Synergistic interactions between the synthetic retinoid tamibarotene and glucocorticoids in human myeloma cells

Abstract

Tamibarotene (TM411) is a synthetic retinoic acid receptor‐α/‐β selective retinoid that is chemically more stable than all‐trans retinoic acid. This study was designed to evaluate the activity of TM411 in multiple myeloma (MM) and the effects of TM411 combined with a glucocorticoid (GC). In vitro, five human myeloma cells were treated with TM411 alone, GC alone, or TM411 + GC. Cell survival was analyzed by the tetrazolium dye assay and the Hoechst 33342/propidium iodide double‐staining method. The effect of TM411 + GC was assessed by the isobologram method. In vivo, the growth‐inhibitory effects of the drugs on RPMI‐8226 cell xenografts established in SCID mice were examined. The effects of the agents on IL‐6‐mediated signaling pathways were also analyzed by Western blotting. TM411 was 2‐ to 10‐fold more potent, in terms of its growth‐inhibitory effect, than all‐trans retinoic acid. The combination of TM411 and GC was found to show a markedly synergistic interaction. While increased expressions of the IL‐6 receptor, phosphorylated MAPK, and Akt were observed after exposure to GC, TM411 attenuated this increase in the expressions, suggesting that such modification of the effect of GC by TM411 might be the possible mechanism underlying the synergistic interaction. Furthermore, TM411 + GC showed a supra‐additive inhibitory effect in a xenograft model as compared with TM411 or GC alone. These results imply that the combination of TM411 + GC might be highly effective against MM, and suggest the need for clinical evaluation of TM411 + GC for the treatment of MM. (Cancer Sci 2009; 100: 1137–1143)

Multiple myeloma (MM) remains an incurable malignant tumor of the plasma cells of the bone marrow, and the five‐year survival rate has been estimated to be 15–20%.^{( 1 )} Although high‐dose chemotherapy with hematopoietic stem‐cell support has been shown to extend the event‐free and overall survival,^{( 2} , ³ , ^{4 )} this aggressive approach is generally not suitable for elderly patients over the age of 65 years, even though they account for more than 60% of all newly diagnosed cases of MM. In this group of patients, conventional chemotherapy with alkylating agents and anthracyclines, and also glucocorticoids (GCs),^{( 5} , ^{6 )} has remained the treatment of choice. New treatments are urgently needed in these patients, and recently, preclinical and clinical trials have shown that novel targeted therapies, including the use of retinoid,^{( 7} , ⁸ , ^{9 )} offer great promise for an improved outcome in patients with MM.^{( 10} , ^{11 )}

Tamibarotene (TM411, retinobenzoic acid) is a synthetic retinoic acid receptor (RAR)‐α/‐β‐selective retinoid, does not bind to RAR‐γ, and is chemically more stable than all‐trans retinoic acid (ATRA) against light, heat, and oxidation (Fig. 1a).^{( 12 )} Therefore, TM411 might be expected to have fewer adverse events related to stimulation of RAR‐γ. It was reported that the adverse events associated with TM411 were generally milder compared with those associated with ATRA therapy in patients with acute promyelocytic leukemia (APL).^{( 13 )} ATRA has previously been shown to inhibit the growth of human myeloma cells.^{( 14} , ¹⁵ , ^{16 )} TM411 has also been shown to have antimyeloma activity,^{( 8 )} and preclinical and early clinical trials of the drug are underway.

Figure 1.

(a) Structural formula of the two retinoids, tamibarotene (TM411) and all‐trans retinoic acid (ATRA). (b) Growth‐inhibitory effect of TM411 and ATRA on RPMI‐8226 cells, determined by MTT assay. The cells were cultured with TM411 or ATRA (0.01–1000 nM) for 96 h. The growth–inhibition curves are shown. Points, mean values of at least three independent cultures; bars, SD.

