Normal Human Colon Research Articles

Preparation of Stereo-Divergent Compounds from the Natural Product Drupacine Based on Complexity-to-Diversity Strategy.

The Complexity-to-Diversity (CtD) strategy was applied to synthesize a 23-member compound collection from the natural product drupacine, including 21 novel compounds. An unusual benzo [d] cyclopenta [b] azepin skeleton was constructed by Von Braun reaction to cleave C-N bond of drupacine. Moreover, compound 10 has potential cytotoxicity to human colon cancer cells with low toxicity to the normal human colon mucosal epithelial cell lines.

Protective Effects of Piceatannol on DNA Damage in Benzo[a]pyrene-Induced Human Colon Epithelial Cells

Evidence shows that the dietary intake of polycyclic aromatic hydrocarbons (PAHs) from food processing induces the cellular DNA damage response and leads to the development of colorectal cancer (CRC). Therefore, protecting from cellular DNA damage might be an effective strategy in preventing CRC. Benzo[a]pyrene (B[a]P) was used as a CRC initiator in the present study. Compared with other stilbenoids, piceatannol (PIC) showed the most effective inhibition of B[a]P-induced cytochrome P450 1B1 (CYP1B1) protein expression in NCM460 normal human colon epithelial cells. PIC treatment alleviated DNA migration and enhanced the expression of DNA-repair-related proteins, including histone 2AX (H2AX), checkpoint kinase 1 (Chk1), and p53, in B[a]P-induced NCM460 cells. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) revealed that PIC exerted antioxidative effects on NCM460 cells by increasing the glutathione (GSH) content and scavenging the excess intracellular reactive oxygen species (ROS) induced by B[a]P. Furthermore, PIC suppressed B[a]P-induced CYP1B1 protein expression and stimulated miR-27b-3p expression. The upregulation of phase II detoxification enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) and quinone oxidoreductase 1 (NQO1), and the antioxidative enzyme, heme oxygenase 1 (HO-1), via the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway was observed in the PIC-treated group. Our results suggest that PIC is a potential CRC-blocking agent due to its ability to alleviate DNA damage, decrease intracellular ROS production, modulate the metabolism and detoxification of B[a]P, and activate the Nrf2 signaling pathway in B[a]P-induced NCM460 cells.

Disruption of retinol-mediated IL-6 expression in colon cancer-associated fibroblasts: new perspectives on the role of vitamin A metabolism

Stromal myo-/fibroblasts (MFs) account for up to 30% of lamina propria cells in the normal human colon and their number is dramatically increased in colon cancer (CRC). Fibroblasts from cancers, also known as cancer-associated fibroblasts (CAFs), differ from normal colonic MF (N-MFs) and support tumor-promoting inflammation, in part due to increased IL-6 secretion. In this editorial, we highlight recent data obtained regarding IL-6 regulation in colorectal cancer CAFs through vitamin A (retinol) metabolism, discuss current limitations in our understanding of the mechanisms leading to the CAF pro-inflammatory phenotype, and discuss potential approaches to target CAF retinoid metabolism during CRC treatment.

Quassinoid analogs exert potent antitumor activity via reversible protein biosynthesis inhibition in human colorectal cancer

