Preclinical analysis of the anti-tumor and anti-metastatic effects of Raf265 on colon cancer cells and CD26(+) cancer stem cells in colorectal carcinoma.

BACKGROUND: In colorectal carcinoma (CRC), activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition, the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore, we hypothesized that an ATP-competitive pan-Raf inhibitor, Raf265, is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS: HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS: Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition, anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally, the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS: This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC, providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC.

Anti-tumor efficacy of a recombinant human arginase in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is considered as auxotrophic for arginine and BCT-100, a new recombinant human arginase, has been synthesized for arginine deprivation to inhibit arginine-dependent tumor growth. The aim of the present study was to evaluate the effects of BCT-100 on the inhibition of in vitro cell proliferation of HCC cell lines and in vivo tumor growth. The molecular mechanism involved was also studied. The anti-tumor efficacy of BCT-100 on cell proliferation, cell cycle distribution and cellular apoptosis were determined in human hepatoma HepG2 and PLC/PRF/5 cells. Protein expression in the Wnt/ß-catenin and Akt signaling pathways were analyzed by western blotting. Tumors were also established subcutaneously and BCT-100, in combination with oxaliplatin, was administrated i.p. to study the anti-tumor growth of the drugs. Treatment with BCT-100 was found to inhibit cell proliferation and enhance caspasedependent cellular apoptosis. Cell cycle arrest at S phase was observed. Inhibition of Wnt/ß-catenin and Akt signaling pathways, with a reduction in survivin and XIAP protein expressions, were also observed. Furthermore, combined treatment of BCT-100 and chemotherapy with oxaliplatin demonstrated synergistic inhibiting effect on tumor growth and the overall survival probability was enhanced as compared with BCT-100 or oxaliplatin treatment alone. These preclinical data demonstrate robust anti-tumor activity of BCT100 in HCC, thus providing the basis for its exploitation as a potential therapeutic agent in arginine-driven tumors. The positive effect of testing BCT100 with oxaliplatin in PLC/PRF/5 tumours also supports the rationale of combining BCT-100 and oxaliplatin in the clinical treatment of HCC.

XIAP-associated factor 1 (XAF1), a novel target of p53, enhances p53-mediated apoptosis via post-translational modification.

The role of X chromosome-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) in mediating apoptosis has been reported but the underlying mechanism remains unclear. The present study was designed to examine the putative interaction between XAF1 and p53 and the functional importance of this interaction in regulation of apoptosis in human gastric and colon cancer cells. We first identified XAF1 as a novel target gene of p53 by the chromatin immunoprecipitation (CHIP) assay and demonstrated that wild-type p53, but not mutant p53, down-regulated XAF1 at both mRNA and protein levels, which acted mostly under the condition of high expression of XAF1 and was associated with the physical interaction between p53 and the XAF1 promoter. We also found that the over-expression of XAF1 led to activation of wild-type p53 via post-translational modification in cells with or without DNA damage, which resulting in p53 nuclear accumulation and its increased transcriptional activity and enhancing p53-dependent apoptosis. These findings suggest that a potential novel feedback loop exists between XAF1 and wild-type p53.

Four and a half LIM protein 2 (FHL2) negatively regulates the transcription of E-cadherin through interaction with Snail1.

E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT), which plays a crucial role in cancer metastasis. We previously demonstrated that four and a half LIM protein 2 (FHL2) inhibited E-cadherin expression and promoted invasive potential and EMT in colon cancer. Here, we aim to further define the mechanism underlying the inhibition of E-cadherin by FHL2 in colon cancer. The expression profiles of FHL2 and Snail1 were first observed by Western blot, immunofluorescence and immunohistochemistry. We found that both the protein level and the cellular localisation of Snail1 were quite similar to FHL2 in colon cancer; reciprocal co-immunoprecipitation assay showed that FHL2 was able to bind Snail1 and its intact structure was required. The expression of FHL2 was positively correlated to Snail1 while negatively to E-cadherin and phospho-Snail1. FHL2 over-expression induced the accumulation of Snail1 in the nucleus. Moreover, dual luciferase assay revealed that FHL2 over-expression decreased while FHL2 siRNA increased the transcriptional activities of two E-cadherin promoter constructs which contained E-box sites (Snail1-binding elements). Mutation of E-boxes increased the transcriptional activities and FHL2 expression was involved in the function of mutation. These results suggested that FHL2 negatively regulated E-cadherin transcriptional activity through interaction with Snail1. Our study established a novel regulatory function of FHL2 and revealed a potential mechanism on promoting the process of EMT.

