Serum angiogenic activity: diagnostic relevance in renal cell carcinoma

OBJECTIVE: Angiogenesis is essential for tumor growth and progression. However, reported data on angiogenic parameters in patients with renal cell carcinoma are contradictory. The objective of this study was to use serum to compare the systemic angiogenic activity in patients with renal cell carcinoma and to determine if pathologic stage and grade correlated to this angiogenesis parameter. METHODS: Serum of 28 patients with a newly diagnosed renal cell carcinoma, 28 healthy volunteers and 9 patients with bladder carcinoma were used for this study. All sera were tested in a 72-hour endothelial cell proliferation assay. In addition the serum concentrations of the angiogenesis stimulators basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were determined using standard ELISA assays. RESULTS: The serum of renal cell carcinoma patients showed a median stimulation of human umbilical vein endothelial cells (HUVEC) of 89.79% (range 58.47-147.95%) and serum of healthy volunteers showed a median stimulation of 95.35% (range 74.64-141.77%) (p > 0.05). In contrast serum of patients with bladder carcinoma showed a median stimulation of 140.16% (range 64.82-200.16%) (p = 0.024). No correlations of the serum angiogenic activity and tumor stage or grade have been found in renal cell carcinoma patients. Furthermore, no correlations for serum bFGF and VEGF concentrations have been found. CONCLUSIONS: Serum angiogenic activity of patients with renal cell carcinoma did not differ significantly from healthy controls, while serum of patients with bladder carcinoma showed a significant increase in endothelial cell stimulation. Furthermore, bFGF and VEGF serum concentrations did not correlate to serum angiogenic activity in patients with renal cell carcinoma. Therefore, the determination of systemic angiogenic parameters, in case of renal cell carcinoma, might not lead to adequate data concerning prognosis or therapeutic effects.     

Comparison of Angiogenic Potential of Human Microvascular Endothelial Cells and Human Umbilical Vein Endothelial Cells

Summary BACKGROUND: Endothelial cells cultured in vitro are commonly used as a model for testing the effects of therapeutic or detrimental agents on endothelium. Cells originating from different vascular beds display, however, a heterogeneity of function and phenotype. Here we compared the production of angiogenic growth factors and the sensitivity to exogenous growth factor stimulation of two popular endothelial cell types. METHODS: Experiments were performed on human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) incubated in optimal conditions for 24–48 h. RESULTS: The profile of the spontaneously produced growth factors differed significantly between the two different cell lines tested. HUVEC did not produce detectable amounts of VEGF, whereas HMEC-1 released 24.9 pg/ml and this amount was significantly increased in response to IL-1. Instead, HUVEC produced high concentrations of soluble form of VEGF receptor-1 (VEGF-R1), whereas the release of VEGF-R1 from HMEC-1 was 10-fold lower. Small amounts of bFGF were found in media from both cell types, but higher levels were detected in HMEC-1 cultures. In contrast, the secretion of interleukin-8 (IL-8) and matrix metalloproteinase-1 (MMP-1) were 30- to 40-fold higher in HUVEC than in HMEC-1. The cell types differed also in their sensitivity to exogenous growth factors. The basal proliferation of HUVEC was very low but could be effectively stimulated by supplementation with VEGF or bFGF. HMEC-1 proliferated spontaneously and their proliferation rate was not further augmented by growth factors. Similarly, the spontaneous outgrowth of capillaries was negligible in HUVEC but well pronounced in HMEC-1. CONCLUSIONS: Production of angiogenic agents and sensitivity to exogenous growth factors is cell-type dependent. HUVEC, which do not release VEGF, can be easily stimulated with exogenous factors, whereas HMEC-1, which are able to produce VEGF, do not respond well to the additional stimulation. Our study demonstrates that conclusions resulting from in vitro experiments performed on only one type of endothelial cells can be misleading.     

