Colorectal Cancer Cell LoVo Research Articles

Gambogic acid reverses oxaliplatin resistance in colorectal cancer by increasing intracellular platinum levels.

Resistance to oxaliplatin (L-OHP) is a major obstacle to successful chemotherapy in colorectal cancer (CRC). In the present study, the ability of gambogic acid (GA) to reverse L-OHP resistance in CRC LoVo cells was investigated. L-OHP-resistant LoVo/L-OHP cells were established by exposing them to increasing concentrations of L-OHP. GA-reversed L-OHP-sensitive LoVo/L-OHP/GA cells were established by exposure to 0.5 µmol/l GA for 2 weeks. A Cell Counting Kit-8 assay was used to assess levels of proliferation. Flow cytometry was applied to detect apoptosis rates. Transwell assays were used to analyse invasion. Inductively coupled plasma mass spectrometry was used to determine intracellular platinum (Pt) content. Western blot analysis was used to reveal the protein levels of Human copper transporter 1 (hCTR1), Copper-transporting p-type adenosine triphosphatases 1 (ATP7A) and Copper-transporting p-type adenosine triphosphatases 2 (ATP7B). LoVo/L-OHP and LoVo/L-OHP/GA cell lines were successfully established, and it was identified that L-OHP inhibited the proliferation of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells in a dose-dependent manner. Compared with the parent LoVo cells, the anti-apoptosis and invasion properties of LoVo/L-OHP cells were enhanced, and were reversed by GA treatment. Intracellular Pt content was highest in the LoVo cells, followed by LoVo/L-OHP/GA cells, and then lowest in the LoVo/L-OHP cells. Downregulated hCTP1 and upregulated ATP7A and ATP7B were associated with L-OHP resistance, and GA reversed the resistance by increasing levels of hCTR1 and decreasing levels of ATP7A and ATP7B. In conclusion, GA has the potential ability to reverse L-OHP resistance in CRC cells by increasing intracellular Pt content, which it achieves by increasing hCTR1 levels and decreasing ATP7A and ATP7B levels. GA may represent a promising treatment agent for L-OHP resistance.

β‑arrestin2 promotes 5‑FU‑induced apoptosis via the NF‑κB pathway in colorectal cancer.

It has been demonstrated that β‑arrestin2 is involved in the initiation and development of many types of cancers. However, its role in colorectal cancer (CRC) remains poorly understood. The present study investigated the role of β‑arrestin2 in CRC using CRC patient tissues as well as the LoVo and HCT116 CRC cell lines. Briefly, significantly higher expression of β‑arrestin2 was observed in CRC tissues compared with normal colon tissues. In addition, the downregulation of β‑arrestin2 reduced 5‑FU‑induced apoptosis in the LoVo cells, while the overexpression of β‑arrestin2 increased the apoptosis of HCT116 cells invitro. Furthermore, the downregulation of β‑arrestin2 reduced the expression of the pro‑apoptotic proteins cleaved‑caspase‑3 and Bax, and increased the expression of the anti‑apoptotic protein Bcl‑2 after 5‑FU treatment. In addition, the expression of p‑p65 was increased after the β‑arrestin2 downregulation and was decreased after the β‑arrestin2 overexpression. However, β‑arrestin2 downregulation had no effect on the proliferation, migration and invasion capacity of the LoVo cells. In conclusion, these results indicated that β‑arrestin2 promoted 5‑FU‑induced CRC cell apoptosis via the NF‑κB pathway and may be used as a prognosis marker for CRC.

Sodium Butyrate Inhibits Colorectal Cancer Cell Migration by Downregulating Bmi-1 Through Enhanced miR-200c Expression.

Short-chain fatty acid sodium butyrate (NaB) is the byproduct of bacterial anaerobic fermentation of dietary fiber in the colon, and has been shown to have an antitumor effect on colorectal cancer (CRC). The miR-200 family is a key regulator of the epithelial-mesenchymal transition (EMT). We investigate the role of miR-200s expression on cell migration in NaB-treated CRC cells. HCT116 and LOVO CRC cells treated with NaB depicted reduced cell proliferation, enhanced apoptosis, and cell cycle arrest. NaB inhibited cell migration in the wound healing and transwell assays, and in spheriod cultures while regulating EMT-related protein expression. NaB reciprocally increased miR-200s but reduced expression of their target genes (Bmi-1, Zeb1, EZH2). Cells transfected with miR-200c shRNA displayed a significant blockade of NaB-induced anti-invasive activity. Upregulation of Bmi-1 expression partially reversed the effect of NaB. In addition to inhibition of tumor growth in vivo, qRT-PCR results showed that NaB increased miR-200c/200b/492 expression in the tumor tissues. Immunohistochemistry and Western blotting results demonstrated that NaB decreased Bmi-1 expression in vivo. NaB inhibits CRC cell migration by enhancing miR-200c expression-mediated downregulation of Bmi-1. These findings support the utility of NaB in colorectal cancer therapy.

