Inhibits Colon Cancer Cell Research Articles

Conditioned media from macrophages of M1, but not M2 phenotype, inhibit the proliferation of the colon cancer cell lines HT-29 and CACO-2

Solid tumors are infiltrated by stroma cells including macrophages and these cells can affect tumor growth, metastasis and angiogenesis. We have investigated the effects of conditioned media (CM) from different macrophages on the proliferation of the colon cancer cell lines HT-29 and CACO-2. CM from THP-1 macrophages and monocyte-derived human macrophages of the M1 phenotype, but not the M2 phenotype, inhibited proliferation of the tumor cells in a dose-dependent manner. Lipopolysaccaharide and interferon γ was used for differentiation of macrophages towards the M1 phenotype and CM were generated both during differentiation (M1DIFF) and after differentiation (M1). M1 and M1DIFF CM as well as THP-1 macrophage CM resulted in cell cycle arrest in HT-29 cells with a decrease of cells in S phase and an increase in G2/M phase. Treatment of HT-29 cells with M1DIFF, but not M1 or THP-1 macrophage CM, resulted in apoptosis of about 20% of the tumor cells and this was accompanied by lack of recovery of cell growth after removal of CM and subsequent culture in fresh media. A protein array was used to identify cytokines released from M1 and M2 macrophages. Among the cytokines released by M1 macrophages, tumor necrosis factor α and CXCL9 were tested by direct addition to HT-29 cells, but neither affected proliferation. Our results indicate that M1 macrophages inhibit colon cancer cell growth and have the potential of contributing to reducing tumor growth in vivo.

PATZ1 interacts with p53 and regulates expression of p53-target genes enhancing apoptosis or cell survival based on the cellular context

PATZ1 is a transcriptional factor functioning either as an activator or a repressor of gene transcription depending upon the cellular context. It appears to have a dual oncogenic/anti-oncogenic activity. Indeed, it is overexpressed in colon carcinomas, and its silencing inhibits colon cancer cell proliferation or increases sensitivity to apoptotic stimuli of glioma cells, suggesting an oncogenic role. Conversely, the development of B-cell lymphomas, sarcomas, hepatocellular carcinomas and lung adenomas in Patz1-knockout (ko) mice supports its tumour suppressor function. PATZ1 role in mouse lymphomagenesis is mainly because of the involvement of PATZ1 in BCL6-negative autoregulation. However, this does not exclude that PATZ1 may be involved in tumorigenesis by other mechanisms. Here, we report that PATZ1 interacts with the tumour suppressor p53 and binds p53-dependent gene promoters, including those of BAX, CDKN1A and MDM2. Knockdown of PATZ1 in HEK293 cells reduces promoter activity of these genes and inhibits their expression, suggesting a role of PATZ in enhancing p53 transcriptional activity. Consistently, Patz1-ko mouse embryonic fibroblasts (MEFs) show decreased expression of Bax, Cdkn1a and Mdm2 compared with wild-type (wt) MEFs. Moreover, Patz1-ko MEFs show a decreased percentage of apoptotic cells, either spontaneous or induced by treatment with 5-fluorouracil (5FU), compared with wt controls, suggesting a pro-apoptotic role for PATZ1 in these cells. However, PATZ1 binds p53-target genes also independently from p53, exerting, in the absence of p53, an opposite function on their expression. Indeed, knockdown of PATZ1 in p53-null osteosarcoma cells upregulates BAX expression and decreases survival of 5FU-treated cells, then suggesting an anti-apoptotic role of PATZ1 in p53-null cancer cells. Therefore, these data support a PATZ1 tumour-suppressive function based on its ability to enhance p53-dependent transcription and apoptosis. Conversely, its opposite and anti-apoptotic role in p53-null cancer cells provides the perspective of PATZ1 silencing as a possible adjuvant in the treatment of p53-null cancer.

Abstract C245: Discovery and characterization of a new Wnt/β-catenin pathway inhibitor targeting tankyrase.

