Physalin A Induces Apoptotic Cell Death and Protective Autophagy in

Article pubs.acs.org/jnp

Physalin A Induces Apoptotic Cell Death and Protective Autophagy in HT1080 Human Fibrosarcoma Cells Hao He,‡,†,§ Ling-He Zang,‡ Yong-Sheng Feng,‡ Jian Wang,§ Wei-Wei Liu,‡ Li-Xia Chen,†,§ Ning Kang,⊥ Shin-ichi Tashiro,∥ Satoshi Onodera,∇ Feng Qiu,*,†,§ and Takashi Ikejima*,‡ †

Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, People’s Republic of China ‡ China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, People’s Republic of China § Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, People’s Republic of China ⊥ Department of Biochemistry and Molecular Biology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, People’s Republic of China ∥ Institute for Clinical and Biomedical Sciences, Kyoto 603-8072, Japan ∇ Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan ABSTRACT: Physalin A (1) is a withanolide isolated from Physalis alkekengi var. f ranchetii. In this study, the selective growth inhibitory effects on tumor cells induced by 1 were screened, and the mechanism was investigated on 1-induced growth inhibition, including apoptosis and autophagy, in human fibrosarcoma HT1080 cells. Apoptosis induced by 1 in HT1080 cells was associated with up-regulation of caspase-3 and caspase-8 expression. However, there were no significant changes in caspase-9, Bid, Bax, and Bcl-2 expression, indicating that 1-induced apoptosis in HT1080 cells occurs mainly through activation of the death receptor-associated extrinsic apoptotic pathways. Autophagy induced by 1 was found to antagonize apoptosis in HT1080 cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor 3methyladenine (3MA). Loss of beclin 1 (as an autophagic regulator) function led to similar results to 3MA. However, 1 did not show inhibitory effects on normal human cells (human peripheral blood mononuclear cells). Taken together, these results suggest that 1 may be a promising agent for the treatment of cancer.

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effectiveness of these methods varies, and the results are not always satisfactory.9 Thus, novel anticancer drugs to combat fibrosarcoma still need to be found. Programmed cell death (PCD), including two classical forms, apoptosis and autophagy, is a crucial mechanism for maintaining cell homeostasis of multicellular organisms.10 Apoptosis, termed type I PCD, is a process of cellular suicide accompanied with many physiological characteristics including nuclear fragmentation, cell shrinkage, chromatin condensation, membrane blebbing, and the formation of apoptotic bodies.11 The superabundance of apoptosis is often associated with neurodegenerative diseases and may result in autoimmune diseases and tumorigenesis.12 Apoptosis is mediated by a family of caspases (cysteine aspartic specific proteases), with two known pathways leading to caspase activation: the death receptor apoptotic pathway (extrinsic pathway) and the

atural products have been the main source of traditional medicine since ancient times. Today, numerous compounds from natural plants are reported to possess growth inhibitory effects on tumor cells. Physalin A (1) is an active withanolide isolated from Physalis alkekengi var. franchetii (Solanaceae) (Chinese name: “Jindenglong”).1 The calyx of this plant has been used as a traditional Chinese medicine for the treatment of cough, sore throat, hepatitis, eczema, dysuria, and tumors.2 Physalins, the major constituents of P. alkekengi, have been studied during the past 20 years and reported to have immunomodulatory,3 antimalarial,4 anti-inflammatory,5 and antitumor activities.6 Compound 1, one of these withanolides, was reported to inhibit human prostate cancer cell growth,7 but no further investigations have been carried out on its effects on other tumor cells, and the mechanisms underlying the growth inhibitory effects of 1 are still unclear. Fibrosarcomas are malignant tumors that arise mostly in soft tissues.8 The current therapeutic approaches for fibrosarcomas are surgery, radiotherapy, and chemotherapy, but the © 2013 American Chemical Society and American Society of Pharmacognosy

