Anticancer, antibacterial and pollutant degradation potential of silver nanoparticles from Hyphaene thebaica

Cancer is among the top global public health burdens leading to millions of deaths each year. The study aims to investigate the antiproliferative effect of Spartium junceum L. flowers on different cancer cell lines. The ethanolic extract of the flowers was prepared in the present study. Phytochemical analysis of the plant extract revealed the presence of several phenolic compounds such as cinnamic acid and its derivatives (chlorogenic, p-coumaric, ferulic acids), protocatechuic acid, epicatechin and luteolin. This extract was tested against human breast (MDA-MB-231) and liver (HepG2) cancer cell lines to find out its antiproliferative activity. It was determined that the extract was effective against both cell lines with IC50 values of 2.37 ± 0.47 and 0.98 ± 0.01 µL/mL for MDA-MB-231 and HepG2, respectively. Particularly, the extract was found to be more effective in the liver cancer cell line than the breast cancer cell line. All these obtained findings led us to believe that this medicinal plant could be a promising antiproliferative agent candidate for the treatment of human liver and breast cancers.

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

Phenolic profiling of edible parts Hyphaene thebaica ( Doum palm) are identified using LC-ESI-MS, the Overall polyphenolic constituents demonstrated by means of LC-ESI-Mass profiling . Twenty three isolated compounds were identified as ; caffeic acid, protocatechuic acid, rhamnetin, catechin, quercitrin, vanillic acid , kaempferol 3-O-acetyleglucoside , cinnamic acid, apigenin-7-O-glucose , intricatin 3-O-htyrosol, luteolin, quercetin ,naringenin , kaempferol ,vanillic acid 4-β-D-glycoside coumaric acid, ferulic acid, luteolin-6-arbinose-8-glucose, p-coumaroyl malic acid eriocitrin, apigenin and hesperetin Iron nanoparticles (FeNps ) of H. thebaica fruit EtOAc extract was freshly prepared and characterized with Dynamic Light Scattering (DLS) with particles size 150.7 nm. The anti-proliferation activity of the crude extract of EtOAc and the synthesized Fe Nps was evaluated using MTT assay on human colon (Caco-2) and liver (HepG2) cancer cell lines. The results declared that half maximal inhibitory concentration (IC50) of EtOAc extract on colon (IC50 35.4 µg/ml) and on liver cancer cell lines (IC50 72.02 µg/ml) while nanoparticles portion of EtOAc was found more pronounced on colon cancer cell lines (IC50 19.44 µg/ml) and on liver (IC50 15.5 µg/ml). So, the Fe Nps of EtOAc of H.thebaica fruit extract with particles size 150.7 nm is more effective as antitumor than the crude EtOAc extract.

Cancer remains the leading cause of death worldwide, with breast cancer being the most prevalent disease diagnosed globally. Liver cancer is the fourth most common cause of death globally, while prostate cancer accounts for the second most frequent malignancy among males worldwide. The main treatment options for cancer include surgery, radiation therapy, and chemotherapy. The stem bark of Tieghemella heckelii has been exploited for its medicinal properties. On a broader scale, research on the anticancer properties of T. heckelii has not been explored. It is therefore important to investigate the anticancer potential of the leaves of T. heckelii. The goal of the study was to evaluate the in vitro anticancer activities of the leaves of T. heckelii on breast, prostate, and liver cancer cell lines. The leaves of T. heckelii were collected from Asantemanso, Akim Oda, in the eastern region of Ghana. Authenticity of the leaves was performed at the Department of Plant and Environmental Biology, University of Ghana. Aqueous and ethanol extraction were performed on the leaves of T. heckelii after grinding into fine particles, followed by low-temperature drying. Breast cancer (MDA-MB-468), liver cancer (HepG2), and normal kidney (Vero E6) cell lines were cultured in DMEM media, while prostate cancer (PC3) cell lines were cultured in RPMI medium. Anticancer activity of the leaves of T. heckelii was conducted on breast (MDA-MB-468), liver (HepG2), and prostate (PC3) cancer cell lines. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Both ethanolic and aqueous extracts demonstrated similar cytotoxicity for the HepG2 cell line, with IC50 values of about 200 μg/mL. selectivity index of > 2 was recorded by all cell lines. When compared to other cell lines, the aqueous extract for prostate cancer showed the lowest IC50 value with a selectivity of 8. In addition, the extracts showed less cytotoxic activity against the normal (Vero) cell line. Leaves of T. heckelii possess cytotoxic properties with notable selectivity against prostate (PC3) and liver (HepG2) cancer cell lines. However, findings should be evaluated in in vivo studies because biological processes can be more complex in living organisms. Further investigation should be conducted to ascertain the bioactive compounds responsible for the anticancer activity.

