Usnic Acid Induce Apoptpsis Via An ROS-Dependent Mitochondrial Pathway In Human Breast Cancer Cells

According to the data released from the International Agency belongs to world health organization for Research on Cancer for research on cancer, GLOBOCAN2012 showed that 14.1 million new cases happened to be cancer and 8.2 million dead. Significantly higher than the GLOBOCAN data released in 2008, 1270 new cancer cases and 7.6 million deaths. The second most commonly diagnosed cancers worldwide were those of the breast(1.7 million, 11.9%) [1]. All above statement declare breast cancer seriously threaten to the health of human. In 2012, It is occurred 1.7 million new cases of breast cancer in the world, the incidence increased more than 20% than it in 2008, and the fatality rate increased 14%. The development of the breast cancer is severe. At present, surgical treatment, radiotherapy, chemotherapy and endocrine treatment become the first line of classic treatment. Although these treatments successfully reduce the mortality rate of patients with breast cancer and prolong the survival period, due to the difference of classification, etiology of breast cancer and the side effects of therapy drugs on the patients also greatly increase the breast cancer mortality. Therefore, looking for a specific plan and therapy targeted the etiology of breast cancer is a problem should be solved by the medical community. Numerous studies announce that substitution extracts from the traditional Chinese herbal has high-efficiency and low-toxicity antitumor effects, usnic acid extract from the lichen plant inhibits the proliferation of abundant tumors, can slow down the growth of breast cancer. But its mechanism and pathway is not clear yet[2]. Objectivethe mechanism and pathway that usnic acid inhibited proliferation of MCF- 7 breast cancer cells are studied in this paper. Usnic acid inhibited the occurrence and development of breast cancer and had the advantages of good drug safety, providing theoretical basis for the choice of clinical treatment of breast cancer. Methods and results:usnic acid induces apoptosis via an ROS- dependent mitochondrial pathway in human breast cancer cells in vitro Human breast cancer cell lines MCF-7, MDA-MB-231, SK-BR-3 and normal human mammary epithelial cell line MCF-10A(ATCC) were cultured in different concentration of usnic acid(0、3.125、6.25、12.5、25、50、100μM)24 and 48 hour. The anti-proliferative activity of UA toward four human breast cell lines, including the cancer cell lines MCF-7, MDA-MB-231, SK-BR-3 and the non-neoplastic cell line MCF-10 A, was detected by the MTT assay. The production of intracellular reactive oxygen species(ROS) was measured using DCFH-DA after culture the MCF-7 cells 24 hour in different construction of usnic acid(0、12.5、25、50 μM). Mitochondrial membrane potential was monitored using the flow cytometry. Apoptosis was quantified by using annexin V-FITC apoptotic detection kit. NAC, the inhibitor of ROS, was used to detective the the influence of apoptosis that usnic acid induces generation of ROS. SP600125, the inhibitor of JNK, was used to detective the the influence of MAPK pathway affect the Usnic acid induces apoptosis via an ROS- dependent mitochondrial pathway. After culture the MCF-7 cells 24 hour, the phosphorylation state of JNK, c-Jun, ERK Bax/Bcl-2、Cytochrome-c、Caspase-3 and PARP was evaluated by Western blot analysis.The result shows that after culture breast cancer cell lines MCF-7, MDA-MB-231, SK-BR-3, MCF-10 A 24hour, IC50 values(the concentration of drug inhibiting 50% of cells) of approximately 34.12±1.25 μM 、 38.41±1.64 μM 、48.07±1.52 μM、87.69±1.04 μM. It illustrates usnic acid inhibited proliferation of breast cancer cells, Cancer cells exhibited time- and concentration-dependent sensitivity to usnic acid, but the influence of the normal breast cells is small. The ratio of apoptosis increase dramatically(P<0.05). The data of DCFH-DA explains the generation of ROS exhibited concentration-dependent sensitivity to usnic acid. The result of flow cytometry states usnic acid can increase the loss of mitochondrial membrane potential. Compared with blank, the data of V-FITC/PI staining shows that ratio of apoptosis increase dramatically after culture the MCF-7 cells with usnic acid 24 hour. The activity of Caspase-3 increase highly that measured by iactivity of Caspase-3 kit. The result of Western blot shows the express of JNK, c-Jun, ERK Bax/Bcl-2、Cytochrome-c、Caspase-3 and PARP increase dramatically, the ratio of Bax /Bcl-2 is increasingly, declare usnic acid induces apoptosis via an ROSdependent mitochondrial pathway in human breast cancer cells.We use NAC and SP600125 can reverse the affect by the usnic acid, stating usnic acid via an ROS-dependent pathway stimulate the MAPK pathway through the mitochondrial pathway, activates the Caspase-3, apopmsises the MCF-7 cells.2.UA inhibited MCF-7 xenograft tumor growth by increasing apoptotic cell death We examined the effect of UA on the growth of MCF-7 cells in nude mice. When the tumor reached 100 mm3, mice were randomly divided into five groups. The mice in the test group first received intraperitoneal(i.p.) injections of UA(25, 50 or 100 mg/kg body weight in sodium chloride) at 2 day intervals for 18 days. The control group was treated with an equal volume of vehicle. The positive group received i.p. injections of cyclophosphamide(CTX)(25 mg kg 1). After 21 days, mice were sacrificed and tumors were removed, weighed, fixed with formaldehyde and embedded in paraffin. The apoptosis is measure by the TUNEL assay.The result illustrates usnic acid inhibited growth curve and weight of the tumor of human breast cancer, and the resistance of caner exhibited concentration-dependent. The effect of the group with the concentration of usnic acid 100mg/kg is better than the group with cyclophosphamide. The result of TUNEL assay reconstructs usnic acid increase the ratio of apoptosis and inhibit the growth of the tumor. From the weight of nude mice, we can see the change of the groups with usnic acid is the similar with the group using sodium chloride, but the weight of the group with cyclophosphamide decrease dramatically. All of above shows usnic acid has high-efficiency and low-toxicity antitumor effects. Conclusion:Usnic acid generate abundant ROS, stimulate the JNK, cause the change of Bcl-2, Bax, Cytochrome-c, Caspase-3, apoptosis via an ROS- dependent mitochondrial pathway in human breast cancer cells in vitro and in vivo. Usnic acid has significant resistance the growth of the breast cancer, is expected to further development of drugs for breast cancer treatment.