Background
Artemisia annua Linné is an annual plant that is chrysanthemum family. This plant is primarily found in the tropical zones of Asia along streets and in fields. Since ancient times,
Artemisia annua Linné has been used as an antipyretic, hemostatic, as a treatment for skin diseases, and an insecticide. In addition, its antibacterial, antiviral and antioxidant properties allow it has been used as a traditional herbalmedicine [
1]. This plant also contains various bioactive compounds [
2‐
5].
The antioxidant activity of phenolic compounds in
Artemisia annua Linné has been reported [
6]. Artemisinin, the main element of sweet wormwood, is being used for medical uses, such as anti-malarial activity [
7‐
9]. In previous studies, they determined that when using
Artemisia annua Linné on extracts from breast cancer, cervical carcinoma cells, stomach cancer, and for cell growth inhibitory effect that there is a cancer cell [
10,
11]. Furthermore, selective necrosis of breast cancer cells was proven to be anti-cancer active. This brought attention to the world to consider taking action with herbal remedies [
12]. Some studies have reported the effect of
Artemisia annua Linné added to food and feed. However, the mechanism of the effects of
Artemisia annua Linné is not well known [
13].
PTEN (Phosphatase and TENsin homolog deleted on chromosome ten), a tumor-suppressorgene with dual lipid and protein phosphatase activity, antagonizes the PI3K/AKT signaling pathway and suppresses cell survival, as well as cell proliferation. Also, PTEN can inhibit the activation of Akt. This effect has been attributed to PTEN reducing the availability of Phosphatidylinositol (3,4,5)-trisphosphate (PIP3; 2–3) [
14‐
16]. The serine/threonine kinase Akt is phosphorylated and activated by PDK1 (phosphoinositide-dependent protein kinase-1) [
17].
PDK1 activation phosphorylates Akt at thr308. Once phosphorylated in T308, phosphorylation additionally occurs at S473by PDK2 [
18]. Akt activation induces different cell survival mechanisms [
19]. Akt plays a central role in many cellular processes that establish survival factor and exert anti-apoptotic activation. Also, Akt activation induces cell cycle progression [
20]. In another case, Akt prevented apoptosis via phosphorylation and translocation of MDM2 (Murine double minute 2) into the nucleus [
21,
22]. MDM2 interacts with p53 and inhibits it. Under normal circumstances, p53 is maintained at very low levels by ubiquitination and degradation [
23]. The p53 gene, a tumor suppressor, plays a key role in the induction of apoptosis and cell cycle arrest in response to a variety of stress genes, including the blocker of cellular DNA damage repair [
24]. p53 is a nuclear DNA-binding phosphor-protein. It is a transcriptional activator that can exert transcriptional repression of specific targeted genes [
25]. Also, p53 interacts directly with cell proliferation-mediated proteins. The direct interaction of p53 activates apoptotic proteins into mitochondrial outer membrane permeabilization (MOMP) [
26].
Mitochondria are well known for playing a key role in activating apoptosis. The mitochondrial apoptosis pathway is mediated via Bcl-2 family proteins [
27,
28]. Bcl-2 family proteins are divided into anti-apoptotic proteins such as Bcl-XL, Bcl-w, Mcl-1 and pro-apoptotic proteins such as Bax, Bak and Bok. In normal cells, Bax exists as a monomer in the cytosol and translocates to mitochondria, experiencing conformational changes to form oligomers during apoptosis. On the other hand, Bak resides on mitochondria. During apoptosis, Bak changes to form oligomers identical to Bax. The activation of Bax and Bak regulates cytochrome c release to cytosol from the mitochondria via alteration of MOMP [
29,
30]. Released cytochrome c induces apoptosis by activating last effectors caspase (caspase-3/−7) [
31].
In this study, we investigated the effects of Artemisia annua Linné extract (AAE) on apoptosis in HCT116colon cancer cells. We suggested that AAE induced apoptosis through PTEN/PDK1/Akt/p53signal pathways and mitochondria-mediated apoptotic proteins.
