HT-29 Colon Cancer Cells Research Articles

The effect of up-regulation of NLE1 gene expression on the invasion and migration of colon cancer cells and its mechanism

ObjectivesTo explore the regulatory effects of up-regulation of NLE1 gene expression in vitro and the mechanism of colon cancer cell invasion and migration. MethodsThe logarithmic growth phase human colonic fibroblasts CCD-18Co, colon cancer cells HT-29, and NLE1 interference group were transfected with NLE1 siRNA to interfere with expressing viral vectors and liposomes. The NLE1 negative control group was added with liposomes, and NLE1 was overexpressed. The expression changes of RNA interference on the target gene were analyzed. ResultsThe expression of NLE1 mRNA in colon cancer cells was significantly higher (P < 0.05). Likewise, the expression of NLE1 protein in colon cancer cells was significantly increased than in control colon cancer cell lines (p < 0.05). Compared with the NLE1 interference group and the NLE1 negative control group, the migration and invasion ability of colon cancer cells in the NLE1 overexpression group were significantly varied (P < 0.05)‘. ConclusionsUp-regulation of the NLE1 gene in colon cancer cells promotes cell migration and invasion of colon cancer cells and inhibits the apoptosis ability of colon cancer cells.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells

Ethnopharmacological relevanceLemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. Materials and methodsWe analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. ResultsLMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 μg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. ConclusionLMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

Abstract 2365: Visualizing exosome structure in Dasatinib resistant colon cancer cells

Abstract Cells release molecules through vesicles or into specific locations through exocytosis. Extracellular vesicle is divided into ectosomes and exosomes. Exosomes has been the main mechanism of discussion in recent studies and cancer research. As Exosomes with a diameter of ~30-160 nm (with an average diameter of 100 nm) carries intracellular molecules such as DNA, RNA, miRNA, proteins, and lipids. The physiological function of exosomes is not fully understood at present, it is important to understand the role of exosome in cancer. Src is highly activated in colon cancer and Src inhibition is the potential treatment strategy in colon cancer. In this study, colon cancer cells HT-29 were used to investigate the difference sizes of exosomes that response to Src inhibitor treatment. In the preliminary results, the exosome from HT-29 cultured medium were isolated and examined by transmission electron microscopy (TEM). The micrograph showed the average size of exosome is around 100 nm. When HT-29 were treated with Dasatinib (DST) the Src inhibitor, the drug resistant (DSTR) cells were grown, the secreted exosome from DSTR were collected and demonstrated the bigger size of exosome with average diameter of 130 nm. The role of the size changes of exosome in response to Dasatinib resistant will be further investigated, however, this study provides the clues that exosome detection can be applied in monitoring drug resistance. Citation Format: Hui Min Koo, Yi-Wen Lu, Yu-Chi Tseng, Kai-Yu Tseng, Yen-Tse Lu, Wei-Ting Chao. Visualizing exosome structure in Dasatinib resistant colon cancer cells [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 2365.

Abstract 5819: Src inhibitor suppresses rab11 mediated collective cell migration in colon cancer cells

Abstract Collective cell migration has been demonstrated the importance in cancer metastasis. Instead of single cell migration, collective cell migration requires E-cadherin for the cohort cell migration. Rab11 the recycling vesicle protein plays the key role in E-cadherin dynamics and mediates collective cell migration. In this study, Src was hypothesized to regulate Rab11 mediated collective cell migration. Our results demonstrated Src inhibitor Dasatinib has less toxicity in HT-29 colon cancer cells, instead, the cell morphology was turned aggregated. The immunofluorescence image also showed E-cadherin polarity was lost after Dasatinib treatment and increased the potential of cell-cell contact inhibition. In transwell assay, Rab11 was found colocalized with E-cadherin in collective migrated cells. Dasatinib treatment can suppress collective cell migration and decrease the interaction of Rab11 and E-cadherin. This study demonstrated the important role of Src in Rab11 mediated collective cell migration through E-cadherin dynamics in colon cancer. Citation Format: Yi-Wen Lu, Xiang-Ling Hou, Hui Min Koo, Wei-Ting Chao. Src inhibitor suppresses rab11 mediated collective cell migration in colon cancer cells [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 5819.

