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1.  Capsaicin-Induced Activation of p53-SMAR1 Auto-Regulatory Loop Down-Regulates VEGF in Non-Small Cell Lung Cancer to Restrain Angiogenesis 
PLoS ONE  2014;9(6):e99743.
Lung cancer is the leading cause of cancer-related deaths worldwide. Despite decades of research, the treatment options for lung cancer patients remain inadequate, either to offer a cure or even a substantial survival advantage owing to its intrinsic resistance to chemotherapy. Our results propose the effectiveness of capsaicin in down-regulating VEGF expression in non-small cell lung carcinoma (NSCLC) cells in hypoxic environment. Capsaicin-treatment re-activated p53-SMAR1 positive feed-back loop in these cells to persuade p53-mediated HIF-1α degradation and SMAR1-induced repression of Cox-2 expression that restrained HIF-1α nuclear localization. Such signal-modulations consequently down regulated VEGF expression to thwart endothelial cell migration and network formation, pre-requisites of angiogenesis in tumor micro-environment. The above results advocate the candidature of capsaicin in exclusively targeting angiogenesis by down-regulating VEGF in tumor cells to achieve more efficient and cogent therapy of resistant NSCLC.
PMCID: PMC4057320  PMID: 24926985
2.  Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment 
BMC Molecular Biology  2012;13:28.
GAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction. Although it serves as one of the first injury markers of β-islets, the mechanisms governing GAD65 expression remain poorly understood. Since the regulation of GAD65 is crucial for the proper functioning of insulin secreting cells, we investigated the stress induced regulation of GAD65 transcription.
The present study shows that SMAR1 regulates GAD65 expression at the transcription level. Using a novel protein-DNA pull-down assay, we show that SMAR1 binding is very specific to GAD65 promoter but not to the other isoform, GAD67. We show that Streptozotocin (STZ) mediated DNA damage leads to upregulation of SMAR1 and p53 expression, resulting in elevated levels of GAD65, in both cell lines as well as mouse β-islets. SMAR1 and p53 act synergistically to up-regulate GAD65 expression upon STZ treatment.
We propose a novel mechanism of GAD65 regulation by synergistic activities of SMAR1 and p53.
PMCID: PMC3459802  PMID: 22978699
SMAR1; Diabetes; GAD65; p53; Streptozotocin
3.  Aqueous Cinnamon Extract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human cervical cancer cell line (SiHa) through loss of mitochondrial membrane potential 
BMC Cancer  2010;10:210.
Chemoprevention, which includes the use of synthetic or natural agents (alone or in combination) to block the development of cancer in human beings, is an extremely promising strategy for cancer prevention. Cinnamon is one of the most widely used herbal medicines with diverse biological activities including anti-tumor activity. In the present study, we have reported the anti-neoplastic activity of cinnamon in cervical cancer cell line, SiHa.
The aqueous cinnamon extract (ACE-c) was analyzed for its cinnamaldehyde content by HPTLC analysis. The polyphenol content of ACE-c was measured by Folin-Ciocalteau method. Cytotoxicity analysis was performed by MTT assay. We studied the effect of cinnamon on growth kinetics by performing growth curve, colony formation and soft agar assays. The cells treated with ACE-c were analyzed for wound healing assay as well as for matrix metalloproteinase-2 (MMP-2) expression at mRNA and protein level by RT-PCR and zymography, respectively. Her-2 protein expression was analyzed in the control and ACE-c treated samples by immunoblotting as well as confocal microscopy. Apoptosis studies and calcium signaling assays were analyzed by FACS. Loss of mitochondrial membrane potential (Δψm) in cinnamon treated cells was studied by JC-1 staining and analyzed by confocal microscopy as well as FACS.
Cinnamon alters the growth kinetics of SiHa cells in a dose-dependent manner. Cells treated with ACE-c exhibited reduced number of colonies compared to the control cells. The treated cells exhibited reduced migration potential that could be explained due to downregulation of MMP-2 expression. Interestingly, the expression of Her-2 oncoprotein was significantly reduced in the presence of ACE-c. Cinnamon extract induced apoptosis in the cervical cancer cells through increase in intracellular calcium signaling as well as loss of mitochondrial membrane potential.