Lupeol alters ER stress-signaling pathway by downregulating ABCG2 expression to induce Oxaliplatin-resistant LoVo colorectal cancer cell apoptosis.

Colorectal cancer (CRC) is one of the most common cancers and causes of cancer-related death. There are several first-line chemotherapeutic drugs used to treat CRC. Oxaliplatin (OXA) is an alkylating cytotoxic agent that is usually combined with other chemotherapeutic drugs to treat stage II and stage III CRC. However, cancer cells commonly acquire multidrug resistance (MDR), which is a major obstruction to cancer treatment. Recent studies have shown that natural components from traditional Chinese medicine or foods that have many biological functions may be new adjuvant therapies in clinical trials. We challenged LoVo CRC cell lines with OXA in a dose-dependent manner to create an OXA-resistant model. The expression of ABCG2 was significantly higher, and levels of endoplasmic reticulum (ER) stress markers were lower than those Parental cells. However, Lupeol, which is found in fruits and vegetables, has been shown to have bioactive properties, including anti-tumor properties that are relevant to many diseases. In our study, Lupeol downregulated cell viability and activated cell apoptosis. Moreover, Lupeol decreased the expression of ABCG2 and activated ER stress to induce OXA-resistant cell death. Importantly, the anti-tumor effect of Lupeol in OXA-resistant cells was higher than that of LoVo Parental cells. In addition, we also confirmed our results with a xenograft animal model, and the tumor size significantly decreased after Lupeol injections. Our findings show that Lupeol served as a strong chemoresistant sensitizer and could be a new adjuvant therapy method for chemoresistant patients.

MiR‑1273g‑3p promotes proliferation, migration and invasion of LoVo cells via cannabinoid receptor 1 through activation of ERBB4/PIK3R3/mTOR/S6K2 signaling pathway.

MicroRNAs (miR) are important in various crucial cell processes including proliferation, migration and invasion. Dysregulation of miRNAs have been increasingly reported to contribute to colorectal cancer. However, the detailed biological function and potential mechanisms of miR‑1273g‑3p in colorectal cancer remain poorly understood. The expression levels of miR‑1273g‑3p in human colorectal cancer LoVo cell lines were detected via reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The target genes of miR‑1273g‑3p were predicted by bioinformatics and verified by a luciferase reporter assay, RT‑qPCR and western blotting. The MTT, wound‑healing and Transwell assays were used to examine the biological functions of miR‑1273g‑3p in LoVo cells. The potential molecular mechanisms of miR‑1273g‑3p on LoVo cell proliferation, migration and invasion was detected by western blotting. The results of the present study demonstrated that miR‑1273g‑3p expression was extensively upregulated in LoVo cells compared with the normal colon epithelial NCM460 cell line. Further studies indicated that miR‑1273g‑3p inhibitor significantly suppressed LoVo cell proliferation, migration and invasion compared with inhibitor control. Following this, the cannabinoid receptor 1 (CNR1) was identified as a direct target gene of miR‑1273g‑3p. Knockdown of CNR1 restored the phenotypes of LoVo cells transfected with miR‑1273g‑3p inhibitor. Furthermore, the potential molecular mechanism of miR‑1273g‑3p on LoVo cell proliferation, migration and invasion may be mediated by activating the Erb‑B2 receptor tyrosine kinase 4 (ERBB4)/phosphoinositide‑3‑kinase regulatory subunit 3 (PIK3R3)/mechanistic target of rapamycin (mTOR)/S6 kinase 2 (S6K2) signaling pathway. These observations indicated that miR‑1273g‑3p promoted the proliferation, migration and invasion of LoVo cells via CNR1, and this may have occurred through activation of the ERBB4/PIK3R3/mTOR/S6K2 signaling pathway, suggesting that miR‑1273g‑3p may serve as a novel therapeutic target for the effective treatment of colorectal cancer.

