Proliferation Of HCT116 Cells Research Articles

Synergistic Chemopreventive Effects of a Novel Combined Plant Extract Comprising Gallic Acid and Hesperidin on Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer with a high mortality rate worldwide. Although gallic acid and hesperidin exert anticancer activity, synergistic effects of gallic acid and hesperidin against CRC remain elusive. This study aims to investigate the therapeutic mechanism of a novel combination of gallic acid and hesperidin against CRC cell growth, including cell viability, cell-cycle-associated proteins, spheroid formation, and stemness. Gallic acid and hesperidin derived from Hakka pomelo tea (HPT) were detected by colorimetric methods and high-performance liquid chromatography using ethyl acetate as an extraction medium. CRC cell lines (HT-29 and HCT-116) treated with the combined extract were investigated in our study for cell viability (trypan blue or soft agar colony formation assay), cell cycle (propidium iodide staining), cell-cycle-associated proteins (immunoblotting), and stem cell markers (immunohistochemistry staining). Compared with other extraction methods, HPT extraction using an ethyl acetate medium exerts the most potent effect on inhibiting HT-29 cell growth in a dose-dependent manner. Furthermore, the treatment with combined extract had a higher inhibitory effect on CRC cell viability than gallic acid or hesperidin alone. The underlying mechanism was involved in G1-phase arrest and Cip1/p21 upregulation that could attenuate HCT-116 cell proliferation (Ki-67), stemness (CD-133), and spheroid growth in a 3D formation assay mimicking in vivo tumorigenesis. Gallic acid and hesperidin exert synergistic effects on cell growth, spheroids, and stemness of CRC and may serve as a potential chemopreventive agent. Further testing for the safety and effectiveness of the combined extract in large-scale randomized trials is required.

Synthesis and In Vitro Biological Evaluation of p-Carborane-Based Di-tert-butylphenol Analogs.

Targeting inflammatory mediators and related signaling pathways may offer a rational strategy for the treatment of cancer. The incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes in dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids is a promising approach. The di-tert-butylphenol derivatives R-830, S-2474, KME-4, and E-5110 represent potent dual COX-2/5-LO inhibitors. The incorporation of p-carborane and further substitution of the p-position resulted in four carborane-based di-tert-butylphenol analogs that showed no or weak COX inhibition but high 5-LO inhibitory activities in vitro. Cell viability studies on five human cancer cell lines revealed that the p-carborane analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb exhibited lower anticancer activity compared to the related di-tert-butylphenols. Interestingly, R-830-Cb did not affect the viability of primary cells and suppressed HCT116 cell proliferation more potently than its carbon-based R-830 counterpart. Considering all the advantages of boron cluster incorporation for enhancement of drug biostability, selectivity, and availability of drugs, R-830-Cb can be tested in further mechanistic and in vivo studies.

Induction of autophagy by oleifolioside A in HCT-116 human colorectal cancer cells

In current study, we addressed the anti-cancer effect of oleifolioside A and its mechanism on the regulation of cell death in HCT-116 human colorectal cancer cells. Oleifolioside A inhibited HCT-116 cell proliferation and caused apoptosis associated with sequential activation of caspases 8 and 3, followed by PARP cleavage. Moreover, anti-LC3-positive granules and the increased LC3-II level were observed in HCT-116 cells treated with oleifolioside A, which is the specific characteristics of autophagy. Treatment of autophagy inhibiors, 3-MA and Wort, markedly accelerated the cell death by oleifolioside A and, furthermore, knockdown of Beclin-1 and Atg7 using shRNA increased oleifolioside A-induced apoptosis, suggesting a cytoprotective function of autophagy against oleifolioside A-triggered apoptosis. Treatment of HCT-116 cells with oleifolioside A time-dependently activated extracellular signal-regulated kinase (ERK). Oleifolioside A-induced autophagy was dramatically inhibited by pretreatment with an ERK inhibitor, U0126, which resulted in a marked reduction in cell viability. These findings indicate that oleifolioside A induce autophagy through ERK activation in HCT-116 cells and that autophagy suppression enhances apoptosis induced by oleifolioside A.

