Inhibition of Morusin From Mulberry Branches As An Agro-Waste to MDA-MB-453 and HCT116 Cancer Cells By Inducing Cell Apoptosis and Disturbing The Cell Cycle

Mulberry tree branches are one of the largest agro-wastes produced in silk industry. How to make full use of this waste has always been one of the most important issues for the silk industry and even the entire biological industry. The paper has rst reported that the inhibition of morusin recovered from mulberry branch barks, a prenylated avonoid, on 20 kinds of tumour cells, of which the IC 50 values of the 80% cells reaches about 15 µM. Second, effects on the proliferation, invasion and apoptosis of two cancer cells were investigated in detail. The experimental results showed that the apoptotic ratio of the high concentration was 77.73% in MDA-MB-453 cells. Western blotting displayed that morusin upregulated E-cadherin and downregulated vimentin and N-cadherin in a dose-dependent manner, and thus reversed epithelial-mesenchymal transition. It could upregulate cleaved Caspase-3 and Bax and downregulate Caspase-3 and Bcl-2, which indicate that the cell apoptosis is induced by morusin. These cancer cells, MDA-MB-453, were blocked in G2 phase, and HCT116 were arrested in S phase when treated with morusin, which is possible that the cell cycle is disturbed. Therefore, morusin could inhibit cancer migration and growth and promote cancer cell apoptosis.


Introduction
The genus Morus consists of 10 to 16 species of deciduous trees in worldwide [ 1] . Various parts of mulberry including leaf Folium mori, fruit Fructus mori, root Cortex mori, branches or stem Ramulus mori, and even fungus hosted in the mulberry can be used as a traditional Chinese medicine (TCM). However, except for its leaves as the silkworm food, the biological utilization of other parts of the mulberry is quite limited, especially the mulberry branches. The branches are one of the largest agro-wastes produced in the sericulture or silk industry. How to make full use of this waste has always been one of the most important issues for the silk industry and even the entire biological industry.
Morusin is thought to restrain the growth and development of human rectal cancer HT-29 cells through the activation of caspase or the suppression of nuclear factor NF-kB [ 9] . It can also reduce the proliferation of human cervical cells, the formation of a tumour sphere and the migration of tumour cells [ 10] . Park et al., chose morusin and TNF-related apoptosis-inducing ligand to treat human glioma cells [ 11] . The antioxidation of morusin involves in blocking phorbol ester-induced malignant transformation in JB6 P+ mouse epidermal cells [ 12] . The latest reports showed that morusin inhibits cell proliferation and tumor growth in human gastric cancer by the down-regulation of c-Myc [ 13] and growth in breast cancer cells in vitro and in vivo [ 14,15] . In addition, blockage of the signal transducer and activator of transcription 3 signalling pathway (STAT3) by morusin induces apoptosis and inhibits invasion in human pancreatic tumour cells [ 16] and human prostatic cells [ 17] . Moreover, morusin can delay the occurrence of convulsions signi cantly and shorten the time of convulsions [ 18] and inhibit the activity of β-  [ 21] .
Our group had investigated in details the difference of morusin in 100 kinds of mulberry branch bark by RP-HPLC-DAD (reverse-phase high-performance liquid chromatography) with diode array detector, and the maximum amount was found to be 585.81 µg/g and 20 times to the minimum [ 22] . Similarly, morusin was found to restrain the proliferation of human hepatocarcinoma Bel-7402 cells, resulting in a survival rate of tumour cells of only 34.56% and an apoptotic rate of cancer cells reaching 81% in high concentrations with a dose-dependent manner [ 23] . It can suppress transplanted H22 hepatocarcinoma in mice [ 24] .
