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

doi:10.21203/rs.3.rs-205057/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-205057/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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 first reported that the inhibition of morusin recovered from mulberry branch barks, a prenylated flavonoid, on 20 kinds of tumour cells, of which the IC₅₀ 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.

General Cell Biology & Physiology

Cancer Biology

morusin

mulberry

cancer cell

inhibitory effects

apoptosis

cell cycle

Morusin, a prenylated flavonoid, recovered from mulberry branch has evidently inhibitory effects on 20 kinds of tumour cells.
The apoptotic ratio of 20 µg/mL morusin was 77.73% in MDA-MB-453 cells.
Morusin could inhibit the migration and growth of MDA-MB-453 and HCT116 cells and promote cancer cell apoptosis.

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.

The main effective constituents of the branch or stem R. mori are flavones, polysaccharides, alkaloids, phenolic acid, terpene and coumarin, which lower blood glucose[^2], regulate blood lipids[^3], possess anti-tumour properties[^4], enhance immunity and possess anti-inflammatory[^5], anticoagulant and antioxidant properties[^6]. Early in 1978, Nomura et al., first separated the morusin from mulberry root bark[^7]. Morusin is a yellow powder, with a molecular weight of 420.459 and molecular formula of C₂₅H₂₄O₆^[8].

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 significantly and shorten the time of convulsions [^18] and inhibit the activity of β-secretase[^19], acetylcholine esterase and butyrylcholin esterase, with IC₅₀ values of 36.4 µM and 24.08 µM[^20], respectively, indicating an effect on Alzheimer’s disease. Its glycoside derivative-morusin-4'-glucoside can also resist the competence of human immunodeficiency virus (HIV)[^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 the present study, IC₅₀values of 20 kinds of tumour cells were first screened with morusin. Then, the research mainly focused on two resistant cell lines, colorectal cancer cells of HCT116 and breast cancer cells of MDA-MB-453. This experiment has explored the morusin sensitivity to resistant cell lines of the two cancer cells in detail.

Materials

Morusin (Chengdu Ruifensi BioTech Co., Ltd., China; Purity≥98%) MCF-7, A549/T, CaEs-17, HePG2, HCCLM3, A2780/T, LLC, SGC-7901/DDP, MDA-MB-231, MCF-7/Adr, MGC-803, HCT-116, NCI-H1299, DU145, SK-MEL-2, SW1116, K562/Adr, CACO-2, HCT-8/V, MDA-MB-453 (Nanjing KeyGen Biotech Co., Ltd., China)

Cell culture

Three breast cancer cells of MCF-7, MDA-MB-453 and MDA-MB-231, human oesophageal cancer cell of CaEs-17, gastric cancer cell of MGC-803, human lung cancer cell of NCI-H1299 and colorectal cancer cell of SW1116 were subcultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with heat inactivated FBS (foetal bovine serum, 10%), penicillin (1%) and streptomycin. These cells were maintained at 37°C in a 5% CO₂incubator. The cells of MDA-MB-453 were suspended, and the others were adherent cells. Culture solution of adherent cells in the process of passage, with PBS flushing one time, and circular shrinkage was observed in microscope and joined medium to stop trypsin digestion. Then, the solution was centrifuged for 5 min (1000 rpm/min) and made cells sedimentation, joined new culture solution, blowed into the cell suspension gently and inoculated cells respectively.

Human hepatoma cell HCCLM3, mouse lung cancer cell LLC and human colon cancer cells HCT-116 and CACO-2 were subcultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing heat inactivated 10% FBS, 1% penicillin and streptomycin. These cells were cultured at 37°C in a 5% CO₂incubator. 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% CO₂incubator. All cells were adherent growth cells, and the culture process was the same as that described above.

