*2.2. AKG Induces Cell Cycle Arrest in the G<sup>1</sup> Phase in OS Cells through Modulation of the Expression of Cell Cycle-Associated Proteins*

To further explore the antiproliferative activity of AKG, the influence of the selected concentrations (10, 25, and 50 mM) of this compound on the distribution of cell cycle phases in both OS cell lines after 48-h incubation was analyzed by flow cytometry. As shown in Figure 2A–D, the AKG treatment at all the concentrations used resulted in accumulation of Saos-2 (Figure 2A,B) and HOS (Figure 2C,D) cells in the G<sup>1</sup> phase with a concomitant reduction of the cell number in the S and G<sup>2</sup> phases. Compared to the control, the highest AKG concentration of 50 mM significantly elevated the G1-fraction from 60.02 ± 0.92% to 72.81 ± 1.58% in the Saos-2 cell culture and from 67.51 ± 0.29 to 74.37 ± 0.61% in the HOS cell culture.

Since AKG caused the cell cycle arrest in the G<sup>1</sup> phase, further studies were conducted to investigate the effect of AKG on the expression of proteins responsible for the transition from the G<sup>1</sup> phase to the S phase of the cell cycle, i.e., cyclin D1 and the cyclin-dependent p21Waf1/Cip1 inhibitor. Changes in the expression of these proteins were evaluated by means of immunoassay methods. Only trace amounts of cyclin D1 were detected in the Saos-2 cells (which is in agreement with a previous study [32]), and its expression did not change significantly after the AKG treatment [data not shown]. In contrast, the HOS cells expressed cyclin D1 and its level was downregulated in the AKG-treated cells in a concentration-dependent manner. The 24-h AKG treatment at the concentrations of 25 and 50 mM decreased its expression by approx. 9% and 33%, respectively (Figure 2E). In turn, the expression of the cyclin-dependent p21Waf1/Cip1 inhibitor in the AKG-treated Saos-2 cells was significantly upregulated in a concentration- and time-dependent manner. After the 6-h AKG treatment at the concentrations of 10, 25, and 50 mM, the expression of p21 Waf1/Cip1 increased by 7%, 14%, and 42%, respectively. The 24-h treatment with the same concentrations of AKG resulted in a greater increase in the expression of this protein by 19%, 39%, and 57% respectively, in comparison with the control levels (Figure 2F). In the case of the HOS cells, only the 6-h incubation with AKG at the concentrations of 25 and 50 mM induced a statistically significant increase in p21Waf1/Cip1 protein expression by 17% and 57%, respectively, compared to the control. In turn, the longer AKG treatment (24 h) resulted in a decrease in the expression of this protein (Figure 2G). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 4 of 21

**Figure 1.** Effect of alpha-ketoglutarate (AKG) on Saos-2 and HOS cell proliferation. The osteosarcoma (OS) cells were treated with increasing concentrations of the compound. Cell proliferation was assessed with the MTT assay after 96 h (**A**,**B**) and the levels of BrdU incorporated into the cells after the 48-h AKG treatment were determined (**C**,**D**). All experiments were repeated independently at least three times, and data (*n* = 24 for each concentration) are expressed as the mean ± SD; \* *p* < 0.05 and \*\*\* *p* < 0.001 in comparison to the control; one-way ANOVA test. **Figure 1.** Effect of alpha-ketoglutarate (AKG) on Saos-2 and HOS cell proliferation. The osteosarcoma (OS) cells were treated with increasing concentrations of the compound. Cell proliferation was assessed with the MTT assay after 96 h (**A**,**B**) and the levels of BrdU incorporated into the cells after the 48-h AKG treatment were determined (**C**,**D**). All experiments were repeated independently at least three times, and data (*n* = 24 for each concentration) are expressed as the mean ± SD; \* *p* < 0.05 and \*\*\* *p* < 0.001 in comparison to the control; one-way ANOVA test.