Received: January 7, 2013 Published: May 6, 2013 880

dx.doi.org/10.1021/np400017k | J. Nat. Prod. 2013, 76, 880−888

Journal of Natural Products

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mitochondrial pathway (intrinsic pathway).13 The death receptor pathway is initiated by the ligation of transmembrane death receptors, such as Fas, which recruits FADD (Fasassociated death domain) and then activates downstream caspase-8 and subsequently effector caspase-3. Activation of caspase-3 leads to apoptosis via cleavage of cellular substrates such as PARP (poly-ADP-ribose polymerase) and inhibitors of caspase-activated DNase (ICAD).14 The mitochondrial pathway is associated with changes in the permeability transition of the outer mitochondrial membrane, and this permeability is controlled mainly by the Bcl-2 family of proteins, such as Bax and Bid, through regulating the formation of apoptotic proteinconducting pores in the mitochondrial membrane. Then, the mitochondria release cytochrome c, which functions to activate caspase-9, thereby promoting the activation of effector caspase3, leading to apoptosis.15,16 Autophagy, termed type II PCD, specific to eukaryotic cells, is a dynamic process involving the sequestration of plasmatic portions and intracellular organelles into double-membrane vacuoles called autophagosomes. Autophagy has many crucial physiological functions and can trigger lysosome-dependent protein degradation, organelle turnover, and removal.17 It can be a pro-survival or a prodeath mechanism depending on the circumstances. Autophagy is a physiological process of cellular suicide and also a survival mechanism under starvation, differentiation, or hormonal stimulation. Increasing evidence has indicated that cross-talk between autophagy and apoptosis is common in mammalian cells. Thus, three types of interplay have been elucidated: both apoptosis and autophagy can act as partners in a coordinated or cooperative manner to induce cell death; autophagy promotes cell survival and antagonizes apoptotic cell death; and autophagy acts to enable apoptosis, not leading to death in itself.18 All the interconnections are supported by findings that the two pathways share key molecular regulators. This crosstalk is quite complex, and sometimes contradictory, and critical to the fate of the cell.19 The present study was designed to detect the cell growth inhibitory effects of 1, the cytotoxicity of this compound on normal human cells, and its mechanisms of induced cell death in human fibrosarcoma HT1080 cells.

manner (Figure 1A). However, the inhibitory ratios were different in different cell lines. HT1080 and A375-S2 cells were

Figure 1. Selective inhibitory effects of physalin A (1) in cancer cells and hPBMC cells. (A) The cell inhibitory ratio of 1 in 10 cell lines (HT1080, A375-S2, HepG2, HeLa, A549, U937, HCT116, A431, MCF7, HL60) was measured using an MTT assay. The cells were treated with 1 at 0−80 μM for 24 h. (B) The growth inhibitory activity of 1 in normal human cells (human peripheral blood mononuclear cells, hPBMC) compared with 5-fluorouracil (5-Fu) and paclitaxel was measured using an MTT assay. The hPBMCs were treated with 1, 5Fu, and paclitaxel at 10−80 μM for 24 and 48 h (means ± SD of three independent experiments; *p < 0.05 vs 1 group).

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the most sensitive tumor cell lines among those used. In this study, human fibrosarcoma HT1080 cells were used therefore to investigate the mechanism of 1-induced cell death in tumor cells. In order to ascertain the cytotoxicity of 1 in normal human cells, human peripheral blood mononuclear cells (hPBMC) were prepared and treated with 0, 10, 20, 40, and 80 μM compound 1 for 24 and 48 h. Also, the cells were treated with the same doses of 5-fluorouracil (5-Fu) and paclitaxel as positive controls. The results showed that the cell viability of hPBMC treated with 1 was better than when treated by 5-Fu and paclitaxel at higher doses (40 or 80 μM). At lower doses

RESULTS AND DISCUSSION Growth Inhibition Effects of Physalin A (1) against Several Different Tumor Cell Lines and Normal Cells. To detect the growth inhibition effects of 1 on different human tumor cell lines (HT1080 fibrosarcoma, A375-S2 melanoma, HepG2 hepatoma, HeLa cervical carcinoma, A549 alveolar basal epithelial, U937 histocytic lymphoma, HCT116 colon cancer, A431 epidermoid carcinoma, MCF7 breast cancer, and HL60 promyelocytic leukemia cells), the cells were cultured with 0, 10, 20, 40, and 80 μM compound 1 for 24 h. The results showed that 1 induced tumor cell death in a dose-dependent 881



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Figure 2. Physalin A (1) induces apoptosis in HT1080 cells. (A) The cells were treated with various doses of 1 for 12, 24, 48, or 72 h. The cell inhibitory ratio was measured using an MTT assay (means ± SD of three independent experiments). (B, C) Cells were incubated with 10 μM 1 for 24 h, and the cellular morphological changes were observed by phase-contrast microscopy (B, 200× magnification, bar = 20 μm) or by fluorescence microscopy with AO staining (C, 200× magnification, bar = 20 μm). Arrows indicate apoptotic bodies. (D) Cells were cultured in the presence of 10 μM 1 for 12, 24, and 36 h, and the apoptotic cells stained with PI (sub-G1 fraction) were measured by flow cytometric analysis (n = 3, mean ± SD; **p < 0.01 vs control group).

