Regulation of Cyclin D1 Degradation by Ubiquitin-Specific Protease 27X Is Critical for Cancer Cell Proliferation and Tumor Growth.

Cyclin D1 (CCND1) is a critical regulator of cell proliferation and its overexpression has been linked to the development and progression of several malignancies. CCND1 overexpression is recognized as a major mechanism of therapy resistance in several cancers; tumors that rely on CCND1 overexpression to evade cancer therapy are extremely sensitive to its ablation. Therefore, targeting CCND1 is a promising strategy for preventing tumor progression and combating therapy resistance in cancer patients. Although CCND1 itself is not a druggable target, it can be targeted indirectly by inhibiting its regulators. CCND1 steady-state levels are tightly regulated by ubiquitin-mediated degradation, and defects in CCND1 ubiquitination are associated with increased CCND1 protein levels in cancer. Here, we uncover a novel function of ubiquitin-specific protease 27X (USP27X), a deubiquitinating enzyme (DUB), in regulating CCND1 degradation in cancer. USP27X binds to and stabilizes CCND1 in a catalytically dependent manner by negatively regulating its ubiquitination. USP27X expression levels correlate with the levels of CCND1 in several HER2 therapy-resistant breast cancer cell lines, and its ablation leads to a severe reduction of CCND1 protein levels, inhibition of tumor growth, and resensitization to targeted therapy. Together, the results presented in our study are the first to expose USP27X as a major CCND1 deubiquitinase and provide a mechanistic explanation for how this DUB fosters tumor growth. IMPLICATIONS: As a deubiquitinating enzyme, USP27X is a druggable target. Our study illuminates new avenues for therapeutic intervention in CCND1-driven cancers.

A Comparative Study of Platelet-Rich Fibrin and Platelet-Rich Fibrin with Hydroxyapatite to Promote Healing of Impacted Mandibular Third Molar Socket.

Aim: The purpose of this study was to compare the efficacy of platelet-rich fibrin (PRF) and platelet-rich fibrin (PRF) with hydroxyapatite (HA) on postoperative pain, swelling, soft tissue healing and osseous regeneration in mandibular third molar extraction socket in human patient. Materials and Methods: This prospective study included total 40 patients who reported to the Department of Oral and Maxillofacial Surgery (OMFS), Teerthanker Mahaveer Dental College and Research Centre (TMDC&RC), Moradabad (U.P.). Twenty patients each in both the groups of PRF and PRF with HA were included for the management of impacted mandibular 3rd molar extraction sockets and were evaluated for effectiveness. Evaluation was done on the basis of following parameters pain and swelling at time interval of 1st, 3rd, 7th day, soft tissue healing at time interval of 3rd, 7th, 14th day and osseous regeneration at time intervals of 1st, 3rd, 6th month postoperatively. Results: Pain and swelling were less in the PRF with HA group when compared to PRF group. Soft tissue healing was better in the PRF with HA group compared to the PRF group. The result of the study shows rapid bone regeneration in the extraction socket treated with the PRF with HA group when compared with the PRF group. Also there was less postoperative discomfort in the PRF with HA-treated group. Conclusion: PRF a mitogenic promoter together with a bone graft forms a scaffolding, promotes early healing thus being creating beneficial for the patient and is also economic.

New sheriff in town: Cyclic peptide inhibitors of a DUB module.

In this issue of Cell Chemical Biology, Morgan et al. (2021) show that cyclic peptides can be potent and highly specific inhibitors for deubiquitinating enzymes. This study identifies the first selective inhibitors of the cancer-associated ubiquitin-specific protease 22 (USP22).

Assessing the Mechanical Properties of a New High Strength Aluminum Hybrid MMC Based on the ANN Approach for Automotive Application.

