Toona Sinensis and Moschus Decoction Induced Cell Cycle Arrest in Human Cervical Carcinoma HeLa Cells

Toona sinensis and Moschus are two herb materials used in traditional Chinese medicine, most commonly for their various biological activities. In this study, we investigated the inhibitory effect of three decoctions from Toona sinensis, Moschus, and Toona sinensis and Moschus in combination on cell growth in several normal and cancer cell lines by cell viability assay. The results showed that the combined decoction exhibited the strongest anticancer effects, compared to two single decoctions. The observations indicated that the combined decoction did not induce cell apoptosis and autophagy in HeLa cells by fluorescence microscopy. Flow cytometry analysis revealed that the combined decoction arrested HeLa cell cycle progression in S-phase. After the decoction incubation, among 41 cell cycle related genes, eight were reduced, while five were increased in mRNA levels by real-time PCR assay. Western blotting showed that there were no apparent changes of protein levels of Cyclin E1, while P27 expression significantly declined and the levels of CDC7 and CDK7 obviously increased. The data suggest that the RB pathway is partially responsible for the decoction-induced S-phase cell cycle arrest in HeLa cells. Therefore, the combined decoction may have therapeutic potential as an anticancer formula for certain cancers.


Introduction
Traditional Chinese medicine (TCM) has been used in clinical practice for thousand years. Compound formula of TCM has been shown to exhibit synergism [1]. TCMs are used to restore overall healthful balance and normal body function in a holistic way due to their moderate treatment effects and lower side effects [2]. These features made themselves popular in China.
Toona sinensis is a type of arbor that is widely distributed in Asia. The leaves and young shoots are used as vegetable in China and Malaysia [3,4]. In fact, it has long been used in TCM for a wide variety of conditions. The leaf extracts showed various biological activities, including anticancer [5][6][7][8][9], antidiabetes [10], and antioxidant [11] effects, as well as inhibiting Leydig cell steroidogenesis [12] and suppressing brain degeneration in senescence-accelerated mice [13].
The bark is used as astringent and depurative, the powdered root is used as a corrective, and the fruits are used as an astringent and for the treatment of eye infection [14,15].
Musk, a ventral glandular secretion of the male musk deer, is also a precious and wide applied material in TCM [16][17][18]. As a major Chinese herbal material, musk was firstly recorded in Shen Nong Ben Cao Jing (The Herbal Classic of the Divine Plowman) in about 2700 BC and has been widely used for thousands of years. Now it is officially listed in Chinese Pharmacopoeia as Moschus, recognized as an important medicinal material with many pharmacological activities, including resuscitation, activating blood to promote menstruation, detumescence, and analgesia [19].
The Toona sinensis and Moschus decoction has been used as a folk medicine for patients with liver cancer to alleviation of the symptom in Gusu province of China, while single Toona sinensis or Moschus shows no effect on 2 Evidence-Based Complementary and Alternative Medicine these patients. The probable mechanism is unclear. In the present study, we investigated the effects of Toona sinensis and Moschus decoction on cell growth inhibition in several normal and cancer cell lines and explored its underlying molecular mechanism in human cervical carcinoma HeLa cells. Our results indicate that Toona sinensis and Moschus decoction inhibits HeLa cell growth by inducing cell cycle arrest at S-phase via regulation of some cell cycle related proteins. The findings provide evidence that Toona sinensis and Moschus decoction may have potential in the therapeutic use for some cancers.

Cell Lines and Cell
Culture. The mouse embryo fibroblast cell line NIH3T3 was obtained from ATCC (Manassas, VA) and human hepatoma cell line SMMC-7721, cervical carcinoma cell line HeLa, and liver cell line QSG-7701 were supplied by the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). They were maintained in RPMI 1640 medium (HeLa, SMMC-7721 and QSG-7701) or Dulbecco's modified Eagle's medium (NIH3T3), supplemented with 10% fetal bovine serum (GIBCO, USA) and 1% penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin, Solarbio Science and Technology, Beijing, China) at 37 ∘ C in humidified air with 5% CO 2 .