The proliferation of MM cells triggered by cytokines such as IL‐6, insulin‐like growth factor‐1, vascular endothelial growth factor, tumor necrosis factor‐α, stromal cell‐derived factor‐1α, and IL‐21 is mediated primarily through the MAPK signaling cascade. The cytokine‐induced survival or resistance to apoptosis in myeloma cells is mediated through the JAK/STAT3 or PI3K/Akt pathways.^{( 10 )} In these signaling cascades, IL‐6‐mediated signaling pathways are presumed to play a major role in the pathogenesis and malignant growth of MM by exerting an anti‐apoptotic effect.^{( 17} , ¹⁸ , ¹⁹ , ^{20 )} TM411, similar to ATRA, has been shown to inhibit IL‐6 signaling.^{( 21} , ²² , ^{23 )}

GCs such as dexamethasone (DEX) and prednisolone (PSL) are involved in the regulation of a variety of biological processes, including immune responses, metabolism, cell growth and proliferation, development, and reproduction. GCs induce apoptosis and have become key chemotherapeutic agents of many hematological malignancies including MM.^{( 24} , ^{25 )} Although these drugs are frequently used to treat MM and are believed to induce apoptosis and cell cycle arrest of the tumor cells,^{( 26 )} the precise mechanism of action has not yet been clearly elucidated. Further, it was reported that GC not only triggered death signaling, but also simultaneously activated the IL‐6 signaling pathway, which protects cells against DEX‐induced apoptosis.^{( 25 )}

In the present study, we evaluated the antitumor effect of the synthetic retinoid TM411 against human myeloma cells, and the combined effects of TM411 + GC, both in vitro and in vivo. In addition, we elucidated the biochemical mechanism underlying the synergistic interaction between the two agents through modulation of expression of the IL‐6 receptor.

Materials and Methods

Agents.  Tamibarotene (TM411, retinobenzoic acid) was provided by TMRC Co. Ltd (Tokyo, Japan). ATRA, DEX, and PSL were obtained from Sigma‐Aldrich Japan (Tokyo, Japan). The drugs were dissolved in DMSO for the in vitro experiments, and TM411 and DEX were dissolved in ethanol and suspended in a 100‐fold volume of PBS for the in vivo experiments.

Cells and culture.  The human myeloma cell lines RPMI‐8226 and U266 (American Type Culture Collection, Rockville, MD, USA), and MM.1S (Northwestern University, Chicago, IL,USA), and KMS‐11 and KMS‐12BM (Kawasaki Medical School, Okayama, Japan) were maintained in RPMI‐1640 (Sigma‐Aldrich Japan) supplemented with 10% heat‐inactivated FBS (Life Technologies, Grand Island, NY, USA), and penicillin–streptomycin (Sigma‐Aldrich Japan).

In vitro growth inhibition assay.  The tetrazolium dye (MTT) assay was used to evaluate the cytotoxicity of the drugs at various concentrations. A 180‐µL volume of an exponentially growing cell suspension (1 to 2 × 10⁴ cells/mL) was seeded into each well of a 96‐well microculture plate containing 10% FBS medium and incubated for 24 h. The cells were exposed to 20 µL of each drug at various concentrations and cultured at 37°C in a humidified atmosphere for 96 h. After the culture period, 20 µL MTT solution (5 mg/mL in PBS) was added to each well and the plates were incubated for a further 4 h at 37°C. After centrifuging the plates at 200 g for 5 min, the medium was aspirated from each well, and 200 µL DMSO was added to each well to dissolve the formazan. The growth‐inhibitory effect of each drug was assessed spectrophotometrically (SpectraMax 190; Molecular Devices, Sunnyvale, CA, USA).

Assessment of effect of TM411 + GC combination in vitro.  The effects of TM411 + GC at the IC₅₀ point were analyzed by the isobologram method^{( 27 )} to evaluate the occurrence of synergism, additivity, or antagonism. For two drugs (A and B) that do not interact, the equation is: (D)_A/(D_x)_A + (D)_B/(D_x)_B = 1. In practice, the concentrations (D_x)_A, (D_x)_B, and (D_x)_A,B for each of the drugs alone and the two drugs combined, respectively, required to produce the same percentage growth inhibition, are obtained from their dose–response curves. The concentrations of drugs A and B are plotted on the X and Y coordinates of the isobologram, respectively. Isoeffect curves are drawn, and the total area enclosed by the lines is considered to represent the ‘envelope of additivity’. When the experimentally determined IC₅₀ of the combination falls to the left side of the envelope, the interaction between the drugs used in combination is considered to be supra‐additive (synergistic). When the experimental data point falls within the envelope, the combination is considered to exert an additive effect, and when it falls to the right side of the envelope but within the square produced by 0–1 IC₅₀ units, the combination is considered to have sub‐additive effects. When the point lies outside the square, the two drugs are considered to exert a protective effect against each other.

