Arrested Cells In G1 Phase Research Articles

Exploring Melezitose as a Potential Therapeutic Agent in Lung Cancer: Inhibitory Effects on Cell Proliferation and EMT-Mediated Signaling in A549 Cells

Background Lung cancer is one of the most common cancers worldwide and a leading cause of cancer-related deaths. The study delves into melezitose, a naturally occurring compound known for its biocompatibility. Purpose This study aims to uncover its therapeutic potential and molecular mechanisms within lung cancer, particularly in A549 cells. Melezitose’s impact on inhibiting cell proliferation and influencing epithelial-mesenchymal transitions (EMT) was the primary focus. Material and Methods In a time-dependent manner, A549 cells, representative of lung adenocarcinoma (LUAD), underwent melezitose treatment. Analysis of cytotoxicity by MTT assay, cell migration assay, and its responsible genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit method. Results The MTT assay indicated a significant reduction in A549 cell growth after 48 hours of melezitose treatment. Additionally, melezitose induced G1 phase cell arrest and stimulated apoptosis in A549 cells. Subsequent determination of IC50 values represents the concentration at which melezitose inhibits 50% of cell growth. The study also investigated EMT-related gene expression like claudin 1 (CLD1), E-cadherin (ECADH), SNAIL1, SLUG, and vimentin (VIM) through RT-PCR. The findings revealed strong binding associations between melezitose and these EMT targets, suggesting a potential regulatory role of melezitose in impeding EMT processes. Conclusion Overall, this study illuminates the significant role of melezitose in lung cancer. Its observed inhibition of lung cancer cell proliferation and its influential impact on EMT-related gene expression highlights. Melezitose has potential as a therapeutic agent, particularly in the context of NSCC. The multifaceted effects of melezitose on A549 cells open promising avenues for advancing our understanding of this disease and developing innovative therapeutic strategies.

Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA

To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism. The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT. Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells. TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.

DDRE-03. THERAPEUTIC TARGETING OF THE ERK AND CDK PATHWAYS IN PRECLINICAL MODELS OF BRAIN METASTASES

Abstract Brain metastases (BM) are the most common neoplasm to affect the adult central nervous system. BM develop in 40-50% of advanced lung adenocarcinoma (LUAD), but the lack of durable response to chemotherapy, immunotherapy, or targeted therapy results in death within a year of BM diagnosis. Several advances have been made in identifying genetic drivers of primary cancers. The cell cycle, RAS and ERK pathways have all been implicated in as critical oncogenic regulators, with aberrations linked to driving the progression and metastasis of LUAD. Abemaciclib is a targeted CDK4/6 inhibitor, and LY3214996 is selective ERK1/2 inhibitor, and have shown efficacy in preclinical tumor models as well as in clinical trials. Furthermore, both therapeutics can interfere with the cell cycle, abemaciclib through targeting CDK4/6 and LY3214996 through cyclinD1. Here we present data assessing abemaciclib and LY3214996, as single and combined agents, in cell lines across different KRAS and CDKN2A mutational backgrounds. Seven days post-intracranial inoculation of NSCLC and NSCLC-BM line, mice received either abemaciclib, LY3214996, or a combination P.O. daily for 21 days, and were monitored pre- and post-treatment for tumor growth with bioluminescent imaging. In vitro we demonstrated a dose-dependent reduction in cell growth with each treatment, as well as cell arrest in G1 phase. In vivo, whereas cell lines with a combined KRAS mutation and CDKN2A mutation/deletion had no significant reduction in BM growth, cell lines with a CDKN2A del or BRAF mutation had significant BM reduction, with single agents and combined treatment. Further research is necessary to elucidate under what genetic contexts abemaciclib, LY3214996 or the combination are most effective. Nonetheless, this work highlights that abemaciclib and LY3214996 should be further explored for CDKN2A or BRAF mutant BM.

Protein Arginine Methyltransferase 5 Promotes Esophageal Squamous Cell Carcinoma Proliferation and Metastasis via LKB1/AMPK/mTOR Signaling Pathway.

