CX-1 Cells Research Articles

Salinomycin and Sulforaphane Exerted Synergistic Antiproliferative and Proapoptotic Effects on Colorectal Cancer Cells by Inhibiting the PI3K/Akt Signaling Pathway in vitro and in vivo.

BackgroundBoth salinomycin (SAL) and sulforaphane (SFN) exert their antitumorigenic effects in various types of cancer We investigated whether combining salinomycin (SAL, an antibiotic ionophore) with sulforaphane (SFN, a phytochemical) exerted synergistic antiproliferative and proapoptotic activities in colorectal cancer (CRC) cells in vitro and in vivo by evaluating the proliferative and apoptotic responses of two CRC cell lines.Materials and MethodsThe combination index (CI) was calculated using the Chou-Talalay method, and the effects of the synergistic combination (CI<1) of lower doses of SAL and SFN were selected for further studies. Anti-tumor effect of the combination of SAL and SFN was tested both in vitro and in vivo.ResultsCotreatment effectively inhibited proliferation, migration and invasion and enhanced apoptosis. The xenograft model also showed similar results. Furthermore, we evaluated the molecular mechanism behind SAL- and SFN-mediated CRC cell apoptosis. The combination treatment induced apoptosis in Caco-2 and CX-1 cells by inhibiting the PI3K/Akt pathway, which increased the expression of the tumor suppressor protein p53. The treatment also decreased the expression of the survival protein Bcl-2 and increased the expression of the proapoptotic protein Bax, which increased the Bax/Bcl-2 ratio, as well as enhanced poly ADP-ribose polymerase (PARP) cleavage. Upon inhibiting the PI3K/Akt pathway with LY294002 prior to cotreatment, we detected enhanced PARP cleavage compared to that in the cotreatment only group.ConclusionWe investigated whether the combination of SAL and SFN had antiproliferative and proapoptotic effects in CRC cells both in vitro and in vivo. Cotreatment also significantly decreased migration and invasion compared to that of the control and SAL or SFN monotherapies. This novel combination of SAL and SFN might provide a potential strategy to treat CRC.

MiR-7112-3p targets PERK to regulate the endoplasmic reticulum stress pathway and apoptosis induced by photodynamic therapy in colorectal cancer CX-1 cells.

Colorectal cancer (CRC) is the third most common malignant tumor worldwide. Photodynamic therapy (PDT) is an emerging modality for the treatment of solid tumors. Sinoporphyrin sodium (DVDMS) is a new photosensitizer with good therapeutic killing effects on cancer cells. Recent findings have shown that microRNAs play important roles in many biological processes. However, the functions of microRNAs in DVDMS-induced PDT remain largely unclear. Proteins involved in endoplasmic reticulum (ER) stress and apoptosis of CX-1 cells treated with DVDMS-PDT were examined by Western blotting and cell viability assays. 15 candidate miRNAs targeting RNA-dependent protein kinase-like ER kinase (PERK) were screened and verified using the TargetScan, miRWalk and miRDB databases. The downstream pathways of candidate miRNAs with high scores were studied by cell transfection, qRT-PCR, Western blotting and dual-luciferase reporter assays. The subcellular location of DVDMS was confirmed by laser confocal microscopy. DVDMS-PDT induced apoptosis via elevated ER stress and activation of the PERK/ATF4/CHOP/caspase cascade pathway in CX-1 cells. The endoplasmic reticulum was involved in the subcellular accumulation of DVDMS in CX-1 cells. Dual-luciferase reporting experiment confirmed that a direct crosslinking between miR-7112-3p and PERK. In addition, miR-7112-3p was highly expressed in CRC tissues compared with peripheral tissues. Our work showed that miR-7112-3p directly targeted PERK and further regulated PERK/ATF4/CHOP/caspase cascade pathway, resulting in enhanced apoptosis in CX-1 cells treated with DVDMS-PDT.

Ferroptosis-inducing agents enhance TRAIL-induced apoptosis through upregulation of death receptor 5.

