SKM-1

Metformin Inhibits Cell Proliferation in SKM-1 Cells

Metformin, a widely used antidiabetic drug, has previously been demonstrated to exert anti-cancer effects in certain hematological malignancies. To evaluate the effects of metformin on the proliferation of SKM-1 cells, we performed a CCK-8 assay. As shown in Fig. 1A, metformin at a concentration of 1 mM has little impact on the proliferation at 24 and 48 h but significantly reduces the proliferation at 72 h; when the concentration reaches 5-10 mM, the proliferation is inhibited remarkably from 48 h; metformin at 15-20 mM slows down the proliferation from 24 h. We further detected the effects of metformin on cell apoptosis in SKM-1 cells. The results of flow cytometry (Fig. 1B and C) show that both early and late apoptotic rates of SKM-1 cells in metformin-treated groups (1 mM, 5 mM, 10 mM, 15 mM, 20 mM) are similar to those in the untreated group.

To elucidate the mechanism by which metformin inhibits the proliferation of SKM-1 cells, we performed flow cytometry to evaluate the cell cycle phases at 48 h. As shown in Fig. 2A and B, more SKM-1 cells are arrested at the G1/G0 phase (P < 0.05) in metformin (5 mM, 20 mM) treated groups compared with those in the untreated group. Meanwhile, metformin reduces the expression of CyclinD1 and CDK4 (Fig. 2C and D).

Fig. 1 Metformin inhibits cell proliferation in SKM-1 cells. (Zhou X, et al., 2019)

Fig. 2 Metformin induces G0/G1 cell cycle arrest in SKM-1 cells. (Zhou X, et al., 2019)

Atg3 Overexpression Enhances Bortezomib-Induced Cell Death in SKM-1 Cells

According to a gene expression microarray, atg3 is downregulated in MDS patients progressing to leukemia, but less is known about the function of Atg3 in the survival and death of MSD/AML cells. To explore the function of the Atg3 protein, SKM-1 cells were transfected with a FLAG-tagged ATG3-overexpressing vector or an empty vector lentivirus. At 72 h after transfection, GFP expression was examined using fluorescence microscopy. The transfection efficiency of each group was above 80% (Fig. 3A). The protein expression was further confirmed by Western blotting. The level of the Atg3 protein was significantly greater in the Atg3 overexpression group (Atg3 OE group) than in the control group and mock group (Fig. 3B and 3C, Fig 3D and 3E).

Three groups of cells were treated with different concentrations of bortezomib (1, 10, 50, and 100 nM) for 24 h. The PI-positive rate of each group was dose-dependent. Compared to the control group and mock group, the Atg3 OE group had a statistically significant increased cell death rate (Fig. 4A and 4B). Then, we treated cells of each group with bortezomib at a concentration of 10 nM. At 24 h following treatment, cells of the Atg3 OE group showed nucleolus pyknosis and nuclear fragmentation (Fig. 4C). Similarly, cell growth was significantly inhibited in the Atg3 OE group 24 h following bortezomib treatment (Fig. 4D). These findings suggest that Atg3 significantly enhances bortezomib-mediated cytotoxicity in SKM-1 cells.

Fig. 3 Lentivirus-mediated Atg3 overexpression in SKM-1 cells. (Zhuang L, et al., 2016)

Fig. 4 Upregulation of autophagy by Atg3 overexpression increases the sensitivity of SKM-1 cells to bortezomib treatment. (Zhuang L, et al., 2016)