Cultured Colon Cancer Cells Research Articles

Abstract 4953: Molecular and cellular localization profiles of tristetraprolin in colorectal cancer: Implications for tumor progression in diverse patient populations

Abstract Introduction: Colorectal cancer (CRC) and other solid tumors rely on production of pro-inflammatory cytokines and growth factors to support their rapid growth. Posttranscriptional control of these factors involves interplay between RNA stabilizing and destabilizing factors. We have observed that the protein tristetraprolin (TTP), a RNA destabilizer, is down-regulated in CRCs and that interleukin (IL)-8, a proinflammatory cytokine, is up-regulated. We hypothesized that loss of negative regulation by TTP selectively favors expression of growth factors and protooncogenes that promote tumor progression. Furthermore, new evidence suggests that TTP is associated estrogen receptor-α (ERα) and functions as a co-repressor of ERα activation interaction with histone deacetylases on the gene promoter; nuclear TTP is linked to this tumor suppressor function. Since CRCs of African Americans (AAs) often present worse outcomes relative to those of Caucasian Americans (CAs), we analyzed the expression of TTP and its targets and assessed its subcellular localization in primary CRCs of AA and CA patients and in cultured colon cancer cells. Its effects on disease progression were also determined. Methods: Paired normal and CRC tissues from 45 patients (AA = 26 and CA = 19), were evaluated by qPCR for mRNA expression levels of TTP, IL-8, and VEGF. Tissue sections were also assayed for immunophenotypic expression by immunohistochemistry, and the staining patterns (nuclear and cytoplasmic) were assessed. To determine TTP effects on the NF-kB pathway, colon cancer cells (CCL235, HCT116 and LoVo) were stimulated with TNF-α, and total RNA was analyzed for TTP, IL-8, VEGF, and cIAP2 expression; interaction of TTP and ERα was also assessed. Results: In 31 of 45 CRCs, there was down-regulated expression of TTP mRNA, and low TTP levels correlated with advanced tumor stage. Low levels of TTP were present in 21 of 26 AAs and in 12 of 19 CAs. There was an inverse correlation between TTP and IL-8 expression in relation to tumor stage. There was an agreement between mRNA expression and immunophenotypic profiles for TTP and IL-8. Furthermore, there was reduced or a lack of nuclear staining of TTP in malignant cells relative to normal/benign epithelial cells. In cultured colon cancer cells, TTP mRNA levels inversely correlated with levels of IL-8, VEGF, and cIAP2 mRNAs, suggesting interactions of TTP with these cell survival factors. Conclusions: Reduced expression of TTP was noted in tumors, especially in CRCs of AAs, and a loss or decreased nuclear expression of TTP was observed in malignant cells. Lower TTP levels correlated with advanced tumor stage. These preliminary findings suggest that TTP negatively regulates the expression of cell survival factors in the NF-kB pathway. This work was supported by funds from the NIH/NCI (a career development supplement to grant U54-CA118948). Citation Format: Esther A. Suswam, Balananda-Dhurjati K. Putcha, Amit K. Tiwari, Trafina Jadhav, Kiera D. Walker, Lualhati Harkins, Samir Amer, Isam-Eldin Eltoum, Sejong Bae, Upender Manne. Molecular and cellular localization profiles of tristetraprolin in colorectal cancer: Implications for tumor progression in diverse patient populations. [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 4953. doi:10.1158/1538-7445.AM2015-4953

Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures.

Epithelial-to-mesenchymal transition (EMT) confers stem cell-like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E-cadherin decreases, resulting in loss of cell-cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real-time PCR and immunofluorescence were performed to investigate the expression of E-cadherin, vimentin, β-catenin, cytokeratin-20 and -18, Twist1, Snail, CD44, cyclooxygenase-2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl-containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E-cadherin epithelial marker, which was instead expressed in cancer and normal colon mucosa of the same patient, while overexpressed vimentin (mesenchymal marker), Twist1, Snail (EMT markers) and COX2. Cytokeratin-18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Oct4 and Nanog, were also observed. Following differentiation with the glycogen synthase kinase 3β (GSK3β) inhibitor LiCl, the cells began to express E-cadherin and, at once, Twist1 and Snail expression was strongly downregulated, suggesting a MET-reverting process. In conclusion, we established primary colon mesenchymal cancer cell cultures expressing mesenchymal and epithelial biomarkers together with high level of EMT transcription factors. We propose that they could represent a good model for studying EMT and its reverting mechanism, the mesenchymal-to-epithelial transition (MET). Our observation indicates that LiCl, a GSK3β inhibitor, induces MET in vitro, suggesting that LiCl and GSK3β could represent, respectively, interesting drug, and target for CRC therapy.

