G1 Block Research Articles

Dicliptera chinensis-derived polysaccharide enhanced the growth activity of submandibular gland cells in vitro after radiotherapy

ObjectiveRadiotherapy for head and neck can damage the salivary gland cells, which can easily result in xerostomia. No effective treatment for radiation-induced salivary gland dysfunction currently exists. Thus, we aimed to study the protective effect of Dicliptera chinensis polysaccharides (DCP) on the prevention of submandibular gland (SMG) cell damage caused by radiotherapy in Sprague-Dawley rats. DesignMechanical enzyme digestion was used to extract primary rat SMG cells. A radiation injury model was established by treating these cells with a dose of 8 Gy, followed by intervention using different DCP concentrations. The cell counting kit 8 assay was used to determine the inhibition rate of SMG cells in each group. The rates of apoptosis and cell cycle progression were detected using flow cytometry. Expression of the Mre11/Rad50/Nbs1 complex (MRN) was detected using western blotting. ResultsDCP increased the proliferation of SMG cells after irradiation, and cell growth activity positively correlated with polysaccharide concentration. Flow cytometry analysis of SMG cell apoptosis revealed that DCP markedly reduced the total apoptosis rate after irradiation, especially the early apoptosis rate. Cell cycle results suggested that DCP reduced the number of cells in the S and G2 phases after irradiation and alleviated the S and G2 blocks. Western blot results indicated that the expression of Mre11, Rad50, and Nbs1 decreased in the radiation-injured group, whereas their expression increased after DCP treatment. ConclusionsDCP can protect the rat SMG cells after radiation and be used as a protective agent against salivary gland cell damage caused by radiotherapy.

Abstract 3762: A novel lncRNA, GTF2A1-AS1, is required for cell cycle progression and cell proliferation

Abstract Long noncoding RNAs (lncRNAs) are major regulators of many cellular processes including cell cycle progression, cell proliferation and tumorigenesis. In this study, we identify a novel lncRNA, GTF2A1-AS1, and reveal its effects on cell cycle progression and cancer growth. Inhibition of GTF2A1-AS1 expression alters cell cycle distribution, leading to a decrease in the number of G1 cells and a concomitant increase in all other stages of the cell cycle, and in particular S phase, suggesting its involvement in the regulation of G1/S transition. Accordingly, knock down of GTF2A1-AS1 enhances G1 phase exit upon release from a late G1 block. In agreement with its suggested role in G1/S transition, prolonged silencing of GTF2A1-AS1 enhances proliferation of human cancer cells. GTF2A1-AS1 RNA is mostly nuclear and its gene is localized upstream to the protein-coding gene GTF2A1. GTF2A1-AS1 functions in trans to repress the expression of EIF5A2 and HOXA13. Knockdown of either EIF5A2 or HOXA13 rescues the effects of GTF2A1-AS1 silencing on cell proliferation, indicating that it functions mainly by modulating the levels of these two proteins. In agreement with the effect of GTF2A1-AS1 silencing on cell cycle progression and cell proliferation, low levels of GTF2A1-AS1 are associated with reduced survival in brain cancer patients. In conclusions, our data identify GTF2A1-AS1 as a novel lncRNA that regulates cell-cycle progression and cell proliferation and has a potential role in human cancer. Citation Format: Yael Goldstein, Tali Nizri-Megnaji, Doron Ginsberg. A novel lncRNA, GTF2A1-AS1, is required for cell cycle progression and cell proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3762.

In vitro evaluation of growth reticence and anticancer potential of 5α,8α-epidioxy-24ᶓ-methylcholesta-6,22-dien-3β-ol and ergosta-5,7,22-trien-3β-ol bioactive isolated from an edible mushroom Lentinus tuberregium (fr.)

