Potassium Bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) Induces Apoptosis and Pyroptosis and Disrupts the P62-HDAC6 Protein Interaction to Suppress the Acetylated Microtubule-dependent Degradation of Autophagosomes*

Background: BpV(phen) is an insulin-mimetic small molecule. Results: We demonstrate that bpV(phen) reduces the stability of p62 in a proteasome-dependent way to activate HDAC6 to inhibit autophagy and induce apoptosis and pyroptosis. Conclusion: We propose that bpV(phen) inhibits autophagy through p62. Significance: We provide insights into a novel function of bpV(phen) and p62 in autophagy. Autophagy is a cellular process that controls and executes the turnover of dysfunctional organelles and misfolded or abnormally aggregated proteins. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activates the initiation of autophagy. Autophagosomes migrate along acetylated microtubules to fuse with lysosomes to execute the degradation of the engulfed substrates that usually bind with sequestosome 1 (SQSTM1, p62). Microtubule-associated protein 1 light chain 3 (LC3) traces the autophagy process by converting from the LC3-I to the LC3-II isoform and serves as a major marker of autophagy flux. Potassium bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) is an insulin mimic and a PTEN inhibitor and has the potential to treat different diseases. Here we show that bpV(phen) enhances the ubiquitination of p62, reduces the stability of p62, disrupts the interaction between p62 and histone deacetylase 6 (HDAC6), activates the deacetylase activity of HDAC6 on α-tubulin, and impairs stable acetylated microtubules. Microtubular destabilization leads to the blockade of autophagosome-lysosome fusion and accumulation of autophagosomes. Autophagy defects lead to oxidative stress and lysosomal rupture, which trigger different types of cell death, including apoptosis and pyroptosis. The consistent results from multiple systems, including mouse and different types of mammalian cells, are different from the predicted function of bpV(phen) as a PTEN inhibitor to activate autophagy flux. In addition, levels of p62 are reduced but not elevated when autophagosomal degradation is blocked, revealing a novel function of p62 in autophagy regulation. Therefore, it is necessary to pay attention to the roles of bpV(phen) in autophagy, apoptosis, and pyroptosis when it is developed as a drug.

Autophagy is a cellular process that controls and executes the turnover of dysfunctional organelles and misfolded or abnormally aggregated proteins. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activates the initiation of autophagy. Autophagosomes migrate along acetylated microtubules to fuse with lysosomes to execute the degradation of the engulfed substrates that usually bind with sequestosome 1 (SQSTM1, p62). Microtubule-associated protein 1 light chain 3 (LC3) traces the autophagy process by converting from the LC3-I to the LC3-II isoform and serves as a major marker of autophagy flux. Potassium bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) is an insulin mimic and a PTEN inhibitor and has the potential to treat different diseases. Here we show that bpV(phen) enhances the ubiquitination of p62, reduces the stability of p62, disrupts the interaction between p62 and histone deacetylase 6 (HDAC6), activates the deacetylase activity of HDAC6 on ␣-tubulin, and impairs stable acetylated microtubules. Microtubular destabilization leads to the blockade of autophagosome-lysosome fusion and accumulation of autophagosomes. Autophagy defects lead to oxidative stress and lysosomal rupture, which trigger different types of cell death, including apoptosis and pyroptosis. The consistent results from multiple systems, including mouse and different types of mammalian cells, are different from the predicted function of bpV(phen) as a PTEN inhibitor to activate autophagy flux. In addition, levels of p62 are reduced but not elevated when autophagosomal degradation is blocked, revealing a novel function of p62 in autophagy regulation. Therefore, it is necessary to pay attention to the roles of bpV(phen) in autophagy, apoptosis, and pyroptosis when it is developed as a drug.
Mammalian cells primarily use the autophagy-lysosome pathway to degrade dysfunctional organelles, misfolded/aggregated proteins, and other macromolecules (1). The autophagy process is regulated by a class III PI3K complex functioning in vesicle nucleation; a serine-threonine kinase complex involved in autophagic induction; and a pair of novel ubiquitin-like protein-conjugating systems, the ATG12 and ATG8 systems, promoting the extension and completion of vesicles (2). Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), 3 a phosphotyrosine phosphatase, activates autophagy initiation by inhibiting PI3K (3). Microtubule-associated protein 1 light chain 3 (LC3) is the mammalian homologue of ATG8, one of the key autophagy markers (4). The LC3 precursor is truncated to form the cytosolic LC3-I and further conjugated with phosphatidylethanolamine to create the membraneassociated LC3-II with the assistance of ATG5 and ATG12 (5,6). The LC3-II-associated isolation membranes target and then completely envelop individual organelles or macromolecules that bind to polyubiquitin-binding protein p62/SQSTM 1 (p62) to form autophagosomes (7,8). Autophagosomes migrate along acetylated microtubules to fuse with lysosomes to form autolysosomes, in which substrates are degraded (9 -11). Inhibition of autophagy initiation reduces the generation of autophagosomes, so the levels of LC3-II and p62 are reduced when lysosomal activity is suppressed. However, inhibition of the degradation of autophagosomes leads to accumulation of LC3-II and p62.
