Substance P Promotes the Progression of Endometrial Adenocarcinoma

2016 ◽  
Vol 26 (5) ◽  
pp. 845-850 ◽  
Author(s):  
Jing Ma ◽  
Shifa Yuan ◽  
Jianxin Cheng ◽  
Shan Kang ◽  
Wenhong Zhao ◽  
...  
Keyword(s):  
Substance P ◽  
Western Blot ◽  
Endometrial Carcinoma ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Endometrial Adenocarcinoma ◽  
Expression Levels ◽  
Ishikawa Cells ◽  
Proliferation And Invasion

ObjectivesIt has been demonstrated that substance P (SP) promotes while neurokinin-1 receptor (NK-1R) antagonist inhibits the proliferation of several human cancer cells. Currently, it is still unknown whether such actions exist in human endometrial carcinoma. This study aimed to explore the role of SP/NK-1R signaling in the progression of endometrial adenocarcinoma.Materials and MethodsThe expression levels of SP and NK-1R in endometrial adenocarcinoma tissues and Ishikawa cell line were detected by real-time quantitative PCR and Western blot analysis. The effects of SP on Ishikawa cells proliferation and invasion were analyzed using MTT assay and transwell matrigel invasion assay, respectively. The expression levels of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) in Ishikawa cells after administration of SP were detected by real-time quantitative RCR and Western blot analysis.ResultsThe expression levels of SP and NK-1R were significantly higher in endometrial adenocarcinoma tissues and Ishikawa cells than in normal endometrium. Substance P significantly enhanced the proliferation and invasion of Ishikawa cells. In addition, SP induced the expression of MMP-9 and VEGF-C in Ishikawa cells, whereas NK-1R antagonist inhibited these effects.ConclusionsSubstance P plays an important role in the development of endometrial carcinoma by inducing the expression of MMP-9 and VEGF-C and promoting cancer cell proliferation and metastasis, which can be blocked by NK-1R antagonist.

scholarly journals Silencing ferritin alleviates atherosclerosis in mice via regulating the expression levels of matrix metalloproteinases and interleukins

2021 ◽  
Author(s):  
Mei Zheng ◽  
Lizhuo Li ◽  
Yuqian Liu ◽  
Yun Liang ◽  
Xiaoyong Qi
Keyword(s):  
Matrix Metalloproteinases ◽  
Western Blot ◽  
Knockout Mice ◽  
Blot Analysis ◽  
High Fat Diet ◽  
Western Blot Analysis ◽  
High Fat ◽  
Expression Levels ◽  
Atherosclerotic Lesions ◽  
Apoe Knockout Mice

This study was conducted to investigate the roles of ferritin in atherosclerosis. The mouse model of atherosclerosis was established by feeding ApoE knockout mice with a high-fat diet. The mice were then treated with ferritin-overexpressing and -silencing constructs, and assessed for interleukins (ILs) and matrix metalloproteinases (MMPs) levels using ELISA and Western blot analysis. After being fed with a high-fat diet, the ApoE knockout mice developed pro-atherogenic lipid profiles with elevated total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C). They also showed increased atherosclerotic lesions including narrowed lumen diameter, reduced lumen area, and increased plaque size. Following injection of the overexpression and silencing constructs, mRNA levels of ferritin were increased and decreased, respectively, and at the same time the atherosclerotic lesions were aggravated and alleviated, respectively. Further analysis indicated that silencing of ferritin gene reduced IL-1β and IL-10 levels while overexpressing ferritin increased them. On other hand, the TNF-α levels showed an opposite trend. MMP8, MMP12 and MMP13 levels were increased or decreased significantly after the mice were injected with ferritin over-expression or silencing vectors, respectively. Western blot analysis showed that compared to the control, overexpressing ferritin resulted in increased expression of p-JNK while silencing ferritin decreased the expression. Meanwhile, the levels of pc-Jun remained unchanged. Our work demonstrates that ferritin can regulate the progress of atherosclerosis via regulating the expression levels of MMPs and interleukins. Silencing ferritin inhibits the development of atherosclerosis and is, therefore, worth being further investigated as a potential therapeutic approach for this disease.

