Establishment of orthotopic transplantation model of human bladder cancer and detection by MRI

Objective： To establish an orthotopic bladder cancer model bearing human bladder cancer for experimental research, and monitor tumor progression by magnetic resonance imaging （MRI）. Methods： The mucosa was mechanically damaged transurethrally under direct vision, and then human bladder cancer cell line T24 was inoculated into the bladders of BALB/c nude mice to establish orthotopic bladder cancer model. To find a suitable concentration of Gd-DTPA for this research. MRI was performed weekly to assess tumor growth, using Gd-DTPA as contrast agent. The pathologic morphology of the bladders and other specimens were observed with HE stain. Results： All the 25 mice developed bladder cancer after inoculation. The best concentration of Gd-DTPA was 1.408 mg/mL. On MRI, no change in the bladders was observed on day 7 after inoculation, filling defect in the bladders, accordant to actual tumor size, was detected on days 14, 21 and 28. Pathologic examination showed that tumor grew in the mucosa or superficial muscle of bladder on day 7, confined in muscle layer on days 14-28, and invaded serosa on day 35. Conclusion： Transurethrally damaged bladder mucosa under direct vision and instilled bladder cancer cell T24, we successfully established an orthotopic bladder cancer model. Tumor growth simulated the progression of human bladder cancer approximately. MRI was a reliable way for dynamic detection of murine orthotopic bladder tumor.     

Study of Benign and Malignant Intraductal Lesions of the Breast by Fiberoptic Ductoscopy

OBJECTIVE To observe and subtype the appearance of intraductal papilloma (lesions) and of infiltrating ductal carcinoma or early infiltrating ductal carcinoma using a fiberoptic ductoscope (FDS) examination, and to discuss the differentiation and diagnosis of benign and malignant tumors by FDS.METHODS The characteristics of FDS images and diagnostic data for 229patients with intraductal papilloma (lesions) and 50 patients with ductal carcinoma, who were confirmed by surgical pathology from October 1998 to December 2003, were analyzed retrospectively.RESULTS The appearance of the lesions observed by FDS were grouped into 4 types: a monothelia (type Ⅰ), polythelia (type Ⅱ), superficies (type Ⅲ)and a mixture (type Ⅳ). Intraductal papillomas (lesions) were more commonly seen in type Ⅰ and Ⅱ, and intraductal carcinomas or early infiltrating ductal carcinomas were more commonly seen in type Ⅲ and Ⅳ;there was a statistically significant difference in the distribution of the ductoscopic types, except in type Ⅱ, between the two types of lesions, P＜0.001. The focal detection rate by FDS for intraductal papilloma and papillomatosis was 99.6% (228/229) and for breast cancer was 96.0% (48/50). The diagnostic accuracy was 97.8% (224/229) and 82.0% (41/50),respectively.CONCLUSION FDS can be a guide for the treament of benign and malignant intraductal tumors, with early discovery and accurate diagnosis.     

Estimation of body composition in muscular dystrophy by MRI and stereology

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Optimising treatment of bone metastases by Aredia™ and Zometa™

