Normal Colonic Cells Research Articles

Evaluation of Toxicity and Antioxidant Activity of the Ethanolic Extract from Ulva Lactuca

Ulva lactuca (sea lettuce) is a popular food for consumers and has high nutritional value. It has also been found to be a source of health-promoting substances, including a high amount of proteins which can be utilized as a future plant-based functional ingredient in the food industry. However, before extensive applications in the aforementioned fields, it is crucial to ensure the extracts’ biological-pharmacological activities and their safety. In this study, we evaluated the cytotoxicity, genotoxicity, and antioxidant activity of the ethanolic extract of the sea lettuce extract.We tested cytotoxicity by MTT assays on 5 cell lines, including fibroblast (L929), macrophages (RAW 264.7), hepatocytes (FL83B), keratinocytes (HaCaT) and normal colon (CCD-18co) cells. Genotoxicity of the extract was tested by comet and micronucleus assays on human lymphoblast cells (TK6). Our results demonstrated at a concentration lower than 50 µg/mL the extract did not show any cytotoxicity as well as genotoxicity. The extract was shown to possess high antioxidant capacity by DPPH and ABTS scavenging at the EC50 value of U. lactuca extracts was 631.84 ± 5.64 and 330.35 ± 19.49 μg/mL, respectively. Therefore, the extract may be safely used as an ingredient for food, that should help to ensure food security for humans in the future, as well as for cosmetic, pharmaceutical and other health-promoting products. HIGHLIGHTS Ulva lactuca extract displayed strong antioxidant activity. Neither cytotoxicity nor genotoxicity to cell lines are seen in the ethanol extract of lactuca. GRAPHICAL ABSTRACT

CGAS regulates metabolic reprogramming independently of STING pathway in colorectal cancer

BackgroundCyclic GMP-AMP synthase (cGAS) is widely acknowledged for detecting cytosolic chromatin fragments and triggering innate immune responses through the production of the second messenger cGAMP, which subsequently activates the adaptor protein STING. However, the role of cGAS in regulating metabolic reprogramming independently of STING activation has not yet been explored. MethodsGene set enrichment pathway analysis (GSEA) based on TCGA transcriptomics, combined with Seahorse metabolic analysis of CRC cell lines and human normal colonic mucosa cell line FHC, was performed to profile the metabolic features in CRC. cGAS doxycycline- (dox) inducible knockout (iKO) CRC sublines were generated to investigate the role of cGAS in CRC. Transcriptome and proteome data from COAD cohorts were utilized to evaluate the RNA and protein expression levels of cGAS in COAD tissues and normal colon tissues. Overall survival information of patients with COAD was used to evaluate the prognostic value of cGAS expression. Colony formation assays were conducted to evaluate the clonogenicity of CRC cells under different situations. Flow cytometry detecting the signal of fluorogenic reactive oxygen species (ROS) probes was performed to evaluate the total cellular and mitochondrial oxidative stress level in CRC cells. A propidium iodide (PI) staining assay was used to evaluate the cell death level in CRC cells. Quantitative PCR (qPCR) was conducted to detect the RNA level of STING pathway downstream target genes. Mass spectrometry was used for the identification of novel binding partners of cGAS in CRC cells. Co-immunoprecipitation (co-IP) was conducted to confirm the interaction between cGAS and NDUFA4L2. ResultsBy integrating metabolic pathway analysis based on TCGA transcriptomics with Seahorse metabolic analysis of a panel CRC cell lines and the human normal colonic mucosa cell line FHC, we demonstrated that CRC cells exhibit typical characteristics of metabolic reprogramming, characterized by a shift from oxidative phosphorylation (OXPHOS) to glycolysis. We found that cGAS is critical for CRC cells to maintain this metabolic switch. Specifically, the suppression of cGAS through siRNA-mediated knockdown or doxycycline-inducible knockout reversed this metabolic switch, resulting in increased OXPHOS activity, elevated production of OXPHOS byproduct reactive oxygen species (ROS), and consequently caused oxidative stress. This disruption induced oxidative stress, ultimately resulting in cell death and reduced cell viability. Moreover, significant upregulation of cGAS in CRC tissues and cell lines and its association with poor prognosis in CRC patients was observed. Subsequently, we demonstrated that the role of cGAS in regulating metabolic reprogramming does not rely on the canonical cGAS-STING pathway. Co-immunoprecipitation combined with mass spectrometry identified NDUFA4L2 as a novel interactor of cGAS. Subsequent functional experiments, including mitochondrial respiration and oxidative stress assays, demonstrated that cGAS plays a crucial role in sustaining elevated levels of NDUFA4L2 protein expression. The increased expression of NDUFA4L2 is essential for cGAS-mediated regulation of metabolic reprogramming and cell survival in CRC cells. ConclusioncGAS regulates metabolic reprogramming and promotes cell survival in CRC cells through its interaction with NDUFA4L2, independently of the canonical cGAS-STING pathway.