Disclosure: P. Ramaraj: None. Our previous in-vitro studies involving vitamins (vitamin-A and vitamin-D3) and steroids (progesterone and RU-486) pointed to a combination of vitamin-A and RU-486 as an efficient combo to decrease human melanoma cell growth. We extended this combo treatment to human liver cancer cell (Hep-G2) line. The aim was to check whether vit-A and RU-486 combo would also decrease human liver cancer cell line growth. Liver cancer (Hep-G2) cells were plated in a 96 well plate overnight. Following day, vit-A and RU-486 were added either alone or in different combinations for 48 hrs. After 48 hrs, cell viability and percentage of cell growth were monitored by the MTT assay. Supernatants were collected for Elisarray and quantitation of cytokine(s) by Elisa. Individual vit-A and RU-486 treatments showed a dose-dependent decrease in cell growth. Though various combinations of vit-A (50, 75, 100 μM) and RU-486 (10, 50 100 μM) also showed a decrease in cell growth, an experimentally significant decrease in cell growth was observed at vit-A 75 μM and RU-486 50 μM combination. This combo resulted in 24% cell growth compared to straight vit-A (75 μM) at 44% cell growth and RU-486 (50 μM) at 35% cell growth. Thus, this combo inhibited liver cancer cell growth also. In the future effect of this combo on normal liver cell line and the cytokine(s) if any, playing a central role in cell growth would be identified by Elisarray and quantitated by Elisa. Presentation: 6/3/2024

MicroRNA-638 induces apoptosis and autophagy in human liver cancer cells by targeting enhancer of zeste homolog 2 (EZH2)

Green nanoparticle synthesis was achieved using environmentally acceptable plant extracts reducing and capping agents. The present study was based on assessments to the anticancer activities to determine the effect of synthesized silver nanoparticles (AgNPs) from three medicinal plants on human liver (HepG2) and prostate (PC3) cancer cell lines. The synthesis of AgNPs using Plumbago zeylanica (Pz), Semecarpus anacardium (Sa) and Terminalia arjuna (Ta) plant extracts in the reaction mixture was monitored by UV-visible spectroscopy. FTIR results clearly illustrated that the plant extracts containing prominent peaks of functional groups and biomolecules viz., tannins, phenols, flavonoids and triterpenoids those act as capping agents and involved in the stabilization of the synthesised silver nanoparticles. Synthesized AgNPs were spherical and cuboid in shape which is determined by SEM. Average size of the AgNPs were between 80-98, 60-95 and 34-70 nm for PzAgNPs, SaAgNPs and TaAgNPs, respectively. Further, the synthesized AgNPs were characterized by XRD, EDX, DLS and Zeta potential analysis. Moreover, the synthesized AgNPs exhibited a dose-dependent cytotoxicity against human liver and prostate cancer cell lines. The inhibitory concentration (IC50) values of HepG2, PC3 and Vero cells were found to be 70.97, 58.61, 96.41; 10.04, 42.77, 83.86; and 28.42, 41.78, 69.48 μg/ml for PzAgNPs, SaAgNPs and TaAgNPs at 48 h incubation. An induction of apoptosis was confirmed by DNA fragmentation, Hoechst, Rhodamine and AO/EtBr staining. The present results strongly suggested that the AgNPs synthesized using P. zeylanica, S. anacardium and T. arjuna extracts showed potential anticancer activity of HepG2 and PC3 cell lines.

