Research ArticleExperimental Studies

Synergistic Effects of a Smac Mimetic with Doxorubicin Against Human Osteosarcoma

ETSUKO KAMATA, TERUYA KAWAMOTO, TAKESHI UEHA, HITOMI HARA, NAOMASA FUKASE, MASAYA MINODA, MASAYUKI MORISHITA, TOSHIYUKI TAKEMORI, SHUICHI FUJIWARA, KOTARO NISHIDA, RYOSUKE KURODA, MASAHIRO KUROSAKA and TOSHIHIRO AKISUE
Anticancer Research November 2017, 37 (11) 6097-6106;

Abstract

Background/Aim: Second mitochondria-derived activator of caspase (Smac) is a proapoptogenic mitochondrial protein that antagonizes inhibitors of apoptosis proteins (IAPs), resulting in induction of apoptosis. In the present study we investigated the effects of a Smac mimetic in combination with doxorubicin against osteosarcoma. Materials and Methods: In vitro effects of the combination of a Smac mimetic AT-406 and doxorubicin on cell proliferation and apoptosis in osteosarcoma cell lines were examined using cell proliferation assays, flow cytometry, and immunoblot analyses. For in vivo experiments, human osteosarcoma xenografts were treated with combination of the two substances, and tumor volume and apoptotic activity in treated tumors were assessed. Results: In vitro studies revealed that combination of the two substances significantly inhibited osteosarcoma proliferation with decreased cIAP1 expression and induced apoptosis in osteosarcoma cells. Combination of the two substances significantly suppressed osteosarcoma growth in vivo. Moreover, decreased cIAP1 expression and increased apoptotic activity were observed in tumors treated by their combination of the substances. Conclusion: The Smac mimetic AT-406 showed an apoptotic effect and a synergistic antitumor effect with doxorubicin on osteosarcoma. The combination of AT-406 and doxorubicin may serve as a novel therapeutic strategy for osteosarcoma treatment.

Osteosarcoma is the most common primary malignant bone tumor in adolescent and young adults. There have been significant advances in adjuvant chemotherapy for osteosarcoma treatment (1-3). Approximately 70% osteosarcoma patients achieve long-term survival with current therapeutic strategies along with systemic therapy to control microscopic metastatic disease. Although some new therapeutic interventions have been tested, the treatment outcome for osteosarcoma shows no great improvement over the last two decades (4-7). Therefore, there is an unmet need for the development of new therapeutic strategies against osteosarcoma.

Apoptosis is a cell death pathway essential for normal tissue homeostasis, cell differentiation, and development (7, 8). There are two major signaling pathways leading to apoptosis-associated caspase activation. First, is the extrinsic death-receptor pathway that is triggered by members of the death receptor superfamily, leading to caspase-8 activation. The second pathway is the intrinsic mitochondrial pathway activated in response to extracellular cues and internal insults (e.g., DNA damage) (7, 8). It results in the release of apoptogenic factors such as cytochrome c or second mitochondria-derived activator of caspase (Smac)/direct IAP-binding protein with low pl (DIABLO) from mitochondria to the cytosol (9). The activity of mature caspases is negatively regulated by their interaction with the inhibitor of apoptosis proteins (IAPs) (10). Human IAP family comprises of the following eight proteins: neuronal apoptosis inhibitory protein (NAIP), cellular IAP 1 (c-IAP1), c-IAP2, X-linked IAP (XIAP), survivin, Apollon/Bruce, Melanoma IAP (ML-IAP/Livin), and IAP-like protein-2 (ILP-2) (11). Overexpression of IAPs is known to enhance resistance to apoptotic stimuli in various malignancies, including sarcomas (10-21). However, very few reports have focused on the role of IAPs in osteosarcoma (13-15).

Smac is a proapoptogenic mitochondrial protein released from mitochondria to the cytosol in response to diverse apoptotic stimuli, including the commonly used chemotherapeutic drugs. In the cytosol, Smac interacts with and antagonizes IAP proteins, thereby allowing activation of caspases and apoptosis (22). Several small-molecule Smac mimetics have been developed over the last decade to abolish drug resistance; these molecules show antitumor effects against various malignancies alone or in combination with chemotherapeutic agents (23-25). However, the effects of Smac on musculoskeletal malignancies are still unknown. In this study, we investigated antitumor effects of a Smac mimetic AT-406 in combination with doxorubicin (DOX) and the mechanisms of cIAP1 degradation by AT-406 in osteosarcoma cells in vitro and in vivo.

