Inhibition Of DNA Binding Activity Research Articles

Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

Leocarpinolide B Attenuates Collagen Type II-Induced Arthritis by Inhibiting DNA Binding Activity of NF-κB.

Rheumatoid arthritis (RA) is a chronic autoimmune disease triggered by a cascading inflammatory response. Sigesbeckia Herba (SH) has long been utilized as a traditional remedy to alleviate symptoms associated with rheumatism. Our previous study found that leocarpinolide B (LB), a sesquiterpene lactone isolated from the whole plant of SH, possesses potent a anti-inflammatory effect on macrophages. This study was designed to evaluate the therapeutic effects of LB on RA, and further investigate the underlying mechanisms. In collagen type II-induced arthritic mice, LB was demonstrated to decrease the production of autoimmune antibodies in serum and inflammatory cytokines in the joint muscles and recover the decreased regulatory T lymphocytes in spleen. Moreover, LB significantly suppressed the inflammatory infiltration, formation of pannus and bone erosion in the paw joints. In vitro testing showed that LB inhibited the proliferation, migration, invasion, and secretion of inflammatory cytokines in IL-1β-induced human synovial SW982 cells. Network pharmacology and molecular docking suggested NF-κB p65 could be the potential target of LB on RA treatment, subsequent experimental investigation confirmed that LB directly interacted with NF-κB p65 and reduced the DNA binding activity of NF-κB in synovial cells. In conclusion, LB significantly attenuated the collagen type II-induced arthritis, which was at least involved in the inhibition of DNA binding activity of NF-κB through a direct binding to NF-κB p65. These findings suggest that LB could be a valuable lead compound for developing anti-RA drugs.

Abstract A095: Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku

Abstract Recent advances in our understanding of the mechanisms of the cellular response to DNA damage (DDR) have opened up an exciting array of opportunities to treat human cancer. The majority of drug discovery efforts have focused on the DDR kinases, ATM, ATR and DNA-PK, each of which is activated in response to different types of DNA damage or genomic perturbations. We have taken the novel approach of drugging this pathway by targeting the DNA binding proteins Replication protein A (RPA) and Ku, which are required to activate the ATR and DNA-PK kinases, respectively. Chemical inhibition of the DNA binding activity functionally abrogates kinase activity and provides a novel mechanism by which DDR kinases can be blocked. The identification of a potent inhibitor of the Ku-DNA interactions was gleaned from a specificity screen and was found to effectively block the Ku-DNA interaction and this inhibits DNA-PK phosphorylation activity. We identified a series of moieties and optimized pharmacophore for potent and specific inhibition of Ku and demonstrate cellular inhibition of Ku DNA binding and DNA-PK activity. Third generation Ku inhibitors were developed to optimize for cellular bioavailability and are able to sensitize cells to DNA double strand break inducing agents, consistent with the role of DNA-PK in the non-homologous end joining repair pathway. We initially identified a series of RPA-DNA interaction inhibitors from an in vitro high throughput screen. Extensive SAR was conducted to optimize for potency, specificity and physicochemical properties and led to the promotion of a lead candidate. We have determined the mechanism of action for single agent anti-cancer activity and combination activity involves chemical exhaustion of RPA. In vivo analysis of the lead candidate in pre-clinical models of lung and ovarian cancer has led to the advancement of a clinical candidate and represent a first in class RPA inhibitor poised for clinical advancement. Together, these data demonstrate the ability to chemically target structure specific protein-DNA interactions. The molecules synthesized to inhibit these interactions share some common features, but can be optimized to obtain high potency and specificity. In addition, chemical modifications can be employed to allow cellular and in vivo bioavailability while retaining potent inhibitory activity. These data reveal the potential to target a new range of macromolecular interactions to expand the anticancer armamentarium to protein-DNA interactions. Citation Format: John Turchi, Katherine S Pawelczak, Navnath Gavande, Pamela S VanderVere-Carozza. Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A095. doi:10.1158/1535-7163.TARG-19-A095

