Augmented Therapeutic Potential of EC-Synthetic Retinoids in Caco-2 Cancer Cells Using an In Vitro Approach.

Colorectal cancer therapies have produced promising clinical responses, but tumor cells rapidly develop resistance to these drugs. It has been previously shown that EC19 and EC23, two EC-synthetic retinoids, have single-agent preclinical anticancer activity in colorectal carcinoma. Here, isobologram analysis revealed that they have synergistic cytotoxicity with retinoic acid receptor (RAR) isoform-selective agonistic retinoids such as AC261066 (RARß2-selective agonist) and CD437 (RARγ-selective agonist) in Caco-2 cells. This synergism was confirmed by calculating the combination index (lower than 1) and the dose reduction index (higher than 1). Flow cytometry of combinatorial IC50 (the concentration causing 50% cell death) confirmed the cell cycle arrest at the SubG0-G1 phase with potentiated apoptotic and necrotic effects. The reported synergistic anticancer activity can be attributed to their ability to reduce the expression of ATP-binding cassette (ABC) transporters including P-glycoprotein (P-gp1), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein-1 (MRP1) and Heat Shock Protein 70 (Hsp70). This adds up to the apoptosis-promoting activity of EC19 and EC23, as shown by the increased Caspase-3/7 activities and DNA fragmentation leading to DNA double-strand breaks. This study sheds the light on the possible use of EC-synthetic retinoids in the rescue of multi-drug resistance in colorectal cancer using Caco-2 as a model and suggests new promising combinations between different synthetic retinoids. The current in vitro results pave the way for future studies on these compounds as possible cures for colorectal carcinoma.

High dose methotrexate in adult Egyptian patients with hematological malignancies: impact of ABCB1 3435C > T rs1045642 and MTHFR 677C > T rs1801133 polymorphisms on toxicities and delayed elimination.

High dose methotrexate (HDMTX) is an essential agent in chemotherapeutic regimens used in various hematological malignancies in Egyptian adults. The research for the impact of gene polymorphism on HDMTX induced toxicities and delayed elimination is an important ongoing objective in many studies, variable and conflicting results produced in the past years to clarify that impact. This study aimed to investigate the role of ABCB1 3435 C > T rs1045642 and MTHFR 677 C > T rs1801133 polymorphisms on HDMTX induced toxicity outcomes and delayed elimination in Egyptian adult patients with hematological malignancies. A prospective, observational cohort study was conducted on a total of 62 Egyptian adult patients with hematological malignancies age ≥ 18-years-old. All demographic, medical, and laboratory data were continuously collected from the patients' medical files in an up-to-date follow-up in selected clinics during the period from April 2018 to March 2020. Venous blood samples were collected for the purpose of genotyping, DNA extraction, and measurement of MTX levels. All the relevant data were statistically analyzed. The studied patients' median age was 25 years old with a range of (18-62) years. Forty-six patients were males with about 74%, and 16 were females with about 26%. Eighty-nine percent of the patients diagnosed with acute lymphoblastic leukemia 'ALL', 5% of the patients had B cell non-hodgkin lymphoma 'B-NHL' and 3% diagnosed with primary central nervous system lymphoma 'PCNSL' and Burkitt's lymphoma 'BL' Hematological, hepatic, renal and gastrointestinal toxicities observed post-HDMTX were recorded with the hematological toxicities toping on all the others, also patients with delayed elimination at 72 hours post the HDMTX dose were determined. Statistical analysis revealed a significant association between ABCB1 3435 C > T rs1045642 and HDMTX delayed elimination with about 10 times higher risk among the minor allele 'T' carriers (p-value = 0.006) (odds ratio [OR]: 10.470; 95% CI: 1.961-55.904). No significant association observed between the studied gene polymorphisms: MTHFR 677 C > T rs1801133, ABCB1 3435 C > T rs1045642, and different toxicity outcomes. According to our best knowledge, this study is the first to conclude a significant association between ABCB1 3435 C > T rs1045642 gene polymorphism and HDMTX delayed elimination at 72 hours post HDMTX infusion; also, it is the first study to analyze the association between ABCB1 3435 C > T rs1045642 polymorphism with HDMTX toxicity and delayed elimination in adult Egyptian patients with hematological malignancies.

Impedimetric Sensors for Cyclocreatine Phosphate Determination in Plasma Based on Electropolymerized Poly(o-phenylenediamine) Molecularly Imprinted Polymers.

