KR20110067817A - Anticancer composition for treating a breast cancer comprising cisplatin and bojungbangdocktang as active ingredients - Google Patents

Abstract

PURPOSE: An anticancer composition containing cisplatin and Bojungbangdocktang is provided to reduce cytotoxicity and apoptosis of normal breast epithelial cells and enhance cytotoxicity and apoptosis of the breast cancers. CONSTITUTION: An anticancer composition for treating breast cancer contains cisplatin and Bojungbangdocktang as an active ingredient. The composition reduces cytotoxicity and apoptosis of normal epithelial cells and enhances cytotoxicity and apoptosis of breast cancer cells.

Description

Anticancer composition for treating breast cancer containing cisplatin and bojeongbangdoktang as active ingredients {Anticancer composition for treating a breast cancer comprising cisplatin and Bojungbangdocktang as active ingredients}

The present invention relates to an anticancer agent composition for treating breast cancer, which contains cisplatin and bojeongbangdogtang as active ingredients. More specifically, the present invention relates to an anticancer composition for treating breast cancer, which contains cisplatin and bojeongbangdoktang as an active ingredient, which lowers cytotoxicity and apoptosis for normal breast epithelial cells and increases cytotoxicity and apoptosis for breast cancer cells.

Cisplatin, cisplatin or cis-diaminedichloridoplatinum (II) (CDDP) is a chemotherapeutic agent used in the treatment of various cancers including sarcoma, small cell lung cancer, ovarian cancer, lymphoma and germ cell tumors. Nevertheless, cisplatin can induce significant side effects such as nephrotoxicity, toxicity, hair loss, electrolyte disorders, nausea and vomiting. Recently, several researchers have reported that the combination of chemotherapy with cisplatin and herbal extracts can reduce side effects and enhance anti-tumor efficacy.

BJBDT, a modified herb prescription of Bangdok-tang and Bajeong-gamgamtang, has been used for the prevention or treatment of cancer in Korea without scientific proof. Thus, the present invention relates to tetrazolium salts for cytotoxicity, 2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide (XTT) assay, apoptosis DAPI Staining and Flow Cytometry for Morphological Changes Human Breast Epithelial Cell MCF by TUNEL Assay, Cell Cycle Assay for Sub-G1 Apoptotic Proteins and Western Blotting for Apoptotic Proteins such as Caspase-3, -9 and PARP The protective effect of Baodbangdogtang on cisplatin-induced cytotoxicity and apoptosis in -10A cells was measured.

Thus, the present inventors have shown that non-toxic concentrations of anti-vessels predicted to prevent cisplatin-induced toxicity and apoptosis in human normal breast epithelial cells, MCF-10A, unlike breast cancer cells MCF-7 and MDA MB-231. Pharmacological activities such as cytotoxicity between breast cancer cells and normal breast epithelial cells of BJBDT, an herbal drug with formation activity, were measured. The present invention has been accomplished by identifying cisplatin-induced cytotoxicity and increasing cytotoxicity and mitochondrial membrane potential (MMP) loss in breast cancer cells MCF-7 cells.

The problem to be solved by the present invention is a non-toxic concentration predicted to prevent cisplatin-induced toxicity and apoptosis in human normal breast epithelial cells MCF-10A, unlike the breast cancer cells MCF-7 and MDA MB-231 To develop anticancer composition for breast cancer treatment by reducing the toxicity of cisplatin by measuring the pharmacological activity such as breast cancer cell and normal breast epithelial cell of BJBDT, an herbal medicine with anti-angiogenic activity will be.

Disclosure of Invention It is an object of the present invention to provide an anticancer composition for treating breast cancer containing cisplatin and bojeongbangdoktang as active ingredients, which lowers cytotoxicity and apoptosis for normal breast epithelial cells and increases cytotoxicity and apoptosis for breast cancer cells.

In this case, the normal breast epithelial cells are MCF-10A cell lines, and breast cancer cells are characterized by being MCF-7 or MDA MB-231 cell lines.

In addition, the effective concentration for selectively increasing cytotoxicity and apoptosis in the breast cancer cell line in vitro of the anticancer agent composition is characterized in that the cisplatin 80 μM, Bojeongbangdaktang 100-200 μg / ml.