The focus point of this current work is to evaluate the anticancer and growth inhibitory efficacy of compounds 5α,8α-epidioxy-24ᶓ-methylcholesta-6,22-dien-3β-ol (LT1), and Ergosta-5,7,22-trien-3β-ol (LT2) of Lentinus tuberregium (Fr.) on three cell lines such as A673 (Rhabdomyosarcoma), MCF7 (breast cancer), and HCT116 (colorectal carcinoma) by MTT assay. LT1 and LT2 exerted maximal growth inhibition in the order as A673 > HCT116 > MCF7. Comparatively, LT1 was more potent in causing cell growth inhibition than LT2 in the A673 cancer cell line. Based on the MTT assay, A673 cells alone proceeded further as a model to evaluate the anticancer potential of LT1 and LT2 at three different semilogarithmic concentrations (3, 10, 30 μM). The cells exposed with compounds at 24 and 48 h were analyzed by flow cytometry. Exposure of LT1 at 3 and 10 μM concentrations for 24 h caused a G2-M arrest. At 10 μM concentration, cells also accumulated in the G0-G1 phase, indicating a G1 block. These effects were only transient as prolonged exposure (48 h) of LT1 treatment brought back the cell population to normalcy. Both the compounds only at 30 μM concentration have the potential to induce a hypodiploid peak (sub G0), indicating an induction of apoptosis which was explicit by nuclear condensation and fragmentation of nuclei in cells. The dose-dependent and compound-specific apoptotic induction was further confirmed by caspase activity higher in LT1 than LT2. The results highlight the significant growth inhibitory activity and anticancer potential of LT1 and LT2 which are recommended for further in-depth analysis.

Phase Ib trial of erdafitinib (E) combined with enfortumab vedotin (EV) following platinum and PD-1/L1 inhibitors for metastatic urothelial carcinoma (mUC) with FGFR2/3 genetic alterations (GAs).

TPS595 Background: Erdafitinib and enfortumab vedotin are available treatment options in mUC patients with somatic FGFR2/3 GAs after progression on platinum-based chemotherapy and PD-1/L1 inhibitors. However, due to decline in their clinical condition, the sequential delivery of these agents is challenging. Tubulin antagonists induce a G2-M cell-cycle block, while FGFR inhibitors cause a G1 block, with studies suggesting that their combination may be additive or synergistic. Retrospective studies suggest that the activity of EV is not compromised by somatic FGFR2/3 GAs. Hence, there is rationale to evaluate the feasibility of the combination of EV and E, to overcome the difficulties of resistance and sequencing these agents in mUC patients with FGFR2/3 GAs. Methods: This is a phase Ib, single arm, multicenter study in patients with mUC harboring somatic FGFR2/3 GAs who have progressed after platinum and PD-1/L1 inhibitor therapies with enrollment of up to 30 patients. Pts are required to have predominant urothelial component, ECOG-PS 0-2, neuropathy ≤ grade 1 and no ophthalmologic conditions precluding treatment with E. FGFR2/3 GAs may be identified by tumor tissue or circulating tumor (ct)-DNA profiling. The primary objective is feasibility and establishing a recommended phase-2 dose (RP2D). Secondary objectives include objective response rate, duration of response, progression-free survival and overall survival. The dose-escalation component will enroll up to 18 pts with 3+3 design (dosing cohorts in table), followed by dose-expansion component of 12 pts. The dose limiting toxicities (DLTs) will be evaluated using a sequential Bayesian toxicity monitoring that allows a maximum DLT rate of 0.33 during the dose-expansion phase. Exploratory biomarker analyses will be performed including 1) tumor PD-L1, Nectin-4 assessment by immunohistochemistry and association with response 2) ct-DNA evaluation at baseline and progression to evaluate resistance pathways 3) pharmacokinetic studies will assess plasma levels of E and free monomethyl auristatin-E (MMAE). This is led by North American Star Consortium as part of Experimental Therapeutics. Clinical trial information: NCT04963153. [Table: see text]

Preparation of Polyethylene Glycol-Poly Lactic Acid Water-Soluble Nano-Particles and Its Effect on the Proliferation, Metastasis, and Angiogenesis of Liver Cancer Cells

