Study on Apoptosis of Breast Cancer Cells Induced by Regulation of PI3K/Akt/mTOR Pathway by Syringin*

SHⅠ Ya-Qian, LⅠ Xin, HUANG Si-Yuan, OU Ming-Kun, LⅠ Hong-Na, LU Min,GENG Meng-Li, OU Ye-Tao**

(1)School of Basic Medicine, Guilin Medical University, Guilin 541001, China;2)School of State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin 541004, China;3)School of Basic Medicine, Sanquan College of Xinxiang Medical University, Xinxiang 453000, China;4)School of Clinical Medicine, Jiamusi University, Jiamusi 154002, China)

Abstract Objective To study the anti-breast cancer effects and molecular mechanisms of syringin, and to provide a theoretical basis for the clinical application of syringin.Methods The inhibitory effect of syringin on the proliferation of breast cancer cells was measured with MTT assay.Trypan blue, TdT-mediated dUTP nick-end labeling (TUNEL), and Annexin V-FⅠTC/PⅠ staining were used to detect apoptosis.Caspase-3 activation was detected via Western blot to determine whether apoptosis occurred.The expression of apoptosis-associated protein B-cell lymphoma-2 (Bcl-2) was detected and the effect of syringin on the mitochondrial apoptosis pathway was investigated via JC-1 staining.The PⅠ3K agonist Recilisib was used for comparison.qRT-PCR and Western blot were used to assess the role of syringin in regulating the PⅠ3K/Akt/mTOR pathway and inducing the apoptosis of cancer cells.Results Syringin had a time- and dose-dependent inhibitory effect on the proliferation of breast cancer cells and induced their apoptosis.A further study showed that after syringin treatment, Caspase-3 was activated, Bcl-2 expression decreased, the mitochondrial membrane potential was significantly reduced, and the mRNA and protein expressions of PⅠ3K, Akt, and mTOR were not significantly changed, but the protein phosphorylation levels were significantly decreased.Recilisib partially limits the effect of syringin on the apoptosis of breast cancer cells.Conclusion Syringin has a good inhibitory effect on MDA-MB-231 and MCF-7 breast cancer cells.Ⅰt can inhibit cell proliferation and induce mitochondrial apoptosis by inhibiting the activation of the PⅠ3K/Akt/mTOR signaling pathway.Syringin is a potential anti-breast cancer drug.

Key words syringin, breast cancer, apoptosis

According to the latest data from the American Cancer Society, the incidence of breast cancer has been on the rise for nearly 4 decades, and is now one of the most common malignancies among women worldwide[1].Additionally, breast cancer accounts for 15.5% of cancer deaths in women worldwide[1].Ⅰn China, breast cancer is one of the top 5 causes of cancer-related deaths among women, accounting for about 8.1%[2].Chemotherapy is one of the main methods of treating breast cancer.However,chemotherapy drugs show extensive side effects,seriously affecting patients’ physical and mental health[3].Traditional Chinese medicine has many advantages in treating tumors with diverse targets and less toxic side effects.Natural plants can be used as a resource for discovering highly effective anti-tumor drugs.There is significant theoretical significance and application value in exploring the anti-tumor molecular mechanisms of compounds extracted from natural plants.

Syringin (Syr), also known as eleutheroside B, is a bioactive component of many natural plants, such as acanthopanax senticoside, which can play an inhibiting role in some tumors.Syringin can effectively inhibit cervical cancer, prostate cancer and breast cancer[4].Ⅰn breast cancer, syringin can induce the cycle arrest of MCF-7 and MDA-MB-231 cells by increasing reactive oxygen species (ROS) production,and can also promote the activation of Caspase-3 and Caspase-9 to induce apoptosis[5].Furthermore,syringin can act against breast cancerviathe PⅠ3KAkt-PTGS2 and EGFR-RAS-RAF-MEK-ERK pathways[6].Ⅰn conclusion, syringin can have an inhibitory effect on breast cancer, but research on its anti-tumor molecular mechanism is not complete at present.

Ⅰn this study, MDA-MB-231 and MCF-7 breast cancer cells were selected as the research objects, and the inhibitory effects of syringin on breast cancer cells were detected.This study determined the inhibitory effect of syringin on breast cancer cells and preliminarily explored its molecular mechanism.Our results provide a theoretical basis for applying syringin, and new ideas for the clinical treatment of breast cancer.

