Role of KCNMA1 gene in breast cancer invasion and metastasis to brain

International audienceBACKGROUND: The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming alpha-subunit of BKCa channels in breast cancer metastasis and invasion. METHODS: We performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX). RESULTS: The Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1. CONCLUSION: Determining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy

Novel Survivin Inhibitor for Suppressing Pancreatic Cancer Cells Growth via Downregulating Sp1 and Sp3 Transciption Factors

Background/Aims: Targeting survivin, an anti-apoptotic protein and mitotic regulator, is considered as an effective therapeutic option for pancreatic cancer (PaCa). Tolfenamic acid (TA) showed anti-cancer activity in pre-clinical studies. A recent discovery demonstrated a copper(II) complex of TA (Cu-TA) can result in higher activity. In this study, the ability of Cu-TA to inhibit survivin and its transcription factors, Specificity protein (Sp) 1 and 3 in PaCa cell lines and tumor growth in mouse xenograft model were evaluated. Methods: Cell growth inhibition was measured in MIA PaCa-2 and Panc1 cells for 2 days using CellTiter-Glo kit. Sp1, Sp3 and survivin expression (by Western blot and qPCR), apoptotic cells and cell cycle phase distribution (by flow cytometry) were evaluated. A pilot study was performed using athymic nude mice [treated with vehicle/Cu-TA (25 or 50 mg/kg) 3 times/week for 4 weeks. Results: The IC50 value for Cu-TA was about half than TA. Both agents repressed the protein expression of Sp1/Sp3/survivin, Cu-TA was more effective than TA. Especially effect on survivin inhibition was 5.2 (MIA PaCa-2) or 6.4 (Panc1) fold higher and mRNA expression of only survivin was decreased. Apoptotic cells increased with Cu-TA treatment in both cell lines, while Panc1 showed both effect on apoptosis and cell cycle (G2/M) arrest. Cu-TA decreased the tumor growth in mouse xenografts (25 mg/kg: 48%; 50 mg/kg: 68%). Additionally, there was no change observed in mice body weights, indicating no overt toxicity was occurring. Conclusion: These results show that Cu-TA can serve as an effective survivin inhibitor for inhibiting PaCa cell growth

Novel Survivin Inhibitor for Suppressing Pancreatic Cancer Cells Growth via Downregulating Sp1 and Sp3 Transcription Factors

Background/Aims: Targeting survivin, an anti-apoptotic protein and mitotic regulator, is considered as an effective therapeutic option for pancreatic cancer (PaCa). Tolfenamic acid (TA) showed anti-cancer activity in pre-clinical studies. A recent discovery demonstrated a copper(II) complex of TA (Cu-TA) can result in higher activity. In this study, the ability of Cu-TA to inhibit survivin and its transcription factors, Specificity protein (Sp) 1 and 3 in PaCa cell lines and tumor growth in mouse xenograft model were evaluated. Methods: Cell growth inhibition was measured in MIA PaCa-2 and Panc1 cells for 2 days using CellTiter-Glo kit. Sp1, Sp3 and survivin expression (by Western blot and qPCR), apoptotic cells and cell cycle phase distribution (by flow cytometry) were evaluated. A pilot study was performed using athymic nude mice [treated with vehicle/Cu-TA (25 or 50 mg/kg) 3 times/week for 4 weeks. Results: The IC50 value for Cu-TA was about half than TA.Both agents repressed the protein expression of Sp1/Sp3/survivin, Cu-TA was more effective than TA. Especially effect on survivin inhibition was 5.2 (MIA PaCa-2) or 6.4 (Panc1) fold higher and mRNA expression of only survivin was decreased. Apoptotic cells increased with Cu-TA treatment in both cell lines, while Panc1 showed both effect on apoptosis and cell cycle (G2/M) arrest. Cu-TA decreased the tumor growth in mouse xenografts (25 mg/kg: 48%; 50 mg/kg: 68%). Additionally, there was no change observed in mice body weights, indicating no overt toxicity was occurring. Conclusion: These results show that Cu-TA can serve as an effective survivin inhibitor for inhibiting PaCa cell growth

Mutational analysis of conserved residues in HhaI DNA methyltransferase

HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids in the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids involved in cofactor S-adenosyl-l-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu100). Characterization of these mutants, by in vitro /in vivo restriction assays and DNA/AdoMet binding studies, indicated that most of the residues present in the AdoMet-binding pocket were not absolutely essential. This study implies plasticity in the recognition of cofactor by HhaI DNA methyltransferase

