Introduction

There are two major types of programmed cell death during tumor therapy: apoptosis and autophagy (Savill and Fadok 2000). Death receptor (extrinsic) and/or mitochondrial (intrinsic) pathway is involved in apoptosis induction (Okada and Mak 2004). Cell shrinkage, chromatin condensation, cytoplasmic membrane blebbing, and formation of apoptotic bodies are canonical morphological changes of apoptotic cells (Burz et al. 2009). Autophagy is a process of cellular suicide with lysosome-dependent protein degradation (Finkel et al. 2007), but it also provides a survival mechanism in the presence of growth factors or under the condition of nutrient deprivation (Kuma et al. 2004). Nitric oxide (NO) is synthesized through the oxidation of L-arginine by nitric oxide synthase (NOS). NO plays an important role in regulation of apoptosis and autophagy process (Meijer and Codogno 2006).

Dihydroartemisinin (DHA) is the main active metabolite of artemisinin, a natural compound from Artemisia annua L. However, DHA has a poor water solubility with low bioavailability (Gabriels and Plaizier-Vercammen 2004). Therefore, a series of derivatives synthesized have been evaluated for their activities against cysteine protease falcipain-2 of Plasmodium falciparum (Luo et al. 2012). The previous study demonstrated that L-A03 (also known as compound 9d), one of such derivatives, had a high activity against recombinant falcipain-2 (Luo et al. 2012). Accumulating evidence shows that the DHA and its derivatives exert anticancer activities in tumor cell lines (Zhong et al. 2016; Chen et al. 2004; Hwang et al. 2010). Breast cancer has been known more than a century but its therapy is still difficult. Recent studies report that series of DHA analogs have obvious activity on human breast cancer MCF-7 cells, but the detailed mechanism remains unknown (Zhong et al. 2016). In this study, we examined the mechanism of cell growth inhibition by L-A03 in human breast cancer MCF-7 cells by focusing on the relationships among apoptosis, autophagy and NO generation.

Materials and methods

Reagents

L-A03 was obtained from Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University. The purity of L-A03 was measured by high performance liquid chromatography and determined to be 99.5%. L-A03 was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution and was diluted to the required concentration, with the DMSO concentration being kept below 0.1%, in RPMI-1640 culture medium (Hyclone, Logan, UT, USA). Ficoll-Hypaque solution (density 1.077 g/mL) was purchased from Tianjin Chuanye Biological Products (Tianjin, China). Fetal bovine serum (FBS) was purchased from TBD Biotechnology Development (Tianjin, China); monodansylcadaverine (MDC), chloroquine (CQ), 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT), dithiothreitol (DTT), 1400 W, L-NAME, SNP, z-VAD (caspase family inhibitor) and anti-LC3 antibodies were purchased from Sigma Chemical (St. Louis, MO, USA). The primary antibodies against FADD, β-actin, caspase-6, caspase-8, Bcl-2, Bax, Bid and PARP, and horseradish-peroxidase-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture

Human breast cancer MCF-7 cell was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 10 μg/ml streptomycin, 100 U/ml penicillin and 0.03% L-glutamine and maintained at 37 °C with 5% CO2 at a humidified atmosphere.

Growth inhibition assay

The growth inhibition effect of L-A03 in MCF-7 cells was measured by MTT assay. The MCF-7 cells were incubated in 96-well tissue culture plates at a density of 5 × 103 cells/well. After 24 h incubation, the cell was treated with or without ZVAD, CQ, DTT, L-NAME, 1400 W, or SNP at the given concentrations for 1 h and subsequently treated with L-A03 for different time periods.