Cellular protein synthesis is accelerated in human colorectal cancer (CRC), and high expression of protein synthesis regulators in CRC patients is associated with poor prognosis. Thus, inhibition of protein synthesis may be an effective therapeutic strategy for CRC. We previously demonstrated that the quassinoid bruceantinol (BOL) had antitumor activity against CRC. Herein, potent tumor growth suppression (>80%) and STAT3 inhibition was observed in two different mouse models following BOL administration. Loss of body and spleen weight was observed but was eliminated upon nanoparticle encapsulation while maintaining strong antitumor activity. STAT3 siRNA knockdown exhibited modest suppression of cell proliferation. Surprisingly, STAT3 inhibition using a PROTAC degrader (SD-36) had little effect on cancer cell proliferation suggesting the possibility of additional mechanism(s) of action for quassinoids. BOL-resistant (BR) cell lines, HCT116BR and HCA7BR, were equally sensitive to standard CRC therapeutic agents and known STAT3 inhibitors but resistant to homoharringtonine (HHT), a known protein synthesis inhibitor. The ability of quassinoids to inhibit protein synthesis was dependent on the structure of the C15 sidechain. Of note, BOL did not inhibit protein synthesis in normal human colon epithelial cells whereas HHT and napabucasin remained effective in these normal cells. Novel quassinoids were designed, synthesized, and evaluated in pre-clinical CRC models. Treatment with the most potent analog, 5c, resulted in significant inhibition of cell proliferation and protein synthesis at nanomolar concentrations. These quassinoid analogs may represent a novel class of protein synthesis inhibitors for the treatment of human CRC.

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

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

Methylation study of tumor suppressor genes in human aberrant crypt foci, colorectal carcinomas, and normal colon.

Aberrant crypt foci (ACF) are the earliest preneoplastic lesions in human colon, identifiable on chromoendoscopic screening. Our objective was to evaluate the %methylation of APC, CDKN2A, MLH1, RASSF1, MGMT, and WIF1 tumor suppressor genes (TSG) in ACF, corresponding colorectal carcinomas (CRC), and normal colonic mucosal controls. In this study, macroscopically normal-appearing mucosal flaps were sampled 5-10 cm away from the tumor mass from 302 fresh colectomy specimens to identify ACF-like lesions. Thirty-five cases with multiple ACFs were selected (n 35) as the main study group, with corresponding sections from CRC (n 35) as disease controls, and mucosal tissue blocks from 20 colectomy specimens (normal controls), operated for non-neoplastic pathologies. Genomic DNA was extracted, and methylation-specific polymerase chain reaction (PCR) was performed on a customized methylation array model. %Methylation data were compared among the groups and with clinicopathological parameters. Selected target mRNA and protein expression studies were performed. %Methylation of TSGs in ACF was intermediate between normal colon and CRC, although a statistically significant difference was observed only for the WIF1 gene (P < 0.01). Also, there was increased nuclear β-catenin expression and upregulation of CD44-positive cancer-stem cells in ACF and CRCs than in controls. Right-sided ACFs and dysplastic ACFs had a higher %methylation of CDKN2A (P < 0.01), whereas hyperplastic ACFs had a higher %methylation of RASSF1 (P 0.04). The topographic characteristics of ACFs did not correlate with TSG %methylation. Early epigenetic methylation of WIF1 gene is one of the mechanisms for ACF development in human colon.

Abstract 300: Mechanisms of growth hormone action in the aging tissue microenvironment

Abstract The aging tissue microenvironment undergoes progressive changes enabling age-related pathologies. Senescent cells increase with age, and the secretome released from senescence cells (SASP) affects neighboring cells, often promoting proliferation, tissue inflammation, angiogenesis, and, depending on tissue type, creating a milieu favoring neoplasia. We showed that local non-pituitary growth hormone (GH) accumulates with age in human colon tissue, is induced in response to DNA damage, and suppresses DNA repair. We now show that GH is a SASP component. To examine SASP-derived GH actions on the tissue microenvironment, we co-cultured intact human 3D intestinal organoids with organoids infected with lentivirus expressing either hGH (lentiGH) or vector (lentiV) for 5 weeks. Using whole-exome sequencing to evaluate somatic mutations and somatic copy number alterations, we found that local paracrine GH emanating from lentiGH organoids increases chromosomal instability in intact organoid cells, inducing somatic mutations including deletions, breakends, duplications, and insertions, as compared to organoids co-cultured with lentiV organoids. Mechanisms for chromosomal instability observed in these organoids likely occur as a consequence of GH-induced suppression of DNA damage repair proteins including pATM, pATR, and pBRCA2, leading to DNA damage accumulation, and, in the long run, favoring cell transformation. KEGG pathway analysis of intact organoids subjected to paracrine GH exposure identified changes in focal adhesion pathways (p=0.0016), consistent with observed increased migration, invasion, and anchorage-independent growth of normal human colon cells (hNCC) exposed to GH. Furthermore, IPA analysis revealed EMT pathway changes (p=0.0018),,also confirmed by showing increased EMT transcription factors Twist2 and Snai1 expression.Although senescence has been defined as irreversible proliferative arrest, recent studies now show that senescence can be reversed. Treating hNCC with etoposide to induce senescence and culturing senescent cells in the presence of GH for 10 days resulted in p53 suppression, enabling cells to re-enter the cell cycle, as evidenced by induced Ki67, a marker of proliferation. These post-senescent cells also acquired the stem cell marker SOX2 and exhibited increased Twist2, consistent with anchorage-independent growth and increased migration as compared to senescence cells not treated with GH. These results identify local paracrine GH as an important determinant of the local tissue microenvironment promoting age-associated changes consistent with a pro-neoplastic milieu. Citation Format: Vera Chesnokova, Svetlana Zonis, Tugce Apaydin, Christian Wang, Robert Barrett, Shlomo Melmed. Mechanisms of growth hormone action in the aging tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 300.