Methods
Reagents and chemicals
AAE was purchased from Daejeon Oriental Herbal Market (Deojun, Korea).3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Pierce lactate dehydrogenase (LDH) Cytotoxicity Assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Muse Annexin V and Dead Cell Assay Kit, The Muse Caspase-3/7 Kit and the Muse MitoPotential Kit were purchased from Millipore (Darmstadt, Germany). MitoTracker was purchased from Molecular Probes (Eugene, OR, USA). Specific antibodies that recognized phosphorylated (p)Akt (Ser473) (4060S), (p)Akt (Tre308) (3038S), (p)PTEN (9549P), (p)PDK1 (3430P), PUMA (4976P) Bax (5023P), Bak (6947), pro-caspase-3 (9665), Bcl-2 (2876) and β-actin (4967) total formed (t)Akt (4060P), (t)PDK1 (3062P) were obtained from Cell Signaling Technology (Beverly, MA, USA). The total formed (t)PTEN (SC-7974) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), (t)MDM2 (NBPI-02158SS) was purchased from Novus Biologicals (Littleton, CO, USA) and (p)MDM2 was purchased from Abcam (Cambridge, MA, USA). LY294002 (PI3K/Akt inhibitor), Pifithrin-α (p53 inhibitor) were purchased from Calbiochem (San Diego, CA, USA), Nutlin-3 (MDM2 inhibitor) and BX-795 (PDK1 inhibitor) were purchased from Sigma-Aldrich (St Louis, MO, USA), BpV (PTEN inhibitor) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse (PA1–30126) and Goat Anti-Rabbit (166–2408) secondary antibodies were purchased from Thermo Fisher Scientific, Inc., and Bio-Rad Laboratories, Inc., (Tokyo, Japan), respectively.
Preparation of Artemisia annua Linné extract
100 g of the powdered AAE was extracted with 800 mL of 95% EthOH for 72 h. The extract was filtered through qualitative filter paper no. 1 (Toyo Roshi Kaisha, Ltd.; Tokyo, Japan) and concentrated with a rotary evaporator to remove the ethanol. AAE was dissolved in dimethyl sulfoxide (DMSO) prior to treatment and stored at −20 °C. The final concentration of AAE in the culture medium was controlled at 30-60 μg/ml.
Cell culture
HCT116 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were grown in RPMI-1640 medium (Hyclone Laboratories Inc.)containing 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C in a 5% CO2 incubator. The cells were sub-cultured by detachment with Trypsin-EDTA (Hyclone Laboratories Inc.)and re-seeded at 1 × 106 cells/mL per 100 mm plate every 48 h.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
HCT116 cells and fibroblast cells (1 × 105) were seeded onto 12-well plates and treated with AAE at concentrations of 30, 40, and 60 μg/mL for 24 h. Certain samples were pre-treated with a respective inhibitor (20 μMLY294002, 20 μM Nutlin, 20 μM pifithrin-α, 1 μM BpV and 5μMBX-)for 30 min prior to treatment with AAE. The selective medium removed and then incubated with20μl of MTT solution (5 mg/ml MTT in PBS) for 1 h. Converted purple formazan dye from MTT was solubilized in DMSO and optical densities were measured at 595 nm.
Lactate dehydrogenase (LDH)assay
Cells were seeded at 2.5 × 105 cells/mL per well in a 96-well plate and incubated for 24 h. The cells were then treated with AAE (30, 40, and 60 μg/mL) and incubated at 37 °C in a 5% CO2 atmosphere. After 24 h, the high control cells (maximum LDH release) were treated with cell lysis solution from the LDH Cytotoxicity Assay Kit for 30 min. The absorbance of the solution in each well was determined using a microplate reader (Bio-Rad Laboratories, Inc.) at 490 and 655 nm.
Cells morphology
Cells were seeded at 1 × 105 cells/mL in 6wellplatesandtreatedwithAAEfora 24 h time period at 30, 40, and 60 μg/ml concentrations.
Hoechst 33342 staining
Cells were seeded at 1 × 104 cells/mL in a 12-well plate with cover glasses and incubated for 24 h. Following incubation, the cells were treated with the AAE (30, 40, and 60 μg/mL) for 24 h at 37 °C in a 5% CO2 atmosphere. Cells were stained with Hoechst33342 for 30 min. Slides were washed with PBS and mounting fluid was poured over them. The slides were covered with a cover slip and sealed with nail polish. Fluorescence was measured by using a fluorescence microscope (Carl Zeiss, Germany).
Cell apoptosis assay
HCT116 cell apoptosis was assayed using the Muse™ Annexin V and Dead Cell Kit (Merck Millipore, Guyancourt, France) according to the user’s guide. A total of 1x105cells were collected by centrifugation (3000 rpm, 5 min) and washed with PBS. Cells were resuspended in a RPMI-medium with 1% bovine serum albumin and 10% FBS, mixed with the Muse™ Annexin V and Dead Cell reagent, and then incubated for 20 min at room temperature in the dark. Assay results were measured using the Muse™ Cell Analyzer.
