Application of Molecular Dynamics Simulations to the Design of Nucleotide Inhibitors Binding to Norovirus Polymerase.

The RNA-dependent RNA polymerase (RdRp) of norovirus is an attractive target of antiviral agents aimed at providing protection against norovirus-associated gastroenteritis. Here, we perform molecular dynamics simulations of the crystal structure of norovirus RdRp in complex with several known binders, as well as free-energy simulations by free-energy perturbation (FEP) to determine binding free energies of these molecules relative to the natural nucleotide substrates. We determine experimental EC50 values and nucleotide incorporation efficiencies for several of these compounds. Moreover, we investigate the mechanism of inhibition of some of these ligands. Using FEP, we screened a virtual nucleotide library with 121 elements for binding to the polymerase and successfully identified two novel chain terminators.

Effect of Different Adjuvants on the Longevity and Strength of Humoral and Cellular Immune Responses to the HCV Envelope Glycoproteins.

Infection by Hepatitis C virus (HCV) can lead to liver cirrhosis/hepatocellular carcinoma and remains a major cause of serious disease morbidity and mortality worldwide. However, current treatment regimens remain inaccessible to most patients, particularly in developing countries, and, therefore, the development of a novel vaccine capable of protecting subjects from chronic infection by HCV could greatly reduce the rates of HCV infection, subsequent liver pathogenesis, and in some cases death. Herein, we evaluated two different semi-synthetic archaeosome formulations as an adjuvant to the E1/E2 HCV envelope protein in a murine model and compared antigen-specific humoral (levels of anti-E1/E2 IgG and HCV pseudoparticle neutralization) and cellular responses (numbers of antigen-specific cytokine-producing T cells) to those generated with adjuvant formulations composed of mimetics of commercial adjuvants including a squalene oil-in-water emulsion, aluminum hydroxide/monophosphoryl lipid A (MPLA) and liposome/MPLA/QS-21. In addition, we measured the longevity of these responses, tracking humoral, and cellular responses up to 6 months following vaccination. Overall, we show that the strength and longevity of anti-HCV responses can be influenced by adjuvant selection. In particular, a simple admixed sulfated S-lactosylarchaeol (SLA) archaeosome formulation generated strong levels of HCV neutralizing antibodies and polyfunctional antigen-specific CD4 T cells producing multiple cytokines such as IFN-γ, TNF-α, and IL-2. While liposome/MPLA/QS-21 as adjuvant generated superior cellular responses, the SLA E1/E2 admixed formulation was superior or equivalent to the other tested formulations in all immune parameters tested.

Titanium dioxide nanoparticles induce COX-2 expression through ROS generation in human periodontal ligament cells.

Titanium dioxide nanoparticles (TiO2-NPs) are used to improve the aesthetic of toothpaste. While TiO2-NPs have been used safely in toothpaste products for a long time, there haven't been studies to determine whether absorption of TiO2-NPs by the mucous membranes in the mouth induces pathogenic conditions. Here, we assessed whether TiO2-NPs induce cyclooxygenase-2 (COX-2) and investigated the molecular mechanisms underlying the pro-inflammatory effect of TiO2-NPs on human periodontal ligament (PDL) cells. Treatment of PDL cells with TiO2-NPs led to induction of both COX-2 mRNA and protein expression. TiO2-NPs stimulated the nuclear translocation of nuclear factor-kappaB (NF-κB) as well as its DNA binding by inducing phosphorylation and subsequent degradation of the inhibitory protein IκBα in PDL cells. TiO2-NPs treatment resulted in rapid activation of extracellular signal-regulated kinase (ERK)1/2 and Akt, which could be upstream of NF-κB. Treatment of PDL cells with both the MEK1/2 inhibitor U0126 and the PI3K inhibitor LY294002 strongly attenuated TiO2-NPs-induced activation of NF-κB, and also the expression of COX-2. PDL cells treated with TiO2-NPs exhibited increased accumulation of intracellular reactive oxygen species (ROS). Pretreatment of cells with ROS scavenger N-acetyl cysteine (NAC) abrogated the stimulatory effect of TiO2-NPs on p65, p50, and COX-2 expression. In conclusion, ROS, concomitantly overproduced by TiO2-NPs, induce COX-2 expression through activation of NF-κB signaling, which may contribute to the inflammatory effect of PDL cells.

Silicon dioxide nanoparticles induce COX-2 expression through activation of STAT3 signaling pathway in HaCaT cells.

