Colorectal Tumor Cells Research Articles

Abstract 2553: Gemcitabine fails to radiosensitize normal intestinal epithelial CCD841 cells at concentrations that promote excellent radiosensitization in colorectal tumor cell lines

Abstract Gemcitabine (2′, 2′-difluroro-2′-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and commonly used in chemoradiotherapy regimens to enhance radiation induced cell killing (radiosensitization), but its use is limited by normal tissue toxicity. We have studied the mechanism by which dFdCyd promotes radiosensitization in an effort to optimize its use in such regimens. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP, which strongly correlates with radiosensitization. DNA replication in the presence of imbalanced dNTPs produces mismatches in DNA, and their persistence underlies the mechanism of radiosensitization by dFdCyd. Importantly, the presence of dFdCyd induced DNA mismatches is necessary for radiosensitization to occur but not for cytotoxicity, and maximal radiosensitization can be elicited at non-toxic concentrations of drug. However, when dFdCyd is currently used as part of chemoradiotherapy regimens, it is administered at the standard and very toxic dose used in monotherapy regimens. The distinction between radiosensitizing and cytotoxic mechanisms of dFdCyd revealed by our studies provides a rationale for optimizing chemoradiotherapy while decreasing normal tissue toxicity. To achieve this, we have evaluated cytotoxicity of dFdCyd + ionizing radiation (IR) in a normal intestinal epithelial cell line, CCD841, as a first approach to identify potential differences in the way tumor versus non-tumor cells respond to dFdCyd ± IR. CCD841 cells were exposed to concentrations of dFdCyd that produce excellent radiosensitization in a variety of colorectal tumor cell lines (10 nM, 30 nM, 100 nM) and produced a range of survival in CCD841 cells (78 ± 17.1%, 46.3 ± 5.5%, 26 ± 3.61%, respectively). Analysis of dNTPs during dFdCyd incubation revealed a profound depletion in dATP. However, all dFdCyd concentrations failed to radiosensitize CCD841 cells (Radiation Enhancement Ratios: 10 nM, 0.92 ± 0.03; 30 nM, 0.82 ± 0.04; 100 nM, 0.67 ± 0.10). Marked differences in cell cycle effects were also noted in CCD841 and tumor cells treated with dFdCyd. While tumor cells accumulate in early S-phase but slowly replicate DNA in the presence of dFdCyd, CCD841 cells arrested right where they were in the cycle at the time of dFdCyd exposure and displayed a striking decrease in DNA synthesis. We propose that this prevents CCD841 cells from undergoing the necessary DNA replication that permits cells to produce the essential DNA mismatches for radiosensitization to occur. Our studies support the use of mechanistically-derived doses and administration schedules for dFdCyd when given with IR to ameliorate normal tissue toxicity while preserving the effectiveness of the chemoradiotherapy regimen. Citation Format: Sheryl A. Flanagan, Jeffrey Ackroyd, Donna S. Shewach. Gemcitabine fails to radiosensitize normal intestinal epithelial CCD841 cells at concentrations that promote excellent radiosensitization in colorectal tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2553. doi:10.1158/1538-7445.AM2015-2553

Abstract 5101: The control of migration and invasion processes in colorectal adenocarcinoma is modulated by prion protein and its ligand STI1/HOP

