Colon Cancer Cell Line HCT116 Research Articles

Combinatorial Inhibition of Complement Factor D and BCL2 for Early-Onset Colorectal Cancer.

The tumor immune microenvironment is distinct between early-onset and late-onset colorectal cancer, which facilitates tumor progression. We previously identified several genes, including complement factor D, as having increased expression in patients with early-onset colorectal cancer. This study aimed to assess and validate the differential expression of immune genes in early-onset and late-onset colorectal cancer. We also aimed to test known drugs targeting genes increased in early-onset colorectal cancer in preclinical mouse models. A retrospective cohort study with analysis was performed using tumor RNA from formalin-fixed paraffin-embedded cell culture and immunohistochemistry to validate gene expression and function and in vivo preclinical tumor study to assess drug efficacy. The Oregon Colorectal Cancer Registry was queried to identify patients with colorectal cancer. The study included 67 patients with early-onset colorectal cancer and 54 patients with late-onset colorectal cancer. Preclinical animal models using the HCT-116 colon cancer cell line were treated with the complement factor D inhibitor danicopan and the BCL2 inhibitor venetoclax, or with vehicle controls. Elevated RNA signatures using NanoString data were evaluated by the retrospective cohort. When inhibiting these markers in the mouse preclinical model, tumor volume and weight were the main outcome measures. After updating our sample size from our previously published data, we found that complement factor D and BCL2, genes with known function and small molecule inhibitors, are elevated in patients with early-onset colorectal cancer. When inhibiting these markers with the drugs danicopan and venetoclax in a mouse model, we found that the combination of these drugs decreased tumor burden but also resulted in toxicity. This study is limited by a small sample size and a subcutaneous tumor model. Combinatorial inhibition of early-onset associated genes complement factor D and BCL2 slows the growth of early-onset colorectal cancer in a mouse preclinical model. See Video Abstract . ANTECEDENTES:El microambiente inmunológico del tumor es distinto entre el cáncer colorrectal de aparición temprana y el de aparición tardía, lo que facilita la progresión de dicho tumor. Anteriormente identificamos varios genes, incluidos el factor D-Complementario, con una mayor expresión en pacientes con cáncer colorrectal de aparición temprana.OBJETIVO:El presente estudio tuvo como objetivo el evaluar y validar la expresión diferenciada de genes inmunes en casos de cáncer colorrectal de aparición temprana y tardía. También nos propusimos evaluar los fármacos conocidos dirigidos sobre los genes aumentados en el cáncer colorrectal de aparición temprana en modelos pre-clínicos en ratones.DISEÑO:Estudio de cohortes con análisis retrospectivo utilizando el ARN tumoral procedente de cultivos celulares fijados con formalina e incluidos en parafina, y el analisis por inmunohistoquímica para validar la expresión y la función genética. Se realizó el estudio pre-clínico de los tumores in vivo para evaluar la eficacia de los fármacos.AJUSTES:Se consultó el Registro de Oregon de casos de Cáncer Colorrectal para encontrar los pacientes afectados.SUJETOS:67 pacientes con cáncer colorrectal de aparición temprana y 54 pacientes con cáncer colorrectal de aparición tardía.INTERVENCIONES (SI LAS HUBIESE):Los modelos animales pre-clínicos que utilizaron la línea celular de cáncer de colon HCT-116 se trataron con el inhibidor del factor D-Complementario o Danicopan y con el inhibidor de BCL-2 o Venetoclax, ambos con control del transportador.PRINCIPALES MEDIDAS DE RESULTADO:Se evaluaron las firmas de ARN elevadas utilizando los datos del NanoString a partir de la cohorte retrospectiva. Al inhibir estos marcadores del modelo pre-clínico en los ratones, el volumen y el peso del tumor fueron las principales medidas de resultado.RESULTADOS:Después de actualizar el tamaño de nuestra muestra a partir de datos publicados con anterioridad, encontramos que el factor D-Complementario y BCL-2, genes con función conocida e inhibidores de moléculas pequeñas, se encuentran elevados en aquellos pacientes con cáncer colorrectal de aparición temprana. Al inhibir estos marcadores con los medicamentos Danicopan y Venetoclax en el modelo de ratones vivos, encontramos que la combinación de estos dos farmacos disminuyó la carga tumoral pero también produjo toxicidad.LIMITACIONES:Estudio limitado por un tamaño de muestra pequeño y el modelo de tumor subcutáneo.CONCLUSIONES:La inhibición combinada de genes asociados de aparición temprana, el factor D-Complementario y el BCL-2, enlentecen el crecimiento del cáncer colorrectal de aparición temprana del modelo preclínico en ratones. (Traducción-Dr. Xavier Delgadillo ).

