血管生成抑制剂治疗结肠癌肝转移的研究进展

血管生成抑制剂与传统的细胞毒化疗药物不同，可作用于包含多种变异的正常细胞。因此，血管生成抑制剂与细胞毒药物临床应用的传统策略不同。许多临床研究正在用一种新的途径评价这些涉及到抑制细胞生长繁殖不同时期的药物。     

Vaccination with autologous endothelium inhibits angiogenesis and metastasis of colon cancer through autoimmunity

Overcoming immune tolerance of tumor angiogenesis should be useful for adjuvant therapy of cancer. We hypothesized that vaccination with autologous endothelium would induce an autoimmune response targeting tumor angiogenesis. To test this concept, we immunized BALB/c mice with a vaccine of glutaraldehyde-fixed murine hepatic sinusoidal endothelial cells (HSEs) in a lung metastasis model of Colon-26 cancer. Vaccination with autologous HSEs induced both preventive and therapeutic anti-tumor immunity that significantly inhibited the development of metastases. ELISA revealed an immunoglobulin response involving IgM and IgG subclasses. These antibodies had a strong affinity for antigens of both murine and human endothelium, and lyzed endothelial cells in the CDC assay. Flow-cytometry and chromium-release cytotoxicity assay revealed a specific CTL response against endothelial cells, which were lyzed in an effector: target ratio-dependent manner. Neither antibodies nor CTLs reacted with Colon26. The effect of autologous HSEs was more pronounced than that of xenogeneic human umbilical vein endothelial cells (HU-VECs), which were tested in the same experimental setting. Our results suggest that vaccination with autologous endothelium can overcome peripheral tolerance of self-angiogenic antigens and therefore should be useful for adjuvant immunotherapy of cancer. (Cancer Sci 2004; 95: 85–90)     

Retinol decreases phosphatidylinositol 3-kinase activity in colon cancer cells

Previously, we showed that retinol inhibited all-trans-retinoic acid (ATRA)-resistant human colon cancer cell invasion via a retinoic acid receptor-independent mechanism. Because phosphatidylinositol 3-kinase (PI3K) regulates cell invasion, the objective of the current study was to determine if retinol affected PI3K activity. Following 24 h of serum starvation, the ATRA resistant human colon cancer cell lines HCT-116 and SW620 were treated with 0, 1, or 10 microM retinol. Thirty minutes of retinol treatment resulted in a significant decrease in PI3K activity in both cell lines. To determine the mechanism by which retinol reduces PI3K activity, the levels and heterodimerization of the regulatory subunit, p85, and the catalytic subunit, p110, of PI3K were examined. Retinol treatment did not alter p85 or p110 protein levels or the heterodimerization of these subunits at any time point examined. To determine if retinol affected the ability of PI3K to phosphorylate the substrate, phosphatidylinositol (PI), PI3K was immunoprecipitated from control cells and incubated with 10 microg PI and increasing concentrations of retinol or 10 microg retinol and increasing concentrations of PI. Retinol decreased PI3K activity in a dose-responsive manner and increased PI suppressed the inhibitory effect of retinol on PI3K activity. Finally, the PI3K inhibitor, LY294002, mimicked the ability of retinol to decrease cell invasion. Computational modeling revealed that retinol may inhibit PI3K activity in a manner similar to that of wortmannin. Thus, a decrease in PI3K activity due to retinol treatment may confer the ability of retinol to inhibit ATRA-resistant colon cancer cell invasion.     

Trastuzumab cotreatment improves survival of mice with PC-3 prostate cancer xenografts treated with the GRPR antagonist 177Lu-DOTAGA-PEG2-RM26