Ferroptosis is considered genetically and biochemically distinct from other forms of cell death. In this study, we examined whether ferroptosis shares cell death pathways with other types of cell death. When human colon cancer HCT116, CX-1, and LS174T cells were treated with ferroptotic agents such as sorafenib (SRF), erastin, and artesunate, data from immunoblot assay showed that ferroptotic agents induced endoplasmic reticulum (ER) stress and the ER stress response-mediated expression of death receptor 5 (DR5), but not death receptor 4. An increase in the level of DR5, which is activated by binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiates apoptosis, was probably responsible for synergistic apoptosis when cells were treated with ferroptotic agent in combination with TRAIL. This collateral effect was suppressed in C/EBP (CCAAT-enhancer-binding protein)-homologous protein (CHOP)-deficient mouse embryonic fibroblasts or DR5 knockdown HCT116 cells, but not in p53-deficient HCT116 cells. The results from in vitro studies suggest the involvement of the p53-independent CHOP/DR5 axis in the synergistic apoptosis during the combinatorial treatment of ferroptotic agent and TRAIL. The synergistic apoptosis and regression of tumor growth were also observed in xenograft tumors when SRF and TRAIL were administered to tumor-bearing mice.

Molecular features of doxorubicin-resistance development in colorectal cancer CX-1 cell line

Background and aimResistance to chemotherapy is the key obstacle to the effective treatment of various cancers. Accumulating evidence suggests significant involvement of the epithelial-to-mesenchymal transition (EMT) in the chemoresistance of most cancer types. This study aimed at analyzing the gene expression profile of doxorubicin (DOX)-resistant colorectal cancer cells CX-1. Materials and methodsDOX-resistant CX-1 cell sublines were acquired by stepwise increment of DOX concentrations in cell growth media. Global gene expression profiling was performed using human gene expression microarrays. The expression levels of individual genes were assessed by means of quantitative PCR (qPCR), while the DNA methylation pattern of several selected genes was determined by methylation-specific PCR. ResultsFour DOX-resistant CX-1 sublines were established as a valuable tool for cell chemoresistance studies. Altered expression of the EMT, cell adhesion and motility, and chemoresistance-related genes was observed in DOX-resistant cells by genome-wide gene expression analysis. Besides, early and significant upregulation of the key EMT genes ZEB1 (5.8×; P<0.001) and CDH2 (6.2×; P=0.044) was identified by qPCR, with subsequent activation of drug transporter gene ABCC1 (3.3×; P=0.007) and cell stemness gene NANOG (2.4×; P=0.008). Downregulation of TET1 (2.1×; P=0.041) and changes in the methylation status of the p16 gene were also involved in the acquisition of cell resistance to DOX. ConclusionThe results of our study suggest possible involvement of the key EMT and drug transporter genes in the early phase of cancer cell chemoresistance development.

Role of AMP-activated protein kinase in cross-talk between apoptosis and autophagy in human colon cancer.

Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and apoptosis.

Role of Bcl-xL/Beclin-1 in synergistic apoptotic effects of secretory TRAIL-armed adenovirus in combination with mitomycin C and hyperthermia on colon cancer cells.

In this study, we attempted to develop a multimodality approach using chemotherapeutic agent mitomycin C, biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L), and mild hyperthermia to treat colon cancer. For this study, human colon cancer LS174T, LS180, HCT116 and CX-1 cells were infected with secretory TRAIL-armed adenovirus (Ad.TRAIL) and treated with chemotherapeutic agent mitomycin C and hyperthermia. The combinatorial treatment caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, dissociation of Bak from Bcl-xL, oligomerization of Bak, alteration of mitochondrial membrane potential, and subsequent cytochrome c release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed the synergistic death effect. Interestingly, Beclin-1 was dissociated from Bcl-xL and overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed dissociation of Beclin-1 from Bcl-xL. A combinatorial treatment of mitomycin C, Ad.TRAIL and hyperthermia induced Beclin-1 cleavage, but the Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells, suppressing the apoptosis induced by the combination therapy. We believe that this study supports the application of the multimodality approach to colon cancer therapy.