Structure, function, and mechanism of HhaI DNA methyltransferases

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase

Water-assisted dual mode cofactor recognition by HhaI DNA methyltransferase

Energetically competent binary recognition of the cofactorS-adenosyl-l-methionine (AdoMet) and the product S-adenosyl-l-homocysteine (AdoHcy) by the DNA (cytosine C-5) methyltransferase (M.HhaI) is demonstrated herein. Titration calorimetry reveals a dual mode, involving a primary dominant exothermic reaction followed by a weaker endothermic one, for the recognition of AdoMet and AdoHcy by M.HhaI. Conservation of the bimodal recognition in W41I and W41Y mutants of M.HhaI excludes the cation-π interaction between the methylsulfonium group of AdoMet and the π face of the Trp<SUP>41</SUP> indole ring from a role in its origin. Small magnitude of temperature-independent heat capacity changes upon AdoMet or AdoHcy binding by M.HhaI preclude appreciable conformational alterations in the reacting species. Coupled osmotic-calorimetric analyses of AdoMet and AdoHcy binding by M.HhaI indicate that a net uptake of nearly eight and 10 water molecules, respectively, assists their primary recognition. A change in water activity at constant temperature and pH is sufficient to engender and conserve enthalpy-entropy compensation, consistent with a true osmotic effect. The results implicate solvent reorganization in providing the major contribution to the origin of this isoequilibrium phenomenon in AdoMet and AdoHcy recognition by M.HhaI. The observations provide unequivocal evidence for the binding of AdoMet as well as AdoHcy to M.HhaI in solution state. Isotope partitioning analysis and preincubation studies favor a random mechanism for M.HhaI-catalyzed reaction. Taken together, the results clearly resolve the issue of cofactor recognition by free M.HhaI, specifically in the absence of DNA, leading to the formation of an energetically and catalytically competent binary complex

Tolfenamic Acid Inhibits Neuroblastoma Cell Proliferation And Induces Apoptosis: A Novel Therapeutic Agent For Neuroblastoma

Current therapeutic options for recurrent neuroblastoma have poor outcomes that warrant the development of novel therapeutic strategies. Specificity protein (Sp) transcription factors regulate several genes involved in cell proliferation, survival, and angiogenesis. Sp1 regulates genes believed to be important determinants of the biological behavior of neuroblastoma. Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to induce the degradation of Sp proteins and may serve as a novel anti-cancer agent. The objective of this investigation was to examine the anti-cancer activity of TA using established human neuroblastoma cell lines. We tested the anti-proliferative effect of TA using SH-SY5Y, CHLA90, LA1 55n, SHEP, Be2c, CMP 13Y, and SMS KCNR cell lines. Cells were treated with TA (0/25/50/100μM) and cell viability was measured at 24, 48, and 72h post-treatment. Selected neuroblastoma cell lines were treated with 50μM TA for 24 and 48h and tested for cell apoptosis using Annexin-V staining. Caspase activity was measured with caspase 3/7 Glo kit. Cell lysates were prepared and the expression of Sp1, survivin, and c-PARP were evaluated through Western blot analysis. TA significantly inhibited the growth of neuroblastoma cells in a dose/time-dependent manner and significantly decreased Sp1 and survivin expression. Apart from cell cycle (G0/G1) arrest, TA caused significant increase in the apoptotic cell population, caspase 3/7 activity, and c-PARP expression. These results show that TA effectively inhibits neuroblastoma cell growth potentially through suppressing mitosis, Sp1, and survivin expression, and inducing apoptosis. These results show TA as a novel therapeutic agent for neuroblastoma. © 2011 Wiley Periodicals, Inc

