Tight association of autophagy and cell cycle in leukemia cells.

BACKGROUND: Autophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet the relationship between these processes is not clearly defined. RESULTS: In exploring this relationship, we observed that the inhibition of autophagy impaired the G2/M phase-arresting activity of etoposide but enhanced the G1 phase-arresting activity of palbociclib. We further investigated the connection of basal autophagy and cell cycle by utilizing the autophagosome tracer dye Cyto-ID in two ways. First, we established a double-labeling flow-cytometric procedure with Cyto-ID and the DNA probe DRAQ5, permitting the cell cycle phase-specific determination of autophagy in live cells. This approach demonstrated that different cell cycle phases were associated with different autophagy levels: G1-phase cells had the lowest level, and G2/M-phase cells had the highest one. Second, we developed a flow-cytometric cell-sorting procedure based on Cyto-ID that separates cell populations into fractions with low, medium, and high autophagy. Cell cycle analysis of Cyto-ID-sorted cells confirmed that the high-autophagy fraction contained a much higher percentage of G2/M-phase cells than the low-autophagy fraction. In addition, Cyto-ID-based cell sorting also proved to be useful for assessing other autophagy-related processes: extracellular flux analysis revealed metabolic differences between the cell populations, with higher autophagy being associated with higher respiration, higher mitochondrial ATP production, and higher glycolysis. CONCLUSION: This work provides clear evidence of high autophagy in G2/M-phase cells by establishing a novel cell sorting technique based on Cyto-ID.

Tumor-Intrinsic PD-L1 Exerts an Oncogenic Function through the Activation of the Wnt/ß-Catenin Pathway in Human Non-Small Cell Lung Cancer.

Programmed death ligand 1 (PD-L1) strongly inhibits T cell activation, thereby aiding tumors in escaping the immune response. PD-L1 inhibitors have proven to be effective in the treatment of different types of cancer, including non-small cell lung cancer (NSCLC). Yet, the knowledge regarding the biological function of tumor-intrinsic PD-L1 in lung cancer remains obscure. In our study, we set the goal of determining the function of PD-L1 using overexpression and knockdown strategies. PD-L1 silencing resulted in decreased migratory and invasive ability of tumor cells, together with attenuated colony-forming capacity. Ectopic expression of PD-L1 showed the opposite effects, along with increased activities of MAPK and Wnt/ß-catenin pathways, and the upregulation of Wnt/ß-catenin target genes. Additionally, overexpression of PD-L1 was associated with dysregulated cellular and exosomal miRNAs involved in tumor progression and metastasis. In primary lung tumors, immunohistochemistry revealed that both PD1 and PD-L1 were highly expressed in squamous cell carcinoma (SCC) compared to adenocarcinoma (p = 0.045 and p = 0.036, respectively). In SCC, PD1 expression was significantly associated with tumor grading (p = 0.016). Taken together, our data suggest that PD-L1 may exert an oncogenic function in NSCLC through activating Wnt/ß-catenin signaling, and may act as a potential diagnostic marker for lung SCC.

Influence of high glucose in the expression of miRNAs and IGF1R signaling pathway in human myometrial explants.

PURPOSE: Several roles are attributed to the myometrium including sperm and embryo transport, menstrual discharge, control of uterine blood flow, and labor. Although being a target of diabetes complications, the influence of high glucose on this compartment has been poorly investigated. Both miRNAs and IGF1R are associated with diabetic complications in different tissues. Herein, we examined the effects of high glucose on the expression of miRNAs and IGF1R signaling pathway in the human myometrium. METHODS: Human myometrial explants were cultivated for 48 h under either high or low glucose conditions. Thereafter, the conditioned medium was collected for biochemical analyses and the myometrial samples were processed for histological examination as well as miRNA and mRNA expression profiling by qPCR. RESULTS: Myometrial structure and morphology were well preserved after 48 h of cultivation in both high and low glucose conditions. Levels of lactate, creatinine, LDH and estrogen in the supernatant were similar between groups. An explorative screening by qPCR arrays revealed that 6 out of 754 investigated miRNAs were differentially expressed in the high glucose group. Data validation by single qPCR assays confirmed diminished expression of miR-215-5p and miR-296-5p, and also revealed reduced miR-497-3p levels. Accordingly, mRNA levels of IGF1R and its downstream mediators FOXO3 and PDCD4, which are potentially targeted by miR-497-3p, were elevated under high glucose conditions. In contrast, mRNA expression of IGF1, PTEN, and GLUT1 was unchanged. CONCLUSIONS: The human myometrium responds to short-term exposure (48 h) to high glucose concentrations by regulating the expression of miRNAs, IGF1R and its downstream targets.

