Long acting octreotide in the treatment of advanced hepatocellular cancer and overexpression of somatostatin receptors: Randomized placebo-controlled trial

AIM: To estimate if and to what extent long acting octreotide (LAR) improves survival and quality of life in patients with advanced hepatocellular carcinoma (HCC). METHODS: A total of 127 cirrhotics, stages A-B, due to chronic viral infections and with advanced HCC, were enrolled in the study. Scintigraphy with 111Indium labeled octreotide was performed in all cases. The patients with increased accumulation of radionuclear compound were randomized to receive either oral placebo only or octreotide/octreotide LAR only as follows: octreotide 0.5mg s.c. every 8 h for 6 wk, at the end of wk 4-8 octreotide LAR 20 mg i.m. and at the end of wk 12 and every 4 wk octreotide LAR 30mg i.m.. Follow-up was worked out monthly as well as the estimation of quality of life (QLQ-C30 questionnaire). Patients with negative somatostatin receptors (SSTR) detection were followed up in the same manner. RESULTS: Scintigraphy demonstrated SSTR in 61 patients. Thirty were randomized to receive only placebo and 31 only octreotide. A significantly higher survival time was observed for the octreotide group (49 ± 6 wk) as compared to the control group (28 ± 1 wk) and to the SSTR negative group (28 ± 2 wk), LR = 20.39, df = 2, P < 0.01. The octreotide group presented 68.5% lower hazard ratio [95% CI (47.4%-81.2%)]. During the f irst year, a 22%, 39% and 43% decrease in the QLQ-C30 score was observed in each group respectively.CONCLUSION: The proposed therapeutic approach has shown to improve the survival and quality of life in SSTR positive patients with advanced HCC.

RELATIONSHIP BETWEEN SOMATOSTATIN RECEPTORS AND ACTIVATION OF HEPATIC STELLATE CELL

Objective To investigate the relationship between expression of somatostatin receptors(SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mR-NA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1-3 mRNA appeared as HSCs became wholly activated, and SSTR1-3 could also be identified on the membrane of activated HSCs in the peri-sinusoid space, fibrous septa, etc. Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

An Experimental Study on Somatostatin Receptors in the Brains of Hepatic Encephalopathy Rats

The present study was undertaken to evaluate the effect of somatostatin (SS) receptor,a brain-gut peptide receptor which is capable of inhibiting central neurons, on the pathogenesis of hepatic encephalopathy (HE).By means of radioligand binding assay, SS receptors in crude synaptosomal membrane of rat brains were investigated in a rat model of HE induced by partial hepatectomy following carbon tetrachloride intoxication and in controls. Binding to SS receptor was studied using125 I-SS as radiolgand Scatchard analysis of binding data was linear, yielding a dissociation constant (Kd) of 3.99 ±0.22 nmol/L and a maximal binding capacity (Bmax) of 238± 14.2 fmol/mg of protein in HE rats.Only increased Bmax values were observed (P< 0.005),while the Kd values were statistically unchanged (P>0.50),in HE rats as compared with those in controls.The results suggest that the changes of SS receptors in brains play a significant role in the pathogenesis of HE.The mechanism of HE induced by the alterations of SS receptors in the brains was discussed in this paper.

Structural Insights into Ligand Recognition and Selectivity of Somatostatin Receptors

Somatostatin is a hormone that regulates multiple hormone releases and cell proliferation in the human body.It does this through a group of somatostatin receptors(SSTRs),of which there are five types:SSTR1-SSTR5.SSTR2 is the most well-known and is often targeted in treating neuroendocrine tumors and acromegaly.

