Tetrandrine induces apoptosis and autophagy in human oral cancer HSC-3 cells via caspase-8, -9 and -3/Beclin-1/ LC3-I, II signaling
pathways
Fu-Shun Yu1, Chun-Shu Yu2, Jaw-Chyun Chen3, Jiun-Long Yang4, Hsu-Feng Lu5,6, Shu-Jen Chang7, Meng-Wei Lin8,*, and Jing-Gung Chung9,10*
1Department of Dentist, China Medical University, Taichung 404, Taiwan; 2School of Pharmacy, China Medical University, Taichung 404, Taiwan; 3Department of Medicinal Botany and Health Applications, Da-Yeh University, Changhua, Taiwan;
4Department of Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan; 5Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan;
6School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei, Taiwan; 7School of Pharmacy, China Medical University, Taichung 404, Taiwan; 8Department of Nursing, Cardinal Tien Junior College of Healthcare and Management, Yilan, Taiwan; 9Department of Biological Science and
Technology, China Medical University, Taichung 404, Taiwan; 10Department of Biotechnology, Asia University, Taichung 413, Taiwan
Running head: Tetrandrine induces autophagy and apoptosis in oral cancer HSC-3 cells
*Correspondence to:
Jing-Gung Chung, Ph.D. Department of Biological Science and Technology, China Medical University. No 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Phone: +886 4 2205 3366 ext 2501; FAX: +886 4 2205 3764, Email: [email protected] Meng-Wei Lin. Department of Nursing, Cardinal Tien Junior College of Healthcare
and Management, Yilan, Taiwan. No 407, Sec. 2, Jianfu Road, Sanxing Township, Yilan County 26646, Taiwan. Phone +886 3 9891178 ext 7911. Email:
Abstract. Tetrandrine is a kind of bisbenzylisoquinoline alkaloid that was found in
the Radix Stephania tetrandra S Moore and it had been reported to induce cytotoxic effects on many human cancer cells. In this study, we investigated the cytotoxic effects of tetrandrine on human oral cancer HSC-3 cells in vitro. Treatments of HSC-3 cells with tetrandrine significantly decreased the percentage of viable cells through the induction of autophagy and apoptosis and these effects are in concentration- dependent manner. To define the mechanism underlying the cytotoxic effects of tetrandrine, we investigated the critical molecular events known to regulate the apoptotic and autophagic machinery. Tetrandrine induced chromatin condensation, internucleosomal DNA fragmentation, activation of caspases-3, -8 and -9, and cleavage of poly (ADP ribose) polymerase that were associated with apoptosis, moreover, as well as enhanced the expression of LC3-I and –II that were associated with the induction of autophagy in HSC-3 cells. Tetrandrine induced autophagy in HSC-3 cells were significantly attenuated by bafilomycin A1 (inhibitor of autophagy) pre-treatment that indicated tetrandrine induced cell death may associated with the formation of autophagy. In conclusion, we suggest that tetrandrine induce cell death may through the induction of apoptosis as well as autophagy in human oral cancer HSC-3 cells via caspases/LC3-I, II signaling pathways
.
Keywords: Oral cancer; HSC-3 cell line; Tetrandrine; Apoptosis; Autophagy
Introduction
About 90% of oral cancer is belongs to squamous cell carcinoma and it is the sixth most common malignancy in the world . In Taiwan, oral cancer is one of the common cancers and this cancer in man is higher than that in women . About 7.9 individuals per 100,000 die annually from oral cancer in Taiwan . It is well known that factors associated with oral cancer including alcohol, smoking, poor oral hygiene, infection, dietary factors, and betel nut chewing . In Taiwan, the major factor is betel nut chewing, however, other factors may also are involved . Oral cancer treatment included surgery, radiation and chemotherapy, or the combination of radiation and chemotherapy, however, they are still inadequate, thus, to find a compounds from natural products are urgent.
