人胰腺癌中bcl—2基因和蛋白的过度表达

目的：分析胰腺癌中bcl-2基因的mRNA转录和蛋白翻译，探讨其在胰腺癌 发生、发展中的作用。方法：以50例胰腺癌组织、15例胰腺癌转移灶组织和10例慢性胰腺炎为研究对象，采用免疫组织化学（SP法）和原位分子杂交法检测bcl-2的表达。结果：Bcl-2蛋白表达与患年龄、性别、TNM分期无关，与肿瘤分化程度、有无转移病灶有关。在胰腺癌组织中的表达低于胰腺炎组织，但明显高于转移灶。免疫组织化学与原位杂交技术检测胰腺癌组织中的bcl-2基因表达结果一致。结论：Bcl-2蛋白可能作用于胰腺细胞癌变阶段，并与胰腺导管上皮癌变有关；bcl-2基因在mRNA和蛋白水平表达是相对稳定的。     

凋亡相关基因bcl-2, bax在人类大肠腺癌中的表达意义

目的　探讨ｂｃｌ２ 和ｂａｘ 在人类大肠腺癌中的表达及其意义．方法　运用ＨＥ 染色和免疫组织化学方法对７２ 例人类大肠腺癌进行研究，并采用５ 例正常人类结肠组织作为对照．结果　①大肠腺癌的细胞凋亡随肿瘤分化的不同，而有显著的差异（ Ｐ＜ ０－０１） ，肿瘤的分化程度越高，凋亡指数ＡＩ 值越大．②ｂｃｌ２ 和ｂａｘ 基因在大肠腺癌中均具有较高程度的表达． ③ｂｃｌ２ 和ｂａｘ 基因均与大肠腺癌的分化程度有关（ Ｐ＜ ０－０５） ，肿瘤分化越低，其表达也就越低． ④ｂｃｌ２ 和ｂａｘ 基因在大肠腺癌组织中阳性反应呈显著正相关（ Ｐ＜ ０－０５ ，γ＝ ０－５２３） ．结论　ＨＥ 染色切片可以识别典型的凋亡细胞，大肠腺癌中的细胞凋亡与肿瘤的恶性程度有关，恶性程度越大，细胞凋亡越少；ｂｃｌ２ 与ｂａｘ 基因均与大肠腺癌的细胞凋亡的调控有关，在此过程中二者也相互作用，并且可能在大肠腺癌分化的中晚期共同调控，以抑制细胞凋亡作用为主．     

Staging and treatment for patients with pancreatic cancer. How small is an early pancreatic cancer?

To determine the tumor size that constitutes early pancreatic cancer, we reviewed and analyzed the English-language and Japanese literature (a total of 25 publications) on small pancreatic cancers less than 2 cm in diameter and/or stage 1 cancers. Reports on in situ carcinoma and intraductal carcinoma of the pancreas were also evaluated. The results were: (1) A total of 302 cases of small pancreatic cancer less than 2 cm in diameter reported at separate institutions were pooled from 15 reports. The rates for patients in stage I and those with no lymph node metastasis averaged 41.7% and 57.9%, respectively. The 5-year postoperative cumulative survival rate (5Y-PCR) was less than 50% in almost all these reports. Similar data were shown in the 7 collective reviews. (2) Another 33 cases of small pancreatic cancer of 1 cm or less in diameter were collected from three reports. The rates for stage I tumor and 5Y-PCR at one institution with two reports were 100% and 100% and the rates in the other report were 85% and 78%, respectively. (3) Twelve cases of in situ carcinoma and intraductal carcinoma of the pancreas were collected from four reports. All of the patients were stage I and were alive with no evidence of tumor recurrence for periods ranging from 6 to 78 months. Small pancreatic cancer less than 1 cm in diameter is better viewed as an early pancreatic cancer, and in situ carcinoma and intraductal carcinoma of the pancreas with minimal invasion to the pancreatic parenchyma may be defined as early pancreatic cancer, regardless of size.     

