Cell Cycle Perturbations Research Articles

First person – Isabella Guardamagna

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Isabella Guardamagna is first author on ‘A functional in vitro cell-free system for studying DNA repair in isolated nuclei’, published in JCS. Isabella conducted the research described in this article while a PhD student in Lucia Anna Stivala's lab at the Immunology and General Pathology Unit, University of Pavia, Italy. She is now a postdoc in the lab of Andrea Ottolenghi, part of the Radiation Biophysics and Radiobiology group at the University of Pavia, Italy, investigating how cancer cells that are resistant to chemotherapy and radiotherapy acquire proliferative advantages in relation to DNA repair mechanisms and cell-cycle perturbation.

Immunomodulation-mediated anticancer activity of a novel compound from Brugmansia suaveolens leaves

Immunomodulation activity-guided fractionation of ethanol extract of Brugmansia suaveolens leaves was carried out to isolate a novel compound SUPH036-022A (1) by co-culturing the test fraction/compound activated PBMC with MCF7 and A549 cancer cell lines. Assessment of immune markers in PBMC, and analysis of apoptosis markers and cell cycle was carried out for cancer cells. The structure of the isolated compound was elucidated by spectral analysis. Compound 1 enhanced the secretion of immune markers, IL-2 and IFN-γ, from PBMC. Further, compound 1 treated PBMC increased cell death in MCF7 and A549 cell lines and induced ROS production and mitochondrial membrane perturbation, leading to apoptosis. Flow cytometry analysis revealed; compound 1 stimulated PBMC to cause a five-fold increase in cell cycle perturbations in the sub-G1 stage of cancer cells as compared to the negative control. The compound, in the absence of PBMC, only had a weak cytotoxic activity against these cell lines. Thus, compound 1 is a novel lead for immunomodulation-mediated anticancer activity.

PRMT5 promotes STAT-1 mediated T cell inflammatory response and is a therapeutic target for acute graft-versus-host disease

Abstract Introduction T cell mediated acute graft-versus-host disease (aGVHD) is the main cause of non-relapse mortality in allogeneic hematopoietic cell transplant recipients. Protein arginine methyltransferase 5 (PRMT5) is a post-translational modifier and promotes activation of memory TH cells. We investigate mechanisms by which PRMT5 regulates T cell function and propose PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods PRMT5 expression and function was evaluated in T cells of healthy and aGVHD mice and humans. We assessed T cell proliferation and effector function, using C220, a novel PRMT5 inhibitor. We tested effects of PRMT5 inhibition using in vivo mouse models of aGVHD, where mice received T cell depleted bone marrow + allogeneic splenocytes. In graft-versus-leukemia (GVL) experiments, P815 tumor cells were administered. Mice were treated with C220 or vehicle and monitored for survival and clinical aGVHD scores. Results PRMT5 expression and function is upregulated in T cells of mice and humans with aGVHD. Inhibition of PRMT5 reduces T cell proliferation, perturbs cell cycle and downregulates STAT-1 mediated interferon response. Administration of C220 improved survival in aGVHD mouse models and maintained tumor specific CTL response contributing to retention of Graft versus leukemia (GVL) effect. Conclusions Inhibition of PRMT5 using C220, down-regulates T cell proliferative and effector response, induces cell-cycle arrest, reduces interferon response and perturbs signaling pathways. C220 shows potent biological activity in vivo by reducing aGVHD clinical severity and prolonging survival in mouse models without compromising GVL. Therefore, PRMT5 is a novel and druggable target for aGVHD.

Design and Synthesis of New Substituted Pyrazolopyridines with Potent Antiproliferative Activity.

