Decrease In c-Myc Research Articles

IMPDH Inhibition Decreases TERT Expression and Synergizes the Cytotoxic Effect of Chemotherapeutic Agents in Glioblastoma Cells.

IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs.

In Vivo Antileukemic Activity of Ost-01 in Acute Myeloid Leukemia (AML): A Novel Natural Product (NP) from Baccharis Coridifolia

Baccharis is the largest genus in the Asteraceae and has been long used by phytomedicine practitioners in South America to treat a variety of medical conditions (i.e., wounds, infections, cancers). Herein, we report on the antileukemic activity of OST-01, a novel NP from Baccharis coridifolia. OST-01 was obtained through maceration of Baccharis coridifolia leaves in ethanol; the starting doses for testing the OST-01 activity against leukemic cells were derived empirically from routinely used dosages of similar plant extracts. OST-01 (0.25-2 µL/mL) inhibits cell growth and induces apoptosis of AML cell lines (MV-4-11, KG-1a, Kasumi-1, and HL-60) and primary CD34 + CD38 ‒ AML blasts [enriched for leukemia stem cells (LSCs)] but did not affect normal human CD34 + CD38 ‒ mononuclear cells [MNCs, enriched for hematopoietic stem cells (HSC)]. Compared to vehicle (V; 1 µl/g/BID PO x 21 days), OST-01 (1 µl/g/BID PO x 21 days) reduced leukemia burden (WBC: 59.4 vs 96.4x10 3/µL, p=0.01; spleen size: 0.38 vs 0.77 g, p<0.0001) and prolonged survival (median: 123 vs 92 days, p<0.0001) of MllPTD/WT/ Flt3ITD/ITD AML mice. In secondary transplant experiments, recipients of bone marrow (BM) MNCs from OST-01-treated donors, lived significantly longer than the recipients of BM MNCs from V-treated donors (median: 58 vs 26 days, p<0.0001), suggesting that OST-01 reduced LSC burden. We confirmed these results in a Cbfb-MYH11 ( CM) knock-in AML mouse model. OST-01-treated mice had a longer survival than V-treated controls (median: 70 vs 58 days, p<0.0001) and secondary recipients of BM MNCs from OST-01-treated donors lived also significantly longer than those of BM MNCs from V-treated donors (median: 63 vs 38 days, p<0.0001). Similar results were also observed in mice engrafted with luciferase-expressing FLT3-ITD+ MOLM-14 AML cells. Bioluminescence imaging indicated that OST-01 reduced leukemic burden compared to V treatment (@day 30: 2.3 vs 4.5x10 6 photons/s/cm 2/sr, p<0.0001); OST-01-treated mice lived significantly longer than V-treated controls (median: 46 vs 32 days, p<0.0001). Of note, no OST-01-related toxicity given within a dose range of 1-3 µl/g/BID, PO x 7, 28 or 56 days was observed in normal B6 mice (n=120 mice). Compared with OST-01 alone or V, combination of OST-01 and the BCL-2 inhibitor, venetoclax (VEN) demonstrated a synergistic activity on AML cell lines and primary blasts (max synergy score: 28.16), and prolonged survival of MllPTD/WT/ Flt3ITD/ITD AML mice (VEN/OST vs OST and VEN, median: 156 vs 123 and 95 days, p=0.0003 and p<0.0001; secondary transplant median: 105 vs 58 and 31 days, p<0.0001 and p<0.0001, respectively) and CM AML mice (VEN/OST vs OST and VEN, median: 85 vs 70 and 58 days, p=0.0005 and p<0.0001; secondary transplant median: 95 vs 62.5 and 48 days, p=0.0003 and p<0.0001, respectively). To gain insights into the mechanism of action of OST-01, we performed RNA-seq on OST-01- or V-treated HL-60 cells and CD34+CD38- AML blasts. By GSEA analysis, we identified 50 enriched Hallmark gene sets (26 upregulated and 24 downregulated) in OST-01- vs V-treated HL-60 cells and 49 (32 upregulated and 17 downregulated) in OST-01- vs V-treated CD34+CD38- AML blasts. The Hallmark gene sets comprising Myc targets were among the most downregulated in OST-01-treated cells. We confirmed these results showing an OST-01-dependent decrease in c-myc, inhibition of c-myc signaling, and deregulation of c-myc-dependent nucleolar structure and ribosome biogenesis in HL-60 cells and CD34+CD38- AML blasts. To this end, OST-01 enhanced ubiquitination and degradation of c-myc and nucleolar proteins (NPM1, NS, nucleolin) and in turn induced nucleolar disruption as shown by immunoblotting, immunoprecipitation, and electron microscope imaging in HL-60 cells and CD34+CD38- AML blasts. Of note, OST-01 also inhibited ErbB3 binding protein 1 (Ebp1)-enhanced ribosomal RNA (rRNA) synthesis through upregulation of expression and function of RNA Polymerase I (Pol I) and DEAD-box helicase 21 (DDX21). In summary, we provide the first report of a significant in vivo antileukemic activity of a novel NP, OST-01. The mechanisms of action of OST-01 are mediated by inhibition of c-myc-dependent signaling, rRNA synthesis and ribosome biogenesis. To date, no preclinical toxicity has been observed. We concluded that OST-01 is a non-toxic, potentially new therapy for AML; translation from the bench to the bedside is underway.

