PA28β regulates cell invasion of gastric cancer via modulating the expression of chloride intracellular channel 1

PA28β is a subunit of proteasome activator PA28. Previous study suggests that PA28β is involved in the invasiveness and metastasis of gastric adenocarcinoma (GA), however, the mechanism is not fully understood. In the present study, we showed that invasive abilities of gastric cancer cells were enhanced when PA28β being down-regulated, and were inhibited when PA28β being overexpressed. To explore the possible mechanism of PA28β associated elevated invasiveness, the protein profiles of PA28β knock down and parental negative control gastric cancer cells were compared using proteomics approach. The results revealed that there were 43 proteins were differentially expressed, among them, chloride intracellular channel 1 (CLIC1) was significantly up-regulated and selected for further functional study. Down-regulation of CLIC1 by RNA interference was able to markedly inhibit cell invasion of PA28β knock down gastric carcinoma cells. In addition, an inverse correlation between PA28β and CLIC1 expressions was also verified in GA tissue samples, suggesting that knockdown of PA28β could enhance tumor invasion and metastasis, at least in part, through up-regulation of CLIC1. Our results provide novel insight into the mechanisms of PA28β related invasiveness and metastasis of GA, and suggest new alternative approaches for GA treatment.     

Overexpression of CLIC1 in human gastric carcinoma and its clinicopathological significance

Gastric cancer is the second most common cancer worldwide and the fifth leading cause of cancer-related death in Taiwan. Identification of biomarkers is essential to improve patient survival. Fifty aberrantly expressed proteins were identified using 2-DE combined with MALDI TOF MS and were grouped based on their function. The overexpression of proteins was confirmed using real-time quantitative RT-PCR, Western blot, and immunohistochemical analysis. The clinicopathological correlations and prognostic significance of these aberrantly expressed proteins were evaluated to determine the novel gastric cancer biomarkers. In this study, expression of chloride intracellular channel 1 (CLIC1) is significantly up-regulated in 67.9% of gastric patients and was selected for further study. The CLIC1 expression in tumor tissues was increased by 1.95-fold (range, 0.01-6.19-fold) compared with that expressed by adjacent noncancerous mucosa. Elevated CLIC1 expression was strongly correlated with lymph node metastasis, lymphatic invasion, perineural invasion, and pathological staging. Additionally, the 5-year survival rate for the low CLIC1 expression group (n = 28; <1.72-fold) was higher than that for the high CLIC1 expression group (n = 28; >or=1.72-fold) (log rank, p = 0.0300). Experimental results indicate that overexpression of CLIC1 is a potential prognostic marker for gastric cancer.     

Protein Kinase C-ζ stimulates colorectal cancer cell carcinogenesis via PKC-ζ/Rac1/Pak1/β-Catenin signaling cascade

Colorectal cancer (CRC) is the second most common cancer in the world and death from CRC accounts for 8% of all cancer deaths both in men and women in the United States. CRC is life-threatening disease due to therapy resistant cancerous cells. The exact mechanisms of cell growth, survival, metastasis and inter & intracellular signaling pathways involved in CRC is still a significant challenge. Hence, investigating the signaling pathways that lead to colon carcinogenesis may give insight into the therapeutic target. In this study, the role of atypical Protein Kinase C (aPKC) on CRC was investigated by using two inhibitors of that protein class: 1) ζ-Stat (8-hydroxynaphthalene-1,3,6-trisulfonic acid) is a specific inhibitor of PKC-ζ and 2) ICA-I 5-amino-1-(2,3-dihydroxy-4-hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide) is a specific inhibitor of PKC-ι. The cell lines tested were CCD18CO normal colon epithelial and LOVO metastatic CRC cells. The inhibition of aPKCs did not bring any significant toxicity on CCD18CO normal colon cell line. Although PKC-ι is an oncogene in many cancers, we found the overexpression of PKC-ζ and its direct association with Rac1. Our findings suggest that the PKC-ζ may be responsible for the abnormal growth, proliferation, and migration of metastatic LOVO colon cancer cells via PKC-ζ/Rac1/Pak1/β-Catenin pathway. These results suggest the possibility of utilizing PKC-ζ inhibitor to block CRC cells growth, proliferation, and metastasis.     

