Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation.

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Bursatella leachii Purple Ink Secretion Concentrate Exerts Cytotoxic Properties against Human Hepatocarcinoma Cell Line (HepG2): In Vitro and In Silico Studies.

Liver cancer is a leading cause of cancer death globally. Marine mollusc-derived drugs have gained attention as potential natural-based anti-cancer agents to overcome the side effects caused by conventional chemotherapeutic drugs during cancer therapy. Using liquid chromatography-mass spectrometry, the main biomolecules in the purple ink secretion released by the sea hare, named Bursatella leachii (B. leachii), were identified as hectochlorin, malyngamide X, malyngolide S, bursatellin and lyngbyatoxin A. The cytotoxic effects of B. leachii ink concentrate against human hepatocarcinoma (HepG2) cells were determined to be dose- and time-dependent, and further exploration of the underlying mechanisms causing the programmed cell death (apoptosis) were performed. The expression of cleaved-caspase-8 and cleaved-caspase-3, key cysteine-aspartic proteases involved in the initiation and completion of the apoptosis process, appeared after HepG2 cell exposure to the B. leachii ink concentrate. The gene expression levels of pro-apoptotic BAX, TP53 and Cyclin D1 were increased after treatment with the B. leachii ink concentrate. Applying in silico approaches, the high scores predicted that bioactivities for the five compounds were protease and kinase inhibitors. The ADME and cytochrome profiles for the compounds were also predicted. Altogether, the B. leachii ink concentrate has high pro-apoptotic potentials, suggesting it as a promising safe natural product-based drug for the treatment of liver cancer.

Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells.

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.

Herbal melanin inhibits colorectal cancer cell proliferation by altering redox balance, inducing apoptosis, and modulating MAPK signaling.

BACKGROUND: Colorectal carcinoma is one of the most deadly cancers that requests effective and safe chemotherapy. Evaluation of natural product-based anticancer drugs as adjuvant treatment with fewer side effects is largely unexplored research fields. Herbal melanin (HM) is an extract of the seed coats of Nigella sativa that modulates an inflammatory response through toll-like receptor 4 (TLR4). This TLR4 receptor is also involved in the modulation of apoptosis. We therefore explored the anticancer potential of HM and specifically its effect on the molecular mechanisms underlying adenocarcinoma and metastatic colorectal cancer (mCRC) cell death in vitro. METHODS: Cell viability was evaluated using the MTT assay. Cellular reactive oxygen species (ROS), glutathione levels, and apoptotic status were assessed using fluorometric and colorimetric detection methods. HM-induced apoptotic and other signaling pathways were investigated using Western blot technology and mitochondrial transition pore assay kit. TLR4 receptor downregulation and blockade were performed using siRNA technology and neutralizing antibody, respectively. RESULTS: Our results showed that HM inhibited the proliferation of the colorectal adenocarcinoma HT29 and mCRC SW620 cell lines. Furthermore, HM enhanced ROS production and decreased glutathione levels. HM-induced apoptosis was associated with mitochondrial outer membrane permeability and cytochrome c release, inhibition of the Bcl2 family proteins, and activation of caspase-3/-7. In addition, HM modulated MAPK pathways by activating the JNK pathway and by inhibiting ERK phosphorylation. TLR4 receptor downregulation enhanced HM-induced apoptosis while TLR4 receptor blockade partially alleviated HM-inhibited ERK phosphorylation. CONCLUSION: Altogether, these findings indicate that HM exerts pro-apoptotic effects and inhibits MAPK pathway through TLR4 in mCRC and colorectal adenocarcinoma cells, suggesting HM as a promising natural-based drug for the treatment of colorectal cancer.

Anastatica hierochuntica (L.) methanolic and aqueous extracts exert antiproliferative effects through the induction of apoptosis in MCF-7 breast cancer cells.

