Multiple myeloma (MM) is a clonal plasma cell disorder affecting the immune system with various systemic symptoms. MM remains incurable even with high dose chemotherapy using conventional drugs, thus necessitating development of novel therapeutic strategies. Gossypol (Gos) is a natural polyphenolic compound extracted from cotton plants, and has been shown to possess anti-neoplastic activity against various tumors. Recent studies have shown that Gos is an inhibitor for Bcl-2 or Bcl-XL acting as BH3 mimetics that interfere interaction between pro-apoptotic BH3-only proteins and Bcl-2/Bcl-XL. Since most of the patients with MM overexpress Bcl-2 protein, we considered Gos might be a promising therapeutic agent for MM. We herein show that Gos efficiently induced apoptosis and inhibited proliferation of the OPM2 MM cell line, in a dose- and time-dependent manner. Gos induced activation of caspase-3 and cytochrome
Multiple myeloma (MM) is a clonal plasma cell disorder affecting both the immune system and bone metabolism, and it remains incurable even with high dose chemotherapy (
Human
Gossypol (Gos) is a promising anticancer agent presently under clinical trial (
In the present study, we tried to elucidate the signaling pathways regulating Gos-induced apoptosis in MM cells. Besides a role as BH3 mimetics, we found that Gos inhibits interleukin (IL)-6 signaling, thereby dephosphorylates Bcl-2 and downregulates Mcl-1. This suggests that inhibition of IL-6 signaling may be an alternative mechanism for Gos-induced apoptosis in MM cells besides the interference of BH3-dependent interaction.
Human myeloma cell line (OPM2) was obtained from the Japan Cancer Research Resources Bank (Tokyo, Japan). Cells were maintained in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO2. Cell morphology was examined by staining cytospin preparation of the cells with Giemsa solution. Viability of the cells was evaluated by trypan blue dye exclusion method.
Gossypol was purchased from Sigma and dissolved in DMSO at a stock concentration of 100 mM, which was stored at −30°C. The pan-caspase inhibitor Z-VAD-FMK, and JAK2 inhibitor AG490 were from Calbiochem (La Jolla, CA, USA).
The antibodies for caspases-3, caspase-8, STAT3, pTyr705-STAT3, pSer727-STAT3, pSer70-bcl2, Bid, Bad, Akt, p38MAPK, pThr180/Tyr182-p38MAPK, cytochrome
Apoptosis was examined by cellular morphology or staining cells with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) by Annexin V staining kit (BD Bioscience Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Stained cells were analyzed by FACSCalibur (Becton-Dickinson, San Jose, CA, USA) with CellQuest software (Becton-Dickinson).
For DNA fragmentation assay, cells were harvested and incubated in a lysis buffer [10 nM Tris-HCl (pH 7.4), 10 mM EDTA, 0.5% Triton 100-X] at 4°C, which were then centrifuged at 15,000 rpm for 15 min at 4°C. Supernatants were collected and incubated with RNase A (Sigma) at 50 μg/ml and proteinase K (Sigma) for 1 h at 37°C. DNA samples were subjected to 2% agarose gel and were visualized by ethidium bromide staining.
For pharmacological inhibition of apoptosis, cells were pre-incubated with 20 μM of pan-caspase inhibitor, Z-VAD-FMK for 2 h prior to addition of Gossypol (5 μM). The final concentration of DMSO in the experiment did not exceed 0.1%. Effect of Z-VAD-FMK was assessed by DNA fragmentation assay.
Cells were washed with PBS and subjected to staining with 40 nM of DioC6 (Sigma-Aldrich Japan, Tokyo, Japan) for 30 min at 37°C. The stained cells were washed with PBS and analyzed by flow cytometry.
Cells were suspended in hypotonic solution [0.1% Triton X-100, 1 mM Tris-HCl (pH 8.0), 3.4 mM sodium citrate, 0.1 mM EDTA] and stained with 50 μg/ml of PI. Cells were analyzed by flow cytometry and the population of cells in each cell cycle phase was determined using ModiFIT software (Becton-Dickinson).
