Lipid storage and lipophagy regulates ferroptosis

The synthesis, storage, and degradation of lipids are highly regulated processes. Impaired lipid metabolism is implicated in inflammation and cell death. Although ferroptosis is a recently described form of regulated cell death driven by lipid peroxidation, the impact of lipid droplets on ferroptosis remains unidentified. Here, we demonstrate that lipophagy, the autophagic degradation of intracellular lipid droplets, promotes RSL3-induced ferroptotic cell death in hepatocytes. Lipid droplet accumulation is increased at the early stage but decreased at the late stage of ferroptosis in mouse or human hepatocytes. Importantly, either genetically enhancing TPD52-dependent lipid storage or blocking ATG5-and RAB7A-dependent lipid degradation prevents RSL3-induced lipid peroxidation and subsequent ferroptosis in vitro and in vivo. These studies support an antioxidant role for lipid droplets in cell death and suggest novel strategies for the inhibition of ferroptosis by targeting the lipophagy pathway.     

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor,saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.     

FTS and 2-DG induce pancreatic cancer cell death and tumor shrinkage in mice

The Ras inhibitor S-trans-trans farnesylthiosalicylic acid (FTS) inhibits active Ras, which controls cell proliferation, differentiation, survival, and metabolism. FTS also inhibits HIF1α expression in cancer cells, leading to an energy crisis. The synthetic glucose analog 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, is selectively directed to tumor cells that exhibit increased glucose consumption. The 2-DG enters tumor cells, where it competes with glucose for glycolytic enzymes. In cancer models, as well as in human phase 1 trials, 2-DG inhibits tumor growth without toxicity. We postulated that under normoxic conditions, tumor cells treated with FTS would be more sensitive than normal cells to 2-DG. We show here that combined treatment with FTS and 2-DG inhibited cancer cell proliferation additively, yet induced apoptotic cell death synergistically both in vitro and in vivo. The induced apoptosis was inferred from QVD-OPH inhibition, an increase in cleaved caspase 3, and loss of survivin. FTS and 2-DG when combined, but not separately, also induced an increase in fibrosis of the tumor tissue, chronic inflammation, and tumor shrinkage. Overall, these results suggest a possible new treatment of pancreatic tumors by the combined administration of FTS and 2-DG, which together induce pancreatic tumor cell death and tumor shrinkage under non-toxic conditions.     

Shuganning injection,a traditional Chinese patent medicine,induces ferroptosis and suppresses tumor growth in triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC), lacking targeted therapies currently, is susceptible to ferroptosis, a recently defined form of cell death.PurposeTo evaluate the anticancer activity of Shuganning injection (SGNI), a traditional Chinese patent medicine, on TNBC cells; To elucidate the mechanism of SGNI induced ferroptosis.MethodsThe anticancer activity of SGNI was examined via in vitro cell proliferation assays and in vivo xenograft growth assay. Ferroptosis was determined by flow-cytometric analysis of lipid ROS, labile iron pool measurement, and propidium iodide exclusion assay. The dependency on heme oxygenase 1 (HO-1) of SGNI induced ferroptosis was confirmed by genetic knockdown and pharmacological inhibition of the protein.ResultsSGNI selectively inhibited the proliferation of TNBC cells compared to non-TNBC breast cancer cells and normal cells. The cell death induced by SGNI in TNBC cells showed distinct morphology from apoptosis and could not be rescued by the pan-caspase inhibitor Z-VAD(OMe)-FMK. On the other hand, SGNI induced cell death was blocked by the lipid ROS scavengers ferrostatin-1 and liproxstatin-1, the acyl-CoA synthetase long chain family member 4 inhibitor rosiglitazone, and the iron chelators 1,10-phenanthroline and deferoxamine. These data indicated that SGNI induced a ferroptotic cell death of TNBC cells. Mechanistically, SGNI induced ferroptosis was dependent on HO-1, which promotes intracellular labile iron pool accumulation, and was alleviated by HO-1 knockdown and inhibition by tin protoporphyrin IX. In line with the in vitro data, SGNI significantly inhibited the xenograft growth of TNBC cell line MD-MB-231 in nude mice.ConclusionCollectively, our study elaborates on a promising regimen for TNBC treatment through induction of ferroptosis by SGNI, a traditional Chinese patent medicine currently available in the clinic, which merits further investigation.     

Characterization and hypoglycemic effect of a polysaccharide extracted from the fruit of Lycium barbarum L.

