mRNA Expression Levels Of C-myc Research Articles

Changes in the molecular nodes of the Notch and NRF2 pathways in cervical cancer tissues from the precursor stages to invasive carcinoma.

Cancer is a multifactorial disease characterized by the loss of control in the expression of genes known as cancer driver genes. Cancer driver genes trigger uncontrolled cell replication, which leads to the development of malignant tumors. A cluster of signal transduction pathways that contain cancer driver genes involved in cellular processes, such as cell proliferation, differentiation, apoptosis and dysregulated organ growth, are associated with cancer initiation and progression. In the present study, three signal transduction pathways involved in cervical cancer (CC) development were analyzed: The Hippo pathway (FAT atypical cadherin, yes-associated protein 1, SMAD4 and TEA domain family member 2), the Notch pathway [cellular-MYC, cAMP response element-binding binding protein (CREBBP), E1A-associated cellular p300 transcriptional co-activator protein and F-Box and WD repeat domain containing 7] and the nuclear factor erythroid 2-related factor 2 (NRF2) pathway [NRF2, kelch-like ECH-associated protein 1 (KEAP1), AKT and PIK3-catalytic subunit α]. Tumor samples from patients diagnosed with various stages of CC, including cervical intraepithelial neoplasia (CIN) 1, CIN 2, CIN 3, in situ CC and invasive CC, were analyzed. The mRNA expression levels were analyzed using reverse transcription-quantitative PCR assays, whereas protein expression levels were assessed through immunohistochemical tissue microarrays. High mRNA expression levels of c-MYC and AKT and low expression levels of NRF2 and KEAP1 were associated with a decreased survival time of patients with CC. Additionally, increased expression levels of c-MYC were detected in the invasive CC stage. At the protein level, increased NRF2 expression levels were observed in all five stages of CC samples compared with those in the cancer-free control samples. AKT1 was found to be dysregulated in the CIN 1 and CIN 2 stages, PI3K in the in situ and invasive stages, and CREBBP in the CIN 3 and in situ stages. In summary, the present study demonstrated significant changes in proteins of the Notch and NRF2 pathways in CC. NRF2 was overexpressed in all cervical cancer stages (cervical intraepithelial neoplasia, in situ CC and invasive CC). The present study makes an important contribution to the possible biomarker proteins to be analyzed for the presence of premalignant and malignant lesions in the cervix.

Abstract 3294: The GSPT1 molecular glue degrader MRT-2359 is active against prostate cancer

Abstract We have previously described our GSPT1 molecular glue degrader MRT-2359, which was optimized to achieve preferential antiproliferative activity in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) expressing high N-MYC or L-MYC mRNA. A clinical trial with this new drug candidate is ongoing (NCT05546268). Here, we present pre-clinical profiling of MRT-2359 across hundreds of cancer cell lines representing multiple cancer types, aiming at identifying other cancers where preferential sensitivity is associated with high expression of a MYC family member. These studies showed that prostate cancer cell lines clearly segregate into groups that are either sensitive or insensitive. Sensitive prostate cancer cell lines could be further divided into two subgroups: (1) androgen receptor positive cell lines displaying higher c-MYC mRNA expression levels than the other prostate cancer cell lines, (2) neuroendocrine prostate cancer cell lines with or without high N-MYC. The latter is in line with our previous observation that the neuroendocrine phenotype is generally associated with heightened sensitivity to MRT-2359 independently of MYC status. The insensitive cell lines were androgen receptor and neuroendocrine negative and displayed lower c-MYC mRNA levels. GSPT1 degradation in androgen receptor-positive cell lines led to rapid and deep loss of c-MYC protein as well as reduction of androgen receptor, including the splice variant AR-V7 that is associated with resistance to agents targeting androgen signaling. In androgen receptor-negative cell lines, the levels of c-MYC protein decreased only modestly upon GSPT1 degradation. Finally, in immunocompromised mice, xenografts of the AR-V7-positive cell line 22RV1 and the neuroendocrine prostate cell line NCI-H660, both of which are sensitive to MRT-2359 in vitro, were treated with several MRT-2359 dose regimens including continuous as well as intermittent (5 days on/9 days off) dosing. Most regimens led to marked tumor regression. Tumors of both models fully regressed after a 4-week course of 10 mg/kg MRT-2359 once daily, and no tumor regrowth could be detected after cessation of treatment. No significant in vivo response was observed for xenografts of the insensitive cell line PC-3. These data support the clinical investigation of MRT-2359 in prostate cancer. Citation Format: Ralph Tiedt, Martin Schillo, Arnaud Osmont, Débora Bonenfant, Rajiv Narayan, Owen Wallace, Markus Warmuth. The GSPT1 molecular glue degrader MRT-2359 is active against prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3294.

