Influence of norcantharidin on proliferation,proliferation-related gene proteins prolifera-ting cell nuclear antigen and Ki-67 of human gallbladder carcinoma GBC-SD cells

BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and should be consi- dered for palliative treatment such as chemotherapy and ra- diotherapy. Unfortunately, reports of chemotherapy and radiotherapy for gallbladder carcinoma are disappointing. We investigated the influence of norcantharidin (NCTD) on proliferation, proliferation-related gene proteins PCNA and Ki-67 of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: GBC-SD cell lines of human gallbladder carci- noma were cultured by the cell culture technique. The ex- periment was divided into NCTD group and control group. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The streptavidin-biotin complex method was used to determine the expressions of prolifera- tion-related gene proteins PCNA and Ki-67 of human gall- bladder carcinoma GBC-SD cells. RESULTS: NCTD inhibited the growth and proliferation of GBC-SD cells from 10 mg/L or after 6 hours in a dose- and time-dependent manner, with the IC50 value of 56.18 μg/ ml at 48 hours. After treatment with NCTD, the expression of PCNA (0.932 ±0.031 vs. 0.318 ±0.023, P<0.001) and Ki-67 (0.964 ±0.092 vs. 0.297 ±0.018, P<0.001) proteins were decreased significantly. CONCLUSION: NCTD inhibits the proliferation of human gallbladder carcinoma GBC-SD cells in vitro and the expres- sion of their proliferation-related gene proteins PCNA and Ki-67.

The potential of carcinoembryonic antigen,p53,Ki-67 and glutathion Stransferase-π as clinico-histopathological markers for colorectal cancer

Objective： Colorectal cancer is one of the major contributors to cancer death worldwide. Lack of reliable colorectal cancer markers has hampered the management of these cancer patients. Our main purpose was to study the correlation between histopathological variables of colorectal adenocarcinomas and identify histopathological markers that are of prognostic value in patients with colorectal cancer. Methods： In the present study, we examined the expression of carcinoembryonic antigen （CEA）, p53, Ki-67 and glutathion Stransferase （GST） -n by using immunohistochemical staining methods in 126 colorectal carcinoma patients and evaluated the lymph node metastasis status in these patients by histopathological examination. Results： The positive rates of CEA, p53, Ki-67 and GST-π expression in the colorectal cancer tissue specimens examined were 95.23%, 55.56%, 53.38% and 82.30%, respectively. Expression of p53 and Ki-67 was significantly correlated with the Dukes stages of the tumor, with higher levels of these proteins in Dukes＇ C and D tumors than those in Dukes＇ A and B tumors. Furthermore, the expression of p53, GST-π and Ki-67 correlated with prognosis of patients with colorectal cancer. Additionally, the expression of p53 in colorectal cancer was closely related to the expression of Ki-67 and the expression of GST-π was directly correlated with that of p53. Conclusion： The expression of CEA, p53, Ki-67 and GST-π was correlated with various clinical features of patients with colorectal cancer. The combined use of these histopathological markers appeared to be a promising tool in predicting the prognosis of patients with this type of cancer.

鼻内翻性乳头状瘤中Ki-67的表达及与微血管密度的关系

1.1临床资料。收集1999年1月～2005年7月手术切除确诊为鼻内翻性乳头状瘤（nasal inverted papilloma,NIP）标本存档蜡块36例,男22例,女8例;鼻息肉（nasal polyps,NP）14例,男9例,女5例：鼻腔鳞状细胞癌（nasal squamous cell caminoma，NSCC）10例，男77例，女3例。

Ki-67在喉鳞状细胞癌的表达及临床病理学意义

目的:探讨Ki-67在喉鳞状细胞癌的表达及临床病理学意义。方法:采用免疫组化技术检测10例声带息肉、20例不典型增生病变和50例喉鳞癌组织中Ki-67的表达。结果:Ki-67阳性表达定位于细胞核,Ki-67在声带息肉组、不典型增生组和喉鳞癌组的阳性表达率分别为10%、25%和78%,统计学上有显著性差异,且Ki-67LI同肿瘤位置、组织学分级、临床分期及淋巴结是否转移相关。结论:Ki-67抗原在喉癌中具有显著的临床病理学意义,能用来反映喉癌的良恶性程度。

Expression of COX-2, PCNA, Ki-67 and p53 in gastrointestinal stromal tumors and its relationship with histopathological parameters