Caspase-3/7 activity analysis
Cells were seeded at 1 × 105 cells/mL per plate in a 6-well plate and incubated for 24 h. Following incubation, the cells were treated with AAE (30, 40, and 60 μg/mL) for 24 h at 37 °C in a 5% CO2 atmosphere. HCT116 cell caspase activity was assayed using the Muse Caspase 3/7 Assay Kit according to the user’s guide. A total of 1 x 105cells were collected by centrifugation (3000 rpm, 5 min) and washed with PBS. Cells were resuspended in a 1X Assay Buffer BA, mixed with the Muse™ Caspase-3/7 reagent, and then incubated for 20 min at 37 °C in a 5% CO2 atmosphere in the dark. After incubation, 150 μL of Muse™ Caspase 7-AAD working solution was added to each tube. The solution was mixed thoroughly by pipetting up and down, also know as vortexing, at a medium speed for 3 to 5 s. It was then incubated at room temperature for 5 min in the dark. Assay results were measured using the Muse™ Cell Analyzer.
Western blotting
Cells were seeded at 1 × 105 cells/mL per plate in a 6-well plate and incubated for 24 h. The cells were then treated with AAE (30, 40, and 60 μg/mL) and incubated at 37 °C in a 5% CO2 atmosphere. Certain samples were pre-treated with the respective inhibitor (20 μMLY294002, 20 μM Nutlin, 20 μM pifithrin-α, 1 μM Bp V and 5 μMBX-795) for 30 min prior to treatment with AAE. Cells were rinsed twice with ice cold PBS and scraped with a lysis buffer (50 mM Tris-HCl pH 8.0, 150 mMNaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM PMSF) and subjected to western blot analysis. The primary antibody was allowed to react overnight at 4 °C and the second antibody reacted for 90 min at room temperature with gentle agitation. Following washing the samples four times with 1X Tris Buffered Saline with Tween 20 (TBST) for 10 min at room temperature, proteins were detected using Super Signal West Pico Chemiluminescent Substrate (PI34080; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and visualized on CP-BU new X-ray film (Agfa HealthCare, Inc., Mortsel, Belgium).
Mitochondrial membrane potential assay
Cells were seeded at 1 × 105 cells/mL per plate in a 6-well plate and incubated for 24 h. Following incubation, the cells were treated with the AAE (30, 40, and 60 μg/mL) for 24 h at 37 °C in a 5% CO2 atmosphere. HCT116 cell caspase activity was assayed using the Muse MitoPotential Kit according to the user’s guide. A total of 1 x 105cells were collected by centrifugation (3000 rpm, 5 min) and washed with PBS. The supernatant was then removed and the cell pellets were stained with the Muse MitoPotential Kit (Merck Millipore, Guyancourt, France) for 25 min at 37 °C.The data was analyzed using the Muse™ Cell Analyzer Assay.
Fraction of mitochondria and cytosol proteins
We used a Mitochondria/Cytosol Fraction Kit (Abcam, Cambridge, MA, USA). Cells were seeded at 1 × 106/ml on a 100 mm plate and incubated for24 h. After incubation, cells were treated with AAE for 24 h at 37 °C in a 5% CO2 atmosphere. Cells were harvested by trypsinization, collected by centrifugation, washed with PBS, and homogenized in an ice cold cytosol extraction buffer mix containing DTT and protease inhibitor using a sonicator. The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C and the supernatants were collected. The supernatants were centrifuged at 13000 rpm for 30 min at 4 °C and collected. The supernatant cytosol proteins and pellets were resuspended with ice cold mitochondria extraction buffer containing DTT and a protease inhibitor for mitochondria proteins.
Immunofluorescence (IF)staining
Cells were seeded at 1 × 104 cells/mL in a 12-well plate with cover glasses and incubated for 24 h. The cells were treated with the AAE (30, 40, and 60 μg/mL) for 24 h at 37 °C in a 5% CO2 atmosphere. The cells were stained with MitoTracker. for 30 min at 37 °C in a 5% CO2 atmosphere. Cells were fixed with 3.7% formaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 20 min. Cells were washed with PBS twice and reacted with cytochrome c, Bax and Bak antibodies overnight at 4 °C. Cells were washed with PBS twice and reacted with a secondary antibody for 1 h 30 min. Fluorescence was detected by confocal microscopy (Olympus; Tokyo, Japan).
