Increase In sub-G1 Population Research Articles

Pyrimidine containing epidermal growth factor receptor kinase inhibitors: Synthesis and biological evaluation.

Structure-based design and synthesis of pyrimidine containing reversible epidermal growth factor receptor (EGFR) inhibitors 1a-d are reported. The compounds (1a-d) inhibited the EGFR kinase activity in vitro with IC50 range 740nm to 3μm. mRNA expression of EGFR downstream target genes, that is twist, c-fos and aurora were found to be altered upon treatment with compounds 1a-d. The compounds 1a-d exhibited excellent anticancer activity at low micromolar level (3.2-9μm) in lung, colon and breast cancer cell lines. Furthermore, compounds induced the alteration in mitochondrial membrane potential and reactive oxygen species level and. Selected compound 1b was found to increase sub-G1 population indicative of cell death, the mode of cell death was apoptotic as evident from Annexin V verses propidium iodide assay. Molecular modelling further helped to investigate the binding recognition pattern of the compounds in ATP binding EGFR domain similar to erlotinib and dissimilar to WZ4002.

Abstract 5059: Preclinical study on the efficacy of Panobinostat in hepatocellular carcinoma

Abstract Background Aberrant regulation of histone deacetylases (HDACs) is known to play a pivotal role in HCC pathogenesis as well as other human malignancies. Panobinostat (LBH589) is a pan-HDAC inhibitor covering a wide range of HDACs (Class I, II and IV) with high inhibitory activity at nanomolar concentration. It has been approved by FDA for treating multiple myeloma and has demonstrated promising anti-proliferative and cytotoxic activity in breast, prostate, colon and pancreatic cancer cell lines. This study investigated in vitro and in vivo effect of Panobinostat in HCC cell lines. Methods Basal expressions of HR23B and HDACs of 7 HCC cell lines (HepG2, PLC/PRF/5, Huh-7, Hep3B, SNU-182, SNU-398 and SNU-449) were determined by western blotting. Their corresponding IC50 for 24, 48 and 72 hours towards Panobinostat were determined by cell viability assay. Huh-7, Hep3B and SNU-449 were selected for further in vitro experiments. Their cell cycle distribution after Panobinostat treatment was evaluated by flow cytometry. Apoptosis was detected by Cell Death Detection ELISA. Huh-7 and Hep3B xenograft model were used for in vivo investigation. Cells were inoculated subcutaneously into the flanks of 3-4 week old male athymic nude mice. When tumors were established, Panobinostat was administrated intraperitoneally at 7.5mg/kg and 15mg/kg five days per week for 2 weeks. Results All cell lines were able to achieve nearly 100% growth inhibition and had displayed a dose- and time-dependent manner towards Panobinostat. Maximum growth inhibition was 20-70% at 24hr compared to over 90% at 72hr. There was significant reduction in cell viability at low nanomolar concentrations (IC50 at 48hr: HepG2=8.81±0.72nM, PLC/PRF/5=18.9±0.74nM, Huh-7=14.01±1.12nM, Hep3B=25.00±3.69nM, SNU-182=73.33±15.52nM, SNU-398=12.86±3.25nM, SNU-449=73.01±9.09nM). Flow cytometry analysis showed Panobinostat induced accumulation of cells at G0/G1 phase in Huh-7 and SNU-449. Meanwhile, an increase in sub G1 population was detected in Hep3B after exposure to 25nM Panobinostat for 48h. Apoptotic induction was further confirmed by cell death detection ELISA and western blotting. Panobinostat promoted apoptosis more remarkable in Hep3B than other 2 cell lines as evidenced by a stronger cleaved PARP expression level. Panobinostat treatment delayed tumor growth in Hep3B (p<0.0005) and Huh-7 (p<0.0005) xenografts compared to vehicle control. The overall weight loss was less than 20% despite of greater drop during middle of the treatment. Conclusion Panobinostat has been demonstrated to inhibit in vitro and in vivo HCC cell growth. Further study on the mechanism behind Panobinostat sensitivity is warranted. The study was supported by Novartis. Citation Format: Chi Tung Choy, Wing Yu Man, Chi Hang Wong, Stephen Lam Chan. Preclinical study on the efficacy of Panobinostat in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5059. doi:10.1158/1538-7445.AM2017-5059

Auraptene Induces Apoptosis via Myeloid Cell Leukemia 1-Mediated Activation of Caspases in PC3 and DU145 Prostate Cancer Cells.