scholarly journals PROTECTIVE EFFECT OF PHYLLANTHUS EMBLICA EXTRACT PREVENT CONTRAST-INDUCED ACUTE KIDNEY INJURY IN RATS

2019 ◽  
pp. 197-201
Author(s):  
SUPRANEE KONGKHAM ◽  
ADIS TASANARONG ◽  
ARUNPORN ITHARAT
Keyword(s):  
Acute Kidney Injury ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Kidney Injury ◽  
Saline Control ◽  
Phyllanthus Emblica ◽  
Rt Pcr ◽  
Expression Levels ◽  
Apoptotic Effect

Objective: The objective of the study was to investigate the anti-apoptosis effect of the extract from Phyllanthus emblica (PE) for the prevention of contrast-induced acute kidney injury (CI-AKI). Methods: Male Sprague Dawley rats were given saline (control) or PE extracts (500 mg/kg/day) for 5 days before the induction of CI-AKI. Renal tissues were collected for an evaluation of gene expression and immunohistochemistry (IHC). To indicate anti-apoptotic effect, the expression levels of Bax, Bcl-2, and caspase in kidney were also determined, using real-time polymerase chain reaction (RT-PCR) and Western blot analysis. Results: In the CI-AKI group, RT-PCR and Western blot analysis revealed that the expression levels of Bax and cleaved-caspase-3 were upregulated in the CI-AKI group, whereas the expression of Bcl-2 was downregulated. However, the pre-treatment with PE increased Bcl-2 expression. Moreover, decreased cleaved-caspases-3 activity was also detected using IHC. Conclusion: These findings suggested that pretreatment with PE extract provided the anti-apoptotic effect against CI-AKI in the rat model.

scholarly journals Qualitative and Quantitative Analysis of IDH1 Mutation in Progressive Gliomas by Allele-Specific qPCR and Western Blot Analysis

2019 ◽  
Vol 18 ◽  
pp. 153303381982839 ◽  
Author(s):  
Moritz Perrech ◽  
Lena Dreher ◽  
Gabriele Röhn ◽  
Pantelis Stavrinou ◽  
Boris Krischek ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Idh1 Mutation ◽  
World Health ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Health Organization

To date, diagnosis of IDH1 mutation is based on DNA sequencing and immunohistochemistry, methods limited in terms of sensitivity and ease of use. Recently, the diagnosis of IDH1 mutation by real-time polymerase chain reaction was introduced as an alternative method. In this study, real-time polymerase chain reaction was validated as a tool for detection of IDH1 mutation, and expression levels were analyzed for correlation with course of the disease. A total of 113 tumor samples were obtained intraoperatively from 84 patients with glioma having a diagnosis of diffuse glioma (World Health Organization II), anaplastic glioma (World Health Organization III), secondary glioblastoma ± chemotherapy, primary glioblastoma ± chemotherapy (World Health Organization IV). Tumor samples were snap frozen and processed for sectioning and RNA and protein isolation. Presence of IDH1 mutation was determined by DNA sequencing. Hereafter, quantitative expression of IDH1 messenger RNA was assessed using real-time polymerase chain reaction with specific primers for IDH1 mutation and –wt; protein expression was verified by Western Blot analysis and immunohistochemistry. Additionally, 19 samples of low-grade glioma and their consecutive high-grade glioma were analyzed at different time points of the disease. IDH1 mutation was identified in 63% of samples by DNA sequencing. In correlation with the real-time polymerase chain reaction results, a cutoff value was determined. Above this threshold, sensitivity and specificity of real-time polymerase chain reaction in detecting IDH1 mutation were 98% and 94%, respectively. Quantitative analysis revealed that IDH1 mutation expression is upregulated in secondary glioblastoma (mean ± standard error of mean: 3.52 ± 0.55) compared to lower grade glioma (II = 1.54 ± 0.22; III = 1.67 ± 0.23). In contrast, IDH1 wt expression is upregulated in all glioma grades (concentration >0.1) compared to control brain tissue (0.007 ± 0.0016). Western Blot analysis showed a high concordance to both sequencing and real-time polymerase chain reaction results in qualitative analysis of IDH1 mutation status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in secondary glioblastoma. In our study, real-time polymerase chain reaction and Western Blot analysis were found to be highly efficient methods in detecting IDH1 mutation in glioma samples. As cost-effective and time-saving methods, real-time polymerase chain reaction and Western Blot analysis may therefore play an important role in IDH1 mutation analysis in the future. IDH1 mutation expression level was found to correlate with the course of disease to a certain extent. Yet, clinical factors as recurrent disease or prior radiochemotherapy did not alter IDH1 mutation expression level.