Tetrandrine, a bisbenzylisoquinoline alkaloid, was obtained from the root of Stephania tetrandra , has been used the treatment of silicosis and arthritis in Chinese medicine in China since ago . Numerous studies have been shown that tetrandrine induced cell death in many human cancer cells . It was reported tetrandrine induced cell death via to induce G0/G1 phase arrest in mouse neuroblastoma cells and human Hep G2 cells . Tetrandrine suppressed T and B cells and to inhibit the production of cytokines and have been used as an adjunct to cisplatin to improve the chemotherapy of ovarian cancer . Tetrandrine have been shown to be a potent autophagy agonist in human hepatocellular carcinoma and may be a promising clinical chemotherapeutic agent . Recently, we also showed that tetrandrine induced apoptosis and autophagy in SAS human oral cancer cells via caspase-dependent and LC3-I and LC3-II-dependent pathways .
Although many studies have been shown that tetrandrine induced apoptosis in many cancer cells including oral cancer, however, the mechanisms involved in autophagy and apoptosis is still unclear, therefore, in the present studies, we further
investigated the effects of tetrandrine on human oral cancer HSC-3 cells. We found that tetrandrine induced HSC-3 cell autophagy and apoptosis via caspases/LC3-I, II/Beclin-1 signal pathways.
Materials and Methods
Chemicals and reagents. Tetrandrine, dimethyl sulfoxide (DMSO), propidium iodide (PI), Triton X-100 and trypan blue were obtained from Sigma Chemical Co. (St.
Louis, MO, USA). DMEM medium, fetal bovine serum (FBS), glutamine Penicillin- streptomycin and Trypsin-EDTA were obtained from Gibco BRL (Invitrogen, Grand Island, NY, USA). The primary antibody such as PARP, caspase 8, -3 and -9, Beclin- 1 were purchased from Santa Cruz Biotechnology. Anti-LC3-I and II, p65, Atg5, Atg7, ULK1, p-ULK1, p-mTOR, p-AKT, Raptor, p-GSK, p-70S6, p-S6 were obtained from Cell Signaling Technology. Beta-Actin was from Sigma-Aldrich.
Secondary antibodies conjugated with horseradish peroxidase (HRP) were from Amersham Pharmacia Biotech.
Measurements of cell morphology and viability. HSC-3 cells (2×105 cells/well) were maintained placed in a 12-well plate in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin and 100U/ml penicillin at 37oC with 5% CO2 for 24 h. Cells were treated with 0, 1, 10, 15, 20, 25, 30 and 40 μM tetrandrine for 24 and 48 h. Cells were examined and photographed under a contrast- phase microscope at 200x for examining cell morphological changes. Then cells were detached with trypsin for 3 min then were collected, washed with PBS, stained with PI for determining the total viability by flow cytometric assay as described previously .
Determination of DNA damage by DAPI staining. HSC-3 cells (2×105 cells/well) were placed on 12-well plates then were incubated with 0, 5, 15, 20, 25 and 30 μM tetrandrine for 48 h. Cells were stained with 4’-6-diamidino-2-phenylindole (DAPI) for DNA condensation examination and were examined and photographed using fluorescence microscopy as described previously .
DNA gel electrophoresis. HSC-3 cells (2×105 cells/well) were placed on 12-well plates then were incubated with 0, 5, 15, 20, 25 and 30 μM tetrandrine for 48 h. DNA was isolated from each treatment using an Easy-DNA Kit (Invitrogen, San Diego, Calif., USA) according to the manufacturer’s instructions. DNA samples were rinsed with 70% ethanol and dried under vacuum then were resuspended in 50 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.5). Electrophoresis was performed at 1.5% agarose gels containing 0.5 μg/ml of ethidium bromide at 15 volt as described previously .
Estimation of apoptotic cells. HSC-3 cells (2×105 cells/well) were placed on 12-well plates then were incubated with 0 and 20 μM tetrandrine for 24 h. Cells from each treatment were collected and was used for measuring apoptosis using annexin V- FITC apoptosis detection kit as described . Harvested cells were stained using PI for 30 min at room temperature in the dark with fluorescein isothiocyanate (FITC)- conjugated annexin V and propidium iodide (PI) in PBS, and analyzed by a two color flow cytometric assay as described previously .
