Colorectal Adenocarcinoma Cells Research Articles

Pan tumor outcomes to immune checkpoint inhibitors in patients of Native American origin in the United States: The multicenter POINT-US study.

2654 Background: Despite receiving identical standard-of-care therapies, epidemiological data indicate that Native American (NA) patients (pts) with cancer have worse outcomes vs. White pts. ICIs have revolutionized the treatment of certain tumors, however pts from certain racial and ethnic groups, including NA pts, are underrepresented in ICI trials. Methods: This is an ongoing multi-center retrospective study across four U.S. academic cancer centers that serve a high volume of NA pts. We included pts with self-reported race as NA in electronic medical records. We evaluated clinical outcomes and immune-related adverse events (irAEs) of 71 NA pts age ≥18 years with various solid tumors treated with ICI-based therapies between 2015 to 2021. IrAEs were defined based on the Common Terminology Criteria for Adverse Events (version 5.0) or RECIST 1.1. as available, was used to determine objective response rate (ORR). Kaplan Meier log-rank method was used to generate median progression free survival (mPFS) and median overall survival (mOS). Results: Median age (range) at ICI initiation was 62 (24 -81) years; 43 (60%) were males, with 26 (37%) females, 2 pts with missing gender data. 55 (77%) of pts had ECOG performance status 0. The distribution of top six tumor types included (number of pts [%]): non-small cell lung cancer (NSCLC) 22 (31%), renal cell carcinoma (RCC) 16 (23%), melanoma 7 (10%), small cell lung cancer (SCLC) 4 (6%), hepatocellular carcinoma 4(6%); colorectal adenocarcinoma, head and neck squamous cell carcinoma, cervical squamous cell carcinoma each had 3 pts (4%). Most pts (51 [72%]) received single agent ICI, 12 (17%) had dual ICI, 4 (6%) had chemotherapy+ ICI, and remaining had either ICI + tyrosine kinase inhibitor or ICI + study drug (4 [6%]). 55% of pts with NSCLC underwent tumor next-generation sequencing (NGS) while <30% of patients with renal cell carcinoma or melanoma had tumor NGS performed. Among 71 pts, 17 (24%) had any grade (G) irAE, 4 (6%) were reported to be ≥G3. The median time to irAE onset was 18 weeks and 3 (4%) pts discontinued ICI due to irAEs. The mPFS, mOS and ORR for the most frequent tumors (N ≥5) are shown. Conclusions: Although limited by sample size, our emerging data provides new insights into ICI efficacy and irAEs in NA pts that have been unknown to date. To our knowledge, this is the first and only attempt to date evaluating ICI outcomes in NA pts. Updated clinical data and tumor genomics will be presented at the meeting. [Table: see text]

Formulating co-loaded nanoliposomes with gallic acid and quercetin for enhanced cancer therapy

Cancer is considered one of the top global causes of death. Natural products have been used in oncology medicine either in crude form or by utilizing isolated secondary metabolites. Biologically active phytomolecules such as gallic acid and quercetin have confirmed antioxidant, anti-bacterial, and neoplastic properties. There is an agreement that microorganisms could mediate oncogenesis or alter the immune system. This research project aims to develop a novel formulation of co-loaded gallic acid and quercetin into nanoliposomes and investigate the efficacy of the free and combined agents against multiple cancerous cell lines and bacterial strains. Thin-film hydration technique was adopted to synthesize the nanocarriers. Particle characteristics were measured using a Zetasizer. The morphology of nanoliposomes was examined by scanning electron microscopy, Encapsulation efficiency and drug loading were evaluated using High-Performance Liquid Chromatography. Cytotoxicity was determined against Breast Cancer Cells MCF-7, Human Carcinoma Cells HT-29, and A549 Lung Cancer Cells. The antibacterial activities were evaluated against Acinetobacter baumannii, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Therapeutic formulas were categorized into groups: free gallic acid, free quercetin, free-mix, and their nano-counterparts. Findings revealed that drug loading capacity was 0.204 for the mix formula compared to 0.092 and 0.68 for free gallic acid and quercetin, respectively. Regarding the Zeta potential, the mix formula showed more amphiphilic charge than the free quercetin and free gallic acid formulas (P-values 0.003 and 0.002 receptively). On the contrary, no significant difference in polydispersity indices was reported. Lung cancerous cells were the most affected by the treatments. The best estimated IC50 values were observed in breast and lung cancer lines for the nano-gallic acid and co-loaded particles. The nano-quercetin formula exhibited the least cytotoxicity with an IC50 value of ≥200 μg/mL in both breast (MCF-7) and colorectal adenocarcinoma cell lines (HT-29) with no activity against the lung. A remarkable improvement in the efficacy of quercetin was measured after mixing it with gallic acid against the breast and lungs. The tested therapeutic agents exhibited antimicrobial activity against gram-positive bacteria. Nano-liposomes can either enhance or reduce the cytotoxicity activity of active compounds depending on the physical and chemical properties of drug-loaded and type of cancer cells.