The onset of the disease is secretive, and the accuracy of early diagnosis is not high. Patients will lose the opportunity of surgical treatment because of severe liver cirrhosis and cancer cell metastasis. Vanib nano-drug was prepared in this study by a polymer, which was used for in vitro research of liver cancer cells. First, polyethylene glycol (PEG)-poly lactic acid (PLA) and Vanib were dissolved in acetone to form a mixed solution, and then, water-soluble nano-particles were obtained by ultrasonic and deionized water. Then, the nano-particles were modified by iRGD and characterized by electron microscopy, DLS, and in vitro release. Besides, CCK-8, flow cytometry apoptosis test, flow cytometry cell cycle test, Transwell cell invasion and metastasis test, and angiogenesis test were applied to detect the effect of the nano-drug on the proliferation, cell metastasis, and angiogenesis of liver cancer cells. The results showed that Vanib nano-particles inhibited the proliferation of liver cancer cells in a dose-dependent manner, and induced apoptosis to G1 block. What’s more, Vanib nano-particles reduced cell viability in a Caspase-dependent manner, thereby inducing cell death. The Vanib nano-particles up-regulated the expression of P21 and also inhibited the cyclins Cyclin E2 and Cycllin B, thereby blocking the cell cycle process. In addition, Vanib nano-particles and free Vanib could inhibit the activity of human umbilical vein endothelial cells (HUVECs) and angiogenesis, which proved that the prepared Vanib nano-particles could effectively curb the growth of liver cancer cells.

RPB5-mediating protein promotes the progression of non-small cell lung cancer by regulating the proliferation and invasion.

BackgroundThis study aimed to investigate the relationship between RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) and clinicopathological characteristics of non-small cell lung cancer (NSCLC) patients by measuring the expression level of RMP in human NSCLC tissues and cell lines. At the same time, we studied the impact of RMP on the biological function of cancer, providing strong support for gene targeted therapy of NSCLC.MethodsReal-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to determine the expression levels of messenger (m)RNA and protein in NSCLC cell lines and tissues. Cell counting kit 8 (CCK8) assay and flow cytometry were selected to detect cell proliferation, cycle and apoptosis. The wound healing assay was chosen to detect the migration and invasion ability of cells. The xenograft model was performed to study the function of RMP in vivo. Immunohistochemical (IHC) staining showed the levels of RMP, Bcl-2, Bax and caspase-3.ResultsFirst, mRNA and protein levels of RMP were relatively overexpressed in NSCLC cells. Compared with the corresponding normal tissues, the mRNA and protein levels of RMP were significantly higher in human NSCLC tissues. Concurrently, we found that the expression of RMP was related to the status of lymph nodes (LNs) in cancer tissues and T stage. Then, RMP overexpression promoted the proliferation of A549. At the same time, RMP provided A549 cells the ability to resist chemotherapy and radiotherapy; when A549 cells were treated with gefitinib and radiation, RMP reduced apoptosis. We also found that RMP can protect A549 from G2 block caused by radiation. Over-irradiated RMP-overexpressed A549 cells had lower Bcl2-associated X protein (Bax) levels and higher B-cell lymphoma 2 (Bcl-2) levels. The migration and invasion ability of A549 cells was increased by RMP. Finally, RMP can promote tumor growth by increasing Bcl-2 levels and decreasing Bax and caspase-3 levels in the xenograft model.ConclusionsThere is potential for RMP to develop into a diagnostic and therapeutic target for NSCLC.

Supramolecular Insights into Domino Effects of Ag@ZnO-Induced Oxidative Stress in Melanoma Cancer Cells.

