Association of interleukin 33 gene polymorphisms with susceptibility and regulation of inflammatory mediators in Systemic lupus erythematosus patients.

BACKGROUND: Studies have indicated the involvement of interleukin (IL)-33 in the pathogenesis of Systemic lupus erythematosus (SLE). This research intended to evaluate the association of IL33 gene rs1929992 and rs7044343 Single nucleotide polymorphisms (SNPs) with risk of SLE. In addition, the association between these SNPs and inflammatory cytokines was determined. METHODS: In this study, 200 SLE cases and 200 healthy subjects were recruited. Using allelic discrimination Real-time PCR, IL33 gene rs1929992 and rs7044343 SNPs were genotyped. The mRNA expression levels of IL-1ß, IL-6, IL-33, TNF-α were determined in the peripheral blood mononuclear cells (PBMCs). The serum levels of cytokines were also measured. RESULTS: The G allele (OR = 1.57, CI: 1.18-2.08, P = 0.0017), GG genotype (OR = 2.52, CI: 1.33-4.77, P = 0.0043), and GA genotype (OR = 2.12, CI: 1.34-3.34, P = 0.0011) of rs1929992 SNP was significantly associated with an increased SLE risk. The C allele (OR = 1.44, CI: 1.08-1.90; P = 0.0105), CC genotype (OR = 2.07, CI: 1.15-3.71; P = 0.0146), and CT genotype (OR = 1.61, CI: 1.02-2.53, P = 0.0395) of rs7044343 was significantly associated with increased SLE risk. The PBMC mRNA expression and serum levels of IL-1ß, IL-6, IL-33, TNF-α were significantly increased in the SLE patients compared to controls. However, there was no significant difference in the mRNA expression and serum levels of IL-1ß, IL-6, IL-33, and TNF-α among the SLE patients with three genotypes for both rs1929992 and rs7044343 polymorphisms. CONCLUSIONS: IL33 gene rs1929992 and rs7044343 SNPs are involved in SLE pathogenesis but they might not influence on the inflammatory pathway.

Evaluation of Apoptotic Gene Expression in Hepatoma Cell Line (HepG2) Following Nisin Treatment.

OBJECTIVE: The present study aims to examine the effects of nisin on the survival and apoptosis of the hepatoma cell line HepG2 and to investigate possible apoptosis pathways activated by nisin. MATERIALS AND METHODS: For this purpose, viability and apoptosis of the cells were accomplished by the nisin treatment using the MTT assay and Annexin-V-fluorescein/propidium iodide (PI) double staining, respectively. Additionally, the human apoptosis PCR array was performed to determine pathways or genes activated by nisin during possible apoptosis. RESULTS: The results of the present study showed that nisin was able to decrease cell viability (IC50 ~ 40 µg/ml) in a dose-dependent manner and could induce apoptosis in HepG2 cells. PCR data indicated a considerable increase in the expression of genes, such as caspase and BCL2 families, involved in the induction of apoptosis. CONCLUSIONS: The data from this study showed that overexpression of genes involved in the intrinsic pathway of apoptosis, especially caspase-9 and BID, increased apoptosis in HepG2 cells treated by nisin, compared to the control group.

Diosgenin and 4-Hydroxyisoleucine from Fenugreek Are Regulators of Genes Involved in Lipid Metabolism in The Human Colorectal Cancer Cell Line SW480.

OBJECTIVE: Diosignin and 4-hydroxy-L-isulosine (4-OH-Ile) are the two active ingredients of Fenugreek (Trigonella foenumgraecum). Thus, in this study, we examined the effects of hydroalcoholic extract of fenugreek seeds (HEFS), diosgenin and 4-OH-Ile on the expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), peroxisome proliferator-activated receptor gamma (PPARγ) and low-density lipoprotein (LDL) receptor (LDLR) which are involved in lipid metabolism in SW480 cell line. MATERIALS AND METHODS: In this experimental study, SW480 cells were cultured in RPMI-1640 medium and treated with HEFS, diosignin, 4-OH-Ile or orlistat for 24 and 48 hours. Inhibitory concentration of 20% (IC20) was calculated using MTT method and cells were then pre-treated with the IC20 concentrations for 24 and 48 hours before RNA extraction and cDNA synthesis. Changes in the expression of ACC, FAS, PPARγ and LDLR genes were assayed by employing the real time-polymerase chain reaction (PCR) method. RESULTS: Our results showed a significant down-regulation in the expression of ACC (P<0.001 and P<0.001 after 24 and 48 hours, respectively) and FAS genes (P<0.001 and P<0.001 after 24 and 48 hours, respectively) in SW480 cells treated with HEFS, diosignin, 4-OH-Ile, or orlistat, but significant up-regulation in the expression of PPARγ (P<0.001 and P<0.001 after 24 and 48 hours, respectively) and LDLR (P=0.005 and P=0.001 after 24 and 48 hours, respectively). CONCLUSION: According to the results of the present study, HEFS, diosgenin and 4-OH-Ile up or down-regulate the expression of some predominant genes involved in lipid metabolism pathway, similar to that observed for orlistat. These types of regulatory effects are presumably proper for the treatment of obesity and overweight.

