The natural compound forskolin synergizes with dexamethasone to induce cell death in myeloma cells via BIM

We have previously demonstrated that activation of the cyclic adenosine monophosphate (cAMP) pathway kills multiple myeloma (MM) cells both in vitro and in vivo. In the present study we have investigated the potential of enhancing the killing of MM cell lines and primary MM cells by combining the cAMP-elevating compound forskolin with the commonly used MM therapeutic drugs melphalan, cyclophosphamide, doxorubicin, bortezomib and dexamethasone. We observed that forskolin potentiated the killing induced by all the tested agents as compared to treatment with the single agents alone. In particular, forskolin had a synergistic effect on the dexamethasone-responsive cell lines H929 and OM-2. By knocking down the proapoptotic BCL-2 family member BIM, we proved this protein to be involved in the synergistic induction of apoptosis by dexamethasone and forskolin. The ability of forskolin to maintain the killing of MM cells even at lower concentrations of the conventional agents suggests that forskolin may be used to diminish treatment-associated side effects. Our findings support a potential role of forskolin in combination with current conventional agents in the treatment of MM.


Primary Patients Samples.
To obtain primary myeloma cells, CD138+ cells were isolated from bone marrow specimens obtained through the Norwegian Myeloma Biobank using RoboSep automated cell separator and Human CD138 Positive Selection Kit (StemCell Technologies). All subjects provided informed consent to participate in the study. This study was carried out in accordance with the approved guidelines by the Regional Ethics Committee.
Bone marrow stromal cells (BMSCs) were prepared as described previously 24 . BMSC (2500 cells/well) were seeded in 96-well plates and allowed to adhere for at least 3 hours before addition of primary myeloma cells (5 × 10 3 cells/well). The cells were cultivated in RPMI medium supplemented with 2% human serum in a total volume of 200 μ L/well, and drugs were added at the concentrations indicated. All samples were run in duplicate.
Small interference RNA transfections. Control  were obtained from Thermo Fisher Scientific Biosciences GmbH (St. Leon-Rot, Germany). OPM-2 cells were transfected with siRNA using Lipofectamine ® RNAiMAX Transfection Reagent (Life technologies) according to manufacturer's protocol. Cells were rested 72 hours before being plated in experimental conditions. H929 cells were transfected with siRNA using Amaxa nucleofection technology (Lonza). Briefly, 2 × 10 6 cells were resuspended in Ingenio TM Electroporation Solution (Mirus Bio LLC, Madisson, WI, USA) and electroporated using program T-16. Cells were rested for 5 hours before being plated in experimental conditions. Knockdown was confirmed by western blot.
Specific cell death was calculated using the following equation: % specific cell death = (% experimental cell death in the drug-treated sample - % spontaneous cell death in the absence drug)/(100 -% spontaneous cell death in the absence of drug) x 100. Analysis of Cell Death by Flow Cytometry. Flow cytometry analysis was performed on a FACSCalibur (BD Biosciences). For determination of cell viability by exclusion of PI, cells in culture were incubated with PI (20 μ g/ml) prior to analysis. Percentage of dead cells was determined by gating for PI positive cells 25,26 . ScanR Viability Assay. This assay was performed as described previously 24 . Briefly, after 3 days of incubation, YO-PRO-1 (1 μ M) (Life Technologies) was added to the wells, and the plates incubated for 30 min at 37 °C. DRAQ5 (2,5 μ M) (eBioscience, San Diego, CA, USA) was added to the wells 15 min before image acquisition without further processing. The ScanR microscope-based screening platform was used for automated image acquisition (Olympus). Images were analyzed using the Olympus ScanR Analysis software, where the cells were classified and gated by size, morphology and fluorescence intensity. Single, circular cells were classified and gated as myeloma cells. YO-PRO-1 is a DNA-binding dye that stains apoptotic cells. Myeloma cells that showed low YO-PRO-1 staining and high intensity DRAQ5 staining were characterized as viable.
