Shamshad Alam

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.

Non-invasive topical delivery of plasmid DNA to the skin using a peptide carrier.

Topical delivery to skin is an essential step in non-invasive application of nucleic acid therapeutics for cutaneous disorders. The barrier posed by different layers of the skin - stratum corneum on top followed by the viable epidermis below - makes it extremely challenging for large hydrophilic molecules like nucleic acids to efficiently enter the uncompromised skin. We report an amphipathic peptide Mgpe9 (CRRLRHLRHHYRRRWHRFRC) that can penetrate the uncompromised skin, enter skin cells and deliver plasmid DNA efficiently as nanocomplexes in vitro and in vivo without any additional physical or chemical interventions prevalent currently. We observe efficient gene expression up to the highly proliferating basal layer of the skin without observable adverse reactions or toxic effects after delivery of reporter plasmids. The entry mechanism of nanocomplexes possibly involves reversible modulation of junction proteins accompanied by transient changes in skin structure. This peptide holds potential to be used as an efficient transporter of therapeutic nucleic acids to the skin.

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.

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

Highlights • Nexrutine has anti-tumor potential in Solt-Farber rat liver tumorigenesis model.• Nexrutine caused decreased cell proliferation in the DEN/2-AAF treated rats.• It decreases cell viability of liver cancer cells and modulates pro- and anti-apoptotic markers.• Nexrutine modulates the cell cycle regulatory proteins and MAPKs.

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.

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

&NA; 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 &mgr;g/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‐3&bgr; and stabilizes &bgr;‐catenin. Overall, our study suggests that the COX‐2/EP2‐EP4/&bgr;‐catenin signaling cascades are involved in the regulation of PAT‐induced intestinal cell proliferation and inflammation. Graphical abstract Figure. No caption available. HighlightsPatulin exposure causes intestinal cell proliferation and inflammation.Patulin causes over‐expression of COX‐2 and PGE2 secretion.EP2 and EP4 receptors are involved in the induction of COX‐2 and PGE2 secretion.PGE2/EP2‐EP4/&bgr;‐catenin signaling regulates patulin induced intestinal toxicity.

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

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.

Aryl Hydrocarbon Receptor Activation Contributes to Benzanthrone-Induced Hyperpigmentation via Modulation of Melanogenic Signaling Pathways

Benzanthrone (BA), an oxidized polycyclic aromatic hydrocarbon (PAH), has been found to be a potential health threat to occupational workers involved in dye manufacturing factories. It has been observed that occupational workers become exposed to BA either during manufacturing, pulverization, or storage and developed various kinds of skin diseases like contact dermatitis, itching, erythema, roughness, and foremost, hyperpigmentation. It has been shown that some environmental organic pollutants (POPs) like dioxins, furans, and polychlorinated biphenyls (PCBs) may act as ligands for the aryl hydrocarbon receptor (AhR) and regulate hyperpigmentation. Here, we hypothesized that BA may also act as a ligand for AhR and possibly regulate the melanogenic pathway to induced hyperpigmentation. Our computation results indicate that BA has a high binding affinity toward AhR for the initiation of melanogenic signaling. Following the in silico predictions, we used primary mouse melanocytes (PMMs) and confirmed that exposure to BA (5, 10, and 25 μM) resulted in an increase in AhR expression, tyrosinase activity, and melanin synthesis. Moreover, to study the physiological relevance of these findings, C57BL/6 mice were topically exposed to BA, and enhanced pigmentation and melanin synthesis were observed. Furthermore, the study was extended to assess the mechanistic aspects involved in BA-induced hyperpigmentation in PMMs as well as in mouse skin. Our results suggest that BA exposure initiates AhR signaling and increases tyrosinase enzyme activity and melanin synthesis. Moreover, the genes that regulate the melanin synthesis, such as TRP-1, TRP-2 and the transcription factor MITF, were also found to be increased. Thus, altogether, we suggest that BA-AhR interactions are critical for BA-induced hyperpigmentation.

157. Breaching the Barrier: Topical Delivery of Peptide-Based Nanocomplexes in Skin

Skin is a dynamic organ known for its protective functions. With a wide range of associated deblitating and untreatable conditions it has been explored for topical delivery of therapeutics for sustainable effects. In spite of the obvious advantages of the organ delivery of various hydrophilic molecules to skin has met with limited success. This could be attributed to the unique lipid composition and compact organization of stratum corneum which impedes the entry of such molecules in skin limiting their potential clinical translations. Recent approaches involve use of minimal invasive methods for macromolecules delivery to skin. However, issues with efficiency, toxicity, robustness and high costs limit their universal use. Thus one of the key challenges in skin biology is to develop noninvasive, non-toxic and efficient methods for delivery of biomolecules to and through the skin. We have developed a peptide-based delivery system for efficient nucleic acid delivery in skin upon topical application as nanocomplexes. Further we have tried to improvise the delivery efficiency using safe enhancers and modified the carrier to attain specific targeting in skin. The peptide is secondary amphipathic in nature that tends to acquire alpha-helical structure in hydrophobic environment and retains in skin till 24hrs as seen through Franz assay. We have also found that upon topical application of peptide either in bare form or as nanocomplex to skin cells or human foreskin tissue it exhibits efficient cellular entry, high transfection efficiency as well as skin penetration ability as assessed by fluorescence and luciferase based assays. Transfection efficiency observed was equivalent to that obtained with commercial agents. In-vivo studies using SKH-1 hairless mice model showed similar activity. To realize the clinical potential of the work we have shown delivery of therapeutically relevant nucleic acids in skin with effective and traceable amounts of therapeutic molecules. The cytotoxicity and dye penetration test analysis of bare peptide and nanocomplex revealed no deleterious effect on skin cells as well as tissue. Mechanistic insights revealed that the entry of the peptide in skin is mediated partially by fluidization of lipids in addition to transient disruption of junctional proteins as seen through FTIR and time lapse studies. To further enhance the transfection efficiency of these nanocomplexes in skin without compromising its integrity, pre-application of silicone oil worked as an effective strategy. We further modified the peptide with a keratinocyte specific ligand and observed high transfection efficiency in selective cellular population. Presently we are validating the same in skin tissue. Overall we describe development of a convenient, clinically effective and possibly patient compliant approach to facilitate delivery of macromolecular therapeutics in skin.