The effects of transplanted mesenchymal stem cells treated with 17-b estradiol on experimental autoimmune encephalomyelitis.

The present study was conducted aimed at exploring the modulatory effects of 17-b estradiol (17-bED) on mesenchymal stem cells (MSCs) in the EAE (experimental autoimmune encephalomyelitis) animal model of multiple sclerosis (MS). Following the isolation of bone marrow-derived MSCs from the bilateral femurs and tibias of the male Wistar rats, the cells were harvested and cultured in the presence of 100 nM 17-bED for 24 h. EAE was induced in male Wistar rats (8-12 weeks old) using guinea pig spinal cord homogenate, in combination with the complete Freund's adjuvant. The MSC therapy was triggered when all of the animals obtained a disability score. The symptoms were monitored on a daily basis throughout the study until the rats were euthanized. The mRNA expression of cytokines, including IL-17, IFN-γ, TNF-α, IL-10, IL-4, and TGF-ß together with MMP8 and MMP9 as the family members of matrix metalloproteinases (MMPs) in the brain and spinal cord tissues were examined using real-time PCR. The levels of splenocytes-originated IL-10 and IFN-γ cytokines were also measured by ELISA. The MTT-based research findings showed that the infiltration of lymphocytes into the spleen decreased considerably. It was also observed that the mRNA expression of proinflammatory cytokines decreased significantly, while the mRNA levels of anti-inflammatory cytokines increased remarkably. It was also found that the mRNA levels of the examined matrix metalloproteinases (MMP8 and MMP9) were downregulated significantly. The findings of the present study indicated that the administration of 17-bED enhanced the efficacy of MSCs transplantation and modulated immune responses relatively in the EAE model, via the regulation of either pro- or anti-inflammatory cytokines and matrix metalloproteinases.

The New Compound of (2R, 4S)-N-(2, 5-difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide to Mediate the Expression of Some Apoptosis Genes by the HepG2 Cell Line

Objectives: Hepatocellular carcinoma is one of the most frequent cancers worldwide, for the treatment of which various therapy protocols and drugs have been introduced; however, none of them has suppressed cancer tissues completely. New research programs have been developed on cancer and the accompanied effects of novel synthesized compounds on cancer cell lines. Our latest reports on the molecular basis of cancer revealed a pattern of changes in gene expression triggered in the cancer pathway. Methods: HepG2 cell lines were cultured under similar conditions in both test and control groups. The IC50 concentration of the (2R, 4S)-N-(2, 5-difluorophenyl)-4-hydroxy-1-(2, 2, 2-trifluoroacetyl) pyrrolidine-2-carboxamide compound was used in the treatment group. After 48 hours from the culture, the expressional profiles of apoptosis pathway genes (84 genes) were studied using the PCR array method. Results: The findings demonstrated that the expression of some apoptosis-related genes pertaining to TNF, BCL2, IAP, and caspase families was regulated by (2R, 4S)-N-(2, 5-difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide. In the same vein, an alteration was observed in the expression of both pro-apoptotic and anti-apoptotic genes associated with the extrinsic and intrinsic apoptosis signaling pathways. Conclusions: According to the data obtained, the pyrrolidine-2-carboxamide compound was demonstrated to be able to regulate the apoptotic activities of HepG2 cells by affecting both pro-apoptotic and anti-apoptotic relevant genes.

A novel copper (II) complex activated both extrinsic and intrinsic apoptotic pathways in liver cancerous cells.