XIAP-associated factor 1 interacts with and attenuates the trans-activity of four and a Half LIM protein 2.

XIAP-associated factor 1(XAF1) is a tumor suppressor with its functional mechanisms not fully understood. The zinc-finger cluster located at the N-terminus is the only domain structure. Four and a half LIM domain protein 2 (FHL2) also contains a tandem zinc finger structure, and its protein functions as an important adaptor and modifier in protein-protein interactions. Both of their structures are relatively simple, while the association between them is still unclear. In this study, we detected the interaction between XAF1 and FHL2 by using the yeast two-hybrid system. We identified FHL2 as a XAF1 binding protein. Furthermore, both XAF1 and FHL2 localized to the cytoplasm, mitochondria, and nucleus of gastric cancer cells. Over-expression of XAF1 excluded FHL2 from the nucleus and suppressed the trans-activity of FHL2 in stimulating the transcriptional activities of ß-catenin and AP-1. In conclusion, our findings unraveled an antagonistic mechanism between a tumor suppressor and an oncoprotein in cancer cells.

Promoter hypermethylation and histone hypoacetylation contribute to pancreatic-duodenal homeobox 1 silencing in gastric cancer.

BACKGROUND AND AIMS: The expression of pancreatic-duodenal homeobox 1 (PDX1) in gastric cancer is aberrantly reduced. The aim of this study was to elucidate the regulation of DNA methylation and histone acetylation at the promoter for PDX1 silencing in gastric cancer. METHODS: PDX1 expression in response to demethylation and acetylation was detected in human gastric cancer cell lines by reverse transcription-polymerase chain reaction (PCR) and western blot. Four CpG islands within the 5'-flanking region of PDX1 gene were analyzed with their transcription activities being detected by dual luciferase assay. Promoter hypermethylation was identified in gastric cancer cell lines and cancer tissues by methylation-specific PCR or bisulfite DNA sequencing PCR analysis. Histone acetylation was determined by chromatin immunoprecipitation (ChIP) assay. RESULTS: Demethylation by 5'-aza-2'-deoxycytidine (5'-aza-dC) and/or acetylation by trichostatin A (TSA) restored PDX1 expression in gastric cancer cells. Hypermethylation was found in four CpG islands in six of seven cancer cell lines. However, only the distal CpG island located in the promoter fragment of PDX1, F383 (c.-2063 to -1681 nt upstream of the ATG start codon) displayed significant transcriptional activity that could be suppressed by SssI methylase and increased by 5'-aza-dC and TSA. More than 70% of the single CpG sites in F383 were methylated with hypermethylation of F383 fragment more common in gastric cancerous tissues compared with the paired normal tissues (P < 0.05). ChIP assay showed F383 was also associated with low hypoacetylation level of the histones. CONCLUSION: Promoter hypermethylation and histone hypoacetylation contribute to PDX1 silencing in gastric cancer.

A subpopulation of CD26+ cancer stem cells with metastatic capacity in human colorectal cancer.

Recent evidence suggests that a subpopulation of cancer cells, cancer stem cells (CSCs), is responsible for tumor growth in colorectal cancer. However, the role of CSCs in colorectal cancer metastasis is unclear. Here, we identified a subpopulation of CD26(+) cells uniformly present in both the primary and metastatic tumors in colorectal cancer patients with liver metastasis. Furthermore, in patients without distant metastasis at the time of presentation, the presence of CD26(+) cells in their primary tumors predicted distant metastasis on follow-up. Isolated CD26(+) cells, but not CD26(-) cells, led to development of distant metastasis when injected into the mouse cecal wall. CD26(+) cells were also associated with enhanced invasiveness and chemoresistance. Our findings have uncovered a critical role of CSCs in metastatic progression of cancer. Furthermore, the ability to predict metastasis based on analysis of CSC subsets in the primary tumor may have important clinical implication as a selection criterion for adjuvant therapy.

Four-and-a-half LIM protein 2 promotes invasive potential and epithelial-mesenchymal transition in colon cancer.

