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Diosgenin induces ROS-dependent autophagy and cytotoxicity via mTOR signaling pathway in chronic myeloid leukemia cells.


Background: Diosgenin, a steroidal saponin isolated from legumes and yams, has been confirmed to possess potent anticancer effect on multifarious tumors including chronic myeloid leukemia (CML).

Purpose: We aimed to further determine the anti-cancer activity of diosgenin and its mechanisms in CML cells.

Methods: The cell vitality was detected by MTT assay. Autophagic flux and reactive oxygen species (ROS) production were analyzed by laser scanning confocal microscope. Apoptosis was observed by flow cytometry. All proteins expression was examined by western blotting.

Results: Autophagy induction was demonstrated by examination of autophagic flux including autophagosomes accumulation, autophagosome-lysosome fusion and degradation of autophagosomes. Moreover, blocking autophagy with inhibitor chloroquine (CQ) and 3-methyladenine (3-MA), enhanced diosgenin-induced apoptosis, indicating the protective effect of autophagy in diosgenin-treated CML cells. Further study suggested that diosgenin-induced autophagy and cytotoxicity were accompanied by reactive oxygen species (ROS) generation and mammalian target of rapamycin (mTOR) signaling pathway inhibition. N-acetyl-L-cysteine (NAC) administration, a scavenger agent of ROS. could down-regulate diosgenin-induced autophagy via reversion of mTOR pathway inhibition.

Conclusion: These results indicate that diosgenin obviously generates ROS and this oxidative pressure not only produces cytotoxic effect on CML cells but also induces autophagy. What's more, autophagy functions as a cytoprotective mechanism to overcome cytotoxicity of diosgenin in tumor cells and inhibition of autophagy can enhance the anti-CML activity of diosgenin.



Chronic myeloid leukemia


Reactive oxygen species



Diosgenin, a natural plant extract mainly from roots of wild yam (Dioscorea villosa), is used as a traditional medicine because of its activities of antitumor, antidiabetes, anti-inflammatory and antiatherosclerosis (Choi et al. 2010; Das et al. 2012; Esfandiarei et al. 2010; Jung et al. 2010; Kalailingam et al. 2014; Raju and Mehta 2009). It has been reported that diosgenin induced proliferation inhibition and apoptosis in multiple cancer cells lines including leukemia (Liu et al. 2004), prostate cancer (Chen et al. 2011), colorectal cancer (Lepage et al. 2011), hepatocellular carcinoma (Kim et al. 2012), lung cancer (Hsieh et al. 2013), and melanoma (Lee et al. 2007). Diosgenin induces proliferation inhibition in human chronic myeloid leukemia (CML) K562 cells via G2/M restraint and apoptosis, with activation of caspase-3, downregulation of Bcl-2, and generation of reactive oxygen species (ROS) (Liu et al. 2004). Its glucoside derivative dioscin reverses adriamycin-induced Multidrug-Resistant (MDR) through down-regulating MDR1 expression via NF-kB signaling pathway inhibition in K562 cells (Wang et al. 2013). Moreover, diosgenin induces megakaryocytic differentiation of human erythroleukemia cells through ERK and sonic hedgehog pathway activation (Fernandez-Zapico et al. 2014).

CML is a lethal hematological malignancy which is defined by the existence of a BCR-ABL oncoprotein. The protein causes a persistent tyrosine kinase activity and gives the character of leukemic cells proliferation and resistance to apoptosis (Lin et al. 2014). The treatment of CML has been significantly improved by tyrosine kinase inhibitor (TKI) therapies (Trela et al. 2014). However, quite a number of patients with CML failed to respond effectively to these drugs because of BCR-ABL kinase-dependent or independent resistance (Eiring et al. 2014). These observations emphasize the need to develop new therapeutic agents and combination approaches.

