Circumvent Cancer Drug Resistance By Induction Of A Necroptotic Cell Death

Background:Cancer drug resistance is a major problem in chemotherapy.The potency of anticancer drugs is largely determined by their efficacies in selectively killing cancer cells and simultaneously inducing drug resistance in cancer cells.Conventional anticancer agents,regardless of their targets and mechanisms,mostly induce apoptosis.Cancer cells are usually sensitive to apoptotic induction initially,but become resistant eventually through dysregulation of apoptotic machinery, manifested by overexpression of anti-apoptotic proteins and defect in apoptotic signaling.Numerous apoptotic inducers are also inducers and substrates of drug transporters,including P-glycoprotein(P-gp),multidrug-resistance associated protein (MRP1),and breast cancer resistance protein(BCRP).Since these drug transporters recognize many structurally and functionally unrelated anticancer agents and efficiently expel intracellular drugs out of cells,the overexpression of these proteins confers cancer cells with a multidrug resistance.Some drug transporters such as P-gp also protect cancer ceils from caspase-dependent cell death.There are several potential pharmacological approaches to overcome cancer drug resistance.To overcome drug transporter(i.g.,P-gp)mediated drug resistance is theoretically achievable,because the targets are few and mechanisms are clear. However,clinically,the efficacies of the specific inhibitors to these drug transporters are yet not conclusive.To overcome the drug resistance relevant to apoptotic defect is much more complicated than to drug transporters.Apoptotic machinery is composed of at least dozens of anti-apoptotic and pro-apoptotic proteins.The balance of anti-and pro-apoptotic proteins contributes to the balance of cell growth and cell death.Many lines of evidence have demonstrated an imbalance with elevated anti-apoptotic and reduced pro-apoptotic activities in cancer cells one way or another,including overexpression of anti-apoptotic proteins(Bcl-2,Bcl-x_L,Mcl-1, c-FLIP,lAPs,heat shock proteins),mutations of pro-apoptotic proteins(P53,Apaf-1, Bax,FAS,FADD,caspase),and loss ofcaspases(caspase 3,caspase 8).In addition, the apoptotic pathways in cancer cells are affected by many oncogenic signals. Therefore,it is highly difficult to treat cancers with apoptotic resistance because of so many potential targets.Many research showed that,besides apoptosis,there are several other programmed cell death pathway distinct form the former.Tumor cells can die by various non-apoptotic programmed cell death pathway,such as necrosis,senescence, autophagy and mitotic catastrophe etc.Since the molecular mechanisms of each death pathway are distinct from each other,no matter how dynamic the resistance occurred along apoptosis,it would be restricted within apoptotic pathway only,and would not affect other death pathways with mechanisms separate from apoptosis. So,it is likely to circumvent apoptotic resistance of cancer cells,if the action of chemotherapeutic agents is to induce non-apoptotic cell death.In addition,this kind of chemotherapeutic agents should not be the substrates of drug transporters(P-gp or MRP1 etc).To develop such class of agents would be beneficial for treating MDR cancers,considering that there are no clinically available drugs against the latter.Objects:The objects of this study is to investigate(1)if shikonin,a naturally occurring naphthoquinone compound,or its analogues,could circumvent multiple drug resistance;(2)the mechanisms whereby shikonin and its analogues circumvent cancer drug resistance;(3)and the potential of this class of compounds in treating drug resistant cancers.Contents:1.Shikonin Overcomes Cancer Drug ResistanceWe established a variety of drug resistant cancer cell lines with overexpression of P-gp,MRP 1,BCRP,Bcl-2,or Bcl-x_L.After treated with shikonin for 72 hours, we calculated the IC₅0 values of these cancer cells.The results demonstrated that the IC₅0 values of drug resistant cancer cells and their parental sensitive cells were not significantly different from each other,indicating that these drug resistant factors did not hamper the efficacy of shikonin.2.Shikonin Circumvents Cancer Drug Resistance by Exclusive Induction of a Neeroptotic Death2.1 Shikonin Exclusive Induction of NecroptosisAfter treating MCF-7 cells with shikonin(2.5-20μM),cells permeable to PI increased proportionally to the increment of both concentration and incubation time, indicating the loss of plasma membrane integrity.Hoechst staining showed dying cells did not exhibit apoptotic nuclear fragmentation.Ultrastructure of shikonin-treated cells demonstrated an extensive dilation of mitochondrion and formation of autophagosomes.The death was neither prevented by the pan-caspase inhibitor z-VAD-fmk nor involved a translocation of apoptosis inducing factor(AIF). Shikonin caused a loss of mitochondrial membrane potential,which was probed by JC-1 and measured by FACScan.When cells were treated with shikonin in the presence of Nec-1,a small molecule specifically inhibiting necroptosis,cells with low mitochondrial membrane potential and positive PI were significantly reduced. The death rate of MCF-7 cells was not significantly inhibited by RNA interference of Atg7--a critical gene in autophagic death.Antioxidants