CN111329848A

CN111329848A - Application of spinosad

Info

Publication number: CN111329848A
Application number: CN202010250655.XA
Authority: CN
Inventors: 张军林; 刘志国; 赖晓晶; 揭晶; 岳硕豪; 熊超; 刘娜
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2020-06-26

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

Application of spinosad

Technical Field

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

Background

Oral cancer is one of the common malignant tumors of the head and the neck, and the incidence rate of the oral cancer is on the increasing trend year by year in the global range. The tongue cancer is the most typical and common one of oral cancer, 90 percent of the oral cancer is squamous cell carcinoma, and the tongue squamous cell carcinoma is up to 50 to 60 percent and is the first place. There are studies showing an approximately new 10990 cases of tongue squamous cell carcinoma worldwide each year. While various therapeutic approaches such as surgery, chemotherapy, radiotherapy, etc. have been used to treat squamous cell carcinoma of the tongue, despite major advances in the state of the art, the prognosis for patients with squamous cell carcinoma of the tongue is still not ideal, with a 5-year survival rate of only about 50%, and local or regional recurrence and cervical lymph node metastasis remain significant challenges for clinical treatment.

Sporothrin (bostrycin) is a natural pigment product with an anthraquinone skeleton, and is firstly discovered by Noda T in 1968, and has great advantages in various aspects such as antibiosis, antitumor and the like. Most of bostrycin research has found extraction in marine endophyte, but bostrycin isolated from nigrospora sphaerica of wormwood has not been studied. The method comprises the steps of separating Chinese mugwort serving as a Chinese authentic crude drug by using endophyte to obtain HCH285 endophyte belonging to Nigrospora sp.nigrospora (Nigrospora oryzae), extracting a metabolite pigment of the HZ 285 endophyte serving as a haematochrome, and performing monomer separation on a main active substance to obtain the bostrycin. At present, the inhibition effect of bostrycin on tongue squamous cell carcinoma is not reported, and the action mechanism of inhibiting tongue carcinoma cells is not clear.

Disclosure of Invention

The inventor of the invention discovers that the spinosad has a good inhibition effect on tongue squamous cell carcinoma cells SCC9 and SCC25, can effectively inhibit the proliferation of SCC9 and SCC25 cells and promote apoptosis, and explores the inhibition effect and action mechanism of the spinosad on tongue carcinoma cells.

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

Further, the tongue cancer cell is a tongue squamous cell carcinoma cell.

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

According to the present invention, the tongue cancer cell apoptosis is via a caspase signaling pathway.

In a second aspect of the present invention, there is provided the use of spinosyns for the preparation of an inducer of mitochondrial apoptosis.

Further, spinosyns induce mitochondrial apoptosis by inducing mitochondrial membrane potential changes.

The invention researches the influence of the streptoverticillium in vitro on the growth of tongue cancer cells and researches the specific mechanism of the influence of the streptoverticillium on the tongue cancer cells. The results show that the colicin can obviously inhibit the proliferation and migration of tongue cancer cells, so that the cell cycle is blocked in the G2/M phase, and meanwhile, the colicin can cause mitochondrial membrane potential change to further cause apoptosis. Sporotrichin induces mitochondrial membrane potential change and BAX expression increase, and induces apoptosis through a mitochondrial apoptosis pathway, thereby inhibiting tongue cancer cell growth.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 shows the results of Bostrycin inhibition of tongue squamous cell carcinoma cell proliferation. FIG. 1A: MTT experiment shows that Bostrycin can obviously inhibit the cell activity of SCC9 cells when the concentration is more than or equal to 4 mu g/mL. FIG. 1B: MTT experiment shows that Bostrycin can obviously inhibit the cell activity of SCC25 cells when the concentration is more than or equal to 4 mu g/mL. FIG. 1C: the clone formation experiment shows that the Bostrycin can obviously inhibit the cell proliferation of SCC9 cells when the concentration is more than or equal to 4 mu g/mL. FIG. 1D: the clone formation experiment shows that the Bostrycin can obviously inhibit the cell proliferation of SCC25 cells when the concentration is more than or equal to 2 mu g/mL. P <0.05, P <0.01, P <0.001 (relative to DMSO).

