Journal of Experimental & Clinical Cancer Research - Latest Articles

The immunosuppressive factors IL-10, TGF-beta, and VEGF do not affect the antigen-presenting function of CD40-activated B cells

Alexander Shimabukuro-Vornhagen — 2012-05-16T00:00:00Z

Background: Progress in recent years strengthened the concept of cellular tumor vaccinations. However, a crucial barrier to successful cancer immunotherapy is tumor-mediated immunosuppression. Tumor-derived soluble factors such as IL-10, TGF-beta, and VEGF suppress effector cells either directly or indirectly by disruption of dendritic cell (DC) differentiation, migration and antigen presentation. Human B cells acquire potent immunostimulatory properties when activated via CD40 and have been shown to be an alternative source of antigen-presenting cells (APCs) for cellular cancer vaccines. Nevertheless, in contrast to DCs little knowledge exists about their susceptibility to tumor derived immunosuppressive factors. Thus, we assessed whether IL-10, TGF-beta, or VEGF do affect key aspects of the immunostimulatory function of human CD40-activated B cells. Methods: Cell surface expression of adhesion and costimulatory molecules and the proliferation capacity of CD40-activated B cells were compared to untreated controls by flow cytometry. Migration towards important chemokines of secondary lymph organs was measured with or without exposure to the immunosuppressive cytokines. Finally, an influence on T cell stimulation was investigated by allogeneic mixed lymphocyte reactions. For statistical analysis Student's t test or two-way analysis of variance followed by Bonferroni's post-hoc test was used to compare groups. P values of <0.05 were considered statistically significant. Results: Neither cell adhesion nor the expression of MHC class II and costimulatory molecules CD80 and CD86 was inhibited by addition of IL-10, TGF-beta, or VEGF. Likewise, the proliferation of CD40-activated B cells was not impaired. Despite being exposed to IL-10, TGF-beta, or VEGF the B cells migrated equally well as untreated controls to the chemokines SLC and SDF-1alpha. Most importantly, the capacity of CD40-activated B cells to stimulate CD4+ and CD8+ T cells remained unaffected. Conclusion: Our findings suggest that key immunostimulatory functions of CD40-activated B cells are resistant to inhibition by the immunosuppressive factors IL-10, TGF-beta, and VEGF. This supports considerations to use ex vivo generated CD40-activated B cells as a promising alternative or additional APC for cellular immunotherapy, especially in settings where these immunosuppressive cytokines are present in tumor environment.

A selective aryl hydrocarbon receptor modulator 3,3'-Diindolylmethane inhibits gastric cancer cell growth

Xiao-Fei Yin — 2012-05-16T00:00:00Z

Background: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor associated with gastric carcinogenesis. 3,3'-Diindolylmethane (DIM) is a relatively non-toxic selective AhR modulator. This study was to detect the effects of DIM on gastric cancer cell growth. Methods: Gastric cancer cell SGC7901 was treated with DIM at different concentrations (0, 1020304050umol/L) with or without an AhR antagonist , resveratrol. The expression of AhR and Cytochrome P4501A1 (CYP1A1)a classic target gene of AhR pathway, were detected by RT-PCR and Western blot; cell viability was measured by MTT assay, and the changes in cell cycle and apoptosis were analyzed by flow cytometry. Results: RT-PCR and western-blot showed that with the increase of the concentration of DIM, AhR protein gradually decreased and CYP1A1 expression increased, suggesting that DIM activated the AhR pathway and caused the translocation of AhR from cytoplasm to nucleus. MTT assay indicated that the viability of SGC7901 cells was significantly decreased in a concentration- and time-dependent manner after DIM treatment and this could be partially reversed by resveratrol. Flow cytometry analysis showed that DIM arrested cell cycle in G1 phase and induced cell apoptosis. Conclusion: Selective aryl hydrocarbon receptor modulator 3,3'-Diindolylmethane inhibits SGC7901 cell proliferation by inducing apoptosis and delaying cell cycle progression. AhR may be a potential therapeutic target for gastric cancer treatment.