Metformin Induces a Caspase 3-Unrelated Apoptosis in Human Colorectal Cancer Cell Lines HCT116 and SW620.

To study the effects of metformin on the proliferation and growth of human colorectal cancer cell lines HCT116 and SW620. The antiproliferative effect of metformin was assayed using an MTS reagent and its ability to inhibit colony formation was demonstrated using a clonogenic assay. Flow cytometry using YO-PRO-1/PI was performed to examine the effects of metformin on apoptosis and cell death of HCT116 and SW620. Caspase 3 activities were measured in caspase-3 activity tests using a caspase-3 activity kit. Furthermore, Western blots were performed with anti-PARP1, anti-caspase 3, and anti-cleaved caspase 3 to confirm whether caspase activation was present or not. Both MTS proliferation assays and clonogenic assays showed that metformin inhibited the proliferation and growth of HCT116 and SW620 cells in a concentration-dependent manner. Flow cytometric analysis identified early apoptosis and metformin-induced cell death in both cell lines. However, caspase 3 activity could not be detected. Cleavage of both PARP1 and pro-caspase 3 was not observed in the Western blot, confirming the absence of caspase 3 activations. This present study suggests a caspase 3-unrelated apoptosis mechanism of metformin-induced cell death in human colorectal cancer cell lines HCT116 and SW620.

WIPI2 enhances the vulnerability of colorectal cancer cells to erastin via bioinformatics analysis and experimental verification.

WD Repeat Domain Phosphoinositide Interacting 2 (WIPI2) is a WD repeat protein that interacts with phosphatidylinositol and regulates multiprotein complexes by providing a b-propeller platform for synchronous and reversible protein-protein interactions assembled proteins. Ferroptosis is a novel iron-dependent form of cell death. It is usually accompanied with the accumulation of membrane lipid peroxides. Our study is to focus on investigating the effect of WIPI2 on the growth and ferroptosis of colorectal cancer (CRC) cells and its potential mechanism. We analyzed the expression of WIPI2 in colorectal cancer versus normal tissues through The Cancer Genome Atlas (TCGA), and the relationship between clinical traits and WIPI2 expression and prognosis was assessed by univariate and multifactorial cox analysis. Next, we constructed the siRNAs targeting the WIPI2 sequence si-WIPI2 to further investigate the mechanism of WIPI2 in CRC cells through vitro experiments. Public data from the TCGA platform showed that WIPI2 expression was significantly elevated in colorectal cancer tissues compared to paracancerous tissues, and high WIPI2 expressionpredicted poor prognosis for CRC patients. Moreover, we found that the knockdown of WIPI2 expression could inhibit the growth and proliferation of HCT116 and HT29 cells. Furthermore, we found that the expression level of ACSL4 decreased and that of GPX4 increased when WIPI2 was knocked down, suggesting that WIPI2 can potentially positively regulate CRC ferroptosis. Meanwhile, both NC and si groups were able to further inhibit cell growth activity, as well as increase WIPI2 and decrease GPX4 expression when treated with Erastin, but the rate of cell viability inhibition and the trend of protein changes were more significantly in the NC group than si groups, which indicated that Erastin induced CRC ferroptosis through the WIPI2/GPX4 pathway thereby enhancing the sensitivity of colorectal cancer cells to Erastin. Our study suggested that WIPI2 had a promotional effect on the growth of colorectal cancer cells, and it also played an important role in the ferroptosis pathway.

Decursin affects proliferation, apoptosis, and migration of colorectal cancer cells through PI3K/Akt signaling pathway