FIG. 2 shows the results of Bostrycin induced block of squamous cell carcinoma of tongue G2/M. FIG. 2A: flow cytometry examined the effect of Bostrycin on the cycle of tongue cancer cell SCC 9. FIG. 2B: statistics show that Bostrycin can induce G2/M phase block of SCC9 cells at the concentration of more than or equal to 4 mu G/mL. FIG. 2C: flow cytometry examined the effect of Bostrycin on the cycle of tongue cancer cell SCC 25. FIG. 2D: statistics show that Bostrycin can induce G2/M phase block of SCC25 cells at the concentration of more than or equal to 8 mu G/mL. P <0.05, P <0.01, P <0.001 (relative to DMSO).

FIG. 3 shows the results of Bostrycin in promoting apoptosis of squamous cell carcinoma of the tongue. FIG. 3A: the effect of Bostrycin on the apoptosis of tongue cancer cell SCC9 was examined by a flow cytometer after annexin V-AlexaFlour488/PI double staining. FIG. 3B: statistics show that Bostrycin can obviously increase the apoptosis of tongue cancer cell SCC9 when the concentration is more than or equal to 4 mu g/mL. FIG. 3C: the influence of Bostrycin on the apoptosis of tongue cancer cell SCC25 was examined by a flow cytometer after double staining with AnnexinV-Alexa flow 488/PI. FIG. 3D: statistics show that Bostrycin can remarkably increase the apoptosis of tongue cancer cell SCC25 when the concentration is more than or equal to 8 mu g/mL. P <0.05, P <0.01, P <0.001 (relative to DMSO).

FIG. 4 shows the results of Bostrycin in promoting apoptosis of squamous cell carcinoma of the tongue. FIG. 4A: AO/EB staining fluorescence map 24h after the treatment of SCC9 cells by Bostrycin; FIG. 4B: AO/EB staining fluorescence map 48h after SCC9 cell treatment by Bostrycin; FIG. 4C: AO/EB staining fluorescence map after 24h of SCC25 cell treated by Bostrycin; FIG. 4D: AO/EB staining fluorescence map 48h after the treatment of SCC25 cells by Bostrycin; FIG. 4E: AO/EB staining flow cytometry detection result chart after 24h of SCC9 cell treated by Bostrycin; FIG. 4F: carrying out statistics on detecting apoptotic cells by using an AO/EB (anaerobic-anoxic-oxic/Epstein) staining flow cytometer after 24 hours of treating SCC9 cells by using Bostrycin; FIG. 4G: AO/EB staining flow cytometer detection result chart after 48h of SCC9 cell treatment by Bostrycin; FIG. 4H: carrying out statistics on detecting apoptotic cells by using an AO/EB (anaerobic-anoxic-oxic/Epstein-Barr) staining flow cytometer after the cells SCC9 are treated by the Bostrycin for 48 hours; FIG. 4I: AO/EB staining flow cytometry detection result chart after 24h of SCC25 cell treated by Bostrycin; FIG. 4J: carrying out AO/EB staining flow cytometry detection on apoptotic cells after 24 hours of SCC25 cell treatment by Bostrycin; FIG. 4K: AO/EB staining flow cytometer detection result chart after 48h of SCC25 cell treatment by Bostrycin; FIG. 4L: and detecting apoptotic cell statistics by AO/EB staining flow cytometry 48 hours after SCC25 cells are treated by Bostrycin. P <0.05, P <0.01, P <0.001 (relative to DMSO).

