Open Access

Hydroxyl safflower yellow B combined with doxorubicin inhibits the proliferation of human breast cancer MCF‑7 cells

  • Authors:
    • Kehao Lin
    • Ze Qin
    • Chuanjun Qu
    • Xiaoyu Chen
    • Qingling Jiang
    • Minjing Li
    • Qiusheng Zheng
    • Defang Li
  • View Affiliations

  • Published online on: March 29, 2021     https://doi.org/10.3892/ol.2021.12687
  • Article Number: 426
  • Copyright: © Lin et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Doxorubicin (DOX) is currently the preferred chemotherapeutic agent for breast cancer, and hydroxyl safflower yellow B (HSYB) has a tumor growth‑inhibiting activity. The present study aimed to investigate the effects of HSYB combined with DOX on the proliferation of human breast cancer MCF‑7 cells and explore the underlying mechanism. MTT and cell colony formation assays revealed that the proliferation rate of MCF‑7 cells was signifiscantly decreased after HSYB and DOX treatment. Combined HSYB and DOX treatment significantly decreased the expression levels of BCL‑2 in MCF‑7 cells, while the expression levels of apoptosis‑associated proteins, including cleaved caspase‑9, BAX and cleaved caspase‑3, were markedly increased. Furthermore, flow cytometry and western blot analysis demonstrated that combined HSYB and DOX treatment stimulated an increase in intracellular reactive oxygen species and promoted the release of cytochrome c, leading to apoptosis. The current data suggested that the combination of HSYB and DOX may have marked antitumor activity.

Introduction

Breast cancer is an invasive cancer, ranking first in the incidence of cancer in women; it accounts for 11.6% of the total cancer incidences, and ~630,000 women worldwide died of breast cancer in 2018, accounting for 15% of cancer-associated deaths in women (1). Compared with the global average, Chinese women have a lower incidence of breast cancer and a higher mortality rate, seriously threatening women's health (2). Breast cancer treatment uses a comprehensive treatment model that includes surgery and chemotherapy (3). The high postoperative recurrence rate and post-surgical complications within a short period following breast cancer surgery are significant (4,5). Therefore, increased attention has been focused on further elucidating the pathogenesis of breast cancer to discover and develop novel treatment methods.

Modern medical research has revealed that the formation and growth of tumors are associated with cellular differentiation and apoptosis (6). Apoptosis-induced inhibition of tumor cell proliferation is currently an effective method for treating tumors (7). After numerous years of development, traditional Chinese medicine has identified a set of unique antitumor methods (8). Modern pharmacological studies have demonstrated that traditional Chinese medicine exerts antitumor effects by inhibiting cellular proliferation, inducing apoptosis, regulating cellular signaling pathways and inhibiting multidrug resistance (911). Furthermore, traditional Chinese medicine is widely used in chemotherapy and radiation therapy, which can effectively extend the life expectancy of patients and improve the overall efficacy of treatments (1214).

As a Chinese herbal medicine, safflower promotes blood circulation and removes blood stasis (15). The red flavonoid hydroxyl safflower yellow A (HSYA) is an effective active ingredient in mitigating numerous different types of tumor, such as liver cancer and glioma (1619). A novel flavonoid, hydroxyl safflower yellow B (HSYB), which is an isomer of HSYA, has been demonstrated to have a well-defined anti-breast cancer effect (20,21). Doxorubicin (DOX), as an anthracycline anticancer drug, is a classic breast cancer chemotherapeutic drug with a high sensitivity to cancer cells that inhibits cell proliferation by inducing apoptosis (22,23). Moreover, combination therapies can exert synergistic therapeutic effects. However, to the best of our knowledge, there are currently no studies on the combination of HSYB and DOX for treating breast cancer. Hence, the present study aimed to investigate the potential synergistic effects of combined DOX and HSYB treatment.

