Treatment Increased Cell Proliferation Research Articles

Role of Histamine and Related Signaling in Kaposi's Sarcoma-Associated Herpesvirus Pathogenesis and Oncogenesis.

Although Kaposi's sarcoma-associated herpesvirus (KSHV) has been reported to cause several human cancers including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), the mechanisms of KSHV-induced tumorigenesis, especially virus-host interaction network, are still not completely understood, which therefore hinders the development of effective therapies. Histamine, together with its receptors, plays an important role in various allergic diseases by regulating different inflammation and immune responses. Our previous data showed that antagonists targeting histamine receptors effectively repressed KSHV lytic replication. In the current study, we determined that histamine treatment increased cell proliferation and anchorage-independent growth abilities of KSHV-infected cells. Furthermore, histamine treatment affected the expression of some inflammatory factors from KSHV-infected cells. For clinical relevance, several histamine receptors were highly expressed in AIDS-KS tissues when compared to normal skin tissues. We determined that histamine treatment promoted KSHV-infected lymphoma progression in immunocompromised mice models. Therefore, besides viral replication, our data indicate that the histamine and related signaling are also involved in other functions of KSHV pathogenesis and oncogenesis.

Neural stem cell-derived extracellular vesicles favour neuronal differentiation and plasticity under stress conditions.

Extracellular vesicles (EVs) are released by all cell types and are involved in intercellular communication. We evaluated if neural stem cells-derived EVs (NSC-EVs) regulate NSCs proliferation and differentiation under control and stress conditions. We found that NSC-EVs treatment increases cell proliferation and promotes neuronal differentiation and plasticity. The fact that nervous tissue poorly recovers after cellular damage, prump us to evaluate the effect of EVs supplementation under oxidative stress and inflammation. We demonstrate that NSC-EVs restore the proliferative potential of the NSCs affected by oxidative stress. In addition, we provide evidence that oxidative stress and inflammation induce neuronal differentiation. Interestingly, the aberrant cell phenotype induced by inflammation is restored by NSC-EVs treatment, suggesting that these vesicles ameliorate the damage burden in neurons and modulate neuronal plasticity. These results contribute to understand the role of the NSCs-derived EVs as key players for brain tissue generation and regeneration and open new pathways to the development of therapies.

Immunomodulatory Effects of an Aqueous Extract of Black Radish on Mouse Macrophages via the TLR2/4-Mediated Signaling Pathway.

Here, we determined the immunostimulatory effects of black radish (Raphanus sativus ver niger) hot water extract (BRHE) on a mouse macrophage cell line (RAW 264.7) and mouse peritoneal macrophages. We found that BRHE treatment increased cell proliferation, phagocytic activity, nitric oxide (NO) levels, cytokine production, and reactive oxygen species synthesis. Moreover, BRHE increased the expression of the following immunomodulators in RAW 264.7 cells and peritoneal macrophages: pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), iNOS, and COX-2. BRHE treatment significantly up-regulated the phosphorylation of components of the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), Akt, and STAT3 signaling pathways. Further, the effects of BRHE on macrophages were significantly diminished after the cells were treated with the TLR2 antagonist C29 or the TLR4 antagonist TAK-242. Therefore, BRHE-induced immunostimulatory phenotypes in mouse macrophages were reversed by multiple inhibitors, such as TLR antagonist, MAPK inhibitor, and Akt inhibitor indicating that BRHE induced macrophage activation through the TLR2/4-MAPK-NFκB-Akt-STAT3 signaling pathway. These results indicate that BRHE may serve as a potential immunomodulatory factor or functional food and provide the scientific basis for the comprehensive utilization and evaluation of black radish in future applications.

Platelet-derived exosomes induce cell proliferation and wound healing in human endometrial cells.

Aim: To investigate the regenerative effects of a platelet-derived purified exosome product (PEP) on human endometrial cells. Materials & methods: Endometrial adenocarcinoma cells (HEC-1A), endometrial stromal cells (T HESC) and menstrual blood-derived stem cells (MenSC) were assessed for exosome absorption and subsequent changes in cell proliferation and wound healing properties over 48 h. Results: Cell proliferation increased in PEP treated T HESC (p<0.0001) and MenSC (p<0.001) after 6 hand in HEC-1A (p<0.01) after 12 h. PEP improved wound healing after 6 hin HEC-1A (p<0.01) and MenSC (p<0.0001)and in T HESC between 24 and 36 h(p<0.05). Conclusion: PEP was absorbed by three different endometrial cell types. PEP treatment increased cell proliferation and wound healing capacity.