Western blot analysis.  Cells were cultured overnight in 10% serum‐containing medium. The cultured cells were washed twice with ice‐cold PBS and lysed with M‐PER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA) containing complete Mini (Roche Diagnostics, Mannheim, Germany) and Phosphatase Inhibitor Cocktail 1/2 (Sigma‐Aldrich Japan). The protein concentration of the supernatants was determined by the BCA protein assay (Pierce Biotechnology). For preparation of the nuclear protein, the CelLytic NuCLEAR Extraction Kit (Sigma‐Aldrich Japan) was used in accordance with the manufacturer's protocol. Then 20 µg samples of the cell lysates protein were electrophoretically separated on a Multigel II Mini (Daiichi Pure Chemicals, Tokyo, Japan) and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was incubated with antibodies against IL‐6Rα, gp130, RAR‐α, RAR‐β, RAR‐γ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt, phospho‐Akt (Ser473), phospho‐p44/42 MAPK (Thr202/204), phospho‐MEK1/2 (Ser217/221), phospho‐Stat3 (Tyr705), BAX, Bcl‐xL, Mcl‐1 (Cell Signaling Technology, Beverly, MA, USA), and β‐actin (Sigma‐Aldrich Japan) as the first antibody, followed by detection using an HRP‐conjugated secondary antibody. The bands were visualized with an ECL detection reagent (Amersham, Piscataway, NJ, USA).

Cell survival assay.  Cell viability was assessed morphologically by staining the nuclei of the cells with Hoechst 33342 and propidium iodide (PI), as previously described.^{( 28 )} Hoechst 33342 and PI were purchased from Molecular Probes (Eugene, OR, USA). After incubation, the cells were collected and stained with Hoechst 33342 and PI for 15 min, then examined by fluorescence microscopy. The cell death induction ratio was calculated as the ratio of the number of cells containing PI‐stained nuclei to the total number of cells (approximately 300–500 cells).

IL‐6 ELISA.  RPMI‐8226 cells were rinsed twice in PBS (pH 7.4) and transferred to fresh culture medium at 1 × 10⁴ cells/mL under serum‐starvation conditions, and equal amounts of the cells were dispensed into three individual dishes. Following incubation for 48 h, aliquots of the medium were analyzed in triplicate using the human IL‐6 ELISA (Bender Medsystems, Burlingame, CA, USA) to determine the levels of IL‐6 secretion in accordance with the manufacturer's protocol.

Xenograft murine model.  Four‐week‐old female mice with SCID were purchased from Charles River (Yokohama, Japan). All mice were maintained in a specific pathogen‐free area in our animal resources facility. Five mice per group were given an s.c. injection into the right flank of 3 × 10⁷ RPMI‐8226 myeloma cells in 100 µL PBS, together with 100 µL Matrigel (BD Biosciences, Bedford, MA, USA). Tumor growth was monitored daily, and the mice were randomized to drug‐treated or control groups when the tumor volume reached approximately 200 mm³. Tumor‐bearing mice were treated with either TM411 (2 mg/kg/day) by i.p. injection daily for 21 consecutive days, DEX (1 mg/kg/day) by i.p. injection on day 0–3, day 7–10, and day14–17, or both agents. In the control group, vehicle alone (PBS 100 µL/day) was injected i.p. by the same schedule as that of TM411. Tumor diameters were measured with calipers three times per week to evaluate the effects of the treatment, and the tumor volume was calculated by the following formula: a × b ²/2 (mm³), where a is the largest diameter of the tumor and b is the shortest diameter. All mice were killed when their tumors reached more than 2 cm in diameter or when the mice became moribund. Survival was evaluated from the first day of treatment until death. The protocol of the experiment was approved by the Committee for Ethics in Animal Experimentation, and conducted in accordance with the Guidelines for Animal Experiments of the National Cancer Center.

Statistical analysis.  The statistical significance of differences among the experimental groups was determined using Student's t‐test. The level of significance was set at P < 0.05. Overall survival in each treatment group was compared using Kaplan–Meier curves and log–rank tests. All analyses were carried out using the SPSS statistical software package (spss version 16.0 for Windows; SPSS, Chicago, IL, USA).

