Bax Expression Levels Research Articles

The Blepharis persica seed hydroalcoholic extract synergistically enhances the apoptotic effect of doxorubicin in human colon cancer and gastric cancer cells.

The goal of this survey is to evaluate the anti-proliferative effects of the hydroalcholic extract of Blepharis persica seeds and its synergic effect on doxorubicin (DOX) in human colon cancer (HT-29) and gastric cancer cell (AGS) lines. 70% Ethanol was used for extraction of B. persica seed. Aluminum-chloride colorimetric and Folin-Ciocalteu reagent methods were used to measure total flavonoid and total phenolic contents of the extract respectively. Gas chromatography-mass spectrometry (GC-MS) analysis of the B. persica extract was performed on GC-MS equipment after silylation. HT-29, AGS, and human fibroblast (SKM) cell lines were treated by different concentration of the B. persica extract, (DOX) and the combination of extraction and DOX. The cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay while the apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. The changes in expression levels of BAX and BCL-2 were determined using Real-Time RT-qPCR. GC-MS analysis of the hydroalcoholic extract from B. persica seeds revealed 24 major components. The MTT assay revealed the cytotoxicity against three cell lines and also it was shown that 125ng/mL of DOX and 0.625mg/mL of B. persica extract had synergistic behavior against HT29 cell line. These results showed B. persica extract induced apoptosis in AGS and HT29 cells and its extract caused dose-dependent increase in up-regulation of BAX level (p < 0.05) and down-regulation of BCL2 (p < 0.05). B. persica showed the synergistic effect in combination with DOX on HT29 cell line. These findings demonstrated a basis for further studies on the characterization and mechanistic evaluation of the bioactive compounds of B. persica extract which had antiproliferative effects on cancer cell lines.

Acupuncture Reduces Apoptosis of Granulosa Cells in Rats with Premature Ovarian Failure Via Restoring the PI3K/Akt Signaling Pathway.

Acupuncture is widely recognized as an effective therapy for premature ovarian failure (POF) in clinical, but information about its potential mechanisms is rarely explored. To investigate the mechanism, fifty SD female rats were randomly divided into normal group, POF group, POF+estradiol-valerate group (abbreviated as estradiol group), and POF+acupuncture group (abbreviated as acupuncture group). The estrous cycle of the rats was tracked by vaginal smears. Their ovaries morphology was observed by hematoxylin-eosin staining. The apoptotic level of granulosa cells was detected by in situ TUNEL fluorescence staining assay. Serum follicle-stimulating hormone (FSH) and estrogen (E2) levels were measured by enzyme-linked-immunosorbent-assay (ELISA). Protein and gene expression of PI3K, Akt, bcl-2, and bax were detected by Western blotting and qPCR. In the acupuncture and estradiol groups, compared with the POF group as controls, the apoptosis number of granulosa cells was significantly decreased (p < 0.05). FSH levels were decreased, while E2 levels were increased (p > 0.05). The gene and protein expression levels of PI3K, Akt, and bcl-2 were increased, while the expression levels of bax were decreased (p < 0.05), and the protein expression level of p-Akt increased. There was no significant difference between the acupuncture group and the estradiol group (p > 0.05). Acupuncture was able to regulate hormone levels in POF rats, up-regulate PI3K/Akt signaling pathway, and reduce the apoptosis of granulosa cells. This may be one of the mechanisms of acupuncture treating premature ovarian failure.

Effects of triclosan on antioxidant- and apoptosis-related genes expression in the gill and ovary of zebrafish.

Triclosan (TCS) is a broad-spectrum antibacterial and anti-fungal agent used in a broad variety of personal care products (PCPs) throughout the world. However, the molecular mechanism of TCS’s effects on the gill and ovary of fish is not clear. In this study, the effects of TCS exposure on expression of antioxidant- and apoptosis-related genes were investigated in the gill and ovary of zebrafish (Danio rerio). Zebrafish were exposed to 0, 17, 34, or 68 µg/l TCS for 42 days. Antioxidant-related genes (SOD, GPx1a, CAT, sMT-B, and MT-2) in the gill were significantly downregulated in the 34 (except GPx1a) and 68 µg/l TCS groups, and these genes (except MT-2) in the ovary were significantly downregulated in the 68 µg/l TCS group. Apoptosis-related gene (Bax and p53) expression level in the gill were significantly downregulated in the 68 µg/l TCS group, while the ratios of BCL-2 to Bax and MDM2 gene were significantly upregulated. The Bax gene in the ovary was significantly upregulated in the 34 and 68 µg/l TCS groups, while the ratio of BCL-2 to Bax was significantly downregulated. Moreover, the p53 gene in the ovary in the 34 µg/l TCS group was significantly upregulated. In addition, the MDA contents in the gill in the 34 and 68 μg/l TCS treated groups and in the ovary in 68 μg/l group were significantly increased. The results showed that the higher dose of TCS might cause oxidative damage in the gills and ovaries and accelerate ROS-dependent ovary apoptosis in zebrafish.