Electron microscopy. HSC-3 cells (2×105 cells/well) were treated with 0 and 20 μM of tetrandrine for 24 h. And then cells were fixed with a solution containing 2.5%
glutaraldehyde and 2% paraformaldehyde (in 0.1 M cacodylate buffer, pH 7.3) for 1 h
then were washed with PBS twice. Cells were postfixed for 30 min in the same buffer containing 1% OsO4. Ultra-thin sections were observed under a transmission electron microscope (JEM-1200EX, JEOL Ltd., Tokyo, Japan) at 100 kV as described previously .
Acridine orange staining. Acridine orange (Sigma) was used to detect and quantify autophagy in HSC-3 cells. HSC-3 cells (2×105 cells/well) placed in 12-well plated then were treated with or without 20 μM tetrandrine for 0, 2, 4 and 6 h. Cells were individually incubated with acridine orange (1 mg/mL) for 15 minutes then were observed and photographed using inverted fluorescence microscope as described previously .
Monodansyl cadaverine (MDC) staining. HSC-3 cells (2×105 cells/well) were treated with 0, 10, 20 and 30 μM tetrandrine for 6 h. Cells were harvested and stained with 1 mg/ml MDC for 20 min and then immediatedly analysed by a fluorescence microscope as described previously .
GFP-LC3 expression. HSC-3 cells (2×105 cells/well) were transfected with pEGFP LC3 for 16 h. Cells were treated with or without 20 μM tetrandrine for 3 and 6 h and were harvested, fixed with 4% paraformaldehyde for 10 min at room temperature.
Cells were stained with LC-3-FITC for 20 min and were analysed by a fluorescence microscope .
Western blotting for examining the levels of proteins associated with autophagy and apoptosis of HSC-3 cells. HSC-3 cells (2×105 cells/well) were maintain in 12-well plates for 24 h and then were pretreated with 0, 10, 15, 20 and 25 μM tetrandrine for
24 h, or were treated with 20 μM tetrandrine for 0, 3, 6, 12, 18 and 24 h, or cells were pretreated with the inhibitors of autophagy 3-MA (2 mM), CQ (30 μM), NAC (10 mM) or BA2 (2 nM) then were treated with 30 μM tetrandrine for 24 h. Cells were collects and lysed in a buffer containing 50 mM Tris-HCI (pH7.4), 125 mM NaCl, 0.1% Triton X-100 and 5 mM EDTA containing both 1% protease inhibitor (Sigma Chemical Co.) and 1% phosphatase inhibitor mixture II (Sigma Chemical Co.) as described previously (24, 25). Then to measure the total amonts of protein by using Bio-protein Kit and 40 μg of each protein were separated by SDS-PAGE on a polyacrylamide gel followed by electrotransfer onto a sequi-blot polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA, USA). The primary antibodies (1:1000 dilution in blocking buffer) anti- PARP, caspase 8, -3 and -9, Beclin-1 were purchased from Santa Cruz Biotechnology. Anti-LC3-I and II, p65, Atg5, Atg7, ULK1, p-ULK1, p-mTOR, p-AKT, Raptor, p-GSK, p-70S6, p-S6 were added to the blots for overnight at 4 oC then were washed and the horseradish peroxidase (HRP)- conjugated secondary antibodies were incubated (1:20,000 in blocking buffer) for 1 h at room temperature as described previously .
Statistical analysis. All the experiments were repeated at least three times. Data are mean ± SD. The difference between the tetrandrine-treated and control groups were analyzed by Student’s t-test, a probability of p<0.05 being considered significant.
Results
Effect of tetrandrine on HSC-3 cell morphological changes and viability were examined and evaluated by microscopy and flow cytometry. HSC-3 cells were treated with various concentration of tetrandrine then were photographed and counted by contrat-phase microscopy and flow cytometric assay, respectively, results are
showing in Figure 1A and B. The results indicated that increasing the concentration of tetrandrine led to increase the cell morphological changes, more floated cells on the well and less whole cells compared to the untreated (control) groups (Fig. 1A).
After calculated the total viable cells indicated that tetrandrine decrease the percentage of viable cells and these effects are in concentration-dependent manner (Fig. 1B). And the 50% of the viable cells were calculated by the treatment of 20 μM of tetrandrine with 24 h and 48 incubations (Fig.1B).
Effect of tetrandrine on HSC-3 cell apoptosis (DNA condension and Fragmentation).