Discovery of Palbociclib as a potent c-Myc G4 stabilizer for lung cancer treatment using molecular docking, molecular dynamics simulation, and in vitro activity evaluation.

Despite significant progress in lung cancer treatment, this disease remains a prevalent and serious global malignancy, leading to high rates of illness and death. Urgent research is needed to discover new or alternative therapies that can improve clinical outcomes for lung cancer patients. In our study, we successfully demonstrated the effectiveness of Palbociclib, a CDK4/6 inhibitor, in suppressing the growth of lung cancer cells. The IC50 values obtained were 11.00μM and 11.74μM for H1299 and A549 cells, respectively. Furthermore, our findings indicate that Palbociclib may possess strong c-Myc G4 stabilizing properties by significantly reducing both protein and mRNA expression levels of c-Myc. Additionally, Palbociclib induces apoptosis and causes cell cycle arrest at the G2/M phase in two cells. Through circular dichroism (CD), molecular docking, and molecular dynamics (MD) simulation, we have provided evidence that Palbociclib enhances the structural stability of c-Myc G4 while exhibiting a high binding affinity to its ligand's binding site on c-Myc G4. These results suggest that Palbociclib holds promise as a novel c-Myc G4 stabilizer for treating cancers associated with abnormal c-Myc activity; further optimization and development are warranted.

Cell proliferation effects of S-allyl-L-cysteine are associated with phosphorylation of janus kinase 2, insulin-like growth factor type-I receptor tyrosine kinase, and extracellular signal-regulated kinase 2 in primary cultures of adult rat hepatocytes

The cell proliferation effect of S-allyl-L-cysteine (SAC) and its mechanisms were examined in primary cultures of adult rat hepatocytes. In serum-free cultivation, SAC (10−6 M)-stimulated hepatocytes showed significant proliferation compared to control at 5-h culture; the effect was dependent on the culture time and the dose of SAC (EC50 value 8.58 × 10−8 M). In addition, SAC-stimulated hepatocytes significantly increased mRNA expression levels of c-Myc and c-Fos at 1 h and cyclin B1 at 3.5 and 4 h, respectively. In contrast, alliin and allicin, structural analogs of SAC, did not show these effects observed with SAC. The SAC-induced hepatocyte proliferation effects were completely suppressed by monoclonal antibodies against growth hormone receptor and insulin-like growth factor type-I (IGF-I) receptor, respectively. Furthermore, the Janus kinase 2 (JAK2) inhibitor TG101209, phospholipase C (PLC) inhibitor U-73122, IGF-I receptor tyrosine kinase (RTK) inhibitor AG538, PI3 kinase inhibitor LY294002, MEK inhibitor PD98059, and mTOR inhibitor rapamycin completely suppressed the SAC-induced hepatocyte proliferation. JAK2 (p125 kDa) phosphorylation in cultured hepatocytes peaked 5 min after SAC stimulation. SAC-induced IGF-I RTK (p95 kDa) and ERK2 (p42 kDa) phosphorylation had slower rises than JAK2, peaking at 20 and 30 min, respectively. These results indicate that SAC promoted cell proliferation by growth hormone receptor/JAK2/PLC pathway activation followed by activation of the IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

Silencing tumor-intrinsic CD73 enhances the chemosensitivity of NSCLC and potentiates the anti-tumoral effects of cisplatin: An in vitro study.

