Dihydrotanshinone I induced apoptosis and autophagy through caspase dependent pathway in colon cancer.
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 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 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-l/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.
Materials and methods
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 CHEM1CON. 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 Q1AGEN Science (Germantown, Germany). Jetprime Transfection reagent was from Polyplus transfection SA (St. Louis, MO, lllkirch 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).
Human colon cancer HCT116 cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD) and was routinely cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS; GIBCO/BRL, NY), 100 mg/1 penicillin G and 100 U/ml streptomycin sulfate at 37[degrees]C in 5% C[O.sub.2].
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 Jetprime[TM] (Polyplus-transfection Inc.) following the protocol provided. All Stars Neg. siRNA AF 555 served as sham control siRNA.
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, ceils were pretreated with Z-VAD-fmk, Z-IETD-FMK or Z-LEHD-FMK (40 [micro]M) for 1 h before DHTS incubation.
Activity assay of caspases
Cells were treated with various concentrations of DHTS (3.1320 [micro]M) for 48 h. For the activity assay, Ac-DEVD-AMC (1 [micro]g/[micro]1), Ac-IETD-AMC (1 [micro]g/[micro]1) or Ac-LEDH-AMC (1 [micro]g/[micro]l) 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[degrees]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.
Detection of AIF, cytochrome c, Bax, Bcl-xl, LC3B-I/II and p62
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[degrees]C for 30 min. After lysis, samples were centrifuged at 16,000 x g at 4[degrees]C for 20 min. The protein in the supernatant was collected and measured with Pierce[R] BCA Protein Assay Kit according to the manufacturer's protocol. Proteins were subjected to SDSPAGE (8-12%) and detected by western blotting. To detect the role of ROS in caspase-3 cleavage and LC3B-1I, cells were pretreated with NAC (2 mM) for 1 h. To detect the autophagic flux, cells were cotreated with bafilomycin Al (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).
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 harvested in lysis buffer containing proteinase and phosphatase inhibitors. Translocation of cytochrome c. AIF, Bax and Bd-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 [micro]M) 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 immunoblotdng 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.
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 (DAP1) 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).
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 x 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 x [width.sup.2]/2. When tumor sizes reach 100 [mm.sup.3] 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[degrees]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.
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.
Results were expressed as the mean [+ or -] 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.
DHTS induced caspase dependent apoptosis in colon cancer cells
DHTS induced a concentration-dependent activation of caspase-3, caspase-9 but not caspase-8. Activation of caspase-3 was found 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. ID).
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-1I accumulation induced by DHTS was decreased by pretreatment with NAC or Z-VAD-fmk (Fig. 2C). Meanwhile, LC3B-I1 accumulation was significantly inhibited by knockdown of caspase-2 (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 A1F. 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 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).
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 l(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 DHTS-induced 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 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 caspase-independent (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 Bedin-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-dinical evidence for the development and application of DHTS as a novel therapeutic agent or adjuvant therapy for the treatment of colon cancer.
Received 2 June 2015
Revised 7 August 2015
Accepted 8 August 2015
Chemical compounds studied in this article: Dihydrotanshinone I (PubChem CID: 11425923)
Conflict of interest
No conflict of interest to disclose.
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).
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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
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: firstname.lastname@example.org, email@example.com (L. Wang).
(1) Lin Wang and Tao Hu contributed equally to this work.
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|Author:||Wang, Lin; Hu, Tao; Shen, Jing; Zhang, Lin; Chan, Ruby Lok-Yi; Lu, Lan; Li, Mingxing; Cho, Chi Hin;|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Nov 15, 2015|
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