Cbx7 promotes the generation of induced pluripotent stem cells.

The iPS cells were discovered in 2006. With their ability to differentiate into cells of all three germ layers, iPS cells have great potential for clinical applications. Oct4, Sox2, c-Myc, and Klf4 were identified as the most effective factors for generating iPS cells. Despite this, iPS cells manufactured with these factors would still be inefficient. As a member of the chromobox family, chromobox protein homolog 7 (Cbx7) binds to PRC1 and PRC2 to inhibit genes involved in differentiation. A decrease in the expression of Cbx7 is observed during embryonic stem cell differentiation. Currently, no report discusses the role of Cbx7 in the production of iPS cells. In this study, we hypothesized that Cbx7 could increase iPS cell generation. We confirmed that Cbx7 is highly expressed in pluripotent stem cells (including ES and iPS cells). In addition, transfecting Cbx7 into fibroblasts increased Oct4, Sox2, c-Myc, and Klf4 expression. Moreover, we describe a novel approach to producing iPS cells using Cbx7 in combination with Oct4, Sox2, c-Myc, and Klf4. In summary, we have demonstrated that Cbx7 enhances the reprogramming of iPS cells and characterized the stemness and pluripotency of iPS cells.

Inhibition of MZF1/c-MYC Axis by Cantharidin Impairs Cell Proliferation in Glioblastoma.

Myeloid zinc finger 1 (MZF1), also known as zinc finger protein 42, is a zinc finger transcription factor, belonging to the Krüppel-like family that has been implicated in several types of malignancies, including glioblastoma multiforme (GBM). MZF1 is reportedly an oncogenic gene that promotes tumor progression. Moreover, higher expression of MZF1 has been associated with a worse overall survival rate among patients with GBM. Thus, MZF1 may be a promising target for therapeutic interventions. Cantharidin (CTD) has been traditionally used in Chinese medicine to induce apoptosis and inhibit cancer cell proliferation; however, the mechanism by which CTD inhibits cell proliferation remains unclear. In this study, we found that the expression of MZF1 was higher in GBM tissues than in adjacent normal tissues and low-grade gliomas. Additionally, the patient-derived GBM cells and GBM cell lines presented higher levels of MZF1 than normal human astrocytes. We demonstrated that CTD had greater anti-proliferative effects on GBM than a derivative of CTD, norcantharidin (NCTD). MZF1 expression was strongly suppressed by CTD treatment. Furthermore, MZF1 enhanced the proliferation of GBM cells and upregulated the expression of c-MYC, whereas these effects were reversed by CTD treatment. The results of our study suggest that CTD may be a promising therapeutic agent for patients with GBM and suggest a promising direction for further investigation.

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer.

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell-mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor-T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

The Effects of Tgfb1 and Csf3 on Chondrogenic Differentiation of iPS Cells in 2D and 3D Culture Environment.

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor ß1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.

The novel application of cordycepin in maintaining stem cell pluripotency and increasing iPS cell generation efficiency.

Maintaining the pluripotency of either embryonic stem (ES) cells or induced pluripotent stem (iPS) cells is a fundamental part of stem cell research. In this study, we reported that cordycepin promoted the expression of pluripotency markers in ES and iPS cells. ES cells treated with cordycepin demonstrated their potential for generating embryoid bodies and differentiating into all three germ layers. The expression levels of phospho-Jak2, phospho-Stat3, integrin αV, and integrin ß5 were increased after cordycepin treatment. Furthermore, the protein expression levels of IL-6 family proteins (IL-6, IL-11, LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF)), and epidermal growth factor (EGF) were also upregulated after cordycepin treatment, but were restored after co-treatment with a Jak2 inhibitor (AG490). The gene expression levels of Yamanaka factors were upregulated in mouse embryonic fibroblasts (MEFs) after cordycepin treatment. Moreover, the generation efficiencies of iPS cells were elevated after cordycepin treatment. We found that iPS cells generated after cordycepin treatment, not only expressed pluripotency markers, but also showed the ability of differentiating into neuron stem/progenitor cells. Taken together, we demonstrated that cordycepin maintained the pluripotency of stem cells via regulation of extracellular matrix (ECM) and Jak2/Stat3 signaling pathway and improved the generation efficiency of iPSCs.