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer and are suitable for targeted radionuclide therapy (TRT). We optimized the bombesin-derived GRPR-antagonist PEG₂-RM26 for labeling with ¹⁷⁷Lu and further determined the effect of treatment with ¹⁷⁷Lu-labeled peptide alone or in combination with the anti-HER2 antibody trastuzumab in a murine model. The PEG₂-RM26 analog was coupled to NOTA, NODAGA, DOTA and DOTAGA chelators. The peptide-chelator conjugates were labeled with ¹⁷⁷Lu and characterized in vitro and in vivo. A preclinical therapeutic study was performed in PC-3 xenografted mice. Mice were treated with intravenous injections (6 cycles) of (A) PBS, (B) DOTAGA-PEG₂-RM26, (C) ¹⁷⁷Lu-DOTAGA-PEG₂-RM26, (D) trastuzumab or (E) ¹⁷⁷Lu-DOTAGA-PEG₂-RM26 in combination with trastuzumab. ¹⁷⁷Lu-DOTAGA-PEG₂-RM26 demonstrated quantitative labeling yield at high molar activity (450 GBq/μmol), high in vivo stability (5 min pi >98% of radioligand remained when coinjected with phosphoramidon), high affinity to GRPR (K_D = 0.4 ± 0.2 nM), and favorable biodistribution (1 hr pi tumor uptake was higher than in healthy tissues, including the kidneys). Therapy with ¹⁷⁷Lu-DOTAGA-PEG₂-RM26 induced a significant inhibition of tumor growth. The median survival for control groups was significantly shorter than for treated groups (Group C 66 days, Group E 74 days). Trastuzumab together with radionuclide therapy significantly improved survival. No treatment-related toxicity was observed. In conclusion, based on in vitro and in vivo characterization of the four ¹⁷⁷Lu-labeled PEG₂-RM26 analogs, we concluded that ¹⁷⁷Lu-DOTAGA-PEG₂-RM26 was the most promising analog for TRT. Radiotherapy using ¹⁷⁷Lu-DOTAGA-PEG₂-RM26 effectively inhibited tumor growth in vivo in a murine prostate cancer model. Anti-HER2 therapy additionally improved survival.     

Increased plasma MMP9 in integrin alpha1-null mice enhances lung metastasis of colon carcinoma cells

Inhibitors of matrix metalloproteinases (MMPs) were developed as anticancer agents based on the observation that MMPs facilitate local tumor spread and metastasis by promoting matrix degradation and cell migration. Unfortunately, these inhibitors were unsuccessful in the clinical treatment of several cancers, including lung cancer. A possible reason contributing to their failure is that MMP activity is critical for the generation of inhibitors of tumor angiogenesis, including angiostatin. Thus, MMPs might play opposing roles in tumor vascularization and invasion. To determine which effect of elevated MMP levels dominates in the progression of metastatic cancer, experimental lung metastasis assays were performed in integrin alpha1-null mice, a genetic model for increased plasma levels of MMP9 and MMP9-generated angiostatin (Pozzi et al., Proc. Natl. Acad. Sci. USA 2000;97:2202-7). We show that while the number of lung colonies in integrin alpha1-null mice was significantly increased compared to their wild-type counterparts, tumor volume was markedly reduced. In vivo treatment with the MMP inhibitor doxycycline resulted in a significant decrease in the number of lung colonies in both genotypes, but the tumors that formed were bigger and more vascularized. Increased tumor vascularization paralleled decreased plasma levels of MMP9 and consequent decreased angiostatin synthesis. These results demonstrate that while inhibition of MMPs prevents and/or reduces tumor invasion and lung metastasis, it has the paradoxical effect of increasing the size and vascularization of metastatic tumors due to decreased generation of inhibitors of endothelial cell proliferation. The continued growth of these large well-vascularized tumors may explain the poor efficacy of MMP inhibitors in lung cancer clinical trials.     

Color Doppler vascularity index can predict distant metastasis and survival in colon cancer patients

