Tanshinone I induces cyclin D1 proteasomal degradation in an ERK1/2 dependent way in human colorectal cancer cells
Mi Kyoung Kim a, Gwang Hun Park a, Hyun Ji Eo a, Hun Min Song a, Jin Wook Lee a,
Min Ji Kwon b, Jin Suk Koo a,b,c, Jin Boo Jeong a,b,c,⁎
a Department of Bioresource Sciences, Andong National University, Andong 760749, Republic of Korea
b Department of Medicinal Plant Resources, Andong National University, Andong 760749, Republic of Korea
c Insititute of Agricultural Science and Technology, Andong National University, Andong 760749, Republic of Korea
Abstract
Tanshinone I (TAN I) as one of the naturally occurring diterpenes from Salvia miltiorrhizae Bunge (Danshen) has been reported to exhibit an anti-cancer activity. However, the underlying mechanisms are still poorly understood. Thus, we performed in vitro study to elucidate the biological mechanism by which TAN I may induce the inhibition of cell growth in human colorectal cancer cells. The treatment of TAN I suppressed the cell proliferation in HCT116 and SW480 cells and decreased the level of cyclin D1 protein. However, the mRNA level of cyclin D1 did not changed by TAN I treatment. Inhibition of proteasomal degradation by MG132 blocked TAN I-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with TAN I. In addition, phosphorylation of cyclin D1 at threonine-286 was increased by TAN I and a point mutation of threonine-286 to alanine attenuated TAN I-mediated cyclin D1 downregulation. Inhibition of ERK1/2 suppressed cyclin D1 phosphorylation and subsequent downregulation by TAN I. From these results, we suggest that TAN I-mediated cyclin D1 downregulation may result from proteasomal degradation through its ERK1/2-mediated phosphorylation of threonine-286. In conclusion, the current study provides new mechanistic link between TAN I, cyclin D1 downregulation and cell growth in human colorectal cancer cells.
1. Introduction
Salvia miltiorrhizae Bunge (Danshen) as one of the most popular herbal medicines has been used for treating cardio- vascular diseases, hepatitis, dysmenorrhea and cerebrovascular diseases in Asian countries [1]. In the past decades, phyto- chemical compounds such as sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids and phenolics have been iden- tified from Danshen. Among these phytochemical compounds, diterpenes are the largest group [1].
Tanshinone I (TAN I), a naturally occurring diterpene, has been known to be one of the bioactive components of Danshen [2]. There are many studies to show the pharmacological properties of TAN I such as protection of hepatocyte injury [3] and enhancement of learning and memory [4].
With regard to the anti-cancer activity, TAN I induces apoptosis in various cancer cells such as lung [5], breast [4], leukemia [6] and colorectal cancers [7]. However, the molecular mechanisms by which TAN I elicits anti-cancer activity remain unknown.
Cyclin D1 has been known to be one of the cell cycle proteins and regulates G1 to S phase in cell cycle through forming the complex with cyclin dependent kinases (CDK) 4 and 6, which results in increase of cell proliferation [8,9]. Thus, cyclin D1 has been regarded as a molecular target for cancer chemoprevention and there are growing evidences to show a relationship between the downregulation of cyclin D1 and cancer prevention [10–13].
Among various cancer types, cyclin D1 is overexpressed by 68.3% of human colorectal cancer cases [14] and abnormal expression of cyclin D1 contributes to tumorigenesis and improves the outcome in colorectal cancer patients [15]. Therefore, it is accepted that control of cyclin D1 level may be a promising colorectal cancer preventive and therapeutic way. In light of the chemopreventive potential of TAN I in colorectal cancer, this study was performed to elucidate the biological mechanism by which TAN I may induce the inhibition of cell growth in human colorectal cancer cells. Here, for the first time, we report that the TAN I induced ERK1/2- dependent proteasomal degradation of cyclin D1 through threonine-286 phosphorylation.
2. Experimental
2.1. Reagents
Tanshinone I (Fig. 1) and 3-(4,5-dimethylthizaol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). Cell culture media, Dulbecco’s Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (DMEM/F-12) was purchased from Lonza (Walkersville, MD, USA). Antibodies against cyclin D1, phospho-cyclin D1 (Thr286), phospho-ERK1/2, total ERK1/2, HA-tag and β-actin were purchased from Cell Signaling (Bervely, MA, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.
