Effect Of Potassium Bisperoxo(1,10-phenanthroline) Oxovanadate (BPV(phen)) On Autophagy And The Mechanism

Cancer is one of the most serious diseases threatening human health.About 20 thousand people die from cancer every day in the world.So far,the mechanism of cancer is not clear,there is no specific treatment for it.Cancer is a multifactorial disease with a complex process,not only related to genetic factors,but also related to various environmental factors,changing autophagy,apoptosis and immune reactions of organism.Autophagy is a special biological phenomenon in eukaryotes,in maintaining cell homeostasis,promoting survival,being involved in various pathophysiological processes.In recent years,autophagy has aroused extensive attention in cancer development and treatment.People are looking forward to interfere with cancer progression through the regulation of the autophagy,even hope to cure cancer thoroughly.As we all know,cancer is a chronic disease,autophagy may play different roles in different stage in cancer processs.In the initial stage of tumorigenesis,autophagy may suppress cancer by improving cell microenvironment.However,at the stage of cancer progression,autophagy may protect cancer cells from metabolic stress,and promote the development,dissemination and metastasis of cancer.However,the effect of autophagy on cancer is direct or indirect,and how to regulate cancer autophagy to improve the outcome of cancer? It is urgent to study.Vanadium is an essential trace element in human body,which has a variety of biological activities.In 1987 Cantley found that vanadate as ATPase inhibiter,then scientists found that vanadate has an insulin-like effect,in recent years found that vanadium compounds anti-inflammatory effect,spermicidal effect.Ghosh Phalguni found vanadium compounds induce apoptosis of testicular cancer cells.Chandler L.Walker reported that vanadium compounds may promote the recovery of cervical spinal cord injury by inhibiting autophagy.How do vanadium compounds affect autophagy pathway is unknown.In this study we want to explore the mechanism of the effect of vanadium compounds on autophagy,in order to provide theoretical basis for the treatment of cancer.The full text is divided into four sections.1.Treatment with BPV(phen)induces cell apoptosis and pyroptosis.To test the function of Potassium Bisperoxo(1,10-phenanthroline)Oxovanadate(BPV(phen)),we treated MEF cells with increasing concentration of BPV(phen)for 48 hrs.A dosage-dependent reduction of cell population and cell viability was detected.Examination of the treated cells by immunoblot revealed an even levels of PCNA and increasing levels of PARP 89 KD fragment.These data suggest that the BPV(phen)exposure helps cell maintain a constant rate of cell proliferation but an increasing rate of apoptosis so that the cell population is reduced.Cell apoptosis was further confirmed by annexin-V flow cytometry.Pyroptosis is described as another type of cell death that is different from apoptosis and characterized by the activation of caspase 1 and release of cellular contents.Treatment with BPV(phen)led to dosage-dependent increases in cellular levels of active caspase 1 with molecular weight of 10 KD and levels of lactate dehydrogenase released in the media.Therefore,BPV(phen)induces both apoptosis and pyroptosis.2.Treatment with BPV(phen)Results in Blockade of the Degradation of Autophagosomes.Because of the close relationship between autophagy and cell death pathways of apoptosis and pyroptosis,we examined the impact of BPV(phen)on autophagy.We injected freely-fed wild-type mice with BPV(phen)and collected liver tissues for autophagy analysis.We found that BPV(phen)induced a significant increase in the levels of LC3-II,suggesting either an activation of autophagy initiation or an inhibition of autophagosomal degradation.To determine the exact mechanism,we treated MEF cells with BPV(phen)in the absence or presence of Bafilomycin A1(BAF)to block the degradation of autophagosomes and observed the similar increases of LC3-II in the absence of BAF but no increase of LC3-II in the presence of BAF,indicating a blockade of the degradation of autophagosomes.The same trends were observed when either He La cells or Hep G2 cells were exposed to BPV(phen).When He La cells stably expressing RFP-LC3 were exposed to BPV(phen),we detected a significant increase in the number of RFP-LC3 punctate foci in the absence of BAF but no increase in the presence of BAF.All results indicate that BPV(phen)induces a