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Abstract

l-Buthionine sulfoximine (l-BSO) is an adjuvant drug that is reported to increase the sensitivity of cancer cells to neoplastic agents. Dendrimers are exceptional drug delivery systems and l-BSO nanoformulations are envisaged as potential chemotherapeutics. The absorption of l-BSO at a low wavelength limits its detection by conventional analytical tools. A simple and sensitive method for l-BSO detection and quantification is now reported. In this study, l-BSO was encapsulated in a folate-targeted generation four polyurea dendrimer (PUREG4-FA2) and its release profile was followed for 24 h at pH 7.4 and 37 °C. The protocol uses in situ l-BSO derivatization, by the formation of a catechol-derived orto-quinone, followed by visible detection of the derivative at 503 nm. The structure of the studied l-BSO derivative was assessed by NMR spectroscopy.

2.1. l-BSO Encapsulation and Release Studies

shows the l-BSO release profile from a l-BSO@PUREG4-FA2 nanoformulation at pH 7.4. The release was followed for 24 h and the results indicate a burst release in the first hours, as expected in this type of delivery system [,]. After 1 h, around 60% of the loaded drug was released to the medium, reaching a plateau after 3 h. After 24 h, 90% of l-BSO was released, meaning that only a residual amount of l-BSO was trapped inside the PUREG4-FA2 dendrimer during this period of time. The quantification of BSO released from the PUREG4-FA2 dendrimer was determined using a calibration curve, obtained from an assay under the same experimental conditions (see ). In more acidic media, a faster release is expected.

3.1. Reagents and Materials

l-Buthionine-(S,R)-sulfoximine (l-BSO) (≥97% purity) was obtained from Sigma-Aldrich. All chemicals and solvents were used as received without further purification. Polyurea (PURE) dendrimer generation four (PUREG4) was synthesized following our reported supercritical-assisted polymerization [].

3.5. Quantification of BSO by UV-Vis Spectroscopy

Using a modified protocol [], l-BSO was derivatized in order to be detected by UV-Vis spectroscopy. l-BSO quantification was performed by adding to the samples 300 µL of catechol 2.25 mM in PBS, followed by 300 µL of sodium periodate 6.75 mM in PBS. After 60 sec, the absorption of the l-BSO derivative (503 nm) was measured in a PerkinElmer Lambda 25 UV-Vis Spectrometer with a slit width of 5 nm at a scan rate of 240 nm min−1 at 25 °C. For the calibration curve, standard solutions of l-BSO were prepared in the concentration range of 0.1–150 µM in PBS and processed using the same protocol. A good correlation coefficient (R2 = 0.997) was obtained (see ).