HSC-3 cells were treated with or without tetrandrine for 48 h then were examined the DNA condensation and fragmentation by using DAPI staining, DNA gel electrophoresis and Annexin V staining which was assayed by flow cytometer and results are showing in Figure 2A, B and C. Results indicated that tetrandrine induced DNA condensation (Fig. 2A), DNA fragmentation was presented that is the characteristics of cell apoptosis (Fig. 2B). Figure 2C showed tetrandrine induced apoptotic cell death in HSC-3 cells. These effects are concentration-dependently manner.
Tetrandrine induce autophagy in HSC-3 cells were examined by Electron microscopy. Cells were treated with or with 20 μM tetrandrine then were washed by PBS and then were incubated with 7.5 mmol/L CBDfor 16 h, then were fixed, embedded, sectioned, mounted, and stained as previously described . Specimens were photographed by using a JEOL 1200EX electron microscope (JEOL Limited) and results are present in Figure 3. Results indicated that tetrandrine induced autophagosomes (autophagy) as showing in arrow in Figure 4
Tetrandrine induces autophagy in HSC-3 cells were examined by acridine orange, MDC and LC-3 staining. To investigate tetrandrine induced cell death whether or not autophagy was involved, we examined the typical hallmarks of autophagy such as acidic vacuoles (AVOs) formations. HSC-3 cells were treated with or without tetrandrine then were stained by acridine orange and results are present in Figure 4A, which indicate that treatment with tetrandrine caused the punctuate accumulation of LC3-II, representing the formation of acidic vacuoles (AVOs) in HSC-3 cells and this effects are concentration-dependent manner. Furthermore, MDC staining also showed increased the dose of tetrandrine led to increase the green fluorescence (Fig. 4B).
Results from Figure 4C also showed that tetrandrine increase the levels of LC-3 in HSC-3 cells.
Tetrandrine affects the levels of proteins associated with apoptosis and autophagy in HSC-3 cells. To confirm the effect of tetrandrine induced autophagy and apoptosis through the effect of associated protein in HSC-3 cells, cells were cultured with various doses of tetrandrine for 24 h or were incubated with 20 μM tetrandrine for 0, 6, 12, 18, and 24 h. The total protein levels from each treatment for autophagy and apoptotic associated proteins expression were assay by western blotting and results are showed in Figure 5A, B, C, and D. Results indicated that the levels of active form of PARP, caspase-3, -8 and -9 are increased (Fig. 5A) and these effects are in dose- dependent manners. Furthermore, cells after were treated with 20 μM tetrandrine then incubated with various time periods led to increased the active form of PARP, caspase-3, -8 and -9 are increased (Fig. 5B) and these effects are in dose-dependent manners. Tetrandrine increased the expression of LC3-I and II (Fig. 5C) and p-ULK1 (Fig. 5D) but inhibited the levels of p65, Atg5 and Atg7 (Fig. 5C), ULK1, p-ULK1, p-mTOR, p-AKT, Raptor, p-GSK, p-70S6 and p-S6 (Fig. 5D) that may have led to
autophagy and apoptosis.
The inhibitor of autophagy affect proteins and proliferation of HSC-3 cells after exposed to tetrandrine. Cells were pretreated with the inhibitors of autophagy such as 3-MA (2 mM), CQ (30 μM), NAC (10 mM) or BA2 (2 nM) then were treated with 20 μM tetrandrine and then to examine the levels of LC3-I and II, procaspase-3 and active form of caspase-3 and PARP and results are showing in Figure 6A, B, C and D.
Results indicated that the inhibitor of autophagy such as BAI (Fig. 6A), 3MC (Fig.
6B), NAC (Fig. 6C) and CQ (Fig. 6D) that all increased the expression of LC3-I and II, active form of caspase-3 and PARP in HSC-3 cells. Cells were pretreated with the inhibitors of autophagy such as 3-MA (2 mM), CQ (30 μM), NAC (10 mM) or BA2 (2 nM) then were treated with or without 20 μM tetrandrine then to measure the total percentage of viable cells and results are showing in Figure7. Results indicated that only BA2 increased the proliferation of HSC-3.