Purine isosteres are often endowed with interesting pharmacological properties, due to their involvement in cellular processes replacing the natural purines. Among these compounds, pyrazolopyridines are under active investigation for potential anticancer properties. Based on previously discovered substituted pyrazolopyridines with promising antiproliferative activity, we designed and synthesized new, suitably substituted analogues aiming to investigate their potential activity and contribute to SAR studies of this class of bioactive compounds. The new compounds were synthesized using suitably substituted 2-amino-4-picolines, which upon ring-closure provided substituted pyrazolo[3,4-c] pyridine-5-carbonitriles that served as key intermediates for the preparation of the target 3,5,7 trisubstituted derivatives. The antiproliferative activity of 31 new target derivatives was evaluated against three cancer cell lines (MIA PaCa-2, PC-3 and SCOV3), whereas cell-cycle perturbations of exponentially growing PC-3 cells, using three selected derivatives were also performed. Eight compounds displayed IC50 values in the low μM range, allowing the extraction of interesting SAR's. Two of the most potent compounds against all cell lines share a common pattern, by accumulating cells at the G0/G1 phase. From this project, a new carboxamidine-substituted hit has emerged. Among the new compounds, those possessing the 3-phenylpyrazolo[3,4-c]pyridine scaffold, proved to be worth investigating and the majority of them showed strong cytotoxic activity against all cell lines, with IC50 values ranging from 0.87-4.3 µM. A carboxamidine analogue that resulted from the synthetic procedure, proved to be highly active against the cancer cells and could be considered as a useful lead for further optimization.

Pyroptotic and apoptotic cell death in iNOS and nNOS overexpressing K562 cells: A mechanistic insight

Previous studies from this lab and others have demonstrated that nitric oxide (NO) in a concentration dependent manner, modulated neutrophil and leukemic cell survival. Subsequent studies delineated importance of iNOS in neutrophil differentiation and leukemic cell death. On the contrary, role of nNOS in survival of these cells remains least understood. Present study was therefore undertaken to assess and compare the role of iNOS and nNOS in the survival of NOS overexpressing myelocytic K562 cells. Cells with almost similar iNOS and nNOS activities displayed comparable cell cycle perturbation, Annexin V positivity, mitochondrial dysfunction, augmented DCF fluorescence, and also attenuated expression of antioxidants. Moreover, induction in cell death was also accompanied by the activation of pJNK/p38MAPK/Erk1/2 and reduction in PI3K/Akt/mTOR signaling. Treatment of NOS isoform overexpressing K562 cells with NAC, a potent free radical scavenger prevented cell death and also the modulations in the signaling proteins. In addition, enhanced expression of CASP1 and CASP4 genes, along with increased Caspase-1 cleavage and increased IL-1β release were significantly more in K562iNOS cells, which indicate priming of these cells for pyroptotic cell death. On the other hand, K562nNOS cells, displayed much enhanced CASP3 gene expression, Caspase-3 cleavage and Caspase-3 activity. Results obtained indicate that similar level of iNOS or nNOS activation in K562 cells, preferred pyroptotic and apoptotic cell death respectively.

The Fluorine Effect in Zwitterionic Half-Sandwich Iridium(III) Anticancer Complexes.

The rational design by the introduction of fluorine into a compound has achieved success in the development of organic anticancer drugs. However, the fluorine effect in metal-based anticancer complexes has rarely been reported. In this contribution, we report the synthesis, characterization, chemical reactivity, and biological activity of a series of half-sandwich zwitterionic iridium(III) complexes containing different substituents in the η5-CpR ring. The molecular structures for complexes Ir1-Ir4 and Ir7 were determined by single-crystal X-ray crystallography techniques. Notably, the asymmetrically substituted fluoro complexes Ir4 and Ir6 in solution show two conformational isomers. These complexes have sufficient stability, exhibit fluorescence emission, and show potent catalytic activity in converting NADH to NAD+. The effect of the substituents in the η5-CpR ring for these zwitterionic complexes on their anticancer activity was systematically investigated. Surprisingly, the presence of fluorinated substituents gives rise to a significant increase in the anticancer activity. The lipophilicity and cellular uptake levels of these complexes appeared to be the primary factors for their cytotoxicity in this system. A microscopic mechanism study showed that the typical complex Ir4 entered A549 cancer cells through an energy-dependent pathway and was mainly located in lysosomes. Furthermore, an increase in ROS level, apoptosis induction, and cell-cycle perturbation together contribute to the anticancer potency of these zwitterionic complexes.

Enhancement of Cytotoxicity of Enediyne Compounds by Hyperthermia: Effects of Various Metal Complexes on Tumor Cells.