Human peripheral blood mononuclear cells (hPBMCs) were isolated from the fresh blood samples of three healthy male volunteers by centrifuging venous blood on Ficoll-hypaque density gradients, and this study was approved by the Ethics Committee of the General Hospital of Shenyang Military Region (201-LS-No.012). 5 × 104 cells per well were seeded in 96-well culture clusters following by drug treatments. The growth inhibition effect was measured by MTT assay. The percentage of cell growth inhibition was calculated as follows:

$${\rm{Cell}}\,{\rm{growth}}\,{\rm{inhibition}}\,\left( {\rm{\% }} \right)\\ {\rm{ = }}\left[ {{\rm{A492}}\left( {{\rm{control}}} \right){\rm{ - A492}}\left( {{\rm{sample}}} \right)} \right]{\rm{/}}\\ \left[ {{\rm{A492}}\left( {{\rm{control}}} \right){\rm{ - A492}}\left( {{\rm{blank}}} \right)} \right]\,{\rm{*}}\,{\rm{100}} $$

Observation of morphological changes

MCF-7 cells were seeded into 6-well culture plates with or without L-A03 and cultured for 24 h. The cellular morphology was observed by using a phase contrast microscopy (Leica, Nussloch, Germany).

Fluorescence morphological examination

The cells were harvested and incubated with 15 μM L-A03, washed with PBS for three times and then stained with 0.05 mM MDC. Cell samples were incubated at 37 °C in the dark for at least 15 min.

Flow cytometric analysis of apoptosis, autophagy and NO generation

MCF-7 cells were pretreated with or without ZVAD, CQ, DTT, L-NAME, 1400 W, or SNP for 1 h before the addition of L-A03. After cultured for 24 h, the cells were harvested and rinsed with PBS two times by centrifugation at 1000 × g for 10 min.

For measuring apoptosis, the pellets were suspended in 1 mL of PI solution containing PI 50 μg/mL, samples were incubated at 4 °C in the dark for at least 30 min and then the samples were analyzed by flow cytometry (FACScan; Becton Dickinson, Sunnyvale, CA, USA) to determine the percentage of cells undergoing apoptosis.

For measuring autophagy, the pellets were suspended with 0.05 mM MDC at 37 °C for 30 min as described previously, and then the samples were analyzed by flow cytometry to determine the percentage of cells undergoing autophagy.

The cells were harvested and rinsed with PBS, then mixed with 10 μM NO specific dye DAF-2DA and incubated at 37 °C for 30 min. The samples were analyzed by FACScan flow cytometry with the excitation wavelength at 480 nm and the emission wavelength at 525 nm.

Western blot analysis

MCF-7 cells were treated with 15 μM L-A03 for 3, 6, 12 and 24 h. The cells were collected, and then western blot analysis was carried out. Briefly, the cell pellets were resuspended with lysis buffer consisting of 50 mM Hepes (PH 7.4), 1% Triton X-100, 2 mM sodium orthovanada, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM PMSF, 10 mg/L aprotinin, 10 mg/L leupeptin (Sigma, MO, USA), and lysed at 4 °C for 1 h. After 12,000 × g centrifugation for 15 min, the protein contents of supernatant were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of the total protein were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and the membranes were soaked in blocking buffer (5% skimmed milk). Proteins were detected using polyclonal antibodies and visualized using anti-rabbit or anti-mouse IgG conjugated with peroxidase (HRP) and 3,3-diaminobenzidine tetrahydrochloride (DAB) as the HRP substrate.

Statistical analysis

All the presented data and results were confirmed in at least three independent experiments. The data were expressed as means ± SD. Statistical comparisons were made by Student’s t-test (Statistics Package for Social Science, SPSS, Chicago, IL). Statistical differences between the different groups were analyzed with post hoc test. P < 0.05 was considered statistically significant.

