Celastrol and Rhynchophylline in the mitigation of simulated muscle atrophy under in vitro.

Muscular atrophy (MA) is a disease of various origins, i.e., genetic or the most common, caused by mechanical injury. So far, there is no universal therapeutic model because this disease is often progressive with numerous manifested symptoms. Moreover, there is no safe and low-risk therapy dedicated to muscle atrophy. For this reason, our research focuses on finding an alternative method using natural compounds to treat MA. This study proposes implementing natural substances such as celastrol and Rhynchophylline on the cellular level, using a simulated and controlled atrophy process. Methods: Celastrol and Rhynchophylline were used as natural compounds against simulated atrophy in C2C12 cells. Skeletal muscle C2C12 cells were stimulated for the differentiation process. Atrophic conditions were obtained by the exposure to the low concertation of doxorubicin and validated by FoxO3 and MAFbx. The protective and regenerative effect of drugs on cell proliferation was determined by the MTT assay and MT-CO1, VDAC1, and prohibitin expression. Results: The obtained results revealed that both natural substances reduced atrophic symptoms. Rhynchophylline and celastrol attenuated atrophic cells in the viability studies, morphology analysis by diameter measurements, modulated prohibitin VDAC, and MT-CO1 expression. Conclusions: The obtained results revealed that celastrol and Rhynchophylline could be effectively used as a supportive treatment in atrophy-related disorders. Thus, natural drugs seem promising for muscle regeneration.

Phytomodulatory proteins isolated from Calotropis procera latex promote glycemic control by improving hepatic mitochondrial function in HepG2 cells.

The medicinal uses of Calotropis procera are diverse, yet some of them are based on effects that still lack scientific support. Control of diabetes is one of them. Recently, latex proteins from C. procera latex (LP) have been shown to promote in vivo glycemic control by the inhibition of hepatic glucose production via AMP-activated protein kinase (AMPK). Glycemic control has been attributed to an isolated fraction of LP (CpPII), which is composed of cysteine peptidases (95%) and osmotin (5%) isoforms. Those proteins are extensively characterized in terms of chemistry, biochemistry and structural aspects. Furthermore, we evaluated some aspects of the mitochondrial function and cellular mechanisms involved in CpPII activity. The effect of CpPII on glycemic control was evaluated in fasting mice by glycemic curve and glucose and pyruvate tolerance tests. HepG2 cells was treated with CpPII, and cell viability, oxygen consumption, PPAR activity, production of lactate and reactive oxygen species, mitochondrial density and protein and gene expression were analyzed. CpPII reduced fasting glycemia, improved glucose tolerance and inhibited hepatic glucose production in control animals. Additionally, CpPII increased the consumption of ATP-linked oxygen and mitochondrial uncoupling, reduced lactate concentration, increased protein expression of mitochondrial complexes I, III and V, and activity of peroxisome-proliferator-responsive elements (PPRE), reduced the presence of reactive oxygen species (ROS) and increased mitochondrial density in HepG2 cells by activation of AMPK/PPAR. Our findings strongly support the medicinal use of the plant and suggest that CpPII is a potential therapy for prevention and/or treatment of type-2 diabetes. A common epitope sequence shared among the proteases and osmotin is possibly the responsible for the beneficial effects of CpPII.

Design, synthesis, DNA binding studies and evaluation of anticancer potential of novel substituted biscarbazole derivatives against human glioma U87 MG cell line.

In this research paper, we report the design and synthesis of novel substituted biscarbazole derivatives which were characterized by 1H and 13C NMR, high resolution mass spectroscopy (HRMS). The SAR study of the compounds is reported based on different substituents and their positions in the biscarbazole scaffold. In vitro cytotoxicity of the compounds was evaluated against human glioma U87 MG cell line by MTT assay for 24 h. The IC50 values of the compounds (30-35, 48-53 and 54-62) were calculated at the concentration range from 1.00 µM to 500 µM. The compound 34 showed the most significant in vitro cytotoxicity (IC50 = 3.9 µM) against human glioma U87 MG cell line and was found to be better than standard drugs used for the treatment of brain tumors such as temozolomide (IC50 = 100 µM) and carmustine (IC50 = 18.2 µM) respectively. To determine the mode of binding of compound 34 with CT-DNA, various biophysical techniques like UV-spectrophotometer, fluorescence, circular dichroism, viscosity, topoisomerase assay and molecular docking analysis, were used. Our results demonstrated groove binding mode of interaction of the compound 34 with CT-DNA with a plausible static bio-molecular quenching rate constant (Kq) 1.7 × 1012 M-1 s-1. The studies of biscarbazole derivatives are anticipated to develop potential novel anticancer agents against brain tumors.