Although auraptene, a prenyloxy coumarin from Citrus species, was known to have anti-oxidant, anti-bacterial, antiinflammatory, and anti-tumor activities, the underlying anti-tumor mechanism of auraptene in prostate cancers is not fully understood to date. Thus, in the present study, we have investigated the anti-tumor mechanism of auraptene mainly in PC3 and DU145 prostate cancer cells, because auraptene suppressed the viability of androgen-independent PC3 and DU145 prostate cancer cells better than androgen-sensitive LNCaP cells. Also, auraptene notably increased sub-G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells as features of apoptosis in two prostate cancer cells compared with untreated control. Consistently, auraptene cleaved poly(ADP-ribose) polymerase, activated caspase-9 and caspase-3, suppressed the expression of anti-apoptotic proteins, including Bcl-2 and myeloid cell leukemia 1 (Mcl-1), and also activated pro-apoptotic protein Bax in both prostate cancer cells. However, Mcl-1 overexpression reversed the apoptotic effect of auraptene to increase sub-G1 population and induce caspase-9/3 in both prostate cancer cells. Taken together, the results support scientific evidences that auraptene induces apoptosis in PC3 and DU145 prostate cancer cells via Mcl-1-mediated activation of caspases as a potent chemopreventive agent for prostate cancer prevention and treatment. Copyright © 2017 John Wiley & Sons, Ltd.

Synergistic Anti-Tumor Efficacy of BET Inhibitors JQ1 and Otx-015 in Combination with Dasatinib in Preclinical Models of T-Cell Lymphomas

Background: Approximately 50% of patients with peripheral T-cell lymphoma (PTCL) enter long-term remission after standard chemotherapy and stem cell transplantation. Patients who do not respond to chemotherapy have few treatment options highlighting the critical need for new effective and targeted therapeutics. Aberrant T cell receptor (TCR) and tyrosine kinase (TK) signaling have been described in PTCL (Agostinelli 2014;Netchiporouka 2014). Single-agent TK inhibitors (TKIs) have significantly improved patient outcomes across multiple tumor subtypes. However, TKI therapy is rarely curative. The recent discovery of a subgroup of PTCL characterized by high levels of GATA3 and c-Myc expression and poor prognosis (Iqbal 2014; Manso 2016), establishes the rationale of targeting c-Myc in PTCLs. Based on the demonstration that pharmacologic inhibition of c-Myc is achievable through targeting bromodomain and extra terminal (BET) family of chromatin adapters, the therapeutic potential of BET inhibition was assessed in a panel of T cell lymphoma and leukemia cell lines. Since expression of c-Myc is regulated by the TCR, we also hypothesized that simultaneous targeting of c-Myc and TCR would significantly enhance the antiproliferative effects of BET inhibitors (BETis) and TKI alone in preclinical models of PTCL.Methods: Five T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR-2, Karpas 299, Sup-T1, HH) were incubated with escalating doses of JQ1 (a small-molecule BETi with the highest affinity for BRD4) and OTX-015 (a BETi with a broader affinity for BRD2, BRD3, BRD4) and the tyrosine-kinase-inhibitor Dasatinib. Analysis of cell viability, cell cycle distribution, apoptosis and mitochondrial depolarization was performed using flow cytometry. Effects of treatments were assessed using gene expression profiling (GEP) and western blotting (WB). Combinations were evaluated using the Chou-Talalay Combination Index (CI), calculated with CompuSyn software (CompuSyn Inc, Paramus, NJ).Results: JQ1 and OTX-015 show antiproliferative activity with IC50 at nanomolar concentrations in all cell lines. As assessed determining viable cells by PI exclusion and flow cytometry, JQ1 and OTX-015 are similarly active in a dose-dependent manner in all cell lines. To understand the activity of JQ1 and OTX-015, we analyzed cell-cycle distribution using flow cytometry. JQ1 and OTX-015 induce a cell cycle arrest with G1-phase accumulation and decrease S-phase with the exception of SUPT1 cells that are characterized by a cell cycle arrest in G2-phase. Minimal increase in the sub-G1 population is observed in all cell lines, suggesting that JQ1 and OTX-015 mainly exert a cytostatic effect. We then examined GATA3 and c-Myc protein levels in all cell lines: varying amounts of GATA3 and c-Myc proteins were observed but a strong correlation between GATA3 and c-Myc expression was detected. After JQ1 and OTX-015 exposure, c-Myc protein level decrease in all cell lines apart from SUP-T1 cell line. Here c-Myc level do not change significantly upon BETis exposure, suggesting that BETis target other pathways relevant for SUP-T1 survival.Dasatinib efficiently inhibits the proliferation in all cell lines at micromolar concentrations in a dose-dependent manner. Dasatinib induces G0/G1-phase arrest and an increase in sub-G1 population indicating a modest induction of apoptosis confirmed by caspase-9 activation and mitochondrial depolarization.Compared to all single agents, combined treatments with sub-optimal concentrations of Dasatinib and JQ1 or OTX-015 exert synergistic lethal activity against all tested cell lines (C.I.<1). To uncover the main biological processes behind the synergistic interactions of BETis and Dasatinib, cell cycle analysis was assessed indicating that both combinations induce a significant increase of sub-G1 population associated with massive mitochondrial depolarization and cleavage of Caspase-9 and PARP.Conclusions: The experiments presented here support the combination of BET inhibitors with the TK inhibitor Dasatinib for PTCLs. Our data suggest a synergistic interaction for the combination of both BETis and Dasatinib in vitro. Mechanistically, combined treatments exert synergistic anti-tumor effects in all cell lines through growth inhibitory effects, direct induction of cell death by promotion of caspase-dependent apoptosis and mitochondrial depolarization. DisclosuresNo relevant conflicts of interest to declare.

HMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia

hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase–PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1–MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells.

Combination effects of arsenic trioxide and fibroblast growth factor receptor inhibitor in squamous cell lung carcinoma

ObjectivesLung cancer remains the top cancer killer worldwide, with squamous cell carcinoma (SCC) as the second commonest histologic subtype. Arsenic trioxide (ATO) was previously shown to suppress growth of lung cancer. Fibroblast growth factor receptor (FGFR) amplification was recently demonstrated in lung SCC, with specific FGFR inhibitor (e.g. PD173074) developed as a potential targeted therapy. Therefore the combination effects of ATO and PD173074 in SCC was studied. Materials and methodsThe combination of ATO/PD173074 was studied in a proof-of-principle model using a lung SCC cell line with FGFR1 overexpression: SK-MES-1. The effects of ATO and/or PD173074 on cell viability and protein expression were studied by MTT assay and Western blot respectively. Cell cycle analysis, phosphatidylserine externalization and mitochondrial membrane depolarization were monitored by flow cytometry. FGFR1 knockdown was performed with siRNAs. Proteasome inhibitor (MG-132) was used to study the degradation mechanism. In vivo effect of ATO and/or PD173074 was investigated using a nude mice xenograft model. ResultsCombined ATO/PD173074 reduced cell viability along with increased sub-G1 population, phosphatidylserine externalization and mitochondrial membrane depolarization more significantly than single treatments. Downregulation of FGFR1, p-Akt, Akt, p-Src, Src, p-c-Raf, c-Raf, Erk and survivin as well as upregulation of p-Erk and cleaved PARP were observed upon ATO and/or PD treatment. MG-132 partially reversed the degradation of Akt, Src, c-Raf and Erk induced by ATO/PD, suggestive of ubiquitin-independent proteasome-dependent degradation. However, the mechanism of FGFR1 downregulation remained unknown. Downregulation of FGFR1, Akt, Src, c-Raf and Erk as well as cleaved PARP elevation induced by ATO and/or PD were confirmed in vivo. ConclusionMassive protein degradation (FGFR1, Akt, Src, c-Raf and Erk) was induced by ATO and/or PD173074 treatment mainly mediated by activation of proteasomal degradation in SCC cell line SK-MES-1 in vitro and in vivo.