Abstract The colorectal cancer is one of the most common types of cancer in the world. Previous results from our group and others revealed the Cellular Prion Protein (PrPC) as a possible therapeutic target in glioblastomas and ovarian tumors. Its activity depends on the interaction with the secreted form of the Stress Inducible Protein 1 (STI1), also known as HSP70/90 Heat Shock Organizing Protein (HOP). In the present work, we aimed to understand the mechanism by which PrPC-STI1/HOP complex can influence the aggressiveness of colorectal tumor cell lines. Cell migration and invasion assays were performed using recombinant STI1/HOP as a chemical attractant in boyden chambers coated with matrigel. The results showed that when stimulated with recombinant STI1/HOP, cells exhibited higher migration and invasion potentials than non-stimulated cells or those that were stimulated with STI1/HOP deletion mutant for the PrPC binding domain (STI1Δ230-245). Moreover, migration and invasion mediated by STI1/HOP were blocked by a STI1/HOP peptide (230-245), which mimics STI1/HOP binding site to PrPC, as well as by a neutralizing anti-PrPC antibody. To confirm the importance of PrPC-STI1/HOP complex in tumor progression and metastasis we generated a stable PrPC-knockdown colorectal adenocarcinoma cell line (WIDr). These cells showed a decreased invasion upon recombinant STI1/HOP stimulus, suggesting an important role of PrPC-STI1/HOP complex in the invasion process. In addition, as ERK signaling pathway has been related to invasion, we evaluated the ERK1/2 phosphorylation pattern in WIDr wild-type cells and their PrPC knockdown (KD) counterpart. Cells where PrPC was impaired showed a reduction in ERK1/2 phosphorylation when compared to those expressing the former protein. Experiments are under way to evaluate if these lower ERK1/2 levels are associated with decreased migration in PrPC-KD cells. In conclusion, our findings pointed that the disruption of PrPC-STI1/HOP complex could be a therapeutic target in colorectal adenocarcinoma. Supported by São Paulo Research Foundation (FAPESP) Citation Format: Tonielli S. Lacerda, Marcos Vinicius S. Dias, Fernanda S. Giudice, Bianca Luise Teixeira, Vilma Regina Martins. The control of migration and invasion processes in colorectal adenocarcinoma is modulated by prion protein and its ligand STI1/HOP. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5101. doi:10.1158/1538-7445.AM2015-5101

Abstract 1070: Epigenetic silencing of galectin-12 in colorectal cancer

Abstract Aim/Background: Galectins constitute a family of ß-galactoside binding proteins some of them known to be involved in colorectal tumorigenesis. Our previous work suggests that sodium butyrate can induce galectin-12 expression in CRC cell lines. How this might be exerted is largely unexplored. In the present study we examined the potential epigenetic regulation of galectin-12 expression in colorectal cancer cell lines and primary tumors. Material and Methods: For DNA-methylation analysis 4 microsatellite unstable (MSI) and 5 microsatellite stable (MSS) human CRC cell lines were treated with 5-Aza-2`deoxycytidine (5-Aza-dC) for 72h. Changes in galectin-12 expression were evaluated by RT-PCR analysis. Galectin-12 promoter methylation was determined by bisulfite treatment and genomic DNA sequencing of single clones in the CRC cell line SW707 as well as in FFPE specimens from 10 primary colorectal tumors (MSI: n = 5; MSS: n = 5). Results: Treatment with 5-Aza-dC led to de novo expression of galectin-12 transcript in 4/9 (44%) of colorectal cancer cell lines. We identified a CpG island in the galectin-12 promoter region that was found to be hypomethylated upon 5-Aza-dC treatment in SW707 cells and to be associated with concomitant de novo galectin-12 mRNA expression. When primary tissues were examined we observed galectin-12 promoter hypomethylation in healthy colon mucosa but significant hypermethylation of this promoter region in corresponding tumor tissue. Conclusions: This study for the first time shows that galectin-12 promoter methylation represses galectin-12 expression in colorectal tumor cells independent of their MSI status. In CRC cell lines this effect can be reversed by butyrate but in human colon primary tumors might be refractory to these effects of butyrate. Re-expression of galectin-12 may be a potential therapeutic strategy to constrain colorectal tumor growth. Citation Format: Eva-Maria Katzenmaier, Johannes Gebert, Matthias Kloor, Jürgen Kopitz. Epigenetic silencing of galectin-12 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1070. doi:10.1158/1538-7445.AM2015-1070

Abstract 2294: Sophorolipid-mediated inhibition of colorectal tumor cell growth in vitro and in vivo

