Galangin induces B16F10 melanoma cell apoptosis via mitochondrial pathway and sustained activation of p38 MAPK

Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent.     

Cucurbitacin B induces rapid depletion of the G-actin pool through reactive oxygen species-dependent actin aggregation in melanoma cells

Cucurbitacin B (CuB), a triterpenoid compound isolated from Cucurbitaceae plants, has been reported as a promising anti-cancer agent, yet its action mechanism is still controversial. In this study, we explored the potential mechanism of CuB in murine B16F10 melanoma cells. Anti-proliferation and anti-invasion effects were assessed in cultured cells, and in vivo anti-tumor activity was evaluated in a murine subcutaneous melanoma model. Flow cytometry was adopted to analyze cell cycle distribution and reactive oxygen species (ROS) levels. Actin levels were determined by western blot analysis, and the profiles of differential expressed proteins were identified by a quantitative proteomic approach. The results showed that CuB exerted inhibitory effects on cell proliferation, colony formation, as well as migration and invasion potential of the melanoma cells. The growth of subcutaneous melanoma was significantly inhibited in mice treated with CuB when compared with control group. Furthermore, CuB treatment caused rapid cell membrane blebbing and deformation, and induced G(2)/M-phase arrest and formation of multiploid cells. Notably, the G-actin pool was rapidly depleted and actin aggregates were formed quickly after CuB treatment. A number of cytoskeleton-regulatory proteins were differentially regulated. Blockage of ROS production significantly reduced the G-actin depletion ability and the anti-tumor activity of CuB. These findings indicate that CuB induces rapid depletion of the G-actin pool through ROS-dependent actin aggregation in melanoma cells, which may at least partly account for its anti-tumor activity.     

Abrogation of histone deacetylases (HDACs) decreases survival of chronic myeloid leukemia cells: New insight into attenuating effects of the PI3K/c-Myc axis on panobinostat cytotoxicity

Although the identification of tyrosine kinase inhibitors (TKIs) has changed the treatment paradigm of many cancer types including chronic myeloid leukemia (CML), still adjustment of neoplastic cells to cytotoxic effects of anticancer drugs is a serious challenge. In the area of drug resistance, epigenetic alterations are at the center of attention and the present study aimed to evaluate whether blockage of epigenetics mechanisms using a pan-histone deacetylase (HDAC) inhibitor induces cell death in CML-derived K562 cells. We found that the abrogation of HDACs using panobinostat resulted in a reduction in survival of the K562 cell line through p27-mediated cell cycle arrest. Noteworthy, the results of the synergistic experiments revealed that HDAC suppression could be recruited as a way to potentiate cytotoxicity of Imatinib and to enhance the therapeutic efficacy of CML. Here, we proposed for the first time that the inhibitory effect of panobinostat was overshadowed, at least partially, through the aberrant activation of the phosphoinositide 3-kinase (PI3K)/c-Myc axis. Meanwhile, we found that upon blockage of autophagy and the proteasome pathway, as the main axis involved in the activation of autophagy, the anti-leukemic property of the HDAC inhibitor was potentiated. Taken together, our study suggests the beneficial application of HDAC inhibition in the treatment strategies of CML; however, further in vivo studies are needed to determine the efficacy of this inhibitor, either as a single agent or in combination with small molecule inhibitors of PI3K and/or c-Myc in this malignancy.     

Sodium valproate potentiates staurosporine‐induced apoptosis in neuroblastoma cells via Akt/survivin independently of HDAC inhibition

Sodium valproate (VPA) has been recently identified as a selective class I histone deacetylase (HDAC) inhibitor and explored for its potential as an anti‐cancer agent. The anti‐cancer properties of VPA are generally attributed to its HDAC inhibitory activity indicating a clear overlap of these two actions, but the underlying mechanisms of its anti‐tumor effects are not clearly elucidated. The present study aimed to delineate the molecular mechanism of VPA in potentiating cytotoxic effects of anti‐cancer drugs with focus on inhibition of HDAC activity. Using human neuroblastoma cell lines, SK‐N‐MC, SH‐SY5Y, and SK‐N‐SH, we show that non‐toxic dose (2 mM) of VPA enhanced staurosporine (STS)‐induced cell death as assessed by MTT assay, PARP cleavage, hypodiploidy, and caspase 3 activity. Mechanistically, the effect of VPA was mediated by down regulation of survivin, an anti‐apoptotic protein crucial in resistance to STS‐mediated cytotoxicity, through Akt pathway. Knock down of class I HDAC isoforms remarkably inhibited HDAC activity comparable with that of VPA but had no effect on STS‐induced apoptosis. Moreover, MS‐275, a structurally distinct class I HDAC inhibitor did not affect STS‐mediated apoptosis, nor decrease the levels of survivin and Akt. Valpromide (VPM), an amide analog of VPA that does not inhibit HDAC also potentiated cell death in NB cells associated with decreased survivin and Akt levels suggesting that HDAC inhibition might not be crucial for STS‐induced apoptosis. The study provides new information on the possible molecular mechanism of VPA in apoptosis that can be explored in combination therapy in cancer. J. Cell. Biochem. 114: 854–863, 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long‐term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK‐2206 in combination with paclitaxel and carboplatin. Although single agent MK‐2206 inhibited phospho‐AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK‐2206 with paclitaxel and carboplatin was cytotoxic in long‐term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase‐dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti‐oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.     

