血管内皮生长因子及其受体与骨肉瘤关系的研究进展

血管内皮生长因子（ＶＥＧＦ）是一种序列高度保守、高度特异性的促血管内皮细胞生长因子,广泛分布于人和动物体内的大脑、肾脏、肝脏、脾脏、胰腺和骨骼等组织中,对内皮细胞具有强烈的促有丝分裂作用,刺激血管内皮细胞增殖和血管通透性增加,促进新生血管形成。ＶＥＧＦ通过与血管内皮细胞表面受体（ＶＥＧＦＲ）特异性结合发挥生物学效应。抑制ＶＥＧＦ及ＶＥＧＦＲ的活性可以减缓或阻滞骨肉瘤侵袭和转移。研究表明,ＶＥＧＦ及ＶＥＧＦＲ对肿瘤血管及淋巴管的生成及肿瘤侵袭和转移起重要作用。本文对ＶＥＧＦ及ＶＥＧＦＲ与骨肉瘤血管与淋巴管生成及其侵袭与转移的关系作一综述。     

Bronchial washing levels of vascular endothelial growth factor receptor-2 (VEGFR2) correlate with overall survival in NSCLC patients

The aim of this study was to define pre-treatment VEGF, VEGFR1 and VEGFR2 levels in serum and bronchial washing samples of NSCLC patients in order to examine their correlation to survival. Forty patients with histologically confirmed NSCLC were enrolled. The results indicated that circulating VEGF was correlated to T-classification, as were the ratios of VEGF/VEGFR2 in serum and washing. Best chemotherapy response was observed at lower serum and washing VEGF concentrations. Higher VEGF levels in washing were associated with worse overall survival and progression-free survival. Similar were the results at high values of VEGF/VEGFR2 ratio in washing. Multivariate analysis revealed VEGFR2 levels in serum and washing as independent markers for overall survival. In conclusion, washing VEGFR2 levels are correlated to overall survival, whereas serum and washing VEGF levels are predictive of chemotherapy response. These could help recognize NSCLC patients who benefit from an aggressive therapeutic approach.     

The clinical toxicity profile of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors; a review

Clinical experience with vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors is rapidly increasing, and some compounds have already been approved for regular anticancer treatment.Apart from their activity, much attention has been focussed on the clinical toxicity profile of these compounds.This review describes the most frequently occurring side-effects of both antibodies and tyrosine kinase inhibitors and discusses some of the underlying mechanisms. Some practical guidelines for treatment of the side-effects are given.     

Immunodetection and quantification of vascular endothelial growth factor receptor-3 in human malignant tumor tissues

Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme-linked immunosorbent assay (ELISA) for the quantification of VEGFR-3 in different human cell and tissue lysates. A combination of the goat polyclonal anti-VEGFR-3 antibody and the mouse monoclonal anti-human VEGFR-3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2-25 ng/ml. The assay was specific for VEGFR-3, with no cross-reactivity to VEGFR-1 or VEGFR-2. Complex formation with VEGF-C and VEGF-D had no effect on the sensitivity of the assay. The VEGFR-3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14-fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR-3 were in the range of 0.6-16.7 ng/mg protein. Importantly, the level of VEGFR-3 protein detected in the ELISA correlated significantly with the number of VEGFR-3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR-3-positive vessel density. The protein levels of VEGFR-3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF-C (p<0.001), or biological active, free VEGF-A (p<0.0001), but not with VEGFR-1 or total VEGF-A. This assay provides a useful tool for the investigations of the expression levels of VEGFR-3 in physiological and pathological processes, particular in cancer and in lymphangiogenesis-related disease.     

Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) regulate colon cancer growth and metastasis. Previous studies utilizing antibodies against the VEGF receptor (DC101) or EGF receptor (C225) have demonstrated independently that these agents can inhibit tumour growth and induce apoptosis in colon cancer in in vivo and in vitro systems. We hypothesized that simultaneous blockade of the VEGF and EGF receptors would enhance the therapy of colon cancer in a mouse model of peritoneal carcinomatosis. Nude mice were given intraperitoneal injection of KM12L4 human colon cancer cells to generate peritoneal metastases. Mice were then randomized into one of four treatment groups: control, anti-VEGFR (DC101), anti-EGFR (C225), or DC101 and C225. Relative to the control group, treatment with DC101 or with DC101+C225 decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells. Although C225 therapy did not change any of the above parameters, C225 combined with DC101 led to a significant decrease in tumour vascularity and increases in tumour cell and endothelial cell apoptosis (vs the DC101 group). These findings suggest that DC101 inhibits angiogenesis, endothelial cell survival, and VEGF-mediated ascites formation in a murine model of colon cancer carcinomatosis. The addition of C225 to DC101 appears to lead to a further decrease in angiogenesis and ascites formation. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon peritoneal carcinomatosis.     

The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma

Vascular endothelial growth factor receptor 1 (VEGFR-1) is present on endothelial cells and subsets of human tumor cells, raising the hypothesis that angiogenic factors may promote tumor growth both by inducing angiogenesis and directly signaling through activation of VEGFR-1 on tumor cells. Here, we report that VEGFR-1 is expressed on a panel of 16 human breast tumor cell lines, and the vasculature and the tumor cell compartment of a subset of breast carcinoma lesions, and that selective signaling through VEGFR-1 on breast cancer cells supports tumor growth through downstream activation of the p44/42 mitogen-activated protein kinase (MAPK) or Akt pathways. Ligand-stimulated proliferation of breast tumor cells was inhibited by specific blockade with an anti-VEGFR-1 neutralizing monoclonal antibody. Treatment with anti-VEGFR-1 mAb significantly suppressed the growth of DU4475, MCF-7, BT-474 and MDA-MB-231 breast xenografts in athymic mice. Histological examination of anti-VEGFR-1 mAb treated tumor xenografts showed a significant reduction of activation of the p44/42 MAPK or Akt pathways in tumor cells resulting in an increase in tumor cell apoptosis. Importantly, cotreatment with mAbs targeting human VEGFR-1 on tumor cells and murine VEGFR-1 on vasculature led to more potent growth inhibition of breast tumor xenografts. The results suggest that VEGF receptors may not only modulate angiogenesis, but also directly influence the growth of VEGF receptor expressing tumors.     

A human IgG-like bispecific antibody co-targeting epidermal growth factor receptor and the vascular endothelial growth factor receptor 2 for enhanced antitumor activity

Both Epidermal Growth Factor Receptor (EGFR) and the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) play critical roles in tumorigenesis. We hypothesized co-targeting EGFR and VEGFR2 using a bispecific antibody might have significant therapeutic potential. Here,we designed and produced a human IgG-like bispecific antibody (Bi-Ab) based on the variable regions of cetuximab (an anti-EGFR antibody) and mAb-04 (an anti-VEGFR2 antibody developed in our lab) . The Bi-Ab was found to inhibit the proliferation, survival and invasion of cancer cells via ablating phosphorylation of receptor and downstream signaling. In vivo efficacy was demonstrated against established HT-29 and SKOV-3 xenografts grown in nude mice. Studies revealed our Bi-Ab was able to restrain xenografted tumor growth and prolong survival of mice through inhibiting cell proliferation,promoting apoptosis and anti-angiogenesis. In contrast to cetuximab or mAb-04 alone, our Bi-Ab exhibits enhanced antitumor activity and has equal or slightly superior activity to their combination (Combi). It shows promise as a therapeutic agent, especially for use against tumors EGFR and/or VEGFR2 over-expressing malignancies.     

