Novel Exopolysaccharide from Marine Bacillus subtilis with Broad Potential Biological Activities: Insights into Antioxidant, Anti-Inflammatory, Cytotoxicity, and Anti-Alzheimer Activity

In the presented study, Bacillus subtilis strain AG4 isolated from marine was identified based on morphological, physiological, phylogenetic characteristics and an examination of 16S rRNA sequences. Novel exopolysaccharide (EPSR4) was extracted and isolated from the Bacillus subtilis strain as a major fraction of exopolysaccharide (EPS). The analysis of structural characterization indicated that EPSR4 is a β-glycosidic sulphated heteropolysaccharide (48.2%) with a molecular weight (Mw) of 1.48 × 104 g/mole and has no uronic acid. Analysis of monosaccharide content revealed that EPSR4 consists of glucose, rhamnose and arabinose monosaccharide in a molar ratio of 5:1:3, respectively. Morphological analysis revealed that EPSR4 possess a high crystallinity degree with a significant degree of porosity, and its aggregation and conformation in the lipid phase might have a significant impact on the bioactivity of EPSR4. The biological activity of EPSR4 was screened and evaluated by investigating its antioxidant, cytotoxicity, anti-inflammatory, and anti-Alzheimer activities. The antioxidant activity results showed that EPSR4 has 97.6% scavenging activity toward DPPH free radicals at 1500 µg/mL, with an IC50 value of 300 µg/mL, and 64.8% at 1500 µg/mL toward hydrogen peroxide free radicals (IC50 = 1500 µg/mL, 30 min). Furthermore, EPSR4 exhibited considerable inhibitory activity towards the proliferation of T-24 (bladder carcinoma), A-549 (lung cancer) and HepG-2 (hepatocellular carcinoma) cancer cell lines with IC50 of 244 µg/mL, 148 µg/mL and 123 µg/mL, respectively. An evaluation of anti-inflammatory activity revealed that EPSR4 has potent lipoxygenase (LOX) inhibitory activity (IC50 of 54.3 µg/mL) and a considerable effect on membrane stabilization (IC50 = 112.2 ± 1.2 µg/mL), while it showed cyclooxygenase (COX2) inhibitory activity up to 125 µg/mL. Finally, EPSR4 showed considerable inhibitory activity towards acetylcholine esterase activity. Taken together, this study reveals that Bacillus subtilis strain AG4 could be considered as a potential natural source of novel EPS with potent biological activities that would be useful for the healthcare system.Faculty of Science, Suez Canal UniversityPrincess Nourah bint Abdulrahman UniversityTaif UniversityPeer Reviewe

Synthesis and evaluation of novel non-viral gene delivery systems for prostate cancer targeting

