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The involvement of cyclin D1 degradation through GSK3[beta]-mediated threonine-286 phosphorylation-dependent nuclear export in anti-cancer activity of mulberry root bark extracts.


Background: Mulberry root bark was shown to induce cyclin D1 proteasomal degradation in the human colorectal cancer cells. Still, the molecular mechanisms whereby mulberry root bark induces cyclin D1 proteasomal degradation remain largely unknown.

Purpose: In this study, the inhibitory effect of mulberry root bark (MRB) on the proliferation of human colorectal cancer cells and the mechanism of action were examined to evaluate its anti-cancer activity. Methods: Anti-proliferative effect was determined by MTT assay. RT-PCR and Western blotting were used to assess the mRNA and protein expression of related proteins.

Results: MRB inhibited markedly the proliferation of human colorectal cancer ceils (HCT116, SW480 and LoVo). In addition, the proliferation of human breast cancer cells (MDA-MB-231 and MCF-7) was suppressed by MRB treatment. However, MRB did not affect the growth of HepG-2 cells as a human hepatocellular carcinoma cell line. MRB effectively decreased cyclin D1 protein level in human colorectal cancer cells and breast cancer ceils, but not in hepatocellular carcinoma cells. Contrast to protein levels, cyclin D1 mRNA level did not be changed by MRB treatment. Inhibition of proteasomal degradation by MG132 attenuated MRB-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with MRB. In addition, MRB increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated MRB-mediated cyclin D1 degradation. Inhibition of GSK3[beta] by LiCl suppressed cyclin D1 phosphorylation and downregulation by MRB. MRB decreased the nuclear level of cyclin D1 and the inhibition of nuclear export by LMB attenuated MRB-mediated cyclin D1 degradation.

Conclusion: MRB has anti-cancer activity by inducing cyclin D1 proteasomal degradation through cyclin D1 nuclear export via GSK3[beta]-dependent threonine-286 phosphorylation. These findings suggest that possibly its extract could be used for treating colorectal cancer.


Mulberry root bark


Cyclin D1 proteasomal degradation

Human colorectal cancer


Cyclin D1 has been regarded as an important regulator of the Gl-to-S phase transition in normal cells. In addition, cyclin D1 functions as a proto-oncogene in cancer cells (Shan et al. 2009). Cyclin D1 overexpression has been observed in many types of human cancer such as lymphoid, breast, esophageal, lung and bladder tumors (Diehl 2002; Landis et al. 2006; Lee and Sicinski 2006; Li et al. 2006; Sherr 1996). Like these literatures, cyclin D1 is important for the development and progression of several cancer cells. In human colon cancer, cyclin D1 has been reported to be overexpressed in 68.3% of cancer case, which indicates that deregulation of cyclin D1 is associated with colon tumorigenesis (Bahnassy et al. 2004; Holland et al. 2001). Therefore, it has been accepted that the control of cyclin D1 level may provide a promising chemopreventive and therapeutic way for human colorectal cancer.

Although both the surgery and adjuvant therapy have been regarded as the most effective treatment for human colon cancer, the complementary and alternative medicine is considered because of ineffectiveness of these therapeutic approaches. Thus, chemoprevention using vegetables, fruits and medicinal plants has received attention as an attractive and promising strategy for human cancer (Wang et al. 2012).

The root bark of mulberry tree (Morns alba L.) as one of the traditional Chinese medicines has been used as antiphlogistic, liver protective, kidney protective, hypotensive, diuretic, anticough and analgesic agent (El-Beshbishy et al. 2006; Fukai et al. 2003). In addition, mulberry root bark has been reported to exert antiviral and antimicrobial activity (Du et al. 2003; Sohn et al. 2004). In anticancer properties, Nam et al. (2002) reported that mulberry root extracts induces apoptotic cell death of different types of cancer cells such as K562 and B380 human leukemia cells and B16 mouse melanoma cells. In addition, our group showed that mulberry root extracts decreases cyclin D1 protein level through the proteasomal degradation in the human colorectal cancer cell line, SW480, which may exert its anti-cancer activity (Eo et al. 2014). Mulberry root bark has been reported to have various active components such as mulberroside A, oxyresveratrol, mulberrofuran G, kuwanon C, kuwanon G, kuwanon H and morusin (Kang et al. 2013). In anticancer activity, morusin induce apoptosis and suppress NF-kB in human colorectal cancer cells (Lee et al. 2008).