Avicularin inhibits cell proliferation and induces cell apoptosis in cutaneous squamous cell carcinoma.

Avicularin (AL), quercetin-3-α-L-arabinofuranoside, has various pharmacological properties such as anticancer and anti-infective effects. However, the potential molecular mechanism via which AL exerts its anticancer activity is not fully understood. Cutaneous squamous cell carcinoma (CSCC) is the second most common skin cancer, where metastasis has resulted in in effective clinical treatments. The aim of the present in vitro study was to investigate the anticancer effects and underlying mechanism of AL on human CSCC. The present results suggested that AL dose-dependently inhibited SCC13 cell viability and induced apoptosis. In addition, the present results suggested that AL induced apoptosis via repression of the mitogen-activated protein kinase kinase (MEK)/NF-κB signal pathway, thereby affecting the expression of apoptosis-related genes. Bax expression level was increased, while Bcl-2 expression level was decreased in SCC13 cells following AL treatment. In addition, the MEK/NF-κB signaling pathway-related genes p-MEK and phosphorylated-p65 were also decreased. The present results suggested that AL treatment increased the expression level of E-cadherin, but decreased the expression levels of N-cadherin, matrix metalloproteinase (MMP)-9 and vimentin in SCC13 cells. Collectively, the present results suggested that AL may have an anti-CSCC effect by inhibiting cell viability, inducing apoptosis and inhibiting epithelial-mesenchymal transition (EMT) of CSCC cells. The mechanism of these anti-CSCC effects was suggested to be via the regulation of apoptosis-related genes and EMT-related genes, and the inhibition of the MEK/NF-κB signaling pathway.

CircularRNA abhydrolase domain containing 16A-microRNA-1237-5p-apoptosis associated tyrosine kinase axis regulates the apoptosis of michigan cancer foundation-7 in breast cancer cells

Objective To investigate the circular RNA abhydrolase domain containing 16A (circRNA ABHD16A)-microRNA (miRNA, miR)-1237-5p-Apoptosis associated tyrosine kinase (AATK) axis on apoptosis of breast cancer cell munggan cancer foundation-7 (MCF-7). Methods The correlation between ABHD16A and miR-1237-5p and miR-1237-5p and AATK was analyzed by CSCD and TargetScan online analysis. The luciferase assay was used to detect whether circRNA ABHD16A targets miR-1237-5p and whether miR-1237-5p targets AATK. The expression of apoptotic marker proteins in AATK and breast cancer cells MCF-7 was detected by Western blot after over-expression or knockdown of circRNA ABHD16A. Cell viability of breast cancer cells MCF-7 was analyzed by methyl thiazolyl tetrazolium (MTT) assay; staining by Annexin-V was performed by MTT assay. The level of apoptosis of breast cancer cells MCF-7 was measured. Student’s t-test analyzed two sets of one-way analysis of variance. Results circRNA ABHD16A targets miR-1237-5p; miR-1237-5p targets the 320-326 and 469-475 regions of the AATK 3’ UTR. When overexpressing ABHD16A, AATK expression increased significantly (t=15.295, P<0.01), apoptosis-related protein bcl-2 antagonist/killer 1 (bak), bcl-2 Associated X Apoptosis Regulator (bax) and cleaved Caspase-9 (cleaved-CAS9) expression levels increased significantly (t=7.403, 8.381, 21.583, P<0.01). The expression of bcl-2 Apoptosis Regulator (bcl-2) was significantly decreased (t=6.301, P<0.01), the activity of MCF7 cells was decreased (t=17.392, P<0.01), and the level of apoptosis was increased (t=8.491, P<0.05). When ABHD16A was knocked down, the expression of AATK was significantly decreased (t=9.290, P<0.01), and the expression levels of apoptosis-related proteins bak, bax and cleaved-CAS9 were significantly decreased (t=8.381, 5.296, 11.492, P<0.01), while the expression of bcl-2 increased significantly (t=5.609, P<0.01), the activity level of MCF7 cells increased (t=5.694, P<0.01), and the level of apoptosis decreased (t=13.502, P<0.01). Conclusion circRNA ABHD16A targets miR-1237-5p and increases the expression of AATK and promotes the apoptosis of breast cancer cells MCF-7. Key words: CircularRNA abhydrolase domain containing 16A; MicroRNA-1237-5p; Apoptosis associated tyrosine kinase; Breast cancer; Michigan cancer foundation-7; Cell apoptosis