This thesis was previously held under moratorium from 18th May 2018 until 18th May 2023Prostate cancer is the second most widespread cancer in men worldwide. Treatment choices are limited to prostatectomy, hormonal and radiotherapy that commonly have deleterious side effects and vary in their efficacy, depending on the stage of the disease. Among novel experimental strategies, gene therapy holds great promise for the treatment of cancer, but its use is currently limited by the lack of delivery systems able to selectively deliver the therapeutic genes to the tumours after intravenous administration without major drawbacks. Given that lactoferrin receptors, prostate specific membrane antigen (PSMA) and integrins are overexpressed on prostate cancer cells, the purpose of this study is to determine whether lactoferrin, Peptide2 (CWQPDTAHHWATL) and Peptide4 (CPRPRGDNPPLTCGGKKK) bearing diaminobutyric polypropylenimine (DAB) based dendriplexs would improve the targeting of therapeutic genes in vitro and in vivo. The chemical and physical characteristics of the synthesised dendrimers were first determined, followed by various in vitro experiments, to assess the improvement in the transfection and cellular uptake of the modified dendriplexes in PC-3, DU145 and LNCaP prostate cancer cell lines. Finally, in vivo studies were carried out using BALB/c nude mice to investigate the anticancer effects of the Lf-bearing dendriplexes encoding TNFα, TRAIL, or IL-12. DAB-Lf significantly increased the cellular uptake of the DNA in all prostate cancer cells. The highest DNA uptake, observed in PC-3 cells, was double than that observed in cells treated with non-targeted dendriplex. The anti-proliferative efficacy of DAB-Lf dendriplex encoding TNFα, TRAIL, or IL-12 was significantly improved compared with unmodified DAB dendriplex. In vivo, intravenous injections of DAB-Lf dendrimer complexed with plasmid DNA encoding TNFα and TRAIL have resulted in complete regression in 70% and 40% of tumours respectively at the end of the experiment in tumour-bearing mice. DAB-PEG2k-Pep2 significantly improved the cellular uptake in LNCaP and DU145 cells, but not in PC-3 cells compared with the non-targeted dendriplex. The therapeutic DNA encoding TNFα was successfully transfected after complexation with DAB-PEG2k-Pep2 in all three cell lines, with significant variance in the cytokine concentration between cell lines. The IC50 of DAB-PEG2k-Pep2 dendriplex encoding TNFα was significantly improved compared with untargeted DAB-PEG dendriplex in LNCaP cells. In conclusion, DAB-Lf is a promising DNA carrier for targeting prostate cancer. This study is one of few showing significant tumour regression after intravenous administration of gene therapy using non-viral vectors as a single therapy approach. Peptide2 was also found to be an effective ligand for PSMA, however more improvement in the formulation is still required to obtain an enhanced therapeutic effect.Prostate cancer is the second most widespread cancer in men worldwide. Treatment choices are limited to prostatectomy, hormonal and radiotherapy that commonly have deleterious side effects and vary in their efficacy, depending on the stage of the disease. Among novel experimental strategies, gene therapy holds great promise for the treatment of cancer, but its use is currently limited by the lack of delivery systems able to selectively deliver the therapeutic genes to the tumours after intravenous administration without major drawbacks. Given that lactoferrin receptors, prostate specific membrane antigen (PSMA) and integrins are overexpressed on prostate cancer cells, the purpose of this study is to determine whether lactoferrin, Peptide2 (CWQPDTAHHWATL) and Peptide4 (CPRPRGDNPPLTCGGKKK) bearing diaminobutyric polypropylenimine (DAB) based dendriplexs would improve the targeting of therapeutic genes in vitro and in vivo. The chemical and physical characteristics of the synthesised dendrimers were first determined, followed by various in vitro experiments, to assess the improvement in the transfection and cellular uptake of the modified dendriplexes in PC-3, DU145 and LNCaP prostate cancer cell lines. Finally, in vivo studies were carried out using BALB/c nude mice to investigate the anticancer effects of the Lf-bearing dendriplexes encoding TNFα, TRAIL, or IL-12. DAB-Lf significantly increased the cellular uptake of the DNA in all prostate cancer cells. The highest DNA uptake, observed in PC-3 cells, was double than that observed in cells treated with non-targeted dendriplex. The anti-proliferative efficacy of DAB-Lf dendriplex encoding TNFα, TRAIL, or IL-12 was significantly improved compared with unmodified DAB dendriplex. In vivo, intravenous injections of DAB-Lf dendrimer complexed with plasmid DNA encoding TNFα and TRAIL have resulted in complete regression in 70% and 40% of tumours respectively at the end of the experiment in tumour-bearing mice. DAB-PEG2k-Pep2 significantly improved the cellular uptake in LNCaP and DU145 cells, but not in PC-3 cells compared with the non-targeted dendriplex. The therapeutic DNA encoding TNFα was successfully transfected after complexation with DAB-PEG2k-Pep2 in all three cell lines, with significant variance in the cytokine concentration between cell lines. The IC50 of DAB-PEG2k-Pep2 dendriplex encoding TNFα was significantly improved compared with untargeted DAB-PEG dendriplex in LNCaP cells. In conclusion, DAB-Lf is a promising DNA carrier for targeting prostate cancer. This study is one of few showing significant tumour regression after intravenous administration of gene therapy using non-viral vectors as a single therapy approach. Peptide2 was also found to be an effective ligand for PSMA, however more improvement in the formulation is still required to obtain an enhanced therapeutic effect