Enediyne natural products are a class of compounds that were recognized for their potential as chemotherapeutic agents many years ago, but found to be highly cytotoxic due to their propensity for low thermal activation. Bergman cyclization of the enediyne moiety produces a diradical intermediate, and may subsequently induce DNA damage and account for the extreme cytotoxicity. While difficulties in controlling the thermal cyclization reaction have limited the clinical use of cyclic enediynes, we have previously shown that enediyne activity, and thus toxicity at physiological temperatures can be modulated by metallation of acyclic enediynes. Furthermore, the cytotoxicity of "metalloenediynes" can be potentiated by hyperthermia. In this study, we characterized a suite of novel metallated enediyne motifs that usually induced little or no cytotoxicity when two different human cancer cell lines were treated with the compounds at 37°C, but showed a significant enhancement of cytotoxicity after cells were exposed to moderate hyperthermia during drug treatment. Cultured U-1 melanoma or MDA-231 breast cancer cells were treated with various concentrations of Cu, Fe and Zn complexes of the enediyne (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine (PyED) and clonogenic survival was assessed to determine the effects of the drugs at 37°C and 42.5°C. Toxicity at 37°C varied for each compound, but hyperthermia potentiated the cytotoxicity of each compound in both cell lines. Cytotoxicity was concentration-, time- and temperature-dependent. Heating cells during drug treatment resulted in enhanced apoptosis, but the role of cell cycle perturbation in the response of the cells to the drugs was less clear. Lastly, we showed that hyperthermia enhanced the number of DNA double-strand breaks (DSBs) induced by the compounds, and inhibited their repair after drug treatment. Thus, thermal enhancement of cytotoxicity may be due, at least in part, to the propensity of the enediyne moiety to induce DSBs, and/or a reduction in DSB repair efficiency. We propose that "tuning" of metalloenediyne toxicity through better-controlled reactivity could have potential clinical utility, since we envision that such compounds could be administered systemically as relatively non-toxic agents, but cytotoxicity could be enhanced in, and confined to a tumor volume when subjected to localized heating.

K092A and K092B, Two Peptides Isolated from the Dogfish (Scyliorhinus canicula L.), with Potential Antineoplastic Activity Against Human Prostate and Breast Cancer Cells.

Cancer therapy is currently a major challenge within the research community, especially in reducing the side effects of treatments and to develop new specific strategies against cancers that still have a poor prognosis. In this context, alternative strategies using biotechnologies, such as marine peptides, have been developed based on their promise of effectivity associated with a low toxicity for healthy cells. The purpose of the present paper is to investigate the active mechanism of two peptides that were isolated from the epigonal tissue of the lesser spotted dogfish Scyliorhinus canicula L., identified NFDTDEQALEDVFSKYG (K092A) and EAPPEAAEEDEW (K092B) on the in vitro growth inhibition of ZR-75-1 mammary carcinoma cells and MDA-Pca-2b prostate cancer cells. The effects of the peptides on cell proliferation and cell death mechanisms were studied by the flow cytometry and immunofluorescence microscopy approaches. The results have shown the onset of both K092A- and K092B-induced early cytoskeleton changes, and then cell cycle perturbations followed by non-apoptotic cell death. Moreover, impedance perturbation and plasma membrane perforation in ZR-75-1 K092A-treated cell cultures and autophagy inhibition in MDA-Pca-2b K092B-treated cells have been observed. In conclusion, these two bioactive peptides from dogfish exhibit antineoplastic activity on the human prostate and breast cancer cells in vitro.