VEGF is upregulated in a neuroblastoma and hepatocyte coculture model

BACKGROUND: We hypothesize that angiogenic factors are altered by the interaction between neuroblastoma cells and host tissues. MATERIALS AND METHODS: Human Chang hepatocytes and human neuroblastoma cells are cultured separately and in a noncontact, coculture system. Immunostaining for VEGF is performed on the cells. ELISA is used to detect vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and interleukin-8 in the conditioned media. Human umbilical vein endothelial cells (HUVEC) are cultured with standard medium (control) and hepatocyte, neuroblastoma, and coculture conditioned media. After 48 and 72 h, cells are counted to determine proliferation. Finally, VEGF-blocking antibody is added to the HUVEC cultures with the conditioned media. RESULTS: VEGF is markedly elevated in the coculture medium compared to the media from hepatocytes or neuroblastoma grown alone [412.2 +/- 52 vs 235 +/- 35 or 74.5 +/- 28.5 (pg/10(6) cells), P < 0.05]. Other growth factors are almost undetectable in any of the media. Immunostaining for VEGF in the cocultured hepatocytes is decreased by almost 50%, but VEGF immunostaining is increased fourfold in the cocultured neuroblastoma cells. A significant increase in cell proliferation is seen at both 48 and 72 h when HUVEC are cultured with the coculture media. Cell proliferation is blocked with the addition of anti-VEGF antibody. CONCLUSION: The interaction of neuroblastoma with hepatocytes results in an increased production of VEGF. It stimulates endothelial cell proliferation and may enhance the tumor's metastatic potential in an autocrine fashion.     

An organ culture system designed to study interaction of fetal rat calvaria with human head and neck squamous cell carcinoma

The development of permanent cell lines of human head and neck squamous cell carcinoma in culture has enabled these cell lines to be used to investigate the interaction of the tumor cells with bone. After the squamous carcinoma lines on fetal rat skulls were implanted the explants with their added tumor were maintained in long-term tissue culture by use of the procedures developed for growing these tumor cells. Results confirm direct interaction with the bone by the malignant cells. Specific surface and cytoplasmic markers have been demonstrated by use of monoclonal antibodies against the tumor cells. Furthermore, tumor angiogenesis without the addition of any endogenous endothelial components has been verified. Investigations into the degree of bone infiltration and susceptibility of these interacting tumor cells to various factors, radiation therapy, and chemotherapeutic agents have been carried out. The establishment of a model system for bony invasion by squamous cell carcinoma of the head and neck permits the investigation of the mechanism of tumor invasion and the study of various potential treatment modalities.     

Activin A Causes Cancer Cell Aggressiveness in Esophageal Squamous Cell Carcinoma Cells

BACKGROUND: Expression of activin A is associated with lymph node metastasis and clinical stage in esophageal cancer. METHODS: To clarify the aggressive behavior of tumors with high activin A expression, we used the beta subunit of activin A to establish stable activin betaA (Act-betaA)-transfected carcinoma cells in two human esophageal carcinoma cell lines, KYSE110 and KYSE140. The biological behavior of these cells was compared with that in mock-transfected cells from the same cell lines. We focused our attention on cell growth and tumorigenesis, and proliferation and apoptosis. RESULTS: Both Act-betaA-transfected carcinoma cell lines showed a higher growth rate than the mock-transfected carcinoma cells. In an in vitro invasion assay and a xenograft analysis, the Act-betaA-transfected carcinoma cells showed far higher proliferation in vitro and a higher potency for tumorigenesis in vivo, respectively. Moreover, in an analysis of apoptosis via Fas stimulation, the Act-betaA-transfected carcinoma cells showed a higher tolerance to apoptosis compared with the mock-transfected carcinoma cells. Moreover, anti-activin-neutralizing antibody-treated squamous cell cancer cell lines inhibited their migration. CONCLUSIONS: Collectively, these data indicate that continuous high expression of activin A in esophageal carcinoma cells is not related to tumor suppression, but rather to tumor progression in vitro and in vivo. The inhibition of activin might be one of the methods to attenuate tumor aggressiveness.     