Synthesis and Biological Evaluation of Imadazo[1,2-a]pyrazines as Anticancer and Antiviral Agents through Inhibition of CDK9 and Human Coronavirus

In this work, novel imadazo[1,2-a]pyrazine derivatives were synthesized and evaluated as CDK9 inhibitors. The results of CDK9 assay showed that the derivatives with pyridin-4-yl in position 2 and benzyl in position 3 of imadazo[1,2-a]pyrazine 3c displayed the most potent CDK9 inhibitory activity with IC50 of 0.16 µM. The anti-proliferative effect of the new compounds was examined against breast cancer (MCF7), colorectal cancer (HCT116), and chronic myelogenous leukaemia (K652) cell lines. The data of MTT assay showed that the cytotoxic effect of the inhibitors is correlated to their inhibitory activity against CDK9. Compound 3c exhibited the most potent cytotoxicity effect with average IC50s of three cell lines of 6.66 µM. The drug likeness properties of 3c were predicated in silico and demonstrated that 3c have reasonable physiochemical and pharmacokinetic properties. Selected derivatives were assessed in antiviral assay against human coronavirus 229E. The results of this assay showed that the derivative with pyridin-4-yl in position 2 and cyclohexyl in position 3 of imadazo[1,2-a]pyrazine 3b exhibited the most potent anti-coronaviral activity with IC50 of 56.96 µM and selectivity index of 7.14. The target predication result revealed that 3b showed high affinity to protease enzyme. Docking studies of 3b with COVID-19 main protease was conducted and showed good binding affinity, which confirmed the in vitro assay data

Anti-Biofilm and Antibacterial Activities of Cycas media R. Br Secondary Metabolites: In Silico, In Vitro, and In Vivo Approaches

Enterococcus species possess many virulence factors that have an essential role in exacerbating the infections caused by them. The current study aimed to evaluate the effect of the secondary metabolites ginkgetin (GINK) and sotetsuflavone (SOTE), isolated from Cycas media R. Br dichloromethane fraction, on Enterococcus faecalis (E. faecalis) isolates for the first time. The antibacterial and antivirulence activities of the isolated compounds were investigated using docking studies and in vitro by determination of the minimum inhibitory concentrations (MICs). Additionally, flow cytometry and scanning electron microscope (SEM) were utilized to assess the effect of SOTE on the tested bacteria. Moreover, crystal violet assay and qRT-PCR were used to test the effect of SOTE on the biofilm-forming ability of E. faecalis isolates. In addition, a systemic infection model was utilized in vivo to investigate the antibacterial activity of SOTE. We found that both GINK and SOTE showed a good affinity for the five proteins enrolled in the virulence of E. faecalis, with SOTE being the highest, suggesting the possible mechanisms for the antivirulence activity of both ligands. In addition, SOTE exhibited a higher antibacterial activity than GINK, as the values of the MICs of SOTE were lower than those of GINK. Thus, we performed the in vitro and in vivo assays on SOTE. However, they did not exhibit any significant variations (p > 0.05) in the membrane depolarization of E. faecalis isolates. Moreover, as evaluated by SEM, SOTE caused distortion and deformation in the treated cells. Regarding its impact on the biofilm formation, it inhibited the biofilm-forming ability of the tested isolates, as determined by crystal violet assay and qRT-PCR. The in vivo experiment revealed that SOTE resulted in a reduction of the inflammation of the liver and spleen with an increase in the survival rate. SOTE also improved the liver-function tests and decreased tumor necrosis factor-alpha using immunostaining and the inflammation markers, interleukins (IL-1β and IL-6), using ELISA. Thus, we can conclude that SOTE could be a promising compound that should be investigated in future preclinical and clinical studies