Background: Esophageal squamous cell carcinoma (ESCC) is the eighth most common cancer in the world. Protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes symmetric and asymmetric methylation on arginine residues of histone and non-histone proteins, is overexpressed in many cancers. However, whether or not PRMT5 participates in the regulation of ESCC remains largely unclear.Methods: PRMT5 mRNA and protein expression in ESCC tissues and cell lines were examined by RT-PCR, western blotting, and immunohistochemistry assays. Cell proliferation was examined by RT-PCR, western blotting, immunohistochemistry assays, MTT, and EdU assays. Cell apoptosis and cell cycle were examined by RT-PCR, western blotting, immunohistochemistry assays, and flow cytometry. Cell migration and invasion were examined by RT-PCR, western blotting, immunohistochemistry assays, and wound-healing and transwell assays. Tumor volume, tumors, and mouse weight were measured in different groups. Lung tissues with metastatic foci, the number of nodules, and lung/total weight were measured in different groups.Results: In the present study, the PRMT5 expression level was dramatically upregulated in ESCC clinical tissues as well as ESCC cell lines (ECA109 and KYSE150). Furthermore, knocking down PRMT5 obviously suppressed cell migration, invasion, proliferation, and cell arrest in G1 phase and promoted cell apoptosis in ESCC cells. Meanwhile, downregulating PRMT5 also increased the expression levels of Bax, caspase-3, and caspase-9, while expression levels of Bax-2, MMP-2, MMP-9, and p21 were decreased, which are members of the cyclin-dependent kinase family. Furthermore, knocking down PRMT5 could increase the expression of LKB1 and the phosphorylation (p)-AMPK expression and decrease the p-mTOR level. Additionally, overexpression of LKB1 could reveal anti-tumor effects in ESCC cell lines by inhibiting ESCC cell, migration, invasion, and proliferation and accelerating cell apoptosis. Besides, upregulating LKB1 expression could increase the levels of Bax, caspase-3, and caspase-9 and weaken the levels of Bax-2, MMP-2, and MMP-9. Moreover, knocking down PRMT5 could weaken the tumor growth and lung metastasis in vivo with upregulating the LKB1 expression and the p-AMPK level and downregulating the p-mTOR expression.Conclusion: PRMT5 may act as a tumor-inducing agent in ESCC by modulating LKB1/AMPK/mTOR pathway signaling.

FOXQ1 promotes proliferation and metastasis of epithelial ovarian cancer via activation of SIRT1/NRF2 signaling pathway

Purpose: To investigate the role of FOXQ1 in the progression of epithelial ovarian cancer and the underlying mechanism.
 Methods: Forkhead Box Q1 overexpression was evaluated by quantitative reverse-transcription (qRTPCR) in clinical epithelial ovarian cancer samples and cell lines. Proliferation, migration, and invasion of cancer cells were determined using CCK8, wound healing and transwell assay.
 Results: FOXQ1 depletion inhibited the proliferation, migration, and invasion of `epithelial ovarian cancer cells. Moreover, FOXQ1 overexpression increased the amount of cells in S phase of the cell cycle, and FOXQ1 knockdown arrested cells inG1 phase. Results from ChIP and luciferase reporter assays showed that FOXQ1 was able to bind SIRT1 promoters. In addition, it was involved in sustaining the stability of nuclear factor erythroid derived 2-like 2 (NRF2) by decreasing its acetylation (p < 0.01), which was mediated by SIRT1. The data also demonstrated that NRF2 promotes proliferation, migration, and invasion of cancer cells upon FOXQ1 overexpression.
 Conclusion: Forkhead Box Q1 contributes to the progression of epithelial ovarian cancer partly via SIRT1/NRF2 signaling pathway, this highlighting a novel strategy for treating epithelial ovarian cancer.

Prolonged cetuximab treatment promotes p27Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma

Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is an efficient anti-tumor therapeutic agent that inhibits the activation of EGFR; however, data related to the cellular effects of prolonged cetuximab treatment are limited. In this study, the long-term cellular outcome of prolonged cetuximab treatment and the related molecular mechanism were explored in a head and neck squamous cell carcinoma cell line constitutively expressing a fluorescent ubiquitination-based cell cycle indicator. Fluorescent time-lapse imaging was used to assess clonal growth, cell motility, and cell-cycle progression. Western blot analysis was performed to measure the level of phosphorylation and protein-expression following cetuximab treatment. Over 5 days cetuximab treatment decreased cell motility and enhanced G1 phase cell arrest in the central region of the colonies. Significantly decreased phosphorylation of retinoblastoma, Skp2, and Akt-mTOR proteins, accumulation of p27Kip1, and induction of type II LC3B were observed over 8 days cetuximab treatment. Results of the present study elucidate the cetuximab-dependent inhibition of cell migration, resulting in high cell density-related stress and persistent cell-cycle arrest at G1 phase culminating in autophagy. These findings provide novel molecular insights related to the anti-tumor effects of prolonged cetuximab treatment with the potential to improve future therapeutic strategy.

