1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through activation of c-jun N-terminal kinase

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and Nur77 and induce receptor-dependent and -independent apoptotic pathways in colon and other cancer cells. Structure-activity studies show that the p-bromo (DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation of Nur77 and PPARgamma, induce expression of CCAAT/enhancer-binding protein homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the position of the phenyl substituents (para >/= meta >/= ortho) and required a free indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose) polymerase (PARP) that is consistent with activation of the extrinsic pathway of apoptosis. These responses were associated with the activation of c-jun N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also observed in athymic nude mice bearing RKO cell xenografts and treated with 30 mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer cells and tumors is related to a novel ER stress-independent activation of JNK.     

Contribution of the orphan nuclear receptor Nur77 to the apoptotic action of IGFBP-3

Tumor suppression by insulin-like growth factor-binding protein-3 (IGFBP-3) has been demonstrated to occur via insulin-like growth factor-dependent and -independent mechanisms in vitro and in vivo. We have recently described IGFBP-3-induced mitochondrial translocation of the nuclear receptors RXRalpha/Nur77 in the induction of prostate cancer (CaP) cell apoptosis. Herein, we demonstrate that IGFBP-3 and Nur77 associate in the cytoplasmic compartment in 22RV1 CaP cells. Nur77 is a major component of IGFBP-3-induced apoptosis as shown by utilizing mouse embryonic fibroblasts (MEFs) derived from Nur77 wild-type and knockout (KO) mice. However, dose-response experiments revealed that a small component of IGFBP-3-induced apoptosis is Nur77 independent. Reintroduction of Nur77 into Nur77 KO MEFs restores full responsiveness to IGFBP-3. IGFBP-3 induces phosphorylation of Jun N-terminal kinase and inhibition of Akt phosphorylation and activity, which have been associated with Nur77 translocation. Finally, IGFBP-3 administration to CaP xenografts on SCID mice induced apoptosis and translocated Nur77 out of the nucleus. Taken together, our results verify an important role for the orphan nuclear receptor Nur77 in the apoptotic actions of IGFBP-3.     

Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.     

TR3/Nur77 in colon cancer cell apoptosis

The orphan nuclear receptor TR3/Nur77 has emerged as a viable candidate in the coordinate regulation of cell proliferation and apoptosis, essential for maintaining normal architecture in rapidly renewing tissues such as the colonic mucosa. TR3 induces apoptosis in a number of cell lineages exposed to proapoptotic stimuli by directly targeting the mitochondria, inducing cytochrome c release. Here we report a distinctly different mechanism of TR3-mediated apoptosis in colon cancer cells. Nucleus-to-cytoplasm translocation of a green fluorescent protein-TR3 construct, but not its direct mitochondrial targeting, was associated with apoptosis induced by the short-chain fatty acid, butyrate. Similar results were observed for the nonsteroidal anti-inflammatory drug, sulindac, and the chemotherapeutic drug, 5-fluorouracil. A mutant TR3 construct lacking DNA-binding ability exerted a potent proapoptotic effect in colon cancer cells that was associated with cytochrome c release, an action dependent upon cytoplasmic localization of the construct, but, again, not its direct mitochondrial targeting. We identified a potential role for BAX recruitment to the mitochondria, secondary to cytoplasmic translocation of TR3, in inducing cytochrome c release and in mediating apoptosis. Therefore, TR3 translocation from the nucleus may initiate the apoptotic cascade in colon cancer cells by stimulating other cytosolic proapoptotic molecules to associate with mitochondria.     

