HCT116 Human Colorectal Carcinoma Research Articles

Organic Moiety on Sn(IV) Does Matter for In Vitro Mode of Action: nBu3Sn(IV) Compounds with Carboxylato N-Functionalized 2-Quinolones Induce Anoikis-like Cell Death in A375 Cells

Objectives: New tributyltin(IV) complexes containing the carboxylate ligands 3-(4-methyl-2-oxoquinolin-1(2H)-yl)propanoic acid (HL1) and 2-(4-methyl-2-oxoquinolin-1(2H)-yl)acetic acid (HL2) have been synthesized. Methods: Their structures have been determined by elemental microanalysis, FT-IR and multinuclear NMR (1H, 13C and 119Sn) spectroscopy and X-ray diffraction study. A solution state NMR analysis reveals a four-coordinated tributyltin(IV) complex in non-polar solvents, while an X-Ray crystallographic analysis confirms a five-coordinated trigonal-bipyramidal geometry around the tin atom due to the formation of 1D chains. A theoretical structural analysis was performed by optimization employing B3LYP-D3BJ functional and 6-311++G(d,p)/def2-TZVP(Sn) basis sets for H, C, N, O/Sn, respectively. The interactions between tin(IV) and surrounding atoms were examined by QTAIM approach. The in vitro antiproliferative activity of the synthesized compounds was evaluated by MTT and CV assays versus MCF-7 (human breast adenocarcinoma), HCT116 (human colorectal carcinoma), A375 (human melanoma), 4T1 (mouse breast carcinoma), CT26 (mouse colon carcinoma) and B16 (mouse melanoma) tumor cell lines. Results: Both synthesized compounds (nBu3SnL1 and nBu3SnL2) exerted powerful micromolar IC50 cytotoxicity values and demonstrated high selectivity toward malignant cells. Both experimental drugs affected cell adhesion and induced anchorage independent apoptosis, a favorable type of cell death with an essential role in cancer dissemination prevention. The BSA-binding affinity of the obtained organotin compounds was followed by spectrofluorometric titration and molecular docking simulations. Conclusions: The tributyltin(IV) compounds selectively induce anoikis-like cell death in A375 cells, also highlighting the importance of the organic moiety on the tin(IV) ion in the mechanism of action.

Upregulation of TET2 and Resistance to DNA Methyltransferase (DNMT) Inhibitors in DNMT1-Deleted Cancer Cells.

Ten-eleven-translocation (TET) 2 is a member of the TET family of proteins (TET1-3). DNMT1 gene deletion confers resistance to DNA methyltransferase (DNMT) inhibitors in colorectal, breast, and ovarian cancer cells. Currently, the effect of DNMT1 gene status on TET2 phenotype following DNMT inhibitor treatment is unclear in human malignancies. Human colorectal carcinoma HCT116 cells (DNMT+/+) and their isogenic DNMT1 knockout (DNMT1-/-) counterpart were treated with DNMT inhibitors. Expression of TET2 and tumor suppressor (p16ink4A and p15ink4B) proteins were examined by Western blot. Apoptosis and CDKN2A promoter demethylation following drug treatment were detected by Annexin-V apoptosis assay and methylation-specific PCR. TET2 expression was robustly increased in DNMT1-/- cells by 0.5 µM and 5 µM decitabine and azacitidine treatment. Augmentation of TET2 expression was accompanied by re-expression of p16ink4A and p15ink4B proteins and CDKN2A promoter demethylation. TET2 upregulation and tumor suppressor re-expression were associated with resistance conferred by DNMT1 deletion. Treatment with 5-aza-4'-thio-2'-deoxycytidine at a low 0.5 µM dose only upregulated TET2 and reduced CDKN2A promoter methylation, and re-expression of p16ink4A in DNMT1-/- cells. DNMT inhibitors showed minimal effects on TET2 upregulation and re-expression of tumor suppressor proteins in cells with intact DNMT1. DNMT1 gene deletion made cancer cells prone to TET2 upregulation and activation of tumor suppressor expression upon DNMT inhibitor challenge. TET2 augmentation is concomitant with resistance to DNMT inhibitors in a DNMT1-deleted state.