Besides suppressing anti-tumoral immune responses, tumor-intrinsic inhibitory immune checkpoints have been implicated in tumor development. Herein, we aimed to investigate the significance of tumor-intrinsic CD73, as an inhibitory immune checkpoint, in non-small cell lung cancer (NSCLC) development and propose a novel therapeutic approach. We investigated the cell viability, chemosensitivity, apoptosis, migration, and the cell cycle of A-549 and NCI-H1299 following treatment with cisplatin and CD73-small interfering RNA (siRNA) transfection. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to study the viability of studied groups and chemosensitivity of tumoral cells. Flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) staining were used to investigate the apoptosis of NSCLC cells. Flow cytometry and the wound-healing assay were used to investigate the cell cycle and migration of NSCLC cells, respectively. The mRNA expression levels of c-Myc, caspase 3, ROCK, and MMP-9 were investigated to study the underlying molecular mechanism. CD73-siRNA transfection has significantly decreased the cell viability and enhanced the chemosensitivity of A-549 and NCI-H1299 cells to cisplatin. CD73-siRNA has considerably stimulated apoptosis, arrested the cell cycle, inhibited tumor migration, downregulated the mRNA expression of c-Myc, MMP-9, and ROCK, and upregulated caspase 3 expression in NSCLC cells. Besides, combined cisplatin therapy with CD73-siRNA transfection has potentiated the aforementioned anti-tumoral effects of cisplatin on NSCLC cells. Besides suppressing anti-tumoral immune responses, tumor-intrinsic CD73 can facilitate NSCLC development, and the combined cisplatin therapy with CD73-siRNA transfection can substantially enhance the chemosensitivity of NSCLC to cisplatin and potentiates cisplatin-induced anti-tumoral effects on NSCLC.

Effects of K-ras gene silence on the expression of oncogenes in HBE cells treated with PM(2.5)

Objective: To explore the effects of K-ras gene on the expressions of oncogenes and cancer suppressor genes in human bronchial epithelial (HBE) cells which were exposed to PM(2.5). Methods: According to the mRNA sequence of K-ras gene provided by GenBank in September 2019, interference sequences were designed and synthesized, and the recombinant lentiviral vector was transfected into HBE cell to construct the K-ras gene-silenced cells. HBE cells and K-ras gene-silenced cells were exposed to 10 μg/ml, 50 μg/ml PM(2.5) suspension and 10 μmol/L Cr(6+). Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of c-myc, c-fos, N-ras, cyclin-D1, p16 and p53 genes, the expression levels of p53 and c-myc proteins were detected by Western blot. Results: In K-ras silenced cell group, K-ras mRNA expression level decreased (80.5%±3.6%) and K-ras protein level decreased (58.9%±4.7%) when compared with the control group (P<0.01) . Compared with the correspoding cell control group without exposure, the mRNA expression levels of c-myc, c-fos, N-ras and cyclin-D1 genes in HBE cell group exposed to different concentrations of PM(2.5), K-ras silenced cell group exposed to different concentrations of PM(2.5), HBE cell group exposed to 10 μmol/L Cr(6+) and K-ras silenced cell group exposed to 10 μmol/L Cr(6+) were increased, the mRNA expressions of p16 and p53 genes were decreased (P<0.01) . Compared with HBE cell group exposed to 10 μg/ml PM(2.5), the mRNA expressions of c-myc, c-fos and p16 genes in K-ras silenced cells exposed to 10 μg/ml PM(2.5) were decreased, and the p53 mRNA level was increased (P<0.01) . Compared with HBE cell group exposed to 50 μg/ml PM(2.5), the mRNA expression levels of c-fos, N-ras, cyclin-D1, p16 and p53 genes in K-ras silenced cell group exposed to 50 μg/ml PM(2.5) were decreased (P<0.01) . Compared with the HBE cell group without exposure, c-myc protein increased and p53 protein decreased in HBE cells exposed to 50 μg/ml PM(2.5) (P<0.05) . Compared with the K-ras silenced cell group without exposure, c-myc protein increased in K-ras silenced cells exposed to 50 μg/ml PM(2.5) (P<0.05) . Conclusion: PM(2.5) can increase the expression levels of oncogenes in HBE cells, and K-ras gene silencing can inhibit the expression levels of oncogenes in HBE cells treated with PM(2.5).

