Phase Arrest In Cells Research Articles

Alisol B 23-acetate induces autophagic-dependent apoptosis in human colon cancer cells via ROS generation and JNK activation.

Alisol B 23-acetate (AB23A), a natural triterpenoid from the rhizome of Alisma orientale, a Chinese medicinal herb, has multiple physiological activities including anticancer. However, its effect on human colon cancer and the underlying mechanism are not clear. Here, we reported for the first time that AB23A induced cell cycle G1 phase arrest and apoptotic cell death in colon cancer cells. Autophagy also occurred in AB23A-treated HCT116 cells as evidenced by the accumulation of microtubule-associated protein 1 light chain 3 form II (LC3-II) and degradation of SQSTM1/p62. An autophagy inhibitor, 3-methyladenine (3-MA) was found to attenuate AB23A-mediated autophagy, apoptosis, and cell death, indicating that AB23A-induced apoptotic response was dependent on the induction of autophagy. In addition, the treatment of HCT116 cells with AB23A resulted in the generation of reactive oxygen species (ROS) and phosphorylation of c-Jun N-terminal kinase (JNK). A ROS scavenger, N-acetylcysteine (NAC) and a JNK-specific inhibitor, SP600125 attenuated AB23A-induced autophagy and apoptotic cell death. Moreover, NAC was able to eliminate AB23A-induced JNK phosphorylation. This finding provides a novel mechanism of action of AB23A in colon cancer HCT116 cells that AB23A induces autophagic-dependent apoptotic cell death in colon cancer cells, at least in part, though the accumulation of intracellular ROS and subsequent activation of JNK.

Polyethylenimine-dermatan sulfate complex, a bioactive biomaterial with unique toxicity to CD146-positive cancer cells.

We report unique bioactivity of a polycation-polyanion complex with potential utility for cancer therapy. A complex of disulfide-crosslinked polyethyleneimine (CLPEI), a polycation used for gene complexation, and dermatan sulfate (DS), an anionic polysaccharide to shield excessive cationic charge of the former, has toxicity to a specific group of cancer cell lines, including B16-F10 murine melanoma, A375SM human melanoma, and PC-3 human prostate cancer cells. These CLPEI-DS-sensitive cells express CD146, which binds to the complex via interaction with DS. There is a positive correlation between toxicity and intracellular level of CLPEI, indicating that the CLPEI-DS-sensitivity is attributable to the increased cellular uptake of CLPEI mediated by the DS-CD146 interactions. In vitro studies show that CLPEI-DS complex causes G0/G1 phase arrest and apoptotic cell death. In syngeneic and allograft models of B16-F10 melanoma, CLPEI-DS complex administered with a sub-toxic level of doxorubicin potentiates the chemotherapeutic effect of the drug by loosening tumor tissues. Given the unique toxicity, CLPEI-DS complex may be a useful carrier of gene or chemotherapeutics for the therapy of CD146-positive cancers.

Hydroquinone induces TK6 cell growth arrest and apoptosis through PARP-1/p53 regulatory pathway.

Hydroquinone (HQ), one of the most important metabolites derived from benzene, induces cell cycle arrest and apoptosis. Poly(ADP-ribose) polymerase-1 (PARP-1) participates in various biological processes, including DNA repair and cell cycle regulation. To explore whether PARP-1 regulatory pathway mediated HQ-induced cell cycle arrest and apoptosis, we assessed the effect of PARP-1 suppression on induction of apoptosis analyzed by FACSCalibur flow cytometer in PARP-1 deficientTK6 cells (TK6-shPARP-1). We observed an increase in the fraction of cells in G1 phase by 7.6% and increased apoptosis by 4.5% in PARP-1-deficient TK6 cells (TK6-shPARP-1) compared to those negative control cells (TK6-shNC cells) in response to HQ treatment. Furthermore, HQ might activate the extrinsic pathways of apoptosis via up-regulation of Fas expression, followed by caspase-3 activation, apoptotic body, and sub G1 accumulation. Enhanced p53 expression was observed in TK6-shPARP-1 cells than in TK6-shNC cells after HQ treatment. In contrast, Fas expression was lower in TK6-shPARP-1 cells than in TK6-shNC cells. Therefore, we conclude that HQ may activate apoptotic signals via Fas up-regulation and p53-mediated apoptosis in TK6-shNC cells. The reduction of PARP-1 expression further intensified up-regulation of p53 in TK6-shPARP-1 cells, resulting in an increased G1→S phase cell arrest and apoptosis in TK6-shPARP-1 cells compared to TK6-shNC cells.

