Association of Mediterranean Diet Adherence with Disease Progression Characteristics, Lifestyle Factors and Overall Survival in Gastric Cancer Patients

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/BACKGROUND: The Mediterranean diet (MD) exerts a protective effect against cancer development and progression; however, the evaluation of its impact on gastric cancer still remains quite scarce. The present study aims to evaluate the association of MD adherence during the lifespan with disease progression characteristics, lifestyle factors and overall survival in gastric carcinoma patients. METHODS: This is an observational, cross-sectional study conducted on 186 gastric cancer patients followed up for a median time interval of 57 months or until death due to cancer disease. Tumor histopathological characteristics were retrieved from patients' medical records, while validated questionnaires assessing, immediately after the time of diagnosis, health-related quality of life, physical activity levels, sleep quality, depression, anxiety and MD adherence during the lifespan were used. RESULTS: Higher MD adherence during the lifespan was significantly associated with younger patients (p = 0.0106), regular smoking (p < 0.0001), abnormal BMI status (p < 0.0001), intestinal-type gastric carcinoma (p = 0.0111), high tumor histopathological grade (p < 0.0001) and earlier disease stage (p < 0.0001). Moreover, patients with elevated MD adherence during their lifespan showed significantly better health-related quality of life (p < 0.0001), higher physical activity levels (p < 0.0001), more adequate sleep quality (p < 0.0001) and lower prevalence of depression (p = 0.0003) and anxiety (p = 0.0006) compared to those with reduced MD adherence. In multiple regression analysis, elevated MD compliance during the lifespan was independently correlated with longer overall patient survival after adjustment for several confounders (Cox regression analysis, p = 0.0001). CONCLUSIONS: Higher MD adherence during the lifespan was associated with less advanced tumor histopathology characteristics and favorable mental and physical lifestyle factors. Moreover, higher MD adherence during the lifespan was also independently correlated with longer overall survival in gastric carcinoma patients. Thus, adopting a healthy dietary pattern like the MD during the lifespan may act as a preventive agent in combination with a healthy lifestyle against gastric cancer development and progression.Peer reviewe

Metformin Delays Satellite Cell Activation and Maintains Quiescence

The regeneration of the muscle tissue relies on the capacity of the satellite stem cell (SC) population to exit quiescence, divide asymmetrically, proliferate, and differentiate. In age-related muscle atrophy (sarcopenia) and several dystrophies, regeneration cannot compensate for the loss of muscle tissue. These disorders are associated with the depletion of the satellite cell pool or with the loss of satellite cell functionality. Recently, the establishment and maintenance of quiescence in satellite cells have been linked to their metabolic state. In this work, we aimed to modulate metabolism in order to preserve the satellite cell pool. We made use of metformin, a calorie restriction mimicking drug, to ask whether metformin has an effect on quiescence, proliferation, and differentiation of satellite cells. We report that satellite cells, when treated with metformin in vitro, ex vivo, or in vivo, delay activation, Pax7 downregulation, and terminal myogenic differentiation. We correlate the metformin-induced delay in satellite cell activation with the inhibition of the ribosome protein RPS6, one of the downstream effectors of the mTOR pathway. Moreover, in vivo administration of metformin induces a belated regeneration of cardiotoxin- (CTX-) damaged skeletal muscle. Interestingly, satellite cells treated with metformin immediately after isolation are smaller in size and exhibit reduced pyronin Y levels, which suggests that metformin-treated satellite cells are transcriptionally less active. Thus, our study suggests that metformin delays satellite cell activation and differentiation by favoring a quiescent, low metabolic state

Metformin perturbs the expression of cyclins and CDKIs.

<p>(a) Western blot analysis of p27, p21, cyclin D3 and p130 in total protein lysate from metformin-treated and control C2C12 cells. When cells reached ~ 60% confluence, were induced to differentiate by serum deprivation and treated in differentiation medium with 100μM, 2mM and 10mM metformin for 3 days. Metformin was refreshed every 24h and crude cell protein extracts were analyzed by SDS PAGE. Tubulin and vinculin were used as loading controls. Numbers above each lane represent the densitometric analysis (b) Cell cycle analysis by DNA staining with propidium iodide. C2C12 cells were cultured for 3 days in GM and treated with 100μM, 2mM and 10mM metformin (c) Flow cytometry analysis of the three different phases of the cell cycle (G0/G1, S, G2/M) is represented by the histogram graph. Quantification of the different phases of the cell cycle was performed after three independent biological replicate experiments (d) Western blot analysis of the G2/M markers cyclin B and Cdc2 kinase in total protein lysate from metformin-treated and control C2C12 cells. Cells were induced to differentiate by serum deprivation and treated with 100μM, 2mM and 10mM metformin for 3 days. Metformin was added fresh every 24h and crude cell protein extracts were analyzed by SDS PAGE. GAPDH and vinculin were used as loading controls. Numbers above each lane represent the densitometric analysis (e) Quantitation of cyclin B and Cdc2 levels after 3 days of metformin treatment. Statistical significance was evaluated by the Student’s t-test (*p<0.05).</p

The metformin effect is reversible.

