Application of Butea superba in the preparation of drugs for treating squamous cell carcinoma of the skin

Abstract

This invention relates to the field of chemical pharmaceutical technology. This invention provides the application of buteaconin in the preparation of drugs for treating cutaneous squamous cell carcinoma. This invention also provides drugs for treating cutaneous squamous cell carcinoma containing buteaconin. The buteaconin of this invention can inhibit the growth of cSCC cells and reduce their migration ability; it can also inhibit the protein expression levels of key tumor proteins TWEAK, Fn14, and cIAP1.

Description

Technical Field

[0001] This invention relates to the field of chemical pharmaceutical technology, and in particular to the application of buteacon in the preparation of drugs for treating squamous cell carcinoma of the skin. Background Technology

[0002] Butein, also known as buteflower extract, is a polyphenolic compound isolated from traditional Chinese medicinal materials such as Dalbergia odorifera, lacquer tree, and the heartwood of Dalbergia odorifera. Studies have shown that butein has various effects, including anti-inflammatory, anti-tumor, antioxidant, and anti-angiogenic properties, and can play a role in the prevention and treatment of various diseases such as inflammatory diseases, tumors, cardiovascular diseases, and diabetes.

[0003] Cutaneous squamous cell carcinoma (cSCC) generally has a good prognosis, but approximately 5%-10% of cSCC cases progress to locally advanced or metastatic disease. Currently, the signaling pathways involved in the pathogenesis of cSCC include: epidermal growth factor receptor (EGFR) pathway, mitogen-activated protein kinase (MAPK) pathway, telomerase pathway, Notch pathway, PRB pathway, CDKA2N pathway, transforming growth factor-β (TGF-β) signaling pathway, PI3K / AKT / mTOR pathway, and nuclear factor-κB (NF-κB) signaling pathway. Dysregulated signaling pathways play a crucial role in the pathogenesis of cSCC, and in-depth research into the various pathways involved in cSCC pathogenesis will help identify new drug targets and develop effective treatments.

[0004] Surgery is the first-line treatment for cSCC, capable of removing the tumor and preventing metastasis. However, surgical resection is usually extensive, and some patients are not candidates for surgery. Radiotherapy and chemotherapy are also important adjuvant therapies for cSCC, controlling local lesions, preventing cancer spread, and reducing tumor size. They are suitable for patients who are not candidates for surgical resection, but radiotherapy and chemotherapy have significant side effects. Targeted therapy and immunotherapy for cSCC have been used in recent years. The EGFR inhibitor cetuximab is one of the systemic therapies that can be used to treat cSCC, but the efficacy of EGFR inhibitors is slightly lower than expected. Some targeted therapies inhibit signaling pathways that lead to cSCC progression, often disrupting skin homeostasis and causing side effects. Several PD-1 inhibitors have been used to treat cSCC; for example, cemiplimab has been shown to be effective in immunocompetent patients with advanced CSCC and metastatic disease. However, immunotherapy has high treatment requirements and is not suitable for immunosuppressed patients, such as those undergoing solid organ transplantation. Therefore, clinical practice needs new, less invasive, less side-effect-prone, and more applicable treatment options. Summary of the Invention

[0005] The purpose of this invention is to provide the application of buteacon in the preparation of drugs for treating cutaneous squamous cell carcinoma. Buteacon can inhibit the growth of cSCC cells and reduce the migration ability of cSCC cells; at the same time, it can also inhibit the protein expression levels of key tumor proteins TWEAK, Fn14, and cIAP1.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of buteacon in the preparation of drugs for treating squamous cell carcinoma of the skin.

[0008] As a preferred method, squamous cell carcinoma of the skin is treated by inhibiting the protein expression levels of TWEAK, Fn14, and cIAP1.

[0009] This invention provides a medicament for treating squamous cell carcinoma of the skin, the medicament comprising butyrine and a pharmaceutically acceptable carrier.