Recent advances have put fundamental focus on the application of copper (II) (Cu [II]) complexes as agents for fighting against cancer. To determine whether [Cu(L)(2imi)] complex as a novel Cu complex can induce apoptosis in HepG2 as cancerous cells and L929 as normal cells via extrinsic or intrinsic apoptotic pathways, both cell lines were treated for 24 and 48 hours at IC50 concentrations of [Cu(L)(2imi)] complex. Then, the expression of some apoptosis-related genes including p53, caspase-8, bcl-2, and bax were assayed by real-time polymerase chain reaction. The [Cu(L)(2imi)] complex seems to inhibit the expression of bcl-2 in complex-treated HepG2 cancerous cells following the 24- and 48-hour treatment. The complex upregulated the p53, bax, and caspase-8 genes, therefore treatment of HepG2 cancerous cells with [Cu(L)(2imi)] complex induces programmed cell death via the upregulation of relative bax/bcl-2 ratio. Finally, this copper complex triggered apoptosis in HepG2 cells via both intrinsic and extrinsic pathway, whereas treatment of normal L929 cells with this complex induce apoptosis only via intrinsic pathway with the upregulation of relative bax/bcl-2 ratio and does not affect the expression level of caspase-8 gene and does not trigger the extrinsic pathway. Finally, these results obtained from present study confirm the role of a novel Cu complex on the induction of apoptosis process in HepG2 and L929 cells by overexpression of bax, inhibition of bcl-2 and increase of the relative bax/bcl-2 ratio. These results support that the [Cu(L)(2imi)] complex is able to induce apoptosis in cancerous cells, therefore, it has a potential for development as a novel anticancer drug.

Evaluating of Induction of Apoptosis by Cornus mass L. Extract in the Gastric Carcinoma Cell Line (AGS)

Aim and objectives: Natural products and derivatives of medicinal vegetation can play an important role to the cure tumor. The Present study was focused to determine the effect of Cornus mass L. extract on the induction of apoptosis in AGS gastric carcinoma cell line in compared to L929 cells. Methods: In this experimental study, AGS and L929 cells were cultured and treated with different concentrations (0­10 mg/ml) of Cornus mass L. extract for 48 and 72 hours. Cell proliferation was assessed by MTT assay. The optical density of the colored solution was quantified at 570 nm wavelengths by an ELISA Reader. Making use of the apoptosis detection kit of Annexin V-FITC, PI and double staining with Annexin V-FITC were carried out for flow cytometry investigations. Data were analyzed by ANOVA. Variations with a P-value less than 0.05 were considered significant. Results: shows a noticeable deviation among various concentrations of extract when cells were treated for 48, 72 h declined cell viability in AGS cell line in comparison L929 cell lines in a dose and time-dependent manner (P < 0.05). This extract also displayed approximately several-fold increased anti-cancer potency in AGS compared to L929 cells. The IC50 value in AGS cells (evaluated after 48,72h) of the extract against AGS cells was 5/44, 2/44 mg/ml (p≤0.05). The analysis results of flow cytometry indicated that apoptosis was induced by the extract in AGS cells treated, compared with L929 cells. Conclusion: Each of our results implicates the reality that Cornus mass L. extract acts as a novel, potent inhibitor of cancer proliferation in in vitro. This may result in developing a promising therapeutic agent for the treatment of indole-sensitive cancers.

Neuroprotective and antihyperalgesic effects of orexin-A in rats with painful diabetic neuropathy.

AIM OF STUDY: Diabetes mellitus is related to the development of neuronal tissue injury in different peripheral and central nervous system regions. A common complication of diabetes is painful diabetic peripheral neuropathy (PDN). We have studied the neuroprotective and anti-nociceptive properties of neuropeptide orexin-A in an animal experimental model of diabetic neuropathy. METHODS: All experiments were carried out on male Wistar rats (220-250 g). Diabetes was induced by a single intraperitoneal injection of 55 mg/kg (i.p.) streptozotocin (STZ). Orexin-A was chronically administrated into the implanted intrathecal catheter (0.6, 2.5 and 5 nM/L, daily, 4 weeks). The tail-flick and rotarod treadmill tests were used to evaluate the nociceptive threshold and motor coordination of these diabetic rats, respectively. Cleaved caspase-3, Bax, Bcl2 and the Bax/Bcl-2 ratio, as the biochemical indicators of apoptosis, were investigated in the dorsal half of the lumbar spinal cord tissue by western blotting method. RESULTS: Treatment of the diabetic rats with orexin-A (5 nM/L) significantly attenuated the hyperalgesia and motor deficit in diabetic animals. Furthermore, orexin-A (5 nM/L) administration suppressed pro-apoptotic cleaved caspase-3 and Bax proteins. Also, orexin-A (5 nM/L) reduced the expression of Bax/Bcl-2 ratio in spinal cord dorsal half of rats with PDN. CONCLUSIONS: Altogether our data suggest that the orexin-A has anti-hyperalgesic and neuroprotective effects in rats with PDN. Cellular mechanisms underlying the observed effects may, at least partially, be related to reducing the neuronal apoptosis.