Breast cancer therapy using anticancer bioactive compounds derived from natural products as adjuvant treatment has gained recognition due to expensive and toxic conventional chemotherapeutic drugs. The whole plant of Anastatica hierochuntica (L.) (A. hierochuntica) has been investigated for its pharmacologically important anticancer properties but without categorizing the biological activities of the plant parts. We assessed the anticancer potential of different parts of A. hierochuntica (seeds, stems and leaves) and explored their mechanisms of action using the human breast cancer cell line, MCF-7. Currently, we investigated the antiproliferative effects of methanolic (MSD, MST, ML) and aqueous (ASD, AST, AL) extracts of A. hierochuntica plant parts on the MCF-7 cells using cell viability assays. Flow cytometry, Western Blot, DNA fragmentation, and gene expression assays were performed to evaluate apoptosis and cell cycle regulatory proteins. The results indicate that the methanolic and aqueous extracts decreased MCF-7 cell viability in a dose-dependent manner. The induction of apoptosis was observed in all the methanolic and aqueous-treated MCF-7 cells. The cell death process was confirmed by the visualization of DNA fragmentation and cleavage of the intrinsic apoptotic pathways, caspase-9 and caspase-3, the key enzyme causing apoptosis hallmarks. In addition, the most pro-apoptotic extracts, ASD and ML, up-regulated the expression of pro-apoptotic Bax, tumor suppressor TP53 genes and the cyclin inhibitor CDKN1A gene. In conclusion, of the aqueous and methanolic extracts of A. hierochuntica plant parts exerting antiproliferative effects through the induction of apoptosis in breast cancer MCF-7 cells, ASD and ML extracts were the most promising natural-based drugs for the treatment of breast cancer.

Risk of Neuropsychiatric Adverse Effects of Lipid-Lowering Drugs: A Mendelian Randomization Study.

Background: Recent studies have highlighted the possible risk of neuropsychiatric adverse effects during treatment with lipid-lowering medications. However, there are still controversies that require a novel genetic-based approach to verify whether the impact of lipid-lowering drug treatment results in neuropsychiatric troubles including insomnia, depression, and neuroticism. Thus, we applied Mendelian randomization to assess any potential neuropsychiatric adverse effects of conventional lipid-lowering drugs such as statins, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, and ezetimibe. Methods: A 2-sample Mendelian randomization study was conducted based on summary statistics from genome-wide association studies for lipids, insomnia, depression, and neuroticism. Single-nucleotide polymorphisms located in or near drug target genes of HMGCR, PCSK9, and NPC1L1 were used as proxies for statins, PCSK9 inhibitors, and ezetimibe therapy, respectively. To assess the validity of the genetic risk score, their associations with coronary artery disease were used as a positive control. Results: The Mendelian randomization analysis showed a statistically significant (P <.004) increased risk of depression after correcting for multiple testing with both statins (odds ratio=1.15, 95% CI: 1.04-1.19) and PCSK9 inhibitor treatment (odds ratio =1.19, 95%CI: 1.1-1.29). The risk of neuroticism was slightly reduced with statin therapy (odds ratio=0.9, 95%CI: 0.83-0.97). No significant adverse effects were associated with ezetimibe treatment. As expected, the 3 medications significantly reduced the risk of coronary artery disease. Conclusion: Using a genetic-based approach, this study showed an increased risk of depression during statin and PCSK9 inhibitor therapy while their association with insomnia risk was not significant.

Biological impact of advanced glycation endproducts on estrogen receptor-positive MCF-7 breast cancer cells.

Diabetes mellitus potentiates the risk of breast cancer. We have previously described the pro-tumorigenic effects of advanced glycation endproducts (AGEs) on estrogen receptor (ER)-negative MDA-MB-231 breast cancer cell line mediated through the receptor for AGEs (RAGE). However, a predominant association between women with ER-positive breast cancer and type 2 diabetes mellitus has been reported. Therefore, we have investigated the biological impact of AGEs on ER-positive human breast cancer cell line MCF-7 using in vitro cell-based assays including cell count, migration, and invasion assays. Western blot, FACS analyses and quantitative real time-PCR were also performed. We found that AGEs at 50-100µg/mL increased MCF-7 cell proliferation and cell migration associated with an enhancement of pro-matrix metalloproteinase (MMP)-9 activity, without affecting their poor invasiveness. However, 200µg/mL AGEs inhibited MCF-7 cell proliferation through induction of apoptosis indicated by caspase-3 cleavage detected using Western blotting. A phospho-protein array analysis revealed that AGEs mainly induce the phosphorylation of extracellular-signal regulated kinase (ERK)1/2 and cAMP response element binding protein-1 (CREB1), both signaling molecules considered as key regulators of AGEs pro-tumorigenic effects. We also showed that AGEs up-regulate RAGE and ER expression at the protein and transcript levels in MCF-7 cells, in a RAGE-dependent manner after blockade of AGEs/RAGE interaction using neutralizing anti-RAGE antibody. Throughout the study, BSA had no effect on cellular processes. These findings pave the way for future studies investigating whether the exposure of AGEs-treated ER-positive breast cancer cells to estrogen could lead to a potentiation of breast cancer development and progression.

