Enhanced hepatoprotective effects of empagliflozin and vitamin D dual therapy against metabolic dysfunction-associated steatohepatitis in mice by boosted modulation of metabolic, oxidative stress, and inflammatory pathways.

Although single treatment with sodium-glucose cotransporter-2 inhibitors (SGLT2i) or vitamin D3 (VD3) inhibited metabolic dysfunction-associated steatohepatitis (MASH) development in diabetic patients, their combination has not been explored previously. Hence, this study investigated the hepatoprotective effects of SGLT2i (empagliflozin) and/or VD3 against MASH in type 2 diabetic mice. Forty Mice were assigned into negative (NC) and positive (PC) controls, SGLT2i, VD3, and SGLT2i + VD3 groups. All animals, except the NC group, received high-fructose/high-fat diet (8 weeks) followed by diabetes induction. Diabetic mice then received another cycle of high-fructose/high-fat diet (4 weeks) followed by 8 weeks of treatment (five times/week) with SGLT2i (5.1 mg/kg/day) and/or VD3 (410 IU/Kg/day). The PC group demonstrated hyperglycaemia, dyslipidaemia, elevated liver enzymes, and increased non-alcoholic fatty liver disease activity score (NAS) with fibrosis. Hepatic glucose transporting molecule (SGLT2) with lipogenesis (SREBP-1/PPARγ), oxidative stress (MDA/H2O2), inflammation (IL1ß/IL6/TNF-α), fibrosis (TGF-ß1/α-SMA), and apoptosis (TUNEL/Caspase-3) markers alongside the PI3K/AKT/mTOR pathway increased in the PC group. Conversely, hepatic insulin-dependent glucose transporter (GLUT4), lipolytic (PPARα/INSIG1), antioxidant (GSH/GPx1/SOD1/CAT), and anti-inflammatory (IL-10) molecules with the inhibitor of PI3K/AKT/mTOR pathway (PTEN) decreased in the PC group. Whilst SGLT2i monotherapy outperformed VD3, their combination showed the best attenuation of hyperglycaemia, dyslipidaemia, and fibrosis with the strongest modulation of hepatic glucose-transporting and lipid-regulatory molecules, PI3K/AKT/mTOR pathway, and markers of oxidative stress, inflammation, fibrosis, and apoptosis. This study is the first to reveal boosted hepatoprotection for SGLT2i and VD3 co-therapy against diabetes-induced MASH, possibly via enhanced metabolic control and modulation of hepatic PI3K/AKT/mTOR, anti-inflammatory, anti-oxidative, and anti-fibrotic pathways.

Improved Glycaemic Control and Nephroprotective Effects of Empagliflozin and Paricalcitol Co-Therapy in Mice with Type 2 Diabetes Mellitus.

Herein, we measured the antidiabetic and nephroprotective effects of the sodium-glucose cotransporter-2 inhibitor (empagliflozin; SGLT2i) and synthetic active vitamin D (paricalcitol; Pcal) mono- and co-therapy against diabetic nephropathy (DN). Fifty mice were assigned into negative (NC) and positive (PC) control, SGLT2i, Pcal, and SGLT2i+Pcal groups. Following establishment of DN, SGLT2i (5.1 mg/kg/day) and/or Pcal (0.5 µg/kg/day) were used in the designated groups (5 times/week/day). DN was affirmed in the PC group by hyperglycaemia, dyslipidaemia, polyuria, proteinuria, elevated urine protein/creatinine ratio, and abnormal renal biochemical parameters. Renal SREBP-1 lipogenic molecule, adipokines (leptin/resistin), pro-oxidant (MDA/H2O2), pro-inflammatory (IL1ß/IL6/TNF-α), tissue damage (iNOS/TGF-ß1/NGAL/KIM-1), and apoptosis (TUNEL/Caspase-3) markers also increased in the PC group. In contrast, renal lipolytic (PPARα/PPARγ), adiponectin, antioxidant (GSH/GPx1/SOD1/CAT), and anti-inflammatory (IL10) molecules decreased in the PC group. Both monotherapies increased insulin levels and mitigated hyperglycaemia, dyslipidaemia, renal and urine biochemical profiles alongside renal lipid regulatory molecules, inflammation, and oxidative stress. While SGLT2i monotherapy showed superior effects to Pcal, their combination demonstrated enhanced remedial actions related to metabolic control alongside renal oxidative stress, inflammation, and apoptosis. In conclusion, SGLT2i was better than Pcal monotherapy against DN, and their combination revealed better nephroprotection, plausibly by enhanced glycaemic control with boosted renal antioxidative and anti-inflammatory mechanisms.