Phytochemical constituents and anticancer activities of Tarchonanthus camphoratus essential oils grown in Saudi Arabia.

Tarchonanthus Camphoratus L. is traditionally known for its various medicinal purposes. In this study, the T. camphoratus essential oil (TCEO) was isolated via steam distillation, and its chemical constituents were determined using GC-MS. The in vitro antiproliferative effects of TCEO on A549, HepG2, MCF-7 cancer cells, and HUVEC non-tumor cells was investigated using an MTT assay. Flow cytometry analysis was conducted to evaluate cell cycle distribution using propidium iodide staining, and cell death mode using Annexin V-FITC/PI assays. The expression of some apoptosis related genes was investigated using qRT-PCR. Major constituents of TCEO included fenchol, borneol, 3-cyclohexene-1-methanol and 3-ethyl-3-methyl. Cell viability test showed that TCEO is highly effective against MCF-7 cells with IC50 12.5 µg/mL. Cell cycle arrest at the G1/S phase, and apoptosis mediation were evident in the presence of TCEO. Gene expression analysis of several pro-apoptotic and anti-apoptotic genes revealed the initiation of apoptosis in TCEO-MCF-7 cells. In conclusion, our study confirms the antiproliferative activity of the T. camphoratus essential oil.

A review on genus Millettia: Traditional uses, phytochemicals and pharmacological activities.

The genus Millettia belongs to Fabaceae includes 200 species which are distributed in tropical and subtropical regions of the world. Plants belong to this genus are used as folkloric medicine, for the treatment of different ailments like in wound healing, boil, sores, skin diseases, snake bite, muscle aches, pains, rheumatic arthritis, and gynaecological diseases. The aim of the review is to provide updated, comprehensive and categorized information on the aspects of ethnobotanical, phytochemical, pharmacological uses and toxicity of genus Millettia in order to identify their therapeutic potential and generate space for future research opportunities. The present study comprises of isolated flavonoids, phenolic compounds, phytosterols, saponins, alkaloids, polysaccharides, terpenoids and resins and pharmacological activities of various Millettia species. The relevant data were searched by using the keyword "Millettia" in different scientific databases like, "Google Scholar"; "NISCAIR repository"; "Pub Med"; "Science Direct"; "Scopus" and the taxonomy is validated by "The Plant List". This review discusses the existing information of the traditional evaluation as well as phytochemical and pharmacological evaluation of the extract and active constituents of the genus "Millettia". This review confirms that several Millettia species have emerged as a high-quality medicine in a traditional system for arthritis, wound healing, inflammation, skin diseases. Numerous conventional uses of Millettia species have been validated by modern pharmacology research. Intensive investigations of the genus Millettia relating to phytochemistry and pharmacology, especially their mechanism of action, safety, and efficacy could be the future research interests by the researcher in the area of phytomedicine.

Thymoquinone-induced antitumor and apoptosis in human lung adenocarcinoma cells.

BACKGROUND: Lung cancer has been associated with the highest cancer-associated mortality rate in the world. Chemotherapeutic management of cancer necessitates introducing new promising agents. Plants represent a rich source of new antineoplastic and chemotherapeutic agents. Thymoquinone (TQ), the main constituent of Nigella sativa (black seed or black cumin), has shown potent antioxidant and anti-inflammatory activities so far. The purpose of the current study was to evaluate the antineoplastic potential of TQ and their underlying mechanisms in A549 cells (human lung cancer cell line). METHOD: The A549 cells were treated with the different concentrations of TQ for three following days. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Necrosis and apoptosis were assessed by fluorescence-activated cell sorter analysis through propidium iodide and annexin V staining and also by assessing caspase-3 and -9 activation. DNA fragmentation was monitored by gel electrophoresis. RESULTS: TQ decreased the viability and increased apoptotic cell death in A549 human lung tumor cells. TQ treatment significantly elevated the Bax/ Bcl-2 ratio in the lung cancer cells. TQ also upregulated p53 expression, another apoptotic modulator in A549 cancer cells. TQ also activated caspase-dependent apoptosis by the activation of caspases-3 and -9. CONCLUSION: Our results proposed that TQ may be a potential new therapeutic agent for the management of lung cancer. TQ promoted apoptosis in A546 lung cancer cells by the activation of p53 and caspase cascade dependent pathways.

