Abstract
Background/Aim: The development of new drugs is urgently needed for new treatment strategies that can improve the prognosis of osteosarcoma (OS). In this study, we attempted to identify combinations of new molecular-targeted agents for OS. Materials and Methods: A library containing 324 compounds was used. For the first screening, MG-63 OS cells were treated with each of the compounds and cell viability was measured. After the best candidate compound was decided, the compound was included in a second screening. The combination of most effective compounds was decided. The antiproliferative effect of the combination was examined and the cell signaling mechanism was evaluated by western blot analysis. 143B OS-bearing mice were used for in vivo antitumor testing. Results: In the first screening, bortezomib was chosen as the effective drug. In the second screening with bortezomib, everolimus was chosen. This combination showed a synergistic inhibitory effect on cell proliferation when compared to monotherapy with each of these drugs alone. Compared to monotherapy, the combination therapy enhanced the levels of cleaved poly (ADP-ribose) polymerase, caspase-3, caspase-8 and caspase-9, phospho-c-Jun N-terminal kinase, and P38. In contrast, the levels of c-MYC proto-oncogene bHLH transcription factor, survivin, and phospho-cyclin D1 were reduced. The combination effectively induced apoptosis and interfered with cell cycle progression. In the in vivo analysis, the combination therapy significantly inhibited tumor growth. Conclusion: The combination of everolimus and bortezomib demonstrated a synergistic effect against OS both in vitro and in vivo. These results indicate that this combination may be useful as a novel therapeutic strategy for OS.
Osteosarcoma (OS) is the most common primary sarcoma of bone of mesenchymal origin in children and adolescents. OS treatment requires a multidisciplinary strategy including surgery and chemotherapy. The major treatment against OS is the combination of doxorubicin, cisplatin, methotrexate and ifosfamide, and these agents have been the gold standard for OS for over 30 years (1, 2). The 5-year survival rate for patients with extremity-localized OS has reached 70% (3). However, despite the success of treatments, 30-40% of patients experience relapse and 88% of relapsed osteosarcoma cases develop lung metastasis (4, 5). Additionally, lung metastasis at initial diagnosis is observed in 10-20% of patients (3, 6). Lung metastasis is the major cause of death and the 5-year overall survival rate for patients with metastatic OS is still only approximately 45%, and little improvement has been seen in recent years (3, 7). The development of new drugs is urgently needed for new treatment strategies that can improve the prognosis of patients with OS.
Recently, molecular-targeted cancer therapy has attracted much attention. In clinical trials for OS, avelumab (programmed death ligand-1 antibody), olaparib (poly ADP-ribose polymerase inhibitor), ataxia telangiectasia mutated and Rad3-related kinase inhibitor, ALMB-0168 (humanized CX43 monoclonal antibody), inhibitor of WEE1 G2 checkpoint kinase, and camrelizumab (programmed cell death protein-1 antibody), among other agents, have been studied (ClinicalTrails.gov IDs: NCT03006848, NCT04417062, NCT03718091, NCT04886765, NCT04833582 and NCT04294511, respectively); however, effective agents for treating OS remain elusive. The development of a new molecular-targeted agent remains a costly and time-consuming process, and it is not uncommon for tested agents to show no clinical efficacy. However, even if a single therapy has only weak efficacy, it is possible that in combination therapy the agent may show a synergistic effect, and the cost of such screening for effective combination therapies is low. The aim of this study was to identify effective combinations of agents from among existing compounds for inducing OS cell death.
For screening, we used the Screening Committee of Anticancer Drugs (SCADS) compound library. Previously, we reported on the compound cucurbitacin I using this SCADS library, which had a broad antiproliferative effect on five OS cell lines of different phenotype 143B, MG63, HOS, SAOS-2, and HUO9 (8). This library contains only 324 anticancer drugs including agents already in clinical use. This number is adequate for screening for synergistic antiproliferative effects from clinical agents. Firstly, we screened the antiproliferative effects using the SCADS library; then we re-screened the drugs for synergistic antiproliferative effects when combined with a drug that showed an antiproliferative effect in the first screening. Secondly, we investigated the mechanism of the synergistic effect. Finally, we examined the inhibitory effect of the combination of agents on tumor growth using an in vivo model. It is expected that this result is informative and useful for OS treatment.
Materials and Methods
OS cell culture. Human OS cell lines MG-63 and 143B (Riken Cell Bank, Ibaraki, Japan) were cultured in minimum essential media (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum, 10 U/ml penicillin, and 10 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.
