Expression Levels Of c-Myc Research Articles

The 5-/12-lipoxygenase-BLT2 cascade induces elevated expression of c-Myc, thus mediating the proliferation and migration of KRAS mutant colorectal cancer cells

ABSTRACTColorectal cancer (CRC) is the most common malignant tumor and one of the leading causes of cancer-related death worldwide. Oncogene KRAS is a commonly mutated in CRC and plays crucial roles in the colorectal tumorigenesis. Emerging evidence suggests that increased expression levels of c-Myc are critical for KRAS-mediated CRC progression. However, it is unclear exactly how c-Myc expression is regulated in CRC. In the present study, we found that blockade of 5/12-lipoxygenase (5/12-LO) or leukotriene B4 receptor (BLT2) markedly reduced c-Myc expression in KRAS-mutant LOVO cells, while the control COLO 320DM cells were not affected. When the 5/12-LO products LTB4 and 12(S)-HETE, ligands of BLT2, were added to LOVO cells, c-Myc expression levels were restored, together suggesting that ‘5/12-LO-BLT2 cascade’ regulates c-Myc expression in KRAS-mutant CRC cells. We also observed that ‘5/12-LO-BLT2’-mediated c-Myc upregulation contributes to cell proliferation by regulating the expression of cyclin D1 in LOVO cells. Moreover, ‘5/12-LO-BLT2-cMyc’ cascade regulates the expression of EMT-related proteins in KRAS-mutant CRC cells, and depletion of both c-Myc and BLT2 using siRNA significantly enhanced the level of E-cadherin and reduced the level of Vimentin in LOVO cells. Taken together, our findings suggest that the BLT2-linked cascade promotes KRAS-mutant CRC cell proliferation and migration through the elevated expression of c-Myc. Thus, our results suggest that BLT2 appears to be an effective therapeutic target for KRAS-mutant CRCs.

Searching for new early detection markers of oral epithelial dysplasia and oral squamous cell carcinoma using oral liquid-based cytology

ObjectiveOral squamous cell carcinomas (SCCs) arise from oral potentially malignant disorders (OPMDs), including oral epithelial dysplasia (OED); however, useful detections markers for early detection of OED and oral SCC are lacking. In this study, we aimed to evaluate the sequential mRNA and protein expression patterns of BRD4, c-MYC, TP53, and MUC21 in human oral samples by liquid-based cytology (LBC) and determine whether these markers are useful for early detection of OED and oral SCC when combined with oral cytological screening. MethodsA total 200 patients were obtained from our hospital between 2020 and 2022. These are samples from same cases with oral cytology and corresponding histological diagnosis. The expression of these markers was examined by immunocytochemistry and mRNA analysis. The percentages of cells with nuclear or cytoplasmic immunostaining were calculated using the labeling index (LI). BRD4, c-MYC, and TP53 mRNA expression levels were upregulated in tissue during progression from a status of negative for intraepithelial lesions or malignancy (NILM) to positive for SCC. BRD4-LI and c-MYC-LI significantly increased in low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL) and SCC samples, whereas the expression of MUC21 mRNA and MUC21-LI gradually decreased. TP53-LI increased only in SCC samples. ConclusionsWe conclude that monitoring BRD4 and c-MYC expression with immunocytochemistry could improve the early detection of OED and oral SCC.

Axatilimab Ameliorates Inflammation and Fibrosis By Targeting Macrophages in a Preclinical Model of Chronic GVHD