Results and discussion

L-A03 inhibits the growth of MCF-7 cells

The MTT assay indicated that L-A03 (the structure has been shown in Fig. 1a) inhibited the growth of MCF-7 cells in a concentration-dependent and time-dependent manner (Fig. 1b). Meanwhile, 5-fluorouracil (5-FU) or paclitaxel (TAX) was used as a positive control (Fig. 1b), suggesting that L-A03 had more obvious anti-tumor activity on MCF-7 cells than positive group. To examine the cytotoxicity of L-A03 on normal cells, hPBMC were prepared and treated with 5, 10, 20, 40, and 80 μM L-A03 for 24 and 36 h. The result demonstrated that L-A03 had low cytotoxicity on human normal cells (Fig. 1c). Taken together, L-A03 induced growth inhibition of MCF-7 cells without significant cytotoxicity on normal cells.

Fig. 1
figure 1

L-A03 inhibits the growth of MCF-7 cells. a The chemical structure of L-A03. b The cell viability of L-A03 in normal human cells (human peripheral blood mononuclear cells, hPBMCs) compared with 5-fluorouracil (5-FU) and paclitaxel (TAX) was determined by MTT assay. The hPBMCs were treated with L-A03, 5-FU and paclitaxel at 5–80 μM for 24 and 36 h. c MTT analysis was applied to measure the inhibitory ratio of MCF-7 cells. The cells were treated with L-A03 at 5–40 μM for 12, 24, and 36 h, 5-FU and paclitaxel for 24 h

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L-A03 induces extrinsic and intrinsic apoptosis in MCF-7 cells

To determine the features of MCF-7 cell death induced by L-A03, morphological changes were observed. In Fig. 2a, 15 μM L-A03 caused significant changes including cell shrinkage and formation of apoptotic bodies. After treatment with 7.5, 15 and 30 μM L-A03, MCF-7 cells were analysed by flow cytometry. The result suggested that L-A03 induced an obvious increase in the percentage of subG0/G1 cells, indicating the induction of apoptosis (Fig. 2b). ZVAD, a pan-caspase inhibitor, which was usually used as an apoptotic inhibitor, attenuated L-A03-induced cell death (Fig. 2c). Western blot analysis showed that L-A03 markedly elevated the expressions of FADD and caspase-8 (Fig. 2d), suggesting that death receptor pathway was triggered in L-A03-treated MCF-7 cells. Moreover, the protein Bid was cleaved by caspase-8 in L-A03-treated cells (Fig. 2e). The rise of the Bax/Bcl-2 ratio also is consistent with apoptosis (Tsujimoto 1998). L-A03 decreased the expression of Bcl-2 and but increased Bax level (Fig. 2e), showing that L-A03 also triggered mitochondrial pathway. Caspase 3 and 6, the downstream executors of apoptosis, were activated in apoptotic cells, but MCF-7 cells were reported to be negative in the expression of caspase 3 (Janicke et al. 1998). Therefore, we examined the level of caspase 6 by Western blot analysis, suggesting that it was activated by L-A03 in MCF-7 cells (Fig. 2e). PARP, a substrate of caspase 6, was cleaved accordingly (Fig. 2e). These results showed that L-A03 caused apoptotic cell death in MCF-7 cells through both extrinsic and intrinsic pathways.

Fig. 2
figure 2

L-A03 induces extrinsic and intrinsic apoptosis in MCF-7 cells. a–e MCF-7 cells treated with different concentrations of L-A03 for 24 h. a The cells were observed with a phase contrast microscope (X 200 magnification, upper panels) and changes of nuclear morphology were detected by AO staining (X 200 magnification, lower panels). Arrows indicate apoptotic cells. b The cells were stained with PI and measured by flow cytometry. The ratio of apoptotic cells was taken as the subG0/G1 ratio. c The cells were incubated with L-A03 (15 μM) and/or in the presence of ZVAD (4 μM). Then, cell viability was measured by MTT assay. d, e The protein levels of FADD, caspase 8 (d), Bid, Bcl-2, Bax, caspase 6, and PARP (e) were detected by western blot. (**P < 0.01 vs. L-A03-treated group)

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Autophagy is induced in L-A03-treated MCF-7 cells

