Monensin, an Antibiotic Isolated from Streptomyces Cinnamonensis, Regulates Human Neuroblastoma Cell Proliferation via the PI3K/AKT Signaling Pathway and Acts Synergistically with Rapamycin.

Neuroblastoma is the most common extracranial childhood tumor and accounts for approximately 15% of pediatric cancer-related deaths. Further studies are needed to identify potential therapeutic targets for neuroblastoma. Monensin is an ionophore antibiotic obtained from Streptomyces cinnamonensis with known antibacterial and antiparasitic effects. No study has reported the effects of monensin on SH-SY5Y neuroblastoma cells by targeting the PI3K/AKT signaling pathway. The aim of this study was to investigate the antiproliferative effects of monensin alone and in combination with rapamycin in human SH-SY5Y neuroblastoma cells mediated by the PI3K/AKT signaling pathway. The effects of single and combination applications of monensin and rapamycin on SH-SY5Y cell proliferation were investigated by XTT, and their effects on the PI3K/AKT signaling pathway by RT-PCR, immunohistochemistry, immunofluorescence, and Western blotting. The combined effects of monensin and rapamycin on SH-SY5Y proliferation were most potent at 72 h (combination index < 1). The combination of monensin and rapamycin caused a significant decrease in the expression of P21RAS, AKT, and MAPK1 genes. Single and combined administrations of monensin and rapamycin caused a significant decrease in PI3K/AKT expression. Our results showed for the first time that monensin exerts an antiproliferative effect by targeting the PI3K/AKT signaling pathway in neuroblastoma cells. It is suggested that monensin and its combination with rapamycin may be an effective therapeutic candidate for treating neuroblastoma.

Glycoside oleandrin downregulates toll-like receptor pathway genes and associated miRNAs in human melanoma cells.

Melanoma accounts for the majority of skin cancer-related deaths. Nerium oleander is a plant known to be toxic and consumed due to the cardiac glycosides it contains. Oleandrin is a cardiac glycoside obtained from of N. oleander. Beside capable of inhibiting proliferation and metastasis of cancer cells, cardiac glycoside derivative compounds cause cardiovascular side effects. Because of cardiovascular toxicity of clinically used cardiac glycosides, it is necessary to investigate cardiac glycoside derivative compounds capable of inhibiting proliferation and metastasis of cancer cells. It is known that oleandrin has anticarcinogenic effects in other cancers. Previous studies have shown that toll-like receptors (TLRs) and their related microRNAs (miRNAs) are associated with cancer. Therefore, aim was to investigate the effect of oleandrin on genes and miRNAs associated with TLRs in A375 melanoma cells in this study. The effects of oleandrin on cell viability, cytokines, apoptosis were evaluated using XTT, ELISA and TUNEL analyses, respectively. The effect of oleandrin on expression of TLR genes and 5 associated miRNAs in A375 cells has been determined by qRT-PCR. In addition, the levels of MyD88, TLR2 and TLR4 proteins were analyzed by western blot method. ELISA indicated that oleandrin treatment (47 nM at 48 h) reduced the level of proinflammatory cytokine IFNG. TUNEL analysis showed that apoptosis rate was significantly increased in the oleandrin dose group. According to qRT-PCR results, there was a significant decrease in IRAK1, IRAK4, MyD88, TLR2-TLR7 and TRAF3 expressions in the oleandrin treated group compared to the control (untreated cell). Also, a significant decrease in TLR4 protein expression has been observed. In addition, oleandrin significantly downregulated the levels of hsa-miRNA-146a-5p and hsa-miRNA-21-5p. In conclusion, it has been observed that oleandrin has an effect on TLR pathway-related genes and miRNAs in melanoma cells. We show that TLRs pathways and hsa-miR-146a-5p and hsa-miR-21-5p can participate in the oleandrin molecular mechanism of action.

The role of ERK-1 and ERK-2 gene polymorphisms in PCOS pathogenesis.