Synthesis, biological evaluation and structure-activity relationship of novel dichloroacetophenones targeting pyruvate dehydrogenase kinases with potent anticancer activity.

Pyruvate dehydrogenase kinases (PDKs) are promising therapeutic targets that have received increasing attentions in cancer metabolism. In this paper, we report the synthesis and biological evaluation of a series of novel dichloroacetophenones as potent PDKs inhibitors. Structure-activity relationship analysis enabled us to identify a potent compound 6u, which inhibited PDKs with an EC50 value of 0.09μM, and reduced various cancer cells proliferation with IC50 values ranging from 1.1 to 3.8μM, while show weak effect against non-cancerous L02cell (IC50>10μM). In the A375 xenograft model, 6u displayed an obvious antitumor activity at a dose of 5mg/kg, but with no negative effect to the mice weight. Molecular docking suggested that 6u formed direct hydrogen bond interactions with Ser75 and Gln61 in PDK1, and meanwhile the aniline skeleton in 6u was sandwiched by the conserved hydrophobic residues Phe78 and Phe65, which contribute to the biochemical activity improvement. Moreover, 6u induced A375cell apoptosis and cell arrest in G1 phase, and inhibited cancer cell migration. In addition, 6u altered glucose metabolic pathway in A375cell by decreasing lactate formation and increasing ROS production and OCR consumption, which could serve as a potential modulator to reprogram the glycolysis pathway in cancer cell.

Nesfatin-1 Promotes Proliferation, Migration and Invasion of HTR-8/SVneo Trophoblast Cells and Inhibits Oxidative Stress via Activation of PI3K/AKT/mTOR and AKT/GSK3β Pathway.

Preeclampsia (PE) is a leading cause of perinatal and maternal mortality. Considering that Nesfatin-1 was reported to be downregulated in serum of PE patients, we aimed to explore the functional role of Nesfatin-1 in trophoblast cells. Cell transfection was conducted to overexpress or inhibit Nesfatin-1, and its expression was measured by quantitative PCR. Cell proliferation, migration, and invasion abilities were determined employing CCK-8, flow cytometry, wound-healing, and transwell assays. Immunofluorescence assay was performed to detect E-cadherin and vimentin. ROS, MDA, and SOD levels were measured using their corresponding commercial kits. Western blot was used to identify the expression of vital kinases in PI3K/AKT/mTOR or GSK3β pathway and invasion-related proteins in trophoblast cells. Nesfatin-1 knockdown significantly suppressed proliferation, migration, and invasion and increased cell arrest in G1 phase, as well as downregulated expressions of MMP2/9 in HTR-8/SVneo cells. Besides, Nesfatin-1 knockdown promoted the expression of E-cadherin and reduced the expression of vimentin. Additionally, the levels of ROS, MDA, and SOD were elevated upon Nesfatin-1 knockdown. On the contrary, Nesfatin-1 overexpression exerted the opposite effects. Nesfatin-1 promoted the activation of PI3K/AKT/mTOR or GSK3β pathway, blocking of which reversed the promotive effects on trophoblast invasion and the inhibitory effects on oxidative stress of Nesfatin-1 in HTR-8/SVneo cells. In short, this study revealed that Nesfatin-1 promoted trophoblast cell proliferation, migration, invasion, and EMT and suppressed oxidative stress by activating PI3K/AKT/mTOR and AKT/GSK3β signaling pathway, laying the foundation for the development of therapeutic strategy for PE by targeting Nesfatin-1.

Piwi-interacting RNAs play a role in vitamin C-mediated effects on endothelial aging.

