CN111329848A - Application of spinosad - Google Patents

Abstract

The invention relates to application of spinosad. The research of the invention shows that the selosporin can obviously inhibit the proliferation and migration of tongue cancer cells, so that the cell cycle is blocked in the G2/M stage, and meanwhile, the change of mitochondrial membrane potential can cause apoptosis. Sporotidine induces mitochondrial membrane potential change and BAX expression increase, and induces apoptosis through a mitochondrial apoptosis pathway, thereby inhibiting tongue cancer cell growth.

Description

Use of capillin

technical field

The present invention belongs to the field of biomedicine, and more particularly, relates to a new application of capillin.

Background technique

Oral cancer is one of the common malignant tumors of the head and neck, and its incidence is increasing year by year worldwide. Among them, tongue cancer is the most typical and common type of oral cancer. 90% of oral cancers are squamous cell carcinomas, and tongue squamous cell carcinomas are as high as 50% to 60%, ranking first. Studies have shown that there are approximately 10,990 new cases of tongue squamous cell carcinoma every year worldwide. Various treatment methods such as surgery, chemotherapy, and radiotherapy have been used to treat tongue squamous cell carcinoma. Although the level of medical technology has made great progress, the prognosis of patients with tongue squamous cell carcinoma is still not ideal, and the 5-year survival rate is only About 50%, local or regional recurrence and cervical lymph node metastasis are still great challenges for clinical treatment.

Bostrycin is a natural pigment product with an anthraquinone skeleton, first discovered by Noda T in 1968, and has shown great advantages in many aspects such as antibacterial and antitumor. Most of the research on bostrycin is found in marine endophytes, but no research has been done on bostrycin isolated from S. wormwood. The endophytic fungi of HCH285 were obtained by isolating the authentic Chinese medicinal material Qi Ai, which belonged to Nigrospora oryzae. The secondary metabolite was red pigment, and the metabolite pigment was extracted. Monomer isolation of the main active substance yields bostrycin. At present, the inhibitory effect of bostrycin on tongue squamous cell carcinoma has not been reported, and the mechanism of inhibition of tongue cancer cells is still unclear.

SUMMARY OF THE INVENTION

The inventors of the present invention have found that caprine has a good inhibitory effect on tongue squamous cell cancer cells SCC9 and SCC25, can effectively inhibit the proliferation of SCC9 and SCC25 cells, and promote cell apoptosis, and explored the volume of Inhibitory effect and mechanism of neomycin on tongue cancer cells.

In order to achieve the above object, the first aspect of the present invention provides the application of caprine in the preparation of tongue cancer cell proliferation inhibitor and/or tongue cancer cell apoptosis promoter.

Further, the tongue cancer cells are tongue squamous cell cancer cells.

Further, the tongue squamous cell carcinoma cells are SCC9 cells and/or SCC25 cells.

According to the present invention, the apoptosis of the tongue cancer cells is via the caspase signaling pathway.

The second aspect of the present invention provides the use of caprines in the preparation of mitochondrial apoptosis inducers.

Further, caprine induces mitochondrial apoptosis by inducing changes in mitochondrial membrane potential.

The present invention studies the effect of caplixin on the growth of tongue cancer cells in vitro, and explores the specific mechanism of the effect of caplixin on tongue cancer cells. The results showed that capilisporin can significantly inhibit the proliferation and migration of tongue cancer cells, arrest the cell cycle in the G2/M phase, and at the same time lead to changes in mitochondrial membrane potential and then apoptosis. Caprine inhibits the growth of tongue cancer cells by inducing mitochondrial apoptotic pathway by inducing changes in mitochondrial membrane potential and increasing BAX expression.

Other features and advantages of the present invention will be described in detail in the detailed description that follows.

Description of drawings

The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of the exemplary embodiments of the present invention in conjunction with the accompanying drawings.

Figure 1 shows the results of Bostrycin inhibiting the proliferation of tongue squamous cell carcinoma cells. Figure 1A: MTT assay showed that Bostrycin significantly inhibited the cell viability of SCC9 cells at a concentration of ≥4 μg/mL. Figure 1B: MTT assay showed that Bostrycin significantly inhibited the cell viability of SCC25 cells at concentrations ≥4 μg/mL. Figure 1C: Colony formation assay showed that Bostrycin significantly inhibited cell proliferation of SCC9 cells at concentrations ≥ 4 [mu]g/mL. Figure 1D: Colony formation assays show that Bostrycin significantly inhibits cell proliferation of SCC25 cells at concentrations ≥ 2 μg/mL. *P<0.05, **P<0.01, ***P<0.001 (vs. DMSO).