Results

TM411 inhibited proliferation of human myeloma cell lines.  We examined the growth‐inhibitory effect of TM411 in the RPMI‐8226, MM.1S, U266, KMS‐11, and KMS‐12BM cell lines by MTT assay. The IC₅₀ value of TM411 was approximately 3 nM for RPMI‐8226 and 10 nM for MM.1S cells. Thus, the growth‐inhibitory effect of TM411 on the two cell lines was 2‐ to 10‐fold more potent than that of ATRA (Fig. 1b). RPMI‐8226 and MM.1S cells were also sensitive to GCs, including PSL and DEX, whereas the U266 and KMS‐12BM cells were resistant to these agents, and the KMS‐11 cells were resistant to TM411 (Table 1).

Table 1.

In vitro growth‐inhibitory effects in human myeloma cells by MTT assay

IC50 (µM)	RPMI‐8226	MM.1S	U266	KMS‐11	KMS‐12BM
TM411	0.0031 ± 0.0018	0.0095 ± 0.0110	37.80 ± 13.80	44.800 ± 26.100	12.2 ± 7.0
ATRA	0.0780 ± 0.0260	0.4200 ± 0.5000	14.00 ± 8.40	N.D.	N.D.
DEX	0.0280 ± 0.0340	0.0190 ± 0.0013	66.36 ± 18.10	0.037 ± 0.023	>100
PSL	0.1100 ± 0.0680	0.0710 ± 0.0190	>100	N.D.	N.D.

Each value is the mean ± SD. ATRA, all‐trans retinoic acid; DEX, dexamethasone; N.D., not done; PSL, prednisolone; TM411, tamibarotene.

Expression levels of RARs observed in myeloma cells.  We examined the expressions of the RARs by Western blot analysis of 20 µg nuclear extracts obtained from the myeloma cells. Comparison of the protein expression levels of RAR‐α and RAR‐β, which represented specific targets of TM411, in the myeloma cells revealed high expression levels of the receptors in the sensitive RPMI‐8226 and MM.1S cells. The expressions were more subtle in the resistant U266 cells, and the resistant KMS‐11 and KMS‐12BM cells showed high expression levels of RAR‐α and RAR‐β. RAR‐γ protein was detected in all five cell lines. (Fig. 2)

Figure 2.

Expression of retinoic acid receptors (RARs) in the three myeloma cell lines, RPMI‐8226, MM.1S, U266, KMS‐11, and KMS‐12BM, determined by Western blot analysis. The nuclear protein was extracted from the myeloma cells and a 20 µg sample of the lysates was subjected to the analysis. The tamibarotene (TM411)‐sensitive RPMI‐8226 and MM.1S cell lines were found to overexpress RAR‐α/‐β, which are target receptors for TM411.

Marked synergistic interaction between TM411 and GCs observed in vitro.  Based on the results of the evaluation of growth inhibition in vitro by the MTT assay, the effect of the combination of TM411 + GC was evaluated against the RPMI‐8226 and MM.1S cells in vitro. The cells were cultured with TM411 for 96 h at various concentrations of TM411, in the presence or absence of GC. The growth rates were expressed as the averages of at least three independent experiments. The effect of combined of TM411 + DEX treatment on the RPMI‐8226 cells is shown in Figure 3. MTT assay revealed that TM411 inhibited RPMI‐8226 cell growth in a dose‐dependent manner; moreover, the drug also synergistically enhanced the growth‐inhibitory effect of DEX (Fig. 3a). The effects of combined TM411 + DEX treatment were further assessed by the in vitro isobologram method. The experimentally determined IC₅₀ values of the combination fell to the left side of the envelope, therefore, the interaction between TM411 and DEX used in combination was considered to be synergistic (Fig. 3b).

Figure 3.

Effects of combined tamibarotene (TM411) + dexamethasone (DEX) treatment on RPMI‐8226 cells, determined by MTT assay. (a) The growth‐inhibition curves for RPMI‐8226 treated with TM411 with or without DEX are shown. Points, mean values of at least three independent cultures; bars, SD. (b) Isoborogram of the interactions between TM411 and DEX in RPMI‐8226 cells. The effect of combined TM411 + DEX treatment was analyzed at the 50% growth inhibition ratio points. The experimentally determined IC₅₀ values of the combination fell to the left side of the envelope, and the drugs were judged to exert synergistic effects.