Interspecies differences in membrane-associated protease activities of thyrocytes and their relevance for thyroid cancer studies

Eleonore Fröhlich — 2012-05-16T00:00:00Z

Background: To understand the role of proteases involved in human thyroid cancer progression and tissue invasion, thyrocytes from other species could potentially be used provided their characteristics are similar. It is not known whether dipeptidyl peptidase IV and aminopeptidase N activities, which are overexpressed in human thyroid cancer, are, as in human, also absent in normal thyrocytes of other species, making them suitable models for studies on the regulation of these proteases. Methods: To assess the role of these proteases, activity was measured in thyroid tissue of human, mouse, rat, porcine, bovine and ovine origin. The lysosomal protease, dipeptidyl peptidase II, was used for comparison. Results: Murine, rat, ovine, bovine and human thyrocytes all lacked dipeptidyl peptidase IV and aminopeptidase N activity, but porcine thyrocytes were found to possess both. In contrast, lysosomal dipeptidyl peptidase II was strongly expressed in all species. These activity patterns were maintained in cultured cells. Cultured porcine thyrocytes formed follicles with typical morphology upon stimulation with TSH but differed from human thyrocytes in their response to thiamazole. Conclusions: These species differences in the expression of dipeptidyl peptidase IV and aminopeptidase N, indicate that porcine thyrocytes cannot be considered appropriate for the study of proteases in human cancer development.

Elevated expression of chloride intracellular channel 1 is correlated with poor prognosis in human gliomas

Liang Wang — 2012-05-11T00:00:00Z

Background Chloride intracellular channel 1 (CLIC1) is expressed ubiquitously in human tissues and is involved in the regulation of cell cycle, cell proliferation and differentiation. Recent studies have shown that CLIC1 is highly expressed in several human malignant tumors. However, its roles in human gliomas are still unclear. The aim of this study was to investigate the clinicopathological significance and prognostic value of CLIC1 expression in human gliomas. Methods CLIC1 expression in human gliomas and nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay and immunohistochemistry. Its association with clinicopathological factors or prognosis in patients with gliomas was statistically analyzed. Results The expression of CLIC1 at both mRNA and protein levels was significantly increased in high-grade (Grade III~IV) glioma tissues compared with that in low-grade (Grade I~II) and nonneoplastic brain tissues, and was up-regulated with ascending tumor World Health Organization (WHO) grades. The elevated expression of CLIC1 protein was also significantly correlated with low Karnofsky performance score (KPS) (P=0.008). Moreover, both univariate and multivariate analysis shown that high CLIC1 expression was significantly associated with poor prognosis in patients with gliomas (P<0.001 and P=0.01, respectively). In particular, the elevated CLIC1 expression also correlated with shorter overall survival in different glioma subgroups stratified according to the WHO grading. Conclusions Our data provide the first evidence that CLIC1 expression might play an important role in the regulation of aggressiveness in human gliomas. The elevated expression of CLIC1 might represent a valuable prognostic marker for this disease.

Claudin-5 is involved in breast cancer cell motility through the N-WASP and ROCK signalling pathways

