Cytotoxic Effect Induced by Sicilian Oregano Essential Oil in Human Breast Cancer Cells.

Origanum vulgare L. is an aromatic plant that exerts antibacterial, antioxidant, anti-inflammatory, and antitumor activities, mainly due to its essential oil (EO) content. In this study, we investigated the possible mechanism underlying the in vitro antitumor activity of EO extracted by hydrodistillation of dried flowers and leaves of Origanum vulgare L. grown in Sicily (Italy) in MDA-MB-231 and MCF-7 breast cancer cell lines. Gas chromatography-mass spectrometry analysis of Oregano essential oil (OEO) composition highlighted the presence of twenty-six major phytocompounds, such as p-cymene, γ-terpinene, and thymoquinone p-acetanisole. OEO possesses strong antioxidant capacity, as demonstrated by the DPPH test. Our studies provided evidence that OEO reduces the viability of both MCF-7 and MDA-MB-231 cells. The cytotoxic effect of OEO on breast cancer cells was partially counteracted by the addition of z-VAD-fmk, a general caspase inhibitor. Caspases and mitochondrial dysfunction appeared to be involved in the OEO-induced death mechanism. Western blotting analysis showed that OEO-induced activation of pro-caspases-9 and -3 and fragmentation of PARP decreased the levels of Bcl-2 and Bcl-xL while increasing those of Bax and VDAC. In addition, fluorescence microscopy and cytofluorimetric analysis showed that OEO induces a loss of mitochondrial membrane potential in both cell lines. Furthermore, we tested the effects of p-cymene, γ-terpinene, thymoquinone, and p-acetanisole, which are the main components of OEO. Our findings highlighted that the effect of OEO on MDA-MB-231 and MCF-7 cells appears to be mainly due to the combination of different constituents of OEO, providing evidence of the potential use of OEO for breast cancer treatment.

Long non-coding RNA H19 enhances the pro-apoptotic activity of ITF2357 (a histone deacetylase inhibitor) in colorectal cancer cells.

Introduction: Long non-coding RNA H19 (lncH19) is highly expressed in colorectal cancer (CRC) and plays critical roles in tumor development, proliferation, metastasis, and drug resistance. Indeed, the expression of lncH19 usually affects the outcomes of chemo-, endocrine, and targeted therapies. ITF2357 (givinostat) is a histone deacetylase inhibitor (HDACi) that revealed a significant anti-tumor action by inducing apoptosis in different tumor models, including leukemia, melanoma, and glioblastoma. However, no data are present in the literature regarding the use of this compound for CRC treatment. Here, we investigate the role of lncH19 in ITF2357-induced apoptosis in CRC cells. Methods: The HCT-116 CRC cell line was stably silenced for H19 to investigate the role of this lncRNA in ITF2357-induced cell death. Cell viability assays and flow cytometric analyses were performed to assess the anti-proliferative and pro-apoptotic effects of ITF2357 in CRC cell lines that are silenced or not for lncH19. RT-PCR and Western blot were used to study the effects of ITF2357 on autophagy and apoptosis markers. Finally, bioinformatics analyses were used to identify miRNAs targeting pro-apoptotic factors that can be sponged by lncH19. Results: ITF2357 increased the expression levels of H19 and reduced HCT-116 cell viability, inducing apoptosis, as demonstrated by the increase in annexin-V positivity, caspase 3 cleavage, and poly (ADP-ribose) polymerase (PARP-1) degradation. Interestingly, the apoptotic effect of ITF2357 was much less evident in lncH19-silenced cells. We showed that lncH19 plays a functional role in the pro-apoptotic activity of the drug by stabilizing TP53 and its transcriptional targets, NOXA and PUMA. ITF2357 also induced autophagy in CRC cells, which was interpreted as a pro-survival response not correlated with lncH19 expression. Furthermore, ITF2357 induced apoptosis in 5-fluorouracil-resistant HCT-116 cells that express high levels of lncH19. Conclusion: This study shows that lncH19 expression contributes to ITF2357-induced apoptosis by stabilizing TP53. Overall, we suggest that lncH19 expression may be exploited to favor HDACi-induced cell death and overcome 5-fluorouracil chemoresistance.