Anti-angiogenic and anti-tumour activities of Lignosus rhinocerus (Cooke) Ryvarden water extracts on HCT116 human colorectal carcinoma cells implanted in chick embryos

Ethnopharmacological relevanceThe sclerotium of Lignosus rhinocerus (Cooke) Ryvarden is used by the local communities in Southeast Asia and China to treat cancer, asthma, fever, and other ailments based on traditional knowledge. The sclerotial water extracts were previously reported to exhibit cytotoxic, apoptotic, and immunomodulatory activities – providing a scientific basis for its use in treating cancer; however, there is still a lack of evidence on its potential anti-angiogenic activity. Aim of the studyThis study aimed to investigate the toxicity, anti-angiogenic, and anti-tumour activities of the hot-water and cold-water extracts of L. rhinocerus using HCT116 human colorectal carcinoma cells implanted in the chick chorioallantoic membrane (CAM) model. Materials and methodsThe toxicity of L. rhinocerus extracts towards the chick embryos was determined 24 h post-treatment. The anti-angiogenic activity of the extracts was then investigated at 0.1–10 μg/embryo (6.7–670 μg/mL) at targeted blood vessels. The anti-tumour effect of selected extracts against the HCT116 human colorectal carcinoma cells xenografted onto the chick embryos was also studied. ResultsThe cold-water extracts of L. rhinocerus displayed strong in ovo toxicity (LC50: 1.2–37.7 μg/mL) while the hot-water extracts are non-toxic up to 670 μg/mL. Among the extracts, the hot-water extracts demonstrated the highest anti-angiogenic activity with 44.0 ± 17.7% reduction of capillary diameter (relative to the saline-treated control). Moreover, treatment of the HCT116 cells xenografted onto the chick embryos with the hot-water extracts resulted in smaller tumour size and lower number of blood vessels compared to the saline-treated control. ConclusionsThe hot-water extracts of L. rhinocerus sclerotium demonstrated anti-angiogenic and anti-tumour activities but most of the cold-water extracts at similar concentrations were devoid of that. Our findings provide further scientific validation of the medicinal use of the sclerotium in treating cancer and thus, expanding our knowledge on the possible mechanism of its anti-cancer effect apart from direct cytotoxicity, induction of apoptosis and immunomodulation that have been studied thus far.

Licochalcone C Inhibits the Growth of Human Colorectal Cancer HCT116 Cells Resistant to Oxaliplatin.

Licochalcone C (LCC; PubChem CID:9840805), a chalcone compound originating from the root of Glycyrrhiza inflata, has shown anticancer activity against skin cancer, esophageal squamous cell carcinoma, and oral squamous cell carcinoma. However, the therapeutic potential of LCC in treating colorectal cancer (CRC) and its underlying molecular mechanisms remain unclear. Chemotherapy for CRC is challenging because of the development of drug resistance. In this study, we examined the antiproliferative activity of LCC in human colorectal carcinoma HCT116 cells, oxaliplatin (Ox) sensitive and Ox-resistant HCT116 cells (HCT116-OxR). LCC significantly and selectively inhibited the growth of HCT116 and HCT116-OxR cells. An in vitro kinase assay showed that LCC inhibited the kinase activities of EGFR and AKT. Molecular docking simulations using AutoDock Vina indicated that LCC could be in ATP-binding pockets. Decreased phosphorylation of EGFR and AKT was observed in the LCC-treated cells. In addition, LCC induced cell cycle arrest by modulating the expression of cell cycle regulators p21, p27, cyclin B1, and cdc2. LCC treatment induced ROS generation in CRC cells, and the ROS induction was accompanied by the phosphorylation of JNK and p38 kinases. Moreover, LCC dysregulated mitochondrial membrane potential (MMP), and the disruption of MMP resulted in the release of cytochrome c into the cytoplasm and activation of caspases to execute apoptosis. Overall, LCC showed anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, inducing ROS generation and disrupting MMP. Thus, LCC may be potential therapeutic agents for the treatment of Ox-resistant CRC cells.

Butyrate-mediated Resistance to Trichostatin A Accompanied by Elevated Expression of Glucose Transporter 3 (GLUT3) in Human Colorectal Carcinoma HCT116 Cells.

