Selenium attenuates the cadmium-induced placenta glucocorticoid barrier damage by up-regulating the expression of specificity protein 1.

Cadmium (Cd) is an environmental pollutant and pregnant women are especially susceptible to the effects of exposure to Cd. Our previous study found Cd can be accumulated in the placenta and causes fetal growth restriction (FGR) through damage the placental glucocorticoid barrier. Selenium (Se), as an essential micronutrient, can allivate Cd-induced toxicity. In this study, we aim to explore the protective mechanism of Se against Cd-induced the placental glucocorticoid barrier damage and FGR. Pregnant Sprague Dawley (SD) rats were exposed to CdCl2 (1 mg/kg/day) and Na2 SeO3 (0.1-0.2-0.3 mg/kg/day) by gavage from gestational day (GD) 0 to GD 19. The results showed that reduced fetal weight, increased corticosterone concentrations in the maternal and fetal serum, and impaired placental labyrinth layer blood vessel development, appeared in pregnant rats after Cd exposure and improved after treated with Se. In cell experiments, we confirmed that Se reduces Cd-induced apoptosis. Moreover, Se can abolish Cd-induced 11ß-HSD2 and specificity protein 1 (Sp1) decreasing in vivo and vitro. In human JEG-3 cells, the knockdown of Sp1 expression by small interfering RNA can suppressed the protective effect of Se on Cd-induced 11ß-HSD2 decreasing. In general, our results demonstrated that Se is resistant to Cd-induced FGR through upregulating the placenta barrier via activation of the transcription factor Sp1.

Inhibition of Sp1-mediated survivin and MCL1 expression cooperates with SLC35F2 and myeloperoxidase to modulate YM155 cytotoxicity to human leukemia cells.

Although YM155 is reported to suppress survivin (also known as BIRC5) expression in cancer cells, its cytotoxic mechanism in human acute myeloid leukemia (AML) cells has not been clearly resolved. In this study, we analyzed the mechanistic pathways that modulate the sensitivity of human AML U937 and HL-60 cells to YM155. YM155 induced apoptosis in AML cells, which was characterized by p38 MAPK phosphorylation and downregulation of survivin and MCL1 expression. Phosphorylated p38 MAPK causes autophagy-mediated Sp1 degradation, thereby inhibiting the transcription of survivin and MCL1. The reduction of survivin and MCL1 levels further facilitated Sp1 protein degradation through autophagy. The restoration of Sp1, survivin, or MCL1 expression protected U937 and HL-60 cells from YM155-mediated cytotoxicity. U937 and HL-60 cells were continuously exposed to hydroquinone (HQ) to generate U937/HQ and HL-60/HQ cells, which showed increased SLC35F2 expression. The increase in SLC35F2 expression led to an increase in the sensitivity of U937/HQ cells to YM155-mediated cytotoxicity, whereas no such effect was observed in HL-60/HQ cells. Of note, myeloperoxidase (MPO) activity in HL-60 and HL-60/HQ cells enhanced YM155 cytotoxicity in these cells, and the enforced expression of MPO also increased the sensitivity of U937 cells to YM155. Taken together, we conclude that p38 MAPK-modulated autophagy inhibits Sp1-mediated survivin and MCL1 expression, which, in turn, leads to the death of U937 and HL-60 cells following YM155 treatment. In addition, our data indicate that SLC35F2 increases the sensitivity of U937 cells to YM155-mediated cytotoxicity, whereas MPO enhances YM155 cytotoxicity in U937 and HL-60 cells.

The reduced lncRNA NKILA inhibited proliferation and promoted apoptosis of chondrocytes via miR-145/SP1/NF-κB signaling in human osteoarthritis.