FIG. 5 shows the results of Bostrycin inhibition of tongue squamous cell carcinoma cell migration. FIG. 5A: the scratch test detects the influence of Bostrycin on the migration of SCC9 cells; FIG. 5B: the scratch test detects the influence of Bostrycin on the migration of SCC25 cells; FIG. 5C: a scratch test detects a statistical graph of the influence of Bostrycin on the migration of SCC9 cells; FIG. 5D: scratch test statistical test of Bostrycin effect on SCC25 cell migration. P <0.05, P <0.01, P <0.001 (relative to DMSO).

FIG. 6 shows the results of Bostrycin increasing mitochondrial apoptosis in squamous cell carcinoma cells of the tongue. FIG. 6A: the effect of Bostrycin on mitochondrial apoptosis of SCC9 cells; FIG. 6B: statistics of mitochondrial apoptosis rate of Bostrycin on SCC9 cells; FIG. 6C: the effect of Bostrycin on mitochondrial apoptosis of SCC25 cells; FIG. 6D: statistics of mitochondrial apoptosis rate of Bostrycin on SCC25 cells. P <0.05, P <0.01, P <0.001 (relative to positive control CCCP).

FIG. 7 shows the results of Bostrycin reducing the expression of a protein associated with proliferation of squamous cell carcinoma cells of the tongue. FIG. 7A: detecting the expression of SCC9 cell-associated protein by Western Blot experiment; FIG. 7B: detecting the expression of SCC25 cell-associated protein by Western Blot experiment; FIG. 7C: SCC9 cell AKT protein expression statistics; FIG. 7D: statistics of SCC25 cell AKT protein expression; FIG. 7E: statistics of SCC9 cell ERK protein expression; FIG. 7F: statistics of SCC25 cell ERK protein expression; FIG. 7G: statistics of BAX protein expression in SCC9 cells; FIG. 7H: statistics of BAX protein expression in SCC25 cells; FIG. 7I: statistics of SCC9 cell PARP protein expression; FIG. 7J: statistics of SCC25 cell PARP protein expression. P <0.05, P <0.01, P <0.001 (relative to DMSO).

Detailed Description

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

(1) Culture of tongue carcinoma cell line cells

Two tongue carcinoma cell lines, SCC9 and SCC25, were purchased from ATCC (USA). The tongue squamous cell carcinoma cell line SCC9 cell and SCC25 cell were both cultured in DME/F12 medium, and 400. mu.g/L hydrocortisone, 60mg/L sodium pyruvate, and,1.2g/L NaHCO₃. The culture medium was supplemented with 10% fetal bovine serum (BBI) and cultured in 5% CO₂An incubator.

(2) Preparation of colistin

The bacterial colony of the endophytic fungi HCH285 is round, white villous hair is grown in the initial stage and is fluffy, the mycelium is gradually red in the later stage, red metabolites can be clearly seen on the back of the bacterial colony, metabolic pigments are extracted, compound separation is carried out by using a semi-prepared liquid phase, a separated monomer substance is optimized, and the endophytic fungi is determined to be the borstrycin by adopting nuclear magnetic resonance structural analysis.

32mg of the colisporine monomer was dissolved in 1mL of DMSO to prepare a stock solution, and 900. mu.L of sterile ddH was added to 100. mu.L of the stock solution₂O is mixed uniformly to obtain 3.2mg/mL of a colicin monomer solution, the colicin monomer solution is sequentially diluted to 1.6mg/mL, 0.8mg/mL, 0.4mg/mL, 0.2mg/mL, 0.1mg/mL and 0.05mg/mL by sterile water gradient, and the colicin monomer solution is added into a cell culture medium according to 1 percent to obtain final concentrations of 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL, 2 mu g/mL, 1 mu g/mL and 0.5 mu g/mL respectively.

(3) Statistical analysis

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

Example 1 cell proliferation assay

MTT test: SCC9 cells and SCC25 cells are inoculated in a 96-well plate at 5000 cells per well, colisporine with different concentrations is added after 12 hours, each well is added with 20 mu L of 5mg/mL MTT solution after 24 hours, 48 hours and 72 hours of treatment respectively, culture is carried out for 4 hours, supernatant is discarded, 150 mu L of DMSO is added into each well to dissolve the formazan, and the absorbance is measured at 490nm of an enzyme labeling instrument after the formazan is completely dissolved.

