Genesis of epicardial adipocytes and its association with progenitor markers, muscarinic receptor type 3 and b-blockers intake in patients with cardiovascular disease

Introduction Epicardial fat thickness or volume was found to be associated with cardiovascular disease (CVD). Our aim was to study the epicardial adipocyte-progenitors' markers and its association with cholinergic or adrenergic activity in patients with cardiovascular disease. Materials and methods We have included epicardial adipose tissue (EAT) biopsies from 29 patients underwent open-heart surgery. From 10 patients (69±5 years old, 31±8 kg/ m2, 40% CAD, 40% HF, 60% AF, 0% T2DM) stromal cells from epicardial and subcutaneous fat were isolated after collagenase activity and cultured for 14 days and then submitted to adipogenesis for next 14 days. Samples from 19 patients (60±9 years old, 29±4 kg/m2, 42% CAD, 37% HF, 32% AF, 32% T2DM, 53% β-blockers) were used for “ex vivo assays”. Explants were split into equal pieces (100 mg), treated with or without acetylcholine (ACh) for 30 min. Afterwards RNA was isolated and cDNA was amplified by real time PCR. We selected adipocytes progenitors (CD36, PREF1, COL1A1), adipocytes markers (ADIPO, FABP4), muscarinic (muscarinic receptor type 2 (CHRM2) and 3 (CHRM2)) and β-adrenergic receptors (ABRD1, ABRD2 and ABRD3). Gene expression was represented regarding ACTB as 2HK/GEN. Results The stromal vascular cells (SVC) from subcutaneous fat (SAT) had higher expression levels of CD36, PREF1 and COL1A1 than SVC from epicardial fat (EAT). It explains the higher adipocytes markers after adipogenesis induction in SAT than EAT cells. However, an upregulation of fibroblasts markers was detected on EAT. The levels of CD36 and PREF1 in SVC were associated with higher adipogenesis. Although CHRM2 was higher in EAT than SAT SVC, the adipogenesis induction upregulated only CHRM3 (1.48±0.065 vs 1.42±0.036 a.u.) in EAT cells. Thus, this receptor was associated with adipocytes markers in epicardial fat (r=0.777 for CD36 and r=0.746 for FABP4) and incremented in epicardial fat biopsies from patients who were taken β-blockers (1.61±0.011 n=10 vs 1.54±0.097 a.u. n=9; p=0.05) and modulated by ACh treatment (p=0.05). Conclusions Our results showed that CD36 and PREF1 in epicardial SVC are adipocytes progenitors. The higher presence of adipocytes markers is associated with higher levels of muscarinic receptor (CHRM3), which are upregulated in epicardial fat from patients who were taken β-blockers and modulated by cholinergic activity. Because a metabolic and lipolytic dysfunction was associated with CHRM3, the sympathetic modulation might play a role in the epicardial adipocytes genesis. Further studies are needed to understand if this mechanism might improve or not future cardiovascular events. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII (PFIS2020)

Natural Phenolic Acid, Product of the Honey Bee, for the Control of Oxidative Stress, Peritoneal Angiogenesis, and Tumor Growth in Mice

Tumor-associated macrophages (TAM) are key regulators of the link between inflammation and cancer, and the interplay between TAM and tumor cells represents a promising target of future therapeutic approaches. We investigated the effect of gallic acid (GA) and caffeic acid (CA) as strong antioxidant and anti-inflammatory agents on tumor growth, angiogenesis, macrophage polarization, and oxidative stress on the angiogenic model caused by the intraperitoneal (ip) inoculation of Ehrlich ascites tumor (EAT) cells (2.5 × 106) in Swiss albino mouse. Treatment with GA or CA at a dose of 40 mg/kg and 80 mg/kg ip was started in exponential tumor growth phase on days 5, 7, 9, and 11. On day 13, the ascites volume and the total number and differential count of the cells present in the peritoneal cavity, the functional activity of macrophages, and the antioxidant and anti-angiogenic parameters were determined. The results show that phenolic acids inhibit the processes of angiogenesis and tumor growth, leading to the increased survival of EAT-bearing mice, through the protection of the tumoricidal efficacy of M1 macrophages and inhibition of proangiogenic factors, particularly VEGF, metalloproteinases -2 and -9, and cyclooxygenase-2 activity.