Dihydroorotate dehydrogenase (DHODH) inhibitors affect ATP depletion, endogenous ROS and mediate S-phase arrest in breast cancer cells

Dihydroorotate dehydrogenase (DHODH) is the key enzyme in de novo biosynthesis of pyrimidine in both prokaryotes and eukaryotes. The de novo pathway of pyrimidine biosynthesis is essential in cancer cells proliferation. Leflunomide is an approved DHODH inhibitor that has been widely used for the treatment of arthritis. Similarly, brequinar sodium is another DHODH inhibitor that showed anti-tumour effect in MC38 colon carcinoma cells when used in combination with fluorouracil. Despite the potential role of DHODH inhibitors in cancer therapy, their mechanisms of action remain obscure and await further elucidation. Here, we evaluated the effect of DHODH inhibitors on the production of ATP and ROS in sensitive and non-sensitive breast cancer cells. Subsequently, the effects of DHODH inhibitors on cell cycle as well as on signalling molecules such as p53, p65 and STAT6 were evaluated in sensitive T-47D and non-sensitive MDAMB-436 cells. The correlations between DHODH protein expression, proliferation speed and sensitivity to DHODH inhibitors were also investigated in a panel of cancer cell lines. DHODH inhibitors-sensitive T-47D and MDAMB-231 cells appeared to preserve ROS production closely to endogenous ROS level whereas the opposite was observed in non-sensitive MDAMB-436 and W3.006 cells. In addition, we observed approximately 90% of intracellular ATP depletion in highly sensitive T-47D and MDAMB-231 cells compared to non-sensitive MDAMB-436 cells. There was significant over-expression of p53, p65 and STAT6 signalling molecules in sensitive cells which may be involved in mediating the S-phase arrest in cell cycle progression. The current study suggests that DHODH inhibitors are most effective in cells that express high levels of DHODH enzyme. The inhibition of cell proliferation by these inhibitors appears to be accompanied by ROS production as well as ATP depletion. The increase in expression of signalling molecules observed may be due to pyrimidine depletion which subsequently leads to cell cycle arrest at S-phase.

Binding, sliding, and function of cohesin during transcriptional activation

The ring-shaped cohesin complex orchestrates long-range DNA interactions to mediate sister chromatid cohesion and other aspects of chromosome structure and function. In the yeast Saccharomyces cerevisiae, the complex binds discrete sites along chromosomes, including positions within and around genes. Transcriptional activity redistributes the complex to the 3' ends of convergently oriented gene pairs. Despite the wealth of information about where cohesin binds, little is known about cohesion at individual chromosomal binding sites and how transcription affects cohesion when cohesin complexes redistribute. In this study, we generated extrachromosomal DNA circles to study cohesion in response to transcriptional induction of a model gene, URA3. Functional cohesin complexes loaded onto the locus via a poly(dA:dT) tract in the gene promoter and mediated cohesion before induction. Upon transcription, the fate of these complexes depended on whether the DNA was circular or not. When gene activation occurred before DNA circularization, cohesion was lost. When activation occurred after DNA circularization, cohesion persisted. The presence of a convergently oriented gene also prevented transcription-driven loss of functional cohesin complexes, at least in M phase-arrested cells. The results are consistent with cohesin binding chromatin in a topological embrace and with transcription mobilizing functional complexes by sliding them along DNA.

CCY-1a-E2 induces G2/M phase arrest and apoptotic cell death in HL-60 leukemia cells through cyclin-dependent kinase 1 signaling and the mitochondria-dependent caspase pathway