Introduction: Chronic graft-versus-host disease (cGVHD) is an immune-mediated serious and life-threatening complication after allogeneic hematopoietic stem cell transplantation, occurring in 30%-70% of patients. The pathophysiology of cGVHD may involve inflammation, cell-mediated immunity, humoral immunity, and fibrosis. Axatilimab is a high-affinity humanized IgG4 (kappa light chain) monoclonal antibody targeting colony-stimulating factor 1 receptor (CSF-1R) that is under investigation for the treatment of cGVHD. CSF-1R is a member of the receptor protein tyrosine kinase family of growth factor receptors and is expressed on monocyte precursors, monocytes, and macrophages. Signaling by colony-stimulating factor 1 (CSF-1) or interleukin-34 (IL-34) through CSF-1R has been shown to be the primary regulatory pathway for determining development and differentiation of the mononuclear phagocytic cell lineage. Recent work has established a role for monocyte-derived macrophages as the pathogenic population of cells that establish the fibrotic niche and drive the fibrotic process. Upon entry of monocytes into tissues, the tissue microenvironment regulates their differentiation, resulting in a heterogeneous pool of cells ranging from pro-inflammatory (often referred to as M1 macrophages) to immune suppressive/wound healing (M2 macrophages). An imbalance in the number and activity of these macrophage populations is believed to result in the excess production of fibrotic growth factors, such as transforming growth factor-β and platelet-derived growth factor, leading to uncontrolled fibroblast activation and subsequent fibrosis. Blocking CSF-1R signaling with axatilimab is expected to reduce the circulating levels of pathogenic monocyte-derived macrophage precursors and inhibit their activation in tissues, thereby providing an opportunity to therapeutically intervene in diseases such as cGVHD. The results described herein support this assertion by demonstrating the direct impact of axatilimab exposure to skewing differentiating monocytes away from a pro-inflammatory and pro-fibrotic phenotype. Methods: Primary human monocytes were differentiated in the presence or absence of ascending doses of axatilimab (0.07-7 μM) into distinct phenotypes: activated (M0), pro-inflammatory (M1-like), and suppressive (M2-like) macrophages. Supernatants and cell pellets were then assessed for secreted protein (Luminex Corporation) and gene expression (NanoString Technologies) profiles, respectively. Data were analyzed in R (version 4.1.1). Results: Gene expression profiles of pro-inflammatory cytokines and chemokines (eg, IL-1β, chemokine CC motif ligand 2 [CCL2], CCL7, CCL24, and CCL22) from primary differentiated macrophage subsets were dramatically reduced in the presence of ascending concentrations of axatilimab. Further, macrophages exposed to axatilimab while differentiated under pro-inflammatory conditions showed increased levels of c-MYC expression compared with control cultures. c-MYC is a transcription factor associated with suppressive M2-like macrophages, which suggests that axatilimab interferes with pro-inflammatory macrophage differentiation. Consistent with this, expression of M2-associated cytokines, including IL-10, IL-5, and IL-4, was enhanced in axatilimab-treated macrophage cultures. Conclusions: Blocking CSF-1R signaling in primary, differentiated macrophages dramatically alters the secretomes and gene expression profiles of these cells. Macrophages are key inducers of fibroblast growth factors, which in turn promote fibrotic disease. These data confirm that production of inflammatory and pro-fibrotic factors by pro-inflammatory macrophages is inhibited in the presence of axatilimab, thereby supporting the notion that axatilimab provides a unique opportunity to therapeutically intervene or prevent fibrosis in cGVHD.

Effect of Quercetin on Fecal Syndecan-2 Gene Methylation Levels in Patients with Colorectal Cancer and Its Potential Mechanism of Action

Colorectal cancer (CRC) is a relatively common malignant tumor of the digestive system. In this research, the impact of quercetin (Que) on fecal syndecan-2 (SDC2) gene methylation levels in CRC patients and its potential mechanisms of action were analyzed. Retrospective analysis was conducted on data from 56 CRC patients admitted to our outpatient department. The methylation status of SDC2 in fecal samples and the sensitivity and specificity of fecal SDC2 gene detection were analyzed. Subsequently, MTT assay was performed to assess the impact of Que on cell viability of SW480 cells during the growth phase. The blank control consisted of 200 μL of L-15 medium, the solvent control group consisted of 200 μL of L-15 medium+150 μL of dimethyl sulfoxide (DMSO), and the experimental group consisted of 200 μL of L-15 medium+different concentrations of Que (5, 10, 20, 40, 80, 120 μmol/L). After SW480 cells were treated with various concentrations of Que, protein expression (EP) levels of C-Myc, B-cell lymphoma (Bcl)-2, phosphorylated STAT3 (p-STAT3), and interleukin (IL)-6 were determined by qRT-PCR and Western blot. The single-gene detection of SDC2 in fecal samples yielded an area under the curve (AUC) of 0.763 (95% confidence interval (CI): 0.623–0.876). As the concentration of Que increased, the OD values decreased. Various concentrations of Que exhibited varying inhibitory effects on SW480 cell proliferation (CP) at different time intervals, with the OD values at 72 h being superior to those at 48 h and 24 h. The OD values at 48 h were superior to those at 24 h, indicating a significant enhancement of SW480 proliferation with prolonged time. As the treatment concentration increased, the cell viability gradually decreased. Que at concentrations ranging from 20 to 160 μmol/L showed a notable inhibition of SW480 CP. With increasing concentrations of Que, protein levels of C-Myc, Bcl-2, p-STAT3, and IL-6 decreased gradually. In summary, Que can inhibit the EP of inflammatory factors in CRC patients, suppress tumor CP by downregulating protein levels of C-Myc, Bcl-2, p-STAT3, and IL-6, and thus exert an anti-CRC effect.

A systems biology approach and in vitro experiment indicated Rapamycin targets key cancer and cell cycle-related genes and miRNAs in triple-negative breast cancer cells.

An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.

Apoptosis Induction by New Coumarin Derivatives in a Mice Model of Breast Cancer.

