Cancer Cell International - Latest Articles

Validation of suitable endogenous control genes for quantitative PCR analysis of microRNA gene expression in a rat model of endometrial cancer

Sanja Jurcevic — 2013-05-16T00:00:00Z

Background: MicroRNAs are small RNA molecules that negatively regulate gene expression by translational inhibition or mRNA cleavage. The discovery that abnormal expression of particular miRNAs contributes to human disease, including cancer, has spurred growing interest in analysing expression profiles of these molecules. Quantitative polymerase chain reaction is frequently used for quantification of miRNA expression due to its sensitivity and specificity. To minimize experimental error in this system an appropriate endogenous control gene must be chosen. An ideal endogenous control gene should be expressed at a constant level across all samples and its expression stability should be unaffected by the experimental procedure. Results: The expression and validation of candidate control genes (4.5S RNA(H) A, Y1, 4.5S RNA(H) B, snoRNA, U87 and U6) was examined in 21 rat cell lines to establish the most suitable endogenous control for miRNA analysis in a rat model of cancer. The stability of these genes was analysed using geNorm and NormFinder algorithms. U87 and snoRNA were identified as the most stable control genes, while Y1 was least stable. Conclusion: This study identified the control gene that is most suitable for normalizing the miRNA expression data in rat. That reference gene will be useful when miRNAs expression are analyzed in order to find new miRNA markers for endometrial cancer in rat.

CpG island hypermethylation-associated silencing of microRNAs promotes human endometrial cancer

Bi-Lan Li — 2013-05-16T00:00:00Z

Background: Endometrial cancer (EC) is the most common gynecologic malignancy, but the molecular events involved in the development and progression of EC remain unclear. This study aimed to explore epigenetic modification of genes and miRNAs involved in EC development. Methods: Ishikawa and AN3CA cells were treated with 5'-Aza-2-deoxycytidine or histone deacetylase inhibitor. The expression of miRNAs and related genes were detected by PCR and Western blot. Promoter methylation was detected by bisulfite specific PCR sequencing. The proliferation, colony formation, cell cycle progression, migration and invasion of EC cells were evaluated by MTT, soft agar assay, flow cytometry, wound healing and invasion assay, respectively. Results: Aberrant expression of miRNAs including miR-200b, miR130a/b, miR-625 and miR-222 was associated with tumorigenesis and metastasis in endometrial cancer. Silencing of miR-130b induced E-cadherin expression, while ectopic expression of miR-130b and knockdown of DICER1 increased the expression of Vimentin, zeb2, N-cadherin, Twist and Snail in EC cells. Furthermore, 5'-Aza-2-deoxycytidine and Histone deacetylase (HDAC) inhibitor inhibited the proliferation, colony formation, migration and invasion of EC cells, accompanied by reduced MMP secretion. Conclusions: Our study provides the first description of epigenetic modification of epithelial mesenchymal transition associated genes and miRNAs in EC cells, which are extensively involved in the regulation of gene expression and subsequent accumulation of malignant features of EC cells.

Expression patterns of Phf5a/PHF5A and Gja1/GJA1 in rat and human endometrial cancer

Eva Falck — 2013-05-15T00:00:00Z

Endometrial adenocarcinoma is the most frequently diagnosed cancer of the female genital tract in the western world. Studies of complex diseases can be difficult to perform on human tumor samples due to the high genetic heterogeneity in human. The use of rat models is preferable since rat has similarities in pathogenesis and histopathological properties to that of human.A genomic region including the highly conserved Phf5a gene associated to development of EAC has previously been identified in an association study. PHF5A has been suggested to acts as a transcription factor or cofactor in the up regulation of expression of Gja1 gene in the presence of estrogen. It has earlier been shown that the Phf5a gene is down regulated in rat EAC derived cell lines by means of expression microarrays.We analyzed the expression of Phf5a and Gja1 by qPCR, and potential relations between the two genes in EAC tumors and non-malignant cell lines derived from the BDII rat model. In addition, the expression pattern of these genes was compared in rat and human EAC tumor samples.Changes in expression for Phf5a/PHF5A were found in tumors from both rat and human even though the observed pattern was not completely consistent between the two species. By separating rat EAC cell lines according to the genetic background, a significant lower expression of Phf5a in one of the two cross backgrounds was revealed, but not for the other. In contrast to other studies, Phf5a/PHF5A regulation of Gja1/GJA1 was not revealed in this study.