Role of Bcl-xL/Beclin-1 in interplay between apoptosis and autophagy in oxaliplatin and bortezomib-induced cell death

Recent studies indicate that a complex relationship exists between autophagy and apoptosis. In this study we investigated a regulatory relationship between autophagy and apoptosis in colorectal cancer cells utilizing molecular and biochemical approaches. For this study, human colorectal carcinoma HCT116 and CX-1 cells were treated with two chemotherapeutic agents—oxaliplatin, which induces apoptosis, and bortezomib, which triggers both apoptosis and autophagy. A combinatorial treatment of oxaliplatin and bortezomib caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment of oxaliplatin and bortezomib promoted the JNK-Bcl-xL-Bax pathway which modulated the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, oligomerization of Bax, alteration of mitochondrial membrane potential, and subsequent cytochrome c release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed cytochrome c release and synergistic death effect. Interestingly, Bcl-xL also affected autophagy through alteration of interaction with Beclin-1. Beclin-1 was dissociated from Bcl-xL and initiated autophagy during treatment with oxaliplatin and bortezomib. However, activated caspase 8 cleaved Beclin-1 and suppressed Beclin-1-associated autophagy and enhanced apoptosis. A combinatorial treatment of oxaliplatin and bortezomib-induced Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133AA/D149A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. In addition, the combinatorial treatment significantly inhibited colorectal cancer xenografts’ tumor growth. An understanding of the molecular mechanisms of crosstalk between apoptosis and autophagy will support the application of combinatorial treatment to colorectal cancer.

Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells

Colorectal cancer is the third leading cause of cancer-related mortality in the world; the main cause of death of colorectal cancer is hepatic metastases, which can be treated with hyperthermia using isolated hepatic perfusion (IHP). In this study, we report that mild hyperthermia potently reduced cellular FLIP(long), (c-FLIPL), a major regulator of the death receptor (DR) pathway of apoptosis, thereby enhancing humanized anti-DR4 antibody mapatumumab (Mapa)-mediated mitochondria-independent apoptosis. We observed that overexpression of c-FLIPL in CX-1 cells abrogated the synergistic effect of Mapa and hyperthermia, whereas silencing of c-FLIP in CX-1 cells enhanced Mapa-induced apoptosis. Hyperthermia altered c-FLIPL protein stability without concomitant reductions in FLIP mRNA. Ubiquitination of c-FLIPL was increased by hyperthermia, and proteasome inhibitor MG132 prevented heat-induced downregulation of c-FLIPL. These results suggest the involvement of the ubiquitin-proteasome system in this process. We also found lysine residue 195 (K195) to be essential for c-FLIPL ubiquitination and proteolysis, as mutant c-FLIPL lysine 195 arginine (arginine replacing lysine) was left virtually un-ubiquitinated and was refractory to hyperthermia-triggered degradation, and thus partially blocked the synergistic effect of Mapa and hyperthermia. Our observations reveal that hyperthermia transiently reduced c-FLIPL by proteolysis linked to K195 ubiquitination, which contributed to the synergistic effect between Mapa and hyperthermia. This study supports the application of hyperthermia combined with other regimens to treat colorectal hepatic metastases.

Repeated batch fermentation with water recycling and cell separation for microbial lipid production

Large waste water disposal was the major problem in microbial lipid fermentation because of low yield of lipid. In this study, the repeated batch fermentation was investigated for reducing waste water generated in the lipid fermentation of an oleaginous yeast Trichosporon cutaneum CX1 strain. The waste fermentation broth was recycled in the next batch operation after the cells were separated using two different methods, centrifugation and flocculation. Two different sugar substrates, glucose and inulin, were applied to the proposed operation. The result showed that at least 70% of the waste water was reduced, while lipid production maintained satisfactory in the initial four cycles. Furthermore, it is suggested that T. cutaneum CX1 cells might produce certain naturally occurring inulin hydrolyzing enzyme(s) for obtaining fructose and glucose from inulin directly. Our method provided a practical option for reducing the waste water generated from microbial lipid fermentation.