2.2. Cell culture and treatment
Human colon cancer cell lines, HCT116 and SW480 were purchased from Korean Cell Line Bank (Seoul, Korea) and grown in DMEM/F-12 supplemented with 10% fatal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained at 37 °C under a humidified atmosphere of 5% CO2. Tanshinone I (TAN I) was dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration did not exceed 0.1% (v/v).
Fig. 1. The chemical structure of tanshinone I.
2.3. Cell proliferation assay
Cell growth was measured using MTT assay system. Briefly, cells were plated onto 96-well plates and grown overnight. The cells were treated with the varying concentrations of TAN I for 24 h. Then, the cells were incubated with 50 μl of MTT solution (1 mg/ml) for an additional 2 h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm.
2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was reverse- transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol for cDNA synthesis. PCR was performed using PCR Master Mix Kit (Promega, Madison, WI, USA) with human primers for cyclin D1 and GAPDH as followed: cyclin D1: forward 5′- aactacctggaccgcttcct-3′ and reverse 5′-ccacttgagcttgttcacca-3′, GAPDH: forward 5′-acccagaagactgtggatgg-3′ and reverse 5′- ttctagacggcaggtcaggt-3′.
2.5. SDS-PAGE and Western blot
After TAN I treatment, cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radio- immunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000×g for 10 min at 4 °C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and trans- ferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room tempera- ture and then incubated with specific primary antibodies in 5% non-fat dry milk at 4 °C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
2.6. Expression vectors
Wild type HA-tagged cyclin D1 and point mutation of T286A of HA-tagged cyclin D1 were provided from Addgene (Cambridge, MA, USA). Transient transfection of the vectors was performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers’ instruction.
2.7. Statistical analysis
All the data are shown as mean ± SEM (standard error of mean). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test. Differences with *P b 0.05 were considered statistically significant.
Fig. 2. Effect of TAN I on cell growth (A) and the level of cyclin D1 (B–D) in human colorectal cancer cells, HCT116 and SW480. (A) HCT116 and SW480 cells were treated with the varying concentrations of TAN I for 24. Cell growth was measured using MTT assay system and expressed as % cell growth. *P b 0.05 compared to cells without TAN I. (B) HCT116 and SW480 cells were plated overnight and then treated with TAN I at the indicated concentrations for 24 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. (C) HCT116 and SW480 cells were plated overnight and then treated with 50 μM of TAN for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot was performed using antibody against cyclin D1. (D) HCT116 and SW480 cells were plated overnight and then treated with TAN I at the indicated concentrations for 24 h. RT-PCR analysis of cyclin D1 gene expression, total RNA was prepared after TAN I treatment for 24 h. Actin and GAPDH were used as internal control for Western blot analysis and RP-PCR, respectively.
3. Results
3.1. Inhibitory effect of TAN I on the cell growth of HCT116 and SW480 cells
To investigate whether TAN I affects the cell growth of human colorectal cancer cells, HCT116 (APC wild type) and SW480 (APC mutant) were treated with the varying concentrations of TAN I for 24 h and the cell growth was evaluated by MTT assay. As shown in Fig. 2A, 6.25 and 12.5 μM of TAN I did not affect the cell growth of HCT116 cells, while more concentrations of TAN I suppressed the cell growth by 14% at 25 μM, 27% at 50 μM and 64% at 100 μM, respectively. In addition, TAN I inhibited the proliferation of SW480 cells by 17% at 6.25 μM, 25% at 12.5 μM, 68% at 25 μM, 77% at 50 μM and 86% at 100 μM, respectively. The results indicate that TAN I suppresses the growth of human colorectal cancer cells in dose- dependent manner and anti-proliferative activity of TAN I is APC-independent.
Fig. 3. Proteasomal degradation of cyclin D1 by TAN I in HCT116 cells. (A) HCT116 cells were plated overnight. The cells were pretreated with MG132 for 2 h and then co-treated with TAN I for the addition 6 h. (B) HCT116 cells were pretreated with DMSO for 50 μM of TAN I for 3 h and then co-treated with 10 μg/ml of cycloheximide (CHX) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1. Actin was used as internal control.