PH-sensitive aminated chitosan/carboxymethyl cellulose/aminated graphene oxide coated composite microbeads for efficient encapsulation and sustained release of 5-fluorouracil

The application of smart pH-sensitive carriers has become an ideal choice for administering drugs with desired release profiles. Although pH-sensitive microbeads offer distinct benefits for delivering anticancer drugs orally, they encounter drawbacks, including low encapsulation efficiency, weak mechanical stability, biocompatibility concerns, and the risk of abrupt release. This study focuses on developing pH-sensitive coated composite microbeads for effective encapsulation and sustained release of 5-fluorouracil (5-FU). Aminated graphene oxide (AmGO) was integrated into carboxymethyl cellulose (CMC) microbeads, which were subsequently coated with an aminated chitosan (AmCs) derivative. Various analysis techniques, including Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), X-ray diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetric analyzer (TGA), zeta potential (ZP), and mechanical testing, were utilized to characterize the microbeads. The AmCs@CMC@AmGO composite microbeads demonstrated a compact structure with enhanced mechanical properties, achieving a maximum Young's modulus value of 35.99 N/mm2 compared to 25.95 N/mm2 for pure CMC microbeads. Moreover, pH-sensitivity and water uptake studies (at pH 1.2 and pH 7.4) revealed significant tunability of the composite microbeads by altering the AmGO and AmCs ratios. The coated composite microbeads encapsulated approximately 86.4 % of 5-FU compared to 47 % for CMC microbeads. The burst release of 5-FU at pH 7.4 was significantly reduced, with sustained release reaching 51 % over 24 h. The predominant release mechanism was Fickian diffusion, which well-described by the Peppas-Sahlin kinetic model. The developed microbeads exposed improved biodegradability and non-toxicity toward normal colon cells, while exhibiting notable toxicity against cancerous cells, emphasizing its potential for anticancer drug delivery.

Food grade titanium dioxide induced endoplasmic reticulum stress in colon cells: Comparison between normal and colorectal carcinoma cells

BackgroundFood-grade titanium dioxide (E171) has been under scrutiny in the last decade since its possible adverse effects; however, the cellular mechanisms underlying E171 toxicity have not been thoroughly described. AimWe aimed to compare the effects of E171 on endoplasmic reticulum (ER) homeostasis in normal and cancer colon cells. Experimental designWe exposed normal, carcinoma, and adenocarcinoma cells to 0.1, 1, 10, 50 and 100 μg/cm2 of E171 for 24, 48 and 72 h, and we evaluated ER stress, cell viability, titanium uptake, intracellular calcium concentration, and gene expression related to unfolded protein response (UPR) and chaperone pathways. ResultsCell viability decreased only after 72 h of exposure to 100 μg/cm2 of E171. Adenocarcinoma cells internalized higher titanium amounts than normal and carcinoma cells, but the effects in ER distribution, intracellular calcium concentration, and gene expression were similar among the three cell lines. The expression of UPR and chaperone pathways were downregulated at the lowest concentrations but upregulated at the highest concentrations in the three cell lines. ConclusionE171 induces ER stress through alterations in ER distribution, intracellular calcium, and UPR and chaperone protein pathways.

Lack of Association Between BsmI and FokI Polymorphisms of the VDR Gene and Sporadic Colorectal Cancer in a Romanian Cohort-A Preliminary Study.