[0010] Preferably, the final concentration of the acetamiprid used is 10 mg / kg to 60 mg / kg.

[0011] Preferably, the dosage form of the drug includes tablets, granules, and capsules.

[0012] By adopting the above technical solution, the present invention has the following beneficial effects:

[0013] The technical solution described in this invention demonstrates that butea superba can serve as a potential traditional Chinese medicine monomer for treating cutaneous squamous cell carcinoma (cSCC). Experiments further show that butea superba treatment can reduce cutaneous squamous cell carcinoma tumor size, inhibit cSCC cell growth, and decrease cSCC cell migration ability. Simultaneously, the protein expression levels of key proteins TWEAK, Fn14, and cIAP1 in cutaneous squamous cell carcinoma tumor tissue were significantly reduced after butea superba treatment. Attached Figure Description

[0014] Figure 1 The following is a description of the body weight of mice in each group in Example 1 of this invention;

[0015] Figure 2 This is a diagram showing the tumor status of each treatment group in Example 1 of the present invention. Figure 2 In the diagram, A represents a photograph of 5 mice in the control group; B represents a photograph of 5 mice in the low-dose group; C represents a photograph of 5 mice in the medium-dose group; D represents a photograph of 5 mice in the high-dose group; E represents a photograph of the tumors of the mice after sacrifice; and F represents the tumor volume growth curve of the mice.

[0016] Figure 3 The image shows the immunofluorescence detection results of each treatment group in Example 3 of the present invention (scale bar is 200 μm);

[0017] Figure 4 The following are the Western Blot experimental results of each treatment group in Example 4 of the present invention;

[0018] Figure 5 Figure 5 shows the cell scratch test results for each treatment group in Example 5 of this invention. Figure 5 In Figure A, A represents cell migration images, and B represents cell migration rate statistics. The left column of Figure A, from top to bottom, shows cell scratch images containing 0 μM / ml, 10 μM / ml, 20 μM / ml, and 40 μM / ml of Butein taken at 0 hours. The right column, from top to bottom, shows cell scratch images containing 0 μM / ml, 10 μM / ml, 20 μM / ml, and 40 μM / ml of Butein taken at 36 hours. Detailed Implementation

[0019] This invention provides the application of buteacon in the preparation of drugs for treating squamous cell carcinoma of the skin.

[0020] In this invention, squamous cell carcinoma of the skin is treated by inhibiting the protein expression levels of TWEAK, Fn14, and cIAP1.

[0021] This invention provides a medicament for treating squamous cell carcinoma of the skin, the medicament comprising butyrine and a pharmaceutically acceptable carrier.

[0022] In this invention, the final concentration of the purpureus is preferably 10 mg / kg to 60 mg / kg, more preferably 20 mg / kg to 50 mg / kg, and even more preferably 40 mg / kg.

[0023] In this invention, the dosage form of the drug includes tablets, granules, and capsules.

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] Twenty female 8-10 week old specific pathogen-free (SPF) grade leukemia laboratory mice (BALB / c-nu) (purchased from Beijing Spaford Biotechnology Co., Ltd.) were selected. After one week of acclimatization culture, 100 μL of LA431 cells (purchased from Wuhan Pronosei Life Sciences Co., Ltd.) suspension (1×10⁻⁶) was added. 8 Inoculate the right axilla of nude mice with a dose of 10 ...

[0027] Ten days after inoculation, the successfully modeled nude mice were randomly divided into a control group, a low-dose Butein group, a medium-dose Butein group, and a high-dose Butein group, with 5 mice in each group. Each group received an intraperitoneal injection of 10 ml of Butein solution at different concentrations: 10 mg / kg for the low-dose group, 20 mg / kg for the medium-dose group, and 40 mg / kg for the high-dose group. The control group received an intraperitoneal injection of an equal volume of physiological saline, once daily for 14 days. (one)

[0029] Starting from the first day of injection, observe the general condition of each group of mice daily and weigh them. Record whether the mice have loose stools, bloody stools, etc., and the degree of such occurrences. Record any mouse deaths.