Tryptanthrin, a potential biofilm inhibitor against toxigenic Vibrio cholerae, modulating the global quorum sensing regulator, LuxO.

Cholera caused by the Gram-negative bacterium Vibrio cholerae still remains a major health burden in developing countries due to its high transmissibility and multidrug resistance. Alternative strategies are in quest to curtail the disease focusing on antivirulent approaches, such as biofilm inhibition, which make bacteria more susceptible to antibiotic therapies. The biofilm state is important for V. cholerae pathogenesis and its persistence in the environment. In the present study, tryptanthrin, a phytochemical, has been identified as possessing strong anti-biofilm activity at sub MIC (2 µg ml-1) against V. cholerae. LuxO was identified as the putative target of tryptanthrin by molecular docking and real time analysis. The phytochemical was identified as safe and possessed synergistic action with ciprofloxacin, a commonly used quinolone antibiotic to treat cholera. Collectively, the study establishes the first report on the anti-biofilm property of tryptanthrin by targeting LuxO, which could serve as a potential antivirulent therapy to combat V. cholerae infections.

Synthesis and Photodynamic Activity of Peri-xanthenoxanthene Derivatives.

Photodynamic therapy (PDT) is a modern cancer therapy. But it is still difficult to obtain ideal photosensitizers. We synthesized six new peri-xanthenoxanthene derivatives rapidly and efficiently using solid-phase carbon-bath microwave irradiation technology, and investigated their in vitro photodynamic antitumor activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our results showed that all compounds exhibited extremely low dark cytotoxicity and good phototoxicity against four human cancer cell lines. In particular, compound 3c showed the best in vitro PDT activity against Hela cells and Bel-7402 cells with IC50 values of 91 and 74 nmol/L, respectively. Its value of 1-octanol/water partition coefficient (log Kow) was 0.5309, suggesting that it is a promising photosensitizer for PDT due to its low dark cytotoxicity, high phototoxicity, and potential water solubility.

Evaluation of cytotoxicity levels of poly vinyl ether silicone, polyether, and poly vinyl siloxane impression materials: An in vitro study.

AIM: To assess the cytotoxicity level of newly introduced poly vinyl ether silicone (PVES) compared to poly vinyl siloxane (PVS) and polyether (PE) elastomeric impression materials. SETTINGS AND DESIGN: Comparative -Invitro study design. MATERIALS AND METHODS: Mouse cell line NIH/3T3 was grown in Dulbecco's modified Eagle's medium. Samples of three elastomers were dissolved in dimethyl sulfoxide and were tested at various concentrations. Twenty-four well plates with NIH/3T3 cells with different concentrations of elastomeric solutions were incubated at 37°C. 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay was performed on day 1, 3, and 7, with a time interval of 15 min, 30 min, 60 min, and 24th h to estimate the cytotoxicity for all three elastomers. STATISTICAL ANALYSIS USED: Kruskal-Wallis ANOVA test and the period effect within the subjects, repeated-measure ANOVA was done using the Greenhouse-Geisser correction method. RESULTS: The mean cell viability (survival rate) of NIH 3T3 cells at the concentrations tested was measured. A repeated-measure Kruskal-Wallis ANOVA determined the mean survival concentration on day 1, 3, and 7. PVES showed significant decrease in the survival rate on day 1 than PVS and PE, while PVS and PE had significant decrease in the survival rates of cells on day 3 and 7 which were statistically significant (P < 0.001). CONCLUSION: PVES shows early cytotoxic signs as compared to PVS and PE, and cell viability for PVS was the highest among all. When making impression with PVES and PE, it is always better to evaluate the impression and gingival sulcus carefully with magnification to prevent adverse reaction, if any material is left inadvertently for longer period of time.

