p21WAF1/Cip1 Regulation by hYSK1 Activates SP-1 Transcription Factor and Increases MMP-2 Expression under Hypoxic Conditions.

The hYSK1, a serine/threonine kinase (STK)-25, has been implicated in a variety of cellular functions including cell migration and polarity. We have recently reported that hYSK1 down-regulated the expression and functions of p16INK4a, a cell cycle regulatory protein, thereby enhancing migration and growth of cancer cells under hypoxic conditions. In this study, we further investigated the mechanisms underlying downregulation of p16INK4a and anti-migratory function of hYSK1. Our study revealed that p21WAF1/Cip1 is a novel binding partner of hYSK1. Moreover, the interaction between hYSK1 and p21WAF1/Cip1 led to the inhibition of SP-1 transcriptional activity, as revealed by a significant down-regulation of SP-1-mediated transactivation of p16INK4a promoter, and accelerated MMP-2 expression. Conversely, the knock-down of hYSK1 enhanced the p16INK4a promoter activity and protein expression, and diminished MMP-2 transcription and protein levels in hypoxic conditions as compared to control. Taken together, hYSK1 blocks the p21WAF1/Cip1 functions by direct interaction and inhibits the p16INK4a expression and induces MMP-2 expression by its regulations of SP-1 transcriptional activity under the hypoxia conditions.

Carnosic acid inhibits STAT3 signaling and induces apoptosis through generation of ROS in human colon cancer HCT116 cells.

Carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., has been reported to possess anticancer activity. However, the molecular mechanisms underlying the anticancer effects of CA remain poorly understood. Our study revealed that CA treatment significantly reduced the viability of human colon cancer HCT116, SW480, and HT-29 cells. Treatment with CA induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, activation of caspase-9, and -3, and the cleavage of PARP in HCT116 cells. CA inhibited the constitutive phosphorylation, the DNA binding and the reporter gene activity of STAT3 in HCT116 cells by blocking the phosphorylation of upstream JAK2 and Src kinases. Moreover, CA attenuated the expression of STAT3 target gene products, such as survivin, cyclin D1, D2, and D3. In STAT3-overexpressed HCT116 cells, CA inhibited cell viability and the expression of cyclin D1 and survivin. Furthermore, CA treatment induced the generation of ROS in these colon cancer cells. Pretreatment of cells with ROS scavenger N-acetyl cysteine abrogated the inhibitory effect of CA on the JAK2-STAT3/Src-STAT3 signaling and rescued cells from CA-induced apoptosis by blocking the induction of p53 and the cleavage of caspase-3 and PARP in HCT116 cells. However, L-buthionine-sulfoximine, a pharmacological inhibitor of GSH synthesis, increased CA-induced ROS production, thereby potentiating apoptotic effect of CA. In conclusion, our study provides the first report that CA induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases, and inhibition of STAT3 signaling pathway. © 2015 Wiley Periodicals, Inc.

Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17.

Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17.

Isoliquiritigenin induces apoptosis and inhibits xenograft tumor growth of human lung cancer cells by targeting both wild type and L858R/T790M mutant EGFR.

Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.

Resolvin D1 stimulates efferocytosis through p50/p50-mediated suppression of tumor necrosis factor-α expression.

Phagocytosis of apoptotic neutrophils, termed efferocytosis, is essential for the resolution of inflammation as it prevents the tissues surrounding the inflamed site from being exposed to the toxic contents of lytic cells. Resolvin D1 (RvD1), endogenously generated from docosahexaenoic acid during resolution of inflammation, is known to stimulate efferocytosis. However, the molecular mechanism underlying RvD1-mediated enhancement of efferocytosis remains largely unresolved. In the present study, murine macrophage-like RAW264.7 cells treated with lipopolysaccharide (LPS) exhibited markedly reduced efferocytic activity, but this was restored by co-incubation with RvD1. RvD1-induced restoration of the efferocytic activity appears to be mediated by downregulation of LPS-induced TNF-α expression. The inhibitory effect of RvD1 on LPS-induced TNF-α expression was associated with enhanced nuclear localization of p50/p50 homodimer and concomitant reduction of p65/p50 heterodimer accumulation in the nucleus. RvD1 triggered phosphorylation and proteasomal degradation of nuclear factor κB1 (NF-κB1) p105 to generate p50, which was subsequently translocated to the nucleus as a p50/p50 homodimer. Knockdown of NF-κB p50 abolished the ability of RvD1 to suppress TNF-α expression and also to restore efferocytosis, suggesting that the replacement of p65/p50 with p50/p50 homodimer in the nucleus is crucial for RvD1-mediated stimulation of efferocytosis. In a murine peritonitis model, intraperitoneal administration of RvD1 abolished the zymosan-A-induced TNF-α production, thereby stimulating efferocytosis. Taken together, these findings indicate that RvD1 expedites resolution of inflammation through induction of efferocytosis by p50/p50-homodimer-mediated repression of TNF-α production.

