An Experimental and Theoretical Study of the 1,3-Dipolar Cycloaddition of Alloxan-Derived Azomethine Ylides to Cyclopropenes

A diastereoselective synthesis of biologically interesting spirobarbiturates has been achieved via [3 + 2] cycloaddition of alloxan-derived azomethine ylides to 3-R-1,2-diphenylcyclopropenes. With this approach, various spirobarbiturate-3-azabicyclo[3.1.0]hexanes and spirobarbiturate-cyclopropa[a]pyrrolizines were obtained in moderate to good yields with excellent diastereoselectivities. DFT calculations (M11 density functional theory) have been carried out to shed light on the molecular mechanism of 1,3-dipolar cycloaddition of alloxan-derived azomethine ylides to cyclopropenes. The cytotoxic activity of some obtained compounds against human erythroleukemia (K562) cell line was evaluated in vitro by MTS-assay.

SETD2 Deficiency and miR-21: Potent Therapeutic Targets in NUT Midline Carcinoma

BackgroundNuclear protein in testis (NUT) midline carcinoma (NMC) is a rare and highly aggressive tumor with the bromodomain containing 4 protein (BRD4)-NUT (NUTM1) gene fusion. Few targeted therapies are available for NMC, and novel therapeutic targets are required. The objectives of our study was to determine gene mutations and microRNA (miRNA) expression levels in NMC and to identify novel therapeutic targets for NMC. Methods Next-generation sequencing (NGS) was used to identify mutations in the NMC cell lines and the tumor sample from a NMC patient; we determined the BRD4-NUT fusion gene in the patient using a digital PCR assay. Trimethylation of lysine 36 on histone H3 expression (H3K36me3) was studied using western blotting. The efficacy of the WEE1 G2 checkpoint kinase (WEE1) inhibitor, AZD1775, was evaluated using the MTS assay. RNA sequencing was performed to determine miRNA expression levels in the NMC cell lines. TaqMan miRNA assays were used to analyze miR-21 expression. The efficacy of miR-21 inhibitor was evaluated using the MTS assay. Results A novel SETD2 mutation (p.Ser2382fs) was identified in the NMC cell lines and the patient tumor. H3K36me3 was depleted in the NMC cells, which was indicative of functional SETD2 loss. The NMC cells were sensitive to AZD1775, an inhibitor for SETD2-deficient cancer. miRNA analysis revealed that miR-21 expression was increased in the NMC cells resistant to the traditional BET-targeted therapy. The miR-21 inhibitor suppressed the proliferation of the NMC cells. ConclusionsSETD2 deficiency and miR-21 may serve as novel therapeutic targets for the treatment of NMC.Trial registrationIn this study, we analyzed the gene alterations in human tumor samples. This study was registered in the UMIN clinical trial registered system (UMIN000043147, January 27, 2021).

Studying the Effect of Curcumin on Hep3B Hepatocarcinoma in Molecular and Biology Research Laboratory at An-Najah National University

Liver cancer is the growth and spread of cells in the liver that are abnormal. In this research, we examined Curcumin's effects on the growth of Hep3B cells liver in Molecular and Biology Research Laboratory at An-Najah National University. Curcumin regarding several theoretical and practical studies starves cancer cells to death. The life cycle of Hep3B cell has been analyzed by MTS assay, which is used to assess cell proliferation, cell viability, cytotoxicity, and flow cytometry. Our results agreed that Curcumin could a potentially efficient medication in the treatment of hepatocellular carcinoma, according to the findings of this study which confirms that curcumin treatment inhibited the growth of Hep3B. Thus, taking curcumin in our food is recommended.