Materials and methods

Chemicals and reagents

HSYB (purity, >95%) was donated by Dr Tao Weiwei of Nanjing University of Traditional Chinese Medicine (Nanjing, China). DOX hydrochloride (purity, >98%; cat. no. 113424) was purchased from Beijing Bailingwei Technology Co., Ltd. Dulbecco's modified Eagle's medium (DMEM) was purchased from Thermo Fisher Scientific, Inc. Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO), Penicillin-Streptomycin, MTT, 0.25% trypsin digestion solution (without phenol red), Hoechst 33258 staining solution, Annexin V-FITC Apoptosis Detection kit, Reactive Oxygen Species Assay kit and Mitochondrial Membrane Potential Assay kit with JC-1 were ordered from Beijing Solarbio Science & Technology Co., Ltd. Anti-cleaved caspase-3 (1:1,000; cat. no. 9662), anti-cleaved caspase-9 (1:1,000; cat. no. 9508), anti-cytochrome c (1:1,000; cat. no. 4280) and anti-BAX (1:1,000; cat. no. 3498) were purchased from Cell Signaling Technology, Inc. Anti-Bcl-2 (1:1,000; cat. no. ab196495), anti-caspase-9 (1:1,000; cat. no. ab25758) and anti-caspase-3 (1:500; cat. no. ab13847) were purchased from Abcam. Primary antibodies against β-actin (1:2,000; cat. no. TA-09) and peroxidase-conjugated goat anti-mouse IgG secondary antibodies (1:25,000; cat. no. ZB2305) were purchased from OriGene Technologies, Inc.

Cell lines and cell culture

MCF-7 cells were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. HaCaT cells were purchased from Procell Life Science & Technology Co., Ltd.. The cells were cultured at 37°C with 5% CO2 in DMEM with 10% FBS and 1% penicillin-streptomycin.

Determination of cellular viability

HaCaT (1.0×104 cells/well) or MCF-7 cells (0.7×104 cells/well) in 96-well plates were treated with DOX (0.1–2 µg/ml), HSYB (5–30 µg/ml) or a combination of the two for 24 h at 37°C. Subsequently, MTT (20 µl) was added to each well and incubated for 4 h at 37°C. DMSO (150 µl) was then directly added to the plate while shaking at low speed for 10 min at room temperature. Finally, the optical density (OD) of the cells at 490 nm was determined using an enzyme-labeling instrument, and the inhibition rate was calculated as follows: (1-OD value of experimental group/OD value of control group) × 100%. In addition, according to the Chou-Talalay combination index (CI) method (24), the CI was calculated to evaluate the effect of drug combination and to test the interaction between two drugs with different concentrations. Data were analyzed using Compusyn 2.0 software (Biosoft) to calculate CI and Fa values. CI<1, CI=1 and CI>1 indicated synergistic, additive and antagonistic effects, respectively.

Colony formation experiments

MCF-7 cells were inoculated into a 6-well plate (200 cells/well) and treated with DOX (0.4 µg/ml), HSYB (6 µg/ml) or a combination of the two for 24 h at 37°C after attachment. The complete fresh medium was replaced every three days. After ~12 days of culture, the cells were fixed at room temperature with 4% paraformaldehyde solution for 20 min. Subsequently, the cells were washed with PBS and treated with crystal-violet staining solution for 10 min at room temperature. Finally, the dye was cleaned with PBS, and the stained cells were imaged using a camera. A colony was defined as >50 cells.

Hoechst 33258 staining detection

An aseptic cover slide was placed into a 6-well plate and inoculated with MCF-7 cells (1.4×105 cells/well). Cells were fixed with 4% paraformaldehyde for 20 min at room temperature after treatment with DOX (0.4 µg/ml), HSYB (6 µg/ml) or a combination of the two for 24 h at 37°C. Subsequently, the cells were washed with PBS and then stained with Hoechst 33258 for 10 min at room temperature. The staining solution was cleaned with PBS, and the slides were observed under an inverted fluorescence microscope (magnification, ×400).

Annexin V-FITC apoptosis detection

MCF-7 cells (1.0×106 cells/group) treated with DOX (0.4 µg/ml), HSYB (6 µg/ml) or a combination of the two drugs were collected and resuspended in a staining buffer (500 µl) containing Annexin V-FITC (5 µl), after which they were tested using a FACSCanto II flow cytometer (Becton, Dickinson and Company). The apoptosis rate was analyzed using the FACSDiva 6.1.3 software (Becton, Dickinson and Company).

Western blot analysis

MCF-7 cells (5.7×106 cells/well) were seeded and treated as aforementioned in Petri dishes (100-mm). Protein was extracted using RIPA lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) after the cells were collected. The protein concentration was detected using a BCA protein assay kit (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.). Proteins (80 µg/lane) were separated via 12% SDS-PAGE and transferred to a PVDF membrane. Protein bands were blocked with 5% BSA (cat. no. 232100; Becton, Dickinson and Company) at room temperature for 3 h and were then incubated overnight with primary antibodies at 4°C. The samples were washed four times with TBS-Tween (TBST; 0.1% of Tween-20) buffer (6 min per wash), incubated with secondary antibodies for 35 min at room temperature and washed four times with TBST buffer (6 min per wash). Finally, the protein bands were incubated with an ECL chemiluminescence working buffer (cat. no. 34580; Thermo Fisher Scientific, Inc.), and western blotting was imaged via a UVP imaging system. ImageJ v1.43 software (National Institutes of Health) was used for densitometry.

