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Dihydrotanshinone I induced apoptosis and autophagy through caspase dependent pathway in colon cancer
Phytomedicine 22 (2015) 1079–1087
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Dihydrotanshinone I induced apoptosis and autophagy through caspase dependent pathway in colon cancer Lin Wang a,1,∗, Tao Hu a,1, Jing Shen a, Lin Zhang a, Ruby Lok-Yi Chan a, Lan Lu a, Mingxing Li a, Chi Hin Cho a, William. Ka Kei Wu b a School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Lo Kwee-Seong Integrated Biomedical Sciences Building, Shatin, NT, Hong Kong, China b Department of Anaesthesia and Intensive Care, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
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Article history: Received 2 June 2015 Revised 7 August 2015 Accepted 8 August 2015
Chemical compounds studied in this article: Dihydrotanshinone I (PubChem CID: 11425923) Keywords: Dihydrotanshinone I Apoptosis Autophagy Caspase Mitochondria Colon cancer
a b s t r a c t Background: Dihydrotanshinone I (DHTS) was previously reported to exhibit the most potent anti-cancer activity among several tanshinones in colon cancer cells. Its cytotoxic action was reactive oxygen species (ROS) dependent but p53 independent. Purpose: To further study the anti-cancer activity of DHTS and its molecular mechanisms of action in colon cancer both in vitro and in vivo. Methods: Caspase activity was detected by fluorescence assay. Apoptosis was detected by flow cytometry and TUNEL assay. Protein levels were analyzed by western blotting. Knockdown of target gene was achieved by siRNA transfection. Formation of LC3B puncta and activation of caspase-3 were detected by confocal fluorescence microscope. In vivo anti-colon cancer activity of DHTS was observed in xenograft tumors in NOD/SCID mice. Results: Anti-colon cancer activity of DHTS by inducing apoptosis and autophagy was observed both in vitro and in vivo. Mitochondria mediated caspase dependent pathway was essential in DHTS-induced cytotoxicity. The apoptosis induced by DHTS was suppressed by knockdown of apoptosis inducing factor (AIF), inhibition of caspase-3/9 but was increased after knockdown of caspase-2. Meantime, knockdown of caspase-2, pretreatment with Z-VAD-fmk or NAC (N-Acety-L-Cysteine) efficiently inhibited the autophagy induced by DHTS. A crosstalk between cytochrome c and AIF was also reported. Conclusion: DHTS-induced caspase and ROS dependent apoptosis and autophagy were mediated by mitochondria in colon cancer. DHTS could be a promising leading compound for the development of anti-tumor agent or be developed as an adjuvant drug for colon cancer therapy. © 2015 Elsevier GmbH. All rights reserved.
Introduction The search for effective regimens with minimal adverse effects for the treatment of colon cancer remains the top priority of cancer research. So far, a number of traditional Chinese medicinal preparations and their components have been reported to exhibit promising anticancer activities, they are potential candidates for anti-cancer drug development. Meanwhile, the mechanisms of beneficial preventive and therapeutic effects achieved by traditional and complementary medicine are currently being deciphered in molecular medicine. Our
Abbreviations: DHTS, dihydrotanshinone I; ROS, reactive oxygen species; DAPI, 4 , 6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP Nick-end Labeling; O.C.T, optimal cutting temperature. ∗ Corresponding author. Tel.: +852 3943 5722; fax: +852 2603 5139. E-mail address: [email protected], [email protected] (L. Wang). 1 Lin Wang and Tao Hu contributed equally to this work. http://dx.doi.org/10.1016/j.phymed.2015.08.009 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
preliminary in vitro studies showed that several tanshinones had potent anti-cancer activity in various colon and liver cancer cells and dihydrotanshinone I (DHTS) was the most potent compound (Wang et al., 2013). The anti-cancer activity of DHTS was reactive oxygen species (ROS) but not p53 dependent. However, the underlying mechanisms of cytotoxic action of DHTS are still not well understood. Programmed cell death, which has been recognized since the 1960s, is any type of cell death during which the cell uses specialized intracellular machinery to kill itself. Two important types of programmed cell death are apoptosis and autophagy. Indeed, current anticancer treatments, including many chemotherapeutic agents as well as ionizing radiation therapy, actually activate apoptosis to utilize the apoptotic machinery to kill cancer cells. However, autophagy can both stimulate and prevent cancer depending on the context. On one hand, cancer cells may utilize autophagy to survive with altered metabolism in the hostile tumor microenvironment, suggesting the potential of autophagy inhibition in cancer therapy. On the other
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hand, high levels of autophagy might directly lead to autophagic cell death in cancers. Apoptosis can be mediated by extrinsic and/or intrinsic pathways, and caspase activation was observed in both pathways (Ouyang et al., 2012). Caspases are synthesized as inactive preforms and cleave to aspartate residues upon activation. Caspases can be divided into two distinct groups, the initiator caspases including caspase-8 and -9 as well as the executioner caspases, such as caspase-3 and -7. Initiator caspases are present in the cell as inactive monomers and their activation is promoted by dimerization, which happens when initiator caspases are recruited to large molecular weight protein complexes that act as signaling platforms (Fan et al., 2005; Lamkanfi and Kanneganti, 2010; Tait and Green, 2010; Woltering, 2010). Caspase2 has been reported as an initiator caspase, its role is very special in apoptosis since caspase-2 gene produces several alternative splicing isoforms (Bouchier-Hayes and Green, 2012). The inclusion of exon 9 leads to an in-frame stop codon in caspase-2 short isoform (casp-2S) mRNA, thus producing a truncated protein that inhibits cell death. Whereas the exclusion of exon 9 results in caspase-2 long isoform (casp-2 L) mRNA, which produces protein product inducing cell death (Brynychova et al., 2013; Han et al., 2013; Iwanaga et al., 2005; Puccini et al., 2013). Caspases cleave a number of different substrates in the cytoplasm or nucleus, leading to many morphologic features of apoptotic cell death. Activation of caspases can be initiated from different entry points, such as the plasma membrane upon ligation of death receptor (receptor mediated pathway) and the mitochondria (mitochondria-mediated pathway), etc. The mitochondrial pathway is initiated by the release of apoptotic factors such as cytochrome c, apoptosis inducing factor (AIF) and endonuclease G from the mitochondrial intermembrane space by mitochondrial outer membrane permeabilization (MOMP), which is a complex process that involves numerous molecular players including Bcl-2 family (Brenner and Grimm, 2006; Kuwana and Newmeyer, 2003). The release of cytochrome c into the cytoplasm subsequently triggers caspase-3/7 activation through formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex. In this study, the anti-cancer activity of DHTS and its molecular mechanisms of action in colon cancer were investigated. DHTS was reported to induce apoptosis and autophagy in colon cancer both in vitro and in vivo. Caspase activation accompanied by the crosstalk between AIF and cytochrome c played the dominant role in DHTSinduced cytotoxicity.
Cells were treated with various concentrations of DHTS (3.13– 20 μM) for 48 h. For the activity assay, Ac-DEVD-AMC (1 μg/μl), Ac-IETD-AMC (1 μg/μl) or Ac-LEDH-AMC (1 μg/μl) and cell lysate were added into Protease Assay Buffer in 96-well plate. Reaction mixtures with lysis buffer were used as negative controls. Cells treated with DMSO (0.1%) were treated as vehicle control. The reaction mixtures were incubated for 1 h at 37 °C. The AMC liberated from the substrates was measured using spectrofluorometer of Victor 2 plate reader (Perkin Elmer, Massachusetts, USA) with an excitation wavelength of 380 nm and an emission wavelength of 430 nm.
Materials and methods
Detection of AIF, cytochrome c, Bax, Bcl-xl, LC3B-I/II and p62
Materials
Expression of AIF, cytochrome c, Bax, Bcl-xl, LC3B-I/II and p62 was detected by western blotting using routine method. In brief, samples from cell culture or tissue were harvested and lysed in protein lysis buffer at 4 °C for 30 min. After lysis, samples were centrifuged at 16,000 × g at 4 °C for 20 min. The protein in the supernatant was collected and measured with Pierce® BCA Protein Assay Kit according to the manufacturer’s protocol. Proteins were subjected to SDS– PAGE (8–12%) and detected by western blotting. To detect the role of ROS in caspase-3 cleavage and LC3B-II, cells were pretreated with NAC (2 mM) for 1 h. To detect the autophagic flux, cells were cotreated with bafilomycin A1 (BAF) (100 nM), a blocker of autophagic degradation. Equal amount of proteins were resolved by SDS–PAGE followed by a standard immunoblotting procedure and developed using an ECL development kit (GE healthcare, UK limited).