3. Discussion

In the present study we isolated gut bacteria from water monitor lizard, identified and cultured it in a minimal media (RPMI) to prepare CM. The CM were examined against several Gram-positive and Gram-negative bacteria to determine their antibacterial activities. Finally, the CM were tested for their cytotoxicity against HaCaT cell lines. The CM from A. hydrophila (CM2) exhibited cytotoxicity against HaCaT cells. This is likely because this bacterium secretes cytotoxic enterotoxins that binds to human, yeast proteins and human intestinal epithelial cell line (HT-29) and induced apoptosis []. These findings are supported by Chopra et al. [] and Krzymińska et al. [], which showed Aeromonas species produce enterotoxins, cytotonic and cytotoxic toxins that cause HEp-2 cell death, lyse red blood cells and abolish tissue culture cell lines. The CM exhibited robust bactericidal activities against selected pathogenic bacteria. Most of the CM showed antibacterial effects even after heat inactivation of these CM at 95 °C for 10 min. This further suggests that the active molecules could be possibly small secondary metabolites. The results from LCMS showed several secondary metabolites with antibacterial properties including flavonoids, alkaloids, terpenes, oxygenated fatty acids, hydroxylated fatty acids and pyrazine derivatives. Similar compounds have been reported for example, 2,5-Dihydroxymethyl-3, 4-dihydroxypyr- rolidine (DMDP) exhibited antiparasitic activity against plant parasite nematode []. Here for the first time, we identified the same compound from WML gut bacteria which suggests that bacteria from gut have the capability to produce anti-nematodal molecules. Similarly, Dehydrocurdione is found in turmeric [], while the same compound has been isolated from gut bacteria with significant antibacterial activity against B. subtilis in the current study. Littlefield-Wyer et al. [] identified 10-Hydroxymyristic acid from aquatic microorganisms, while similar molecule is shown to be synthesized by gut bacteria in this study. Several identified molecules with no reported biological activities are S-Methyl-1-thio-D-glycerate, omega-Hydroxymoracin N, 1,3,8-Trihydroxy-4-methyl-2,7-diprenylxanthone, Desmethyl-maprotiline glucuronide, Desmethyl-nortriptyline glucuronide, (3b,21b)-12-Oleanene-3,21,28-triol 28-[arabinosyl-(1->3)-arabinosyl-(1->3)-arabinoside], Chondrilla-sterol 3-[glucosyl-(1->2)-glucosyl-(1->2)-glucoside], 4-Methyl-dibenzothiophene, 1,2-Epoxy-3,4-butanediol 4-methane-sulfonate and 1R,2R)-3-oxo-2-pentyl-cyclopentanehexanoic acid. Further identification, characterization and their functional studies could be a major breakthrough in development of novel drugs against multi-drug-resistant bacteria. In future studies, individual compound(s) will be isolated and screened against MDR bacteria and human cell lines to determine their translational value in therapy.

Abstract

Cerebral ischemia/reperfusion (I/R) injury causes cognitive deficits, excitotoxicity, neuroinflammation, oxidative stress and brain edema. Vitamin K2 (Menaquinone 4, MK-4) as a potent antioxidant can be a good candidate to ameliorate I/R consequences. This study focused on the neuroprotective effects of MK-4 for cerebral I/R insult in rat’s hippocampus. The rat model of cerebral I/R was generated by transient bilateral common carotid artery occlusion for 20 min. Rats were divided into control, I/R, I/R+DMSO (solvent (1% v/v)) and I/R+MK-4 treated (400 mg/kg, i.p.) groups. Twenty-four hours after I/R injury induction, total brain water content, superoxide dismutase (SOD) activity, nitrate/nitrite concentration and neuronal density were evaluated. In addition to quantify the apoptosis processes, TUNEL staining, as well as expression level of Bax and Bcl2, were assessed. To evaluate astrogliosis and induced neurotoxicity by I/R GFAP and GLT-1 mRNA expression level were quantified. Furthermore, pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α were measured. Seven days post I/R, behavioral analysis to quantify cognitive function, as well as Nissl staining for surviving neuronal evaluation, were conducted. The findings indicated that administration of MK-4 following I/R injury improved anxiety-like behavior, short term and spatial learning and memory impairment induced by I/R. Also, MK-4 was able to diminish the increased total brain water content, apoptotic cell density, Bax/ Bcl2 ratio and GFAP mRNA expression following I/R. In addition, the high level of nitrate/nitrite, IL-6, IL-1β and TNF-α induced by I/R was reduced after MK-4 administration. However, MK-4 promotes the level of SOD activity and GLT-1 mRNA expression in I/R rat model. The findings demonstrated that MK-4 can rescue transient global cerebral I/R consequences via its anti-inflammatory and anti-oxidative stress features. MK-4 administration ameliorates neuroinflammation, neurotoxicity and neuronal cell death processes and leads to neuroprotection.

Animals

172 Adult male Wistar rats with weighing 250–300 gr selected at random from the animal facility of Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran. Animals were housed in standard cages under controlled room temperature (22–24°C) and humidity (45–50%) and exposed to 12:12 h light–dark cycle conditions (lights on at 08:00 am) (). The experimental procedures were approved by the National Food Chain Safety and conducted according to Institutional Animal Ethics Committee guidelines for the care and use of laboratory animals [, ].