Colony formation experiments: inoculating 1000 cells/dish in a 35mm culture dish, adding spinosad after 12h, treating for 24h, changing the liquid, culturing for 7 days, then removing the supernatant, fixing the supernatant with methanol/acetic acid fixing liquid at room temperature for 30min, and dyeing with 2% crystal violet for 1 h. The photographs were taken after 3 washes in PBS and counted using Image J software.

As a result: sporotidine significantly inhibited the proliferation of tongue cancer cells SCC9 and SCC 25.

As shown in fig. 1A-1B. Wherein, FIG. 1A is directed to SCC9 cells and FIG. 1B is directed to SCC25 cells. SCC9 cells and SCC25 cells were treated with different concentrations of spinosin for 24h, 48h, and 72h, respectively. The results show that the effect of the treatment for different times is small, the effect of different concentrations of the colicin on tongue cancer cells is large, the activity of SCC9 cells is obviously reduced when the concentration is 4-8 mu g/mL, and the IC50 is 5.37 mu g/mL when the colicin is used for treating SCC9 cells for 24 hours. The activity of SCC25 cells is obviously reduced when the concentration of the colicin is 2-4 mug/mL, and the IC50 of the colicin treated SCC25 cells is 3.50 mug/mL when the cells are treated for 24 hours.

As shown in fig. 1C-1D. Wherein, fig. 1C is directed to SCC9 cells and fig. 1D is directed to SCC25 cells. Colony formation experiment results show that the spinosad can obviously inhibit the proliferation of SCC9 cells when the concentration is greater than or equal to 4 mu g/mL, the inhibition rate of the spinosad on the clonal formation of SCC9 cells is 45.8% when the concentration is 4 mu g/mL, and the inhibition rate of the spinosad on the clonal formation of SCC9 cells is 66.9% when the concentration is 8 mu g/mL; the colicin obviously inhibits the proliferation of SCC25 cells when the content is more than or equal to 2 mu g/mL, the inhibition rate of the colicin on the clone formation of SCC25 cells is 43.7% when the content is 2 mu g/mL, the inhibition rate of the colicin on the clone formation of SCC25 cells is 73.3% when the content is 4 mu g/mL, and the inhibition rate of the colicin on the clone formation of SCC25 cells is 95.0% when the content is 8 mu g/mL.

Example 2 cell cycle experiments

SCC9 cells and SCC25 cells were inoculated on a 35mm dish, cultured to 70% confluence, added with spinosad, cultured for 24h, collected, pre-cooled 70% ethanol-20 ℃ fixed overnight, collected, washed once with PBS, added with 80. mu.L of Propidium Iodide (PI) with a concentration of 100. mu.g/mL, 0.25% Triton-100, RNase with a final concentration of 20. mu.g/mL, incubated at 4 ℃ in the dark for 30min, and detected using a flow cytometer.

As a result: sporotidine increased G2/M arrest in tongue cancer cells SCC9 and SCC 25.