HPLC grade authentic standard of DHTS was purchased from Chengdu Congon Bio-tech Co., Ltd. (Sichuan, China). Z-VAD-FMK (pan caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), Ac-DMQD-CHO (caspase-3 inhibitor IV) and Z-LEHD-FMK (caspase-9 inhibitor) were from Calbiochem (Darmstadt, Germany). Ac-DEVD-AMC (caspase3/7 substrate), Ac-IETD-AMC (caspase-8 substrate) and Ac-LEDHAMC (caspase-9 substrate) were from EMD Millipore (Darmstadt, Germany). NucBuster TM Protein Extraction Kit was from EMD Biosciences (Darmstadt, Germany). Mitochondria extraction kit for cells was from Millipore (Billerica, MA). Primary antibody of GAPDH was purchased from CHEMICON. Primary antibody of AIF was from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were from Cell Signaling. FlexiTube siRNA for human AIFM1 (Gene accession: NM_001130846), HS-Cap2-10 (Gene accession: NM_001224) and All Stars Neg. SiRNA AF 555 were purchased from QIAGEN Science (Germantown, Germany). Jetprime Transfection reagent was from Polyplus transfection SA (St. Louis, MO, Illkirch FRANCE). Antibodies of Alexa-Fluor 488 goat anti-rabbit, Alexa-Fluor 488 goat anti-mouse, Alexa-Fluor 555 donkey anti-mouse were obtained from Molecular
Probes (Eugene, OR). Unless otherwise specified, all chemicals used in this study were purchased from Sigma (St. Louis, MO). Cell culture Human colon cancer HCT116 cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD) and was routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; GIBCO/BRL, NY), 100 mg/l penicillin G and 100 U/ml streptomycin sulfate at 37 °C in 5% CO2 . RNA interference The expression of AIF or caspase-2 was efficiently lowered using predesigned target-specific siRNA purchased from Qiagen Science. FlexiTube siRNA was transfected into cells using JetprimeTM (Polyplus-transfection Inc.) following the protocol provided. All Stars Neg. siRNA AF 555 served as sham control siRNA. Apoptosis analysis Cells were seeded and cultured overnight in 24-well plate. DHTS were then added into the medium except the control and vehicle control groups. Cells with 0.1% (v/v) DMSO served as vehicle control. Apoptosis was detected after 24 h treatment by flow cytometry. To verify the role of AIF and caspase-2 in the apoptotic activity of DHTS, apoptosis in cells with AIF or caspase-2 knockdown was determined. The role of caspases in apoptotic activity of DHTS was also defined. In this regard, cells were pretreated with Z-VAD-fmk, Z-IETD-FMK or Z-LEHD-FMK (40 μM) for 1 h before DHTS incubation. Activity assay of caspases
Detection for the translocation of cytochrome c, AIF, Bax, Bcl-xl and RAPR cleavage Cell fractions including mitochondrial protein, cytoplasmic protein and nuclear protein were extracted according to protocols provided by respective kits. For isolation of whole-cell protein, cells were
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Fig. 1. Caspase dependent apoptosis induced by DHTS in HCT116 cells. (A) DHTS induced concentration and ROS dependent caspase activation. Ac-DEVD-AMC (substrate of caspase3/7), Ac-IETD-AMC (substrate of caspase-9) or Ac-LEDH-AMC (substrate of caspase-8) and cell lysate were added into Protease Assay Buffer in 96-well culture plate. Measure the 7-amino-4-methylcoumarin (AMC) liberated using spectrofluorometer (Victor 2 plate reader, Perkin Elmer, Massachusetts, USA) with an excitation wavelength of 380 nm and an emission wavelength of 430 nm. The representative results from three independent experiments were shown. Each value was presented as Mean ± S.E.M of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicated significant difference among treatments. Caspase-3 cleavage was detected by western blotting following routine method with GAPDH as an internal control. The representative results were shown; (B) Apoptosis induced by DHTS was completely prevented by Z-VAD-fmk; (C) Apoptosis induced by DHTS was significantly inhibited by pretreatment of Z-LEHD-fmk but only was partially inhibited by Z-IETD-fmk; D. Apoptosis induced by DHTS was significantly increased by caspase-2 knockdown. Cells treated with 0.1% (v/v) DMSO served as vehicle control. After treatment with DHTS (6.25 μM) for 24 h, cells were collected and resuspended in binding buffer. To evaluate the effect of caspase activation on the apoptosis induced by DHTS, cells were pretreated with 40 μM of Z-VAD-fmk (pan caspase inhibitor), Z-LEHD-fmk (caspase-9 inhibitor) or Z-IETD-fmk (caspase-8 inhibitor) for 1 h. To evaluate the role of caspase-2 in apoptotic activity of DHTS, apoptosis were detected in HCT116 cells with caspase-2 knockdown. Apoptosis in colon cancer cells were analyzed by flow cytometry (BD LSRFortessa Cell Analyzer) by double stained with FITC Annexin V and PI. Percentage of the cells in early apoptosis (FITC+/PI-) in each group was calculated and compared using Flowjo7.6.1 software. Each value was presented as Mean ± S.E.M of three independent experiments. ∗∗∗P < 0.001 indicated significant difference among treatments.
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Detection of the translocation of cytochrome c and AIF by immunofluorescence The translocations of cytochrome c and AIF were further observed by immunofluorescence as previously described. Briefly, cells were fixed with 4% formalin and incubated with primary antibody followed by a secondary fluorescent antibody. Alexa Fluor anti-mouse 568 or Alexa Fluor anti-rabbit 488 were used as a secondary antibody. In addition, 4 , 6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. Expression of cytochrome c and AIF was evaluated with an Olympus FV1000 laser confocal microscope (Olympus, Richmond Hill, ON, Canada). Animal experimentation All experiments were performed under Laboratory Animal Ethics Committee approval (The Chinese University of Hong Kong; Ref No: 12/085/MIS-5). Xenograft tumors were established in male NOD/SCID mice (6–7-week-old, Jackson Laboratories) by s.c. injections of 5 × 106 HCT116 cells into lower back areas of the mice. Monitoring of tumor growth was performed twice per week. The tumor size was measured by a caliper as length × width2 /2. When tumor sizes reach 100 mm3 at day 14 after injection, mice were randomly assigned into two groups. DHTS therapy was administered i.p. every other day as a single dose of 10 mg/kg for 19 days. The mixture of 4% DMSO and 0.1% PEG800 was administered i.p. to mice as the vehicle control group. Body weight and general conditions of mice were assessed at the times of drug administration. Finally, all mice were sacrificed and underwent necropsy. Tumors were removed and weighed, fixed in 4% paraformaldehyde and embedded in paraffin or O.C.T (optimal cutting temperature) and stored at –80 °C for further analyses. Determination of cell apoptosis in xenograft of colon cancer by Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling (TUNEL) TUNEL assay was performed with the Dead End Tm Colorimetric TUNEL System (Roche) following the protocol provided. Sections embedded in paraffin were observed with a Olympus FV1000 laser confocal microscope (Olympus, Richmond Hill, ON, Canada). The percentage of TUNEL positive cells was calculated in at least four fields for each slide.
Fig. 2. Pro-cell death of autophagy induced by DHTS in HCT116 cells. (A) DHTS induced concentration-dependent LC3B-II accumulation; (B) DHTS induced time-dependent LC3B-II accumulation and p62 decrease; (C) LC3B-II accumulation induced by DHTS was decreased by pretreatment of NAC or Z-VAD-fmk; (D) LC3B-II accumulation induced by DHTS was decreased after caspase-2 knockdown; (E) Autophagic flux was increased by DHTS after 24 h as determined by LC3-II turnover assay; (F) Inhibition of autophagy by 3-MA increased the viability of colon cancer cells. The expression of LC3BII and p62 was determined by western blotting following routine method. To detect the role of ROS and caspase in LC3B-II accumulation, cells were pretreated with NAC (2 mM) for 1 h. To detect the autophagic flux, cells were cotreated with bafilomycin A1 (BAF) (100 nM). Viability of colon cancer cells was determined by MTT assay. Each value was presented as Mean ± S.E.M of three independent experiments. ∗∗ P < 0.01, compared with the DHTS alone group.
Detection of LC3B-II puncta and cleaved caspase-3 by immunofluorescence For the fluorescence staining of tissue sections, slides were incubated with primary antibody followed by a secondary fluorescent antibody. Alexa Fluor anti-rabbit 488 was used as the secondary antibody. In addition, 4 , 6-diamidino-2-phenylindole (DAPI) was employed to stain cell nuclei. The mounted slides were subjected to microscopic analysis under Olympus FV1000 laser confocal microscope (Olympus, Richmond Hill, ON, Canada). The percentage of the positive cells was evaluated in at least four fields for each slide. Statistical analysis
harvested in lysis buffer containing proteinase and phosphatase inhibitors. Translocation of cytochrome c, AIF, Bax and Bcl-xl was detected in respective cell fractions. To identify the role of caspase in translocation of AIF, cells were pretreated with Z-VAD-FMK (40 μM) for 1 h. Release of cytochrome c and PARP cleavage were also observed in cells with AIF knockdown. Equal amount of proteins were resolved by SDS–PAGE followed by a standard immunoblotting procedure. GADPH, Lamin A/C and cytochrome oxidase subunit IV (Cox IV) were used as loading reference for cytoplasmic protein, nuclear protein and mitochondrial protein, respectively.