Epithelial Membrane Protein 2 Suppresses Non-Small Cell Lung Cancer Cell Growth by Inhibition of MAPK Pathway.

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs.

The sirtuin 1/2 inhibitor tenovin-1 induces a nonlinear apoptosis-inducing factor-dependent cell death in a p53 null Ewing's sarcoma cell line.

The sirtuin 1/2 inhibitor tenovin-1 activates p53 and may have potential in the management of cancer. Here, we investigated the responsiveness of Ewing's sarcoma cells to tenovin-1. We examined its effects in two Ewing's sarcoma cell lines with different p53 status, i.e. in p53 wild-type and p53 null cells. Effects were assessed by flow cytometric analyses of cell death, mitochondrial membrane depolarization and reactive oxygen species (ROS) generation, by caspase 3/7 activity measurement, by mRNA expression profiling and by immunoblotting. Tenovin-1 elicited caspase-mediated cell death in p53 wild-type cells, but caspase-independent cell death in p53 null cells. Remarkably, it induced a nonlinear concentration response in the latter: low concentrations of tenovin-1 were much more effective than were higher concentrations. Tenovin-1's effects in p53 null cells involved gene expression changes of Bcl-2 family members, mitochondrial membrane depolarization, nuclear translocation of apoptosis-inducing factor, ROS formation and DNA damage; all these effects followed a bell-shaped pattern. In conclusion, our results provide new insights into tenovin-1's mode of action by demonstrating that it can induce different pathways of cell death.

Tribbles 2 mediates cisplatin sensitivity and DNA damage response in epithelial ovarian cancer.

Aim was to identify methylated genes with functional involvement in cisplatin-resistance development of epithelial ovarian cancer (EOC). Genome-wide analyses of hypermethylated CpG-islands in resistant cell lines in combination with qRT-PCR analyses were used to identify epigenetically silenced genes. EOC-Type-II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in-silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5'-Aza-Cytidine treatment in resistant cells but a cisplatin-dependent, prominent upregulation on mRNA level in sensitive cells, only. Re-expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA-damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin-dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt-DNA-adduct formation in TRIB2 re-expressing cells did not translate in higher levels of dsDNA damage (yH2AX-foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC-Type-II patients from Jena (n = 38) and the TCGA (n = 149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log-rank p < 0.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin-resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC.

Changes of Proteases, Antiproteases, and Pathogens in Cystic Fibrosis Patients' Upper and Lower Airways after IV-Antibiotic Therapy.

BACKGROUND: In cystic fibrosis (CF) the upper (UAW) and lower airways (LAW) are reservoirs for pathogens like Pseudomonas aeruginosa. The consecutive hosts' release of proteolytic enzymes contributes to inflammation and progressive pulmonary destruction. Objectives were to assess dynamics of protease : antiprotease ratios and pathogens in CF-UAW and LAW sampled by nasal lavage (NL) and sputum before and after intravenous- (IV-) antibiotic therapy. METHODS: From 19 IV-antibiotic courses of 17 CF patients NL (10 mL/nostril) and sputum were collected before and after treatment. Microbiological colonization and concentrations of NE/SLPI/CTSS (ELISA) and MMP-9/TIMP-1 (multiplex bead array) were determined. Additionally, changes of sinonasal symptoms were assessed (SNOT-20). RESULTS: IV-antibiotic treatment had more pronounced effects on inflammatory markers in LAW, whereas trends to decrease were also found in UAW. Ratios of MMP-9/TIMP-1 were higher in sputum, and ratios of NE/SLPI were higher in NL. Remarkably, NE/SLPI ratio was 10-fold higher in NL compared to healthy controls. SNOT-20 scores decreased significantly during therapy (P = 0.001). CONCLUSION: For the first time, changes in microbiological patterns in UAW and LAW after IV-antibiotic treatments were assessed, together with changes of protease/antiprotease imbalances. Delayed responses of proteases and antiproteases to IV-antibiotic therapy were found in UAW compared to LAW.

Assessment of Mitochondrial Dysfunctions After Sirtuin Inhibition.

Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), with the latter being grouped into four phylogenetic classes. The class III of the HDAC family, the sirtuins (SIRTs), contributes to gene expression, genomic stability, cell metabolism, and tumorigenesis. Thus, several specific SIRT inhibitors (SIRTi) have been developed to target cancer cell proliferation. Here we provide an overview of methods to study SIRT-dependent cell metabolism and mitochondrial functionality. The chapter describes metabolic flux analysis using Seahorse analyzers, methods for normalization of Seahorse data, flow cytometry and fluorescence microscopy to determine the mitochondrial membrane potential, mitochondrial content per cell and mitochondrial network structures, and Western blot analysis to measure mitochondrial proteins.

