Prognostic significance of FOXA1 and BRCA1 expression in triple negative breast cancer

AIM: To investigate the expression of forkhead box protein A1(FOXA1) BRCA1 protein,P53 and vascular endothelial growth factor (VEGF) in triple negative breast cancer (TNBC) and non-TNBC, and the relevance with the clinicopathological parameters for evaluating the prognosis. METHODS: The tumor samples were collected from 113 cases of breast cancer patients in the First Affiliated Hospital of Jinan University,and divided into TNBC group, luminal subtype group and HER-2 overexpression subtype group by the immunohistochemical results of estrogen receptor, progesterone receptor and HER-2. EnVision two-step method was used to detect the expression of FOXA1, BRCA1, P53 and VEGF in the tumor samples. RESULTS: Total FOXA1 positive expression rate was 63.7% (72/113), with 45.2% (19/42) in TNBC, 88.0% (44/50) in luminal subtype and 42.9% (9/21) in HER-2 overexpression subtype.The statistically sigfnificant difference among the 3 groups was observed (P<0.01). Total BRCA-1 positive expression rate was 47.8% (54/113), with 66.7% (28/42) in TNBC, 44.0% (22/50) in luminal subtype and 19.0% (4/21) in HER-2 overexpression subtype.The statisticallysignificant difference among the 3 groups was also observed (P<0.01). In the cases of clinical stages Ⅰ~Ⅱand histological grades 1~2, FOXA1 positive rate was higher than the FOXA1 negative rate (P<0.01). Negative correlations between FOXA1 positive rate and expression of P53/VEGF, and between FOXA1 positive rate and the recurrence rate were found (P<0.05). In the cases of clinical stages Ⅱ~Ⅲ and histological grades 2~3, the BRCA1 positive rate was higher than the BRCA1 negative rate (P<0.05). Positive correlations between BRCA-1 positive rate and the expression of P53/VEGF, and between BRCA1 positive rate and the recurrence rate were also observed (P<0.05). CONCLUSION: Expression of FOXA1 and BRCA1 in breast cancer is different. BRCA1 may be an adverse prognostic indicator for triple negative breast cancer.     

Prognostic significance of CUEDC2 expression in hepatocellular carcinoma

AIM: To investigate the expression of CUE domain-containing 2 (CUEDC2) in hepatocellular carcinoma (HCC) and to analyze its clinical prognostic significance. METHODS: Total 186 formalin-fixed paraffin-embedded tissues obtained from surgical HCC with detailed clinicopathological and follow-up data were used. The expression of CUEDC2 was detected by immunohistochemistry. The relationships between the expression of CUEDC2 and clinicopathological characteristics and prognosis were analyzed. RESULTS: The positive rate of CUEDC2 in HCC was 85.5% (159/186), among which, the low expression was 52.2% (97/186) and the high expression was 47.8% (89/186). CUEDC2 expression was correlated with serum alpha-fetal protein (AFP) level, tumor size, tumor number, tumor differentiation and TNM stage (P<0.05). Kaplan-Meier survival curves showed that the patients with high expression of CUEDC2 were associated with significantly shorter overall survival and recurrence-free survival than those with low CUEDC2 expression (P<0.05). Multivariate Cox regression analysis revealed 3 independent prognostic factors including CUEDC2 expression, serum AFP and tumor number. CONCLUSION: CUEDC2 was expressed in most HCC tissues, which was relevant to tumor growth, tumor differentiation and prognosis. CUEDC2 could be a novel valuable molecular marker to predict the HCC prognosis.     