Pectin mediated gold nanoparticles induces apoptosis in mammary adenocarcinoma cell lines

Pectin and its several modified forms have shown remarkable impact in therapeutic use against various cancers. In the present study, pectin, an anionic polysaccharide isolated from Musa paradisiaca is employed for the synthesis of gold nanoparticles at ambient temperature conditions. The synthesized nanoparticles were characterized using microscopic and spectroscopic studies and its anti-cancer potential was evaluated in mammary adenocarcinoma cell lines MCF-7 and MDA-MB-231. Apoptosis induction was evident from increase in sub-G1 population studied using flow cytometry analysis. DNA damage followed by cell death in pectin mediated gold nanoparticles (p-GNPs) treated cells was confirmed by Comet assay. Uptake of p-GNPs by cancer cells (MCF-7 and MDA-MB-231) was analyzed using FE-SEM which revealed the presence of p-GNPs as aggregates over the surface of cells with loss in cellular integrity compared to control cells.

Inactivation of hypoxia-induced YAP by statins overcomes hypoxic resistance tosorafenib in hepatocellular carcinoma cells.

Sorafenib is a multikinase inhibitor used as a first-line treatment for advanced hepatocellular carcinoma (HCC), but it has shown modest to low response rates. The characteristic tumour hypoxia of advanced HCC maybe a major factor underlying hypoxia-mediated treatment failure. Thus, it is urgent to elucidate the mechanisms of hypoxia-mediated sorafenib resistance in HCC. In this study, we found that hypoxia induced the nuclear translocation of Yes associate-Protein (YAP) and the subsequent transactivation of target genes that promote cell survival and escape apoptosis, thereby leading to sorafenib resistance. Statins, the inhibitors of hydroxymethylglutaryl-CoA reductase, could ameliorate hypoxia-induced nuclear translocation of YAP and suppress mRNA levels of YAP target genes both in vivo and in vitro. Combined treatment of statins with sorafenib greatly rescued the loss of anti-proliferative effects of sorafenib under hypoxia and improved the inhibitory effects on HepG2 xenograft tumour growth, accompanied by enhanced apoptosis as evidenced by the increased sub-G1 population and PARP cleavage. The expression levels of YAP and its target genes were highly correlated with poor prognosis and predicted a high risk of HCC patients. These findings collectively suggest that statins utilization maybe a promising new strategy to counteract hypoxia-mediated resistance to sorafenib in HCC patients.

Induction of apoptosis and downregulation of ERα in DMBA-induced mammary gland tumors in Sprague-Dawley rats by synthetic 3,5-disubstituted isoxazole derivatives.

Isoxazole derivatives are an important group of chemotherapeutic prototypes. In the current study, we have synthesized few isoxazole derivatives and tested them for their antiproliferative properties in cancer cell lines such as MCF7 and HeLa. The lead compound, 3-(3,4-dimethoxyphenyl)-5-(thiophen-2-yl)isoxazole (2b), showed considerable inhibition of proliferation of MCF7 and HeLa cells with the IC50 values of 19.5 and 39.2µM, respectively. Cell cycle analyses and annexin-FITC staining in 2b-treated breast adenocarcinoma cells (MCF7) showed increased sub-G1 population and apoptosis. Furthermore, we tested the tumor inhibitory effect of 2b and estrogen receptor expression profile in DMBA-induced mammary tumors in Sprague-Dawley rats. The gross morphology of tumor studies was investigated by histopathology and ERα protein expression was evaluated by immunohistochemistry, which showed tumor regression and downregulation of ERα in tumor cells. The present results implicate that compound 2b could be used for the further derivatization for the treatment of breast cancer.