MEGF6 Promotes the Epithelial-to-Mesenchymal Transition via the TGFβ/SMAD Signaling Pathway in Colorectal Cancer Metastasis

Background/Aims: Colorectal cancer (CRC) is a malignancy that has high morbidity and mortality and is initiated from accumulative genetic events. Although much effort has been made to elucidate the genetic mechanism underlying this disease, it still remains unknown. Here, we discovered a novel role for multiple epidermal growth factor-like domains protein 6 (MEGF6) in CRC, namely, that it induces the epithelial-to-mesenchymal transition (EMT) to promote CRC metastasis via the transforming growth factor beta (TGFβ)/SMAD signaling pathway. Methods: RNA sequencing data from the Gene Expression Omnibus database were analyzed using R software. Based on The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) cohort, the clinical significance of MEGF6 was investigated. HCT8R, HCT116, and LoVo CRC cells were transfected with small interfering RNA against MEGF6, and their proliferation and sensitivity to fluorouracil were evaluated with the MTT cell proliferation and colony formation assays. Proteins associated with cell growth were detected by western blot analysis. The apoptosis of cells was evaluated by Annexin V/propidium iodide staining, and transwell assays were performed to assess the involvement of MEGF6 in cell migration. Markers of EMT and TGFβ/SMAD signaling were evaluated by quantitative PCR and western blotting, and the correlation between MEGF6 and these markers was assessed in the TCGA colon and renal adenocarcinoma cohort. Results: The results showed that MEGF6 was upregulated in HCT8R cells. In addition, MEGF6 was significantly overexpressed in tumor tissue and predicted a poor survival in the TCGA-COAD cohort. Moreover, MEGF6 accelerated CRC cell growth and inhibited apoptosis, and promoted CRC metastasis by inducing the EMT. Finally, we found that TGFβ/SMAD signaling triggered the expression of Slug, which regulates the MEGF6-mediated EMT. Conclusions: MEGF6 may serve as an oncogene to promote cell proliferation and inhibit apoptosis. MEGF6 can also accelerate cell migration via TGFβ/SMAD signaling-mediated EMT.

Rubus sanctus Schreb. root extract alters the MicroRNA expression and inhibits tumor activities of colorectal cancer cell lines

Background: Colorectal cancer (CRC) is one of the most common cancers in the world. Although surgical and screening techniques have vastly improved in the last 30 years, chemotherapeutics have not advanced sufficiently for successful treatment. Objective: The aim of this study is to investigate the microRNA (miRNA) expression changes and anticancer agent potential of Rubus Sanctus Schreb. root extract (RRE) on LoVo and HT-29 colorectal adenocarcinoma cell lines. Materials and Methods: LoVo and HT-29 CRC cell lines treated with different concentrations of RRE to find growth inhibitory effect with WST-1 assay. Fifty percent growth inhibition and 25% growth inhibition concentrations further evaluated with annexin V, total caspase, cell cycle, and migration assays. Real-time polymerase chain reaction was used to investigate the expression differences in miRNA after extract treatment. Results: Cell proliferation was reduced 77.98% in HT-29 cells after RRE treatment ( P P P P P Conclusion: Our data show for the first time that RRE has the capability to inhibit CRC cell proliferation and invasion and alter epigenetic mechanisms. Although further studies should be conducted on this topic, RRE is thought to be a potential candidate for the future studies regarding new therapy options. Abbreviation used: RRE: Rubus Sanctus Schreb. root extract; CRC: Colorectal cancer; GI 50 , GI 25: 50% and 25% growth inhibition; miRNA: MicroRNA; MGMT: O-6-Methylguanine-DNA methyltransferase; UTR: Untranslated region; Akt: Protein kinase B; PI3K: Phosphoinositide 3-kinase; NF-κB: Nuclear factor kappaB; iNOS: Inducible nitric oxide synthase; COX-2: cyclooxygenase 2; TNFα: tumor necrosis factor alpha; IL-6: Interleukin 6; DMEM: Dulbecco's Modified Eagle's medium; RPMI-1640: Roswell Park Memorial Institute 1640 medium; HPLC: High-performance liquid chromatography; UV-Vis: Ultraviolet-visible; FBS: Fetal bovine serum; OD: Optical density; RFU: Relative fluorescence unit; PE: Phycoerythrin; 7-AAD: 7-Aminoactinomycin D; PBS: Phosphate-buffered saline; EGFR: Epidermal growth factor receptor; 5-FU: Fluorouracil; MSI: Microsatellite instability; NSCLC: Nonsmall cell lung cancer; TGF-β: Transforming growth factor beta; EMT: Epithelial–mesenchymal transition

A fluorogenic probe for sensitive detection of MMP-2/9 activities in clinical colorectal cancer tissues