Green coating of metal and metal oxide nanomaterials is currently recognized as an eco-friendly route to magnify their biological efficacy and reduce risks due to low biocompatibility. In this study, bio-fabricated metallic silver nanoparticles (AgNPs) were synthesized using three medicinal plant extracts, i.e. Eclipta prostrata, Moringa oleifera and Thespesia populnea and then tested for their cytotoxic activity against human prostate (PC3) and liver (HepG2) cancer cell lines. The green fabricated AgNPs were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, zeta potential analysis, dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Biofabricated AgNPs exhibited dose-dependent increase in cell toxicity on human prostate cancer, liver cancer and African monkey kidney cell lines. IC50 values of PC3, HepG2 and Vero cells varied depending upon the source used for nanoparticle synthesis. DNA fragmentation, Hoechst, rhodamine and AO/EtBr staining assays confirmed nano-triggered apoptosis of treated cells. The main achievement of this study is that nanofabrication routes relying to E. prostrata, M. oleifera and T. populnea medicinal plant extracts to fabricate Ag nanocomplex within 2 h duration can represent a novel cancer nanodrug and effective way to boost their anticancer efficacy.

This study determined the bioactive compounds and evaluated the antioxidant, antimicrobial, and cytotoxicity activities of Bidens pilosa aqueous and ethanolic extracts against bacterial and fungal pathogens and various human cancer cell lines. The B. pilosa leaves were extracted using ethanol and water as solvents. The aqueous (BA) and ethanolic (BE) extracts of B. pilosa were subjected to qualitative phytochemical screening. The phosphomolybdate method was used to determine the total antioxidant activity of the extracts. The antimicrobial activity of the extracts against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida tropicalis was determined using the Kirby-Bauer method. Lastly, the brine shrimp lethality test (BSLT) and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay in the human breast, colon, and liver cancer cell lines were used to evaluate the cytotoxicity activities of the more potent extract. Steroids, flavonoids, tannins, saponins, alkaloids, and cyanogenic glycosides were present in B. pilosa leaf extracts. BE showed higher antioxidant and antimicrobial activities than BA. However, BSLT results showed BA was more toxic than BE based on their lethal concentration 50 (LC50) values after 24- hour exposure. MTT assay also showed that BA was more potent against the colon cancer (HCT116) cell line, followed by breast (MCF-7) and liver (Hep G2) cancer cell lines. The B. pilosa extracts possess potent antioxidant, antimicrobial, and anticancer activities. These biological activities are due to the presence of various natural products present in its leaves. Further investigations are needed to understand the pharmacological properties of B. pilosa fully.

A novel series of nitrostyrene-based spirooxindoles were synthesized via the reaction of substituted isatins 1a-b, a number of α-amino acids 2a-e and (E)-2-aryl-1-nitroethenes 3a-e in a chemo/regio-selective manner using [3+2] cycloaddition (Huisgen) reaction under microwave irradiation conditions. The structure elucidation of all the synthesized spirooxindoles were done using 1H and 13C NMR and HRMS spectral analysis. The single crystal X-ray crystallographic study of compound 4l was used to assign the stereochemical arrangements of the groups around the pyrrolidine ring in spiro[pyrrolidine-2,3'-oxindoles] skeleton. The in vitro anticancer activity of spiro[pyrrolidine-2,3'-oxindoles] analogs 4a-w against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines shows promising results. Out of the 23 synthesized spiro[pyrrolidine-2,3'-oxindoles], while five compounds (4c, 4f, 4m, 4q, 4t) (IC50 = 34.99-47.92 µM; SI = 0.96-2.43) displayed significant in vitro anticancer activity against human lung (A549) cancer cell lines, six compounds (4c, 4f, 4k, 4m, 4q, 4t) (IC50 = 41.56-86.53 µM; SI = 0.49-0.99) displayed promising in vitro anticancer activity against human liver (HepG2) cancer cell lines. In the case of lung (A549) cancer cell lines, these compounds were recognized to be more efficient and selective than standard reference artemisinin (IC50 = 100 µM) and chloroquine (IC50 = 100 µM; SI: 0.03). However, none of them were found to be active as compared to artesunic acid [IC50 = 9.85 µM; SI = 0.76 against lung (A549) cancer cell line and IC50 = 4.09 µM; SI = 2.01 against liver (HepG2) cancer cell line].

