Proliferation Of HCT116 Cells Research Articles

Negative regulation of HDAC3 transcription by histone acetyltransferase TIP60 in colon cancer.

Colon cancer is the third most common cancer globally. The expression of histone deacetylase 3 (HDAC3) is upregulated, whereas the expression of tat interactive protein, 60kDa (TIP60) is downregulated in colon cancer. However, the relationship between HDAC3 and TIP60 in colon cancer has not been clearly elucidated. We investigated whether TIP60 could regulate the expression of HDAC3 and suppress colon cancer cell proliferation. RNA sequencing data (GSE108834) showed that HDAC3 expression was regulated by TIP60. Subsequently, we generated TIP60-knockdown HCT116 cells and examined the expression of HDAC3 by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined the expression pattern of HDAC3 in various cancers using publicly available datasets. The promoter activity of HDAC3 was validated using a dual-luciferase assay, and transcription factors binding to HDAC3 were identified using GeneCards and Promo databases, followed by validation using chromatin immunoprecipitation-quantitative polymerase chain reaction. Cell proliferation and apoptosis were assessed using colony formation assays and fluorescence-activated cell sorting analysis of HCT116 cell lines. In response to TIP60 knockdown, the expression level and promoter activity of HDAC3 increased. Conversely, when HDAC3 was downregulated by overexpression of TIP60, proliferation of HCT116 cells was inhibited and apoptosis was promoted. TIP60 plays a crucial role in the regulation of HDAC3 transcription, thereby influencing cell proliferation and apoptosis in colon cancer. Consequently, TIP60 may function as a tumor suppressor by inhibiting HDAC3 expression in colon cancer cells.

Interferon-induced transmembrane protein 2 is a prognostic marker in colorectal cancer and promotes its progression by activating the PI3K/AKT pathway

BackgroundInterferon-induced transmembrane protein 2 (IFITM2) is involved in repressing viral infection. This study aim to investigate the expression of IFITM2 in colorectal cancer (CRC) and explore its effect on cell proliferation, migration, and invasion.MethodsWe analyzed The Cancer Genome Atlas (TCGA) database for IFITM2 expression in colorectal cancer and used western blots to detect IFITM2 protein in specimens and cell lines of colorectal cancers. To assess the association between IFITM2 and clinical features, both univariate and multivariate cox regression analysis were conducted. Kaplan–Meier plots were used in the TCGA database to assess IFITM2 gene expression's prognostic significance. Silencing IFITM2 in SW480 and HCT116 cells was achieved by transient transfection with siRNA. Proliferation of CRCs was examined using Cell Counting Kit-8. The effect of IFITM2 on the migration and invasion of CRC cells was studied using wound healing and transwell assays. Gene set enrichment analysis (GSEA) was used to examine IFITM2-associated pathways and Western blotting was used to confirm it.ResultsIFITM2 was over-expressed in the CRC tissues and cells, with high IFITM2 expression related to the tumor N, M, and pathologic stages. The presence of IFITM2 significantly impacted patient survival in CRC. The proliferation of SW480 and HCT116 cells was suppressed when IFITM2 was silenced, resulting in weakened migration and invasion of CRC cells. GSEA analysis showed that IFITM2 was positively related to the phosphoinositide 3-kinase (PI3K)/AKT pathway, and western blot results confirmed that IFITM2 activated it.ConclusionsIFITM2 was over-expressed in CRC and modulated the PI3K/AKT pathway to promote CRC cells proliferation and metastasis.

Novel miR-490-3p/hnRNPA1-b/PKM2 axis mediates the Warburg effect and proliferation of colon cancer cells via the PI3K/AKT pathway.

Heterogeneous ribonucleoprotein A1 (hnRNPA1) has been reported to enhance the Warburg effect and promote colon cancer (CC) cell proliferation, but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated. To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway. Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b. The relationship between the expression values and the clinicopathological features of the patients was investigated. Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction, while differences in protein expression were analyzed using western blot. Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays, and cell cycle and apoptosis were detected using flow cytometric assays. The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay. The Warburg effect was evaluated by glucose uptake and lactic acid production assays. The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls (P < 0.05). Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC, including stage I, II-III, and IV. Furthermore, the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification. HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway, thereby promoting proliferation of HCT116 and SW620 cells. However, the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b, effectively blocking the Warburg effect. These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.

Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits.

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

SLC38A3 Promotes the Proliferation and Migration of Tumor Cells and Predicts Poor Prognosis in Colorectal Cancer.

Previous studies have revealed that abnormal expressions of membrane transporters were associated with colorectal cancer (CRC). We herein performed a comprehensive bioinformatics analysis to identify the key transporter protein-related genes involved in CRC and potential mechanisms. Differentially expressed transporter protein-related genes (DE-TPRGs) were identified from CRC and normal samples using The Cancer Genome Atlas database. SLC38A3 expression was validated by immunohistochemistry and RT-qPCR, and the potential mechanism was explored. A total of 63 DE-TPRGs (29 up-regulated and 34 down-regulated) were screened. Inside, ABCC2, ABCG2, SLC4A4, SLC9A3, SLC15A1, and SLC38A3 were identified as hub genes. SLC38A3 is indeed upregulated in colorectal cancer patients. Furthermore, we found that knockdown of SLC38A3 inhibited the proliferation and migration of HCT116 cells, and Hsp70 ATPase activator could rescue it. Overall, SLC38A3 is a novel potential biomarker involved in CRC progression and promotes the proliferation and migration of tumor cells by positively regulating the function of Hsp70.

NLRX1: a key regulator in mitochondrial respiration and colorectal cancer progression.

Colorectal cancer (CRC) is a prevalent and aggressive malignancy with high mortality rates and significant risks to human well-being. Population-wide screening for tumor suppressor genes and oncogenes shows promise for reducing the incidence and fatality of CRC. Recent studies have suggested that NLRX1, an innate immunity suppressor, may play a role in regulating chronic inflammation and tumorigenesis. However, further investigation is needed to understand the specific role of NLRX1 in CRC. To evaluate the impact of NLRX1 on migration, invasion, and metastasis, two human colon cancer cell lines were studied in vitro. Additionally, a knockout mouse tumor-bearing model was used to validate the inhibitory effect of NLRX1 on tumor emergence and progression. The Seahorse XF96 technology was employed to assess mitochondrial function and glycolysis in colorectal cancer cells overexpressing NLRX1. Moreover, public databases were consulted to analyze gene and protein expression levels of NLRX1. Finally, the results were validated using a series of CRC patient samples. Our findings demonstrate that downregulation of NLRX1 enhances proliferation, colony formation, and tumor-forming capacity in HCT116 and LoVo cells. Conversely, overexpression of NLRX1 negatively impacts basal respiration and mitochondrial ATP-linked respiration in both cell lines, resulting in a notable decrease in maximal respiration during the standard mitochondrial stress test. Furthermore, analysis of data from the TCGA database reveals a significant reduction in NLRX1 expression in colon and rectal cancer tissues compared to normal tissues. This result was validated using clinical samples, where immunohistochemistry staining and western blotting demonstrated a notable reduction in NLRX1 protein levels in CRC compared to adjacent normal tissues. The decreased expression of NLRX1 may serve as a significant prognostic indicator and diagnostic biomarker for CRC patients.

Role of the 3-mercaptopyruvate sulfurtransferase in colon/colorectal cancers

The 3-mercaptopyruvate sulfurtransferase (MPST) is a protein persulfidase, occurring mainly in mitochondria. Although function of this protein in cancer cells has been already studied, no clear outcome can be postulated up to now. Therefore, we focused on the determination of function of MPST in colon (HCT116 cells)/colorectal (DLD1 cells) cancers. In silico analysis revealed that in gastrointestinal cancers, MPST together with its binding partners can be either of a high risk or might have a protective effect. Silencing of MPST gene resulted in decreased ATP, while acetyl-CoA levels were elevated. Increased apoptosis was detected in cells with silenced MPST gene, which was accompanied by decrease in mitochondrial membrane potential, but no changes in IP3 receptor’s protein. Mitochondria underwent activation of fission and elevated DRP1 expression after MPST silencing. Proliferation and migration of DLD1 and HCT116 cells were markedly affected, showing the importance of MPST protein in colon/colorectal cancer development.