Tumor necrosis factor alpha induces BID cleavage and bypasses antiapoptotic signals in prostate cancer LNCaP cells

Survival of cancer cells in response to therapy, immune response, or metastasis depends on interactions between pro- and antiapoptotic signals. Two major proapoptotic pathways have been described: (a) a death receptor pathway; and (b) a mitochondrial pathway. We reported previously that Akt and the epidermal growth factor (EGF) receptor send separate, redundant survival signals that act to inhibit the mitochondrial proapoptotic pathway in prostate cancer LNCaP cells. However, it was unclear at what level the pro- and antiapoptotic signals interact in these cells, and it was also unclear whether these signals would inhibit the death receptor pathway. We found that EGF can protect LNCaP cells from apoptosis induced by LY294002 but not from tumor necrosis factor a (TNF-alpha)-induced apoptosis. Furthermore, TNF-alpha induced apoptosis under conditions in which Akt was active. Treatment with TNF-alpha resulted in activation of caspase 8 and cleavage of BID, which in turn induced cytochrome c release and caspase 9-dependent activation of effector caspases. Thus, proapoptotic signals induced by both TNF-alpha and LY294002 converge on mitochondria and trigger cytochrome c release. Because EGF can inhibit cytochrome c release induced by LY294002 but not cytochrome c release induced by TNF-alpha, we suggest that the EGF survival mechanism operates on the mitochondrial pathway at a site upstream of cytochrome c release. The ability of TNF-alpha to bypass survival signals from activated EGF receptor and Akt in prostate cancer cells makes death receptor signaling a promising avenue for therapeutic intervention.     

Rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase C

Signaling pathways involved in survival responses may attenuate the apoptotic response to the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon carcinomas. In six lines examined, three were sensitive (GC(3)/c1, VRC(5)/c1, HCT116), HT29 demonstrated intermediate sensitivity, and RKO and HCT8 were resistant to TRAIL-induced apoptosis. Calphostin c [an inhibitor of classic and novel isoforms of protein kinase C (PKC)] sensitized five of six cell lines to TRAIL, whereas Go6976, (inhibitor of classic PKC isoforms), did not influence TRAIL sensitivity. Rottlerin, an inhibitor of novel isoforms of PKC, specifically PKC delta, sensitized five of six cell lines to TRAIL-induced apoptosis, suggesting that PKC delta may be involved in the mechanism of TRAIL resistance. Transfection of HCT116 with a proapoptotic cleaved fragment of PKC delta or an antiapoptotic full-length PKC delta did not influence the sensitivity of HCT116 to TRAIL. Furthermore, the incubation of HCT116 or RKO with phorbol myristate acetate for 16 h, which down-regulated the expression of novel PKC isoforms, also did not influence sensitivity to TRAIL either in the absence or presence of rottlerin. However, after 15-min incubation with rottlerin, mitochondrial membrane potential (Delta psi m) was dramatically reduced in RKO cells, and, in cells subsequently treated with TRAIL, rapid apoptosis was evident within 8 h. Calphostin c, but not Go6976, also caused a decrease in Delta psi m. In RKO, rottlerin induced the release of cytochrome c, HtrA2/Omi, Smac/DIABLO, and AIF from the mitochondria, potentiated in combination with TRAIL, with concomitant caspase activation and down-regulation of XIAP. In HT29, the release of proapoptotic factors was demonstrated only when rottlerin and TRAIL were combined, and Bcl-2 overexpression inhibited this release and the induction of apoptosis. TRAIL-induced apoptosis was not influenced by rottlerin or Bcl-2 overexpression in type I (GC(3)/c1) cells. Data suggest that rottlerin affects mitochondrial function independent of PKC delta, thereby sensitizing cells to TRAIL, and that mitochondria constitute an important target in overcoming inherent resistance to TRAIL in colon carcinomas.     

Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt

Proapoptotic nuclear receptor family member Nur77 translocates from the nucleus to the mitochondria, where it interacts with Bcl-2 to trigger apoptosis. Nur77 translocation is induced by certain apoptotic stimuli, including the synthetic retinoid-related 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN)/CD437 class. In this study, we investigated the molecular mechanism by which AHPN/CD437 analog (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces Nur77 nuclear export. Our results demonstrate that 3-Cl-AHPC effectively activated Jun N-terminal kinase (JNK), which phosphorylates Nur77. Inhibition of JNK activation by a JNK inhibitor suppressed 3-Cl-AHPC-induced Nur77 nuclear export and apoptosis. In addition, several JNK upstream activators, including the phorbol ester TPA, anisomycin and MAPK kinase kinase-1 (MEKK1), phosphorylated Nur77 and induced its nuclear export. However, Nur77 phosphorylation by JNK, although essential, was not sufficient for inducing Nur77 nuclear export. Induction of Nur77 nuclear export by MEKK1 required a prolonged MEKK1 activation and was attenuated by Akt activation. Expression of constitutively active Akt prevented MEKK1-induced Nur77 nuclear export. Conversely, transfection of dominant-negative Akt or treatment with a phosphatidylinositol 3-kinase (PI3-K) inhibitor accelerated MEKK1-induced Nur77 nuclear export. Furthermore, mutation of an Akt phosphorylation residue Ser351 in Nur77 abolished the effect of Akt or the PI3-K inhibitor. Together, our results demonstrate that both activation of JNK and inhibition of Akt play a role in translocation of Nur77 from the nucleus to the cytoplasm.     

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes induce peroxisome proliferator-activated receptor gamma-mediated growth inhibition, transactivation, and differentiation markers in colon cancer cells

1,1-Bis(3'indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF)), p-t-butyl (DIM-C-pPhtBu), and p-phenyl (DIM-C-pPhC6H5) groups induce peroxisome proliferator-activated receptor gamma (PPARgamma)-mediated transactivation in HT-29, HCT-15, RKO, and SW480 colon cancer cell lines. Rosiglitazone also induces transactivation in these cell lines and inhibited growth of HT-29 cells, which express wild-type PPARgamma but not HCT-15 cells, which express mutant (K422Q) PPARgamma. In contrast, DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 inhibited growth of both HT-29 and HCT-15 cells with IC50 values ranging from 1 to 10 micromol/L. Rosiglitazone and diindolylmethane (DIM) analogues did not affect expression of cyclin D1, p21, or p27 protein levels or apoptosis in HCT-15 or HT-29 cells but induced keratin 18 in both cell lines. However, rosiglitazone induced caveolins 1 and 2 in HT-29 but not HCT-15 cells, whereas these differentiation markers were induced by DIM-C-pPhCF3 and DIM-C-pPhC6H5 in both cell lines. Because overexpression of caveolin 1 is known to suppress colon cancer cell and tumor growth, the growth inhibitory effects of rosiglitazone and the DIM compounds are associated with PPARgamma-dependent induction of caveolins.     

Apoptosis induced by 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile in human leukemia cells involves ROS-mitochondrial mediated death signaling and activation of p38 MAPK

Benzothiazoles are multitarget agents with broad spectrum of biological activity. 2-Acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile (AMBAN) is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects towards tumor cells. The aim of the present study was to examine the antiproliferative and apoptosis inducing activities of AMBAN towards human leukemia HL60 and U937 cells. Further, the molecular mechanism involved in AMBAN-induced apoptosis was investigated. Benzothiazole inhibited the growth and induced programmed cell death of HL60 and U937 cells. In addition, AMBAN elevated the level of reactive oxygen species, decreased the mitochondrial membrane potential, activated caspases 9 and 3, induced the cytochrome c release and PARP cleavage and led to intranucleosomal DNA fragmentation. Further, p38 MAPK was associated with the apoptotic activity of AMBAN. It can be concluded that AMBAN-induced apoptosis in HL60 and U937 cells through mitochondrial/caspase 9/caspase 3-dependent pathway.     