Recent studies suggest that cancer cell death accompanied by organelle dysfunction might be a promising approach for cancer therapy. The Golgi apparatus has a key role in cell function and may initiate signaling pathways to mitigate stress and, if irreparable, start apoptosis. It has been shown that Golgi disassembly and fragmentation under oxidative stress act as indicators for stress-mediated cell death pathways through cell cycle arrest in the G2/M phase. The present study shows that UV-induced reactive oxygen species (ROS) generation by Ag@ZnO nanoparticles (NPs) transform the Golgi structures from compressed perinuclear ribbons into detached vesicle-like structures distributed in the entire cytoplasm of melanoma cells. This study also demonstrates that Ag@ZnO NP-induced Golgi fragmentation cooccurs with G2 block of cell cycle progression, preventing cells from entering the mitosis phase. Additionally, the increased intracellular ROS production triggered by Ag@ZnO NPs upon UV exposure promoted autophagy. Taken together, Ag@ZnO NPs induce stress-related Golgi fragmentation and autophagy, finally leading to melanoma cell apoptosis. Intracellular oxidative stress generated by Ag@ZnO NPs upon UV irradiation may thus represent a targeted approach to induce cancer cell death through organelle destruction in melanoma cells, while fibroblast cells remained largely unaffected.

Abstract 3912: The PI3K/mTOR dual inhibitor PF-04691502 shows antitumor activity in Sezary cells and in a xenograft mouse model

Abstract Introduction: Cutaneous T-cell Lymphomas (CTCLs) are extranodal non-Hodgkin’s lymphomas deriving from neoplastic transformation of T lymphocytes that home to the skin. Mycosis fungoides (MF) is the most diffuse form, usually having an indolent course but presenting in some cases as an aggressive disease with a poor prognosis. Sézary syndrome (SS) is a rare and aggressive variant, with erythroderma, leukemia, and lymph node involvement. Despite recent advances, CTCLs are still incurable cancers and the clinical symptoms are highly disabling. Among the landscape of genomic alterations identified in CTCL, we and others have described alterations of the PI3K pathway members (loss of PTEN, LKB1 and PDCD4, and gain of p70S6K). As a consequence, several SS patients show hyper-activation of PI3K/AKT/mTOR pathway. Hence, inhibition of this pathway represents a potential therapeutic choice against CTCL. Objective: In this work, we investigated the therapeutic potential of the dual-PI3K/mTOR inhibitor PF-04691502 (hereafter PF-502) against CTCL. Materials and Methods: PF-502 therapeutic efficacy was evaluated in four CTCL cell lines and in patient-derived SS cells, for its ability to inhibit cell growth, to induce apoptosis and to inhibit chemotaxis toward the chemokine SDF-1. Moreover, to evaluate PF-502 therapeutic effects in vivo we used a xenograft mouse model, consisting of nude mice transplanted with the CTCL-derived HH cell line. Results: PF-502 inhibited the cell growth of all the CTCL cell lines (0.07-0.32 µM, IC50 range), and of tumor cells derived from six SS patients (2.3 µM, mean IC50). The mechanism of action of PF-502 in CTCL cell lines was a combination of apoptosis induction and G1 block, while in SS patients it was mostly based on a strong apoptosis induction. Of note, PF-502 showed an only modest effect in T-lymphocytes from 3 healthy donors. PF-502 inhibited also cell migration toward the chemokine SDF-1, in both the systems. This result might have an important clinical implication because we have recently demonstrated that malignant lymphocytes are activated by the skin microenvironment. Thus, blocking the homing to the skin could affect the disease course. Finally, PF-502 has a robust antitumor activity in the xenograft mouse model, by reducing tumor volume (400 mm3 in treated mice vs 936 mm3 in controls) as well as tumor weight (0.2 g in treated mice vs 0.56 g in controls). Moreover, the Kaplan-Meier curve, in these mice, revealed that PF-502 treatment prolongs survival (P<0.005). Conclusions: Our results strongly support the therapeutic potential of targeting PI3K/AKT/mTOR pathway in CTCL. Citation Format: Antonella Bresin, Cristina Cristofoletti, Francesca Monzo, Elisabetta Caprini, Mauro Helmer Citterich, Alessandra Frezzolini, Alessandro Monopoli, Roberto Benucci, Maria Cantonetti, Enrico Scala, Giandomenico Russo, Maria Grazia Narducci. The PI3K/mTOR dual inhibitor PF-04691502 shows antitumor activity in Sezary cells and in a xenograft mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3912.

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.