Results were expressed as the mean ± SEM. Statistical analysis was performed with an Analysis of Variance (ANOVA) followed by Dunnett test or Tukey’s t-test. P values less than 0.05 were considered to be statistically significant. Results DHTS induced caspase dependent apoptosis in colon cancer cells DHTS induced a concentration-dependent activation of caspase3, caspase-9 but not caspase-8. Activation of caspase-3 was found
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Fig. 3. DHTS induced significant translocation of cytochrome c, AIF, Bax and Bcl-xl. (A) Time-dependent translocation of cytochrome c induced by DHTS; (B) Time-dependent translocation of AIF induced by DHTS; (C) Time-dependent translocation of cytochrome c and AIF was detected by immunofluorescence; (D) Time-dependent translocation of Bcl-xl and Bax induced by DHTS; (E) Translocation of Bax to mitochondria and Bcl-xl to cytosol observed at 24 h after treatment. HCT116 cells were treated with DHTS (20 μM) for different timepoints. Protein of cell fractions was isolated using Mitochondria extraction kit and NucBuster Protein Extraction Kit according to the manufacturer’s protocol. Expression of AIF, cytochrome c, Bcl-xl and Bax in the cell fractions was detected by western blotting following the routine method. Expression of GAPDH, COXIV and LaminA/C was determined as loading reference for cytosolic protein, mitochondrial protein and nuclear protein, respectively. The gray value of the protein band was analyzed by Quantity one and the relative expression of AIF and cytochrome c was statistically analyzed. #p < 0.05, ##p < 0.01, ###p < 0.001 versus control. For immunofluorescence detection, cells were treated with DHTS (20 μM) for certain hours before the termination of incubation. To evaluate the role of Z-VAD-fmk in the translocation of AIF and cytochrome c, cells were pretreated with Z-VAD-fmk (40 μM) for 1 h. Then, the fixed cells were incubated with primary antibody in blocking buffer overnight at 4 °C and then incubated with fluorochromeconjugated Alexa Fluor® 488 or fluorochrome-conjugated Alexa Fluor® 568 secondary antibodies. To normalize the measurements to the number of cells present in each well, a solution of DAPI was added to a final concentration of 0.1 μg/ml. The signals were detected by a FV1000 confocal fluorescence microscope (Olympus, Richmond Hill, ON, Canada). The representative pictures were shown. 400×, Scale bar = 20 μm.
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Fig. 4. Apoptosis induced by DHTS was inhibited by AIF knockdown. (A) Apoptosis induced by DHTS was partly inhibited by AIF knockdown; (B) PARP cleavage and accumulation of cytochrome c were inhibited by AIF knockdown. At the end of the treatment with DHTS (12.5 μM) for 24 h, apoptosis in the cells with or without transfection of AIF siRNA was detected by the FACSCalibur System and CellQuest program. Cells treated with 0.1% (v/v) DMSO served as the vehicle control. Cells transfected with negative siRNA served as sham control. To evaluate the role of caspase, cells of AIF knockdown was pretreated with Z-VAD-fmk (40 μM) for 1 h. Percentage of the cells in early apoptosis (FITC+/PI-) in each group was calculated and compared. PARP cleavage and accumulation of cytochrome c in cytosol were detected by western blotting. Each value was presented as Mean ± S.E.M of three independent experiments. ∗∗∗ P < 0.001 indicated significant difference compared with the vehicle control group.
to be ROS dependent, which was in accordance with ROS dependent apoptosis induced by DHTS as previously reported (Fig. 1A). The pro-apoptotic action of DHTS was almost completely suppressed by Z-VAD-fmk (pan-caspase inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) but only was partially suppressed by Z-IETD-fmk (caspase-8 inhibitor) in HCT116 cells (Fig. 1B–C). Significant apoptosis after silencing caspase-2 expression suggested that caspase-2 played a role as an apoptosis suppressor in colon cancer cells. The pro-apoptotic activity of DHTS was further enhanced to a significant high level upon caspase-2 deficiency (Fig. 1D). DHTS induced autophagic cell death in colon cancer cells DHTS induced time- and concentration-dependent LC3B-II accumulation and time-dependent degradation of p62 in HCT116 cells (Fig. 2A–B). LC3B-II accumulation induced by DHTS was decreased by
pretreatment with NAC or Z-VAD-fmk (Fig. 2C). Meanwhile, LC3B-II accumulation was significantly inhibited by knockdown of caspase2 (Fig. 2D). Autophagic flux was increased in colon cancer cells after treatment with DHTS for 24 h as determined by LC3-II turnover assay (Fig. 2E). Besides, the cell viability was increased after inhibition of DHTS-induced autophagy by 3-MA, an autophagy inhibitor (Fig. 2F). DHTS induced the translocation of AIF, cytochrome c, Bax and Bcl-xl DHTS induced a time-dependent translocation of cytochrome c and AIF. The translocation of AIF to cytosol and nucleus was significantly prevented by Z-VAD-fmk (Fig. 3A–B). The leakage of cytochrome c occurred before the release of AIF from mitochondria, which was confirmed by the results from the immunofluorescence measurement (Fig. 3C). A time-dependent translocation of Bcl-xl from mitochondria to cytosol was also detected after treatment with
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Fig. 5. Anti-colon cancer activity of DHTS in xenograft of colon cancer. (A) Tumor growth was inhibited by DHTS treatment; (B) No significant change of the body weight by DHTS treatment; (C) Tumor volume decreased by DHTS treatment; (D) Tumor weight decreased by DHTS treatment; (E) LC3B-II accumulation and p62 decrease induced by DHTS; (F) PARP cleavage induced by DHTS; (G) Apoptosis and caspase-3 activation induced by DHTS; (H) Increase of LC3B puncta by DHTS. NOD/SCID mice (male, 4–6 weeks) were subcutaneously inoculated 5 × 106 of HCT116 cells. After injection for 2 weeks, the mice were random allocated into three groups. The mice in the treatment groups were i.p. injected with DHTS at 10 mg/kg every other day. The mice in vehicle control group were i.p. injected with the solvent at the same time. The animals were sacrificed at day 19. Tumor growth and body weight were evaluated during treatment. Expression of LC3B-II, p62 and PARP cleavage was detected by western blotting following routing method. Formation of LC3B puncta, in situ apoptosis and caspase-3 cleavage were observed by immunofluorescence with confocal microscopy. 4 ,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei in the sections. Apoptosis in situ was detected by TUNEL assay. The percentage of the positive cells was calculated in at least four fields for each slide. ∗ P < 0.05, ∗∗ P < 0.01 indicated significance compared with the vehicle control group.
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DHTS for 3 h, which was reversed after 12 h of treatment. Meanwhile, time-dependent decrease of Bax from cytosol was detected. After 24 h, obvious translocation of Bax from cytosol to mitochondria was detected (Fig. 3D–E). Apoptosis induced by DHTS was partially inhibited by AIF knockdown The apoptosis induced by DHTS was partly inhibited by knockdown of AIF (Fig. 4A). In this regard, the release of cytochrome c and PARP cleavage were partially but significantly decreased by knocking down of AIF (Fig. 4B). Inhibition of tumor growth by DHTS in xenograft of colon cancer DHTS at the dose of 10 mg/kg significantly inhibited tumor growth, tumor size and tumor weight in comparison to those of the vehicle control group. Meanwhile, the body weight of the animals after DHTS treatment was not notably decreased (Fig. 5A–B). Their average tumor volume was 45% of that of the vehicle control group, and their average weight was 48% of that of the control group at day 19 (Fig. 5C–D). PARP cleavage and accumulation of LC3B-II with decreased p62 were detected (Fig. 5 E–F). An increase of LC3B puncta, significant induction of apoptosis and caspase-3 activation were also detected in xenograft of colon cancer treated by DHTS (Fig. 5G–H).
significant decrease of DHTS-induced cytochrome c release from mitochondria after AIF knockdown in HCT116 cells. At the same time, AIF release was reported to be either caspase-dependent or caspaseindependent (Singh et al., 2010). For instance, caspases-3 and -7 are crucial for apoptosis and contribute to some mitochondrial events including the translocation of AIF (Arnoult et al., 2002; Lakhani et al., 2006). Our results demonstrated that Z-VAD-fmk indeed inhibited the translocation of AIF. Taken together, translocation of AIF and activation of caspases were necessary for each other during DHTSinduced apoptosis in HCT116 cells. As for the regulatory role of caspases in autophagy, it has been reported that consecutive activation of caspases inhibited autophagy via Beclin-1 cleavage, thus enhancing apoptosis at the final stage (Pan et al., 2015). In our study, however, inhibitors of caspase-3/7 decreased the accumulation of LC3B-II, which was also significantly inhibited by caspase-2 knockdown. Given the anti-apoptotic activity of caspase-2 in colon cancer, it is therefore speculated that caspase2 played a key role in maintaining the balance of apoptosis and autophagy both in physiological condition and after exposure to DHTS. Taken together, these findings provide pre-clinical evidence for the development and application of DHTS as a novel therapeutic agent or adjuvant therapy for the treatment of colon cancer. Conflict of interest No conflict of interest to disclose.