Cells were lysed in a lysis buffer [1% NP40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 40 mM Tris-HCl (pH 8.0) and 150 mM NaCl] at 4°C for 15 min. Mitochondria and cytosol were fractionated using the Mitochondria/Cytosol Fractionation kit (BioVision Inc., Mountain View, CA, USA). Cell lysates (15 μg of protein/lane) were fractionated in SDS-polyacrylamide gels and were transferred onto the nylon membranes (Immobilon-P; Millipore, Bedford, MA, USA). Membranes were probed with primary antibodies and horseradish peroxidase labeled secondary antibodies as described previously. Bound antibodies were detected by enhanced chemiluminescence (ECL) kit (Amersham, Buckinghamshire, UK).
Data are expressed as mean ± standard deviation (SD) Statistical analyses were performed by unpaired Student’s t-test. P-values <0.05 were considered statistically significant.
We first examined the effect of Gos on the proliferation of MM cell line, OPM2 cells. As shown in
Next we examined the effect of Gos on the cell cycle status of OPM2 cells. Cells were incubated with Gos and the cell cycle status was examined by flow cytometry at various time-points (
To investigate the molecular mechanism of Gos-induced cell cycle arrest in OPM2 cells, the expression of cell cycle-associated genes was examined by western blotting. As shown in
It is well known that the activation of caspases plays a pivotal role in the apoptosis-signaling pathway (
To examine the activation of mitochondrial apoptosis pathway by Gos, we next examined the mitochondrial changes evoked by Gos treatment. Mitochondrial changes during apoptosis include permeability transition pore opening and the collapse of the mitochondrial transmembrane potential (ΔΨm), which results in the release of cytochrome
We have also reconfirmed the activation of death receptor mediated pathways as shown by the activation of caspase-8 in Gos-induced apoptosis by checking the expression of Bid protein, since Bid is a substrate of activated caspase-8. As shown in
We next examined the expression of apoptosis-associated proteins during Gos-induced apoptosis in OPM2 cells. In addition to the decrease of Bid protein as shown above, MCL-1, one of the major anti-apoptotic proteins in hematopoietic cells, clearly decreased by 24 h of Gos-treatment. In contrast, however, Gos did not affect the levels of pro-apoptotic Bax and Bad protein as well as anti-apoptotic Bcl-2 and Bcl-XL proteins (
Although protein levels did not change by Gos-treatment, we suspected that BCL-2 might be functionally impaired, since anti-apoptotic function of BCL-2 can be regulated by phosphorylation at serine 70 (Ser70) (
Recent studies have shown that ERK1/2, p38MAPK, protein kinase C (PKC) or protein phosphatase (PP) 2A were involved in the phosphorylation or dephosphorylation of BCL-2, respectively (
It is well known that interleukin (IL)-6 plays a critical role in the proliferation of myeloma cells including OPM2 (
To elucidate the acting point of Gos-mediated inhibition of IL-6 signaling, we examined whether inhibition of JAK2 was sufficient to cause deregulation of apoptosis-associated proteins evoked by Gos stimulation. As shown in
These data suggest that Gos-induced deregulation of Bcl-2 and Mcl-1 was mediated through inhibition of IL-6 signaling at the level leading to JAK2 activation.
Gos is a natural small molecule inhibitor for Bcl-2 or Bcl-XL that has been shown to act as a potent inducer of apoptosis in multiple tumor cell types. Previous studies have shown that Gos downregulated the expression of Bcl-2/Bcl-XL/Mcl-1 proteins in multiple tumor cell lines. In contrast, however, our data indicated that Gos treatment did not affect the protein levels of Bcl-2 or Bcl-XL. Instead, Gos attenuated Ser-70 phosphorylation of Bcl-2, which is critical for executing its full and potent anti-apoptotic effects (
We also demonstrated that another anti-apoptotic protein, Mcl-1, was downregulated by Gos treatment. This should be due to the inhibition of IL-6 signaling by Gos as well, since Mcl-1 is a downstream target of IL-6 (
Gos shares a structural profile of BH3 mimetics with other inhibitors for Bcl-2/Bcl-XL such as Genasense, TW-37, Obatoclax or ABT-263 that are presently under clinical trials (
Molecular mechanism of Gos-mediated inhibition of IL-6 signaling is not clear at present. In cytokine signaling, ligand-induced dimerization of receptor chains provokes activation of Jak kinases, which then leads to phosphorylation of receptor chains and initiates downstream signaling cascades (
During the preparation of this report, we noticed that another group reported apoptosis inducing effect of Gos in MM cells (
In conclusion, we demonstrated that Gos, a natural BH3 mimetics, induces apoptosis in MM cells not only through displacement of BH3-only proteins from Bcl-2/Bcl-XL, but also via the inhibition of IL-6 signaling. Future study will focus on the mechanism how Gos inhibits IL-6 signaling in MM cells. It is expected that this allows us to obtain vital information on novel strategies for IL-6 inhibition, which leads to a development of new drugs for MM treatment.