Materials and Methods

Reagents. Doxorubicin (Adriamycin; 14-hydroxydaunomycin, HCl) was obtained from Merck (Darmstadt, Germany) and AT-406 was purchased from Active Biochem (Maplewood, NJ, USA). Stock solution of AT-406 was prepared in dimethyl sulfoxide, followed by immediate storage at −80°C. The stock solution was diluted in culture medium for in vitro study or saline for in vivo experiments prior to use.

Cell lines. Two human osteosarcoma cell lines (KHOS/NP and MG63) were used in this study. The cell line KHOS/NP was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and the MG63 cell line was obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT (Ibaraki, Japan). Cells were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 100 U/mL penicillin/streptomycin solution (Sigma-Aldrich) at 37°C in a humidified 5% CO2 atmosphere. For all experiments, DMEM containing 10% FBS without the antibiotic solution was used.

Animal studies. All animal experiments were approved by the Kobe University Animal Experimentation Regulations (Permission no. P160307). Male BALB/c nude mice (5-week old) were purchased from CLEA Japan Inc. (Tokyo, Japan) and maintained in a facility under specific pathogen-free conditions. Animals were fed with pathogen-free laboratory chow and allowed free access to autoclaved water in an air-conditioned room with a 12-h light/dark cycle. For in vivo experiments, KHOS/NP cells were implanted into the dorsal subcutaneous area of mice (n=24) at 2.0×106 cells in 500 μl phosphate-buffered saline (PBS), as previously described (26). Mice were randomly divided into following treatment groups: control (n=6), AT-406 (n=6), DOX (n=6), and combination (AT-406 + DOX; n=6).

A week after cell implantation, treatment commenced by an intraperitoneal injection of AT-406 (50 mg/kg) for AT-406 group, DOX (3 mg/kg) for DOX group, AT-406 (50 mg/kg) and DOX (3 mg/kg) for combination group, or saline for control group, twice a week for 2 weeks. Tumor volume was calculated twice a week, as previously described using the formula V=π/6 × a2 × b, where a and b represent the shorter and longer dimension of the tumor, respectively (26). The body weight of each mouse was regularly monitored. At the end of the experiments, all tumors were excised and immediately stored at −80°C. Apoptotic activity in treated tumors was evaluated by flow cytometric assays, immunoblot analyses, and immunofluorescence staining.

Cell proliferation assays. To evaluate the effect of the combination of Smac mimetic AT-406 and DOX against osteosarcoma cell growth in vitro, we performed the WST-8 cell proliferation assays using the Cell Counting Kit-8 (CCK-8; Dojindo Inc., Kumamoto, Japan). Cells were seeded in 96-well culture plates at a density of 5,000 cells/well in 100 μl culture medium, followed by treatment with the combination of 200 nM DOX and various concentrations of AT-406 (0-500 μM). At the indicated incubation times, 10 μl of CCK-8 solution was added into each well and incubated for 1 h. The optical density of the solution was measured at a wavelength of 450 nm using a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA) and the relative number of viable cells in each well calculated.

Flow cytometric analysis. Flow cytometry was performed to investigate the apoptotic activity in cultured cells or implanted tumors. Briefly, cells were collected from cultured cells or implanted tumors, suspended in 1% paraformaldehyde in PBS, and resuspended in ice cold ethanol at 1×106 cells/ml. Each cell pellet was labeled using the APO-DIRECT Kit according to the manufacturers' protocol (BD Pharmingen, Franklin Lakes, NJ). Fluorescent intensity was analyzed using the BD FACSVerse™ (BD Biosciences, Franklin Lakes, NJ, USA).

Immunoblot analysis. Cell lysates were extracted from cells or implanted tumors using the whole cell lysis buffer (Mammalian Protein Extraction reagent, Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, USA). Protein concentration was quantified using the bicinchoninic acid (BCA) Protein Assay reagent (Bio-Rad, Hercules, CA, USA). Samples containing equal amounts of proteins were separated on a 7.5-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were incubated overnight at 4°C with the following primary antibodies in the CanGet Signal Solution 1 (TOYOBO Co., LTD, Osaka, Japan): anti-human cIAP1 (1:1,000) (Cell Signaling Technology), anti-human cleaved caspase-8 (1:1,000) (Cell Signaling Technology), anti-human cleaved caspase-3 (1:1,000) (Cell Signaling Technology), anti-human cleaved PARP (1:1,000) (Cell Signaling Technology), and anti-human α-tubulin antibody (1:10,000) (Sigma-Aldrich). Following washes, membranes were incubated with appropriate secondary antibody conjugated to horseradish peroxidase and exposed to the enhanced chemiluminescence (ECL) Plus western blot detection system reagent (GE Healthcare Biosciences, Piscataway, NJ, USA). Protein expression was detected by the Chemilumino analyzer LAS-3000 mini (Fujifilm, Tokyo, Japan). Membranes were reprobed with anti-human α-tubulin antibody (Sigma-Aldrich) to confirm equal protein loading.