BACKGROUND AND AIMS: Cancer invasion and metastasis may associate with the phenotype transition called epithelial-mesenchymal transition (EMT). We aim to evaluate the impact of four-and-a-half LIM protein 2 (FHL2) on EMT and invasion of colon cancer. METHODS: The functional role of FHL2 in EMT was determined by overexpression or small interfering RNA-mediated depletion of FHL2. Mechanisms of FHL2 on expression or activity of E-cadherin and beta-catenin were assessed. RESULTS: FHL2 was highly expressed in primary and metastatic colon cancer but not in normal tissues. FHL2 was critical for cancer cell adhesion to extracellular matrix, migration and invasion. FHL2 expression was stimulated by transforming growth factor (TGF)-beta1. Moreover, FHL2 acted as a potent EMT inducer by stimulating vimentin and matrix metalloproteinase-9 expressions and causing a loss of E-cadherin, whereas those alterations of EMT markers were not affected by silencing of Smad molecules (typical TGF-beta signal mediators) in FHL2 stable transfectant cells. Therefore, FHL2 induced EMT in a TGF-beta-dependent and Smad-independent manner. FHL2 downregulated E-cadherin expression and inhibited the formation of membrane-associated E-cadherin-beta-catenin complex. FHL2 also stabilized nuclear beta-catenin, resulting in enforcement of beta-catenin transactivation activity. CONCLUSION: FHL2 is a potent EMT inducer and might be an important mediator for invasion and/or metastasis of colon cancer.

Trend of colorectal cancer in Hong Kong: 1983-2006.

BACKGROUND AND AIM: The incidence of colorectal cancer (CC) is increasing in many Asian countries, but decreasing in western countries. The present study examined the local incidence of CC in the past few decades. METHODS: A population based study, using data from Hong Kong (HK) Cancer Registry, was carried out to examine the trends of CC in different age groups in HK. Comparison with other countries was made. RESULTS: The crude rate of CC in HK increased from 29.6/100,000 in 1983 to 57.1/100,000 in 2006. Age standardized rate (ASR) increased by less than 20%. It was markedly smaller than the 190% increase in crude rate. ASR progressively increased in males. In females, ASR peaked in 1994 and declined in the last decade. In most countries, the risk of CC was higher and increasing in males, but stable or decreasing in females. With respect to age, increasing risk was noted in males above 60 years old and females above 70 years old. However, a declining rate was noted in those below 50 years old. The decrease was over 40% in the 30-34 years group over the past two decades. CONCLUSIONS: Increasing incidence of CC in HK was mostly in the older and male population, but not in the younger age group.

Promising multifunctional anti-Alzheimer's dimer bis(7)-Cognitin acting as an activator of protein kinase C regulates activities of alpha-secretase and BACE-1 concurrently.

We have recently demonstrated that bis(7)-Cognitin, a promising multifunctional anti-Alzheimer's dimer, can remarkably reduce the generation of amyloid beta peptide (Abeta) by inhibiting beta-secretase (BACE-1) and activating alpha-secretase activity. In this study, the mechanism(s) underlying bis(7)-Cognitin's regulation of the activity of these two proteases was further investigated. In N2a cells stably expressing human amyloid precursor protein with the Swedish mutation (APPswe), the reduction in Abeta production induced by 1microM bis(7)-Cognitin was not altered by the co-pretreatment of muscarinic and nicotinic cholinergic receptor antagonists, indicating that the regulation of APP processing by this dimer is independent of cholinergic transmission. Furthermore, bis(7)-Cognitin (0.1-3microM) significantly increased protein kinase C (PKC) activity in cells and in vitro in a concentration-dependent manner. Administration of a PKC activator, phorbol 12-myristate 13-acetate (PMA), concentration-dependently increased the alpha-secretase cleavage products, and reduced the BACE-1 cleavage products. In addition, the inhibition of PKC prevented PMA- or bis(7)-Cognitin-induced alterations in alpha-secretase and BACE-1 activities, eliminating reductions in Abeta production seen with PMA or the dimer. These results strongly suggest that bis(7)-Cognitin may reduce the biosynthesis of Abeta by inhibiting BACE-1 and activating alpha-secretase concurrently through the direct activation of PKC. Combined with previous findings of direct inhibition of AChE and BACE-1 by this dimer, this work indicates that strategy may have potential to provide new insights into designing novel drugs that target multiple steps of aberrant APP processing to treat Alzheimer's disease.

Adenovirus-mediated down-regulation of X-linked inhibitor of apoptosis protein inhibits colon cancer.