We investigated the effects of decursin on the proliferation, apoptosis, and migration of colorectal cancer HT29 and HCT116 cells through the phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway. Decursin(10, 30, 60, and 90 μmol·L~(-1)) was used to treat HT29 and HCT116 cells. The survival, colony formation ability, proliferation, apoptosis, wound hea-ling area, and migration of the HT29 and HCT116 cells exposed to decursin were examined by cell counting kit-8(CCK8), cloning formation experiments, Ki67 immunofluorescence staining, flow cytometry, wound healing assay, and Transwell assay, respectively. Western blot was employed to determine the expression levels of epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), vimentin, B-cell lymphoma/leukemia-2(Bcl-2), Bcl-2-associated X protein(Bax), tumor suppressor protein p53, PI3K, and Akt. Compared with the control group, decursin significantly inhibited the proliferation and colony number and promoted the apoptosis of HT29 and HCT116 cells, and it significantly down-regulated the expression of Bcl-2 and up-regulated the expression of Bax. Decursin inhibited the wound healing and migration of the cells, significantly down-regulated the expression of N-cadherin and vimentin, and up-regulated the expression of E-cadherin. In addition, it significantly down-regulated the expression of PI3K and Akt and up-regulated that of p53. In summary, decursin may regulate epithelial-mesenchymal transition(EMT) via the PI3K/Akt signaling pathway, thereby affecting the proliferation, apoptosis, and migration of colorectal cancer cells.

TBX2‐WNT3A Axis Mediates the Development of Colorectal Cancer In Vitro and In Vivo

AbstractThis study is designed to explore the roles of the T‐Box transcription factor 2 (TBX2)‐Wnt family member 3A (WNT3A) axis in colorectal cancer. Clinical specimens are collected from the colorectal cancer patients and the volunteers. TBX2 overexpression and knockout stable cell lines and WNT3A knockdown cells are constructed. The xenograft animal model is established by using those stable cell lines. Cell proliferation, migration, and invasion are evaluated. Quantitative reverse transcription polymerase chain reaction and Western blotting are used to determine the expression levels of target biomarkers. ChIP analysis is utilized to assess the enrichment of TBX2 on the promoter site of WNT3A. An elevation of TBX2 is observed in colorectal cancer specimens and TBX2 expression is positively correlated to cell proliferation, migration, and invasion. Besides, TBX2 regulates the expressions of biomarkers relevant to epithelial‐mesenchymal transition (EMT) and WNT3A signaling pathways. Interestingly, WNT3A also promotes the HCT116 cell proliferation, migration, invasion by the regulation of EMT‐related biomarkers. The in vivo study shows that the TBX2‐WNT3A axis promotes tumor growth. TBX2‐WNT3A axis promotes the development of colorectal cancer.

Circ_0060967 contributes to colorectal cancer progression by sponging miR-1184 to up-regulate SRC proto-oncogene

Background and study aimsCircular RNAs (circRNAs) are closely associated with cancer pathogenesis. The purpose of our current study was to explore the role and mechanism of circ_0060967 in colorectal cancer (CRC) development. Patients and methodsHuman CRC specimens and paired healthy tissues were used to examine variable expression. The expression of circ_0060967 and microRNA (miR)-1184 was examined by quantitative reverse transcription-PCR. The protein levels of proliferating cell nuclear antigen, BCL2-associated X, apoptosis regulator (Bax), proto-oncogene nonreceptor tyrosine kinase Src (SRC), nuclear factor-κB inhibitor alpha (IκBα), phosphorylated-IκBα (p-IκBα), RELA proto-oncogene, nuclear factor-κB subunit (p65), and phosphorylated-p65 (p-p65) were determined by western blot. Proliferation and motility of HCT-116 and SW480 CRC cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and transwell assays, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation assay were used to determine the binding relation between miR-1184 and circ_0060967 or SRC. Animal studies were used to detect the role of circ_0060967 in CRC cell tumorigenicity. ResultsCirc_0060967 abundance was enhanced in human CRC tissue samples versus paired normal colorectal tissues and in HCT-116 and SW480 CRC cells versus normal HCO cells. Decreased expression of circ_0060967 could suppress cell growth, motility, and invasiveness of CRC cells in vitro and tumor growth in vivo. Circ_0060967 sponged miR-1184, and miR-1184 targeted SRC. Furthermore, we also found circ_0060967 affected cell growth by modulating miR-1184/SRC axis in CRC. ConclusionThis study demonstrates a novel circ_0060967/miR-1184/SRC regulatory cascade in affecting CRC cell malignant behaviors, which can have a broad effect on the field of molecularly targeted therapeutics.