Our previous study demonstrated that 2-[(3-methoxybenzyl)oxy]benzaldehyde(CCY-1a-E2) is a potent compound that acts against multiple human leukemia cell lines. CCY-1a-E2 was also shown to have efficacious anti‑leukemic activityinvivo. However, the molecular mechanism of action of CCY‑1a‑E2 attributed to its anticancer effect remains poorly understood. In the present study, CCY‑1a‑E2 suppressed cell viability in multiple leukemia cell lines (HL‑60, K562, KG‑1 and KG‑1a) via inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. CCY‑1a‑E2 exhibited a marked toxic effect on HL‑60 cells and displayed low cytotoxicity in normal human peripheral blood mononuclear cells(PBMCs). Results from flow cytometric analysis indicated that CCY‑1a‑E2 promoted G2/M phase arrest and promoted apoptosis in the HL‑60 cells. CCY‑1a‑E2 treatment upregulated cyclinB, cyclin‑dependent kinase1(CDK1), cell division cycle25C(cdc25C) and p21 protein expression. CCY‑1a‑E2 caused apoptotic cell death and DNA fragmentation as determined by 4',6‑diamidino‑2‑phenylindole(DAPI) staining and DNA gel electrophoresis. Elevated activities of caspase‑8, ‑9 and‑3 were observed during CCY‑1a‑E2‑induced cell apoptosis; their specific inhibitors were found to block CCY‑1a‑E2‑induced apoptosis, respectively. Moreover, CCY‑1a‑E2 time‑dependently disrupted the mitochondrial membrane potential(ΔΨm), and it enhanced the protein levels of Fas/CD95, cytochromec, Bax, cleaved PARP, aswellas attenuated Bcl‑2 expression in the HL‑60 cells. Our results provide direct evidence that supports the future potential therapeutic application of CCY-1a-E2 in leukemia.

Abstract 2065: Anti-FSHR antibody Fab’ fragment conjugated immunoliposomes loaded with cyclodextrin-paclitaxel complex for improved in vitro efficacy on ovarian cancer cells

Abstract Purpose of the study was to prepare and characterize cyclodextrin-paclitaxel (CD-PTX) complex encapsulated immunoliposomes (ILs) conjugated with Fab’ fragment of anti-FSHR antibody and to determine their in vitro efficacy for cellular uptake, cytotoxicity, anti-metastatic activity and cell apoptosis on ovarian cancer (OC) cells. CD-PTX complex was formulated using Heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) to improve aqueous solubility and liposomal loading of PTX. The complex was encapsulated in PEGylated liposomes (PLs) using reverse phase evaporation technique. ILs were prepared by covalent conjugation of Fab’ fragments of anti-FSHR antibody to functionalized PLs via thioether linkage which was confirmed by SDS-PAGE analysis. The ILs were characterized for size, zeta potential, entrapment, loading efficiency and in vitro drug release profile. ILs were also evaluated for uptake efficiency and in vitro cytotoxicity in FSHR-expressing Caov-3 OC cells. To establish anti-metastatic effect, the ILs, PLs and equal amounts of PTX solution were studied on Caov-3 cells by in vitro wound scratch assay. The effect of PTX solution, PLs and ILs on Caov-3 cell apoptosis and cell cycle was examined using FACS technique. Increased aqueous solubility of 11 mg/ml was achieved for PTX using DMBCD at molar ratio of 20 and the cytotoxic properties of CD-PTX complex was also retained similar to PTX as studied in Caov-3 cells. The PLs and ILs were found to be nanosized with optimum entrapment and improved loading efficiency of PTX. The in vitro release profiles exhibited controlled release of PTX from PLs (21.235±0.423%) and ILs (18.219±0.603%) after completion of 48 h. The IC50 values from MTT assay suggested that ILs was 3.7 and 8.1 times more cytotoxic than PLs and PTX solution respectively for Caov-3 cells after 24 h period. The ILs showed more anti-migration effect and less covered wound (39.66±5.2%) at a concentration of 3nM as compared with PTX solution (70.45±7.12%) and PLs (61.38±4.41%). After 24 h treatment no major distinction in% apoptosis was observed between PTX solution, PLs and ILs. However, after 48 h treatment, PLs and ILs respectively showed about 5.36% and 9.12% higher apoptosis when compared to PTX solution. In Caov-3 cells, after 48 h the ILs at 3nM concentration showed increased G2 phase cell arrest as compared to PLs which indicates the superiority of ILs. To conclude, ILs displayed improved intracellular uptake and enhanced cytotoxic as well as anti-metastatic effects compared to PTX solution and PLs in OC cells. The results clearly highlight the importance of FSHR as one of the prominent targets for OC therapy and also the potential of anti-FSHR Ab Fab’ conjugated nanocarriers to reduce the limitations associated with OC chemotherapy. Citation Format: Priyanka Bhatt, Rohan Lalani, Rajashree Mashru, Ambikanandan Misra. Anti-FSHR antibody Fab’ fragment conjugated immunoliposomes loaded with cyclodextrin-paclitaxel complex for improved in vitro efficacy on ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2065.