Assessment of HDAC Inhibitor-Induced Endoplasmic Reticulum (ER) Stress.

The endoplasmic reticulum (ER) is a multifunctional cell organelle which is important for the folding and processing of proteins. Different endogenous and exogenous factors can disturb the ER homeostasis, causing ER stress and activating the unfolded protein response (UPR) to remove misfolded proteins and aggregates. ER stress and the UPR are associated with several human diseases, such as diabetes, Alzheimer's or Parkinson's disease, and cancer. Histone deacetylase inhibitors (HDACi) are used to treat cancer and were shown to induce ER stress/to modulate the UPR, although the exact mechanism is not fully understood and needs further research. Several approaches to monitoring ER stress exist. Here we describe methods including qPCR, Western blot, transmission electron microscopy, and fluorescence microscopy to analyze changes in mRNA and protein expression levels as well as defects in ER structures after HDAC inhibitor-induced ER stress.

Synergistic anticancer activity of combined ATR and ribonucleotide reductase inhibition in Ewing's sarcoma cells.

PURPOSE: Ewing's sarcoma is a highly malignant childhood tumour whose outcome has hardly changed over the past two decades despite numerous attempts at chemotherapy intensification. It is therefore essential to identify new treatment options. The present study was conducted to explore the effectiveness of combined inhibition of two promising targets, ATR and ribonucleotide reductase (RNR), in Ewing's sarcoma cells. METHODS: Effects of the ATR inhibitor VE821 in combination with the RNR inhibitors triapine and didox were assessed in three Ewing's sarcoma cell lines with different TP53 status (WE-68, SK-ES-1, A673) by flow cytometric analysis of cell death, mitochondrial depolarisation and cell cycle distribution as well as by caspase 3/7 activity determination, by immunoblotting and by real-time RT-PCR. Interactions between inhibitors were evaluated by combination index analysis. RESULTS: Single ATR or RNR inhibitor treatment produced small to moderate effects, while their combined treatment produced strong synergistic ones. ATR and RNR inhibitors elicited synergistic cell death and cooperated in inducing mitochondrial depolarisation, caspase 3/7 activity and DNA fragmentation, evidencing an apoptotic form of cell death. All effects were independent of functional p53. In addition, VE821 in combination with triapine increased p53 level and induced p53 target gene expression (CDKN1A, BBC3) in p53 wild-type Ewing's sarcoma cells. CONCLUSION: Our study reveals that combined targeting of ATR and RNR was effective against Ewing's sarcoma in vitro and thus rationalises an in vivo exploration into the potential of combining ATR and RNR inhibitors as a new strategy for the treatment of this challenging disease.

Histone deacetylase inhibitors enhance the anticancer activity of nutlin-3 and induce p53 hyperacetylation and downregulation of MDM2 and MDM4 gene expression.

Nutlin-3, a small-molecule MDM2 inhibitor, restores p53 function and is, thus, an appealing candidate for the treatment of cancers retaining wild-type p53. However, nutlin-3 applied as single agent may be insufficient for cancer therapy. Therefore, we explored whether the anticancer activity of nutlin-3 could be enhanced by combination with histone deacetylase inhibitors (HDACi), i.e. vorinostat, sodium butyrate, MS-275 and apicidin. We found that nutlin-3 and HDACi cooperated to induce cell death in the p53 wild-type cell lines A549 and A2780, but not in the p53 null cell line PC-3, as assessed by Alamar Blue assay and flow cytometric analyses of propidium iodide uptake and mitochondrial depolarization. Combination index analysis showed that the effect was synergistic. For comparison, we tested nutlin-3 in combination with paclitaxel, revealing that nutlin-3 antagonized the cytotoxic activity of paclitaxel. To shed light on the underlying mechanism of the synergistic action of nutlin-3 and HDACi, we determined the acetylation status of p53 by immunoblotting and the mRNA levels of MDM2 and MDM4 by real-time RT-PCR. We observed vorinostat to induce p53 hyperacetylation, to reduce the constitutive gene expression of MDM2 and MDM4, and to counteract the nutlin-3-induced upregulation of MDM2 gene expression. In conclusion, our study shows that HDACi amplify the antitumor activity of nutlin-3-possibly by inducing p53 hyperacetylation and/or MDM2 and/or MDM4 downregulation-suggesting that treatment with a combination of nutlin-3 and HDACi may be an effective strategy for treating tumors with wild-type p53.

Global metabolic alterations in colorectal cancer cells during irinotecan-induced DNA replication stress.