Novel 1,2,4-triazole-maleamic acid derivatives: synthesis and evaluation as anticancer agents with carbonic anhydrase inhibitory activity

Hybrid compounds with 1,2,4-triazole ring and a hydrazone moiety were designed and synthesized by amine acylation of uncommon triaminogunidine derivatives using maleic anhydride. Synthesized compounds were characterized by various spectral techniques, including FTIR, 1H NMR, 13C NMR, and HRMS. Furthermore, the proposed structure of TM-2 was resolved by single-crystal X-ray analysis. The compounds were evaluated for their anticancer activity in vitro against the colon cancer cell line HCT116 and the ovarian cancer cell line A2780. Among them, some compounds, TM-3, TM-4, and TM-14, exhibited remarkable antiproliferative activity in the cell line A2780, with IC50 values of 6.11, 5.15, and 5.93 µM, respectively. Further investigation revealed that these compounds could inhibit cancerous cell growth by inducing an increase in caspase-3 activity. In addition, antioxidant capabilities, interaction with plasmid pBR322 DNA, and inhibitory effects against two physiologically and pharmacologically relevant human carbonic anhydrase (hCA) isoforms, namely hCA I and hCA II, of the synthesized compounds were also assessed. Finally, computational studies, such as DFT calculation, molecular docking, and in silico predictions of ADME/T analysis, were conducted to gain an understanding of the specific reactive sites of the compounds, the binding modes in the active sites, and the pharmacokinetic parameters of the newly synthesized hybrid compounds, which were calculated to establish their drug-likeness properties.

Design, synthesis and characterization of novel 1,5- and 2,5-coumarin-4-yl-methyl regioisomers of 5-pyrazol-3-yl-tetrazoles as promising anticancer and antifungal agents

A novel series of compounds was synthesized using 3-arylsydnone 1a-b. Initially, 3-arylsydnone was converted into 1-aryl-1H-pyrazole-3-carbonitriles 2a-bvia [3+2] cycloaddition with acrylonitrile by ring transformation which once again underwent [2+3] cycloaddition with sodium azide to afford 5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles 3a-b.These were subjected to N-alkylation with 4-bromomethyl coumarins 4c-e to afford regioisomers viz., 2,5- and 1,5-disubstituted tetrazole derivatives 5f-k and 6f-k. Single crystal X-ray analysis of compound 6f was carried out. All the synthesized compounds were characterized using various techniques including 1H NMR, 13C NMR, FT-IR, elemental analysis, and mass spectroscopy.These previously unknown compounds were screened for their anticancer activity against HCT-116 colon cancer and non-cancerous L929 cell line at a single high dose (10−5 M) concentration assay. Among the tested compounds, 6g and 5f have exhibited excellent activity with very low IC50 valuei.e.,27.85 µg/mLand 37.86 µg/mL respectively, comparable with standard Cisplatin. In the non-cancerous L929 cell line, the tested compound 6f was proven to be less toxic, and all the tested compounds exhibited toxicity at higher concentrations comparable with standard Cisplatin. Further, the in vitro antifungal activity against Candida albicans for these compounds 5f-k and 6f-k revealed potential to moderate activities compared to the standard. Moreover, docking analysis was performed to know the exact binding mode of the newly synthesized compounds with N-myristoyltransferase (PDB ID: 1IYL) from Candida albicanswhich was selected as the antifungal targets. The drug-likeness of the compound was assessed using Molinspiration, while the toxicity profile was evaluated using Protox.

Gfi-1 modulates HMGB1-Mediated autophagy to overcome oxaliplatin resistance in colorectal cancer

BackgroundResistance to oxaliplatin (L-OHP) is a major barrier in the treatment of colorectal cancer (CRC). Autophagy is the main cause of L-OHP tolerance in CRC cells. MethodThe human colon cancer cell lines HCT116 and SW480 were treated with L-OHP to obtain the drug-resistant cell lines HCT116/L-OHP and SW480/L-OHP, respectively. To probe the relationship between autophagy and L-OHP tolerance of growth factor independent 1 (Gfi-1) and high-mobility group protein 1 (HMGB1) in CRC cells, gene knockout or overexpression was performed, and Western blotting was used to determine the levels of drug tolerance interrelated proteins. Transwell and CCK-8 assays were employed to analyze the proliferation of cancer cells. Immunofluorescence detection of LC3 reflected autophagy levels. Finally, the relationship between Gfi-1 and HMGB1 was detected by chromatin immunoprecipitation (ChIP). ResultCompared to normal CRC cells, L-OHP-tolerant CRC cells exhibited greater autophagy (8.2 times greater in HCT116/L-OHP cells and 7.4 times greater in SW480/L-OHP cells). In addition, we detected low levels of Gfi-1 (0.6-fold for HCT116/L-OHP cells and 0.4-fold for SW480/L-OHP cells), and OE-Gfi-1 decreased HMGB1 levels (0.6-fold for HCT116/L-OHP + OE-Gfi-1 cells and 0.5-fold for SW480/L-OHP + OE-Gfi-1 cells). The inhibition of Gfi-1 further enhanced cell viability (1.7 times in HCT116+sh-Gfi-1 cells and 1.2 times in SW480+sh-Gfi-1 cells) and invasion (1.8 times in HCT116+sh-Gfi-1 cells and 2.1 times in SW480+sh-Gfi-1 cells) in CRC cells, thus promoting oxaliplatin resistance in these cells. The autophagy inhibitor 3-MA reversed the above effects. Furthermore, we noted that Gfi-1 can restrain HMGB1 expression by binding to its promoter (0.5 times in HCT116+OE-Gfi-1 cells and 0.5 times in SW480+OE-Gfi-1 cells). The inhibitory influence of 3-MA on HMGB1 reversed the influence of Gfi-1 on autophagy and malignant progression in CRC cells. ConclusionOur study suggested that Gfi-1 inhibited HMGB1 to reduce CRC autophagy levels, increasing CRC sensitivity to L-OHP.