Aluminum-based composites with characteristics such as low density and high strength to weight ratio have been identified to be one of the best-emerging alternatives. The lightweight composite is gaining popularity, particularly in the automotive industry. The composite's qualities make it a prospective material to replace significant materials that are now used in the automobile industry. For lightweight products, various weight reduction solutions were proposed. In the present work, one such lightweight composite was fabricated by using a stir casting process, which includes reinforcement powders viz. carbon nanotube and fly ash to pure aluminum. The use of fly ash helps in reducing the overall associated cost of the material as well as provides low density. The work aims to identify the amount of fly ash (by weight %) suitable to avail good mechanical properties. In concern with the mechanical properties, density, yield strength, ultimate tensile strength, and wear resistance of the composite specimen were examined. Moreover, the artificial neural network was adopted to identify minimum volumetric wear for a given set of conditions. From the results, it was perceived that with the increase in fly ash content, the volumetric wear of the fabricated composite decreases. However, with the increase in load and speed, the volumetric wear rate increases.

Safety studies of Nexrutine, bark extract of Phellodendron amurense through repeated oral exposure to rats for 28 days.

Nexrutine (NX), a marketable herbal extract from a traditional Chinese herbal plant, Phellodendron amurense, is majorly used for the resolution of inflammation, gastroenteritis, and some tissue-specific cancer. Strategies for the identification of the safety of anticancer solutions of plant origin are an important area of study. The present investigation assesses the single and repeated dose (28 days) toxicity of NX following OECD guidelines 425 and 407, respectively. Briefly, to identify acute toxic properties of NX, a dose of 2000 mg/kg b. wt was administered once orally. Simultaneously, repeated dose toxicity was evaluated through daily administration of the three different doses (250, 500, 750 mg/kg b. wt) of NX for 28days. The single administration of NX showed no signs of toxicity and morbidity, suggesting LD50 of NX more than 2000 mg/kg b. wt. Furthermore, repeated dose exposure of NX for 28 days did not show any sign of toxicity. Hematology, serum biochemistry, and histopathological analysis also did not show any significant abnormalities. However, a marginal decrease in triglyceride, cholesterol, and glucose levels along with mild tubular degeneration in the kidney was also noticed in the high dose NX treatment group. Overall, the findings of the study suggest that NX is safe for use up to 500 mg/kg b.wt.

To study the usefulness and comparison of myocardial strain imaging by 2D and 3D echocardiography for early detection of cardiotoxicity in patients undergoing cardiotoxic chemotherapy.

BACKGROUND: Chemotherapy-induced cardiotoxicity constitutes subclinical myocardial dysfunction, arrhythmias, pericarditis, coronary vasospasm, and significant symptomatic heart failure. Anthracyclines pose higher risk for long-term cardiac dysfunction, with increased incidences of morbidity and mortality. Hence, early detection of chemotherapy-induced cardiac dysfunction may prompt an earlier treatment modification. AIM: To evaluate global, longitudinal, radial, and circumferential strain changes in adult patients undergoing anthracycline chemotherapy along with the usefulness of three-dimensional (3D) echocardiography as the new modality over two-dimensional (2D) echocardiography. METHODS: This was a single centre, prospective, observational study that included asymptomatic patients free from any cardiac signs and symptoms attributable to heart failure, who underwent potentially cardiotoxic chemotherapy for malignancy from December 2017 to November 2018 at a tertiary care centre in India. Baseline demographics were recorded, and 2D and 3D echocardiography was performed at baseline and after completion of four cycles of chemotherapy. RESULTS: All the 55 patients received a cumulative dose of doxorubicin of less than 550 mg/m2. Follow-up period from the beginning of doxorubicin therapy was 108 ± 14 days. 9 patients were excluded from the study due to poor 3D images, so data analysis was done only for 46 patients. In 2D echocardiography, only global longitudinal strain (GLS) was observed to be significantly reduced (Δ18.33%; P < 0.001). 2D ejection fraction (EF) did not show significant change (Δ0.67%; P = 0.176), while by 3D echo, EF reduced significantly (Δ3.55%; P < 0.001). 3D global longitudinal (Δ29.19%; P < 0.001), circumferential (Δ30.65%; P < 0.001), area (Δ21.61%; P < 0.001), and radial (Δ29.66%; P < 0.001) strains were observed to be significantly reduced at follow-up. CONCLUSION: Myocardial dysfunction induced by cardiotoxic chemotherapy can be detected earlier by using 2D GLS, 3D volumetric analysis, and 3D strain analysis by calculating global, longitudinal, radial, and circumferential strain changes. 3D echocardiographic assessment seems to be more accurate in picking out small changes in left ventricular functions, but at the cost of slightly poor image quality as compared to the 2D echocardiography. These newer techniques could potentially improve the ability for early detection of subclinical abnormalities of LV function in patients undergoing cardiotoxic chemotherapy and thus early initiation of treatment could be possible.