表达VEGFR-2的减毒鼠伤寒沙门氏菌疫苗诱导特异性抗胶质瘤血管免疫应答

背景与目的:血管内皮生长因子(vascular endothelialgrowth factor,VEGF)及其主要受体血管内皮生长因子受体-2(vascular endothelialgrowth factorreceptor-2,VEGFR-2)在肿瘤新生血管和肿瘤基质形成过程中起着重要作用。本研究的目的是观察表达鼠VEGFR-2的重组减毒沙门氏疫苗菌诱导的抗血管特异性免疫应答及抗胶质瘤作用。方法:构建真核表达载体pcDNA3.1-VEGFR2,通过电转化法将pcDNA3.1-VEGFR2导入减毒鼠伤寒沙门氏菌SL7207中,经由胃管饲予C57BL/6J小鼠,对小鼠进行基因免疫。采用ELISA法检测免疫小鼠血清中特异性抗VEGFR2-IgG抗体,分离免疫小鼠的脾细胞,分析重组疫苗菌免疫后小鼠体内的特异性细胞毒性T细胞(cytotoxic T lym phocyte,CTL)应答。用携带pcDNA3.1-VEGFR2的重组沙门氏菌免疫治疗胶质瘤荷瘤小鼠,通过测量荷瘤小鼠肿瘤大小,检测肿瘤微血管密度及肿瘤细胞凋亡,评价重组疫苗菌的抗血管及肿瘤生长抑制作用。结果:重组疫苗菌免疫后小鼠产生了高水平的抗VEGFR2-IgG抗体,诱导小鼠脾淋巴细胞产生针对VEGFR2的特异性CTL活性。重组疫苗菌的免疫能够明显抑制胶质瘤的生长。NaH CO3对照组、载体对照组、重组疫苗菌组的平均微血管密度分别为26.5±5.8、27.2±4.5、8.8±1.9,平均凋亡细胞数分别为4.41.2、3.     

RNA干扰VEGFR-2表达对Calu-1细胞增殖、迁移、侵袭力及放射效应影响

目的 研究VEGFR-2对肺癌细胞系Calu-1细胞增殖、迁移、侵袭及联合放射后凋亡率影响, 并探讨其可能机制。方法 siRNA敲低Calu-1细胞中的VEGFR-2基因, 借助实时荧光定量PCR和蛋白印迹法检测VEGFR-2表达水平的变化;将细胞分为对照组、VEGF组、VEGFR-2基因敲低组和基因敲低加VEGF组。利用CCK8法、细胞划痕实验及Transwell实验分别检测细胞增殖、迁移和侵袭能力变化, 利用蛋白印迹法检测VEGFR-2及下游相关信号通路蛋白表达水平变化;各组细胞联合放射后, 检测细胞凋亡。结果 RNA干扰VEGFR-2后Calu-1细胞中VEGFR-2的mRNA水平和蛋白水平均降低(P=0.001、0.000);RNA干扰VEGFR-2后Calu-1细胞的增殖、迁移、侵袭能力均降低(P=0.000、0.000、0.031);RNA干扰VEGFR-2后Calu-1细胞中Akt、ERK1/2、p38的蛋白磷酸化水平均降低(P=0.336、0.986、0.553);RNA干扰VEGFR-2后联合放射后Calu-1细胞的凋亡率增加(P=0.012), RNA干扰VEGFR-2后HIF-1α蛋白表达被抑制(P=0.016)。结论 VEGFR-2基因表达敲低后显著抑制了Calu-1细胞的多项生理功能, 并提高了放射后细胞凋亡率。     

Tumor oxygenation in hormone-dependent tumors during vascular endothelial growth factor receptor-2 blockade, hormone ablation, and chemotherapy

Tumor oxygenation is critical for tumor survival as well as for response to therapy, e.g., radiation therapy. Hormone ablation therapy in certain hormone-dependent tumors and antiangiogenic therapy lead to vessel regression and have also shown beneficial effects when combined with radiation therapy. These findings are counterintuitive because vessel regression should reduce oxygen tension (pO2) in tumors, decreasing the effectiveness of radiotherapy. Here we report on the dynamics of pO2 and oxygen consumption in a hormone-dependent tumor following hormone ablation and during treatment with an anti-VEGFR-2 monoclonal antibody (mAb) or a combination of doxorubicin and cyclophosphamide; the latter combination is not known to cause vessel regression at doses used clinically. Androgen-dependent male mouse mammary carcinoma (Shionogi) was implanted into transparent dorsal skin-fold chambers in male severe combined immunodeficient mice. Thirteen days after the tumors were implanted, mice were treated with antiangiogenic therapy (anti-VEGFR-2 mAb, 1.4 mg/30 g body weight), hormone ablation by castration, or doxorubicin (6.5 mg/kg every 7 days) and cyclophosphamide (100 mg/kg every 7 days). A non-invasive in vivo method was used to measure pO2 profiles and to calculate oxygen consumption rates (Q(O2)) in tumors. Tumors treated with anti-VEGFR-2 mAb exhibited vessel regression and became hypoxic. Initial vessel regression was followed by a "second wave" of angiogenesis and increases in both pO2 and Q(O2). Hormone ablation led to tumor regression followed by an increase in pO2 coincident with regrowth. Chemotherapy led to tumor growth arrest characterized by constant Q(O2) and elevated pO2. The increased pO2 during anti-VEGFR-2 mAb and hormone ablation therapy may explain the observed beneficial effects of combining antiangiogenic or hormone therapies with radiation treatment. Thus, understanding the microenvironmental dynamics is critical for optimal scheduling of these treatment modalities.     