Natural plant products, especially steroid compounds, have a potential role in exploiting innovative chemotherapy drugs for various cancers (Burgett et al. 2011). In our researches on antitumor activity of diosgenin, we observed that diosgenin induced autophagy as well as cytotoxicity and apoptosis in CML cells. Autophagy is important in regulating cellular energetic balance (Martinez-Lopez et al. 2013). It can be markedly upregulated by environmental stimuli such as nutrient and growth factor starvation, chemotherapeutic agents and oxidative stress, and in most cases protects cells against stressful situations (Russell et al. 2013). Accumulating evidence suggests that autophagy plays an important role in CML and other stem cell driven malignancies, and might also serve as a therapeutic target in CML cells that are resistant to TKI treatment (Helgason et al. 2013). Despite some researches on anti-CML activities and mechanisms of diosgenin, the exact mechanism by which diosgenin induces autophagy and its role in CML therapy have not been elucidated.

In this study, we elucidate that diosgenin not only produces cytotoxic effect on CML cells but also induces autophagy, and inhibition of autophagy potentiated diosgenin-induced cell death in CML cells. We further explore the molecular mechanisms, that is, induction of cytotoxicity and autophagy mediated by ROS generation and mTOR signaling pathway. Taken together, our results demonstrate the multiple roles of diosgenin and combination of its antineoplastic activity with autophagic inhibitor is a prospective new treatment strategy for CML.

Materials and methods

Chemicals and antibodies

Diosgenin was obtained from Nanjing Spring & Autumn Biotech Co.Ltd (Jiangsu, China) and its purity analyzed by high performance liquid chromatography was over 98%. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), chloroquine (CQ) and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein (FITC)-Annexin V Apoptosis Detection kit was purchased from BD Bioscience (Franklin Lakes, NJ, USA). 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), BCA Protein Quantitation Kit and N-acetyl-L-cysteine (NAC) were purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu Province, China). EGFP-LC3 was obtained from Addgene (Cambridge, MA, USA). Cyto-ID[R] Autophagy Detection Kit was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Lyso-Tracker Red was purchased from Invitrogen (San Diego, CA, USA). All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell lines and culture

The CML cell lines K562 and Ba[F.sub.3]-WT were obtained from Cell Bank of Chinese Academy of Sciences, Shanghai Branch (Shanghai, China). All cells were cultured in RPMi-1640 (CORNING) medium contains 10% fetal bovine serum (Gibco), penicillin (100 IU/ml), and streptomycin (100g/ml) at 37[degrees]C in a 5% C02 atmosphere incubator. Cells were used when reached in the logarithmic phase.

Determination of cell viability (MTT assay)

Cell viability was determined by the MTT cytotoxicity assay. The cells seeded in 96-well plates at a density of 4 x [10.sup.3] cells/well were pretreated with or without optimal concentration of CQ or 3MA for 2 h, and then treated with diosgenin for indicated concentrations and times. Thereafter, the cells were incubated with MTT (0.5mg/ml) for 4h at 37[degrees]C. After formazan was fully dissolved in 100 [micro]l of 20% SDS in dimethyl formamide/[H.sub.2]O (1:1, v/v), the optical density (OD) was measured by microplate reader.

Confocal immunofluorescence

K562 and Ba[F.sub.3]-WT cells were seeded in 6-well culture plates at a density of 1 x [10.sup.5] and then treated with 20[micro]M of diosgenin for 48 h, with cells treated with 50 nM autophagy inducer rapamycin for 6h as positive controls. Thereafter, cells were washed twice with culture medium and then treated with nuclear dye Hoechst 33342 and autophagy detection kit Cyto-ID[R] green dye, or ROS detection kit Mito Sox[TM] red dye at 37[degrees]C for 15 min. After incubation, cells were immediately analyzed by fluorescence microscope. All the procedures were kept in the dark place.

