阅读说明：本技术 乙酰紫草素在制备抗癌药物中的应用 (Application of acetylshikonin in preparation of anti-cancer drugs ) 是由 黄晓颖 杨乐和 赵承光 唐叶萌 周峰 郑丹丹 盛珏琦 王娴 姚丹 王良兴 于 2021-09-27 设计创作，主要内容包括：本发明公开了一种乙酰紫草素在制备抗癌药物中的应用,所述的抗癌药物包括活性组分和药用辅料；所述的活性组分包含乙酰紫草素；所述的抗癌药物用于治疗肺癌。本发明提供了乙酰紫草素的新抗癌用途,对肺癌尤其是小细胞肺癌具有良好的抗癌作用。(The invention discloses an application of acetylshikonin in preparing an anticancer medicament, wherein the anticancer medicament comprises an active component and a pharmaceutic adjuvant; the active component comprises acetylshikonin; the anticancer drug is used for treating lung cancer. The invention provides a new anticancer application of acetylshikonin, which has good anticancer effect on lung cancer, in particular small cell lung cancer.)

1. The application of the acetylshikonin in preparing the anticancer drug is characterized in that the anticancer drug comprises an active component and a pharmaceutic adjuvant;

the active component comprises acetylshikonin;

the anticancer drug is used for treating lung cancer.

2. The use of acetylshikonin in the preparation of an anticancer agent, as claimed in claim 1, wherein said anticancer agent is used for treating small cell lung cancer.

3. Use of acetylshikonin in the preparation of an anticancer drug according to claim 1, characterized in that said anticancer drug is used for the treatment of lung adenocarcinoma.

4. The use of acetylshikonin in the preparation of an anticancer drug as claimed in claim 1, wherein said anticancer drug is used for inhibiting proliferation of lung cancer cells, reducing invasion and metastasis of lung cancer cells, and inducing apoptosis of lung cancer cells.

5. The use of acetylshikonin in the preparation of an anticancer drug as claimed in claim 1, wherein said lung cancer cell is PC-9, H1975 or a549 cell.

6. The use of acetylshikonin in the preparation of an anticancer agent as claimed in claim 1, wherein said active ingredient is composed of acetylshikonin and other anticancer agents;

the other anticancer drugs are cisplatin, oxitinib or erlotinib.

7. The use of acetylshikonin in the preparation of an anticancer agent as claimed in claim 1, wherein said other anticancer agent is erlotinib;

the molar ratio of the acetylshikonin to the erlotinib is 5: 0.5 to 10.

8. The use of acetylshikonin in the preparation of an anticancer agent as claimed in claim 1, wherein said other anticancer agent is cisplatin;

the molar ratio of the acetylshikonin to the cisplatin is 1-2.5: 0.5 to 10.

9. The use of acetylshikonin in the preparation of an anticancer agent as claimed in claim 1, wherein said other anticancer agent is oxitinib;

the molar ratio of the acetylshikonin to the oxitinib is 0.5-1: 1 to 10.

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to application of acetylshikonin in preparation of an anti-cancer drug.

Background

Lung cancer is the most common cause of cancer-related death worldwide. 1800 million people are diagnosed with lung cancer each year, and 1600 million people die of the disease. The 5-year survival rate varies between 4% and 17% depending on stage and regional differences. In China, the incidence and the death rate of lung cancer are the first, and the death rate of lung cancer is far ahead, and reaches 71 ten thousands, accounting for 23.8 percent of the total number of cancer deaths. Non-Small Cell Lung Cancer (NSCLC) is the most common aggressive Lung Cancer, accounting for about 85% of Lung Cancer, with Lung adenocarcinoma being the most common subtype of NSCLC and the proportion thereof increasing year by year. Although the treatment of NSCLC has made a significant breakthrough in recent years, and the treatment has been developed from traditional surgical treatment, chemotherapy and radiotherapy to precise molecular targeted therapy and immunotherapy, the 5-year survival rate of most NSCLC patients is still lower than 16.1% due to metastasis when diagnosed, and has not been significantly improved in recent ten years.