Toxic or beneficial? What is the role of food‐relevant selenium species selenoneine?

Toxic or beneficial? What is the role of food‐relevant selenium species selenoneine?

Optimization of parameters for stir-frying of Kansui Radix with vinegar based on conversion of toxic components

This study aims to optimize the parameters for stir-frying of Kansui Radix with vinegar based on the conversion of representative toxic diterpenes, which is expected to serve as a reference for the standardized production of Kansui Radix stir-fried with vinegar. To be specific, the toxic components [3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol(3-O-EZ), kansuiphorin C(KPC)] in Kansui Radix and the products(ingenol, 20-deoxyingenol) after the stir-frying with vinegar were selected. The toxicity to intestine and water-draining activity were evaluated with NCM460(normal human colon mucosal epithelial cell line) and HT-29(a human colorectal adenocarcinoma cell line). An HPLC method was then developed to assess the conversion of toxic components. On this basis, temperature, time, and amount of vinegar for the processing of Kansui Radix were optimized with the Box-Behnken design and the content of ingenol and 20-deoxyingenol as evaluation index. The results showed that after the stir-frying of Kansui Radix with vinegar, 3-O-EZ and KPC were first converted to monoester 3-O-(2'E,4'Z-decadienoyl)ingenol(3-EZ) and 5-O-benzoyl-20-deoxyingenol(5-O-Ben) and finally to almost non-toxic ingenol and 20-deoxyingenol, respectively. Meanwhile, the water-draining activity was retained. Six compounds had a good linear relationship with the peak area in the corresponding concentration ranges(R~2≥0.999 8), and the average recovery fell in the range of 98.20%-102.3%(RSD≤2.4%). The content of representative diterpenes and intermediate products was 14.78%-24.67% lower in the Kansui Radix stir-fried with vinegar than in the Kansui Radix, while the content of the conversed products was 14.37%-71.37% higher. Among the process parameters, temperature had significant influence on the total content of products, followed by time. The optimal parameters were 210 ℃, 15 min, and 30% vinegar. The relative error between the experimental results and the predicted values was 1.68%, indicating that the process was stable and reproducible. The strategy of screening optimal parameters for stir-frying of Kansui Radix with vinegar based on the transformation of toxic components can help improve the production stability, reduce the toxicity, and ensure the efficacy of Kansui Radix stir-fried with vinegar, which can serve as a reference for the process optimization of similar toxic Chinese medicinals.

"Cytotoxic Activity of Some Water Soluble Curcumin Conjugates on Human Colorectal Adenocarcinoma and Human Hepatocellular Carcinoma Cells"

"Cytotoxic Activity of Some Water Soluble Curcumin Conjugates on Human Colorectal Adenocarcinoma and Human Hepatocellular Carcinoma Cells"

The biochemical characteristics of viable but nonculturable state Yersinia enterocolitica induced by lactic acid stress and its presence in food systems