AIM: To investigate the expression of Cyclooxygenase-2 (COX-2), proliferating cell nuclear antigen (PCNA), Ki-67 and p53 in gastrointestinal stromal tumors (GISTs) and its relationship with histopathological parameters. METHODS: Twenty-five GISTs were examined by light microscopy and immunohistochemistry. c-kit, CD34, SMA, S-100 protein, COX-2, PCNA, Ki-67 and p53 were detected immunohistochemically and the relationship was evaluated among histopathologic parameters such as mitotic index (MI), tumor grade, tumor size, COX-2, PCNA, Ki-67 and p53. RESULTS: COX-2 protein expression was found in 19 of 25 (76%) of the tumors, and expression was noted in the cytoplasm of the tumor cells. p53 was significantly related to MI and tumor grade but no relationship was found between COX-2, proliferation markers and MI, tumor grade and tumor size. CONCLUSION: COX-2 is expressed in most GISTs and it may play an important role in the proliferation and progression of these tumors or a useful marker to identify GIST. Although immunohistochemical assessment of p53 can be used for distinguishing the risk groups of GISTs, tumor size and mitotic rate should be considered at the same time.

The predictive value of vascular endothelial growth factor and Ki-67 expression on neoadjuvant therapy in rectal cancer

Objective： To investigate the expressions of vascular endothelial growth factor （VEGF） and proliferating cell nuclear antigen （Ki-67） in patients with rectal adenocarcinoma and their associations with neoadjuvant therapy. Methods： The expressions of Ki-67 and VEGF in 32 cases of rectal adenocarcinoma, including both pretreatment tumor biopsies and postoperative specimen, were detected by immunohistochemistry using specific antibodies, and were correlated with clinicopathological factors. Results： The intensity of VEGF staining was significantly correlated with lymph nodal metastasis （P =0.033）, depth of tumor invasion （P =0.007） and tumor stage （P= 0.016）, but not with histological types, tumor sizes, patients＇ ages and genders （P 〉 0.05）. Low level of VEGF expression had significant correlation with the high sensitivity of response to neoadjuvant therapy （P = 0.016）. The transient increase of VEGF expression could be seen after neoadjuvant therapy （P = 0.035）. Ki-67 labeling index （Ki-67-LI） was significantly correlated with lymph node metastasis （P = 0.028）, but not correlated to tumor sizes, patients＇ ages and genders （P 〉 0.05）. Tumors with lower Ki-67-LI were more sensitive to neoadjuvant therapy （P = 0.032）. The Ki-67 level decreased after neoadjuvant therapy, but no statistical significance was found （P 〉 0.05）. Conclusion： Our results demonstrate that the expression of VEGF and Ki-67 in pretreatment rectal adenocarcinoma biopsies may be predictive of tumor response to neoadjuvant therapy.

Effect of Wuziyanzong pill on levels of sex hormones, and expressions of nuclear- associated antigen Ki-67 and androgen receptor in testes of young rats

OBJECTIVE: To evaluate the effects of Wuziyanzong pill on the levels of serum follicle stimulating hormone(FSH), luteinizing hormone(LH), testosterone(T) and the expressions of nuclear-associated antigen Ki67(Ki67), androgen receptor(AR) in testes of young rats.METHODS: Sixteen 20-day-old Wistar male rats were randomly divided into control and treatment group(n = 8). Rats in treatment group were administered Wuziyanzong pill by gavage; rats in control group administered the same volume of saline. After 10 days of treatment, the rats were killed, and then serum and testes were taken. The levels of FSH, LH and T were measured by radioimmunoassay(RIA). The histology of seminiferous tubule was observed by hematoxylin-eosin(HE) staining. The expression of Ki67 was detected by immunohistochemical assay(IHC). The m RNA level of Ki67, ARand CK-18 was detected with quantitative real-time polymerase chain reaction(Q-RT-PCR), the protein level of AR and CK-18 were tested by Western blot.RESULTS: Compared with control group, T level in treatment group increased significantly(P < 0.05).HE staining showed that both leydig cells and germ cells increased in treatment group. Expressions of Ki67 and AR became higher after treatment. There were no changes in CK-18 expression.CONCLUSION: Wuziyanzong pill can up-regulate AR level to promote germ cell proliferation and differentiation in young male rats.

