Harmine Hydrochloride Induced G2/M Phase Cell Cycle Arrest, Autophagy and Apoptosis in Human Leukemia K562 Cells through Modulation of the MAPK Pathway


 Background

Previous studies have indicated that harmine hydrochloride (HAR-HC) has anti-tumor characteristics. However, its potential impact on human leukemia cells is unknown. In this study, we explored the potential mechanism of HAR-HC effects on human leukemia cells in vitro.
Methods

MTT assay was used to detect cell viability; A flow cytometer was used to analyze the cell cycle; Anexinn V-FITC/PI was used to detect cell apoptosis; Western blotting assay was used to analyze the expression of related proteins.
Results

The result of flow cytometry suggested G2/M phage arrest in K562 cells induced by HAR-HC. The expression levels of Cyclin E2, Cyclin D1, Bcl-2, Bcl-xL, Mcl-1, pro-caspase-3, and PARP decreased and the expression levels of Cyclin A2, Cyclin B1, p21, Myt-1, p-cdc2 (Tyr15), cleaved -caspase-3 and cleaved-PARP increased. Moreover, the expression of p-JNK and p-ERK1/2 increased and autophagy was induced in the HAR-HC treatment group. Additionally, HAR-HC facilitated autophagy by activating the ERK1/2 pathway.
Conclusion

HAR-HC induced G2/M phase cell cycle arrest, autophagy and apoptosis by activating the JNK, and ERK1/2 pathways in the human leukemia K562 cells.


Conclusion
HAR-HC induced G2/M phase cell cycle arrest, autophagy and apoptosis by activating the JNK, and ERK1/2 pathways in the human leukemia K562 cells.

Background
Chronic myeloid leukemia (CML) is a malignant tumor that affects blood and bone marrow. The disease is characterized by markedly increased but immature peripheral blood granulocytes. The presence of the Ph chromosome and/or BCR-ABL fusion genes substantially affects the survival and prognosis of patients [1]. Tyrosine kinase inhibitors are the rst-line molecular targeted therapy for chronic myeloid leukemia in the clinic [2]. Tyrosine kinase inhibitors can speci cally block the binding of ATP to ABL kinase; thus, tyrosine residues cannot be phosphorylated and the proliferation of the BCR-ABL positive cells is inhibited. However, a wide use of tyrosine kinase inhibitors is associated with frequent drug resistance [3] and severe systemic toxicity. Moreover, these drugs cannot completely cure chronic myeloid leukemia. Patients may relapse due to drug resistance or a gene mutation. These factors may limit the use of tyrosine kinase inhibitors. At present, there are no speci c targeted therapies for chronic myeloid leukemia. Allogeneic hematopoietic stem cell transplantation is the only way to cure CML; however, limited availability of suitable donors severely affects the therapeutic options of the patients. Thus, it is necessary to nd other therapeutic drugs to improve the cure rate of CML.
Harmine is prevalent in nature. It has various types of pharmacological activators including antimicrobial, antifungal, antitumor, cytotoxic, antispasmodic, antioxidant, antimutagenic, antigenotoxic and hallucinogenic properties [4]. Harmine hydrochloride is a water-soluble nitro-carlin alkaloid with the same properties as harmine. Previous studies have shown that HAR-HC has various antitumor effects. It has been reported that harmine activated intrinsic and extrinsic pathways of apoptosis in B16F-10 melanoma [5]. Additionally, our previous study revealed that harmine induced cell cycle arrest and mitochondrial pathway-mediated cellular apoptosis in SW620 cells by inhibiting the AKT and ERK signaling pathway [6].
Our previous results revealed that harmine hydrochloride triggers the G2 phase arrest and apoptosis in MGC-803 and SMMC-7721 Cells by up-regulating p21, activating caspase-8/Bid, and down-regulating the ERK/Bad pathway [7]. At present, the effect of HAR-HC on chronic myeloid leukemia cells is poorly understood. Therefore, we studied the effect of HAR-HC and its mechanism in K562 cells.