Combination therapy of mesenchymal stromal cells and sulfasalazine attenuates trinitrobenzene sulfonic acid induced colitis in the rat: The S1P pathway.

Adipose derived mesenchymal stem cells (ASCs) transplantation is a novel immunomodulatory therapeutic tool to ameliorate the symptom of inflammatory bowel disease (IBD). The objective of this study was to investigate the therapeutic effects of combined sufasalazine and ASCs therapy in a rat model of IBD. After induction of colitis in rats, ASCs were cultured and intraperitoneally injected (3 × 106 cells/kg) into the rats on Days 1 and 5 after inducing colitis, in conjunction with daily oral administration of low dose of sulfasalazine (30 mg/kg). The regenerative effects of combination of ASCs and sulfasalazine on ulcerative colitis were assessed by measuring body weight, colonic weight/length ratio, disease activity index, macroscopic scores, histopathological examinations, cytokine, and inflammation markers profiles. In addition, western blot analysis was used to assess the levels of nuclear factor-kappa B (NF-κB) and apoptosis related proteins in colitis tissues. Simultaneous treatment with ASCs and sulfasalazine was associated with significant amelioration of disease activity index, macroscopic and microscopic colitis scores, as well as inhibition of the proinflammatory cytokines in trinitrobenzene sulfonic acid (TNBS)-induced colitis. Moreover, combined ASCs and sulfasalazine therapy effectively inhibited the NF-κB signaling pathway, reduced the expression of Bax and prevented the loss of Bcl-2 proteins in colon tissue of the rats with TNBS-induced colitis. Furthermore, combined treatment with ASCs and sulfasalazine shifted inflammatory M1 to anti-inflammatory M2 macrophages by decreasing the levels of MCP1, CXCL9 and increasing IL-10, Arg-1 levels. In conclusion, combination of ASCs with conventional IBD therapy is potentially a much more powerful strategy to slow the progression of colitis via reducing inflammatory and apoptotic markers than either therapy alone.

Comparison of the effects of 17ß- estradiol treated and untreated mesenchymal stem cells on ameliorating animal model of multiple sclerosis.

OBJECTIVES: The current investigation was undertaken to evaluate the effects of 17ß- estradiol (17ß-ED) on the potential of the mesenchymal stem cells (MSCs) for modulation of immunity responses in an animal model of multiple sclerosis (MS). MATERIALS AND METHODS: After isolation of MSCs, cells were cultured in presence of 100 nM 17ß-ED for 24 hr. Modeling of experimental autoimmune encephalomyelitis (EAE) was achieved by using guinea pig spinal cord homogenate, in addition to complete Freund's adjuvant in male Wistar rats. The processes of cell therapy were started following 12 days post-immunization. This duration allows all animals to develop a disability score. The achieved EAE clinical symptoms were regularly monitored every day until day 36, when all of examined rats were euthanized. RESULTS: Cell therapy in the EAE rats with 17ß-ED-primed MSCs exhibited more desirable consequences, which in turn lead to regression of the cumulative clinical score and neuropathological changes that are more than the therapy with untreated MSCs. The serum measures of myeloperoxidase (MPO), nitric oxide (NO) as well as splenocytes-originated pro-inflammatory interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α) were significantly decreased in EAE rats treated by 17ß-ED primed-MSCs compared to EAE rats that received untreated MScs. CONCLUSION: Combination of 17ß-ED and MSCs more effectively improved the signs and symptoms of EAE.