In the last decades, numerous studies have focused on the search for new agents to suppress the growth of cancer cells. In this study, we investigated the effect of two novel synthetic coumarin derivatives, namely 2-amino-4-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile and 2-amino-4-(4-hydroxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile, on the induction of apoptosis in breast cancer in a mouse model. Breast cancer was induced in BALB/c mice, which were randomly divided into six groups and then underwent the experiment. The groups and treatments included A1: coumarin A with a low dose (10 µm), A2: coumarin A with a high dose (1 mM), B1: coumarin B with a low dose (10 µm), B2: coumarin B with a high dose (1 mM), D: doxorubicin, and C: cancer control/ treatment with normal saline. The samples underwent treatments for 5 weeks. Animals were euthanized, and tissue samples, including the lung, liver, and tumor mass, were collected for histopathological examination. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine some apoptotic markers, such as BCL-2, caspase-9, COX-2, and c-Myc. The qRT-PCR presented that both coumarin compounds could significantly alter the expression levels of BCL-2, caspase-9, COX-2, and c-Myc. Consistent with these results, histopathological observations showed a significant reduction in pathological lesions and severity of malignancy of the tumor mass, as well as a decrease in microscopic metastases in the lung and liver. This suggests that the present new coumarin compounds may induce apoptosis in breast cancer cells by altering some apoptosis-related genes that may play a chemotherapeutic role in breast cancer therapy in the future.

Prognostic and therapeutic implications of iron-related cell death pathways in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is diverse in terms of its molecular abnormalities and clinical outcomes. Iron homeostasis and cell death pathways play crucial roles in cancer pathogenesis, including AML. The objective of this study was to examine the clinical significance of genes involved in iron-related cell death and apoptotic pathways in AML, with the intention of providing insights that could have prognostic implications and facilitate the development of targeted therapeutic interventions. Gene expression profiles, clinical information, and molecular alterations were integrated from multiple datasets, including TCGA-LAML and GSE71014. Our analysis identified specific molecular subtypes of acute myeloid leukemia (AML) displaying varying outcomes, patterns of immune cell infiltration, and profiles of drug sensitivity for targeted therapies based on the expression of genes involved in iron-related apoptotic and cell death pathways. We further developed a risk model based on four genes, which demonstrated promising prognostic value in both the training and validation cohorts, indicating the potential of this model for clinical decision-making and risk stratification in AML. Subsequently, Western blot analysis showed that the expression levels of C-Myc and CyclinD1 were significantly reduced after CD4 expression levels were knocked down. The findings underscore the potential of iron-related cell death pathways as prognostic biomarkers and therapeutic targets in AML, paving the way for further research aimed at understanding the molecular mechanisms underlying the correlation between iron balance, apoptosis regulation, and immune modulation in the bone marrow microenvironment.

Effects of adriamycin on cell differentiation and proliferation in rat testis

ABSTRACT Although adriamycin (ADR) is used to treat many cancers, it can be toxic to healthy organs including the testis. We investigated the effects of ADR on pluripotency in rat testis. Testicular damage was induced by either cumulative or single dose single dose administration of ADR in Wistar albino rats. Rats were divided randomly into three groups: untreated control, cumulative dose ADR group (2 mg/kg ADR every three days for 30 days) and single dose ADR group (15 mg/kg, single dose ADR). Testicular damage was evaluated and seminiferous tubule diameters were measured using light microscopy. Expression levels of Oct4, Sox2, Klf4, c-Myc, Utf1 and Dazl were assessed by immunohistochemistry and real time PCR. Serum testosterone levels were measured using ELISA assay. Histopathologic scores were lower and mean seminiferous tubule diameters were less compared to the ADR groups. Oct4, Sox2, Klf4 and Utf1 expressions were decreased significantly in spermatogenic cells of both cumulative and single dose ADR groups compared to the control group. We found that c-Myc expression in spermatogenic and Leydig cells were increased significantly in both ADR groups compared to the control group. Dazl expression was decreased in the cumulative adriamycin group compared to the control group, but increased in the single dose ADR group compared to both the control and cumulative ADR groups. Serum testosterone levels were decreased in both ADR groups compared to the control group. Our findings suggest that ADR is detrimental to regulation and maintenance of pluripotency in rat testis.

Silencing of keratin 15 impairs viability and mobility while facilitating the doxorubicin chemosensitivity by inactivating the β‑catenin pathway in liver cancer.

