A Study On The Novel Anticancer Activities Of Honokiol

BackgroundHonokiol,a naturally occurring phenyl lignan present in Magnolia grandiflora, is a representative with a pair of hydroxy groups(-OH)in its structure.It is of multiple medicinal uses against microbial infection,anxiety,oxidative stress,platelet aggregation,among others.Previous reports have demonstrated the anticancer activities of HNK by induction of apoptosis in cancer cell lines.Recently,two important studies from Dana-Farber cancer institute of Harvad University demonstrated that HNK efficiently induced apoptosis in apoptotic resistant B-cell chronic lymphocytic leukemia(B-CLL)from B-CLL patients and multiple myeloma (MM)cells from relapsed refractory MM patients.Honokiol qualifies as a "promiscuous" rather than a "selective" agent based on its known mulitiple pharmacologic effect."Promiscuity" is not unique to honokiol because many known successful drugs as well as a number of promising natural anticancer agents such as thiladomide and asprin which are promiscuous.The best known example of a widely used drug that can be classified as promiscuous type is aspirin.In addition to its pain-relieving and antiarthritic effects,aspirin exhibits many other pharmacologic effects including blood thinning,reduction of platelet aggregation,prevention of preeclampsia(a hypertensive disorder during pregnancy),and anticancer effects.Likewise,recent studies including those from our own laboratory have revealed that many promising anticancer nature product(e.g,schisandrin B and shikonin)targeted multiple signal transduction pathways in various cell types to inhibit cancer cell growth in vitro and in vivo.Because the etiology and pathogenesis of cancer is highly complex involving abnormalities in multiple cellular checkpoints and signal transduction pathways,we are in agreement with the idea that promiscuity may be of advantage as potential anticancer agents.The indirect evidence to support this notion is that some recently developed anticancer agents with exceptionally high selectivity have failed to live up to the expectations(e.g.,specific epidermal growth factor receptor inhibitor Iressa).Cancer drug resistance is the major obstacle to the successful cancer chemotherapy.Drug resistnce is directly or indirectly associated with 90%of cancer related death.The most important causes of cancer drug resistance are(1) Overexpressed drug transporter,such as P-gp,MRP-1,BCRP;(2)Abnormal apoptotic machinery:conventional anticaneer agents,regardless of their targets and mechanisms,mostly induce apoptosis,cancer cells are usually sensitive to apoptotic induction initially,but become resistant eventually because its heterogeneity and genomic instability.Thus,the potency of anticancer drugs is largely determined by the resistance machinery of the cancer cell.More and more evidence(Such as MDR reversal agents,Iressa,Gleevac)have indicated that the best way to overcome conventional cancer drug resistance was to bypass the resistance,which is also one of the the strategy to develop new generation of anticancer agent.In this study we report that honokiol could circumvent the conventional drug resistance,and kill cancer cell by activating a programmed cell death associated with mitochondria PT pore.The study from primary leukemia cells and in vivo model are also involved in this work.To our best knowledge,this is the first report to document all these results.ObjectsTo investigate the effect of honokiol on MDR cancer cells;To investigate whether honokiol could induce a nonapoptotic cell death on cancer cells;To analyze the mechanism of the nonapoptotic cell death triggered by honokiol;To evaluate the effect of honokiol on primary cancer cells and its assoicated mechanism;we also determined the effect(lifespan,tumor size,and invasion)of bonokiol on female NOD/SCID mice bearing implanted HL60 cells.Results1.The anticancer effect of honokiol on MDR and sensitive cancer cells(1)Using four pairs of MDR and their parental cancer cell lines,K562/ADR and K562,HL60/ADR and HL60,MCF-7/ADR and MCF-7,Beap37/ADR and Bcap37, we determined the drug sensitivity of all these cell lines using MTT assay. Paticularly important,we found honokiol had the same efficacy in killing MDR cells and their parental drug-sensitive cells.Combined with our previous report,we believe honokiol is not the substrate of the MDR pump;(2)Caspase-3 is the most important apoptotic marker,thus we examined if it was activated in the cells treated with honokiol.There was no caspase-3 activation in HL60,MCF-7 and their MDR cells.Consistently,no apoptotie morphological changes under electronic microscopy were found.Thus,we thought that honokiol may trigger cell death other than apoptosis;(3)Using drug-sensitive parental cells as representatives to explore the truth of killing effect of honokiol,we adopt Hoechst staining to measure apoptotic cell death and Trypan blue exclusion assay to measure the total cell death. The discrepancy between the apoptotic death rate and total death rate was confirmed, which led to the conclusion that honokiol could induce nonapoptotic cell death in cancer cells.2.Honokiol induces a regulated nonapoptotic cell death in cancer cells Here,we show that honokiol can induce a cell death distinct from apoptosis in