CD95-mediated apoptosis in Burkitt's lymphoma B-cells is associated with Pim-1 down-regulation.

B-cells of the high-grade non-Hodgkin lymphoma Burkitt's lymphoma (BL) overexpress survival oncoproteins, including the proviral integration site for Moloney murine leukaemia virus kinase (Pim)-1, and become apoptosis resistant. Activated death receptor CD95 after ligation with anti-CD95 monoclonal antibody (mAb) resulted in the regression of BL via induction of apoptosis, suggesting a decrease of survival protein expression. Here, CD95-mediated apoptotic pathways in BL B-cell lines (Raji and Daudi) following treatment with anti-CD95 mAb was investigated with the cause-and-effects on pim-1 gene expression, in comparison with leukemic cell line (K562) used as CD95-negative cells. Immunohistochemical staining for CD95 and Pim-1 was performed, and the effects of anti-CD95 mAb on apoptotic signalling using western blotting, on caspase activity and cell survival of BL B-cell and leukemic cell lines were determined. We showed that Raji cells expressed more CD95 receptors than Daudi cells. Half of each population underwent apoptosis accompanied by decreased cell viability after anti-CD95 mAb treatment. Distinct extrinsic and intrinsic CD95-mediated apoptotic pathways in Raji and Daudi cells were revealed by high caspase activity and mitochondrial outer membrane permeabilization, respectively. We observed decreased Pim-1 transcript and protein expression levels with increased heat-shock protein (Hsp)70 and decreased Hsp90 expression in anti-CD95 mAb-treated cells. Throughout the study, K562 cells did not undergo apoptosis upon anti-CD95 mAb treatment. Pim-1 knockdown following to stable transfection with plasmid vectors induced apoptosis and decreased viability of BL and K562 cells. Therefore, CD95-mediated apoptosis induces Pim-1 down-regulation in BL B-cells, but Pim-1 down-regulation cannot fully eradicate BL and leukaemia.

Advanced glycation endproducts increase proliferation, migration and invasion of the breast cancer cell line MDA-MB-231.

Diabetic patients have increased likelihood of developing breast cancer. Advanced glycation endproducts (AGEs) underlie the pathogenesis of diabetic complications but their impact on breast cancer cells is not understood. This study aims to determine the effects of methylglyoxal-derived bovine serum albumin AGEs (MG-BSA-AGEs) on the invasive MDA-MB-231 breast cancer cell line. By performing cell counting, using wound-healing assay, invasion assay and zymography analysis, we found that MG-BSA-AGEs increased MDA-MB-231 cell proliferation, migration and invasion through Matrigel™ associated with an enhancement of matrix metalloproteinase (MMP)-9 activities, in a dose-dependent manner. Using Western blot and flow cytometry analyses, we demonstrated that MG-BSA-AGEs increased expression of the receptor for AGEs (RAGE) and phosphorylation of key signaling protein extracellular signal-regulated kinase (ERK)-1/2. Furthermore, in MG-BSA-AGE-treated cells, phospho-protein micro-array analysis revealed enhancement of phosphorylation of the ribosomal protein 70 serine S6 kinase beta 1 (p70S6K1), which is known to be involved in protein synthesis, the signal transducer and activator of transcription (STAT)-3 and the mitogen-activated protein kinase (MAPK) p38, which are involved in cell survival. Blockade of MG-BSA-AGE/RAGE interactions using a neutralizing anti-RAGE antibody inhibited MG-BSA-AGE-induced MDA-MB-231 cell processes, including the activation of signaling pathways. Throughout the study, non-modified BSA had a negligible effect. In conclusion, AGEs might contribute to breast cancer development and progression partially through the regulation of MMP-9 activity and RAGE signal activation. The up-regulation of RAGE and the concomitant increased phosphorylation of p70S6K1 induced by AGEs may represent promising targets for drug therapy to treat diabetic patients with breast cancer.

Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway.

C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in Matrigel(TM) with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.

Variations in DEPDC5 gene and its association with chronic hepatitis C virus infection in Saudi Arabia.