Abstract Background: Sophorolipids (SL), are amphiphilic biosurfactant molecules which consist of a sophorose molecule with 2 variable chain length (C10 - C22) They contain double bonds at the 3” 4” positions and fatty acids at the 1” positions. SL exist in either a lactonic (SLL; open ring) or acidic (SLA; closed ring) forms. SL is produced in crude mixtures by the yeast Candidia bombicola in economically viable amounts, with variable levels of anti-proliferative activity on tumour cell lines in vitro. As biosurfactants are well tolerated in the GI tract and currently used in a variety of food products, we tested the hypotheses that purified forms of either SLL or SLA have differential effects on colo-rectal tumour versus “normal” cells as well as in a well-established model of pre-cancerous lesions; viz the Apcmin+/− mouse. Methodology: The colo-rectal cancer cell lines HT29, HT115, HCT116, Caco-2 in addition to CCD-841 colonic epithelium and MRC5 lung fibroblasts were exposed to 10 - 100 μg/ml of either SLL or SLA (96% or 94%) pure respectively by HPLC/MS analysis) for 24 hours following serum starvation and an MTT assay performed. The mechanism of cell death in cell lines was assessed by acridine orange/ethidium bromide staining followed by microscopic examination. Five-week old APCmin+ mice or wild-type littermate mice were treated orally with 50mg/kg of either SLL or SLA, or vehicle-only control every other day for 14 weeks. Weights, water and food consumption were measured on a daily basis. On completion of the experiment, mice were euthanized, the digestive tract was excised, washed and fixed with 10% BFS. Polyp size, number and location were recorded and samples were blocked for paraffin embedding, sectioning and H&E/immunofluorescence staining and analysis. Results and Discussion: In vitro, SLL caused a decrease in colorectal cancer cell viability at >70μg/ml. However, it also caused an unfavourable effect on the colonic epithelium and lung fibroblast cell lines by reducing cell viability at a lower concentration (<10μg/ml). In-vivo, SLL treatment exacerbated the growth of polyps along the digestive tract of APCmin+ mice. SLL treatment resulted in an increase in polyp number and size compared to vehicle treated APCmin+ mice. SLL treated mice presented with reduced haematocrits (28.5% vs 34.5% p<0.03) and increased spleen weights (0.56g vs 0.70g p<0.01) compared to vehicle treated control APCmin+ mice. SLA induced a dose dependent decrease in cell viability of colo-rectal cancer cell lines (20μg/ml) without affecting the viability of the colonic epithelial and lung cell lines. SLA selectively induced cell death in the colo-rectal cancer cells by means of necrosis. However the effects of SLA in vivo are yet to be established. Contrary to current literature, purified SLA appears to have an advantage over the SLL form in vitro. To date, this is the first study investigating anti-cancer effects of sophorolipids in vivo. Citation Format: Breedge Callaghan, Sophie Roelants, Niki Baccile, Helen Lydon, Inge Van Bogaert, Ibrahim M. Banat, Roger Marchant, Christopher A. Mitchell. Sophorolipid-mediated inhibition of colorectal tumor cell growth in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2294. doi:10.1158/1538-7445.AM2015-2294

Design and radio-synthesis of somatostatin receptors targeted 68Ga-DOTA-Benereotide for non-invasive PET imaging

Here we reported the synthesis, radiolabeling and evaluation of a novel positron emission tomography (PET) tracer DOTA-Benerotide labeled with 68Ga with potential application in SSTR1,2,3,5 positive tumors for PET imaging. Radiolabeling of DOTA-Benerotide with 68Ga achieved its highest efficiency at pH 3.5 and 100 °C for 20 min. It also showed excellent in vitro stability in different buffers. Micro-PET imaging of human colorectal adenocarcinoma tumor cell (HT-29) bearing mice clearly delineated tumors at 4 h after tail vein injection of 68Ga-DOTA-Benerotide tracer. Further studies are needed to identify the mechanism of cell/organ uptake of this new SSTR PET imaging tracer.

Abstract A13: A positive feedback loop for transcriptional regulation of beta-catenin that favors sustained colorectal cancer cell invasion