MiR-197靶向JAK2调节人皮肤鳞状细胞癌细胞增殖侵袭能力的研究

目的:探讨miR-197通过调控JAK2对人皮肤鳞状细胞癌细胞A431增殖和侵袭的影响。方法:收集2018年7月-2019年5月于笔者医院经手术切除且被证实为皮肤鳞状细胞癌的组织标本以及对应的癌旁组织标本各40例,RT-qPCR检测miR-197在皮肤鳞状细胞癌组织、癌旁组织、人正常皮肤细胞系HaCaT、人皮肤鳞状细胞癌细胞株A431、A431-miR-197-siRNA和A431-miR-197-siRNA-NC中的表达情况;Western-Blot检测JAK2在各细胞株中的表达;生物信息学预测及双荧光素酶报告基因实验验证miR-197和JAK2的靶向关系;MTS法检测miR-197对A431增殖能力的影响;Transwell检测miR-197对皮肤鳞状细胞癌细胞侵袭能力的影响;IFA检测miR-197对上皮间质转化(EMT)相关分子N-cadherin表达的影响。结果:miR-197在皮肤鳞状细胞癌组织和皮肤鳞状细胞癌细胞株A431中的表达水平分别高于癌旁组织和人正常皮肤细胞系HaCaT,差异均具有统计学意义(P<0.05);与A431和A431-miR-197-siRNA-NC相比,稳定下调细胞系A431-miR-197-siRNA中miR-197、JAK2的表达水平均显著下降(P<0.05)。经Target Scan数据库分析发miR-197和JAK2有互补结合位点。下调miR-197能显著降低皮肤鳞状细胞癌细胞的增殖和侵袭能力,同时抑制N-cadherin的表达水平。结论:下调miR-197能够靶向抑制皮肤鳞状细胞癌中JAK2的表达和降低EMT相关分子N-cadherin的表达,进而抑制皮肤鳞状细胞癌细胞的EMT进程以及增殖侵袭能力。     

Cytokine modulation of endothelial cell sensitivity to photodynamic therapy

The purpose of this study was to determine if recombinant angiogenic cytokines modulate the sensitivity of endothelial cells to the toxic effects of chloroaluminum sulphonated phthalocyanine (AlSPc) photodynamic therapy (PDT). Bovine pulmonary artery endothelial cells in 24-well tissue culture plates were pretreated for 24 hr with AlSPc and either acidic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), tumor necrosis factor-α (TNF), interleukin-1-α (IL-1), or transforming growth factor-β (TGF) followed by argon-pumped dye laser. Endothelial cell damage was monitored with ⁵¹chromium release. FGF, TGF, and, to a lesser extent, IL-1, enhanced the PDT-mediated damage to endothelial cells, whereas PDGF and TNF did not significantly modulate toxicity. The enhanced endothelial cell damage was seemingly not related to rate of cell proliferation or amount of photoactive drug uptake by the EC. These results suggest that presence of tumor secreted cytokines may enhance PDT-mediated toxicity of tumor associated endothelial cells. © 1993 Wiley-Liss, Inc.     

UCN-01 (7-hydroxystaurosporine) induces apoptosis and G1 arrest of both primary and metastatic oral cancer cell lines in vitro.

