The Roles Of PARP Inhibitor In Autophagy Induced By Doxorubicin In Multiple Myeloma Cells

Background and Aim: Autophagy is a dynamic process of protein degradation, which is typically observed during nutrient deprivation. It is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy in cancer cells causes death or protects cells is controversial. In multiple studies, autophagy has been inhibited pharmacologically or genetically, resulting in contrasting outcomes—survival or death—depending on the specific context.The nuclear enzyme PARP convertsβ-nicotinamide adenindinucleotide(NAD+) into polymers of poly(ADP ribose)(PAR), which participate in regulating nuclear homeostasis.Many different cell insults infringing DNA damage have been shown to be able to activate PARP. So PARP has been implicated in different pathways leading to cell death and its inhibition has different results in different types of cells.The aim of this study is to investigate the functions and mechanisms of PARP inhibitor ANI(4-amino-1,8-naphthalimide) in autophagy induced by doxorubicin in multiple myeloma(MM) cells.Methods and Results: 1. Doxorubicin induced significant cell death in a concentration-dependent manner and zVAD.fmk (a pan-caspase inhibitor) blocked DOXO-indcued cell death (P<0.05), cell survival was determined using the MTT assays.Data were presented as means±S.D. of three independent experiments (P<0.05 comparing with the untreated control group, t-test). 2.①DOXO-induced PARP activation. Cells were treated with DOXO up to 5 hours. The formation of PAR polymer was detected by western blot.②DOXO induced intracellular ATP depletion. Cells were treated with DOXO up to 1 hour. The cellular ATP level was measured as described in Materials and Methods. The ATP level was presented as percentage to the untreated control group. Data were shown as means±S.D. of three independent experiments. 3.①Doxorubicin induced functional autophagy. Conversion of LC3-I to LC3-II was induced by DOXO treatment. H929 cells were treated with DOXO for various time and subjected to western blot for detection.②3MA blocked DOXO-induced high ATP level. Cells were incubated with autophagy inhibitor 3MA (10 mM) and DOXO (2.2μM×24 hr) and detected by cellular ATP measurement. 4. ANI, a potent inhibitor of PARP, significantly blocked DOXO-induced LC3-I to LC3-II conversion. Cells were treated with DOXO (2.2 uM 24 h) with or without 1 h pretreatment of ANI (10umol/l). Cells were collected and subjected to western blot for analysis, cellular ATP measurement and MTT assay.Conclusions: 1.Doxorubicin can induce apoptosis of MM cells in a dose-dependent manner.2.DOXO-induced PARP activation.3. Doxorubicin induces functional autophagy which has a high intracellular ATP level to prevent cells from death. 4. Non-inhibitory concentration of ANI combined with doxorubicin can significantly enhance the pro-apoptotic activity of doxorubicin in MM cells.