Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway
- Rishi Kumar Gara†1, 2,
- Vikas Kumar Srivastava†3,
- Shivali Duggal†3,
- Jaspreet Kaur Bagga1,
- MLB Bhatt3,
- Sabyasachi Sanyal4 and
- Durga Prasad Mishra1Email author
© Gara et al.; licensee BioMed Central. 2015
Received: 10 August 2014
Accepted: 6 March 2015
Published: 1 April 2015
Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.
The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.
The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.
Prostate cancer is one of the leading causes of urino-genital cancer related deaths in men . Despite the initial response to androgen deprivation, the disease gradually progresses to a hormone-refractory state due to cumulative genetic alterations resulting in progressive clinical deterioration . Despite the recent advances in diagnostic methods and improvement in treatment strategies mostly using androgen pathway inhibitors, the prognosis of hormone refractory prostate cancers in advanced stages remains largely unsatisfactory [3,4]. Therefore there is a urgent need for expedited development of effective therapeutic agents against hormone refractory prostate cancers.
Aberrant accumulation of unfolded/misfolded proteins and lipids or sudden changes of the endoplasmic reticulum Ca2+ homeostasis leads to a cellular adaptive response known as endoplasmic reticulum stress (ER stress). However, excessive ER stress is believed to be intricately associated with oxidative stress and mitochondrial dysfunction, resulting in apoptotic cell death [5-7]. Therefore the therapeutic modulation of the pro-apoptotic ER stress could be a potential strategy for chemo-sensitization of hormone refractory prostate cancer cells [8-10].
Shikonin, a natural naphthaquinone compound from the herb Lithospermum erythrorhizon is known to act on a variety of molecular targets associated with carcinogenesis and shows similar potency towards drug sensitive and drug-resistant cancer cell lines [11-17]. Furthermore, Shikonin is used as a food additive in many countries and has favorable toxicity, pharmacokinetic and pharmacodynamic profiles [15,16,18]. However its effects on pro-apoptotic-ER stress in hormone refractory prostate cancer cells is unknown. Therefore in the present study, we examined the effects of Shikonin on DU-145 and PC-3 prostate cancer cells and investigated the molecular mechanisms involved in the process.
Materials and reagents
Hormone refractory prostate cancer cell lines DU-145, PC-3 and PrEC, a normal prostate cell type were purchase from ATCC (ATCC; Manassas, VA, USA) and Lonza (Walkersville, MD USA) respectively. The details of the cell lines used in this study are summarized in the (Additional file 1: Table S1). RPMI-1640 media and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Life Technologies, Inc., Rockville, MD, U.S.A.). Shikonin and Salubrinal (ER stress inhibitor) were purchased from Calbiochem (San Diego, CA, U.S.A.). 4′,6-diamidino-2-phenylindole (DAPI), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl- benzimidazolylcarbocyanine iodide (JC-1) were obtained from Invitrogen (Carlsbad, CA, U.S.A.). Trypsin, streptomycin, penicillin, N-acetyl cysteine (NAC), glutathione (GSH) and Catalase were obtained from Sigma Chemical Co. The antibodies used in this study were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, U.S.A.). Caspase colorimetric assay kits were purchased from Millipore (Billerica, CA, USA). Rest of the chemicals used in the study were from Sigma (St. Louis, MO, U.S.A.) unless otherwise stated.
Cell culture and treatment
DU-145, PC-3 and PrEC cells were grown in RPMI 1640 medium (Life Technologies, Inc., Rockville, MD) with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Inc.) or DMEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) at 37°C with 5% CO2 incubator. Stock of Shikonin was prepared in DMSO and stored in −20°C, cells were treated with different concentration and time periods with Shikonin for different experiments.
Cell viability assay
Cell viability was measured using the CCK-8 assay kit in (PC-3 and DU-145) hormone refractory prostate cancer cells and PrEC cells as per the manufacturer’s instructions. Cells were treated with Shikonin for various time points, at the end of treatment, the absorbance was read using a Fluostar Omega Spectrofluorimeter (BMG Technologies, Offenburg, Germany). All the experiments were repeated at least thrice.