Antidiarrheal and Antibacterial Activities of Monterey Cypress Phytochemicals: In Vivo and In Vitro Approach

Monterey cypress (Cupressus macrocarpa) is a decorative plant; however, it possesses various pharmacological activities. Therefore, we explored the phytochemical profile of C. macrocarpa root methanol extract (CRME) for the first time. Moreover, we investigated its antidiarrheal (in vivo), antibacterial, and antibiofilm (in vitro) activities against Salmonella enterica clinical isolates. The LC-ESI-MS/MS analysis of CRME detected the presence of 39 compounds, besides isolation of 2,3,2″,3″-tetrahydro-4′-O-methyl amentoflavone, amentoflavone, and dihydrokaempferol-3-O-α-l-rhamnoside for the first time. Dihydrokaempferol-3-O-α-l-rhamnoside presented the highest antimicrobial activity and the range of values of MICs against S. enterica isolates was from 64 to 256 µg/mL. The antidiarrheal activity of CRME was investigated by induction of diarrhea using castor oil, and exhibited a significant reduction in diarrhea and defecation frequency at all doses, enteropooling (at 400 mg/kg), and gastrointestinal motility (at 200, 400 mg/kg) in mice. The antidiarrheal index of CRME increased in a dose-dependent manner. The effect of CRME on various membrane characters of S. enterica was studied after typing the isolates by ERIC-PCR. Its impact on efflux and its antibiofilm activity were inspected. The biofilm morphology was observed using light and scanning electron microscopes. The effect on efflux activity and biofilm formation was further elucidated using qRT-PCR. A significant increase in inner and outer membrane permeability and a significant decrease in integrity and depolarization (using flow cytometry) were detected with variable percentages. Furthermore, a significant reduction in efflux and biofilm formation was observed. Therefore, CRME could be a promising source for treatment of gastrointestinal tract diseases

Elucidation of the Metabolite Profile of Yucca gigantea and Assessment of Its Cytotoxic, Antimicrobial, and Anti-Inflammatory Activities

The acute inflammation process is explained by numerous hypotheses, including oxidative stress, enzyme stimulation, and the generation of pro-inflammatory cytokines. The anti-inflammatory activity of Yucca gigantea methanol extract (YGME) against carrageenan-induced acute inflammation and possible underlying mechanisms was investigated. The phytochemical profile, cytotoxic, and antimicrobial activities were also explored. LC-MS/MS was utilized to investigate the chemical composition of YGME, and 29 compounds were tentatively identified. In addition, the isolation of luteolin-7-O-β-d-glucoside, apigenin-7-O-β-d-glucoside, and kaempferol-3-O-α-l-rhamnoside was performed for the first time from the studied plant. Inflammation was induced by subcutaneous injection of 100 μL of 1% carrageenan sodium. Rats were treated orally with YGME 100, 200 mg/kg, celecoxib (50 mg/kg), and saline, respectively, one hour before carrageenan injection. The average volume of paws edema and weight were measured at several time intervals. Levels of NO, GSH, TNF-α, PGE-2, serum IL-1β, IL-6 were measured. In additionally, COX-2 immunostaining and histopathological examination of paw tissue were performed. YGME displayed a potent anti-inflammatory influence by reducing paws edema, PGE-2, TNF-α, NO production, serum IL-6, IL-1β, and COX-2 immunostaining. Furthermore, it replenished the diminished paw GSH contents and improved the histopathological findings. The best cytotoxic effect of YGME was against human melanoma cell line (A365) and osteosarcoma cell line (MG-63). Moreover, the antimicrobial potential of the extract was evaluated against bacterial and fungal isolates. It showed potent activity against Gram-negative, Gram-positive, and fungal Candida albicans isolates. The promoting multiple effects of YGME could be beneficial in the treatment of different ailments based on its anti-inflammatory, antimicrobial, and cytotoxic effects

Promising Antiviral Activity of Agrimonia pilosa Phytochemicals against Severe Acute Respiratory Syndrome Coronavirus 2 Supported with In Vivo Mice Study