However, the specific mechanism by which mulberry root extracts can induce cyclin D1 proteasomal degradation still remains unanswered. Here, we propose a novel mechanism involved in the induction of cyclin D1 proteasomal degradation by mulberry root bark. Mulberry root bark induces cyclin D1 proteasomal degradation through GSK3[beta]-dependent threonine-286 phosphorylation of cyclin D1 and subsequent cyclin D1 nuclear export.

Materials and methods


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). PD98059, SB203580, SP600125, LiCl, BAYU-7082, MG132, cycloheximide (CHX), leptomycin B (LMB) and 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against cyclin D1, phospho-cyclin D1 (Thr286), HA-tag, CDK4, CDK6 and /S-actin were purchased from Cell Signaling (Bervely, MA, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.

Sample preparation

Mulberry root barks were kindly provided by the Bonghwa Alpine Medicinal Plant Experiment Station, Korea. One kilogram of mulberry root barks was extracted with 1000 ml of 80% methanol with shaking for 24 h. After 24 h, the methanol-soluble fraction was filtered and concentrated to approximately 200 ml volume using a vacuum evaporator and then fractionated with 200 ml of petroleum ether three times, and then 200 ml of ethyl acetate three times in a separating funnel. The ethyl acetate fraction was separated from the mixture, evaporated by a vacuum evaporator, and prepared aseptically and kept in a refrigerator.

Cell culture and treatment

Human colon cancer cell lines such as HCT116 (APC wild type, [beta]-catenin mutant and p53 wild type) and, SW480 (APC mutant, [beta]-catenin wild type and p53 mutant) and LoVo (APC mutant, [beta]-catenin wild type and p53 wild type), breast cancer cell lines such as MDA-MB-231 (ER-negative and p53 mutant) and MCF-7 (ERpositive and p53 wild type), hepatocellular carcinoma cell line such as HepG-2 (p53 wild type), and colon normal cells (CCD-18co) 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 [micro]g/ml streptomycin. The cells were maintained at 37[degrees]C under a humidified atmosphere of 5% C[O.sub.2]. Mulberry root bark extract (MRB) 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).

Cell proliferation assay

Cell growth was measured using MTT assay system. Briefly, cells were plated onto 96-well plated and grown overnight. The cells were treated with 0, 1, 5, 10 and 20 [micro]g/ml of MRB for 24 h. Then, the cells were incubated with 50 [micro]l 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.

Isolation of nuclear fraction

Nuclear fractions were prepared following the manufacturer's protocols of nuclear extract kit (Active Motif, Carlsbad, CA, USA). Briefly, the cells were washed with ice-cold PBS containing phosphatase inhibitors and harvested with 1 x hypotonic buffer for 15 min at 4[degrees]C. After adding detergent, the cells were centrifuged at 15,000 rpm for 30 min. After centrifugation, nuclear fractions were collected by suspending nuclear pellet with lysis buffer and centrifugation.

SDS-PAGE and Western blot

After MRB treatment, cells were washed with lx phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation 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 15,000g for 10 min at 4[degrees]C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred 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 temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4[degrees]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.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

After MRB treatment, total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 pig) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer's protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with human primers for cyclin D1 and GAPDH as followed : cyclin Dl: forward 5'-AACTACCTGGACCGCTTCCT-3' and reverse 5'-CCACTTGAGCTTGTTCACCA-3', GAPDH: forward 5'-ACCCAGAAGACTGTGGATGG-3' and reverse 5 -TTCTAGACGGCAGGTCAGGT-3'.

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.

Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS analysis of mulberry root bark extract was performed on an Agilent 6890 GC/5973N mass selective detector (MSD) (Agilent Technologies, Palo Alto, CA, USA); a high resolution capillary column HP-5MS (30 m x 0.25 mm x 0.25 [micro]m) was used. The oven temperature was initially set at 100[degrees]C for 5 min, then increased to 300[degrees]C at a flow rate of 5[degrees]C/min and was maintained for 10 min. The front inlet temperature was maintained at 280[degrees]C. Split injection was conducted with a split ratio of 10:1, and helium was used as the carrier gas at a flow rate of 1 ml/min; 1 [micro]l of the sample was injected for analysis.