As shown in fig. 2A-2D, wherein fig. 2A and 2B are directed to SCC9 cells and fig. 2C and 2D are directed to SCC25 cells. After the colicin is used for treating the tongue cancer cells for 24 hours, the colicin increases 2.0% of the sub-G1 stage, reduces 13.2% of the G0/G1 stage, reduces 4.6% of the S stage and increases 1.9% of the G2/M stage of SCC9 cells when the colicin is 2 mug/mL; at 4 mu G/mL, the sub-G1 phase of SCC9 cells is increased by 6.5%, the G0/G1 phase is decreased by 13.3%, the S phase is decreased by 5.8%, and the G2/M phase is increased by 3.4%; at 8. mu.g/mL, the sub-G1 phase of SCC9 cells is increased by 7.3%, the G0/G1 phase is decreased by 12.9%, the S phase is decreased by 10.0%, and the G2/M phase is increased by 8.8%. When the concentration of the colicin is 2 mu G/mL, the sub-G1 phase of SCC25 cells is increased by 2.4%, the G0/G1 phase is reduced by 16.7%, the S phase is increased by 3.5%, and the G2/M phase is increased by 2.7%; at 4 mu G/mL, the sub-G1 phase of SCC25 cells is increased by 10.1%, the G0/G1 phase is decreased by 16.0%, the S phase is decreased by 0.3%, and the G2/M phase is increased by 4.4%; at 8. mu.g/mL, the sub-G1 phase of SCC25 cells is increased by 8.3%, the G0/G1 phase is decreased by 17.2%, the S phase is decreased by 2.8%, and the G2/M phase is increased by 11.7%. The result shows that after the asbest is used for treating the tongue cancer cells for 24 hours, the sub-G1 stage is increased along with the increase of the concentration, which indicates that the asbest can increase the tongue cancer cell apoptosis; the increase in G2/M phase indicates that spinosad inhibits cell proliferation by blocking cell G2/M phase.

Example 3 apoptosis test Using apoptosis kit double staining

SCC9 cells and SCC25 cells were seeded in a 24-well plate, cultured to 70% confluence, added with colistin, cultured for 24h, and then collected and stained with Annexin V-Alexa Fluor 488/PI apoptosis kit (Yeasen Biotech, shanghai, china), and apoptosis was detected by flow cytometry.

And in the AO/EB experiment, SCC9 cells and SCC25 cells are respectively inoculated in a 12-well plate, and are cultured overnight until the confluence is 70 percent, and are added with the gelonin and are cultured for 24 hours and 48 hours respectively. Discarding the culture medium, taking 2mg/mL AO and EB, respectively taking 1 μ L AO and 1 μ L EB, adding 1mL PBS to dilute, mixing well, using as-prepared, adding 100 μ L staining working solution into each well, standing at room temperature for 2-5min, and taking a picture with a fluorescence microscope.

And respectively inoculating SCC9 cells and SCC25 cells in 12-well plates, culturing overnight until the confluence is 70%, adding the spinosad, and culturing for 24h and 48h respectively. Collecting cells, taking 2mg/mL AO and EB, respectively taking 1 muL AO and 1 muL EB, adding 1mL PBS to dilute and uniformly mixing, using in situ, adding 100 muL staining working solution suspension cells into each tube, and performing flow detection.

As a result: spinosin significantly increased apoptosis of tongue cancer cells SCC9 and SCC 25.

As shown in fig. 3A-3D, wherein fig. 3A and 3B are directed to SCC9 cells and fig. 3C and 3D are directed to SCC25 cells. After cells are treated by adopting the colicin for 24 hours, the apoptosis of SCC9 and SCC25 is detected by PI and Annexin V-Alexa Fluor 488 double staining, and the results show that the colicin increases 3.7% of the apoptosis of SCC9 at 2 mu g/mL, 10.5% of the apoptosis of SCC9 at 4 mu g/mL and 38.2% of the apoptosis of SCC9 at 8 mu g/mL; sporotidine increased apoptosis in SCC25 at 2. mu.g/mL by 0.8%, in SCC25 at 4. mu.g/mL by 3.1%, and in SCC25 at 8. mu.g/mL by 24.6%. The colicin has obvious increase on early apoptosis and late apoptosis of SCC9 and SCC25 cells.

