Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5

Angiogenesis and signaling through the RAF/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK cascade have been reported to play important roles in the development of hepatocellular carcinomas (HCC). Sorafenib (BAY 43-9006, Nexavar) is a multikinase inhibitor with activity against Raf kinase and several receptor tyrosine kinases, including vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), FLT3, Ret, and c-Kit. In this study, we investigated the in vitro effects of sorafenib on PLC/PRF/5 and HepG2 HCC cells and the in vivo antitumor efficacy and mechanism of action on PLC/PRF/5 human tumor xenografts in severe combined immunodeficient mice. Sorafenib inhibited the phosphorylation of MEK and ERK and down-regulated cyclin D1 levels in these two cell lines. Sorafenib also reduced the phosphorylation level of eIF4E and down-regulated the antiapoptotic protein Mcl-1 in a MEK/ERK-independent manner. Consistent with the effects on both MEK/ERK-dependent and MEK/ERK-independent signaling pathways, sorafenib inhibited proliferation and induced apoptosis in both HCC cell lines. In the PLC/PRF/5 xenograft model, sorafenib tosylate dosed at 10 mg/kg inhibited tumor growth by 49%. At 30 mg/kg, sorafenib tosylate produced complete tumor growth inhibition. A dose of 100 mg/kg produced partial tumor regressions in 50% of the mice. In mechanism of action studies, sorafenib inhibited the phosphorylation of both ERK and eIF4E, reduced the microvessel area (assessed by CD34 immunohistochemistry), and induced tumor cell apoptosis (assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling) in PLC/PRF/5 tumor xenografts. These results suggest that the antitumor activity of sorafenib in HCC models may be attributed to inhibition of tumor angiogenesis (VEGFR and PDGFR) and direct effects on tumor cell proliferation/survival (Raf kinase signaling-dependent and signaling-independent mechanisms).     

The antitumor effect of a novel angiogenesis inhibitor (an octahydronaphthalene derivative) targeting both VEGF receptor and NF-κB pathway

Development of a novel type of angiogenesis inhibitor will be essential for further improvement of therapeutics against cancer patients. We examined whether an octahydronaphthalene derivative, AMF-26, which was screened as an inhibitor of intercellular adhesion molecule-1 (ICAM-1) production stimulated by inflammatory stimuli in vascular endothelial cells, could block angiogenesis in response to vascular endothelial growth factor (VEGF) and/or inflammatory cytokines. Low dose AMF-26 effectively inhibited the tumor necrosis factor-α (TNF-α)- or the interleukin-1β (IL-1β)-induced production of ICAM-1 in human umbilical vascular endothelial cells (HUVECs). We found that the TNF-α-induced phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and nuclear translocation of p65 were impaired by AMF-26 in both endothelial cells and cancer cells. AMF-26 was found to inhibit the phosphorylation of VEGF receptor 1 (VEGFR1), VEGFR2 and the downstream signaling molecules Akt, extracellular signal-regulated kinase (ERK)1/2 stimulated by VEGF in HUVECs. Therefore, the VEGF-induced proliferation, migration and tube formation of vascular endothelial cells was highly susceptible to inhibition by AMF-26. Oral administration of AMF-26 significantly blocked VEGF- or IL-1β-induced angiogenesis in the mouse cornea, and also tumor angiogenesis and growth. Together, our results indicate that AMF-26 inhibits angiogenesis through suppression of both VEGFR1/2 and nuclear factor-κB (NF-κB) signaling pathways when stimulated by VEGF or inflammatory cytokines. AMF-26 could be a promising novel candidate drug for cancer treatments.     

BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis

The RAS/RAF signaling pathway is an important mediator of tumor cell proliferation and angiogenesis. The novel bi-aryl urea BAY 43-9006 is a potent inhibitor of Raf-1, a member of the RAF/MEK/ERK signaling pathway. Additional characterization showed that BAY 43-9006 suppresses both wild-type and V599E mutant BRAF activity in vitro. In addition, BAY 43-9006 demonstrated significant activity against several receptor tyrosine kinases involved in neovascularization and tumor progression, including vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, platelet-derived growth factor receptor beta, Flt-3, and c-KIT. In cellular mechanistic assays, BAY 43-9006 demonstrated inhibition of the mitogen-activated protein kinase pathway in colon, pancreatic, and breast tumor cell lines expressing mutant KRAS or wild-type or mutant BRAF, whereas non-small-cell lung cancer cell lines expressing mutant KRAS were insensitive to inhibition of the mitogen-activated protein kinase pathway by BAY 43-9006. Potent inhibition of VEGFR-2, platelet-derived growth factor receptor beta, and VEGFR-3 cellular receptor autophosphorylation was also observed for BAY 43-9006. Once daily oral dosing of BAY 43-9006 demonstrated broad-spectrum antitumor activity in colon, breast, and non-small-cell lung cancer xenograft models. Immunohistochemistry demonstrated a close association between inhibition of tumor growth and inhibition of the extracellular signal-regulated kinases (ERKs) 1/2 phosphorylation in two of three xenograft models examined, consistent with inhibition of the RAF/MEK/ERK pathway in some but not all models. Additional analyses of microvessel density and microvessel area in the same tumor sections using antimurine CD31 antibodies demonstrated significant inhibition of neovascularization in all three of the xenograft models. These data demonstrate that BAY 43-9006 is a novel dual action RAF kinase and VEGFR inhibitor that targets tumor cell proliferation and tumor angiogenesis.     

Antitumor activities of synthetic and natural stilbenes through antiangiogenic action

We reported that the antitumor and antimetastatic actions of resveratrol might be due to the inhibition of tumor‐induced angiogenesis. To search for anticancer agents with stronger activity than resveratrol, we examined the antiangiogenic effects of 21 synthetic and/or natural stilbenes. Among these 21 stilbenes, 2,3‐, 3,4‐, and 4,4′‐dihydroxystilbene inhibited the pro‐matrix metalloproteinase (pro‐MMP)–9 production in colon 26 cells at 5–25 µM, vascular endothelial growth factor (VEGF)–induced human umbilical vein endothelial cell (HUVEC) migration at 10 and 25 µM, and VEGF‐induced angiogenesis at 5–50 µM. Resvertarol inhibited the pro‐MMP‐9 production and VEGF‐induced angiogenesis at 25 or 50 µM. Thus, the inhibition of pro‐MMP‐9 production in colon 26 cells and VEGF‐induced angiogenesis by three dihydroxystilbenes were greater than those of resveratrol. The three dihydroxystilbenes (8 mg/kg, intraperitoneal injection) inhibited the tumor‐induced neovascularization in colon 26–packed chamber‐bearing mice and the tumor growth in colon 26–bearing mice. Furthermore, the three dihydroxystilbenes inhibited VEGF‐induced VEGFR‐2 phosphorylation. On the other hand, the three dihydroxystilbenes had no effect on VEGFR‐1 and‐2 expression, and VEGF‐induced VEGFR‐1 phosphorylation in HUVECs. These findings suggest that the inhibition of tumor‐induced neovascularization by these three dihydroxystilbenes may be due to the inhibition of VEGF‐induced endothelial cell migration and VEGF‐induced angiogenesis through the inhibition of VEGF‐induced VEGFR‐2 phosphorylation in endothelial cells and pro‐MMP‐9 expression in colon 26 cells. (Cancer Sci 2008; 99: 2083–2096)     

Barbigerone, an isoflavone, inhibits tumor angiogenesis and human non-small-cell lung cancer xenografts growth through VEGFR2 signaling pathways

Purpose

We previously reported that barbigerone (BA), an isoflavone isolated from Suberect Spatholobus, exhibited inhibitory effects on proliferation of many cancer cell lines in vitro. The objective of this study was to explore whether BA could effectively suppress tumor angiogenesis and tumor growth.

Methods

Zebrafish model and Matrigel assay were performed to access the anti-angiogenesis effects of BA. A549 and SPC-A1 tumor xenografts in mice models were used to examine the antitumor activity of BA. The anti-angiogenic effects and underlying mechanisms were also investigated using human umbilical vein endothelial cells (HUVECs) and A549 cells.

Results

In zebrafish model, 2.5???mol/L of BA significantly inhibited angiogenesis. Intravenous administration of BA effectively inhibited the tumor growth of A549 and SPC-A1 xenograft models in mice. The anti-angiogenic effect was indicated by CD31 immunohistochemical staining, Matrigel plug assay, and mouse aortic ring assay. BA could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, and capillary-like tuber formation of HUVECs in a dose-dependent manner, suggesting that BA inhibited tumorigenesis by targeting angiogenesis. Western blots revealed that BA directly inhibited the phosphorylation of VEGFR2, followed by inhibiting the activations of its downstream protein kinases, including ERK, p38, FAK, AKT, and expression of iNOS, but had no effect on COX2. Additionally, BA could also down-regulate VEGF secretion in A549 cancer cells, which may correlate with the suppression of ERK, AKT activation, indicating that BA inhibits tumor angiogenesis and tumor growth through VEGFR2 signaling pathways.

