Paris saponin-induced autophagy promotes breast cancer cell apoptosis via the Akt/mTOR signaling pathwayZhan-Zhi Xie a, 1, Man-Mei Li c, 1, Peng-Fei Deng a, Sheng Wang a, Lei Wang c, Xue-Ping Lu a, Liu-Bing Hu a,Zui Chen a, Hui-Yang Jie a, Yi-Fei Wang a, Xiao-Xiao Liu b, ∗∗, Zhong Liu a, ∗
a Guangzhou Jinan Biomedicine Research and Development Center, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology,Jinan University, Guangzhou 510632, Chinab Guangdong Institute for Drug Control, Guangzhou 510632, Chinac College of Pharmacy, Jinan University, Guangzhou 510632, China
A R T I C L E I N F O
Article history:Received 17 November 2016Received in revised form 8 December2016Accepted 10 January 2017Available online xxx
Keywords:Paris saponinApoptosisAutophagy
A B S T R A C T
Paris saponins possess anticancer, anti-inflammatory, and antiviral effects. However, the anticancer effect of Parissaponins has not been well elucidated and the mechanisms underlying the potential function of Paris saponins in can-cer therapy are needed to be further identify. In this study, we report that saponin compounds isolated from Paris poly-phylla exhibited antitumor activity against breast cancer cell lines, MCF-7 and MDA-MB-231. Paris saponin XA-2 in-duced apoptosis in both cell lines, as evidenced by the activation of caspases and cleavage of Poly (ADP-ribose) poly-merase. The ability of XA-2 to induce autophagy was confirmed by acridine orange staining, accumulation of autophago-some-bound Long chain 3 (LC3)-II, and measurement of autophagic flux. XA-2-induced autophagy was observed topromote apoptosis by the combined treatment of breast cancer cell lines with XA-2 and autophagy inhibitors 3-methy-ladenine and bafilomycin A1, respectively. Moreover, we report a decrease in the levels of Akt/mTOR signaling pathwayproteins, such as the phosphorylated forms of Akt, mTOR, P70S6K, and eukaryotic translation initiation factor 4E-bind-ing protein 1 (4EBP1). Taken together, these results provide important insights explaining the anticancer activity of Parissaponins and the potential development of XA-2 as a new therapeutic agent.
Breast cancer has become the most commonly diagnosed cancerand the second leading cause of cancer-related death among women[1,2]. Although a concerted effort has been made to treat breast can-cer, particularly in the form of improved surgical methods for lumpec-tomy, mastectomy, and radiation, as well as hormone and chemother-apies, the available drugs exhibit low efficiency [3,4]. Accordingly,
additional agents and therapeutic approaches should be developed toovercome this malignancy.
Growing evidence indicates that autophagy affects various biologi-cal activities, such as survival, apoptosis, and inflammatory responses;as well as diseases, such as neurological disorders, myocardial dis-ease, and cancer [5,6]. Apoptosis is generally recognized as a form ofprogrammed cell death that involves the suicide and cracking of cellsin response to a number of stimuli, such as growth factor deprivation,antitumor drugs, and ionizing radiation, with the aim of preventingdamage, stress, or the accumulation of non-functional cells in the tis-sue [7,8]. Activation of apoptotic pathways serves as a potential anti-cancer strategy by repressing many types of tumor cells [9,10].
Autophagy represents a conserved process whereby non-essentialintracellular components are transported to the lysosomes for degra-dation in response to a variety of stress stimuli, such as nutrientor growth factor deprivation, reactive oxygen species, damaged or-ganelles, DNA damage, hypoxia, protein aggregates, and intracellularmicroorganisms [11,12]. In cancer cells, autophagy has a dual effectas a mechanism to protect or kill cells. However, chemotherapy-in-duced autophagy stimulates a pro-survival response in cancer cells todevelop drug resistance. Autophagy can also enhance treatment effi-cacy, leading to cancer cell death. In this context, the developmentof new anticancer drugs requires a better understanding of which au-tophagy mechanism influences a particular type of cancer [5]. Usu
ally, autophagy can be detected with the help of specific protein mark-ers, such as Light chain 3 (LC3), Sequestosome 1 (SQSTM1)/P62,and autophagosome staining [13]. Autophagy-associated genes, suchas ATG3, ATG7, and ATG8, regulate the synthesis and activation ofLC3, which is converted from the cytosolic LC3-I to the autophago-some-bound LC3-II form [14]. SQSTM1/P62 is a scaffold proteinpotentially involved in delivering ubiquitinated substrates to the au-tophagosome.