Cytotoxic activity of pentachlorophenol and its active metabolites in SH-SY5Y neuroblastoma cells

As knowledge regarding mechanisms of pentachlorophenol (PCP) toxicity in neuronal cell lines is limited, the aim of the study was to evaluate the effects of PCP and its active metabolites, tetrachloro-1,4-benzoquinone (TCBQ) and tetrachlorohydroquinone (TCHQ) in human neuroblastoma SH-SY5Y cells. All compounds induced cytotoxic effects in time- and dose-dependent manners, and resulted in differential modes of cell death. Reduced mitochondrial membrane potential (ΔᴪM) and oxidative damage lead to apoptosis and necrosis following TCBQ and PCP exposure, respectively. Time-dependent investigations revealed transient ΔᴪM recovery in TCHQ exposed cells, and redox stress. Sufficient ΔᴪM recovery allowed apoptosis completion in TCHQ exposed cells, whereas overwhelming metabolic and oxidative stress saw a conversion from apoptotic to necrotic-like cell death. The onset of mitochondrial dysfunction preceded that of redox damage for all compounds, indicating that oxidative damage is secondary to ΔᴪM insult. Cytotoxic events were further linked to cell cycle. S phase and G2/M blocks were observed after 12 h exposure to TCBQ and TCHQ, respectively, while a G1 block occurred after 24 h exposure to PCP. This study provides new insight regarding time-dependant toxic effects of PCP and its metabolites in human neuronal cells.

Fanconi Anaemia-Like Mph1 Helicase Backs up Rad54 and Rad5 to Circumvent Replication Stress-Driven Chromosome Bridges.

Homologous recombination (HR) is a preferred mechanism to deal with DNA replication impairments. However, HR synapsis gives rise to joint molecules (JMs) between the nascent sister chromatids, challenging chromosome segregation in anaphase. Joint molecules are resolved by the actions of several structure-selective endonucleases (SSEs), helicases and topoisomerases. Previously, we showed that yeast double mutants for the Mus81-Mms4 and Yen1 SSEs lead to anaphase bridges (ABs) after replication stress. Here, we have studied the role of the Mph1 helicase in preventing these anaphase aberrations. Mph1, the yeast ortholog of Fanconi anaemia protein M (FANCM), is involved in the removal of the D-loop, the first JM to arise in canonical HR. Surprisingly, the absence of Mph1 alone did not increase ABs; rather, it blocked cells in G2. Interestingly, in the search for genetic interactions with functionally related helicases and translocases, we found additive effects on the G2 block and post-G2 aberrations between mph1Δ and knockout mutants for Srs2, Rad54 and Rad5. Based on these interactions, we suggest that Mph1 acts coordinately with these helicases in the non-canonical HR-driven fork regression mechanism to bypass stalled replication forks.

Effects of magnetic dihydroartemisinin nano-liposome in inhibiting the proliferation of head and neck squamous cell carcinomas

BackgroundDihydroartemisinin (DHA) was one of the most potent anticancer artemisinin-like compounds that had been proved by many researchers, but its application was limited by its own characteristics. PurposeMagnetic DHA nano-liposomes (DHA-MLPs) were developed to improve the targeting antitumor efficiency and bioavailability of DHA, and their physical properties were characterized. Study design and methodsLiposomes were prepared by thin film dispersion and orthogonal experimental design was used to optimize the formula. The magnetic targeting and antitumor effects of DHA-MLPs in the externally applied magnetic field was investigated in vitro and in vivo. ResultsThe mean particle size of DHA-MLPs was 209.10 ± 4.92 nm, the charge potential was -37.13 ± 1.01 mV, the encapsulation efficiency (E.E.%) was 82.12 ± 0.91%, and the saturation magnetization at room temperature was 11.84 emu g−1. Targeting DHA-MLPs as well as free DHA could lead to cell cycle G1 block and apoptosis of HNSCC tumor cells in vitro. The tumor volumes of targeting DHA-MLPs treated mouse group were distinctly decreased than that in the control group, free DHA group and non-targeting DHA-MLPs group (P < 0.05). It was observed from iron staining intensity that DHA-MLPs had significant targeting effect in magnetic field (P < 0.05). ConclusionThis novelty liposome could strengthen the ability of DHA in tumor suppression, by increasing the targeted delivery of DHA and biocompatibility, optimize the bioefficacy of DHA.

