Novel Realgar-indigo Naturalis Formula For Treating Acute Promyelocytic Leukemia Cell Line Of NB₄ And HL-60 And Its Mechanism

Realgar-indigo naturalis pills?RIF?have been clinically used to treat acute promyelocytic leukemia?APL?.However,the intensive use of realgar was limited by its large dose in prescription,high toxicity and low bioavailability.In order to resolve this problem,realgar microbial transforming solution?RTS?was obtained by transforming realgar powder with Acidithiobacillus ferrooxidans?At.f?and a novel Formulae Realgar bioleaching solution?FRBS?were prepared after replacement the realgar powder with RTS In this study.In present work,FRBS were further investigated on its therapeutic efficacy for treating acute promyelocytic leukemia cell line of NB₄ and HL-60,its mechanism and its acute toxicity.The results showed that the inhibitory rates of FRBS against NB₄ and HL-60 cells are 32.5% and 46.3%,respectively after treatment for 12 hours.The FRBS with 20 ?g/ml of arsenic exhibited more significant activity of anti-cancer in NB₄ cell line than RIF and original RIF with 0.8 mg/ml of arsenic.Similar results were found in HL-60 cell line.Besides,no significant effect was founded on the normal cells of HUVEC and RAW264.7 after treatment with FRBS containing 12?g/ml of arsenic for 24 hours.After treatment NB₄ and HL-60 cells with FRBS and RIF,a clear DNA ladder at a range from 180 bp to 200 bp could be detected by agarose gel electrophoresis.In contrast to RIF,FRBS induced more DNA ladders.The flow cytometry analysis showed that FRBS promoted the cell cycle arrest on G2 phase for NB₄ at early stage and for HL-60 at later stage.Also,FRBS significantly induced higher frequent apoptosis than RIF.The nuclear fragmentation feature was observed directly by fluorescence microscopy.Hochest33258 probe was employed to stain the nuclear of NB₄ and HL-60,and apoptotic bodies were founded after treatment with FRBS containing 2?g/ml of arsenic,but not RIF.The results of western blotting indicated that the expression of caspase-3,caspase-9 and Cyt-c increased in a time-dependent manner.The expression of Cyt-c in NB₄ cell and the expression of caspase-3 and caspase-9 in HL-60 cells were significantly higher after treating with FRBS than RIF.Acute toxicity test results showed that median lethal dose?LD50?was obtained after mice introgastric administrated with the tested drugs.LD50 for FRBS,RIF and RTS was 19.32 mg/kg,13.16 mg/kg and 13.07 mg/kg,respectively.No remarkable change was identified in the morphology of heart,liver,spleen,lung and kindney samples by microscopy after FRBS treatment.Additionally,no difference was detected among control and FRBS treating mice by blood routine examination.FRBS specifically inhibited the proliferation of NB₄ and HL-60 cells,but not normal cells of HUVEC and RAW264.7.Further,FRBS induced G2 cell cycle arrest,DNA degradation and apoptotic body appearance,and increased the expression of caspase-3,caspase-9,Cyt-c in NB₄ and HL-60 cells,finally led to cell apoptosis.When the arsenic concentrations of FRBS is lower than RIF by 40-fold,its apoptosis induction was significantly higher than the RIF;The related toxicity results showed that the toxicity of FRBS was significantly lower than RIF.All these results supported the notion that RTS replacing raw realgar powder of RIF is feasible,FRBS is promising to be an efficient novel realgar-indigo naturalis with low toxicity.