The viable but nonculturable (VBNC) state is adopted by many foodborne pathogenic bacteria to survive in adverse conditions. This study found that lactic acid, a widely used food preservative, can induce Yersinia enterocolitica to enter a VBNC state. Y. enterocolitica treated with 2 mg/mL lactic acid completely lost culturability within 20 min, and 10.137 ± 1.693 % of the cells entered a VBNC state. VBNC state cells could be recovered (resuscitated) in tryptic soy broth (TSB), 5 % (v/v) Tween80−TSB, and 2 mg/mL sodium pyruvate-TSB. In the VBNC state of Y. enterocolitica induced by lactic acid, the intracellular adenosine triphosphate (ATP) concentration and various enzyme activities were decreased, and the reactive oxygen species (ROS) level was elevated, compared with uninduced cells. The VBNC state cells were significantly more resistant to heat and simulated gastric fluid than uninduced cells, but their ability to survive in a high−osmotic−pressure environment was significantly less than that of uninduced cells. The VBNC state cells induced by lactic acid changed from long rod-like to short rod-like, with small vacuoles at the cell edges; the genetic material was loosened and the density of cytoplasm was increased. The VBNC state cells had decreased ability to adhere to and invade Caco−2 (human colorectal adenocarcinoma) cells. The transcription levels of genes related to adhesion, invasion, motility, and resistance to adverse environmental stress were downregulated in VBNC state cells relative to uninduced cells. In meat-based broth, all nine tested strains of Y. enterocolitica entered the VBNC state after lactic acid treatment; among these strains, only VBNC state cells of Y. enterocolitica CMCC 52207 and Isolate 36 could not be recovered. Therefore, this study is a wake−up call for food safety problems caused by VBNC state pathogens induced by lactic acid.

Germination and Simulated Gastrointestinal Digestion of Chickpea (Cicer arietinum L.) in Exhibiting In Vitro Antioxidant Activity in Gastrointestinal Epithelial Cells.

Plant-based proteins, in particular pulse proteins, have grown in popularity worldwide. Germination, or sprouting, is an effective method to release peptides and other dietary compounds. However, the combination of germination and gastrointestinal digestion in enhancing the release of dietary compounds with potential health-beneficial biological activity has yet to be entirely elucidated. The present study illustrates the impact of germination and gastrointestinal digestion on the release of dietary compounds with antioxidant activity from chickpeas (Cicer arietinum L.). Germination up to 3 days (D0 to D3) increased the peptide content by denaturing chickpea storage proteins and increased the degree of hydrolysis (DH) in the gastric phase. The antioxidant activity was measured at three different dosages (10, 50, and 100 μg/mL) and compared between D0 and D3 on human colorectal adenocarcinoma cells (HT-29). A significant increase in antioxidant activity was observed in the D3 germinated samples in all three tested dosages. Further analysis identified 10 peptides and 7 phytochemicals differentially expressed between the D0 and D3 germinated samples. Among the differentially expressed compounds, 3 phytochemicals (2',4'-dihydroxy-3,4-dimethoxychalcone, isoliquiritigenin 4-methyl ether, and 3-methoxy-4,2',5'-trihydroxychalcone) and 1 peptide (His-Ala-Lys) were identified only in the D3 samples, indicating their potential contribution towards the observed antioxidant activity.

Optimization of process factor and characterization of vinegar-like beverage production via spontaneous fermentation from pineapple peel waste

A feasible approach to compensating for pineapple wastage is by utilizing the waste. In the present study, pineapple peels were subjected to spontaneous fermentation to produce a vinegar-like beverage. Based on the central composite design (CCD) approach, optimization of the process factor recorded a maximum total acid yield, Yp/s, and total acidity of 0.49 and 3.03%, respectively. Furthermore, the beverage possessed increased tartaric, citric, ascorbic, acetic, and ferulic acids at a maximum of 1.196%. The cytotoxicity activity toward the human colorectal adenocarcinoma cell line documented a half maximal inhibitory concentration (IC50) at 3.4% v/v of the beverage. This study showcased optimized vinegar-like beverage production by indigenous microorganisms (IMO) with pineapple peel. The beverage contained improved organic and phenolic acids contents and antioxidant potential, which could be employed as a possible human colorectal cancer cure.