Lipid metabolism alterations in the neuronal response to A53T α-synuclein and Fe-induced injury.

Pathological α-synuclein (α-syn) overexpression and iron (Fe)-induced oxidative stress (OS) are involved in the death of dopaminergic neurons in Parkinson's disease (PD). We have previously characterized the role of triacylglycerol (TAG) formation in the neuronal response to Fe-induced OS. In this work we characterize the role of the α-syn variant A53T during Fe-induced injury and investigate whether lipid metabolism has implications for neuronal fate. To this end, we used the N27 dopaminergic neuronal cell line either untransfected (UT) or stably transfected with pcDNA3 vector (as a transfection control) or pcDNA-A53T-α-syn (A53T α-syn). The overexpression of A53T α-syn triggered an increase in TAG content mainly due to the activation of Acyl-CoA synthetase. Since fatty acid (FA) ß-oxidation and phospholipid content did not change in A53T α-syn cells, the unique consequence of the increase in FA-CoA derivatives was their acylation in TAG moieties. Control cells exposed to Fe-induced injury displayed increased OS markers and TAG content. Intriguingly, Fe exposure in A53T α-syn cells promoted a decrease in OS markers accompanied by α-syn aggregation and elevated TAG content. We report here new evidence of a differential role played by A53T α-syn in neuronal lipid metabolism as related to the neuronal response to OS.

Antioxidant and anti-inflammatory activities of Lonicera japonica Thunb. var. sempervillosa Hayata flower bud extracts prepared by water, ethanol and supercritical fluid extraction techniques.

Lonicera japonica Thunberg (LJ) has long been used as an antipyretic, anti-inflammatory and anti-infectious agent in East Asia. The subspecies L. japonica Thunb. var. sempervillosa Hayata (LJv) is a variant that mainly grows in Taiwan. This study examined the antioxidant and anti-inflammatory activities of the extracts from the flower buds of these two species. The extracts were obtained by three extraction methods: water extraction, ethanol extraction, and supercritical-CO2 fluid extraction (SFE). The antioxidant activities of dry LJ (dLJ) extracts were superior to those of LJv extracts. Water extracts possessed higher activities than that prepared by ethanol or SFE. The total polyphenols content, total flavonoids content, and the amount of chlorogenic acid and luteolin-7-O-glucoside were all higher in the water extracts compared to the other two. The SFE extracts of these two species all exhibited excellent anti-inflammatory activities. Although the water and ethanol extracts of dLJ extracts had higher anti-inflammatory activity than that of LJv extracts, the SFE extracts prepared from fresh LJv flower buds (fLJv) exhibited the highest activity among all extracts. The SFE effectively isolates the bioactive components of L. japonica and can obtain the L. japonica extracts with high anti-inflammatory activity.

Calcium electroporation in three cell lines: a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions.

BACKGROUND: Electroporation with calcium (calcium electroporation) can induce ATP depletion-associated cellular death. In the clinical setting, the cytotoxic drug bleomycin is currently used with electroporation (electrochemotherapy) for palliative treatment of tumors. Calcium electroporation offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium electroporation and electrochemotherapy. METHODS: The effects of calcium electroporation and bleomycin electroporation (alone or in combination) were compared in three different cell lines (DC-3F, transformed Chinese hamster lung fibroblast; K-562, human leukemia; and murine Lewis Lung Carcinoma). Furthermore, the effects of electrical pulsing parameters and calcium compound on treatment efficacy were determined. RESULTS: Electroporation with either calcium or bleomycin significantly reduced cell survival (p<0.0001), without evidence of a synergistic effect. Cellular death following calcium or bleomycin treatment occurred at similar applied voltages, suggesting that similar parameters should be applied. At equimolar concentrations, calcium chloride and calcium glubionate resulted in comparable decreases in cell viability. CONCLUSIONS: Calcium electroporation and bleomycin electroporation significantly reduce cell survival at similar applied voltage parameters. The effect of calcium electroporation is independent of calcium compound. GENERAL SIGNIFICANCE: This study strongly supports the use of calcium electroporation as a potential cancer therapy and the results may aid in future clinical trials.

