Use of dihydroartemisinin as a drug for treating triple-negative breast cancer in combination with cisplatin

Abstract

The present invention discloses a use of dihydroartemisinin in manufacturing a drug for treating triple-negative breast cancer, wherein the use refers to administering dihydroartemisinin in combination with cisplatin to triple-negative breast cancer patients, combined with radiotherapy, to enhance the sensitivity of triple-negative breast cancer to radiation. The use improves the sensitivity of triple-negative breast cancer cells to radiation rays by combining dihydroartemisinin (DHA) and cisplatin (DDP), thereby improving the efficacy of radiotherapy and reducing the side effects of radiotherapy.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to use of dihydroartemisinin as a medicament for treating triple-negative breast cancer in combination with cisplatin. Background Art

[0002] Triple negative breast cancer (TNBC) is a type of breast cancer with a very poor prognosis in clinical practice, and is prone to brain metastasis, bone metastasis, etc. Triple negative breast cancer refers to breast cancer in which the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor are all negative. It is a special type of breast cancer. The 5-year survival rate of triple negative breast cancer stage II is around 50%-60%. Triple negative breast cancer stage II means that the tumor is >2cm and is combined with lymph node metastasis. In this case, triple negative breast cancer requires postoperative adjuvant chemotherapy. Of course, the first step is radical surgical resection, followed by local radiotherapy and systemic chemotherapy. Chemotherapy requires 4-6 cycles, and finally an observation strategy can be adopted.

[0003] At present, the treatment of triple-negative breast cancer mainly includes surgical treatment, chemotherapy, radiotherapy, endocrine therapy, targeted therapy, etc.

[0004] Surgical treatment: For early-stage triple-negative breast cancer, surgical treatment is the main treatment method. The commonly used procedure is modified radical mastectomy. After surgery, radiotherapy, chemotherapy and other treatments are also needed according to the patient's condition.

[0005] Chemotherapy is the use of chemotherapeutic drugs to kill cancer cells and is currently one of the most effective means of treating cancer. Chemotherapy drugs can act on the DNA of cancer cells, causing DNA damage and cancer cell death, thereby achieving the effect of treatment. Commonly used chemotherapy drugs include cisplatin, carboplatin, paclitaxel, docetaxel, etc.

[0006] Radiotherapy is the use of high-energy rays to kill cancer cells. It can be divided into radical radiotherapy, palliative radiotherapy, preoperative radiotherapy, and postoperative radiotherapy.

[0007] Endocrine therapy refers to the use of drugs or hormones to suppress ovarian function, thereby regulating the hormone levels in the patient's body and reducing the recurrence rate of breast cancer after surgery. Commonly used drugs include tamoxifen citrate tablets, toremifene citrate tablets, letrozole tablets, etc.

[0008] Targeted therapy refers to the use of targeted drugs to selectively inhibit the growth and spread of tumor cells, thereby achieving the purpose of treatment. Commonly used targeted drugs include trastuzumab, pertuzumab, lapatinib, etc.

[0009] Chemotherapy and radiotherapy are still the main means of treating triple-negative breast cancer. However, chemotherapy can still produce drug resistance, and radiotherapy can kill normal cells, damaging normal tissue cells while killing cancer cells. How to increase the sensitivity of tumor tissue to radiation and reduce the radiation dose to reduce damage to normal tissue has always been a problem that people have been exploring.

[0010] Cancer cells' resistance to radiotherapy often leads to radiotherapy failure, affecting the treatment effect. In addition, the breast is close to important organs such as the heart and lungs. Simply increasing the radiation dose to improve the local control rate will lead to serious side effects such as cardiac toxicity and radiation pneumonitis.

[0011] At present, cisplatin (DDP) is used as a first-line chemotherapy drug in clinical practice in combination with radiotherapy to improve the effect of radiotherapy, but it has strong hepatotoxicity, renal toxicity and ototoxicity, and has not shown the highest therapeutic potential. In this context, it is urgent to develop new treatment methods with high efficiency and low toxicity. The combination of traditional Chinese medicine and first-line chemotherapy drugs for the treatment of tumors is a current research hotspot. The combination of drugs can not only reduce the dose of traditional chemotherapy drugs, reduce toxic and side effects, but also improve the efficacy of radiotherapy.