Conclusions

These findings suggest that BA may be a novel candidate in inhibiting tumor angiogenesis and NSCLC tumor growth.     

WMJ-S-001, a novel aliphatic hydroxamate derivative,exhibits anti-angiogenic activities via Src-homology-2-domain-containing protein tyrosine phosphatase 1

Angiogenesis, one of the major routes for tumor invasion and metastasis represents a rational target for therapeutic intervention. Recent development in drug discovery has highlighted the diverse biological and pharmacological properties of hydroxamate derivatives. In this study, we characterized the anti-angiogenic activities of a novel aliphatic hydroxamate, WMJ-S-001, in an effort to develop novel angiogenesis inhibitors. WMJ-S-001 inhibited vascular endothelial growth factor (VEGF)-A-induced proliferation, invasion and endothelial tube formation of human umbilical endothelial cells (HUVECs). WMJ-S-001 suppressed VEGF-A-induced microvessel sprouting from aortic rings, and attenuated angiogenesis in in vivo mouse xenograft models. In addition, WMJ-S-001 inhibited the phosphorylations of VEGFR2, Src, FAK, Akt and ERK in VEGF-A-stimulated HUVECs. WMJ-S-001 caused an increase in SHP-1 phosphatase activity, whereas NSC-87877, a SHP-1 inhibitor, restored WMJ-S-001 suppression of VEGFR2 phosphorylation and cell proliferation. Furthermore, WMJ-S-001 inhibited cell cycle progression and induced cell apoptosis in HUVECs. These results are associated with p53 phosphorylation and acetylation and the modulation of p21 and survivin. Taken together, WMJ-S-001 was shown to modulate vascular endothelial cell remodeling through inhibiting VEGFR2 signaling and induction of apoptosis. These results also support the role of WMJ-S-001 as a potential drug candidate and warrant the clinical development in the treatment of cancer.     

Liposomal honokiol inhibits VEGF‐D‐induced lymphangiogenesis and metastasis in xenograft tumor model

Lymph nodes metastasis of tumor could be a crucial early step in the metastatic process. Induction of tumor lymphangiogenesis by vascular endothelial growth factor‐D may play an important role in promoting tumor metastasis to regional lymph nodes and these processes can be inhibited by inactivation of the VEGFR‐3 signaling pathway. Honokiol has been reported to possess potent antiangiogenesis and antitumor properties in several cell lines and xenograft tumor models. However, its role in tumor‐associated lymphangiogenesis and lymphatic metastasis remains unclear. Here, we established lymph node metastasis models by injecting overexpressing VEGF‐D Lewis lung carcinoma cells into C57BL/6 mice to explore the effect of honokiol on tumor‐associated lymphangiogenesis and related lymph node metastasis. The underlying mechanisms were systematically investigated in vitro and in vivo. In in vivo study, liposomal honokiol significantly inhibited the tumor‐associated lymphangiogenesis and metastasis in Lewis lung carcinoma model. A remarkable delay of tumor growth and prolonged life span were also observed. In in vitro study, honokiol inhibited VEGF‐D‐induced survival, proliferation and tube‐formation of both human umbilical vein endothelial cells (HUVECs) and lymphatic vascular endothelial cells (HLECs). Western blotting analysis showed that liposomal honokiol‐inhibited Akt and MAPK phosphorylation in 2 endothelial cells, and downregulated expressions of VEGFR‐2 of human vascular endothelial cells and VEGFR‐3 of lymphatic endothelial cells. Thus, we identified for the first time that honokiol provided therapeutic benefit not only by direct effects on tumor cells and antiangiogenesis but also by inhibiting lymphangiogenesis and metastasis via the VEGFR‐3 pathway. The present findings may be of importance to investigate the molecular mechanisms underlying the spread of cancer via the lymphatics and explore the therapeutical strategy of honokiol on antilymphangiogenesis and antimetastasis. © 2008 Wiley‐Liss, Inc.     