Effect of a Copper (II) Complex on The Induction of Apoptosis in Human Hepatocellular Carcinoma Cells

Objectives: In the present study, we aimed to identify the anti-proliferative potential of [Cu(L)(2imi)] complex [L = 2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato) and 2imi = 2-methyl imidazole] against HepG2 cells as an in vitro model of human hepatocellular carcinoma and normal mouse fibroblast L929 cells. Methods: The cytotoxic and apoptotic effects of [Cu(L)(2imi)] complex on HepG2 cells and normal fibroblasts (L929) were examined by MTT assay and flow cytometry, respectively. Results: Cytotoxicity induced by [Cu(L)(2imi)] complex was time dependent. Also, there was a positive correlation between cytotoxicity and an increase in Cu complex concentration. For HepG2 cells, the cell viability percentage was 50% at 58 µg/mL after 24 h treatment, whereas in the same concentration and conditions, the viability percentage was surprisingly higher (about 100%) for L929 cells. Also, after 48 h treatment, the viability percentage of HepG2 cells at 55 µg/mL concentration was 50% in contrast with 89.3% for L929 cells in the same conditions. Flow cytometry findings suggest that [Cu(L)(2imi)] complex is capable of decreasing cancer cell viability through apoptosis and did not efficiently activate the necrosis process. Conclusions: Finally, we found that [Cu(L)(2imi)] complex possess the potential for development as an anti-cancer drug for human hepatocellular carcinoma.

Nisin Induces Cytotoxicity and Apoptosis in Human Asterocytoma Cell Line (SW1088)

Background: Nisin is a member of the group of anti-microbial peptides which are considered as bacteriocins, but it possesses a vast range of activities. Astrocytoma is among the most prevalent types of brain tumor globally. Considering all facts about this peptide, the aim of the present study was the evaluation of any impact of nisin on proliferation and apoptosis of an astrocytoma cell line (SW1088). Methods: The SW1088 cell line was purchased from the Pasteur Institute of Iran and treated with various concentrations of Nisin. Nisin-induced cell toxicity and apoptosis were detected by both MTT assay and annexin V-FITC /propidium iodide (PI) staining. Result: In current study we observed that the cell death and apoptosis were significantly increased following nisin treatment, as compared to the control group. Conclusion: These results open a new window for establishment promising approaches with the concept of anti-cancer therapy by nisin in the future.

The cytotoxicity effects of a novel Cu complex on MCF-7 human breast cancerous cells.

A variety of biological activities, such as anti-microbial and anti-tumor properties was reported for 1,10-phenanthroline and its copper complexes. In this study, the anti-proliferative activity of a novel  [Cu(L)(phen)] complex was investigated on MCF-7 breast cancer cells using MTT assay. Since chemotherapy is lake of ability to distinguish between normal cells from cancerous cells, therefore we also investigated the effect of  [Cu(L)(phen)] complex on normal L929 cells. The results showed that following 24 and 48 h exposure of cells with  [Cu(L)(phen)] complex, the IC50 values for MCF-7 were significantly lower than that recorded for L929 and normal cells were less sensitive than cancerous cells to the complex. Additionally, the  [Cu(L)(phen)] complex displayed a time- and concentration-dependent cytotoxic response, with MCF-7 and L929 cells. Also flow cytometry findings suggest that  [Cu(L)(phen)] complex is capable of decreasing cancer cell viability through apoptosis and did not efficiently activate the necrosis process.

Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line

Background: Apoptosis is suppressed in cancer tissues and tumor cell lines because anti-apoptosis genes are overexpressed. The inhibitor of apoptosis proteins (IAP) gene family contributes to control of apoptosis. The expression profile of eight genes of the IAP family in biopsies from patients with a history of bladder cancer and normal bladder tissues, as well as a bladder tumor cell line (5637), was assessed in the present study. Methods: Cancer tissue samples were obtained at surgery and the 5637 tumor cell line was cultured in RPMI1640 medium. Beyond tumor margins were selected as normal tissue. Expressional profile of interested genes was obtained by using specific primers and the real-time PCR method. Results: The results showed that expression of seven of the studied genes was up-regulated in cancer tissues and the cell line whereas BIRC4 (XIAP) was down-regulated in both. Conclusions: The results showed that these genes were expressed to a greater extent in cancer tissue and cancer cells than in normal tissues. The data suggested that over-expression of anti-apoptotic genes such as IAP family members, can trigger cells to escape from apoptosis.