OBJECTIVE: Our aim was to clarify the in vitro antiproliferative effects of UCN-01 on human oral squamous cell carcinoma (OSCC) cell lines. STUDY DESIGN: Cell growth was measured by MTT assay, and cell cycling was assessed by flow cytometry. Changes in the levels of protein and protein phosphorylation were analyzed by Western blotting. In addition, tumor cell apoptosis was assessed by propidium iodide (PI) and annexin double-staining. RESULTS: UCN-01 significantly inhibited the proliferation of all the OSCC cell lines, with a 50% inhibition concentration of about 300 nmol/L, and induced G1 arrest in these cell lines in a dose-dependent manner. Primary and metastatic oral cancer cell lines had different sensitivities to UCN-01. Our results showed that HSC-3 cells (primary-type OSCC) are less sensitive than LMF4 cells (metastatic-type OSCC) to UCN-01. In addition, the induction of p21 in OSCCs was found to be important for the suppression of tumor growth. CONCLUSION: The results of this study suggest that UCN-01 induces apoptosis and G1 arrest in OSCCs, albeit with different sensitivity of the primary and metastatic cell lines to UCN-01.     

A Novel Effect of β-Adrenergic Receptor on Mammary Branching Morphogenesis and its Possible Implications in Breast Cancer

Understanding the mechanisms that govern normal mammary gland development is crucial to the comprehension of breast cancer etiology. β-adrenergic receptors (β-AR) are targets of endogenous catecholamines such as epinephrine that have gained importance in the context of cancer biology. Differences in β₂-AR expression levels may be responsible for the effects of epinephrine on tumor vs non-tumorigenic breast cell lines, the latter expressing higher levels of β₂-AR. To study regulation of the breast cell phenotype by β₂-AR, we over-expressed β₂-AR in MCF-7 breast cancer cells and knocked-down the receptor in non-tumorigenic MCF-10A breast cells. In MCF-10A cells having knocked-down β₂-AR, epinephrine increased cell proliferation and migration, similar to the response by tumor cells. In contrast, in MCF-7 cells overexpressing the β₂-AR, epinephrine decreased cell proliferation and migration and increased adhesion, mimicking the response of the non-tumorigenic MCF-10A cells, thus underscoring that β₂-AR expression level is a key player in cell behavior. β-adrenergic stimulation with isoproterenol induced differentiation of breast cells growing in 3-dimension cell culture, and also the branching of murine mammary epithelium in vivo. Branching induced by isoproterenol was abolished in fulvestrant or tamoxifen-treated mice, demonstrating that the effect of β-adrenergic stimulation on branching is dependent on the estrogen receptor (ER). An ER-independent effect of isoproterenol on lumen architecture was nonetheless found. Isoproterenol significantly increased the expression of ERα, Ephrine-B1 and fibroblast growth factors in the mammary glands of mice, and in MCF-10A cells. In a poorly differentiated murine ductal carcinoma, isoproterenol also decreased tumor growth and induced tumor differentiation. This study highlights that catecholamines, through β-AR activation, seem to be involved in mammary gland development, inducing mature duct formation. Additionally, this differentiating effect could be resourceful in a breast tumor context.     

The influence of stromal cells on the MTT assay (I)—In vitro chemosensitivity of the tumor and stromal cells to mitomycin C—

To clarify the influence of stromal cells on the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay), a gastric carcinoma cell line (KATO-III) and a human fibroblast cell line (IMR-90) were subjected to a colorimetric assay, in which the chemosensitivity to mitomycin C was assessed in different mixtures of the cell lines. KATO-III was found to be highly sensitive to mitomycin C at 10 μg/ml, whereas IMR-90 was insensitive to mitomycin C at the same concentration. When the mixtures of these two cell lines were tested by the assay, a mixture of more than 25 per cent stromal cells reduced the sensitivity of KATO-III to mitomycin C. This suggested that the stromal cells in fresh surgical specimens might reduce the apparent sensitivity of the tumor cells.     