OBJECTIVE: Growing evidence has shown that long non-coding RNAs (lncRNAs) play some roles in the progression of osteoarthritis. In this study, we investigated the functions and mechanisms of lncRNA NKILA (NKILA) of chondrocytes in human osteoarthritis (OA). PATIENTS AND METHODS: RT-PCR was used to detect the expressions of NKILA and miR-145 in OA tissues. After transfection of NKILA overexpression lentivirus (LV-NKILA) and NKILA downregulation lentivirus (LV-shNKILA) into primary chondrocytes, MTT assay was carried out to measure the cell proliferation of chondrocytes. The expressions of SP1, Bcl-2, Bax, cleaved caspase-3 and NF-κB signaling factors were detected by Western blot. Moreover, luciferase assay was performed to explore the binding site of NKILA and miR-145, miR-145 and SP1. Finally, JSH, a NF-κB signaling inhibitor, was added into chondrocytes transfected with LV-shNKILA or miR-145 mimic to detect that NKILA functions via miR-145/SP1/NF-κB signaling pathway. RESULTS: We found that NKILA and SP1 were significantly reduced, miR-145 was increased in cartilage tissues of OA patients. After LV-NKILA transfection, the proliferation ability of chondrocytes was improved and cell apoptosis was inhibited; however, the proliferation ability of chondrocytes was repressed, and cell apoptosis was increased in LV-sh NKILA group. MiR-145 was predicted to be a potential target of NKILA and luciferase gene reporter assay confirmed that NKILA could directly bind with miR-145. Furthermore, SP1 was predicted to be a target gene of miR-145 and luciferase gene reporter assay proved that miR-145 could directly bind with SP1. Finally, we added JSH, a NF-κB signaling inhibitor, into chondrocytes with LV-shNKILA or miR-145 mimic. Results showed that the repressed SP1 was reversed after the addition of JSH in both LV-shNKILA and miR-145 mimic group. Further, the repressed proliferation capacities and promoted cell apoptosis were also reversed after the addition of JSH. CONCLUSIONS: According to the results, this study uncovers NKILA is reduced in human osteoarthritic cartilage tissues. Furthermore, we firstly uncover that the reduced NKILA could function as a ceRNA to improve miR-145, which inhibited SP1 expression and regulated NF-κB signaling pathway, thereby promoting tissue inflammation, and inhibiting proliferation and promoting apoptosis of chondrocytes. Thus, it may be used as a promising prognostic marker and a potential target for osteoarthritis.

MiR-29c protects against inflammation and apoptosis in Parkinson's disease model in vivo and in vitro by targeting SP1.

MicroRNAs (miRNAs) have been shown to have complicated implications in the pathogenesis of Parkinson's disease (PD). However, the role of miR-29c and the underlying mechanism in the development of PD remain not well understood. In this work, the MPTP-treated mice or MPP+ -intoxicated SH-SY5Y cells were established as an in vivo or in vitro PD model. Then the specific agomir of miR-29c was employed to examine its biological function on PD progress. We found that miR-29c was down-expressed but SP1 was high-expressed in substantia nigra pars compacta (SNpc) of MPTP-induced PD mice. Overexpression of miR-29c attenuated dopaminergic neuron loss and α-synuclein accumulation in SNpc of PD mice. Furthermore, the increments of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) and TUNEL-positive apoptotic cells in MPTP-treated mice were ameliorated by miR-29c. Similarly, in SH-SY5Y cell models of PD, we also found that miR-29c inhibited inflammatory cytokine production, reduced apoptotic rate and suppressed pro-apoptotic regulator activity. In addition, the increased expression of SP1 in PD models was found to be inhibited by miR-29c. Luciferase reporter assay confirmed that SP1 was complementary with miR-29c. Knockdown of SP1 with siRNA restored α-synuclein accumulation, inflammation and apoptosis in MPP+ -induced SH-SY5Y cells. Collectively, this current work presents that miR-29c may directly target SP1 to protect against the neuroinflammatory and apoptotic responses in PD, providing a potential biomarker for PD diagnosis and treatment.

HPV8 activates cellular gene expression mainly through Sp1/3 binding sites.