Foodomics-Based Approaches Shed Light on the Potential Protective Effects of Polyphenols in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic and progressive inflammatory disorder affecting the gastrointestinal tract (GT) caused by a wide range of genetic, microbial, and environmental factors. IBD is characterized by chronic inflammation and decreased gut microbial diversity, dysbiosis, with a lower number of beneficial bacteria and a concomitant increase in pathogenic species. It is well known that dysbiosis is closely related to the induction of inflammation and oxidative stress, the latter caused by an imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity, leading to cellular ROS accumulation. ROS are responsible for intestinal epithelium oxidative damage and the increased intestinal permeability found in IBD patients, and their reduction could represent a potential therapeutic strategy to limit IBD progression and alleviate its symptoms. Recent evidence has highlighted that dietary polyphenols, the natural antioxidants, can maintain redox equilibrium in the GT, preventing gut dysbiosis, intestinal epithelium damage, and radical inflammatory responses. Here, we suggest that the relatively new foodomics approaches, together with new technologies for promoting the antioxidative properties of dietary polyphenols, including novel delivery systems, chemical modifications, and combination strategies, may provide critical insights to determine the clinical value of polyphenols for IBD therapy and a comprehensive perspective for implementing natural antioxidants as potential IBD candidate treatment.

MCL1 Inhibition Overcomes the Aggressiveness Features of Triple-Negative Breast Cancer MDA-MB-231 Cells.

Triple-Negative Breast Cancer (TNBC) is a particularly aggressive subtype among breast cancers (BCs), characterized by anoikis resistance, high invasiveness, and metastatic potential as well as Epithelial-Mesenchymal Transition (EMT) and stemness features. In the last few years, our research focused on the function of MCL1, an antiapoptotic protein frequently deregulated in TNBC. Here, we demonstrate that MCL1 inhibition by A-1210477, a specific BH3-mimetic, promotes anoikis/apoptosis in the MDA-MB-231 cell line, as shown via an increase in proapoptotic markers and caspase activation. Our evidence also shows A-1210477 effects on Focal Adhesions (FAs) impairing the integrin trim and survival signaling pathways, such as FAK, AKT, ERK, NF-κB, and GSK3ß-inducing anoikis, thus suggesting a putative role of MCL1 in regulation of FA dynamics. Interestingly, in accordance with these results, we observed a reduction in migratory and invasiveness capabilities as confirmed by a decrease in metalloproteinases (MMPs) levels following A-1210477 treatment. Moreover, MCL1 inhibition promotes a reduction in EMT characteristics as demonstrated by the downregulation of Vimentin, MUC1, DNMT1, and a surprising re-expression of E-Cadherin, suggesting a possible mesenchymal-like phenotype reversion. In addition, we also observed the downregulation of stemness makers such as OCT3/4, SOX2, NANOG, as well as CD133, EpCAM, and CD49f. Our findings support the idea that MCL1 inhibition in MDA-MB-231 could be crucial to reduce anoikis resistance, aggressiveness, and metastatic potential and to minimize EMT and stemness features that distinguish TNBC.

Oncogenic BRAF and p53 Interplay in Melanoma Cells and the Effects of the HDAC Inhibitor ITF2357 (Givinostat).

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.

A Deadly Liaison between Oxidative Injury and p53 Drives Methyl-Gallate-Induced Autophagy and Apoptosis in HCT116 Colon Cancer Cells.