Western blot analysis

K562 and Ba[F.sub.3]-WT cells were suspended in RIPA Cell lysis buffer. An equal quantity of total protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were terminated by TBST including 5% nonfat milk and followed by incubating with primary and secondary antibodies. Immunoreactive proteins were visualized using an enhanced chemiluminescent detection kit (Pierce, Rockford, IL, USA). Intensities in the resulting bands were quantified by the IQuantTL software (GE Healthcare, USA).

Measurement of intracellular ROS

Intracellular ROS generation was detected in CML cell lines by Reactive Oxygen Species Assay Kit. Dichlorofluorescein diacetate (DCFH-DA), a cell permeable and nonfluorescent dye can be oxidized to fluorescent carboxy dichlorofluorescein (DCF) by intracellular ROS. Briefly, K562 and Ba[F.sub.3]-WT cells were stimulated with diosgenin (20 [micro]M) and co-incubated with 10 [micro]M DCFH-DA at 37[degrees]C for 24 h, then collected and washed twice with phosphate buffered saline (PBS). To quantify the level of intracellular ROS, DCF fluorescence intensity was measured by Tecan Infinite[R] 200 PRO microplate reader at 488 nm for excitation wavelength and 525 nm for emission wavelength.

Apoptosis assay

Annexin V-FITC/PI Detection Kit was used for the determination of cell apoptosis. After designated treatment, cells were harvested and washed twice with ice-cold PBS, then re-suspended in 500 [micro]l binding buffer at a concentration of 1 x [10.sup.6] cells/ml. According to the manufacturer's description, cells were incubated in the dark with 5 [micro]l Annexin V-FITC (fluorescein isothiocyanate) and 5 [micro]l PI (propidium iodide) for 15 min at 37 C. The rate of apoptosis was immediately analyzed by FACS Calibur flow cytometer.

Transmission electron microscopy analysis

After treated by diosgenin for 48 h, K562 and Ba[F.sub.3]-WT cells were harvested and fixed in 2.5% phosphate-buffered glutaraldehyde for transmission electron microscopy (TEM) assay (Song et al. 2015). Samples were detected with a JEM 1410 transmission electron microscope at 80 kV.

Microscopy and photography

Cells were incubated with different concentrations of diosgenin with or without autophagy inhibitors. After treated for 24 h or 48 h, cells were observed by an inverted microscope.

Statistical analysis

We used GraphPad Prism 5 to perform statistical analysis and the results were expressed as mean [+ or -] S.D. Multiple sets of data were compared using one-way ANOVAs or two-tailed Student's t test. Values of P < 0.05 were considered to be statistically significant.


Cytotoxic effect of diosgenin on CML cells

The structure of diosgenin is shown in Fig. 1A. Cells were challenged with diosgenin (0-30 [micro] M) and the cytotoxicity was quantified by MTT assay. Diosgenin inhibited cell viability in a dose- and time-dependent manner (Fig. 1C and D). Moreover, morphological analysis by microscope (Fig. 1B) indicated apparent morphology changes, including decreased volume, changed shape and reduced number of cells after incubation with diosgenin. Cell apoptosis was also analyzed with flow cytometry after double staining with Annexin-V and PI. According to Fig. 4D and 4E, the early apoptotic cells were elevated after exposure of CML cells to 20 [micro]M of diosgenin for 24 h. These results demonstrate that diosgenin induces the cytotoxicity of CML cells in both dose- and time-dependent manners.