Tumor suppressor p16(INK4a) inhibits cancer cell growth by downregulating eEF1A2 through a direct interaction.

The tumor suppressor protein p16(INK4a) is a member of the INK4 family of cyclin-dependent kinase (Cdk) inhibitors, which are involved in the regulation of the eukaryotic cell cycle. However, the mechanisms underlying the anti-proliferative effects of p16(INK4a) have not been fully elucidated. Using yeast two-hybrid screening, we identified the eukaryotic elongation factor (eEF)1A2 as a novel interacting partner of p16(INK4a). eEF1A2 is thought to function as an oncogene in cancers. The p16(INK4a) protein interacted with all but the D2 (250-327 aa) domain of eEF1A2. Ectopic expression of p16(INK4a) decreased the expression of eEF1A2 and inhibited cancer cell growth. Furthermore, suppression of protein synthesis by expression of p16(INK4a) ex vivo was verified by luciferase reporter activity. Microinjection of p16(INK4a) mRNA into the cytoplasm of Xenopus embryos suppressed the luciferase mRNA translation, whereas the combination of p16(INK4a) and morpholino-eEF1A2 resulted in a further reduction in translational activity. We conclude that the interaction of p16(INK4a) with eEF1A2, and subsequent downregulation of the expression and function of eEF1A2 is a novel mechanism explaining the anti-proliferative effects of p16(INK4a).

[6]-shogaol inhibits growth and induces apoptosis of non-small cell lung cancer cells by directly regulating Akt1/2.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which cause aberrant activation of diverse cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target-oriented virtual screening revealed that the ginger components, including [6]-shogaol, [6]-paradol and [6]-gingerol, seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, [6]-shogaol showed the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, [6]-shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. In NCI-H1650 lung cancer cells, [6]-shogaol reduced the constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and decreased the expression of cyclin D1/3, which are target proteins in the Akt signaling pathway. The induction of apoptosis in NCI-H1650 cells by [6]-shogaol corresponded with the cleavage of caspase-3 and caspase-7. Moreover, intraperitoneal administration of [6]-shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude mice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1 and phosphorylated Akt and STAT3 and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly indicates that [6]-shogaol can be exploited for the prevention and/or treatment of NSCLC.

Rutin inhibits UVB radiation-induced expression of COX-2 and iNOS in hairless mouse skin: p38 MAP kinase and JNK as potential targets.

Exposure to ultraviolet B (UVB) radiation, a complete environmental carcinogen, induces oxidative and inflammatory skin damage, thereby increasing the risk of skin carcinogenesis. The antioxidant and anti-inflammatory activities of a wide variety of plant polyphenols have been reported. Rutin (3-rhamnosyl-glucosylquercetin), a polyphenol present in many edible plants, possesses diverse pharmacological properties including antioxidant, anti-inflammatory, antimutagenic and anticancer activities. The present study was aimed to investigate the effects of rutin on UVB-induced inflammation in mouse skin in vivo. Topical application of rutin onto the dorsal skin of female HR-1 hairless mice 30 min prior to UVB irradiation diminished epidermal hyperplasia and the levels of proteins modified by 4-hydroxynonenal, which is a biochemical hallmark of lipid peroxidation. Topical application of rutin also significantly inhibited UVB-induced expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), two representative inflammatory enzymes, in hairless mouse skin. Rutin inhibited the DNA binding of activator protein-1 (AP-1) and phosphorylation of signal transducer and activator of transcription-3 (STAT3) in mouse skin exposed to UVB. Moreover, rutin attenuated UVB-induced phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun-N-terminal kinase (JNK). Pharmacological inhibition of p38 MAP kinase and JNK decreased UVB-induced expression of COX-2 in mouse skin. Taken together, these findings suggest that rutin exerts anti-inflammatory effects in UVB-irradiated mouse skin by inhibiting expression of COX-2 and iNOS, which is attributable to its suppression of p38 MAP kinase and JNK signaling responsible for AP-1 activation.