Shed syndecan-2 enhances tumorigenic activities of colon cancer cells.

Because earlier studies showed the cell surface heparan sulfate proteoglycan, syndecan-2, sheds from colon cancer cells in culture, the functional roles of shed syndecan-2 were assessed. A non-cleavable mutant of syndecan-2 in which the Asn148-Leu149 residues were replaced with Asn148-Ile149, had decreased shedding, less cancer-associated activities of syndecan-2 in vitro, and less syndecan-2-mediated metastasis of mouse melanoma cells in vivo, suggesting the importance of shedding on syndecan-2-mediated pro-tumorigenic functions. Indeed, shed syndecan-2 from cancer-conditioned media and recombinant shed syndecan-2 enhanced cancer-associated activities, and depletion of shed syndecan-2 abolished these effects. Similarly, shed syndecan-2 was detected from sera of patients from advanced carcinoma (625.9 ng/ml) and promoted cancer-associated activities. Furthermore, a series of syndecan-2 deletion mutants showed that the tumorigenic activity of shed syndecan-2 resided in the C-terminus of the extracellular domain and a shed syndecan-2 synthetic peptide (16 residues) was sufficient to establish subcutaneous primary growth of HT29 colon cancer cells, pulmonary metastases (B16F10 cells), and primary intrasplenic tumor growth and liver metastases (4T1 cells). Taken together, these results demonstrate that shed syndecan-2 directly enhances colon cancer progression and may be a promising therapeutic target for controlling colon cancer development.

Abstract C77: Tristetraprolin suppression is associated with advanced stage colorectal cancer

Abstract Background: Interleukin (IL)-8, vascular endothelial growth factor (VEGF), and IL-6 contribute to the colorectal cancer (CRC) progression by inhibiting apoptosis and by promoting angiogenesis and tumor proliferation. We have found that the tristetraprolin (TTP) gene attenuates these processes [J Neurooncol. 2013; 113(2):195-205]. TTP expression is lost or reduced in many cancers, including CRCs, and loss of TTP is thought to contribute to tumorigenesis. We hypothesized that low TTP levels favor expression of growth factors and correlate with CRC progression. In addition, we suggest that TTP modulates CRC growth through negative regulation on cell survival and/or anti-apoptotic factors in the NF-kB pathway. We tested this hypothesis by analyzing mRNA expression of TTP and its targets in primary CRCs of African American (AA) and Caucasian American (CA) patients. Methods: We analyzed frozen primary tissues from 45 CRC patients (AA=26 and CA=19), each with corresponding normal (benign/control) tissue. cDNAs were reverse-transcribed from total RNA; mRNA levels of TTP and its target genes (IL-8, VEGF, IL-6) were quantified by the qPCR sybr-green method. Expression levels were normalized to GAPDH. To assess TTP effects on the NF-kB pathway, colon cancer cells (CCL235, HCT116, SW480, and LoVo) were stimulated with TNF-α for 0-24 hr, and total RNA was analyzed for TTP, IL-8, IL-6, VEGF, and cIAP2 expression by qRT-PCR. Levels of HuR mRNA in cells were also assessed. Extracts from the cells were immunoblotted with anti-TTP and antiHuR antibodies. Results: We observed down-regulated expression of TTP mRNA in primary CRCs (31 of 45), and decreased TTP levels correlated with advanced tumor stage. Low levels of TTP were found in 21 of 26 AAs and 12 of 19 CAs. In both racial groups, there was an inverse correlation between TTP and IL-8 expression in relation to tumor stage. Studies with cultured colon cancer cells demonstrated that TTP mRNA levels inversely correlated with levels of IL-8, IL-6, VEGF, and cIAP2 mRNAs, suggesting interactions of TTP with cell survival factors. Western blot analyses confirmed TTP expression levels in these cells. Conclusions: For both racial groups, TTP expression was lower in tumor tissues relative to normal tissues; the difference was more pronouced in CRCs of AAs. Further, lower TTP levels correlated with advanced tumor stage; and TTP negatively regulated the expression of IL-8, VEGF, and cIAP2 in cultured cells. These studies were supported by a pre-pilot project of the UAB/TU/MSM Partnership grant of NIH/NCI, U54-CA 118948. Citation Format: Esther A. Suswam, Balanada Dhurjarti Kumar Putcha, Kiera D. Walker, LaJessica Johnson, Jasmine Howard, Edward E. Partridge, Mona N. Fouad, Sejong Bae, Upender Manne. Tristetraprolin suppression is associated with advanced stage colorectal cancer. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C77. doi:10.1158/1538-7755.DISP13-C77

Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src.

Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.

5‐Demethylnobiletin inhibits colon carcinogenesis in azoxymethane/dextran sulfate sodium‐treated mice (123.3)

Our previous studies have demonstrated inhibitory effects of 5‐demethylnobiletin (5DN, a unique citrus flavonoid) in cultured colon cancer cells. Herein, we determined effects of 5DN on colitis‐associated colon carcinogenesis in azoxymethane/Dextran Sulfate Sodium ‐treated mice. Our results demonstrated that oral administration of 5DN significantly lowered colon tumor incidence by 54% and decreased overall tumor size by 49%. immunohistochemical analysis showed that 5DN had strong anti‐proliferative and pro‐apoptotic effects on colonic tumors. ELISA results demonstrated that 5DN suppressed the levels of IL‐1 and IL‐6 in colonic tissue by 89% and 87%. Immunoblot assay showed that 5DN significantly decreased the expression level of iNOS and COX‐2 in mice colon. HPLC analysis showed that oral administration of 5DN resulted in high levels of 5DN and its metabolites, i.e. 5,3'‐didemethylnobiletin, 5,4'‐didemethylnobiletin, and 5,3', 4’‐tridemethylnobiletin in the colonic tissues. Cell culture study showed that these metabolites, especially 5,3’‐didemethylnobiletin had much stronger inhibition on human colon cancer cells than 5DN. Flowcytometry analysis indicated that 5DN and its metabolites induced extensive cell cycle arrest and apoptosis. In conclusion, our results demonstrated that oral administration of 5DN significantly inhibited colitis‐associated colon carcinogenesis in mice and these effects can be partially attributed to the colonic metabolites of 5DN.Grant Funding Source: This work was supported by funding from NIH, AICR and USDA.

Delayed Administration of a Single Dose of Lithium Promotes Recovery from AKI

Evidence suggests that glycogen synthase kinase 3β (GSK3β) contributes to AKI; however, its role in post-AKI kidney repair remains uncertain. Here, delayed treatment with a single dose of lithium, a selective inhibitor of GSK3β and a US Food and Drug Administration-approved mood stabilizer, accelerated recovery of renal function, promoted repopulation of renal tubular epithelia, and improved kidney repair in murine models of cisplatin- and ischemia/reperfusion-induced AKI. These effects associated with reduced GSK3β activity and elevated expression of proproliferative molecules, including cyclin D1, c-Myc, and hypoxia-inducible factor 1α (HIF-1α), in renal tubular epithelia. In cultured renal tubular cells, cisplatin exposure led to transient repression of GSK3β activity followed by a prolonged upregulation of activity. Rescue treatment with lithium inhibited GSK3β activity, enhanced nuclear expression of cyclin D1, c-Myc, and HIF-1α, and boosted cellular proliferation. Similarly, ectopic expression of a kinase-dead mutant of GSK3β enhanced the expression of cyclin D1, c-Myc, and HIF-1α and amplified cellular proliferation after cisplatin injury, whereas forced expression of a constitutively active mutant of GSK3β abrogated the effects of lithium. Mechanistically, GSK3β colocalized and physically interacted with cyclin D1, c-Myc, and HIF-1α in tubular cells. In silico analysis revealed that cyclin D1, c-Myc, and HIF-1α harbor putative GSK3β consensus phosphorylation motifs, implying GSK3β-directed phosphorylation and subsequent degradation of these molecules. Notably, cotreatment with lithium enhanced the proapoptotic effects of cisplatin in cultured colon cancer cells. Collectively, our findings suggest that pharmacologic targeting of GSK3β by lithium may be a novel therapeutic strategy to improve renal salvage after AKI.