Zebrafish embryonic development-interfering macrolides from Streptomyces californicus impact growth and mitochondrial function in human colorectal cancer cells

Abstract Microorganisms are now recognized as a keysource of medicine. In this study, we aim to identify and characterize target-specific inhibitors from actinobacteria using zebrafish embryo developmental defect as readouts. From our preliminary screen, we evaluated 176 Malaysian actinobacteria extracts and from these, 15 extracts (8.52%) were observed to be toxic to embryos at 20 μg/mL at 72-h post fertilization (hpf). Detailed characterization of these 15 toxic hits uncovered one extract derived from Streptomyces californicus TY004-069 that arrested zebrafish embryonic development at 4–6 hpf after 24 h treatment, a phenotype indicative potentially targeting mitochondrial activity. Subsequent validation on one of the three isolated compounds from this extract, dimeric dinactin (DD) revealed its ability to disrupt mitochondrial membrane potential (MMP) in human colorectal HCT116 and HT29 cancer cells in a dose-dependent manner and impacting cell proliferation through a cell cycle G1 block. Overall, using zebrafish phenotypic assay, we identified a potential mitochondrial function inhibitor from actinobacteria that warrants further detailed analysis and also demonstrated that macrolides may hold promise as therapeutic molecules for human diseases including cancer.

Multipronged activity of combinatorial miR-143 and miR-506 inhibits Lung Cancer cell cycle progression and angiogenesis in vitro

Lung cancer (LC) is the leading cause of cancer-related deaths. Downregulation of CDK1, 4 and 6, key regulators of cell cycle progression, correlates with decreased LC cell proliferation. Enforced expression of miRNAs (miRs) is a promising approach to regulate genes. Here, we study the combinatorial treatment of miR-143 and miR-506 to target the CDK1, 4/6 genes, respectively. We analyzed the differential expression of CDK genes by qPCR, and western blot, and evaluated changes in the cell cycle distribution upon combinatorial treatment. We used an antibody microarray analysis to evaluate protein expression, focusing on the cell cycle pathway, and performed RNA-sequencing for pathway analysis. The combinatorial miR treatment significantly downregulated CDK1, 4 and 6 expression, and induced a shift of the cell cycle populations, indicating a G1 and G2 cell cycle block. The two miRs induces strong cytotoxic activity, with potential synergism, and a significant Caspase 3/7 activation. We identified a strong inhibition of tube formation in the presence or absence VEGF in an in vitro angiogenesis model. Together with the pathways analysis of the RNA-sequencing data, our findings establish the combinatorial miR transfection as a viable strategy for lung cancer treatment that merits further investigation.

Equol Enhances Apoptosis-inducing Activity of Genistein by Increasing Bax/Bcl-xL Expression Ratio in MCF-7 Human Breast Cancer Cells

ABSTRACTAnticancer activities of soy isoflavones, such as genistein and equol, a bioactive metabolite of daidzein, have been extensively studied because of possible involvement in the prevention of breast cancer. However, their interactions still remain unclear. We investigated here whether cytotoxic activity of genistein was enhanced by equol, using estrogen receptor positive MCF-7, HER2-positive SK-BR-3, and triple-negative MDA-MB-468 cell lines. Although cytotoxicity of genistein did not significantly differ between three subtypes of breast cancer cells, cytotoxic activities of genistein were significantly enhanced in combination with 50 μM equol in MCF-7 cells, but not in SK-BR-3 and MDA-MB-468 cells. In fluorescence activated cell sorting (FACS) analyses, MCF-7 cells were arrested at the G2/M by genistein but at G1/S by equol. Combination treatment arrested cells at G2/M but abolished equol-induced G1 block, indicating an antagonistic activity of genistein against equol in cell-cycle progression. Although apoptosis was not so evident with genistein alone, the combination made a drastic induction of apoptosis, accompanied by the increase of Bax/Bcl-xL expression ratio, without affecting the activities of Akt and mTOR. Taken together, these data suggest that enhancement of genistein activity by equol would be mainly mediated by augmented induction of apoptosis rather than arrest or delay of the cell cycle.