Low-level laser irradiation effect on endothelial cells under conditions of hyperglycemia

Diabetes mellitus is considered to be a very serious lifestyle disease leading to cardiovascular complications and impaired wound healing observed in the diabetic foot syndrome. Chronic hyperglycemia is the source of the endothelial activation. The inflammatory process in diabetes is associated with the secretion of inflammatory cytokines by endothelial cells, e.g., tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). The method of phototherapy using laser beam of low power (LLLT—low-level laser therapy) effectively supports the conventional treatment of diabetic vascular complications such as diabetic foot syndrome. The aim of our study was to evaluate the effect of low-power laser irradiation at two wavelengths (635 and 830 nm) on the secretion of inflammatory factors (TNF-α and IL-6) by the endothelial cell culture—HUVEC line (human umbilical vein endothelial cell)—under conditions of hyperglycemia. It is considered that adverse effects of hyperglycemia on vascular endothelial cells may be corrected by the action of LLLT, especially with the wavelength of 830 nm. It leads to the reduction of TNF-α concentration in the supernatant and enhancement of cell proliferation. Endothelial cells play an important role in the pathogenesis of diabetes; however, a small number of studies evaluate an impact of LLLT on these cells under conditions of hyperglycemia. Further work on this subject is warranted.     

Platelet-derived growth factor and epidermal growth factor play a major role in human colonic fibroblast repair activities

BACKGROUND AND AIMS: Ulceration is a common feature of inflammatory bowel diseases, where subepithelial cell growth is frequently necessary for resolution. In order to further understand the role of colonic fibroblasts in this process, we have used an in vitro model of wound repair to study the response of human colonic fibroblasts to several growth factors expressed in colonic tissues. METHODS: Proliferation was determined by [(3)H]thymidine incorporation into DNA in subconfluent fibroblast cultures. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of growth factors secreted by fibroblasts was studied in conditioned medium by heparin affinity chromatography and immunodetection with specific antibodies. RESULTS: Serum and platelet-derived growth factor (PDGF-BB) induced a dramatic increase in both colonic fibroblast proliferation and closure of wounded cell monolayers. Epidermal growth factor (EGF) stimulated both fibroblast activities, but the effect was less potent. However, colonic fibroblasts did not respond to transforming growth factor-beta(1). Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was reverted by the addition of suramin. Furthermore, a PDGF-like factor was isolated from colonic fibroblast-conditioned medium. CONCLUSIONS: EGF and PDGF-BB promote human colonic fibroblast-dependent wound repair activities. Human colonic fibroblasts may exert an autocrine regulation via the production of growth factors.     

The influence of stromal cells on the MTT assay (I)--In vitro chemosensitivity of the tumor and stromal cells to mitomycin C

To clarify the influence of stromal cells on the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay), a gastric carcinoma cell line (KATO-III) and a human fibroblast cell line (IMR-90) were subjected to a colorimetric assay, in which the chemosensitivity KATO-III was found to be highly sensitive to mitomycin C at 10 micrograms/ml, whereas IMR-90 was insensitive to mitomycin C at the same concentration. When the mixtures of these two cell lines were tested by the assay, a mixture of more than 25 per cent stromal cells reduced the sensitivity of KATO-III to mitomycin C. This suggested that the stromal cells in fresh surgical specimens might reduce the apparent sensitivity of the tumor cells.     

Renal cell carcinoma associated with prominent angioleiomyoma-like proliferation: Report of 5 cases and review of the literature

Five cases of renal cell carcinoma of clear cell type are presented, Fuhrmann's grade 2, associated with a peculiar stromal proliferation having angioleiomyoma-like features. This proliferation was particularly prominent at the interphase between the tumor edge and the surrounding normal tissues, in which it acquired the configuration of a tumor capsule. Four similar cases were taken from the literature. We postulate that this angioleiomyoma-like change is a tumor epiphenomenon and that it represents yet another manifestation of the well-documented capacity of renal cell carcinoma to induce vascular proliferation, probably through the secretion of angiogenic and other growth factors by the tumor cells.     

