Trigonelline, a plant derived alkaloid prevents ultraviolet-B-induced oxidative DNA damage in primary human dermal fibroblasts and BALB/c mice via modulation of phosphoinositide 3-kinase-Akt-Nrf2 signalling axis.

BACKGROUND: DNA is the main target for UV-B-irradiation-induced skin photodamage and accounts for 90 % of all the non-melanoma skin cancers. PURPOSE: In this study, we explored the mechanistic basis of photoprotective effect of Trigonelline, a naturally occurring alkaloid from the Trigonella foenum-graecum, against UV-B-induced oxidative DNA Damage Response using Primary Human Dermal Fibroblasts (HDFs) and BALB/C mice as models of skin photodamage. METHODS: Primary HDFs were subjected to UV-B exposure (10 mJ/cm2) with or without TG for 24 h. Effect of UV-B exposure and TG treatment was evaluated by analyzing the cell survival, cellular morphology, oxidative stress & DNA damage response markers by performing biochemical studies, florescent microscopy & protein expression studies. In in-vivo study, TG pre-treated BALB/c mice were -irradiated with 180 mJ/cm2 of UV-B dose thrice a week on alternative days for four months, followed by topical application of different concentrations of TG. The photodamage caused by UV-B exposure and its ameleoriation by topical treatment of TG was studied by physical and morphological appearance and analyzing the oxidative stress & DNA damage response markers from skin. RESULTS: We found that TG significantly alleviates UV-B-induced cell death effects in HDFs. TG protects HDF cells and BALB/c mice from UV-B-induced DNA damage by regulating the expression profile of key protein markers of DNA damage which include P53, ATM, ATR, ϒH2AX, Chk1 and Chk2. We found that TG offers geno-protection to UV-B-irradiated HDFs by alleviating CPD induction, reducing the number of TUNEL positive cells and by decreasing the expression levels of DNA damage marker protein ϒH2AX in immunocytochemistry. Further, we found that TG prevents the UVB induced oxidative stress by activating the PI3K-AKT-Nrf2 signalling pathway. On employing PI3K inhibitor, LY294002, we found the expression of ϒH2AX and p-P53 is significantly increased compared to UV-B treated only, indicating that TG mediates the geno-protection against UV-B irradiation via PI3K-AKT-Nrf2 signalling pathway. CONCLUSION: Current study presents for the first time the photo-protective role of TG against UV-B-induced oxidative DNA damage and provides its mechanistic insights also and provide strong evidence for TG to be carried forward as a potential remedial and cosmeceutical agent against UV-B-induced skin photodamage disorders.

Synthesis and anti-melanoma effect of 3-O-prenyl glycyrrhetinic acid against B16F10 cells via induction of endoplasmic reticulum stress-mediated autophagy through ERK/AKT signaling pathway.

Melanoma is an aggressive form of cancer with poor prognosis and survival rates and limited therapeutic options. Here, we report the anti-melanoma effect of 3-O-prenyl glycyrrhetinic acid (NPC-402), a derivative of glycyrrhtinic acid, from a reputed medicinal plant Glycyrrhiza glabra against B16F10 cells. We studied the cytotoxic effect of NPC-402 on melanoma cells and investigated the role of mitogen-activated protein (MAP) kinase, AKT axis, and endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated autophagy as the involved signaling cascade by studying specific marker proteins. In this study, 4-phenylbutyric acid (4PBA, a chemical chaperone) and small interference RNA (siRNA) knockdown of C/EBP Homologous Protein (CHOP)/growth arrest- and DNA damage-inducible gene 153(GAD153) blocked NPC-402-mediated autophagy induction, thus confirming the role of ER stress and autophagy in melanoma cell death. NPC-402 induced oxidative stress and apoptosis in melanoma cells, which were effectively mitigated by treatment with N-acetylcysteine (NAC). In vivo studies showed that intraperitoneal (i.p.) injection of NPC-402 at 10 mg/kg (5 days in 1 week) significantly retarded angiogenesis in the Matrigel plug assay and reduced the tumor size and tumor weight without causing any significant toxic manifestation in C57BL/6J mice. We conclude that NPC-402 has a high potential to be developed as a chemotherapeutic drug against melanoma.

Frizzled receptors in melanomagenesis: From molecular interactions to target identification.

Frizzled (FZD) proteins are receptors for the WNT family ligands. Inherited human diseases and genetic experiments using knockout mice have revealed a central role of FZDs in multiple aspects of embryonic development and tissue homeostasis. Misregulated FZD signaling has also been found in many cancers. Recent studies on three out of the ten mammalian FZDs in melanoma have shown that they promote tumor cell proliferation and invasion, via the activation of the canonical WNT/ß-catenin or non-canonical PCP signaling pathway. In this concise review, we summarize our current knowledge of individual FZDs in melanoma, discuss the involvement of both the canonical and non-canonical pathways, and describe ongoing efforts to target the FZD receptors for melanoma treatment.

Inhibition of Ultraviolet-B Radiation Induced Photodamage by Trigonelline Through Modulation of Mitogen Activating Protein Kinases and Nuclear Factor-κB Signaling Axis in Skin.

