Anticancer peptides from induced tumor-suppressing cells for inhibiting osteosarcoma cells

Abstract

Osteosarcoma (OS) is the most frequent primary bone cancer, which is mainly suffered by children and young adults. While the current surgical treatment combined with chemotherapy is effective for the early stage of OS, advanced OS preferentially metastasizes to the lung and is difficult to treat. Here, we examined the efficacy of ten anti-OS peptide candidates from a trypsin-digested conditioned medium that was derived from the secretome of induced tumor-suppressing cells (iTSCs). Using OS cell lines, the antitumor capabilities of the peptide candidates were evaluated by assaying the alterations in metabolic activities, proliferation, motility, and invasion of OS cells. Among ten candidates, peptide P05 (ADDGRPFPQVIK), a fragment of aldolase A (ALDOA), presented the most potent OS-suppressing capabilities. Its efficacy was additive with standard-of-care chemotherapeutic agents such as cisplatin and doxorubicin, and it downregulated oncoproteins such as epidermal growth factor receptor (EGFR), Snail, and Src in OS cells. Interestingly, P05 did not present inhibitory effects on non-OS skeletal cells such as mesenchymal stem cells and osteoblast cells. Collectively, this study demonstrated that iTSC-derived secretomes may provide a source for identifying anticancer peptides, and P05 may warrant further evaluations for the treatment of OS.

Keywords: ALDOA; EGFR; Osteosarcoma; induced tumor-suppressing cells; peptide.

Figure 1

Anti-tumor action of P02, P04, and P05. MSC = mesenchymal stem cell, and CN = control. The single and double asterisks indicate P < 0.05 and 0.01, respectively. A, B. Reduction of MTT-based viability and scratch-based motility of U2OS, TT2, and MG63 osteosarcoma cells in response to 10 and 50 µg/ml P02, P04, and P05. C, D. No significant effects on MTT-based viability and scratch-based motility of MSCs in response to 10 and 50 µg/ml P02, P04, and P05.

Figure 2

A Dose-dependent effects of P02, P04, and P05 on cell viability and motility. CN = control. The single and double asterisks indicate P < 0.05 and 0.01, respectively. A. Effects of P02, P04, and P05 on MTT-based viability of TT2 osteosarcoma cells. B, C. Combined effects of P02, P04, and P05 on MTT-based viability and scratch-based motility of TT2 osteosarcoma cells.

Figure 3

Effects of P05 on proliferation, invasion, and gene expression, and its combined effects with the selected chemotherapeutic agents. CN = control, Cis = Cisplatin, and Dox = Doxorubicin. The double asterisk indicates P < 0.01. A, B. Effects of 25 µg/ml P05 on proliferation and transwell invasion of TT2 osteosarcoma cells. C. Decrease in the levels of p-Src, EGFR, and Snail, and an increase in cleaved caspase 3 (c-Cas-3) in TT2 osteosarcoma cells by the application of 25 µg/ml P05. D. Additive anti-tumor effects of P05 with Cisplatin, Doxorubicin, or Taxol.

Figure 4

Anti-tumor effects of P02, P04, and P05 on MDA-MB-231 breast cancer cells. CN = control. The single and double asterisks indicate P < 0.05 and 0.01, respectively. A-D. Reduction in MTT-based viability, scratch-based motility, Transwell invasion, and EdU-based proliferation, respectively. E. Downregulation of oncoproteins such as EGFR, p-Src, and Snail by P05.

Figure 5

Double-edged role of ALDOA to osteosarcoma and MSCs. CN = control, pl = plasmid transfection, MSC = mesenchymal stem cell, and CM = conditioned medium. The double asterisk indicates P < 0.01. A. Increase in MTT-based viability and the level of EGFR by overexpressing ALDOA in TT2 osteosarcoma cells. B. Overexpression of ALDOA in MSCs and the elevation of EGFR. C-F. Reduction in MTT-based viability, EdU-based proliferation, scratch-based motility, and transwell invasion, respectively, by ALDOA-overexpressing MSC-derived conditioned medium.

Figure 6

Possible involvement of EGFR in the action of ALDOA and P05. CN = control. The double asterisk indicates P < 0.01. A, B. Predicted interactions of ALDOA-EGFR and P05-EGFR. C, D. Reciprocal immunoprecipitation of EGFR and ALDOA using TT2 derived cell lysates. E. Suppression of ALDOA-driven reduction in MTT-based viability of TT2 OS cells by silencing EGFR.

Figure 7

P05’s protective effects on bone and the proposed mechanism of P05’s action. CN = control. The double asterisk indicates P < 0.01. A. Reduction in multi-nucleated RANKL-stimulated osteoclasts in response to P05. B. Reduction in EGFR, M-CSF, OPN, IL-1β, IL6, and p-Src by the administration of 25 µg/mL of P05 in the ex vivo bone culture. C. Proposed mechanism of the anti-tumor action of P05.