TSP1 promotes fibroblast proliferation and extracellular matrix deposition via the IL6/JAK2/STAT3 signalling pathway in keloids.

Keloids are benign fibroproliferative diseases with abnormally proliferated bulges beyond the edge of the skin lesions, and they are characterized by uncontrolled fibroblast proliferation and excessive extracellular matrix deposition in the dermis. However, the definite mechanisms that increase fibroblast proliferation and collagen deposition in keloids remain unclear. Thrombospondin 1 (TSP1) has been suggested to play an important role in wound healing and fibrotic disorders, but its role in keloids is unknown. In this study, we aimed to clarify the specific role of TSP1 in keloids and explore the potential mechanism. Our results demonstrated that TSP1 was highly expressed in keloid lesions compared to normal skin. Knockdown of TSP1 in keloid fibroblasts decreased cell proliferation and collagen I deposition. Exogenous TSP1 treatment increased cell proliferation and collagen I deposition in normal fibroblasts. We further investigated the underlying mechanism and found that TSP1 promoted fibroblast proliferation and extracellular matrix deposition by upregulating the IL6/JAK2/STAT3 pathway. Moreover, we verified that TSP1 expression was positively correlated with IL6/STAT3 signalling activity in keloids. Taken together, our findings indicate that TSP1 promotes keloid development via the IL6/JAK2/STAT3 signalling pathway and blocking TSP1 may represent a potential strategy for keloid therapy.

Cold atmospheric plasma modification and electrical conductivity induction in gelatin/polyvinylidene fluoride nanofibers for neural tissue engineering.

This research follows some investigations through neural tissue engineering, including fabrication, surface treatment, and evaluation of novel self-stimuli conductive biocompatible and degradable nanocomposite scaffolds. Gelatin as a biobased material and polyvinylidene fluoride (PVDF) as a mechanical, electrical, and piezoelectric improvement agent were co-electrospun. In addition, polyaniline/graphene (PAG) nanoparticles were synthesized and added to gelatin solutions in different percentages to induce electrical conductivity. After obtaining optimum PAG percentage, cold atmospheric plasma (CAP) treatment was applied over the best samples by different plasma variable parameters. Finally, the biocompatibility of the scaffolds was analyzed and approved by in vitro tests using two different PC12 and C6 cell lines. In the present study the morphology, FTIR, dynamic light scattering, mechanical properties, wettability, contact angle tests, differential scanning calorimetric, rate of degradation, conductivity, biocompatibility, gene expression, DAPI staining, and cell proliferation were investigated. The PAG percentage optimization results revealed fiber diameter reduction, conductivity enhancement, young's modulus improvement, hydrophilicity devaluation, water uptake decrement, and degradability reduction in electrospun nanofibers by increasing the PAG concentration. Furthermore, ATR-FTIR, FE-SEM, AFM, and contact angle tests revealed that helium CAP treatment improves scaffold characterizations for 90s in duration time. Furthermore, the results of the MTT assay, FE-SEM, DAPI staining, and RT-PCR revealed that samples containing 2.5% w/w of PAG are the most biocompatible, and CAP treatment increases cell proliferation and improves neural gene expression in the differentiation medium. According to the results, the samples with the 2.5% w/w of PAG could provide a suitable matrix for neural tissue engineering in terms of physicochemical and biological.

Crosstalk between progesterone receptor membrane component 1 and estrogen receptor α promotes breast cancer cell proliferation

Progesterone (P4) and estradiol (E2) have been shown to stimulate and regulate breast cancer proliferation via classical nuclear receptor signaling through progesterone receptor (PR) and estrogen receptor α (ERα), respectively. However, the basis of communication between PR/ERα and membrane receptors remains largely unknown. Here, we aim to identify classical and nonclassical endocrine signaling mechanisms that can alter cell proliferation through a possible crosstalk between PR, ERα, and progesterone receptor membrane component 1 (PGRMC1), a membrane receptor frequently observed in breast cancer cells. While P4 and E2 treatment increased cell proliferation of ER+/PR+/PGRMC1 overexpressing breast cancer cells, silencing ERα and PR or treatment with selective estrogen receptor modulator (SERM) tamoxifen, or (PR-antagonist) RU-486 decreased cell proliferation. All four treatments rapidly altered PGRMC1 mRNA levels and protein expression. Furthermore, P4 and E2 treatments rapidly activated EGFR a known interacting partner of PGRMC1 and its downstream signaling. Interestingly, downregulation of ERα by tamoxifen and ERα silencing decreased the expression levels of PGRMC1 with no repercussions to PR expression. Strikingly PGRMC1 silencing decreased ERα expression irrespective of PR. METABRIC and TCGA datasets further demonstrated that PGRMC1 expression was comparable to that of ERα in Luminal A and B breast cancers. Targeting of PR, ERα, and PGRMC1 confirmed that a crosstalk between classical and nonclassical signaling mechanisms exists in ER+ breast cancer cells that could enhance the growth of ER+/PR+/PGRMC1 overexpressing tumors.