NXPH4 can be used as a biomarker for pan-cancer and promotes colon cancer progression.

NXPH4 promotes cancer proliferation and invasion. However, its specific role and mechanism in cancer remain unclear. Transcriptome and clinical data for pan-cancer were derived from the TCGA database. K-M survival curve and univariate Cox were used for prognostic analysis. CIBERSORT and TIMER algorithms were employed to calculate immune cell infiltration. Gene set enrichment analysis (GSEA) was employed for investigating the function of NXPH4. Western blot verified differential expression of NXPH4 in colon cancer. Functional assays (CCK-8, plate clonogenicity assay, wound healing assay, and Transwell assay) confirmed the impact of NXPH4 on proliferation, invasion, and migration of colon cancer cells. Dysregulation of NXPH4 in pan-cancer suggests its potential as a diagnostic and prognostic marker for certain cancers, including colon and liver cancer. High expression of NXPH4 in pan-cancer might be associated with the increase in copy number and hypomethylation. NXPH4 expression in pan-cancer is substantially linked to immune cell infiltration in the immune microenvironment. NXPH4 expression is associated with the susceptibility to immunotherapy and chemotherapy. Western blot further confirmed the higher expression of NXPH4 in colon cancer. Knockdown of NXPH4 significantly suppresses proliferation, invasion, and migration of colon cancer cell lines HT-29 and HCT116, as validated by functional assays.

Abstract LB322: ARC2-001, a CAF-targeting drug exhibits strong anticancer effect in in vivo setting

Abstract For more effective tumor growth suppression, a combinatorial treatment of CAF-targeting agent and other well-validated therapies such as chemotherapy, radiotherapy, and immune checkpoint therapy should be considered. Thus, we hypothesized that co-administration of the CAF-targeting drug, ARC2-001 (the in vitro-approved CAF-targeting candidate) and clinically practiced anticancer drugs could be useful strategy for treating under clinical settings. However, it is essentially important to evaluate this using well-established pre-clinical models. We thoroughly verified the anticancer effect of ARC-001 using two in vivo model systems. ( 2nd- in vivo systems for CAF targeted drugs Research). 1. (Subcutaneous model) Immune-deficient mice (NOD/SCID): in which human colon cancer cell line HCT116 was co-injected with human CAF (CAFs were obtained from stage IV CRC patients) followed by administration of ARC2-001 alone or in combination with 5-fluorouracil (5FU). 2. (Subcutaneous and Orthotopic model) Wild-type mice (C57BL/6): in which mouse colon cancer cells was co-injected with mouse colon CAFs (both cancer cells and CAFs were obtained from chemically-induced colorectal cancer model with rich desmoplastic stroma) followed by administration of ARC2-001 alone or in combination with aPD-1. We observed a magnificent inhibition of tumor growth and metastasis in the combined treatment group over singular treatment groups in both models. This denotes that ARC2-001 largely augments the anti-cancer effect of widely employed anticancer drugs, such as 5FU, and thus can be exploited as an adjuvant for enhanced colorectal cancer chemotherapy.Collectively, our results evidenced the necessity of considering CAF-targeting agents for a more regulated therapeutic strategy to treat desmoplastic cancers. Citation Format: So-Young Yeo, Mohamed Farh, InSuk Sohn, Seok-Hyung Kim, Keun-Woo Lee. ARC2-001, a CAF-targeting drug exhibits strong anticancer effect in in vivo setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB322.

DNA-DEPENDENT PROTEIN KINASE INHIBITOR INDUCES APOPTOSIS IN COLON CANCER CELLS

Objectives: Among all types of DNA damage, DNA double-strand breaks (DSBs) are considered the most deleterious form induced by either endogenous factors (oxi-dative damages, mismatches, altered chromatin structures, and missing, or modified nucleotides) or exogenous factors, i.e., ultraviolet (UV) radiation, ionizing radiation (IR), and chemicals or drugs. DNA-dependent protein kinase (DNA-PK) plays a crucial role in repairing DSBs through non-homologous end joining (NHEJ). Cells lacking DNA-PK exhibit heightened sensitivity to IR and various DNA-damaging agents. The inhibition of DNA-PK further intensifies cellular susceptibility to IR and DNA-damaging agents. Several small molecules that inhibit DNA-PK have been developed. This study aimed to evaluate the effect of DNA-PK inhibitor (DNA-PKi) NU7441 on the HCT116 cell line. Methods: DNA-PKi NU7441 was used to assess the effect on anti-proliferation and induction of apoptosis on the HCT116 colorectal cancer cell line. Cells were cultured under standard conditions; crystal violet and apoptosis assay were applied to evaluate cell proliferation and apoptosis. Data were analysed using GraphPad Prism 8.4. Results: DNA-PKi effectively inhibited HCT116 colon cancer cell growth via crystal violet assay (p < 0.01). In addition, DNA-PKi also induced programmed cell death in the HCT116 cell line (p < 0.05). Conclusion: DNA-PKi NU7441 suppressed cell proliferation and induced apoptosis in the HCT116 colon cancer cell line.