The purpose of this study was to investigate the clinical usefulness of the color Doppler vascularity index (CDVI) in patients with colon cancer before surgery. Forty-four patients with sonographically visible tumor mass of colon cancer were investigated. The CDVI of each tumor was determined using transabdominal color Doppler ultrasound. The CDVI was defined as the ratio of the number of the colored pixels within a tumor section to the number of total pixels in that specific tumor section and was calculated by using Encomate software (Electronic Business Machine Co. Ltd., Taipei, Taiwan). The correlation between the CDVI and clinicopathological factors, mode of recurrence, and patient survival was studied. For comparison, microvessel density (the mean number of microvessels in three areas of highest vascular density at x200 magnification) of the tumors of these 44 patients was also evaluated by using immunohistochemical staining of surgical specimens with anti-CD34. The microvessel density was not correlated with Dukes' classification, clinicopathological factors, and survival. The CDVI was significantly higher in the patients with lymph node metastases and vascular invasion than in those without such metastases and invasion (P = 0.006 and P = 0.0098, respectively). Moreover, in patients with a high CDVI (> 15%) and positive vascular invasion, survival was significantly poorer than in those with low CDVI (< or = 15%) and negative invasion (P = 0.0037 and 0.0039, respectively). Multivariate analysis indicated that liver metastasis, vascular invasion, and CDVI are independent prognostic factors in the patients with colon cancer. According to the mode of recurrence in 36 patients who underwent curative resection, the frequency of the distant organ recurrence was significantly higher in the high CDVI group (40%) than in the low CDVI group (0%). The CDVI is a good preoperative indicator of recurrence and patient survival in colon cancer. Thus, the CDVI may be helpful in stratifying patients for adjuvant therapy.     

人血管能抑素抑制小鼠Lewis肺癌移植瘤生长、转移及血管新生

目的:探讨人血管能抑素(canstatin)对小鼠Lewis肺癌移植瘤生长、转移和血管新生的影响.方法:将pCMV-Script/canstatin及空载体pCMV-Script通过电穿孔的方法转染A549细胞,G418筛选获得阳性克隆.RT-PCR检测转染后细胞中canstatin mRNA的表达,Western blotting检测转染后细胞中canstatin蛋白的表达.建立Lewis肺癌小鼠移植瘤模型,观察pCMV-Script/canstatin组A549细胞培养上清对小鼠Lewis肺癌移植瘤的治疗作用,免疫组化检测各治疗组荷瘤小鼠移植瘤的微血管密度.结果:pCMV-Script/canstatin转染A549细胞在G418筛选后成功形成克隆,转染的A549细胞能有效表达canstatin mRNA和蛋白.pCMV - Script/canstatin治疗组小鼠肿瘤体积明显小于pCMV-Script组和NS组[(1.47 ±0.21)cm3 vs(2.43 ±0.15) cm3、(2.53 ±0.18) cm3,P ＜0.01);pCMV-Script/canstatin组、pCMV - Script组和NS组的肺转移结节数分别为(3.00±1.00)、(7.80±1.48)、(7.60 ±2.41)个,pCMV-Script/canstatin组肿瘤转移受到显著的抑制(P＜0.01);pCMV-Script/canstatin组小鼠的肿瘤组织微血管数明显少于pCMV-Script组和NS组[(84.40 ±8.83) vs (188.68 ±11.15)、(190.24±12.91)个,P＜0.01].结论:pCMV-Script/canstatin能在A549细胞中表达并分泌至细胞外,canstatin可明显抑制Lewis肺癌移植瘤的生长、转移和血管新生.     

Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis

Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewis^a and sialyl Lewis^x. The sialyl Lewis^a and sialyl Lewis^x determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewis^a and sialyl Lewis^x are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewis^a/x determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system.     

IDIBELL cancer conference on metastasis and angiogenesis

The IDIBELL Cancer Conference (ICC) on Metastasis and Angiogenesis was held in Barcelona, Spain, on May 26-27, 2011. The program content was developed by Dr. Manel Esteller, director of the Cancer Epigenetics and Biology Program (PEBC-IDIBELL), Dr. Oriol Casanovas and Dr. Francesc Vi?als Canals of the Catalan Institute of Oncology (ICO-IDIBELL), and Dr. Danny R. Welch from the University of Kansas Cancer Center. The topics discussed during the meeting included the latest advances in epigenetic control of metastasis and tumor cell invasion, and molecular mechanisms of angiogenesis and tumoral angiogenesis, and were presented by invited keynote speakers. One issue that recurred throughout the meeting was the increased appreciation of tumor-stromal/microenvironment interactions and how the tumor cells respond to these signals in the cancer dissemination process.     