Caspase-2 is involved in cell death induction by taxanes in breast cancer cells

Michael Jelínek — 2013-05-15T00:00:00Z

Background: We studied the role of caspase-2 in apoptosis induction by taxanes (paclitaxel, novel taxane SB-T-1216) in breast cancer cells using SK-BR-3 (nonfunctional p53, functional caspase-3) and MCF-7 (functional p53, nonfunctional caspase-3) cell lines. Results: Both taxanes induced apoptosis in SK-BR-3 as well as MCF-7 cells. Caspase-2 activity in SK-BR-3 cells increased approximately 15-fold within 48 h after the application of both taxanes at the death-inducing concentration (100 nM). In MCF-7 cells, caspase-2 activity increased approximately 11-fold within 60 h after the application of taxanes (300 nM). Caspase-2 activation was confirmed by decreasing levels of procaspase-2, increasing levels of cleaved caspase-2 and the cleavage of caspase-2 substrate golgin-160. The inhibition of caspase-2 expression using siRNA increased the number of surviving cells more than 2-fold in MCF-7 cells, and at least 4-fold in SK-BR-3 cells, 96 h after the application of death-inducing concentration of taxanes. The inhibition of caspase-2 expression also resulted in decreased cleavage of initiator caspases (caspase-8, caspase-9) as well as executioner caspases (caspase-3, caspase-7) in both cell lines after the application of taxanes. In control cells, caspase-2 seemed to be mainly localized in the nucleus. After the application of taxanes, it was released from the nucleus to the cytosol, due to the long-term disintegration of the nuclear envelope, in both cell lines. Taxane application led to some formation of PIDDosome complex in both cell lines within 24 h after the application. After taxane application, p21WAF1/CIP1 expression was only induced in MCF-7 cells with functional p53. However, taxane application did not result in a significant increase of PIDD expression in either SK-BR-3 or MCF-7 cells. The inhibition of RAIDD expression using siRNA did not affect the number of surviving SK-BR-3 and MCF-7 cells after taxane application at all. Conclusion: Caspase-2 is required, at least partially, for apoptosis induction by taxanes in tested breast cancer cells. We suggest that caspase-2 plays the role of an apical caspase in these cells. Caspase-2 seems to be activated via other mechanism than PIDDosome formation. It follows the release of caspase-2 from the nucleus to the cytosol.

Depleting NFAT1 expression inhibits the ability of invasion and migration of human lung cancer cells

Ji-fu Liu — 2013-05-12T00:00:00Z

Background: Nuclear factor of activated T-cells (NFAT) is a general name applied to a family of transcription factors shown to be important in immune response. One or more members of the NFAT family are expressed in most cells of the immune system. NFAT1 is considered to involve in the development of cardiac, skeletal muscle, nervous systems, and tumorigenesis. Methods: In the current study, we analyzed MEKK1 expression in 159 surgically resection non-small cell lung cancer patient's samples by immunohistochemistry and determined its role in SK-EMS-1 cells via RNAi experiment. Results: The abilities of invasion, motility, and adhesion of SK-EMS-1 cells were detected by transwell assay, wound healing assay and adhesion assay, respectively. The result showed NFAT1 was highly expressed in lung tumor tissues instead of adjacent lung tissues (54.1% vs 8.8%, p < 0.05); its overexpression was positively correlated with lymph node metastasis (p < 0.05). Depleting its expression in SK-EMS-1 cells can inhibit its invasion and migration abilities significantly (p < 0.05); and also can reduce proliferation of lung cancer cells (p < 0.05). Conclusion: Our study showed NFAT1 plays an important role in origination, invasion and metastasis of non-small lung cancer cells; its underlying action mechanism needs further study.