Isotretinoin and Thalidomide Down-Regulate c-MYC Gene Expression and Modify Proteins Associated with Cancer in Hepatic Cells.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer. The number of cases is increasing and the trend for the next few years is not encouraging. HCC is usually detected in the advanced stages of the disease, and pharmacological therapies are not entirely effective. For this reason, it is necessary to search for new therapeutic options. The objective of this work was to evaluate the effect of the drugs isotretinoin and thalidomide on c-MYC expression and cancer-related proteins in an HCC cellular model. The expression of c-MYC was measured using RT-qPCR and western blot assays. In addition, luciferase activity assays were performed for the c-MYC promoters P1 and P2 using recombinant plasmids. Dose-response-time analyses were performed for isotretinoin or thalidomide in cells transfected with the c-MYC promoters. Finally, a proteome profile analysis of cells exposed to these two drugs was performed and the results were validated by western blot. We demonstrated that in HepG2 cells, isotretinoin and thalidomide reduced c-MYC mRNA expression levels, but this decrease in expression was linked to the regulation of P1 and P1-P2 c-MYC promoter activity in isotretinoin only. Thalidomide did not exert any effect on c-MYC promoters. Also, isotretinoin and thalidomide were capable of inducing and repressing proteins associated with cancer. In conclusion, isotretinoin and thalidomide down-regulate c-MYC mRNA expression and this is partially due to P1 or P2 promoter activity, suggesting that these drugs could be promising options for modulating the expression of oncogenes and tumor suppressor genes in HCC.

Effect of mir-129 on the Sensitivity Enhancement of Methotrexate and Migration Inhibition in Osteosarcoma Cancer Cells

Background: MicroRNAs have been recently declared to be contributed to the various aspects of osteosarcoma cells, including growth and survival, apoptosis, invasion, and chemoresistance. Objectives: The present study aimed to investigate the potentiating effects of miR-129 on the chemosensitivity of Saose-2 osteosarcoma cells to methotrexate (MTX) and underlying mechanisms. Methods: Saose-2 cells were transfected with miR-129 mimics using Lipofectamine. The cytotoxic effects of miR-129 and MTX on Saose-2 cells were measured using MTT assay. Scratch wound healing assay was used to evaluate cell migration. The apoptosis rate of cancer cells was also measured using ELISA Cell Death Assay and flow cytometry. The mRNA expression levels of target genes were measured using quantitative RT-PCR. Results: miR-129 mimic transfection significantly increased the expression levels of this miRNA in Saose-2 cells (P&lt;0.05). The combination of MTX with miR-129 transfection led to enhanced cytotoxic effects of MTX in lower concentrations. In addition, miR-129 significantly increased MTX-induced apoptosis levels and decreased invasion behavior in Saose-2 cells. The mRNA expression levels of c-Myc, K-Ras, CXCR4, MMP9, and ADAMTS, as main genes involved in chemoresistance and invasion, were downregulated in miR-129 transfected cells. Conclusion: The obtained results revealed the importance of miR-129 in the sensitivity of osteosarcoma cells to MTX and its underlying mechanisms. Therefore, miR-129 might be an appropriate candidate for reversing MTX resistance in osteosarcoma cells.

PIM1 overexpression in T-cell lymphomas protects tumor cells from apoptosis and confers doxorubicin resistance by upregulating c-myc expression.