Metastatic bone disease develops as a result of the many interactions between tumour cells and bone cells. This leads to disruption of normal bone metabolism, with the increased osteoclast activity seen in most, if not all, tumor types providing a rational target for treatment. The clinical course of metastatic bone disease in multiple myeloma, breast and prostate cancers is relatively long, with patients experiencing sequential skeletal complications over a period of several years. These include bone pain, fractures, hypercalcaemia, and spinal cord compression, all of which may profoundly impair a patient's quality of life. External beam radiotherapy and systemic endocrine and cytotoxic treatments are the mainstay of treatment in advanced cancers. However, it is now clear that the bisphosphonates provide an additional treatment strategy, which reduces both the symptoms and complications of bone involvement. Pamidronate (Aredia(TM)) is the most widely evaluated bisphosphonate and is recommended for most patients with multiple myeloma or breast cancer with bone metastases. Current research aims include the evaluation of new potent bisphosphonates such as zoledronic acid (Zometa(TM)). It is hoped that this compound is not only more convenient and easier to administer but also more effective in inhibiting skeletal morbidity. Zometa may also have some direct anticancer activity. Preclinical studies with Zometa have demonstrated its potential in malignant bone disease. Clinical studies in treatment of hypercalcemia of malignancy have been completed, as have Phase I and II trials in patients with cancer and pre-existing bone metastases. Three randomized, double-blind, controlled Phase III trials are now ongoing to establish the efficacy and safety of Zometa in treatment of bone metastases in patients with osteolytic and osteoblastic lesions. Additionally, new specific molecules such as osteoprotogerin have been developed that are based on our improved understanding of the cellular signalling mechanisms involved in cancer induced bone disease. These potent molecules are now entering clinical trials. Ongoing research is aimed at trying to define the optimum route, dose, schedule and type of bisphosphonate in metastatic bone disease and their use in the prevention and treatment of osteoporosis in cancer patients. In vitro suggestions of direct anti-cancer activity and some promising clinical data in early breast cancer have resulted in considerable interest in the possible adjuvant use of bisphosphonates to inhibit the development of bone metastases.     

Suppression of Glioma-Cell Survival by Antisense and Dominant-Negative AKT2 RNA

OBJECTIVE Overexpression of growth factors and their receptors such as PDGF, FGF, VEGF, IGF, EGF, TGFα etc. play a critical role in the development and progression of malignant gliomas. AKT, one of the most potent downstream signaling effectors of these growth factor receptors is usually overactivated in malignant gliomas. The present study was undertaken to investigate the effects of antisense and dominant negative AKT2 RNA on the survival of glioma cells with overexpression of AKT2. METHODS Antisense and dominant negative AKT2 constructs (AS-AKT2, DN-AKT2) were transfected into human glioblastoma cell line T J905 with overexpression of AKT2. Using Western blotting, MTT assay, Ki67 labeling index (Ki67 LI), flow cytometry and the TUNEL method, the expression of AKT2 and GFAP, the proliferation rate and apoptosis of glioma cells transfected with AS-AKT2 or DN-AKT2 were compared to those characteristics of parental and glioma cells transfected with an empty vector. RESULTS Cell proliferation was inhibited in glioma cells transfected with ASAKT2 and DN-AKT2 RNA, while GFAP expression and apoptosis were markedly increased in those cells. CONCLUSION AKT is an important mediator in the growth signaling pathway of malignant gliomas and is a potential promising therapeutic target for malignant gliomas.     

Analysis of Inactivation of hMLH1 by Promoter Hypermethylation and Microsatellite Instability in Gastric Carcinogenesis

OBJECTIVE To study the microsatellite instability (MSI) and methylation state of the hMLH1 gene promoter and their mechanisms underlying the development of gastric cancer. METHODS Forty-one gastric cancer samples were obtained from patients undergoing surgery and 46 chronic atrophic gastritis tissues with dysplasia or intestinal metaplasia (IM) were obtained from patients undergoing gastro-endoscopy. Fourteen normal gastric mucosal samples were used as controls. Genomic DNA was extracted from the samples and 5 microsatellite markers were used to measure MSI. Methylation-specific PCR (MSP) was used to screen the methylation state of the samples. DNA sequencing and immunohistochemistry were performed to verify the results. RESULTS MSI was identified in 22 out of the 41 (53.7%) gastric cancers, of which 8 cases showed high-level MSI (2 or more loci altered) and 14 showed low-level MSI (1 locus altered). MSI was also detected in 12 out of 46 (26.1%) pre-cancerous lesions of the stomach, whereas it was not seen in the normal tissue. Moreover, hMLH1 hypermethylation was detected in 17 out of the 41 (41.5%) gastric cancers, 9 out of the 46 (19.6%) pre-cancerous lesions and 0 out of the 14 normal tissue. Significant differences in frequency of MSI and hMLH1 promoter methylation were observed among gastric cancers, precancerous lesions and normal gastric tissue. Gastric samples with MSI had a tendency to be hypermethylated in the hMLH1 promoter. DNA sequencing and immunohistochemistry results also confirmed that hMLH1 promoter methylation could lead to loss of the hMLH1 protein and gene silence which sequentely resulted in gene mismatch and MSI. CONCLUSION Accumulation of MSI and hMLH1 promoter methylation may be important early molecular events during gastric carcinogenesis and may contribute to the acquisition of a transformed cell phenotype and the development of gastric cancer.     