Abstract 5820: Chloroquine inhibited Rab11 mediated E-cadherin transport in collective colon cell migration in Dasatinib resistant cells

Abstract In has been demonstrated epithelial mesenchymal transition (EMT) is related to E-cadherin expression level. Recently, studies have been demonstrated the collective cancer cell migration which expresses high E-cadherin is a powerful mechanism of metastasis. Therefore, the role of E-cadherin in cancer metastasis is still not clear. Endocytosis was shown to regulate E-cadherin turnover on the membrane and the function of lysosome is also associated with cell vesicle trafficking. The past studies indicated Src can regulate vesicle trafficking. In this study, HT29 colon cancer cells were treated with Src inhibitor, Dasatinib (DST), to investigate the role of lysosome in regulating E-cadherin dynamics through vesicle trafficking in collective cell migration. In the result, the recycling protein Rab11 and lysosome marker Lamp1 were found localized with E-cadherin in the cell-cell contacts, however, Dasatinib sensitive (DSTS) cells showed less colocalization of Rab11 with E-cadherin. When cells were treated with lysosome inhibitor chloroquine, Rab11 were accumulated in the cell-cell contacts and reduced collective cell migration in Dasatinib resistant (DSTR) cells. These results suggest lysosome inhibition can suppress Rab11 mediated vesicle trafficking of E-cadherin transport for collective cell migration, and chloroquine has therapeutic potential in colon cancer treatment. Citation Format: Xiang-Ling Hou, Hui Min Koo, Chia-Wen Lin, Chien-Chi Huang, Yu-Chi Tseng, Kai-Yu Tseng, Wei-Ting Chao. Chloroquine inhibited Rab11 mediated E-cadherin transport in collective colon cell migration in Dasatinib resistant cells [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 5820.

Benzotriazole Substituted 2-Phenylquinazolines as Anticancer Agents: Synthesis, Screening, Antiproliferative and Tubulin Polymerization Inhibition Activity.

Development of anticancer agents targeting tubulin protein. Tubulin protein is being explored as an important target for anticancer drug development. Ligands binding to the colchicine binding site of the tubulin protein act as tubulin polymerization inhibitors and arrest the cell cycle in the G2/M phase. Synthesis and screening of benzotriazole-substituted 2-phenyl quinazolines as potential anticancer agents. A series of benzotriazole-substituted quinazoline derivatives have been synthesized and evaluated against human MCF-7 (breast), HeLa (cervical) and HT-29 (colon) cancer cell lines using standard MTT assays. ARV-2 with IC50 values of 3.16 μM, 5.31 μM, 10.6 μM against MCF-7, HELA and HT29 cell lines, respectively displayed the most potent antiproliferative activities in the series while all the compounds were found non-toxic against HEK293 (normal cells). In the mechanistic studies involving cell cycle analysis, apoptosis assay and JC-1 studies, ARV-2 and ARV-3 were found to induce mitochondria-mediated apoptosis. The benzotriazole-substituted 2-phenyl quinazolines have the potential to be developed as potent anticancer agents.

Anticancer activity of lycopene in HT-29 colon cancer cell line

An inverse association between serum lycopene levels and the risk of cancers has been pointed out by many prospective and retrospective epidemiological studies which prompted more studies to be performed on animal models and cell cultures in order to test this hypothesis. The aim of the present study was to evaluate the antiproliferative and pro-apoptotic effect of lycopene on colon cancer HT-29 cell line. The effect of lycopene on the viability of HT-29 cell line was investigated using XTT assay. The levels of Bcl-2, cleaved caspase 3, BAX, cleaved PARP, and 8-oxo-dG in lycopene-treated HT-29 cells were measured using ELISA. Gamma-H2AX and cytochrome c expression was assessed semi-quantitatively using immunofluorescence staining. Lycopene at doses of 10 and 20 μM produced a significant antiproliferative effect on HT-29 cells compared to the control (p < 0.05). The IC50 value of lycopene in HT-29 cells was found to be 7.89 μM for 24 h. Lycopene (7.89 μM) significantly elevated cleaved caspase 3 (p <0.01), BAX, and cleaved PARP, 8-oxo-dG levels (p<0.05). The levels of γ-H2AX foci are significantly higher while the levels of cytochrome-c are lower (p<0.05) in lycopene-treated HT-29 cells. These results indicate that lycopene has an antiproliferative apoptotic and genotoxic effect on HT-29 colon cancer cell line.

