Cyclic AMP induces apoptosis in multiple myeloma cells and inhibits tumor development in a mouse myeloma model

Forskolin and rolipram (Sigma; Saint Louis, MO, USA) were diluted in dimethyl sulfoxide (DMSO), 8CPT-cAMP (Biolog, Bremen, Germany) was diluted in distilled water, whereas prostaglandin E₂ (Cayman, Ann Arbor, MI, USA) was diluted in ethanol. Propidium iodide, DMSO, saponin, paraformaldehyde and bovine serum albumin (BSA) were purchased from Sigma. The cationic fluorescent carbocyanine dye 5,5',6,6'-Tetrachloro-1,1',3,3' -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was from Calbiochem (San Diego, CA, USA).

Antibodies against caspase 3 (8G10), caspase 9 (the mouse-specific 9504 and the human-specific 9502) and PARP were purchased from Cell Signaling Technologies (Danvers, MA, USA). P53 (fl393) antibody was purchased from Santa Cruz Biotechnology (Fremont, CA, USA). Antibody against GAPDH (Sigma) was used as a loading control. Anti-goat and anti-mouse HRP-conjugated secondary antibodies were purchased from Bio-Rad (Hercules, CA, USA).

Cells were irradiated using a ¹³⁷Cs source at 4.3 Gy/min.

The BCP-ALL cell line Reh [23] was cultured as previously described [19]. The transplantable BALB/c mineral oil-induced plasmacytoma cell line, MOPC315 [21], was used to generate a subline, MOPC315.4, that grew well in vitro and in vivo [24]. A subline of MOPC315.4, MOPC315.BM (Bogen et al., unpublished), was used for the present experiments. Some experiments employed MOPC315.BM labeled with the fluorescent protein DsRed. For simplicity, the MOPC315.BM subline will be referred to as MOPC315 throughout the paper. The cells were cultured in vitro in RPMI 1640 (Invitrogen, Paisley, UK) containing 2 mM L-glutamine, supplemented with MEM non essential amino acid (Sigma), 1 mM sodium pyruvate (Sigma), 50 μM monothioglycerol (Sigma), 12 μg/ml gentamycin (Sigma) and 10% heat-inactivated FBS (Lonza, Verviers, Belgium). The human multiple myeloma cell line, INA-6 cells, was a kind gift from Dr. M. Gramatzki (Erlangen, Germany) and were cultured in RPMI 1640 (Invitrogen) containing 2 mM L-glutamine (Invitrogen), supplemented with 1 ng/ml Il-6 (Invitrogen), 12 μg/ml gentamycin (Sigma) and 10% heat-inactivated FBS (Lonza). The U266 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and was cultured in RPMI 1640 (Invitrogen) containing 2 mM L-glutamine (Invitrogen), supplemented with 15% heat-inactivated FBS (Sigma), 100 U/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen).

Flow cytometry analysis was performed on a FACS Calibur (Becton-Dickinson). For determination of cell viability by exclusion of propidium iodide (PI), 500 μl of cell culture were incubated with 20 μg/ml PI for 10 min at room temperature prior to analysis. The cationic fluorescent carbocyanine dye, 5,5',6,6'-tetrachloro-1,1',3,3' -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used to assess changes in the mitochondrial membrane potential (ΔΨm) observed in apoptotic cells. Cells were incubated for 15 min at 37°C with 15 μg/ml JC-1 before analysis. For determination of apoptotic cells, TUNEL assays were performed by using an In Situ Cell Death Detection Kit, Fluorescein from Roche (Mannheim, Germany). Briefly, cells were washed in ice cold PBS before being fixed with 4% paraformaldehyde and permeabilized with 0,1% saponin. Cells were washed in ice cold PBS before incubation in the TUNEL reaction mix for 1 h at 37°C. After washing the cells 3 times, the cells were analyzed by flow cytometry.

Cells were lysed in RIPA buffer (50 mM Tris [pH7.5], 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 10 μg/ml leupeptin, and 0.5% aprotinin) and an equal amount of proteins (50 μg) was separated by SDS-PAGE (Bio-Rad) electrophoresis. After transfer to a nitrocellulose membrane (GE Healthcare) using a semidry transfer cell (Bio-Rad), proteins were detected by standard immunoblotting procedures. In brief, the nitrocellulose membranes were washed in Tris buffered saline and 0.1% Tween (TBST) and incubated in blocking solution (5% non-fat dry milk in TBST or 5% BSA in TBST) at room temperature. After washing, the membranes were incubated overnight at 4°C with primary antibodies diluted in blocking solution. After washing in TBST, the membranes were incubated for 1 h with HRP-conjugated secondary antibody diluted in blocking solution, followed by a final washing at room temperature. Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, UK) or the SuperSignal^® west Dura Extended Duration substrate (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol.