Silicon dioxide nanoparticles (SiO2-NPs) are widely used in biomedicines and consumer products, such as sunscreens and cosmetics. However, SiO2-NPs can cause adverse effects on human health, depending on the size and concentration of nanoparticles. The present study was aimed at investigating the molecular mechanism underlying SiO2-NPs-induced inflammation in human keratinocyte (HaCaT) cells. Incubation of HaCaT cells with SiO2-NPs induced the expression of cyclooxygenase-2 (COX-2) mRNA and protein. Treatment of cells with SiO2-NPs also induced the phosphorylation, DNA binding and the reporter gene activity of signal transducer and activator of transcription 3 (STAT3). Transfection of cells with STAT3 siRNA abrogated SiO2-NPs-induced COX-2 expression. Moreover, SiO2-NPs enhanced the phosphorylation of Janus kinase2 (JAK2), Src and Akt. Pharmacological inhibition of either JAK2, Src or Akt abrogated SiO2-NPs-induced STAT3 transcriptional activity and the expression of COX-2. Treatment with LY294002 also attenuated SiO2-NPs-induced Src phosphorylation, while, JAK2 phosphorylation was not changed. In addition, SiO2-NPs generated reactive oxygen species (ROS) and treatment of N-acetyl cysteine (NAC) attenuated the phosphorylation of JAK2, Src, Akt and STAT3, as well as the expression of COX-2 in SiO2-NPs-treated HaCaT cells. Taken together, our study provides the first report that SiO2-NPs induce COX-2 expression in HaCaT cells by activating the STAT3 signaling through ROS-mediated phosphorylation of upstream kinases, Akt/Src and JAK2.

Role of the E2 Hypervariable Region (HVR1) in the Immunogenicity of a Recombinant Hepatitis C Virus Vaccine.

Current evidence supports a protective role for virus-neutralizing antibodies in immunity against hepatitis C virus (HCV) infection. Many cross-neutralizing monoclonal antibodies have been identified. These antibodies have been shown to provide protection or to clear infection in animal models. Previous clinical trials have shown that a gpE1/gpE2 vaccine can induce antibodies that neutralize the in vitro infectivity of all the major cell culture-derived HCV (HCVcc) genotypes around the world. However, cross-neutralization appeared to favor certain genotypes, with significant but lower neutralization against others. HCV may employ epitope masking to avoid antibody-mediated neutralization. Hypervariable region 1 (HVR1) at the amino terminus of glycoprotein E2 has been shown to restrict access to many neutralizing antibodies. Consistent with this, other groups have reported that recombinant viruses lacking HVR1 are hypersensitive to neutralization. It has been proposed that gpE1/gpE2 lacking this domain could be a better vaccine antigen to induce broadly neutralizing antibodies. In this study, we examined the immunogenicity of recombinant gpE1/gpE2 lacking HVR1 (ΔHVR1). Our results indicate that wild-type (WT) and ΔHVR1 gpE1/gpE2 antigens induced antibodies targeting many well-characterized cross-genotype-neutralizing epitopes. However, while the WT gpE1/gpE2 vaccine can induce cross-genotype protection against various genotypes of HCVcc and/or HCV-pseudotyped virus (HCVpp), antisera from ΔHVR1 gpE1/gpE2-immunized animals exhibited either reduced homologous neutralization activity compared to that of the WT or heterologous neutralization activity similar to that of the WT. These data suggest that ΔHVR1 gpE1/gpE2 is not a superior vaccine antigen. Based on previously reported chimpanzee protection data using WT gpE1/gpE2 and our current findings, we are preparing a combination vaccine including wild-type recombinant gpE1/gpE2 for clinical testing in the future.IMPORTANCE An HCV vaccine is an unmet medical need. Current evidence suggests that neutralizing antibodies play an important role in virus clearance, along with cellular immune responses. Previous clinical data showed that gpE1/gpE2 can effectively induce cross-neutralizing antibodies, although they favor certain genotypes. HCV employs HVR1 within gpE2 to evade host immune control. It has been hypothesized that the removal of this domain would improve the production of cross-neutralizing antibodies. In this study, we compared the immunogenicities of WT and ΔHVR1 gpE1/gpE2 antigens as vaccine candidates. Our results indicate that the ΔHVR1 gpE1/gpE2 antigen confers no advantages in the neutralization of HCV compared with the WT antigen. Previously, we showed that this WT antigen remains the only vaccine candidate to protect chimpanzees from chronic infection, contains multiple cross-neutralizing epitopes, and is well tolerated and immunogenic in humans. The current data support the further clinical development of this vaccine antigen component.