Figure 2 shows the results of Bostrycin-induced G2/M arrest in tongue squamous cell carcinoma cells. Figure 2A: The effect of Bostrycin on the cycle of tongue cancer cells SCC9 detected by flow cytometry. Figure 2B: Statistics show that Bostrycin can induce G2/M phase arrest in SCC9 cells at concentrations ≥4 μg/mL. Figure 2C: The effect of Bostrycin on the cycle of tongue cancer cells SCC25 detected by flow cytometry. Figure 2D: Statistics show that Bostrycin can induce G2/M phase arrest in SCC25 cells at concentrations ≥8 μg/mL. *P<0.05, **P<0.01, ***P<0.001 (vs. DMSO).

Figure 3 shows the results of Bostrycin promoting apoptosis of tongue squamous cell carcinoma cells. Figure 3A: The effect of Bostrycin on the apoptosis of tongue cancer cell SCC9 was detected by flow cytometry after AnnexinV-AlexaFlour 488/PI double staining. Figure 3B: Statistics show that Bostrycin can significantly increase the apoptosis of tongue cancer cells SCC9 when the concentration is ≥ 4 μg/mL. Figure 3C: The effect of Bostrycin on the apoptosis of tongue cancer cell SCC25 was detected by flow cytometry after AnnexinV-Alexa Flour 488/PI double staining. Figure 3D: Statistics show that Bostrycin can significantly increase the apoptosis of tongue cancer cells SCC25 when the concentration is ≥8 μg/mL. *P<0.05, **P<0.01, ***P<0.001 (vs. DMSO).

Figure 4 shows the results of Bostrycin promoting apoptosis of tongue squamous cell carcinoma cells. Figure 4A: AO/EB staining fluorescence image of SCC9 cells treated with Bostrycin for 24 hours; Figure 4B: AO/EB staining fluorescence image of SCC9 cells treated with Bostrycin for 48 h; Figure 4C: AO/EB staining fluorescence image of SCC25 cells treated with Bostrycin for 24 h; Figure 4D : AO/EB staining fluorescence image of SCC25 cells treated with Bostrycin for 48h; Figure 4E: AO/EB staining flow cytometry results of Bostrycin treatment of SCC9 cells for 24h; Figure 4F: AO/EB staining flow cytometry after Bostrycin treatment of SCC9 cells for 24h Statistics of apoptosis cells detected by cytometry; Figure 4G: AO/EB staining flow cytometry detection results of SCC9 cells treated with Bostrycin for 48 hours; Figure 4H: Apoptotic cells detected by AO/EB staining flow cytometry after Bostrycin treatment of SCC9 cells for 48 hours Statistics; Figure 4I: AO/EB staining flow cytometry detection results of SCC25 cells treated with Bostrycin for 24 hours; Figure 4J: AO/EB staining flow cytometry detection of apoptotic cells after Bostrycin treatment of SCC25 cells for 24 hours; Figure 4K: Bostrycin Figure 4L: AO/EB staining flow cytometry detection results of apoptotic cells after 48h treatment of SCC25 cells with Bostrycin. *P<0.05, **P<0.01, ***P<0.001 (vs. DMSO).

Figure 5 shows the results of Bostrycin inhibiting migration of tongue squamous cell carcinoma cells. Figure 5A: Scratch test to detect the effect of Bostrycin on the migration of SCC9 cells; Figure 5B: Scratch test to detect the effect of Bostrycin on the migration of SCC25 cells; Figure 5C: Statistical chart of the scratch test to detect the effect of Bostrycin on the migration of SCC9 cells; Figure 5D: Scratch Statistical chart of the effect of Bostrycin on the migration of SCC25 cells detected by the trace test. *P<0.05, **P<0.01, ***P<0.001 (vs. DMSO).