Paris polyphyllin, a plant from the Liliaceae family, used to be akey ingredient of traditional Chinese medicine for hundreds of years.Steroidal saponins have been reported to be the main active compo-nents of Paris polyphyllin [15]. Many studies have reported that Parissaponins possess anticancer, anti-inflammatory, and antiviral effects[16]. In particular, Paris saponins have been used for several years totreat leukemia, as well as pancreas, liver, and other tumors [17]. How-ever, the anticancer effect of Paris saponins has not been well eluci-dated and the mechanisms underlying the potential function of Parissaponins in cancer therapy are needed to be further identify. In thisstudy, we demonstrated the anticancer effect of seven saponins iso-lated from Paris polyphalla. Mechanistically, we demonstrated thatsaponin XA-2 induced apoptosis in the MCF-7 and MDA-MB-231breast cancer cell lines. XA-2 also promoted apoptosis by induction ofautophagy. This was likely mediated by the inhibition of Akt/mTORsignaling pathway.
2. Materials and methods
2.1. Reagents
All reagents and chemicals were obtained from standard commer-cial sources. Antibodies against LC3; SQSTM1/P62; the phospho-rylated forms of Akt, mTOR, eukaryotic translation initiation fac-tor 4E-binding protein 1 (4EBP1), P70S6K; poly (ADP-ribose) poly-merase (PARP); Caspase 3; Caspase 9; and glyceraldehyde 3-phos-phate dehydrogenase (GAPDH) were purchased from Cell SignalingTechnology (Beverly, MA, USA). Anti-mouse IgG and anti-rabbitIgG were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-Di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),3-methyladenine (3-MA) and bafilomycin A1 (BFA) were purchasedfrom Sigma. The Annexin V-fluorescein isothiocyanate (FITC)/pro-pidium iodide (PI) apoptosis detection kit was purchased from Be-yotime (Shanghai, China). Paris saponins were isolated from Parispolyphalla as described previously [18].
2.2. Cell culture
Human breast cancer cell lines MDA-MB-231 and MCF-7 wereobtained from the Cell Bank of the Chinese Academy of Sciences(Shanghai, China). Cells were cultured in DMEM (Gibco, ThermoScientific, Waltham, MA, USA) supplemented with 10% fetal bovineserum (Gibco), 10 μg/mL streptomycin and 100 U/mL penicillin(complete medium) at 37 °C in a humidified atmosphere containing5% CO2.
2.3. Growth inhibition assay
Growth inhibition was detected using the MTT assay as describedby Mossman [19]. MDA-MB-231 and MCF-7 cells were seeded in96-well plates at a density of 4 × 103 cells per well in medium and al-lowed to attach for about 12 h. Cells were then treated with a rangeof XA-2 concentrations. After 48 h, the medium was removed, 50 μLMTT was added to the wells, and cells were incubated for 4 h. After
medium removal, 100 μL dimethyl sulfoxide was added to each welland the plates were incubated for 20 min. Cell viability was assessedby measuring absorbance at 570 nm using a microplate reader(BIO-RAD, Hercules, CA, USA). The IC50 (half maximal inhibitoryconcentration) values for each cell line were determined by comparingtreated and untreated cells.
To assess the effect of autophagy inhibitors, 4 × 103 cells/wellwere pre-treated with 2 mM 3-MA or 3 nM BFA for 4 h and thencell viability was measured with the MTT assay as described above.Data were fitted using GraphPad Prism 4 Software (GraphPad Inc., LaJolla, CA, USA).
2.4. Flow cytometric analysis of cell apoptosis
MDA-MB-231 and MCF-7 cells (2 × 105) were seeded in 60-mmpetri dishes for 24 h prior to the experiment. Cells were treated with1.25, 2.5, and 5 μM XA-2 for 48 h, after which they were harvested,washed with ice-cold phosphate-buffered saline (PBS), and stainedwith Annexin-V-FITC/PI according to the manufacturer's instructions.Annexin-V-FITC and PI fluorescence were monitored at 630 nm and525 nm, respectively, using a FACSCalibur flow cytometer (BD,Franklin Lakes, NJ, USA). Data were fitted using FlowJo software(FlowJo LLC, Ashland, OR, USA).