The underlying mechanisms that mediate the effects of vitamin C on endothelial cell aging are widely unknown. To investigate whether Piwi-interacting RNAs (piRNAs) are involved in this process, an endothelial aging model was induced in vitro using H2O2 in human umbilical vein endothelial cells (HUVECs) and then treated with vitamin C (VC). Untreated HUVECs without H2O2 exposure were used to serve as the negative control group. Cell cycle, cell viability, and aging-associated protein expression were assessed, and RNA sequencing was performed to reveal the piRNA profile. Functional and regulatory networks of the different piRNA target genes were predicted by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis. H2O2 induced G1 phase cell arrest, decreased cell viability, and upregulated the senescence marker p16 in HUVECs. We found that VC treatment inhibited G1 phase cell arrest, increased the number of cells in the S and G2/M phases, increased cell viability, and decreased p16 expression. The piRNA expression profiles revealed that a large proportion of piRNAs that were differentially expressed in H2O2-treated HUVECs were partly normalized by VC. Furthermore, a number of piRNAs associated with the response to VC in H2O2-treated HUVECs were linked with senescence and cell cycle-related pathways and networks. These results indicate that the ability of VC to attenuate H2O2-mediated endothelial cell senescence may be associated with changes in expression of piRNAs that are linked to the cell cycle.

Inhibition of Obtusifolin on retinal pigment epithelial cell growth under hypoxia.

To explore the effect of Obtusifolin on retinal pigment epithelial cell growth under hypoxia. In vitro chemical hypoxia model of ARPE-19 cells was established using cobalt chloride (CoCl2). Cell viability was tested by cell counting kit-8 (CCK-8) assay. Western blot and real-time quantitative polymerase chain reaction were applied to detect proteins and mRNAs respectively. Flow cytometry was used to examine the cell cycle. Secretion of vascular endothelial growth factor (VEGF) was tested by using enzyme linked immunosorbent assay (ELISA). Under the chemical hypoxia model established by CoCl2, hypoxia inducible factor-1α (HIF-1α) mRNA and protein levels was up-regulated. Cell viability was increased and the proportion of S phase was higher. Obtusifolin could reduce cell viability under hypoxic conditions and arrest cells in G1 phase. Obtusifolin reduced the expression of Cyclin D1 and proliferating cell nuclear antigen (PCNA) in the hypoxic environment and increased the expression of p53 and p21. The levels of VEGF, VEGFR2 and eNOS proteins and mRNA were significantly increased under hypoxia while Obtusifolin inhibited the increasing. Obtusifolin can inhibit cell growth under hypoxic conditions and down-regulate HIF-1/VEGF/eNOS secretions in ARPE-19 cells.

ESRP1 Induces Cervical Cancer Cell G1-Phase Arrest Via Regulating Cyclin A2 mRNA Stability.

Accumulating evidence indicates that epithelial splicing regulatory protein 1 (ESRP1) can inhibit the epithelial-to-mesenchymal transition (EMT), thus playing a central role in regulating the metastatic progression of tumors. However, it is still not clear whether ESRP1 directly influences the cell cycle, or what the possible underlying molecular mechanisms are. In this study, we showed that ESRP1 protein levels were significantly correlated with the Ki-67 proliferative index (r = −0.521; p < 0.01), and that ESRP1 overexpression can significantly inhibit cervical carcinoma cell proliferation and induced G1-phase arrest by downregulating cyclin A2 expression. Importantly, ESRP1 can bind to GGUGGU sequence in the 3′UTR of the cyclin A2 mRNA, and ESRP1 overexpression significantly decreases the stability of the cyclin A2 mRNA. In addition, our experimental results confirm that ESRP1 overexpression results in enhanced CDC20 expression, which is known to be responsible for cyclin A2 degradation. This study provides the first evidence that ESRP1 overexpression induces G1-phase cell cycle arrest via reducing the stability of the cyclin A2 mRNA, and inhibits cervical carcinoma cell proliferation. The findings suggest that the ESRP1/cyclin A2 regulatory axis may be essential as a regulator of cell proliferation, and may thus represent an attractive target for cervical cancer prevention and treatment.

Abstract 1869: Mechanistic evaluation of ergosterol peroxide onin vitrobreast cancer models

