KCC1调控ERK通路对子宫内膜癌细胞侵袭能力影响的观察

目的:探讨K-CL共转运体(KCC)1基因在调节子宫内膜癌侵袭能力的过程中与细胞外信号调节激酶(ERK)转导途径相互作用机制。方法:将pcDNA-KCC1表达载体转染子宫内膜癌HEC-1B细胞,蛋白质印迹法检测KCC1、p-ERK1/2表达的变化;Transwell侵袭小室检测细胞侵袭能力的变化;ERK抑制剂U0126处理转染后的细胞,再通过上述方法检测p-ERK1/2表达及细胞侵袭能力的变化。结果:转染pcDNA-KCC1表达载体后,转染组细胞KCC1、p-ERK1/2蛋白表达明显上调(P<0.05),侵袭至滤膜下的细胞数由26.7±2.3增加到50.3±3.1,P<0.05;应用ERK抑制剂U0126处理实验组后,p-ERK1/2蛋白表达明显下调,侵袭至滤膜下的细胞数由50.3±3.1降低到30.8±5.9,P<0.05。结论:KCC1通过参与ERK信号转导途径调节子宫内膜癌细胞的侵袭能力。     

Increased platinum accumulation in SA-1 tumour cells after in vivo electrochemotherapy with cisplatin

Electrochemotherapy is an anti-tumour treatment that utilizes locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. The aim of our study was to determine whether anti-tumour effectiveness of electrochemotherapy with cisplatin is a consequence of increased plasma membrane permeability caused by electroporation that enables cisplatin binding to DNA. For this purpose, anti-tumour effectiveness of electrochemotherapy was evaluated on SA-1 tumours treated with electric pulses 3 min after intravenous injection of cisplatin (4 mg kg(-1)). Anti-tumour effectiveness was correlated with platinum accumulation in tumours and the amount of platinum bound to DNA, as determined by atomic absorption spectrometry. In tumours treated with electrochemotherapy, cell kill was increased by a factor of 20 compared with treatment with cisplatin only, as determined from tumour growth curves. The amount of platinum bound to DNA and platinum content in the tumours treated by electrochemotherapy was approximately two times higher than in cisplatin-treated tumours. Based on our results, we conclude that in vivo application of electric pulses potentiates anti-tumour effectiveness of cisplatin by electroporation that consequently results in cisplatin increased delivery into the cells. In addition, besides electroporation, immune system and tumour blood flow changes could be involved in the observed anti-tumour effectiveness of electrochemotherapy.     

Fos-related antigen 1 (Fra-1) pairing with and transactivation of JunB in GBM cells

Fos-related antigen 1 (Fra-1) plays an important role in maintenance/progression of various cancers, including glioblastoma multiforme (GBM). In this study, we used both shRNA and siRNA to examine the effect of fra-1 knockdown in GBM cells over-expressing Fra-1. Furthermore, we analyzed both the expression of JunB and its knockdown, a previously identified target for Fra-1, and also examined its potential association with Fra-1. When using fra-1 shRNA and siRNA, we found that GBM cells has Fra-1 levels diminished together with the levels of JunB, but Fra-1 remains unchanged in cells with junB knockdown. This is accompanied by dramatic changes in cell morphology and significant alteration in their migration. We next uncovered that the expression of JunB increased in response to ectopic Fra-1 and also to EGF-induced signaling, similarly to Fra-1. This was associated with an avid pairing between phosphorylated Fra-1 and JunB. Importantly, we found that Fra-1 paired with JunB binds to an AP-1 site in the junB gene promoter. JunB knockdown did not affect Fra-1 and the changes in cell morphology did not fully replicate that seen with Fra-1 knockdown. Thus, Fra-1 takes part in a control of architecture and migratory nature of GBM cells. Moreover, Fra-1 is a phosphorylated factor that transactivates JunB with which it makes effectively AP-1 pairs in GBM cells.     

Bmi-1 cooperates with H-Ras to transform human mammary epithelial cells via dysregulation of multiple growth-regulatory pathways

Elevated expression of Bmi-1 is associated with many cancers, including breast cancer. Here, we examined the oncogenic potential of Bmi-1 in MCF10A cells, a spontaneously immortalized, nontransformed strain of human mammary epithelial cells (HMEC). Bmi-1 overexpression alone in MCF10A cells did not result in oncogenic transformation. However, Bmi-1 co-overexpression with activated H-Ras (RasG12V) resulted in efficient transformation of MCF10A cells in vitro. Although early-passage H-Ras-expressing MCF10A cells were not transformed, late-passage H-Ras-expressing cells exhibited features of transformation in vitro. Early- and late-passage H-Ras-expressing cells also differed in levels of expression of H-Ras and Ki-67, a marker of proliferation. Subsets of early-passage H-Ras-expressing cells exhibited high Ras expression and were negative for Ki-67, whereas most late-passage H-Ras-expressing cells expressed low levels of Ras and were Ki-67 positive. Injection of late-passage H-Ras-expressing cells in severe combined immunodeficient mice formed carcinomas with leiomatous, hemangiomatous, and mast cell components; these tumors were quite distinct from those induced by late-passage cells co-overexpressing Bmi-1 and H-Ras, which formed poorly differentiated carcinomas with spindle cell features. Bmi-1 and H-Ras co-overexpression in MCF10A cells also induced features of epithelial-to-mesenchymal transition. Importantly, Bmi-1 inhibited senescence and permitted proliferation of cells expressing high levels of Ras. Examination of various growth-regulatory pathways suggested that Bmi-1 overexpression together with H-Ras promotes HMEC transformation and breast oncogenesis by deregulation of multiple growth-regulatory pathways by p16(INK4a)-independent mechanisms.     