Colorectal cancer (CRC) is a major public health problem worldwide, currently ranking third in cancer incidence and second in mortality. Multiple genes and environmental factors have been involved in the complex and multifactorial process of CRC carcinogenesis. VDR is an intracellular hormone receptor expressed in both normal epithelial and cancer colon cells at various levels. Several VDR gene polymorphisms, including FokI and BsmI, have been evaluated for their possible association with CRC susceptibility. The aim of our study was to investigate these two SNPs for the first time in Romanian CRC patients. FokI (rs228570 C>T) and BsmI (rs1544410 A>G) were genotyped by real-time polymerase chain reaction (RT-PCR) in 384-well plates using specific TaqMan predesigned probes on a ViiA™ 7 RT-PCR System. A total of 441 subjects (166 CRC patients and 275 healthy controls) were included. No statistically significant difference was observed between CRC patients and controls when we compared the wild-type genotype with heterozygous and mutant genotypes for both FokI (OR 0.85, 95% CI: 0.56-1.28; OR 0.95, 95% CI: 0.51-1.79, respectively) and BsmI (OR 0.97, 95% CI: 0.63-1.49; OR 1.10, 95% CI: 0.65-1.87, respectively) or in the dominant and recessive models. Also, we compared allele frequencies, and no correlation was found. Moreover, the association between these SNPs and the tumor site, TNM stage, and histological type was examined separately, and there was no statistically significant difference. In conclusion, our study did not show any association between FokI and BsmI SNPs and CRC susceptibility in a Romanian population. Further studies including a larger number of samples are needed to improve our knowledge regarding the influence of VDR polymorphism on CRC susceptibility.

Mitoception, or transfer of normal cell mitochondria to cancer cells, reverses remodeling of store-operated Ca2+ entry in tumor cells

Most cancer cells show the Warburg effect, the rewiring of aerobic metabolism to glycolysis due to defective mitochondrial ATP synthesis. As a consequence, tumor cells display enhanced mitochondrial potential (∆Ψ), the driving force for mitochondrial Ca2+ uptake. Mitochondria control the Ca2+-dependent inactivation of store-operated channels (SOCs), leading to enhanced and sustained store-operated Ca2+ entry (SOCE) involved in cancer hallmarks. We asked here whether the transfer of mitochondria (mitoception) from normal cells to tumor cells may reverse SOCE remodeling in cancer cells. For this end, we labeled mitochondria in normal NCM460 human colonic cells, isolated them and transferred them to tumor HT29 cells. We tested the viability and efficiency of mitoception using flow cytometry and confocal microscopy, as well as calcium imaging to investigate the effects of mitoception on SOCE. Our results show that mitoception of tumor HT29 cells with normal mitochondria restores a low ∆Ψ and SOCE. Conversely, self-mitoception of tumor HT29 cells with tumor cell mitochondria increases further ∆Ψ and SOCE, thus excluding the possibility that effects of mitoception are due to increased mitochondrial mass. Strikingly, mitoception of normal NCM460 cells with tumor cell mitochondria has no effects on either ∆Ψ or SOCE. These results are consistent with the previous proposal that transformed mitochondria may modulate SOC channels involved in SOCE. Further research is warranted to test whether mitoception of cancer cells with normal mitochondria may reverse Ca2+ remodeling associated to cancer.

New S-substituted-3-phenyltetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one scaffold with promising anticancer activity profile through the regulation and inhibition of mutated B-RAF signaling pathway.

Novel 3-phenyltetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives were synthesized and screened for their antiproliferative activity against a panel of 60 cancer cell lines. Derivatives 5b, 5f, and 9c showed significant antitumor activity at a single dose with mean growth inhibition of 55.62%, 55.79%, and 71.40%, respectively. These compounds were further investigated against HCT-116, colon cancer cell line, and FHC, normal colon cell line. Compound 9c showed the highest activity with IC50 = 0.904 ± 0.03 µM and SI = 20.42 excelling doxorubicin which scored IC50 = 2.556 ± 0.09 µM and SI = 6.19. Compound 9c was also the most potent against B-RAFWT and mutated B-RAFV600E with IC50 = 0.145 ± 0.005 and 0.042 ± 0.002 µM, respectively in comparison with vemurafenib with IC50 = 0.229 ± 0.008 and 0.038 ± 0.001 µM, respectively. The cell cycle analysis showed that 9c increased the cell population and induced an arrest in the cell cycle of HCT-116 cancer cells at the G0-G1 stage with 1.23-fold. Apoptosis evaluation showed that compound 9c displayed an 18.18-fold elevation in total apoptosis of HCT-116 cancer cells in comparison to the control. Compound 9c increased the content of caspase-3 by 3.52-fold versus the control. A molecular modeling study determined the binding profile and interaction of 9c with the B-RAF active site.