(10 or 20 μM), the cell viability of hPBMC treated with 1 was also better than or similar to those treated with 5-Fu and paclitaxel (Figure 1B), indicating that the cytotoxicity of 1 is lower than the commonly used anticancer drugs 5-Fu and paclitaxel. Taken together, 1 induced cell growth inhibition selectively in tumor cells and without significant cytotoxicity toward normal human cells, indicating good selectivity. Physalin A (1)-Induced Apoptotic Cell Death in HT1080 Cells. Compound 1 inhibited HT1080 cell growth in a time- and dose-dependent manner with an IC50 value (at 24 h) of 10.7 ± 0.91 μM (Figure 2A). The inhibitory effects of 5-fluorouracil and paclitaxel on HT1080 cells were also screened, with a 24 h IC50 of 70.8 ± 6.62 μM and 6.0 ± 0.23 μM, respectively. To determine the features of HT1080 cell death, morphological changes were observed. Compared with the control group, treatment with 1 caused significant morphological changes, including the appearance of membrane blebbing and granular apoptotic bodies (Figure 2B,C). Flow cytometric analysis showed a significant increase in the

percentage of sub-G1 cells after treatment with 1 (Figure 2D). These results suggested that 1 induces apoptotic cell death in HT1080 cells. Inhibitory Effects and Apoptosis Induced in HT1080 Cells by Physalin A (1) Depend on Caspase-3 and Caspase-8, but Are Not Correlated with Caspase-9. To investigate the role of 1-induced apoptosis, HT1080 cells were pretreated with z-DEVD-FMK (caspase-3 inhibitor, 20 μM), zIETD-FMK (caspase-8 inhibitor, 10 μM), and z-LEHD-FMK (caspase-9 inhibitor, 20 μM) before treatment with 1. The concentrations of these inhibitors were determined according to previous studies.20,21 The cell inhibitory ratio of the 1-treated group was decreased markedly by z-DEVD-FMK and z-IETDFMK, but was without obvious change after a z-LEHD-FMK pretreatment (Figure 3A). Next, it was examined whether the Fas-mediated death pathway is activated in 1-treated cells, and the expression of Fas, FADD, caspase-8, and caspase-3 was detected by Western blot analysis. Fas and FADD expression were elevated markedly. A time-dependent cleavage of 882



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Figure 3. The inhibitory effects and apoptosis induced by physalin A (1) depend on caspase-3 and caspase-8, but are not correlated with caspase-9. (A) The cells were treated with 10 μM 1 for 24 h, in the presence or absence of z-DEVD-FMK (20 μM), z-IETD-FMK (10 μM), or z-LEHD-FMK (20 μM), and the inhibitory ratio was measured using an MTT assay. (B, C) Cells were incubated with 10 μM 1 for 6, 12, 18, and 24 h, followed by Western blot analysis for detection of Fas, FADD, caspase-8, caspase-3, PARP, and ICAD levels (B), and the protein levels of caspase-9, Bid, cytochrome c (Cyto.c), Bax, and Bcl-2 were also detected by Western blot analysis (C). β-Actin was used as an equal loading control. Band density of the specific protein was analyzed with Quantity One image software, and the results are expressed as average density to β-actin (n = 3, means ± SD; *p < 0.05, **p < 0.01 vs 1 group, #p < 0.05, ##p < 0.01 vs control group).

procaspase-3 and procaspase-8 was also observed in 1-treated cells. Moreover, activated caspase-3 also cleaved poly-ADPribose polymerase, which is required for repairing DNA damage.22 In addition, the apoptosis was enhanced further by down-regulation of ICAD, causing the release of caspaseactivated DNase (CAD), which triggered DNA fragmentation in the nuclei (Figure 3B).23 It is known that Bax, Bcl-2, Bid, and caspase-9 play essential roles in the mitochondrial apoptotic pathway.15,16 Caspase-8 cleaves cytosolic Bid to truncated tBid, which is translocated to the mitochondria, resulting in the release of cytochrome c. The latter normally functions with other molecules to form a caspase-9-activating complex, which induces apoptosis in the caspase-dependent pathway. The rise of the Bax to Bcl-2 ratio also results in apoptosis. Bcl-2 is a potent inhibitor of apoptotic cell death, while Bax accelerates apoptosis by contributing to permeabilization of the outer mitochondrial membrane. In this