Statistical analysis

All the data are shown as mean [+ or -] SEM (standard error of mean). Statistical analysis was performed with one-way ANOVA followed by Dunnett's test. Differences with *P < 0.05 were considered statistically significant.


Effect of MRB on the proliferation of human cancer cells

The effects of MRB on cell viability were evaluated in colon cancer cells (HCT116, SW480 and LoVo), human breast cancer cells (MCF-7 and MDA-MB-231) and hepatocellular carcinoma cells (HepG-2) by MTT assay. As shown in Fig. 1A, cell proliferation of human colon cancer cells was significantly reduced by the treatment of 10 and 20 pg/ml of MRB for 24 h. In addition, MRB suppressed the proliferation of MDA-MB-231 and MCF-7 cells (Fig. IB). However, the proliferation of HepG-2 cells did not be affected by MRB treatment (Fig. 1C). In addition, MRB induced minimal inhibition of cell growth in CCD-18co (colon normal cells) (Fig. ID). These data indicate that MRB's anti-cancer activity may be cancer-specific.

Downregulation of cyclin D1 protein level by MRB treatment, but not mRNA level

To evaluate if MRB affects cyclin D1 expression in human colorectal cancer cell lines, HCT116, SW480 and LoVo, Western blot analysis was performed at 24 h after MRB treatment. As shown in Fig. 2A, MRB treatment at 1 and 5 [micro]g/ml did not affect cyclin D1 expression, while cyclin D1 protein level was decreased at 10 [micro]g/ml of MRB. In time-course experiment (Fig. 2B), MRB started to decrease cyclin D1 protein level at 10 h in HCT116 cells, 3 h in LoVo cells, and 6 h in SW480 cells. To determine if MRB-mediated down-regulation of cyclin D1 protein level results from transcriptional regulation, mRNA level of cyclin D1 was evaluated by RT-PCR. However, MRB treatment did not affect cyclin D1 mRNA in HCT116, SW480 and LoVo cells (Fig. 2C). In addition, we assessed the cyclin Dl-downregulatory effect of MRB on other cancer cell lines such as human breast cancer cells (MCF-7 and MDA-MB-231) and hepatocellular carcinoma cells (HepG-2). As shown in Fig. 2D, MRB down-regulated cyclin D1 protein level in MDAMB-231 and MCF-7. While cyclin D1 in HepG-2 cells did not be changed by MRB treatment. For promoting cell cycle progression, cyclin D1 binds to cyclin-dependent kinase (CDK) 4/6 as the binding partners of cyclin D1 and subsequently phosphorylates and inactivates the retinoblastoma protein (Kato et al. 1993; Lundberg and Weinberg 1998; Weinberg 1995). To evaluate whether MRB affects the expressions of CDK4 or 6, HCT116, LoVo and SW480 cells were treated with MRB for 24 h. As a result, CDK4 expression was significantly attenuated in the cells treated with 10 [micro]g/ml of MRB (Fig. 2E). However, CDK6 expression did not be changed by MRB (Fig. 2F).

MRB-mediated cyclin D1 downregulation is involved in the proteasomal degradation

Our findings for MRB-mediated downregulation of cyclin D1 protein level but not mRNA level indicate that MRB may affect the decrease of protein stability of cyclin Dl. MG132 has been used for evaluating cyclin D1 proteasomal degradation because it effectively abolishes cyclin D1 degradation following the inhibition of protein synthesis (Alao et al. 2006; Lin et al. 2006). Thus, the cells were pretreated with MG132 and then co-treated with MRB to evaluate MRB affects cyclin D1 proteasomal degradation. As shown in Fig. 3A-C, pre-treatment of MG132 blocked MRB-induced decrease of cyclin D1 protein level in HCT116, SW480 and LoVo cells. In addition, cydoheximide is commonly used in studies on protein stability and degradation (Alao 2007). To verify these results, the cells were-pretreated with DMSO or MRB, and then exposed to cycloheximide. As shown in Fig. 3D and E, MRB treatment decreased half-life of cyclin D1 protein in HCT116 and SW480 cells. These data suggest that downregulation of cyclin D protein level by MRB is involved in the proteasomal degradation.