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

[Corrigendum] Carnosic acid induces apoptosis through inactivation of Src/STAT3 signaling pathway in human renal carcinoma Caki cells.

After carefully checking the original data of ROS generation experiment, we found that in Fig. 3E the x-axis was incorrently labeled. ISL should be substituted with CA. The corrected version of Fig. 3E is shown below. We apologize for the error and appreciate the opportunity to correct the scientific record. All authors agree with this correction. [the original article was published in the Oncology Reports 35: 2723-2732, 2016; DOI: 10.3892/or.2016.4642].

Fraxetin Induces Heme Oxygenase-1 Expression by Activation of Akt/Nrf2 or AMP-activated Protein Kinase α/Nrf2 Pathway in HaCaT Cells.

BACKGROUND: Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. METHODS: Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2',7'-dichlorofluorescin diacetate staining. RESULTS: Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase (AMPK)α and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and AMPKα abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. CONCLUSIONS: Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or AMPKα/Nrf2 pathway in HaCaT cells.

Carnosic acid induces apoptosis through inactivation of Src/STAT3 signaling pathway in human renal carcinoma Caki cells.

Carnosic acid (CA), the major bioactive compound of Rosmarinus officinalis L., has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of CA remain poorly understood. In the present study, we investigated that CA significantly reduced the viability of human renal carcinoma Caki cells. CA-induced apoptosis was connected with the cleavage of caspase-9, -7 and -3, and that of PARP. Moreover, CA increased the expression of pro-apoptotic protein Bax and diminished the expression of anti-apoptotic protein Bcl-2 and Bcl-xL, thereby releasing cytochrome c into the cytosol. Treatment with CA in Caki cells also induced the expression of p53 and its target gene product, p27, through down-regulation of Murine double minute-2 (Mdm2). Furthermore, CA generated reactive oxygen species (ROS), and pretreatment with ROS scavenger N-acetyl cysteine (NAC) abrogated CA-induced cleavage of PARP and expression of p53. One of the key oncogenic signals is mediated through signal transducer and activator of transcription-3 (STAT3), which promotes abnormal cell proliferation. Incubation of cells with CA markedly diminished the phosphorylation of STAT3 and its upstream, Src, and reduced the expression of STAT3 responsive gene products, such as D-series of cyclins and survivin. Taken together, the present study revealed that CA induced apoptosis in Caki cells by induction of p53 and suppression of STAT3 signaling.

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.

Transducer of ERBB2.1 (TOB1) as a Tumor Suppressor: A Mechanistic Perspective.

Transducer of ERBB2.1 (TOB1) is a tumor-suppressor protein, which functions as a negative regulator of the receptor tyrosine-kinase ERBB2. As most of the other tumor suppressor proteins, TOB1 is inactivated in many human cancers. Homozygous deletion of TOB1 in mice is reported to be responsible for cancer development in the lung, liver, and lymph node, whereas the ectopic overexpression of TOB1 shows anti-proliferation, and a decrease in the migration and invasion abilities on cancer cells. Biochemical studies revealed that the anti-proliferative activity of TOB1 involves mRNA deadenylation and is associated with the reduction of both cyclin D1 and cyclin-dependent kinase (CDK) expressions and the induction of CDK inhibitors. Moreover, TOB1 interacts with an oncogenic signaling mediator, ß-catenin, and inhibits ß-catenin-regulated gene transcription. TOB1 antagonizes the v-akt murine thymoma viral oncogene (AKT) signaling and induces cancer cell apoptosis by activating BCL2-associated X (BAX) protein and inhibiting the BCL-2 and BCL-XL expressions. The tumor-specific overexpression of TOB1 results in the activation of other tumor suppressor proteins, such as mothers against decapentaplegic homolog 4 (SMAD4) and phosphatase and tensin homolog-10 (PTEN), and blocks tumor progression. TOB1-overexpressing cancer cells have limited potential of growing as xenograft tumors in nude mice upon subcutaneous implantation. This review addresses the molecular basis of TOB1 tumor suppressor function with special emphasis on its regulation of intracellular signaling pathways.

Phosphorylation of Smac by Akt promotes the caspase-3 activation during etoposide-induced apoptosis in HeLa cells.

