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Tumor Biology

, Volume 37, Issue 4, pp 4479–4491 | Cite as

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

  • Zahra Shahsavari
  • Fatemeh Karami-Tehrani
  • Siamak Salami
  • Mehran Ghasemzadeh
Original Article

Abstract

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.

Keywords

RIP1K RIP3K Triple negative breast cancer cell line ROS Necroptosis Apoptosis 

Abbreviations

ER

Estrogen receptor

PR

Progesterone receptor

HER-2

HER-2/neu receptor

Z-VAD-FMK

Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone

Nec-1

Necrostatin-1

ROS

Reactive oxygen species

Δψm

Mitochondrial membrane potential

FITC

Fluorescein isothiocyanate

PI

Propidium iodide

MTT

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

Introduction

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 [4]. 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 [7]. 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) [8]. 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 [9]. Necroptosis is completely different from apoptosis; therefore, the barriers to apoptosis are no longer problems for necroptosis [10].

Breast cancer subtypes are based on the estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu receptor (HER-2) expressions [11]. 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 [12]. 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].

It has been shown that execution of necroptosis involves reactive oxygen species (ROS) production which results in mitochondrial dysfunction [29], although not essential for all cases of necroptosis. In the cell death signaling pathways, mitochondrial ROS elevation could serve as a second messenger [30]. In cancer cells, induction of intrinsic ROS by drugs leads to cell cytotoxicity that essentially disrupts tumor cells or blocks their proliferation [31]. Shikonin, a naphthoquinone pigment (Fig. 1), is the most important pharmacologically active substance from the dried root of Lithospermum erythrorhizon which increases intracellular ROS levels and causes mitochondrial injury [32, 33].
Fig. 1

Structure of shikonin (C16H16O5)

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).

Cell culture

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

Viability of the cells was determined by the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The assay is based on the reduction of yellow tetrazolium MTT by the alive cells [23]. Briefly, adherent cells were detached by treatment with 0.25 % trypsin/0.02 % EDTA, and an aliquot of 5–7.5 × 103 cells was placed in each well (200 μl) of a 96-well plate (Thermo Scientific, Germany). Cells were allowed to attach overnight and then were stimulated with different concentrations of shikonin (0.5, 2.5, 5, 10, 15, 20, 25 μM) for 6, 12, 24, or 48 h. Each well was incubated with 20 μl MTT at 37 °C for 4 h. The supernatant was then removed and 200 μl DMSO was added into each well in order to solubilize the blue-purple crystals of formazan. Absorbance was measured at 570 nm with a microplate reader (Tecan, Austria). Each assay was done at least six times, with three replicates each. The viability was evaluated based on a comparison with untreated cells. IC50 values represent the shikonin concentrations required to inhibit 50 % of cell proliferation and were calculated by GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). The survival rate was calculated according to the following formula:
$$ \mathrm{Survival}\kern0.5em \mathrm{rate}=\left(\mathrm{Absorbance}\;\mathrm{of}\;\mathrm{treatment}/\mathrm{Absorbance}\;\mathrm{of}\;\mathrm{control}\right)\times 100\;\%. $$

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 [31]. 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 [34]. 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 [27]. 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 [35]. 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 [28]. 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.

Statistical analysis

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.

Results

Cell viability assay

The effects of shikonin to determine the viability of the breast cancer cell line were examined by the MTT assay. We first determined the effect of shikonin on MDA-MB-468 cell proliferation by treating cells with shikonin at various concentrations (2.5, 5, 10, 15, 20, and 25 μM) for 6, 12, 24, or 48 h. As shown in Fig. 2, shikonin significantly inhibited the viability of MDA-MB-468 cells in a time- and dose-dependent manner. Shikonin treatment resulted in a dose-dependent reduction in cell viability when compared with the control cells. The cytotoxic effect was more evident at 48 h in the cell line. The effective dose of shikonin that inhibited 50 % of growth (IC50) of MDA-MB-468 cells after 12 h of treatment was 3.586 μM. The concentration of 5 μM was used as the optimum concentration in the subsequent experiments. Therefore, MDA-MB-468 cells are exquisitely sensitive to cell death induced by shikonin treatment, and shikonin inhibits the proliferation of triple negative breast cancer cell line.
Fig. 2

