The vascular disrupting agent combretastatin A-4 phosphate causes prolonged elevation of proteins involved in heme flux and function in resistant tumor cells

Abstract

Vascular disrupting agents (VDAs) represent a promising class of anti-cancer drugs for solid tumor treatment. Here, we aim to better understand the mechanisms underlying tumor reccurrence and treatment resistance following the administration of a VDA, combretastatin A-4 phosphate (CA4P). Firstly, we used photoacoustic tomography to noninvasively map the effect of CA4P on blood oxygen levels throughout subcutaneous non-small cell lung cancer (NSCLC) tumors in mice. We found that the oxygenation of peripheral tumor vessels was significantly decreased at 1 and 3 hours post-CA4P treatment. The oxygenation of the tumor core reduced significantly at 1 and 3 hours, and reached anoxia after 24 hours. Secondly, we examined the effect of CA4P on the levels of proteins involved in heme flux and function, which are elevated in lung tumors. Using immunohistochemistry, we found that CA4P substantially enhanced the levels of enzymes involved in heme biosynthesis, uptake, and degradation, as well as oxygen-utilizing hemoproteins. Furthermore, measurements of markers of mitochondrial function suggest that CA4P did not diminish mitochondrial function in resistant tumor cells. These results suggest that elevated levels of heme flux and function contribute to tumor regrowth and treatment resistance post-VDA administration.

Elevated Heme Synthesis and Uptake Underpin Intensified Oxidative Metabolism and Tumorigenic Functions in Non–Small Cell Lung Cancer Cells

“…Hemoproteins and enzymes that are required for OXPHOS are also protumorigenic. Interestingly, a recent study from the authors' lab showed that viable NSCLC tumor cells resistant to the vascular disrupting agent, combretastatin A-4 phosphate, exhibit further elevated levels of hemoproteins and proteins and enzymes involved in heme metabolism (32).…”

“…IHC was carried out exactly as described previously (32). Paraffin-embedded tumor tissues from mice described above were used.…”

Oxygen-Enhanced and Dynamic Contrast-Enhanced Optoacoustic Tomography Provide Surrogate Biomarkers of Tumor Vascular Function, Hypoxia, and Necrosis

“…In 4 animals, both tumors could not be visualized in a single imaging slice, resulting in 4 tumors being excluded, leaving 12 tumors for analysis. Combretastatin 4A phosphate (CA4P, C7744; Sigma-Aldrich), a potent Vascular Disrupting Agent with well-established efficacy in preclinical models, was used (30,41,42). When tumors reached approximately 8 mm linear dimension, mice were randomly allocated into two groups: treated (CA4P, 8 mL/kg of 12.5 mg/mL solution dosed intraperitoneally to achieve a dose of 100 mg/kg, n ¼ 7 tumors, 4 mice) and vehicle (PBS 8 mL/kg intraperitoneally, n ¼ 5 tumors, 4 mice).…”

“…Imaging studies were performed both before and 4 hours after administration of the vascular disrupting agent combretastatin A4 phosphate. The 4-hour time point was chosen so as to observe the induced vascular disruption prior to the development of substantial tumor necrosis (30).…”

“…These imaging biomarkers provide complementary hemodynamic information to those measured clinically with DCE-based techniques and also the label-free MRI-based techniques introduced above. OT has been shown to monitor the evolution of tumor vasculature during disease development (27,28) and to detect response to vascular-targeted therapies (29,30). OT has also been combined with DCE ultrasound (31,32), showing relationships between hemoglobin parameters and perfusion metrics.…”