RIP1K and RIP3K provoked by shikonin induce cell cycle arrest in the triple negative breast cancer cell line, MDA-MB-468: necroptosis as a desperate programmed suicide pathway
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Resistance to cell death and reprogramming of metabolism are important in neoplastic cells. Increased resistance to apoptosis and recurrence of tumors are the major roadblocks to effective treatment of triple negative breast cancer. It has been thought that execution of necroptosis involves ROS generation and mitochondrial dysfunction in malignant cells. In this study, the effect of shikonin, an active substance from the dried root of Lithospermum erythrorhizon, on the induction of necroptosis or apoptosis, via RIP1K-RIP3K expressions has been examined in the triple negative breast cancer cell line. The expression levels of RIP1K and RIP3K, caspase-3 and caspase-8 activities, the levels of ROS, and mitochondrial membrane potential have been studied in the shikonin-treated MDA-MB-468 cell line. An increase in the ROS levels and a reduction in mitochondrial membrane potential have been observed in the shikonin-treated cells. Cell death has mainly occurred through necroptosis with a significant increase in the RIP1K and RIP3K expressions, and characteristic morphological changes have been observed. In the presence of Nec-1, caspase-3 mediating apoptosis has occurred in the shikonin-treated cells. The current findings have revealed that shikonin provoked mitochondrial ROS production in the triple negative breast cancer cell line, which works as a double-edged executioner’s ax in the execution of necroptosis or apoptosis. The main route of cell death induced by shikonin is RIP1K-RIP3K-mediated necroptosis, but in the presence of Nec-1, apoptosis has prevailed. The present results shed a new light on the possible treatment of drug-resistant triple negative breast cancer.
KeywordsRIP1K RIP3K Triple negative breast cancer cell line ROS Necroptosis Apoptosis
Reactive oxygen species
Mitochondrial membrane potential
In normal development, programmed cell death is an important process to eliminate damaged cells. Multiple mechanisms have been identified for the regulation of cell death including necrosis, apoptosis, and autophagy [1, 2, 3]. Activation of the TNF receptor, resulting in cell death, may be executed through apoptosis or necroptosis . Necroptosis is a kind of cell death that can occur when caspase activity has been blocked [5, 6]. Receptor-interacting protein 1 kinase (RIP1K) is a member of the RIP kinase family (including RIP1K, RIP2K, RIP3K, RIP4K, RIP5K, RIP6K, and RIP7K) that harbors a conserved kinase domain . RIP1K is a multifunctional signal transducer enzyme that is involved in NF-kB activation, apoptosis, and necroptosis. When apoptosis is blocked, RIP1 kinase and thereby necroptosis are activated by the death receptor ligands, such as TNF-α and Fas. Necrostatin-1 (Nec-1) could inhibit necroptosis. In some cell types, such as fibrosarcoma cells, necroptosis is activated via inhibition of caspases by carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) (pan-caspase inhibitor) . Following TNF stimulation, complex I mediates NF-kB activation and MAPK signaling, resulting in cell inflammation and survival. Under cIAP depletion, cytosolic complex II initiates FADD-caspase-8-mediated cell death either independent (complex IIa) or dependent (complex IIb) on RIP1K activity. Typically, RIPK1 and RIPK3 are inactivated by caspase-8-mediated cleavage that results in the suppression of necroptosis. In the absence or inhibition of caspase-8, complex II initiates necroptosis, which is dependent on RIPK1-RIPK3-MLKL . Necroptosis is completely different from apoptosis; therefore, the barriers to apoptosis are no longer problems for necroptosis .