Engineering 5-flourouracil and leucovorin-loaded vesicular systems for possible colon specific delivery: In vitro evaluation and real time cell assay against HCT-116 colon cell lines

5-flourouracil (5-FU) is typically modulated with leucovorin (LEU) in clinical practice to improve clinical efficacy and patient survival rates. However, this combination has undesirable side effects and makes 5-FU more toxic. Hence, integrating a vesicular system (proniosomes) with another delivery vehicle may improve drug targeting, while resolving the aforementioned drawbacks. This study aimed to engineer 5-FU/LEU proniosomes for possible delivery to the colon. The modified slurry approach was used to create drug-loaded proniosomes (150 mg/9 g of carrier) using both water-soluble (dextrin (DEX) and lactose (LAC)) and insoluble (Neusilin FH2 (NEU)) carriers. The powdered formulations were filled into Eudragit S100 (10 %)-coated capsules or Eudragit FS 30D capsules for enteric- or colon-specific delivery. In vitro evaluations (flow properties, powder X-ray diffractometry (XRD) analysis, particle size analysis, entrapment efficiency, drug release, scanning electron microscopy (SEM), polydispersity index, Fourier transform infrared spectroscopy (FTIR), and stability studies) were performed on the formulations. An in vitro cytotoxicity test [real-time cell assay (RTCA)] against HCT-116 colon cancer cell lines was performed using the optimized formulation. In vitro evaluations showed that the nanoparticles had good physicochemical properties. RTCA studies showed sustained cell death with the formulations compared to the pure drug and placebo. The sequential drug release of the colon-targeted capsules containing 5-FU and LEU- loaded proniosomes showed negligible drug release in SGF (pH 1.2) and phosphate buffer solution (pH 6.8) (approximately 11 %) but profound drug release (>80 %) at pH 7.4. Drug-loaded proniosomes engineered for colon targeting (Eudragit S100 (10 %) capsules or Eudragit FS 30D capsules) showed good colon-specific targeting and favorable in vitro cytotoxicity profiles.

Exploring the antibiofilm effects on Escherichia coli biofilm associated with colon cancer and anticancer activities on HCT116 cell line of bee products

The association between dysbiotic microbiota biofilm and colon cancer has recently begun to attract attention. In the study, the apitherapeutic effects of bee products (honey, bee venom, royal jelly, pollen, perga and propolis) obtained from the endemic Yığılca ecotype of Apis mellifera anatoliaca were investigated. Antibiofilm activity were performed by microplate assay using crystal violet staining to measure adherent biofilm biomass of Escherichia coli capable of forming biofilms. Bee venom showed the highest inhibition effect (73.98%) at 50% concentration. Honey, perga and royal jelly reduced biofilm formation by >50% at all concentrations. The antiproliferation effect on the HCT116 colon cancer cell line was investigated with the water‑soluble tetrazolium salt‑1 assay. After 48 h of honey application at 50% concentration, cell proliferation decreased by 86.51%. The high cytotoxic effects of royal jelly and bee venom are also remarkable. Additionally, apoptotic pathway analysis was performed by ELISA using caspase 3, 8 and 9 enzyme-linked immunosorbent assay kits. All bee products induced a higher expression of caspase 9 compared with caspase 8. Natural products that upregulate caspase proteins are promising therapeutic targets for proliferative diseases.

CACNA2D1 regulates the progression and influences the microenvironment of colon cancer.

Calcium voltage-gated channel auxiliary subunit alpha 2/delta 1 (CACNA2D1), a gene encoding a voltage-gated calcium channel, has been reported as an oncogene in several cancers. However, its role in colon cancer (CC) remains unclear. This study aimed to investigate the function of CACNA2D1 and its effect on the microenvironment in CC. Immunohistochemistry (IHC) analysis was performed on samples collected from 200 patients with CC who underwent curative colectomy. Knockdown experiments were performed using CACNA2D1 siRNA in the human CC cell lines HCT116 and RKO, and cell proliferation, cycle, apoptosis, and migration were then analyzed. The fibroblast cell line CCD-18Co was co-cultured with CC cell lines to determine the effect of CACNA2D1 on fibroblasts and the relationship between CACNA2D1 and the cancer microenvironment. Gene expression profiles of cells were analyzed using microarray analysis. IHC revealed that high CACNA2D1 expression was an independent poor prognostic factor in patients with CC and that CACNA2D1 expression and the stroma are correlated. CACNA2D1 depletion decreased cell proliferation and migration; CACNA2D1 knockdown increased the number of cells in the sub-G1 phase and induced apoptosis. CCD-18Co and HCT116 or RKO cell co-culture revealed that CACNA2D1 affects the cancer microenvironment via fibroblast regulation. Furthermore, microarray analysis showed that the p53 signaling pathway and epithelial-mesenchymal transition-associated pathways were enhanced in CACNA2D1-depleted HCT116 cells. CACNA2D1 plays an important role in the progression and the microenvironment of CC by regulating fibroblasts and may act as a biomarker for disease progression and a therapeutic target for CC.