The Lithospermum erythrorhizon is a traditional Chinese herbal medicine in China, and the Lithospermum erythrorhizon is sweet, salty and cold in nature, enters heart and liver meridians, and has the functions of cooling blood, activating blood, detoxifying and promoting eruption. Modern pharmacological research shows that the compound has the effects of resisting bacteria, diminishing inflammation, resisting tumors and the like, and the active ingredients of the compound are mainly distributed in roots. The radix Arnebiae contains naphthoquinone small molecule compounds such as shikonin, isovaleryl shikonin, acetylshikonin, beta-dimethylacryl alkannin, beta-dimethylacryl shikonin, etc. Acetylshikonin is a naphthoquinone known for its anti-inflammatory potential, however, its anti-cancer effect has not been fully studied.

Disclosure of Invention

The invention provides an application of acetylshikonin in preparing an anti-cancer drug, and provides more and better alternatives for treating cancers.

An application of acetylshikonin in preparing anticancer drugs, wherein the anticancer drugs comprise active components and pharmaceutic adjuvants;

the active component comprises acetylshikonin;

the anticancer drug is used for treating lung cancer.

Preferably, the anticancer drug is used for treating small cell lung cancer.

As a further preference, the anticancer drug is used for treating lung adenocarcinoma.

Test results show that the anticancer drug can be used for inhibiting the proliferation of lung cancer cells, reducing the invasion and metastasis of the lung cancer cells and inducing the apoptosis of the lung cancer cells. Preferably, the lung cancer cell is a PC-9, H1975 or A549 cell.

Preferably, the active component consists of acetylshikonin and other anticancer drugs;

the other anticancer drugs are cisplatin, oxitinib or erlotinib.

Preferably, the other anticancer drug is erlotinib;

the molar ratio of the acetylshikonin to the erlotinib is 5: 0.5 to 10. More preferably, the concentration of the acetylshikonin is 5 mu M, the concentration of the erlotinib is 0.5-10 mu M, and the combination of the acetylshikonin and the erlotinib can have more than moderate synergistic effect; as a further preference, the concentration of acetylshikonin is 5. mu.M and the concentration of erlotinib is 10. mu.M, when the combination of acetylshikonin and erlotinib has a highly synergistic effect.

Preferably, the other anticancer drug is cisplatin;

the molar ratio of the acetylshikonin to the cisplatin is 1-2.5: 0.5 to 10. More preferably, the concentration of the acetylshikonin is 1-2 mu M, the concentration of the cisplatin is 0.5-10 mu M, and the combination of the acetylshikonin and the cisplatin has at least high synergistic effect; preferably, the concentration of the acetylshikonin is 2.5 mu M, the concentration of the cisplatin is 0.5-5 mu M, and the combination of the acetylshikonin and the cisplatin has moderate synergistic effect.

Preferably, the other anticancer drug is oxitinib;

the molar ratio of the acetylshikonin to the oxitinib is 0.5-1: 1-10; more preferably, the concentration of the acetylshikonin is 0.5 mu M, the concentration of the cisplatin is 1-2.5 mu M, and the combination of the acetylshikonin and the oxitinib has moderate synergistic effect.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a new anticancer application of acetylshikonin, which has good anticancer effect on lung cancer, in particular small cell lung cancer;

(2) the invention further provides a combination of the acetylshikonin and other anticancer drugs, and the combination of the acetylshikonin and the other anticancer drugs can generate synergy, so that the anticancer effect is further improved.

Drawings

FIG. 1 shows the result of the inhibition of NSCLC cell proliferation by acetylshikonin in example 1 of the present invention;

FIG. 2 shows the effect of acetylshikonin on the invasion of NSCLC in example 2 of the present invention;

FIG. 3 is a graph showing the effect of acetylshikonin on apoptosis of NSCLC cells in example 3 of the present invention;

FIG. 4 shows the effect of acetylshikonin in combination with cisplatin, oxitinib and erlotinib in example 4 of the present invention.