Development of Magnetic Levitation 3-D Cultures of Breast and Colon Cancer Cells Using Carbon Encapsulated Cobalt Magnetic Nanoparticles

Innovative technologies are required for creation of in vitro models that mimic in vivo systems and can be used in drug discovery and development, disease models for cancer, stem cell research, and tissue engineering. Therefore, we established nanomagnetically levitated three-dimensional (3-D) cultures of breast and colon cancer cells using carbon encapsulated cobalt nanoparticles.

Expression of Toll-Like Receptors on Human Rectal Adenocarcinoma Cells

The innate immune system uses Toll-like receptors (TLR) to detect the presence of pathogen patterns thus allowing for rapid host defense responses. Stimulation of TLR results in inflammatory response and regulatory cytokine production affecting acquired immunity. The aim of the study was an evaluation of TLR2 and TLR4 expression on the surface of human colon cancer cells in primary culture with or without autologous peripheral blood mononuclear cells. Surgical specimens of colon cancer were processed to obtain cancer cells. Cancer cells separation was conducted first by mechanical tissue disintegration and than by gradient centrifugation to obtain 95 % cell confluence. By staining the isolated cells the pathologist determined them as adenocarcinoma. Colon cancer cells were then co-cultured in 24 h culture alone or together with autologous lymphocytes. Reverse-transcription polymerase chain reaction was performed for detection of TLR2 and TLR4 mRNA in colon cancer and normal colon epithelial cells using commercially available primers. Resting as well as phytohemagglutinin or lipopolysaccharide (LPS) stimulated cells were tested. Receptor proteins on cancer cells were examined by immunohistochemistry. TLR4 mRNA was detected in cancer cells. Autologous lymphocytes do not exert any effect on these receptors expression. TLR4 mRNA expression was not observed in normal colon epithelial cells. TLR2 mRNA was present on LPS stimulated cancer cells as well as on resting and stimulated lymphocytes. Expression of TLR2 and TLR4 receptor proteins on colon cancer cells were confirmed by immunohistochemistry. TLR4 may be responsible for uncontrolled tumor growth under LPS stimulation in human colon environment.

Abstract A108: Induction of mitochondrial dysfunction as a strategy for targeting tumor cells in hypoxic microenvironment.

Abstract A fundamental problem in cancer drug discovery is to identify compounds that eliminate dormant malignant cells responsible for tumor relapse. Abnormal vascularization of solid tumors leads to the generation of tissue microenvironments that are chronically starved of oxygen and nutrients. Cells residing in such environments are slowly growing or quiescent and display altered phenotypic characteristics when compared to cells located in more vascularized regions. Such non-dividing cells are often resistant to mainstay standard chemotherapies that rely on DNA replication and cell division to elicit their anti-tumor effect. The altered phenotype of quiescent cells enables them to survive chemotherapeutic regimes and reseed nascent tumors following secession of chemotherapy. Indeed resistance to agents such as doxorubicin, cisplatin and vinblastine has been correlated with poor vasculature, tumor relapse and poor patient survival. Thus there is a need to alter the scope of cancer drug discovery and focus more on screening for agents that can exploit the altered phenotype of quiescent metabolically stressed cells to eliminate tumors. Cell-based screening for novel anticancer drugs is typically performed using monolayer cultures of tumor cells, however such monolayer cultures do not represent the characteristics of 3-D solid tumors, frequently leading to the failure of subsequent in vivo models. The multicellular tumor spheroid model is of intermediate complexity between in vivo tumors and in vitro monolayer cultures and is more suitable for drug screening and evaluation. Spheroids are known to be more resistant to drug effects compared to monolayer cultures. Resistance is due not only to pharmacokinetic obstacles limiting drug penetrance to inner layers, but also to multi-cellular interactions leading to altered expression of genes and proteins regulating drug response. An additional benefit of growing cells three- dimensionally is the opportunity to explore the core regions’ potential vulnerability related to hypoxia and nutrient defiency and to reflect the heterogeneous milieu in tumor microregions relative to the supply. Poorly vascularized and perfused tumor microareas in many aggressive cancers have limited access not only to oxygen but also to glucose. Core regions are also associated with acidic pHsince these tumor cells change their metabolism towards increased glycolysis, resulting in increased lactic acid production. We here employed spheroid cultures of HCT116 colon cancer cells to screen a diverse chemical library with the aim to find compounds with cytotoxic activity in core, hypoxic, regions. The screen identified a compound (VLX600) demonstrating anti-cancer activity with a large therapeutic window both in vitro and in vivo. Here we characterized the mechanism of action and potential of VLX600 as anti-cancer therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A108. Citation Format: Xiaonan Zhang, Mårten Fryknäs, Emma Hernlund, Walid Fayad, Angelo De Milito, Maria Hägg Olofsson, Stig Linder. Induction of mitochondrial dysfunction as a strategy for targeting tumor cells in hypoxic microenvironment. [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 A108.