Shikonin induces cell death in MDA-MB-468 cell line in a dose- and time-dependent manner. MDA-MB-468 cells were treated with shikonin in increasing concentrations (2.5, 5, 10, 15, 20, and 25 μM) for 6, 12, 24, or 48 h as indicated. The cell viability was determined by the MTT assay. Viability of untreated cells was set at 100 %. Data shown are mean ± SD of triplicate samples at six independent experiments with similar results. *P < 0.05, **P < 0.01, and ***P < 0.001 denote means significantly different from untreated cells

Shikonin induced cell death modalities in breast cancer cells

In order to examine whether the effects of shikonin on the cells were associated with the induction of apoptosis, FITC-conjugated annexin V and PI staining was used to distinguish apoptotic cells. Cells were incubated with shikonin, 5 μM, for 12 h and the percentages of apoptotic cells were measured. Control cells were negative for both annexin V-FITC and PI (Fig. 3a). A significant increase has been shown in the percentage of apoptosis in MDA-MB-468 cells (P ≤ 0.05) (Fig. 3b). Necroptosis is optimally induced when the apoptotic machinery is interrupted. To delineate the cell death modes by shikonin, MDA-MB-468 cells were treated with shikonin in combination with either Z-VAD-FMK or Nec-1. Taken together, shikonin induces a dominant necroptosis in triple negative breast cancer cells. Importantly, this shikonin-induced necroptosis observed was inhibited in the presence of the RIPK1 kinase inhibitor Nec-1. Nec-1 converts shikonin-induced necrosis to apoptosis. The addition of Z-VAD-FMK to cells under apoptosis-inducing condition reduced apoptosis. No significant alteration was found in the shikonin- and shikonin/Z-VAD-FMK-treated cells at the early apoptosis stage compared to control cells, but the early apoptosis stage of shikonin/Nec-1-treated cells increased significantly. Similarly, the cells at the late apoptosis stage were also attenuated by Z-VAD-FMK. There was no significant difference between the percentages of necrosis in the shikonin/Nec-1-treated cells with the control cells. This suggested that Nec-1 effectively blocked shikonin-induced necrosis.
Fig. 3

Shikonin induced cell modes in MDA-MB-468 cell line. a Shikonin (5 μΜ) treated MDA-MB-468 cells in the absence or presence of Nec-1 (50 μΜ) and or Z-VAD-FMK (20 μM) for 12 h. Nec-1 and Z-VAD-FMK were pretreated for 3 h prior to shikonin treatment. b After 12 h of incubation with shikonin, both the percentages of necrotic cells (stained with PI only) and late apoptotic cells (stained with both annexin V and PI) increased significantly. The necrotic cells were significantly inhibited by pretreatment with Nec-1 and early and late apoptotic cells suppressed by preincubation with Z-VAD-FMK. Induction of necrosis by shikonin was blocked by Nec-1 in the breast cancer cells. Additionally, late apoptotic cells reduced when they were pretreated with Z-VAD-FMK. Data are representative of three independent experiments. *P < 0.05 denotes a mean significantly different from untreated cells

Shikonin upregulates RIP1K and RIP3K expression

We compared RIPK1 and RIPK3 protein expressions in MDA-MB-468 cell line and found that RIP1K and RIP3K proteins were ubiquitously detected in this triple negative cancer cells tested. In this study, RIP1K was upregulated significantly after shikonin treatment. Similarly, the RIP3K expression in MDA-MB-468 cells was upregulated significantly by 5 μM shikonin after 12 h (Fig. 4, P < 0.05).
Fig. 4