Simultaneous saccharification and microbial lipid fermentation of corn stover by oleaginous yeast Trichosporon cutaneum

Simultaneous saccharification and fermentation (SSF) is the most commonly practiced operation in lignocellulose bioconversion to avoid the sugar product inhibition to cellulase enzymes. In this study, for the first time SSF was tested on microbial lipid fermentation using the diluted acid pretreated and biodetoxified corn stover. The results show that SSF was effective than the separate hydrolysis and fermentation (SHF) on lipid accumulation of Trichosporon cutaneum CX1 cells in both the small scale (5L) and the enlarged scale (50L) bioreactors. The solutions for the oxygen transfer and the lipid extraction in SSF practically worked well. The process parameters were optimized and the lipid yield obtained were 3.03g/L in the 5L, and 3.23g/L in the 50L, respectively. The result also shows that the cellulase enzyme could be partially recycled in the SSF. The study provided a practical and efficient way for microbial lipid production from lignocellulose material.

Abstract 5646: Allele-specific shRNA to Nanog family members inhibits 3-D growth of colorectal carcinoma through the intrinsic apoptotic pathway

Abstract Nanog and NanogP8 modulate stemness of human colorectal cancer (CRC) as measured by directly affecting spherogenicity, tumorigenicity and metastatic potential. As a result, we tested whether targeting NanogP8 (the dominant Nanog member expressed in human CRC lines and liver metastases) by specific lentiviral (LV) shRNA inhibits the growth of CRC in 3-D spheroids in suspension culture. We first designed allele-specific shRNAs to NanogP8 and Nanog, genes that differ only in 5 nucleotides in a 915 nt ORF, and demonstrated that the shRNA to NanogP8 (shNp8-1) and Nanog (shNG-1) specifically inhibited their target in PA-1, a human embryonal carcinoma line expressing only Nanog, or CX-1, a CRC that expresses predominantly NanogP8. When Clone A or CX-1 CRC cells transfected with Luc2 were cultured for 24 hours in suspension in serum-free medium (SFM), transduced with LV shNP8-1, shNG-1 or a control shRNA (shNeg) at a multiplicity of infection (MOI) 8 and cultured for another 72 hours in SFM, all of the LV vectors transduced 40 - 75% of the cells in 3-D constructs. shNp8-1 transduction significantly reduced the mass of cells and luciferase activity by 50%. Apoptosis was induced because CRC treated with shNp8-1 had significantly more annexin V+ cells than did shNEG or untreated parental cells. Previous work in our lab demonstrated that caspase 8 was important in anoikis - the apoptosis caused by suspension of cells whose growth generally requires adherence. Caspase 3, 8 and 9 activity was measured by fluorogenic substrates in Clone A and CX-1 cells at 4 days of suspension culture, 3 days after LV treatment. Caspase 3 activity was increased by shNp8-1 by 2.5 to 3-fold in both CX-1 and Clone A compared to untreated cells. shNp8-1 treatment increased Caspase 9 activity by ∼50% in both CRC lines compared to untreated cells whereas shNEG and shNG-1 transduction did not increase Caspase 9 activity significantly. Treatment with a Caspase 9 peptidomimetic blocked apoptosis and increased CX-1 survival significantly. When cells were harvested at the end of the 4 day apoptosis assay and replated in a standard colony forming assay, CX-1 cells transduced with shNp8-1 LV produced 33% fewer colonies than did the shNEG and shNG-1 controls. These results indicate that shNp8-1 transduction may activate apoptosis through a Caspase 9-dependent pathway and that this treatment may have long term effects that inhibit tumor regrowth after treatment. Supported by Center for Cancer Research funds and Department of Defense IDEA grant W81XWH-11-1-0327. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5646. doi:1538-7445.AM2012-5646

Abstract 5206: Nanog family members are essential for spherogenicity and metastasis in colorectal carcinoma