Effects of curcumin on lipid peroxidation and antioxidant enzymes in kidney, liver, brain and testis of mice bearing Ehrlich Solid Tumor

Introduction: Cancer is the second most common cause of death in the world. Several natural products have been studied for anticancer activity and for prevention or repair of oxidative injury. Curcumin is one of the natural products of high medicinal interest. This study was performed to investigate effects of curcumin on lipid peroxidation and antioxidant enzymes in tissues of mice bearing Ehrlich solid tumor. Materials and Methods: Forty mice were distributed to four groups as healthy control and treatments that received 1x106 Ehrlich ascites tumor (EAT) cells and EAT cells plus 25 mg/kg/day or 50 mg/kg/day curcumin with a single subcutaneous injection. The liver, kidney, brain and testis tissues were collected for the MDA, SOD and CAT analyses. Results: Tumor development increased MDA levels in liver (p=0.001), kidney (p<0.001) and testis (p<0.01) and curcumin reduced liver MDA. Liver and kidney SOD activities were increased by both levels of curcumin (p=0.001) but 50 mg/kg/day curcumin increased brain SOD activity (p<0.001). The kidney CAT activity was increased by 50 mg/kg/day curcumin (p<0.001). Discussion: This study showed that curcumin suppresses tumor progression, and alleviates the lipid peroxidation and improves antioxidant status in the tissues of solid tumor-bearing mice.

Rhamnetin improves antioxidant status in the liver of Ehrlich solid tumor bearing mice

Objective: Rhamnetin, a flavanol, is in the subclasses of the flavonoids existing in plants. The antioxidant properties of several plants containing flavonoids have been extensively studied in several diseases including cancer. This study investigated the effects of rhamnetin on tumor masses, oxidant and antioxidant status in the livers of mice bearing Ehrlich solid tumor. Material and Methods: Fifty male Balb/C mice weighing 25-30 g were used in the study. Ten mice were kept for Ehrlich ascites tumor (EAT) cells production and the remaining mice were randomly assigned to four groups containing 10 mice in each as healthy control and treatments receiving 1x106 EAT cells and EAT cells plus 100 µg/kg/day or 200 µg/kg/day rhamnetin via subcutaneous route. The tumor inhibition rates of rhamnetin treatments were calculated. The livers were analyzed for malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels. Results: Compare to tumor control, both levels of rhamnetin suppressed tumor masses throughout the experiment. The MDA levels were increased whereas SOD and CAT activities were reduced by EAT cells injection in the liver of mice. The 100 µg/kg/day rhamnetin treatment decreased MDA level but 200 µg/kg/day rhamnetin had no significant effect on increased MDA level. The reduced liver SOD (p<0.001) and CAT (p<0.01) activities were elevated by both levels of rhamnetin injection. Conclusions: The results of this study have revealed that rhamnetin suppresses tumor progression and improves antioxidant status in the livers of solid tumor-bearing mice.

The Anti-Tumoral Effects of Wolfberry (Lycium barbarum) on Experimentally Induced Ehrlich Ascites Carcinoma in Mice

Wolfberry (Lycium barbarum) shows strengthen immune system, antioxidant and anticancerogenic effect. In this research, the effects of Wolfberry's extract fractions, cultivated in Kayseri, investigated on Ehrlich ascites tumor (EAT) using in vivo and in vitro techniques. For in vivo study, 200 mg/kg fractions of Wolfberry extract (above and below 50 kDa) were injected ip to EAT injected Balb/C mice. EAT cells were also cultured in the presence of Wolfberry's extract fractions (1500, 2000 µg/ml) to examine cell vitality and apoptosis using the Muse Cell Analyzer. Histopathological examination of intra-abdominal organs showed that there were decrease in the EAT cells adherence in the Wolfberry applied tissue groups when compared to the control. According to in vitro results, the both extract fractions (above and below 50 kDa) increased apoptosis in cancer cells. However, total apoptotic cell percentage was higher and statistically significant in groups above 50 kDa (1500, 2000 mg/ml) (p < 0.05). Previous literature and the results of the present study reveal that the consumption of wolfberry—which is also produced in Turkey—might be effective in preventing the formation of cancer and the deceleration of its progress.

