CHD4 Predicts Aggressiveness in PTC Patients and Promotes Cancer Stemness and EMT in PTC Cells.

Chromodomain-helicase-DNA-binding protein 4 (CHD4), a core subunit of the nucleosome remodeling and deacetylation (NuRD) complex is highly expressed in several cancers. However, its role in the pathogenesis and progression of papillary thyroid carcinoma (PTC) has not been investigated. We investigated the prognostic significance of CHD4 in a large cohort of Middle Eastern PTC patients and explored the functional role of CHD4 in regulating cancer stemness and EMT in PTC cells. CHD4 overexpression was observed in 45.3% (650/1436) of PTCs, and was associated with aggressive clinico-pathological parameters and worse outcome. Functional analysis using PTC cell lines showed that forced expression of CHD4 promoted cell proliferation, spheroid growth, migration, invasion and progression of epithelial to mesenchymal transition (EMT) in PTC cells whereas its knockdown reversed the effect. Methylation of E-cadherin was associated with loss of expression in CHD4 expressing cells, while CHD4 depletion reactivated E-cadherin expression. Most importantly, knockdown of mesenchymal transcriptional factors, Snail1 or Zeb1, attenuated the spheroid growth in CHD4 expressing PTC cells, showing a potential link between EMT activation and stemness maintenance in PTC. These findings suggest that CHD4 might be a promising therapeutic target in the treatment of patients with an aggressive subtype of PTC.

MED12 is recurrently mutated in Middle Eastern colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a common cancer and a leading cause of cancer deaths. Previous studies have identified a number of key steps in the evolution of CRC but our knowledge of driver mutations in CRC remains incomplete. Recognising the potential of studying different human populations to reveal novel insights in disease pathogenesis, we conducted genomic analysis of CRC in Saudi patients. DESIGN: In the discovery phase of the study, we conducted whole genome sequencing of tumour and corresponding germline DNA in 27 patients with CRC. In addition to known driver mutations, we identified three MED12 somatic mutations. In the replication phase, we employed a next-generation sequencing approach to capture and sequence MED12 and other candidate genes in a larger sample of 400 patients with CRC and confirmed the enrichment for recurrent MED12 mutations. RESULTS: In order to gain insight into a plausible biological mechanism for the potential role of MED12 mutations in CRC, we studied CRC cell lines that differ substantially in the expression level of MED12, and found the latter to be correlated inversely with transforming growth factor (TGF)-ß signalling and directly with apoptosis in response to chemotherapeutic agents. Importantly, these correlations were replicated when MED12 expression was experimentally manipulated. CONCLUSIONS: Our data expand the recently described role of MED12 as a tumour suppressor in other cancers to include CRC, and suggest TGF-ß signalling as a potential mediator of this effect.

Nuclear factor erythroid 2-related factor 2 enhances carcinogenesis by suppressing apoptosis and promoting autophagy in nickel-transformed cells.

Nickel-containing compounds are widely used in industry. Nickel is a known human carcinogen that primarily affects the lungs. Proposed mechanisms of nickel-induced carcinogenesis include disruption of cellular iron homeostasis, generation of reactive oxygen species (ROS), and induction of hypoxia signaling. However, the precise molecular mechanisms of nickel-induced malignant transformation and tumor development remain unclear. This study shows that the transcription factor Nrf2 is highly expressed in lung tumor tissue and in nickel-transformed human lung bronchial epithelial BEAS-2B cells (NiT cells). Additionally, constitutively high levels of Nrf2 play a critical role in apoptosis resistance in NiT cells. Basal ROS levels were extremely low in NiT cells and were correlated with elevated expression levels of both antioxidant enzymes (e.g. catalase and superoxide dismutases) and antiapoptotic proteins (e.g. Bcl-2 and Bcl-xL). These processes are tightly controlled by Nrf2. Autophagy inhibition, induced pharmacologically or genetically, enhanced Ni2+-induced apoptosis, indicating that the induction of autophagy is the cause of apoptosis resistance in NiT cells. Using similar approaches, we show that in NiT cells the inhibition of apoptosis decreases autophagy. We have shown that Stat3, which is up-regulated by Nrf2, controls autophagy induction in NiT cells. Colony formation and tumor growth were significantly attenuated by knockdown of Nrf2 or Bcl-2. Taken together, this study demonstrates that in NiT cells constitutively high Nrf2 expression inhibits apoptosis by up-regulating antioxidant enzymes and antiapoptotic proteins to increase autophagy via Stat3 signaling. These findings indicate that the Nrf2-mediated suppression of apoptosis and promotion of autophagy contribute to nickel-induced transformation and tumorigenesis.