Diabetes mellitus is a group of complicated metabolic disorders characterized by high blood glucose level and inappropriate insulin secreting capacity due to decreased glucose metabolism and pancreatic β cell mass or dysfunction of β cells. Thus, improving glucose metabolism and preserving β cell mass and function might be useful for the treatment of diabetes. In this study, a novel acidic polysaccharide LBP-s-1 extracted from Lycium barbarum L. was obtained by purification using macroporous resin and ion-exchanged column. Monosaccharide composition analysis indicated that LBP-s-1 was comprised of rhamnose, arabinose, xylose, mannose, glucose, galactose, galacturonic acid in the molar ratio of 1.00:8.34:1.25:1.26:1.91:7.05:15.28. The preliminary structure features of LBP-s-1 were investigated by FT-IR, ¹H NMR and ¹³C NMR. In vitro and in vivo hypoglycemic experiments showed that LBP-s-1 had significant hypoglycemic effects and insulin-sensitizing activity through increasing glucose metabolism and insulin secretion and promoting pancreatic β cell proliferation. Preliminary mechanisms were also elucidated.     

Atractylenolide I inhibits colorectal cancer cell proliferation by affecting metabolism and stemness via AKT/mTOR signaling

BackgroundAtractylenolide I (ATL-1) is a natural herbal compound used in traditional Chinese medicine that has exhibited anti-cancer properties. The anti-tumorigenic activity of ATL-1 against colorectal cancer (CRC) and the underlying signaling pathways involved in its mechanisms are examined here.HypothesisATL-1 exerts therapeutic effect against CRC by disrupting glucose metabolism and cancer stem cell maintenance via AKT/mTOR pathway regulation.Study designIn vitro studies were performed in COLO205 and HCT116 CRC cell lines and in vivo studies were conducted in a mouse xenograft model of CRC tumor.MethodsCRC cells were treated with ATL-1 at various concentrations, with or without inhibitors of AKT or mTOR. Cell proliferation, apoptosis, invasion, stemness maintenance, glucose metabolism, and AKT/mTOR signaling were evaluated. CRC tumor-xenografted mice were treated with an AKT inhibitor and/or ATL-1, and glucose metabolism and stemness maintenance were examined in tumor tissues.ResultsATL-1 significantly inhibited the invasion of CRC cells by inducing their apoptosis, possibly via the excessive production of reactive oxygen species. Glucose metabolism (Warburg effect) was also altered and stem-like traits were suppressed by ATL-1. In addition, ATL-1 effectively acted as an inhibitor or AKT/mTOR by downregulating the phosphorylation of proteins related to the AKT/mTOR pathway. In vivo studies showed that tumor weight and volume were reduced by ATL-1 and that aerobic glycolysis, stemness maintenance, and AKT/mTOR activation were impaired by ATL-1 in colorectal tumors.ConclusionsATL-1 acts as an effective agent to suppress colorectal tumor progression, mainly by inhibiting CRC cell proliferation through altering apoptosis, glucose metabolism, and stem-like behavior. These processes were mediated by the AKT/mTOR signaling pathway both in vitro and in vivo. ATL-1 may be a potential agent to be used in molecular-targeted strategies for cancer treatment.     

Long noncoding RNA ZEB1-AS1 attenuates ferroptosis of gastric cancer cells through modulating miR-429/BGN axis

Gastric cancer (GC) is the fifth utmost common malignant cancer type globally, in which ferroptosis acts a critical function in the progress of GC. Long noncoding RNA ZEB1-AS1 has been recognized in numerous cancers, but the role of ZEB1-AS1 in ferroptosis remains obscure. Hence, we investigated the efficacy of ZEB1-AS1 on ferroptosis of GC cells. The cell growth and viability were analyzed via cell counting kit assay and xenograft tumor model in vivo and in vitro, respectively. The RNA and protein expression were measured by qRT-PCR and western blot analysis assay, respectively. The levels of Fe²⁺, malondialdehyde (MDA), and lipid reactive oxygen species (ROS) were tested to determine ferroptosis. The erastin and RSL3 were used to induce ferroptosis. The mechanism was analyzed via luciferase reporter gene and RIP assays. The treatment of ferroptosis inducer Erastin and RSL3 suppressed the viability of GC cells and the ZEB1-AS1 overexpression rescued the phenotype in the cells. The levels of Fe²⁺, MDA, and ROS were enhanced through the depletion of ZEB1-AS1 in Erastin/RSL3 treated GC cells. ZEB1-AS1 directly sponged miR-429 in GC cells and miR-429 targeted BGN in GC cells, and the inhibition of miR-429 rescued ZEB1-AS1 depletion-inhibited BGN expression. We validated that miR-429 induced and BGN-repressed ferroptosis in cancer cells. The BGN overexpression and miR-429 suppression could reverse the efficacy of ZEB1-AS1 on proliferation and ferroptosis in cancer cells. The expression of ZEB1-AS1 and BGN was enhanced and miR-429 expression was decreased in clinical GC tissues. ZEB1-AS1 attenuated ferroptosis of cancer cells by modulating miR-429/BGN axis.     