The anticancer agent composition on the other hand reduces cisplatin-induced apoptotic bodies in normal breast epithelial cells, decreases apoptotic protein sub-G1 DNA content in cisplatin-treated human normal breast epithelial cells, caspase-3 and- 9 and block activation of poly (ADP-ribose) polymerase (PARP) cleavage to selectively increase cytotoxicity and apoptosis in breast cancer cells.

The effects of the present invention is to protect the cisplatin-induced cytotoxicity in normal breast epithelial cells MCF-10A cells and the cytotoxicity and mitochondrial membrane potential in breast cancer cells MCF-7 cells when administered in combination with chemotherapeutic agents such as cisplatin (MMP) Increased loss of cisplatin-induced cytotoxicity and apoptosis in MCF-10A cells, normal breast epithelial cells, by co-administration of cisplatin and jungbangdogang in combination with chemotherapeutic cisplatin alone in breast cancer treatment. It is to provide an anticancer composition for treating breast cancer in combination with cisplatin and bojungbangdoktang, which can reduce the normal cytotoxicity of cisplatin.

Cisplatin is widely used as a cancer chemotherapeutic agent for the treatment of various human cancers, but it causes significant side effects such as nephrotoxicity and hepatotoxicity due to damage to normal cells.

Therefore, the present invention provides anti-angiogenic activity of non-toxic concentrations that can suppress cisplatin-induced cytotoxicity and apoptosis in human normal breast epithelial cells MCF-10A, which are different from breast cancer cells MCF-7 and MDA MB-231. The pharmacological activity of BJBDT, a herbal medicine, was measured.

Bojungbangdok-tang protected cisplatin-induced cytotoxicity in human normal breast epithelial MCF-10A cells and increased cytotoxicity and mitochondrial membrane potential (MMP) loss in breast cancer cells MCF-7 cells. In addition, 4 ', 6-diamidino-2-phenylindole (DAPI) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays indicated that Bajeongbangdaktang was a cisplatin-treated control. Compared to cisplatin-induced apoptotic bodies in MCF-10A cells.

In line with this, Bojungbangdoktang not only reduces apoptotic protein sub-G1 DNA content in cisplatin-treated human normal breast epithelial MCF-10A cells, but also caspase-3 and -9 and poly (ADP-ribose) polymerase. (PARP) blocked the activation of cleavage. Therefore, the present invention shows that Bojungbangdok-tang can prevent cisplatin-induced cytotoxicity and apoptosis in human normal breast epithelial cells MCF-10A breast cells as adjuvant of chemotherapeutic agent for cancer treatment.

The present invention is described in more detail below.

Bojeongbangdoktang Protects Cisplatin-Induced Cytotoxicity in Human Normal Breast Epithelial Cells MCF-10A Cells

In order to investigate whether Bojeongbangdoktang has a cytotoxic effect on human MCF-10A breast epithelial cells, the present invention performed an XTT assay. MCF-10A cells were treated with Bojungbangdok-tang (0, 25, 50, 100 or 200 μg / ml) for 24 hours and cell viability was evaluated. As shown in FIG. 1A, Bojeongbangdok-tang showed no cytotoxicity against human normal breast epithelial MCF-10A cells up to a concentration of 200 μg / ml. In contrast, cisplatin showed significant cytotoxicity against human normal breast epithelial MCF-10A cells with an IC ₅₀ of ˜80 μM. Parallel assays were performed to test the effect of Baodbangdangtang on cisplatin-induced cytotoxicity in MCF-10A cells. Cells were treated with Baodbangdogang (0, 25, 50, 100 or 200 μg / ml) with or without cisplatin (80 μM). In particular, the cell viability of human normal breast epithelial cells MCF-10A treated with cisplatin was increased by Baodbangdangtang treatment in a dose-dependent manner, indicating the protective effect of Baodbangdangtang on cisplatin-induced cytotoxicity.