To assess apoptosis after a combined treatment with XA-2 and au-tophagy inhibitors 3-MA and BFA, we pre-treated MDA-MB-231 andMCF-7 cells with 2 mM 3-MA and 3 nM BFA for 4 h, and then added5 μM XA-2. Apoptosis was detected by flow cytometry as describedabove.
2.5. Western blots assay
After treatment with different concentrations of XA-2 for 48 h (seeabove), cells were harvested and washed twice with ice-cold PBS.Cell lysates were incubated in RIPA buffer containing a protease in-hibitor cocktail (1 mM phenylmethanesulfonyl fluoride and 1 μg/mLleupeptin) and a phosphatase inhibitor cocktail (1 mM sodium fluorideand 1 mM sodium orthovanadate), cracked by ultrasonication, andcentrifuged at 12,000 rpm for 15 min. Supernatants were collected andequal amounts of denatured proteins were resolved by SDS-PAGE.Proteins were transferred onto nitrocellulose membranes, which wereblocked with 5% nonfat milk at room temperature for 1 h and thenincubated with primary antibodies overnight at 4 °C. The membraneswere washed three times with Tris-buffered saline-5% Tween 20 so-lution and incubated with a horseradish peroxidase-conjugated sec-ondary antibody at room temperature for 1 h. Protein bands were vi-sualized by enhanced chemiluminescence (Millipore, Waltham, MA,USA) and analyzed by densitometry.
2.6. Acridine orange staining
MCF-7 and MDA-MB-231 cells (2 × 105) were seeded in 60-mmpetri dishes for 24 h prior to the experiment. After treatment with5 μM XA-2 for 48 h, cells were harvested, washed twice with ice-coldPBS, and incubated with 1 μg/mL acridine orange for 15 min at 37 °C.Subsequently, cells were washed three times with ice-cold PBS andthen observed under a fluorescence microscope (IX71; Olympus,Tokyo, Japan).
2.7. Transmission electron microscopy analysis
MCF-7 and MDA-MB-231 cells (2 × 105) were seeded in 60-mmpetri dishes for 24 h prior to the experiment. After treatment with
5 μM XA-2 for 48 h, cells were harvested, washed twice with ice-coldPBS, and fixed with 2.5% modified glutaraldehyde for 4 h. Cells werethen post-fixed with 1% osmium tetroxide, dehydrated in graded se-ries of ethanol, and embedded in Durcupanresin. Ultrathin 50-nm sec-tions were cut with an ultramicrotome and stained with 2% (w/v)uranyl acetate and lead citrate. Samples were examined on a transmis-sion electron microscope (Tokyo, Japan).
2.8. Statistical analysis
Data were expressed as mean ± standard deviation of three in-dependent experiments. Statistical differences were determined byone-way ANOVA followed by post-hoc Bonferroni test if the overallP value was less than 0.05. P < 0.05 was assumed to indicate a statis-tically significant difference.
3. Results
3.1. Effect of XA-2 on the growth of MCF-7 and MDA-MB-231 cells
The chemical structures of Paris saponins are shown in Fig. 1. Theeffect of these compunds on cell viability was examined in breast can-cer cell lines MCF-7 and MDA-MB-231 by MTT assay. As shownin Table 1 and Fig. 2, XA-1-XA-5 exhibited growth inhibitory ef-fects on MCF-7 and MDA-MB-231 cell lines with IC50 values in thesubmicromolar range after 48 h of treatment. The most potent com-pound was XA-2 with IC50 values of 2.54 and 2.28 μM for MCF-7 andMDA-MB-231 cells, respectively.
3.2. XA-2 induces apoptosis in MCF-7 and MDA-MB-231 cells
Apoptosis is a major form of cell death induced by chemotherapeu-tic agents [20]. To investigate the role of XA-2 in this process, MCF-7and MDA-MB-231 cells were treated with indicated concentrations ofXA-2, and cell apoptosis was assessed via Annexin-V-FITC/PI stain-ing and flow cytometric analysis. Notably, both cell lines showed sig-nificant dose-dependent increases in apoptotic cell ratios followingtreatment with XA-2 (Fig. 3A and B). Thus, at an XA-2 concentra-tion of 5 μM, apoptotic cell ratios were 50% and 77% for MCF-7 andMDA-MB-231 cells, respectively, compared with the control.