[0030] The mice did not exhibit loose stools, bloody stools, or die. Their weight is as follows: Figure 1 . (two)

[0032] Starting from the first day of injection, the longest diameter (L, mm) and shortest diameter (S, mm) of the tumor were measured daily using calipers, and the volume V (mm²) was calculated. 3 Then, on day 21 after injection, nude mice in each group were sacrificed by cervical dislocation. The tumor tissue of the mice was weighed and its size and weight were recorded. One part was fixed and embedded in 10% formaldehyde solution to make paraffin sections, and the other part was directly placed in cryovials and stored at -80°C.

[0033] V(mm 3 )=L×S2 ×π / 6

[0034] Table 1. Tumor tissue quality in each group of mice

[0035] serial number Control 10mg / kg 20mg / kg 40mg / kg 1 0.73g 0.56g 0.23g 0.13g 2 0.54g 0.87g 0.34g 0.17g 3 0.62g 0.6g 0.17g 0.24g 4 1.3g 0.45g 0.35g 0.09g 5 1.45g 0.36g 0.31g 0.24g

[0036] The results are as follows Figure 2 Compared to the control group in the first row, the tumor size in the other three experimental groups was significantly reduced. This preliminarily indicates that Butein has the ability to inhibit cSCC growth, and the inhibitory effect of the high-dose Butein group is significant.

[0037] Example 2

[0038] Immunofluorescence assay of TWEAK, Fn14, TRAF1, TRAF2, and ki67 protein expression levels:

[0039] Tumor tissue was paraffin-embedded, sectioned, dewaxed, and hydrated. Sections were placed in a citrate-buffered retrieval chamber and microwaved for antigen retrieval. After natural cooling, the sections were washed three times with PBS buffer, 5 min each time. The sections were then incubated in 3% hydrogen peroxide solution at room temperature in the dark for 25 min, followed by three washes with PBS, 5 min each time. A histochemistry circle was drawn around the section with a histochemistry pen, and 3% BSA was added evenly to cover the tissue within the histochemistry circle. The section was blocked at room temperature for 30 min. The blocking solution was discarded, and primary antibody was added to the section. The section was then incubated overnight at 4°C in a humidified chamber. The slides were washed three times with PBS, 5 min each time. After slightly drying the sections, secondary antibody was added to cover the tissue, and the section was incubated at room temperature for 50 min. The slides were washed three times with PBS, 5 min each time. DAB staining was performed, observed under a microscope, and the process was stopped at the appropriate time. The slides were rinsed thoroughly with tap water. Hematoxylin counterstaining was performed at room temperature for 30 seconds, followed by rinsing with tap water. The slides were rinsed with tap water until the blue color returned, for 15 min. After dehydration with graded ethanol, the slides were cleared with xylene. They were then mounted with neutral resin. The slides were scanned using an automated slide scanner, and the images were exported and analyzed using ImageJ software to identify positive areas. Immunohistochemical images were observed and photographed using an optical microscope.

[0040] The results are as follows Figure 3 As shown, the protein expression levels of key tumor proteins TWEAK, Fn14, and cIAP1 decreased with increasing Butein dosage, indicating that the expression levels of some tumor-related proteins were reduced in tumor tissues treated with Butein, and that Butein has the ability to inhibit tumor-related proteins.

[0041] Example 3

[0042] Western blot experiments were used to determine the protein expression levels of TWEAK, Fn14, and cIAP1.