Urocortin-1 promotes colorectal cancer cell migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway

PurposeTo investigate the effect of urocortin-1 (UCN-1) on growth, migration, and apoptosis in colorectal cancer (CRC) in vivo and vitro and the mechanism by which UCN-1 modulates CRC cells in vitro.MethodsThe correlation between UCN-1 and CRC was evaluated using The Cancer Genome Atlas (TCGA) database and a tissue microarray. The expression of UCN-1 in CRC cells was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. In vitro, the influence of UCN-1 on the proliferation, apoptosis, and migration of HT-29, HCT-116, and RKO cells was explored using the celigo cell counting assay or cell counting kit-8 (CCK8), flow cytometry, and wound healing or Transwell assays, respectively. In vivo, the effect of UCN-1 on CRC growth and progression was evaluated in nude mice. The downstream pathway underlying UCN-1-mediated regulation of CRC was determined using the phospho-kinase profiler array in RKO cells. Lentiviruses were used to knockdown or upregulate UCN-1 expression in cells.ResultsBoth the TCGA and tissue microarray results showed that UCN-1 was strongly expressed in the tissues of patients with CRC. Furthermore, the tissue microarray results showed that the expression of UCN-1 was higher in male than in female patients, and high expression of UCN-1 was associated with higher risk of lymphatic metastasis and later pathological stage. UCN-1 knockdown caused a reduction in CRC cell proliferation, migration, and colony formation, as well as an increase in apoptosis. In xenograft experiments, tumors generated from RKO cells with UCN-1 knockdown exhibited reduced volumes and weights. A reduction in the expression of Ki-67 in xenograft tumors indicated that UCN-1 knockdown curbed tumor growth. The human phospho-kinase array showed that the p53 signaling pathway participated in UCN-1-mediated CRC development. The suppression in migration and proliferation caused by UCN-1 knockdown was reversed by inhibitors of p53 signal pathway, while the increase in cell apoptosis was suppressed. On the other hand, overexpression of UCN-1 promoted proliferation and migration and inhibited apoptosis in CRC cells. Overexpression of p53 reversed the effect of UCN-1 overexpression on CRC development.ConclusionUCN-1 promotes migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.

Indoxyl sulfate contributes to colorectal cancer cell proliferation and increased EGFR expression by activating AhR and Akt.

Although patients with chronic kidney disease (CKD) have a higher risk of colorectal cancer (CRC) aggravation, the connection between these two diseases is not well understood. Recent studies have shown that both CKD and CRC aggravation are closely related to an increased abundance of indole-producing Fusobacterium nucleatum in the gut. The indole absorbed from the gut is eventually metabolized to indoxyl sulfate in the liver. Since indoxyl sulfate is involved not only in accelerating CKD progression but also in the initiation and development of its associated complications, the present study aimed to clarify whether indoxyl sulfate induces the proliferation of CRC cells. This study found that indoxyl sulfate induced the proliferation of CRC-derived HCT-116 cells by activating the aryl hydrocarbon receptor (AhR) and the proto-oncogene Akt. The AhR antagonist CH223191 and Akt inhibitor MK2206 suppressed indoxyl sulfate-induced proliferation of HCT-116 cells. We also found that indoxyl sulfate upregulated epidermal growth factor receptor (EGFR) expression, which is associated with poor prognosis of CRC, whereas CH223191 and MK2206 repressed EGFR expression. Furthermore, indoxyl sulfate increased the sensitivity of CRC cells to EGF by upregulating EGFR expression. These findings suggest that indoxyl sulfate may be an important link between CKD and CRC aggravation.