Discussion
Tetrandrine had been reported to exhibit anticancer activities and to inhibit cell cycle progress at G1 phase and to induce apoptosis in various cancer cells . It was also reported that tetrandrine induced cell cycle arrest and apoptosis may through inhibit Akt and activate GSK-3β or may through PI3K/AKT/GSK3beta pathway and identify GSK3beta as an important mediator in the processes . However, the effects of tetrandrine on human oral cancer HSC-3 cells have not been reported. Herein, in the present study, we investigated the effect of tetrandrine on human oral cancer HSC-3 cells in vitro and further to investigate the molecular mechanism and possible signal pathways of tetrandrine action. Our findings indicated that tetrandrine induced cell death of HSC-3 cells through the autophagy and induction of apoptosis.
In order to further understand the possible pathways of apoptosis in HSC-3 cells after exposed to tetrandrine, we examined the percentage of apoptosis based on DAPI staining (Fig. 2A) and annexin V staining assay (Fig. 2C), both clear showed that tetrandrine induced apoptosis and these effects are dose-dependent manners. We also confirmed it by using DNA gel electrophoresis assay (Fig. 2B). Furthermore, we also found that tetrandrine also induced autophagy (Fig. 3) in HSC-3 cells based on the observations of autophagosomes.
Tetrandrine-induced apoptosis of HSC-3 cells are in concentration- and time- dependent manners that mean the higher concentrations and prolonged exposure eliciting significantly higher percentage of apoptosis. Western blotting indicated that tetrandrine increased the active form of caspase-8, -9 and -3 (Fig. 5A and B) that demonstrated tetrandrine induced apoptosis was via caspases-dependently.
In addition, we also found other dose of tetrandrine induced higher cytotoxic that of apoptosis that may indicated tetrandrine induced cell death also involved independent of apoptosis this is in agreement with other reports showed in colon cancer cells . Other reports showed that tetrandrine induced apoptosis through caspase-dependent apoptosis or caspase-independent apoptosis or ROS-dependent that dependent cell lines. Tetrandrine treatment decreased the percentage of viable HSC-3 cells through the induction of apoptosis, which was associated with caspase-3, -8 and -9 activation and PPAR cleavage (Fig. 5A and B) that mean the dysfunction of mitochondria was involved. It is well documented that some of the anticancer drugs were used to treat cancer patients may induced cancer cell apoptosis and it also involved in dysfunction of mitochondria then lead to the activation of caspase-9 followed the activation of caspase-3 to cause cancer cell apoptosis . These observations indicated that tetrandrine induced apoptosis through caspases- and mitochondria-dependent pathways.
We further investigated the development of autophagy in HSC-3 cells after exposed to tetrandrine and results from EM examination (Fig. 3) and acridine orange staining (Fig.4A) showed that tetrandrine induced autophagy in HS-3 cells. Due to the molecular mechanism of tetrandrine induced apoptosis have been shown in numerous studies, thus, we further investigated the molecular mechanism of autophagy from HSC-3 cells after incubated with various concentration of tetrandrine. Rapamycin, a clinical used anticancer drug, can induce autophagic cancer cell death, thus, autophagy may be a critical mechanism for cancer treatment . Results from Figure 5C and Figure 5D showed LC3 I and II were elevated and AOs development (Fig. 4A), respectively, that indicated that tetrandrine induced autophagy in HSC-3 cells. HSC-3 cells were pretreated with the scavenger of reactive oxygen species (ROS) but did not see significant affect the LC3-1 and II, cleave caspase-3 and PARP that may suggest that tetrandrine induce autophagy in HSC-3 cells was not involved in ROS production. However, it is well known that ROS play an important role in the development of autophagy and apoptosis . The further investigation is needed in future.
Atg5 and Atg7 both are essential autophagy regulators . Herein, our results showed that tetrandrine treatment on HSC-3 cells led to increase the levels of Atg5 and Atg7 (Fig. 5C) at earlier treated time 3 h then after 6h start to decrease. Thus, we may suggest tetrandrine induced autophagy at earlier time then to induce apoptosis in HSC-3 cells. Further investigations are needed to examine the up-regulation of Atg5 and Atg7.
