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Abstract

Treating neuropathic pain remains challenging, and therefore new pharmacological strategies are urgently required. Here, the enhancement of glycinergic neurotransmission by either facilitating glycine receptors (GlyR) or inhibiting glycine transporter (GlyT) function to increase extracellular glycine concentration appears promising. Propacetamol is a N,N-diethylester of acetaminophen, a non-opioid analgesic used to treat mild pain conditions. In vivo, it is hydrolysed into N,N-diethylglycine (DEG) and acetaminophen. DEG has structural similarities to known alternative GlyT1 substrates. In this study, we analyzed possible effects of propacetamol, or its metabolite N,N-diethylglycine (DEG), on GlyRs or GlyTs function by using a two-electrode voltage clamp approach in Xenopus laevis oocytes. Our data demonstrate that, although propacetamol or acetaminophen had no effect on the function of the analysed glycine-responsive proteins, the propacetamol metabolite DEG acted as a low-affine substrate for both GlyT1 (EC50 > 7.6 mM) and GlyT2 (EC50 > 5.2 mM). It also acted as a mild positive allosteric modulator of GlyRα1 function at intermediate concentrations. Taken together, our data show that DEG influences both glycine transporter and receptor function, and therefore could facilitate glycinergic neurotransmission in a multimodal manner.

1. Introduction

To foster a fast translation to clinical application, we searched for substances that might have potential effects on glycine sensitive proteins, but that are already used in clinics. We focused on substances that display, by themselves or by one of their metabolites, structural similarities to known GlyT1 substrates (i.e., glycine, sarcosine, or N-ethylglycine). Here, we identified propacetamol, a N,N-diethylglycine ester of acetaminophen that was developed initially to increase its solubility for a possible intravenous application and to reduce hepatotoxicity by prolonged release of acetaminophen [,]. After systemic application of propacetamol, it is cleaved by esterase to acetaminophen and N’N’-Diethyl-glycine (). In this study we assessed the effect of N,N-diethylglycine (DEG) on both glycine transporter (GlyT) and glycine receptor (GlyR) function in a Xenopus laevis oocyte expression system by a two-electrode voltage clamp approach. We demonstrate that DEG, but not acetaminophen or propacetamol, functions as a low affinity alternative substrate for both GlyT1 and GlyT2. Moreover, DEG shows partial agonistic activity on homomeric GlyRα1, but not on GlyRα2 or GlyRα3. Taken together, our data raise the possibility that propacetamol, in addition to its well-characterized function as a COX inhibitor via acetaminophen, might influence glycine-dependent neurotransmission via a multimodal mechanism.

2.4. Electrophysiology

All two-electrode-voltage-clamp (TEVC) recordings were performed using a TurboTec-03X amplifier (npi elctronics, Tamm, Germany), and a custom-made recording chamber with the membrane potential set to −50 mV. Only oocytes showing a stable leak current <300 nA at the beginning of the experiment were used. Oocytes were superfused with ND96-Ca2+. Solutions containing substances at the indicated concentration for 20 s followed by a washout for at least 30 s or until stable baseline currents were observed. All measurements were performed at room temperature (21–23 °C). Current traces were evaluated using the software Cell Works and Cell Works Reader (npi electronics, Tamm, Germany). Excel (Microsoft, Redmond, WA, USA) and Graphpad Prism 7 (Graphpad Software, Inc., San Diego, CA, USA) were used for statistical analysis. Data are presented as mean ± SEM in dose-response curves or boxplots with whiskers from minimum to maximum with median and quartiles.

3.2. GlyT Responses to Propacetamol and Paracetamol and DEG

To test if propacetamol or acetaminophen had any effect on the activity of GlyT1 or GlyT2, GlyT-expressing oocytes were superfused with each of these substances. Neither propacetamol nor acetaminophen alone produced any current response. Alkaline pretreatment, however, which is predicted to cause hydrolysis of the propacetamol (10 mM) to acetaminophen and DEG, resulted in significant inward currents in both GlyT1 and GlyT2-expressing oocytes, reaching 20–40% of the maximum current amplitude. Currents of slightly larger amplitudes (50–70%) were observed after the application of 10 mM DEG alone. Adding 20 µM glycine to 10 mM acetaminophen and 10 mM propacetamol solution each did not significantly alter the current response of any GlyT compared to 20 µM glycine solution alone (A,B).