Keratin 15 (KRT15) regulates the invasion as well as the stemness and is associated with tumor size and metastasis of several gastrointestinal cancers apart from liver cancer. The present study aimed to explore the effect of KRT15 knockdown on liver cancer malignant behaviors and its interaction with the β-catenin pathway. Small interfering (si)-KRT15 and si-negative control (NC) were transfected into liver cancer cell lines, followed by the addition or not of CHIR-99021 (a β-catenin agonist). Cell viability, invasion, apoptosis, and the half maximal inhibitory concentration (IC50) value of doxorubicin (Dox) were then assessed. The present study illustrated that KRT15 gene and protein expression levels were upregulated in most liver cancer cell lines (Huh7, PLC, Hep3B and HepG2) compared to the normal liver cell line THLE-2. si-KRT15 reduced cell viability and invasive cell count while promoting the apoptosis rate in Huh7 and HepG2 cells. In addition, si-KRT15 also reduced the IC50 value of Dox. Furthermore, si-KRT15 inactivated the β-catenin pathway as reflected by β-catenin, cyclin D1 and c-Myc expression levels in Huh7 and HepG2 cells. Subsequently, CHIR-99021 treatment increased the cell viability and invasive cell count while reducing the apoptosis rate in Huh7 and HepG2 cells. Concurrently, the IC50 value of Dox was also increased. Notably, CHIR-99021 treatment attenuated the effect of si-KRT15 on mediating the aforementioned Huh7 and HepG2 cell malignant behaviors and Dox chemosensitivity. In conclusion, KRT15 knockdown suppressed viability and mobility but facilitated Dox chemosensitivity via inactivating the β-catenin pathway in liver cancer, suggesting its potential as a target for liver cancer treatment.

Targeted inhibition of mTOR by BML-275 induces mitochondrial-mediated apoptosis and autophagy in prostate cancer

Prostate cancer (PCa) is the most frequently diagnosed cancer among men and the second leading cause of death in Western countries. Clinically, screening drugs and develop developing new therapeutics to treat PCa is of great significance. In this study, BML-275 was demonstrated to exert potent antitumor effects in PCa by antagonizing mTOR activity. In cultured PCa cells, BML-275 treatment reduced the expression levels of c-Myc and survivin, promoted the activation of p53, and thereby induced p21/cyclin D1/CDK4/6-dependent cell cycle G1/S arrest. As a result, BML-275 inhibited cellular proliferation and induced mitochondrial-mediated apoptosis. In addition, BML-275 treatment triggered autophagy. Interestingly, EACC-mediated suppression of autophagy did not affect BML-275-induced proliferation and apoptosis. Nude mouse tumorigenic experiments also confirmed that BML-275 inhibited PCa growth, induced PCa cell apoptosis and autophagy. Mechanistically, the activities of PI3K/AKT and AMPK pathways were downregulated by BML-275 treatment in vitro and in vivo. Importantly, mTOR, a common downstream negative protein of PI3K/AKT and AMPK signaling, was induced to inactivate, which may be associated with the induction of apoptosis and autophagy. The pharmacological activation of mTOR by MHY1485 abolished the induction of apoptosis and autophagy of BML-275. Molecular docking results showed that BML-275 can bind to the FKRP12-rapamycin binding site on mTOR protein, and thereby may have the same inhibitory activity on mTOR as rapamycin. Thus, these findings indicated that BML-275 induces mitochondrial-mediated apoptosis and autophagy in PCa by targeting mTOR inhibition. BML-275 may be a potential candidate for the treatment of PCa.

Apoptosis Induction by New Coumarin Derivatives in a Mice Model of Breast Cancer

In the last decades, numerous studies have focused on the search for new agents to suppress the growth of cancer cells. In this study, we investigated the effect of two novel synthetic coumarin derivatives, namely 2-amino-4-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile and 2-amino-4-(4-hydroxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile, on the induction of apoptosis in breast cancer in a mouse model. Breast cancer was induced in BALB/c mice, which were randomly divided into six groups and then underwent the experiment. The groups and treatments included A1: coumarin A with a low dose (10 µm), A2: coumarin A with a high dose (1 mM), B1: coumarin B with a low dose (10 µm), B2: coumarin B with a high dose (1 mM), D: doxorubicin, and C: cancer control/ treatment with normal saline. The samples underwent treatments for 5 weeks. Animals were euthanized, and tissue samples, including the lung, liver, and tumor mass, were collected for histopathological examination. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine some apoptotic markers, such as BCL-2, caspase-9, COX-2, and c-Myc. The qRT-PCR presented that both coumarin compounds could significantly alter the expression levels of BCL-2, caspase-9, COX-2, and c-Myc. Consistent with these results, histopathological observations showed a significant reduction in pathological lesions and severity of malignancy of the tumor mass, as well as a decrease in microscopic metastases in the lung and liver. This suggests that the present new coumarin compounds may induce apoptosis in breast cancer cells by altering some apoptosis-related genes that may play a chemotherapeutic role in breast cancer therapy in the future.