Vitamin D and docosahexaenoic acid inhibit proliferation of the ovarian cancer cell line OVCAR4.

Ovarian cancer is one of the deadliest cancers in women. Improved preventative, diagnostic, and therapeutic strategies are needed. Certain dietary patterns and nutrients such as vitamin D and omega-3 fatty acids are associated with reduced cancer risk, but their effects on ovarian cancer remain to be fully elucidated, and their combined effects have not been explored. To determine the individual and combined effects of the active vitamin D metabolite, calcitriol, and the omega-3 fatty acid, docosahexaenoic acid, on cell growth, and the abundance of the vitamin D receptor (VDR), proteins that modulate cell cycle progression, and apoptotic markers. OVCAR4 cells, a model of ovarian cancer, were treated with calcitriol, and docosahexaenoic acid, either alone or in combination. Effects on cell growth were determined by the sulforhodamine B assay. Changes in VDR, the cell cycle promotor c-Myc, the cell cycle inhibitor p27 and cleaved PARP, were determined by Western blotting. While OVCAR4 cell growth was inhibited by individual treatment with either calcitriol or docosahexaenoic acid, the combined treatment revealed enhanced growth inhibition as compared to either treatment alone. Furthermore, long-term treatment (12 days) yielded stronger growth inhibition at lower concentrations as compared to short-term treatments (3 days). Accompanying this growth inhibition was a decrease in c-Myc, and an increase in p27. The observed reduction in cell growth mediated by calcitriol and docosahexaenoic acid highlights the need for further research utilizing these nutrients, alone and especially in combination, to support ovarian cancer prevention and treatment.

EGFL7 drives the evolution of resistance to EGFR inhibitors in lung cancer by activating NOTCH signaling

Accumulating evidence supports evolutionary trait of drug resistance. Like resilience in other systems, most tumor cells experience drug-tolerant state before full resistance acquired. However, the underlying mechanism is still poorly understood. Here, we identify that EGF like domain multiple 7 (EGFL7) is a responsive gene to epidermal growth factor receptor (EGFR) kinase inhibition during a period when tumors are decimated. Moreover, our data reveal that the adaptive increase of EGFL7 during this process is controlled by the depression of nonsense-mediated mRNA decay (NMD) pathway. Upregulation of EGFL7 activates NOTCH signaling in lung cancer cells, which slows down the decrease of c-Myc caused by EGFR inhibition, thereby helping the survival of cancer cells. Our data, taken together, demonstrate that EGFL7 is a driver gene for resistance to EGFR kinase inhibition, and suggest that targeting EGFL7/NOTCH signaling may improve the clinical benefits of EGFR inhibitors in patients with EGFR mutant tumors.

Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

BackgroundIsocitrate dehydrogenase-2 (IDH2) is a mitochondrial enzyme that catalyzes the metabolic conversion between isocitrate and alpha-ketoglutarate (α-KG) in the TCA cycle. IDH2 mutation is an oncogenic event in acute myeloid leukemia (AML) due to the generation of 2-hydroxyglutarate. However, the role of wild-type IDH2 in AML remains unknown, despite patients with it suffer worse clinical outcome than those harboring mutant type.MethodsIDH2 expression in AML cell lines and patient samples was evaluated by RT-qPCR, western blotting and database analyses. The role of wild-type IDH2 in AML cell survival and proliferation was tested using genetic knockdown and pharmacological inhibition in AML cells and animal models. LC–MS, GC–MS, isotope metabolic tracing, and molecular analyses were performed to reveal the underlying mechanisms.ResultsWe found that wild-type IDH2 was overexpressed in AML and played a major role in promoting leukemia cell survival and proliferation in vitro and in vivo. Metabolomic analyses revealed an active IDH2-mediated reductive TCA cycle that promoted the conversion of α-KG to isocitrate/citrate to facilitate glutamine utilization for lipid synthesis in AML cells. Suppression of wild-type IDH2 by shRNA resulted in elevated α-KG and decreased isocitrate/citrate, leading to reduced lipid synthesis, a significant decrease in c-Myc downregulated by α-KG, and an inhibition of AML viability and proliferation. Importantly, pharmacological inhibition of IDH2 showed significant therapeutic effect in mice inoculated with AML cells with wt-IDH2 and induced a downregulation of C-MYC in vivo.ConclusionsWt-IDH2 is an essential molecule for AML cell survival and proliferation by promoting conversion of α-KG to isocitrate for lipid synthesis and by upregulating c-Myc expression and could be a potential therapeutic target in AML.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