HL60.After exposure to honokiol,HL-60 exhibited the following characteristics: (1)Using FCM detection,the death characterized with a rapid loss of integrity of plasma membrane which is both time and concentration dependent and without externalization of phosphatidyl serine;(2)Significant change of cell cycle (hypodiploid peak)and DNA ladder,the hallmarks of apoptosis,were not observed. Consistently,the broad caspase inhibitor zVAD-fmk failed to prevent the cell death; (3)Apoptosis-inducing factor(AIF)was not translocated to nuclei in HNK-treated HL60 cells;(4)Honokiol could overcome Bcl-2 or Bcl-X_L mediated apoptotic resistance,because they can not affect the cell death;(5)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 ratios increased even after drug was removed,but still below the death ratio without wash-out.This assay prove that the cell death was regulative;(6)Cyclosporin A(CsA),an inhibitor of mitochondria permeability transition pore,effectively prevented HNK-induced cell death;(7)HNK induces similar cell death in MCF-7 cell line.3.Mitochondrial permeability transition pore is the key to the novel killing mechanism by honokiolSince ultrastructural studies revealed the damaged mitochondria in HNK-treated cells, we hypothesized that HNK-induced death was probably due to the mitochondrial dysfunction.Mitochondria plays a central role in cell death.It serves as an integrator of upstream death signaling.(1)The Mitochondrial memberane potential (MMP)was examined by JC-1 staining via FCM detection after treatment with 15μg/ml of 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 15μg/ml of HNK for 1,3,6,12 hours.The HNK treated HL60 cells showed increased ROS production in all the time points,while CsA could partially inhibit ROS generation;(2)The opening of PT pore was examined by Calcein-AM staining and observed by confocal microscope after treatment with 15μg/ml of HNK for 6 hours.The green fluorescence in HNK group was weak compared to control, indicating the opening of PT pore,while CsA could partially inhibit this opening. We presumed that CsA,an inhibitor of cyclophilin D(an essential component of mitochondria permeability transition pore),effectively prevented HNK-induced cell death;(3)Inhibition of cyclophilin D by RNA interference blocked honokiol-induced cell death,indicating that the cell death was mechanistically associated with the mitochondria permeability transition(PT)pore regulated by cyclophilin D;(4)Upregulation of cyclophilin D was observed in HL60,MCF-7 with the susceptibility to the death,but not in K562 which underwent apoptosis after exposure to honokiol.4.The study of the anticancer efficacy of honokiol on primary leukemia cellsTo further explore the potential of HNK-induced cell death in clinical application, primary leukemia cells from fifteen untreated patients with acute leukemia were collected.(1)The primary cells were incubated in vitro with various concentrations of HNK for indicated hours and assayed for cell death and LC₅₀.The concentration of HNK that caused death of 50%of the cells(LC₅₀)(from most of the samples)after 24 hours of incubation with HNK was 30μg/ml.Although HNK was cytotoxic toward normal peripheral blood mononuclear cells(PBMCs)(48 hour LC₅₀is 52μg/ml),primary cancer cells were significantly more susceptible to HNK;(2) Consistent with the result in HL60 cells,in ten patients,HNK induced a dominant caspase-independent and CsA-inhibitable cell death characterized with a loss of plasma membrane integrity(PI positivity)but without a significant externalization of phosphatidyl serine(Annexin V-FITC positive).HNK also significantly upregulated CypD in AML cells like HL60;(3)With the colony forming assay,we found after fourteen days culture in Methocult,the number of CFU derived from the honokiol pretreatment is the same as the Ara C pretreatment.The results indicated that the CypD associated death in human primary AML cells may have potential clinical significance.5.The pilot study of the in vivo effect of honokiol on female NOD/SCID mice bearing implanted HL60 cellsFurthermore,a prolonged survival time was observed after treatment with HNK in SCID mice transplanted(i.p.or i.v.)with human HL60 cells engrafts.In the i.v. group,the Median survival time(MST)of HNK group was 29 days,compared with the control group(MST 17 days).Consistenly,in the i.p.group,the Median survival time(MST)of HNK group was 37.5 days,compared with the control group(MST 24.5 days).Although in the i.p.group,there was no statical difference between control and honokiol groups on the mean volume of HL60 implanted tumor(P＞0.05), honokiol treatment could mitigate the wide spread invasion of the implanted leukemic cells.Conclusions1.Honokiol has the same therapeutic efficacy to MDR and sensitive cancer cells.2.Honokiol can bypass cancer apoptotic resistance by induction of a regulated nonapoptotic cell death in cancer cells.3.Honokiol induces a mitochondrial permeability transition pore dependent nonapoptotic cell death.4.Honokiol can also induce a cell death distinct from apoptosis in primary leukemic cells via mitochondrial permeability transition pore5.Honokiol could significantly prolong the lifespan of the NOD/SCID mice bearing implanted HL60 cells,it could also mitigate invasion of the HL60 cells。...