Xenograft model
Five-week-old male Balb/c nudemice were obtained from SLC (SLC; Tokyo, Japan) Five mice made up the control group while five other mice made up the experimental and delivery groups for each concentration. For tumor induction, HCT116 human colon cancer cells (2.5 × 105 cells/0.1 ml) were subcutaneously injected into the left flank of the mice (each group had 10 animals). One week after the injection of cells, the mice were co-treated with AAE20, 40 mg/kg/day and 0.2cm3 PBS/DMSO for 21 days. Tumor size was measured by taking two perpendicular diameter measurements, using a caliper, every 2 days. The tumor volume was calculated using the following formula: V = 1/2 (length x width). The body weight of each animal was measured at a set time, once per week. After the 3-week treatment, the tumor was removed and frozen in liquid nitrogen for western blot analysis or fixed with formalin for immunohistochemistry, TUNEL and H&E staining. All of the animal experiments were approved by the Ethics Committee for Animal Experimentation of Hannam University (Daejeon, Korea, HNU 2016–9).
TUNEL assay
Levels of apoptosis in distal colon tissue were determined using the TdT-mediated dUTP nickend labeling (TUNEL)method. Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5 μm thick slices. Tissue sections were processed according to manufacturer’s instructions for the Apop Tag Peroxidase In Situ Apoptosis Detection Kit (Vector Laboratories; Burlingame, CA, USA).
Immunohistochemistry
Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5 μm thick slices. Consecutive thin cryosections (5 μm) of OCT compound (Sakura Finetek; Torrance, CA,USA) embedded tumor tissues were fixed in acetone at 4 °C for 10 min. After washing in PBS, the sections were treated with3% H2O2 for 10 min to block endogenous peroxidase activity. The sections were then blocked with normal rabbit serum. Last, the sections were blocked and washed in PBS and incubated with specific antibodies overnight at 4 °C. Negative controls were incubated with the primary normal serum IgG for the species from which the primary antibody was obtained.
Statistical analysis
Cell viability was statistically analyzed using unpaired SPSS Student’s ANOVA-tests and t-tests (SPSSChicago, IL, USA). p < 0.05 was considered statistically significant.
Discussion
From old times, Artemisia annua Linné has been known for its anti-cancer, anti-viral and anti-bacterial properties, but the mechanisms were unknown. In this study, we investigated the effects of apoptosis and signal pathway via extract from Artemisia annua Linné in HCT116 colon cancer cells.
In this study, we focused on the effects of AAE on the induction of apoptosis. First, in order to determine the influence of AAE on cell viability, we performed a MTT assay and LDH assay after treatment with AAE. We confirmed that there is a range of cell damage contributing to significant inhibition of cell proliferation depending on the treatment.
Apoptosis at the mitochondrial level is completely dependent on Bax and Bak, as deficiency in the genes encoding these two proteins renders cells resistant to apoptosis and concomitant release of cytochrome c through the outer mitochondrial membrane. Cytochrome c, as a pro-apoptotic protein, plays an important role in triggering programmed cell death [
32]. The release of cytochrome c from mitochondria directly triggers caspase-3 activation through formation of the cytochrome c containing apoptosome complex [
33]. To confirm the apoptosis mechanism, we performed a Hoechst staining, an Annexin V/Dead cell staining, caspase activity, western blot, MitoPotential staining, fraction western blot and IF staining. Taking all of the results together, AAE induced apoptotic cell death. This cell death is the AAE controlled cytochrome c released from mitochondria to cytoplasm by formation of Bax/Bak oligomeric complexes and led to translocation onto the mitochondria outer membrane. Released cytochrome c by AAE treatment was an induced caspase-3 activity and this activity caused the apoptotic cell death.
Former studies demonstrated special compound-induced apoptosis via a p53-independent manner in HCT116 cells [
34]. Thus, in order to confirm the association of AAE-induced apoptosis and p53, we treated Pifithrin-α (p53 inhibitor) in HCT116 cells. Our results showed an AAE-induced apoptosis via a p53-independent manner, was in cells treated with only AAE.
Our in vivo results showed AAE-induced apoptosis in a mouse xenograft model. The ratio of tumor growth was reduced in the AAE-injected group compared to the control and delivery groups. Also, the AAE-injected group revealed a growth of pro-apoptosis proteins and p53 expressed in a p53-independent manner.
When AAE-induced apoptosis occurred by p53-independent pathway, we used BpV (PTEN inhibitor), and BX-795 (PDK1 inhibitor) to determine the directly regulating proteins and signal pathway by AAE. In the MTT assay, through Annexin V staining and MitoPotential staining, we confirmed that AAE-induced apoptotic cell death is mitochondria-mediated apoptotsis by PTEN independent, PDK1 dependent pathways. Through the western blot on equal terms, we detected that AAE-induced apoptosis induced activation of apoptosis related proteins by regulating the PDK1 directly.