T-cell lymphomas (TCLs) are a malignancy characterized by tumor aggression and resistance to traditional chemotherapy. Disruption of the extrinsic cell death pathway is essential for resistance to chemotherapy. PIM1 serves as a crucial modulator in cancers. However, the role of PIM1 in TCLs remains unclear. In this study, we studied the roles of PIM1 in established T-lymphoma cell lines Jurkat and HUT-78. CCK-8 assay was conducted to evaluate cell survival and flow cytometry was performed to evaluate cell death of TCL cells. siRNAs were used to knockdown the expression of PIM1 and c-myc. qRT-PCR was used to evaluate the mRNA expression levels of c-myc and PIM1. Western blot analysis was used to evaluate the protein expression levels of PIM1, c-myc, STAT3, and phospho-STAT3. Doxorubicin was used to determine the effect of PIM1 on apoptosis. Our results showed that PIM1 expression was markedly enhanced and induced c-myc expression in TCL cells. Doxorubicin inhibited the expressions of c-myc and PIM1, and triggered the extrinsic cell death of TCLs by suppressing the JAK-STAT3 signaling pathway. Moreover, PIM1 silencing via siRNA suppressed c-myc expression, promoted the cell death of TCLs, and increased doxorubicin sensitivity. Conversely, PIM1 overexpression in TCL cells induced c-myc expression, suppressed TCL cell death, and promoted doxorubicin resistance. Collectively, our results demonstrate that PIM1 overexpression in TCLs participates in cancer cell protection from apoptosis and leads to doxorubicin resistance by inducing c-myc expression, indicating that PIM1 may be a promising target in TCL treatment.

Oncogene RNA helicase DDX6 promotes the process of c-Myc expression in gastric cancer cells.

Human DEAD-box RNA helicase gene DDX6 was cloned from B-cell lymphoma cell line RC-K8. Previously, we reported that DDX6 acts as oncogene in several cancers such as colorectal cancer and hepatocellular carcinoma. However, the detailed mechanism of DDX6 action in carcinogenesis is largely unknown. In this study, we examined the functions of DDX6 in clinical gastric cancer (GC) samples and GC cells. DDX6 protein expression levels of cancer samples were higher than those of the adjacent normal tissues in 25 clinical GC samples (median value: 1.4 times higher). Also, the results of an RNA immunoprecipitation-assay (RIP-assay) showed that DDX6 associated with c-Myc mRNA. Moreover, enforced overexpression of DDX6 promoted both mRNA and protein expression of c-Myc in GC cells. On the other hand, the gene silencing of DDX6 induced growth suppression through down-regulation of c-Myc in GC cells grown in either two or three dimensions. Furthermore, c-Myc mRNA expression levels of cancer samples were higher than those of the adjacent normal tissues in DDX6 up-regulated-GC clinical samples. Our findings in this study suggested that DDX6 acted as oncogene in GC cells through promotion of c-Myc expression by association with the mRNA of c-Myc.

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line.

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines.

The evaluation of the anti-cancer activity of ixazomib on Caco2 colon solid tumor cells, comparison with bortezomib

Proteasome inhibition has recently emerged as a clinically effective anticancer therapeutic approach. The first proteasome inhibitor, bortezomib (Velcade, PS-341), and new proteasome inhibitors including ixazomib have become more important in the development of targeted cancer therapies. Under physiological conditions, MLN9708 (ixazomib citrate), the stable citrate ester drug substance, hydrolyzes rapidly to MLN2238 (ixazomib), the biologically active boronic acid. It is a second-generation proteasome inhibitor, similar to the well-known proteasome inhibitor bortezomib, which is currently being investigated in phase 3 trials as a treatment for multiple Myeloma. Despite the proven efficacy of these drugs in hematologic malignancies, clinical activity is limited to solid tumors such as colon adenocarcinoma. This study is the first to investigate and compare the antiproliferative and apoptotic effects of MLN2238 and bortezomib on human colon adenocarcinoma Caco2 cells. The antiproliferative effects of MLN2238 and bortezomib were determined using WST-1; apoptotic effects of this drug were determined by caspase-3 and a mitochondrial membrane potential (JC-1) activity assay. Expression levels associated with proteasome inhibition and apoptosis of NF-κB and c-myc mRNA were evaluated by RT-PCR. At 24 and 48 h, MLN2238 showed significant time- and concentration-dependent antiproliferative and apoptotic effects on Caco2 cells. Depending on increasing mitochondrial depolarization and caspase-3 activation, MLN2238 induced apoptosis at level similar to that of bortezomib. In addition, MLN2238 downregulated NF-κB and c-myc mRNA expression levels. For the first time, MLN2238 was shown to induce antiproliferative and apoptotic effects on human colon adenocarcinoma cells that are comparable with those of bortezomib; these in vitro data in Caco2 cells support the development of MLN2238 for colon cancer.