YWHAE promotes proliferation, metastasis, and chemoresistance in breast cancer cells.

Breast cancer is the most common female-specific malignancy in Taiwan and developed countries worldwide, and its incidence continues to grow. 14-3-3ε (YWHAE), which belong to 14-3-3 family, it has been reported up-regulated in breast cancer tissues. However, the clinical implication and function of YWHAE in breast cancer remains unclear. In this study, we investigated the prognostic value of the YWHAE in human breast cancer. Immunohistochemistry was used to analyze YWHAE expression in breast cancer tissues. Cell model was applied to examine the functions of YWHAE. The chemotherapeutic agents were used to evaluate the effect of YWHAE in breast cancer cell lines. YWHAE expression was associated with tumor size, lymph node metastasis, and poor patient survival in patients with breast cancer. YWHAE overexpression significantly increased the proliferation, migration, and invasion abilities of breast cancer cells. Knockdown of YWHAE expression reduced the expression of Snail and Twist in breast cancer cells. We also found that YWHAE was responsible for the resistance of breast cancer cells to chemotherapeutic agents, and knockdown of YWHAE enhanced sensitivity to multiple chemotherapeutic agents in breast cancer cells. Taken together, our findings indicated that YWHAE promoted cancer progression and chemoresistance in breast cancer cells and can be a potential therapeutic target for breast cancer.

Increase of Meningitis Risk in Stroke Patients in Taiwan.

BACKGROUND AND PURPOSE: The blood-brain barrier (BBB) not only provides a physical obstruction but also recruits and activates neutrophils in cases of infection. Hemorrhagic or ischemic stroke reportedly induces the disruption of the BBB. However, few studies have reported a correlation between the incidence of meningitis in patients with a history of stroke. This study tested the hypothesis that patients with a history of stroke may be more vulnerable to meningitis. METHODS: Stroke and age-matched comparison (n = 29,436 and 87,951, respectively) cohorts were recruited from the Taiwan National Health Insurance database (2000-2011). Correlations between the two cohorts were evaluated by Cox proportional hazard regression model, Kaplan-Meier curve, and log-rank tests. RESULTS: The incidence of meningitis was higher in the stroke cohort compared to that in the comparison cohort [hazard ratio (HR), 2.89; 95% confidence interval (CI), 2.23-3.74, p < 0.001]. After adjusting for age, sex, and comorbidities, the estimated HR in the stroke cohort was 2.55-fold higher than that in the comparison cohort (CI, 1.94-3.37; p < 0.001). Notably, patients who had experienced hemorrhagic stroke had a higher incidence rate of meningitis than those with a history of ischemic stroke, except for patients older than 75 years (incidence rates in hemorrhagic/ischemic stroke patients, 3.14/1.48 in patients younger than 45 years, 1.52/0.41 in 45- to 64-year group, 1.15/0.90 in 65- to 74-year group, 0.74/0.93 in patients older than 75 years). Moreover, stroke patients who had undergone head surgery had the highest meningitis risk (adjusted HR, 8.66; 95% CI, 5.55-13.5; p < 0.001) followed by stroke patients who had not undergone head surgery (adjusted HR, 2.11; 95% CI, 1.57-2.82; p < 0.001). CONCLUSION: Our results indicated that stroke patients have higher risks of meningitis. Compromised BBB integrity in stroke patients may lead to increased vulnerability to infectious pathogens. In summary, our study provided new evidence of the clinical relationship between stroke and meningitis, and our findings suggest the need for precautions to prevent meningitis in stroke patients.

3'-Hydroxy-4'-methoxy-ß-methyl-ß-nitrostyrene inhibits tumor growth through ROS generation and GSH depletion in lung cancer cells.