The authors would like to thank Chika Saito for excellent technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports and Technology of Japan; the Ministry of Health, Labor, and Welfare of Japan (KAKENHI 24591409). This study was also supported in part by the National Cancer Research and Development Fund (26-A-4).
Growth inhibition and apoptosis induction of OPM2 cells by Gossypol. (A) OPM2 cells were treated with various concentrations (0–20 μM) of Gos for the time indicated (24–72 h). Viability of the cells was exmined by trypan blue dye exclusion method. Results were expressed as the mean ± SD (n=3). (B) Morphological changes of OPM2 cells by Gos-treatment. Cells were treated with 5 μM of Gos for 24 h, cytospun onto glass slides, and assessed for their morphology by Giemsa staining (original magnification, ×1,000). (C) Assessment of apoptosis by Annexin V/PI staining. OPM2 cells were treated with 5 μM of Gos for 0, 12, 24 or 48 h, and analyzed as described in Materials and methods. Representative figures from three independent experiments are shown.
Cell cycle analyses of OPM2 cells treated with Gossypol. (A) Cell cycle analysis of OPM2 cells with Gos. Cell were cultured with 5 μM of Gos for 0–24 h and then stained with PI as described in Materials and methods. DNA content was analyzed by flow cytometry. Representative figures from three independent experiments are shown. (B) Expression of cell cycle related proteins in OPM2 cells treated with 5 μM of Gos for the indicated times. β-actin was used as a loading control.
Effects of Gossypol on caspase activation. (A) Western blot analysis of caspase-3 and -8. OPM2 cells were treated with 5 μM of Gos for the indicated times. Activation of caspases as shown by the appearance of cleaved caspase-3 (upper panel) or caspase-8 (lower panel) is shown by the arrows. (B) DNA fragmentation assays. DNA extracted from OPM2 cells treated with 5 μM of Gos was subjected to agarose gel electrophoresis. DNA smear and ladder formation was observed from 12 h of Gos-treatment, which was effectively blocked by a pan-caspase inhibitor, Z-VAD-FMK (24 h + zVAD-FMK).
Gossypol activates mitochondrial dysfunction pathway of apoptosis. (A) Flow cytometric analysis of mitochondrial transmembrane potential (ΔΨm). OPM2 cells were treated with 5 μM of Gos for 0–12 h, and mitochondrial membrane potential was measured by DioC6 fluorescence. (B) Release of cytochrome
Expression of apoptosis associated proteins and phosphorylation of Bcl-2 during Gos-induced apoptosis. OPM2 cells were treated with 5 μM of Gos for 0–24 h and the expression of apoptosis associated proteins (A) or Ser-70 phosphorylation of Bcl-2 (B) was examined by western blot analysis. Blots are representative of three independent experiments.
Expression of PKC and PP2A during Gos-induced apoptosis. OPM2 cells were treated with 5 μM of Gos for 0–24 h, and the protein levels of PKCα and three isoforms of PP2A (PP2A/A, PP2A/B56α and PP2A/C) was examined by western blot analysis. Blots are representative of three independent experiments.
Gossypol inhibits IL-6 signaling. OPM2 cells were treated with 5 μM of Gos for 0–24 h, and the phosphorylation of signaling subunit of IL-6 receptor, gp130 and downstream signaling molecules, JAK2, STAT3, ERK1/2 and p38MAPK was examined by western blot analysis. Blots are representative of three independent experiments.
JAK2 inhibition mimics the effect of Gossypol on IL-6 signaling in OPM2 cells. OPM2 cells were treated with various concentrations of JAK2 inhibitor, AG490, for 24 h. Phosphorylation status and protein levels of JAK2, STAT3, p38MAPK, Bcl-2 or Mcl-1 were examined by western blot analysis. Results are representative of three independent experiments.