TZT-1027 (Soblidotin), an antimicrotubule agent, has been demonstrated to show potent antitumor effects, though the relationships among antitumor effect, cytotoxicity and anti-vascular effect of TZT-1027 have not been studied. We established in vivo human lung vascular-rich tumor models using a vascular endothelial growth factor-secreting tumor (SBC-3/VEGF). SBC-3/VEGF tumors exhibited a high degree of angiogenesis in comparison with the mock transfectant (SBC-3/Neo) tumors in a dorsal skinfold chamber model and grew much faster and larger than SBC-3/Neo tumors in the tumor growth study. The antitumor activity of antimicrotubule agents, including TZT-1027, was evaluated in both early-and advanced-stage SBC-3/Neo and SBC-3/VEGF tumor models to elucidate the relationship between the antitumor activity and anti-vascular effect of these agents. TZT-1027 exhibited potent antitumor activity against both early-and advanced-stage SBC-3/Neo and SBC-3/VEGF tumors, whereas combretastatin A4 phosphate did not. Vincristine and docetaxel exhibited potent antitumor activity against early-stage SBC-3/Neo and SBC-3/VEGF tumors, and advanced-stage SBC-3/Neo tumors, but did not exhibit activity against advanced-stage SBC-3/VEGF tumors. The difference in antitumor activity between these agents could be ascribed to differences in direct cytotoxicity and anti-vascular effect. Furthermore, a prominent accumulation of erythrocytes in the tumor vasculature, followed by leakage and scattering of these erythrocytes from the tumor vasculature, was observed after TZT-1027 administration to mice bearing advanced-stage SBC-3/VEGF tumors. These findings strongly suggest that TZT-1027 has a potent anti-vascular effect, in addition to direct cytotoxicity. (Cancer Sci 2003; 94: 826-833) ngiogenesis is the process whereby capillaries sprout from pre-existing blood vessels. The overall process is complicated, involving many cell types and biological functions, including endothelial cell activation, migration and proliferation.1) Previous studies have demonstrated that tumor angiogenesis is required for the growth and metastasis of primary solid tumors.2) This angiogenesis is influenced by the balance between the activation of promoting and inhibiting factors. The activation of promoting factors, such as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF), released from hypoxic tumor cells, leads to neovascularization, while the activation of inhibiting factors generally causes tumors to undergo necrotic cell death.3) The importance of angiogenesis in human tumors was proved by recent reports demonstrating the vessel density in the tumor mass to be a prognostic factor for survival in people with various types of tumor. [4][5][6][7][8]

TZT‐1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit et al. , is a potent antimicrotubule agent. We have compared the activity of TZT‐1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT‐1027 and dolastatin 10 inhibited microtubule polymerization concentration‐dependently at 1–100 μ M with IC 50 values of 2.2±0.6 and 2.3±0.7 μ M , respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1–3 μ M with IC 50 values of 2.7±0.6, 1.6±0.4 and 1.6±0.2 μ M , respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 μ M or more. TZT‐1027 also inhibited monosodium glutamate‐induced tubulin polymerization concentration‐dependently at 0.3–10 μ M , with an IC 50 of 1.2 μ M , whereas VLB was only effective at 0.3–3 μ M , with an IC 50 of 0.6 μ M , and caused so‐called “aggregation” of tubulin at 10 μ M. Scatchard analysis of the binding data for [ 3 H]VLB suggested one binding site (K d 0.2±0.04 μ M and B max 6.0±0.26 n M /mg protein), while that for [ 3 H]TZT‐1027 suggested two binding sites, one of high affinity (K d 0.2±0.01 μ M and B max 1.7±0.012 n M /mg protein) and the other of low affinity (K d 10.3±1.46 μ M , and B max 11.6±0.83 n M /mg protein). [ 3 H]TZT‐1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [ 3 H]VLB was completely displaced by dolastatin 10 and TZT‐1027. Furthermore, TZT‐1027 prevented [ 3 H]VLB from binding to tubulin in a non‐competitive manner according to Lineweaver‐Burk analysis. TZT‐1027 concentrationdependently inhibited both [ 3 H]guanosine 5′‐triphosphate (GTP) binding to and GTP hydrolysis on tubulin. VLB inhibited the hydrolysis of GTP on tubulin concentration‐dependently to a lesser extent than TZT‐1027, but no inhibitory effect of VLB on [ 3 H]GTP binding to tubulin was evident even at 100 μ M . Thus, TZT‐1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT‐1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mo...

TZT-1027, a newly synthesized dolastatin 10 derivative, is a potent antitumor agent which inhibits microtubule polymerization and perturbs microtubule dynamics. In this report, we investigated whether TZT-1027 inhibited the growth of various human cancer cells, and the cell death caused by TZT-1027 was due to apoptosis. In addition, we elucidated the apoptosis machinery induced by treatment with TZT-1027. The 50% growth-inhibitory concentrations (IC50 values) of TZT-1027 on cancer cells derived from various sources were not more than 5.9 ng/ml. TZT-1027 showed superior cytotoxicity than any other antitumor agents. Next, we evaluated morphological nuclear change, namely, chromatin condensation and DNA fragmentation. We used three cancer cell lines derived from different types in view of having apoptosis related protein, human leukemia HL-60 (in the presence of both Caspase-3 and Bcl-2), human breast cancer MCF-7 (in the absence of Caspase-3), and human prostate cancer DU145 (in the absence of Bcl-2). TZT-1027 induced DNA fragmentation in the presence but not absence of Caspase-3. Nevertheless, apoptic chromatin condensation was observed in all cancer cells even if there was no Caspase-3. Furthermore, we examined whether TZT-1027, microtubule-disrupting agent, influenced cell cycle progression. Flow cytometric analysis revealed the cells treated with TZT-1027, and with the other antimicrotubule agents, to be arrested at the G2/M phase and subsequently to show fragmented DNA smaller than that of G1 phase cells. Moreover, we tested TZT-1027 for its ability to induce Bcl-2 phosphorylation in human cancer cell lines. TZT-1027 and other agents which interacted with microtubules induced Bcl-2 phosphorylation, whereas DNA-damaging agents did not. The present results suggested an association of the growth-inhibitory effect of TZT-1027 with the induction of apoptosis and indicated that the apoptosis induced by TZT-1027 was followed by G2/M arrest even if there was no Caspase-3 or Bcl-2.

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