<p>(a) C2C12 myoblasts were treated with 10mM metformin in GM for 48h and subsequently let recover for additional 48h after replacing the medium containing the drug with fresh GM. After 48h of recovery, the total cell number was counted in the treated and control sample. Statistical significance was evaluated by the Student’s t-test (*p<0.05) (b) C2C12 induced to differentiate and treated with 10mM metformin for 72h let recover their differentiation potential for additional 48h in fresh DM. Protein extracts were analyzed by Western Blot. Tubulin and vinculin were used as loading controls. Numbers above each lane represent the densitometric analysis (c) The expression of the myogenic markers MyoD and MyHC and the formation of myotubes was analyzed by immunofluorescence microscopy. C2C12 myoblasts were treated with 100μM, 500μM, 2mM, 5mM and 10mM metformin in DM for 3 days and let recover for additional 72h (d) C2C12 myoblasts treated with 10mM metformin for 72h in differentiation medium, were subsequently cultured for additional 48h in fresh DM without metformin. The expression of Myf5 and p130 was measured by Western Blot. Tubulin and vinculin were used as loading controls. Numbers above each band represent the densitometric analysis.</p

The recovery of C2C12 cells from metformin inhibition is not mediated by p38 activation.

<p>(a) C2C12 cells induced to differentiate by serum deprivation and treated in differentiation medium with 100μM, 2mM and 10mM metformin for 3 days. Metformin was refreshed every 24h and the levels of phospho-p38 and total p38 protein were analyzed in crude cell protein extracts by SDS PAGE. Tubulin was used as loading control. Numbers above each lane represent the densitometric analysis. (b) Western blot analysis and quantitation graph of phospho-p38 and total p38 protein in protein extracts from C2C12 myoblasts treated with 10mM metformin in DM for 72h and subsequently cultured for additional 8, 24 and 48 hours in fresh DM. Vinculin was used as loading control. Numbers above each lane represent the densitometric analysis (c) Western blot analysis of total P70S6K, RPS6 and their phosphorylation levels. C2C12 myoblasts were treated with 10mM metformin in DM for 72h and subsequently cultured for additional 8, 24 and 48 hours in fresh DM. Vinculin was used as loading control. Numbers above each lane represent the densitometric analysis.</p

High doses of metformin inhibit the proliferation of C2C12 cells without inducing apoptosis.

<p>(a) C2C12 cells were treated with different doses of metformin in growth medium (GM) and the total number of cells was counted after 1, 2, 3 and 4 days of treatment by immunofluorescence microsopy. The initial number of plated cells was the same in each growth condition. Statistical significance was evaluated by the Student’s t-test (*p<0.05) (b) TUNEL assay. C2C12 cells were treated with 100μM, 2mM and 10mM metformin for 48h. As positive control for the TUNEL assay C2C12 myoblasts were incubated with DNΑse I before staining and, as negative control, cells were stained with the label solution without the addition of the reaction enzyme terminal deoxynucleotidyl transferase (TdT). (c) Total protein extracts of C2C12 myoblasts treated with 100μM, 2mM and 10mM were analyzed by SDS-PAGE for the expression of the apoptotic markers cl-caspase 3 and cl-caspase 7. For the induction of apoptosis in the positive control was used staurosporine 1μM for 4 hours. GAPDH is used as loading control (d) Proliferating C2C12 myoblasts, were plated at the same initial number (4*10⁴ in GM in 9,5 cm² area wells), incubated with 100μM, 2mM and 10mM metformin for 48h. The percentage of cells expressing Ki67 was measured by Cell Profiler cell image analysis software. Statistical significance was evaluated by the Student’s t-test (*p<0.05).</p

Proposed model consistent with the presented data.

<p>The model represents the causal relationships that can explain the experimental observations obtained by treating the cells with high doses of metformin (5 and 10mM), as extracted from the SIGNOR database, available at <a href="http://signor.uniroma2.it/" target="_blank">http://signor.uniroma2.it</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182475#pone.0182475.ref054" target="_blank">54</a>]. Activations and inhibitions are depicted as blue arrowhead and red hammerhead, respectively. Direct relationships are represented by continuous lines while indirect ones by dashed lines. Proteins are labeled with their UniprotKB gene names. The correspondence with the common name used in the text is the following: PRKAA1 = Ampk, NAMPT = Nampt, SIRT1 = Sirt1, PPARGC1A = PGC1a, FOXO3 = Foxo3, MYOD1 = MyoD, MYOG = myogenin, CCND3 = cyclin D3, CDKN1A = p21, RBL2 = p130, CDKN1B = p27.</p

Metformin inhibits myogenic differentiation of C2C12 cells in a dose-dependent manner.

<p>(a) Immunofluorescence microscopy for the expression of the myogenic markers Myosin Heavy Chain (MHC) and MyoD. C2C12 induced to differentiate by serum deprivation in differentiation medium (DM) and treated for 3 days with different doses of metformin (100μM, 500μM, 2mM, 5mM and 10mM) were analyzed by immunofluorescence technique. The percentage of the cells positive for MyoD and the fusion index were determined using the Cell Profiler cell image analysis software. The fusion index was calculated as the % of the nuclei inside myotubes compared to the total number of nuclei. Only myotubes with at least three nuclei inside a continuous cell membrane were considered. Statistical significance was evaluated by the Student’s t-test (*p<0.05) (b) Western blot analysis of MyoD, myogenin, MHC and Myf5 in C2C12 myoblasts treated with different doses of metformin (100μM, 2mM and 10mM) for 3 days. Tubulin and vinculin are used as loading controls. Numbers above each band represent the quantitation of the densitometric analysis.</p