Apoptosis In Human Colorectal Cancer Cells Research Articles

Decursin Induces G1 Cell Cycle Arrest and Apoptosis through Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress in Human Colorectal Cancer Cells in In Vitro and Xenograft Models.

Decursin, a coumarin isolated from Angelica gigas Nakai, possesses anti-inflammatory and anti-cancer properties. However, the molecular mechanisms underlying its anti-cancer effects against human colorectal cancer (CRC) are unclear. Therefore, this study aimed to evaluate the biological activities of decursin in CRC in vitro and in vivo and to determine its underlying mechanism of action. Decursin exhibited anti-tumor activity in vitro, accompanied by an increase in G1 cell cycle arrest and apoptosis in HCT-116 and HCT-8 CRC cells. Decursin also induced the production of reactive oxygen species (ROS), thereby activating the endoplasmic reticulum (ER) stress apoptotic pathway in CRC cells. Furthermore, the role of ROS in decursin-induced apoptosis was investigated using the antioxidant N-acetyl-L-cysteine. Inhibiting ROS production reversed decursin-induced ER stress. Moreover, decursin significantly suppressed tumor growth in a subcutaneous xenograft mouse model of HCT-116 and HCT-8 CRC cells without causing host toxicity. Decursin also decreased cell proliferation, as documented by Ki-67, and partly increased cleaved caspase 3 expression in tumor tissues by activating ER stress apoptotic pathways. These findings suggest that decursin induces cell cycle arrest and apoptosis in human CRC cells via ROS-mediated ER stress, suggesting that decursin could be a therapeutic agent for CRC.

P53-armed oncolytic adenovirus induces autophagy and apoptosis in KRAS and BRAF-mutant colorectal cancer cells.

Colorectal cancer (CRC) cells harboring KRAS or BRAF mutations show a more-malignant phenotype than cells with wild-type KRAS and BRAF. KRAS/BRAF-wild-type CRCs are sensitive to epidermal growth factor receptor (EGFR)-targeting agents, whereas KRAS/BRAF-mutant CRCs are resistant due to constitutive activation of the EGFR-downstream KRAS/BRAF signaling pathway. Novel therapeutic strategies to treat KRAS/BRAF mutant CRC cells are thus needed. We recently demonstrated that the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 exhibit therapeutic potential against KRAS-mutant human pancreatic cancer cells. In this study, we evaluated the therapeutic potential of OBP-301 and OBP-702 against human CRC cells with differing KRAS/BRAF status. Human CRC cells with wild-type KRAS/BRAF (SW48, Colo320DM, CACO-2), mutant KRAS (DLD-1, SW620, HCT116), and mutant BRAF (RKO, HT29, COLO205) were used in this study. The antitumor effect of OBP-301 and OBP-702 against CRC cells was analyzed using the XTT assay. Virus-mediated modulation of apoptosis, autophagy, and the EGFR-MEK-ERK and AKT-mTOR signaling pathways was analyzed by Western blotting. Wild-type and KRAS-mutant CRC cells were sensitive to OBP-301 and OBP-702, whereas BRAF-mutant CRC cells were sensitive to OBP-702 but resistant to OBP-301. Western blot analysis demonstrated that OBP-301 induced autophagy and that OBP-702 induced autophagy and apoptosis in human CRC cells. In BRAF-mutant CRC cells, OBP-301 and OBP-702 suppressed the expression of EGFR, MEK, ERK, and AKT proteins, whereas mTOR expression was suppressed only by OBP-702. Our results suggest that p53-armed oncolytic virotherapy is a viable therapeutic option for treating KRAS/BRAF-mutant CRC cells via induction of autophagy and apoptosis.