Autophagy induced by diosgenin in CML cells

Three well-established methods were adopted to detect autophagosome formation to determine whether diosgenin induces autophagy in CML cells (Mizushima et al. 2010). First, the number of autophagic vacuoles presenting in cells was investigated using TEM analysis. Fig. 2A showed an accumulation of double-membrane-enclosed autophagosome in K562 and Ba[F.sub.3]-WT cells after exposure to 20 [micro]M of diosgenin for 48 h, whereas no autophagosome was observed in untreated cells. Second, the CytoID[R] Green dye autophagy detection kit was used to detect LC3-II, the protein bound on the membrane of autophagosomes with fluorescence microscopy. After treatment with 20 [micro]M of diosgenin for 48 h, K562 and Ba[F.sub.3]-WT cells exhibited significant green fluorescence, the same as the positive control cells treated with 50 nM rapamycin. Meanwhile, the negative controls showed no specific fluorescence (Fig. 2B-D). To further characterize diosgenin-induced autophagy in CML cells, we used western blot analysis to examine the conversion of the microtubule-associated protein 1 light chain 3 (LC3) to autophagosome-associated LC3-II. Fig. 2E showed an apparent conversion of endogenous LC3-I to LC3-II in a dose-dependent manner. Moreover, Fig. 2F revealed that the accumulation of LC3-II was remarkably increased after 48 h treatment with diosgenin, which indicated autophagosome formation.

Altogether, these observations suggest that diosgenin induces autophagy in K562 and Ba[F.sub.3]-WT CML cells.

Autophagic flux in diosgenin-treated CML cells

It has been reported that autophagy is a dynamic process and the level of LC3-II and the number of autophagosomes are insufficient for evaluating autophagic activity (Meijer 2014; Mizushima et al. 2010; Shen and Codogno 2014). In the following experiments, we first used LC3 turnover assay by a lysosomotropic reagent chloroquine (CQ), which inhibits autophagosome-lysosome fusion, to measure autophagic flux. Western blot analysis indicated that LC3-II accumulation was proportional with time both in the presence and absence of CQ. LC3-II levels in the presence of CQ were larger than that in the absence of CQ especially in 24 h and 48 h of diosgenin treatment, and the differences between them indicated that LC3 delivered to lysosomes for degradation (Fig. 3A). Furthermore, the results of Cyto-ID[R] and Lyso Tracker staining showed three stages of autophagic flux in diosgenin-treated CML cells of formation and accumulation of autophagosomes at 12 h (green fluorescence), autophagosome-lysosome fusion at 24 h (yellow fluorescence) and degradation of autophagosomes by lysosomes at 48 h (red fluorescence) (Fig. 3B-D). These data strongly demonstrate that autophagic flux is simulated by diosgenin in CML cells.

Blocking autophagy enhances diosgenin-induced cytotoxicity and apoptosis in CML cells

Previous studies show that autophagy may act as cytoprotective role in tumor cells under metabolic stress and the cytotoxicity of chemotherapy (Avalos et al. 2014; Lorin et al. 2013; Zeng et al. 2013). To find out the role of autophagy in diosgenin-treated CML cells, we inhibited autophagy using CQ and 3-MA, and analyzed the effects on diosgenin-induced cell cytotoxicity and apoptosis. As a lysosome inhibitor, CQ led to the aggregation of autophagosomes and increased LC3-II level in diosgenin-treated K562 and Ba[F.sub.3]-WT cells (Fig. 4A). However 3-MA, an inhibitor of P13K which inhibits autophagosomes accumulation, decreased LC3-I1 level by blocking the conversion of LC3-I to LC3-I1 (Fig. 4B). The results indicated that diosgenin-induced autophagy was successfully inhibited by 3-MA and CQ.

Compared with cells that incubated with diosgenin alone, combined treatment with autophagy inhibitor CQ or 3-MA obviously increased diosgenin-induced cytotoxicity in K562 and Ba[F.sub.3]-WT cells (Fig. 4C). We also examined the changes of diosgenin-induced apoptosis in the presence and absence of CQ or 3-MA to further exploit the role of autophagy using Annexin V-F1TC/PI double staining assay. Diosgenin in combination with CQ or 3-MA induced a higher scale of apoptotic cells (Fig. 4D and E) and more cleavage of poly-ADP-ribose polymerase (PARP) (Fig. 4F) as compared with diosgenin treatment alone, whereas treatment with CQ or 3-MA alone showed limited apoptosis-inducing effects on K562 and Ba[F.sub.3]-WT cells. In addition, microscope observation showed that diosgenin combined with CQ or 3-MA induced more obvious morphology changes including cell shrinkage, abnormity, and death as compared with diosgenin treatment alone (Fig. 4G).