Abstract 2352: Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells

Abstract Background. Cell cycle is regulated by cyclin-dependent kinases (CDKs) activity, whose deregulation can lead to uncontrolled proliferation and cancer. Other CDKs are engaged in the regulation of transcription and post-transcriptional mRNA processing through the phosphorylation of the C-terminal domain of RNA polymerase II, such as CDK9 and CDK12. Inhibitors of cell cycle CDKs have been developed as anticancer agents and some of them are under clinical validation (e.g. palbociclib). While recent data would suggest that inhibition of CDK9 is feasible and has antitumor effect, the data on the therapeutic role of CDK12 inhibition are very scanty. Methods. Ovarian cancer cell lines were maintained in RPMI medium supplemented with 5% glutamine and 10% FBS. Cells were treated with different drug concentrations and after 72 hours cell survival was evaluated by MTS assay (Promega). IC50 values were calculated by interpolation method. Cell cycle analysis and apoptosis were performed with standard flow cytometric methods. A2780 and SKOV3 ovarian cancer cell lines knocked out for CDK12 were generated with CRISPR/CAS9 genome editing tool. Results. The cytotoxicity of palbociclib (a CDK4/6 inhibitor) and LDC000067 (a CDK9 inhibitor) was tested in a panel of ovarian cancer cell lines (A2780, SKOV3, OVCAR3, OVCAR5, OVCAR8, OVCA432, OVCA433, IGROV1, EFO27). Sensitivity of cells was similar for palbociclib and LDC000067, ranging from 10 to 33 and from 8 to 60 μM, respectively. A preferential G1 block was observed with palbociclib, while LDC000067 caused a S-G2 block. A higher induction of apoptosis was observed after LDC000067 than after palbociclib treatment in both A2780 and SKOV3. The palbociclib-induced G1 block was associated with decreased Rb phosphorylation, while no modulation of the Ser2 in the carboxyterminal domain of RNA polymerase II was observed after LDC000067 treatment. We generated CDK12 knocked out cells transfecting CRISPR/CAS9 engineered plasmid in both A2780 and SKOV3 ovarian cancer lines. The biological and pharmacological characterization of these clones is under study. Conclusions. Palbociclib and LDC000067 showed a dose dependent cytotoxic effect in the panel of ovarian cancer cell lines tested and were active in the μM range. Preliminary data of treatment induced cell cycle perturbation and apoptosis suggest that the two drugs behave in a different manner and have distinct molecular effects on cells. Citation Format: Rosaria Chilà, Nicolò Panini, Eugenio Erba, Giovanna Damia, Massimo Broggini. Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells [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 2352. doi:10.1158/1538-7445.AM2017-2352

Evaluation of sheep fed on multinutritional blocks with garlic extract

The aim of this study was to evaluate the effects of supplementation with three formulations of nutritional blocks and its consumption by 60 sheep from the Pantaneiro genetic group. The animals were weighed (35 kg ± 4.69) and separated into three groups of 20 each: G1 (control block), G2 (block with palatalising), and G3 (block + garlic extract). The solidified mineral supplement of silage of pearl millet (Pennisetum glaucum) provided ad libitum had an estimated consumption of 0.6%/kg/day. The sheep were kept in pastures in a grazing system cultivated with the grass Brachiaria brizantha ‘Marandu’. The best performance was obtained by G2 (P < 0.05) for weight gain, with 105 g of feed conversion ratio 75% more efficient than G1 and 107% more efficient than G3. The provision of multinutrient blocks for sheep kept in pastures of U. brizantha ‘Marandu’ contributed to the improvement of the animal's nutritional status. Among the formulations tested, the block with palatalising showed the highest rate of food conversion. The block with garlic extract had an inhibitory effect on reinfection by gastrointestinal nematodes after 30 days.

