2023 Volume 46 Issue 6 Pages 773-780
This study aimed to determine the effect of brazilin on the invasion and metastasis of breast cancer. The breast cancer MDA-MB-231 and 4T1 cells were treated with brazilin to investigate proliferation and invasion using cell proliferation assay, wound healing assay, transwell assay. BALB/C mice were randomized into normal, model, positive control, and Sappan L. extract groups (n = 6/group). The mice were injected with 4T1 cells via caudal veins to establish a lung metastasis model and via subcutaneous injection to establish a xenograft model. Metastatic nodules on the lung surface, survival rates and visceral indices were evaluated. Subcutaneous tumor volumes and weights were measured. Brazilin inhibited the proliferation of breast cancer cells and significantly inhibited the wound healing, migration, and invasion of MDA-MB-231 and 4T1 cells. Compared with the normal group, the average survival days and spleen index in the model group were significantly decreased, but the lung index and number of pulmonary metastatic nodules were significantly increased. Compared with the model group, the average survival and spleen index of dose groups were significantly increased, and the lung index, the number of pulmonary metastatic nodules, and tumor volume and weight were significantly decreased. Brazilin significantly inhibits the proliferation and metastasis of breast cancer. This study might suggest a new therapeutic agent for breast cancer.
Breast cancer is a malignancy of the breast tissue and is the most common malignancy diagnosed in women worldwide.1,2) The estimated global breast cancer incidence was 2088849 cases in 2018.3) Risk factors for breast cancer include genetic causes, increased age, reproductive history, hormone exposure, lifestyle factors, medical history, and radiation exposure.4,5) The management of breast cancer is multidisciplinary and includes surgery, chemotherapy, targeted therapy, endocrine therapy, and radiotherapy.2,6,7) In the United States of America, the 5-year survival after a breast cancer diagnosis is 99% for women with localized disease, 85% for women with regional spread, and 27% for women with distant metastases.8) In addition, women with estrogen receptor-positive cancers treated with endocrine therapy have a 13–41% risk of recurrence 5–20 years after diagnosis.8) Therefore, there is still room for improving the current treatment strategies, especially for women with advanced disease.
Sappan L., the heartwood of Caesalpinia sappan L., is mainly distributed in Yunnan, Hainan, and Guangxi (China).9) In the Chinese Pharmacopeia, Sappan L. has a bitter, salty, slightly spicy, and flat taste, has the functions of blood-activating and freeing collateral vessels, and is mainly used for treating a fracture, tendon injury, and fall injury.10–12) Its bacteriostatic13) and blood glucose regulation14,15) effects have been confirmed. Recently, Sappan L. has been reported with an antitumor effect.10,16) Brazilin is one of the most important antitumor components in Sappan L.10,17) It is known as an anticancer reagent and is effective in several cancer types.18) Yang et al.10) showed that brazilin arrested T24 and BTT cells in the G2 phase and inhibited their proliferation.10) Zhang et al. found that brazilin played the role of antitumor activity by inducing c-FOS and GADD45β expression.11,19)
Brazilin has been reported to have effects on tongue cancer,20) cervical cancer,21) colon cancer,22) head and neck cancer,23) and bone cancer.24) Breast cancer is the cancer with the highest morbidity and mortality rate in women, severely threatening people’s lives,25,26) but only a few studies are available regarding the effect of brazilin on breast cancer. Indeed, a study suggested that brazilin inhibits metastatic breast cancer by inhibiting the tumor necrosis factor (TNF)-α axis.27) Brazilin suppresses DNA methyltransferase 1 (DNMT1) expression and restores p21 in MCF7 breast cancer cells.28)
Still, the effects of brazilin in breast cancer remain mostly unknown. Therefore, this study aimed to investigate the inhibitory effect of brazilin on the invasion and metastasis of breast cancer cells.