Compounds. The SCADS compound library, containing 324 compounds in four 96-well microplates (http://gantoku-shien.jfcr.or.jp/), was obtained through a Grant-in-Aid for Scientific Research in the Priority Area “Cancer” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The compounds were provided at a concentration of 100 μM in 100 μl of solution containing 50% methanol.
Bortezomib and everolimus (Sigma-Aldrich, St. Louis, MO, USA) were initially dissolved in dimethyl sulfoxide (DMSO), and stored at −20°C. For the experiments, bortezomib and everolimus were diluted with culture media to the final concentrations needed.
Compound screening. For compound screening, monolayers of MG-63 cells at a density of 3.0×104 cells/well (100 μl) were treated with either the diluent control (DMSO) or 1 μM of each of the compounds in the 96-well plates of the SCADS library. After 24 h of treatment, cell viability was measured using Cell-Titer 96® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA). In the first screening, the cell viability was calculated from the absorbance as (absorbance with compound)/(absorbance without compound), and a color-coded table was prepared: White indicating that less than 30% of the cells remained viable; gray indicating that 30-50% of the cells remained viable; and dark gray indicating that more than 50% of the cells remained viable. From these data, the first candidate, bortezomib, was chosen because it is the only agent that has been used clinically.
Bortezomib (10 nM) was added into all wells of a SCADS library plate with 1 μM of each candidate compound in a second screening. After 24-h treatment, cell viability was measured as described above. The cell viability was calculated from the absorbance as (absorbance under treatment with compound and bortezomib)/(absorbance under treatment with compound without bortezomib), and a color-coded table was prepared similar to that in the first screening: White indicating that less than 15% of the cells remained viable; gray indicating that 15-20% of the cells remained viable; and dark gray indicating that more than 20% of the cells remained viable. From these data, clinical agents everolimus, lapatinib and vorinostat were highlighted as potential candidates for use in combination with bortezomib. Two of these, everolimus and lapatinib, are related to the phosphatidylinositol 3-kinase (PI3K)/AKT/mechanistic target of rapamycin kinase (mTOR) pathway. The downstream protein of this pathway, mTOR, was chosen as a target. Therefore, the second candidate agent, everolimus, was used.
Evaluation of synergistic effects. To evaluate the synergistic effect of bortezomib and everolimus, MG-63 and 143B cells were exposed to media containing different concentrations of everolimus (mTOR complex 1 inhibitor; 5-30 μM), rapamycin (inhibitor of mTOR complex 1 and 2; 5-30 μM), or DMSO (negative control) with or without bortezomib (5 or 50 nM) for 24 h and cell viability was then measured as described above.
Western blot analysis. After treatment with or without everolimus (10-20 μM) and bortezomib (5 nM) for 6 or 12 h, cells were lysed with radioimmunoprecipitation buffer (Millipore-Upstate, Temecula, CA, USA) supplemented with a protease inhibitor cocktail, 0.5 mM phenylmethylsulfonyl fluoride, and 0.2 mM Na3VO4. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and samples were adjusted to the same protein concentration before loading onto a nitrocellulose membrane. Western blotting was then performed. The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), and used at the indicated dilutions: Rabbit antibody to cleaved caspase-3 (Asp175) (#9661; 1:100), mouse monoclonal antibody to caspase-8 (1C12) (#9746; 1:500), rabbit monoclonal antibody to cleaved caspase-9 (Asp315) (D8I9E) (#20750; 1:1,000), rabbit monoclonal antibody to cleaved PARP (Asp214) (D64E10) XP® (#5625; 1:1,000), rabbit monoclonal antibody to phospho-p44/42 mitogen-activated protein kinase (MAPK) (ERK1/2) (Thr202/Tyr204) (D13.14.4E) XP® (#4370; 1:2,000), rabbit antibody to phospho- stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK; Thr183/Tyr185) (#9251; 1:200), XP® rabbit monoclonal antibody phospho-p38 MAPK (Thr180/Tyr182) (D3F9) (#4511; 1:200), rabbit monoclonal antibody to c-MYC proto-oncogene bHLH transcription factor (c-MYC; D85C12; #5605; 1:1,000), rabbit monoclonal antibody to survivin (#2808; 1:1,000), rabbit monoclonal antibody to MCL-1 apoptosis regulator, BCL2 family member (MCL-1; #5453; 1:1,000), rabbit antibody to phospho-AKT serine/threonine kinase 1 (AKT; (#4060; 1:1,000), and rabbit antibody phospho-cyclin D1 (#3300; 1:1,000), rabbit polyclonal antibody to BCL2-interacting killer (BIK) was obtained from Abcam (ab52182; 1:500; Abcam, Oxford, UK). β-Actin (ab8229; 1:500) protein was assayed as a loading control and was also from Abcam. After hybridization with a horseradish peroxidase-conjugated secondary antibody, bands were visualized using the ECL Prime western blotting system (Cytiva, Little Chalfont, UK).