Abstract Liver is the most common site for colorectal cancer (CRC) distant metastasis and hepatic metastasis is one of the main causes of death in CRC patients. Inhibition of liver metastases in patients with CRC may increase the survival of patients. Our postulate is that genes involved in early embryogenesis, Nanog, Oct4 and SOX2, may be reactivated and control the malignant properties of metastasis and resistance to chemo- and radiotherapy. Nanog is a family of 11 pseudogenes with the original parent gene on chromosome 12. In order to identify which gene product is expressed in human CRC, we developed a novel endonuclease restriction enzyme assay, which was used in six human CRC cell lines. Monolayer cultures of CRC cells express Nanog, NanogP8, and NanogP7 but spheroids cultured in serum-free medium only express NanogP8 which was confirmed by direct sequencing. In addition, two potential DNA methylation regions are detected in the promoters of Nanog and NanogP8 by sodium bisulfite sequencing of genomic DNA in hES and CRC CloneA cells. In hES cells, the methylation status of Nanog and NanogP8 was about 30% and 60%, respectively. In contrast, in CloneA cells, it was 65% for Nanog and about 40% for NanogP8 and lower for CX-1 CRC cells. These results suggest that NanogP8 is re-activated in CRC through promoter demethylation in CRC spheroids that are enriched for putative stem cells. Overexpression of NanogP8 in CloneA and CX-1 significantly increased the spherogenicity of single CRC cells. In addition, lentiviral shRNA to Nanog inhibited spherogenicity in vitro and tumorigenicity in vivo whereas shRNA to OCT4 and SOX2 actually increased spherogenicity and tumorigenicity. Lentiviral shRNA inhibition of Nanog prevented liver colonies after intrasplenic injection of CX-1 cells whereas 45% of NOD/SCID mice developed experimental metastases after injection of the untreated CX-1 cells or CX-1 cells treated with negative control shRNA. As a result, targeting Nanog and its family members expressed in CRC may block the progression of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5206. doi:10.1158/1538-7445.AM2011-5206

Valproate inhibits colon cancer growth through cell cycle modification in vivo and in vitro

Valproate (VPA) is a well-characterized histone deacetylase inhibitor with anti-neoplastic properties. We analyzed the growth blocking effects and the molecular mode of action of this compound in colorectal cancer cells in vitro and in vivo. Caco-2, SW-480, CX-1 or WIDR cell lines were exposed to VPA (0.25-2 mM) for various time periods. Cell growth, cell cycle progression and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide dye reduction assay and flow cytometry. Cell cycle- and apoptosis-regulating proteins and histone acetylation were assessed by Western blotting. In vivo tumor growth and regulating protein expression under VPA were investigated in a subcutaneous xenograft tumor model. VPA inhibited the growth of all cell lines with cell cycle arrest paralleled by the up-regulation of H3 and H4 acetylation. In vivo tumor growth was substantially depressed by VPA (200 mg/kg bw). Cell cycle proteins (cdk1, cdk2, cdk4, cyclin D, cyclin E, p19, p21 and p27) were differentially altered by VPA. Predominantly cdk1 was decreased and p27 was up-regulated in all models. Apoptosis-related proteins were altered in vivo with up-regulation of bax and down-regulation of bcl-2. VPA exerts anti-neoplastic activity in colorectal tumor cell lines in vitro and in vivo by altering cell cycle regulation.

ChemInform Abstract: Sulfonylureas: A New Class of Cancer Chemotherapeutic Agents.

This study summarizes the antitumor properties of a number of sulofenur thiophene analogs against subcutaneously implanted 6C3HED lymphosarcoma with structural modification of the aryl moiety of the sulfonamide portion of the diarylsulfonylureas. The spectrum of activity of N-(p-chlorophenyl)-N'- [(5-methoxy-2-thienyl)sulfonyl]urea in the HXGC3, VRC5, CX-1, and LX-1 cell lines is also presented.

Effect of hyperthermia in combination with TRAIL on the JNK‐Bim signal transduction pathway and growth of xenograft tumors

Approximately 25% of patients with colorectal cancer develop metastases to the liver, and surgery is currently the best treatment available. But there are several patients who are unresectable, and isolated hepatic perfusion (IHP) offers a different approach in helping to treat these patients. IHP is a method used for isolating the liver and delivering high doses of chemotherapeutic agents. The efficacy of IHP has been improved by combining hyperthermia not only with chemotherapeutics but with other deliverable agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we used human colorectal cancer CX-1 cells and treated them with hyperthermia and TRAIL, causing cytotoxicity. We were able to demonstrate that the numbers of live cells were significantly reduced with hyperthermia and 10 ng/ml of TRAIL combined. We also showed that the effect of hyperthermia on TRAIL in our studies was enhancement of the apoptotic pathway by the promotion of JNK and Bim(EL) activity as well as PARP cleavage. We have also used our CX-1 cells to generate tumors in Balb/c nude mice. With intratumoral injections of TRAIL combined with hyperthermia at 42 degrees C, we were able to show a delayed onset of tumor growth in our xenograft model.

