Knockdown of IGF—IR by Antisense Oligodeoxynucleotide auguments the sensitivity of bladder cancer cells to MMC

AND AIM: Transitional cell carcinoma (TCC) of the bladder represents the fifth most prevalent malignancy in Western population, with peak incidence found in males of the 50-to 70- year-old age group. A major problem in the management of bladder cancer is the low sensitivity of a large proportion (approximately 40%) among bladder tumors to chemotherapy and the high risk for recurrence of bladder tumors after transurethral resection. So drug resistance, especially in its multiple type forms, remains a major and difficult problem to resolve in bladder cancer therapy. This phenomenon has often been ascribed to strictly pharmacolo-gic factors, such as the overexpression of multidrug transporters P-glycoprotein, multidrug resistance related protein (MRP), and other variables closely implicated DNA repair and induction/modulation of apoptosis, such as P53 and the Bcl-protein family. Furthermore, it has been recently shown that certain growth factors(IGFs etc) may be involved in the mechanism of drug resistance. Clearly, these findings suggest the design of new strategies that might improve bladder tumor response to chemotherapy. Results have previously shown that human bladder tumor cell lines may be adapted to grow in the complete absence of serum or any other growth supplement and that this can be explained on the basis of autocrine stimulation. The acquirement of autonomous growth capacity was likely to be an important element in the oncogenesis of bladder tumors. Furthermore, criss-cross experiments showed that supernatants stimulated not only proliferation of the autologous cell line of bladder cancer, but also growth of the other bladder cancer cell lines, suggesting the production of common autocrine factors in bladder tumor cells. Some factors or their receptors involved in autocrine loop mechanism of bladder tumor cells have been confirmed, such as IL-6, the epidermal growth factor receptor, IFN-beta, transferrins-like substance etc. But certain factors which may play key roles in autocrine mechanisms of bladder cancer cells remain to be definite and their physiological mechanism remain to be fully understood. More are still needed to know about the role of IGF1R signaling in bladder cancers. The following problems remain to be investigated in bladder cancer cells, about which the present studies are concerned: ①Is IGFs/IGF-IR signaling pathway involved in autocrine growth of human bladder cancer cells and how does bladder instillation drugs such as MMC affect the autocrine expression of bladder cancer cells? ②Can targeting against IGF1R gene can significantly enhance drug sensitivity of urinary bladder cancer cells to chemotherapy? ③What potential intracellular signaling mechanisms are involved in IGF1R blockage? ④May IGF1R self-stablized antisense ODN serves as a potential therapeutic approach to bladder cancer? To investigate whether IGF-1R was involved in drug resistance of bladder cancer cells. METHODS: RT-PCR was used to detect the mRNA expression of IGF-I, IGF-Ⅱ, and IGF-IR in T24 cells and normal urothelial cells. Flow cytometry and MTT tests were used to assess the effect of antisense oligodeoxynucleotide (ODN) on drug sensitivities and apoptosis of T24 cells to mitomycin (MMC). Western blot was used to analyze the effect of ODN on expression of IGF-IR protein. RESULTS: mRNA of IGF-I, IGF-Ⅱ, and IGF-IR were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; knockdown of IGF1R by antisense ODN significantly inhibited the growth of bladder cancer cells and enhanced sensitivity and apoptosis of T24 cells to MMC. CONCLUSION: These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.     

Reversion of multidrug-resistance by proteasome inhibitor bortezomib in K562/DNR cell line

