METHODS: The structures of all synthesized compounds were characterized by physicochemical properties and spectral means (IR and NMR). The synthesized compounds were evaluated for their in vitro antimicrobial activity against Gram-positive (B. subtilis), Gram-negative (P. aeruginosa and E. coli) bacterial and fungal (C. albicans and A. niger) strains by tube dilution method using ciprofloxacin, amoxicillin and fluconazole as standards. In-vitro antioxidant and anti-urease screening was done by DPPH assay and indophenol method, respectively. The in-vitro anticancer evaluation was carried out against MCF-7 and HCT116 cancer cell lines using 5-FU as standards.
RESULTS, DISCUSSION AND CONCLUSION: The biological screening results reveal that the compounds T5 (MICBS, EC = 24.7 µM, MICPA, CA = 12.3 µM) and T17 (MICAN = 27.1 µM) exhibited potent antimicrobial activity as comparable to standards ciprofloxacin, amoxicillin (MICCipro = 18.1 µM, MICAmo = 17.1 µM) and fluconazole (MICFlu = 20.4 µM), respectively. The antioxidant evaluation showed that compounds T2 (IC50 = 34.83 µg/ml) and T3 (IC50 = 34.38 µg/ml) showed significant antioxidant activity and comparable to ascorbic acid (IC50 = 35.44 µg/ml). Compounds T3 (IC50 = 54.01 µg/ml) was the most potent urease inhibitor amongst the synthesized compounds and compared to standard thiourea (IC50 = 54.25 µg/ml). The most potent anticancer activity was shown by compounds T2 (IC50 = 3.84 μM) and T7 (IC50 = 3.25 μM) against HCT116 cell lines as compared to standard 5-FU (IC50 = 25.36 μM).
Objective: This study aimed to determine the effects of selected phytoestrogens on annexin A1 (ANXA1) expression, mode of cell death and cell cycle arrest in different human leukemic cell lines.
Methods: Cells viability were examined by MTT assay and ANXA1 quantification via Enzyme-linked Immunosorbent Assay. Cell cycle and apoptosis were examined by flow cytometer and phagocytosis effect was evaluated using haematoxylin-eosin staining.
Results: Coumestrol significantly (p
Methods: Streptomyces strains' growth curves, namely SUK 12 and SUK 48, were measured and P. falciparum 3D7 IC50 values were calculated. Metabolomics analysis was conducted on both strains' mid-exponential and stationary phase extracts.
Results: The most successful antiplasmodial activity of SUK 12 and SUK 48 extracts shown to be at the stationary phase with IC50 values of 0.8168 ng/mL and 0.1963 ng/mL, respectively. In contrast, the IC50 value of chloroquine diphosphate (CQ) for antiplasmodial activity was 0.2812 ng/mL. The univariate analysis revealed that 854 metabolites and 14, 44 and three metabolites showed significant differences in terms of strain, fermentation phase, and their interactions. Orthogonal partial least square-discriminant analysis and S-loading plot putatively identified pavettine, aurantioclavine, and 4-butyldiphenylmethane as significant outliers from the stationary phase of SUK 48. For potential isolation, metabolomics approach may be used as a preliminary approach to rapidly track and identify the presence of antimalarial metabolites before any isolation and purification can be done.
Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.
Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.
Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.
MATERIALS AND METHODS: This study introduced a simple and green synthesis of Fe3O4 NPs using a low-cost stabilizer of plant waste extract rich in polyphenols content with a well-known antioxidant property as well as anticancer ability to eliminate colon cancer cells. Herein, Fe3O4 NPs were fabricated via a facile co-precipitation method using the crude extract of Garcinia mangostana fruit peel as a green stabilizer at different weight percentages (1, 2, 5, and 10 wt.%). The samples were analyzed for magnetic hyperthermia and then in vitro cytotoxicity assay was performed.