AIMS: Members of the ß-nitrostyrene family are known to suppress tumor growth, with the underlying mechanisms of ß-nitrostyrene remain mostly unclear. Herein, we synthesized a ß-nitrostyrene derivative, 3'-hydroxy-4'-methoxy-ß-methyl-ß-nitrostyrene (CYT-Rx20), and explored its anticancer activities in human lung cancer cells in vitro and in vivo. MAIN METHODS: Cell viability was measured by XTT assay. Apoptosis was detected by Annexin V/PI staining. Caspase activation was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by flow cytometry. GSH level was detected by ELISA assay. KEY FINDINGS: In this study, we found that CYT-Rx20 significantly reduced cell viability, accompanied by G2/M arrest in lung cancer cells. Increased protein levels of cleaved-caspase families indicated apoptotic cell death upon CYT-Rx20 treatment. Furthermore, increased level of intracellular reactive oxygen species (ROS), loss of mitochondrial membrane potential (ΔΨm), glutathione (GSH) depletion and inhibition of GSH reductase were observed after CYT-Rx20 treatment. The effects of CYT-Rx20 on cell viability and the loss of ΔΨm were significantly reversed when cells were pretreated with thiol antioxidants NAC, GSH, or 2-ME. Finally, xenograft animal study demonstrated that CYT-Rx20 significantly suppressed lung tumor growth in vivo. SIGNIFICANCE: Our data demonstrated that CYT-Rx20 triggered apoptotic cell death in lung cancer cells and suppressed lung tumor growth through GSH depletion, suggesting that CYT-Rx20 may have the potential to be further developed as an anticancer compound for treating lung cancer.

VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis.

Abnormal lipoprotein profiles are associated with breast cancer progression. However, the mechanisms linking abnormal lipoprotein levels to breast cancer progression, especially metastasis, remain unclear. Herein, we found that L1 and L5 subfractions of LDL and VLDL, but not HDL, enhanced breast cancer cell viability. L1, L5, and VLDL also increased the in vitro tumorigenesis of breast cancer cells in anchorage-independent soft agar assay. In addition, L1, L5, and VLDL, but not HDL, increased the levels of mesenchymal markers Slug, Vimentin, and ß-Catenin, and promoted breast cancer cell migration and invasion. L1, L5, and VLDL increased Akt Ser473 phosphorylation and promoted cell migration, which were reversed by the PI3K/Akt inhibitor wortmannin. Further in vitro angiogenesis assay and cytokine array analysis demonstrated that L1, L5, and VLDL enhanced secretion of angiogenic factors in breast cancer cells and promoted angiogenic activity. However, only VLDL reduced anchorage-dependent cell death and promoted lung metastasis in nude mice. In summary, our data suggest that L1, L5, and especially VLDL promote breast cancer progression and metastasis through Akt-induced EMT and angiogenesis, and provide a novel mechanism of how dyslipoproteinemia promotes breast cancer progression.

The Synthetic ß-Nitrostyrene Derivative CYT-Rx20 Inhibits Esophageal Tumor Growth and Metastasis via PI3K/AKT and STAT3 Pathways.