RNA干扰介导的Adrml基因沉默对大肠癌细胞增殖的抑制作用

目的 探讨RNA干扰使Adrml基因沉默后对大肠癌细胞增殖的影响.方法 构建靶向Adrml基因的shRNA真核表达载体,转染大肠癌细胞RKO,筛选Adrml基因沉默的稳定克隆.实验细胞分为3组,即稳定转染Adrml-shRNA的实验组、仅含大肠癌RKO细胞的空白对照组和转染空载体的阴性对照组.采用Western blot法检测Adrml蛋白的表达水平.通过软琼脂细胞集落形成实验观察3组细胞的集落形成能力.采用四甲基偶氮唑蓝(MTT)法和原位末端标记(TUNEL)法检测细胞的增殖和凋亡水平.应用流式细胞仪检测细胞周期的变化情况.结果 实验组大肠癌RKO细胞中,Adrml蛋白的表达受到明显抑制.实验组细胞的非贴壁依赖生长能力明显下降,偶见个别集落形成.实验组细胞的凋亡百分率为(12.4±1.1)%,明显高于阴性对照组[(1.3±0.2)%,P＜0.05].实验组G_0/G_1期和S/G_2期细胞所占的比例分别为(41.2±1.1)%和(58.8±1.1)%,实验组细胞被阻滞在G_1期.Adrml基因沉默能明显增强5-氟尿嘧啶(5-Fu)对大肠癌RKO细胞的生长抑制作用,并引起大肠癌RKO细胞大量凋亡.结论 RNA干扰介导的Adrml基因沉默能诱导大肠癌细胞发生凋亡并出现细胞周期阻滞,从而抑制肿瘤细胞的增殖.对于Adrml基因高表达的大肠癌患者,RNA干扰Adrml基因的表达并结合传统化疗有望成为新的治疗手段.     

Dual roles of Nur77 in selective regulation of apoptosis and cell cycle by TPA and ATRA in gastric cancer cells

Nur77 is an orphan receptor. Although Nur77 affects cell proliferation and apoptosis through its capability of binding to a variety of response elements and regulating their transactivation activities, the intrinsic function of Nur77 is not yet fully understood; in particular, its regulation of apoptosis and proliferation has been characterized as cell type-dependent and agent context-dependent. In this study, Nur77 can be seen to regulate apoptosis via its expression and translocation, rather than its transactivation activity in gastric cancer cells. Nur77 was constitutively expressed in BGC-823 cells. The tetradecanoylphorbol-1,3-acetate (TPA) treatment not only resulted in up-regulation of the Nur77 mRNA level, but also led to translocation of Nur77 protein from the nucleus to the mitochondria, and caused the release of cytochrome c. This TPA-induced translocation of Nur77 was in association with the initiation of apoptosis in gastric cancer cells. Although all-trans retinoic acid (ATRA) could not induce apoptosis in BGC-823 cells due to failure of stimulating Nur77 translocation, expression of Nur77 in the nucleus was required for cell growth inhibition by ATRA. Transfection of antisense Nur77 receptor into BGC-823 cells resulted in resistance of cell growth against ATRA inhibition, and the cells were still arrested in the S phase. Furthermore, the action of Nur77 in TPA-induced apoptosis was mediated through a protein kinase C signaling pathway, while mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways were responsible for the regulation of Nur77 mRNA expression. Taken together, the data revealed the dual functioning mechanisms of Nur77 in gastric cancer cells in response to TPA and ATRA.     

Oncogenic microRNA‐27a is a target for anticancer agent methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate in colon cancer cells

Methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate (CDODA‐Me) is a synthetic derivative of glycyrrhetinic acid, a triterpenoid phytochemical found in licorice extracts. CDODA‐Me inhibited growth of RKO and SW480 colon cancer cells and this was accompanied by decreased expression of Sp1, Sp3 and Sp4 protein and mRNA and several Sp‐dependent genes including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1 or Flt‐1). CDODA‐Me also induced apoptosis, arrested RKO and SW480 cells at G₂/M, and inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. CDODA‐Me decreased expression of microRNA‐27a (miR‐27a), and this was accompanied by increased expression of 2 miR‐27a‐regulated mRNAs, namely ZBTB10 (an Sp repressor) and Myt‐1 which catalyzes phosphorylation of cdc2 to inhibit progression of cells through G₂/M. Both CDODA‐Me and antisense miR‐27a induced comparable responses in RKO and SW480 cells, suggesting that the potent anticarcinogenic activity of CDODA‐Me is due to repression of oncogenic miR‐27a. © 2009 UICC     

