Normal Intestinal Cells Research Articles

The treatment efficacy of three-layered functional polymer materials as drug carrier for orthotopic colon cancer

Colorectal cancer (CRC) is a worldwide disease posing serious threats to people’s life. Surgery and postsurgical chemotherapy are still the first choices to control the rapid progression of cancer. However, tumor recurrence and even distant metastasis are prone to occur. As a result, it is in urgent demand to find a new method to control CRC progression while inhibiting distant metastasis. On this basis, this study developed the three-layered functionalized hydrogel-fibrous membrane-hydrogel composite materials. The Chinese traditional drugs 20 (S)-ginsenoside Rg3 (Rg3) and chemotherapeutic agent 5-fluorouracil (5-Fu) were loaded in the inner hydrogel and middle fibrous membrane and could be constantly released at the same time and space. The outer hydrogel was decorated with phenylboronic acid (PA) to interact with sialic acid expressed on the CRC cell surface. The composite materials possessed biocompatibility and showed no toxicity to normal human intestinal mucosa endothelial cells HIEC. According to the results, the cell viability of CT26 could be significantly decreased in vitro. The three-layered functionalized materials inhibited the original tumor progression and distant tumor metastasis to the liver in an orthotopic colon cancer mouse model by increasing the caspase3 expression and inhibiting the expressions of Bcl-2, Ki-67, and VEGF. In addition, the functions of major organs were not significantly damaged. Our study provides a safe and efficacious method of this three-layered functionalized hydrogel-fibrous membrane-hydrogel composite materials for CRC treatment.

Abstract A013: Single-cell RNA sequencing of dedifferentiating cells from a mouse model that display oncogenic plasticity: Implications in understanding colon cancer stem cells

Abstract Characterization of colon cancer stem cells in vivo is extremely challenging. Our mouse model provides the opportunity to do so, as it displays oncogenic dedifferentiation. In this mouse model, Wnt and TGF signaling are deregulated by simultaneously mutating their transcriptional effectors, b-catenin and Smad4 respectively. This mouse model referred to as b-catenin gain-of-function and Smad4 loss-of-function (b-cateninGOF-Smad4LOF) conditional mutant displays oncogenic dedifferentiation in the intestinal epithelium. Within a week of inducing the mutation, villi cells (the mature epithelial compartment) express proliferative and stem cell markers; eventually developing adenomas (reported previously). Two striking observations, however, remain unexplained: firstly, fate reversal to stemness is seen only in a minor fraction of the epithelial cells despite the mutation being pan-epithelial; secondly, when the mutation is induced in a subset of stem cells, selective pressure is seen against the retention of mutant stem cells in the crypt s(progenitor compartment), and adenomas occur preferentially near the luminal epithelium. Our goal is to a) determine the basis for the selective pressure against mutant stem cells, and b) the differences between the normal and the b-cateninGOF-Smad4LOF mutant intestinal stem cells. To determine the gene expression profile unique to the dedifferentiating cells, we performed scRNA-seq on the dedifferentiating cells. To sort these cells–which express CD44 or proliferative markers–we integrated a reporter gene that expresses red fluorescent protein (RFP) into the b-cateninGOF-Smad4LOF mice. FACS for CD44 and RFP was performed seven days after the mutation to isolate the dedifferentiating cells from the mutant villi and the proliferating and/or CD44 positive cells in the mutant crypts. scRNA-seq confirmed enrichment of stem cell markers in the dedifferentiating cells. Gene set enrichment analyses revealed significant enrichment for the molecular signatures associated with Myc targets and active OXPHOS and Mtorc1 signaling in the dedifferentiating cells. The increase in OXPHOS is contrary to the expected metabolism in tumors. However, bulk RNA-seq for differential gene expression in the mutant villi epithelium and molecular analyses show enrichment of hypoxia gene targets and transient increase in hypoxia. These contradictory results are consistent with the spatial and temporal heterogeneity of hypoxia and metabolism observed in tumors and support the notion that intestinal stem cells rely on OXPHOS using the metabolites derived from the glycolytic metabolism of the neighboring cells. To determine the differences between normal intestinal stem cells and mutant stem cells, we cultured epithelium in 3D-matrigel to derive organoids from wild-type and mutant epithelium. The mutant organoids show higher expression of the CD44 splice variant implicated in cancer. Since stem cells are attributed to the organoid formation, our data indicate that stem cells from the mutant cells are oncogenic stem cells. Citation Format: Ansu O. Perekatt, Kylee Wrath, Zahra Hashemi, Dahlia Matouba, Christina Li. Single-cell RNA sequencing of dedifferentiating cells from a mouse model that display oncogenic plasticity: Implications in understanding colon cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A013.