Preclinical characterization of anlotinib,a highly potent and selective vascular endothelial growth factor receptor‐2 inhibitor

Abrogating tumor angiogenesis by inhibiting vascular endothelial growth factor receptor‐2 (VEGFR2) has been established as a therapeutic strategy for treating cancer. However, because of their low selectivity, most small molecule inhibitors of VEGFR2 tyrosine kinase show unexpected adverse effects and limited anticancer efficacy. In the present study, we detailed the pharmacological properties of anlotinib, a highly potent and selective VEGFR2 inhibitor, in preclinical models. Anlotinib occupied the ATP‐binding pocket of VEGFR2 tyrosine kinase and showed high selectivity and inhibitory potency (IC₅₀ <1 nmol/L) for VEGFR2 relative to other tyrosine kinases. Concordant with this activity, anlotinib inhibited VEGF‐induced signaling and cell proliferation in HUVEC with picomolar IC₅₀ values. However, micromolar concentrations of anlotinib were required to inhibit tumor cell proliferation directly in vitro. Anlotinib significantly inhibited HUVEC migration and tube formation; it also inhibited microvessel growth from explants of rat aorta in vitro and decreased vascular density in tumor tissue in vivo. Compared with the well‐known tyrosine kinase inhibitor sunitinib, once‐daily oral dose of anlotinib showed broader and stronger in vivo antitumor efficacy and, in some models, caused tumor regression in nude mice. Collectively, these results indicate that anlotinib is a well‐tolerated, orally active VEGFR2 inhibitor that targets angiogenesis in tumor growth, and support ongoing clinical evaluation of anlotinib for a variety of malignancies.     

Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor‐associated endothelial cells, where it modulates tumor‐promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti‐VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti‐VEGFR2‐AF, which shows excellent VEGFR2 binding activity. Anti‐VEGFR2‐AF bound Ig‐like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF‐A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti‐VEGFR2‐AF inhibited capillary structure formation and exerted Ab‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC‐3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC‐3 xenograft mouse model, treatment with anti‐VEGFR2‐AF repressed tumor growth and angiogenesis as effectively and safely as US FDA‐approved anti‐VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti‐VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL‐60 human leukemia‐xenografted mice, anti‐VEGFR2‐AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti‐VEGFR2‐AF has strong potential as a cancer therapy that could directly target VEGFR2‐expressing tumor cells in addition to its anti‐angiogenic action.     

Angiogenesis inhibition by vascular endothelial growth factor receptor-2 blockade reduces stromal matrix metalloproteinase expression, normalizes stromal tissue, and reverts epithelial tumor phenotype in surface heterotransplants

Inhibition of vascular endothelial growth factor (VEGF) signaling, a key regulator of tumor angiogenesis, through blockade of VEGF receptor (VEGFR)-2 by the monoclonal antibody DC101 inhibits angiogenesis, tumor growth, and invasion. In a surface xenotransplant assay on nude mice using a high-grade malignant squamous cell carcinoma cell line (A-5RT3), we show that DC101 causes vessel regression and normalization as well as stromal maturation resulting in a reversion to a noninvasive tumor phenotype. Vessel regression is followed by down-regulation of expression of both VEGFR-2 and VEGFR-1 on endothelial cells and increased association of alpha-smooth muscle actin-positive cells with small vessels indicating their normalization, which was further supported by a regular ultrastructure. The phenotypic regression of an invasive carcinoma to a well-demarcated dysplastic squamous epithelium is accentuated by the establishment of a clearly structured epithelial basement membrane and the accumulation of collagen bundles in the stabilized connective tissue. This normalization of the tumor-stroma border coincided with down-regulated expression of the stromal matrix metalloproteinases 9 and 13, which supposedly resulted in attenuated turnover of extracellular matrix components permitting their structural organization. Thus, in this mouse model of a human squamous cell carcinoma cell line, blockade of VEGF signaling resulted in the reversion of the epithelial tumor phenotype through stromal normalization, further substantiating the crucial role of stromal microenvironment in regulating the tumor phenotype.     