Piceatannol inhibits phorbol ester-induced expression of COX-2 and iNOS in HR-1 hairless mouse skin by blocking the activation of NF-κB and AP-1.

OBJECTIVES: The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory activity of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) in mouse skin in vivo. METHODS: Female HR-1 hairless mice were topically treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) with or without piceatannol pretreatment. Epidermal protein expression was assessed by Western blot analysis. The cyclooxygenase-2 (COX-2) expression was detected by immunohistochemistry. The DNA binding of nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) was examined by the electrophoretic mobility gel shift assay. The catalytic activity of IκBα kinase-ß (IKKß) was measured by in vitro kinase assay. RESULTS: Pretreatment with piceatannol attenuated TPA-induced expression of COX-2 and inducible nitric oxide synthase (iNOS) in mouse skin. Piceatannol diminished nuclear translocation and the DNA binding of NF-κB through the blockade of phosphorylation and subsequent degradation of IκBα. Piceatannol attenuated the catalytic activity of IKKß and inhibited the phosphorylation of mitogen-activated protein (MAP) kinases in TPA-treated mouse skin. In addition, piceatannol decreased TPA-induced expression of c-Fos and the DNA binding of AP-1. CONCLUSION: Piceatannol inhibits TPA-induced COX-2 and iNOS expression by blocking the activation of NF-κB and AP-1 via suppression of the IKKß activity and phosphorylation of MAP kinases, which provides a mechanistic basis of its anti-inflammatory effects in mouse skin.

Targeting FGFR1 by ß,ß-dimethylacrylalkannin suppresses the proliferation of colorectal cancer in cellular and xenograft models.

BACKGROUND: Colorectal cancer (CRC) continues to be a major global health challenge, ranking as a top cause of cancer-related mortality. Alarmingly, the five-year survival rate for CRC patients hovers around a mere 10-30 %. The disruption of fibroblast growth factor receptor (FGFRs) signaling pathways is significantly implicated in the onset and advancement of CRC, presenting a promising target for therapeutic intervention in CRC management. Further investigation is essential to comprehensively elucidate FGFR1's function in CRC and to create potent therapies that specifically target FGFR1. PURPOSE: This study aims to demonstrate the oncogenic role of FGFR1 in colorectal cancer and to explore the potential of ß,ß-dimethylacrylalkannin (ß,ß-DMAA) as a therapeutic option to inhibit FGFR1. METHODS: In this research, we employed a comprehensive suite of techniques including tissue array, kinase profiling, computational docking, knockdown assay to predict and explore the inhibitor of FGFR1. Furthermore, we utilized kinase assay, pull-down, cell proliferation tests, and Patient derived xenograft (PDX) mouse models to further investigate a novel FGFR1 inhibitor and its impact on the growth of CRC. RESULTS: In our research, we discovered that FGFR1 protein is markedly upregulated in colorectal cancer tissues, suggesting a significant role in regulating cellular proliferation, particularly in patients with colorectal cancer. Furthermore, we conducted a computational docking, kinase profiling analysis, simulation and identified that ß,ß-DMAA could directly bind with FGFR1 within ATP binding pocket domain. Cell-based assays confirmed that ß,ß-DMAA effectively inhibited the proliferation of colon cancer cells and also triggered cell cycle arrest, apoptosis, and altered FGFR1-mediated signaling pathways. Moreover, ß,ß-DMAA effectively attenuated the development of PDX tumors in mice that were FGFR1-positive, with no notable toxicity observed. In summary, our study highlights the pivotal role of FGFR1 in colorectal cancer, suggesting that inhibiting FGFR1 activity could be a promising strategy for therapeutic intervention. We present strong evidence that targeting FGFR1 with ß,ß-DMAA is a viable approach for the management of colorectal cancer. Given its low toxicity and high efficacy, ß,ß-DMAA, as an FGFR1 inhibitor, warrants further investigation in clinical settings for the treatment of FGFR1-positive tumors.

Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo.

Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.

Repurposing pentamidine for cancer immunotherapy by targeting the PD1/PD-L1 immune checkpoint.

Immunotherapy has emerged as an effective therapeutic approach to several cancer types. The reinvigoration of tumor-infiltrating lymphocyte-mediated immune responses via the blockade of immune checkpoint markers, such as program cell death-1 (PD-1) or its cognate ligand PD-L1, has been the basis for developing clinically effective anticancer therapies. We identified pentamidine, an FDA-approved antimicrobial agent, as a small-molecule antagonist of PD-L1. Pentamidine enhanced T-cell-mediated cytotoxicity against various cancer cells in vitro by increasing the secretion of IFN-γ, TNF-α, perforin, and granzyme B in the culture medium. Pentamidine promoted T-cell activation by blocking the PD-1/PD-L1 interaction. In vivo administration of pentamidine attenuated the tumor growth and prolonged the survival of tumor-bearing mice in PD-L1 humanized murine tumor cell allograft models. Histological analysis of tumor tissues showed an increased number of tumor-infiltrating lymphocytes in tissues derived from pentamidine-treated mice. In summary, our study suggests that pentamidine holds the potential to be repurposed as a novel PD-L1 antagonist that may overcome the limitations of monoclonal antibody therapy and can emerge as a small molecule cancer immunotherapy.

Redox modulation of p53: mechanisms and functional significance.

The tumor suppressor protein p53 functions as a stress-responsive transcription factor. In response to oxidative, nitrosative, and electrophilic insults, p53 undergoes post-translational modifications, such as oxidation and covalent modification of cysteines, nitration of tyrosines, acetylation of lysines, phosphorylation of serine/threonine residues, etc. Because p53 plays a vital role in the transcriptional regulation of genes encoding proteins involved in a wide spectrum of biochemical processes including DNA repair, cell-cycle regulation, and programmed cell death, the redox-modification of p53 appears to be an important determinant of cell fate. This review highlights the redox regulation of p53 and its consequences on cellular function.

Ultraviolet B radiation activates NF-κB and induces iNOS expression in HR-1 hairless mouse skin: role of IκB kinase-ß.

Exposure to ultraviolet B (UVB) radiation is known to cause inflammatory tissue damage and skin cancer. One of the molecular links between inflammation and cancer is the eukaryotic transcription factor nuclear factor-kappaB (NF-κB), which is known to regulate expression of various pro-inflammatory genes including inducible nitric oxide synthase (iNOS). The present study was aimed at elucidating the molecular mechanisms underlying UVB-induced NF-κB activation and iNOS expression in hairless mouse skin. Irradiation of male HR-1 hairless mouse skin with UVB (5 kJ/m(2) ) resulted in increased degradation of IκBα, nuclear translocation of p65 and p50, and the DNA binding of NF-κB. Exposure to UVB radiation induced the phosphorylation and the catalytic activity of an upstream kinase IκB kinase-ß (IKKß). Pharmacological inhibition of IKKß attenuated UVB-induced NF-κB activation in mouse skin. Irradiation of mouse skin with UVB also increased phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Pretreatment with SC-514, a specific inhibitor of IKKß, attenuated UVB-induced phosphorylation of ERK and p38 MAP kinase. A kinetic study showed that UVB significantly increased the expression of iNOS in mouse skin at 6 h postirradiation, which was abrogated by pretreatment with SC-514. In conclusion, the upstream kinase IKKß is involved in UVB-induced activation of MAP kinases and NF-κB, and expression of iNOS in mouse skin.

Nrf2-Keap1 signaling as a potential target for chemoprevention of inflammation-associated carcinogenesis.