Abstract PR012: Aberrant cell state plasticity mediates colorectal cancer initiation

Abstract Cell state plasticity – the ability of cells to acquire new states via differentiation programs - is an important feature of embryogenesis, allowing for tissue specification during development, and adult homeostasis, enabling adaptive responses to physiological cues and pathological states. Epigenetic governance and gene regulation endow epithelia with restricted cell state plasticity that enable tissue homeostasis while avoiding properties that may facilitate neoplasia. There is emerging evidence that aberrant expansion of the normally restricted capability for cell state plasticity to escape terminal differentiation is a key aspect of cancer initiation. The nongenetic factors and specific programs that mediate aberrant cell state plasticity require deeper characterization to understand this elusive aspect of cancer pathogenesis. Previously challenging to study, cell state plasticity can now be investigated using single cell technology, offering unprecedented molecular resolution. We applied state-of-the-art methods on primary human tissue and derivative organoids as well as different mouse models to (a) characterize aberrant transcriptional cell states enabling cancer initiation and (b) define a functional mediator of expanded cell state plasticity. Using genetically engineered and carcinogen-induced murine models of intestinal neoplasia, we demonstrate that inappropriate Sox9 expression and loss of post-mitotic villus differentiation are early events preceding cancer development. By applying single cell RNA-sequencing (scRNA-seq) to flow-sorted epithelial cells from the Lgr5Cre; Apcf/f; R26tdT genetic model, we observed that preneoplastic cells express specific intestinal stem cell genes but are otherwise transcriptionally rewired, evading resemblance to normal intestinal cell types; we therefore refer to these cells as aberrant stem cell-like (AbSC). We further analyzed the AbSC transcriptional state, searching for features that explain its distinguished gene expression profile, and found that it is characterized by impaired differentiation, enhanced regenerative capacity, and reactivation of fetal genes. The evaluation of preneoplastic colonic lesions from the carcinogen-induced mouse model and a patient with familial adenomatous polyposis (FAP) by scRNA-seq confirmed these findings. Notably, while chromatin accessibility increased at regenerative genes, new accessibility was observed at fetal intestinal genes by ATAC-seq. Genetic inactivation of Sox9 prevented adenoma formation in ApcKO mice, obstructed emergence of the aberrant transcriptional state, including genes reserved for fetal intestinal development, and restored multi-lineage differentiation by scRNA-seq. SOX9 knockdown in human adenoma organoids compromised expression of fetal genes and induced differentiation. These studies indicate that cancer initiation requires aberrant phenotypic plasticity mediated by unabated regenerative activity and developmental reprogramming. Citation Format: Pratyusha Bala, Jonathan Rennhack, Clare Morris, Daulet Aitymbayev, Sydney M. Moyer, Gina N. Duronio, William Hahn, Nilay S. Sethi. Aberrant cell state plasticity mediates colorectal cancer initiation [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR012.

Quantitative Phase Imaging Detecting the Hypoxia-Induced Patterns in Healthy and Neoplastic Human Colonic Epithelial Cells.