Abstract Background: Wnt/β-catenin pathway controls many biological processes including cell proliferation and tissue development. In Wnt pathway, β-catenin is a key downstream factor which interacts with transcription factor T-cell factor (TCF) in the nucleus and induces expression of TCF responsive genes. β-catenin degradation is promoted by a cytoplasmic complex of APC, Axin and Gsk3β. In many cancers, constitutive activation of Wnt/β-catenin pathway is observed due to mutations of the related genes and overexpression of Wnt ligands and receptors. For example, truncating mutation of the tumor suppressor APC are the most prevalent in colon cancer. APC deficiency leads to accumulation of nuclear β-catenin and promotes the transcription of downstream target genes. Hence, Wnt/β-catenin pathway inhibitor can be an attractive therapeutic agent for cancer patients. Results: To screen for Wnt/β-catenin pathway inhibitor, a reporter-based screen using a colon cancer cell line harboring APC mutation was performed. We identified a Wnt/β-catenin pathway inhibitor K-756 with an IC50 of 110 nM, which is a distinct chemical structure from previously reported Wnt/β-catenin pathway inhibitors. The WNT/β-catenin downstream gene expression profile of K-756 treated cells was highly similar to that of β-catenin siRNA treated cells. K-756 upregulated Axin 1 and 2 protein levels, suggesting that K-756 inhibited Wnt/β-catenin pathway by stabilization of Axin 1 and 2, which led to degradation of active β-catenin. Axin protein levels are suppressed by tankyrase-mediated poly(ADP-ribosyl)ation. To examine whether K-756 inhibits tankyrase, tankyrase enzyme activity assay was performed. K-756 inhibited tankyrase 1 with an IC50 of 31 nM and tankyrase 2 with an IC50 of 36 nM, but did not inhibit other poly-ADP-ribose polymerase family members even at 10 μM, suggesting that K-756 is a selective TNKS1/2 inhibitor. Co-crystal structure of K-756 with TNKS1 was analyzed to reveal the binding site. K-756 bound to ADP-ribose binding pocket, which was different from that of XAV939, a known TNKS1/2 inhibitor. Using β-catenin siRNA, we have identified several colon cancer cell lines as Wnt pathway dependent cell lines. K-756 inhibited Wnt pathway signal and cell growth in the cell lines. Oral dosing of K-756 resulted in inhibition of the reporter activity and Wnt downstream genes in a colon cancer xenograft mouse model. Conclusions: We have screened Wnt/β-catenin pathway inhibitor by reporter assay and downstream gene profile analysis. The target of K-756 was revealed as TNKS1/2 and the binding state was identified. K-756 inhibited colon cancer cell growth via Wnt/β-catenin pathway inhibition. Moreover, K-756 exhibited Wnt/β-catenin signal inhibition in vivo. Taken together, K-756 represents a new TNKS inhibitor and has a potential to be an important lead for further development of antitumor agents targeting the Wnt/β-catenin pathway regulation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C245. Citation Format: Ryoko Okada, Yuichi Takahashi, Yasuo Watanabe, Hiroshi Ishida, Junichi Saito, Ryuichiro Nakai. Discovery and characterization of a new Wnt/β-catenin pathway inhibitor targeting tankyrase. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C245.

Highlights of This Issue

Despite the breakdown of the blood-brain barrier in brain metastases of breast cancer, traditional chemotherapy does not typically accumulate at sufficient concentrations to induce cytotoxicity. Here, Mittapalli and colleagues have designed a nanoconjugate of hyaluronic acid and paclitaxel that is small enough to passively get through the leaky tumor vasculature and to enter into the tumor interstitial space. Once there, the hyaluronic polymers promote CD44 receptor-mediated internalization of the nanoconjugate. Once the nanoconjugate is taken up into the cell, paclitaxel remains cytotoxic. This nanoconjugate system reduced lesion burden in the brain and improved overall survival in a preclinical model of brain metastases of breast cancer.Members of conventional drug classes targeting the MET pathway have faced multiple challenges, including specificity, selectivity, partial agonism, and HGF-dependent activity. Olwill and colleagues discovered and characterized for the first time a novel MET inhibitor (PRS-110) based on a new class of biopharmaceuticals called Anticalins. The monovalent binding nature of Anticalins makes them exquisitely suited for the antagonism of receptor tyrosine kinases, unlike bivalent mAbs, which can lead to receptor activation. The manuscript demonstrates the drug-like properties of this novel inhibitor, which supports its evaluation in a future clinical trial.Synthetic cannabinoids (CB), such as WIN 55,212-2, inhibit the growth of solid tumors and mechanisms can be CB receptor-dependent or -independent. Sreevalson and Safe showed that WIN 55,212-2 inhibited colon cancer cell growth and survival, and an important underlying pathway for these responses was due to downregulation of the specificity protein (Sp) transcription factors Sp1, Sp3, Sp4, and Sp-regulated genes. The mechanism was CB receptor-independent and due to induction of protein phosphatase 2A-mediated disruption of miR-27a-ZBTB10 (an Sp repressor). The design of future applications of WIN 55,212-2 for individual or combined chemotherapies can now be guided by these novel mechanistic insights.MicroRNA-141 (miR-141) was shown to play an important role in tumorigenesis. However, the exact role of miR-141 in pancreatic cancer has not yet been elucidated. In the present study, Zhao and colleagues discovered that downregulation of miR-141 in pancreatic cancer was correlated with poor overall survival and MAP4K4 was the direct target of miR-141. Restoration of miR-141 increased the chemosensitivity in vitro. Moreover, overexpression of miR-141 or knockdown of MAP4K4 inhibited the tumor growth of mice xenografts in vivo. All these findings suggest that miR-141 targets MAP4K4 and acts as a tumor suppressor in pancreatic cancer cells.