The aim of study was to investigate the correlation of GLUT3 upregulation and butyrate-mediated acquired chemoresistance. A butyrate-resistant CRC cell model was established from parental (PT) HCT116 cells by gradually increasing the concentration of sodium butyrate (NaBu), followed by evaluation of resistance to butyrate and trichostatin A (TSA) by the MTT method. The expression of SLC2A3 gene and GLUT3 protein were assessed by semi-quantitative RT-PCR and western blotting, respectively. The correlation of GLUT3 and butyrate-induced acquired chemoresistance was investigated using SLC2A3 silencing. Butyrate-resistant (BR) HCT116 cells were more tolerant to butyrate-induced cell death and also resist to 750 and 1000 nM TSA when compared with HCT116-PT cells (p <0.05). Long-term exposure to butyrate revealed that upregulation of the SLC2A3 gene was significantly increased by more than 20 fold (p < 0.01), and that of GLUT3 was elevated by approximately 2 fold (p < 0.05) in HCT116-BR cells. Silencing of the SLC2A3 gene increased the sensitivity of HCT116-BR cells to the effects of TSA. Upregulation of GLUT3 is associated with resistance to butyrate and TSA. GLUT3 is a molecular target for the detection of chemoresistant CRC cells and thus a potential target for diagnostic strategies.

Anticancer Potential of Farnesol Against Human Osteosarcoma Saos-2 Cells and Human Colorectal Carcinoma HCT-116 Cells.

Increased colorectal carcinoma (CRC) and osteosarcomaprevalence, low survival rate, poor prognosis, and the limitations of existing anticancer therapies like side effects of drugs, non-specificity, short half-life, etc., pose a need for novel anticancer drugs.Farnesol, an organic sesquiterpene compound, found in the essential oils of various plants has been shown to possess antioxidant, anti-inflammatory, and anticancer properties. However, the anticancer effect of farnesol againstCRCand osteosarcoma has not yet been adequately elucidated. The aim of the study was to analyze the anticancer effects of farnesol against human osteosarcoma and CRC cell lines. Human osteosarcoma (Saos-2) and colorectal carcinoma (HCT-116) cell lines were procured and cultured at37oC and 5% CO2. The cells were treated with 10, 20, 40, 60, 80, and 100 µM/ml and 20, 40, 60, 80, 100, and 120 µM/ml of farnesol for 24 hours, respectively.3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromideassay was performed to assess the cytotoxicity of farnesol on Saos-2 and HCT-116 cells. Acridine orange/ethidium bromidestaining was carried out to analyze apoptosis.4',6-diamidino-2-phenylindolestaining was done to observe the nuclear changes.Dichloro-dihydro-fluorescein diacetatestaining was performed to assess the farnesol-induced reactive oxygen species (ROS)-mediated cell death. Farnesol reduced the viability and proliferation of Saos-2 and HCT-116 cells in a dose-dependent manner. Farnesol was able to alter the cellular and nuclear morphology of Saos-2 and HCT-116 cells, promoting cell death. Farnesol-induced apoptosis inhuman osteosarcoma and colorectal carcinoma cell lines. Early apoptosis was observed in farnesol-treated HCT-116 cells. Additionally, ROS-mediated apoptotic cell death was reported in Saos-2 cells. Farnesol has the potential to induce cytotoxicity against human osteosarcoma and CRC cell lines.

Driver gene KRAS aggravates cancer-associated stroke outcomes

The incidence of cancer-associated stroke has increased with the prolonged survival times of cancer patients. Recent genetic studies have led to progress in cancer therapeutics, but relationships between oncogenic mutations and stroke remain elusive. Here, we focused on the driver gene KRAS, which is the predominant RAS isoform mutated in multiple cancer types, in cancer associated stroke study. KRASG13D/− and parental human colorectal carcinoma HCT116 cells were inoculated into mice that were then subjected to a photochemically-induced thrombosis model to establish ischemic stroke. We found that cancer inoculation exacerbated neurological deficits after stroke. Moreover, mice inoculated with KRASG13D/− cells showed worse neurological deficits after stroke compared with mice inoculated with parental cells. Stroke promoted tumor growth, and the KRASG13D/− allele enhanced this growth. Brain RNA sequencing analysis and serum ELISA showed that chemokines and cytokines mediating pro-inflammatory responses were upregulated in mice inoculated with KRASG13D/− cells compared with those inoculated with parental cells. STAT3 phosphorylation was promoted following ischemic stroke in the KRASG13D/− group compared with in the parental group, and STAT3 inhibition significantly ameliorated stroke outcomes by mitigating microglia/macrophage polarization. Finally, we compared the prognosis and mortality of colorectal cancer patients with or without stroke onset between 1 January 2007 and 31 December 2020 using a hospital-based cancer registry and found that colorectal cancer patients with stroke onset within 3 months after cancer diagnosis had a worse prognosis. Our work suggests an interplay between KRAS and ischemic stroke that may offer insight into future treatments for cancer-associated stroke.