Herbal Extract and Treatments.
The raw herbs of Toona sinensis bark and Moschus were purchased from Gansu province of China. Both of them were identified by Professor Guichen Chen from Northwest Institute of Plateau Biology, Chinese Academy of Sciences. The bark of Toona sinensis was firstly washed to remove all contaminants and then cut into small pieces. The decoction was prepared by mixing Toona sinensis bark and Moschus 50 : 1 weight ratio. The mixture was allowed to soak with 10-fold of water for 2 h, followed by extraction at 100 ∘ C for 3 h. The water extract was then dried at 50-60 ∘ C and stored at room temperature. The similar amount of Toona sinensis bark and Moschus was extracted as controls according to the above procedure, respectively. For all experiments, the herbal preparation was weighed out and dissolved in culture medium to a concentration of 5 mg/mL and was immediately added at the indicated concentrations in cultured cells.

Cell Viability Assay. Cell viability was evaluated by
Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to the manufacturer's instruction. Cells were plated in 96-well micro-well plates in 0.1 mL volumes of medium with 10% FBS, at a density of 1 × 10 5 cells/well. Absorbance was measured at 450 nm with a iMark Microplate Absorbance Reader (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Hoechst 33258
Staining. The cells at 2 × 10 5 cells/mL were seeded in 24-well plates and treated with different concentrations of the decoctions for 48 h. They were fixed in 4% paraformaldehyde in 4 ∘ C refrigerator for 30 min and then stained with 5 g/mL Hoechst 33258, a DNA-specific fluorescent dye, for 10 min at 37 ∘ C. The stained cells were observed under a fluorescence microscope (Olympus BX51, Tokyo, Japan).

MDC Autophagy Staining.
For autophagic fluorescence observation, cells were grown on coverslips and treated with the decoction. After 48 h, cells were stained with 0.05 mM MDC at 37 ∘ C for 1 h. The cellular fluorescence was observed using Nikon Eclipse Ti-S inverted fluorescence microscope (Tokyo, Japan) with 490 nm band-pass excitation filter and 515 nm long-pass barrier filter.
2.6. Cell Cycle Analysis. Cellular DNA content was determined by flow cytometry. After incubation with the decoction for 24 h, cells were harvested, washed twice with ice-cold PBS, and fixed with 70% ethanol overnight at −20 ∘ C. Cells were then washed twice with ice-cold PBS and resuspended in PBS. RNA was digested with RNase A (100 g/mL) and DNA was stained with PI (50 g/mL). After incubation for 30 min in the dark at 37 ∘ C, stained cells were analyzed by a FACScan flow cytometry and CellQuest analysis software (Becton Dickinson, San Jose, CA).

RNA Extraction and
Real-Time PCR. The total RNA extraction was performed using TRNzol Reagent (Tiangen, Beijing, China) according to the manufacturer's instructions. Complimentary DNA (cDNA) was prepared from the total RNA using FastQuant RT kit (With gDNase) (Tiangen, Beijing, China).
The real-time PCR analysis was performed in triplicate assays using SuperReal PreMix Plus (SYBR Green) according to the manufacturer's instructions (Tiangen, Beijing, China) in a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). The reactions were as follows: activation of the Taq DNA polymerase at 95 ∘ C for 15 min and 40 cycles of 95 ∘ C for 10 s and 60 ∘ C for 32 s. We quantified 41 genes based on a cell cycle-associated cDNA microarray (CTBioscience, Changzhou, China). The symbols of the 41 genes are listed in Table 1. Gapdh, -actin, B2M, HPRT1, and OAZ1 were selected as internal controls. The relative changes in gene expression were calculated using the equation: 2 −ΔΔCT , as Livak described [20].

Western Blot.
After cells were lysed with ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Changzhou, China) for 30 min on ice, lysates were centrifuged at 13,000 ×g with 20 min and the supernatants were used as total cell lysates. Protein concentration was determined by Bradford protein assay (Bio-Rad, USA). A quantity of 50 g total protein per lane was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Amersham Hybond-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked with 5% milk powder in 0.05% Tween-TBS, incubated with the specific antibodies, including Cyclin E1, p27, and CDK7 and CDC7 rabbit monoclonal antibodies (Epitomics, Burlingame, CA, USA). Detection of the target proteins on the membranes was performed using  the ECL western blotting detection reagents. Signal density was detected using the MF-ChemiBIS Family including gel capture software (DNR Bio-Image system, Ltd., Jerusalem, Israel) and quantitatively analyzed by densitometry using the Image J software.