Materials And Methods
Reagents and antibodies.
Cell lines and cell culture.
The human cell lines human leukemia K562 and normal liver epithelial LO2 cells were obtained from Department of Biochemistry, Basic Medical College, Jinan University (Guangdong, China). LO2 cells were cultured in high glucose DMEM containing 10% FBS. Chronic leukemia K562 and lymphoma Raji cells were cultured in RPMI-1640 medium with 7% FBS. The cells were maintained at 37°C in a humidi ed incubator with 95% air and 5% CO 2 .
Cell viability assay. Cells (6 × 10 3 /ml) were seeded in 96-well plates overnight and then treated with various concentrations of HAR-HC (0, 5, 10, 20, 40 and 80µM). After incubation of 24,48 or 72 h, 90 µL fresh medium without FBS and with 10 µL of MTT was added to each well for 4 h at 37°C. Then, discarded the supernatant by centri and 150 µL DMSO was added to each well. Finally, a microplate reader was used to measure the absorbance at 570 nm in each well.
Cell cycle analysis.
Cells were seeded at a density of 5.0×10 5 per well in six-well plates. The cells were exposed to various concentrations of HAR-HC (0, 10, 20 and 40 µM) for 24 h. The cells were harvested, washed twice with cold PBS and xed with 70% cold ethanol overnight at 4°C. Cells were subjected to the cell cycle distribution analysis after 30 min of incubation with RNase and PI in the dark. A ow cytometer was used to analyze the samples.

Cell apoptosis analysis.
Cells were seeded at a density of 5.0×10 5 per well in six-well plates. After 24 h, the cells were exposed to various concentrations of HAR-HC (0, 10, 20 and 40 µM) for 24 h. The cells were harvested, washed twice with cold PBS. Then, 5µL of Anexinn V-FITC/PI and 5µL PI were added to the cells and incubated for 15 min in the dark at room temperature following the manufacturer's protocol. A ow cytometer was used to analyze the samples.
Western blotting assay.
Cellular proteins were extracted after treatment with a cell-lysis buffer which containing proteinase inhibitors on ice for 30 min and the samples were centrifuged at 12,000 rpm at 4℃ for 15 min. Then, the total protein was quanti ed using a BCA protein assay kit according to the manufacturer' instructions.
The samples were subjected to 10% or 15% SDS-PAGE, and the protein bands were transferred to polyvinylidene di uoride (PVDF) membranes. After blocking with 5% dried nonfat milk, the membranes were incubated overnight at 4°C with an appropriate primary antibody. Then, the membranes were washed three times in Tris-buffered saline with tween 20 (TBST) and incubated for 1 h at room temperature with a secondary antibody conjugated to horseradish peroxidase. After three washes in TBST, the bands on the membranes were visualized with an ECL chemiluminescence reagent and X-ray lm (Kodak, Rochester, NY, USA) as described previously.

Statistical analysis.
Statistical analysis was done by GraphPad software, version 5. Data are presented as the means ± standard error of the means (SEM) or standard Deviation (SD). Dunnett-t test was applied to assess the statistical signi cance. p value < 0.05 were considered signi cant.

Results
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,  These results showed that HAR-HC inhibited K562 and Raji cells' proliferation. In addition, we analyzed the cell cycle distribution by ow 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 D 1 , cyclin D 3 and cyclin E 2 , while the levels of cyclin A 2 and cyclin B 1 increased (Fig. 2C). And the expression levels of the p21, Myt1, p-cdc2 (Tyr 15 ) 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.
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 signi cantly reduced. However, the expression of Bax was not signi cantly in uenced (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 [8]. As shown in Fig. 4A, the downregulation of p62 and the increase of LC3 / conversion indicated that HAR-HC upregulated the formation of autophagosomes in a dosedependent manner in K562 cells. To con rm 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-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][10][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-speci c 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-was upregulated in the HAR-HC group; the downregulation of p62 and the upregulation of LC3-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 cleavedcaspase-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.