In vitro and in vivo accumulation of the anticancer Ru complexes [RuII(cym)(HQ)Cl] and [RuII(cym)(PCA)Cl]Cl.

The cellular accumulation and the underlying mechanisms for the two ruthenium-based anticancer complexes [RuII(cym)(HQ)Cl] 1 (cym = η6-p-cymene, HQ = 8-hydroxyquinoline) and [RuII(cym)(PCA)Cl]Cl 2 (PCA = N-fluorophenyl-2-pyridinecarbothioamide) were investigated in HCT116 human colorectal carcinoma cells. The results showed that the cellular accumulation of both complexes increased over time and with higher concentrations, and that 2 accumulates in greater quantities in cells than 1. Inhibition studies of selected cellular accumulation mechanisms indicated that both 1 and 2 may be transported into the cells by both passive diffusion and active transporters, similar to cisplatin. Efflux experiments indicated that 1 and 2 are subjected to efflux through a mechanism that does not involve p-glycoprotein, as addition of verapamil did not make any difference. Exploring the influence of the Cu transporter by addition of CuCl2 resulted in a higher accumulation of 1 and 2 whilst the amount of Pt detected was slightly reduced when cells were treated with cisplatin. Complexes 1 and 2 were further explored in zebrafish where accumulation and distribution were determined with ICP-MS and LA-ICP-MS. The results correlated with the in vitro observations and zebrafish treated with 2 showed higher Ru contents than those treated with 1. The distribution studies suggested that both complexes mainly accumulated in the intestines of the zebrafish.

Effect of sonochemically synthesized Ni0.4Cu0.2Zn0.4DyxFe2-xO4 nanospinel ferrites on human colorectal carcinoma and human embryonic kidney cells

Dysprosium-substituted nickel-copper-zinc ferrite Ni0.4Cu0.2Zn0.4DyxFe2-xO4 (x ≤ 0.10) nanospinel ferrites (NSFs) (Dy-NiCuZn NSFs) were prepared by an ultrasonication assisted sol–gel chemical approach. The synthesized Dy-NiCuZn NSFs displayed a Porous Microsphere Assembly (PMA) based on Scanning Electron Microscopy (SEM) results. X-ray diffraction (XRD) results revealed the cubic spinel structures of the NSFs and their crystallite sizes ranged between 17.19 and 30.22 nm. TEM analysis revealed the presence of Dy-NiCuZn NSFs with a 15.2 nm size in diameter. BET surface analysis calculations showed that the pore size of NSFs varies between 3.0 nm and 4.0 nm. A microporous structure was confirmed in the NSFs, and their surface area generally decreased (247 to 201 m2/g) as the Dy ratio increased in the NSFs. Antiproliferative activities of NSFs were studied on HCT-116 human colorectal carcinoma cells (HCT-116) and human embryonic kidney cells. Inhibitory concentration values of NSFs were determined to lie between 145 µg/mL to 408 µg/mL for HCT-116 cells. A noteworthy reduction in cell viability after the treatments of cells with NSFs was detected. NSFs-treated cells showed nuclear disintegration and chromatic fragmentation which are signs of apoptosis or programmed cell death in human colorectal cancer cells.

Nine undescribed oxidized ergosterols from the endophytic fungus Penicillium herquei and their cytotoxic activity