Tyrosine kinase inhibition of multiple angiogenic growth factor receptors improves survival in mice bearing colon cancer liver metastases by inhibition of endothelial cell survival mechanisms

Redundant mechanisms mediate colon cancer angiogenesis. Targeting multiple angiogenic factors simultaneously may improve survival of mice with colon cancer metastases. BALB/c mice underwent splenic injection with CT-26 colon cancer cells to generate liver metastases and received administration of either vehicle alone or a tyrosine kinase inhibitor for vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor receptors (SU6668). Mice were sacrificed when they became moribund as determined by a blinded observer. In a parallel experiment, groups of mice were sacrificed at earlier time points to better define the kinetics of the effect of SU6668 on angiogenic parameters over time. SU6668 increased median survival by 58% (P < 0.001) and led to a progressive increase in tumor cell and endothelial cell apoptosis that increased over time. In addition, pericyte vessel coverage and tumor vascularity were significantly decreased in mice treated with SU6668. Based on current knowledge of endothelial cell survival, these data suggest that SU6668 may prevent tumor endothelial cell survival directly (vascular endothelial growth factor) and indirectly (pericyte coverage) by affecting endothelial cell survival mechanisms.     

Hepatic gene expression of NK4, an HGF-antagonist/angiogenesis inhibitor, suppresses liver metastasis and invasive growth of colon cancer in mice

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancer cells by enhancing invasion and metastasis. We earlier found that NK4, a four-kringle fragment of HGF, functions as both an HGF antagonist and an angiogenesis inhibitor. We have now carried out studies to determine if hydrodynamics-based delivery and expression of the NK4 gene would inhibit liver metastasis and invasive growth of colon carcinoma cells in mice. When the naked plasmid for NK4 was introduced into mice by hydrodynamics-based gene delivery, a high level of expression of NK4 was predominant in the liver. After intrasplenic inoculation of MC-38 murine colon carcinoma cells, the cells formed numerous metastatic nodules in the liver and showed invasive growth behavior. On the other hand, when mice were given the NK4 plasmid, hepatic gene expression of NK4 inhibited the liver metastasis and subsequent growth associated with a decrease in microvessel density. Likewise, intrahepatic invasion of cancer cells was inhibited by NK4 gene expression, and this anti-invasive effect was associated with in situ inhibition of c-Met receptor tyrosine phosphorylation. Moreover, NK4 gene expression prolonged survival of these mice. Taken together with the knowledge that the majority of deaths from colon cancer are due to liver metastasis, the potential therapeutic use of hepatic gene expression of NK4 for metastatic colon cancer treatment can be given consideration.     

黄连素改善小鼠结肠腺癌恶病质实验研究

目的通过小鼠结肠腺癌恶病质模型实验探索传统中药提取物黄连素改善恶病质状态的作用及机制。方法将40只BALB/c小鼠随机分成5组,每组8只。即非荷瘤健康对照组、肿瘤恶病质组、黄连素低剂量干预组、黄连素中剂量干预组、黄连素高剂量干预组,使用小白鼠结肠腺癌细胞株CT-26皮下注射BALB/c小鼠,构建荷瘤小鼠肿瘤恶病质模型。黄连素低、中、高剂量干预组小鼠分别给予黄连素12.5 mg/（kg·d）、25 mg/（kg·d）、50mg/（kg·d）灌胃,连续15天。健康对照组、肿瘤恶病质组给予等量生理盐水灌胃。观察低、中、高剂量黄连素对恶病质小鼠的体质量、摄食量、饮水量以及血清中三酰甘油、血糖及总蛋白、白蛋白水平的影响,并比较各组肿瘤组织炎性细胞因子白细胞介素1（interleukin-1,IL-1）、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）水平以及肝、肾组织形态。观察不同剂量黄连素纠正恶病质的疗效及对肝肾的保护作用。结果与肿瘤恶病质组比较,黄连素干预各组能抑制癌症恶病质小鼠体质量下降,增加小鼠的摄食量、饮水量（均P〈0.05）;可升高血中总蛋白、白蛋白、血糖浓度,降低血清中三酰甘油的异常升高（均P〈0.05）;可降低肿瘤组织中的IL-1、IL-6及TNF-α表达水平（均P〈0.05）;可改善肝、肾组织形态。结论黄连素能有效改善小鼠结肠腺癌恶病质状态,以黄连素中、高剂量组疗效更佳,其机制可能与其有效降低细胞因子（IL-1、IL-6及TNF-α）表达相关。     