In Human hepatoma cell HCCLM3, mouse lung cancer cell LLC and human colon cancer cells HCT-116 and CACO-2 were subcultured in Dulbecco's Modi ed Eagle's Medium (DMEM) containing heat inactivated 10% FBS, 1% penicillin and streptomycin. These cells were cultured at 37°C in a 5% CO 2 incubator. LLC was the hemiparietal cell, and the other cells were adherent. The culture process was the same as that described above.
Human hepatoma cell (Hep-G2), human prostatic cancer cell (DU145) and human malignant melanoma cell (SK-MEL-2) were subcultured in extreme magnetoelectric (EME) medium supplemented with heat inactivated FBS (10%), 1% penicillin and streptomycin. Cells were maintained at 37°C in a 5% CO 2 incubator. All cells were adherent growth cells, and the culture process was the same as that described above.
MTT & CCK8 assay. The logarithmic growth cells were collected, and the appropriate concentration of cell suspension was selected. After each hole in the 96-well plate was added to 90 µl of cell suspension, it was cultivated overnight at 37°C in a 5% CO 2 Incubator (HERACELL 150i type of Thermo Scienti c). When cells were in the adherent condition, the control (normal) group and the morusin (sample) groups were respectively set up, each group with 4 compound holes, with 10 µl of morusin added to each experimental well and 10 µl of complete medium added to the control group. Then, they were cultured in the incubator for 24 h, 48 h and 72 h, added 20 µl MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5mg/ml, Sigma) solution or 10 µl CCK (Cell Counting Kit 8, Nanjing KeyGen BioTech Co., Ltd., China) solution and cultivated for an additional 4 h. Finally, the culture solution was removed, and formazan crystals were dissolved in 150 µL of DMSO (dimethyl sulfoxide). Absorbance was then recorded at a 450 nm or 495 nm wavelength by a spectrophotometer (SpectraMax M5, Molecular Devices Company, USA). Tumour inhibitory rate and cell viability were calculated as follows: Hoechst staining 1. The ordinary cover glass was soaked in 70% ethanol for 5 min or longer, washed in PBS times, and then washed with the cell culture medium. The control group and morusin groups were set up, and the cover glass was placed into the 6-well plate, which was approximately 50 to 80% full, and the cells were cultivated overnight.
3. The xed liquid was discarded, the cells were washed twice (6 min) with 4°C precooling PBS, and then all liquid was absorbed.
4. Hoechst 33258 staining solution (0.5 mL) was added to each hole and then dyed for 15 min at room temperature in the dark.
5. The staining liquid was discarded, and the cells were washed twice (10 min) with 4°C precooling PBS.
6. A drop of anti-uorescence quenching sealant was placed on the glass slide, covered with the cell of cover glass, avoiding the bubble as much as possible. min. Then, the secondary antibodies were bioconjugated with horseradish peroxidase at a dilution (1:10000) for 1 h at room temperature and washed 4 times with TBST. The β-tubulin was used as the internal reference. The enhanced chemiluminescence (ECL) method was applied for development, and cassette exposure was used for development and xation. Bands were visualised using a UVP detection system and band intensity that was quanti ed using LAB WORKS 4.6. All experimental results were repeated 3 times.

