Journal of Experimental & Clinical Cancer Research - Latest articles

Immunomodulatory effects of recombinant BCG expressing pertussis toxin on TNF-alpha and IL-10 in a bladder cancer model

2008-11-28

Background: Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a rBCG-S1PT strain that induced a stronger cellular immune response than BCG. This preclinical study was designed to compare the modulatory effects of BCG and rBCG-S1PT on bladder TNF-alpha and IL-10 expression and evaluate antitumour activity. Methods: For Experiment I, the MB49 bladder cancer cell line was used in C57BL/6 mice. Chemical cauterization of the bladder was performed to promote intravesical tumor implantation. Mice were treated by intravesical instillation with BCG, rBCG-S1PT or PBS once a week for four weeks. After 35 days the bladders were removed and weighed. TNF-alpha and IL-10 cytokine responses were measured by qPCR. Experiment II was performed the same as Experiment I, except the animals were not challenged with MB49 tumor cells. Results: rBCG-S1PT immunotherapy resulted in bladder weight reduction, compared to the BCG and control group. There were increases in TNF-alpha in the BCG-treated group, as well as increases in TNF-alpha and IL-10 mRNA in the rBCG-S1PT group. Conclusions: These data indicate a significant reduction of bladder tumor volume for the rBCG group, compared to the BCG and PBS groups. This suggests that rBCG could be a useful substitute for wild-type BCG and that the potential modulation between TNF-alpha and IL-10 cytokine production may have therapeutic value.

Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro

Pei-Xin Dong, Nan Jia, Zhu-Jie Xu, Ying-Tao Liu, Da-Jin Li and You-Ji Feng — 2008-11-27

Background: IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells. Methods: We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay. Results: IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAi-mediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration. Conclusions: Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.

Modulating effect of the PI3-kinase inhibitor LY294002 on cisplatin in human pancreatic cancer cells

2008-11-25

Background: Chemoresistance is a serious problem in pancreatic cancer, but the mechanism of resistance and strategies against the resistance have not been elucidated. We examined the potential of the phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor LY294002 to enhance the anti-tumor effect of cisplatin and investigated the mechanism of chemoresistance in pancreatic cancer cells using a combination therapy of cisplatin and LY294002, both in vitro and in vivo. Methods: Cisplatin and LY294002, individually or in combination, were given to AsPC-1 and PANC-1 cell lines. Tumor growth, DNA fragments, and Akt phosphorylation were examined in vitro. To examine the therapeutic effect of cisplatin and LY294002, individually or combination an AsPC-1 tumor xenograft model was prepared for in vivo study. Results: Cisplatin induced growth inhibition and Akt phosphorylation in pancreatic cancer cells. LY294002 also inhibited cell proliferation but without showing Akt phosphorylation. However, the combination therapy markedly increased cleavage of caspase-3 and cytoplasmic histone-associated DNA fragments compared to the results with cisplatin alone. In the in vivo study, blocking the PI3K/Akt cascade with LY294002 increased the efficacy of cisplatin-induced inhibition of tumor growth in nude mice, suppressing half the tumor growth with cisplatin alone. There were no detectable side effects in mice treated with combination therapy. Conclusions: Our studies suggest that the PI3K/Akt pathway plays an important role in cisplatin of pancreatic cancer cells. The augmentation of cisplatin with PI3K/Akt inhibitor may resolve the chemoresistance problem of cisplatin, and this might be a plausible strategy for achieving tolerance for chemotherapeutic agents in pancreatic cancer therapy.

The withdrawal from oncogenetic counselling and testing for hereditary and familial breast and ovarian cancers. A descriptive study of an Italian sample

2008-11-24

Background: Oncogenetic counselling is seldom followed through with, even when individuals are eligible according to the test criteria. The basic variables which influence the decision to undergo the genetic counselling process are: the risk perception, expected benefit or limitations of genetic testing, general psychological distress or cancer-specific distress, lack of trust in one's emotional reactions when faced with negative events, expected level of family support and communications within the family. The aim of this study was to describe the psychosocial variables of an Italian sample that gives up genetic counselling. Methods: From May 2002 to December 2006 a psychological questionnaire was sent out to one hundred and six subjects, who autonomously requested the first genetic consultation, and never ask to have a second visit and the family tree drawn up in order to inquire about their eligibility for genetic testing. Statistical analysis was performed by Pearson chi-square test, t-test and Spearman RHO coefficient. Results: The survey presents a lack of emotional cohesion and structured roles and rules within the family system and a positive correlation between the number of children, anxiety and risk perception. The main reasons for giving up on counselling were a sense that testing was a waste of time and the inability to handle emotionally the negative consequences of the outcome of test. The subjects who maintained that test and an early diagnosis were a "waste of time" experienced more anxiety. Conclusions: The study revealed the importance to pay attention to the whole persona and their family system as well as provide information highlighting usefulness of early diagnosis.