The human papillomavirus type 8 (HPV8) is associated with skin cancer development. The goal of this study was to investigate the effects of HPV8 oncoproteins on cellular gene expression and the identification of key regulators. We performed affymetrix microarray analyses to identify differentially expressed genes and common sequence motifs and identified Sp1/3 binding sites as being crucial. In transient transfection assays, we confirmed that HPV8-E7 stimulates the activity of Sp1/3 promoters. Interestingly, the HPV8-E7L23A mutant, which cannot trigger keratinocyte invasion was unable to activate fibronectin gene expression. In skin models or HPV8 positive skin cancers we found a peculiar deposition of fibronectin in the dermal compartment, and a correlation of Sp3 and fibronectin in the nucleus of HPV8-positive keratinocytes. Taken together, we identified that HPV8-E7 exerts control over cellular gene expression through Sp1/3 binding motifs, which may contribute to HPV8-mediated keratinocyte transformation and subsequent fibronectin-dependent invasion.

Resistin impairs activation of protein C by suppressing EPCR and increasing SP1 expression.

Endothelial cells are vital to blood coagulation and maintain whole body hemostasis. Binding of endothelial cells to endothelial protein C receptor (EPCR) and thrombomodulin (TM) is essential to the formation of activated protein C (APC), one of the key factors regulating blood coagulation. In our study, we showed that resistin, an adipocyte hormone, suppresses thrombin-induced protein C activation in endothelial cells. Resistin treatment results in a reduction in EPCR expression, but not TM. Mechanistically, we demonstrate that resistin induces expression of the nuclear transcription factor SP-1, which could lead to downregulation of EPCR. Both inhibition and silencing of SP1 protein abolishes abnormal APC generation induced by resistin. Collectively, our data support a new role of resistin in disturbing APC formation.

MicroRNA-296 Targets Specificity Protein 1 to Suppress Cell Proliferation and Invasion in Cervical Cancer.

Cervical cancer is the third most commonly diagnosed malignancy and the fourth leading cause of cancer-related deaths in women worldwide. MicroRNA-296 (miR-296) is aberrantly expressed in a variety of human cancer types. However, the expression levels, biological roles, and underlying molecular mechanisms of miR-296 in cervical cancer remain unclear. This study aimed to detect miR-296 expression in cervical cancer and evaluate its roles and underlying mechanisms in cervical cancer. This study demonstrated that miR-296 was significantly downregulated in cervical cancer tissues and cell lines. Restoring the expression of miR-296 inhibited the proliferation and invasion of cervical cancer cells. Moreover, miR-296 directly targeted the 3'-untranslated regions of specificity protein 1 (SP1) and decreased its endogenous expression at both the mRNA and protein levels. Similar to induced miR-296 expression, SP1 knockdown suppressed the proliferation and invasion of cervical cancer cells. Besides, resumption expression of SP1 rescued the tumor-suppressing roles of miR-296 in cervical cancer. These results indicated that miR-296 may act as a tumor suppressor in cervical cancer by directly targeting SP1. Therefore, SP1 may be developed as a therapeutic target for the treatment of patients with this malignancy.

DCLK1 promotes epithelial-mesenchymal transition via the PI3K/Akt/NF-κB pathway in colorectal cancer.

Double cortin-like kinase 1 (DCLK1) plays important roles during the epithelial-mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF-κB-dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF-κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF-κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.

Tumor suppressor protein p53-mediated repression of human mitotic centromere-associated kinesin gene expression is exerted via down-regulation of Sp1 level.

The repressive role of p53 on the human mitotic centromere-associated kinesin (MCAK) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53‒/‒) than in HCT116 (p53+/+) cells. Enforced down-regulation of p53 levels either in HCT116 (p53+/+) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at -173/-166 and p53-RE2 at -245/-238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at -93/-84 and GC2 at -119/-110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53+/+) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53-/-) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants (p53V143A, p53R248W, and p53R273H) failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation.

MicroRNA-337 inhibits cell proliferation and invasion of cervical cancer through directly targeting specificity protein 1.