Hypoxia is a frequent phenomenon during carcinogenesis and may lead to functional and structural changes in proliferating cancer cells. Colorectal cancer (CRC) is one of the most common neoplasms in which hypoxia is associated with progression. The aim of this study was to assess the optical parameters and microanatomy of CRC and the normal intestinal epithelium cells using the digital holotomography (DHT) method. The examination was conducted on cancer (HT-29, LoVo) and normal colonic cells (CCD-18Co) cultured in normoxic and hypoxic environments. The assessment included optical parameters such as the refractive index (RI) and dry mass as well as the morphological features. Hypoxia decreased the RI in all cells as well as in their cytoplasm, nucleus, and nucleoli. The opposite tendency was noted for spheroid-vesicular structures, where the RI was higher for the hypoxic state. The total volume of hypoxic CCD-18Co and LoVo cells was decreased, while an increase in this parameter was observed for HT-29 cells. Hypoxia increased the radius and cell volume, including the dry mass of the vesicular content. The changes in the optics and morphology of hypoxic cells may suggest the possibility of using DHT in the detection of circulating tumor cells (CTCs).

Polystyrene micro and nano-particles induce metabolic rewiring in normal human colon cells: A risk factor for human health.

Polystyrene is a thermoplastic polymer widely used in commercial products. Like all plastics, polystyrene can be degraded into microplastic and nanoplastic particles and ingested via food chain contamination. Although the ecological impact due to plastic contamination is well known, there are no studies indicating a carcinogenic potential of polystyrene microplastics (MPs) and nanoplastics (NPs). Here, we evaluated the effects of the MPs and NPs on normal human intestinal CCD-18Co cells. Our results show that internalization of NPs and MPs induces metabolic changes under both acute and chronic exposure by inducing oxidative stress, increasing glycolysis via lactate to sustain energy metabolism and glutamine metabolism to sustain anabolic processes. We also show that this decoupling of nutrients mirrors the effect of the potent carcinogenic agent azoxymethane and HCT15 colon cancer cells, carrying out the typical strategy of cancer cells to optimize nutrients utilization and allowing metabolic adaptation to environmental stress conditions. Taken together our data provide new evidence that chronic NPs and MPs exposure could act as cancer risk factor for human health.

Anticancer Potential of Post-Fermentation Media and Cell Extracts of Probiotic Strains: An In Vitro Study.

Simple SummaryProbiotics and their metabolites are very important for human health. The aim of this research was to determine probiotic strains with the strongest inhibitory properties against intestinal cancer cells. As a result of the screening, it was possible to find two strains, i.e., Lactiplantibacillus plantarum 0991 and Levilactobacillus brevis 0983, that could inhibit the proliferation of cancer cells by induction of oxidative stress and programmed cell death. Both strains exhibit interesting anticancer properties and potential as functional food ingredients; however, the results must be confirmed in further research.Background: Lactic acid bacteria (LAB), many of which are probiotics, can produce health-promoting metabolites (postbiotics). Purpose: To assess the mechanism of antiproliferative action of postbiotics, post-fermentation media (PFM) and cell extracts (CEs) of several strains of LAB were studied against colon (Caco-2), and cervix (HeLa) cancer cell lines, as well as normal intestine (IEC-6) cells, were used as a comparison. Methods: Postbiotics of various LAB (n = 39) were screened for their antiproliferative activity. The effect of PFM and CEs on reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP production, phosphatidylserine (PS) externalisation, and apoptosis-related caspases 3/7 and 9 activation was assayed. Results: PFM and CEs showed strong dose-dependent antiproliferative activity against Caco-2 cells, up to 77.8 ± 0.8% and 58.4 ± 1.6% for PFM and CEs, respectively. Stronger inhibitory activity against cancerous (Caco-2 and HeLa) cells than against normal (IEC-6) cells was observed. PFM were more inhibitory than CEs, and both generated oxidative stress in Caco-2 cells. PFM of L. plantarum 0991 and L. brevis 0983 induced apoptosis in Caco-2 cells by the mitochondrial signalling pathway. Conclusions: Anticancer activity of PFM and CEs of LAB, as well as the ability of apoptosis induction, is strain-specific.

Aging of intestinal stem cells.

SummaryThe intestine is one of the organs that relies on stem cell function for maintaining tissue homeostasis. Recent findings on intestinal aging show that intestinal architecture, such as villus length, crypt size, and cell composition changes in the aged crypts. The correspondent decline in the regenerative capacity of the intestine is mainly due to a decline in intestinal stem cell function upon aging, as the underlying mechanisms of aging intestinal stem cells are beginning to unravel. This review summarizes our current knowledge on stem cell-intrinsic mechanisms of aging of intestinal stem cells and their connection to extrinsic factors, such as niche cells and microbiota and will introduce recent approaches to attenuate or even revert the aging of intestinal stem cells.