Prognostic factors of survival time after haematopoietic stem cell transplant in acute lymphoblastic leukemia patients: Cox proportional hazard versus accelerated failure time models

2008-11-23

Background: The aim of this study is to evaluate the prognostic factors of overall survival (OS) after haematopoietic stem cell transplant (HSCT) in acute lymphoblastic leukaemia (ALL) patients using accelerated failure time (AFT), Cox proportional hazard (PH), and Cox time-varying coefficient models. Methods: 206 patients were enrolled after HSCH in Shariati Hospital between 1993 and 2007. There was evidence of marked departures from the proportional hazards assumption with two prognostic factors, relapse and chronic graft-versus-host disease (cGVHD) (P<.001). Performance among AFT and Cox's models was assessed using explained variation and goodness of fit methods. Discrimination among the exponential, Weibull, generalized gamma (GG), log-logistic, and lognormal distributions was done using maximum likelihood and Akaike information criteria. Results: The 5-year OS was 52% (95%CI: 47.3-56.7). Peak mortality hazard occurred at months 6-7 after HSCT followed by a decreasing trend. In univariate analysis, the data was better fitted by GG distribution than by other distributions. Univariate analysis using GG distribution showed a positive association between OS with acute graft-versus-host disease (aGVHD) (P=.021), no relapse (P<.001), cGVHD (P<.001), neutrophil recovery (P<.001) and platelet recovery (P<.001). Based on Cox PH models; however cGVHD and relapse were the predictive factors of OS (P<.001). Multivariate analysis indicated that, OS is related to relapse (P<.001) and platelet recovery (P=.037), where predictive power of Weibull AFT models was superior to Cox PH model and Cox with time-varying coefficient (R-Squared =0.46 for AFT, R-Squared=.21 for Cox PH and R-Squared=.34 for Cox time- varying coefficient). Cox-Snell residual shows Weibull AFT fitted to data better than other distributions in multivariate analysis. Conclusions: We concluded that AFT distributions can be a useful tool for recognizing prognostic factors of OS in acute lymphoblastic leukemia patients. Key words: acute lymphoblastic leukaemia * prognostic factors * accelerated failure time models * Cox proportional hazard models * Cox time-varying coefficients

Immunohistochemical expression of promyelocytic leukemia body in soft tissue sarcomas

2008-11-23

Background: The function of promyelocytic leukemia (PML) bodies is not well known but plays an important role in controlling cell proliferation, apoptosis and senescence. This study was undertaken to analyze the clinical significance of PML body expression in primary tumor samples from malignant fibrous histiocytoma (MFH) and liposarcoma patients. Methods: We studied MFH and liposarcoma samples from 55 patients for PML bodies. Fluorescent immunostaining of PML bodies was performed in the paraffin-embedded tumor sections. Results: PML body immunostaining was identified in 63.9 % of MFH and 63.2 % of liposarcoma samples. PML body expression rates of all sarcoma cells were 1.5+/-1.8 % (range: 0-7.0) in MFH and 1.3+/-1.4 % (0-5.2) in liposarcoma samples. PML body expression (p = 0.0053) and a high rate of PML body expression (p = 0.0012) were significantly greater prognostic risk factors for death than the other clinical factors in MFH patients. All liposarcoma patients without expression of PML were disease free at the end of the study. Conclusion: Our study suggests that the presence of PML bodies may indicate a poor prognosis for MFH and liposarcoma patients.

Inhibition of hepatocelluar carcinoma MAT2A and MAT2betagene expressions by single and dual small interfering RNA

Wang Qun, Liu Quan yan, Liu Zhi Su, Qian Qun, Sun Quan and Pan Ding-yu — 2008-11-21

RNA interference (RNAi) has been successfully applied in suppression of hepatic cancer genes. In hepatocelluar carcinoma cell , one methionine adenosyltransferase (MAT) isozyme , MATII was found to have two catalytic subunits which were encoded by MAT2A and MAT2betarespectively. During tumorigeness of hepatocelluar carcinoma, expressions of the two genes were discovered to be increased combining with a switch of MAT (form MATI to MATII), To figure out the role played by MATII in hepatic cancer, In this study, for the first time we established a dual small interfering RNA (siRNA) expression system, which could simultaneously express two different siRNA molecules specifically targeting two genes. To test the effectiveness of this system, we applied this approach to express simultaneously two different siRNA duplexes that specifically target MAT2A and MAT2betagenes of hepatocelluar carcinoma respectively in HepG2 cell. Results indicated that dual siRNA could simultaneously inhibit the expression of MAT2A and MAT2betagene by 89.5% and 97.8% respectively, In addition, dual siRNA molecules were able to significantly suppress growth of hepatocelluar carcinoma cell in vitro as well as induce apoptosis which was involved in arrest cell cycle at the G1/ S checkpoint and the expressions of p21 p27 and Bax.