Abstract 1301: Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku

Abstract Recent advances in our understanding of the mechanisms of the cellular response to DNA damage (DDR) have opened up an exciting array of opportunities to treat human cancer. The majority of drug discovery efforts have focused on the DDR kinases, ATM, ATR and DNA-PK, each of which is activated in response to different types of DNA damage or genomic perturbations. We have taken the novel approach of drugging this pathway by targeting the DNA binding proteins Replication protein A (RPA) and Ku, which are required to activate the ATR and DNA-PK kinases, respectively. Chemical inhibition of the DNA binding activity functionally abrogates kinase activity and provides a novel mechanism by which DDR kinases can be blocked. The identification of a potent inhibitor of the Ku-DNA interactions was gleaned from a specificity screen and was found to effectively block the Ku-DNA interaction and this inhibits DNA-PK phosphorylation activity. We identified a series of pharmacophores and moieties for potent and specific inhibition of Ku and demonstrate cellular inhibition of Ku DNA binding and DNA-PK activity. Third generation Ku inhibitors were developed to optimize for cellular bioavailability and are able to sensitize cells to DNA double strand beaks inducing agents, consistent with the role of DNA-PK in the non-homologous end joining repair pathway. We initially identified a series of RPA-DNA interaction inhibitors from an in vitro high throughput screen. Extensive SAR was conducted to optimize for potency, specificity and physicochemical properties and led to the promotion of a lead candidate. We have determined the mechanism of action for single agent anti-cancer activity and combination activity involves chemical exhaustion of RPA. In vivo analysis of the lead candidate in pre-clinical models of lung and ovarian cancer has led to the advancement of a clinical candidate and represent a first in class RPA inhibitor poised for clinical advancement. Together, these data demonstrate the ability to chemically target structure specific protein-DNA interactions. The molecules synthesized to inhibit these interactions share some common features, but can be optimized to obtain high potency and specificity. In addition, chemical modifications can be employed to allow cellular and in vivo bioavailability while retaining potent inhibitory activity. These data reveal the potential to target a new range of macromolecular interactions to expand the anticancer armamentarium to protein-DNA interactions. Citation Format: John J. Turchi, Pamela S. VanderVere-Carozza, Navnath S. Gavande, Katherine S. Pawelczak. Targeting protein-DNA interactions in the DNA damage response: Lead identification and optimization for novel inhibitors of RPA and Ku [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1301.

Involvement of pRb-E2F pathway in green tea extract-induced growth inhibition of human myeloid leukemia cells

Both inhibitory and stimulatory effect of EGCG on cancer cells have been reported, which often is linked to receptor tyrosine kinase signaling. In this study, we present evidence that green tea extract and its chemical component, Epigallocatechin-3-gallate (EGCG), inhibit growth of human myeloid leukemia cells through the regulation of pRb synthesis and formation of pRb-E2F complexes. Addition of green tea extract to the culture of TF-1a and MV4-11 myeloid leukemia cells significantly inhibited their proliferation with a substantial portion of cell death being observed. The green tea extract and EGCG had no significant effect on the expression of G1 CDKs and the CDK inhibitors but downregulated the formation of pRb-CDKs. Surprisingly, the expression of pRb was markedly upregulated while the phosphorylation of pRb downregulated. The upregulation of pRb was blocked by pre-treatment with cycloheximide, a protein synthesis inhibitor, suggesting a requirement of protein synthesis. In agreement with these results, pRb-E2F complexes were upregulated and E2F DNA binding activity decreased. Since both TF-1a and MV4-11 are factor-independent cell lines, the upregulation of pRb-E2F complexes and inhibition of DNA binding activity by green tea extract is most likely through a receptor tyrosine kinase-independent pathway. We also found that the stem/progenitor cells derived from these two leukemia cell lines are more sensitive to the inhibitory effect of green tea extract. Our result suggests that concentrated green tea extract and EGCG may have potential for clinical investigation as an inducer of cancer cell death.

Green Tea Bioactive Polyphenol Epigallocatechin-3-O-Gallate in Osteoarthritis : Current Status and Future Perspectives

Green Tea Bioactive Polyphenol Epigallocatechin-3-O-Gallate in Osteoarthritis : Current Status and Future Perspectives

A transcriptional repressive role for epithelial-specific ETS factor ELF3 on oestrogen receptor alpha in breast cancer cells.

