Galectins constitute a family of lectins that specifically exhibit the affinity for β-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC50 between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH2-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.
Galectins constitute a family of animal lectins that show affinity for β-galactosides and share conserved amino-acid sequences in their carbohydrate recognition domains.1, 2 Fourteen mammalian galectins, galectin-1 to -14, are classified into three subtypes according to their structure, that is, prototype, chimera, and tandem-repeat type. The tandem-repeat type galectins with two carbohydrate recognition domains that recognize different sugar binding target molecules are capable of cross-linking with a wider variety of glycoconjugates than other subtypes.3 Galectin-9 is a tandem-repeat type galectin, and has been shown to be involved in a variety of cellular functions, such as cell adhesion, proliferation, and apoptosis.4, 5, 6, 7, 8, 9, 10 Moreover, recent studies have indentified anti-cancer properties of galectin-9 against several cancers.11, 12, 13, 14, 15 In this study, we show the anti-multiple myeloma (MM) activity of a recombinant mutant form of human galectin-9 (hGal9) through the activation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, those share crucial roles in the survival and death of myeloma cells.16
Materials and methods
Cell lines and reagents
MM cell lines IM9, KMS-12-BM, AMO-1, NCI-H929, and RPMI8226 were maintained as described earlier.17 The IM9 subline with lower sensitivity to bortezomib (Bor) (IM9-Bor), established by Dr Terui Y, was maintained with 20 nM Bor. The recombinant hGal9, which is highly stable against proteolysis, was synthesized by Galpharma (Kagawa, Japan).18 JNK-inhibitor VIII, SB203580, an inhibitor for p38 MAPK, Z-VAD-FMK, Z-IETD-FMK, and Z-LEHD-FMK were purchased from Calbiochem (San Diego, CA, USA) and lactose was purchased from Sigma (St Louis, MO, USA).
Primary human myeloma samples
Studies with patient samples were approved by the Ethics Board of our institute. Bone marrow (BM) mononuclear cells were labeled with anti-CD138 MicroBeads and were positively isolated with the MiniMACS separator (Miltenyi Biotec KK, Tokyo, Japan). More than 98% of the cells isolated were morphologically confirmed as plasma cells after Wright-Giemsa staining, and were cultured with 20 ng/ml interleukin-6.
Cell surface binding affinity of hGal9
Cells were incubated with biotinylated human Gal-9 NC (hGal9NC) (Galpharma) in 2.0% FCS-containing PBS buffer supplemented with 2 μg/ml Streptavidin-FITC. For negative control, cells were incubated only with Streptavidin-FITC. Cells were washed twice and subjected to flow cytometric analyses.
Assays for growth inhibition and apoptosis
Growth-inhibitory effect of hGal9 was analyzed with a modified MTT assay using Cell Counting Kit-8 (Dojindo, Japan) or by direct counting of cell number. Data represent the results of means±standard errors of three independent experiments. To determine apoptosis, cells were counterstained with propidium iodide and Annexin V-FITC. Mitochondrial outer membrane potential (MOMP) was assayed using MitoScreen (JC-1) Kit (Beckton Dickinson, San Diego, CA, USA).
IM9 and KMS-12-BM were treated with or without 100 nM hGal9 for 6 h, and total RNA was isolated. Gene expression was analyzed with Affymetrix Gene Chip arrays and GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA, USA), and array data analysis was carried out with Affymetrix GeneChip operating software version 1.0. Genes showing at least a 2.0-fold difference in expression levels between control and hGal9-treated cells were considered to be modulated by hGal9. Data were also analyzed with the Ingenuity pathway analysis software (Ingenuity Systems, Mountain View, CA, USA).
Quantitative RT-PCR (RQ-PCR)
RQ-PCR was performed as was described earlier.19 Primers used were as follows: c-jun 5′-IndexTermtgactgcaaagatggaaacg-3′ and 5′-IndexTermccgttgctggactggattat-3′, jun-D 5′-IndexTermctcaaggacgagccacagac-3′ and 5′-IndexTermtggctgaggactttctgctt-3′.
Western blotting was performed as was described elsewhere.19, 20 Primary antibodies (Abs) used were those against JNK, p38 MAPK, phosphorylated p38 MAPK, caspase-3, caspase-8, caspase-9 (Cell Signaling Technologies, Beverly, MA, USA), H2AX, phosphorylated H2AX (Upstate Biotechnology, Inc., Lake Placid, NY, USA), phosphorylated JNK, c-Jun, Jun-D (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-tubulin (Sigma).