Breast cancer MDA-MB-231 and 4T1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (BBI Life Sciences Corp., Shanghai, China) containing 10% fetal bovine serum (GIBCO, Invitrogen Inc., Carlsbad, CA, U.S.A.) and 1% streptomycin (Solarbio Science & Technology Co., Ltd., Beijing, China) at 37 °C, saturated humidity, and 5% CO2 in an incubator (Thermo Fisher Scientific, Waltham, MA, U.S.A.).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) AssayMDA-MB-231 and 4T1 cells in the logarithmic growth phase were seeded in two sterile 96-well plates (1 × 105 and 0.5 × 105 cells/well) on a super-clean workbench and were cultured with 100 µL of culture medium overnight. The cells were observed to adhere to the wall on the next day, and the original medium was gently discarded. The treatment groups were successively added with 200 µL of brazilin at different concentrations (purity >98%, provided by Prof. Shengwan Zhang, School of Life Sciences, Shanxi University, China), with five replicates for each group. The control group was not added with brazilin. Blank wells were set. After incubation, the original culture medium in the 96-well plate was gently discarded, and all wells were added with 100 µL of medium containing MTT (final concentration of 0.5 mg/mL) and cultured for 4 h. The medium in each well was carefully discarded, and 150 µL of dimethyl sulfoxide (DMSO) was added in the dark. The formazan crystals were fully dissolved by oscillating for 10 min in a microplate analyzer. The absorbance (A) value was measured at 490 nm to calculate the inhibition rate and IC50.
Inhibition rate (%) = (Mean A of negative control group-Mean A of treatment group)/Mean A of negative control group × 100%
 |
where Xm: immunoglobulin (Ig) maximum dose; I: Ig (maximum dose/adjacent dose); P: sum of positive reaction rates; Pm: maximum positive reaction rate; Pn: minimum positive reaction rate
Wound Healing AssayMDA-MB-231 and 4T1 cells in the logarithmic growth phase were cultured into two 6-well plates at 7 × 105 cells/well. The cells were cultured overnight and were observed to adhere to the walls. The original medium was discarded. A 10-µL pipette tip was used to draw four evenly-spaced straight lines perpendicular to the black line at the bottom of the 6-well plate. The wells were rinsed with phosphate buffered saline (PBS) three times. The treatment group received 2 mL of serum-free medium containing 10 µg/mL of brazilin, and the control group was added with an equivalent volume of serum-free medium for starvation. The experiments were performed in triplicates. The cells were cultured for 12, 24, and 48 h, respectively. The cell status and movement of each group were observed under the microscope and photographed. Cell migration rate was calculated as follows:
Cell migration rate (%) = (Migration distance in treatment group/Migration distance in the control group) × 100%
Transwell Cell Migration and Invasion AssaysMDA-MB-231 and 4T1 cells in the logarithmic growth phase were cultured into two 6-well plates at 7 × 105 cells/well for 24 h. The cells were starved with DMEM containing 2% serum and RPMI-1640 medium for 24 h. Then, the effects of brazilin at final concentrations of 10 and 15 µg/mL on cell migration and invasion were detected. A blank control was set up.
Preparation of the Brazilin ExtractSappan L. decoction pieces were cut to 2-3-cm-long pieces, immersed in 56% ethanol with a solid:liquid ratio of 1 : 8 at room temperature for 1 h, and extracted by heating and reflux for 1.8 h. The extraction procedures were repeated, and the extract solutions were combined for further heating and reflux four times. The solutions were combined to concentrate at 1 g/mL, stored at 0–5 °C for 48 h, and filtered. Twice the amount of petroleum ether : ethyl acetate = 3 : 1 was added into a 500 mL beaker and stirred using a magnetic stirrer at 450 rpm for 15 min). The mixture was placed in a 500 mL separatory funnel for phase separation to collect the aqueous phase. The aqueous phase was added with two times the amount of ethyl acetate into a 500-mL beaker, stirred using a magnetic stirrer at 450 rpm for 15 min, and placed in a 500-mL separatory funnel for phase separation. The organic phase was collected in a 50 mL conical flask, added with anhydrous sodium sulfate (q.s.) for sealing and drying, and transferred to a rotary evaporator under negative pressure for evaporation (60 °C, 0.08 MPa) to obtain an orange powdery crude extract. The crude extract was dissolved and filtered by twice the amount of water, extracted twice by adding five times the amount of extractant (ethyl acetate : petroleum ether = 2 : 1), and stirred at 450 rpm for 15 min. After phase separation, the organic phase was collected, added with anhydrous sodium sulfate for drying, and then transferred to a rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) under negative pressure for evaporation (40 °C, 0.08 MPa) to obtain an orange-yellow powdery extract.
