|Year : 2015 | Volume
| Issue : 2 | Page : 454-458
Ligustrazine induces apoptosis of breast cancer cells in vitro and in vivo
Jun Pan1, Jiang-Feng Shang2, Guo-Qin Jiang1, Zhi-Xue Yang1
1 Department of General Surgery, the 2nd Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, China
2 Department of Thyroid and Breast Surgery, The No. 1 People's Hospital of Changshu, Changshu, Jiangsu Province 215500, China
|Date of Web Publication||7-Jul-2015|
Department of General Surgery, The 2nd Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, Jiangsu 215004
Source of Support: None, Conflict of Interest: None
Introduction: Ligustrazine, the active ingredient present in Umbelliferae plant roots used in Chinese medicine, plays a vital role in reversing multidrug resistance in tumors. This study aims to investigate its anticancer activity and underlying mechanisms in human breast cancer cells in vitro and in vivo.
Materials and Methods: Human breast cancer cell line MDA-MB-231 was incubated with different concentrations of ligustrazine. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; and cell apoptosis and cell cycle were assessed by flow cytometry. Subcutaneous MDA-MB-231 xenograft tumors were established in nude BALB/c mice. Ligustrazine was intraperitoneally administered. The tumor growth was monitored.
Results: In vitro, ligustrazine inhibited cell survival in a dose-and time-dependent manner, and induced apoptosis, as indicated by a dramatic increase in sub-G0/G1 cells. The in vivo results were consistent with the in vitro ones: Administration of ligustrazine substantially inhibited tumor growth, which also indicated that ligustrazine inhibited proliferation of MDA-MB-231 cells in vitro.
Conclusions: Ligustrazine causes apoptotic death and tumor regression in human breast cancer cells in vitro and in vivo models.
Keywords: Apoptosis, breast cancer, cell growth inhibition, ligustrazine
|How to cite this article:|
Pan J, Shang JF, Jiang GQ, Yang ZX. Ligustrazine induces apoptosis of breast cancer cells in vitro and in vivo. J Can Res Ther 2015;11:454-8
FNx01Jun Pan and Jiangfeng Shang contributed equally to this work.
| > Introduction|| |
Breast cancer is the most frequently diagnosed cancer, and the leading cause of cancer death in women worldwide, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008. About half of the breast cancer cases and 60% of cancer deaths are estimated to occur in economically, developing countries.  Conventional drug treatment has obvious beneficial effects, but it is also associated with serious side-effects. Therefore, searching for drugs with high selectivity and low toxicity has become the recent focus of attention. One of the drugs in focus is ligustrazine, which is the active ingredient of extracts from Umbelliferae plant roots used in traditional Chinese medicine.  Ligustrazine possesses a wide range of biological properties such as anticoagulation, inhibition of platelet aggregation,  dilation of blood vessels, improvement of microcirculation, protection brain function,  and protection of vascular endothelium. It also has confirmed effects on reversing multidrug resistance (MDR) in tumor cells. , The mechanism underlying the effect of ligustrazine on leukemia, gastric carcinoma, and melanoma cells has been reported. ,, However, the effects of ligustrazine on cell proliferation and apoptosis in breast cancer cells have rarely been reported. Zhang et al. reported that tetramethylpyrazine has potential application in the treatment of chemotherapy-resistant MCF-7 cells in vitro.  In this investigation, we observed the effects of ligustrazine on proliferation and apoptosis in human MDA-MB-231 breast cancer cells.
| > Materials and methods|| |
Mice, cell lines, and reagents
Female nude BALB/c mice (aged 6-8 weeks) were purchased from ***SLAC Laboratory Animal Co. Ltd. The human breast cancer cell line MDA-MB-231 was obtained from the cell bank of the ***Academy of Sciences. The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). Ligustrazine was purchased from ***Pharmaceutical Co. Ltd. (Batch No.: 091202A1). It was dissolved in distilled water to produce a stock solution.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay
MDA-MB-231 cells were seeded in 200 μL cell culture medium, poured into 96-well plates, and cultured for 24 h. Then, the medium was replaced with fresh medium alone or cell culture medium containing different concentrations of ligustrazine (0.25, 0.5, 1.0, 1.5, and 2.0 mg/mL). After incubation for 24 h, 48 h, or 72 h, MTT (5 mg/mL, 20 μL) was added to each well; this was followed by 4 h of incubation. The medium was then discarded, and 150 μL of dimethylsulfoxide was added into each well; this was followed by an additional 20 min of incubation. The optical density (OD) at 490 nm was measured spectrophotometrically. The cell viability index was calculated according to the formula: Experimental OD value/control OD value × 100%. The experiments were conducted in triplicate.