Relationship between c-erbB-2, BCSG1 expression and recurrence,metastasis in breast cancer

AIM: To assess the significance of c-erbB-2, BCSG1 (breast cancer specific gene-1) expression and other parameters in recurrence or metastasis of breast cancer. METHODS: The expression of c-erbB-2, BCSG1, and ER, PR, MVD, VEGF, VEGF-C, FLT-4, LVD were determined with the SP immunohistochemical method in 58 cases of invasive breast cancer patients occurred over 5 years. The cases were used to analyze the effect of c-erbB-2, BCSG1, VEGF-C and ER, PR, MVD, VEGF, FLT-4, LVD expression on clinical-pathological manifestations and prognosis in breast cancer. RESULTS: The expression rates of c-erbB-2, BCSG1, VEGF-C, LVD were respectively 25.9%, 62.1%, 36.2%, 32.8% in association with the lymph node metastasis and recurrence of breast cancer (P<0.05), the expression rate of MVD was also increased significantly (P＜0.05). CONCLUSION: The c-erbB-2, BCSG1, VEGF-C, LVD are highly expressed and strongly correlated with the lymph node metastasis and recurrence of breast cancer, of which BCSG1 may be used as a predictor of prognosis.     

Detection of bcl-2 methylation and the relationship between bcl-2 methylation and expression,prognostic factors in breast cancer

AIM: To establish methylation-specific PCR (MSP) method for detecting bcl-2 gene, and to study the relationship between bcl-2 methylation and expression, prognostic factors in breast cancer. METHODS: The primer of bcl-2 gene for MSP was designed. The methylations in CpG island of bcl-2 gene in 54 cases of breast cancer were detected by using MSP. The expressions of bcl-2, PCNA, ER and PR in 54 cases of breast cancer were detected by using SP immunohistochemical technique. RESULTS: The overall positive rate of bcl-2 methylation was 29.6% in breast cancer. There was a significant negative correlation between the methylation of bcl-2 and the expression of bcl-2 (P＜0.01). The methylation of bcl-2 coincided with those bad prognostic factors such as high PCNA label index (LI), ER-and PR-(P＜0.01). CONCLUSIONS: This study established the MSP method for detecting bcl-2 gene. The results of MSP and sequence analysis testified that the design of the MSP primer of bcl-2 gene in this study was successful. The methylation of bcl-2 would become the marker indicating bad prognosis of breast cancer.     

Relationship between expression of somatostatin and DNA ploidy in breast cancer

AIM: To investigate the relationship between somatostatin and the pathologic type, estrogen receptor,DNA ploidy of nuclei in tumor cells of breast cancer.METHODS: 67 cases of primary breast cancer and 25 cases of benign breast tumor were examined by immunohistochemical stretomyces avidin peroxidase method. 26 cases of breast cancer selected at random were analyzed by flow cytometry.RESULTS: Somatostatin expressed significantly higher in low malignant breast cancer than that in high malignant breast cancer (P<0.05). Most of cancers with positive staining of somatostatin were diploidy,most of cancers with negative staining were aneuploidy,there had significant difference between two groups (P<0.05).CONCLUSION: Somatostatin may delay the progress of breast cancer,and somatostatin levels in cancer tissues may become a useful indicator for assessing prognosis of patients with breast cancer.     

Relationship between zinc and zinc transporter expression in breast cancer MCF-7 cells

AIM: To study the changes of zinc transporter gene expression in MCF-7 cell line exposed to ZnCl₂ and TPEN. METHODS: Human breast cancer cell line MCF-7 was exposed to different concentrations of ZnCl₂ (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT and levels of zinc transporter mRNA by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations. RESULTS: MCF-7 cells viability rate was significantly decreased when exposed to ZnCl₂ (with 150 μmol/L and 200 μmol/L) and TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl₂ and decreased when exposed to TPEN. ZnT-1 mRNA level was increased along with the increasing concentration of ZnCl₂ but decreased when exposed to TPEN. The expressions of ZIP2 and ZIP10 were increased along with the increasing concentration of TPEN. CONCLUSION: ZnT-1 gene expression is induced by zinc supplement and repressed by zinc deficiency. ZIP2 and ZIP10 gene expressions are induced by zinc deficiency.     