The Role of miR-34a in Tritiated Water Toxicity in Human Umbilical Vein Endothelial Cells.

In this work, we investigated the toxic effects of tritiated water (HTO) on the cardiovascular system. We examined the role of microRNA-34a (miR-34a) in DNA damage and repair in human umbilical vein endothelial cells (HUVECs) exposed to HTO. Cell proliferation capacity was evaluated by cell counting, and miR-34a expression was detected using quantitative PCR (QT-PCR). The Comet assay and γ-H2AX immunostaining were used to measure DNA double-strand breaks (DSBs). Reverse transcription polymerase chain reaction was used to measure the expression level of c-myc messenger RNA (mRNA). The cells exposed to HTO showed significantly lower proliferation than the control cells over 3 days. The DNA damage in the HTO group was more severe than that in the control group, at each time point examined. The expression of miR-34a mimics caused increased DNA DSBs whereas that of the miR-34a inhibitor caused decreased DNA DSBs. The proliferation viability was the opposite for the miR-34a mimics and inhibitor groups. The expression levels of c-myc mRNA in cells transfected with miR-34a mimics were lower than that in cells transfected with the miR-34a-5p inhibitor, at 0.5 hours and 2 hours after transfection. In summary, miR-34a mediates HTO toxicity in HUVECs by downregulating the expression of c-myc.

Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells

Inhibition of the proteasome has emerged as a clinically effective anticancer therapeutic approach in recent years. Bortezomib (Velcade®) showed extremely high potency against a wide range of cancer cell lines. Ixazomib (MLN9708-MLN2238), the second-generation proteasome inhibitor, selectivity and potency were similar to that of bortezomib, is currently being investigated in phase I studies. It shows superior antitumor activity in hematologic malignancy, especially multiple myelomas. In this study, for the first time, we evaluated and compared the antiproliferative and apoptotic effects of the novel proteasome inhibitor MLN2238 (the active form of MLN9708) with bortezomib using in vitro chronic myeloid leukemia. Cytotoxic and apoptotic effects of MLN2238 and bortezomib were determined by trypan blue dye exclusion assays, WST-1 cell proliferation assay, increased AnnexinV-PI binding capacity, changes in caspase-3 activity and loss of mitochondrial membrane potential (JC-1). Associated with proteasome pathway NFκB1 and c-myc mRNA expression levels were examined by the qRT-PCR method. We observed that cytotoxic and apoptotic effects on K562 cells were started at 5 μm of MLN2238 and 1 μm of bortezomib after 24 and 48 h. Also, MLN2238 and bortezomib downregulated NFκB1 and c-myc mRNA expression at 24 h. Our result revealed that MLN22238 and bortezomib had significant cytotoxic and apoptotic effects on K562 cells. Here, we first demonstrate in vitro data that support the development of MLN2238, by direct comparison with bortezomib on K562 cells.

Chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish

The potential interference of endocrine disrupting chemicals (EDCs) on aquatic animals and humans has drawn wide attention in recent years. Reports have shown that some organophosphorus pesticides were a kind of EDCs, but their effects on fish species are still under research. In present study, flow cytometry data of HEC-1B cell line showed that chlorpyrifos (CPF) could increase cell proliferation index like 17β-estradiol (E2), but the effect of CPF was weaker than of E2 in the same concentration. Moreover, CPF altered the expression pattern of estrogen-responsive gene VTG and ERα in zebrafish embryos. When exposed to CPF at various concentrations (0, 0.10, 0.25, 0.50, 0.75 and 1.00mg/L) for 48h during the embryo stage, compared with controls, the hatching rate of treated groups significantly increased at the same time and the hatching rate of embryos was proportional to CPF concentration. The mRNA expression levels of c-myc, cyclin D1, Bax and Bcl-2, which are closely related to cell proliferation and cell apoptosis, were disturbed by CPF in zebrafish embryos after exposure treated for 48h. In addition, acridine orange (AO) staining of zebrafish embryos showed that cell apoptosis was appeared in the 0.75, 1.00mg/L CPF treated groups. Taken together, the results obtained in the present study indicated that chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish.