Oestrogen receptor-α (ERα) is a ligand-dependent transcription factor that primarily mediates oestrogen (E2)-dependent gene transcription required for mammary gland development. Coregulators critically regulate ERα transcription functions by directly interacting with it. In the present study, we report that ELF3, an epithelial-specific ETS transcription factor, acts as a transcriptional repressor of ERα. Co-immunoprecipitation (Co-IP) analysis demonstrated that ELF3 strongly binds to ERα in the absence of E2, but ELF3 dissociation occurs upon E2 treatment in a dose- and time-dependent manner suggesting that E2 negatively influences such interaction. Domain mapping studies further revealed that the ETS (E-twenty six) domain of ELF3 interacts with the DNA binding domain of ERα. Accordingly, ELF3 inhibited ERα's DNA binding activity by preventing receptor dimerization, partly explaining the mechanism by which ELF3 represses ERα transcriptional activity. Ectopic expression of ELF3 decreases ERα transcriptional activity as demonstrated by oestrogen response elements (ERE)-luciferase reporter assay or by endogenous ERα target genes. Conversely ELF3 knockdown increases ERα transcriptional activity. Consistent with these results, ELF3 ectopic expression decreases E2-dependent MCF7 cell proliferation whereas ELF3 knockdown increases it. We also found that E2 induces ELF3 expression in MCF7 cells suggesting a negative feedback regulation of ERα signalling in breast cancer cells. A small peptide sequence of ELF3 derived through functional interaction between ERα and ELF3 could inhibit DNA binding activity of ERα and breast cancer cell growth. These findings demonstrate that ELF3 is a novel transcriptional repressor of ERα in breast cancer cells. Peptide interaction studies further represent a novel therapeutic option in breast cancer therapy.

Lipoteichoic acid isolated from Lactobacillus plantarum down-regulates UV-induced MMP-1 expression and up-regulates type I procollagen through the inhibition of reactive oxygen species generation

Ultraviolet (UV) irradiation from the sun is the primary environmental factor that causes human skin aging. UV irradiation induces the expressions of matrix metalloproteinases (MMPs) and extracellular matrix degrading enzymes. Among the members of MMP family, MMP-1 is an interstitial collagenase that degrades the collagen triple helix. We investigated the effect of Lactobacillus plantarum, well known as useful microorganism, on UV-induced-MMP-1 expression in human dermal fibroblasts. Human dermal fibroblasts (HDF) was pre-stimulated with lipoteichoic acid isolated from L. plantarum followed by UV irradiation. Secreted protein level of MMP-1 was evaluated by Western blot analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) from the cell lysates was also examined by western blotting. Electrophoretic mobility-shift assay (EMSA) was used to detect the activated transcription factor, AP-1 and NF-κB. The detection of type 1 procollagen was carried with Procollagen type 1 C-peptide (PIP) EIA kit. The generation of reactive oxygen species (ROS) by LTA and UV irradiation was examined by Griess reagent assay and fluorescence microscope. We found that lipoteichoic acid (LTA), a cell-wall component of Gram-positive bacteria, isolated from L. plantarum, inhibited MMP-1 expression. Pretreatment with LTA from L. plantarum (pLTA) reduced MMP-1 expression in a dose-dependent manner and inhibited activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). It also led to the inhibition of DNA binding activity of activator protein-1 (AP-1) and of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB). Furthermore, LTA promoted type 1 procollagen synthesis and reduced the generation of ROS induced by UV irradiation. Our study demonstrates that pLTA inhibits degradation of collagen and promotes its synthesis and that pLTA contributes to a decrease in ROS production. Therefore, pLTA from L. plantarum has potential abilities to prevent and treat skin photo-aging.