Epoxyquinol B shows antiangiogenic and antitumor effects by inhibiting VEGFR2, EGFR, FGFR, and PDGFR

Angiogenesis is the development of new blood vessels to provide oxygen and nutrients and is indispensable for solid tumor growth. Therefore, the inhibition of angiogenesis is an important modality for cancer chemotherapy. Here we report the antiangiogenic mechanism and antitumor effects of epoxyquinol B (EPQB), which was isolated from fungal metabolites. Short-term treatment of EPQB resulted in the reduction of tumor growth and the number of blood vessels directed to the tumor in a murine xenografts model. Furthermore, EPQB inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation in human umbilical vein endothelial cells (HUVECs) without cytotoxicity. VEGF-stimulated phosphorylation of VEGF receptor 2 (VEGFR2), phospholipase Cgamma-1 (PLCgamma1), and p44/42 MAP kinases (ERK) was inhibited by EPQB in a dose-dependent manner, and in vitro assay using kinase domain of VEGFR2 showed that EPQB covalently bound and inhibited the VEGFR2 kinase. Its binding site on VEGFR2 was different from SU5614, a well-known VEGFR2 kinase inhibitor. Interestingly, EPQB inhibited growth factor-induced activation of not only VEGFR2 but also epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR), suggesting that EPQB is a novel multiple kinase inhibitor. These findings suggest that EPQB would be a good lead compound for the development of potent antiangiogenic and antitumor drugs.     

Cancer antineovascular therapy with liposome drug delivery systems targeted to BiP/GRP78

Angiogenesis is crucial for tumor growth and hematogenous metastasis. Specifically expressed and functional protein molecules in angiogenic endothelial cells, especially on the plasma membrane, may be molecular targets for antiangiogenic drugs and drug delivery systems (DDS) in cancer therapy. To discover such target molecules, we performed subcellular proteome analysis of human umbilical vein endothelial cells (HUVECs) treated with or without vascular endothelial growth factor (VEGF) using 2‐dimensional difference in‐gel electrophoresis (2D‐DIGE) and matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry (MALDI‐TOF/TOF‐MS). Among the identified proteins, BiP/GRP78, a molecular chaperone, was highly expressed in the membrane/organelle fraction of HUVECs after VEGF treatment. The involvement of BiP in VEGF‐induced angiogenesis was examined by RNA interference. BiP knockdown significantly suppressed VEGF‐induced endothelial cell proliferation and VEGF‐induced phosphorylation of extracellular‐regulated kinase 1/2, phospholipase C‐γ, and VEGF receptor‐2 in HUVECs. Cell surface biotinylation analysis revealed that the cell surface expression of BiP was elevated in VEGF‐activated HUVECs. Aiming to apply BiP to a target molecule in liposomal DDS, we developed liposomes modified with the WIFPWIQL peptide, which has been shown to bind to BiP, and investigated its potential for cancer therapy. The WIFPWIQL‐modified liposomes (WIFPWIQL liposomes) were significantly taken up by VEGF‐activated HUVECs as compared to peptide‐unmodified liposomes. WIFPWIQL liposomes appeared to accumulate in tumor endothelial cells in vivo. WIFPWIQL liposomes containing doxorubicin significantly suppressed tumor growth and prolonged the survival of colon26 NL‐17 carcinoma cell‐bearing mice. In summary, BiP may regulate VEGF‐induced endothelial cell proliferation through VEGFR‐2‐mediated signaling and be an effective target molecule for cancer antineovascular therapy.     

TKI-31 inhibits angiogenesis by combined suppression signaling pathway of VEGFR2 and PDGFRbeta

Tyrosine kinases have been strongly implicated as therapeutic targets that influence the angiogenic process in growing tumors. In this study, we revealed that TKI-31 is a potent broad spectrum tyrosine kinase inhibitor, which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor beta (PDGFRbeta) and also inhibits kinases of other class, such as c-Kit and c-Src on molecular base, but showed no activity against vascular endothelial growth factor receptor 1 (VEGFR1) and epidermal growth factor receptor (EGFR). TKI-31 inhibits VEGF-induced phosphorylation of VEGFR2 in endothelial cells as well as PDGF(BB)-induced phosphorylation in fibroblast cells, and leading to the inhibition of down-stream signaling triggered by these receptors such as PI3K/Akt/mTOR, MAPK42/44(ERK) and paxillin. TKI-31 also inhibited VEGF-induced endothelial cells proliferation, migration and their differentiation into capillary-like tube formation. Its anti-angiogenic property was further confirmed by the inhibition of neovascularization on CAM, in vivo. These results collectively highlight the therapeutic potential of this compound for the treatment of solid tumors and other diseases where angiogenesis plays an important role.     

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.     