Study of the expression of the gene encoding Ki-67 antigen in human pancreatic cancer using non-radioactive in situ hybridization and immunohistochemistry

bjective To investigate, at transcriptional and translational level in situ, whether the gene expression of the Ki 67 protein in pancreatic carcinoma specimens is altered to get insight of the gene structure and function. Methods Forty pancreatic cancer, 5 normal pancreatic and 4 chronic pancreatitis tissues were used in this experiment. A 435 bp cDNA fragment located in codon 2, exon 13 of Ki 67 antigen gene was amplified by the polymerase chain reaction (PCR). The DIG labeled cRNA probes were transcribed using a commercial DIG RNA labeling kit. Localization of the Ki 67 protein and the specific mRNA was performed by combining immunohistochemistry (ICH) with DIG labeled in situ hybridization (ISH). Results Successful localization of the Ki 67 protein mRNA in pancreatic tissue sections, routinely formalin fixed and paraffin embedded , was first accomplished in this experiment. Analysis of the Ki 67 mRNA transcription in 17 pancreatic cancer specimens with Ki 67 ICH labeling index >20% revealed stronger mRNA signals in poorly differentiated specimens with Ki 67 index >50% than in well differentiated cases with the ICH labeling index of 20% 50%. A high expression of both the mRNA and the protein was observed in pancreatic adenocarcinomas with poor differentiation. Conclusions This is the first study in which the abnormal overexpression of the gene encoding Ki 67 protein was detected not only at the protein level, but also at the mRNA level in pancreatic tumours. The abnormal overexpression of the Ki 67 protein might be correlated with the central part, exon 13, of the gene.

Automated quantification of Ki-67 index associates with pathologic grade of pulmonary neuroendocrine tumors

Background: Classification of the pulmonary neuroendocrine tumor (pNET) categories is a step-wise process identified by presence of necrosis and number of mitoses per 2 mm^2. In neuroendocrine tumor pathology, Ki-67 was first described as a prognostic factor in the pancreas and incorporated into the grading system of digestive tract neuroendocrine neoplasms in the 2010 WHO classification. However, the significance of Ki-67 in pNETs was still a controversial issue. This study was to investigate the potentially diagnostic value of Ki-67 in pNETs. Methods: We retrieved 159 surgical specimens of pNETs, including 35 typical carcinoids (TCs), 2 atypical carcinoid (ACs), 28 largecell neuroendocrine carcinomas (LCNECs), 94 small-cell lung cancers (SCLCs). Manual conventional method (MCM) and computer-assisted image analysis method (CIAM) were used to calculate the Ki-67 proliferative index. In CIAM, 6 equivalent fields lly annotated for digital image analysis. Results: The Ki-67 index among the 4 groups with ranges of 0.38% to 12.66% for TC, 4.34% to 29.48% for AC, 30.67% to 93.74% for LCNEC, and 40.71% to 96.87% for SCLC. The cutoff value of Ki-67 index to distinguish low grade with high grade was 30.07%. For the univariate survival analyses in pNETs, both the overall survival and progression-free survival correlated with Ki-67 index. In addition, the Ki-67 index performed by CIAM was proved to be of great positive correlation with MCM.(500 ×500 μm) at 10× magnification were manua Conclusions: Ki-67 index counted by CIAM is a reliable method and can be a useful adjunct to classify the low- and high-grade NETs.

Effect of preoperative transcatheter arterial chemoembolization on proliferation of hepatocellular carcinoma cells

AIM： To evaluate the effect of preoperative transcatheter arterial chemoembolization （TACE） on proliferation of hepatocellular carcinoma （HCC） cells. METHODS： A total of 136 patients with HCC underwent liver resection. Of 136 patients, 79 patients received 1 to 5 courses of TACE prior to liver resection （TACE group）, who were further subdivided into four groups： Group A （n = 11） who received i to 4 courses of chemotherapy alone; Group B （n = 33） who received i to 5 courses of chemotherapy combined with iodized oil; Group C （n = 23） who received i to 3 courses of chemotherapy combined with iodized oil and gelatin sponge; and Group D （n = 12） who received 1 to 3 courses of chemotherapy combined with iodized oil, ethanol and gelatin sponge. The other 57 patients only received liver resection （non- TACE group）. The expressions of Ki-67 and proliferating cell nuclear antigen （PCNA） protein were detected in the liver cancer tissues by immunohistochemical method. RESULTS： The Ki-67 protein expression was significantly lower in Groups C and D as compared with non-TACE group （31.35% ± 10.85% vs 44.43% ± 20.70%, 30.93% ± 18.10% vs 44.43% ± 20.70%, respectively, P 〈 0.05）. The PCNA protein expression was significantly lower in Groups C and D as compared with non-TACE group （49.61% ± 15.11% vs 62.92% ± 17.21%, 41.16% ± 11.83% vs 62.92% ± 17.21%, respectively, P 〈 0.05）. The Ki-67 protein expression was significantly higher in Group A as compared with non-TACE group （55.44% ± 13.72% vs 44.43% ± 20.70%, P 〈 0.05）. The PCNA protein expression was significantly higher in Groups A and B as compared with non-TACE group （72.22% ± 8.71% vs 62.92% ± 17.21%, 69.91% ± 13.38% vs 62.92% ± 17.21%, respectively, P 〈0.05）. CONCLUSION： Preoperative multi-material TACE suppresses the proliferation of HCC cells, while a single material embolization and chemotherapy alone enhance the proliferation of HCC cells.