Persistent inflammatory tissue damage is causally associated with each stage of carcinogenesis. Inflammation-induced generation of reactive oxygen species, reactive nitrogen species, and other reactive species not only cause DNA damage and subsequently mutations, but also stimulate proliferation of initiated cells and even metastasis and angiogenesis. Induction of cellular cytoprotective enzymes (e.g., heme oxygenase-1, NAD(P)H:quinone oxidoreductase, superoxide dismutase, glutathione-S-transferase, etc.) has been shown to mitigate aforementioned events implicated in inflammation-induced carcinogenesis. A unique feature of genes encoding these cytoprotective enzymes is the presence of a cis-acting element, known as antioxidant response element (ARE) or electrophile response element (EpRE), in their promoter region. A stress-responsive transcription factor, nuclear factor erythroid-2-related factor-2 (Nrf2), initially recognized as a key transcriptional regulator of various cytoprotective enzymes, is known to play a pivotal role in cellular defense against inflammatory injuries. Activation of Nrf2 involves its release from the cytosolic repressor Kelch-like ECH-associated protein-1 (Keap1) and subsequent stabilization and nuclear localization for ARE/EpRE binding. Genetic or pharmacologic inactivation of Nrf2 has been shown to abolish cytoprotective capability and to aggravate experimentally induced inflammatory injuries. Thus, Nrf2-mediated cytoprotective gene induction is an effective strategy for the chemoprevention of inflammation-associated carcinogenesis.

Acetylshikonin suppressed growth of colorectal tumour tissue and cells by inhibiting the intracellular kinase, T-lymphokine-activated killer cell-originated protein kinase.

BACKGROUND AND PURPOSE: Overexpression or aberrant activation of the T-lymphokine-activated killer cell-originated protein kinase (TOPK) promotes gene expression and growth of solid tumours, implying that TOPK would be a rational target in developing novel anticancer drugs. Acetylshikonin, a diterpenoid compound isolated from Lithospermum erythrorhizon root, exerts a range of biological activities. Here we have investigated whether acetylshikonin, by acting as an inhibitor of TOPK, can attenuate the proliferation of colorectal cancer cells and the growth of patient-derived tumours, in vitro and in vivo. EXPERIMENTAL APPROACH: Targets of acetylshikonin, were identified using kinase profiling analysis, kinetic/binding assay, and computational docking analysis and knock-down techniques. Effects of acetylshikonin on colorectal cancer growth and the underlying mechanisms were evaluated in cell proliferation assays, propidium iodide and annexin-V staining analyses and western blots. Patient-derived tumour xenografts in mice (PDX) and immunohistochemistry were used to assess anti-tumour effects of acetylshikonin. KEY RESULTS: Acetylshikonin directly inhibited TOPK activity, interacting with the ATP-binding pocket of TOPK. Acetylshikonin suppressed cell proliferation by inducing cell cycle arrest at the G1 phase, stimulated apoptosis, and increased the expression of apoptotic biomarkers in colorectal cancer cell lines. Mechanistically, acetylshikonin diminished the phosphorylation and activation of TOPK signalling. Furthermore, acetylshikonin decreased the volume of PDX tumours and reduced the expression of TOPK signalling pathway in xenograft tumours. CONCLUSION AND IMPLICATIONS: Acetylshikonin suppressed growth of colorectal cancer cells by attenuating TOPK signalling. Targeted inhibition of TOPK by acetylshikonin might be a promising new approach to the treatment of colorectal cancer.

Multidirectional tumor-suppressive activity of AIMP2/p38 and the enhanced susceptibility of AIMP2 heterozygous mice to carcinogenesis.