Biological functionality and characteristics of Jerusalem artichoke (Helianthus tuberosus L.) tuber extracts

AbstractJerusalem artichoke tuber extracts (JAE) bioactivity including anticancer, antimicrobial, and digestion-inhibiting properties were investigated. The findings showed that the extracts were able to inhibit cancer growth in the HT-29 colon cancer cell line (HT-29 cc cell line) in a dose-dependent form. The suppression of cell proliferation rose to about 78.05 ± 3.9 percent at a dose of 250 μg mL−1. The Annexin V assay showed dose-dependent DNA fragmentation and detected late apoptotic induction in the HT-29 cc cell line. Depending on the concentration, the extract was able to stop the cell cycle in the HT-29 cc cell line at the G1 phase. Also, JAE prevented the HT-29 cc cell line growth, which resulted in programmed cell death. Additionally, the extracts are potential antibacterial agents and may inhibit lipase and α-amylase.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Quercetin and Its Fermented Extract as a Potential Inhibitor of Bisphenol A-Exposed HT-29 Colon Cancer Cells’ Viability

Bisphenol A (BPA) promotes colon cancer by altering the physiological functions of hormones. Quercetin (Q) can regulate signaling pathways through hormone receptors, inhibiting cancer cells. The antiproliferative effects of Q and its fermented extract (FEQ, obtained by Q gastrointestinal digestion and in vitro colonic fermentation) were analyzed in HT-29 cells exposed to BPA. Polyphenols were quantified in FEQ by HPLC and their antioxidant capacity by DPPH and ORAC. Q and 3,4-dihydroxyphenylacetic acid (DOPAC) were quantified in FEQ. Q and FEQ exhibited antioxidant capacity. Cell viability with Q+BPA and FEQ+BPA was 60% and 50%, respectively; less than 20% of dead cells were associated with the necrosis process (LDH). Treatments with Q and Q+BPA induced cell cycle arrest in the G0/G1 phase, and FEQ and FEQ+BPA in the S phase. Compared with other treatments, Q positively modulated ESR2 and GPR30 genes. Using a gene microarray of the p53 pathway, Q, Q+BPA, FEQ and FEQ+BPA positively modulated genes involved in apoptosis and cell cycle arrest; bisphenol inhibited the expression of pro-apoptotic and cell cycle repressor genes. In silico analyses demonstrated the binding affinity of Q > BPA > DOPAC molecules for ERα and ERβ. Further studies are needed to understand the role of disruptors in colon cancer.

Uncoupled nitric oxide synthase activity promotes colorectal cancer progression.

Increased levels of reactive oxygen/nitrogen species are one hallmark of chronic inflammation contributing to the activation of pro-inflammatory/proliferative pathways. In the cancers analyzed, the tetrahydrobiopterin:dihydrobiopterin ratio is lower than that of the corresponding normal tissue, leading to an uncoupled nitric oxide synthase activity and increased generation of reactive oxygen/nitrogen species. Previously, we demonstrated that prophylactic treatment with sepiapterin, a salvage pathway precursor of tetrahydrobiopterin, prevents dextran sodium sulfate-induced colitis in mice and associated azoxymethane-induced colorectal cancer. Herein, we report that increasing the tetrahydrobiopterin:dihydrobiopterin ratio and recoupling nitric oxide synthase with sepiapterin in the colon cancer cell lines, HCT116 and HT29, inhibit their proliferation and enhance cell death, in part, by Akt/GSK-3β-mediated downregulation of β-catenin. Therapeutic oral gavage with sepiapterin of mice bearing azoxymethane/dextran sodium sulfate-induced colorectal cancer decreased metabolic uptake of [18F]-fluorodeoxyglucose and enhanced apoptosis nine-fold in these tumors. Immunohistochemical analysis of both mouse and human tissues indicated downregulated expression of key enzymes in tetrahydrobiopterin biosynthesis in the colorectal cancer tumors. Human stage 1 colon tumors exhibited a significant decrease in the expression of quinoid dihydropteridine reductase, a key enzyme involved in recycling tetrahydrobiopterin suggesting a potential mechanism for the reduced tetrahydrobiopterin:dihydrobiopterin ratio in these tumors. In summary, sepiapterin treatment of colorectal cancer cells increases the tetrahydrobiopterin:dihydrobiopterin ratio, recouples nitric oxide synthase, and reduces tumor growth. We conclude that nitric oxide synthase coupling may provide a useful therapeutic target for treating patients with colorectal cancer.