Cutaneous photodamage is incited via exposure of ultraviolet-B (UV-B) radiation to skin, characterized by the manifestation of oxidative stress, inflammation, collagen degradation and apoptosis which translates to external aging signs such as wrinkle formation and leathery skin appearance. Meanwhile, it increases cellular susceptibility to photocarcinogenesis. Several studies have accumulated evidence regarding the usage of natural agents in reversing the clinical signs of photoaging as well as preventing photo-toxicity at molecular level. In this study, we have explored the therapeutic potential of natural agent Trigonelline (TG) against UV-B radiation mediated skin photodamage. Various parameters modulated by the exposure of UV-B radiation were investigated in human skin cells and chronic photodamage mice model (Balb/c). We found that TG alleviates UV-B radiation induced photodamage in human skin cells and Balb/c skin mice. TG treatment in UV-B irradiated skin cells abates UV-B radiation mediated phototoxicity, oxidative stress, inflammation and apoptosis. At molecular level, we observed TG treatment significantly prevents the reactive oxygen species (ROS) generation and lipid peroxidation, restores collagen synthesis and matrix metalloproteinase (MMPs) levels. The in vitro findings were replicated in the in vivo model. We found that the TG acts potentially via modulation of ROS-MAPKs-NF-κB axis. Collectively, we propose that TG acts antagonistically against UV-B mediated skin damage and has strong potential to be developed as a therapeutic and cosmetical agent against photodamage disorders.

Glycyrrhizic Acid Prevents Oxidative Stress Mediated DNA Damage Response through Modulation of Autophagy in Ultraviolet-B-Irradiated Human Primary Dermal Fibroblasts.

BACKGROUND/AIMS: Excessive exposure to UV radiation negatively affects the human skin, characterized by photo-damage (premature aging & carcinogenesis). UV-B radiation causes about 90% of non-melanoma skin cancers by damaging de-oxy ribonucleic acids (DNA). We have previously reported that UV-B radiation induces skin photodamage through oxidative & Endoplasmic Reticulum (ER) stresses and Glycyrrhizic acid (GA), a natural triterpene, protects skin cells against such stresses. UV-B radiation elicits signalling cascade by activation of proteins involved in sensing, signalling, and repair process of DNA damage. In this study, we explored the effects & mechanisms of Glycyrrhizic acid (GA) against UV-B -induced photodamage using a well established cellular model. METHODS: We used primary human dermal fibroblasts as a cellular model. The cells were cultured in the presence or absence of GA for 3,6, & 24 h. Effect of UV-B was assessed by examining cell viability, cell morphology, oxidative stress, ER stress, DNA damage & cellular autophagy levels through biochemical assays, microscopy & protein expression studies. RESULTS: In this study, we have determined the effect of GA on autophagy mediated DNA damage response system as the main mechanism in preventing photodamage due to UV-B -irradiation to primary human dermal fibroblasts (HDFs). GA treatment to UV-B exposed HDFs, significantly inhibited cell death, oxidative & ER stress responses, prevented Cyclobutane Pyrimidine dimer (CPD) DNA adduct formation, and DNA fragmentation via modulation of UV-B induced autophagic flux. Present results showed that GA treatment quenched reactive oxygen species (ROS), relieved ER stress response, improved autophagy (6 hr's post-UV-B -irradiation) and prevented UV-B induced DNA damage. CONCLUSION: The present study links autophagy induction by GA as the main mechanism in the prevention of DNA damage and provides a mechanistic basis for the photoprotective effect of GA and suggests that GA can be potentially developed as a promising agent against UV-B induced skin photo-damage.

Chemical chaperone 4-phenyl butyric acid (4-PBA) reduces hepatocellular lipid accumulation and lipotoxicity through induction of autophagy.

Defective autophagy has been linked to lipotoxicity in several cellular models. We aimed to investigate autophagy in lipid-stimulated hepatoma (Huh7) cells and tested whether 4-phenyl butyric acid (4-PBA), a chemical chaperone, has a beneficial role in hepatic fat accumulation and lipotoxicity. We report that long-term (24 h) exposure of hepatocytes to palmitate block autophagic flux that leads to lipid accumulation and cell death. Western blotting analysis showed increased accumulation of SQSTM1/p62, and decreased expression of Beclin1 and Atg7 in palmitate-treated cells. Autophagy inhibition by 3-methyladenine (3-MA) in palmitate-treated cells neither increased SQSTMI/p62 accumulation nor cell death, thus suggesting complete blockade of autophagy by palmitate. 4-PBA reduced lipid accumulation and cell death that were associated with restoration of autophagy. siRNA-mediated knockdown of Atg7 and presence of autophagy inhibitors, 3-MA and chloroquine, resulted in the decrease in lipid-lowering effect of 4-PBA, suggesting that 4-PBA mediates its lipid-lowering effect via autophagy. Apoptotic parameters, including altered Bcl2:Bax ratio and PARP1 cleavage induced by palmitate, were improved by 4-PBA. Our results indicate that palmitate impairs autophagy and increases lipid accumulation in Huh7 cells, whereas 4-PBA plays a protective role in lipid accumulation and lipotoxicity through activation of autophagy.