α-Mangostin: A Dietary Antioxidant Derived from the Pericarp of Garcinia mangostana L. Inhibits Pancreatic Tumor Growth in Xenograft Mouse Model

Pancreatic cancer (PC) is the most aggressive malignant disease, ranking as the fourth most leading cause of cancer-related death among men and women in the United States. In this study, we provide evidence of chemotherapeutic effects of α-mangostin, a dietary antioxidant isolated from the pericarp of Garcinia mangostana L. against human PC. The chemotherapeutic effect of α-mangostin was determined using four human PC cells (PL-45, PANC1, BxPC3, and ASPC1). α-Mangostin resulted in a significant inhibition of PC cells viability without having any effects on normal human pancreatic duct epithelial cells. α-Mangostin showed a dose-dependent increase of apoptosis in PC cells. Also, α-mangostin inhibited the expression levels of pNF-κB/p65Ser552, pStat3Ser727, and pStat3Tyr705. α-Mangostin inhibited DNA binding activity of nuclear factor kappa B (NF-κB) and signal transducer and activator 3 (Stat3). α-Mangostin inhibited the expression levels of matrix metallopeptidase 9 (MMP9), cyclin D1, and gp130; however, increased expression of tissue inhibitor of metalloproteinase 1 (TIMP1) was observed in PC cells. In addition, i.p. administration of α-mangostin (6 mg/kg body weight, 5 days a week) resulted in a significant inhibition of both primary (PL-45) and secondary (ASPC1) human PC cell-derived orthotopic and ectopic xenograft tumors in athymic nude mice. No sign of toxicity was observed in any of the mice administered with α-mangostin. α-Mangostin treatment inhibited the biomarkers of cell proliferation (Ki-67 and proliferating cell nuclear antigen [PCNA]) in the xenograft tumor tissues. We present, for the first time, that dietary antioxidant α-mangostin inhibits the growth of PC cells in vitro and in vivo. These results suggest the potential therapeutic efficacy of α-mangostin against human PC.

Anticancer Activity of Phyllanthus emblica Linn. (Indian Gooseberry): Inhibition of Transcription Factor AP-1 and HPV Gene Expression in Cervical Cancer Cells

Plant products of Phyllanthus emblica Linn. are traditionally consumed for its immense nutritive and medicinal values. However, the molecular mechanism(s) by which it exerts it effects is less understood. In this study, we investigated mechanism of action of P. emblica fruit extract (PE) by studying its effect on activator protein-1 (AP-1) activity and human papillomavirus (HPV) transcription that are essential for tumorigenicity of cervical cancer cells. PE resulted in a dose-and time-dependent inhibition of DNA binding activity of constitutively active AP-1 in both HPV16-positive (SiHa) and HPV18-positive (HeLa) cervical cancer cells. PE-induced AP-1 inhibition was found mediated through downregulation of constituent AP-1 proteins, c-Jun, JunB, JunD, and c-Fos; however, the kinetics of their inhibition varied in both the cell types. Inhibition of AP-1 by PE was accompanied by suppression of viral transcription that resulted in growth inhibition of cervical cancer cells. Growth inhibitory activity of PE was primarily manifested through induction of apoptotic cell death. These results suggest that P. emblica exhibits its anticancer activities through inhibition of AP-1 and targets transcription of viral oncogenes responsible for development and progression of cervical cancer thus indicating its possible utility for treatment of HPV-induced cervical cancers.

Apoptosis Induced by Knockdown of uPAR and MMP-9 is Mediated by Inactivation of EGFR/STAT3 Signaling in Medulloblastoma

