**Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma E**ff**ects through Inhibition of Cell Proliferation, Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism, and Suppression of TGF-**β **and VEGF Production and Metastatic Potential of Cells**

**Katarzyna Kaławaj <sup>1</sup> , Adrianna Sławi ´nska-Brych <sup>2</sup> , Magdalena Mizerska-Kowalska <sup>1</sup> , Aleksandra Zurek ˙ <sup>1</sup> , Agnieszka Bojarska-Junak <sup>3</sup> , Martyna Kandefer-Szersze ´n <sup>1</sup> and Barbara Zdzisi ´nska 1,\***


Received: 10 November 2020; Accepted: 8 December 2020; Published: 10 December 2020

**Abstract:** Osteosarcoma (OS) is the most common type of primary bone tumor. Currently, there are limited treatment options for metastatic OS. Alpha-ketoglutarate (AKG), i.e., a multifunctional intermediate of the Krebs cycle, is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor progression. There is growing evidence suggesting that AKG may represent a novel adjuvant therapeutic opportunity in anti-cancer therapy. The present study was intended to check whether supplementation of Saos-2 and HOS osteosarcoma cell lines (harboring a TP53 mutation) with exogenous AKG exerted an anti-cancer effect. The results revealed that AKG inhibited the proliferation of both OS cell lines in a concentration-dependent manner. As evidenced by flow cytometry, AKG blocked cell cycle progression at the G<sup>1</sup> stage in both cell lines, which was accompanied by a decreased level of cyclin D1 in HOS and increased expression of p21Waf1/Cip1 protein in Saos-2 cells (evaluated with the ELISA method). Moreover, AKG induced apoptotic cell death and caspase-3 activation in both OS cell lines (determined by cytometric analysis). Both the immunoblotting and cytometric analysis revealed that the AKG-induced apoptosis proceeded predominantly through activation of an intrinsic caspase 9-dependent apoptotic pathway and an increased Bax/Bcl-2 ratio. The apoptotic process in the AKG-treated cells was mediated via c-Jun N-terminal protein kinase (JNK) activation, as the specific inhibitor of this kinase partially rescued the cells from apoptotic death. In addition, the AKG treatment led to reduced activation of extracellular signal-regulated kinase (ERK1/2) and significant inhibition of cell migration and invasion in vitro concomitantly with decreased production of pro-metastatic transforming growth factor β (TGF-β) and pro-angiogenic vascular endothelial growth factor (VEGF) in both OS cell lines suggesting the anti-metastatic potential of this compound. In conclusion, we showed the anti-osteosarcoma potential of AKG and provided a rationale for a further study of the possible application of AKG in OS therapy.

**Keywords:** alpha-ketoglutarate; cell cycle; apoptosis; JNK; cell migration; cell invasion; TGF-β; VEGF

#### **1. Introduction**

Osteosarcoma (OS) is a malignant mesenchymal-origin primary tumor of bone. Its histological hallmark is the production of osteoid or immature bone by neoplastic cells [1]. Although bone tumors are relatively rare overall (less than 1% and 3–5% of all newly diagnosed malignant cancers in adults and children, respectively), OS is the most common bone cancer in children and adolescents [2]. OS has a bimodal age incidence distribution worldwide with the first peak in teenagers (at the age of 10–14 in females and 15–19 in males) and the second peak in the elderly [3]. The survival rates may vary depending on various factors (e.g., age, sex, disease stage, localization, country); in children and adolescents, they are similar in most countries, ranging from 55% to 75% [4]. However, the 5-year overall survival rates of OS patients with distant metastasis and/or relapsed OS is low, i.e., approximately 30% in individuals with lung metastasis [5]. The current OS treatment combines surgery with chemotherapy, which was introduced in the 1980s and resulted in significant improvement in OS patients' survival rates over the 1990s [4]. Most importantly, for the past 20 years, the survival rates in OS patients did not change essentially and no successful targeted therapies of this cancer have been developed so far [6]. Therefore, there is still a need for novel OS treatment strategies.