Abstract 4248: NANOG depletion decreases cell proliferation rate in colorectal carcinoma cells and tumor growth in nude mice

Abstract Nanog is a critical transcription factor involved in maintaining embryonic stem cells in a pluripotent, dedifferentiated state and expression of NANOG or its nearly identical retrogene NANOGP8 is increased in human gastric, hepatocellular, and colon cancer. Our postulate is that NANOG/NANOGP8 is critical for the functions of putative CSC in colorectal carcinoma (CRC): self-renewal, pluripotency, spherogenicity, tumorigenicity and resistance to chemotherapy. Five human CRC lines are being used in this study (HCC 2998, KM-12c, Clone A, MIP-101, CX-1). CX-1 cells are more aggressive than the other CRC because they form more spheroids from single cells and are highly metastatic after intrasplenic compared to the other CRC that are tumorigenic but weakly metastatic. qRT-PCR analysis of spheroids of CX-1 and Clone A cultured in suspension in serum free medium indicated that NANOG transcript levels were increased by 43- and 12-fold, respectively, relative to monolayer cultures while the relative increase in transcript levels of several cancer stem cell markers such as EPCAM, ALDH1A1, ALDH3A1, CD44, CD133, and CD166 was less than 5-fold except for POU5F1 and SOX2 in Clone A, which were 5- and10-fold respectively. NANOG promoter activity was low in monolayer culture in CRC stable transfectants with a GFP NANOG promoter, but consistently increased as CRC detached and formed spheroids in serum free media. Stable transfection with lentiviral NANOG shRNA decreased NANOG transcript levels and spherogenicity of Clone A and CX1 cells compared to vector controls in a single cell spherogenicity assay in serum-free medium. NANOG downregulation also induced a S phase cell cycle arrest with inhibition of ATM and WEE1 expression with associated reduction in phosphorylation of other key cell-cycle checkpoint proteins. Finally, marked tumor growth retardation was observed in NOD/SCID mice injected subcutaneously with NANOG shRNA transfected cells compared to parental or vector controls. NANOG shRNA transfected cells were also more resistant to treatment with Topotecan, a camptothecin. In summary, our data suggest that expression of NANOG/NANOGP8 occurs in human CRC and may be critical for such functions of putative CSC as spherogenicity, cell proliferation, tumor formation and resistance to chemotherapy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4248.

Abstract 4249: Shape change induces nitric oxide and stemness in colorectal cancer cells

Abstract Shape changes in cancer cells profoundly alter the expression of genes that affect malignant properties. We postulated that shape change may increase expression of NANOG, an embryonic stem cell transcription factor expressed in human colorectal carcinoma (CRC) that regulates pluripotency and proliferation. Since shape change also increases intracellular nitric oxide (iNO) expression, we further postulated that iNO modulates NANOG expression. Clone A and CX-1 human CRC lines were cultured in serum-containing medium in monolayer cultures or in spheroids in serum free medium in low attachment plates. NANOG promoter activity was assessed in stable transfectants expressing promoter-driven GFP. iNO concentrations were determined by fluorescence cytometry with DAF-FM diacetate while Nanog gene and protein expression was analyzed by qRT-PCR and densitometry of western blots, respectively. Spherogenicity was measured by the percentage of spheroids formed from single cells in serum-free medium. Monolayer cultures with flat, spreading cells had low NANOG gene and protein expression, promoter activity and iNO whereas spheroids displayed high NANOG gene expression, promoter activity and iNO. 7 day monolayer cultures initiated with 104 cells/ml (Sparse) grew horizontally compared to cultures initiated with 106 cells/ml (Dense) that grew vertically and formed colonies. Sparse cultures had low NANOG gene expression, low NANOG promoter activity and low iNO. Dense cultures had high NANOG gene expression, NANOG promoter activity and iNO. Since NO stimulates cGMP production, the effects of the NO synthase inhibitor L-NMMA, NOC (a NO donor), 8-Br-cGMP (cGMP agonist) and ODQ (cGMP inhibitor) were tested on CRC monolayers. Physiologic concentrations of NOC increased NANOG and Oct4 gene and protein expression 3-fold in highly metastatic CX-1 cells but not in weakly metastatic Clone A cells. NOC also increased CD44 in CX-1 but not Clone A. cGMP may regulate NANOG expression since 8-Br-cGMP increased NANOG and Oct4 protein expression up to 3-fold in CX-1 but not in Clone A and without affecting CD44 expression. ODQ inhibited NANOG expression. L-NMMA stimulated both NANOG protein expression and the spherogenicity of CX-1 but not Clone A. NOC and 8-Br-cGMP increased inducible NO synthase (NOS2) but not NOS3 gene expression 3-fold in both spheroids and monolayers while ODQ decreased NOS2 levels. Assays are in progress to test the effects of NOC and the cGMP modulators on spherogenicity. Shape change, iNO and cGMP enhance NANOG, NOS2 and CD44 expression to alter CRC cancer stem cell function. cGMP may be an important regulator of stemness genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4249.

