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.     

Role of leukotriene B4 in celecoxib-mediated anticancer effect

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has anticancer effect on many cancers associated with chronic inflammation by both COX-2-dependent and COX-2-independent mechanisms. The non-COX-2 targets of celecoxib, however, are still a matter of research. Leukotriene B₄ (LTB₄) has been implicated in prostate and colon carcinogenesis, but little is known about the potential role of LTB₄ in celecoxib-mediated anticancer effect. In this study, we evaluated whether LTB₄ was involved in celecoxib-mediated inhibitory effect on human colon cancer HT-29 cells and human prostate cancer PC-3 cells. Our data showed that survival of both cell lines was obviously suppressed after celecoxib treatment for 72 h in a concentration-dependent manner. However, only in HT-29 cells, this inhibitory effect could be reversed by LTB₄, which promoted survival of HT-29 cells rather than PC-3 cells. Consistent with these results, lioxygenase (LOX) potent inhibitor nordihydroguaiaretic acid (NDGA) had a higher inhibitory effect on HT-29 cells than PC-3 cells. Additionally, ELISA results showed that celecoxib could suppress expression of LTB₄ in both cell lines, whereas, inhibition of PGE₂ was only detected in HT-29 cells. These results indicate that the anticancer effect of celecoxib is COX-2-independent in HT-29 and PC-3 cells and in HT-29 cells primarily via down-regulating LTB₄ production.     

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

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

Activation of P27kip1-cyclin D1/E-CDK2 pathway by polysaccharide from Phellinus linteus leads to S-phase arrest in HT-29 cells

Our previous study showed that polysaccharide (P1) from Phellinus linteus exhibits a significant inhibitive activity on human colorectal carcinoma cells (HT-29). However its novel molecular mechanism remains unknown. To obtain insights into P1’s mechanism of action, we examined its effects on cell proliferation in vitro and in vivo, cell cycle distribution, apoptosis, autophagy, and expression of several cell cycle interrelated proteins in HT-29 cells. Interestingly, we found that volume and weight of the solid tumor significantly decreased in P1 (200 mg/kg)-treated mice compared with the control. However, slightly increased the body weight of the P1 treated tumor-bearing mice, with no significant increased ALT, AST levels in serum and LPO concentration in liver and kidney indicated that P1 has no toxicity to mammals at a dose of 200 mg/kg. Furthermore, P1 caused a significantly dose-dependent increase in the S-phase cell cycle, but no apoptosis and autophagy in HT-29 cells. RT-PCR and Western blot results showed significantly down-regulated expressions of cyclin D1, cyclin E, and CDK2, as well as increased expressions of P27kip1 in P1 (100 μg/mL)-treated HT-29 cells. These results suggested that the activation of P27kip1-cyclin D1/E-CDK2 pathway is involved in P1-induced S-phase cell cycle arrest in HT-29 cells.     

Protease-activated receptor 2 in colon cancer: trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation

Several lines of evidence suggest that tumor-derived trypsin contributes to the growth and invasion of cancer cells. We have recently shown that trypsin is a potent growth factor for colon cancer cells through activation of the G protein-coupled receptor protease-activated receptor 2 (PAR2). Here, we analyzed the signaling pathways downstream of PAR2 activation that lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events upon activation of PAR2 by the serine protease trypsin or the specific PAR2-activating peptide (AP2): (i) a matrix metalloproteinase-dependent release of transforming growth factor (TGF)-alpha, as demonstrated with TGF-alpha-blocking antibodies and measurement of TGF-alpha in culture medium; (ii) TGF-alpha-mediated activation of epidermal growth factor receptor (EGF-R) and subsequent EGF-R phosphorylation; and (iii) activation of ERK1/2 and subsequent cell proliferation. The links between these events are demonstrated by the fact that stimulation of cell proliferation and ERK1/2 upon activation of PAR2 is reversed by the metalloproteinase inhibitor batimastat, TGF-alpha-neutralizing antibodies, EGF-R ligand binding domain-blocking antibodies, and the EGF-R tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGF-R appears to be a major mechanism whereby activation of PAR2 results in colon cancer cell growth. By using the Src tyrosine kinase inhibitor PP2, we further showed that Src plays a permissive role for PAR2-mediated ERK1/2 activation and cell proliferation, probably acting downstream of the EGF-R. These data explain how trypsin exerts robust trophic action on colon cancer cells and underline the critical role of EGF-R transactivation.     

Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling

Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation.     

Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C₁₆-alkyl chain attached to the γ-carboxylic acid and one of the analogues had an additional benzyl group attached to the α-carboxylic acid. The cytotoxicity of the γ-alkylated compound towards KATO III (IC₅₀ = 55 nM) and HT-29 (IC₅₀ = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the α-carboxyl group made the compound nontoxic. The γ-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A₂ (sPLA₂), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA₂ hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA₂. However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA₂ hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems.     

Fluorescent-Antibody Targeting of Insulin-Like Growth Factor-1 Receptor Visualizes Metastatic Human Colon Cancer in Orthotopic Mouse Models

Fluorescent-antibody targeting of metastatic cancer has been demonstrated by our laboratory to enable tumor visualization and effective fluorescence-guided surgery. The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of metastatic colon cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24–31) was conjugated with 550 nm, 650 nm or PEGylated 650 nm fluorophores. Subcutaneous, orthotopic, and liver metastasis models of colon cancer in nude mice were targeted with the fluorescent IGF-1R antibodies. Western blotting confirmed the expression of IGF-1R in HT-29 and HCT 116 human colon cancer cell lines, both expressing green fluorescent protein (GFP). Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery.     

激活FXR抑制结肠癌细胞浸润转移及MMP-7的表达

目的:探讨法尼酯X受体(FXR)特异性激动剂GW4064抑制结肠癌细胞浸润转移的机制。方法:在体外培养人结肠癌细胞HT-29,应用GW4064作用于结肠癌细胞,以四唑氮蓝还原法(MTT)检测细胞活性的变化。用transwell小室研究结肠癌细胞的迁移及浸润。用RT-PCR检测FXRm RNA及MMP-7mRNA表达的变化,用western blot检测FXR及MMP-7蛋白表达的变化。结果:MTT结果显示GW4064作用于人结直肠HT-29细胞的生长抑制率呈浓度依赖性;transwell小室结果显示GW4064抑制结肠癌细胞的浸润转移,与对照组相比,差异具有统计学意义(P0.05),RT-PCR及Western blot显示GW4064促进FXR m RNA及蛋白表达,抑制MMP-7mRNA及蛋白的表达,与对照组相比差异有统计学意义(P0.05)。结论:GW4064抑制结肠癌细胞的生长及转移,上调HT-29细胞FXR m RNA及蛋白的表达,下调HT-29细胞MMP-7 m RNA及蛋白的表达。FXR被激活后抑制结肠癌细胞转移,MMP-7可能是其作用通路之一。     

Concomitant supplementation of lycopene and eicosapentaenoic acid inhibits the proliferation of human colon cancer cells

Several studies indicated that people who live in the Mediterranean region have very low rates of chronic diseases such as cardiovascular disease and cancer. It is well known that Mediterranean-style diet is rich in vegetables, tomato, fruit, fish and olive oil. These important dietary components may contribute to lower risk of cancer. Lycopene, a major component in tomato, exhibited potential anticarcinogenic activity. Previous studies showed that consumption of fish containing eicosapentaenoic acid (EPA) correlated with reduced risk of cancer. However, the combined effects of lycopene and EPA on the proliferation of human colon cancer have not been studied well yet. Thus, we investigated the anticancer properties and therapeutic potential of lycopene and EPA in human colon cancer HT-29 cells. In this study, we determined the combined effects of lycopene and EPA on the proliferation of human colon cancer HT-29 cells. We demonstrated that low concentration of lycopene and EPA could synergistically inhibit the proliferation of colon cancer cells. The inhibitory mechanism was associated with suppression of phosphatidylinositol 3-kinase/Akt signaling pathway. Furthermore, treatment of lycopene and EPA also synergistically blocked the activation of downstream mTOR molecule. Immunocytochemical staining results revealed that lycopene and EPA could also up-regulate the expression of apoptotic proteins such as Bax and Fas ligand to suppress cell survival. In conclusion, our novel findings suggest that lycopene and EPA synergistically inhibited the growth of human colon cancer HT-29 cells even at low concentration. The inhibitory effects of lycopene and EPA on cell proliferation of human colon cancer HT-29 cells were, in part, associated with the down-regulation of the PI-3K/Akt/mTOR signaling pathway.     