Immunofluorescence staining. Immunofluorescence staining was performed to evaluate cellular apoptosis in treated osteosarcoma cells and implanted tumors using the APO-DIRECT Kit (BD Pharmingen). In vitro, cells from AT-406, DOX, AT-406 + DOX, and control groups were fixed in 4% paraformaldehyde for 30 min at room temperature. Cells were incubated in the prepared DNA Labeling Solution (APO-DIRECT Kit, BD Pharmingen) for 1 h and subjected to nuclear staining using propidium iodide. Stained cells were assessed using the BZ-8000 confocal microscope (Keyence, Osaka, Japan). For in vivo experiments, tumor tissue samples were embedded in the optimal cutting temperature compound (Sakura Finetek Co., Tokyo, Japan) and 10-μm thick sections were prepared on a cryostat and stored frozen at −80°C. Sections were incubated with an anti-actin antibody (Sigma-Aldrich) diluted in PBS for 30 min at 37°C. After washing, sections were incubated with the APO-DIRECT Kit reagents (BD Pharmingen) in PBS for 30 min in a dark humid chamber at 37°C. The nucleus was stained with the 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using the BZ-8000 confocal microscope.

Figure 1.
Figure 1.

Effects of the combination of the Smac mimetic (AT-406) and doxorubicin (DOX) on cell proliferation in human osteosarcoma cell lines in vitro. In vitro effects of the combination of AT-406 (0-500 μM) and DOX (200 nM) against two osteosarcoma cell lines, KHOS/NP (A) and MG63 (B), were assessed by WST-8 assays at 24, 48, and 72 h following treatment. Data represent mean±SEM of at least three independent experiments (*p<0.05 vs. 0 μM AT-406; †p<0.05 vs. 1 μM AT-406; ‡p<0.05 vs. 1 and 10 μM AT-406; §p<0.05 vs. 1, 10, and 100 μM AT-406).

Figure 2.
Figure 2.
Figure 2.

Effects of the combination of the Smac mimetic (AT-406) and doxorubicin (DOX) on apoptotic activity in human osteosarcoma cell lines in vitro. (A, B) Flow cytometric analyses for DNA fragmentation in cells after 48 h of treatment with control, AT-406 alone (200 μM), DOX alone (200 nM), and combination of AT-406 and DOX. (C, D) Immunoblot analyses for expression of cIAP-1 and cleaved forms of caspase-3, caspase-8, and PARP in cells after 48 h of treatment. (E, F) Immunofluorescence staining for DNA fragmentation in cells after 48 h of treatment (green, apoptosis nuclear [APO-DIRECT]; blue, nuclear [DAPI]).

Statistical analysis. All experiments were performed independently at least thrice and data presented as the mean±standard error of the mean (SEM). Significance of differences between groups was evaluated using a two-tailed Student's t-test and analysis of variance (ANOVA) with post-hoc test to compare for continuous values. A value of p<0.05 was considered statistically significant.

Results

Smac mimetic AT-406 showed synergistic antitumor effects with DOX against human osteosarcoma cells in vitro. To examine the effects of the combination of the Smac mimetic AT-406 and DOX against osteosarcoma cells (KHOS/NP and MG63) in vitro, we assessed cell viability and apoptotic activity in these cells following treatment with 200 nM DOX and various concentrations of AT-406 (0-500 μM). AT-406 showed a synergistic antitumor effect with DOX, as cell viability was significantly decreased in both osteosarcoma cell lines treated with 200 nM DOX and 10 μM or more AT-406 in a dose- and time-dependent manner (Figure 1A and B). Flow cytometric analyses revealed that the apoptotic activity was significantly increased in cells subjected to combination treatment compared to the control cells and those treated with AT-406 or DOX alone (Figure 2A and B). Immunoblot analyses showed that cIAP1 expression was decreased in cells treated with AT-406 and combination and that expressions of cleaved forms of caspase-8, caspase-3, and PARP were strongly increased after combination treatment (Figure 2C and D). In addition, immunofluorescence staining revealed increased number of apoptotic cells in both cell lines following combination treatment (Figure 2E and F). These results suggest that AT-406 displayed a synergistic antitumor effect with DOX against human osteosarcoma cells in vitro mediated by apoptosis via decreased cIAP1 expression.