could suppress DCFH oxidation in shikonin-treated cells,but did not prevent cellular PI permeability.Similarly,shikonin could induce a cell death of drug-resistant cells(MCF-7 and HEK293 overexpressing P-gp,BCRP,Bcl-2,or Bcl-X_L)which could be effectively prevented by Nec-1.2.2 Necroptosis Circumvents Cancer Drug Resistance in vitro and in vivoThe potency of shikonin against MCF-7/Adr,MCF-7/mx,MCF-7/Bcl-2, MCF-7/Bcl-x_L was not significant different from MCF-7 in a vitro study.Similar results were obtained with HEK293/Bcl-2 and HEK293/Bcl-X_L.In a pilot animal study,MCF-7 or MCF-7/Adr were injected(s.c.)into one flank of 5-week-old nude female mice.On day 7 after inoculation,mice received vehicle control or shikonin(2.5 mg/kg/d,5 days,i.p.).The tumor weight inhibition rate of MCF-7 and MCF-7/Adr was 43±10%and 57±14%,respectively,clearly indicating that the potency of shikonin was not significantly affected by P-glycoprotein, consistent with the in vitro data.The dead cancer cells in the xenograft were morphologically similar to necroptosis.3.Shikonin Circumvents Cancer Drug Resistance by Alternative Induction of a Necroptotic Cell Death3.1 Shikonin alternatively induces Apoptosis or NecroptosisWhen treated with 1.25-5μM shikonin,HL60 cells exhibited apoptotic characteristics including nuclear fragmentation,caspase activation,and apoptotic morphology.While treated with 10-20μM shikonin,HL60 cells had necroptotic characteristics such as mitochondrion dilation and formation of autophagosomes,and cell death inhibitable by Nec-1.These results indicated that shikonin induced a dominant apoptotic death at low concentration and induce a dominant necroptotic death at high concentration in certain types of cells.Similar results were obtained with HL60/Adr,HL60/Bcl-2,K562 and K562/Adr.3.2 Necroptosis Circumvents Apoptotic Resistance in Tumor CellsAfter treated with various concentration of shikonin,we used Hoechst/tryphan blue staining method to calculate the apoptotic/necroptotic rates of HL60,HL60/Adr, HL60/Bcl-2,K562 or K562/Adr cells.The results demonstrated that drug resistant tumor cells were more resistant to shikonin-induced apoptosis but had the same susceptibility to shikonin-induced necroptosis versus its parental sensitive cells.3.3 Shikonin Alternatively Induces Necroptosis in Primary Leukemia Cells.Shikonin demonstrated a strong potency against primary leukemia cells which were isolated from bone marrow of 15 CML or AL patients.Like in HL60 or K562, shikonin can induce apoptosis or necroptosis in primary leukemia cells depending on the concentration.4.Preliminary Study on Molecular Mechanism of Necroptosis4.1 Neerostatin-1 Enhances Shikonin-induced ApoptosisAfter treated with shikonin in absence or presence of Nec-1,we calculated the apoptotic/necrotic rates of HL60,HL60/Adr,K562 or K562/Adr cells by Hoechst/tryphan blue stain.The results showed that there was less necroptotic but more apoptotic cell rate in Nec-1 pre-treated cells,which indicated that Nec-1 can convert shikonin induced necroptosis to apoptosis.Furthermore,we found the activity of caspase 3,8,9 in shikonin-treated cells was increased by Nec-1.4.2 Role of Mitochondrion in NecroptosisAfter HL60 or HL60/adr was treated with various concentrations of shikonin, we checked the mitochondria membrane potential(MMP)of cells probed with JC-1 by flow cytometry analyses.The results demonstrated that shikonin-induced neeroptosis was accompanied by a rapid MMP dissipation but not shikonin-induced apoptosis.This MMP dissipation was possibly resulted from inner mitochondrial membrane(IM)permeabilization,as supported by the calcein quenching assay and electron microscope.5.Shikonin Analogues Circumvents Cancer Drug Resistance by Induction of NeeroptosisFourteen shikonin analogues were chemically synthesized on the basis of shikonin as a leader.These chemicals,like shikonin,could overcome cancer drug resistance by induction of neeroptosis.We determined the IC₅₀values of K562, K562/Adr,MCF-7,MCF-7/Adr,HL60 and HL60/Adr against these agents by MTT assay.The results demonstrated that anti-cancer activity of these agents had no significant difference between drug resistant cell lines and parental drug-sensitive lines.Conclusions:1.Shikonin is a leader that has the capacity to circumvents cancer drug resistance.2.Shikonin can exclusively induce neeroptosis in some types of cancer cells,such as MCF-7 and HEK293.3.Shikonin can alternatively induce apoptosis or neeroptosis on cancer cells,such as HL60 and K562.The selection of cell death pathway is dependent on concentration of shikonin.4.Shikonin-induced necroptosis circumvents cancer drug resistance mediated by overexpression of drug transporters,such as P-gp,MRP 1 and BCRP.5.Shikonin-induced necroptosis circumvents cancer drug resistance mediated by apoptotic resistance.6.In the presence of necrostatin-1,a small molecule specifically inhibiting necroptosis,shikonin induced cell death could be converted to apoptosis.7.The mechanism whereby shikonin induces necroptosis is associated with the inner mitochondrial membrane permeabilization.8.Shikonin analogues circumvents cancer drug resistance by induction of necroptosis.9.We propose here necroptosis induction is an alternative choice to circumvents cancer drug resistance mediated by overexpression of drug transporter or apoptotic resistance.