Inhibition of autophagy triggers melatonin-induced apoptosis in glioblastoma cells

BackgroundAutophagy is considered to be another restorative focus for the treatment of brain tumors. Although several research have demonstrated that melatonin induces autophagy in colon cancer and hepatoma cells, there has not been any direct evidence of whether melatonin is capable of inducing autophagy in human glioma cells.ResultsIn the present research, we report that melatonin or its agonist, agomelatine, induced autophagy in A172 and U87-MG glioblastoma cells for a concentration-and time-dependent way, which was significantly attenuated by treatment with luzindole, a melatonin receptor antagonist. Furthermore, by suppressing autophagy at the late-stage with bafilomycin A1 and early stage with 3-MA, we found that the melatonin-induced autophagy was activated early, and the autophagic flux was complete. Melatonin treatment alone did not induce any apoptotic changes in the glioblastoma cells, as measured by flow cytometry. Western blot studies confirmed that melatonin alone prominently upregulated the levels of Beclin 1 and LC3 II, which was accompanied by an increase in the expression of Bcl-2, whereas it had no effect on the expression of Bax in the glioblastoma cells. Remarkably, co-treatment with 3-MA and melatonin significantly enhanced the apoptotic cell population in the glioblastoma cells, along with a prominent decrease in the expression of bcl-2 and increase in the Bax expression levels, which collectively indicated that the disruption of autophagy triggers the melatonin-induced apoptosis in glioblastoma cells.ConclusionsThese results provide information indicating that melatonin may act as a common upstream signal between autophagy and apoptosis, which may lead to the development of new therapeutic strategies for glioma.

Effect of Down-Regulating the CD59 by RNAi Lentivirus on the Expression of Acute T-lineage Leukemia Jurkat Cell Line

To analyze the effect of down-regulating the CD59 gene expression by RNAi lentivirus as vector on Jurkat cell line of acute T-lineage leukemia. The expression of CD59 in Jurkat cell line of acute T-line leukemia was induced to decrease by RNAi lentivirus as vector. The transfection of RNA lentivirus and the localization of CD59 molecule were analyzed by laser confocal technique. The relative expression of CD59 gene in blank control, negative control and RNAi lentivirus transfected group was detected by real-time fluorescence quantitative PCR, and the enzyme-linked immunosorbent assay was used to detect the expression of TNF-β and IL-3 in supernatants of cultured cells in 3 groups. The expression levels of apoptosis-related molecules including Caspase-3, Survivin, BCL-2 and BCL-2-associated X protein (BAX) were measured by Western blot. The transfection efficiency for Jurkat cells was higher than 90%. CD59 was mainly located on the cell membrane. Compared with the blank control group and the negative control group, the expression level of CD59 mRNA and protein in the RNAi lentivirus transfected group significantly decreased (P<0.05). Compared with the blank control group and the negative control group, the expression of TNF-β and IL-3 in the RNAi lentivirus transfected group were significantly higher and lower (P<0.05) respectively. The expression levels of Survivin and BCL-2 in the RNAi lentivirus transfected group were significantly lower than those in the blank control group and the negative control group, while the expression levels of Caspase-3 and BAX in the RNAi lentivirus transfected group were significantly higher than those in the blank control group and the negative control group (P< 0.05). The down-regulation of CD59 gene expression induced by RNAi lenti-virus can decrease the expression of proliferation and differentiation-promoting molecule such as IL-3 and increase the expression of TNF-related factor in Jurkat cell line of acute T-lineage leukemia, which also can increase the expression of apoptosis-related proteins such as Caspase-3 and BAX, and decrease the expression of anti-apoptosis-related proteins such as Survivin and BCL-2.

