ABSTRACT
Metformin (MET), an oral antidiabetic drug, was reported to possess promising anticancer effects. We hypothesized that MET encapsulation in unique nanospanlastics would enhance its anticancer potential against HEP-2 cells. Our results showed the successful fabrication of Nano-MET spanlastics (d = 232.10 ± 0.20 nm; PDI = 0.25 ± 0.11; zeta potential = (-) 44.50 ± 0.96; drug content = 99.90 ± 0.11 and entrapment efficiency = 88.01 ± 2.50%). MTT assay revealed the enhanced Nano-MET cytotoxicity over MET with a calculated IC50 of 50 µg/mL and > 500 µg/mL, respectively. Annexin V/PI apoptosis assay showed that Nano-MET significantly decreased the percentage of live cells from 95.49 to 93.70 compared to MET and increased the percentage of cells arrested in the G0/G1 phase by 8.38%. Moreover, Nano-MET downregulated BCL-2 and upregulated BAX protein levels by 1.57 and 1.88 folds, respectively. RT-qPCR revealed that Nano-MET caused a significant 13.75, 4.15, and 2.23-fold increase in caspase-3, -8, and - 9 levels as well as a 100 and 43.47-fold decrease in cyclin D1 and mTOR levels, respectively. The proliferation marker Ki67 immunofluorescent staining revealed a 3-fold decrease in positive cells in Nano-MET compared to the control. Utilizing the combined Pathway-Enrichment Analysis (PEA) and Reactome analysis indicated high enrichment of certain pathways including nucleotides metabolism, Nudix-type hydrolase enzymes, carbon dioxide hydration, hemostasis, and the innate immune system. In summary, our results confirm MET cytotoxicity enhancement by its encapsulation in nanospanlastics. We also highlight, using PEA, that MET can modulate multiple pathways implicated in carcinogenesis.
ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common cancers reported worldwide with poor morbidity and high mortality rates. HCC is a very vascular solid tumour as angiogenesis is not only a key driver for tumour progression but also an exciting therapeutic target. Our research investigated the use of fucoidan, a sulfated polysaccharide readily abundant in edible seaweeds commonly consumed in Asian diet due to their extensive health benefits. Fucoidan was reported to possess a strong anti-cancer activity, but its anti-angiogenic potential is still to be fully unraveled. Our research investigated fucoidan in combination with sorafenib (an anti-VEGFR tyrosine kinase inhibitor) and Avastin® (bevacizumab, an anti-VEGF monoclonal antibody) in HCC both in vitro and in vivo. In vitro on HUH-7 cells, fucoidan had a potent synergistic effect when combined with the anti-angiogenic drugs and significantly reduced HUH-7 cell viability in a dose dependent manner. Using the scratch wound assay to test cancer cell motility, sorafenib, A + F (Avastin and fucoidan) or S + F (sorafenib and fucoidan) treated cells consistently showed an unhealed wound and a significantly smaller %wound closure (50%-70%) versus untreated control (91%-100%) (p < 0.05, one-way ANOVA). Using RT-qPCR; fucoidan, sorafenib, A + F and S + F significantly reduced the expression of the pro-angiogenic PI3K/AKT/mTOR and KRAS/BRAF/MAPK pathways by up to 3 folds (p < 0.05, one-way ANOVA versus untreated control). While ELISA results revealed that in fucoidan, sorafenib, A + F and S + F treated cells, the protein levels of caspases 3, 8, and 9 was significantly increased especially in the S + F group showing 40- and 16-times higher caspase 3 and 8 protein levels, respectively (p < 0.05, one-way-ANOVA versus untreated control). Finally, in a DEN-HCC rat model, H&E staining revealed larger sections of apoptosis and necrosis in the tumour nodules of rats treated with the combination therapies and immunohistochemical analysis of the apoptotic marker caspase 3, the proliferation marker Ki67 and the marker for angiogenesis CD34 showed significant improvements when the combination therapies were used. Despite the promising findings reported herein that highlighted a promising chemomodulatory effect of fucoidan when combined with sorafenib and Avastin, further investigations are required to elucidate potential beneficial or adversary interactions between the tested agents.
ABSTRACT
Natural products are considered as a good source of antifibrotic agents, but identifying and isolating bioactive molecule(s) is still challenging. Fortunately, numerous computational techniques have evolved to save time and efforts in this field. The aim of the current study was to utilize several cheminformatics software to study the chemical and biological features of the bark of Eucalyptus globulus cultivated in Egypt. Sirius software, with the aid of online databases, was used to process liquid chromatography-mass spectrometry (LC-MS) chemical profiling and predict precise molecular formulae, chemical classes, and structures. Accordingly, 37 compounds were tentatively identified, including 15 reported here for the first time from this species. Also, the BioTransformer tool was successfully applied for in silico virtual study of the human metabolism of these compounds, and 1960 different products were obtained through various metabolic pathways. Finally, an electronic library of the identified compounds and their metabolites were developed and docked in silico against eight different protein targets that are involved in the liver fibrosis process. The results revealed that the extract may have a potential hepatoprotective effect through several mechanisms and that the metabolites have the highest binding affinities to the relevant enzymes than their parent compounds. The extract was found to show potent cytotoxic activity against the liver cancer cell lines HEPG2 and HUH-7, and its absorption was enhanced through nanoformulation, as proved using the ex vivo everted gut sac method.