Carboxypeptidase A6 suppresses the proliferation and invasion of colorectal cancer cells and is negatively regulated by miR-96–3p

BackgroundColorectal cancer (CRC) is a common malignant tumor, and this study aims to explore the role and the regulatory mechanism of carboxypeptidase A6 (CPA6) in CRC cells. MethodsSpecific shRNA targeting CPA6 mRNA was transfected into NCM460 and HT29 cells to down-regulate CPA expression, and expression plasmid was transfected into HCT116 cells to exogenously overexpress CPA6. The dual luciferase assay was used to detect the direct binding of miR-96–3p to CPA6 3′UTR. Phosphorylation and activation of Akt were detected using Western blot. Cells were treated with miR-96–3p mimics, Akt inhibitor (MK-2206) or agonist (SC79) for rescue experiments. The cell functions were evaluated using CCK-8, clone formation, transwell, and Western blot assays. Xenograft tumor assay was also used to analyze the effect of altered CPA6 expression on tumor growth. ResultsKnockdown of CPA6 promoted the proliferation, clone formation, migration, and invasion of NCM460 and HT29 cells in vitro, and the tumor growth of nude mouse xenograft tumor in vivo. Moreover, over-expression of CPA6 significantly inhibited the malignant proliferation and invasion of HCT116 cells in vitro, and the tumor growth of xenograft tumor in vivo. Furthermore, miR-96–3p could directly regulate CPA6 expression by targeting its 3′UTR, and miR-96–3p mimics rescued the inhibitory effects of CPA6 overexpression on the malignant proliferation and invasion of CRC cells. Finally, CPA6 knockdown enhanced Akt/mTOR phosphorylation and activation, while CPA6 overexpression inhibited Akt/mTOR activation. The regulatory effect of CPA6 on Akt/mTOR signaling was naturally regulated by miR-96–3p. Akt inhibitor or agonist rescued the effects of CPA6 knockdown or overexpression on proliferation and EMT of colon cancer cells. ConclusionCPA6 has a significant tumor suppressive effect on CRC by inhibiting the activation of Akt/mTOR signaling, and miR-96–3p negatively regulates the expression of CPA6.

Bromodomain-containing protein 9 activates proliferation and epithelial-mesenchymal transition of colorectal cancer via the estrogen pathway in vivo and in vitro.

Bromodomain-containing protein 9 (BRD9) has been reported to be upregulated in multiple malignancies and facilitate cancer progression. However, there is a paucity of data relating to its expression and biological role in colorectal cancer (CRC). Therefore, this current study examined the prognostic role of BRD9 in CRC and the underlying mechanisms involved. Real-time polymerase chain reaction (PCR) and Western blotting were used to examine the expression of BRD9 in paired fresh CRC and para-tumor tissues from colectomy patients (n=31). Immunohistochemistry (IHC) was performed to assess BRD9 expression in 524 paraffin-embedded archived CRC samples. The clinical variables are including age, sex, carcinoembryonic antigen (CEA), location of tumor, T stage, N stage, and TNM classification. The effect of BRD9 on the prognosis of CRC patients was explored by Kaplan-Meier and Cox regression analyses. Cell counting kit 8 (CCK-8), clone formation assay, transwell assay, and flow cytometry were used to determine CRC cell proliferation, migration, invasion, and apoptosis, respectively. Xenograft models in nude mice were established to investigate the role of the BRD9 in vivo. BRD9 mRNA and protein expression levels were significantly upregulated in CRC cells compared to normal colorectal epithelial cells (P<0.001). IHC analysis of 524 paraffin-embedded archived CRC tissues showed that high BRD9 expression was significantly associated with TNM classifications, CEA, and lymphatic invasion (P<0.01). Univariate and multivariate analyses indicated that BRD9 [hazard ratio (HR): 3.04, 95% confidence interval (CI): 1.78-5.20; P<0.01] expression and sex (HR: 6.39, 95% CI: 3.94-10.37; P<0.01) were independent prognostic factors for overall survival in the entire cohort. Overexpressing BRD9 promoted CRC cell proliferation, while silencing BRD9 inhibited the proliferation of CRC cells. Furthermore, we showed that BRD9 silencing significantly inhibited epithelial-mesenchymal transition (EMT) via the estrogen pathway. Finally, we demonstrated that silencing BRD9 significantly inhibited the proliferation and tumorigenicity of SW480 and HCT116 cells in vitro and in vivo in nude mice (P<0.05). This study demonstrated that BRD9 high could be an independent prognostic risk factor for CRC. Furthermore, the BRD9/estrogen pathway may contribute to the proliferation of CRC cells and EMT, suggesting that BRD9 may be a novel molecular target in the therapeutic treatment of CRC.