A chemical investigation of the EtOAc extract of the endophytic fungus Penicillium herquei led to the isolation of nine undescribed oxidized ergosterols, penicisterols A–I (1–9), along with ten known analogs (10–19). Their structures and absolute configurations were elucidated by a combination of spectroscopic data analysis, quantum-chemical electronic circular dichroism (ECD) calculations and comparisons, [Rh2(OCOCF3)4]-induced ECD experiments, DFT-calculated 13C chemical shifts and DP4+ probability analysis. Compound 1 was a rare example of ergosterol in which the bond between C-8 and C-9 was cleaved to form an enol ether. Moreover, compound 2 possessed a rare (2,5-dioxo-4-imidazolidinyl)-carbamic acid ester group substituted at C-3. All undescribed oxidized ergosterols (1–9) were evaluated for their cytotoxic activity against five cancer cell lines including 4T1 (mouse breast carcinoma), A549 (human pulmonary carcinoma), HCT-116 (human colorectal carcinoma), HeLa (human cervical carcinoma) and Hepg2 (human hepatoma carcinoma) cells. Compounds 2 and 3 displayed moderate cytotoxic activity against 4T1, A549 and HeLa cells, with IC50 values ranging from 17.22 to 31.35 μM.

Design, Synthesis, Anticancer Activity and Molecular Docking of New 1,2,3-Triazole-Based Glycosides Bearing 1,3,4-Thiadiazolyl, Indolyl and Arylacetamide Scaffolds.

New 1,3,4-thiadiazole thioglycosides linked to a substituted arylidine system were synthesized via heterocyclization via click 1,3-dipolar cycloaddition. The click strategy was used for the synthesis of new 1,3,4-thiadiazole and 1,2,3-triazole hybrid glycoside-based indolyl systems as novel hybrid molecules by reacting azide derivatives with the corresponding acetylated glycosyl terminal acetylenes. The cytotoxic activities of the compounds were studied against HCT-116 (human colorectal carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines using the MTT assay. The results showed that the key thiadiazolethione compounds, the triazole glycosides linked to p-methoxyarylidine derivatives and the free hydroxyl glycoside had potent activity comparable to the reference drug, doxorubicin, against MCF-7 human cancer cells. Docking simulation studies were performed to check the binding patterns of the synthesized compounds. Enzyme inhibition assay studies were also conducted for the epidermal growth factor receptor (EGFR), and the results explained the activity of a number of derivatives.

In vitro anticancer and antiproliferative activity of crocin on HCT-116 cells

One of the world’s main causes of cancer-related fatalities worldwide is colorectal cancer. The adherent epithelial cells known as HCT116 (human colorectal carcinoma) are frequently employed to examine inflammatory responses in colonic epithelial cells. They are derived from the human colorectal cancer cell line. Crocin, a potent antioxidant, anticarcinogenic was tested in this study to see how it affected the HCT-116 human colorectal cancer cell line. MTT analysis was used to assess cell viability. Crocin's antiproliferative activity to inhibit the proliferation of HCT 116 cancer cells was examined. Crocin displayed the outstanding activities for MTT test, IC50= 10.57 μL/mL for 24 hours was calculated as IC50= 3.29 μL/mL for 48 hours on HCT 116 cell line. Total antioxidant status (TAS) and total oxidant status (TOS) in the prepared cell lysate were determined by using commercial kits. Crocin is thought to be a high potential agent to be used in treatment.

Ilimaquinone (Marine Sponge Metabolite) Induces Apoptosis in HCT-116 Human Colorectal Carcinoma Cells via Mitochondrial-Mediated Apoptosis Pathway.

Ilimaquinone (IQ), a metabolite found in marine sponges, has been reported to have a number of biological properties, including potential anticancer activity against colon cancer. However, no clear understanding of the precise mechanism involved is known. The aim of this study was to examine the molecular mechanism by which IQ acts on HCT-116 cells. The anticancer activity of IQ was investigated by means of a cell viability assay followed by the determination of induction of apoptosis by means of the use of acridine orange–ethidium bromide (AO/EB) staining, Annexin V/PI double staining, DNA fragmentation assays, and TUNEL assays. The mitochondrial membrane potential (ΔΨm) was detected using the JC-1 staining technique, and the apoptosis-associated proteins were analyzed using real-time qRT-PCR. A molecular docking study of IQ with apoptosis-associated proteins was also conducted in order to assess the interaction between IQ and them. Our results suggest that IQ significantly suppressed the viability of HCT-116 cells in a dose-dependent manner. Fluorescent microscopy, flow cytometry, DNA fragmentation and the TUNEL assay in treated cells demonstrated apoptotic death mode. As an additional confirmation of apoptosis, the increased level of caspase-3 and caspase-9 expression and the downregulation of Bcl-2 and mitochondrial dysfunction were observed in HCT-116 cells after treatment with IQ, which was accompanied by a decrease in mitochondrial membrane potential (ΔΨm). Overall, the results of our studies demonstrate that IQ could trigger mitochondria-mediated apoptosis as demonstrated by a decrease in ΔΨm, activation of caspase-9/-3, damage of DNA and a decrease in the proportion of Bcl-2 through the mitochondrial-mediated apoptosis pathway.