Cyclocreatine and its water-soluble derivative, cyclocreatine phosphate (CCrP), are potent cardioprotective drugs. Based on recent animal studies, CCrP, FDA-awarded Orphan Drug Designation, has a promising role in increasing the success rate of patients undergoing heart transplantation surgery by preserving donor hearts during transportation and improving the recovery of transplanted hearts in recipient patients. In addition, CCrP is under investigation as a promising treatment for creatine transporter deficiency, an X-linked inborn error resulting in a poor quality of life for both the patients and the caregiver. A newly designed molecularly imprinted polymer (MIP) material was fabricated by the anodic electropolymerization of o-phenylenediamine on screen-printed carbon electrodes and was successfully applied as an impedimetric sensor for CCrP determination to dramatically reduce the analysis time during both the clinical trial phases and drug development process. To enhance the overall performance of the proposed sensor, studies were performed to optimize the electropolymerization conditions, incubation time, and pH of the background electrolyte. Scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the behavior of the developed ultrathin MIP membrane. The CCrP-imprinted polymer has a high recognition affinity for the template molecule because of the formation of 3D complementary cavities within the polymer. The developed MIP impedimetric sensor had good linearity, repeatability, reproducibility, and stability within the linear concentration range of 1 × 10-9 to 1 × 10-7 mol/L, with a low limit of detection down to 2.47 × 10-10 mol/L. To verify the applicability of the proposed sensor, it was used to quantify CCrP in spiked plasma samples.

Restoring NK cells functionality via cytokine activation enhances cetuximab-mediated NK-cell ADCC: A promising therapeutic tool for HCC patients.

Natural Killer (NK) cells are considered the first line of defense against viral infections and tumors. Several factors affect NK cytotoxic activity rendering it dysfunctional and thereby impeding the ability to scavenge abnormal cells as a part of immune escaping mechanisms induced by different types of cancers. NK cells play a crucial role augmenting the activity of various types of anticancer mAb since dysfunctional NK cells are the main reason for the low response to these therapies. To this light, we examined the phenotypic characters of the circulating NK cells isolated from HCC patients compared to healthy controls. Then, dysfunctional NK cells, from HCC patients, were reactivated with cytokines cocktail and their cytotoxic activity with the anti-EGFR mAb "cetuximab" was investigated. This showed a downregulation of patients NK cells activating receptors (NKP30, NKP46, NKG2D and CD16) as well as CD56 and up-regulation of NKG2A inhibitory receptor. We also reported an increase in aberrant CD56- NK cells subset in peripheral blood of HCC patients compared to healthy controls. Thus, confirming the dysfunctionality of peripheral NK cells isolated from HCC patients. Cytokines re-activation of those NK cells lead to upregulation of NK activating receptors and downregulation of inhibitory receptor. Moreover, the percentage of aberrant CD56- NK cells subset was reduced. Here, we proved that advanced HCC patients have an increased percentage of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for the low cytotoxicity noticed in those patients. A significant improvement in the cytotoxicity against HCC was noticed upon using reactivated NK cells combined with cetuximab. Therefore, this study highlights the potential recruitment of NK immune cells along with cetuximab to enhance cytotoxicity against HCC.

Interrupted Crosstalk between Natural Killer Cells and Anti-epidermal Growth Factor Receptor: A Possible Role in Hepatocellular Carcinoma Treatment Failure.

Hepatocellular carcinoma (HCC) is a major health problem worldwide. Most patients are diagnosed for the first time at late stages, which leads to very poor prognosis. It is challenging to discover strategies for treatment at these advanced stages. Recently, monoclonal antibodies (mAbs) targeting specific cellular signaling pathways in HCC have been developed. Unfortunately, they still have a low survival rate, and some of them failed clinically to produce effective responses even if they showed very good results against HCC in preclinical studies. This review focuses on and discusses the possible causes for the failure of mAbs, precisely anti-Epidermal Growth Factor Receptor (EGFR) mAb and the crosstalk between this mAb and patients' NK cells.

Revealing the Potential Application of EC-Synthetic Retinoid Analogues in Anticancer Therapy.