Pneumococcal Surface Protein A: A Promising Candidate for the Next Generation of Pneumococcal Vaccines.

Streptococcus pneumoniae is the bacterium that causes pneumococcal disease which often results in pneumonia, meningitis, otitis media, septicemia and sinusitis. Pneumonia, particularly, is a significant cause of worldwide morbidity and a global health burden as well. Treatment often relies on antimicrobials, to which the pathogen is frequently mutating and rendering infective. Consequently, vaccination is the most effective approach in dealing with pneumococcal antimicrobial resistance (AMR). Unfortunately, the current pneumococcal polysaccharide and conjugate vaccines have a narrow serotype coverage. Therefore, the current need for vaccines with a broader serotype coverage cannot be overstated. Pneumococcal Surface Protein A and C are potential vaccine candidate antigens present in over 90% of the strains from clinical isolates as well as laboratory non-encapsulated strains. Pneumococcal Surface Protein A is an active virulent factor that pneumococci use to evade complement-mediated host immune responses and has been shown to elicit immune responses against pneumococcal infections. This review explores the potential utilization of Pneumococcal Surface Protein A to immunize against S. pneumoniae.

Ethyl benzoate bearing pyrrolizine/indolizine moieties: Design, synthesis and biological evaluation of anti-inflammatory and cytotoxic activities.

Two new series of ethyl benzoate bearing pyrrolizine and indolizine moieties 8-11 were synthesized and evaluated for their anti-inflammatory and anticancer activities. Among these derivatives, compounds 9a, 10b and 11b displayed in vivo anti-inflammatory and analgesic activity comparable to ibuprofen. The acute ulcerogenicity and histopathological studies revealed better GIT safety profile than ibuprofen. Mechanistic study of these compounds revealed inhibitory activity against COX-1/2 with preferential inhibition of COX-2. Evaluation of cytotoxic activity of the new compounds using MTT assay revealed potent to moderate activity against three human (MCF-7, A2780 and HT29) cancer cell lines (IC50 = 0.02-23.35 µM). Compounds 9a, 10a,b and 11a,b exhibited high cytotoxic selectivity against MCF-7 cells (SI = 4-84). Although the indolizine bearing derivatives 8-11b exhibited higher selectivity to COX-2 than their corresponding pyrrolizine analogs 8-11a, but they were less active and selective against MCF-7 cells. Cell cycle analysis and annexin V-FITC/PI assay revealed G1 cell cycle arrest and induction of apoptosis in MCF-7 cells by compound 9a. The docking study revealed nice fitting of the new compounds into the active site of COX-1/2 with higher affinity to COX-2. Compounds 8-11 displayed drug-likeness score in the range of 0.67-1.56 compared to 1.06 for licofelone. These results suggested that compounds 9a, 10b and 11b could be promising agents in future research as anti-inflammatory and anticancer agents.

Synergistic Anti Leukemia Effect of a Novel Hsp90 and a Pan Cyclin Dependent Kinase Inhibitors.

Acute myeloid leukemia (AML) is among the top four malignancies in Saudi nationals, and it is the top leukemia subtype worldwide. Resistance to available AML drugs requires the identification of new targets and agents. Hsp90 is one of the emerging important targets in AML, which has a central role in the regulation of apoptosis and cell proliferation through client proteins including the growth factor receptors and cyclin dependent kinases. The objective of the first part of this study is to investigate the putative Hsp90 inhibition activity of three novel previously synthesized quinazolines, which showed HL60 cytotoxicity and VEGFR2 and EGFR kinases inhibition activities. Using surface plasmon resonance, compound 1 (HAA2020) showed better Hsp90 inhibition compared to 17-AAG, and a docking study revealed that it fits nicely into the ATPase site. The objective of the second part is to maximize the anti-leukemic activity of HAA2020, which was combined with each of the eleven standard inhibitors. The best resulting synergistic effect in HL60 cells was with the pan cyclin-dependent kinases (CDK) inhibitor dinaciclib, using an MTT assay. Furthermore, the inhibiting effect of the Hsp90α gene by the combination of HAA2020 and dinaciclib was associated with increased caspase-7 and TNF-α, leading to apoptosis in HL60 cells. In addition, the combination upregulated p27 simultaneously with the inhibition of cyclinD3 and CDK2, leading to abolished HL60 proliferation and survival. The actions of HAA2020 propagated the apoptotic and cell cycle control properties of dinaciclib, showing the importance of co-targeting Hsp90 and CDK, which could lead to the better management of leukemia.