blockade of the degradation of autophagosomes.3.Treatment with BPV(phen)Reduces the Stability of P62 in a Proteasome-dependent Way.P62,another important marker of autophagy flux,was widely considered to act as a receptor to bring ubiquitinated proteins into autophagosome for degradation.It was expected to be elevated when the degradation of autophagosomes is inhibited.Surprisingly,we found a dosage-dependent decrease in the levels of P62 based on both immunoblot analysis and immunofluorescent staining of He La cells.Further analysis revealed that it was the stability of P62 that was significantly decreased in the presence of BPV(phen).P62 was reported to bind with polyubiquitinated protein aggregates and help them be packed into autophagosomes and then degraded with the bound aggregates in lysosomes.Inhibition of lysosomal activity with Bafilomycin A1(BAF)is predicted to increase the levels of P62.However,the decrease in levels of P62 in the presence of BPV(phen)was restored only partially by inhibition of lysosomal activity with BAF but more dramatically by inhibition of proteasomal activity of 26 S complex with MG-132.P62 was not associated with RFP-LC3 punctate foci in the presence of MG-132 but colocalized with the punctate foci in the presence of BAF.Thus,BPV(phen)exposure reduces the stability of p62 not through autophagy.Exposure to BPV(phen)led to a non-specific enhancement in the levels of total ubiquitinated proteins and a specific enhancement of P62 ubiquitination.Blocking proteasomal activity with MG132 caused an accumulation of poly-ubiquitinated P62.Comparing to the control,MG132 treatment led to accumulation of more ubiquitinated P62 with low molecular weight,suggesting those ubiquitinated P62 with high molecular weight cannot be degraded through proteasomes.Thus,BPV(phen)treatment promoted proteasomal degradation of poly-ubiquitinated P62.4.The mechanism of BPV(phen)on autophagy.In addition to serving as a substrate receptor,P62 was recently reported to interact with histone deacetylase 6(HDAC6)to suppress its deacetylase activity on ?-tubulin.We detected no change in the levels of total ?-tubulin but significant decreases in the levels of acetylated ?-tubulin when the P62 levels were suppressed in He La cells with two different P62-specific si RNA molecules.Overexpression of P62 exhibited the opposite impact on the levels of acetylated tubulin.The levels of acetylated microtubules were dramatically reduced while the general microtubules remained constant.Acetylated microtubules are stable and required for the trafficking of autophagosomes to fuse with lysosomes.The reduction in levels of acetylated microtubules led to an increase of LC3-II in the absence of BAF but even levels of LC3-II in the presence of BAF.Overexpression of P62 accelerated the degradation of autophagosomes and led to reduction in LC3-II levels in the absence of BAF.Thus,degradation of P62 leads to a release of HDAC6 activity that causes deacetylation of ?-tubulin,destabilization of microtubules and blockade of the degradation of autophagosomes.Treatment with BPV(phen)leads to reduction of P62 and release of HDAC6 to deacetylate the acetylated ?-Tubulin.To check how BPV(phen)impacts autophagy through P62,we tested the impact of BPV(phen)on the interaction of P62 with HDAC6.When He La cells were exposed to BPV(phen),the levels of HDAC6 were slightly reduced but the levels of acetylated ?-tubulin were dramatically reduced although the total levels of ?-tubulin were unchanged.The levels of acetylated microtubules in He La cells were also impaired in the presence of BPV(phen).We detected much weaker interaction between P62 and HDAC6 in the presence of BPV(phen)than in the absence of BPV(phen).We reasoned that such weak interaction might have been caused by reduced input levels of HDAC6 and P62.We treated the cells with BAF and MG-132 and restored the levels of both proteins to be close to the levels of untreated cells,the weak interaction was not improved at all.Therefore,BPV(phen)exposure has disrupted the interaction between P62 and HDAC6 and leads to release of HDAC6 to deacetylate ?-tubulin,destabilize microtubules and impair the degradation of autophagosomes.In summary,BPV(phen)induces cell apoptosis and pyroptosis,and inhibits the degradation of basic level autophagosome.The mechanism is BPV(phen)leads to reduction of P62 and release of HDAC6 to deacetylate the acetylated ?-Tubulin resulting in blockade of autophagosome-lysosome fusion and accumulation of autophagosomes.