Glioma-produced extracellular matrix influences brain tumor tropism of human neural stem cells

Summary A major obstacle in the treatment of gliomas is the invasive capacity of the tumor cells. Previous studies have demonstrated the capability of neural stem cells (NSCs) to target these disseminated tumor cells and to serve as therapeutic delivery vehicles. Less is known about the factors involved in brain tumor tropism of NSCs and their interactions within the tumor environment. As gliomas progress and invade, an extensive modulation of the extracellular matrix (ECM) occurs. Tumor-ECM derived from six glioblastoma cell lines, ECM produced by normal human astrocytes and purified ECM compounds known to be upregulated in the glioma environment were analyzed for their effects on NSCs motility in vitro. We found that tumor-produced ECM was highly permissive for NSC migration. Laminin was the most permissive substrate for human NSC migration, and tenascin-C the strongest inducer of a directed human NSC migration (haptotaxis). A positive correlation between the degree of adhesion and migration of NSCs on different ECM compounds exists, as for glioma cells. Our in vitro data suggest that the ECM of malignant gliomas is a modulator of NSC migration. ECM proteins preferentially expressed in areas of glioma cell invasion may provide a permissive environment for NSC tropism to disseminated tumor cells. ^† These authors contributed equally to this work.     

Rescue of hypoxia-inducible factor-1alpha-deficient tumor growth by wild-type cells is independent of vascular endothelial growth factor

In tumors, rapid cell proliferation associated with deficient vascularization leads to areas of hypoxia.Tumor hypoxia has direct consequences on clinical and prognostic parameters and is a potential therapeutic target. The hypoxic response depends critically on hypoxia-inducible factor-1alpha (HIF-1alpha) in pathological (e.g., tumorigenesis) as well as physiological (e.g., development and wound healing) processes. By s.c. injection of HIF-1alpha(-/-) embryonic stem (ES) cells in nude mice, we were able to demonstrate the role of HIF-1alpha in cell differentiation of teratocarcinomas. HIF-1alpha(+/+) tumors grow fast and preferentially form neuronal tissue, whereas HIF-1alpha(-/-) tumors show delayed growth and favorably form mesenchyme-derived tissue. Mixing wild-type and HIF-1alpha(-/-) ES cells in the same tumor at a ratio as low as 1:100, we showed that HIF-1alpha(+/+) cells can rescue the growth of mixed tumors although these tumors are not significantly different phenotypically or genotypically from the original HIF-1alpha(-/-) tumors. Interestingly, these results are not restricted to teratocarcinomas: they were confirmed with mixtures of Hepa1/Hepa1C4 cells (where HIF-1beta is mutated), demonstrating that growth changes are not related to differences in differentiation observed within teratocarcinomas. We also showed that despite lower mRNA expression, vascular endothelial growth factor protein status in HIF-1alpha(-/-) and mixed tumors does not significantly differ from the HIF-1alpha(+/+) tumors. Moreover, we demonstrated that tumor vascularization remains proportional to vascular endothelial growth factor protein levels, but that hypoxic up-regulation of this growth factor is not the decisive factor influencing tumor growth. Differences in levels of apoptosis are not responsible for alteration in growth because poly(ADP-ribose) polymerase cleavage, a hallmark of the apoptotic process, was similar in HIF-1alpha(+/+), HIF-1alpha(-/-), and mixed tumors. Our data demonstrate that the HIF-1alpha-dependent response of a few cells is capable of sustaining the growth of the whole tumor, probably through the secretion of factors up-regulated under low oxygen conditions.     

Negligible radiation protection of endothelial cells by vascular endothelial growth factor

Glioblastoma multiforme (GBM) is a radioresistant tumor. Tumor neoangiogenesis is an important mechanism for tumor sustenance. Angiogenesis is primarily mediated by vascular endothelial growth factor (VEGF), and earlier studies have suggested that VEGF protects human umbilical vein endothelial cells (HUVECs) against high doses of radiation. We tried to extend these findings to other endothelial cell lines and clinically relevant irradiation doses. Therefore, four different endothelial cell lines (HUVEC-C, primary HUVEC-P, an immortalized HUVEC cell line: EC-RF24, and bovine retina endothelial cells: BREC) were cultured without or with recombinant human VEGF165 (rhVEGF165). Cells were irradiated with gamma-rays from a 137Cs-source. Radiosensitivity was determined by proliferation or clonogenic assay. Apoptosis was assayed by flow cytometric determination of the sub-G1 population or by counting nuclear fragmentation. We found that the biologically active rhVEGF165 was able to improve clonogenic survival of HUVEC-C after 2 and 5 Gy. However, rhVEGF165 could not significantly alter the radiosensitivity of all cell lines studied in proliferation assays. rhVEGF165 only slightly reduced apoptosis in HUVEC-C after 3 Gy. In conclusion, the radioprotective effect from rhVEGF165 was found on different endothelial cell lines after clinically relevant radiation doses was negligible. We therefore hypothesize that the high VEGF-levels found in GBM in vivo do not reduce the radiosensitivity of endothelial cells, which is thought to contribute to the strong radioresistance of the tumor vasculature.     