Abstract A51: Cancer stem cell marker ALDH1A3 is associated with increased resistance of Lovo-1 colon cancer cells to autophosphorylation inhibitor of FAK.

Abstract Introduction: Focal Adhesion Kinase is an important survival signal in cancer. Recently, we demonstrated that the autophosphorylation inhibitor of FAK, Y15 effectively inhibited colon cancer cell viability in vitro and blocked xenograft tumor growth in vivo. Among several colon cancer cell lines, Lovo-1 was more resistant to Y15 than other cell lines. To reveal signaling pathways responsible for increased resistance of Lovo-1 cells to autophosphorylation inhibitor of FAK, we performed microarray gene profile study in SW620 and Lovo-1 cells treated with Y15 inhibitor. Experimental Procedures: MTT assay and clonogenicity assays were used to detect the effect of Y15 on viability and colony formation. Western blotting was used to detect protein expression. IlluminaRef 8 whole genome gene expression microarray, bioinformatics and statistical analyses were performed to detect significantly affected genes by Y15 in two colon cancer cell lines: SW620 and Lovo-1 cells. PicoProbe ALDH activity assay kit (Biovision) was used to detect ALDH activity. ALDH1A3 siRNA was used to inhibit ALDH1A3 expression. The specific ALDH inhibitor, diethylaminobenzaldehyde was used to inhibit ALDH activity. Results: MTT, clonogenicity assays and Western blotting with Y397-FAK antibody demonstrated that Lovo-1 colon cancer cells were more resistant to Y15 treatment than SW620 colon cancer cells. The microarray analysis detected 12 up- and 4 significantly down-regulated genes that were affected >2-fold (p<0.05) in Lovo-1, but not in SW620 cells. Among unique genes up-regulated by Y15 in Lovo-1 cells, a stem cell marker- ALDH1A3 was detected to be up-regulated >2-fold. ALDH activity kit demonstrated high activity of ALDH in Lovo-1 colonospheres, and the spheres were resistant to Y15 treatment. Treatment with ALDH1A3 siRNAs sensitized Lovo-1 cells to Y15 inhibitor and decreased cell viability more effectively than each agent alone. The same result was obtained with ALDH inhibitor, DEAB in combination with Y15 inhibitor. Conclusion: The microarray study revealed several important genes up- or down-regulated in Lovo-1 cancer cells in response to Y15 inhibitor compared to SW620 cells. The Lovo-1 cells formed spheres with increased ALDH activity. A cancer stem cell marker, ALDH1A3 was associated with increased resistance of Lovo-1 cancer cells to Y15. In addition, down-regulation of ALDH1A3 and FAK effectively decreased viability of Lovo-1 colon cancer cells, resistant to FAK inhibitor that can be used as a novel combination therapy approach in resistant cell lines. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A51. Citation Format: Baotran Ho, Melissa Heffler, Jeffrey Conroy, Liu Song, Dan Wang, William G. Cance, Vita M. Golubovskaya. Cancer stem cell marker ALDH1A3 is associated with increased resistance of Lovo-1 colon cancer cells to autophosphorylation inhibitor of FAK. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A51.

(E)-2,4-Bis(p-hydroxyphenyl)-2-butenal inhibits tumor growth via suppression of NF-κB and induction of death receptor 6

The Maillard reaction products are known to be effective in chemoprevention. Here, we focused on the anti-cancer effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal on in vitro and in vivo colon cancer. We analysed the anti-cancer activity of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal on colon cancer cells by using cell cycle and apoptosis analysis. To elucidate it's mechanism, NF-κB DNA binding activity, docking model as well as pull-down assay. Further, a xenograft model of colon cancer was studied to test the in vivo effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal. (E)-2,4-Bis(p-hydroxyphenyl)-2-butenal inhibited colon cancer cells (SW620 and HCT116) growth followed by induction of apoptosis in a concentration-dependent manner via down-regulation of NF-κB activity. In docking model as well as pull-down assay, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal directly binds to three amino acid residues of IKKβ, thereby inhibited IKKβ activity in addition to induction of death receptor 6 (DR6) as well as their target apoptotic genes. Finally, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal suppressed anchorage-independent cancer cell growth, and tumor growth in xenograft model accompanied with apoptosis through inhibition of IKKβ/NF-κB activity, and overexpression of DR6. These results suggest that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal inhibits colon cancer cell growth through inhibition of IKKβ/NF-κB activity and induction of DR6 expression.