Suppression of Cellular Apoptosis Susceptibility (CSE1L)Inhibits Proliferation and Induces Apoptosis in ColorectalCancer Cells

The cellular apoptosis susceptibility (CSE1L) gene has been demonstrated to regulate multiple cellularmechanisms including the mitotic spindle check point as well as proliferation and apoptosis. However, theimportance of CSE1L in human colon cancer is largely unknown. In the present study, we examined expressionlevels of CSE1L mRNA by semiquantitative RT-PCR. A lentivirus-mediated small interfering RNA (siRNA)was used to knock down CSE1L expression in the human colon cancer cell line RKO. Changes in CSE1L targetgene expression were determined by RT-PCR. Cell proliferation was examined by a high content screeningassay. In vitro tumorigenesis was measured by colony-formation assay. Cell cycle distribution and apoptosiswere detected by flow cytometric analysis. We found CSE1L mRNA to be expressed in human colon cancer cells.Using a lentivirus based RNAi approach, CSE1L expression was significantly inhibited in RKO cells, causingcell cycle arrest in the G2/M and S phases and a delay in cell proliferation, as well as induction of apoptosisand an inhibition of colony growth capacity. Collectively, the results suggest that silencing of CSE1L may be apotential therapeutic approach for colon cancer.     

p53 dependence of Fas induction and acute apoptosis in response to 5-fluorouracil-leucovorin in human colon carcinoma cell lines.

We examined the patterns of induction of apoptosis, Fas expression, and the influence of the status of the p53 tumor suppressor gene, in response to treatment of human colon carcinoma cell lines to 5-fluorouracil (FUra) combined with leucovorin (LV) under conditions of both DNA-directed (HT29, VRC5/c1, and RKO) and RNA-directed (HCT8 and HCT116) cytotoxicity. Acute apoptosis was induced in cell lines expressing wtp53 (RKO, HCT8, and HCT116), independent of the mechanism of FUra action. In HT29 cells that expressed mp53, apoptosis was a delayed event. Cell lines undergoing DNA-directed FUra cytotoxicity demonstrated marked accumulation of cells in S-phase (HT29 and RKO), whereas those lines undergoing RNA-directed cytotoxicity (HCT8 and HCT116) demonstrated marked cell cycle phase arrest in G2-M, both reversible by dThd. dThd partially protected HCT8 and HCT116 cells from FUra-LV-induced apoptosis but had no influence on FUra-LV-induced loss in clonogenic survival. In cells expressing wtp53, the Fas death receptor was induced in response to FUra-LV treatment. FUra-LV sensitized RKO cells to the anti-Fas monoclonal antibody CH-11 that was completely reversed by dThd, demonstrating the involvement of DNA damage in FUra-LV-induced, Fas-dependent sensitization to CH-11. In contrast, FUra-LV sensitized HCT116 cells to CH-11-induced apoptosis, which was not dThd reversible. Transduction of HT29 cells with Ad-wtp53 induced elevated Fas expression and sensitized the cells to FUra-LV-induced apoptosis. Data indicate that the presence of a wtp53 gene determines FUra-LV-induced Fas expression, the kinetics of FUra-LV-induced apoptosis and not the extent of apoptosis induced, both being independent of the mechanism of FUra action. Therefore, in colon carcinomas that express wtp53, the approach to sensitize tumors to Fas-mediated apoptosis may be further enhanced from the effect of FUra-LV in elevating Fas expression in a p53-dependent manner.