Discussion
At present, conventional chemotherapy is one of the main methods of cancer treatment. Most chemotherapeutic drugs have a low response rate and poor e cacy in the clinic. More importantly, severe toxicity and resistance of chemotherapy reduce the life quality of patients and the effectiveness of chemotherapeutic drugs. In recent years, numerous natural products and their derivatives have received considerable attention from pharmacologists and chemists studying the use in the treatment of tumors [12][13][14]. In this study, the anticancer activity of HAR-HC was detected. The results showed that HAR-HC induced the G2/M phase arrest and caspase-dependent apoptosis and autophagy in K562 cells through modulation of the MAPK signaling pathway.
In this study, a G2/M phase arrest treated with HAR-HC was detected in K562 cells by ow cytometry. We demonstrated that HAR-HC decreased the expression levels of Cyclin D1, D3 and Cyclin E2 and increased the expression levels of Cyclin B 1 , Cyclin A 2, Myt-1, p21 and p-cdc2 Tyr15 in K562 cells. Up-regulation of p21 inhibited the activation of the cdc2 and Cyclin B 1 complexes, while Myt-1 reduced activity of cdc2 by phosphorylating Tyr 15 leading to G2/M phase in K562 cells. ERK1/2 signal pathways regulate cell growth, proliferation and differentiation and the JNK, p38 signal pathways play important roles in stress responses including in ammation and apoptosis [15][16][17][18]. We detected that the expression levels of anti-apoptosis proteins (Bcl-2, Bcl-xL and Mcl-1) were downregulated and enhanced the ratio of Bax/Bcl-2, Bax/Bcl-xL. HAR-HC promoted activation of caspase-3 and PARP in K562 cells. While we added z-DEVD-fmk, a caspase 3 inhibitor, before the treatment of HAR-HC, and the expression levels of cleaved-caspase 3 and cleaved-PARP were reversed. The results showed that HAR-HC induced apoptosis via caspase-dependent manner. Studies have shown that JNK activation can induce apoptosis [7,9]; moreover, SP600125, a JNK-speci c inhibitor, partially but signi cantly rescued the cells from death and signi cantly repressed caspase-3 activity [19,20]. Some studies demonstrated that ERK1/2 is also important for induction of apoptosis [21][22][23]. However, recent studies demonstrated that ERK1/2 can suppress apoptosis via phosphorylation of the pro-apoptotic Bcl-2 family proteins [17,18,23]. To determine the detailed mechanism of HAR-HC induction of apoptosis, we investigated the role of HAR-HC in MAPK signaling, and found that HAR-HC treatment activated JNK and ERK in K562 cells.
Autophagy is an intracellular metabolic process. In the progress of autophagy, cytoplasmic LC3-binds to phosphatidyl ethanolamine and is transformed into LC3-. In the present study, we demonstrated that HAR-HC treatment increased the expression of LC3-/LC3-ratio and decreased the express levels of p62. These results suggested that HAR-HC could induce autophagy in K562 cells. Autophagy plays a dual role in the survival and death of cancer cells [24,25]. To further con rm the relationship between pro-survival and pro-apoptotic effect of HAR-HC inducing autophagy in K562 cells, an autophagy inhibitor (CQ) was used. The results showed that CQ treatment increased apoptosis in the CQ and HAR-HC combination group. This result revealed that autophagy may play a pro-survival role in HAR-HC-induced apoptosis. ERK signaling pathway is involved in regulation of autophagy and apoptosis [12,16]. In the experiment, we found that autophagy was inhibited and apoptosis was increased in U0126 and HAR-HC combination group. ERK1/2 activation and autophagy played a pro-survival role in the HAR-HC-induced K562 cells.

Conclusion
In conclusion, our research demonstrated that HAR-HC induced G2/M phase arrest in K562 cells via the up-regulation of p21, activation of Myt1 and phosphorylation of cdc2. Additionally, HAR-HC induced mitochondria-related apoptosis and autophagy by modulating the expression of Bcl-2 protein family, activating the JNK and ERK1/2 signaling pathways. ERK1/2 activation reduced apoptosis and increased autophagy in HAR-HC treated K562 cells.