Anti-Angiogenic Effects of Some (5-hydroxy-4-methyl-2-((1-methyl-1H-indol-3-yl)methyl)-Thieno[2,3-d] Pyrimidine Derivatives

Some previously reported pyrimidine candidates 1–7 were founded to have potent tumor inhibition activities on diffrrent models of human primary and secondary brain tumors and in the same time these derivatives were founded to have potent anti-angiogenic effects on Ehrlich Ascites Tumor [EAT] cells in-vivo. These pyridine derivatives were founded to be selectively inhibit the p110α isoform. These pyridine derivatives were potent inhibited the histone deactylase. Also, they are inhibited the Sphingosine kinase SK1 activities.

SYNERGIC CARCINOSTATIC EFFECTS OF ASCORBIC ACID AND HYPERTHERMIA ON EHRLICH ASCITES TUMOR CELL

Aim: In this study, we evaluated the carcinostatic effects of combined ascorbic acid (AsA) and a capacitive-resistive electric transfer (CRet) hyperthermic apparatus-induced hyperthermic treatment on Ehrlich ascites tumor (EAT) cells. Materials and Methods: EAT cells were exposed to various AsA (0–10 mM) concentrations for 1 h; they subsequently underwent CRet treatment for 15 min at 42 °C. Cell viability was assessed by the WST-8 assay 24 h after the combined treatment. Reactive oxygen species involvement was evaluated using catalase and tempol; caspase-3/7 activation was determined by their fluorescent substrates; cell proliferation were estimated by time-lapse observation. The effect on the cell cycle was analyzed by flow cytometry. Results: Combined AsA and CRet treatment synergistically suppressed cell viability compared with either treatment alone, and these synergistically carcinostatic effects were evident even at noncytotoxic concentrations of AsA alone (≤ 2 mM). The carcinostatic effects of combined AsA and CRet treatment were attenuated in a dose-dependent manner by catalase addition, but not by the superoxide anion radical scavenger tempol. Time-lapse observation revealed that combined AsA and CRet treatment activated caspase-3/7 at 10–24 h after treatment, accompanied by significant cell growth suppression. Cell cycle analysis revealed that the rate of sub-G1-phase (apoptotic) cells was drastically increased at 12 h and 24 h, and that the G2/M-phase cells gradually increased at 6–24 h after treatment. Conclusion: These results indicate that combined AsA and CRet treatment synergistically inhibits EAT cell growth through G2/M arrest and apoptosis induction via H2O2 generation at lower AsA concentrations; this carcinostatic effect cannot be exert ed by AsA alone.

Modulation of Cytokines Production by Indomethacin Acute Dose during the Evolution of Ehrlich Ascites Tumor in Mice

The aim of the present study was to investigate the influence of a nonselective COX1/COX2 inhibitor (indomethacin) on tumor growth of Ehrlich Ascites Tumor (EAT) in mice, using as parameters the tumor growth and cytokine profile. Mice were inoculated with EAT cells and treated with indomethacin. After 1, 3, 6, 10, and 13 days the animals were evaluated for the secretion of TNFα, IL-1α, IL-2, IL-4, IL-6, IL-10, and IL-13 and PGE2level in peritoneal cavity. The results have shown that EAT induces PGE2production and increases tumor cells number from the 10th day. The cytokine profile showed EAT induces production of IL-6 from 10th day and of IL-2 on 13th day; the other studied cytokines were not affected in a significant way. The indomethacin treatment of EAT-bearing mice inhibited the tumor growth and PGE2synthesis from the 10th day. In addition, the treatment of EAT-bearing mice with indomethacin has stimulated the IL-13 production and has significantly inhibited IL-6 in the 13th day of tumor growth. Taken together, the results have demonstrated that EAT growth is modulated by PGE2and the inhibition of the tumor growth could be partly related to suppression of IL-6 and induction of IL-13.