Furthermore, the present invention evaluated the effect of Bojeongbangdok-tang on breast cancer cells MCF-7 and MDA MB-231. As shown in FIG. 1B and FIG. 1C, Bojeongbangdok-tang showed weak cytotoxicity against both MCF-7 and MDA MB-231 cells by XTT analysis, but was more sensitive to MCF-10A cells. Cisplatin (80 μM) reduced cell viability by 53.98% and 41.39% in breast cancer cells MCF-7 and MDA MB-231 cells, respectively. On the other hand, contrary to the results of MCF-10A cells, Bojeongbangdoktang significantly increased cisplatin-induced cytotoxicity in breast cancer cells MCF-7 and MDA MB-231 cancer cells, which was cisplatin-induced cells against breast cancer cells. Shows the synergistic anti-tumor effect of Bojungbangdok-tang on toxicity.

Morphological Changes of MCF-10A Cells Exposed to Baodbangdangtang and / or Cisplatin As Shown in FIG. 1D to Determine the Protective Effect of Bodbangdangtang on Cisplatin-Induced Cytotoxicity in Human Normal Breast Epithelial Cells Was observed under inverted microscopy. Cells treated with cisplatin up to 80 μM exhibited dead cell morphology with swelling and apoptotic contraction compared to untreated controls. In contrast, cells treated with Bojeongbangdok-tang at concentrations up to 200 μg / ml were protected by this herb mixture and showed complete cell morphology. Induction, which demonstrated the protective effect of Cijungplatok-tang on cisplatin-induced cytotoxicity in normal MCF-10A cells.

Baodbangbang-tang inhibits apoptotic morphological changes in cisplatin-treated human normal breast epithelial cells MCF-10A cells

In addition, the present invention assessed whether Bajeongbangdok-tang protects cisplatin-induced apoptosis in human normal breast epithelial MCF-10A cells using DAPI staining assay. Cisplatin is known to induce apoptotic apoptosis in cancer cells. Consistent with these results, as shown in FIG. 2A, DAPI staining showed significant changes in Bojeongbangdaktang-treated cells, whereas apoptosis morphological features such as apoptotic bodies, cell contractions, and chromatin condensation (arrows) were seen in cisplatin-treated controls. Did not exist.

Flow cytometry TUNEL analysis showed the same results as nuclear nuclei staining. Cisplatin increased TUNEL-positive cell populations undergoing apoptosis, while Baodbangdangtang had no significant change (0.43%) compared to the control (0.10%). As expected, Bojeongbangdogtang significantly reduced the TUNEL-positive cell population of MCF-10A cells exposed to Bojeongbangdogtang and cisplatin (4.00%) (FIG. 2B). Quantitative graphs show the percentage of nucleated cells (FIG. 2C).

Bojungbangdang-tang reduces cisplatin-treated human normal breast epithelial cells MCF-10A cells in the sub-G1 apoptosis portion

Cell cycle analysis was performed to further confirm the protective effect of Bojeongbangdak-tang on apoptosis in cisplatin-treated human normal breast epithelial cells MCF-10A cells. DNA content after PI staining was analyzed by flow cytometry. As shown in FIG. 3A, the sub-G1 content of DNA representing the apoptotic portion was increased in cisplatin-treated cells (25.7%) compared to the untreated control (1.29%). In contrast, apoptotic sub-G1 was unchanged (1.31%) in Baodbangdogang-treated cells. Consistent with the TUNEL and DAPI staining data, Baodbangdangtang significantly reduced the sub-G1 apoptosis portion in cisplatin-treated MCF-10A cells.

However, while Bojungbangdog-tang did not affect cisplatin-induced apoptosis in MCF-7 breast cancer cells, cisplatin significantly increased the DNA content of sub-G1, which was cisplatin-induced apoptosis between normal and cancer cells. It shows the differential effect of Bojeongbangdak-tang on apoptosis.

The present invention also attempted Annexin / PI staining to measure early and late apoptosis in MCF-10A cells treated with Baodbangdang-tang and / or cisplatin. After staining cells with Annexin V-FITC and PI, Bajeongbangdoktang showed a protective effect only on late apoptosis, not early apoptosis induced by cisplatin (FIG. 3C).

Thus these results again show the protective effect of Baodbangdang-tang on cisplatin-induced apoptosis in MCF-10A cells.