Expression of exon 13 from the Ki-67 gene in human cells and tissues by digoxigenin-labelled mRNA in situ hybridization

OBJECTIVE: To get insight on the regulatory mechanism of Ki-67 gene expression in malignant cell cycle. METHODS: Non-radioactive in situ hybridization (ISH) was undertaken, combined with immunohistochemistry to study the Ki-67 gene transcription and translation in various human cells and tissues. HeLa cells and fresh colon cancer cells, tonsil, normal pancreas and pancreatic cancer tissues were used in this study. A 435 bp cDNA fragment located in exon 13 of the Ki-67 antigen gene was amplified by polymerase chain reaction (PCR). Digoxigenin-labelled antisense and sense RNA probes were prepared for detecting Ki-67 mRNA, combined with MIB-1 immunohistochemistry. RESULTS: Successful localization of Ki-67 mRNA in human HeLa cells, colon cancer cells, tissues specimen of the tonsil and pancreatic cancer tissue sections was accomplished by digoxigenin-labelling in situ hybridization technique. ISH to colon cancer cells and pancreatic cancer tissue slides showed that much stronger cytoplasm and perinuclear mRNA signals of the Ki-67 gene were present in malignant cells than in normal cells, which was in accordance with MIB-1 nuclear protein signals. CONCLUSIONS: A sensitive and practical in situ hybridization method for the analysis of Ki-67 antigen mRNA in human cell and tissue was developed. Abnormal transcription of exon 13 of Ki-67 gene might be responsible for malignant cell proliferation in colon and pancreatic cancer.

Insulin receptor substrate 1 may play divergent roles in human colorectal cancer development and progression

BACKGROUND Despite effective prevention and screening methods,the incidence and mortality rates associated with colorectal cancer(CRC)are still high.Insulin receptor substrate 1(IRS-1),a signaling molecule involved in cell proliferation,survival and metabolic responses has been implicated in carcinogenic processes in various cellular and animal models.However,the role of IRS-1 in CRC biology and its value as a clinical CRC biomarker has not been well defined.AIM To evaluate if and how IRS-1 expression and its associations with the apoptotic and proliferation tumor markers,Bax,Bcl-xL and Ki-67 are related to clinicopathological features in human CRC.METHODS The expression of IRS-1,Bax,Bcl-xL and Ki-67 proteins was assessed in tissue samples obtained from 127 patients with primary CRC using immunohistochemical methods.The assays were performed using specific antibodies against IRS-1,Bax,Bcl-xL,Ki-67.The associations between the expression of IRS-1,Bax,Bcl-xL,Ki-67 were analyzed in relation to clinicopathological parameters,i.e.,patient age,sex,primary localization of tumor,histopathological type,grading,staging and lymph node spread.Correlations between variables were examined by Spearman rank correlation test and Fisher exact test with a level of significance at P<0.05.RESULTS Immunohistochemical analysis of 127 CRC tissue samples revealed weak cytoplasmatic staining for IRS-1 in 66 CRC sections and strong cytoplasmatic staining in 61 cases.IRS-1 expression at any level in primary CRC was associated with tumor grade(69%in moderately differentiated tumors,G2 vs 31%in poorly differentiated tumors,G3)and with histological type(81.9%in adenocarcinoma vs 18.1%in adenocarcinoma with mucosal component cases).Strong IRS-1 positivity was observed more frequently in adenocarcinoma cases(95.1%)and in moderately differentiated tumors(85.2%).We also found statistically significant correlations between expression of IRS-1 and both Bax and Bcl-xL in all CRC cases examined.The relationships between studied proteins were related to clinicopathological parameters of CRC.No significant correlation between the expression of IRS-1 and proliferation marker Ki-67,excluding early stage tumors,where the correlation was positive and on a high level(P=0.043,r=0.723).CONCLUSION This study suggests that IRS-1 is co-expressed with both pro-and antiapoptotic markers and all these proteins are more prevalent in more differentiated CRC than in poorly differentiated CRC.