Next, we analyzed caspase activation and PARP cleavage inXA-2-treated cells. As shown in Fig. 3C, the levels of cleaved Cas-pase-3 and -9 increased significantly as the concentration of XA-2 in-creased. Additionally, as shown in Fig. 3C, XA-2 treatment led to spe-cific proteolytic cleavage of PARP, indicated by the presence of 116to 89 kDa PARP fragments. These results suggest that caspase activa-tion and PARP cleavage may be responsible for XA-2-mediated apop-tosis in MCF-7 and MDA-MB-231 cells.
3.3. XA-2 induces autophagy in MCF-7 and MDA-MB-231 cells
Growing evidence highlights the important roles of drug-inducedautophagy in anticancer therapies [20]. Here, we examined the ex-pression of LC3, a widely used autophagy marker. During autophagy,LC3 is converted from the cytosolic LC3-I form to an autophago-some-bound LC3-II form [21]. As shown in Fig. 4A, LC3-II accumu-lated in MCF-7 and MDA-MB-231 cells in a dose-dependent manner
with the treatment of XA-2 for 48 h. In addition, a decrease in theexpression level of SQSTM1/P62, another autophagy marker, wasobserved in cells following treatment with XA-2. XA-2-induced au-tophagy was also demonstrated by the formation of characteristicacidic vesicular organelles visible by acridine orange staining (Fig.4C), and the presence of autophagic vacuoles by electron microscopy(Fig. 4D). These results show that XA-2 could induce autophagy inMCF-7 and MDA-MB-231 cells.
3.4. The Akt/mTOR signaling pathway is involved in XA-2-inducedautophagy
The Akt/mTOR signaling pathway has been shown to play a majorrole in regulating autophagy in cancer cells [22]. We treated MCF-7and MDA-MB-231 cells with different concentrations of XA-2 andanalyzed them after 48 h. As shown in Fig. 5, Akt phosphorylationlevel declined after XA-2 treatment, suggesting the involvement ofAkt signaling in XA-2-induced autophagy. Furthermore, XA-2 sup-pressed the phosphorylation level of mTOR, the downstream proteinof Akt. The phosphorylation status of P70S6K and 4E-BP1, the sub-strates of mTOR was then examined. It was observed that the phos-phorylated forms of P70S6K and 4EBP1 decreased in both cell linesafter treatment with XA-2. These results indicate that the Akt/mTORsignaling cascade is involved in XA-2-induced autophagy in MCF-7and MDA-MB-231 cells.
3.5. Effects of XA-2-induced autophagy on apoptosis in breastcancer cells
To explore whether XA-2-induced autophagy had a protective orharmful effect, MCF-7 and MDA-MB-231 cells were pre-culturedwith two different autophagy inhibitors, 3-MA and BFA, before treat-ment with XA-2. The MTT assay suggested that 3-MA, a commonlyused inhibitor of starvation or rapamycin-induced autophagy [23],prevented XA-2-dependent growth inhibition of MCF-7 andMDA-MB-231 cell lines (Fig. 6A). This finding suggests thatXA-2-induced autophagy could promote apoptosis. To verify this hy-pothesis, flow cytometry was used to analyze apoptosis induced by thecombination of XA-2 and 3-MA. The results confirmed that XA-2-in-duced autophagy had an apoptosis-promoting effect in both breastcancer cell lines (Fig. 6C and E), reflecting previous reports on therole of 3-MA on cell survival [24]. Finally, BFA also prevented thegrowth inhibitory effect of XA-2 and attenuated XA-2-triggered apop-tosis in MCF-7 and MDA-MB-231 cells (Fig. 6B, D, and E). Thesefindings indicate that XA-2-induced autophagy promotes apoptosis inbreast cancer cells.
4. Discussion
In addition to their traditional use, natural products have played im-portant roles in medical discovery as sources of bioactive compounds[25]. Rhizoma paridis is the root of either Paris polyphylla Smith var.chinensis (Franch.) Hara or Paris polyphylla Smith var. yunnanen-sis (Franch.) Hand-Mazz. Rhizoma paridis has been reported to ex-ert numerous pharmacological effects, and was shown to inhibit tumorgrowth in hepatic, gastric, or nasopharyngeal carcinoma models [26].Furthermore, Paris chinensis dioscin is reported to induce G2/M cellcycle arrest and apoptosis in human gastric cancer SGC-7901 cells[27]. Dioscin can restore the activity of adriamycin, an anticanceragent, in multidrug-resistant human leukemia K562/adriamycin cellsvia a mechanism involving Nuclear factor-kappa beta signaling inhi-bition and the consequent downregulation of multidrug resistance pro-tein 1 [28]. Paris Saponin II suppresses the growth of human ovar-ian cancer xenografts by modulating vascular endothelial growth fac-tor-mediated angiogenesis and tumor cell migration [29]. These find-ings highlight the potential therapeutic application of XA-2 as an anti-cancer agent for the treatment of breast tumors and further warrant invivo research.