Detailed Description

The invention is further described with reference to specific examples.

MTT method for detecting the survival rate and the death rate of the human non-small cell lung cancer cells. NSCLE cells (4.5 × 103 cells/well) were seeded onto 96-well plates and treated appropriately in different media. After 48H, MTT solution was added at a rate of 25. mu.L/well, and incubated in a CO2 incubator at a temperature of about 37 ℃ for 4 hours. Formalin crystals were dissolved in 150. mu.L of dimethyl sulfoxide and the Optical Density (OD) of the MTT solution was recorded using a microplate reader (490nm absorbance). Cell viability and IC50 values were calculated according to GraphPad Pro Prism 8.0.

Colony formation assay: human NSCLC cells (1000 cells/well) were seeded overnight in six-well plates at 37 ℃ under an atmosphere of 5% CO 2. Dimethyl sulfoxide (control), acetylshikonin (0.5 μ M,1 μ M,2 μ M or 2.5 μ M,5 μ M,10 μ M) was added to the cells for 7 days. The medium was replaced with fresh medium every 2 days to keep the cells growing for 7 days. Colonies were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed 3 times with PBS, and stained with crystal violet for 10 minutes. Each experiment was performed in triplicate for three independent experiments.

EDU experiment: EdU staining with BeyoClick to study cell proliferation^TMEdU cell proliferation kit containing Alexa Fluor 594 (Beyotime, china, cat. No. C00788L). Cells were first washed three times with PBS for five minutes each. Fresh medium was supplemented with a defined amount of EDU. CO of cells at 37 ℃₂The culture was carried out in an incubator for 1 hour. After incubation, the cells were washed again twice with PBS for 10min each. Cells were fixed with 4% paraformaldehyde for 30min at room temperature and then stained with DAPI for 10 min. After washing again with PBS, the cells were observed under an inverted microscope.

TRANSWELL experiment: studies were performed using a transwell filter (BD Biosciences, usa) to observe cell migration according to the manufacturer's instructions. Cell density of 1X 10³Add the upper chamber containing serum free medium. Medium containing 10% FBS was added to the lower chamber. After 48 hours of drug treatment, cells were fixed with 4% paraformaldehyde, and unpulverized cells were removed from the upper surface of the filter. Cells of the inferior surface were stained with 1% crystal violet for 15 minutes at room temperature in the dark. The number of migrated cells was recorded and counted under a light microscope. The images were taken under an inverted microscope.

Scratch test: mixing cells (5X 10)⁵Cells/well) were seeded onto 6-well plates and allowed to adhere overnight. At 80-9At 0% confluence, a "reference line" was drawn at the bottom of the plate using a sterile 10 μ L pipette tip. After three washes with Phosphate Buffered Saline (PBS), the cells were further incubated with inhibitors or controls (DMSO) in culture medium to check for cell migration without cell growth. Micrographs of cell migration across the reference line were taken with a digital camera in different areas after 0, 24 and 48 hours of treatment, respectively. Mobility was quantified by the cell migration distance moving from the reference line towards the center compared to the control group. All experiments were repeated 3 times.

Apoptosis experiments: h1975 cells were seeded into six-well culture plates and allowed to grow to 80% confluence in complete medium. Cells were then treated with dimethylsulfoxide (control), acetylshikonin (0.5 μ M,1 μ M,2 μ M) for 48 hours to assess the effect of these compounds on apoptosis. Cells were harvested, washed twice in ice-cold PBS and then resuspended in binding buffer according to the instructions of the apoptosis kit. The treated cells (as described above) were incubated simultaneously with fluorescein-labeled annexin V and PI. Annexin V binding buffer was then added to the mixture, and fluorescence was measured on a FACSCalibur (BD Biosciences, Baltimore, MD, USA). Data were analyzed using Flowjo software.