Objective:To observe the reversion of multi-drug resistance by proteasome inhibitor bortezomib in K562/DNR cell line and to analyze the possible mechanism of reversion of multidrug-resistance.Methods:MTT method was used to determine the drug resistance of K562/DNR cells and the cellular toxicity of bortezomib.K562/DNR cells were cultured for 12 hours,24 hours and 36 hours with 100 μg/ml DNR only or plus 4 μg/L bortezomib.The expressions of NF-κB,IκB and P-gp of K562/DNR were detected with Western blot method,the activity of NF-κB was tested by ELISA method and the apoptosis rate was observed in each group respectively.Results:The IC50 of DNR on cells of K562/S and K562/DNR groups were 1.16 μg/ml and 50.43 μg/mL,respectively.The drug-resistant fold was 43.47.The IC10 of PS-341 on Cell strain K562/DNR was 4 μg/L.Therefore,4 μg/L was selected as the concentration for PS-341 to reverse drug-resistance in this study.DNR induced down-regulation of IκB expression,up-regulation of NF-κB and P-gp expression.After treatment with PS-341,a proteasome inhibitor,the IκB degradation was inhibited,IκB expression increased,NF-κB and P-gp expression decreased in a time dependent manner.Compared to DNR group,the NF-κB p65 activity of DNR+PS-341 group was decreased.Compared to corresponding DNR group,DNR induced apoptosis rate increases after addition of PS-341 in a time dependent manner.Conclusion:Proteasome inhibitor bortezomib can convert the leukemia cell drug resistance.The mechanism may be that bortezomib decreases the degradation of IκB and the expression of NF-κB and P-gp,therefore induces the apoptosis of multi-drug resistant cells.     

Correlation between Expression of P38 MAPK-Signaling and uPA in Breast Cancer

OBJECTIVE To study the expression of phosphorylated p38 mitogen-activated protein kinase(p-p38)and uPA and the correlation of their expression with breast cancer clinicopathological characteristics,and to investigate the role of the p38MAPK-signaling pathway in regulating uPA expression in breast cancer cells. METHODS Immunohistochemistry(S-P) was used to test the expression of p-p38 and uPA in 60 specimens of breast cancer tissues.Western blots were adopted to detect expression of the p-p38 and uPA proteins in MDA-MB-231 and MCF-7 breast cancer cells,and uPA expression a er treatment with SB203580,a specific inhibitor of p38 MAPK. RESULTS The positive rate of the p-p38 protein and uPA protein expression in the breast cancer tissues was 56.7% and 60.0%,respectively.The expression of p-p38 was positively related to the expression of uPA(r=0.316,P<0.05).The expression of p-p38 and uPA was related to lymph node metastas is and the TNM stage(P<0.05),but it was not related to the patient’s age or tumor size(P>0.05).The expression of p-p38 and uPA in MDA-MB-231 cells was higher than that in MCF-7 cells.SB203580 inhibited the p38 MAPK pathway and reduced uPA protein expression. CONCLUSION The p38 MAPK-signaling pathway promotes breast cancer malignant progression by up-regulating uPA expression,and it may be an important process in breast cancer invasion and metastasis.     

结直肠癌中NF-κB表达的研究

Objective: The aim of the study was to explore the protein level of NF-κB change in carcinoma and different grades of tumor cells differentiation tissue in colorectal cancer patients. Methods: This was a comparative study between normal and carcinoma tissues and in different grades of tumor cells differentiation tissue in colorectal cancer patients. Ex-pression of NF-κΒ were assessed by immunohistochemical method using rabbit polyclonal antibodies against human p65 NF-κΒ proteins. Results: There was none or...     

肺癌患者外周血血小板活化标志物水平及其临床意义

Objective： To investigate the relationship between the activation markers of platelets and the lung cancer. Methods： Based on international stages of lung cancer in 1997, lung cancer patients of 120 cases diagnosed by pathology as well as with operation indication were selected as the experimental group. During the process of experiment, 60 cases concluded as healthy in the physical examination were chosen as control group. The activation markers of platelets were detected by FCM method. The experimental result would be processed by SPSS 11.5. Results： The level of activation markers of platelets in peripheral blood of lung cancer patients was significantly higher than those healthy people （P 〈 0.01）. The level of activation markers of platelets in peripheral blood of lung cancer patients on the seventh postoperative day was significantly lower than that before operation and on the first postoperative day （P 〈 0.01）. The level of activation markers of platelets in peripheral blood of lung cancer patients was closely related to the size of the primary tumor, lymph node status and stages, but not to the grade of cell differentiation, type of tumor, age, sex of the patients （P 〉 0.05）. Conclusion： Elevation of the level of activation markers of platelets in peripheral blood exists in lung cancer patients and the levels of activation marker of platelets plays an important role in tumor growth and lymphatic metastasis. The levels of activation markers of platelets maybe a predictor for prognosis.     

Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells

Objective To explore the interaction of Anxa2 with P-Glycoprotein(P-gp) in the migration and invasion of the multidrug-resistant (MDR) human breast cancer cell line MCF-7/ADR. Methods A pair of short hairpin RNA(shRNA) targeting P-gp was transfected into MCF-7/ADR cells,and monoclonal cell strains were screened.The expression of P-gp was detected by Western blot.Transwell chambers were used to observe the cell migration capacity and invasion ability.The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses. Results P-gp expression was significantly knocked down,and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells(P<0.05).There was a close interaction between Anxa2 and P-gp. Conclusions MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities.The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells.The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells.     

核转录因子-κB在胰腺癌中的表达及其与上皮细胞间质转化的关系

 Objective　To investigate the expression of NF-κB and epithelial-mesenchymal transition(EMT) related markers E-cadherin and Vimentin proteins in human pancreatic cancer tissues and its relation with the malignant features． Methods　The expression of NF-κB、E-cadherin and Vimentin proteins in 62 cases of pancreatic cancer tissues were detected by using immunohistochemistry and compared with the clinicopathological data of pancreatic cancer. Results　The positive expression rate of NF-κB was 81 ％ (50/62), Vimentin protein increased of expression was 61 ％ (38/62), and E-cadherin protein loss of expression was 55 ％ (34/62) in pancreatic cancer. The positive expression rate of NF-κB was significantly related with the lymph node metastasis (χ2=11.761, P ＜0.05), distant metastasis(χ2＝9.225, P ＜0.05), the absent expression of epithelial marker E-cadherin protein (r =0．352, P <0．05) and the positive expression of mesenchymal marker Vimentin protein (r =0．343, P <0．05), but there was no relation with the patients gender, age, tumor location, tumor type and tumor differentiation (P ＞0．05)． In addition, the significant correlation of E-cadherin expression loss and Vimentin expression with tumor lymph node metastasis and distant metastasis was found (χ2＝6.914, 4.984, 7.753, 5.144, P <0.05). Conclusion　The overexpression of NF-κB in pancreatic cancer may accelerate invasion and metastasis of pancreatic cancer through inducing EMT．     

Down-Regulation of Notchl and NF-κB by Curcumin in Breast Cancer Cells MDA-MB-231

Objective:To test whether the down-regulation of Notch1 gene expression by curcumin could inhibit cell growth and induce apoptosis,which may be associated mechanistically with the down-regulation of NF-κB in breast cancer cells. Methods:Breast cancer cell lines MDA-MB-231 were cultured in vitro and treated with different dosages of curcumin(0,1.25,5.0,20.0μmol/L)for dose-dependent assay and different time(0,24,48,72 h)at the dosage of 5.0μmol/L for time course assay.The changes of the mRNA and protein expression of Notch1 and NF-κB were measured by RT-PCR and Western Blot,and MTT assay was used to measure the change of proliferation. Results:The mRNA and protein levels of Notch 1 and NF-κB were decreased significantly in human breast cancer cell line with the increase of dosage of curcumin(P＜0.05),and with the extension of time course(P＜0.05).These changes suggested a dose- and time-dependent manner.The proliferation rate of cells also was significantly inhibited(P＜0.05). Conclusion:The current results show that the Notch-1 signaling pathway is associated mechanistically with NF-κB activity during curcumin-induced cell growth inhibition and apoptosis of breast cancer cells.These results suggest that the down-regulation of Notch signaling by curcumin may be a novel strategy for the treatment of patients with breast cancer.     