In vitro cell apoptosis assay
MDA-MB-231 cells were treated with ligustrazine at a concentration of 0.5 mg/mL, 1 mg/mL, or 1.5 mg/mL. The control group cells were only cultured in RPMI 1640 medium with 10% FBS. After treatment of 48 h with ligustrazine, the cells were collected. The cell suspension (100 μL/tube) was incubated with annexin-V FITC (5 μL) and propidium iodide (PI) (5 μL) for 10 min at 0°C in the dark. The percentage of apoptotic cells was determined by flow cytometry analysis using the FACScan flow cytometer (BD Pharmingen) with[TAG:2][/TAG:2]
CellQuest software (BD Pharmingen). All experiments were repeated in triplicate.
In vitro cell cycle assay
MDA-MB-231 cells were treated with ligustrazine at a final concentration of 0.5 mg/mL, 1 mg/mL, or 1.5 mg/mL. Cells in the control group were cultured in RPMI 1640 with 10% FBS. After treatment for 48 h, the cells were centrifuged, fixed in 70% ethanol for at least 24 h, washed with PBS, and treated with RNA enzyme (50 μg/mL). Then, the cells were exposed to 400 μL PI (50 μg/mL) at room temperature for 30 min. The cell cycle distribution was examined with BD FACSArray™ Bioanalyzer (San Jose, CA, USA). The experimental results were analyzed using the ModFit software with the flow cytometry. The experiment was conducted in triplicate.
In vitro morphological observation
After treatment with ligustrazine at a final concentration of 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, or 2 mg/mL for 48 h, the morphology of the MDA-MB-231 cells was examined under an inverted light microscope (Carl Zeiss, Inc.).
In vivo experiment
All surgical procedures were conducted, and care was administered to the animals in accordance with the institutional guidelines. Tumors were established by subcutaneous injection of 1 × 10 7 MDA-MB-231 cells into the flanks of mice. Tumor volumes were estimated according to the formula of V = a 2 × b × π/6, where a is the short axis and b is the long axis.  When the tumors reached a volume of 100 mm 3 at about 1 week, the experimental MDA-MB-231 nude mice were randomly, divided into the saline group, high-dose ligustrazine group, low-dose ligustrazine group, and cyclophosphamide (CTX) group. The mice in the saline group were given isotonic saline (0.2 mL intraperitoneally [ip]) injections once a day for 3 weeks; mice in the CTX group were given diluted CTX injection (100 mg/kg; 0.2 mL ip) once a week for 3 weeks; mice in the high-dose ligustrazine group were given 200 mg/kg (0.2 mL ip) ligustrazine once a day for 3 weeks; mice in the low-dose ligustrazine group were given 50 mg/kg (0.2 mL ip) ligustrazine once a day for 3 weeks. The experiment was terminated at the end of the third week. The mice were sacrificed after neck dislocation; partial growth of the transplanted tumor was observed, and tumor tissue was taken.
Data were expressed as the mean ± SEM of at least three independent experiments. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni's multiple comparison test. P < 0.05 was considered statistically significant.
| > Results|| |
0Ligustrazine reduces breast cell viability
Ligustrazine could remarkably inhibit the proliferation of MDA-MB-231 cells in vitro in a dose-and time-dependent manner at concentrations ≥0.50 mg/mL [Table 1]. However, the inhibitory effect of ligustrazine at low concentrations (≤0.25 mg/mL) was weak, even after prolonged incubation. Logarithmic regression analysis was used to calculate the minimum concentration of ligustrazine that resulted in 50% inhibition of cell proliferation (IC 50 ). This was found to be 1.80 mg/mL at 24 h, 1.57 mg/mL at 48 h and 1.25 mg/mL at 72 h.
