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Honokiol inhibits the growth of SKBR3 cells

Background Breast cancer is one of the most malignant tumors in the reproductive system and has a poor prognosis. Finding drugs with high efficiency, low side-effects, and low cost has become a research hotspot. Methods In the present study, we treated SK-BR-3 cells with different doses of honokiol. Crystal violet staining method was used to detect changes in the total number of living cells; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect the effect of honokiol on SK-BR-3 cell proliferation. Cell migration ability change was determined by wound healing assay. Cell invasion ability change was determined by Transwell migration assay. Flow cytometry was used to detect the apoptotic rate of SK-BR-3 cells, and Western blot was used to detect the expression levels of proliferation-associated protein (PCNA); migration- and invasion-related protein matrix metalloproteinase-2 (MMP-2); vimentin; apoptosis-related proteins Bcl-xl, caspase 3, and cleaved caspase 3 (CC3); and β-catenin and its downstream target molecule c-Myc. Results Compared with the control group, different doses of honokiol have different degrees of inhibitory effects on cells, including proliferation and invasion and migration (P<0.01). After treatment with 50 or 60 µmol·L–1 honokiol, the apoptotic rate of SK-BR-3 cells increased (both P<0.01); PCNA expression was significantly downregulated (P<0.01). Intracellular accumulation of apoptosis-related proteins Bcl-xl and caspase-3 decreased but C-C3 increased. We also found downregulation of MMP-2 expression, a protein related to invasion and migration (P<0.01), and a decrease in the expression levels of the Wnt/β-catenin signaling pathway-related proteins β-catenin and c-Myc (P<0.01). Conclusions Honokiol can promote the apoptosis of SK-BR-3 cells and can inhibit the proliferation, migration, and invasion of human breast cancer SK-BR-3 cells. The underlying mechanism may be through inhibiting the activation of the Wnt signaling pathway.

also an issue for patients. Therefore, it is important to find a drug with high efficiency, low-side effects, and low cost for the treatment of breast cancer.
Honokiol is a biologically active ingredient isolated from the Chinese herbal medicine magnolia, which can effectively inhibit the growth of tumor cells. According to the literature, honokiol can induce apoptosis in pancreatic and gastric cancer cells (4,5), and can also inhibit the growth and metastasis of melanoma (6). Current breast cancer research is limited to the effects of honokiol on the two human epidermal growth factor receptor 2 (HER2)-negative cell lines MDA-MB-231 and MCF-7 (7,8). The role and mechanism of honokiol in breast cancer SK-BR-3 cells have not yet been reported. SK-BR-3 cells were isolated from the pleural effusion of a 43 white female breast cancer patient in 1970, It is an epithelioid adherent cell. As a landmark breast cancer cell, SK-BR-3 cells have been used as experimental objects in many studies (9)(10)(11). SK-BR-3 cells are breast cancer cells with high HER2 expression. HER2-positive breast cancer has a high rate of recurrence and metastasis, as well as poor prognosis (12). Finding anti-tumor drugs that can effectively inhibit the growth of SK-BR-3 cells is important for the treatment of breast cancer. Therefore, in the present study, we used different concentrations of honokiol to treat breast cancer SK-BR-3 cells to observe its effects on the proliferation, apoptosis, invasion, and migration of SK-BR-3 cells, and to detect the changes in expression in Wnt signaling pathway-related proteins β-catenin and C-Myc. This can provide new evidence that honokiol can be used as an anti-tumor drug to effectively treat breast cancer. We present the following article in accordance with the MDAR reporting checklist (available at http://dx.doi.org/10.21037/tcr-20-3110).

Crystal violet staining
SK-BR-3 cells with good morphology and growth were selected; 3×10 4 cells (500 μL) were inoculated in a 24-well plate, and the plate was shaken. After 12 h, when the cells had grown to 40%, they were treated with honokiol at concentrations of 40, 50, and 60 μmol·L -1 . After processing for 48 and 72 h, the medium was discarded from the 24-well plate. A total of 500 μL of 4% paraformaldehyde solution was added and fixed in a 37 ℃ incubator for 25 min. The paraformaldehyde was discarded and 700 μL crystal violet staining solution was added to each well and left for 10 min under dark conditions before the crystal violet staining solution was discarded. The dye was washed, and the plate was scanned after drying. A total of 200 μL of 10% glacial acetic acid solution was then added to each well and shaken at room temperature for 15 min. Absorbance of each well at a wavelength of 590 nm was detected with an enzyme-linked immunoassay, and the cell proliferation inhibition rate was calculated using the following equation: Proliferation inhibition rate (%)=(control group absorbance value -medication group absorbance value)/control group absorbance value × 100%.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
In total, 5,000 cells (constant volume to 100 μL) were counted and inoculated evenly on 96-well plates. Cells were treated with different concentrations of honokiol for 48 and 72 h, and then 10 μL MTT solution was added to each well, place the 96-well plate flat in the incubator and continued to incubate for 4 h. A total of 100 μL DMSO was added to each well, protected from light, shake the 96-well plate 10 min at room temperature, set the wavelength to 492 nm, and measure the absorbance of each well with an enzyme-linked immunoassay to reflect the cell proliferation activity.