Abstract Ganoderma lucidum extract (GLE), an extensively studied mushroom for its anti-cancer and anti-inflammatory properties, has been shown to possess physiologically active compounds such as polysaccharides, triterpenoids and sterols. In our previous studies, we reported that commercially available GLE whole mushroom extract selectively inhibits cancer cell viability, induces apoptosis, reduces invasion, and regulates key signaling molecules in various cell models of breast cancer. Hence, we sought to elucidate the compounds responsible for the observed anti-cancer activity. Among the isolated compounds from GLE, we identified a steroidal compound, Ergosterol Peroxide (EP), which has shown antiproliferative activity against cancer. EP is a member of natural endoperoxides, featuring the peroxy- warhead that can lead to hydroxyperoxy radicals or be activated and react as nucleophile, leading to significant biological cytotoxicity against various cancer models (e.g. ovarian, liver, colon). We hypothesized that EP would promote cell death mechanisms in the aggressive breast cancer phenotype. Thus, in this study, we evaluated EP’s anti-cancer potential using immunoblotting, colony formation, cell motility, cell cycle and reactive oxygen species (ROS) production assays against human breast cancer SUM-149, MCF-7, MDA-MB-231 cellular models. We show that EP displays anti-proliferative effects through cell arrest in G1 phase, and apoptosis induction via caspase 3/7 activation. EP induced ROS and decreased the migratory and invasive phenotype of these cancer cells. Moreover, it inhibited the expression of total AKT1, AKT2, BCL-2, BCL-XL, Cyclin D1 and c-Myc. Our combined results indicate that EP from GLE is a promising molecular scaffold for further exploration as an anti-cancer agent,particularly in aggressive breast cancer models. Citation Format: Camille L. Santiago, Fatima Rivas, Taotao Ling, Ivette J. Suarez-Arroyo, Gabriela Ortiz, Mercedes Y. Lacourt, Anibal Valentin, Walter H. Lang, Michelle M. Martínez-Montemayor. Mechanistic evaluation of ergosterol peroxide onin vitrobreast cancer models [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 1869.

Abstract 1871: Tannic acid: A natural anticancer agent for non-small cell lung cancer

Abstract Background Lung cancer is the leading cause of cancer deaths across globe. Statistically, 70-80% of lung cancer cases fall under non-small cells lung cancer (NSCLC) category. Recently, usage of natural compounds (e.g. EGCG, curcumin, resveratrol, apigenin, etc.,) for cancer therapy has gained lots of attention. However, poor dissolution profiles of these natural compounds lead to unfavorable pharmacological doses and administration. For this purpose, we have candidate, a water-soluble natural polyphenol, Tannic acid (TA, C76H52O46), which, a natural polyphenol that is richly found in plants such as green tea. This molecule exhibits anti-oxidant and anti-proliferative activities, which is being used as an alternative therapeutic option in cancer therapy. However, role of TA in NSCLC was not examined. Therefore, the aim of present study is to assess the molecular effects of TA in NSCLC cells. Methods For this study, two NSCLC cell lines (A549 and H1299) and one normal lung cell line (BEAS-2B) were used. We investigated the anti-proliferative effects of TA on NSCLC cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium (MTT), and colony formation assays. The anti-invasiveness and anti-migratory potential of TA was evaluated through Matrigel and Boyden chamber studies, respectively. Cell cycle and apoptotic effects of TA was evaluated through flow cytometry and western blot studies. The quantification of secreted VEGF was assessed by ELISA. Also, for affirmation results of gene expression profiles we performed q-PCR studies. Results Cell proliferation study demonstrated a dose dependent anti-cancer effects of TA on NSCLC cells. However, TA had no significant toxicity effects on human bronchial epithelial cells. Similarly, colony formation results exhibited dose dependence reduction in response to escalated dose of TA exposure. Both the invasion and migration results confirm its anti-metastatic role. Extracellular VEGF quantification showed lower VEGF secretion with TA treatments compared to the control cells. The cell cycle analysis evidently showed TA efficiently induced G1 phase cell arrest in NSCLC cells which was further confirmed through western blotting studies of relevant proteins (Cyclin D1, P53, p21, p18, BAX, BCL-2). We also achieved to show the decreased expression of VEGFR2, p-AKT, and cleaved caspase 7 indicating TA induces apoptosis. Conclusion Overall, these results demonstrate illustrate anti-cancer activity of TA by targeting the VEGF related pathways in NSCLC. This approach can lead to an alternative therapeutic strategy for lung cancer, with ideal PK/PD profiles. Citation Format: Elham Hatami, Prashanth K.B. Nagesh, Pallabita Chowdhury, Advit Bhaskar Shetty, Manish K. Tripathi, Subhash Chauhan, Meena Jaggi, Murali Yallapu. Tannic acid: A natural anticancer agent for non-small cell lung cancer [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 1871.

Cyclin-dependent kinase inhibitor 2B gene is associated with the sensitivity of hepatoma cells to Sorafenib.