BACKGROUND: Variations at DEPDC5 gene have been recently reported as genetic markers associated with hepatocellular carcinoma (HCC) progression in chronic HCV-infected patients. This study was conducted to assess the association of DEPDC5 variants with advanced liver cirrhosis and HCC development among chronic HCV-infected patients in Saudi Arabian population. METHODS: Six-hundred and one HCV-infected patients were genotyped for DEPDC5 polymorphisms (rs1012068 and rs5998152), in comparison with 592 non-infected healthy control subjects. The allelic frequency and genotype distribution of both DEPDC5 polymorphisms were determined followed by haplotype frequency estimation and multiple logistic regression analysis. RESULTS: The frequency of the risk alleles of both rs1012068 and rs5998152 was shown to be more in healthy control subjects than in patients (p = 0.0001, OR = 0.704, CI = 0.591-0.839; p = 0.002, OR = 0.761, CI = 0. 0.639-0.907, respectively). Also, our results revealed that GT for SNP rs1012068 (OR =1.715; 95% CI 1.132-2.597; p = 0.0104) and CT for SNP rs5998152 (OR = 1.932; 95% CI 1.276-2.925; p = 0.0017) showed significant association with development of cirrhosis compared with the GG and CC genotypes, respectively. The data also revealed that subjects with the T allele of both SNPs appeared to have a lower susceptibility to HCV-related cirrhosis/HCC than those with the G allele of rs1012068 (p = 0.038, OR = 1.353, 95 % CI 1.017-1.800) and C allele of rs5998152 (p = 0.043, OR = 1.342, 95 % CI 1.010-1.784). Haplotype analysis showed that a combination of T-T alleles of rs1012068 and rs5998152 was significantly associated with liver cirrhosis (frequency = 71.3% and p = 0.027) and with cirrhosis/HCC (frequency = 71.4% and P = 0.045). Also, multiple logistic regression analysis showed that rs5998152 (OR = 2.844, 95% CI 1.333-6.069 and p = 0.007), rs1012068 (OR = 2.793, 95% CI 1.316-5.928 and p = 0.010), age (OR = 1.029, 95% CI 1.001-1.057 and p = 0.041) and HCV genotypes (OR = 0.247, 95% CI 0.097-0.630 and p = 0.003) were independently associated with chronicity of HCV infection. CONCLUSION: Genetic variations in DEPDC5 gene region may influence HCV-associated liver cirrhosis and/or HCC development.

Identification of Anastatica hierochuntica L. Methanolic-Leaf-Extract-Derived Metabolites Exhibiting Xanthine Oxidase Inhibitory Activities: In Vitro and In Silico Approaches.

There is a growing interest in the discovery of novel xanthine oxidase inhibitors for gout prevention and treatment with fewer side effects. This study aimed to identify the xanthine oxidase (XO) inhibitory potential and drug-likeness of the metabolites present in the methanolic leaf extract of Anastatica (A.) hierochuntica L. using in vitro and in silico models. The extract-derived metabolites were identified by liquid-chromatography-quadrupole-time-of-flight-mass-spectrometry (LC-QTOF-MS). Molecular docking predicted the XO inhibitory activity of the identified metabolites and validated the best scored in vitro XO inhibitory activities for experimental verification, as well as predictions of their anticancer, pharmacokinetic, and toxic properties; oral bioavailability; and endocrine disruption using SwissADMET, PASS, ProTox-II, and Endocrine Disruptome web servers. A total of 12 metabolites, with a majority of flavonoids, were identified. Rutin, quercetin, and luteolin flavonoids demonstrated the highest ranked docking scores of -12.39, -11.15, and -10.43, respectively, while the half-maximal inhibitory concentration (IC50) values of these metabolites against XO activity were 11.35 µM, 11.1 µM, and 21.58 µM, respectively. In addition, SwissADMET generated data related to the physicochemical properties and drug-likeness of the metabolites. Similarly, the PASS, ProTox-II, and Endocrine Disruptome prediction models stated the safe and potential use of these natural compounds. However, in vivo studies are necessary to support the development of the prominent and promising therapeutic use of A. hierochuntica methanolic-leaf-extract-derived metabolites as XO inhibitors for the prevention and treatment of hyperuricemic and gout patients. Furthermore, the predicted findings of the present study open a new paradigm for these extract-derived metabolites by revealing novel oncogenic targets for the potential treatment of human malignancies.

Bioportide: an emergent concept of bioactive cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

Updates on Triple-Negative Breast Cancer in Type 2 Diabetes Mellitus Patients: From Risk Factors to Diagnosis, Biomarkers and Therapy.