Surgical procedure for induction global cerebral I/R model

Rats were anesthetized by an intraperitoneal (i.p.) injection of Xylazine 20 mg/kg and Ketamine 100 mg/kg. The body temperature was monitored and maintained between 37.1°C and 37.3°C under free-regulating conditions with using a thermometer and heating pad during and after the surgery up to the animals recovered from anesthesia. After separation of the vagus nerve, bilateral common carotid arteries occlusion (two-vessel occlusion; 2-VO) was conducted by 20 min clamping [, , ]. After 20 min, clamps were removed for reperfusion onset either for 24 hours or for 7 days depends on different experimental sets. After surgery, animals were placed in home cages with free access to food and water. Some of the rats were removed from experiments because of: 1) death induced by ischemia, 2) movement impairment, 3) visual deficiency, 4) difficulties in reperfusion (not operated in both common carotids) and 5) seizure.

Experimental design

216 animals were divided in 4 groups as follows: Age-matched control group, I/R injury group (sham control group), I/R injury + 2 times DMSO i.p. injection (solvent of MK-4, 1% v/v) [], I/R injury + 400 mg/kg MK-4 (2 times 200 mg/kg i.p. injections, immediately and 2h after reperfusion). This dosage was selected empirically. At first, the dose of 100 mg/kg MK-4 i.p. was selected due to the previous investigations [–], however, the mortality rate of ischemic animals was high (equal I/R group). To this reason, we had to enhance the treatment dose to 400 mg/kg in two injections (200 mg/kg i.p. immediately and 200 mg/kg i.p. 2h after reperfusion). The high number of survived rats was observed subsequently. Besides, earlier studies showed that a high dose of vitamin K2 has very low toxicity [, ]. Behavioral tests were conducted 7 days post reperfusion in healthy animals. TUNEL and Nissl histological staining were performed 24h and 7 days after reperfusion respectively. The brain volume changes for total brain water content assessment was measured 24 hours after reperfusion. In addition, 24h after I/R injury ELISA was used for detection of pro-inflammatory cytokines including TNF-α, IL-1β, IL-6 and real-time PCR was utilized for evaluation of mRNA level of GFAP, Bax, Bcl-2 and GLT-1 (EAAT2). Then, the activity of SOD enzyme and NO concentration were assessed ().

Abstract

All‐trans‐retinal (atRAL) is a highly reactive carbonyl specie, known for its reactivity on cellular phosphatidylethanolamine in photoreceptor. It is generated by photoisomerization of 11‐cis‐retinal chromophore linked to opsin by the Schiff's base reaction. In ABCA4‐associated autosomal recessive Stargardt macular dystrophy, atRAL results in carbonyl and oxidative stress, which leads to bisretinoid A2E, accumulation in the retinal pigment epithelium (RPE). This A2E‐accumulation presents as lipofuscin fluorescent pigment, and its photooxidation causes subsequent damage. Here we describe protection against a lethal dose of atRAL in both photoreceptors and RPE in primary cultures by a lipidic polyphenol derivative, an isopropyl‐phloroglucinol linked to DHA, referred to as IP‐DHA. Next, we addressed the cellular and molecular defence mechanisms in commonly used human ARPE‐19 cells. We determined that both polyunsaturated fatty acid and isopropyl substituents bond to phloroglucinol are essential to confer the highest protection. IP‐DHA responds rapidly against the toxicity of atRAL and its protective effect persists. This healthy effect of IP‐DHA applies to the mitochondrial respiration. IP‐DHA also rescues RPE cells subjected to the toxic effects of A2E after blue light exposure. Together, our findings suggest that the beneficial role of IP‐DHA in retinal cells involves both anti‐carbonyl and anti‐oxidative capacities.