(1) Background and Aim: All-trans retinoic acid (ATRA) induces differentiation and inhibits growth of many cancer cells. However, resistance develops rapidly prompting the urgent need for new synthetic and potent derivatives. EC19 and EC23 are two synthetic retinoids with potent stem cell neuro-differentiation activity. Here, these compounds were screened for their in vitro antiproliferative and cytotoxic activity using an array of different cancer cell lines. (2) Methods: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, AV/PI (annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)), cell cycle analysis, immunocytochemistry, gene expression analysis, Western blotting, measurement of glutamate and total antioxidant concentrations were recruited. (3) Results: HepG2, Caco-2, and MCF-7 were the most sensitive cell lines; HepG2 (ATRA; 36.2, EC19; 42.2 and EC23; 0.74 µM), Caco-2 (ATRA; 58.0, EC19; 10.8 and EC23; 14.7 µM) and MCF-7 (ATRA; 99.0, EC19; 9.4 and EC23; 5.56 µM). Caco-2 cells were selected for further biochemical investigations. Isobologram analysis revealed the combined synergistic effects with 5-fluorouracil with substantial reduction in IC50. All retinoids induced apoptosis but EC19 had higher potency, with significant cell cycle arrest at subG0-G1, -S and G2/M phases, than ATRA and EC23. Moreover, EC19 reduced cellular metastasis in a transwell invasion assay due to overexpression of E-cadherin, retinoic acid-induced 2 (RAI2) and Werner (WRN) genes. (4) Conclusion: The present study suggests that EC-synthetic retinoids, particularly EC19, can be effective, alone or in combinations, for potential anticancer activity to colorectal cancer. Further in vivo studies are recommended to pave the way for clinical applications.

Gasdermin D restricts Burkholderia cenocepacia infection in vitro and in vivo.

Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1ß) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.

Chitinase 3-like-1, Tolloid-like protein 1, and intergenic gene polymorphisms are predictors for hepatocellular carcinoma development after hepatitis C virus eradication by direct-acting antivirals.

Hepatocellular carcinoma (HCC) is a major cause of cancer death in Egypt. There is still a risk for HCC development even after eradicating hepatitis C virus (HCV) by direct-acting antivirals (DAAs). Chitinase-3-like-protein-1 (CHI3L1), a biomarker for predicting many diseases, plays an essential role in inflammation, angiogenesis, and antiapoptosis. Tolloid-like protein 1 (TLL1) may be involved in hepatic fibrogenesis and carcinogenesis. This study aimed to determine the role and combined effect of CHI3L1 (rs880633), TLL1 (rs1503298), and an intergenic (rs597533) polymorphisms on the risk of developing HCC in Egyptian patients after achieving sustained virological response (SVR) by DAAs. Blood samples were collected from 68 HCC patients, 77 non-HCC subjects, and 80 healthy controls. The DNA was extracted and analyzed for rs880633, rs1503298, and rs597533 using Genotyping TaqMan™ assay. The result of the present study showed a significant difference in genotypes and alleles frequencies in both (rs880633) and (rs597533) in HCC group as compared to healthy control and also as compared to the non-HCC group. However, regarding to (rs1503298) genotypes and alleles between the HCC and non-HCC groups, there were no significant differences. Combined polymorphism in more than one gene simultaneously showed a higher risk to HCC after SVR than an individual locus. Both allelic and genotypic variations of the CHI3L1 gene (rs880633) and an intergenic (rs597533) seemed to be significant predictors confirming a great risk for HCC susceptibility in Egyptian patients achieved SVR. Patients with a polymorphism in more than one gene showed an increased risk to HCC after SVR rather than individual locus.

Targeting NUPR1 for Cancer Treatment: A Risky Endeavor.

NUPR1 is a transcription factor that has attracted great attention because of its various roles in cancer. Several studies were carried out to determine its molecular targets and mechanism of action to develop novel therapies against cancer. Here, we shed light on the role of NUPR1 in different types of cancer. NUPR1 regulates a complex network of pathways that may be affected by its silencing, which can cause varying effects. Its role in some types of cancer has been reported but remains incompletely understood, whereas its roles in other types of cancers have not been reported yet. Therefore, targeting NUPR1 for cancer treatment remains challenging and risky.

Histone H3K27M Mutation Overrides Histological Grading in Pediatric Gliomas.