ABSTRACT
Hepatocellular carcinoma (HCC), one of the most prevalent types of cancers worldwide, continues to maintain high levels of resistance to standard therapy. As clinical data revealed poor response rates, the need for developing new methods has increased to improve the overall wellbeing of patients with HCC. Furthermore, a growing body of evidence shows that cancer metabolic changes are a key feature of many types of human malignancies. Metabolic reprogramming refers to cancer cells' ability to change their metabolism in order to meet the increased energy demand caused by continuous growth, rapid proliferation, and other neoplastic cell characteristics. For these reasons, metabolic pathways may become new therapeutic and chemopreventive targets. The aim of this study was to investigate the metabolic alterations associated with metformin (MET), an anti-diabetic agent when combined with two antifolate drugs: trimethoprim (TMP) or methotrexate (MTX), and how metabolic changes within the cancer cell may be used to increase cellular death. In this study, single drugs and combinations were investigated using in vitro assays including cytotoxicity assay (MTT), RT-qPCR, annexin V/PI apoptosis assay, scratch wound assay and Seahorse XF analysis, on a human HCC cell line, HepG2. The cytotoxicity assay showed that the IC50 of MET as single therapy was 44.08 mM that was reduced to 22.73 mM and 29.29 mM when combined with TMP and MTX, respectively. The co-treatment of both drugs increased p53 and Bax apoptotic markers, while decreased the anti-apoptotic marker; Bcl-2. Both combinations increased the percentage of apoptotic cells and halted cancer cell migration when compared to MET alone. Furthermore, both combinations decreased the MET-induced increase in glycolysis, while also inducing mitochondrial damage, altering cancer cell bioenergetics. These findings provide an exciting insight into the anti-proliferative and apoptotic effects of MET and anti-folates on HepG2 cells, and how in combination, may potentially combat the aggressiveness of HCC.
ABSTRACT
Isoflavones are considered one of the most extensively studied plant-derived phytoestrogenic compounds. Of these, Biochanin A (Bio-A), a natural isoflavone abundant in cabbage, alfalfa, and red clover, has drawn a lot of attention. As reported in multiple studies, Bio-A possesses a promising anticancer activity against estrogen receptor-positive (ER+) breast cancer. The current study investigated the working hypothesis that Bio-A could synergistically enhance the potency of 5-fluorouracil (5-FU) in ER+ breast cancer. The hypothesis was tested both in vitro on hormone receptor-positive (MCF-7) and triple-negative breast cancer cells (MDA-MB231). Additionally, in vivo studies were performed in the Ehrlich solid-phase carcinoma mouse model. The in vitro cytotoxicity studies revealed that Bio-A synergistically increased the potency of 5-FU in both MCF-7 and MDA-MB231 cell lines. The synergistic effect of 5-FU/Bio-A combination was verified in vivo. The combination therapy (where 5-FU was used at one fourth its full dose) led to a significant 75% reduction in tumor volume after two treatment cycles. This was in addition to producing a significant 2.1-fold increase in tumor necrosis area% compared to mock-treated control. In conclusion, the current study presents the first preclinical evidence for the potential merit of 5-FU/Bio-A combination for the treatment of ER+ breast cancer. The synergistic antitumor effect of Bio-A/ 5-FU combination can be, at least partly, attributed to Bio-A-mediated suppression of ER-α/Akt axis and the augmentation of 5-FU-mediated proapoptotic effects. © 2022 John Wiley & Sons, Ltd.
Subject(s)
Carcinoma , Isoflavones , Animals , Apoptosis , Cell Line, Tumor , Drug Synergism , Fluorouracil/pharmacology , Genistein/pharmacology , Humans , Isoflavones/pharmacology , MiceABSTRACT
Magnetic hyperthermia (MH) harnesses the heat-releasing properties of superparamagnetic iron oxide nanoparticles (SPIONs) and has potential to stimulate immune activation in the tumor microenvironment whilst sparing surrounding normal tissues. To assess feasibility of localized MH in vivo, SPIONs are injected intratumorally and their fate tracked by Zirconium-89-positron emission tomography, histological analysis, and electron microscopy. Experiments show that an average of 49% (21-87%, n = 9) of SPIONs are retained within the tumor or immediately surrounding tissue. In situ heating is subsequently generated by exposure to an externally applied alternating magnetic field and monitored by thermal imaging. Tissue response to hyperthermia, measured by immunohistochemical image analysis, reveals specific and localized heat-shock protein expression following treatment. Tumor growth inhibition is also observed. To evaluate the potential effects of MH on the immune landscape, flow cytometry is used to characterize immune cells from excised tumors and draining lymph nodes. Results show an influx of activated cytotoxic T cells, alongside an increase in proliferating regulatory T cells, following treatment. Complementary changes are found in draining lymph nodes. In conclusion, results indicate that biologically reactive MH is achievable in vivo and can generate localized changes consistent with an anti-tumor immune response.