Cervical cancer is the fourth most commonly occurring malignancy in females worldwide. Accumulated studies have demonstrated that the aberrant expression of microRNAs plays important roles in tumorigenesis and tumor development and potentially serves as therapeutic targets in various cancers including cervical cancer. Therefore, the identification of specific microRNAs contributed to cervical cancer formation and progression would provide critical clues for the treatments for patients with this disease. In this study, we aimed to detect microRNA-337 expression pattern and investigate the biological roles of microRNA-337 in the regulation of the malignant phenotypes of cervical cancer and its underlying mechanisms. We found that microRNA-337 expression was significantly downregulated in cervical cancer tissues and cell lines. In addition, its aberrant expression levels were positively correlated with tumor size, International Federation of Gynecology and Obstetrics stage, and lymph node metastasis of cervical cancer. The ectopic expression of microRNA-337 suppressed cell proliferation and invasion of cervical cancer in vitro. Furthermore, specificity protein 1 was identified as a direct target of microRNA-337 in cervical cancer. The expression of specificity protein 1 increased in cervical cancer tissues and negatively correlated with microRNA-337 expression level. Moreover, rescue experiments revealed that upregulation of specificity protein 1 could rescue the effects of microRNA-337 on cervical cancer cells. Taken together, these findings collectively demonstrate that microRNA-337 exerts its tumor-suppressing roles in cervical cancer by directly targeting specificity protein 1, thereby indicating a potential novel potential therapeutic target for patients with cervical cancer.

Glyceraldehyde-3-phosphate dehydrogenase promotes cancer growth and metastasis through upregulation of SNAIL expression.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in multiple cellular functions including metabolism and gene transcription. Our previous study showed that GAPDH expression was elevated in colon cancer and further upregulated in liver metastatic tissues, suggesting a possilbe role of GAPDH in promoting cancer metastasis. The present study was designed to investigate the underlying mechanism, using multiple experimental approaches including genetic silencing of GAPDH expression by short hairpin RNA (shRNA) and biochemcial/molecular analyses of the key events involved in glycolytic metabolism and epithelial-mesenchymal transition (EMT). We showed that silencing of GAPDH expression resulted in a significant reduction of glycolysis in colon cancer cell lines, accompanied by a decrease in cell proliferation and an apparent change in cell morphology associated with alterations in actin expression and phalloidine staining patterns. Furthermore, GAPDH suppression also caused a downregulation of gene expression involved in cancer stem-like cells and EMT. CHIP assay and co-immunoprecipitation revealed that GAPDH physically interacted with the transcriptional factor Sp1 and enhance the expression of SNAIL, a major regulator of EMT. Suppression of GAPDH expression resulted in a signficant decrease in SNAIL expression, leading to inhibition of EMT and attenuation of colon cancer cell migration in vitro and reduced metastasis in vivo. Overall, the present study suggests that GAPDH plays an important role in cancer metastasis by affecting EMT through regulation of Sp1-mediated SNAIL expression.

MicroRNA-892b influences proliferation, migration and invasion of bladder cancer cells by mediating the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 pathways.

Cancers often utilize microRNAs to suppress tumor suppressor genes, thus facilitating their potential for growth and invasion. In the present study, we report the novel findings that miR-892b inhibits proliferation, migration and invasion of bladder cancer cells. The basal expression level of miR­892b was significantly lower in 3 different bladder cancer cell lines than in normal human urothelial cells. Transfection of miR-892b mimics to bladder cancer cells resulted in dose­dependent growth arrest. Flow cytometric analysis of the cell cycle showed that miR-892b-transfected bladder cancer cells were subject to arrest in the G1 phase, which was due to the downregulation of cyclin D1 and CDK6 followed by upregulation of p19ARF. In addition, overexpression of miR-892b impeded the migration and invasion of EJ cells. Expression of MMP-9 in EJ cells was blocked by transfection of miR-892b; the effect was regulated, at least in part, by activation of the Sp-1 transcription factor. Overall, we verified that miR-892b regulates the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 signaling networks in bladder cancer cells and may provide a treatment option for advanced-stage bladder cancers.

