Triple combination of irradiation, chemotherapy (pemetrexed), and VEGFR inhibition (SU5416) in human endothelial and tumor cells.
OBJECTIVE
This is the first preclinical report evaluating a trimodal therapy consisting of irradiation, chemotherapy, and antiangiogenesis in the context of a multimodal anticancer strategy. The combination of the folate antimetabolite pemetrexed, SU5416, a receptor tyrosine kinase inhibitor of VEGFR2, and irradiation was investigated in human endothelial cells and tumor cell lines.


METHODS
Primary isolated human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HDMEC), and human glioblastoma (U87) and prostate cancer cells (PC3) were exposed to pemetrexed (2 h) alone and in combination with SU5416 (2 h). When combined with irradiation up to 8 Gy, fixed concentrations of pemetrexed (1.06 muM) and SU5416 (1.0 muM) were used. Proliferation and clonogenic assays were conducted with endothelial and tumor cells. The migration/invasion ability of endothelial cells and the ability to produce tubular structures were tested in Matrigel and tube formation assays. Apoptosis was measured by sub-G1 DNA and caspase-3 flow cytometry. To investigate underlying cell signaling, immunocytochemistry was used to detect Akt survival signaling involvement.


RESULTS
Triple combination using only a low-toxicity drug exposure of pemetrexed and SU5416 results in greater response than each treatment alone or than each combination of two modalities in all tested endothelial and tumor cell models. Triple combination substantially inhibits proliferation, migration/invasion, tube formation, and clonogenic survival. Triple combination also induced the highest rate of apoptosis in HDMEC and HUVEC as indicated by sub-1 G1 and caspase-3 assessment. Interestingly, triple combination therapy also reduces proliferation and clonogenic survival significantly in U87 and PC3 tumor cell lines. SU5416 potently inhibited Akt phosphorylation which could be induced by radiation and radiochemotherapy in human endothelial cells.


CONCLUSIONS
Our findings demonstrate the high antiendothelial/antitumoral efficacy of the concurrent administration of irradiation, chemotherapy, and angiogenesis inhibition in vitro. A potential explanation for the favorable combination would be that VEGF signaling inhibition downregulates Akt survival signaling upon activation by radiation and/or chemotherapy. The data also suggest that endothelial cell apoptosis may have an important role in the benefits of the presented therapy.
Bischof
Marc
M
Department of Radiation Oncology, University of Heidelberg, Medical School, Heidelberg, Germany.
Abdollahi
Amir
A
Gong
Ping
P
Stoffregen
Clemens
C
Lipson
Kenneth E
KE
Debus
J Urgen
JU
Weber
Klaus J
KJ
Huber
Peter E
PE
D020533:Angiogenesis Inhibitors / Q000627:therapeutic use*; D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000528:radiation effects; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D003131:Combined Modality Therapy / Q000379:methods*; D042783:Endothelial Cells / Q000187:drug effects / Q000528:radiation effects; D005971:Glutamates / Q000627:therapeutic use*; D006147:Guanine / Q000031:analogs & derivatives* / Q000627:therapeutic use*; D006801:Humans; D007211:Indoles / Q000627:therapeutic use*; D009369:Neoplasms / Q000098:blood supply / Q000188:drug therapy / Q000532:radiotherapy / Q000628:therapy*; D009389:Neovascularization, Pathologic / Q000517:prevention & control; D000068437:Pemetrexed; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011758:Pyrroles / Q000627:therapeutic use*; D014410:Tumor Stem Cell Assay / Q000379:methods
D020533:Angiogenesis Inhibitors; D000964:Antimetabolites, Antineoplastic; D005971:Glutamates; D007211:Indoles; D011758:Pyrroles; D000068437:Pemetrexed; D006147:Guanine; C116890:Semaxinib; D011505:Protein-Tyrosine Kinases