3.2. Effect of TAN I on the level of cyclin D1 in HCT116 and SW480 cells
To investigate whether TAN I affects the level of cyclin D1 protein in human colorectal cancer cells, HCT116 and SW480 cells were treated with the varying concentrations of TAN I for 24 h. As shown in Fig. 2B, TAN I decreased cyclin D1 protein level at dose-dependent manner in both HCT116 and SW480 cells. In a time-course experiment (Fig. 2C), cyclin D1 protein was completely decreased at 6 h after TAN I treatment in HCT116 cells, while TAN I slightly decreased cyclin D1 protein at 1 h and cyclin D1 protein was completely decreased by TAN I at 10 h after treatment in SW480 cells. To elucidate that TAN I-mediated down-regulation of cyclin D1 protein results from the transcriptional effect, mRNA level of cyclin D1 was evaluated by RT-PCR. As shown in Fig. 2D, TAN I did not affect mRNA level of cyclin D1. These results indicate that TAN I may decrease protein stability of cyclin D1.
3.3. Cyclin D1 proteasomal degradation by TAN I through therionine-286 phosphorylation
To confirm that TAN I affects cyclin D1 proteasomal degradation, HCT116 cells were pretreated with MG132 as a proteasome inhibitor and then co-treated with TAN I. As shown in Fig. 3A, cyclin D1 was reduced by TA N I in the cells with DMSO, while pre-treatment of MG132 blocked TAN I-induced downregulation of cyclin D1. To verify these results, the cells were pre-treated with DMSO or TAN I and then exposed to cycloheximide (CHX) for indicated times. As shown in Fig. 3B,TAN I treatment decreased half-life of cyclin D1 protein in HCT116 cells.
Fig. 4. Effect of TAN I on threonine-286 phosphorylation and proteasomal degradation of cyclin D1 in HCT116 cells. (A) HCT116 cells were plated overnight and then treated with 50 μM of TAN I for the indicated times. (B) HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 and then treated with 50 μM of TAN I for 6 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against phospho-cyclin D1 (Thr286) or HA-tag. Actin was used as internal control.
Cyclin proteasomal degradation is associated with its phosphorylation on threonine-286 [16]. Thus, we tested if TAN I phosphorylates cyclin D1 at threonine-286. As a result (Fig. 4A), cyclin D1 begun to be phosphorylated at threonine- 286 by TAN I at 1 h after treatment. To verify that threonine-286 phosphorylation of cyclin D1 results in cyclin D1 proteasomal degradation by TAN I, HCT116 cells were transfected with HA-wild type cyclin D1 or HA-T286A cyclin D1. As shown in Fig. 4B, cyclin D1 degradation by TAN I was observed in wild type cyclin D1-transfected cells. However, it was partially attenuated in T286A cyclin D1-transfected cells. Overall, these data proposed that downregulation of cyclin D1 by TAN I may depend on proteolytic proteasomal degradation via threonine-286 phosphorylation.
3.4. ERK1/2 activation contributes to TAN-induced threonine-286 phosphorylation of cyclin D1 and subsequent proteasomal degradation
To determine the upstream kinases involved in TAN I- mediated proteasomal degradation of cyclin D1, HCT116 cells were pretreated with PD98059 as an ERK1/2 inhibitor and then exposed to TAN I. As a result (Fig. 5A), inhibition of ERK1/2 blocked cyclin D1 proteasomal degradation by TAN I. We also tested that ERK1/2 affects cyclin D1 phosphorylation on threonine-286 by TAN I and found ERK1/2 inhibition attenu- ated TAN I-induced phosphorylation of threonine-286 (Fig. 5B). In addition, we found that TAN I phosphorylated ERK1/2 indicating ERK1/2 activation (Fig. 5C). However, other kinases such as p38, GSK3β and NF-κB did not affect cyclin D1 proteasomal degradation by TAN I (data not shown). These findings indicate that ERK1/2 activation by TAN I at least in part contributes to TAN I-mediated cyclin D1 proteasomal degradation.