Transcriptomic and proteomic insights into patulin mycotoxin-induced cancer-like phenotypes in normal intestinal epithelial cells.

Patulin (PAT) is a natural contaminant of fruits (primarily apples) and their products. Significantly, high levels of contamination have been found in fruit juices all over the world. Several in vitro studies have demonstrated PAT's ability to alter intestinal structure and function. However, in real life, the probability of low dose long-term exposure to PAT to humans is significantly higher through contaminated food items. Thus, in the present study, we have exposed normal intestinal cells to non-toxic levels of PAT for 16weeks and observed that PAT had the ability to cause cancer-like properties in normal intestinal epithelial cells after chronic exposure. Here, our results showed that chronic exposure to low doses of PAT caused enhanced proliferation, migration and invasion ability, and the capability to grow in soft agar (anchorage independence). Moreover, an in vivo study showed the appearance of colonic aberrant crypt foci (ACFs) in PAT-exposed Wistar rats, which are well, establish markers for early colon cancer. Furthermore, as these neoplastic changes are consequences of alterations at the molecular level, here, we combined next-generation RNA sequencing with liquid chromatography mass spectrometry-based proteomic analysis to investigate the possible underlying mechanisms involved in PAT-induced neoplastic changes.

ORAL ADMINISTRATION OF NANOLIPOSOMES LOADED M13 MAY TREAT ULCERATIVE COLITIS AND COLITIS-ASSOCIATED CANCER WITHOUT CAUSING ANY SIDE EFFECTS

Abstract Intestinal inflammation, especially chronic (such as inflammatory bowel disease [IBD]), is an indispensable participant in the colonic neoplastic process, fostering the proliferation, survival, and migration of colonic tumor cells. Molecularly targeted therapeutics (e.g. anti-TNF-α agents) that exploit the targets involved in the pathophysiology of inflammatory responses in IBD are the most potent drugs currently available to treat IBD, which may also present anti-colon cancer effects. Unfortunately, most of these drugs were administered systemically in a high dose, and their use is limited by severe side effects. There is an unmet need for a targeted carrier system capable of delivering drugs specifically and effectively to inflamed colonic regions over a prolonged period. Such a system could significantly reduce drug dosage and side effects that limit existing treatments. The goal of the present study is to engineer natural “nanoliposomes” from the lipids of ginger-derived-nanoparticles and load with an anti-inflammatory drug candidate (M13), test its anticancer activity, and evaluate its safety. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay demonstrated that M13, a stable metabolite from 6-Shogaol as demonstrated by the incubation with intestinal microsomes, was more cytotoxic against cancerous intestinal cells (e.g. HCT-116, Caco-2/BBe, and human colon cancer stem cell) than normal intestinal cells (e.g. HIEC-6, FHC, and human colon stem cell). The results suggested that M13 has more a better ability to specifically target cancerous intestinal cells over normal intestinal cells. Protein point hybridization data showed that nanoliposomes-loaded M13 decreased the expression levels of proteins involved in colon cancer progressions such as Survivin, EGFR, and BCL-x in colon cancer cells. Further, orally administered nanoliposomes-loaded M13 presented potent wound-healing/anti-inflammatory effects in the DSS-induced mouse model of colitis and anti-cancer effects in the AOM/DSS-induced colon cancer model. We also demonstrated that nanoliposomes-loaded M13 treatment did not affect blood. In addition, we examined the anti-inflammatory activity of M13 on active ulcerative mucosal biopsies (Reprocell tissue protocol TPS-008) from a patient (42-year-old white male) undergoing therapeutic resection for ulcerative colitis. The results of the RT2 Profiler PCR Array demonstrated that M13 had very potent anti-inflammatory activity and reduced the expression levels of the pro-inflammatory cytokines involved in IBD, including TNF-α, IL-1β, IL-6, and IL-23A, in the M13-treated human ulcerative biopsies compared to the untreated controls. Our proposed targeted therapy using nanoliposomes that deliver M13 to the colon might be used to treat ulcerative colitis and colitis-associated cancer without causing any side effects.