Cell proliferation can be modulated by receptor tyrosine kinase-like orphan receptor 2 in the silkworm, Bombyx mori

The receptor tyrosine kinase-like orphan receptor 2 (Ror2) is involved in the Wnt/β-catenin signaling pathway which regulates cell proliferation, polarity, differentiation, migration, metabolism and survival. However, the function of Ror2 in the silkworm Bombyx mori is still undisclosed. In the present investigation, we have made an effort to clone the silkworm Ror2 gene (BmRor2). The sequence analysis showed that the open reading frame (ORF) was 1914 bp in size and encoded a protein with the conserved domains of Ror2 protein. The qRT-PCR results indicated that the BmRor2 gene expression level was the highest in the head among all identified tissues on 3rd day of the fifth instar larvae. In the gonads of the different development stages, the BmRor2 gene expression level was highest on the 4th day of the fourth instar larvae. The immunofluorescence assay indicated that the BmRor2 protein was located at the cytomembrane. The effects of BmRor2 protein on the expression levels of genes related to TGF-β, Hippo, JAK-STAT and Notch signaling pathways were investigated by qRT-PCR. The expression levels of crumbs (crb), warts (wts), α-catenin (cat), four-jointed (fj), decapentaplegic (dpp), kibra ortholog (kibra), serrate (serr) and c-myc (myc) genes were down-regulated, whereas, suppressor of cytokine signaling 2 (socs 2) gene expression was up-regulated in the cultured BmN cells after the BmRor2 expression level was up-regulated. Further, cell proliferation was demoted and the size of cells was decreased when BmRor2 expression level was elevated. Our current finding recommended that BmRo2 can regulate TGF- β, Hippo, JAK-STAT, and Notch signaling pathways, and affect cell proliferation and size.

Cytotoxic Effect of L-Methioninase from Brevibacterium linens BL2 in Combination with Etoposide against Glioblastoma Cells

L-methioninase degrades methionine, which is essential in methionine-dependent cancer cells, resulting in specific cell death. Normal cells can synthesize their own methionine amino acids even in the absence of exogenous methionine. This selective targeting of cancer cells makes L-methioninase a promising therapeutic candidate for cancer. In this study, L-methioninase was partially purified from Brevibacterium linens BL2. The specific activity of the enzyme was found as 3.055 units/mg. IC50 values (24 h) of the enzyme were 5.792 units/mL for U87MG cell line and 5.215 units/mL for T98G cell line. When L-methioninase and etoposide were used in combination, synergistic cytotoxic and cell migration inhibition effects on U87MG and T98G cells alongside decreased cytotoxic activity on the Mouse Embryonic Fibroblast and HaCaT cells compared to etoposide alone were observed. Additionally, colony numbers of U87MG cells were significantly reduced by L-methioninase and etoposide administration after 21 days of incubation. Furthermore, L-methioninase suppressed the expression levels of survivin and c-Myc while increasing the expression level of Caspase-3 in both glioblastoma cell lines. These effects were enhanced when etoposide was used in combination with etoposide. This investigation reveals that the L-methioninase enzyme not only exhibited cytotoxic effects on U87MG and T98G cells but also enhanced the anti-proliferative effects of etoposide when used in combination while also demonstrating fewer adverse effects on normal cells.

Linggui Zhugan decoction delays ventricular remodeling in rats with chronic heart failure after myocardial infarction through the Wnt/β-catenin signaling pathway

ContextTraditional Chinese medicine plays an important role in the prevention and treatment of heart failure (HF). Linggui Zhugan decoction has been approved for clinical treatment of chronic HF. However, the mechanism is still unclear. ObjectiveThe effect of Linggui Zhugan decoction on the Wnt/β-catenin signaling pathway in rat myocardium was studied to investigate the mechanism by Linggui Zhugan decoction effects ventricular remodeling in rats with heart failure after myocardial infarction. MethodA rat model of HF after myocardial infarction was prepared by ligating the left anterior descending coronary artery. After 6 weeks of intervention with Linggui Zhugan decoction, the effect of Linggui Zhugan decoction on the cardiac function of chronic HF model rats was observed. Myocardial infarct size was measured by triphenyl tetrazolium chloride (TTC) staining. Enzyme linked immunosorbent assays (ELISAs) were used to measure NT-proBNP and sST-2 concentrations in rat serum. Hematoxylin and eosin (H&E) staining, and Masson's trichrome staining were used to observe the morphology of myocardial tissue; immunohistochemistry was used to detect the protein expression of type I collagen and type III collagen in myocardial tissue; and mRNA expression levels of Wnt3a, GSK-3β, β-catenin, and c-Myc in the infarct marginal zone were detected using PCR. Protein expression of Wnt3a, p-GSK-3β, GSK-3β, and β-catenin in the infarct marginal zone was detected using western blot. ResultsCompared with the control, Linggui Zhugan decoction reduced the levels of serum ST-2 and NT-proBNP, improved cardiac function, and reduced the deposition of collagen fiber. In addition, Linggui Zhugan decoction inhibited the expression of Wnt3a, p-GSK-3β, and β-catenin in cardiomyocytes. ConclusionLinggui Zhugan decoction inhibits the expression of several key proteins in the Wnt/β-catenin signaling pathway, delays cardiomyocyte hypertrophy and fibrosis, and improves cardiac function.

