Relationship between vessel density and expression of vascular endothelial growth factor and basic fibroblast growth factor in small cell lung cancer in vivo and in vitro.

In 21 human small cell lung cancer (SCLC) cell lines, we determined the expression of mRNA and secreted protein levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The VEGF expression was highly variable between cell lines, with a > 100-fold variation, under identical in vitro conditions. The bFGF expression in cell lines was generally very low. Nine of the cell lines were further analyzed during growth as solid tumor xenografts in nude mice (in vivo). A more uniform VEGF protein expression was present in vivo. Compared with the variable in vitro expression, VEGF was relatively up-regulated in the tumor lines CPH 54A and CPH 54B and down-regulated in GLC 3. One line, DMS 79, had a high VEGF expression in vivo as well as in vitro. The vessel density was determined by Chalkley point counting on CD31 immunostained cryosections of tumors of each of the nine SCLC lines. We found a strong positive correlation between vessel density and tissue VEGF protein expression (r(s) = 0.75; P = 0.02) and a comparatively strong negative correlation (r(s) = -0.80; P = 0.01) between vessel density and tissue bFGF expression. No significant correlation was present between vessel density and in vitro VEGF expression. We conclude that VEGF and bFGF expression is dependent on microenvironmental conditions, as well as cell line-specific factors, and that a strong positive correlation exists between in vivo VEGF expression and vessel density, whereas high tissue levels of bFGF are not correlated with higher vessel densities in SCLC xenografts.     

Cell cycle basis for the onset and progression of c-Myc-induced, TGFalpha-enhanced mouse mammary gland carcinogenesis

Using single and double transgenic mouse models, we investigated how c-Myc modulates the mammary epithelial cell cycle to induce cancer and how TGFalpha enhanced the process. In c-myc transgenic mice, c-myc expression was high in the hyperplastic mammary epithelium and in the majority of tumor areas. However, the tumors displayed focal areas of low expression of c-myc but high rates of proliferation. In contrast to E2F1 and cyclin A2, which were induced and co-localized with c-myc expression, induction of cyclins D1 and E occurred only in these tumor foci. Overexpression of cyclin D1 also occurred in the hyperplastic epithelium of tgfalpha-single and tgfalpha/c-myc-double transgenic mice. In tgfalpha/c-myc tumors, cells positive for cyclins D1 and E were randomly spread, without showing a reciprocal relationship to c-myc expression. In contrast to c-myc tumors, most tgfalpha/c-myc tumors showed undetectable levels of retinoblastoma protein (pRB), and the loss of pRB occurred in some cases at the mRNA level. These results suggest that E2F1 and cyclin A2 may be induced by c-Myc to mediate the onset of mammary cancer, whereas overexpression of cyclins D1 and E may occur later to facilitate tumor progression. TGFalpha may play its synergistic role, at least in part, by inducing cyclin D1 and facilitating the loss of pRB.     

Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis

Metastasis is the primary cause of death in patients with breast cancer. Overexpression of c-myc in humans correlates with metastases, but transgenic mice only show low rates of micrometastases. We have generated transgenic mice that overexpress both c-myc and vascular endothelial growth factor (VEGF) (Myc/VEGF) in the mammary gland, which develop high rates of pulmonary macrometastases. Gene expression profiling revealed a set of deregulated genes in Myc/VEGF tumors compared to Myc tumors associated with the increased metastatic phenotype. Cross-comparisons between this set of genes with a human breast cancer lung metastasis gene signature identified five common targets: tenascin-C(TNC), matrix metalloprotease-2, collagen-6-A1, mannosidase-alpha-1A and HLA-DPA1. Signaling blockade or knockdown of TNC in MDA-MB-435 cells resulted in a significant impairment of cell migration and anchorage-independent cell proliferation. Mice injected with clonal MDA-MB-435 cells with reduced expression of TNC demonstrated a significant decrease (P<0.05) in (1) primary tumor growth; (2) tumor relapse after surgical removal of the primary tumor and (3) incidence of lung metastasis. Our results demonstrate that VEGF induces complex alterations in tissue architecture and gene expression. The TNC signaling pathway plays an important role in mammary tumor growth and metastases, suggesting that TNC may be a relevant target for therapy against metastatic breast cancer.     

Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans.