Baodbangbang-tang dissociation of mitochondrial membrane potentials, activation of caspase-9 and -3 and cleavage of PARP cleavage in cisplatin-treated MCF-10A cells

Changes in mitochondrial membrane potential are involved in the execution of apoptosis. The present invention measured mitochondrial membrane potential in cisplatin-treated MCF-10A cells in the presence or absence of Baodbangdang-tang by flow cytometry after tetramethylhodamine (TMRE) staining. As shown in FIGS. 4A and B, cisplatin increased fluorescence intensity reflecting mitochondrial membrane potential to 86.00 ± 0.33% at 80 μM compared to untreated control (97.12 ± 2.40%). On the other hand, Baodbangbang-tang blocked the loss of cisplatin-induced mitochondrial membrane potential when MCF-10A cells were preincubated for 1 hour with Baodbangbangtang (200 μg / ml) and treated with cisplatin (80 μM). Treatment with poison (200 μg / ml) alone did not affect 1 h mitochondrial membrane potential in MCF-10A cells (97.49 ± 0.81%). More interestingly, Bojungbangdogang synergistically increased cisplatin-induced mitochondrial membrane potential loss in MCF-7 breast cancer cells (FIG. 4C), indicating that Bojungbangdogang can selectively affect normal and cancer cells. .

Consistent with the results of apoptosis assay, Western blotting data indicated that Baodbangdoktang protected cisplatin-induced PARP cleavage and activation of caspase-3 or -9 in a concentration dependent manner in MCF-10A cells, with cisplatin at 80 μM concentration Induces PARP cleavage and activation of caspase-3 or -9. In contrast, Bojeongbangdoktang did not show any significant changes in PARP cleavage and caspase-3 or -9 activation in MCF-10A cells.

Although chemotherapy is one of the most useful methods for the treatment of cancer, the cytotoxicity of chemotherapy can lead to side effects such as immunosuppression and myelosuppression, nausea and vomiting, hair loss, anemia and digestive disorders. Thus, damage to normal tissues by anti-tumor drugs may be a major limitation in the treatment of patient resistant cancer.

Recent studies have shown the anticancer efficacy of herbal medicines and compounds thereof to reduce side effects and improve the therapeutic effect of anti-angiogenic agents. In addition, synergistic anti-tumor effects have been reported by combined treatment with herbal medicines and anticancer drugs. For example, Hau, D.M. et al., Am. J. Chin. Med. 21 (1), 51-58, 1993 show that the combination of mitomycin C with herbal Shi-Hung-One (SHO) enhances the therapeutic effect in sarcoma 180 tumor cells, Cao, Y. et al., World J. Gastroenterol. 11 (33), 5218-5220, 2005 reported that the Chinese medicine Bushen huayu jiedu regimen (BSHYJDR) in combination with cisplatin could inhibit transplanted liver cancer in mice. Similarly, the combination chemotherapeutic agent of the Chinese herbal compound hydroxycamptothecin with oxiliplatin has been proposed for adjuvant treatment of human colon cancer.

Most of these studies focus on the synergistic effects of combination therapy in cancer cells by activation of apoptosis signaling. In the present invention, we demonstrated for the first time the protective effect of Korean herbivore Jungbangdeoktang against cisplatin-induced damage as well as apoptosis on human normal breast MCF-10A epithelial cells. The data of the present invention demonstrated that Bajeongbangdok-tang prevents cisplatin-induced cytotoxicity against MCF-10A cells by XTT assay. Direct observation by inverted microscopy also demonstrated that Baodbangdogang clearly blocks morphological changes, including cisplatin-induced swelling and apoptotic contraction. In contrast, Bojeongbangdok-tang synergistically increased cisplatin-induced cytotoxicity against MCF-7 and MDA-231 breast cancer cells, suggesting possible combination treatment of cisplatin and bojeongbangdoktang. Moreover, by DAPI and TUNEL analysis, Bojeongbangdok-tang reduced apoptotic morphological changes such as apoptotic bodies and nuclear fission in cisplatin-treated MCF-10A cells. Cell cycle analysis also showed that the cisplatin-induced sub-G1 apoptosis portion was significantly reduced by Baodbangdangtang, indicating the protective effect of Baodbangdangtang from cisplatin-induced apoptosis in MCF-10A cells.