Methyl gallate (MG), which is a gallotannin widely found in plants, is a polyphenol used in traditional Chinese phytotherapy to alleviate several cancer symptoms. Our studies provided evidence that MG is capable of reducing the viability of HCT116 colon cancer cells, while it was found to be ineffective on differentiated Caco-2 cells, which is a model of polarized colon cells. In the first phase of treatment, MG promoted both early ROS generation and endoplasmic reticulum (ER) stress, sustained by elevated PERK, Grp78 and CHOP expression levels, as well as an upregulation in intracellular calcium content. Such events were accompanied by an autophagic process (16-24 h), where prolonging the time (48 h) of MG exposure led to cellular homeostasis collapse and apoptotic cell death with DNA fragmentation and p53 and γH2Ax activation. Our data demonstrated that a crucial role in the MG-induced mechanism is played by p53. Its level, which increased precociously (4 h) in MG-treated cells, was tightly intertwined with oxidative injury. Indeed, the addition of N-acetylcysteine (NAC), which is a ROS scavenger, counteracted the p53 increase, as well as the MG effect on cell viability. Moreover, MG promoted p53 accumulation into the nucleus and its inhibition by pifithrin-α (PFT-α), which is a negative modulator of p53 transcriptional activity, enhanced autophagy, increased the LC3-II level and inhibited apoptotic cell death. These findings provide new clues to the potential action of MG as a possible anti-tumor phytomolecule for colon cancer treatment.

The Histone Deacetylase Inhibitor ITF2357 (Givinostat) Targets Oncogenic BRAF in Melanoma Cells and Promotes a Switch from Pro-Survival Autophagy to Apoptosis.

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy.

Parthenolide and Its Soluble Analogues: Multitasking Compounds with Antitumor Properties.

Due to its chemical properties and multiple molecular effects on different tumor cell types, the sesquiterpene lactone parthenolide (PN) can be considered an effective drug with significant potential in cancer therapy. PN has been shown to induce either classic apoptosis or alternative caspase-independent forms of cell death in many tumor models. The therapeutical potential of PN has been increased by chemical design and synthesis of more soluble analogues including dimethylaminoparthenolide (DMAPT). This review focuses on the molecular mechanisms of both PN and analogues action in tumor models, highlighting their effects on gene expression, signal transduction and execution of different types of cell death. Recent findings indicate that these compounds not only inhibit prosurvival transcriptional factors such as NF-κB and STATs but can also determine the activation of specific death pathways, increasing intracellular reactive oxygen species (ROS) production and modifications of Bcl-2 family members. An intriguing property of these compounds is its specific targeting of cancer stem cells. The unusual actions of PN and its analogues make these agents good candidates for molecular targeted cancer therapy.

Bio-Waste Products of Mangifera indica L. Reduce Adipogenesis and Exert Antioxidant Effects on 3T3-L1 Cells.

Several studies highlighted the beneficial value of natural compounds in the prevention and treatment of obesity. Here, we investigated the anti-obesity effects of extracts of peel and seed of mango (Mangifera indica L.) cultivated in Sicily (Italy) in 3T3-L1 cells. Mango Peel (MPE) and Mango Seed (MSE) extracts at a 100 µg/mL concentration significantly reduced lipid accumulation and triacylglycerol contents during 3T3-L1 adipocyte differentiation without toxicity. HPLC-ESI-MS analysis showed that both the extracts contain some polyphenolic compounds that can account for the observed biological effects. The anti-adipogenic effect of MPE and MSE was the result of down-regulation of the key adipogenic transcription factor PPARγ and its downstream targets FABP4/aP2, GLUT4 and Adipsin, as well SREBP-1c, a transcription factor which promotes lipogenesis. In addition, both MPE and MSE significantly activated AMPK with the consequent inhibition of Acetyl-CoA-carboxylase (ACC) and up-regulated PPARα. The addition of compound C, a specific AMPK inhibitor, reduced the effects of MPE and MSE on AMPK and ACC phosphorylation, suggesting a role of AMPK in mediating MPE and MSE anti-lipogenic effects. Notably, MPE and MSE possess an elevated radical scavenging activity, as demonstrated by DPPH radical scavenging assay, and reduced ROS content produced during adipocyte differentiation. This last effect could be a consequence of the increase in the antioxidant factors Nrf2, MnSOD and HO-1. In conclusion, MPE and MSE possesses both anti-adipogenic and antioxidant potential, thus suggesting that the bio-waste products of mango are promising anti-obesity natural compounds.

The Beneficial Effects of Essential Oils in Anti-Obesity Treatment.

