Metastatic Human Colon Cancer Cell Research Articles

Anti-Cancer Mechanisms of Diarylpentanoid MS17 (1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3-one) in Human Colon Cancer Cells: A Proteomics Approach.

Diarylpentanoids are synthesized to overcome curcumin's poor bioavailability and low stability to show enhanced anti-cancer effects. Little is known about the anti-cancer effects of diarylpentanoid MS17 (1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one) in colon cancer cells. This study aimed to elucidate molecular mechanisms and pathways modulated by MS17 in colon cancer based on proteomic profiling of primary SW480 and metastatic SW620 colon cancer cells. Cytotoxicity and apoptotic effects of MS17 were investigated using MTT assay, morphological studies, and Simple Western analysis. Proteomic profiling using LC/MS analysis identified differentially expressed proteins (DEPs) in MS17-treated cells, with further analysis in protein classification, gene ontology enrichment, protein-protein interaction network and Reactome pathway analysis. MS17 had lower EC50 values (SW480: 4.10 µM; SW620: 2.50 µM) than curcumin (SW480: 17.50 µM; SW620: 13.10 µM) with a greater anti-proliferative effect. MS17 treatment of 1× EC50 induced apoptotic changes in the morphology of SW480 and SW620 cells upon 24 h treatment. A total of 24 and 92 DEPs (fold change ≥ 1.50) were identified in SW480 and SW620 cells, respectively, upon MS17 treatment of 2× EC50 for 24 h. Pathway analysis showed that MS17 may induce its anti-cancer effects in both cells via selected DEPs associated with the top enriched molecular pathways. RPL and RPS ribosomal proteins, heat shock proteins (HSPs) and ubiquitin-protein ligases (UBB and UBC) were significantly associated with cellular responses to stress in SW480 and SW620 cells. Our findings suggest that MS17 may facilitate the anti-proliferative and apoptotic activities in primary (SW480) and metastatic (SW620) human colon cancer cells via the cellular responses to stress pathway. Further investigation is essential to determine the alternative apoptotic mechanisms of MS17 that are independent of caspase-3 activity and Bcl-2 protein expression in these cells. MS17 could be a potential anti-cancer agent in primary and metastatic colon cancer cells.

Sulforaphane has an additive anticancer effect to FOLFOX in highly metastatic human colon carcinoma cells.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Patients with CRC may need chemotherapy (CTx) in a neoadjuvant, adjuvant or palliative setting through the course of the disease. Unfortunately, its effect is limited by chemoresistance and chemotoxicity. Novel more effective and non‑toxic CTx regimens are needed to further improve CRC treatment outcomes. Thus, the present study was designed to test the hypothesis that non‑toxic sulforaphane (SF) is effective against CRC and has additive effects in combination with conventional 5‑fluorouracil, oxaliplatin and folinic acid (FOLFOX) CTx invitro. Highly metastatic human colon cancer cells, CX‑1, and fibroblasts were treated with FOLFOX ± SF. Cell viability was assessed using an MTT assay. The level of apoptosis and the expression of apoptotic proteins were measured by TUNEL assay and quantitative PCR analysis. Aldehyde dehydrogenase isoform 1 (ALDH1) and multidrug resistance protein 2 (MRP2) levels were evaluated. The ability of cells to form spheroids was measured in three‑dimensional cell culture. SF alone and in combination with FOLFOX effectively decreased the viability of the CX‑1 cells, promoted apoptosis within the CX‑1 cells, prevented cellular spheroid formation and decreased ALDH1 activity. However, SF promoted MRP2 expression and protein levels. In conclusion, SF together with conventional FOLFOX has additive anticancer effects against highly metastatic human CRC invitro.

The Curcumin Analogue, MS13 (1,5-Bis(4-hydroxy-3- methoxyphenyl)-1,4-pentadiene-3-one), Inhibits Cell Proliferation and Induces Apoptosis in Primary and Metastatic Human Colon Cancer Cells.