BackgroundMedulloblastoma is a highly invasive cancer of central nervous system diagnosed mainly in children. Matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator receptor (uPAR) are over expressed in several cancers and well established for their roles in tumor progression. The present study is aimed to determine the consequences of targeting these molecules on medulloblastoma progression.Methodology/Principal FindingsRadiation is one of the foremost methods applied for treating cancer and considerable evidence showed that radiation elevated uPAR and MMP-9 expression in medulloblastoma cell. Therefore efforts are made to target these molecules in non-irradiated and irradiated medulloblastoma cells. Our results showed that siRNA-mediated knockdown of uPAR and MMP-9, either alone or in combination with radiation modulated a series of events leading to apoptosis. Down regulation of uPAR and MMP-9 inhibited the expression of anti-apoptotic molecules like Bcl-2, Bcl-xL, survivin, XIAP and cIAPI; activated BID cleavage, enhanced the expression of Bak and translocated cyctochrome C to cytosol. Capsase-3 and -9 activities were also increased in uPAR- and MMP-9-downregulated cells. The apoptosis induced by targeting MMP-9 and uPAR was initiated by inhibiting epidermal growth factor receptor (EGFR) mediated activation of STAT3 and NF-κB related signaling molecules. Silencing uPAR and MMP-9 inhibited DNA binding activity of STAT3 and also reduced the recruitment of STAT3 protein at the promoter region of Bcl-2 and survivin genes. Our results suggest that inhibiting uPAR and MMP-9 reduced the expression of anti-apoptotic molecules by inactivating the transcriptional activity of STAT3. In addition, treating pre-established medulloblastoma with siRNAs against uPAR and MMP-9 both alone or in combination with radiation suppressed uPAR, MMP-9, EGFR, STAT3 expression and induced Bak activation leading to apoptosis.Conclusion/SignificanceTaken together, our results illustrated that RNAi mediated targeting of uPAR and MMP-9 might have therapeutic potential against medulloblastoma.

Deficiency of C–C chemokine receptor 5 suppresses tumor development via inactivation of NF–ĸB and inhibition of monocyte chemoattractant protein-1 in urethane-induced lung tumor model

To evaluate the significance of C-C chemokine receptor type 5 (CCR5) in lung tumor development, we compared carcinogen-induced tumor growth in CCR5 knockout (CCR5(-/-)) mice and wild-type (CCR5(+/+)) mice. CCR5(-/-) mice showed reduced urethane (1g/kg)-induced tumor incidence when compared with those of CCR5(+/+) mice. We investigated the activation of nuclear factor-kappaB/STAT3 since these are implicated transcription factors in the regulation of genes involving tumor growth. Significant inhibition of DNA-binding activity of nuclear factor-kappaB and STAT3, and the translocation of p50 and p65 into the nucleus and the phosphorylation of IĸB were found in the lungs of CCR5(-/-) mice compared with the lungs of CCR5(+/+) mice. Expression of apoptotic protein such as cleaved caspase-3, cleaved PARP and Bax was elevated, whereas the expression levels of survival protein such as Bcl-2 and cIAP1 was decreased in the lungs of CCR5(-/-) mice. Interestingly, we found that the level of monocyte chemoattractant protein-1 (MCP-1), a tumor growth-promoting cytokine, was significantly reduced in the lung tumor tissue and blood of CCR5(-/-) mice compared with the level in CCR5(+/+) mice. In addition, CCR5 small interfering RNA (siRNA) and inhibitor of MCP-1 blocked lung cancer cell growth, which was abolished by the addition of MCP-1 protein in cultured lung cancer cells. Moreover, inactivation of CD8(+) cytotoxic T cell and dendritic cells was significantly increased in the blood, lung tumors and spleens of CCR5(-/-) mice compared with that of CCR5(+/+) mice. Therefore, these results showed that CCR5 deficiency suppressed lung tumor development through the inhibition of nuclear factor-kappaB/STAT3 pathways and the downregulation of MCP-1 in the carcinogen-induced lung tumor model.

Deficiency of C-C Chemokine Receptor 5 Suppresses Tumor Development via Inactivation of NF-κB and Upregulation of IL-1Ra in Melanoma Model

To evaluate the relevance of C-C chemokine receptor type 5 (CCR5) expression and tumor development, we compared melanoma growth in CCR5 knockout (CCR5−/−) mice and wild type (CCR5+/+) mice. CCR5−/− mice showed reduced tumor volume, tumor weight, and increased survival rate when compared to CCR5+/+ mice. We investigated the activation of NF-κB since it is an implicated transcription factor in the regulation of genes involving cell growth, apoptosis, and tumor growth. Significant inhibition of DNA binding activity of NF-κB, and translocation of p50 and p65 into the nucleus through the inhibition of phosphorylation of IκB was found in the melanoma tissues of CCR5−/− mice compared to melanoma tissues of CCR5+/+ mice. NF-κB target apoptotic protein expression, such as cleaved caspase-3, cleaved PARP, and Bax, was elevated, whereas the survival protein expression levels, such as Bcl-2, C-IAP1, was decreased in the melanoma tissues of CCR5−/− mice. Interestingly, we found that the level of IL-1Ra, a tumor growth suppressive cytokine, was significantly elevated in tumor tissue and spleen of CCR5−/− mice compared to the level in CCR5+/+ mice. Moreover, infiltration of CD8+ cytotoxic T cell and CD57+ natural killer cells was significantly increased in melanoma tumor and spleen tissue of CCR5−/− mice compared to that of CCR5+/+ mice. Therefore, these results showed that CCR5 deficiency caused apoptotic cell death of melanoma through inhibition of NF-κB and upregulation of IL-1Ra.