Mitochondria play a pivotal role in the apoptosis signaling pathway. During apoptotic apoptosis, cytochrome c is generally released from the mitochondria into the cytoplasm after cell exposure to apoptotic stimuli. Release of cytochrome c prior to the loss of mitochondrial membrane potential is essential for energy (ATP) production and cell homeostasis preservation. Mitochondrial-dependent apoptosis was exerted by cisplatin. The decrease in mitochondrial membrane potential was also related to US ginseng-induced apoptosis of SW-480 colon cancer cells, and the Korean traditional herb, Euonymus alatus (Thunb.) Sieb, is a mitochondria as an oxidant in human leiomyoma smooth muscle cells. Apoptosis was induced through the pathway.

Cisplatin in the present invention induced mitochondrial membrane potential loss at 80 μM compared to untreated control in MCF-10A cells, indicating that mitochondrial membrane potential change is associated with cisplatin-induced apoptosis in MCF-10A cells. As expected, treatment of Bajeongbangdok-tang alone restored cisplatin-induced mycochondrial membrane potential loss without altering mitochondrial membrane potential in MCF-10A cells.

The loss of mycochondrial membrane potential in the apoptosis pathway is explained by two different mechanisms. One is that cytochrome c release precedes the mitochondrial membrane potential loss, and the other is that cytochrome c release follows mitochondrial expansion, outer membrane rupture and mitochondrial membrane potential loss. Mitochondrial regulation in apoptosis is also controlled by bcl-2 family proteins such as bcl-2, bcl-xL, bax and bak. Therefore, further studies are needed to determine the regulatory mechanisms responsible for the loss of mitochondrial membrane potential in cisplatin-treated MCF-10A cells in the presence or absence of Baodbangdogang.

Various cellular stimuli cause caspase activation belonging to the cysteine protease family. Caspase-3 and -9 are important components in the mitochondrial initiation pathway. When caspase is activated, various cellular proteins are targeted and eventually lead to apoptosis. Poly (ADP-ribose) polymerase (PARP) is the most well known substrate of caspase and is divided into 85 kDa fragments in a complete form of 116 kDa.

Correspondingly, the data of the present invention showed that cisplatin, but not Baodbangdogang, showed the activation of caspase-3 and -9 in MCF-10A cells. Moreover, the combination of cisplatin and bojeongbangdoktang blocked the activation of caspase-3 and -9 as well as PARP cleavage in cisplatin-treated MCF-10A cells.

Previously, the present inventors have indicated that Bojeongbangdok-tang is a potent angiogenesis inhibitor that blocks vascular endothelial growth factor (VEGF) and its receptor signaling pathway in human umbilical vein endothelial cells at non-toxic concentrations, and Bojungbangdaktang is anti-angiogenic. It has been reported that the activity can exert anti-tumor activity against cancer cells.

In the present invention, the present inventors found that Bojeongbangdok-tang enhances the anti-tumor effect of cisplatin on breast cancer cells MCF-7 and MDA MB-231. In conclusion, these findings indicate that the herbal medicinal product, Jeongbangbangdoktang, can prevent cisplatin-induced cytotoxicity and apoptosis in human normal breast epithelial MCF-10A cells.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. no. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

Suitable dosages of the pharmaceutical compositions of the invention can be determined in a variety of ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. It may be prescribed. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.01-1000 mg / kg body weight per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. It is preferable that the route of administration is determined according to the type of the disease to which the pharmaceutical composition of the present invention is applied.

The concentrations of cisplatin and bojeongbangdogtang, the active ingredients included in the composition of the present invention, are determined in consideration of the purpose of treatment, the condition of the patient, the period of time, the degree of disease, and the like, and are not limited to a specific range of concentrations.