Phosphorylation (Thr286)-dependent degradation of cyclin D1 by MRB

Since cyclin D1 degradation is associated with threonine-286 phosphorylation, we investigated whether MRB affects threonine-286 phosphorylation of cyclin D1 by Western blot in HCT116 and SW480 cells. As shown in Fig. 4A, MRB rapidly phosphorylated cyclin D1- threonine-286 at 1 h after treatment. For the contribution of cyclin D1 phosphorylation to MRB-induced cyclin D1 degradation, HCT116 cells were transfected with the expression vectors, HA-tagged wild type cyclin D1 or HA-tagged T286A cyclin D1 and then treated with MRB. As a results (Fig. 4B), MRB treatment resulted in the downregulation of HA-tag in the cells transfected with HA-tagged wild type cyclin D1 expression vector. However, T286A transfection abolished MRB-mediated downregulation of HA-tag. These data indicate that MRB-induced cyclin D1 degradation may be dependent on threonine-286 phosphorylation.

Involvement of GSK3[beta] in MRB-mediated cyclin D1 degradation

To investigate the upstream kinases associated with MRB-mediated cyclin D1 degradation, HCT116 and SW480 cells were pretreated with PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor) or SP600125 (JNK inhibitor), and then co-treated with MRB. As shown in Fig. 5A-F, inhibition of ERK1/2, p38 or JNK did not affect MRB-mediated cyclin D1 degradation. In addition, we investigated whether other kinases such as I[kappa]cK-[alpha] and GSK[beta] affects cyclin D1 degradation, and found that MRB induced cyclin D1 degradation irrespective of the inhibition of 1/cK-a by BAY11-7082 in HCT116 and SW480 cells (Fig. 6A and B). However, GSK[beta] inhibition by LiCl attenuated MRB-mediated cyclin D1 degradation in HCT116 and SW480 cells (Fig. 6C and D). We also found that GSK[beta] inhibition by LiCl suppressed MRB-mediated threonine-286 phosphorylation of cyclin D1 (Fig. 6E).

Induction of cyclin D1 nuclear export by MRB

There is growing evidence that GSK[beta]-dependent phosphorylation of cyclin D1 at threonine-286 phosphorylation promotes the nudear-to-cytoplasmic redistribution of cyclin D1 and subsequently cyclin D1 is degraded (Alt et al. 2000). In addition, mutation of threonine-286 of cyclin D1 to alanine (T286A-cydin D1) has been reported to prevent nuclear-to-cytoplasmic redistribution of cyclin D1 (Diehl et al. 1998). To investigate whether MRB-mediated cyclin D1 phosphorylation at threonine-286 affects cyclin D1 nuclear export, HCT116 cells were transfected with wild type-cyclin D1 or T286A-cyclin D1 expression vector and then were treated with MRB for 3 h. As shown in Fig. 7A and B, MRB treatment attenuated the nuclear cyclin D1 level in the cells transfected with wild type-cyclin D1, while mutation of threonine-286 to alanine blocked MRB-mediated decrease of the nuclear cyclin D1. Next, HCT116 cells were pre-treated with leptomycin B (LMB, nuclear export inhibitor) and then co-treated with MRB to elucidate whether MRB-mediated nuclear export of cyclin D1 results in cyclin D1 proteasomal degradation. As a result (Fig. 7C), the inhibition of cyclin D1 nuclear export by LMB blocked MRB-mediated cyclin D1 proteasomal degradation.

Components of MRB identified by GC-MS

We identified the potential medicinal components of MRB using GC-MS. Compounds were identified by comparisons with those in the library (in the library search program hits that were >90% probable were viewed as likely hits). As shown in Fig. 8, the analysis yielded 3 compounds with anti-cancer properties: linoleic acid, [alpha]-amyrin and lupeol.


Various herbal extracts used as oriental traditional medicines have provided the benefits such as minimal side effects and preliminary knowledge for developing novel anticancer agents (Cragg and Newman 2005; Huang et al. 2006). Mulberry (Morus alba L.) root bark has long been used in Chinese medicine to treat fever, protect the liver, improve eyesight, strengthen joints, facilitate discharge of urine, and lower blood pressure (Zhishen et al. 1999).