Abstract Wnt/beta-catenin signaling is a branch of a functional network that is involved in a broad range of biological systems and deregulation of components involved in Wnt/beta-catenin signaling has been implicated in a wide spectrum of cancers. We have shown earlier that beta-catenin, is up regulated in the invasion front compared to the inner parts of the tumor in a murine model of colorectal liver metastasis and clinical specimens. This activity is due to the fact that beta-catenin acts as co-transcription factor of its own promoter contributing to the dynamic changes of beta-catenin levels upon the confrontation of tumor cells with the host microenvironment. To examine the putative role of the host cells for transcriptional activation of beta-catenin and its promoter, co-culture experiments and application of recombinant proteins or small molecule inhibitors were performed. To find out which cells play a role in activation of the beta-catenin promoter both hepatocytes and hepatic stellate cells were co-cultured with a colorectal tumor cell line LS174T that was stably transfected with a SEAP reporter gene driven by a fragment of the beta-catenin promoter. We could demonstrate that co-culture of LS174T cells with the hepatocytes (AML12) results in an increase of the SEAP reporter indicating an activation of the beta-catenin promoter. In contrast, co-culturing of the LS174T with hepatic stellate cells (LX-2) caused a decrease of reporter gene expression. Treatment of LS174T beta-catenin cells with FCS showed a 5-fold up-regulation of the beta-catenin promoter as compared to serum-free conditions concluding that fetal calf serum contains factors that activate the beta-catenin promoter. Application of different cytokines and growth factors that are involved in tumor formation and metastasis did not lead to any alteration of the reporter gene expression. Upon application of several small molecule inhibitors, Wortmannin and LY492004 that inhibits the PI3 kinase showed a pronounced up-regulation of the beta-catenin promoter in a concentration dependent manner. PI3K is known to activate Akt that in turn stabilizes beta-catenin and translocates to the nucleus. Inhibition of the above described PI3K-akt- beta-catenin pathway by application of PI3K inhibitors leads to the activation of the beta-catenin-promoter indicating the presence of a feed-back loop that leads to increased beta-catenin transcription in response to decreased beta-catenin protein levels that in turn results in sustained up regulation of the invasiveness of CRC cells. These results warrant for the development of small molecule inhibitors targeting beta-catenin rather than the PI3K, Wnt/beta-catenin signaling pathways. Citation Format: Franziska Ehrmann, Linda Frank, Peter Schirmacher, Karsten Brand, Obul R. Bandapalli. A positive feedback loop for transcriptional regulation of beta-catenin that favors sustained colorectal cancer cell invasion. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A13.

Targeting MYC Translation in Colorectal Cancer

There is a great interest in finding ways to inhibit the expression or activity of the "undruggable" MYC, a master regulator of transcription and one of the most deadly oncoproteins in human cancer. In this issue of Cancer Discovery, Wiegering and colleagues find a way of inhibiting translation of MYC in colorectal cancer cells by directly targeting the translation initiation factor eIF4A, resulting in inhibition of MYC-dependent proliferation of colorectal tumor cells in vitro and in vivo.

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation.

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-xL leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling

ObjectiveColorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour...

Chemopreventive effect of α-viniferin in azoxymethane-induced mouse colorectal tumor and Caco-2 cells

Chemopreventive effect of α-viniferin in azoxymethane-induced mouse colorectal tumor and Caco-2 cells

PWE-256 Enhanced radiosensitivity of colorectal cancer cells by targeting BCL-3

Introduction Colorectal cancer remains the second most common cause of cancer related death in the UK. The successful management of rectal cancer has improved significantly with changes to surgical technique and the addition of pre-operative chemo-radiotherapy. A significant proportion of patients fail to respond to neo-adjuvant therapy. BCL-3 expression (an NF-KB homodimeric binding protein) is associated with poor prognosis in colorectal cancer patients, 1 and has been shown to drive expression of Cancer Stem Cell (CSC) markers in colorectal cancer. 2,3,4 Importantly evidence shows that CSCs contribute to tumour development and therapeutic resistance. 5 It was hypothesised that expression of BCL-3 could be driving cell survival after radiation, and therefore represent a novel biomarker for therapeutic response. Method Established colorectal cells lines LS174T and HCA7 were cultured in flasks/plates. Suppression of BCL-3 expression was achieved by transfection with short interfering RNA (siRNA), cells were irradiated with 1–2.5Gy γ-radiation (Cs-137 source). Cell yield and apoptosis were determined. Cell viability was assessed using a crystal violet assay, protein analysis by Western Blotting. Results BCL-3 suppression results in reduced cell yield and increased apoptosis in LS174T and HCA7 cells. Suppression of BCL-3 expression combined with γ-irradiation results in a significant decrease in cell viability of both LS174T (40% reduction in cell viability at 1Gy, p = 0.028; 54% reduction in cell viability at 2.5Gy, p = 0.005) and HCA7 cells (33% reduction in cell viability at 1Gy, p = 0.017; 50% reduction in cell viability at 2.5Gy, p = 0.031). Conclusion We have shown for the first time that suppressing BCL-3 expression results in radio-sensitisation in an in-vitro colorectal cancer model, the mechanisms of which are currently under investigation. Work is underway to validate this research with in-vivo tissue samples; the aim is to determine whether BCL-3 expression levels correlate with therapeutic response to radiotherapy in rectal cancer patients and whether BCL-3 levels could be used to stratify patient treatment. Disclosure of interest None Declared. References Puvvada SD et al . Oncology. 2010;78:181–188 Al-Kharusi, MR et al . Carcinogenesis. 2013;34:1150–1157 Smartt, HJ et al . Gut. 2012;61:1306–1314 Urban BC et al . BCL-3 promotes colorectal tumour cell survival through activation of the AKT signalling pathway. In revision. 2015 Pattabiraman, DR et al . Nature reviews. Drug discovery. 2014;13:497–512

