Carvacrol induces apoptosis in human breast cancer cells via Bcl-2/CytC signaling pathway

doi:10.21203/rs.3.rs-2105532/v1

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Carvacrol induces apoptosis in human breast cancer cells via Bcl-2/CytC signaling pathway

https://doi.org/10.21203/rs.3.rs-2105532/v1

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Objective To elucidate the molecular mechanisms underlying carvacrol-induced apoptosis in human breast cancer cells.

Methods The antiproliferative effect of carvacrol on human breast cancer cell line, HCC1937, was determined using MTT assay, and ultrastructural changes in the treated cells were observed using uranium acetate/lead citrate double staining. Cell cycle analysis was performed using propidium iodide (PI) staining and apoptotic cells were detected using flow cytometric Annexin V-FITC/PI assay. Further, the expression of Bcl-2/CytC signaling pathway-related proteins (Bcl-2, CytC, and Caspase-3) was detected using western blot technique.

Results Carvacrol exhibited antiproliferative activity against HCC1937 cells in a concentration-dependent manner, with an IC₅₀ of 320 mM. Ultrastructure analysis revealed that treatment with carvacrol induced morphologic hallmarks of apoptosis in HCC1937 cells. Carvacrol increased the distribution of HCC1937 cells in the G₀/G₁ phase and decreased the distribution of HCC1937 cells in the S and G₂/M phases in a concentration-dependent manner. Moreover, the compound increased the rate of apoptosis and protein expression of Bax, CytC, and Caspase-3 while decreasing the protein levels of Bcl-2 in a dose-dependent manner.

Conclusion Carvacrol can inhibit proliferation and induce apoptosis in human breast cancer HCC1937 cells. This effect may be mediated by the activation of the Bcl-2/CytC mitochondrial apoptosis pathway.

Carvacrol

Breast cancer

Antiproliferative

Apoptosis

Bcl-2

Cytochrome c

Breast cancer is a malignant tumor arising from breast epithelial tissue, and its incidence is increasing year by year. Breast cancer is associated with a high mortality rate and poses a great threat to the lives and health of women ^[1]. At present, surgery, radiotherapy and chemotherapy, endocrine therapy, and targeted therapy are the main strategies used for treating breast cancer. However, their overall curative effect remains unsatisfactory. Moreover, surgery, radiotherapy, and chemotherapy can cause severe adverse effects, bringing pain and discomfort among patients ^[2]. Owing to recent developments in Chinese medicine, the anti-cancer potential of traditional Chinese medicines and their compounds have received great attention from researchers.

One such compound is carvacrol, a natural monoterpene phenol. It is considered to be a safe food additive and is used in the manufacture of sweets, chewing gum, and beverages ^[3]. However, studies have shown that carvacrol also has antimicrobial, anti-inflammatory, anti-oxidant, and antiangiogenic properties ^[4]. Although carvacrol is known to exert anti-cancer effects against breast cancer ^[5], the mechanisms underlying these effects remain unclear. Therefore, this aspect warrants further investigation.

The imbalance between cancer cell proliferation and apoptosis contributes to tumorigenesis and tumor growth, The B-cell lymphoma-2/CytC (B-cell lymphoma-2/CytC, Bcl-2/CytC) pathway, a mitochondrial apoptosis pathway, is known to play an important role in the process of cancer cell apoptosis ^[6-7] . Ye Min et al. ^[8] showed that inhibiting the expression of Bcl-2 family proteins can inhibit the proliferation of human breast cancer cells and induce cell apoptosis.

To date, the role of the Bcl-2/CytC pathway and its regulation in the effects of carvacrol on breast cancer has not been reported. Therefore, this study aimed to investigate the antiproliferative and apoptotic effects of carvacrol on HCC1937 human breast cancer cells as well as to elucidate the underlying molecular mechanisms involved in the induced cell death.

1.1 Cell line and cell culture

HCC1937 human breast cancer cells, purchased from the Beijing Beina Chuanglian Biotechnology Research Institute, were thawed and resuscitated. They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 100 IU/ml penicillin/streptomycin. The cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

1.2 Main reagents and instruments

The RPMI-1640 medium and Protein Extraction Kit were purchased from American Image (Wuhan) Technology Co., Ltd.