BACKGROUND: Metabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently. METHODS: We incubated p53-positive and p53-negative permanent CRC cells and short-term cultured primary CRC cells with the topoisomerase-1 inhibitor irinotecan and other drugs that cause DNA replication stress and consequently DNA damage. We analyzed pro-apoptotic mitochondrial membrane depolarization and cell death with flow cytometry. We evaluated cellular metabolism with immunoblotting of electron transport chain (ETC) complex subunits, analysis of mitochondrial mRNA expression by qPCR, MTT assay, measurements of oxygen consumption and reactive oxygen species (ROS), and metabolic flux analysis with the Seahorse platform. Global metabolic alterations were assessed using targeted mass spectrometric analysis of extra- and intracellular metabolites. RESULTS: Chemotherapeutics that cause DNA replication stress induce metabolic changes in p53-positive and p53-negative CRC cells. Irinotecan enhances glycolysis, oxygen consumption, mitochondrial ETC activation, and ROS production in CRC cells. This is connected to increased levels of electron transport chain complexes involving mitochondrial translation. Mass spectrometric analysis reveals global metabolic adaptations of CRC cells to irinotecan, including the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. P53-proficient CRC cells, however, have a more active metabolism upon DNA replication stress than their p53-deficient counterparts. This metabolic switch is a vulnerability of p53-positive cells to irinotecan-induced apoptosis under glucose-restricted conditions. CONCLUSION: Drugs that cause DNA replication stress increase the metabolism of CRC cells. Glucose restriction might improve the effectiveness of classical chemotherapy against p53-positive CRC cells. The topoisomerase-1 inhibitor irinotecan and other chemotherapeutics that cause DNA damage induce metabolic adaptations in colorectal cancer (CRC) cells irrespective of their p53 status. Irinotecan enhances the glycolysis and oxygen consumption in CRC cells to deliver energy and biomolecules necessary for DNA repair and their survival. Compared to p53-deficient cells, p53-proficient CRC cells have a more active metabolism and use their intracellular metabolites more extensively. This metabolic switch creates a vulnerability to chemotherapy under glucose-restricted conditions for p53-positive cells.

Molecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffolds.

Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. However, its efficacy can be impaired by insufficient numbers of donor HSCs. A promising strategy to overcome this hurdle is the use of an advanced ex vivo culture system that supports the proliferation and, at the same time, maintains the pluripotency of HSCs. Therefore, we have developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) that model the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized HSC culture medium allow the amplification of high numbers of undifferentiated HSCs. After 14 days in vitro cell culture, we performed transcriptome and proteome analysis. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Thus, we have shown that the 3D PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSCs ex vivo effectively.

Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing's sarcoma cells.

INTRODUCTION: Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells. METHODS: Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis. RESULTS: AUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect. CONCLUSION: Our study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.

Mechanistic insights into p53-regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells.

Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.

Anticancer effects of the PLK4 inhibitors CFI-400945 and centrinone in Ewing's sarcoma cells.

PURPOSE: Polo-like kinase 4 (PLK4) inhibitors, such as CFI-400945 and centrinone, are emerging as promising antineoplastic agents. However, their effectiveness against Ewing's sarcoma, a highly aggressive childhood cancer, remains to be established. METHODS: CFI-400945 and centrinone were tested in three Ewing's sarcoma cell lines with different TP53 status. Effects were assessed by flow-cytometric analyses of cell death, dissipation of the mitochondrial transmembrane potential and cell cycle distribution, by cell viability assay as well as by caspase 3/7 activity measurement, by immunoblotting and by immunofluorescence microscopy. RESULTS: CFI-400945 and centrinone elicited cell death in p53 wild-type and mutant Ewing's sarcoma cells. Both agents induced mitochondrial membrane depolarisation, caspase 3/7 activation, PARP1 cleavage and DNA fragmentation, indicating an apoptotic form of cell death. In addition, the PLK4 inhibitors induced a G2/M cell cycle arrest, particularly when cell killing was attenuated by the pan-caspase inhibitor z-VAD-fmk. Moreover, CFI-400945 treatment produced polyploidy. CONCLUSION: Our findings show that PLK4 inhibitors were effective against Ewing's sarcoma cells in vitro and thus provide a rationale for their evaluation in vivo.