Low-density lipoprotein upregulate SR-BI through Sp1 Ser702 phosphorylation in hepatic cells.

Scavenger receptor class B type I (SR-BI) is one of the key proteins in the process of reverse cholesterol transport (RCT), and its major function is to uptake high density lipoprotein (HDL) cholesterol from plasma into liver cells. The regulation of SR-BI expression is important for controlling serum lipid content and reducing the risks of cardiovascular diseases. Here we found that SR-BI expression was significantly increased by LDL in vivo and in vitro, and the transcription factor specific protein 1 (Sp1) plays a critical role in this process. Results from co-immunoprecipitation experiments indicate that the activation of SR-BI was associated with Sp1-recruited protein complexes in the promoter region of SR-BI, where histone acetyltransferase p300 was recruited and histone deacetylase HDAC1 was dismissed. As a result, histone acetylation increased, leading to activation of SR-BI transcription. With further investigation, we found that LDL phosphorylated Sp1 through ERK1/2 pathway, which affected Sp1 protein complexes formation in SR-BI promoter. Using mass spectrometry and site directed mutagenesis, a new Sp1 phosphorylation site Ser702 was defined to be associated with Sp1-HDAC1 interaction and may be important in SR-BI activation, shedding light on the knowledge of delicate mechanism of hepatic HDL receptor SR-BI gene modulation by LDL.

miRNA-223 inhibits epithelial-mesenchymal transition in gastric carcinoma cells via Sp1.

Sp1 plays critical roles in epithelial-mesenchymal transition (EMT) of certain cancer. However, few studies have indicated whether Sp1 is involved in the EMT of gastric cancer, and whether abnormal expression of Sp1 in gastric cancer EMT is regulated in a post-transcriptional manner, and the involvement of miRNAs in this regulation. In this study, we selected 20 cases of gastric cancers, their liver metastases and para-carcinoma tissues to examine the levels of Sp1 protein and mRNA by immunohistochemistry and fluorescent PCR, which showed that Sp1 was increased in gastric cancers and their metastases compared with adjacent tissues, but there was no difference in Sp1 mRNA between these three groups, suggesting changes in Sp1 may be attributed to inactivation of post-transcriptional regulation. We verified by a luciferase reporter system that miRNA-223 binds to 3'-UTR of Sp1 gene and inhibits its translation, in agreement with negative correlation between miRNA-223 and Sp1 protein levels in gastric cancer cells. By employing TGF-ß1 to induce MGC-803, BGC-823 and SGC-7901, we successfully built cellular EMT model. Then, we overexpressed miRNA-223 in the model by using a lentiviral system, which diminished EMT indicators and suppressed proliferation and invasion ability, and induced apoptosis. Finally, we verified the specificity of the regulation pathway miRNA-223/Sp1/EMT. These findings suggest that low expression of miRNA-223 in gastric cancer cells is an important cause for EMT. miRNA-223 specifically regulates the EMT process of gastric cancer cells through its target gene Sp1. Overexpression of miRNA-223 in these cells inhibits EMT via the miRNA-223/Sp1/EMT pathway.

Sp1 and COX2 expression is positively correlated with a poor prognosis in pancreatic ductal adenocarcinoma.

Previous studies showed that celecoxib, a cyclooxygenase-2 (COX2) inhibitor, can inhibit angiogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC) via the suppression of specificity protein 1 (Sp1). In this study, we investigated the prognostic value of Sp1 and COX2 in 88 PDAC patients. Our study showed there was a positive correlation between Sp1 and COX2 expression (P=0.001) by using the Spearman's rank test. Pearson Chi-square test revealed that Sp1 and COX2 expression were positively associated with lymph node metastasis (P<0.05, both). In addition, the Kaplan-Meier analysis showed that patients with Sp1- or COX2-positive expression exhibited poorer overall survival (OS) than those with Sp1- or COX2-negative expression (P<0.05, all). Most importantly, Sp1- and COX2-negative patients had the best OS (P=0.01). In multivariate analysis, Sp1 expression (P=0.03), COX2 expression (P=0.04), and nuclear grade (P=0.009) were found to be independent predictors for OS. Moreover, we confirmed that Sp1 could upregulate the expression of COX2 in PDAC cell lines by western blot analysis, and both are of important prognostic value in PDAC.

