Rebound increase in microRNA levels at the end of 5-FU-based therapy in colorectal cancer patients.

Treatment with 5-fluorouracil (5-FU) based therapy is still used for colorectal cancer (CRC). Epigenetics has become a focus of study in cancer because of its reversibility besides its known regulatory functions. In this study, we will monitor the change in microRNAs (miRNAs) levels with 5-FU-based therapy at baseline and after 3 and 6 months of treatment to be correlated with their prognostic potential. The expression levels of 5 miRNAs, namely miRNA223-3p, miRNA20a-5p, miRNA17-5p, miRNA19a-3p, and miRNA7-5p, were measured in the peripheral blood of 77 CRC patients, along with the expression of 3 proteins PTEN, ERK, and EGFR. At baseline, CRC patients had significantly higher levels of circulating miRNAs than healthy controls. This level was reduced after 3 months of 5-FU-based therapy, then increased after 6 months significantly in responder patients compared to non-responders. MiRNA19a-3p showed that significant pattern of change in the subgroups of patients with high ERK, EGFR, and PTEN protein levels, and its 6 months level after 5-FU-based therapy showed significance for the hazard of increased risk of disease recurrence and progression.

A Single-Shot Harmonic Imaging Approach Utilizing Deep Learning for Medical Ultrasound.

Tissue Harmonic Imaging (THI) is an invaluable tool in clinical ultrasound owing to its enhanced contrast resolution and reduced reverberation clutter in comparison to fundamental mode imaging. However, harmonic content separation based on high pass filtering suffers from potential contrast degradation or lower axial resolution due to spectral leakage. Whereas nonlinear multi-pulse harmonic imaging schemes, such as amplitude modulation and pulse inversion, suffer from a reduced framerate and comparatively higher motion artifacts due to the necessity of at least two pulse echo acquisitions. To address this problem, we propose a deep-learning-based single-shot harmonic imaging technique capable of generating comparable image quality to pulse amplitude modulation methods, yet at a higher framerate and with fewer motion artifacts. Specifically, an asymmetric convolutional encoder-decoder structure is designed to estimate the combination of the echoes resulting from the half-amplitude transmissions using the echo produced from the full amplitude transmission as input. The echoes were acquired with the checkerboard amplitude modulation technique for training. The model was evaluated across various targets and samples to illustrate generalizability as well as the possibility and impact of transfer learning. Furthermore, for possible interpretability of the network, we investigate if the latent space of the encoder holds information on the nonlinearity parameter of the medium. We demonstrate the ability of the proposed approach to generate harmonic images with a single firing that are comparable to those from a multi-pulse acquisition.

The prognosis of lipid reprogramming with the HMG-CoA reductase inhibitor, rosuvastatin, in castrated Egyptian prostate cancer patients: Randomized trial.

AIM: The role of surgical castration and rosuvastatin treatment on lipid profile and lipid metabolism related markers was evaluated for their prognostic significance in metastatic prostate cancer (mPC) patients. METHODS: A total of 84 newly diagnosed castrated mPC patients treated with castration were recruited and divided into two groups: Group I served as control (statin non-users) while group II treated with Rosuvastatin (20 mg/day) for 6 months and served as statin users. Prostate specific antigen (PSA), epidermal growth factor receptor (EGFR), Caveolin-1 (CAV1), lipid profile (LDL, HDL, triglycerides (TG) and total cholesterol (TC)) and lipid metabolism related markers (aldoketoreductase (AKR1C4), HMG-CoA reductase (HMGCR), ATP-binding cassette transporter A1 (ABCA1), and soluble low density lipoprotein receptor related protein 1 (SLDLRP1)) were measured at baseline, after 3 and 6 months. Overall survival (OS) was analyzed by Kaplan-Meier and COX regression for prognostic significance. RESULTS: Before castration, HMG-CoA reductase was elevated in patients <65 years (P = 0.009). Bone metastasis was associated with high PSA level (P = 0.013), but low HMGCR (P = 0.004). Patients with positive family history for prostate cancer showed high levels of EGFR, TG, TC, LDL, alkaline phosphatase (ALP), but low AKR1C4, SLDLRP1, CAV1 and ABCA-1 levels. Smokers had high CAV1 level (P = 0.017). After 6 months of castration and rosuvastatin administration, PSA, TG, LDL and TC were significantly reduced, while AKR1C4, HMGCR, SLDLRP1, CAV1 and ABCA-1 were significantly increased. Overall survival was reduced in patients with high baseline of SLDLRP1 (>3385 pg/ml, P = 0.001), PSA (>40 ng/ml, P = 0.003) and CAV1 (>4955 pg/ml, P = 0.021). CONCLUSION: Results of the current study suggest that the peripheral lipidogenic effects of rosuvastatin may have an impact on the treatment outcome and survival of castrated mPC patients. TRAIL REGISTRATION: This trial was registered at the Pan African Clinical Trial Registry with identification number PACTR202102664354163 and at ClinicalTrials.gov with identification number NCT04776889.