Evidence for an upper temperature limit for thermotolerance development in L1A2 tumour cells in vitro

The response of LI A2 cells in vitro to water bath hyperthermia was investigated. In previous studies in the temperature range 40–5–45 0°C., an Arrhenius plot for heat killing of L1A2 cells was linear, and thermotolerance did not develop during continuous heating. The present Arrhenius analysis was extended to the range 38–45°C., and the Arrhenius plot was biphasic with an inflection point at 40–5°C., below which the activation energy was significantly increased from 724 to 923 kJ/mol (P< 0 001). In order to test for the development of thermotolerance during continuous heating, the cells were heated in the range 38–41°C for 10 h, followed immediately by a graded test treatment at 42°C. Thermotolerance developed below 40–5°C as shown by an increased D₀ of the 42°C survival curve, but not at 40 5 and 4FC. Preheating for 90min at 42°C followed by a 10 h incubation at 37°C resulted in maximal thermotolerance with a thermotolerance ratio of approximately 4–3, a ratio also obtained if the cells were incubated for 10 h at temperatures of 38–40°C. No thermotolerance was observed at incubation temperatures of 40 5°C and above. Thus, in the L1A2 cells 40°C is the upper temperature permissive for thermotolerance development, and the data support the assumption that the inflection point on the Arrhenius plot reflects the upper limit for thermotolerance development.     

Evidence for an upper temperature limit for thermotolerance development in L1A2 tumour cells in vitro

The response of L1A2 cells in vitro to water bath hyperthermia was investigated. In previous studies in the temperature range 40.5-45.0 degrees C, an Arrhenius plot for heat killing of L1A2 cells was linear, and thermotolerance did not develop during continuous heating. The present Arrhenius analysis was extended to the range 38-45 degrees C, and the Arrhenius plot was biphasic with an inflection point at 40.5 degrees C, below which the activation energy was significantly increased from 724 to 923 kJ/mol (P less than 0.001). In order to test for the development of thermotolerance during continuous heating, the cells were heated in the range 38-41 degrees C for 10 h, followed immediately by a graded test treatment at 42 degrees C. Thermotolerance developed below 40.5 degrees C as shown by an increased D0 of the 42 degrees C survival curve, but not at 40.5 and 41 degrees C. Preheating for 90 min at 42 degrees C followed by a 10 h incubation at 37 degrees C resulted in maximal thermotolerance with a thermotolerance ratio of approximately 4.3, a ratio also obtained if the cells were incubated for 10 h at temperatures of 38-40 degrees C. No thermotolerance was observed at incubation temperatures of 40.5 degrees C and above. Thus, in the L1A2 cells 40 degrees C is the upper temperature permissive for thermotolerance development, and the data support the assumption that the inflection point on the Arrhenius plot reflects the upper limit for thermotolerance development.     

Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1

Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion. As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts. We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2. These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH.     

Genistein cooperates with the histone deacetylase inhibitor vorinostat to induce cell death in prostate cancer cells

ABSTRACT: BACKGROUND: Among American men, prostate cancer is the most common, non-cutaneous malignancy that accounted for an estimated 241,000 new cases and 34,000 deaths in 2011. Previous studies have suggested that Wnt pathway inhibitory genes are silenced by CpG hypermethylation, and other studies have suggested that genistein can demethylate hypermethylated DNA. Genistein is a soy isoflavone with diverse effects on cellular proliferation, survival, and gene expression that suggest it could be a potential therapeutic agent for prostate cancer. We undertook the present study to investigate the effects of genistein on the epigenome of prostate cancer cells and to discover novel combination approaches of other compounds with genistein that might be of translational utility. Here, we have investigated the effects of genistein on several prostate cancer cell lines, including the ARCaP-E/ARCaP-M model of the epithelial to mesenchymal transition (EMT), to analyze effects on their epigenetic state. In addition, we investigated the effects of combined treatment of genistein with the histone deacetylase inhibitor vorinostat on survival in prostate cancer cells. METHODS: Using whole genome expression profiling and whole genome methylation profiling, we have determined the genome-wide differences in genetic and epigenetic responses to genistein in prostate cancer cells before and after undergoing the EMT. Also, cells were treated with genistein, vorinostat, and combination treatment, where cell death and cell proliferation was determined. RESULTS: Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 muM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1). In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation. CONCLUSION: Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways. In addition, genistein cooperates with vorinostat to induce cell death in prostate cancer cell lines with a greater effect on early stage prostate cancer.     