Abstract

Supplementation with [6S]-5-methyltetrahydrofolic acid (MTHF) is recommended as an alternative to folic acid (FA) in prenatal supplements. This study compared equimolar gestational FA and MTHF diets on energy regulation of female offspring. Wistar rats were fed an AIN-93G diet with recommended (2 mg/kg diet) or 5-fold (5X) intakes of MTHF or FA. At weaning, female offspring were fed a 45% fat diet until 19 weeks. The 5X-MTHF offspring had higher body weight (>15%), food intake (8%), light-cycle energy expenditure, and lower activity compared to 5X-FA offspring (p < 0.05). Both the 5X offspring had higher plasma levels of the anorectic hormone leptin at birth (60%) and at 19 weeks (40%), and lower liver weight and total liver lipids compared to the 1X offspring (p < 0.05). Hypothalamic mRNA expression of leptin receptor (ObRb) was lower, and of suppressor of cytokine signaling-3 (Socs3) was higher in the 5X-MTHF offspring (p < 0.05), suggesting central leptin dysregulation. In contrast, the 5X-FA offspring had higher expression of genes encoding for dopamine and GABA- neurotransmitter receptors (p < 0.01), consistent with their phenotype and reduced food intake. When fed folate diets at the requirement level, no differences were found due to form in the offspring. We conclude that MTHF compared to FA consumed at high levels in the gestational diets program central and peripheral mechanisms to favour increased weight gain in the offspring. These pre-clinical findings caution against high gestational intakes of folates of either form and encourage clinical trials examining their long-term health effects when consumed during pregnancy.

2.1. Animals and Diets

First-time pregnant (2–3 days) Wistar rats weighing 170–250 g (Charles River Farms, QC, Canada) were singly housed in a 12 h dark-light cycle (lights on at 0700 hrs at 22 ± 1 °C) and provided ad libitum food and water throughout the study. The protocol (# 20011892) was approved by the University of Toronto Animal Care Committee. Upon arrival, dams (n = 16–18/group) were randomly allocated to one of four custom-made dietary interventions (Research Diets Inc., New Brunswick, NJ, USA) that were provided only during their 3-week pregnancy: an AIN-93G diet (energy density 4.0 kcal/g) containing FA at either recommended (1X-FA, 2 mg/kg diet, control) or high (5X-FA, 10 mg/kg diet) quantities for rodents or [6S]-5-methyltetrahydrofolic acid calcium salt (Metafolin®, Merck & Cie, Schaffhausen, Switzerland) at equimolar recommended (1X-MTHF, 2.1 mg/kg diet) or high quantities (5X-MTHF, 10.4 mg/kg diet). Diets were kept in the dark at 4 °C until freshly provided to the animals weekly. Diets were analytically tested for long-term stability []. FA and MTHF were provided at equimolar levels in the diet due to a suggested comparable bioavailability of MTHF at equimolar levels to FA. Equimolar levels are used in clinical trials comparing folate status and homocysteine level maintenance [,]. The FA form was used as a reference to calculate the MTHF equimolar dose equivalent as only the synthetic folate form contains a tolerable upper level (1000 µg/day). The recommended (1X) FA dose provides 2 mg/kg of FA (4000 kcal) and represents the basal requirement for rodents for adequate growth rate []. This dose reflects recommendations of 0.4 mg of FA/day for women at low risk of neural-tube defects planning a pregnancy []. The supplemented FA diet contains 5X the basal requirement for rodents or 10 mg/kg of FA (4000 kcal). This dose reflects FA intakes commonly consumed by pregnant women in North America during the post-fortification era at >2-fold above basal dietary requirements for humans [,]. As MTHF is incorporated at the equivalent dose as FA in supplements, intakes may soon increase beyond recommendations [].

3.1. X-MTHF Offspring Gained more Weight and Ate more Food Than 5X-FA Offspring

A Dose×Form interaction effect was also found on measures of cumulative food intake up to 19 weeks post-weaning (Dose p = 0.7, Form p = 0.05, Dose×Form p = 0.02, B). The 5X-MTHF female offspring consumed ~8% more food compared to the 5X-FA offspring, but food intakes of the 1X folate offspring were similar to both 5X groups. Cumulative food intake and total weight gain of the offspring were also strongly and positively correlated within each group (1X-FA r = 0.89, p < 0.001, 1X-MTHF r = 0.90, p < 0.001, 5X-FA r = 0.71, p < 0.01; 5X-MTHF r = 0.72, p < 0.01).