Fig. 2. Effect of XA-2 on MCF-7 and MDA-MB-231 cells. Cells (4 × 104/mL) were treated with different concentrations of XA-2 (0, 1.56, 3.125, 6.25, 12.5, 25, and 50 μM) for48 h. The MTT assay was used to detect viability of (A) MCF-7 and (B) MDA-MB-231 cells. Data are presented as the mean ± SD of three independent experiments.
Fig. 3. XA-2 induces apoptosis of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. MCF-7 and MDA-MB-231 cells (4 × 104/mL) were treated with different con-centrations of XA-2 (0, 1.25, 2.5, and 5 μM) for 48 h. Flow cytometry was used to detect apoptotic cells based on the Annexin V-FITC/PI assay among (A) MCF-7 and (B)MDA-MB-231 cells. (C) Western blots were used to measure the levels of apoptosis-related proteins: cleaved Caspase-3, cleaved Caspase-9, and cleaved PARP. GAPDH was usedas control. All data are representatives of three independent experiments or presented as the mean ± SD of three independent experiments. *, P < 0.05.
Fig. 4. XA-2 induces autophagy of MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were treated with different concentrations of XA-2 (0, 1.25, 2.5, and 5 μM) for48 h (A, B) Western blots were used to measure the levels of autophagy markers: (A) cytoplasmic LC3-I and autophagosome-bound LC3-II; (B) SQSTM1/P62. GAPDH was used ascontrol. (C) Untreated and XA-2-treated MCF-7 and MDA-MB-231 cells were stained with acridine orange to detect autophagosomes (see arrows). (D) Untreated and XA-2-treatedMCF-7 and MDA-MB-231 cells were visualized by transmission electron microscopy to confirm the presence of autophagosomes. All data are representatives of three independentexperiments.
Fig. 5. XA-2 downregulates the Akt/mTOR signaling pathway in MCF-7 and MDA-MB-231 cells. Cells were treated with different concentrations of XA-2 (0, 1.25, 2.5, and 5 μM)for 48 h. Western blots were performed to detect the expression levels of Akt, p-Akt, p-mTOR, p-P70S6K, and p-4EBP1 in MCF-7 and MDA-MB-231 cells. GAPDH was used ascontrol. All data are representatives of three independent experiments.
Apoptosis is a form of programmed cell death based on the degra-dation of cellular constituents by a series of cysteine proteases calledcaspases. Caspase activation involves two main molecular pathways,known as the extrinsic and intrinsic apoptotic pathways [30]. The lat-ter causes the release of pro-apoptotic proteins into the cytosol, andis characterized by mitochondrial permeabilization [31]. A growingnumber of studies have confirmed apoptosis as the prevailing anti-cancer mechanism elicited by chemotherapy.
We report that XA-2 substantially decreased cell viability ofMCF-7 and MDA-MB-231 cells (Fig. 2). Moreover, XA-2 was ableof inducing apoptosis in both breast cancer cell lines (Fig. 3A andB), upregulating caspase signaling proteins (Caspase-3 and -9), andactivating the downstream cellular death substrate PARP (Fig. 3C).These results indicate that XA-2 stimulates apoptosis in MCF-7 andMDA-MB-231 cells.
Fig. 6. Autophagy inhibitors 3-MA and BFA affect XA-2-induced cell growth and apoptosis in MCF-7 and MDA-MB-231. Cells (4 × 104/mL) were added to 96-well plates,pre-cultured with either 2 mM 3-MA or 3 nM BFA for 4 h, and then treated with 5 μM XA-2. Af
ter 48 h, the MTT assay was used to detect viability of cells pre-cultured with (A) 3-MA or (B) BFA. Flow cytometry was used to visualize apoptotic cells based on the AnnexinV-FITC/PI assay, following pre-treatment with (C) 3-MA or (D) BFA, as specified above. (E) Apoptotic ratios of the cultures shown in panels C and D were calculated. Data arepresented as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01.