Identification of the minimal melanocyte-specific promoter in the melanocortin receptor 1 gene

2008-11-18

Background: The understanding of cutaneous pigmentation biology is relevant from the biologic and clinical point of view. The binding of alpha-melanocortin and its specific receptor, on the plasma membrane of melanin synthetizing cells, plays a crucial role in melanins biosynthesis. Furthermore, loss of MC1R function is associated with an increased incidence of melanoma and non-melanoma skin cancer. The expression of the alpha-melanocortin receptor gene is highly controlled but, at the present , region responsible for tissue-specific activity of the gene promoter has not been identified. Methods: We have cloned the genomic sequences upstream the human MC1R coding gene. A DNA fragment of 5 kilobases upstream the human MC1R encoding sequence was placed in front of a reporter gene and several deletion mutants of such fragment have been prepared. These constructs have been tested for the ability to drive the melanocyte-specific gene expression of the reporter gene using transfection experiments in melanocyte and non-melanocyte cell lines. From these experiments we identified a DNA fragment with the ability to drive the gene transcription in a tissue-specific way and we used this small DNA fragment in DNA-protein interaction assays. Results: We show that the 150 base pairs upstream the MC1R gene initiation codon are able to drive the melanocyte-specific gene transcription. Furthermore, we provide experimental evidences suggesting that on such minimal melanocyte-specific gene promoter can assemble tissue-specific complexes. Conclusions: The present results strongly imply that the transcriptional regulation of the melanocyte-specific MC1R gene requires an internal promoter located in the 150 base pairs upstream the initiation codon.

Classification of tumours.

2008-11-14

Tumours are classified according to the most differentiated cells with the exception of carcinomas where a few tumour cells show neuroendocrine differentiation. In this case these cells are regarded as redifferentiated tumour cells, and the tumour is not classified as neuroendocrine. However, it is now clear that normal neuroendocrine cells can divide, and that continuous stimulation of such cells results in tumour formation, which during time becomes increasingly malignant. To understand tumourigenesis, it is of utmost importance to recognize the cell of origin of the tumour since knowledge of the growth regulation of that cell may give information about development and thus possible prevention and prophylaxis of the tumour. It may also have implications for the treatment. The successful treatment of gastrointestinal stromal tumours by a tyrosine kinase inhibitor is an example of the importance of a correct cellular classification of a tumour. In the future tumours should not just be classified as for instance adenocarcinomas of an organ, but more precisely as a carcinoma originating from a certain cell type of that organ.

Id-1: Regulator of EGFR and VEGF and potential target for colorectal cancer therapy

Ibrahim Meteoglu, Nezih Meydan and Muhan Erkus — 2008-11-12

Background: The helix-loop-helix transcription factor Id-1 (an inhibitor of differentiation and DNA binding) plays a role in development and progression of many tumours. Id-1 is known to exert its effects on the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF). The aim of this study was to reveal whether there was a relationship between Id-1 and EGFR and VEGF in colorectal carcinoma. Methods: Tumour and non-tumour tissue specimens from 46 cases of colorectal carcinoma were exposed to immunohistochemical staining for Id-1, EGFR and VEGF. The relationship between the degree of staining and tumour grade, tumour stage and all tumour markers was investigated. Results: Tumour cells showed positive staining for Id-1 in 43 cases (93.5%), for EGFR in 41 cases (89%) and for VEGF in 42 cases (91%). There was a significant relation between the tumour grade and the degree of staining for Id-1, EGFR and VEGF. The relation between the tumour stage and the degree of staining for Id-1, EGFR and VEGF was also significant. There was a significant relation between Id-1 expression and EGFR and VEGF expressions. Non-tumoural tissue specimens were not stained with Id-1 and EGFR antibodies in any of the cases, but stained with VEGF antibody in 3 cases. Conclusion: This study revealed that Id-1, EGFR and VEGF took part in development and progression of colorectal carcinomas and that Id-1 was associated with regulations of EGFR and VEGF. The results of this study support the idea that not only EGFR and VEGF but also Id-1 could be new targets in cancer treatment.