Fatty acid synthase inhibitor cerulenin suppresses liver metastasis of colon cancer in mice

Fatty acid synthase (FAS) is highly expressed in many kinds of human cancers, including colorectal cancer (CRC), and we have investigated the potential use of FAS inhibitors for chemoprevention of liver metastasis of CRC in mice. Expression of FAS was evaluated in murine CRC cell lines Colon 26 and CMT 93. Cerulenin, a natural inhibitor of FAS, induced apoptosis in these cell lines. The ability of cerulenin to prevent development of liver metastatic lesions in Colon 26 was evaluated. The numbers and sizes of liver metastatic CRC tumors were significantly reduced by treating mice with cerulenin. Cerulenin treatment was associated with reduced levels of phosphorylated Akt in Colon 26 cells, suggesting that inhibition of this signal transduction pathway might be involved in the chemopreventive activity of this compound. Based on studies in mouse models, inhibiting FAS would be an effective strategy to prevent and retard growth of liver metastatic tumors of CRC that have high expression of this enzyme. (Cancer Sci 2010; 00: 000–000)     

Down-regulation of frizzled-7 expression decreases survival, invasion and metastatic capabilities of colon cancer cells

Background:

The canonical Wnt signalling pathway is activated in most sporadic colorectal cancers (CRCs). We previously reported that FZD7 functions as a receptor for the canonical Wnt signalling pathway in colon cancer cells.

Methods and results:

In this study, we examined the function of FZD7 in survival, invasion and metastatic capabilities of colon cancer cells. FZD7_siRNA transfection decreased cell viability of HT-29 and HCT-116 colon cancer cells. Expression of c-Jun, phosphorylation of JNK and c-Jun, and activation of RhoA were suppressed after FZD7_siRNA transfection into HCT-116 cells. In vitro invasion activity and Wnt target gene expression were also reduced in HCT-116 cells transfected with FZD7_siRNA. Liver metastasis of stable FZD7_siRNA HCT-116 cell transfectants in scid mice was decreased to 40–50% compared to controls. The mRNA levels of FZD7 in 135 primary CRC tissues were examined by real-time PCR. FZD7 mRNA levels were significantly higher in stage II, III or IV tumours than in non-tumour tissues (P<0.005), and overall survival was shorter in those patients with higher FZD7 expression (P<0.001).

Conclusion:

These data suggest that FZD7 may be involved in enhancement of survival, invasion and metastatic capabilities of colon cancer cells through non-canonical Wnt signalling pathways as well as the canonical pathway.     

Angiotensin II type I antagonist prevents pulmonary metastasis of murine renal cancer by inhibiting tumor angiogenesis

Angiotensin II (AII) is a potent vasoconstrictor peptide from the renin-angiotensin system in the kidney. The AII type 1 receptor (AT1R) is reportedly expressed in several tumors including renal cell carcinoma, and AII is involved in tumor angiogenesis. We p.o. administered the long-acting AT1R antagonist, candesartan (10 mg/kg), to the 16 days mouse renal cancer lung metastasis model to test the preventive effects in tumor metastasis. Pulmonary metastases of renal cancer showed prominent AT1R expression in both mice and humans, and candesartan treatment dramatically prevented lung metastatic nodules (14.9 +/- 1.8; P < 0.0001; n = 12) in mice along with the inhibition of neovascularization and vascular endothelial growth factor expression, compared with control metastatic mice (123.3 +/- 8.6; n = 13). Candesartan is widely used clinically, so it seems to be a reasonable therapy for patients with lung metastases of renal cell carcinoma.