Autophagy is an evolutionarily conserved process that allows forself-digestion of cells. Accordingly, cytoplasmic material is se-questered within autophagosomes and is incorporated in the lysoso-mal compartment. We verified the effect of XA-2 on autophagy ofMCF-7 and MDA-MB-231 cells through different methods and theuse of LC3, a reliable autophagy marker. Western blot revealed thedose-dependent accumulation of the autophagosome-bound LC3-IIform (Fig. 4A). Another autophagy marker, SQSTM1/P62, was shownto decrease as the concentration of XA-2 increased (Fig. 4B). Thesefindings suggest that XA-2 induces autophagy of MCF-7 andMDA-MB-231 cells. Furthermore, acridine orange staining and trans-mission electron microscopy confirmed the existence of au-tophagolysosomes in response to XA-2 treatment (Fig. 4C and D).Taken together, these results indicate that XA-2 promotes autophagyin breast cancer cells.
Studies have reported that autophagy can be upregulated in manycancer cells under stress conditions, such as chemotherapy or inani-tion, but the exact effect of autophagy in tumor cell death remains un-known [32,33]. On the one hand, autophagy degrades damaged, aged,or non-functional proteins, organelles, and cytoplasm components intotheir constituent blocks: nucleosides, fatty acids, and amino acids.This allows the cell to maintain the steady state, generate energy, andenable organelle replacement. On the other hand, autophagy can pro-mote elimination of damaged cells by apoptosis [34]. Accordingly,we investigated the association between apoptosis and autophagy inbreast cancer cells treated with a combination of XA-2 and autophagyinhibitors 3-MA and BFA. 3-MA is an inhibitor of Phosphatidylinos-itol-3-kinase. The combined treatment with XA-2 and either 3-MAor BFA decreased viability of both breast cancer cell lines, suggest-ing that XA-2-induced autophagy contributed to its anticancer func-tion (Fig. 6A and B). To further illustrate this assumption, we usedflow cytometry to confirm the apoptotic ratios observed with the MTTassay (Fig. 6C–E). Taken together, these results suggest that XA-2-in-duced apoptosis promotes autophagy in breast cancer cells.
Given that both apoptosis and autophagy could be induced byXA-2 in MCF-7 and MDA-MB-231 cells, we investigated their mu-tual relationship. Several studies have reported that chemotherapeuticdrugs can induce both apoptosis and autophagy of cancer cells [35].Therefore, it would be beneficial to identify which is the main au-tophagy pathway induced by such drugs during cancer therapy. Akt/mTOR is essential for many cellular reactions and is important in can-cer, diabetes, and aging [36]. In tumor cells, it regulates cell prolif-eration, survival, and metabolism [37]. This pathway consists of thedownstream mediator Akt and mTOR [37]. Recently, the Akt/mTORsignaling pathway has been reported to negatively regulate autophagy[38]. In the present study, we sought to detect the main proteins of thispathway, such as the phosphorylated forms of Akt, mTOR, P70S6K,and 4EBP1. The levels of all of them decreased with increasing XA-2concentration. These results suggest that the Akt/mTOR signalingpathway is involved in XA-2-induced autophagy, and regulates apop-tosis in breast cancer cells. This result does not preclude the involve-ment of other processes, such as phosphorylation of AMP-activatedprotein kinase by upstream kinases (e.g., LKB1, CaMKK, or TAK1)in XA-2-induced autophagy of MCF-7 and MDA-MB-231 cells [39].However, the detailed roles of networks controlling XA-2-induced au-tophagy, and crosstalk and back-regulations between XA-2-inducedapoptosis and autophagy require further studies.
In summary, our datas reveal that seven saponin compounds iso-lated from Paris polyphylla exhibited antitumor activity against breastcancer cells. Mechanistical studies demonstrated that Paris saponinXA-2 could induce apoptosis in both cell lines. Moreover, XA-2 in-hibited the Akt/mTOR signaling pathway and facilitated autophagy.Also, XA-2-induced autophagy could promote the apoptosis inMCF-7 and MDA-MB-231 cell lines. The present study provides im-portant insights explaining the anticancer activity of Paris saponinsand their potential development as new therapeutic agents.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (81302654, 81673670), the Science andTechnology Program of Guangzhou (201610010108 and201300000041) and the Fundamental Research Funds for the CentralUniversities (21615413).
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