The interaction of butyrate with TNF-alpha during differentiation and apoptosis of colon epithelial cells: role of NF-kappaB activation

We demonstrated that TNF-alpha suppressed differentiation and potentiated cell death induced by butyrate (NaBt) in both adenocarcinoma HT-29 and fetal FHC human colon cells in vitro. Since TNF-alpha is a typical activator of NF-kappaB pathway, we studied the role of NF-kappaB activation in cell differentiation and death during the TNF-alpha and NaBt co-treatment. TNF-alpha induced rapid NF-kappaB activation in both HT-29 and FHC cell lines and this effect was differently modulated by NaBt in these two cell lines. In HT-29 cells, NaBt potentiated NF-kappaB activity induced by TNF-alpha after 4h treatment. However, this initial potentiation of NF-kappaB activity was not observed in FHC cells. During additional time of TNF-alpha and NaBt co-treatment, NaBt decreased the TNF-alpha-mediated NF-kappaB activity in both cell types. We also detected a different response of HT-29 and FHC cells after the pre-treatment with the NF-kappaB inhibitor parthenolide. Our results indicated that NaBt-mediated differentiation and apoptosis of colon epithelial cells can be modulated by TNF-alpha. Furthermore, we found significant differences in the mechanism of the NaBt and TNF-alpha co-treatment effects between cells of non-cancer and cancer origin, suggesting that the NF-kappaB pathway may be more effectively involved in these processes in cancer cells.     

Activation of proteinase-activated receptor 1 promotes human colon cancer cell proliferation through epidermal growth factor receptor transactivation

Serine proteases are now considered as crucial contributors to the development of human colon cancer. We have shown recently that thrombin is a potent growth factor for colon cancer cells through activation of the aberrantly expressed protease-activated receptor 1 (PAR1). Here, we analyzed the signaling pathways downstream of PAR1 activation, which lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events on activation of PAR1 by thrombin or specific activating peptide: (a) a matrix metalloproteinase-dependent release of transforming growth factor-alpha (TGF-alpha) as shown with TGF-alpha blocking antibodies and measurement of TGF-alpha in culture medium; (b) TGF-alpha-mediated activation of epidermal growth factor receptor (EGFR) and subsequent EGFR phosphorylation; and (c) activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and subsequent cell proliferation. The links between these events are shown by the fact that stimulation of cell proliferation and ERK1/2 on activation of PAR1 is reversed by the MMP inhibitor batimastat, TGF-alpha neutralizing antibodies, EGFR ligand binding domain blocking antibodies, and the EGFR tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGFR seems to be a major mechanism whereby activation of PAR1 results in colon cancer cell growth. Finally, PAR1 activation induces Src phosphorylation, which is reversed by using the Src tyrosine kinase inhibitor PP2, suggesting that Src activation plays a permissive role for PAR1-mediated ERK1/2 activation and cell proliferation probably acting downstream of the EGFR. These data explain how thrombin exerts robust trophic action on colon cancer cells and underline the critical role of EGFR transactivation.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells

Quercetin has chemoprotective properties in experimental colon cancer models, and in vitro studies have demonstrated that quercetin inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 receptor tyrosine kinases have been associated with the development of human colon cancer, and the expressions of both receptors are high in HT-29 cells. In this study, we assessed quercetin regulation of HT-29 and SW480 cell apoptosis and the influence of quercetin on the protein expression of ErbB2, ErbB3, Akt, Bax and Bcl-2. We cultured HT-29 cells in the presence of various concentrations (0, 25, 50, or 100 micromol/L) of quercetin or rutin. Quercetin inhibited HT-29 cell growth in a dose-dependent manner, whereas rutin had no effect on the cell growth. DNA that was isolated from cells treated with 50 micromol/L of quercetin exhibited an oliogonucleosomal laddering pattern characteristic of apoptotic cell death. Western blot analysis of cell lysates revealed that Bcl-2 levels decreased dose-dependently in cells treated with quercetin, but Bax remained unchanged. Quercetin increased levels of cleaved caspase-3 and the 89-kDa fragment of poly (ADP-ribose) polymerase. In addition, phosphorylated Akt levels were markedly lower in cells treated with 25 micromol/L quercetin, but total Akt levels decreased only at 100 micromol/L quercetin. Furthermore, a dose-dependent decrease in ErbB2 and ErbB3 levels was detected in quercetin-treated cells. The results obtained using SW480 cells were similar to those obtained with HT-29 cells. In conclusion, we have shown that quercetin inhibits cell growth and induces apoptosis in colon cancer cells, and that this may be mediated by its ability to down-regulate ErbB2/ErbB3 signaling and the Akt pathway.     

Calcitriol sensitizes colon cancer cells to H2O2-induced cytotoxicity while inhibiting caspase activation

The anti-cancer activity of calcitriol, the active metabolite of Vitamin D, in the colon is usually attributed to its anti-proliferative and pro-differentiative actions. The levels of reactive oxygen species (ROS) are high in colon carcinomas due to increased aerobic metabolism and exposure to various anti-cancer modalities. We examined whether calcitriol modulates the response of colon cancer cells to the cytotoxic action of the common mediator of ROS injury, H2O2. Pretreatment with calcitriol (100 nM, 48 h) sensitized HT-29 colon cancer cells to cell death induced by acute exposure to H2O2 or chronic exposure to the H2O2 generating system, glucose/glucose-oxidase. Although the morphological features of H2O2-induced HT-29 cell death are consistent with apoptosis, we detected no executioner caspase activation in response to cytotoxic concentrations of H2O2 and treatment with a pan-caspase inhibitor did not affect H2O2-induced cytotoxicity nor its enhancement by calcitriol. Conversely, exposure of HT-29 cells to sub-toxic concentrations of H2O2 resulted in low executioner caspase activation that was inhibited by pretreatment with calcitriol. The sensitization of colon cancer cells to ROS-induced cytotoxicity may contribute to its assumed action as a chemopreventive agent and to its therapeutic potential alone or in combination with other anti-cancer modalities.     

Cytotoxic benzil and coumestan derivatives from Tephrosia calophylla

A benzil, calophione A, 1-(6′-Hydroxy-1′,3′-benzodioxol-5′-yl)-2-(6″-hydroxy-2″-isopropenyl-2″,3″-dihydro-benzofuran-5″-yl)-ethane-1,2-dione and three coumestan derivatives, tephcalostan B, C and D were isolated from the roots of Tephrosia calophylla. Their structures were deduced from spectroscopic data, including 2D NMR ¹H-¹H COSY and ¹³C-¹H COSY experiments. Compounds were evaluated for cytotoxicity against RAW (mouse macrophage cells) and HT-29 (colon cancer cells) cancer cell lines and antiprotozoal activity against various parasitic protozoa. Calophione A exhibited significant cytotoxicity with IC₅₀ of 5.00 (RAW) and 2.90 μM (HT-29), respectively.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.     

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPARγ is expressed at considerable levels in human colon cancer cells. This suggests that PPARγ expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPARγ expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPARγ mRNA and protein in these cells were in the order HT-29>LOVO>Caco-2>DLD-1. We also found that PPARγ overexpression promoted cell growth inhibition in PPARγ lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPARγ expression and the cells' sensitivity for proliferation.