[0012] Artemisinin is a sesquiterpene lactone compound with a peroxide bridge extracted and separated from the leaves of Artemisia annua L., a plant of the Asteraceae family, by Chinese pharmacists. Its chemical structure has been improved and modified, and a variety of derivatives such as artesunate, artemether, arteether and dihydroartemisinin (DHA) have been developed. DHA has the characteristics of good absorption, rapid metabolism and excretion, anticancer activity, and obvious tumor selectivity, and extremely low toxicity to normal cells. At present, DHA is used in basic and clinical research as an anticancer drug and therapeutic sensitizer, but there is no report on the radiosensitization effect of DHA combined with cisplatin on malignant tumors. Summary of the invention

[0013] The purpose of the present invention is to provide a use of dihydroartemisinin in the manufacture of a drug for treating triple-negative breast cancer, wherein the use refers to the combined use of dihydroartemisinin and cisplatin for triple-negative breast cancer patients.

[0014] The purpose of the present invention is to enhance the radiotherapy treatment effect of triple-negative breast cancer cells. By using dihydroartemisinin (DHA) and cisplatin (DDP) in combination, the sensitivity of triple-negative breast cancer cells to radiation rays is increased, thereby improving the efficacy of radiotherapy and / or reducing the side effects of radiotherapy.

[0015] The invention discloses a use of dihydroartemisinin in the manufacture of a drug for treating triple-negative breast cancer, wherein the use refers to the combined use of dihydroartemisinin and cisplatin for administration to patients with triple-negative breast cancer.

[0016] The above-mentioned use of the present invention, the said combined medication, comprises the simultaneous administration of dihydroartemisinin and cisplatin, or the administration at intervals in no particular order.

[0017] The above-mentioned use of the present invention, the combined use of dihydroartemisinin and cisplatin can be used as a radiotherapy sensitizer for triple-negative breast cancer.

[0018] The use of the present invention comprises the following steps: using a flow cytometer to detect the effects of dihydroartemisinin (DHA), cisplatin (DDP) and cimetidine sodium (CNMa) on the cell cycle and apoptosis of BT549 cells when used alone or in combination; using a cell scratch healing experiment and a Transwell experiment to detect the effects of DHA, DDP and CNMa on the migration and invasion ability of BT549 cells when used alone or in combination; and detecting the content of reactive oxygen species (ROS) in BT549 cells when DHA, DDP and CNMa are used alone or in combination.

[0019] Therefore, the application of dihydroartemisinin combined with cisplatin can be used as a radiosensitizer to improve the radiosensitivity of triple-negative breast cancer BT549 cells, thereby improving the radiotherapy efficacy, and / or reducing the radiation dose to reduce the killing effect on normal cells, that is, reducing the side effects of radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison diagram of the experimental effect of the combined use of DHA and DDP on BT549 cell apoptosis in Example 1;

[0021] Figure 2 This is a comparison chart of the experimental effects of the combined use of DHA and DDP on the cell cycle arrest of BT549 cells in Example 2;

[0022] Figure 3 This is a comparative diagram of the effect of the combined use of DHA and DDP on the content of reactive oxygen species (ROS) in BT549 cells in Example 3;

[0023] Figure 4 This is a comparison chart of the movement and migration ability of each group of cells in the BT549 cell scratch healing experiment using DHA and DDP in Example 4;

[0024] Figure 5 This is a comparison chart of the inhibitory effects of the combined use of DHA and DDP on the invasion ability of BT549 cells in each group in the Transwell experiment in Example 5;

[0025] Figure 6 This is a comparison chart of the experimental effects of different ratios of DHA and DDP combined on BT549 cell cycle arrest in Example 6;

[0026] Figure 7This is a comparison diagram of the apoptosis effects of different ratios of DHA and DDP combined on BT549 cells in Example 6;

[0027] Figure 8 This is a comparison chart of the effects of different ratios of DHA and DDP combined in Example 6 on BT549 cells in a 24-hour scratch test;

[0028] Fig. 9 This is a comparison chart of the inhibitory effects of different ratios of DHA and DDP combined in Example 6 on the invasion ability of BT549 cells in a Transwell experiment;

[0029] Fig.10 This is a comparison chart of the effects of different ratios of DHA and DDP combined in Example 6 on the content of reactive oxygen species (ROS) in BT549 cells. DETAILED DESCRIPTION

[0030] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of the present invention in any way.

[0031] The experimental materials of the following examples are:

[0032] Triple-negative breast cancer BT549 cells were purchased from Wuhan Pronocell Life Science Co., Ltd. and routinely cultured in the laboratory of Chongqing Medical University under the conditions of 37°C, 5% CO2, 95% humidity, and Pronocell BT549 cell-specific culture medium (RPMI-1640+10% FBS+10ug / ml Insulin+1% P / S).