Cell death increased by combined TM411 + GC treatment.  To detect apoptosis and necrosis morphologically, Hoechst 33342 and PI nuclear staining was used. Cells were treated with either 6 nM TM411 (concentration twofold the IC₅₀), 100 nM PSL (concentration twofold the IC₅₀), 3 nM TM411 (IC₅₀) combined with 50 nM PSL (IC₅₀), or vehicle control for 24, 48, or 72 h. Then we randomly selected three visual fields for microscopy, and counted the total number of cells, and the number of apoptotic or necrotic cells. The cell death rate was determined as the average from three independent experiments. The cell death induction ratio was significantly higher in cultures treated with the combination of TM411 + PSL than in the cultures treated with TM411 or PSL alone (Fig. 4).

Figure 4.

The cytotoxixcity of tamibarotene (TM411) in the presence or absence of prednisolone (PSL) was evaluated morphologically by the Hoechst 33342/propidium iodide double‐staining method. RPMI‐8226 cells were cultured in either control medium, or medium containing TM411 (6 nM; concentration twofold the IC₅₀), PSL (100 nM; concentration twofold the IC₅₀), or TM411 (3 nM; IC₅₀) in combination with PSL (50 nM; IC₅₀) for 24, 48 or 72 h. At the end of the incubation period, the cells were stained with Hoechst 33342 (blue) and propidium iodide (red) for 15 min and examined under a fluorescence microscope. (a) Fragmented cells stained blue or red with condensed nuclei correspond to apoptotic cells, whereas cells stained blue with round nuclei correspond to viable cells. (b) We counted the total number of cells and number of apoptotic cells in each visual field, and calculated the cell death induction ratio as the ratio of the number of apoptotic cells to the total number of cells in three visual fields. The cell death induction ratio at 72 h was significantly higher in the TM411 + PSL group than in the PSL alone group (P = 0.019). Colums, mean values of three independent cultures; bars, SD. *Significant difference (P < 0.05) compared to the control; **significant difference (P < 0.05) compared to the PSL alone group.

TM411 inhibited myeloma cell growth associated with downregulation of IL‐6‐related molecules.  Because previous studies have shown that IL‐6 is a major growth factor for myeloma cells both in vitro and in vivo,^{( 29} , ³⁰ , ³¹ , ^{32 )} we examined the modulation of IL‐6‐related molecules by combined TM411 + DEX treatment. We first examined the effect of TM411, DEX, and combined TM411 + DEX treatment on the level of IL‐6 secretion from the RPMI‐8226 cells using ELISA. The IL‐6 secretion showed a trend towards decrease with the treatment, however, no significant differences were observed among the groups (Fig. 5a). We next examined the expression of IL‐6 receptor‐α and gp130 by Western blotting. IL‐6 receptor‐α expression in the RPMI‐8226 cells was downregulated by TM411. Although increased expression of IL‐6 receptor‐α was observed after exposure to DEX, TM411 inhibited this increase of expression induced by DEX. However, TM411 had no inhibitory effect on the increased expression of gp130 induced by DEX (Fig. 5b).

Figure 5.

Tamibarotene (TM411) modulates the expression of IL‐6‐related molecules. (a) Supernatant was collected from RPMI‐8226 cells treated with TM411 (3 nM, IC₅₀; 100 nM, IC₈₀) in the presence or absence of dexamethasone (DEX) (30 nM, IC₅₀; 100 nM, IC₈₀) for 24 or 48 h. IL‐6 was measured by ELISA (n = 3 experiments each). No significant change in the amount of IL‐6 secreted was observed after exposure to the drugs. Columns, mean values of three independent cultures; bars, SD. (b) RPMI‐8226 cells were treated with TM411 (3 nM) in the presence or absence of DEX (30 nM) for 24 h. A 20‐µg sample of the cell lysates was subjected to Western blot analysis to assess the protein expressions of the IL‐6 receptor (IL‐6R)‐α, gp130, and β‐actin (control). Treatment with DEX increased the expressions of both IL‐6R‐α and gp130. TM411 inhibited the expression of IL‐6R‐α. The expression of IL‐6R‐α induced by DEX was also inhibited by TM411.