Inhibitory Effects of Different Forms of Tocopherols, Tocopherol Phosphates, and Tocopherol Quinones on Growth of Colon Cancer Cells

Tocopherols are the major source of dietary vitamin E. In this study, the growth inhibitory effects of different forms of tocopherols (T), tocopheryl phosphates (TP), and tocopherol quinones (TQ) on human colon cancer HCT116 and HT29 cells were investigated. δ-T was more active than γ-T in inhibiting colon cancer cell growth, decreasing cancer cell colony formation, and inducing apoptosis; however, α-T was rather ineffective. Similarly, the rate of cellular uptake also followed the ranking order δ-T > γ-T ≫ α-T. TP and TQ generally had higher inhibitory activities than their parent compounds. Interestingly, the γ forms of TP and TQ were more active than the δ forms in inhibiting cancer cell growth, whereas the α forms were the least effective. The potencies of γ-TQ and δ-TQ (showing IC50 values of ∼0.8 and ∼2 μM on HCT116 cells after a 72 h incubation, respectively) were greater than 100-fold and greater than 20-fold higher, respectively, than those of their parent tocopherols. Induction of cancer cell apoptosis by δ-T, γ-TP, and γ-TQ was characterized by the cleavage of caspase 3 and PARP1 and DNA fragmentation. These studies demonstrated the higher growth inhibitory activity of δ-T than γ-T, the even higher activities of the γ forms of TP and TQ, and the ineffectiveness of the α forms of tocopherol and their metabolites against colon cancer cells.

Clinical significance of microRNA-148a in patients with early relapse of stage II stage and III colorectal cancer after curative resection

Development of robust prognostic/predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of microRNAs might be a simple and reliable biomarker to detect postoperative early relapse in patients with CRC after radical resection. We used microRNA arrays and identified that microRNA-148a (miRNA-148a) had substantially different expression levels in early and nonearly relapsed stage II and III CRC tissues. The validation study, which included 55 early relapsed patients and 55 nonearly relapsed patients, further confirmed overexpression of miRNA-148a in nonearly relapsed samples. Subsequently, we explored whether the serum level of miRNA-148a can be used to predict early CRC recurrence. The invitro and invivo effects of miRNA-148a were examined by cell proliferation, migration, and invasion, as well as cell cycles, and xenograft in null mice. Last, miRNA-148a was investigated as a potential biomarker for identifying early relapse. Cellular studies demonstrated that the overexpression of miRNA-148a inhibited colon cancer cell proliferation and migration but not invasion. The cell cycle studies also revealed that miRNA-148a caused an accumulation of the G2 population. Moreover, lower levels of miRNA-148a expression were associated with significantly shorter disease-free survival rates and poorer overall survival rates. This study showed that miRNA-148a can inhibit tumorigenesis and reduce the early recurrence of CRC. These findings suggest that miRNA-148a may have potential clinical applications for predicting the early relapse of patients with CRC after radical resection.

Expression of miR-126 suppresses migration and invasion of colon cancer cells by targeting CXCR4

A previous study demonstrated that miR-126 expression was significantly downregulated in highly metastatic colon cancer cells. This study was to investigate the biological function of miR-126 and its regulation of target genes in colon cancer cells. Quantitative PCR was used to detect miR-126 expression in colon cancer SW480 and SW620 cells. MTT assay was to measure the changed cell viability after miR-126 mimics transfection. Wound healing and Transwell migration and invasion assays measured capacity of tumor cell migration and invasion of SW480 and SW620 cells after miR-126 transfection. Luciferase reporter assay and Western blot were used to assess both transcriptional and expression levels of one of the miR-126 target genes (i.e., CXCR4). Levels of miR-126 expression were lower in colon cancer SW480 and SW620 cells than in the adjacent normal epithelial tissues (P < 0.05). Transfection of miR-126 mimics significantly reduced colon cancer cell viability compared to NC cells (P < 0.05). The wound healing and Transwell migration and invasion assays showed that miR-126 mimics inhibited SW480 and SW620 cell migration and invasion capacity. Bioinformatics predicted that CXCR4 is one of the miR-126 target genes. Indeed, luciferase reporter assay and Western blot confirmed that CXCR4 is a miR-126 target gene. Expression of miR-126 inhibited colon cancer cell viability and reduced tumor cell migration and invasion capacity by its negative regulation of CXCR4 expression.

Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways.

Tolfenamic acid inhibits colon cancer cell and tumor growth and induces degradation of specificity protein (Sp) transcription factors

Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) that inhibits lung, esophageal, breast and pancreatic cancer cell and tumor growth, and this study investigated the anticancer activity of TA in colon cancer. TA inhibited growth and induced apoptosis in RKO, SW480, HT-29, and HCT-116 colon cancer cells, and TA (50 mg/kg/d) also inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. TA downregulated expression of Sp proteins (Sp1, Sp3, and Sp4) in colon cancer cells and this was accompanied by decreased expression of several Sp-regulated growth promoting (cyclin D1, hepatocyte growth factor receptor), angiogenic (vascular endothelial growth factor (VEGF) and its receptor 1), survival (survivin and bcl-2), and inflammatory (NFκBp65/p50) gene products. The mechanism of TA-mediated effects on Sp proteins was due to activation of caspases. These results now extend the number of NSAIDs that may have clinical potential for colon cancer chemotherapy and show that the anticancer activity of TA is due, in part, to targeting Sp transcription factors.

MicroRNA-19a targets tissue factor to inhibit colon cancer cells migration and invasion

The over-expression of tissue factor (TF) and its roles in colon cancer progression have attracted much attention. However, the mechanisms regulating TF expression have not yet been shown in detail. In this study, we over-expressed miR-19a, miR20a and miR-106b in colon cancer cells, and evaluated their impact on TF expression and cellular function. We provide evidence demonstrating that miR-19a inhibited TF expression in vitro. Luciferase reporter assay confirmed that TF was a direct target of miR-19a because the miR-19a mediated repression of luciferase activity was abolished by mutation of the putative binding site. Moreover, miR-19a suppressed colon cancer cell migration and invasion. This effect was due to the indirect down-regulation of matrix metalloproteinase 9. Finally, we investigated the relevance of TF and miR-19a expression in a total of 48 paired colon cancer samples and revealed that miR-19a was inversely correlated with TF expression in stages I and II cases. Therefore, our results suggested that miR-19a was capable of suppressing TF expression in vitro and inhibiting cell migration and invasion. Although it was not the unique mechanism responsible for the expression of TF in vivo, miR-19a was inversely correlated with TF expression in early stage colon cancer patients.

Resveratrol 3‐O‐d‐glucuronide and resveratrol 4′‐O‐d‐glucuronide inhibit colon cancer cell growth: Evidence for a role of A3 adenosine receptors, cyclin D1 depletion, and G1 cell cycle arrest

Resveratrol is a plant-derived polyphenol with chemotherapeutic properties in animal cancer models and many biochemical effects in vitro. Its bioavailability is low and raises the possibility that the metabolites of resveratrol have biological effects. Here we investigate the actions of resveratrol 3-O-D-glucuronide, resveratrol 4-O-D-glucuronide, and resveratrol 3-O-Dsulfate on the growth of colon cancer cells in vitro. The growth of Caco-2, HCT-116, and CCL-228 cells was measured using the neutral red and MTT assays. Resveratrol and each metabolite inhibited cell growth with IC50 values of 9.8–31 μM. Resveratrol caused S phase arrest in all three cell lines. Resveratrol 3-O-D-glucuronide and resveratrol 4-O-D-glucuronide caused G1 arrest in CCL-228 and Caco-2 cells. Resveratrol 3-O-D-sulfate had no effect on cell cycle. Growth inhibition was reversed by an inhibitor of AMP-activated protein kinase (compound C) or an adenosine A3 receptor antagonist (MRS1191). The A3 receptor agonist 2Cl-IB-MECA inhibited growth and A3 receptors were detected in all cell lines. The resveratrol glucuronides also reduced cyclin D1 levels but at higher concentrations than in growth experiments and generally did not increase phosphorylated AMP-activated protein kinase. Resveratrol glucuronides inhibit cell growth by G1 arrest and cyclin D1 depletion, and our results strongly suggest a role for A3 adenosine receptors in this inhibition.

Abstract 1855: Regulation of proliferation of colon cancer cells by compounds affecting glycolysis including 3-bromopyruvate, 2-deoxyglucose and biguanides.