Purpose: The sensitivity of advanced hepatocellular carcinoma (HCC) to Sorafenib is low. The purpose of this study was to investigate the effects of cyclin-dependent kinase inhibitor 2B (CDKN2B) gene on the prognosis of HCC and the sensitivity of HCC cells to Sorafenib.Patients and methods: Streptavidin-perosidase (SP) staining was performed to determine the expression of CDKN2B in HCC tissues and adjacent tissues. The cell counting kit-8 (CCK-8) assay was carried out to determine cell viability. CDKN2B mRNA and protein were tested by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. CDKN2B gene was silenced or over-expressed in the cells by plasmid transfection technique. Flow cytometry was carried out to detect cell cycle and apoptosis.Results: SP staining results showed that CDKN2B was positive in adjacent tissues and in HCC tissues from partial response (PR) patients, CDKN2B was slightly positive in stable disease (SD) patients, but negative in progression disease (PD) patients. The survival rate of patients with low expression of CDKN2B was low. Up-regulation of CDKN2B expression could promote the pro-apoptotic effect of Sorafenib and cell arrest in G1 phase. When the CDKN2B gene expression was down-regulated, the cell apoptosis rate and the proportion of cells treated with Sorafenib in G1 phase decreased. Silencing CDKN2B reversed CDKN2B overexpression caused by Sorafenib.Conclusion: CDKN2B genes were lowly expressed in tumor tissues from HCC patients who were treated with Sorafenib and had a poor prognosis. Up-regulation of CDKN2B promoted sensitivity of HCC to Sorafenib, and similarly down-regulation of CDKN2B reduced the sensitivity.

Silencing of Long Non-Coding RNA Myocardial Infarction-Associated Transcript Suppresses Progression of Gastric Cancer

Background: Long noncoding RNAs (lncRNAs) have been consistently demonstrated to be involved in gastric cancer (GC) as either tumor oncogenes or tumor suppressors. However, the detailed role of MIAT in GC remains poorly understood. Methods: The expression of MIAT in GC tissues was measured by In situ hybridization (ISH) assay. Cell proliferation, apoptosis, cycle, migration and invasion assays were performed to analyze the biological functions of MIAT in GC cells. Besides, western blotting was used to evaluate the role of MIAT in the expressions of P16, COX-2 and MMP-9. Results: In the present study, we identified that MIAT was up-regulated in GC tissues. Furthermore, silencing MIAT significantly suppressed GC cells proliferation, migration and invasion, promoted GC cells apoptosis, and induced GC cells cycle arrest in G1 phase. Additionally, knockdown of MIAT notably up-regulated the protein level of P16 and down-regulated the protein levels of COX-2 and MMP-9. Conclusion: These observations imply that silencing MIAT inhibits the proliferation, migration and invasion, promotes apoptosis, and induces cell arrest in G1 phase, partially through up-regulating the expression level of P16 and down-regulating the expression levels of COX-2 and MMP-9, indicating that MIAT may a novel biomarker and therapeutic target for GC.

Antioxidant, Anti-Lung Cancer, and Anti-Bacterial Activities of Toxicodendron vernicifluum.

This work tested antioxidant, anti-lung cancer, and antibacterial activities by in vitro, in vivo, and computational experiments for the metabolites extracted from the bark, seed, and stem of Toxicodendron vernicifluum. The results showed that all the extracts significantly scavenged 1,2-diphenyl-1-picrylhydrazyl (DPPH) in a dose-dependent manner. But, the total phenol content (TPC) ranged from 2.12 to 89.25% and total flavonoids content (TFC) ranged from 1.02 to 15.62% in the extracts. The methanolic bark extract (MBE) exhibited higher DPPH scavenging activity than the other extracts, probably due to the higher content of the TPC and TFC present in it. Among the extracts, only the MBE showed anti-lung cancer activity at an acceptable level with a therapeutic index value (22.26) against human lung carcinoma. This was due to the cancer cell death in A549 induced by MBE through reactive oxygen species (ROS) generation, apoptosis, and cell arrest in G1 phase and inhibition of anti-pro-apoptotic protein survivin. Among the extracts, MBE showed significantly higher antibacterial activity as evident through the higher zone of inhibition 13 ± 0.5 mm against methycilin resistant strain of Staphylococcus aureus (MRSA), Salmonila enteria subp. enterica, and P. aeruginosa, 11 ± 0.3 mm against E. coli and 10 ± 0.2 mm against B. cereus. The MBE also showed an excellent antibacterial activity with lower minimal inhibitory concentration (MIC). Particularly, the MBE showed more significant antibacterial activity in MRSA. The in vivo antibacterial activity of the MBE was further tested in C. elegans model. The treatment of the MRSA induced cell disruption, damage and increased mortality of C. elegans as compared to the untreated and MBE treated C. elegans with normal OP50 diet. Moreover, the MBE treatment enhanced the survival of the MRSA infected C. elegans. The compounds, such as 2,3,3-trimethyl-Octane and benzoic from the MBE, metabolized the novel bacterial topoisomerases inhibitor (NBTI) and MRSA related protein (PBP2a). Overall the T. vernicifluum is potentially bioactive as evident by antioxidant, anti-lung cancer, and antibacterial assays. Further studies were targeted on the purification of the novel compounds for the clinical evaluation.