Cell proliferation assay
Cellular proliferation was measured by measurement of bromodeoxyuridine (BrdU) incorporation into DNA using a nonradioactive colorimetric assay using ELISA (Roche Applied Science, Indianapolis, IN) as per the manufacturer’s instructions. All the experiments were repeated at least thrice.
Assessment of DNA fragmentation was done using the TUNEL assay according to a previously standardized procedure . Briefly, cells were harvested and fixed in freshly prepared 4% para-formaldehyde in PBS for 30 min at 4°C and then in 70% ethanol for 1 h at 4°C. Subsequently the fixed cells were permeabilized using 0.2% Triton X-100 in 0.1% sodium citrate. The DNA labeling mixture containing terminal deoxynucleotidyl transferase was then added. Cells were incubated overnight at room temperature and washed twice with PBS. Controls were resuspended in the TUNEL reaction mixture containing fluorescent dUTP without terminal deoxynucleotidyl transferase. Finally the analysis was carried out in a BD LSR flow cytometer (Becton–Dickinson, San José, CA).
Measurement of reactive oxygen species
For measurement of reactive oxygen species, the cell permeant probe CM-H2DCFDA was used. The dye was dissolved in dimethyl sulfoxide, and dilutions were made in culture medium. Cells were seeded overnight in 6-well plates with various treatments. At the end of treatments the cells were incubated with 20 μM of the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA) for 30 min. At the end of the incubation period adherent cells were trypsinized and collected. After washing twice with phosphate-buffered saline (PBS, pH 7.4) the fluorescence was monitored at an excitation wavelength of 488 nm and an emission wavelength of 530 nm in a Fluostar Omega Spectrofluorimeter (BMG Technologies, Offenburg, Germany) over a period of time. For each experiment, fluorometric measurements were performed in triplicate and expressed as fluorescence intensity units.
Measurement of intracellular Ca2+concentration
The intracellular Ca2+ levels in DU-145 and PC-3 cells were determined using flourimetry with the Fluo-4 AM dye (Invitrogen Carlsbad, CA, U.S.A.). Cells were cultured in specialized 96-well plates and loaded with 5 μM of Fluo-4 AM fluorescent dye for 30 min at 25°C. Experiments were performed in Hanks’ Balanced Salt Solution (HBSS) solution containing (mM); NaCl, 142; KCl, 5.6; MgCl2, 1; CaCl2, 2; Na2HPO4, 0.34; KH2PO4,0.44; HEPES, 10; glucose, 5.6; buffered to pH 7.4 with NaOH. The Ca2+ −free HBSS had the same constituents as HBSS solution, but with no CaCl2 and with EGTA 1 mM added to eliminate any possible calcium contamination. Fluorescence measurements were performed at an excitation of 488 nm and an emission of 522 nm Fluostar Omega Spectrofluorimeter (BMG Technologies, Offenburg, Germany). All the experiments were repeated at least thrice.
Calpain activity assays
DU-145 and PC-3 cells were cultured on 24-well plates and pretreated with BAPTA, a Ca2+ chelator or calpeptin and an inhibitor of calpain for 1 h. Then, cells were loaded with 40 M Suc- Leu-Leu-Val-Tyr-AMC calpain protease substrate (Biomol, USA,) and treated with shikonin to the indicated time at 37°C in a humidified 5% CO2 incubator. Proteolysis of the fluorescent probe was monitored by a Fluostar Omega Spectrofluorimeter (BMG Technologies, Offenburg, Germany) with filter settings of 360 nm for excitation and 460 nm for emission. All the experiments were repeated at least thrice.