RESULTS: The XRD planes of the samples were corresponding to the standard magnetite Fe3O4 with high crystallinity. From TEM analysis, the green synthesized NPs were spherical with an average size of 13.42±1.58 nm and displayed diffraction rings of the Fe3O4 phase, which was in good agreement with the obtained XRD results. FESEM images showed that the extract covered the surface of the Fe3O4 NPs well. The magnetization values for the magnetite samples were ranging from 49.80 emu/g to 69.42 emu/g. FTIR analysis verified the functional groups of the extract compounds and their interactions with the NPs. Based on DLS results, the hydrodynamic sizes of the Fe3O4 nanofluids were below 177 nm. Furthermore, the nanofluids indicated the zeta potential values up to -34.92±1.26 mV and remained stable during four weeks of storage, showing that the extract favorably improved the colloidal stability of the Fe3O4 NPs. In the hyperthermia experiment, the magnetic nanofluids showed the acceptable specific absorption rate (SAR) values and thermosensitive performances under exposure of various alternating magnetic fields. From results of in vitro cytotoxicity assay, the killing effects of the synthesized samples against HCT116 colon cancer cells were mostly higher compared to those against CCD112 colon normal cells. Remarkably, the Fe3O4 NPs containing 10 wt.% of the extract showed a lower IC50 value (99.80 µg/mL) in HCT116 colon cancer cell line than in CCD112 colon normal cell line (140.80 µg/mL).
DISCUSSION: This research, therefore, introduced a new stabilizer of Garcinia mangostana fruit peel extract for the biosynthesis of Fe3O4 NPs with desirable physiochemical properties for potential magnetic hyperthermia and colon cancer treatment.
Materials and Methods: Dried root of P. glabra was extracted under reflux with methyl alcohol, fractionated through the vacuum liquid chromatography technique, and evaporated and then purified the compounds using column chromatography and preparative thin-layer chromatography. THP-1 cells were treated with amentoflavone, 5,7,4'-hydroxyflavonoid, and stigmasterol with various concentrations (0-30 µg/mL) and then incubated with MTS reagent for 2h. Treatment was done for 24, 48, and 72h. Then, effects of these compounds were also tested on PGE2, TNF-α, and IL-6 expression in human THP-1-derived macrophage cells for 24h.
Results: Three new compounds such as amentoflavone, 5,7,4'-hydroxyflavonoid, and stigmasterol were isolated. After 24h of incubation, a significant decrease in cell viability was reported with IC50 values of amentoflavone, 5,7,4'- hydroxyflavonoid, and stigmasterol (21 µg/mL ≡ 38 M), (18 µg/mL ≡ 66 M) and (20 µg/mL ≡ 48.5 M), respectively. Whereas for 48 and 72h treatment showed a less decreased cell viability compared with 24h treatment. These compounds also showed a significant reduction in the production of TNF-α, IL-6, and PGE2 in a dose-dependent manner.
Conclusions: The isolated new compounds showed significant cytotoxicity and anti-inflammatory effects.
Materials and Methods: SF1 was produced by optimized methodology for bioassay-guided fractionation. Fourier transform infrared (FTIR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) were carried out to characterize the SF1. SF1 was screened for cytotoxicity activity toward HeLa, SiHa, and normal cells (NIH) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The anticancer mechanism of SF1 was evaluated toward SiHa cells, which showed highest cytotoxicity toward SF1 treatment. The mechanism includes cell cycle progression and protein expression, which was detected using specific antibody-conjugated fluorescent dye, p53-FITC, by flow cytometry.
Results: Major constituents of SF1 were alkaloids with amines as functional group. SF1 showed highest cytotoxic activity against SiHa (half-maximal inhibitory concentration [IC50] < 10 µg/mL) compared to HeLa cells. Cytoselectivity of SF1 was observed with no IC50 detected on normal NIH cells. On flow cytometry analysis, SF1 was able to induce apoptosis on SiHa cells by arresting cell cycle at G1/S and upregulation of p53 protein.
Conclusion: SF1 showed anticancer activity by inducing apoptosis through arrested G1/S cell cycle checkpoint-mediated mitochondrial pathway.