Abstract 1478: Chronic perfluorooctanesulfonic acid exposure promotes proliferation of colorectal cancer cells

Abstract Background: Chronic exposure to long-term exposures to per- and polyfluoroalkyl substances (PFAS), widely known as “forever chemicals”, has been associated with multiple negative health outcomes including increased risk of cancer. Perfluorooctanesulfonic acid (PFOS), a “long-chain” subtype of PFAS, has high bioaccumulation potential with a long elimination half-life. PFOS is frequently detected in drinking water leading to its high absorption through the gastrointestinal tract. Recent studies demonstrate that PFAS exposures promote intestinal inflammation and gut barrier dysfunction. However, how a long-term PFOS exposure affects colorectal cancer (CRC) progression is not known. Therefore, the purpose of this study is to delineate the effect of PFOS exposure on CRC cell proliferation and test the potential mitigation strategy for the harmful effects of PFOS. Methods: SW480 and HCT116 cells have been treated with 1 ug/mL PFOS and proliferation was measured at 1, 2, and 3 months using the Presto Blue Cell Viability Reagent fluorescence assay. Proliferation markers were assessed by qPCR and Western blot. Control and PFOS exposed cells were treated with sulforaphane, a nuclear factor erythroid 2-related factor 2 (Nrf-2) inducer, at concentration 5, 10 and 20 uM for 72 hours. Results: We show that chronic, low-dose PFOS exposure promotes proliferation of SW480 and HCT116 cell lines starting at 3 months since the first exposure. The increase in proliferation is associated with upregulation of Cyclin D, pAkt and FASN. We also show that sulforaphane significantly decreases proliferation of HCT116 and SW480 cells, and its efficacy is significantly higher in PFOS exposed CRC cells. The current studies show no effect on sulforaphane on normal colon epithelium cells. Conclusion: Our studies suggest that chronic PFOS exposure promotes proliferation of CRC cells by increasing pro-carcinogenic gene expression and signaling. Furthermore, our findings suggest that supplementation of diet sulforaphane can potentially mitigate the harmful effects from PFOS exposure. Our studies warrant further investigation of the mechanisms behind the effect of PFOS exposure on cancer progression that would guide development of effective intervention strategies to mitigate the harmful effects of the exposure to “forever chemicals”. Citation Format: Jerika Durham, Josiane Weber Tessmann, Bernhard Hennig, Yekaterina Zaytseva. Chronic perfluorooctanesulfonic acid exposure promotes proliferation of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1478.

Abstract 7278: Discovery of a novel WRN inhibitor, ZM-3329 that efficiently inhibits MSI-H tumor growth

Abstract Microsatellite instability-high status (MSI-H) is typically associated with endometrial and gastrointestinal cancers. These tumors harbor large numbers of frameshift and single-nucleotide variants and are characterized by a high tumor mutational burden (TMB). The clinical benefits of immune checkpoint inhibitor (ICI) therapy on MSI-H cancers were well-demonstrated, however, 50% of these cancers are intrinsically resistant to PD-1 therapies and resistance inevitably develops with prolonged treatment. Thus, a novel efficient therapeutic strategy for MSI-H cancer is still needed. Werner syndrome ATP-dependent helicase (WRN) is a member of the RecQ helicases family and has recently emerged as a promising synthetic-lethal target in the treatment of MSI-H tumors. In MSI-H tumors, microsatellite sequences consisting of repeated nucleotides are unstable, and replication slippage causes a unique secondary structure that hinders the progression of replication forks. With its helicase activity, WRN unwinds this secondary structure to restart DNA synthesis and sustain MSI-H tumor cell growth. WRN knockdown or helicase enzyme-dead effectively inhibits cell proliferation and tumor growth in multiple preclinical MSI-H models. Here we identified ZM-3329, a novel and highly potent WRN inhibitor. Helicase activities were efficiently inhibited by ZM-3329 with IC50 less than 30 nM, and equal potency was identified on HCT-116 cell proliferation inhibition. In a panel of 124 tumor cell lines, ZM-3329 only specifically inhibited the growth of MSI-H but had no significant effects on the MSS (Microsatellite stable) tumor cell lines, indicating high selectivity against MSS. Additionally, ZM-3329 strongly inhibited the growth of MSI-H patient-derived organoids. The intracellular mechanism of action study revealed that inhibition of WRN by ZM-3329 in MSI-H cells led to the accumulative DNA damage signatures which was demonstrated by the increase of γH2AX and P21 expression, and arrested cell cycle to G2/M. ZM-3329 demonstrated favorable pharmacokinetic profiles in pre-clinical species, supporting oral administration in humans. ZM-3329 showed robust anti-tumor activities in multiple MSI-H xenograft models with significant tumor regression observed. Meanwhile, mice were tolerant well in all tested doses, and no abnormality was observed. In conclusion, a novel WRN inhibitor was developed, and robust antitumor activities were demonstrated in xenograft tumors and PDOs with different tissue types. In terms of synthetic lethality between MSI-H and WRN as well as broad anti-tumor activities across different tumor types, ZM-3329 would be developed as a tissue-agnostic therapy for MSI-H patients. Citation Format: Feng Zhou, Guimei Yang, Yajing Liu, Liting Xue, Yao Guo, Zhengtao Li, Weikun Wang, Jian Li, Renhong Tang. Discovery of a novel WRN inhibitor, ZM-3329 that efficiently inhibits MSI-H tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7278.