Leflunomide Inhibits c-Myc Signaling and Impairs Myeloma Cell Growth

Introduction:Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide (Lef), a commercially available oral immunosuppressive agent that has been FDA-approved for treatment of rheumatoid arthritis was evaluated as a potential MM therapeutic. In vitro anti-MM activity of Lef has previously been reported; however, the molecular mechanisms of action of Lef in MM are incompletely understood. Inhibition of Src kinase-family members and blocking of dihydroorotate dehydrogenase, an enzyme involved in pyrimidine synthesis are among the few known targets of Lef. To identify novel direct targets of Lef and to further our understanding of Lefs' mode of action on MM cells, we performed in vitro kinase screening assays and RNA/miRNA sequencing analysis of cells treated with or without Lef.Methods and Results:Our pre-clinical studies with teriflunomide (Ter), the active metabolite of Lef, showed that it inhibited growth of MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226, OPM-2, L-363) and primary MM patients' (CD138+) plasma cells at IC50s ranging from 30 uM to 140 uM. Pharmacokinetics analysis accompanying a phase 1 dose-escalation clinical trial in MM at our institute has demonstrated that Ter serum concentrations of 500 uM can be obtained. We also found that Ter induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <150 uM. At higher in vitro concentrations (100 uM to 200 uM), Ter induces apoptosis in MM cells. Moreover, Ter synergized with dexamethasone as well as lenalidomide in in vitro growth inhibition of MM cell line MM.1S. In vivo mouse studies with Lef as single agent or in combination with the immune modulatory drug lenalidomide or with the proteasome inhibitor ixazomib are currently ongoing and results will be presented at the meeting. To identify novel direct targets of Ter, a screening assay described by Anastassiadis et al. (PMCID: PMC3230241) was applied to test the inhibitory activity of Ter on 600 known kinases. We identified 55 kinases that were inhibited >50% at 200 uM Ter. Subsequent dose-response kinase assays identified, among others, Aurora A and PIM family members, all of which are involved in MM pathogenesis, as novel direct targets of Lef, with IC50s ranging from 35 uM to 53 uM. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon Lef exposure, 4 MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary MM cells were exposed to 200 µM Ter or DMSO control for 24h before extraction, purification and sequencing of mRNA and microRNA. RNA-sequencing analysis and DAVID functional annotation revealed that similar changes were present between patient samples and cell lines. Pathways that were predominantly affected by Ter included purine/pyrimidine metabolism, DNA-replication and cell-cycle regulation. Gene set enrichment analysis revealed that c-Myc, an oncogene that contributes to the malignant phenotype of MM and a known promising target in MM, signaling was impaired upon Ter treatment, in MM.1S cells. Differential expression analysis, real-time PCR and western blot analysis showed that c-Myc mRNA, c-Myc protein and c-Myc-regulated miRNAs belonging to the oncomir-1 cluster miR-17-92 were all down-regulated upon Ter exposure in the majority of the MM cells. Since PIM family members are involved in c-Myc signaling in cancer cells, we performed in silico molecular docking studies using Ter/PIM. Our results suggest that the active metabolite of Lef, Ter, can directly bind to the PIM family of proteins by binding to the ATP-binding site, thereby inhibiting its kinase activity. Since pan-PIM inhibitors such as PIM447 have been reported to be cytotoxic for myeloma cells due to decrease in c-Myc levels, these results suggest that decrease in c-Myc induced by Ter may be due to PIM inhibition.Conclusion:Leflunomide impairs in vitro MM cell growth - at least in part - through inhibition of c-Myc signaling. A clinical trial with leflunomide in MM has been initiated and a separate abstract with encouraging initial results has been submitted for presentation at this meeting. DisclosuresKrishnan:Onyx: Speakers Bureau; Sutro: Consultancy; Janssen: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau. Rosenzweig:Celgene: Speakers Bureau.

Combined Treatment with JQ1 and Curcumin Synergically Modulates Apoptosis, Cell Cycle Features, Micrornas and Gene Expression in High-Risk Pediatric Acute Myeloid Leukemia (AML)