The sodium borate relieves the hypertrophic damage induced during pregnancy, it improves contractibility, reduces oxidative stress and stimulates cell proliferation

IntroductionFetal and postnatal hypertrophy develop in response to such different exposures or illnesses the mother suffers during gestation as anti-infectious and physical agents, obesity, hypertension, diabetes, and even advanced maternal age. This gives rise to high comorbidities in the newborn; therefore, looking for alternatives that contribute to cardiac homeostasis is quite necessary to inhibit the overgrowth of myocytes. Boron-derivative compounds could play a key role in exerting a repairing effect on chronic cardiac damage induced during gestation. MethodologyThe cardiotoxic effect of 6.4, 12 and 100 mg/kg of sodium tetraborate administered by oral delivery route to healthy pregnant mice was assessed. After that, the use of the chemical compound was tested in the treatment of pregnant mice previously subjected to isoproterenol (fetal hypertrophy model) on the fifth day post coitus. Prior to the sacrifice of the pups of mice an electrocardiography (ECG) was done. Morphological and histological changes of heart were assessed in newborn pups. As a damage marker, the concentration of p38 nitrogen-activated protein kinases were evaluated by using Western Blot and the levels of malondialdehyde (MDA) as well as glutathione antioxidants (GSH) and glutathione peroxidase (GPx) were tested by spectrometry. Moreover, the mRNA expression for early response genes (c-jun, c-fos y c-myc), late response (GATA-4, Mef2c, NFAT) and heart damage (ANP and BNP) was measured by qPCR real time. ResultsThe supply of 6,4 and 12 mg/kg-sodium tetraborate favored ventricular remodeling with histological alterations. By comparison, 100 mg/kg of sodium tetraborate administered during the fetal stage did not alter neither the cardiac morphology of six-week old pups nor the p38/P-p38MAPK ratio remained the same and no oxidative stress was observed.When pregnant females treated with isoproterenol were treated with 100 mg/kg sodium tetraborate during the fetal stage, an improvement in contractility was detected in the pups with an actual reduction in myocardial fibrosis and oxidative stress, but cardiac mass increased. In addition, the expression levels of c-jun, c-myc, GATA-4, MEF2c and ANP mRNA declined in comparison with CTR. However, the hypertrophic damage mechanism was sustained by c-fos, NFAT and BNP expressions. ConclusionsThe set of results achieved suggests that high concentrations of sodium tetraborate have no cardiotoxic effects. Furthermore, sodium tetraborate mitigates hypertrophy induced during pregnancy, thereby improving contractibility, reducing oxidative stress and stimulating cell proliferation. Therefore, sodium tetraborate could be an excellent prophylactic treatment administered by delivery oral route during pregnancy when there is a risk of developing fetal left ventricular hypertrophy (LVH).

MiR-455-3p inhibits gastric cancer progression by repressing Wnt/β-catenin signaling through binding to ARMC8

BackgroundGlobally, gastric cancer (GC) is one of the world’s most widespread malignancies, with persistent high mortality and morbidity rates. Increasing evidence now suggests that microRNAs (miRNAs) participate in many biological processes, with miR-455-3p having key roles in the progression of diverse cancers. Nevertheless, miR-455-3p function and expression in GC remain unclear.MethodsWe explored miR-455-3p expression in GC using quantitative polymerase chain reaction (qPCR). To further examine the effect of miR-455-3p in GC, after transfecting miR-455-3p mimics or inhibitors into GC cells, 5-ethynyl-2’-deoxyuridine (EdU) incorporation and colony formation assays were performed to examine cell proliferation. Flow cytometry was used to detect apoptosis, and expression levels of Bax, Bcl-2, Snail, N-cadherin, E-cadherin, and Caspase-3 were assessed by western blotting (WB). Using online databases and luciferase assays, we identified armadillo repeat-containing protein 8 (ARMC8) as a promising target of miR-455-3p. A mouse tumor model was established to investigate actions of miR-455-3p in vivo. Expression levels of C-myc, cyclinD1, and β-catenin were examined using WB and immunofluorescence.ResultsMiR-455-3p expression was attenuated in GC tissue and cell lines. MiR-455-3p overexpression inhibited GC cell proliferation, epithelial-mesenchymal transition (EMT), as well as facilitated apoptosis, while suppression of miR-455-3p had the opposite effects. From luciferase assays, we confirmed that ARMC8 was a novel and direct downstream target gene of miR-455-3p, and that the tumor suppressive role of miR-455-3p was in part reversed due to ARMC8 overexpression. Moreover, miR-455-3p inhibited GC growth in vivo via ARMC8. We also observed that miR-455-3p repressed canonical Wnt pathway activation by binding to ARMC8.ConclusionsMiR-455-3p exerted tumor inhibitory effects in GC by targeting ARMC8. Therefore, intervening in the miR-455-3p/ARMC8/Wnt/βcatenin axis could be a promising novel treatment strategy for GC.