[0043] Protein extraction: Tumor tissue was removed from -80℃ and placed in liquid nitrogen. The tissue tube was held with large forceps, and 0.02-0.025g (≤0.1g) of tissue was cut off with a sterile scalpel and weighed on weighing paper. The tissue was then placed into a 1.5mL centrifuge tube, labeled, and the remaining kidney tissue was returned to the tissue tube and placed in liquid nitrogen for subsequent cryopreservation. Calculate the required doses of lysis buffer (RIPA), protease inhibitor (PMSF), and phosphatase inhibitor; add the calculated RIPA, PMSF, and phosphatase inhibitor to an EP tube containing weighed tumor tissue, place it in an ultrasonic homogenizer, sonicate for 8 seconds each time, place on ice and let stand for 15 minutes, sonicate again, centrifuge at 12000 r / min for 10 minutes, place on ice, collect the supernatant into a new centrifuge tube and label it; determine the protein concentration of each group using a BCA kit (purchased from Yamei ZJ102L), and denature the protein by adding 5× Loading Buffer and placing it in a 95℃ protein heater for 15 minutes, then store at -20℃.

[0044] Electrophoresis: Prepare a 10% SDS-PAG gel, add the calculated protein groups to the SDS-PAG gel, and set the electrophoresis apparatus voltage to 80V-100V;

[0045] Electric rotation: constant voltage 100V, 50-90min;

[0046] Sealing: 5% skim milk powder, sealed at room temperature for 90 minutes;

[0047] Apply primary antibody: Add an appropriate volume of primary antibody and incubate overnight on a shaker at 4°C.

[0048] Secondary antibody application: On the second day, wash the membrane three times with TBST for 5 minutes each time, add an appropriate volume of secondary antibody, and incubate at room temperature for 60 minutes; then develop and preserve the developed bands using the ECL method.

[0049] V(RIPA) = Tissue weight (g) × 10000

[0050] V(PMSF) = V(phosphatase inhibitor) = V(RIPA) / 100

[0051] The results are as follows Figure 4 As shown, the protein expression levels of key tumor proteins TWEAK, Fn14, and cIAP1 decreased with increasing dose, indicating that the expression levels of some tumor-related proteins were reduced in tumor tissues treated with Butein, and Butein has the ability to inhibit tumor-related proteins.

[0052] Example 4

[0053] The effect of Butein on cell migration levels was detected by cell scratch assay.

[0054] Cells in the logarithmic growth phase were digested with 0.05% trypsin containing EDTA (Pronotin PB180219) at 37°C for 6 minutes to form a single-cell suspension, which was then seeded into 6-well culture plates at a density of 5 x 102 cells per well. 5 Cells were seeded at 6 wells per well, with the seeding principle being 100% confluence achieved overnight. The final total culture medium volume per well was 2 mL. Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The next day, Butein at concentrations of 10 μM / ml, 20 μM / ml, and 40 μM / ml were added to the 6-well plates. Using a 200 μL pipette tip, parallel or perpendicular to the marker lines on the back of the 6-well plate, the marker lines were scratched using the plate cap or ruler. The scratch lines on the back of the 6-well plate were then wiped away. The cells were photographed under a 10x microscope, ensuring the scratches were centered and perpendicular. The distance between the two ends of the cell scratches was observed; a larger distance indicated a stronger inhibitory effect of Butein on cancer cell migration.

[0055] The results are as follows Figure 5 As shown, compared with the control group in the first row, the Butein experimental group significantly reduced the migration ability of cSCC cells A431.

[0056] In summary, the technical solution of this invention demonstrates that butea superba can serve as a potential traditional Chinese medicine monomer for the treatment of cutaneous squamous cell carcinoma (cSCC). Experiments further show that butea superba treatment can reduce the size of cutaneous squamous cell carcinoma tumors, inhibit the growth of cSCC cells, and decrease the migration ability of cSCC cells. Simultaneously, the protein expression levels of key proteins TWEAK, Fn14, and cIAP1 in cutaneous squamous cell carcinoma tumor tissues treated with butea superba are significantly reduced.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of buteacon in the preparation of drugs for treating cutaneous squamous cell carcinoma.

2. The application according to claim 1, characterized in that, Treatment of squamous cell carcinoma of the skin by inhibiting the protein expression levels of TWEAK, Fn14, and cIAP1.

Images