JNK supports survival in melanoma cells by controlling cell cycle arrest and apoptosis

JNK1/2 proteins belong to the family of stress-activated protein kinases. They play a complex role in growth regulation, inducing either cell death or growth support. In this report, we provide evidence that, in human melanoma cells, JNK inhibition with the small molecule inhibitor SP600125 induces either predominantly a G2/M arrest or apoptosis depending on the cell line. In 1205Lu cells, JNK inhibition induced cell cycle arrest through p53-dependent induction of p21 Cip1/Waf1 expression, while in WM983B cells, induction of apoptosis by JNK inhibition was accompanied by p53, Bad and Bax induction, not p21 Cip1/Waf1. JNK inhibition with the small molecule inhibitor SP600125 slowed growth of all cell lines, although the effect was markedly greater in cells exhibiting high phospho- (P-)JNK1 levels. Specific gene knockdown of JNK1 by means of siRNA oligonucleotides inhibited cell growth only in melanoma cell lines exhibiting high P-JNK1 levels. siRNAs directed against JNK2 did not reduce cell growth in any of the cell lines tested. Together, our findings demonstrate that JNK, and in particular the JNK1 isoform, support the growth of melanoma cells, by controlling either cell cycle progression or apoptosis depending on the cellular context.     

Valproic acid induces functional heat-shock protein 70 via Class I histone deacetylase inhibition in cortical neurons: a potential role of Sp1 acetylation

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat-shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. This study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly up-regulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors – sodium butyrate, trichostatin A, and Class I HDAC-specific inhibitors MS-275 and apicidin, – all mimicked the ability of VPA to induce HSP70. Pre-treatment with phosphatidylinositol 3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the phosphatidylinositol 3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.     

Induction of autophagy by valproic acid enhanced lymphoma cell chemosensitivity through HDAC-independent and IP3-mediated PRKAA activation

Autophagy is closely related to tumor cell sensitivity to anticancer drugs. The HDAC (histone deacetylase) inhibitor valproic acid (VPA) interacted synergistically with chemotherapeutic agents to trigger lymphoma cell autophagy, which resulted from activation of AMPK (AMP-activated protein kinase) and inhibition of downstream MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling. In an HDAC-independent manner, VPA potentiated the effect of doxorubicin on lymphoma cell autophagy via reduction of cellular inositol 1,4,5 trisphosphate (IP3), blockade of calcium into mitochondria and modulation of PRKAA1/2-MTOR cascade. In murine xenograft models established with subcutaneous injection of lymphoma cells, dual treatment of VPA and doxorubicin initiated IP3-mediated calcium depletion and PRKAA1/2 activation, induced in situ autophagy and efficiently retarded tumor growth. Aberrant genes involving mitochondrial calcium transfer were frequently observed in primary tumors of lymphoma patients. Collectively, these findings suggested an HDAC-independent chemosensitizing activity of VPA and provided an insight into the clinical application of targeting autophagy in the treatment of lymphoma.     

VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation

Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway.     

The histone deacetylase inhibitor vorinostat prevents TNFα-induced necroptosis by regulating multiple signaling pathways

Histone deacetylase (HDAC) inhibitors are novel anticancer reagents that have recently been reported to have anti-inflammatory and neuroprotective effects; however, the mechanisms underlying their activities are largely undefined. The data from this study show that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) can protect L929 cells from TNFα-induced necroptosis. This effect involves multiple mechanisms, including the upregulation of cFLIP_L expression, the enhanced activation of NFκB and p38 MAPK, and the inactivation of JNK. In addition, SAHA could initiate cell autophagy by inhibiting Akt and mTOR, which also play important roles in protecting cells from necroptosis. Because cell necroptosis is important for inflammation-related deterioration and neurodegenerative disease, our results indicate that preventing cell necrosis may be an important mechanism through which HDAC inhibitor compounds exert their anti-inflammatory or neuroprotective effects.     