|Table 1: Inhibition rate in MDA - MB - 231 cells treated with different concentrations of ligustrazine|
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Ligustrazine induces cell apoptosis
Ligustrazine has been reported to reverse MDR and induce cell death in leukemia, gastric cancer, and melanoma. To determine whether ligustrazine can also induce cell death in breast cancer cells, we exposed human breast cancer cells (MDA-MB-231) to different dosages of ligustrazine, and the flow cytometry analysis was used to measure the apotosis rates. As shown in [Figure 1], treatment with ligustrazine at a concentration of 0.5 mg/mL resulted in an apoptosis rate of 6.59 ± 2.90%, which was significantly higher than that in untreated cells (P < 0.05). Compared with the untreated cells, treatment with 1 mg/mL and 1.5 mg/mL of ligustrazine resulted in significantly higher apoptosis rates (14.85 ± 3.41% and 22.22 ± 2.98%, respectively) (P < 0.05). Consistently, cell cycle distribution analysis demonstrated that treatment with 0.5 mg/mL, 1.0 mg/mL, and 1.5 mg/mL ligustrazine for 48 h resulted in a marked increase in the sub-G 0 /G 1 population (54.71 ± 3.83%, 64.17 ± 4.01%, 75.06 ± 4.32%, respectively) than control group (P < 0.05) [Figure 2].
|Figure 1: Apoptosis rate of MDA-MB-231 cells treated with different concentrations of ligustrazine. Ligustrazine increased the in vitro apoptosis rates of MDA-MB-231 cells in a dose-dependent manner. Dot plots represent the flow cytometric results for MDA-MB-231 cells incubated with ligustrazine at different concentrations. The abscissa shows the number of Annexin V-positive cells, and the ordinate, propidium iodide-positive cells|
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|Figure 2: Cell cycle distribution of MDA-MB-231 cells treated with different concentrations of ligustrazine. Cells in sub-G1/G0 phase are marked. The shaded portion indicates the S cycle|
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Ligustrazine affected cell morphology
With increasing concentrations of ligustrazine, the number of attached and adhesive cells showed a decrease under the inverted light microscope [Figure 3]. Cytoplasmic vacuoles were observed in cells treated with l mg/mL ligustrazine (data not shown).
|Figure 3: Cell morphology of MDA-MB-231 cells treated with different concentrations of ligustrazine as observed by inverted light microscopy. The magnification of the pictures is ×10. The number of attached and adhesive MDA-MB-231 cells showed a decrease with increasing concentrations of ligustrazine|
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Ligustrazine inhibited the growth of MDA-MB-231 breast cancer cells in vivo
Tumors in the control group mice grew remarkably fast, reaching a volume of 1.31 ± 0.23 cm 3 just 1 week after injection. Administration of CTX at a dose of 100 mg/kg per week resulted in a reduction in tumor volume (0.51 ± 0.20 cm 3 ), which was significantly different from the tumor volume in the control mice that were treated with isotonic saline. Simultaneously, the tumors of mice treated with 50 mg/kg and 200 mg/kg of ligustrazine were significantly (P < 0.01) smaller than those of the control group mice, reaching a volume of only 0.86 ± 0.27 cm 3 and 0.81 ± 0.21 cm 3 respectively. However, there was no significant difference in tumor reduction between the mice given the two doses of ligustrazine, which may be due to a limited animal number that we were observing, or due to the absorption of the drug is limited in vivo, especially in a high dosage [Figure 4].
|Figure 4: Tumor volume of nude rice after ligustrazine treatment. *Indicates a significant difference at P < 0.01, compared with control. The tumors of mice treated with 50 mg/kg and 200 mg/kg of ligustrazine were significantly (P < 0.01) smaller than those of the control group mice, reaching a volume of only 0.86 ± 0.27 cm3 and 0.81 ± 0.21 cm3 respectively. In contrast, there was no significant difference in tumor reduction between the mice given the two doses of ligustrazine|
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| > Discussion|| |
A typical feature of tumor cells is their ability to continuously divide and proliferate and therefore, inhibition of cell proliferation and induction of apoptosis is the main objective in the treatment of malignant tumors. We have studied the in vivo and in vitro effects of ligustrazine, an active ingredient in extracts used in Chinese medicine, apoptosis of human breast cancer cell line MDA-MB-231. The effects of ligustrazine have not been widely studied in breast cancer treatment previously.