Immunoblot Analysis. 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 Na 3 VO 4 , 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 10 μ g/ml leupeptin, and 0.5% aprotinin) and equal amounts of proteins were separated by SDS-PAGE (Bio-Rad). After transfer to a nitrocellulose membrane (GE Healthcare, Amersham, UK) using a semi-dry 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) at room temperature. After washing, the membranes were incubated overnight at 4 °C with primary antibodies diluted in 5% non-fat dry milk in TBST or 5% BSA in TBST. After washing in TBST, the membranes were incubated for 1 hour 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 SuperSignal ® West Dura Extended Duration substrate (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol. Images were acquired with a Syngene ChemiGenius camera and presented by the GeneSnap software tool (Syngene, Cambridge, UK); the resulting SGD files were converted to JPEG files for image presentation. Densitometric analyses were performed on the primary SGD files to preserve maximal information, using the GeneTools 4.01 program (Syngene). Determination of synergism by the combination index (CI) method. The interaction of forskolin and dexamethasone was determined by combination index (CI) method 27 using the CompuSyn software program (ComboSyn Inc., Paramus, NJ, USA) to determine whether the combination was antagonistic, additive or synergistic. Data from cell viability by exclusion of PI were expressed as the fractional inhibition by the individual drugs or the combination in drug-treated cells, and CI values between two drugs were generated. CI values greater than 1 indicate antagonism, CI value of 1 indicates an additive effect, and CI values of less than 1 indicate synergy.

Results
Forskolin in combination with therapeutic agents: effects on death of multiple myeloma cell lines. In order to determine the effects of forskolin and the different therapeutic agents on death of human myeloma cell lines (HMCLs), incorporation of PI was measured after 72 hours of treatment. As shown in Fig. 1, forskolin alone induced cell death in a dose-dependent manner in the five HMCLs tested, with U266, OPM-2 and INA-6 being more sensitive than H929 and RPMI 8226 cells. Hence, in the highly forskolin-sensitive cell line U266, forskolin more than doubled the extent of basal cell death at a concentration of 1 μ M in U266 or 5 μ M in H929, whereas the percentage of dead cells more than tripled when forskolin was used at a concentration of 5 μ M in U266 and 50 μ M in H929.
We next evaluate the benefit of combining forskolin with different agents that are all frequently utilized in treatment of MM 28 . First, we assessed the death of U266 and H929 cells treated with selected concentration of the DNA damaging agents melphalan, cyclophosphamide and doxorubicin as well as to the proteasome inhibitor bortezomib using ranges that are commonly applied in preclinical models [29][30][31][32] . Both cell lines responded to the different drugs per se with variable sensitivity (Fig. 2a-d). Notably, only 2 μ M melphalan was required to obtain 70% cell death in H929 cells, whereas a ten times higher concentration of melphalan was needed to reach the same level of cell death in U266 (Fig. 2a). We next combined forskolin with these agents using a suboptimal concentration of forskolin together with a low dose of the particular agent. The level of cell death obtained by these treatments was compared to a single higher dose of that same agent. The rationale behind these experiments was to explore the possibility that the combined treatment with forskolin would make it possible to lower the concentrations of the therapeutic agents and still obtain a beneficial level of cell death. If so, this would potentially reduce the side effects of the conventional agents in a clinical setting. In U266 cells, 5 μ M of forskolin significantly increased the level of melphalan-induced cell death from approximately 30% (in the presence of 2 μ M melphalan alone) to 60% (when forskolin was combined with 2 μ M of melphalan) (Fig. 2e, left panel). Notably, the combination of melphalan (2 μ M) and forskolin (5 μ M) enhanced the cell death to the same extent as a single high dose (10 μ M) of melphalan alone. Forskolin also significantly improved the cell death induced by a single lower dose of melphalan in H929 cells (Fig. 2e, right panel), but in these cells a higher concentration (50 μ M) of forskolin was required. An even lower concentration of forskolin (1 μ M) was sufficient to enhance the death of U266 cells induced by 4 μ M of cyclophosphamide from 30% to 50% (Fig. 2f, left panel). Again the combined treatment with forskolin induced the same level of cell death as a five times higher concentration of cyclophosphamide alone. Comparable results were obtained upon treatment with doxorubicin (Fig. 2g). Hence, in both cell lines, forskolin significantly enhanced the cell death induced by 50 nM of doxorubicin (from 25% to 45%), and the combination with forskolin induced the same level of cell death as the three times higher