[0033] Basal culture medium RPMI-1640 (Gibco), fetal bovine serum (Biosharp), PBS (Biosharp), 0.1% crystal violet stain (Biosharp), 4% paraformaldehyde (Biosharp), 0.25% Trypsin-EDTA (Gibco), 0.25% Trypsin-EDTA Free (Biosharp), Matrigel 5ml (Corning, USA). DHA (10μM in DMSO) and cisplatin were purchased from Shanghai McLean, and sodium glycidyl bisoprolol for injection was purchased from Shanghai Fudan Fuhua. Six-well plates, 24-well plates, and 96-well plates were purchased from Anhui Zhenxuan. Cell apoptosis kit, cell cycle kit, and ROS detection kit were purchased from Dalian Meilun. CCK-8 kit was purchased from Shanghai Puyu. Transwell 8μm chamber was purchased from Corning, USA.

[0034] The radiation source was Varian linear accelerator 6Mv 6Gy X-ray, the dose rate was 4Gy / min, and the source surface distance was 100cm.

[0035] Example 1 Cell apoptosis experiment

[0036] Flow cytometry was used to detect cell apoptosis

[0037] The triple-negative breast cancer BT549 cells in the logarithmic growth phase were inoculated in a six-well plate. When the cell growth density reached 80%, a total radiation dose of 40 Gy of X-rays was used (2 Gy / day, 20 consecutive days, a dose rate of 4 Gy / min, and a source surface distance of 100 cm). After the last irradiation, the cells were cultured for 24 hours, and the drugs DHA, DDP, CNMa, DHA+DDP, DHA+CNMa and blank control were added to the groups and treated with X-rays (2 Gy / day, a dose rate of 4 Gy / min, and a source surface distance of 100 cm) for 24 hours. The cells were digested with EDTA-free trypsin (Biosharp) and centrifuged, washed and centrifuged twice with PBS, and the cells were resuspended in 1X Bingding buffer to adjust the cell concentration to 1×10 5 / mL, add 5μl Annexin-V FITC and 10μl PI, incubate at room temperature in the dark for 15 min, add 400μl 1X Binding buffer, and load on flow cytometer within 1 hour.

[0038] Apoptosis is a programmed death regulated by genes. Changes in the expression of important targets of the apoptosis pathway in tumor cells may be a mechanism of primary or acquired drug resistance in tumor cells. Tumor treatment targeting the induction of apoptosis is a major research hotspot.

[0039] The experimental results are as follows Figure 1 As shown, the results showed that DHA induced cell apoptosis slightly better than DDP, CNMa and blank control group. In combination therapy, DHA+DDP induced apoptosis significantly increased, and DHA+CNMa was higher than single drug, but inferior to DHA+DDP.

[0040] Example 2 Cell cycle experiment

[0041] Cell cycle analysis by flow cytometry

[0042] Cells in the logarithmic growth phase were inoculated into six-well plates and cultured for 24 hours. After the cells adhered to the wall, they were treated with DHA, DDP, CNMa, DHA+DDP, DHA+CNMa and blank control for 24 hours. The cells were digested with trypsin and collected by centrifugation, washed with PBS and collected by centrifugation twice, and fixed with 75% ethanol at 4°C overnight. Washed with pre-cooled PBS, added with staining working solution (500μl Buffer, 25μl 20XPI, 10μl 50X RNase A), incubated at 37°C in the dark for 30 minutes, stored at 4°C in the dark, and loaded on the flow cytometer within 24 hours.

[0043] At present, it is generally recognized in international radiation oncology that cells in the G1 and G2 phases of the cell cycle are sensitive to radiation, while cells in the S phase are insensitive to radiation because they can perform DNA double-strand repair in response to radiation stress, that is, cell damage is relatively small. Therefore, measuring the cell cycle can determine the sensitivity of cells to radiation.

[0044] The experimental results are as follows Figure 2 As shown,

[0045] The proportion of cells in each cycle in each drug experimental group is:

[0046] DHA DDP CNMa Dip G1: 54.69% Dip G1: 57.13% Dip G1: 43.62% Dip S: 32.95% Dip S: 30.32% Dip S: 24.60% Dip G2: 12.36% Dip G2: 12.55% Dip G2: 31.78%

[0047] DHA+DDP DHA+CNMa Control group Dip G1: 83.69% Dip G1: 65.37% Dip G1: 43.3% Dip S: 14.95% Dip S: 2.05% Dip S: 52.95% Dip G2: 1.36% Dip G2: 32.58% Dip G2: 3.75%

[0048] The results showed that compared with single drugs, DHA and DDP were significantly better than CNMa in improving the radiation sensitivity of BT549 cells, DDP was slightly higher than DHA, and there was no significant difference between CNMa and the blank control group. In combination therapy, DHA+DDP was far better than DHA+CNMa, and far better than single drugs.