Subject(s)
Hyperthermia, Induced , Magnetite Nanoparticles , Ferric Compounds , Humans , Hyperthermia , Magnetic Fields , MagneticsABSTRACT
BACKGROUND: The monocyte monolayer assay (MMA) is an in-vitro assay that can predict the outcome of blood transfusion of antigen positive units when serologically compatible blood is not available. MATERIALS AND METHODS: Fifty-four patients testing positive by the antibody screening test using gel agglutination were further examined by the alloantibody identification panel to determine alloantibody specificity. After determining and categorizing the antibodies, patients' samples were examined using the MMA to determine the clinical significance of the detected alloantibodies. We also tested 2 seeding methods (24-well cell culture plates versus 8-well chamber-slides) and 3 visualization/staining techniques (unstained phase contrast, Leishman and Giemsa staining). RESULTS: 35 out of the 54 cases (64.8%) had a monocyte index of >5% which is predictive of occurrence of hemolytic reaction after transfusion; 23 cases with antibodies known to be clinically significant [anti-C, anti-E, anti-c, anti-K, anti-Fy(a), anti Fy(b), anti-JK(b)], 2 with Anti-M specificity, 7 cases with autoantibodies and 3 cases with multiple antibodies. On the other hand, 19 out of the 54 (35.2%) cases included in the study showed a monocyte index of <5% which is predictive of absence of hemolytic reaction after transfusion. The 8-well chamber-slides were better than the 24-well culture plates, as the latter showed a lot of un-phagocytosed RBCs in the background. Also, Leishman staining was better than Giemsa staining with better and clearer differentiation between the RBCs, monocytes and phagocytic vacuoles. CONCLUSION: MMA can be used as a surrogate cross-match test for the selection of blood units in cases where antigen-negative blood units are not available.
Subject(s)
Blood Group Antigens/immunology , Blood Grouping and Crossmatching/methods , Isoantibodies/analysis , Monocytes/immunology , Transfusion Reaction/prevention & control , Cell Culture Techniques , Humans , Isoantibodies/immunologyABSTRACT
The magnetic properties and safety of dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) have facilitated their clinical use as MRI contrast agents and stimulated research on applications for SPIONs in particle imaging and magnetic hyperthermia. The wider clinical potential of SPIONs, however, has been limited by their rapid removal from circulation via the reticuloendothelial system (RES). We explored the possibility of extending SPION circulatory time using fucoidan, a seaweed-derived food supplement, to inhibit RES uptake. The effects of fucoidan on SPION biodistribution were evaluated using ferucarbotran, which in its pharmaceutical formulation (Resovist) targets the RES. Ferucarbotran was radiolabeled at the iron oxide core with technetium-99m (99mTc; t1/2 = 6 h) or zirconium-89 (89Zr; t1/2 = 3.3 days). Results obtained with 99mTc-ferucarbotran demonstrated that administration of fucoidan led to a 4-fold increase in the circulatory half-life (t1/2 slow) from 37.4 to 150 min (n = 4; P < 0.0001). To investigate whether a longer circulatory half-life could lead to concomitant increased tumor uptake, the effects of fucoidan were tested with 89Zr-ferucarbotran in mice bearing syngeneic subcutaneous (GL261) tumors. In this model, the longer circulatory half-life achieved with fucoidan was associated with a doubling in tumor SPION uptake (n = 5; P < 0.001). Fucoidan was also effective in significantly increasing the circulatory half-life of perimag-COOH, a commercially available SPION with a larger hydrodynamic size (130 nm) than ferucarbotran (65 nm). These findings indicate successful diversion of SPIONs away from the hepatic RES and show realistic potential for future clinical applications.
ABSTRACT
The rapid reticuloendothelial system (RES) mediated clearance of superparamagnetic iron oxide nanoparticles (SPIONs) from circulation is considered a major limitation of their clinical utility. We aimed to address this by using dextran sulfate 500 (DSO4 500), a Kupffer cell blocking agent, to prolong SPIONs circulatory time. Blood concentrations of SPIONs are difficult to quantify due to the presence of haemoglobin. We therefore developed methods to functionalise SPIONs with near-infrared (NIR) dyes in order to trace their biodistribution. Two SPIONs were investigated: Nanomag®-D-spio-NH(2) and Ferucarbotran. Nanomag®-D-spio-NH(2) was functionalised using NHS (N-hydroxysuccinimide) ester NIR dye and Ferucarbotran was labelled using periodate oxidation followed by reductive amination or a combination of EDC (ethyl(dimethylaminopropyl) carbodiimide )/NHS and click chemistries. Stability after conjugation was confirmed by dynamic light scattering (DLS), superconducting quantum interference device (SQUID) and transmission electron microscopy (TEM). In vivo experiments with the functionalised SPIONs showed a significant improvement in SPIONs blood concentrations in mice pre-treated with dextran sulfate sodium salt 500 (DSO4 500).