Abstract 1958: MicroRNA Mir-27b Rescues Impaired Angiogenic Function of Endothelial Progenitor Cells and Accelerates Wound Healing in Type 2 Diabetes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jie-Mei Wang ◽  
Jun Tao ◽  
Alex F Chen
Keyword(s):  
Type 2 Diabetes ◽  
Wound Healing ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Real Time Pcr ◽  
Western Blot Analysis ◽  
Type 2 Diabetic

Endothelial progenitor cells (EPCs) play a key role in angiogenesis, which is dysfunctional in diabetes. MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level. However, whether miRNAs regulate EPC-mediated angiogenesis in diabetes is unknown. We tested the hypothesis that mir-27b rescues impaired EPC angiogenesis in vitro and in vivo via suppressing anti-angiogenic molecule thrombospondin-1 (TSP-1) in type 2 diabetes. Bone marrow-derived EPCs from adult male (C57BLKS/J, 9 weeks) type 2 diabetic db/db and their normal littermates db/+ mice (glucose 371.8±37.8 vs. 167.5±21.3 mg/dL, n=38, p<0.05) were used. miRNA processing enzyme Dicer in EPCs was decreased by >40% in db/db vs. db/+ mice (Western blot analysis, n=4 p<0.01), paralleled with >66% reduction of mir-27b expression (real-time PCR, n=4, p<0.05). Both TSP-1 mRNA and protein in EPCs were significantly higher in db/db vs. db/+ mice (real-time PCR, 130.1%, n=4, p<0.05, Western blot analysis, 127.4%, n=4 p<0.05), which were suppressed upon mir-27b mimic transfection (by 75%, real-time PCR and 69%, Western blot analysis, n=4 – 6, p<0.01). EPC-induced angiogenesis was decreased by >70% in db/db vs. db/+ mice (Matrigel tube formation assay, n=4, p<0.05), which was rescued upon mir-27b mimic transfection or silencing TSP-1 expression by its siRNA (both n=4, p<0.05). Furthermore, inhibition of mir-27b in normal EPCs increased their TSP-1 protein by 117.5% (n=6, p<0.05) and impaired their angiogenesis by 81.5% (n=4, p<0.01), both were reversed by silencing TSP-1 expression by its siRNA. Finally, excisional wound closure was markedly delayed in db/db vs. db/+ mice (4-mm punch biopsy, n=4, p<0.05), accompanied by impaired wound angiogenesis (perfusion index by Laser Doppler, n=4, p<0.05). Cell therapy of diabetic EPCs (3×10 5 cells) transfected with mir-27b mimic onto diabetic wounds significantly accelerated their closure rates (n=4, p<0.05 vs. diabetic EPCs alone), with a concomitant augmentation of in vivo wound angiogenesis (n=4, p<0.05). Mir-27b rescues impaired EPC angiogenesis and accelerates wound healing in type 2 diabetic mice, at least in part, via suppressing TSP-1 expression. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

scholarly journals Glucocorticoids differentially regulate Na-bile acid cotransport in normal and chronically inflamed rabbit ileal villus cells

2010 ◽  
Vol 298 (5) ◽  
pp. G675-G682 ◽  
Author(s):  
Steven Coon ◽  
Ramesh Kekuda ◽  
Prosenjit Saha ◽  
Uma Sundaram
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bile Acid ◽  
Western Blot ◽  
Bowel Disease ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Kinetic Studies ◽  
Rabbit Ileum ◽  
Expression Levels ◽  
Inflammatory Bowel

Previous studies have demonstrated that apical Na-bile acid cotransport (ASBT) is inhibited during chronic ileitis by both a decrease in the affinity as well as a decrease in the number of cotransporters. Methylprednisolone (MP), a commonly used treatment for inflammatory bowel disease (IBD, e.g., Crohn's disease), has been shown to reverse the inhibition of several other Na-solute cotransporters during chronic enteritis. However, the effect of MP on ASBT in the chronically inflamed ileum is not known. MP stimulated ASBT in villus cells from the normal rabbit ileum by increasing the cotransporter expression without a change in the affinity of the cotransporter for bile acid. Western blot studies demonstrated an increase in cotransporter expression. MP reversed the inhibition of ASBT in villus cells from the chronically inflamed ileum. Kinetic studies demonstrated that the mechanism of MP-mediated reversal of ASBT inhibition was secondary to a restoration of both affinity as well as cotransporter numbers. Western blot analysis demonstrated restoration of cotransporter numbers after MP treatment of rabbits with chronic ileitis. Thus MP stimulates ASBT in the normal ileum by increasing cotransporter numbers. MP reverses the inhibition of ASBT during chronic ileitis. However, MP restores the diminished affinity as well as cotransporter expression levels during chronic ileitis. Thus MP differentially regulates ASBT in the normal and in the chronically inflamed ileum.