Radioresistant tumor cell lines derived from head and neck radiation failures

We studied the in vitro radiobiological parameters of 16 human head and neck squamous cell carcinoma tumor cell lines cultured from patients who suffered local failure after a curative course of radiotherapy. The radiobiological parameters determined included D0, n, and D. When compared with in vitro radiobiological parameters of tumor cells cultured from head and neck cancer patients prior to radiotherapy, human sarcoma cell lines, and normal human diploid fibroblasts studied in our laboratory (as well as other human tumor cell lines reported in the literature), tumor cells derived from radiotherapy failures on average are resistant to the cytotoxic effects of ionizing radiation.     

改良MTT法检测胃癌乳腺癌体外药物敏感性

采用改良的MTT法对53例胃癌、乳腺癌进行体外药敏试验。结果表明，改良的MTT法，即多次贴壁分离癌组织中夹杂的成纤维细胞，且细胞悬液培养过夜后又能排出污染的标本，因此提高了体外药敏的准确性。改良的MTT法优点有：（1）获得了较纯化的癌细胞；（2）培养过夜能使受损伤的细胞恢复活性，增加癌细胞含量，提高实验成功率。     

Interferon sensitivity of fibroblast cell cultures derived from patients with neoplasms of the head and neck

Interferon (IFN) is a protein with antiviral activity that has been shown to inhibit the growth of many different types of cells. We have measured the IFN sensitivity of nine cell cultures isolated from patients with squamous cell carcinoma and one with malignant melanoma of the head and neck. Normal-appearing fibroblast cultures isolated from these tissues appear quite sensitive to the antiviral effects of IFN. When the encephalomyocarditis virus yield reduction assay is used, these diploid cells are as sensitive to IFN-alpha as are newborn foreskin fibroblast cultures. A similar antiviral effect is seen with IFN-gamma. These cells are relatively insensitive to the antigrowth effect of both IFN preparations as measured by 3H thymidine incorporation and direct observations of cell growth. This is the same relative sensitivity as fibroblasts derived from normal patients. Since these cells are at least 100 times less sensitive to the antigrowth action of the IFNs, it appears unlikely that the IFNs play a significant role in the control of normal fibroblast growth, in contrast to the sensitivity of malignant cell lines.     

Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo

BACKGROUND/PURPOSE: Preventing tumors from forming new blood vessels appears to be an effective new anticancer approach. Antiangiogenic therapy usually is cytostatic, however, and, therefore, long-term angiogenesis inhibition is likely to be required. The objective of this study was to determine if sustained gene therapy-mediated expression of these agents from tumor cells could restrict tumor growth in vivo. METHODS: Two replication-defective retroviral vectors were made, one encoding both the soluble, truncated vascular endothelial growth factor receptor (VEGF-R2), flk-1, together with green fluorescent protein (GFP), and the other encoding GFP alone. These vectors were then used to transduce murine neuroblastoma cells (NXS2). Stable, high expression of the flk-1 transgene was confirmed in the former population of cells by Western analysis. Flk-1 protein was isolated from cell culture supernatants and tested in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays to confirm that functional protein was being made. Finally, in vivo activity was assessed by injecting 10(6) tumor cells subcutaneously into SCID mice and monitoring subsequent tumor growth. RESULTS: Purified flk-1 (0.1 micromol/L) was able to inhibit basic fibroblast growth factor (bFGF) stimulated HUVEC proliferation by 44% and VEGF-stimulated migration by 30%. In vitro growth rates for the transduced cell lines were similar to the unmodified cell line. In vivo, however, after 23 days, tumors from flk-1 expressing neuroblastoma cells were less than 33% the average volume of tumors from cells expressing only the GFP transgene (mean volume, 1.9 cm(3) v 5.8 cm(3), P<.001). GFP expression alone had no effect on tumor growth when compared with unmodified tumor cells. CONCLUSIONS: Engineered expression of flk-1, a competitive inhibitor of VEGF, by tumor cells results in the production of an inhibitor of endothelial cell proliferation and migration that greatly restricts the growth of the tumor cells in vivo. Gene therapy-mediated delivery of angiogenesis inhibitors may provide an alternative approach to treating refractory tumors such as neuroblastoma.