Loxoprofen sodium suppresses mouse tumor growth by inhibiting vascular endothelial growth factor

There is increasing evidence to suggest the anti-tumor effects of non-steroidal anti-inflammatory drugs (NSAIDs). In this study it was shown that the most popular NSAID in Japan, loxoprofen sodium (LOX), inhibited in vivo growth of implanted Lewis lung carcinoma (LLC), whereas LOX did not affect the proliferation and viability of LLC cells in vitro. Intratumoral vessel density in LOX-treated mice was significantly lower than that of mice without treatment. Intratumoral expressions of vascular endothelial growth factor (VEGF) mRNA were attenuated by the LOX treatment. LOX suppressed both intratumoral and systemic VEGF protein in LLC-implanted mice. LOX also inhibited tubular formation of primary cultured human umbilical vein endothelial cells, presumably due to the inhibition of VEGF. In patients with advanced non-small cell lung cancer, LOX medication (120 mg/day) for a week significantly decreased the plasma VEGF level. These results suggest that LOX may have potent anti-cancer effects in patients with advanced NSCLC.     

Tumor-derived expression of vascular endothelial growth factor is a critical factor in tumor expansion and vascular function

There is considerable controversy concerning the importance of tumor-derived angiogenic factors to the neovascularization of solid tumors. Tumor, endothelial, and stromal expression of vascular endothelial growth factor (VEGF) have been hypothesized to be critical for tumor angiogenesis. To determine the relative contribution of tumor versus nontransformed tissue expression of VEGF to tumor growth, we used gene targeting and cre-loxP recombination to generate embryonic stem cell lines in which VEGF can be conditionally deleted. These lines were used to derive mouse embryonic fibroblast lines with null mutations in both alleles of VEGF. Upon immortalization and H-ras transformation, we used these VEGF null fibroblasts to make fibrosarcomas in immunocompromised mice. We report that tumorigenic VEGF expression is critical for ras-mediated tumorigenesis, and the loss of tumorigenic expression causes dramatic decreases in vascular density and vascular permeability and increases in tumor cell apoptosis.     

Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor

Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy.     

Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.     

Placental growth factor is a survival factor for tumor endothelial cells and macrophages

The vascular endothelial growth factor (VEGF)-related factor, placental growth factor (PlGF),has been shown recently to play an important role in pathological VEGF-driven angiogenesis.In this study, we examine the effects of mPlGF/PlGF-2 overexpression in tumors grown from glioma cells containing a tetracycline-regulated mPlGF cDNA. Overexpression of mPlGF leads to increased tumor growth and vascular survival. When tetracycline is used to abruptly withdraw mPlGF overexpression, we see increased apoptosis in both vascular cells and macrophages. In addition, PlGF-2 induces survival gene expression and inhibits apoptosis in vitro. Thus, we propose that PlGF-2 contributes to tumor angiogenesis by providing increased survival function to endothelial cells and macrophages.     

Overexpression of vascular endothelial growth factor by MCF-7 breast cancer cells promotes estrogen-independent tumor growth in vivo

Alteration of the phenotype of breast cancers from estrogen-dependent to estrogen-independent growth often leads to the failure of antiestrogenic tumor therapies. We report that overexpression of vascular endothelial growth factor (VEGF) by estrogen-dependent MCF-7 breast cancer cells could abolish estrogen-dependent tumor growth in ovariectomized mice. In the absence of estrogen, MCF-7 VEGF-expressing tumors with increased vessel density showed growth kinetics similar to, or even greater than, that of parental MCF-7 tumors with estrogen supplementation. Overexpression of VEGF by MCF-7 cells or treatment on parental MCF-7 cells with recombinant VEGF also stimulated cell proliferation in culture. Our data suggest that VEGF stimulation of MCF-7 tumor angiogenesis and growth is mediated by both autocrine and paracrine mechanisms.     