5. Conclusions

MTHF compared to FA consumed at high levels in the gestational diet program central and peripheral mechanisms to favour increased weight gain in the female offspring. These pre-clinical findings caution against high gestational intakes of folates of either form, and encourage clinical trials examining their long-term health effects when consumed during pregnancy.

Abstract

The probiotic Bacillus subtilis strain 29784 (Bs29784) has been shown to improve performance in broilers. In this study, we used a metabolomic and 16S rRNA gene sequencing approach to evaluate effects of Bs29874 in the broiler intestine. Nicotinic acid and hypoxanthine were key metabolites that were produced by the strain in vitro and were also found in vivo to be increased in small intestinal content of broilers fed Bs29784 as dietary additive. Both metabolites have well-described anti-inflammatory effects in the intestine. Furthermore, Bs29784 supplementation to the feed significantly altered the ileal microbiome of 13-day-old broilers, thereby increasing the abundance of genus Bacillus, while decreasing genera and OTUs belonging to the Lactobacillaceae and Enterobacteriacae families. Moreover, Bs29784 did not change the cecal microbial community structure, but specifically enriched members of the family Clostridiales VadinBB60, as well as the butyrate-producing families Ruminococcaceae and Lachnospiraceae. The abundance of various OTUs and genera belonging to these families was significantly associated with nicotinic acid levels in the cecum, suggesting a possible cross-feeding between B. subtilis strain 29784 and these beneficial microbes. Taken together, the data indicate that Bs29784 exerts its described probiotic effects through a combined action of its metabolites on both the host and its microbiome.

2.1. Bacterial Strain and Growth Conditions

Bs29784 is a commercially available probiotic for broilers (Alterion® NE, Adisseo, Commentry, France). The commercial product contains 1010 CFU/g spores of B. subtilis strain 29784 and is mixed at 1 g/kg in the feed that is supplied to broilers. For in vitro experiments, a pure culture of Bs29784 was obtained by inoculating the commercial probiotic in Luria–Bertani (LB) broth (Sigma-Aldrich, St. Louis, MO, USA). The bacteria were grown overnight at 37 °C under aerobic conditions. Bacteria were plated on LB plates and their identity was confirmed via matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) [] and Sanger sequencing of the 16S region []. Bacterial growth was determined in LB broth over a 24-hour time span (grown in triplicate). Bacterial supernatant was obtained by centrifugation (5 min, 13,300 rpm) and filtered using a Polyvinylidene difluoride (PVDF) membrane filter (0.22 µm × 13 mm diameter, Kynar 500®, Arkema, Amsterdam, The Netherlands). Blank samples (medium without bacteria) were incubated simultaneously with the bacterial samples and processed in the same way to serve as controls. Samples were stored at −80 °C until metabolomic analysis.

2.2. Animal Trial

The study was undertaken following the guidelines of the ethics committee of the Faculty of Veterinary Medicine, Ghent University, in accordance with the EU Directive 2010/63/EU. One-day-old Ross 308 broiler chicks were obtained from a local hatchery and divided into 2 groups of 5 birds consisting of (1) a control group that received a standard commercial diet and (2) a group that received a standard commercial diet supplemented with the commercial Bs29784 probiotic at a dose of 1010 CFU/kg feed (FARM 1&2 mash, Versele-laga, Deinze, Belgium). Animals were housed on a solid floor covered with wood shavings at a density of 5 birds/m2. Animals were subjected to a light schedule of 12 h light and 12 h dark. All broilers were given water and feed ad libitum. At 13 days of age, all birds were weighed, the birds were euthanized, and digestive content from the jejunum, ileum and cecum was collected. These samples were frozen in liquid nitrogen directly after sampling and stored at −20 °C until further processing. The material from the 3 sections was used for metabolomic analysis and Bacillus quantification, while the ileal and cecal content was used for 16S sequencing. At 13 days of age, no differences in bodyweight could be observed, with an average bodyweight of 273.4 g ± 19.54 g (mean ± SD) for the control group and 254.3 g ± 38.37 g for the Bs29784-supplemented group (p = 0.358).