C-myc in whitefish (Coregonus lavaretus): structure, expression, and insights into possible posttranscriptional regulatory mechanism.

c-myc has a crucial function in growth control, differentiation, and apoptosis of vertebrate cells. Despite the important role of c-myc in mediating the biological effects, studies of c-myc gene expression and factors that control it in organisms other than mammals, such as fish, have been rare. In the current study, we asked whether c-myc mRNA of whitefish, a feasible organism for pollution monitoring in aquatic systems and a model in toxicological research, contains activity sites for regulatory motifs in its 5'- and 3'-UTRs, similar to those found in mammals. We were particularly interested in whether miRNA-34, a known negative regulator of c-myc's in mammals, is able to regulate c-myc in fish. To answer these questions, we determined the mRNA sequence of whitefish c-myc and inferred the structure of the protein that it codes for. We found that the active sites of mRNA and structures of the inferred c-myc protein are similar to those found in mammals and other fish. Remarkably, levels of c-myc mRNA expression were very high in ovaries compared to other tissues of whitefish, thus corroborating previous data in fish. Using bioinformatic searches on c-myc 3'-UTR, we confirmed the presence of two miRNA-34a(miR-34a)response elements. Luciferase reporter assay showed that activity of reporters containing either the miR response elements or entire c-myc 3'-UTR was significantly reduced (p<0.001) by ectopic expression of miR-34a. Therefore, we further investigated possible involvement of miR-34a in c-myc gene silencing by profiling the expression of both genes in livers of whitefish treated for 8, 24, 48h with MC-LR, a potent c-myc inducer in mammals. Although the difference was only significant at p=0.08, the expression of c-myc mRNA in challenged whitefish after 24h of the treatment was notably higher than that in livers of control fish. Concurrently, we noticed slight but significant up-regulation of miR-34a after 24 and 48h of the challenge (p<0.05); however, we found no significant correlation of the c-myc mRNA levels and miR-34a expression. Together, these results suggest that miR-34a might regulate c-myc gene expression in whitefish liver; however, their involvement in MC-LR hepatotoxicity should be clarified in future studies.

The effect of caralluma fimbriata extract on metabolic parameters in high-fat fed wistar rats

The effect of caralluma fimbriata extract on metabolic parameters in high-fat fed wistar rats

Epigenetic mechanisms regulate placental c-myc and hTERT in normal and pathological pregnancies; c-myc as a novel fetal DNA epigenetic marker for pre-eclampsia.

Placental development is known for its resemblance with tumor development, such as in the expression of oncogenes (c-myc) and telomerase (hTERT). The expression of c-myc and hTERT is up-regulated during early pregnancy and gestational trophoblastic diseases (GTDs). To determine the role of DNA methylation [via methylation-sensitive high resolution melting (MS-HRM)] and histone modifications [via chromatin immunoprecipitation (ChIP assay)] in regulating the differential expression of c-myc and hTERT during normal gestation and their dysregulation during placental disorders, we obtained placental samples from 135 pregnant women, in five groups: normal first, second and third trimester (n = 30 each), pre-eclamptic pregnancy (n = 30) and molar pregnancy (n = 15). Two placental cell lines (JEG-3 and HTR-8/SVneo) and isolated first-trimester cytotrophoblasts were also studied. Quantitative RT-PCR revealed decreased mRNA expression levels of c-myc and hTERT, which were associated with a higher level of H3K9me3 (1.5-fold, P < 0.05) and H3K27me3 (1.9-fold, P < 0.05), respectively, in third-trimester placental villi versus first-trimester villi. A significantly lower level of H3K27me3 in molar placenta was associated with a higher mRNA expression of c-myc and hTERT. The development of pre-eclampsia (PE) was associated with increased methylation (P < 0.001) and H3K27me3 (P < 0.01) at the c-myc promoter and reduced H3K9me3 (P < 0.01) and H3K27me3 (P < 0.05) at the hTERT promoter. Further, mRNA expression of c-myc and hTERT was strongly correlated in molar villi (r = 0.88, P < 0.01) and JEG-3 cells (r = 0.99, P < 0.02). Moreover, on the basis of methylation data, we demonstrate the potential of c-myc as a fetal DNA epigenetic marker for pre-eclamptic pregnancies. Thus we suggest a role for epigenetic mechanisms in regulating differential expression of c-myc and hTERT during placental development and use of the c-myc promoter region as a potential fetal DNA marker in the case of PE.