The Akt, family of serine/threonine protein kinases functions as key regulators of multiple aspects of cell behavior, such as survival, proliferation, migration, and carcinogenesis. Notably, Akt exerts its anti-apoptotic effects through the phosphorylation of numerous substrates related with cell cycle, genome stability, and cancer development. In this report, nevertheless, we focused our view on the novel role of Akt which involves in a pro-apoptotic action by phosphorylating second mitochondria derived activator of caspases (Smac) protein during etoposide-induced apoptotic processes. Our data reveals that Akt could bind to and phosphorylate Smac at serine residue 67, which enhances the ability of Smac to interact with the cytosolic X-chromosome linked IAP (XIAP) protein. The cellular interaction of wild-type Smac with XIAP was enhanced with similar activation kinetics of Akt activity, while this interaction was markedly attenuated in cells expressing the phosphorylation-defective mutant S67A-Smac during etoposide-induced apoptosis. Moreover, we provide the evidence indicating that the phosphorylation of Smac at ser-67 markedly upregulates the caspase-3 activity by promoting the interaction of Smac with XIAP. Taken together, we propose that the phosphorylation of Smac by Akt might be a novel mechanism that involves in amplification of caspase cascade pathway during etoposide-induced apoptosis in HeLa cells.

Carnosol: a phenolic diterpene with cancer chemopreventive potential.

Cancer is an unbeaten health challenge for the humankind. After striving for decades to find a cancer cure, attention has now been shifted to reduce the morbidity and mortality from cancer by halting the course of tumor development. Numerous bioactive phytochemicals, especially those present in edible and non-edible plant species, have been reported to reduce the risk of many cancers. Multiple lines of evidence suggest that carnosol, a phenolic diterpene present in rosemary (Rosmarinus officinalis L.), holds the promise of preventing certain types of cancer. A remarkable progress has been made in delineating the biochemical mechanisms underlying the chemopreventive effects of carnosol. Results from in vitro cell culture studies as well as animal model experiments have revealed that carnosol inhibits experimentally induced carcinogenesis and exhibits potent anti-oxidative, anti-inflammatory, antiproliferative and apoptosis inducing properties. Moreover, carnosol enhances the sensitivity of chemoresistant cancer cells to chemotherapeutic agents. The purpose of this review is to shed light on the detailed mechanistic aspects of cancer chemoprevention with carnosol.

Methanol extract of Flacourtia indica aerial parts induces apoptosis via generation of ROS and activation of caspases in human colon cancer HCT116 cells.

Different plant parts of Flacourtia indica have long been used in Ayurvedic medicine. Previous studies have demonstrated that the methanolic extract of F. indica possess anti-inflammatory properties. The present study was aimed at investigating the anticancer effects of methanol extract of Flacourtia indica (FIM) aerial parts in human colon cancer (HCT116) cells. Treatment of cells with FIM at a concentration of 500 µg/ml for 24 hours significantly reduced cell viability and induced apoptosis, which was associated with the increased cytoplasmic expression of cytochrome c, activation of caspase-3, and the cleavage of poly-(ADP-ribose) polymerase. Incubation with FIM also inhibited the levels of Bcl-2, Bcl-xl and survivin, which are the markers of cell proliferation, whereas the expression of Bax remained unchanged. Treatment with FIM led to the generation of reactive oxygen species (ROS) in a concentration-dependent manner. Pharmacological inhibition of ROS generation by pretreatment of cells with N-acetyl cysteine abrogated FIM-induced apoptosis in HCT116 cells. Thus, these results demonstrate that FIM has anti-proliferative and pro-apoptotic effects in HCT116 cells and the effects are, at least in part, due to the ROS dependent activation of caspases.

SMAD4 suppresses AURKA-induced metastatic phenotypes via degradation of AURKA in a TGFß-independent manner.