To determine the effect of L-A03 on autophagy in MCF-7 cells, the cells were observed by monodansylcadaverine (MDC) staining. As shown in Fig. 3a, L-A03 caused a significant increase in the number of MDC-labeled autophagolysosomes. Flow cytometric analysis also showed that the percentage of MDC positive ratio was enhanced by L-A03 treatment (Fig. 3b). Western blot analysis revealed that L-A03 induced the conversion from LC3 I to LC3 II (Fig. 3c). Since L-A03 induced both apoptosis and autophagy in MCF-7 cells, flow cytometric analysis was applied to compare the occurrence timings between them. The results showed that L-A03 caused significant apoptosis at 12 h, but an obvious autophagy was already observed at 3 h (Fig. 3d). These results demonstrated that L-A03-induced autophagy was an earlier event than apoptosis induction.

Fig. 3
figure 3

Autophagy is induced in L-A03-treated MCF-7 cells. a Cells were cultured with 15 μM L-A03 for 24 h and then observed by fluorescence microscopy with MDC staining (X 200 magnification). b–c The cells were treated with L-A03 (15 μM) for 3, 6, and 12 h. b A positive ratio of MDC staining by flow cytometric analysis was applied to detect autophagy induction. c The protein level of LC3 was detected by western blot analysis. d The subG0/G1 and MDC-positive ratio were measured by flow cytometric analysis after staining with PI and MDC, respectively. (*P < 0.05, **P < 0.01, # P < 0.05 vs. control group)

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NO generation decreases the process of apoptosis

Previous studies reported that NO contributed to apoptosis and autophagy in several tumor cells (Fan et al. 2012; Zang et al. 2012; He et al. 2014). Flow cytometric analysis after DAF-2DA staining indicated that L-A03 induced the generation of NO, and the DAF-2T positive ratio increased in a dose-dependent manner (Fig. 4a). As shown in Fig. 4b, DTT (a NO scavenger), L-NAME and 1400 W (inhibitors of NOS) significantly suppressed the accumulation of NO in L-A03-treated cells. Meanwhile, the activation of caspase-6 and the cleavage of PARP were enhanced by DTT, L-NAME or 1400 W in MCF-7 cells after treatment with L-A03 (Fig. 4c). However, SNP (a NO donor) played an opposite role. It elevated the NO level but attenuated the process of apoptosis (Fig. 4d, e). These results indicated that L-A03-induced NO generation inhibited apoptosis induction in MCF-7 cells.

Fig. 4
figure 4

NO generation decreases the process of apoptosis. a MCF-7 cells were treated with various dose of L-A03 for 24 h followed by flow cytometric analysis of NO changes after DAF-2DA staining. b–c Cells were pretreated with DTT (200 µM), 1400 W (150 µM) or L-NAME (250 µM) for 1 h prior to 15 µM L-A03 treatment for 24 h. b The DAF-2T positive ratio was determined by flow cytometric analysis with DAF-2DA staining. c The protein levels of caspase-6 and PARP were detected by western blot analysis. d–e The cells were pretreated with SNP (5 µM) for 1 h prior to 15 µM L-A03 treatment for 24 h. d The DAF-2T positive ratio was determined by flow cytometric analysis with DAF-2DA staining. E: The protein levels of caspase-6 and PARP were detected by Western blot analysis. (*P < 0.05, **P < 0.01 vs. L-A03-treated group)

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Autophagy blocks NO generation and upregulates apoptosis induction

Since the induction of autophagy was an earlier event than apoptosis (Fig. 3d). CQ (an autophagy inhibitor) was applied to affect the apoptotic ratio in the relationship between autophagy and apoptosis. Flow cytometric and western blot assay suggested that L-A03-induced apoptotic cells were decreased in the presence of CQ (Fig. 5b, c). These results demonstrated that autophagy was indispensable for apoptosis induction. Since NO generation could decrease the process of apoptosis (Fig. 4), CQ was used to investigate the relationship between NO and autophagy. As shown in Fig. 5a, DAF-2T positive ratio was upregulated in the presence of CQ, indicating that autophagy induction blocked NO generation. Subsequently, inhibitors (DTT, L-NAME or 1400 W) or activator (SNP) of NO generation were used to examine the conversion from LC3 I to LC3 II. Interestingly, inhibition or activation of NO generation had no significant effect on autophagy (Fig. 5d, e). These results indicate that autophagy induction is an upstream event for the generation of NO. Taken together, L-A03-induced autophagy contributes to the process of apoptosis via blocking NO generation in MCF-7 cells (Fig. 5f).