Overexpression of C-MYC oncogene in prostate cancer predicts biochemical recurrence

Alterations of chromosome 8, including amplification at 8q24 harboring the C-MYC oncogene, have been noted as one of the most common chromosomal abnormalities in prostate cancer (CaP) progression. However, the frequency of C-MYC alterations in CaP has remained uncertain. A recent study, using a new anti-MYC antibody, described prevalent upregulation of nuclear C-MYC protein expression as an early oncogenic alteration in CaP. Further, we have recently reported regulation of C-MYC expression by ERG and a significant correlation between C-MYC overexpression and TMPRSS2-ERG fusion in early stage CaP. These emerging data suggest that increased C-MYC expression may be a critical and early oncogenic event driving CaP progression. In this study, we assessed whether C-MYC mRNA overexpression in primary prostate tumors was predictive of more aggressive tumor or disease progression. Our approach was to quantitatively determine C-MYC mRNA expression levels in laser capture micro-dissected tumor cells and matched benign epithelial cells in a radical prostatectomy cohort with long follow-up data available. On the basis of our results, we conclude that elevated C-MYC expression in primary prostate tumor is biologically relevant and may be a predictor of future biochemical recurrence.

Combined analysis of cell growth and apoptosis-regulating proteins in HPVs associated anogenital tumors

BackgroundThe clinical course of human papillomavirus (HPV) associated with Bowenoid papulosis and condyloma acuminatum of anogenital tumors are still unknown. Here we evaluated molecules that are relevant to cellular proliferation and regulation of apoptosis in HPV associated anogenital tumors.MethodsWe investigated the levels of telomerase activity, and inhibitor of apoptosis proteins (IAPs) family (c-IAP1, c-IAP2, XIAP) and c-Myc mRNA expression levels in 20 specimens of Bowenoid papulosis and 36 specimens of condyloma acuminatum in anogenital areas. Overall, phosphorylated (p-) AKT, p-ribosomal protein S6 (S6) and p-4E-binding protein 1 (4EBP1) expression levels were examined by immunohistochemistry in anogenital tumors both with and without positive telomerase activity.ResultsPositive telomerase activity was detected in 41.7% of Bowenoid papulosis and 27.3% of condyloma acuminatum compared to normal skin (p < 0.001). In contrast, the expression levels of Bowenoid papulosis indicated that c-IAP1, c-IAP2 and XIAP mRNA were significantly upregulated compared to those in both condyloma acuminatum samples (p < 0.001, p < 0.001, p = 0.022, respectively) and normal skin (p < 0.001, p = 0.002, p = 0.034, respectively). Overall, 30% of Bowenoid papulosis with high risk HPV strongly promoted IAPs family and c-Myc but condyloma acuminatum did not significantly activate those genes. Immunohistochemically, p-Akt and p-S6 expressions were associated with positive telomerase activity but not with p-4EBP1 expression.ConclusionCombined analysis of the IAPs family, c-Myc mRNA expression, telomerase activity levels and p-Akt/p-S6 expressions may provide clinically relevant molecular markers in HPV associated anogenital tumors.