Application of ligustrazine has been shown to have beneficial effects. Zhang et al. reported that ligustrazine used in combination with 5-FU, mitomycin, and cisplatin can improve the inhibition of MKN45 cell proliferation.  The IC 50 of doxorubicin and vincristine in K562/ADM cell inhibition showed a significant reduction after the addition of ligustrazine to the treatment regime.  There is also an evidence for the inhibitory effects of ligustrazine on cell growth of lung cancer and leukemia cells.  Liu and Cui  established a human small lung cancer mouse model, and treated the C57BL nude mice with ip injection of 100 mg/kg and 200 mg/kg daily doses of ligustrazine. They found that ligustrazine restrained the growth of the lung cancer cells in vivo by down-regulating gene and protein expression of vascular endothelial growth factor. Liang and Yang reported that administration of 100 μg/mL of ligustrazine can sensitize HL-60 and K562 leukemia cells to erythromycin, indicating its inhibitory effects on HL-60 and K562 cells. , Similar to the results of these studies, we found that ligustrazine could remarkably inhibit the proliferation of MDA-MB-231 cells in a dose-and time-dependent manner at concentrations ≥0.50 mg/mL.
With regard to the effects of ligustrazine on the cell cycle, Zhang et al.  suggested that ligustrazine induces cell cycle arrest in human small cell lung cancer cell line H446 by stalling cell growth at the S phase and simultaneously, inhibiting cell mitosis and DNA synthesis. We found that in MDA-MB-231 cells, ligustrazine can block the cell cycle at the G0/G1 phase and thus inhibit DNA synthesis.
A large number of studies have shown that anticancer drugs can induce tumor cell apoptosis.  Fu and Li  showed that ligustrazine induced the apoptosis of Lewis lung cancer cells in mice. In this study, we found that the number of attached and adhesive cells decreased with increasing concentrations of ligustrazine, which was also involved in the apoptosis.  Moreover, ligustrazine could induce apoptosis of MDA-MB-231 cells in a dose-dependent manner in vitro by the flow cytometry analysis. The members of the Bcl-2 family are the most prominent regulators of apoptosis. It is located on the membrane of mitochondria, is an anti-apoptotic protein, while Bax is a pro-apoptotic one by inhibiting the function of Bcl-2.  Hu and Li  reported ligustrazine down-regulated the expression of Bcl-2, and decreased the rate of Bcl-2/Bax so as to promote the apoptosis of lung cancer cells A549. Han et al.  also found ligustrazine induced human prostate cancer PC3 cells by down-regulating the expression of Bcl-2, and up-regulating that of Bax through the PI3K/Akt/mTOR pathway. It needs to be further determined whether ligustrazine-induced breast cancer cell apoptosis is caused by downregulation of the rate of Bcl-2/Bax or additional molecular mechanisms.
Xu and Yan reported the dose-dependent effect of ligustrazine on a C57BL rat model of lung cancer (doses: 50, 100, and 200 mg/kg per day).  After 21 days, substantial tumor inhibition was noted, but it was not significantly, different among the doses (45.5%, 43.1%, and 43.0% respectively). This is in agreement with our results that there is a significant difference in tumor inhibition between the ligustrazine-treated and control mice. However, there was no statistically significant difference between the high-and low-dose ligustrazine groups. Our data in vitro clearly, demonstrated a dose dependent effect for 50-200 mg/kg of ligustrazine [Figure 1] and [Figure 2], so we speculate that the in vitro and in vivo difference may be due to the limited absorption of the agent by the body, particular in a high concentration; or due to the quick degradation of the agent and reaching a stable level in vivo. In addition, we treated only six animals for each group, and the marginal differences between the two groups may not be able to produce a statistical significance. Therefore, in the future, we may measure the drug concentration in the animals and expose more animals with a range of dosages of ligustrazine.
Many other beneficial effects of ligustrazine have also been reported. Wan et al established a mouse Lewis lung model and demonstrated that the mice administered ligustrazine had higher body weight (2.81 ± 0.25 g) compared to the mice administered other chemotherapy treatment (1.02 ± 0.22 g).  This valuable result implies that ligustrazine results in an increase in bodyweight and thus improves the quality of life. This indicates that ligustrazine is safe, has low toxicity, and provides enhanced immunity.
Together with above paramount reports, our current study indicates the potential benefits of using ligustrazine in the treatment of breast cancer. Further investigation however needs to be conducted into the mechanism, by which ligustrazine inhibits breast cancer cell growth.
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