The ß-nitrostyrene family have been implicated for anti-cancer property. However, the pharmacological role of ß-nitrostyrene in esophageal cancer remain unclear. Here, a ß-nitrostyrene derivative, CYT-Rx20, was synthesized and assessed for its anti-cancer activities and underlying mechanism in esophageal cancer. CYT-Rx20 induced cytotoxicity in esophageal cancer cells by promoting apoptosis through activation of caspase cascade and poly(ADP-ribose) polymerase (PARP) cleavage. Besides, CYT-Rx20 inhibited esophageal cancer cell migration and invasion by regulating the expression of epithelial to mesenchymal transition (EMT) markers. CYT-Rx20 decreased cell viability and migration through suppression of the PI3K/AKT and STAT3 pathways. Of note, the cytotoxicity and anti-migratory effect of CYT-Rx20 were enhanced by co-treatment with SC79 (AKT activator) or colivelin (STAT3 activator), suggesting the dependency of esophageal cancer cells on AKT and STAT3 for survival and migration, an oncogene addiction phenomenon. In xenograft tumor-bearing mice, CYT-Rx20 significantly reduced tumor growth of the implanted esophageal cancer cells accompanied by decreased Ki-67, phospho-AKT, and phospho-STAT3 expression. In orthotopic esophageal cancer mouse model, decreased tumor growth and lung metastasis with reduced Ki-67 and phospho-STAT3 expression were observed in mice treated with CYT-Rx20. Together, our results suggest that CYT-Rx20 is a potential ß-nitrostyrene-based anticancer compound against the tumor growth and metastasis of esophageal cancer.

Extracellular Visfatin-Promoted Malignant Behavior in Breast Cancer Is Mediated Through c-Abl and STAT3 Activation.

PURPOSE: Visfatin is an adipocytokine involved in cellular metabolism, inflammation, and cancer. This study investigated the roles of extracellular visfatin in breast cancer, and explored underlying mechanisms in clinical and experimental settings. EXPERIMENTAL DESIGN: Associations of serum visfatin with clinicopathologic characteristics and patient survival were assessed with Cox regression models and Kaplan-Meier analyses. Effects of extracellular visfatin on cultured breast cancer cells were examined, followed by in vivo investigation of tumor growth and metastasis in xenograft animal models. Imatinib and Stattic were used to inhibit c-Abl and STAT3 activation, respectively. RESULTS: Breast cancer patients with high serum visfatin levels were associated with advanced tumor stage, increased tumor size and lymph node metastasis, and poor survival. Elevated phosphorylation of c-Abl and STAT3 in breast tumor tissues were correlated with high serum visfatin levels in patients. Visfatin-promoted in vitro cell viability and metastatic capability were suppressed by imatinib (c-Abl inhibitor) and Stattic (STAT3 inhibitor). Increased in vivo cell invasiveness was observed in zebrafish xenografted with visfatin-pretreated breast cancer cells. Tumor growth and lung metastasis occurred in visfatin-administered mice xenografted with breast cancer cells. Tail vein-injected mice with visfatin-pretreated breast cancer cells showed increased lung metastasis, which was suppressed by imatinib. CONCLUSIONS: Serum visfatin levels in breast cancer patients reveal potential prognostic values, and our findings that visfatin promoted breast cancer through activation of c-Abl and STAT3 may provide an important molecular basis for future design of targeted therapies that take into account different serum visfatin levels in breast cancer. Clin Cancer Res; 22(17); 4478-90. ©2016 AACR.

The synthetic ß-nitrostyrene derivative CYT-Rx20 induces breast cancer cell death and autophagy via ROS-mediated MEK/ERK pathway.

The ß-nitrostyrene family has been shown to suppress cancer cell proliferation and induce programmed cell death. However, mechanisms underlying ß-nitrostyrenes remain less evaluated. Here, we synthesized a ß-nitrostyrene derivative, CYT-Rx20, and characterized its anticancer effect and involving mechanisms in breast cancer. We found that CYT-Rx20 arrested breast cancer cells at G2/M phase and decreased cell viability by activating the caspase cascade, accompanying with increases of poly (ADP-ribose) polymerase (PARP) cleavage and γ-H2AX expression. On the other hand, up-regulation of Beclin-1, ATG5, and LC-3 was observed in CYT-Rx20-induced autophagy, which was evidently shown by transmission electron microscopy. In addition to these, CYT-Rx20-induced breast cancer cell death, intracellular reactive oxygen species (ROS) formation and expression of phospho-ERK1/2, Beclin-1, and LC-3 were significantly reversed in the presence of N-acetyl-l-cysteine (NAC), a thiol antioxidant. Furthermore, the cytotoxicity of CYT-Rx20 was enhanced by co-treatment with the autophagy inhibitor chloroquine or bafilomycin A1, suggesting that an incomplete autophagy process could deteriorate CYT-Rx20-induced cytotoxicity. In nude mice xenograft study, CYT-Rx20 significantly reduced orthotopic tumor growth. Immunohistochemical analysis revealed elevated expression of phospho-ERK1/2 and LC-3 in tumor tissues of the mice treated with CYT-Rx20. Together, we propose that CYT-Rx20 may have potential to be further developed into a ß-nitrostyrene-based anticancer compound for the treatment of breast cancer.