Breast cancer subtypes are based on the estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu receptor (HER-2) expressions . Breast cancer has been also classified into five biologically distinct intrinsic subtypes namely luminal A, luminal B, HER2-enriched, basal-like, and normal-like. Poor prognosis breast cancer, basal-like, occurs more commonly in younger women. This subtype lacks ER, PR, and HER2 expression [12, 13]. Approximately 15 % of globally diagnosed breast cancers are designated as ER−, PR−, and Her2− [14, 15, 16]. MDA-MB-468 is categorized as a triple negative breast cancer cell line [17, 18, 19]. Although this subtype benefits from chemotherapy, less toxic and more sensitive treatment is required . Several different molecular studies have been reported on the induction of apoptosis in basal-like breast cancers [12, 20, 21, 22, 23]. Increased resistance to apoptosis and recurrence of tumors are the major roadblocks to effective treatment of triple negative breast cancer [24, 25]. Inactivation or downregulation of proapoptotic effectors and upregulation of antiapoptotic factors are mechanisms which elicit resistance to apoptosis in advanced cancer cells. Therefore, new approaches are essential for the treatment of advanced triple negative breast cancers which are resistant to current therapies [26, 27, 28].
In the current investigation, the effect of shikonin on the induction of necroptosis or apoptosis through RIP1K and RIP3K expressions was evaluated in the triple negative breast cancer cell line, MDA-MB-468.
Materials and methods
Reagents and antibodies
Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham (DMEM/Ham’s F12), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco (Grand Island, NY, USA). Rabbit anti-actin (Santa Cruz, CA, USA) and rabbit anti-RIP1K and rabbit anti-RIP3K (Cell Signaling Technology Inc., Danvers, MA) were used, as primary antibodies for Western blotting. The secondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit (Bio-Rad, Hercules, CA, USA). Shikonin and Nec-1 were purchased from CalBiochem (EMD Chemicals, Inc., San Diego, CA, USA). The purity of shikonin was ≥98 % by HPLC. The pan-caspase inhibitor, Z-VAD-FMK, was from BD Biosciences (Becton Dickinson, San Jose, CA). Protein concentration was measured using the Bradford kit provided by Bio-Rad Laboratories (Hercules, CA, USA). ECL Advance Western Blotting Detection Kit was obtained from Amersham Company (Buckinghamshire, UK). Polyvinylidene fluoride transfer membrane (PVDF) and complete protease inhibitor (cocktail tablets) were obtained from Roche (Life Science, USA). Other reagents and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The Caspase-3 Fluorometric Assay Kit was bought from BioVision, Inc. (Milpitas, CA, USA). The CaspGLOW™ Fluorescein Active Caspase-8 Staining Kit was from eBioscience, Inc. (San Diego, CA USA). Marker Gene™ Live Cell Fluorescent Reactive Oxygen Species Detection Kit was obtained from Marker Gene Technologies, Inc. (Eugene, OR). JC-1 Mitochondrial Membrane Potential Assay Kit was obtained from Cayman Chemical Company (Ann Arbor, MI, USA).
MDA-MB-468 breast cancer cell line was obtained from the Iranian Biological Resource Center (IBRC, Tehran, Iran). MDA-MB-468 was cultured in DMEM-Ham’s F12 supplemented with 10 % FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, at 37 °C, 5 % CO2 and humidified atmosphere. The cells were provided with fresh medium every 2 to 3 days. Cells were harvested at 70–90 % confluence with trypsin 0.25 % ⁄EDTA 0.02 % and were either used fresh or frozen on liquid nitrogen (stored at −70 °C). Shikonin was dissolved at a concentration of 50 mM in DMSO as a stock solution and stored in the dark at −20 °C. Nec-1 and Z-VAD-FMK were dissolved in DMSO to a storage concentration of 1 mM. Different concentrations (μM) of shikonin, Nec-1, and Z-VAD-FMK for cell line treatment were prepared in the cultured medium. Control cells were incubated with a volume of DMSO equal to that added to the cultures that received drugs. The maximum final concentration of DMSO was less than 0.1 % for each treatment.