Discussion Acknowledgments The present study suggests that apoptosis and autophagy synchronously contribute to the anti-colon cancer activity of DHTS both in vitro and in vivo through ROS and caspase dependent pathway. Moreover, we firstly reported that caspase-2 served as a stimulator of autophagy but an inhibitor of apoptosis induced by DHTS in HCT116 cells, which was in accordance with the other reports on the role of caspase-2 as a tumor suppressor (Aksenova et al., 2013) . Time-dependent translocation of AIF and cytochrome c from mitochondria induced by DHTS was observed in vitro. Apoptosis could be induced by AIF and EndoG mediated caspase independent pathway (Daugas et al., 2000; Lorenzo et al., 1999; Walsh et al., 2008). Although DHTS was found to induce the translocation of AIF, it was not the primary pathway since caspase inhibitor almost completely suppressed the apoptosis induced by DHTS. However, the regulatory role of AIF on cytochrome c mediated pathway was reported in this study (Fig. 4). We demonstrated that DHTS stimulated the translocation of both cytochrome c and AIF in HCT116 cells, which occurred synchronously with the translocation of Bcl-xl and Bax. Bax plays an important role in apoptosis initiation as Bax multimers could function as pores in the mitochondria to facilitate the release of apoptotic proteins. Whereas Bcl-xl is the most potent anti-apoptotic protein in colon cancer cells by occupying the binding site of Bax (Ming et al., 2006) and blocking the permeability transition pore via binding to voltage-dependent anion channel 1(VDAC) (Arbel et al., 2012). Therefore, the opening of “Bax pore” probably facilitated the initiation of apoptosis induced by DHTS in colon cancer. Activation of caspase-9/3 but not caspase-8 was involved in DHTSinduced apoptosis. Arnoult et al. reported that AIF released from mitochondria by general apoptosis inducers in HeLa and Jurkat cell lines was suppressed or delayed by caspase inhibitors (Arnoult et al., 2003). It was believed that AIF accumulation in cytoplasm also contributed to the apoptotic process with evidence of the AIF targets such as HSP70 and eIF3g in the cytoplasm, showing that nucleases are not the single role of AIF in apoptosis. In some cases, AIF was reported as an essential apoptotic factor released from mitochondria in a cytochrome c or ROS dependent caspase activation cascade (Joza et al., 2001; Sevrioukova, 2011; Thayyullathil et al., 2008). In accordance with these reports, our study also showed a
This work was supported by Innovation and Technology Support Programme, Tier 3/Seed Projects, Innovation and Technology Commission, Hong Kong (ITS/212/12) and General Research Fund, Hong Kong Research Grant Council, Hong Kong (CUHK463613). References Aksenova, V.I., Bylino, O.V., Zhivotovskii, B.D., Lavrik, I.N., 2013. [Caspase-2: what do we know today?]. Mol. Biol. 47, 187–204. Arbel, N., Ben-Hail, D., Shoshan-Barmatz, V., 2012. Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein. J. Biol. Chem. 287, 23152–23161. Arnoult, D., Gaume, B., Karbowski, M., Sharpe, J.C., Cecconi, F., Youle, R.J., 2003. Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J. 22, 4385–4399. Arnoult, D., Parone, P., Martinou, J.C., Antonsson, B., Estaquier, J., Ameisen, J.C., 2002. Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli. J. Cell Biol. 159, 923–929. Bouchier-Hayes, L., Green, D.R., 2012. Caspase-2: the orphan caspase. Cell Death Differ. 19, 51–57. Brenner, C., Grimm, S., 2006. The permeability transition pore complex in cancer cell death. Oncogene 25, 4744–4756. Brynychova, V., Hlavac, V., Ehrlichova, M., Vaclavikova, R., Pecha, V., Trnkova, M., Wald, M., Mrhalova, M., Kubackova, K., Pikus, T., Kodet, R., Kovar, J., Soucek, P., 2013. Importance of transcript levels of caspase-2 isoforms S and L for breast carcinoma progression. Future Oncol. 9, 427–438. Daugas, E., Susin, S.A., Zamzami, N., Ferri, K.F., Irinopoulou, T., Larochette, N., Prevost, M.C., Leber, B., Andrews, D., Penninger, J., Kroemer, G., 2000. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. Faseb J. 14, 729–739. Fan, T.J., Han, L.H., Cong, R.S., Liang, J., 2005. Caspase family proteases and apoptosis. Acta Biochim. et Biophys. Sin. 37, 719–727. Han, C., Zhao, R., Kroger, J., Qu, M., Wani, A.A., Wang, Q.E., 2013. Caspase-2 short isoform interacts with membrane-associated cytoskeleton proteins to inhibit apoptosis. PloS One 8, e67033. Iwanaga, N., Kamachi, M., Aratake, K., Izumi, Y., Ida, H., Tanaka, F., Tamai, M., Arima, K., Nakamura, H., Origuchi, T., Kawakami, A., Eguchi, K., 2005. Regulation of alternative splicing of caspase-2 through an intracellular signaling pathway in response to pro-apoptotic stimuli. J. Lab. Clin. Med. 145, 105–110. Joza, N., Susin, S.A., Daugas, E., Stanford, W.L., Cho, S.K., Li, C.Y., Sasaki, T., Elia, A.J., Cheng, H.Y., Ravagnan, L., Ferri, K.F., Zamzami, N., Wakeham, A., Hakem, R., Yoshida, H., Kong, Y.Y., Mak, T.W., Zuniga-Pflucker, J.C., Kroemer, G., Penninger, J.M., 2001. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410, 549–554. Kuwana, T., Newmeyer, D.D., 2003. Bcl-2-family proteins and the role of mitochondria in apoptosis. Curr. Opin. Cell Biol. 15, 691–699.
L. Wang et al. / Phytomedicine 22 (2015) 1079–1087 Lakhani, S.A., Masud, A., Kuida, K., Porter Jr., G.A., Booth, C.J., Mehal, W.Z., Inayat, I., Flavell, R.A., 2006. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311, 847–851. Lamkanfi, M., Kanneganti, T.-D., 2010. Caspase-7: a protease involved in apoptosis and inflammation. Int. J. Biochem. Cell Biol. 42, 21–24. Lorenzo, H.K., Susin, S.A., Penninger, J., Kroemer, G., 1999. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ. 6, 516–524. Ming, L., Wang, P., Bank, A., Yu, J., Zhang, L., 2006. PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells. J. Biol. Chem. 281, 16034–16042. Ouyang, L., Shi, Z., Zhao, S., Wang, F.T., Zhou, T.T., Liu, B., Bao, J.K., 2012. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 45, 487–498. Pan, W.R., Chen, Y.L., Hsu, H.C., Chen, W.J., 2015. Antimicrobial peptide GW-H1-induced apoptosis of human gastric cancer AGS cell line is enhanced by suppression of autophagy. Mol. Cell. Biochem. 400, 77–86. Puccini, J., Dorstyn, L., Kumar, S., 2013. Caspase-2 as a tumour suppressor. Cell Death Differ. 20, 1133–1139.
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Sevrioukova, I.F., 2011. Apoptosis-inducing factor: structure, function, and redox regulation. Antioxid. Redox. Signal. 14, 2545–2579. Singh, M.H., Brooke, S.M., Zemlyak, I., Sapolsky, R.M., 2010. Evidence for caspase effects on release of cytochrome c and AIF in a model of ischemia in cortical neurons. Neurosci. Lett. 469, 179–183. Tait, S.W.G., Green, D.R., 2010. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell. Biol. 11, 621–632. Thayyullathil, F., Chathoth, S., Hago, A., Patel, M., Galadari, S., 2008. Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspase-dependent and -independent apoptosis in L929 cells. Free Radic. Biol. Med. 45, 1403–1412. Walsh, J.G., Cullen, S.P., Sheridan, C., Luthi, A.U., Gerner, C., Martin, S.J., 2008. Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc. Natl. Acad. Sci. U S A 105, 12815–12819. Wang, L., Yeung, J.H., Hu, T., Lee, W.Y., Lu, L., Zhang, L., Shen, J., Chan, R.L., Wu, W.K., Cho, C.H., 2013. Dihydrotanshinone induces p53-independent but ROS-dependent apoptosis in colon cancer cells. Life Sci. 93, 344–351. Woltering, E.J., 2010. Death proteases: alive and kicking. Trends Plant Sci. 15, 185–188.