In this study, the following effects of mulberry root bark extracts (MRB) were examined: inhibition of the proliferation of the human colorectal cancer cells and cyclin D1 downregulation. MRB dose-dependently suppressed the growth of human colorectal cancer cells including HCT116, SW480 and LoVo. In addition, the proliferation of human breast cancer cells such as MDA-MB-231 and MCF-7 was inhibited by MRB treatment. However, MRB did not affect the proliferation of HepG-2 cells as a human hepatocellular carcinoma cell line.

Cyclin D1 as one of the proto-oncoprotein is a crucial regulator in the cell growth, and its overexpression is frequently observed in many different cancer cells, which indicates that the overexpression of cyclin D1 has been associated with the development and progression of cancer (Alao 2007). In addition, cyclin D1 has been regarded as a useful marker of enhanced proliferation of cancer cells (Mamay et al. 2001). The present results showed that MRB decreased cyclin D1 protein level in HCT116, SW480, LoVo, MDAMB-231 and MCF-7. However, cyclin D1 protein level of HepG-2 cells did not be changed by MRB treatment, which was similar to the effect of MRB on the cell growth inhibition. We do not know why MRB has no effect on the decrease of cyclin D1 in HepG-2 cells. However, we don't exclude two hypotheses. Firstly, HepG-2 cells could overcome the treatment concentration of MRB (10 [micro]g/ml). Secondly, MRB could not affect Hepatitis B virus protein (HBx). HBx has been known to contribute centrally to the pathogenesis of hepatocellular carcinoma (HCC). HBx can induce cyclin D1 protein nuclear accumulation and subsequently stabilizes cyclin D1 protein, which accelerate hepatocarcinogenesis (Chen et al. 2015).

In addition, we found that MRB significantly attenuated CDK4 expression. CDK 4 has been reported to be essential to Gl/S progression by binding to cyclins D (Malumbres and Barbacid 2009). Thus CDK4 downregulation has been regarded to be a potential anti-cancer target. From our result, CDK 4 may be a molecular target for MRB-mediated cell growth arrest.

Cyclin D1 level can be regulated by multiple mechanisms. One is through transcriptional regulation. The present results showed that cyclin D1 mRNA level did not be changed by MRB treatment, which indicates that MRB-mediated downregulation may be independent on transcription. Another mechanism to regulating cyclin D1 level is through the activation of proteasomal degradation and cyclin D1 proteasomal degradation has been regarded as one of important anti-cancer mechanisms, previously reported with curcumin (Mukhopadhyay et al. 2002), retinoic acid (Spinella et al. 1999) and troglitazone (Huang et al. 2005). In our results, MRB-mediated cyclin D1 downregulation was attenuated in presence of MG132, as a proteasome inhibitor. In addition, MRB treatment decreased half-life of cyclin D1 protein in the cells exposed to CHX. These results indicate that MRB-mediated cyclin D1 downregulation may be attributed to activating proteasomal degradation.

We also observed that MRB induced the threonine-286 phosphorylation of cyclin D1 and the mutation of threonine-286 of cyclin D1 to alanine (T286A) blocked MRB-mediated cyclin D1 degradation. Indeed, cyclin D1 proteasomal degradation is associated with its threonine-286 phosphorylation (Diehl et al. 1997). Our findings indicate that proteasomal degradation of cyclin D1 by MRB may depend on threonine-286 phosphorylation.

There is growing evidence that threonine-286 phosphorylation can be regulated by ERK1/2, p38, JNK, GSK3[beta] and I[kappa]K-[alpha]; (Diehl et al. 1998; Kwak et al. 2005; Okabe et al. 2006; Thoms et al. 2007). The present results showed that GSK3[beta] inhibition by LiCl attenuated MRB-mediated threonine-286 phosphorylation of cyclin D1 and subsequent cyclin D1 proteasomal degradation. However, inhibition of other kinases such as ERK1/2, p38, JNK and I[kappa]K-[alpha] did not affect cyclin D1 degradation by MRB. These results indicate that GSK3[beta] may be an upstream kinase involved in MRB-mediated cyclin D1 degradation.