The anti-proliferative effects of type I IFN involve STAT6-mediated regulation of SP1 and BCL6.

Type I IFN-induced STAT6 has been shown to have anti-proliferative effects in Daudi and B cells. IFN-sensitive (DS) and IFN-resistant (DR) subclones of Daudi cells were used to study the role of STAT6 in the anti-proliferative activities. Type I IFN significantly increased STAT6 mRNA and protein expression in DS but not DR cells. STAT6 knockdown significantly reduced the sensitivity to IFN in both cell lines. The molecular targets and functional importance of IFN-activated STAT6 were performed by chromatin immunoprecipitation-on-chip (ChIP-on-chip) experiments in type I IFN-treated Daudi cells. Two target genes (Sp1 and BCL6) were selected from the ChIP-on-chip data. IFN-induced STAT6 activation led to Sp1 upregulation and BCL6 downregulation in DS cells, with only minimal effects in DR cells. siRNA inhibition of STAT6 expression resulted in decreased Sp1 and BCL6 mRNA and protein levels in both DS and DR cells. IFN treatment did not increase Sp1 and BCL6 expression in a STAT2-deficient RST2 cell line, and this effect was mitigated by plasmid overexpression of STAT2, indicating that STAT2 is important for STAT6 activation. These results suggest that STAT6 plays an important role in regulating Sp1 and BCL6 through STAT2 to exert the anti-proliferative effects of type I IFN.

ß-lapachone suppresses the proliferation of human malignant melanoma cells by targeting specificity protein 1.

ß-lapachone (ß-lap), a novel natural quinone derived from the bark of the Pink trumpet tree (Tabebuia avellanedae) has been demonstrated to have anticancer activity. In this study, we investigated whether ß-lap exhibits anti-proliferative effects on two human malignant melanoma (HMM) cell lines, G361 and SK-MEL-28. The effects of ß-lap on the HMM cell lines were investigated using 3-(4,5-dimethylthiazol-2-yl)­5-(3-carboxymethoxyphenyl)­2-(4-sulfophenyl-2H-tetrazolium (MTS) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V and Dead cell assay, mitochondrial membrane potential (MMP) assay and western blot analysis. We demonstrated that ß-lap significantly induced apoptosis and suppressed cell viability in the HMM cells. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly downregulated by ß-lap in a dose- and time-dependent manner. Furthermore, ß-lap modulated the protein expression level of the Sp1 regulatory genes including cell cycle regulatory proteins and apoptosis-associated proteins. Taken together, our findings indicated that ß-lap modulates Sp1 transactivation and induces apoptotic cell death through the regulation of cell cycle- and apoptosis-associated proteins. Thus, ß-lap may be used as a promising anticancer drug for cancer prevention and may improve the clinical outcome of patients with cancer.

Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells.