Environmental Factors In Causing Human Cancers: Emphasis On Tumorigenesis

The environment and dietary factors play an essential role in the etiology of cancer. Environmental component is implicated in ~80 % of all cancers; however, the causes for certain cancers are still unknown. The potential players associated with various cancers include chemicals, heavy metals, diet, radiation, and smoking. Lifestyle habits such as smoking and alcohol consumption, exposure to certain chemicals (e.g., polycyclic aromatic hydrocarbons, organochlorines), metals and pesticides also pose risk in causing human cancers. Several studies indicated a strong association of lung cancer with the exposure to tobacco products and asbestos. The contribution of excessive sunlight, radiation, occupational exposure (e.g., painting, coal, and certain metals) is also well established in cancer. Smoking, excessive alcohol intake, consumption of an unhealthy diet, and lack of physical activity can act as risk factors for cancer and also impact the prognosis. Even though the environmental disposition is linked to cancer, the level and duration of carcinogen-exposure and associated cellular and biochemical aspects determine the actual risk. Modulations in metabolism and DNA adduct formation are considered central mechanisms in environmental carcinogenesis. This review describes the major environmental contributors in causing cancer with an emphasis on molecular aspects associated with environmental disposition in carcinogenesis. © 2012 International Society of Oncology and BioMarkers (ISOBM)

Water-assisted dual mode cofactor recognition by HhaI DNA methyltransferase

Energetically competent binary recognition of the cofactorS-adenosyl-l-methionine (AdoMet) and the product S-adenosyl-l-homocysteine (AdoHcy) by the DNA (cytosine C-5) methyltransferase (M.HhaI) is demonstrated herein. Titration calorimetry reveals a dual mode, involving a primary dominant exothermic reaction followed by a weaker endothermic one, for the recognition of AdoMet and AdoHcy by M.HhaI. Conservation of the bimodal recognition in W41I and W41Y mutants of M.HhaI excludes the cation−π interaction between the methylsulfonium group of AdoMet and the π face of the Trp41 indole ring from a role in its origin. Small magnitude of temperature-independent heat capacity changes upon AdoMet or AdoHcy binding by M.HhaI preclude appreciable conformational alterations in the reacting species. Coupled osmotic-calorimetric analyses of AdoMet and AdoHcy binding by M.HhaI indicate that a net uptake of nearly eight and 10 water molecules, respectively, assists their primary recognition. A change in water activity at constant temperature and pH is sufficient to engender and conserve enthalpy−entropy compensation, consistent with a true osmotic effect. The results implicate solvent reorganization in providing the major contribution to the origin of this isoequilibrium phenomenon in AdoMet and AdoHcy recognition by M.HhaI. The observations provide unequivocal evidence for the binding of AdoMet as well as AdoHcy to M.HhaI in solution state. Isotope partitioning analysis and preincubation studies favor a random mechanism for M.HhaI-catalyzed reaction. Taken together, the results clearly resolve the issue of cofactor recognition by free M.HhaI, specifically in the absence of DNA, leading to the formation of an energetically and catalytically competent binary complex

Tolfenamic Acid Suppresses Cytochrome P450 2E1 Expression In Mouse Liver

Non-steroidal anti-inflammatory drugs (NSAIDs) play a significant role in the chemoprevention of cancer. We recently showed the chemopreventive response of a NSAID, 2-[(3-chloro-2-methylphenyl)amino]benzoic acid) known as tolfenamic acid (TA) in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats. Pre-clinical studies showed that TA inhibits Specificity protein (Sp) transcription factors and acts as an anti-cancer agent in several cancer models; however the pertinent mechanisms associated with its chemopreventive response in esophageal cancer are not known. Since the bioactivation of carcinogens through cytochrome P450 (CYP) is critical for the induction of cancer, we have studied the effect of TA on critical CYP isozymes in mouse liver samples. Athymic nude mice were treated with vehicle (corn oil) or TA (50 mg kg -1, 3 times per week) for 4 weeks. Protein extracts (whole cell lysates and microsomal fractions) were prepared from liver tissue and the expression of various CYP isozymes was determined by Western blot analysis. Rat (Sprague-Dawley) livers were harvested and primary hepatocyte cultures were treated with vehicle (DMSO) or TA (50 μM) and cell viability was assessed at 2 and 5 days post-treatment. TA caused remarkable decrease in the expression of CYP2E1 in both liver lysates and sub-cellular fraction, while its response on other tested isozymes was marginal. TA did not affect the body weight of animals (mice) and viability of rat hepatocytes. These results demonstrate that TA modulates the expression of CYP2E1 which is associated with the bioactivation of carcinogens without causing apparent toxicity. These data suggest that TA-induced inhibition of CYP2E1 attenuates the bioactivation of carcinogens potentially leading to the chemoprevention of NMBA-induced esophageal tumorigenesis in rats. © 2012 The Royal Society of Chemistry