Therapeutic Effects of LS-13 on Glioma in Vivo and in Vitro.
CNS neuroscience & therapeutics(2014)
摘要
Glioma is the most common primary brain tumor, accounting for 40–50% of all malignant brain tumors 1. In most cases, glioma is locally invasive. The resulting lack of boundary with surrounding healthy tissues is a major barrier for surgical resection. Treatment options include surgical resection followed by chemotherapy (e.g., with temozolomide) and irradiation. These treatments could prolong patient survival, but only to a limited extent, and at the cost of reduced life quality 2. Angiogenesis is an important pathological progress in the growth, development, and invasion of solid tumors 3. For glioma, new blood vessels must be formed to allow further growth when the tumor is 1–2 mm in diameter. As a result, interventions that inhibit angiogenesis could inhibit glioma growth 4. LS-13 (molecular formula shown in Figure 1, from Tongli Pharmaceutical Technology Co., Ltd., Suzhou, China) is a novel small-molecule compound capable of inhibiting vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 kinases (IC50 at 3.1 and 3.4 nM, respectively; unpublished observation). We hypothesized that the inhibitory action of LS-13 on glioma is mediated by its effects on angiogenesis. In the first step of the present study, we examined the potential effects of LS-13 on glioma, on the proliferation of human umbilical vein endothelial cells (HUVEC). The results revealed concentration-dependent inhibition of LS-13 on HUVEC proliferation. In a scratch assays using cultured human U251 glioma cells, LS-13 significantly inhibited cell migration at a concentration of 10 μM. In the next step, we conducted a transwell migration assay using HUVEC and U251 cells in logarithmic growth phase. Cells were starved overnight prior to the incubation with LS-13 in the upper chamber. The results showed that LS-13 incubation of 12 h inhibited the migration of HUVEC and U251 cells in a concentration-dependent manner (Figure 2). In the next set of experiments, U251 cells were transplanted subcutaneously to 4-to 6-week-old nude mice before treatment with LS-13, when tumor size get to 100 mm3, then daily oral gavage at doses of 12.5, 25, 50, and 100 mg/kg/day over a 14-day period. The body weight and signs of stress were monitored daily. Tumor size was measured at different time points and estimated using an electronic caliper with the following equation: tumor size (cm3) = 1/2(length [cm]) × (width [cm])2. The relative tumor volume (RTV) and relative tumor proliferation rate (T/C [%]) were then calculated. At doses of 50 and 100 mg/kg, LS-13 decreased RTV significantly (P < 0.05 vs. negative control group; Figure 3). A dose of 12.5 mg/kg, LS-13 decreased RTV significantly after Day 10 (P < 0.05), but at a higher dose of 25 mg/kg, LS-13 started to decreased RTV since Day 3 (P < 0.05; Figure 3). At a dose of 12.5 mg/kg, T/C was >40%. At a higher dose of 25, 50, and 100 mg/kg, T/C was <40%, suggesting that the lowest dose needed to inhibit glioma is 25 mg/kg. The body weight was not significantly reduced by LS-13 at any doses (P > 0.05). In the last step of our experiments, we examined the effects of LS-13 (50 mg/kg/day via oral garage) in combination with doxorubicin (2 mg/kg twice every week, intravenously) over a 14-day period in nude mice bearing U251 glioma xenograft. RTV was significantly decreased by LS-13 treatment alone, doxorubicin alone, as well as the combined treatments (P < 0.05 vs. negative control group). RTV in the combined treatment group was significantly lower than in the mice receiving LS-13 alone (P < 0.05), but not in the mice receiving doxorubicin alone (P < 0.05). T/C was 27.31% for LS-13 alone, 14.49% for doxorubicin alone, and 10% for combined treatments. The body weight was not affected significantly by any of the treatment (P > 0.05). Angiogenesis is a complex process that involves VEGF and its receptor VEGFR. Bevacizumab is an angiogenesis inhibitor and specifically VEGF-A inhibitor 5. It has been approved by the US Food and Drug Administration (FDA) for recurrent glioblastoma. Bevacizumab does not prolong patient survival, but could improve quality of life considerably 6, 7. The VEGFR family includes VEGFR-1, VEGFR-2, and VEGFR-3. VEGFR-2 has high affinity for VEGF and strong kinase activity. It is primarily expressed in vascular endothelial cells and critically implicated in endothelial cell proliferation vascular permeability, neo-angiogenesis 8. VEGFR-3 is mainly expressed in lymphatic endothelia, as well as on the surfaces of tumor vascular endothelial cells. VEGFR-3 is associated with the sprouting of blood vessels and the formation of new branches 9. Simultaneous blockade of VEGFR-2 and VEGFR-3 is more effective in inhibiting tumors than VEGFR-2 blockade alone 8, 9. LS-13 inhibits the kinase activity of both VEGFR-2 and VEGFR-3. The results of this study support this mode of action. Drug resistance is a formidable obstacle to tumor targeted therapy 10. Drugs that target multiple molecular target and drug combinations are one strategy to minimize such a problem. In this regard, LS-13 has clear advantage: It targets both VEGFR2 and VEGFR3. We advocate further research of LS-13 as an addition to bevacizumab, the only approved targeted drug for glioma. The authors declare no conflict of interest.
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