Human equilibrative nucleoside transporter 1 level does not predict prognosis in pancreatic cancer patients treated with neoadjuvant chemoradiation including gemcitabine

Background

Gemcitabine is a key drug for the treatment of pancreatic cancer. Human equilibrative nucleoside transporter 1 (hENT1) is a major transporter responsible for gemcitabine uptake into cells. This study was conducted to elucidate the association between expression level of hENT1 and outcome for pancreatic cancer patients treated with neoadjuvant therapy including gemcitabine.

Methods

Sixty-three patients who underwent neoadjuvant chemoradiation followed by curative surgery for pancreatic ductal adenocarcinomas were included. Immunohistochemistry was performed using resected specimens and the staining intensity of hENT1 was scored as having no staining, low staining, or high staining; the former two were defined as negative expression of hENT1. The association between expression level of hENT1 and overall survival was evaluated by Cox proportional regression model.

Results

Expression level of hENT1 was evaluated as positive in 22 (35%) patients, and as negative in 41 (65%) patients. Univariate analysis showed that regional lymph node metastasis, vascular permeation, and perineural invasion are prognostic factors; however, expression level of hENT1 did not reach statistical significance. Multivariate analysis showed only vascular permeation as a prognostic factor.

Conclusions

Expression level of hENT1 was not associated with prognosis for pancreatic cancer patients who were treated with neoadjuvant chemoradiation including gemcitabine.     

High expression of MMP28 indicates unfavorable prognosis in pancreatic cancer

To investigate the expression pattern and diagnostic performance of matrix metalloproteinase 28 (MMP28) in pancreatic cancer (PC).The RNA-seq data of PC and normal pancreas tissue were acquired from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression. Clinical information of PC that included prognostic data was obtained from TCGA. Later, Fisher exact test was applied for comparison of different clinicopathological features between high and low expression of MMP28 in PC. Afterwards, Kaplan-Meier survival analysis and Cox analysis (univariate and multivariate analysis) were used to explore the prognostic performance of MMP28 in PC cohort. Finally, gene set enrichment analysis (GSEA) revealed the potential signaling pathways related to high expression of MMP28 in PC.Upregulation of MMP28 was identified in PC tissue compared to normal pancreas tissue (P < .001). Overexpression of MMP28 was related to histological grade (P < .001), M classification (P = .014), and survival status (P = .028). Kaplan-Meier survival analysis revealed that high level of MMP28 implied unfavorable prognosis in PC (P = .002). Multivariate analysis confirmed that MMP28 was an independent risk factor in PC (hazard rate = 1.308, P = .018). Our GSEA analysis found that signaling pathways including glycolysis, p53 pathway, notch signaling, estrogen response late, cholesterol homeostasis, estrogen response early, mitotic spindle, and transforming growth factor beta signaling were enriched in the group with higher MMP28 expression.High expression of MMP28 could be identified in PC, which also served as an independent risk element for PC.     

The patterns of coexpression of tumor-associated antigens CA 19-9, TAG-72, and DU-PAN-2 in human pancreatic cancer

Coexpression of CA 19-9, DU-PAN-2, and TAG-72 was examined by a multilabeling immunohistochemical procedure in 31 surgically resected human pancreatic carcinomas. CA 19-9 was expressed in 74%, DU-PAN-2 in 84%, and TAG-72 in 65% of the cases. CA 19-9 and DU-PAN-2 were coexpressed in 16 cases (52%), CA 19-9 and TAG-72 in 10 cases (32%), DU-PAN-2 and TAG-72 in 8 cases (26%), and all three antigens in 10 tumors (32%). With the combination of the three antibodies, all 31 tumors were labeled. However, heterogeneity in antigen expression existed and the antibodies against CA 19-9, DU-PAN-2, and TAG-72 depicted 55%, 49%, and 35% of the tumor cells, respectively. The average number of cells coexpressing CA 19-9 and DU-PAN-2, CA 19-9 and TAG-72, DU-PAN-2, and TAG-72 was 22%, 11%, and 10%, respectively. Only about 3% of tumor cells coexpressed all three antigens, whereas 8% of tumor cells did not express any of the antigens. There was no correlation between the patterns of antigen expression and age or sex. However, there was a tendency of reduced CA 19-9, DU-PAN-2, and TAG-72 expression in less differentiated tumor areas. The results show that: 1) pancreatic cancer cells coexpress two or three antigens in different proportions; and 2) although the sensitivity for pancreatic cancer reaches 100% by all three antibodies, a remarkable heterogeneity exists and a minor fraction of tumor cells does not seem to produce any of these antigens.     