The pharmaceutical compositions of the present invention may be formulated in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. It can be made or prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Example 1 Preparation of Baoding Gasoktang

Bojeongbangdogang is composed of 12 g of Astragalus membranaceous Bunge, 9 g of Panax gineseng CA Mey., 9 g of Poria cocos Wolf., 9 g of Dolichos lablab Linne, 9 g of Atractylodes japonica Koidzumi. It consists of eight herbs including g, Polygonatum sibiricum Delar, 9 g, 12 g of Coiz lacrymajobi Linne and 12 g of hemp ( Dioscorea batatas Decaisne) (Table 1).

Herbal ingredients were identified by Professor Paik Nam-in, Department of Pharmacy, Kyung Hee University and deposited in the Herb Container of Cancer Prevention Materials Research Center with the certificate name. Bojungbangdogang was placed in 3 volumes of 95% ethanol for 2 days at room temperature and concentrated by rotary evaporator. The concentrated extract was lyophilized by freeze dryer (EYELA, Japan) and powder extract (yield = 3.73%) was used for in vitro and in vivo experiments. The HPLC pattern of Bojeongbangdok-tang was confirmed for quality control.

Example 2 Cell Culture

MCF-10A, MCF-7 and MDA MB-231 cells were purchased from the American Type Culture Collection (ATCC). MCF-10A cells are maintained in Dulbecco's modified Eagle's medium (DMEM) / F12 supplemented with 10% fetal calf serum, epidermal growth factor (EGF), insulin, hydrocortisone, cholera toxin, 2 μιη L-glutamine and penicillin / streptomycin It became. MCF-7 and MDA MB-231 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 μιη L-glutamine and penicillin / streptomycin.

Example 3 Drug Treatment

Cells were treated with Baodbangdogtang and / or cisplatin for 24 hours. In the case of the combination treatment, the cells were pretreated for 1 hour with Bajeongbangdogtang and then treated with cisplatin for 24 hours.

Example 4 Cytotoxicity Assay

Cytotoxicity of Bojungbangdok-tang and / or cisplatin is described in Jost, LM et al., J. Immunol. It was evaluated by XTT analysis as described in Methods 147 (2), 153-165, 1992. Cells were dispensed on 96-well microplates at a density of X 10 ⁴ cells per well in 100 μl RPMI 1640 medium. After 24 hours of incubation at 37 ° C. in a humidified incubator, cells were exposed to various concentrations of Baodbangdogang (0, 25, 50, 100 or 200 μM) and / or cisplatin (80 μM) in serum-free medium. After incubation, 50 μl of XTT (1 mg / ml in phosphate buffered saline) mixture containing phenazine methosulfate (PMS) (1.53 mg / ml in PBS) was added to the cells at a ratio of 100: 1. Cells were incubated at 37 ° C. for 2 hours and optical density was measured at 450 nm using a microplate reader (Molecular Devices Co.). Cell viability was calculated as the ratio of viable cells in the drug-treated group to the untreated control group by the following equation:% cell viability = [OD (isorhamnetin)-OD (blank) / OD (control)- OD] X 100.

Example 5 DAPI Staining

Nucleus staining was performed using 4 ', 6-diimidino-2-phenylindole (DAPI). Briefly, cells were fixed at 4 ° C. for 25 minutes using 4% methanol-free formaldehyde and encapsulated in DAPI (VECTOR Lab, VECTASHIELD Hard Set Mounting Medium). Stained cells were visualized under Axio vision 4.0 fluorescence microscopy (Carl Zeiss Inc.) at 460 nm.

Example 6 TUNEL Analysis

Individual apoptotic apoptosis was observed using the DeadEnd ^™ Fluorescence TUNEL Assay Kit by manufacturer's protocol. MCF-10A cells treated for 24 hours with 200 μg / ml Baodbangdogang and / or 80 μM cisplatin were incubated and washed with cold PBS. Cells were dispensed on poly-L-lysine-coated slides, fixed for 15 minutes with 4% paraformaldehyde and washed twice with PBS for 3 minutes. The cells permeated by soaking the slides in 0.2% Triton X-100 for 5 minutes were allowed to equilibrate for 5 minutes at room temperature with 100 μl of equilibration buffer after washing with PBS. After removal of the equilibration buffer, cells were added with 50 μl of TdT enzyme buffer containing fluorescent-12-dUTP and covered with plastic coverslips, and slides were incubated at 37 ° C. for 1 hour. After washing with PBS, cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson).