Cell cycle, apoptosis, cellular uptake and whole-transcriptome microarray gene expression analysis of HeLa cells treated with a ruthenium(II)-arene complex with an isoquinoline-3-carboxylic acid ligand

Ruthenium(II)-arene complexes are promising drug candidates for the therapy of solid tumors. In previous work, seven new compounds of the general formula [Ru(η6-p-cymene)(L1–7)Cl] were synthesized and characterized, of which the complex with L=isoquinoline-3-carboxylic acid (RuT7) was two times as active on HeLa cells compared to normal cell line MRC-5, as indicated by IC50 values determined after 48h of incubation (45.4±3.0 vs. 84.2±5.7μM, respectively). In the present study, cell cycle analysis of HeLa cells treated with RuT7 showed S phase arrest and an increase in sub-G1 population. The apoptotic potential of the title compound was confirmed with the Annexin V-FITC/PI assay together with a morphological evaluation of cells using fluorescent microscopy. Analysis of the intracellular accumulation of ruthenium showed 8.9ng Ru/106 cells after 6h of incubation. To gain further insight in the molecular mechanism of action of RuT7 on HeLa cells, a whole-transcriptome microarray gene expression analysis was performed. Analysis of functional categories and signaling and biochemical pathways associated with the response of HeLa cells to treatment with RuT7 showed that it leads the cells through the intrinsic (mitochondrial) apoptotic pathway, via indirect DNA damage due to the action of reactive oxygen species, and through direct DNA binding of RuT7. Statistical analysis for enrichment of gene sets associated with known drug-induced toxicities identified fewer associated toxicity profiles in RuT7-treated cells compared to cisplatin treatment. Altogether these results provide the basis for further development of RuT7 in animal and pre-clinical studies as a potential drug candidate.

The effects of the mycotoxin austdiol on cell cycle progression, cytotoxicity and genotoxicity in Chinese hamster ovary (CHO-K1) cells

Austdiol is a mycotoxin mainly produced byAspergillus ustus andMycoleptodiscus indicus. These fungi are found in rye, oats, barley, corn and feed grains; thus, as a potential contaminant of human food and animal feed, this mycotoxin is of great concern. As such, the elucidation of the cytotoxicity and mutagenicity of austdiol is important. In this study, austdiol was purified from a rice-oat solid medium culture ofM. indicus using chromatographic separation techniques. Chinese hamster ovary (CHO-K1) cells were then used to study the effect of austdiol on mammalian cell cycle, clonogenicity and DNA damage. Austdiol induced cell cycle arrest in G2/M phase, with a decreased S phase population and increased sub-G1 population. Austdiol also increased the polyploid population. These events resulted in cell death detected 7 days after treatment by clonogenic assay. DNA damage represents the main mechanism of action of austdiol, which induces DNA breaks and increases the frequency of micronuclei and nucleoplasmic bridges in binucleated cells in a CHO-K1 cell line. Moreover, cells exposed to austdiol and doxorubicin (DXR) combined treatments presented a reduced number of colonies and increased frequencies of micronuclei and nucleoplasmic bridges compared with negative control and cells treated with austdiol or DXR alone.

C-Jun N-terminal Kinase-Dependent Endoplasmic Reticulum Stress Pathway is Critically Involved in Arjunic Acid Induced Apoptosis in Non-Small Cell Lung Cancer Cells.

Though arjunic acid, a triterpene isolated from Terminalia arjuna, was known to have antioxidant, antiinflammatory, and cytotoxic effects, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the molecular antitumor mechanism of arjunic acid was examined in A549 and H460 non-small cell lung cancer (NSCLC) cells. Arjunic acid exerted cytotoxicity by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and significantly increased sub-G1 population in A549 and H460 cells by cell cycle analysis. Consistently, arjunic acid cleaved poly (ADP-ribose) polymerase (PARP), activated Bax, and phosphorylation of c-Jun N-terminal kinases (JNK), and also attenuated the expression of pro-caspase-3 and Bcl-2 in A549 and H460 cells. Furthermore, arjunic acid upregulated the expression of endoplasmic reticulum (ER) stress proteins such as IRE1 α, ATF4, p-eIF2α, and C/EBP homologous protein (CHOP) in A549 and H460 cells. Conversely, CHOP depletion attenuated the increase of sub-G1 population by arjunic acid, and also JNK inhibitor SP600125 blocked the cytotoxicity and upregulation of IRE1 α and CHOP induced by arjunic acid in A549 and H460 cells. Overall, our findings suggest that arjunic acid induces apoptosis in NSCLC cells via JNK mediated ER stress pathway as a potent chemotherapeutic agent for NSCLC.