Cell growth inhibition and induction of apoptosis by snake venom toxin in ovarian cancer cell via inactivation of nuclear factor κB and signal transducer and activator of transcription 3

Snake venom toxin from Vipera lebetina turanica induces apoptosis in many cancer cell lines, but there is no study about the apoptotic effect of snake venom toxin on human ovarian cancer cells. In this study, we investigated the apoptotic effect of snake venom toxin in human ovarian cancer PA-1 and SK-OV3 cells. Snake venom toxin dose dependently (0∼10 μg/mL) inhibited ovarian cancer cell growth with IC(50) values 4.5 μg/mL in PA-1 cells, and 6.5 μg/mL in SK-OV3 cells. Our results also showed that apoptotic cell death increased by snake venom toxin in a dose dependent manner (0∼10 μg/mL). Consistent with increased cell death, snake venom toxin increased the expression of pro-apoptotic protein Bax and caspase-3, but down-regulated anti-apoptotic protein Bcl-2. Untreated ovarian cancer cells showed a high DNA binding activity of nuclear factor B (NF-κB), but it was inhibited by snake venom toxin accompanied by inhibition of p50 and p65 translocation into the nucleus as well as phosphorylation of inhibitory κB. Snake venom toxin also inhibited DNA binding activity of the signal transducer and activator of transcription 3 (STAT3). Moreover, the combination treatment of NF-κB (salicylic acid, 1 or 5 μM) and STAT3 (stattic, 1 μM) with snake venom toxin (1 μg/mL) further enhanced cell growth inhibitory effects of snake venom toxin. These results showed that snake venom toxin from Vipera lebetina turanica caused apoptotic cell death of ovarian cancer cells through the inhibition of NF-κB and STAT3 signal, and suggested that snake venom toxin may be applicable as an anticancer agent for ovarian cancer.

18α-Glycyrrhetinic Acid Down-Regulates Expression of Type I and III Collagen via TGF-Β1/Smad Signaling Pathway in Human and Rat Hepatic Stellate Cells

Objective: To investigate the effects of 18α-glycyrrhetinic acid (18α-GA) on the expression of type I and III collagen in human and rat hepatic stellate cells (HSC) and to explore the role of TGF-β1/Smad signaling pathway involved.Methods: Following 18α-GA treatment, the cell viability and cell growth were detected to determine the optimal concentration of 18α-GA. The expressions of TGF-β1/Smad signaling-related genes including type I and III collagen in human and rat HSCs before and after 18α-GA treatment were measured by real time PCR. The expression of related proteins was verified by western blot assay. The phosphorylation level of Smad2 and Smad3 was detected by immunocytochemistry. The DNA binding activities of SP-1, AP-1 and NF-κB were measured by both EMSA and ArrayStar transcription factor activity assay.Results: 18α-GA could decrease the mRNA and protein expression of Smad3, type I and III collagen, increase the Smad7 expression in human and rat HSCs (P<0.05), and reduce phosphorylation level of Smad3 at 24 h and 48 h after treatment. The DNA binding activities of transcription factors were suppressed by 18α-GA in human and rat HSCs at 24 h, and the activities reduced in a time dependent manner with the lowest activities at 48 h, especially for SP-1.Conclusion: 18α-GA could inhibit the mRNA and protein expression of type I and III collagen in human and rat HSCs, which may be attributed to down-regulation of Smad3, up-regulation of Smad7, and inhibition of DNA binding activities of SP-1, AP-1 and NF-κB.