HPLC was used for the quantitative determination of brazilin (brazilin standard provided by LookChem, CAS No. 474-07-7) (1525, Waters, Milford, MA, U.S.A.). HPLC conditions: column Agela Venusil XBP-C18 (4.6 × 150 mm, 5 µm, Agela); mobile phase A: methanol; phase B: 0.2% formic acid solution (v/v); gradient elution; flow rate: 1.0 mL/min; column temperature: 35 °C; volume: 20 µL; detection wavelength: 285 nm. Gradient elution procedure: 0–30 min, 18% A; 30–50 min, A from 18 to 50%; 50–55 min, 50% A.
AnimalsThirty-six 7-week-old SPF female BALB/C mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Jing) 2016-0011). The mice were raised in the experimental animal center of Shanxi Cancer Hospital (SYXK (Jin) 2019-0003) at 20–26 °C, 40–60% humidity, 12/12 h lighting/dark cycle, with free drinking water and sterile full nutritional pellet feed (SBF Beijing Biotechnology Co., Ltd., China).
Preparation of Breast Tumor Lung Metastasis Model in MouseThe mice were randomly divided into six groups: normal, model, negative control, and high-dose, medium-dose, and low-dose Sappan L. extract groups (n = 6, each group). Single-cell suspension was prepared using 4T1 cells in the logarithmic growth phase at 2 × 106/mL with PBS. Except for the normal group, the mice were injected with 4T1 cells (100 µL) via tail vein to establish a breast cancer lung metastasis model.
Two days after cell injection, the normal and model groups were intraperitoneally injected with saline (0.2 mL) once a day. The positive control group was intraperitoneally injected with fluorouracil 125 mg/kg once a week, at 0.1 mL/10 g. The high-, medium-, and low-dose Sappan L. extract groups were intraperitoneally injected with 200, 100, and 50 mg/kg brazilin, once a day, at 0.1 mL/10 g, consecutive for 3 weeks.
The hair, activity, mental state, reaction ability, diet, and other general conditions were recorded every morning and evening. The mice were weighed every 3 d during the experiment, and the overall survival was recorded. The observation lasted 35 d.
The survival rate was calculated by the Kaplan–Meier method, and the effects of the extract on the survival rate and median survival were compared and analyzed.
Life extension rate (%) = Mean overall survival in treatment group/Mean overall survival in model group × 100%.
After the mice were sacrificed, the kidney, spleen, and lung were taken, and the blood on the organ surfaces was removed with filter paper. The organs were weighed, and the kidney, spleen, and lung indexes were calculated.
Visceral index = Visceral weight (g)/body weight (g)×100%.
The total number of metastatic nodules on the lung surface was counted. The lung was fixed in Bouin fixative solution (saturated picric acid : formaldehyde : glacial acetic acid = 70 : 25 : 5) for 24 h and immersed in anhydrous ethanol for decolorization. The number of pulmonary metastatic nodules was counted, and the diameter of the metastatic tumor was measured under an anatomical microscope. The classification and counting were carried out according to the following equation:
Total number of metastatic nodules on lung surface = I × 1 + II × 2 + III × 3 + IV × 4.
(Four grades according to the diameter of pulmonary nodules: I: <0.5 mm; II: 0.5–0.9 mm; III: 1–2 mm; IV: >2 mm).
Lung metastasis inhibition rate = (Number of lung metastases in model group-Number of lung metastases in the treatment group)/Number of lung metastases in model group × 100%.