Astrid Escudero-Esparza — 2012-05-04T00:00:00Z

Background Recent studies have shown dysregulation in TJ structure of several cancers including breast. Claudin-5 is a protein member of the TJ structure expressed in both endothelial and epithelial cells. This study examined the level of expression and distribution of Claudin-5 in human breast cancer tissues and the effect of knockdown and forced expression of Claudin-5 in the MDA-MB-231 breast cancer cell line.Methods Immunohistochemistry and quantitative-PCR were used to analyse patient tissue samples. The Claudin-5 gene was cloned and overexpressed or knocked down using ribozyme technology in human breast cancer cells. Changes in function were assessed using in vitro assays for invasion, growth, adhesion, wounding, motility, transepithelial resistance and electric cell-substrate impedance sensing. Changes in cell behaviour were achieved through the use of Hepatocyte Growth factor (HGF) which we have shown to affect TJ function and expression of TJ proteins. In addition, an in vivo model was used for tumour growth assays. Results data was analyzed using a Students two sample t-test and by Two-way Anova test when the data was found to be normalized and have equal variances. In all cases 95% confidence intervals were used.Results Patients whose tumours expressed high levels of Claudin-5 had shorter survival than those with low levels (p=0.004). Investigating in vitro the effect of altering levels of expression of Claudin-5 in MDA-MB-231cells revealed that the insertion of Claudin-5 gene resulted in significantly more motile cells (p<0.005). Low levels of Claudin-5 resulted in a decrease in adhesion to matrix (p<0.001). Furthermore, a possible link between Claudin-5 and N-WASP, and Claudin-5 and ROCK was demonstrated when interactions between these proteins were seen in the cells. Moreover, followed by treatment of N-WASP inhibitor (Wiskostatin) and ROCK inhibitor (Y-27632) cell motility was assessed in response to the inhibitors. Results showed that the knockdown of Claudin-5 in MDA-MB-231 masked their response after treatment with N-WASP inhibitor; however treatment with ROCK inhibitor did not reveal any differences in motility in this cell line.Conclusions This study portrays a very new and interesting role for Claudin-5 in cell motility involving the N-WASP signalling cascade indicating a possible role for Claudin-5 in the metastasis of human breast cancer.

The role of estrogen receptor alpha in mediating chemoresistance in breast cancer cells

Jiang Zhinong — 2012-05-03T00:00:00Z

IntroductionPrevious studies suggested that estrogen receptor alpha (ERalpha) plays an important role in the chemoresistance of breast cancers. However, large random trials failed to demonstrate any benefit of the concurrent estrogen antagonist tamoxifen on the chemotherapy efficacy. Thus, in the present study, the importance of the role of ERalpha in the chemoresistance of breast cancer cells was investigated. Methods: The ERalpha-transfected Bcap37 cells and natural ERalpha-positive T47D breast cancer cells were treated using chemotherapeutic agents with or without 17-beta estradiol (E2) pretreatment. Their viabilities were assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. The dead cell rates were determined using propidium iodide dye exclusion tests, and the expression levels of Bcl-2 and Bax were detected through Western blot analysis. The effects of E2 on the growth of breast cancer cells were also determined via cell growth curve and cell cycle analysis. Results: ERalpha activation by E2 increased the sensitivity of natural ERalpha-positive T47D breast cancer cells to chemotherapeutic agents. However, the increase in ERalpha expression in ERalpha-negative Bcap37 breast cancer cells also significantly increased their resistance. These phenomena cannot be explained by asserting that ERalpha mediated the chemoresistance of breast cancer cells by regulating the expression of Bcl-2 and Bax. Our findings show that ERalpha activation upregulated the expression of Bcl-2 in natural ERalpha-positive T47D breast cancer cells, whereas ERalpha activation by E2 downregulated and upregulated the Bcl-2 and Bax expression levels, respectively, in ERalpha-transfected Bcap37 cells. This phenomenon was due to the influence of ERalpha on the growth of breast cancer cells. Specifically, ERalpha activation enhanced the growth of natural ERalpha-positive breast cancer cells and thus increased their sensitivity to chemotherapeutic agents. However, ERalpha activation also inhibited the growth of ERalpha-transfected Bcap37 cells and increased the resistance of cancer cells to chemotherapeutic agents. Chemoresistance of ERalpha-transfected Bcap37 cells was only due to the specific growth inhibition by E2, which is not applicable to common ERalpha-positive breast cancer cells. Conclusions: Although ERalpha was associated with chemoresistance of breast cancers, ERalpha itself did not mediate this resistance process.

Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

John Hornick — 2012-05-02T00:00:00Z

Background: Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Results: Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, alpha-tocopherol (alpha-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though alpha-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and alpha-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Conclusions: Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and alpha-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways.