PURPOSE: Specificity protein 1 (SP1) is an oncogenic transcription factor overexpressed in various human malignancies. This study sought to examine SP1 expression in malignant pleural mesotheliomas (MPM) and ascertain the potential efficacy of targeting SP1 in these neoplasms. EXPERIMENTAL DESIGN: qRT-PCR, immunoblotting, and immunohistochemical techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, ß-galactosidase, and Apo-BrdUrd techniques were used to assess proliferation, migration, clonogenicity, senescence, and apoptosis in MPM cells following SP1 knockdown, p53 overexpression, or mithramycin treatment. Murine subcutaneous and intraperitoneal xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblotting, and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 overexpression and correlate these changes with SP1 and p53 levels within target gene promoters. RESULTS: MPM cells and tumors exhibited higher SP1 mRNA and protein levels relative to control cells/tissues. SP1 knockdown significantly inhibited proliferation, migration, and clonogenicity of MPM cells. Mithramycin depleted SP1 and activated p53, dramatically inhibiting proliferation and clonogenicity of MPM cells. Intraperitoneal mithramycin significantly inhibited growth of subcutaneous MPM xenografts and completely eradicated mesothelioma carcinomatosis in 75% of mice. Mithramycin modulated genes mediating oncogene signaling, cell-cycle regulation, senescence, and apoptosis in vitro and in vivo. The growth-inhibitory effects of mithramycin in MPM cells were recapitulated by combined SP1 knockdown/p53 overexpression. CONCLUSIONS: These findings provide preclinical rationale for phase II evaluation of mithramycin in patients with mesothelioma.

Sp1 upregulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes.

Type XI collagen is a cartilage-specific extracellular matrix, and is important for collagen fibril formation and skeletal morphogenesis. We have previously reported that NF-Y regulated the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes (Hida et. al. In Vitro Cell. Dev. Biol. Anim. 2014). However, the mechanism of the Col11a1 gene regulation in chondrocytes has not been fully elucidated. In this study, we further characterized the proximal promoter activity of the mouse Col11a1 gene in chondrocytes. Cell transfection experiments with deletion and mutation constructs indicated that the downstream region of the NF-Y binding site (-116 to +1) is also necessary to regulate the proximal promoter activity of the mouse Col11a1 gene. This minimal promoter region has no TATA box and GC-rich sequence; we therefore examined whether the GC-rich sequence (-96 to -67) is necessary for the transcription regulation of the Col11a1 gene. Luciferase assays using a series of mutation constructs exhibited that the GC-rich sequence is a critical element of Col11a1 promoter activity in chondrocytes. Moreover, in silico analysis of this region suggested that one of the most effective candidates was transcription factor Sp1. Consistent with the prediction, overexpression of Sp1 significantly increased the promoter activity. Furthermore, knockdown of Sp1 expression by siRNA transfection suppressed the proximal promoter activity and the expression of endogenous transcript of the mouse Col11a1 gene. Taken together, these results indicate that the transcription factor Sp1 upregulates the proximal promoter activity of the mouse Col11a1 gene in chondrocytes.

Combination of tolfenamic acid and curcumin induces colon cancer cell growth inhibition through modulating specific transcription factors and reactive oxygen species.

Curcumin (Cur) has been extensively studied in several types of malignancies including colorectal cancer (CRC); however its clinical application is greatly affected by low bioavailability. Several strategies to improve the therapeutic response of Cur are being pursued, including its combination with small molecules and drugs. We investigated the therapeutic efficacy of Cur in combination with the small molecule tolfenamic acid (TA) in CRC cell lines. TA has been shown to inhibit the growth of human cancer cells in vitro and in vivo, via targeting the transcription factor specificity protein1 (Sp1) and suppressing survivin expression. CRC cell lines HCT116 and HT29 were treated with TA and/or Cur and cell viability was measured 24-72 hours post-treatment. While both agents caused a steady reduction in cell viability, following a clear dose/ time-dependent response, the combination of TA+Cur showed higher growth inhibition when compared to either single agent. Effects on apoptosis were determined using flow cytometry (JC-1 staining to measure mitochondrial membrane potential), Western blot analysis (c-PARP expression) and caspase 3/7 activity. Reactive oxygen species (ROS) levels were measured by flow cytometry and the translocation of NF-kB into the nucleus was determined using immunofluorescence. Results showed that apoptotic markers and ROS activity were significantly upregulated following combination treatment, when compared to the individual agents. This was accompanied by decreased expression of Sp1, survivin and NF-kB translocation. The combination of TA+Cur was more effective in HCT116 cells than HT29 cells. These results demonstrate that TA may enhance the anti-proliferative efficacy of Cur in CRC cells.