SP-8356 (A Verbenone Derivative) Inhibits Proliferation, Suppresses Cell Migration and Invasion and Decreases Tumor Growth of Osteosarcoma: Role of PGC-1α/TFAM and AMPK-Activation

Document Type : Original Article


1 Department of Orthopedics and Traumatology, Hanzhong Hospital of TCM, Shaanxi, China

2 Department of Orthopedics, Hanzhong Central Hospital, Hantai District, Hanzhong, Shaanxi, Chinaal

3 Department of Orthopedics, Hanzhong Central Hospital, Hantai District, Hanzhong, Shaanxi, China


Objective: Osteosarcoma (OS) is an uncommon sarcoma with osteoid formation in conjunction with malignant
mesenchymal cells on histological examination. SP-8356 has been reported to exhibit anti-cancer properties in human
cancers. However the impact of SP-8356 on OS is largely unknown. The metabolic pathways are coordinated by AMPactivated
protein kinase (AMPK), which maintains a balance between the supply and demand of nutrients and energy.
This study aimed to investigate effect of SP-8356 on proliferation and apoptosis of OS cells and tumor growth in mice.
Furthermore, involvement of PGC-1α/TFAM and AMPK-activation was studied.
Materials and Methods: In the experimental study, Saos-2 and MG63 cells were cultured with SP-8356 for 24
hours and analysed for cellular proliferation using MTT assay. DNA fragmentation was studied using ELISA based
kit. Furthermore, transwell chambers assay was used to determine cell migration and cell invasion. Targeted protein
expression levels were assessed using western blotting. For in vivo studies, mice (5-6 weeks old) were implanted with
either Saos-2 or MG63 cells on dorsal surface subcutaneously and they were administered with SP-8356 (10 mg/kg)
for two weeks prior to bone tumor induction.
Results: We found that SP-8356 exerted anti-proliferative effects on Saos-2 and MG63 cells. Furthermore, SP-8356
treatment significantly restricted migration and invasion of Saos-2 and MG63 cells. Compared to the control, SP-8356
significantly reduced apoptotic cell death, while it increased PGC-1α and TFAM expressions. Without affecting body
weight, SP-8356 significantly reduced tumor development in mice, as compared to the control group.
Conclusion: SP-8356 was found to inhibit proliferation, suppressed cells migration and invasion and decreased OS
tumor growth. Furthermore, SP-8356 was found to act through PGC-1α/TFAM and AMPK activations. SP-8356 can be
therefore used as therapeutic agent for OS treatment.