ORAL ADMINISTRATION OF NANOLIPOSOMES LOADED M13 MAY TREAT ULCERATIVE COLITIS AND COLITIS-ASSOCIATED CANCER WITHOUT CAUSING ANY SIDE EFFECTS

Intestinal inflammation, especially chronic (such as inflammatory bowel disease [IBD]), is an indispensable participant in the colonic neoplastic process, fostering the proliferation, survival, and migration of colonic tumor cells. Molecularly targeted therapeutics (e.g. anti-TNF-α agents) that exploit the targets involved in the pathophysiology of inflammatory responses in IBD are the most potent drugs currently available to treat IBD, which may also present anti-colon cancer effects. Unfortunately, most of these drugs were administered systemically in a high dose, and their use is limited by severe side effects. There is an unmet need for a targeted carrier system capable of delivering drugs specifically and effectively to inflamed colonic regions over a prolonged period. Such a system could significantly reduce drug dosage and side effects that limit existing treatments. The goal of the present study is to engineer natural “nanoliposomes” from the lipids of ginger-derived-nanoparticles and load with an anti-inflammatory drug candidate (M13), test its anticancer activity, and evaluate its safety. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay demonstrated that M13, a stable metabolite from 6-Shogaol as demonstrated by the incubation with intestinal microsomes, was more cytotoxic against cancerous intestinal cells (e.g. HCT-116, Caco-2/BBe, and human colon cancer stem cell) than normal intestinal cells (e.g. HIEC-6, FHC, and human colon stem cell). The results suggested that M13 has more a better ability to specifically target cancerous intestinal cells over normal intestinal cells. Protein point hybridization data showed that nanoliposomes-loaded M13 decreased the expression levels of proteins involved in colon cancer progressions such as Survivin, EGFR, and BCL-x in colon cancer cells. Further, orally administered nanoliposomes-loaded M13 presented potent wound-healing/anti-inflammatory effects in the DSS-induced mouse model of colitis and anti-cancer effects in the AOM/DSS-induced colon cancer model. We also demonstrated that nanoliposomes-loaded M13 treatment did not affect blood. In addition, we examined the anti-inflammatory activity of M13 on active ulcerative mucosal biopsies (Reprocell tissue protocol TPS-008) from a patient (42-year-old white male) undergoing therapeutic resection for ulcerative colitis. The results of the RT2 Profiler PCR Array demonstrated that M13 had very potent anti-inflammatory activity and reduced the expression levels of the pro-inflammatory cytokines involved in IBD, including TNF-α, IL-1β, IL-6, and IL-23A, in the M13-treated human ulcerative biopsies compared to the untreated controls. Our proposed targeted therapy using nanoliposomes that deliver M13 to the colon might be used to treat ulcerative colitis and colitis-associated cancer without causing any side effects.

Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer.

Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer.

In Vitro Selective Antibacterial and Antiproliferative Effects of Ethanolic Extracts from Cambodian and Philippine Plants Used in Folk Medicine for Diarrhea Treatment.