PURPOSE: The overexpression of c-myc associated with uncontrolled cell proliferation is a frequent genetic event in androgen-refractory prostatic neoplasia. The purpose of this study was to evaluate the bioavailability and efficacy of a novel antisense phosphorodiamidate morpholino oligomer directed against c-myc, AVI-4126, in PC-3 androgen-independent human prostate cancer xenograft murine model and its safety in a Phase I human clinical study. EXPERIMENTAL DESIGN: AVI-4126 administration in athymic mice bearing s.c. PC-3 xenografts was carried out to determine the bioavailability, tolerance, antitumor activity, and histological changes induced by targeted inhibition of c-Myc expression using a specific morpholine antisense oligomer. The Phase I safety study involved a single center, open label, dose-escalating design in healthy volunteers after i.v. administration of AVI-4126. RESULTS: The data reveal that AVI-4126 targets and inhibits c-myc translation in a sequence-specific manner and causes significant growth inhibition and apoptosis in prostate cancer cells and in s.c. tumor xenografts. A 75-80% reduction in tumor burden was observed in AVI-4126-treated animals compared with the scrambled oligomer and saline control groups. Histologically, tumors grown in the athymic mice treated with AVI-4126 were less cellular and vascular than those in control mice and showed an increased level of cellular degeneration, cytoplasmic vacuoles, and hyperchromatic nuclei. Phase I safety trials in humans via i.v. route of administration showed no toxicity or serious adverse events. CONCLUSIONS: The present study demonstrates that inhibition of c-Myc expression by antisense phosphorodiamidate morpholino oligomer is a promising new and safe therapeutic strategy for prostate cancer.     

n-3 PUFAs reduce VEGF expression in human colon cancer cells modulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-1alpha induction pathway

n-3 Polyunsaturated fatty acids (PUFAs) inhibit the development of microvessels in mammary tumors growing in mice. Human colorectal tumors produce vascular endothelial growth factor (VEGF) whose expression is up-regulated in tumor cells by both cyclooxygenase-2 (COX-2) and PGE(2) and directly correlated to neoangiogenesis and clinical outcome. The goal of this study was to examine the capability of n-3 PUFAs to regulate VEGF expression in HT-29 human colorectal cells in vitro and in vivo. Constitutive VEGF expression was augmented in cultured HT-29 cells by serum starvation and the effects of eicosapentaenoic (EPA) or docosahexaenoic acid (DHA) on VEGF, COX-2, phosphorylated extracellular signal-regulated kinase (ERK)-1 and -2 and hypoxia-inducible-factor 1-alpha (HIF-1alpha) expression and PGE(2) levels were assessed. Tumor growth, VEGF, COX and PGE(2) analysis were carried out in tumors derived from HT-29 cells transplanted in nude mice fed with either EPA or DHA. Both EPA and DHA reduced VEGF and COX-2 expression and PGE(2) levels in HT-29 cells cultured in vitro. Moreover, they inhibited ERK-1 and -2 phosphorylation and HIF-1alpha protein over-expression, critical steps in the PGE(2)-induced signaling pathway leading to the augmented expression of VEGF in colon cancer cells. EPA always showed higher efficacy than DHA in vitro. Both fatty acids decreased the growth of the tumors obtained by inoculating HT-29 cells in nude mice, microvessel formation and the levels of VEGF, COX-2 and PGE(2) in tumors. The data provide evidence that these n-3 PUFAs are able to inhibit VEGF expression in colon cancer cells and suggest that one possible mechanism involved may be the negative regulation of the COX-2/PGE(2) pathway. Their potential clinical application as anti-angiogenic compounds in colon cancer therapy is proposed.     

Imatinib mesylate efficiently achieves therapeutic intratumor concentrations in vivo but has limited activity in a xenograft model of small cell lung cancer.

