Anti-tumor efficacy of CKD-516
We evaluated the anti-tumor efficacy of CKD-516 at two doses (3 mg/kg and 5 mg/kg) in H520 xenograft mice. In group 1 mice treated with 3 mg/kg of CKD-516, tumor growth was delayed until day 3. After that, tumor began to regrow. In group 2 mice treated with 5 mg/kg of CKD-516, tumor growth was inhibited from day 3. Compared to the control, at the completion of drug administration, tumor sizes were reduced by 39.5% and 81.2% in groups 1 and 2, respectively (Fig. 1A). Additionally, we stained tumor tissues with Hoechst 33342 dye to examine morphological changes of blood vessels caused by CKD-516. Under a fluorescence microscope, we found that the morphology of blood vessels in mice treated with CKD-516 showed obvious changes (Fig. 1B). Based on these results, we selected the dose of CKD-516 at 3 mg/kg for the next study.
Anti-tumor efficacy of short-term treatment with CKD-516 alone or in combination with radiation
We evaluated the anti-tumor efficacy of short-term administration with CKD-516 alone or in combination with radiation. At 24 hours after the completion of administration schedule, CKD-516 did not reduce tumor size. However, IR reduced it by 27.8% compared to the control (Fig. 2A). When CKD-516 was combined with IR, tumor size was reduced by 28.6% in CKD-516 + IR (d1) and 27.9% in CKD-516 + IR (d1, 5). We also checked tumor size at 72 hours after the end of drug administration. CKD-516 reduced tumor size more at 72 hours than that at 24 hours. Both IR alone and CKD-516 + IR (d1) delayed tumor growth. On the contrary, in CKD-516 + IR (d1, 5), tumor did grow again at 72 hours (Fig. 2A). When tumor sizes were compared among four groups at the end of treatment, IR alone and CKD-516 + IR (d1) significantly reduced the tumor by 55.5% (p = 0.0062) and 58.5% (p = 0.0051), respectively. Notable body weight loss was observed in IR alone, CKD-516 + IR (d1), and CKD-516 + IR (d1, 5) groups by 15.4%, 13.7%, and 11.5%, respectively. In contrast, no changes in body weight were observed in the CKD-516 alone group (Fig. 2B). We counted the number of blood vessels and tumor necrosis area as well as tumor volume. As shown in Fig. 2C, the number of blood vessels stained with CD31 antibody was significantly diminished in CKD-516 group (52.1%, p = 0.0001) compared with the control. Both CKD-516 + IR (d1) and CKD-516 + IR (d1, 5) groups showed much more reduction in the number of blood vessels by 64.8% (p < 0.005) and 59.1% (p = 0.00016), respectively. We also analyzed tumor necrosis area in tumor tissue stained with H & E. IR significantly induced necrosis by 60.4% compared to the control (p = 0.004). CKD-516 + IR (d1) produced the most extensive tumor necrosis which was significantly increased by 66.0% (p = 0.02) compared to the control (Fig. 2D). However, tumor necrosis in CKD-516 alone or CKD-516 + IR (d1, 5) group did not significantly differ from that in the control.
Sustained tumor necrosis and hypoxia after short-term treatment with CKD-516 in combination with radiation
We investigated post-treatment effects of IR, CKD-516, and their combinations on tumor necrosis and hypoxic environment. The calculated tumor necrosis area (%) was the largest in IR alone (37.3%) at 24 hours from the beginning of treatment. However, no further change was detected at 72 hours (37.1%) (Fig. 3A). Both CKD-516 and CKD-516 + IR (d1) produced more tumor necrosis area by 41.3% (p = 0.049) and by 47.1% (p = 0.004), respectively, at 72 hours. At 24 hours after the end of treatment, calculated hypoxic area was in the following order: 56.6% in CKD-516 + IR (d1), 42.0% in CKD-516 + IR (d1, 5), and 34.4% in IR alone. At 72 hours, the hypoxic area was rapidly declined from 34.3% to 6.9% in IR alone (p = 0.0003). However, it was increased from 56.6% to 64.9% in CKD-516 + IR (d1).