Alternariol induced proliferation in primary mouse keratinocytes and inflammation in mouse skin is regulated via PGE2/EP2/cAMP/p-CREB signaling pathway.

Alternariol (AOH) is a mycotoxin that contaminates various food stuffs as well as animal feed and may cause toxicity after consumption. However, a dermal toxic potential of AOH has not been explored so far. In the present study, skin toxicity after topical exposure of AOH and the involved mechanism/s are revealed. Single topical application of different AOH doses (12.5, 25, 50 µg/animal) caused increased bi-fold thickness as well as hyperplasia and higher production of prostaglandin E2 (PGE2) along with cAMP in the skin demonstrating its inflammatory potential. Western blot analysis showed that exposure of AOH lead to phosphorylation of CREB and increased the expression of COX-2, cyclin D1 as well as prostanoid EP2 receptor. Further studies on primary mouse keratinocytes (PMK) revealed that very low concentrations of AOH (50-500 nM) resulted in significant PMK proliferation. Additionally, using specific antagonist or agonist of prostanoid receptors, we delineated that EP2 receptor play a key role in AOH-induced PMKs proliferation. Collectively, our findings show that AOH can lead to dermal toxicity in mice by activating the EP2/cAMP/p-CREB signaling cascade.

Hydroxyl Group Difference between Anthraquinone Derivatives Regulate Different Cell Death Pathways via Nucleo-Cytoplasmic Shuttling of p53.

BACKGROUND: Despite a number of measures having been taken for cancer management, it is still the second leading cause of death worldwide. p53 is the protein principally being targeted for cancer treatment. Targeting p53 localization may be an effective strategy in chemotherapy as it controls major cell death pathways based on its cellular localization. Anthraquinones are bioactive compounds widely being considered as potential anticancer agents but their mechanism of action is yet to be explored. It has been shown that the number and position of hydroxyl groups within the different anthraquinones like Emodin and Chrysophanol reflects the number of intermolecular hydrogen bonds which affect its activity. Emodin contains an additional OH group at C-3, in comparison to Chrysophanol and may differentially regulate different cell death pathways in cancer cell. OBJECTIVE: The present study was aimed to investigate the effect of two anthraquinones Emodin and Chrysophanol on induction of different cell death pathways in human lung cancer cells (A549 cell line) and whether single OH group difference between these compounds differentially regulate cell death pathways. METHODS: The cytotoxic effect of Emodin and Chrysophanol was determined by the MTT assay. The expression of autophagy and apoptosis marker genes at mRNA and protein level after treatment was checked by the RT-PCR and Western Blot, respectively. For cellular localization of p53 after treatment, we performed immunofluorescence microscopy. RESULTS: We observed that both compounds depicted a dose-dependent cytotoxic response in A549 cells which was in concurrence with the markers associated with oxidative stress such as an increase in ROS generation, decrease in MMP and DNA damage. We also observed that both compounds up-regulated the p53 expression where Emodin causes nuclear p53 localization, which leads to down-regulation in mTOR expression and induces autophagy while Chrysophanol inhibits p53 translocation into nucleus, up-regulates mTOR expression and inhibits autophagy. CONCLUSION: From this study, it may be concluded that the structural difference of single hydroxyl group may switch the mechanism from one pathway to another which could be useful in the future to improve anticancer treatment and help in the development of new selective therapies.

ZnO Nanoparticles Modified with an Amphipathic Peptide Show Improved Photoprotection in Skin.