Over-expressed human TREK-1 inhibits CHO cell proliferation via inhibiting PKA and p38 MAPK pathways and subsequently inducing G1 arrest.

Recent studies have shown that the two-pore-domain potassium channel TREK-1 is involved in the proliferation of neural stem cells, astrocytes and human osteoblasts. In this study, we investigated how TREK-1 affected the proliferation of Chinese hamster ovary (CHO) cells in vitro. A CHO cell line stably expressing hTREK-1 (CHO/hTREK-1 cells) was generated. TREK-1 channel currents in the cells were recorded using whole-cell voltage-clamp recording. The cell cycle distribution was assessed using flow cytometry analysis. The expression of major signaling proteins involved was detected with Western blotting. CHO/hTREK-1 cells had a high level of TREK-1 expression, reached up to 320%±16% compared to the control cells. Application of arachidonic acid (10 μmol/L), chloroform (1 mmol/L) or etomidate (10 μmol/L) substantially increased TREK-1 channel currents in CHO/hTREK-1 cells. Overexpression of TREK-1 caused CHO cells arresting at the G1 phase, and significantly decreased the expression of cyclin D1. The TREK-1 inhibitor l-butylphthalide (1-100 μmol/L) dose-dependently attenuated TREK-1-induced G1 phase cell arrest. Moreover, overexpression of TREK-1 significantly decreased the phosphorylation of Akt (S473), glycogen synthase kinase-3β (S9) and cAMP response element-binding protein (CREB, S133), enhanced the phosphorylation of p38 (T180/Y182), but did not alter the phosphorylation and expression of signal transducer and activator of transcription 3 (STAT3). TREK-1 overexpression suppresses CHO cell proliferation by inhibiting the activity of PKA and p38/MAPK signaling pathways and subsequently inducing G1 phase cell arrest.

The Mechanism in Gastric Cancer Chemoprevention by Allicin.

Gastric cancer remains high prevalence and fatality rates in China even though its morbidity has been decreased drastically. Allicin, which is from an assistance food-garlic (Allium Sativum L), was found to be effective in gastric cancer treatment. It is a defensive substance with a board biological properties: inhibition of bacteria, fungus, virus, controlled hypertension, diabetes, and chemoprevention of several cancers, etc. Experiments have shown that allicin can be chemopreventive to gastric cancer by inhibiting the growth of cancer cells, arresting cell cycle at G2/M phase, endoplasmic reticulum (ER) stress, and mitochondria-mediated apoptosis, which includes the caspase-dependent/-independent pathways and death receptor pathway. Those mechanisms probably involve in modulating enzymatic activity, restraining DNA formation, scavenging free radicals, and affecting cell proliferation and even tumor growth. Therefore, this review is focus on the mechanism of allicin in gastric cancer.

KLF8 knockdown triggered growth inhibition and induced cell phase arrest in human pancreatic cancer cells

BackgroundThe transcription factor Krüppel-like factor 8 (KLF8) plays an important role in tumor development and growth, but its role in pancreatic cancer (PC) is not clear. MethodsKLF8 expression in human PC cell lines and tumor tissues was measured by quantitative real-time polymerase chain reaction and Western blot analyses. The effects of lentivirus mediated knockdown of KLF8 on proliferation and growth in Panc-1 pancreatic cancer cells were examined. ResultsKLF8 was overexpressed in 5 pancreatic cancer cell lines and in samples from patients with PC. In Panc-1 cells, KLF8 knockdown inhibited cell proliferation, tumorigenicity, and induced G2/M phase arrest. KLF8 knockdown suppressed PC tumor growth in nude mice model. Western blot analysis showed that KLF8 knockdown in Panc-1 cells down-regulated the expression of CDK1/CDC2, cyclin B1, and cyclin D1 and up-regulated the expression of p21, and p27. ConclusionsOverexpression of KLF8 may contribute to the progression of pancreatic cancer, and downregulation of KLF8 expression by lentivirus-delivered shRNA is a novel therapeutic approach for PC.

Biofunctionalization of Selenium Nanoparticle with Dictyophora Indusiata Polysaccharide and Its Antiproliferative Activity through Death-Receptor and Mitochondria-Mediated Apoptotic Pathways.