Pharmacokinetics and disposition of 4′-O-tetrahydropyranyladriamycin in mice by HPLC analysis

Summary The plasma level of 4-O-tetrahydropyranyladriamycin (THP) declined rapidly after IV injection to mice, with a t_1/2() of 0.453 min. Only 1.2 g/ml THP was detected 2 min after injection of 5 mg/kg. The drug was immediately transferred to various tissues, where the drug levels were much higher than the plasma concentration. In the lung and spleen, 8.26 and 13.6 g/g THP was present, respectively, 2 h after administration. Major metabolites of THP were 13-dihydro-THP, ADM, 7-deoxyadriamycinone, and 7-deoxy-13-dihydro-adriamycinone. Only 1.12% of the dose had been recovered in the urine by 48 h after injection as THP and its metabolites, according to analysis by HPLC fluorospectroscopy. The observed disposition of THP was compared with that of adriamycin (ADM). The plasma disappearance and tissue transfer of THP were faster than those of ADM. THP levels in the spleen and lung were higher and those in the heart and liver were lower than the corresponding ADM levels. Drug levels declined more quickly in most tissues in the case of THP than of ADM. Tissue distributions after single bolus and multiple injections were also compared and discussed.     

Treatment of inoperable hepatocellular carcinoma by intra-arterial Lipiodol and 4′-epidoxorubicin

Summary A total of 30 patients presenting with inoperable hepatocellular carcinoma (HCC) were treated with intrahepatic arterial Lipiodol (5 ml) and 4-epidoxorubicin (90 mg/m²) once every 4 weeks. The treatment results included no complete response, 2 partial responses, 6 cases of static disease and 19 cases of progressive disease. The median survival was 18.9 weeks. All patients had died by the time of this writing, with survival duration ranging from 4.1 to 87.3 weeks. Toxicities were minimal and included anaemia and alopecia. As compared with a historic control group that had received the same dose of intravenous 4-epidoxorubicin, the treatment group showed similar response rates but developed fewer toxicities. There was no significant survival benefit over the control group. We concluded that although this form of treatment had comparable activity and produced fewer side effects, it provided no survival benefit over intravenous treatment. The slight prolongation of survival achieved in the treatment group as compared with the control arm might have been due to case selection.     

Identification and Charaterization of Genes Encoding Melanoma Antigens Recognized by Tumor-infiltrating Lymphocytes

Adoptive transfer of tumor infiltrating lymphocytes (TILs) into the autologous patient with melanoma resulted in the objective regression of tumor, suggesting that these TILs recognize     

Association between biomarkers of obesity and risk of high-grade prostatic intraepithelial neoplasia and prostate cancer – Evidence of effect modification by prostate size

Prostate enlargement is common with aging and obesity. We investigated the association between obesity and prostate cancer controlling for differential detection related to prostate enlargement. In an analysis of 500 men, we found body mass index, waist–hip ratio, and blood leptin levels were significantly associated with high-grade PC, but only among men without prostate enlargement. Leptin was also significantly associated with high-grade prostatic intraepithelial neoplasia (HGPIN) in the absence of prostate enlargement. Our results suggest obesity advances prostate carcinogenesis, and that detection biases at prostate biopsy may explain past inconsistencies in the association between obesity and PC.     