HIF-1α Promotes Epithelial-Mesenchymal Transition and Metastasis through Direct Regulation of ZEB1 in Colorectal Cancer

It is well recognized that hypoxia-inducible factor 1 alpha (HIF-1α) is involved in cancer metastasis, chemotherapy and poor prognosis. We previously found that deferoxamine, a hypoxia-mimetic agent, induces epithelial-mesenchymal transition (EMT) in colorectal cancer. Therefore, here we explored a new molecular mechanism for HIF-1α contributing to EMT and cancer metastasis through binding to ZEB1. In this study, we showed that overexpression of HIF-1α with adenovirus infection promoted EMT, cell invasion and migration in vitro and in vivo. On a molecular level, HIF-1α directly binding to the proximal promoter of ZEB1 via hypoxia response element (HRE) sites thus increasing the transactivity and expression of ZEB1. In addition, inhibition of ZEB1 was able to abrogate the HIF-1α-induced EMT and cell invasion. HIF-1α expression was highly correlated with the expression of ZEB1 in normal colorectal epithelium, primary and metastatic CRC tissues. Interestingly, both HIF-1α and ZEB1 were positively associated with Vimentin, an important mesenchymal marker of EMT, whereas negatively associated with E-cadherin expression. These findings suggest that HIF-1α enhances EMT and cancer metastasis by binding to ZEB1 promoter in CRC. HIF-1α and ZEB1 are both widely considered as tumor-initiating factors, but our results demonstrate that ZEB1 is a direct downstream of HIF-1α, suggesting a novel molecular mechanism for HIF-1α-inducing EMT and cancer metastasis.     

Ferroptosis is involved in alcohol-induced cell death in vivo and in vitro

ABSTRACT

A critical pathogenic factor in the development of lethal liver failure is cell death induced by the accumulation of lipid reactive oxygen species. In this study, we discovered and illuminated a new mechanism that led to alcoholic liver disease via ferroptosis, an iron-dependent regulated cell death. Study in vitro showed that both necroptosis inhibitor and ferroptosis inhibitors performed significantly protective effect on alcohol-induced cell death, while apoptosis inhibitor and autophagy inhibitor had no such effect. Our data also indicated that alcohol caused the accumulation of lipid peroxides and the mRNA expression of prostaglandin-endoperoxide synthase 2, reduced the protein expression of the specific light-chain subunit of the cystine/glutamate antiporter and glutathione peroxidase 4. Importantly, ferrostatin-1 significantly ameliorated liver injury that was induced by overdosed alcohol both in vitro and in vivo. These findings highlight that targeting ferroptosis serves as a hepatoprotective strategy for alcoholic liver disease treatment.     

The organotelluride catalyst LAB027 prevents colon cancer growth in the mice

Organotellurides are newly described redox-catalyst molecules with original pro-oxidative properties. We have investigated the in vitro and in vivo antitumoral effects of the organotelluride catalyst LAB027 in a mouse model of colon cancer and determined its profile of toxicity in vivo. LAB027 induced an overproduction of H₂O₂ by both human HT29 and murine CT26 colon cancer cell lines in vitro. This oxidative stress was associated with a decrease in proliferation and survival rates of the two cell lines. LAB027 triggered a caspase-independent, ROS-mediated cell death by necrosis associated with mitochondrial damages and autophagy. LAB027 also synergized with the cytotoxic drug oxaliplatin to augment its cytostatic and cytotoxic effects on colon cancer cell lines but not on normal fibroblasts. The opposite effects of LAB027 on tumor and on non-transformed cells were linked to differences in the modulation of reduced glutathione metabolism between the two types of cells. In mice grafted with CT26 tumor cells, LAB027 alone decreased tumor growth compared with untreated mice, and synergized with oxaliplatin to further decrease tumor development compared with mice treated with oxaliplatin alone. LAB027 an organotelluride catalyst compound synergized with oxaliplatin to prevent both in vitro and in vivo colon cancer cell proliferation while decreasing the in vivo toxicity of oxaliplatin. No in vivo adverse effect of LAB027 was observed in this model.     