Small molecules targeting histone demethylase genes (KDMs) inhibit growth of temozolomide-resistant glioblastoma cells.

In glioblastoma several histone demethylase genes (KDM) are overexpressed compared to normal brain tissue and the development of Temozolomide (TMZ) resistance is accompanied by the transient further increased expression of KDM5A and other KDMs following a mechanism that we defined as “epigenetic resilience”. We hypothesized that targeting KDMs may kill the cells that survive the cytotoxic therapy.We determined the effect of JIB 04 and CPI-455, two KDM inhibitors, on glioblastoma cells and found that both molecules are more effective against TMZ-resistant rather than native cells.Because of its lower IC50, we focused on JIB 04 that targets KDM5A and other KDMs as well. We have shown that this molecule activates autophagic and apoptotic pathways, interferes with cell cycle progression, inhibits cell clonogenicity and dephosphorylates Akt thus inactivating a potent pro-survival pathway. We performed combination temozolomide/JIB 04 in vitro treatments showing that these two molecules, under certain conditions, have a strong synergic effect and we hypothesize that JIB 04 intercepts the cells that escape the G2 block exerted by TMZ. Finally we studied the permeability of JIB 04 across the blood-brain barrier and found that this molecule reaches bioactive concentration in the brain; furthermore a pilot in vivo experiment in an orthotopic GB xenograft model showed a trend toward longer survival in treated mice with an Hazard Ratio of 0.5.In conclusion we propose that the combination between cytotoxic drugs and molecules acting on the epigenetic landscape may offer the opportunity to develop new therapies for this invariably lethal disease.

Rare Coumarins Induce Apoptosis, G1 Cell Block and Reduce RNA Content in HL60 Cells

AbstractThe rare coumarins stenocarpin, stenocarpin isobutyrate, oficinalin, oficinalin isobutyrate, 8-methoxypeucedanin and the known xanthotoxin, isoimperatorin, bergapten, peucedanin and 8–methoxyisoimperatorin were isolated fromPeucedanum luxuriansTamamsch. (Apiaceae) and identified by means of spectral data (1D and 2D NMR). Their immunomodulating activity was evaluated by flow cytometry and their influence on HL60 cells as well as on PHA-stimulated PBLs was tested. All tested coumarins induce apoptosis (maximal in the 48 h culture) and decrease cell proliferation in a time- and dose-dependent manner, especially in HL60 cells. They also induce partial G1 block, but only in HL60 cells (at 100 µM concentrations). Dose-dependent reduction of RNA content was also found in G1 cells treated by the coumarins. All of the tested coumarins also possessed immunomodulatory activities. Bergapten and xanthotoxin were found to be the best candidates for further evaluation as anti-cancer drugs.

T Cells Encountering Myeloid Cells Programmed for Amino Acid-dependent Immunosuppression Use Rictor/mTORC2 Protein for Proliferative Checkpoint Decisions

Modulation of T cell proliferation and function by immunoregulatory myeloid cells are an essential means of preventing self-reactivity and restoring tissue homeostasis. Consumption of amino acids such as arginine and tryptophan by immunoregulatory macrophages is one pathway that suppresses local T cell proliferation. Using a reduced complexity in vitro macrophage-T cell co-culture system, we show that macrophage arginase-1 is the only factor required by M2 macrophages to block T cells in G1, and this effect is mediated by l-arginine elimination rather than metabolite generation. Tracking how T cells adjust their metabolism when deprived of arginine revealed the significance of macrophage-mediated arginine deprivation to T cells. We found mTORC1 activity was unaffected in the initial G1 block. After 2 days of arginine deprivation, mTORC1 activity declined paralleling a selective down-regulation of SREBP target gene expression, whereas mRNAs involved in glycolysis, gluconeogenesis, and T cell activation were unaffected. Cell cycle arrest was reversible at any point by exogenous arginine, suggesting starved T cells remain poised awaiting nutrients. Arginine deprivation-induced cell cycle arrest was mediated in part by Rictor/mTORC2, providing evidence that this nutrient recognition pathway is a central component of how T cells measure environmental arginine.