Growth of 143B xenografts in athymic nude mice in vivo. All animal experiments were performed according to protocols approved (approval number: 27-27) by the Institutional Animal Care and Use Committee of Mie University. 143B Cells were inoculated subcutaneously into the back of female nude mice (2.0×106 cells/mouse). Mice were randomly assigned to each of the following experimental groups, with n=5 per group: (i) Control group administered saline with DMSO (diluent-specific control); (ii) bortezomib-treated group injected intraperitoneally with 0.5 mg/kg of bortezomib twice a week; (iii) everolimus-treated group administered 1.0 mg/kg of everolimus orally twice a week; and (iv) combination-treated group administered both bortezomib and everolimus. The tumor size of the mice was measured twice a week, and the tumor volume was calculated using the following formula: V=lw2/2, where (l) is the length, (w) the width, and (V) is the volume, as described previously (8). The treatment was stopped at 28 days.
Statistical analysis. Data are expressed as the mean±standard deviation. A nonparametric analysis of variance (ANOVA) test (Mann–Whitney) was used to compare the differences between two groups. A repeated ANOVA or one-way ANOVA was used to compare the tumor volume. A value of p<0.05 was considered to be statistically significant.
Results
Compound screening. The antiproliferative effect of 324 compounds on MG-63 cells was screened. Twenty compounds were categorized into the white color-coded group, i.e. that reducing viability the most (Figure 1). Among these compounds was cucurbitacin I, which has been reported by our group to be an effective antitumor agent for OS (8). In the present study, a proteasome inhibitor, bortezomib, was chosen as the first compound because it is already used in a clinical setting.
Initial screening of the Screening Committee of Anticancer Drugs (SCADS) library. MG-63 cells were treated for 24 h with 1 μM of each compound from the SCADS library, and the cell viability was evaluated. Compounds were categorized into color-coded groups according to cell viability when compared to the control. White: <30% viable cells, gray: 30-50% viable cells, dark gray: >50% viable cells. The 20 compounds that were categorized into the white color-coded group are listed. Bortezomib was chosen as the first candidate compound.
Second screening for a synergistic effect. To find a compound that has a synergistic effect with bortezomib, compounds were screened with low-dose bortezomib in MG-63 cells. Twenty-one compounds were categorized into the white color-coded group (Figure 2). In this study, the mechanistic target of rapamycin (mTOR) inhibitor everolimus was chosen as the second compound because it is also already used in a clinical setting.
Second screening of the Screening Committee of Anticancer Drugs (SCADS) library. MG-63 cells were treated for 24 h with bortezomib and each compound in the library, and the cell viability was evaluated. Compounds were categorized into color-coded groups according to the cell viability when compared to the control. White: <15% viable cells, gray: 15-20% viable cells, dark gray: >20% viable cells. The 21 compounds that were categorized into the white color-coded group are listed. Everolimus was chosen as the second candidate compound.
Evaluation of the synergistic effect. The mTOR inhibitors everolimus and rapamycin were used. The antiproliferative effect was compared in MG-63 (Figure 3A) and 143B (Figure 3B) cells. At 5, 10, and 20 μM, everolimus showed a more effective antiproliferative effect than rapamycin in MG-63 cells; 30 μM everolimus alone reduced the cell viability of MG-63 and 143B cells to 77.7% and 85.7%, respectively, when compared to the controls. Cells treated with 5 nM bortezomib exhibited almost the same level of cell viability as the untreated control cells. In the presence of 5 nM bortezomib, everolimus significantly reduced cell viability, more than with the same dose of everolimus alone. Combined therapy of 30 μM bortezomib and 5 nM everolimus reduced cell viability to 37.4% and 48.1% in MG-63 and 143B cells, respectively, when compared to the untreated controls (Figure 3). Thus, the combination of everolimus and bortezomib had a synergistic antiproliferative effect on the OS cell lines.