α-Hederin induces human colorectal cancer cells apoptosis through disturbing protein homeostasis

Protein homeostasis and quality control are crucial for normal cellular activities, and a severe imbalance in protein homeostasis can lead to cell death. α-Hederin, a pentacyclic triterpenoid saponin isolated from Fructus Akebia, has a clear role in promoting colorectal cancer (CRC) cell apoptosis and has been recently used for CRC therapy. However, whether the pro-apoptotic activity of α-hederin in CRC cells involves disturbing protein homeostasis remains unknown. Here, we aimed to uncover the underlying molecular mechanism of α-hederin-induced apoptosis in CRC cells. Cell viability and proliferation were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) and 5-ethynyl-2′-deoxyuridine (EdU) assays, respectively. Apoptosis was detected using flow cytometry and western blotting. Autophagic flux was examined by western blotting and AdPlus-mCherry-GFP-LC3B adenovirus infection assays, and western blotting and immunofluorescence staining were performed to detect the expression of proteins in related pathways. The results showed that α-hederin significantly inhibited the growth and promoted the apoptosis of human CRC cells. Furthermore, α-Hederin induced endoplasmic reticulum (ER) stress, but inhibited proteasomal degradation. Also, the autophagic flux was blocked by α-hederin although this drug promoted autophagosome formation, and the lysosomal degradation was inhibited. Expression of p-JNK and p-p38 were increased by α-hederin. So, our findings provide strong evidence that α-hederin disrupts protein homeostasis by blocking ER-associated degradation (ERAD) and autophagic flux, thereby contributing to apoptosis. PERK-eIF2α-ATF4-CHOP and IRE1-ASK1-JNK/p38 signal pathway may be involved in those regulation. Our results make it a promising alternative or adjunct therapeutic candidate for CRC.

FUT2 promotes the tumorigenicity and metastasis of colorectal cancer cells via the Wnt/β‑catenin pathway.

The incidence of colorectal cancer (CRC), a leading cause of cancer‑related mortality, has increased globally. Fucosyltransferase 2 (FUT2), catalyzing the α1, 2‑linked fucose in mammals, has been reported to be overexpressed in several malignant cancers, including CRC. However, the effects of FUT2 on CRC remain largely unknown. Herein, it was determined that the FUT2 expression levels in CRC tissues were higher than those in adjacent non‑tumor tissues, whereas no association with tumor stage was revealed. The results of biological functional analysis revealed that FUT2 knockdown inhibited the proliferation, migration and invasion of human CRC cells. Moreover, the knockdown of FUT2 arrested the CRC cells at the G0/G1 phase and promoted the apoptosis of human CRC cells. Western blot analysis demonstrated that the expression levels of β‑catenin, C‑myc and cyclin D1 were decreased by FUT2 knockdown in CRC cells, whereas the expression of glycogen synthase kinase‑3β and the phosphorylation levels of β‑catenin were increased. Additionally, Wnt2 was fucosylated by FUT2 in CRC cells. Furthermore, the knockdown of FUT2 inhibited the growth of human CRC invivo. Overall, the findings of the present study suggest that FUT2 may be used as a potential diagnostic biomarker and therapeutic target for CRC treatment.

Hydroxy-γ-sanshool from Zanthoxylum bungeanum (prickly ash) induces apoptosis of human colorectal cancer cell by activating P53 and Caspase 8.

Sanshools, long-chain polyunsaturated amides in Zanthoxylum bungeanum (prickly ash), have important bioactivity. The objective was to assess inhibitory effects and molecular mechanisms of sanshools isolated from supercritical fluid (SCF) extract on human colon adenocarcinoma cells (HCT-116) cultured in vitro. Cells were exposed to various concentrations (0, 50, 90, or 130 μM) of sanshools for 24 or 48 h, with assessment of apoptosis and cell cycle arrest as well as regulatory gene and protein expression associated with apoptosis and the cell cycle. Sanshools profoundly inhibited growth of HCT-116 cells, with hydroxy-γ-sanshool (HRS) being the optimal active component (IC50 = 88.01 μM) inhibiting cell proliferation and having no cytotoxic effect to normal cells (IC50 = 481.52 μM) by CCK-8 assay. In HCT-116 cells, HRS inhibited cell growth, induced morphological distortion, and arrested the cell cycle at G1 phase (50.31 ± 4.13% vs. 72.16 ± 8.14% in Control and 130 μM HRS, respectively), and also caused programmed cell death in a dose-dependent manner. The percentage of apoptotic cells were remarkably increased after treated with HRS (6.2, 11.9, 19.8, and 30.7% for 0, 50, 90, and 130 μM, respectively). Moreover, in HCT-116 cells, HRS significantly inhibited mRNA and protein levels of Cyclin D1, CDK4, PCNA, and increased mRNA and protein levels of P21, P53, Fas, and Caspase 8. Furthermore, inhibitors of P53 and Caspase 8 proteins significantly mitigated the HRS-induced cell cycle arrest and apoptosis. In conclusion, our study provides evidence that HRS induced human colorectal cancer cell apoptosis by up-regulating P53 and Caspase 8.