Abstract In earlier work we found that incubation of colon cancer cells with 2-deoxyglucose had an additive inhibitory effect on proliferation with the biguanides, metformin and phenformin, and blocked the acidification of the medium caused by incubation of cells with biguanides. In the present work, studies were performed with Caco-2, HCT116, HT29 and SW1116 colon cancer cells. Metabolism of glucose and acidification of the medium was greater with the more rapidly proliferating cells being greatest with HCT116 cells and least with SW1116 cells. We have seen that, as with 2-deoxyglucose, 3-bromopyruvate can also prevent the lowering of medium pH caused by biguanide treatment. . Inhibition of glucose metabolism paralleled the decrease in acidification of the medium. 3-bromopyruvate inhibited colon cancer cell proliferation. Additive effects with metformin and 3-bromopyruvate were not observed in any of the four colon cancer cell lines but some additive effects were seen with phenformin and 3-bromopyruvate. An additive effect was more notable in combined treatment with 3-bromopyruvate and 2-deoxyglucose. Induction of alkaline phosphatase activity serves as a marker of differentiation in colon cancer cells. The induction of alkaline phosphatase activity by butyrate was not consistently affected by combination with other agents that modified glucose metabolism. The drug combinations that were examined inhibited proliferation in wild type and P53 null HCT116 cells and affected colon cancer lines with different growth rates. Although metformin and phenformin increase glucose metabolism by colon cancer cells while 3-bromopyruvate and 2-deoxyglucose inhibit glucose metabolism, all these compounds have potential inhibitory action on the proliferation of colon cancer cells. Citation Format: Michael A. Lea, Mehreen Qureshi, Michael Buxhoeveden, Nicolette Gengel, Jessica Kleinschmit, Charles desBordes. Regulation of proliferation of colon cancer cells by compounds affecting glycolysis including 3-bromopyruvate, 2-deoxyglucose and biguanides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1855. doi:10.1158/1538-7445.AM2013-1855

Abstract 3280: The sphingosine-1-phosphate receptor modulator FTY720 synergistically inhibits colon cancer cell growth with 5-fluorouracil, irinotecan and oxaliplatin.

Abstract Introduction: Despite the appearance of new cancer chemotherapeutics during the last decade, the five-year survival rate of advanced colon cancer remains low. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, plays critical roles in cancer biology by regulating cell proliferation, migration, metastasis and angiogenesis. S1P exerts most of its functions via its specific cell surface receptors. The immunosuppressive drug, FTY720 (fingolimod), is a functional antagonist of S1P receptor 1 and is approved by FDA for treatment of multiple sclerosis. Although there have been some reports on the effects of FTY720 on breast and prostate cancers, its effects on colon cancer have not yet been examined. The aim of this work was to determine whether FTY720 suppresses survival of colon cancer cell lines either alone or in combination with 5-fluorouracil (5-FU), irinotecan, or oxaliplatin the most commonly used chemotherapies for colon cancer. Methods: The effects of FTY720 on cell survival of multiple cancer types were assessed with the NCI-60 panel. Colon cancer cell lines, human well-differentiated (DLD-1), human moderately differentiated (HT29), and human poorly differentiated (HCT-116) were seeded at a density of 2000 cells per well in 96-well culture plates and FTY720 and 5-FU, or irrinotecan, or oxaliplatin or both were added after 24 hours. Effects on cell viability were examined by WST-8 assay 48 hours later. Results: Among the 9 types of cancers tested in the NCI-60 cell line panel, colon cancer and leukemia were the only ones in which all cell lines responded to FTY720. FTY720 completely suppressed growth of DLD-1 and HCT-116 cells at a concentration of 5 μM or greater in a dose dependent manner. The half maximal (50%) inhibitory concentrations (IC50) of FTY720 were less than 1.8 uM in the 3 colon cancer cell lines. The IC50 values for 5-FU, irinotecan, and oxaliplatin alone were about 8.0 uM, 5.8uM,and 1.1uM respectively, in the same 3 colon cancer cell lines. The combined treatment with FTY720 and 5FU synergistically suppressed survival. Also the combination treatment of FTY720 and irinotecan synergistically suppressed survival of these cell lines, except for HT29 cells. As for the combination treatment with FTY720 and oxaliplatin, it synergically suppressed survival HT29 cells. Conclusion: Our results demonstrate that FTY720 has a synergistic cell killing effect with 5-FU, irinotecan, and oxaliplatin on colon cancer cell lines. Further studies will focus on delineating the mechanisms determining susceptibility to these drugs. This work was supported by NIH (R01CA160688 to KT and R01CA61774 to SS). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. Citation Format: Tomoyoshi Aoyagi, Akimitsu Yamada, Masayuki Nagahashi, Sheldon Milstien, Sarah Spiegel, Kazuaki Takabe. The sphingosine-1-phosphate receptor modulator FTY720 synergistically inhibits colon cancer cell growth with 5-fluorouracil, irinotecan and oxaliplatin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3280. doi:10.1158/1538-7445.AM2013-3280

Abstract 2578: Berberine suppress the colon cancer proliferation through activating AMPK.