The cytotoxic and apoptotic effects of turmeric (Curcuma longa) on colon cancer have been well documented but specific structural modifications of curcumin have been shown to possess greater growth-suppressive potential on colon cancer than curcumin. Therefore, the aim of this study is to identify the anti-cancer properties of curcumin analogue-MS13, a diarylpentanoid on the cytotoxicity, anti-proliferative and apoptotic activity of primary (SW480) and metastatic (SW620) human colon cancer cells. A cell viability assay showed that MS13 has greater cytotoxicity effect on SW480 (EC50: 7.5 ± 2.8 µM) and SW620 (EC50: 5.7 ± 2.4 µM) compared to curcumin (SW480, EC50: 30.6 ± 1.4 µM) and SW620, EC50: 26.8 ± 2.1 µM). Treatment with MS13 at two different doses 1X EC50 and 2X EC50 suppressed the colon cancer cells growth with lower cytotoxicity against normal cells. A greater anti-proliferative effect was also observed in MS13 treated colon cancer cells compared to curcumin at 48 and 72 h. Subsequent analysis on the induction of apoptosis showed that MS13 treated cells exhibited morphological features associated with apoptosis. The findings are also consistent with cellular apoptotic activities shown by increased caspase-3 activity and decreased Bcl-2 protein level in both colon cancer cell lines. In conclusion, MS13 able to suppress colon cancer cell growth by inhibiting cell proliferation and induce apoptosis in primary and metastatic human colon cancer cells.

Anticancer Activities of Meroterpenoids Isolated from the Brown Alga Cystoseira usneoides against the Human Colon Cancer Cells HT-29.

Colorectal cancer (CRC) is one of the most common types of cancers and a leading cause of cancer death worldwide. The current treatment for CRC mainly involves surgery, radiotherapy, and chemotherapy. However, due to the side effects and the emergence of drug resistance, the search for new anticancer agents, pharmacologically safe and effective, is needed. In the present study, we have investigated the anticancer effects of eight algal meroterpenoids (AMTs, 1-8) isolated from the brown seaweed Cystoseira usneoides and their underlying mechanisms of action using HT-29, a highly metastatic human colon cancer cell line. All the tested meroterpenoids inhibited the growth of HT-29 malignant cells and were less toxic towards non-cancer colon cells, with the AMTs 1 and 5 exhibiting selectivity indexes of 5.26 and 5.23, respectively. Treatment of HT-29 cells with the AMTs 1, 2, 3, 4, 5, and 7 induced cell cycle arrest in G2/M phase and, in some instances, apoptosis (compounds 2, 3, and 5). Compounds 1-8 also exhibited significant inhibitory effects on the migration and/or invasion of colon cancer cells. Mechanistic analysis demonstrated that the AMTs 1, 2, 5, 6, 7, and 8 reduced phosphorylation levels of extracellular signal-regulated kinase (ERK) and the AMTs 2, 3, 4, 5, 7, and 8 decreased phosphorylation of c-JUN N-terminal kinase (JNK). Moreover, the AMTs 1, 2, 3, 4, 7, and 8 inhibited phosphorylation levels of protein kinase B (AKT) in colon carcinoma cells. These results provide new insights into the mechanisms and functions of the meroterpenoids of C. usneoides, which exhibit an anticancer effect on HT-29 colon cancer cells by inducing cell cycle arrest and apoptosis via the downregulation of ERK/JNK/AKT signaling pathways.

Effects of α-lactalbumin and sulindac on primary and metastatic human colon cancer cell lines

Abstract Background HAMLET (human α-lactalbumin made lethal to tumor cells) and sulindac are active ingredients which are used in the treatment of cancers. Under both in vivo and in vitro conditions, HAMLET binds to the surface of tumor cells and enters the cells. Sulindac is one of nonsteroidal anti-inflammatory drugs. It inhibits the growth of tumor cells by inducing the apoptosis. In our study, we aimed to investigate effects of α-lactalbumin and sulindac on COLO-320 primary and COLO-741 metastatic human colon carcinoma cell lines via mitochondrial apoptotic pathway by indirect immunocytochemistry and TUNEL assays. Materials and methods The effects of α-lactalbumin and sulindac were assessed by using MTT assay at 24, 48 and 72 h and apoptosis markers caspase-3, caspase-9 and cytochrome-c were detected using immunocytochemistry and TUNEL methods. Results It was appeared that α-lactalbumin and sulindac may triggger mechanisms of apoptosis in both primary and metastatic colon carcinoma cell lines and the primary colon carcinoma cell line was affected more than the metastatic cells. Conclusion It is proposed that α-lactalbumin and sulindac can be used in cancer treatments and future in vivo experiments.

Isorhamnetin glycoside isolated from Opuntia ficus-indica (L.) MilI induces apoptosis in human colon cancer cells through mitochondrial damage

This work aimed to evaluate the mechanisms involved in the apoptosis induction of isorhamnetin-3-O-glucosyl-pentoside (IGP) in metastatic human colon cancer cells (HT-29). To achieve this, we assessed phosphatidylserine (PS) exposure, cell membrane disruption, chromatin condensation, cell cycle alterations, mitochondrial damage, ROS production, and caspase-dependence on cell death. Our results showed that IGP induced cell death on HT-29 cells through PS exposure (48%) and membrane permeabilization (30%) as well as nuclear condensation (54%) compared with control cells. Moreover, IGP treatment induced cell cycle arrest in G2/M phase. Bax/Bcl-2 ratio increased and the loss of mitochondrial membrane potential (63%) was observed in IGP-treated cells. Finally, as apoptosis is a caspase-dependent cell death mechanism, we used a pancaspase-inhibitor (Q-VD-OPh) to demonstrate that the cell death induced by IGP was caspase-dependent. Overall these results indicated that IGP induced apoptosis through caspase-dependent mitochondrial damage in HT-29 colon cancer cells.

Cytotoxic, Antiproliferative and Apoptosis-inducing Activity of L-Amino Acid Oxidase from Malaysian Calloselasma rhodostoma on Human Colon Cancer Cells.

The aim of this study was to investigate the cytotoxic, antiproliferative activity and the induction of apoptosis by L-amino acid oxidase isolated from Calloselasma rhodostoma crude venom (CR-LAAO) on human colon cancer cells. CR-LAAO was purified using three chromatographic steps: molecular exclusion using G-50 gel filtration resin, ion-exchange by MonoQ column and desalted on a G25 column. The purity and identity of the isolated CR-LAAO was confirmed by SDS-PAGE and LC-MS/MS. CR-LAAO demonstrated time- and dose-dependent cytotoxic activity on SW480 (primary human colon cancer cells) and SW620 (metastatic human colon cancer cells) with an EC50 values of 6 μg/ml and 7 μg/ml at 48 hr, respectively. Quantification of apoptotic cells based on morphological features demonstrated significant increase in apoptotic cell population in both SW480 and SW620 cells which peaked at 48 hr. Significant increase in caspase-3 activity and reduction in Bcl-2 levels were demonstrated following CR-LAAO treatment. These data provide evidence on the potential anticancer activity of CR-LAAO from the venom of C. rhodostoma for therapeutic intervention of human colon cancer.

Cistanche tubulosa induces reactive oxygen species-mediated apoptosis of primary and metastatic human colon cancer cells

Colon cancer is the third most common cancer worldwide. Conventional therapies have shown moderate efficacy with severe adverse effects, therefore there is an urgent need for safer alternatives. In this study, Cistanche tubulosa, local name Thanoon, was considered as a potential phytotherapeutic strategy because of its known high therapeutic potential in traditional medicine and wide abundance in the Middle East region. Bioactive compounds were extracted from powdered C. tubulosa and tested for their anticancer properties against four colon cancer cell lines including two derived from primary tumor (CaCo2 and HCT116) and two derived from metastatic site (LoVo and SW620). Effect of C. tubulosa on induction of apoptosis and cellular redox homeostasis were also investigated. C. tubulosa exhibited a concentration and time-dependent inhibition of proliferation of all tested cancer cell lines by more than 60% upon 72 hours treatment with 1 mg/mL of crude extract. Inhibition of proliferation was marked by induction of apoptosis, intracellular reactive oxygen species production and mitochondrial superoxides. This data suggest that C. tubulosa is a promising candidate for additive anti-colon cancer therapy. This is the first study showing anticancer bioactivity of C. Tubulosa against colon cancer cells.

Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression

BackgroundCeramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis.MethodsThe activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models.ResultsExposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model.ConclusionWe have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.

Restoration of PTEN activity decreases metastases in an orthotopic model of colon cancer

BackgroundMutational loss of tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) is associated with malignant progression in many cancers, including colorectal cancer (CRC). PTEN is involved in negatively regulating the phosphatidylinositol 3-kinase/AKT oncogenic signaling pathway and has been implicated in the metastatic colonization process. Few in vivo models are available to study CRC metastasis. The purpose of this study was to determine the effect of restoring PTEN activity on metastases in an orthotopic murine model. MethodsGreen fluorescent protein labeled TENN, a highly metastatic human colon cancer cell line with mutational loss of PTEN gene and TENN clones (with restoration of PTEN gene) tumors were orthotopically implanted onto the colons of BALB/c nude mice and allowed to develop primary and metastatic tumors. Seven weeks post-implantation, mice were euthanized and organs extracted for examination. ResultsBoth TENN and TENN clone cell lines demonstrated 100% primary invasion. However, compared with the parental TENN cells, which demonstrated 62% metastases to both lungs and liver, TENN clone cells showed an approximately 50% reduction in metastasis, with only 31.6% liver metastasis and no metastasis to the lungs (P = 0.02). ConclusionsOur study shows that reactivation of PTEN tumor suppressor pathway leads to a 50% reduction in CRC metastasis without affecting primary tumor formation. Importantly, PTEN restoration also changed the organotropic homing from liver and lung metastasis to liver metastasis only. This in vivo study demonstrates that PTEN might act specifically as a metastasis suppressor and, thus, efforts to target the phosphatidylinositol 3-kinase/PTEN pathway are legitimate.

Genistein reduced colon cancer metastasis to liver through regulating the expression of metastasis‐related genes in xenographed mice model

This study aimed at investigating the effect of genistein (Gen) on liver metastases derived from colon cancer. Male athymic nude mice were fed with western diet (W; calories%, fat 39%, carb 50%), W diet supplemented with 100ppm Gen (GL) or 500ppm Gen (GH). After 16 weeks, all the animals were intrasplenically injected with metastatic human colon cancer cells SW620 and sacrificed 8 weeks later. GL and GH mice showed lower liver metastases (36% and 17%) compared to W group (55% of mice with metastases). Mean size of metastases in the W group (4.7 mm2) was larger than that in GL (2.1 mm2) and GH (2.0 mm2) groups. To understand the molecular mechanism of the anti‐metastatic function of Gen, the expression of multiple metastasis‐related genes were determined by real‐time PCR. mRNA of NDRG1 gene in livers was greatly decreased by 50% and 60% in GL (p=0.005) and GH group (p=0.001) compared to W group. As NDRG1 has been reported as a biomarker for metastasis and poor prognosis in hepatocellular carcinoma, the down‐regulation of NDRG1 by Gen supplementation was corresponding to decreased liver metastasis. In summary, our study indicated that dietary supplementation of Gen suppressed colon cancer metastasis, which is associated with altered expression of metastasis‐related genes by the treatment.Grant Funding Source: university of illinois

Novel signaling role of G protein‐coupled receptor kinase‐5 in human colon cancer cell line

In this study we investigated the role and mechanisms by which G protein coupled receptor kinase‐5, a serine/threonine kinase, modulates proliferation and chemokine gene expression in human colon cancer cells in the absence and presence of Toll‐like receptor‐4 stimulation. For this, we used lentiviral based RNAi against GRK5 to knockdown GRK5 in metastatic human colon cancer cell line SW620 and examined the effects on proliferation, chemokine gene expression, and signaling in the absence and presence of lipopolysaccharide (LPS), a Toll‐like receptor‐4 ligand. Knockdown of GRK5 in SW620 cells enhanced proliferation compared to the control cells under unstimulated conditions. Compared to this, LPS treatment had very little effect on proliferation in control cells, whereas proliferation was significantly inhibited in GRK5 knockdown cells. Even though GRK5 knockdown did not affect basal mRNA expression of either CXCL8 or MIP2α, LPS‐induced CXCL8 and MIP2α were markedly inhibited in the GRK5 knockdown cells. Additionally our signaling data provide evidence that GRK5 regulates NFκB and MAPK signalling differentially under basal and TLR4 stimulated conditions. Taken together, our results provide evidence that GRK5 regulates proliferation and chemokine gene expression in the absence and presence of TLR4 stimulation via distinct signaling mechanisms in human colon cancer epithelial cells.

Silencing of Wnt5a during colon cancer metastasis involves histone modifications

Colorectal cancer (CRC) is the third most common cancer in the United States. Approximately 90% of colon cancer deaths arise from the metastasis of primary tumors. Aberrant expression of Wnt5a, one of the WNT signaling factors, has been reported during colon cancer development and progression. We found that both mRNA and protein expression of Wnt5a were decreased in the highly metastatic human colon cancer cell line SW620 compared with the non-metastatic human colon cancer cell SW480. This study tested the hypothesis that the silencing of Wnt5a in metastatic human colon cancer cells is related to altered epigenetic modifications. Wnt5a expression was not responsive to DNA methyltransferase inhibitor 5-aza-cytidine treatment. However, histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate (NaBt) significantly increased Wnt5a mRNA expression in SW620. Importantly, lower transcription of Wnt5a in SW620 than SW480 corresponded to multiple histone modifications, including lower levels of acetylated histone H3, H4 and H3K4me2 and higher levels of H3K27me3 in the promoter region. The increase of H3Ac, H4Ac and H3K4me2 after NaBt treatment in SW620 confirmed the involvement of histone modifications in the transcriptional regulation of Wnt5a. Additionally, NaBt treatment increased β-catenin signaling and diminished the difference in cell adhesion ability between non-metastatic SW480 and metastatic SW620, suggesting that the HDAC inhibitor plays critical roles in the WNT signaling pathway and cell physiology that relate to metastasis. In conclusion, our study suggests the importance of Wnt5a in colon cancer metastasis and also indicates that Wnt5a silencing in the highly invasive human colon cancer cell line might result from transcriptional regulation of the gene by histone modifications.

Genistein represses invasion in an in vitro colon cancer metastasis model by transcriptionally upregulating metastasis repressor NDRG1

Cancer metastasis accounts for most cancer deaths. Genistein, a biologically active soy isoflavone, has been indicated as an inhibitor of cancer progression. In this study, we tested our hypothesis that genistein can repress colon cancer metastasis in vitro by modifying the expression of metastasis suppressor NDRG1. Cell models SW480 and SW620 which are poorly and highly metastatic human colon cancer cell lines respectively, were used. Genistein treatment reduced the proliferation and wound healing ability of both cell lines. Interestingly, genistein specifically increased the adhesion of the metastatic cell line SW620 and inhibited the migration of SW620. Moreover, genistein increased the mRNA expression of NDRG1 in both cell lines. The low NDRG1 gene expression in SW620 compared to that in SW480 is associated with altered chromatin configuration of the gene. In conclusion, genistein treatment increased expression of several genes responsible for metastasis repression in SW620. Future research will focus on illustrating the epigenetic mechanisms underlining the metastasis inhibiting effects by genistein.Grant Funding Source : University of Illinois at Urbana‐Champaign

High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde

BackgroundTumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL.ResultsPC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.ConclusionThis study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and combination of TRAIL therapy with agent that inhibits the DNA-PKcs/Akt signaling pathway might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.

Abstract 4543: Targeting the NADPH binding site of dihydrofolate reductase for cancer therapy

Abstract Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate into tetrahydrofolate using NADPH as the cofactor. Folate-dependent one carbon metabolism is essential for the de novo synthesis of purines, pyrimidines and certain amino acids. Thus, DHFR plays an essential role in production and maintaining reduced folates for cellular proliferation, and consequently it has been a critical target for chemotherapy. Methotrexate (MTX), an antifolate that targets DHFR, has been used for decades in the clinic. However, both intrinsic and acquired resistance occurs due to decreased uptake, gene amplification and protein mutation. We have initiated studies to find an inhibitor that targets the cofactor NADPH binding site that would bypass these resistance mechanisms to MTX in contrast to analogs that target the dihydrofolate binding site. Fourteen NAD/NADP analogues were screened by the MTT assay in Chinese hamster ovary (CHO) cells which lack endogenous DHFR expression but were stably transfected with a DHFR-EGFP fusion construct driven by a CMV promoter (DG44 DHFR-EGFP). Two compounds, thionicotinamide adenine dinucleotide (NAD-S) and thionicotinamide adenine dinucleotide phosphate (NADP-S), had IC50 values of 1 μM. NAD-S and NADP-S were also found to be potent inhibitors of two other cancer cell lines, C85 (metastatic human colon cancer cell line) and CCRF-CEM (human T-cell lymphoblastic leukemia cell line). Surprisingly, both NAD-S and NADP-S decreased the levels of DHFR in CEM-CCRF cells. Pulse-chase experiments showed that the half life of DHFR is significantly reduced upon treatment of cells with 10 μM NADP-S. Furthermore, this NADP analog caused an increase in cells at the G1 phase (11% at 10 μM and 44% at 40 μM NADP-S treatment) that was reversible by removing NADP-S from the cell culture. Because NADP-S decreased DHFR levels, the combination of NADP-S with MTX was tested and found to significantly reduce colony formation, and was synergetic in vitro (Chou-Talalay analysis). We conclude that NADPS has a novel mechanism of action, i.e., it causes degradation of DHFR, likely by competing with NADPH binding, known to stabilize DHFR. 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 4543.

Probiotic Lactobacilli Decrease Invasion of Metastatic Human Colon Cancer Cells In Vitro

Colon cancer metastasis involves loss of intercellular junctions and cell invasion through the colonic epithelial basement membrane, a process mediated by matrixmetalloproteinases (MMP). Probiotic bacteria have been shown to improve colonic epithelial barrier function and decrease MMP activity suggesting anti‐metastatic potential. The objective of this study was to explore anti‐metastatic activity of probiotics Lactobacillus casei (L. casei) and Lactobacillus rhamnosus (L. rhamnosus) in terms of decrease in cell invasion. This was assessed by an in vitro assay, measuring the invasion of metastatic human colon cancer cell line HCT‐116 through Matrigel‐coated membranes in Boyden chambers. HCT‐116 cells were plated on top of the membrane with probiotic CFS from either L. casei or L. rhamnosus at 25% (v/v) in DMEM. Bacterial growth medium (MRS) at 25% (v/v) in DMEM was used as the control. Cell invasion was measured after 24 h of treatment; by fixing, staining and counting the cells present on the bottom side of the membrane. Treatment of HCT‐116 cells with CFS from L. casei and L. rhamnosus resulted in a significant decrease in cell invasion by 72% +/− 4.25 and 50% +/− 17.65 respectively, of control. These data indicate that probiotic CFS contain anti‐metastatic bioactive compounds that may be contributing to the decrease in cell invasion in vitro. Funding source: Texas State University Research Enhancement Program.Grant Funding Source: Texas State University Research Enhancement Program

CXCR4 Regulates the Early Extravasation of Metastatic Tumor Cells In Vivo

Recent studies have demonstrated that the chemokine receptor CXCR4 plays a crucial role in organ-specific metastasis formation. Although a variety of studies showed the expression of chemokine receptors, in particular, CXCR4, by gastrointestinal tumors, the precise mechanisms of chemokine receptor-mediated homing of cancer cells to specific sites of metastasis remained elusive. Here, we used liver metastatic human HEP-G2 hepatoma and HT-29LMM colon cancer cells expressing functional CXCR4 to dissect the metastatic cascade by intravital fluorescence microscopy. Immunohistochemistry revealed that the CXCR4 ligand CXCL12 is expressed by endothelial cells and likely Kupffer cells lining the liver sinusoids. Tumor cell adhesion and extravasation in vivo was quantitatively analyzed using intravital fluorescence microscopy. Treatment of cells with an anti-CXCR4 antibody did not affect cell adhesion but significantly impaired tumor cell extravasation (HEP-G2; isotype control: 22.3% ± 4.3% vs anti-CXCR4: 6.0% ± 5.0%, P < .001). In addition, pretreatment of tumor cells with the ligand CXCL12 enhanced the activation of the small GTPases Rho, Rac, and cdc42 as well as tumor cell extravasation without affecting tumor cell adhesion within liver sinusoids. Taken together, the findings of the present study provide first in vivo insights into the early events of chemokine ligand/receptor-mediated liver metastasis formation of tumor cells and define tumor cell extravasation rather than tumor cell arrest as the rate-limiting event.

Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rγnull (NOG) mice

To examine the drug efficacy of a novel farnesyltransferase inhibitor (FTI), CH4512600, in vivo, we developed a reliable liver metastasis model of human colon cancer using NOD/Shi-scid IL2Rgamma(null) (NOG) mice. Eleven human colon cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOG mice. When inoculated with 10(4) COLO320DM, HCT 116, HT-29, WiDr, LoVo and LS174T cells, liver metastasis was evident in 100% (6/6), 100% (6/6), 88.9% (8/9), 87.5% (7/8), 83.3% (5/6) and 50.0% (3/6) of the NOG mice, respectively. CaCo2, COLO201, LS123, SW48 and SW1417 showed no metastasis when seeded at 10(4) cells even in NOG mice. The mRNA expression levels and genetic mutations of N, H and K-RAS genes, which directly affect the levels of cellular RAS protein that would be molecular target for FTI, were also examined in these six metastatic human colon cancer cell lines for molecular biological and genotypic characteristics. Only three cell lines had a point mutation in the RAS oncogene. LS174T cell line had a point mutation of the K-RAS gene at codon 12 (gly12 --> asp; G12D), and HCT 116 and LoVo cell lines had a point mutation of the K-RAS gene at codon 13 (gly13 --> asp; G13D). Relative gene expression levels of N, H and K-RAS genes in the HCT 116 cell line were 2.6-5.0-fold lower than that of LS174T and LoVo cell lines. We selected HCT 116 cell line from our liver metastasis model for evaluation of FTI CH4512600 efficacy in vivo. Using the NOG mouse liver metastasis model, we demonstrated the effectiveness of FTI CH4512600 to suppress tumor growth in vivo and to prolong mouse survival significantly from 36.9+/-2.9 to 50.3+/-9.4 days.

Focal adhesion kinase mediates defects in the force-dependent reinforcement of initial integrin–cytoskeleton linkages in metastatic colon cancer cell lines

Micro-environmental clues, including the biophysical interpretation of the extracellular matrix, are critical to proliferation, apoptosis and migration. Here, we show that metastatic human colon cancer cell lines display altered matrix interaction. Interaction of colon cancer cells with collagen I depends on integrins (mainly α 1/ β 1) but metastatic cells display delayed spreading and reduced extension of lamellipodia. In addition, cells show defective strengthening of integrin–cytoskeleton linkages upon mechanical stimulation, as determined by laser trapping experiments and binding of large beads to the cell surface. However, adhesion to pliable surfaces is ameliorated in metastatic variants. These changes are caused by constitutive activation of focal adhesion kinase (FAK) and can be modulated by changing expression and/or activity of FAK via RNA-interference or expression of inhibitory constructs, respectively. In addition, consistent with defective strengthening of integrin–cytoskeleton linkages, metastatic cell lines show reduced random motility. Taken together these data suggest that constitutive activation of FAK causes defects in spreading, reinforcement of integrin–cytoskeleton linkages and migration and at the same time could ameliorate the adhesion of metastatic cells to suboptimal surfaces.