The combination of AT-406 and DOX suppressed in vivo osteosarcoma growth mediated by increased apoptotic activity. We evaluated the in vivo antitumor activity of the combination of AT-406 and DOX using human osteosarcoma xenografts. The combination treatment significantly suppressed in vivo osteosarcoma tumor growth as compared with the untreated control (Figure 3A and B). At the end of experiments, the tumor volume in the combination group was 61.2% of that in the control group (Figure 3B) (p<0.05). No significant loss in body weight was observed during the experimental period (Figure 3C). In comparison to the untreated control and AT-406 or DOX alone, combination treatment resulted in a significant increase in the apoptotic activity, as evident from the flow cytometric analyses (Figure 4A). Moreover, immunoblot analyses showed that cIAP1 expression was decreased in AT-406 and combination groups but not in control and DOX groups. In comparison to other treatment groups, combination treatment resulted in increased expression of cleaved forms of caspase-3, caspase-8, and PARP (Figure 4B). Immunofluorescence staining revealed an increased number of apoptotic cells in osteosarcoma tumor tissues following combination treatment (Figure 4C). These results suggest that AT-406 exhibits a synergistic antitumor effect with DOX against human osteosarcoma in vivo.

Figure 3.
Figure 3.

In vivo antitumor activity of the combination of the Smac mimetic (AT-406) and DOX against human osteosarcoma xenografts. Tumor volume (mm3) (A, B) and body weight (g) (C) in mice treated with AT-406 (50 mg/kg) only, DOX (3 mg/kg) only, combination of AT-406 and DOX, and control were monitored for 14 days. Data represent mean±SEM of at least three independent experiments (*p<0.05).

Discussion

Osteosarcoma – the most common primary solid malignant tumor of the bone – comprises of about 20% of primary bone sarcomas (1-5). A critical problem associated with osteosarcoma is the frequent formation of micrometastases in the lung prior to diagnosis (5). Substantial improvements in surgery and chemotherapy have increased the survival rate of patients with localized disease. However, the prognosis of patients with metastatic or recurrent disease is still poor owing to the lack of second-line chemotherapies (4-6). This necessitates the development of better and safer chemotherapeutic strategies against osteosarcomas.

Figure 4.
Figure 4.

Effects of the combination of the Smac mimetic (AT-406) and DOX on apoptosis in osteosarcoma in vivo. (A) DNA fragmentation in tumor tissues after 14 days of treatment was assessed by flow cytometry. (B) Immunoblot analyses for expressions of cIAP-1 and cleaved forms of caspase-3, caspase-8, and PARP in tumor tissues after 14 days of treatment. (C) Immunofluorescence staining for DNA fragmentation in tumor tissues after 14 days of treatment (green, apoptosis nuclear [APO-DIRECT]; blue, nuclear [DAPI]).

During cancer progression, apoptosis is frequently blocked in several ways such as through the overexpression of anti-apoptotic molecules (7-9). Overexpression of IAPs contributes to cancer progression by inducing resistance to apoptotic stimuli in various malignancies (10-21). Several studies have focused on the roles of IAPs as well as the therapeutic strategies targeting IAPs in various malignancies (22-25, 27, 28). However, very few reports have focused on the role of IAPs in osteosarcoma (13-15). IAPs belong to a family of anti-apoptotic proteins. The first identified human IAP was NAIP isolated based on its contribution to neurodegenerative disorder (29). Four human IAPs, including cIAP1, cIAP2, XIAP, and survivin, have been isolated thereafter, all of which have been shown to counter cell death by inhibiting apoptotic activity (30, 31). cIAP1 and cIAP2 were originally identified as binding partners for the tumor necrosis factor (TNF)-associated factors 1 and 2 (TRAF1 and TRAF2) (32). Several studies have suggested that both cIAPs are critical regulators of TNFα-mediated activation of nuclear factor-kappa B (NF-ĸB), resulting in apoptosis inhibition (33-35). Of these, cIAP1 was reported to be involved in the development of osteosarcoma (13). Hence, we focused on cIAP1 as a potent therapeutic target for osteosarcoma treatment.