BackgroundDespite recent progresses in the treatment of childhood AML, the outcome of high-risk patients (carrying selected molecular aberrations and/or having high levels of minimal residual disease at the end of therapy) remains dismal. In particular, patients harboring FLT3-ITD or some translocations involving MLL (such as MLL-AF10 or MLL AF4) still have a poor prognosis, mainly due to leukemia recurrence.A key feature of FLT3-ITD and MLL-rearranged leukemia is cMYC overexpression, a well-known oncogene often involved in cancer. JQ1 is a BET inhibitor, preventing cMYC expression through the binding to the BET bromodomain protein, BRD4. Moreover, Curcumin is a compound able to induce epigenetic changes, including inhibition of histone-acetyltransferases (HATs), resulting in a decrease of acetylated lysine residues, a target for BRD4 bound on DNA.AimWe evaluated whether the combination of JQ1 and Curcumin has a potential synergic effect on AML cell lines or primary blasts harboring FLT3-ITD and MLL-rearrangement. These experimental data could be instrumental for developing novel treatment options alternative or complementary to standard chemotherapy.MethodsCell lines with FLT3-ITD and/or MLL fusion proteins used in this study were: MV-4-11, THP1, MOLM-13 and RS-4-11 [ALL cell line harboring t(4;11)]. Cells were all treated with JQ1 [250nM], while the dose of Curcumin varied according to the MLL-rearrangement (5μM for MV-4-11 and RS-4-11 carrying MLL-AF4 rearrangement and 10μM for THP1 and MOLM-13 harboring MLL-AF9 rearrangement). After 48 hours of either single-drug or combined treatment (J+C), western blot, XTT assay, RT-qPCR and Annexin V FACS analysis were carried out to assess proliferation rates, apoptosis and expression of their related genes, as well as the expression of cMYC and AcH3K14. Capacity to form colonies after either single or combined drug exposure was tested on bone marrow derived CD34+ cells from 4 patients with either MLL-rearranged acute leukemia (AML MLL-AF9; ALL MLL-AF4; AML MLL-AF4) or FLT3-ITD using Methocult media.miRNA expression profile of 20 BM samples of either FLT3-ITD or MLL-rearranged AML collected at both diagnosis (Dx) and at relapse (Rel) was assessed with Nanostring platform. Results were compared with those obtained analyzing BM samples of 8 healthy controls (HCs) and of 12 patients with the same molecular lesions who did not relapse (NRel). Expression levels of the modulated miRNAs were also evaluated on the cell lines reported above. A supplemental miRNA profiling analysis was conducted comparing cells harboring FLT3-ITD with those carrying MLL-rearrangements with respect to HCs.ResultsIn comparison with treatment with either of the 2 agents, the J+C combination resulted into to a synergic effect on apoptosis rate of MOLM-13 cells and of RS-4-11 cells. In particular, the apoptosis rates of the former cell line were 16% with JQ1, 10% with Curcumin and 56% with J+C, while those of the latter cell line were 20% with JQ1, 4% with Curcumin and 52% with J+C, respectively. The XTT metabolic assay showed significant reduction in 24 and 72-hour proliferation rates after both single-agent and combined treatments in all cell lines compared to DMSO (2WayANOVA; P<0.05)RT-qPCR and western blot experiments showed an increased expression of BCL2, cleaved CASPASE-3, cleaved PARP1 and p21 levels, as well as a decrease of cMYC and AcH3K14. Single-agent and combined treatment also resulted into a significant decrease of number of colonies in all the 4 primary cultured patient blasts (Fisher's exact t-test; P<0.05). 118 miRNAs were found to be significantly modulated in samples of Rel and NRel groups compared with those of HCs, while 18 miRNAs were found to be modulated in either MLL-rearranged or FLT3-ITD AML as described in the Figure1, 2, and Table 1, 2. We also noticed an opposite expression trend for miR-99 and miR-34a in MLL-AF4 AML and MLL-AF9 AML, and significant modulations of expression after single-agent and J+C treatment for miR-99, miR-34a, miR-451 and miR-196a in our cell lines.ConclusionsOur data demonstrate that J+C exert an intense synergic effect on AML cell lines and primary blasts, leading to increased apoptosis, reduced proliferation rate, modulation in expression of some key genes, such as cMYC, BCL2 and AcH3K14. We also identified distinct miRNA signatures in patients collected at relapse as compared to those obtained at diagnosis or HCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

(\u2013)-Epigallocatechin-3-gallate induces apoptosis and differentiation in leukaemia by targeting reactive oxygen species and PIN1

(–)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

NGF/TRKA Decrease miR-145-5p Levels in Epithelial Ovarian Cancer Cells.