Detection of reactive oxygen species (ROS) in cells

MCF-7 cells (1.8×105 cells/well) were plated in Petri dishes (30-mm). Cells were treated with DOX (0.4 µg/ml), HSYB (6 µg/ml) or a combination of the two for 24 h at 37°C. The cells were detached from the plate with EDTA-containing trypsin and resuspended in serum-free medium containing a DCFH-DA probe (10 µmol/l). Subsequently, the cells were transferred to a thermostatic chamber at 37°C for 20 min. Finally, the cells were washed three times with TBST buffer, and the DCF fluorescence intensity of the treated cells was determined using a FACSCanto II flow cytometer. The DCF fluorescence intensity was analyzed using the FACSDiva 6.1.3 software (Becton, Dickinson and Company). In addition, aseptic cover slides were placed into a 6-well plate and inoculated cells were imaged under an inverted fluorescence microscope (magnification, ×200). The dye solutions used for ROS detection were from a Reactive Oxygen Species Assay kit (cat. no. CA1410; Beijing Solarbio Science & Technology Co., Ltd.).

Measurement of mitochondrial membrane potential (MMP)

As aforementioned, DOX- and HSYB-treated cells were treated with EDTA-containing trypsin. The cells were resuspended in complete medium (500 µl) containing JC-1 staining working solution (500 µl) and then incubated at 37°C for 20 min. Next, cells were washed twice with the JC-1 staining buffer (1X). Finally, the JC-1 staining buffer (500 µl) was used to resuspend the cells, which were subsequently analyzed using a FACSCanto II flow cytometer. The ratio of red to green fluorescence was analyzed using the FACSDiva 6.1.3 software.

Statistical analysis

The SPSS 21.0 software (IBM Corp.) was used to analyze the data presented as the mean ± SD. Significant differences were determined using an unpaired Student's t-tests or one-way ANOVA followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Combination of HSYB and DOX has a synergistic effect on MCF-7 cells

The viability of HSYB and/or DOX-treated MCF-7 cells was assessed using the MTT method to investigate whether there was a synergistic effect of HSYB and DOX. First, MCF-7 cells were incubated with different concentrations of HSYB (0–30 µg/ml) or DOX (0–2 µg/ml) for 24 h. The inhibitory effect of HSYB and DOX on breast cancer MCF-7 cells was significantly decreased in a concentration-dependent manner (Fig. 1A and B). The IC50 values of HSYB and DOX were 22.12 µg/ml and 6.85 µg/ml, respectively, after 24 h of drug administration. Subsequently, the effect of the combination of HSYB and DOX for 24 h on the viability of MCF-7 cells was detected via MTT assays. The Chou-Talalay method and Compusyn 2.0 software were used to calculate the combination index (CI). The present results demonstrated that HSYB synergistically enhanced the efficacy of DOX against breast cancer MCF-7 cells. When HSYB (6 µg/ml) was combined with DOX (0.4 µg/ml), the CI value was the smallest, and the effect of coadministration was the most obvious (CI<1; Table SI). Therefore, these concentrations were used for subsequent experiments. Compared with HSYB (6 µg/ml) or DOX (0.4 µg/ml) alone, inhibition of cellular proliferation was significantly enhanced after combined treatment with HSYB and DOX (Fig. 1C).

Furthermore, the effects of HSYB, DOX or combination therapy on cell colony formation were studied. There were fewer cell colonies in the DOX or HSYB groups than in the untreated (control) group, and after combined HSYB and DOX treatment, the number of cell colonies was markedly decreased (Fig. 1D). Therefore, the combination of HSYB and DOX enhanced the inhibition of MCF-7 cell proliferation. In addition, the normal human keratinocyte HaCaT cell line was chosen as a control group. The results revealed that DOX (0.1–2 µg/ml) significantly inhibited the proliferation of HaCaT cells and had a higher cytotoxicity in HaCaT cells than in MCF-7 cells (Fig. S1A). Additionally, the results revealed that a high concentration (25 µg/ml) of HSYB significantly inhibited the proliferation of HaCaT cells and had a higher cytotoxicity in these cells than in MCF-7 cells; however, a low concentration (5 µg/ml) of HSYB significantly promoted the proliferation of HaCaT cells (Fig. S1B). Based on these data, a lower concentration (6 µg/ml) of HSYB was selected for the combined treatment with HSYB and DOX.

