Harmine hydrochloride inhibited the proliferation and induced the G2/M cell cycle arrest in K562 cells.
HAR-HC is a derivative of the natural beta-carboline alkaloid with a better water solubility than harmine (Fig. 1A). We examined the effects of HAR-HC on cell viability in K562, Raji and LO2 cells. After cells were exposed to indicated concentrations (0, 5, 10, 20, 40 and 80 µM) of HAR-HC for various times (24, 48 and 72 h), it demonstrated a remarkable inhibition effect on the proliferation of K562 cells with the IC50 values ranged from 30 µM to 70 µM, and the IC 50 values of Raji cells ranged from 30 µM to35 µM of (Fig. 1B, 1C, Table 1), while the IC 50 values of HAR-HC on LO2 cells at 24 h and 48 h were > 80 µM (Table.1). These results showed that HAR-HC inhibited K562 and Raji cells’ proliferation.
In addition, we analyzed the cell cycle distribution by flow cytometry. Compared with the control, HAR-HC treatment resulted in a decrease in the G1 phase and an accumulated population in the G2 phase at 24 h, while the fraction of the S phase cells was approximately the same in the untreated and treated groups (Fig. 2A and 2B). We also found that HAR-HC treatment decreased the expression levels of cyclin D1, cyclin D3 and cyclin E2, while the levels of cyclin A2 and cyclin B1 increased (Fig. 2C). And the expression levels of the p21, Myt1, p-cdc2 (Tyr15) proteins were increased (Fig. 2D). Thus, we concluded that HAR-HC increased the protein levels of p21 and Myt1 leading to the G2/M phase arrest in K562 cells.
Harmine hydrochloride induced apoptosis in K562 cells.
To further investigate the potential mechanism of HAR-HC regulation of K562 cells, we detected apoptotic cells by the Annexin V-FITC/PI double staining assay. The results showed that the apoptosis in K562 cells increased with increasing concentrations of HAR-HC (Fig. 3A). Hence, we determined whether mitochondria-associated apoptosis pathway was involved in the HAR-HC group. The expression levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1 were significantly reduced. However, the expression of Bax was not significantly influenced (Fig. 3B). After treatment with HAR-HC for 24 h, the expression levels of cleaved caspase-3 and cleaved PARP were increased in K562 cells (Fig. 3C). To further investigate whether HAR-HC-induced cell apoptosis was caspase dependent, z-DEVD-fmk, a caspase 3 inhibitor was used. The results showed that z-DEVD-fmk reversed the changes in the expression levels of cleaved-caspase 3 and cleaved-PARP. These results indicated that HAR-HC induced apoptosis in K562 cells through the mitochondria-associated apoptosis pathway (Fig. 3D).
HAR-HC induced autophagy in K562 cells.
Autophagy is the cellular pathway the clearance of ubiquitinated proteins in the lysosomes during nutrient deprivation or cellular stress . As shown in Fig. 4A, the downregulation of p62 and the increase of LC3Ⅰ/Ⅱ conversion indicated that HAR-HC upregulated the formation of autophagosomes in a dose-dependent manner in K562 cells. To confirm that autophagy is induced by HAR-HC, CQ, an inhibitor of autophagy was added to K562 cells for 1 h prior to HAR-HC exposure. Western blot showed that the expression levels of p62 and LC3-Ⅱ were reversed by CQ (Fig. 4B), suggesting that HAR-HC induced autophagy in K562 cells.
Autophagy played a protective effect in K562 cells.
We found that the expression levels of cleaved -caspase-3 and cleaved -PARP were increased in the HAR-HC group and the expression levels of cleaved-caspase-3 and cleaved-PARP were even further increased in HAR-HC and CQ combination group (Fig. 4C). These results showed that HAR-HC-elicited autophagic response played a protective effect, and inhibition of autophagy could sensitize K562 cells to HAR-HC.
HAR-HC-treated K562 cells activated the MAPK signaling pathway.
Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases mainly involved in embryonic development, cell differentiation, cell proliferation and cell death[9–11]. ERK was considered to be the classical MAPK pathway participating in cell growth, proliferation and differentiation. However, the role of ERK1/2 in inducing cell apoptosis remains controversial. The results showed that the expression levels of p-JNK and p-ERK were increased in K562 cells after HAR-HC treatment (Fig. 5A).
Activating of ERK1/2 pathway induced autophagy, inhibit apoptosis in K562 cells. To verify between activation of ERK pathway and autophagy in HAR-HC treatment cells, a MEK/ERK- specific inhibitor U0126 was used. The results showed that the expression levels of p-ERK1/2 were increased in the HAR-HC group and were reversed in HAR-HC and U0126 combination group (Fig. 5B). We also found that the expression of p62 was downregulated and the expression of LC3-Ⅱ was upregulated in the HAR-HC group; the downregulation of p62 and the upregulation of LC3-Ⅱ were reversed in the HAR-HC and U0126 combination group (Fig. 5B). That meant activation of ERK pathway induced autophagy, and inhibition of ERK pathway blocked autophagy in K562 cells.
We also investigated the relationship between the activation of ERK pathway and apoptosis in K562 cells treated with HAR-HC. As shown in Fig. 5C, western blotting assay revealed that the expression levels of cleaved-caspase-3 and cleaved-PARP were increased in the HAR-HC group and the expression of cleaved-caspase-3 and cleaved-PARP were even further increased in the HAR-HC and U0126 combination group. These results demonstrated that apoptosis was enhanced by inhibition of ERK1/2 pathway, suggesting that activating of the ERK1/2 pathway reduced apoptosis and promoted cell survival in HAR-HC-treated K562 cells.