Synthesis and toxicity characterization of carbon coated iron oxide nanoparticles with highly defined size distributions.

BACKGROUND: Iron oxide nanoparticles hold great promise for future biomedical applications. To this end numerous studies on iron oxide nanoparticles have been conducted. One aspect these studies reveal is that nanoparticle size and shape can trigger different cellular responses through endocytic pathways, cell viability and early apoptosis. However, systematic studies investigating the size dependence of iron oxide nanoparticles with highly defined diameters across multiple cells lines are not available yet. METHODS: Iron oxide nanoparticles with well-defined size distributions were prepared. All samples were thoroughly characterized and the cytotoxicity for four standard cell lines (HeLa Kyoto, human osteosarcoma (U2OS), mouse fibroblasts (NIH 3T3) and mouse macrophages (J7442)) where investigated. RESULTS: Our findings show that small differences in size distribution (ca. 10nm) of iron oxide nanoparticles do not influence cytotoxicity, while uptake is size dependent. Cytotoxicity is dose-dependent. Broad distributions of nanoparticles are more easily internalized as compared to the narrow distributions for two of the cell lines tested (HeLa Kyoto and mouse macrophages (J7442)). CONCLUSION: The data indicate that it is not feasible to probe changes in cytotoxicity within a small size range (10nm). However, TEM investigations of the nanoparticles indicate that cellular uptake is size dependent. GENERAL SIGNIFICANCE: The present work compares narrow and broad distributions for various samples of carbon-coated iron oxide nanoparticles. The data highlights that cells differentiate between nanoparticle sizes as indicated by differences in cellular uptake. This information provides valuable knowledge to better understand the interaction of nanoparticles and cells.

Photodynamic activity of the boronated chlorin e6 amide in artificial and cellular membranes.

Photodynamic tumor-destroying activity of the boronated chlorin e6 derivative BACE (chlorin e6 13(1)-N-{2-[N-(1-carba-closo-dodecaboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester), previously described in Moisenovich et al. (2010) PLoS ONE 5(9) e12717, was shown here to be enormously higher than that of unsubstituted chlorin e6, being supported by the data on much higher photocytotoxicity of BACE in M-1 sarcoma cell culture. To validate membrane damaging effect as the basis of the enhanced tumoricidal activity, BACE was compared with unsubstituted chlorin e6 in the potency to photosensitize dye leakage from liposomes, transbilayer lipid flip-flop, inactivation of gramicidin A ionic channels in planar lipid membranes and erythrocyte hemolysis. In all the models comprising artificial and cellular membranes, the photodynamic effect of BACE exceeded that of chlorin e6. BACE substantially differed from chlorin e6 in the affinity to liposomes and erythrocytes, as monitored by fluorescence spectroscopy, flow cytometry and centrifugation. The results support the key role of membrane binding in the photodynamic effect of the boronated chlorin e6 amide.

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice.

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p<0.01) and IL-1ß (p<0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p<0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.

Quercetin modulates OTA-induced oxidative stress and redox signalling in HepG2 cells - up regulation of Nrf2 expression and down regulation of NF-κB and COX-2.

BACKGROUND: Ochratoxin A (OTA), a mycotoxin, causes extensive cell damage, affecting liver and kidney cells. OTA toxicity is fairly well characterized where oxidative stress is believed to play a role, however, the sequence of molecular events after OTA-exposure, have not been characterized in literature. Further, antidotes for alleviating the toxicity are sparsely reported. The aim of this study was to understand the sequence of some molecular mechanisms for OTA-induced toxicity and the cytoprotective effect of quercetin on OTA-induced toxicity. METHODS: Time course studies to evaluate the time of intracellular calcium release and ROS induction were carried out. The time of activation and induction of two key redox- sensitive transcription factors, NF-κB and Nrf-2 were determined by nuclear localization and expression respectively. The time of expression of inflammatory marker COX-2 was determined. Oxidative DNA damage by comet assay and micronucleus formation was studied. The ameliorative effect of quercetin on OTA-induced toxicity was also determined on all the above-mentioned parameters. RESULTS: OTA-induced calcium release, ROS generation and activated NF-κB nuclear translocation and expression. Pre-treatment with quercetin ameliorated ROS and calcium release as well as NF-κB induction and expression. Quercetin induced Nrf-2 nuclear translocation and expression. Quercetin's anti-inflammatory property was exhibited as it down regulated COX-2. Anti-genotoxic effect of quercetin was evident in prevention of DNA damage and micronucleus formation. CONCLUSION: Quercetin modulated OTA-induced oxidative stress and redox-signaling in HepG2 cells. GENERAL SIGNIFICANCE: The results of the study demonstrate for the first time that quercetin prevents OTA-induced toxicity in HepG2 cells.