Plumbagin induces ferroptosis in colon cancer cells by regulating p53-related SLC7A11 expression

ObjectiveThis study examined the mechanism through which plumbagin induces ferroptosis of colon cancer cells. MethodsCCK-8 assay was performed to examine the viability of colon cancer cells (SW480 and HCT116 cells) after they were treated with 0-, 5-, 10-, 15- and 20-μmol/L plumbagin. Colony formation assay and Transwell assay were used to examine the effects of 15-μmol/L plumbagin on the proliferation, invasive ability. The ferroptosis of SW480 and HCT116 cells and the expression of p-p53, p53 and SLC7A11 were analysed. The effects of blocking necrosis, apoptosis and ferroptosis on the anti-cancer effects of plumbagin were examined. After p53 was silenced, the effects of plumbagin on proliferation, invasion, ferroptosis and SLC7A11 expression were assessed. A tumour-bearing nude mouse model was used to examine the effects of p53 silencing and/or plumbagin on tumour growth, ferroptosis and SLC7A11 expression. ResultsPlumbagin inhibited the proliferation of SW480 and HCT116 cells and their invasive and colony-forming abilities. It increased Fe2+ levels but significantly decreased GSH and GPX4 levels. When ferroptosis was inhibited, the effects of plumbagin on colon cancer cells were significantly alleviated. Plumbagin promoted the expression and phosphorylation of p53 and inhibited the mRNA and protein levels of SLC7A11. Silencing of p53 counteracted the effects of plumbagin on the ferroptosis and biological behaviour of SW480 and HCT116 cells. In mouse models of colon cancer, silencing of p53 attenuated the tumour-suppressing effects of plumbagin as well as its inhibitory effects on the protein level of SLC7A11 and restored the expression of GSH and GPX4. ConclusionPlumbagin promotes ferroptosis and inhibits cell proliferation and invasion by decreasing the protein expression of SLC7A11 through p53.

Integrative approach using network pharmacology, bioinformatics, and experimental methods to explore the mechanism of cantharidin in treating colorectal cancer.

Cantharidin, a terpenoid produced by blister beetles, has been used in traditional Chinese medicine to treat various ailments and cancers. However, its biological activity, impact, and anticancer mechanisms remain unclear. The Cantharidin chemical gene connections were identified using various databases. The GSE21815 dataset was used to collect the gene expression information. Differential gene analysis and gene ontology analyses were performed. Gene set enrichment analysis was used to assess the activation of disease pathways. Weighted gene co-expression network analysis and differential analysis were used to identify illness-associated genes, examine differential genes, and discover therapeutic targets via protein-protein interactions. MCODE analysis of major subgroup networks was used to identify critical genes influenced by Cantharidin, examine variations in the expression of key clustered genes in colorectal cancer vs. control samples, and describe the subject operators. Single-cell GSE188711 dataset was preprocessed to investigate Cantharidin's therapeutic targets and signaling pathways in colorectal cancer. Single-cell RNA sequencing was utilized to identify 22 cell clusters and marker genes for two different cell types in each cluster. The effects of different Cantharidin concentrations on colorectal cancer cells were studied in vitro. One hundred and ninety-seven Cantharidin-associated target genes and 480 critical genes implicated in the development of the illness were identified. Cantharidin significantly inhibited the proliferation and migration of HCT116 cells and promoted apoptosis at certain concentrations. Patients on current therapy develop inherent and acquired resistance. Our study suggests that Cantharidin may play an anti-CRC role by modulating immune function.

