Expressions of GRP78 and Bax associate with differentiation,metastasis, and apoptosis in non-small cell lung cancer

The purpose of this study was to detect the expressions of GRP78 and Bax in human non-small cell lung cancer (NSCLC) tissues, to analyze their correlations with carcinogenesis and the development of NSCLC, and to investigate the relationship of GRP78 expression to metastasis and apoptosis in the NSCLC cell line HCC827. The positive expression rates of GRP78 and Bax in NSCLC lung tissues were 59.7% and 34.7% by RT-PCR, respectively. The mRNA and protein expression levels of GRP78 in NSCLC tissues were significantly higher than that in the relatively normal surrounding lung tissues (p < 0.05); the lesser the degree of tumor differentiation was, the higher the mRNA and protein expression levels of GRP78 were (p < 0.05). The mRNA and protein expression levels of GRP78 from patients in advanced pathological stages (III–IV) were significantly higher than the corresponding levels in patients in early pathological stages (I–II) (p < 0.05); the mRNA and protein expression levels of GRP78 in patients with positive lymph node metastasis were significantly higher than those in patients with negative lymph node metastasis (p < 0.05). The mRNA and protein expression levels of Bax in the above cases showed the opposite trend of the mRNA and protein expression levels of GRP78. However, the mRNA and protein expression levels of both GRP78 and Bax were independent of the patient’s sex, the patient’s age, the tumor size and the histological type (adenocarcinoma or squamous cell carcinoma) of NSCLC (p > 0.05). The mRNA expression level of GRP78 and the mRNA expression level of Bax in human NSCLC tissues were negatively correlated (r = −0.353, p = 0.002). After transfection of GRP78 siRNA in HCC827 cells, the GRP78 protein expression level was significantly decreased (p < 0.01), while the Bax protein expression level was significantly increased (p < 0.01); the number of cells that passed through the Transwell chamber was significantly less in the non-transfected control group compared to the transfected control group (p < 0.01). The number of apoptotic cells was significantly greater in the non-transfected control group compared to the transfected control group (p < 0.01). The expression levels of GRP78 and Bax were related to the carcinogenesis, development and metastasis of NSCLC. GRP78 expression with siRNA interference in the human NSCLC cell line HCC827 can reduce metastasis and promote apoptosis in HCC827 cells.     

Overexpression of osteopontin in hepatocellular carcinoma and its relationships with metastasis,invasion of tumor cells

Osteopontin (OPN) plays an important role in metastasis and relapse of human cancer. However, the whole story of OPN relating to cancer has been far from clear untill now. To investigate the expression of OPN in hepatocellular carcinoma (HCC) and its relationships with recurrence and metastasis of HCC, normal and malignant liver tissues from patients with HCC were analyzed using immunohistochemical staining. OPN expression was inhibited by small interfering RNA (siRNA) in HCC cells lines, and then colony formation and matrigel invasion were examined. The results showed that expression of OPN was associated with metastasis of HCC with a positive rate of OPN in the tissue of HCC (70.00%), which was highly more obvious than those in paracarcinoma tissue and normal liver tissue (P < 0.01). In HCC cell lines, OPN depletion could reduce formed colony and metastasizing numbers in vitro. In conclusion, Expression of OPN in the tissue of HCC is related to metastasis or metastases. Specific siRNA could decrease expressions of OPN at both mRNA and protein levels, and abates the invasiveness of hepatocellular carcinoma cells, suggesting that OPN might be a promising agent for treatment of metastasis and recurrence of HCC.     