R-phycoerythrin alginate/shellac beads by external gelation: Process optimization and the effects of gastrointestinal digestion for nutraceutical applications

This study aimed to evaluate an alginate/shellac mixture as wall material to develop an aqueous phycoerythrin (R-PE) encapsulation system by external gelation and to determine the effect of encapsulation on the biological properties of R-PE released during simulated gastrointestinal digestion. The Taguchi method was implemented to formulate beads with a high R-PE encapsulation efficiency (EE). The effect of the variables: feeding flow (90 and 20 mL h−1), distance (5 and 10 cms), and CaCl2 (5 and 15 g L−1) were optimized, and the bead size, sphericity factor (SF), and total R-PE content were characterized. Finally, the optimal alginate/shellac beads were subjected to in vitro dynamic gastrointestinal digestion. The results show that the beads formed under optimal conditions reached an EE value of 97.5 %. The CaCl2 concentration and feeding flow most affected the R-PE EE. The release of R-PE from alginate/shellac was affected at acid pH 1; however, the concentration was under 10 % of the total R-PE content showing promising targeting properties in terms of time and pH. The R-PE extracts and capsules were partially degraded in gastric and intestinal conditions; the mouth did not detect signals from the protein profile. The encapsulation of alginate/shellac led to higher R-PE contents at the end of digestion than non-encapsulated R-PE, suggesting a protective role. Significantly, from permeate streams, equivalent to the absorption of encapsulated R-PE, the bioavailability was 2.5 times higher than non-encapsulated R-PE. NMR results indicate the presence of R-PE and its methyl amino acids and oligosaccharides between 0.5 and 2.5 and 3.8–6.8 ppm, respectively. Cytotoxicity activity was observed for the R-PE extract on the HCT-116 human colon cancer cell line. The alginate/shellac as a wall material and ionic gelation technology used may determine the release of the R-PE pigment at an intestinal site and its antiproliferative effect on health.

MITF regulates the subcellular location of HIF1α through SUMOylation to promote the invasion and metastasis of daughter cells derived from polyploid giant cancer cells.

High concentrations of cobalt chloride (CoCl2) can induce the formation of polyploid giant cancer cells (PGCCs) in various tumors, which can produce daughter cells with strong proliferative, migratory and invasive abilities via asymmetric division. To study the role of hypoxia‑inducible factor (HIF) 1α in the formation of PGCCs, colon cancer cell lines Hct116 and LoVo were used as experimental subjects. Western blotting, nuclear and cytoplasmic protein extraction and immunocytochemical experiments were used to compare the changes in the expression and subcellular localization of HIF1α, microphthalmia‑associated transcription factor (MITF), protein inhibitor of activated STAT protein4 (PIAS4) and von Hippel‑Lindau disease tumor suppressor (VHL) after treatment with CoCl2. The SUMOylation of HIFα was verified by co‑immunoprecipitation assay. After inhibiting HIF1α SUMOylation, the changes in proliferation, migration and invasion abilities of Hct116 and LoVo were compared by plate colony formation, wound healing and Transwell migration and invasion. In addition, lysine sites that led to SUMOylation of HIF1α were identified through site mutation experiments. The results showed that CoCl2 can induce the formation of PGCCs with the expression level of HIF1α higher in treated cells than in control cells. HIF1α was primarily located in the cytoplasm of control cell. Following CoCl2 treatment, the subcellular localization of HIF1α was primarily in the nuclei of PGCCs with daughter cells (PDCs). After treatment with SUMOylation inhibitors, the nuclear HIF1α expression in PDCs decreased. Furthermore, their proliferation, migration and invasion abilities also decreased. After inhibiting the expression of MITF, the expression of HIF1α decreased. MITF can regulate HIF1α SUMOylation. Expression and subcellular localization of VHL and HIF1α did not change following PIAS4 knockdown. SUMOylation of HIF1α occurs at the amino acid sites K391 and K477 in PDCs. After mutation of the two sites, nuclear expression of HIF1α in PDCs was reduced, along with a significant reduction in the proliferation, migration and invasion abilities. In conclusion, the post‑translation modification regulated the subcellular location of HIF1α and the nuclear expression of HIF1α promoted the proliferation, migration and invasion abilities of PDCs. MITF could regulate the transcription and protein levels of HIF1α and participate in the regulation of HIF1α SUMOylation.

Green synthesis of silver nanoparticles using Illicium verum extract: Optimization and characterization for biomedical applications

Abstract The synthesis of noble metal nanoparticles is currently experiencing substantial development and considerable attention. Plant extracts are commonly used for the biological synthesis of nanoparticles because they contain biologically active constituents. In our present study, silver nanoparticles (AgNPs) were synthesized using an aqueous Illicium verum (Star anise) extract to evaluate their antimicrobial, antioxidant, and cytotoxicity activities. For maximum yields of AgNPs, the extract (2.5 ml), silver ions (500 µM), and pH (8) were shown to be the ideal nanoparticle production parameters. The visual colour shifted from pale brown to dark brown when the ultraviolet-visible spectrophotometer was used to validate the synthesis of AgNPs. A transmission electron microscope was utilized to evaluate nanoparticles’ physical nature. The presence of silver metal with face-centred cubic symmetry was confirmed by X-ray diffraction analysis. Fourier-transform infrared spectroscopy was used to identify the functional groups in charge of reducing silver ions (Ag+) and the stability of AgNPs produced using the I. verum aqueous extract. The agar well diffusion method investigated the antibacterial activity of I. verum silver nanoparticles (Iv-AgNPs) against pathogenic bacteria and fungi. At higher doses (100 µg·mL−1), the highest zone of inhibition was observed, and spherical AgNPs demonstrated the antibacterial activity. The I. verum extract and Iv-AgNPs enhanced (70%) their free radical scavenging activity at 500 µg·mL−1 according to the 2,2-diphenyl-1-picrylhydrazyl assay. Moreover, the cytotoxicity of Iv-AgNPs against the HCT-116 human colon cancer cell line indicated cell inhibition in a dose-dependent manner. Ultimately, the findings of this study indicate that techniques used to produce AgNPs are environmental friendly, cost-effective, harmless, uncomplicated, and can effectively tackle a broad spectrum of medical and nutritional concerns.

