Combinatorial
therapies using the
proteasome inhibitor,
bortezomib, have been found to induce synergistic apoptosis in
cancer cells grown as monolayers; however, three-dimensional spheroid culture may be a better model for the multicellular resistance found in solid
tumors, such as
lung cancer. We tested the combinatorial apoptotic strategy of using
bortezomib together with
TNF-related apoptosis-inducing ligand (TRAIL), both in monolayers and in spheroids of A549
lung cancer cells. Indeed,
bortezomib plus TRAIL induced synergistic apoptosis in A549 cells grown as monolayers, but had little effect on A549 cells grown as three-dimensional multicellular spheroids. The acquired resistance of spheroids was not due to a limitation of diffusion, to survival pathways, such as
NF-kappaB or PI3K/Akt/mTOR, or to the up-regulation of
FLIP(S) (Fas-associated death domain-like
IL-1 beta-converting enzyme inhibitory
protein, short). We then investigated a role for the Bcl-2 family of anti- and proapoptotic
proteins. When cells formed spheroids, antiapoptotic Bcl-2 increased, whereas antiapoptotic Mcl-1 decreased.
ABT-737, a small molecule that inhibits Bcl-2, but not Mcl-1, abolished the multicellular resistance of A549 spheroids to
bortezomib plus TRAIL. In another
lung cancer cell line, H1299, acquisition of multicellular resistance in spheroids was also accompanied by an increase in Bcl-2 and decrease in Mcl-1. In H1299 spheroids compared with those of A549, however, Mcl-1 remained higher, and Mcl-1 knockdown was more effective than
ABT-737 in removing multicellular resistance. Our study suggests that the balance of Bcl-2 family
proteins contributes to the acquired multicellular resistance of spheroids, and suggests a possible target for improving the response of
lung cancer to
bortezomib therapies.