Developing a novel fluorogenic probe which can be specifically recognized by active matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2/9) to rapidly and accurately detect the clinical colorectal cancer is significant but remains a great challenge. Herein, we have designed and synthesized a novel fluorescence resonance energy transfer (FRET)-based probe Dab-GPLGVRGY-FITC for specific detection of MMP-2/9 activity. The probe was prepared by conjugating fluorescein-N3 (FITC-N3) and fluorescent quencher dabcyl-NHS to MMP-2/9-responsive peptide substrate GPLGVRGY through copper (I)-catalyzed ″click″ and amidation reactions, respectively. To assess the specificity and sensitivity of the probe for active MMP-2/9 in vitro , the enzymatic assays were firstly performed with recombinant MMP-2/9. Fluorescent signal change of the mixed solution of probe and MMP-2 with and without treatment of MMP-2 specific inhibitor was recorded at 520 nm with fluorescence spectrometer. The capability of probe for specific detection of endogenous MMP-2/9 activity was further evaluated with human colorectal cancer cell LoVo and 30 clinical colorectal cancer tissue samples. The in vitro enzymatic assay results indicated that the probe showed good specificity and sensitivity for recombinant MMP-2/9 comparing to other proteins. The detection limit for recombinant MMP-2 is estimated to be 14 ng mL−1. Besides, the confocal laser scanning microscopy (CLSM) imaging results showed that enhanced green fluorescence signals were collected for LoVo cells upon treatment with probe. However, most of the fluorescence was readily competed away by pretreatment of the probe with inhibitor. In sharp contrast, only weak fluorescence was observed for normal gastric mucosa epithelial GES-1 cells. Additionally, the lysates of 15 clinical colorectal tissues displayed ~5 to 9-fold fluorescence enhancements after treatment of the probe in comparison with normal gastritis tissues. However, the fluorescence intensities of 15 peficancerous tissue lysates was only ~0.9 to 3-fold higher than normal tissues. Most notably, in comparison with normal gastritis tissue, strong green fluorescence was observed for both colorectal tissue and peficancerous tissue once the probe was sprayed onto the tissue surface. In summary, this probe shows good specificity and sensitivity for the detection of active MMP-2 both in cancer cell and clinical cancer tissue, which may have great potential for early diagnosis of MMP-2/9 active colorectal cancer in clinic.

18β-glycyrrhetinic acid exhibits potent antitumor effects against colorectal cancer via inhibition of cell proliferation and migration.

Accumulating evidence shows that 18β-glycyr-rhetinic acid (GRA) has antitumor activities in breast, ovarian cancer and leukemia, while its role in colorectal cancer remains unknown. In the present study, we investigated the effect of GRA in colorectal cancer cells LoVo, SW480 and SW620 and studied the underlying molecular mechanisms. Results showed that GRA had potent inhibitory effects on colorectal cancer cell proliferation in a dose- and time-dependent manner invitro and invivo. Growth inhibition was mediated by pro-apoptosis, as evident from AnnexinV-FITC staining, the reduced expression of survivin and the induced expression of cleaved PARP. Furthermore, GRA treatment resulted in marked reduction of cell migration, invasion and wound healing capability, accompanying by the downregulated MMP expression. Moreover, GRA decreased the protein levels of p-PI3K, p-AKT, p-STAT3, p-JNK, p-p38 and p-NF-κB p65, of which the phosphorylation of PI3K and STAT3 decreased as early as 2h after the GRA treatment. These results suggest that regulation of the apoptosis, invasion and migration of colorectal cancer cells by GRA might be through suppressing PI3K and STAT3 signaling pathways. the present study indicated that GRA could be a potential effective therapy for patients with colorectal cancer.

Effect of microRNA-371-5p on human colorectal cancer cell proliferation and apoptosis