Expression of cytokeratin 19 and matrix metalloproteinase 2 predicts lymph node metastasis in hepatocellular carcinoma

The purpose of this study was to assess the value of cytokeratin 19 (CK19) and matrix metalloproteinase 2 (MMP-2) in predicting lymph node metastasis (LNM) and survival after curative resection in hepatocellular carcinoma (HCC) patients. Expression of CK19 and MMP-2 in tumor tissue was assessed through immunohistochemical staining of tissue microarrays (TMAs), which were constructed using samples from HCC patients with (n = 123) and without (n = 145) LNM. Positive CK19 expression was correlated with LNM (P < 0.001), satellite lesions (P = 0.016), and lymph node location (P = 0.039). High MMP-2 expression correlated with LNM (P < 0.001), UICC T stage (P = 0.023), and Edmondson grade (P = 0.022). Moreover, CK19 expression correlated with MMP-2 expression (P = 0.033). CK19 and MMP-2 expression were predictive of HCC LNM (AUC: 0.640; 95% CI: 0.572–0.707; P < 0.001 and AUC: 0.611; 95% CI: 0.544–0.679; P = 0.002, respectively). CK19 and MMP-2 expression were independent prognostic factors for disease-free survival (P = 0.031 and P = 0.012, respectively) and overall survival (P = 0.013 and P = 0.018, respectively) in HCC patients with LNM. CK19 expression (P < 0.001), MMP-2 expression (P = 0.006), and UICC T stage (P < 0.001) were independent risk factors for developing LNM in HCC. These findings show that CK19 and MMP-2 expression may be beneficial in predicting HCC LNM and survival.     

Epigenetic silencing of DACH1 induces the invasion and metastasis of gastric cancer by activating TGF‐β signalling

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non‐tumour tissues were examined. Methylation‐specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re‐expression was induced by 5‐Aza‐2′‐deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non‐tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial–mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)‐β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1‐expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF‐β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.     

Abnormal expression of NRF-2α in hepatocellular carcinoma identified with a newly prepared monoclonal antibody against human NRF-2α protein

Human nuclear respiratory factor 2 alpha subunit (NRF-2α) is fundamentally important to cell function and the development. We aimed to establish the monoclonal antibody (MAb) against the human NRF-2α protein and to investigate its distribution in human hepatocellular carcinoma (HCC) and tumor-adjacent tissues. The 6× His-NRF-2α fusion protein was successfully induced and purified. One monoclonal antibody (MAb) against human NRF-2α, 1-D10-E1-B11-G3 (IgG1), effective in detecting the recombinant and the cellular protein, was characterized. Using immunohistochemical analysis, the expression of NRF-2α was investigated in 38 cases of HCC specimens and 14 cases of tumor-adjacent specimens. Staining was found positive in 9 cases of HCC tissues (23.7%) and 8 cases of normal hepatic tissues (57.1%). The higher-grade frequency of expression of NRF-2α in tumor-adjacent tissues was significantly higher (P < 0.01) than that in tumor tissues, suggesting that NRF-2α may play important roles in carcinogenesis of HCC.     

Expression of connective tissue growth factor and interleukin-11 in intratumoral tissue is associated with poor survival after curative resection of hepatocellular carcinoma

In the present study, we evaluated the prognostic value of intratumoral and peritumoral expression of connective tissue growth factor (CTGF), transforming growth factor-beta 1 (TGF-β1), and interleukin-11 (IL-11) in patients with hepatocellular carcinoma (HCC) after curative resection. Expression of CTGF, TGF-β1, and IL-11 was assessed by immunohistochemical staining of tissue microarrays containing paired tumor and peritumoral liver tissue from 290 patients who had undergone hepatectomy for histologically proven HCC. The prognostic value of these and other clinicopathologic factors were evaluated. The median follow-up time was 54.3 months (range, 4.3–118.3 months). High intratumoral CTGF expression was associated with vascular invasion (P = 0.015), intratumoral IL-11 expression correlated with higher tumor node metastasis (TNM) stage (P = 0.009), and peritumoral CTGF overexpression correlated with lack of tumor encapsulation (P = 0.031). Correlation analysis of these proteins revealed that intratumoral CTGF and IL-11 correlated with high intratumoral TGF-β1 expression (r = 0.325, P < 0.001; and r = 0.273, P < 0.001, respectively). TNM stage (P < 0.001), high intratumoral CTGF levels (P = 0.010), and intratumoral IL-11 expression (P = 0.015) were independent prognostic factors for progression-free survival (PFS). Vascular invasion (P = 0.032), TNM stage (P < 0.001), high intratumoral CTGF levels (P = 0.036), and intratumoral IL-11 expression (P = 0.013) were independent prognostic factors for overall survival (OS). High intratumoral CTGF and intratumoral IL-11 expression were associated with PFS and OS after hepatectomy, and the combination of intratumoral CTGF with IL-11 may be predictive of survival.     