6686 Age-Associated Local GH Adversely Impacts the Microenvironmental Epithelial Landscape

Abstract Disclosure: V.M. Chesnokova: None. S. Zonis: None. T. Apaydin: None. C. Wang Valencia: None. R. Ainsworth: None. R. Barrett: None. A. Kettenbach: None. S. Melmed: None. The tissue microenvironmental homeostasis is disrupted with age-related changes. Knowledge of mechanisms enabling the age-associated tissue landscape is limited. Senescent cells increase with age, and the secretome released from senescence cells (SASP) affects neighboring cells, often creating a milieu favoring neoplasia. We previously showed that local non-pituitary epithelial growth hormone (GH) is induced and secreted from senescent cells, accumulates with age, and suppresses DNA repair, leading to abundant DNA damage. To examine SASP-derived autocrine GH actions, we analyzed normal human colon cells (hNCC) infected with lentivirus expressing hGH (lentiGH) or vector (lentiV). Mass spectrometry revealed significant changes in the Rho signaling pathway reflecting cell cytoskeletal rearrangements. To examine paracrine GH action, we co-cultured intact human 3D intestinal organoids with organoids infected with lentiGH for 5 weeks. Using whole-exome sequencing we found that local paracrine GH emanating from lentiGH organoids increases neighboring intact organoid cell chromosomal instability, inducing somatic mutations, including deletions, breakends, duplications, and insertions. Mechanisms for observed chromosomal instability include sequelae of GH-mediated suppressed DNA damage repair proteins including pATM, pATR, and pBRCA2, leading to DNA damage accumulation and favoring cell transformation over the long term. Furthermore, enriched gene ontology and KEGG pathway analysis of intact organoids exposed to paracrine GH identified distorted extracellular matrix (ECM) gene expression and focal adhesion pathways. We confirmed these genomic changes with Western blotting showing altered proteins associated with ECM and those responsible for cell structural rearrangements. Moreover, phospho-omics of GH-exposed cells revealed significant phosphorylation changes in proteins associated with cell skeletal rearrangement and cell migration pathways. We found that GH triggers these changes by EMT activation as shown by suppressing E-cadherin and inducing Twist2 and Snai1 transcription factors. Together, all these changes are consistent with observed increased migration, invasion, and anchorage-independent growth of hNCC infected with lentiGH or co-cultured with GH-secreting hNCC or with GH-secreting normal colon fibroblasts. As paracrine GH also facilitates metastases after intra-splenic injection of senescent cells in nude mice carrying xenografts secreting GH, our results indicate that accumulated and locally secreted GH in aging tissue enables a microenvironmental landscape favoring epithelial cell transformation. Presentation: 6/1/2024

Extracellular vesicle-mediated delivery of miR-766-3p from bone marrow stromal cells as a therapeutic strategy against colorectal cancer

ObjectiveAs colorectal cancer (CRC) remains one of the leading causes of cancer-related deaths, understanding novel therapeutic mechanisms is crucial. This research focuses on the role of extracellular vesicles (EVs) from bone marrow stromal cells (BMSCs) in delivering miR-766-3p to CRC cells, targeting the MYC/CDK2 signaling axis.MethodsDifferentially expressed genes between BMSCs-EVs and CRC were identified using the Gene Expression Omnibus database. miR-766-3p target genes were predicted via TargetScan and RNAInter, with protein interactions analyzed using the STRING database. The analysis included RT-qPCR and Western blot on samples from 52 CRC patients. Characterization of BMSCs-EVs was followed by their functional assessment on CRC cell lines and the normal colon cell line CCD-18CO, evaluating cellular uptake, proliferation, migration, invasion, and apoptosis.ResultsmiR-766-3p was confirmed in BMSCs-EVs and found underexpressed in CRC. BMSCs-EVs transported miR-766-3p to CRC cells, inhibiting their proliferation, migration, and invasion while promoting apoptosis. miR-766-3p targeted MYC, leading to decreased CDK2 transcription. Overexpression of MYC in HCT-116 cells counteracted these effects. In vivo studies showed that BMSCs-EVs carrying miR-766-3p hindered tumor growth.ConclusionThe study demonstrates the efficacy of BMSCs-EVs in delivering miR-766-3p to CRC cells, leading to the suppression of the MYC/CDK2 signaling pathway and hindering cancer progression.