Citreorosein Inhibits Production of Proinflammatory Cytokines by Blocking Mitogen Activated Protein Kinases, Nuclear Factor-κB and Activator Protein-1 Activation in Mouse Bone Marrow-Derived Mast Cells

Citreorosein (CIT), an anthraquinone component of Polygoni cuspidati (P. cuspidati) radix, suppressed gene expression of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in mouse bone marrow-derived mast cells (BMMCs) stimulated with phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187. To investigate the molecular mechanisms underlying CIT inhibition of the pro-inflammatory cytokine production, its effects on the activation of both nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) were assessed. CIT attenuated phosphorylation of the MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAP kinase and c-Jun NH(2)-terminal kinase (JNK). Furthermore, CIT strongly inhibited DNA binding activity of NF-κB through the inhibition of phosphorylation and degradation of inhibitor of kappaB (IκB) as well as activator protein-1 (AP)-1 through the reduction of phosphorylation of c-Jun. These results demonstrate that CIT inhibits proinflammatory cytokines production through the inhibition of both MAPKs and AKT-mediated IκB kinase (IKK) phosphorylation and subsequent inhibition of transcription factor NF-κB activation, thereby attenuating the production of proinflammatory cytokines.

Inhibition of Matrix Metalloproteinase-1 Induced by Oxidative Stress in Human Keratinocytes by Mangiferin Isolated fromAnemarrhena asphodeloides

Oxidative stress is related to the synthesis of matrix metalloproteinases (MMPs), which cause skin aging. The protective effects of mangiferin derived from Anemarrhena asphodeloides were investigated against hydrogen peroxide (H(2)O(2))-induced damage using human skin keratinocyte (HaCaT) cells. Mangiferin was found to scavenge intracellular reactive oxygen species (ROS), superoxide radicals, and hydroxyl radicals. ROS regulate MMPs gene expression and activation of proenzymes. Mangiferin inhibited H(2)O(2)-induced MMP-1 gene expression and protein levels as well as its activity. Moreover, it abrogated mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway and stress-activated protein kinase/extracellular signal-regulated kinase (SEK)-c-JUN N-terminal kinase (JNK) pathway, which are induced by H(2)O(2) treatment. And, it inhibited DNA binding activity of activator protein-1 (AP-1), a transcription factor of MMP-1 and downstream of ERK and JNK. Finally, it protected the human skin keratinocytes from H(2)O(2)-induced cell death. Taken together, these results indicate that mangiferin attenuated H(2)O(2)-induced MMP-1 activation via inhibition of ERK and JNK pathway and AP-1.

Inhibition of DNA binding activity of cAMP response element-binding protein by 1,2-naphthoquinone through chemical modification of Cys-286

1,2-Naphthoquinone (1,2-NQ) is an atmospheric electrophile that reacts covalently with protein thiols. Our previous study revealed that exposure of bovine aortic endothelial cells to 1,2-NQ causes covalent modification of cAMP response element-binding protein (CREB), thereby inhibiting its DNA binding activity and substantial gene expression of B-cell lymphoma-2 (Bcl-2) that is regulated by this transcription factor. In this study, we identified the modification sites of CREB that are associated with the decreased transcriptional activity. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis indicated that three amino acids (Cys-286, Lys-290, and Lys-319) were irreversibly modified by 1,2-NQ. Mutational analysis revealed that electrophilic modification of Cys-286, but not the other two amino acids, at the DNA binding domain is essential for the reduced CREB activity. Substitution of Cys-286 with tryptophan (C286W), which mimics CREB modification by 1,2-NQ, supported this notion. These results suggest that the covalent interaction of CREB with 1,2-NQ through Cys-286 blocks the DNA binding activity of CREB, resulting in the repression of CREB-regulated genes.