Berberine is a suppressor of Hedgehog signaling cascade in colorectal cancer

BackgroundColorectal cancer (CRC) is a malignant affliction that burdens people globally. Overactivated Hedgehog signal is highly implicated in CRC pathogenesis. Phytochemical berberine exerts strong potency on CRC, with molecular mechanism elusive. PurposeWe sought to study berberine's anti-CRC action and explore its underlying mechanism based on Hedgehog signaling cascade. MethodsIn CRC HCT116 cells and SW480 cells treated with berberine, the proliferation, migration, invasion, clonogenesis, apoptosis and cell cycle were measured, with determination of Hedgehog signaling pathway activity. Following establishment of mouse model of HCT116 xenograft tumor, the efficacies of berberine on carcinogenesis, pathological manifestation and malignant phenotypes of CRC were examined, with analysis of Hedgehog signaling axis in HCT116 xenograft tumor tissues. Additionally, toxicological study of berberine was conducted on zebrafish. ResultsBerberine was discovered to suppress the proliferation, migration, invasion and clonogenesis of HCT116 cells and SW480 cells. Furthermore, berberine caused cell apoptosis and blockaded cell cycle at phase G0/G1 in CRC cells, with dampened Hedgehog signaling cascade. In HCT116 xenograft tumor of nude mice, berberine inhibited tumor growth, alleviated pathological score, and promoted apoptosis and cell cycle arrest in tumor tissues, through constraining Hedgehog signaling. The toxicological study of berberine on zebrafish indicated that berberine incurred damage to the liver and heart of zebrafish at high dosage and prolonged administration. ConclusionsTaken together, berberine may inhibit the malignant phenotypes of CRC through diminishing Hedgehog signaling cascade. However, the potential adverse reactions should be taken into account upon abuse of berberine.

N-methylsansalvamide elicits antitumor effects in colon cancer cells in vitro and in vivo by regulating proliferation, apoptosis, and metastatic capacity.

N-methylsansalvamide (MSSV), a cyclic pentadepsipeptide, was obtained from a strain of Fusarium solani f. radicicola. The current study investigated the anti-colorectal cancer effect of MSSV. MSSV exhibited the inhibition of the proliferation in HCT116 cells via induction of G0/G1 cell cycle arrest by downregulating CDK 2, CDK6, cyclin D, and cyclin E, and upregulating p21WAF1 and p27KIP1. Decreased phosphorylation of AKT was observed in MSSV-treated cells. Moreover, MSSV treatment induced caspase-mediated apoptosis through elevating the level of cleaved caspase 3, cleaved PARP, cleaved caspase 9, and pro-apoptotic Bax. MSSV revealed the declined MMP-9 level mediated by reduction in the binding activity of AP-1, Sp-1, and NF-κB motifs, which led to the migration and invasion of HCT116 cells. In vitro metabolism with rat liver S9 fractions was performed to examine the effect of MSSV metabolites. The metabolic process enhanced the inhibitory effect of MSSV on the HCT116 cell proliferation via decline of cyclin D1 expression and AKT phosphorylation. Finally, oral administration of MSSV inhibited the tumor growth of HCT116 xenograft mice. These results suggest that MSSV is a potential anti-tumor agent in colorectal cancer treatment.

Raw Lacquer Extract from Toxicodendron vernicifluum in Combination with ONC201 Enhances the Inhibitory Effects on Colorectal Cancer Cell Activity.