ACTL8 expression and its correlation with clinicopathological features and prognosis in breast cancer

AIM: To investigate the actin-like protein 8 (ACTL8) expression and its relationship with clinicopathological features and prognosis in breast cancer.METHODS: The expression of ACTL8 in human normal mammary epithelial cell line MCF-10A and 5 breast cancer cell lines was detected by Western blot. The expression of ACTL8 was also investigated by immunohistochemistry in 6 cases of breast cancer specimens with adjacent normal tissues. The data in 488 cases of breast specimens from TCGA dataset were downloaded, and the relationship between the mRNA expression of ACTL8 and the clinicopathological features and prognosis was analyzed.RESULTS: The expression of ACTL8 in 4 breast cancer cell lines was significantly higher than that in breast epithelial cell line MCF-10A.The level of the ACTL8 expression in breast tumors was significantly higher than that in the corresponding adjacent normal breast tissues. The mRNA expression of ACTL8 was correlated with age, tumor size, clinical TNM stage and lymph node metastasis of breast cancer patients (P < 0.05). The high expression level of ACTL8 mRNA indicated a poor prognosis of breast cancer patients. CONCLUSION: ACTL8 protein is highly expressed in breast cancer specimens and is closely correlated with the clinicopathological features and prognosis, suggesting that ACTL8 is a prognostic marker for breast cancer or a potential new target for treatment of breast cancer.     

Relationship between apoptosis,proliferation and expression,mutation of related genes in breast cancer

AIM:To study the relationship between apoptosis, proliferation and expression,mutation of related genes in breast cancer.METHODS:Methods of TUNEL, immunohistochemical S-P and PCR-SSCP were used respectively to study apoptotic index (AI), mitotic index(MI), expression of Bcl-2,p53,c-erbB-2,PCNA,Ki67,TopoⅡ and mutation of p53 in 54 cases of breast cancer.RESULTS:AI and MI were 9.40±3.78 and 5.96±2.36, respectively. There was a significant direct correlation between them(r=0.46.P＜0.01).High expression of Bcl-2,PCNA,Ki67,TopoⅡ coincided with high AI,MI(P＜0.01). High expression of p53,c-erbB-2 and mutation of p53 coincided with high MI(P＜0.01). Type of p53 mutation coincided with AI(P＜0.05).CONCLUSION:Disturbance of gene control between apoptosis and proliferation is related with expression,mutation of related genes in breast cancer.     

Changes of MDR1 and MRP gene expression in primary breast cancer after neoadjuvant chemotherapy

AIM: To investigate the expression of drug resistance genes, MDR1 and MRP, in patients with primary breast cancer after neoadjuvant chemotherapy. METHODS: MDR1 and MRP gene expression were detected by semi-quantitative RT-PCR in 20 patients with primary breast cancer before and after chemotherapy. RESULTS: Before chemotherapy, MDR1 and MRPexpression could be detected in 15 cases (75%) and 18 cases (90%), respectively. After chemotherapy, expression of MDR1 was not significantly different from that before chemotherapy, but expression of MRPwas significantly different from that before chemotherapy. CONCLUSION: Drug resistance gene MRP, but not MDR1 expression is enhanced in patients with primary breast cancer subjected to neoadjuvant chemotherapy.     

Effects of intermittent high glucose on apoptosis and PTEN expression in islet cells

AIM: To investigate whether the increase in PTEN expression is related to apoptosis, and whether it is regulated by reactive oxygen species(ROS). METHODS: The rat islet cells were divided into constant low glucose group (group L), constant high glucose group (group H), glucose fluctuation group (group F), low glucose after high glucose group (group HL) and low glucose after fluctuation group (group FL). The ROS level, apoptotic rate, intracellular calcium, insulin release and PTEN protein expression were analyzed. RESULTS: Compared with groups H and L, the insulin secretion decreased, and intracellular calcium, ROS level, PTEN protein expression and apoptotic rate increased in group F (P<0.05). Compared with group H, the intracellular calcium, ROS level, PTEN protein expression and apoptotic rate in group HL decreased, but were still higher than those in group L (P<0.05). Compared with group F, the intracellular calcium, ROS level, PTEN protein expression and apoptotic rate in group FL decreased, but were still higher than those in group L (P<0.05). CONCLUSION: Glucose fluctuation can cause the apoptosis of islet cells more easily than constant high glucose. This may be related to the change of intracellular calcium and increase in oxidative stress which promotes PTEN expression. The recovery of glucose level to some extent relieves oxidative stress, decrease PTEN expression and reduce cell damage.     