3-2(4,5-Dimethylthiazole-2)-2-tetramethylazothiazole blue (MTT), the propidium iodide (PI) dye, and the Annexin V-FITC/PI apoptosis detection kit were purchased from Shanghai Jingke Chemical Technology Co., Ltd.

The rabbit anti-human Bcl-2, anti-human CytC, anti-human Caspase-3, anti-human β-actin polyclonal antibodies, and the goat anti-rabbit HRP secondary antibody were purchased from Wuhan Yipu Biotechnology Co., Ltd.

The carbon dioxide cell incubator (model BBD6220) was purchased from Thermo Fisher Scientific, USA.

1.3 MTT assay for the detection of HCC1937 cell proliferation

HCC1937 cells were seeded at 2.5×10⁴ cells/well in a 24-well plate and incubated for 24 h. Then, the cells were treated with varying concentrations (0-80 mM) of carvacrol for 24 h. Following treatment, 20 μL MTT (5 g/L) and 150 μL DMSO were added to each well. The plate was shaken to dissolve the added reagents. The inhibition of cell proliferation was calculated as follows:

Cell proliferation inhibition rate (%) = [(OD of the blank control group – OD of the experimental group)/OD of the blank control group] × 100

The non-toxic dose with no effect on cell proliferation was selected for subsequent experiments.

1.4 Transmission electron microscopy for examining cellular ultrastructure

HCC1937 cells were trypsinized and centrifuged for 10 min at 1000 rpm following treatment for 24 h. After discarding the supernatant, 0.25% glutaraldehyde was slowly added along the centrifuge tube wall. After ethanol dehydration, the cells were embedded in paraffin and sectioned. The sections were double stained with uranyl acetate and lead citrate, and the changes in the internal structure of the cells were observed using transmission electron microscopy (HDMI3800-A; Shenzhen Seepack Optical Instrument Co., Ltd., Shenzhen, china).

1.5 PI staining for cell cycle analysis

After treatment for 24 h, HCC1937 cells were trypsinized and collected for centrifugation (1000 rpm for 10 min). Then, the cells were resuspended in phosphate-buffered saline (PBS). Pre-chilled 70% absolute ethanol was added to fix the cells at 4°C. Subsequently, the mixture was centrifuged, and the supernatant was discarded. After washing, the cell pellet was resuspended in PBS, and 50 μL·mL^-1 PI dye was added. After 10 min, the cell cycle distribution was detected using a flow cytometer (CytoFLEX; Beckman Coulter Life Sciences, USA). The percentage of cells in the G₁ phase, S phase, and G₂/M phase for each treatment was calculated.

1.6 Annexin V-FITC/PI double staining for apoptosis detection

HCC1937 cells treated for 24 h were washed with PBS, trypsinized and centrifuged at 1000 rpm for 10 min. The supernatant was discarded and the pellet was resuspended with fresh media to obtain a cell suspension, which was adjusted to a concentration of 1×10⁶ cells/mL. Then, 100 μL of the cell suspension was mixed with 5 μL Annexin V-FITC and 10 μL of 20 μg/mL PI and incubated for 30 min. Apoptotic cells were detected using flow cytometry. Percentage of apoptosis rate was calculated using the following equation:

Apoptosis rate(%) = (number of apoptotic cells/total number of cells) × 100

1.7 Western blot for the detection of Bcl-2/CytC pathway-related protein expression

Following treatment with increasing concentrations of carvacrol for 24 h, HCC1937 cells were lysed with RIPA solution on ice and incubated for 20 min. The mixture was centrifuged at 3000 rpm for 10 min and the supernatant was obtained to detect the protein levels. Then, 20 μg of protein were mixed with an equal amount of loading buffer. The proteins were denatured in a boiling water bath, separated using electrophoresis, and transferred onto a membrane. The membrane was blocked with 5% skimmed milk powder for 2 h and then washed with TBST. Then, it was incubated overnight with antibodies against Bax, CytC, Caspase-3, and the internal control β-actin (1:500) at 4℃. After washing with TBST, the HRP-conjugated goat anti-rabbit secondary antibody (1:1000) was added and incubated at 37°C for 1.5 h. Protein bands were developed, the relative expression of each protein was calculated.