Effect of goji berry on the formation of extracellular senile plaques of Alzheimer’s disease

BACKGROUND: Alzheimer’s disease (AD) is the most common neurodegenerative disease and a major source of morbidity and mortality. Currently, no therapy nor drug can cure or modify AD progression, but recent studies suggest that nutritional compounds in certain foods can delay or prevent the onset of AD. Diets with high antioxidants is one of the examples which is believed to influence AD pathogenesis through direct effect on amyloid beta levels. Compared to other fruits and vegetables, Goji berry (GB) has high levels of polyphenolic substances with antioxidant activities which have shown some positive effects on cognitive function while its mechanism on neuroprotection is yet to be explored. We investigated whether GB would decrease the quantity of amyloid beta in cell culture model of AD. OBJECTIVE: To assess the protective effects of GB against amyloid beta toxicity in M17 cells using different techniques. METHODS: Goji berry powder (GBP) at different concentrations was treated with 20μM amyloid beta-induced neuronal cells. MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetra zolium), bicinchoninic acid (BCA) assay, Western blot analysis, enzyme-linked immunosorbent assay (ELISA) and atomic force microscopy (AFM) were performed to identify how GB affected amyloid beta. RESULTS: MTS assay indicated that GBP significantly increased cell viability up to 105% when GBP was at 1.2μg/ mL. Western blot showed significant reduction of amyloid beta up to 20% in cells treated with 1.5μg/ mL GBP. GBP at 1.5μg/ mL was the most effective concentration with 17% reduction of amyloid beta in amyloid beta-induced neuronal cells compared to control (amyloid beta only) based on ELISA results. AFM images further confirmed increasing GBP concentration led to decreased aggregation of amyloid beta. CONCLUSION: GB can be a promising anti-aging agent and warrants further investigating due to its effect on reduction of amyloid beta toxicity.

Biological evaluation of Safrole oil and Safrole oil Nanoemulgel as antioxidant, antidiabetic, antibacterial, antifungal and anticancer

Background Safrole is a natural compound extracted from various plants, and has shown various biological activities. The current study aimed to investigate the antioxidant, antidiabetic, antimicrobial, and anticancer activity of safrole oil and to study the influence of safrole nanoemulgel on these activities. Methods The antioxidant and antidiabetic in-vitro assays were conducted using standard biomedical methods. The safrole oil nanoemulgel was developed using a self-emulsifying technique. Then the antimicrobial activity of the safrole oil and safrole nanoemulgel were performed on different microbial species, and cytotoxicity was determined against Hep3B cancer cell lines using the MTS assay. Results Safrole oil showed moderate antioxidant activity compared with standard Trolox, with IC50 value 50.28 ± 0.44 and 1.55 ± 0.32 μg/ml, respectively. Moreover, it had potent α-amylase inhibitory activity (IC50 11.36 ± 0.67 μg/ml) compared with Acarbose (IC50 value 5.88 ± 0.63). The safrole nanoemulgel had pseudo-plastic behaviour, droplet sizes below 200 nm, a polydispersity index (PDI) below 0.3, and a zeta potential of less than − 30 mV. Safrole oil has potential antimicrobial and anticancer activities, and these activities were improved with safrole nanoemulgel. Conclusion The safrole oil may be applied for the prevention and treatment of oxidative stress, diabetes, different microbial species and cancer, and these activities could be improved by nano-carriers.