Changes of dendritic cells subsets and their HLA-DR expression in peripheral blood of breast cancer patients

AIM: To explore the changes of the subsets and HLA-DR expression of dendritic cells and their concerning cytokine levels in peripheral blood of patients with breast cancer. METHODS: The subsets of the precussors of dendritic cells (pDC) in the peripheral blood of 57 cases of patients with breast cancer before operation and a week or six months after operation and 20 cases of healthy controls were analyzed by four-color FCM. The levels of IL-12p40, IL-10, IFN-γ and IL-4 in the plasmas were tested by ELISA. RESULTS: Among 57 cases of patients with breast cancer, 2 cases in Ⅲ phase and 4 cases in Ⅳphase expressed deficiency of pDC, the ratios of pDC₁/pDC₂ in the other cases inⅠ, Ⅱ, Ⅲ, Ⅳ phase were respectively 1.62±0.59, 1.41±0.63, 0.91±0.32, 0.81±0.29 before operation, which were markedly lower than those in controls (1.94±0.44). The ratios of pDC₁/pDC₂ in the cases inⅠ, Ⅱ, Ⅲ phase were 1.71±0.47, 1.52±0.54, 1.04±0.36 a week after operation, which were the same as those in pre-operation, but markedly lower than those in controls. The ratios of pDC₁/pDC₂ in the cases inⅠ, Ⅱ, Ⅲ phase were 1.92±0.72, 1.63±0.65, 1.28±0.34 six months after operation, which were markedly higher than those in pre-operation, meanwhile, to compare with controls, those were still lower for patients in Ⅱ, Ⅲ phase except in Ⅰphase. No difference between patients and controls in the expression of HLA-DR of pDCs and the levels of IL-12p40, IL-10, IFN-γ, IL-4 in plasmas and the ratios of IL-12p40/ IL-10, IFN-γ/ IL-4 was observed. CONCLUSION: The ratios of pDC₁/pDC₂ in peripheral blood of patients with breast cancer inⅠ-Ⅳ phase are decreased. Parts of patients in Ⅲ, Ⅳ phase are deficiency of pDCs. HLA-DR expression of DCs and the ability of DCs which secret the concerning cytokines do not change as pDC subsets change. pDC subsets improve markedly inⅡ, Ⅲ phase patients and recover to the normal level inⅠphase patients after operation.     

Dynamic expression of PTEN in liver tissue of rats with hepatic fibrosis

AIM: To investigate the dynamic expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in liver tissue in the process of hepatic fibrosis in rats. METHODS: The rat model of hepatic fibrosis used in this study was induced by common bile duct ligation (BDL). Hematoxylin and eosin (HE) and Masson’s trichrome staining were used for observing the histological changes in hepatic fibrosis tissue. At 4 time points, the expressions of PTEN protein and mRNA in hepatic tissues of rats were detected by immunohistochemical staining, Western blotting and real-time fluorescent quantitative PCR assay, respectively. RESULTS: The rat model of hepatic fibrosis was established successfully. With each consecutive week after BDL, increased fibrosis, degeneration and necrosis were found in rat liver cells. Not surprisingly, a disruption of normal architecture and a decrease in normal hepatic cells was concomitantly observed. The immunohistochemical staining indicated that there was extensive expression of PTEN in liver tissues of normal rats, it expressed mainly in the cytoplasm, and with the aggravation of hepatic fibrosis, the expression of PTEN in liver tissues decreased gradually (P<0.01). Western blotting and real-time fluorescent quantitative PCR at weekly time points (1, 2, 3, and 4 weeks) after BDL showed that, the expression of PTEN protein and mRNA in fibrotic rat liver tissue decreased gradually with increasing severity of hepatic fibrosis (P<0.01). Furthermore, all values from BDL rats were significantly lower than those from the sham operation group (P<0.01). CONCLUSION: The expressions of PTEN protein and mRNA in fibrotic liver tissue of rat decrease gradually with the progression of hepatic fibrosis, and increasing severity of fibrosis correlated well with decreasing PTEN expression.     