Effect of Down-Regulating the CD59 by RNAi Lentivirus on the Expression of Acute T-lineage Leukemia Jurkat Cell Line

To analyze the effect of down-regulating the CD59 gene expression by RNAi lentivirus as vector on Jurkat cell line of acute T-lineage leukemia. The expression of CD59 in Jurkat cell line of acute T-line leukemia was induced to decrease by RNAi lentivirus as vector. The transfection of RNA lentivirus and the localization of CD59 molecule were analyzed by laser confocal technique. The relative expression of CD59 gene in blank control, negative control and RNAi lentivirus transfected group was detected by real-time fluorescence quantitative PCR, and the enzyme-linked immunosorbent assay was used to detect the expression of TNF-β and IL-3 in supernatants of cultured cells in 3 groups. The expression levels of apoptosis-related molecules including Caspase-3, Survivin, BCL-2 and BCL-2-associated X protein (BAX) were measured by Western blot. The transfection efficiency for Jurkat cells was higher than 90%. CD59 was mainly located on the cell membrane. Compared with the blank control group and the negative control group, the expression level of CD59 mRNA and protein in the RNAi lentivirus transfected group significantly decreased (P<0.05). Compared with the blank control group and the negative control group, the expression of TNF-β and IL-3 in the RNAi lentivirus transfected group were significantly higher and lower (P<0.05) respectively. The expression levels of Survivin and BCL-2 in the RNAi lentivirus transfected group were significantly lower than those in the blank control group and the negative control group, while the expression levels of Caspase-3 and BAX in the RNAi lentivirus transfected group were significantly higher than those in the blank control group and the negative control group (P< 0.05). The down-regulation of CD59 gene expression induced by RNAi lenti-virus can decrease the expression of proliferation and differentiation-promoting molecule such as IL-3 and increase the expression of TNF-related factor in Jurkat cell line of acute T-lineage leukemia, which also can increase the expression of apoptosis-related proteins such as Caspase-3 and BAX, and decrease the expression of anti-apoptosis-related proteins such as Survivin and BCL-2.

Investigating the mechanisms behind extensive death in human cancer cells following nanoparticle assisted photo-thermo-radiotherapy.

We have recently reported the synthesis and characterization of gold-coated iron oxide nanoparticle and demonstrated such a nanoparticle (Au@Fe2O3 NP) was able to significantly enhance the lethal effects of photo-thermo-radiotherapy. The purpose of this study was to determine the mechanisms behind such an enhancement by investigating the changes induced in cancer cell viability, proliferation, and morphology as well as monitoring the alteration of some genes which play important role in the process of cell death. Using MTT assay and transmission electron microscopy (TEM), the KB cells viability and morphology were assessed after treating with various combinations of NPs, photothermal therapy (PTT), and radiotherapy (RT). Clonogenic assay was used to assess the proliferation ability of treated KB cells. Nanoparticle internalization into the cells was investigated by TEM and inductively coupled plasma (ICP). During the treatment procedures, temperature changes were monitored using an IR-camera. Furthermore, the changes occurred in Bax, BCL2 and HSP70 genes expression level were measured using real-time PCR. The results showed that combination of NP, PTT, and RT caused more cell death compared to PTT or RT alone. Following such a combination therapy, massive cell injury was detected. We also observed an extensive increase in Bax/Bcl2 ratio and HSP70 expression for the KB cells treated by combination therapy procedure. Our results showed that massive cell injury and apoptosis induction are the main reasons of extensive cell death observed in cancer cells when a nanoparticle assisted photo-thermo-radiotherapy procedure is applied.