SETD2 and miR-21 as therapeutic targets for NUT midline carcinoma

Background:Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare and highly aggressive tumor with the bromodomain containing 4 (BRD4)-NUT (NUTM1) gene fusion. BRD4 is a member of the bromodomain and extra-terminal domain (BET) family of proteins, and BET inhibitors have been investigated in NMC clinical trials. However, few targeted therapies are available for NMC, and novel therapeutic targets remain to be determined. We determined the role of two epigenetic regulators as possible therapeutic targets for NMC. Methods:We performed next-generation sequencing (NGS) in NMC cell lines (HCC2429 and Ty82). H3K36me3 expression was studied using western blotting. The efficacy of AZD1775, a WEE1 inhibitor, was evaluated using the MTS and γH2AX assays. We established an NMC cell line that was resistant to BET inhibitors. The sensitivity of the cells resistant to AZD1775 was analyzed using the MTS assay. RNA sequencing was performed to determine miRNA expression levels. TaqMan miRNA assays were used to analyze miR-21 expression. The efficacy of the miR-21 inhibitor was evaluated using the MTS assay. We established a digital PCR (dPCR) assay to detect NUT gene rearrangements to identify patients with NMC. Using NGS, a patient with NMC was identified with the SETD2 mutation.Results:SETD2 mutation (p.Ser2382fs) was determined in NMC cells, in which H3K36me3 expression was depleted, indicating SETD2 loss. NMC cells were sensitive to the WEE1 inhibitor, AZD1775 in cancer cells with SETD2 deficiency. γH2AX expression was increased in NMC cells treated with AZD1775. The efficacy of the combination of AZD1775 and JQ-1 was additive. We established NMC cells that were resistant to BET inhibitors. The resistant cells were also sensitive to AZD1775. miRNA analysis revealed increased miR-21 expression in BET-inhibitor-resistant NMC cells. MiR-21 regulated the growth of NMCs. The miR-21 inhibitor suppressed the growth of BET-inhibitor-resistant cells. Thirty-two clinical samples were analyzed and NMC was identified using digital PCR assays. Tumors with the SETD2 mutation were analyzed using NGS.Conclusions:We report for the first time SET domain-containing protein 2 (SETD2) deficiency and miR-21 as novel therapeutic targets for NMC. SETD2 loss and miR-21 may be therapeutic targets for NMC.Trial registrationIn this study, we analyzed the gene alterations in human tumor samples. This study was registered in the UMIN clinical trial registered system (UMIN000043147, January 27, 2021).

Macrophage supernatant infected with Leishmania major mediates the cytology of fibroblast cells in skin wounds

Objective: Healing of Cutaneous Leishmaniasis relies on the effective and modulates protective immune responses. Although the immune system is necessary to eliminate the parasite, it could be considered as the main cause of ulcers. Therefore, main aim of this study was to explore the possible regulatory functions of macrophage supernatant infected with Leishmania major on the fibroblast cells. Materials and Methods: In this experimental study, different concentrations of infected macrophage supernatant extract (50, 100, 150, 200, and 250μg/mL) were tested at different times (6, 24, 48, and 72h) and the effect of the leishmanicidal extract on fibroblast cells was determined by MTS assay. Also, the flow-cytometry technique was used for the investigation of apoptosis induction percentage. Results: MTS assay showed that the leishmanicidal effect of infected macrophage supernatant extract was dependent on the concentration and the time of treatment. So, the best efficacy was observed in 200 μg/mL with 72 hours exposure time. Flow cytometry analysis showed that the infected macrophage supernatant extract could induce apoptosis in cultured fibroblasts. Conclusions: We have demonstrated that reduction of survival rate and induction of apoptosis in fibroblasts displayed a similar manner to keratinocytes when exposed to infected macrophages with L. major. Our data suggest that such a phenomenon can be the underlying cause of lesions with scarring, and future, the mechanism remains to be elucidated.

Analysis of Cytotoxicity of Selected Asteraceae Plant Extracts in Real Time, Their Antioxidant Properties and Polyphenolic Profile