The potential of dietary nanoparticles to enhance allergenicity of milk proteins: an in vitro investigation.

In recent years, the popularity of dietary nanoparticles (NPs) in the food industry as additives has raised concerns because of the lack of knowledge about potential adverse health outcomes ensuing from the interactions of NPs with components of the food matrix and gastrointestinal system. In this study, we used a transwell culture system that consisted of human colorectal adenocarcinoma (Caco-2) cells in the apical insert and Laboratory of Allergic Diseases 2 mast cells in the basal compartment to study the effect of NPs on milk allergen delivery across the epithelial layer, mast cell responses and signaling between epithelial and mast cells in allergenic inflammation. A library of dietary particles (silicon dioxide NPs, titanium dioxide NPs and silver NPs) that varied in particle size, surface chemistry and crystal structures with or without pre-exposure to milk was used in this investigation. Milk-interacted particles were found to acquire surface corona and increased the bioavailability of milk allergens (casein and β-lactoglobulin) across the intestinal epithelial layer. The signaling between epithelial cells and mast cells resulted in significant changes in the early phase and late-phase activation of the mast cells. This study suggested that antigen challenge in mast cells with the presence of dietary NPs may cause the transition of allergic responses from an immunoglobulin E(IgE)-dependent mechanism to a mixed mechanism (both IgE-dependent and IgE-independent mechanisms).

Biotransformation of white and black mustard grains through germination and enzymatic hydrolysis revealed important metabolites for antioxidant properties and cytotoxic activity against Caco-2 cells

Germination and enzymatic hydrolysis are biological processes with well-recognized positive effects on phenolic composition and antioxidant potential. This study aimed to apply those processes to white (Sinapsis alba) and black (Brassica nigra) mustard grains and to analyze the influences on the total phenolic content (TPC); phenolic and peptide profile determined by ultra-performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS); antioxidant potential (DPPH, ABTS, and FRAP assays); and cytotoxicity against Caco-2, a human colorectal adenocarcinoma cell line. Enzyme combinations for hydrolysis were different for each mustard grain, but for both species, enzymatic hydrolysis and germination showed a positive effect on antioxidant properties. From UPLC-HRMS analysis and molecular network studies, 14 peptides and 17 phenolic compounds were identified as metabolites released from mustard after processes application, which were strongly correlated with increased antioxidant activity. In addition, enzymatic hydrolysis applied in germinated mustard grains for both mustards increased the cytotoxic activity against Caco-2 human colorectal adenocarcinoma cell line.

Effectiveness and Durability of a Quaternary Ammonium Compounds-Based Surface Coating to Reduce Surface Contamination.

Foodborne diseases are of major concern as they have a significant impact on public health, both socially and economically. The occurrence of cross-contamination of food in household kitchens is a serious threat and the adoption of safe food practices is of paramount importance. This work aimed to study the effectiveness and durability of a commercial quaternary ammonium compound-based surface coating which, according to the manufacturer, retains its antimicrobial activity for 30 days, and is suitable for all types of hard surfaces for the prevention and/or control of cross-contamination. For that, its antimicrobial efficacy, killing contact time and durability on three different surfaces-polyvinyl chloride, glass, and stainless-steel-against three pathogens-Escherichia coli ATCC 25922, Acinetobacter baumannii ESB260 and Listeria monocytogenes Scott A-were tested according to the current antimicrobial treated surfaces efficacy test (ISO22196:2011). The results showed that the antimicrobial coating was effective against all pathogens with a reduction of >5.0 log CFU/cm2 in less than one minute for the three surfaces, but its durability was less than one week on all surfaces cleaned in the usual manner. Additionally, trace amounts (≤0.2 mg/kg) of the antimicrobial coating, which may migrate into food when contacting the surface, did not show cytotoxicity to human colorectal adenocarcinoma cells. The suggested antimicrobial coating has the potential to significantly reduce surface contamination, ensure surface disinfection and reduce the likelihood of cross-contamination in domestic kitchens, although it is less durable than suggested. The use of this technology in household settings is an attractive complement to the existing cleaning protocols and solutions that are already in place.

Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy.

In recent years, receptor-mediated drug delivery has gained major attention in the treatment of cancer. The pathogen-derived Shiga Toxin B subunit (STxB) can be used as a carrier that detects the tumor-associated glycosphingolipid globotriaosylceramide (Gb3) receptors. While drug conjugation via lysine or cysteine offers random drug attachment to carriers, click chemistry has the potential to improve the engineering of delivery systems as the site specificity can eliminate interference with the active binding site of tumor ligands. We present the production of recombinant STxB in its wild-type (STxBwt) version or incorporating the noncanonical amino acid azido lysine (STxBAzK). The STxBwt and STxBAzK were manufactured using a growth-decoupled Escherichia coli (E. coli)-based expression strain and analyzed via flow cytometry for Gb3 receptor recognition and specificity on two human colorectal adenocarcinoma cell lines-HT-29 and LS-174-characterized by high and low Gb3 abundance, respectively. Furthermore, STxBAzK was clicked to the antineoplastic agent monomethyl auristatin E (MMAE) and evaluated in cell-killing assays for its ability to deliver the drug to Gb3-expressing tumor cells. The STxBAzK-MMAE conjugate induced uptake and release of the MMAE drug in Gb3-positive tumor cells, reaching 94% of HT-29 cell elimination at 72 h post-treatment and low nanomolar doses while sparing LS-174 cells. STxBAzK is therefore presented as a well-functioning drug carrier, with a possible application in cancer therapy. This research demonstrates the feasibility of lectin carriers used in delivering drugs to tumor cells, with prospects for improved cancer therapy in terms of straightforward drug attachment and effective cancer cell elimination.

Proliferation Inhibitory Activity of Quinones from Blaps rynchopetera Defense Secretion on Colorectal Tumor Cells.

To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines. Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively. MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC50) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC50 values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC50 values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G1 phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells. Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.

Physicochemical characterization and cancer cell antiproliferative effect of silver-doped magnesia nanoparticles

Silver-doped magnesia nanoparticles (Ag/MgO) were synthesized using the precipitation method and characterized by various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), Brunner-Emmett-Teller (BET) surface area measurements, and dispersive X-ray spectroscopy (EDX). The morphology of Ag/MgO nanoparticles was determined by transmission and scanning electron microscopy, which revealed cuboidal shaped nanoparticles with sizes ranging from 31 to 68 nm and an average size of 43.5 ± 10.6 nm. The anticancer effects of Ag/MgO nanoparticles were evaluated on human colorectal (HT29) and lung adenocarcinoma (A549) cell lines, and their caspase-3, -8, and -9 activities, as well as Bcl-2, Bax, p53, cytochrome C protein expressions were estimated. Ag/MgO nanoparticles showed selective toxicity towards HT29 and A549 cells while remaining relatively innocuous towards the normal human colorectal, CCD-18Co, and lung, MRC-5 cells. The IC50 values of Ag/MgO nanoparticles on the HT29 and A549 cells were found to be 90.2 ± 2.6 and 85.0 ± 3.5 μg/mL, respectively. The Ag/MgO nanoparticles upregulated caspase-3 and -9 activities, downregulated Bcl-2, upregulated Bax and p53 protein expressions in the cancer cells. The morphology of the Ag/MgO nanoparticle treated HT29 and A549 cells was typical of apoptosis, with cell detachment, shrinkage, and membrane blebbing. The results suggest that Ag/MgO nanoparticles induce apoptosis in cancer cells and exhibit potential as a promising anticancer agent.

In Vitro Cytotoxic Potential of Selected Jordanian Flora and Their Associated Phytochemical Analysis.