Cancer: a ¿stem-cell¿ disease?

Shi-Ming Tu — 2013-05-06T00:00:00Z

Background: Nowadays, we believe that cancer is a genetic disease. We focus on the genetic targets and epigenetic changes in a tumor. Remarkably, many crucial signal pathways in a malignant cell involve “stem-ness” genes. The prevalence of stem-ness in cancer suggests that cancer has a stem-cell origin and is a stem-cell disease.Presentation of the hypothesisThe observation that many innate stem-ness properties are easily interchangeable with malignant hallmarks needs to be further elucidated. There appears to be a malignant potential in every stem cell and a stem cell potential in every malignant cell. I hypothesize that cancer is a stem-cell disease rather than a genetic disease.Testing the hypothesisWe will use homeobox genes to endow a certain progenitor cell with specific stem-ness properties and confer different stem-cell phenotypes to the particular cell type in a hierarchical manner. We will demonstrate that an earlier homeobox gene plus a genetic defect (such as Pten loss) tend to form a more virulent tumor, while a later homeobox gene plus the same genetic defect tend to express a more indolent phenotype. Importantly, we will show that in clinically relevant cancer subtypes, those with worse clinical outcomes may paradoxically harbor fewer genetic mutations than those with better outcomes do.Implications of the hypothesisThe recognition that cancer is a stem-cell disease will instigate major paradigm shifts in our basic understanding of cancer. Many fundamental principles of oncology, such as multistep carcinogenesis, need to be reconciled. The realization that cancer is a stem-cell disease will also have profound clinical implications on personalized care. Many aspects of our current clinical trials need to be reevaluated.

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay

Jo-Anne de la Mare — 2013-05-01T00:00:00Z

Background: The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents. Methods: In this study, the tumoursphere assay was validated in MCF-7 cells and used to screen novel marine algal compounds for potential anti-cancer stem cell (CSC) activity in vitro. Results: MCF-7 breast cancer cells were observed to generate tumourspheres or mammospheres after 3-5 days growth in anchorage-independent conditions and an apparent enrichment in potential CSCs was observed by an increase in the proportion of CD44high/CD24low marker-bearing cells and Oct4 expression compared to those in the bulk population grown in regular adherent conditions. Using this assay, a set of algal metabolites was screened for the ability to inhibit mammosphere development as a measure of potential anti-CSC activity. We report that the polyhalogenated monoterpene stereoisomers RU017 and RU018 isolated from the red alga Plocamium cornutum, both of which displayed no cytotoxicity against either adherent MCF-7 breast cancer or MCF-12A non-transformed breast epithelial cells, were able to prevent MCF-7 mammosphere formation in vitro. On the other hand, neither the brown algal carotenoid fucoxanthin nor the chemotherapeutic paclitaxel, both of which were toxic to adherent MCF-7 and MCF-12A cells, were able to inhibit mammosphere formation. In fact, pre-treatment with paclitaxel appeared to enhance mammosphere formation and development, a finding which is consistent with the reported resistance of CSCs to traditional chemotherapeutic agents. Conclusion: Due to the proposed clinical significance of CSC in terms of tumour initiation and metastasis, the identification of agents able to inhibit this subpopulation has clinical significance.

Independent of ErbB1 gene copy number, EGF stimulates migration but is not associated with cell proliferation in non-small cell lung cancer