Morphological transformation of mouse and rat embryo cells in vitro by an agent from S37 ascites tumour

When normal rat and mouse embryo cells were treated with a cell free extract of S37 ascites tumour, morphological transformations occurred in both. The transformed cells readily induced lymphosarcoma-type tumours in the mice inoculated when newborn or young adults (8-12 weeks old), but not so readily in rats.     

Phosphatidylinositol 3'-kinase and MAPK/ERK kinase 1/2 differentially regulate expression of vascular endothelial growth factor in human malignant astrocytoma cells

Malignant astrocytomas are characterized by extensive vascularization attributed to increased expression of the angiogenic cytokine vascular endothelial growth factor (VEGF). VEGF is elevated in astrocytomas under normal oxygen conditions and undergoes induction in hypoxic stress. Prior studies have shown that both the phosphatidylinositol 3'-kinase (PI3-kinase) and MEK1/2 (MAPK/ERK kinase 1/2) pathways promote proliferation of astrocytoma cells and growth of astrocytic tumors. Whether these pathways regulate growth by modulating angiogenesis as well as proliferation is not clear. In this study, pharmacologic inhibitors were used to specifically inhibit PI3-kinase and MEK1/2 activity in human malignant astrocytoma cell lines, and their effects on VEGF expression were determined. Northern blot analysis of VEGF messenger RNA (mRNA) from cells treated with inhibitors demonstrated cell line-specific responses. The PI3-kinase pathway regulated both the normoxic expression and hypoxic induction of VEGF in 2 cell lines, whereas MEK1/2 regulated only the normoxic expression in the same 2 lines. The third cell line showed no change in VEGF mRNA with inhibition of either of these 2 pathways. This study suggests that modulation of signaling pathways implicated in proliferation of astrocytoma cell lines may have varying effects in vivo depending on the role these pathways play in regulating tumor angiogenesis.     

High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells

The cAMP-protein kinase A (PKA) pathway is the major signal transduction pathway involved in melanocyte-stimulating hormone receptor-mediated signaling and melanin production, whereas its role in the control of melanocyte proliferation is still controversial. In this study, we evaluated the effects of selective activation of the different PKA regulatory subunits type 1A (R1A) and type 2B (R2B) on melanocyte proliferation. Immunohistochemistry demonstrated that normal melanocytes lacked R1A protein whereas this subunit was highly expressed in all human melanomas studied (N=20) and in six human melanoma cell lines. Pharmacological activation of the R2 subunits by the cAMP analogue 8-Cl-cAMP inhibited proliferation and increased caspase-3 activity by 68.77+/-10.5 and 72+/-9% respectively, in all cell lines with the exception of the only p53-mutated one. Similar effects were obtained by activating R2 subunits with other analogues and by silencing R1A expression. The antiproliferative and proapoptotic effects of 8-Cl-cAMP were comparable to those observed with commonly used antitumoral drugs. Moreover, 8-Cl-cAMP potentiated the effects of these drugs on both cell proliferation and caspase-3 activity. In conclusion, this study first reports that human melanomas are characterized by a high R1/R2 ratio and that pharmacological and genetic manipulations able to revert this unbalanced expression cause significant antiproliferative and proapoptotic effects in melanoma cells.     

Detection of circulating tumour cells in peripheral blood with an automated scanning fluorescence microscope

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.     

Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer

Background:

To investigate the clinical relevance of CK-19mRNA-positive circulating tumour cells (CTCs) detected before the initiation of front-line treatment in patients with metastatic breast cancer (MBC).

Methods:

The presence of CTCs was detected in 298 patients with MBC using a real-time PCR (RT-PCR assay. In 44 patients, the detection of CTCs was evaluated by both the CellSearch and the RT-PCR assay. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated.

Results:

There was a strong correlation between the detection of CTCs by both assays. CK-19mRNA-positive CTCs were detected in 201 (67%) patients and their detection was independent of various patients'' clinico-pathological characteristics. The median progression-free survival (PFS; 9.2 vs 11.9 months (mo), P=0.003) and the overall survival (OS; 29.7 vs 38.9 mo, P=0.016) were significantly shorter in patients with detectable CK-19mRNA-positive CTCs compared with patients without detectable CTCs. Multivariate analysis demonstrated that oestrogen receptor status, performance status and detection of CTCs were emerged as independent prognostic factors associated with decreased PFS and OS.

Conclusion:

The detection of CK-19mRNA-positive CTCs in patients with MBC before front-line therapy could define a subgroup of patients with dismal clinical outcome.