Novel N , N ‐disubstituted benzimidazolium salts were efficaciously synthesized in moderate to high yields and identified via 1 H NMR and 13 C NMR analyses. These compounds were tested on human liver cancer, prostate cancer, and normal embryonic kidney cell lines for 72 h. The results demonstrated that these compounds had antiproliferative activity. In particular, it was found that one of the compounds, 1‐(3‐chlorobenzyl)‐3‐(3‐methylbenzyl)‐1 H ‐benzo[ d ]imidazol‐3‐ium chloride, showed very high activity against liver cancer cell line and the IC 50 value of this compound was almost twice as low as the IC 50 value of cisplatin. The anticancer activity potential of the compounds was explored through computational methods to support the experimental study results. The binding potential of the compounds to human sulfotransferase 1A1 (SULT1A1) was investigated through molecular docking and molecular dynamics simulation. Their electrochemical properties were computed via density functional theory. The molecular docking study exhibited that 1‐(3‐methylbenzyl)‐3‐(4‐nitrobenzyl)‐1 H ‐benzo[ d ]imidazol‐3‐ium chloride had the highest potential to bind to SULT1A1. The molecular dynamics study showed that the synthesized compounds formed a stable complex. Furthermore, the density functional theory study exhibited that 1‐(3‐chlorobenzyl)‐3‐(4‐fluorobenzyl)‐1 H ‐benzo[ d ]imidazol‐3‐ium chloride might have the highest chemical stability.

An accumulating body of evidence reports the synthesis and biomedical applications of silver nanoparticles. However, the studies regarding the use of maleic acid and citric acid in the synthesis of nano-sized silver particles (AgNPs) and micro-sized silver particles (AgMPs) as well as their antibacterial, antifungal, and anticancer activities have not been reported. In the current study, we synthesized AgNPs and AgMPs using maleic acid and citric acid as capping agents and have characterized them by UV-Vis, energy-dispersive X-Ray spectroscopy (EDS), X-Ray diffraction (XRD), and scanning electron microscope (SEM) analysis. The capped silver particles were examined for their antimicrobial activity and cytotoxicity against bacteria, fungi, and brine shrimp. Additionally, the anticancer activity of these particles was tested against human breast and liver cancer cell lines. The free radical scavenging activity of capped silver particles was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. SEM analysis revealed a round plate-like morphology of maleic acid capped particles with an average size of 39 ± 4 nm, whereas citric acid capped particles display flower-shaped morphology with rough surfaces and an average size of 250 ± 5 nm. The uncapped AgMPs were hexagonal with 500 ± 4 nm size. EDS and XRD analysis confirmed the presence of Ag and face-centered cubic crystalline nature, respectively. Functionally, capped silver particles exhibited antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus) and Gram-negative bacteria (Salmonella setubal, Enterobacter aerogenes, and Agrobacterium tumefaciens). The bactericidal activity was more active against Gram-negative bacteria with minimum inhibitory concentration (MIC) as low as 5 ppm as compared to 25 ppm for Gram-positive. Similarly, the silver particles demonstrated antifungal activity by inhibiting the growth of five fungal strains (Mucor species, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, and Fusarium solani) up to 50% at the concentration of 500 ppm. Additionally, these particles showed substantial toxicity against brine shrimp and also significantly inhibited the proliferation of breast cancer (MCF7) and liver cancer (HePG2) cell lines (IC50 8.9–18.56 µM). Uncapped AgMPs were less effective, inhibiting only the proliferation of MCF7 cells with IC50 46.54 µM. Besides cytotoxicity, these particles acted as potential antioxidants, showing free radical scavenging up to 74.4% in a concentration-dependent manner. Taken together, our results showed that the modifiers affect the shape and size of silver particles and may, in part, contribute to the antimicrobial and antioxidant activity of silver particles. However, the contribution of maleic acid and citric acid in enhancing the antimicrobial, anticancer, and antioxidant potential independent of silver nano and microparticles needs to be studied further. In vivo experiments may determine the therapeutic effectiveness of silver particles capped with these modifiers.