Subcutaneous Xenotransplantation Mouse ModelThe 15 female BALB/c mice (7 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were randomly divided into three groups: the model group, the positive control group, and the Sappan L. extract group. A total of 1 × 106 breast cancer 4T1 cells were subcutaneously injected into the right armpit of each mouse. When the tumor size reached 80 mm3, mice in the model group were intraperitoneally injected with 0.2 mL normal saline once daily; mice in the positive control group were intraperitoneally injected with fluorouracil (125 mg/kg, 0.1 mL/10 g body weight) once a week; mice in the Sappan L. extract group were intraperitoneally injected with the extract (100 mg/kg, 0.1 mL/10 g body weight) once a day. The general condition of the animals was observed daily, such as the hair, mobility, mental state, responsiveness, and diet. The tumor size was measured regularly during the experiment, and mice were weighed every three days. Tumor volume was calculated as 1/2 × tumor length × tumor width2. The experiment was terminated when the tumor’s maximum side length reached 2 cm. The study was approved by the Laboratory Animal Management Committee at the Shanxi Cancer Institute following the National Research Council Guide for the Care and Use of Laboratory Animals in China (Approval No. 2022052 and No. 2021020).
Statistical AnalysisSPSS 21 (IBM, Armonk, NY, U.S.A.) was used to analyze the data. Measurement data were expressed as means ± standard deviation. Pearson correlation analysis was used. Data with a normal distribution were analyzed by one-way ANOVA. Least significant difference (LSD) test was used for homogeneous variance, and Dunnett’s T3 test was used for heterogeneous variance. A nonparametric test was used for data with a non-normal distribution. p-Values <0.05 were considered statistically significant.
The inhibition rates of brazilin on MDA-MB-231 and 4T1 cells for 24h were investigated at the concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL (Fig. 1A). The proliferation of the cells was significantly inhibited by brazilin at different concentrations, and the effect increased with concentration (4T1 cells: p = 0.003, 0.006, 0.007, 0.002, 0.002, and 0.002, respectively, vs. 0 µg/mL; MDA-MB-231 cells: p = 0.002, 0.003, 0.0006, 0.001, 0.001, and 0.001, respectively, vs. 0 µg/mL) The calculated IC50s of brazilin on MDA-MB-231 and 4T1 for 24 h were 15.65 and 1.08 µg/mL, respectively. The inhibition rates of brazilin on the cells were investigated at IC50 for different times (3, 6, 12, 24, 48 h). As shown in Fig. 1B, brazilin had a significant inhibitory effect on breast cancer cells in a time-dependent manner (4T1 cells: p = 0.003, 0.002, 0.003, 0.003, and 0.002, respectively, vs. 0 h; MDA-MB-231 cells: p = 0.003, 0.003, 0.002, 0.006, and 0.002, respectively, vs. 0 h).
Dose-responsive curves are presented. ANOVA with the LSD or Dunnett’s T3 post hoc test. * p < 0.05 vs. blank controls. (B) Effects of different treatment times of brazilin on the proliferation inhibition rate of breast cancer cells. Time-responsive curves are presented.
The results of the wound healing assay are shown in Figs. 2A and B. In MDA-MB-231 and 4T1 cells, brazilin at 10 µg/mL decreased the cell migration rate compared with the control group (4T1 cells: p = 0.018 for 24 h and 0.032 for 48 h vs. 0 h; MDA-MB-231 cells: p = 0.015 for 12 h and 0.021 for 24 h vs. 0 h).
(B) Effect of brazilin on the wound healing capacity of breast cancer 4T1 cells. (C) Effect of brazilin on the Transwell migration of breast cancer MDA-MB-231 cells. (D) Effect of brazilin on the Transwell migration of breast cancer 4T1 cells. (E) Effect of brazilin on the Transwell invasion of breast cancer MDA-MB-231 cells. (F) Effect of brazilin on the Transwell invasion of breast cancer 4T1 cells. ANOVA with the LSD or Dunnett’s T3 post hoc test. * p < 0.05 vs. blank controls.