study, no apparent change was observed in the expression levels of pro-caspase-9, and the cleaved form of caspase-9 also could not be detected in 1-treated HT1080 cells. Meanwhile, there was no change of cytosolic Bid and cytochrome c expression; changes of Bax and Bcl-2 were also minimal, all the results indicating that 1-induced apoptosis might occur through a caspase-9-independent pathway (Figure 3C). Physalin A (1) Time Dependently Induced Autophagy in HT1080 Cells. To determine the effect of 1 on autophagy in HT1080 cells, the cells were observed with monodansylcadaverine (MDC) staining. Compared with the control group, 1 treatment caused a marked increase in the number of MDClabeled autophagolysosomes in the cells. Flow cytometric analysis also showed that the MDC-positive cells caused by treatment with 1 were increased in a time-dependent manner (Figures 4A,B). Western blot analysis revealed that the upregulation of beclin 1 and the conversion from LC3 I to LC3 II 883



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Figure 4. Physalin A (1) induces autophagy in HT1080 cells. (A) Cells were cultured with 10 μM 1 for 24 h and then observed by fluorescence microscopy with MDC staining (C, 200× magnification, bar = 20 μm). Arrows indicate cells containing autophagolysosomes. (B) The cells were treated with 10 μM 1 for 12, 24, and 36 h, and quantitative analysis detected a positive ratio of MDC staining by flow cytometric analysis, n = 3, means ± SD. (C) HT1080 cells were lysed after treatment with 10 μM 1 for 6, 12, 18, and 24 h, and the protein levels of beclin 1 and LC3 were detected by Western blot analysis. β-Actin was used as an equal loading control. Band density of the specific protein was analyzed with Quantity One image software, and the results are expressed as average density to β-actin (n = 3, means ± SD; **p < 0.01 vs control group).

were detected in 1-treated cells in a time-dependent manner. These results demonstrated that 1 induces autophagy in HT1080 cells (Figure 4C). Inhibition of Autophagy with 3MA Increased Apoptosis, while Augmentation of Autophagy by Rapamycin Decreased Apoptosis in Physalin A (1)-Treated HT1080 Cells. Compound 1 is therefore able to induce apoptosis and autophagy in HT1080 cells. To find out the relationship between apoptosis and autophagy, the specific autophagic inhibitor 3-methyladenine (3MA) and the autophagic agonist rapamycin were applied. An MTT assay showed that 1 inhibited cell growth significantly, but this inhibition was partially augmented by 3MA and was reversed by rapamycin (Figure 5A). Compared with the group treated by 1 alone, the morphological change of autophagic vacuole formation and autophagy ratio analyzed by flow cytometry were markedly decreased in the presence of 3MA, while they were increased by pretreatment with rapamycin. No change was observed in the group treated by 3MA and rapamycin alone (Figure 5B,C). As for apoptosis, flow cytometric analysis showed that 3MA pretreatment caused a marked increase, whereas rapamycin pretreatment induced a significant decrease in the apoptotic ratio compared with the group treated by 1 (Figure 5D). The effect of 3MA and rapamycin on 1-induced autophagy was

detected by beclin 1 expression and LC3 conversion, which are autophagic markers. The up-regulation of beclin 1 levels and the conversion from LC3 I to LC3 II were reversed by 3MA but enhanced by rapamycin in 1-treated HT1080 cells (Figure 5E). Subsequently, the influence of 3MA and rapamycin on 1induced apoptosis was examined also by the detection of apoptosis-related proteins. Treatment with 1 elevated the expression level of Fas and FADD, whereas these enhancements were further up-regulated by 3MA but down-regulated by rapamycin pretreatment. The cleavage of procaspase-3, procaspase-8, and PARP was observed also in 1-treated cells, and 3MA pretreatment enhanced the cleavage, whereas rapamycin pretreatment decreased cleavage. In addition, the down-regulation of ICAD induced by 1 treatment was augmented by 3MA and suppressed by rapamycin (Figure 5F). Taken together, 3MA suppressed and rapamycin augmented autophagy induced by 1. In turn, 3MA increased and rapamycin decreased the apoptotic ratio, indicating that autophagy can antagonize apoptosis in 1-treated HT1080 cells. Autophagy Antagonized Apoptosis in Physalin A (1)Treated HT1080 Cells. To elucidate the role of autophagy in 1-induced apoptosis, the expression of the autophagy-associated marker protein beclin 1 in HT1080 cells was knocked down by application of short interfering RNA (siRNA) [Figure 6A (a)]. 884