Alpha-ketoglutarate (AKG) or 2-oxoglutarate is known mainly as an intermediate of the tricarboxylic acid (TCA) cycle that serves the production of the energetic molecule ATP [7], although it can also be synthesized via other biosynthetic pathways in cells [8]. In the TCA cycle, AKG is formed as a product of isocitrate oxidative decarboxylation catalyzed by NADP-dependent isocitrate dehydrogenase isoforms (IDH1-3) [9]. AKG is characterized as a metabolite with pleiotropic activity due to its metabolic and non-metabolic functions associated with direct involvement in different cellular processes as a biosynthetic substrate, a co-substrate of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), or a signaling molecule [8]. Mounting evidence suggests that AKG is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor development [10]. Most importantly, it is involved in the regulation of hypoxic response and epigenetic modifications, i.e., two main phenomena that drive oncogenic transformation. AKG is a co-substrate for prolyl hydroxylases (PHDs) belonging to the 2-OGDD family, which regulate the stability of the hypoxia-inducible factor (HIF-1), i.e., an important transcription factor in cancer development and progression. Thus, the abundance of AKG may be a determinant of the HIF-1 stabilization/activity through the regulation of PHD activity [11,12]. Moreover, other AKG-dependent enzymes from the OGDD family, namely ten-eleven translocation hydroxylases (TET1-3) and Jumonji C domain-containing lysine demethylases (KDM2-7), are involved in DNA and histone demethylation, respectively, and take part in shaping the cellular epigenetic landscape that is important in cancerogenesis [13]. What is more, mutations in genes encoding TCA cycle enzymes, such as succinate dehydrogenase (SDH), fumarate hydratase (FH), and IDHs, may be present in several cancers, leading to the accumulation of appropriate metabolites, i.e., succinate, fumarate, and D-2-hydroxyglutarate, respectively, inside cells. All these metabolites may act as competitive inhibitors of 2-OGDD enzymes (including PHDs, TETs, and KDMs), and they have been termed "oncometabolites" due to their role in the metabolic reprogramming of cells and progression toward malignancy [14]. It is suggested that an increase in the level of AKG in cells may result in the reverse of 2-OGDD inhibition by oncometabolites and exert an anticancer effect [11,15–19].

Recent in vitro and in vivo experiments suggest that an increase in the intracellular level of AKG through different strategies, e.g., exogenous supplementation, alpha-ketoglutarate dehydrogenase (KGD; an enzyme that catalyses the oxidative decarboxylation of AKG to succinyl-Co-A in the TCA cycle) inactivation or even IDH overexpression, may lead to downregulation of HIF-1 or upregulation of

epigenetic enzymes and prevent/inhibit tumor progression or show direct anticancer effects [17,19–24]. A more recent study has also proposed therapeutic strategies to increase AKG intracellular levels as a mechanism of engagement of latent tumor-suppressive pathways in p53-deficient cancers. It has been shown that AKG is an effector molecule of p53-mediated tumor suppression and its accumulation in p53-deficient tumors can antagonize malignant progression [25].

OS is a cancer with numerous chromosomal abnormalities, gene mutations, and epigenetic defects, e.g., hypermethylation at promoter CpG islands of the key (Rb and p53) tumor suppressor pathways [26]. However, no mutations in the TCA enzymes have been identified so far [27,28] except for one study that has demonstrated IDH2 mutations in OS tissues [29]. Nevertheless, a few studies have shown that IDH1/IDH2 expression inversely correlated with the pathological grade and metastasis in OS [30,31], and the expression of IDH1 was lower in OS than normal bone tissue [24], suggesting that interference in the level of AKG abundance-regulating enzymes may represent a potential target in the OS therapy. Given these reports, our present study was intended to check whether supplementation of OS cells with exogenous AKG exerted an anti-osteosarcoma effect.

#### **2. Results**

#### *2.1. AKG Inhibits Proliferation of OS Cells*

Since AKG (supplemented as an alpha-ketoglutarate disodium salt dihydrate) was described to exhibit direct antiproliferative activity [22], we evaluated its influence on cell proliferation in two OS cell lines, i.e., Saos-2 (p53-null cell line) and HOS (p53 mutant). The OS cells were cultured in a complete growth medium with AKG, which was used at concentrations ranging from 2.5 to 200 mM established on the basis of research carried out by other authors [20–22]. The results showed a concentration-dependent ability of AKG to inhibit proliferation in both cell lines. Incubation of the Saos-2 and HOS cells in a growth medium with increasing concentrations of AKG for 96 h resulted in a similar degree of inhibition of proliferation of both types of OS cells evaluated by the MTT assay (Figure 1A,B). To verify the antiproliferative effect of AKG against OS cells, measurement of DNA synthesis (with the BrdU assay) was additionally performed after 48-h incubation with AKG. As shown in Figure 1C,D, AKG induced a concentration-dependent decrease in OS cell proliferation. The lowest concentrations inducing significant inhibition of the BrdU incorporation into the DNA of dividing cells were 5 mM and 10 mM of AKG for Saos-2 and HOS, respectively. However, the IC<sup>50</sup> values for both cell lines were very similar and amounted to 35.41 ± 0.17 mM and 35.37 ± 0.19 mM for the Saos-2 and HOS cells, respectively.