Resveratrol induces human colorectal cancer cell apoptosis by activating the mitochondrial pathway via increasing reactive oxygen species.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer‑related mortality worldwide according to Global Cancer Statistics2018. Resveratrol (RSV) is a phenolic compound that possesses anticancer functions against various types of cancer, including breast and gastric cancer. However, the functions and mechanism underlying RSV in CRC are not completely understood. The present study aimed to investigate the anticancer effects and mechanism underlying RSV in CRC cells by conducting Cell Counting Kit‑8, apoptosis, reactive oxygen species (ROS) and western blotting assays. The results suggested that RSV dose‑dependently inhibited CRC cell viability, and increased cell apoptosis and ROS levels compared with the control group. The protein expression levels of Bax, cytochromec, cleaved caspase‑9 and cleaved caspase‑3 were upregulated, whereas Bcl‑2 expression levels were downregulated in RSV‑treated CRC cells compared with control cells. The results indicated that RSV might activate the mitochondrial apoptotic pathway by increasing ROS release. The present study suggested that RSV possessed antitumour activity against CRC by modulating an ROS‑mediated mitochondrial apoptotic pathway.

Gracillin shows potent efficacy against colorectal cancer through inhibiting the STAT3 pathway.

Colorectal cancer (CRC) accounts for about 10% of all annually diagnosed cancers and cancer‐related deaths worldwide. STAT3 plays a vital role in the occurrence and development of tumours. Gracillin has shown a significant antitumour activity in tumours, but its mechanism remains unknown. The human CRC cell lines HCT116, RKO, and SW480 and immunodeficient mice were used as models to study the effects of gracillin on cell proliferation, migration and apoptosis. These were evaluated by cell viability, colony formation, wound‐healing migration and cell apoptosis assays. Luciferase reporter assay, and immunostaining and western blot analyses were used to explore the specific mechanism through which gracillin exerts its effects. Gracillin significantly reduces viability and migration and stimulates apoptosis in human CRC cells. It also significantly inhibits tumour growth with no apparent physiological toxicity in animal model experiments. Moreover, gracillin is found to inhibit STAT3 phosphorylation and STAT3 target gene products. In addition, gracillin inhibits IL6‐induced nuclear translocation of P‐STAT3. Gracillin shows potent efficacy against CRC by inhibiting the STAT3 pathway. It should be further explored as a unique STAT3 inhibitor for the treatment of CRC.

Long intergenic non-protein coding RNA-467 targets microRNA-451a in human colorectal cancer.

Accumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) are frequently overexpressed in colorectal cancer (CRC). However, few related lncRNA signatures have been established for predicting CRC metastasis. The purpose of the present study was to identify lncRNAs that serve key roles in the metastasis of human CRC, and their potential downstream targets. A total of 31 human CRC biopsy samples were collected, and the expression of long intergenic non-protein coding RNA-467 (linc00467) and its association with clinical characteristics were evaluated. Consequently, linc00467 was revealed to be overexpressed in human CRC tissues, and its expression was significantly associated with metastasis and Tumor-Node-Metastasis stage. In HT29 and HCT116 cells, linc00467-knockout was revealed to decrease cellular proliferation and increase apoptosis (P<0.05). Finally, the downstream target of linc00467 in CRC promotion was predicted using bioinformatics analysis. The results demonstrated that linc00467 targets and regulates the expression of microRNA (miR)-451a, promoting carcinogenesis and metastasis in CRC. In conclusion, the results of the present study indicate that increased linc00467 expression promotes metastasis by targeting miR-451a, which ultimately increases cellular proliferation and inhibits apoptosis in human CRC cells.