Bacterial diarrhea remains a global health problem, especially in developing tropical countries. Moreover, dysbiosis caused by diarrheagenic bacteria and inappropriate antimicrobial treatment has been associated with intestinal carcinogenesis. Despite the rich tradition of the use of herbs for the treatment of gastrointestinal disorders in Cambodian and Philippine folk medicine, many of them have not yet been systematically studied for their in vitro selective inhibitory effects on intestinal bacteria and cells. In the present study, in vitro inhibitory activities of 35 ethanolic extracts derived from 32 Cambodian and Philippine medicinal plants were determined by broth microdilution method against 12 pathogenic bacteria. Furthermore, cytotoxicity against intestinal cancer cells (Caco-2 and HT-29) using thiazolyl blue tetrazolium bromide cytotoxicity assay and safety to six beneficial intestinal bacteria (bifidobacteria and lactobacilli) and intestinal normal cells (FHs 74 Int) were determined for the antimicrobially active extracts. Selectivity indices (SIs) were calculated among the averages of minimum inhibitory concentrations (MICs), half-maximal inhibitory concentrations (IC50), and 80% inhibitory concentrations of proliferation (IC80) for each type of the tested agents. The extracts of Artocarpus blancoi (Elmer) Merr. (Moraceae), Ancistrocladus tectorius (Lour.) Merr. (Ancistrocladaceae), and Pentacme siamensis (Miq.) Kurz (Dipterocarpaceae) produced significant growth-inhibitory effects (MICs = 32–512 μg/ml) against intestinal pathogenic bacteria at the concentrations nontoxic to normal intestinal cells (IC80 values >512 μg/ml; SIs = 0.11–0.2). Moreover, the extract of P. siamensis (Miq.) Kurz was relatively safe to beneficial bacteria (MICs ≥512 μg/ml; SI = 0.1), and together with A. blancoi (Elmer) Merr., they selectively inhibited intestinal cancer cells (IC50 values ≥51.98 ± 19.79 μg/ml; SIs = 0.3 and 0.6). Finally, a strong selective antiproliferative effect on cancer cells (IC50 values 37.89 ± 2.68 to 130.89 ± 13.99 μg/ml; SIs = 0.5) was exerted by Ehretia microphylla Lam. (Boraginaceae), Lagerstroemia cochinchinensis Pierre ex Gagnep. (Lythraceae), and Melastoma saigonense (Kuntze) Merr. (Melastomataceae) (leaves with flower buds). The results suggest that the above-mentioned species are promising materials for the development of new selective antibacterial and antiproliferative agents for the treatment of infectious diarrhea and associated intestinal cancer diseases. However, further research is needed regarding the isolation and identification of their active constituents.

Long noncoding RNA plasmacytoma variant translocation 1 promotes progression of colorectal cancer by sponging microRNA-152-3p and regulating E2F3/MAPK8 signaling.

The purpose of this study was to investigate the pathogenesis of colorectal cancer (CRC) and the effects of the long noncoding RNA plasmacytoma variant translocation 1 (PVT1) on CRC progression. Bioinformatics analysis verified PVT1 expression in tumor and normal tissues. Quantitative PCR and western blotting were used to measure mRNA and protein levels, respectively. The MTT, Transwell, colony formation, and in vivo assays were used to assess the effects of PVT1 on proliferation, migration, and invasion by CRC cells. Both PVT1 and microRNA (miR)‐152‐3p were shown to be colocalized in CRC cells using FISH assay. The target genes of miR‐152‐3p were predicted and verified by bioinformatics analysis, luciferase assay, and RNA pull‐down assay. The ChIP assay revealed that E2F3 binds with the promoter of MAPK8. We found that PVT1 was overexpressed in CRC specimens, and its expression was higher in CRC cells than normal intestinal cells. Overexpression of PVT1 enhanced the proliferation, migration, and invasion of CRC cells, whereas PVT1 knockdown inhibited these processes. MicroRNA‐152‐3p was a target of PVT1, and E2F3 was a target of miR‐152‐3p. Rescue experiments confirmed the interaction between miR‐152‐3p and PVT1 and between miR‐152‐3p and E2F3. Luciferase and ChIP assay results confirmed that E2F3 modulates the transcriptional activation of MAPK8. Long noncoding RNA PVT1 activated E2F3 signaling by sponging miR‐152‐3p. The PVT1/miR‐152‐3p/E2F3/MAPK8 axis promoted CRC progression.

Therapeutic Deep Eutectic Systems towards the Treatment of Tuberculosis and Colorectal Cancer: Opportunities and Challenges