Human lung cancer A549/Taxol cells, human ovarian cancer A2780/Taxol cells, human gastric cancer SGC-7901/DDP cells, human breast cancer MCF-7/Adr cells, human leukaemia K562/Adr cells and human colorectal HCT-8/V cells were subcultured in RPMI-1640 medium containing heat inactivated 10% FBS, 1% penicillin and streptomycin. Cells were maintained at 37°C in a 5% CO₂incubator. These cells joined Taxol, DDP, Adr 2 µg/ml, respectively.

Experimental groups

The test was divided into six different concentration groups: normal group (no morusin), 1.25, 2.5, 5, 10 and 20 µg/mL (these concentrations are final concentrations).

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% CO₂ Incubator (HERACELL 150i type of Thermo Scientific). 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:

Tumour inhibitory rate (%) = [1-A (morusin) / A (control)] Í 100%

Cell viability (%) = [A (morusin) – A (control)] / [A (no morusin) – A (control)] Í 100%

A (morusin): the absorbance of the wells containing CCK solution, cells and different doses of morusin

A (control): the absorbance of the wells containing CCK solution, complete medium and no cells

A (no morusin): the absorbance of the wells containing CCK solution, cells and no morusin

Cell apoptosis and cell cycle

The logarithmic growth cells were added to a 6-well plate in an incubator overnight. The morusin was added to the corresponding drug medium, and the control group was set. After cells were mixed with morusin for 48 h, the cells were collected with 0.25% trypsin enzyme [without ethylenediaminetetraacetic acid (EDTA)].

Cells were centrifuged for 5 min, and the supernatant was abandoned and the precipitate was resuspended by complete medium. The 100 µl of MUSE^TMAnnexin & Dead Cell Kit (Nanjing Fcmacs Biotech Co., Ltd., China) reagents were loaded into 1.5 ml centrifuge tubes, and 100 µl of cell suspension was shifted to each tube, which was slightly mixed and incubated for 20 min at room temperature, and the cell apoptosis was measured by MUSE^TMflow cytometry (Becton-Dickinson, San Jose, CA).
Cell cycle immobilisation: cell pellets were collected by centrifuge and resuspended in PBS (phosphate-buffered saline) with 0. 2 mL of ice-cold 70% ethanol and then kept at -20°C overnight. Immobilised cells were centrifuged for 5 min at 300×g speed, and cleaned in 1×PBS, then resuspended by 200 µL of MUSE^TM Cell Cycle Kit (Nanjing Fcmacs Biotech Co., Ltd., China) reagents and incubated for 30 min at room temperature. Cell apoptosis was measured by MUSE^TMFlow Cytometry (Becton-Dickinson, San Jose, CA, USA).

Hoechst staining

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.
Corresponding concentrations of morusin (including 5, 10 and 20 µg/ml) and complete medium were combined in morusin group wells and blank control group well. After cultivation for 48 h, the culture solution was discarded and 0.5 mL of fixed liquid (4% formaldehyde solution) was added and fixed for 15 min.
The fixed liquid was discarded, the cells were washed twice (6 min) with 4°C precooling PBS, and then all liquid was absorbed.
Hoechst 33258 staining solution (0.5 mL) was added to each hole and then dyed for 15 min at room temperature in the dark.
The staining liquid was discarded, and the cells were washed twice (10 min) with 4°C precooling PBS.
A drop of anti-fluorescence quenching sealant was placed on the glass slide, covered with the cell of cover glass, avoiding the bubble as much as possible.
Ultraviolet excitation was used to observe and photograph the cells under the inverted fluorescence microscope.

Western Blotting

The expression of caspase-3, cleaved caspase-3, E-cadherin, N-cadherin, Vimentin, Bax, Bcl-2 and b-tubulin in samples and in cells irradiated by 6 MV x-ray in different groups were detected by WB. Total proteins from cells were centrifuged for 20 min at 13,000´g. Proteins were then shifted to polyvinylidene difluoride membranes (Millipore, Shanghai, China), closed with 5% non-fat milk in tris-buffered saline with Tween (TBST, 20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) and buffered for 1 h. The target proteins were detected with different antibodies at 4°C overnight, and washed 4 times with TBST for 30 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 fixation. Bands were visualised using a UVP detection system and band intensity that was quantified 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 final results, with P values less than 0.05 as significant and P values less than 0.01 as very significant.