In vivo anti-myeloma activity of hGal9
Animal studies were performed according to the guidelines of the institutional review board. In all, 5 × 106 IM9 cells were subcutaneously injected into the right hind flank of ten 6-week-old nude mice, which had been whole-body irradiated with 2 Gy 24 h before tumor implantation. Tumor volume was calculated with the formula V=0.5a × b2, where a is the long and b is the short diameter of the tumor. Mice were randomized into two groups, five mice for hGal9 treated and five mice for control, when the mean tumor volume reached 400 mm3. In all, 100 μg/body/day of hGal9 or PBS was administered to the respective groups for 14 consecutive days through intrapenitoneal injection. Volume of tumors of hGal9-treated mice was compared with that of vehicle-treated controls with the Mann–Whitney U-test. A P-value of <0.05 was considered statistically significant.
Anti-myeloma effect of hGal9 through induction of apoptosis in vitro
hGal9 displays a growth-inhibitory effect on IM9, KMS-12-BM, AMO-1, and NCI-H929, in a time- and concentration-dependent manner at IC50s of 75. 1, 77.2, 84.1, and 280.0 nM, respectively (Figure 1a; Supplementary Figure 1). The only exception was RPMI8226, against which >1 μM hGal9 did not have any anti-proliferative effect. As was shown by the increased number of Annexin V-positive cells in hGal9-treated cells (Figure 1b), the anti-myeloma effect of hGal9 involved the induction of apoptosis. The apoptotic induction by hGal9 was accompanied by the loss of MOMP and the processing of caspase-3, -8, and -9 (Figures 1c and d). The blockade of caspase activation by the pretreatment with either Z-VAD-FMK, a pan-caspase inhibitor, or Z-IETD-FMK, an inhibitor for caspase-8, partly prevented the cell death induction by hGal9, whereas the pretreatment with Z-LEHD-FMK, an inhibitor for caspase-9, did not protect cells from hGal9-induced cell death in KMS-12-BM and IM9 (Figure 1e and data not shown).
Cell surface binding is the prerequisite for the anti-MM activity of hGal9
The addition of 25 mM lactose completely abrogated the anti-myeloma activity of hGal9 (Figure 2a). The cell lines with stronger affinity for hGal9 are more sensitive to the anti-proliferative effect of hGal9, whereas hGal9-resistant RPMI8226 has the lowest affinity for hGal9 (Figures 2b and c).
JNK and p38 MAPK signaling pathways in the anti-myeloma effect of hGal9
Microarray analyses showed that 16 genes, including c-jun and junD, were upregulated by hGal9 in KMS-12-BM and IM9. The upregulation of c-jun and junD genes by hGla9 was confirmed by RQ-PCR (Supplementary Table 1; Supplementary Figure 2), and the upregulation of c-Jun and JunD proteins by hGal9 was also confirmed (Figure 3a). The Ingenuity pathway analysis suggested the relationship between the anti-MM effect of hGal9 and the cross-talk networks of JNK and p38 MAPK signaling pathways (Supplementary Figure 3), and, indeed, JNK and p38 were phosphorylated by treatment with hGal9 in both KMS-12-BM and IM9. hGal9 also induced H2AX phosphorylation, which is critical for apoptosis by JNK activation or p38 MAPK activation (Figure 3a).21, 22, 23, 24 Conversely, neither JNK nor p38 activation was observed in myeloma cell lines those are resistant to hGal9-induced cell death (Supplementary Figure 7). Next, KMS-12-BM and IM9 cells were preincubated with 20 μM JNK-inhibitor VIII and/or 20 μM SB203580 for 2 h and then treated with 100 nM hGal9 for 48 h. Neither JNK-inhibitor VIII nor SB203580 alone decreased the cell viability of two cell lines. The blockade of the JNK or p38 MAPK pathway partially eliminates the cytotoxic effect of hGal9 on both cell lines, whereas the concomitant blockade of both kinases did not further increase the number of viable cells (Figures 3b and c), indicating that the activation of the JNK and p38 MAPK pathways at least partly mediates the anti-MM activity of hGal9 in a complementary manner.
The effects of hGal9 on primary myeloma cells isolated from MM patients
We examined the effect of hGal9 on myeloma cells freshly isolated from 10 patients. Three patients were treatment-naive, five relapsed after hematopoietic stem cell transplantation, one was treated with a Bor-containing regimen, and one was with a thalidomide-containing regimen. Five had chromosome abnormality t(4;14), seven had 13q chromosomal deletion, and four had both. Two had 17p chromosomal deletion (Supplementary Table 2). More than 80% of the untreated cells remained alive, whereas the 48-h treatment with hGal9 significantly reduced the number of viable cells in a dose-dependent manner in all samples examined (Figure 4).