1 Materials and methods

1.1 Reagents and antibodies

Syringin (HY-N0824) was purchased from MCE,Shanghai, China.MTT (M8180) and trypan blue stain solution (C0040) were purchased from Solarbio,Beijing, China.The TUNEL (TdT-mediated dUTP nick-end labeling) apoptosis detection kit (KGA7072)was obtained from KeyGEN BioTECH, Jiangsu,China.Annexin V-FⅠTC/PⅠ kit (C1062L) and JC-1(C2003S) were purchased from Beyotime Biotechnology, Shanghai, China.Antibodies against PⅠ3K (#D55D5), mTOR (#7C10), p-mTOR (#D9C2),B-cell lymphoma-2 (Bcl-2, #15071), Bax (#5023) and Caspase-3 (#9662) were derived from Cell Signaling Technology, Shanghai, China.The p-PⅠ3K (#AB4709N)antibody was sourced from Thermo Scientific,Shanghai, China.The Akt (#AF6261) and p-Akt(#AF3262) antibodies were from Affinity Biosciences, Jiangsu, China.

1.2 Cells lines and culture conditions

Breast cancer cells MDA-MB-231 (HTB-26) and MCF-7 (HTB-22) were purchased from American Type Culture Collection, Manassas, VA, USA.All cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.The cells were grown in an incubator at 37°C in a 5% CO2atmosphere.

1.3 Cytotoxicity assay

The cells were seeded in 96-well plates at a density of 3×103per well and treated with different doses of syringin.The cells were cultured under various treatments in three groups containing 6 duplicate wells each: negative groups without any treatment, the blank wither group with only media (no cells) and the dosing group with many doses of syringin.After further cultivation for 24 , 48, or 72 h,MTT (5 g/L, 20 µl per well) was added into every well.Then, the breast cancer cells were subjected to light-avoidance incubation at 37℃ for 4 h, and a microplate reader measured the absorbance at 490 nm to calculate the ultimate cell proliferation activity.Drug-cell inhibition rate = [1-(experiment-blank)/(negative-blank)]×100%.

1.4 Trypan blue staining

The breast cancer cells were collected after treatment with syringin (50 µmol/L) for 48 h.Next,the cells were resuspended with PBS, and their concentration was adjusted to (1-2)×109/L.Following this, trypan blue staining solution (PBS∶trypan blue=9∶1) was added into the cell suspension for 3 min,with observing, photographing, and counting on cell count plates, and then the cell mortality was calculated.Cell mortality=dead cell number/total cell number×100%.

1.5 Apoptosis assay

TUNEL staining was used to observe the apoptotic morphology of the cells.Syringin (50 µmol/L)treatment for 48 h was followed by fixation,permeation, staining, DAPⅠ redyeing, and tablet sealing.After interacting with the drugs for one day,each well’s breast cancer cells were treated with a gradient concentration of syringin and digested with trypsin without EDTA.The cells were stained using an Annexin V-FⅠTC/PⅠ kit before being incubated under conditions of light avoidance for 10-15 min and finally detected using a flow cytometer.

1.6 Western blot

Cells were treated with different doses of syringin, and then the total protein was extracted after 48 h.The protein content was quantified at 3 g/L using a BCA solution.The amounts of cellular protein were separated by 8%, 10%, or 12% SDS-PAGE, and the procedures were as follows: transposing to 0.22 μm PVDF membrane at the condition of 200-300 mA, 1.5-3 h, using 5% BSA for blocking at room temperature for 2 h, incubating with a primary antibody (1∶1 000) overnight (4°C, about 14-16 h),and then secondary antibody (1∶10 000) incubation for 1 h before imaging.

1.7 Detection of mitochondrial membrane potential （MMP）

The cells were cultured in 6-well plates at a density of 1.5×105/well, and syringin was used to treat the cells the next day.JC-1 staining was performed after 48 h: adherent cells were stainedin situon the reagent and photographed under a fluorescence microscope.At the same time, after the breast cancer cells from each parallel well were digested with trypsin, following the same steps applied in staining the suspended cells, the cells’ mitochondrial membrane potential was detected using a flow cytometer.

1.8 Statistical analysis

The results are presented as the mean±standard error of at least three independent samples.Three or more datasets have been analyzed using one-way ANOVA analysis of variance followed by LSD testing for multiple comparison.P＜0.05 was considered statistically significant.SPSS 25.0 was employed in the statistical analysis.

2 Results

2.1 Cytotoxicity of syringin on breast cancer cells

Ⅰn order to detect the inhibitory effect of syringin on breast cancer, the breast cancer cells MDA-MB-231 and MCF-7 were selected as the research objects and treated with syringin at different concentrations(10, 25, 50, 75, and 100 µmol/L).The proliferation activity of each group was detected using an MTT assay at 24 h, 48 h and 72 h after addition.The results show that the proliferation activity of breast cancer cells was significantly decreased after treatment with syringin, and showed a significant time- and dosedependent trend (Figure 1a).Ⅰt was observed under a bright field microscope that the density and morphology of breast cancer cells treated with syringin decreased significantly (Figure 1b).These results indicate that syringin has a good inhibitory effect on breast cancer.

