BMT-1 was obtained from Chembridge, Inc. (San Diego, CA, USA). Dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., St. Louis, MO, USA) was used to dissolve BMT-1.

Caspase-3, cleaved capase-3, caspase-8, cleaved capase-8, poly(ADP-ribose) polymerase (PARP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Beverly, MA, USA). The H⁺/K⁺-ATPase β-subunit (ATP4B) antibody was purchased from Abnova (Abnova, Taipei, Taiwan). The mitochondrial staining kit was purchased from Sigma (Sigma-Aldrich Co.). The H⁺/K⁺-ATPase kit was from Nanjing Jiancheng Biochemical Institute (Nanjing, China).

The MM cell lines, MM1.S, MM1.R, RPMI8226 and U-266, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). The cell lines were maintained in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (both from Gibco, Gaithersburg, MD, USA), L-glutamine and antibiotics (Gibco Laboratories, Grand Island, NY, USA). The MM cell liness were treated with varying concentrations of BMT-1 for the indicated times as described in the figure legends.

Cell growth inhibition was performed in accordance with the protocol from the Drug Evaluation Branch, National Cancer Institute (17,18). Briefly, cells were plated at an appropriate density in 96-well plates in RPMI-1640 with 10% (v/v) FBS and allowed to attach overnight. The cells were exposed to different drug dilutions for 48 h. Using the absorbance measurements [time zero (Tz), control growth (C) and test growth in the presence of drug at the four concentrations (Ti)]; the percentage of growth was calculated at each of the drug concentration levels. The percentage of growth inhibition was calculated as: [(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti≥Tz and [(Ti − Tz)/Tz] × 100 for concentrations for which Ti≤Tz. Growth inhibition of 50% (GI₅₀) was calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in net protein increase.

To detect the effect of BMT-1 in primary cells, peripheral blood mononuclear cells (PBMCs) were isolated from whole human blood following informed consent. The cells were treated with different concentrations of BMT-1 for 72 h. PBMCs were counted and analyzed using CellQuest software BD Accuri C6 Flow Cytometer and analyzed using CFlow software (version 1.0.243.1) (both from BD Accuri, Ann Arbor, MI, USA). The study protocol was approved by the Institutional Review Board (IRB) of Chengdu Medical College.

Cells (5×10⁵) treated with varying concentrations of BMT-1 were stained with JC-1 from the mitochondrial staining kit (Sigma) at 37°C for 20 min following the manufacturer’s procedures. Changes in cellular mitochondrial membrane potential were quantified by measuring the fluorescence intensity emission ratios at 590 nm (red)/525 nm (green) using the BD Accuri C6 Flow Cytometer and CFlow software (version 1.0.227.4; BD Accuri). The widely used mitochondrial membrane potential disruptor valinomycin was used as a positive control.

MM cells (5×10⁵) were cultured for 24 h in media alone or with varying concentrations of BMT-1. The cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), fixed with 70% (v/v) ethanol for 2 h, and pretreated with 100 μg/ml RNAse (Sigma) for 1 h. Cells were stained with propidium iodide (PI) (20 μg/ml; Sigma), and the cell cycle profile was determined using the BD Accuri C6 Flow Cytometer and CFlow software (version 1.0.227.4). Dual staining with Fluos-labeled Annexin V and PI was carried out to detect the induction of apoptotic cell death. Following treatment of 2×10⁵ cells with a test agent for 24 h, cells were washed with PBS and re-suspended in 100 μl binding buffer containing Annexin V-Fluos and PI (Annexin V-Fluos staining kit; Roche). After 15 min of incubation at room temperature, cells were analyzed on a BD Accuri C6 Flow Cytometer for the presence of Annexin V-Fluos-positive/PI-negative apoptotic cell populations.

MM cells were treated with varying concentrations of BMT-1 for the indicated times, as described in the figure legends. Cells were lysed for 30 min in ice-cold lysis buffer (BioTeke Corporation, Beijing, China). The cell lysates were cleared by centrifugation for 15 min at 12,000 × g and used for immunoprecipitation. Proteins (50 μg/lane) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane. The membranes were blocked with a 5% (w/v) milk solution for 1 h and incubated in the following primary antibodies: caspase-3, cleaved capase-3, caspase-8, cleaved capase-8, PARP and GAPDH.

The effects of BMT-1 on cytosolic pH were evaluated by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM (Beyotime Institute of Biotechnology, China). Approximately 1×10⁶ cells were incubated at 37°C for 30 min in 1 ml RPMI containing 1 μmol/l BCECF-AM. The cells were then washed in Hank’s balanced salt solution (HBSS), placed on ice, and analyzed with a FACSCalibur equipped with a 488-nm argon laser collecting the emission of BCECF-AM in the FL1 and FL2 channels (19,20). The relative cytosolic pH of individual cells was displayed in a two-dimensional dot-plot showing their fluorescence intensity at 533/30 nm (FL1, base) and 585/40 nm (FL2, acid).

Cells (5×10⁷) were collected by centrifugation at 600 × g for 5 min at 4°C, and washed with ice-cold 0.9% (w/v) saline solution. Cells were then homogenized on ice with a dounce tissue grinder, and the supernatant was transferred into tubes with final BMT-1 concentrations of 0, 25, 50 and 100 μM. To analyze the activity of H⁺/K⁺-ATPase, the OD value at 660 nm was detected according to the procedure of the H⁺/K⁺-ATPase kit (Nanjing Jiancheng Biochemical Institute, Nanjing, China) using the formula: Activity of H⁺/K⁺-ATPase (U/mg-prot) = (OD_test − OD_control)/OD_standard × standard concentration (0.5 μmol/ml) × (60 min/10 min) × dilution ratio (4.8)/protein concentration (mg/ml).