LYW-6, a novel cryptotanshinone derived STAT3 targeting inhibitor, suppresses colorectal cancer growth and metastasis

The constitutive activation of signal transducer and activator of transcription 3(STAT3) is associated with aggressive development and metastasis in colorectal cancer (CRC), but STAT3-targeting drugs remain elusive in clinic. Here, structure-based strategy was used to remodel the natural compound cryptotanshinone into a more effective STAT3 inhibitor LYW-6. Using the Biolayer Interferometry assay, we observed that LYW-6 exhibited specific interactions with STAT3(KD = 6.6 ± 0.7 μM). Western blot analysis and electrophoretic mobility shift assays (EMSA) showed that LYW-6 inhibited the phosphorylation of STAT3 tyrosine 705 (Tyr-705) and had slight effects on STAT1 and STAT5 phosphorylation. Western blot analysis on the upstream kinases of STAT3 confirmed that the inhibitory mechanism on p-STAT3 was independent of upstream kinases. Further investigation demonstrated that LYW-6 downregulated the expression of downstream oncogenes to inhibit cell viability, cell cycle development, and potently increased cell apoptosis in human CRC cells. The invasion and metastasis linked signaling was also blocked by LYW-6 treatment. LYW-6 was found to reduce the metastasis foci in lung on tail-lung metastasis models. In addition, it was observed that LYW-6 markedly diminished STAT3 phosphorylation in tumor tissue and significantly inhibited tumor growth on xenograft models. Tumor development on chemically-induced colorectal cancer model also significantly inhibited by LYW-6 treatment. These findings provided adequate evidence that STAT3 inhibitor LYW-6 might be a potential candidate agent for CRC treatment.

&lt;p&gt;Rhein sensitizes human colorectal cancer cells to EGFR inhibitors by inhibiting STAT3 pathway&lt;/p&gt;

BackgroundActivation of epidermal growth factor receptor (EGFR) has been reported in a variety of cancer types, including colorectal cancer (CRC), and represents a potential chemotherapeutic drug target. EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been increasingly applied in the clinical treatment of CRC, but development of drug resistance during the treatment has greatly limited their application. Signal transducer and activator of transcription 3 (STAT3) and its mediated signal transduction pathway play an important role in the occurrence, development and metastasis of CRC, and are related to the development of EGFR-TKI resistance in CRC.MethodsCell viability, colony formation and cellular morphology were examined to evaluate the potent antiproliferative effect of the STAT3 inhibitor napabucasin, LY5 and rhein on the human CRC cell lines HCT116, SW620, RKO and DLD-1. Flow cytometry-based analysis was employed to determine whether rhein can affect the cell cycle and apoptosis. The expression level of phosphorylated STAT3 (P-STAT3), and cell cycle- and apoptosis-related proteins BCL2, CDC2 BAX, Cyclin D1 and Cyclin B1 were detected by Western blot analysis.ResultsThis study revealed that rhein can significantly reduce cell viability and stimulate apoptosis in human CRC cells in a dose-dependent manner. In addition, rhein induced cell cycle arrest at the G2/M phase in CRC cells and dose-dependently inhibited the expression of cell cycle-related proteins. Additionally, it was found that napabucasin, LY5 and rhein considerably sensitized cells to the EGFR-TKI erlotinib, thus suppressing CRC cell proliferation. Rhein also inhibited the phosphorylation of its downstream target STAT3. Inhibition of STAT3 and EGFR phosphorylation was also observed after treatment with a combination of rhein and EGFR inhibitors.ConclusionThis study confirmed the synergistic effect of STAT3 inhibitor and EGFR inhibitor in CRC cell lines. Additionally, we found that rhein sensitizes human CRC cells to EGFR-TKIs by inhibiting STAT3 pathway. When combined with EGFR-TKIs, rhein may be a novel STAT3 inhibitor in CRC.

Formononetin promotes apoptosis of colorectal cancer cells via activation of mitochondria-dependent MAPK pathway