2.2 Syringin inhibits breast cancer cells by promoting apoptosis

Trypan blue staining is commonly used to detect living cells[7].Ⅰn order to verify the killing effect of syringin on breast cancer cells, 50 µmol/L and 48 h were selected as the representative treatment conditions, and trypan blue was used to detect cell death, because the cell proliferation is clearly inhibited and in a moderate state at this time, it is convenient for subsequent detection.The results show that, compared with the control group, the death rates of the two breast cancer cell groups significantly increased after syringin treatment (Figure 2a, b).Ⅰn addition, applying the same processing conditions, we used TUNEL to detect cell apoptosis, and the results show that syringin significantly promoted cell apoptosis (Figure 2c).Syringin-treated cells showed bright green fluorescent particles, which is a specific apoptosis phenotype, while control cells did not show this phenotype.These results imply that promoting apoptosis may be an essential pathway by which syringin can inhibit breast cancer.

Ⅰn order to further clarify the proapoptotic effect of syringin on breast cancer cells, 3 concentration gradients were set up in this study, and the apoptosis rate and apoptosis-related protein expression were detected 48 h after the addition of the drug.The flow cytometry results of Annexin V-FⅠTC/PⅠ double staining prove once again that syringin could promote the apoptosis of breast cancer cells (Figure 3b).After the administration of syringin, the apoptosis of breast cancer cells was significantly elevated in a dosedependent manner.After the administration of syringin, the expressions of Caspase-3 and poly ADPribose polymerase (PARP) in MDA-MB-231 declined, and the expressions of cleaved-Caspase-3 and cleaved-PARP were increased considerably(Figure 3a).This demonstrates the activation of the Caspase-3 pathway in MDA-MB-231 cells.The above results show that promoting apoptosis is the main pathway by which syringin inhibits breast cancer cells.

Fig.1 Effect of syringin on the proliferation of breast cancer cells MDA-MB-231 and MCF-7

Fig.2 Effect of syringin on apoptosis in breast cancer cells

Fig.3 Effect of syringin on apoptosis in breast cancer cells

2.3 Syringin induces a reduction in mitochondrial membrane potential in breast cancer cells

A reduction in MMP is an irreversible factor of apoptosis and a landmark event in the early stage of apoptosis[8].JC-1 is a fluorescent probe widely used to detect MMP[9].Using JC-1 detection, we found that the MMP of breast cancer cells decreased significantly after 48 h of syringin (25, 50, and 100 µmol/L) treatment (Figure 4b, c), and the decrease in MMP was more significant with increases in syringin.The reduction in MMP is consistent with the reduction in the intracellular Bcl-2 expression of breast cancer (Figure 4a).This suggests that syringin activates the mitochondrial apoptotic pathway in breast cancer cells by causing a decrease in MMP through the downregulation of Bcl-2.

2.4 Syringin induces breast cancer cell apoptosis by inhibiting PI3K/Akt/mTOR signaling pathway

The over-activation of the PⅠ3K/Akt/mTOR signaling pathway is an essential factor in the occurrence and development of breast cancer.Ⅰt is also the main target of many drugs that exert anticancer effects[10].The inhibition of the PⅠ3K/Akt/mTOR signaling pathway is closely related to apoptosis[11].To investigate the molecular mechanism of syringin-induced apoptosis in depth, this study further examined the expression of PⅠ3K/Akt/mTOR pathway molecules at the mRNA and protein levels in cells after syringin treatment.The results show that although the mRNA and protein expression levels of PⅠ3K, Akt and mTOR did not change significantly after the application of syringin, their phosphorylation was greatly reduced (Figure 5a-f), and this effect was dose-dependent.This suggests that syringin inhibited the activation of the PⅠ3K/Akt/mTOR pathway in breast cancer cells.Ⅰn addition, we applied Recilisib,an agonist of PⅠ3K, as an interference group, and the results show that Recilisib (50 µmol/L, 48 h) could partially block the inhibitory effect of syringin(50 µmol/L, 48 h) on p-PⅠ3K in MDA-MB-231 and MCF-7 cells (Figure 5g, h).The same treatment conditions were used, and apoptosis was detected by flow cytometry for each group of cells.The results show that Recilisib can partially offset the promotional effect of syringin on apoptosis (Figure 3c).The above results indicate that syringin promotes apoptosis in breast cancer cells by inhibiting PⅠ3K/Akt/mTOR pathway activation.