Puerarin blocks the aging phenotype in human dermal fibroblasts.

Dermal fibroblast aging contributes to aging-associated functional defects in the skin since dermal fibroblasts maintain skin homeostasis by interacting with the epidermis and extracellular matrix. Here, we found that puerarin, an isoflavone present in Pueraria lobata (Kudzu), can prevent the development of the aging-phenotype in human dermal fibroblasts. Normal human dermal fibroblasts (NHDFs) were subcultivated and high-passage cells were selected as senescent cells, whereas low-passage cells were selected as a young cell control. Puerarin treatment increased cell proliferation and decreased the proportion of senescence-associated beta-galactosidase-positive cells in a high-passage culture of NHDFs. Moreover, puerarin treatment reduced the number of smooth muscle actin (SMA)-positive myofibroblasts and the expression of a reticular fibroblast marker, calponin 1 (CNN1), which were induced in high-passage NHDFs. Fulvestrant, an estrogen receptor antagonist, blocked the puerarin-mediated downregulation of SMA and CNN1. Our results suggest that puerarin may be a useful functional food that alleviates aging-related functional defects in dermal fibroblasts.

Abstract 3435: Characterizing the dependence of AP-1 transcription factors on Estrogen Receptor Alpha transcriptional activity and Fulvestrant sensitivity in ovarian cancer cells

Abstract Ovarian cancer is the leading cause of death among gynecologic malignancies. Epidemiological evidence has suggested a role for steroid hormones in the pathogenesis of ovarian cancer, however, their precise role in ovarian cancer remains unknown. We hypothesized that Estrogen Receptor α (ERα) drives a transcriptional program that is sufficient to promote ovarian cancer cell proliferation in ER+ ovarian cancers. Preliminary data generated in our laboratory has suggested that estradiol (E2) treatment increases cell proliferation in PEO1 and OVKATE cells, ERα+ ovarian cancer cell lines. This increase in cell proliferation can be inhibited by co-administration with the selective estrogen receptor modulator, Tamoxifen, and the selective estrogen receptor degrader, Fulvestrant. To further interrogate the mechanism of action of ERα in ovarian cancer, we performed RNA-seq on PEO1 cells treated with Vehicle, 10 nM E2, 100 nM Fulvestrant, and E2+Fulvestrant for 4 and 24 hours. We identified 86 significantly differentially expressed genes following 4 hr E2 treatment and 659 significantly differentially expressed genes following 24 hr E2 treatment. Fulvestrant inhibited the majority of E2-induced differentially expressed genes, confirming that these genes are dependent upon ERα. Gene Set Enrichment Analysis (GSEA) indicated that the Hallmark early and late estrogen responses and G2/M checkpoint as positively enriched in our dataset, indicating that classical ER pathways are intact in these cells. To further characterize the transcriptional role of ERα in ovarian cancer cells, ChIP-seq was performed on PEO1 cells treated with Vehicle, E2, Tamoxifen, or E2+Tamoxifen for 45 minutes. E2 treatment robustly increased ER recruitment to its regulatory regions when compared to Vehicle or Tamoxifen alone. Motif analysis of these ERα binding sites demonstrated a significant enrichment in members of the AP-1 transcription factor family, but not in known ERα cofactors FOXA1 and GATA3. Furthermore, using GIGGLE analysis to identify similar ChIP-seq datasets to the PEO1 dataset, we identified FOSL1, FOSL2, JUND, JUN, and FOS as among the datasets with the highest similarity scores to the E2 PEO1 dataset. In order to identify potential combinatorial endocrine therapies for ovarian cancer, we performed a genome-wide CRISPR screen in the presence of Fulvestrant in PEO1 cells. Among the top positively selected genes in the presence of Fulvestrant was the AP-1 family member, FOSL2, indicating that loss of FOSL2 contributes to Fulvestrant resistance and further confirmed the role of AP-1 factors in estrogen function. We also identified genes that confer sensitivity to Fulvestrant treatment; the E2F, PI3K/mTOR, and steroid hormone biosynthesis pathways are enriched in the Fulvestrant sensitizer genes. Further analysis is currently underway to determine synergistic combinations with Fulvestrant for the treatment of ovarian cancer. Citation Format: Irene I. Lee, Myles Brown. Characterizing the dependence of AP-1 transcription factors on Estrogen Receptor Alpha transcriptional activity and Fulvestrant sensitivity in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3435.