What if a new technology based on therapeutic deep eutectic systems would disrupt the current treatment of major economic and socially burden diseases? The classical definition of eutectic systems is that they are the combination of two or more compounds that interact via hydrogen bonds, from which results a melting temperature depression in comparison with that of its individual components. Therapeutic deep eutectic systems are defined as eutectic systems in which at least one of the individual components is an active pharmaceutical ingredient, or a eutectic system in which the active pharmaceutical ingredient is dissolved. Current literature reports on tuberculosis have been mostly based on the most common anti-tuberculosis drugs prescribed. Using eutectic systems based on naturally occurring molecules known for their anti-microbial activity may also present a promising therapeutic strategy able to cope with the prevalence of Mycobacterium tuberculosis and prevent the appearance of multidrug resistance strains. With regards to colorectal cancer, literature has been unravelling combinations of terpenes with anti-inflammatory drugs that are selectively cytotoxic towards colorectal cancer cells and do not compromise the viability of normal intestinal cells. This technology could contribute to preventing tumor growth and metastasis while providing a patient compliance therapeutics, which will be crucial to the success of overcoming the challenges presented by cancers.

Methanolic extract of coconut (Cocos nucifera L.) haustorium mitigates pro-oxidant-mediated apoptotic cell death via Nrf-2 pathway and lipopolysaccharide-induced cytokine release in cells

Cocos nucifera L is well-known for its edible and medicinal uses; multiple products from the different parts of the plant are being used by various communities in India. A less explored product is the coconut haustorium, the solid endosperm portion, which is highly nutritious. The present study first time evaluated the antioxidant, anti-inflammatory and cytoprotective properties of methanolic extract of Coconut haustorium (CHE). The cytoprotective effect of CHE was evaluated in hydrogen peroxide/malondialdehyde-induced toxicity IEC-6 (normal intestinal cell). Initially the biologically safe concentration of CHE and IC50 value of hydrogen peroxide and malondialdehyde was estimated by MTT assay. Treatment with H2O2 and MDA led to glutathione depletion and reduction in the catalase and glutathione reductase activity; which resulted in the increased oxidative stress and thereby cell death. Pretreatment with the different concentrations (10, 20 and 40 µg/mL) of CHE offered significant and dose-dependent protection against the toxicity induced by hydrogen peroxide and malondialdehyde. It is observed that the CHE pretreatment restored the antioxidant enzyme activities and GSH levels back to normal; the improvement in antioxidant status may be through the upregulation of Nrf2/HO1 pathway as observed in CHE pretreated cells. The anti-inflammatory potential was evaluated in the lipopolysaccharide (LPS-1 µg/mL) induced model in macrophages (Raw 264.7 cells). CHE pretreatment reduced the LPS- induced release of cytokines such as IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α). The results indicated the antioxidant, anti-inflammatory and cytoprotective effect of less explored haustorium from Coconut.

Single-cell DNA and RNA sequencing reveals the dynamics of intra-tumor heterogeneity in a colorectal cancer model

BackgroundIntra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. Mouse models that recapitulate cancer development are useful for controlled serial time sampling.ResultsWe performed single-cell exome and transcriptome sequencing of 200 cells to investigate how ITH is generated in a mouse colorectal cancer model. In the model, a single normal intestinal cell is grown into organoids that mimic the intestinal crypt structure. Upon RNAi-mediated downregulation of a tumor suppressor gene APC, the transduced organoids were serially transplanted into mice to allow exposure to in vivo microenvironments, which play relevant roles in cancer development. The ITH of the transcriptome increased after the transplantation, while that of the exome decreased. Mutations generated during organoid culture did not greatly change at the bulk-cell level upon the transplantation. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes after the transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages.ConclusionsThis study revealed an evolutionary dynamics of single-cell RNA and DNA heterogeneity in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.

LEF1 silencing sensitizes colorectal cancer cells to oxaliplatin, 5-FU, and irinotecan

Colorectal cancer (CRC) is the third most prevalent cancer all around the world. Chemotherapy plays an essential role in the treatment of CRC while Oxaliplatin, Irinotecan, and 5 – fluorouracil (5-FU) are the most commonly used chemotherapeutic drugs. However, chemo-resistance is a major obstacle to successful therapy. It has been shown that inhibition of Wnt signaling pathway can sensitize the cells to chemotherapy. Lymphoid enhancer factor (LEF1) is a member of TCF/LEF transcription family mediating Wnt nuclear responses. The long isoform of LEF1 is highly expressed in colorectal cancer cells compared to the normal intestinal cells, in which expression of the short isoform is dominant. We found that the downregulation of long isoforms of LEF1 makes CRC cell lines more sensitive to the effect of chemotherapeutic drugs. This sensitivity is imposed by reduced proliferation, increased apoptosis, or cell cycle arrest. Our results also demonstrated that there is a balance in the expression of long, and short isoforms of LEF1. In summary, we showed the role of LEF1 in chemo-resistance of colorectal cancer cells to Oxaliplatin, Irinotecan and 5-FU.