INTRODUCTION

In the present study, we investigated the protective effect of the medicinal chemical compound, isopropyl‐phloroglucinol‐DHA (IP‐DHA), also called lipophenol, against atRAL‐related carbonyl and oxidative stresses (COS). We first analysed primary cultures of outer retina to demonstrate the dose‐dependent protective effect against lethal dose of atRAL. We then used ARPE‐19 cells as a standard cellular model to study the mechanisms of cell death and protection. We demonstrate that each of the structural part, that is isopropyl and PUFA, is essential for the full action of the lipophenol with selectivity for LA and DHA. We compared the capacities of phloroglucinol and IP‐DHA to reverse the effects of atRAL and finally discuss how to understand the enhanced protective effects of IP‐DHA compared to phloroglucinol.

Cell cultures and atRAL treatment

Primary rat RPE and mouse neural retina (NR) cultures were obtained as previously described. RPE cells were cultured for 3 days until they reached 80%‐85% confluency, and NR was cultured for 10 days until glial cells were confluent in the bottom layer and neural cells with a neurite outgrowth in the upper layer. Human RPE‐like cells, ARPE‐19, were seeded at 100 000 cells/cm2 and grown to confluency before being assayed as instruct by ATCC.

Cell cultures and atRAL treatment

Pre‐ and co‐treatment procedures with lipophenol were carried out. During pre‐treatments, rat RPE primary cultures received a medium containing lipophenol at different concentrations (40‐320 μmol/L) for 24 hours. The medium was then removed and replaced by a serum‐free culture medium containing 25 μmol/L atRAL for 4 hours. During co‐treatments, RPE and NR cells received a serum‐free medium containing 25 μmol/L atRAL and/or IP‐DHA (10‐320 μmol/L) for 4 hours. NR cells were refreshed with serum‐free medium the next day.

A2E treatment

ARPE‐19 cells were plated into 96‐well plates (4 × 104 cells/well) and cultured for 24 hours to confluence prior to lipophenol treatment. The cell cultures were treated with serum‐free DMEM/F12 medium without phenol red containing IP‐DHA at different concentrations (0‐80 μmol/L) for 1 hour. A2E was then added to a final concentration of 20 μmol/L for 6 hours before rinsing with medium. Control cells were incubated with 0.2% DMSO with or without A2E. The cells were exposed to intense blue light (4600 lux) for 30 minutes to induce phototoxicity of A2E and incubated at 37°C. Irradiation was achieved using a LED device with blue emission wavelengths from 430 to 470 nm and a dimmable luminance (Roleadro lighting). Control ambient white light was less than 300 lux. Without blue light exposure, A2E‐loaded cell survival was not affected. The cell viability was determined 16‐20 hours later using a MTT colorimetric assay. Results are expressed in percentage of viable cells normalized to control conditions in the absence of lipophenol and stressor.

Abstract

Recently, a study published in “Toxicology In Vitro” (Kate Samardzic and Kenneth J. Rodgers) was entitled: “Cytotoxicity and Mitochondrial Dysfunction Caused by the Dietary Supplement L-Norvaline”. The title may be greatly overstated, and here we provide several arguments showing that norvaline is not as toxic as reported.

Abstract

Tocotrienol, an analogue of vitamin E has been known for its numerous health benefits and anti-cancer effects. Of the four isoforms of tocotrienols, gamma-tocotrienol (γT3) has been frequently reported for their superior anti-tumorigenic activity in both in vitro and in vivo studies, when compared to its counterparts. In this study, the effect of γT3 treatment in the cytoplasmic and nuclear fraction of MDA-MB-231 human breast cancer cells were assessed using the label-free quantitative proteomics analysis. The cytoplasmic proteome results revealed the ability of γT3 to inhibit a group of proteasome proteins such as PSMA, PSMB, PSMD, and PSME. The inhibition of proteasome proteins is known to induce apoptosis in cancer cells. As such, the findings from this study suggest γT3 as a potential proteasome inhibitor that can overcome deficiencies in growth-inhibitory or pro-apoptotic molecules in breast cancer cells. The nuclear proteome results revealed the involvement of important nuclear protein complexes which hardwire the anti-tumorigenesis mechanism in breast cancer following γT3 treatment. In conclusion, this study uncovered the advancing roles of γT3 as potential proteasomes inhibitor that can be used for the treatment of breast cancer.