Electrochemotherapy induces tumor regression and decreases the proliferative index in canine cutaneous squamous cell carcinoma

Canine cutaneous squamous cell carcinoma (cSCC) is the most common skin cancer in dogs, and, due to its low metastatic rate, local treatments, such as electrochemotherapy (ECT), promote disease control or even complete remission (CR). This study aimed to evaluate the gene and protein expression of Bcl-2 and Bcl-2 associated X protein (BAX), the proliferative index and clinical parameters in dogs with cSCC subjected to ECT. A prospective nonrandomized clinical study was performed using dogs with naturally occurring cSCC that was treated with ECT. Eighteen lesions from 11 dogs were selected. The tumor size at day 0 (D0) had no impact on survival or prognosis (P > 0.05). Tumor samples had a lower proliferative index after ECT (D21) than before ECT (P = 0.031). The survival of subjects with Ki67 values lower and higher than the Ki67 median value were not significantly different (P > 0.05). Regarding apoptotic markers, there were no significant differences in the gene and protein expression levels of BAX or Bcl-2 at D0 and D21 (P > 0.05) or in the overall survival of subjects with different levels of apoptotic markers. In conclusion, there was no change in BAX or Bcl-2 gene and protein expression in response to ECT at the time points evaluated, but ECT was able to reduce tumor volume and cellular proliferation in cSCC.

Sperm miR-15a and miR-29b are associated with bull fertility.

MicroRNAs modulate male fertility by regulating gene expression. In this study, dynamics of sperm miR-15a, miR-29b and miR-34a from high fertility (HF) and low fertility (LF) bulls using RT-qPCR were evaluated. Bioinformatic tools were employed to ascertain genes of interest of the sperm miRNAs. The expression levels of p53, BCL2, BAX and DNMT1 in bull spermatozoa were determined by immunoblotting. MicroRNA levels of miR-15a and miR-29 were higher in LF sires when compared with those present in HF bulls. Expression levels of miR-34a did not differ between the two groups. We found an inverse correlation between miR-15a and bull fertility. MiR29-b was also negatively associated with fertility scores. BCL2 and DNMT1 were higher in HF bulls while BAX was higher in the LF group. Our data showed a positive correlation between BCL2 and bull fertility. In addition, DNMT1 was positively associated with bull fertility. Furthermore, levels of BAX were negatively linked with bull fertility scores. Identification of miRNAs found in the spermatozoa of sires with different in vivo fertility helps understand the alterations in the fertilising capacity from cattle and other mammals. These potential biomarkers can be used in reproductive biotechnology as fertility markers to assess semen quality and predict male fertility.

In vitro supplementation of trans-10, cis-12 conjugated linoleic acid ameliorated deleterious effect of heat stress on bovine oocyte developmental competence

Environmental stresses, such as heat stress (HS), have been shown to have diverse effects on the developmental competence of oocytes. The aim of this study was to determine the effect of exogenous conjugated linoleic acid (CLA) supplementation in maturation medium on bovine oocyte maturation and developmental competence under HS condition. Accordingly, cumulus-oocyte complexes (COCs) were cultured at 41 °C and 38.5 °C for the first and second 12 h of maturation in the presence of 0 (PC), 50 (CLA50-HS) and 100 (CLA100-HS) μM CLA. Also, a group of COCs were cultured at 38.5 °C for 24 h of maturation without CLA supplementation as negative control (NC). Nuclear maturation, level of intracellular glutathione (GSH), reactive oxygen species (ROS) content, cleavage and blastocyst rates as well as relative expression of BAX, and BCL2 genes in blastocysts were investigated. Our finding for the PC and NC groups revealed that HS decreased the percentage of MII oocytes, cleavage and blastocyst rates (P < 0.05). Moreover, HS lead to an increase in ROS levels and relative expression of BAX gene, decreased the intracellular content of GSH and relative expression of BCL2 gene (P < 0.05). However, the cleavage and blastocyst rates tended to increase in the CLA-supplemented groups compared to PC group (p < 0.10). Also, ROS and GSH levels in the matured oocytes decreased and increased in the CLA50-HS group compared to the PC group (P < 0.05), respectively. The ratio of expression levels of BAX to BCL2 genes was not different between the PC and CLA50-HS groups (P > 0.05). These findings suggest that HS has undesirable effects on the maturation competence of bovine oocyte and subsequent embryo development while administration of CLA can ameliorate some of adverse effects of HS.