Cinnamon could be used as a potent chemopreventive drug in cervical cancer.
PMCID: PMC2893107  PMID: 20482751
4.  Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study 
BMC Cancer  2009;9:350.
Imaging tools such as scanning electron microscope (SEM) and atomic force microscope (AFM) can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections.
We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK) 293, human breast cancer (MCF-7) and mouse melanoma (B16F1) cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies.
Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins.
Tumor suppressor protein SMAR1 might be used as a phenotypic differentiation marker between cancerous and non-cancerous cells.
PMCID: PMC2765988  PMID: 19799771
5.  Stabilization of SMAR1 mRNA by PGA2 involves a stem–loop structure in the 5′ UTR 
Nucleic Acids Research  2007;35(18):6004-6016.
Prostaglandins are anticancer agents known to inhibit tumor cell proliferation both in vitro and in vivo by affecting the mRNA stability. Here we report that a MAR-binding protein SMAR1 is a target of Prostaglandin A2 (PGA2) induced growth arrest. We identify a regulatory mechanism leading to stabilization of SMAR1 transcript. Our results show that a minor stem and loop structure present in the 5′ UTR of SMAR1 (ϕ1-UTR) is critical for nucleoprotein complex formation that leads to SMAR1 stabilization in response to PGA2. This results in an increased SMAR1 transcript and altered protein levels, that in turn causes downregulation of Cyclin D1 gene, essential for G1/S phase transition. We also provide evidence for the presence of a variant 5′ UTR SMAR1 (ϕ17-UTR) in breast cancer-derived cell lines. This form lacks the minor stem and loop structure required for mRNA stabilization in response to PGA2. As a consequence of this, there is a low level of endogenous tumor suppressor protein SMAR1 in breast cancer-derived cell lines. Our studies provide a mechanistic insight into the regulation of tumor suppressor protein SMAR1 by a cancer therapeutic PGA2, that leads to repression of Cyclin D1 gene.
PMCID: PMC2094063  PMID: 17726044
6.  p53 Target Gene SMAR1 Is Dysregulated in Breast Cancer: Its Role in Cancer Cell Migration and Invasion 
PLoS ONE  2007;2(8):e660.
Tumor suppressor SMAR1 interacts and stabilizes p53 through phosphorylation at its serine-15 residue. We show that SMAR1 transcription is regulated by p53 through its response element present in the SMAR1 promoter. Upon Doxorubicin induced DNA damage, acetylated p53 is recruited on SMAR1 promoter that allows activation of its transcription. Once SMAR1 is induced, cell cycle arrest is observed that is correlated to increased phospho-ser-15-p53 and decreased p53 acetylation. Further we demonstrate that SMAR1 expression is drastically reduced during advancement of human breast cancer. This was correlated with defective p53 expression in breast cancer where acetylated p53 is sequestered into the heterochromatin region and become inaccessible to activate SMAR1 promoter. In a recent report we have shown that SMAR1 represses Cyclin D1 transcription through recruitment of HDAC1 dependent repressor complex at the MAR site of Cyclin D1 promoter. Here we show that downmodulation of SMAR1 in high grade breast carcinoma is correlated with upregulated Cyclin D1 expression. We also established that SMAR1 inhibits tumor cell migration and metastases through inhibition of TGFβ signaling and its downstream target genes including cutl1 and various focal adhesion molecules. Thus, we report that SMAR1 plays a central role in coordinating p53 and TGFβ pathways in human breast cancer.
PMCID: PMC1924604  PMID: 17668048
7.  Tumor Suppressor SMAR1 Mediates Cyclin D1 Repression by Recruitment of the SIN3/Histone Deacetylase 1 Complex†  
Molecular and Cellular Biology  2005;25(19):8415-8429.