#### *2.3. AKG Induces Cell Death in OS Cells through Apoptosis via an Intrinsic Caspase-Dependent Pathway*

*2.2. AKG Induces Cell Cycle Arrest in the G1 phase in OS Cells through Modulation of the Expression of Cell Cycle-Associated Proteins*  To further explore the antiproliferative activity of AKG, the influence of the selected concentrations (10, 25, and 50 mM) of this compound on the distribution of cell cycle phases in both OS cell lines after 48-h incubation was analyzed by flow cytometry. As shown in Figure 2A–D, the AKG treatment at all the concentrations used resulted in accumulation of Saos-2 (Figure 2A,B) and HOS (Figure 2C,D) cells in the G1 phase with a concomitant reduction of the cell number in the S and G2 phases. Compared to the control, the highest AKG concentration of 50 mM significantly elevated the G1-fraction from 60.02 ± 0.92% to 72.81 ± 1.58% in the Saos-2 cell culture and from 67.51 ± 0.29 to Since the cell growth inhibition by AKG may have been also a result of the induction of cell death via apoptosis and/or necrosis, the cells were analyzed using Annexin V-FITC/PI double staining and flow cytometry. As shown in Figure 3A–D, after 72-h treatment, AKG was found to induce apoptosis in both Saos-2 and HOS cells, whereas necrosis was only slightly increased when the Saos-2 cells were incubated with 50 mM of AKG (Figure 3B). Significant induction of apoptosis was observed even at 5 mM of AKG. The percentage of Saos-2 cells undergoing apoptosis increased significantly from 0.5 ± 0.01% in the control to 7.5 ± 0.29%, 8.2 ± 0.28, 9.6 ± 0.22%, and 12.1 ± 0.22% after the incubation with 5, 10, 25, or 50 mM of the AKG, respectively (Figure 3B). Similarly, the percentage of HOS cells

Changes in the expression of these proteins were evaluated by means of immunoassay methods. Only trace amounts of cyclin D1 were detected in the Saos-2 cells (which is in agreement with a previous study [32]), and its expression did not change significantly after the AKG treatment [data not shown]. In contrast, the HOS cells expressed cyclin D1 and its level was downregulated in the AKG-treated cells in a concentration-dependent manner. The 24-h AKG treatment at the concentrations of 25 and 50 mM decreased its expression by approx. 9% and 33%, respectively (Figure 2E). In turn, the expression of the cyclin-dependent p21Waf1/Cip1 inhibitor in the AKG-treated Saos-2 cells was significantly upregulated in a concentration- and time-dependent manner. After the 6-h AKG

Since AKG caused the cell cycle arrest in the G1 phase, further studies were conducted to

74.37 ± 0.61% in the HOS cell culture.

3D).

undergoing apoptosis increased from 1.0 ± 0.16% in the control to 5.0 ± 0,58%, 5.8 ± 0.16%, 8.3 ± 0.30%, and 12.0 ± 0.28% after the incubation with 5, 10, 25, or 50 mM of the compound, respectively (Figure 3D).

Moreover, to identify the mechanism of AKG-induced apoptosis in OS cells, the activation of effector caspase-3 was evaluated by flow cytometry. As shown in Figure 4A–D, in both OS cell lines, the 72-h AKG treatment resulted in a concentration-dependent increase in the number of cells with active caspase 3.