Conditioned media from adipocytes promote proliferation,migration, and invasion in melanoma and colorectal cancer cells

Epidemiological evidence suggests that obesity can significantly increase the risk of various cancers, although the mechanisms underlying this link are completely unknown. Here, we analyzed the effect of adipocytes on melanoma and colon cancer cells proliferation, migration, and invasion. The potential effects of conditioned media (CM) obtained from differentiated mouse 3T3-L1 cells and human adipose tissue-derived mesenchymal stem cells (hAMSC) on the proliferation, migration, and invasion of B16BL6 melanoma and colon 26-L5 cancer cells were investigated. The 3T3-L1 and hAMSC CM increased cell proliferation, migration, and invasion in both the cell lines. In addition, adipocytes CM increased matrix metalloproteinase 9 (MMP-9) and MMP-2 activity in both B16BL6 and colon 26-L5 cells. These effects were found to be associated with an increased expression of various oncogenic proteins in B16BL6 and colon 26-L5 cells. Also, adipocyte CM induced Akt and mTOR activation in both tumor cell lines, and the pharmacological inhibition of Akt and mTOR blocked the CM induced Akt as well as mTOR activation and CM-stimulated melanoma and colon cancer cell proliferation, migration, and invasion. These data suggest that adipocyte promotes melanoma and colon cancer progression through modulating the expression of diverse proteins associated with cancer growth and metastasis as well as modulation of the Akt/mTOR signaling.     

MAPK‐mediated upregulation of fibrinogen‐like protein 2 promotes proliferation,migration, and invasion of colorectal cancer cells

Fibrinogen‐like protein 2 (FGL2) has been reported to play a key role in the development of human cancers. However, it is still unmasked whether FGL2 plays a potential role in colorectal carcinogenesis. In this study, the messenger RNA and protein expression levels were measured by quantitative real‐time polymerase chain reaction and western blot. Cell counting kit‐8 assay, transwell migration, and invasion assay were carried out to evaluate the proliferation, migration, and invasion of LOVO and SW620 cells. FGL2 was upregulated in colorectal cancer (CRC) tissues, as well as cell lines. Mitogen‐activated protein kinase (MAPK) signaling was activated in CRC tissues and cell lines. FGL2 was confirmed to be downregulated by MAPK signaling inhibitor U0126. Further, we determined that knockdown of FGL2 caused a reduction of proliferation, migration, and invasion in LOVO and SW620 cells. Consistently, treatment of LOVO and SW620 cells with U0126 led to a decrease in cell proliferation, migration, and invasion. However, these changes initiated by U0126 were abolished by FGL2 overexpression. To conclude, MAPK‐mediated upregulation of FGL2 promotes the proliferation, migration, and invasion of CRC cells.     

弱碱性消癌液抑制乳腺癌细胞生长、转移并介导其消亡

为了研究新的肿瘤治疗方法,设计了1种弱碱性消癌液（weak alkaline cancer-eliminating liquid,WACEL）,测试WACEL是否抑制癌细胞的生长转移及消亡.在体外,检测WACEL对3种细胞株,即子宫颈鳞癌细胞SiHa、非小细胞肺鳞癌细胞H1299、人乳腺癌细胞MDA-MB-231的生长增殖、细胞周期、细胞凋亡、侵袭转移的影响. 在体内,建立人乳腺癌裸鼠颈背皮下移植瘤模型,观察WACEL对裸鼠皮下肿瘤生长转移的作用. 研究结果发现,WACEL明显抑制3种细胞系的生长增殖,G2/M期细胞增多,出现G2/M期阻滞,阻止细胞周期的进程. 细胞形态呈圆状,细胞核浓缩,caspase-3活性检测增加,线粒体的膜电位降低,细胞凋亡;活细胞数目减少,细胞膜破裂,发生消亡现象,癌细胞迁移明显减少（P﹤0.001）.目标基因SCCA1、cyclinB1、MMP-2以及MMP-9 的mRNA表达水平下调,caspase-3的mRNA表达水平上调;SCCA1、cyclinB1和MMP-2蛋白表达下调,caspase-3蛋白表达上调.体内动物实验发现,处理组的肿瘤生长较对照组明显缓慢,肺组织HE染色未见明显癌细胞转移,而对照组可见癌细胞转移. WACEL能抑制乳腺癌细胞的生长、转移并介导其消亡.本研究系统分析WACEL与癌细胞本身及其pH微环境之间的相互作用机制,发现其改变癌细胞所处的微环境的重要性.     