Paris polyphylla Sm. Induces Reactive Oxygen Species and Caspase 3-Mediated Apoptosis in Colorectal Cancer Cells In Vitro and Potentiates the Therapeutic Significance of Fluorouracil and Cisplatin.

Paris polyphylla Sm. (Melanthiaceae) is an essential, vulnerable herb with a wide range of traditional applications ranging from fever to cancer in various communities. The use of P. polyphylla in India is limited to traditional healers. Here, we demonstrated that P. polyphylla extract (PPE) has good phenol, flavonoid, saponin, and steroidal saponin content and anti-oxidant activity with IC50 35.12 ± 6.1 μg/mL in DPPH and 19.69 ± 6.7 μg/mL in ABTS. Furthermore, PPE induces cytotoxicity in HCT-116 with IC50 8.72 ± 0.71 μg/mL without significant cytotoxicity inthe normal human colon epithelial cell line, CCD 841 CoN. PPE inhibits the metastatic property and induces apoptosis in HCT-116, as measured by Annexin V/PI, by increasing the production of reactive oxygen species (ROS) and caspase 3 activation. PPE acts synergistically with 5FU and cisplatin in HCT-116 and potentiates their therapeutic significance. Steroidal saponins with anticancer activities were detected in PPE by HR-LCMS. The present study demonstrated that PPE induces apoptosis by increasing ROS and activating caspase 3, which was attributed to steroidal saponins. PPE can be used as a potential natural remedy for colon cancer.

Increased ENT2 expression and its association with altered purine metabolism in cell lines derived from different stages of colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent malignant cancer types worldwide. Although the purine metabolism pathway is vital for cancer cell survival, little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with purine metabolites. The aim of the present study was to evaluate the levels of hypoxanthine phosphoribosyl transferase (HPRT), hypoxanthine and uric acid (UA), as well as xanthine oxidase (XO) activity, and investigate their association with ENT2 expression levels in a normal human colon cell line and CRC cell lines derived from different stages of CRC. These analyses were performed using the normal colon CCD-841CoN cell line and a panel of human CRC cell lines comprising SW480, HCT15 and HCT116, which represent Dukes' B, C and D stages, respectively. Reverse transcription-quantitative PCR was performed to determine the level of ENT2 mRNA expression. In cells of all CRC stages, the levels of HPRT and hypoxanthine were significantly higher (P<0.05), while XO activity and UA levels were significantly decreased (P<0.05), compared with those in the CCD-841CoN cell line. ENT2 expression was found to be elevated in cells derived from all stages of CRC. The Dukes' D stage cell line had higher levels of HPRT and hypoxanthine, although its ENT2 level was not significantly lower than that of the Dukes' B and C stage cell lines. Increased levels of HPRT and hypoxanthine in various stages of CRC may indicate an increase in the activity of the salvage pathway. The increased expression of ENT2 implies the importance of the ENT2 protein in facilitating hypoxanthine transport, which is required for enhanced DNA synthesis via hypoxanthine recycling. In conclusion, ENT2 may have potential as a target in the development of CRC therapeutics.