Fatty acid synthase inhibitor orlistat impairs cell growth and down-regulates PD-L1 expression of a human T-cell leukemia line

Fatty Acid Synthase (FASN) is responsible for the de novo synthesis of fatty acids, which are involved in the preservation of biological membrane structure, energy storage and assembly of factors involved in signal transduction. FASN plays a critical role in supporting tumor cell growth, thus representing a potential target for anti-cancer therapies. Moreover, this enzyme has been recently associated with increased PD-L1 expression, suggesting a role for fatty acids in the impairment of the immune response in the tumor microenvironment. Orlistat, a tetrahydrolipstatin used for the treatment of obesity, has been reported to reduce FASN activity, while inducing a sensible reduction of the growth potential in different cancer models. We have analyzed the effect of orlistat on different features involved in the tumor cell biology of the T-ALL Jurkat cell line. In particular, we have observed that orlistat inhibits Jurkat cell growth and induces a perturbation of cell cycle along with a decline of FASN activity and protein levels. Moreover, the drug produces a remarkable impairment of PD-L1 expression. These findings suggest that orlistat interferes with different mechanisms involved in the control of tumor cell growth and can potentially contribute to decrease the tumor-associated immune-pathogenesis.

Amelioration of ochratoxin-A induced cytotoxicity by prophylactic treatment of N-Acetyl-L-Tryptophan in human embryonic kidney cells

Ochratoxin A (OTA) is known to induce nephro-toxicity via induction of cellular redox homeostasis perturbation, mitochondrial hyperpolarisation and depolarization, protein synthesis inhibition leading to apoptosis. In the present study, protective efficacy of N-Acetyl-L-Tryptophan (NAT) against OTA induced toxicity was evaluated using Human Embryonic Kidney (HEK-293) cells. Cells were treated with NAT (0–200 μg/ml) before OTA treatment (0–20 μg/ml) and protective efficacy of NAT was evaluated using MTT and SRB assay. OTA-induced intracellular ROS generation and its inhibition by NAT (2.5 μg/ml) pre-treatment was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe. Effects of NAT pre-treatment on OTA treated cells were also evaluated in terms of cell cycle perturbations and mitochondrial membrane potential disturbance using flowcytometry. Results of the study demonstrated significant (∼89 % cell growth in comparison to 50% in OTA alone group; P < 0.05) protection by NAT to the HEK-293 cells against OTA mediated cell death in terms of cell viability. Further, significant reduction in ROS levels and mitochondrial membrane potential disturbance was also observed in NAT pre-treated plus OTA cells as compared to only OTA treated cells. Significant (p < 0.05) arrest in G0 and S phase of cell cycle was observed in OTA treated cells that was found to be inhibited by NAT pre-treatment to OTA treated cells. Also, molecular docking analysis demonstrated higher probability of NAT to bind with OTA binding pocket on phenylalanyl t-RNA synthetase, resulting in inhibition of OTA incorporation in the newly synthesized peptides and thus may ameliorate OTA induced protein synthesis inhibition. Conclusively, present study suggested that NAT offers protection against OTA toxicity in HEK-293 cells by counterbalancing oxidative stress, cell cycle regulation, mitochondrial membrane potential stabilization, protein synthesis inhibition and cell death retardation.

18F-FDG-induced DNA damage, chromosomal aberrations, and toxicity in V79 lung fibroblast cells

18F-FDG PET/CT imaging is used in the diagnosis of diseases, including cancers. The principal photons used for imaging are 511 ke V gamma photons resulting from positron annihilation. The absorbed dose varies among body organs, depending on administered radioactivity and biological clearance. We have attempted to evaluate DNA double-strand breaks (DSB) and toxicity induced in V79 lung fibroblast cells in vitro by 18F-FDG, at doses which might result from PET procedures. Cells were irradiated by 18F-FDG at doses (14.51 and 26.86 mGy), comparable to absorbed doses received by critical organs during PET procedures. The biological endpoints measured were formation of γ-H2AX foci, mitochondrial stress, chromosomal aberrations, and cell cycle perturbation. Irradiation induced DSB (γH2AX assay), mitochondrial depolarization, and both chromosome and chromatid types of aberrations. At higher radiation doses, increased aneuploidy and reduced mitotic activity were also seen. Thus, significant biological effects were observed at the doses delivered by the 18F-FDG exposure and the effects increased with dose.