Abstract LB-400: Tannic acid induces prostate cancer cell death via unfolded protein response (UPR) and modulation of CHOP

Abstract Objectives: Endoplasmic reticulum (ER) is an intricate organelle that is crucial for cellular function and survival. Cellular environments that interfere with ER functioning can lead to the accumulation of unfolded proteins, which are sensed by transmembrane sensors that instigate the unfolded protein response (UPR) to reinstate ER proteostasis. When the UPR is perturbed or not sufficient to deal with the stress conditions, apoptotic cell death is ensued. Often, prostate tumor cells (PrCa) are exposed to intrinsic and external factors that alter protein homeostasis producing endoplasmic reticulum (ER) stress. IRE1, PERK, and ATF6 are key signaling stems of the UPR during ER stress. At molecular level, dimerization of PERK kinase phosphorylates eIF2α, resulting in translation attenuation. This selectively enhances translation of the ATF4 transcription factor. Also, activated IRE1 stimulates the nuclear translocation of XBP1. Both ATF4 and XBP1 induces expression of UPR genes. The UPR stimulates and protects tumor cells against stressful conditions within the tumor microenvironment. Therapeutic intervention on this aspect ER Stress mediated apoptosis is much needed. Here, we examined the mechanistic role of tannic acid (TA) through novel targeting inhibition of UPR via modulation of ER stress in PrCa cell death. Methods: In vitro therapeutic perspective of tannic acid was evaluated using clinically relevant human prostate cancer cell line models (C4-2, PC3 and DU145) through cell proliferation and clonogenic assays. The anti-metastatic potential of TA in PrCa cells was determined using invasive and migration studies. The phase arrest and concomitant apoptosis in PrCa cells were examined using Propidium Iodide based flow cytometric studies. TA induced ER stress-mediated UPR cellular apoptotic activity was confirmed using Western blot, real-time studies, and nuclear distortion studies. Results: Protein profiling results have showed yin approach of inhibition ER stress during TA treatments with the inhibition of CHOP protein. Also, we observed TA treated PrCa cells was able to induce ER stress response proteins, such as PERK and IRE1. Its downstream signaling events include the induction of XBP1, EIF2α expression key mediators by alteration of Bcl2/Bax ratio from shifting survival towards apoptosis. With this line we assessed the expression profile of apoptosis-associated markers such as Bid and Bim (upregulated), and Bcl-2 and Bcl-xL which ratifies the induction of apoptosis during treatments. TA exhibited prominent growth arrest at G1 phase and increase of concentration led to elevated sub G1, annotating the increase of apoptotic cells. The expression of P18 and P21 were increased during TA treatments and cyclin D1 expression was inhibited supporting the G1 phase cell arrest. The expression of MMP2 and MMP9 were reduced, signifying the superior anti-metastatic ability of this compound. Cell proliferation studies showed a dose-dependent inhibitory profile of TA. Distinct results of anti-migratory and anti-metastatic effects of TA was observed during treatments. Conclusion: This study suggests that TA can efficiently induce UPR and promote ER stress signaling in cells that renders apoptosis in PrCa cells by the over expression of CHOP. We believe this novel therapeutic paradigm have clinical significance in advanced PrCa. Citation Format: Elham Hatami, Prashanth Kumar Bhusetty Nagesh, Pallabita Chowdhury, Vivek K. Kashyap, Sheema Khan, Bilal Hafeez, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. Tannic acid induces prostate cancer cell death via unfolded protein response (UPR) and modulation of CHOP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-400.