MicroRNA-17-5p targets expression of cancer-associated genes in breast cancer cells

Although there are several studies, biological function and therapeutic potential of miR-17-5p in breast cancer carcinogenesis remains muchly elusive. Specifically, its interaction with several cancer-associated genes remains unexplored in breast cancer. Here, we aimed to demonstrate the biological function and therapeutic potential of miR-17-5p in breast cancer through determination of the interactions between cancer-associated genes and miR-17-5p. MFC-7 breast cancer cells used in the study and cells were transfected with miR-17-5p miRNA mimics to ectopically overexpress miR-17-5p. MiR-17-5p expression levels and expression changes of cancer-associated genes were determined by using qPCR method. Expression levels of miR-17-5p were significantly enhanced following 72 h of mimic transfections as compared to scrambled control and blank control. Overexpression of miR-17-5p led to differential expression of several cancer-associated genes. Notably, BECN, CDKN2B and AIFM genes were significantly altered. Although not significant, expression levels of BAX, BCLXL, VIM, BCL2, MTOR, RB1, AKT1, XIAP, P53, and PTEN genes were found to be slightly elevated whereas expression levels of BCL-2, VIM and AKT1 were found to be decreased. Results of the present study indicate that miR-17-5p can act as both tumor suppressor and tumor promoter oncomiR in breast cancer.

Cuscuta chinensis flavonoids alleviate bisphenol A-induced apoptosis of testicular cells in male mice offspring.

Bisphenol A (BPA) is a widespread environmental endocrine disruptor that has multiple effects on reproductive organ development. To investigate the effect of Cuscuta chinensis flavonoids (CCFs) on testicular apoptosis induced by BPA in male mice offspring, pregnant mice were administered intragastrically with BPA and CCF at gestation day (GD) 0.5-17.5. The testes of male offspring (F1 males) were collected at post-natal day (PND) 21 and PND 56 for the detection of related indicators. The results showed that compared with the BPA group, the testicular index in CCF groups was significantly increased at PND 21 (p<.01). For the mice of different concentrations of CCF groups, the expression levels of bax, caspase-9 and caspase-7 proteins were significantly decreased at PND 21 and PND 56, while the expression level of bcl-2 protein was significantly increased, and testicular apoptotic cells were also decreased significantly (p<.01 or p<.05). Forty mg/kg CCF has no significant difference compared with the control group. The results indicated that CCF could protect the testis development of F1 male mice by alleviating the apoptosis of testicular cells induced by BPA.

Involvement of apoptosis in the protective effects of Dracocephalum moldavaica in cerebral ischemia reperfusion rat model

ABSTRACTAn extract of Dracocephalum moldevica (DML) was found to exert protective effects on cerebral ischemia-reperfusion injury (CIRI); however, the mechanisms underlying the observed actions of this plant-derived mixture remain to be determined. Thus, the aim of this study was to examine the influence of DML on CIRI rat model induced by middle cerebral artery occlusion (MCAO). The following parameters were measured: (1) viable neurons in the infarcted area using Nissl staining; and (2) immunohistochemistry and Western blot were employed to determine protein expression levels of p53, bcl-2 associated X protein (bax) and B-cell lymphoma-2 (bcl-2), three biomarkers of apoptosis. MCAO significantly decreased the number of viable cortical pyramidal neurons in the infarcted area, while treatment with DML extract significantly elevated the number of viable neurons. MCAO was found to significantly elevate in gene expression levels of p53 and protein expression levels bax accompanied by diminished protein expression levels of bcl-2. Prior administration of DML extract produced marked reduction in gene expression levels of p53 and protein expression levels bax but increased in protein expression levels of bcl-2. Data suggested apoptosis was initiated in MCAO and that DML was effective in treating CIRI via an anti-apoptotic action as evidenced by inhibition of gene expression levels of p53 and protein expression levels of bax with concomitant elevation in protein expression levels of bcl-2. Our findings suggest that extract of DML may prove beneficial in treatment of cerebrovascular disorders.