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Dihydrotanshinone I induced apoptosis and autophagy through caspase dependent pathway in colon cancer Lin Wang a,1,∗, Tao Hu a,1, Jing Shen a, Lin Zhang a, Ruby Lok-Yi Chan a, Lan Lu a, Mingxing Li a, Chi Hin Cho a, William. Ka Kei Wu b a School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Lo Kwee-Seong Integrated Biomedical Sciences Building, Shatin, NT, Hong Kong, China b Department of Anaesthesia and Intensive Care, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
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Article history: Received 2 June 2015 Revised 7 August 2015 Accepted 8 August 2015
Chemical compounds studied in this article: Dihydrotanshinone I (PubChem CID: 11425923) Keywords: Dihydrotanshinone I Apoptosis Autophagy Caspase Mitochondria Colon cancer
a b s t r a c t Background: Dihydrotanshinone I (DHTS) was previously reported to exhibit the most potent anti-cancer activity among several tanshinones in colon cancer cells. Its cytotoxic action was reactive oxygen species (ROS) dependent but p53 independent. Purpose: To further study the anti-cancer activity of DHTS and its molecular mechanisms of action in colon cancer both in vitro and in vivo. Methods: Caspase activity was detected by fluorescence assay. Apoptosis was detected by flow cytometry and TUNEL assay. Protein levels were analyzed by western blotting. Knockdown of target gene was achieved by siRNA transfection. Formation of LC3B puncta and activation of caspase-3 were detected by confocal fluorescence microscope. In vivo anti-colon cancer activity of DHTS was observed in xenograft tumors in NOD/SCID mice. Results: Anti-colon cancer activity of DHTS by inducing apoptosis and autophagy was observed both in vitro and in vivo. Mitochondria mediated caspase dependent pathway was essential in DHTS-induced cytotoxicity. The apoptosis induced by DHTS was suppressed by knockdown of apoptosis inducing factor (AIF), inhibition of caspase-3/9 but was increased after knockdown of caspase-2. Meantime, knockdown of caspase-2, pretreatment with Z-VAD-fmk or NAC (N-Acety-L-Cysteine) efficiently inhibited the autophagy induced by DHTS. A crosstalk between cytochrome c and AIF was also reported. Conclusion: DHTS-induced caspase and ROS dependent apoptosis and autophagy were mediated by mitochondria in colon cancer. DHTS could be a promising leading compound for the development of anti-tumor agent or be developed as an adjuvant drug for colon cancer therapy. © 2015 Elsevier GmbH. All rights reserved.
Introduction The search for effective regimens with minimal adverse effects for the treatment of colon cancer remains the top priority of cancer research. So far, a number of traditional Chinese medicinal preparations and their components have been reported to exhibit promising anticancer activities, they are potential candidates for anti-cancer drug development. Meanwhile, the mechanisms of beneficial preventive and therapeutic effects achieved by traditional and complementary medicine are currently being deciphered in molecular medicine. Our
Abbreviations: DHTS, dihydrotanshinone I; ROS, reactive oxygen species; DAPI, 4 , 6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP Nick-end Labeling; O.C.T, optimal cutting temperature. ∗ Corresponding author. Tel.: +852 3943 5722; fax: +852 2603 5139. E-mail address: [email protected], [email protected] (L. Wang). 1 Lin Wang and Tao Hu contributed equally to this work. http://dx.doi.org/10.1016/j.phymed.2015.08.009 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
preliminary in vitro studies showed that several tanshinones had potent anti-cancer activity in various colon and liver cancer cells and dihydrotanshinone I (DHTS) was the most potent compound (Wang et al., 2013). The anti-cancer activity of DHTS was reactive oxygen species (ROS) but not p53 dependent. However, the underlying mechanisms of cytotoxic action of DHTS are still not well understood. Programmed cell death, which has been recognized since the 1960s, is any type of cell death during which the cell uses specialized intracellular machinery to kill itself. Two important types of programmed cell death are apoptosis and autophagy. Indeed, current anticancer treatments, including many chemotherapeutic agents as well as ionizing radiation therapy, actually activate apoptosis to utilize the apoptotic machinery to kill cancer cells. However, autophagy can both stimulate and prevent cancer depending on the context. On one hand, cancer cells may utilize autophagy to survive with altered metabolism in the hostile tumor microenvironment, suggesting the potential of autophagy inhibition in cancer therapy. On the other
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hand, high levels of autophagy might directly lead to autophagic cell death in cancers. Apoptosis can be mediated by extrinsic and/or intrinsic pathways, and caspase activation was observed in both pathways (Ouyang et al., 2012). Caspases are synthesized as inactive preforms and cleave to aspartate residues upon activation. Caspases can be divided into two distinct groups, the initiator caspases including caspase-8 and -9 as well as the executioner caspases, such as caspase-3 and -7. Initiator caspases are present in the cell as inactive monomers and their activation is promoted by dimerization, which happens when initiator caspases are recruited to large molecular weight protein complexes that act as signaling platforms (Fan et al., 2005; Lamkanfi and Kanneganti, 2010; Tait and Green, 2010; Woltering, 2010). Caspase2 has been reported as an initiator caspase, its role is very special in apoptosis since caspase-2 gene produces several alternative splicing isoforms (Bouchier-Hayes and Green, 2012). The inclusion of exon 9 leads to an in-frame stop codon in caspase-2 short isoform (casp-2S) mRNA, thus producing a truncated protein that inhibits cell death. Whereas the exclusion of exon 9 results in caspase-2 long isoform (casp-2 L) mRNA, which produces protein product inducing cell death (Brynychova et al., 2013; Han et al., 2013; Iwanaga et al., 2005; Puccini et al., 2013). Caspases cleave a number of different substrates in the cytoplasm or nucleus, leading to many morphologic features of apoptotic cell death. Activation of caspases can be initiated from different entry points, such as the plasma membrane upon ligation of death receptor (receptor mediated pathway) and the mitochondria (mitochondria-mediated pathway), etc. The mitochondrial pathway is initiated by the release of apoptotic factors such as cytochrome c, apoptosis inducing factor (AIF) and endonuclease G from the mitochondrial intermembrane space by mitochondrial outer membrane permeabilization (MOMP), which is a complex process that involves numerous molecular players including Bcl-2 family (Brenner and Grimm, 2006; Kuwana and Newmeyer, 2003). The release of cytochrome c into the cytoplasm subsequently triggers caspase-3/7 activation through formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex. In this study, the anti-cancer activity of DHTS and its molecular mechanisms of action in colon cancer were investigated. DHTS was reported to induce apoptosis and autophagy in colon cancer both in vitro and in vivo. Caspase activation accompanied by the crosstalk between AIF and cytochrome c played the dominant role in DHTSinduced cytotoxicity.
Cells were treated with various concentrations of DHTS (3.13– 20 μM) for 48 h. For the activity assay, Ac-DEVD-AMC (1 μg/μl), Ac-IETD-AMC (1 μg/μl) or Ac-LEDH-AMC (1 μg/μl) and cell lysate were added into Protease Assay Buffer in 96-well plate. Reaction mixtures with lysis buffer were used as negative controls. Cells treated with DMSO (0.1%) were treated as vehicle control. The reaction mixtures were incubated for 1 h at 37 °C. The AMC liberated from the substrates was measured using spectrofluorometer of Victor 2 plate reader (Perkin Elmer, Massachusetts, USA) with an excitation wavelength of 380 nm and an emission wavelength of 430 nm.
Materials and methods
Detection of AIF, cytochrome c, Bax, Bcl-xl, LC3B-I/II and p62
Materials
Expression of AIF, cytochrome c, Bax, Bcl-xl, LC3B-I/II and p62 was detected by western blotting using routine method. In brief, samples from cell culture or tissue were harvested and lysed in protein lysis buffer at 4 °C for 30 min. After lysis, samples were centrifuged at 16,000 × g at 4 °C for 20 min. The protein in the supernatant was collected and measured with Pierce® BCA Protein Assay Kit according to the manufacturer’s protocol. Proteins were subjected to SDS– PAGE (8–12%) and detected by western blotting. To detect the role of ROS in caspase-3 cleavage and LC3B-II, cells were pretreated with NAC (2 mM) for 1 h. To detect the autophagic flux, cells were cotreated with bafilomycin A1 (BAF) (100 nM), a blocker of autophagic degradation. Equal amount of proteins were resolved by SDS–PAGE followed by a standard immunoblotting procedure and developed using an ECL development kit (GE healthcare, UK limited).