Mesenchymal stem cells- derived exosomes inhibit the expression of Aquaporin-5 and EGFR in HCT-116 human colorectal carcinoma cell line

BackgroundAquaporins are channel proteins, form pores in the membrane of biological cells to facilitate the transcellular and transepithelial water movement. The role of Aquaporins in carcinogenesis has become an area of interest. In this study, we aimed to investigate the effects of adipose-derived mesenchymal stem cells secreted exosomes on the expression of aquaporin 5 and EGFR genes in the HCT-116 tumor cell line.Methods and resultsSurface antigenic profile of Ad-MSCs was evaluated using specific markers. Exosomes were purified from the Ad-MSc supernatant while the quality and the shape of isolated exosomes were assessed by western blot and transmission electron microscopy (TEM) respectively. HCT-116 cells were co-cultured with MSC-conditioned medium (MSC-CM) and/or with 100 μg/ml of MSC-derived exosomes for 48 h and. Real-time PCR was carried out to determine the expression of aquaporin5 and EGFR in HCT-116. Relative expression levels were calculated using the 2-ΔΔct method.Our result showed that AQP5 and EGFR mRNA levels were significantly reduced in CM and/or exosomes treated HCT116 compare to the control group (P-value < 0.05).ConclusionThe current study showed that MSC derived exosomes could inhibit expression of two important molecules involved in tumor progression. Hence it seems MSCs-derived exosomes may hold a hopeful future as drug delivery vehicles which need the furtherer investigation.

Intensification of resveratrol cytotoxicity, pro-apoptosis, oxidant potentials in human colorectal carcinoma HCT-116 cells using zein nanoparticles

Resveratrol (RSV), a non-flavonoid stilbene polyphenol, possesses anti-carcinogenic activities against all the major stages of cancer. Zein nanoparticles (ZN NPs) have been utilized successfully in delivery of variant therapeuticals by virtue of their histocompatible nature. The goal of this work was to comparatively explore the antiproliferative, pro-apoptotic and oxidative stress potentials of RSV-ZN NPs versus RSV against human colorectal carcinoma HCT-116 cells. ZN-RSV NPs were developed and assayed for particle size analysis and RSV diffusion. The selected formula obtained 137.6 ± 8.3 nm as mean particle size, 29.4 ± 1.8 mV zeta potential, 92.3 ± 3.6% encapsulation efficiency. IC50 of the selected formula was significantly lower against HCT-116 cells versus Caco-2 cells. Also, significantly enhanced cellular uptake was generated from RSV-ZN NPs versus free RSV. Enhanced apoptosis was concluded due to increased percentage cells in G2-M and pre-G1 phases. The pro-apoptotic potential was explained by caspase-3 and cleaved caspase-3 increased mRNA expression in addition to NF-κB and miRNA125b decreased expression. Biochemically, ZN-RSV NPs induced oxidative stress as demonstrated by enhanced reactive oxygen species (ROS) generation and endothelial nitric oxide synthase (eNOS) isoenzyme increased levels. Conclusively, ZN-RSV NPs obtained cell cycle inhibition supported with augmented cytotoxicity, uptake and oxidative stress markers levels in HCT-116 tumor cells in comparison with free RSV. These results indicated intensified chemopreventive profile of RSV due to effective delivery utilizing ZN nano-dispersion against colorectal carcinoma HCT-116 cells.

Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor-mediated extrinsic apoptosis, specifically in cancer cells, and Bid (BH3-interacting domain death agonist) plays an important role in TRAIL-induced apoptosis. Ferroptosis is a newly defined form of regulated cell death known to be distinct from other forms of cell death. However, our previous studies have shown that ferroptosis shares common pathways with other types of programmed cell death such as apoptosis. In this study, we investigated the role of Bid in the crosstalk between the ferroptotic agent-induced endoplasmic reticulum (ER) stress response and TRAIL-induced apoptosis. When human colorectal carcinoma HCT116 cells were treated with the ferroptosis-inducing agents artesunate and erastin in combination with TRAIL, TRAIL-induced activation of caspase-8 was enhanced, and subsequently, the truncation of Bid was increased. Similar results were observed when ovarian adenocarcinoma OVCAR-3 cells were treated with the ferroptotic agents in combination with TRAIL. Results from studies with Bid mutants reveal that the truncation of Bid and the presence of intact BH3 domains are critical for synergistic apoptosis. Nonfunctional Bid mutants were not able to activate the mitochondria-dependent apoptosis pathway, which is required for the conversion of p19 to p17, the active form of caspase-3. These results indicate that Bid plays a critical role in the crosstalk between the ferroptotic agent-induced ER stress response and TRAIL-induced apoptosis.

Preparation, Characterization and In vitro Biological activity of 5-Fluorouracil Loaded onto poly (D, L-lactic-co-glycolic acid) Nanoparticles

Nanoscale devices offer a lot of potential in drug delivery because of their small size. The goal of this work was to increase the oral bioavailability of the anti-cancer hydrophilic drug as 5-fluorouracil (5-FU) by incorporating it into poly (D, L-lactide-co-glycolide) nanoparticles (PLGNPs) using the double emulsion process, 5-FU- PLGNPs nanoparticles were created. Various factors, such as drug, polymer, and stabilizer concentrations, were investigated for assembly in order to arrive at the most effective formulation of 5-FU-PLGNPs. PLGNPs had a drug encapsulation efficiency of 9.75 to 24.8%. The prepared nanoparticles had a spherical shape and an average size of 212.3–285 nm, as shown by TEM. The dispersion of the drug into the prepared PLGNPs was confirmed by XRPD and FTIR. The optimized nanoparticles (F225) had high encapsulation efficiency 24.8 ± 0.21%, low particles size 212.3 ± 48.2 nm with an appropriate PDI value of 0.448, and ZP of − 48.3 ± 2.7 mV. The molecular dispersion of the medication within the system was validated by thermal behavior studies (DSC). In vitro drug release from the best-selected formulations revealed a sustained release of nanoparticles, with slower release reported when lower PVA concentrations were utilized. Three 5-FU-PLGNPs formulations were tested for anticancer efficacy against cell cultures of HCT-116 (human colorectal carcinoma), MCF-7 (human breast carcinoma), and HepG2 (human hepatocellular carcinoma). The created formulations were examined for in vitro cytotoxic activity, revealing that they appeared to be promising effective anticancer formulations when compared to the positive controlled (doxorubicin).

Abstract LB045: β-catenin antagonist peptide, ST316, attenuates Wnt-dependent oncogenic activity

Abstract Constitutive activation of the Wnt/β-catenin signaling pathway underlines many breast and colorectal cancers, promoting cancer cell survival, proliferation and migration. B-cell CLL/lymphoma 9 protein (BCL9), a co-activator of β-catenin transcriptional activity, is overexpressed in many tumors and facilitates β-catenin nuclear localization. Disruption of the β-catenin and BCL9 interaction has been shown genetically to suppress oncogenic Wnt/β-catenin transcription, without impacting β-catenin homeostatic functions, and represents a powerful approach to inhibit this previously ‘undruggable’ target. Here, we investigate the impact of ST316, a peptide antagonist of β-catenin that inhibits its interaction with BCL9, on cells displaying hyperactivation of the Wnt/β-catenin pathway. Administration of ST316 to HCT116 human colorectal carcinoma cells, which carry a degradation-escape, gain-of-function mutation in β-catenin, dramatically reduced nuclear β-catenin localization. Furthermore, ST316-mediated loss of nuclear β-catenin was associated with a significant decrease in the expression of Wnt/β-catenin target genes in HCT116 cells stimulated with Wnt3a, including Axin2, hTCF7, Fox01 and CDK4. ST316 reduced β-catenin target gene expression by up to ∼70%, whereas no impact was observed following administration of a negative control peptide. A significant anti-proliferative and dose-dependent cytotoxic effect was observed on HCT116 cells 48 hours post ST316 exposure, with an EC50 of 4.4 µM, and on patient-derived, APC-mutant colorectal adenocarcinoma and breast carcinoma cells 72 hours post ST316 exposure, with EC50 values ranging from 1.5 to 2.6 µM. To extend these findings, we investigated the impact of ST316 on HCT116 tumors in vivo. ST316 (5 mg/kg) administered three times weekly by subcutaneous injection resulted in 99.6% tumor growth inhibition vs. vehicle controls (p&amp;lt;0.05), while a negative control peptide had no significant impact on tumor growth. These data demonstrate the potential of ST316 as a potent therapeutic candidate for Wnt/β-catenin-dependent cancers. Citation Format: Lila Ghamsari, Erin Gallagher, Emad Darvishi, Siok Leong, Mark Koester, Rick Ramirez, Jerel Gonzales, Gene Merutka, Barry Kappel, Jim A. Rotolo. β-catenin antagonist peptide, ST316, attenuates Wnt-dependent oncogenic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB045.