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%. Fig 2 shows that the average of IC₅₀ (50% inhibition of cell growth) was 14.79 µM. These results indicated that morusin has inhibited effects on many cancer cells and induced maximal cytotoxicity against cancer cells.

The inhibition ratio of MDA-MB-453 and HCT116 cells

Two cells, MDA-MB-453 and HCT116, were treated with different concentrations of morusin. The inhibition ratio was detected after 12 h，24 h，48 h by MTT or CCK8 assay, respectively. As illustrated in Fig 3, the inhibition ratio of both cancer cells was elevated with the increasing time, indicating that the higher the concentration, the higher the inhibition ratio. MDA-MB-453 cells were processed in 5 µg/ml of morusin for 48 h, and the inhibition ratio was approximately 3 times higher than that after a 24-h treatment. The inhibition ratio in 20 µg/ml of morusin peaked at 83.57% and 94.02% after 12 h and 48 h, respectively. After 12 hours, the inhibition ratio of HCT116 cells reached 90.81% in 20 µg/ml of morusin. Therefore, the results suggested that morusin induced inhibition effect in a concentration- and time-dependent manner.

The influence of morusin on cancer cell apoptosis

Annexin V/7-AAD dyeing can quantitative analyse cell apoptosis, including early and late apoptotic cells, viable cells and necrotic cells. Both cells were processed in different concentrations of morusin for 48 h, and the apoptotic ratio increased in a dose-independent manner. Fig 4 shows that the apoptotic ratio of MDA-MB-453 was 77.73% 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 significantly 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

Table 1. Changes of the cell cycle in MDA-MB-453 cells induced by morusin

Concentration	G₁(%)	S (%)	G₂ (%)
Control	47.469±5.0985	38.490±4.278	14.04±1.793
2.5 (µg/ml)	42.129±1.258*	41.223±0.872	16.646±1.100
5 (µg/ml)	43.047±1.360*	40.756±0.405	16.196±1.357
10 (µg/ml)	26.425±5.166*	31.849±7.982	41.681±10.237

*P＜0.05, compared with the control.

Table 2. Changes of the cell cycle in HCT116 cells induced by morusin

Group	G₁(%)	S (%)	G₂ (%)
Control	57.561±1.227	27.037±0.332	15.417±1.100
2.5 (µg/ml)	58.391±0.994	26.464±0.552	15.144±0.601
5 (µg/ml)	58.749±1.241	25.798±0.820	15.453±0.928
10 (µg/ml)	38.114±10.202	43.316±5.567	18.570±4.718

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 flow cytometry. The number of cells in the G1 phase was significantly 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 influences 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 significantly 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-cadherin expression was increased and Vimentin was reduced in HCT116 cells in a dose-dependent manner. The WB results indicated that morusin could improve EMT by three cell surface proteins, E-cadherin, N-cadherin and Vimentin. Consequently, morusin could inhibit the migration, invasion and metastasis of tumour cells.

The expression of caspase-3-related proteins

Csapase-3 is an important protein in the process of cell apoptosis and widely expressed in normal human tissues and a variety of tumour tissues. As represented in Fig 12, two cells of MDA-MB-453 and HCT116 were treated with three concentrations morusin (2.5, 5, 10 µg/mL) for 48 h. The caspase-3 expression was downregulated, and the cleaved caspase-3 was upregulated significantly in a dose-dependent manner. The result suggested that morusin would inhibit tumour cell growth and induce cell death.