Clinicopathological pattern and Annexin A2 and Cdc42 status in patients presenting with differentiation and lymphnode metastasis of esophageal squamous cell carcinomas

Annexin A2 and Cdc42 were identified by 2-dimensional electrophoresis (2-DE) and MALDI-TOF–MS between esophageal squamous cell carcinomas (ESCC) and corresponding normal esophagus mucosa in our previous study. To assess clinico-pathological pattern and Annexin A2 and Cdc42 status with respect to cell differentiation and lymphnode metastasis in patients with ESCC. The expression of Annexin A2 and Cdc42 in 22 pairs of fresh ESCC and matched tissues were detected by qRT-PCR and western blot, respectively. And it was further confirmed by immunohistochemistry with 175 pairs of formalin-fixed, paraffin-embedded ESCC. Results showed that Annexin A2 expression was significantly down-regulated, and Cdc42 was up-regulated in ESCC compared to matched control on both mRNA and protein level (P < 0.05), which was in accordance with our previous results on proteomics data. Additionally, Annexin A2 and Cdc42 expression was significantly correlated with lymphoid node metastasis (P < 0.05) and pathological differentiation (P < 0.05). Taken together, we proposed that the aberrant expression of Annexin A2 and Cdc42 played a role in carcinogenesis, differentiation and metastasis of ESCC, which implied its potential target for clinical biomarkers in differentiation and lymph node metastasis.     

The expression of HIF-1α in primary hepatocellular carcinoma and its correlation with radiotherapy response and clinical outcome

The purpose of this study was to evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) protein expression in hepatocellular carcinoma (HCC), and responses of abdominal metastatic lymph nodes (LNs) from HCC patients treated with external beam radiotherapy (EBRT). HIF-1α immunohistochemical staining was performed on tissue microarrays (TMAs) of primary HCC specimens from 69 HCC patients with abdominal LN metastases. All patients received abdominal metastatic LN EBRT at the Department of Radiation Oncology at Zhongshan Hospital. A receiver-operating characteristic (ROC)-based approach and logistical regression analysis were used to determine the predictive value of HIF-1α expression in primary tumors with HCC metastatic LN EBRT response. Kaplan–Meier curves and log-rank tests were used to analyze patient survival. Cox proportional hazards regression model was used to analyze independent prognostic factors. HIF-1α expression was correlated with blood hemoglobin (Hb: r = −0.280, P = 0.020), response of abdominal metastatic LNs to EBRT (r = 0.286, P = 0.017), locoregional recurrence (r = 0.278, P = 0.021), and cancer-specific deaths (r = 0.298, P = 0.013). HIF-1α expression was predictive of EBRT response of metastatic LNs [area under the curve (AUC): 0.646; 95% confidence interval (CI): 0.499–0.793; P = 0.047], locoregional recurrence (AUC: 0.657; 95% CI: 0.509–0.805; P = 0.049) and cancer-specific deaths (AUC: 0.671; 95% CI: 0.531–0.812; P = 0.035). Patients with tumors exhibiting high HIF-1α expression had significantly poorer overall survival (OS) than those with low tumor expression of HIF-1α (P = 0.016). Multivariate analysis showed that Hb (P = 0.035), vascular invasion (P = 0.026), Child-Pugh score (P < 0.001), intrahepatic tumor control (P < 0.001), and HIF-1α (P = 0.020) were independent prognosis factors for OS of HCC patients after receiving abdominal metastatic LN EBRT. HIF-1α expression in primary HCCs was associated with EBRT response of abdominal metastatic LNs and poor prognosis.     