scholarly journals MiR-221/222 Promote Dexamethasone Resistance of Multiple Myeloma through Inhibition of Autophagy By Targeting ATG12

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4469-4469
Author(s):  
Jian Xu ◽  
Yan Su ◽  
Aoshuang Xu ◽  
Fengjuan Fan ◽  
Haifan Huang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Combined Treatment ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Expression Levels

Abstract Dexamethasone (Dex) is the most widely used chemotherapeutic drug in the treatment of multiple myeloma (MM). Inherent or acquired resistance to Dex is broadly associated with poor prognosis in MM. Many microRNAs are aberrantly expressed in MM, including miR-221/222, which have been reported to act as oncogenes in many cancer types. Recently, accumulating evidence has shown that miR-221/222 are involved in the development of chemoresistance in a variety of cancers. However, there is still a lack of valuable data regarding the role of miR-221/222 in the chemoresistance of MM. Here, we first evaluated the expression levels of miR-221/222 in plasma cells (PCs) from MM patients by qRT-PCR analysis. The results showed that miR-221/222 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and they were further upregulated in PCs from patients with relapsed MM. In addition, we found that the expression levels of miR-221/222 were inversely correlated with Dex-sensitivity of human MM cell lines (HMCLs). Importantly, enforced expression of miR-221/222 dramatically reduced the sensitivity of Dex-sensitive HMCLs to Dex, while inhibition of miR-221/222 re-sensitized Dex-resistant HMCLs to Dex. Previous studies have shown that Dex-induced cell death in lymphoid leukemia is mediated through initiation of autophagy. To study whether autophagy was involved in Dex-induced cell death in MM cells, HMCLs were exposed to Dex, and then autophagy in these cells was evaluated by the transmission electron microscopy and western blot analysis. The results showed that Dex induced the occurrence of autophagy in Dex-sensitive HMCLs, but not in Dex-resistant HMCLs. Moreover, pharmacological inhibitors of autophagy could significantly reduce Dex-induced cell death in Dex-sensitive HMCLs. These results reveal that autophagy is critical for the induction of cell death following Dex treatment in MM. MicroRNAs have been reported to play an important role in regulating autophagy. We therefore examined whether miR-221/222 can regulate autophagy in MM cells. Low miR-221/222 expressing MM.1S (Dex-sensitive) or high miR-221/222 expressing MM.1R (Dex-resistant) cells were transfected with agomir-221/222 or antagomir-221/222, respectively, and then the level of autophagy was evaluated. The results showed that overexpression of miR-221/222 reduced the level of autophagy in MM.1S cells, while inhibition of miR-221/222 elevated the level of autophagy in MM.1R cells. Using microRNA target prediction bioinformatics tools and dual-luciferase reporter assay, we confirmed that autophagy-related gene 12 (ATG12) was a novel target gene of miR-221/222. Indeed, miR-221/222 could negatively regulate the expression of ATG12 at both the mRNA and protein levels in MM cells. In addition, knockdown of ATG12 by siRNA markedly reduced the autophagy-inducing and Dex-sensitizing activity of miR-221/222 antagomirs in MM.1R cells. Of note, in MM.1S cells, Dex treatment could further decreased the expression of miR-221/222, accompanied by upregulated expression of ATG12, whereas silencing the expression of ATG12 could significantly inhibited Dex-induced autophagy and cell death. Thus, these results suggest that ATG12 is a key player in miR-221/222-mediated autophagy inhibition and Dex-resistance. Next, we evaluated whether miR-221/222 could regulate autophagy and Dex-sensitivity of MM cells invivo. NOD/SCID mice were subcutaneously injected with MM.1R cells to establish Dex-resistant MM xenografts. Combined treatment with antagomir-221/222 plus Dex showed a remarkable reduction of tumor size compared to antagomir-221/222 or Dex alone (397.6±55.08 mm3 VS 895.8±72.44 mm3 VS 987.3±68.49 mm3). Immunohistochemistry and western blot analysis of the retrieved xenografted tumors showed that combination treatment with antagomir-221/222 plus Dex induced upregulation of ATG12, as well as extended autophagy with increased p62 degradation and Beclin-1 expression. In conclusion, our data reveal that upregulation of miR-221/222 promotes Dex resistance of MM cells through inhibition of autophagy by targeting ATG12. Therefore, miR-221/222-ATG12 autophagy-regulatory axis may potentially be applied in glucocorticoid resistance prediction and treatment. Disclosures No relevant conflicts of interest to declare.