Abstract Background and Objectives: Numerous clinical studies and in vitro study have shown efficacy of Berberine, an isoquinoline alkaloid isolated from plants, in treating multiple diseases. Berberine possesses anti-diabetic, anti-inflammatory and anti-tumor properties. AMP-activated protein kinase (AMPK) is a cellular energy sensor that exists in almost all eukaryotes and is a potential therapeutic target for cancer. Berberine displays beneficial effects in the treatment of diabetes and obesity at least in part via stimulation of AMPK activity. Herein, this study explores whether the therapeutic role of Berberine on colon cancer is linked to the activation of AMPK signal pathway. Methods: In vitro studies: Three human colorectal cancer cell (CRC) lines (HCT116, SW480, LOVO) were treated with Berberine (0μM to 100μM) and cell proliferation assay was used to evaluate the inhibition effects on cell growth. The CRC lines were then treated with Berberine (0 to 60μM), for 24h or 48h, after which AMPK activation was measured by western blotting. In vivo studies: we used the azoxymethane-dextran sulfate sodium (AOM/DSS) mouse model of colitis induced colon cancer to explore the effects of Berberine on colon carcinogenesis. After DSS exposure, the FVB mice were randomly divided into two groups (N=19) and treated with Berberine (40mg/kg) or water for 10 weeks. After 10 weeks, all the mice were euthanized, the colon tissues were collected and the tumor numbers and tumor sizes were observed under microscopy. Results: In vitro studies: Berberine inhibited the growth of the three CRC lines with an IC50 around 30μM. Berberine activated AMPK in a dose-dependent and time-dependent manner. In addition, mTOR, a downstream target of AMPK, was down-regulated by Berberine in a dose-dependent and time-dependent manner. Furthermore, 4EBP1 and p70s6kinase, downstream target of mTOR, were also down-regulated by Berberine treatment in three colon cancer cell lines, whereas the MAPK pathway was not affected by Berberine. In vivo studies: Berberine treated mice showed a significant reduction of tumor numbers (60% reduction, p=0.013) and in tumor size (49% reduction for &amp;lt;2mm, p=0.034; 94% for 2-4mm, p=0.006; and 100% for &amp;gt;4mm p=0.024) compare to vehicle treated mice. Conclusions: We have provided evidence that Berberine inhibits colon cancer cell growth and decrease AOM/DSS induced tumor numbers and tumor size. Our data suggest that Berberine suppresses colon epithelial proliferation via inhibition of mTOR through the activation of AMPK. Similar anticancer activity has been reported for Metformin, another antidiabetic drug that activates AMPK and inhibits mTOR signaling. Metformin is currently being tested in clinical trials for preventing cancer. In conclusion, Berberine might offer a safe and promising candidate for chemoprevention of colon cancer. Citation Format: Weidong Li, Baojin Hua, Hongsheng Lin, Wei Hou, Libin Jia, Matthew R. Young, Nancy H. Colburn. Berberine suppress the colon cancer proliferation through activating AMPK. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2578. doi:10.1158/1538-7445.AM2013-2578

Abstract 2583: Regulation of AKT-FoxO signaling by PKG2 inhibits colon cancer cell growth.

Abstract Signaling through cGMP has emerged as a potentially important suppressor of tumorigenesis in the colon, but the mechanism is poorly defined. Studies of guanylyl-cyclase (GCC) knockout mice indicate that cGMP signaling suppresses proliferation in the colon epithelium, and more recently that inhibition of AKT has an important role. Type-2 cGMP-dependent protein kinase (PKG2) is a central effector of cGMP in the colon epithelium and is a likely mediator of the homeostatic effects of cGMP signaling. The present study sought to determine whether PKG2 can regulate AKT signaling in colon cancer cells and in the colon mucosa. Activation of PKG2 in LS174T and HT29 colon cancer cells inhibited cell proliferation and increased G1 arrest. PKG2 activation also inhibited AKT activity in these cells as determined by immunoblotting for phospho-AKT and downstream phospho-S6. Suggestive of a mechanism, analysis of upstream components revealed that PKG2 increased the levels of phosphatase and tensin homolog (PTEN). Central to the growth-promoting role of AKT signaling is the inactivation of forkhead transcription factors (FoxO), which regulate growth-inhibitory and pro-apoptotic target gene expression. Consistent with the inhibition of AKT signaling, activation of PKG2 significantly increased luciferase reporter activity driven by FoxO-responsive elements in colon cancer cells. This activation was associated with increased levels of several FoxO target genes, including catalase, p27kip. The regulation of AKT/FoxO pathway by PKG2 in vivo was supported by increased phospho-AKT levels, and reduced FoxO target gene expression in the colon mucosa of Prkg2-/- mice compared to wild type siblings. Moreover, activation of cGMP signaling in the colon mucosa led to increased expression of FoxO targets, and suppression of both proliferation and redox stress. Taken together these data define a novel signaling pathway in the colon epithelium, where of PKG2 leads to activation of FoxO. The established tumor suppressor role of the FoxO proteins highlights the potential therapeutic role for cGMP signaling in the prevention of colon cancer. Citation Format: Rui Wang, In-Kiu Kwon, Muhan Hu, Darren D. Browning. Regulation of AKT-FoxO signaling by PKG2 inhibits colon cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2583. doi:10.1158/1538-7445.AM2013-2583