Nerve Growth Factor (NGF) and its high-affinity receptor tropomyosin receptor kinase A (TRKA) increase their expression during the progression of epithelial ovarian cancer (EOC), promoting cell proliferation and angiogenesis through several oncogenic proteins, such as c-MYC and vascular endothelial growth factor (VEGF). The expression of these proteins is controlled by microRNAs (miRs), such as miR-145, whose dysregulation has been related to cancer. The aims of this work were to evaluate in EOC cells whether NGF/TRKA decreases miR-145 levels, and the effect of miR-145 upregulation. The levels of miR-145-5p were assessed by qPCR in ovarian biopsies and ovarian cell lines (human ovarian surface epithelial cells (HOSE), A2780 and SKOV3) stimulated with NGF. Overexpression of miR-145 in ovarian cells was used to evaluate cell proliferation, migration, invasion, c-MYC and VEGF protein levels, as well as tumor formation and metastasis in vivo. In EOC samples, miR-145-5p levels were lower than in epithelial ovarian tumors. Overexpression of miR-145 decreased cell proliferation, migration and invasion of EOC cells, changes that were concomitant with the decrease in c-MYC and VEGF protein levels. We observed decreased tumor formation and suppressed metastasis behavior in mice injected with EOC cells that overexpressed miR-145. As expected, ovarian cell lines stimulated with NGF diminished miR-145-5p transcription and abundance. These results suggest that the tumoral effects of NGF/TRKA depend on the regulation of miR-145-5p levels in EOC cells, and that its upregulation could be used as a possible therapeutic strategy for EOC.

Synergic pro-apoptotic effects of Ferulic Acid and nanostructured lipid carrier in glioblastoma cells assessed through molecular and Delayed Luminescence studies

Herein, we assessed the effect of Ferulic Acid (FA), a natural antioxidant with anti-cancer effect, on the human glioblastoma cells through molecular and Delayed Luminescence (DL) studies. DL, a phenomenon of ultra-week emission of optical photons, was used to monitor mitochondrial assessment. The effect of FA loaded in nanostructured lipid carriers (NLCs) was also assessed. To validate NLCs as a drug delivery system for glioblastoma treatment, particular attention was focused on their effect. We found that free FA induced a significant decrease in c-Myc and Bcl-2 expression levels accompanied by the apoptotic pathway activation. Blank NLCs, even if they did not induce cytotoxicity and caspase-3 cleavage, decreased Bcl-2, ERK1/2, c-Myc expression levels activating PARP-1 cleavage. The changes in DL intensity and kinetics highlighted a possible effect of nanoparticle matrix on mitochondria, through the involvement of the NADH pool and ROS production that, in turn, activates ERK1/2 pathways. All the effects on protein expression levels and on the activation of apoptotic pathway appeared more evident when the cells were exposed to FA loaded in NLCs. We demonstrated that the observed effects are due to a synergic pro-apoptotic influence exerted by FA, whose bio-availability increases in the glioblastoma cells, and NLCs formulation.

Moringa oleifera Aqueous Leaf Extract Induces Cell-Cycle Arrest and Apoptosis in Human Liver Hepatocellular Carcinoma Cells

Aim: Hepatocellular carcinoma is one of the leading global epidemics. A medicinal tree, Moringa oleifera (MO), has been part of traditional treatments including cancer therapies. We investigated the apoptosis inducing effects of MO crude aqueous leaf extract (MOE) in human liver hepatocellular carcinoma (HepG2) cells.Methods: HepG2, PBMCs and Hek293 cell viability was evaluated using MTT assay. Oxidative stress and DNA damage was determined using TBARS and comet assays, respectively. Apoptosis was assessed by caspase-9, -3/7 activities and ATP levels (luminometry). Cell cycle, γH2AX, and cleaved PARP-1 were determined (flow cytometry). Protein expression of c-myc, Bax, p-Bcl2, Smac/DIABLO, Hsp70, SRp30a and cleaved PARP-1 was assessed using western blotting.Results: MOE displayed minimal toxicity in PBMCs and Hek293 cells for 24 h. HepG2 cells were exposed to MOE (24 h) and an IC50 (4.479 mg/mL) was determined. MOE significantly increased lipid peroxidation, DNA damage and γH2AX levels. A significant decrease in G1, S and G2-M phase was seen. Significant increase in SRp30a protein expression activated caspase-9. Caspase-9 and -3/7 was significantly increased with significant decrease in ATP levels. Apoptosis was confirmed with significant decrease in c-myc, p-Bcl2 and Hsp70 protein expression and a significant increase in Bax, Smac/DIABLO and PARP-1 cleavage.Conclusion: MOE induces cell-cycle arrest and apoptosis in cancerous HepG2 cells.

Down-Regulation of Nfatc1 Suppresses Proliferation, Migration, Invasion, and Warburg Effect in Prostate Cancer Cells.

BackgroundProstate cancer (PCa), accounting for 28% of all male cancer cases, is the second leading cause of cancer-related death among men. NFATc1, belonging to the NFAT family, is overexpressed in PCa and is correlated with the risk of recurrence after radical prostatectomy.Material/MethodsIn the present study, the expression of NFATc, c-myc, and PKM2 in PCa cells was regulated by lentiviruses and then detected by real-time PCR and Western blot analysis. Further, proliferation, invasion, and migration assays were performed. The glucose consumption and lactate production were assessed by biochemical detection.ResultsWe found that NFATc1 down-regulation significantly suppressed the proliferation and Warburg effect of PCa cells, concurrent with a decrease of c-myc and PKM2 expression. Likewise, the abilities of migration and invasion were also inhibited in NFATc1-silenced PCa cells. In addition, NFATc1 down-regulation-induced inhibition of cell proliferation, migration, invasion, and Warburg effect were counteracted by up-regulation of c-myc or PKM2. The expression of PKM2 was positively regulated by NFATc1 and c-myc expression.ConclusionsThese results indicate that NFATc1 down-regulation can suppress the proliferation, Warburg effect, and migration and invasion abilities of PCa cells, probably by regulating c-myc and PKM2 expression. NFATc1 may be a potential therapeutic target for PCa and could be used as a diagnosis or prognosis indicator of PCa.