Seseli tortuosum L. subsp. tortuosum Essential Oils and Their Principal Constituents as Anticancer Agents.

Seseli tortuosum L. subsp. tortuosum, belonging to the Apiaceae family, is a species that grows in Europe, mainly in the Mediterranean regions. The history of its application in traditional medicine highlights its various biological properties. Trying to explore the phytochemistry and pharmacological aspects of this species, the essential oils (EOs) extracted from flowers, stems, and roots of a locally wild accession, never previously investigated, growing in Sicily, Italy, were investigated. The chemical composition of all EOs, obtained by the hydrodistillation method, was evaluated by GC-MS. The most abundant class of all investigated samples was that of monoterpene hydrocarbons (79.98-91.21%) with p-cymene, α-pinene, β-pinene, and β-ocimene as major compounds. These EOs, and their main components, were tested for their possible anticancer activity. Obtained data provided evidence that among the different EOs tested, at the dose of 100 μg/mL, those extracted from stems and roots were particularly effective, already at 24 h of treatment, in reducing the cell viability of 42% and 95%, respectively, in HCT116 colon cancer cell line. These EOs also exerted a remarkable cytotoxic effect that was accompanied by morphological changes represented by cell shrinkage as well as a reduction in residual cell population. Differently, modest effects were found when EOs extracted from flowers were tested in the same experimental conditions. The evaluation of the phytocompounds mainly represented in the EOs extracted from different parts of the plant and tested in a range of concentrations between 20 and 200 μg/mL, revealed that α-pinene, β-pinene, and p-cymene exerted only modest effects on cell viability. Differently, a remarkable effect was found when β-ocimene, the most abundant phytocomponent in EOs from roots, was tested on colon cancer cells. This phytocompound, among those identified in EOs from Seseli tortuosum L. subsp. tortuosum, was found to be the most effective in reducing colon cancer cell viability with IC50 = 64.52 μg/mL at 24 h of treatment. All together, these data suggest that β-ocimene could be responsible for the effects observed in colon cancer cells.

Exploring the Molecular Targets and Therapeutic Potential of Coptisine in Colon Cancer: A Network Pharmacology Approach.

Colon cancer is a frequent malignancy, and surgery is still the primary therapy for people with colon cancer. Other treatments, including radiation, chemotherapy, and biologic therapy, may be utilized as a supplement. Chemotherapy, a prominent treatment for colon cancer, has failed to provide positive outcomes. This necessitates the development of more effective and less harmful treatment drugs. Coptisine was discovered to inhibit the development of colon cancer cell line HCT-116 in vivo, decrease the growth of HCT-116 cells, and cause apoptosis in vitro in colon cancer. Coptisine (COP) has shown antitumor activity in colon cancer, but its molecular mechanism and its molecular targets have not been fully understood. In this study, the biological behavior was verified in vitro. The targets of Huanglian alkaloids on colon cancer were predicted, and the protein-protein interaction (PPI) network was constructed. The core targets of safranine for colon cancer were extracted and analyzed by GO and KEGG enrichment to identify the possible molecular mechanisms of safranine treatment. Western blot was used to detect the changes of related pathway proteins in colon cancer cells. The differential expression of hub genes in colon cancer was analyzed using the GEPIA2 website. The binding ability of safranine to the target was verified by molecular docking. Finally, the targets were preliminarily verified by q-PCR analysis. Coptisine can inhibit the survival, migration, and proliferation of colon cancer cells DLD1 and HCT-116. Based on network pharmacology, ninety-one targets for colon cancer were screened. ESR1, ALB, AR, CDK2, PARP1, HSP90AB1, IGF1R, CCNE1, and CDC42 were found in the top 10. Enrichment analysis showed that these targets were mainly related to pathways in cancer, FC γ R-mediated phagocytosis, prostate cancer, progesterone-mediated oocyte maturation, the oestrogen signal pathway, proteoglycan in cancer and the PI3K-Akt signal pathway. WB results showed that after the treatment of colon cancer DLD1 cells with coptisine, the expression of P-AKT and AKT decreased, that of its downstream protein Bcl-2 decreased, and that of BAX increased. Differential expression analysis of hub genes showed that CCNE1, CDK2, HSP90AB1, and CHEK2 were upregulated in colon cancer samples, and molecular docking showed that these targets had a good ability to bind to coptisine. After the treatment of colon cancer DLD1 cells with coptisine, q-PCR results showed that CCNE1 and HSP90AB1 were significantly downregulated, while CDK2 and CHEK2 had no significant changes. Coptisine may be a candidate drug for the treatment of colon cancer, and its therapeutic effect may be related to the cancer pathway and PI3K-Akt signalling pathway. CCNE1 and HSP90AB1 may be potential targets of coptisine in the treatment of colon cancer.