The tetraindole SK228 reverses the epithelial-to-mesenchymal transition of breast cancer cells by up-regulating members of the miR-200 family.

The results of recent studies have shown that metastasis, the most common malignancy and primary cause of mortality promoted by breast cancer in women, is associated with the epithelial-to-mesenchymal transition (EMT). The results of the current study show that SK228, a novel indole containing substance, exhibits anti-cancer activity. In addition, the effects of SK228 on the regulation of EMT in breast cancer cells as well as the underlying mechanism have been explored. SK228 was observed to induce a fibroblastoid to epithelial-like change in the appearance of various breast cancer cell lines and to suppress the migration and invasion of these cancer cells in vitro. Moreover, expression of E-cadherin was found to increase following SK228 treatment whereas ZEB1 expression was repressed. Expression of other major EMT inducers, including ZEB2, Slug and Twist1, is also repressed by SK228 as a consequence of up-regulation of members of the miR-200 family, especially miR-200c. The results of animal studies demonstrate that SK228 treatment leads to effective suppression of breast cancer growth and metastasis in vivo. The observations made in this investigation show that SK228 reverses the EMT process in breast cancer cells via an effect on the miR-200c/ZEB1/E-cadherin signalling pathway. In addition, the results of a detailed analysis of the in vivo anti-cancer activities of SK228, carried out using a breast cancer xenograft animal model, show that this substance is a potential chemotherapeutic agent for the treatment of breast cancer.

Synthesis and evaluation of the cytotoxicities of tetraindoles: observation that the 5-hydroxy tetraindole (SK228) induces G2 arrest and apoptosis in human breast cancer cells.

Current chemical and biological interest in indole-3-carbinol (I3C) and its metabolites has resulted in the discovery of new biologically active indoles. As part of a program aimed at the development of indole analogues, tetraindoles 1-15 were prepared and their antiproliferative effects on human breast cancer cells were evaluated. The results show that the 5-hydroxy-tetraindole 8 (SK228) has optimum antiproliferative activity against breast adenocarcinoma (MCF 7 and MDA-MB-231) cells and that this activity involves G(2)-phase arrest of the cell cycle with a distinctive increase in the expression of cyclin B1 and phospho-cdc2. Further observations suggest that 5-hydroxy-tetraindole 8 induces apoptosis through externalization of membrane phosphatidylserine, DNA fragmentation, and activation of caspase-3. Given the fact that I3C and its metabolites have been shown to improve therapeutic efficacy and to have a broad range of antitumor activities in human cancer cells, the current findings have important pharmacological relevance as they open a promising route to the development of a potential chemotherapeutic application of tetraindoles as agents for the treatment of breast cancer.

Inhibition of glutathione S-transferase M1 by new gabosine analogues is essential for overcoming cisplatin resistance in lung cancer cells.

A new class of human GST inhibitors has been identified via rational design approach; we report their discovery, synthesis, inhibitory activity, and synergetic effect in combination with cisplatin against A549 lung cancer cell line. The results of this effort show that the lead 4-O-decyl-gabosine D (24) has optimum synergetic effect in A549 human lung adenocarcinoma epithelial cell and that this activity involves inhibition of glutathione S-transferase M1, apparently consistent with siRNA-mediated knockdown of GSTM1 gene.

Preparation and characterization of amino-linked heterocyclic carbene palladium, gold, and silver complexes and their use as anticancer agents that act by triggering apoptotic cell death.