Single extract of Forsythia Suspense versus the prepared drug in pieces: comparison of their anti-inflammatory, antitumor and antibacterial effects in zebrafish

To compare the anti-inflammatory, antitumor and anti-bacterial effects of the single extract (in granules) and the prepared drug in pieces of Forsythia Suspense (Lianqiao, a traditional Chinese herbal medicine). In zebrafish embryo models of CuSO4 exposure, tail transection and LPS microinjection-induced inflammation, the anti-inflammatory effects of 10 μg/mL DEX, single extract of Forsythia Suspense, and the water extract of the prepared drug (400, 600, and 800 μg/mL) were evaluated by observing neutrophil counts, RT- qPCR, HE staining and survival analysis. Zebrafish embryo models bearing different human tumor cell xenografts were used to assess the anti-tumor effect of the drugs in different dosage forms by fluorescence staining and HE staining. The microbroth dilution method was used to evaluate the antibacterial efficacy of the drugs. In the zebrafish embryo models of inflammation, both of the two dosage forms of Forsythia Suspense significantly inhibited neutrophil aggregation, reduced the mRNA expressions of TNF-α, IL-6, P38, Jnk, Erk and P65, and increased the survival rate of zebrafish. They both showed obvious inhibitory effects against xenografts of different human cancer cells including colon cancer cells (HCT116), pancreas adenocarcinoma cells (PANC-1), lung cancer cells (A549), liver cancer cells (Hep3B) and cervical carcinoma cells (Hela) in zebrafish embryos, and exhibited strong anti-bacterial effects at the concentration of 15.63 mg/mL. The two dosage forms of Forsythia Suspense have similar anti-inflammatory, antitumor and antibacterial effects, but their effects for inhibiting IL-6, P65, and Jnk mRNA expressions and HCT116 cell proliferation differ significantly at low doses in zebrafish.

Cinobufotalin regulates the USP36/c-Myc axis to suppress malignant phenotypes of colon cancer cells in vitro and in vivo.

Ubiquitin-specific protease 36 (USP36) has been reported to exhibit oncogenic effects in various malignancies, but the function of USP36 in colon cancer progression remains indefinite. Herein, we aimed to determine the role and mechanism of USP36 in malignant phenotypes of colon cancer cells and explore the potential drug targeting USP36. Bioinformatics analyses indicated that USP36 is highly expressed and significantly related to tumor stages in colon cancer. Besides, USP36 was further up-regulated in oxaliplatin (Oxa)-resistant colon cancer cells. Colony formation, Edu staining, Transwell, wound healing, sphere formation, and CCK-8 assays were conducted and showed that the proliferation, Oxa-resistance, migration, stemness, and invasion of HCT116 cells were promoted after overexpressing USP36, while suppressed by USP36 knockdown. Mechanically, USP36 enhances c-Myc protein stabilization in HCT116 cells via deubiquitination. AutoDock tool and ubiquitin-AMC hydrolysis assay identified cinobufotalin (CBF), an anti-tumor drug, maybe a USP36 inhibitor by inhibiting its deubiquitination activity. CBF significantly prohibited proliferation, migration, invasion, and stemness of HCT116 cells and reversed Oxa-resistance, whereas enforced expression of USP36 blocked these effects. Moreover, in vivo analyses confirmed the oncogenic role of USP36 and the therapeutic potential of CBF in the malignancy of colon cancer. In conclusion, CBF may be a promising therapeutic agent for colon cancer due to its regulation of the USP36/c-Myc axis.

Kurarinone targets JAK2-STAT3 signaling in colon cancer-stem-like cells.