Inhibition of TGF-β and EGF pathway gene expression and migration of oral carcinoma cells by mucosa-associated lymphoid tissue 1

Background:

Expression of mucosa-associated lymphoid tissue 1 (MALT1) is inactivated in oral carcinoma patients with worse prognosis. However, the role in carcinoma progression is unknown. Unveiling genes under the control of MALT1 is necessary to understand the pathology of carcinomas.

Methods:

Gene data set differentially transcribed in MALT1-stably expressing and -marginally expressing oral carcinoma cells was profiled by the microarray analysis and subjected to the pathway analysis. Migratory abilities of cells in response to MALT1 were determined by wound-healing assay and time-lapse analysis.

Results:

Totally, 2933 genes upregulated or downregulated in MALT1-expressing cells were identified. The subsequent pathway analysis implicated the inhibition of epidermal growth factor and transforming growth factor-β signalling gene expression, and highlighted the involvement in the cellular movement. Wound closure was suppressed by wild-type MALT1 (66.4%) and accelerated by dominant-negative MALT1 (218.6%), and the velocities of cell migration were increased 0.2-fold and 3.0-fold by wild-type and dominant-negative MALT1, respectively.

Conclusion:

These observations demonstrate that MALT1 represses genes activating the aggressive phenotype of carcinoma cells, and suggest that MALT1 acts as a tumour suppressor and that the loss of expression stimulates oral carcinoma progression.     

siRNA-Mediated Suppression of Synuclein γ Inhibits MDA-MB-231 Cell Migration and Proliferation by Downregulating the Phosphorylation of AKT and ERK

Purpose

Synuclein-γ (SNCG), which was initially identified as breast cancer specific gene 1, is highly expressed in advanced breast cancers, but not in normal or benign breast tissue. This study aimed to evaluate the effects of SNCG siRNA-treatment on breast cancer cells and elucidate the associated mechanisms.

Methods

Vectors containing SNCG and negative control (NC) siRNAs were transfected into MDA-MB-231 cells; mRNA levels were determined by real-time polymerase chain reaction. Cell proliferation was evaluated using the MTT assay, cell migration was assessed by the Transwell assay, apoptosis and cell cycle analyses were conducted with the flow cytometer, and Western blot analysis was performed to determine the relative levels of AKT, ERK, p-AKT, and p-ERK expression.

Results

SNCG mRNA levels were significantly reduced in MDA-MB-231 cells transfected with SNCG siRNA. Our results indicate that in SNCG siRNA-treated cells, cell migration and proliferation decreased significantly, apoptosis was induced, and the cell cycle was arrested. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA-treated groups, than in the control and NC groups.

Conclusion

SNCG siRNA could decrease the migration and proliferation of breast cancer cells by downregulating the phosphorylation of AKT and ERK.     

Expression of TGF-β1 and the mechanism of invasiveness and metastasis induced by TGF-β1 in breast cancer

Objective To investigate the expression of MMP-2,TIMP-2,TGF-β1 and TGF-βRI and the relationship among them in breast cancer.Methods The protein expression of MMP-2,TIMP-2,TGF-β1 and TGF-β1R1 was detected on tissue chips by S-P immunohistochemical staining in 160 cases of breast carcinoma.Results The positive rates of TGF-β1,TGF-β1 mRNA,MMP-2,MMP-9,TIMP-1 and TIMP-2 expression were 73.7%,56.2%,96.9%,95.0%,87.5% and 89.4%,respectively.Axillary lymph node metastasis and TNM staging(P ＜0.01 and P ＜0.01,respectively)were positively correlated to the expression of TGF-β1.Relase-free survival of TGF-β1 positive group was lower than that of TGF-β1 negative group(P = 0.023).The expression of M MP-2 or M M P-9 was positively correlated to that of TGF-β1 (r=0.170,P＜0.05;r =0.221,P＜0.01)and was negatively correlated to that of TGF-β1 mRNA(r =-0.126,P ＞0.05;r = 0.019,P ＞ 0.05).Conclusion The expression of TGF-β1 may be closely correlated with the invasion and metastasis of breast cancer.TGF-β1-induced invasiveness and metastasis of breast cancer cells are mediated by MMP-2 and MMP-9.     