4. Discussion
Tanshinones including tanshinone I (TAN I), tanshinone IIA, cryptotanshinone and dihydrotanshinone have been known to be the major diterpenes isolated from S. miltiorrhiza Bunge (Danshen) [17] and reported to show cytotoxic effects on various human carcinomas such as the colon, ovary, lung and mouth [18,19]. Anti-cancer studies of tanshinones were mostly confined to tanshinone IIA. Tanshinone I has been contained in the ethanolic fraction of crude Danshen by ~2% [20], which indicates its high content in this plant as well as its clinical safety [21]. Recently, TAN I has been reported to attenuate lung tumorigenesis and the growth of lung cancer through inhibiting VEGF, cyclin A and cyclin B expression [22], and downregulating Aurora A function [23]. In addition, TAN I exerts the inhibition of cell cycle and PI3K/Akt/mTOR signaling pathway [24].
Fig. 5. Attenuation of TAN I-mediated cyclin D1 downregulation by ERK1/2 inhibition. (A, B) HCT116 cells were pretreated with 50 μM of a selective inhibitor of ERK1/2, PD98059 for 2 h and then co-treated with 50 μM of TAN I for 6 h (For cyclin D1) or 1 h (For p-cyclin D1). (C) HCT116 cells were plated overnight and then treated with 50 μM of TAN I at the indicated concentrations for 24 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1, p-cyclin D (Thr286), p-ERK1/2 and ERK1/2.
Interestingly, there is growing evidence showing that TAN I exerts higher activity than tanshinone IIA in the cell growth inhibition and induction of apoptosis in human prostate and colon cancer cells [25,26]. Thus, we evaluated the anti-cancer activity and molecular mechanism of TAN I in human colorectal cancer cells in this study.
Cyclin D1, the focused in this study has been reported to overexpressed, detected in 68.3% of human colorectal cancer cases [14] and has been regarded as the important target for cancer chemoprevention against human colorectal cancer.Cyclin D1 expression can be regulated through the transcriptional regulation or proteasomal degradation. In the current study, TAN I decreased the level of cyclin D1 protein, while cyclin D1 mRNA did not changed by TAN I. These findings indicate that TAN I-mediated downregulation of cyclin D1 may be independent on transcription. MG132-mediated inhibition of proteasomal degradation blocked TAN I-induced cyclin D1 downregulation and the half-life of cyclin D1 was attenuated in the cells treated with TAN I. These findings suggest that TAN I-induced downregulation may result from cyclin D1 proteasomal degradation. Cyclin D1 proteasomal degradation has been regarded as one of the important anti-cancer mechanisms [13,27,28]. Therefore, cyclin D1 proteasomal degradation may be one of the molecular targets for the anti- cancer activity of TAN I.
Mutated cyclin D1 observed in human cancer cells disrupts threonine-286 phosphorylation resulting in inhibiting cyclin D1 proteasomal degradation [29] and this phosphorylation is associated with cyclin D1 proteasomal degradation [16]. We found that TAN I phosphorylated threonine-286 of cyclin D1 in time-course experiments. The mutation of threonine-286 to alanine (T286A) attenuated TAN I-mediated cyclin D1 proteasomal degradation. Therefore, cyclin D1 proteasomal degradation by TAN I may result from its threonine-286 phosphorylation.
Cyclin D1 proteasomal degradation can be regulated by the upstream kinases such as ERK1/2 [30], p38 [31], GSK3β [32] and NF-κB [33]. In the current study, we found that TAN I induces ERK1/2 phosphorylation, and inhibition of ERK1/2 activation attenuates TAN I-mediated cyclin D1 phosphoryla- tion on threonine-286 and subsequent cyclin D1 proteasomal degradation. From these findings, it is indicated that ERK1/2 activation by TAN I at least in part contributes to TAN I- mediated cyclin D1 proteasomal degradation.In conclusion, TAN I-induced proteasomal degradation of cyclin D1 might inhibit the cell proliferation in human colorectal cancer cells. Furthermore, the current study provides information on molecular events for the anti-cancer activity of TAN I.
Acknowledgments
This work was supported by a grant from 2014 Research Fund of Andong National University (2014-0169) and by the BK21 PLUS program of the Ministry of Education of the Republic of Korea.
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