Resveratrol downregulates ENaCs through the activation of AMPK in human colon cancer cells

Epithelial sodium channels (ENaCs) are critically engaged in a number of hallmarks of cancer progression, including proliferation, migration, invasion and apoptosis. Thus, the inhibition of ENaCs possesses therapeutic potential in cancer. Resveratrol, a natural polyphenol with anti-carcinogenic activity, is a potent activator of 5′ AMP-activated protein kinase (AMPK) which reduces the abundance of ENaCs in the cell membrane by causing the internalization of the β subunit. However, the effect of resveratrol on ENaCs in cancer cells is unknown. Therefore, in this study, we aimed to investigate the effects of resveratrol on ENaCs in human colon cancer cells HCT116 and HT29. The influences of resveratrol either alone or together with AMPK inhibitor compound C (CC), and ENaC inhibitor amiloride on cell viability were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression levels of phospho-AMPK Thr172 and βENaC in cells were determined by immunofluorescence staining, and the expression of apoptotic markers Caspase-3 and Caspase-9 were analyzed by qRT-PCR. Resveratrol was found to activate AMPK in a dose-dependent manner. Both AMPK activation by resveratrol and ENaC inhibition by amiloride decreased cell viability and increased apoptosis significantly. AMPK activation also reduced βENaC expression in cells. Our results suggest that ENaC inhibition through AMPK activation might be a potential mechanism underlying the anti-cancer effects of resveratrol.

Punicalagin is cytotoxic to human colon cancer cells by modulating cell proliferation, apoptosis, and invasion.

Purpose: The purpose of this study was to explore the anticancer effect of punicalagin, an abundant bioactive tannin compound isolated from Punica granatum L., on three colon cancer cell lines, namely, HCT 116, HT-29, and LoVo.Research Design: Normal and colon cancer cells were treated with different concentrations of punicalagin for different periods. Data Collection and Analysis: Cell viability was measured with a CCK-8 assay. Programmed cell death and invasion were analyzed using an annexin V and cell death kit and a cell invasion analysis kit. The expression of active caspase-3, MMP-2, MMP-9, Snail, and Slug were measured by Western blot.Results: The results of the cell viability analysis showed that punicalagin was cytotoxic to colon cancer cells, but it was not to normal cells in a dose- and time-dependent manner. Additionally, punicalagin induced apoptosis in colon cancer cells (shown by the cumulative percentage of colorectal cancer cells in early and late apoptosis). It was found that caspase-3 activity increased following punicalagin treatment. Western blot results also showed that punicalagin increased the expression of activated caspase-3. In contrast, punicalagin inhibited the invasion of colon cancer cells. Further, treatment of colon cancer cells with punicalagin suppressed the expression of MMP-2, MMP-9, Snail, and Slug. Conclusions: These results showed that the activation of caspase-3 and the inhibition of MMP-2, MMP-9, Snail and Slug were involved in the effects of punicalagin on colon cancer cells.