Cables1 is a tumor suppressor gene that regulates intestinal tumor progression in ApcMin mice

The transformation of colonic mucosal epithelium to adenocarcinoma requires progressive oncogene activation and tumor suppressor gene inactivation. Loss of chromosome 18q is common in colon cancer but not in precancerous adenomas. A few candidate tumor suppressor genes have been identified in this region, including CABLES1 at 18q11.2−12.1. This study investigates the role of CABLES1 in an in vivo mouse model of intestinal adenocarcinoma and in human colon cancer cell culture. ApcMin/+ mice were crossed with mice harboring targeted inactivation of the Cables1 gene (Cables1−/−). The intestinal tumor burden and tumor expression of β-catenin and PCNA was compared in Cables1+/+ApcMin/+ and Cables1−/−ApcMin/+ mice. β-catenin activity in human colon cancer cells with CABLES1 inactivation and intestinal progenitor cell function in Cables1−/− mice were assayed in vitro. The mean number of small intestinal tumors per mouse was 3.1 ± 0.6 in Cables1+/+ ApcMin/+ mice, compared with 32.4 ± 3.5 in the Cables1−/−ApcMin/+ mice (P < 0.0001). Fewer colonic tumors were observed in Cables1+/+ApcMin/+ mice (mean 0.6 ± 0.1) compared with the Cables1−/−ApcMin/+ mice (mean 1.3 ± 0.3, P = 0.01). Tumors from Cables1−/−ApcMin/+ mice demonstrated increased nuclear expression of β-catenin and an increased number of PCNA-positive cells. In vitro studies revealed that CABLES1 deficiency increased β-catenin dependent transcription and increased intestinal progenitor cell activity. Loss of Cables1 enhances tumor progression in the ApcMin/+ mouse model and activates the Wnt/β-catenin signaling pathway. Cables1 is a tumor suppressor gene on chromosome 18q in this in vivo mouse model and likely has a similar role in human colon cancer.

Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methodsWe examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n=43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. ResultsHuman colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000pg/ml within 48h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. ConclusionsOur findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is independent of its primary function in the induction of angiogenesis.

Activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in cultured human colon cancer cells

Here we report that activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in both primary cultured human colon cancer cells and cell lines. Knocking-down of AMPKα by the target shRNA significantly inhibits plumbagin-induced cytotoxicity in cultured colon cancer cells, while forced activation of AMPK by introducing a constitutively active AMPK (CA-AMPK), or by the AMPK activator, inhibits HT-29 colon cancer cell growth. Our Western-blots and immunoprecipitation (IP) results demonstrate that plumbagin induces AMPK/Apoptosis signal regulating kinase 1 (ASK1)/TNF receptor-associated factor 2 (TRAF2) association to activate pro-apoptotic c-Jun N-terminal kinases (JNK)-p53 signal axis. Further, after plumbagin treatment, activated AMPK directly phosphorylates Raptor to inhibit mTOR complex 1 (mTORC1) activation and Bcl-2 expression in colon cancer cells. Finally, we found that exogenously-added short-chain ceramide (C6) enhances plumbagin-induced AMPK activation and facilitates cell apoptosis and growth inhibition. Our results suggest that AMPK might be the key mediator of plumbagin's anti-tumor activity.

FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF−/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.

Abstract 3999: Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling.

Abstract Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling Iryna Kolosenko1, Mårten Fryknäs2, Hanif Rassoolzadeh1, Paola Pellegrini1, Slavica Brnjic1, Per Johnsson1, Giuseppe Di Lernia1, Dan Grandér1, Rolf Larsson2, Katja Pokrovskaja Tamm1, Stig Linder1, Angelo De Milito1 1 Cancer Center Karolinska, Dep. Oncology-Pathology, Karolinska Institute, Stockholm, Sweden 2 Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University Hospital, 751 85 Uppsala, Sweden Solid tumors contain microenvironments characterized by hypoxia and limited availability of nutrients. Such metabolically compromised environments show an increased level of resistance to many chemotherapeutic agents. Multicellular tumor spheroids (MCS) contain hypoxic and nutrient-starved cell populations and provide a model to study multicellular therapy-resistance. We here used 3D cultures of HCT116 colon cancer cells as a model to identify factors related to multicellular drug-resistance. Microarray analysis revealed a dramatic increase in the expression of interferon-alpha (IFNα)-regulated genes in the 3D as compared to 2D cultures. This finding is of significance since an IFNα-related gene signature has previously been associated with therapy resistance in several human cancers. IFNα is known to regulate gene transcription through transcription complex ISGF3 consisting of STAT1, STAT2 and IRF9, as well as through STAT1 homodimers. We found that IRF9, but not STAT1, was consistently induced in colon cancer cell lines grown in 3D as compared to the 2D cultures. Upregulation of IFN-induced genes was observed during crowding of monolayer HCT116 cultures, providing both mechanistic insight as well as a convenient experimental model. STAT1 knock-down experiments showed that induction of IRF9 occurred independently of STAT1. Interestingly, over-expression of IRF9 rendered monolayer 2D HCT116 cells significantly more resistant to cisplatin, docetaxel and etoposide. Vice versa, inhibition of IRF9 expression significantly increased drug-sensitivity of monolayer HCT116 cells to cisplatin. Our data further support an association of IFNα-related gene signature and drug resistance and suggest an important role for the STAT1-independent IRF9 upregulation associated with therapy resistance in cancer. Citation Format: Iryna Kolosenko, Mårten Fryknäs, Hanif Rassoolzadeh, Paola Pellegrini, Slavica Brnjic, Per Johnsson, Giuseppe Di Lernia, Dan Grander, Rolf Larsson, Katja Pokrovskaja, Stig Linder, Angelo De Milito De Milito. Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling. [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 3999. doi:10.1158/1538-7445.AM2013-3999

Nuclear expression of Glycogen synthase kinase‐3β and lack of membranous β‐catenin is correlated with poor survival in colon cancer

Dysregulation of Wnt/β-catenin signaling is a hallmark of colon cancer. Glycogen synthase kinase-3β (GSK-3β) can be a positive regulator of survival and proliferation of cultured colon cancer cell but its role in clinical colon cancer is unknown. Our objectives were to evaluate the role of GSK-3β in colon cancer. A tumor tissue microarray of primary colon cancers and metastases was used to evaluate expression and subcellular localization of GSK-3β and β-catenin. In total, 85 primary colon cancer samples were evaluated by immunohistochemistry. Immunoreactivity was correlated to known markers of adverse prognosis. Overall survival was the primary end-point. We found nuclear accumulation of GSK-3β in 39% (33/85) of evaluated tumors. Nuclear GSK-3β was significantly associated with shorter overall survival (p = 0.008), larger tumor size (p = 0.015), distant metastasis (p = 0.029) and loss of membranous β-catenin (p = 0.007). Loss of membranous β-catenin occurred in 37% (30/82) of the tumors and was associated with poor survival (p = 0.016). The combination of nuclear GSK-3β and lack of membrane β-catenin occurred in a total of 26% of the studied tumors (21/61) and was significantly and independently associated with poor prognosis. Our results suggest that nuclear expression of GSK-3β and loss of membrane β-catenin identify a subset of colon carcinomas with worse prognosis.