Evaluation of Markers of Oxidative Stress,Antioxidant Function and Astrocytic Proliferation in the Striatum and Frontal Cortex of Parkinson’s Disease Brains

Dopaminergic neurons die in Parkinson’s disease (PD) due to oxidative stress and mitochondrial dysfunction in the substantia nigra (SN). We evaluated if oxidative stress occurs in other brain regions like the caudate nucleus (CD), putamen (Put) and frontal cortex (FC) in human postmortem PD brains (n = 6). While protein oxidation was elevated only in CD (P < 0.05), lipid peroxidation was increased only in FC (P < 0.05) and protein nitration was unchanged in PD compared to controls. Interestingly, mitochondrial complex I (CI) activity was unaffected in PD compared to controls. There was a 3–5 fold increase in the total glutathione (GSH) levels in the three regions (P < 0.01 in FC and CD; P < 0.05 in Put) but activities of antioxidant enzymes catalase, superoxide dismutase, glutathione reductase and glutathione-s-tranferase were not increased. Total GSH levels were elevated in these areas because of decreased activity of gamma glutamyl transpeptidase (γ-GT) (P < 0.05) activity suggesting a decreased breakdown of GSH. There was an increase in expression of glial fibrillary acidic protein (GFAP) (P < 0.001 in FC; P < 0.05 in CD) and glutathione peroxidase (P < 0.05 in CD and Put) activity due to proliferation of astrocytes. We suggest that increased GSH and astrocytic proliferation protects non-SN brain regions from oxidative and mitochondrial damage in PD.     

Effect of Different Iodine Nutrition on Cerebellum Pcp-2 in Rat Offspring During Lactation

The thyroid functions of breastfed infants, as well as (indirectly) the development of their central nervous system, are dependent on the iodine status of the lactating mother. Purkinje cell protein-2 is a cell-specific marker of the cerebellum Purkinje cell and is a suitable indicator for observing the postnatal development of the cerebellum after birth. We measured the Purkinje cell protein-2 mRNA and protein levels in the rat cerebellum in the critical postnatal (14 days after birth) and maturation periods (28 days after birth) to determine the effect of different nutritional iodine levels on cerebellum growth in the offspring during lactation. We found that severe iodine deficiency resulted in thyroid dysfunction in lactating rats and their offspring on both 14 and 28 days, showing maternal total T₄ 16.7 ± 12.0 vs 36.4 ± 15.0, P < 0.05 (14 days) and 22.6 ± 18.7 vs 53.4 ± 9.4, P < 0.01 (28 days), and neonatal total T₄ 10.6 ± 2.3 vs 16.4 ± 4.7, P < 0.01(14 days) and 12.8 ± 2.9 vs 16.7 ± 3.4, P < 0.05 (28 days), respectively. The Purkinje cell protein-2 mRNA and its protein levels in offspring rats were significantly reduced that showed Purkinje cell protein-2 mRNA 1.12 ± 0.04 vs 2.25 ± 0.53, P < 0.05 (14 days) and 1.74 ± 0.94 vs 8.69 ± 2.71, P < 0.01 (28 days). However, mild iodine deficiency and excessive iodine maintained almost normal thyroid function in maternal and neonatal rats and normal Purkinje cell protein-2 mRNA and protein levels in offspring’s cerebellum. We conclude that severe iodine deficiency could significantly reduce Purkinje cell protein-2 mRNA and its protein levels, indicating that the cerebellum development was retarded, but mild iodine deficiency and excessive iodine could maintain them at an approximately normal level by the mother’s and offspring’s compensations, especially by the mother’s mammary glands.     