ZnO nanoparticles of different sizes were functionalized with an amphipathic peptide, and its effect on nanoparticle stabilization and UV photoprotective activity was studied in this article. The peptide-modified nanoparticles exhibited lower aggregation, significant reduction in Zn2+ leaching in vitro and even inside the cells for smaller particle sizes, reduced photocatalytic activity, and reduced cellular toxicity under UV-B treated conditions. In addition, the peptide-modified 60 nm ZnO nanoparticles showed lower genotoxicity, lower oxidative stress induction levels, less DNA damage responses, and less immunogenic potential than the bare counterparts in the presence of UV-B rays. They localized more in the stratum corneum and epidermis ex vivo, indicating better retention in epidermis, and demonstrated improved UV-B protection and/or skin integrity in SKH-1 mice in vivo compared to unmodified nanoparticles and commercial UV-protective agents tested. To our knowledge, this is the first report on the application of peptide-modified ZnO nanoparticles for improved photoprotection.

COX-2/EP2-EP4/ß-catenin signaling regulates patulin-induced intestinal cell proliferation and inflammation.

Patulin (PAT), a mycotoxin, is a natural contaminant that is produced by certain species of Penicillium, Aspergillus and Byssochlamys. The major contamination of PAT is in apple and apple based products. PAT is known to cause glutathione depletion, oxidative DNA damage and cell proliferation. Recently, in vitro studies have indicated that PAT can also increase the intestinal epithelial permeability, modulate tight junctions and decrease transepithelial electrical resistance. Nonetheless, no previous study has evaluated the mechanisms responsible for PAT-induced intestinal toxicity or its relevance to the in vivo situation. Here, Wistar rats were orally treated with 100 µg/kg body weight (b.wt.) of PAT, either alone or along with 100 mg/kg b. wt. of celecoxib for 3 days. We found that PAT exposure led to significantly higher levels of PGE2 in serum and intestinal tissue and high expression of COX-2 and Ki-67 compared to controls. Interestingly, our results showed that celecoxib treatment could decrease the PAT-induced PGE2 and reduce the PAT-induced intestinal damage. To study the mechanistic aspect, normal rat intestinal epithelial cells (IEC-6) were treated with non-toxic concentrations (100 nM, 250 nM and 500 nM) of PAT for 6 h. It was observed that PAT exposure caused enhanced proliferation, higher expression of COX-2, and EP2 and EP4 receptors, along with increased PGE2 secretion. Additionally, PAT exposure caused enhanced Akt expression, which in turn inhibits GSK-3ß and stabilizes ß-catenin. Overall, our study suggests that the COX-2/EP2-EP4/ß-catenin signaling cascades are involved in the regulation of PAT-induced intestinal cell proliferation and inflammation.

Protective Activity of Silk Sericin against UV Radiation-Induced Skin Damage by Downregulating Oxidative Stress.

Topical delivery of potential antioxidants protects the skin against ultraviolet (UV) radiation-induced oxidative damage through maintaining redox balance. Sericin, one of the major components of silk, possesses antioxidant property along with skin-protective activity against UVB radiation-induced damage. However, the protective activity of silk sericin (SS) extracted from different sources has not been explored against UVA and UVB radiation-induced oxidative damage. In the present study, we have systematically investigated the protective activity of sericin against UVA and UVB radiation-induced skin damage. MTT and neutral red assays showed that Philosamia ricini sericin (PRS) and Antheraea assamensis sericin (AAS) (10 µg/mL) treatment prior to UVA (12 J/cm2) and UVB (120 mJ/cm2) irradiations enhanced the viability of human keratinocytes. Examination of cell cycle arrest and apoptotic/necrotic cell death using flow cytometry showed that sericin treatment before UVA and UVB irradiation protected the cells from apoptotic cell death by arresting the cell cycle at G1 phase. Sericin pretreatment downregulated the interleukin (IL)-6 and IL-8, upregulated p53 and decrease the dysregulation of Bcl-2/Bax gene expression. AAS treatment prior to UVB irradiation significantly reduced skin inflammation, DNA fragmentation, and lipid peroxidation in the female SKH-1 hairless mouse skin. Altogether, our results substantiate the use of AAS in effectively ameliorating UVA and UVB radiation-induced skin damage, which holds prospects as a potent antioxidant supplement in the preparation of skin care products.

Topical application of Nexrutine inhibits ultraviolet B-induced cutaneous inflammatory responses in SKH-1 hairless mouse.