HPLC grade authentic standard of DHTS was purchased from Chengdu Congon Bio-tech Co., Ltd. (Sichuan, China). Z-VAD-FMK (pan caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), Ac-DMQD-CHO (caspase-3 inhibitor IV) and Z-LEHD-FMK (caspase-9 inhibitor) were from Calbiochem (Darmstadt, Germany). Ac-DEVD-AMC (caspase3/7 substrate), Ac-IETD-AMC (caspase-8 substrate) and Ac-LEDHAMC (caspase-9 substrate) were from EMD Millipore (Darmstadt, Germany). NucBuster TM Protein Extraction Kit was from EMD Biosciences (Darmstadt, Germany). Mitochondria extraction kit for cells was from Millipore (Billerica, MA). Primary antibody of GAPDH was purchased from CHEMICON. Primary antibody of AIF was from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were from Cell Signaling. FlexiTube siRNA for human AIFM1 (Gene accession: NM_001130846), HS-Cap2-10 (Gene accession: NM_001224) and All Stars Neg. SiRNA AF 555 were purchased from QIAGEN Science (Germantown, Germany). Jetprime Transfection reagent was from Polyplus transfection SA (St. Louis, MO, Illkirch FRANCE). Antibodies of Alexa-Fluor 488 goat anti-rabbit, Alexa-Fluor 488 goat anti-mouse, Alexa-Fluor 555 donkey anti-mouse were obtained from Molecular
Probes (Eugene, OR). Unless otherwise specified, all chemicals used in this study were purchased from Sigma (St. Louis, MO). Cell culture Human colon cancer HCT116 cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD) and was routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; GIBCO/BRL, NY), 100 mg/l penicillin G and 100 U/ml streptomycin sulfate at 37 °C in 5% CO2 . RNA interference The expression of AIF or caspase-2 was efficiently lowered using predesigned target-specific siRNA purchased from Qiagen Science. FlexiTube siRNA was transfected into cells using JetprimeTM (Polyplus-transfection Inc.) following the protocol provided. All Stars Neg. siRNA AF 555 served as sham control siRNA. Apoptosis analysis Cells were seeded and cultured overnight in 24-well plate. DHTS were then added into the medium except the control and vehicle control groups. Cells with 0.1% (v/v) DMSO served as vehicle control. Apoptosis was detected after 24 h treatment by flow cytometry. To verify the role of AIF and caspase-2 in the apoptotic activity of DHTS, apoptosis in cells with AIF or caspase-2 knockdown was determined. The role of caspases in apoptotic activity of DHTS was also defined. In this regard, cells were pretreated with Z-VAD-fmk, Z-IETD-FMK or Z-LEHD-FMK (40 μM) for 1 h before DHTS incubation. Activity assay of caspases
Detection for the translocation of cytochrome c, AIF, Bax, Bcl-xl and RAPR cleavage Cell fractions including mitochondrial protein, cytoplasmic protein and nuclear protein were extracted according to protocols provided by respective kits. For isolation of whole-cell protein, cells were
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Fig. 1. Caspase dependent apoptosis induced by DHTS in HCT116 cells. (A) DHTS induced concentration and ROS dependent caspase activation. Ac-DEVD-AMC (substrate of caspase3/7), Ac-IETD-AMC (substrate of caspase-9) or Ac-LEDH-AMC (substrate of caspase-8) and cell lysate were added into Protease Assay Buffer in 96-well culture plate. Measure the 7-amino-4-methylcoumarin (AMC) liberated using spectrofluorometer (Victor 2 plate reader, Perkin Elmer, Massachusetts, USA) with an excitation wavelength of 380 nm and an emission wavelength of 430 nm. The representative results from three independent experiments were shown. Each value was presented as Mean ± S.E.M of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicated significant difference among treatments. Caspase-3 cleavage was detected by western blotting following routine method with GAPDH as an internal control. The representative results were shown; (B) Apoptosis induced by DHTS was completely prevented by Z-VAD-fmk; (C) Apoptosis induced by DHTS was significantly inhibited by pretreatment of Z-LEHD-fmk but only was partially inhibited by Z-IETD-fmk; D. Apoptosis induced by DHTS was significantly increased by caspase-2 knockdown. Cells treated with 0.1% (v/v) DMSO served as vehicle control. After treatment with DHTS (6.25 μM) for 24 h, cells were collected and resuspended in binding buffer. To evaluate the effect of caspase activation on the apoptosis induced by DHTS, cells were pretreated with 40 μM of Z-VAD-fmk (pan caspase inhibitor), Z-LEHD-fmk (caspase-9 inhibitor) or Z-IETD-fmk (caspase-8 inhibitor) for 1 h. To evaluate the role of caspase-2 in apoptotic activity of DHTS, apoptosis were detected in HCT116 cells with caspase-2 knockdown. Apoptosis in colon cancer cells were analyzed by flow cytometry (BD LSRFortessa Cell Analyzer) by double stained with FITC Annexin V and PI. Percentage of the cells in early apoptosis (FITC+/PI-) in each group was calculated and compared using Flowjo7.6.1 software. Each value was presented as Mean ± S.E.M of three independent experiments. ∗∗∗P < 0.001 indicated significant difference among treatments.
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Detection of the translocation of cytochrome c and AIF by immunofluorescence The translocations of cytochrome c and AIF were further observed by immunofluorescence as previously described. Briefly, cells were fixed with 4% formalin and incubated with primary antibody followed by a secondary fluorescent antibody. Alexa Fluor anti-mouse 568 or Alexa Fluor anti-rabbit 488 were used as a secondary antibody. In addition, 4 , 6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. Expression of cytochrome c and AIF was evaluated with an Olympus FV1000 laser confocal microscope (Olympus, Richmond Hill, ON, Canada). Animal experimentation All experiments were performed under Laboratory Animal Ethics Committee approval (The Chinese University of Hong Kong; Ref No: 12/085/MIS-5). Xenograft tumors were established in male NOD/SCID mice (6–7-week-old, Jackson Laboratories) by s.c. injections of 5 × 106 HCT116 cells into lower back areas of the mice. Monitoring of tumor growth was performed twice per week. The tumor size was measured by a caliper as length × width2 /2. When tumor sizes reach 100 mm3 at day 14 after injection, mice were randomly assigned into two groups. DHTS therapy was administered i.p. every other day as a single dose of 10 mg/kg for 19 days. The mixture of 4% DMSO and 0.1% PEG800 was administered i.p. to mice as the vehicle control group. Body weight and general conditions of mice were assessed at the times of drug administration. Finally, all mice were sacrificed and underwent necropsy. Tumors were removed and weighed, fixed in 4% paraformaldehyde and embedded in paraffin or O.C.T (optimal cutting temperature) and stored at –80 °C for further analyses. Determination of cell apoptosis in xenograft of colon cancer by Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling (TUNEL) TUNEL assay was performed with the Dead End Tm Colorimetric TUNEL System (Roche) following the protocol provided. Sections embedded in paraffin were observed with a Olympus FV1000 laser confocal microscope (Olympus, Richmond Hill, ON, Canada). The percentage of TUNEL positive cells was calculated in at least four fields for each slide.
Fig. 2. Pro-cell death of autophagy induced by DHTS in HCT116 cells. (A) DHTS induced concentration-dependent LC3B-II accumulation; (B) DHTS induced time-dependent LC3B-II accumulation and p62 decrease; (C) LC3B-II accumulation induced by DHTS was decreased by pretreatment of NAC or Z-VAD-fmk; (D) LC3B-II accumulation induced by DHTS was decreased after caspase-2 knockdown; (E) Autophagic flux was increased by DHTS after 24 h as determined by LC3-II turnover assay; (F) Inhibition of autophagy by 3-MA increased the viability of colon cancer cells. The expression of LC3BII and p62 was determined by western blotting following routine method. To detect the role of ROS and caspase in LC3B-II accumulation, cells were pretreated with NAC (2 mM) for 1 h. To detect the autophagic flux, cells were cotreated with bafilomycin A1 (BAF) (100 nM). Viability of colon cancer cells was determined by MTT assay. Each value was presented as Mean ± S.E.M of three independent experiments. ∗∗ P < 0.01, compared with the DHTS alone group.
Detection of LC3B-II puncta and cleaved caspase-3 by immunofluorescence For the fluorescence staining of tissue sections, slides were incubated with primary antibody followed by a secondary fluorescent antibody. Alexa Fluor anti-rabbit 488 was used as the secondary antibody. In addition, 4 , 6-diamidino-2-phenylindole (DAPI) was employed to stain cell nuclei. The mounted slides were subjected to microscopic analysis under Olympus FV1000 laser confocal microscope (Olympus, Richmond Hill, ON, Canada). The percentage of the positive cells was evaluated in at least four fields for each slide. Statistical analysis
harvested in lysis buffer containing proteinase and phosphatase inhibitors. Translocation of cytochrome c, AIF, Bax and Bcl-xl was detected in respective cell fractions. To identify the role of caspase in translocation of AIF, cells were pretreated with Z-VAD-FMK (40 μM) for 1 h. Release of cytochrome c and PARP cleavage were also observed in cells with AIF knockdown. Equal amount of proteins were resolved by SDS–PAGE followed by a standard immunoblotting procedure. GADPH, Lamin A/C and cytochrome oxidase subunit IV (Cox IV) were used as loading reference for cytoplasmic protein, nuclear protein and mitochondrial protein, respectively.