Dose response for cell viability. The effect of combination therapy with an inhibitor of mechanistic target of rapamycin kinase, everolimus or rapamycin, and bortezomib was analyzed in MG-63 (A) and 143B (B) cells for 24 h treatment. Everolimus and rapamycin were used at a concentration of 0, 5, 10, 20, or 30 μM. Bortezomib was used at a concentration of 5 or 50 nM.
Cell signaling mechanism. Cells were treated with 10 or 20 μM everolimus and 5 nM bortezomib for 6 h or 12 h. Cleaved PARP was expressed in the cells treated with 20 μM everolimus for 6 h. Dual therapy with everolimus and bortezomib enhanced the levels of cleaved caspase-3 and cleaved PARP. This indicates that the combination therapy induced strong apoptotic signals. The enhanced activation of caspases 8 and 9 indicate the involvement of both the extrinsic and intrinsic pathways of apoptosis (Figure 4). In addition, enhanced expression of pJNK and p38 was observed (Figure 4), and they are also associated with the induction of apoptosis. The levels of c-MYC and survivin, which are important proteins for cell proliferation, were reduced after treatment with the combination for 12 h. In addition, the expression of p-AKT and cyclin D1 was reduced at 12 h (Figure 4). This indicates that the combination therapy interfered with the cell cycle.
Cell signaling. The 143B cell line was used for western blot analysis. Cells were treated with 0, 10, or 20 μM everolimus with and without 5 nM bortezomib for 6 or 12 h. BIK: BCL2-interacting killer; ERK: extracellular signal regulated kinase; JNK: phospho-c-Jun N-terminal kinaser; MCL1 apoptosis regulator, BCL2 family member; MYC: MYC proto-oncogene bHLH transcription facto; PARP: poly (ADP-ribose) polymerase.
In vivo effect of everolimus and bortezomib. To evaluate the synergistic effect of everolimus and bortezomib in vivo, we examined the time-dependent changes in 143B xenografts. In the absence of the compounds, the growth of 143B tumors was highly aggressive. Treatment with everolimus or bortezomib alone slightly reduced tumor growth, while combination treatment with everolimus and bortezomib significantly inhibited tumor growth (Figure 5).
In vivo antitumor effect. Athymic nude mice with 143B xenografts were treated with the vehicle (Cont), single therapy of bortezomib or everolimus, or a combination therapy of everolimus and bortezomib (Dual). The effect on tumor growth was assayed. Data are the mean tumor volumes±standard error of five mice per group. The Dual group was significantly different from each group at *p<0.005 by repeated analysis of variance; and at #p<0.05 at 31 days by Mann–Whitney test.
Discussion
The PI3K/AKT/mTOR pathway has been analyzed and reported in many malignant tumors, both experimentally and clinically. Among 56 OS cases, 24% of the patients were identified to have alterations, including mutations and deletions, in the PI3K/mTOR pathway (9). Phosphatase and tensin homolog (PTEN) deletion was observed in OS tissues (10, 11). The expression of phospho-AKT in tissues both before and after chemotherapy has been reported to be associated with a poor prognosis in OS (12). In addition, expression of mTOR or p70S6 kinase was reported to be significantly associated with poor disease-free and overall survival in OS (13, 14). The therapeutic potential of PI3K/AKT/mTOR pathway inhibitors for OS his been demonstrated. BYL719, a specific PI3Kα inhibitor, was shown to reduce OS cell proliferation and migration in vitro, and tumor progression in vivo (15). In addition, inhibitors of pyruvate dehydrogenase kinase (PDK)1 (16) and PDK2 (17) were reported to suppress proliferative activity in OS cells. NVP-BEZ235, which targets the ATP-binding sites of PI3K and mTOR enzymes, was shown to inhibit cell proliferation and tumor progression both in vitro and in vivo (18). AIM2, dieckol and rhaponticin induced OS cell apoptosis via PI3K/AKT/mTOR pathway suppression (19-21). In addition, rapamycin treatment was shown to suppress lung colonization, and prolong survival in a mouse model of OS (22). As such, the PI3K/AKT/mTOR pathway appears to play an important role in tumor growth in OS. However, clinically, everolimus monotherapy had only weak antitumor effects in clinical settings. In a phase II study of everolimus in 41 patients with advanced bone and soft-tissue sarcoma, the results were one partial response, 18 cases of stable disease, and 20 of progressive disease (23).