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters.

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection.

Seroprevalence of transfusion-transmitted infections among blood donors in Makkah, Saudi Arabia.

INTRODUCTION: Blood donation is vital for healthcare; however, transfusion-transmitted infections (TTIs) pose a serious risk. This study investigated the seroprevalence of TTIs among Saudi blood donors. METHODOLOGY: This retrospective study included male blood donors aged ≥ 18 years who donated blood at Al-Noor Specialist Hospital in Makkah from January 2017 to December 2022. The blood units were screened for hepatitis B surface antigen (HBsAg) and core antibodies (HBc-IgG), hepatitis C antibodies (HCV-Abs), syphilis, HIV-1 antigen/antibody (HIV-1 Ag/Ab), human T-lymphotropic virus 1, 2 (HTLV-1/2), and malaria. RESULTS: There were 40,287 donors with an average age of 44.33 ± 18.12 years, and 62.3% (n = 25103) were Saudis. The overall rate of TTIs seropositivity was 7.4% (n = 2953); HBc-IgG (6.1%; n = 2473) was the most common, followed by HCV-Abs (0.4%; n = 177), and syphilis (0.34%; n = 136). All cases were negative for malaria, whilst HIV and HTLV positive donors were 0.06% (n = 24) and 0.13% (n = 52), respectively. Syphilis was more prevalent among non-Saudis (0.24%; n = 83) than among Saudis (0.1%; n = 53), whereas anti-HBc antibodies seropositivity was significantly higher among Saudi (3.4%; n = 1373) than non-Saudi donors (2.7%; n = 1100). CONCLUSIONS: Hepatitis B virus was the most frequently detected bloodborne pathogen, followed by hepatitis C virus and syphilis. Hepatitis B virus was also more prevalent among Saudi donors, whilst expatriates had higher rates of syphilis. Additional prospective multicenter studies are needed to accurately determine the prevalence of TTIs in Saudi Arabia.

Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells.

BACKGROUND: Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. METHODS: Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. RESULTS: Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2. CONCLUSION: In summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent.

The Quality of Blood Donation Services and Its Association with Blood Donors' Trust and Loyalty at Makkah Blood Donation Centers in Saudi Arabia: A Cross-Sectional Study.

The current cross-sectional study was conducted to determine the quality of blood donation services and its association with blood donors' trust and loyalty at Makkah blood donation centers in Saudi Arabia. A total of 373 healthy blood donors aged ≥18 years who visited blood donation centers in Makkah, Saudi Arabia, between 1st and 28th February 2023 were recruited using a census sampling method. A pre-tested and validated Arabic language questionnaire was employed. The study survey included a checklist of sociodemographic variables (seven items), as well as seven-point Likert-scale questions on the quality of blood donation services (21 items), questions to assess the participant's level of trust in blood donation centers (4 items), and questions to evaluate the level of loyalty to blood donations (4 items). SPSS (version 24) was used for data analysis. A total of 373 blood donors were included in this study. Of them, 240 (64.3%) were males and 133 (35.7%) were females. The vast majority of the study participants, 330 (88.5%), had a high educational level. The overall average agreement score for the quality of blood donation services was 71.7%. Furthermore, the overall average item agreement score for trust in blood donation centers and places was 83.0%, while the overall average item agreement score for loyalty to blood donation was 72.1%. Moreover, after adjustment for potential confounding factors, high levels of quality in blood donation services were associated with high levels of trust and loyalty among the blood donors (OR: 1.518, CI 95%: 0.321-0.864 and OR: 2.466, CI 95%: 0.285-0.763, respectively) (p-value < 0.05 for all). The overall quality of, trust in, and loyalty to blood donation services were 71.7%, 83.0%, and 72.1%, respectively. In addition, high levels of quality in blood donation services could improve blood donors' trust and loyalty levels at Makkah blood donation centers in Saudi Arabia.