Wound healing assay
The cells were inoculated in 6-well plates until the cells cover the entire plate. A 200 μL pipette tip was used to make a vertical scratch on the cell culture plate. Floating cells were removed by washing with phosphate-buffered saline (PBS). A total of 2 mL of serum-free medium was slowly added along the wall, and the SK-BR-3 cells were treated with concentrations of 40, 50, 60 μmol·L -1 honokiol. A blank control and DMSO control were set, and pictures were taken at 0 and 48 h to compare the position of the cell.

Flow cytometry cell apoptosis analysis
Cells were treated with different concentrations of honokiol.
After 48 h, the cells were digested and collected with trypsin without ethylenediaminetetraacetic acid. The cells were resuspended in PBS after centrifugation and then treated with Annexin V-allophycocyanin (APC; BD Pharmingen, USA) and propidium iodide (PI; BD Pharmingen, USA) under dark conditions. Positive cells were identified as apoptotic by flow cytometry.

Statistical analysis
Each experiment was repeated 3 times independently. Data are expressed as mean ± standard deviation. Oneway analysis of variance was used to analyze the datasets in the group. Use GraphPad Prism 8 software (GraphPad Software, USA) for date processing. P<0.05 indicated a statistically significant difference.

Honokiol inhibits the proliferation of human SK-BR-3 cells
After treating SK-BR-3 cells with different concentrations of honokiol (40, 50, 60 μmol·L -1 ) for 48 h and 72 h, the total number of cells was detected by crystal violet staining.
The results are shown in ( Figure 1A). After treatment with 40 μmol·L -1 honokiol, the total number of cells decreased compared with the control group. When treated with 50 and 60 μmol·L -1 honokiol, the total number of cells further decreased (P<0.01). Further MTT measurement results are shown in ( Figure 1B). Compared with the blank control group, 40, 50, and 60 μmol·L -1 honokiol was found to significantly inhibit the proliferation activity of SK-BR-3 cells (P<0.01). We recorded the morphology of SK-BR-3 cells after treatment with different concentrations of honokiol ( Figure 1C), we found that as the treatment concentration increases, the overall number of cells decreases, and the cell state gradually deteriorates. Specifically, vacuoles appear in the cells, the cell shape becomes round, and some cells are broken. In addition, we treated SK-BR-3 cells with different concentrations of honokiol for 48 h to extract total protein ( Figure 1D). When SK-BR-3 cells were treated with 50 and 60 μmol·L -1 honokiol, the expression level of cell proliferation-related protein PCNA was significantly lower than that of the blank control group (P<0.01). These results indicate that honokiol can inhibit the proliferation of human breast cancer SK-BR-3 cells, and the inhibitory effect is concentration dependent.

Honokiol promotes apoptosis of SK-BR-3 cells
To investigate the effect of honokiol on the degree of SK-BR-3 cell apoptosis. After treating SK-BR-3 cells with different concentrations of honokiol (40, 50, 60 μmol·L -1 ) for 48 h, apoptosis was detected by flow cytometry (Figure 2A). The apoptotic rate of the 50 and 60 μmol·L -1 honokiol treatment groups was significantly higher than that of the blank control group (P<0.01). The results of Western blot are shown in Figure 2B. After treatment of SK-BR-3 cells with 50 and 60 μmol·L -1 honokiol, the expression level of the apoptosis-related protein CC3 significantly increased, while the expression level of caspase 3 decreased; the anti-

Honokiol inhibits the invasion and migration of SK-BR-3 cells
After treating SK-BR-3 cells with different concentrations of honokiol (40, 50, 60 μmol·L -1 ) for 48 h, wound healing assay was used to detect the change of cell migration ability ( Figure 3A). Compared to the blank control cells, honokiol was found to inhibit the ability of cell migration and was concentration dependent. Transwell analysis showed that, compared with the blank control group, the number of cells passing through the compartment membrane in the honokiol treatment group (40, 50, and 60 μmol·L -1 ) was significantly reduced (P<0.01) ( Figure 3B). Western blot was performed to further confirm that honokiol can reduce the invasion-and migration-related proteins expressed by cells, such as vimentin and MMP-2 (P<0.05) ( Figure 3C). Taken together, these data demonstrate that honokiol can inhibit the invasion and migration ability of SK-BR-3 cells.