Western blotting analysis
The protein content of the control and treated cell extracts was measured by the Bradford assay. The western blotting was done as per a previously standardized protocol . Briefly, each of the samples 50 μg of protein were electrophoresed on 10–12% SDS–PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked, incubated with primary antibodies at the appropriate concentration, and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:5000 dilution). Labeled bands were detected by Immobilion western chemiluminescence horseradish peroxidase kit and images were captured. Densitometric analysis for determination of relative protein expression was done using a Doc image system (Bio-Rad, Laboratory, UK) with β-actin as loading control.
Measurement of mitochondrial membrane potential
The integrity of mitochondrial membrane potential (Δψ) was measured by JC-1 (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-benzimidazolylcarbocyanine iodide; T-3168; Molecular Probes, Eugene, OR), a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (527 nm) to red (590 nm). With normal mitochondrial function, Δψ is high and red fluorescence is predominant. After injury to mitochondria, Δψ is reduced and an increase in green fluorescence is observed. At the end of the treatments, cells were washed and incubated with JC-1 dye at the concentration of 10 μg/ml in media for 15 min at 37°C. The presence of depolarized mitochondria was identified by fluorimetry. The ratio of the reading at 590 nm to the reading at 530 nm (590:530 ratio) was considered as the relative (Δψ) value. All the experiments were repeated thrice.
Caspase activity assay
To measure caspase-9 and caspase-3 activities, cell lysates were prepared from cells treated with Shikonin for 24 h or with various treatments and analyzed using the caspase-9 and caspase- 3 colorimetric activity assay kits as per the manufacturer’s instructions. All the experiments were repeated thrice.
The data shown are a summary of the results from at least three independent experiments and are presented as the means ± standard error (SEM.). A statistical evaluation of the results was performed with one-way analysis of variance (ANOVA). The results were considered significant at a value of p < 0.05.
Shikonin inhibits proliferation of prostate cancer cells without affecting normal prostate epithelial cells
Shikonin treatment induces ROS generation, intracellular calcium and ROS-dependent mitochondrial apoptosis in prostate cancer cells
Shikonin treatment induces oxidative stress mediated induction of ER stress related protein expression
Shikonin regulates endoplasmic reticulum stress through modulation of intracellular calcium in prostate cancer cells
Inhibition of ER stress response by salubrinal attenuated shikonin induced effects
Shikonin effectively induces apoptosis, necrosis and necroptosis in cancer cells [10,14,23]. Shikonin is known to selective killing of prostate cancer cell types, while sparing normal cells . However nothing was known regarding the effects of Shikonin on regulation of the ROS-Ca2+-pro apoptotic-ER stress in prostate cancer cells. Using in vitro assays we report that the natural naphthoquinone Shikonin induces cell death in DU-145 and PC-3 prostate cancer cell lines through the modulation of the ER stress and the mitochondrial apoptotic pathway. Furthermore, for the first time showed that Shikonin induces apoptosis in prostate cancer cells through the ROS mediated-intracellular Ca2+-proapoptotic ER stress associated mitochondrial dysfunction.
Previous studies have established that increase in the intracellular Ca2+levels lead to the activation of ROS and is considered as a second messenger in ER stress signaling . In this study, treatment of DU-145 and PC-3 cells with Shikonin resulted in marked increase in the levels of intracellular Ca2+ within 60 min and ROS within 90 min. The majority of Ca2+ remains in the ER, but ER stress leads to the release of Ca2+ . Moreover, pretreatment of cells with antioxidants like NAC, GSH or Catalase markedly decreased the levels of intracellular Ca2+, suggesting that intracellular ROS were directly involved in the cytotoxic action of Shikonin (Figures 2 and 3). These data indicated that Shikonin induces production of ROS and activates ER stress and the subsequent release of Ca2+ in both DU-145 and PC-3 cells lead to cell death. Our result is consistent with previous reports that ROS activates ER dependent apoptosis in cancer cells [27-29]. Our results also indicated that the treatment of these cells with Shikonin induceds ER stress leading to the elevation in the levels of GRP78/Bip and CHOP/GADD153, and the phosphorylation of PERK and eIF2α (Figure 4A), while pretreatment with antioxidants attenuated these effects (Figure 4B).
