Background
Under normal conditions, programmed cell death (PCD) maintains a balance between cell death with survival; however, once the equilibrium becomes disturbed, PCD plays a key role in determining the fate of cancer cells [
1,
2]. “Necroptosis” is a type of PCD, which is characterized by the swelling of the cytosol and organelles and rupture of the plasma membrane, with a subsequent loss of the intracellular contents.
Autophagy involves in cellular homeostasis and differentiation, as well as in tissue remodeling, aging, cancer, and other diseases. This process can be activated by a lack of nutrients and growth factors in the extracellular microenvironment, but it can also contribute to programmed cell death under specific environmental conditions [
3‐
5]. Autophagy is an important, well-established cell survival mechanism, especially in cells under stress conditions, such as starvation [
6,
7]. On the other hand, autophagy has been implicated in the cell death process, either in apoptosis, or in non-apoptotic or necrotic cell death, including autophagic cell death [
8].
Necrosis is another programmed and regulated cell death process [
9]. Among various forms of necrotic cell death, necroptosis and PARP-mediated necrosis have emerged as two important forms, and our understanding of the molecular mechanisms and biological functions of these processes has increased. Necroptosis refers to a specific form of caspase-independent, non-apoptotic or necrotic cell death that is triggered by cell death ligands (TNF-α and FasL) via cell death receptors and a unique downstream signaling pathway [
10,
11]. At present, the relationship between autophagy and necroptosis is rather complex.
Shikonin, purified from
Lithospermum erythrorhizon, has been used for thousands of years in traditional Chinese medicine for the treatment of burns, carbuncles, measles, macular eruptions, and sore throat [
12,
13]. Recent studies have demonstrated that shikonin has significant anti-tumor potential, inducing apoptosis and necroptosis in cancer cell lines of various types, including breast cancer, hepatocellular carcinoma, glioma, osteosarcoma, and leukemia [
14‐
17]. However, the effect on lung cancer cell lines remains unknown. In this study, we hypothesized that autophagy plays an important role in the outcome of necroptosis following shikonin treatment. Furthermore, we tested whether shikonin can induce necroptosis and autophagy in non-small cell lung cancer (NSCLC) cells and whether the inhibition of autophagy can drive lung cancer cells to necroptosis.
Methods
Materials
Roswell Park Memorial Institute Medium 1640 (RPMI 1640), fetal bovine serum, and antibiotics (penicillin and streptomycin) were obtained from GIBCO BRL Co. (Grand Island, NY, USA). Shikonin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide (PI), and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against caspase-3, poly(ADP-ribose) polymerase (PARP), AMPK, mTOR, Beclin, ATG5, LC3B, p62/SQSTM1, and GFP were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against RIP1 were purchased from Abcam, Inc. (Cambridge, UK). 3-Methyl adenine (3-MA) and bafilomycin A were purchased from Sigma-Aldrich. Caspase inhibitor Z-VAD-FMK, Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK were purchased from R&D Systems (Minneapolis, MN, USA). Necrostatin-1 (Nec-1) was purchased from Tocris Bioscience (Ellisville, MO, USA). Anti-rabbit IgG-conjugated horseradish peroxidase (HRP) antibodies and enhanced chemiluminescence (ECL) kits were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).
Cell culture
A549 human lung cancer cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and grown in RPMI 1640 containing 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum. The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37 °C and maintained in log-phase growth.
Multiplexed viability and cytotoxicity assays
The CellTox Green Cytotoxicity Assay (Promega, Madison, WI, USA) was used to measure the DNA in dead cells and the CellTiter-Glo 2.0 Assay (Promega) was then used to measure ATP as a marker of viable cells. A549 cells were seeded in 96-well plates at 20,000 cells/well in 50 µL and incubated at 37 °C for 24 h in a 5% CO2 incubator, followed by the addition of 50 μL/well CellTox Green Cytotoxicity Assay Reagent (Promega). After 15 min of incubation at room temperature, the resulting fluorescence was measured in the 485Ex 530Em channels using a SpectraMax M3 Plate Reader. After the readings were obtained, 100 µL of CellTiter-Glo 2.0 assay reagent was added to all wells and the resulting luminescence was measured using a SpectraMax M3 Plate Reader after 10 min of incubation at 37 °C.