UNLABELLED: SMAD4 has been suggested to inhibit the activity of the WNT/ß-catenin signaling pathway in cancer. However, the mechanism by which SMAD4 antagonizes WNT/ß-catenin signaling in cancer remains largely unknown. Aurora A kinase (AURKA), which is frequently overexpressed in cancer, increases the transcriptional activity of ß-catenin/T-cell factor (TCF) complex by stabilizing ß-catenin through the inhibition of GSK-3ß. Here, SMAD4 modulated AURKA in a TGFß-independent manner. Overexpression of SMAD4 significantly suppressed AURKA function, including colony formation, migration, and invasion of cell lines. In addition, SMAD4 bound to AURKA induced degradation of AURKA by the proteasome. A luciferase activity assay revealed that the transcriptional activity of the ß-catenin/TCF complex was elevated by AURKA, but decreased by SMAD4 overexpression. Moreover, target gene analysis showed that SMAD4 abrogated the AURKA-mediated increase of ß-catenin target genes. However, this inhibitory effect of SMAD4 was abolished by overexpression of AURKA or silencing of AURKA in SMAD4-overexpressed cells. Meanwhile, the SMAD4-mediated repression of AURKA and ß-catenin was independent of TGFß signaling because blockage of TGFßR1 or restoration of TGFß signaling did not prevent suppression of AURKA and ß-catenin signaling by SMAD4. These results indicate that the tumor-suppressive function of SMAD4 is mediated by downregulation of ß-catenin transcriptional activity via AURKA degradation in a TGFß-independent manner. IMPLICATIONS: SMAD4 interacts with AURKA and antagonizes its tumor-promoting potential, thus demonstrating a novel mechanism of tumor suppression.

Thymoquinone induces apoptosis in human colon cancer HCT116 cells through inactivation of STAT3 by blocking JAK2- and Src­mediated phosphorylation of EGF receptor tyrosine kinase.

Thymoquinone (TQ), a compound isolated from black seed oil (Nigella sativa), has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of TQ remain poorly understood. In the present study, we found that TQ significantly reduced the viability of human colon cancer HCT116 cells in a concentration- and time-dependent manner. Treatment of cells with TQ induced apoptosis, which was associated with the upregulation of Bax and inhibition of Bcl-2 and Bcl-xl expression. TQ also activated caspase-9,-7, and -3, and induced the cleavage of poly-(ADP-ribose) polymerase (PARP). Pretreatment with a pan-caspase inhibitor, z-VAD-fmk, abrogated TQ-induced apoptosis by blocking the cleavage of caspase-3 and PARP. Treatment of cells with TQ also diminished the constitutive phosphorylation, nuclear localization and the reporter gene activity of signal transducer and activator of transcription-3 (STAT3). TQ attenuated the expression of STAT3 target gene products, such as survivin, c-Myc, and cyclin-D1, -D2, and enhanced the expression of cell cycle inhibitory proteins p27 and p21. Treatment with TQ attenuated the phosphorylation of upstream kinases, such as Janus-activated kinase-2 (JAK2), Src kinase and epidermal growth factor receptor (EGFR) tyrosine kinase. Pharmacological inhibition of JAK2 and Src blunted tyrosine phosphorylation of EGFR and STAT3, while treatment with an EGFR tyrosine kinase inhibitor gefitinib inhibited phosphorylation of STAT3 without affecting that of JAK2 and Src in HCT116 cells. Collectively, our study revealed that TQ induced apoptosis in HCT116 cells by blocking STAT3 signaling via inhibition of JAK2- and Src-mediated phosphorylation of EGFR tyrosine kinase.

Targeting Nrf2-Keap1 signaling for chemoprevention of skin carcinogenesis with bioactive phytochemicals.

The incidence of skin cancer is increasing worldwide. Over the last several decades, attention has been focused on understanding the molecular basis of skin carcinogenesis and identifying substances for use in chemoprevention of skin cancer. Reactive oxygen species generated by chemical carcinogens or UV irradiation play a key role in skin tumorigenesis. Multiple lines of evidence suggest that cellular antioxidant and/or phase-2 detoxification enzymes, collectively known as cytoprotective proteins, can protect against skin carcinogenesis. A redox sensitive transcription factor, nuclear factor-erythroid related factor-2 (Nrf2), is a master regulator of transcriptional activation of genes encoding cytoprotective proteins. Many chemopreventive phytochemicals are known to activate Nrf2 either by oxidative or covalent modification of its cytosolic repressor Kelch-like ECH-associated protein (Keap1) or by phosphorylation of Nrf2. Upon activation, Nrf2 translocates to the nucleus and binds to the antioxidant response element (ARE) located in the promoter regions of genes encoding cytoprotective proteins. Mice genetically deficient in Nrf2 are highly susceptible to chemically induced skin tumorigenesis and are less responsive to the cytoprotective effects of some chemopreventive phytochemicals. This article highlights the Nrf2-ARE signaling as a prime target for chemoprevention of skin cancer with some phytochemicals.