Overexpression of caudal-related homeobox transcription factor 2 inhibits the growth of transplanted colorectal tumors in nude mice.

Caudal-related homeobox transcription factor 2 (CDX2) is a transcription factor, which is specifically expressed in the adult intestine. It is essential for the development and homeostasis of the intestinal epithelium and its functions as a tumor suppressor have been demonstrated in the adult colon. The present study aimed to examine the inhibitory effects of the overexpression of CDX2 on subcutaneously-transplanted tumors, derived from LoVo colon cancer cells, in nude mice, and to provide experimental evidence for the biotherapy of colon cancer. A pEGFP-C1-CDX2 eukaryotic expression vector was transfected into the LoVo cells via lipofection, and LoVo cells stably-expressing CDX2 (pEGFP-C1-CDX2 cells) were obtained using G418 selection. A nude mouse subcutaneously-transplanted tumor model was established by inoculating the nude mice with the pEGFP-C1-CDX2 cells, and the effects of overexpression of CDX2 on transplanted tumor growth in the LoVo cells were observed. Western blotting results demonstrated that the protein expression of CDX2 in the LoVo cells was higher in the pEGFP-C1-CDX2 cell group, compared with that in the pEGFP-C1 cell group and the untreated cell group. At 20 days post-inoculation with either pEGFP-C1-CDX2 or pEGFP-C1, the transplanted tumor masses were significantly lower in the pEGFP-C1-CDX2 group, compared with those in the pEGFP-C1 and untreated groups. Immunohistochemistry revealed that the expression levels of CDX2 and matrix metalloproteinase-2 (MMP-2) were detected in each group, and the protein expression of CDX2 was increased in the tumor tissues from the nude mice in the pEGFP-C1-CDX2 group. However the expression of MMP-2 was downregulated in the tumor tissues of the nude mice in the pEGFP-C1-CDX2 group. Taken together, these data suggested that pEGFP-C1-CDX2 cells exhibited suppressed tumor growth in vivo. Overexpression of CDX2 was observed in transplanted tumors in the pEGFP-C1-CDX2 group, and the gene expression of MMP-2 was reduced. These results indicate that CDX2 inhibited the growth of colorectal tumor cells, possibly by downregulating the gene expression.

Increased HMGB1 and cleaved caspase-3 stimulate the proliferation of tumor cells and are correlated with the poor prognosis in colorectal cancer.