The members of the Bcl-2 family of proteins are important regulators of apoptotic cell death . Although the involvement of Bcl-2 proteins in ER stress-induced cell death is clear, the mechanism by which they are regulated by ER stress is not well understood. Until recently, Bcl-2 proteins were thought to regulate the mitochondrial-mediated apoptotic pathway exclusively [38,39]. One of the recent studies linked ER stress-induced cell death to the Bcl-2 family of proteins showed that overexpression of Bcl-2 or the deficiency of Bax and Bak conferred protection against lethal ER stress . Stress signals are relayed from the ER to mitochondria, and ER stress induced apoptosis, similar to mitochondrial-mediated apoptosis regulated by the Bcl-2 family of proteins . The ratio of the Bax/Bcl-2 is critical for the induction of apoptosis . ER stress inducers including cellular stress inducers have also been shown to induce a change in the conformation of Bax, resulting in its accumulation on the mitochondria, and to induce the release of cytochrome c from the mitochondria into the cytosol [31,41,42]. Cytosolic cytochrome c then binds to Apaf-1, leading to the activation of caspase-3 and PARP protein . The present results also indicate that the reduced expression of antiapoptotic Bcl-2 protein and the increased expression of the pro-apoptotic Bax protein facilitated the Shikonin-mediated cell death of these cells (Figure 6), which increases the ratio of Bax/Bcl-2. This may be responsible for the concomitant execution phase of apoptosis observed in these cells, which included the disruption of the mitochondrial membrane (Figure 6). As the level of cytochrome c increases in the cytosol, it leads to the activation of the procaspase-9 and caspase- 3 . Activated caspase-3 is the key executioner of apoptosis and the cleaved caspase-3 leads to the cleavage and inactivation of key cellular proteins such as PARP [45,46]. The present results revealed that the treatment of prostate cancer cells with Shikonin led to the activation of caspase-9, caspase-3, and PARP (Figure 1). Salubrinal was identified as a selective inhibitor of phosphatases that act on eIF2α thereby maintaining protein phosphorylation and offering protection from the adverse effects of ER stress. Inhibition of ER stress in DU-145 and PC-3 cells by salubrinal in the present study led to the increased expression of Bcl-2 and caspase-9, and caspase-3 activities and the decreased expression of Bax (Figure 6B), caspase dependent cell death (Figure 7A) and DNA damage as evident by the nuclear condensation indicated by DAPI staining (Figure 7B). Natural compounds like shikonin might have multiple cellular targets in order to achieve their biological beneficial effects such as tumor growth inhibition [14,16]. A previous study using prostate cancer cells had indicated that the tumor proteasomal chymotrypsin subunit is one of the cellular as well as in vivo biological targets of shikonin . Our results are consistent with is study as it demonstrates that shikonin with established proteasomal inhibitory activity induces apoptosis through induction of reactive oxygen species and endoplasmic reticulum stress-in prostate cancer cells  (Figure 7C).
Taken together our studies suggest that (i) Shikonin inhibits proliferation of hormone refractory prostate cancer cells through the induction of ROS, mediated activation of ER stress and intracellular Ca2+, and (ii) induction of mitochondrial apoptotic pathway mediated through the enhanced expression of Bax, disruption of the mitochondrial membrane potential, PARP cleavage and activation of caspase-9 and caspase-3. This ability of Shikonin to induce dual pathways of cell death underscores its potential as a chemotherapeutic agent against hormone refractory prostate cancers need for further evaluation using in vivo models.
We would like to thank all the members of the D.P. Mishra laboratory for the helpful discussions. R.K.G. and V.K.S. are supported by senior research fellowships from the UGC and ICMR, respectively. This study was supported by the Grants from the Department of Science and Technology, India (GAP-0056) and CSIR-Network Project PROGRAM (BSC0101).
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