Erlotinib inhibits colon cancer metastasis through inactivation of TrkB-dependent ERK signaling pathway

The distal metastasis is the main cause of death in patients with colon cancer. Tyrosine receptor kinase B (TrkB) and ERK signals may be the potential targets for the treatment of colon cancer metastasis. This study aims to investigate whether erlotinib inhibits distant metastasis of colon cancer by regulating TrkB and ERK signaling pathway. Human colon adenocarcinoma cell lines (SW480 and Caco-2) pretreated with exogenous C-X-C motif chemokine ligand 8 (CXCL8) were used to assess the suppressive effect of erlotinib on tumor metastasis, including anoikis, epithelial-mesenchymal transformation (EMT), migration, and invasion. Through TrkB overexpression, Akt suppression, and ERK suppression, the roles of TrkB, Akt, and ERK in erlotinib-induced metastasis inhibition of colon cancer cells were explored. The results showed that erlotinib alleviated CXCL8-induced metastasis of the colon cancer cells. Overexpression of TrkB in colon cancer cells eliminated the effect of erlotinib on anoikis, inhibition of EMT, migration, and invasion, and downregulation of p-ERK and p-Akt. Furthermore, the inhibition of ERK activation instead of Akt activation was found to participate in erlotinib-mediated metastasis resistance, including anoikis, inhibition of EMT, migration, and invasion. In conclusion, erlotinib inhibits colon cancer cell anoikis resistance, EMT, migration, and invasion by inactivating TrkB-dependent ERK signaling pathway.     

Chloride intracellular channel 1 identified using proteomic analysis plays an important role in the radiosensitivity of HEp‐2 cells via reactive oxygen species production

The nature of the molecules underlying the radioresistance phenotype of laryngeal cancer cells remains to be established. We initially generated radioresistant laryngeal cancer cell lines from human HEp‐2 cells with fractionated radiation. These RR‐HEp‐2 cells and isolated clones displayed more radioresistant and anti‐apoptotic phenotypes than parental HEp‐2 cells after radiation. Characteristics of RR‐Hep‐2 cell lines were confirmed by upregulation of radioresistance‐related genes, such as epidermal growth factor receptor, Hsp90, and Bcl‐xl. Subsequently, we examined proteome changes between HEp‐2 and RR‐HEp‐2 cells and identified 16 proteins showing significantly altered expression levels. Interestingly, protein expression of chloride intracellular channel 1 (CLIC1) was markedly suppressed in RR‐HEp‐2 cells, compared with non‐irradiated control cells. Suppression of CLIC1 with an indanyloxyacetic acid‐94 or small interfering RNA led to radioresistance in HEp‐2 cells by suppressing the radiation‐induced cellular ROS level. However, ectopic overexpression of CLIC1 induced radiosensitivity in RR‐HEp‐2 cells via induction of ROS level after radiation, suggesting that the protein acts as a positive regulator of ROS production. Our results collectively indicate that suppression of CLIC1 contributes to acquisition of the radioresistance phenotype of laryngeal cancer cells via inhibition of ROS production, implying that this protein is an important candidate molecule for radiotherapy in radioresistant laryngeal cancer cells.     

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.     