Combination of HSYB and DOX induces apoptosis in MCF-7 cells

The synergistic effects of HSYB and DOX on MCF-7 apoptosis and cellular morphology were further investigated. Using Hoechst 33258 staining, it was revealed that the HSYB and DOX combined-treatment group exhibited typical apoptotic morphological characteristics compared with the control and the single drug groups, with the combined therapy group exhibiting a higher concentration of nuclear chromatin and the formation of apoptotic bodies (Fig. 2A).

The apoptotic rate of the HSYB and DOX combined- treatment group in MCF-7 cells was further evaluated by Annexin V-FITC staining. Treatment with DOX or HSYB alone significantly increased apoptosis compared with the control group, but the combined use of DOX and HSYB further significantly increased apoptosis (Fig. 2B and C). The apoptotic rate of MCF-7 cells increased from 4.2 to 14.1, 9.2 and 53.7% after treatment with DOX, HSYB and a combination of HSYB and DOX, respectively (Fig. 2C). Therefore, the present results confirmed that DOX combined with HSYB significantly induced apoptosis of MCF-7 cells.

Combination of HSYB and DOX affects the level of apoptosis-associated molecular proteins in MCF-7 cells

Western blotting was employed to identify the synergistic effects of HSYB and DOX on the expression levels of signaling molecules associated with apoptosis in MCF-7 cells. BAX expression in MCF-7 cells in the single drug groups was significantly upregulated compared with in the control group, while the levels of BCL-2 and BCL-2/BAX were significantly decreased, and this effect was even more marked in the HSYB and DOX combined-treatment group (Fig. 3A-D). Further research revealed that the ratios of cleaved caspase-3/total caspase-3 and cleaved caspase-9/total caspase-9 in the HSYB and DOX combined-treatment group were significantly higher compared with in the untreated and single drug groups (Fig. 3E-G). These findings demonstrated that a combination of HSYB and DOX had the strongest regulatory effect on apoptotic proteins, indicating that a combination of HSYB and DOX synergistically induced apoptosis of MCF-7 cells.

Combination of HSYB and DOX increases ROS levels in MCF-7 cells and activates the mitochondrial apoptotic pathway

To further determine whether the synergistic effects of HSYB and DOX in inducing apoptosis of MCF-7 cells were associated with ROS and the mitochondrial apoptotic pathway, ROS levels were detected by DCFH-DA staining and the MMP by JC-1 staining. The ROS levels of MCF-7 cells treated with HSYB and/or DOX were higher than in control cells, and the combined-treatment group exhibited the highest ROS levels (Fig. 4A and B). Furthermore, MMP was significantly lower in cells treated with HSYB and/or DOX than in the control group, and the combined-treatment group had the most pronounced effect (Fig. 4C and D). Additionally, the synergistic effects of HSYB and DOX on cytoplasmic cytochrome c (Cyto-c) levels in MCF-7 cells were detected by western blotting. It was revealed that the Cyto-c levels of MCF-7 cells in the single drug groups were higher than in the control group, with a further significant increase in the combined-treatment group (Fig. 4E and F). The current results confirmed that a combination of HSYB and DOX promoted ROS levels in MCF-7 cells and activated the mitochondrial apoptotic pathway.

Discussion

In most countries globally, breast cancer has the highest incidence and mortality rates among female-specific types of cancer (1). DOX represents the first-line drug for breast cancer treatment; however, it also induces side effects, such as cardiac toxicity (25,26). Safflower can be used for treating angina pectoris or cardiovascular/cerebrovascular diseases, and the effective component, HSYA, exerts antitumor effects (18,27,28). Recently, combinations of different drugs have been widely used to treat tumors and have yielded effective results (29). A number of studies have obtained encouraging results on the combination of Chinese herbal medicine and chemotherapeutic drugs to treat tumors, indicating that traditional Chinese medicine may be a safe and effective auxiliary approach for tumor treatment (13,3032). The present study revealed that HSYB, a flavonoid component in safflower, combined with DOX significantly inhibited breast cancer MCF-7 cell proliferation.

Apoptosis is a form of programmed cell death that requires the participation of various complex factors and is an indispensable component in multicellular life processes (33,34). Numerous studies have demonstrated that apoptosis is affected by various apoptosis-associated factors, including BCL-2 (35,36). Furthermore, BCL-2 and BAX serve opposite roles in the process of apoptosis (37). BCL-2 inhibits apoptosis and promotes cell proliferation (38,39) while BAX is mostly distributed in the cytoplasm, it can destroy the integrity of the mitochondrial membrane and promote apoptosis after being activated (4042).