Differential responsiveness of human hepatoma cells versus normal hepatocytes to TRAIL in combination with either histone deacetylase inhibitors or conventional cytostatics.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer because it elicits cell death in many tumor cells while sparing most normal cells. Liver cancer, however, is largely resistant to TRAIL and, thus, requires sensitization for TRAIL-mediated cytotoxicity. Sensitization may be achieved by cotreatment with chemotherapeutic agents. In this study, we comparatively investigated the treatment efficacy of TRAIL in combination with histone deacetylase inhibitors (HDI) versus TRAIL in combination with conventional cytostatics in the hepatocellular carcinoma cell line HepG2 and in the childhood hepatoblastoma cell line Huh6. We found that TRAIL resistance could be overcome by cotreatment with the HDI vorinostat, sodium butyrate and MS-275, but not by cotreatment with the cytostatics carboplatin and etoposide. However, TRAIL combination treatment bears the risk of sensitizing otherwise TRAIL-resistant normal cells. We thus explored a potential cytotoxic effect of combined HDI/TRAIL treatment in normal hepatocytes: TRAIL in conjunction with HDI did not impose any cytotoxicity on the non-malignant cells. In searching for the determinants of HDI-mediated TRAIL sensitization in hepatoma cells, we observed that HDI treatment did not increase cell-surface expression of proapoptotic TRAIL receptors. Instead, HDI treatment enhanced TRAIL-induced cleavage of Bid. In conclusion, our data suggest that HDI are potent sensitizers to TRAIL in hepatoma cells and that the combination of HDI and TRAIL is selectively active in hepatoma cells without affecting normal hepatocytes, indicating that the combination of HDI and TRAIL may be an effective approach for the treatment of advanced liver cancer.

Histone deacetylase inhibitors and aspirin interact synergistically to induce cell death in ovarian cancer cells.

Histone deacetylase inhibitors (HDIs) as well as non-steroidal anti-inflammatory drugs including aspirin show promise as antineoplastic agents. The treatment with both HDIs and aspirin can result in hyperacetylation of proteins. In this study, we investigated whether HDIs and aspirin interacted in inducing anticancer activity and histone acetylation. We found that the HDIs, suberoylanilide hydroxamic acid and sodium butyrate, and aspirin cooperated to induce cell death in the ovarian cancer cell line, A2780. The effect was synergistic, as evidenced by CI-isobologram analysis. However, aspirin had no effect on histone acetylation, neither in the absence nor presence of HDIs. To gain insight into the mechanism underlying the synergistic action of HDIs and aspirin, we employed the deacetylated metabolite of aspirin, salicylic acid, and the cyclooxygenase-1- and -2-selective inhibitors, SC-560 and NS-398, respectively. We found that HDIs and salicylic acid interacted synergistically, albeit less efficiently than HDIs and aspirin, to induce cancer cell death, suggesting that the acetyl and the salicyl moiety contributed to the cooperative interaction of aspirin with HDIs. SC-560 and NS-398 had little effect both when applied alone or in conjunction with HDIs, indicating that the combinatorial effect of HDIs and aspirin was not the result of cyclo-oxygenase inhibition. In conclusion, our study demonstrates that HDIs and aspirin synergize to induce cancer cell death and, thus, provides a rationale for a more in-depth exploration into the potential of combining HDIs and aspirin as a strategy for anticancer therapy.

Synergistic activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid and the bisphosphonate zoledronic acid against prostate cancer cells in vitro.

Bisphosphonates are widely used agents for the treatment of malignant bone disease. They inhibit osteoclast-mediated bone resorption and can have direct effects on cancer cells. In this study, we investigated whether the anticancer activity of the third-generation bisphosphonate zoledronic acid (ZOL) could be enhanced by combination with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). We found that ZOL and SAHA cooperated to induce cell death in the prostate cancer cell lines LNCaP and PC-3. The effect was synergistic, as evidenced by combination index isobologram analysis. ZOL and SAHA synergized to induce dissipation of the mitochondrial transmembrane potential, to activate caspase-3, and to trigger DNA fragmentation, showing that the combination of ZOL and SAHA resulted in the initiation of apoptosis. Because ZOL acts by inhibiting the mevalonate pathway, thereby preventing protein prenylation, we explored whether the mevalonate pathway was also the target of the cooperative action of ZOL and SAHA. We found that geranylgeraniol, but not farnesol, significantly reduced ZOL/SAHA-induced cell death, indicating that the synergistic action of the agents was due to the inhibition of geranylgeranylation. Consistently, a direct inhibitor of geranylgeranylation, GGTI-298, synergized with SAHA to induce cell death, whereas an inhibitor of farnesylation, FTI-277, had no effect. In addition, SAHA synergized with mevastatin, an inhibitor of the proximal enzyme in the mevalonate pathway. These in vitro findings provide a rationale for an in vivo exploration into the potential of combining SAHA and ZOL, or other inhibitors of the mevalonate pathway, as an effective strategy for anticancer therapy.