噬菌体随机十二肽库淘选及鉴定胃癌特异性结合肽

目的利用噬菌体展示随机十二肽库淘选出与胃癌BGC823细胞特异性结合的多肽,为寻找胃癌早期诊断和治疗的标志物打下基础。方法以胃癌细胞BGC823为靶细胞,胃黏膜永生化上皮细胞GES为吸附细胞,在常温下对噬菌体随机十二肽库进行三轮消减淘选,挑取单克隆、扩增纯化噬菌体,并经ELISA和免疫组化法初步鉴定噬菌体克隆亲和力;测序阳性克隆,合成荧光素FITC标记多肽,鉴定其与胃癌细胞和胃癌组织的亲和力及特异性。结果三轮淘选后,经ELISA法初步鉴定,随机挑选的20个单克隆中11号(GC-11)多肽对BGC823细胞亲和力最高。细胞、组织免疫荧光实验进一步证明,荧光素标记的多肽FITC-GC-11对BGC823细胞和胃癌组织具有高亲和力。结论利用噬菌体随机十二肽库成功淘选出与胃癌细胞BGC823和胃癌组织具有较高亲和力的多肽GC-11。     

肝癌特异性噬菌体多肽的筛选和初步鉴定

目的 利用噬菌体展示肽库筛选与肝癌HepG2细胞特异性结合的多肽,为筛选及明确新的肝癌早期诊断和治疗标志物打下基础.方法 以肝癌细胞HepG2为靶细胞,LO-2为吸附细胞,在37℃条件下对噬菌体随机12肽库进行多轮减性筛选,挑取单克隆扩增并鉴定.利用ELISA初步鉴定克隆亲和力,测定阳性克隆DNA测序并进行同源性及氨基酸分析.结果 经过3轮减性筛选发现,随机挑选的30个单克隆中,其中ZS-9对HepG2具有较高亲和力,氨基酸测序结果表明,该序列与美国国立生物技术信息中心(NCBI)GenBankDNA序列数据库和Swiss-Prot蛋白数据库中的已知基因和蛋白无同源性,而且,国内外文献均未见报道,表明笔者筛选到一新的肝癌相关抗原的配体.结论 利用噬菌体随机12肽库成功筛选到与肝癌细胞HepG2具有较高亲和力的多肽,为筛选鉴定新的肝癌特异的标志物奠定工作基础,也为肝癌的早期诊断和靶向治疗进一步研发确定了靶标.     

利用噬菌体随机七肽库技术筛选人膀胱癌相关表面标志物Her2、Survivin表位模拟肽

目的 利用噬菌体随机七肽库技术筛选人膀胱癌相关表面标志物Her2、Survivin表位模拟肽.方法 分别以Her2、Survivin多克隆抗体为固相筛选分子,对噬菌体随机七肽库进行3轮生物淘洗,随机挑选单克隆噬菌体进行酶联免疫吸附试验(ELISA)分析检测其结合力,竞争抑制实验鉴定其阳性克隆,对结合力较好的噬菌体提取DNA并测序,寻找比较保守的核心序列,合成多肽,最后鉴定合成多肽的结合力.结果经噬菌体随机七肽库3轮淘洗后,特异性噬菌体模拟肽得到富集,分别挑选Her2、Survivin20个克隆,经DNA测序分析,Her2较保守的序列为ACT ACG GCT GAG AAT AAT GAG,Survivin为TCT CCT CCT CAT CTT TCG CAG,合成多肽,Her2为Thr-Thr-Ala-Glu-Asn-Asn-Glu(TTAENNE),Survivin为Ser-Pro-Pro-His-Leu-Ser-Gln(SPPHLSQ),ELISA分析具有良好结合力.结论 利用噬菌体随机七肽库成功筛选到人膀胱癌相关表面标志物Her2、Survivin表位模拟肽,从而为今后膀胱癌多肽疫苗的研制提供良好的基础和条件.     