Aminoacyl-transfer ribonucleic acid (tRNA) synthetases-interacting multifunctional protein (AIMP) 2 is a factor associated with the macromolecular protein synthesis machinery consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors. However, it was shown to work as a multifaceted regulator through the versatile interactions with diverse signal mediators. For instance, it can mediate pro-apoptotic response to DNA damage and tumor necrosis factor-alpha (TNF-alpha) stimulus and growth-arresting signal by transforming growth factor (TGF)-beta. Considering that these pathways are critically implicated in the control of tumorigenesis, AIMP2 is expected to work as a potent tumor suppressor with broad coverage against different cancer types. Here we investigated whether AIMP2 would give gene dosage effect on its pro-apoptotic and anti-proliferative activities using the wild-type, hetero- and homozygous AIMP2 cells and whether AIMP2 would be critical in preventing tumorigenesis using different in vivo tumor models. Both the apoptotic responses to DNA damage and TNF-alpha and sensitivity to growth arresting TGF-beta signal were reduced in AIMP2 hetero- and homozygous cells compared with the wild-type cells in dose-dependent manner. In all the in vivo carcinogenesis experiments, reduction of AIMP2 level in heterozygous AIMP2 mice provided higher susceptibility to tumor formation. Thus, this work proves the functional significance of AIMP2 in determination of cell proliferation and death, and as a haploinsufficient tumor suppressor.

Resveratrol and piceatannol inhibit iNOS expression and NF-kappaB activation in dextran sulfate sodium-induced mouse colitis.

Inflammatory tissue injury has been implicated in tumor promotion and progression. 3,5,4'-trihydroxy-trans-stilbene (resveratrol) and 3,4,3', 5'-tetrahydroxy-trans-stilbene (piceatannol), 2 structurally related plant polyphenols, have been reported to possess antioxidant, anti-inflammatory, and chemopreventive properties. This study was aimed at investigating the possible protective effects of resveratrol and piceatannol against dextran sulfate sodium (DSS)-induced inflammation in mouse colonic mucosa. Administration of DSS (2.5%) in drinking water for 7 days to male ICR mice resulted in colitis and elevated expression of inducible nitric oxide synthase (iNOS) and activation of nuclear factor-kappa B (NF-kappaB), a major transcription factor known to upregulate proinflammatory gene expression. Phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-3 (STAT3) was also enhanced after DSS treatment. Oral administration of resveratrol or piceatannol (10 mg/kg body weight each) for 7 constitutive days attenuated the DSS-induced inflammatory injury, upregulation of iNOS expression, and activation of NF-kappaB, STAT3, and ERK.

Role of Nrf2-mediated heme oxygenase-1 upregulation in adaptive survival response to nitrosative stress.

Nitrosative stress caused by reactive nitrogen species such as nitric oxide and peroxynitrite overproduced during inflammation leads to cell death and has been implicated in the pathogenesis of many human ailments. However, relatively mild nitrosative stress may fortify cellular defense capacities, rendering cells tolerant or adaptive to ongoing and subsequent cytotoxic challenges, a phenomenon known as 'preconditioning' or 'hormesis'. One of the key components of cellular stress response is heme oxygenase-1 (HO-1), the rate limiting enzyme in the process of degrading potentially toxic free heme into biliverdin, free iron and carbon monoxide. HO-1 is upregulated by a wide array of stimuli and has antioxidant, anti-inflammatory and other cytoprotective functions. This review is intended to provide readers with a welldocumented account of the research done in the area of cellular adaptive survival response against nitrosative stress with special focus on the role of HO-1 upregulation, especially through activation of the transcription factor, Nrf2.

Targeting ROCK/LIMK/cofilin signaling pathway in cancer.

Rho-associated coiled-coil-containing protein kinase (ROCK)/Lin11, Isl-1 and Mec-3 kinase (LIMK)/cofilin-signaling cascades are stimulated by receptor tyrosine kinases, G protein-coupled receptors, integrins and its ligands, growth factors, hormones, fibronectin, collagen, and laminin. Activated signaling cascades can cause transit from normal cells to cancer cells by modulating actin/filament dynamics. In various cancers including breast, prostate, and colorectal cancers, high expression or activity of each cascade protein is significantly associated with poor survival rate of patients as well as aggressive metastasis. Silencing ROCK, LIMK, or cofilin can abrogate their activities and inhibit cancer cell growth, invasion, and metastasis. Therefore ROCK/LIMK/cofilin signaling proteins might be good candidates to develop cancer prevention strategies or therapeutics. Currently, netarsudil, a ROCK inhibitor, is only used in clinical patients for glaucoma or ocular hypertension, but not for cancer. In this review, we will discuss comprehensive ROCK/LIMK/cofilin signaling pathway in cancers and its inhibitors for developing cancer therapy.