The expression of Bcl-2 family

Bcl-2 and Bax are the most representative genes of apoptosis inhibition and promotion in the Bcl-2 family. Bax is the main factor in the regulation of Bcl-2 activation [²⁷]. The two cells of MDA-MB-453 and HCT116 were treated with morusin (2.5, 5, 10 µg/mL) for 48 h. The expression of Bax (pro-apoptotic protein) was increased, and the expression of Bcl-2 was decreased significantly in a dose-dependent manner (Fig 13). The above results indicated that increasing of Bax level and decreasing of Bcl-2 level may lead to tumour cell apoptosis.

Breast cancer is now the most frequently to be diagnosed cancer [²⁸] and 9.6% of all deaths breast cancer worldwide [²⁹]. Colon cancer is also one of the most widespread malignant tumours [³⁰]. Chemoprevention using natural drugs is considered a hopeful anti-tumour approach [³¹]. Phytochemicals with antioxidant and anti-inflammatory abilities have been suggested to play an important role in the treatment and prevention of tumour [³²]. Cancer cells usually show resistance to the drugs, which is the main cause of treatment failure [³³]. Overcoming drug resistance will improve prognosis and survival. Pokorna et al reported that morusin has therapeutic potential for the treatment of chronic inflammatory diseases such as fibrosis or cancer [³⁴].

Cytotoxicity assays were used to verify whether morusin could be a therapeutic agent for two cancer cells. The results of IC50 indicated that morusin has good effects on inhibiting the growth of many types of tumour cells. The two cells of MDA-MB-453 and HCT116 were treated with three concentrations of morusin. With the addition of morusin, the inhibited effects gradually increased. There are marked inhibitory effects when tumour cells treated by high concentration (20 µg/ml) of morusin long time (48 h), breast cancer cells almost died and the inhibition ratio up to 94.02%. The above results verified that morusin can be used to treat cancer from a cytological perspective.

The disappearance of E-cadherin function is a clinically important indicator for poor prognosis and metastasis [³⁵, ³⁶, ³⁷]. The function and expression of E-cadherin, an epithelial cell-cell adhesion molecule, is lost, whereas the expression of N-cadherin, a mesenchymal cell-cell adhesion molecule, is induced, a process also known as the cadherin switch [³⁸]. Our results indicated that morusin can upregulate E-cadherin, downregulate Vimentin and N-cadherin and prevent EMT occurrence. Thereby, morusin reverses EMT and inhibiting tumour cell invasion and migration.

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 confirmed 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 double-stained 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.

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 findings further supported that morusin can be used as a potential anticancer drug and could be an attraction for further anti-tumour compounds.

Acknowledgments

The authors gratefully acknowledge the earmarked fund for the China Agriculture Research System (CARS-18-ZJ0502) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China.