Figure 6 shows the results that Bostrycin increases mitochondrial apoptosis in tongue squamous cell carcinoma cells. Figure 6A: The effect of Bostrycin on mitochondrial apoptosis in SCC9 cells; Figure 6B: Statistics on the mitochondrial apoptosis rate of SCC9 cells by Bostrycin; Figure 6C: The effect of Bostrycin on mitochondrial apoptosis in SCC25 cells; Figure 6D: The rate of mitochondrial apoptosis in SCC25 cells by Bostrycin statistics. *P<0.05, **P<0.01, ***P<0.001 (vs. positive control CCCP).

Figure 7 shows the results of Bostrycin reducing the expression of proliferation-related proteins in tongue squamous cell carcinoma cells. Figure 7A: Western Blot assay detects SCC9 cell-related protein expression; Figure 7B: Western Blot assay detects SCC25 cell-related protein expression; Figure 7C: SCC9 cell AKT protein expression statistics; Figure 7D: SCC25 cells AKT protein expression statistics; Figure 7E: SCC9 Figure 7F: Statistics of ERK protein expression in SCC25 cells; Figure 7G: Statistics of BAX protein expression in SCC9 cells; Figure 7H: Statistics of BAX protein expression in SCC25 cells; Figure 7I: Statistics of PARP protein expression in SCC9 cells; Figure 7J: SCC25 Cellular PARP protein expression statistics. *P<0.05, **P<0.01, ***P<0.001 (vs. DMSO).

Detailed ways

Preferred embodiments of the present invention will be described in more detail below. While preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

(1) Culture of tongue cancer cell lines

Two tongue cancer cell lines, SCC9 and SCC25, were purchased from ATCC (USA). Tongue squamous cell carcinoma cell lines SCC9 cells and SCC25 cells were cultured in DME/F12 medium supplemented with 400 μg/L hydrocortisone, 60 mg/L sodium pyruvate, and 1.2 g/L NaHCO ₃ . The medium was supplemented with 10% fetal bovine serum (BBI) and cultured in a 5% CO ₂ incubator.

(2) Preparation of cyclosporine

The endophytic fungal colony of HCH285 is round, with white fluffy hairs and fluffy growth in the early stage of growth. In the later stage, the mycelium gradually turns red, and red metabolites can be clearly seen on the back of the colony. The compound was isolated, and the monomer substance was optimized and isolated, and the structure analysis of nuclear magnetic resonance was used to determine that it was bostrycin.

Take 32 mg of triclosporin monomer and add 1 mL of DMSO to dissolve it as a storage solution, take 100 μL of the storage solution and add 900 μL of sterile ddH ₂ O and mix to obtain 3.2 mg/mL of triclosporin monomer solution, followed by sterile water. Gradient dilution to 1.6mg/mL, 0.8mg/mL, 0.4mg/mL, 0.2mg/mL, 0.1mg/mL and 0.05mg/mL, all were added to the cell culture medium according to 1%, the final concentration was 32μg/mL, respectively mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, and 0.5 μg/mL.

(3) Statistical analysis

In the following examples, all values are expressed as mean ± standard error. To compare the differences between the two groups of data, a two-tailed t-test followed by analysis of variance (ANOVA) was used. P values < 0.05 were considered significant differences.

Example 1 Cell Proliferation Experiment

MTT experiment: SCC9 cells and SCC25 cells were seeded in 96-well plates at 5,000 cells/well, and after 12 h, different concentrations of convolvosporin were added, and 20 μL of 5 mg/mL MTT solution was added to each well after 24 h, 48 h, and 72 h of treatment, respectively. 4h, discard the supernatant, add 150 μL of DMSO to each well to dissolve the formazan, and measure the absorbance at 490 nm with a microplate reader after the formazan is completely dissolved.

Colony formation experiment: cells were seeded in a 35mm petri dish at 1000 cells/dish, and after 12 hours, conchlosporin was added, and the medium was changed after 24 hours of treatment. % crystal violet staining for 1 h. After washing 3 times with PBS, take pictures and count with Image J software.

Results: Caprine significantly inhibited the proliferation of tongue cancer cells SCC9 and SCC25.