The expression of RIP1K and RIP3K in shikonin-treated MDA-MB-468 cells. In a six-well plate, the cells were cultured at 80 % confluence and then treated with 5 μM shikonin for 12 h. After treatment, cytosolic protein was extracted to assess RIP1K and RIP3K expressions. As an internal loading control, β-actin was used. Fifty micrograms of protein was loaded onto a 10 % SDS-polyacrylamide gel and analyzed by immunoblotting with antibodies against RIP1K, RIP3K, or β-actin. The protein expressions were subsequently quantified by densitometric analysis. After repeating the experiments three times, similar results were obtained. *P < 0.05 denotes a mean significantly different from untreated cells

Cell cycle analysis

Flow cytometric cell cycle analysis was performed on triple negative breast cancer cells after 12 h of treatment with 5 μM shikonin (Fig. 5a). Shikonin significantly increased the percentage of cells in the sub-G1 and super-G2 phases (Fig. 5b), representing an increase in cell death, consistent with the induction of apoptosis and necroptosis. Z-VAD-FMK significantly attenuated sub-G1 and increased sub-G2 amounts of shikonin-treated cells. Necroptotic cells were accumulated in the super-G2 phase of the cell cycle. In the presence of Nec-1, sub-G1 increased and super-G2 decreased, respectively. These results showed that shikonin is a potent inducer of cell death modes of apoptosis and necroptosis in triple negative breast cancer cells.
Fig. 5

Shikonin induces cell cycle arrest, apoptosis, or necroptosis death, in MDA-MB-468 cells. a Typical DNA content histograms of cells treated with shikonin for 12 h. b Statistical analysis of cell cycle distribution of cells after treatment with shikonin for 12 h. Data points are means of at least three independent experiments. *P < 0.05 denotes a mean significantly different from untreated cells

Caspase-3 and caspase-8 activities in shikonin-mediated apoptosis

Following treatment of MDA-MB-468 with 5 μM of shikonin, an increase in the activity of caspase-8 (Fig. 6) and caspase-3 (Fig. 7) has been observed (P < 0.01, compared with that of the controls). Inhibition of caspase by Z-VAD-FMK (20 μM) suppressed shikonin-induced apoptosis (P < 0.01), while in the presence of Nec-1 (50 μM), elevation of caspase-3 and caspase-8 activity has been shown.
Fig. 6

Detection of active caspase-8 in breast cancer cells. MDA-MB-468 cells were seeded on the same day in a Costar black wall/clear bottom 24-well plate. The cells were treated with shikonin at the final concentration of 5 μM in the absence or presence of Nec-1 (50 μΜ) and Z-VAD-FMK (20 μM) for 24 h, while the untreated cells were used as control. Nec-1 and Z-VAD-FMK were pretreated for 3 h before shikonin treatment. Fluorescence intensity measurement has been made at Ex = 485 nm and Em = 535 nm. ****P < 0.0001 denotes a mean significantly different from untreated cells

Fig. 7

Specific activity of caspase-3 in MDA-MB-468 cells. Following treatment with shikonin, caspase activity was quantified by an enzymatic assay. The activity of caspase-3 is increased after shikonin treatment. Z-VAD-FMK decreased caspase-3-specific activity and Nec-1 increased that. Protein level was measured by the Bradford method. ****P < 0.0001 denotes a mean significantly different from untreated cells, ns not significantly different from untreated cells

ROS generation plays critical roles in shikonin-induced cell death

After treatment with shikonin, the intracellular ROS was determined by the DCFH-DA probe. In MDA-MB-468 cells, after treatment by shikonin, a significant increase was observed in the ROS levels when compared with that of control cells (shown in Fig. 8). Taking the above results together, exposure of cells to shikonin led to mitochondrial dysfunction that resulted in ROS production via RIP1K, which contributed to necroptosis. We observed increased ROS production shortly after cellular shikonin uptake, and ROS levels continuously increased for at least 1 h after exposure to shikonin. Thus, these data indicated that shikonin-induced necroptosis was associated as well with oxidative stress. Thus, shikonin is indeed a potent ROS inducer.
Fig. 8