Example 7 Cell Cycle Analysis

To determine apoptosis Hibasami, H. et al., J. Mol. Med. Cell cycle analysis was performed using propidium iodide (PI) staining as described in 15 (5), 805-809, 2005. After cell treatment with 200 μg / ml Baodbangdogang and / or 80 μM cisplatin, cells were harvested, washed with cold PBS and fixed in 75% ethanol at −20 ° C. After washing twice with cold PBS, the fixed cells were resuspended in 100 μl PBS containing 10 μl of RNase A (10 mg / ml) at room temperature under dark conditions for 30 minutes. The cells were then stained by adding 400 μl of PI (50 μg / ml) at room temperature under dark conditions for 30 minutes. FITC Annexin V (BD Pharmingen) and PI (BD Pharmingen) staining were also performed to distinguish apoptosis or necrotic cells. DNA content of the stained cells was analyzed using CellQuest software with FACSVantage (Becton Dickinson) flow cytometer.

Example 8 Mitochondrial Membrane Potential Measurement

Cells (1 × 10 ⁶ cells) were treated with 200 μg / ml Bodogbangtang and / or 80 μM cisplatin for 24 hours, washed with cooled PBS and 150 nM of fluorescence potential-dependent indicator tetramethylpodamine ethyl ester ( TMRE) was stained by adding at 37 ° C. for 30 minutes. Mitochondrial membrane potential (MMP) was then detected by flow cytometry (FACS Vantage SE, Becton Dickinson) at 582 nm.

Example 9 Western Blot Analysis

MCF-10A (5 × 10 ⁵ cells) treated with Bajungbangdangtang and / or cisplatin was harvested and washed with cold PBS. Cell pellets were prepared on 50 μl of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na ₃ VO ₄ , 1 mM NaF, Protease inhibitor cocktail) for 20 minutes. Lysates were centrifuged at 14,000 xg for 20 minutes at 4 ° C. Protein content in the supernatant was then measured using the Bio-Rad DC Protein Assay Kit II. Lysates containing 20 μg of protein were mixed with 4 × NuPAGE SDS sample buffer and boiled for 5 minutes. Proteins were separated on 4-12% NuPAGE Bis-Tris gels for 90 min at 300 mA with 1X NuPAGE MES SDS electrophoresis buffer and then electrophoresed on Hybond ECL transfer membrane with transfer buffer (25 mM Tris, 250 mM glycine, 20% methanol). Moved. The membrane was blocked with 5% skim milk powder in TBS buffer (TBST) containing 0.1% Tween 20 for 90 minutes at room temperature. Membranes were rabbit anti-mouse split caspase-9 (1: 1000 dilution), rabbit anti-mouse split caspase-3 (1: 1000 dilution) and rabbit anti-mouse PARP in TBST containing 5% skim milk powder Immunoblot overnight at 4 ° C. with primary antibody (1: 1000 dilution). After three consecutive washes with TBST for 10 min, the membrane was either goat anti-mouse IgG HRP conjugated secondary antibody (1: 1000 dilution) or goat anti-rabbit IgG HRP conjugated secondary in TBST containing 3% skim milk powder. Incubated with antibody (1: 1000) for 90 minutes at room temperature. After three consecutive washes with TBST for 10 min, the protein was developed using ECL Westerton Blotting Detection Kit (Amersham Pharmacia Biotech) and exposed to X-ray film (Agfa, Japan). Protein content was normalized by probe of the same membrane with anti-β-actin antibody. Membranes previously used for β-actin detection are soaked at 65 ° C. for 30 minutes in stripping buffer (62.5 mM Tris-HCl, pH 6.8, 150 mM NaCl, 2% SDS, 100 mM β-mercaptoethanol) Hybridized to anti-β-actin (1: 20,000 dilution). Primary antibodies of cleaved caspase-9, cleaved caspase-3 and PARP were purchased from Cell Signaling. β-actin antibody was purchased from Sigma.