Abstract B51: The pan deacetylase inhibitor panobinostat shows cytotoxic activity as monotherapy and synergism with topoisomerase inhibitors in cervical cancer cells

Abstract Cervical carcinoma a tumor related to high risk human papillomavirus (HPV) is one of the most common malignancies for women of reproductive age worldwide. It is established now that genetic alterations together with epigenetic changes are associated with cancer progression. Epigenetic alterations have been identified both in the genome of host cell and HPV during cancer progression. The use of epigenetic drugs, methylation inhibitors and/or histone deacetylases inhibitors (HDACi) for cancer treatment, therefore utilizes the reversible nature of epigenetic aberrations, which would re-expresses the silenced tumor suppressor genes and consequently exert their anticancer effects. Studies showing the aberrant recruitment and/or over-expression of HDACs in many human cancers, and promising anticancer results through induction of cell cycle arrest, apoptosis and autophagy with HDACi have been reported. Combination of HDACi with other anticancer drugs that target genomic DNA have shown synergistic effects and enhanced apoptosis because of the increased accessibility of these agents to the chromatin. Panobinosat (LBH589), is a non-selective HDACi, showing anticancer activity against both hematological and solid malignancies. However the anticancer effect of panobinostat in cervical cancer cells has not been tested. Therefore, in the present study, we investigated the effects of panobinostat on cell growth and death in human cervical cells alone and in combination with topoisomerase inhibitors. HeLa and SiHa cells were selected for this study, they represents useful cellular models for cervical carcinoma, as both lines contain an integrated form of HPV 18 and HPV 16, respectively. MTT assay was used to evaluate the effect of panobinostat, cells were incubated with escalating concentration of panobinostat for 24, 48 and 72 hours. The results showed time and dose dependent decrease in cell proliferation of both HeLa and SiHa cells. The data also indicate that panobinostat is more potent in SiHa than in HeLa cells for inhibiting cell proliferation. The IC50 at 72 h in SiHa cells is approximately 50 nM and for HeLa cells is approximately 150 nM. Cell cycle analysis, revealed G1 arrest in HeLa cells but G2-M arrest in SiHa cells after 24 h of treatment. However increase in sub-G1 population was significant in both cell lines. These results demonstrate that panobinostat exerts cytotoxic effects on cervical cancer cells. Next we determined the combined anticancer effect between panobinostat and topoisomerase I and II inhibitors (topotecan and etoposide) using Chou and Talalay combination index (CI). Three different treatment schedules were explored, either simultaneous or sequential (with 24 h delay between the two agents). It was found that simultaneous treatment was synergistic for panobinostat and topotecan/etoposide combination in both cell lines. Flow cytometry analysis showed that topoisomerase inhibitors induced G2+M arrest, and the sub-G1 population was markedly increased in combination in comparison to single agents. Annexin V-FITC and PI double staining and DNA fragmentation showed enhanced cell death in combination treatment and demonstrated that apoptosis was the main mechanism for the observed synergistic interaction. Induction of apoptosis was found to be correlated to enhanced ROS levels in combination treatment. Furthermore, coadministration of antioxidant L-NAC substantially blocked the panobinostat and topotecan/etoposide mediated ROS levels. In conclusion, to our knowledge, this study is the first to show, the antiproliferative effect of panobinostat, and a synergistic cytotoxicity effect between panobinostat and topoisomerase inhibitors in a cervical cancer cell model. Citation Format: Lubna Wasim, Madhu Chopra. The pan deacetylase inhibitor panobinostat shows cytotoxic activity as monotherapy and synergism with topoisomerase inhibitors in cervical cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B51.

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

Diacerein-mediated inhibition of IL-6/IL-6R signaling induces apoptotic effects on breast cancer.