Roles of ROS, Nrf2, and autophagy in cadmium-carcinogenesis and its prevention by sulforaphane.

Environmental and occupational exposures to cadmium increase the risk of various cancers, including lung cancer. The carcinogenic mechanism of cadmium, including its prevention remains to be investigated. Using fluorescence and electron spin resonance spin trapping, the present study shows that in immortalized lung cells (BEAS-2BR cells), exposure cadmium generated reactive oxygen species (ROS). Through ROS generation, cadmium increased the protein level of TNF-α, which activated NF-κB and its target protein COX-2, creating an inflammatory microenvironment. As measured by anchorage-independent colony formation assay, cadmium induced malignant cell transformation. Inhibition of ROS by antioxidants inhibited transformation, showing that ROS were important in the mechanism of this process. The inflammatory microenvironment created by cadmium may also contribute to the mechanism of the transformation. Using tandem fluorescence protein mCherry-GFP-LC3 construct, the present study shows that cadmium-transformed cells had a property of autophagy deficiency, resulting in accumulation of autophagosomes and increased p62. This protein upregulated Nrf2, which also upregulated p62 through positive feed-back mechanism. Constitutive Nrf2 activation increased its downstream anti-apoptotic proteins, Bcl-2 and Bcl-xl, resulting in apoptosis resistance. In untransformed BEAS-2BR cells, sulforaphane, a natural compound, increased autophagy, activated Nrf2, and decreased ROS. In cadmium-transformed BEAS-2BR cells, sulforaphane restored autophagy, decreased Nrf2, and decreased apoptosis resistance. In untransformed cells, this sulforaphane induced inducible Nrf2 to decrease ROS and possibly malignant cell transformation. In cadmium-transformed cells, it decreased constitutive Nrf2 and reduced apoptosis resistance. The dual roles of sulforaphane make this natural compound a valuable agent for prevention against cadmium-induced carcinogenesis.

Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium.

Hexavalent chromium (Cr(VI))-containing compounds are well established environmental carcinogens. Most mechanistic investigations of Cr(VI)-induced carcinogenesis focus on oxidative stress and various cellular responses, leading to malignant cell transformation or the first stage of metal-induced carcinogenesis. The development of malignantly transformed cells into tumors that require angiogenesis is the second stage. This study focuses on the second stage, in particular, the role of EGF receptor (EGFR) signaling in angiogenesis and tumorigenesis of Cr(VI)-transformed cells. Our preliminary studies have shown that EGFR is constitutively activated in Cr(VI)-transformed cells, in lung tissue from Cr(VI)-exposed animals, and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. Using in vitro and in vivo models, the present study has investigated the role of EGFR in angiogenesis of Cr(VI)-transformed cells. The results show that Cr(VI)-transformed cells are angiogenic. Hypoxia-inducible factor-1α, pro-angiogenic protein matrix metalloproteinase 1, and VEGF are all highly expressed in Cr(VI)-transformed cells, in lung tissue from animals exposed to Cr(VI), and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. p38 MAPK is also activated in Cr(VI)-transformed cells and in human lung tumor tissue. Inhibition of EGFR reduces p38 MAPK, resulting in decreased expression of hypoxia-inducible factor-1α, metalloproteinase 1, and VEGF, leading to suppressions of angiogenesis and tumorigenesis. Overall, the present study has demonstrated that EGFR plays an important role in angiogenesis and tumorigenesis of Cr(VI)-transformed cells.