Methylselenol, a selenium metabolite, inhibits colon cancer cell growth in vitro and in vivo

Methylselenol is hypothesized to be a critical selenium (Se) metabolite for certain anticancer activities in vivo but its role in colon cancer prevention remains to be characterized. This study tested the hypothesis that methylselenol inhibits the growth of colon cancer cells and tumors. We found that submicromolar methylselenol exposure inhibited cell growth and led to an increase in the G1 and G2 fractions with a concomitant drop in the S‐phase, and an induction of apoptosis in cancerous colon HCT116 cells, but to a much lesser extent in noncancerous NCM460 colon cells. Furthermore, in agreement with the fact that blockade of extracellular‐regulated kinase1/2 (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) pathways suppresses the growth of colon tumors, we demonstrated that methylselenol inhibited ERK1/2 and p38 MAPK in cancerous colon HCT116 cells but also to a lesser extent in NCM460 cells. Consistent with these in vitro observations, a daily single oral dose (1 to 3 mg Se/kg body weight) of methylseleninic acid, a methylselenol precursor, for 5 weeks inhibits the growth of colon cancer xenografts in a C57/BL mouse model. Taken together, methylselenol's strong potential of inhibiting colon cancer cell proliferation, may be an important mechanism by which Se exerts its anticancer activity in colon cancer prevention.

Resveratrol metabolites inhibit human metastatic colon cancer cells progression and synergize with chemotherapeutic drugs to induce cell death

Resveratrol (RSV) has been proposed to prevent tumor growth; nevertheless, these preventive effects are controversial since RSV pharmacokinetics studies show a low bioavailability. Recent clinical trials show that patients with colorectal cancer and receiving oral RSV have high levels of RSV conjugates in the colorectum, mainly RSV-3-O-sulfate (R3S), RSV-3-O-glucuronide, and RSV-4'-O-glucuronide. However, their potential biological activity has not yet been established. This study thus investigated in human colorectal cancer cell lines whether RSV main metabolites retain anticarcinogenic properties as their parental molecule. Proliferation, apoptosis assays and cell cycle analysis were performed to study the effect of RSV, R3S, RSV-3-O-glucuronide, or RSV-4'-O-glucuronide alone or of a mixture of the three metabolites. R3S inhibits colon cancer cells proliferation and an accumulation of cells in S phase. Interestingly, the mixture induced a synergistic effect. This process was associated with an induction of DNA damages and apoptotic process, which allowed sensitization of colon cancer cells to the anticancer drugs. Altogether, our data provide significant new insight into the molecular mechanism of RSV and support the notion that despite low bioavailability in vivo, RSV biological effects could be mediated by its metabolites.

CInQ-03, a novel allosteric MEK inhibitor, suppresses cancer growth in vitro and in vivo

The mitogen-activated protein kinase kinase 1 and 2 signaling pathway is a major component of the RAS (Rat sarcoma)/RAF (Radpidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERKs (Extracellular signal-regulated kinases) signaling axis that regulates tumorigenesis and cancer cell growth. MEK is frequently activated in various cancers that have mutations in the KRAS and BRAF oncogenes. Therefore, MEK has been suggested as a therapeutic target for inhibitor development against tumors that are dependent on the activating mutations in mitogen-activated protein kinase signaling. Herein, we report the discovery of three novel MEK inhibitors, herein referred to as CInQ-01, CInQ-03 and CInQ-06. All three inhibitors were highly effective in suppressing MEK1 and MEK2 in vitro kinase activity as well as anchorage-dependent and anchorage-independent cell growth. The inhibitory activity was associated with markedly reduced phosphorylation of ERKs and ribosomal S6 kinases. Furthermore, administration of CInQ-03 inhibited colon cancer cell growth in an in vivo xenograft mouse model and showed no skin toxicity. Overall, these results suggest that these novel MEK inhibitors might be used for chemotherapy or prevention.