An Inducible Caspase-9 Delivering System Using Mesenchymal Stem Cells for Lung Cancer Treatment

We plan to deliver an apoptosis-inducing protein, inducible caspase 9 (iC9), to lung cancer cells. Delivery will be made via an adenoviral vector encoding iC9, which itself will be carried to the tumor by mesenchymal stem cells (MSCs) since MSCs selectively accumulate in the lung vasculature and the tumor bed following intravenous infusion. Caspase-9 is a member of a family of caspase proteases that are essential mediators of apoptosis. We have previously shown that an inducible version of caspase-9, can be used as a suicide gene to induce apoptosis in adoptively transferred T cells. The inducible caspase-9 molecule (iC9) was made by the fusion of human caspase-9 to a modified human FK-binding protein (FKBP), allowing conditional dimerization. In the presence of a small molecule chemical inducer of dimerization (CID), the dimerization of iC9 is enforced, which activates the intrinsic apoptotic pathway, leading to cell death. We will encode the iC9 gene in an adenovirus vector (AdV) and use MSC as vehicles to deliver this iC9 AdV to lung tumor cells. To allow AdV production, the MSC were also transduced by a retrovirus with a plasmid encoding E1A gene. E1A MSCs efficiently produced iC9 adenovirus that infected several different lung cancer cell line sufficiently well to produce high levels of apoptosis by CID treatment. Some lung cancer cell lines, such as A549, were more resistant, so that only 50% or less of infected cells were killed by apoptosis upon iC9 activation. To determine the mechanism of partial resistance, we used the proteasome Inhibitor, Bortezomib. In the cell line A549, Bortezomib or CID alone each induced low-level apoptosis, while the combination induced synergistic apoptosis. We used Western blotting to analyze components of the caspase pathway to discover the molecular basis of this synergy, and showed maximum cleavage of caspase-9 and caspase-3 only when iC9 transduced A549 cells were treated with the combination. This suggests that the cleaved form of caspase-3 is usually degraded by proteasome in A549 cells. Therefore addition of Bortezomib results in stabilization of cleaved caspase-3which is accompanied by increase in death. Thus Bortezomib may enhance iC9 induced death in otherwise resistant human tumor cells and combination therapy of MSC Ad-iC9 with CID and Bortezomib may useful in treating human lung tumors.