Insights from the molecular docking analysis of EGFR antagonists.

Overexpression of the epidermal growth factor receptor (EGFR) has been shown to be a critical factor in tumor development and cancer progression. Although established EGFR inhibitors have been effective in the treatment of cancer, they are associated with several side effects. As a result, there is an urgent need to develop novel EGFR inhibitors that can effectively target the receptor while causing no adverse side effects. Here, the bioactive compounds of Glycyrrhiza glabra and established EGFR inhibitors have been screened against the EGFR catalytic site. The compounds LTS0058805, LTS0114552, LTS0128805, LTS0174203, LTS0007447, and LTS0164690 exhibited binding energies to the EGFR that were comparable to those of established EGFR inhibitors. Further, these hit compounds were observed to interact with critical residues of the EGFR, suggesting their potential as inhibitors of the receptor. In addition, these hits possess good drug-like properties and merit further exploration for their potential application in cancer management.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Vitamin D and calcium co-therapy mitigates pre-established cadmium nephropathy by regulating renal calcium homeostatic molecules and improving anti-oxidative and anti-inflammatory activities in rat.

BACKGROUND: Cadmium (Cd) is a major environmental pollutant and chronic toxicity could induce nephropathy by increasing renal oxidative stress and inflammation. Although vitamin D (VD) and calcium (Ca) prophylactic treatments attenuated Cd-induced cell injury, none of the prior studies measure their renoprotective effects against pre-established Cd-nephropathy. AIMS: To measure the alleviating effects of VD and/or Ca single and dual therapies against pre-established nephrotoxicity induced by chronic Cd toxicity prior to treatment initiation. METHODS: Forty male adult rats were allocated into: negative controls (NC), positive controls (PC), Ca, VD and VC groups. The study lasted for eight weeks and all animals, except the NC, received CdCl2 in drinking water (44 mg/L) throughout the study. Ca (100 mg/kg) and/or VD (350 IU/kg) were given (five times/week) during the last four weeks to the designated groups. Subsequently, the expression of transforming growth factor-ß (TGF-ß1), inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), VD synthesising (Cyp27b1) and catabolizing (Cyp24a1) enzymes with VD receptor (VDR) and binding protein (VDBP) was measured in renal tissues. Similarly, renal expression of Ca voltage-dependent channels (CaV1.1/CaV3.1), store-operated channels (RyR1/ITPR1), and binding proteins (CAM/CAMKIIA/S100A1/S100B) were measured. Serum markers of renal function alongside several markers of oxidative stress (MDA/H2O2/GSH/GPx/CAT) and inflammation (IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 were also measured. RESULTS: The PC group exhibited hypovitaminosis D, hypocalcaemia, hypercalciuria, proteinuria, reduced creatinine clearance, and increased renal apoptosis/necrosis with higher caspase-3 expression. Markers of renal tissue damage (TGF-ß1/iNOS/NGAL/KIM-1), oxidative stress (MDA/H2O2), and inflammation (TNF-α/IL-1ß/IL-6) increased, whilst the antioxidants (GSH/GPx/CAT) and IL-10 decreased, in the PC group. The PC renal tissues also showed abnormal expression of Cyp27b1, Cyp24a1, VDR, and VDBP, alongside Ca-membranous (CaV1.1/CaV3.1) and store-operated channels (RyR1/ITPR1) and cytosolic Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B). Although VD was superior to Ca monotherapy, their combination revealed the best mitigation effects by attenuating serum and renal tissue Cd concentrations, inflammation and oxidative stress, alongside modulating the expression of VD/Ca-molecules. CONCLUSIONS: This study is the first to show improved alleviations against Cd-nephropathy by co-supplementing VD and Ca, possibly by better regulation of Ca-dependent anti-oxidative and anti-inflammatory actions.

Profiling estrogen, progesterone, and androgen receptors in colorectal cancer in relation to gender, menopausal status, clinical stage, and tumour sidedness.