Potassium bisperoxo(1,10-phenanthroline)oxovanadate (K[VO(O 2 ) 2 C 12 H 8 N 2 ]⅐3H 2 O or bpV(phen)) is a peroxovanadium small molecule with insulin-mimetic properties (12). It has been demonstrated to activate insulin receptor kinase and to inhibit the dephosphorylation of autophosphorylated insulin receptors and cause significant decreases in circulating insulin and plasma glucose levels (13). It has also been characterized as a potent protein phosphotyrosine phosphatase inhibitor exhibiting anti-tumor activity (14). It inhibits PTEN to induce cardioprotection against ischemia-reperfusion injury (15).
Because of the potential of bpV(phen) to treat different diseases and its inhibition of PTEN which has been suggested to regulate autophagy through PI3K, we wanted to investigate whether bpV(phen) affects autophagy. Here we report that bpV(phen) promotes not only cell apoptosis but also pyroptosis, resulting in overall cell death. Surprisingly, it exhibited no impact on autophagy initiation but blocked autophagosomal degradation by reducing the stability of p62 to activate HDAC6 to impair the fusion of autophagosomes and lysosomes, supported by acetylated microtubules.
Cell Culture, Cell Transfection with siRNA, Coimmunoprecipitation Assay, Immunoblot Analysis, and Fluorescence Microscopy-HepG2 cells, HeLa cells, HeLa cells stably expressing ERFP-LC3, and mouse embryonic fibroblasts (MEFs) were established and cultured as described previously (17,18). HeLa cells were transfected with random or p62-specific siRNA molecules packed with Oligofectamine in a similar way as we described previously (17,18). Cell lysates were prepared from attached cells, and coimmunoprecipitation was performed as described previously (17,18). Cell lysates with the same amounts of total proteins were subjected to immunoprecipitation with equal amounts of specific antibodies and control antibodies (IgG) from the same species. Lysates and/or immunoprecipitates were subjected to immunoblot analyses as reported previously (17,18). Immunofluorescence staining was performed, and images were captured with a laser-scanning microscope in a similar way as described previously (17,18).
Cell Proliferation Assay-The cell proliferation rate was assessed using the CellTiter 96 non-radioactive cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Promega) according to the protocols of the manufacturer. Briefly, 10,000 cells were seeded in a well on 96-well plates (Corning) containing 100 l of culture medium plus different concentrations of bpV(phen) and grown at 37°C for 24 h. Then 15 l of dye solution was added to each well, plates were incubated continuously at 37°C for 4 h, the reaction was terminated with 100 l of stop solution, and optical densities at 570 nm were determined.
Detection of Apoptotic Cells-Apoptosis was evaluated using the Annexin V/FITC apoptosis detection kit. At first, cells were treated with different concentrations of bpV(phen) for 48 h and harvested by centrifugation twice at 1000 rpm (5 min for each spin). Cells were then washed twice (3 min for each wash) in binding buffer. 1 ϫ 10 6 cells were resuspended in 1 ml of binding buffer containing 1.25 l of Annexin V-FITC (BD Biosciences) and 10 l of propidium iodide (PI), and incubated for 15 min at room temperature in the dark. Finally, cell cycle analysis was performed by flow cytometry (BD Calibar). Scatter plots were performed against the intensities of FITC fluorescence and PI fluorescence. The scatter plots were divided into four quadrants: the left lower quadrant (Annexin V-FITC (Ϫ) and PI (Ϫ)) representing viable cells, the left upper quadrant (Annexin V-FITC (Ϫ) and PI (ϩ)) representing necrotic cells, the right lower quadrant (Annexin V-FITC (ϩ) and PI (Ϫ)) representing early apoptotic cells, and the right upper quadrant (Annexin V-FITC (ϩ) and PI (ϩ)) representing late apoptotic cells.
Lactate Dehydrogenase Release Assay-To quantify the extent of pyroptosis after bpV(phen) exposure, release of lactate dehydrogenase in cell culture supernatants was determined using the lactate dehydrogenase activity assay kit from Sigma-Aldrich according to the instructions of the manufacturer. Briefly, HeLa cells were cultured in the presence of different concentration of bpV(phen) for 48 h. Then 50 l of cell culture supernatant was collected, added to the kit reagent, and incubated for 10 min at room temperature. After addition of stop solution, fluorescence intensity was measured using a microplate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm.