[0049] Example 3 Cellular Reactive Oxygen Species Experiment

[0050] Flow cytometry was used to detect cellular reactive oxygen species

[0051] Cells in the logarithmic growth phase were inoculated into six-well plates. After adherence, they were divided into groups and treated with DHA, DDP, CNMa, DHA+DDP, DHA+CNMa and blank control, and treated with X-rays. When the cell confluence was 50%-70%, 1 ml of DCFH-DA (10 μM) probe was added to each well for loading, and the cells were incubated at 37°C in the dark for 30 min, washed three times with basal medium, incubated for 2 h with drug induction, and the cells were collected by trypsin digestion and centrifugation and loaded on the flow cytometer within 1 h.

[0052] ROS is the initiating factor of cell radiation damage. Radiation acts on intracellular biomacromolecules such as proteins or DNA, as well as water molecules, generating free radicals or ROS, leading to lipid peroxidation or damage to membrane protein structure, and oxidative stress causing cell damage.

[0053] The results are as follows Figure 3 As shown in the figure, the results showed that ROS were generated inside the cells of each group 2 hours after drug and radiation treatment. The peak of the combined drug treatment shifted to the right compared with the single drug treatment, and the peak of the control group shifted significantly to the right; DHA+DDP was slightly better than DHA+CNMa.

[0054] Example 4 Cell healing experiment

[0055] Cells in the logarithmic growth phase were inoculated into six-well plates with a cell confluence of 100%. The cells were scratched vertically with a 200 μl pipette tip, washed with PBS, cultured with basal medium, added with drugs, treated with X-rays, and photographed at regular intervals.

[0056] Cells migrate after being stimulated by both intracellular and extracellular stimuli, and participate in tumor deterioration and metastasis. Cell migration is an important means of evaluating the efficacy of tumor treatment.

[0057] The experimental results are as follows Figure 4 As shown, the results showed that there was no significant difference in the 24H healing migration area among the three single-drug treatment groups, the healing migration area of ​​the three single-drug treatment groups was smaller than that of the control group, and the healing migration area of ​​the DHA+DDP combination group was much smaller than that of the single-drug group and the control group, and smaller than that of the DHA+CNMa group.

[0058] Example 5 Transwell experiment

[0059] Cells in logarithmic growth phase were taken and starved for 24 hours with basal medium. 50,000 cells / well were inoculated in the matrix gel-coated chamber and cultured for 24 hours. DHA, DDP, CNMa, DHA+DDP, DHA+CNMa and blank control were added to the groups and treated with X-rays for 24 hours. The cells were fixed with 4% paraformaldehyde for 30 minutes, washed with PBS, stained with 0.1% crystal violet for 30 minutes, washed with PBS 3 times, and photographed with an upright microscope after drying.

[0060] Tumor cell invasion is the basis for the in situ breakthrough of the basement membrane, invasion of lymphatic vessels and blood vessels, and distant metastasis. The ability of tumor cells to secrete hydrolases to remove movement disorders was determined by using Transwell chamber polycarbonate membrane and matrigel to simulate the extracellular matrix in vivo.

[0061] The experimental results are as follows Figure 5 As shown, the results showed that DHA and DDP inhibited tumor invasion better than CNMa, but the difference between DHA and DDP was not statistically significant (P>0.05). The three single-drug treatment groups had better ability to inhibit tumor invasion than the control group. Combination therapy could further inhibit cell invasion, and the DHA+DDP group was the best.

[0062] Example 6 Effect of different ratios of DHA and DDP combined on radiosensitization

[0063] Referring to the methods of Experimental Examples 1-5, DHA and DDP were used in combination at different weights, such as 1:9, 2:8, 3:7, 4:6, and 5:5 (ug / mL), to perform cell cycle arrest experiments, cell apoptosis experiments, cell scratch (24H scratch) experiments, Transwell experiments, and reactive oxygen species experiments on BT549 cells. The results of the cell cycle experiments are shown in Tables 1 and Figure 6The results of cell apoptosis experiment, cell scratch (24H scratch) experiment, Transwell experiment and reactive oxygen species experiment are as follows Figure 7-10 shown.

[0064] Table 1

[0065]

[0066] The above experimental results show that the combined use of DHA and DDP in different ratios such as 1:9, 2:8, 3:7, 4:6, and 5:5 has a sensitizing effect on BT549 cell radiation, but the sensitizing effects of 3:7, 4:6, and 5:5 are better, and the sensitizing effect of 3:7 is the best.

Claims

1. Use of a dihydroartemisinin and cisplatin combination in the manufacture of a radiosensitizer for treating triple-negative breast cancer, wherein the weight ratio of the dihydroartemisinin and cisplatin is 3:7.

Images