hen YC, Tien YJ, Chen CH, Beltran FN, Amor EC, Wang RJ, Wu DJ, Mettling C, Lin YL, Yang WC. (2013). Morus alba and active compound oxyresveratrol exert anti-inflammatory activity via inhibition of leukocyte migration involving MEK/ERK signaling. BMC Complement Altern Med. 23;13:45. doi: 10.1186/1472-6882-13-45. PMID: 23433072; PMCID: PMC3639811.
iu HY, Wang J, Ma J, Zhang YQ. (2016). Interference effect of oral administration of mulberry branch bark powder on the incidence of type II diabetes in mice induced by streptozotocin. Food Nutr Res. Jun 1;60:31606. doi: 10.3402/fnr.v60.31606. PMID: 27257845; PMCID: PMC4891971.
ha JY, Kim YT, Kim HS, Cho YS. (2008). Antihyperglycemic effect of stem bark powder from paper mulberry (Broussonetia kazinoki Sieb.) in type 2 diabetic Otsuka Long-Evans Tokushima fatty rats. J Med Food. 11(3):499-505. doi: 10.1089/jmf.2007.0028. PMID: 18800898.
ZHANG, M., WANG, R.-R., CHEN, M., ZHANG, H.-Q., Sun, S., ZHANG, L.-Y., (2009). A New Flavanone Glycoside with Anti-proliferation Activity from the Root Bark of Morus alba. Chin. J. Nat. Med. 7, 105–107. https://doi.org/10.1016/S1875-5364(09)60046-7.
hoi E M, Hwang J K. (2005). Effects of Morus alba, leaf extract on the production of nitric oxide, prostaglandin E 2, and cytokines in RAW264.7 macrophages. Fitoterapia, 76(7):608-613.
ang S, Fang M, Ma YL, Zhang YQ. (2014). Preparation of the Branch Bark Ethanol Extract in Mulberry Morus alba, Its Antioxidation, and Antihyperglycemic Activity In Vivo. Evid Based Complement Alternat Med. 2014; 2014: 569652. doi: 10.1155/2014/569652.
omura, T., Fukai, T., Yamada, S., Katayanagi, M., (1978). Studies on the constituents of the cultivated mulberry tree. I. Three new prenylflavones from the root bark of Morus alba L. Chem. Pharm. Bull. (Tokyo) 26, 1394–1402. https://doi.org/10.1248/cpb.26.1394
chida, A., Mizutani, H., Ohshima, S., Oonishi, I., Hano, Y., Fukai, T., Nomura, T., (1996). Morusin and Morusin Dimethyl Ether. Acta Crystallogr. C. 52: 1713–1716. https://doi.org/10.1107/S0108270196001382
ee, 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-κB activity in human colorectal cancer HT-29 cells. Biochem. Biophys. Res. Commun. 372, 236–42. https://doi.org/10.1016/j.bbrc.2008.05.023
ang, li, Guo, H., Yang, L., Dong, L., Lin, C., Zhang, J., Lin, P., Wang, X., (2013). Morusin inhibits human cervical cancer stem cell growth and migration through attenuation of NF-(B activity and apoptosis induction. Mol. Cell. Biochem. 379. https://doi.org/10.1007/s11010-013-1621-y
ark, D., Ha, I., Park, S.-Y., Choi, M., Lim, S.-L., Kim, S.-H., Lee, J.-H., Ahn, K., Yun, M., Lee, S.-G., (2016). Morusin Induces TRAIL Sensitization by Regulating EGFR and DR5 in Human Glioblastoma Cells. J. Nat. Prod. 79. https://doi.org/10.1021/acs.jnatprod.5b00919
heng, P., Hu, C., Wang, C., Lee, Y., Chung, W., & Tseng, T. (2017). Involvement of the antioxidative property of morusin in blocking phorbol ester-induced malignant transformation of JB6 P+ mouse epidermal cells. Chem-Biol Interactions, 264, 34–42.
ang, F., Zhang, D., Mao, J., Ke, X., Zhang, R., & Yin, C. (2017). Morusin inhibits cell proliferation and tumor growth by down-regulating c-Myc in human gastric cancer. Oncotarget, 8(34), 57187–57200.
seng, T.-H., Chuang, S.-K., hu, chao chin, Chang, C.-F., Huang, Y.-C., Lin, C.-W., Lee, Y., 2010. The synthesis of morusin as a potent antitumor agent. Tetrahedron 66, 1335–1340. https://doi.org/10.1016/j.tet.2009.12.002