Expression of hypoxia-related molecules in mice after short-term treatment of CKD-516 in combination with radiation
We evaluated protein expression levels of hypoxia-related molecules (HIF1α, Glut-1, VEGF, and Ki-67) affecting the maintenance of hypoxic microenvironment in mice treated with CKD-516, IR, or their combinations (Fig. 4A). The expression of HIF1α, a classic marker for hypoxic condition, was the highest in CKD-516 group (57.6%) at 24 hours after drug administration (Fig. 4B). However, at 72 hours after treatment, it was the highest in IR group (68.1%). VEGF expression in IR alone was increased by 34.8% at 72 hours. In CKD-516 + IR (d1) group, it was significantly diminished from 22.0% to 7.0% (p = 0.019). The expression of Glut-1 was decreased as much as 20–30% for the analyzed area in all treatment groups at 24 hours after the end of drug administration. In IR alone, Glut-1 expression was decreased by 50.2% from 24 hours to 72 hours. It was greatly diminished in CKD-516 + IR (d1) group (81%, p = 0.0039). Ki-67 expression was the lowest (16.3%) in the CKD-516 alone group among four groups at 24 hours after the beginning of drug administration. However, at 72 hours, its expression was significantly declined in CKD-516 + IR (d1) and CKD-516 + IR (d1, 5) groups (86%, p = 0.0036 and 50.8%, p = 0.027, respectively).
Delayed tumor growth after long-term treatment of CKD-516 in combination with radiation
We evaluated delayed tumor growth, tumor necrosis, and tumor hypoxia after long-term treatment with a combination of CKD-516 with IR compared to results from short-term treatment with a combination of CKD-516 and IR. Because weight loss and skin rash due to IR were frequently observed in short-term treatment, IR dose was decreased from 4 Gy to 2 Gy in the long-term combination schedule. Regarding tumor growth inhibition (TGI), both IR alone and CKD-516 + IR remarkably reduced tumor size (56.2%, p = 0.0091 and 71.2%, p = 0.007, respectively) at the end of administration (Fig. 5A and Table 1). Additionally, we found sustained tumor growth delay even at 72 hours after the end of treatment, especially in the group of CKD-516 + IR (33.0% vs. 37.6%). No significant differences in body weight were found between CKD-516 and control groups (Fig. 5B). However, both IR alone and CKD-516 + IR groups displayed gradual decrease of body weight when the administration schedule progressed. We measured the number of blood vessels at 72 hours after the end of administration. Compared to the control, CKD-516, IR, and CKD-516 + IR significantly decreased the number of blood vessels by 38.4% (p = 0.003), 72.9% (p = 0.0002), and 84.2% (p = 0.0006), respectively (Fig. 5C). Conversely, tumor necrosis area was significantly expanded to 67% in IR group, 82% in CKD-516 group, and 84% in CKD-516 + IR group compared to the control (p = 0.02, p = 0.005, and p = 0.004, respectively) (Fig. 5D).
Table 1
Tumor growth inhibition by CKD-516 combined with IR in long-term administration
Group (n = 10) | Radiation (Gy/kg/day) | CKD-516 (mg/kg/day) | TGI (%) |
24 h | 72 h |
Vehicle | - | - | - | - |
IR | 2 | - | 51.9 | 56.2* |
CKD-516 | - | 3 | - | - |
CKD-516 + IR | 2 | 3 | 61.0 | 71.2* |
Expression of hypoxia-related molecules in mice after long-term treatment of CKD-516 in combination with IR
HIF1α expression was significantly increased by 64.0% in CKD-516 group (p = 0.0022) and by 65.0% in CKD-516 + IR group (p = 0.0047) compared to the control (Fig. 6A). VEGF expression levels in both IR and CKD-516 groups were similar to those in the control. However, it was significantly diminished by 41.0% in CKD-516 + IR group (p = 0.046) (Fig. 6B). Glut-1 expression was increased in both IR and CKD-516 groups. However, it showed no significant change in CKD-516 + IR group (Fig. 6C). Ki-67 expression was greatly diminished by 4.3%, 4.4%, and 5.2% in IR, CKD-516, and CKD-516 + IR groups, respectively (data not shown).