The global emergence of the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused the entire world’s attention toward searching for a potential remedy for this disease. Thus, we investigated the antiviral activity of Agrimonia pilosa ethanol extract (APEE) against SARS-CoV-2 and it exhibited a potent antiviral activity with IC50 of 1.1 ± 0.03 µg/mL. Its mechanism of action was elucidated, and it exhibited a virucidal activity and an inhibition of viral adsorption. Moreover, it presented an immunomodulatory activity as it decreased the upregulation of gene expression of COX-2, iNOS, IL-6, TNF-α, and NF-κB in lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells. A comprehensive analysis of the phytochemical fingerprint of APEE was conducted using LC-ESI-MS/MS technique for the first time. We detected 81 compounds and most of them belong to the flavonoid and coumarin classes. Interestingly, isoflavonoids, procyanidins, and anthocyanins were detected for the first time in A. pilosa. Moreover, the antioxidant activity was evidenced in DPPH (IC50 62.80 µg/mL) and ABTS (201.49 mg Trolox equivalents (TE)/mg) radical scavenging, FRAP (60.84 mg TE/mg), and ORAC (306.54 mg TE/g) assays. Furthermore, the protective effect of APEE was investigated in Lipopolysaccharides (LPS)-induced acute lung injury (ALI) in mice. Lung W/D ratio, serum IL-6, IL-18, IL-1β, HO-1, Caspase-1, caspase-3, TLR-4 expression, TAC, NO, MPO activity, and histopathological examination of lung tissues were assessed. APEE induced a marked downregulation in all inflammation, oxidative stress, apoptosis markers, and TLR-4 expression. In addition, it alleviated all histopathological abnormalities confirming the beneficial effects of APEE in ALI. Therefore, APEE could be a potential source for therapeutic compounds that could be investigated, in future preclinical and clinical trials, in the treatment of patients with COVID-19

Regression of prostate tumors after intravenous administration of lactoferrin-bearing polypropylenimine dendriplexes encoding TNF-α, TRAIL, and interleukin-12

The possibility of using gene therapy for the treatment of prostate cancer is limited by the lack of intravenously administered delivery systems able to safely and selectively deliver therapeutic genes to tumors. Given that lactoferrin (Lf) receptors are overexpressed on prostate cancer cells, we hypothesized that the conjugation of Lf to generation 3-diaminobutyric polypropylenimine dendrimer would improve its transfection and therapeutic efficacy in prostate cancer cells. In this study, we demonstrated that the intravenous administration of Lf-bearing DAB dendriplexes encoding TNFα resulted in the complete suppression of 70% of PC-3 and 50% of DU145 tumors over one month. Treatment with DAB-Lf dendriplex encoding TRAIL led to tumor suppression of 40% of PC-3 tumors and 20% of DU145 tumors. The treatment was well tolerated by the animals. Lf-bearing generation 3-polypropylenimine dendrimer is therefore a highly promising delivery system for non-viral gene therapy of prostate cancer

In Vitro and In Vivo Antitumor Activity of Indolo[2,3-<i>b</i>] Quinolines, Natural Product Analogs from Neocryptolepine Alkaloid

Neocryptolepine (5-methyl-5H-indolo[2,3-b] quinoline) analogs were synthesized and evaluated in vitro and in vivo for their effect versus Ehrlich ascites carcinoma (EAC). The analogs showed stronger cytotoxic activity against EAC cells than the reference drug. The in vivo evaluation of the target compounds against EAC-induced solid tumor in the female albino Swiss mice revealed a remarkable decrease in the tumor volume (TV) and hepatic lipid peroxidation. A noticeable increase of both superoxide dismutase (SOD) and catalase (CAT) levels was reported (p p < 0.001) with the elevation of the responsiveness of lymphocytes to phytohemagglutinin (PHA). These results indicate that these naturally-based neocryptolepine alkaloids exhibit marked antitumor activity in vivo and represent an important lead in the development of natural-based anticancer drugs