Triple-negative breast cancer (TNBC) is usually the most malignant and aggressive mammary epithelial tumor characterized by the lack of expression for estrogen receptors and progesterone receptors, and the absence of epidermal growth factor receptor (HER)2 amplification. Corresponding to 15-20% of all breast cancers and well-known by its poor clinical outcome, this negative receptor expression deprives TNBC from targeted therapy and makes its management therapeutically challenging. Type 2 diabetes mellitus (T2DM) is the most common ageing metabolic disorder due to insulin deficiency or resistance resulting in hyperglycemia, hyperinsulinemia, and hyperlipidemia. Due to metabolic and hormonal imbalances, there are many interplays between both chronic disorders leading to increased risk of breast cancer, especially TNBC, diagnosed in T2DM patients. The purpose of this review is to provide up-to-date information related to epidemiology and clinicopathological features, risk factors, diagnosis, biomarkers, and current therapy/clinical trials for TNBC patients with T2DM compared to non-diabetic counterparts. Thus, in-depth investigation of the diabetic complications on TNBC onset, development, and progression and the discovery of biomarkers would improve TNBC management through early diagnosis, tailoring therapy for a better outcome of T2DM patients diagnosed with TNBC.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS.

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.

Stimulatory effects of Lycium shawii on human melanocyte proliferation, migration, and melanogenesis: In vitro and in silico studies.

There is no first-line treatment for vitiligo, a skin disease characterized by a lack of melanin produced by the melanocytes, resulting in an urgent demand for new therapeutic drugs capable of stimulating melanocyte functions, including melanogenesis. In this study, traditional medicinal plant extracts were tested for cultured human melanocyte proliferation, migration, and melanogenesis using MTT, scratch wound-healing assays, transmission electron microscopy, immunofluorescence staining, and Western blot technology. Of the methanolic extracts, Lycium shawii L. (L. shawii) extract increased melanocyte proliferation at low concentrations and modulated melanocyte migration. At the lowest tested concentration (i.e., 7.8 µg/mL), the L. shawii methanolic extract promoted melanosome formation, maturation, and enhanced melanin production, which was associated with the upregulation of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 melanogenesis-related proteins, and melanogenesis-related proteins. After the chemical analysis and L. shawii extract-derived metabolite identification, the in silico studies revealed the molecular interactions between Metabolite 5, identified as apigenin (4,5,6-trihydroxyflavone), and the copper active site of tyrosinase, predicting enhanced tyrosinase activity and subsequent melanin formation. In conclusion, L. shawii methanolic extract stimulates melanocyte functions, including melanin production, and its derivative Metabolite 5 enhances tyrosinase activity, suggesting further investigation of the L. shawii extract-derived Metabolite 5 as a potential natural drug for vitiligo treatment.

SARS-CoV-2 modulates inflammatory responses of alveolar epithelial type II cells via PI3K/AKT pathway.

Background: SARS-CoV-2 infects through the respiratory route and triggers inflammatory response by affecting multiple cell types including type II alveolar epithelial cells. SARS-CoV-2 triggers signals via its Spike (S) protein, which have been shown to participate in the pathogenesis of COVID19. Aim: Aim of the present study was to investigate the effect of SARS-CoV2 on type II alveolar epithelial cells, focusing on signals initiated by its S protein and their impact on the expression of inflammatory mediators. Results: For this purpose A549 alveolar type II epithelial cells were exposed to SARS CoV2 S recombinant protein and the expression of inflammatory mediators was measured. The results showed that SARS-CoV-2 S protein decreased the expression and secretion of IL8, IL6 and TNFα, 6 hours following stimulation, while it had no effect on IFNα, CXCL5 and PAI-1 expression. We further examined whether SARS-CoV-2 S protein, when combined with TLR2 signals, which are also triggered by SARS-CoV2 and its envelope protein, exerts a different effect in type II alveolar epithelial cells. Simultaneous treatment of A549 cells with SARS-CoV-2 S protein and the TLR2 ligand PAM3csk4 decreased secretion of IL8, IL6 and TNFα, while it significantly increased IFNα, CXCL5 and PAI-1 mRNA expression. To investigate the molecular pathway through which SARS-CoV-2 S protein exerted this immunomodulatory action in alveolar epithelial cells, we measured the induction of MAPK/ERK and PI3K/AKT pathways and found that SARS-CoV-2 S protein induced the activation of the serine threonine kinase AKT. Treatment with the Akt inhibitor MK-2206, abolished the inhibitory effect of SARS-CoV-2 S protein on IL8, IL6 and TNFα expression, suggesting that SARS-CoV-2 S protein mediated its action via AKT kinases. Conclusion: The findings of our study, showed that SARS-CoV-2 S protein suppressed inflammatory responses in alveolar epithelial type II cells at early stages of infection through activation of the PI3K/AKT pathway. Thus, our results suggest that at early stages SARS-CoV-2 S protein signals inhibit immune responses to the virus allowing it to propagate the infection while in combination with TLR2 signals enhances PAI-1 expression, potentially affecting the local coagulation cascade.

