Chemico-biological aspects of (-)-epigallocatechin-3-gallate (EGCG) to improve its stability, bioavailability and membrane permeability: Current status and future prospects.

Natural products have been a bedrock for drug discovery for decades. (-)-Epigallocatechin-3-gallate (EGCG) is one of the widely studied natural polyphenolic compounds derived from green tea. It is the key component believed to be responsible for the medicinal value of green tea. Significant studies implemented in in vitro, in cellulo, and in vivo models have suggested its anti-oxidant, anti-cancer, anti-diabetic, anti-inflammatory, anti-microbial, neuroprotective activities etc. Despite having such a wide array of therapeutic potential and promising results in preclinical studies, its applicability to humans has encountered with rather limited success largely due to the poor bioavailability, poor membrane permeability, rapid metabolic clearance and lack of stability of EGCG. Therefore, novel techniques are warranted to address those limitations so that EGCG or its modified analogs can be used in the clinical setup. This review comprehensively covers different strategies such as structural modifications, nano-carriers as efficient drug delivery systems, synergistic studies with other bioactivities to improve the chemico-biological aspects (e.g., stability, bioavailability, permeability, etc.) of EGCG for its enhanced pharmacokinetics and pharmacological properties, eventually enhancing its therapeutic potentials. We think this review article will serve as a strong platform with comprehensive literature on the development of novel techniques to improve the bioavailability of EGCG so that it can be translated to the clinical applications.

Ginger extract activates caspase independent paraptosis in cancer cells via ER stress, mitochondrial dysfunction, AIF translocation and DNA damage.

The rhizome of ginger (Zingiber officinale) a common culinary agent is also known for its medicinal activity. We have earlier reported that pure 6-shogaol, an important component of ginger induces paraptosis in triple negative breast cancer (MDA-MB-231) and non small cell lung (A549) cancer cells. However, the chemopreventive potential of the whole ginger extract in food remains to be elucidated. Here, we demonstrate for the first time that ginger extract (GE) triggers similar anticancer activity/paraptosis against the same cell lines but through different molecular mechanisms. Q-TOF LC-MS analysis of the extract showed the presence of several other metabolites along with 6-shogaol and 6-gingerol. GE induces cytoplasmic vacuolation through ER stress and dilation of the ER. Drastic decrease in the mitochondrial membrane potential and ATP production along with the excess generation of ROS contributed to mitochondrial dysfunction. Consequently, GE caused the translocation of apoptosis inducing factor to the nucleus leading to the fragmentation of DNA. Taken together, these show a novel mechanism for ginger extract induced cancer cell death that can be of potential interest for cancer preventive strategies.

Proteasomal dysfunction and ER stress triggers 2'-hydroxy-retrochalcone-induced paraptosis in cancer cells.

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2'-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

An α, ß-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of ß5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells.

Protein S-palmitoylation is markedly inhibited by 4″-alkyl ether lipophilic derivatives of EGCG, the major green tea polyphenol: In vitro and in silico studies.

S-palmitoylation is a dynamic lipid-based protein post-translational modification facilitated by a family of protein acyltransferases (PATs) commonly known as DHHC-PATs or DHHCs. It is the only lipid modification that is reversible, and this very fact uniquely qualifies it for therapeutic interventions through the development of DHHC inhibitors. Herein, we report that 4″-alkyl ether lipophilic derivatives of EGCG can effectively inhibit protein S-palmitoylation in vitro. With the help of metabolic labeling followed by copper(I)-catalyzed azide-alkyne cycloaddition Click reaction, we demonstrate that 4″-C14 EGCG and 4″-C16 EGCG markedly inhibited S-palmitoylation in various mammalian cells including HEK 293T, HeLa, and MCF-7 using both in gel fluorescence as well as confocal microscopy. Further, these EGCG derivatives were able to attenuate the S-palmitoylation to the basal level in DHHC3-overexpressed cells, suggesting that they are plausibly targeting DHHCs. Confocal microscopy data qualitatively reflected spatial and temporal distribution of S-palmitoylated proteins in different sub-cellular compartments and the inhibitory effects of 4″-C14 EGCG and 4″-C16 EGCG were clearly observed in the native cellular environment. Our findings were further substantiated by in silico analysis which revealed promising binding affinity and interactions of 4″-C14 EGCG and 4″-C16 EGCG with key amino acid residues present in the hydrophobic cleft of the DHHC20 enzyme. We also demonstrated the successful inhibition of S-palmitoylation of GAPDH by 4″-C16 EGCG. Taken together, our in vitro and in silico data strongly suggest that 4″-C14 EGCG and 4″-C16 EGCG can act as potent inhibitors for S-palmitoylation and can be employed as a complementary tool to investigate S-palmitoylation.