No cross-resistance between hGal9 and Bor in IM9 cells
IM9-Bor was approximately four times more resistant to Bor with an IC50 of 22.3 nM compared with parental IM9 with an IC50 of 5.4 nM. By contrast, both cell lines showed the similar cell surface binding affinity for hGal9, and hGal9 was effective without any statistically significant difference against both IM9 and IM9-Bor cells with respective IC50s of 75.1 and 112.6 nM (Figures 5a and b; Supplementary Figure 4).
hGal9 inhibits the proliferation of IM9 cells in vivo
As shown in Figure 6, hGal9 treatment for 14 consecutive days significantly delayed the tumor growth of IM9 cells (P<0.05; Mann–Whitney U-test), thus constituting evidence for the direct in vivo anti-myeloma activity of hGal9.
Here, we showed that the anti-myeloma activity of hGal9 was mediated by the activation of JNK and p38 MAPK signaling pathways. Reportedly, JNK activation is one of the crucial pathways for apoptosis induction by the leading anti-MM agents such as proteasome inhibitors or IMiDs, or various new candidate agents for MM.16, 25, 26, 27, 28, 29 Also, various new candidate anti-MM agents, such as the histone deacetylase inhibitor PXD101, induce apoptosis by activating the p38 MAPK pathway in myeloma cells.30, 31, 32, 33 These findings lead us to suggest that both JNK and p38 pathways activation are the logical molecular targets for the development of new therapeutic strategies for MM. On the other hand, although earlier studies suggest that NF-κB signaling inactivation or the activation of Ca2+-calpain-caspase-1 pathway are involved in the anti-proliferative effect of hGal9 in other cancers,13, 14, 34 those were not the case in the myelomas (data not shown). These indicate that, as hGal9 recognizes various β-galactosides-containing surface molecules that may differ among cell types, its primary molecular targets may also differ according to cancer type. Also, our study reveals that hGal9-induced apoptosis involves both caspase-dependent and -independent pathways, and suggests that caspase-8 activation has more dominant role than caspase-9 as the initiator caspase in cell death induced by hGal9 in myeloma cells. No specific effect of hGal9 was observed on the expression levels of Bcl-2 family protein, the crucial regulators for intrinsic apoptosis pathway, or death receptor molecules, such as Fas or DR5 (Supplementary Figure 5). Thus, we speculate that hGal9 may activate an apoptotic pathway, such as endoplasmic reticulum stress pathway, which activates JNK and p38 and then activate caspase-8 independently from classical intrinsic or extrinsic apoptosis pathway,35 and, consequently, may activate caspase-9 and caspase-3 in cascade.
There is an urgent need for the development of new therapeutic strategies for MM patients. hGal9-induced cell death in all 10 primary human myeloma cells tested, even in cells from treatment-resistant patients with poor prognosis chromosomal abnormalities. Compared with cell lines, a higher dose of hGal9 seems to be required for killing primary myeloma cells, presumably because these cells may become quiescent and less sensitive to any cytotoxic stimuli ex vivo. Also, hGal9 was found to inhibit proliferation of IM9-Bor to a similar degree as that seen in bortezomib-sensitive parental IM9 cells. hGal9 was also found to be safe for mice, did not result in body weight reduction during treatment (data not shown), and did not significantly inhibit normal human CFU-GM formation (Supplementary Figure 6). Thus, hGal9 may be a new candidate nti-MM agent even for high-risk MM patients. Although the ‘subcutaneous xenografted’ myeloma model used in this study showed the in vivo bioactivity of hGal9, this model has the potential limitation for recapitulating MM, which normally represents multiple tumors in systemic BM sites. It is urgently desired to explore the anti-myeloma effect of hGal9 at multiple BM sites.
In conclusion, we have shown the activity of hGal9 against myeloma cell lines and primary human MM cells both in vitro and in vivo, and that the activation of JNK/p38-H2AX pathways is involved in this anti-myeloma activity. Further clinical study of hGal9 as a new anti-MM agent thus certainly seems to be warranted.
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This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by grants from the Kobayashi Foundation of Innovative Cancer Chemotherapy, the Award in Aki's Memory from International Myeloma Foundation, and the Japan Leukaemia Research Fund (MT and JK).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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