Background: Although estrogen (ERα/ERß), progesterone (PGR), and androgen (AR) receptors are pathologically altered in colorectal cancer (CRC), their simultaneous expression within the same cohort of patients was not previously measured. Methods: ERα/ERß/PGR/AR proteins were measured in archived paired normal and malignant colon specimens (n =120 patients) by immunohistochemistry, and results were analyzed by gender, age (≤50 vs. ≥60 years), clinical stages (early-stage I/II vs. late-stage III/IV), and anatomical location (right; RSCs vs. left; LSCs). Effects of 17ß-estradiol (E2), progesterone (P4), and testosterone alone or combined with the specific blockers of ERα (MPP dihydrochloride), ERß (PHTPP), PGR (mifepristone), and AR (bicalutamide) on cell cycle and apoptosis were also measured in the SW480 male and HT29 female CRC cell lines. Results: ERα and AR proteins increased, whilst ERß and PGR declined markedly in malignant specimens. Moreover, male neoplastic tissues showed highest AR expression, whilst ERß and PGR weakest alongside ERα strongest expression was seen in cancerous tissues from women aged ≥60 years. Late-stage neoplasms also revealed maximal alterations in the expression of sex steroid receptors. By tumor location, LSCs disclosed significant elevations in ERα with marked declines in PGR compared with RSCs, and ERα strongest alongside PGR weakest expression was detected in advanced LSCs from women aged ≥60 years. Late-stage LSCs from females aged ≥60 years also showed weakest ERß and strongest AR expression. In contrast, male RSC and LSC tissues exhibited equal ERß and AR expression in all clinical stages. ERα and AR proteins also correlated positively, whereas ERß and PGR inversely, with tumor characteristics. Concomitantly, E2 and P4 monotherapies triggered cell cycle arrest and apoptosis in the SW480 and HT29 cells, and while pre-treatment with ERα-blocker enhanced the effects of E2, ERß-blocker and PGR-blocker suppressed the E2 and P4 anti-cancer actions, respectively. In contrast, treatment with the AR-blocker induced apoptosis, whilst co-treatment with testosterone hindered the effects. Conclusions: This study advocates that protein expression of sex steroid receptors in malignant tissues could represent prognostic markers, as well as hormonal therapy could provide an alternative strategy against CRC, and their efficacies could be dependent on gender, clinical stage, and tumor location.

Deadenylation of cytoplasmic mRNA by the mammalian Ccr4-Not complex.

The Ccr4-Not complex is one of the major deadenylase factors present in eukaryotic cells. This multi-subunit protein complex is composed of at least seven stably associated subunits in mammalian cells including two enzymatic deadenylase subunits: one DEDD (Asp-Glu-Asp-Asp)-type deadenylase (either CNOT7/human Caf1/Caf1a or CNOT8/human Pop2/Caf1b/Calif) and one EEP (endonuclease-exonuclease-phosphatase)-type enzyme (either CNOT6/human Ccr4/Ccr4a or CNOT6L/human Ccr4-like/Ccr4b). Here, the role of the human Ccr4-Not complex in cytoplasmic deadenylation of mRNA is discussed, including the mechanism of its recruitment to mRNA and the role of the BTG/Tob proteins.

Enhanced anti-cancer effects of oestrogen and progesterone co-therapy against colorectal cancer in males.

Although ovarian sex steroids could have protective roles against colorectal cancer (CRC) in women, little is currently known about their potential anti-tumorigenic effects in men. Hence, this study measured the therapeutic effects of 17ß-oestradiol (E2) and/or progesterone (P4) against azoxymethane-induced CRC in male mice that were divided into (n = 10 mice/group): negative (NC) and positive (PC) controls, E2 (580 µg/Kg/day; five times/week) and P4 (2.9 mg/Kg/day; five times/week) monotherapies, and concurrent (EP) and sequential (E/P) co-therapy groups. Both hormones were injected intraperitoneally to the designated groups for four consecutive weeks. Similar treatment protocols with E2 (10 nM) and/or P4 (20 nM) were also used in the SW480 and SW620 human male CRC cell lines. The PC group showed abundant colonic tumours alongside increased colonic tissue testosterone levels and androgen (AR) and oestrogen (ERα) receptors, whereas E2 and P4 levels with ERß and progesterone receptor (PGR) decreased significantly compared with the NC group. E2 and P4 monotherapies equally increased ERß/PGR with p21/Cytochrome-C/Caspase-3, reduced testosterone levels, inhibited ERα/AR and CCND1/survivin and promoted apoptosis relative to the PC group. Both co-therapy protocols also revealed better anti-cancer effects with enhanced modulation of colonic sex steroid hormones and their receptors, with E/P the most prominent protocol. In vitro, E/P regimen showed the highest increases in the numbers of SW480 (2.1-fold) and SW620 (3.5-fold) cells in Sub-G1 phase of cell cycle. The E/P co-therapy also disclosed the lowest percentages of viable SW480 cells (2.8-fold), whilst both co-therapy protocols equally showed the greatest SW620 apoptotic cell numbers (5.2-fold) relative to untreated cells. Moreover, both co-therapy regimens revealed maximal inhibitions of cell cycle inducers, cell survival markers, and AR/ERα alongside the highest expression of cell cycle suppressors, pro-apoptotic molecules, and ERß/PGR in both cell lines. In conclusion, CRC was associated with abnormal levels of colonic sex steroid hormones alongside aberrant protein expression of their receptors. While the anti-cancer effects of E2 and P4 monotherapies were equal, their combination protocols showed boosted tumoricidal actions against CRC in males, possibly by promoting ERß and PGR-mediated androgen deprivation together with inhibition of ERα-regulated oncogenic pathways.