Caspase 8 assay
A549 cells were dispensed in culture medium at 2 × 104 cells/well in white-walled 96-well luminometer plates and incubated for 24 h at 37 °C in the presence or absence of test materials, followed by the addition of 100 μL/well Caspase-Glo 8 Reagent (Promega). After 30 min of incubation at room temperature, the luminescence intensity was measured using a SpectraMax M3 Plate Reader.
MTT assay
After cells were treated with the specified drugs, MTT was added to the cell suspension and incubated for 4 h. The cells were then washed three times with phosphate-buffered saline (PBS; pH 7.4), and the insoluble formazan product was dissolved in dimethyl sulfoxide. The optical density (OD) at 595 nm in each well was measured using a microplate reader (Titertek Multiskan; Flow Laboratories, North Ryde, New South Wales, Australia). The OD resulting from formazan production in control cells was defined as 100% cell viability, and all other measurements were expressed as a percentage of the control cell value.
Annexin V/PI assay
Annexin V-FITC and PI staining were used to analyze whether A549 cells were undergoing early/late apoptosis or necroptosis. Cells in the exponential growth phase (2.5 × 105 cells) were seeded in 35-mm2 dishes and were incubated at 37 °C for the indicated times in the presence or absence of specified test drugs. The cells, both adherent and floating, were then harvested and analyzed by the annexin V assay according to the manufacturer’s instructions. Pelleted cells were briefly washed with PBS and resuspended in annexin-binding buffer. They were then incubated with annexin V-FITC and PI for 15 min at room temperature. After incubation, the stained cells were analyzed using a FACSCalibur system and Cell Quest software (Becton–Dickinson, San Jose, CA, USA).
Tumor xenograft studies in nude mice
Five- to six-week-old BALB/c athymic nude mice (Charles River, Tokyo, Japan) were housed in cages with HEPA-filtered air (12-h light/dark cycle). Food and autoclaved water were provided ad libitum. A549 cells were injected subcutaneously (s.c.) into both hind legs of each mouse. When the implanted tumors reached a volume of 90–130 mm3, the mice were randomly assigned to one of two groups (n = 5 per group). For the experimental group, shikonin (2.0 mg/kg, diluted PBS) was injected intraperitoneally once per da. The control group received PBS alone. Animals were monitored for 14 days or until the tumors reached a volume of 1300 mm3.
Tumor orthotopic studies in nude mice
Nude mice were intraperitoneally injected with thiopental sodium (0.08 mL/kg of body weight) to induce anesthesia; subsequently, the mice were placed in the right lateral decubitus position. Then, 50 μL of the A549 single cell suspension (1.5 × 106) prepared using a 1-mL injector was rapidly inoculated percutaneously into the upper margin of the sixth intercostal rib on the left anterior axillary line to a depth of about 5 mm. The needle was then promptly removed. The mice were maintained in the left lateral decubitus position after injection and were observed until complete recovery.
Micro-CT imaging analysis
Mice with lung cancer underwent micro-CT scanning weekly after cell inoculation. Whole lungs were scanned for the detection of tumors at 20× magnification. For each tumor, micro-CT images were used to reconstruct three-dimensional images (axial, coronal, and 3D). Tumor size was evaluated using imaging software (Xelis; Infinitt, Seoul, Korea). The diameter of the tumor was defined as the maximum diameter of the tumor in a 2D plane. The tumor volume was evaluated using volume analysis software (VGStudio MAX, Heidelberg, Germany). Small tumors were not included in the data analysis owing to the inability to measure the size of these tumors.
H&E staining
At the end of the study, the whole tumor was harvested from each mouse and fixed in 100 mL of buffered formalin for 24 h. Formalin-fixed tissue was paraffin-embedded, sectioned (3–5 μm), and stained with H&E. Sections were evaluated for necrotic degree of tumors and counted under a microscope.