噬菌体展示的肽库中N-甲基-D-天门冬氨酸受体2B亚基模拟抗原表位的筛选

目的 从噬菌体展示的随机十二肽库中筛选N-甲基-D-天门冬氨酸受体（NMDAR）2B亚基模拟抗原表位。方法以NMDAR2B单克隆抗体为配基，免疫亲和筛选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机十二肽。经过3轮筛选，从第3轮洗脱物中随机挑选12个单克隆噬菌体扩增后进行ELISA鉴定，用酶标仪测定450nm处的吸光值（A450）。对这12个单克隆噬菌体分别进行扩增、纯化，并对DNA测序，以确定插入十二肽的氨基酸序列。通过细胞竞争ELISA法分析阳性单克隆噬菌体对NMDAR2B天然抗原表位与其特异性抗体结合的竞争性抑制。结果经过3轮筛选后能与NMDAR2B单克隆抗体特异性结合的噬菌体得到了有效富积，12个单克隆噬菌体中有9个单克隆噬菌体的A450高于其它3个。DNA测序结果表明这9个单克隆噬菌体表达了一个共同氨基酸序列：SHPPVMPWPTST，将其命名为阳性克隆噬菌体，该阳性克隆噬菌体可以竞争性抑制细胞表面天然抗原与特异性抗体的结合，抑制率（45土3）％，具有抗原模拟性。结论从噬菌体展示的随机十二肽库中成功筛选到了能与NMDAR2B特异性抗体结合的短肽，该肽模拟了天然抗原的某个表位。     

膀胱癌细胞系T24特异性导向小分子多肽的筛选

目的膀胱原位癌和微小转移灶的诊断和治疗目前尚未取得令人满意的效果,寻找高特异性和高结合力的肿瘤导向多肽已成为提高诊断和治疗效率的研究热点。本研究旨在利用噬菌体随机肽库获得与膀胱尿路上皮细胞癌细胞株T24特异结合的小分子多肽序列,以期作为膀胱癌靶向诊断和治疗的导向载体。方法利用噬菌体随机7肽库对肿瘤细胞进行4轮全细胞筛选,分析筛选后单克隆对膀胱癌细胞的特异性结合能力。提取单克隆DNA并测序,得出多肽序列进行对比分析。结果经过4轮筛选后所挑选的20个单克隆均显示与膀胱癌细胞有较高的特异性结合,并测序得到四条重复性高的多肽序列(-SNARGTE-、-VSEKNRQ-、-DSIYNAR-、-DWS-GACS-)。结论通过噬菌体随机肽库对膀胱癌细胞进行全细胞筛选得到的噬菌体多肽能与膀胱癌细胞T24特异性结合,初步可作为膀胱癌靶向诊断和导向药物研究的载体。     

人膀胱癌P-糖蛋白结合肽的筛选

目的：寻找人膀胱癌P-糖蛋白特异性结合肽。方法：利用表达获得的P-糖蛋白胞外段融合蛋白为靶蛋白，采用酶联板法筛选噬菌体随机12肽库，免疫细胞化学方法进行鉴定。结果：从噬菌体随机肽库中筛选获得与P-糖蛋白特异性结合的噬菌体阳性克隆，测序获得特异性结合肽序列。免疫细胞化学结果显示：筛选得到的噬菌体阳性克隆可与耐药细胞BIU-87／ADM结合，而与敏感细胞BIU-87不结合。筛选获得的结合肽具有亲和力，表现出一定的肿瘤特异性。结论：P-糖蛋白结合肽的筛选，为人膀胱癌多药耐药的靶向治疗提供了实验依据。     

miR-3189-3p在肾癌组织中的表达及对肾癌细胞增殖和侵袭的影响

目的:探讨miR-3189-3p在肾癌组织和癌旁组织中的表达差异及其对肾癌细胞生物学功能的影响。方法:通过实时荧光定量聚合酶链反应(qRT-PCR)检测miR-3189-3p在肾癌组织及癌旁组织、肾癌细胞株(Caki-1、ACHN、A498、OS-RC-2)和正常肾小管上皮细胞HK-2中的表达。分别转染miR-NC或m...     

肾癌组织消减文库的构建与肾癌特异表达基因克隆

目的 构建人肾癌组织与正常肾组织差异表达的cDNA消减文库,从文中克隆鉴定出肾癌特异性表达的基因奠定基础。方法 应用抑制性消减杂交技术,分别从肾癌及正常肾组织中提取poly(A) RNA;依次合成单链及双链cDNA,分别与2种不同的接头衔接,再与正常肾cDNA进行2次消减杂交及2次抑制性PCR;将产物T／A载体连接接构建成功cDNA消减文库。结果 构成功具有高消减效率的人肾癌组织cDNA消减文库,文库扩增后得到350个阳性克隆,其中95％克隆均含50－400bp插入片段。结论 应用抑制性消减杂交技术所构建的人肾癌组织cDNA消减文库为进一步大批量筛选、克隆肾癌特异性表达的基因奠定了基础。     

miR-1303靶向抑制LPAR3对肾癌786-O细胞增殖和迁移的影响及机制研究

目的:探讨微小RNA(miRNA)-1303通过靶向调控溶血磷脂酸受体3(LPAR3)的表达抑制肾癌786-O细胞增殖和迁移的机制。方法:采用实时荧光定量聚合酶链反应(qRT-PCR)检测肾癌细胞株(A498、ACHN、786-O、OS-RC-2)及正常肾小管上皮细胞HK-2中miR-1303的相对表达量。将miR-1...     

血清肝癌特异性结合多肽的筛选

目的 从噬菌体展示肽库中筛选出能够用于血清学筛查的多肽.方法 利用M13噬菌体随机展示肽库,对15例临床确诊肝癌患者的血清和15例正常体检者的血清进行4轮吸附-洗脱筛选.挑取筛选后的噬菌体单克隆,用血清试验筛选特异性及阳性率较高的单克隆.目标单克隆通过DNA测序确定其表达的多肽序列,进而人工合成带FITC标记的多肽,再对血清进行鉴定.结果 随机挑取的50个经过4轮筛选的噬菌体单克隆.通过血清试验,从中筛选出9个特异性和确诊率较高的单克隆,其中单克隆ZH-3对肝癌血清确诊率达到46.7%.ZH-3经DNA测序分析,确定其表达的多肽序列为:SAHGTSTGVPWP.结论 ZH-3多肽在肝癌患者血清中有一定的检出率,提示ZH-3多肽能够为研发肝癌诊断试剂奠定基础.

Abstract：

Objective To find the liver cancer specific peptide for serological screen of liver cancer patients via screening phage-display peptide library. Methods Fifteen sera from liver cancer patients and physical examinates were collected for the four-round screening with Ph. D. 12TM phage display peptide kit. Highly specific phage monoclones were selected based on the ELISA results of the serological assay. The peptide labeled with FITC was synthesized according to the DNA sequencing of the optimal monoclone and tested with serum via fluorescent imagery. Results Nine highly specific monoclones were found among 50 selected ones after 4 rounds of screenings. The positive rate of the optimal monoclone,ZH-3, reached 46.7 %. The peptide sequence of ZH-3 was concluded by DNA sequencing as SAHGTSTGVPWP. Desirable specificity and affinity were also shown in the serum of liver cancer patients. Conclusion The peptide ZH-3 can be used as a diagnostic reagent for liver cancer.     

人结肠癌血管特异结合短肽的噬菌体肽库筛选及鉴定

目的 建立小鼠结肠癌模型,筛选并鉴定能够与人结肠癌血管内皮细胞特异结合的噬菌体呈现短肽.方法 建立小鼠荷瘤模型.鉴定阳性噬菌体的体内归巢能力及内皮细胞结合能力.人工合成噬菌体呈现短肽,通过竞争结合实验观察短肽对其呈现噬菌体的竞争结合抑制作用.并检测短肽与共培养内皮细胞及结肠癌血管的特异性结合能力.结果 鉴定出两种外源肽噬菌体,称为pCV1及pCV2.体内实验显示,pCV1、pCV2均能特异性归巢于人结肠癌移植瘤组织,滴度比值pCV1/pCont、pCV2/pCont分别为15.9、20.1倍,明显高于对照组织.体外细胞酶联免疫吸附试验(ELISA)结果显示pCV2在Co-HUVECs上的结合显著高于在HUVECs上的结合,其比值达2.61.人工合成短肽CV2能特异性竞争抑制pCV2向结肠癌移植瘤内的归巢及与Co-HUVECs的特异性结合.免疫荧光染色结果显示,FITC-CV2特异性结合于Co-HUVECs的胞膜与核周胞质,以及结肠癌移植瘤的血管组织.结论 得到两个能特异性结合于结肠癌移植瘤的噬菌体单克隆pCV1及pCV2.pCV2能够特异性结合于Co-HUVECs,pCV2所呈现的环状九肽CV2介导了它与Co-HUVECs的特异结合.短肽CV2具有与Co-HUVECs及结肠癌移植瘤血管特异性结合的能力,有可能用于结肠癌的血管靶向治疗.

Abstract：

Objective To select, identify and analyze a peptide binding specifically to blood vessels of human colon cancer by phage displayed peptide library in vivo. Methods Animal models were established using sub-renal capsular assay (SRCA) in immunosuppressive mice implanted with human colon cancer xenografts. The phage displayed peptide library was injected intravenously into mice. After 4 rounds of selection, 20 clones were picked up randomly and sequenced individually. The homing ability to human colon cancer xenografts and co-cultured human umbilical vein endothelial cells ( Co-HUVECs with human colon cancer Lovo cells) of the positive phage clones were determined by in vivo binding assay and in vitro cell enzyme linked immunosorbent assay ( ELISA ). The binding ability to Co-HUVECs of peptide-displayed phage clone was identified by immunocytochemical stain. Peptide displayed on the phage was synthesized and competitive binding assays were performed to observe the competitive inhibition effect of the peptide with their phage counterpart. Immunofluorescence microscopy was used to study the binding of synthesized peptides to Co-HUVECs and vascular vessels in colon cancer. Results Two peptide sequences were obtained finally and named CV1 and CV2. In vivo binding assay showed that the homing ability to human colon cancer xenografts of peptides of CV1 or CV2 was higher than that of control organs. In vitro cell ELISA suggested that CV2 phage preferably binded to Co-HUVECs rather than control HUVECs. Then CV2 phage clone was identified further. Immunocytochemical staining revealed that CV2 phage preferably binded to Co-HUVECs rather than the control. Competition binding assays demonstrated a significant competition between the synthesized peptide CV2 and the phage displaying CV2 while binding to Co-HUVECs or human colon cancer xenografts. Under the immunofluorescence microscopy, fluorescence-labeled CV2 peptide was seen on the membrane and in the perinuclear cytoplasm of Co-HUVECs, and bound to colon cancer xenografts rather than control organs. Conclusion Two phage clones displaying CV1 and CV2 peptide could target to human colon cancer xenografts. The peptide CV2 and its displayed phage were identified binding preferably with Co-HUVECs. And the cyclic nonapeptide (CV2) was binding site of the CV2 phage with Co-HUVECs. Synthesized nonapeptide CV2 had specificity to Co-HUVECs and colon cancer vascular endothelial cells. The peptide CV2 could be used in target therapy of tumor angiogenesis.     

Construction and preliminary screening of a human phage single-chain antibody library associated with gastric cancer.

BACKGROUND: The aim of this study was to construct a phage library of human single-chain antibodies associated with gastric cancer and screen such a library for CEA binding scFv. MATERIALS AND METHODS: The cDNA library of antibody variable regions was constructed using mRNA from metastatic lymph nodes or spleen of patients with stomach cancer by RT-PCR. These cDNA were assembled into a single-chain format and cloned into phagemid pCANTAB-5 and then transformed into Escherichia coli TG1. The scFv gene library was rescued by M13KO7 helper phage. CEA and the viable CEA-positive gastric cancer cell line MKN-28 were used to screen the phage antibody library. Indirect and tumor cell ELISA was used to determine the specificity of phage antibody. Fixed cell immunofluorescence and live cell FACS analysis were used to further characterize the binding of phage scFv. RESULTS: After transformation into E. coli TG1, 2.5 x 10(7) cfu/microg ampicillin-resistant clones grew. Sequences of those positive insert clones showed that the V(H) genes were derived from the V(H) III subgroup, while the V(L) genes belonged to the V(kappa) III subgroup. After four rounds of panning, the titer of eluted binding phage increased 135- to 158-fold and ELISA results showed that 20/95 clones can bind CEA and 47/95 clones can bind fixed tumor cells. Immunofluorescence and FACS analysis results showed that these phage scFv fragments could bind CEA-positive cells. CONCLUSIONS: We successfully constructed a human phage antibody library from lymph nodes of stomach cancer patients. Such kinds of library prove useful for generating tumor-antigen-specific human antibody fragments.     

Identification of chondrocyte‐binding peptides by phage display

As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage‐binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12‐amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild‐type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue‐specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC‐tagged peptides were synthesized based on the sequence of cartilage‐binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1053–1058, 2013     

Mimotope selection of blood group A antigen from a phage display 15-mer peptide library

We select the peptide mimics of blood group A antigen by a monoclonal anti-A from a phage display 15-mer peptide library. Monoclonal anti-A was used in biopanning a phage display 15-mer peptide library. After four rounds of panning, ELISA was carried out to confirm the positive phage clones. The exogenous DNAs of the positive phages were sequenced and the corresponding amino acid sequences were deduced. Both the synthesized peptide and the phage clones were used to bind to anti-A in competitive ELISA. Erythrocyte agglutination inhibition tests were carried out to determine the mimic ability of the free synthesized peptide to the natural blood group A antigen. Computer softwares were used to simulate the interaction between the peptide and anti-A. After four rounds of biopanning, the eluted phage reached an enrichment of approximately 1600 times. Thirty-seven phage clones were chosen randomly and amplified. There were eleven clones that interacted specifically with anti-A in ELISA. DNA sequencing of the inserted oligonucleotide revealed that nine clones present a same peptide — TRWLVYFSRPYLVAT (named TRW) and each of the other two clones presented a different peptide. The synthesized free peptide TRW could inhibit the interaction of both phage displayed peptide and group A red blood cell with anti-A in competitive ELISA and hemagglutination inhibition test. Both the peptide TRW and the natural group A antigen were docked into a same cavity of anti-A in a computer simulation assay. The results indicate that peptide TRW can mimic blood group A antigen. It may be used as a proxy of natural blood group A antigen in clinical application.     

circSIPA1L1对肾癌细胞增殖、迁移、侵袭的作用及机制研究

目的探讨环状RNA信号诱导增殖相关基因1(circSIPA1L1)对肾癌细胞增殖、迁移、侵袭的作用及相关机制。方法该研究于2019年1—12月完成。采用实时定量逆转录聚合酶链反应(RT-qPCR)法检测天津市第四中心医院55例肾癌患者肾癌、癌旁组织,以及正常肾细胞KiMA和肾癌细胞A498、OSRC2中circSIPA1L1、miR-22-3p的表达;采用脂质体法将circSIPA1L1干扰载体阴性对照(si-NC组)、circSIPA1L1干扰载体(si-circSIPA1L1组)、si-circSIPA1L1+miR-22-3p抑制载体质粒阴性对照(anti-miR-NC组)、si-circSIPA1L1+miR-22-3p抑制载体质粒(anti-miR-22-3p组)分别转染至A498、OSRC2细胞;双荧光素酶报告基因实验验证靶向关系;克隆形成实验、Transwell实验检测细胞的增殖、迁移和侵袭。将稳定转染sh-circSIPA1L1和sh-NC的A498细胞按照2×10⁶个/0.2 ml接种于裸鼠背部皮下建立移植瘤模型,分别为sh-circSIPA1L1组和sh-NC组,行裸鼠成瘤实验检测肿瘤的形成能力。结果肾癌组织中circSIPA1L1表达量(3.89±1.35)高于癌旁组织(1.09±0.44),miR-22-3p表达量(0.44±0.19)低于癌旁组织(1.02±0.30),肾癌组织和癌旁组织中circSIPA1L1和miR-22-3p表达量的差异均有统计学意义(P<0.05)。肾癌细胞A498、OSRC2中circSIPA1L1的表达量(4.61±0.33、3.86±0.23)高于正常肾细胞KiMA细胞(1.00±0.13),miR-22-3p的表达量(0.34±0.05、0.40±0.04)低于KiMA细胞(1.00±0.08),差异均有统计学意义(P<0.05)。si-circSIPA1L1组A498、OSRC2细胞克隆数量[(130.67±15.04)、(99.00±14.80)个]低于si-NC组[(314.33±29.57)、(234.67±21.50)个];细胞迁移数量[(108.33±17.01)、(85.67±11.93)个]低于si-NC组[(265.00±20.00)、(210.33±18.58)个];细胞侵袭数量[(84.00±12.00)、(66.00±10.15)个]低于si-NC组[(210.33±18.58)、(173.00±17.52)个],差异均有统计学意义(P<0.05)。双荧光素酶报告基因实验结果显示circSIPA1L1靶向负调控miR-22-3p表达。si-circSIPA1L1+anti-miR-22-3p组A498、OSRC2细胞克隆数量[(234.20±21.90)、(185.06±20.72)个]高于si-circSIPA1L1+anti-miR-NC组[(134.65±26.55)、(106.14±16.38)个];细胞迁移数量[(187.02±23.54)、(117.86±15.09)个]高于si-circSIPA1L1+anti-miR-NC组[(110.59±12.12)、(91.70±14.83)个];细胞侵袭数量[(168.23±11.69)、(103.70±9.23)个]高于si-circSIPA1L1+anti-miR-NC组[(90.46±11.53)、(61.35±9.10)个],差异均有统计学意义(P<0.05)。sh-NC组、sh-circSIPA1L1组第35天裸鼠肿瘤体积分别为(578.65±68.67)mm3和(242.56±42.35)mm3;裸鼠肿瘤重量分别为(0.68±0.06)g和(0.38±0.04)g,差异均有统计学意义(P<0.05)。结论circSIPA1L1在肾癌组织中高表达,可促进癌细胞增殖、迁移、侵袭和肿瘤生长,其作用机制与直接靶向负调控miR-22-3p相关。     

Targeting urothelium: ex vivo assay standardization and selection of internalizing ligands

PURPOSE: With the goal of targeting the human bladder using phage display technology we designed and tested a tissue binding assay on intact urothelium ex vivo. This approach may form the molecular basis for clinical development of peptide or peptidomimetic guided intravesical compounds. MATERIALS AND METHODS: We screened 2 phage display random peptide libraries on human urothelium. Select peptides were tested for their binding ability to human urothelium, 2 human transitional cell carcinoma cell lines and a nontransitional cell carcinoma cell line. Next we standardized an ex vivo binding assay, validated binding of selected phage to whole urothelium, and evaluated whether receptor mediated internalization into urothelium derived cells occurred. Finally we tested if the presence of the glycosaminoglycan layer had any effect on the binding of the urothelium targeted phage. RESULTS: Phage selected and recovered in the screening were isolated and sequenced. Displayed peptide sequences were searched against online protein databases. Five classes of peptide motifs were characterized based on their ability to bind to normal urothelium but not to control cell lines. Remarkable consistency and reproducibility were observed in the ex vivo binding assays. Two classes of peptide motifs sharing the sequence Ile/Leu-Ser-Gly-Leu bound to normal urothelium and to 2 transitional cell carcinoma cells but not to nontransitional cell carcinoma cells in a glycosaminoglycan independent manner and mediated internalization into cells of urothelial origin. CONCLUSIONS: We introduce a strategy for screening combinatorial peptide libraries on bladder mucosa, a standard model for ex vivo intact urothelium binding assays and a panel of urothelium binding peptides that may be suitable for translation into targeted intravesical therapy applications.     

Human renal cell cancer proliferation in tissue culture is tonically inhibited by opioid growth factor

PURPOSE: Peptide growth factors alter cellular events by binding to specific receptors. One group of peptides, the endogenous opioids, is important in the growth of normal and neoplastic tissue. [Met5]enkephalin, also termed opioid growth factor (OGF), is a tonically active inhibitory factor that interacts with the OGF receptor, OGFr, formerly identified as Greek zeta (zeta) and appears to be autocrine produced by human cancer cells. This study examined the hypothesis that OGF directly inhibits proliferation of renal cell carcinoma in tissue culture. MATERIALS AND METHODS: Human renal cancer cells (Caki-2) were grown using routine tissue culture techniques. A variety of natural and synthetic opioids including OGF, opioid antagonists, and opioid antibodies were added to renal cancer cell cultures to determine role of these peptides in renal cell carcinoma. The experiments were repeated in serum-free media, and with 4 other human renal cancer cell lines: Caki-2, A498, SN12C, and ACHN. Immunocytochemistry was performed to examine the presence of OGF and its receptor. RESULTS: OGF was the most potent opioid peptide to influence human renal cell carcinoma. OGF depressed growth within 12 hours of treatment, with cell numbers subnormal by up to 48% of control levels. OGF action was receptor mediated, reversible, not cytotoxic, neutralized by antibodies to the peptide, and detected in the human renal cell carcinoma lines examined. OGF appeared to be autocrine produced and secreted, and was constitutively expressed. Both OGF and its receptor were detected in these cells. CONCLUSION: OGF tonically inhibits renal cancer cell proliferation in tissue culture, and may play a role in the pathogenesis and management of human renal cell cancer.