Interestingly, we observed that MRB decreases the nuclear level of HA-tag in the cells transfected with HA-tagged wild type cyclin D1 expression vector. However, MRB did not affected HA-tag level in the cells transfected with HA-tagged T286A cyclin D1 expression vector. In addition, the pretreatment of LMB as a nuclear export inhibitor blocked cyclin D1 proteasomal degradation by MRB. Alt et al. reported that GSK3[beta]-dependent phosphorylation of cyclin D1 on threonine-286 regulates its nuclear export (Alt et al. 2000) and mutation of GSK3[beta] phosphorylation site within cyclin D1 to alanine prevents GSK3[beta]-dependent nudear-to-cytoplasmic redistribution of cyclin D1 such that the T286A mutant of cyclin D1 is constitutively nuclear (Diehl et al. 1998). Thus, our findings indicate that MRB-mediated nuclear decrease of cyclin D1 may contribute to cyclin D1 degradation. Kim and Diehl reported that overexpression of cyclin D1 is not sufficient to drive oncogenic transformation although cyclin D1 overexpression is clearly implicated in cancer, but nuclear retention of cyclin D1 may be critical for the manifestation of its oncogenicity (Kim and Diehl 2009).

We showed that 3 compounds such as linoleic acid, [alpha]-amyrin and lupeol in MRB identified by GC-MS analysis were reported to have anti-cancer effects. Indeed, linoleic acid, [alpha]-amyrin and lupeol have been reported to exert anti-cancer effects (Khiev et al. 2009; Lu et al. 2010; Pitchai et al. 2014; Siveen et al. 2014). However, further studies are required to determine the role of the components from MRB that are responsible for cyclin D1 proteasomal degradation. In addition, the effect of anti-cancer in in vivo experiment remains to be verified.


In conclusion, MRB may induce cyclin D1 proteasomal degradation through cyclin D1 nuclear export via GSK3[beta]-dependent threonine-286 phosphorylation and MRB-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells. These findings can provide detailed account of preclinical studies conducted to determine the utility of MRB as a therapeutic and chemopreventive agent for the treatment of human colorectal cancer.

Conflict of interest

The authors declare that they have no competing interest.


Article history:

Received 17 August 2015

Revised 4 December 2015

Accepted 5 December 2015

Abbreviations: CDK, cyclin-dependent kinase; CHX, cycloheximide; ERK1/2, extracellular signal-regulated kinasesl/2; GSK3[beta], glycogen synthase kinase 3 beta: 1[kappa]/K-[alpha], 1[kappa]B kinase-[alpha]; JNK, c-Jun N-terminal kinases; LMB, leptomycin B; MRB, mulberry root bark extracts; MTT, 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide.

Hyun Ji Eo, Gwang Hun Park, Jin Boo Jeong *

Department of Bioresource Sciences, Andong National University, Andong 760749, Republic of Korea

* Corresponding author. Tel.: +82 54 820 7757; fax: +82 54 820 6252.

E-mail address: (J.B. Jeong).


This work was supported by a grant from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2053448).


Alao, J.P., 2007. The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol. Cancer 6, 24.

Alao, J.P., Stavropoulou, A.V., lam, E.W., Coombes, R.C., Vigushin, D.M., 2006. Histone deacetylase inhibitor, trichostatin A induces ubiquitin-dependent cyclin D1 degradation in MCF-7 breast cancer cells. Mol. Cancer 5, 8.

Alt, J.R., Cleveland, J.L., Hannink, M., Diehl, J.A., 2000. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev 14, 3102-3114.

Bahnassy, AA, Zekri, A.R., El-Houssini, S., El-Shehaby, A.M., Mahmoud, M.R., Abdallah, S., El-Serafi, M., 2004, Cyclin A and cyclin D1 as significant prognostic markers in colorectal cancer patients. BMC Gastroenterol 23, 22-24.

Chen, X., Zhang, L., Zheng, S., Zhang, T., Li, M., Zhang, X., Zeng, Z., McCrae, M.A., Zhao, J., Zhuang, H., Lu, F., 2015. Hepatitis B virus X protein stabilizes cyclin D1 and increases cyclin D1 nuclear accumulation through ERK-mediated inactivation of GSK-3/5. Cancer Prev. Res. 8, 455-463.

Cragg, G.M., Newman, D.J., 2005. Plants as a source of anti-cancer agents. J. Ethnopharmacol. 100, 72-79.

Diehl, JA, 2002. Cycling to cancer with cyclin D1. Cancer Biol. Ther. 1, 226-231.

Diehl, JA., Cheng, M., Roussel, M.F., Sherr, C.J., 1998. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12, 3499-3511.