Objective To observe the effect of microRNA (miRNA, miR)-371-5p on human colorectal cancer LoVo and SW620 cell proliferation and apoptosis. Methods Constructed miR-371-5p overexpression lentivirus vector and stable gene silencing miR-371-5p vector [miR-371-5p/short hairpin RNA (shRNA)], and were transfected into LoVo and SW620 cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) detected the expression of miR-371-5p, cell counting kit-8 (CCK-8), colony formation assay, soft agar colony formation assay, flow cytometry detected the proliferation and apoptosis of LoVo and SW620 cells. Results The expression of miR-371-5p in LoVo and SW620 cells transfected miR-371-5p (14.25±0.25, 10.01±0.19) were higher than that of negative control group and blank control group (1.03±0.02, 1.04±0.03; 1.00±0.00, 1.00±0.00; F=21.426, 8.957, P=0.000, 0.003), The expression of miR-371-5p in LoVo and SW620 cells transfected miR-371-5p/shRNA (0.08±0.00, 0.11±0.01) were lower than that of negative control group and blank control group cells (0.97±0.02, 1.04±0.03; 1.00±0.00, 1.00±0.00; F=15.231, 8.537, P=0.000, 0.005). After LoVo and SW620 cells transfected 3 d and 7 d, the proliferation of miR-371-5p transfected cell was lower than that of negative control group and blank control group (F=5.932, 19.324, 7.256, 35.846, P=0.003, 0.000, 0.000, 0.000); the proliferation of transfected miR-371-5p/shRNA cell was higher than that of negative control group and blank control group (F=37.342, 43.254, 43.242, 105.231, P=0.003, 0.000, 0.000, 0.000). The cell clone number of miR-371-5p transfected LoVo and SW620 were lower than that of negative control group and blank control group cells (F=5.432, 4.023, P=0.004, 0.022); the cell clone number of miR-371-5p/shRNA transfected LoVo and SW620 were higherthan that of negative control group and blank control group cells (F=3.976, 4.732, P=0.015, 0.011). The cell colony number of miR-371-5p transfected LoVo and SW620 were lower than that of negative control group and blank control group cells (F=5.647, 4.023, P=0.007, 0.015); the cell colony number of miR-371-5p/shRNA transfected LoVo and SW620 were higher than that of negative control group and blank control group cells (F=4.231, 4.231, P=0.012, 0.013). The difference of cell apoptosis rate between miR-371-5p transfected cells, miR-371-5p/shRNA transfected cells and the negative control group and blank control group were no statistically significant (F=0.883, 1.017, 1.165, 0.954, P=0.742, 0.474, 0.483, 0.621). Conclusion MiR-371-5p can inhibite the proliferation of colorectal cancer LoVo and SW620 cells, has little effect on apoptosis. Key words: MicroRNA-371-5p; Colorectal cancer; Proliferation; Apoptosis

Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma.

Angiopoietin-2 (Ang-2) has been investigated in cancer primarily in terms of its angiogenic function, and its role as an oncogene has yet to be elucidated. The current study hypothesized that Ang-2 may be an oncogene and have a function in tumor progression. An investigation of the function of Ang-2 in the LoVo colorectal cancer (CRC) cell line in vitro, which expresses a high level of Ang-2, was performed by knocking down endogenous expression with a targeted short hairpin RNA. The aggressive phenotypic effects of Ang-2 on experimental and control group cells were assessed using cell proliferation, migration and invasion assays. The association between Ang-2 expression levels and clinicopathological factors was evaluated in 415 CRC tissues using immunohistochemistry. Suppressing Ang-2 expression decreased cellular proliferation, invasion and migration in an in vitro study. Ang-2 overexpression was observed in 46% of patients with CRC and was significantly associated with pT (P=0.048), pN (P<0.001), venous invasion (P=0.023), lymphatic invasion (P<0.001) and tumor-node-metastasis stage (P=0.022). Furthermore, Ang-2 overexpression was an independent prognostic factor in pN stages 1 and 2. These results reveal that Ang-2 may be an oncogene in colorectal carcinogenesis and its expression may exert aggressive phenotypic effects during tumor progression. In addition, Ang-2 expression may serve as a prognostic marker and a potential drug target.

Down regulation TUG1 in LoVo colorectal cancer cells and its affection on cell proliferation and migration

Down regulation TUG1 in LoVo colorectal cancer cells and its affection on cell proliferation and migration

Effect of ASCT2 gene knock-down by shRNA on biological behaviors of colorectal cancer cells

To investigate the effect of ASCT2 gene (glutamine transporter) knock-down by shRNA on biological behaviors of colorectal cancer cells. shRNA was transfected into colorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine. ASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells(P<0.01). MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells (A490) and decrease the number of invasive Lovo and SW480 cells from the membrane (both P<0.01). The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells (both P<0.01). ASCT2 plays an oncogenic role in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.

MicroR-545 mediates colorectal cancer cells proliferation through up-regulating epidermal growth factor receptor expression in HOTAIR long non-coding RNA dependent.