Traditional medicines are a significant source of phytochemicals with potential anticancer effects. Ten Jordanian plants were chosen to be tested for cytotoxicity on human colorectal (HT-29) and breast adenocarcinoma (MCF-7) cell lines. The ethanol extracts were screened for their potential cytotoxic effects using a Sulforhodamine B (SRB) colorimetric assay, using doxorubicin as positive control. Plants extracts exhibiting marked cytotoxic activity were further investigated by qualitative and quantitative phytochemical methods. Total phenolics were quantified using the Folin-Ciocalteu reagent, while flavonoids were quantified using aluminum chloride. The total saponins of the n-butanol fraction were estimated using diosgenin as a standard. The total alkaloids and total terpenoids were also evaluated using the gravimetric method. As results, Senecio leucanthemifolius (IC50: 13.84 μg/mL) and Clematis cirrhosa (IC50: 13.28 μg/mL) exhibited marked cytotoxic effects on human colorectal adenocarcinoma (HT-29) cell lines. Total phenolics, flavonoids, saponins, alkaloids, and terpenoids found in Senecio leucanthemifolius were (91.82, 14.90, 14.27, 101, and 135.4 mg/g of dry extract), respectively. They were revealed to be (68.18, 7.16, 31.25, 73.6, and 180 mg/g of dry extract) in Clematis cirrhosa, respectively. Senecio leucanthemifolius and Clematis cirrhosa have been found to possess cytotoxicity against colorectal (HT-29). In conclusion, the findings of this study offer a new perspective on Jordanian plant extracts anticancer activity research.

Studies on the Anticancer and Antioxidant Activities of Resveratrol and Long-Chain Fatty Acid Esters.

Resveratrol (RES) is gaining recognition as a natural bioactive compound. To expand the possible applications of RES with its enhanced bioactivity as well as to increase the health benefits of long-chain fatty acids, a lipophilization process of RES was performed using three fatty acids: palmitic acid (PA), oleic acid (OA), and conjugated linoleic acid (CLA). The obtained mono-, di-, and tri-esters of RES were evaluated for their anticancer and antioxidant properties against lung carcinoma (A549), colorectal adenocarcinoma (HT29), and pancreatic ductal adenocarcinoma (BxPC3) cell lines. Human fibroblast (BJ) cells were used as a control. Several parameters were investigated: cell viability and apoptosis, including the expression of major pro- and anti-apoptotic markers, as well as the expression of superoxide dismutase, a key enzyme of the body's antioxidant barrier. Three of the obtained esters: mono-RES-OA, mono-RES-CLA, and tri-RES-PA, which significantly reduced the tumor cell viability up to 23%, at concentrations 25, 10, 50 μg/mL, respectively, turned out to be particularly interesting. The above-mentioned resveratrol derivatives similarly increased the tumor cells' apoptosis by modifying their caspase activity of pro-apoptotic pathways (p21, p53, and Bax). Moreover, among the mentioned esters, mono-RES-OA induced apoptosis of the analyzed cell lines most strongly, reducing the number of viable cells up to 48% for HT29 cells versus 36% for pure RES. Furthermore, the selected esters exhibited antioxidant properties towards the normal BJ cell line by regulating the expression of major pro-antioxidant genes (superoxide dismutases-SOD1 and SOD2) without the effect on their expression in the tumor, and therefore reducing the defense of cancer cells against increased oxidative stress induced by high ROS accumulation. The obtained results indicate that the esters of RES and long-chain fatty acids allow enhancement of their biological activity. The RES derivatives have the potential for being applied in cancer prevention and treatment, as well as for oxidative stress suppression.

Hydroxyanthracene derivates citotoxicity: A differential evaluation between single molecule and whole plant extract.