Synergistic in-vitro effects of combining an antiglycolytic, 3-bromopyruvate, and a bromodomain-4 inhibitor on U937 myeloid leukemia cells.

This project investigated the in-vitro effects of a glycolytic inhibitor, 3-bromopyruvate (3-BrP), in combination with and a new in silico-designed inhibitor of the bromodomain-4 (BRD-4) protein, ITH-47, on the U937 acute myeloid leukemia cell line. 3-BrP is an agent that targets the altered metabolism of cancer cells by interfering with glucose metabolism in the glycolytic pathway. ITH-47 is an acetyl-lysine inhibitor that displaces bromdomain 4 proteins from chromatin by competitively binding to the acetyl-lysine recognition pocket of this bromodomain and extraterminal (BET) BRD protein, thereby preventing transcription of cancer-associated genes and further cell growth. Cell growth studies determined the IC50 after 48 h exposure for 3-BrP and ITH-47 to be 6 and 2 μmol/l, respectively. When combined, 2.4 and 1 μmol/l of 3-BrP and ITH-47, respectively, inhibited 50% of the cell population, yielding a synergistic combination index of 0.9. Subsequent mechanistic studies showed that the IC50 concentrations of ITH-47 and 3-BrP and the combination increased observable apoptotic bodies and cell shrinkage in U937 cells treated for 48 h. Cell cycle analysis showed an increase in the sub-G1 fraction in all treated cells, suggesting that cell death was increased in the treated samples. Annexin-V-FITC apoptosis analysis showed a statistically significant increase in the number of cells in early and late apoptosis, indicating that cell death occurred through apoptosis and not necrosis. Only U937 cells exposed to ITH-47 showed a decrease in mitochondrial membrane potential compared with the vehicle control. Reactive oxygen species production was decreased in all treated samples. ITH-47-exposed cells showed a decrease in c-Myc, Bcl-2, and p53 gene expressions. 3-BrP-treated cells showed an increase in c-myc and p53 gene expressions. The combination of ITH-47 and 3-BrP lead to downregulation of c-myc and Bcl-2 genes. ITH-47 exposure conditions yielded a marked decrease in c-myc protein levels as well as a decrease in Ser70 phosphorylated Bcl-2. Analysis of 3-BrP and the combination of ITH-47 and 3-BrP test conditions indicated an increase in p53 protein levels. This novel study is the first to investigate the in-vitro synergistic therapeutic effect of ITH-47 and 3-BrP. The current study contributes toward unraveling the in-vitro molecular mechanisms and signal transduction associated with a novel combination of BRD inhibitors and antiglycolytic agents, providing a basis for further research on these combinations.

Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest.

Diosmetin, a plant flavonoid, has been shown to exert promising effects on prostate cancer cells as an anti-proliferative and anticancer agent. In this study, using western blot analysis for protein expression and flow cytometry for cell cycle analysis, we determined that the treatment of the LNCaP and PC-3 prostate cancer cells with diosmetin resulted in a marked decrease in cyclin D1, Cdk2 and Cdk4 expression levels (these proteins remain active in the G0-G1 phases of the cell cycle). These changes were accompanied by a decrease in c-Myc and Bcl-2 expression, and by an increase in Bax, p27Kip1 and FOXO3a protein expression, which suggests the potential modulatory effects of diosmetin on protein transcription. The treatment of prostate cancer cells with diosmetin set in motion an apoptotic machinery by inhibiting X-linked inhibitor of apoptosis (XIAP) and increasing cleaved PARP and cleaved caspase-3 expression levels. On the whole, the findings of this study provide an in-depth analysis of the molecular mechanisms responsible for the regulatory effects of diosmetin on key molecules that perturb the cell cycle to inhibit cell growth, and suggest that diosmetin may prove to be an effective anticancer agent for use in the treatment of prostate cancer in the future.