Synthesis, molecular docking study, and biological evaluation and of new thiadiazole and thiazole derivatives incorporating isoindoline-1,3-dione moiety as anticancer and antimicrobial agents

To highlight the importance of thiazole and thiadiazole derivatives in the progression of cancer and microbial treatments and to aid in drug design, we have synthesized a new series of 1,3,4-thiadiazole and 1,3-thiazole derivatives. These were created by a two-step reaction process: initially, 2-(4-(2-bromoacetyl)phenyl)isoindoline-1,3-dione was reacted with potassium thiocyanate, and then the resulting thiocyanate intermediate was coupled with aryl diazonium salts to produce iminothiadiazole derivatives. These derivatives served as crucial intermediates for further synthesizing a range of thiadiazole derivatives using different reagents. Additionally, treating 2-(4-(2-bromoacetyl) phenyl)isoindoline-1,3-dione with thiourea resulted in aminothiazole derivatives. These were further coupled with arenediazonium chloride to form 5-arylazoaminothiazole derivatives. All the synthesised compounds were characterised using IR, 1H NMR and 13C NMR spectrum data, as well as physical data. The assessment of the synthesized compounds on the HCT-116 human colon cancer cell line has yielded promising results. Specifically, compounds 3a, 3b, 4b, 5b, 6b, and 9b have shown noteworthy efficacy, suggesting their potential as anticancer agents. These compounds have demonstrated a greater potency compared to the standard drug, doxorubicin, highlighting their significance in cancer treatment research. The study assessed the activity of various newly synthesized compounds against diverse microorganisms, including bacteria and fungi. Notably, two of these compounds, specifically 4b and 7b, exhibited significant efficacy against both gram-positive and gram-negative bacteria, surpassing the performance of a standard antibacterial reference agent. Furthermore, molecular docking of new products revealed interactions with enzyme binding sites, aligning with in vitro findings. Additionally, in-silico studies confirmed their favourable oral bioavailability through ADME profiling.

Antimicrobial, Anticancer, and Antioxidant Activities of Maize and Clover Pollen Grains Extracts: A Comparative Study with Phytochemical Characterizations.

The failure to treat infectious diseases due to the continual emergence of drug-resistant microbes poses a huge and serious challenge for human health globally. Currently, the discovery and development of natural therapeutic compounds are attracting considerable attention from researchers worldwide. In this project, two types of pollen grains (maize and clover) were evaluated for potential antimicrobial activities. Extracts of both pollen grains were purified using HPLC, which has been shown to have numerous phenolic and flavonoid compounds. Pyro catechol and methyl gallate were detected in high concentrations (1145.56 and 1056.57 µg/mL, respectively) in the maize extract, while caffeic acid, quercetin, and kaempferol (464.73, 393.05, and 390.93 µg/mL, respectively) were among the compounds observed at high concentrations in the clover pollen grains extract. Staphylococcus aureus, Escherichia coli, Salmonella typhi, and Candida albicans were more sensitive to the clover pollen grains extract with inhibition zones of 22 ± 0.2, 18 ± 0.1, 29 ± 0.3, and 42 ± 0.4 mm compared to the size of the inhibitory zones caused by the maize pollen grains extract (19 ± 0.3, 15 ± 0.4, 27 ± 0.1, and 22 ± 0.4 mm, respectively). Moreover, lower MIC values for the clover pollen grains extract were recorded against C. albicans (1.97 ± 0.04 µg/mL), S. aureus (62.5 ± 1.00 µg/mL), and E. coli (62.5 ± 0.07 µg/mL) than the MICs caused by the maize pollen grains extract. The use of a transmission electron microscope revealed that the E. coli that had been treated with the clover pollen grains extract showed changes in its cell walls compared to that treated with the maize pollen grains extract. The clover pollen grains extract exhibited a stronger antioxidant potential, with an IC50 value of 22.18 µg/mL, compared to an IC50 value of 54.85 µg/mL for the maize pollen grains extract, via a DPPH scavenging assay. Regarding anticancer activity, the maize pollen grains extract was revealed to be more effective in terms of inhibiting the human colon cancer cell line HCT-116, with an IC50 value of 67.02 ± 1.37 µg/mL, compared with the observed toxicity caused by the clover extract, with an IC50 value of 75.03 ± 1.02 µg/mL. Overall, the clover pollen grains extract demonstrated potent antibacterial and antioxidant activities, but not anticancer activity, when compared to the maize grains extract. Thus, the current findings related to both types of pollen grains (clover and maize) highlight their potential therapeutic applications for the treatment of certain infectious diseases and malignancies.