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Figure 5. The inhibitory effects and apoptotic ratio are enhanced by 3MA but inhibited by rapamycin in physalin A (1)-treated HT1080 cells. (A) Cells were incubated with 10 μM 1 in the presence or absence of 3MA (1 mM) or rapamycin (1 nM) for 24 h, and the inhibitory ratio was measured using an MTT assay. (B−D) Cells were pretreated with 3MA (1 mM) or rapamycin (1 nM) for 1 h prior to 10 μM 1 treatment for 24 h; then the autophagolysosome was observed by fluorescent microscopy of MDC staining (B). The MDC positive ratio was determined by flow cytometric analysis with MDC staining (C). The apoptotic ratio was determined by flow cytometric analysis with PI staining (D). (E) Effects of 3MA (1 mM) and rapamycin (1 nM) on expression of beclin 1 and conversion from LC3 I to LC3 II detected by Western blot analysis. (F) The cells were treated with 1 mM 3MA and 1 nM rapamycin for 1 h and then co-incubated with 1 for another 24 h. Protein levels of Fas, FADD, caspase-8, caspase-3, PARP, and ICAD were detected by Western blot analysis. β-Actin was used as an equal loading control. Band density of the specific protein was analyzed with Quantity One image software, and the results are expressed as average density to β-actin (n = 3, means ± SD; *p < 0.05, **p < 0.01 vs 1 group, #p < 0.05, ##p < 0.01 vs control group). 885



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Figure 6. Physalin A (1)-induced autophagy antagonized apoptosis in HT1080 cells. (A) (a) HT1080 cells were transfected with either nonspecificsiRNA (NS-siRNA) or beclin 1-siRNA, and beclin 1 protein was detected 24 h after transfection by Western blot analysis. (b) The transfected HT1080 cells were treated with 1 for 24 h and stained with MDC, then examined by flow cytometric analysis. (B) The transfected cells were incubated with 10 μM 1 for 24 h, and the apoptotic ratio was determined by flow cytometric analysis with PI staining. (C) (a) The transfected HT1080 cells were lysed after treatment with 10 μM 1 for 24 h; then the expression of beclin 1 and conversion from LC3 I to LC3 II were detected by Western blot analysis. (b) The protein levels of Fas, FADD, caspase-8, caspase-3, PARP, and ICAD were also detected by Western blot analysis. β-Actin was used as an equal loading control. Band density of the specific protein was analyzed with Quantity One image software, and the results are expressed as average density to β-actin (n = 3, means ± SD; *p < 0.05, **p < 0.01 vs NS-siRNA control group, #p < 0.05, ##p < 0.01 vs 1 with NSsiRNA group). 886



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Growth Inhibition Assay. HT1080, A375-S2, HepG2, HeLa, A549, U937, HCT116, A431, MCF7, and HL60 cells were incubated in 96-well cell culture clusters (Corning, NY, USA) at a density of 0.8 × 104 cells per well and cultured for 24 h. Thereafter, the cells were treated with various concentrations of 1 for the indicated time periods. In addition, HT1080 cells were treated with or without z-DEVD-FMK, z-IETD-FMK, z-LEHD-FMK, 3MA, or rapamycin at a given concentration for 1 h prior to 1 treatment for 24 h. After this, for adhesion cell lines, the cells were rinsed with ice-cold PBS twice and incubated with 5.0 mg/mL MTT solution at 37 °C for 2 h, and the resulting crystals were dissolved in 150 μL of DMSO. For suspension cell lines, 20 μL of MTT solution was added and the cells were incubated for an additional 2 h at 37 °C. The plate was then centrifuged at 2000g (DL-4000B; An Ting, Shanghai, China) for 10 min, the supernatant was discarded, and the deposited formazan formed in the cells was dissolved with 150 μL of DMSO. All optical densities were measured in the MTT assay using a microplate reader (Thermo Multiskan MK3, Thermo Scientific, Helsinki, Finland). The percentage of cell growth inhibition was calculated as follows:

Beclin 1 knockdown led to a decreased level of autophagy (marked by MDC staining) [Figure 6A (b)]. After transfection, the apoptotic ratio was increased significantly in the 1-treated beclin 1-siRNA group when compared to the nonspecific siRNA (NS-siRNA) group (Figure 6B). The changes at the protein level were also evaluated. 1-induced up-regulation of beclin 1 was reversed after transfection, and the conversion from LC3 I to LC3 II was also suppressed, demonstrating that the autophagy induced by 1 was markedly inhibited [Figure 6C (a)]. Next, the loss of beclin 1 further enhanced 1-induced upregulation of Fas and FADD, and the cleavage of procaspase-3, procaspase-8, and PARP was also augmented in 1-treated cells. Otherwise, the down-regulation of ICAD caused by 1 treatment was enhanced compared to the NS-siRNA group [Figure 6C (b)]. These results further demonstrated autophagy antagonized apoptosis in HT1080 cells treated with 1. In this investigation, 1 showed significant inhibition on the tumor cell line used. Also, the cytotoxicity of 1 on normal human cells (hPBMC) was lower than the commonly used anticancer drugs 5-Fu and paclitaxel. Therefore, 1 can be considered a lead compound for anticancer drug development.

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cell growth inhibition (%) = (A492control − A492sample)/(A492control − A492blank ) × 100 Preparation of Human Peripheral Blood Mononuclear Cells. Fresh human peripheral blood were collected from three healthy male volunteer subjects by the General Hospital of Shenyang Military Region; all the subjects involved gave written consent, and the study was approved by the Ethics Committee of the General Hospital of Shenyang Military Region (201-LS-No.012). The fresh blood samples were separated using 50 mL sterile conical centrifuge tubes. Each was diluted 1:3 in PBS and mixed well. Ficoll-Hypaque solution was slowly layered underneath the blood/PBS mixture by placing the tip of the pipet containing the solution at the bottom of the sample tube; 3 mL of Ficoll-Hypaque solution per 10 mL of blood/PBS mixture was used. Then, this was centrifuged for 30 min at 900g. The hPBMC were harvested by careful pipetting of the corresponding density band and washed in PBS (400g, 10 min) to remove platelets. The hPBMC were resuspended in complete RPMI-1640, and the cells were counted and incubated in 96-well culture clusters, followed by an MTT assay.25,26 Observation of Morphological Changes. HT1080 cells were seeded into 24-well culture plates (Corning, NY, USA) with or without 1 for 24 h. The cellular morphology was observed by using a phase contrast microscope (Leica, Nussloch, Germany). Fluorescent Microscopy of Apoptotic (with AO) and Autophagy (with MDC) Staining. HT1080 cells (2 × 104 cells/ well) were inoculated in 24-well culture plates and cultured for 24 h. Then, the cells were treated with or without 3MA or rapamycin at the given concentration for 1 h prior to 1 treatment for 24 h. After this, the cells were stained with 20 μg/mL AO in a dark place for 15 min or stained with 0.05 mM MDC (a marker for autophagic vacuoles27) at 37 °C for 1 h. Fluorescent changes were observed by an Olympus IX70 reverse fluorescence microscope (Olympus, Tokyo, Japan). siRNA Transfection. One day before transfection, 5 × 105 HT1080 cells were inoculated in a 75 mL culture bottle and incubated overnight. Then, 48 μL of siRNA duplex was diluted with 800 μL of diluent, and 16 μL of TranSmarter was diluted with 800 μL of the same diluent. The siRNA solution was added to the TranSmarter solution dropwise, mixed by pipet, and then incubated at room temperature for 45 min. The cells were washed once with the same diluent, and the mixture was added dropwise into the culture and mixed by gentle shaking of the flask. The cells were incubated at 37 °C, 5% CO2, for 7 h; then the transfection mixture was removed and replaced with normal growth medium. The transfected cells were maintained for 24 h before harvesting. Flow Cytometric Analysis of Apoptosis and Autophagy. HT1080 cells were dispensed in a 25 mL culture bottle at a density of 3 × 105 per bottle and then incubated for 24 h. Then, the cells were treated with or without 3MA or rapamycin at given concentrations 1 h prior to the 1 treatment for the indicated time periods. The cells were harvested and rinsed with PBS. For measuring apoptosis, the collected