BACKGROUND: Ovulation is regulated by extracellular signal-regulated kinase-1 (ERK-1) and ERK-2 signaling mechanisms, and ERK-1/2 kinases modulates the function of most of the LH-regulated genes. Defective ERK kinase signaling that is secondary to a genetic problem contributes to both ovulatory dysfunction and metabolic problems in polycystic ovary syndrome (PCOS). We planned to investigate ERK-1 and ERK-2 gene polymorphisms in PCOS for the first time in the Turkish population. METHODS: One hundred two PCOS patients and 102 healthy controls were recruited for this patient control study. HOMA-IR, Ferriman-Gallwey score (FGS), waist-to-hip ratio (WHR), and body mass index (BMI) were assessed. Lipid profile levels, CRP, and total testosterone were determined. ERK-2 rs2276008 (G > C) and ERK-1 rs11865228 (G > A) SNPs were analyzed with a real-time PCR system. RESULTS: ERK-1 and ERK-2 genotypes were found to differ between the PCOS and control groups. In patients with PCOS, ERK-1 GA and ERK-2 GC genotypes were different in terms of BMI, FGS, HOMA-IR, CRP, total testosterone, and total cholesterol levels. CONCLUSIONS: ERK-1 and ERK-2 genes are involved in PCOS pathogenesis. BMI, FGS, HOMA-IR, and CRP levels are related to the heterozygote polymorphic types of ERK-1 and ERK-2 genes.

Granzyme B levels and granzyme B polymorphisms in peripheral blood of patients with endometriosis: a preliminary study.

The chronic course of endometriosis suggests that the immune system may play a role in its aetiology. There may be resistance to cell lysis, as well as an immune defect underlying endometriosis. Granzyme B is a serine protease that is secreted by Natural Killer (NK) cells and cytotoxic T lymphocytes during a cellular immune response and can induce apoptosis. The aim of this study was to evaluate the relationship between both Granzyme B levels and Granzyme B gene polymorphisms in endometriosis patients. Women between the ages of 20 - 45 were included in the study. The patients were divided into two groups: those diagnosed with endometriosis and those who had not been diagnosed with endometriosis. In the blood samples, Granzyme B gene polymorphisms and serum levels of Granzyme B were studied. There was no difference between the groups in terms of median Granzyme B levels and the presence of AA, AG, and GG genotypes. There was a difference in median granzyme levels for the control group; the GG genotype was found at a lower frequency. The immune defect within endometriosis-related immune cells may not be exclusively due to Granzyme B. Other mediators that are secreted from immune cells may have additive effects.IMPACT STATEMENTWhat is already known on this subject? NK cells are cytotoxic and inhibit the implantation of autologous endometrial cells that are spilled into the peritoneum by retrograde menstruation. Thus, a reduction in NK cell activity may facilitate the progression of endometriosis. The literature review reveals that there are studies suggesting that NK cell activity may be insufficient in endometriosis. Granzyme B is a serine protease that is secreted by NK cells and cytotoxic T lymphocytes during a cellular immune response.What do the results of this study add? Granzyme B is one of the cytotoxic granules in NK and cytotoxic T lymphocyte cells and its genetic polymorphisms were tested in endometriosis. We found that median Granzyme B levels were significantly different in patients with the GG genotype in the control group, compared to those with the AA and AG genotype. However, this difference was not detected between the control and endometriosis groups.What are the implications of these findings for clinical practice and/or further research? Our results contribute to uncovering the pathogenesis of endometriosis since there are no previous studies in the literature regarding this topic. Although we did not find a difference, our results will inform further studies made on this topic. Studies with different molecules and an increased number of patients are needed. The immune defect of endometriosis may not be due exclusively to Granzyme B. Other mediators that are secreted from immune cells may have mutual effects and interactions.

Investigation of possible effects of apigenin, sorafenib and combined applications on apoptosis and cell cycle in hepatocellular cancer cells.

Hepatocellular carcinoma (HCC) is the most common type of liver tumors. There is only one chemodrug for treatment called sorafenib that is an effective multikinase inhibitor. However, most of the patients gain resistance to sorafenib treatment in six months. Thus, there is a limitation for treatment of HCC. Apigenin is a natural flavonoid that has been used for many years as an antioxidant and anti-inflammatory agent. The aim of this study is to investigate the combined therapeutic effects of sorafenib and apigenin upon apoptosis and cell cycle on HepG2 cell line. Cytotoxic effects of sorafenib and apigenin on HepG2 cells were determined by XTT assay. Effects of single and combined treatment on cell migration, invasion and colony formation were analysed by wound healing, transwell matrigel invasion assay and colony formation assay, respectively. TUNEL assay was performed for analyse apoptosis rates. Expression changes of genes related with apoptosis and cell cycle were analysed by quantitative real-time PCR. Combined treatment of sorafenib and apigenin has more decreasing effects on cell viability than single treatment groups. Also, combination group caused significant increase of apoptotic cells. Migration and invasion capability of cells in combined treatment group are decreased. Lastly, quantitative real-time PCR results showed that combination of both drugs arrested cell cycle and increased apoptotic gene expressions more than single treatment groups. This is the first study that investigating the combined treatment of sorafenib and apigenin on HCC in vitro. By combined treatment, apigenin potentiates sorafenib cytotoxicity on HepG2 cells. Effects of combined treatment on migration, invasion, apoptosis and gene expressions showed that may sorafenib and apigenin have synergistic effect.