2.1. Cell Line and Treatment Conditions

The MDA-MB-231 cells were grown in T75 flasks in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) in a humidified atmosphere of 5% carbon dioxide in air at 37 °C, until it reaches 70–90% confluency. A total of 5 × 105 cells were seeded in T75 flask containing 10 mL of complete media. Following cell adherence, the cells were treated with IC50 concentration, 5.8 µg × mL−1 of γT3, as established previously []. Following 24 h of γT3 treatment, the cells were washed with 1× PBS solution. A cell scrapper was used to dislodge the attached cells. The cells were collected in 5 mL PBS and washed with two volumes of ice-cold PBS and centrifuged at 250 × g for 5 min at 4 °C. The supernatant was discarded, and the pellets were resuspended in 40 mL of ice-cold PBS and centrifuged as before. The washing step was repeated twice. The pellet was subjected to two separate processing steps to extract (i) cytoplasmic proteins and (ii) nuclear proteins.

Abstract

One of the omega-3 essential fatty acids, docosahexaenoic acid (DHA), is a significant constituent of the cell membrane and the precursor of several potent lipid mediators. These mediators are considered to be important in preventing or treating several diseases. Resolvin D5, an oxidized lipid mediator derived from DHA, has been known to exert anti-inflammatory effects. However, the detailed mechanism underlying these effects has not yet been elucidated in human monocytic THP-1 cells. In the present study, we investigated the effects of resolvin D5 on inflammation-related signaling pathways, including the extracellular signal-regulated kinase (ERK)-nuclear factor (NF)-κB signaling pathway. Resolvin D5 downregulated the production of interleukin (IL)-6 and chemokine (C-C motif) ligand 5 (CCL5). Additionally, these inhibitory effects were found to be modulated by mitogen-activated protein kinase (MAPK) and NF-κB in lipopolysaccharide (LPS)-treated THP-1 cells. Resolvin D5 inhibited the LPS-stimulated phosphorylation of ERK and translocation of p65 and p50 into the nucleus, resulting in the inhibition of IL-6 and CCL5 production. These results revealed that resolvin D5 exerts anti-inflammatory effects in LPS-treated THP-1 cells by regulating the phosphorylation of ERK and nuclear translocation of NF-κB.

Effects of Resolvin D5 on IL-6 and CCL5 Production in LPS-Treated THP-1 Cells

To identify whether resolvin D5 has anti-inflammatory effects in LPS-stimulated THP-1 cells, the cells were pre-treated with resolvin D5 for 1 h, and then with LPS for 24 h. The RT-qPCR analyses revealed that at concentrations of 20–40 μM, resolvin D5 significantly inhibited the IL-6 and CCL5 mRNA levels following LPS treatment (). There was no contamination of LPS during resolvin D5 purification because resolvin D5 was extracted from the reaction solution not from the recombinant cells, and resolvin D5 itself did not induce IL-6 or CCL5 mRNA levels (data not shown), suggesting that there was no immunotoxicity in the resolvin D5-treated cells. Furthermore, ELISA results also showed that the secreted protein level of IL-6 in the THP-1 cell supernatants was reduced by resolvin D5. However, unlike the IL-6 level, the secreted CCL5 level was not inhibited notably (). Taking all results from and , it was revealed that resolvin D5 neither exerted cytotoxicity nor immunotoxicity and suppressed the expression or production of pro-inflammatory cytokine IL-6 and chemokine CCL5.

Effects of Resolvin D5 on MAPK Signaling in LPS-Treated THP-1 Cells

To identify whether resolvin D5 alters the LPS stimulation of MAPK signaling, several key MAPK members were examined including ERK, JNK, and p38. The phosphorylation of ERK was attenuated by resolvin D5 dose-dependently in THP-1 cells, while that of JNK and p38 was not altered (). Subsequently, the production of CCL5 and IL-6 was evaluated after the treatment with the ERK inhibitor, PD98059, to determine whether CCL5 and IL-6 expressions were modulated via ERK. Pre-treatment with PD98059 (20 μM) for 1 h before LPS treatment in the absence or presence of resolvin D5 (40 μM) attenuated the production of CCL5 and IL-6. These data indicated that LPS-induced CCL5 and IL-6 secretion was reduced by resolvin D5 via inhibition of the ERK/MAPK signaling pathway ().