Suppression of growth and invasive behavior of human prostate cancer cells by ProstaCaid™: mechanism of activity

Since the use of dietary supplements as alternative treatments or adjuvant therapies in cancer treatment is growing, a scientific verification of their biological activity and the detailed mechanisms of their action are necessary for the acceptance of dietary supplements in conventional cancer treatments. In the present study we have evaluated the anti-cancer effects of dietary supplement ProstaCaid? (PC) which contains mycelium from medicinal mushrooms (Ganoderma lucidum, Coriolus versicolor, Phellinus linteus), saw palmetto berry, pomegranate, pumpkin seed, green tea [40% epigallocatechin-3-gallate (EGCG)], Japanese knotweed (50% resveratrol), extracts of turmeric root (BCM-95?), grape skin, pygeum bark, sarsaparilla root, Scutellaria barbata, eleuthero root, Job's tears, astragalus root, skullcap, dandelion, coptis root, broccoli, and stinging nettle, with purified vitamin C, vitamin D3, selenium, quercetin, citrus bioflavonoid complex, β sitosterolzinc, lycopene, α lipoic acid, boron, berberine and 3.3'-diinodolymethane (DIM). We show that PC treatment resulted in the inhibition of cell proliferation of the highly invasive human hormone refractory (independent) PC-3 prostate cancer cells in a dose- and time-dependent manner with IC50 56.0, 45.6 and 39.0 μg/ml for 24, 48 and 72 h, respectively. DNA-microarray analysis demonstrated that PC inhibits proliferation through the modulation of expression of CCND1, CDK4, CDKN1A, E2F1, MAPK6 and PCNA genes. In addition, PC also suppresses metastatic behavior of PC-3 by the inhibition of cell adhesion, cell migration and cell invasion, which was associated with the down-regulation of expression of CAV1, IGF2, NR2F1, and PLAU genes and suppressed secretion of the urokinase plasminogen activator (uPA) from PC-3 cells. In conclusion, the dietary supplement PC is a promising natural complex with the potency to inhibit invasive human prostate cancer.     

Molecular Mechanism of Differentiation and Apoptosis of U937 Cells Induced by 12-O-Tetradecanoylphorbol-13-Acetate

OBJECTIVE To explore the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on differentiation, apoptosis and related molecular mechanisms in U937 myelomonocytic leukemia cells.METHODS Morphological changes were analyzed by phase contrast and light microscopy, expression of the monocytic differentiation maker CD11b by direct immunofluorescence staining, cell cycle distribution and apoptosis by flow cytometry, and expression of bcl-2, Bax, survivin and p21Cip1/Waf1proteins by Western analysis.RESULTS Treatment of U937 cells with 10 nmol/L TPA induced cell adherence. The adherent cells showed G0/G1 cell cycle arrest (69.0% at 24h vs 52.1% control; P< 0.01),and morphologic changes and increased expression of the monocytic differentiation marker CD11b (63.0% at 72 h vs15.3% control; P< 0.01 ). In addition to these effects, about 20% of the cells still remained in suspension and exhibited a time-dependent increasing apoptosis, which reached 70.3% after 72 h of treatment ( P< 0.01 ). TPA treatment for 24 h induced expression of p21Cip1/Waf1 in the adherent cells, but not in the non-adherent cells. Furthermore, bcl-2 and survivin expression declined in 24 h-TPA-treated non-adherent cells compared with untreated control and adherent cells, whereas no change in the expression of Bax was detected.CONCLUSION TPA induces both differentiation and apoptosis in U937 cells,which may be related to the upregulation of p21Cip1/Waf1 and downregulation of bcl-2 and survivin expression.