The functional impact of recently discovered miRNAs in human cancer remains to be clarified. One miRNA in colorectal cancer which has attracted attention is miR-545. In this study, we examined the function of miR-545 in proliferation of colorectal cancer cells. Expressions of HOTAIR, miRNA-545, and epidermal growth factor receptor (EGFR) mRNA were measured in 100 paired cancerous and non-cancerous tissues as well as in SW480 and LOVO colorectal cancer cell (CRC) lines by quantitative RT-PCR. The relative protein level of EGFR was measured using western blotting. Effects of miRNA-545 and HOTAIR on gastric cancer cells were studied by overexpression and RNA interference approaches. Insight of mechanism of promotion cancer by miR-545 was gained from luciferase reporter assay and gene expression analysis. CRC proliferation was evaluated using clone formation and MTT assay. Differential expressions of HOTAIR, miR-545, and EGFR were observed in cancerous tissues in comparison to non-cancerous tissues. By expressional management of miR-545, we observed that miR-545 negatively regulated cell proliferation. Also luciferase reporter assay revealed that miR-545 inhibited regulated EGFR expression by affecting its 3'-UTR activity. In addition, miR-545 expression was suppressed by HOTAIR overexpression whereas enhanced by HOTAIR silence. Suppression of EGFR expression by miR-545 mimic was abrogated by HOTAIR overexpression. Monitoring of tumor growth in mice showed that miR-545 overexpression suppressed LOVO tumor growth. Our data suggested that HOTAIR long non-coding RNA mediates microR-545 regulating colorectal cancer cells proliferation.

WNT5A Promoter Methylation Is Associated with Better Responses and Longer Progression-Free Survival in Colorectal Cancer Patients Treated with 5-Fluorouracil-Based Chemotherapy.

Aberrant activation of the canonical WNT or WNT/β-catenin signaling pathway plays a pivotal role in multiple types of cancers. WNT5A, a nontransforming WNT protein suppressing the Wnt/β-catenin signaling pathway, is frequently detected to be hypermethylated in colorectal cancer (CRC). In this study, we investigated the prognostic value of WNT5A methylation in human patients and its potential underlying molecular mechanisms in cultured human CRC cells. We measured WNT5A mRNA level using qRT-PCR and DNA methylation using methylation-specific PCR (MSP) in HCT116, HT29, SW620, HCT8, LoVo, SW480, and Rko CRC cells. 5-FU-mediated tumor suppression was determined by measuring cell viability using MTT assay in cultured CRC cells. We also determined whether WNT5A methylation was associated with drug response and progression-free survival in CRC patients (n = 126) treated with 5-FU-based chemotherapy as first-line treatment. WNT5A expression inversely correlated with methylation status of CRC cells. Moreover, WNT5A expression was restored upon demethylation in hypermethylated cells. 5-FU-induced tumor suppression (reduced cell viability) was reduced by WNT5A overexpression in hypermethylated HCT116 and SW620 cells and enhanced by WNT5A downregulation in unmethylated LoVo and SW480 cells. In 5-FU-treated CRC patients, WNT5A methylation status is associated with better drug response and longer progression-free survival, suggesting that 5-FU is more effective in CRC with WNT5A hypermethylation. WNT5A methylation status is prognostic and is useful as a potential drug selection biomarker in CRC.

Sufentanil attenuates oxaliplatin cytotoxicity via inhibiting connexin 43‑composed gap junction function.

Comprehensive strategies for the treatment of colorectal cancer (CRC) have become increasingly important. One of the most important factors is pain relief. Therefore, patients with CRC are concurrently treated with analgesics and chemotherapeutic agents; however, the effects of analgesics on the therapeutic activity of chemotherapeutic agents remain largely unknown. The present study investigated the effects of three widely used analgesics in clinics: Fentanyl, remifentanil and sufentanil, on the cytotoxicity of oxaliplatin, a commonly used chemotherapeutic agent for CRC. Furthermore, the underlying mechanisms of those effects in association with connexin 43 (Cx43)‑composed gap junction (GJ) function were analyzed. The Lovo, Colo320, HCT116 and HT29 human CRC cell lines, with or without Cx43 expression, were used to examine the effects of the three analgesics on the cytotoxicity of oxaliplatin. The results demonstrated that in the cell lines expressing Cx43 (Lovo and Colo320), the cytotoxicity of oxaliplatin was attenuated and Cx43 GJ function was inhibited. Sufentanil, not fentanyl or remifentanil, inhibited Cx43 GJ function effectively, and reduced the cytotoxicity of oxaliplatin. In contrast, these effects were not observed in the other two colon cancer cell lines not expressing Cx43 (HCT116 and HT29). These results suggested that alternation of Cx43 GJ function may regulate the cytotoxicity of oxaliplatin in regard to CRC. Furthermore, sufentanil, not fentanyl or remifentanil, suppressed the cytotoxicity of oxaliplatin through inhibition of Cx43 GJ function. These results may be beneficial for the treatment of CRC and reduction of treatment resistance.