The results of the Transwell migration are shown in Figs. 2C and D. The number of MDA-MB-231 permeating the membrane in the control group was 374.8 ± 14.7. After treatment with 10 and 15 µg/mL brazilin, the number decreased to 155.0 ± 24.2 (p = 0.008) and 56.2 ± 28.8 (p = 0.005), respectively. The number of 4T1 penetrating the membrane in the control group was 494.2 ± 29.3, and those in the low- and high-dose brazilin groups were lower (257.6 ± 19.5 and 124.4 ± 20.6, p = 0.005 and 0.003, respectively). These results suggest that brazilin significantly inhibited the migration of MDA-MB-231 cells.
Influence on the Invasion of Breast Cancer CellsThe transwell invasion assay was used to detect the effect of brazilin on the invasion of MDA-MB-231 cells. As shown in Fig. 2E, the number of MDA-MB-231 permeating the membrane in the control group was 436.2 ± 58.3. After treatment with 10 and 15 µg/mL brazilin, the number decreased (246.2 ± 35.2 and 115.2 ± 19.2, p = 0.008 and 0.005, respectively). As shown in Fig. 2F, the number of 4T1 cells penetrating the membrane in the control group was 479.4 ± 41.4, and those numbers were 361.2 ± 16.3 (p = 0.005) and 162.8 ± 19.2 (p = 0.003) after treatment with 10 and 15 µg/mL brazilin, respectively.
Brazilin AssayThe peak time of the standard brazilin was about 14 min, and the relative assay of the component was 99.10% (Supplementary Fig. S1). The main active component in Sappan L. extract was 68.39%.
Sappan L. Improves the Condition of Mice with Breast Cancer Lung MetastasesIn the initial modeling of the mice with breast cancer lung metastasis, the diet, behavior, and mental state in each group were normal. The mice in the normal group survived well, and their activity and diet were normal. The mice in the model group showed decreased activity, poor mental state, malaise, reduced food intake, and lack of luster. Compared with the model group, the mice in the fluorouracil group and the high, medium, and low-dose groups were in a better state, with a relatively reduced diet, trance, and slow movement.
The effects of the extract on the body weight changes are shown in Fig. 3A. The body weight in the normal group increased gradually. Compared with the normal group, the model control group had a good survival in the early stage, and the body weight increased slowly without significant difference. The mice in the control group had loose and dull fur, and their body weight decreased sharply until death, without significant difference. Compared with the model group, the weight of the different dose groups changed steadily without significant difference. The results showed that the Sappan L. extract had no significant effect on the body weight of mice.
(B) Effect of Sappan L. extract on survival rate of mice. (C) Effect of Sappan L. extract on the spleen, kidney, and lung indexes. (D) Effect of Sappan L. extract on the number of metastatic nodules on the lung surface of mice. # p < 0.05 vs. normal; * p < 0.05 vs. model.
The effect of the extract on the overall survival is shown in Table 1. Compared with the normal group, the average survival days in the model group were significantly decreased (p = 0.012). Compared with the model group, the average survival days of the high-, medium-, and low-dose groups were significantly increased (p = 0.043, 0.026, and 0.033, respectively), and the life extension rates were 40.0, 92.0, and 44.0%, respectively. The overall survival of the fluorouracil group was slightly increased, without significant difference (p = 0.054). The results showed that the Sappan L. extract effectively prolonged the survival of mouse models of metastatic breast cancer, and the concentration of 100 mg/kg performed the best.