concentration of doxorubicin alone (Fig. 2g). Forskolin also significantly improved bortezomib-induced cell death in both cell lines (Fig. 2h). It is noteworthy that in most of the cases we presently have analyzed, even a low concentration of forskolin alone was nearly as effective as the combination with a low concentration of the therapeutic agent. The exceptions, i.e. where there was a statistical significant higher cell death obtained by combining forskolin with a given agent as compared to forskolin alone, are indicated by asterisks in Fig. 2. The combination of forskolin and dexamethasone synergistically enhanced the death of HMCLs. Unlike bortezomib and the different DNA damaging agents tested, the glucocorticoid dexamethasone alone had no or modest effect in U266 and H929 respectively (Fig. 3a). The OPM-2 and the RPMI8226 MM cell line, however, were sensitive to dexamethasone treatment (Fig. 3a). Remarkably, dexamethasone was the only agent that was found to induce strong synergy at a low concentration of forskolin. Hence, in H929 cells, 1 μ M of forskolin and 0.1 μ M of dexamethasone alone did not induce any cell death, whereas the combination between these two compounds strongly enhanced the cell death from approximately 20% to 70% (Fig. 3b). The same combination also enhanced cell death in OPM-2 cells as compared to single agents alone (Fig. 3b). More moderate effect was obtained in RPMI8226 and INA-6 cell lines. Dexamethasone had no effect alone or in combination with forskolin in U266 cells (Fig. 3b). The combinatorial effect of forskolin with dexamethasone was evaluated by the CI method, and synergy was observed across a wide range of concentrations for the four MM cell lines tested (Fig. 3c). However, it is noteworthy that not all cell lines responded to the same extent. Hence, synergistic killing was stronger in H929 and OPM-2 cells as compared to the killing of RPMI8226 and INA-6 cells.
In order to establish that apoptosis was the mode of cell death induced by forskolin in combination with dexamethasone, apoptosis in terms of caspase activation was assessed in H929 and OPM-2 cells. Hence, caspase activation was determined by western blot analysis of cleaved caspase 3 and the caspase substrate poly (ADP-Ribose) polymerase (PARP). In accordance with the results on PI-incorporation, the western blot analysis showed that forskolin synergized with dexamethasone also in terms of caspase activation (Fig. 3d). Hence, the combination of 1 μ M of forskolin and 0.1 μ M of dexamethasone strongly enhanced the cleavage of both caspase 3 and PARP, whereas each of the compounds alone had negligible effects.   33,34 . In addition, BIM has been suggested to play an important role in glucocorticoid-mediated apoptosis 35 . There are three splice variants of BIM protein (BIM EL , BIM L and BIM S ) and all of them are pro-apoptotic. We therefore assessed the expression of all 3 isoforms of BIM after dexamethasone and forskolin treatment in H929 cells. Interestingly, treatment of H929 cells with combined low doses of forskolin and dexamethasone triggered a four time increase in BIM EL expression levels compared to control (Fig. 4), and the combination was significantly more potent to increase BIM EL levels than dexamethasone or forskolin alone. BIM L and BIM S were also strongly enhanced by forskolin plus dexamethasone treatment. In contrast, examination of the other BCL-2 family members BCL-2, BCL-XL, and BAX revealed only minor alterations in protein expression (data not shown). ERK1/2 has been shown to promote BIM degradation 36 . Consistently, we observed that forskolin alone or in combination with dexamethasone inhibited the activating phosphorylation of ERK (Fig. 4), suggesting that BIM upregulation could result from inhibition of ERK signaling.
To determine whether BIM mediated the apoptosis induced by the combination of forskolin and dexamethasone, BIM expression was knocked down by using the siRNA strategy. Quantification of protein bands by densitometry revealed that knock down reduced the levels of BIM EL by 40% in OPM-2 cells and 30% in H929 cells as compared with control siRNA, and the levels of BIM L and BIM S were similarly knocked down (data not shown). Importantly, the knock down of BIM inhibited the apoptotic cell death induced by the combination of forskolin and dexamethasone in both H929 and OPM-2 MM cells. Hence, the induced activation of caspases, as envisioned by proteolytic cleavage of PARP and caspase 3, was markedly inhibited upon knock down of BIM both in H929 cells (Fig. 5a) and to a greater extent in OPM-2 (Fig. 5b). Furthermore, the cell death induced by the combination of forskolin and dexamethasone was significantly reduced by knocking down BIM in H929 cells as compared with cells treated with control siRNA (Fig. 5c). Knocking down BIM also decreased the cell death induced by combining forskolin and dexamethasone in OPM-2 cells, but the effect was less pronounced (data not shown). Taken together, these results suggest that apoptosis induced by the combination of forskolin and dexamethasone is at least in part mediated via BIM.