Obesity is a complex disease caused by an excessive amount of body fat. Obesity is a medical problem and represents an important risk factor for the development of serious diseases such as insulin resistance, type 2 diabetes, cardiovascular disease, and some types of cancer. Not to be overlooked are the psychological issues that, in obese subjects, turn into very serious pathologies, such as depression, phobias, anxiety, and lack of self-esteem. In addition to modifying one's lifestyle, the reduction of body mass can be promoted by different natural compounds such as essential oils (EOs). EOs are mixtures of aromatic substances produced by many plants, particularly in medicinal and aromatic ones. They are odorous and volatile and contain a mixture of terpenes, alcohols, aldehydes, ketones, and esters. Thanks to the characteristics of the various chemical components present in them, EOs are used in the food, cosmetic, and pharmaceutical fields. Indeed, it has been shown that EOs possess great antibiotic, anti-inflammatory, and antitumor powers. Emerging results also demonstrate the anti-obesity effects of EOs. We have examined the main data obtained in experimental studies and, in this review, we summarize the effect of EOs in obesity and obesity-related metabolic diseases.

The Good and Bad of Nrf2: An Update in Cancer and New Perspectives in COVID-19.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a well-known transcription factor best recognised as one of the main regulators of the oxidative stress response. Beyond playing a crucial role in cell defence by transactivating cytoprotective genes encoding antioxidant and detoxifying enzymes, Nrf2 is also implicated in a wide network regulating anti-inflammatory response and metabolic reprogramming. Such a broad spectrum of actions renders the factor a key regulator of cell fate and a strategic player in the control of cell transformation and response to viral infections. The Nrf2 protective roles in normal cells account for its anti-tumour and anti-viral functions. However, Nrf2 overstimulation often occurs in tumour cells and a complex correlation of Nrf2 with cancer initiation and progression has been widely described. Therefore, if on one hand, Nrf2 has a dual role in cancer, on the other hand, the factor seems to display a univocal function in preventing inflammation and cytokine storm that occur under viral infections, specifically in coronavirus disease 19 (COVID-19). In such a variegate context, the present review aims to dissect the roles of Nrf2 in both cancer and COVID-19, two widespread diseases that represent a cause of major concern today. In particular, the review describes the molecular aspects of Nrf2 signalling in both pathological situations and the most recent findings about the advantages of Nrf2 inhibition or activation as possible strategies for cancer and COVID-19 treatment respectively.

Tributyltin(IV) Butyrate: A Novel Epigenetic Modifier with ER Stress- and Apoptosis-Inducing Properties in Colon Cancer Cells.

Organotin(IV) compounds are a class of non-platinum metallo-conjugates exhibiting antitumor activity. The effects of different organotin types has been related to several mechanisms, including their ability to modify acetylation protein status and to promote apoptosis. Here, we focus on triorganotin(IV) complexes of butyric acid, a well-known HDAC inhibitor with antitumor properties. The conjugated compounds were synthesized and characterised by FTIR spectroscopy, multi-nuclear (1H, 13C and 119Sn) NMR, and mass spectrometry (ESI-MS). In the triorganotin(IV) complexes, an anionic monodentate butyrate ligand was observed, which coordinated the tin atom on a tetra-coordinated, monomeric environment similar to ester. FTIR and NMR findings confirm this structure both in solid state and solution. The antitumor efficacy of the triorganotin(IV) butyrates was tested in colon cancer cells and, among them, tributyltin(IV) butyrate (BT2) was selected as the most efficacious. BT2 induced G2/M cell cycle arrest, ER stress, and apoptotic cell death. These effects were obtained using low concentrations of BT2 up to 1 µM, whereas butyric acid alone was completely inefficacious, and the parent compound TBT was poorly effective at the same treatment conditions. To assess whether butyrate in the coordinated form maintains its epigenetic effects, histone acetylation was evaluated and a dramatic decrease in acetyl-H3 and -H4 histones was found. In contrast, butyrate alone stimulated histone acetylation at a higher concentration (5 mM). BT2 was also capable of preventing histone acetylation induced by SAHA, another potent HDAC inhibitor, thus suggesting that it may activate HDACs. These results support a potential use of BT2, a novel epigenetic modulator, in colon cancer treatment.