Statistical Analysis
The experimental data were recorded and analysed using Origin 8.5 software. The result as mean standard deviation (±SD) and analysis of variance (ANOVA) was used to evaluate the difference between multiple groups. SigmaScan Pro 5 software was used for WB light density determination, and X ±S (n = 3) expressed the nal results, with P values less than 0.05 as signi cant and P values less than 0.01 as very signi cant.

Results
The inhibtion ratio of different concentration of morusin to 20 cancer cells In order to screen the possible therapeutic effects of morusin in drug-resistant cancer cells, 20 kinds of cancer cells were treated for 48 h by adding morusin. The results, which were obtained with MTT or CCK8 (Fig 1), indicated that morusin inhibited cell viability in all cancer cells in a dose-and/or time-dependent manner. The inhibition rate of all high concentration groups reached as high as 84%. .73% in the 20 µg/mL morusin group, which was evidently higher than that of the control group (P<0.01). The apoptotic ratio in the 20 µg/mL morusin group was 4.5-fold in comparison with that of the control group (Fig 5), and the apoptotic ratios in the 5 µg/mL and 10 µg/mL morusin groups were signi cantly higher than that of the control group (P<0.05). The apoptotic effect was much more noticeable in two cancer cells of MDA-MB-453 and HCT116.

Hoechst staining
Hoechst staining was used to investigate the nucleus of apoptosis. MDA-MB-453 cells and HCT116 cells were treated with three concentrations of morusin for 48 h. As shown in Fig 6a and Fig 7a, the nucleus morphology of two kinds of cells were expressed as oval or round, and the chromatin was distributed equally in the nucleus. With the addition of different concentrations of morusin, the chromatin condensation of the cells was scattered and normal nuclear structure was lost, which resulted in apoptotic cell death (Fig 6d and Fig 7d).
Changes of cancer cell cycle  The cell cycle regulation mechanism is closely related to tumourigenesis. Cell cycle arresting induces cell apoptosis and the relation of cell cycle and cell apoptosis is mediated by some cytokines such as c-myc, p53, pRb, RAS, PKC, PKA, Bcl-2, NF-κB, CDK [ 25 ]. After the cancer cells of MDA-MB-453 were mixed with three concentrations of morusin for 48 h, the cell cycle was measured via ow cytometry. The number of cells in the G1 phase was signi cantly reduced by almost half (Table 1) in the 10 µg/ml morusin group in comparison with that of the control group. The number of cells increased in the G2 phase and remained mainly unchanged in the S phase (Fig 8), suggesting that morusin has growth inhibition on MDA-MB-453 cells by perturbation at the G2 phase. As shown in Fig 9 and Table 2, HCT116 cells were processed with morusin for 48 h, and the amount of cells was reduced in the G1 phase, enhanced in S phase and basically unchanged in the G2 phase in the 10 µg/ml morusin group. Obvious arrest was seen in the S phase at 10 µg/ml of morusin. The results demonstrated that the cells of HCT116 were accumulated in the S phase. In general, morusin in uences DNA replication, disturbs the cell cycle and induces cell death.
The expression of EMT-related regulatory proteins The embryonic programme 'EMT' (epithelial-mesenchymal transition) is believed to promote malignant tumour progression [ 26 ]. The cells of MDA-MB-453 were treated with three concentrations of morusin (2.5, 5, 10 µg/mL) for 48 h, respectively. Fig 10 shows that E-cadherin expression was increased and the expression of N-cadherin and Vimentin were reduced in a dose manner. The increase of E-cadherin strengthened cell adhesion; thus it inhibited cancer occurrence. E-cadherin expression in the 10 µg/mL morusin group was signi cantly higher than that of the control group (p 0.01), and the expression of Vimentin in the 10 µg/mL morusin group was lower than that of the control group (p 0.01). Similarly, as shown in Fig 11, E-  In the test, after 48 h, the cancer cells of HCT116 were accumulated in the S phase, and the cancer cells of MDA-MB-453 were accumulated in the G2 phase. It has been well con rmed that cell growth and proliferation of mammalian cells occurs via cell cycle progression and that the inhibition of the cell cycle has been recognised as a target for anti-cancer drugs [ 39 , 40 ].
The triggering of tumour cell apoptosis is a foundation of many cancer therapies [ 41 ]. Our results showed that morusin could induce cancer cell apoptosis and that the apoptotic rate was positively correlated with the dosage of morusin. The nucleus apoptosis of MDA-MB-453 and HCT116 cells can be observed by Hoechst staining. The results also indicated that morusin could induce cell apoptosis by destroying the nuclear structure. In the current study, the results of the apoptotic cells to be Annexin V-FITC/PI doublestained after morusin treatment also indicated that morusin induced the apoptotic death of human HCC cells [ 42 ].
All above results showed that morusin can inhibit cancer cell growth and induce cell apoptosis, which provided an important experimental basis for the study of the apoptosis of breast and colon cancer cells.

Conclusion
The present experimental results indicated that morusin has the promising cytotoxic activities against many human cancer cells through inhibiting cell growth in a dose-and time-dependent manner, inducing morphological changes and arresting normal progression of cell cycle. Moreover, with the apoptotic mechanism, morusin results in the two cells of MDA-MB-453 and HCT116 upregulating protein expression of cleaved caspase-3 and Bax and down-regulating expression of caspase-3 and Bcl-2, which triggered cell apoptosis. In general, these ndings further supported that morusin can be used as a potential anticancer drug and could be an attraction for further anti-tumour compounds.