Protection from Cr(VI)-induced malignant cell transformation and tumorigenesis of Cr(VI)-transformed cells by luteolin through Nrf2 signaling.

Cr(VI) is a well known environmental carcinogen, but its mechanism of action and the measures required to mitigate its effects remain to be investigated. Our previous studies showed that exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) caused malignant transformation, that these transformed cells progressed through tumorigenesis, and that luteolin, a natural compound, inhibited both of these processes. The present study investigates the underlying mechanisms by which luteolin protects cells against Cr(VI)-induced transformation and tumorigenesis. The present study shows that luteolin activates inducible Nrf2 to inhibit Cr(VI)-generated reactive oxygen species (ROS) in normal BEAS-2B cells. The decreased ROS level is likely responsible for the protective effect of luteolin against Cr(VI)-induced malignant cell transformation in normal cells. By contrast, in cells that have been transformed by Cr(VI), Nrf2 is constitutively activated, and its target proteins, heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and superoxide dismutase 1/2 (SOD1/SOD2) are all constitutively activated, and ROS levels are low. Bcl-2, an anti-apoptotic protein and target protein of Nrf2 is elevated. Cr(VI)-transformed BEAS-2B cells develop apoptosis resistance, increasing the survival of these transformed cells. Luteolin decreases interactions between Nrf2 and the antioxidant response element sites of its target anti-apoptotic and antioxidant proteins, Bcl-2, Bcl-XL, and HO-1, which results in decreased constitutive Nrf2 activation. The decreased constitutive Nrf2 activation, decrease in Nrf2 target proteins and consequent apoptosis resistance by luteolin are possible mechanisms that mediate the protective effect of luteolin in Cr(VI)-transformed cells.

XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis.

BACKGROUND: Breast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women. Despite improvement in diagnosis, the survival rate of this disease has still not improved. X-linked Inhibitor of Apoptosis (XIAP) has been shown to be over-expressed in various cancers leading to poor overall survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored. METHODS: We examined the expression of XIAP in more than 1000 Middle Eastern breast cancer cases by immunohistochemistry. Apoptosis was measured by flow cytometry. Protein expression was determined by western blotting. Finally, in vivo studies were performed on nude mice following xenografting and treatment with inhibitors. RESULTS: XIAP was found to be over-expressed in 29.5% of cases and directly associated with clinical parameters such as tumor size, extra nodal extension, triple negative breast cancer and poorly differentiated breast cancer subtype. In addition, XIAP over-expression was also significantly associated with PI3-kinase pathway protein; p-AKT, proliferative marker; Ki-67 and anti-apoptotic marker; PARP. XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; embelin and found that embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. Finally, breast cancer cells treated with combination of embelin and PI3-kinase inhibitor; LY294002 synergistically induced apoptosis and caused tumor growth regression in vivo. CONCLUSION: These data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells.

Antioncogenic and Oncogenic Properties of Nrf2 in Arsenic-induced Carcinogenesis.