High expression of TMEM40 is associated with the malignant behavior and tumorigenesis in bladder cancer

BackgroundBladder cancer (BCa) is one of the most common cancers in the urinary system among the world. Previous studies suggested that TMEM40 expression level was significantly associated with clinicopathological parameters including histological grade, clinical stage and pT status of bladder cancer. However, the molecular mechanism of TMEM40 in BCa remains poorly understood.MethodsReal-time quantitative RT-PCR (qRT-PCR) and western blot (WB) were used to examine the expression levels of TMEM40 in BCa tissues, paired non-cancer tissues and cell lines. A series of experiments, including CCK-8, wound healing, flow cytometry, transwell and EdU assays were performed to assess the effects of TMEM40 on cell proliferation, cell cycle and apoptosis, migration and invasion. In addition, tumor growth was evaluated in vivo using a xenogenous subcutaneously implant model. All statistical analyses were executed by using the SPSS 20.0 software. All experimental data from three independent experiments were analyzed by Student’s t test and results were expressed as mean ± standard deviation.ResultsIn this study, we identified the role of TMEM40 in the tumorigenesis of bladder cancer and found that it was upregulated in bladder cancer tissues and cell lines, compared with their normal counterparts. The results demonstrated that effective silence of TMEM40 expression suppressed cell proliferation, blocked G1-to-S cell cycle transition, and inhibited cell migration and invasion in human bladder 5637 and EJ cell lines. Consistently, in vivo data showed that TMEM40 silencing could dramatically decreased tumor growth. Further study revealed that TMEM40 knockdown resulted in accumulation of p53 and p21 protein and decrease of c-MYC and cyclin D1 protein.ConclusionThese data suggest that TMEM40 represents a potential oncogene, which exert a crucial role in the proliferation and apoptosis via the p53 signaling pathway in BCa, thus probably serve as a novel candidate biomarker and a potential therapeutic target for patients with BCa.

Differential Behavior of Normal and Fibrotic Fibroblasts under the Synergistic Influence of Micropillar Topography and the Rigidity of Honey/Silk-Fibroin Substrates.

We report differential proliferation behavior of normal and fibrosis associated human oral fibroblasts on micropillar honey embedded silk fibroin substrates (HSF). Oral fibroblasts of different origins manifest differences in proliferation rate, morphology, and the cytoskeletal arrangement on HSF substrates with distinct topography (H, D, and S), stiffness, and honey concentration. It is observed that the proliferation rate is maximized for normal and inhibited for fibrosis associated fibroblasts on a HSF substrate surface with moderate height of ∼8.5 μm and 2% honey concentration. Molecular expression analysis reveals decrease in c-myc and p53 expression in later cells validating the inhibition of their proliferation rate, which is further correlated with the decreased Col I and Col III expression on this substrate. A substrate with enhanced interspacing and intermediate mechanical stiffness (0.57 ± 0.32 μN/nm) favors strong adhesion and stable cell-matrix interaction for normal cells, while exhibiting negative influence on fibrotic fibroblasts with poor adhesion and spreading capability. Decrease in vimentin, fibronectin expression, and cytoskeleton reorganization justify the poor stability of later cells on the optimized substrate, thereby allowing selective modulation of normal and fibrosis associated fibroblasts under the synergistic influence of honey concentration, topography, and rigidity of HSF substrates. The work highlights the possible therapeutic efficacy of honey based micropatterned substrates as smart patches for fast wound healing and in minimizing the chances of recurrence of precancer post oral tumor resection surgeries.

Reducing VDAC1 expression induces a non-apoptotic role for pro-apoptotic proteins in cancer cell differentiation

Proteins initially identified as essential for apoptosis also mediate a wide range of non-apoptotic functions that include cell cycle progression, differentiation and metabolism. As this phenomenon was mostly reported with non-cancer cells, we considered non-conventional roles for the apoptotic machinery in the cancer setting. We found that treating glioblastoma (GBM) tumors with siRNA against VDAC1, a mitochondrial protein found at the crossroads of metabolic and survival pathways and involved in apoptosis, inhibited tumor growth while leading to differentiation of tumor cells into neuronal-like cells, as reflected in the expression of specific markers. Although VDAC1 depletion did not induce apoptosis, the expression levels of several pro-apoptotic regulatory proteins were changed. Specifically, VDAC1 deletion led to up-regulation of caspases, p53, cytochrome c, and down-regulation of SMAC/Diablo, AIF and TSPO. The down-regulated group was highly expressed in U-87MG xenografts, as well as in GBMs from human patients. We also showed that the rewired cancer-cell metabolism resulting from VDAC1 depletion reinforced cell growth arrest and differentiation via alterations in the transcription factors p53, c-Myc, HIF-1α and NF-κB. The decrease in c-Myc, HIF-1α and NF-κB levels was in accord with reduced cell proliferation, whereas increased p53 expression promoted differentiation. Thus, upon metabolic re-programing induced by VDAC1 depletion, the levels of pro-apoptotic proteins associated with cell growth decreased, while those connected to cell differentiation increased, converting GBM cells into astrocyte- and neuron-like cells. The results reveal that in tumors, pro-apoptotic proteins can perform non-apoptotic functions, acting as regulators of cell growth and differentiation, making these molecules potential new targets for cancer therapy. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi.