Plants from Asteraceae family are widely used for their therapeutic effects in the treatment of gastrointestinal diseases, but the consequences of excessive intake still need to be studied. The aims of this study were the evaluation of cytotoxicity, measurement of antioxidant properties and determination of polyphenolic profile of Tanacetum vulgare L. (tansy), Achillea millefolium L. (yarrow) and Solidago gigantea Ait. (goldenrod) ethanolic extracts. The cytotoxicity of extracts was monitored by xCELLigence system in real time by using porcine intestinal epithelial cell line (IPEC-1) and by measurement of changes in metabolic activity ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay). The antioxidant properties were measured by spectrophotometric methods and polyphenolic profiles were determined by HPLC-DAD for 50% ethanol extracts (10% w/v). Strong cytotoxic effect was recorded for tansy and yarrow extracts (125–1000 µg/mL) by xCELLigence system and MTS assay. Conversely, a supportive effect on cell proliferation was recorded for goldenrod extracts (125 µg/mL) by the same methods (p < 0.001). The antioxidant activity was in good correlation with total polyphenolic content, and the highest value was recorded for goldenrod leaves, followed by tansy leaves, goldenrod flowers and yarrow leaf extracts. The goldenrod extracts were abundant with flavonoids, whereas phenolic acid derivatives predominated in the polyphenolic profile of tansy and yarrow.

A Simple Liquid-Liquid Fractionation (LLF) Method for Isolating Deoxyandrographolide dan Andrographolide from Herbs of Andrographis paniculata (Burm., F) Ness and Its Cytotoxic Activity on 3T3-L1 Preadipocyte Cells

The main bitter constituents of sambiloto (Androgaphis paniculata (Burm., F) Ness) are diterpene lactones, namely andrographolide and deoxyandrographolide which have been reported to have antidiabetic, cytotoxic, antiatherosclerosis, anti-inflammatory, and antimicrobial activity. There are many studies that performed the isolation of deoxyandrographolide and andrographolide from A. paniculata herbs, but most of them included several steps that make them not efficient. This research was conducted to do an isolation of deoxyandrographolide and andrographolide through liquid-liquid fractionation (LLF) due to its simplicity, low cost, and time efficient. The extraction of deoxyandrographolide and andrographolide from the herbs was carried out using chloroform as the solvent by using Soxhlet apparatus, and LLF was performed to isolate the compounds. The identities of the compounds were confirmed by TLC scanner compared to its standard references. Hence, these present methods were successfully isolated and determined deoxyandrographolide and andrographolide of A. paniculata. The compounds were also showed relatively moderate cytotoxicity on 3T3-L1 cell lines using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, with LC50 of deoxyandrographolide and andrographolide; 29.3173 µg/mL and 37.7011 µg/mL, respectively.

Combined Treatment with Low Cytotoxic Ethyl Acetate Nepenthes Extract and Ultraviolet-C Improves Antiproliferation to Oral Cancer Cells via Oxidative Stress

Ultraviolet-C (UVC) irradiation provides an alternative radiotherapy to X-ray. UVC sensitizer from natural products may improve radiotherapy at low cytotoxic side effects. The aim of this study is to assess the regulation for oral cancer cell proliferation by a combined treatment of UVC and our previously reported anti-oral cancer natural product (ethyl acetate extract of Nepenthes adrianii × clipeata; EANA). The detailed possible UVC sensitizing mechanisms of EANA such as effects on cell proliferation, cell cycle, apoptosis, and DNA damage are investigated individually and in combination using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay, flow cytometry, and western blotting at low dose conditions. In a 24 h MTS assay, the low dose EANA (5 μg/mL) and low dose UVC (12 J/m2) individually show 80% and combinedly 57% cell proliferation in oral cancer Ca9-22 cells; but no cytotoxicity to normal oral HGF-1 cells. Mechanistically, low dose EANA and low dose UVC individually induce apoptosis (subG1 accumulation, pancaspase activation, and caspases 3, 8, 9), oxidative stress (reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential depletion), and DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). Moreover, the combined treatment (UVC/EANA) synergistically induces these changes. Combined low dose treatment-induced antiproliferation, apoptosis, oxidative stress, and DNA damage were suppressed by the ROS scavenger N-acetylcysteine. In conclusion, UVC/EANA shows synergistic antiproliferation, oxidative stress, apoptosis, and DNA damage to oral cancer cells in an oxidative stress-dependent manner. With the selective killing properties of low dose EANA and low dose UVC, EANA provides a novel UVC sensitizing agent to improve the anti-oral cancer therapy.