**5. Conclusions**

In conclusion, our data demonstrated the anti-osteosarcoma effects of AKG supplementation in an in vitro study. AKG was able to modulate the expression of cell cycle-associated proteins (cyclin D1, p21Waf1/Cip1) and arrest cell cycle progression at the G<sup>1</sup> phase, which resulted in inhibition of OS cell proliferation. Moreover, the AKG-induced activation of the JNK pathway, augmentation of the Bax/Bcl-2 ratio, and activation of caspase-9 and -3 led to the induction of apoptotic cell death in the OS cells. The inhibition of the ERK pathway by AKG may also be involved both in the pro-apoptotic effect of AKG and in the anti-metastatic potential of AKG linked with inhibition of OS cell motility and invasion by this compound. The anti-osteosarcoma potential of AKG was also attributed to its inhibitory influence on the production and release of cytokines such as pro-metastatic TGF-β and

pro-angiogenic VEGF. These results may thus provide a rationale for further in vivo study of the possible application of AKG in osteosarcoma therapy.

**Author Contributions:** Conceptualization, B.Z.; methodology and investigations, K.K., A.S.-B., M.M.-K., A.Z., ˙ A.B.-J. and B.Z.; data analysis and interpretation, K.K., A.S.-B. and B.Z.; writing—original draft preparation, B.Z.; writing—review & editing, B.Z. and M.K.-S.; founding acquisition, M.K.-S.; project administration, B.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by research Grant No. 2013/11/B/NZ4/04557 from the State Funds for Scientific Research National Science Centre, Poland.

**Conflicts of Interest:** The authors declare no conflict of interest.