Effect of Dehydrocostus Lactone on Proliferation of K562 Cells and Its Mechanism

To explore the inhibitory effect of dehydrocostus lactone on the proliferation of human chronic myeloid leukemia K562 cells and the underlying mechanisms. Cell viability was evaluated by CCK-8 assay. Flow cytometry was used to assess the effect of dehydrocostus lactone on the cell cycle and apoptosis of K562 cells. The levels of BCR/ABL-STATs-related molecules were analyzed by using Western blot. The CCK-8 assay showed that dehydrocostus lactone at graded concentration of 4, 6, 8, 10, 12 μmol/L could significantly inhibit the proliferation of K562 cells after exposure for 24 h. The proliferation inhibition rate was (24.32±3.05%), (42.91±3.89%), (46.35±4.93%), (77.06±5.42%) and (89.04±4.25%) respectively, showing statistically significantly different from (2.08±0.27%) in the control (P＜0.05). Also, the treatment with 5 and 10 μmol/L of dehydrocostus lactone induced K562 cell apoptosis, the apoptotic rate of K562 cells was significantly higher than that the control group (P＜0.05), and up-regulated the expression level of BAX and p21. Furthermore, dehydrocostus lactone (5 and 10 μmol/L) also increased the percentage of cells in G2/M [(8.53±1.71)% to (17.42±2.72) and (31.79±4.38%)](P＜0.05). The study results also revealed that dehydrocostus lactone significantly inhibited the expression of BCR/ABL STAT3, STAT5, CyclinB1, CDK1 and BCL-2, and up-regulated the expression level of BAX and p21. Dehydrocostus lactone can suppress the proliferation of K562 cells and induce the apoptosis of K562 cells through BCR/ABL-STAT signaling pathways.

Bax expression is optimal at low oxygen tension and constant agitation

Bax is a pro-apoptosis protein that translocates from the cytosol to the mitochondria membrane upon initiation of programed cell death. Bax subsequently disrupts the mitochondria membrane, resulting in the release of cytochrome C which activates the downstream caspases. The structure of inactive Bax has been solved, but despite intensive investigation, the mechanism by which it regulates apoptosis is not established. The low yield of Bax expression in E. coli hampers efforts to elucidate the mechanism. Thus, we undertook a systematic study aimed at improving the yield of Bax. Bacteria were grown in a computer-controlled fermenter and expression was induced by addition of Isopropyl ß-d-1-thiogalactopyranoside (IPTG). The Bax expression level decreased continuously when the dissolved oxygen level was kept at 30%, which is non-limiting for E. coli. Alternatively, when oxygen input was decreased with constant agitation and air flow (or kLa), Bax yield increased by a factor of three. To make sure the short chain fatty acids generated during micro-aerobic fermentation had no adverse effect, their concentrations were closely monitored with HPLC and their effect on cell growth and Bax expression were investigated additionally using shake flasks. Through proteomic analysis using Tandem Mass Tag (TMT) labeling, we identified degradation pathway within E. coli cells as a potential player behind the lower expression level.

Zearalenone induces apoptosis in bovine mammary epithelial cells by activating endoplasmic reticulum stress

Zearalenone (ZEA) is a common mycotoxin produced by fungi within the genus Fusarium. However, few studies have examined the direct effects of the toxin on the mammary glands. In the present study, the effects of ZEA treatment on bovine mammary epithelial cells (MAC-T) from dairy cows were investigated. The cells were treated with different concentrations of ZEA to evaluate the effect of the toxin on cell viability, intracellular reactive oxygen species (ROS) concentrations, mitochondrial membrane potential, endoplasmic reticulum (ER) stress, and the expression of apoptosis-related genes. The results indicated that different concentrations (5, 10, 15, 20, 25, 30, 50, 60, or 100 μM) of ZEA were able to inhibit growth of MAC-T cells. After exposing the MAC-T cells to 30 μM ZEA, compared with the control group, ROS levels increased, mitochondrial membrane potential decreased, and mRNA expression of the ER-specific stress-related genes GRP78, HSP70, ATF6, EIF2A, ASK1, and CHOP was upregulated in the ZEA-treated group. Further, we analyzed the increase in apoptotic rate by flow cytometry. At the mRNA level, compared with the control group, the expression of the apoptosis-promoting gene BAX was increased in the ZEA-treated group, the expression of the inhibitory gene BCL2 decreased, and the expression of the gene CASP3 increased. We observed a significant increase in caspase-3 activity in ZEA-treated MAC-T cells. Furthermore, the apoptotic rate of the cells in the ZEA group treated with 4-phenylbutyric acid (ER stress inhibitor) decreased and the mRNA expression levels of ER stress markers GRP78 and CHOP decreased. Compared with the ZEA treatment group, the mRNA expression level of the apoptosis-related gene BAX was decreased and the expression level of BCL2 was increased in the ZEA + 4-phenylbutyric acid cotreatment group. These findings indicate that ZEA-induced ER stress increases apoptosis in MAC-T cells. The treatment of MAC-T cells with ZEA reduced cell viability, increased ROS content, decreased mitochondrial membrane potential, increased ER stress marker expression, and induced apoptosis.