Cell viability assay
Assessment of cell death
To quantify the cell death modality, annexin V/propidium iodide (PI) staining assay was applied. Briefly, 3 × 105 cells were plated and pretreated with 50 μM Nec-1 or 20 μM Z-VAD-FMK for 3 h and then the cells were treated with shikonin (5 μM for 24 h). After treatment, cells were washed twice with phosphate buffered saline (PBS), mixed with 500 μl of binding buffer, and stained with 5 μl of annexin V-fluorescein isothiocyanate (FITC) and 5 μl of PI (PI 50 μg/ml) for 10 min at room temperature in the dark. Apoptotic cells were quantified by a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA); 1 × 104 cells were counted for each sample. Both early apoptotic (annexin V-positive, PI-negative) and late apoptotic (double positive of annexin V and PI) cells were detected. The percentages of cells in each quadrant were analyzed using the Flowing Software version 2.4.1 (Turku Centre for Biotechnology University of Turku, Finland). The experiments were repeated at least three times.
Western blot analysis
MDA-MB-468 cells (1–3 × 105) were treated with 5 μM shikonin for 12 h in a six-well plate. After treatment, cells were washed with PBS and resuspended in a digestion buffer [150 mM NaCl, 25 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1.0 % Triton X-100, 1.0 % sodium deoxycholate, 0.1 % SDS] containing a cocktail of complete protease inhibitors on ice for 1 h. Cell lysates were centrifuged at 14,000g for 15 min at 4 °C. The supernatants were collected and the protein concentration was determined by the Bradford assay. A total volume of 50 μg protein in 62.5 mM loading buffer (pH 6.8, containing 25 % glycerol, 2 % sodium dodecyl sulfate (SDS), 0.01 % bromophenol blue, and 5 % β-mercaptoethanol) was boiled for 5 min and loaded into each lane of 10 % SDS-PAGE gel. The polypeptides were electrotransferred to a PVDF. Nonspecific binding was blocked with 5 % skimmed milk in TBST (10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05 % Tween 20) at 4 °C for 6 h. Then, the membranes were incubated with either RIP1K or RIP3K, and the β-actin primary antibody was diluted in 0.7 % skimmed milk in TBST overnight at 4 °C and then washed with TBST solution. Following washing, the secondary antibodies were labeled with HRP, diluted in 0.7 % skim milk in TBST, and added to the membrane for 2 h at room temperature. They were washed then with TBST solution. The antigen-antibody complexes were detected by enhanced chemiluminescence using a ChemiDoc™ Imaging System (Bio-Rad Laboratories, CA). Western blot bands were measured with the Image Lab 4.1 software (National Institutes of Health, USA) to analyze the integrated density value (IDV). The average IDV values of RIP1K and RIP3K with β-actin were compared and the average relative value was obtained.
Cell cycle analysis
In ethanol-fixed cells, the fluorescent molecule, PI, binds to double-stranded DNA in the nucleus. In DNA-damaged cells, sub-G1 stage, the PI fluorescence intensity was weaker than that of normal cells . In the present study, 3 × 105 cells in each well were grown in 12-well plates overnight. Cells were treated with 5 μM shikonin for 12 h. Cells were washed twice with PBS and resuspended in 0.3 ml of PBS. For cell fixation, 0.7 ml of cold 70 % ethanol was added to samples on ice for at least 3 h. Cells were washed with PBS and resuspended in 0.25 ml of PBS, with 5 μl of 10 mg/ml RNase A and Triton X-100 (0.1 %). Cells were incubated at 37 °C for 1 h and 10 μl of 50 μg/ml PI was added. The DNA content of the cells was measured using a FACS Calibur flow cytometer. Apoptotic cells were considered to constitute the sub-G1 population, and the percentage of nonapoptotic cells in each phase of the cell cycle was determined. Cytographs were analyzed using FlowJo v 10.0.8.