生长抑素Ⅱ型受体SSTR2蛋白的理化性质及生物信息学分析

利用生物信息学方法在线分析生长抑素Ⅱ型受体SSTR2的理化性质、信号肽、跨膜结构、分泌蛋白类型、亚细胞定位、磷酸化位点修饰、空间结构及蛋白互作网络等,并通过MEGA5.0软件建立SSTR2蛋白的系统进化树。结果显示,该基因编码369个氨基酸,分子式为C_(1898)H_(2984)N_(470)O_(513)S_(23),相对分子质量为41332.79,等电点理论值为9.15,不稳定系数为37.16。SSTR2是一种碱性稳定亲水蛋白,无信号肽,存在7个跨膜区,作用于质膜结构;二级结构主要为α-螺旋,属7tmGPCRs超家族,且含有1个7tmA_SSTR2结构域;与SSTR2相互作用的蛋白质包括CORT、SST、NPY、GHRL、GNAI1、GNAI2、GNAI3、SHANK1、HIVEP2。SSTR2基因及其编码蛋白在进化上高度保守,可能参与G蛋白偶联受体信号通路,进而发挥其作用。本研究通过对SSTR2蛋白的理化性质及生物学功能进行分析,为深入研究该蛋白在疾病发生中的机制以及为靶向治疗神经内分泌肿瘤等肿瘤药物研发提供理论依据。

Gene transfer of somatostatin receptor type 2 by intratumoral injection inhibits established pancreatic carcinoma xenografts

AIM: To investigate the therapeutic effect of somatostatin receptor type 2 (SSTR2) gene transfection on pancreatic carcinoma xenografts in vivo in experimental cancers. METHODS: Human pancreatic cancer cell line Panc-1 was inoculated subcutaneously into the back of nude mice. When tumor nodules were grown as large as about 5 mmx5 mm days after inoculation, the mice were randomly divided into 3 groups (6 mice in each group). Group Ⅰ served as untreated control group. Group Ⅱ received an intratumoral injection of a combination of human cytomegalovirus promoter-6C (pCMV-6C) and lipofectamine 2000. Group Ⅲ received an intratumoral injection of a combination of pCMV-6C-SSTR2 and lipofectamine 2000. The rate of tumor growth was compared among these three groups. The expression of SSTR2 in these tumors was detected by immunohistochemistry and Western-blot. Apoptosis index (AI) in these tumors was examined by using TUNEL in situ. RESULTS: Intratumoral injection of a combination of pCMV-6C-SSTR2 and lipofectamine 2000 resulted in the expression of SSTR2 protein. The tumor size and weight in group Ⅲ (0.318±0.098 cm3, and 0.523±0.090 g, respectively) were significantly lower than those in group I (2.058±0.176 cms, and 1.412±0.146 g, respectively) and group Ⅱ (2.025±0.163 cm3, and 1.365±0.116 g, respectively) (P<0.05) The AI in group Ⅲ (1.47±0.13%) was significantly higher than that in groupⅠ(0.56±0.09%) and group Ⅱ (0.57±0.11%) (P<0.05). But there were no significant differences between groups Ⅰ and Ⅱ. CONCLUSION: Our data demonstrate that re-expression of SSTR2 gene has antitumor effects on experimental pancreatic cancer. Restoration of SSTR2 gene expression through gene transfer in vivo might be a potential gene therapy strategy for human pancreatic cancer.

Relationship between somatostatin receptor subtype expression and clinicopathology,Ki-67,Bcl-2 and p53 in colorectal cancer

AIM： To study the SSTR1, 2, 3, 4, 5 expression and their relationships with clinico-pathological factors, cell proliferation, Bcl-2 and p53 expression in colorectal cancer cells. METHODS： Immunohistochemical staining of five SSTR subtypes, Ki-67, Bcl-2 and p53 was performed by the standard streptavidin-peroxidase （SP） technique for the paraffin sections of 127 colorectal cancers, and expression of five SSTR subtypes in 40 specimens of normal colorectal mucosae was detected with the same method. RESULTS： Positive staining for five SSTR subtypes was observed in colorectal cancer cells and normal colorectal mucosae. SSTR1 was the most predominant subtype in both colorectal cancer and normal colorectal mucosa, and the second was SSTR5 or SSTR2. As compared with normal colorectal mucosa, SSTR4 was more frequently expressed in colorectal cancer cells （2.5% vs 18.9%, P〈 0.05）; the expression of SSTR2, 4, 5 in moderately to well differentiated colorectal adenocarcinoma was significantly higher than that in poorly differentiated ones （P〈 0.05）, the SSTR1 expression in colorectal cancer with positive lymph node metastasis was significantly higher than that with negative lymph node metastasis （72.2% and 54.5%, P〈 0.05）. In addition, in the ulcerative type of colorectal cancer, SSTR2 expression was obviously decreased （P 〈 0.05）; the correlation did not reach a statistical significance between the five SSTR subtypes expression and Dukes＇stages （P〉 0.05）, but the frequency of SSTR1 expression increased with Dukes＇ stage, while SSTR3 and SSTR5 expression decreased with Dukes＇ stage. Moreover, there was no correlation between expression of the five SSTR subtypes and other clinicopathological factors such as age, sex, tumor site, tumor depth, distant metastasis. The proliferative indexes in colorectal cancer cells with negative expression of SSTR2 and SSTR3 were significantly higher than that with positive expression （P〈0.05）. The Bcl-2 expression in colorectal cancer cells with positive expression of SSTR1, 2, 3, 5 was significantly lower than that with negative expression （P〈 0.05）. There was no correlation between five SSTR subtypes and p53 expression. CONCLUSION： The most predominant SSTR subtype is SSTR1, and the second is SSTR2 or SSTR5, Five SSTR subtypes play different roles in the development of colorectal cancer, SSTR2 and SSTR3 can inhibit the proliferation and promote apoptosis of tumor cells.