NDRG1对人肠癌细胞失巢凋亡的影响

目的:探讨NDRG1对体外培养的人肠癌细胞系失巣凋亡的影响。方法:采用慢病毒系统将NDRG1表达单元转入人肠癌细胞系SW620、HCT8中,建立相应的过表达稳定细胞系;通过siRNA的方法干扰HCT116和LOVO细胞系中NDRG1的表达,分别在非贴壁培养的情况下培养48小时,采用流式细胞术和TUNEL染色检测细胞的凋亡情况。结果:在贴壁培养条件下,NDRG1过表达并没有显著影响肠癌细胞的生长及增殖,而NDRG1特异性siRNA干扰HCT116细胞中NDRG1的表达后,其凋亡率无明显变化(P0.05)。在悬浮培养条件下,NDRG1过表达的肠癌细胞的失巢凋亡率显著低于正常对照组(P0.05),而用三种不同的siRNA干扰HCT116及LOVO细胞中NDRG1的表达后,其失巢凋亡率均显著高于正常对照组(P0.05)。结论:NDRG1在体外可抑制人肠癌细胞的失巢凋亡。     

Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.     

Silencing of Discoidin Domain Receptor-1 (DDR1) Concurrently Inhibits Multiple Steps of Metastasis Cascade in Gastric Cancer

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by interacting with the surrounding collagen matrix. In this study, we investigated the expression and role of DDR1 in human gastric cancer metastasis. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1-expressing MKN74 gastric cancer cells and corresponding DDR1-silenced cells. The effects of DDR1 on tumor growth and metastasis were examined in orthotopically implanted and experimental liver metastasis models in nude mice. The expression of DDR1 in surgical specimens was analyzed by immunohistochemistry. DDR1 was expressed in human gastric cancer cell lines, and its expression in human gastric tumors was associated with poor prognosis. Among seven gastric cancer cell lines, MKN74 expressed the highest levels of DDR1. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumors significantly reduced angiogenesis and lymphangiogenesis, thereby leading to reductions in lymph node metastasis and liver metastasis. In a model of experimental liver metastasis, DDR1-silenced cells almost completely inhibited liver colonization and metastasis. DDR1 deficiency led to reduced expression of the genes encoding vascular endothelial growth factor (VEGF)-A, VEGF-C, and platelet-derived growth factor-B. These results suggest that DDR1 is involved in gastric cancer tumor progression and that silencing of DDR1 inhibits multiple steps of the gastric cancer metastasis process.     

Interleukin 4 inhibits hepatocyte growth factor-induced invasion and migration of colon carcinomas

Hepatocyte growth factor (HGF) is known to have a number of biological properties including promoting tumor progression of human carcinomas. Metastasis involves a number of events that are attributed to induction by paracrine factors such as HGF. Identification of natural inhibitors of these events would allow better control of tumor progression. Recently we demonstrated that interleukin 4 (IL-4) can regulate proliferation of various human carcinoma cell lines. In the present study, we used established human colon carcinoma cell lines and primary colon carcinoma cell cultures to determine if IL-4 could regulate HGF-induced cell proliferation and other events of tumor progression such as MMP (matrix metalloproteinases)-1, -2, and -9 production, cell migration and cell-matrix invasive activity. All colon carcinoma cell lines expressed HGF and IL-4 receptors. IL-4 significantly inhibited HGF-induced proliferation of one cell line. Cell-matrix invasion was significantly enhanced by HGF (0.1–10 ng/ml); IL-4 (1–10 U/ml) significantly inhibited HGF-induced invasion in a dose-dependent manner. IL-4 also inhibited HGF-induced cell-matrix invasion of metastatic colon carcinoma cells and HGF-induced cell migration. HGF enhanced MMP-1, -2, and -9 production by cell lines. This effect could be inhibited by IL-4. These findings indicate that IL-4 is a potent inhibitor of HGF-induced invasion and metastasis-related functions of human colon carcinoma cells. © 1996 Wiley-Liss, Inc.     

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.     

Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5‘-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.