Transition metal complexes bearing amino linked N-heterocyclic carbenes (NHC) were prepared and evaluated for their antiproliferative activities in human cancer cells. The optimum antiproliferative activity, observed for the gold complex 3 in U-87 MG cells, was found to involve S-phase arrest of the cell cycle. The results indicate that 3 induces apoptosis through a p53-bak pathway, a finding that could serve as a new strategy to reduce the resistance of cancer cells to p53-induced apoptosis.

A novel sialyltransferase inhibitor AL10 suppresses invasion and metastasis of lung cancer cells by inhibiting integrin-mediated signaling.

Aberrant sialylation catalyzed by sialyltransferases (STs) is frequently found in cancer cells and is associated with increased cancer metastasis. However, ST inhibitors developed till now are not applicable for clinical use because of their poor cell permeability. In this study, a novel ST inhibitor AL10 derived from the lead compound lithocholic acid identified in our previous study is synthesized and the anti-cancer effect of this compound is studied. AL10 is cell-permeable and effectively attenuates total sialylation on cell surface. This inhibitor shows no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of alpha-2,3-ST-overexpressing A549 and CL1.5 human lung cells. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of various integrin molecules and attenuated activation of the integrin downstream signaling mediator focal adhesion kinase. More importantly, AL10 significantly suppresses experimental lung metastasis in vivo without affecting liver and kidney function of experimental animals as determined by serum biochemical assays. Taken together, AL10 is the first ST inhibitor, which exhibits potent anti-metastatic activity in vivo and may be useful for clinical cancer treatment.

Overcoming the drug resistance in breast cancer cells by rational design of efficient glutathione S-transferase inhibitors.

A new type of competitive human GST inhibitors has been developed via the bioisostere and structure activity profile strategies; we report their discovery, preparation, inhibitory activity, and synergetic effect in combination with chemotherapy drugs against breast cancer cells.

Synthesis and structure-activity relationships of novel furazan-3,4-diamide analogs as potent anti-cancer agents.

This study describes the synthesis and structure-activity relationships of a series of furazan-3,4-diamide analogs. 1,2,5-Oxadiazole ring and electron-withdrawing substituent on the phenyl ring are proposed to be the important elements which contribute to a significant extent maximal potency of anti-proliferation effect.

p16 inhibits matrix metalloproteinase-2 expression via suppression of Sp1-mediated gene transcription.

Previous studies demonstrate that p16, a cyclin-dependent kinase inhibitor and a tumor suppressor, may inhibit matrix metalloproteinase-2 (MMP-2) expression in human cancer cells to suppress tumor invasion and metastasis. However, the detailed mechanism is still unclear. Our results show that p16 inhibits MMP-2 expression via transcriptional repression. Promoter deletion and mutation analysis indicates that p16 acts through the Sp1 transcription factor-binding site located between -72 and -64 bp region from the transcriptional start site of the human MMP-2 promoter to repress gene expression. DNA affinity precipitation assay (DAPA) and chromatin immuno-precipitation (CHIP) assay demonstrate that Sp1 proteins constitutively bind to this consensus sequence in vitro and in vivo. p16 attenuates Sp1 binding to the MMP-2 promoter to suppress gene transcription and overexpression of Sp1 may counteract p16-induced downregulation of MMP-2. CyclinA/CDK complex may directly phosphorylate Sp1 and enhance its DNA-binding activity. Thus, we investigated the effect of p16 on the interaction between cyclin A and Sp1. Our results indicate that p16 induces downregulation of cyclin A and CDK2, reduces the interaction between cyclin A and Sp1, and attenuates phosphorylation of Sp1. Ectoexpression of cyclin A counteracts p16-mediated inhibition of DNA binding of Sp1 and activates MMP-2 promoter activity and mRNA expression. Collectively, our results suggest that p16 suppresses MMP-2 by blocking Sp1-mediated gene transcription.