BackgroundDying tumor cells after irradiation could promote the proliferation of living tumor cells might cause tumor relapse and treatment failure. Our previous study showed that activated caspase-3 after irradiation probably participates in tumor repopulation. In this study, we investigated whether high mobility group box 1(HMGB1) is also involved in tumor repopulation.MethodsColorectal tumor cells were irradiated. The cleaved caspase-3 (CC3) in irradiated tumor cells and HMGB1 in the supernatant of irradiated tumor cells were detected by Western blot. A large number of irradiated colorectal tumor cells (feeder cells) were then co-cultured with a small number of luciferase-labeled living colorectal tumor cells (reporter cells) and proliferation of reporter cells was measured by bioluminescence imaging. The CC3 and HMGB1 protein expression in colorectal tumor and peritumoral tissues were detected by immunohistochemistry and their correlation with prognosis were analyzed.ResultsThe irradiated colorectal tumor cells underwent apoptosis and necrosis and produced CC3 in tumor cells and HMGB1 in the supernatant of cultured cells. The increased expression of secretory HMGB1 correlated with CC3 level and proliferating cell nuclear antigen (PCNA) after irradiation in vitro. The irradiated dying cells remarkably stimulated living tumor cell proliferation. Interestedly, immunohistochemistry staining showed that positive HMGB1, CC3, and Ki67 expression were significantly higher in colorectal tumor tissues than in peritumoral tissues (p <0.01). The Kaplan-Meier survival analysis revealed that high HMGB1, CC3, and Ki67 levels were significantly associated with poor prognosis (p <0.05, p <0.01). Multivariate analysis using Cox proportional hazards model showed that TNM staging and HMGB1 were independent prognostic factors in patients with colorectal cancer (CRC) (p <0.01, p <0.001).ConclusionBoth apoptotic and necrotic cells could stimulate proliferation of living tumor cells, and the increased expression of CC3 and HMGB1 in tumor cells could be new markers for poor prognosis in colorectal cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0166-1) contains supplementary material, which is available to authorized users.

5-Fluorouracil sensitizes colorectal tumor cells towards double stranded DNA breaks by interfering with homologous recombination repair.

Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

Transcriptional mimicry by tumor-associated stroma.

Recent molecular classification of colorectal cancer (CRC) has identified a poor-prognosis transcriptional subtype associated with mesenchymal traits. New studies used CRC transcriptomic data to show that tumor-associated stroma mimics the gene signature of epithelial-to-mesenchymal transition (EMT) and found no evidence for EMT of colorectal tumor cells.

MicroRNA-124 inhibits cancer cell growth through PTB1/PKM1/PKM2 feedback cascade in colorectal cancer

Altered levels and functions of microRNAs (miRs) have been associated with carcinogenesis. In this study, we investigated the role of miR-124 in colorectal adenoma (CRA) and cancer (CRC). The expression levels of miR-124 were decreased in CRA (81.8%) and CRC (57.6%) in 55 clinical samples. The ectopic expression of miR-124 induced apoptosis and autophagy in colon cancer cells. Also, miR-124 targeted polypyrimidine tract-binding protein 1 (PTB1), which is a splicer of pyruvate kinase muscles 1 and 2 (PKM1 and PKM2) and induced the switching of PKM isoform expression from PKM2 to PKM1. Also, siR-PTB1 induced drastic apoptosis in colon cancer cells. Furthermore, we found that the ectopic expression of miR-124 enhanced oxidative stress and the miR-124/PTB1/PKM1/PKM2 axis constituted a feedback cascade. Finally, we showed that intratumor injection of miR-124 and siR-PTB1 induced a tumor-suppressive effect in xenografted mice. The axis was established by both in vitro and in vivo experiments to function in human colorectal cancer cells. These findings suggest that miR-124 acts as a tumor-suppressor and a modulator of energy metabolism through a PTB1/PKM1/PKM2 feedback cascade in human colorectal tumor cells.

Pilot trial of combined BRAF and EGFR inhibition in BRAF-mutant metastatic colorectal cancer patients.

BRAF-mutant metastatic colorectal cancer (mCRC) forms an aggressive subset of colorectal cancer with minimal response to selective RAF inhibitors. Preclinical data show that reactivation of EGFR signaling occurs in colorectal tumor cells treated with RAF inhibitors and that the addition of an EGFR inhibitor enhances antitumor activity. These data suggest that combined therapy with RAF and EGFR inhibitors could be an effective strategy for treating BRAF V600E mCRC. We undertook a pilot trial to assess the response rate and safety of the BRAF inhibitor vemurafenib combined with anti-EGFR antibody panitumumab in patients with BRAF-mutant mCRC. Patients received standard approved doses of panitumumab and vemurafenib. Fifteen patients were treated. Performance status was Eastern Cooperative Oncology Group (ECOG) 0 in 4 patients (27%) and ECOG 1 in 11 patients (73%). All patients had progressed through at least one standard treatment regimen, and 8 (53%) had received previous fluoropyrimidine, oxaliplatin, and irinotecan chemotherapy. Treatment was well tolerated, with less cutaneous toxicity than would be expected with either agent, and no cases of keratoacanthomas/squamous cell carcinomas. Tumor regressions were seen in 10 of 12 evaluable patients with partial responses in 2 patients (100% and 64% regression lasting 40 and 24 weeks, respectively), and stable disease lasting over 6 months in 2 patients. Combined RAF and EGFR inhibition is well tolerated, with less cutaneous toxicity than would be expected with either agent, and results in modest clinical activity in this highly aggressive and chemoresistant subset of CRC.