i, H., Wang, Q., Dong, L., Liu, C., Sun, Z., Gao, L., Wang, X., (2015). Morusin suppresses breast cancer cell growth in vitro and in vivo through C/EBPβ and PPARγ mediated lipoapoptosis. J. Exp. Clin. Cancer Res. CR 34. https://doi.org/10.1186/s13046-015-0252-4
HYPERLINK "https://xueshu.baidu.com/s?wd=author%3A%28C%20Kim%29%20&tn=SE_baiduxueshu_c1gjeupa&ie=utf-8&sc_f_para=sc_hilight%3Dperson" \t "_blank" Kim, C., Jin, HK., Oh, EY., Nam, D., Ahn, KS. (2016). Blockage of STAT3 Signaling Pathway by Morusin Induces Apoptosis and Inhibits Invasion in Human Pancreatic Tumor Cells. Pancreas 45(3): 409-419
im, S.-L., Park, S.-Y., Kang, S., Park, D., Kim, S.-H., Um, J.-Y., Jang, hyeung jin, Lee, J.-H., Jeong, C.-H., Jang, J.-H., Ahn, K., Lee, S.-G., (2015). Morusin induces cell death through inactivating STAT3 signaling in prostate cancer cells. Am. J. Cancer Res. 5, 289–99.
upta, G., Dua, K., Kazmi, I., Anwar, F., (2013). Anticonvulsant activity of Morusin isolated from Morus alba: Modulation of GABA receptor. Biomed. Aging Pathol. 4(1): 29-32. https://doi.org/10.1016/j.biomag.2013.10.005
ho, J., Ryu, Y., Curtis-Long, M., Kim, J.Y., Kim, D., Lee, S., Lee, W., Park, K., (2011). Inhibition and Structural Reliability of Prenylated Flavones from the Stem Bark of Morus lhou on β-Secretase (BACE-1). Bioorg. Med. Chem. Lett. 21, 2945–8. https://doi.org/10.1016/j.bmcl.2011.03.060
im, J.Y., Lee, W., Kim, Y.S., Curtis-Long, M., Lee, B., Ryu, Y., Park, K., (2011). Isolation of Cholinesterase-Inhibiting Flavonoids from Morus lhou. J. Agric. Food Chem. 59, 4589–96. https://doi.org/10.1021/jf200423g
usum, M., Klinbuayaem, V., Bunjob, M., Sangkitporn, S., (2004). Preliminary efficacy and safety of oral suspension SH, combination of five Chinese medicinal herbs, in people living with HIV/AIDS; the phase I/II study. J. Med. Assoc. Thail. Chotmaihet Thangphaet 87, 1065–70
a, B., Cui, F., & Zhang, Y.-Q. (2013). Rapid Analysis of morusin from Ramulus mori by HPLC-DAD, Its distribution in vivo and differentiation in the cultivated mulberry species. Food Nutr Sci, 4(2), 189–194. http://doi.org/10.4236/fns.2013.42026
ing B, Lv Y, Zhang Y Q. (2016). Anti-tumor effect of morusin from the branch bark of cultivated mulberry in Bel-7402 cells via the MAPK pathway. Rsc Advances, 6(21):17396-17404.
an, L.-Z., Ma, B., & Zhang, Y.-Q. (2014). Preparation of morusin from Ramulus mori and its effects on mice with transplanted H22 hepatocarcinoma. BioFactors, 40(6), 636–45. http://doi.org/10.1002/biof.1191
ermeulen K, Berneman Z N, Bockstaele D R V. (2003). Cell cycle and apoptosis. 2(3):291-299.
urk, U., Schubert, J., Wellner, U., Schmalhofer, O., Vincan, E., Spaderna, S., Brabletz, T., (2008). A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells.
ossé, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., Borner, C., (1998). BCL-2 prolongs cell survival after BAX-induced release of cytocrome c. Nature 391, 496–9. https://doi.org/10.1038/35160
erlay, J., Shin, H.R., Bray, F., Forman, D., Parkin, D.M., (2013). Cancer incidence and mortality worldwide: IARC CancerBase No. Int. Agency Res. Cancer 2.
an, L., Strasser-Weippl, K., Li, J.-J., Louis, J., Finkelstein, D., Yu, K.-D., Chen, W.-Q., Zhao, N., Goss, P., (2014). Breast cancer in China. Lancet Oncol. 15, e279–e289. https://doi.org/10.1016/S1470-2045(13)70567-9