Anticancer properties of curcumin-treated Lactobacillus plantarum against the HT-29 colorectal adenocarcinoma cells

Probiotic bacteria with functions of importance to the health and well-being of the host exhibit various medicinal properties including anti-proliferative properties against cancer cells. There are observations demonstrating probiotic bacteria and their metabolomics can be different in various populations with different eating habits. Here, Lactobacillus plantarum was treated with curcumin (the major compound of turmeric), and its resistance to the curcumin was determined. After then the cell-free supernatants of untreated bacteria (CFS) and bacteria treated with curcumin (cur-CFS) were isolated and their anti-proliferative properties against HT-29 colon cancer cells were compared. The ability of L. plantarum treated with curcumin to combat a variety of pathogenic bacterial species and its ability to survive in acidic conditions were evidence that the probiotic properties of the bacterium were unaffected by the curcumin treatment. L. plantarum treated with curcumin and intact L. plantarum were both able to live in acidic conditions, according to the results of the resistance to low pH test. The MTT result showed that CFS and cur-CFS dose-dependently decreased the growth of HT29 cells with a half-maximal inhibitory concentration of 181.7 and 116.3 µL/mL at 48 h, respectively. Morphological alteration of DAPI-stained cells also exhibited significant fragmentation in the chromatin within the nucleus of cur-CFS-treated cells compared to CFS-treated HT29 cells. Moreover, flow cytometry analyses of apoptosis and cell cycle confirmed DAPI staining and MTT assay results and stipulated the increased occurrence of programmed cell death (apoptosis) in cur-CFS-treated cells (~ 57.65%) compared to CFS-treated cells (~ 47%). These results were more confirmed with qPCR and exhibited the upregulation of Caspase 9–3 and BAX genes, and downregulation of the BCL-2 gene in cur-CFS- and CFS-treated cells. In conclusion, turmeric spice and curcumin may affect the metabolomics of probiotics in intestinal flora which could subsequently influence their anticancer properties.

DNA groove binder and significant cytotoxic activity on human colon cancer cells: Potential of a dimeric zinc (II) phthalocyanine derivative

The interaction of a multi-component system consisting of benzene-1,4-diyldimethanimine-bridged dimeric zinc-phthalocyanine groups (4OMPCZ) with calf thymus DNA (ct-DNA) was investigated using UV–Vis absorption, fluorescence emission spectroscopy methods, and viscosity measurements. The binding constant, Kb, which is an important parameter to gain information about the binding mode, was found as 9.7 × 107 M−1 from the UV–Vis absorption studies. Another important spectrophotometric tool is competitive displacement assays with Ethidium bromide and Hoechst 33342. Through this experiment, a higher KSV value was obtained with Hoechst for the phthalocyanine derivative, 4OMPCZ, and the ct-DNA complex than with ethidium bromide. Additionally, molecular docking studies were conducted to calculate the theoretical binding constant and visualize the interactions of 4OMPCZ with a model DNA. According to docking results, although the interactions are mainly located in the major groove of the DNA helix, due to the wrapping, these interactions can also be extended to the minor groove of the DNA. Spectrophotometric, molecular docking, and viscosity studies revealed that the interaction of 4OMPCZ with DNA is likely to be via the major and minor grooves. The in vitro cytotoxic activity of 4OMPCZ was evaluated by MTT assay on human colon cancer cells (HT29) after 72 h of treatment. 4OMPCZ indicated significant cytotoxic activity when stimulated with UV light compared to the standard chemotherapy drugs, fluorouracil (5-FU), and cisplatin on HT29 colon cancer cells. The IC50 value of 4OMPCZ displayed considerably lower concentrations compared to the standard drugs, 5-FU, and cisplatin.

In vitro Digestion of Phaseolus vulgaris L. Cooked Beans Induces Autophagy in Colon Cancer Cells.

Phaseolus vulgaris L. (common bean) contains high levels of proteins, unsaturated fatty acids, minerals, fibers, and vitamins, and for this reason, it represents an essential component of the diet. More than 40,000 varieties of beans have been recognized and are staple foods in the traditional cuisine of many countries. In addition to its high nutritional value, P. vulgaris is also characterized by its nutraceutical properties and favors environmental sustainability. In this manuscript, we studied two different varieties of P. vulgaris, Cannellino and Piattellino. We investigated the effects of traditional processing (soaking and cooking) and in vitro gastrointestinal digestion of beans on their phytochemical composition and anticancer activity. Using HT29 and HCT116 colon cancer cell lines, we showed that the extract obtained after gastrointestinal digestion of cooked beans (the bioaccessible fraction, BF) induces cell death through the induction of the autophagic process. We demonstrated that the BF of Cannellino and Piattellino beans at the concentration of 100 μg/mL reduces cell vitality, measured by MMT assay, of both HT29 (88.41% ± 5.79 and 94.38% ± 0.47) and HCT116 (86.29% ± 4.3 and 91.23% ± 0.52) cell lines. Consistently, the treatment of HT29 cells with 100 μg/mL of Cannellino and Piattellino BFs reduced clonogenicity by 95% ± 2.14 and 96% ± 0.49, respectively. Moreover, the activity of extracts appeared to be selective for colon cancer cells. The data shown in this work further confirm P. vulgaris to be among foods with beneficial effects for human health.