Purpose: To investigate whether formononetin exhibits antitumor activity in colorectal cancer cell lines via the mitochondria-dependent mitogen-activated protein kinase (MAPK) pathway. Methods: Human colorectal cells were treated with various doses of formononetin for 24 h, followed by Cell Counting Kit-8 (CCK-8) assay and western blot. Human colorectal cells were incubated with equivalent vehicle (DMSO) or 100 μM formononetin for 24 h, followed by nuclear staining with propidium iodide (PI) and diamidino-2-phenylindole (DAPI) for analyses of apoptosis. Human colorectal cells were incubated with equivalent vehicle or 100 μM formononetin for 24 h followed by analysis of cell migration and invasion. Human colorectal cells were incubated with equivalent vehicle (DMSO) or 100 μM formononetin for various duration (3, 6, 12, and 24 h), followed by detection of intracellular reactive oxygen species (ROS) level and measurement of mitochondrial membrane potential (Δψm) to monitor mitochondria functionality. Results: In human colorectal cancer cell lines SW1463 and T84, formononetin (> 20 μM) significantly inhibited cell growth (p < 0.05) in a dose-dependent manner, noticeably induced apoptosis, and suppressed cell migration and invasion. Western blot analysis revealed that formononetin treatment caused significantly increased levels of proapoptotic proteins, and suppression of cell proliferationrelated protein and matrix metallopeptidases (MMP) levels. Formononetin also induced mitochondrial depolarization and ROS generation in a time-dependent manner, indicating that formononetin mediates human colorectal cancer cell apoptosis via activation of MAPK pathway in a dose-dependent manner. Conclusion: Formononetin induces human colorectal cancer cell apoptosis via mitochondriadependent MAPK pathway, thus lending experimental support for the clinical application of formononetin for colorectal cancer therapy. Keywords: Formononetin, Colorectal cancer, Mitochondria, Reactive oxygen species, Cytochrome C, Mitogen-activated protein kinase

MiR-17-3P regulates the proliferation and survival of colon cancer cells by targeting Par4.

Colorectal cancer (CRC) is a common malignancy worldwide. However, the pathogenesis by which CRC progression occurs remains to be elucidated. The present study investigated the role of miRNA (miR)‑17‑3P in the regulation of CRC cell survival. Firstly, miR‑17‑3P expression was aberrantly upregulated in human CRC tumor tissues compared with controls. Further results demonstrated that the proliferation capacity of human CRC SW480 and LoVo cells was significantly increased by an miR‑17‑3P specific mimic, and was inhibited by miR‑17‑3P silencing. Conversely, the apoptosis of human CRC cells was remarkably decreased by miR‑17‑3P mimic, and enhanced by miR‑17‑3P suppression compared with control. Additionally, it was observed that there was a potential binding site of miR‑17‑3P on the 3'‑untranslated region of Prostate apoptosis responde‑4 (Par4) and miR‑17‑3P may directly target Par4 mRNA. In human CRC cells, an miR‑17‑3P inhibitor significantly upregulated Par 4 expression, however the miR‑17‑3P mimic reduced Par4expression. Furthermore, Par4 expression exhibited an inhibitory effect on the proliferation of CRC cells transfected with miR‑17‑3P mimic, and exhibited a promoting role in the repressed apoptosis by miR‑17‑3P mimic. Inconclusion, the results of the present study demonstrated that miR‑17‑3P is important in CRC cell survival by targeting Par4, indicating a novel finding regarding human CRC progression.

Epigenetic silencing of tumor suppressor candidate 3 confers adverse prognosis in early colorectal cancer.

Colorectal cancer (CRC) is a biologically and clinically heterogeneous disease. Even though many recurrent genomic alterations have been identified that may characterize distinct subgroups, their biological impact and clinical significance as prognostic indicators remain to be defined. The tumor suppressor candidate-3 (TUSC3/N33) locates to a genomic region frequently deleted or silenced in cancers. TUSC3 is a subunit of the oligosaccharyltransferase (OST) complex at the endoplasmic reticulum (ER) which catalyzes bulk N-glycosylation of membrane and secretory proteins. However, the consequences of TUSC3 loss are largely unknown. Thus, the aim of the study was to characterize the functional and clinical relevance of TUSC3 expression in CRC patients’ tissues (n=306 cases) and cell lines. TUSC3 mRNA expression was silenced by promoter methylation in 85 % of benign adenomas (n=46 cases) and 35 % of CRCs (n =74 cases). Epidermal growth factor receptor (EGFR) was selected as one exemplary ER-derived target protein of TUSC3-mediated posttranslational modification. We found that TUSC3 inhibited EGFR-signaling and promoted apoptosis in human CRC cells, whereas TUSC3 siRNA knock-down increased EGFR-signaling. Accordingly, in stage I/II node negative CRC patients (n=156 cases) loss of TUSC3 protein expression was associated with poor overall survival. In sum, our data suggested that epigenetic silencing of TUSC3 may be useful as a molecular marker for progression of early CRC.

TanshinoneI induces human colorectal cancer cell apoptosis: The potential roles of AuroraA-p53 and survivin-mediated signaling pathways.