Natural compounds are known to regulate stemness/self-renewal properties in colon cancer cells at molecular level. In the present study, we first time studied the colon cancer stem-like cells targeting potential of Kurarinone (KU) and explored the underlying mechanism. Cytotoxic potential of KU was checked in colon cancer cells. Colonosphere formation assay was performed to check the spheroid formation reduction potential of KU in HCT-116 cells by using phase-contrast microscopy. Stemness/self-renewal marker expression was studied at mRNA and protein levels in colonosphere. The qRT-PCR, western blot analysis, and flow cytometer techniques were used to assess the effect of KU treatment on cell cycle progression and apoptosis induction in colon cancer cells and colonosphere. Further, effect of KU treatment on pSTAT3 status and its nuclear translocation was also studied. KU treatment significantly decreased HCT-116 cell proliferation and reduced sphere formation potential at IC30 (8.71 µM) and IC50 (20.34 µM) concentrations compared to respective vehicle-treated groups, respectively. KU exposure significantly reduced the expression of CD44, c-Myc, Bmi-1, and Sox2 stemness/self-renewal markers in colonosphere in a dose-dependent manner. KU treatment inhibits JAK2-STAT3 signaling pathway by reducing pSTAT3 levels and its nuclear translocation in HCT-116 cells and colonosphere at IC50 concentration. KU treatment significantly decreased the expression of CCND1 and CDK4 cell cycle-specific markers and arrested the HCT-116 cells and colonosphere in G1-phase. Further, KU treatment increased Bax/Bcl-2 ratio, apoptotic cell population, cleaved caspase 3, and PARP-1 in HCT-116 cells and colonosphere. In conclusion, KU treatment decreases stemness/self-renewal, induces cell cycle arrest and apoptosis in HCT-116 colonosphere by down-regulating CD44-JAK2-STAT3 axis. Thus, targeting stemness/self-renewal and other cancer hallmark(s) by KU through CD44/JAK2/STAT3 signaling pathway might be a novel strategy to target colon cancer stem-like cells.

DPY30 knockdown suppresses colorectal carcinoma progression via inducing Raf1/MST2-mediated apoptosis

Colorectal Carcinoma (CRC) is one of the most common malignant tumors of the digestive tract, with a high mortality rate. DPY30 is one of the core subunits of the histone methyltransferase complex, which was involved in many cancer processes. However, the role of DPY30 in the occurrence and progression of CRC remains unclear. In this study, we sought to evaluate the role and mechanism of DPY30 in CRC cells apoptosis. Here, we identified that knockdown of DPY30 significantly inhibited the HT29 and HCT116 cells proliferation in vitro. Moreover, the knockdown of DPY30 significantly increased the apoptosis rate and promoted the expression of apoptosis-related proteins in CRC cells. Meanwhile, DPY30 knockdown promoted CRC cells apoptosis through endogenous programmed death and in a caspase activation-dependent manner. Furthermore, RNA-seq analysis revealed that the action of DPY30 is closely related to the apoptosis biological processes, and screened its potential effectors Raf1. Mechanistically, DPY30 downregulation promotes MST2-induced apoptosis by inhibiting Raf1 transcriptional activity through histone H3 lysine 4 trimethylation (H3K4me3). In vivo experiments showed that DPY30 was correlated with Raf1 in nude mouse subcutaneous xenografts tissues significantly. Clinical colorectal specimens further confirmed that overexpression of DPY30 in malignant tissues was significantly correlated with Raf1 level. The vital role of the DPY30/Raf1/MST2 signaling axis in the cell death and survival rate of CRC cells was disclosed, which provides potential new targets for early diagnosis and clinical treatment of CRC.

ZMIZ1 Regulates Proliferation, Autophagy and Apoptosis of Colon Cancer Cells by Mediating Ubiquitin-Proteasome Degradation of SIRT1.

There are nearly 1.15 million new cases of colon cancer, as well as 586,858 deaths from colon cancer worldwide in 2020. The aim of this study is to reveal whether ZMIZ1 can control the fate of colon cancer cells and the mechanism by which it functions. Specific shRNA transfection was used to knock down the expression of ZMIZ1 in colon cancer cell lines (HCT116 and HT29), and cell proliferation was detected using EdU and CCK-8 reagents, apoptosis by flow cytometry, and autophagy by western blot. The interaction of ZMIZ1 and SIRT1 was analyzed. Knockdown of ZMIZ1 significantly inhibited autophagy and proliferation, and induced apoptosis of HCT116 and HT29 cells. The mRNA level of SIRT1 was not affected by ZMIZ1 knockdown, but the protein level of SIRT1 was significantly decreased and the protein level of the SIRT1-specific substrate, acetylated FOXO3a, was reduced. Immunoprecipitation assays identified the interaction between SIRT1 and ZMIZ1 in HCT116 and HT29 cells. ZMIZ1 increased intracellular ubiquitination of SIRT1. Knockdown or pharmacological inhibition of SIRT1 neutralized the effects of ZMIZ knockdown on proliferation, autophagy and apoptosis in HCT116 and HT29 cells. ZMIZ1 may control the fate of colon cancer cells through the SIRT1/FOXO3a axis. Targeting ZMIZ1 would be beneficial for the treatment of colon cancer.