Lumbrokinase effects on pro- and anti-apoptotic gene expression in Wistar rats with testicular torsion.

BackgroundTesticular reperfusion is believed to be the mechanism by which testicular injury occurs in the ischemic testis. This study was performed to determine the therapeutic efficacy of lumbrokinase for treating ischemia-reperfusion (IR) injury-induced bilateral testicular torsion.MethodsTwenty-four male rats were equally divided into the following groups: torsion only (T), torsion plus lumbrokinase (TL), torsion-detorsion only (TD) and torsion-detorsion plus lumbrokinase (TDL) groups. The right testicle in each groups sample was rotated 720° for 4 h, followed by orchiectomy. The rats in the TD (TD and TDL) groups additionally underwent detorsion for 1 h after the initial rotation. Testicular tissues were collected for measuring anti-apoptotic B-cell lymphoma-2 (BCL-2) and pro-apoptotic BCL-2-associated X protein (BAX) gene expression levels using real-time polymerase chain reaction.ResultsPro- and anti-apoptotic gene expression levels were increased in the TD groups. Lumbrokinase was significantly effective in lowering BAX expression levels, particularly those in the TDL group compared with those in the TD group (P<0.05). Lumbrokinase did not significant change BCL-2 expression levels.ConclusionThe administration of lumbrokinase before orchiectomy can protect against IR-induced testicular damage by reducing pro-apoptotic gene expression levels.

Curcumin protects against palmitic acid-induced apoptosis via the inhibition of endoplasmic reticulum stress in testicular Leydig cells

BackgroundPalmitic acid (PA) is a common saturated fatty acid that induces apoptosis in various types of cells, including testicular Leydig cells. There is evidence suggesting that PA is increased in patients with obesity and that PA-induced cell apoptosis may play an important role in obesity-related male infertility. Curcumin, a natural polyphenol, has been reported to exert cytoprotective effects in various cell types. However, the cytoprotective effect of curcumin against PA-induced apoptosis in Leydig cells remains unknown. Therefore, the current study was performed to investigate the protective effects of curcumin in response to PA-induced toxicity and apoptosis in murine Leydig tumor cell line 1 (MLTC-1) cells and explore the mechanism underlying its anti-apoptotic action.MethodsMLTC-1 cells were cultured in Roswell Park Institute-1640 medium and divided into five groups. First four groups were treated with 50–400 μM PA, 400 μM PA + 5–40 μM curcumin, 400 μM PA + 500 nM 4-phenylbutyric acid (4-PBA, an endoplasmic reticulum (ER) stress inhibitor), and 500 nM thapsigargin (TG, an ER stress inducer) + 20 μM curcumin, respectively, followed by incubation for 24 h. Effects of PA and/or curcumin on viability, apoptosis, and ER stress in MLTC-1 cells were then determined by cell proliferation assay, flow cytometry, and western blot analysis. The fifth group of MLTC-1 cells was exposed to 400 μM of PA and 5 IU/mL of human chorionic gonadotropin (hCG) for 24 h in the absence and presence of curcumin, followed by measurement of testosterone levels in cell-culture supernatants by enzyme-linked immunosorbent assay (ELISA). Rats fed a high-fat diet (HFD) were treated with or without curcumin for 4 weeks, and the testosterone levels were detected by ELISA.ResultsExposure to 100–400 μM PA reduced cell viability, activated caspase 3, and enhanced the expression levels of the apoptosis-related protein BCL-2-associated X protein (BAX) and ER stress markers glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in MLTC-1 cells. Treating cells with 500 nM 4-PBA significantly attenuated PA-induced cytotoxicity through inhibition of ER stress. Curcumin (20 μM) significantly suppressed PA- or TG-induced decrease in cell viability, caspase 3 activity, and the expression levels of BAX, CHOP, and GRP78. In addition, treating MLTC-1 cells with 20 μM curcumin effectively restored testosterone levels, which were reduced in response to PA exposure. Similarly, curcumin treatment ameliorated the HFD-induced decrease in serum testosterone level in vivo.ConclusionsThe present study suggests that PA induces apoptosis via ER stress and curcumin ameliorates PA-induced apoptosis by inhibiting ER stress in MLTC-1 cells. This study suggests the application of curcumin as a potential therapeutic agent for the treatment of obesity-related male infertility.