Upregulation of GCLC is Responsible for SFN‐induced Tumor Cell Proliferation

Sulforaphane (SFN), a sulfur‐containing compound that belongs to the isothiocyanate class of dietary phytochemicals found in cruciferous vegetables, has been known to cause a hormetic effect on cancer cell growth. We previously reported that SFN at low doses stimulated proliferation of HCT116 human colorectal carcinoma cells through Nrf2 activation in vitro and in vivo. Recently, we discovered that brusatol, a nuclear factor erythroid 2‐related factor 2 (Nrf2) inhibitor, significantly suppressed the SFN‐induced proliferation of HCT116 cells. Therefore, we hypothesized that SFN‐induced proliferation of HCT116 cells is mediated by an Nrf2 downstream gene(s). Transcriptomic profiling revealed a set of differentially expressed genes (DEGs) involved in glutathione (GSH) metabolism as compared between wildtype (WT) and Nrf2 knockout (KO) HCT116 cells treated with or without SFN. In particular, we found that glutamate‐cysteine ligase catalytic subunit (GCLC) was highly elevated in SFN‐treated WT HCT116 cells among the DEGs, but not in SFN‐treated Nrf2 KO HCT116 cells. To confirm if upregulation of γ‐glutamate‐cysteine ligase (γ‐GCL) is associated with SFN‐induced proliferation of HCT116 cells, inhibiting γ‐GCL by buthionine sulfoximine (BSO, a selective γ‐GCL inhibitor) and silencing GCLC by short hairpin RNA (shRNA) were carried out. The results showed that SFN‐induced proliferation of HCT116 cells was significantly reduced by γ‐GCL inhibition and abolished by GCLC knockdown. Furthermore, we confirmed that BSO suppressed SFN‐induced tumor growth of WT HCT116 xenograft mice and the tumor growth was not stimulated in both Nrf2 KO and GCLC shRNA HCT16 xenograft mice by SFN at a low dose. Collectively, these findings indicate that upregulation of GCLC‐dependent GSH biosynthesis by SFN promotes proliferation of HCT116 cells and thus GCL could be a potential target for chemotherapy.

Luteolin Synergistically Enhances Antitumor Activity of Oxaliplatin in Colorectal Carcinoma via AMPK Inhibition.

Luteolin is a naturally-occurring polyphenolic compound that is known to have antioxidative and antitumor activities in vitro. This study aimed to examine the in vivo anticancer efficacy of luteolin in conjunction with oxaliplatin treatment using a colorectal carcinoma xenograft mouse model. HCT116 human colorectal carcinoma cells were subcutaneously implanted into BALB/c nude mice, followed by the intraperitoneal administration of luteolin at a dose of 50 mg/kg body weight (BW)/day with or without oxaliplatin at a dose of 10 mg/kg BW/day three times per week for a total of 3 weeks. The combined luteolin and oxaliplatin treatment resulted in the synergistic suppression of the growth of HCT116 xenograft tumors when compared to treatment with luteolin or oxaliplatin alone. In addition, the combined treatment significantly increased the expression of cleaved PARP and p53 in the xenograft tumors compared with the vehicle control, but only marginally affected the level of heme oxygenase-1 (HO-1). These results indicated that luteolin treatment retarded oxaliplatin-induced tumor growth by facilitating apoptotic cell death and inhibiting HO-1-mediated cytoprotection. Therefore, these findings suggest the synergistic potential of dietary luteolin in conjunction with conventional chemotherapy for the treatment of colorectal cancer.