PURPOSE: Despite recent advances in cancer therapy, long-term survival in small cell lung cancer (SCLC) remains uncommon, underscoring the need for novel therapeutic approaches. Previous studies have identified constitutive expression of the receptor tyrosine kinase, c-Kit, and its ligand, stem cell factor, in a substantial proportion of SCLC specimens. The purpose of this study was to determine whether imatinib mesylate, an inhibitor of c-Kit, could achieve therapeutic concentrations in tumors and in brain (a frequent site of SCLC metastasis) and interfere with SCLC tumor growth in vivo. EXPERIMENTAL DESIGN: Human SCLC tumor cell lines with constitutive c-kit expression and tyrosine phosphorylation (NCI-H209, NCI-H526, and NCI-H1607) were used to establish SCLC tumor xenografts in NCr nude (nu/nu)-immunodeficient mice. SCLC tumor-bearing mice were randomly assigned to imatinib or control (water) administered twice a day by oral gavage. Imatinib concentrations in plasma, brain, and tumor were quantitated and correlated with tumor response. RESULTS: Therapeutic concentrations of imatinib were achieved in plasma and tumor xenografts but not in the brain. Imatinib blocked the constitutive activation of c-kit in SCLC tumor cell lines in vitro but had a negligible effect on SCLC xenograft growth in vivo. CONCLUSIONS: Orally administered imatinib rapidly reaches therapeutic concentrations in SCLC xenografts, suggesting the feasibility of combining imatinib with other novel or traditional chemotherapeutic agents in SCLC or other solid tumors. The c-Kit signaling pathway does not appear to play a critical role in SCLC proliferation and viability in vivo, however, suggesting that imatinib is unlikely to be effective as monotherapy for SCLC.     

Additive and differential biological activity of alpha-interferon A, difluoromethylornithine, and their combination on established human lung cancer cell lines

The effect of human recombinant leukocyte interferon A (IFN-alpha A) and DL-alpha-difluoromethylornithine (DFMO) as single drugs and in combination on the in vitro growth, cell cycle distribution, activity of the enzyme L-dopa decarboxylase, and expression of the c-myc and N-myc oncogenes was studied in human lung cancer cell lines. In vitro growth activities were tested in concentrations ranging from 10 to 50,000 IU/ml for IFN-alpha A and from 0.1 to 10 mM for DFMO by means of the soft agarose clonogenic assay using continuous drug exposure. Ten well established small cell lung cancer (SCLC) cell lines including five cell lines of the classic and five of the variant phenotype, two cell lines derived from adenocarcinoma of the lung, and one large cell lung cancer cell line were included in the study. We found that IFN-alpha A inhibited the growth only of the variant phenotype of SCLC with an approximate drug concentration yielding a 50% inhibition of colony growth of 1000 IU/ml. None of the SCLC classic cell lines was inhibited significantly. The growth inhibition of IFN-alpha A correlated with the proliferation rate of the tumor. IFN-alpha A inhibited one of two adenocarcinoma cell lines and 0 of 1 large cell lung cancer cell line. DFMO inhibited the colony formation of 10 of 10 SCLC cell lines, 2 of 2 adenocarcinoma cell lines, and 0 of 1 large cell lung cancer cell line with a drug concentration yielding a 50% inhibition of colony growth of 1 mM. No difference between the classic and variant phenotypes of SCLC was found. The combination of IFN-alpha A and DFMO resulted in an additive cytostatic effect in all cell lines tested. The same result, i.e., an additive cytostatic effect, was obtained for two SCLC cell lines that were tested in liquid culture. Neither single drugs nor their combination led to an accumulation of cells in a particular phase of the cell cycle nor did it affect the activity of the SCLC classic marker enzyme L-dopa decarboxylase. In addition, IFN-alpha A, DFMO, and their combination did not affect the expression of the c-myc and N-myc oncogenes in cell lines NCI-N417 and NCI-H526, respectively, following 4, 24, and 72 h of continuous drug exposure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of p64c-myc and neuroendocrine properties define three subclasses of small cell lung cancer

Twelve human small cell lung cancer (SCLC) cell lines and 6 non-SCLC cell lines were analysed with respect to expression of the c-myc, c-myb, and c-raf1 protooncogenes at the protein level. Analysis of p64c-myc protein expression in 12 SCLC cell lines resulted in the observation that it is present at high levels not only in cells with low, but also in those with moderate neuroendocrine differentiation. Neuroendocrine differentiation was based on parameters such as growth rate, colony formation, L-Dopa decarboxylase (DDC) activity, bombesin, and neurotensin described before. Surprisingly, in two cell lines with low neuroendocrine differentiation but without c-myc protein expression (SCLC-86M1 and NCI-H526) p75c-myb expression was observed which may therefore be able to substitute for the p64c-myc protein. Analysis of p74c-raf1 expression did not result in correlation with any growth or differentiation parameter since it was expressed at low levels in 11 out of 12 cases. We conclude that SCLC in vitro can be classified in three rather than two previously defined subclasses. In addition to the classic subclass with slow growth, high neuroendocrine differentiation, and absent or very low p64c-myc expression and the variant subclass with fast growth, absent to very low neuroendocrine differentiation, and high p64c-myc expression, we suggest a third subclass designated as transitional with moderate growth, moderate neuroendocrine differentiation, and high p64c-myc expression. Data on a small number of non-SCLC cell lines tested showed that high levels of p64c-myc correlate with high in vitro growth rates. This indicates that high p64c-myc levels may be associated with high proliferative activity, and lack of differentiation in lung cancer in general. The p74c-raf1 protein was found in all non-SCLC cell lines. Whether this classification of SCLC cell lines is applicable to SCLC in vivo remains to be determined.     