As shown in Figures 1A-1B. Among them, Fig. 1A is for SCC9 cells, and Fig. 1B is for SCC25 cells. SCC9 cells and SCC25 cells were treated with different concentrations of caprine for 24h, 48h and 72h, respectively. The results showed that different treatment time had little effect, and different concentrations of caplixin had a great effect on tongue cancer cells. When the concentration was 4-8 μg/mL, the viability of SCC9 cells was significantly reduced. The IC50 at 24h was 5.37μg/mL. The viability of SCC25 cells was significantly reduced when the concentration of convolvosporin was 2-4 μg/mL, and the IC50 was 3.50 μg/mL when SCC25 cells were treated with convolvosporin for 24 h.

As shown in Figures 1C-1D. Among them, Figure 1C is for SCC9 cells, and Figure 1D is for SCC25 cells. The results of the colony formation experiment showed that convolvosporin significantly inhibited the proliferation of SCC9 cells when the concentration was greater than or equal to 4 μg/mL, the inhibition rate of SCC9 cell clone formation was 45.8% at 4 μg/mL, and the clone formation of SCC9 cells was inhibited by 8 μg/mL at 8 μg/mL. The formation inhibition rate was 66.9%; conchlosporin significantly inhibited the proliferation of SCC25 cells at 2 μg/mL or more, and the inhibition rate of SCC25 clone formation at 2 μg/mL was 43.7%, and at 4 μg/mL SCC25 cells The colony formation inhibition rate was 73.3%, and the colony formation inhibition rate for SCC25 cells was 95.0% at 8 μg/mL.

Example 2 Cell cycle experiments

SCC9 cells and SCC25 cells were inoculated in a 35mm dish, cultured to 70% confluency, added with cyclosporine, cultured for 24 h, collected cells, fixed overnight in pre-cooled 70% ethanol at -20 °C, collected cells, washed once with PBS, added 80 μL propidium iodide (PI) at a concentration of 100 μg/mL, 0.25% Triton-100, and RNase at a final concentration of 20 μg/mL were incubated at 4°C for 30 min in the dark, and detected by flow cytometry.

Results: Caprine increased G2/M arrest in tongue cancer cells SCC9 and SCC25.

As shown in Figures 2A-2D, wherein Figures 2A and 2B are for SCC9 cells, and Figures 2C and 2D are for SCC25 cells. After 24h of caprolactin treatment of tongue cancer cells, at 2 μg/mL, caprine at 2 μg/mL increased the sub-G1 phase of SCC9 cells by 2.0%, decreased G0/G1 phase by 13.2%, S phase decreased by 4.6%, G2/M 1.9% increase in phase; 6.5% increase in sub-G1 phase of SCC9 cells at 4 μg/mL, 13.3% decrease in G0/G1 phase, 5.8% decrease in S phase, and 3.4% increase in G2/M phase; SCC9 at 8 μg/mL Cells in sub-G1 phase increased by 7.3%, G0/G1 phase decreased by 12.9%, S phase decreased by 10.0%, and G2/M phase increased by 8.8%. At 2 μg/mL, caprine increased 2.4% in sub-G1 phase of SCC25 cells, decreased by 16.7% in G0/G1 phase, increased by 3.5% in S phase, and increased by 2.7% in G2/M phase; at 4 μg/mL, SCC25 The sub-G1 phase of cells increased by 10.1%, the G0/G1 phase decreased by 16.0%, the S phase decreased by 0.3%, and the G2/M phase increased by 4.4%; at 8 μg/mL, the sub-G1 phase of SCC25 cells increased by 8.3%, G0/G1 17.2% decrease in S phase, 2.8% decrease in S phase, and 11.7% increase in G2/M phase. The results showed that the sub-G1 phase increased with the increase of concentration after 24 hours of caprolactin treatment of tongue cancer cells, indicating that caprolidin could increase the apoptosis of tongue cancer cells; the increase of G2/M phase indicated that the sub-G1 phase increased. Sporine inhibits cell proliferation by blocking the G2/M phase of cells.

Example 3 Experiment of apoptosis detection by double staining of cell apoptosis kit

SCC9 cells and SCC25 cells were seeded in 24-well plates, cultured to 70% confluency, added with cyclosporine, and after culturing for 24 h, the cells were collected and stained with Annexin V-Alexa Fluor 488/PI apoptosis kit (Yeasen Biotech, Shanghai, China). china), and apoptosis was detected by flow cytometry.