Selection and their antitumor activity of antisense oligonucleotides targeting messenger RNA of vascular endothelial growth factor receptor 2

Objective： To select the antisense oligonucleotides （asONs） which hybridize with the mRNA of vascular endothelial growth factor receptor2 （VEGFR2, also named as kinase insert domain-containing receptor：KDR） in an effective and specific way, and to investigate their antitumor activity in MCF-7 cells. Methods： The effective antisense oligonucleotides were chosen by computer prediction combined with oligonucleotide microarrays. The inhibition effect on MCF-7 cells proliferation was measured by MTT; and VEGFR2 expression was surveyed by Western-blotting and RT- PCR. Results： Using predicting secondary structure of VEGFR2 mRNA with RNA folding program, computer prediction designed 30 antisense oligonucleotide probes that were directed to local loose regions of RNA structure. In 30 probes, 4（4/30, 13.33%） antisense oligonucleotides showed strong hybridization intensities in oligonucleotide microarrays test and were selected. All these antisense oligonucleotides targeting 4 different sites of VEGFR2 mRNA lowered the level of VEGFR2 mRNA and protein present in MCF-7 cells. Proliferation of MCF-7 cells was reduced by 4 antisense oligonucleotides, respectively, in which asON1 was the most effective, with the inhibitory rates being 53.06% at 0.8 I.tmol/L. Conclusion： Combination of computer prediction with oligonucleotide microarrays is an effective way in selecting optimal antisense oligonucleotides. The antisense oligonucleotides showed good correlation between their antitumor activity and the hybridization intensities. The antisense oligonucleotides targeting VEGFR2 mRNA demonstrated prominent antitumor role in vitro.     

术前化疗对乳腺癌组织VEGF表达和血管生成的影响

目的研究术前化疗对乳腺癌血管内皮生长因子(VEGF)表达、肿瘤组织微血管密度的影响.方法36例术前化疗的乳腺癌和32例未做术前化疗的乳腺癌标本,进行VEGF、CD34免疫组织化学标记,并进行微血管计数和统计学处理.结果术前化疗组VEGF阳性率为47.22%(17/36),对照组53.13%(17/32),微血管计数化疗组和对照组分别为23.67±13.45,26.12±11.32,两者差异均无显著性.化疗组VEGF阳性和阴性的标本,MVC均值差异无显著性;对照组VEGF阳性和阴性的标本,MVC均值差异有显著性.结论常规术前化疗并未显示出明显的血管生成抑制作用,也未显示出明显的血管内皮生长因子表达的改变.     

Reduced serum vascular endothelial growth factor receptor-2 (sVEGFR-2) and sVEGFR-1 levels in gastric cancer patients

The relationship between gastric cancer and serum vascular endothelial growth factor receptor-1 (sVEGFR-1) and sVEGFR-2, which are soluble form receptor proteins of vascular endothelial growth factor (VEGF), has not been extensively studied. VEGF, sVEGFR-1 and sVEGFR-2 were measured in the sera obtained before surgical operation from 164 gastric cancer patients and from 164 healthy controls matched for age and gender. Compared with controls, the cases showed elevated VEGF (P < 0.01) and reduced sVEGFR-1 (P = 0.07) and sVEGFR-2 (P = 0.02). The difference in VEGF levels was small among men and when the outcome was early cancer. The difference in sVEGFR-1 levels was significant or borderline significant only in men and when the outcome was diffuse type cancer. The difference in sVEGFR-2 levels was significant only in men and when the outcome was advanced or diffuse type cancer. The sensitivities and specificities of VEGF, sVEGFR-1 and sVEGFR-2 were all approximately 60%. For diffuse type cancer, sVEGFR-2 showed a sensitivity of 62.4% and a specificity of 63.4%, which was similar to serum pepsinogen. In conclusion, elevated VEGF and reduced sVEGFR-1 and sVEGFR-2 in serum are characteristic of gastric cancer patients, and the value of serum sVEGFR-2 in the diagnosis of diffuse type gastric cancer should be further evaluated.     