Astragali Radix-Curcumae Rhizoma combination inhibits proliferation, migration, and invasion of colon cancer HT-29 cells by regulating EMT

This study aims to investigate the effect of Astragali Radix-Curcumae Rhizoma(AC) combination on the proliferation, migration, and invasion of colon cancer HT-29 cells based on epithelial-mesenchymal transition(EMT). HT-29 cells were respectively treated with 0, 3, 6 and 12 g·kg~(-1) AC-containing serum for 48 h. The survival and growth of cells were measured by thiazole blue(MTT) colorimetry, and the proliferation, migration, and invasion of cells were detected by 5-ethynyl-2'-deoxyuridine(EdU) test and Transwell assay. Cell apoptosis was examined by flow cytometry. The BALB/c nude mouse model of subcutaneous colon cancer xenograft was established, and then model mice were classified into blank control group, 6 g·kg~(-1) AC group, and 12 g·kg~(-1) AC group. The tumor weight and volume of mice were recorded, and the histopathological morphology of the tumor was observed based on hematoxylin-eosin(HE) staining. The expression of apoptosis-associated proteins B-cell lymphoma-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), and cleaved caspase-3, and EMT-associated proteins E-cadherin, MMP9, MMP2 and vimentin in HT-29 cells and mouse tumor tissues after the treatment of AC was determined by Western blot. The results showed that cell survival rate and the number of cells at proliferation stage decreased compared with those in the blank control group. The number of migrating and invading cells reduced and the number of apoptotic cells increased in the administration groups compared with those in the blank control group. As for the in vivo experiment, compared with the blank control group, the administration groups had small tumors with low mass and shrinkage of cells and karyopycnosis in the tumor tissue, indicating that the AC combination may improve EMT. In addition, the expression of Bcl2 and E-cadherin increased and the expression of Bax, caspase-3, cleaved caspase-3, MMP9, MMP2, and vimentin decreased in HT-29 cells and tumor tissues in each administration group. In summary, the AC combination can significantly inhibit the proliferation, invasion, migration, and EMT of HT-29 cells in vivo and in vitro and promote the apoptosis of colon cancer cells.

Uncovering the effects and molecular mechanism of Astragalus membranaceus (Fisch.) Bunge and its bioactive ingredients formononetin and calycosin against colon cancer: An integrated approach based on network pharmacology analysis coupled with experimental validation and molecular docking.

Colon cancer is a highly malignant cancer with poor prognosis. Astragalus membranaceus (Fisch.) Bunge (Huang Qi in Chinese, HQ), a well-known Chinese herbal medicine and a popular food additive, possesses various biological functions and has been frequently used for clinical treatment of colon cancer. However, the underlying mechanism is not fully understood. Isoflavonoids, including formononetin (FMNT) and calycosin (CS), are the main bioactive ingredients isolated from HQ. Thus, this study aimed to explore the inhibitory effects and mechanism of HQ, FMNT and CS against colon cancer by using network pharmacology coupled with experimental validation and molecular docking. The network pharmacology analysis revealed that FMNT and CS exerted their anticarcinogenic actions against colon cancer by regulating multiple signaling molecules and pathways, including MAPK and PI3K-Akt signaling pathways. The experimental validation data showed that HQ, FMNT and CS significantly suppressed the viability and proliferation, and promoted the apoptosis in colon cancer Caco2 and HT-29 cells. HQ, FMNT and CS also markedly inhibited the migration of Caco2 and HT-29 cells, accompanied by a marked increase in E-cadherin expression, and a notable decrease in N-cadherin and Vimentin expression. In addition, HQ, FMNT and CS strikingly decreased the expression of ERK1/2 phosphorylation (p-ERK1/2) without marked change in total ERK1/2 expression. They also slightly downregulated the p-Akt expression without significant alteration in total Akt expression. Pearson correlation analysis showed a significant positive correlation between the inactivation of ERK1/2 signaling pathway and the HQ, FMNT and CS-induced suppression of colon cancer. The molecular docking results indicated that FMNT and CS had a strong binding affinity for the key molecules of ERK1/2 signaling pathway. Conclusively, HQ, FMNT and CS exerted good therapeutic effects against colon cancer by mainly inhibiting the ERK1/2 signaling pathway, suggesting that HQ, FMNT and CS could be useful supplements that may enhance chemotherapeutic outcomes and benefit colon cancer patients.