Autophagy Enhances the Aggressiveness of Human Colorectal Cancer Cells and Their Ability to Adapt to Apoptotic Stimulus

Objective To investigate LC3B-Ⅱand active caspase-3 expression in human colorectal cancer to elucidate the role of autophagy and to explore the relationship of autophagy with apoptosis in human colorectal cancer. Methods LC3B expression was detected by immunohistochemistry in 53 human colorectal cancer tissues and 20 normal colon tissues.The protein levels of LC3B-Ⅱand active caspase-3 were also determined by Western blot analysis in 23 human colorectal cancer tissues and 10 normal colon tissues. Results LC3B was expressed both in cancer cells and normal epithelial cells.LC3B expression in the peripheral area of cancer tissues was correlated with several clinicopathological factors,including tumor differentiation(P=0.002),growth pattern of the tumor margin (P=0.028),pN(P=0.002),pStage(P=0.032),as well as vessel and nerve plexus invasion(P=0.002).The protein level of LC3B-Ⅱin cancer tissue was significantly higher than in normal tissue(P=0.038),but the expression of active forms of procaspase-3 in cancer tissue was lower(P=0.041).There was a statistically significant positive correlation between the expression levels of LC3B-Ⅱand the active forms of procaspase-3(r=0.537,P=0.008). Conclusions Autophagy has a prosurvival role in human colorectal cancer.Autophagy enhances the aggressiveness of colorectal cancer cells and their ability to adapt to apoptotic stimulus.     

Overexpression of HOXB9 promotes metastasis and indicates poor prognosis in colon cancer简

Objective：Homeobox B9 （HOXB9） is proposed to be involved in tumor angiogenesis and metastasis.We investigated the role of HOXB9 in the progression of colon cancer.Methods：HOXB9 expression was investigated by immunohistochemically and Western blotting in 128 colon cancer patients and the results were analyzed statistically associated with clinicopathological data and survival of the patients.The effect of HOXB9 on cell invasion and metastases abilities were analyzed in vitro and in vivo.Results：HOXB9 is overexpressed in colon cancer tissues and significandy correlated with metastasis and poor survival of patients （P＜0.05,respectively）.Additionally,high levels of expression of HOXB9 were observed in metastatic lymph nodes.The down-regulation of HOXB9 expression can inhibit the migration and invasive ability of colon cancer cells,while exogenous expression of HOXB9 in colon cancer cells enhanced cell migration and invasiveness.Moreover,stable knockdown of HOXB9 reduced the liver and lung metastasis of colon cancer in vivo.Conclusions：HOXB9 may play an important role in the invasion and metastasis of colon cancer cells and may be a useful biomarker for metastasis and prognostic of colon cancer.     

Evaluate the expression of uPA, PAI-1 in human gastric cancer and its correlation with the angiogenesis by the application of tissue microarray

OBJECTIVE To investigate the expression of urokinase-type plasminogen （uPA）, its inhibitor-1 （PAI-1） mRNA and its protein in human gastric cancer and to find out the relationship among the tumor differentiation, angiogenesis, and other clinical pathologic factors. METHODS In situ hybridization （ISH） was used to get the uPA, PAI-lmRNA in 110 cases with human gastric cancer in 2-tissue microarray （TMA）. Immunohistochemical staining （S-P method） for uPA, PAI-1 protein and CD34 were performed in the 110 cases in 2 TMA. RESULTS The expression of the uPA, PAI-lmRNA and their protein happened in the cytoplasm of gastric cancer cells were induced by the poor differentiation of the GC, and the expression of uPA had an increasing trend while the expression of the PAI-1 had a decreasing trend. The microvessel density （MVD） had a positive correlation with the clinical stages and the significant relationship with the lymph node metastasis （P 〈 0.05）. The MVD in uPA positive group was significantly higher than those in uPA negative group （P 〈 0.05）. The expression of PAI-1 has no correlation neither with the clinical stages nor the lymph node metastasis. CONCLUSION The uPA play an important role in invasion and metastasis of GC through promoting angiogenesis. Interdicting the secretion and function of the uPA may allow the target therapy against the tumor invasion. As a new high-throughput technology, the tissue microarray is a valuable way to be used in clinical treatment.     