Abstract

Virgin olive oil (VOO) is one of the key components of the Mediterranean diet owing to the presence of monounsaturated fatty acids and various bioactive compounds. These beneficial traits, which are usually associated with the cultivar genotype, are highlighting the demand of identifying characteristics of olive oil that will ensure its authenticity. In this work, the fatty acid (FA) composition of 199 VOO samples from Koroneiki, Megaritiki, Amfissis, and Manaki cultivars was determined and studied by chemometrics. Olive cultivar greatly influenced the FA composition, namely, oleic acid (from 75.36% for Amfissis to 65.81% for Megaritiki) and linoleic acid (from 13.35% for Manaki to 6.70% for Koroneiki). Spearman’s rho correlation coefficients revealed differences and similarities among the olive oil cultivars. The use of the forward stepwise algorithm identified the FAs arachidonic acid, gadoleic acid, linoleic acid, α-linolenic acid, palmitoleic acid, and palmitic acid as the most significant for the differentiation of samples. The application of linear and quadratic cross-validation discriminant analysis resulted in the correct classification of 100.00% and 99.37% of samples, respectively. The findings demonstrated the special characteristics of the VOO samples derived from the four cultivars and their successful botanical differentiation based on FA composition.

2.2. Fatty Acid Composition

The dominant FA was oleic acid (C18:1) with the highest concentrations observed in Amfissis (75.36 ± 1.14%) and Koroneiki (74.70 ± 1.58%) VOO samples. Lower levels were detected in Megaritiki (65.81 ± 2.75%) and Manaki (70.15 ± 1.72%) cultivars. Adversely, for the polyunsaturated FA, linoleic acid (C18:2), the highest levels were observed in the Manaki (13.35 ± 1.07%) and Megaritiki (12.51 ± 2.02%) cultivars. The major saturated FAs were palmitic acid (C16:0), determined in the highest concentration in Megaritiki (15.53 ± 1.01%), and stearic acid (C18:0) in Koroneiki (2.78 ± 0.28%) and Manaki (2.63 ± 0.15%) samples. Interestingly, the highest levels of oleic/linoleic acid (C18:1/C18:2) and MUFA/PUFA ratios were observed in the Koroneiki cultivar which indicates a high performance against oxidative deterioration [].

2.2. Fatty Acid Composition

ANOVA and subsequent post-hoc tests between FA in association with botanical origin were performed (). Statistically significant differences were observed among the VOOs based on the levels of palmitoleic acid (16:1) and gadoleic acid (20:1), indicating a strong contribution on the discrimination between the four cultivars. Other FA such as arachidonic acid (20:4) and linoleic acid (18:2) were also significant for the differentiation of VOOs from Koroneiki, Megaritiki, and Amfissis cultivars. From the obtained results it is evident that a further application of chemometric methods to FA composition could be successful for the classification of samples with regard to cultivar.

2.3. Correlation Coefficients

Correlations of FA concentrations from the combination of the four olive oil cultivars as well as in each cultivar separately () were studied using Spearman’s rho correlation coefficients to designate potent similarities and differences between the cultivars. From the combined FA results of the four cultivars, strong positive correlations (p ≤ 0.01) were observed (a) between palmitic acid (16:0)—palmitoleic acid (16:1), arachidic acid (20:0)—behenic acid (22:0), and arachidic acid (20:0)—stearic acid (18:0), while strong negative correlations were observed between gadoleic acid (20:1)—palmitoleic acid (16:1) and gadoleic acid (20:1)—palmitic acid (16:0). A medium to strong antagonistic relationships was detected among oleic acid (18:1)—linoleic acid (18:2), oleic acid (18:1)—palmitoleic acid (16:1), and oleic acid (18:1)—stearic acid (18:0) (). The correlations between palmitic acid (16:0)—palmitoleic acid (16:1) and arachidic acid (20:0)—stearic acid (18:0) have been previously reported from Stefanoudaki et al. [] and Kritioti et al. [] who studied olive oil samples from Mastoides, Koroneiki, and Cypriot (ladoelia) cultivars. The antagonistic relationship between oleic acid (18:1)—stearic acid (18:0) has also been reported by Kritioti et al. [].

