HEK293 and MCF-7 cell lines, suggesting the generality of this kind of cell death. |
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