B7-H1在膀胱癌患者外周血树突状细胞中的表达及其临床意义(英文)

Objective:The aim of this study was to study the expression and the clinical significance of B7-H1 on dendritic cells (DCs) in peripheral blood from patients with bladder cancer. Methods:Peripheral blood mononuclear cell were disparted from 30 bladder cancer patients and 7 healthy controls by density gradient centrifugation and then co-cultured. The expression of B7-H1 on DCs were analyzed by flow cytometry. Results:Expression of B7-H1 on DCs in bladder cancer was higher than healthy controls (P<0.01). And the expression were strongly associated with the pathological grade and clinical stage of bladder cancer (P<0.05). Conclusion:The up-regulation of B7-H1 on DCs was strongly associated with neoplastic progression of bladder cancer. B7-H1/programmed death (PD)-1 signal pathway may also play an important role in immune escape of bladder cancer during initial phase of T cell immune response.     

NF-κB and MMP-2 expression regulated by Lrig1 through the MEK-ERK signaling pathway in Hazaks' esophageal squamous cell carcinoma北大核心CSCD

Objective: This work aimed to investigate the expression levels of Lrig1, NF-κB, and MMP-2 in Hazak's esophageal squamous cell carcinoma (ESCC), as well as to explore the relationship of Lrig1 expression through the PI3K-AKT and MEK-ERK signaling pathways with clinicopathological indices. Methods: First, RT-PCR analysis was performed to study the expression levels of Lrig1, NF-κB, and MMP-2 in 48 ESCC and noncancerous specimens. Second, some key genes of the PI3K and MEK signaling pathways such as PI3K, AKT1, MEK1, MEK2, MEK5, ERK1, and ERK2 were detected in Lrig1-positive and -negative tissues to identify the possible signaling pathway of Lrig1-regulated NF-κB and MMP-2 expression. Results: The expression of NF-κB (P<0.001, P=0.014) and MMP-2 (P=0.003, P=0.045) was significantly correlated with Lrig1 in cancer and distal normal tissues. Lrig1 may be negatively correlated with NF-κB and MMP-2 at the protein level. MEK5 (P<0.001) and ERK2 (P=0.009) expression was associated with Lrig1 in cancer tissues. PI3K expression was correlated with Lrig1 in the distal normal tissues (P<0.001). The coordinate expression ratio of Lrig1 to NF-κB and MMP2 reached 52.1% (25/48). This co-expression may be related to lymph node metastasis (P=0.020) but not to other clinicopathological features. Conclusions: These findings suggest that MMP-2 and NF-κB expression is related to Lrig1 in Hazak's ESCC, and that MEK and ERK2 mRNA expression are related to Lrig1. The co-expression of NF-κB, MMP-2, and Lrig1 may promote lymph node metastasis in ESCC among Hazak people. Lrig1 may regulate the expression of target genes not through the PI3K-AKT pathway but through the MEK-ERK signaling pathway. Further related studies are needed in the future.     

Arsenic Trioxide Induced Differentiation and Apoptosis in Human Nasopharyngeal Carcinoma Xenografts in BALB／C Nude Mice

To study the effect of arsenic trioxide (As2O3) on human poorly differentiated nasopharyngeal cancer cell line, CSNE-1, in vivo and its possible mechanism of action. Methods: CSNE-1 cells were established as xenografts in BALB/C nude mice. The tumor-bearing mice were treated with As2O3 at the dose of 5 mg/kg every day. The tumor growth was observed by tumor-growth curve. Morphologic changes were studied under light microscopy and electron microscopy. TUNEL was used to detect apoptosis. The expression of PCNA, p53, Bcl-2 and Bax were determined by immunohistochemistry. Results: The cell growth and proliferate activity were significantly inhibited by As203 at the dose of 5 mg/kg every day. Morphologic changes such as the formation of keratinization of tumor cells, decreased ratio of nuclear/cytoplasm, increased organelle and plasmic fibril in cytoplasm were identified. Cytodesma, desmosomes and micro-process were seen under light microscopy and transmission electron microscopy, which revealed that the cancer cells underwent differentiation. In addition, remarkable cell apoptosis were observed by TUNEL assay. Over expression of p53 and Bax was detected in the As203 treatment group when compared with control group. Conclusion: As203 inhibited proliferation of human poorly differentiated nasopharyngeal cancer cell CSNE-1 by inducing differentiation and apoptosis, which may be related to the up-regulation of p53 and Bax expression.     