Survivin-2B induces apoptosis of human breast cancer cells

AIM：To explore the role of survivin-2B in the process of tumor cell apoptosis. METHODS：The survivin-2B gene was cloned into pcDNA3.1 vector and the recombinant plasmid pcDNA3.1-survivin-2B was obtained. Human breast cancer MCF7 cells were transfected with pcDNA3.1 and pcDNA3.1-survivin-2B using Lipofectamine 2000. The cell cycle was determined by propidium iodide staining, and the apoptosis was detected by annexin V/7-AAD staining 48 h after transfection. Meanwhile, tatal RNA was extrated and multiplex polymerase chain reaction based on GenomeLab GeXP Genetic Analysis System was performed to detect the expression of 21 tumor-related genes. RESULTS：Flow cytometry analysis indicated that over-expression of survivin-2B promoted the apoptosis and cell cycle arrest of MCF7 cells. Compared with control group, totally 10 differential expressed genes were related to the over-expressed survivin-2B, among which 2 were up-regulated and 8 were down-regulated. The expression of aldehyde dehydrogenase 4 family member A1 (ALDH4A1) was 48% down-regulated, and the expression of protein regulator of cytokinesis 1 (PRC1) was 1.08 folds up-regulated. CONCLUSION：Survivin-2B induces the expression changes of some tumor-related genes, which results in the apoptosis and G2/M arrest of MCF7 cells.     

Relationship between uPA and NF-κB p65 expression and its clin ical significance in breast cancer

AIM:To evaluate the relationship between u PA and NF-κB p65 expression and its clinical significance in breast cancer.METHODS:The uPA mRNA was measured by real-time fluorescent quan t itative PCR in 46 cases of breast cancer tissues and their adjacent counterparts .NF-κB p65 were measured using immunohistochemistry.RESULTS:The expression of uPA gene was elevated in 63% of cases ,and there was a strong correlation between NF-κB p65 and uPA expression (r ＝0.451，P<0.01).In addition,the expression of uPA was associated with num bers of axillary lymph node involvement and tumor size.However,no significance was found in uPA expression in the age,pathological type and distant metastasi s organ classification.CONCLUSION:The uPA gene is highly expressed in most of the brea st cancer,and there is strong correlation to NF-κB p65 expression,number of l ymph nodes involvement and tumor size.     

Effect of curcumin on expression of p21 and CD44V6 in human breast cancer xenografts

AIM:To study the effect of curcumin on the expression of p21 and CD44V₆ in breast carcinoma in nude mice.METHODS:Nude mice were xenografted with human breast cancer cell line MCF-7 and randomly divided into 2 groups (n=4 in each group): control group and curcumin group. In latent period,the percentage of tumor development was observed. Tumors were measured and the surface areas were calculated. RT-PCR was performed to detect the expression level of cyclin D1,p21 and CD44V₆ mRNA. RESULTS:The tumor surface areas in the curcumin group were significantly lower than those in control group. In curcumin treatment group,the expression of p21 was up-regulated while cyclin D1 was nearly not changed. The expression of CD44V₆ was significantly down-regulated in curcumin group.CONCLUSION:Curcumin inhibits the expression of CD44V₆ and up-regulates the expression of p21 in nude mice bearing human breast cancer cell line MCF-7.