Organoids transplantation as a new modality to design epithelial signature to create a membrane-protective sulfomucin-enriched segment.

The organoids therapy for ulcerative colitis (UC) is under development. It is important to dissect how the engrafted epithelium can provide benefits for overcoming the vulnerability to inflammation. We mainly focused on the deliverability of sulfomucin, which is reported to play an important role in epithelial function. We analyzed each segment of colon epithelium to determine differences in sulfomucin production in both mice and human. Subsequently, we transplanted organoids established from sulfomucin-enriched region into the injured recipient epithelium following dextran sulfate sodium-induced colitis and analyzed the engrafted epithelium in mouse model. In human normal colon, sulfomucin production was increased in proximal colon, whereas it was decreased in the inflammatory region of UC. In murine colon epithelium, increased sulfomucin production was found in cecum compared to distal small intestine and proximal colon. RNA sequencing analysis revealed that several key genes associated with sulfomucin production such as Papss2 and Slc26a1 were enriched in isolated murine cecum crypts. Then we established murine cecum organoids and transplanted them into the injured epithelium of distal colon. Although the expression of sulfomucin was temporally decreased in cecum organoids, its secretion was restored again in the engrafted patches after transplantation. Finally, we verified a part of mechanisms controlling sulfomucin production in human samples. This study illustrated the deliverability of sulfomucin in the disease-relevant grafting model to design sulfomucin-producing epithelial units in severely injured distal colon. The current study is the basis for the better promotion of organoids transplantation therapy for refractory UC.

Effects of Curcumin and Soy Isoflavones on Genomic Instability of Human Colon Cells NCM460 and SW620.

Curcumin (CUR) and soy isoflavones (SIs) are two plant-based polyphenols that have attracted much attention, because of their extensive anticancer and health maintenance effects. However, the relevant molecular mechanisms are still uncertain. Genomic instability (GIN) refers to a combination of gene abnormal amplification, sequence deletion, ectopic, and other types of gene damage in cells, and it is one of the main factors causing cells to lose normal physiological functions. Therefore, we used the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay as the main research method to analyze the effects of CUR and SIs on the GIN of human normal colon cells NCM460 and colon cancer cells SW620. Results show that CUR (12.5 μM) could reduce the apoptosis of NCM460 and maintain its genomic stability while inhibiting the proliferation of SW620 and promoting its apoptosis. There was no difference in the promoting effect of GIN between SW620 and NCM460 using SIs (3.125-50 μM). When the two polyphenols (v/v = 1/1, 1.5625-6.25 μM) were mixed, they could promote the proliferation and GIN of the NCM460 and SW620 cells, but we did not find that combining the two produced a better effect on the cells. In conclusion, CUR has more prominent health and anticancer effects, and it may become a dietary recommendation for daily health maintenance and a potential adjuvant drug for cancer treatment.

P918 Clostridium butyricum inhibits epithelial-mesenchymal transition of intestinal carcinogenesis through downregulating METTL3