TM411 modulated IL‐6‐triggered signaling cascades.  Given that TM411 inhibited the expression of IL‐6 receptor‐α, we examined the effects on the IL‐6‐triggered signaling cascades. RPMI‐8226 cells were cultured with TM411 for 24 h. We showed that treatment with DEX alone increased the expression of phosphorylated MAPK, MEK, and Akt, which might be protective signals against DEX‐induced apoptosis,^{( 25 )} in the RPMI‐8226 cells stimulated or not stimulated with IL‐6 (100 ng/mL). The increase in the expressions of phosphorylated MAPK and Akt induced by DEX was attenuated by combined treatment with TM411 (Fig. 6). The findings suggest that such modification by TM411 of the effects of DEX might be the possible mechanism underlying the synergistic effect of TM411 and GC.

Figure 6.

Regulation of IL‐6‐mediated signaling cascades. (a) RPMI‐8226 cells were pretreated with tamibarotene (TM411) (3 nM, IC₅₀) in the presence or absence of dexamethasone (DEX) (30 nM, IC₅₀) for 24 h. Samples (20 µg) of the cell lysates were subjected to Western blot analysis to assess the phosphorylation status and protein expression levels of STAT3, MAPK, MEK, Akt, BAX, Bcl‐xL, Mcl‐l and β‐actin. The phosphorylations of MAPK and Akt induced by DEX were inhibited by TM411.

TM411 inhibited human myeloma cell growth and enhanced cytotoxicity of GC in vivo.  In order to confirm whether combined TM411 + GC treatment exerted a synergistic effect, the growth‐inhibitory effect of the combination was evaluated in vivo using a xenograft murine model. The mice were given i.p. injections of either TM411 (2 mg/kg/day) on days 0–20, or DEX (1 mg/kg/day) on days 0–3, 7–10, and 14–17, both TM411 and DEX, or only vehicle control for 21 days (Fig. 7a), the first treatment day being defined as day 0. Treatment with TM411 or DEX alone suppressed the tumor growth compared with that in the control. Moreover, combined TM411 + DEX treatment induced more potent growth inhibition of the tumor (Fig. 7b,c). No significant body weight loss was observed in any of the groups (Fig. 7d). We also observed significantly prolonged survival in the animals treated with the combination than in the other groups (Fig. 7e). These results confirm the activity of TM411 as an antimyeloma agent with less toxicity and also the more marked growth‐inhibitory effect of combined TM411 + DEX use against a tumor xenograft of MM.

Figure 7.

Combined effects of tamibarotene (TM411) + dexamethasone (DEX) on RPMI‐8226 tumor xenografts in vivo. (a) Treatment schedule. (b) Mice were randomized to four groups (five mice/group). The tumor volume was calculated as described in Materials and Methods. Each data point represents the mean tumor volume of the five mice in each group. Significant growth inhibition was observed in the mice treated with TM411 + DEX. (c) Histogram of the mean tumor volume and statistical analysis on day 21. Columns, mean values of five mice; bars, SD. *Significant difference (P < 0.05) compared to the control; **significant difference compared to the TM411 alone group (P < 0.01). (d) Body weight change was evaluated three times per week. Points, mean values of five mice in each treatment group; bars, SD. (e) Overall survival was evaluated from the first day (day 0) of treatment until death using Kaplan–Meier curves. The TM411‐ and DEX‐treated mice showed longer overall survival than the control mice (P = 0.012 and 0.080, respectively); moreover, the mice treated with TM411 + DEX also showed longer survival than those treated with TM411 (P = 0.017) or DEX (P = 0.048) alone.

Discussion

Retinoids have been indicated to have antitumor activity.^{( 33 )} In particular, ATRA has been used successfully in patients with APL, and significant clinical responses have also been obtained in other malignancies. The growth‐inhibitory effect of ATRA on MM cells has also been shown.^{( 15} , ³⁴ , ^{35 )} However, ATRA alone was found to be ineffective in pretreated MM patients with toxicity,^{( 7 )} therefore, it was not developed for clinical use. Tamibarotene (TM411) is a synthetic RAR‐α/‐β selective retinoid^{( 12 )} approved for use in the treatment of relapsed or refractory APL in Japan. Compared to ATRA, which is indicated for the first‐line treatment of APL, TM411 is characterized by greater chemical stability, is a more potent inducer of differentiation in APL cells, and shows a lower potential for drug resistance.^{( 12 )} Furthermore, the adverse events of this drug have also been shown to be milder than those of ATRA in clinical trials.^{( 13 )} TM411 is being investigated in clinical trials for the treatment of MM, with the objective of confirming the greater therapeutic advantage of this drug as compared to ATRA.