In Vivo and In Vitro Enhanced Tumoricidal Effects of Metformin, Active Vitamin D3, and 5-Fluorouracil Triple Therapy against Colon Cancer by Modulating the PI3K/Akt/PTEN/mTOR Network.

Chemoresistance to 5-fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, active vitamin D3 (VD3), and/or metformin single/dual/triple regimens as complementary/alternative therapies. Ninety male mice were divided into: negative and positive (PC) controls, and 5-FU, VD3, Met, 5-FU/VD3, 5-FU/Met, VD3/Met, and 5-FU/VD3/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. Similar regimens were also applied in the SW480 and SW620 CRC cell lines. The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR, and reduced p21/PTEN/cytochrome C/caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/VD3/Met being the most efficacious regimen. All protocols decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, and increased proapoptotic molecules with an apoptosis index, and 5-FU/VD3/Met revealed the strongest effects. In vitro, all therapies equally induced G1 phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in the G0/G1 phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/VD3/Met disclosed the lowest viable SW620 cell percentages (81%). Moreover, 5-FU/VD3/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2), and the PI3K/Akt/mTOR molecules alongside the highest expression of cell cycle inhibitors (p21/p27), proapoptotic markers (BAX/cytochrome C/caspase-3), and PTEN in both cell lines. In conclusion, metformin monotherapy was superior to VD3, whereas the 5-FU/Met protocol showed better anticancer effects relative to the other dual therapies. However, the 5-FU/VD3/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.

Enhanced in vitro tumoricidal effects of 5-Fluorouracil, thymoquinone, and active vitamin D3 triple therapy against colon cancer cells by attenuating the PI3K/AKT/mTOR pathway.

AIMS: This study measured the effects of 5-Fluorouracil (5-FU), calcitriol (VD3), and/or thymoquinone (TQ) single/dual/triple therapies on cell cycle progression, apoptosis, inhibition of the PI3K/AKT/mTOR pathway, and oxidative stress against colorectal cancer (CRC). MAIN METHODS: The HT29, SW480 and SW620 cell lines were treated with 5-FU (50 µM), VD3 (25 µM), and TQ (75 µM), alone or combined for 12 h, prior to cell cycle/apoptosis analyses. KEY FINDINGS: TQ monotherapy had greater anticancer effects to active VD3 or 5-FU, revealing higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3 and increased levels of total glutathione, with inhibitions in CCND1/CCND3/BCL-2 and PI3K/AKT/mTOR molecules, alongside higher rates of apoptosis in HT29, SW480 and SW620 cells (P < 0.005 for all markers). Additionally, all combination protocols revealed enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway, higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3, and better anti-oxidant effects, than the monotherapies. Although TQ/5-FU and TQ/VD3 co-therapies were better relative to the VD3/5-FU regimen, the best tumoricidal effects were observed with triple therapy in the HT29 and SW480 cell lines, possibly by boosted attenuations of the PI3K/AKT/mTOR oncogenic pathway. In contrast, TQ single treatment was more effective than the triple therapy regimen in metastatic SW620 cells, suggesting that this protocol would be more useful therapeutically in late-stage CRC. SIGNIFICANCE: In conclusion, this study is the first to demonstrated enhanced anti-tumorigenic effects for VD3, TQ, and 5-FU triple therapy against CRC cells and could represent the best strategy for treating early stages of malignancy, whereas TQ monotherapy could be a better approach for treating metastatic forms of the disease.