In vitro the behaviors of metastasis with suppression of VEGF in human bone metastatic LNCaP-derivative C4-2B prostate cancer cell line

Lei Yang — 2012-05-01T00:00:00Z

Background: Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. VEGF is believed to implicate poor prognosis in various cancers. The overexpression of VEGF may be an early step in the process of metastasis. Methods: ELISA was used to investigate the levels of VEGF, bFGF and IL8 in human bone metastatic LNCaP-derivative C4-2B prostate cancer cell line and its parental cell line, LNCa-P, and to determine the effect of bevacizumab on reducing the level of VEGF. Cell proliferation assay, invasion assay and in vitro angiogenesis assay were performed under the condition with bevacizumab or control IgG. Results: Human bone metastatic LNCaP-derivative C4-2B prostate cancer cell line expressed a higher level of VEGF than its parental primary prostate cancer cell line LNCaP. The effect of bevacizumab is dose-dependent and time-dependent: 100 ug/mL of bevacizumab and 3-day treatment was more effective than low-dose and lesser-day treatment for decreasing the level of VEGF. Bevacizumab is able to suppress cell proliferation, angiogenesis and invasion in human bone metastatic C4-2B prostatic cancer cell line. Conclusions: The overexpression of VEGF can be inhibited by bevacizumab in human bone metastatic cancer cell line. The behaviors of metastasis involving proliferation, angiogenesis and invasion are suppressed by anti-VEGF therapy.

Knockdown of glucose-regulated protein 78 decreases the invasion, metalloproteinase expression and ECM degradation in hepatocellular carcinoma cells

Hongdan Li — 2012-04-30T00:00:00Z

Background: We have reported previously that overexpression of glucose-regulated protein 78 (GRP78) promotes the invasion of hepatocellular carcinoma. However, whether GRP78 knockdown affects the extracellular matrix degradation has not been elucidated. Here we are going to determine whether GRP78 knockdown affect the ECM degradation and the role of MMP-2 and MMP-9 in these process in hepatocellular carcinoma cells. Methods: Human hepatocellular carcinoma cell line SMMC7721 and HepG2 were cultured in DMEM supplemented with 10% FBS, RT-PCR and western blot were used to detect the endogenous expression of GRP78, MMP-2, MMP-9 and TIMP-2 in SMMC7721 and HepG2. GRP78 shRNAs were transfected using lipofection2000. Transwell assay and wound healing assay were used to analyze the invasion of each transfectant. Gelatin zymography and FITC-gelatin degradation assay were employed to investigate the capabilities of ECM degradation of each transfectant. MTT assay was used to determine the proliferation status. Western blot were employed to detect the expression of matrix metalloproteinase 2(MMP-2), MMP-9, MMP-14, and tissue inhibitor of metalloproteinases 2(TIMP-2), focal adhesion kinase (FAK), ERK1/2, JNK and Src. Results: According to the expression levels of GRP78, MMP-2, MMP-9, MMP-14 and TIMP-1 in hepatocellular carcinoma cell lines SMMC7721 and hepG2, we used SMMC7721 as the in vitro invasion model for further functional analysis. Using this model, we found that GRP78 knockdown decreased the invasion of tumor cells, and this inhibitory effect was independent of cell proliferation. In hepatocellular carcinoma cells, Grp78 knockdown inhibited ECM degradation and the decreased activity and expression of MMP-2, but not MMP-9 contributed largely to this impact. Further analysis revealed that the decreased activity and expression of MMP-2 is mediated by JNK. Conclusion: Knockdown of GRP78 decreases ECM degradation, and downregulates the expression and activity of MMP-2 and TIMP-2. These results further demonstrate that GRP78 is a potential target for inhibiting the invasion of hepatocellular carcinoma cells.

Circulating microRNAs in cancer: origin, function and application

Ruimin Ma — 2012-04-30T00:00:00Z

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level. The dysregulation of miRNAs has been linked to a series of diseases, including various types of cancer. Since their discovery in the circulation of cancer patients, there has been a steady increase in the study of circulating miRNAs as stable, non-invasive biomarkers. However, the origin and function of circulating miRNAs has not been systematically elucidated. In this review, we summarize the discovery of circulating miRNAs and their potential as biomarkers. We further emphasize their possible origin and function. Finally, we discuss the application and existing questions surrounding circulating miRNAs in cancer diagnostics. Although several challenges remain to be concerned, circulating miRNAs could be useful, non-invasive biomarkers for cancer diagnosis.