Effects of Promethazine on Proliferation and Apoptosis of Myocardial Cells in Rats with Myocardial Ischemia-Reperfusion Injury Through PI3K/Akt Signaling Pathway

Objective: To assess promethazine's effect on myocardial cells in rats with myocardial ischemiareperfusion injury (MIRI). Methods: The rat MIRI model was established and treated as the ischemia group. MIRI rats were treated with promethazine and included as the drug group. Rats only undergoing thoracotomy were enrolled as the control group. The physiological function of heart was assessed using the ultrasound cardiotachograph, and the apoptosis and proliferation of myocardial cells were detected using TUNEL assay and Ki67 staining, respectively. Moreover, the expressions of Caspase-3, Bcl-2, PI3K, GSK-3, PDK-1 and PKB were determined via Western blotting and qPCR. Results: There were significant differences in cardiac function indexes [left ventricular enddiastolic diameter (LVEDd), left ventricular end systolic diameter (LVESd), ejection fraction (EF) and fractional shortening (FS)] among the three groups (p= 0 002, 0.004, 0.025 and 0.012), and ischemia group had the highest LVEDd [(8.73± 0.31) mm] and LVESd [(7.98± 0.37) mm] and lowest EF [(42± 3.8)%] and FS [(40.3± 2.8)%]. The number of apoptotic myocardial cells was significant higher in ischemia group than control ( p&lt; 0 05), while it was significantly declined after treatment with promethazine ( p&lt; 0 05). Caspase-3 was significantly upregulated and Bcl-2 was downregulated in ischemia group which were all significantly reversed in drug group. Besides, Ki67 level was significantly reduced in ischemia group compared to control and higher in drug group than ischemia group, indicating that drug treatment increased cell proliferation ability. The levels of PI3K, GSK-3 and PKB in myocardial tissues were significantly declined in ischemia group and elevated after the treatment with promethazine without difference of PDK-1 level in myocardial tissues among the three groups. Conclusion: Promethazine inhibits apoptosis and promotes proliferation of myocardial cells in MIRI rats through PI3K/Akt signaling pathway.