P-427 Control of in vitro follicle activation by inhibition of YAP/TAZ activity in mouse ovaries

Abstract Study question Does verteporfin, an inhibitor of YAP/TAZ activity, prevent from the impacts of chemotherapy exposure on signaling pathways governing follicle activation and survival? Summary answer Verteporfin treatment prevents from the chemotherapy-induced impairments of the Hippo pathway but also of PI3K and survival pathways. What is known already The Hippo pathway is a crucial regulator of the ovarian reserve. Disruption of this pathway occurring in non-physiological contexts, such as ovarian processing during ovarian tissue preservation or after chemotherapy exposure, leads to massive uncoordinated follicular growth and depletion of the ovarian stockpile. To prevent from the harmful impact of chemotherapy exposure, several studies assessed the potential of inhibition of the PI3K pathway, the major signaling pathway involved in physiological follicle activation. However, the protective effect of this inhibition appeared to be moderate in vitro, suggesting the involvement of other disrupted mechanisms, as Hippo pathway. Study design, size, duration This study was performed on post-natal day 3 mouse ovaries. Half-cut ovaries were first treated with 0, 0.2, 1.5 and 3 µM Verteporfin (VERT) during 3 hours in vitro culture to establish the efficient dose of VERT. Whole ovaries were then cultured for 24 and 48 hours and exposed to 10 µM 4-hydroperoxycyclophosphamide (4HC) and/or 3 µM VERT to assess the effect of this inhibitor on follicle activation and survival pathways. (N = 3-5) Participants/materials, setting, methods To assess the impacts of sectioning and VERT treatment on follicle activation, gene expression analyses (RT-qPCR) were used to assess Hippo, PI3K/AKT/mTOR and apoptosis signaling pathways. The potential preventive effect of VERT co-treatment with 4HC exposure was evaluated by gene (RT-qPCR) and protein expression (western blot) analyses of these three pathways, and completed with histological staining for DNA damages (TUNEL). Main results and the role of chance Following exposure to increased concentration of VERT for 3 hours, a significant impact of the inhibitor was observed at 3µM on Ccn2 (p = 0.003) and Cmyc (p = 0.045) expression levels compared to control, without impacting on apoptosis genes expression. Exposure to 10 µM 4HC induced a higher YAP/pYAP protein ratio and Ccn2 expression level compared to control after 24 and 48 hours of culture, confirming the Hippo pathway disruption. VERT co-treatment was able to prevent from this increase of protein levels and gene expression, although the impact on Ccn2 expression was moderated after 48 hours of culture. Assessment of PI3K signaling revealed an induction of mTOR signaling following 4HC exposure at both time of culture as shown by the higher pRPS6/RPS6 ratio compared to control. Surprisingly, VERT co-treatment significantly decreased this level compared to 4HC alone at 24 and 48 hours of culture. This result suggests an indirect impact of this inhibitor on the PI3K pathway. Despite Bax and Bcl2 gene levels remained stable among conditions, ovaries exposed to 4HC had a significant higher level of DNA damages at both culture timepoints compared to control. Notably, VERT co-treatment was able to decrease the 4HC-induced gonadotoxicity at 48 hours of culture. Limitations, reasons for caution This study evaluated the impact of an inhibitor to control follicle activation in mouse model, and was limited to the assessment of two main signaling. Although we completed our study with apoptosis analyses, the results should be interpreted with caution as other pathways may be involved into the activation process. Wider implications of the findings Our results suggest that verteporfin is a promising inhibitor to control acute signaling pathway impairments under unphysiological conditions. Moreover, this study sustains the presence of a potential interaction between the two main signaling pathways regulating follicle activation, PI3K and Hippo pathways. Trial registration number Not applicable

ZERDEÇALIN BİYOAKTİF BİLEŞİĞİ KURKUMİN, GEMSİTABİNİN PROSTAT KANSERİ HÜCRELERİNDEKİ ANTİ-MALİGNANT ÖZELLİĞİNİ GELİŞTİREBİLİR

Objective: The aim of this study was to investigate the possible synergistic effect of curcumin on the anticancer features of gemcitabine on prostate cancer cells. Material and Method: The human prostate adenocarcinoma cell line LNCaP was used in the studies. The effect of the co-administration of gemcitabine and curcumin on the viability of LNCaP cells was investigated by the WST-1 assay. Autophagy, ubiquitin-proteasome system (UPS), unfolded protein response (UPR) and cell death-associated proteins, androgenic signaling, proto-oncogenic, angiogenic and epithelial-mesenchymal transition (EMT) associated protein levels were investigated by immunoblotting studies. Result and Discussion: Our results showed that curcumin potentiated the anticancer effects of gemcitabine on LNCaP cells. Co-administration of curcumin and gemcitabine strengthened the suppressive effect of gemcitabine on cell viability. Moreover, co-administration modulated the autophagy, more strongly stimulated UPS and UPR, suppressed androgenic signaling, led to the activation of cell death-related poly [ADP-ribose] polymerase 1 (PARP-1) and caspase-3 and strongly suppressed the expression levels of proto-oncogenic c-Myc and angiogenic vascular endothelial growth factor-A (VEGF-A). In addition, it was determined that co-administration negatively regulated EMT by stimulating E-cadherin expression and suppressing N-cadherin level. These results suggest that the combined usage of gemcitabine and curcumin may offer a potent therapeutic approach to prostate cancer by enhancing the anticancer effects of gemcitabine.