Bio-functionalized nanoparticles with semiconducting/metallic core encapsulated in a bio- or bio-derived materials are promising for applications in biology and especially in cancer diagnostic and healing. In this report, we report a facile, single-step, first-time synthesis and in-situ functionalization strategy for the preparation of monodispersed selenium nanoparticles (SeNPs) functionalized using a novel polysaccharide (DP1) extracted from Dictyophora indusiata (a fungus). The DP1 functionalized SeNPs (DP1-SeNPs), where DP1 is attached to the surface via Se-O bond as well as physic-sorption had, an average diameter of 89 nm, and were highly uniform, extremely stable compared to bare SeNPs. Detailed investigation of the biological properties of DP1-SeNP illustrated that they exhibit unprecedented, enhanced, and selective antiproliferative activity through inducing cell apoptosis confirmed by nuclear condensation, DNA cleavage, and accumulation of S phase cell arrest. The mechanism of the induced apoptosis was found to be a combination of the activation of caspases 3, 8, and 9, the Fas-associated death domain protein (FADD), reactive oxygen species (ROS) overproduction, as well as mitochondrial dysfunction. It is envisioned that the reported DP1-SeNPs will offer a new phase space for high-efficiency anticancer treatment with little side effect.

Chelidonine induces mitotic slippage and apoptotic-like death in SGC-7901 human gastric carcinoma cells.

The aim of the present study was to investigate the effect of chelidonine on mitotic slippage and apoptotic-like death in SGC-7901 human gastric cancer cells. The MTT assay was performed to detect the antiproliferative effect of chelidonine. Following treatment with chelidonine (10 µmol/l), the ultrastructure changes in SGC-7901, MCF-7 and HepG2 cells were observed by transmission electron microscopy. The effects of chelidonine on G2/M phase arrest and apoptosis of SGC-7901 cells were determined by flow cytometry. Indirect immunofluorescence assay and laser scanning confocal microscopy (LSCM) were used to detect the phosphorylation level of histone H3 (Ser10) and microtubule formation was detected using LSCM following immunofluorescent labeling. Subsequent to treatment with chelidonine (10 µmol/l), expression levels of mitotic slippage-associated proteins, including BUB1 mitotic checkpoint serine/threonine kinase B (BubR1), cyclin-dependent kinase 1 (Cdk1) and cyclin B1, and apoptosis-associated protein, caspase-3 were examined by western blotting at 24, 48 and 72 h. The half maximal inhibitory concentration of chelidonine was 23.13 µmol/l over 48 h and chelidonine induced G2/M phase arrest of cells. The phosphorylation of histone H3 at Ser10 was significantly increased following treatment with chelidonine for 24 h, indicating that chelidonine arrested the SGC-7901 cells in the M phase. Chelidonine inhibited microtubule polymerization, destroyed microtubule structures and induced cell cycle arrest in the M phase. Giant cells were observed with multiple micronuclei of varying sizes, which indicated that following a prolonged arrest in the M phase, the cells underwent mitotic catastrophe. Western blotting demonstrated that the protein expression levels of BubR1, cyclin B1 and Cdk1 decreased significantly between 48 and 72 h. Low expression levels of BubR1 and inactivation of the cyclin B1-Cdk1 complex results in the cells being arrested at mitosis and leads to mitotic slippage. In addition, apoptotic morphological changes in multinucleated cells were observed, the apoptosis rates increased gradually with administration of chelidonine in a time-dependent manner and the protein levels of caspase-3 increased significantly between 24 and 72 h. Thus, chelidonine induces mitotic slippage, and apoptotic-like death occurs in SGC-7901 cells undergoing mitotic catastrophe. Gastric cancer is a common malignancy, and ranks second in overall cancer-associated mortalities worldwide. The present study demonstrated that chelidonine induces M phase arrest and mitotic slippage of SGC-7901 human gastric carcinoma cells via downregulating the expression of BubR1, Cdk1 and cyclin B1 proteins. With the prolongation of chelidonine treatment, the giant cells with multiple micronuclei underwent mitotic slippage and were maintained in the G1 phase and did not survive. A number of multinucleated cells underwent apoptosis via a caspase-dependent signaling pathway. The current study proposes that chelidonine induces mitotic slippage and apoptotic-like death of SGC-7901 cells.