All of these results elucidate that autophagy plays a cytoprotective role in diosgenin-induced cell death in CML cells, and inhibition of autophagy enhances cell cytotoxicity, morphology changes and apoptosis induced by diosgenin.

Intracellular ROS controls diosgenin-induced autophagy and cytotoxicity

As it has been reported that diosgenin provoked cytotoxicity by inducing the production of ROS in HepG2 cells (Kim et al. 2012), we investigated the ability of diosgenin-induced ROS in CIVIL cells. Cells were incubated with 20 [micro]M of diosgenin with or without N-acetyl-cysteine (NAC) and analyzed for the presence of ROS by relative DCF fluorescence intensity. As shown in Fig. 5A, diosgenin markedly induced ROS generation in K562 and Ba[F.sub.3]-WT cells as compared with controls. As a potent antioxidant, NAC could not only inhibit oxidative stress via directly scavenging ROS but also remarkably down-regulated diosgenin-induced ROS (P < 0.01). Clearing ROS by NAC could also terminate diosgenin-induced cytotoxicity (Fig. 5B)

A growing amount of evidence in recent years confirms that ROS production induced by diverse conditions of stress represents an important mediator of autophagy (Filomeni et al. 2014). Hence, the involvement of ROS in diosgenin-induced autophagy was also examined. Western blot analysis indicated that the levels of LC3-II stimulated by diosgenin were obviously down-regulated by antioxidant NAC in K562 and Ba[F.sub.3]-WT cells (Fig. 5C), suggesting that removing ROS led to inhibition of diosgenin-induced autophagy. We also proved intracellular ROS and autophagy generation process by co-staining with ROS detection kit Mito Sox[TM] red dye and autophagy detection kit Cyto-ID[R] green dye. As shown in Fig. 5D and Fig. SI, at first the red fluorescence representing ROS generation was enhanced gradually after 12-18 h of diosgenin treatment. Then both K562 and Ba[F.sub.3]-WT cells exhibited significant green fluorescence which represents autophagy production in 24-48 h of diosgenin treatment. These data strongly suggest the role of intracellular ROS as the upstream stimuli, regulating both autophagy and cytotoxicity in this model.

The mTOR signaling pathway is included in diosgenin-induced autophagy in CML cells

It has been demonstrated that ROS can induce cellular damage, then inhibit mammalian target of rapamycin complex 1 (mTORC1) and induce autophagy (Alexander et al. 2010). The mTOR pathway is not only an important regulator of protein synthesis and cell growth but also a key negative regulator of autophagy (Dunlop and Tee 2014; Heras-Sandoval et al. 2014). mTOR positively regulates protein translation through the phosphorylation of two major protein targets, protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Neufeld 2012). mTOR can be phosphorylated at serine 2448 to form p-mTOR-S2448 which inhibits the induction of autophagy (Nazio and Cecconi 2013).

To confirm whether mTOR pathway was involved in autophagy induced by diosgenin, we evaluated the level of phosphorylated mTOR (p-mTOR-S2448) in diosgenin-treated K562 and Ba[F.sub.3]-WT cells. Western blot manners showed that diosgenin down-regulated the level of p-mTOR-S2448 in a dose- and time-dependent manner in K562 and Ba[F.sub.3]-WT cells. The level of phosphorylated p70S6K and 4E-BP1, two downstream substrates of mTOR, also significantly decreased after dose- and time-dependently exposure to diosgenin (Fig. 6A and B). Moreover, Fig. 6C showed that the levels of p-mTOR-S2448, p-p70S6K-S371 and p-4E-BPl-T45 were all reversed in response to the removal of intracellular ROS by NAC. These findings declare that mTOR pathway is involved in ROS-dependent autophagy and cytotoxicity induced by diosgenin in CML cells.