Abstract Background Colorectal cancer (CRC) is related to gut microbiota dysbiosis, especially butyrate-producing bacteria reduction. Our previous study suggested administration of Clostridium butyricum (C. butyricum), a butyrate-producing bacterium, exerts a crucial effect against CRC. Methyltransferase-like 3 (METTL3) contributes to tumorigenic epigenetic regulation. We aimed to investigate the effects of C. butyricum on METTL3 in the prevention of CRC. Methods TCGA and CancerSEA databases were used to investigate METTL3 co-expressed genes, and the GO and KEGG enrichment analysis was conducted on METTL3. Tissue specimens of human normal colonic tissue, adenoma and carcinoma were obtained and examined the expression of METTL3, EMT-associated markers, and VM formation. We overexpressed METTL3 in CRC cells to assess cell migration and tube formation. Meanwhile, the effects of C. butyricum on METTL3, epithelial-mesenchymal transition (EMT), and vasculogenic mimicry (VM) formation in CRC cells were evaluated. Furthermore, a murine xenograft model was established to provide further evidence of the inhibition of C. butyricum on tumor growth. Results Database analysis suggested that METTL3 showed a positive correlation with proliferation, EMT, DNA repair, metastasis, and invasion. The expression of METTL3 gradually increased from human normal colon tissue, adenoma to carcinoma, and was positively correlated with VM formation and EMT. METTL3 overexpression promoted the proliferation, migration, and invasion of CRC cells and induced VM formation. C. butyricum could downregulate METTL3 expression in CRC cells and decrease the expression of vimentin and vascular endothelial growth factor receptor 2 to reduce EMT and VM formation. Moreover, C. butyricum alleviated the pro-oncogenic effect of METTL3 overexpressing plasmid in CRC cells. Accordingly, C. butyricum downregulated the expression of METTL3 in tumors and prevented EMT in nude mice. Conclusion C. butyricum could inhibit EMT and VM of intestinal carcinogenesis through downregulating METTL3. These findings broaden our understanding of probiotics supplement in the prevention and treatment of CRC.

Benzenesulfonyl thiazoloimines as unique multitargeting antibacterial agents towards Enterococcus faecalis

New efficient antimicrobial agents are urgently needed to combat invasive multidrug-resistant pathogens infections. Structurally unique benzenesulfonyl thiazoloimines (BSTIs) were exploited as novel potential antibacterial victors to confront terrific drug resistance. Some developed BSTIs exerted effectively antimicrobial efficacy against the tested strains. Notably, 2-pyridyl BSTI 14d exhibited good antibacterial activity against E. faecalis with MIC value of 1 μg/mL, which was superior to sulfathiazole and norfloxacin. The most active compound 14d not only showed rapid bactericidal properties and impeded E. faecalis biofilm formation to effectually relieve the development of drug resistance, but also performed low toxicity toward human red blood cells, human normal squamous epithelial cells and human non-neoplastic colon epithelial cells. Mechanistic investigation demonstrated that molecule 14d could exert efficient membrane destruction leading to the leakage of intracellular materials and metabolism inhibition, cause oxidative damage of E. faecalis through accumulation of excess reactive oxygen species and reduction of glutathione activity, and intercalate into DNA to hinder replication of DNA. Molecular docking indicated that the formation of 14d-dihydrofolate synthetase supramolecular complex could hinder the function of this enzyme. ADME analysis displayed that compound 14d possessed promising pharmacokinetic properties. These findings suggested that the newly developed benzenesulfonyl thiazoloimines with multitargeting antibacterial potential provided a new possibility for evading resistance.

A new modality for cholesterol impact tracking in colon cancer development - Raman imaging, fluorescence and AFM studies combined with chemometric analysis.

Colorectal cancer (CRC) is the third most common cancer worldwide. Obesity, alcohol consumption, smoking, high consumption of red or processed meat and a diet with low fibre, fruit, and vegetable intake increase CRC risk. Despite advances in surgery (the basic treatment for recovery), chemotherapy, and radiotherapy, CRC remains the second leading cause of cancer-related deaths in the world. Therefore the social importance of this problem stimulates research aimed at developing new tools for rapid CRC diagnosis and analysis of CRC risk factors. Considering the association between the cholesterol level and CRC, we hypothesize that cholesterol spectroscopic and AFM (atomic force microscopy) studies combined with chemometric analysis can be new, powerful tools used to visualize the cholesterol distribution, estimate cholesterol content and determine its influence on the biochemical and nanomechanical properties of colon cells. Our paper presents the analysis of human colon tissues: normal and cancer and human colon single cells normal CCD18-Co and cancer CaCo-2 in the physiological state and CaCo-2 upon mevastatin supplementation. Based on vibrational features we have shown that Raman spectroscopy and imaging allow cholesterol content in human colon tissues and human colon single cells of both types to be tracked and allow the effectiveness of mevastatin in the mevalonate pathway modulation and disruption of the cholesterol level to be proven. All observations have been confirmed by chemometric analysis including principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA). The positive impact of statins on cholesterol content has also been studied by using fluorescence microscopy and atomic force microscopy (AFM). A significant increase in Young modulus as a mechanomarker for CaCo-2 human cancer colon cells upon mevastatin supplementation compared to CCD18-Co human normal colon cells was observed. This paper is one of the first reports about the use of Raman spectroscopic techniques in cholesterol investigations and the first one about cholesterol investigation using Raman spectroscopy (RS) on human cells ex vivo in the context of colon cancer development.