In the present study, we illustrated the cytotoxic activity of TM411 against human myeloma cells. It is well known that IL‐6 signaling plays an important role in the growth of MM, both in vitro and in vivo. ^{( 15} , ¹⁷ , ³⁶ , ³⁷ , ³⁸ , ^{39 )} IL‐6 acts as a potent survival and anti‐apoptotic factor in MM through activation of multiple signaling cascades. Previous studies have shown that IL‐6 induces growth and survival of human myeloma cells by way of the MAPK,^{( 40 )} JAK/STAT,^{( 31} , ⁴¹ , ^{42 )} and PI3K/Akt kinase pathways, which inhibit apoptosis and the appearance of drug resistance in MM cells.^{( 32} , ^{43 )} Our data indicated that TM411 induced IL‐6 receptor‐α downregulation and inhibited its downstream signaling molecules, such as MAPK and Akt. These effects might be responsible for the observed growth‐inhibitory effect of the drug on myeloma cells in vitro and in vivo. Similarly, the inhibitory effect of ATRA on the growth of MM cells was determined to be through the regulation of an IL‐6 receptor‐related signaling pathway.^{( 15} , ^{44 )}

The in vitro and in vivo experiments in our study showed the synergistic potential of the combination of TM411 + GC. GCs such as DEX and PSL induce apoptosis through transcriptional activation of death‐specific genes in hematological cells, including leukemia, lymphoma, and myeloma.^{( 26 )} GC not only triggers death signaling, but also simultaneously activates a protective signal, whereby increased IL‐6 receptor expression on the MM cell surface facilitates enhanced IL‐6 binding and related protection against GC‐induced apoptosis.^{( 25 )} IL‐6 not only triggers myeloma cell growth through the MAPK signaling cascade,^{( 40 )} but also blocks DEX‐induced apoptosis by activation of the PI3K/Akt pathway,^{( 32 )} therefore, our data suggested that the inhibitory effect of TM411 on GC‐mediated anti‐apoptotic signaling molecules might represent synergistic interaction.

Increased phosphorylation of MAPK, MEK, and Akt was observed in RPMI‐8226 cells exposed to GC. Activation of the MAPK and Akt pathways by GCs might elicit a survival response against apoptosis^{( 30} , ³² , ⁴⁵ , ^{46 )} and such activation was inhibited partially by TM411. Increase in the expression of phosphorylated STAT3 was observed only when the cells were exposed to TM411 in combination with a GC. Although STAT3 is one of the key molecules that promote cell growth in MM, the reason for this modification occurring only in the combination setting is unclear. We examined the expression of the downstream molecules of STAT3 such as pro‐apoptotic protein BAX, anti‐apoptotic protein Bcl‐xL and Mcl‐1 by Western blot analysis. As a result, we observed that the expression of Bcl‐xL was decreased in the combination of TM411 + DEX compared with the treatment of TM411 or DEX alone, and those of Mcl‐1 was decreased in the combination compared with the treatment of TM411 alone without IL‐6 stimulation. We suggested the treatment of TM411 combined with DEX might promote apoptosis compared with DEX alone, but could not show the significant modulations of STAT3‐related molecules by these drugs, therefore further study is required.

In younger patients with MM, high‐dose chemotherapy with hematopoietic stem cell transplantation is a useful approach with survival benefit, but is generally not suitable for patients older than 65 years because of its severe toxicity. Recently, novel biologically‐based treatments such as thalidomide,^{( 47} , ⁴⁸ , ⁴⁹ , ^{50 )} lenalidomide,^{( 51} , ⁵² , ^{53 )} and bortezomib^{( 54} , ^{55 )} have been shown to exert marked activity against MM in clinical trials, but specific toxicity profiles, such as myelosuppression and neurotoxicity, remain clinical problems.^{( 10 )} The present data suggest that TM411 given in combination with a GC is one of the most promising therapeutic regimens for MM, especially in high‐risk patients, however, further studies are required to verify the feasibility of use of the combination in the clinical setting.