Effects of probiotic Lactobacillus acidophilus and Lactobacillus casei on colorectal tumor cells activity (CaCo-2).

The probiotic microorganisms are live normal flora that provide nutritional benefits. When probiotic administered in adequate amounts, they also confer a health benefit on the host. Different mechanisms of probiotic effects include the following: stimulating the immune system, modifying the composition of normal intestinal flora and preventing the carcinogenic activity of fecal enzymes. In this study, direct effects of probiotic lactobacilli on tumor cells were investigated. Supernatants and bacterial extracts of two standard Lactobacillus species (L. acidophilus and L. casei) were prepared and CaCo-2 cells were treated with them. Probiotic effects on cell proliferation, necrosis, apoptosis, migration and invasion were assessed. The supernatants of Lactobacilli decreased cell proliferation and increased cell apoptosis, however, no significant effect on cell necrosis was reported. In contrast, Lactobacilli extract, reduced cell proliferation and increased cell apoptosis. Lactobacilli extract also led to cell necrosis. Furthermore, both supernatants and cell extracts of the probiotic agents resulted in decreased cells' migration and invasion. In this study, it was shown that Lactobacilli probiotics useful effects are not confined to the enhancement of the immune system; however, they effectively suppress the malignant phenotypes of colorectal cancer cells.

The Cytotoxicity of Kahweol in HT-29 Human Colorectal Cancer Cells Is Mediated by Apoptosis and Suppression of Heat Shock Protein 70 Expression.

Although coffee is known to have antioxidant, anti-inflammatory, and antitumor properties, there have been few reports about the effect and mechanism of coffee compounds in colorectal cancer. Heat shock proteins (HSPs) are molecular chaperones that prevent cell death. Their expression is significantly elevated in many tumors and is accompanied by increased cell proliferation, metastasis and poor response to chemotherapy. In this study, we investigated the cytotoxicity of four bioactive compounds in coffee, namely, caffeine, caffeic acid, chlorogenic acid, and kahweol, in HT-29 human colon adenocarcinoma cells. Only kahweol showed significant cytotoxicity. Specifically, kahweol increased the expression of caspase-3, a pro-apoptotic factor, and decreased the expression of anti-apoptotic factors, such as Bcl-2 and phosphorylated Akt. In addition, kahweol significantly attenuated the expression of HSP70. Inhibition of HSP70 activity with triptolide increased kahweol-induced cytotoxicity. In contrast, overexpression of HSP70 significantly reduced kahweol-induced cell death. Taken together, these results demonstrate that kahweol inhibits colorectal tumor cell growth by promoting apoptosis and suppressing HSP70 expression.

CEA-targeted nanoparticles allow specific in vivo fluorescent imaging of colorectal cancer models.

Fluorescent imaging of colorectal tumor cells would improve tumor localization and allow intra-operative staging, facilitating stratification of surgical resections thereby improving patient outcomes. We aimed to develop and test fluorescent nanoparticles capable of allowing this in vivo. Dye-doped silica nanoparticles were synthesized. Anti-CEA (carcinoembryonic antigen) or control IgGs were conjugated to nanoparticles using various chemical strategies. Binding of CEA-targeted or control nanoparticles to colorectal cancer cells was quantified in vitro, and in vivo after systemic-delivery to murine xenografts. CEA-targeted, polyamidoamine dendrimer-conjugated, nanoparticles, but not control nanoparticles, allowed strong tumor-specific imaging. We are the first to demonstrate live, specific, in vivo imaging of colorectal cancer cells using antibody-targeted fluorescent nanoparticles. These nanoparticles have potential to allow intra-operative fluorescent visualization of tumor cells.