The determination of the potential anticancer effects of Coriandrum sativum in PC-3 and LNCaP prostate cancer cell lines.

Coriander (Coriandrum sativum L.) is such an herb from the Apiaceae family, used both for its medicinal and nutritional properties for many centuries. In this study, the effects of C. sativum extract on gene expression, viability, colony formation, migration, and invasion of PC-3 and LNCaP prostate cancer cell lines have been investigated. The half maximal inhibitory concentration (IC50 ) dose in PC-3 and LNCaP cells was detected to be 2 and 5 mg/mL at the 24th hour, respectively. C. sativum extracts have been observed to cause a significant decrease in the expression of Akt and Bcl-2 in the PC-3 cells and just Akt in LNCaP cells while increasing in the expression of p53, caspase-9, caspase-10, PTEN, DR5, TRADD, PUMA, and NOXA. DR4 expression was increased in LNCaP cell line but not PC-3, and APAF and BID had increased expression in PC-3 but not the LNCaP cells. Our observations have shown that C. sativum extract decreased colony formation while inhibiting cell invasion and migration. Cell migration was hindered in PC-3 but not the LNCaP cells. In conclusion, this data present a valuable addition to the very limited data available out there on the potential use of C. sativum in prostate cancer treatment.

Investigation of the Anticancer Mechanism of Isoorientin Isolated from Eremurus Spectabilis Leaves via Cell Cycle Pathways in HT-29 Human Colorectal Adenocarcinoma Cells.

OBJECTIVE: Isoorientin (ISO) is a flavonoid compound extracted from plant species. The goal of this study was to determine the potential antiproliferative effects of ISO in HT-29 human colorectal adenocarcinoma cell line in vitro, specifically on cell viability, apoptosis, and cell cycle pathways. MATERIALS AND METHODS: The cytotoxic effect of ISO isolated from E. spectabilis was measured using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in HT-29 cell lines. Total RNA was isolated using Tri-Reagent protocol. The effects of ISO on apoptosis-related gene were detected using real-time polymerase chain reaction (RT-PCR). The findings were analyzed using "Delta-Delta CT" ΔΔCT method and evaluated using a computer program. Volcano plot analysis was used for comparing groups and the data obtained were statistically analyzed using Student t test. RESULTS: According to XTT result analysis, the 50% inhibitory concentration (IC50) value of ISO was 125 µM at the 48th h in HT-29 cells. The RT-PCR analysis in HT-29 cells showed that Cyclin D1 (CCND1 ), Cyclin-dependent kinase 6 (CDK6), BAX, BCL-2, Checkpoint kinase 1-2 (CHEK1, CHEK2) and Excision repair cross-complementing 1 (ERCC1) expressions were reduced in ISO-treated cells compared with those in the control group of cells. P53, P21, Caspase-3 (CASP-3), Caspase-8 (CASP-8), and Caspase-9 (CASP-9) gene expressions were increased Ataxia Telengiectasia and Rad-3 related (ATR) was activated in the ISO-treated group of cells compared with those in the control group of cells (p<0.05). CONCLUSION: ISO affected the proliferation of colorectal cancer (CRC) cells via cell cycle pathways. It also altered apoptosis gene expression. These results demonstrated that ISO can be a therapeutic agent for CRC treatment; however, more studies are needed to investigate its mechanism of actions.

Human TLR gene family members are differentially expressed in patients with urothelial carcinoma of the bladder.