Fusobacterium nucleatum Increases Proliferation of Colorectal Cancer Cells and Tumor Development in Mice by Activating Toll-Like Receptor 4 Signaling to Nuclear Factor−κB, and Up-regulating Expression of MicroRNA-21

Nearly 20% of the global cancer burden can be linked to infectious agents. Fusobacterium nucleatum promotes tumor formation by epithelial cells via unclear mechanisms. We aimed to identify microRNAs (miRNAs) induced by F nucleatum and evaluate their ability to promote colorectal carcinogenesis in mice. Colorectal cancer (CRC) cell lines were incubated with F nucleatum or control reagents and analyzed in proliferation and would healing assays. HCT116, HT29, LoVo, and SW480 CRC cell lines were incubated with F nucleatum or phosphate-buffered saline (PBS [control]) and analyzed for miRNA expression patterns and in chromatin immunoprecipitation assays. Cells were incubated with miRNAs mimics, control sequences, or small interfering RNAs; expression of reporter constructs was measured in luciferase assays. CRC cells were incubated with F nucleatum or PBS and injected into BALB/C nude mice; growth of xenograft tumors was measured. C57BL adenomatous polyposis colimin/+, C57BL miR21a-/-, and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane and dextran sodium sulfate to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by enzyme-linked immunosorbent assay. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. Fusobacterium nucleatum DNA in 90 tumor and matched nontumor tissues from patients in China were explored for the expression correlation analysis; levels in 125 tumor tissues from patients in Japan were compared with their survival times. Fusobacterium nucleatum increased proliferation and invasive activities of CRC cell lines compared with control cells. CRC cell lines infected with F nucleatum formed larger tumors, more rapidly, in nude mice than uninfected cells. Adenomatous polyposis colimin/+ mice gavaged with F nucleatum developed significantly more colorectal tumors than mice given PBS and had shorter survival times. We found several inflammatory factors to be significantly increased in serum from mice given F nucleatum (interleukin 17F, interleukin 21, and interleukin 22, and MIP3A). We found 50 miRNAs to be significantly up-regulated and 52 miRNAs to be significantly down-regulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (>4-fold). Inhibitors of miR21 prevented F nucleatum from inducing cell proliferation and invasion in culture. miR21a-/- mice had a later appearance of fecal blood and diarrhea after administration of azoxymethane and dextran sodium sulfate, and had longer survival times compared with control mice. The colorectum of miR21a-/- mice had fewer tumors, of smaller size, and the miR21a-/- mice survived longer than control mice. We found RASA1, which encodes an RAS GTPase, to be one of the target genes consistently down-regulated in cells that overexpressed miR21 and up-regulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB. Levels of F nucleatum DNA and miR21 were increased in tumor tissues (and even more so in advanced tumor tissues) compared withnon-tumor colon tissues from patients. Patients whose tumors had high amounts of F nucleatum DNA and miR21 hadshorter survival times than patients whose tumors had lower amounts. We found infection of CRC cells with F nucleatum to increase their proliferation, invasive activity, and ability to form xenograft tumors in mice. Fusobacterium nucleatum activates Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB and increased expression of miR21; this miRNA reduces levels of the RAS GTPase RASA1. Patients with both high amount of tissue F nucleatum DNA and miR21 demonstrated a higher risk for poor outcomes.

Effect of API-1 and FR180204 on cell proliferation and apoptosis in human DLD-1 and LoVo colorectal cancer cells.

The activation of the phosphatidylinositol-3 kinase/v-akt murine thymoma viral oncogene homolog (Akt) and mitogen activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathways are implicated in the majority of cancers. Selective inhibition of Akt and ERK represents a potential approach for cancer therapy. Therefore, the present study aimed to investigate the apoptotic and anti-proliferative effects of the novel and selective Akt inhibitor 4-amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide (API-1) and selective ERK1/2 inhibitor FR180204 (FR) alone and in combination on colorectal cancer (CRC) cells (DLD-1 and LoVo). In addition, the effects of API-1 and FR on Akt and ERK signaling pathways were also investigated. The effects of the agents on DLD-1 and LoVo cells were evaluated in terms of cell viability, cytotoxicity, DNA synthesis rate, DNA fragmentation and caspase-3 activity levels. In addition, quantitative reverse transcription-polymerase chain reaction and western blot analysis were performed to examine relevant mRNA and protein levels. The present study observed that the combination of FR with API-1 resulted in significant apoptosis and cytotoxicity compared with any single agent alone in a time-dependent manner in these cells. Also, treatment with FR and API-1 in combination decreased the expression levels of B-cell lymphoma-2 (BCL2), Bcl-2-like1, cyclin D1 and cMYC, and increased the expression levels of BCL2-associated X protein and BCL2 antagonist/killer via phosphorylated Akt and phosphorylated ERK1/2 downregulation. The combination of Akt and ERK1/2 inhibitors resulted in enhanced apoptotic and anti-proliferative effects against CRC cells. The present study hypothesizes that the combination of FR and API-1 in CRC cells may contribute toward potential anti-carcinogenic effects. Additional analyses using other cancer cell lines and animal models are required to confirm these findings in vitro and in vivo.