In the AO/EB experiment, SCC9 cells and SCC25 cells were respectively inoculated into 12-well plates, and cultured overnight to 70% confluence, followed by the addition of triclosporin, and cultured for 24h and 48h, respectively. Discard the culture medium, take 2 mg/mL AO and EB, take 1 μL AO and 1 μL EB respectively, add 1 mL PBS to dilute, mix well, prepare for use now, add 100 μL staining working solution to each well, and place at room temperature for 2-5 min to take photos with a fluorescence microscope.

SCC9 cells and SCC25 cells were respectively inoculated in 12-well plates, cultured to 70% confluence overnight, and conchrysporin was added, and cultured for 24h and 48h, respectively. Collect the cells, take 2 mg/mL AO and EB, take 1 μL AO and 1 μL EB respectively, add 1 mL PBS to dilute and mix well, prepare for the current use, add 100 μL staining working solution to each tube to suspend cells, and perform flow detection.

Results: Caprine significantly increased the apoptosis of tongue cancer cells SCC9 and SCC25.

As shown in Figures 3A-3D, wherein Figures 3A and 3B are directed to SCC9 cells, and Figures 3C and 3D are directed to SCC25 cells. The cells were treated with caplixin for 24 h, and the apoptosis of SCC9 and SCC25 cells was detected by double staining with PI and Annexin V-Alexa Fluor 488. Apoptosis of SCC9 cells increased 10.5% at 4 μg/mL and 38.2% to SCC9 cells at 8 μg/mL; convolvosporin increased apoptosis of SCC25 cells by 0.8% at 2 μg/mL, and at 4 μg/mL 3.1% increase in apoptosis of SCC25 cells at 8 μg/mL and 24.6% increase in apoptosis of SCC25 cells at 8 μg/mL. Capilisporin significantly increased both early and late apoptosis of SCC9 and SCC25 cells.

As shown in Figures 4A-4L, Figures 4A-4B and 4E-4H are directed to SCC9 cells, and Figures 4C-4D and 4I-4L are directed to SCC25 cells. The cells were treated with capilisporin for 24h and 48h, respectively, and their apoptosis was detected by AO/EB staining. The fluorescence was observed under a microscope. When the cells were treated with 2μg/mL of caprinesporin, a small amount of red fluorescence appeared, and at 4μg/mL, red fluorescence appeared. Fluorescence increased, and a large amount of red fluorescence appeared at 8 μg/mL. It showed that the apoptosis rate also increased gradually with the increase of the concentration of spinosporin. The results of AO/EB staining detected by flow cytometry showed that the red fluorescence shifted to the right, indicating that the red fluorescence increased with the increase of the concentration of convolvin. When SCC9 cells were treated with caplixin for 24h, the concentration of 2μg/mL of caprinesporin increased the apoptosis of SCC9 cells by 14.93%, and the concentration of 4μg/mL of caprinesporin increased the apoptosis of SCC9 cells by 23.4% , the concentration of 8 μg/mL conchlosporin increased the apoptosis of SCC9 cells by 71.8%, of which 4 μg/mL and 8 μg/mL conchlosporin treated SCC9 cells for 24h compared with the control group. Significant difference; volume When the SCC9 cells were treated with cyclosporine for 48h, the concentration of 2 μg/mL of cyclosporine increased the apoptosis of SCC9 cells by 5.30%, and the concentration of 4 μg/mL of cyclosporine increased the apoptosis of SCC9 cells by 38.10%. The concentration of 8 μg/mL conchrysporin increased the apoptosis of SCC9 cells by 48.23%, of which 4 μg/mL and 8 μg/mL conchlosporin treated SCC9 cells for 48h compared with the control group, there was a significant difference; convoluted line When SCC25 cells were treated with sporin for 24 h, the concentration of 2 μg/mL conchrysporin increased the apoptosis of SCC25 cells by 16.77%, and the concentration of 4 μg/mL was increased by 33.07% on the apoptosis of SCC25 cells. The apoptosis of SCC25 cells was increased by 37.57% at 8 μg/mL convolvosporin, and the SCC25 cells treated with 8 μg/mL convolvosporins for 24 h had a significant difference compared with the control group; At 48 h, the apoptosis of SCC25 cells was increased by 3.73% with a concentration of 2 μg/mL of convolvosporin, and by 26.03% with a concentration of 4 μg/mL, and by 26.03% with a concentration of 8 μg/mL The apoptosis of SCC25 cells was increased by 56.17% with cyclosporine, among which 4 μg/mL and 8 μg/mL of cyclosporine treated SCC25 cells for 48h had significant difference compared with the control group.