Administration of VEGF receptor tyrosine kinase inhibitor increases VEGF production causing angiogenesis in human small-cell lung cancer xenografts

Angiogenesis is mediated mainly by vascular endothelial growth factor (VEGF), and VEGF causes rapid growth in cancers, including human small-cell lung cancer (SCLC). The anti-angiogenic strategy of treating cancer using VEGF receptor (VEGFR) inhibition is currently of great interest. We tested the effects of the VEGFR2 tyrosine kinase inhibitor (TKI) vandetanib on the proliferation of two kinds of SCLC cell lines: SBC-1 cells, with detectable VEGFR2 expression and MS-1-L cells, without detectable VEGFR2 expression. To evaluate the anti-tumor and anti-angiogenic effects of vandetanib in vivo, we grafted SBC-1 and MS-1-L cells into mice. After a 3-week treatment, we measured the tumor size and histologically evaluated necrosis and apoptosis using H&E and TUNEL staining, respectively. The microvessels in the xenografts were also quantified by immunostaining of CD31. Vandetanib did not affect the proliferation of SBC-1 cells, but stimulated the growth of MS-1-L cells. In the SCLC xenograft model, vandetanib inhibited growth and tumor angiogenesis in a dose-dependent manner in SBC-1 xenografts. Vandetanib inhibited the growth of MS-1-L xenografts at a low dose (<12.5 mg/kg/day), but it did not affect tumor size or change microvessel counts at a higher dose. Interestingly, secretion of VEGF increased significantly in the MS-1-L cell line in the presence of a high dose of vandetanib in vitro. The effects of vandetanib on tumor angiogenesis were different in SBC-1 and MS-1-L cell lines. Production of angiogenic factors such as VEGF by the tumor potentially stimulates tumor angiogenesis and results in the acquisition of resistance to VEGFR TKI.     

Circulating angiogenic growth factor levels in mice bearing human tumors using Luminex Multiplex technology

Tumor angiogenesis is essential for tumor growth and metastasis formation. Luminex methodology was used to measure the levels of four angiogenic cytokines in cell culture medium and in the plasma of mice bearing human tumors. We obtained plasma and conditioned culture medium from 12 different human tumor cell lines. Tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-beta) were determined by the Luminex FlowMetrix assay. VEGF, TNF-alpha, and bFGF were undetectable in non-tumor-bearing animals. HS746T gastric cancer and Caki-1 renal cell cancer cells in culture produced high levels of VEGF (1000 and 450 pg/10(6) cells, respectively). High levels of TGF-beta were produced by HS746T gastric carcinoma and Calu-6 non-small-cell lung carcinoma (3000 and 1000 pg/10(6) cells, respectively). Caki-1 renal cell carcinoma and Calu-6 non-small-cell lung carcinoma cells in culture produced high levels of bFGF (42 and 10 pg/10(6) cells, respectively). Caki-1, SW2 SCLC, HCT-116 and HT-29 colon tumors produced high plasma levels of VEGF (200, 220, 42, and 151 pg/ml, respectively) and TGF-beta (31, 36, 45, 32 pg/ml, respectively). A positive linear correlation was seen between tumor volume and VEGF in SW2 (r=0.87) and Caki-1 (r=0.47) tumors, and a moderate correlation in HCT116 tumors (r=0.3). Angiogenic profiles in the plasma of nude mice bearing human tumors may be useful to identify appropriate biomarkers for antiangiogenic therapy, as diagnostic and prognostic tools, and to monitor the responses of individual tumors to antiangiogenic therapy.     