Astaxanthin extenuates the inhibition of aldehyde dehydrogenase and Klotho protein expression in cyclophosphamide-induced acute cardiomyopathic rat model.

This study evaluated the mechanistic sequel of aldehyde dehyrogenase (ALDH2) and Klotho protein in cyclophosphamide (CP)-induced cardiotoxicity in rats and the protective effect of astaxanthin (AST) against that sequel. A total of 40 male Wistar albino rats were divided into four groups of 10 animals each: Group 1 was injected intraperitoneally (i.p.) with normal saline for 10 successive days. Group 2 was injected with normal saline for 5 days before and after a single dose of CP (200 mg/kg, i.p.). Group 3 received AST (50 mg/kg/day, i.p.) for 10 days. Group 4 received CP as group 2 and AST as group 3. After the last dose of the treatment protocol, serum was separated to measure cardiotoxicity indices and the left ventricle was then dissected for mRNA and protein expression studies and histopathological examinations. Treatment with CP significantly increased serum lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), and troponin, while significantly decreased soluble α Klotho protein and caused histopathological lesions in cardiac tissues. In cardiac tissues, CP significantly decreased gene expression of ALDH2, Klotho protein, mTOR, IGF, AKT, AMPK, BCL2, but significantly increased expression of BAX and caspase-8. Interestingly, administration of AST in combination with CP completely reversed all the biochemical, histopathological and gene expression changes induced by CP to the control values. The current study suggests that inhibition of ALDH2, Klotho protein, mTOR, and AMPK signals in cardiac tissues may contribute to CP-induced acute cardiomyopathy. AST supplementation attenuates CP-induced cardiotoxicity by modulating ALDH2 and Klotho protein expression in heart tissues, along with its downstream apoptosis effector markers.

Upregulation of FOXP3 is associated with severity of hypoxia and poor outcomes in COVID-19 patients.

The levels of messenger RNA (mRNA) transcription of FOXP3, IFN-γ, TNF, IL-6 and COX-2 from both COVID-19 infected and control subjects were evaluated using SYBRTM green real-time polymerase chain reaction (RT-PCR). Severe/critical cases showed significantly lower lymphocyte counts and higher neutrophil counts than the mild or moderate cases. There were significantly lower levels of mRNA expressions of IFN-γ, TNFα and FOXP3 in COVID-19 patients than in the control group. On the other hand, IL-6 and COX-2 expressions were significantly higher in patients suffering from severe disease. FOXP3 expressions were correlated with the severities of hypoxia and were excellent in predicting the disease severity. This was followed by the IL-6, COX-2 and TNFα expressions. FOXP3 expression was the only biomarker to show a significant correlation with patient mortality. It was concluded that SARS-CoV-2 infection is associated with the downregulation of FOXP3 and upregulations of IL-6 and COX-2.

Fluoropyrimdine therapy induced alterations in interleukins expression in colorectal cancer patients.

This study monitored the changes in the expression of inflammatory IL-6 and IL-1ß during the treatment period of Fluoropyrimidine (FP) based therapy. RNA was extracted from the peripheral blood of 102 CRC patients before treatment with FP therapy, and from 48 and 32 patients after 3 and 6 months of treatment, respectively. The genetic transcription of IL-6 and IL-1ß was determined by real time PCR. Patients were stratified according to their levels of IL-6 and IL-1ß genes expression for subgroup and survival analyses. Baseline CRC patients showed overexpression of IL-6 and IL-1ß compared to healthy control. FP therapy significantly induced IL-6 and IL-1ß expression. Subgroup analysis showed that patients with right colon tumors had significant elevation in both IL-6 and IL-1ß with FP therapy. FP therapy significantly induced IL-1ß expression in patients ⩽45 years, smokers, with high baseline level of CA19.9, right colon tumors, low grade pathology, T3 tumors and positive lymph nodes. Survival analysis showed that baseline levels of interleukins expression had insignificant effect on overall survival and event free survival. FP therapy has an impact on the level of interleukins expression declared in certain clinicopathological subgroups of CRC patients, but without a prognostic significance on patients' survival.