Redox Imbalance and Mitochondrial Release of Apoptogenic Factors at the Forefront of the Antitumor Action of Mango Peel Extract.

Today, an improved understanding of cancer cell response to cellular stress has become more necessary. Indeed, targeting the intracellular pro-oxidant/antioxidant balance triggering the tumor commitment to cell demise could represent an advantageous strategy to develop cancer-tailored therapies. In this scenario, the present study shows how the peel extract of mango-a tropical fruit rich in phytochemicals with nutraceutical properties-can affect the cell viability of three colon cancer cell lines (HT29, Caco-2 and HCT116), inducing an imbalance of cellular redox responses. By using hydro-alcoholic mango peel extract (MPE), we observed a consistent decline in thiol group content, which was accompanied by upregulation of MnSOD-a mitochondrial scavenger enzyme that modulates the cellular response against oxidative damage. Such an effect was the consequence of an early production of mitochondrial superoxide anions that appeared after just 30 min of exposure of colon cancer cells to MPE. The effect was accompanied by mitochondrial injury, consisting of the dissipation of mitochondrial membrane potential and a decrease in the level of proteins localized in the mitochondrial membrane-such as voltage-dependent anion-selective channel (VDAC1), mitofilin, and some members of Bcl-2 family proteins (Mcl-1, Bcl-2 and Bcl-XL)-with the mitochondrial release of apoptogenic factors (cytochrome C and AIF). The analysis of the cytotoxic effects exerted by the different constituents of MPE (gallic acid, mangiferin, citric acid, quinic acid, pentagalloyl glucose, and methyl gallate) allowed us to identify those phytochemicals responsible for the observed anticancer effects, sustaining their future employment as chemopreventive or therapeutic agents.

ROS-Dependent ER Stress and Autophagy Mediate the Anti-Tumor Effects of Tributyltin (IV) Ferulate in Colon Cancer Cells.

Organotin compounds represent potential cancer therapeutics due to their pro-apoptotic action. We recently synthesized the novel organotin ferulic acid derivative tributyltin (IV) ferulate (TBT-F) and demonstrated that it displays anti-tumor properties in colon cancer cells related with autophagic cell death. The purpose of the present study was to elucidate the mechanism of TBT-F action in colon cancer cells. We specifically show that TBT-F-dependent autophagy is determined by a rapid generation of reactive oxygen species (ROS) and correlated with endoplasmic reticulum (ER) stress. TBT-F evoked nuclear factor erythroid-2 related factor 2 (Nrf2)-mediated antioxidant response and Nrf2 silencing by RNA interference markedly increased the anti-tumor efficacy of the compound. Moreover, as a consequence of ROS production, TBT-F increased the levels of glucose regulated protein 78 (Grp78) and C/EBP homologous protein (CHOP), two ER stress markers. Interestingly, Grp78 silencing produced significant decreasing effects on the levels of the autophagic proteins p62 and LC3-II, while only p62 decreased in CHOP-silenced cells. Taken together, these results indicate that ROS-dependent ER stress and autophagy play a major role in the TBT-F action mechanism in colon cancer cells and open a new perspective to consider the compound as a potential candidate for colon cancer treatment.

p62: Friend or Foe? Evidences for OncoJanus and NeuroJanus Roles.

p62 is a versatile protein involved in the delicate balance between cell death and survival, which is fundamental for cell fate decision in the context of both cancer and neurodegenerative diseases. As an autophagy adaptor, p62 recognizes polyubiquitin chains and interacts with LC3, thereby targeting the selected cargo to the autophagosome with consequent autophagic degradation. Beside this function, p62 behaves as an interactive hub in multiple signalling including those mediated by Nrf2, NF-κB, caspase-8, and mTORC1. The protein is thus crucial for the control of oxidative stress, inflammation and cell survival, apoptosis, and metabolic reprogramming, respectively. As a multifunctional protein, p62 falls into the category of those factors that can exert opposite roles in the cells. Chronic p62 accumulation was found in many types of tumors as well as in stress granules present in different forms of neurodegenerative diseases. However, the protein seems to have a Janus behaviour since it may also serve protective functions against tumorigenesis or neurodegeneration. This review describes the diversified roles of p62 through its multiple domains and interactors and specifically focuses on its oncoJanus and neuroJanus roles.