Diosgenin has been extensively studied for its anti-cancer potentials including induce cell growth inhibition and apoptosis of various cancer cells (Chen et al. 2011; Kim et al. 2012; Lepage et al. 2011; Liu et al. 2004). Its antiproliferative activity on human CIVIL has been investigated by Liu et al. (Liu et al. 2004). However, the effects of diosgenin on the autophagy-related researches of human CML cells and the underlying mechanism have not been elucidated.

In the present study, we first examined the diosgenin-induced autophagic response in K562 and Ba[F.sub.3]-WT cells. We confirmed it by the formation of autophagosome through TEM, LC3-positive autophagy-like vacuoles through Cyto-ID[R] Green dye, and the increased conversion of LC3-I to LC3-II through western blot analysis. Many anticancer natural products were reported to induce autophagy. For example, dioscin, a glucoside derivative of diosgenin, caused autophagy as a cytoprotective mechanism against apoptosis in human lung cell lines (Hsieh et al. 2013), while celastrol induced autophagic cell death in human osteosarcoma cells.

As autophagy involves dynamic and complicated processes (Meijer 2014; Shen and Codogno 2014), therefore, to further confirm the diosgenin-induced autophagy in CML cells, we also examine the autophagic flux using LC3 turnover assay and costained with Cyto-ID[R] Green and Lyso-Tracker Red dyes. LC3 turnover assay is one of the primary methods to evaluate autophagic flux, which is based on the observation that LC3-II is degraded in autolysosomes. Lysosomotropic reagent CQ, which inhibits autophagosome-lysosome fusion, can block LC3-II degradation, resulting in the accumulation of LC3-II (Rubinsztein et al. 2009). Therefore, combination with CD reflects the autophagic flux during diosgenin treatment. We also measure the autophagic flux using co-staining of Cyto-ID[R] Green and Lyso-Tracker Red. The green fluorescent signal of Cyto-ID[R] is based on cytosolic compartments which can be specifically labeled with minimal staining of lysosomes and endosomes (Guo et al. 2015). Besides, Lyso-Tracker as a lysosomotropic probe sends the red fluorescent signal (Wang et al. 2014). Co-staining assay here is used to measure autophagic flux. In the early stage of diosgenin treatment (before 24 h), the green and yellow fluorescence represents the accumulation of autophagosomes and autophagosome-lysosome fusion. The red fluorescence at 48 h indicates that the autophagic substrates have been delivered into the lysosome and degraded. All of these data confirm the autophagic flux in CML cells induced by diosgenin.

The role of autophagy in tumor is complicated and involves several, sometimes paradoxical functions (Roy et al. 2014). We observed that combined treatment with autophagy inhibitor CQ or 3-MA increased diosgenin-induced cell death, and microscope analysis showed more obvious morphology changes including cell shrinkage, fragmentation, and death as compared with diosgenin-treated alone. Remarkably, CQ. and 3-MA treatment also enhanced diosgenin-induced apoptosis as evidenced by increased AnnexinV positive cells and PARP cleavage. All of these results demonstrate that autophagy promotes cell survival under the cytotoxicity of diosgenin in K562 and Ba[F.sub.3]-WT cells. Blocking autophagy can enhance the efficacy of diosgenin on CML cells and this may be a potential new therapeutic strategy for CML.