Abstract GMM-038: DECIPHERING THE ROLE OF ESTROGEN RECEPTOR ALPHA IN OVARIAN CANCER

Abstract Ovarian cancer is the leading cause of death among gynecologic malignancies. Despite years of research, the 5-year survival rate for ovarian cancer patients has remained poor and the standard of care is largely the same as it was decades ago. Epidemiological evidence and previous studies have suggested a role for steroid hormones in the pathogenesis of ovarian cancer, however, the role of steroid hormones in ovarian cancer remains understudied. We hypothesized that Estrogen Receptor α (ERα) is transcriptionally active and drives a transcriptional program that is sufficient to promote ovarian cancer cell proliferation and survival in a subset of ER+ ovarian cancers. Preliminary data generated in our laboratory has suggested that estradiol (E2) treatment increases cell proliferation in PEO1 cells, an ERα+ ovarian cancer cell line. This increase in cell proliferation can be inhibited by co-administration with the selective estrogen receptor modulator, Tamoxifen, and the selective estrogen receptor degrader, Fulvestrant. To further interrogate the mechanism of action of ERα in ovarian cancer, we performed RNA-seq on PEO1 cells treated with Vehicle, 10 nM E2, 100 nM Fulvestrant, and E2+Fulvestrant for 24 hours. We identified 659 significantly differentially expressed genes following E2 treatment. Fulvestrant inhibited the majority of E2-induced differentially expressed genes, confirming that these genes are dependent upon ERα. Gene Set Enrichment Analysis (GSEA) indicated that the Hallmark early and late estrogen responses are enriched in the E2 dataset, confirming that classical ER activity is intact in these cells. Furthermore, we identified G2/M checkpoint as positively enriched in our dataset, indicating that proliferation genes are upregulated by ERα in these cells. Additionally, we found that apoptosis was negatively enriched in our dataset, indicating that apoptosis pathway genes are suppressed by E2 treatment. To further characterize the transcriptional role of ERα in ovarian cancer cells, ChIP-seq was performed on PEO1 cells treated with Vehicle, E2, Tamoxifen, or E2+Tamoxifen for 45 minutes. E2 treatment robustly increased ER recruitment to its regulatory regions (25,717 binding sites) when compared to Vehicle (8,370 binding sites) or Tamoxifen (18,935 binding sites) alone. Motif analysis of these ERα binding sites demonstrated a significant enrichment in members of the AP-1 transcription factor family, but not in known ERα cofactors FOXA1 and GATA3. FOXA1 and GATA3 are important regulators of ERα-dependent transcription in breast cancer cells; our results may suggest that mechanisms governing ERα transcriptional activity are lineage dependent. Finally, Binding and expression target analysis (BETA) was performed in order to predict the activating and repressing function of ERα. This analysis demonstrated that ERα has significant repressive function in PEO1 cells, indicating that ER may be acting as a strong transcriptional repressor in ovarian cancer cells. Future investigation in this project will center on the role of AP-1 in mediating ERα-dependent transcription. Additionally, we will use CRISPR/Cas9 screening to determine genetic sensitizers to ER antagonists. We believe that these studies will provide additional drug targets that may suggest a combinatorial therapeutic approach in conjunction with endocrine therapies for the treatment of ovarian cancer. Citation Format: Irene I Lee, Myles Brown. DECIPHERING THE ROLE OF ESTROGEN RECEPTOR ALPHA IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-038.

Oncogenic Role of Secreted Engrailed Homeobox 2 (EN2) in Prostate Cancer.

Engrailed variant-2 (EN2) has been suggested as a potential diagnostic biomarker; however, its presence and functional role in prostate cancer (PCa) cells is still controversial or unknown. Here, we analyzed 1) the expression/secretion profile of EN2 in five independent samples cohorts from PCa patients and controls (prostate tissues and/or urine) to determine its utility as a PCa biomarker; and 2) the functional role of EN2 in normal (RWPE1) and tumor (LNCaP/22Rv1/PC3) prostate cells to explore its potential value as therapeutic target. EN2 was overexpressed in our two cohorts of PCa tissues compared to control and in tumor cell lines compared with normal-like prostate cells. This profile was corroborated in silico in three independent data sets [The Cancer Genome Atlas(TCGA)/Memorial Sloan Kettering Cancer Center (MSKCC)/Grasso]. Consistently, urine EN2 levels were elevated and enabled discrimination between PCa and control patients. EN2 treatment increased cell proliferation in LNCaP/22Rv1/PC3 cells, migration in RWPE1/PC3 cells, and PSA secretion in LNCaP cells. These effects were associated, at least in the androgen-sensitive LNCaP cells, with increased AKT and androgen-receptor phosphorylation levels and with modulation of key cancer-associated genes. Consistently, EN2 treatment also regulated androgen-receptor activity (full-length and splicing variants) in androgen-sensitive 22Rv1 cells. Altogether, this study demonstrates the potential utility of EN2 as a non-invasive diagnostic biomarker for PCa and provides novel and valuable information to further investigate its putative utility to develop new therapeutic tools in PCa.

Abstract P5-05-12: Progesterone drives ER-positive and triple negative breast cancer cell proliferation through progesterone receptor membrane component 1