| Group | Administration time/d | Average survival days/d | Life extension rate/% |
|---|---|---|---|
| Normal | — | 35.0 ± 0 | — |
| Model | — | 16.67 ± 4.16# (p = 0.012) | — |
| 125 mg/kg 5-FU | 3 | 21.00 ± 1.00 (p = 0.054) | 25.97 |
| 200 mg/kg Sappan L. extract | 21 | 23.33 ± 3.05* (p = 0.043) | 39.95 |
| 100 mg/kg Sappan L. extract | 21 | 32.00 ± 2.64* (p = 0.026) | 91.96 |
| 50 mg/kg Sappan L. extract | 21 | 24.00 ± 3.00* (p = 0.033) | 43.97 |
# p < 0.05 vs. the normal group; * p < 0.05 vs. the model group. x̄ ± s, n = 6.
The effect of the extract on the survival rate is shown in Fig. 3B. The results showed that all mice in the normal group survived during the experiment. The mice in the model group began to die on day 11, and all were dead on day 25, with a median overall survival of 16 d. In the fluorouracil group, one mouse died on day 12, most died after day 20, for a median overall survival of 21 d. The mice in the high-dose group began to die on day 15 and were dead by day 32, for a median overall survival of 23 d. The mice in the medium-dose group began to die on day 31, and one was surviving at the end, for a median overall survival of 31 d. The mice in the low-dose group died from day 17, and one was surviving at the end, for a median overall survival of 19 d. The study indicated that the Sappan L. extract significantly prolonged the overall survival of the mice, especially in the medium-dose group.
The effect of the extract on the visceral organ index is shown in Fig. 3C. Compared with the normal group, the spleen index in the model group was significantly decreased (p = 0.045), the kidney index was slightly decreased, but the lung index was significantly increased (p = 0.032), possibly due to the lung metastases resulting in lung weight gain. Compared with the model group, the spleen index in the high and medium-dose groups increased significantly (p = 0.042 and 0.038, respectively), the kidney index in each dose group changed slightly, and the lung index decreased significantly (p = 0.042 and 0.048, respectively). The possible reason was that the extract inhibited the lung metastasis of breast tumors and significantly reduced the lung index in the dose groups compared with the model group.
The effect on the number of metastatic nodules on the lung surface is shown in Fig. 3D and Table 2. Compared with the normal group, the number of pulmonary metastatic nodules in the model group increased significantly. Compared with the model group, the number in the high, medium, and low-dose groups decreased significantly (p = 0.024, 0.031, and 0.025, respectively), and that in the fluorouracil group also decreased significantly (p = 0.038). The results indicated that the extract significantly inhibited the number of metastatic nodules on the lung surface of mice.
| Group | Total number of metastatic pulmonary nodules | Lung metastatic inhibition rate/% |
|---|---|---|
| Model | 105.67 ± 3.78 | — |
| 125 mg/kg 5-FU | 65.67 ± 5.01* (p = 0.038) | 37.85 |
| 200 mg/kg Sappan L. extract | 48.33 ± 3.05* (p = 0.024) | 54.26 |
| 100 mg/kg Sappan L. extract | 55.67 ± 3.29* (p = 0.031) | 47.32 |
| 50 mg/kg Sappan L. extract | 46.33 ± 4.16* (p = 0.025) | 56.16 |
* p < 0.05 vs. the model group. x̄ ± s, n = 6.
Having observed that medium-dose Sappan L. was more effective than other doses at extending the overall survival and inhibiting tumor metastasis in breast cancer metastasis model mice, we examined its effect on breast cancer xenograft growth. During the experiment, there were no abnormalities in feeding, drinking, hair, or defecation in the animals. The weight of the animals increased over time in each group, but there was no statistical difference between groups. Tumor growth was slower in the positive control group and the Sappan L. extract group than in the model group. Tumor mass was significantly reduced in the positive control group and the Sappan L. extract group when compared with the model group, with a 63.37% reduction (p = 0.015) in tumor volume and 66.67% (p = 0.011) reduction in tumor weight following fluorouracil treatment and 50.62% (p = 0.023) and 54.16% (p = 0.035) following Sappan L. extract treatment (Table 3, Fig. 4). These data suggest that Sappan L. extract had a similar anti-tumor effect to fluorouracil on breast cancer.
| Group | Volume (cm3) | Volume reduction rate (%) | Weight (g) | Weight reduction rate (%) |
|---|---|---|---|---|
| Model | 2.43 ± 0.71 | — | 2.88 ± 1.63 | — |
| 125 mg/kg 5-FU | 0.89 ± 0.53* (p = 0.015) | 63.37 | 0.96 ± 0.561* (p = 0.011) | 66.67% |
| 100 mg/kg Sappan L. extract | 1.20 ± 0.47* (p = 0.023) | 50.62 | 1.32 ± 1.20* (p = 0.035) | 54.16% |
* p < 0.05 vs. the model group. x̄ ± s, n = 5.