The purpose of the present research was to explore the therapeutic impact of raw lacquer extract from Toxicodendron vernicifluum on colorectal cancer cells and to investigate the outcome of raw lacquer extract and ONC201 co-treatment on the activity of colorectal cancer cells. The cells of HCT116 were treated with raw lacquer extract, ONC201, or co-treatment. Subsequently, MTT, trypan blue staining, colony formation, annexin V/propidium iodide staining, wound healing, and transwell assays were performed to assess the effects of raw lacquer extract, ONC201 and the synthesis effect of co-treatment on cell activity, survival, proliferation, apoptosis, migration, and invasion in HCT116 cells. Western blotting and immunostaining assay were also performed to detect the expressions of tumor necrosis factor-related apoptosis-inducing ligand, death receptor-5, cleaved caspase-8, p-mTOR/mTOR, and p-S6K/S6K in cells. The results showed that ONC201 and raw lacquer extract had effective anti-cancer effects on HCT116 cells. ONC201 and raw lacquer extract treatment on colorectal cancer cells inhibited cell viability and growth, as well as induced cell apoptosis and cell death of HCT116. The migration and invasion of HCT116 cells were also inhibited. Significantly, raw lacquer extract and ONC201 cotreatment further enhanced the anti-colorectal cancer cell activity in HCT116 cells. Western blotting and immunostaining assay showed that raw lacquer extract in combination with ONC201 induced tumor necrosis factor-related apoptosis-inducing ligand/death receptor-5 expression activation, inhibited the expression of cleaved caspase-8/procaspase-8, and reduced the expression of p-mTOR/mTOR and p-S6K/S6K. These results indicated that raw lacquer extract in combination with ONC201 enhanced the inhibitory effects on colorectal cancer cell activity.

Expression and prognostic value of PRDX family in colon adenocarcinoma by integrating comprehensive analysis and in vitro and in vivo validation.

The peroxiredoxin family, a crucial regulator of redox reactions, is strongly associated with various tumorigenesis. However, the role of peroxiredoxin4 (PRDX4) in colon adenocarcinoma (COAD) remains poorly understood. Multicenter databases, including GEPIA, HPA, UALCAN, cBioPortal, cancerSEA, STRING, CCLE, and LinkedOmics, comprehensively analyzed transcriptional expression, prognostic value, genetic alterations, signaling pathways, and associated genes of the PRDXs in COAD patients. Colony formation, transwell, flow cytometry, sphere formation, and xenograft assays were performed to validate further in vitro and in vivo. Members of the PRDX family were differentially expressed in COAD, with each member showing varying degrees of genetic alterations. Intriguingly, only PRDX4 significantly correlated with COAD prognosis and stage. The single-cell sequencing suggested that PRDX4 is positively correlated with proliferation, apoptosis, and invasion, whereas negatively correlated with stemness. Moreover, PRDX4 involved in a series of critical biological processes, such as cell growth. Furthermore, in vivo and in vitro analyses indicated that knocking down PRDX4 inhibits the proliferation and invasion of HCT116 cells while promoting apoptosis and stemness. We identified PRDX4 expression as a novel potential prognostic marker in COAD.

Redundant roles of extra-cellular signal-regulated kinase (ERK) 1 and 2 in the G1-S transition and etoposide-induced G2/M checkpoint in HCT116 cells.

The extracellular signal-regulated kinase (ERK) 1 and 2 intracellular signaling pathways play key roles in a variety of cellular processes, such as proliferation and differentiation. Dysregulation of ERK1/2 signaling has been implicated in many diseases, including cancer. Although ERK1/2 signaling pathways have been extensively studied, controversy remains as to whether ERK1 and ERK2 have specific or redundant functions. In this study, we examined the functional roles of ERK1 and ERK2 in cell proliferation and cell cycle progression using an auxin-inducible degron system combined with gene knockout technology. We found that ERK1/2 double depletion, but not ERK1 or ERK2 depletion, substantially inhibited the proliferation of HCT116 cells during G1-S transition. We further demonstrated that ERK1/2-double-depleted cells were much more tolerant to etoposide-induced G2/M arrest than ERK1 or ERK2 single-knockout cells. Together, these results strongly suggest the functional redundancy of ERK1 and ERK2 in both the G1-S transition under physiological conditions and the DNA damage-induced G2/M checkpoint. Our findings substantially advance understanding of the ERK1/2 pathways, which could have strong implications for future pharmacological developments.

Hexafluoro-2-propanol represses colorectal cancer proliferation by regulating transaminases.