Hydroxyanthracene derivates (HADs) are a group of natural or synthetic compounds with a wide range of biological activities (for instance, anti-inflammatory, antibacterial, and antiarthritic). In addition, because of their properties for helping the normal bowel function, HADs are widely used in constipation as pharmacological drugs and nutritional supplements. Nevertheless, during the past years, a safety usage of HAD products has been under consideration because some studies reported that HADs are not lacking toxicity (i.e., genotoxic and carcinogenic activity). Thus, the first objective of this study is to shed light on the large variability in composition of botanical food supplements containing HAD by a systematic analysis of the qualitative and quantitative composition of a cohort of extracts and raw materials of plants with high levels of anthraquinones commercially available (Cassia angustifolia, Rhamnus purshiana, Rhamnus frangula, Rheum palmatum, and Rheum raponticum). To date, the investigation of HAD toxicity was based on in vitro and in vivo studies conducted mainly on the use of the single molecules (emodin, aloe-emodin, and rhein) rather than on the whole plant extract. The qualitative-quantitative characterization was the starting point to select the most appropriate products to be used as treatment for our in vitro cell studies. Thus, the second objective of this study is the investigation, for the first time, of the toxic events of HAD used as single molecule in comparison with the whole plant extracts containing HAD in an intestinal in vitro model using human colorectal adenocarcinoma cells (Caco-2). In addition, a shotgun proteomics approach was applied to profile the differential protein expression in the Caco-2 cells after a single-HAD or whole-plant extract treatment to fully understand the potential targets and signaling pathways. In conclusion, the combination of a detailed phytochemical characterization of HAD products and a largely accurate analysis of the proteomic profile of intestinal cells treated with HAD products provided the opportunity to investigate their effects in the intestinal system.

Abstract 4119: Anti-KRAS antibody aggregates KRAS in the cytoplasm of live ex vivo cultured human colorectal adenocarcinoma cells and prevents its trafficking to the plasma membrane

Abstract Worldwide, colorectal cancer (CRC) is the third highest incidence cancer and a leading cause of cancer mortality. Metastasis to distal organ is the major cause of cancer mortality. However, chemotherapeutic treatment of metastatic CRC has a dismayed success rate of less than 30%. Patients who are resistant to chemotherapy and who are KRAS wildtype are sometimes offered anti-EGFR as a second line therapy. Nevertheless, only about ½ of these patients respond and those who do inevitably develop resistance within months due to selection for downstream KRAS mutations. Moreover, most (80%) sporadic CRCs are microsatellite-stable and are refractory to immune checkpoint blockade therapy. Hence, there is an unmet need to identify novel therapeutics for CRC. KRAS is a gatekeeper gene in colorectal tumorigenesis but is ‘undruggable’ due to its structure not being easily amenable to inhibitor docking. Focus has been diverted to develop small molecule inhibitors for its downstream effectors such as ERK/MAPK and AKT. Despite intense research efforts for the past few decades, however, no small molecule inhibitor has been in clinical use for CRC. Recently developed Sotorasib inhibitor specific for KRAS G12C mutations (rare in CRC) is not effective for CRC. Antibodies have high affinity to their target proteins without having to bind to specific pocket and are fully human in nature and hence less toxic. Antibody targeting KRAS itself is thus an attractive alternative. We developed a transient ex vivo patient-derived matched mucosa-tumor primary culture to assess whether anti-KRAS antibody can be internalized to bind and inactivate KRAS. We showed that anti-KRAS antibody can enter live matched mucosa-tumor cells and specifically aggregate KRAS in the cytoplasm, thus hindering its trafficking to the inner plasma membrane. The mis-localization of KRAS reduces KRAS dwelling time at the site where it tethers to activate downstream effectors. We previously showed that expression of SOX9 was KRAS-mutation-dependent and possibly a better effector than ERK in CRC. In the present study, we showed that anti-KRAS antibody treated tumor cells have less intense SOX9 cytoplasmic and nuclear staining compared to untreated cells suggesting down-regulation of KRAS signaling. Our results demonstrated that anti-KRAS antibody can be internalized and specifically inhibits KRAS function in live tumor cells. With an efficient intracellular antibody delivery system, this can be further developed as combinatorial therapeutics for CRC and other KRAS-driven cancers. Citation Format: Peh Yean Cheah, Kuen Kuen Lam, Yee Syuen Low, Michelle Lo, Michelle Wong, Choong Leong Tang, Emile Tan, Aik Yong Chok, Issac Seow En, Siew Heng Wong. Anti-KRAS antibody aggregates KRAS in the cytoplasm of live ex vivo cultured human colorectal adenocarcinoma cells and prevents its trafficking to the plasma membrane. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4119.

Green Synthesized Gold and Silver Nanoparticles Increased Oxidative Stress and Induced Cell Death in Colorectal Adenocarcinoma Cells.