A Novel Antisense Long Noncoding RNA Modulates NR4A1 Protein Level in Myeloid Malignancies

Introduction: The advent of whole transcriptome sequencing technologies revealed that most of the human genome is transcribed, producing a large repertoire of long noncoding RNAs (lncRNAs). They are known to participate in epigenetic regulation at different levels and alter mRNA stability of coding genes, emerging as key players in carcinogenesis. In the present study, we identified a novel unspliced long noncoding RNA that is transcribed from the opposite strand on the NR4A1 gene locus. The tumor suppressor NR4A1 has been associated with the regulation of apoptosis and proliferation in various tumors and shows a reduced expression in myelodysplastic syndrome (MDS). Additionally, the abrogation of this locus in murine models caused the development of acute myeloid leukemia (AML). We hypothesized that lncNR4A1 plays an important role in hematopoietic malignancy pathogenesis and possibly regulates its coding counterpart.Methods: Expression of lncNR4A1 and its sense gene NR4A1 were evaluated in CD34+ cells from bone marrow of normal donors (n=7) and patients (n= 27; MDS= 14 [low‐risk=8, high‐risk=6], AML with myelodysplastic related changes [AML‐MRC]=4 and de novo AML=9) by qRT-PCR. Results were expressed as mean (minimum-maximum) after appropriate statistical analysis. The orientation and full-length of lncNR4A1 were confirmed by strandspecific RT-PCR, RACE-PCR and sequencing techniques. For functional analysis, lncNR4A1 was inhibited with a lentiviral vector system in U937 cells. Apoptosis was evaluated by Annexin‐V/PI and the cell cycle analysis was performed applying PI incorporation by flow cytometer analysis. Mitochondrial activity was evaluated with MTT assays. Western blot was used to measure the NR4A1 protein expression and for monitoring key proteins in the cell cycle signaling pathway.Results:NR4A1 mRNA was significantly decreased in bone marrow/CD34+ from patients with MDS and AML compared to healthy donors (p<0.05), consistent with previous studies. In contrast, the long noncoding expression was significantly increased in patients with low-risk MDS (4.60[0.20-10.05]) and high-risk MDS (8.40[2.54-16.87]; p=0.008) compared to healthy donors (1.22[0.02-4.11]). Next, we examined the pattern of expression of the non-coding transcript compared with NR4A1 gene expression from each MDS patients and healthy donors. Interestingly the results showed higher levels of noncoding expression when compared to NR4A1 gene expression in MDS patients, however, noncoding expression was less abundant compared with coding gene expression in all of healthy donors samples (low-risk MDS 3.55[-6.25 to 14.54]; p=0.020 and high-risk MDS 17.43[2.15 to 50.17] p=0.002; donors -13.06 [-33.3 to -3.22]). The strand-oriented RT-PCR show that lncNR4A1 is transcribed in the antisense direction relative to protein-coding mRNA encoded in NR4A1 locus in two different leukemia cell lines. Using a combined approach involving RACE with primer-walking PCR and sequencing, we extend the sequence resulting in a full-length lncNR4A1 transcript of 5581 nt, overlapping the exons 3,4,5 and 6 of the coding gene. The knockdown of lncNR4A1 with shRNA did not affect the abundance of NR4A1 mRNA, but unexpectedly increased the NR4A1 protein expression levels. Noncoding depletion reduced significantly the cell viability compared with control cells (p=0.007). In addition to reducing cell proliferation, lncNR4A1 sh-mediated depletion induced the increase on the sub-G1 percentage population cells with a subsequent decrease of DNA synthesis S phase (p=0.027) compared with control cells. Moreover, lncNR4A1 knockdown increased p21 protein levels which correlates with G1 phase cell arrest, and with blocking the S phase entrance and cell growth. We also detected a slight Cyclin A down-regulation after noncoding inhibition which explains the reduction of G1-S transition. No significant difference was found in cell death between sh-noncoding and control cells.Conclusions: Our results showed a post-transcriptional modulation of the NR4A1 by a novel long noncoding RNA and suggested a putative oncogenic role of lncNR4A1 in hematologic malignancy. Since NR4A1 is a potent tumor suppressor for leukemia and MDS, the inhibition of lncNR4A1 could emerge as a new therapeutic strategy by upregulation of NR4A1 protein. DisclosuresNo relevant conflicts of interest to declare.