Effects of Resolvin D5 on Transcription Factor Activities

Transcription factors are involved in the regulation of the expression of various genes via their phosphorylation and translocation. Therefore, the translocation of several transcription factors from the cytosol to the nucleus was investigated. NF-κB is a well-known transcription factor; the nuclear fractionation results revealed that the LPS-induced translocation of NF-κB was decreased by resolvin D5 ().

Discussion

The ubiquitous transcription factor NF-κB is important in inflammation, and responsible for the modulation of genes encoding proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL-12, inducible enzymes such as cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) [, ]. It is activated via signaling cascades triggered by extracellular stimuli such as LPS. Cell culture studies have shown that the ω-3 fatty acids EPA and DHA inhibit the LPS-induced production of TNF-α, IL-1, IL-6, COX-2, and iNOS in monocytes and macrophages. Additionally, ω-3 fatty acids inhibit the LPS-induced activation of NF-κB [-]. Resolvin D5, a lipid mediator that is biosynthesized from DHA, also inhibited the expression of inflammatory mediators via the NF-κB pathway in LPS-stimulated THP-1 cells. The nuclear translocation of the NF-κB subunits, p65 and p50, were upregulated after LPS stimulation; however, resolvin D5 treatment attenuated this translocation (). Other transcription factors involved in inflammation, such as activated protein-1 (AP-1) and STAT3, were also examined; resolvin D5 did not affect the translocation of these transcription factors. To determine the relationship between NF-κB and ERK, LPS-treated THP-1 cells were treated with the ERK inhibitor PD98059 in the presence or absence of resolvin D5. Indeed, the translocation of p65 was inhibited by PD98059, and it was revealed that NF-κB was regulated via the ERK signaling pathway (). To confirm the effects of resolvin D5 on NF-κB transcriptional activation, a transient transfection and luciferase assay were performed. The results revealed an effective inhibitory effect of resolvin D5 on the transcriptional level of NF-κB after LPS stimulation ().

Abstract

The modification of lipid composition allows cells to adjust membrane biophysical properties in response to changes in environmental temperature. Here, we use adaptive laboratory evolution (ALE) in the presence of myriocin, a sphingolipid (SLs) biosynthesis inhibitor, to remodel the lipid profile of an industrial yeast strain (LH) of Saccharomyces cerevisiae. The approach enabled to obtain a heterogeneous population (LHev) of myriocin‐tolerant evolved clones characterized by its growth capacity at high temperature. Myriocin exposure also caused tolerance to soraphen A, an inhibitor of the acetyl‐CoA carboxylase Acc1, the rate‐limiting enzyme in fatty acid de novo production, supporting a change in lipid metabolism during ALE. In line with this, characterization of two randomly selected clones, LH03 and LH09, showed the presence of lipids with increased saturation degree and reduced acyl length. In addition, the clone LH03, which displays the greater improvement in fitness at 40°C, exhibited higher SL content as compared with the parental strain. Analysis of the LH03 and LH09 genomes revealed a loss of chromosomes affecting genes that have a role in fatty acid synthesis and elongation. The link between ploidy level and growth at high temperature was further supported by the analysis of a fully isogenic set of yeast strains with ploidy between 1N and 4N which showed that the loss of genome content provides heat tolerance. Consistent with this, a thermotolerant evolved population (LH40°) generated from the parental LH strain by heat‐driven ALE exhibited a reduction in the chromosome copy number. Thus, our results identify myriocin‐driven evolution as a powerful approach to investigate the mechanisms of acquired thermotolerance and to generate improved strains.