Measurement of caspase-3/DEVDase activity
The influence of shikonin on caspase-3 activity in MDA-MB-468 was detected using Caspase-3/CPP32 Fluorometric Assay Kit (BioVision Research Products, Mountain View, CA, USA). Cleavage of DEVD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin) was detected for caspase 3 activity. DEVD-AFC emits a blue light (λ max = 400 nm) but free AFC, upon cleavage of the substrate by CPP32, emits a yellow-green light (λ max = 505 nm). Cultured cells in a 96-well plate were pretreated with 20 μM Z-VAD-FMK or 50 μM Nec-1, 3 h prior to treatment with 5 μM shikonin. After 12 h of treatment, 0.5–2 × 105 cells were lysed in 50 μl chilled cell lysis buffer on ice, for 10 min. The amount of protein in each well was measured using the Bradford method . After the addition of 50 μl of 2× reaction buffer (containing 10 mM DTT), cell lysate from shikonin-treated or control cells were incubated at 37 °C for 2 h with 50 μM DEVD-AFC substrate. Samples were read at 400 nm excitation filter and 505 nm emission filter with a fluorescent microplate reader (BioTek Synergy HT, Winooski, VT, USA), and the results were expressed as fold increase over the basal level (control cells).
Fluorescein-active caspase-8 staining assay
Caspase activation plays a central role in the cell apoptosis . The fluorescent marker, FITC-IETD-FMK, is a labeled, cell-permeable, nontoxic inhibitor that binds irreversibly to activated caspase-8 in living cells. Cultured cells in a 24-well plate were pretreated with 20 μM Z-VAD-FMK or 50 μM Nec-1, 3 h prior to treatment with 5 μM shikonin. After 24 h of treatment, according to the manufacturer’s protocol, cellular active caspase-8 was determined. Briefly, after treatment of the cells with shikonin, 300 μl each of the induced and control aliquots was transferred into Eppendorf tubes, and 1 μl of FITC-IETD-FMK was added to each tube and incubated for 0.5–1 h in a 37 °C incubator with 5 % CO2. Control cells were unlabeled. Cells were centrifuged at 3000 rpm for 5 min and the supernatant was removed. Cells were resuspended in 0.5 ml of wash buffer and centrifuged again. Cells were resuspended in 100 μl of wash buffer and then the cell suspension was transferred to each well of the black microtiter plate. The fluorescence intensity was measured at 485 nm excitation and 535 nm emission.
Detection of intracellular reactive oxygen species
For the assessment of intracellular ROS formation in cultured breast cancer cells, a fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used . DCFH-DA was dissolved in DMSO at a concentration of 10 mM. To assay ROS, cells were plated in a 96-well plate. Assay was administered with DCFH-DA (20 μM working substrate solution) in a culture medium. After incubation at 37 °C for 45 min, the substrate solution was removed and the cells were washed with PBS. Shikonin was added to wells at a concentration of 5 μM to generate ROS for 4 h. Untreated cells were used as control. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a fluorescent microplate reader (BioTek Synergy HT, Winooski, VT, USA). The ROS levels were expressed as RFU. All experiments were performed at least in triplicate.
Assessment of mitochondrial membrane potential (Δψm)
JC-1, a lipophilic cationic dye, was used to evaluate the effect of shikonin on the potential of mitochondrial membrane. A decrease in the red/green fluorescence intensity ratio shows mitochondrial depolarization . Briefly, cells were seeded into a white 96-well plate at a density of 5–7.5 × 103 cells/well in 200 μl media. Following 24 h of incubation, shikonin was added at a concentration of 5 μM and the cells were incubated for further 12 h, respectively (50 μM Nec-1 and/or 20 μM Z-VAD-FMK was added 3 h before stimulation with shikonin). The assay was terminated by aspirating the media and the addition of 10 μl of staining solution, and the plate was incubated for 15 min at 37 °C, 5 % CO2. The plate was centrifuged for 5 min at 400g and the supernatant was removed. Two hundred microliters of assay buffer was added. After another centrifuge step for 5 min at 400g, the supernatant was removed and 100 μl assay buffer was added. Analysis was performed on a fluorescent microplate reader with the filters set to 485 nm excitation/528 nm emission (green) and 530 nm excitation/590 nm emission (red). Data were presented as the ratio of fluorescence intensity of green (590 nm, emission of J-aggregate form) to fluorescence intensity of red (530 nm, emission of JC-1 monomeric form). All experiments were performed at least in triplicate.