Modulation of angiogenesis and tumorigenicity of human melanocytic cells by vascular endothelial growth factor and basic fibroblast growth factor

Human melanoma cells express two prominent angiogenic factors, e.g., vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF/fibroblast growth factor-2). In this study, we report on the relative contribution of these two factors to in vitro and in vivo growth of a tumorigenic melanoma cell line (WM164) and nontumorigenic, immortalized melanocytes (FM516SV). Overexpression of either cytokine significantly boosted tumorigenicity of WM164 cells in immunodeficient SCID mice. Attempting to overexpress bFGF antisense sequences produced no viable clones confirming earlier reports that autocrine bFGF is obligatory to melanoma cell survival and growth. By contrast, down-regulation of endogenous VEGF production did not affect growth of WM164 cells in vitro. In vivo expansion of WM164 cells expressing VEGF antisense was delayed but not abrogated. Forced expression of either bFGF or VEGF in immortalized but nontumorigenic melanocytes did not induce sustained tumor growth in vivo highlighting that neither of the two factors is sufficient for induction of tumorigenicity in this model system. Overexpression of either cytokine in WM164 cells led to the development of atypical large vessels but not to an increase in microvessel density. Taken together our results confirm an essential autocrine role of bFGF in human melanoma and indicate a beneficial but nonessential role of VEGF in the tumorigenic phenotype of human melanoma cells.     

Induced apoptosis in human prostate cancer cells by blocking of vascular endothelial growth factor by siRNA

Introduction

Vascular endothelial growth factor (VEGF) regulates several cell functions including; proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules. The purpose of this study was to evaluate the potency of specific short-interfering RNA (siRNA) to suppress human VEGF expression by siRNA and investigate the effects of VEGF down-regulation on the cell proliferation and apoptosis of the human prostate cancer cell lines DU-145.

Methods

Transfection was performed using X-tremeGENE siRNA transfection reagent. At different time intervals, transfected cells were harvested and total RNA was extracted for RT-PCR. The VEGF content in supernatants were measured by ELISA. Inhibition of cell growth by hVEGF-siRNA was measured by using cell proliferation ELISA BrdU assay. Apoptotic cells were evaluated by using annexin-V-FITC apoptotic detection method.

Results

Transfection of hVEGF-siRNA resulted in statistically significant inhibition of hVEGF-mRNA that in turn caused a marked reduction in the expression of hVEGF. The cell growth was assessed every 24?h for 4?days after siRNA treatment resulted in a marked inhibition of cell proliferation as compared to scramble siRNA. The results of apoptosis showed that approximately 15?% of the cells treated with control-siRNA manifested evident apoptotic changes after 24?hpt, whereas DU-145 cells treated with hVEGF-siRNA significantly were positive, that is to say, 53?% at 72?hpt 23.9?±?2.78?% (P?<?0.001) and 13?±?1.57?% at 96?hpt.

Conclusion

Our findings indicate that siRNA are effective in eliciting the RNAi pathway in cancerous cells and that specific siRNA efficiently down-regulate VEGF expression. They could decrease VEGF production and induce apoptosis, which may also be linked to the inhibition of cancerous cell proliferation. Therefore, it can be concluded that siRNA-mediated suppression of VEGF represents a powerful tool against prostate cancer cell proliferation. VEGF down-regulation exerts a direct anti-apoptotic function in the DU-145 cell lines and promises the development of drugs for cancer therapy.     

肿瘤细胞系血管内皮生长因子及其受体共表达的研究

背景与目的：血管内皮生长因子（vascular endothelial growth factor,VEGF)旁分泌在肿瘤血管新生中的作用已得到证实,但其自分泌作用尚不清楚。本研究的目的是分析VEGF及其受体（Flt-1和KDR）基因在恶性肿瘤细胞系中的共表达。方法：以看家基因为内标,采用半定量逆转录－聚合酶链反应分析VEGF及其受体基因在30种肿瘤细胞系和4种内皮细胞中的表达水平。结果：在29种肿瘤细胞系和3种内皮细胞系检测到中度以上的VEGF基因表达,而人脐静脉内皮细胞仅有低表达;Flt-1基因表达分别见于50％（6／12）的血液肿瘤,28％（5／18）的实体瘤细胞和2种内皮细胞;仅在16．7％（2／12）的血液肿瘤,33．3％（6／18）实体瘤细胞和2种内皮细胞检测到KDR基因表达;而ECV304细胞并无Flt-1或KDR基因的表达。结论：VEGF基因高表达是肿瘤细胞的重要特征,而VEGF及其受体共表达表明肿瘤细胞系中存在自分泌途径。