Honokiol interferes with the Wnt/β-catenin signaling pathway in SK-BR-3 cells
It has been reported in the literature that the activation of the Wnt signaling pathway is involved in the development of a number of cancers. Therefore, in the present study, we used Western blot to detect the effect of different concentrations of honokiol on Wnt signaling pathway activity in SK-BR-3 cells (Figure 4). Compared with the blank control group, after SK-BR-3 cells were treated with 50 and 60 μmol·L -1 honokiol, the expression level of β-catentin and its downstream protein c-Myc was significantly downregulated (P<0.01). We also tested the protein expression of GSK-3β and phosphorylated Gsk-3β, the expression level of GSK-3β has not change but phosphorylated GSK-3β expression has decreased. Taken together, these data demonstrate that honokiol can effectively inhibit the occurrence and development of breast cancer by promoting apoptosis of SK-BR-3 cells while inhibiting their proliferation, migration, and invasion. The mechanism may be interference with the Wnt signaling pathway.

Discussion
Breast cancer is a malignant tumor that occurs in the epithelial tissue of the breast; it has a high rate of recurrence and a poor prognosis (13). Finding drugs with high efficiency, low side-effects, and low cost has become a research hotspot. The present study focused on the multiple inhibitory effects of honokiol on human breast cancer SK-

BR-3 cells and their specific mechanisms.
Apoptosis is a cell process; it is the autonomous and orderly death of cells controlled by genes. Its purpose is to maintain the stability of the cell's internal environment. Promoting tumor apoptosis is a relatively simple and efficient method to treat cancer patients (14). The Bcl-2 protein family is closely related to cell apoptosis and also affects the development of cancer. Bcl-xl is a classic anti-apoptotic protein, and its expression changes affect tumor cell apoptosis (15). Apoptosis is also regulated by the caspase cascade. When caspase 3 receives the apoptotic signal, self-splicing occurs, causing cell apoptosis (16). The proliferation-related protein PCNA is the core of many important cellular processes in tumor cells, and its main role is to control tumor progression and prognosis (17). A large number of studies have confirmed that there are abnormal expressions of PCNA in the occurrence and development of various tumors (18)(19)(20). In the present study, we used crystal violet staining, MTT assay, and Western blot to confirm that 50 and 60 μmol·L -1 honokiol can inhibit the proliferation of human breast cancer SK-BR-3 cells. Subsequently, flow cytometry was used to detect the level of apoptosis, and Western blot was used to detect apoptosis and proliferation-related proteins, such as Bclxl, caspase 3, CC3, and PCNA, to explore the effects of honokiol on apoptosis and the proliferation of human breast cancer SK-BR-3 cells. Flow cytometric results showed that 50 and 60 μmol·L -1 honokiol can cause obvious apoptosis of human breast cancer SK-BR-3 cells. Western blot also confirmed that when breast cancer cells were treated with 50 and 60 μmol·L -1 honokiol for 48 h, the expression level of the anti-apoptosis-related protein Bcl-xl was significantly reduced, the expression level of pro-apoptotic protein (CC3) increased, and the expression level of caspase 3 decreased. Honokiol was found to promote SK-BR-3 through cell apoptosis and inhibit its proliferation.
Epithelial-mesenchymal transition is a common biologic process of cancer cells. Epithelial cells undergo partial procedures to transform into a mesenchymal cell phenotype; the expression of vimentin is beneficial to the metastasis of tumor cells (21). MMPs are closely related to the metastasis of tumor cells. They help tumor cells to migrate by breaking down MMP proteins (22). In the present study, the wound healing assay, Transwell assay, and Western blot were used to detect the effect of honokiol on the migration and invasion of human breast cancer SK-BR-3 cells and the expression of its related proteins MMP-2 and vimentin. Experimental results showed that honokiol has a significant inhibitory effect on the migration and invasion of human breast cancer SK-BR-3 cells. Previously published studies have confirmed that the Wnt signaling pathway is involved in controlling the process and prognosis of breast cancer (23,24). In the Wnt pathway, GSK-3β is upstream of β-catenin and regulates stability of β-catenin, when Serine 9 of GSK-3β is phosphorylated, its activity decreases, results in reduced β-catein degradation and Wnt pathway is activated (25). The downstream c-Myc protein is closely related to the occurrence and development of breast cancer (26). In the present study, Western blot confirmed that honokiol can downregulate the expression level of β-catentin protein in the Wnt signaling pathway, and at the same time downregulate the expression of its downstream protein c-Myc. We also detected the protein expression of GSK-3β and its phosphorylated form. Western blot showed that the expression level of phosphorylated GSK-3β (Ser9) was down-regulated, while the level of total GSK-3β expression was not significantly different, which interferes with the Wnt signaling pathway in breast cancer cells, thereby inhibiting breast cancer development. In summary, we found that certain concentrations of honokiol can effectively inhibit the occurrence and development of human breast cancer, as well as the proliferation, migration, and invasion of human breast cancer SK-BR-3 cells by effectively interfering with the Wnt/β-catenin signaling pathway. The results of the present study provide new and effective evidence for honokiol as a safe and effective antibreast cancer drug. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.