Diehl, J.A., Zindy, F., Sherr, C.J., 1997. Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev 11, 957-972.

Du, J., He, Z.D., Jiang, R.W., Ye, W.C., Xu, H.X., But, P.P., 2003. Antiviral flavonoids from the root bark of Morns alba L. Phytochemistry 62, 1235-1238.

El-Beshbishy, HA., Singab, A.N., Sinkkonen, J., Pihlaja, K., 2006. Hypolipidemic and antioxidant effects of Morns alba L. (Egyptian mulberry) root bark fractions supplementation in cholesterol-fed rats. Life Sci 78, 2724-2733.

Eo, H.J., Park, J.H., Park, G.H., Lee, M.H., Lee, J.R., Koo, J.S., Jeong, J.B., 2014. Anti-inflammatory and anti-cancer activity of mulberry (Moras alba L.) root bark. BMC Complement. Altern. Med 14, 200.

Fukai, T., Satoh, K., Nomura, T., Sakagami, H., 2003. Antinephritis and radical scavenging activity of prenylflavonoids. Fitoterapia 74, 720-724.

Holland, TA., Elder, J., McCloud, J.M., Hall, C., Deakin, M., Fryer, A.A., Elder, J.B., Hoban, P.R., 2001. Subcellular localisation of cyclin D1 protein in colorectal tumours is associated with p21(WAF1/CIP1) expression and correlates with patient survival. Int. J. Cancer 95, 302-306.

Huang, J.W., Shiau, C.W., Yang, Y.T., Kulp, S.K., Chen, K.F., Brueggemeier, R.W., Shapiro, C.L., Chen, C.S., 2005. Peroxisome proliferator-activated receptor gamma-independent ablation of cyclin D1 by thiazolidinediones and their derivatives in breast cancer cells. Mol. Pharmacol. 67, 1342-1348.

Huang, Y.T., Huang, D.M., Chueh, S.C., Teng, C.M., Guh, J.H., 2006. Alisol B acetate, a triterpene from Alismatis rhizoma, induces Bax nuclear translocation and apoptosis in human hormone-resistant prostate cancer PC-3 cells. Cancer Lett 231, 270-278.

Kang, K.B., Lee, D.Y., Kim, T.B., Kim, S.H., Kim, H.J., Kim, J., Sung, H.S., 2013, Prediction of tyrosinase inhibitory activities of Moras alba root bark extracts from HPLC fingerprints. Macrochem. J. 110, 731-738.

Kato, J., Matsushime, H., Hiebert, S.W., Ewen, M.E., Sherr, C.J., 1993. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev 7, 331-342.

Khiev, P., Cai, X.F., Chin, Y.W., Ahn, K.S., Lee, H.K., Oh, S.R., 2009. Cytotoxic terpenoids from the methanolic extract of Bridelia cambodiana. J. Korean Soc. Appl. Biol. Chem 52, 626-631.

Kim, J.K., Diehl, JA., 2009. Nuclear cyclin D1: an oncogenic driver in human cancer. J. Cell. Physiol. 220, 292-296.

Kwak, Y.T., Li, R., Becerra. C.R., Tripathy, D.. Frenkel, E.P., Verma, U.N., 2005. IkappaB kinase alpha regulates subcellular distribution and turnover of cyclin D1 by phosphorylation. The J. Biol. Chem. 280, 33945-33952.

Landis, M.W., Pawlyk, B.S., Li, T., Sicinski, P., Hinds, P.W., 2006. Cyclin D1-dependent kinase activity in murine development and mammary tumorigenesis. Cancer Cell 9, 13-22.

Lee, J.C., Won, S.J., Chao, C.L., Wu, F.L., Liu, H.S., Ling, P., Lin, C.N., Su, C.L., 2008. Morusin induces apoptosis and suppresses NF-kappaB activity in human colorectal cancer HT-29 cells. Biochem. Biophys. Res. Commun. 372, 236-242.

Lee, Y.M., Sicinski, P., 2006. Targeting cyclins and cyclin-dependent kinases in cancer: lessons from mice, hopes for therapeutic applications in human. Cell Cycle 5, 2110-2114.

Li, Z., Wang, C., Prendergast, G.C., Pestell, R.G., 2006. Cyclin D1 functions in cell migration. Cell Cycle 5, 2440-2442.