Ablation of the Ferroptosis Inhibitor Glutathione Peroxidase 4 in Neurons Results in Rapid Motor Neuron Degeneration and Paralysis

Glutathione peroxidase 4 (GPX4), an antioxidant defense enzyme active in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that GPX4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis and death. Pathological inspection revealed that the paralyzed mice had a dramatic degeneration of motor neurons in the spinal cord but had no overt neuron degeneration in the cerebral cortex. Consistent with the role of GPX4 as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis, including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplementation with vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Gpx4 ablation. Also, lipid peroxidation and mitochondrial dysfunction appeared to be involved in ferroptosis of motor neurons induced by Gpx4 ablation. Taken together, the dramatic motor neuron degeneration and paralysis induced by Gpx4 ablation suggest that ferroptosis inhibition by GPX4 is essential for motor neuron health and survival in vivo.     

Directly targeting glutathione peroxidase 4 may be more effective than disrupting glutathione on ferroptosis-based cancer therapy

BackgroundCancer is one of the major threats to human health and current cancer therapies have been unsuccessful in eradicating it. Ferroptosis is characterized by iron-dependence and lipid hydroperoxides accumulation, and its primary mechanism involves the suppression of system X_c⁻-GSH (glutathione)-GPX4 (glutathione peroxidase 4) axis. Co-incidentally, cancer cells are also metabolically characterized by iron addiction and ROS tolerance, which makes them vulnerable to ferroptosis. This may provide a new tactic for cancer therapy.Scope of reviewThe general features and mechanisms of ferroptosis, and the basis that makes cancer cells vulnerable to ferroptosis are described. Further, we emphatically discussed that disrupting GSH may not be ideal for triggering ferroptosis of cancer cells in vivo, but directly inhibiting GPX4 and its compensatory members could be more effective. Finally, the various approaches to directly inhibit GPX4 without disturbing GSH were described.Major conclusionsTargeting system X_c⁻ or GSH may not effectively trigger cancer cells' ferroptosis in vivo the existence of other compensatory pathways. However, directly targeting GPX4 and its compensatory members without disrupting GSH may be more effective to induce ferroptosis in cancer cells in vivo, as GPX4 is essential in preventing ferroptosis.General significanceCancer is a severe threat to human health. Ferroptosis-based cancer therapy strategies are promising, but how to effectively induce ferroptosis in cancer cells in vivo is still a question without clear answers. Thus, the viewpoints raised in this review may provide some references and different perspectives for researchers working on ferroptosis-based cancer therapy.     

Tumor Suppressor WWOX Contributes to the Elimination of Tumorigenic Cells in Drosophila melanogaster

WWOX is a >1Mb gene spanning FRA16D Common Chromosomal Fragile Site, a region of DNA instability in cancer. Consequently, altered WWOX levels have been observed in a wide variety of cancers. In vitro studies have identified a large number and variety of potential roles for WWOX. Although its normal role in vivo and functional contribution to cancer have not been fully defined, WWOX does have an integral role in metabolism and can suppress tumor growth. Using Drosophila melanogaster as an in vivo model system, we find that WWOX is a modulator of TNFα/Egr-mediated cell death. We found that altered levels of WWOX can modify phenotypes generated by low level ectopic expression of TNFα/Egr and this corresponds to altered levels of Caspase 3 activity. These results demonstrate an in vivo role for WWOX in promoting cell death. This form of cell death is accompanied by an increase in levels of reactive oxygen species, the regulation of which we have previously shown can also be modified by altered WWOX activity. We now hypothesise that, through regulation of reactive oxygen species, WWOX constitutes a link between alterations in cellular metabolism observed in cancer cells and their ability to evade normal cell death pathways. We have further shown that WWOX activity is required for the efficient removal of tumorigenic cells from a developing epithelial tissue. Together these results provide a molecular basis for the tumor suppressor functions of WWOX and the better prognosis observed in cancer patients with higher levels of WWOX activity. Understanding the conserved cellular pathways to which WWOX contributes provides novel possibilities for the development of therapeutic approaches to restore WWOX function in cancer.     