Pediatric high-grade gliomas (HGG) are rare aggressive tumors that present a prognostic and therapeutic challenge. Diffuse midline glioma, H3K27M-mutant is a new entity introduced to HGG in the latest WHO classification. In this study we evaluated the presence of H3K27M mutation in 105 tumor samples histologically classified into low-grade gliomas (LGG) (n = 45), and HGG (n = 60). Samples were screened for the mutation in histone H3.3 and H3.1 variants to examine its prevalence, prognostic impact, and assess its potential clinical value in limited resource settings. H3K27M mutation was detected in 28 of 105 (26.7%) samples, and its distribution was significantly associated with midline locations (p-value < 0.0001) and HGG (p-value = 0.003). Overall and event- free survival (OS and EFS, respectively) of patients with mutant tumors did not differ significantly, neither according to histologic grade (OS p-value = 0.736, EFS p-value = 0.75) nor across anatomical sites (OS p-value = 0.068, EFS p-value = 0.153). Detection of H3K27M mutation in pediatric gliomas provides more precise risk stratification compared to traditional histopathological techniques. Hence, mutation detection should be pursued in all pediatric gliomas. Meanwhile, focusing on midline LGG can be an alternative in lower-middle-income countries to maximally optimize patients' treatment options.

Role of Diosmin in protection against the oxidative stress induced damage by gamma-radiation in Wistar albino rats.

The benchmark of this study is to evaluate the radio protective efficiency of diosmin, a natural citrus flavone of hesperidin derivative on radiation-induced damage in wistar albino rats. Rats orally administered two diosmin doses (100 and 200 mg/kg body wt.) for a month (every other day) prior to exposure to high gamma radiation single dose (8Gy) or cumulative dose (10Gy). To evaluate the radio protective efficiency of diosmin various biochemical estimations, histopathological alterations as well as comet assay and caspase-3 activity for assessment of apoptosis were performed. Results indicated that radiation-induced decline in the levels of antioxidant parameters (SOD and GSH), increased lipid peroxidation, DNA damage and apoptosis were improved by pre-administration of diosmin. Diosmin dose (200 mg/kg body wt.) restored the antioxidant status to near normal and reduced lipid peroxidation, DNA and tissue damage. These results were confirmed by histopathological examinations, which showed that pre-administration of diosmin protected the liver and kidney of albino rats against gamma-irradiation induced damage. Hence, it has been illustrated that diosmin might be an effective radio protector against radiation-induced damage in rats. Moreover, diosmin alone pretreated group did not show any biochemical alterations or DNA damage indicating the protective nature of the drug.

Inflammasome Genes' Polymorphisms in Egyptian Chronic Hepatitis C Patients: Influence on Vulnerability to Infection and Response to Treatment.

Chronic inflammation is a pivotal contributor to the liver damage mediated by hepatitis C virus (HCV). The NOD-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome is activated by HCV in both hepatocytes and Kupffer cells. The aim of our study was to investigate the association of nine single-nucleotide polymorphisms in four inflammasome genes (NLRP3, CARD8, IL-1ß, and IL-18) with the susceptibility to HCV infection and outcome of interferon treatment in 201 Egyptian chronic hepatitis C patients and 95 healthy controls. The genotyping was conducted using TaqMan predesigned SNP assay. In the comparative analysis, the CC genotype of the NLRP3 rs1539019 was found to be associated with the lower risk to chronic HCV infection (OR: 0.33, 95% CI: 0.17-0.62). This association was also found for the CA genotype and the A allele of the NLRP3 rs35829419 (OR: 0.18 and 0.22, respectively), in addition to the GG genotype and G allele of IL-18 rs1946518 (OR: 0.55 and 0.61, respectively). In contrast, the AA genotype of the IL-1ß rs1143629 was significantly more frequent in HCV patients (OR: 1.7, 95% CI: 1-2.86). Notably, the frequency of the AA genotype of NLRP3 rs1539019 was significantly higher in patients with lack of response (NR) to the interferon treatment (OR: 1.95, 95% CI: 1-3.7). A similar association was found for both the CC genotype and C allele of the NLRP3 rs35829419 (OR: 2.78 and 2.73, respectively) and for the TT genotype and T allele of CARD8 rs2043211 (OR: 2.64 and 1.54, respectively). Yet, the IL-1ß (rs1143629, rs1143634) and IL-18 (rs187238, rs1946518) polymorphisms did not show any significant association with response to interferon treatment. In conclusion, this study reports, for the first time, the association of genetic variations in NLRP3 with hepatitis C susceptibility and response to treatment in Egyptian patients. However, further large-scale studies are recommended to confirm our findings.