Morphologic analysis: Hoechst 33258, PI, and JC-1 staining
MDA-MB-468 cells (1–5 × 105 cells/well) were allowed to grow on coverslips in eight-well culture plates for 24 h. The cells were then treated with shikonin at 10 μM concentration for 6 h at 37 °C. Cells growing on glass coverslips were fixed in methanol/acetic acid (3/1) for 10 min at 37 °C. The fixed cells were incubated with Hoechst 33258 (1 mg/ml) for 10 min in a CO2 incubator at 37 °C. For PI staining, fixed cells were resuspended in 0.25 ml of PBS, with 5 μl of 10 mg/ml RNAse A at 37 °C for 1 h. Then, MDA-MB-468 cells were incubated with PI (1 mg/mL) for 10 min at room temperature. After a final wash in PBS, the samples were visualized with a fluorescence microscope (Nikon, Melville, USA). Excitation wavelength of 330–380 nm was applied for the Hoechst dye and 450–490 nm for PI. Apoptotic cells were identified based on morphologic changes in their nuclear assembly by observing chromatin condensation and fragment staining by the Hoechst dye. Necroptotic cells were identified based on the positive staining with PI and without apoptotic nuclear morphology with the Hoechst dye. After cells were treated with 5 μM shikonin, the cultured MDA-MB-468 cells were loaded with the mitochondrial potential indicator JC-1 at 5 μM for 30 min at 37 °C. Regions of high mitochondrial polarization are indicated by red fluorescence due to J-aggregate formation by the concentrated dye. Depolarized regions are indicated by the green fluorescence of the JC-1 monomers. Healthy cells with mainly JC-1 J-aggregate can be detected with fluorescence ex/em = 540/570 or ex/em = 590/610 nm. Apoptotic or unhealthy cells with mainly JC-1 monomers can be detected with ex/em = 485/535 nm.
IC50 value was determined using the GraphPad Prism statistical software 6 (CA, USA). To compare the data, one-way analysis of variance (ANOVA) was used followed by Dunnett’s post hoc test. In all cases, the mean ± SD of at least three independent experiments was presented; P < 0.05 was taken as the level of significance.
Cell viability assay
Shikonin induced cell death modalities in breast cancer cells
Shikonin upregulates RIP1K and RIP3K expression
Cell cycle analysis
Caspase-3 and caspase-8 activities in shikonin-mediated apoptosis
ROS generation plays critical roles in shikonin-induced cell death
Reduction of mitochondrial membrane potential
RIP1 and RIP3 are regarded as crucial modulators of necroptosis. Like TNF stimulation, surprisingly, shikonin treatment also induced pronecrotic complex formation between RIP1K and RIP3K, although it was less substantial than TNF stimulation . In one study, the protein levels of RIP1K and RIP3K were significantly increased in osteosarcoma cell lines, K7 and U2OS, after treatment with shikonin in a concentration-dependent manner . Here, we have also showed that the expression of RIP1K and RIP3K was upregulated by shikonin. In some studies, the antiproliferative and apoptotic property of shikonin and its derivatives was shown on some tumor cells, including breast cancer, hepatocellular carcinoma, malignant melanoma, colorectal carcinoma, oral squamous cell carcinoma, and chronic myelogenous leukemia . Recent studies have also shown that shikonin was specifically accumulated in the mitochondria and deregulated cellular Ca2+ and ROS levels, which disrupted the mitochondrial membrane . Our findings revealed that shikonin acts as a double edge executioner’s ax, which disrupts the triple negative breast cancer cells either by necroptosis or apoptosis. When the apoptotic pathway was attenuated or blocked by Z-VAD-FMK, the necroptotic pathway becomes dominant. In the presence of Nec-1, a RIP1K inhibitor, the necroptotic pathway was blocked and apoptosis is a selective route for cell death. Although shikonin has induced both routes of cell death, indeed it is a potent necroptotic inducer and could be a good candidate for the treatment of apoptotic resistant triple negative breast cancer. Along with RIP1K deubiquitination, shikonin induced a typical caspase-3-dependent apoptotic cell death in MDA-MB-468 cell line. Other investigations have also shown that shikonin induced caspase-dependent apoptosis in other cancer cells [41, 43, 44].