Hepatocellular carcinoma (HCC) most prevalent form of liver cancer and poses a few available treatments and is a major worldwide health burden. Monacolin X, a natural compound derived from the marine sponge-associated symbiont Monascus ruber, has garnered attention for its potential anticancer and anti-angiogenesis properties. This current study aimed to investigate the anticancer and apoptosis-inducing effects of Monacolin X against the human liver cancer (HepG2) cell line invitro. This present study utilized various assays to assess cytotoxicity by MTT assay, apoptosis induction, DCFH-DA staining to measure the intracellular ROS levels, and apoptosis-and inflammation-related gene expression changes induced by the Monacolin X. The MTT results discovered dose-dependent cytotoxicity in HepG2 cells with an IC50 value of 72.4 μM. The apoptosis-inducing effect of Monacolin X was evident through propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) staining, accompanied by increased intracellular ROS levels and downregulated expression of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and modulation of key apoptosis regulators (Bax and Bcl-2) as determined by qPCR analysis. In conclusion, these observations suggest a mechanism whereby Monacolin X has potent anticancer activity against the HepG2 cell line, and further investigation will be required to determine the molecular pathways responsible for the potential therapeutic effects for the clinical implications in liver cancer.

Titanium (IV)–dithiophenolate complex chitosan nanocomposites (DBT–CSNPs) are featured by their antibacterial activities, cytotoxicity, and capacity to bind with DNA helixes. In this study, their therapeutic effects against rat liver damage induced by carbon tetrachloride (CCl4) and their anti-proliferative activity against human liver cancer (HepG2) cell lines were determined. Results of treatment were compared with cisplatin treatment. Markers of apoptosis, oxidative stress, liver functions, and liver histopathology were determined. The results showed that DBT–CSNPs and DBT treatments abolished liver damage induced by CCl4 and improved liver architecture and functions. DNA fragmentation, Bax, and caspase-8 were reduced, but Bcl-2 and the Bcl-2/Bax ratios were increased. However, there was a non-significant change in the oxidative stress markers. DBT–CSNPs and DBT inhibited the proliferation of HepG2 cells by arresting cells in the G2/M phase and inducing cell death. DBT–CSNPs were more efficient than DBT. Low doses of DBT and DBT–CSNPs applied to healthy rats for 14 days had no adverse effect. DBT and DBT–CSNP treatment gave preferable results than the treatment with cisplatin. In conclusion, DBT–CSNPs and DBT have anti-apoptotic activities against liver injuries and have anti-neoplastic impacts. DBT–CSNPs are more efficient. Both compounds can be used in pharmacological fields.

Disclosure: P. Ramaraj: None. Our previous in-vitro studies involving vitamins (vitamin-A and vitamin-D3) and steroids (progesterone and RU-486) pointed to a combination of vitamin-A and RU-486 as an efficient combo to decrease human melanoma cell growth. We extended this combo treatment to human liver cancer cell (Hep-G2) line. The aim was to check whether vit-A and RU-486 combo would also decrease human liver cancer cell line growth. Liver cancer (Hep-G2) cells were plated in a 96 well plate overnight. Following day, vit-A and RU-486 were added either alone or in different combinations for 48 hrs. After 48 hrs, cell viability and percentage of cell growth were monitored by the MTT assay. Supernatants were collected for Elisarray and quantitation of cytokine(s) by Elisa. Individual vit-A and RU-486 treatments showed a dose-dependent decrease in cell growth. Though various combinations of vit-A (50, 75, 100 μM) and RU-486 (10, 50 100 μM) also showed a decrease in cell growth, an experimentally significant decrease in cell growth was observed at vit-A 75 μM and RU-486 50 μM combination. This combo resulted in 24% cell growth compared to straight vit-A (75 μM) at 44% cell growth and RU-486 (50 μM) at 35% cell growth. Thus, this combo inhibited liver cancer cell growth also. In the future effect of this combo on normal liver cell line and the cytokine(s) if any, playing a central role in cell growth would be identified by Elisarray and quantitated by Elisa. Presentation: 6/3/2024