Molecular biomarkers of cell proliferation in ameloblastomas

Cell proliferation is a vital biological process that is important for all living organisms because of its role in growth and the maintenance of tissue homeostasis.The control of this important process differs greatly among benign and malignant neoplasms,and the evaluation of cell proliferation in neoplasms has become a common tool used by pathologists to provide useful information pertaining to diagnosis,clinical behavior,and treatment.The usefulness of information regarding cell proliferation has led to numerous studies on the value of these methods for diagnosing different types of tumors and for clinical decision making.Ameloblastomas are no exception.This review discusses the use of several classical molecular proliferation markers,including Ki-67,proliferating cell nuclear antigen,cyclin D1 and DNA topoisomeraseⅡalpha,to characterize ameloblastomas and proposes the use of new proliferation markers used previously to characterize other neoplasms.The use of these biomark-ers offers valuable opportunities to evaluate the biological behavior of this type of odontogenic tumor.

Cell Replication and Angiogenesis in Central Nervous System Tumors and Their Relationship with the Expression of Tissue Prolactin and Hyperprolactinemia

This study aimed to assess the effect of intracellular prolactin (ICPRL) and hyperprolactinemia on cell replication, using an immunohistochemical (IHC) technique for Ki-67 and Mcm-2, and angiogenesis, using IHC for endoglin CD-105, in central nervous system (CNS) tumors. This cross-sectional study included 79 cases of surgically excised primary CNS tumors of neuroepithelial origin (41.8% of all cases: 10.2% astrocytomas, 24% glioblastomas and 7.6% oligodendrogliomas) and meningeal origin (58.2% of all cases). Ki-67 and Mcm-2 indexes were calculated as a percentage of marked cells. The medians for Ki-67 and Mcm-2 indexes were significantly lower in meningiomas than in glioblastomas (p S = 0.60) replication markers. There were no significant differences in vascular density between the different histological types. Immunohistochemistry for ICPRL was positive in 45.6% of the tumors. Serum prolactin (PRL) was elevated in 30.6% of the cases. Multiple regression analysis revealed no important correlation of ICPRL and serum PRL on Ki-67 and Mcm-2 indexes or vascular density. The analysis of the combined impact of ICPRL and serum PRL variables revealed a trend towards an increase in microvessel density in tumor tissue and a significant increase in cell replication markers (p = 0.009 for Ki-67 and p = 0.05 for Mcm-2). PRL in tumor tissue may be one of the modulating factors of cell proliferation in the CNS.

Telomerase expression in sebaceous carcinoma of the eyelid

Background In humans telomerase is expressed in most ca ncers and immortal cell lines, and astivation of telomerase may play important r oles in tumorigenesis and immortalization. This study was to investigate the rol es of telomerase activity (TA) and human telomerase RNA (hTR) in sebaceous carcinoma of the eyelid.Methods The telomerase repeated amplification protocal (TRAP) was used to demonstrate telomerase activity in 12 cases of sebaceous carcinoma of the eyelid. In situ hybridiza tion (ISH) was used to demonstrate the expression of hTR in 55 cases of paraffin-embedded sebaceous carcinoma of the eyelid, and the results were compared with the proliferative index determined by Mib-1 immuno-labeling, histological patterns and r ecurrence of the tumor.Results Different telomerase activity was shown in the 12 cases of sebaceous carcinoma of the eyelid. The positive expression of hTR was 85.5% (47/55) in tumor cells, but not in the adjacent tissues. The positive expression of hTR was correlated with the proliferative activity (as assessed by Mib-1 immunolabelling, r=0.942, P<0.001) and the dif ferentiation of sebaceous carcinoma of the eyelid (χ 2=17.621, P<0.001), but not si gnificantly related to tumor recurrence. The level of hTR expression increased with the de crease of differentiation of sebaceous carcinoma of the eyelid.Conclusions The results suggest that the up-regulation of telo merase expression plays some roles in tarsal gland carcinogenesis, and the express ion of hTR is a useful marker for malignant degree of sebaceous carcinoma of the eyelid.