Evaluation of novel pyrrolopyrimidine derivatives as antiviral against gastroenteric viral infections.

Viral gastroenteritis is a major global public-health threat. All age groups are susceptible for this infection, but its most serious consequences affect children. Rotavirus, Coxsackievirus and Adenovirus are the most common viruses that cause gastroenteritis. Herein, we synthesized novel pyrrole, pyrrolo[2,3­d]pyrimidine and pyrrolo[3,2­e][1,2,4]triazolo[4,3­c]pyrimidine derivatives. The non-toxic doses of these compounds were determined using BGM cell lines. We examined all the new compounds for their anti-viral activities against Rotavirus Wa strain and Coxsackievirus B4. Compounds 2a, 2d, 5a, 5c, 5d, 7b, 7j, 7n, 14b, 14c, 14e and 14f exhibited significant antiviral activity. We interpreted the action of these compounds using molecular docking against the homology models of viral polymerase enzymes of these viruses. RMSD value of 5d/Coxsackievirus was higher than the RMSD value for 5d/rotavirus and hence better as a stability parameter, which can be correlated to the biological activity.

Discovery, Structure-Activity Relationship, and Binding Mode of an Imidazo[1,2-a]pyridine Series of Autotaxin Inhibitors.

Autotaxin (ATX) is a secreted enzyme playing a major role in the production of lysophosphatidic acid (LPA) in blood through hydrolysis of lysophosphatidyl choline (LPC). The ATX-LPA signaling axis arouses a high interest in the drug discovery industry as it has been implicated in several diseases including cancer, fibrotic diseases, and inflammation, among others. An imidazo[1,2-a]pyridine series of ATX inhibitors was identified out of a high-throughput screening (HTS). A cocrystal structure with one of these compounds and ATX revealed a novel binding mode with occupancy of the hydrophobic pocket and channel of ATX but no interaction with zinc ions of the catalytic site. Exploration of the structure-activity relationship led to compounds displaying high activity in biochemical and plasma assays, exemplified by compound 40. Compound 40 was also able to decrease the plasma LPA levels upon oral administration to rats.

Novel antiviral compounds against gastroenteric viral infections.

Viral gastroenteritis is a serious viral infection which affects a large number of individuals around the world, most of them being children. The infection may occur due to different viruses, for example, coxsackievirus, adenovirus, and rotavirus. There is no available cure for such infections, and the treatment mainly depends on hospitalization and administration of nutritional supports. A new antiviral agent against gastroenteritis viral infection will be a breakthrough in healthcare. Pyrrole and pyrrolopyrimidine derivatives are well known for their biological activity as antibacterial, antifungal, and anticancer agents. These compounds also proved to possess antiviral activity. Here, we synthesized novel pyrrole and pyrrolopyrimidine compounds and examined their antiviral activity. We synthesized several new pyrrole, pyrrolo[2,3-d]pyrimidine, and pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. The characterization of all synthesized compounds was based on microanalysis and spectral data. Moreover, we determined the non-toxic doses of these compounds on BGM, Hep-2, and MA-104 cells. We tested all the synthesized compounds for their antiviral activities against coxsackievirus B4, adenovirus type 7, and rotavirus Wa strain. Several compounds exhibited significant activities as antiviral agents.

Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug design to solve pharmacokinetic issues in a series of potent 1,2,4-oxadiazole EthR inhibitors.

Mycobacterial transcriptional repressor EthR controls the expression of EthA, the bacterial monooxygenase activating ethionamide, and is thus largely responsible for the low sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. We recently reported structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors leading to the discovery of potent ethionamide boosters. Despite high metabolic stability, pharmacokinetic evaluation revealed poor mice exposure; therefore, a second phase of optimization was required. Herein a structure-property relationship study is reported according to the replacement of the two aromatic heterocycles: 2-thienyl and 1,2,4-oxadiazolyl moieties. This work was done using a combination of structure-based drug design and in vitro/ex vivo evaluations of ethionamide boosters on the targeted protein EthR and on the human pathogen Mycobacterium tuberculosis. Thanks to this process, we identified compound 42 (BDM41906), which displays improved efficacy in addition to high exposure to mice after oral administration.

A llama-derived gelsolin single-domain antibody blocks gelsolin-G-actin interaction.

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca(2+)-activated gelsolin (K (d) approximately 10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca(2+) (K (d) approximately 5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.