BACKGROUND: Ultraviolet B (UVB) radiation is the major contributor to skin inflammation which leads to the development of skin cancer. Hence, in this study, we studied the effect of Nexrutine (NX) on UVB-induced cutaneous inflammation and its mediators. METHODS: Ultraviolet absorption spectra of NX were measured by spectrophotometer. To conduct the photoprotective studies, SKH-1 hairless mice were topically treated with NX, 30 minutes before to the UVB (180 mJ/cm2 ) exposure. Twenty hours of post-UVB irradiation, mouse skin was used for edema measurements, H & E staining, myeloperoxidase (MPO) activity, and estimation of plasma cytokines. In addition, expression levels of inflammatory cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were also determined by Western blot analysis. RESULTS: Nexrutine displayed absorbance over the UVB spectrum. NX significantly decreased the UVB-induced epidermal edema, skin thickness, leukocyte infiltration, number of the sunburn, and TUNEL-positive cells. NX treatment also decreased the number of mast cells, MPO activity, expression of pro-inflammatory cytokines, and inflammation mediator protein in mouse skin. CONCLUSION: These results provide evidences that NX inhibits the UVB-induced cutaneous inflammatory responses in SKH-1 mouse skin.

Non-invasive Oil-Based Method to Increase Topical Delivery of Nucleic Acids to Skin.

Topical delivery of nucleic acids to skin has huge prospects in developing therapeutic interventions for cutaneous disorders. In spite of initial success, clinical translation is vastly impeded by the constraints of bioavailability as well as stability in metabolically active environment of skin. Various physical and chemical methods used to overcome these limitations involve invasive procedures or compounds that compromise skin integrity. Hence, there is an increasing demand for developing safe skin penetration enhancers for efficient nucleic acid delivery to skin. Here, we demonstrate that pretreatment of skin with silicone oil can increase the transfection efficiency of non-covalently associated peptide-plasmid DNA nanocomplexes in skin ex vivo and in vivo. The method does not compromise skin integrity, as indicated by microscopic evaluation of cellular differentiation, tissue architecture, enzyme activity assessment, dye penetration tests using Franz assay, and cytotoxicity and immunogenicity analyses. Stability of nanocomplexes is not hampered on pretreatment, thereby avoiding nuclease-mediated degradation. The mechanistic insights through Fourier transform infrared (FTIR) spectroscopy reveal some alterations in the skin hydration status owing to possible occlusion effects of the enhancer. Overall, we describe a topical, non-invasive, efficient, and safe method that can be used to increase the penetration and delivery of plasmid DNA to skin for possible therapeutic applications.

UVB irradiation-enhanced zinc oxide nanoparticles-induced DNA damage and cell death in mouse skin.

UV-induced reactive oxygen species (ROS) have been implicated in photocarcinogenesis and skin aging. This is because UV-induced ROS can induce DNA damage that, if unrepaired, can lead to carcinogenesis. Sunscreens contain UV attenuators, such as organic chemical and/or physical UV filters, which can prevent all forms of damage from UV irradiation. In recent years, the effective broad-spectrum UV attenuation properties of ZnO-nanoparticles (ZnO-NPs) have made them attractive as active components in sunscreens and other personal care products. As the use of ZnO-NPs in sunscreens is on the rise, so is public concern about their safety, particularly with exposure to sunlight. Therefore, in the present study, using various experimental approaches, we investigated the possible toxic effects resulting from exposure to UVB and ZnO-NPs in primary mouse keratinocytes (PMKs) as well as in the skin of SKH-1 hairless mice. The findings of the present study demonstrated that co-exposure to UVB and ZnO-NPs: (1) translocated the ZnO-NPs into the nucleus of PMKs; (2) caused enhanced generation of ROS; (3) induced more severe DNA damage as evident by alkaline comet assay and immunocytochemistry for γ-H2AX and 8-hydroxy-2'-deoxyguanosine (8-OHdG); and (4) subsequently caused much more pronounced cell death in PMKs. Further, to elucidate the physiological relevance of these in vitro findings, SKH-1 hairless mice were topically treated with ZnO-NPs and after 30min irradiated with UVB (50mJ/cm(2)). Interestingly, we found that co-exposure of ZnO-NPs and UVB caused increased oxidative DNA damage and cell death, indicated by immunostaining for 8-OHdG and TUNEL assay in sections of exposed mouse skin. Thus, collectively, our findings suggest that UVB exposure increases ZnO-NPs-mediated oxidative stress and oxidative damage, thereby enhancing ZnO-NPs-induced cell death.