2.3. Correlation Coefficients

A high degree of positive correlation coefficients were observed between palmitic (16:0)—palmitoleic acid (16:1) for all cultivars, especially in Manaki VOO samples (d). Differences were observed in arachidic acid (20:0)—behenic acid (22:0), medium to strong correlations were observed in Megaritiki and Amfissis, while in Koroneiki and Manaki were low positive (). Positive correlations of arachidic acid (20:0)—stearic acid (18:0) were detected in all cultivars except from Manaki where the significance level was above 0.01. Strong negative correlations were observed between gadoleic acid (20:1)—palmitoleic acid (16:1) in Manaki and Megaritiki while in Koroneiki and Amfissis the correlations were insignificant. Strong negative correlations were also observed in gadoleic acid (20:1)—palmitic acid (16:0) in all cultivars except for Koroneiki samples. Medium to strong negative correlations of oleic acid (18:1)—palmitoleic acid (16:1) were detected in all cultivars. Strong antagonistic relationships were detected among oleic acid (18:1)—linoleic acid (18:2) in all cultivars except Amfissis, and medium antagonistic relationships were among oleic acid (18:1)—stearic acid (18:0) for Koroneiki and Megaritiki VOO samples. Summarizing the above results, common correlation patterns were detected in the four cultivars for the FA palmitic acid (16:0)—palmitoleic acid (16:1) and oleic acid (18:1)—palmitoleic acid (16:1), while several differences were observed among the remaining FA of this study depicting the differences in each cultivar.

2.4. Principal Component Analysis

The variables that contributed primarily in component 1 () were palmitic (16:0), palmitoleic (16:1), oleic (18:1), stearic (18:0), gadoleic (20:1), arachidic (20:0), behenic (22:0), and linoleic acid (18:2), with the highest contribution observed from palmitoleic (16:1) and oleic (18:1) acid. Arachidonic acid (20:4) mainly contributed to the formation of the second axis, followed by stearic (18:0), lignoceric (24:0), and gadoleic acid (20:1). α-linolenic acid (18:3) had a major contribution in the third component (), palmitic (16:0), linoleic acid (18:2), and lignoceric acid (24:0). Based on the first two principal components a good separation has been achieved for the Megaritiki and Amfissis cultivars. Thus, the FA that participate in the formation of the two axes are successfully contributing to the differentiation of the VOO samples from Megaritiki and Amfissis. Nevertheless, VOO samples from the Manaki cultivar were not clearly separated from the Koroneiki samples. Consequently, other chemometric methods should be applied for their differentiation.

Abstract

Sydnone methides are described from which only one single example has been mentioned in the literature so far. Their deprotonation gave anions which can be formulated as π‐electron rich anionic N‐heterocyclic carbenes. Sulfur and selenium adducts were stabilized as their methyl ethers, and mercury, gold as well as rhodium complexes of the sydnone methide carbenes were prepared. Sydnone methide anions also undergo C−C coupling reactions with 1‐fluoro‐4‐iodobenzene under Pd(PPh3)4 and CuBr catalysis. 77Se NMR resonance frequencies and 1 J C4‐Se as well as 1 J C4‐H coupling constants have been determined to gain knowledge about the electronic properties of the anionic N‐heterocyclic carbenes. The carbene carbon atom of the sydnone methide anion 3 j resonates at δ=155.2 ppm in 13C NMR spectroscopy at −40 °C which is extremely shifted upfield in comparison to classical N‐heterocyclic carbenes.

Abstract

The rise in cancer cases in recent years is an alarming situation worldwide. Despite the tremendous research and invention of new cancer therapies, the clinical outcomes are not always reassuring. Cancer cells could develop several evasive mechanisms for their survivability and render therapeutic failure. The continuous use of conventional cancer therapies leads to chemoresistance, and a higher dose of treatment results in even greater toxicities among cancer patients. Therefore, the search for an alternative treatment modality is crucial to break this viscous cycle. This paper explores the suitability of curcumin combination treatment with other cancer therapies to curb cancer growth. We provide a critical insight to the mechanisms of action of curcumin, its role in combination therapy in various cancers, along with the molecular targets involved. Curcumin combination treatments were found to enhance anticancer effects, mediated by the multitargeting of several signalling pathways by curcumin and the co-administered cancer therapies. The preclinical and clinical evidence in curcumin combination therapy is critically analysed, and the future research direction of curcumin combination therapy is discussed.