ROS are critical factors that mediate cell necrosis via death receptor such as TNFR1 . During the early phase of apoptotic progression, shikonin has the ability to generate large amounts of intracellular ROS and was consequently accompanied by the disturbance of mitochondrial transmembrane potential in the hepatoma SK-Hep-1 cells . From the mechanistically point of view, we found that the primary effect of shikonin, through RIP1K and RIP3K activation, is the direct targeting of the mitochondria. A dose-dependent overproduction of ROS and the breakdown of the mitochondrial membrane potential are some strong lines of evidence that support the mitocan action of shikonin. It has been shown that an increase in the levels of intracellular ROS and mitochondrial injury may result in the oxidative DNA damage, inhibition of cancer cell migration, and cell cycle arrest . Shikonin-induced mitochondrial ROS might immediately destroy the mitochondrial function and then induces the intrinsic apoptotic pathway. Shikonin could act as a prooxidant agent and was likely binding to complex II because TTFA prevented the shikonin-induced ROS elevation . Although ROS production is not essential in all instances of necroptosis, increasing mitochondrial ROS production can act as a second messenger in the signaling pathways leading to cell death . Parallel with the results of other investigations, we have observed an increase in the amount of ROS in the presence of shikonin which implies two separate cell death paths by increasing intrinsic oxygen-free radicals. It has been shown that Nec-1 protected cells from necrosis by blocking the mitochondrial membrane potential reduction . In the current study, shikonin has significantly reduced the mitochondrial membrane potential, following RIP1K involvement in complex II. Mitochondrial membrane potential has been increased in the Nec-1-pretreated cells, which improves mitochondrial function and provides another proof for the involvement of the mitochondria in the shikonin-induced cell death by mediating RIP1K-RIP3K. It has been demonstrated that shikonin could arrest the cell cycle at G1/G0 and induces apoptosis in the tumor cells and was associated with a disturbance of cell cycle regulation . In the current study, it has been shown that the sub-G1 or sub-G2 cell population has increased in the presence of Nec-1 or Z-VAD-FMK, respectively. In the presence of Nec-1, the cells treated with shikonin in sub-G1 stages were increased, which indicates that the cells were in the apoptotic phase. However, by the addition of Z-VAD-FMK to the cell milieu, the sub-G2 stage has been increased which probably represents those in the necroptotic phase. These results provide more lines of evidence for the dual action of shikonin mediating RIP1K-RIP3K on the induction of cell death, necroptosis, or apoptosis. Since there is no unique biochemical evidence for necroptosis, morphological alteration could help to evaluate cell death modality . Caspase activation, chromatin condensation, nuclear fragmentation, DNA cleavage, formation of apoptotic bodies, and rapid and efficient phagocytosis are remarkable points in the cell apoptosis. In the cell necroptosis, no massive caspase activation, no chromatin condensation, intact nuclei, spillage of cell content, phagocytosis by macropinocytosis, lysosomal leakage, and an oxidative burst have been observed . In our study, the morphological findings have supported a dominant necroptosis by shikonin via RIP1K-RIP3K expressions in MDA-MB-468 cells.
As a conclusion, the current findings have indicated that the main route of shikonin-induced cell death goes through RIP1K-RIP3K-mediated necroptosis. It circumvents the effect of strong road blockers of necroptosis such as Nec-1 by induction of caspase-dependent apoptosis. Shikonin provokes ROS production in the mitochondria of triple negative MDA-MB-468 cells, which works as a double-edged executioner’s ax in the execution of cell death, necroptosis, or apoptosis. Although the precise molecular mechanism of shikonin on the induction of cell death has remained to be further elucidated, the current results shed a new light on the treatment of drug-resistant triple negative breast cancers.
Part of this work was supported by a Ph.D. grant from Tarbiat Modares University. The authors would like to express their gratitude to Professor Peter Vandenabeele for his valuable comments. The sincere cooperation of Mrs. Batoul Etemadi-kia, lab expert, is much obliged.
Conflicts of interest
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