Nexrutine inhibits azoxymethane-induced colonic aberrant crypt formation in rat colon and induced apoptotic cell death in colon adenocarcinoma cells.

Colon cancer is the third most common cause of death in the United States. Therefore, new preventive strategies are warranted for preventing colon cancer. Nexrutine (NX), an herbal extract from Phellodendron amurense, has been shown to have anti-inflammatory, anti-microbial and anti-cancer activity for various tissue specific cancers, but its chemopreventive efficacy has not been evaluated against colon cancer. Here, we explored the mechanism of chemopreventive/chemotherapeutic efficacy of NX against colon cancer. We found that dietary exposure of NX significantly reduced the number of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats. In addition, significant inhibition in AOM-induced cell proliferation and reduced expression of the inflammatory markers COX-2, iNOS as well as the proliferative markers PCNA and cyclin D1 were also seen. Moreover, NX exposure significantly enhanced apoptosis in the colon of AOM treated rats. Furthermore, in in vitro studies, NX (2.5, 5, 10 µg/ml, 48 h) decreased cell survival and colony formation while inducing G0/G1 cell cycle arrest and apoptosis in colon adenocarcinoma cells COLO205 and HCT-15. However, NX had minimal cytotoxic effect on IEC-6 normal rat intestinal cells, suggesting its high therapeutic index. NX treatment also modulates the level of Bax and Bcl-2 proteins along with cytochrome c release, cleavage and enhanced expression of poly (adenosine diphosphate-ribose) polymerase as well as the catalytic activity of caspase 3 and caspase 9 in both COLO205 and HCT-15 cells. Based on these in vivo and in vitro findings, we suggest that NX could be useful candidate agent for colon cancer chemoprevention and treatment. © 2015 Wiley Periodicals, Inc.

Dietary administration of Nexrutine inhibits rat liver tumorigenesis and induces apoptotic cell death in human hepatocellular carcinoma cells.

Epidemiological studies suggested that plant-based dietary supplements can reduce the risk of liver cancer. Nexrutine (NX), an herbal extract from Phellodendronamurense, has been shown to have anti-inflammatory, anti-microbial and anti-tumor activities. In the present study, we have shown the anti-tumor potential of NX against Solt-Farber model with elimination of PH, rat liver tumor induced by diethylnitrosoamine (DEN) as carcinogen and 2-acetylaminofluorene (2-AAF) as co-carcinogen. The elucidation of mechanistic pathways was explored in human liver cancer cells. Dietary intake of NX significantly decreased the cell proliferation and inflammation, as well as increased apoptosis in the liver sections of DEN/2-AAF-treated rats. Moreover, NX (2.5-10 µg/ml) exposure significantly decreased the viability of liver cancer cells and modulated the levels of Bax and Bcl-2 proteins levels. NX treatment resulted in increased cytochrome-c release and cleavage of caspases 3 and 9. In addition, NX decreased the expression of CDK2, CDK4 and associated cyclins E1 and D1, while up-regulated the expression of p21, p27 and p53 expression. NX also enhanced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and JNK1/2. Collectively, these findings suggested that NX-mediated protection against DEN/2-AAF-induced liver tumorigenesis involves decrease in cell proliferation and enhancement in apoptotic cell death of liver cancer cells.

EGFR-mediated Akt and MAPKs signal pathways play a crucial role in patulin-induced cell proliferation in primary murine keratinocytes via modulation of Cyclin D1 and COX-2 expression.

Patulin (PAT), a present day major contaminant of commercial apple and apple products is reported to be carcinogenic, embryotoxic, and immunotoxic. While oral and inhalation are considered to be the most prevalent routes of exposure to this toxin, exposure through skin is now being extensively investigated. Our previous study showed that short-term dermal exposure to PAT resulted in toxicological injury to the skin, while long-term exposure induced skin tumorigenesis. In this study, we explore the mechanism involve in proliferation of mouse keratinocytes by PAT. Our study revealed that PAT rapidly induces phosphorylation of EGFR, activation of the Ras/MAPKs, and Akt pathways. This in-turn leads to the activation of NF-κB/AP-1 transcription factors which then binds to the promoter region of the cell growth regulatory genes Cyclin D1 and COX-2 inducing their expression leading ultimately to PMKs proliferation. Inhibition of EGFR or the Ras/MAPKs, PI3/Akt pathways with different pharmacological inhibitors or knockdown of NF-κB, c-jun, c-fos, Cyclin D1, and COX-2 with siRNA inhibited PAT-induced PMKs proliferation.

Protective effect of topical application of α-tocopherol and/or N-acetyl cysteine on argemone oil/alkaloid-induced skin tumorigenesis in mice.

Since bioantioxidants in plasma of Epidemic Dropsy patients [a condition caused by consumption of adulterated mustard oil with argemone oil (AO)] were found to be significantly decreased, the beneficial effect of N-acetyl cysteine (NAC) and α-tocopherol (TOCO) against AO- or sanguinarine (SANG)-induced tumorigenicity was undertaken in mice. Topical application of TOCO and NAC either alone or in combination showed significant protection against AO/TPA- and SANG/TPA-induced skin tumorigenicity. Histopathological findings suggest that papillomatous growth in AO/TPA- and SANG/TPA-treated animals were substantially protected following topical application of TOCO or NAC. Further, treatment of TOCO and NAC either alone or in combination to AO/TPA- or SANG/TPA-induced mice significantly decreased lipid peroxidation, along with significant revival in glutathione (GSH) content and activities of tyrosinase, histidase, catalase, SOD, GSH peroxidase, and GSH reductase in skin. In vitro studies showed that TOCO and/or NAC significantly decreased the AO and SANG induced cell proliferation and activation of ERK, p38, JNK MAPKs and NF-κB signaling in HaCaT cells. In summary, TOCO and NAC may be useful in preventing the tumorigenic response of AO and SANG probably by acting as scavenger of free radicals and inhibiting MAPKs and NF-κB signaling.

Ochratoxin A-induced cell proliferation and tumor promotion in mouse skin by activating the expression of cyclin-D1 and cyclooxygenase-2 through nuclear factor-kappa B and activator protein-1.

Our prior studies have indicated that ochratoxin A (OTA), a mycotoxin, has skin tumor initiating activity. In the present investigation, skin tumor promoting activity of OTA and the mechanism/(s) involved therein was undertaken. A single topical application of OTA (100 nmol/mouse) caused significant enhancement in short-term markers of skin tumor promotion such as ornithine decarboxylase activity, DNA synthesis, hyperplasia as well as expression of cyclin-D1 and COX-2 in mouse skin. In a two-stage mouse skin tumorigenesis protocol, twice-weekly exposure of OTA (50 nmol/mouse) to 7,12-dimethylbenz[α]anthracene (120 nmol/mouse) initiated mice skin for 24 weeks leads to tumor formation. Further, exposure of primary murine keratinocytes (PMKs) with non-cytotoxic dose of OTA (5.0 µM) caused (i) significant enhancement of DNA synthesis, (ii) enhanced phosphorylation and subsequent activation of epidermal growth factor receptor (EGFR) and its downstream signaling pathways viz Akt, ERK1/2, p38 and JNK mitogen-activated protein kinases (MAPKs), (iii) overexpression of c-jun, c-fos, cyclin-D1 and COX-2 and (iv) increased binding of nuclear factor-kappaB (NF-κB) and AP-1 transcription factors to the promoter region of cyclin-D1 and COX-2 genes. It was also observed that knocking down the messenger RNA expression of NF-κB, c-jun, c-fos, cyclin-D1 and COX-2 results in significant inhibition in OTA-induced PMKs proliferation. These results suggest that OTA has cell proliferative and tumor-promoting potential in mouse skin, which involves EGFR-mediated MAPKs and Akt pathways along with NF-κB and AP-1 transcription factors and that cyclin-D1 and COX-2 are the target genes responsible for tumor-promoting activity of OTA.