Animal Experimentation-Animal protocols were approved by the Institutional Animal Care and Use Committee, Institute of Biosciences and Technology, Texas A&M Health Science Center. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (Publication 86-23, revised 1985). Three pairs of 1-month-old, freely fed female wild-type C57BL/6 mice were subjected to subcutaneous injection of 2.5 mol/30 g body weight in a total of 250 l of saline (or saline only for the control group) as described previously (19).

Treatment with bpV(phen) Induces Cell Apoptosis and
Pyroptosis-To test the function of bpV(phen), we treated MEF cells with increasing concentrations of bpV(phen) for 48 h. As a PTEN inhibitor, bpV(phen) enhanced the levels of phosphorylated AKT (Fig. 1A). A dose-dependent reduction in cell population and cell viability was detected (Fig. 1, B and C). Examination of the treated cells by immunoblot revealed an even level of proliferating cell nuclear antigen and increasing levels of 89-kD PARP fragments (Fig. 1, D and E). Proliferating cell nuclear antigen is a cellular marker for proliferation. PARP is cleaved into an 89-kD fragment by the apoptosis-specific caspase 3, and levels of such fragments serve as markers of apoptosis. These data suggest that bpV(phen) exposure helps the 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 (Fig. 1F). Pyroptosis is another type of cell death that is different from apoptosis and is characterized by the activation of caspase 1 and release of cellular contents. Treatment with bpV(phen) led to dose-dependent increases in cellular levels of active caspase 1 with a molecular weight of 10 kDa and levels of lactate dehydrogenase released in the medium (Fig. 1, G-I). Therefore, bpV(phen) induces both apoptosis and pyroptosis.
Treatment with BpV(phen) Results in Blockade of the Degradation of Autophagosomes-Because of the close relationship between autophagy and the cell death pathways of apoptosis and pyroptosis (20,21), 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 (Fig. 2, A and B), 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 similar increases of LC3-II in the absence of BAF but no increase of LC3-II in the presence of BAF (Fig. 2, C and D), indicating a blockade of the degradation of autophagosomes. The same trends were observed when either HeLa cells (Fig. 2, E and F) or HepG2 cells (Fig. 2, G and H) were exposed to bpV(phen). When HeLa 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 (Fig. 2, I and J). All results indicate that bpV(phen) induces a blockade of the degradation of autophagosomes.
Treatment with bpV(phen) Reduces the Stability of p62 in a Proteasome-dependent Way-p62, another important marker of autophagy flux, has been widely considered to act as a receptor to bring ubiquitinated proteins into the autophagosome for degradation (7). It is expected to be elevated when the degradation of autophagosomes is inhibited. Surprisingly, we found a dose-dependent decrease in the levels of p62 on the basis of both immunoblot analysis (Fig. 3, A and B) and immunofluorescence staining (Fig. 3C) of HeLa cells. Further analysis revealed that it was the stability of p62 that was decreased significantly in the presence of bpV(phen) (Fig. 3, D and E). p62 has been reported to bind with polyubiquitinated protein aggregates and help them be packed into autophagosomes and then degraded with the bound aggregates in lysosomes (7,22). Inhibition of lysosomal activity with BAF was 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 the lysosomal activity with BAF but more dramatically by inhibition of the proteasomal activity of the 26S complex with MG-132 (Fig. 3, F and G). P62 was not associated with RFP-LC3 punctate foci in the presence of MG-132 but colocalized with punctate foci in the presence of BAF (Fig. 3H). Therefore, bpV-(phen) exposure does not reduce the stability of p62 through autophagy. Exposure to bpV(phen) led to a nonspecific enhancement in the levels of total ubiquitinated proteins and a specific enhancement of p62 ubiquitination. Blocking proteasomal activity with MG-132 caused an accumulation of polyubiquitinated p62 (Fig. 3I). Compared with the control, MG-132 treatment led to accumulation of more ubiquitinated p62 with a low molecular weight, suggesting that ubiquitinated p62 with a high molecular weight cannot be degraded through proteasomes (Fig. 3I). Therefore, bpV(phen) treatment promoted the proteasomal degradation of poly-ubiquitinated p62.