Results were expressed as the mean ± SEM. Statistical analysis was performed with an Analysis of Variance (ANOVA) followed by Dunnett test or Tukey’s t-test. P values less than 0.05 were considered to be statistically significant. Results DHTS induced caspase dependent apoptosis in colon cancer cells DHTS induced a concentration-dependent activation of caspase3, caspase-9 but not caspase-8. Activation of caspase-3 was found
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Fig. 3. DHTS induced significant translocation of cytochrome c, AIF, Bax and Bcl-xl. (A) Time-dependent translocation of cytochrome c induced by DHTS; (B) Time-dependent translocation of AIF induced by DHTS; (C) Time-dependent translocation of cytochrome c and AIF was detected by immunofluorescence; (D) Time-dependent translocation of Bcl-xl and Bax induced by DHTS; (E) Translocation of Bax to mitochondria and Bcl-xl to cytosol observed at 24 h after treatment. HCT116 cells were treated with DHTS (20 μM) for different timepoints. Protein of cell fractions was isolated using Mitochondria extraction kit and NucBuster Protein Extraction Kit according to the manufacturer’s protocol. Expression of AIF, cytochrome c, Bcl-xl and Bax in the cell fractions was detected by western blotting following the routine method. Expression of GAPDH, COXIV and LaminA/C was determined as loading reference for cytosolic protein, mitochondrial protein and nuclear protein, respectively. The gray value of the protein band was analyzed by Quantity one and the relative expression of AIF and cytochrome c was statistically analyzed. #p < 0.05, ##p < 0.01, ###p < 0.001 versus control. For immunofluorescence detection, cells were treated with DHTS (20 μM) for certain hours before the termination of incubation. To evaluate the role of Z-VAD-fmk in the translocation of AIF and cytochrome c, cells were pretreated with Z-VAD-fmk (40 μM) for 1 h. Then, the fixed cells were incubated with primary antibody in blocking buffer overnight at 4 °C and then incubated with fluorochromeconjugated Alexa Fluor® 488 or fluorochrome-conjugated Alexa Fluor® 568 secondary antibodies. To normalize the measurements to the number of cells present in each well, a solution of DAPI was added to a final concentration of 0.1 μg/ml. The signals were detected by a FV1000 confocal fluorescence microscope (Olympus, Richmond Hill, ON, Canada). The representative pictures were shown. 400×, Scale bar = 20 μm.
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Fig. 4. Apoptosis induced by DHTS was inhibited by AIF knockdown. (A) Apoptosis induced by DHTS was partly inhibited by AIF knockdown; (B) PARP cleavage and accumulation of cytochrome c were inhibited by AIF knockdown. At the end of the treatment with DHTS (12.5 μM) for 24 h, apoptosis in the cells with or without transfection of AIF siRNA was detected by the FACSCalibur System and CellQuest program. Cells treated with 0.1% (v/v) DMSO served as the vehicle control. Cells transfected with negative siRNA served as sham control. To evaluate the role of caspase, cells of AIF knockdown was pretreated with Z-VAD-fmk (40 μM) for 1 h. Percentage of the cells in early apoptosis (FITC+/PI-) in each group was calculated and compared. PARP cleavage and accumulation of cytochrome c in cytosol were detected by western blotting. Each value was presented as Mean ± S.E.M of three independent experiments. ∗∗∗ P < 0.001 indicated significant difference compared with the vehicle control group.
to be ROS dependent, which was in accordance with ROS dependent apoptosis induced by DHTS as previously reported (Fig. 1A). The pro-apoptotic action of DHTS was almost completely suppressed by Z-VAD-fmk (pan-caspase inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) but only was partially suppressed by Z-IETD-fmk (caspase-8 inhibitor) in HCT116 cells (Fig. 1B–C). Significant apoptosis after silencing caspase-2 expression suggested that caspase-2 played a role as an apoptosis suppressor in colon cancer cells. The pro-apoptotic activity of DHTS was further enhanced to a significant high level upon caspase-2 deficiency (Fig. 1D). DHTS induced autophagic cell death in colon cancer cells DHTS induced time- and concentration-dependent LC3B-II accumulation and time-dependent degradation of p62 in HCT116 cells (Fig. 2A–B). LC3B-II accumulation induced by DHTS was decreased by
pretreatment with NAC or Z-VAD-fmk (Fig. 2C). Meanwhile, LC3B-II accumulation was significantly inhibited by knockdown of caspase2 (Fig. 2D). Autophagic flux was increased in colon cancer cells after treatment with DHTS for 24 h as determined by LC3-II turnover assay (Fig. 2E). Besides, the cell viability was increased after inhibition of DHTS-induced autophagy by 3-MA, an autophagy inhibitor (Fig. 2F). DHTS induced the translocation of AIF, cytochrome c, Bax and Bcl-xl DHTS induced a time-dependent translocation of cytochrome c and AIF. The translocation of AIF to cytosol and nucleus was significantly prevented by Z-VAD-fmk (Fig. 3A–B). The leakage of cytochrome c occurred before the release of AIF from mitochondria, which was confirmed by the results from the immunofluorescence measurement (Fig. 3C). A time-dependent translocation of Bcl-xl from mitochondria to cytosol was also detected after treatment with
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Fig. 5. Anti-colon cancer activity of DHTS in xenograft of colon cancer. (A) Tumor growth was inhibited by DHTS treatment; (B) No significant change of the body weight by DHTS treatment; (C) Tumor volume decreased by DHTS treatment; (D) Tumor weight decreased by DHTS treatment; (E) LC3B-II accumulation and p62 decrease induced by DHTS; (F) PARP cleavage induced by DHTS; (G) Apoptosis and caspase-3 activation induced by DHTS; (H) Increase of LC3B puncta by DHTS. NOD/SCID mice (male, 4–6 weeks) were subcutaneously inoculated 5 × 106 of HCT116 cells. After injection for 2 weeks, the mice were random allocated into three groups. The mice in the treatment groups were i.p. injected with DHTS at 10 mg/kg every other day. The mice in vehicle control group were i.p. injected with the solvent at the same time. The animals were sacrificed at day 19. Tumor growth and body weight were evaluated during treatment. Expression of LC3B-II, p62 and PARP cleavage was detected by western blotting following routing method. Formation of LC3B puncta, in situ apoptosis and caspase-3 cleavage were observed by immunofluorescence with confocal microscopy. 4 ,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei in the sections. Apoptosis in situ was detected by TUNEL assay. The percentage of the positive cells was calculated in at least four fields for each slide. ∗ P < 0.05, ∗∗ P < 0.01 indicated significance compared with the vehicle control group.
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DHTS for 3 h, which was reversed after 12 h of treatment. Meanwhile, time-dependent decrease of Bax from cytosol was detected. After 24 h, obvious translocation of Bax from cytosol to mitochondria was detected (Fig. 3D–E). Apoptosis induced by DHTS was partially inhibited by AIF knockdown The apoptosis induced by DHTS was partly inhibited by knockdown of AIF (Fig. 4A). In this regard, the release of cytochrome c and PARP cleavage were partially but significantly decreased by knocking down of AIF (Fig. 4B). Inhibition of tumor growth by DHTS in xenograft of colon cancer DHTS at the dose of 10 mg/kg significantly inhibited tumor growth, tumor size and tumor weight in comparison to those of the vehicle control group. Meanwhile, the body weight of the animals after DHTS treatment was not notably decreased (Fig. 5A–B). Their average tumor volume was 45% of that of the vehicle control group, and their average weight was 48% of that of the control group at day 19 (Fig. 5C–D). PARP cleavage and accumulation of LC3B-II with decreased p62 were detected (Fig. 5 E–F). An increase of LC3B puncta, significant induction of apoptosis and caspase-3 activation were also detected in xenograft of colon cancer treated by DHTS (Fig. 5G–H).
significant decrease of DHTS-induced cytochrome c release from mitochondria after AIF knockdown in HCT116 cells. At the same time, AIF release was reported to be either caspase-dependent or caspaseindependent (Singh et al., 2010). For instance, caspases-3 and -7 are crucial for apoptosis and contribute to some mitochondrial events including the translocation of AIF (Arnoult et al., 2002; Lakhani et al., 2006). Our results demonstrated that Z-VAD-fmk indeed inhibited the translocation of AIF. Taken together, translocation of AIF and activation of caspases were necessary for each other during DHTSinduced apoptosis in HCT116 cells. As for the regulatory role of caspases in autophagy, it has been reported that consecutive activation of caspases inhibited autophagy via Beclin-1 cleavage, thus enhancing apoptosis at the final stage (Pan et al., 2015). In our study, however, inhibitors of caspase-3/7 decreased the accumulation of LC3B-II, which was also significantly inhibited by caspase-2 knockdown. Given the anti-apoptotic activity of caspase-2 in colon cancer, it is therefore speculated that caspase2 played a key role in maintaining the balance of apoptosis and autophagy both in physiological condition and after exposure to DHTS. Taken together, these findings provide pre-clinical evidence for the development and application of DHTS as a novel therapeutic agent or adjuvant therapy for the treatment of colon cancer. Conflict of interest No conflict of interest to disclose.