Novel hKDR mouse model depicts the antiangiogenesis and apoptosis-promoting effects of neutralizing antibodies targeting vascular endothelial growth factor receptor 2

Vascular endothelial growth factor receptor 2 (VEGFR2)/KDR plays a critical role in tumor growth, diffusion, and invasion. The amino acid sequence homology of KDR between mouse and human in the VEGF ligand-binding domain was low, thus the WT mice could not be used to evaluate Abs against human KDR, and the lack of a suitable mouse model hindered both basic research and drug developments. Using the CRISPR/Cas9 technique, we successfully inserted different fragments of the human KDR coding sequence into the chromosomal mouse Kdr exon 4 locus to obtain an hKDR humanized mouse that can be used to evaluate the marketed Ab ramucirumab. In addition, the humanized mAb VEGFR-HK19 was developed, and a series of comparative assays with ramucirumab as the benchmark revealed that VEGFR-HK19 has higher affinity and superior antiproliferation activity. Moreover, VEGFR-HK19 selectively inhibited tumor growth in the hKDR mouse model but not in WT mice. The most important binding epitopes of VEGFR2-HK19 are D257, L313, and T315, located in the VEGF binding region. Therefore, the VEGFR2-HK19 Ab inhibits tumor growth by blocking VEGF-induced angiogenesis, inflammation, and promoting apoptosis. To our best knowledge, this novel humanized KDR mouse fills the gaps both in an animal model and the suitable in vivo evaluation method for developing antiangiogenesis therapies in the future, and the newly established humanized Ab is expected to be a drug candidate possibly benefitting tumor patients.     

Bevacizumab‐enhanced antitumor effect of 5‐fluorouracil via upregulation of thymidine phosphorylase through vascular endothelial growth factor A/vascular endothelial growth factor receptor 2‐specificity protein 1 pathway

Bevacizumab (Bv) can be used synergistically with fluoropyrimidine‐based chemotherapy to treat colorectal cancer. Whether and how it affects the delivery of fluoropyrimidine drugs is unknown. The present study aimed to explore the effect of Bv on the delivery of 5‐fluorouracil (5‐FU) to tumors and the underlying mechanism from metabolic perspective. Bv enhanced the anti‐tumor effects of 5‐FU in LoVo colon cancer xenograft mice and increased the 5‐FU concentration in tumors without affecting hepatic 5‐FU metabolism. Interestingly, Bv remarkably upregulated thymidine phosphorylase (TP) in tumors, which mediated the metabolic activation of 5‐FU. Although TP is reported to promote angiogenesis and resistance, the combination of Bv and 5‐FU resulted in anti‐angiogenesis and vessel normalization in tumors, indicating that the elevated TP mainly contributed to the enhanced response to 5‐FU. Bv also induced TP upregulation in LoVo cancer cells. Treatment with vascular endothelial growth factor receptor 2 (VEGFR2) antagonist apatinib and VEGFR2 silencing further confirmed TP upregulation. Bv and apatinib both enhanced the cytotoxicity of 5‐FU in LoVo cells, but there was no synergism with adriamycin and paclitaxel. We further demonstrated that the effect of Bv was dependent on VEGFR2 blockade and specificity protein 1 activation via MDM2 inhibition. In summary, Bv enhanced the accumulation of 5‐FU in tumors and the cytotoxicity of 5‐FU via TP upregulation. We provide data to better understand how Bv synergizes with 5‐FU from metabolic perspective, and it may give clues to the superiority of Bv in combination with fluoropyrimidine drugs compared to other chemotherapeutic drugs in colon cancer.