Effects Of Curcumin Analog EF24 On Apoptotic Induction In Senescent Cells And Malignant Melanoma Cells

Objectives:It was demonstrated that curcumin had effects on anti-aging and anti-cancer,but the mechanism was partly unknown and the low bioavailability of curcumin limited its clinical applications.So we screened curcumin analogs for identifying the most effective and safest ones as promising senolytics and antineoplastic agents,and further explored the molecular mechanism of inducing apoptosis of senescent cells(SCs)and malignant melanoma cells(MMCs).Methods:Using replicative exhaustion,ionizing radiation(IR)and ectopic Ras expression(Ras)induced SCs,and MMCs from different cell types,we screened the effects of curcumin analogs in inducing cell death in SCs and melanoma cells.We also compared its anti-tumor activity to that of commonly used anti-malignant melanoma agents using flow cytometry.Then we identified the molecular mechanisms on reducing survival of SCs and MMCs by Western Blot and polymerase chain reaction(PCR).Results:Our results showed that EF24 significantly inhibited the viability of IR-induced WI-38 SCs with the half maximal effective concentration(EC50)value of 1.62 ?M,which was lower than that of HO-3867,2-HBA and DIMC(3.85 ?M,3.85?M and 15.18 ?M),respectively.EC50 values in replicative and IR-induced senescent human pulmonary fibroblasts(WI-38 and IMR-90),human umbilical vein endothelial cells(HUVEC),human renal epithelial cells(HREC)and human preadipocytes were 1.26/1.74 ?M,0.33/1.72 ?M,0.77/2.09 ?M,0.60/1.15 ?M,and 4.60/5.04 ?M,separately,which were lower than 4.32 ?M,10.88 ?M,1.39 ?M,3.25?M,and 8.92 ?M in normal cells,respectively.Besides,EF24 also inhibited the viability of MMCs in a time-dependent manner.The EC50 values of EF24 in A-375,CHL-1,MeWo and SK-MEL-28 cells were 0.15 ?M,0.92 ?M,1.00 ?M and 1.02 ?M,which was lower than that of cisplatin,dacarbazine and vemurafenib in these four MMCs,separately.To explore the mechanism of EF24 eliminating SCs and MMCs,we tested the rate of apoptosis on SCs and MMCs after EF24 treatment.EF24 promoted the apoptosis of SCs and MMCs,and the effect of apoptosis was blocked by pretreatment of pan-caspase inhibitor(QVD).Morever,we discovered that 1)Bcl-xl and Mcl-1 proteins were degraded in a proteasome-dependent manner in SCs;2)the expression of Bcl-2 protein was decreased through inhibition of NF-?B signaling pathway,and caspase-3 and PARP were cleaved into activated caspase-3 and PARP in MMCs.Since senescent cells depend on Bcl-xL for survival,and ABT263,a Bcl-2/Bcl-xL inhibitor,is the most potent reagent to kill SCs,we combine EF24 and ABT263 as co-senolytics.The coefficient of drug interaction(CDI)values of 2.5?M EF24 in combination with ABT263(at least 0.625 ?M)was less than 0.3,indicating strong synergistic effect.Whereas,EF24 neither increased ROS production in SCs and MMCs,nor changed mRNA level of BCL-XL,MCL-1 and BCL-2 in SCs.Conclusion:EF24 is a potential senolytic agent with high selectivity and safety,which targets elimination of SCs.It is prossibly used to delay aging,extend healthspan,prevent and treat aging-related diseases in senior subjects.EF24 is also a promising anti-cancer compound with high efficiency and safety,which may be used in patients with drug resistance of targeted therapies and senescence-associated side effects after radiotherapy and chemotherapy in malignant melanoma.