The Clinical Significance of Promoter Methylation of Fluoropyrimidine Metabolizing and Cyclooxygenase Genes in Colorectal Cancer.

AIMS: This study investigated the impact of promoter methylation of flouropyrimidine (FP) metabolizing and cyclooxygenase 2 (COX2) genes on their mRNA expression and on the clinical outcome of colorectal cancer (CRC) patients. METHODS: Methylation specific-PCR and real time-PCR of thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and COX2 were performed at baseline and after 3 and 6 months of FP therapy. Pairwise comparisons were conducted between the subgroups of CRC patients. The event free survival (EFS) and the hazard of progression were estimated by univariate and multivariate analyses. RESULTS: At baseline CRC patients, both TS and TP were overexpressed, in spite of the unmethylation of TS and the full methylation of TP genes. Significant downexpression of DPD and COX2 were associated their promoter's methylation. At the end of FP therapy, TS, DPD and COX2 were overexpressed by 7.52, 2.88 and 3.45 folds, respectively, while TP was downexpressed by 0.54 fold. However, no change was observed in the methylation status of genes with FP therapy. Pairwise comparisons revealed significant difference in the expression and the methylation status of genes according to the clinicopathological characters of CRC patients either at baseline or after FP therapy. The overexpression of DPD and COX2 genes were indicators for a poor EFS of CRC patients. Also, the high level of COX2 expression was found to be significantly correlated with the hazard of progression (HR = 1.73, 95% CI = 1.02-3.03). CONCLUSION: The promoter methylation of FP metabolizing and COX2 genes has significant impact on the expression and the treatment outcome of CRC patients.

Epigenetic immunomodulatory effect of eugenol and astaxanthin on doxorubicin cytotoxicity in hormonal positive breast Cancer cells.

BACKGROUND: Hormonal receptor positive (HR+) breast cancer is the most commonly diagnosed molecular subtype of breast cancer; which showed good response to doxorubicin (DOX)-based chemotherapy. Eugenol (EUG) and astaxanthin (AST) are natural compounds with proved epigenetic and immunomodulatory effects in several cancer cell lines. This study has been initiated to investigate the molecular mechanism (s) whereby EUG and AST could enhance DOX cytotoxicity in MCF7 cells. METHODS: Cytotoxic activity of DOX alone and combined with either 1 mM EUG or 40 µM AST was performed using sulphorhodamine-B assay in MCF7 cells. Global histones acetylation and some immunological markers were investigated using ELISA, western blotting and quantitative RT-PCR techniques. Functional assay of multidrug resistance was performed using rhodamine 123 and Hoechst 3342 dyes. Flow cytometry with annexin V and propidium iodide were used to assess the change in cell cycle and apoptosis along with the expression of some differentiation, apoptosis and autophagy proteins. RESULTS: DOX alone resulted in concentration-dependent cytotoxicity with IC50 of 0.5 µM. Both EUG and AST significantly increased DOX cytotoxicity which is manifested as a significant decrease in DOX IC50 from 0.5 µM to 0.088 µM with EUG and to 0.06 µM with AST. Combinations of DOX with 1 mM EUG or 40 µM AST significantly increased the level of histones acetylation and histone acetyl transferase expression, while reduced the expression of aromatase and epidermal growth factor receptor (EGFR) when compared with 0.25 µM DOX alone. Also both combinations showed higher uptake of rhodamine but lower of Hoechst stains, along with increased the percentage of caspase 3, and decreased the expression of CK7 and LC3BI/II ratio. EUG combination induced IFγ but reduced TNFα causing shifting of cells from G2/M to S and G0/ G1 phases. Combination of DOX with EUG induced apoptosis through the higher BAX/ BCl2 ratio, while with AST was through the increase in caspase 8 expressions. CONCLUSION: EUG and AST potentiated the anticancer activity of DOX through epigenetic histones acetylation along with the immunonomodulation of different apoptotic approaches in MCF7 cells.

The Prognostic Significance of 5-Fluorouracil Induced Inflammation and Immuno-Modulation in Colorectal Cancer Patients.