Bufadienolides from Venenum Bufonis Inhibit mTOR‐Mediated Cyclin D1 and Retinoblastoma Protein Leading to Arrest of Cell Cycle in Cancer Cells

Objective Bufadienolides, the main components in Venenum Bufonis secreted from toads, have been proved to be with significant anticancer activity aside from the positive inotropic action as cardenolides. Here an underlying anticancer mechanism was further elucidated for an injection made from Venenum Bufonis containing nine bufadienolides. Methods One solution reagent and cell cycle analyses were for determining effect of bufadienolides on cancer cells. Western blotting was used for protein expression. Results Bufadienolides inhibit cell proliferation and arrest cells in G1 phase. Bufadienolides also inhibit the mammalian target of rapamycin (mTOR) signaling pathway, which is evidenced by the data that bufadienolides inhibit type I insulin-like growth factor- (IGF-1-) activated phosphorylation of mTOR by a concentration- and time-dependent way, as well as phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Subsequent results indicated that cyclin D1 expression and phosphorylation of retinoblastoma protein (Rb)—two characterized regulators in cell cycle of G1—are also inhibited and the process is dependent on mTOR pathway. Conclusion Bufadienolides inhibit proliferation partially due to arresting cell cycle in G1 phase, which is mediated by inhibiting mTOR-cyclin D1/Rb signal pathway.

Experimental study of inhibition of tendon fibroblast growth by chitosan through the pathway of TGF-β1/Smad3 mediated by miR-29b

Objective To study the molecular mechanism of chitosan inhibiting fibroblast growth after tendon repair. Methods The rats with Achilles tendon injury were treated with chitosan during operation, and the sliding distance and total collagen content were examined 8 weeks postoperatively. Simultaneously, the fibroblasts in the repaired tendon tissue were separated and treated with different concentrations of chitosan. The survival rate, apoptosis and cell cycle of the cells were examined. In addition, the expression of miRNAs was quantified by real-time PCR and expression of TGF-β1, Smad3 and P21were quantified by western blot. Results Chitosan can increase the tendon gliding distance, reduce the content of collagen fibers, inhibit the growth of fibroblasts and arrest cells in G1 phase of the growth cycle. Chitosan also up-regulated the expression of miR-29b and P21 but down-regulated the levels of TGF-β1and Smad3 in repaired tendon tissue and in vitro fibroblasts. In fibroblasts, miR-29b inhibitor can reverse the expression changes of miR-29b、TGF-β1、Smad3 and P21 induced by chitosan. Conclusion Chitosan can promote the repair of Achilles tendon injury in rats. miR-29b causes fibroblasts staying at G1 phase by down-regulating the expression of TGF-β1and Smad3 and up-regulating the expression of P21. It may be one of the mechanisms of chitosan playing a positive role in the process of tendon repair. Key words: Tendon injuries; Chitosan; Fibroblasts; miR-29b

Cell Cycle Arrest as a Therapeutic Target of Acute Kidney Injury.

The current lack of complete understanding of the pathogenesis of acute kidney injury (AKI) is a significant barrier to its early diagnosis and treatment. Cell cycle arrest plays an important role in the protection of renal tubular epithelial cells and maladaptive repair following AKI. G1 phase cell arrest serves as a protective mechanism following AKI, avoiding replication of damaged DNA. Insulinlike growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinase-2 (TIMP-2) are closely associated with G1 cell cycle arrest during the very early phase of cellular damage and can serve as an ideal biomarker to predict AKI. However, sustained cell cycle arrest after severe AKI may result in cell senescence and maladaptive repair, with typical characteristics of the development of cell cycle arrest in the gap 2 (G2) or mitotic (M) phase. Markers of cell cycle arrest signal and spread the "alarm" from the site of injury to adjacent cells in an autocrine or paracrine manner, giving rise to abnormal amplification and release of profibrogenic factors, activation of pericytes/perivascular fibroblasts, and eventually fibrosis. Therefore, cell cycle regulation has become a potentially new target for the prevention and treatment of AKI. In this review, we summarize the characteristics of the cell cycle following AKI and the markers of cell cycle arrest that enable the early detection of AKI. We also discuss how to prevent the progression of chronic kidney disease (CKD) by regulating cell cycle arrest.