Furthermore, results from Figure 5 also showed that tetrandrine in the earlier treated time at 6h led to increase Beclin -1 (Fig. 5C ) and p-mTOR (Fig. 5D) but after 12 h start to decrease that may followed by apoptosis occur. Our results also showed that tetrandrine decreased the expression of p-Akt (S473) (Fig. 5D). Other studies
have pointed out that agent inhibit Akt and its down-regulation of mTOR that may to regulate autophagy then cell go survival or death .
In summary, our studies provided the first findings demonstrated that tetrandrine induce cytotoxic effects on human oral cancer HSC-3 cells, the induction of cell death through the autophagy development and the apoptotic activity. The development of autophagy based on the expressions of autophagy hallmarkers such as IC3-I and II in HSC-3 cells at earlier treatment of tetrandrine. Tetrandrine induced apoptosis through caspases activation in HSC-3 cells. Based on these observations, we may suggest that tetrandrine may be a potential agent to treat human oral cancer in future.
Acknowledgements
This work was supported by the grant-in-aid from the National Science Council, Republic of China (Taiwan) (NSC101-2320-B039-028-MY2).
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Figure legends
Figure 1. Tetrandrine induced cell morphological changes and decrease the
percentage of HSC-3 human oral cancer viable cells. Cells (2×105 cells/well) were incubated with or without 0, 1, 10, 15, 20, 25, 30 or 40 μM tetrandrine for 24 h and 48 h then were examined and photographed by phase contrast microscope (A) and were collected to measure total percentage of viable cells by flow cytometry (B) as described in Materials and Methods. Data represents mean ± S.D. of three experiments.
Figure 2. Tetrandrine induced apoptosis in HSC-3 human oral cancer cells. Cells
(2×105 cells/well) were incubated with or without 0, 5, 15, 20, 25 or 30 μM tetrandrine for 24 h then were harvested for DAPI staining (A), DNA gel electrophoresis (B) and stained with Annexin IV (C) to measure the percentage of apoptotic cells by flow cytometry as described in Materials and Methods. Data represents mean ± S.D. of three experiments.
Figure 3. Tetrandrine induce autophagy in HSC-3 cancer cells were examined by
Electron microscopy. HSC-3 cells (2×105 cells/well) were treated with or without 20 μM tetrandrine for 16 h, then fixed, embedded, sectioned, mounted, and stained as described in Materials and Methods. Specimens were photographed by using a JEOL 1200EX electron microscope.
Figure 4. Tetrandrine induced autophagy in HSC-3 cancer cells. HSC-3 cells (2×105
cells/well) were treated with or without 20 μM tetrandrine for various time periods.
Cells were stained by using acridine orange (A), MDC (B) and GFP-LC3B (C) then were examined and photographed as described in Materials and Methods.
Figure 5. Tetrandrine affected the autophagic and apoptotic associated protein levels
in HSC-3 cancer cells. Cells (2×105 cells/well) were incubated with 0, 10, 15, 20, 25 μM tetrandrine for 24 h or were treated with 20 μM tetrandrine for 0, 3, 6, 12, 18 and 24 h. Cells were harvested, quantited the total protein and associated protein were examined by using Western blotting as described in Materials and Methods. The concentration of tetrandrine (A): cleaved caspase-3, -8, -9 and PARP. The treatment time of tetrandrine (B): cleaved caspase-3, -8, -9 and PARP. (C): LC3-I and –II, p62, Atg5, Beclin-1 and Ath7. (D): p-ULK1, ULK1, p-mTOR, pAKT (S473), Raptor, p- GSK3b, p-70S6 and pS6.
Figure 6. Inhibitors of autophagy affected the effects of tetrandrine on autophagic
and apoptotic associated proteins levels in HSC-3 cells. Cells (2×105 cells/well) were pretreated with the inhibitors of autophagy including BA1 3-MA (A), BA1 (B). NAC (C) and CQ (D) then were treated with 20 μM tetrandrine for 24 h then were collected for examining the levels of IC3-1 and II, procaspase-3 and cleavage caspase-3 and
PARP as described in Materials and Methods. Or cells were harvested for examining the cell relative proliferation (E). Data represents mean ± S.D. of three experiments.