VIP增强VEGF mRNA在非小细胞肺癌细胞中的表达

背景与目的研究发现血管内皮生长因子(VEGF)和血管活性肠肽(VIP)均对肿瘤的生长具有促进作用,但VIP通过何种方式来促进肿瘤的生长还不清楚。本研究的目的是观察VIP对非小细胞肺癌(NSCLC)细胞中VEGFmRNA表达的影响。方法应用反转录聚合酶链式反应(RTPCR)技术检测VEGFmRNA在NSCLC细胞系和小细胞肺癌(SCLC)细胞系中的表达及VIP对其表达的影响。结果在NSCLC细胞系A549、GLC82、H157、H460和SCLC细胞系H446中检测到了VEGFmRNA的表达。VIP能促进VEGFmRNA在A549和H157细胞中的表达,在VIP作用8h和16h时,VEGFmRNA的表达水平达到最高,显著高于VIP作用0h时(P<0.01)。结论VIP可能通过增强VEGFmRNA在肺癌细胞中的表达和分泌,促进肺癌新生血管的生成,参与肿瘤的生长。     

Malignant transformation of host cells by a human small cell lung cancer xenografted into nude mice

Malignant transformation of mouse host cells by a human small cell lung cancer (SCLC) was demonstrated by short-term in vitro cultivation of the tumor cells from a xenograft at two different transplant generations. Isoenzyme (LDH) and chromosome analysis showed that out of the 3 cell lines established from this tumor, 1 retained a human karyotype similar to that of the xenograft and 2 were murine transformed cell lines. These murine cell lines produced fibro-sarcoma-like tumors when injected into nude mice. Because of the early in vitro emergence of murine transformed cell populations, it is likely that the transformation process had occurred in vivo. Since in our experience the induction of transformation of host murine cells, also observed directly in vivo, is more frequent with SCLC than other histotypes (lung and colorectal adenocarcinoma), it is suggested that the known production of growth factor by these tumors may contribute to this transformation.     

EGCG对胃癌血管新生中VEGF信号通路的多靶点作用

目的：探讨EGCG对胃癌血管生成抑制作用及其信号通路。方法：建立裸鼠异位胃癌模型,经腹腔注射EGCG,检测肿瘤生长及肿瘤组织微血管密度;不同浓度EGCG处理胃癌细胞24 h,检测胃癌VEGF蛋白和mRNA表达及VEGF分泌;同时不同浓度EGCG处理脐静脉内皮细胞,检测内皮细胞生长、迁移和体外小管形成。结果：EGCG显著抑制胃癌生长和肿瘤血管生成,平均肿瘤抑制率60.4%;EGCG显著抑制胃癌VEGF蛋白、mRNA表达和VEGF分泌;EGCG时间和剂量依赖性地抑制VEGF诱导的内皮细胞增殖,同时也剂量依赖性地抑制VEGF诱导的内皮细胞的迁移和小管生成。结论：EGCG多靶点作用于VEGF信号通路,抑制胃癌生长和血管生成。     

TSG101在肺癌组织和细胞系中的表达及意义

背景和目的在小鼠NIH3T3成纤维细胞中通过随机纯合子敲除法证实,破坏肿瘤易感基因TSG101(Tumor Susceptibility Gene101)导致裸鼠的自发性肺转移性肿瘤,从而TSG101作为侯选的肿瘤抑制基因被认识。随后,越来越多的研究证实TSG101并非是严格意义上的肿瘤抑制基因,在原发乳腺癌并没有检测到基因内部的序列的丢失或异常,相反发现存在异常的TSG101选择性剪接体。本研究目的旨在研究肺癌细胞系和组织中TSG101的表达情况,分析TSG101及异常的TSG101选择性剪接体在肺癌的发生发展过程中可能所起的作用。方法采用免疫组织化学方法(SP法)检测79例肺鳞癌和肺腺癌组织及相应癌旁正常肺组织中TSG101的表达情况。采用RT-PCR实验分析,检测出在7种肺癌细胞系中野生型和异常剪接转录产物表达情况。采用WesternBlot方法检测肺癌细胞系中蛋白质表达情况。结果TSG101在肺鳞癌和肺腺癌中的蛋白水平显著低于其在相应正常肺组织中的表达。七种细胞系中普遍存在由于异常的选择性剪接而缩短的TSG101转录产物,和野生性转录产物同时表达。通过统计学分析发现在小细胞肺癌细胞系中存在更多的异常的转录产物。通过WesternBlot分析同样发现小细胞肺癌细胞系同样与其他非小细胞肺癌细胞系在蛋白水平上存在差异。结论尽管在非小细胞肺癌中作为侯选的肿瘤抑制因子,但是普遍存在的TSG101异常转录产物也许和肺癌的类型相关,可作为肺癌恶性进展的生物学标志。