An increasing amount of evidence in recent years suggests that ROS are involved in intracellular signal transducers sustaining autophagy (Levonen et al. 2014). Our experiments showed that diosgenin markedly induced the ROS generation in CML cells, and removing ROS by antioxidant NAC led to inhibition of diosgenin-induced autophagy as evidenced by down-regulation of LC3-II. A gradually enhanced ROS signaling was observed by fluorescence microscope in the early stage of diosgenin treatment, where after CML cells exhibited significant green fluorescence which represents autophagy production in 24-48 h of diosgenin treatment. The results of MTT assay showed NAC could also reverse diosgenin-induced cytotoxicity. These results strongly suggest that ROS act as early inducers of autophagy and cytotoxicity upon diosgenin treatment, this oxidative stress not only produces cytotoxic effect on CML cells but also induces autophagy which functions as a survival mechanism to overcome cytotoxicity of diosgenin in tumor cells.

Recent researches prove that elevated ROS inhibit mTORC1 and induce autophagy, and repression of mTORC1 in response to oxidative stress can be relieved with the ROS scavenger NAC (Alexander et al. 2010; Filomeni et al. 2014). mTOR signaling pathway positively regulates protein translation through phosphorylation of p70S6K and 4E-BP1, and negatively regulates autophagy via phosphorylation of mTORC1 at serine 2448 (Dunlop and Tee 2014; Nazio and Cecconi 2013). On the basis of above experiment results, we guess that mTOR signaling pathway plays a significant role in the ROS-dependent autophagy and cytotoxicity induced by diosgenin. Next investigations corroborate our view. Upon diosgenin treatment, the dose and time-dependent reduction of mTOR phosphorylation, as well as the phosphorylation substrates of mTOR (pP70S6K-S371 and p-4EBPl-T45) in K562 and Ba[F.sub.3]-WT cells were observed by western blot analysis. At the same time, removal of intracellular ROS by NAC reversed the phosphorylation levels. These results elucidate that mTOR signaling pathway is involved in ROS-dependent autophagy induced by diosgenin in CML cells.

In conclusion, our study demonstrates that diosgenin can simultaneously induce cytotoxicity and autophagy in K562 and Ba[F.sub.3]-WT cells, and autophagy plays a cytoprotective role. Further investigations suggest that diosgenin strongly elevates ROS level, and this oxidative stress not only produces cytotoxic effect on CML cells but also induces autophagy which functions as a survival mechanism to overcome cytotoxicity of diosgenin in CML cells via mTOR signaling pathway (Fig. 7). This compelling evidence highlights that, combined with autophagic inhibitors, diosgenin is a promising candidate for development of antitumor drugs targeting CML.


Article history:

Received 7 December 2015

Revised 18 January 2016

Accepted 26 January 2016

Conflict of interest

The authors declare no conflict of interest.


This study was supported by National Natural Science Foundation of China (No. 81572979), National Key Basic Research Program of China (2015CB931800), and School of Pharmacy, Fudan University & The Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.01.010.


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Shanshan Jiang (a),(1), Jiajun Fan (a),(1), Qian Wang (a), Dianwenju (a), Meiqing Feng (a), Jiyang Li (a), Zhong- bin Guan (b), Duopeng An (a), Xin Wang (a), Li Ye (a),*

(a) Department of Biosynthesis & Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China

(b) Shanghai Institute For Food And Drug Control, Shanghai, China

Abbreviations: CML, chronic myeloid leukemia; CQ, chloroquine; 3-MA, 3-methyladenine; ROS, reactive oxygen species; mTOR, mammalian target of rapamycin; NAC, N-acetyl-L-cysteine; MDR, multidrug-resistant; TKI, tyrosine kinase inhibitor; BCA, bicinchoninic acid; PVDF, poiyvinylidene fluoride; TEM, transmission electron microscopy; p70S6K, protein S6 kinase; 4E-BP1, 4E-binding protein 1.

* Corresponding author. Tel.; +86 21 5198 0035; fax: +86 21 5198 0036.

E-mail address: (L Ye).

(1) Shanshan Jiang and Jiajun Fan contributed equally to this work.
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Author:Jiang, Shanshan; Fan, Jiajun; Wang, Qian; Ju, Dianwen; Feng, Meiqing; Li, Jiyang; Guan, Zhong-bin; A
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Mar 15, 2016
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