Abstract 1689: Resveratrol inhibits epithelial-to-mesenchymal transition and metastasis in colorectal cancer through regulating Snail/E-cadherin expression by TGFβ1/Smads signaling pathway

Abstract Resveratrol has been an ideal alternative drug in the therapy of different cancers including colorectal cancer (CRC). Since the underlying mechanisms of resveratrol on the invasion and metastasis of CRC have not been fully elucidated, and epithelial-to-mesenchymal transition (EMT) is a key process associated with the progression of CRC, here we aimed to investigate the potential mechanism of resveratrol. We investigated the anticancer effect of resveratrol against LoVo cells in vitro and in vivo. In vivo, the impact of resveratrol on invasion and metastasis was investigated by mice tail vein injection model and mice orthotopic transplantation tumor model. In vivo imaging was applied to observe the lungs metastases, and hemaoxylin-eosin (HE) staining was used to evaluate metastatic lesions from lung metastases or hepatic metastases. In vitro, impact of resveratrol on the migration and invasion of LoVo cells was evaluated by transwell assay. Inhibition effect of resveratrol on TGF-β-induced EMT was examined by morphological observation. Epithelial phenotype marker E-cadherin and mesenchymal phenotype marker Vimentin were detected by western blot and immunofluorescence. The promoter activity of E-cadherin was measured using a dual-luciferase assay kit. The mRNA expression of Snail and E-cadherin was measured by RT-PCR. We demonstrated that, resveratrol inhibited the lung metastases of CRC LoVo cells in vivo. In addition, resveratrol reduced the rate of lung metastases and hepatic metastases in mice orthotopic transplantation. In vitro, TGF-β1-induced EMT promoted the invasion and metastasis of CRC, reduced the expression of E-cadherin and elevated the expression of Vimentin, and activated the TGF-β1/Smads signaling pathway. But resveratrol could inhibit the invasive and migratory ability of LoVo cells in a concentration-dependent manner, increased the expression of E-cadherin, repressed the expression of Vimentin, as well as the inhibition of TGF-β1/Smads signaling pathway. Meanwhile, resveratrol reduced the level of EMT-inducing transcription factors Snail and the transcription of E-cadherin during the initiation of TGF-β1-induced EMT. Our new findings provided evidence that, resveratrol could inhibit EMT in CRC through TGF-β1/Smads signaling pathway mediated Snail/E-cadherin expression, and this might the potential mechanism of resveratrol on the inhibition of invasion and metastases in CRC. Citation Format: Qing Ji, Zhifen Han, Lihong Zhou, Hua Sui, Xuan Liu, Jianlin Ren, Fenggang Hou, Ronghua Zhao, Qi Li. Resveratrol inhibits epithelial-to-mesenchymal transition and metastasis in colorectal cancer through regulating Snail/E-cadherin expression by TGFβ1/Smads signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1689.

Effect of peroxisome proliferator-activated receptor-γ coactivator-1α on metastasis and anoikis resistance in colorectal cancer

To investigate the role of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in the metastasis of colorectal cancer and underlying mechanisms. A colorectal cancer LoVo cell line transfected with a lentivirus vector stably expressinga shRNA targeting PGC-1αwas established. Cell proliferation was detected by CCK8 array. Migration and invasion were determined by Transwell assay. The expressions of epithelial-mesenchymal transition (EMT) markers were examined by western blot analysis. Down-regulation of PGC-1α significantly inhibited the migration (74.4±4.5 versus 31.4±3.8,P<0.05), invasion (55.0±7.7 versus 17.6±5.0,P<0.05) and anoikis resistance (32.3±4.3)% versus (54.3±4.8)%,P<0.05, of LoVo cells. However, knockdown of PGC-1α had little effect on cell proliferation. Moreover, knockdown of PGC-1α induced EMT of LoVo cells by up-regulating E-cadherin and down-regulating vimentin. Alternatively, the expression of PGC-1α was induced by cell detachment. PGC-1αexpression was also higher in the colorectal cancer tissues than that in para-cancerous tissues, and its expression in the invading front area was higher than that in the tumor center area. PGC-1α plays an important role in the metastasis of colorectal cancer, which may promote the invasion and anoikis resistance of colorectal cancer cells through EMT induction.