Polyphyllin I induces apoptosis and autophagy via modulating JNK and mTOR pathways in human acute myeloid leukemia cells

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Highlights

  • PPI was found to induce apoptosis and autophagy in AML cell lines and primary cells.

  • PPI-induced JNK phosphorylation was found to be a dependent factor for apoptosis.

  • PPI-induced mTOR phosphorylation was found to be a key factor for autophagy.

  • A cross-talk between apoptosis and autophagy was investigated.

Abstract

Polyphyllin I (PPI), a bioactive component extracted from Paris polyphylla, was reported to have potent anticancer activities in previous studies. However, there were few reports on the effects and underlying mechanism of PPI in human acute myeloid leukemia cells. The present study demonstrated that PPI had an inhibitory effect through inducing apoptosis and autophagy in THP-1 and NB4 cells. PPI induced apoptosis via activating JNK pathway, as evidenced by the decreased Bcl-2 levels and increased Bax, cleaved-caspase-3 and phosphorylated-JNK expressions. In addition, PPI promoted autophagy as evidenced with increased expressions of LC3-II and Beclin-1 in western blot and autophagic vacuoles in MDC staining, which was associated with the inhibition of AKT-mTOR pathway. Furthermore, JNK inhibitor SP600125 and autophagy inhibitor 3-MA were employed to evaluate the role of apoptosis and autophagy in PPI-induced cell death. We found that autophagy and apoptosis were both causes of cell death induced by PPI. These data suggested that PPI could be a potent therapeutic agent for the treatment of human acute myeloid leukemia.

Introduction

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, originating from the hematopoietic bone marrow cells and featuring highly heterogeneous clinical and molecular biological characteristics [1]. Currently, high-dose chemotherapy followed by hematopoietic stem cell transplantation remained the most successful therapeutic strategy in AML and could achieve complete remission (CR) in many cases. Albeit the high relapse rate after transplant in many patients has become problematic and limited the survival of patients. Data showed that ~80% of AML patients relapsed after CR and the 5-year survival rate of AML is only ~30% [2]. Therefore, novel effective therapeutic approaches to treat AML remain imminently needed.

In recent years, increasing interest in natural products, such as certain bioactive components in anti-tumor herbal regimes of traditionalmedicine has attributed to several potential benefits including novel therapeutical drug targets, good effectiveness in some patients and better drug safety [3]. For example, Sphaeranthus indicus found mostly in southern India has been reported to induce apoptosis in human leukemia HL-60 cells [4]. Timosaponin AIII isolated from A. asphodeloides originated from Europe has been showed to inhibit cell growth in gastric cancer cell lines and reverse multi-drug resistance in human leukemia K562_ADM cells [5,6]. Uncaria tomentosa found in South America has been used in traditional medicine to prevent and treat inflammation. Recently, it has been found to possess potent anti-cancer activities in human breast cancer cells and reduce the adverse effects of chemotherapy [7,8]. The rhizome of Paris polyphylla, named as Chong-lou in traditional medicine, has been traditionally used to manage parotitis, sore throat, hemostasis, snakebite and malignant tumors [9]. Previous studies showed that Polyphyllin I (PPI) (Fig. 1), a steroidal saponin isolated from Paris polyphylla, has potent anti-cancer activity in various cancer cell lines with anti-tumor activities including induction of cell cycle arrest, apoptosis induction, anti-angiogenesis effect and reverse of drug-resistance [[10], [11], [12], [13]]. However, the antitumor activity of PPI in hematopoietic malignancies, such as AML, remains to be determined.

In a cell-type dependent manner, there are two major pathways of programmed cell death, including apoptosis and autophagy [14]. Accumulating evidence has suggested that the pathogenesis of AML is closely associated with signaling pathways regulating apoptosis and/or autophagy [15]. Mitogen-activated protein kinases (MAPKs) signaling pathways, particularly the c-jun NH2-terminal kinase (JNK), has been shown to regulate apoptosis in AML cells [16,17]. Autophagy is an evolutionarily conserved cellular degradation system to maintain the normal physiological state of cells via cell fate decision [18]. It is well known that the mammalian target of rapamycin (mTOR) pathway is one of the major upstream targets of autophagy [19]. Currently, the functional correlation between apoptosis and autophagy remains controversial [20].

In the present study, we investigated the cytotoxic effect of PPI on THP-1, NB4 and HL-60 human AML cells in vitro and any possible underlying molecular mechanisms associated with apoptosis and/or autophagy signaling pathways. PPI was found to significantly inhibit the proliferation of THP-1 and NB4 cells and induce apoptotic cell death. The participation of JNK and mTOR signaling in PPI-induced apoptosis/autophagy were demonstrated. A selective JNK inhibitor SP600125 or an autophagy inhibitor 3-methyladenine (3-MA) were added to the PPI-treated THP-1 cells and their respective effects were studied. In parallel, the cytotoxic potential of PPI was further evaluated in the primary AML cells from patients and white blood cells from healthy volunteers. Together, our research work demonstrated the potential therapeutical effect of PPI to combat AML cells but not the normal hematopoietic cells, and could inspire further studies to explore more about this novel potential drug candidate for hematopoietic malignancies.

Section snippets

Chemicals and reagents

PPI was purchased from China Food and Drug Administration (Beijing, China). RPMI1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). RIPA lysis buffer and PVDF membrane was purchased from Beyotime Institute of Biotechnology (Shanghai, China). CCK8 cell activity assay kit and AnnexinV FITC/PI apoptosis assay kit were purchased from Sigma (St. Louis, MO, USA). Rabbit monoclonal antibodies specific for cleaved-caspase 3,

PPI suppressed the proliferation of AML cell lines and PBMCs

THP-1, NB4, HL-60 cells and PBMC cells harvested from human acute myleoid leukemia subjects were used to evaluate the anti-proliferative effects of PPI. Firstly, the CCK8 viability assay confirmed that PPI significantly inhibited the growth of THP-1 cells in a dose-dependent manner (Fig. 2A and B). The 72h IC50 for the THP-1, NB4 and HL-60 cells were (3.91 ± 0.15) μM, (5.68 ± 0.19) μM and (5.35 ± 0.19) μM Secondly, the effect of PPI was tested in PBMC cells and compared between cells harvested

Discussion

AML is a hematological malignancy occurring in adults and is characterized by excessive proliferation of abnormal immature hematopoietic cells. At present, despite of the growing technical maturation of different transplantation methods, chemotherapy drugs remain heavily needed in various circumstances. For example, AML induction treatment typically uses a combination of cytarabine and anthracycline (daunorubicin or idarubicin) and AML consolidation treatments may use cytarabine, etoposide,

Conclusion

In summary, our study demonstrated that PPI inhibits the proliferation of human acute myeloid leukemia cells by inducing apoptosis and autophagy. PBMC cells from AML subjects and healthy volunteers were also tested and a major cytotoxic effect of PPI on leukemic cells but not normal cells was observed. PPI triggered apoptosis through the JNK pathway while downregulating Bcl-2 level, increasing Bax, cleaved-PARP and cleaved-caspase-3 expression. PPI activated autophagy through inhibiting

Conflicts of interest

The authors declare that they have no conflict of interest.

Declaration of interests

The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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