茉莉酸甲酯对人胰腺癌细胞株HS766T抑制作用的研究

[目的]观察茉莉酸甲酯对人胰腺癌细胞株HS766T的抑制增殖及诱导凋亡的作用。[方法]用0~10mmol/L浓度的茉莉酸甲酯体外处理人胰腺癌细胞株HS766T,分别在24h、48h、72h、96h用MTT法检测其细胞生长活性;PI单染色法检测其细胞周期;Annexin V/PI双染色法检测其细胞凋亡;RT-PCR、Western Blot检测基因Bcl-2、Bax及其蛋白的表达量。[结果]用0~10mmol/L茉莉酸甲酯处理胰腺癌细胞24h、48h、72h、96h后,细胞抑制率分别为:14.40%~71.41%、23.62%~89.68%、29.30%~94.76%、37.50%~94.74%;经0.001、0.01mmol/L的茉莉酸甲酯分别作用24h、48h后,与对照组相比,茉莉酸甲酯处理组处于G0/G1/S期的比例升高,G2/M期的比例降低;同时人胰腺癌细胞在0.001、0.01 mmol/L茉莉酸甲酯处理24h、48h后,对照组凋亡率为1.2%、1.6%,0.001mmol/L浓度组凋亡率为12.3%、16.8%,0.01mmol/L浓度组凋亡率为14.5%、23.2%;RTPCR检测显示Bax和Bcl-2在不同浓度和时间的扩增倍数分别为:1.18~1.48、0.92~0.33;Western Blot检测结果显示,Bax表达呈递增而Bcl-2表达呈递减的规律。[结论]不同浓度的茉莉酸甲酯对人胰腺癌细胞株HS766T细胞均有抑制作用,且呈明显的浓度依赖性,48h前细胞抑制率与药物浓度呈正相关,并可降低抑凋亡基因Bcl-2及升高促凋亡基因Bax的表达,从而抑制该细胞株体外生长和促进其凋亡。     

Vitamins A and D but not E and K decreased the cell number in human pancreatic cancer cell lines

Background: The four fat‐soluble vitamins A, D, E and K have been tested in experimental and human studies to assess their influence on the growth of cancer cells of different origins. Receptors for vitamin A and D have been detected on pancreatic cancer cells, and analogues of these reduced the cell number in vitro. The aim of the present study was to evaluate the effect of fat‐soluble vitamins on the growth of pancreatic cancer cells. Methods: The seven cell lines used were established from patients operated on for pancreatic adenocarcinoma. The effect of incubation with the vitamin A analogues all‐trans‐retinoic acid (atRA;tretinoin) and 9‐cis‐retinoic acid (9‐cis‐RA), the synthetic vitamin D analogue EB 1089, vitamin E succinate and K ₁ on the cell number was examined by the XTT method. Results: The vitamin A and D analogues decreased the pancreatic cancer cell number when high concentrations of 10 ^?5 – 10 ^?4 M were administered. A combination of retinoids and the vitamin D analogue EB 1089 did not enhance the effect. Vitamin E succinate inhibited cell growth to a small extent (maximal 26%) in 3 out of 7 cell lines, whereas vitamin K ₁ increased the pancreatic cancer cell number in 3 out of 7 cell lines. Conclusion: High concentrations of vitamin A and D analogues decreased the cell number in pancreatic cancer cell lines. Vitamin E succinate and K ₁ did not have this effect. In the treatment of pancreatic cancer, further exploration of vitamin D analogues could be fruitful.     