EXPERIMENTAL SECTION

General Experimental Procedures. Compound 1 was isolated from Physalis alkekengi var. f ranchetii in our laboratory1 and was identified by comparing its physical and spectroscopic (1H NMR, 13C NMR) data with those reported in the literature.24 The purity was measured by HPLC [column: Aglient Zorbax SB-C18, 4.6 × 150 mm, 5 μm; solvent phase: methanol−H2O (60:40)] and determined to be 98.0%. Compound 1 was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution and diluted in RPMI 1640 medium or Minimum Essential Medium (MEM) before the experiments. The DMSO concentration was kept below 0.05% in all cell cultures used and did not exert any detectable effect on cell growth or cell death. FicollHypaque solution (density 1.077 g/mL) was purchased from Tianjin Chuanye Biological Products (Tianjin, People’s Republic of China). Fetal bovine serum (FBS) was obtained from TBD Biotechnology Development (Tianjin, People’s Republic of China); RPMI 1640 medium and MEM medium were obtained from Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), monodansylcadaverine (MDC), acridine orange (AO), 3-methyladenine (3MA), and rapamycin were purchased from Sigma (St. Louis, MO, USA). Caspase-3 inhibitor (zDEVD-FMK), caspase-8 inhibitor (z-IETD-FMK), and caspase-9 inhibitor (z-LEHD-FMK) were obtained from Calbiochem (La Jolla, CA, USA). Nonspecific-siRNA and beclin 1-siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TranSmarter was from Abmart (Shanghai, People’s Republic of China). Polyclonal antibodies against Fas, FADD, caspase-3, caspase-8, caspase-9, PARP (poly-ADP-ribose polymerase), ICAD (inhibitor of caspase-activated DNase), β-actin, Bax, Bcl-2, Bid, cytochrome c, LC3, beclin 1, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrochemiluminescence (ECL) reagent was from Thermo Scientific (Rockford, IL, USA). Cell Culture. Human fibrosarcoma HT1080 cells, human melanoma A375-S2 cells, human hepatoma HepG2 cells, human cervical carcinoma HeLa cells, adenocarcinomic human alveolar basal epithelial A549 cells, human histocytic lymphoma U937 cells, human colon cancer HCT116 cells, human epidermoid carcinoma A431 cells, human breast cancer MCF7 cells, and human promyelocytic leukemia HL60 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in RPMI 1640 or MEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2 in a humidified atmosphere. The cells in the exponential phase of growth were used in the experiments. 887



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cells were fixed with 500 μL of PBS and 10 mL of 70% ethanol at 4 °C for 18 h; then after washing twice with PBS, they were suspended with 1 mL of PI solution (PI 50 μg/mL and RNase A 1 mg/mL). For measuring autophagy, collected cells were suspended with 0.05 mM MDC at 37 °C for 1 h. Then the samples were analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Western Blot Analysis. HT1080 cells were treated with 10 μM 1 for the indicated time periods or treated with 10 μM 1 in the presence or absence of 3MA or rapamycin for 24 h. Both adherent and floating cells were collected and lysed in lysis buffer (50 mM Hepes pH 7.4, 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM egtazic acid, 1 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin) at 4 °C for 60 min. Lysates were centrifuged at 12000g for 15 min, and the supernatants were determined by the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein were separated by 12% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. The membranes were soaked in blocking buffer (5% skim milk). Proteins were detected with the primary antibodies against Fas, FADD, caspase-3, caspase-8, caspase-9, PARP, ICAD, β-actin, Bid, cytochrome c, Bax, Bcl-2, LC3, and beclin 1, followed by HRP-conjugated secondary antibody, and visualized by using ECL as the HRP substrate. Band density of the specific protein was analyzed with Quantity One image software. Statistical Analysis. All the present data and results were confirmed in at least three independent experiments and were expressed as means ± SD. Statistical comparisons were made using Student’s t-test and one-way ANOVA followed by Tukey’s post hoc test. p < 0.05 was considered to represent a statistically significant difference.

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AUTHOR INFORMATION

Corresponding Author

*(F. Qiu) Tel: +86 24 23986463. Fax: +86 24 23993994. Email: [email protected]. (T. Ikejima) Tel: +86 24 23844463. Fax: +86 24 23844463. E-mail: [email protected]. com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No. 31270399 and No. 81274039) and the Fund of the Educational Department of Liaoning Province, People’s Republic of China (L2011174 and L2011177).

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