Epistatic interaction of Arg72Pro TP53 and −710 C/T VEGFR1 polymorphisms in breast cancer: predisposition and survival

Arginine deprivation is a promising strategy for treating ASS-negative malignant tumors including melanoma. However, autophagy can potentially counteract the effectiveness of this treatment by acting as a pro-survival pathway. By combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with arginine deprivation using ADI-PEG20 (pegylated arginine deiminase), we achieved enhanced apoptosis and accelerated cell death in melanoma cell lines. This implies a switch from autophagy to apoptosis. In our current investigation, we found that TRAIL could induce the cleavage of two key autophagic proteins, Beclin-1 and Atg5, in the combination treatment. Using specific inhibitors for individual caspases, we found that caspase-8 inhibitor could completely abolish the cleavage. Furthermore, caspase-8 inhibitor was able to fully reverse the enhanced cytotoxicity induced by TRAIL. Inhibitors for caspase-3, 6, 9, and 10 were able to block the cleavage of these two autophagic proteins to some extent and correspondingly rescue cells from the cytotoxicity of the combination of TRAIL and arginine deprivation. In contrast, calpain inhibitor could not prevent the cleavage of either Beclin-1 or Atg5, and was unable to prevent cell death. Overall, our data indicate that the cleavage of Beclin-1 and Atg5 by TRAIL-initiated caspase activation is one of the mechanisms that lead to the enhancement of the cytotoxicity in the combination treatment.     

Valproic acid enhances fludarabine-induced apoptosis mediated by ROS and involving decreased AKT and ATM activation in B-cell-lymphoid neoplastic cells

Histone deacetylase (HDAC) inhibitors have been shown synergize with a number of cytotoxic drugs in leukemic cells. In chronic lymphocytic leukemia (CLL), the first line therapy is based on the combination of fludarabine, a nucleoside analogue, and rituximab, an anti-CD20 monoclonal antibody, and there are presently no HDAC inhibitors are used to manage CLL. In the present study, we found that the addition of valproic acid (VPA), a HDAC inhibitor, increases cell death in B-cell-neoplasm-derived cell lines, BJAB, NALM-6 and I-83. This increased apoptosis caused release of mitochondrial cytochrome c, activation of caspases, and increased reactive oxygen species (ROS). The addition of a ROS scavenger inhibited cell death induced by the VPA–fludarabine combination. In contrast, blocking the death receptor pathway failed to inhibit VPA increased fludarabine induced apoptosis. Combination of VPA and fludarabine treatment decreased both total and phosphorylated levels of AKT, an important anti-apoptotic protein, and ATM, a pivotal protein in DNA damage response. Chemical inhibition of AKT or ATM was sufficient to enhance fludarabine-induced apoptosis. We next examined patient samples from a local clinical trial where relapsed CLL patients were treated with VPA and examined the effects of VPA on AKT and ATM in vivo. After 30 days, there was a reduction in ATM levels in three out of the four patients treated, while AKT phosphorylation was reduced only in one patient. Taken together, VPA reduces ATM levels, thereby increasing ROS-dependent cell death via the mitochondrial apoptotic pathway when combined with fludarabine.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF^V600E melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF^V600E melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF^V600E melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.     

Combination of valproic acid and ATRA restores RARβ2 expression and induces differentiation in cervical cancer through the PI3K/Akt pathway

Epigenetic silencing of the tumor suppressor gene, RARβ2, through histone deacetylation has been established as an important process of cervical carcinogenesis. This pivotal role has led to the suggestion that a combination of retinoids selective for RARβ2 with histone deacetylase (HDAC) inhibitors may have therapeutic potential. Valproic acid (VPA), a HDAC inhibitor, has a critical role in the regulation of gene expression through histone acetylation and causes transformed cells to undergo growth arrest, differentiation, and apoptosis. Therefore, we hypothesized that the combination of VPA and ATRA could restore RARβ2 expression, thus resulting in enhanced anti-neoplastic activity in cervical cancer. Here, we show that VPA combined with ATRA led to hyperacetylation of histone H3 and a significant alteration of gene expression in cervical cancer cells, including RARβ2 gene expression, which was upregulated 50- to 90-fold. The combination therapy effectively inhibited the growth of cervical cancer cells more than the single agent treatment both in vitro and in vivo. The additive effects were associated with a significant upregulation of p21(CIP1) and p53 as well as a pronounced decrease in p-Stat3. Furthermore, the combined treatment led to cell cycle arrest predominantly at the G1 phase, and it preferentially induced cell differentiation rather than apoptosis in cervical cancer cells. The differentiation program was determined by the presence of E-cadherinmediated adhesion and activation of the PI3K/Akt pathway. Taken together, these results provide new insight into the mechanisms of enhanced antitumor activity of the HDAC inhibitor and ATRA regimen, thus offering a new therapeutic strategy for cervical cancer patients.     

Coordinate Autophagy and mTOR Pathway Inhibition Enhances Cell Death in Melanoma

The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation. Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma. Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy. Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent. We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis. Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts. These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.