Colorectal cancer (CRC) is one of the most common malignancies worldwide and a leading cause of cancer death. Despite decades of intensive investigations, effective interventional options are still limited and patient prognosis remains poor. TanshinoneI, an active compound from traditional Chinese herbal medicine Salvia miltiorrhiza Bunge, has been shown to inhibit cell growth of leukemia, lung, and breast cancers. However, whether and how TanshinoneI exerts similar effects on CRC needs to be elucidated. TanshinoneI induced CRC cell apoptosis was characterized and the roles of AuroraA-p53 and survivin-mediated pathways were analyzed in different CRC cell lines. TanshinoneI markedly inhibited CRC cell growth and induced apoptosis in CRC cells with functional p53 protein. Interestingly, TanshinoneI did not exert as much inhibitory effect on normal colon epithelial cells or CRC cells with mutant p53, indicating relative selectivity toward colorectal cancer cells with full presence of p53. In tse cells with wild-type p53, data showed that TanshinoneI mediated AuroraA inhibition results in p53 upregulation, which is required for cell apoptosis. In CRC cells with mutant p53 protein (not able to localize to the nucleus), however, AuroraA knockdown failed to induce CRC cell apoptosis. Instead, data showed that protein level of survivin decreased following TanshinoneI treatment. These observations were further substantiated by the pivotal role of survivin in TanshinoneI mediated apoptosis in CRC cells with p53 mutant. TanshinoneI, a novel natural compound, exerts significant inhibitory effect on CRC cell growth via a mechanism involving either AuroraA-p53 axis or survivin-involving mechanism depending on different intrinsic characteristics of tumor cells.

Hedyotis diffusa Willd. extract suppresses proliferation and induces apoptosis via IL-6-inducible STAT3 pathway inactivation in human colorectal cancer cells.

Recent studies have indicated that the inflammatory microenvironment plays a significant role in colorectal cancer (CRC). The interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway mediates the proliferative and anti-apoptotic activities required for oncogenesis under inflammatory conditions; thus, suppressing tumor growth by targeting the IL-6/STAT3 pathway is a promising therapeutic strategy for CRC. Our previous study reported that the ethanol extract obtained from Hedyotis diffusa Willd. (EEHDW) can induce apoptosis, and inhibit the proliferation of colon cancer cells and tumor angiogenesis by modulating various signaling pathways; however, less is known regarding the activity of EEHDW in a cancer-promoting inflammatory environment. Therefore, the present study investigated whether EEHDW inhibits the growth of the CRC HT-29 cell line via the IL-6/STAT3 signaling pathway. Pretreatment of HT-29 cells with IL-6 led to an increase in cell viability, colony formation and phosphorylated STAT3 (p-STAT3) expression. Treatment of these cells with EEHDW prior to IL-6 stimulation resulted in a significant reduction in the IL-6-induced phosphorylation of STAT3. In addition, EEHDW treatment significantly reduced the mRNA expression levels of cyclin D1, cyclin-dependent kinase 4 and B-cell lymphoma-2 (Bcl-2), and upregulated the expression levels of Bcl-2-associated X protein (P<0.05), which are important target genes of the IL-6/STAT3 pathway. These findings strongly indicated that EEHDW suppresses tumor cell growth and induces the apoptosis of human CRC cells via inactivation of the IL-6/STAT3 signaling pathway.

Caffeic acid derivatives inhibit the growth of colon cancer: involvement of the PI3-K/Akt and AMPK signaling pathways.