Normally, BCL-2 expression is relatively stable (43). When BAX is highly expressed, numerous BAX/BAX homodimers are formed to induce apoptosis (44). By contrast, when BCL-2 expression is upregulated, BCL-2/BAX heterodimers are formed (45). Many BAX/BAX homodimers are dissociated, decreasing the responsiveness of cells to apoptotic signals, which inhibits apoptosis (46). Therefore, the ratio between these two antagonistic proteins is key to cell survival (44,46). With the decrease in BCL-2/BAX levels, the permeability of the mitochondrial membrane is altered, Cyto-c and apoptosis-promoting factors are released into the cytoplasm, and the apoptotic promoter caspase-9 is activated, directly promoting apoptosis by activating the downstream effector caspase-3 (47,48). In the present study, the BCL-2/BAX ratio was significantly decreased, the level of Cyto-c was increased and the levels of the apoptotic promoter (cleaved caspase-9) and the apoptotic effector (cleaved caspase-3) were also upregulated after combined treatment with HSYB and DOX.

ROS are closely associated with the induction and regulation of apoptosis. Increased intracellular ROS levels can cause cellular damage, alter mitochondrial membrane permeability and decrease MMP, thereby promoting Cyto-c release into the cytoplasm and inducing apoptosis (49,50). In the present study, the combination of HSYB and DOX in MCF-7 cells increased ROS levels and decreased the MMP, as determined by fluorescence microscopy and flow cytometry. Overall, the current results revealed that a combination of HSYB and DOX synergistically induced apoptosis of MCF-7 cells by increasing ROS levels, downregulating BCL-2/BAX ratio, promoting Cyto-c release into the cytoplasm and subsequently activating caspase-3 and caspase-9.

However, the present study presents some limitations. First, the antitumor experiments testing HSYB and DOX were only performed in a single cell line, the human breast cancer MCF-7 cell line. Therefore, future studies should investigate the effects of HSYB and DOX in other breast cancer cells in vitro and in mouse models in vivo. Second, the effects of the combination of HSYB and DOX on MCF-7 cell invasion and migration were not assessed, which will require further investigation in future studies.

In conclusion, the current study revealed that the combination of HSYB and DOX synergistically induced apoptosis and enhanced antitumor effects in breast cancer MCF-7 cells, indicating that the combination of HSYB and DOX may be a new alternative for clinical treatment of breast cancer.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was funded by the National Natural Science Foundation of China (grant no. 31870338), the Key Research and Development Program of Shandong Province of China (grant no. 2019GSF108214), Taishan Scholars Construction Engineering of Shandong Province (grant no. tsqn201812099), the Dominant Disciplines' Talent Team Development Scheme of Higher Education of Shandong Province (grant no. 2016052410), the Introduction and Cultivation Project for Young Creative Talents of Higher Education of Shandong Province (grant no. 20191008189) and the Guidance Plan of Science and Technology Research and Development of Shijiazhuang (grant no. 171561533).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

KL, ZQ, QZ and DL designed the experiments. KL and DL obtained the experimental data and wrote the manuscript. KL and CQ collected cell samples for Hoechst 33258 staining, apoptosis and western blot analysis. CQ, XC, QJ and ML interpreted and analyzed the data. KL and QJ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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May-2021
Volume 21 Issue 5

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Spandidos Publications style
Lin K, Qin Z, Qu C, Chen X, Jiang Q, Li M, Zheng Q and Li D: Hydroxyl safflower yellow B combined with doxorubicin inhibits the proliferation of human breast cancer MCF‑7 cells. Oncol Lett 21: 426, 2021
APA
Lin, K., Qin, Z., Qu, C., Chen, X., Jiang, Q., Li, M. ... Li, D. (2021). Hydroxyl safflower yellow B combined with doxorubicin inhibits the proliferation of human breast cancer MCF‑7 cells. Oncology Letters, 21, 426. https://doi.org/10.3892/ol.2021.12687
MLA
Lin, K., Qin, Z., Qu, C., Chen, X., Jiang, Q., Li, M., Zheng, Q., Li, D."Hydroxyl safflower yellow B combined with doxorubicin inhibits the proliferation of human breast cancer MCF‑7 cells". Oncology Letters 21.5 (2021): 426.
Chicago
Lin, K., Qin, Z., Qu, C., Chen, X., Jiang, Q., Li, M., Zheng, Q., Li, D."Hydroxyl safflower yellow B combined with doxorubicin inhibits the proliferation of human breast cancer MCF‑7 cells". Oncology Letters 21, no. 5 (2021): 426. https://doi.org/10.3892/ol.2021.12687