Hexafluoro-2-propanol (HFIP) is a metabolite of sevoflurane used as part of the general anaesthesia technique. This study aims to investigate the effect of HFIP in colorectal cancer (CRC) and the regulation of associated genes. The differential expressed genes (DEGs) with HFIP treatment were analysed based on GEO dataset GSE56256. Due to the restricted number of studies performing RNA-Seq in CRC with HFIP treatment, we selected a CRC patient cohort (GSE23878) to obtain DEGs that were compared with DEGs from GSE56256. The DEGs with opposite expression patterns in these two GEO datasets indicate that the genes mediate the function of HFIP, thereby repress the progression of CRC. The DEGs from these two GEO datasets were analysed independently. We obtained 10 up-regulated genes in GSE23878 (CRC patient cohort) were also down-regulated in GSE56256 (HFIP cohort), therefore, these 10 genes may function as tumor suppressors in CRC and may be involved in the function of HFIP. STRING analysis demonstrated an interaction in GPT2, PSAT1 and SLC7A11. The high expression of GPT2, PSAT1 and SLC7A11 were confirmed in TCGA-COAD datasets and in CRC cells. HFIP treatment repressed GPT2, PSAT1 and SLC7A11, which resulted in an inhibited proliferation and colony formation ability of DLD-1 and HCT-116 cells. Silencing GPT2, PSAT1 and SLC7A11 showed similar effect compared to HFIP treatment. Overexpression of GPT2, PSAT1 and SLC7A11 abrogated the effect of HFIP. In conclusion, the sevoflurane metabolite HFIP plays a cancer suppression role depending on cell proliferation in colorectal cancer through the down-regulation of GPT2, PSAT1 and SLC7A11 expression.

Lappaol F regulates the cell cycle by activating CDKN1C/p57 in human colorectal cancer cells

Context Lappaol F (LAF), a natural lignan from Arctium lappa Linné (Asteraceae), inhibits tumor cell growth in vitro and in vivo. The underlying mechanism involves the suppression of the Yes-associated protein. However, the specific role of LAF in cell cycle regulation remains unknown. Objective This study determined the molecular mechanism by which LAF regulates cell cycle progression. Materials and methods Various colon cancer cell lines (SW480, HCT15, and HCT116) were treated with LAF (25, 50, and 75 μmol/L) for 48 h. The effects of LAF on cell proliferation and cell cycle were determined using sulforhodamine B and flow cytometry assays. Differentially expressed proteins (DEPs) were identified using quantitative proteomics. Bioinformatic analysis of DEPs was conducted via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Expression levels of DEPs in the cell cycle pathway were analyzed using RT-qPCR and western blotting. Results LAF suppressed the proliferation of SW480, HCT15, and HCT116 cells (IC50 47.1, 51.4, and 32.8 μmol/L, respectively) and induced cell cycle arrest at the S phase. A total of 6331 proteins were identified and quantified, of which 127 were differentially expressed between the LAF-treated and untreated groups. GO and KEGG enrichment analyses revealed that DEPs mainly participated in the cell cycle. CDKN1C/p57 showed the most significant differential expression, with the highest fold-change (3.155-fold). Knockdown of CDKN1C/p57 attenuated the S phase cell cycle arrest and proliferation inhibition induced by LAF. Conclusion LAF exerts antitumor effects via S phase arrest by activating CDKN1C/p57 in colorectal cancer cells.

Silenced ANP32A inhibits the growth, invasion and migration of colorectal cancer in vitro via the inactivation of AKT pathway