Obesity hormones and itaconate mediating inflammation in human colon cancer cells – Another lead to early-onset colon cancer?

BackgroundChronic inflammation is a key feature of obesity and a hallmark of colon cancer (CC). The obesity-related hormones leptin and adiponectin alter inflammatory gene profiles in cancer, but their specific role in CC is unclear. We have previously studied the effects of leptin and the macrophage-specific mediator itaconate on M2-like macrophages. This current study evaluates their effects on CC cells. MethodsHT-29 CC cells (derived from a young patient, stage III CC) were treated with either leptin, adiponectin, 4-octyl itaconate (OI) or dimethyl itaconate (DI). Gene expression after treatment was analyzed at four time points (3, 6, 18, and 24 h). ResultsCCL22 was upregulated after treatment with adiponectin (at 18 h [FC 16.3, p < 0.001]). IL-8 expression increased following both adiponectin (at 3 h [FC 68.1, p < 0.001]) and leptin treatments (at 6 h [FC 7.3, p < 0.001]), while OI induced downregulation of IL-8 (at 24 h [FC −5.0, p < 0.001]). CXCL10 was upregulated after adiponectin treatment (at 6 h [FC 3.0, p = 0.025]) and downregulated by both OI and DI at 24 h, respectively (OI [FC −10.0, p < 0.001]; DI [FC −10.0, p < 0.001]). IL-1β was upregulated after adiponectin treatment (at 3 h [FC 10.6, p < 0.001]) and downregulated by DI (at 24 h [FC −5.0, p < 0.001]). TNF-α expression was induced following adiponectin (at 6 h [FC 110.7, p < 0.001]), leptin (at 18 h [FC 5.8, p = 0.027]) and OI (at 3 h [FC 91.1, p = 0.001]). PPARγ was affected by both OI (at 3 h [FC 10.1, p = 0.031], at 24 h [FC −10.0, p = 0.031]) and DI (at 18 h [FC −1.7, p = 0.033]). ConclusionsObesity hormones directly affect inflammatory gene expression in HT29 CC cells, potentially enhancing cancer progression. Itaconate affects the prognostic marker PPARγ in HT29 CC cells. Leptin, adiponectin and itaconate may represent a link between obesity and CC.

In situ crosslinked dialdehyde guar gum-chitosan Schiff-base hydrogels for dual drug release in colorectal cancer therapy

This work advocates that in situ crosslinked chitosan-dialdehyde guar gum (CsDAGG) hydrogels can be effectually used for dual drug release in colorectal cancer therapy. Herein, we designed various grades of CsDAGG hydrogels where DAGG was accomplished by periodate oxidation of guar gum. With increasing DAGG content, a diminution in swelling and a more compact matrix was evident. In this study, curcumin in combination with aspirin were chosen for investigation in colon carcinoma. The hydrogels ensured the protection of the drugs from absorption in stomach and small intestine and enabled a controlled release at the targeted site of colorectum. The exposure of the aforesaid combined drug formulation further demonstrated enhanced anticancer effects in HT-29 colon cancer cells. The findings altogether suggested the high potency of the developed hydrogels for the co-administration of drugs that can provide chemotherapy and pain relief simultaneously in colorectal cancer combination therapy.