Adult BALB/c nude mice (purchased from Charles River, Germany) were injected subcutaneously in the interscapular region with 5 × 10⁵ tumor MOPC315.DsRed cells suspended in 100 μL PBS. Two days after injection of the cells, 5 mice were injected intraperitoneally with 4-5 mg/kg forskolin diluted in a PBS/DMSO solution (15:0.1), and 5 mice were injected with the vehicle. In a separate experiment, forskolin (or vehicle) was injected 3 times on days 2, 4 and 6. Tumor growth was followed daily by palpation and imaging. Mice with tumor diameters of 15-20 mm were killed by cervical dislocation. The study was approved by the National Committee for Animal Experiments.

Mice were anaesthetized with 2.5% isoflurane (Baxter As, Norway). Immediately afterwards, they were placed in a light-sealed imaging chamber and kept anaesthetized throughout the imaging period.

Images were acquired using a combination of excitation (30 nm passband) and emission (20 nm passband) filters on an IVIS Spectrum Imaging System (Caliper Life Sciences). The following spectral channels were used (excitation:emission center wavelength in nm): 465:540, 465:580, 535:600 and 570:620. Spectral images were recorded in units of photons/second/cm²/sr and imported as 32 bit floating point TIFF files into Mathematica 5.2 (Wolfram Research) for further processing. Images were scaled with an excitation light correction factor [25] yielding normalized fluorescence efficiency (NFE) images for further processing. Background reference autofluorescence spectrum was recorded from the interscapular region on day 0 before MOPC315 injection. A reference MOPC315.DsRed spectrum was determined from a region containing a localized tumor (day 5) with the reference autofluorescence subtracted. MOPC315.DsRed specific signal was determined by linear (pseudo-inverse) unmixing [26], yielding DsRed fluorescence maps, which were thresholded, intensity color-coded and overlaid a white light illuminated image. Quantification of MOPC315.DsRed fluorescence was done by computing the total DsRed fluorescence for above-threshold pixels for each animal.

We have previously shown that in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, elevated intracellular levels of cAMP prevent apoptosis induced by a variety of DNA-damaging cytotoxic agents, including ionizing radiation (IR). We have also demonstrated that destabilization of p53 is a key feature in this process [20]. Now we have compared the effects of the adenylyl cyclase-activating diterpine forskolin [27] on IR-mediated cell death of the BCP-ALL cell line Reh and the plasmacytoma cell line MOPC315. Figure 1A (left panel) shows that forskolin (50 μM) alone had no effect on the viability of the Reh cells, but unsurprisingly prevented IR-induced apoptosis, as measured 24 h later. In sharp contrast, forskolin did not prevent IR-mediated death of the myeloma cells (Figure 1A, right panel), but rather potentiated the effect of irradiation. More intriguingly, after 24 hours of sole forskolin treatment, the percentage of dead MOPC315 cells increased from ~16% to ~50%.

To confirm the differential effect of forskolin on IR-treated Reh cells and MOPC315 cells, the effect of elevating cAMP on p53 expression was analyzed. In accordance with our previous result [20], forskolin prevented the IR-induced stabilization of p53 in Reh cells (Figure 1B). In contrast forskolin had no effect on p53 induced by IR in MOPC315 cells. Importantly, forskolin alone decreased the p53 levels in MOPC315 cells, indicating that induction of p53 is not involved in cAMP-mediated cell death of MOPC315 cells. Myeloma cells were notably more sensitive to irradiation than Reh cells; only 2 Gy was used to obtain similar death in MOPC315 cells compared to 10 Gy in Reh cells.

MOPC315 cells were treated for 24 h with increasing doses of forskolin, or with 50 μM of forskolin, at various time points. Cell death was measured by incorporation of PI. Forskolin induced death of MOPC315 cells was both dose- and time-dependent (Figure 2A and 2B, respectively). Death occurred at doses as low as 0.1 μM forskolin, and statistically significant death could be detected already after 8 h with 50 μM forskolin.