Effect of UV irradiation on colorectal cancer cells with acquired TRAIL resistance

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. TRAIL shows strong cytotoxicity to many cancer cells but minimal cytotoxicity to most normal cells. Interestingly, our recent studies have demonstrated that pretreatment with TRAIL induces acquired resistance to TRAIL (Song et al. 2007 J Biol Chem 282: 319). Acquired TRAIL resistance develops within 1 day and gradually decays within 5 days after TRAIL treatment. In our current study, we examined whether human colorectal carcinoma CX-1 cells with acquired TRAIL resistance are resistant to UV irradiation as well. CX-1 cells were treated with 200 ng/ml TRAIL for 6 h and incubated various times (0.25-5 days) and then challenged to UV irradiation. Unexpectedly, we observed an increase in apoptosis in acquired TRAIL resistant cells after UVC as well as UVB exposure. This was due to an increase in caspase activation which was mediated through cytochrome c release. These results suggest that cells with acquired TRAIL resistance are sensitive to UV irradiation.

Effect of hyperthermia and chemotherapeutic agents on TRAIL‐induced cell death in human colon cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL-induced apoptosis. We observed in human colorectal cancer cell line CX-1 that TRAIL-induced apoptotic death and also that mild hyperthermia promoted TRAIL-induced apoptotic death through caspase activation and cytochrome-c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX-1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP-1 and several caspases and antiapoptotic proteins were performed. Activities of casapse-8, caspase-9, and caspase-3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non-apoptotic, more efficiently. We demonstrate here that CX-1 cells exposed to 42 degrees C and chemotherapeutic agents were sensitized and died by apoptotic and non-apoptotic cell death even in low concentration (10 ng/ml) of TRAIL.

The Host Inflammatory Response Promotes Liver Metastasis by Increasing Tumor Cell Arrest and Extravasation

Inflammation can play a regulatory role in cancer progression and metastasis. Previously, we have shown that metastatic tumor cells entering the liver trigger a proinflammatory response involving Kupffer cell-mediated release of tumor necrosis factor-alpha and the up-regulation of vascular endothelial cell adhesion receptors, such as E-selectin. Here, we analyzed spatio-temporal aspects of the ensuing tumor-endothelial cell interaction using human colorectal carcinoma CX-1 and murine carcinoma H-59 cells and a combination of immunohistochemistry, confocal microscopy, and three-dimensional reconstruction. E-selectin expression was evident mainly on sinusoidal vessels by 6 and 10 hours, respectively, following H-59 and CX-1 inoculation, and this corresponded to a stabilization of the number of tumor cells within the sinuses. Tumor cells arrested in E-selectin(+) vessels and appeared to flatten and traverse the vessel lining, away from sites of intense E-selectin staining. This process was evident by 8 (H-59) and 12 (CX-1) hours after inoculation, coincided with increased endothelial vascular cell adhesion molecule-1 expression, and involved tumor cell attachment in areas of intense vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 expression. Nonmetastatic (human) MIP-101 and (murine) M-27 cells induced a weaker response and could not be seen to extravasate. The results show that metastatic tumor cells can alter the hepatic microvasculature and use newly expressed endothelial cell receptors to arrest and extravasate.