Lin, D.I., Barbash, O., Kumar, K.G., Weber, J.D., Harper, J.W., Klein-Szanto, A.J., Rustgi. A., Fuchs, S.Y., Diehl, J.A., 2006. Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex. Mol. Cell 24, 355-366.

Lu, X.F., He, C.Q., Yu, H.N., Ma, Q,, Shen, S.R., Das, U.N., 2010. Colorectal cancer cell growth inhibition by linoleic acid is related to fatty acid composition changes. J. Zhejiang Univ. Sci. B 11, 923-930.

Lundberg, A.S., Weinberg, R.A., 1998. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell. Biol. 18, 753-761.

Malumbres, M., Barbacid, M., 2009. Cell cycle, CDKs and cancer: changing paradigm. Nat. Rev. Cancer 9, 153-166.

Mamay, C.L., Schauer, I.E., Rice, P.L., Dwyer-Nield, L.D., You, M., Sclafani, RA., Malkinson, A.M., 2001. Cyclin D1 as a proliferative marker regulating retinoblastoma phosphorylation in mouse lung epithelial cells. Cancer Lett 168, 165-172.

Mukhopadhyay, A., Banerjee, S., Stafford, L.J., Xia, C., Liu, M., Aggarwal, B.B., 2002. Curcumin-induced suppression of cell proliferation correlates with downregulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation. Oncogene 21, 8852-8861.

Nam, S.Y., Yi, H.K., Lee, J.C., Kim, J.C., Song, C.H., Park, J.W., Lee, D.Y., Kim, J.S., Hwang, P.H., 2002. Cortex mori extract induces cancer cell apoptosis through inhibition of microtubule assembly. Arch. Pharm. Res. 25, 191-196.

Okabe, H., Lee, S.H., Phuchareon, J., Albertson, D.G., McCormick, F., Tetsu, 0., 2006. A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer cell proliferation. PloS one 1, e128.

Pitchai, D., Roy, A., Ignatius, C., 2014. In vitro evaluation of anticancer potentials of lupeol isolated from Elephantopus scaber L. on MCF-7 cell line. J. Adv. Pharm. Technol. Res. 5,179-184.

Shan, J., Zhao, W., Gu, W., 2009. Suppression of cancer cell growth by promoting cyclin D1 degradation. Mol. Cell 36, 469-476.

Sherr, C.J., 1996. Cancer cell cycles. Science 274, 1672-1677.

Siveen, K.S., Nguyen, A.H., Lee, J.H., Li, F., Singh, S.S., Kumar, A.P., Low, G., Jha, S., Tergaonkar, V., Ahn, K.S., Sethi, G., 2014. Negative regulation of signal transducer and activator of transcription-3 signalling cascade by lupeol inhibits growth and induces

apoptosis in hepatocellular carcinoma cells. Brit. J. Cancer 111, 1327-1337.

Sohn, H.Y., Son, K.H., Kwon, C.S., Kwon, G.S., Kang, S.S., 2004, Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Moras alba L., Moras mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai. Phytomedicine 11, 666-672.

Spinella, M.J., Freemantle, S.J., Sekula, D., Chang, J.H., Christie, A.J., Dmitrovsky, E., 1999. Retinoic acid promotes ubiquitination and proteolysis of cyclin D1 during induced tumor cell differentiation. J. Biol. Chem. 274, 22013-22018.

Thoms, H.C., Dunlop, M.G., Stark, L.A., 2007. p38-mediated inactivation of cyclin D1/cyclin-dependent kinase 4 stimulates nucleolar translocation of RelA and apoptosis in colorectal cancer cells. Cancer Res 67, 1660-1669.

Wang, H., Khor, T.O., Shu, L., Su, Z.Y., Fuentes, F., Lee, J.H., Kong, A.N., 2012. Plants vs. cancer: a review on natural phytochemicals in preventing and treating cancers and their draggability. Anti-Cancer Agents Med. Chem 12, 1281-1305.

Weinberg, R.A., 1995. The retinoblastoma protein and cell cycle control. Cell 81, 323-330.

Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavonoid contents in mulberry and their scavenging effects in superoxide radicals. Food Chem 64, 555-559.
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Author:Eo, Hyun Ji; Park, Gwang Hun; Jeong, Jin Boo
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Feb 15, 2016
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