u, J., Qiu, T., Pengtao, P., Yu, D., ju, Z., Qu, X., Gao, X., Mao, C., Wang, L., (2011). Detection of Serum Anti-P53 Antibodies from Patients with Colorectal Cancer in China Using a Combination of P53-and phage-ELISA: Correlation to Clinical Parameters. Asian Pac. J. Cancer Prev. APJCP 12, 2921–4.
otecha R, Takami A, Espinoza J L. (2016). Dietary phytochemicals and cancer chemoprevention: a review of the clinical evidence. Oncotarget, 7(32):52517.
i Won Lee, HyongJoo Lee. (2010). The roles of polyphenols in cancer chemoprevention. BioFactors, 26(2):105-121.
eetla, C., Bhave, R., Vijayaraghavalu, S., Stine, A., Kooijman, E., Labhasetwar, V., (2010). Drug Resistance in Breast Cancer Cells: Biophysical Characterization of and Doxorubicin Interactions with Membrane Lipids. Mol. Pharm. 7, 2334–48. https://doi.org/10.1021/mp100308n
okorna, M., Rotrekl, D., Gajdziok, J., & Karel, S. (2017). Prenylated flavonoid morusin protects against TNBS-induced colitis in rats. PLOS ONE, 18(2), e0182464.
issell M J, Radisky D. (2001). Putting tumours in context. Nature Reviews Cancer, 1(1):46-54.
avallaro U, Christofori G. (2004). Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nature Reviews Cancer, 4(2):118.
erl, A.-K., Wilgenbus, P., Dahl, U., Semb, H., Christofori, G., (1998). A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 392, 190–3. https://doi.org/10.1038/32433
ilmaz M, Christofori G. (2009). EMT, the cytoskeleton, and cancer cell invasion. Cancer & Metastasis Reviews, 28(2):15-33.
chwartz G K, Shah M A. (2005). Targeting the Cell Cycle: A New Approach to Cancer Therapy. Journal of Clinical Oncology, 23(36):9408-9421.
im, S.-J., Min, H.-Y., Chung, H.-J., Park, E.-J., Hong, J.-Y., Kang, Y.-J., Shin, D.-H., Jeong, L., Lee, S., (2008). Inhibition of cell proliferation through cell cycle arrest and apoptosis by thio-Cl-IB-MECA, a novel A3 adenosine receptor agonist, in human lung cancer cells. Cancer Lett. 264, 309–15. https://doi.org/10.1016/j.canlet.2008.01.037
ohnstone R W, Frew A J, Smyth M J. (2008). The TRAIL apoptotic pathway in cancer onset, progression and therapy. Nature Reviews Cancer, 8(10):782.
ao, L., Wang, L., Sun, Z., Li, H., Wang, Q., Yi, C., & Wang, X. (2017). Morusin shows potent antitumor activity for human hepatocellular carcinoma in vitro and in vivo through apoptosis induction and angiogenesis inhibition. Drug Design, Dev Therapy, 11: 1789–1802.

GraphicAbstract.png
Morusin, a prenylated flavonoid recovered from mulberry branches, has inhibitory effects on 20 kinds of cancer cells, of which a 20 µg/mL of morusin could induce 77.73% of apoptotic ratio of MDA-MB-453 cells.

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Novelty Of Statement

Introduction

Methods And Materials

Materials

Cell culture

Experimental groups

Cell apoptosis and cell cycle

Hoechst staining

Western Blotting

Statistical Analysis

Results

The inhibtion ratio of different concentration of morusin to 20 cancer cells

The inhibition ratio of MDA-MB-453 and HCT116 cells

The influence of morusin on cancer cell apoptosis

Hoechst staining

Changes of cancer cell cycle

The expression of EMT-related regulatory proteins

The expression of caspase-3-related proteins

Discussion

Conclusion

Declarations

Acknowledgments

References

Supplementary Files

Status:

Version 1