Mechanism Of The Intermediate Metabolites Of Arsenic Trioxide And Cryptotanshinone Induced-apoptosis In The Breast Cancer MCF-7Cell

Objective: To investigate the effect and mechanism of sodium arsenite(iAsIII), and its intermediate metabolites such as MMAIIIand DMAIII, whichcombine with cryptotanshinone (CPT) respectively on human breast cancerMCF-7cells.Methods: Breast cancer MCF-7cells, cryptotanshinone, three arseniccompounds, arsenite (iAsIII) and its intermediate metabolites MMAIIIandDMAIII, are used for present study. MTT assay is used to detect the vitalityand viabilit of MCF-7cells, which are treated with three differentconcentrations of arsenic compounds and cryptotanshinone. According to theresults of MTT assay, we select concentration of three arsenic compounds andcryptotanshinone, and then we set the final concentration of arseniccompounds which is1M and the concentration range for cryptotanshinone is10-20M. Then MTT assay is used to detect the vitality and viabilit ofMCF-7cells continuously, which are dealed with the combination of threearsenic compounds with cryptotanshinone respectively. Finally, we select themost suitable concentration of drugs: arsenic compounds concentration of1M and cryptotanshinone concentration of15M. So MCF-7cells are dividedinto eight groups randomly:Con group, CPT group, iAsIIIgroup, iAsIII+CPT group, MMAIIIgroup, MMAIII+CPT group, DMAIIIgroup, DMAIII+CPT group. Cells are treated with1M of arsenic compounds and15M ofcryptotanshinone (CPT) individually and jointly. Flow cytometry,Western-blot, et al, are used to detect drug induced-apoptosis on MCF-7cellsand then according to the experimental results to select the group with themost obvious effect on MCF-7cells to study its mechanism of action.Results: MTT assay shows that there has no significant change about the vitality and viability when we use lonely at the low concentrations of the drug(arsenic compounds concentrations below5M, Cryptotanshinoneconcentration below15M). In the high concentrations (arsenic compoundsconcentrations higher than5M, cryptotanshinone concentrations higher than20M), the cells are obvious dead. However, when MCF-7cells were treatedwith combination of low doses of arsenic compounds (concentration1M)and cryptotanshinone (15M), cells vitality and viability have significantchange, and the most obvious effect is combination of MMAIIIand CPT, thecell survival rate is equivalent to5-7times the Con group. Cell apoptosis isdetected by flow cytometry, the24hours rate of MMAIII+CPT group havesignificant change and cell apoptosis rate reached34.84±3.423%, whileapoptosis rate of which MCF-7cells were treated with iAsIII, MMAIII, DMAIII,CPT lonely and iAsIIIand DMAIIIrespectively combine with CPT have nosignificant change. After24hours of treatment with MMAIIIand CPT,Western-blot assay shows that mitochondrial pro-apoptotic proteins Bak、Baxtransfer from the cytoplasm to the mitochondria, and induce a lot ofCytochrome C to release from mitochondria into the cytoplasm. Andapoptosis-related proteins PARP, Caspase9are activated, its cleavagefragments are increased, while the other experimental groups or a controlgroup have no significant change. Therefore, we select the most notable effecton MCF-7cells experimental group: MMAIII+CPT group, to investigate theinduction of apoptosis-related mechanisms. First, we examined the change ofthe endoplasmic reticulum stress-related proteins: PERK, ASK1and itsdownstream protein: ATF4, CHOP and JNK. Western-blot assay shows thatPERK, ASK1are activated, and then phosphate PERK and phosphate ASK1have a transient rise, and reach the highest in6hours. There is a rising trendabout the downstream protein ATF4, CHOP and JNK. Then we detect theprotein of MAPK pathway, such as p-Erk and p-P38. We find the changes ofp-Erk and p-P38in24h. When we pre-treat cells with JNK inhibitor(SP600125) for45min, the effect of the combination of MMAIIIand CPT areinhibited of reducing. Western-blot assay shows that the cleavage fragment of apoptosis-related proteins PARP and Caspase9are reduced. And there has alittle change when we pre-tread Caspase inhibitor (Z-VAD-FMK) for30min.And then, the change of12h ROS is detected by flow cytometry, we find thatwhen we pre-treat with NAC for1.5hours and then treat with MMAIIIandCPT, ROS is reduced significantly. Then we detect ROS colocalization withendoplasmic reticulum and mitochondria by confocal immunofluorescence for12h. The result shows a good colocalization. Western-blot assay shows thatcells are treated with NAC, MMAIIIand CPT for24hours, the activation ofapoptosis-related protein PARP, Caspase9are inhibited, and its cleavagefragments are also significantly reduced, while the other groups have nosignificant effect.Conclusion: According to above results, we know that when cells aretreated with sodium arsenite (iAsIII) and its intermediate metabolites: trivalentmonomethylarsonous acid (MMAIII), trivalent dimethylarsinous acid (DMAIII)and cryptotanshinone (CPT) lonely, there have no change obvious. Thecombination of CPT and MMAIIIis the most obvious apoptosis-effect on cells.The combination of MMAIIIand CPT induces apoptosis by ROS-dependentand Caspase-dependent mitochondria pathway and endoplasmic reticulumpathway directly——PERK/eif2/ATF4and ASK1/JNK.