A further study was undertaken to resolve which pathway, receptor- or mitochondria-dependent, was involved in the AKG-induced caspase 3 activation in the OS cells. Since a higher number of cells with activated caspase 3 was observed in the Saos-2 culture, this cell line was chosen to examine the activation of initiator caspase-8 (extrinsic pathway) and caspase-9 (intrinsic pathway) with the use of immunoblotting and flow cytometry methods. As shown in Figure 5A–C, AKG caused a slight increase in the active forms of caspase-8, in comparison with the control, after the 72-h treatment, although only in a small percentage of cells. In contrast, an AKG concentration-dependent decrease in the procaspase-9 levels and an increase in its active form were observed in the Saos-2 cells (Figure 5D), and a large number of cells exhibited the presence of the active form of this caspase (Figure 5E,F). Since these results indicated that the AKG-treatment activated predominantly the intrinsic apoptotic pathway, the expression of pro-apoptotic and anti-apoptotic proteins associated with mitochondrial membrane integrity were further assessed by Western blot analysis at 72 h. As shown in Figure 5G,H, the exposure to AKG triggered a significant increase in the amount of the pro-apoptotic Bax protein and a decrease (although to a lesser extent) in the expression of Bcl-2 (apoptosis inhibitor) in the Saos-2 cells. This suggests that the AKG treatment may result in the predominance of pro-apoptotic signals through an increase in the Bax/Bcl-2 ratio. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 5 of 21 treatment at the concentrations of 10, 25, and 50 mM, the expression of p21 Waf1/Cip1 increased by 7%, 14%, and 42%, respectively. The 24-h treatment with the same concentrations of AKG resulted in a greater increase in the expression of this protein by 19%, 39%, and 57% respectively, in comparison with the control levels (Figure 2F). In the case of the HOS cells, only the 6-h incubation with AKG at the concentrations of 25 and 50 mM induced a statistically significant increase in p21Waf1/Cip1 protein expression by 17% and 57%, respectively, compared to the control. In turn, the longer AKG treatment (24 h) resulted in a decrease in the expression of this protein (Figure 2G).

**Figure** *2.* Effect of AKG on cell cycle distribution and expression of cell cycle-associated proteins in Saos-2 and HOS cultures. After the treatment with various concentrations of AKG for 48 h, the cells were stained with propidium iodide and analyzed by flow cytometry. Representative DNA histograms for Saos-2 (**A**) and HOS (**C**) cell lines with statistical analysis of the percentages of cells in the G1, S, and G2 phases in Saos-2 (**B**) and HOS (**D**) cultures. The levels of cyclin D1 in HOS cells (**E**) were measured after 24-h, while p21Waf1/Cip1 in the Saos-2 (**F**) and HOS (**G**) cells after 6-h and 24-h incubation without or with AKG (10, 25, and 50 mM) (with the ELISA assay). Data are expressed as means ± SD for at least three independent experiments. (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 in comparison to the control; one-way ANOVA test. **Figure 2.** Effect of AKG on cell cycle distribution and expression of cell cycle-associated proteins in Saos-2 and HOS cultures. After the treatment with various concentrations of AKG for 48 h, the cells were stained with propidium iodide and analyzed by flow cytometry. Representative DNA histograms for Saos-2 (**A**) and HOS (**C**) cell lines with statistical analysis of the percentages of cells in the G1, S, and G2 phases in Saos-2 (**B**) and HOS (**D**) cultures. The levels of cyclin D1 in HOS cells (**E**) were measured after 24-h, while p21Waf1/Cip1 in the Saos-2 (**F**) and HOS (**G**) cells after 6-h and 24-h incubation without or with AKG (10, 25, and 50 mM) (with the ELISA assay). Data are expressed as means ± SD for at least three independent experiments. (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 in comparison to the control; one-way ANOVA test.

in both Saos-2 and HOS cells, whereas necrosis was only slightly increased when the Saos-2 cells were incubated with 50 mM of AKG (Figure 3B). Significant induction of apoptosis was observed even at 5 mM of AKG. The percentage of Saos-2 cells undergoing apoptosis increased significantly from 0.5 ± 0.01% in the control to 7.5 ± 0.29%, 8.2 ± 0.28, 9.6 ± 0.22%, and 12.1 ± 0.22% after the incubation with 5, 10, 25, or 50 mM of the AKG, respectively (Figure 3B). Similarly, the percentage of HOS cells undergoing apoptosis increased from 1.0 ± 0.16% in the control to 5.0 ± 0,58%, 5.8 ± 0.16%, 8.3 ± 0.30%, and 12.0 ± 0.28% after the incubation with 5, 10, 25, or 50 mM of the compound, respectively (Figure

Since the cell growth inhibition by AKG may have been also a result of the induction of cell death

*2.3. AKG Induces Cell Death in OS Cells through Apoptosis Via an Intrinsic Caspase-Dependent Pathway* 