The proteasome is a large multi-protease complex that is involved in the controlled degradation of more than 80% of cellular proteins, as well as in maintaining cellular protein homeostasis (24). Inhibition of proteasome activity induces protein overload and endoplasmic reticulum stress, finally leading to cell death (25, 26). Bortezomib (first-generation), carfilzomib (second-generation), and the first oral proteasome inhibitor, ixazomib, are therapeutic agents for multiple myeloma and lymphoma (27-32). In OS, bortezomib induces apoptosis and suppresses tumor growth (33-35). Harris et al. compared the antitumor activity against OS between bortezomib and ixazomib, and reported that both drugs inhibited the growth of pulmonary metastases (36). Clinically, proteasome inhibitors have the potential to be a treatment for OS. A clinical trial of bortezomib was performed as a multicenter phase II study in patients with recurrent or metastatic sarcomas (37). In that study, eight out of the 21 evaluable patients achieved the best clinical result of stable disease while 13 patients had progressive disease. Only one patient with extra-skeletal osteogenic sarcoma was enrolled, and the result was not indicated in the article.
A number of molecular-targeted agents are currently being produced. Studies of monotherapy using these agents are essential to estimate their therapeutic effects and the degree of involvement of the target molecule in the tumor growth. While antibody products cover a single target, molecular targeted compounds usually have several targets to bind. The addition of an inhibitory targeted agent as a combination therapy is not unethical, and is easier than the development of a new agent. For the development of more effective treatments, combination therapies have attracted much attention, and various combinations have been reported, including dual therapy with everolimus and bortezomib. Li et al. demonstrated that the synergistic anti-myeloma effects of everolimus with bortezomib in a xenograft mouse model involved the enhancement of apoptosis (38). In the present study, we performed compound screening and successfully identified the synergistic effect of everolimus and bortezomib against OS. Bortezomib (5 nM) alone did not reduce the cell viability of the OS cell lines, but in combination with everolimus, it significantly reduced the cell viability. This synergistic effect mainly resulted from activation of apoptosis pathways, reduced expression of c-MYC and survivin, and interference in the cell cycle. Additionally, this combination suppressed tumor growth in an in vivo xenograft model.
Furthermore, clinical trials of this combination for malignant neoplasms have already been performed: A phase I/II study for Waldenstrom macroglobulinemia (39, 40), and a phase I study for non-Hodgkin’s lymphoma (41). In the macroglobulinemia study, everolimus was given at 5 or 10 mg daily, bortezomib at 1.3 or 1.6 mg/m2, and rituximab at 375 mg/m2; the results were two complete responses, 21 partial responses, 17 minimal responses, five with stable disease and one with progressive disease. In the non-Hodgkin’s lymphoma study, everolimus was used at three doses (5 mg every other day, 5 mg every day, or 10 mg every day), and the dose of bortezomib was 0.7, 1.0, or 1.3 mg/m2; the results were one complete response, three partial responses, nine patients with stable disease, and 12 with progressive disease. The most common adverse events were fatigue (63%), anemia and leukopenia (52%), neutropenia (48%), and diarrhea (43%). Significant thrombocytopenia was observed, and the incidence of grade 3 thrombocytopenia was 57%. One case of cardiac dysfunction in the absence of acute coronary syndrome was encountered, and the patient recovered after cessation of the drugs. The clinical side-effects of this combination were not unacceptable, and this combination therapy could not be used clinically for patients with OS.
In this study, we successfully demonstrated the synergistic effect of the combination of everolimus and bortezomib against OS both in vitro and in vivo. These results indicate that this combination of targeted therapeutic agents may be useful as a novel therapeutic strategy for OS.
Acknowledgements
The Authors would like to express their thanks for the SCADS Inhibitor Kit of the Screening Committee of Anticancer Drugs that was obtained through a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Mr. Takahiro Iino and Ms. Kei Chiba for their excellent technical assistance.
Footnotes
Authors’ Contributions
All Authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization: K.A. Methodology: T.O. Investigation: K.N., K.K., Y.M. and K.Y. Formal analysis: K.A. Writing – original draft: K.A. Writing – review and editing: T.N. and Y.A. Visualization: K.A. Supervision: S.A.
Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received May 23, 2022.
- Revision received July 13, 2022.
- Accepted July 21, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