28P - GLPG 1790, a new selective EPHA2 inhibitor, is active in colorectal cancer cell lines belonging to the CMS4/mesenchymal-like subtype

BackgroundEPHA2 tyrosine kinase receptor is implicated in tumor progression, stemness phenotype and resistance to treatment in a wide range of cancers. We investigated the effects of GLPG1790, a new selective Eph receptor inhibitor, in colorectal cancer (CRC) across the 4 consensus molecular subtypes (CMS). MethodsWe tested the antiproliferative effect of GLPG 1790 used alone or in combination with either 5-fluorouracil, oxaliplatin or SN38 (the active metabolite of irinotecan) in a panel of 11 CRC cell lines encompassing the 4 consensus molecular subtypes (CMS). Cell cycle analysis was performed in order to understand possible cell cycle perturbation after treatment. Pathway analysis using western blot (WB) was also performed. We then evaluated the expression of stemness genes upon treatment using qRT-PCR. ResultsGLPG 1790 is active in CRC cell lines, with the strongest activity in the cell lines from the CMS4/mesenchymal-like cluster. Combination with chemotherapeutics is not synergistic according to Chou-Talalay model. The selective inhibitor elicits a persistent inactivation of EPHA2 receptor, associated to G0-G1 cell cycle block in the sensitive cell lines. Furthermore, GLPG 1790 is able to decrease the expression of cancer stem cell genes in cell lines belonging to the CMS4 group. ConclusionsEPHA2 blockade using the selective inhibitor GLPG 1790 has a strong antiproliferative effect in the chemorefractory subgroup of CMS4/mesenchymal-like CRC cell lines, associated to a G0-G1 cell cycle arrest.The stronger efficacy of GLPG1790 on the mesenchymal-like subtype is probably due to the impairment of cancer cell stemness and induction of cell differentiation after treatment. Legal entity responsible for the studyThe authors. FundingUniversità della Campania "Luigi Vanvitelli"; Galapagos NV (drug supply); Associazione Italiana per la Ricerca sul Cancro (AIRC). DisclosureP.P. Vitiello: Travel/Accommodation/Expenses: Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche; Research grant/Funding (institution): Servier; Research grant/Funding (institution): Ipsen; Travel/Accommodation/Expenses: BMS; Travel/Accommodation/Expenses: Sanofi. C. Cardone: Research grant/Funding (institution): Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Ipsen; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche. D. Ciardiello: Travel/Accommodation/Expenses: Sanofi. L. Poliero: Travel/Accommodation/Expenses: BMS. C. Borrelli: Travel/Accommodation/Expenses: BMS. N. Zanaletti: Travel/Accommodation/Expenses: BMS. P. Vitale: Travel/Accommodation/Expenses: BMS. T. Troiani: Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Travel/Accommodation/Expenses: Amgen; Travel/Accommodation/Expenses: Servier; Travel/Accommodation/Expenses: Sanofi; Travel/Accommodation/Expenses: Novartis. F. Ciardiello: Advisory/Consultancy, Research grant/Funding (institution): Amgen; Advisory/Consultancy, Research grant/Funding (institution): Bayer; Advisory/Consultancy, Research grant/Funding (institution): Roche; Advisory/Consultancy, Research grant/Funding (institution): Merck; Advisory/Consultancy: Servier; Advisory/Consultancy: Pfizer; Research grant/Funding (institution): Ipsen. E. Martinelli: Honoraria (self), Research grant/Funding (institution): Amgen; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Honoraria (institution): Servier. All other authors have declared no conflicts of interest.

P2.14-50 Low Temperature Plasma Needle Induces Cell Cycle Arrest of Epithelial Lung Cancer Cells in Vitro via a p21-Dependent Pathway