Fig. 5
figure 5

Autophagy blocks NO generation and upregulates apoptosis induction. a–c In the absence or presence of 10 µM chloroquine (CQ), cells were incubated with 15 µM L-A03 for 24 h. a The NO level was determined by flow cytometric analysis with DAF-2DA staining. b The cells were stained with PI and measured by flow cytometric analysis. c The protein levels of caspase-6 and PARP were determined by Western blot analysis. d–e The cells were treated with 15 µM L-A03 in the presence or absence of DTT, 1400 W, L-NAME d, and SNP e. The protein level of LC3 was detected by Western blot analysis. Density of protein bands was quantified with Quantity One image software and the results were expressed as average density to β-actin. f A schematic diagram of L-A03-induced autophagy, NO generation and apoptosis. (**P < 0.01 vs. L-A03-treated group, NS: no significant)

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L-A03 had significant activity against cysteine protease falcipain-2 of Plasmodium falciparum (Luo et al. 2012), and our preliminary study also showed that it exhibited high antitumor effect on several cancer cell lines (supplementary Tab. 1). The data suggested that MCF-7 was one of the sensitive tumor cell lines to L-A03. Besides, breast cancer was one of the most common cancers worldwide and the main cause of death by cancer among females (Jemal et al. 2005). Therefore, the anti-tumor mechanism of L-A03 in MCF-7 cells was investigated in this study. The results demonstrated that autophagy, NO generation and apoptosis were involved in L-A03-treated MCF-7 cells. Moreover, autophagy promoted apoptosis induction via repression of NO generation.

The relationship between autophagy and apoptosis is not simple neither in parallel nor anti-parallel. Autophagy not only provides a survival mechanism but leads to cell death. Autophagy is necessary for apoptotic cell death, playing an upstream role in apoptosis induction (Eisenberg-Lerner et al. 2009). In some cases, autophagy plays a protective role against the process of apoptosis (He et al. 2013a, b). In this study, L-A03-induced autophagy contributed to apoptosis induction in MCF-7 cells.

There are many important regulators in the induction of apoptosis and autophagy. It was reported that NO, a highly reactive free radical, is involved in apoptosis and autophagy (Janssen et al. 1997; Swanlund et al. 2008). Several cases showed that NO generation promoted apoptotic cell death (Fan et al. 2012). On the contrary, the generation of NO antagonized silibinin-induced apoptosis in MCF-7 cells (Wang et al. 2010), so did L-A03. NO promotes autophagy induction in one case but inhibits it in another case (Barsoum et al. 2006; Sarkar et al. 2011). Moreover, we reported that autophagy downregulated the synthesis of NO in physalin A-treated human melanoma A375-S2 cells.(He et al. 2014). Surprisingly, L-A03-induced NO generation had no effect on autophagy, but autophagy decreased the level of NO, indicating that autophagy was an upstream event to NO induction. Together, the present results indicate that L-A03 might be a potent antitumor derivative of DHA. Consequently, much further work should be done to clarify mechanisms of the cross-talks among autophagy, NO and apoptosis.

Conclusion

Above results demonstrate that autophagy induction and NO generation deficiency could contribute to apoptotic cell death in L-A03-treated MCF-7 cells, but autophagic cells repress the generation of NO. This present study provides a new insight into the anti-tumor effect of L-A03 on human breast cancer.