Abstract Introduction: The role of progesterone and its receptors in breast cancer progression continue to be studied but remain controversial. Progesterone membrane receptors with the ability to regulate kinase signals, mediating breast cancer proliferation have been demonstrated. Increased expression of the Progesterone Receptor Membrane Component 1 (PGRMC1), a heme – binding protein with the ability to interact and stabilize epidermal growth factor receptor (EGFR) is frequently found in breast cancer tissue. Some evidence suggests that progesterone can stimulate and regulate breast cancer cell proliferation. The basis of the signaling mechanisms by which Progesterone exerts its function remains largely unknown. Both the nuclear and membrane progesterone receptors could play a significant role in the development and progression of breast cancer and both could become viable therapeutic options. We, aim to investigate the role of PGRMC1 in progesterone driven breast cancers. Materials and Methods: Human breast tissues were utilized to identify PGRMC1 expression along with a panel of normal and breast cancer cell lines. Two breast cancer cell lines (ZR-75-1 and MDA-MB-468) were selected and treated with progesterone and AG-205 (PGRMC1 inhibitor) at different concentrations to assess optimum dosage. We performed MTS assay, qRT-PCR, Western blot, immunofluorescence, immunohistochemistry and flow cytometry for measuring cell proliferation, apoptosis and key markers involved in these processes. We also performed an in silico analysis to compare the expression of PGRMC1 in various cell lines and breast cancer tissues. Results: Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal tissue. Increased PGRMC1 expression was observed specifically in ZR-75-1 and MDA-MB-468 cells by qRT-PCR, western blot and immunofluorescence, these results were validated and compared to microarray-based gene expression analysis of breast cell lines and breast tumor data sets. Progesterone treatment increased cell proliferation in a dose dependent manner while AG-205 decreased cell proliferation in a dose dependent manner in ZR-75-1 and MDA-MB-468. Minimal effects of AG-205 were observed in normal breast epithelial cells. AG-205 also, induced apoptosis in both ZR-75-1 and MDA-MB-468 cell lines. Furthermore, short-term treatment of progesterone increased both mRNA and protein expression of PGRMC1. Key markers of cell proliferation (pAKT, CCND1, pEGFR, pmTOR) and apoptosis (PTEN, Bcl2, Bax, Bim) revealed that PGRMC1 facilitated the proliferative effect of progesterone. Interestingly progesterone increases phosphorylation of EGFR and treatment of AG-205 alters EGFR expression in a dose dependent manner. Conclusion: Our data demonstrates that PGRMC1 plays a prominent role in regulating progesterone driven cell proliferation in both ER-positive and triple negative breast cancer cells. These initial findings uncover the potential of PGRMC1 as a therapeutic target for breast cancers. Citation Format: Pedroza DA, Subramani R, Galvez A, Lakshmanaswamy R. Progesterone drives ER-positive and triple negative breast cancer cell proliferation through progesterone receptor membrane component 1 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-12.

Anti-Inflammatory Effect of Ginsenoside Rh2-Mix on Lipopolysaccharide-Stimulated RAW 264.7 Murine Macrophage Cells.

Ginsenoside Rh2, a protopanaxadiol saponin from ginseng, has been reported to have strong anti-inflammatory activity. However, the concentration of ginsenoside Rh2 is very low (>0.001%) in the total ginseng extracted, which is not enough for production despite its high pharmacological effects. Thus, in this study, we evaluated the anti-inflammatory effect of ginsenoside Rh2-mix (GRh2-mix) on lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. From the high-performance liquid chromatography analysis, it was confirmed that the GRh2-mix was mainly composed of 20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3. The LPS-stimulated RAW 264.7 cells were treated with different concentrations of GRh2-mix (100, 200, 400, 500 μg/mL). The cell counting kit-8 assay showed that the GRh2-mix treatment increased cell proliferation in LPS-stimulated RAW 264.7 murine macrophage cells. The GRh2-mix inhibited nitric oxide production in a dose-dependent manner, suggesting an anti-inflammatory effect. Furthermore, reverse transcription polymerase chain reaction and Western blot results also indicated that the GRh2-mix suppressed inflammatory genes such as iNOS, TNF-α, COX-2, IL-1β, IL-6, and NF-κB. In summary, these results suggest that the GRh2-mix exhibits anti-inflammatory activity via the downregulation of the NF-κB pathway and has high efficiency with a simple production procedure.

Lipolysis-stimulated lipoprotein receptor overexpression is a novel predictor of poor clinical prognosis and a potential therapeutic target in gastric cancer.

The prognosis of patients with advanced gastric cancer (GC) remains poor despite the recent advances in molecular targeted therapies, and the search for biomarkers that can predict prognosis and additional new agents with acceptable toxicity profiles are needed. Lipolysis-stimulated lipoprotein receptor (LSR) is a lipoprotein receptor that binds to triglyceride-rich lipoproteins and related to some malignancies. Herein, we examined the association between LSR expression and the prognosis of patients with GC, and investigated the antitumor effect of a previously developed anti-human LSR monoclonal antibody (#1–25). We first performed immunohistochemical analysis of LSR protein expression in GC and normal tissues, and then examined its association with the prognosis of 110 patients with GC. LSR was overexpressed in most of primary GC and metastatic tumors, but not in normal tissues. Patients with strong LSR expression (N = 80, 72.7%) had significantly poorer overall survival (OS) than those with weak expression (P = 0.017). Multivariate analysis identified strong LSR (as well as pT) as independent and significant prognostic factors for OS. Next, we demonstrated that very low density lipoprotein (VLDL) treatment increases cell proliferation in LSR-expressing GC cell lines in vitro; LSR inhibition using #1–25 inhibited VLDL-induced proliferation by suppressing JAK/STAT and PI3K signaling. In vivo, we demonstrated a marked antitumor effect of #1–25 in 2 distinct GC cell line xenograft mice models. Our findings suggest that LSR plays a key functional role in GC development, and that this antigen can be therapeutically targeted to improve GC treatment.