Matrix attachment region binding proteins have been shown to play an important role in gene regulation by altering chromatin in a stage- and tissue-specific manner. Our previous studies report that SMAR1, a matrix-associated protein, regresses B16-F1-induced tumors in mice. Here we show SMAR1 targets the cyclin D1 promoter, a gene product whose dysregulation is attributed to breast malignancies. Our studies reveal that SMAR1 represses cyclin D1 gene expression, which can be reversed by small interfering RNA specific to SMAR1. We demonstrate that SMAR1 interacts with histone deacetylation complex 1, SIN3, and pocket retinoblastomas to form a multiprotein repressor complex. This interaction is mediated by the SMAR1(160-350) domain. Our data suggest SMAR1 recruits a repressor complex to the cyclin D1 promoter that results in deacetylation of chromatin at that locus, which spreads to a distance of at least the 5 kb studied upstream of the cyclin D1 promoter. Interestingly, we find that the high induction of cyclin D1 in breast cancer cell lines can be correlated to the decreased levels of SMAR1 in these lines. Our results establish the molecular mechanism exhibited by SMAR1 to regulate cyclin D1 by modification of chromatin.
PMCID: PMC1265755  PMID: 16166625
8.  SMAR1 and Cux/CDP modulate chromatin and act as negative regulators of the TCRβ enhancer (Eβ) 
Nucleic Acids Research  2004;32(16):4862-4875.
Chromatin modulation at various cis-acting elements is critical for V(D)J recombination during T and B cell development. MARβ, a matrix-associated region (MAR) located upstream of the T cell receptor β (TCRβ) enhancer (Eβ), serves a crucial role in silencing Eβ-mediated TCR activation. By DNaseI hypersensitivity assays, we show here that overexpression of the MAR binding proteins SMAR1 and Cux/CDP modulate the chromatin structure at MARβ. We further demonstrate that the silencer function of MARβ is mediated independently by SMAR1 and Cux/CDP as judged by their ability to repress Eβ-dependent reporter gene expression. Moreover, the repressor activity of SMAR1 is strongly enhanced in the presence of Cux/CDP. These two proteins physically interact with each other and colocalize within the perinuclear region through a SMAR1 domain required for repression. The repression domain of SMAR1 is separate from the MARβ binding domain and contains a nuclear localization signal and an arginine–serine (RS)-rich domain, characteristic of pre-mRNA splicing regulators. Our data suggest that at the double positive stage of T cell development, cis-acting MARβ elements recruit the strong negative regulators Cux and SMAR1 to control Eβ-mediated recombination and transcription.
PMCID: PMC519105  PMID: 15371550
9.  Stimulation of Tat-independent transcriptional processivity from the HIV-1 LTR promoter by matrix attachment regions 
Nucleic Acids Research  2003;31(12):3248-3256.
The chromatin environment and the sites of integration in the host genome are critical determinants of human immunodeficiency virus (HIV) transcription and replication. Depending on the chromosomal location of provirus integration within the genome, HIV-1 long terminal repeat (LTR)-mediated transcription may vary from 0- to 70-fold. Cis-elements such as topoisomerase II cleavage sites, Alu repeats and matrix attachment regions (MARs) are thought to be targets for retroviral integration. Here we show that a novel MAR sequence from the T-cell receptor β locus (MARβ) and the IgH MAR mediate transcriptional augmentation when placed upstream of the HIV-1 LTR promoter. The effect of transcriptional augmentation is seen in both transient and stable transfection, indicating its effect even upon integration in the genome. MAR-mediated transcriptional elevation is independent of Tat, and occurs synergistically in the presence of Tat. Further, we show that MAR-mediated transcriptional elevation is specific to the HIV-1 LTR and the Moloney murine leukemia virus LTR promoter. In a transient transfection assay using over-expressed IκB, the inhibitor of NF-κB, we show that MAR-induced processive transcription is NF-κB dependent, signifying the role of local enhancers within the LTR promoter. Furthermore, by RNase protection experiments using proximal and distal probes, we show that MAR-mediated transcriptional upregulation is more prominent at the distal rather than the proximal end, thus indicating the potential role of MARs in promoting elongation.
PMCID: PMC162244  PMID: 12799452

Results 1-9 (9)