AIM: The change in the levels of peripheral inflammatory markers together with EGFR in relation to 5- fluorouracil (5-FU) therapy was evaluated for their prognostic significance in colorectal cancer (CRC) patients. PATIENTS AND METHODS: Expression levels of COX2, IL6, IL1ß, EGFR, IL10, and TNFα were determined with quantitative real-time PCR (qPCR) in the peripheral blood of 90 CRC patients. The inflammatory response was correlated with patients' clinical features, disease-free survival (DFS), and overall survival (OS). RESULTS: After 6 months of 5-FU therapy, increased inflammatory response was found to be associated with smoking, T3 or T4 tumors, performance status (PS) III, positive lymph nodes, distant metastasis, and gastrointestinal (GIT) toxicity. The combination of COX2 with interleukins in a predictive equation for DFS was significant in patients with over-expression of EGFR. DFS and OS rates were reduced in patients with increased COX2, IL6, IL10, and TNFα expression with 5-FU therapy. Significant hazard of disease progression was associated with smoking (HR=1.27, P=0.004), 5-FU induction of COX2, and IL6 expression (HR=1.35, P=0.001 and HR=1.27, P=0.004, respectively). Moreover, smoking, 5-FU induction of IL6, TNFα, and IL10 expression are found to be independent prognostic factors for OS (P=0.003, 0.003, 0.002, and 0.002, respectively). CONCLUSION: The peripheral effects of 5-FU therapy have shown a significant impact on the treatment outcome of CRC patients.

Impact of Global DNA Methylation in Treatment Outcome of Colorectal Cancer Patients.

Background: Global DNA methylation has an impact in cancer pathogenesis and progression. This study aimed at investigating the impact of global DNA methylation in treatment outcome of Colorectal Cancer (CRC). Patients and Methods: Global DNA methylation was measured by LC/MS/MS in peripheral blood leucocytes of 102, 48, and 32 Egyptian CRC patients at baseline and after 3 and 6 months of Fluoropyrimidine (FP) therapy respectively, in addition to 32 normal healthy matched in age and sex. The genetic expressions of DNA methyl transferases (DNMTs) were determined and correlated with patients' survival using univariate and multivariate methods of analyses. Results: Egyptian CRC patients had significant global hypomethylation of 5mC level and 5mC % with overexpression of DNMT3A and DNMT3B. Significant higher 5mC levels were shown in patients > 45 years, male gender, T2 tumors, stage II, negative lymph nodes, and absence of metastasis. FP therapy significantly reduced DNA methylation particularly in the subgroups of patients with high DNA methylation level at baseline and good prognostic features. After 3 years of follow up, patients with 5mC % > 8.02% had significant poor overall survival (OS) while, significant better event-free survival (EFS) was found in patients with 5mC level > 0.55. High initial CEA level and presence of metastasis were significantly associated with hazards of disease progression and death. Conclusion: Global DNA methylation has a significant impact on the treatment outcome and survival of Egyptian CRC patients treated with FP- based therapy.

The Interaction between Adult Cardiac Fibroblasts and Embryonic Stem Cell-Derived Cardiomyocytes Leads to Proarrhythmic Changes in In Vitro Cocultures.

Transplantation of stem cell-derived cardiomyocytes is one of the most promising therapeutic approaches after myocardial infarction, as loss of cardiomyocytes is virtually irreversible by endogenous repair mechanisms. In myocardial scars, transplanted cardiomyocytes will be in immediate contact with cardiac fibroblasts. While it is well documented how the electrophysiology of neonatal cardiomyocytes is modulated by cardiac fibroblasts of the same developmental stage, it is unknown how adult cardiac fibroblasts (aCFs) affect the function of embryonic stem cell-derived cardiomyocytes (ESC-CMs). To investigate the effects of aCFs on ESC-CM electrophysiology, we performed extra- and intracellular recordings of murine aCF-ESC-CM cocultures. We observed that spontaneous beating behaviour was highly irregular in aCF-ESC-CM cocultures compared to cocultures with mesenchymal stem cells (coefficient of variation of the interspike interval: 40.5 ± 15.2% versus 9.3 ± 2.0%, p = 0.008) and that action potential amplitude and maximal upstroke velocity (V max) were reduced (amplitude: 52.3 ± 1.7 mV versus 65.1 ± 1.5 mV, V max: 7.0 ± 1.0 V/s versus 36.5 ± 5.3 V/s), while action potential duration (APD) was prolonged (APD50: 25.6 ± 1.0 ms versus 16.8 ± 1.9 ms, p < 0.001; APD90: 52.2 ± 1.5 ms versus 43.3 ± 3.3 ms, p < 0.01) compared to controls. Similar changes could be induced by aCF-conditioned medium. We conclude that the presence of aCFs changes automaticity and induces potentially proarrhythmic changes of ESC-CM electrophysiology.