Early detection of pancreatic cancer: a possibility in some cases but not a reality in most

Pancreatic cancer is notoriously difficult to diagnose until a late stage when curative options are no longer available. Owing to its relatively low incidence and the lack of sensitivity of current diagnostic tool, screening of pancreatic cancer in the general population is not recommended. However, in high‐risk individuals, especially those with well‐described genetic syndromes and a strong family history of pancreatic cancer, screening can be carried out. Detection of a lesion of the diameter < 1 cm without lymph node involvements and subsequent removal of the tumor results in long‐term cure of the cancer. Endoscopic ultrasound (EUS) is the only diagnostic tool that is able to detect such small lesions. EUS is often combined with endoscopic retrograde cholangiography to augment the diagnostic yield. The conundrum in clinical practice is to differentiate between a malignant and a benign lesion. Resection of the pancreas constitutes major surgery with a high morbidity and mortality. The need continues, therefore, to find even more accurate imaging modalities to diagnose small pancreatic cancers with confidence.     

Overexpression of pancreatitis-associated protein (PAP) in human pancreatic ductal adenocarcinoma

Pancreatitis-associated protein (PAP) is almost absent in normal pancreas, but is strongly induced in acute pancreatitis. PAP mRNA is also expressed in cancer cells, including pancreatic ductal adenocarcinoma. However, the clinicopathological significance of PAP in human pancreatic cancer is not clear. We examined PAP expression in pancreatic tissues from individuals with pancreatic ductal adenocarcinoma using immunohistochemistry. PAP was overexpressed in 79% (30 of 38) of pancreatic ductal adenocarcinoma, 19% (7 of 36) of chronic pancreatitis, and 29% (2 of 7) of mucinous cystadenoma. PAP was found in malignant ductular structures in pancreatic carcinomas as well as in benign proliferating ductules and acinar cells in chronic pancreatitis. It was not expressed in normal pancreas. The incidence of PAP overexpression was significantly higher in pancreatic cancer than in the other pancreatic diseases (P < 0.01). PAP overexpression was significantly correlated with nodal involvement, distant metastasis (P < 0.05), and short survival (P < 0.01) in pancreatic cancer. These results suggest that overexpression of PAP in human pancreatic ductal adenocarcinoma indicates tumor aggressiveness.     

靶向HIF-2 siRNA对人胰腺癌BXPC-3细胞增殖和凋亡的影响

目的观察靶向缺氧诱导因子2(HIF-2)的小干扰RNA(siRNA)对人胰腺癌BXPC-3细胞增殖和凋亡的影响。方法将BXPC-3细胞随机分为A、B、C组,A组转染HIF-2 siRNA、B组转染非特异性siRNA、C组不转染,分别予常氧、缺氧环境下培养;采用MTT法检测培养24、48、72、96 h时BXPC-3细胞的光密度(OD)值观察增殖情况,用流式细胞仪检测培养48 h时BXPC-3细胞凋亡率。结果在缺氧培养48 h时,A、B、C组细胞OD值分别为0.54±0.07、0.69±0.09、0.72±0.13,72 h时分别为0.55±0.11、0.88±0.07、0.88±0.05,96 h时分别为0.56±0.12、0.93±0.11、0.94±0.09;A组与B、C组比较,P均<0.05。在缺氧培养48 h时,A、B、C组细胞凋亡率分别为21.37%±0.17%、7.12%±0.03%、7.24%±0.07%;A组与B、C组比较,P均<0.05。在常氧状态下,各组细胞OD值及凋亡率无明显差别。结论缺氧状态下,靶向HIF-2 siRNA可抑制人胰腺癌BXPC-3细胞增殖,诱导其凋亡。     

Neutral lipid isolated from endosperm of Job's tears inhibits the growth of pancreatic cancer cells via apoptosis, G2/M arrest, and regulation of gene expression