Inhibition of PRAME Expression Causes Cell Cycle Arrest and Apoptosis In Leukemic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1695-1695
Author(s):  
Norina Tanaka ◽  
Yan-Hua Wang ◽  
Masayuki Shiseki ◽  
Minoko Takanashi ◽  
Toshiko Motoji
Keyword(s):  
Cell Cycle ◽  
Acute Leukemia ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
S Phase ◽  
Leukemic Cells ◽  
Rt Pcr

Abstract Abstract 1695 Introduction: The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples. Methods: The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained. Results: A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18). Conclusions: Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy. Disclosures: No relevant conflicts of interest to declare.

Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4213-4213
Author(s):  
Priya Khoral ◽  
Robert J Guo ◽  
Jahangir Abdi ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Western Blot ◽  
Real Time ◽  
Cell Lines ◽  
Blot Analysis ◽  
Drug Combination ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of Electroacupuncture on Ovarian Expression of the Androgen Receptor and Connexin 43 in Rats with Letrozole-Induced Polycystic Ovaries

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Ge Xu ◽  
Andong Zhang ◽  
Jiandang Liu ◽  
Xi Wang ◽  
Jiwei Feng ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Connexin 43 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Endocrine Dysfunction ◽  
Rt Pcr

Background. Polycystic ovarian syndrome (PCOS) occurs in women of reproductive age and is often characterized by reproductive and endocrine dysfunction. Androgens play a major role in PCOS, and previous studies reported abnormal expression of Connexin 43 (Cx43) in animal models of PCOS, suggesting an association of Cx43 with PCOS pathogenesis. Experimental and clinical evidence indicated that acupuncture may be a safe and effective approach for treating reproductive and endocrine disorders in women with PCOS. This study aimed to determine the effects of electroacupuncture (EA) on PCOS and its relationship with the expression of the androgen receptor (AR) and Cx43. Methods. In total, 30 female Sprague Dawley rats (6 weeks old) were randomly divided into three groups: control group, letrozole (LE) group, and LE + EA group. Rats were administered LE solution (1.0 mg/kg) for 21 consecutive days to induce PCOS. For the LE + EA group, additional EA treatment was conducted (2 Hz, 20 min/d) with “Guanyuan” (CV3) for 14 consecutive days. After hematoxylin-eosin staining, the ovarian structure was observed with an optical microscope, and serum levels of the following hormones were examined via enzyme-linked immunosorbent assay (ELISA): testosterone (T), estradiol (E2), sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH); luteinizing hormone (LH), insulin (INS), anti-Müllerian hormone (AMH), and inhibin B (INHB). Fasting blood glucose (FBG) levels were evaluated using glucose oxidase-peroxidase. Ovarian mRNA and protein expressions of AR and Cx43 were determined by real-time RT-PCR and Western blot analysis. Results. EA was found to restore the cyclicity and ovarian morphology in the PCOS rat model. Serum derived from the LE + EA group showed significant decreases in the levels of T, free androgen index (FAI), LH, LH/FSH ratio, AMH, INHB, and fasting serum insulin (FINS), and significant increases in the levels of E2, FSH, and SHBG. Western blot analysis showed a decreased protein expression of ovarian AR and Cx43; real-time RT-PCR showed reduced expression of ovarian mRNA levels of AR and Cx43. Conclusions. In conclusion, our results showed that EA can ease hyperandrogenism and polycystic ovary morphology in PCOS rats. Furthermore, EA counteracted the letrozole-induced upregulation of AR and Cx43. These results suggested that acupuncture can break the vicious cycle initiated by excessive androgen secretion and may be an effective treatment method for improving the reproductive and endocrine dysfunction caused by PCOS.

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