Water Soluble Antioxidative Crude Polysaccharide From Russula senecis Elicits TLR Modulated NF-κB Signaling Pathway and Pro-inflammatory Response in Murine Macrophages.

Russula senecis has recently been reported as a new addition to macrofungal flora of West Bengal. Besides, it also emerged as a seasonal health promoting nutrient to local ethnic people and enlisted for the first time as tribal food in our previous publication. In this context, the present work was designed to establish such usefulness scientifically and to meet the aim, crude polysaccharide, Rusenan, was prepared using conventional heated water reflux. Initially, the polymers were characterized to determine chemical composition and for that spectrophotometry along with Fourier-transform infrared spectroscopy (FT-IR), high-performance thin-layer chromatography (HPTLC), and gas chromatography-mass spectrometry (GC-MS) were performed. Analysis indicated that Rusenan was consisted mainly of carbohydrate conjugated with trace amount of protein. Furthermore, glucose was detected as the major monosaccharide (mainly in β-type glycosidic linkage) while other monomers were presented in the order of galactose > mannose > xylose > rhamnose. Conversely, antioxidant potential was determined following eight in vitro systems where the fraction evidenced strong superoxide, hydroxyl, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, high affinity to Fe2+ as well as instant ability to donate electron with EC50 values ranging from 80 to 3885 μg/ml concentration. In addition, effect on murine macrophages was also investigated where the polysaccharide treatment increased cell proliferation, phagocytic activity, filopodia or lamellipodia formation, nitric oxide (NO) production and reactive oxygen species (ROS) synthesis. Thereafter, through reverse transcriptase polymerase chain reaction (RT-PCR) analysis, significant increase in the expression of Toll like receptor (TLR)-4, TLR-2 and nuclear factor kappa B (NF-κB) was observed; as a result alleviated level of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, IκB-α, and interferon (IFN)-γ were also noticed explaining definite immune-stimulatory activity of the fraction. Thus, overall finding suggests that R. senecis can be considered as a functional food and may be used in preparation of dietary supplement to enhance general health.

Abstract 3731: Deciphering the transcriptional role of estrogen receptor alpha in ovarian cancer

Abstract Ovarian cancer is the leading cause of death among gynecologic malignancies. Despite years of research, the 5-year survival rate for ovarian cancer patients has remained poor and the standard of care is largely the same as it was decades ago. Epidemiological evidence and previous studies have suggested a role for steroid hormones in the pathogenesis of ovarian cancer, however, the role of steroid hormones in ovarian cancer remains understudied. We hypothesized that Estrogen Receptor α (ERα) is transcriptionally active and drives a transcriptional program that is sufficient to promote ovarian cancer cell proliferation and survival in a subset of ER+ ovarian cancers. Preliminary data generated in our laboratory has suggested that estradiol (E2) treatment increases cell proliferation in PEO1 cells, an ERα+ ovarian cancer cell line. This increase in cell proliferation can be inhibited by co-administration with the selective estrogen receptor modulator, Tamoxifen, and the selective estrogen receptor degrader, Fulvestrant. To further interrogate the mechanism of action of ERα in ovarian cancer, we performed RNA-seq on PEO1 cells treated with Vehicle, 10 nM E2, 100 nM Fulvestrant, and E2+Fulvestrant for 24 hours. We identified 659 significantly differentially expressed genes following E2 treatment. Fulvestrant inhibited the majority of E2-induced differentially expressed genes, confirming that these genes are dependent upon ERα. Gene Set Enrichment Analysis (GSEA) indicated that the Hallmark early and late estrogen responses are enriched in the E2 dataset, confirming that classical ER activity is intact in these cells. Furthermore, we identified G2/M checkpoint as positively enriched in our dataset, indicating that proliferation genes are upregulated by ERα in these cells. Additionally, we found that apoptosis was negatively enriched in our dataset, indicating that apoptosis pathway genes are suppressed by E2 treatment. Future investigation in this project will center on exploring the mechanisms of ERα transcriptional activity through examining the ERα-dependent cistrome, regulation of target genes, and its interactions with other cofactors. These data will give us insight into how ER is regulated and what pathways and processes ER is driving. Additionally, we will use CRISPR/Cas9 screening to uncover the key genes downstream of ER that are executing the estrogen-dependent effects on proliferation and survival. We believe that these studies will provide additional drug targets that may suggest a combinatorial therapeutic approach in conjunction with endocrine therapies for the treatment of ovarian cancer. Citation Format: Irene Lee, Myles Brown. Deciphering the transcriptional role of estrogen receptor alpha in ovarian cancer [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 3731.

MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway

BackgroundThe granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches.ResultsThe role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling pathway, granulosa cells were treated with activin A. Activin A treatment increased cell proliferation and downregulation of both miRNA-424/503 members and its target gene, indicated the presence of negative feedback loop between activin A and the expression of miRNA-424/503.ConclusionThis study suggests that the miRNA-424/503 cluster members are involved in regulating bovine granulosa cell proliferation and cell cycle progression. Further, miRNA-424/503 cluster members target the SMAD7 and ACVR2A genes which are involved in the activin signalling pathway.

BMP4 enhances hepatocellular carcinoma proliferation by promoting cell cycle progression via ID2/CDKN1B signaling.

Bone morphogenetic protein-4 (BMP4) plays a crucial role in carcinogenesis, but the effects and signaling mechanisms of BMP4 in hepatocellular carcinoma (HCC) are not clearly clarified. The present study aimed to identify the roles of BMP4 in the proliferation of human HCC. In this study, BMP4 expression and its correlation with clinicopathological characteristics and the survival of HCC patients were analyzed in two independent cohorts consisting of 310 subjects. Functional analysis of BMP4 on HCC proliferation was performed in vitro and in vivo in human HCC specimens, HCC cells of Bel-7402 and HCCLM3, and subcutaneous tumor model. The downstream signaling targets of BMP4 in HCC were investigated by PCR Array and Western blot. The results indicated that BMP4 expression was significantly increased in HCC tissues and closely related with unfavorable prognosis of HCC. BMP4 treatment increased cell proliferation and promoted G1/S cell cycle progression. In vivo subcutaneous tumor of nude mice model supported that BMP4 overexpression promoted the growth of HCC cells and BMP4 knockdown hold the opposite trend. Id2 was directly upregulated by BMP4, resulting in the mediated expression of cell cycle regulatory protein of CDKN1B. Blocking of Id2 attenuated BMP4-induced proliferation, confirming the important roles of Id2 in BMP4-mediated proliferation in HCC. So BMP4 is overexpressed in HCC tissues and acts as a poor prognostic factor of HCC patients. BMP4-induced ID2/CDKN1B signaling facilitates proliferation of HCC.

Increase of proliferation and DNA damage in mouse hepatocytes treated with diethylnitrosamine

As diethylnitrosamine (DEN) effect on cell proliferation, DNA damage and stem cell marker(s) expression have been largely unknown in mouse normal hepatocytes (AML-12 cells) cultured over a short-term period, this study was conducted to examine the cell proliferation, Ataxia telangiectasia mutated (ATM) and epithelial cell adhesion molecule (EpCAM) and Neighbor of Punc E 11 (Nope) expression in AML-12 cells treated with DEN for 24 and 48 h. Cells were treated with DEN (25-800 μg/mL) and cell phenotype was determined, and the MTT assay was used to quantify the proliferation of cells treated with DEN. Expression and distribution of ATM in AML-12 cells were determined by indirect immunofluorescence microscopy. And Western blot analysis of EpCAM and Nope was performed. Cell viability was significantly increased in response to all doses of DEN treatment compared to control at 24 h (p&lt;0.05 or p&lt;0.01). However, there was no significant increase at 48 h, even though it showed increased trend. Immunofluorescence staining of ATM showed that there was an increase of ATM expression at doses of 50, 100 and 200 μg/mL of DEN treatment, showing strong nuclear staining. Furthermore, Western blot analysis showed that DEN treatment showed increased trend of EpCAM and Nope expression. Taken together, DEN treatment increased cell proliferation in AML- 12 cells, and it was associated with increased ATM expression.