Dickkopf 1 is expressed in normal fibroblasts during early stages of colorectal tumorigenesis.

Colorectal cancer progression from adenoma to cancer is a time-intensive process; however, the interaction between normal fibroblasts (NFs) with early colorectal tumors, such as adenomas, remains unclear. Here, we analyzed the response of the microenvironment during early tumorigenesis using co-cultures of organoids and NFs. Colon normal epithelium, adenoma, cancer organoid, and NFs were established and co-cultured using Transwell inserts. Microarray analysis of NFs was performed to identify factors expressed early in tumor growth. Immunostaining of clinical specimens was performed to localize the identified factor. Functional analysis was performed using HCT116 cells. Serum DKK1 levels were measured in patients with colorectal cancer and adenoma. Colorectal organoid-NF co-culture resulted in increased organoid diameter and cell viability in normal epithelial and adenomatous organoids but not in cancer organoids. Microarray analysis of NFs revealed 18 genes with increased expression when co-cultured with adenoma and cancer organoids. Immunohistochemical staining revealed DKK1 expression in the tumor stroma from early tumor growth. DKK1 stimulation reduced HCT116 cell proliferation, while DKK1 silencing by siRNA transfection increased cell proliferation. Serum DKK1 level was significantly higher in patients with advanced cancer and adenoma than in controls. Serum DKK1 level revealed area-under-the-curve values of 0.78 and 0.64 for cancer and adenoma, respectively. These findings contribute valuable insights into the early stages of colorectal tumorigenesis and suggest DKK1 as a tumor suppressor. Additionally, serum DKK1 levels could serve as a biomarker to identify both cancer and adenoma, offering diagnostic possibilities for early-stage colon tumors. The present study has a few limitations. We considered using DKK1 as a candidate gene for gene transfer to organoids and NFs; however, it was difficult due to technical problems and the slow growth rate of NFs. Therefore, we used cancer cell lines instead. In addition, immunostaining and ELISA were based on the short-term collection at a single institution, and further accumulation of such data is desirable. As described above, most previous reports were related to advanced cancers, but in this study, new findings were obtained by conducting experiments on endoscopically curable early-stage tumors, such as adenomas.

Novel sulfonyl-substituted tetrandrine derivatives for colon cancer treatment by inducing mitochondrial apoptosis and inhibiting PI3K/AKT/mTOR pathway

Tetrandrine (TET) possesses multiple pharmacological activities and could suppress tumor proliferation via PI3K pathway inhibition. However, inferior antitumor activity and potential toxicity limit its clinical application. In the present study, a series of 14-sulfonamide and sulfonate TET derivatives were designed, synthesized, and evaluated for biological activities. Through structural-activity relationship studies, compound 3c with α, β-unsaturated carbonyl group exhibited the most potent activity against all tested tumor cell lines (including Hela, HCT116, HepG2, MCF-7, and SHSY5Y), as well as negligible toxicity against normal cell lines LO2 and HEK293. Additionally, compound 3c effectively inhibited HCT116 and CT26 cell proliferation in vitro with increased cell proportion in the G2/M phase, activated the mitochondrial apoptosis pathway, and induced colon cancer cell apoptosis by suppressing the PI3K/AKT/mTOR pathway. The further molecular docking results confirmed that compound 3c is potentially bound to multiple residues in PI3K with a stronger binding affinity than TET. Ultimately, compound 3c dramatically suppressed tumor growth in the CT26 xenograft tumor model, without noticeable visceral toxicity detected in the high-dose group. In summary, compound 3c might present new insights for designing new PI3K inhibitors and be a potential candidate for colon cancer treatment.