Abstract 4787: MMP-2 RNA interference leads to decreased NF-κB activation and induced JNK-mediated apoptosis by modulating TRADD-TRAF2-FADD interactions in glioma

Abstract MMP-2 belongs to a family of extracellular matrix metalloproteinases (MMPs) that play a pivotal role in the degradation of the ECM, thereby enhancing the invasive and metastatic potential of cancer cells. Our study unveiled a new mechanism of how MMP-2 RNA interference inhibits NF-κB activation. Essentially, siRNA against MMP-2 (pM) modulated the interactions between the adaptor protein TRADD-TRAF2 and TRADD-FADD, which consequently hindered the nuclear localization and DNA binding activity of NF-κB. This, in turn, led to apoptosis. Furthermore, pM treatment led to RIP cleavage and remarkable increases in the expression levels of FADD and activated MAP kinases, p38 MAPK and JNK. The prolonged activation of JNK resulted in phosphorylation of c-Jun. Treatments with specific JNK1 inhibitor (SP600125) reduced and significantly reversed the pro-apoptotic effect of pM, thereby suggesting a role of JNK in pM-induced cell death. Supplementation of recombinant human MMP-2 to pM-transfected cells restored the inhibited DNA binding activity of NF-κB and rescued cells from the pro-apoptotic effect of pM. Here, we demonstrate that the simultaneous inhibition of NF-κB activation and sustained activation of JNK play significant roles in pM-mediated apoptosis in human xenograft glioma cells. In addition, our results show that the specific downregulation of MMP-2 diminished intracranial tumor growth in vivo. The inhibition of tumor growth after pM treatment was associated with decreased MMP-2 expression, inhibited NF-κB activation, and enhanced phospho-JNK levels. In conclusion, the present study elucidates an underlying mechanism of pM-induced cell death in vitro as well as decreased tumor development in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4787. doi:10.1158/1538-7445.AM2011-4787

Abstract 4203: EF24, a novel curcumin analogue inhibits oncogenic activation induced by radiation abscopal effect in distant mice lungs

Abstract Ascertaining the ionizing radiation (IR)-induced carcinogenesis through an abscopal effect would raise a paradigm shift in our understanding of the radiobiological effects and radiation carcinogenesis. EF24, a synthetic analog of curcumin with improved physicochemical properties has been shown to possess potent anticancer activity, both in vitro as well as in vivo. In this study, we investigated the oncogenic activation after IR in an organ (lung) distant from the exposure field. C57/BL6 mice either mock irradiated, exposed to single dose IR (SDR: 2 or 10Gy) or fractionated IR (FIR, 2 Gy/day for 5 days) with or without EF24 (i.p. 200μg/Kg, 3h prior to IR) treatment were sacrificed 1h through 2 weeks post-irradiation. All IR exposures were limited to the lower abdomen area (1cm diameter), while the rest of the animal was protected by a specially designed cerrobend shield. DNA binding activity of AP-1, E2F1, cFOS and FRA1 were examined using EMSA analysis. Transcriptional activation of 88 oncogenes was assessed using QPCR profiling. Phosphorylation of CREB and JNK were examined using immunoblotting. Cellular localization of JunB, JunC and JunD were analyzed by immunohisto staining. IR significantly induced AP-1, E2F1, cFOS and FRA1 DNA binding activity in lungs distant from the exposure fields. EF24 completely and persistently (up to 2wks) suppressed IR-induced distant effect on AP-1, E2F1, cFOS and FRA1 DNA binding activity. Second, EF24 completely suppressed all 52, 31 and 64 oncogenes that were significantly upregulated after 2 Gy, 10Gy and 2Gyx5D exposures. We observed a similar inhibitory effect of oncogene transcriptome even after 2wk with EF24 treatment. Third, a profound increase in JunB, JunC and JunD levels observed in distant lung tissue after IR were significantly inhibited by EF24. Fourth, we observed a profound inhibition of IR-induced CREB phosphorylation with EF24 treatment in distant lungs. Taken together, these data clearly demonstrated an induced abscopal carcinogenic response in mice lungs after clinically relevant doses of IR exposure. The findings further imply that systemic administration of EF24 could potentially inhibit DNA binding activity, transactivation and translation of oncogenes and thereby mitigate IR-induced oncogenic activation at distant site. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4203. doi:10.1158/1538-7445.AM2011-4203