BACKGROUND: The neutral lipid isolated from the endosperm of Job's tears (NLEJ) has been known to possess an anticancer activity with relatively low toxicity. The present study was designed to examine its antiproliferative effects in the PaTu-8988 and SW1990 human pancreatic cancer cells and to investigate its potential mechanism(s). METHODS: Pancreatic cancer cells were treated with NLEJ to evaluate cell viability, cell cycle progression, nuclear morphology, DNA fragmentation and annexin V binding analysis. Regulation of gene expression was determined by using cDNA microarrays and western immunoblotting. RESULTS: The NLEJ induced a dose- and time-dependent inhibition of proliferation in both PaTu-8988 and SW1990 cell lines. Further studies were carried out with only the PaTu-8988 cells. Flow cytometry analysis showed that NLEJ blocked cell cycle progression at the G(2)/M phase. There was also an increase in annexin V binding and DNA fragmentation, indicative of apoptosis. The cDNA microarray analysis with cell cycle- and apoptosis-targeted arrays showed that the expression signals of 24 genes were found to be significantly altered at 24 h of NLEJ treatment. These genes are involved in cell cycle control (e.g. p21, p27, CDK2, and cyclins), apoptosis regulation (e.g. bcl-2 and bax), and signal transduction (e.g. ATM, RAD50, and p53). Some of these results were confirmed by western blot analysis. CONCLUSIONS: These data show that NLEJ inhibits pancreatic cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene expression in vitro. Therefore, NLEJ might be a chemotherapeutic agent against pancreatic cancer.     

膈下逐瘀汤加减方对人肝癌细胞bax、bcl-2、P53基因表达调控的影响

目的：探讨膈下逐瘀汤加减方（GXZY）时人肝癌细胞bax、bcl-2、P53基因表达调控的影响。方法：制备5组试验血清：不合药物血清（无药血清组），阳性对照药物复方斑蝥胶囊含药血清（阳性血清组）和GXZY低、中、高浓度含药血清（5％血清组、10％血清组、15％血清组）。检测各组药物血清及对照血清对sMMC-7721细胞增殖、细胞周期、肝癌相关基因（bax、bcl-2、P53）表达的影响。结果：GXZY各血清组与阳性血清组抑制人肝癌细胞SMMC-7721增殖、调控癌基因与抑癌基因、促进癌细胞凋亡均具有显著统计学意义（P〈0．05或P〈0．01），其中GXZY高浓度血清组的作用优于阳性血清组。结论：膈下逐瘀汤加减方能够抑制人肝癌细胞增殖，促进细胞凋亡。     

Antineoplastic activity of 2-methoxyestradiol in human pancreatic and gastric cancer cells with different multidrug-resistant phenotypes

BACKGROUND AND AIM: Chemoresistance often leads to loss of the last treatment option for cancer. 2-Methoxyestradiol (2-ME2) has been shown to inhibit tumor growth. The aim was to examine the efficacy of 2-ME2 on multidrug-resistant human cells from pancreatic and gastric cancer. METHODS: We investigated the impact of 2-ME2 on multidrug-resistant cells derived from human pancreatic and gastric cancer cells that were positive or negative for the MDR1-gene. RESULTS: In pancreatic cancer cells, growth inhibition was 57% in parental, 72% in MDR1-negative and 87% in MDR1-positive cells after 1 micromol/L 2-ME2. In gastric cancer cells we found a growth inhibition of 75% in parental, 82% in MDR1-positive and 95% in MDR1-negative cells. Strong induction of apoptosis was induced after a low dose of 2-ME2. No significant difference in the amount of apoptotic cells was observed between parental and multidrug-resistant cells of both tumor types. The number of apoptotic cells after 2-ME2 ranged from 7.5% in parental gastric cancer cells to 20.1% in MDR1-negative gastric cancer cells. CONCLUSION: 2-ME2 may therefore have clinical application for chemoresistant cancer.     