Forskolin enhances cell death mediated by therapeutic agents on primary cells from multiple myeloma patients.
To support the notion that forskolin can be of potential use in MM therapy, CD138+ B cells isolated from ten patients with MM were co-cultured with bone marrow stromal cells and treated with forskolin in combination with melphalan, cyclophosphamide, bortezomib or dexamethasone, respectively. The cell death of primary myeloma cells was assessed after 72 h hours of treatment by using an automated fluorescence microscope platform as previously described 24 . In accordance with the results obtained on cell lines (Figs 2 and 3), forskolin increased the activity of melphalan (Fig. 6a), bortezomib (Fig. 6c) and dexamethasone (Fig. 6d). The effect was statistically significant with low concentrations of bortezomib and melphalan, and with the two concentrations of dexamethasone that were tested. Forskolin also increased the activity of cyclophosphamide, however without reaching statistical  Cells were stained with DRAQ5/YO-PRO-1, and cell viability was evaluated by using the ScanR microscope as previously described 24 . *P < 0.05, **P < 0.01. Panel e: results from panel d presented as bar lines for each patients. significance (Fig. 6b). It is noteworthy that the combination of forskolin and dexamethasone was the most efficient treatment combination in terms of killing the primary MM cells. Hence, 5 μ M of forskolin in combination with 1 μ M of dexamethasone reduced the viability of the MM cells to a larger extent than even a ten times higher concentration of dexamethasone alone (Fig. 6d). It should be noted that not all patient-derived MM cells responded to the combination of forskolin and dexamethasone. Hence, forskolin had no effect on dexamethasone-mediated killing of the three patient-derived cells MMP5, MMP6 and MMP10, and only minor potentiation of dexamethasone-induced killing was observed in MMP7 and MMP8 (Fig. 6e). Importantly however, forskolin potentiated the effect of dexamethasone in 5 of the 10 patient-derived MM cells, and in these cells the combination of dexamethasone and forskolin was always more effective than even a ten times higher dose of dexamethasone alone (Fig. 6e).
As it is well established that BMSC may confer treatment resistance to MM cells 24 , it should be emphasized that forskolin alone or in combination with the conventional anticancer drugs in the present study was able to kill the primary MM cells even in the presence of BMSCs.

Discussion
Despite the development of new combination therapies during the past 15 years, the majority of multiple myeloma patients relapse, their tumor cells become chemoresistant, and they die from the disease. Furthermore, chemotherapeutic agents used for the treatment of MM have substantial side effects including peripheral neuropathy, thromboembolism, neutropenia, anemia, fatigue and gastrointestinal complications 6 . Hence, there is a need for new treatment strategies aiming at reducing side effects and enhancing the efficiency of existing MM therapies.
We previously showed that single treatment by forskolin kills MM cells in vitro and delays tumor growth in vivo 8 . Combined with the notion that clinical studies have reported limited side effects of forskolin 20,23 , we wished to elucidate the potential of using forskolin in combination with existing therapies for MM. In the present study, we report that combination of forskolin with the common MM therapeutic agents melphalan, cyclophosphamide, doxorubicin, bortezomib, and dexamethasone significantly increased apoptosis compared to single agent treatment. In two different MM cell lines, combination of forskolin with low doses of DNA damaging agents induced the same extent of cell death as two to five times higher doses of the single agent treatment alone. Interestingly, dexamethasone and forskolin had a strong synergistic effect on cell death in H929 and OPM-2 cells. This is in line with a previous report demonstrating synergistic inhibition of MM cells between dexamethasone and PDE inhibitors and/or activators of the G protein coupled receptor adenosine A2A both of which can enhance cAMP levels in the cells 13,37 . It should be noted that the combination of forskolin and dexamethasone was not equally potent in all MM cell lines. Hence, despite the fact that the Chou-Talalay method revealed synergistic killing also of RPMI8226 and INA-6 cells, these cells were much less responsive to the combination of forskolin and dexamethasone than were H929 and OPM2-cells.