Acridine orange staining
Autophagy is characterized by the formation and promotion of acidic vesicular organelles (AVOs). In acridine orange-stained cells, the cytoplasm and nucleus exhibit bright green and dim red fluorescence, whereas acidic compartments exhibit bright red or orange fluorescence, as described previously [
18]. Following drug treatment, 5 µg/mL acridine orange (A1301; Invitrogen, Carlsbad, CA, USA) was added to the culture medium, and the cells were incubated at 37 °C for 15–30 min. The cells were then trypsinized, washed twice with cold PBS, and observed under a confocal microscope. Fluorescence imaging was performed using a blue bandpass filter with 490 nm excitation, and the fluorescence of the green and orange channels was recorded and merged.
Transient transfection
Adenoviral GFP-LC3B was kindly provided by Dr. Xiao-Ming Yin (University of Pittsburgh School of Medicine, Pittsburgh, PA, USA). After the cells were washed with OPTI-MEM (Invitrogen), DNA was transfected into cells using Lipofectamine™ 2000 according to the manufacturer’s protocol (Invitrogen). After incubation for 4 h, the medium was replaced with complete medium containing 10% serum and antibiotics. The cells were incubated for 24 h and treated as indicated in the figure legends. Images were obtained using a confocal microscope (FluoView™ FV1000; Olympus, Tokyo, Japan).
Western blotting
Cells were harvested and lysed in radioimmunoprecipitation assay buffer [50 mM Tris–Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL each of aprotinin and leupeptin, and 1 mM Na3VO4]. After centrifugation at 12,000×g for 30 min, the supernatant was collected, and the protein concentration was determined by the Bradford method (Bio-Rad Protein Assay; Hercules, CA, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and were subsequently transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in TBS-T [25 mM Tris (pH 7.6), 138 mM NaCl, and 0.05% Tween-20] for 1 h and probed with primary antibodies (at 1:1000–1:5000). After washing, the membranes were incubated with the relevant HRP-conjugated secondary antibody (at 1:2000–1:10,000). Immunoreactive signals were detected using an ECL detection system.
Gene silencing
Pooled small interfering RNA (siRNA) oligonucleotides against ATG5 were purchased from Cell Signaling Technology. siRNA against RIP1 was purchased from Ambion (Austin, TX, USA). Cells were transfected with 100 nM pooled oligonucleotide mixture using Lipofectamine2000 (Invitrogen) according to the manufacturer’s protocol. Twenty-four hours after transfection, media were removed and cells were treated with the indicated drugs. Gene silencing efficacy by siRNA was assessed by a western blot analysis.
Statistical analysis
Each experiment was performed at least three times, and all values are expressed as the mean ± SD of triplicate samples. The Student’s t test was used to determine statistical significance. Values of p < 0.05 were considered statistically significant.
Discussion
In the present study, we attempted to address the intricate relationship between autophagy and necroptosis, focusing on the roles of autophagy in necroptosis by examining the effects of shikonin treatment. We demonstrated an anti-tumor effect of shikonin and that suppressing autophagy enhances shikonin-induced necroptosis in A549 cells. Necroptosis has been shown to be dependent on RIP3, which is activated following phosphorylation by the serine/threonine kinase RIP1 [
19]. RIP1 kinase activity is crucial for necroptosis [
20]; the allosteric RIP1 kinase inhibitor (necrostatin-1) inhibits death receptor-induced necroptosis in various cellular models [
21]. In our study, we found that treatment with shikonin significantly increased the levels of the RIP1 protein in a concentration-dependent manner. These results, in accordance with previous data [
13], indicated that shikonin induces cell death in A549 cells via the RIP1-dependent necroptosis pathway.
Necroptosis is a type of regulated cell death characterized by the loss of plasma-membrane integrity, organelle and cell swelling, and by consequent cell lysis [
22]. Although the exact machinery controlling necroptosis is not completely understood, several key signaling molecules downstream of the death receptor have been identified, including RIP1 [
23], RIP3 [
24,
25], and JNK [
26]. Several key pro-apoptosis factors have also been identified as important negative regulators of TNF-induced necroptosis. For instance, FADD and caspase 8, two essential components in the extrinsic apoptosis pathway are known to suppress necroptosis via the cleavage of RIP1 [
27], while cIAP is able to block both apoptosis and necroptosis [
28].