In addition to the activation of adenylyl cyclase, forskolin has been reported to modulate other cellular processes, such as ion channels [28, 29]. We tested the effect of other cAMP increasing agents to verify that forskolin-induced cell death was mediated by intracellular accumulation of cAMP. This included the cell membrane permeable cAMP analog 8-chlorophenylthio-cAMP (8CPT-cAMP) and prostaglandin E2 (PGE2), which increases intracellular levels of cAMP through the activation G-protein-coupled receptors [30, 31]. MOPC315 cells were killed in a dose-dependent manner by 8CPT-cAMP or PGE2 (Figure 2C and 2D, respectively), supporting the notion that forskolin kills the MOPC315 cells via induction of cAMP.

To verify that the killing of murine multiple myeloma cells by cAMP was applicable to human myeloma cells, the human multiple myeloma cell line U266 and the IL-6-dependent human myeloma cell line INA-6 were included. The cells were treated for 24 h with increasing doses of forskolin or 8CPT-cAMP, and cell death was assessed by PI incorporation. Both human multiple myeloma cell lines were sensitive to intracellular elevation of cAMP (Figure 3A, B and 3C), indicating that elevation of intracellular levels of cAMP indeed also induces cell death in human multiple myeloma cells. By using a cAMP Biotrack enzymeimmunoassay (EIA), we verified that forskolin increased the intracellular cAMP concentrations in these two cell lines (data not shown). To further confirm the involvement of the cAMP signaling pathway in killing multiple myeloma cells, INA-6 cells were treated for 24 hours with a low dose of forskolin in combination with rolipram, an inhibitor of PDE4. Rolipram or a low dose of forskolin alone induced little or no cell death, whereas a combination of the two compounds markedly increased cell death (Figure 3D).

To ascertain whether cAMP induces cell death by apoptosis, MOP315 cells treated with forskolin or 8CPT-cAMP were analyzed for DNA fragmentation by TdT-mediated dUTP nick end labeling (TUNEL) technique, or by analysis of changes in mitochondrial membrane potential (Δ Ψm) by staining the cells with JC-1. Forskolin and 8CPT-cAMP induced similar percentage of dead cells whether cell death was measured as percentage of cells with fragmented DNA (TUNEL assay), by changes in mitochondrial membrane potential (JC-1 staining), or by simple incorporation of PI (Figure 4A), clearly suggesting that cAMP induces apoptotic death of multiple myeloma cells.

To verify cAMP induced death of myeloma cells to be apoptoic, we investigated the downstream events following mitochondrial depolarization. Mitochondrial outer membrane permeabilization results in the release of cytochrome C from the intermembrane space into the cytosol, triggering the assembly of the caspase-activating complex that mediates autocleavage and activation of caspase 9 [32]. Once activated, caspase 9 activates downstream effector caspases such as caspase 3 provoking the cleavage of several proteins, such as PARP, which ultimately leads to cell destruction [33]. MOPC315 cells and INA-6 cells were treated with 50 μM forskolin or vehicle, and expression of cleaved caspase 3, caspase 9 and PARP were examined by western blot analysis. Forskolin induced profound cleavage of caspase 9, caspase 3 and PARP in INA-6 cells, and to a lesser extent in MOPC315 cells (Figure 4B), confirming that cAMP indeed kills human and murine multiple myeloma cells by activating the apoptotic machinery

Having shown that elevation of intracellular cAMP kills multiple myeloma cells in vitro, we explored the therapeutic potential of cAMP-elevating compounds on tumor growth in vivo, taking advantage of a previously established mouse model for multiple myeloma based on subcutaneous injection of MOPC315 cells [24] pre-labeled with the fluorescent protein DsRed [34]. BALB/c nude mice were subcutaneously injected between the shoulders with 5 × 10⁵ MOPC315 cells stably transfected with the gene encoding the fluorescent protein DsRed (MOPC315.DsRed cells). Two days after inoculation of the cells, 5 mice were intraperitoneally injected with a single dose of forskolin (4-5 mg/kg), whereas 5 mice were injected with the same volume of vehicle. Tumor size was followed daily by in vivo imaging of DsRed fluorescence using an IVIS Spectrum Imaging System from Caliper Life Sciences. All 10 mice eventually developed tumors, but a single dose of forskolin substantially delayed the tumor growth in vivo (Figure 5A). Similar results were obtained in a separate experiment where mice were injected 3 times with forskolin or vehicle on days 2, 4, and 6 after tumor cells injection (Figure 5B). Statistical differences between vehicle treated and forskolin treated mice is achieved (p < 0.05) from day 6 after tumor cell injection. Figure 5C shows in vivo images of mice taken at day 7. Together, these results suggest that cAMP-elevating compounds may indeed have a therapeutic potential in treatment of multiple myeloma.