Adaptive evolution in the presence of myriocin

We used a robust industrial baker's yeast strain named L'Hirondelle (LH) in order to force by ALE changes in the lipid metabolism. For this, yeast was propagated by successive batch refreshments maintained constantly in the presence of myriocin at 30°C during 50 generations (see Fig. A). In these experiments, a chronic‐medium dose of 1.2 μM myriocin was used to lower but not completely inhibit SPT activity (Huang et al., ; Fig. B). Moreover, a short‐term experiment was chosen because deleterious mutations, which could affect negatively important traits of industrial strains, such as cell size, fitness or carbon‐source utilization (Aguilera et al., ; Wenger et al., ; Çakar et al., ; Strucko et al., ), have been reported to accumulate over time (Tenaillon et al., ). In general, the rates of spontaneous mutation are higher for neutral and deleterious mutations and lower for beneficial mutations (Barrick and Lenski, ; Van den Bergh et al., ). In this manner, we seek to generate evolved clones containing stable compensatory mutations conferring increased SL biosynthesis (see Fig. B) or altered PL asymmetry, but still maintaining key industrial properties. We found that the doubling time decreased gradually over the course of the experiment (data not shown), indicating that the original yeast population was capable of adapting to optimize growth in the presence of the drug. As shown in Fig. C, the 50‐generations evolved population (LHev) grew in a solid myriocin‐containing YPD medium, while the parental strain did not. As expected, tolerance to myriocin was also observed for 10 individual adaptive clones isolated from the terminal population. In addition, all of them showed a similar growth (Fig. C), suggesting that adaptation‐driving mutations were distributed across the population (Tenaillon et al., ).

Myriocin‐driven evolution alters yeast growth at different temperatures

We asked whether the changes in myriocin tolerance observed in the evolved population were relevant for the physiological response to thermal stress. As shown in Fig. , growth at 30°C, the optimal temperature for S. cerevisiae of the yeast population was unaffected by the evolutionary experiment. On the contrary, the terminal population exhibited increased growth at 40°C as compared with the parental, whereas a trade‐off in cell proliferation under cold conditions was observed (Fig. ). Likewise, most of the selected clones under study (LH01–LH10) exhibited increased fitness at 40°C (data not shown) although again the phenotype differed quantitatively among individuals as it is illustrated for two randomly selected clones, LH03 and LH09 (Fig. ). Thus, the clone LH09 exhibited only a slight growth advantage at 40°C, as compared with the parental LH strain, while the LH03 clone grew much faster and had a similar behaviour to that of the LHev population. Finally, these phenotypes correlated again with a loss in cold growth (Fig. ). We conclude that myriocin‐drive evolution improved yeasts' ability to adapt to increased temperature with no apparent trade‐offs at the ancestral optimal temperature of 30°C. The results also highlight how tolerance to high temperature is associated with fitness impairments under cold‐stressful conditions.

3.1. Effect of Beta- and Gamma- Tocotrienols on the Cell Proliferation of MDA-MB-231 and MCF7 cells

Using WST-1 as a cell proliferation reagent, the percent proliferation of the MDA-MB-231 cell line treated with different concentrations of beta-T3 (10–50 µM) or gamma-T3 (10–50 µM) for 24 and 48 h was calculated and the results showed a significant dose- and time-dependent decrease in the proliferation of both cell lines; however, the effect was more prominent with beta-T3 treatment. Beta-tocotrienol induced a significant progressive decrease in percentage of proliferating MDA cells, with an IC50 of 29.99 μM and 21.14 μM after 24 and 48 h respectively (A). On the other hand, gamma-tocotrienol induced a significant progressive decrease in cell proliferation of MDA cells starting from 30 μM with an IC50 of 39.04 μM and 30.98 μM after 24 and 48 h respectively (B). The IC50 concentrations of beta-T3 were lower than that of the gamma derivative after both 24 and 48 h treatments, indicating a significant higher potency of beta-T3 on MDA cells at 20, 30 and 40 μM (C,D).