MDA-MB-468 cells generated ROS after treatment with shikonin. Cells were loaded with 2′,7′-dichlorofluorescin diacetate at 20 μM final concentration for 30 min before treating with shikonin (5 μM) for 4 h. After treatment with shikonin, ROS levels were measured at 485 nm excitation with 528 nm emission in a microplate reader. Data points represent RFU (relative fluorescence unit) of three independent experiments. **P < 0.01 denotes a mean significantly different from untreated cells

Reduction of mitochondrial membrane potential

Following treatment of MDA-MB-468 with 5 μM shikonin for 12 h, a significant decrease in the ratio between red and green fluorescence has been noticed (Fig. 9) which implies that mitochondrial membrane potential has been depleted (P < 0.0001). These data suggest that the mitochondria are engaged in shikonin-induced apoptosis and necroptosis.
Fig. 9

Effects of shikonin on mitochondrial transmembrane potential in MDA-MB-468 cell line. After treatment with shikonin (5 μM) and in the absence or presence of Nec-1 and Z-VAD-FMK for 12 h, cells were stained with JC-1, which has a strong red fluorescence in healthy mitochondria. Shikonin induced a decrease of the red JC-1 fluorescence after 12 h of treatment, indicating a breakdown of the mitochondrial membrane potential. In healthy cells with high mitochondrial ΔΨm, JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the other hand, in apoptotic or unhealthy cells with low ΔΨm, JC-1 remains in the monomeric form, which shows only green fluorescence. Data represent the average values from triplicates of three independent experiments, ****P ≤ 0.0001 is significant. Statistical analysis was performed by ANOVA

Morphological analysis

For clarifying the death mode in MDA-MB-468 cells treated with shikonin, we performed a morphological investigation although it is difficult to precisely identify between necroptosis, apoptosis, and necrosis in various methods until recently. As shown in Fig. 10a, cells were incubated with 5 μM shikonin for 12 h in the absence or presence of Z-VAD-FMK and Nec-1 and then were observed by a phase-contrast microscope. Shikonin-induced cell death was confirmed by PI staining characterized by a loss of plasma membrane integrity and morphology of necrotic death. MDA-MB-468 cells were incubated with 5 μM shikonin for 12 h for appropriate intervals (Fig. 10b). To detect apoptosis, nuclear condensation was observed by Hoechst 33258 staining. MDA-MB-468 cells were incubated with 10 μM shikonin for 12 h. There were changes in cell morphology, i.e., increase in the chromatin condensation or fragmentation and plasma membrane blebbing compared with those of controls (Fig. 10c). Shikonin-treated cells exhibited apoptotic nuclear fragmentation (pointed by the arrowhead in the figure). In addition, majority of cells had a typical necrotic cell death morphology (shikonin/Z-VAD-FMK-treated cells), as manifested by the extensive vesiculation of cytoplasmic organelles and rupture of the plasma membrane. Thus, based on these morphologic findings, cell death caused by shikonin might be associated with apoptosis and necroptosis. To check the status of the mitochondria, MDA-MB-468 cells were stained with JC-1. Cells were incubated with 5 μM shikonin for 12 h. Shikonin induced a decrease of the red JC-1 fluorescence after 12 h of treatment, and shikonin accumulated in the mitochondria indicating a breakdown of the mitochondrial membrane potential (Fig. 10d).
Fig. 10

Microscopic morphology of MDA-MB-468 after shikonin treatment. a Shikonin-induced cell death morphology in MDA-MB-468 cells. Cells were incubated with 5 μM shikonin for 12 h in the absence or presence of Z-VAD-FMK and Nec-1. b Shikonin-induced cell death is characterized by a loss of plasma membrane integrity and morphology of necrotic death. MDA-MB-468 cells were incubated with 5 μM shikonin for 12 h for appropriate intervals. The integrity of the plasma membrane was examined by a PI exclusion assay. c Detection of typical features of apoptosis nuclear condensation by Hoechst 33258 staining. MDA-MB-468 cells were incubated with 5 μM shikonin for 12 h. There were changes in cell morphology, i.e., increase in the chromatin condensation or fragmentation (dashed arrow) and plasma membrane blebbing compared with those of controls. d Distribution of JC-1, J-aggregate fluorescence in normal and treated cells. MDA-MB-468 cells were stained with JC-1. Cells were incubated with 5 μM shikonin for 12 h. Shikonin induced a decrease of the red JC-1 fluorescence after 12 h of treatment. Shikonin accumulated in the mitochondria indicating a breakdown of the mitochondrial membrane potential