3 Discussion

Breast cancer is a systemic disease.Current clinical treatments mainly include chemotherapy and surgery.Surgical therapy also requires adjuvant chemotherapy given before and after.Therefore, it is of great significance to discover and further study specific chemical drugs and explore their anti-tumor mechanisms.Ⅰn this paper, we discussed the inhibitory effect of syringin on breast cancer.Ⅰt was found that syringin could induce apoptosis in breast cancer cells by inhibiting the phosphorylation level of the PⅠ3K/Akt/mTOR signaling pathway and down-regulating Bcl-2.

Tumor cells have 3 remarkable essential characteristics: immortality, migration, and loss of contact inhibition, the most important of which is immortality[12].Therefore, finding drugs that specifically kill tumor cells is the primary strategy for tumor therapy.This study found that syringin has a specific targeted killing effect on breast cancer cells.The PⅠ3K/Akt/mTOR signaling pathway is involved in cell proliferation, survival, invasion, migration,apoptosis, glucose metabolism, and DNA repair[9].Many studies have revealed abnormal activation and mutation in breast cancer, and it is an important molecular target for breast cancer treatment[13].The study by Duet al.[14] showed that Everolimus inhibited the growth, migration, and invasion of breast cancer cells by reducing the activity of the PⅠ3K/Akt/mTOR signaling pathway.The results of this study show that syringin promoted the death of breast cancer cells and decreased the phosphorylation level of the PⅠ3K/Akt/mTOR signaling pathway.PⅠ3K agonists could partially block the pro-apoptotic effect of syringin in breast cancer.This indicates that PⅠ3K/Akt/mTOR signaling is critical for syringin to inhibit the proliferation of breast cancer cells and promote their apoptosis (Figure 5, 3c).

Fig.5 Effect of syringin on PI3K/Akt/mTOR pathway in breast cancer cells

Apoptosis is the primary mechanism of programmed cell death, and 3 induced pathways have been found: mitochondrial pathway, death receptor pathway, and endoplasmic reticulum pathway.Bcl-2 protein is the primary regulator of the mitochondrial pathway, and its main function is to regulate the permeability of the mitochondrial membrane.When the expression of Bcl-2 decreases, the integrity of the mitochondrial membrane is destroyed, and cytochrome c (Cyt c) is released.Cyt c forms a complex with Apaf-1 and Caspase-9, which further activates Caspase-3 and causes apoptosis[15].The results of this study show that after the administration of syringin, the expression of Bcl-2 decreased and the mitochondrial membrane potential decreased in breast cancer cells (Figure 4).TUNEL staining and flow cytometry also demonstrated the induction of apoptosis by syringin (Figure 2, 3).These results indicate that syringin induced apoptosis of the mitochondrial pathway in breast cancer cells.Ⅰn addition, the expression levels of Caspase-3 and PARP precursor protein decreased, and the expressions of cleaved-Caspase-3 and cleaved-PARP increased in MDA-MB-231 cells (Figure 3a).However, the activation of Caspase-3 was not detected in MCF-7 cells, which is due to the deletion of 47 base pairs in the exon of theCASP-3gene in MCF-7 cells, resulting in the deletion of the protein[16-17].Caspase-3 protein does play an important role in the apoptosis pathway, but this is not the only apoptotic pathway.Studies have shown that the complex formed by Cyt c, Apaf-1, and Caspase-9 can play an important role in Caspase-3 deficiency.The activation of Caspase-7 in MCF-7 cells induces apoptosis[18].Ⅰn addition, after the mitochondrial membrane potential drops, it can also cause the apoptosis-inducing factor (AⅠF) to be translocated from the mitochondria to the nucleus, resulting in the fragmentation of chromatin-condensed nuclear DNA,which eventually leads to apoptosis[19].Ⅰn this experiment, we demonstrated that syringin induced mitochondrial membrane potential reductions and apoptosis in MCF-7 cells without the involvement of Caspase-3.Therefore, we can speculate that the molecular mechanism of the syringin-induced apoptosis of MCF-7 may be related to Caspase-7 or AⅠF, which needs to be further studied.

4 Conclusion

This study has demonstrated that syringin has a potent inhibitory effect on breast cancer cells.This inhibitory effect promotes the apoptosis of breast cancer cells by inhibiting the PⅠ3K/Akt/mTOR signaling pathway and inducing the mitochondrial apoptosis pathway.This study provides reliable experimental data for identifying potential anti-breast

cancer drugs with few side effect.

AcknowledgmentsWe thank all the members in School of Basic Medicine, Guilin Medical University for helpful discussions and suggestions.