Exploring the Potential of Synthetic Cannabinoids: Modulation of Biological Activity of Normal and Cancerous Human Colon Epithelial Cells.

Colorectal cancer (CRC) is a global problem. Oncology currently practices conventional methods of treating this carcinoma, including surgery, chemotherapy, and radiotherapy. Unfortunately, their efficacy is low; hence, the exploration of new therapies is critical. Recently, many efforts have focused on developing safe and effective anticancer compounds. Some of them include cannabinoids. In the present study, we obtained cannabinoids, such as cannabidiol (CBD), abnormal cannabigerol (abn-CBG), cannabichromene (CBC), and cannabicitran (CBT), by chemical synthesis and performed the biological evaluation of their activity on colon cancer cells. In this study, we analyzed the effects of selected cannabinoids on the lifespan and metabolic activity of normal colonic epithelial cells and cancer colon cells. This study demonstrated that cannabinoids can induce apoptosis in cancer cells by modulating mitochondrial dehydrogenase activity and cellular membrane integrity. The tested cannabinoids also influenced cell cycle progression. We also investigated the antioxidant activity of cannabinoids and established a relationship between the type of cannabinoid and nitric oxide (NO) production in normal and cancerous colon cells. To conclude, it seems that, due to their interesting properties, the cannabinoids studied may constitute an interesting target for further research aimed at their use in alternative or combined therapies for human colon cancer.

Microbially mediated phenolic catabolites exert differential genoprotective activities in normal and adenocarcinoma cell lines

Age-associated decline of nuclear factor erythroid 2-related factor 2 (Nrf2) activity and DNA repair efficiency leads to the accumulation of DNA damage and increased risk of cancer. Understanding the mechanisms behind increased levels of damaged DNA is crucial for developing interventions to mitigate age-related cancer risk. Associated with various health benefits, (poly)phenols and their microbially mediated phenolic catabolites represent a potential means to reduce DNA damage. Four colonic-microbiota-derived phenolic catabolites were investigated for their ability to reduce H2O2-induced oxidative DNA damage and modulate the Nrf2-Antixoidant Response Element (ARE) pathway, in normal (CCD 841 CoN) and adenocarcinoma (HT29) colonocyte cell lines. Each catabolite demonstrated significant (p < .001) genoprotective activity and modulation of key genes in the Nrf2-ARE pathway. Overall, the colon-derived phenolic metabolites, when assessed at physiologically relevant concentrations, reduced DNA damage in both normal and adenocarcinoma colonic cells in response to oxidative challenge, mediated in part via upregulation of the Nrf2-ARE pathway.

Evaluating sacha inchi (Plukenetia volubilis) oil stability and physicochemical properties: A comparison between conventional extraction and supercritical fluids

This study aimed to compare the effects of two extraction techniques (conventional n-hexane and supercritical CO2) on the oil extraction yields, fatty acids profile, anti-hyaluronidase activity, oxidative stability, and in vitro bioactivities of oils from Sacha Inchi (Plukenetia volubilis). Higher oil extraction yield (99 %) was achieved using the SC-CO2, although similar fatty acids profiles were depicted between both treatments (p < 0.05). The SC-CO2 oil presented higher anti-hyaluronidase (31 %) activity, but lower oxidative stability (5.05 h) compared to the solvent extraction (10 %, and 5.3 h, respectively). In vitro assays further revealed that the best human normal colon cells (FHC) cell viability (100 %), anti-inflammatory (50 % lower NO production), and antioxidant (20 % ROS reduction) activities were consistently observed in both extraction treatments at concentrations of 50 μg/mL and higher. These findings highlight the potential of supercritical CO2 extraction in yielding Sacha Inchi oil with enhanced bioactive properties without the disadvantages of the use of organic solvents extraction.