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer.

BACKGROUND: Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling. METHODS: Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots. RESULTS: Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity. CONCLUSION: In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies. GENERAL SIGNIFICANCE: Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.

Green tea phenolics inhibit butyrate-induced differentiation of colon cancer cells by interacting with monocarboxylate transporter 1.

Diet has a significant impact on colorectal cancer and both dietary fiber and plant-derived compounds have been independently shown to be inversely related to colon cancer risk. Butyrate (NaB), one of the principal products of dietary fiber fermentation, induces differentiation of colon cancer cell lines by inhibiting histone deacetylases (HDACs). On the other hand, (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two abundant phenolic compounds of green tea, have been shown to exhibit antitumoral properties. In this study we used colon cancer cell lines to study the cellular and molecular events that take place during co-treatment with NaB, EC and EGCG. We found that (i) polyphenols EC and EGCG fail to induce differentiation of colon adenocarcinoma cell lines; (ii) polyphenols EC and EGCG reduce NaB-induced differentiation; (iii) the effect of the polyphenols is specific for NaB, since differentiation induced by other agents, such as trichostatin A (TSA), was unaltered upon EC and EGCG treatment, and (iv) is independent of the HDAC inhibitory activity of NaB. Also, (v) polyphenols partially reduce cellular NaB; and (vi) on a molecular level, reduction of cellular NaB uptake by polyphenols is achieved by impairing the capacity of NaB to relocalize its own transporter (monocarboxylate transporter 1, MCT1) in the plasma membrane. Our findings suggest that beneficial effects of NaB on colorectal cancer may be reduced by green tea phenolic supplementation. This valuable information should be of assistance in choosing a rational design for more effective diet-driven therapeutic interventions in the prevention or treatment of colorectal cancer.

Negative modulation of mitochondrial oxidative phosphorylation by epigallocatechin-3 gallate leads to growth arrest and apoptosis in human malignant pleural mesothelioma cells.

Increasing evidence reveals a large dependency of epithelial cancer cells on oxidative phosphorylation (OXPHOS) for energy production. In this study we tested the potential of epigallocatechin-3-gallate (EGCG), a natural polyphenol known to target mitochondria, in inducing OXPHOS impairment and cell energy deficit in human epitheliod (REN cells) and biphasic (MSTO-211H cells) malignant pleural mesothelioma (MMe), a rare but highly aggressive tumor with high unmet need for treatment. Due to EGCG instability that causes H2O2 formation in culture medium, the drug was added to MMe cells in the presence of exogenous superoxide dismutase and catalase, already proved to stabilize the EGCG molecule and prevent EGCG-dependent reactive oxygen species formation. We show that under these experimental conditions, EGCG causes the selective arrest of MMe cell growth with respect to normal mesothelial cells and the induction of mitochondria-mediated apoptosis, as revealed by early mitochondrial ultrastructure modification, swelling and cytochrome c release. We disclose a novel mechanism by which EGCG induces apoptosis through the impairment of mitochondrial respiratory chain complexes, particularly of complex I, II and ATP synthase. This induces a strong reduction in ATP production by OXPHOS, that is not adequately counterbalanced by glycolytic shift, resulting in cell energy deficit, cell cycle arrest and apoptosis. The EGCG-dependent negative modulation of mitochondrial energy metabolism, selective for cancer cells, gives an important input for the development of novel pharmacological strategies for MMe.

Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis.

Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433-451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3-30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1-3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS-MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell-cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.