Abstract B83: The histone deacetylase inhibitor, panobinostat (LBH589) exerts antiproliferative effect as single agent and synergizes with topotecan or etoposide in cervical cancer cells

Abstract Cervical carcinoma is one of the most common malignancies of women worldwide. Genetic alterations together with epigenetic changes have been identified both in the genome of host cell and human papillomavirus (HPV) during cervical cancer progression. Studies showing the aberrant recruitment and /or overexpression of histone deacetylases (HDACs) in many human cancers, and promising anticancer results through induction of cell cycle arrest, apoptosis and autophagy with HDAC inhibitors (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 nonselective 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. MTT assay was used to evaluate the antiproliferative effect of panobinostat on HeLa and SiHa cell lines. Flow cytometry was used for analyzing cell cycle kinetics using PI staining and apoptosis using annexin V-FITC. Agarose gel electrophoresis was done for DNA fragmentation. DCF-DA assay was used to measure ROS levels. Combination index (CI) of panobinostat with topoisomerase inhibitors was calculated at fixed ratio following three different treatment schedules, either simultaneous or sequential (with 24 h delay between the two agents) according to Chou and Talalay using CompuSyn software. The results showed that panobinostat is more potent in SiHa than in HeLa cells for inhibiting cell proliferation. Cell cycle analysis, revealed G1 arrest in HeLa cells but G2-M arrest in SiHa cells after 24 h of treatment. These results demonstrate that panobinostat exerts antiproliferative effects on cervical cancer cells. In combination it was found that simultaneous treatment was synergistic for panobinostat and topotecan/etoposide combination in both cell lines having CI&amp;lt;1. Topoisomerase inhibitors induced G2+M and late S phase arrest, in Hela and Siha cell lines, respectively. The hypodiploid sub-G1 population and annexin V-FITC and PI positive cells were markedly increased in combination in comparison to single agents. Clear ladder pattern was seen 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 histone deacetylase inhibitor, panobinostat (LBH589) exerts antiproliferative effect as single agent and synergizes with topotecan or etoposide in cervical cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B83.

PTEN regulates DNA replication progression and stalled fork recovery.

Faithful DNA replication is a cornerstone of genomic integrity. PTEN plays multiple roles in genome protection and tumor suppression. Here we report on the importance of PTEN in DNA replication. PTEN depletion leads to impairment of replication progression and stalled fork recovery, indicating an elevation of endogenous replication stress. Exogenous replication inhibition aggravates replication-originated DNA lesions without inducing S-phase arrest in cells lacking PTEN, representing replication stress tolerance. Our analysis reveals the physical association of PTEN with DNA replication forks and PTEN-dependent recruitment of Rad51. PTEN deletion results in Rad51 dissociation from replication forks. Stalled replication forks in Pten null cells can be reactivated by ectopic Rad51 or PTEN, the latter facilitating chromatin loading of Rad51. These data highlight the interplay of PTEN with Rad51 in promoting stalled fork restart. We propose that loss of PTEN may initiate a replication stress cascade that progressively deteriorates through the cell cycle.

The Effects of Morphology and Linker Length on the Properties of Peptide–Lanthanide Upconversion Nanomaterials as G2 Phase Cell Cycle Inhibitors

AbstractIn this work, the shape effect of our same‐sized hybrid nanomaterials conjugated with two different Plk1‐specific peptides had been investigated in terms of their photophysical properties, cellular uptake efficiencies, and selective inhibitory effects towards cancer and normal cells. The results clearly indicate that our spherical NaGdF4@SiO2‐P1 nanoparticles achieve the best performance for in vitro imaging, G2 phase cell arrest, and, hence, cell cycle inhibition.

MiR-128-3p suppresses hepatocellular carcinoma proliferation by regulating PIK3R1 and is correlated with the prognosis of HCC patients.

microRNAs (miRNAs) are known to be involved in the pathogenesis of hepatocellular carcinoma (HCC). miR-128-3p was recently reported to be deregulated in several types of cancer. However, the biological function and potential mechanisms of miR-128-3p in HCC remain unknown. In the present study, we found that miR-128-3p was frequently downregulated in HCC tissues and cell lines by qRT-PCR analysis. Moreover, functional assays showed that overexpression of miR-128-3p markedly suppressed HCC cell proliferation by inducing G1 phase cell arrest and migration. Mechanistically, miR-128-3p was confirmed to regulate PIK3R1 (p85α) expression thereby suppressing phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation using qRT-PCR and western blot analysis. Furthermore, correlation analysis and Kaplan-Meier estimates revealed an inverse correlation between miR-128-3p and p85α as well as a shorter disease-free survival (DFS) period after HCC resection in patients with low miR-128-3p expression. Hence, we conclude that miR-128-3p, which is frequently downregulated in HCC, inhibits HCC progression by regulating PIK3R1 and PI3K/AKT activation, and is a prognostic marker for HCC patients.