Abstract P5-04-13: Pin1 negatively impacts Smad3 tumor suppression in triple negative breast cancer cell lines

Abstract Introduction: Triple negative breast cancer (TNBC) is an aggressive subtype associated with poor outcomes. Accordingly, there is an urgent need to develop novel and targeted therapeutics for patients with this disease subtype. Cyclins D and E and the corresponding activation of CDK4/2 represent promising therapeutic targets for the treatment of TNBC. CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, to promote the transition from tumor suppressive to oncogenic TGFβ activity in cyclin-overexpressing breast cancers. We identified a Smad3 interaction with Pin1, a cis-trans isomerase also overexpressed in aggressive breast cancers and associated with CDK-mediated Smad3 phosphorylation. Smad3 interaction with Pin1 can influence protein function and fidelity through recruitment of Smurf2 and subsequent proteasomal degradation. Based on these findings, we hypothesized that inhibition of the CDK-mediated Smad3-Pin1 interaction would stabilize Smad3 protein expression and restore tumor-suppressive Smad3 activity. Methods: Pin1 expression was knocked-down (KD) in MDA-MB-231 TNBC cells by transfecting with Pin1-targeting siRNA (siPin1) or control non-specific siRNA (siNS). KD efficiency was confirmed by immunoblotting. To assay Smad3 transcriptional activity with Pin1 KD, luciferase reporter studies were performed. Also, following Pin1 KD, immunoblotting was used to determine expression of Smad3 and associated protein targets. MTS assays were utilized to determine cellular proliferation after Pin1 KD. Transwell migration assays were used to assay the effect of Pin1 KD or CDK2 inhibitor treatment, which blocked non-canonical Smad3 Thr179 phosphorylation, on TNBC cell migration. Results: KD of Pin1 expression in TNBC cell lines resulted in an increase in Smad3 transcriptional activity compared to control cells, and correlated with an increase in expression of cdki p15 and a decrease in c-myc, Smad3-target genes and cell cycle regulators. Additionally, Pin1 KD resulted in a significant decrease in TNBC cell proliferation compared to siNS control TNBC cells. Smad3 protein levels increased following Pin1 KD, suggesting Pin1 action may negatively impact Smad3 stability. We also found that KD of Pin1 or treatment with a CDK2 inhibitor, which blocked Smad3 noncanonical Thr179 phosphorylation, resulted in significantly reduced TNBC cell migration. Conclusions: Inhibiting the Smad3-Pin1 interaction by knock-down of Pin1 expression in TNBC cells restored Smad3 transcriptional activity, which correlated to an increase in expression of the Smad3 associated protein cdki p15, decrease in c-myc, and a decrease in cellular proliferation. Additionally, Pin1 KD enhanced Smad3 protein levels, suggesting a role of Pin1 in mediating Smad3 stability. Inhibiting the Smad3-Pin1 interaction with Pin1 KD or CDK2 inhibitor treatment also reduced TNBC cell migration. Collectively, these data suggest that the Smad3-Pin1 interaction, facilitated by noncanonical CDK-mediated Smad3 phosphorylation, is associated with pro-tumorigenic and pro-migratory TGFβ signaling, and inhibition of this interaction may provide an important therapeutic option for TNBC patients. Citation Format: Thomas AL, Hamdan R, Hong A, Rosenthal E, Thomas AJ, Jeruss JS. Pin1 negatively impacts Smad3 tumor suppression in triple negative breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-13.

Linc-RoR promotes c-Myc expression through hnRNP I and AUF1

Linc-RoR was originally identified to be a regulator for induced pluripotent stem cells in humans and it has also been implicated in tumorigenesis. However, the underlying mechanism of Linc-RoR-mediated gene expression in cancer is poorly understood. The present study demonstrates that Linc-RoR plays an oncogenic role in part through regulation of c-Myc expression. Linc-RoR knockout (KO) suppresses cell proliferation and tumor growth. In particular, Linc-RoR KO causes a significant decrease in c-Myc whereas re-expression of Linc-RoR in the KO cells restores the level of c-Myc. Mechanistically, Linc-RoR interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) I and AU-rich element RNA-binding protein 1 (AUF1), respectively, with an opposite consequence to their interaction with c-Myc mRNA. While Linc-RoR is required for hnRNP I to bind to c-Myc mRNA, interaction of Linc-RoR with AUF1 inhibits AUF1 to bind to c-Myc mRNA. As a result, Linc-RoR may contribute to the increased stability of c-Myc mRNA. Although hnRNP I and AUF1 can interact with many RNA species and regulate their functions, with involvement of Linc-RoR they would be able to selectively regulate mRNA stability of specific genes such as c-Myc. Together, these results support a role for Linc-RoR in c-Myc expression in part by specifically enhancing its mRNA stability, leading to cell proliferation and tumorigenesis.