Low temperature plasma sources which operate at atmospheric pressure produce reactive oxygen species (ROS) and reactive nitrogen species which can cause cancer cell death. In this study we investigated the effect of our low temperature plasma needle system on a non-small cell lung cancer cell line A549 and studied its mechanism of action. The housing of the pen lookalike atmospheric plasma source (plasma needle) was made of Teflon. A Pyrex glass tube (o.d. 6 mm and i.d. 4 mm) through which 1 slm of helium was released as the feeding gas was positioned within the housing. The central electrode, a tungsten wire, was powered with a 13.56 MHz sine wave and placed within a ceramic tube inside the glass tube. The central electrode was sticking 1 mm outside of the ceramic and glass tubes, so the discharge occurred on its tip as a weak glow. The cytotoxic activity was determined using MTT assay. The potential of inducing cell cycle perturbations and apoptosis, and changes in the level of ROS was investigated by flow cytometry. The influence of plasma treatment on growth inhibition of multicellular tumor spheroids (MCTS) was also investigated. Evaluation of gene expression was performed by qPCR. All experiments were performed in triplicate, with statistical significance set at p<0.05. Our plasma needle exerted a cytotoxic effect with lower sensitivity towards BEAS-2B normal cells than towards A549 cells. A decrease in the number of cells in the G1 phase (up to 45%), increase in the G2 phase (up to 27%), and an increase in the sub-G1 phase (up to 12%) with fragmented DNA was detected upon treatment. The plasma treatment exerted a mild apoptotic effect (around 15% of apoptotic cells), and an increase in the level of ROS in a power dependent manner. There was no significant reduction in growth of MCTS after plasma treatment under investigated experimental conditions. A statistically non-significant proapoptotic effect (increase in Bax, decrease in Bcl2 and decrease in SKP2) was observed at the genetic level. A significant overexpression of the cyclin-dependent kinase inhibitor 1 (p21) was also observed at the genetic level in a power dependent manner. Our low temperature plasma needle induced cell cycle arrest of epithelial lung cancer cells through overexpression of p21. The effect of combined plasma treatment with existing treatment modalities (cisplatin, PARP inhibitors) is currently under in vitro investigation.

Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.

Vorinostat Potentiates 5-Fluorouracil/Cisplatin Combination by Inhibiting Chemotherapy-Induced EGFR Nuclear Translocation and Increasing Cisplatin Uptake.

The 5-fluorouracil/cisplatin (5FU/CDDP) combination is one of the most widely used treatment options for several solid tumors. However, despite good anticancer responses, this regimen is often associated with high toxicity and treatment resistance. In our study, we evaluated whether the histone deacetylase inhibitor (HDACi), vorinostat, may induce synergistic antitumor and proapoptotic effects in combination with 5FU/CDDP in squamous cancer cell models. We demonstrated in cancer cell lines, including the intrinsic CDDP-resistant Cal27 cells, that simultaneous exposure to equitoxic doses of vorinostat plus 5FU/CDDP results in strong synergistic antiproliferative and proapoptotic effects related to cell-cycle perturbation and DNA damage induction. These effects were confirmed in vivo in both orthotopic and heterotopic xenograft mouse models of Cal27 cells. Mechanistically, vorinostat reverted 5FU/CDDP-induced EGFR phosphorylation and nuclear translocation, leading to the impairment of nuclear EGFR noncanonical induction of genes such as thymidylate synthase and cyclin D1. These effects were exerted by vorinostat, at least in part, by increasing lysosomal-mediated EGFR protein degradation. Moreover, vorinostat increased platinum uptake and platinated DNA levels by transcriptionally upregulating the CDDP influx channel copper transporter 1 (CTR1). Overall, to our knowledge, this study is the first to demonstrate the ability of vorinostat to inhibit two well-known mechanisms of CDDP resistance, EGFR nuclear translocation and CTR1 overexpression, adding new insight into the mechanism of the synergistic interaction between HDACi- and CDDP-based chemotherapy and providing the rationale to clinically explore this combination to overcome dose-limiting toxicity and chemotherapy resistance.

Cell Cycle Perturbation Induces Collagen Production in Fibroblasts.

Myocardial infarction (MI) occurs when the heart muscle is severely damaged due to a decrease in blood flow from the coronary arteries. During recovery from an MI, cardiac fibroblasts become activated and produce extracellular matrices, contributing to the wound healing process in the damaged heart. Inappropriate activation of the fibroblasts leads to excessive fibrosis in the heart. However, the molecular pathways by which cardiac fibroblasts are activated have not yet been fully elucidated.Here we show that serum deprivation, which recapitulates the cellular microenvironment of the MI area, strikingly induces collagen production in C3H/10T1/2 cells. Based on transcriptomic and pharmacological studies, we found that cell cycle perturbation is directly linked to collagen production in fibroblasts. Importantly, collagen synthesis is increased independently of the transcriptional levels of type I collagen genes. These results reveal a novel mode of fibroblast activation in the ischemic area, which will allow us to gain insights into the molecular mechanisms underlying cardiac fibrosis and establish a basis for anti-fibrotic therapy.