  1. Mirabello L, Troisi RJ, Savage SA. International osteosarcoma incidence patterns in children and adolescents, middle ages and elderly persons. Int J Cancer. 2009; 125(1): 229-234.
  2. Eilber F, Giuliano A, Eckardt J, Patterson K, Moseley S, Goodnight J. Adjuvant chemotherapy for osteosarcoma: a randomized prospective trial. J Clin Oncol. 1987; 5(1): 21-26.
  3. Kager L, Zoubek A, Dominkus M, Lang S, Bodmer N, Jundt G, et al. Osteosarcoma in very young children: experience of the cooperative osteosarcoma study group. Cancer. 2010; 116(22): 5316-5324.
  4. Morrow JJ, Khanna C. Osteosarcoma genetics and epigenetics: emerging biology and candidate therapies. Crit Rev Oncog. 2015; 20(3-4): 173-197.
  5. Misaghi A, Goldin A, Awad M, Kulidjian AA. Osteosarcoma: a comprehensive review. SICOT J. 2018; 4: 12.
  6. Chan DC. Mitochondria: dynamic organelles in disease, aging, and development. Cell. 2006; 125(7): 1241-1252.
  7. Neuzil J, Dong LF, Rohlena J, Truksa J, Ralph SJ. Classification of mitocans, anti-cancer drugs acting on mitochondria. Mitochondrion. 2013; 13(3): 199-208.
  8. Yamada S, Nomoto S, Fujii T, Kaneko T, Takeda S, Inoue S, et al. Correlation between copy number of mitochondrial DNA and clinico-pathologic parameters of hepatocellular carcinoma. Eur J Surg Oncol. 2006; 32(3): 303-307.
  9. Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, et al. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum Mol Genet. 2004; 13(9): 935-944.
  10. Scarpulla RC. Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network. Biochim Biophys Acta. 2011; 1813(7): 1269-1278.
  11. LeBleu VS, O’Connell JT, Gonzalez Herrera KN, Wikman H, Pantel K, Haigis MC, et al. PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis. Nat Cell Biol. 2014; 16(10): 992-1003, 1-15.
  12. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell. 1998; 92(6): 829-839.
  13. Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, et al. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum Mol Genet. 2004; 13(9): 935-944.
  14. Polychronopoulos P, Magiatis P, Skaltsounis AL, Myrianthopoulos V, Mikros E, Tarricone A, et al. Structural basis for the synthesis of indirubins as potent and selective inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinases. J Med Chem. 2004; 47(4): 935-946.
  15. Bajpai J, Susan D. Adjuvant chemotherapy in soft tissue sarcomas… Conflicts, consensus, and controversies. South Asian J Cancer. 2016; 5(1): 15-19.
  16. Masuike Y, Tanaka K, Makino T, Yamasaki M, Miyazaki Y, Takahashi T, et al. Esophageal squamous cell carcinoma with low mitochondrial copy number has mesenchymal and stem-like characteristics, and contributes to poor prognosis. PLoS One. 2018; 13(2): e0193159.
  17. Cantó C, Auwerx J. PGC-1alpha, SIRT1 and AMPK, an energy sensing network that controls energy expenditure. Curr Opin Lipidol. 2009; 20(2): 98-105.
  18. Xie H, Lev D, Gong Y, Wang S, Pollock RE, Wu X, et al. Reduced mitochondrial DNA copy number in peripheral blood leukocytes increases the risk of soft tissue sarcoma. Carcinogenesis. 2013; 34(5): 1039-1043.
  19. O’Hara R, Tedone E, Ludlow A, Huang E, Arosio B, Mari D, et al. Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution. Genome Res. 2019; 29(11): 1878-1888.
  20. Reznik E, Miller ML, Şenbabaoğlu Y, Riaz N, Sarungbam J, Tickoo SK, et al. Mitochondrial DNA copy number variation across human cancers. Elife. 2016; 5: e10769.
  21. Araujo LF, Siena ADD, Plaça JR, Brotto DB, Barros II, Muys BR, et al. Mitochondrial transcription factor A (TFAM) shapes metabolic and invasion gene signatures in melanoma. Sci Rep. 2018; 8(1): 14190.
  22. Mander S, Kim DH, Thi Nguyen H, Yong HJ, Pahk K, Kim EY, et al. SP-8356, a (1S)-(-)-verbenone derivative, exerts in vitro and in vivo anti-breast cancer effects by inhibiting NF-κB signaling. Sci Rep. 2019; 9(1): 6595.
  23. Ju C, Song S, Hwang S, Kim C, Kim M, Gu J, et al. Discovery of novel (1S)-(-)-verbenone derivatives with anti-oxidant and antiischemic effects. Bioorg Med Chem Lett. 2013; 23(19): 5421- 5425.
  24. Ahmad Waza A, Andrabi K, Ul Hussain M. Adenosine-triphosphate-sensitive K+ channel (Kir6.1): a novel phosphospecific interaction partner of connexin 43 (Cx43). Exp Cell Res. 2012; 318(20): 2559-2566.
  25. Fu B, Yin G, Song K, Mu X, Xu B, Zhang X. Indirubin-3’-Oxime (IDR3O) inhibits proliferation of osteosarcoma cells in vitro and tumor growth in vivo through AMPK-activation and PGC- 1α/TFAM up-regulation. Dokl Biochem Biophys. 2020; 495(1): 354-360.
  26. Wang L, Xue GB. Catalpol suppresses osteosarcoma cell proliferation through blocking epithelial-mesenchymal transition (EMT) and inducing apoptosis. Biochem Biophys Res Commun. 2018; 495(1): 27-34.
  27. Xie L, Ji T, Guo W. Anti-angiogenesis target therapy for advanced osteosarcoma (Review). Oncol Rep. 2017; 38(2): 625- 636.
  28. Valkenburg KC, de Groot AE, Pienta KJ. Targeting the tumour stroma to improve cancer therapy. Nat Rev Clin Oncol. 2018; 15(6): 366-381.
  29. Liu R, Fu C, Sun J, Wang X, Geng S, Wang X, et al. A new perspective for osteosarcoma therapy: proteasome inhibition by MLN9708/2238 successfully induces apoptosis and cell cycle arrest and attenuates the invasion ability of osteosarcoma cells in vitro. Cell Physiol Biochem. 2017; 41(2): 451-465.
  30. Kim DH, Yong HJ, Mander S, Nguyen HT, Nguyen LP, Park HK, et al. SP-8356, a (1S)-(-)-verbenone derivative, inhibits the growth and motility of liver cancer cells by regulating NF-κB and ERK signaling. Biomol Ther (Seoul). 2021; 29(3): 331-341.
  31. Bartke T, Siegmund D, Peters N, Reichwein M, Henkler F, Scheurich P, et al. p53 upregulates cFLIP, inhibits transcription of NF-kappaB-regulated genes and induces caspase-8-independent cell death in DLD-1 cells. Oncogene. 2001; 20(5): 571-580.
  32. Chaitanya GV, Steven AJ, Babu PP. PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal. 2010; 8: 31.
  33. Mander S, Kim DH, Thi Nguyen H, Yong HJ, Pahk K, Kim EY, et al. SP-8356, a (1S)-(-)-verbenone derivative, exerts in vitro and in vivo anti-breast cancer effects by inhibiting NF-κB signaling. Sci Rep. 2019; 9(1): 6595.
  34. Fares J, Fares MY, Khachfe HH, Salhab HA, Fares Y. Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther. 2020; 5(1): 28.
  35. Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol. 2011; 13(9): 1016-1023.
  36. He G, Zhang YW, Lee JH, Zeng SX, Wang YV, Luo Z, et al. AMP-activated protein kinase induces p53 by phosphorylating MDMX and inhibiting its activity. Mol Cell Biol. 2014; 34(2): 148- 157.
  37. Dowling RJ, Zakikhani M, Fantus IG, Pollak M, Sonenberg N. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res. 2007; 67(22): 10804-10812.
  38. Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K, Hara K, et al. A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells. 2003; 8(1): 65-79.