Role of fibrogenic markers in chronic hepatitis C and associated hepatocellular carcinoma

Detection and follow up of fibrogenesis in chronic hepatitis C (CHC) is mandatory for early treatment and risk stratification. The current study included 120 patients with CHC, of whom 30 had liver cirrhosis (LC) and 30 had hepatocellular carcinoma (HCC). 15 wedge liver biopsies, taken during laparoscopic cholecystectomy, were included as normal controls. Cases were subjected to laboratory investigations, serologic markers for viral hepatitis and assessment of circulating levels of hyaluronic acid (HA) and platelet-derived growth factor (PDGF). Immunohistochemical expression of connective tissue growth factor (CTGF), PDGF and transforming growth factor-β1 (TGF-β1) was also carried out. A significant increase (p < 0.01) in serum HA was noticed in CHC, LC and HCC compared to controls. Although, a significant decrease in serum PDGF was detected in CHC and LC compared to controls, HCC values were comparable. A significant up-regulation of CTGF was detected in CHC, LC and HCC (p < 0.01) in contrast to its limited mild expression in normal livers. Intense PDGF positive staining was noticed in CHC, LC and HCC compared to scattered faint expression in controls. The significant expression and marked intensity of PDGF staining matched the progress to tumorigenesis. A positive TGF-β1 immunostaining was also noticed in CHC, LC and HCC. An intense and extensive cytoplasmic expression of TGF-β1 was encountered in patients with LC revealing that CTGF, PDGF and TGF-β1 act synergistically in LC. Data revealed that HA and CTGF may be implicated as important diagnostic parameters for assessment of hepatic fibrosis and PDGF for monitoring malignant transformation in CHC.     

Protection of Selenium on Hepatic Mitochondrial Respiratory Control Ratio and Respiratory Chain Complex Activities in Ducklings Intoxicated with Aflatoxin B<Subscript>1</Subscript>

To investigate the protection of selenium on hepatic mitochondrial functions, 90 7-day-old ducklings were randomly divided into three groups (groups I–III). Group I was used as a blank control. Group II was administered with aflatoxin B₁ (0.1 mg/kg body weight). Group III was administered with aflatoxin B₁ (0.1 mg/kg body weight) plus selenium (sodium selenite, 1 mg/kg body weight). All treatments were given once daily for 21 days. The results showed that the activities of hepatic mitochondrial complexes I–IV in group II ducklings significantly decreased when compared with group I (P < 0.01). Furthermore, the activities of hepatic mitochondrial complexes I–IV in group III significantly increased when compared with group II (P < 0.05). The hepatic mitochondrial respiratory control ratio (RCR) in group II ducklings significantly decreased when compared with group I (P < 0.01). In addition, the hepatic mitochondrial RCR in group III significantly increased when compared with group II (P < 0.05). These results revealed that the aflatoxin B₁ significantly induced hepatic mitochondrial dysfunction in the activities of hepatic mitochondrial respiratory chain complexes I–IV and the RCR in ducklings. However, sodium selenite could significantly ameliorate the negative effect induced by aflatoxin B₁.     

T细胞分化蛋白2在肝细胞癌中的表达及其临床意义

目的:观察T细胞分化蛋白2(Mal2)在肝细胞癌(HCC)中的表达并探讨其临床意义。方法:采用免疫组织化学染色方法检测226例HCC患者的肿瘤组织和86例正常肝组织中Mal2蛋白的表达情况,并分析其与HCC临床病理指标的关系。通过生存分析比较不同Mal2表达水平患者的生存情况,并分析影响HCC患者生存情况的危险因素。结果:Mal2蛋白在HCC肿瘤组织中的表达水平显著高于正常肝组织(P0.05)。Mal2蛋白表达水平增高与HCC患者血管侵犯、淋巴结转移和较高的TNM分期相关。生存分析表明Mal2蛋白高表达组患者术后生存率显著低于低表达组患者(P0.05)。Mal2阳性表达、血管癌栓形成、淋巴结转移及较高的TNM分期是影响HCC患者术后生存时间的独立危险因素。结论:Mal2蛋白在HCC组织中呈过表达趋势,且与肿瘤转移密切相关,可能在HCC的诊断与预后判断中有一定的应用价值。     

Influence of Selenium on Hepatic Mitochondrial Antioxidant Capacity in Ducklings Intoxicated with Aflatoxin B<Subscript>1</Subscript>

The aim of the study was to investigate the effect of selenium on hepatic mitochondrial antioxidant capacity in ducklings administrated with aflatoxin B₁ (AFB₁). Ninety 7-day-old ducklings were randomly divided into three groups (groups I–III). Group I was used as a blank control. Group II was administered with AFB₁ (0.1 mg/kg body weight). Group III was administered with AFB₁ (0.1 mg/kg body weight) plus selenium (sodium selenite, 1 mg/kg body weight). All treatments were given once daily for 21 days. The results showed that the activities of mitochondrial superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) in group II ducklings significantly decreased when compared with group I (P < 0.01). Furthermore, the content of hepatic mitochondrial malondialdehyde (MDA) significantly increased (P < 0.01). However, the activities of hepatic mitochondrial SOD, CAT, GSH-Px, and GR in group III ducklings significantly increased when compared with group II (P < 0.05). In addition, the content of hepatic mitochondrial MDA significantly decreased (P < 0.01). These results revealed that AFB₁ significantly induced hepatic mitochondrial antioxidant dysfunction. However, sodium selenite could significantly ameliorate the negative effect induced by AFB₁.     

Clinical implications of activated leukocyte cell adhesion molecule expression in breast cancer

In the study, we enrolled 150 breast cancer cases to investigate the expression status of activated leukocyte cell adhesion molecule (ALCAM), and the relationships between ALCAM expression and clinical-pathological characteristics and prognosis of breast cancer. It was observed that ALCAM was expressed at higher levels in breast cancer tissue compared to levels observed for tumor-adjacent tissue. Compared to cancers with low membranous ALCAM expression, cancers with high membranous ALCAM expression were prone to lymph node metastasis (χ² = 15.910, P = 0.010) and metastasis in general (χ² = 5.211, P = 0.029). High cytoplasmic ALCAM expression was noticeably correlated with local recurrence (χ² = 7.379, P = 0.012), especially for short-term recurrence (interval <2 years) (χ² = 5.562, P = 0.037), while not associated to long-term local recurrence (interval >2 years). The content of ALCAM protein is closely associated with the expression of estrogen receptor (ER) (P = 0.024). The disease-free survival of patients with high cytoplasmic ALCAM expression was significantly shorter compared to the cases with low cytoplasmic ALCAM expression (P = 0.036). In conclusion, ALCAM expressed at high levels in breast cancer. High membranous expression of ALCAM probably resulted in weakened adherent ability and metastasis. In addition, high cytoplasmic ALCAM expression strengthened invasive ability of malignant cells and then promoted tumor development.     

The effect of formalin fixation on the levels of brain transition metals in archived samples

Reports that iron, zinc and copper homeostasis are in aberrant homeostasis are common for various neurodegenerative diseases, particularly for Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease. Manipulating the levels of these elements in the brain through the application of chelators has been and continues to be tested therapeutically in clinical trials with mixed results. Much of the data indicating that these metals are abnormally concentrated in Alzheimer’s disease and Parkinson’s disease brain tissue was generated through the analysis of post-mortem human tissue which was archived in formalin. In this study, we evaluated the effect of formalin fixation of brain on the levels of three important transition metals (iron, copper, and zinc) by atomic absorption spectroscopy. Paired brain specimens were obtained at autopsy for each case; one was conserved by formalin archival (averaging four years), the other was rapidly frozen. Both white and grey matter samples were analyzed and the concentrations of iron and zinc were found to decrease with fixation. Iron was reduced by 40% (P < 0.01), and zinc by 77% (P < 0.0001); copper concentrations increased by 37% (P < 0.05) by the paired T-test. The increase in copper is likely due to contamination from trace copper in the formalin. These results indicate that transition metal data obtained from fixed tissue may be heavily distorted and care should be taken in interpreting this data.