Discussion

Resistance to cell death and reprogramming of metabolism are important in neoplastic cells [32, 35]. The most aggressive and treatment-resistant breast cancer is the ER, PR, and Her2-negative subtype. Therefore, alternative approaches that trigger nonapoptotic cell death are sought to compensate for apoptotic resistant cells [26]. Developing a class of anticancer agents that target the weak point of cancer by induction of necroptosis may significantly improve the effectiveness of chemotherapy, especially the drug-resistant cancers [10, 36]. Therefore, in the present study, we assessed the effects of RIP1K and RIP3K expressions by shikonin on the induction of two programmed cell death pathways, apoptosis and necroptosis, in the triple negative breast cancer cells, MDA-MB-468, and then explored the possible mechanisms (illustrated in Fig. 11) [4, 37]. It has been reported that TNF-α stimulation may result in the induction of cell death through two ways, apoptosis or necroptosis [38]. Shikonin interferes with energy-generating mitochondrial processes that consequently result in the accumulation of ROS, destabilization of the mitochondria, and induction of apoptosis. In a clinical trial, growth of the tumor was suppressed by shikonin in patients with advanced lung cancer. It has also been shown that shikonin reduced serum estrogen and progesterone in a MCF-7 human breast cancer cell xenograft model [39] with no drug resistance in MCF-7 breast cancer cells [40].
Fig. 11

The TNF-α death complexes induced by shikonin. TNF-α stimulation of TNFR1 results in the formation of complex I at the cytoplasmic membrane that includes TRADD, TRAF2, RIP1K, and cIAP1. RIP1K, FADD, and caspase-8 are involved in the formation of complex IIa that activates caspase cascade in apoptosis. Caspase-8 cleavage of RIP1K and RIP3K inhibits necroptotic signaling. Some factors like caspases (3, 8, 9, etc.), Bax, and ATP increased during apoptosis but Bcl-2 and Δψm decreased. Inhibition of caspase-8 by Z-VAD-FMK results in RIP1K and RIP3K interaction and complex IIb formation that mediates necroptosis. Kinase activity of RIP1K is required for complex IIb formation, which is blocked by Nec-1. This complex has been proposed to adopt an amyloid structure. When lethal signals prevail, mitochondrial outer membrane permeabilization occurs and leads to Δψm dissipation, arrest of mitochondrial ATP synthesis, and Δψm-dependent transport activities. Moreover, the respiratory chains get uncoupled, leading to ROS overgeneration. Induction of ROS by shikonin could trigger multiple signaling cascades in drug-induced apoptosis and necroptosis. Phosphorylation sites are marked by the letter P. Z-VAD-FMK N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, Nec-1 necrostatin-1, ROS reactive oxygen species, Δψm mitochondrial transmembrane 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 [38]. 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 [41]. 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 [42]. 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 [32]. 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 [38]. 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 [31]. 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 [32]. 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 [31]. 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 [45]. 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 [46]. 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 [47]. 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 [48]. 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 [48]. 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.

Notes

Acknowledgments

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

None

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Zahra Shahsavari
    • 1
  • Fatemeh Karami-Tehrani
    • 1
  • Siamak Salami
    • 2
  • Mehran Ghasemzadeh
    • 3
  1. 1.Cancer Research Laboratory, Department of Clinical Biochemistry, Faculty of Medical ScienceTarbiat Modares UniversityTehranIran
  2. 2.Department of Clinical Biochemistry, Faculty of Medical ScienceShahid Beheshti University of Medical SciencesTehranIran
  3. 3.Blood Transfusion Research CenterHigh Institute for Research and Education in Transfusion MedicineTehranIran

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