Similar to the relationship between autophagy and apoptosis, there is evidence suggesting that autophagy is capable of either promoting [
29], suppressing [
30,
31], or having no effect on necroptosis [
32]. A combination of rapamycin and the glucocorticoid dexamethasone triggers autophagy-dependent cell death, with characteristic features of necroptosis in acute lymphoblastic leukemia cells, suggesting that autophagy promotes necroptosis in this particular system [
29]. Basit et al. [
33] demonstrated that recruitment of FADD to Obatoclax-induced autophagosomes directly activated RIPK1 and RIPK3, and that, importantly, caspase inhibition was not required for necroptosis in this setting. In contrast, the majority of studies have tended to demonstrate that autophagy is able to inhibit necroptosis in various systems, such as L929 cells, lymphocytes, or human cancer cells stimulated by TNFα, antigens, or starvation [
30,
34]. At present, the molecular links between autophagy and necroptosis in these studies remain elusive. Here, we used zVAD, widely used as a general caspase inhibitor, as an example to further address the intricate relationship between autophagy and necroptosis. Holler et al. [
21] reported that zVAD greatly sensitizes L929 cells to TNFα-induced necrosis, suggesting that zVAD itself possesses pro-necrotic function in addition to the inhibition of caspases. We also showed that zVAD induces necroptosis since zVAD-induced cell death is inhibited by necrostatin 1 via the suppression of RIP1.
In addition to necroptosis, PARP-mediated cell death is another important form of programmed necrosis. PARP is an energetically expensive process, causing the rapid deletion of intracellular ATP and eventually necrotic cell death [
35]. PARP-mediated necrotic cell death also involves the translocation of apoptosis-inducing factor from mitochondria to nuclei in response to DNA damage [
36]. PARP is readily activated by DNA damage, mainly DNA strand breaks, in response to oxidative stress or DNA-damaging agents [
37]. Since autophagy is inducible by the disturbance of cellular energy homeostasis, the involvement of autophagy in PARP-mediated cell death has attracted substantial attention. Our results showed that autophagic inhibitors increase the expression of cleaved PARP, suggesting that the induction of autophagy by shikonin is related, at least in part, to PARP-mediated cell death in A549 cells.
Shikonin, a naturally occurring naphthoquinone, has been reported to induce necroptotic cell death in cancer cells, including those resistant to drugs and apoptosis [
23,
24]. However, previous studies have not examined whether necroptosis is also induced in NCSCL cells. Flow cytometry with Annexin V and PI double staining is an effective method to quantitatively distinguish between necrosis and apoptosis. The flow cytometry data in this study showed that incubation with Nec-1 before exposure to shikonin substantially reduced the number of necrotic cells, while Z-VAD-FMK, a general inhibitor of apoptosis, had no obvious effect. Despite previous results indicating that Nec-1 suppresses apoptosis in a mouse traumatic brain injury model, it is generally considered a specific inhibitor of necroptosis and has been used to differentiate necroptosis from apoptosis. Our results showed that Nec-1 did not inhibit apoptosis caused by shikonin.
In the present study, A549 cells treated with the autophagy inhibitors 3-MA and bafilomycin A displayed increased expression of cleaved PARP, caspase-3, and RIP1, suggesting that autophagy serves a protective role. These findings are further supported by our observation that shikonin-induced necroptosis was significantly increased in ATG5-silenced A549 cells. We also observed that pretreatment with 3-MA was associated with a decrease in LC3B formation and an increase in p62 levels, essentially reversing the effect of shikonin and blocking autophagy. Accumulation of p62 facilitates autophagic clearance [
38], and evidence indicates that p62 levels are regulated by autophagy and accumulate in autophagy-deficient cells [
39]. We found that pretreatment with bafilomycin A, which blocks subsequent autophagosomal degradation, increased the formation of LC3B in A549 cells after treatment with shikonin. Cross-talk occurring between these signaling pathways is not entirely clear and requires further investigation.
Authors’ contributions
The authors have made the following declarations about their contributions: Conceived and designed: HJK, KEH, HRK, and YSK. Development of methodology: HJK, DSP, HYJ, HRK, and YSK. Acquisition of data: HJK, KEH, HYJ, HRK, and YSK. Analysis and interpretation of data: KEH, DSP, SHO, HYJ, KHY, ETJ, HRK, and YSK. Writing, review, and revision of the manuscript: HJK, KEH, HRK, and YSK. Study supervision: HRK and YSK. All authors read and approved the final manuscript.