Persian shallot, Allium hirtifolium Boiss, induced apoptosis in human hepatocellular carcinoma cells.

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 149 µg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24 h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.

Down-regulation of anti-apoptotic genes in tumor cell lines is facilitated by suppression of OCT4B1.

PURPOSE: The OCT4B1 as a variant of OCT4 is expressed in both cancer cells and tissues. The anti-apoptotic property of this variant aid cancer cells to escape from apoptosis. Therefore, the aim of the present study was to determine the effects of OCT4B1 suppression on regulation of 25 genes involved in anti-apoptotic pathway in tumor cell lines. MATERIAL AND METHODS: AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cells were transfected with specific OCT4B1 siRNA and a scramble siRNA by siRNA silencing gene technology, using Lipofectamine 2000 commercial kit. The real-time PCR technique was employed to examine and calculate fold changes of evaluated genes using the 2-ΔΔCT formula. RESULTS: Present results demonstrated that 22 (88%) of interested genes were similarly down-regulated in all three examined cell lines. Our results also indicated that three genes (CASP2, IGF1R,TNF) were up-regulated. The CFLAR gene was down-regulated in AGS, while it was inversely up-regulated in 5637 and U87MG cells. CONCLUSIONS: It may possibly be concluded that suppression of OCT4B1 can lead to apoptosis in tumor cell lines and this is at least facilitated via down-regulation of examined anti-apoptotic genes. Accordingly, suppression of OCT4B1 may probably be considered as useful tool in cancer therapy and research.

Down-regulation of HSP40 gene family following OCT4B1 suppression in human tumor cell lines.

OBJECTIVES: The OCT4B1, as one of OCT4 variants, is expressed in cancer cell lines and tissues more than other variants and plays an important role in apoptosis and stress (heat shock protein) pathways. The present study was designed to determine the effects of OCT4B1 silencing on expressional profile of HSP40 gene family expression in three different human tumor cell lines. MATERIALS AND METHODS: The OCT4B1 expression was suppressed by specific siRNA transfection in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines employing Lipofectamine reagent. Real-time PCR array technique was employed for RNA qualification. The fold changes were calculated using RT(2) Profiler PCR array data analysis software version 3.5. RESULTS: Our results indicated that fifteen genes (from 36 studied genes) were down-regulated and two genes (DNAJC11 and DNAJC5B) were up-regulated in all three studied tumor cell lines by approximately more than two folds. The result of other studied genes (19 genes) showed different expressional pattern (up or down-expression) based on tumor cell lines. CONCLUSION: According to the findings of the present study, we may suggest that there is a direct correlation between OCT4B1 expression in tumor cell lines (and tissues) and HSP40 family gene expressions to escape from apoptosis and cancer expansion.

Altered Expression of High Molecular Weight Heat Shock Proteins after OCT4B1 Suppression in Human Tumor Cell Lines.

OBJECTIVE: OCT4B1, a novel variant of OCT4, is expressed in cancer cell lines and tis- sues. Based on our previous reports, OCT4B1 appears to have a crucial role in regulating apoptosis as well as stress response [heat shock proteins (HSPs)] pathways. The aim of the present study was to determine the effects of OCT4B1 silencing on the expression of high molecular weight HSPs in three different human tumor cell lines. MATERIALS AND METHODS: In this experimental study, OCT4B1 expression was suppressed in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines using RNAi strategy. Real-time polymerase chain reaction (PCR) array was em- ployed for expression level analysis and the fold changes were calculated using RT2 Pro- filer PCR array data analysis software version 3.5. RESULTS: Our data revealed up-regulation of HSPD1 (from HSP60 family) as well as HSPA14, HSPA1L, HSPA4, HSPA5 and HSPA8 (from HSP70 family) following OCT4B1 knock-down in all three cell lines. In contrast, the expression of HSP90AA1 and HSP90AB1 (from HSP90 family) as well as HSPA1B and HSPA6 (from HSP70 family) was down-regulated under similar conditions. Other stress-related genes showed varying ex- pression pattern in the examined tumor cell lines. CONCLUSION: Our data suggest a direct or indirect correlation between the expression of OCT4B1 and HSP90 gene family. However, OCT4B1 expression was not strongly corre- lated with the expression of HSP70 and HSP60 gene families.

Altered expression of apoptotic genes in response to OCT4B1 suppression in human tumor cell lines.

OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1.