Targeting colon cancer and normal cells with cold plasma-activated water: Exploring cytotoxic effects and cellular responses

Plasma-activated water (PAW), generated by cold plasma, is emerging as a potential treatment for colon cancer. This study focused on its anticancer effects against HCT-116 colon cancer cells, emphasizing the role of pH and conductivity variations due to plasma–fluid reactions. These changes suggest a chemical transformation in PAW, leading to increased acidity and ion presence. The cytotoxic impact of PAW on HCT-116 cells was analyzed using methods like enzyme-linked immunosorbent assay and microscopic evaluation. PAW exhibited cytotoxicity against HCT-116 cells, but also affected normal colon cells, posing a challenge for selectivity. An 18 h exposure duration was identified as a balance between cancer cell eradication and normal cell preservation. Observed morphological changes indicated apoptotic characteristics in PAW-treated cells, hinting at mechanisms of cancer cell death. PAW-induced reactive oxygen species release mirrored cellular stress, with early apoptotic markers, DNA fragmentation, and increased heat shock proteins (HSPs) signifying complex cellular responses. These findings suggest that PAW can trigger apoptosis and cellular stress pathways cancer cells. However, further studies are necessary for its potential as a cancer therapy.

RREB1-mediated SUMOylation enhancement promotes chemoresistance partially by transcriptionally upregulating UBC9 in colorectal cancer.

Chemoresistance is a main cause of chemotherapy failure and tumor recurrence. The effects of global protein SUMOylation on chemoresistance in colorectal cancer (CRC) remains to be investigated. Herein, we have proposed that the elevated SUMO2/3-modified proteins confer 5-fluorouracil (5-FU) chemoresistance acquisition in CRC. The SUMOylation levels of global proteins in CRC cell lines were elevated compared with normal colon cell line NCM460. 5-FU treatment obviously reduced SUMOylation of global proteins in 5-FU-sensitive CRC cells including HT29, HCT116 and HCT-8. However, in 5-FU-resistant HCT-8/5-FU cells, the expression level of SUMO2/3-modified proteins was increased under 5-FU exposure in a concentration-dependent manner. 5-FU treatment combined with SUMOylation inhibitor ML-792 significantly increased the sensitivity of 5-FU-resistant cells to 5-FU and reduced colony formation numbers in HCT-8/5-FU cells. And UBC9-mediated SUMOylation elevation contributes to 5-FU resistance in HCT116 cells. Moreover, we also identified RREB1 as a regulator of SUMOylation profiling of global cellular proteins via directly binding to the promoter of UBC9. Overexpression of RREB1 promoted 5-FU resistance in CRC, which was partially abolished by treatment of inhibitor ML-792. In conclusion, RREB1-enhanced protein SUMOylation contributes to 5-FU resistance acquisition in CRC.

Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer

BackgroundTreatment with regorafenib, a multiple-kinase inhibitor, to manage metastatic colorectal cancers (mCRCs) shows a modest improvement in overall survival but is associated with severe toxicities. Thus, to reduce regorafenib-induced toxicity, we used regorafenib at low concentration along with a dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition to enhance antitumor activity. The therapeutic efficacy and safety of the combination treatment was evaluated with CRC models.MethodsThe cytotoxicity of JAK/HDACi, regorafenib, and their combination were tested with normal colonic and CRC cells exhibiting various genetic backgrounds. Kinomic, ATAC-seq, RNA-seq, cell cycle, and apoptosis analyses were performed to evaluate the cellular functions/molecular alterations affected by the combination. Efficacy of the combination was assessed using patient-derived xenograft (PDX) and experimental metastasis models of CRC. To evaluate the interplay between tumor, its microenvironment, and modulation of immune response, MC38 syngeneic mice were utilized.ResultsThe combination therapy decreased cell viability; phosphorylation of JAKs, STAT3, EGFR, and other key kinases; and inhibited deacetylation of histone H3K9, H4K8, and alpha tubulin proteins. It induced cell cycle arrest at G0-G1 phase and apoptosis of CRC cells. Whole transcriptomic analysis showed that combination treatment modulated molecules involved in apoptosis, extracellular matrix-receptor interaction, and focal adhesion pathways. It synergistically reduces PDX tumor growth and experimental metastasis, and, in a syngeneic mouse model, the treatment enhances the antitumor immune response as evidenced by higher infiltration of CD45 and cytotoxic cells. Pharmacokinetic studies showed that combination increased the bioavailability of regorafenib.ConclusionsThe combination treatment was more effective than with regorafenib or JAK/HDACi alone, and had minimal toxicity. A clinical trial to evaluate this combination for treatment of mCRCs is warranted.