Interleukin-6 (IL-6) signaling network has been implicated in oncogenic transformations making it attractive target for the discovery of novel cancer therapeutics. In this study, potent antiproliferative and apoptotic effect of diacerein were observed against breast cancer. In vitro apoptosis was induced by this drug in breast cancer cells as verified by increased sub-G1 population, LIVE/DEAD assay, cell cytotoxicity and presence of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, as well as downregulation of antiapoptotic proteins Bcl-2 and Bcl-xL and upregulation of apoptotic protein Bax. In addition, apoptosis induction was found to be caspase dependent. Further molecular investigations indicated that diacerein instigated apoptosis was associated with inhibition of IL-6/IL-6R autocrine signaling axis. Suppression of STAT3, MAPK and Akt pathways were also observed as a consequence of diacerein-mediated upstream inhibition of IL-6/IL-6R. Fluorescence study and western blot analysis revealed cytosolic accumulation of STAT3 in diacerein-treated cells. The docking study showed diacerein/IL-6R interaction that was further validated by competitive binding assay and isothermal titration calorimetry. Most interestingly, it was found that diacerein considerably suppressed tumor growth in MDA-MB-231 xenograft model. The in vivo antitumor effect was correlated with decreased proliferation (Ki-67), increased apoptosis (TUNEL) and inhibition of IL-6/IL-6R-mediated STAT3, MAPK and Akt pathway in tumor remnants. Taken together, diacerein offered a novel blueprint for cancer therapy by hampering IL-6/IL-6R/STAT3/MAPK/Akt network.

MTR-10PHARMACOLOGICAL NOTCH BLOCKADE IN GLIOMAS INDUCES AUTOPHAGY AND COMBINATION TREATMENT WITH AN AUTOPHAGY INHIBITOR INCREASES TUMOR CELL DEATH

BACKGROUND: Induction of autophagy has been identified as a potential mechanism for chemotherapeutic resistance, and its manipulation has increasingly been explored as a treatment strategy in many cancers. However, there is minimal data on the effects of the Notch pathway on autophagy in cancer, and none in gliomas. We examined if Notch pathway blockade using a gamma-secretase inhibitor (GSI) MRK003 would induce autophagy in glioma neurosphere lines and studied the effects of chloroquine (CQ), a late-stage autophagy inhibitor, in combination with the GSI. METHODS: 500,000 cells from 2 neurosphere lines (JHH520 and HSR-GBM1), maintained in Neural Stem Cell medium with EGF and FGF, were treated with DMSO, 0.5-5 µM levels of MRK003, and/or 10 µM CQ for 24-72 hours. Western blotting was performed to detect alterations in protein levels of LC3B, pAkt, Akt, p62 and cleaved PARP. MTS, Ki-67, Annexin V, cell cycle and soft agar assays were performed to look at the combinatorial effects of MRK003 and CQ. RESULTS: An LC3B-II band, suggesting induction of autophagy, was observed after Notch pathway blockade in a dose dependent manner in the 2 neurosphere lines. Reduction of pAkt was seen in GBM1, suggesting mTORC2 inhibition. Decreased growth (MTS assay both p <0.001), proliferation (Ki-67 assay: both p <0.05), increased apoptosis (increased sub-G1 population: JHH520 p <0.005, HSR-GBM1 p <0.05; increased Annexin V percentage: JHH520 p <0.05, HSR-GBM1 p <0.01, increased cleaved PARP protein expression), and decreased clonogenicity (soft agar assay) were seen after combination MRK003 and CQ treatment compared to DMSO. CONCLUSIONS: Induction of autophagy, possibly through mTORC2 inhibition, occurs after Notch inhibition in glioma neurospheres using a GSI. Inhibition of autophagy using CQ caused increased cytotoxicity. These results suggest a possible resistance mechanism to Notch inhibition in malignant gliomas and pave the way for a new treatment strategy.

Anticancer Effects of Curcuma C20-Dialdehyde against Colon and Cervical Cancer Cell Lines.

Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

Abstract 4556: Identification of novel agent for HCC treatment by GNMT-promoter orientated cell-based drug screening platform