Our previous studies and those of others have indicated that X-linked inhibitor of apoptosis protein (XIAP) holds promise as a target gene in colon cancer gene therapy. In this study, we constructed an adenoviral vector to deliver small hairpin RNA (shRNA) against XIAP (XIAP-shRNA) into colon cancer cells and tested its therapeutic efficacy in vitro and in vivo. We first confirmed an overexpression of XIAP in colon cancer cells and human cancer tissues. We then designed XIAP-small interfering RNA (siRNA) and confirmed the knockdown effect of these siRNAs in colon cancer cells. The sequences of the effective siRNAs were converted into shRNA and then packed into replication-deficient adenoviral vectors using BLOCK-iT Adenoviral RNAi Expression System to generate Adv-XIAP-shRNA. Infection of HT29 and HCT116 cells with Adv-XIAP-shRNA led to enhanced caspase-3 activity, which was associated with increased apoptosis and reduced cell proliferation. The therapeutic effect of Adv-XIAP-shRNA was then tested in xenograft tumors in nude mice. We showed that treatment of the xenograft tumors derived from HCT116 cells with Adv-XIAP-shRNA resulted in a retardation of tumor growth, which was associated with enhanced apoptosis, increased caspase-3 activity, and reduced expression of proliferating cell nuclear antigen in the tumor tissues. Treatment of xenograft tumors with Adv-XIAP-shRNA did not affect the expressions of inflammatory cytokines in tumor-bearing mice. Thus, Adv-XIAP-shRNA-mediated down-regulation of XIAP exerts a therapeutic effect in colon cancer by promoting apoptosis and inhibiting proliferation of colon cancer cells, and the antitumor effect of Adv-XIAP-shRNA was unlikely to be related to virus-induced immune response.

Mecamylamine prevents neuronal apoptosis induced by glutamate and low potassium via differential anticholinergic-independent mechanisms.

Neuronal loss via apoptosis caused by various stimuli may be the fundamental mechanism underlying chronic and acute neurodegenerative diseases. A drug inhibiting neuronal apoptosis may lead to a practical treatment for these diseases. In this study, treatment with mecamylamine, a classical antagonist of nicotinic acetylcholine receptors (nAChRs), prevented neuronal apoptosis induced by 75 microM glutamate and by low potassium (LK) in cerebellar granule neurons (CGNs) with EC(50)s of 35 and 293 microM, respectively. Two other antagonists of nAChRs, dihydro-beta-erythroidine and tubocurarine, failed to inhibit these two kinds of apoptosis. Mecamylamine inhibited the NMDA (30 microM)-evoked current and competed with [(3)H]MK-801. Furthermore, two inhibiters of the c-Jun N-terminal kinase (JNK) pathway prevented LK-induced apoptosis. Mecamylamine reversed the phosphorylation levels of JNK and c-Jun as well as the expression of c-Jun caused by LK in a Western blot assay. In addition, the JNK/c-Jun pathway was not involved in glutamate-induced cell death of CGNs. Our results suggest that mecamylamine prevents glutamate-induced apoptosis by blocking NMDA receptors at the MK-801 site and LK-induced apoptosis by inhibiting the activation of the JNK/c-Jun pathway.

Synergistic neuroprotection by bis(7)-tacrine via concurrent blockade of N-methyl-D-aspartate receptors and neuronal nitric-oxide synthase.

The excessive activation of the N-methyl-D-aspartate receptor (NMDAR)/nitric oxide (NO) pathway has been proposed to be involved in the neuropathology of various neurodegenerative disorders. In this study, NO was found to mediate glutamate-induced excitotoxicity in primary cultured neurons. Compared with the NO synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), and the NMDAR antagonist memantine, bis(7)-tacrine was found to be more potent in reducing NO-mediated excitotoxicity and the release of NO caused by glutamate. Moreover, like L-NMMA but not like 5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and memantine, bis(7)-tacrine showed greater neuroprotection and inhibition on NO release when neurons were pretreated for a prolonged time between 0 and 24 h and remained quite potent even when neurons were post-treated 1 h after the glutamate challenge. Bis(7)-tacrine was additionally found to be as moderately potent as memantine in competing with [(3)H]MK-801, inhibiting NMDA-evoked currents and reducing glutamate-triggered calcium influx, which eventually reduced neuronal NOS activity. More importantly, at neuroprotective concentrations, bis(7)-tacrine substantially reversed the overactivation of neuronal NOS caused by glutamate without interfering with the basal activity of NOS. Furthermore, in vitro pattern analysis demonstrated that bis(7)-tacrine competitively inhibited both purified neuronal and inducible NOS with IC(50) values at 2.9 and 9.3 microM but not endothelial NOS. This result was further supported by molecular docking simulations that showed hydrophobic interactions between bis(7)-tacrine and three NOS isozymes. Taken together, these results strongly suggest that the substantial neuroprotection against glutamate by bis(7)-tacrine might be mediated synergistically through the moderate blockade of NMDAR and selective inhibition of neuronal NOS.