Cell-based high-throughput proliferation and cytotoxicity assays for screening traditional Chinese herbal medicines

A simple, reliable, high-throughput screening method was developed and used to assess the pharmaceutical effects of extracts of traditional Chinese herbal medicines (TCHMs). This method is based on 3-dimensional (3-D) cultures of mouse embryonic stem (mES) and human colon cancer and breast cancer cells expressing enhanced green fluorescent protein (EGFP) in polyethylene terephthalate (PET) fibrous scaffolds on modified 384-well plates with online monitoring of culture fluorescence for dynamic responses of cells to drugs present in culture media. Cell responses to deoxycholic acid and the extracts of 3 TCHMs (Ganoderma lucidum spores, Ginkgo biloba, and Epimedium brevicornum) at various concentrations were investigated for their effects on proliferation and cytotoxicity. The screening results, i.e., the growth responses of cancer cells to those drugs, were consistent with what have been reported in the literature, confirming the reliability of the new screening approach. Different from previous screening methods for both TCHMs and western medicines that used animal models or 2-D cell-based assays with single cell lines, this 3-D cell-based screening method employs both cancer and normal cells and thereby provides a way for quick, direct evaluation of the anticancer effects of TCHMs. This method also offers assessment on the side effects of TCHMs.

Arene–RuII Complexes of Curcumin Exert Antitumor Activity via Proteasome Inhibition and Apoptosis Induction

Organometallic ruthenium(II) complexes of general formula [(η(6)-arene)Ru(curcuminato)Cl], with arene being p-iPrC6 H4Me (1), C6H6 (2), and C6Me6 (3), were synthesized, characterized, and evaluated for their antitumor effects. Specifically, we explored their ability to regulate the proteasome, a validated pharmacological target in cancer treatment. Ruthenium complexes inhibited isolated proteasomes to various extents, with the biological activity of these complexes depending on the nature of the bound arene; in particular, [(η(6)-arene)Ru(curcuminato)Cl] 2 suppressed proteasomal activities more potently than 1, 3, or free curcumin. Each complex also inhibited proteasomes in cultured colon cancer cells and consequently triggered apoptosis, with the [(η(6)-benzene)Ru(curcuminato)Cl] complex 2 being the most active. The influence on the oxidative status of HCT116 cells and the DNA binding ability of the [(η(6)-arene)Ru(curcuminato)Cl] complexes were studied. Complex 2 showed the highest antioxidant capacity; moreover, complexes 1 and 2 were shown to bind isolated DNA with higher affinity (up to threefold) than free curcumin. Collectively, our results demonstrate that the complexation of curcumin with ruthenium(II) is a promising starting point for the development of curcumin-based anticancer drugs.

The Multi-Targeted Kinase Inhibitor Sunitinib Induces Apoptosis in Colon Cancer Cells via PUMA

Constitutive activation of pro-survival kinases has become a promising target of small molecules with an increasing interest in developing multi-targeted agents. The mechanisms underlying the responsiveness to most agents targeting cancer specific survival pathways are still poorly understood but critical for their clinical application. In this study, we found that sunitinib, a small molecule inhibitor of multiple tyrosine kinases including VEGFRs and PDGFRs induces apoptosis and inhibits cell growth in colon cancer cells in cell culture and xenograft models via the BH3-only protein PUMA. Sunitinib treatment induced PUMA transcription via the AKT/FoxO3a axis. PUMA, BH3 mimetics, or 5-Flurourical sensitized colon cancer cells to sunitinib-induced apoptosis. Furthermore, PUMA was induced by sunitinib treatment in xenograft tumors, and deficiency in PUMA significantly suppressed the anti-tumor effects of sunitinib. Our study suggests that PUMA-mediated apoptosis is important for the therapeutic responses to sunitinib, and activation of the mitochondrial pathway by BH3 mimetics or PUMA manipulation may be useful for improving the antitumor activity of sunitinib. Modulation of PUMA and selective Bcl-2 family members might be potential biomarkers for predicting sunitinib responses.

The TNF family member APRIL promotes colorectal tumorigenesis

The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in Apc(Min) mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis.