IL-4/Stat6 activities correlate with apoptosis and metastasis in colon cancer cells

IL-4-induced Stat6 signaling is active in a variety of cell types and plays a role in cell proliferation/growth and resistance to apoptosis. Using EMSA, we identified differential IL-4/Stat6 activities in colorectal cancer cell lines, HT-29 being active Stat6^high phenotype and Caco-2 being defective Stat6^null phenotype, respectively. Active Stat6^high HT-29 cells exhibited resistance to apoptosis by flowcytometry and aggressive metastasis by Transwell assay compared with defective Stat6^null Caco-2 cells. Comparing one another using RT-PCR, Stat6^high HT-29 cells expressed more mRNA of anti-apoptotic and pro-metastatic genes Survivin, MDM2, and TMPRSS4, while Stat6^null Caco-2 cells expressed more mRNA of pro-apoptotic and anti-metastatic genes BAX, CAV1, and P53, respectively. This is the first study describing correlations of IL-4/Stat6 activities with apoptosis and metastasis in colon cancer. These findings, together with the observation of constitutive Stat6 activation in many human malignancies, suggest that Stat6 activities could be a biomarker for cancer cell’s invasive/metastatic capability.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

Molecular signaling cascade in DNA bisintercalator, echinomycin-induced apoptosis of HT-29 cells: evidence of the apoptotic process via activation of the cytochrome c-ERK-caspase-3 pathway

The DNA-interactive drug, echinomycin, is a potent antitumor agent, which is able to induce apoptosis in a multitude of cancer cell lines. Previously, we showed that echinomycin strongly inhibited the growth of a variety of cancer cell lines, and the activation of mitogen-activated protein kinases (MAPK) in human colon cancer cells (HT-29). However, little information currently exists regarding the details of intracellular signaling pathways such as the MAPK, mitochondrial, and caspase pathways. In order to clarify this issue, we verified the plausible molecular signaling cascade by performing an immunobiochemical apoptosis experiment involving the mitochondrial and caspase pathways. The apoptotic process of HT-29 cells was accompanied by the activation of procaspase-9, -3 and cytochrome c release. Both caspase and MAPK inhibitors were used in the determination of the specific roles of MAPK and caspase in echinomycin-induced apoptosis. ERK (PD98059) or caspase-3-specific (Z-DEVD-FMK) inhibitors were discovered to significantly attenuate echinomycin-induced apoptosis. PD98059 treatment or overexpression of kinase-inactive ERK did not alter the echinomycin-induced cytochrome c release into the cytosol, but did diminish the activation of procaspase-3. Also, Z-DEVD-FMK was found to have no effect on either cytochrome c release or ERK activation. Taken together, these results indicate that cytochrome c release, and the activation of ERK and caspase-3 in the final apoptosis pathway are all relevant factors in echinomycin-induced apoptosis. To our knowledge, this study is the first to delineate the echinomycin's direct detrimental effects on colon cancer cells.     

Caco-2 and LS174T cell lines provide different models for studying mucin expression in colon cancer

To compare the differences in MUC2 and MUC5AC mRNA among four colon cancer cell lines and to identify the best in vitro models for studying mucin expression, quantitative real-time polymerase chain reaction was used to measure the expression of MUC2 and MUC5AC mRNA in Caco-2, HT29, LoVo, and LS174T cell lines. The levels of MUC2 mRNA expression in the four colon cancer cell lines ranked in order of mRNA abundance were: LS174T > LoVo > HT-29 > Caco-2. In contrast to MUC2, the abundances of MUC5AC mRNA were in the order: Caco-2 > HT-29 > LS174T > LoVo. Caco-2 (highest level of MUC5AC mRNA) and LS174T (highest level of MUC2 mRNA) were used to investigate the phenotypes. Morphologically, Caco-2 cells were larger with low electron density mucus-storing vacuoles, many cell surface microvilli, and no obvious intercellular spaces between cells, compared to LS174T cells. The proliferative and invasive capacities of LS174T cells were significantly higher than those of Caco-2 cells. Caco-2 and LS174T cells provide excellent in vitro models for studying mucin expression in colon cancer.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

Synthesis of 1′,2′-methano-2′,3′-dideoxynucleosides as potential antivirals

The synthesis of constrained nucleosides has become an important tool to understand the SAR in the interaction between biological and synthetic nucleotides in the context of antisense oligonucleotide therapy. The incorporation of a cyclopropane into a furanose ring of a nucleoside induces some degree of constrain without affecting significantly the steric environment of a nucleoside. Here, we report a new, short and stereocontrolled synthesis of two constrained nucleosides analogues, 1′,2′- methano-2′,3′-dideoxyuridine 9, and the corresponding cytidine analog 12. X-ray crystallography revealed that the furanose ring in the constrained uridine and cytidine analogues was flattened with virtual loss of pseudorotation. The phosphoramidate esters of the novel constrained uridine and cytidine nucleosides, intended as prodrugs, were tested in cell-based assays for viral replication across the herpes virus family and HIV inhibition courtesy of Merck laboratories, Rahway. They were also tested in antiproliferative assays against colorectal and melanoma cell lines. Unfortunately, none of the compounds showed activity in these assays.     

Synthesis and in vitro growth inhibitory activity of novel silyl- and trityl-modified nucleosides

Seventeen silyl- and trityl-modified (5′-O- and 3′,5′-di-O-) nucleosides were synthesized with the aim of investigating the in vitro antiproliferative activities of these nucleoside derivatives. A subset of the compounds was evaluated at a fixed concentration of 100 μM against a small panel of tumor cell lines (HL-60, K-562, Jurkat, Caco-2 and HT-29). The entire set was also tested at varying concentrations against two human glioma lines (U373 and Hs683) to obtain GI₅₀ values, with the best results being values of ∼25 μM.     

The Chemopotential Effect of Annona muricata Leaves against Azoxymethane-Induced Colonic Aberrant Crypt Foci in Rats and the Apoptotic Effect of Acetogenin Annomuricin E in HT-29 Cells: A Bioassay-Guided Approach

Annona muricata has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of A. muricata leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome c from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of A. muricata leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the anticancer activity of A. muricata leaves.     

Upregulation of activin A gene by butyrate in human colon cancer cell lines

Activin A has been reported to play a role in the progression of colorectal cancer. Because dietary fiber protects against colorectal cancer, we hypothesized that butyrate, a fermentation product of dietary fiber, may affect the expression of activin A in colon cancer cells. Semiquantitative RT-PCR demonstrated that the activin A gene was upregulated by sodium butyrate in the human colon cancer cell lines HT-29 and Caco-2 in a concentration- and time-dependent manner. However, the activin A gene did not respond to sodium butyrate in the human normal colonic cell line FHC, rat normal intestinal epithelial cell (IEC) line IEC-6, and the explant of rat colon. Flow cytometry and agarose gel electrophoresis of genomic DNA revealed that cell cycle arrest and apoptosis were induced by sodium butyrate but not exogenous activin A in HT-29 cells, indicating that activin A could not act as an autocrine factor in colon cancer cells. By assuming that activin A promotes colorectal cancer spread as a paracrine factor, our findings suggest that butyrate could act as a tumor promoter in some circumstances.     

NO-mesalamine protects colonic epithelial cells against apoptotic damage induced by proinflammatory cytokines

The activation of a self-amplifying cascade of caspases, of which caspase-8 is the apical protease, mediates Fas-, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-, and TNF-alpha-induced apoptosis in colon cell lines. Nitric oxide (NO) protects from apoptosis induced by Fas and TNF-alpha. We examined whether NCX-456, an NO-releasing derivative of mesalamine, protects colon epithelial cells from cytokine-induced apoptosis. Caco-2 and HT-29 cell lines express death factor receptors and are driven to apoptosis in response to incubation with Fas-agonistic antibody, TNF-alpha/interferon-gamma, and TRAIL. The two novel observations reported here are that 1) cotreatment of cells with NCX-456, but not mesalamine, resulted in concentration-dependent protection against death factor-induced apoptosis and inhibition of caspase activity, and 2) exposure to dithiothreitol, an agent that effectively removes NO from thiol groups, resulted in a 70% recovery of caspase activity, which is consistent with S-nitrosation as a major mechanism for caspase inactivation. These data suggest that caspase S-nitrosation represents a mechanism for protection of colonic mucosal epithelial cells from death factor-induced death.     

Anti-tumoral activity of imidazoquines,a new class of antimalarials derived from primaquine

The growth inhibitory activity of imidazoquines, antimalarial imidazolidin-4-ones derived from primaquine, on human cancer cell lines HT-29, Caco-2, and MCF-7 has been evaluated. Primaquine, N-dipeptidyl-primaquine derivatives, and other quinolines have been included in the study for comparison purposes. Primaquine and some of its derivatives were significantly active against the MCF-7 human breast cancer cell line, so these compounds might represent useful leads targeted at the development of novel specific agents against breast cancer. Conversely, all compounds were generally inactive against HT-29, with only one of the imidazoquines having IC₅₀ below 50 μM. Activities against the Caco-2 cell line were modest and did not follow any defined trend.     

Anticancerogenic effect of a novel chiroinositol-containing polysaccharide from Bifidobacterium bifidum BGN4

Strains of bifidobacteria have many health-promotion effects. Whole cells or cytoplasm extracts of Bifidobacterium bifidum BGN4, isolated from human feces, inhibited the growth of several cancer cell lines. The polysaccharide fraction (BB-pol) extracted from B. bifidum BGN4 had a novel composition, comprising chiroinositol, rhamnose, glucose, galactose, and ribose. Three human colon cancer cell lines were treated with BB-pol: HT-29, HCT-116, and Caco-2. Trypan blue exclusion assay and BrdU incorporation assay showed that BB-pol inhibited the growth of HT-29 and HCT-116 cells but did not inhibit the growth of Caco-2 cells.     

Synthesis of azole nucleoside analogues of D-pinitol as potential antitumor agents

A convenient strategy is reported for the synthesis of azole nucleoside analogues of D-pinitol (=3-O-methyl-D-chiro-inositol). The key intermediate 3-O-methyl-4,5-epoxy-D-chiro-inositol was obtained in excellent yield via an epoxidation from mono-methanesulfonate of D-pinitol. The process of opening of the epoxy ring by azole-bases appeared strongly regioselective in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene. All newly synthesized carbocyclic azole nucleosides were assayed against lung and bladder cancer in vitro. Only the triazole and benzotriazole nucleoside analogues inhibited the growth of human lung cancer cell lines (PG) with EC(50) of 11.3 and 22.6 microM, respectively, and showed much less inhibitory activity against human bladder cell lines (T(24)).     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

Hyperthermia in combination with oxidative stress induces autophagic cell death in HT-29 colon cancer cells

The purpose of this study was to evaluate the mechanism of ROS-induced hyperthermic cell death in a colon cancer cell line. HT-29 colon cancer cells were exposed to heat (43 degrees C) in the presence of tert-butyl hydroperoxide (t-BOOH). t-BOOH combined with hyperthermia significantly decreased cell viability as compared with t-BOOH or hyperthermia alone. This decrease in cell numbers was associated with retardation in the S phase transit and not through apoptosis. Cell death was noted to be accompanied by specific features characteristic of autophagy: the presence of cytoplasmic autophagic vacuoles; autophagosome membrane association of microtubule-associated protein light chain 3; accumulation of acidic vesicular organelles; and increased incorporation of MDC in the autophagosome. Thermal sensitization through modulation of cellular ROS may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.     

Bisnaphthalimidopropyl spermidine induces apoptosis within colon carcinoma cells

Bisnaphthalimido compounds bisintercalate to DNA via the major groove and are potentially potent cancer therapeutics. We incorporated natural polyamines as linkers connecting the two-naphthalimido ring moieties to create a series of novel soluble cytotoxic bisnaphthalimidopropyl polyamines (BNIPPs). Here, we determined the cytotoxicity of bisnaphthalimidopropyl spermidine (BNIPSpd) towards Caco-2 and HT-29 colon adenocarcinoma cells revealing an IC₅₀ value of 0.15 and 1.64 μM after 48 h exposure within Caco-2 and HT-29 cells, respectively. After 4 h, ≥0.5 μM BNIPSpd treatment-induced significant DNA damage. After 24 h exposure a concentration-dependent increase in active caspase-3 expression, chromatin condensation and internucleosomal DNA fragmentation identified apoptosis as the principal manifestation for the cytotoxicity within both cell lines. By 24 h exposure, there was also a significant decline in cellular spermine and spermidine levels. It is concluded that bisnaphthalimidopropyl spermidine (BNIPSpd) toxicity primarily results from apoptosis and that BNISpd has potential to be further developed as an anti-tumour agent.     

PTB-Associated Splicing Factor (PSF) Is a PPARγ-Binding Protein and Growth Regulator of Colon Cancer Cells

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that plays an essential role in cell proliferation, apoptosis, and inflammation. It is over-expressed in many types of cancer, including colon, stomach, breast, and lung cancer, suggesting that regulation of PPARγ might affect cancer pathogenesis. Here, using a proteomic approach, we identify PTB-associated splicing factor (PSF) as a novel PPARγ-interacting protein and demonstrate that PSF is involved in several important regulatory steps of colon cancer cell proliferation. To investigate the relationship between PSF and PPARγ in colon cancer, we evaluated the effects of PSF expression in DLD-1 and HT-29 colon cancer cell lines, which express low and high levels of PPARγ, respectively PSF affected the ability of PPARγ to bind, and expression of PSF siRNA significantly suppressed the proliferation of colon cancer cells. Furthermore, PSF knockdown induced apoptosis via activation of caspase-3. Interestingly, DLD-1 cells were more susceptible to PSF knockdown-induced cell death than HT-29 cells. Our data suggest that PSF is an important regulator of cell death that plays critical roles in the survival and growth of colon cancer cells. The PSF-PPARγ axis may play a role in the control of colorectal carcinogenesis. Taken together, this study is the first to describe the effects of PSF on cell proliferation, tumor growth, and cell signaling associated with PPARγ.