Mitochondria-targeting EGCG derivatives protect H9c2 cardiomyocytes from H2O2-induced apoptosis: design, synthesis and biological evaluation.

Pathologies related to cardiovascular diseases mostly emerge as a result of oxidative stress buildup in cardiomyocytes. The heavy load of mitochondrial oxidative phosphorylation in cardiac tissues corresponds to a surge in oxidative stress leading to mitochondrial dysfunction and cellular apoptosis. Thus, scavenging the reactive oxygen species (ROS) linked to mitochondria can significantly improve cardio-protection. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea has been extensively studied for its profound health-beneficial activities. Herein, we designed and synthesized a series of mitochondrial-targeting EGCG derivatives, namely MitoEGCGn (n = 4, 6, 8) by incorporating triphenylphosphonium ion onto it using different linkers. MitoEGCGn were found to be non-toxic to H9c2 rat cardiomyocyte cells even at higher doses in comparison to its parent molecule EGCG. Interestingly, MitoEGCG4 and MitoEGCG6 protected the H9c2 cardiomyocyte cells from the oxidative damage induced by H2O2 whereas EGCG was found to be toxic and ineffective in protecting the cells from H2O2 damage. MitoEGCG4 and MitoEGCG6 also protected the cells from the H2O2-induced disruption of mitochondrial membrane potential as well as activation of apoptosis as revealed by pro-caspase 3 expression profile, DNA fragmentation assay, and AO/EtBr staining. Taken together, our study shows that the mitochondria targeting EGCG derivatives were able to effectively combat the H2O2-induced oxidative stress in H9c2 cardiomyocytes. They eventually augmented the mitochondrial health of cardiomyocytes by maintaining the mitochondrial function and attenuating apoptosis. Overall, MitoEGCG4 and MitoEGCG6 could provision a cardioprotective role to H9c2 cardiomyocytes at the time of oxidative insults related to mitochondrial dysfunction-associated injuries.

In situ global proteomics profiling of EGCG targets using a cell-permeable and Click-able bioorthogonal probe.

Despite possessing a wide spectrum of biological activities, molecular targets of EGCG remain elusive and as a result, its precise mode of action is still unknown. Herein, we have developed a novel cell-permeable and Click-able bioorthogonal probe for EGCG, YnEGCG for in situ detection and identification of its interacting proteins. The strategic structural modification on YnEGCG allowed it to retain innate biological activities of EGCG (IC50 59.52 ± 1.14 µM and 9.07 ± 0.01 µM for cell viability and radical scavenging activity, respectively). Chemoproteomics profiling identified 160 direct EGCG targets, with H:L ratio ≥ 1.10 from the list of 207 proteins, including multiple new proteins that were previously unknown. The targets were broadly distributed in various subcellular compartments suggesting a polypharmacological mode of action of EGCG. GO analysis revealed that the primary targets belonged to the enzymes that regulate key metabolic processes including glycolysis and energy homeostasis, also the cytoplasm (36 %) and mitochondria (15.6 %) contain the majority of EGCG targets. Further, we validated that EGCG interactome was closely associated with apoptosis indicating its role in inducing toxicity in cancer cells. For the first time, this in situ chemoproteomics approach could identify a direct and specific EGCG interactome under physiological conditions in an unbiased manner.