Brazilin extracted from Sappan L. inhibits the growth of various cancer cell types,10,11,13,14,16,19–24,27,28) but its effects on breast cancer are poorly known. Therefore, this study aimed to investigate the inhibitory effect of brazilin on the invasion and metastasis of breast cancer cells. The results indicate that brazilin significantly inhibits the proliferation and metastasis of breast cancer cells. The Sappan L. extract could significantly inhibit the metastasis of breast tumors, prolong the overall survival, and suppress tumor growth in mice. Brazilin might be a treatment against breast cancer.
The cell proliferation assay, wound healing assay, and Transwell migration and invasion assays showed that brazilin inhibited the proliferation of breast cancer cells in a dose and time-dependent manner. Moreover, compared with the control group, after 12, 24, and 48 h treatment with 10 µg/mL of brazilin, the motility, migration, and invasion of the breast cancer cells were significantly inhibited. Thus, brazilin can inhibit the proliferation and metastasis of different breast cancer cells, as shown in tongue cancer,20) cervical cancer,21) colon cancer,22) head and neck cancer,23) and bone cancer.24)
The Sappan L. extract had no significant effect on the body weight of mice, suggesting low toxicity. Compared with the control group, the average survival days and spleen index in the model group were significantly decreased, but the lung index and the number of pulmonary metastatic nodules were significantly increased. Compared with the model group, the average survival and spleen index of the three dose groups were significantly increased compared with the model group, and the number of pulmonary metastatic nodules was significantly decreased. Furthermore, the extract significantly reduced the volume and weight of tumor xenografts in mice compared to normal saline. Thus, the extract may inhibit the proliferation of breast cancer cells, decreasing lung metastases.
Above all, the Sappan L. extract can significantly inhibit the growth and metastasis of breast tumors and prolong the overall survival of mice, with the best performance at a medium dose.
This study only examined the in vitro and in vivo effects of brazilin on breast cancer cells. No mechanistic exploration was performed. Future studies should examine the mechanisms leading to cancer cell inhibition by brazilin. Currently, the available studies suggest that brazilin might exert its effects through c-FOS and GADD45β,11,19) the TNF-α axis,27) and DNMT1 suppression.28) The mechanisms of brazilin in breast cancer cells should be further studied before planning clinical trials.
In conclusion, brazilin significantly inhibits the proliferation and metastasis of breast cancer cells. The Sappan L. extract could significantly inhibit the growth and metastasis of breast tumors and prolong the overall survival of mice. The study provides a theoretical reference for the development of brazilin as an anticancer drug and new ideas for the treatment of breast cancer. These results suggest a new therapeutic agent for treating breast cancer.
This work was supported by The National Natural Science Foundation of China (#82003810), The “SIGEYIPI” Project of Health Commission of Shanxi Province (#2020SYS25) and The Shanxi Provincial Science and Technology Innovation Youth Talent Team Project (#202204051001033).
Conceptualization: Xihua Yang and Shengwan Zhang; Data curation: Yongqin Liang and Yongming Yang; Formal analysis: Lili Zhao and Lixia Chen; Funding acquisition: Xihua Yang; Project administration: Xianping Liu and Lei Yan; Resources: Huayi Zhang and Jing Wang; Roles/Writing—original draft: Xihua Yang, Yongqin Liang; Writing—review & editing: Shengwan Zhang, Xianping Liu and Huayi Zhang.
The authors declare no conflict of interest.
All data generated or analysed during this study are included in this published article.
This article contains supplementary materials.
