Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growth
Graphical abstract
Introduction
Osteosarcoma (OS) is the most common form of primary malignant bone tumor, which is locally destructive by producing malignant osteoid or immature bone. Accounting for approximately 60% of malignant bone tumors, OS is the most prevalent among children and young adults [1], [2], [3]. Although some options including complete resection of the tumor tissues and chemotherapy with high-dose methotrexate, doxorubicin, and cisplatin (sometimes with ifosfamide) are available for the treatment of OS, about 30% of OS patients would not able to survive over five years. Because OS has a high potential for pulmonary metastasis [4], [5], [6], most OS patients may eventually die of pulmonary metastases. Therefore, there is a clear need for the development of new and more effective therapeutics to combat malignant OS [7], [8].
MicroRNAs (miRNAs or miRs) are a large family of small noncoding RNAs (ncRNAs) that are derived from human genome and responsible for posttranscriptional regulation of numerous target genes underlying various cellular processes including cancer cell proliferation and tumor progression [9], [10], [11]. With an improved understanding of miRNA cancer biology, there has been a growing interest in developing miRNA-based therapies [12], [13], [14]. Of particular note, some tumor suppressing miRNAs, such as miR-34a, depleted in cancerous tissues may be reintroduced into cancer cells to control cancer cell growth and tumor progression [15], [16], [17]. Indeed miR-34a is a direct target of p53 and it exhibits strong antiproliferative activity against a variety of cancer cells through targeting of many (proto-) oncogenes, such as NAD-dependent deacetylase sirtuin-1 (SIRT1), cyclin-dependent kinase 6 (CDK6), hepatocyte growth factor receptor c-MET, and cell surface glycoprotein CD44, which regulate apoptosis, cell cycle, invasion, and other cellular processes [18], [19], [20], [21]. Co-administration of miR-34a also increases the sensitivity of human carcinoma cells to chemotherapeutics, e.g., bladder cancer cells to cisplatin [22], and triple negative breast cancer cells to doxorubicin [23]. Additionally, a liposome-formulated miR-34a, namely “MRX34”, has entered Phase I clinical trials for the treatment of unresectable primary liver cancer [24].
Recent studies have also revealed that miR-34a is significantly downregulated in human OS tissues, and a lower miR-34a level may forecast a notably poor disease-free survival rate in OS patients [25], [26], [27]. Furthermore, restoration of miR-34a function in OS cells using synthetic miR-34a mimics reduces cell proliferation in intro, and forced expression of miR-34a in OS cells not only inhibits cell proliferation in vitro but also represses tumorigenesis in vivo [26], [27], [28], [29]. Nevertheless, there is no report thus far on the utility of systemic administration of a miR-34a agent or combination of miR-34a and chemotherapeutic drug for the treatment of OS in a whole body system.
Aiming to develop miRNA-based therapy, we have developed a novel approach to bioengineering large quantities of miR-34a prodrug [30], [31]. These genetically engineered miRNA agents are distinguished from synthetic miRNA agents (e.g., miRNA mimics or pre-miRNAs) for being produced and folded within live cells, which should better capture the function and safety properties of natural RNAs [30], [32], [33]. Indeed biological miR-34a prodrug is selectively processed to mature miR-34a in human lung carcinoma cells, and consequently reduces cancer cell proliferation in vitro and inhibits xenograft tumor growth in vivo [30]. However, bioengineered miR-34a prodrug often exerts a partial inhibition against human carcinoma cells, similar as synthetic miR-34a agents. On the other hand, high-dose doxorubicin chemotherapy may completely inhibit cancer cell growth but also produce toxic effects [34]. Therefore, in the present study, we aimed to evaluate the utility of combination therapy with bioengineered miR-34a prodrug and doxorubicin for the treatment of OS. Acting through RNA interference and DNA intercalation, miR-34a and doxorubicin combination treatment could produce synergistic effects in the control of cancer cell growth (Fig. 1), and thus much lower and safe doses may be used to achieve the same efficacy and minimize or avoid toxicity [35], [36], [37]. We then delineated the synergism and mechanistic actions of combination therapy in the inhibition of OS cell proliferation in human OS cells in vitro, and defined the effectiveness and safety profiles of miR-34a prodrug and doxorubicin co-administered intravenously in an orthotopic OS xenograft tumor mouse models in vivo.
Section snippets
Materials
Doxorubicin hydrochloride salt (>98%) was purchased from Sigma–Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Trizol reagent, BCA Protein Assay Kit, and Lipofectamine 2000 were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). RPMI 1640 medium, trypsin and phosphate-buffered saline were bought from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA). Fetal bovine serum was purchased from GBICO BRL (Rockville, MD, USA). RIPA lysis
Co-administration of bioengineered miR-34a prodrug and doxorubicin synergistically inhibits osteosarcoma cell proliferation, and the best outcomes can be achieved when cells are treated at earlier stage
To critically assess whether miR-34a prodrug and doxorubicin produce synergistic effects in the suppression of OS cancer cell proliferation, we conducted combination analysis using Chou–Talalay method [38]. Our data showed that single drug treatment and all the four different Schedules of combination treatments exhibited dose-dependent antiproliferative effects against human OS 143B (Fig. 2A–D) and MG63 (Fig. 3A–D) cells. The synergism between doxorubicin and miR-34a prodrug was indicated by
Discussion
Given the fact that miR-34a exhibits antiproliferative activity via RNA interference and doxorubicin exerts anticancer activity through DNA intercalation, it was reasoned that combination therapy with miR-34a and doxorubicin could produce synergistic effects. Using Chou–Talalay approach [38], our comprehensive studies clearly demonstrated a synergism between doxorubicin and bioengineered miR-34a prodrug for the inhibition of human OS cell proliferation, which were attributable to much greater
Conflict of interests
The authors declare that they have no conflict of interests.
Authors’ contributions
All authors participated in research design, performed data analyses, and contributed to writing, revising, and final approval of the manuscript. Y.Z., M.-J.T., W.-P.W., Q.-X.C., and J.-X.Q. conducted the experiments. A.-M.Y. and A.-X.Y. contributed to new reagents and analytical tools.
Acknowledgements
A.-X. Yu is supported by the Outstanding Medical Academic leader Program of Hubei Province. A.-M. Yu is supported by NIH grants numbers U01CA175315 and R41AA024029. Y Zhao is funded by the Chinese Scholarship Council (No. 201406270084). The authors appreciate the access to shared resources funded by the UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (NCI P30CA093373) and thank Drs. Hongyong Zhang, Wenwu Xiao, and Tsung-Chieh Shih for their
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