Camila Lauand — 2013-04-30T00:00:00Z

Background: Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene. ErbB1 encodes epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, involved mainly in cell proliferation and survival. EGFR overexpression has been associated with more aggressive disease, poor prognosis, low survival rate and low response to therapy. ErbB1 amplification and mutation are associated with tumor development and are implicated in ineffective treatment. The aim of the present study was to investigate whether the ErbB1 copy number affects EGFR expression, cell proliferation or cell migration by comparing two different cell lines. Methods: The copies of ErbB1 gene was evaluated by FISH. Immunofluorescence and Western blotting were performed to determine location and expression of proteins mentioned in the present study. Proliferation was studied by flow cytometry and cell migration by wound healing assay and time lapse. Results: We investigated the activation and function of EGFR in the A549 and HK2 lung cancer cell lines, which contain 3 and 6 copies of ErbB1, respectively. The expression of EGFR was lower in the HK2 cell line. EGFR was activated after stimulation with EGF in both cell lines, but this activation did not promote differences in cellular proliferation when compared to control cells. Inhibiting EGFR with AG1478 did not modify cellular proliferation, confirming previous data. However, we observed morphological alterations, changes in microfilament organization and increased cell migration upon EGF stimulation. However, these effects did not seem to be consequence of an epithelial-mesenchymal transition. Conclusion: EGFR expression did not appear to be associated to the ErbB1 gene copy number, and neither of these aspects appeared to affect cell proliferation. However, EGFR activation by EGF resulted in cell migration stimulation in both cell lines.

Changes in the MALT1-A20-NF-¿B expression pattern may be related to T cell dysfunction in AML

Li Shi — 2013-04-30T00:00:00Z

To elucidate the characteristics of T-cell receptor (TCR) signal transduction in T-cells from acute myeloid leukemia (AML), the mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1 (MALT1), A20, NF-κB and MALT1-V1 gene expression levels in CD3+ T cells sorted from the peripheral blood of patients with AML were analyzed by real-time PCR. A significantly lower MALT1 and A20 expression level was found in T cells from patients with AML compared with healthy controls (p = 0.045, p < 0.0001); however, the expression level of MALT1-V1 (variant 1) was significantly higher in the AML group than in the healthy control group (p = 0.006), and the expression level of NF-κB was increased in the AML group. In conclusion, the characteristics of the expression pattern of MALT1-A20-NF-κB and the distribution of MALT1 variants in T cells from AML were first characterized. Overall, low TCR-CD3 signaling is related to low MALT1 expression, which may related to T cell immunodeficiency, while the up-regulation of MALT1-V1 may play a role in overcoming the T cell activity by downregulating A20 in patients with AML, which may be related to a specific response to AML-associated antigens.

Targeting PI3K/Akt represses Hypoxia inducible factor1¿ activation and sensitizes Rhabdomyosarcoma and Ewing¿s sarcoma cells for apoptosis

Mehtap Kilic-Eren — 2013-04-16T00:00:00Z

Background: Hypoxia inducible factor-1 α (HIF-1α) has been identified as an important novel target in apoptosis resistance of pediatric tumors such as Rhabdomyosarcoma (RMS) and Ewing’s sarcoma (ES). Evidence suggests that PI3K/Akt signaling plays a role in regulation of HIF-1α activation as well as apoptosis resistance in various adult tumors. However the relevance of PI3K/Akt signaling in HIF-1bα activation and apoptosis resistance in childhood tumors has not been addressed yet. Thus, this study was to investigate whether PI3K/Akt signaling is involved in hypoxia induced activation of HIF-1α as well as in resistance to hypoxia-induced apoptosis in childhood tumors such as RMS and ES. Methods: Constitutive activation of PI3K/Akt signaling was analyzed by Western blotting. Hypoxic activation of HIF-1α was determined by Western Blot analysis and electrophoretic mobility shift assay. Apoptosis was determined by flow cytometric analysis of the propidium iodine stained nuclei of cells treated with PI3K inhibitor LY294002 in combination with either TNF-related apoptosis-inducing ligand (TRAIL) or doxorubicin. Results: This study demonstrated that PI3K/Akt signaling was constitutively activated in RMS and ES cell lines, A204 and A673, respectively. Targeting PI3K/Akt signaling by the inhibitor LY294002 (30 μM) significantly decreased the protein expression as well as DNA binding activity of HIF-1α and restored the apoptosis-inducing ability of cells in hypoxia Additionally, pretreatment with LY294002 sensitized A204 and A673 cells to TRAIL or doxorubicin induced apoptosis under hypoxia. Conclusion: These results suggest that the constitutively active PI3K/Akt signaling contributes to hypoxic activation of HIF-1α as well as HIF1α-mediated apoptosis resistance in RMS and ES cells under hypoxia.