Combination of metformin/efavirenz/fluoxetine exhibits profound anticancer activity via a cancer cell-specific ROS amplification

ABSTRACT The possible anticancer activity of combination (M + E + F) of metformin (M), efavirenz (E), and fluoxetine (F) was investigated in normal HDF cells and HCT116 human colon cancer cells. Metformin increased cellular FOXO3a, p-FOXO3a, AMPK, p-AMPK, and MnSOD levels in HDFs but not in HCT116 cells. Cellular ATP level was decreased only in HDFs by metformin. Metformin increased ROS level only in HCT116 cells. Transfection of si-FOXO3a into HCT116 reversed the metformin-induced cellular ROS induction, indicating that FOXO3a/MnSOD is the key regulator for cellular ROS level. Viability readout with M, E, and F alone decreased slightly, but the combination of three drugs dramatically decreased cell survival in HCT116, A549, and SK-Hep-1 cancer cells but not in HDF cells. ROS levels in HCT116 cells were massively increased by M + E + F combination, but not in HDF cells. Cell cycle analysis showed that of M + E + F combination caused cell death only in HCT116 cells. The combination of M + E + F reduced synergistically mitochondrial membrane potential and mitochondrial electron transport chain complex I and III activities in HCT116 cells when compared with individual treatments. Western blot analysis indicated that DNA damage, apoptosis, autophagy, and necroptosis-realated factors increased in M + E + F-treated HCT116 cells. Oral administration with M + E + F combination for 3 weeks caused dramatic reductions in tumor volume and weight in HCT116 xenograft model of nude mice when compared with untreated ones. Our results suggest that M + E + F have profound anticancer activity both in vitro and in vivo via a cancer cell-specific ROS amplification (CASRA) through ROS-induced DNA damage, apoptosis, autophagy, and necroptosis.

Raman Spectroscopy and Imaging Studies of Human Digestive Tract Cells and Tissues—Impact of Vitamin C and E Supplementation

Cancers of digestive tract such as colorectal cancer (CRC) and gastric cancer (GC) are the most commonly detected types of cancer worldwide and their origin can be associated with oxidative stress conditions. Commonly known and followed antioxidants, such as vitamin C and E, are widely considered as potential anti-cancer agents. Raman spectra have great potential in the biochemical characterization of matter based on the fact that each molecule has its own unique vibrational properties. Raman spectroscopy allows to precisely characterize components (proteins, lipids, nucleic acids). The paper presents the application of the Raman spectroscopy technique for the analysis of tissue samples and cells of the human colon and stomach. The main goal of this study is to show the differences between healthy and cancerous tissues from the human digestive tract and human normal and cancer colon and gastric cell lines. The paper presents the spectroscopic characterization of normal colon cells, CCD-18 Co, in physiological and oxidative conditions and effect of oxidative injury of normal colon cells upon supplementation with vitamin C at various concentrations based on Raman spectra. The obtained results were related to the Raman spectra recorded for human colon cancer cells-CaCo-2. In addition, the effect of the antioxidant in the form of vitamin E on gastric cancer cells, HTB-135, is presented and compared with normal gastric cells-CRL-7869. All measured gastric samples were biochemically and structurally characterized by means of Raman spectroscopy and imaging. Statistically assisted analysis has shown that normal, ROS injured and cancerous human gastrointestinal cells can be distinguished based on their unique vibrational properties. ANOVA tests, PCA (Principal Component Analysis) and PLSDA (Partial Least Squares Discriminant Analysis) have confirmed the main role of nucleic acids, proteins and lipids in differentiation of human colon and stomach normal and cancer tissues and cells. The conducted research based on Raman spectra proved that antioxidants in the form of vitamin C and E exhibit anti-cancer properties. In consequence, conducted studies proved that label-free Raman spectroscopy may play an important role in clinical diagnostic differentiation of human normal and cancerous gastrointestinal tissues and may be a source of intraoperative information supporting histopathological analysis.