Design and Synthesis of Quinoxaline Hybrids as Modulators of HIF-1a, VEGF, and p21 for Halting Colorectal Cancer.

A library of 16 3-benzyl-N 1-substituted quinoxalin-2-ones was synthesized as N 1-substituted quinoxalines and quinoxaline-triazole hybrids via click reaction. These compounds were tested for their anticancer activity via MTT assay on HCT-116 and normal colonocyte cell lines to assess their cytotoxic potentials and safety profiles. Overall, compounds 6, 9, 14, and 20 were found to be promising anticolorectal cancer agents; they exhibited remarkable cytotoxicity (IC50 0.05-0.07 μM) against HCT-116 cells within their safe doses (EC100) on normal colon cells. Their pronounced anticancer activities were observed as severe morphological alterations and shrinkage of the treated cancer cells. Besides, qRT-PCR analysis was conducted showing the potential of the promising hits to downregulate HIF-1a, VEGF, and BCL-2 as well as their ability to enhance the expression of proapoptotic genes p21, p53, and BAX in HCT-116 cells. In silico prediction revealed that most of our compounds agree with Lipinski and Veber parameters of rules, in addition to remarkable medicinal chemistry and drug-likeness parameters with no CNS side effects. Interestingly, docking studies of the compounds in the VEGFR-2' active site showed significant affinity toward the essential amino acids, which supported the biological results.

Comprehensive Toxicological Assessment of Halobenzoquinones in Drinking Water at Environmentally Relevant Concentration.

Halobenzoquinones (HBQs), an emerging unregulated category of disinfection byproduct (DBP) in drinking water, have aroused an increasing concern over their potential health risks. However, the chronic toxicity of HBQs at environmentally relevant concentrations remains largely unknown. Here, the occurrence and concentrations of 13 HBQs in drinking water from a northern megacity in China were examined using ultrahigh performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS). Four HBQs, including 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ), 2,3,6-trichloro-1,4-benzoquinone (TriCBQ), and 2,5-dibromo-1,4-benzoquinone (2,5-DBBQ), were detected beyond 50% occurrence frequency and at median concentrations from 4 to 50 ng/L. The chronic toxicity of these four HBQs to normal human colon and liver cells (FHC and THLE-2) was investigated at these concentrations. After 90 days of exposure, 2,5-DBBQ and 2,6-DCBQ induced the highest levels of oxidative stress and deoxyribonucleic acid (DNA) damage in colon and liver cells, respectively. Moreover, 2,5-DBBQ and 2,6-DCBQ were also found to induce epithelial-mesenchymal transition (EMT) in normal human liver cells via the extracellular signal regulated kinase (ERK) signaling pathway. Importantly, heating to 100 °C (boiling) was found to efficiently reduce the levels of these four HBQs in drinking water. These results suggested that environmentally relevant concentrations of HBQs could induce cytotoxicity and genotoxicity in normal human cells, and boiling is a highly efficient way of detoxification for HBQs.

Dual ionotropic crosslinked biopolymer magnetic nanocomposites as 5‐Fluorouracil nanocarrier for potential colorectal cancer treatment