Arsenic (As(3+)) is a carcinogen with considerable environmental and occupational relevancy. The present study shows that As(3+)-transformed human lung bronchial epithelial BEAS-2B cells (AsT cells) exhibit the property of apoptosis resistance. The level of basal reactive oxygen species (ROS) is very low in AsT cells in correlation with elevated expressions of both antioxidant enzymes and antiapoptotic proteins. Nuclear factor erythroid 2-related factor (Nrf2) and p62 are constitutively expressed. These two proteins up-regulate antioxidant enzymes and antiapoptotic proteins. The knockdown of Nrf2 or p62 by small interfering RNA (siRNA) enhanced both ROS levels and As(3+)-induced apoptosis in transformed cells. AsT cells have autophagy deficiency as evidenced by reduced formation of microtubule-associated protein 1 light chain 3 (LC3)-II, GFP-LC3 puncta, and autophagy flux. Results obtained using a soft agar assay and shRNA Nrf2-transfected cells show that Nrf2 plays an antioncogenic role before transformation, whereas this transcription factor plays an oncogenic role after transformation. In addition, depletion of Nrf2 by shRNA dramatically inhibited growth and proliferation of transformed cells. Furthermore, the Nrf2 protein levels and antiapoptotic and antioxidant enzyme levels are higher in lung adenocarcinoma than in normal tissues. Collectively, this study demonstrates that a constitutively high level of Nrf2 in AsT cells up-regulates the antioxidant proteins catalase and superoxide dismutase as well as the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decreased ROS generation and increased apoptotic resistance, cell survival and proliferation, and tumorigenesis.

Constitutive activation of epidermal growth factor receptor promotes tumorigenesis of Cr(VI)-transformed cells through decreased reactive oxygen species and apoptosis resistance development.

Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing to Cr(VI)-induced tumorigenesis.

Different roles of ROS and Nrf2 in Cr(VI)-induced inflammatory responses in normal and Cr(VI)-transformed cells.

Hexavalent chromium (Cr(VI)) is classified as a human carcinogen. Cr(VI) has been associated with adenocarcinomas and squamous cell carcinoma of the lung. The present study shows that acute Cr(VI) treatment in human bronchial epithelial cells (BEAS-2B) increased inflammatory responses (TNF-α, COX-2, and NF-кB/p65) and expression of Nrf2. Cr(VI)-induced generation of reactive oxygen species (ROS) are responsible for increased inflammation. Despite the fact that Nrf2 is a master regulator of response to oxidative stress, silencing of Nrf2 in the acute Cr(VI) treatment had no effect on Cr(VI)-induced inflammation. In contrast, in Cr(VI)-transformed (CrT) cells, Nrf2 is constitutively activated. Knock-down of this protein resulted in decreased inflammation, while silencing of SOD2 and CAT had no effect in the expression of these inflammatory proteins. Results obtained from the knock-down of Nrf2 in CrT cells are very different from the results obtained in the acute Cr(VI) treatment. In BEAS-2B cells, knock-down of Nrf2 had no effect in the inflammation levels, while in CrT cells a decrease in the expression of inflammation markers was observed. These results indicate that before transformation, ROS plays a critical role while Nrf2 not in Cr(VI)-induced inflammation, whereas after transformation (CrT cells), Nrf2 is constitutively activated and this protein maintains inflammation while ROS not. Constitutively high levels of Nrf2 in CrT binds to the promoter regions of COX-2 and TNF-α, leading to increased inflammation. Collectively, our results demonstrate that before cell transformation ROS are important in Cr(VI)-induced inflammation and after transformation a constitutively high level of Nrf2 is important.

Nrf2/p62 signaling in apoptosis resistance and its role in cadmium-induced carcinogenesis.

The cadmium-transformed human lung bronchial epithelial BEAS-2B cells exhibit a property of apoptosis resistance as compared with normal non-transformed BEAS-2B cells. The level of basal reactive oxygen species (ROS) is extremely low in transformed cells in correlation with elevated expressions of both antioxidant enzymes (catalase, SOD1, and SOD2) and antiapoptotic proteins (Bcl-2/Bcl-xL). Moreover, Nrf2 and p62 are highly expressed in these transformed cells. The knockdown of Nrf2 or p62 by siRNA enhances ROS levels and cadmium-induced apoptosis. The binding activities of Nrf2 on the antioxidant response element promoter regions of p62/Bcl-2/Bcl-xL were dramatically increased in the cadmium-exposed transformed cells. Cadmium exposure increased the formation of LC3-II and the frequency of GFP-LC3 punctal cells in non-transformed BEAS-2B cells, whereas these increases are not shown in transformed cells, an indication of autophagy deficiency of transformed cells. Furthermore, the expression levels of Nrf2 and p62 are dramatically increased during chronic long term exposure to cadmium in the BEAS-2B cells as well as antiapoptotic proteins and antioxidant enzymes. These proteins are overexpressed in the tumor tissues derived from xenograft mouse models. Moreover, the colony growth is significantly attenuated in the transformed cells by siRNA transfection specific for Nrf2 or p62. Taken together, this study demonstrates that cadmium-transformed cells have acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decrease in ROS generation, apoptotic resistance, and increased cell survival, proliferation, and tumorigenesis.