LncRNA CRNDE promotes the progression and angiogenesis of pancreatic cancer via miR-451a/CDKN2D axis

BackgroundThe lncRNA colorectal neoplasia differentially expressed (lncRNA CRNDE) has been reported to play a pivotal role in various cancers. However, the expression and function of CRNDE in pancreatic cancer remain unclear. The objective of this study was to investigate the effects of CRNDE on pancreatic cancer and the underlying mechanisms.MethodsThe expression of CRNDE in pancreatic cancer tissues and cell lines was determined by RT-qPCR. Proliferation and angiogenesis were detected by MTT, colony formation, transwell and tube formation assays in vitro and in vivo. ELISA assay was used to detect the secretion of VEGFA. IHC was performed to test the expression levels of Ki67 and CD31. The binding sites between CRNDE, CDKN2D and miR-451a were predicted by bioinformatics analysis. Dual luciferase reporter and RNA immunoprecipitation assays were conducted to confirm the interaction with each other.ResultsThe results showed that CRNDE was significantly up-regulated in pancreatic cancer tissues as well as cell lines. CRNDE overexpression promoted the progression and angiogenesis of pancreatic cancer cells in vitro and in vivo. Moreover, we identified that CRNDE functioned as a sponge for miR-451a and CRNDE overexpression inhibited the expression of miR-451a. Furthermore, we confirmed that miR-451a directly interacted with CDKN2D and negatively regulated CDKN2D expression. In addition, CRNDE was found to positively regulate CDKN2D expression and mediate pancreatic cancer cell proliferation and angiogenesis through miR-451a/CDKN2D axis.ConclusionCRNDE modulates cell proliferation and angiogenesis via miR-451a/CDKN2D axis in pancreatic cancer, which provides a potential therapeutic target for pancreatic cancer treatment.     

ROS-Mediated Autophagy Induced by Dysregulation of Lipid Metabolism Plays a Protective Role in Colorectal Cancer Cells Treated with Gambogic Acid

Gambogic acid (GA), the main active component of gamboge resin, has potent antitumor activity both in vivo and in vitro. However, the underlying molecular mechanisms remain unclear. In this study, we found that GA could initiate autophagy in colorectal cancer cells, and inhibition of the autophagy process accelerated the effect of proliferative inhibition and apoptotic cell death induced by GA, implying a protective role of autophagy. Two-dimensional electrophoresis-based proteomics showed that GA treatment altered the expression of multiple proteins involved in redox signaling and lipid metabolism. Functional studies revealed that GA-induced dysregulation of lipid metabolism could activate 5-lipoxygenase (5-LOX), resulting in intracellular ROS accumulation, followed by inhibition of Akt-mTOR signaling and autophagy initiation. Finally, results using a xenograft model suggested ROS-induced autophagy protect against the antitumor effect of GA. Taken together, these data showed new biological activities of GA against colorectal cancer underlying the protective role of ROS-induced autophagy. This study will provide valuable insights for future studies regarding the anticancer mechanisms of GA.     

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.Subject terms: Macroautophagy, Macroautophagy     

Human mesenchymal stem cells are resistant to cytotoxic and genotoxic effects of cisplatin in vitro

Mesenchymal stem cells (MSCs) are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS) is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.     

C6-ceramide synergistically potentiates the anti-tumor effects of histone deacetylase inhibitors via AKT dephosphorylation and ��-tubulin hyperacetylation both in vitro and in vivo

Histone deacetylase inhibitors (HDACIs) have shown promising anti-tumor effects for a variety of malignancies, however, many tumors are reportedly resistant to them. In this study, we made a novel discovery that co-administration of HDACIs (Trichostatin A (TSA) and others) and exogenous cell-permeable short-chain ceramide (C6) results in striking increase in cancer cell death and apoptosis in multiple cancer cells. These events are associated with perturbations in diverse cell signaling pathways, including inactivation of Akt/mTOR and increase in α-tubulin acetylation (both in vivo and in vitro). TSA interacts in a highly synergistic manner with C6-ceramide to disrupt HDAC6/protein phosphatase 1 (PP1)/tubulin complex, to induce α-tubulin hyperacetylation, and to release and activate PP1, which then leads to AKT dephosphorylation and eventually causes cancer cell death. Interestingly, TSA itself results in short-term ceramide accumulation, which as a result of metabolic (glycosylation) removal, does not result in evident increase of cancer cell death. However, adding C6-ceramide led to a very pronounced increase in ceramide level and marked increase in cell death. Importantly, the effective synergistic anti-tumor activity of TSA plus C6-ceramide is also seen in in vivo mice xenograft pancreatic and ovarian cancer models, indicating that this regimen (HDACI plus C6-ceramide) may represent a more effective form of therapy against pancreatic and ovarian carcinoma.