Guipi decoction effects on brain somatostatin levels and receptor mRNA expression in rats with spleen deficiency

BACKGROUND： Somatostatin is abundant in the hypothalamus, cerebral cortex, limbic system, and mesencephalon. Somatostatin mRNA expression in the brain of rats with spleen deficiency is noticeably reduced, as well as attenuation of cognitive function. OBJECTIVE： To observe the interventional effect of Guipi decoction on somatostatin level and somatostatin receptor 1 （SSTRl） mRNA expression in different encephalic regions of rats with spleen deficiency, and to compare the interventional effects of Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet. DESIGN： A randomized controlled observation. SETTING： Basic Medical College, Beijing University of Traditional Chinese Medicine. MATERIALS： Fifty adult Wistar male rats, of clean grade, weighing （160 ± 10） g, were provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Somatostatin 1 polyclonal anti-rabbit antibody and SSTRl in situ hybridization kit were provided by Department of Neuroanatomy, Shanghai Second Military Medical University of Chinese PLA. The drug for developing rat models of spleen deficiency was composed of Dahuang, Houpu and Zhishi, and prepared at 2：1：1. Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet recipes were made according to previous studies. METHODS： This study was performed at the Basic Medical College, Beijing University of Traditional Chinese Medicine from March 2002 to March 2005. The rats were randomly divided into 5 groups, with 10 rats in each group： normal, model, Guipi decoction, Chaihu Shugan powd.er, and Tianwang Buxin pellet groups. Rat models of the latter 4 groups were developed by methods of purgation with bitter and cold nature drugs, improper diet, and overstrain. The rats received 7.5 g/kg of the drugs each morning and were fasted every other day, but were allowed free access to water at all times. The rats were forced to swim in 25 ℃ water until fatigued. Rats in the normal group were intragastrically administered the same amount of normal saline. Rats in the Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet groups were intragastrically administered 7.5 g/kg Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet, respectively, every afternoon. All rats were treated for 6 weeks. MAIN OUTCOME MEASURES： Somatostatin protein and SSTRI mRNA expression in the ventral nucleus of hypothalamus, hippocampal CAl region, and cortex of prefrontal lobe were determined by immunohistochemistry and in situ hybridization, respectively. RESULTS： Fifty rats were included in the final analysis. In the model group, expression of somatostatin protein and SSTRl mRNA in the ventral nucleus of hypothalamus, hippocampal CAl region, and cortex of prefrontal lobe were significantly less than in the normal group （P 〈 0.01）. Above-mentioned indices were identical in the Chaihu Shugan powder and model groups. However, expression of somatostatin protein and SSTRl mRNA were significantly higher in the Guipi decoction group compared to model group （P 〈 0.01）. In the Tianwang Buxin pellet group, SSTRl mRNA expression in rat ventral nucleus of hypothalamus and somatostatin level in rat hippocampal CAl region and cortex of prefrontal lobe, as well as ventral nucleus of hypothalamus, were significantly higher compared to model group （P 〈 0.01 ）. CONCLUSION： Somatostatin level and SSTRl mRNA expression in rats with spleen deficiency were lower than in normal rats. Guipi decoction and Tianwang Buxin pellet up-regulated somatostatin level and SSTRl mRNA expression.