Lipid nanoparticles containing PlncRNA-1 promote apoptosis of liver cancer cells by down-regulating Wnt/beta-catenin signaling

PlncRNA-1 has an anti-cancer property. Liposome nanoparticles (LNPs) are used as carriers of targeted drugs and contribute to tumor-targeted drug delivery. However, the interaction between LNPs containing PlncRNA-1 and β-catenin, a key component of Wnt signaling pathway, and its role in liver cancer have not yet been confirmed. Human liver cancer cells were exposed to LNPs containing PlncRNA-1 at different concentrations followed by analysis of cell proliferation, cell cycle distribution and apoptosis. Wnt/β-catenin pathway-related gene expression was determined. LNPs containing PlncRNA-1 dose-dependently suppressed the proliferation of liver cancer HepG2 cells with IC50 at 24 h, 48 h and 72 h of 12.8±0.67 μmol/L, 8.8±0.43 μmol/L and 4.6±0.42 μmol/L, respectively. PlncRNA-1-loaded LNPs (5, 10, and 20 μmol/L) dose-dependently arrested HepG2 cells in G2/M phase and induced apoptosis (p &gt; 0.05). Administration of LNPs carrying PlncRNA-1 decreased the expression of overall, cytoplasmic, and nuclear β-catenin, thereby suppressing Wnt/β-catenin pathway. Protein expression levels of GSK-3 (p-tyr216) and Axin-2 were increased, while protein expression levels of Dvl-2, Dvl-3, GSK-3β, c-myc and survivin were significantly decreased. Invasion and metastasis of malignant tumors is an important factor. Inhibiting invasion of cancer cells effectively might improve the prognosis of cancer patients. LNPs carrying PlncRNA-1 promoted cell cycle transition and cell apoptosis by decreasing the expression of Dvl-2, Dvl-3, intracellular β-catenin, cytoplasmic β-catenin, and total β-catenin protein, which may relate to the inhibition of Wnt/β-catenin pathway.

MESP1-knockdown inhibits the proliferation and epithelial mesenchymal transition of hepatocellular carcinoma and enhances the tumor-suppressive effect of 5-fluorouracil

Primary liver hepatocellular carcinoma (HCC) is the third most deadly malignancy worldwide,in part, because it is often diagnosed at an advanced stage. Thus, molecular markers are needed to aid in the early diagnosis and treatment of HCC. Expression of abnormal mesoderm posterior-1 (MESP1) promotes tumorigenesis; however,its role in the regulation of HCC proliferation, apoptosis,and invasion is unknown. Here,we analyzed data in The Cancer Genome Atlas (TCGA)and Genotype Tissue Expression (GTEx) databases on the pan-cancer expression of MESP1 and its relationship with clinical characteristics and prognosis of patients with HCC. The expression of MESP1 was measured in 48 HCC tissues using immunohistochemical staining,and the results were correlated with clinical stage, tumor differentiation, tumor size,and metastasis. MESP1 expression was downregulated using small interfering RNA (siRNA) in the HCC cell lines HepG2 and Hep3B,and cell viability, proliferation,cell cycle, apoptosis,and invasion were analyzed. Finally,we also evaluated the tumor suppression effect of MESP1 downregulation combined with 5-fluorouracil (5-FU) treatment. Our results showed that MESP1 is a pan-oncogene associated with poor prognosis in patients with HCC. siRNA-induced downregulation of MESP1 expression in HepG2 and Hep3B cells exhibited downregulation of β-catenin and GSK3β expression 48h after transfection, along with an increase in apoptosis rate, arrest in the G1-S phase,and a decrease in mitochondrial membrane potential. Moreover,the expression levels of c-Myc, PARP1, bcl2, Snail1, MMP9, and immune checkpoint genes (TIGIT, CTLA4,LAG3,CD274,and PDCD1) were downregulated, while those of caspase3 and E-cadherin were upregulated. Tumor cells also showed decreased migration ability. Furthermore, siRNA interference of MESP1 expression combined with 5-FU-treatment of HCC cells significantly enhanced the G1-S phase block and apoptosis. MESP1 showed an aberrant high expression in HCC and was associated with poor clinical outcomes; therefore, MESP1 may be a potential target for the diagnosis and treatment of HCC.