Therapeutic Enhancement of Verteporfin-Photodynamic Therapy by Inhibiting Phosphatidylinositol 3-Kinase Pathway Signaling or Augmenting Endoplasmic Reticulum Stress

Photodynamic therapy (PDT) induces cell injury and death through generation of reactive oxygen species (ROS) after light activation. Verteporfin is a photosensitizer that has been approved for the treatment of age-related macular degeneration and is under investigation for targeting tumors and tumor associated vasculature. In our study, verteporfin-PDT induced rapid apoptotic cell death in Simian Virus 40 transformed mouse endothelial cells (SVEC) by activating mitochondria-initiated cell death pathways. Activation of pro-survival phosphatidylinositol 3- kinase/mechanistic target of rapamycin (PI3K/mTOR) signaling pathway was also observed after verteporfin-PDT, hindering the treatments effectiveness. Furthermore, activation of PI3K/mTOR signaling was correlated with cell growth after PDT. Thus, the goal of this study is to test the hypothesis that the therapeutic outcome of verteporfin-PDT can be enhanced by inhibiting the PDT-induced pro-survival PI3K/mTOR signaling pathway. We found that the combination of verteporfin-PDT and a dual PI3K/mTOR pathway inhibitor, BEZ235, synergistically enhanced the treatment outcome. Through the addition of BEZ235, compensatory post-PDT upregulation of the PI3K/mTOR pathway was inhibited, as evidenced by a dramatic reduction in the phosphorylation of s6 proteins. Cell apoptosis and proliferation assays show that this combination treatment promotes a higher degree of cell apoptosis, and a greater inhibition of cell proliferation in endothelial cells. We observed a strong synergistic effect of PDT combined with BEZ235. However, we did not see as much of an enhancement with more selective PI3K pathway inhibitors, including pan-PI3K inhibitor BKM120 and p110α isoform-selective inhibitor BYL719, suggesting that the inhibition of mTOR pathway plays a more prominent role in enhancement of vascular PDT. This concept was further explored with the use of two mTOR inhibitors, mTOR complex 1 (mTORC1) inhibitor rapamycin, and mTOR complex 1 and 2 (mTORC1/C2) inhibitor AZD2014. Although PDT in combination with AZD2014 did show a stronger therapeutic effect than PDT combined with rapamycin in the short-term, the long-term response of these two combination treatments were similar. Both combination treatments caused an enhanced mitochondrial-mediated apoptosis and greater cell growth inhibition. These results indicate that, to target the PI3K/mTOR pro-survival pathway for PDT enhancement, sustained inhibition of mTORC1 is necessary and broader inhibition of mTORC1/C2 is likely more therapeutically beneficial. The other part of this study is on the endoplasmic reticulum (ER) stress and unfolded protein response (UPR) induced by verteporfin-PDT. An interesting finding is that verteporfin was preferentially associated with the UPR sensor protein (IRE1α and PERK) in the ER and verteporfin-PDT induced aggregation of these proteins. Despite the aggregation of UPR sensor proteins, PDT was shown to activate the UPR response pathways. PDT combined with ER stress inducer thapsigargin induced more cell apoptosis by activating mitochondria-mediated apoptotic pathway. These results highlight the importance of mitochondria in determining cell death induced by verteporfin-PDT and support the combination of PDT and other ER-stress inducers for enhanced outcomes. In conclusion, we found that verteporfin-PDT activated the pro-survival PI3K/mTOR signaling pathway and induced ER stress in endothelial cells. Inhibition of PI3K/mTOR pathway with targeted therapeutics, particularly mTOR inhibitors, not only induced more cell apoptosis but also led to a sustained inhibition of cell proliferation. Augmenting of PDT-induced ER stress with additional ER stress inducers was also effective for improving PDT response. Our study shows that therapeutic enhancement of verteporfin-PDT can be achieved by either inhibiting a prosurvival pathway activated by PDT or further augmenting PDT-induced cell stress