Virgin coconut oil complements with its polyphenol components mitigate sodium fluoride toxicity in vitro and in vivo

Virgin coconut oil (VCO), prepared from fresh coconut kernel without any chemical refining, is an emerging functional food. The pharmacological benefits of VCO are believed to be due to the natural combination of phenolics. Although cell culture studies have demonstrated the antioxidant activity of VCO under different oxidative stress conditions, a valid in vivo demonstration of the antioxidant activity of VCO is yet to come. Sodium fluoride (NaF), an environmental pollutant, is widely used to induce oxidative stress in cell culture models and rodents to test the antioxidant potential of several compounds. Herein, VCO and its polyphenolic (VCOP) and non-phenolic oil fraction (VCOF) were individually tested in fluoride-exposed normal intestinal cells (IEC-6) and mice to address their contribution to the documented antioxidant potential. It was found that pretreatment of VCOP (40 µg/mL) was effective in mitigating the fluoride-induced cell death when compared to VCO (200 µg/mL) and VCOF (160 µg/mL). Further, exposure to fluoride (10 mM), increased the intracellular ROS measured based on the dichlorofluorescein (DCF) fluorescence, and this, in turn, was significantly reduced when the cells were supplemented with VCOP. Oral administration of VCO (2 mL/kg bwt) reversed the drop in the hepatic catalase and SOD activities to near normal with a minimal level of lipid peroxidation in fluoride intoxicated mice. However, VCOP and VCOF were less effective in lowering the fluoride-induced increase in hepatic oxidative stress markers. It is reasoned that the oil components of VCO complement the natural antioxidant molecules resulting in an overall increase in their bioavailability.

Mechanism of dimerization and structural features of human LI-cadherin

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion–free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin–dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.

Long noncoding RNA TUG1 regulates the progression of colorectal cancer through miR-542-3p/TRIB2 axis and Wnt/\u03b2-catenin pathway

BackgroundColorectal cancer (CRC) is one of the third normal malignancy worldwide. Taurine-upregulated gene 1 (TUG1), a member of long noncoding RNAs (lncRNAs), has been reported to be involved in various cancers. However, the mechanism underlying TUG1 in the progression of CRC remains unclear.MethodsThe expression of TUG1, microRNA-542-3p (miR-542-3p), and tribbles homolog 2 (TRIB2) in CRC tissues and cells (LoVo and HCT116) were detected by quantitative real-time PCR (qRT-PCR). Methyl thiazolyl tetrazolium (MTT), transwell and flow cytometry assays were employed to evaluate the effects of TUG1 in CRC cells. The interaction between miR-542-3p and TUG1 or TRIB2 were verified by dual-luciferase reporter assay. A xenograft tumor model in nude mice was established to investigate the biological role of TUG1 in CRC in vivo.ResultsTUG1 was increased in CRC tissues and cells (LoVo and HCT116) in contrast with adjacent normal tissues and normal intestinal mucous cells (CCC-HIE-2). Downregulation of TUG1 or TRIB2 suppressed the proliferation, migration, invasion, and induced apoptosis in CRC cells. And knockdown of TUG1 repressed tumor growth in vivo. Besides, overexpression of TRIB2 reversed the effects of TUG1 depletion on the progression of CRC. Meanwhile, TUG1 interacted with miR-542-3p and TRIB2 was a target of miR-542-3p. Furthermore, miR-542-3p knockdown or TRIB2 overexpression partly reversed the suppression effect of TUG1 depletion on the Wnt/β-catenin pathway.ConclusionsTUG1 served as a tumor promoter, impeded the progression of CRC by miR-542-3p/TRIB2 axis to inactivate of Wnt/β-catenin pathway, which providing a novel target for CRC treatment.