2. Immunomodulatory Effects of Curcumin

In some initial studies, curcumin supplementation showed no effect on the activity of natural killer (NK) cells in rats when given at doses of up to 40 mg/kg for up to five weeks []. However, in another study, Yadav et al. explored the immunomodulatory effects of curcumin and showed that this compound can increase NK cell cytotoxicity in vitro and that this effect can be further enhanced when curcumin was given along with IFN-γ treatment []. Increasing evidence indicates that curcumin exhibits its anti-inflammatory effects through the downregulation of NLRP3 inflammasomes []. Inflammasomes are multiprotein complexes that are responsible for innate immunity. The NLRP3, as a component of inflammasomes, can trigger immune response after detecting the products of damaged cells []. Curcumin can suppress NLRP3 inflammasome activation through regulation of the NF-κB signaling, leading also to suppression of IL-1β secretion []. For example, in malignant mesothelioma, curcumin has been found to exhibit its anticancer properties through regulation of signaling pathways affecting inflammasomes, such as IL-1 and NF-κB [].

2. Immunomodulatory Effects of Curcumin

Other studies have also determined the anti-inflammatory and anticancer properties of curcumin. In a clinical investigation where human chronic myelogenous leukemia cells (K562) were studied, curcumin treatment resulted in significant downregulation of IL-6, TLRs, IL-3 and STAT-1 []. In another randomized double-blind placebo-controlled trial, 80 patients with solid tumors were randomly assigned to 180 mg/day of curcuminoids supplementation or matched placebo for 8 weeks. All mediators of inflammation, including interleukins, TNF-α and monocyte chemotactic protein-1 were significantly lower in the curcuminoids versus placebo group []. When curcumin was given as part of a botanical drug containing multiple polyphenols, named APG-157 in a double-blind, randomized phase 1 clinical trial, where 12 patients with oral cancer were enrolled, reduced IL-1β, IL-6, and IL-8 concentrations were found in the saliva 24 h after treatment [].

2. Immunomodulatory Effects of Curcumin

Other studies have also determined the anti-inflammatory and anticancer properties of curcumin. In a clinical investigation where human chronic myelogenous leukemia cells (K562) were studied, curcumin treatment resulted in significant downregulation of IL-6, TLRs, IL-3 and STAT-1 []. In another randomized double-blind placebo-controlled trial, 80 patients with solid tumors were randomly assigned to 180 mg/day of curcuminoids supplementation or matched placebo for 8 weeks. All mediators of inflammation, including interleukins, TNF-α and monocyte chemotactic protein-1 were significantly lower in the curcuminoids versus placebo group []. When curcumin was given as part of a botanical drug containing multiple polyphenols, named APG-157 in a double-blind, randomized phase 1 clinical trial, where 12 patients with oral cancer were enrolled, reduced IL-1β, IL-6, and IL-8 concentrations were found in the saliva 24 h after treatment [].

2. Immunomodulatory Effects of Curcumin

Other studies have also determined the anti-inflammatory and anticancer properties of curcumin. In a clinical investigation where human chronic myelogenous leukemia cells (K562) were studied, curcumin treatment resulted in significant downregulation of IL-6, TLRs, IL-3 and STAT-1 []. In another randomized double-blind placebo-controlled trial, 80 patients with solid tumors were randomly assigned to 180 mg/day of curcuminoids supplementation or matched placebo for 8 weeks. All mediators of inflammation, including interleukins, TNF-α and monocyte chemotactic protein-1 were significantly lower in the curcuminoids versus placebo group []. When curcumin was given as part of a botanical drug containing multiple polyphenols, named APG-157 in a double-blind, randomized phase 1 clinical trial, where 12 patients with oral cancer were enrolled, reduced IL-1β, IL-6, and IL-8 concentrations were found in the saliva 24 h after treatment [].

3. Lung Cancer

Curcumin exhibits its therapeutic potential in lung cancer treatment by acting on the Wnt/β-catenin-dependent pathway in the human lung cancer cell line A549 []. Curcumin also suppresses the vascular endothelial growth factor (VEGF0 and NF-κB expression in the same cell line []. In addition, curcumin has been shown to inhibit the expression of enhancer of zeste homolog 2 (EZH2) in lung cancer cells, which was followed by a decrease in the expression of the Notch 1 gene []. Curcumin has exhibited cytotoxicity against non-small-cell lung cancer (NSCLC) through inhibition of cell proliferation, increased apoptosis, and G2/M arrest. Curcumin also induces ROS production, thus activating the DNA damage signaling pathway [].