A new EGFR inhibitor induces apoptosis in colon cancer cells

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, mAbs and EGFR tyrosine kinase inhibitors seemed to be the most promising. However they have demonstrated scarce utility in therapy, the former being effective only at toxic doses, the latter resulting inefficient in colon cancer. This paper presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphthoquinone core as shikonin, an agent with great anti-tumor potential. In HT29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 microM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer.     

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21^/Cip and p27^/Kip1. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.     

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27^Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 431–440, 1998     

Sodium butyrate induces G2 arrest in the human breast cancer cells MDA-MB-231 and renders them competent for DNA rereplication

When exposed to sodium butyrate (NaBut), exponentially growing cells accumulate in G1 and G2 phases of the cell cycle. In the human breast cancer cell line MDA-MB-231, an arrest in G2 phase was observed when the cells were released from hydroxyurea block (G1/S interface) in the presence of NaBut. The inhibition of G2 progression was correlated with increased contents both of total p21(Waf1) and of p21(Waf1) associated with cyclin A and with an inhibition of cyclin A- and B1-associated histone H1 kinase activities measured in cell lysates, as well as with dephosphorylation of the RB protein. A decrease in the cell contents of cyclins A and B1 was also observed but this decrease was preceded by p21(Waf1) accumulation. When NaBut was removed from the culture medium of cells blocked in G2 phase, p21(Waf1) level decreased and, instead of proceeding to mitosis, these cells resumed a progression toward DNA rereplication. These results suggest that the induction of p21(Waf1) by NaBut leads to the inhibition of the sequential activation of cyclin A- and B1-dependent kinases in this cell line, resulting in the inhibition of G2 progression and rendering the cells competent for a new cell division cycle.     

Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

Recently, salidroside (p-hydroxyphenethyl-β-d-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) were sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.     

Inhibition of cyclooxygenase-1 lowers proliferation and induces macroautophagy in colon cancer cells

Evolving evidence supports that cyclooxygenase-1 (COX-1) takes part in colon carcinogenesis. The effects of COX-1 inhibition on colon cancer cells, however, remains obscured. In this study, we demonstrate that COX-1 inhibitor sc-560 inhibited colon cancer cell proliferation with concomitant G₀/G₁-phase cell cycle arrest. The anti-proliferative effect was associated with down-regulation of c-Fos, cyclin E₂ and E₂F-1 and up-regulation of p21^Waf1/Cip1 and p27^Kip1. In addition, sc-560 induced macroautophagy, an emerging mechanism of tumor suppression, as evidenced by the formation of LC3⁺ autophagic vacuoles, enhanced LC3 processing, and the accumulation of acidic vesicular organelles and autolysosomes. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the processing of LC3 induced by sc-560. To conclude, this study reveals the unreported relationship between COX-1 and proliferation/macroautophagy of colon cancer cells.     

A novel histone deacetylase inhibitor Chidamide induces apoptosis of human colon cancer cells

Many studies have demonstrated that histone deacetylase (HDAC) inhibitors induce various tumor cells to undergo apoptosis, and such inhibitors have been used in different clinical trials against different human cancers. In this study, we designed and synthesized a novel HDAC inhibitor, Chidamide. We showed that Chidamide was able to increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, which resulted in arresting colon cancer cells at the G1 phase of the cell cycle and promoting apoptosis. As a result, the proliferation of colon cancer cells was suppressed in vitro. Our data support the potential application of Chidamide as an anticancer agent in treating colon cancer. Future studies are needed to demonstrate its in vivo efficacy.     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.     

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.     

MicroRNA-5p and -3p co-expression and cross-targeting in colon cancer cells

Background

Two mature miRNA species may be generated from the 5’ and 3’ arms of a pre-miRNA precursor. In most cases, only one species remains while the complementary species is degraded. However, co-existence of miRNA-5p and -3p species is increasingly being reported. In this work, we aimed to systematically investigate co-expression of miRNA-5p/3p in colon cancer cells in a genome-wide analysis, and to examine cross-targeting of the dysregulated miRNAs and 5p/3p species.

Results

Four colon cancer cell lines were examined relative to two normal colon tissues. Of the 1,190 miRNAs analyzed, 92 and 36 were found to be up- or down-regulated, respectively, in cancer cells. Nineteen co-expressed miRNA-5p/3p pairs were further identified suggesting frequent 5p/3p co-accumulation in colon cancer cells. Of these, 14 pairs were co-up-regulated and 3 pairs were co-down-regulated indicating concerted 5p/3p dysregulation. Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process. Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor. Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study. The findings of let-7d-5p/3p and multiple other miRNAs targeting IGF1R, KRAS and other metastasis-related factors suggest that 5p/3p miRNAs contribute to cross-targeting of multiple cancer-associated factors and processes possibly to evade functional abolishment when any one of the crucial factors are inactivated.

Conclusions

miRNA-5p/3p species are frequently co-expressed and are coordinately regulated in colon cancer cells. In cancer cells, multiple cross-targeting by the miRNAs, including the co-existing 5p/3p species, frequently occurs in an apparent safe-proof scheme of miRNA regulation of important tumorigenesis processes. Further systematic analysis of co-existing miRNA-5p/3p pairs in clinical tissues is important in elucidating 5p/3p contributions to cancer pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0095-x) contains supplementary material, which is available to authorized users.     