Example 4 Scratch test

SCC9 cells and SCC25 cells were inoculated in a 24-well plate. After 12 hours, when the cell density exceeded 90%, two vertical lines were drawn with a yellow pipette tip, and the medium was replaced with 1% serum medium. 12h to take pictures at the same location. Image J counts the area of the trace area and calculates the statistical migration rate.

Results: Caprine inhibited the migration of tongue cancer cells SCC9 and SCC25.

As shown in Figures 5A-5D, wherein Figures 5A and 5C are directed to SCC9 cells, and Figures 5B and 5D are directed to SCC25 cells. Scratch test results showed that capillin could significantly inhibit the migration of tongue squamous cell carcinoma cells SCC9 and SCC25. At 12 h, the migration rate of convolvosporin at a concentration of 2 μg/mL decreased by 10.8% on SCC9 cells and by 47.1% on SCC25 cells, and the concentration of convolvosporin at a concentration of 4 μg/mL on SCC9 The migration rate of the cells was reduced by 96.5%, the migration rate of the SCC25 cells was reduced by 77.1%, and the concentration of 8 μg/mL convolvosporin was reduced by 92.2% for the SCC9 cells and 92.2% for the SCC25 cells. 90.6%; At 24h, the migration rate of Conchrysporin at a concentration of 2 μg/mL decreased by 32.3% for SCC9 cells and 60.4% for SCC25 cells, and the concentration of Conchrysporin at a concentration of 4 μg/mL decreased by 32.3% The migration rate of SCC9 cells was reduced by 108.1%, and the migration rate of SCC25 cells was reduced by 92.3%. The rate is reduced by 96.6%. At low concentrations, 2 μg/mL effectively reduced the migration rate of tongue cancer cells, and SCC25 cells were more sensitive. With the increase of the concentration, the effect of SCC9 cells is more than that of SCC25 cells, which may be related to the fact that the IC50 of SCC25 cells is lower than that of SCC9 cells.

Example 5 JC-1 mitochondrial apoptosis detection

SCC9 cells and SCC25 cells were inoculated in a 24-well plate, and after 12 h, when the cell density was about 70%, different concentrations of cyclosporine were added for treatment for 24 h, and positive control CCCP was added for treatment for 20 min. Cells were collected, stained with JC-1, and incubated in a cell incubator at 37°C for 20 minutes for flow cytometry detection.

Results: Trichosporine reduced mitochondrial membrane potential.

JC-1 assay was used to detect the effect of Bostrycin on mitochondrial membrane potential of tongue cancer cells, using flow cytometry green fluorescence. The results are shown in Figures 6A-6D, wherein Figures 6A-6B are for SCC9 cells and Figures 6C-6D are for SCC25 cells. The mitochondrial apoptosis rate of SCC9 cells was increased by 0.75% with the concentration of 2 μg/mL convolvosporin, and 7.8% with the concentration of 4 μg/mL, and 7.8% with the concentration of 8 μg/mL. The mitochondrial apoptotic rate of SCC9 cells was increased by 20.9% with conchlosporin; the mitochondrial apoptotic rate of SCC25 cells was increased by 4.1% with the concentration of 2 μg/mL The mitochondrial apoptosis rate of SCC25 cells was increased by 39.8%, and the mitochondrial apoptosis rate of SCC25 cells was increased by 14.6% with the concentration of 8 μg/mL convolvosporin. The results showed that mitochondrial apoptosis of SCC25 cells was more sensitive to cyclosporine, and the apoptosis rate was much higher than that of SCC9 cells when the concentration was 4 μg/mL, while when SCC25 cells were treated at 8 μg/mL, the detection value was caused by the death of most of the cells. low.

Example 6 Western Blot detection of proliferation and apoptosis-related protein expression

SCC9 cells and SCC25 cells were cultured in a 35mm dish to a confluence of 70%, and the cells were collected after 24 hours of culture with cyclosporine, and the total protein was extracted for cryopreservation. After polyacrylamide gel electrophoresis, transfer to NC membrane, cut the corresponding band, and incubate the corresponding primary antibody at 4°C overnight. The primary antibody was recovered, and the membrane was washed and incubated with the secondary antibody for 1 h before developing.