EFFECTS AND MECHANISMS OF ENDOSTATIN ON THE GROWTH OF OVARIAN CANCER SKOV3 CELLS IN VITRO AND IN VIVO

Objective： To evaluate the inhibitory effect of Endostatin on ovarian cancer cell line SKOV3 and to investigate the possible mechanism of the inhibition. Methods： Using MTT, transmission electron microscope （TEM） and immunocytochemistry, the effects of Endostatin on the proliferation of SKOV3 cells were studied. Nude mice were subcutaneously implanted with SKOV3 cells. The cell apoptosis of implanted tumor was detected by TUNEL and TEM. The expressions of bcl-2 and bax in implanted tumor tissues were measured by RT-PCR and immunohistochemistry. Results： Endostatin significantly inhibited the proliferation of SKOV3 cells in vitro （P〈0.01） and induced cell apoptosis, whereas the expressions of bcl-2 and bax were not changed obviously in SKOV3 cell treated with Endostatin. The mean tumor weight of Endostatin treated group was markedly lower than that of PBS control group （P〈0.05）. The expression of bcl-2 was down-regulated in Endostatin treated group, but bax was not influenced. Conclusions： The results demonstrated that Endostatin might have anti-tumor effect on ovarian carcinoma. One of the important mechanisms of Endostatin effect of anti-angiogenic and anti-tumor activities might involve regulating the bcl-2/bax expression and inducing apoptosis.     

Lipopolysaccharide enhances the inhibition of NF-κB expression in NNK-mediated peritoneal macrophages

Objective： The aim of the study was to investigate the effect of lipopolysaccharide （LPS） on the expression of nuclear factor kappa B （NF-κB） in 4-（methylitrosamino）-1-（3-pyridyl）-1-butanone （NNK）-mediated primary mouse peritoneal macrophages in vitro. Methods： The activity of peritoneal rnacrophages treated with different concentrations of LPS was detected by MTT assay in rider to find the optimal concentration. Peritoneal macrophages were also treated with NNK （100-500 μM）, with or without LPS for 9 h. The expression of NF-κB was demonstrated via immunocytochemistry （ICC） and Western- blot, respectively. Results： The concentration of LPS at 25 μg/mL was found to be the optimal concentration to improve the activity of peritoneal macrophages （P 〈 0.01）. Simultaneously, LPS （25 μg/mL） increased the expression of NF-κB in both the nucleus and cytoplasm and facilitated transfer of NF-κB to the nucleus. NNK treatment significantly inhibited the expression of NF-κB in a concentration-dependent manner, among the LPS-stimulated or unstimulated peritoneal macrophages, especially when cotreated with LPS （25 μg/mL, P 〈 0.01 ）. Furthermore, NNK treatment （500 μM） with LPS yielded a significant decrease in NF-κB translocation to nucleus and inhibited the expression of NF-κB （P 〈 0.005）. Conclusion： LPS enhances the suppression of NF-κB expression in NNK-mediated mouse peritoneal macrophages, which may provide a theoretical basis for the inhibition of cancer.     

Celecoxib up-regulates expression of■chain of TIL receptor complex in human cervical cancer

Tumor-infiltrating lymphocytes (TIL) were once considered as the effector cells in the immune attack against cancer.However,TIL had not been shown significant tumor cytolytic activity in vitro.This was later found to be due to down-regulation of theζchain of T cell receptor complex. Withoutζchain, signal transduction necessary for T cell activation would not occur. This is one of the reasons why tumor cells could evade immune surveillance of the host.A study by Ferrandina et al.from University of Rome has made clear the mechanism of tumor-induced down regulation ofζchain and how to restore its expression (Clin Cancer Res 12:2055-2060,2006).The authors