1.8 Statistical analysis

All experiments were performed in triplicate, unless otherwise stated, and results were expressed as means ± standard deviation (SD). All data sets were normally distributed before further analyzed using SPSS 22.0 software (SPSS Inc., Chicago, Illinois, USA). Comparisons between multiple groups were performed using a one-way analysis of variance (ANOVA) and further pairwise comparisons were performed using the SNK-q test. A p-value less than 0.05 (p < 0.05) was considered as statistically significant..

2.1 Carvacrol affects the proliferation of HCC1937 cells

The antiproliferative activity of carvacrol against HCC1937 cells was assessed using MTT assay. According to MTT assay, The IC50 of carvacrol was found to be 320 µM (at 24 h). the concentration of carvacrol, which was nontoxic to cells, was selected for subsequent experiments. the cells were treated with 0, 40, 60, and 80 mM carvacrol, respectively. Results showed that the proliferation inhibition rate of HCC1937 cells was higher in the 40, 60, and 80 mM carvacrol groups than in the blank control group (P < 0.05). This inhibition rate increased in a concentration-dependent manner. Details are provided in Figure 1.

2.2 Carvacrol affects the cell cycle of HCC1937 cells

To further investigate the mechanism of inhibition of HCC1937 cell proliferation by carvacrol, cell cycle distribution was analyzed by flow cytometry. The distribution of HCC1937 cells in the G₀/G₁ phase was higher in the 40, 60, and 80 mM carvacrol groups than in the blank control group, while the distribution of cells in the S phase and G₂/M phase was lower. These differences appeared to be dose-dependent (P < 0.05). These findings indicated that carvacrol could induce G0/G1 phase arrest, inhibit DNA synthesis in the S phase and induce apoptosis of HCC1937 cells, Details are provided in Figure 2 and Figure 3.

2.3 Carvacrol induces ultrastructural changes in HCC1937 cells

The ultrastructural changes in HCC1937 cells following treatment were observed under a transmission electron microscope. The untreated (control) HCC1937 cells had large and clear nuclei and were rich in organelles and matrix (Figure 4A). The mitochondria were round or oval in shape, and the nuclear chromatin was evenly distributed. In contrast, the treated HCC1937 cells had lost their microvilli, and their cytoplasm had degenerated (Figure 4B-D). The inner and outer mitochondrial membranes were fused and shrunken. Mitochondrial cristae were diminished and disordered, and swelling and vacuolar changes were observed. Moreover, chromatin condensation, which is an important morphological hallmark of apoptosis was also seen in the treated cells. These ultrastructural changes observed after carvacrol treatment in HCC1937 were concentration-dependent.

2.4 Carvacrol induces apoptosis in HCC1937 cells

Apoptotic cell death in HCC1937 cells induced by carvacrol was detected by Annexin V and PI staining. Flow cytometric analysis revealed that the apoptosis rate in HCC1937 cells increased with increasing concentrations of carvacrol (P < 0.05). After 24 h treatment with carvacrol, the number of apoptotic cells were increased by 1.6- to 4-fold of the amount in control, suggesting that the carvacrol induced apoptosis in a dose- dependent manner. Details are illustrated in Figure 5 and Figure 6.

2.5 Carvacrol affects the Bcl-2/CytC pathway in HCC1937 cells

Compared with the blank control group(0 mM), the 40, 60, and 80 mM carvacrol groups showed lower protein levels of Bcl-2 and higher protein levels of Bax, CytC, and Caspase-3. These differences showed a dose-dependent decrease in Bcl-2 protein expression and a dose-dependent increase in Bax, CytC, and Caspase-3 expression (P < 0.05). This indicated that carvacrol could inhibit breast cancer proliferation and induce apoptosis. Details are provided in Figure 7 and Figure 8.

Breast cancer is the most common malignant tumor of the breast ductal epithelium in women. The incidence of breast cancer is increasing year by year, making breast cancer one of the main causes of tumor-related deaths in women worldwide ^[9-10]. Currently, surgery, radiotherapy, and chemotherapy are the main strategies for the treatment of breast cancer. However, surgery causes pain and affects the patient’s quality of life, while radiotherapy and chemotherapy are associated with severe adverse effects and the risk of drug resistance ^[11-12]. At present, it is challenging to identify new and effective clinical treatments for breast cancer. Traditional Chinese medicines have few side effects and a low toxicity profile, and some also exert anti-tumor effects^[13]. Carvacrol, a monoterpene phenol commonly found in plant volatile oils, has been found to inhibit the proliferation of breast cancer cells and induce apoptosis, however, the specific mechanisms underlying its effects are still unclear. The excessive proliferation of cancer cells and reduced rates of apoptosis play a key role in tumorigenesis and tumor growth^[14-15].