Distinct novel quinazolinone exhibits selective inhibition in MGC-803 cancer cells by dictating mutant p53 function

The mutant p53 proteins and their corresponding cellular response can be manipulated by novel quinazolinone derivatives 4–8 (a–i) in p53 mutant cancer cells. Of the two most potent compounds, 4a exhibited promising broad-spectrum anti-cancer effects, whereas 6c showed selective and exclusive inhibition activity in p53 mutant cancer cell lines but low toxicity to wild-type p53 cancer cell A375 and normal lung fibroblast WI-38 cells. Furthermore, 6c exhibited a more sophisticated mechanism for cell-destructive response by causing S/G2 phase arrest effect and cell size reduction. Compared with the cellular response of 6b and genetic background of cell lines studied, p53 mutation was found to be the key factor and main target for 6c evoked cell-destructive response. Molecular mechanism studies indicated that p53 phosphorylation and acetylation dual-targeting inhibitor 6c exerted anti-cancer activities with a special mechanism in evoking cell apoptosis by arresting mutant p53 function to trigger the deregulation of Cdk2 caused Bim-mediated apoptosis. To the best of our knowledge, 6c is the first quinazolinone derivative to dictate mutant p53 function for apoptotic cell death.

P16 Gene Transfer Induces Centrosome Amplification and Abnormal Nucleation Associated with Survivin Downregulation in Glioma Cells

Objective: In human glioma cells, p16 gene transfer induced G1/S arrest, increased radiosensitivity and abnormal nucleation (especially bi- and multinucleation). Survivin suppression caused G2/M arrest, radiosensitization and an increase in aneuploidy accompanied by centrosome amplification. Abnormal nucleation and aneuploidy represent chromosome instability (CIN), and it is well known that centrosome amplification leads to CIN. However, little has been reported that suggests that transferring p16 causes centrosome overduplication during the G1/S phase. Methods: The p16 gene was transferred into p16-null human glioma cell lines (U251MG and D54MG) using adenovirus with or without irradiation. Centrosome amplification was evaluated by immunofluorescence. We also investigated the DNA replication licensing factor CDT1, its inhibitor geminin and survivin expression as regulators of chromosomal segregation. Results: p16 gene transfer with radiation initiated the greatest degree of centrosome overduplication. CDT1 showed low levels, geminin was unchanged and survivin decreased in Ax-hp16-infected cells with radiation. Those changes of factors affecting DNA licensing or chromosomal segregation might contribute to CIN. Conclusion: p16 transfer caused centrosome amplification even in G1/S phase-arrested cells. This suggests that p16 is involved in abnormal nucleation and radiosensitization in human glioma cells.

Antiproliferative activity and induction of apoptosis by Annona muricata (Annonaceae) extract on human cancer cells.

BackgroundAnnona muricata (A. muricata) is widely distributed in Asia, Africa and South America. Different parts of this plant are used to treat several diseases in Cameroon. The aim of this study is to determine the in vitro anti-proliferative effects and apoptotic events of A. muricata extracts on HL-60 cells as well as to quantify its phenols content.MethodsThe cell viability was measured by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay while the changes in morphology of HL-60 cells, membrane mitochondrial potential (MMP) and the cell cycle were used for assessment apoptosis induction.ResultsThe results show that the concentration of phenols, flavonoids and flavonols in the extracts varied depending on the part of the plant. All the extracts tested inhibited the proliferation of HL-60 cells in a concentration dependent manner with IC50 varied from 6–49 μg/mL. The growth inhibition of the cells by extracts was associated with the disruption of MMP, reactive oxygen species (ROS) generation and the G0/G1 cell arrest.ConclusionThese findings suggest that the extracts from A. muricata have strong antiproliferation potential and can induce apoptosis through loss of MMP and G0/G1 phase cell arrest.