AbstractThis study aimed to synthesize dual‐crosslinked chitosan‐alginate nanocomposites (Cs‐Alg/IO NCs) using green‐synthesized iron oxide nanoparticles (IO, Fe3O4 NPs) for drug delivery application. The NCs, formed via ionotropic gelation with sodium tripolyphosphate (TPP) and calcium chloride (CaCl2) as crosslinking agents for Cs and Alg, respectively, and further characterized for physiochemical properties. The presence of Fe3O4 NPs and 5‐Fluorouracil (5FU) increased hydrodynamic size (153.12 ± 0.57 to 207.64 ± 3.59 nm). Morphological studies revealed NC agglomeration within nanoscales. 5FU‐loaded Cs‐Alg/IO NCs (Cs‐Alg/IO 5FU NCs) exhibited superparamagnetic behavior and sustained drug release under various pH conditions. Cytotoxicity assays on colon cancer cells (HCT116 and HT‐29) and normal colon cells (CCD‐112) indicated higher cancer cell selectivity (selectivity index, SI = 1.91) in 2D models. Elevated IC50 values in 3D models were linked to lower drug loading capacity (LC) and encapsulation efficiency (EE). Overall, the study highlights the potential of dual‐crosslinked biopolymer NCs with green‐synthesized Fe3O4 NPs for developing an anticancer drug delivery system in colorectal cancer treatment.Highlights Synthesis of 5FU‐loaded dual crosslinked Cs‐Alg/IO NCs and Cs‐Alg IPN. Physicochemical characterization of cross‐linked magnetic nanocarriers. Investigation of sustained drug release using superparamagnetic nanocarriers. Evaluation of selective cytotoxicity for Cs‐Alg/IO 5FU NCs.

Non-pituitary growth hormone enables colon cell senescence evasion.

DNA damage-induced senescence is initially sustained by p53. Senescent cells produce a senescence-associated secretory phenotype (SASP) that impacts the aging microenvironment, often promoting cell transformation. Employing normal non-tumorous human colon cells (hNCC) derived from surgical biopsies and three-dimensional human intestinal organoids, we show that local non-pituitary growth hormone (npGH) induced in senescent cells is a SASP component acting to suppress p53. npGH autocrine/paracrine suppression of p53 results in senescence evasion and cell-cycle reentry, as evidenced by increased Ki67 and BrdU incorporation. Post-senescent cells exhibit activated epithelial-to-mesenchymal transition (EMT), and increased cell motility. Nu/J mice harboring GH-secreting HCT116 xenografts with resultant high GH levels and injected intrasplenic with post-senescent hNCC developed fourfold more metastases than did mice harboring control xenografts, suggesting that paracrine npGH enables post-senescent cell transformation. By contrast, senescent cells with suppressed npGH exhibit downregulated Ki67 and decreased soft agar colony formation. Mechanisms underlying these observations include npGH induction by the SASP chemokine CXCL1, which attracts immune effectors to eliminate senescent cells; GH, in turn, suppresses CXCL1, likely by inhibiting phospho-NFκB, resulting in SASP cytokine downregulation. Consistent with these findings, GH-receptor knockout mice exhibited increased colon phospho-NFκB and CXCL1, while GH excess decreased colon CXCL1. The results elucidate mechanisms for local hormonal regulation of microenvironmental changes in DNA-damaged non-tumorous epithelial cells and portray a heretofore unappreciated GH action favoring age-associated epithelial cell transformation.

Lineage tracing reveals heterogeneity within tumor-reactive CD8 T cells in mismatch repair-proficient (MMR-p) CRC

Abstract While CRC develop in the colon and rectum, little is known about the relationship between tissue resident memory (TRM) CD8 T cells and newly recruited tumor-reactive T cells. We compared the phenotype of CD8 T cells from paired adjacent normal colon (NC) and tumor by flow cytometry and observed a higher T cell infiltration in the tumor with an enrichment in CD8 T cells. Interestingly, a subset of CD103+ CD8 T cells in NC expressed CD39, a phenotype reminiscent of tumor-reactive T cells, but those cells lacked PD-1 and Ki-67. To better understand the relationship between T cells from NC and tumor and to uncover the differentiation path of tumor-reactive CD8 T cells, we performed scRNA-seq and scTCR-seq together with CITE-seq on paired NC and tumor from five patients. We confirmed that DP CD8 T cells from NC were distinct from tumor-infiltrating DP CD8 T cells (DP CD8 TILs). In contrast, the latter represented an heterogenous population with cells at different stages of differentiation as illustrated by the detection of cells sharing the same TCRs in multiple cell clusters. To further appreciate the differentiation path of neoantigen-reactive CD8 TILs, we performed neoantigen discovery on three of the five patients. We are now evaluating whether T cell differentiation and activation are linked to functional avidity and cellular location in the tumor. This work provides essential information regarding the differentiation of tumor-reactive CD8 TILs in MMR-p CRC tumors.