To elucidate the potential mechanism underlying osteosarcoma

To elucidate the potential mechanism underlying osteosarcoma cell growth decrease by CDK4 inhibition, flow cytometry analysis was used to determine SCH772984 TFA and apoptosis in human osteosarcoma cells after palbociclib treatment. The results showed that osteosarcoma cells were arrested in G1 phase of the cell cycle after CDK4 inhibition by palbociclib. Cell apoptosis determination simultaneously revealed that CDK4 inhibition dramatically induced cell apoptosis in osteosarcoma cells. Hence, we propose that CDK4 inhibition by palbociclib decreases osteosarcoma cell proliferation and growth through induction of cell apoptosis via arresting the cell cycle in G1 phase. TMA analysis revealed that CDK4 expression positively correlated with metastasis in osteosarcoma patients. We further assessed the influence of CDK4 inhibition on osteosarcoma cell migration in vitro. Treatment of osteosarcoma cells with lower doses of palbociclib illustrated that CDK4 inhibition reduced osteosarcoma cell migration, suggesting that CDK4 may be a contributor to osteosarcoma metastasis, which is the main obstacle in the treatment of osteosarcoma. Taken together, our current study demonstrates that CDK4 is highly expressed in osteosarcoma, and elevated CDK4 expression correlates with metastasis and poor outcome in osteosarcoma patients. CDK4 inhibition decreases osteosarcoma cell proliferation and growth through apoptosis induction via cell cycle arrest. These findings, together with the potency of palbociclib, highlight CDK4 as a potential therapeutic target and palbociclib as a promising candidate agent for osteosarcoma treatment. The following are the supplementary data related to this article.
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CDKs and their Regulation CDKs are multifunctional enzymes that can modify various protein substrates involved in the cell cycle. Human cells have 13 CDKs that interact with at least 29 cyclins or cyclin-related proteins, which allows the association of individual CDKs with a circumscribed subset of cyclins to create a large combinatorial repertoire of active CDK complexes [1]. As schematically shown in Figure 1, CDKs interacting with multiple cyclins (CDK1, 2, 4, 6) primarily regulate the cell cycle, whereas the CDKs activated by a single cyclin (CDK7, 9) are typically involved in the regulation of transcription. In the cell cycle, the activities of CDKs are positively regulated at several levels including the association with a specific cyclin and phosphorylation by a CDK-activating protein kinase (CAK) complex composed of CDK7, cyclin H, and MAT1 (menage à trois 1) 2, 3 (Figure 1A). CDK activity is negatively regulated by two families of inhibitory proteins (Figure 1A). Members of the CIP (CDK interacting protein) and KIP (kinase inhibitory protein) family (composed of p21CIP1, p27KIP1, and p57KIP2) bind to CDK/cyclin complexes and are able to inhibit CDK–cyclin once these complexes have already formed [4]. By contrast, INK4 (inhibitor of CDK4) family members (including p16INK4A, p15INK4B, p18INK4C, and p19INK4D) interact with monomeric CDK4 or CDK6, and thus prevent the activation by D-type cyclins or by CAK [5] (Figure 1A). Once assembled with the correct cyclin and phosphorylated at a threonine in the activation loop (also called the T-loop), the activated CDK4/6 phosphorylates its target proteins at serines or threonines such a retinoblastoma (RB) protein in the G1 phase of the cell cycle 6, 7 (Figure 1B, upper left panel). Genetic and biochemical experiments identified CDK7 as the main CAK with the ability to phosphorylate the T-loops of all CDKs [8]. In addition, CDK7 is a subunit of the general transcription factor complex TFIIH (transcription factor II human). Within this complex CDK7 phosphorylates Ser5 and Ser7 contained in the C-terminal domain (CTD) of RNA polymerase (Pol) II and thus contributes to co-transcriptional capping, promoter-proximal pausing, and productive elongation 9, 10. CDK7 also phosphorylates and activates CDK9, which phosphorylates Ser2 in the RNA Pol II CTD, a modification that is essential for promoter-distal transcription elongation [11] (Figure 1B, upper right panel).