Statistical analysis

All data are expressed as mean ± SD. Statistically significant differences between Bojeongbangdok-tang and the control group were calculated by the Student test.

1 shows that Bojeongbangdok-tang protects cisplatin-induced cytotoxicity against MCF-10A breast epithelial cells but increases cisplatin-induced cytotoxicity against MCF-7 and MDA MB-231 breast cancer cells. MCF-10A (A), MCF-7 (B) and MDA MB-231 (C) cells were 24 hours with 0, 25, 50, 100 or 200 μg / ml of Baodbangdogang and / or cisplatin (80 μM). Was processed. Cell viability was measured by XTT assay. Data represent mean ± SD. Asterisks indicate statistical significance by Student t-test ( ^* p <0.05, ^** p <0.01, ^*** p <0.001). In FIG. 1 (D) morphological changes of MCF-10A cells exposed to Baodbangdogang (0, 50, 100 or 200 μg / ml) and / or cisplatin were observed under inverted microscope (200 ×).

Figure 2 shows that Bajeongbangdok-tang reduces cisplatin-induced apoptosis in MCF-10A cells. MCF-10A cells were exposed to cisplatin (80 μM) and / or Baodbangdogang (200 μg / ml) for 24 hours. DAPI staining assay (A) and TUNEL assay (B) using flow cytometry were performed. Arrows indicate apoptotic bodies (A). In FIG. 1 (C), the graph shows the percentage of cell nuclei in MCF-10A cells.

Figure 3 shows that Bojeongbangdok-tang reduces the sub-G1 apoptosis portion in cisplatin-treated MCF-10A cells by PI staining (A and B). MCF-10A (A) and MCF-7 (B) cells were treated with cisplatin (40 or 80 μM) and / or Baodbangdogang (200 μg / ml) for 24 hours. After being fixed in 75% ethanol the cells were stained with PI and the sub-G1 DNA content was analyzed by flow cytometry. 3 (C) shows Annexin V / PI double staining; MCF-10A cells were treated with Baodbangdogang and / or cisplatin for 24 hours and stained with FITC Annexin V and PI solution for 15 minutes at room temperature. Apoptosis and necrotic cells were measured by flow cytometry.

Figure 4 shows that Bajeongbangdok-tang cleaves mitochondrial membrane potential loss, caspase-9 and -3 activation and PARP cleavage in cisplatin-treated MCF-10A cells (A-C). Mitochondrial membrane potentials: MCF-10A (A-graph, B- histogram) and MCF-7. In FIG. 4C, cells were treated with cisplatin (40 or 80 μM) and / or Baodbangdogang (50, 100 or 200 μg / ml) for 24 hours. Cells were stained with tetramethyltamine ethyl ester (TMRE) at 37 ° C. for 30 minutes and mitochondrial membrane potential (MMP) was detected by flow cytometry. 4 (D) shows western blotting: Cell lysates were measured for expression of PARP, cleaved caspase-3, -9 and β-actin.

Claims (4)

Anticancer composition for the treatment of breast cancer containing cisplatin and bojeongbangdoktang as active ingredients which lowers cytotoxicity and apoptosis for normal breast epithelial cells and increases cytotoxicity and apoptosis for breast cancer cells According to claim 1, wherein the normal breast epithelial cells are MCF-10A cell line, breast cancer cells are anti-cancer composition for the treatment of breast cancer, characterized in that the MCF-7 or MDA MB-231 cell line [Claim 2] The anticancer composition for treating breast cancer according to claim 1, wherein an effective concentration for selectively increasing cytotoxicity and apoptosis in the in vitro breast cancer cell line of the anticancer agent composition is cisplatin 80 µM and Bojungbangdaktang 100-200 µg / ml. The method of claim 1, wherein the anticancer composition reduces cisplatin-induced apoptotic bodies in normal breast epithelial cells and apoptotic protein sub-in cisplatin-treated human normal breast epithelial cells. Breast cancer treatment characterized by decreasing G1 DNA content and blocking the activation of caspase-3 and -9 and poly (ADP-ribose) polymerase (PARP) cleavage to selectively increase cytotoxicity and apoptosis in breast cancer cells Anticancer composition for

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