To investigate the effect of ANP32A silencing on invasion and migration of colon cancer cells and the influence of the activity of AKT signaling pathway on this effect. Colorectal cancer HCT116 and SW480 were transfected with a small interfering RNA targeting ANP32A via a lentiviral vector. At 24, 48 and 72 h after the transfection, the changes in cell proliferation and AKT activity in the cells were detected using MTT assay and Western blotting, respectively. HCT116 and SW480 cells were treated with the AKT agonist SC79 or its inhibitor MK2206 for 24, 48, 72 and 96 h, and the changes in cell migration and invasion ability were analyzed using Transwell chamber assay and cell proliferation was assessed using MTT assay. The effects of SC79 and MK2206 on migration and invasion abilities of HCT116 and SW480 cells with or without ANP32A silencing were examined using wound healing and Transwell chamber assays, and the changes in the expression of metadherin (MTDH), a factor associated with cells invasion and migration, was detected with Western blotting. Lentivirus-mediated ANP32A silencing significantly down-regulated the activity of AKT and inhibited the proliferation of both HCT116 and SW480 cells (P < 0.01). The application of AKT inhibitor MK2206 obviously inhibited the proliferation, invasion and migration of the colorectal cancer cells (P < 0.05), while the AKT agonist SC79 significantly promoted the invasion and migration of the cells (P < 0.01). In HCT116 and SW480 cells with ANP32A silencing, treatment with MK2206 strongly enhanced the inhibitory effects of ANP32A silencing on cell invasion and migration (P < 0.05) and the expression of MTDH, while SC79 partially reversed these inhibitory effects (P < 0.01). ANP32A silencing inhibits invasion and migration of colorectal cancer cells possibly by inhibiting the activation of the AKT signaling pathway.

β-Sitosterol blocks the LEF-1-mediated Wnt/β-catenin pathway to inhibit proliferation of human colon cancer cells

ObjectivesThis study aimed to investigate the LEF-1-mediated Wnt/β-catenin pathway for its biological functions and prognostic value in colon cancer (CC). Furthermore, the potential molecular mechanism of β-sitosterol in CC was investigated in vitro. MethodsClinical information and gene expression profiles from CC patients were obtained based on Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. In addition, we applied R software “Limma” package for the differential analysis of LEF-1 between cancer and para-carcinoma tissue samples. Kaplan–Meier (KM) survival analysis was adopted for analyzing whether LEF-1 was of prognostic significance. Moreover, gene set enrichment analysis (GSEA) was adopted for pathway enrichment analysis and visualization. In addition, CCK8, plate cloning, scratch and high-content screening (HCS) imaging assays were performed to examine the therapeutic efficacy of β-sitosterol in human CC HCT116 cells. siRNA technology was employed to knock down LEF1 expression in HCT116 cells. qRT-PCR and Western-blot (WB) analysis were carried out to analyze the HCT-116 mRNA and protein expression levels, respectively. ResultsLEF-1 was up-regulated within CC and acted as an oncogenic gene. LEF-1 up-regulation predicted the dismal prognostic outcome and activated the Wnt/β-catenin pathway. β-sitosterol effectively suppressed HCT116 cells proliferation and invasion. For the mechanism underlying β-sitosterol, β-sitosterol was found to significantly down-regulate LEF-1 gene and protein expression and disrupt Wnt/β-catenin pathway transmission in HCT116 cells. After suppressing LEF-1 expression, its downstream targets including C-myc, Survivin and CCND1 were also down-regulated. ConclusionAccording to our results, LEF-1 down-regulation can effectively block Wnt/β-catenin pathway, inhibit CC cell growth and migration. Collectively, β-sitosterol can be used to treat CC, which can provide anti-tumor activity by targeting LEF-1.

LWJ-M30, a conjugate of DM1 and B6, for the targeted therapy of colorectal cancer with improved therapeutic effects.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide as well as a significant cause of mortality. The conventional treatment could cause serious side effects and induce drug resistance, recurrence and metastasis of cancers. Hence, specific targeting of cancer cells without affecting the normal tissues is currently an urgent necessity in cancer therapy. The emerging of peptide-drug conjugates (PDC) is regarded as a promising approach to address malignant tumors. LWJ-M30, a conjugate of DM1 and B6 peptide, targeted transferrin receptors (TfRs) on the surface of the CRC cells, showing a powerful anti-cancer effect. LWJ-M30 significantly inhibited the HCT116 cells proliferation and migration in vitro. LWJ-M30 also dramatically decreased the level of polymeric tubulin, while the disruption of microtubules caused the cell cycle to be arrested in the G2/M phase. LWJ-M30 induced the HCT116 cells apoptosis both in vivo and in vitro. The results in vivo demonstrated that LWJ-M30 could inhibit the HCT116 growth without affecting the mouse body weight. Taking these results together, our data indicated that LWJ-M30 could improve the therapeutic effects of DM1 while reducing the systemic toxicity in normal tissues.