Abstract Glycine N-methyltransferase (GNMT) a tumor suppressor gene for hepatocellular carcinoma (HCC) plays very important role in liver detoxification and tumorigenisis. GNMT expression is highly down-regulated in HCC. Recent studies have shown that overexpression of GNMT reduce tumor growth and sensitize HuH-7 cells to rapamycin treatment in vitro and in vivo. Therefore, enhancing GNMT expression is an attractive approach for HCC therapy. In present study we developed a GNMT-promoter driven luciferase reporter assay as a drug screen platform for the identification of novel drugs for prevention and treatment of HCC. We identified crude extract 317 as highest activator of GNMT promoter activity from a Chinese medicine library containing 472 herbal extracts. Partial purified fraction “F2-6” was isolated by bioassay-guided fractionation from crude extract 317 and validated for enhancement of endogenous GNMT expression at both mRNA and protein levels. Fraction F2-6 inhibited growth of liver cancer cell lines in vitro. Moreover, orally administrated F2-6 can delay and reduce growth of HCC xenograft tumor in vivo. Next, we identified the active component in fraction F2-6 named as compound P. Furthermore, we demonstrated that combination of sorafenib with compound P led to additive toxicity on HCC cells in vitro and in vivo. The cDNA microarray and real time PCR analysis showed that compound P altered cell cycle-associated genes such as CDK6, CCND1, CDKN1C, MYC and PLK1. Finally, Compound P treatment induced apoptosis in huh7 cells were confirmed by increased sub-G1 population and caspase 3/7 activity. Collectively, our results suggest that compound P has potential to be a novel agent for prevention and treatment of HCC. Citation Format: Rajni Kant, Lucas Tung, Chung-Kuang Lu, Yi-Ming Chen, Chia-Hung Yen. Identification of novel agent for HCC treatment by GNMT-promoter orientated cell-based drug screening platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2015-4556

Abstract 5375: Combined treatment of trichostatin A enhances cytotoxic effects of sunitinib on renal cell carcinoma cells

Abstract Introduction: Sunitinib (SU) is one of the multi-targeted tyrosine kinase inhibitors (TKIs) and currently approved for use in advanced renal cell carcinoma (RCC) by inhibition of angiogenic signaling. Although it is a really attractive drug for patients who cannot have surgical treatment, SU treatment hasn't been always successful because of frequent resistance and severe side effects. Combination therapy is one of the strategies to solve these problems. The combinations of SU and IFN-α or mTOR inhibitors in the treatment of RCC have been already explored. However, both of which resulted in failure due to dose-limiting toxicities. Therefore, it is important to establish a novel combination strategy for RCC treatment. In the context of seeking for a prominent combination agent for SU, we paid attention to epigenetic agents. There has been several reports where epigenetic agents were proven to induce apoptotic cells in combination with chemotherapeutic agents, such as gemcitabine and sorafenib. To investigate whether epigenetic agents could be a sensitizer of SU as well in RCC cells, we used trichostain A (TSA), a well-known and strong inhibitor of histone deacetylase (HDAC), and also examined what the mechanism is. Methods: We used 786-O, ACHN and Caki-1 RCC cell lines. The cells were treated with SU alone, TSA alone or the combination, then cytotoxicity was measured by the MTT cytotoxicity method under normoxic or hypoxic conditions. To explore the effect on cell cycle after the combination treatment, flowcytometry was performed. The expression of RTK signaling proteins and p21, which is one of the major targets of TSA, were detected by western blotting. Results: TSA significantly enhanced SU cytotoxicity in all of RCC cells, especially in 786-O. VEGF protein expression, a major angiogenic factor showed increasing trend in 786-O after 8 h of hypoxic exposure, which was decreased by the combined treatment. Flowctometry has revealed that suggested apoptotic cell population (sub-G1) was significantly higher in 1 μM SU and 0.5 μM TSA combination group compared with control or single SU treatment group. On the other hand, ACHN cells didn't show significant increase of sub-G1 population although cell cycle-arrest at S and G2/M phase was observed in combined treatment group (2.5 μM SU and 0.25 μM TSA). Additionally, cell cycle regulator protein, p21 was significantly increased in both 786-O and ACHN cells in TSA concentration dependent manner, which was accompanied with increase of acetylated histone H3. Conclusion: These findings suggest that combined treatment of SU and TSA is effective against RCC cells. The combination possibly enhances apoptosis or growth inhibition, where increase of p21 by TSA might be involved. Citation Format: Hiromi Sato, Tatsuro Kashiba, Miaki Uzu, Takuya Fujiwara, Yukihiro Shibata, Rina Suzuki, Katsunori Yamaura, Akihiro Hisaka. Combined treatment of trichostatin A enhances cytotoxic effects of sunitinib on renal cell carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5375. doi:10.1158/1538-7445.AM2015-5375

Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observed in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER+ and ER− breast cancer cell lines.