Discussion Acknowledgments The present study suggests that apoptosis and autophagy synchronously contribute to the anti-colon cancer activity of DHTS both in vitro and in vivo through ROS and caspase dependent pathway. Moreover, we firstly reported that caspase-2 served as a stimulator of autophagy but an inhibitor of apoptosis induced by DHTS in HCT116 cells, which was in accordance with the other reports on the role of caspase-2 as a tumor suppressor (Aksenova et al., 2013) . Time-dependent translocation of AIF and cytochrome c from mitochondria induced by DHTS was observed in vitro. Apoptosis could be induced by AIF and EndoG mediated caspase independent pathway (Daugas et al., 2000; Lorenzo et al., 1999; Walsh et al., 2008). Although DHTS was found to induce the translocation of AIF, it was not the primary pathway since caspase inhibitor almost completely suppressed the apoptosis induced by DHTS. However, the regulatory role of AIF on cytochrome c mediated pathway was reported in this study (Fig. 4). We demonstrated that DHTS stimulated the translocation of both cytochrome c and AIF in HCT116 cells, which occurred synchronously with the translocation of Bcl-xl and Bax. Bax plays an important role in apoptosis initiation as Bax multimers could function as pores in the mitochondria to facilitate the release of apoptotic proteins. Whereas Bcl-xl is the most potent anti-apoptotic protein in colon cancer cells by occupying the binding site of Bax (Ming et al., 2006) and blocking the permeability transition pore via binding to voltage-dependent anion channel 1(VDAC) (Arbel et al., 2012). Therefore, the opening of “Bax pore” probably facilitated the initiation of apoptosis induced by DHTS in colon cancer. Activation of caspase-9/3 but not caspase-8 was involved in DHTSinduced apoptosis. Arnoult et al. reported that AIF released from mitochondria by general apoptosis inducers in HeLa and Jurkat cell lines was suppressed or delayed by caspase inhibitors (Arnoult et al., 2003). It was believed that AIF accumulation in cytoplasm also contributed to the apoptotic process with evidence of the AIF targets such as HSP70 and eIF3g in the cytoplasm, showing that nucleases are not the single role of AIF in apoptosis. In some cases, AIF was reported as an essential apoptotic factor released from mitochondria in a cytochrome c or ROS dependent caspase activation cascade (Joza et al., 2001; Sevrioukova, 2011; Thayyullathil et al., 2008). In accordance with these reports, our study also showed a
This work was supported by Innovation and Technology Support Programme, Tier 3/Seed Projects, Innovation and Technology Commission, Hong Kong (ITS/212/12) and General Research Fund, Hong Kong Research Grant Council, Hong Kong (CUHK463613). References Aksenova, V.I., Bylino, O.V., Zhivotovskii, B.D., Lavrik, I.N., 2013. [Caspase-2: what do we know today?]. Mol. Biol. 47, 187–204. Arbel, N., Ben-Hail, D., Shoshan-Barmatz, V., 2012. Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein. J. Biol. Chem. 287, 23152–23161. Arnoult, D., Gaume, B., Karbowski, M., Sharpe, J.C., Cecconi, F., Youle, R.J., 2003. Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J. 22, 4385–4399. Arnoult, D., Parone, P., Martinou, J.C., Antonsson, B., Estaquier, J., Ameisen, J.C., 2002. Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli. J. Cell Biol. 159, 923–929. Bouchier-Hayes, L., Green, D.R., 2012. Caspase-2: the orphan caspase. Cell Death Differ. 19, 51–57. Brenner, C., Grimm, S., 2006. The permeability transition pore complex in cancer cell death. Oncogene 25, 4744–4756. Brynychova, V., Hlavac, V., Ehrlichova, M., Vaclavikova, R., Pecha, V., Trnkova, M., Wald, M., Mrhalova, M., Kubackova, K., Pikus, T., Kodet, R., Kovar, J., Soucek, P., 2013. Importance of transcript levels of caspase-2 isoforms S and L for breast carcinoma progression. Future Oncol. 9, 427–438. Daugas, E., Susin, S.A., Zamzami, N., Ferri, K.F., Irinopoulou, T., Larochette, N., Prevost, M.C., Leber, B., Andrews, D., Penninger, J., Kroemer, G., 2000. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. Faseb J. 14, 729–739. Fan, T.J., Han, L.H., Cong, R.S., Liang, J., 2005. Caspase family proteases and apoptosis. Acta Biochim. et Biophys. Sin. 37, 719–727. Han, C., Zhao, R., Kroger, J., Qu, M., Wani, A.A., Wang, Q.E., 2013. Caspase-2 short isoform interacts with membrane-associated cytoskeleton proteins to inhibit apoptosis. PloS One 8, e67033. Iwanaga, N., Kamachi, M., Aratake, K., Izumi, Y., Ida, H., Tanaka, F., Tamai, M., Arima, K., Nakamura, H., Origuchi, T., Kawakami, A., Eguchi, K., 2005. Regulation of alternative splicing of caspase-2 through an intracellular signaling pathway in response to pro-apoptotic stimuli. J. Lab. Clin. Med. 145, 105–110. Joza, N., Susin, S.A., Daugas, E., Stanford, W.L., Cho, S.K., Li, C.Y., Sasaki, T., Elia, A.J., Cheng, H.Y., Ravagnan, L., Ferri, K.F., Zamzami, N., Wakeham, A., Hakem, R., Yoshida, H., Kong, Y.Y., Mak, T.W., Zuniga-Pflucker, J.C., Kroemer, G., Penninger, J.M., 2001. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410, 549–554. Kuwana, T., Newmeyer, D.D., 2003. Bcl-2-family proteins and the role of mitochondria in apoptosis. Curr. Opin. Cell Biol. 15, 691–699.
L. Wang et al. / Phytomedicine 22 (2015) 1079–1087 Lakhani, S.A., Masud, A., Kuida, K., Porter Jr., G.A., Booth, C.J., Mehal, W.Z., Inayat, I., Flavell, R.A., 2006. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311, 847–851. Lamkanfi, M., Kanneganti, T.-D., 2010. Caspase-7: a protease involved in apoptosis and inflammation. Int. J. Biochem. Cell Biol. 42, 21–24. Lorenzo, H.K., Susin, S.A., Penninger, J., Kroemer, G., 1999. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ. 6, 516–524. Ming, L., Wang, P., Bank, A., Yu, J., Zhang, L., 2006. PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells. J. Biol. Chem. 281, 16034–16042. Ouyang, L., Shi, Z., Zhao, S., Wang, F.T., Zhou, T.T., Liu, B., Bao, J.K., 2012. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 45, 487–498. Pan, W.R., Chen, Y.L., Hsu, H.C., Chen, W.J., 2015. Antimicrobial peptide GW-H1-induced apoptosis of human gastric cancer AGS cell line is enhanced by suppression of autophagy. Mol. Cell. Biochem. 400, 77–86. Puccini, J., Dorstyn, L., Kumar, S., 2013. Caspase-2 as a tumour suppressor. Cell Death Differ. 20, 1133–1139.
1087
Sevrioukova, I.F., 2011. Apoptosis-inducing factor: structure, function, and redox regulation. Antioxid. Redox. Signal. 14, 2545–2579. Singh, M.H., Brooke, S.M., Zemlyak, I., Sapolsky, R.M., 2010. Evidence for caspase effects on release of cytochrome c and AIF in a model of ischemia in cortical neurons. Neurosci. Lett. 469, 179–183. Tait, S.W.G., Green, D.R., 2010. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell. Biol. 11, 621–632. Thayyullathil, F., Chathoth, S., Hago, A., Patel, M., Galadari, S., 2008. Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspase-dependent and -independent apoptosis in L929 cells. Free Radic. Biol. Med. 45, 1403–1412. Walsh, J.G., Cullen, S.P., Sheridan, C., Luthi, A.U., Gerner, C., Martin, S.J., 2008. Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc. Natl. Acad. Sci. U S A 105, 12815–12819. Wang, L., Yeung, J.H., Hu, T., Lee, W.Y., Lu, L., Zhang, L., Shen, J., Chan, R.L., Wu, W.K., Cho, C.H., 2013. Dihydrotanshinone induces p53-independent but ROS-dependent apoptosis in colon cancer cells. Life Sci. 93, 344–351. Woltering, E.J., 2010. Death proteases: alive and kicking. Trends Plant Sci. 15, 185–188.