An extract from the frass of swallowtail butterfly (Papilio machaon) larvae inhibits HCT116 colon cancer cell proliferation but not other cancer cell types

BackgroundThe frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system.ResultsThe components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we explored the metabolic enzyme candidates in A. keiskei-fed larvae by RNA-seq analysis. We found that the A. keiskei-fed larval midgut might have different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems, and we found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut.ConclusionsThese findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

Abstract C093: Unlocking the potential of Metformin: Revealing its impact on colorectal cancer cells at a molecular level

Abstract Metformin is a drug commonly used to treat type-2 diabetes, but recent studies suggest that it may also have anti-cancer effects by influencing cell cycle arrest and apoptosis. While previous studies have focused on metformin's effect on mTor, the downstream genetic pathways underlying its anti-proliferation mechanism remain unclear. Identifying these pathways may help predict patient response and allow for tailored treatment plans. This study aims to investigate the early genetic pathways involved in colon cancer using an in-vitro cell model and assess the impact of metformin on cell cycle and cancer proliferation in colon cancer cells, taking into account the p53/p21 status. Four different metformin concentrations were used on the HCT116 colon cancer cell line and its genetic modified forms. To determine cell viability and to measure cell cycle and apoptosis, MTT assay and flow cytometer were used. RNA-seq was performed on HCT116 after 24 hours of metformin treatment and pathway analysis was conducted using the Pathway Studio software. Results showed a significant decline in cell viability in response to metformin treatment, with an increase in the apoptosis phase. Transcriptomic analysis revealed that over 3,000 genes were significantly down-regulated, while 1,200 genes were up-regulated. Metformin promotes cell death and fragmentation at the SubG0 level, particularly in the case of p21 null, but p53 likely plays a primary role in driving apoptosis in colorectal cancer cells. In conclusion, metformin has potential in inhibiting proliferation and inducing apoptosis in colorectal cancer cells, affecting molecular pathways involved in the disease. However, further investigation through next-generation sequencing and molecular targeting is necessary to fully understand the disease at different levels. Citation Format: Jamila Hijazi, Pierre Khoueiry, Assaad Eid, Nadine Darwiche, Georges Nemer. Unlocking the potential of Metformin: Revealing its impact on colorectal cancer cells at a molecular level [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C093.

Abstract A114: Naturally occurring LAG3-blocking recombinant antibodies as a novel class of checkpoint inhibitors

Abstract LAG3 is a promising cancer immunotherapeutic target due to its negative regulatory role on T cells, NK cells, and B cells. Using our proprietary technology [US patent US9810694; 2017], we have isolated, sequenced, and pre-examined the potential immunological capability of fully human, naturally occurring anti-LAG3 antibodies. The best candidate, monoclonal antibody IG2 with molecular weight of 150 kDa and affinity of 4.4 nM, a complete recombinant reproduction of the antibody isolated from the human donor, was selected from 6 candidates for future research. In vitro allogeneic mixed lymphocyte reaction (MLR) assays using human T cells were performed to evaluate lymphocyte responses to stimulation with IG2 (5 μg/ml) or regular RPMI media alone. LAG3 blockade systematically resulted in an enhancement of CD3+ T cell proliferation by 45%. The main impact was on proliferation of CD4+ T cells with 50% increase. The count of CD8+ T cells also increased by 36%. Another assay evaluated IFNγ expression in NK92 cells treated with anti-LAG3 antibody. NK92 cells were starved without IL-2 for 24 hours in complete NK cell media and then cultivated with IG2 (5 μg/ml and 10 μg/ml) or media for 24 hours. Even short-term cultivation led to an increase in IFNg expression by 50%. Increasing the dose of the antibody did not affect the level of IFNγ expression. We did not observe any antibody dependent cellular cytotoxicity (ADCC) in co-cultures of NK92 cells with 786-O (renal), H358 (lung) or Hct116 (colon) cancer cell lines. Several studies demonstrate that NK cells can stimulate the expression of pro-imflammatory/pro-tumorigenic cytokines, IL-6 and IL-8, by tumor cells. Therefore, we evaluated the effects of IG2 on those genes’ expression induced by NK cells. Clear cell RCC cells (786-O) and NSCLC (H358) cell lines were co-cultured with NK92 cells for 2 hours and overnight with IG2 (1 mcg/ml) or media only. Co-cultivation of NK cells, cancer cells, and IG2 did not result in robust up-regulation of IL-6 and IL-8 by tumor cells. In order to evaluate the direct impact of the IG2 antibody on tumor cells, we incubated 786-O renal carcinoma cells with escalating concentrations of anti-LAG3 antibodies for 72 hours followed by Cell Titer Blue analysis when compared to un-treated cells (0 ug/ml). As expected, there was no direct cytotoxic effect of the antibody on tumor cells. Finally, assuming that novel naturally occurring antibodies may lead to fewer adverse events, we evaluated the preliminary toxicity in vivo. Eighteen mice were randomized in a 1:1 ratio to IG2 antibody group (1 mg/kg, intravenously, twice a week, 6 weeks) or to the untreated control group. The use of the antibody at this dose did not lead to the development of toxicity and a change in the weight of the animals compared to the control group. The results of the first studies demonstrate the potential activity and safety of a recombinant anti-LAG3 antibody reproduced from a human donor. Citation Format: Ilya Tsimafeyeu, Petr Makhov, Gennady Bratslavsky. Naturally occurring LAG3-blocking recombinant antibodies as a novel class of checkpoint inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A114.