Somatostatin receptor scintigraphy in the follow up of neuroendocrine neoplasms of appendix

BACKGROUND Neuroendocrine tumors of appendix(ANETs)known as carcinoids,are rare endocrine neoplasms originated from enterochromaffin cells of gastrointestinal tract.ANETs are the third most frequent(16.7%)gastrointestinal neuroendocrine tumors,with the incidence of 0.08-0.2 cases/100000 during one year.Incidental ANETs occur in 0.2%-0.7%of emergency surgical resections because of suspected appendicitis which is usually the first manifestation of ANET.Although there are a lot of papers about application of somatostatin receptor scintigraphy in gastrointestinal neuroendocrine tumors,there are very rare sporadic cases described about ANETs particularly.AIM To establish the role of somatostatin receptor scintigraphy(SRS)in the management of patients with neuroendocrine tumors of appendix(ANET).METHODS The total of 35 patients was investigated,23 females and 12 males,average age(43.7±17.3 years).All patients had histological diagnosis of ANET(34 carcinoids of appendix and one tubular carcinoid).Majority of tumors have been found incidentally during surgery of:Acute appendicitis(n=15),perforated appendicitis(n=2),ileus(n=3),hysterectomy(n=3),ruptured ovarian cyst(n=2),caecal volvulus(n=1),while 9 patients had diagnosis of appendiceal tumor before the surgery.Seventeen patients had tumor grade(G)G1,12 G2 and 6 G3.The right hemicolectomy was performed in 13,while the rest of the patients had appendectomy only.SRS was done early(2 h)and late(24 h)after i.v.application of 740 MBq technetium-99 m ethylenediamine-N,N’-diacetic acid Hydrazinonicotinyl-Tyr3-Octreotide(technetium-99 m-Tektrotyd,Polatom,Poland).SRS was performed for restaging in all the patients after surgery.RESULTS There were 12 true positive(TP),19 true negative,3 false positive and 1 false negative SRS result.Sensitivity of the method was 92.31%,specificity was 86.36%,positive predictive value was 80.00%,negative predictive value was 95.00%and accuracy 88.57%.Receiver operating characteristics analysis showed that SRS scintigraphy is a good test for detection TP cases[area under the curve of 0.850,95%confidence interval(CI):0.710-0.990,P<001].Single photon emission computed tomography contributed diagnosis in 7 TP findings.In 10 patients Krenning score was 4 and in 2 was 3.In 8 patients SRS significantly changed the management of the patients(in two surgery was repeated,in 4 somatostatin analogues and in two peptide receptor radionuclide therapy).Median progression-free survival in SRS positive patients was 52 months(95%CI:39.7-117.3 mo)while in SRS negative patients it was 60 months(95%CI:42.8-77.1 mo),without statistically significant difference between the two groups(P=0.434).CONCLUSION In conclusion,our results confirmed the value of SRS in the follow-up of the patients with ANET after surgery,if recurrences or metastases are suspected.

Somatostatin receptor subtype 2-mediated scintigraphy and localization using ^(99m)Tc-HYNIC-Tyr^3-octreotide in human hepatocellular carcinoma-bearing nude mice

AIM: To investigate the uptake of 99mTc-HYNIC-Tyr3-octreotide (99mTc-HYNIC-TOC) in human hepatocellular carcinoma (HCC), which can provide the localizable diagnosis in hepatic carcinoma. METHODS: The expression of somatostatin receptor 2 (SSTR2) messenger RNA (mRNA) in human HCC cell line HepG2 was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Uptake of 99mTc-HYNIC-TOC was evaluated in the human HCC implanted into BALB/c nude mice. ANMIS2000 nuclear medicine analysis system was used to calculate the ratio of 99mTc uptake between tumor tissue and vital organs. RESULTS: We demonstrated the expression of SSTR2 mRNA in human HCC cell line HepG2 by RT-PCR. The size of the RT-PCR products was 364 bp detected by sequence analysis of the human SSTR2 mRNA. Scintigraphy proved that 99mTc-HYNIC-TOC was uptaken in the tumor tissue, liver and kidney of the tumor-bearing mice. CONCLUSION: Based on expression of the SSTR2 mRNA in human HCC, 99mTc-HYNIC-TOC can markedly bind with and be uptaken by human HCC tissues as compared with normal liver tissue. The significant retention of radionudide in kidney and bladder is probably related to non-specific peptide uptake in the tubulus cells of kidney and possibly due to excretion by kidney. Our results show that localizable diagnosis and targeting radiotherapy with radionuclide-labeled somatostatin analog for HCC are of great value to be further studied.

Somatostatin receptor subtypes in hormone-refractory （castration-resistant） prostatic carcinoma

The aim of this study was to examine the tissue expression and Iocalisation of the somatostatin receptors （SSTRs） in hormone-refractory （HR） prostate cancer （PCa）. Five SSTRs were evaluated immunohistochemically in 20 radical prostatectomies （RPs） with Gleason score （GS） 3＋3=6 PCa, in 20 RPs with GS 4＋4=8 and 4＋5=9 PCa, and 20 transurethral resection of the prostate specimens with HR PCa. The mean values in the cytoplasm （all five SSTRs were expressed）, membrane （only SSTR3 and SSTR4 were expressed） and nuclei （only SSTR4 and SSTR5 were expressed） of the glands in HR PCa were 20-70% lower than in the other two groups, the differences being statistically significant. All five SSTRs were expressed in the smooth muscle and endothelial cells of HR PCa, the mean values being lower than in the other two groups. In conclusion, this study expands our knowledge on the expression and Iocalisation of five SSTRs in the various tissue components in the HR PCa compared with hormone-sensitive PCa.

过表达SSTR2通过多种信号途径抑制SSTR2表达阳性的肿瘤生长

目的:研究转基因SSTR2对有不同内源性SSTRs表达谱的癌细胞增殖抑制及其潜在信号途径。方法:在裸鼠身上接种过表达SSTR2或LacZ的capan-2cell和A549细胞,观察肿瘤生长情况。在裸鼠身上构建capan-2移植瘤模型,通过皮下注射携带SSTR2基因的腺病毒,观察肿瘤生长情况。免疫印迹法分析可能涉及的信号途径。结果:过表达SSTR2能够抑制具有不同内源性SSTRs表达谱肿瘤的生长,包括capan-2具有内源性SSTR2表达。SSTR2过表达明显影响了凋亡途径、MAPK途径以及血管生成中的一些组件。结论:SSTR2有希望成为使用转基因治疗多数癌症的候选基因。

环磷酸腺苷信号通路参与调控生长抑素受体5激活对垂体催乳素腺瘤激素分泌的抑制作用

目的探究环磷酸腺苷(cAMP)信号通路在生长抑素受体5(SSTR5)激活对垂体催乳素腺瘤激素分泌抑制作用中的调控机制.方法使用构建的SSTR5过表达GH3细胞系作为垂体催乳素腺瘤的体外细胞模型,采用不同浓度SSTR5激动剂BIM23052联合cAMP激动剂Forskolin(5μmol/L)或百日咳毒素(10 nmol/L)进行细胞刺激,或联合转染过表达环磷腺苷效应元件结合蛋白(CREB)作为干预.通过荧光素酶报告基因系统检测不同刺激组间PRL-luc、环磷酸腺苷反应元件(CRE)-Luc、Pit1-Luc、AP1-Luc和雌激素反应元件(ERE)-Luc启动子活性,Renilla荧光素酶基因用作内参以标准化转染效率.组间统计分析采用Student's t检验和单向方差分析.结果为验证GH3细胞中SSTR5过表达的有效性,使用cAMP响应元件报告载体进行监测,GH3SSTR5细胞中CRE报告基因活性显著低于GH3mock组[(74.87±6.66)%比(100.00±5.21)%,t=5.15,P<0.05],提示SSTR5过表达显著抑制CRE转录活性.不同浓度BIM23052(0、0.1、1.0、10.0、100.0nmol/L)联合Forskolin(5μmol/L)预处理GH3SSTR5细胞,CRE报告基因活性分别为(100.00±2.23)%、(17.79±2.30)%、(6.25±0.37)%、(25.27±2.99)%、(44.60±3.25)%,提示BIM23052降低了Forskolin诱导的CRE转录活性,呈现U型剂量响应曲线,在1.0 nmol/L时抑制效果最强(t=71.68,P<0.01).此外,Pit1启动子包含CRE元件,与未添加BIM23052和Forskolin组[CRE报告基因活性(100.00±4.24)%]比较,不同浓度BIM23052(0、0.1、1.0、10.0、100.0 nmol/L)联合Forskolin(5μmol/L)预处理GH3SSTR5细胞,Pit1报告基因活性分别为(317.91±9.03)%、(278.11±4.94)%、(250.72±14.33)%、(176.21±8.88)%、(162.34±7.54)%,表明BIM23052处理同样降低了GH3SSTR5细胞中Forskolin诱导的Pit1启动子活性,且这种降低具有剂量依赖性(P<0.01).然而,BIM23052和百日咳毒素处理对Prl启动子上的其他响应元件,如AP1或ERE(雌激素反应元件),并未表现出明显抑制作用(P>0.05).过表达CREB后,Prl启动子活性从空白转染组[Ctrl组(100.00±12.01)%比BIM组(63.02±15.35)%,t=5.10,P<0.01]到CREB过表达组[Ctrl组(102.17±11.80)%比BIM组(106.27±14.62)%,t=0.58,P>0.05].结论在垂体催乳素腺瘤中,SSTR5通过抑制cAMP-CREB途径来调控催乳素合成.

SSTR2A、SSTR5与EGFR蛋白在非小细胞肺癌中的表达及其意义

背景与目的生长抑素受体(somatostatin receptor,SSTR)作为基因的肿瘤标记物之一,正逐步受到研究者的重视。随着SSTR5种亚型先后被克隆,人们对SSTR家族各成员的氨基酸序列、分子生物学特性、在正常组织和肿瘤组织中的分布和表达及其特异性配体等方面的研究也进一步深入。本研究探讨SSTR亚型SSTR2A、SSTR5与表皮生长因子受体(epidermal growth factor receptor,EGFR)在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达及其意义,并探讨其相关性。方法用免疫组化法(SP法)检测SSTR2A、SSTR5和EGFR三种蛋白在62例NSCLC组织和7例癌旁肺组织中的表达情况,并进行预后随访。结果在62例NSCLC组织中,SSTR2A蛋白阳性表达30例,占48.4%;SSTR5阳性表达44例,占71.0%。SSTR2A、SSTR5蛋白表达与NSCLC的TNM分期有密切关系(P<0.05),但与患者的年龄、性别、吸烟与否、病理类型、肿瘤大小和淋巴结转移均无明显关系(P>0.05)。EGFR蛋白在癌旁肺组织中无一例表达,而在NSCLC组织中有35例阳性,占56.5%。EGFR蛋白的表达与NSCLC患者的年龄、性别、吸烟与否、肿瘤组织类型、肿瘤大小、TNM分期、病理分级及淋巴结转移均无明显关系(P>0.05)。SSTR2A、SSTR5蛋白表达与EGFR蛋白的表达呈负相关关系。SSTR2A、SSTR5蛋白阳性表达者的3年生存率分别为64.52%和65.91%,阴性表达者为45.16%和22.22%,两者之间差异显著(P<0.05);EGFR蛋白阳性表达者的3年生存率为30.77%,阴性表达者为69.44%,两者之间差异显著(P<0.05)。结论NSCLC组织中SSTR和EGFR表达均显著增高,而且SSTR与EGFR的表达呈负相关,检测SSTR2A、SSTR5和EGFR表达对NSCLC淋巴结转移、病理分期及预后的评估有一定的临床意义。

放射性碘标记RC-160对A549-hSSTR2细胞受体内化及杀伤作用的研究

目的研究^(125)I-伐普肽(RC-160)对转染人生长抑素2型受体(hSSTR2)基因的肺腺癌A549细胞(A549-hSSTR2)受体内化的规律,以及^(131)I-RC-160对 A549-hSSTR2细胞的杀伤作用。方法采用放射性配基结合分析法,以^(125)I-RC-160为放射性配基,测定 A549-hSSTR2细胞及未转染 hSSTR2基因的 A549(A549-pc3)细胞与^(125)I-RC-160在37℃温育不同时间(0.25,0.5,1,4,8,20和24 h)的内化率;采用四甲基偶氮唑蓝(MTT)法测不同浓度^(131)I-RC-160、Na^(131)I、RC-160对 A549-hSSTR2细胞及A549-pc3细胞作用24,48,72和96 h 的杀伤作用。结果 37℃条件下温育,^(125)I-RC-160迅速与 A549-hSSTR2受体结合并使受体发生内化。在温育1h 时,A549-hSSTR2细胞结合(膜结合+内化)的放射性计数为总计数的(18.2±1.9)%,明显高于 A549-pc3组[(5.7±1.4)%,P<0.01]。1 h 后,A549-hSSTR2细胞膜受体内化率(内化部分放射性计数与总放射性计数比)随时间的延长继续增加,膜结合率(膜结合部分放射性与总放射性计数比)随时间的延长逐渐减少。至24 h 时,受体内化率达(13.0±1.1)%,膜结合率为(3.9±2.2)%。^(131)I-RC-160对 A549-hSSTR2细胞的杀伤作用较对 A549-pc3细胞明显增强,并呈一定的剂量-效应和时间-效应关系,在96 h 时,3700 kBq/ml ^(131)I-RC-160对A549-hSSTR2的抑制率达(78.8±5.9)%。结论 ^(125)I-RC-160可以使转染 hSSTR2基因的细胞膜受体内化;^(125)I-RC-160对 A549-hSSTR2细胞的杀伤作用较强,这为外源性受体基因介导核素靶向性内照射治疗肿瘤提供了实验依据。

生长抑素类似物通过细胞生长抑制和细胞溶解途径以SSTR2依赖的方式抑制癌细胞增殖

通过在两个具有不同内源性生长抑素受体(SSTR)表达谱的癌细胞系capan-2和A549中过表达SSTR2,用生长抑素类似物(SSA)奥曲肽或者伐普肽(RC-160)处理实验组的癌细胞,对过表达SSTR2和生长抑素类似物的抗肿瘤增殖效果通过细胞增殖实验进行了研究,而且进一步通过免疫印记的方法研究了SSA/SSTR2涉及的信号通路.结果表明,过表达SSTR2明显抑制了内源性SSTR2表达阳性和阴性癌细胞增殖,而单独使用奥曲肽或者伐普肽对癌细胞增殖影响甚微.然而,在过表达SSTR2的癌细胞中奥曲肽或者伐普肽则可明显抑制癌细胞的增殖,而且具有剂量依赖性.深入研究发现SSA/SSTR2是通过细胞周期阻滞和促凋亡来抑制细胞增殖.结果提示SSTR2可作为候选基因用于本身SSTR2表达阳性或阴性肿瘤的基因治疗,细胞内SSTR2的水平可能是影响肿瘤进程和SSA治疗效果的一个关键因素.

肽受体放射性核素治疗中国晚期胃肠胰神经内分泌瘤患者的前瞻性Ⅱ期临床研究

目的前瞻性研究肽受体放射性核素(177Lu-Dotatate)治疗中国晚期胃肠胰神经内分泌瘤(gastroenteropancreatic neuroendocrine tumor,GEP-NET)患者的有效性和安全性。方法本研究为前瞻性、单臂、单中心Ⅱ期临床研究,纳入2018年2月至2023年5月北京大学肿瘤医院收治的入组分化良好、生长抑素受体阳性、经过治疗进展的晚期GEP-NET患者32例。入组的患者均接受每8~12周1次,每次150~200 mCi的177Lu-Dotatate治疗。主要研究终点为客观缓解率(overall response rate,ORR)和安全性,次要研究终点为无进展生存(progressive free survival,PFS)时间。结果32例患者接受177Lu-Dotatate的平均累积剂量为(623±171)mCi,其中2例患者无法评价疗效(1例失访,1例未完成治疗)。在可评估疗效的30例患者中,ORR为53.3%,疾病控制率为93.3%,中位PFS时间为24.5个月(95%CI为17.0~31.9个月),中位总生存时间未达到。除2例(6.7%)患者出现3级血液学毒性,其余患者均未出现≥3级不良事件。结论肽受体放射性核素治疗中国晚期GEP-NET患者有效率高,且安全性良好,是一种十分有前景的治疗手段。

人类大肠癌SSTR2 mRNA表达及其与细胞增殖相关性研究

目的了解人类大肠癌组织中生长抑素受体亚型2(SSTR2)mRNA的表达情况,并探讨其与细胞增殖的关系。方法收集60例大肠癌标本,采用多相寡核苷探针原位杂交法检测SSTR2mRNA表达,免疫组化法检测增殖细胞核抗原(PCNA)表达,并用图像分析仪进行SSTR2mRNA相对含量测定。结果SSTR2mRNA在早期和分化好的大肠癌表达的阳性率和相对含量均明显高于晚期和分化差的病变,并发现大肠癌SSTR2mRNA表达与PCNA表达呈负相关(P<0·05)。结论SSTR2mRNA表达与大肠癌的分化程度和临床分期有关;SSTR2mRNA是阻滞肿瘤细胞增殖的因素之一,对大肠癌具有反映恶性程度及估计预后的价值。

生长抑素受体亚型SSTR2和SSTR5 mRNA在肝癌组织中的表达及其临床意义

目的:分析肝癌组织中生长抑素受体亚型SSTR2和SSTR5 mRNA表达状况及其与肿瘤的关联性,为应用生长抑素及其类似物诊断和治疗肝癌奠定理论基础。方法:肝癌患者肝组织标本22例,采用RT-PCR检测并鉴定肝癌癌组织、癌旁组织及正常肝组织SSTR2和SSTR5 mRNA表达率,分析表达率与肿瘤特征之间的关系。结果:SSTR2 mRNA肝癌癌组织的表达率为81.8%(18/22),表达水平为118.27±17.25,与正常肝组织[36.4%(8/22),69.49±19.43]比较差异有显著性(P<0.05);与癌旁组织[72.7%(16/22),111.18±23.74]比较差异无显著性(P>0.05);癌旁组织与正常肝组织比较差异有显著性(P<0.05)。SSTR5 mRNA表达率和表达水平肝癌癌组织[18.2%(4/22),51.67±30.32]、癌旁组织[18.2%(4/22),49.56±27.61]及正常肝组织[9.1%(2/22),48.73±15.72]三组间两两比较差异均无显著性(P>0.05)。高分化肝癌组织中[SSTR2 100%(18/18),SSTR5 25%(4/16)]和无淋巴结转移的肝癌组织中表达率[SSTR2 25.0%(4/16),SSTR5 100%(4/4)]分别高于低分化的表达率[0(0/4),0(0/6)]和有淋巴结转移的表达率[0(0/6),0(0/18)](P<0.05);Ⅰ、Ⅱ期[SSTR2 85.7%(12/14),SSTR5 21.4%(3/14)]与Ⅲ、Ⅳ期[75.0%(6/8),12.5%(1/8)]表达率比较差异无显著性。结论:SSTR2 mRNA亚型在肝癌中呈高表达,其表达率、表达水平与癌组织分化程度、有无淋巴结转移有关联性,与肝癌的分期无关联。

hsstr2基因转染肿瘤细胞的受体显像研究

目的将人生长抑素受体2亚型(hsstr2)基因转染至该受体表达阴性的肿瘤细胞,并进行该受体介导的肿瘤显像。方法应用逆转录-聚合酶链反应(RT-PCR)方法获得hsstr2基因并构建真核表达载体,将hsstr2基因转染至肺腺癌A549细胞系(hsstr2表达阴性),用RT—PCR及流式细胞术检测受体表达情况;采用高表达细胞株进行该受体体外结合特性研究,建立裸鼠肿瘤模型,探讨该受体介导的肿瘤显像方法。结果获得经测序完全正确的hsstr2基因,RT-PCR及流式细胞术鉴定选取高表达的细胞株,体外结合实验测得平衡解离常数Kd=5.65×10-10mol/L,最大结合容量Bmax=2.01×104结合位点/细胞,半数抑制浓度IC50=1.88×10-9mol/L。裸鼠肿瘤模型显像示,静脉注射显像剂99Tcm-RC-160后0.5-1 h肿瘤显影明显,24 h后肿瘤部位第2次显影。结论将hsstr2转染至该受体表达阴性的肿瘤细胞并进行受体介导的肿瘤显像是可行的,适宜显像时间为0.5-1 h。