Rhizophora mucronata Lam. (Mangrove) Bark Extract Reduces Ethanol-Induced Liver Cell Death and Oxidative Stress in Swiss Albino Mice: In Vivo and In Silico Studies.

The bark extract of Rhizophora mucronata (BERM) was recently reported for its prominent in vitro protective effects against liver cell line toxicity caused by various toxicants, including ethanol. Here, we aimed to verify the in vivo hepatoprotective effects of BERM against ethanol intoxication with the prediction of potential targets employing in silico studies. An oral administration of different concentrations (100, 200 and 400 mg/kg body weight) of BERM before high-dose ethanol via intraperitoneal injection was performed in mice. On day 7, liver sections were dissected for histopathological examination. The ethanol intoxication caused liver injury and large areas of necrosis. The pre-BERM administration decreased the ethanol-induced liver damage marker tumor necrosis factor-alpha (TNF-α) expression, reduced hepatotoxicity revealed by nuclear deoxyribonucleic acid (DNA) fragmentation and decreased oxidative stress indicated by malondialdehyde and glutathione contents. Our in silico studies have identified BERM-derived metabolites exhibiting the highest predicted antioxidant and free radical scavenger activities. Molecular docking studies showed that most of the metabolites were predicted to be enzyme inhibitors such as carbonic anhydrase inhibitors, which were reported to stimulate the antioxidant defense system. The metabolites predominantly presented acceptable pharmacokinetics and safety profiles, suggesting them as promising new antioxidant agents. Altogether, the BERM extract exerts antioxidative activities and shows promising hepatoprotective effects against ethanol intoxication. Identification of related bioactive compounds will be of interest for future use at physiological concentrations in ethanol-intoxicated individuals.

Achillea fragrantissima (Forssk.) Sch.Bip Flower Dichloromethane Extract Exerts Anti-Proliferative and Pro-Apoptotic Properties in Human Triple-Negative Breast Cancer (MDA-MB-231) Cells: In Vitro and In Silico Studies.

The aggressive triple-negative breast cancer (TNBC) is a challenging disease due to the absence of tailored therapy. The search for new therapies involves intensive research focusing on natural sources. Achillea fragrantissima (A. fragrantissima) is a traditional medicine from the Middle East region. Various solvent extracts from different A. fragrantissima plant parts, including flowers, leaves, and roots, were tested on TNBC MDA-MB-231 cells. Using liquid chromatography, the fingerprinting revealed rich and diverse compositions for A. fragrantissima plant parts using polar to non-polar solvent extracts indicating possible differences in bioactivities. Using the CellTiter-Glo™ viability assay, the half-maximal inhibitory concentration (IC50) values were determined for each extract and ranged from 32.4 to 161.7 µg/mL. The A. fragrantissima flower dichloromethane extract had the lowest mean IC50 value and was chosen for further investigation. Upon treatment with increasing A. fragrantissima flower dichloromethane extract concentrations, the MDA-MB-231 cells displayed, in a dose-dependent manner, enhanced morphological and biochemical hallmarks of apoptosis, including cell shrinkage, phosphatidylserine exposure, caspase activity, and mitochondrial outer membrane permeabilization, assessed using phase-contrast microscopy, fluorescence-activated single-cell sorting analysis, Image-iT™ live caspase, and mitochondrial transition pore opening activity, respectively. Anticancer target prediction and molecular docking studies revealed the inhibitory activity of a few A. fragrantissima flower dichloromethane extract-derived metabolites against carbonic anhydrase IX, an enzyme reported for its anti-apoptotic properties. In conclusion, these findings suggest promising therapeutic values of the A. fragrantissima flower dichloromethane extract against TNBC development.