WB experiment: cancer cells were seeded at a density of 500000 cells/well in 6-well plates and then cultured overnight. Dimethyl sulfoxide (control), acetylshikonin (0.5,1,2 μ M or 2.5,5,10 μ M) was added to 6-well plates. Treated cells were washed with ice cold PBS and lysed with 1% benzenesulfonyl fluoride in RIPA lysis buffer (total protein extraction kit). After high speed centrifugation to obtain supernatant, the proteins were collected and quantified with an enhanced BCA protein assay kit. Subsequently, approximately 60. mu.g of protein was loaded onto 8%, 10% or 12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and separated. Then, they were transferred to PVDF membranes. The membrane was washed three times with TBST and blocked with 5% fresh skim milk for 1.5 hours at room temperature. The blot was incubated overnight with the specific primary antibody at 4 ℃. After washing with TBST, the membrane was incubated with the relevant secondary antibody for 60 minutes at room temperature. Immunoreactive bands were observed using the Omni ECL femtosecond photochemiluminescence kit (epiczyme, shanghai, china). Visualization of the bands was analyzed using ImageJ computer software.

Example 1

The MTT test, colony formation test and EDU test show that acetylshikonin has dose-dependent and time-dependent cytotoxicity and anti-proliferation effect on NSCLC cells. The effect of acetylshikonin on NSCLC cell growth was examined by EDU and colony formation experiments. EDU cell proliferation assays suggest that acetylshikonin may inhibit NSCLC cell proliferation dose-dependently; the results of the colony formation experiments show that acetylshikonin obviously inhibits the colony formation number of PC-9, A549 and H1975 cells (FIG. 1). These results indicate that acetylshikonin can inhibit NSCLC cell proliferation.

Example 2

This example further observes the effect of acetylshikonin on NSCLC invasion. In the TRANSWELL experiment, the invasive transfer capacity of PC-9, H1975 and A549 cells was greatly reduced by the addition of drugs (FIG. 2). The results of the scratch healing assay of PC-9 cells further support the role of acetylshikonin in metastasis. Furthermore, we further examined invasion-migration proteins suggesting that acetylshikonin can induce a decrease in the expression of the proteins SNAIL and MMP 2.

Example 3

This example further analyzed the effect of acetylshikonin on NSCLC apoptosis using flow cytometry. Annexin fluorescein isothiocyanate (Annexin V-FITC) is used as a marker of apoptosis, and Propidium Iodide (PI) is used as a marker of cell viability. The results show that the acetylshikonin can promote H1975 apoptosis (figure 3), and further detection of the apoptotic proteins suggests that the acetylshikonin can induce the expression increase of the apoptotic proteins Cl-PARP, BAX and Cl-Caspase3 and inhibit the expression of the anti-apoptotic protein Bcl-2.

Example 4

FIG. 4 is a graph showing the synergy between acetylshikonin and cisplatin, and between oxitinib and erlotinib, which can be judged according to the CI value, and the drug combination index of 5 μ M acetylshikonin and 10 μ M erlotinib combined in PC-9 cells is 0.493, and the synergy between acetylshikonin and cisplatin is high. 5 mu M acetylshikonin and 0.5 mu M erlotinib respectively at 1 mu M,2.5 mu M and 5 mu M, the drug combination indexes are respectively 0.617, 0.509, 0.637 and 0.719, and moderate synergy is shown. The acetylshikonin and cisplatin showed strong synergistic effect against a549 cells. Whereas acetylshikonin and cisplatin showed a high degree of synergy against H460 cells. Both acetylshikonin and oxitinib have moderate synergy against H1975 cells.

The joint index (CI) can judge the interaction of the two, and when the CI is more than or equal to 0.9 and less than or equal to 1.1, the two are shown as the superposition; when CI is more than or equal to 0.8 and less than 0.9, the two are indicated to have low degree of synergistic action; when CI is more than or equal to 0.6 and less than 0.8, the two are indicated to be moderate synergistic effect; when CI is more than or equal to 0.4 and less than 0.6, the two are expressed as high synergistic action; when CI is more than or equal to 0.2 and less than 0.4, the two are expressed as strong synergistic action.