Timp3 deficiency affects the progression of DEN-related hepatocellular carcinoma during diet-induced obesity in mice.

Obesity and low-grade inflammation are associated with an increased risk of hepatocellular carcinoma (HCC), a leading cause of cancer-related death worldwide. The tissue inhibitor of metalloproteinase (TIMP) 3, an endogenous inhibitor of protease activity that represents a key mediator of inflammation, is reduced in inflammatory metabolic disorders and cancer. In contrast, Timp3-deficient mice (Timp3-/-) are highly resistant to developing HCC in response to a diethylnitrosamine (DEN); therefore, we aimed to elucidate the biological role of genetic loss of Timp3 in obesity-related hepatocarcinogenesis. Fourteen-day-old male wild-type (wt) and Timp3-/- mice were injected with 25mg/kg DEN or an equal volume of saline. After 4weeks, mice were randomized into two dietary groups and fed either normal or high-fat diet and allowed to grow until 32weeks of age. Liver histological features were analyzed, and differentially expressed genes in the liver were quantified. In Timp3-/- mice fed with the obesogenic diet, despite the increase in liver steatosis and inflammation, both the number of tumors and the total tumor size are significantly reduced 30weeks post-DEN injection, compared to control mice. Moreover, Timp3 deletion in hepatocarcinogenesis during obesity is associated with a reduction in FoxM1 transcriptional activity through H19/miR-675/p53 pathway. This study suggests that Timp3 ablation leads to cell cycle perturbation, at least in part by repressing FoxM1 transcriptional activity through H19/miR-675/p53 pathway.

Radiosensitizing Effect of Novel Phenylpyrimidine Derivatives on Human Lung Cancer Cells via Cell Cycle Perturbation.

Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need to develop radiosensitizers to improve the therapeutic outcomes of radiotherapy in cancer patients. Our previous efforts to identify novel radiosensitizers, using high-throughput screening targeting p53 and Nrf2 revealed a promising N-phenylpyrimidin-2-amine (PPA) lead compound. In the present study, 17 derivatives of this lead compound were examined, and it was found that 4-(4-fluorophenyl)-N-(4-nitrophenyl)-6-phenylpyrimidin-2-amine (PPA5), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-methoxy-N-methyl -benzamide (PPA13), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA14), 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA15), and 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)-N-methylbenzamide (PPA17) inhibited cell viability by more than 50%, with a marked increase in the proportion of cells arrested at the G2/M phase of cell cycle. Among these compounds, PPA15 markedly increased the sub-G1 cell population and increased the levels of cyclin B1 and the phosphorylation levels of cyclin-dependent kinase (CDK) 1. Combined treatment with radiation and PPA14 or PPA15 significantly decreased clonogenic survival. An in vitro kinase assay revealed that PPA15 inhibited multiple CDKs involved in cell cycle regulation. Compared with drug or radiation treatment alone, combined treatment with PPA15 and radiation resulted in the suppression of A549 tumor growth in mice by 59.5% and 52.7%, respectively. Treatment with PPA15 alone directly inhibited tumor growth by 25.7%. These findings suggest that the novel pan CDK inhibitor, PPA15, may be a promising treatment to improve the effectiveness of radiotherapy for the treatment of cancer. SIGNIFICANCE STATEMENT: Several inhibitors of CDK have been successfully evaluated in combination with other chemotherapeutics in clinical trials, but negative side effects have partially restricted their clinical use. In this study, we identified a novel pan-CDK inhibitor to increase radiosensitivity, and we hope this work will encourage the development of promising small-molecule radiosensitizers.