PURPOSE: Toll-like receptors (TLRs) have an important role in the activation of both innate and adaptive immunity in response to pathogens and endogenous danger signals from damaged or dying cells. The aim of this study was to determine the relationship between urothelial carcinoma (UC) and TLR expression. BASIC PROCEDURES: Real-time polymerase chain reaction evaluation was made of the messenger RNA expression of TLRs 1-10 in 24 UC samples and 46 nontumoral bladder tissue samples. The levels of proinflammatory cytokines (IL-1ß, IL-6, and IL-8) in the urine samples were also determined with enzyme-linked immunosorbent assay. MAIN FINDINGS: TLR2-7 and TLR10 expressions were significantly higher in UC than in the control group (P<0.05 for all comparisons). No concordance was found between matched tumor tissue and urine samples in terms of TLR expression. IL-1ß, IL-6, and IL-8 levels were significantly higher in urine specimens of patients with UC (P = 0.033, P = 0.001, and P = 0.008, respectively). PRINCIPAL CONCLUSIONS: The results of this study demonstrated that the TLR gene expression profiles reflect the heterogeneity within UC. These results might also prompt further investigation to better understand the role of the TLR gene family expression in the tumor progression of UC.

MicroRNA-125b as a new potential biomarker on diagnosis of renal ischemia-reperfusion injury.

BACKGROUND: Acute renal failure is commonly seen in the perioperative period. Ischemia-reperfusion (IR) injury plays a major role in acute renal failure and delayed graft function. MicroRNAs (miRs), which are pivotal modulators of cell activities, offer a major opportunity for affective diagnosis and treatment strategies because they are tissue specific and in the center of gene expression modulation. The effect of bardoxolone methyl (BM) on miR-21, miR-223-5p, and miR-125b in renal IR injury was evaluated in this study. METHODS: Wistar-Albino rats (12-16 wk old, weighing 300-350 g) were used in the study. Rats (n = 6) were randomized into three groups (control, IR, and BM + IR). Tissue levels of miRs were analyzed with reverse transcription polymerase chain reaction. RESULTS: Significant reduction of urea and total oxidant status, increase of total antioxidant status, and oxidative stress index were identified in the IR + BM group compared with the IR group. Significant increases of miR-21 (2842.82-fold) and miR-125b (536.8-fold) were identified in the IR group compared with the control group; however, miR-223-5p levels did not show any significant difference. Also, miR-21 and miR-125b were significantly reduced in the IR + BM group compared with the IR group. Reduced histopathologic changes were observed in the IR + BM group. A significant decrease in the number of tunel-positive cells was identified in the IR + BM group compared with the IR group. CONCLUSIONS: miR-125b was significantly increased in IR injury; thus, miR-125b can be a potential novel marker that can be used in diagnosis and treatment of renal IR injury. BM reduces miR-21 and miR-125b in case of IR injury and makes functional and histopathologic repairs.

Anti-tumor effects of bemiparin in HepG2 and MIA PaCa-2 cells.

Recent researches have demonstrated improved survival in oncologic patients treated with low molecular weight heparins (LMWHs) which are anticoagulant drugs. We evaluated "second generation" LMWH bemiparin and its in vitro anti-tumor effects on HepG2 hepatocellular carcinoma and MIA PaCa-2 cancer cells. The aim of the study is to investigate anti-cancer mechanism of bemiparin in HepG2 and Mia-Paca-2 cancer cells. Cytotoxic effects of bemiparin were determined by XTT assay. IC50 dose of bemiparin was found to be 200 IU/mL in the 48th hour in the MiaPaCa-2 cell line and 50 IU/mL in the 48th hour in the HepG2 cell line. CCND1 (cyclin D1), CDK4, CDK6, p21, p16, p53, caspase-3, caspase-9, caspase-8, Bcl-2, BID, DR4, DR5, FADD, TRADD, Bax, gene mRNA expressions were evaluated by Real-time PCR. Real-time PCR analysis showed that CCND1 expression was reduced in HepG2 dose the group cells when compared with the control group cells and p53, caspase-3, caspase p21, caspase-8 and expressions were increased in the dose group cells when compared with the control group cells. CCND1, CDK4 and CDK6 expressions were reduced in MIA PaCa-2 dose group cells when compared with the control group cells and p53 expression was increased in the dose group cells when compared with the control group cells. Other expressions of genes were found statistically insignificant both of cell lines. It was found that bemiparin in HepG2 and MIA PaCa-2 cells suppressed invasion, migration, and colony formation by using matrigel invasion chamber, and colony formation assay, respectively. In conclusion, it is thought that bemiparin indicates anti-tumor activity by affecting cell cycle arrest, apoptosis, invasion, migration, and colony formation on cancer cells.