Figure 2.

Dual PI3K/mTOR inhibition, combined with autophagy inhibition, enhances apoptosis of myeloma cells. Given that PI-103 induces autophagy in myeloma cells, we were interested to understand the effect on cells when autophagy was blocked. (a) The combination ofPI-103, with a constant subtoxic concentration of Bfa, over 24 h effectively inhibited the proliferation of MM.1S and H929 (left panels) at all PI-103 concentrations tested, as determined by triplicate MTT assays. **P<0.01. The combination was no more effective in U266 than PI-103 alone (right panel). (b) Initiation of apoptosis in MM.1S (left) and U266 (right) was confirmed by trypan blue exclusion and Annexin V/PI (propidium iodide) staining over a 24 h period with PI-103 and Bfa. Results represent the average of three independent experiments. (c) Specificity of the observed effect with PI-103 and Bfa was demonstrated by triplicate MTT assay by treating cells for 24 h with a second PI3K/mTOR inhibitor, BEZ235, in combination with another autophagy inhibitor, CHQ. When CHQ was applied at a constant subtoxic concentration, it significantly inhibited proliferation of MM.1S, but not U266, at all tested concentrations of BEZ235. (d) This was confirmed, in triplicate, by Annexin V/PI staining, indicating that apoptosis is enhanced by combination treatment in MM.1S but not U266. (e) In order to delineate whether it was the inhibition of PI3K or mTOR that was more important for the observed effect, we performed triplicate MTT assays with the mTOR inhibitor, Rapamycin, in combination with Bfa for 24 h. The combination significantly enhanced apoptosis at most of the tested Rapamycin concentrations in MM.1S but not in H929 or U266. (f) Left, PI-103 had minimal effect on patient-derived BMSCs (BMSC 1-3) seeded at two different densities (5 × 10⁴ (5) and 10 × 10⁴ (10) cells per well) as assessed by 24 h Wst1 assays. Right, Using conditioned medium from patient BMSCs, 24 h MTT assays were performed to determine the efficacy of the combination in MM.1S in the presence of bone marrow cytokines. Representative results from one of the three experiments are shown. CM, conditioned medium; normal, normal growth medium. (g) The chymotrypsin-like activity of the proteasome was determined in triplicate in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Bortezomib (Bort) was used as a positive control. *P<0.05; **P<0.01. (h) Detection of UPR markers, XBP1s, CHOP and ATF4, by RT-PCR in MM.1S and U266 following 6 and 24 h treatment with PI-103, Bfa or both. Data were normalized using actin. Representative plots are shown.(i) Western blot analysis was carried out at 24 h in MM.1S (left) and U266 (right) treated with PI-103, Bfa or both. Drug concentrations were as follows. PI-103: MM.1S, 2 μmol/l; U266, 5.5 μmol/l. Bfa: MM.1S and H929, 100 nmol/l; U266, 200 nmol/l. BEZ235: MM.1S, 800 nmol/l; U266, 25 μmol/l. CHQ: MM.1S and U266, 500 nmol/l. Bortezomib was used at 8 nmol/l for both cell lines.