Forskolin also enhanced melphalan-and dexamethasone-induced cell death in patient-derived primary MM cells. As it is well established that BMSCs may confer treatment resistance to MM cells 24,38,39 , the ability of forskolin to kill primary myelomas was particularly interesting given the fact that the cells were co-cultured with BMSCs. Importantly, forskolin did not significantly decrease the viability of the BMSCs (data not shown) supporting the perception of low toxicity of forskolin on normal cells. It should be remarked that for four out of the ten MM patients primary cells we tested, forskolin alone had little or no effect on cell death, supporting the notion that forskolin should always be combined with conventional chemotherapy in clinical settings.
In an attempt to elucidate the mechanisms whereby forskolin may interfere with the various MM therapeutic agents, we investigated steps in the apoptotic pathway as well as upstream signaling events. The ability of forskolin alone or in combination with dexamethasone to kill MM cells was confirmed by assessing the cleavage of caspase 3 and PARP. Upstream activation of the intrinsic apoptotic pathway is regulated by BCL-2 family members. In this study, we demonstrated that forskolin plus dexamethasone treatment rapidly increases the levels of the pro-apoptotic BCL-2 member BIM. Notably, knocking down BIM partially prevented the cell death as well the caspase activation induced by the combination of forskolin and dexamethasone, suggesting the involvement of BIM in the apoptotic cell death. The present findings are in accordance with previous studies showing that BIM mRNA and protein expression is increased in response to activation of the cAMP/PKA pathway in certain T lymphoid cell lines 40,41 , and that knock down of BIM with shRNA blunts cAMP-promoted apoptosis in the S49 murine T-lymphoma cell line 41 . Microarray analyses have also demonstrated that glucocorticoids can upregulate BIM levels in lymphoid cells 35,42 . Hence, BIM has been suggested to be a convergence point for the killing of lymphoid cells by glucocorticoids and treatments that raise the levels of cAMP 40 . Here we show for the first time that forskolin, a natural compound that raises intracellular levels of cAMP, is able to enhance the expression of BIM also in MM cells, and that forskolin synergizes with dexamethasone to induce BIM upregulation and apoptosis in MM cells.
How forskolin upregulates BIM remains unclear. The pro-apoptotic activity of BIM is regulated by transcriptional as well as posttranscriptional mechanisms. The forkhead-like transcription factor FOXO3a is a key transcriptional regulator of BIM 43 , whereas posttranscriptional regulation of BIM occurs through a series of phosphorylation events. ERK phosphorylates FOXO3a thereby increasing its degradation via an MDM2-dependent proteasomal pathway 44 . In addition, ERK has been shown to regulate phosphorylation of BIM EL on serine 69, hence promoting its degradation via the proteasomal pathway 36 . In agreement with these results we found that ERK phosphorylation is inhibited by forskolin, thus providing a possible mechanism whereby forskolin may upregulate BIM.
Interactions between MM cells and the bone marrow microenvironment (BMM) activate a multitude of proliferative and anti-apoptotic signaling pathways, including the Ras-Raf-MAPK kinase (MEK)extracellular signal-regulated kinase (ERK) pathway and the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway 45 . We have previously reported that forskolin kills U266 cells by inhibiting the JAK-STAT pathway 10 , and here we could show that forskolin alone or in combination with dexamethasone, melphalan or bortezomib ( Fig. 4 and data not shown) also inhibit ERK-phosphorylation. Combined disruption of ERK-signaling and the JAK-STAT pathway is required to induce apoptosis of MM cells in the presence of BMSCs 46 . Hence, by demonstrating the inhibition of both signaling pathways, the results of the present study support the clinical potential of combining forskolin with conventional therapeutic agents.
Natural compounds such as forskolin are often considered to be affordable and, although not necessarily true, to have low general toxicity 47 . As many natural compounds have the advantage of targeting multiple signaling pathways [48][49][50][51] , a growing number of clinical studies are now evaluating their potentials in cancer treatment in combination with conventional chemotherapies 52 . The present study opens for clinical studies along the same lines. The natural compound forskolin has for centuries been applied in traditional medicine 15 , and its safety has also been documented in conventional modern medicine 23 . We suggest that forskolin, by targeting multiple signaling pathways in MM cells, might reduce the doses of chemotherapeutic agents required to kill such cells in vivo and thereby limit the severe side effects associated with current conventional therapies of MM.