Smac is a proapoptogenic mitochondrial protein and an endogenous antagonist of IAPs, which is released into the cytosol from mitochondria in response to diverse apoptotic stimuli such as the commonly used chemotherapeutic drugs (22-25, 36). In the cytosol, Smac binds to IAPs to induce their degradation, activates caspase-8 and caspase-3, promotes PARP cleavage, and stimulates cell apoptosis (22). Several efforts have resulted in the design and development of small-molecule Smac mimetics to abolish drug resistance (23-25, 36-40). Moreover, studies have reported the enhanced antitumor activity of chemotherapeutic agents by Smac mimetics in various malignant cells (36-40). Several Smac mimetics, including AT-406 (38-43), are currently undergoing clinical trials for treatment of various cancers alone or in combination with anticancer drugs (38-46). Smac mimetics are reported to exhibit synergistic effect in combination with DOX and facilitate cIAP1 degradation (36-38). DOX, an anthracycline antibiotic, has been widely used for treatment of various malignancies such as lymphomas, leukemia, osteosarcomas as well as lung, breast, and ovarian cancers (47, 48). DOX is one of the most widely used anticancer drugs and the most important key drug used in multidrug chemotherapy regimen in combination with methotrexate and cisplatin for osteosarcoma treatment (3, 49). However, studies have reported resistance to apoptotic effects of DOX in some cancers (48, 50). And, despite its highly beneficial anticancer effects, DOX has the serious adverse effect of cardiotoxicity in the clinical use. To overcome drug resistance and/or to increase chemosensitivity, we focused on Smac mimetics known to enhance apoptotic effects of anticancer agents against various cancer cells and evaluated the effects of the Smac mimetic AT-406 against human osteosarcoma cells.

Here, we revealed that AT-406 in combination with DOX showed a significant increase in cell growth inhibition and induced apoptotic activity through caspase-8 activation via decreased expression of cIAP1 in human osteosarcoma cells. In addition, in vivo tumor volume of human osteosarcoma was significantly reduced by the combination of AT-406 with the low doses of DOX, which did not cause apparent loss in body weight. The findings strongly suggest that Smac mimetics could augment the efficacy of DOX and/or other currently used chemotherapeutic agents, and could permit lower doses of DOX to prevent its unfavorable side effects in osteosarcoma treatment.

To the best of our knowledge, this is the first study targeting cIAP1 through combination treatment with DOX for osteosarcoma. The present study highlights the synergistic effect of Smac mimetic with DOX against osteosarcoma progression and apoptosis via cIAP1 degradation in vitro and in vivo. However, this study had several limitations. In this study, we found that a Smac mimetic could show an apoptotic effect and a synergistic antitumor effect with DOX using only two osteosarcoma cell lines. And, we did not assess the antitumor effects of a Smac mimetic with other chemotherapeutic agents than DOX. Although further studies are needed to determine whether Smac mimetics have sufficient effects in human osteosarcoma and to elucidate the mechanisms mediating the antitumor effects of Smac, the findings in this study strongly indicate that inhibition of IAPs using the Smac mimetic may be considered as a potent therapeutic target for the treatment of osteosarcoma.

Conclusion

Smac mimetics may enhance the anticancer activity mediated by chemotherapeutics against human osteosarcoma and may be considered a potent therapeutic agent in combination with conventional chemotherapy for human osteosarcoma treatment.

Acknowledgements

The Authors would like to thank Minako Nagata, Maya Yasuda, and Kyoko Tanaka for their expert technical assistance.

Footnotes

  • Conflicts of Interest

    The Authors have no conflict of interest to declare.

  • Received June 12, 2017.
  • Revision received June 29, 2017.
  • Accepted June 30, 2017.

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Synergistic Effects of a Smac Mimetic with Doxorubicin Against Human Osteosarcoma
ETSUKO KAMATA, TERUYA KAWAMOTO, TAKESHI UEHA, HITOMI HARA, NAOMASA FUKASE, MASAYA MINODA, MASAYUKI MORISHITA, TOSHIYUKI TAKEMORI, SHUICHI FUJIWARA, KOTARO NISHIDA, RYOSUKE KURODA, MASAHIRO KUROSAKA, TOSHIHIRO AKISUE
Anticancer Research Nov 2017, 37 (11) 6097-6106;
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