We first examined the growth inhibitory effect of BMT-1 on MM cell lines. By using the CCK-8 assay, we found that BMT-1 markedly inhibited the growth of U266, RPMI-8226, MM.1S and MM.1R cells with IC₅₀ values of 889 to 1476 nM (Fig. 1B). To examine the inhibition of BMT-1 on cancer cell growth that was not caused by toxicity, we performed in vitro analyses using cultured PBMCs in the absence and presence of BMT-1. As shown in Fig. 1C, non-significant lymphotoxicity was observed after exposure to BMT-1 at a concentration of 10 μM. Overall, these results suggest that BMT-1 inhibits the growth of MM cells that is not related to toxicity.

To further confirm that BMT-1 inhibits proliferation of MM cells through induction of cell cycle arrest, we analyzed the cell cycle distribution after PI staining. The percentage of MM1.S and MM1.R cells in the sub-G1 (apoptotic cells) phase was significantly elevated as the concentration of BMT-1 increased. For example, 10.3±3.6 and 14.8±4.6% of MM1.S cells existed in the sub-G1 region following treatment with 2 and 4 μM BMT-1 for 24 h, respectively (Fig. 2A and B). Collectively, we observed a significant increase in the sub-G1 cell population indicating the occurrence of apoptosis that was associated with a reduction in the G0–G1 fraction.

To confirm whether the growth inhibition of BMT-1 was caused by apoptosis, the percentage of Annexin V-positive cells was measured using flow cytometry. A dose-dependent increase in the population of apoptotic cells was observed (Fig. 3A). BMT-1 at a concentration of 4 μM induced 17.5 and 19.3% apoptosis in the MM1.S and MM1.R cells, respectively (cells positive for Annexin V, negative for PI) after 24 h when compared with 4.6% (MM1.S) and 5.1% (MM1.R) in the negative control (DMSO). To evaluate the contribution of active caspases in BMT-1-induced apoptosis, we evaluated cells carrying activated caspase-3 and PARP and found that pro-caspase-3 and pro-PARP were cleaved to yield smaller fragments in MM cells following BMT-1 treatment (Fig. 3B). These data indicated that BMT-1 indeed induced apoptosis in cancer cells, which was consistent with the results of the growth-inhibition assay.

The effect of BMT-1 on mitochondrial membrane potential was monitored. Any event that dissipates mitochondrial membrane potential prevents the accumulation of JC-1 in the mitochondria and thus, the dye is dispersed throughout the entire cell leading to a shift from red (J-aggregates) to green fluorescence (JC-1 monomers). As shown in Fig. 4A, after an 8-h drug exposure, BMT-1 depolarized the mitochondrial membranes in a concentration-dependent manner, a time-point when no cellular morphological change could be observed. Most control cells fluoresced red, while BMT-1-treated cells turned green. During mitochondrial-dependent apoptosis, the instability of mitochondria initiates the formation of the apoptosome and the sequential activation of caspase-8 and -9 (21,22). The present study found that BMT-1 induced the loss of mitochondrial membrane potential resulting in activation of caspase-8 and -9 (Fig. 4B). These results suggest that the mitochondrial dependent pathway is involved in BMT-1-trigged MM cell apoptosis.

As the energy power plants of the cell, mitochondria generate ATP by utilizing the proton electrochemical gradient potential (23). Thus, H⁺/K⁺-ATPase is extremely important to mitochondria since it uses energy to transport protons from the matrix of the mitochondrion to the inner and outer mitochondrial membranes. Therefore, H⁺/K⁺-ATPase inhibition disrupts the proton electrochemical gradient to induce a loss of mitochondrial function. In the present study, BMT-1 strongly depolarized mitochondrial membranes. Furthermore, all of the currently marketed H⁺/K⁺-ATPase inhibitors (e.g., omeprazole, lansoprazole and pantoprazole) are benzimidazole derivatives (24–26). Therefore, we proposed that BMT-1 would also inhibit H⁺/K⁺-ATPase activity.

The expression of H⁺/K⁺-ATPase in MM cells was determined by western blot analysis (Fig. 5A). To further confirm whether inhibition of H⁺/K⁺-ATPase activity is induced by BMT-1, the homogenized MM cells were treated with different concentrations of BMT-1 for 30 min and the activity of H⁺/K⁺-ATPase was detected. Compared with the control group (0 μM), BMT-1 at every concentration and lansoprazole (LAN), a positive inhibitor of H⁺/K⁺-ATPase, both inhibited H⁺/K⁺-ATPase from the MM1.S and MM1.R cells (Fig. 5B), while the activity of H⁺/K⁺-ATPases was not inhibited by the negative control inhibitor bortezomib (BOR). H⁺/K⁺-ATPases are ion pumps that use the energy of ATP hydrolysis to transport protons (H⁺) in exchange for potassium ions against their concentration gradients. Thus, the inhibition of H⁺/K⁺-ATPases by BMT-1 could block H⁺ extrusion, resulting in proton accumulation and intracellular acidification, which would terminate or limit the growth of cancer cells (19,27). To explore this possibility, the effect of BMT-1 on intracellular pH changes was examined; we used BCECF-AM as a pH-dependent fluorescent dye. Flow cytometry was used to measure intracellular fluorescence of BCECF-labeled MM cells, which were exposed to BMT-1. Treated cells exhibited a strong decrease in the FL1/FL2 ratio (indicating an acidic cytosolic pH), whereas little change in the emission ratio of control cells was observed. Hence, the results indicated that the intracellular pH was reduced in the cells exposed to BMT-1 (Fig. 5C).