Blockage of Nrf2 and autophagy by L-selenocystine induces selective death in Nrf2-addicted colorectal cancer cells through p62-Keap-1-Nrf2 axis

Persistent Nrf2 activation is typically noted in many cancers, including colorectal cancer (CRC), aiding cancer cells in overcoming growth stress and promoting cancer progression. Sustained Nrf2 activation, which is beneficial for cancer cells, is called “Nrf2 addiction”; it is closely associated with malignancy and poor prognosis in patients with cancer. However, Nrf2 inhibitors may have adverse effects on normal cells. Here, we found that the selenocompound l-selenocystine (SeC) is selectively cytotoxic in the Nrf2-addicted CRC cell line WiDr cells, but not in non–Nrf2-addicted mesenchymal stem cells (MSCs) and normal human colon cells. Another CRC cell line, C2BBe1, which harbored lower levels of Nrf2 and its downstream proteins were less sensitive to SeC, compared with the WiDr cells. We further demonstrated that SeC inhibited Nrf2 and autophagy activation in the CRC cells. Antioxidant GSH pretreatment partially rescued the CRC cells from SeC-induced cytotoxicity and Nrf2 and autophagy pathway inhibition. By contrast, SeC activated Nrf2 and autophagy pathway in non–Nrf2-addicted MSCs. Transfecting WiDr cells with Nrf2-targeting siRNA decreased persistent Nrf2 activation and alleviated SeC cytotoxicity. In KEAP1-knockdown C2BBe1 cells, Nrf2 pathway activation increased SeC sensitivity and cytotoxicity. In conclusion, SeC selectively attacks cancer cells with constitutively activated Nrf2 by reducing Nrf2 and autophagy pathway protein expression through the P62–Nrf2–antioxidant response element axis and eventually trigger cell death.

Circ_0000799 promotes proliferation and invasion in colorectal cancer and epithelial-mesenchymal transition

The current study aimed to investigate the effect of circ_0000799 on the biological function of colorectal cancer (CRC) cells and its mechanism. First, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed for detecting the expression of circ_0000799, miR-647, and miR-1243 in surgically resected specimens from hospitalized CRC patients, CRC-adjacent normal tissues (Normal group), human normal colon epithelial cells (FHC group), and CRC cell lines (HCT116, HT29, SW480, SW620). The cell proliferation, viability, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation assay, transwell assay in HCT116 and SW480 cells with overexpression or inhibition of circ_0000799. The targeting relationship between circ_0000799 and miR-647 was verified by dual-luciferase reporter assay. The expression of epithelial-mesenchymal transition (EMT) proteins (E-cadherin, vimentin, and N-cadherin) was tested by western blot. The expression level of circ_0000799 was significantly increased in CRC tissues and cells. Overexpression of circ_0000799 significantly increased cell proliferation rate, viability, invasion, and the EMT process, whereas knockdown of circ_0000799 inhibited the biological performance of CRC cells. Bioinformatic analysis suggested that miR-647 was regulated by circ_0000799, and a dual-luciferase reporter assay further showed a targeting relationship between the two. In addition, circ_0000799 was negatively correlated with miR-647 expression in CRC. Our findings suggest that circ_0000799 promotes proliferation and invasion in CRC and EMT. These effects of circ_0000799 may be achieved by negatively regulating miR-62.