Role of reactive oxygen species in arsenic-induced transformation of human lung bronchial epithelial (BEAS-2B) cells.

Arsenic is an environmental carcinogen, its mechanisms of carcinogenesis remain to be investigated. Reactive oxygen species (ROS) are considered to be important. A previous study (Carpenter et al., 2011) has measured ROS level in human lung bronchial epithelial (BEAS-2B) cells and arsenic-transformed BEAS-2B cells and found that ROS levels were higher in transformed cells than that in parent normal cells. Based on these observations, the authors concluded that cell transformation induced by arsenic is mediated by increased cellular levels of ROS. This conclusion is problematic because this study only measured the basal ROS levels in transformed and parent cells and did not investigate the role of ROS in the process of arsenic-induced cell transformation. The levels of ROS in arsenic-transformed cells represent the result and not the cause of cell transformation. Thus question concerning whether ROS are important in arsenic-induced cell transformation remains to be answered. In the present study, we used expressions of catalase (antioxidant against H2O2) and superoxide dismutase 2 (SOD2, antioxidant against O2(-)) to decrease ROS level and investigated their role in the process of arsenic-induced cell transformation. Our results show that inhibition of ROS by antioxidant enzymes decreased arsenic-induced cell transformation, demonstrating that ROS are important in this process. We have also shown that in arsenic-transformed cells, ROS generation was lower and levels of antioxidants are higher than those in parent cells, in a disagreement with the previous report. The present study has also shown that the arsenic-transformed cells acquired apoptosis resistance. The inhibition of catalase to increase ROS level restored apoptosis capability of arsenic-transformed BEAS-2B cells, further showing that ROS levels are low in these cells. The apoptosis resistance due to the low ROS levels may increase cells proliferation, providing a favorable environment for tumorigenesis of arsenic-transformed cells.

Ethanol enhances arsenic-induced cyclooxygenase-2 expression via both NFAT and NF-κB signalings in colorectal cancer cells.

Arsenic is a known carcinogen to humans, and chronic exposure to environmental arsenic is a worldwide health concern. As a dietary factor, ethanol carries a well-established risk for malignancies, but the effects of co-exposure to arsenic and ethanol on tumor development are not well understood. In the present study, we hypothesized that ethanol would enhance the function of an environmental carcinogen such as arsenic through increase in COX-2 expression. Our in vitro results show that ethanol enhanced arsenic-induced COX-2 expression. We also show that the increased COX-2 expression associates with intracellular ROS generation, up-regulated AKT signaling, with activation of both NFAT and NF-κB pathways. We demonstrate that antioxidant enzymes have an inhibitory effect on arsenic/ethanol-induced COX-2 expression, indicating that the responsive signaling pathways from co-exposure to arsenic and ethanol relate to ROS generation. In vivo results also show that co-exposure to arsenic and ethanol increased COX-2 expression in mice. We conclude that ethanol enhances arsenic-induced COX-2 expression in colorectal cancer cells via both the NFAT and NF-κB pathways. These results imply that, as a common dietary factor, ethanol ingestion may be a compounding risk factor for arsenic-induced carcinogenesis/cancer development.

Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin.

Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm(2)) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways.

Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin.

Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE2 and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2'-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways.

Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways.

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5µM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis.