Anti-metastatic and Anti-invasive Effects of Polymeric Arg-Gly-Asp (RGD) Peptide, Poly(RGD), and Its Analogues

We have investigated the anti-metastatic and anti-invasive activities of polypeptide analogues based on the Arg-Gly-Asp (RGD) adhesive signal in fibronectin, poly(RGD), poly(RGDS)[Arg-Gly-Asp-Ser] and poly(RGDT)[Arg-Gly-Asp-Thr]. These polypeptides containing repetitive RGD sequences were able to inhibit experimental and spontaneous lung metastases of B16-BL6 cells more effectively than the corresponding monomer peptides. In the spontaneous metastasis model, multiple i.v. administrations of these polymeric peptides before or after surgical excision of the primary tumor resulted in a significant reduction of lung tumor colonies. However, there was no significant difference in ability to inhibit spontaneous lung metastasis among poly(RGD), poly(RGDS) and poly(RGDT), although the carboxy-terminal amino acid residue (i.e., Xaa in -RGDXaa-) has been shown to play an important role in the expression of cell adhesive character. The treatment with poly(RGD) substantially prolonged the survival time for mice injected s.c. with B16-BL6 melanoma as compared with the untreated control. We also found that the polypeptides were potently able to inhibit the invasion and migration of tumor cells in vitro . Since these polypeptide analogues showed no antigenicity in the host and had no toxic effect on tumor cells in vitro , they may be potentially useful in the prevention of cancer metastasis.     

Inhibition of Tumor Angiogenesis by a Synthetic Cell-adhesive Polypeptide Containing the Arg-Gly-Asp (RGD) Sequence of Fibronectin, Poly(RGD)

We have investigated the anti-angiogenic effect of a polymeric peptide based on the Arg-Gly-Asp (RGD) core sequence of fibronectin as a monomer unit, i.e., poly(RGD), in syngeneic mice and in vitro . Single intratumoral administration of poly(RGD) on day 0, 1 or 7 after tumor implantation achieved a significant reduction of B16-BL6 melanoma colonization in the lungs, but did not affect the size of the primary tumor at the time of amputation. The number of capillary blood vessels oriented toward the tumor mass increased during the early growth phase after the intradermal inoculation of the tumor. Poly(RGD) significantly inhibited the formation of tumor neovascularization when co-injected with the tumor cells or separately injected intratumorally or intravenously on day 1 or 3 after tumor inoculation. This inhibitory effect of poly(RGD) was dose-dependent. Poly(RGD) was able to inhibit the haptotactic migration of endothelial cells along a gradient of substratum-immobilized fibronectin but not laminin. Tumor-conditioned medium (CM) by itself did not act as a chemoattractant when it was added in the lower compartment of a Transwell chamber, but promoted the endothelial cell migration to immobilized fibronectin or laminin. Poly(RGD) inhibited the enhanced cell migration to fibronectin but not to laminin in response to CM. Thus, poly(RGD)-mediated inhibition of tumor metastasis may be partly due to the inhibition of tumor-induced angiogenesis at primary and secondary sites.     

Antimetastatic Effect of a Novel Indolocarbazole (NB-506) on IMC-HM Murine Tumor Cells Metastasized to the Liver

IMC-HM cells were isolated from spontaneously induced ascitic IMC carcinoma cells that had been maintained intraperitoneally in CDF₁ mice. Metastasis to the liver of subcutaneously implanted IMC-HM cells was detected 10 days after implantation into the flanks of mice (day 10), but metastasis to other organs was limited. Thereafter, however, tumor cells spread rapidly to lymph nodes, lung, spleen, ovary and other organs, and the mice died on day 13 to 18. We report here, together with the properties of IMC-HM cells, the effects of adriamycin, cisplatin, etoposide and a new indolocarbazole antitumor compound (NB-506) on this model of metastasis. Although these anticancer agents all inhibited the growth of the subcutaneous tumors, their effects on the life span of the tumor-bearing mice varied. Treatment with NB-506, started on day 1, more than doubled the survival period at doses 30 mg/m² to 900 mg/m². Further, treatment with NB-506, started on day 4 after resection of the primary tumor, inhibited growth of the metastasized tumor in the liver and other organs. Etoposide also increased the life span at a limited range of doses. However, the life-prolonging effects of adriamycin and cisplatin were marginal. These results demonstrate that IMC-HM carcinoma is a good model for spontaneous metastasis to the liver followed by lethal spread to many organs. Moreover, NB-506 was found to be highly effective against the growth not only of subcutaneous tumors, but also of tumors metastasized to the liver.     

乙酰肝素酶的表达与肝癌侵袭转移及预后的关系

目的探讨乙酰肝素酶(Heparanase Hpa)在肝癌中的表达及其与肝癌临床病理特征的关系。方法分别采用RT-PCR和免疫组织化学法检测Hpa mRNA及Hpa蛋白在肝细胞肝癌、癌旁肝组织及肝癌细胞株中的表达。结果肝癌Hpa mRNA的表达显著高于癌旁及正常肝组织(P<0.05),肝癌组织中Hpa mRNA的表达与肿瘤包膜受侵、门脉受侵、肝内转移、TNM分期、复发有关(P<0.05)。肝癌Hpa蛋白的表达与包膜和门脉是否受侵、Edmondson分级、有无肝内转移、TNM分期及复发有关(P<0.05)。结论Hpa mRNA及蛋白在肝癌中高表达,并与肝癌的侵袭转移、复发、预后有关。     

Inhibition of Pulmonary Metastases and Tumor Cell Invasion in Experimental Tumors by Sodium d-Glucaro-δ-lactam (ND2001)

Sodium d -glucaro-δ-lactam (ND2001) inhibited spontaneous pulmonary metastases of the highly metastatic B16 melanoma variant with a maximal inhibition rate of 99.5%, and 6 of 7 animals remained metastasis-free. Likewise, ND2001 inhibited the spontaneous pulmonary metastases of both Lewis lung carcinoma (3LL) with a rate of 98.0% (3 of 5 animals remaining metastasis-free) and rat KDH-8 liver carcinoma with a rate of 82.5% (3 of 7 animals remaining metastasis-free), although it was unable to inhibit the metastases of mouse BMT-11 fibrosarcoma and rat SST-2 breast carcinoma. Pretreatment with ND2001 in vitro inhibited the pulmonary metastases of the B16 variant and 3LL cells, which indicates direct action upon the cancer cells. When the invasive activity of cancer cells was measured by the Boyden chamber method, the number of invading B16 variant or 3LL cells was reduced with maximal inhibition rates of 93.0% or 89.9%, respectively, but pretreatment with ND2001 failed to reduce the invasive activity of BMT-11 or SST-2 cells. ND2001 showed neither cytocidal nor antitumor activity. These results suggest that ND2001 inhibited pulmonary metastases at the invasive step into the basement membrane by directly changing some property of the tumor cells.     

Antimetastatic effect of a lipophilic ascorbic acid derivative with antioxidation through inhibition of tumor invasion

Purpose: Ascorbic acid (AA), the natural antioxidant, has been demonstrated to exert an antimetastatic action; however, AA is quite unstable in physiological condition. The aim of the present study is to investigate the stability, the antioxidation and the antimetastatic effects of three lipophilic AA derivatives in vitro as well as in vivo. Methods: The 95D cells were treated with ascorbic acid-2-O-phosphate-6-O-laureate (AA2P6L), ascorbic acid-2-O-phosphate-6-O-myristate (AA2P6M) and ascorbic acid-2-O-phosphate-6-O-stearate (AA2P6S). AA derivatives’ stability in medium under cell culture condition, in the presence and in the absence of 95D cells, was assessed by high-performance liquid chromatography assay. Cell viability and intracellular oxidative stress were measured by MTT assay and CDCFH assay, respectively. Wound healing assay and cell adhesion assay were used to investigate the antimetastatic activities against 95D cells in vitro, and the C57BL/6 mice model was used to evaluate the antimetastatic action in vivo. Results: All the three AA derivatives exhibited excellent stability, significantly different from AA. Results of MTT assay showed that IC₅₀ values of the cytotoxicity of those AA derivatives, namely AA2P6L, AA2P6M and AA2P6S, were 38.46, 28 and 22.97 μg/ml, while the CDCFH assay indicated that EC₅₀ values of antioxidant effects on 95D cells were 31.12, 33.51 and 38.31 μg/ml, respectively. Through the ratio of IC₅₀ vs EC₅₀ for AA derivatives, AA2P6L was demonstrated to be the most effective AA derivative, which retained the antioxidant ability as well as low cytotoxicity. AA2P6L dose-dependently inhibited 95D cells’ migration and adhesion, by 50% at the concentration of 20 and 57 μg/ml, respectively. In the animal experiment, intraperitoneal administration of 75 mg/kg AA2P6L decreased the number of metastatic nodules by 62% and elevated the survival rate of C57BL/6 mice about onefold compared to the control group. Conclusion: AA2P6L, a lipophilic AA derivative with antioxidation, is shown to be a potent antimetastatic agent through the inhibition of tumor invasion. These results support future investigations on the feasibility of cancer chemotherapy with AA2P6L.     

尿激酶型纤溶酶原激活剂对鼻咽癌侵袭和转移的影响

目的　研究尿激酶型纤溶酶原激活剂 (uPA )在鼻咽癌侵袭和转移中的作用。方法　应用逆转录聚合酶反应 (RT -PCR )方法检测uPA在鼻咽癌细胞系 (CNE -2Z)及其克隆株 (CNE -2Z -H 5、CNE -2Z -H 5 -9)中的表达 ;应用酶联吸附法(ELISA )测定uPA在CNE -2Z、CNE -2Z -H 5、CNE -2Z -H 5 -9中的分泌量 ;采用异质黏附抑制实验 ,观察uPA在鼻咽癌细胞侵袭和转移过程中的作用。结果　uPA在CNE -2Z -H 5 -9中表达水平最高 ,在CNE -2Z中表达水平最低 ,在CNE -2Z -H 5中表达水平处于前两者之间 ;抗uPA抗体抑制CNE -2Z -H 5 -9的异质黏附能力。结论　uPA可能促进鼻咽癌细胞的侵袭和转移     

人肺癌裸鼠移植瘤中uPA系统的表达及意义

研究ｕＰＡ系统４种主要成分（ｕＰＡ、ＰＡＩ－１、ＰＡＩ－２、ｕＰＡＲ）在肺癌细胞裸鼠移植瘤侵袭转移过程中的作用。方法将４株肺癌细胞（１株肺巨细胞癌、２株肺腺癌、株肺鳞癌）接种入裸鼠体内,建立体内侵袭转移模型;应用免疫组化,蛋白免疫印迹等方法检测移植瘤组织中ｕＰＡ系统各因子的表达。     

联合检测suPAR、SccAg在宫颈癌中的意义

目的探讨suPAR、SccAg在宫颈癌中的变化及监测意义。方法采用ELISA方法测定57例宫颈鳞癌患者血清中suPAR、SccAg值,以30例子宫肌瘤的正常宫颈做对照。结果宫颈癌患者治疗前血清中suPAR、SccAg测定值明显高于正常宫颈的血清测定值（P<0.01）,宫颈癌Ⅰ～Ⅱ期血清suPAR、SccAg测定值明显低于Ⅲ～Ⅳ期者,淋巴结无转移者明显低于有转移者（P<0.01）,治疗后无复发者明显低于复发者（P<0.01）,而治疗后复发、术中残留对其影响不显著（P>0.05）。在宫颈癌中,suPAR、SccAg表达的阳性率分别为75.64%、61.69%。结论suPAR、SccAg在宫颈癌呈高表达,提示其可能是宫颈癌发生发展中的特征之一,有待成为术后监测、术后随访及是否追加放疗的参考指标,为判断预后、评估治疗提供帮助。     

uPAR表达对鼻咽癌侵袭和转移的影响

目的　探讨尿激酶型纤溶酶原激活剂受体 (uPAR)对鼻咽癌侵袭和转移的影响。方法　以鼻咽癌细胞系 (CNE 2Z)及其克隆株 (CNE 2Z H 5 ,CNE 2Z H 5 9)作为研究材料 ,应用逆转录聚合酶反应 (RT PCR )检测uPAR在基因转录的表达 ;应用蛋白免疫印迹 (Westen Blotting)方法检测uPAR蛋白水平的表达 ;通过异质黏附抑制实验 ,观察uPAR在鼻咽癌细胞侵袭和转移过程中的作用。结果　uPAR在CNE 2Z H 5 9中表达最高 ,其次为CNE 2Z H 5 ,在CNE 2Z中表达最低 ;uPAR抗体能抑制CNE 2Z H 5 9的异质黏附能力。结论　uPAR可能促进鼻咽癌细胞的侵袭和转移     

胶原酶A与大肠癌侵袭转移性的关系研究

目的 揭示胶原酶Ａ（ＭＭＰ－２）与大肠癌分期、临床表现的关系，探讨以胶原酶Ａ在肿瘤组织中的含量及活性比值来判定大肠癌侵袭转移的可能性及方法。方法 采用明胶酶谱法检测３２例大肠癌组织和１７例癌旁组织及７例外伤或尸检的正常大肠组织中ＭＭＰ－２的酶原和酶含量，并计算其活性比值。结果 ＭＭＰ－２含量及活性在癌、癌旁及正常大肠组织中存在显著性差异（Ｐ〈０．０１），且与大肠癌Ｄｕｋｅ’ｓ分期（Ｐ〈０．０１）、     

RAGE在胃癌组织中的表达及与肿瘤侵袭转移的关系

目的 研究RAGE蛋白在胃癌组织中的表达及其与胃癌的侵袭和转移的关系。方法 选取50例胃癌组织标本,采用SP免疫组化法检测RAGE表达情况。选取9例新鲜手术切除胃癌标本用于蛋白质印迹（western blot）检测。结果 RAGE在正常组织表达于内皮、平滑肌细胞、单核吞噬细胞。胃癌组织中表达明显增多（P〈0.05）。RAGE在低分化腺癌中的表达明显升高,RAGE的表达和有无淋巴结转移呈负相关（γs=-0.399,P=0.004）。有淋巴结转移组明显高于无淋巴结转移组（P〈0.05）。RAGE蛋白表达的相关性分析表明浸润深度与RAGE蛋白的表达呈正相关（γs=0.385,P=0.06）。不同浸润深度的RAGE的表达差异有显著性意义（P〈0.05）。Western blot结果 分析表明：胃癌的组织学类型与RAGE的表达呈正相关（γs=0.744,P=0.001）。在正常组织、癌旁组织、高分化、中分化、低分化癌组织中,RAGE蛋白含量呈依次上升趋势（P〈0.05）。结论 RAGE在正常胃组织低水平表达,在胃癌组织中的表达明显增强。RAGE的表达与胃癌的侵袭转移密切相关。     

基质溶素(MMP-7)与喉癌浸润转移及预后的相关性研究

基质溶素（matrilysin,matrix metalloproteinases-7,MMP-7）在肿瘤浸润转移中具有重要作用,并与多种肿瘤的预后有关。本研究旨在探讨MMP-7的表达在喉癌浸润转移中的意义及其与预后的关系。     

Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

Polyethylene glycol (PEG) modification of a chimeric Fab'' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab'' to F(ab'')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab'' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.     

High Induction of Poly(ADP-ribose) Polymerase Activity in Bleomycin-resistant HeLa Cells

Poly(ADP-ribose) polymerase activity was measured in bleomycin (BLM) - resistant HeLa (HeLa-BLM^r) and the parental HeLa cells after BLM treatment. HeLa-BLM^r cells, which had been subcultured in growth medium containing 1 μg/ml of BLM, showed a 3.75-fold higher enzyme activity than did HeLa cells, but this activity was decreased to the same level as that of HeLa cells after 48 h of BLM-free cultivation. When HeLa and HeLa-BLM^r cells after a 48-h cultivation in BLM-free growth medium were treated with BLM, the enzyme activity was induced at a higher level (2.5–3.8 times) in HeLa-BLM^r than in HeLa cells and was inhibited markedly in HeLa-BLM^r and slightly in HeLa cells by nicotinamide, an inhibitor of this enzyme. The BLM-induced cell killing by nicotinamide was highly potentiated (about 18 times) in HeLa-BLM^r as compared to HeLa cells.     

Poly(ADP-ribose) glycohydrolase inhibitor as chemosensitiser of malignant melanoma for temozolomide

Disruption of poly(ADP-ribose) polymerase (PARP) pathways by inhibitors of PARP catalytic domain has been shown to increase the anti-tumour activity of temozolomide (TMZ). Since PARP is inhibited by poly(ADP)ribosylation, herein we tested whether inhibition of poly(ADP-ribose) glycohydrolase (PARG) might enhance TMZ efficacy. The PARG inhibitor N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide (GPI 16552) was administered in combination with TMZ to mice injected subcutaneously or intracranially with B16 melanoma cells. The ability of treatment to reduce melanoma metastatic spreading and invasion of the extracellular matrix was also tested. The results indicated that combined treatment with GPI 16552 and TMZ significantly reduced melanoma growth, increased life-span of mice bearing tumour at the CNS site, and decreased the ability of melanoma cells to form lung metastases and to invade the extracellular matrix. In conclusion, PARG inhibition represents an alternative strategy to enhance TMZ efficacy against melanoma in peripheral as well as at CNS site.     

The potential for enhanced tumour localisation by poly(ethylene glycol) modification of anti-CEA antibody.

Attachment of poly(ethylene glycol) (PEG) to proteins can greatly alter their pharmacological properties, including extending the plasma half-life and reducing immunogenicity, both of which are potentially beneficial to tumour targeting. IgG, F(ab'')2 and Fab'' fragments of the anti-CEA antibody A5B7 were chemically modified with PEG (M(r) 5,000), labelled with 125I and their pharmacokinetics compared with the unmodified forms in the LS174T colonic xenograft in nude mice. PEG modification of the intact antibody had little effect on biodistribution, although tumour localisation was slightly reduced. In contrast, similar modification of F(ab'')2 and Fab''A5B7 significantly prolonged plasma half-life and increased radioantibody accumulation in the tumour and to a lesser extent in normal tissues, but reduced tissue to blood ratios. Prior to modification, Fab'' A5B7 (M(r) 50,000) cleared more rapidly from the circulation than F(ab'')2 (M(r) 100,000), but after PEG attachment their biodistributions converged, while the tumour to blood ratios were reduced and resembled that of the intact antibody. The enhanced tumour accumulation, reduced normal tissue to blood ratios and potentially reduced immunogenicity of fragments after PEG attachment may therefore prove superior to either unmodified fragments or intact antibody for antibody-targeted therapy, although the increased plasma half-life may necessitate the use of a clearance mechanism.     

Characterization and anticancer activity of the micelle-forming polymeric anticancer drug adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer

Adriamycin (ADR), an anthracycline anticancer drug, was bound to the poly(aspartic acid) chain of poly(ethylene glycol)-poly(aspartic acid) block copolymer by amide bond formation between an amino group of Adriamycin and the carboxyl groups of the poly(aspartic acid) chain. The polymeric drug thus obtained was observed to form a micelle structure possessing diameter of approximately 50 nm, with a narrow distribution, in phosphate-buffered saline and to show excellent water solubility despite a large amount of ADR introduction. Further, it was able to be stored in lyophilized form without losing its water solubility in the redissolving procedure. Increased stability of the bound Adriamycin molecules in phosphate-buffered saline and elimination of binding affinity for bovine serum albumin due to the micelle formation were further advantages of this polymeric drug. In vivo high anticancer activity of this micelle-forming polymeric drug against P 388 mouse leukemia was obtained with less body weight loss than that seen with free ADR, due to low toxicity as compared with free ADR.     

胃泌素释放肽及其受体预测小细胞肺癌远处转移的价值分析

目的探讨小细胞肺癌肿瘤标志物与预后,远处转移的关系以及预测远处转移的价值。方法回顾性分析73例小细胞肺癌患者临床及病理资料,接受手术治疗者38例(52.1%),局部放疗者35例(47.9%)。用免疫组织化学法检测神经元特异性烯醇化酶(NSE)、胃泌素释放肽(GRP)、胃泌素释放肽受体(GRPR)的蛋白表达,结合肿瘤体积重新分级,分析其与小细胞肺癌患者预后、远处转移的关系。结果 NSE、GRP、GRPR蛋白在小细胞肺癌组织中的阳性表达率分别为71.2%、79.5%、75.3%,GRP与GRPR的表达存在相关性(P=0.000)。GRP、GRPR蛋白不同表达水平患者间的预后及远处转移差异均有统计学意义(P<0.05)。GRP与GRPR蛋白表达预测小细胞肺癌远处转移的敏感度均为71.4%,特异性分别为83.9%、90.3%。结论 GRP、GRPR是敏感度及特异性均较好的预测小细胞肺癌侵袭性及远处转移的重要指标。