**Figure 3.** Effect of AKG on apoptosis induction in Saos-2 and HOS cell lines. After the 72-h exposure to the different concentrations of AKG, the cells were stained with annexin (An) V-FITC)/propidium iodide (PI) and examined with flow cytometry. The representative dot plots indicate the percentage of An- /PI+ necrotic cells (Q1), An+/PI+ late apoptotic cells (Q2), An<sup>−</sup>/PI<sup>−</sup> viable cells (Q3), and An+/PI<sup>−</sup> early apoptotic cells (Q4) in AKG-treated Saos-2 (**A**) and HOS (**C**) cell cultures. Histogram representation of the quantitative percentage of total apoptotic cells (early + late apoptosis) and necrotic cells in the control and AKG-treated Saos-2 (**B**) and HOS (**D**) cell cultures. All experiments presented in this figure were repeated independently at least three times, and data (*n* = 12 for each concentration) are expressed as mean ± SD; \*\*\* *p* < 0.001 in comparison to the control; one-way **Figure 3.** Effect of AKG on apoptosis induction in Saos-2 and HOS cell lines. After the 72-h exposure to the different concentrations of AKG, the cells were stained with annexin (An) V-FITC)/propidium iodide (PI) and examined with flow cytometry. The representative dot plots indicate the percentage of An−/PI<sup>+</sup> necrotic cells (Q1), An+/PI<sup>+</sup> late apoptotic cells (Q2), An−/PI<sup>−</sup> viable cells (Q3), and An+/PI<sup>−</sup> early apoptotic cells (Q4) in AKG-treated Saos-2 (**A**) and HOS (**C**) cell cultures. Histogram representation of the quantitative percentage of total apoptotic cells (early + late apoptosis) and necrotic cells in the control and AKG-treated Saos-2 (**B**) and HOS (**D**) cell cultures. All experiments presented in this figure were repeated independently at least three times, and data (*n* = 12 for each concentration) are expressed as mean ± SD; \*\*\* *p* < 0.001 in comparison to the control; one-way ANOVA test.

#### ANOVA test. *2.4. AKG Modulates the Phosphorylation of Mitogen-Activated Protein Kinases and Induces Apoptosis in OS Cells through a c-Jun N-Terminal Protein Kinase (JNK)-Dependent Mechanism*

Moreover, to identify the mechanism of AKG-induced apoptosis in OS cells, the activation of effector caspase-3 was evaluated by flow cytometry. As shown in Figure 4A–D, in both OS cell lines, the 72-h AKG treatment resulted in a concentration-dependent increase in the number of cells with active caspase 3. To explore the involvement of mitogen-activated protein kinases (MAPKs) in AKG-induced OS cell apoptosis, phosphorylation of JNK, extracellular signal-regulated kinase (ERK1/2), and p38 was examined with the quantitative ELISA method. As shown in Figure 6A, the AKG treatment reduced ERK1/2 phosphorylation in a concentration-dependent manner within 6 and 24 h in the Saos-2 cells. In contrast, AKG remarkably augmented the level of phospho-JNK in a concentration-dependent manner, but not phospho-p38 (Figure 6B,C).

JNK is a stress-activated kinase, and a signaling pathway with the participation of this kinase regulates e.g., apoptosis [33]. To clarify whether the AKG-activated JNK signaling pathway was engaged in the apoptotic process, the Saos-2 cells were cultured with AKG in the presence of a specific inhibitor of JNK (SP600125). After 72 h, the percentage of apoptotic cells was measured with FACS, whereas the JNK phosphorylation status was evaluated after 24 h with ELISA. As shown in Figure 6D, the percentage of apoptotic cells declined from 22.52 ± 1.8% after the treatment with 50 mM AKG alone to 13.1 ± 1.2% when the cells were co-treated with AKG and 5 µM of SP600125. It was found that the inhibition of JNK phosphorylation by SP600125 completely inhibited the activation of JNK induced by this compound (Figure 6E) and partially reduced the level of AKG-induced apoptosis in the Saos-2 cells (Figure 6F). These data may therefore support the observation that the AKG-induced apoptosis in the Saos-2 cells was mediated partially through the activation of the JNK signaling pathway.