Highly chemoselective turn-on fluorescent probe for ferrous (Fe2+) ion detection in cosmetics and live cells.

In modern society, the use of cosmetics has increased extensively; unfortunately, so-called several toxic metal salts are present as the colorant or filler in cosmetics. The ferrous ion (Fe2+) is one of the metal ions used in cosmetics as a colorant. Ferrous ion (Fe2+) is a vital component in live cells. Considering the adverse effect of high doses of ferrous ions in cosmetics and live cells, we developed a turn-on fluorescent probe PFe(II) for quantitative estimation of ferrous ion (Fe2+) in cosmetics and monitoring of labile ferrous (Fe2+) ion in live cells. The fluorescent probe PFe(II) showed a visual color change from colorless to orange in the presence of ferrous ion (Fe2+) in the cosmetics. We observed that UV-absorption increased at 390 nm upon incubation with ferrous ion (Fe2+). The probe PFe(II) has provided quantitative information on ferrous ion (Fe2+) in various cosmetics, kajol, lip balm, face foundation, mascara, eyeliner, lipliner, face makeup, sindoor, lipstick, nail polish in ppm level through the fluorescence signaling at 460 nm.The probe PFe(II) provided information on labile Fe2+ ion pool via a fluorescence imaging. It is a new addition to the diagnostic inventory for detecting ferrous ion in live cells and cosmetics.

In-vitro evaluation on drug release kinetics and antibacterial activity of dextran modified polyurethane fibrous membrane.

pH stimuli drug release nanofibrous membranes of polyurethane/dextran were developed for tailoring of antibacterial wound dressings. Incorporation of dextran in polyurethane (PU) showed increment in hydrophilicity, vapour transmission rate, percentage sorption values, and biodegradability. Dextran also acts as reinforcement filler in PU matrix. Dextran induces a high degree of platelet adhesion and hemostasis potential which is essential for promoting the wound healing process. Moreover, 20 wt% dextran loaded membranes (PU/20D) exhibited enhanced cell proliferation, attachment and viability against 3T3 fibroblasts. Curcumin loaded PU/20 dextran membrane exhibited pH-controlled drug release potency and synergistic antibacterial activity against gram-positive bacteria. It is confirmed that, PU/20D membranes could promote, pH-controlled drug release and synergistic antibacterial activity for a promising wound dressing material.

Plumbagin induces paraptosis in cancer cells by disrupting the sulfhydryl homeostasis and proteasomal function.

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.

Direct readout protonophore induced selective uncoupling and dysfunction of individual mitochondria within cancer cells.

Concurrently, manipulation of mitochondrial activity and its monitoring have enormous significance in cancer therapy and diagnosis. In this context, a fluorescent probe MitoDP has been developed for validating H2S mediated protonophore (2,4-dinitrophenol, DNP) induced mitochondrial membrane potential change, ROS formation and ATP depletion in cancer cells. The extent of protonophore activation for mitochondrial dysfunction is monitored through fluorescence signalling at 450 nm. The current study provides a proof for the concept of endogenous H2S-mediated controlled and spatial release of bioactive agents, or toxins specifically in mitochondria of cancer cells.

A bioorthogonal fluorescent probe for mitochondrial hydrogen sulfide: new strategy for cancer cell labeling.

We report the application of a chemodosimeter 'turn on' fluorescent probe for detecting endogenous H2S formation in cancer cells. Mito-HS showed a bathochromic shift in the UV-vis-absorption spectrum from 355 nm to 395 nm in the presence of H2S. Furthermore, it showed an ∼43-fold fluorescence enhancement at λem = 450 nm in the presence of H2S (200 µM). The cancer cell-specific fluorescence imaging reveals that Mito-HS has the ability to distinguish cancer cells from normal cells based on the level of endogenous H2S formation. In due course, Mito-HS would be a powerful cancer biomarker based on its ability to estimate endogenous H2S formation in living cells.