Suppression of p62 Causes Deacetylation of ␣-Tubulin and Inhibition of the Degradation of Autophagosomes-In addition to serving as a substrate receptor, p62 has recently been reported to interact with HDAC6 to suppress its deacetylase activity on ␣-tubulin (23). We detected no change in the levels of total ␣-tubulin but significant decreases in the levels of acetylated ␣-tubulin when p62 levels were suppressed in HeLa cells with two different p62-specific siRNA molecules (Fig. 4, A-C). Overexpression of p62 exhibited the opposite impact on the levels of acetylated tubulin (Fig. 4D). The levels of acetylated microtubules were reduced dramatically, whereas the general microtubules remained constant (Fig. 4E). Acetylated microtubules are stable and required for the trafficking of autophagosomes to fuse with lysosomes (11). 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 (Fig. 4, A-C). Overexpression of p62 accelerated the degradation of autophagosomes and led to a reduction in LC3-II levels in the absence of BAF (Fig. 4D). Therefore, 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 decipher how bpV(phen) impacts autophagy through p62, we tested the impact of bpV(phen) on the interaction of p62 with HDAC6. When HeLa cells were exposed to bpV(phen), the levels of HDAC6 were reduced slightly, but the levels of acetylated ␣-tubulin were reduced dramatically, although the total levels of ␣-tubulin were unchanged (Fig. 5A). The levels of acetylated microtubules in HeLa cells were also impaired in the presence of bpV(phen) (Fig. 5B). We detected a much weaker interaction between p62 and HDAC6 in the presence of bpV-(phen) than in the absence of bpV(phen) (Fig. 5C). We reasoned that such a 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, but the weak interaction was not at all improved (Fig. 5C). Therefore, bpV(phen) exposure disrupted the interaction between p62 and HDAC6 and led to the release of HDAC6 to deacetylate ␣-tubulin, destabilized microtubules, and impaired the degradation of autophagosomes.

Discussion
p62 protein contains multiple domains involving different functions, such as the LC3-interacting region and ubiquitinassociated domain (24). It is generally considered a substrate  OCTOBER 23, 2015 • VOLUME 290 • NUMBER 43 receptor that connects the polyubiquitinated protein aggregates and dysfunctional mitochondria to LC3-II and facilitates their packaging into autophagosomes (7,8,25). Specific phosphorylation of serine 403 in the ubiquitin-associated domain leads to enhancement of its association with the polyubiquitinated cargo for autophagosomal engulfment (24). The sequence 163-225 of p62 has been identified as the HDAC6interactive domain (23). Serine 207 in the HDAC6-interactive domain of p62 is highly phosphorylated in the presence of a proteasomal inhibitor, and inhibition of autophagy with bafilomycin A1 has been reported to show no accumulation of phosphorylated p62 at serine 207 (24). Treatment with bpV(phen) seems to enhance the phosphorylation of p62 at serine 207 and disrupt the interaction with HDAC6. Further analysis of the impact of bpV(phen) on the p62-HDAC6 interaction will clarify the mechanism in detail. Interestingly, proteasomal inhibition did not cause any alternation in the phosphorylation of the tyrosine residue in p62 protein (24). Therefore, the impact of bpV(phen) on autophagy seems to be not through its PTEN inhibition because PTEN inhibition may activate autophagy initiation.

Impact of bpV(phen) on Autophagy and Cell Death
p62 has been reported to bind with ubiquitinated proteins and be engulfed and degraded in the autophagosome. It is unknown whether p62 is required to be ubiquitinated for lysosomal degradation (26). Cullin-3 (Cul-3) is an E3 ubiquitin ligase that catalyzes the ubiquitination of p62 and promotes the degradation of p62 through proteasomes (27). Exposure to bpV(phen) may activate E3 ubiquitin ligases such as Cul-3 to promote the ubiquitination of p62 or promote the modification of p62 into a better substrate of ubiquitin ligase. The report that proteasomal inhibition with MG-132 did not cause any altera-tion in the ubiquitination of p62 protein (24) suggests that the ubiquitination of p62 is a specific effect of bpV(phen) exposure.
Overactive autophagy leads to the depletion of cellular contents and, eventually, cell death. In this sense, autophagy is classified as type II programmed cell death (28). Instead, autophagy has a pro-survival function when cells are under metabolic stress (29,30). Its dysfunction causes failure of the cells to sustain metabolic homeostasis and survive (31). If the autophagic process is blocked before autophagosomal formation, then the fragmented mitochondria will release cytochrome c and other small molecules to induce conventional apoptosis, which is usually associated with diverse forms of aggregation and perinuclear clustering of the dysfunctional mitochondria. The dead cell debris generated from apoptosis and autophagy is still contained in an intact plasma membrane and taken up by phagocytosis (32,33). If the process is blocked before autolysosome formation or if autophagosomes are not degraded efficiently, then the accumulated mitochondria may become damaged by their own production of superoxide, start to leak electrons, lose their membrane potentials, and even induce robust oxidative stress (1,34) or lysosomal rupture (35). Both oxidative stress and lysosomal rupture, in turn, activate the NLRP3 inflammasome, which results in direct activation of caspase 1 (36). Activation of caspase 1 subsequently induces the secretion of potent pro-inflammatory cytokines and, eventually, an inflammatory form of cell death referred to as pyroptosis of the cell and other cells in the environment (21,(37)(38)(39)(40)(41). Although bpV-(phen) has been suggested as a drug for different types of diseases, it is necessary to consider the potential side effects caused by its impact on autophagy, apoptosis, and pyroptosis.