Up-regulation of CHAF1A,a poor prognostic factor,facilitates cell proliferation of colon cancer

Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) was significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.     

Histone deacetylase inhibitor BL1521 induces a G1-phase arrest in neuroblastoma cells through altered expression of cell cycle proteins

Histone deacetylase inhibitors (HDACi) have been discovered as potential drugs for cancer treatment. The effect of BL1521, a novel HDACi, on the cell cycle distribution and the induction of apoptosis was investigated in a panel of MYCN single copy and MYCN amplified neuroblastoma cell lines. BL1521 arrested neuroblastoma cells in the G1 phase and induced up to 30% apoptosis. Downregulation of CDK4, upregulation of p21(WAF1/CIP1) and an increase of hypophosphorylated retinoblastoma protein were observed, indicating a possible mechanism for the cell-cycle arrest. BL1521 also induced downregulation of p27, which may underlie the observed induction of apoptosis.     

MicroRNA expression profile of colon cancer stem-like cells in HT29 adenocarcinoma cell line

Increasing evidence has suggested cancer stem cells (CSCs) are considered to be responsible for cancer formation, recurrence, and metastasis. Recently, many studies have also revealed that microRNAs (miRNAs) strongly implicate in regulating self renewal and tumorigenicity of CSCs in human cancers. However, with respect to colon cancer, the role of miRNAs in stemness maintenance and tumorigenicity of CSCs still remains to be unknown. In the present study, we isolated a population of colon CSCs expressing a CD133 surface phenotype from human HT29 colonic adenocarcinoma cell line by Flow Cytometry Cell Sorting. The CD133⁺ cells possess a greater tumor sphere-forming efficiency in vitro and higher tumorigenic potential in vivo. Furthermore, the CD133⁺ cells are endowed with stem/progenitor cells-like property including expression of “stemness” genes involved in Wnt2, BMI1, Oct3/4, Notch1, C-myc and other genes as well as self-renewal and differentiation capacity. Moreover, we investigated the miRNA expression profile of colon CSCs using miRNA array. Consequently, we identified a colon CSCs miRNA signature comprising 11 overexpressed and 8 underexpressed miRNAs, such as miR-429, miR-155, and miR-320d, some of which may be involved in regulation of stem cell differentiation. Our results suggest that miRNAs might play important roles in stemness maintenance of colon CSCs, and analysis of specific miRNA expression signatures may contribute to potential cancer therapy.     

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.     

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of ³⁵S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.     

Anandamide inhibits Cdk2 and activates Chk1 leading to cell cycle arrest in human breast cancer cells

This study was designed to determine the molecular mechanisms underlying the anti-proliferative effect of the endocannabinoid anandamide on highly invasive human breast cancer cells, MDA-MB-231. We show that a metabolically stable analogue of anandamide, Met-F-AEA, induces an S phase growth arrest correlated with Chk1 activation, Cdc25A degradation and suppression of Cdk2 activity. These findings demonstrate that Met-F-AEA induced cell cycle blockade relies on modulated expression and activity of key S phase regulatory proteins. The observed mechanism of action, already reported for well-known chemotherapeutic drugs, provides strong evidence for a direct role of anandamide related compounds in the activation of cell cycle checkpoints.     

结肠癌患者血清及癌组织中VEGF的表达水平及临床意义

目的探讨结肠癌患者血清及癌组织中血管内皮生长因子（VEGF）的表达水平及临床意义。方法选择不同TNM分期的结肠癌患者88例,同时收集因外伤而切除部分结肠组织的患者43例作为对照组,采用酶联免疫吸附法（ELISA）检测不同患者在结肠癌手术前后血清中VEGF水平,利用实时定量PCR,免疫印迹及免疫组织化学染色法测定癌组织及正常对照组中VEGF表达水平,分析VEGF在血清及癌组织中表达水平与临床病理因素相关性。结果 TNM各期结肠癌患者手术前后血清VEGF水平均高于对照组,且差异有统计学意义（P〈0.05）。各期结肠癌患者术前血清VEGF表达高于术后表达水平,差异有统计学意义（P〈0.05）。与对照组相比,结肠癌组织中VEGF表达水平明显高于对照组,差异有统计学意义（P〈0.01）。结论血清及癌组织中VEGF表达水平与结肠癌的临床病理分级及血管浸润呈正相关,有望作为结肠癌病理分级和手术疗效及预后的评价指标。     

Lactoferrin induces growth arrest and nuclear accumulation of Smad-2 in HeLa cells

Lactoferrin (Lf) is a multifunctional glycoprotein. Due to its anti-inflammatory and anti-cancer properties and the resulting therapeutical potential, lactoferrin is at present focus of a variety of research areas. The regulation of cell growth represents one of the prominent performances of lactoferrin. In this study we found lactoferrin to inhibit proliferation of the human epithelial cancer cell line HeLa. The extent of this growth inhibition was comparable to the one induced by the transforming-growth-factor-beta-1 (TGFβ1). In contrast to other cell lines where lactoferrin stimulates growth, lactoferrin failed to activate the MAP kinases ERK1/2 or p38 in HeLa cells. However, by immunocytochemistry and cell fractionation experiments, we found that lactoferrin is capable of activating the TGFβ/Smad-2 pathway. The nuclear accumulation of Smad-2 induced by Lf was comparable in magnitude to the one induced by TGFβ1.