Tributyltin(IV) ferulate, a novel synthetic ferulic acid derivative, induces autophagic cell death in colon cancer cells: From chemical synthesis to biochemical effects.

Ferulic acid (FA) is a natural phenolic phytochemical that has low toxicity and exhibits therapeutic effects against various diseases, behaving as an antioxidant. FA also displays modest antitumor properties that have been reported at relatively high concentrations. With the aim of improving the anti-tumor efficacy of FA, we synthesized the novel compound tributyltin(IV) ferulate (TBT-F). The coordination environment at the tin center was investigated spectroscopically. Following synthesis, chemical characterization and computational analysis, we evaluated TBT-F effects in colon cancer cells. The results showed that TBT-F, at nanomolar range concentrations, was capable of reducing the viability of HCT116, HT-29 and Caco-2 colon cancer cells. On the other hand, FA was completely inefficacious at the same treatment conditions. Cell viability reduction induced by TBT-F was associated with G2/M cell cycle arrest, increase in membrane permeabilization and appearance of typical morphological signs. TBT-F-induced cell death seemed not to involve apoptotic or necroptotic markers whereas autophagic vacuoles appearance and increase in LC3-II and p62 autophagic proteins were observed after treatment with the compound. The autophagy inhibitor bafylomicin A1 markedly prevented the effect of TBT-F on colon cancer cells, thus indicating that autophagy is triggered as a cell death process. Taken together, our results strongly suggest that the novel ferulic derivative TBT-F is a promising therapeutic agent for colon cancer since it is capable of triggering autophagic (type-II) cell death that may be important in case of resistance to classic apoptosis.

WIN55,212-2-Induced Expression of Mir-29b1 Favours the Suppression of Osteosarcoma Cell Migration in a SPARC-Independent Manner.

WIN55,212-2 (WIN) is a synthetic agonist of cannabinoid receptors that displays promising antitumour properties. The aim of this study is to demonstrate that WIN is able to block the migratory ability of osteosarcoma cells and characterize the mechanisms involved. Using wound healing assay and zymography, we showed that WIN affects cell migration and reduces the activity of the metalloproteases MMP2 and MMP9. This effect seemed to be independent of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein involved in tissue remodeling and extracellular matrix deposition. SPARC release was indeed prevented by WIN, and SPARC silencing by RNA interference did not influence the effect of the cannabinoid on cell migration. WIN also increased the release of extracellular vesicles and dramatically upregulated miR-29b1, a key miRNA that modulates cell proliferation and migration. Interestingly, reduced cell migration was observed in stably miR-29b1-transfected cells, similarly to WIN-treated cells. Finally, we show the absence of SPARC in the extracellular vesicles released by osteosarcoma cells and no changes in SPARC level in miR-29b1 overexpressing cells. Overall, these findings suggest that WIN markedly affects cell migration, dependently on miR-29b1 and independently of SPARC, and can thus be considered as a potential innovative therapeutic agent in the treatment of osteosarcoma.

The Anti-Cancer Effect of Mangifera indica L. Peel Extract is Associated to γH2AX-mediated Apoptosis in Colon Cancer Cells.

Ethanolic extracts from Mangifera indica L. have been proved to possess anti-tumor properties in many cancer systems. However, although most effects have been demonstrated with fruit pulp extract, the underlying molecular mechanisms of mango peel are still unclear. This study was designed to explore the effects of mango peel extract (MPE) on colon cancer cell lines. MPE affected cell viability and inhibited the colony formation trend of tumor cells, while no effects were observed in human dermal fibroblasts used as a non-cancerous cell line model. These events were a consequence of the induction of apoptosis associated to reactive oxygen species (ROS) production, activation of players of the oxidative response such as JNK and ERK1/2, and the increase in Nrf2 and manganese superoxide dismutase (MnSOD). Significantly, mango peel-activated stress triggered a DNA damage response evidenced by the precocious phosphorylation of histone 2AX (γH2AX), as well as phosphorylated Ataxia telangiectasia-mutated (ATM) kinase and p53 upregulation. Mango peel extract was also characterized, and HPLC/MS (High Performance Liquid Chromatography/Mass Spectrometry) analysis unveiled the presence of some phenolic compounds that could be responsible for the anti-cancer effects. Collectively, these findings point out the importance of the genotoxic stress signaling pathway mediated by γH2AX in targeting colon tumor cells to apoptosis.