Statistical Analysis.
Values were expressed as means ± standard deviations. Statistical analysis was performed using Student's t-test. Values of < 0.05 were considered to be statistically significant.

The Toona Sinensis and Moschus Decoction Inhibits Cell
Proliferation. Using the WST-8 based Colorimetric Assay Cell Counting Kit-8, the effect of the Toona sinensis and Moschus decoction on cell viability was measured. As shown in Figure 1(a), cells proliferation of human cervical carcinoma cell line HeLa and hepatoma cell line SMMC-7721 was inhibited by 50%, 70%, 46%, and 62% after 50 g/mL decoction treatments for 48 h and 72 h, respectively. Compared to the two carcinoma cells, mouse embryo fibroblast cell line NIH3T3 and normal liver cell line QSG-7701 showed that cell viability decreased by 29% and 38% at 50 g/mL decoction treatment for 72 h (Figure 1(a)). Obviously, the cell growth inhibitory effects of the decoction on the two cancer cells were stronger than those on the normal cells. Furthermore, the data indicates that the decoction inhibited the growth of cancer cells in a concentration-and time-dependent manner (Figure 1(a)).
At the same time, cells were treated by the single Toona sinensis or Moschus decoction. As shown in Figure 1(b), after the treatment of 50 g/mL single Toona sinensis decoction for 72 h, the percentages of cell viability were 70%, 71%, 99%, and 99.8% in HeLa, SMMC-7721, NIH3T3, and QSG-7701 cells, respectively. According to the weight ratio of the Toona sinensis and Moschus decoction, the single Moschus concentration was set as 0.5 g/mL and 1 g/mL. As shown in Figure 1(c), after the treatment of 1 g/mL single Moschus for 24 h, the percentages of cell viability were 122.8%, 88.8%, 93.3%, and 81.1% in HeLa, SMMC-7721, NIH3T3, and QSG-7701 cells, respectively. In contrast to NIH3T3 and QSG-7701 cells, HeLa and SMMC-7721 cells showed no statistical decrease in the percentage of cell viability (Figure 1(c)). In order to further understand the cell growth inhibition of Moschus, we used

The Toona Sinensis and Moschus Decoction Does Not
Induce Cell Apoptosis and Autophagy. We sought to explore a possible explanation for its inhibition of cancer cell growth. Hoechst 33258, a DNA-specific fluorescent dye, was chosen to observe the morphologic characteristics of apoptosis in HeLa cells. As shown in Figure 2(a), no apoptotic cells can be found after the decoction treatment for 48 h by fluorescence microscopy. Based on the above observations, which indicate that apoptosis is not the cause of cell death induced by the decoction, we next questioned if it induces autophagy, a second form of cell death, in HeLa cells. To address this issue, vital staining of HeLa cells treated with the decoction for 48 h was performed with MDC dye, a selective fluorescent marker of autophagic vesicles. The dye shows diffuse staining in nonautophagic cells but exhibits punctuate vesicular staining when autophagy is induced. As shown in Figure 2(b), no acidic vesicles were observed. Likewise, the decoction did not induce autography in HeLa cells.

The Toona Sinensis and Moschus Decoction Induces Cell
Cycle Arrest at S-Phase. To examine whether the Toona sinensis and Moschus decoction, induced growth inhibition was associated with cell cycle regulation, the cell cycle distribution was analyzed by flow cytometry. After HeLa cells were incubated with the decoction for 24 h, cells were harvested and further prepared for cell cycle analysis. Figures 3(a) and 3(b) showed that cells accumulated in the S phase of the cell cycle after the decoction treatment, whereas the percentage of cells in G 0 /G 1 phase reduced significantly. Especially, the percentage of cells in S phase increased by 68% when the decoction concentration was 50 g/mL, compared to that of the untreated control ( Figure 3).

Changes of Cell Cycle Related Genes mRNA and Proteins
Expression. In order to determine whether the Toona sinensis and Moschus decoction was able to regulate cell cycle related genes expression, real-time PCR experiment was performed in the cell extracts prepared from HeLa cells. The results showed that the decoction incubation reduced the levels of CCNA1, CCND1, CCND2, CCNH, CDKN1B, MCM2, ORC6L, and WEE1 and increased the levels of CDC7, CDK6, CDK7, E2F3, and RB1 (Table 1). Taken together, among 41 detected genes, the mRNA levels of 13 genes were changed by the decoction. Next, western blotting with specific antibodies was done to detect the changes of related proteins expression levels. We found reduction of p27 and increase of CDK7 after the treatment of the decoction (Figure 4). The level of Cyclin E1 treated with the decoction showed no significant alteration as compared to the untreated control ( Figure 4). In summary, the decoction regulates mRNA and subsequent protein levels of certain cell cycle-related genes to induce S-phase cell cycle arrest.