Phenethyl Isothiocyanate-Conjugated Chitosan Oligosaccharide Nanophotosensitizers for Photodynamic Treatment of Human Cancer Cells.

The aim of this study is to synthesize phenethyl-conjugated chitosan oligosaccharide (COS) (abbreviated as ChitoPEITC) conjugates and then fabricate chlorin E6 (Ce6)-incorporated nanophotosensitizers for photodynamic therapy (PDT) of HCT-116 colon carcinoma cells. PEITC was conjugated with the amine group of COS. Ce6-incorporated nanophotosensitizers using ChitoPEITC (ChitoPEITC nanophotosensitizers) were fabricated by dialysis method. 1H nuclear magnetic resonance (NMR) spectra showed that specific peaks of COS and PEITC were observed at ChitoPEITC conjugates. Transmission electron microscope (TEM) confirmed that ChitoPEITC nanophotosensitizers have spherical shapes with small hydrodynamic diameters less than 200 nm. The higher PEITC contents in the ChitoPEITC copolymer resulted in a slower release rate of Ce6 from nanophotosensitizers. Furthermore, the higher Ce6 contents resulted in a slower release rate of Ce6. In cell culture study, ChitoPEITC nanophotosensitizers showed low toxicity against normal CCD986Sk human skin fibroblast cells and HCT-116 human colon carcinoma cells in the absence of light irradiation. ChitoPEITC nanophotosensitizers showed a significantly higher Ce6 uptake ratio than that of free Ce6. Under light irradiation, cellular reactive oxygen species (ROS) production of nanophotosensitizers was significantly higher than that of free Ce6. Especially, PEITC and/or ChitoPEITC themselves contributed to the production of cellular ROS regardless of light irradiation. ChitoPEITC nanophotosensitizers showed significantly higher PDT efficacy against HCT-116 cells than that of free Ce6. These results indicate that ChitoPEITC nanophotosensitizers have superior potential in Ce6 uptake, ROS production and PDT efficacy. In the HCT-116 cell-bearing mice tumor-xenograft model, ChitoPEITC nanophotosensitizers efficiently inhibited growth of tumor volume rather than free Ce6. In the animal imaging study, ChitoPEITC nanophotosensitizers were concentrated in the tumor tissue, i.e., fluorescence intensity in the tumor tissue was stronger than that of other tissues. We suggest that ChitoPEITC nanophotosensitizers are a promising candidate for the treatment of human colon cancer cells.

NFK prevent postoperative abdominal adhesion through downregulating the TGF-β1 signaling pathway.

Postoperative abdominal adhesions (PAAs) represent a frequent condition occurring in more than 90% of patients undergoing abdomen and pelvic surgeries, which can cause chronic abdominal pain, female infertility, and repeated bowel obstruction, requiring repetitive surgical interventions causing morbidity and mortality, as well as high costs. It is therefore of paramount clinical importance and significance to develop practical and reliable strategies for preventing the occurrence of PAAs. In this study, we demonstrated that Nianfukang (NFK, composed of polyethylene glycol 1450 and diclofenac sodium) is highly effective in preventing PAAs, likely by reducing leukocytes and inflammatory factors in the abdominal cavity, and inhibiting intestinal fibrosis in a rat model of PAAs induced by postoperative cecum scraping. We further uncovered that NFK downregulates the expression of TGF-β1, a key factor for adhesion formation, to suppress the TGF-β1/TGF-βRIII/Smad2 signaling pathway, thereby inhibiting the proliferation and migration of fibroblasts and provided evidences for the involvement of the TGF-β1/TGF-βRIII/Smad2 axis in the prevention of PAAs in normal human colon fibroblast CCD-18Co. Our findings support NFK as a potential anti-adhesive product that has the advantages of significant effectiveness, safety profile, and low cost, as well as clear mechanism of action.