BackgroundThe aberrant regulation of phosphatidylinositide 3-kinases (PI3-K)/Akt, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (m-TOR) signaling pathways in cancer has prompted significant interest in the suppression of these pathways to treat cancer. Caffeic acid (CA) has been reported to possess important anti-inflammatory actions. However, the molecular mechanisms by which CA derivatives including caffeic acid phenethyl ester (CAPE) and caffeic acid phenylpropyl ester (CAPPE), exert inhibitory effects on the proliferation of human colorectal cancer (CRC) cells have yet to be elucidated.Methodology/Principal FindingsCAPE and CAPPE were evaluated for their ability to modulate these signaling pathways and suppress the proliferation of CRC cells both in vitro and in vivo. Anti-cancer effects of these CA derivatives were measured by using proliferation assays, cell cycle analysis, western blotting assay, reporter gene assay and immunohistochemical (IHC) staining assays both in vitro and in vivo. This study demonstrates that CAPE and CAPPE exhibit a dose-dependent inhibition of proliferation and survival of CRC cells through the induction of G0/G1 cell cycle arrest and augmentation of apoptotic pathways. Consumption of CAPE and CAPPE significantly inhibited the growth of colorectal tumors in a mouse xenograft model. The mechanisms of action included a modulation of PI3-K/Akt, AMPK and m-TOR signaling cascades both in vitro and in vivo. In conclusion, the results demonstrate novel anti-cancer mechanisms of CA derivatives against the growth of human CRC cells.ConclusionsCA derivatives are potent anti-cancer agents that augment AMPK activation and promote apoptosis in human CRC cells. The structure of CA derivatives can be used for the rational design of novel inhibitors that target human CRC cells.

Identification of differential proteins in colorectal cancer cells treated with caffeic acid phenethyl ester.

To investigate the molecular mechanisms of the anti-cancer activity of caffeic acid phenethyl ester (CAPE). Protein profiles of human colorectal cancer SW480 cells treated with or without CAPE were analysed using a two-dimensional (2D) electrophoresis gel-based proteomics approach. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250. Digital images were taken with a GS-800 Calibrated Densitometer, and image analysis was performed using PDQuest 2-D Analysis software. The altered proteins following CAPE treatment were further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry following a database search. The identified proteins were validated by Western blot and immunofluorescence assay. CAPE induced human colorectal cancer cell apoptosis. Four up-regulated proteins and seven down-regulated proteins in colorectal cancer cells treated with CAPE were found. The identified down-regulated proteins in CAPE-treated colorectal cancer cells were Triosephosphate Isomerase (Tim), Proteasome subunit alpha 4 (PSMA4) protein, Guanine nucleotide binding protein beta, Phosphoserine aminotransferase 1 (PSAT1), PSMA1, Myosin XVIIIB and Tryptophanyl-tRNA synthetase. Notably, CAPE treatment led to the down-regulation of PSAT1 and PSMA1, two proteins that have been implicated in tumorigenesis. The identified up-regulated proteins were Annexin A4, glyceraldehyde-3-phosphate dehydrogenase, Glucosamine-6-phosphate deaminase 1 (GNPDA1), and Glutathione peroxidase (GPX-1). Based on high match scores and potential role in cell growth control, PSMA1, PSAT1, GNPDA1 and GPX-1 were further validated by Western blotting and immunofluorescence assay. PSMA1 and PSAT1 were down-regulated, while GNPDA1 and GPX-1 were up-regulated in CAPE-treated colorectal cancer cells. These differentiated proteins in colorectal cancer cells following CAPE treatment, may be potential molecular targets of CAPE and involved in the anti-cancer effect of CAPE.

3,3′-Diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4 in human colorectal cancer cells

3,3′-Diindolylmethane (DIM) is a major in vivo condensation product of indole-3-carbinol, which is present in cruciferous vegetables. Although these compounds have been widely implicated in antitumorigenic and proapoptotic properties in animal as well as in vitro models of cancer, the underlying cellular mechanisms regulated by DIM are only partially understood. Activating transcription factor 3 (ATF3) is a member of the ATF/c-AMP response element-binding (CREB) subfamily of the basic-region leucine zipper family and has been known to induce apoptosis in human colorectal cancer (CRC) cells. The present study was performed to elucidate the molecular mechanism of ATF3 induction by DIM in human CRC cells. The DIM treatment induced apoptosis and induced ATF3 gene expression at protein and messenger RNA levels. DIM increased ATF3 promoter activity, and the region of −84 to +34 within ATF3 promoter was responsible for promoter activation by DIM. This region contained an ATF binding site. Deletion and point mutation of the ATF binding site (−23 to −16) abolished ATF3 promoter activation by DIM, and overexpression of ATF4 enhanced ATF3 transactivation. Chromatin immunoprecipitation assay confirmed the binding of ATF4 in the ATF3 promoter. Inhibition of ATF4 expression by small interference RNA results in repression of DIM-induced ATF3 expression. The current study demonstrates that DIM stimulates ATF3 expression through ATF4-mediated pathway and subsequently induces apoptosis in human CRC cells.