Ethanol-Mediated Stress Promotes Autophagic Survival and Aggressiveness of Colon Cancer Cells via Activation of Nrf2/HO-1 Pathway.

Epidemiological studies suggest that chronic alcohol consumption is a lifestyle risk factor strongly associated with colorectal cancer development and progression. The aim of the present study was to examine the effect of ethanol (EtOH) on survival and progression of three different colon cancer cell lines (HCT116, HT29, and Caco-2). Our data showed that EtOH induces oxidative and endoplasmic reticulum (ER) stress, as demonstrated by reactive oxygen species (ROS) and ER stress markers Grp78, ATF6, PERK and, CHOP increase. Moreover, EtOH triggers an autophagic response which is accompanied by the upregulation of beclin, LC3-II, ATG7, and p62 proteins. The addition of the antioxidant N-acetylcysteine significantly prevents autophagy, suggesting that autophagy is triggered by oxidative stress as a prosurvival response. EtOH treatment also upregulates the antioxidant enzymes SOD, catalase, and heme oxygenase (HO-1) and promotes the nuclear translocation of both Nrf2 and HO-1. Interestingly, EtOH also upregulates the levels of matrix metalloproteases (MMP2 and MMP9) and VEGF. Nrf2 silencing or preventing HO-1 nuclear translocation by the protease inhibitor E64d abrogates the EtOH-induced increase in the antioxidant enzyme levels as well as the migration markers. Taken together, our results suggest that EtOH mediates both the activation of Nrf2 and HO-1 to sustain colon cancer cell survival, thus leading to the acquisition of a more aggressive phenotype.

Sicilian Litchi Fruit Extracts Induce Autophagy versus Apoptosis Switch in Human Colon Cancer Cells.

Litchi chinensis Sonnerat is a tropical tree whose fruits contain significant amounts of bioactive polyphenols. Litchi cultivation has recently spread in Sicily where the climate conditions are particularly favorable for this crop. Recent findings have shown that Litchi extracts display anti-tumor and pro-apoptotic effects in vitro, but the precise underlying mechanisms have not been fully elucidated. In this study, we report for the first time the effects of Sicilian litchi fruit extracts on colon cancer cells. The results indicated that litchi exocarp, mesocarp and endocarp fractions reduce the viability and clonogenic growth of HT29 cells. These effects were due to cell cycle arrest in the G2/M phase followed by caspase-dependent cell death. Interestingly, litchi exocarp and endocarp triggered a precocious autophagic response (16⁻24 h), which was accompanied by an increase in the level of autophagy related 1/autophagy activating kinase 1 (ATG1/ULK1), beclin-1, microtubule associated protein 1 light chain 3 (LC3)-II and p62 proteins. Autophagy inhibition by bafilomycin A1 or beclin-1 silencing increased cell death, thus suggesting that autophagy was initially triggered as a pro-survival response. Significant effects of Litchi extracts were also observed in other colon cancer cells, including HCT116 and Caco-2 cells. On the other hand, differentiated Caco-2 cells, a model of human enterocytes, appeared to be insensitive to the extracts at the same treatment conditions. High-Performance Liquid Chromatography⁻Electrospray Ionization-Quadrupole-Time-Of-Flight HPLC/ESI/Q-TOF evidenced the presence of some polyphenolic compounds, specifically in exocarp and endocarp extracts, that can account for the observed biological effects. The results obtained suggest a potential therapeutic efficacy of polyphenolic compounds purified from Sicilian Litchi fractions for the treatment of colon cancer. Moreover, our findings indicate that modulation of autophagy can represent a tool to improve the effectiveness of these agents and potentiate the anti-tumor response of colon cancer cells.