Results: Caprine increased apoptosis of tongue cancer cells SCC9 and SCC25 through caspase signaling pathway.

Western Blot was used to detect the expression of proliferation and apoptosis-related proteins. The results are shown in Figures 7A-7J. With the increase of Bostrycin concentration, the expression of p-AKT increased, and the expression of ERK gradually decreased, indicating that Bostrycin may be mediated by AKT The pathway inhibited cell proliferation and the expression of BAX increased, indicating that Bostrycin may promote apoptosis through mitochondrial apoptosis pathway.

In the present invention, monomer bostrycin is used as a material to study its inhibitory effect on tongue squamous cell carcinoma SCC9 and SCC25, and to explore its action mechanism. Studies have shown that bostrycin can effectively inhibit the proliferation of tongue squamous cell carcinoma. The results of MTT assay show that the IC50 of bostrycin is 5.37μg/ml when it acts on SCC9 cells for 24 hours, and the IC50 when it acts on SCC25 cells for 24 hours is 3.50μg/ml. It can be seen that bostrycin has a good in vitro anticancer activity on tongue squamous cell carcinoma cells, and has a good inhibitory effect on the proliferation of SCC25 cells. Cell cycle experiments showed that caprines inhibited the proliferation of tongue cancer cells by inducing the G2/M phase arrest of tongue cancer cells. The cycle detection also found that with the increase of caprine concentrations, the small peak of apoptosis in sub-G1 phase was significantly increased. increased, indicating that caprine may induce apoptosis in tongue cancer cells. The AnnexinV-Alexa Flour 488/PI double staining and AO/EB experiments showed that bostrycin cells can promote the apoptosis of tongue squamous cell carcinoma cells. Apoptosis increased by 10.5%, 38.2% for SCC9 cells at 8 μg/mL, 3.1% for SCC25 cells at 4 μg/mL, and 24.6% for SCC25 cells at 8 μg/mL. It can be seen that bostrycin can significantly increase the apoptosis of tongue cancer cells, and the increase in the apoptosis rate of SCC9 cells is higher than that of SCC25 cells. The results of AO/EB experiments showed that the proportion of red fluorescence increased with the increase of bostrycin concentration, and the increase of red fluorescence observed by fluorescence microscope was consistent with the results of flow cytometry. This indicated that with the increase of bostrycin concentration, the apoptosis rate of tongue squamous cell carcinoma cells gradually increased, and the apoptosis rate of SCC9 cells was higher than that of SCC25 cells, which was consistent with the conclusion of AnnexinV-Alexa Flour 488/PI double staining experiment.

In conclusion, bostrycin has a good inhibitory effect on tongue squamous cell carcinoma in vitro, the inhibitory effect on SCC25 cell proliferation is better than that of SCC9 cells, and the increase of SCC9 cell apoptosis is better than that of SCC25 cells. Western Blot detected the expression of proliferation and apoptosis-related proteins. The results showed that bostrycin could significantly increase the expression of p-AKT and decrease the expression of ERK protein, which may be a way for bostrycin to reduce the proliferation of tongue squamous cell carcinoma cells. In order to investigate whether bostrycin affects cell apoptosis through mitochondrial apoptosis pathway, the expression of BAX protein was detected. The results showed that BAX expression was increased, which indicated that bostrycin might affect cell apoptosis through mitochondrial apoptosis pathway.

Various embodiments of the present invention have been described above, and the foregoing descriptions are exemplary, not exhaustive, and not limiting of the disclosed embodiments. Numerous modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims (6)

1. Application of caprine in the preparation of tongue cancer cell proliferation inhibitor and/or tongue cancer cell apoptosis promoter. 2. The use according to claim 1, wherein the tongue cancer cells are tongue squamous cell cancer cells. 3. The use according to claim 2, wherein the tongue squamous cell carcinoma cells are SCC9 cells and/or SCC25 cells. 4. The use according to claim 1, wherein apoptosis of the tongue cancer cells is via the caspase signaling pathway. 5. Application of caprine in the preparation of mitochondrial apoptosis inducer. 6. The use according to claim 5, wherein caprine induces mitochondrial apoptosis by inducing changes in mitochondrial membrane potential.

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