Exploring the radiosensitizing potential of magnetotherapy: a pilot study in breast cancer cells

Aim: To explore the influence of electromagnetic fields (EMFs) on the cell cycle progression of MDA-MB-231 and MCF-7 breast cancer cell lines and to evaluate the radiosensitizing effect of magnetotherapy during therapeutic co-exposure to EMFs and radiotherapy.Material and methods: Cells were exposed to EMFs (25, 50 and 100 Hz; 8 and 10 mT). In the co-treatment, cells were first exposed to EMFs (50 Hz/10 mT) for 30 min and then to ionizing radiation (IR) (2 Gy) 4 h later. Cell cycle progression and free radical production were evaluated by flow cytometry, while radiosensitivity was explored by colony formation assay.Results: Generalized G1-phase arrest was found in both cell lines several hours after EMF exposure. Interestingly, a marked G1-phase delay was observed at 4 h after exposure to 50 Hz/10 mT EMFs. No cell cycle perturbation was observed after repeated exposure to EMFs. IR-derived ROS production was enhanced in EMF-exposed MCF-7 cells at 24 h post-exposure. EMF-exposed cells were more radiosensitive in comparison to sham-exposed cells.Conclusions: These results highlight the potential benefits of concomitant treatment with magnetotherapy before radiotherapy sessions to enhance the effectiveness of breast cancer therapy. Further studies are warranted to identify the subset(s) of patients who would benefit from this multimodal treatment.

PF169 LONG‐TERM INHIBITION OF CHK1/CHK2 KINASES MODIFY ACUTE LYMPHOBLASTIC LEUKEMIA CELL LINE RESPONSE TO DNA DAMAGING AGENTS

Background:Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR‐pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR‐related genes drastically affect the effectiveness of DNA‐damaging treatments in cancer cells. For this reasons selective DDR‐inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR‐inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR‐pathways in cancer cells.Aims:The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR‐related kinase, the cell cycle checkpoint kinase 1 (CHK1) and 2 (CHK2), in B‐ALL cell lines.Methods:The resistant cell lines were obtained from B‐ALL NALM‐6 cell line by prolonged incubation with a CHK1/CHK2 inhibitor, PF‐00477736. Parental cells were progressively incubated with increasing concentration of PF‐00477736 (from 100 nM to 10uM). The functional characterization of resistant cell lines was evaluated in vitro in term of response to different chemotherapy agents (doxorubicin, methotrexate and clofarabine) in term of reduction of cell viability, induction of apoptosis, cell cycle perturbation and activation of DDR pathways. The molecular characterization of resistant cell lines was evaluated in term of Copy Number Alteration (SNP‐array, Affimetrix).Results:Starting from NALM‐6 parental cell lines, we generated two resistant models, N6R‐PF4 and N6R‐PF8. N6R‐PF8 was generated form N6R‐PF4 cells after a prolong incubation with increased PF‐00477736 concentration. In term of reduction of cell viability and induction of apoptosis the resistant cell lines were not only significantly unresponsive to PF‐00477736 (increasing the IC50 up to 8‐fold) but also to different DNA damaging agents in comparison to parental NALM‐6. The cell cycle profile revealed that the resistant cell lines had a significant reduction in the percentage of cells in S phase and a significant increase of cells in G2/M phase. SNP microarray highlighted different alterations in DDR‐related genes and in particular in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN = 1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, BIRC3, CARD17, CASP1, CASP12 and CASP5) were detectable only in NALM‐6 parental cell lines and were copy number neutral in the two resistant models. Immunoblotting analysis confirmed that in the resistant cell lines the ATM/CHK2 down‐stream pathways was significant over‐expressed and activated in comparison to the parental NALM‐6. By chromosome binding analysis we identified that NALM‐6 cells were composed by a predominant clone with ATM locus CN loss and by a sub‐clone (45,X,‐Y,t(5;12)(q33;p13),t(7;19)(q11;p13)) with ATM locus in CN neutral. The treatment with PF‐00477736 progressively selected the sub‐clone, reaching 60% of clones in N6R‐PF4 and 100% of clone in N6R‐PF8.Summary/Conclusion:We showed that the higher protein expression of ATM and CHK2 affected not only the sensitivity to PF‐00477736 but also compromised the efficacy of conventional chemotherapy. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF‐00477736.