Immunolocalization of pS2, a putative growth factor, in pancreatic carcinoma

pS2 is a 60 amino acid secretory polypeptide which belongs to a newly described family of trefoil-shaped growth factors. It is widely distributed throughout the gastrointestinal tract, particularly adjacent to damaged mucosa, and is also expressed by some epithelial tumours such as breast carcinoma. The aim of this study was to examine the expression of pS2 in pancreatic cancer. The presence of pS2 was analysed immunohistochemically using two antibodies, a polyclonal (pNR-2) and a monoclonal (pS2^TM) in 42 cases of pancreatic adenocarcinoma and 10 cases of ampullary carcinoma. The findings were compared with chronic pancreatitis and normal pancreas. No immunostaining was seen in normal pancreas, with the exception of one area of ductular proliferation, and although 8/10 cases of chronic pancreatitis expressed pS2, it was focal and confined to the occasional duct. In contrast, a significant proportion of malignant cells in 23/42 (55%) of pancreatic adenocarcinoma and 8/10 (80%) of ampullary tumours expressed immunoreactive pS2. The finding of pS2 expression in more than 50% of pancreatic and ampullary carcinomas in contrast to the findings seen in chronic pancreatitis and normal pancreas suggests that pS2 may play an important role in the growth of these highly malignant tumours.     

Role of cyclooxygenase-2 and inducible nitric oxide synthase in pancreatic cancer

BACKGROUND AND AIM: Recently, it has been recognized that both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) produce important endogenous factors of human tumor progression. However, the clinicopathological and biological significance of the expression of COX-2 and iNOS in pancreatic cancer remains unclear. The objective of this study is to find the possible roles and clinical significance of COX-2 and iNOS expression in pancreatic cancer. METHODS: Seventy-two pancreatic adenocarcinoma tissue specimens were obtained through surgical resection. We investigated the immunohistochemical expression of COX-2 and iNOS in respect to variable clinicopathological characteristics, proliferation activity (by Ki-67 expression), apoptosis (by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling stain), and microvessel density (by CD34 expression; angiogenesis). RESULTS: Immunohistochemical investigations demonstrated immunolabeling of tumor cells with the primary antibodies, bovine anti-iNOS and anti-COX-2 antibodies. The COX-2 and iNOS positive rates were 41.7 and 66.7%, respectively. There was significant correlation between positive COX-2 and positive iNOS expression (P = 0.043). The proliferation index (Ki-67 labeling index) was higher in COX-2 positive specimens compared to COX-2 negative specimen (P = 0.015). The apoptotic index of positive iNOS expressions was significantly higher than negative expressions (P < 0.001). The expression of COX-2 and iNOS proteins did not correlate with age, sex, serum bilirubin, CA-19-9, location, size, American Joint Committee on Cancer stage, differentiation, distant metastasis, patient survival, or microvessel density. CONCLUSIONS: Although the pattern of positive expression was similar in both enzymes, the effect on tumor progression differed; iNOS expression may play a role in apoptosis of tumor cell, while COX-2 expression may contribute to tumor proliferation. However, COX-2 and iNOS expression is not related to prognosis in patients with pancreatic cancer.     

p53, p21(Waf1/Cip1) and cyclin D1 protein expression and prognosis in esophageal cancer

Recently, various cell cycle regulators have been investigated as biological markers of malignant potential. These regulators might influence the survival rate and the effect of adjuvant therapies. In this study, we analyzed p53, p21(Waf1/Cip1) and cyclin D1 expression in 64 esophageal cancer patients and the relationship between clinicopathologic parameters and patient survival. The positive expression rate was 48.4%, 42.2% and 43.8% in the p53, p21 and cyclin D1 groups respectively. Multivariant analysis revealed that tumor depth, chemotherapy, p53, p21 and cyclin D1 expression showed significant values. p53- and cyclin D1-negative patients had a worse prognosis. p21-positive patients had a better prognosis. In stage 0, I and II patients, there was a significant difference between p53-positive and -negative, p21-positive and -negative, and cyclin D1-positive and -negative groups. In stage III and IV patients, there was no significant difference between any two groups. However, a significant difference was seen in the p21 group: among patients who received adjuvant chemotherapy, the p21-positive group had a 5-year survival rate of 50% compared with 13.4% in the p21-negative group (not significant).