(Pre‐)Clinical Pharmacology and Activity of Pazopanib,a Novel Multikinase Angiogenesis Inhibitor

Pazopanib is a recently approved, novel tyrosine kinase inhibitor specifically designed to impair angiogenesis by abrogating vascular endothelial growth factor receptor 2 (VEGFR‐2) to exert its function. Pazopanib inhibits VEGF‐induced endothelial cell proliferation in vitro and angiogenesis in vivo and demonstrates antitumor activity in mouse models. Furthermore, the pazopanib concentration resulting in maximal inhibition of VEGFR‐2 phosphorylation in vivo was in line with the steady‐state concentration required to inhibit growth of tumor xenografts, suggesting that pazopanib's mechanism of action is indeed through VEGFR‐2 inhibition. In a phase I trial, a generally well‐tolerated dose was identified at which the majority of patients achieved pazopanib plasma concentrations above the concentration required for maximal in vivo inhibition of VEGFR‐2 phosphorylation in preclinical models. Administered as monotherapy, evidence of antitumor activity was observed in phase II studies in several tumor types, including soft tissue sarcoma, renal cell cancer (RCC), ovarian cancer, and non‐small cell lung cancer. Recently, the U.S. Food and Drug Administration granted approval for treatment with pazopanib in patients with RCC based on the longer progression‐free survival time observed with this agent in a placebo‐controlled, randomized trial. This review summarizes the preclinical and clinical pharmacokinetics and pharmacodynamics of pazopanib, as well as data on clinical activity, that ultimately resulted in its recent approval.     

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.     

Antitumor activity of the MEK inhibitor trametinib on intestinal polyp formation in ApcΔ716 mice involves stromal COX‐2

Extracellular signal‐regulated kinase is an MAPK that is most closely associated with cell proliferation, and the MEK/ERK signaling pathway is implicated in various human cancers. Although epidermal growth factor receptor, KRAS, and BRAF are considered major targets for colon cancer treatment, the precise roles of the MEK/ERK pathway, one of their major downstream effectors, during colon cancer development remain to be determined. Using Apc^Δ716 mice, a mouse model of familial adenomatous polyposis and early‐stage sporadic colon cancer formation, we show that MEK/ERK signaling is activated not only in adenoma epithelial cells, but also in tumor stromal cells including fibroblasts and vascular endothelial cells. Eight‐week treatment of Apc^Δ716 mice with trametinib, a small‐molecule MEK inhibitor, significantly reduced the number of polyps in the large size class, accompanied by reduced angiogenesis and tumor cell proliferation. Trametinib treatment reduced the COX‐2 level in Apc^Δ716 tumors in vivo and in primary culture of intestinal fibroblasts in vitro. Antibody array analysis revealed that trametinib and the COX‐2 inhibitor rofecoxib both reduced the level of CCL2, a chemokine known to be essential for the growth of Apc mutant polyps, in intestinal fibroblasts in vitro. Consistently, trametinib treatment reduced the Ccl2 mRNA level in Apc^Δ716 tumors in vivo. These results suggest that MEK/ERK signaling plays key roles in intestinal adenoma formation in Apc^Δ716 mice, at least in part, through COX‐2 induction in tumor stromal cells.     

Vascular endothelial growth factor tyrosine kinase inhibitor AZD2171 and fractionated radiotherapy in mouse models of lung cancer

The vascular endothelial growth factor receptor (VEGFR) tyrosine kinases are being explored as targets for antiangiogenic cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We investigated the combination of the potent VEGFR tyrosine kinase inhibitor AZD2171 and ionizing radiation in cell culture and mouse models of lung cancer. We show that ionizing radiation induces expression of phosphorylated VEGFR-2 (Flk-1) in endothelial cells and that this phosphorylation is inhibited by AZD2171. Human umbilical vascular endothelial cells become more sensitive to radiation after treatment with AZD2171 as determined by clonogenic assay. Matrigel assay showed a decrease in in vitro endothelial tubule formation with AZD2171/radiation combination treatment. When similar combination was applied to the H460 lung cancer xenograft model in nude mice, loss of radiation-induced phosphorylated Flk-1 was observed in the combination treatment group, which also showed a large decrease in tumor vascular density by staining of the von Willebrand factor. H460 tumor growth delay was enhanced in the combination treatment group compared with the groups treated with AZD2171 or radiation alone. Additionally, after therapy, Ki67 index showed >4-fold reduction of tumor proliferation in the combination therapy group, which also showed increased intratumoral apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. In conclusion, AZD2171 sensitizes lung tumor xenografts to radiation and inhibits angiogenesis both in vitro and in vivo. When used as a radiation enhancer, AZD2171 has the potential to improve tumor growth delay by inhibiting tumor proliferation and promoting apoptosis. Clinical trials are needed to determine the potential of this combination therapy in patients with locally advanced lung cancer.     

Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model

Although gemcitabine has been approved as the first-line chemotherapeutic reagent for pancreatic cancer, its response rate is low and average survival duration is still only marginal. Because epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) modulate tumor progression, we hypothesized that inhibition of phosphorylation of all three on tumor cells, tumor-associated endothelial cells, and stroma cells would improve the treatment efficacy of gemcitabine in an orthotopic pancreatic tumor model in nude mice and prolong survival. We implanted L3.6pl, a human pancreatic cancer cell, in the pancreas of nude mice. We found that tumor-associated endothelial cells in this model highly expressed phosphorylated EGFR, VEGFR, and PDGFR. Oral administration of AEE788, a dual tyrosine kinase inhibitor against EGFR and VEGFR, decreased phosphorylation of EGFR and VEGFR. PDGFR phosphorylation was inhibited by STI571. Although i.p. injection of gemcitabine did not inhibit tumor growth, its combination with AEE788 and STI571 produced >80% inhibition of tumor growth and prolonged survival in parallel with increases in number of tumor cells and tumor-associated endothelial cell apoptosis, decreased microvascular density, decreased proliferation rate, and prolonged survival. STI571 treatment also decreased pericyte coverage on tumor-associated endothelial cells. Thus, inhibiting phosphorylation of EGFR, VEGFR, and PDGFR in combination with gemcitabine enhanced the efficacy of gemcitabine, resulting in inhibition of experimental human pancreatic cancer growth and significant prolongation of survival.     

AEE788, a dual tyrosine kinase receptor inhibitor, induces endothelial cell apoptosis in human cutaneous squamous cell carcinoma xenografts in nude mice.

PURPOSE: We investigated whether concomitant blockade of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways by AEE788, a dual inhibitor of EGFR and VEGFR tyrosine kinases, would inhibit the growth of cutaneous squamous cell carcinoma (SCC) cells and human cutaneous cancer xenografts in nude mice. EXPERIMENTAL DESIGN: We examined the effects of AEE788 on the phosphorylation of EGFR and VEGFR-2 in cutaneous SCC cells expressing EGFR and VEGFR-2 and cutaneous SCC cell growth and apoptosis. We assessed the in vivo antitumor effects of AEE788 in a xenograft model in nude mice. AEE788 (50 mg/kg) was given orally thrice weekly to mice that had been s.c. injected with Colo16 tumor cells. Mechanisms of in vivo AEE788 activity were determined by immunohistochemical analysis. RESULTS: Treatment of cutaneous SCC cells with AEE788 led to dose-dependent inhibition of EGFR and VEGFR-2 phosphorylation, growth inhibition, and induction of apoptosis. In mice treated with AEE788, tumor growth was inhibited by 54% at 21 days after the start of treatment compared with control mice (P < 0.01). Immunohistochemical analysis revealed that AEE788 inhibited phosphorylation of EGFR and VEGFR and induced apoptosis of tumor cells and tumor-associated endothelial cells. CONCLUSIONS: In addition to inhibiting cutaneous cancer cell growth by blocking EGFR and VEGFR signaling pathways in vitro, AEE788 inhibited in vivo tumor growth by inducing tumor and endothelial cell apoptosis.     

VEGFR2 Expression in Head and Neck Squamous Cell Carcinoma Cancer Cells Mediates Proliferation and Invasion

Vascular endothelial growth factor 2 (VEGFR2) was initially identified as a receptor of VEGF on endothelial cells with a role in regulating angiogenesis during organism development and tumorigenesis. Previously, in cancer tissue, VEGFR2 has been reported to be expressed in endothelial cells. In our research, we found that VEGFR2 was expressed not only in endothelial cells but also cancer cells in head and neck squamous cell carcinomas (HNSCCs). Knockdown of VEGFR2 in Hep2 cells could arrest the cell cycle in G0/G1, leading to a decrease in proliferation. We also present evidence that MAPK/ERK signal pathways and expression of CDK1 downstream of VEGFR2 might regulate proliferation and cell cycle arrest. Furthermore, we discovered that down-regulate VEGRF2 in Hep2 cells could significantly affect the invasion ability. Taken together, our data suggest that VEGFR2 might regulate proliferation and invasion in HNSCC cancer cells in vivo.     

AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.