Discussion
Different from the orthodox medicine focusing on a specific pathogenic process [21], TCMs have been recognized as a popular complementary and alternative medicines, emphasizing on individualized diagnosis and treatment of patients; maximizing the body's inherent healing ability; treatment of the "whole" person by addressing their physical, mental, and spiritual attributes [22,23]. Multiple agents contained in each formula, at least in some formulas, could hit multiple targets and exert synergistic therapeutic efficacies [24,25]. In our study, it is consistent that the Toona sinensis and Moschus decoction shows stronger anticancer effects than either single Toona sinensis decoction or Moschus one by cell viability assay.
Our subsequent investigation was focused on the mechanisms of the Toona sinensis and Moschus decoctioninduced anticancer effects in HeLa cells. The major findings of this study were that the decoction induces cell cycle arrest in S-phase. Moreover, we showed that the cell-cycle arrest was accompanied by down-regulation of CCNA1, CCND1, CCND2, CCNH, CDKN1B, MCM2, ORC6L, and WEE1 and upregulation of CDC7, CDK6, CDK7, E2F3, and RB1. Western blot also confirmed the consistent changes of several corresponding proteins. It is well known that eukaryotic cell cycle is driven by an ordered activation of a family of protein kinases, cyclin-dependent kinases, which are composed of a catalytic subunit CDK and a positive regulatory subunit cyclin [26]. Each CDK/cyclin complex is considered to play specialized roles at a defined stage of cell cycle, through phosphorylation of its specific substrate  proteins [27][28][29][30]. Our results demonstrated that there were no apparent changes of both the mRNA and protein levels of Cyclin E1 in the decoction treated cells. In contrast, P27 expression significantly declined and the level of CDK7 obviously increased in accordance to cell cycle arrest in the S-phase. All these results indicate that the effect of the Toona sinensis and Moschus decoction on cell cycle arrest may be a transcriptional event regulating the certain cell cycle related proteins. A schematic presentation of cell cycle arrest at Sphase in our study is shown in Figure 5. It seems that the RB pathway (CDK inhibitor-Cyc/CDK-RB-E2F3) is partially responsible for the decoctioninduced S-phase cell cycle arrest in HeLa cells.
In Moschus, many active compounds have been identified, such as muscone, stanolone, muscopyridine, androstane-3, 17-dione, hydroxymuscopyridine, and cholic acid. They are mainly classified as three major types including flavonoides, alkaloids, and steroids [31]. Hitherto, no reports show that these individual active ingredients exhibit effect on cell cycle progression. Regarding Toona sinensis, a number of compounds, including gallic acid, retinoid, vitamins B and C, o-coumaric acid, methyl gallate, ethyl gallate, kaempferol, kaempferol-3-O--D-glucoside, quercetin, quercitrin, quercetin-3-O--D-glucoside, and rutin, were identified [32,33]. Among them, gallic acid has been shown to block the growth of DU145 cells at G2/M phases [34]. According to our results, the decoction induces S-phase cell cycle arrest in HeLa cells, which suggests that the cell cycle arrest is induced by compound(s) other than gallic acid. It is known that the use of complex medicinal formulation exhibits synergism of the multiple ingredients or drug-drug interactions [1]. The effect may not be achieved by individual active ingredients. Therefore, future studies in identifying the complex interactions of different chemical components of the Toona sinensis and Moschus will be helpful to evaluate the anticancer effect of the decoction.

Conclusion
This study is the first to report the anticancer activity of decoction of Toona sinensis and Moschus in combination. Furthermore, we have shown that the Toona Sinensis and Moschus decoction exhibits its anticancer effects by inducing cell cycle arrest in S-phase via mRNA regulation of cell cycle related proteins in HeLa cells. Our data provide strong evidence that the Toona sinensis and Moschus decoction could be potentially utilized for the treatment of some cancers.