The research investigated the effect of gold (Au-CM) and silver nanoparticles (Ag-CM) phytoreduced with Cornus mas fruit extract (CM) on a human colorectal adenocarcinoma (DLD-1) cell line. The impact of nanoparticles on the viability of DLD-1 tumor cells and normal cells was evaluated. Oxidative stress and cell death mechanisms (annexin/propidium iodide analysis, caspase-3 and caspase-8 levels, p53, BCL-2, BAX, NFkB expressions) as well as proliferation markers (Ki-67, PCNA and MAPK) were evaluated in tumor cells. The nanoparticles were characterized using UV-Vis spectroscopy and transmission electron microscopy (TEM) and by measuring zeta potential, hydrodynamic diameter and polydispersity index (PDI). Energy dispersive X-ray (EDX) and X-ray powder diffraction (XRD) analyses were also performed. The nanoparticles induced apoptosis and necrosis of DLD-1 cells and reduced cell proliferation, especially Ag-CM, while on normal cells, both nanoparticles maintained their viability up to 80%. Ag-CM and Au-CM increased the expressions of p53 and NFkB in parallel with the downregulation of BCL-2 protein and induced the activation of caspase-8, suggesting the involvement of apoptosis in cell death. Lipid peroxidation triggered by Ag-CM was correlated with tumor cell necrosis rate. Both nanoparticles obtained with phytocompounds from the CM extract protected normal cells and induced the death of DLD-1 tumor cells, especially by apoptosis.

Phytochemical Analysis, In vitro Assessment of Antioxidant Properties and Cytotoxic Potential of Thymus capitatus Essential Oil

Essential oils (EOs) of medicinal plants became a matter of interest in scientific research because of their potential of utilization for several therapeutic purposes, The EO of Thymus capitatus has been traditionally used in treatment of several conditions related to skin, muscles, and respiratory system, to reduce fever, and to stimulate menstrual flow in addition to its anti-bacterial properties. To chemically analyze the components of the EO of Thymus capitatus and to evaluate its anti-cancerous and anti-oxidant activity on breast cancer cells (T47D), colorectal adenocarcinoma cells (CACO2), and normal human fibroblasts (MRC5). The EOs were extracted by hydro-distillation. Gas chromatography and mass spectrometry were used to analyze the extracts. Cell viability was assessed using three different assays; MTT, trypan blue and neutral red assays. Antioxidant activity was assessed using DPPH scavenging assay. Cell apoptosis was assessed using DNA fragmentation tests and Annexin V-propidium iodide assay. Morphological analysis of cells was assessed using light microscopy and DAPI staining. Twenty-two different compounds were identified in the essential oil of T. capitatus leaves including Thymol (44.34%) and Carvacrol (38.89%) in addition to 20 minor components. The oil extract exhibited a significant inhibitory action on the three cell lines. The viability assays showed significantly higher (p<0.05) IC50 in the fibroblast cell line than the other two cell lines. The selectivity index was higher in T47D cell line compared to CACO II cell line. For apoptosis assessment, DNA fragmentation in T47D cells was significantly correlated with the extract concentration. The membrane polarity was disrupted in an increasing manner (15.60±1.13, 35.73±1.69 and 46.53±2.51) as the extract concentration (20.22, 40.45, and 80.9µg/ml, respectively) increased. On the cellular level, morphological changes including cell shrinkage, chromatin condensation and nuclear fragmentation in addition to marginalization of chromatin, vacuolization of cytoplasm and formation of micronuclei was clearly visible. The extract showed high anti-oxidant activity with an IC50 of 6.4µg/ml. Anti-oxidant activity was dose-dependent; double IC50 (80.9µg/ml) exhibited the highest antioxidant activity whereas the lowest antioxidant activity was observed for the ½ IC50 (20.225µg/ml). The oil extract exerted an inhibitory action lipid peroxidation. Thymus capitatus EO is a potential anti-oxidant and anti-neoplastic agent against breast and colon carcinomas due to its high selectivity for cancer cells compared to normal cell lines.