In the present study, transmission electron microscopy revealed that, unlike the blank control group(0 mM), the 40, 60, and 80 mM carvacrol groups contained HCC1937 cells with lost microvilli, cytoplasmic degeneration, vacuoles, chromatin shrinkage, and apoptotic morphology. These ultrastructural changes observed in HCC1937 cells were concentration-dependent. Moreover, carvacrol increased the cell proliferation inhibition rate, G0/G1 cell distribution ratio, and cell apoptosis rate in a dose-dependent manner. These findings indicated that carvacrol could inhibit the proliferation of HCC1937 cells, induce G0/G1 phase arrest, inhibit DNA synthesis in the S phase, and subsequently cause apoptosis of HCC1937 cells. The observed anti-tumor effects of carvacrol were consistent with the results from Wang Xuan ^[⁵^]and ^[16], who showed that carvacrol could inhibit the proliferation of breast cancer cells and induce cell apoptosis.

Studies show that mitochondria play a key role in the process of apoptosis. Mitochondria are key regulators of energy metabolism and contribute to cellular aging ^[17-18]. Bcl-2 family proteins — such as the apoptosis inhibitor Bcl-2 and the pro-apoptotic protein Bax — play an important role in the mitochondrial apoptosis pathway. Bax regulates signal transmission in the mitochondria by forming homodimers or heterodimers, The low expression of Bcl-2 is correlated with a high expression of Bax. Bax can destroy the mitochondrial membrane and promote the release of CytC, The subsequent activation of the Caspase cascade upregulates the downstream pro-apoptotic gene Caspase-3, leading to cell apoptosis ^[19-20]. Zhang Limei et al. ^[21] showed that the activation of Bcl-2/Bax/Caspase-3 signaling could significantly inhibit cell proliferation and induce apoptosis in breast cancer cells. In the present study, we observed that compared with the blank control group(0 mM), the 40, 60, and 80 mM carvacrol groups showed a dose-dependent decrease in Bcl-2 protein expression and a dose-dependent increase in Bax, CytC, and Caspase-3 expression. This indicated that carvacrol could inhibit breast cancer proliferation and induce apoptosis, likely via the activation of the intrinsic pathway that is activated by the release of the Bcl-2 family proteins from the mitochondria.

In summary, carvacrol can inhibit the proliferation of HCC1937 cells and induce their apoptosis in a dose-dependent manner. These effects are likely mediated by the activation of the Bcl-2/CytC mitochondrial apoptosis pathway (intrinsic pathway). However, the specific regulatory mechanisms that control Bcl-2/CytC pathway activation during the carvacrol-induced apoptosis of breast cancer cells and whether the extrinsic pathway was activated by the activation of transmembrane death receptors remain to be elucidated, further investigation is needed.

Declaration of Competing Interest

The authors declare that there is no conflict of interest.

Funding

The research was supported by the UMS Research Grant Scheme (GUG0505-2/2020) from the Universiti Malaysia Sabah and Gansu Province Higher Education Innovation Fund project (2021B-279、2022A-129).

Availability of materials and data

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Carvacrol induces apoptosis in human breast cancer cells via Bcl-2/CytC signaling pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

1.1 Cell line and cell culture

1.2 Main reagents and instruments

1.3 MTT assay for the detection of HCC1937 cell proliferation

1.4 Transmission electron microscopy for examining cellular ultrastructure

1.5 PI staining for cell cycle analysis

1.6 Annexin V-FITC/PI double staining for apoptosis detection

1.7 Western blot for the detection of Bcl-2/CytC pathway-related protein expression

1.8 Statistical analysis

Results

2.1 Carvacrol affects the proliferation of HCC1937 cells

2.2 Carvacrol affects the cell cycle of HCC1937 cells

2.3 Carvacrol induces ultrastructural changes in HCC1937 cells

2.4 Carvacrol induces apoptosis in HCC1937 cells

2.5 Carvacrol affects the Bcl-2/CytC pathway in HCC1937 cells

Discussion

Conclusion

Declarations

References

Additional Declarations

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