Chemopreventive effects of vitamin D3 and its analogue, paricalcitol, in combination with 5-fluorouracil against colorectal cancer: The role of calcium signalling molecules.

BACKGROUND: Although vitamin D (VD) is chemoprotective and enhances 5-fluorouracil (5-FU) cytotoxicity against colorectal cancer (CRC), little is known about its potential calcium (Ca2+)-mediated anti-tumorigenic actions. Therefore, this study compared between VD and its non-calcaemic analogue, Paricalcitol (Pcal), ± 5-FU in relation to chemoprevention and Ca2+-mediated apoptosis in vivo and in vitro. METHODS: Seventy male mice were distributed to: negative controls, positive controls (PC), VD, Pcal, 5-FU, VD + 5-FU and Pcal+5-FU groups. All groups, except negative, received two consecutive azoxymethane (AOM)-injections (10 mg/Kg/week) for CRC induction. VD3 (1000 IU/kg; three times/week) and Pcal (1.25 µg/kg; three times/week) injections started week-16 post-AOM and for 10 weeks. Three successive 5-FU cycles began at week-21 (50 mg/Kg/week). Similar protocols with VD3, Pcal and/or 5-FU were applied in the HT29 colon cancer cells. RESULTS: The PC group had abundant malignant tumours, markedly elevated proliferation markers (survivin/CCND1) and declines in cyclin-dependent kinase-inhibitor-1A, pro-apoptotic molecules (p53/BAX/cytochrome_C/caspase-3), tissue Ca2+ concentrations and Ca2+-dependent proteins (CaSR/CAM/CAMKIIA). All monotherapies equally reduced tumour numbers and proliferation markers whilst promoting the anti-tumorigenic molecules. VD and/or 5-FU, but not Pcal monotherapy, enhanced Ca2+ levels and Ca2+-related molecules (CaSR/CAM/CAMKIIA/BAX/cytochrome_C) in vivo and in vitro. However, VD + 5-FU co-therapy showed the lowest tumour numbers, the highest cell numbers in sub-G1 phase of cell cycle, alongside the most effective modulations of oncogenes, tumour suppressors and Ca2+-related molecules at the gene and protein levels in vivo and in vitro. CONCLUSIONS: VD3 was superior than Paricalcitol in potentiating 5-FU cytotoxicity, possibly by upregulating several Ca2+-related molecules involved in tumour suppression.

Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status.

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (n = 90 patients) alongside fresh (n = 40 patients) specimen cohorts. Activin ß-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours (n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The ßA-subunit and activin-A increased, whilst ßB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro, activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein.

Effects of ß-Blockers on the Sympathetic and Cytokines Storms in Covid-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative virus in the development of coronavirus disease 2019 (Covid-19) pandemic. Respiratory manifestations of SARS-CoV-2 infection such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) leads to hypoxia, oxidative stress, and sympatho-activation and in severe cases leads to sympathetic storm (SS). On the other hand, an exaggerated immune response to the SARS-CoV-2 invasion may lead to uncontrolled release of pro-inflammatory cytokine development of cytokine storm (CS). In Covid-19, there are interactive interactions between CS and SS in the development of multi-organ failure (MOF). Interestingly, cutting the bridge between CS and SS by anti-inflammatory and anti-adrenergic agents may mitigate complications that are induced by SARS-CoV-2 infection in severely affected Covid-19 patients. The potential mechanisms of SS in Covid-19 are through different pathways such as hypoxia, which activate the central sympathetic center through carotid bodies chemosensory input and induced pro-inflammatory cytokines, which cross the blood-brain barrier and activation of the sympathetic center. ß2-receptors signaling pathway play a crucial role in the production of pro-inflammatory cytokines, macrophage activation, and B-cells for the production of antibodies with inflammation exacerbation. ß-blockers have anti-inflammatory effects through reduction release of pro-inflammatory cytokines with inhibition of NF-κB. In conclusion, ß-blockers interrupt this interaction through inhibition of several mediators of CS and SS with prevention development of neural-cytokine loop in SARS-CoV-2 infection. Evidence from this study triggers an idea for future prospective studies to confirm the potential role of ß-blockers in the management of Covid-19.