MET is a potential target for use in combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.

MET, a cell surface receptor for hepatocyte growth factor, is involved in the development of triple-negative/basal-like breast cancer (TNBC/BLBC). However, its utility as a therapeutic target in this subtype of breast cancer is poorly understood. To evaluate MET fully as a potential therapeutic target for TNBC/BLBC, we investigated the relationship between MET expression and clinical outcomes of patients with breast cancer and the functional effect of MET inhibition. Using automated immunohistochemistry (Ventana), we analyzed MET expression in 924 breast cancer patients with relevant clinicopathologic parameters. BLBC showed the strongest relationship with MET expression (57.5%, p < 0.001). High expression of MET in breast cancer resulted in poor overall survival (p = 0.001) and disease-free survival (DFS, p = 0.010). MET expression was relatively high in TNBC cell lines, and the silencing of MET via small interfering RNA reduced cell proliferation and migration. We observed reduced TNBC cell viability after treatment with the MET inhibitor PHA-665752. In the most drug-resistant cell line, MDA-MB-468, which showed elevated epidermal growth factor receptor (EGFR) expression, silencing of EGFR resulted in increased sensitivity to PHA-665752 treatment. We confirmed that PHA-665752 synergizes with the EGFR inhibitor erlotinib to decrease the viability of MDA-MB-468 cells. TNBC patients coexpressing MET and EGFR showed significantly worse DFS than that in patients expressing EGFR alone (p = 0.021). Our findings strongly suggest that MET may be a therapeutic target in TNBC and that the combined therapy targeting MET and EGFR may be beneficial for the treatment of TNBC/BLBC patients.

Carnosol induces apoptosis through generation of ROS and inactivation of STAT3 signaling in human colon cancer HCT116 cells.

Carnosol, an active constituent of rosemary, has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of carnosol remain poorly understood. In the present study, we found that carnosol significantly reduced the viability of human colon cancer (HCT116) cells in a concentration- and time-dependent manner. Treatment of cells with carnosol induced apoptosis, which was associated with activation of caspase-9 and -3 and the cleavage of poly-(ADP-ribose) polymerase (PARP). Incubation with carnosol elevated the expression of Bax and inhibited the levels of Bcl-2 and Bcl-xl. Carnosol induced expression of p53 and inhibited that of murine-double minute-2 (Mdm2). Moreover, carnosol generated reactive oxygen species (ROS), and pretreatment with N-acetyl cysteine abrogated carnosol-induced cleavage of caspase-3 and PARP. The constitutive phosphorylation, the DNA binding and reporter gene activity of signal transducer and activator of transcription-3 (STAT3) was diminished by treatment with carnosol. To further elucidate the molecular mechanisms of STAT3 inactivation, we found that carnosol attenuated the phosphorylation of Janus-activated kinase-2 (Jak2) and Src kinase. Pharmacological inhibition of Jak2 and Src inhibited STAT3 phosphorylation. Furthermore, carnosol attenuated the expression of STAT3 target gene products, such as survivin, cyclin-D1, -D2, and -D3. Taken together, our study provides the first report that carnosol induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases and inhibition of STAT3 signaling pathway.

Mechanistic perspectives on cancer chemoprevention/chemotherapeutic effects of thymoquinone.

The bioactive natural products (plant secondary metabolites) are widely known to possess therapeutic value for the prevention and treatment of various chronic diseases including cancer. Thymoquinone (2-methyl-5-isopropyl-1,4-benzoquinone; TQ), a monoterpene present in black cumin seeds, exhibits pleiotropic pharmacological activities including antioxidant, anti-inflammatory, antidiabetic and antitumor effects. TQ inhibits experimental carcinogenesis in a wide range of animal models and has been shown to arrest the growth of various cancer cells in culture as well as xenograft tumors in vivo. The mechanistic basis of anticancer effects of TQ includes the inhibition of carcinogen metabolizing enzyme activity and oxidative damage of cellular macromolecules, attenuation of inflammation, induction of cell cycle arrest and apoptosis in tumor cells, blockade of tumor angiogenesis, and suppression of migration, invasion and metastasis of cancer cells. TQ shows synergistic and/or potentiating anticancer effects when combined with clinically used chemotherapeutic agents. At the molecular level, TQ targets various components of intracellular signaling pathways, particularly a variety of upstream kinases and transcription factors, which are aberrantly activated during the course of tumorigenesis.