4A-4L, wherein FIGS. 4A-4B, 4E-4H are directed against SCC9 cells and FIGS. 4C-4D, 4I-4L are directed against SCC25 cells. The cells are respectively treated by the colicin for 24h and 48h, the apoptosis is detected by AO/EB staining, the fluorescence is observed under a microscope, when the cells are treated by the colicin with the concentration of 2 mu g/mL, a small amount of red fluorescence appears, when the red fluorescence increases with the concentration of 4 mu g/mL, and when the red fluorescence appears with the concentration of 8 mu g/mL. Indicating that the apoptosis rate gradually increases with the increase of the concentration of the spinosad. The AO/EB staining result detected by the flow cytometer shows that the red fluorescence is shifted to the right, which indicates that the red fluorescence is increased along with the increase of the concentration of the spinosad. When the colicin is used for treating the SCC9 cells for 24 hours, the 2 mug/mL concentration of the colicin increases 14.93 percent of the apoptosis of SCC9 cells, the 4 mug/mL concentration of the colicin increases 23.4 percent of the apoptosis of SCC9 cells, and the 8 mug/mL concentration of the colicin increases 71.8 percent of the apoptosis of SCC9 cells, wherein the 4 mug/mL and 8 mug/mL concentrations of the colicin have significant difference compared with a control group when the 4 mug/mL and 8 mug/mL colicin are used for treating the SCC9 cells for 24 hours; when the colicin is used for treating the SCC9 cells for 48 hours, the 2 mug/mL concentration of the colicin increases 5.30% of the apoptosis of SCC9 cells, the 4 mug/mL concentration of the colicin increases 38.10% of the apoptosis of SCC9 cells, and the 8 mug/mL concentration of the colicin increases 48.23% of the apoptosis of SCC9 cells, wherein the 4 mug/mL and 8 mug/mL concentrations of the colicin have significant difference compared with a control group when the 4 mug/mL and 8 mug/mL concentrations of the colicin are used for treating the SCC9 cells for 48 hours; when the colicin is used for treating the SCC25 cell for 24h, the 2 mug/mL concentration of the colicin increases the apoptosis of the SCC25 cell by 16.77%, the 4 mug/mL concentration of the colicin increases the apoptosis of the SCC25 cell by 33.07%, and the 8 mug/mL concentration of the colicin increases the apoptosis of the SCC25 cell by 37.57%, wherein the 8 mug/mL concentration of the colicin has a significant difference compared with a control group when the 8 mug/mL concentration of the colicin is used for treating the SCC25 cell for 24 h; when the colicin is used for treating the SCC25 cells for 48h, the 2 mug/mL concentration of the colicin increases 3.73% of the apoptosis of SCC25 cells, the 4 mug/mL concentration of the colicin increases 26.03% of the apoptosis of SCC25 cells, and the 8 mug/mL concentration of the colicin increases 56.17% of the apoptosis of SCC25 cells, wherein the 4 mug/mL and 8 mug/mL concentrations of the colicin have significant difference compared with a control group when the 4 mug/mL and 8 mug/mL colicin are used for treating the SCC25 cells for 48 h.

Example 4 scratch test

And inoculating SCC9 cells and SCC25 cells in a 24-well plate, after 12 hours, drawing 2 vertical lines with a yellow tip, changing the vertical lines into 1% serum culture medium, adding the spinosad with different concentrations, and taking pictures at the same position every 12 hours. And Image J counts the area of the scratch region and calculates the statistical migration rate.

As a result: sporotysin inhibits the migration of tongue cancer cells SCC9 and SCC 25.

As shown in fig. 5A-5D, wherein fig. 5A, 5C are directed to SCC9 cells and fig. 5B, 5D are directed to SCC25 cells. The scratching experiment result shows that the spinosad can obviously inhibit the migration of tongue squamous cell carcinoma cells SCC9 and SCC 25. At 12h, the migration rate of 2 mug/mL of colicin to SCC9 cells is reduced by 10.8%, the migration rate of 25 cells is reduced by 47.1%, the migration rate of 4 mug/mL of colicin to SCC9 cells is reduced by 96.5%, the migration rate of 25 cells is reduced by 77.1%, the migration rate of 8 mug/mL of colicin to SCC9 cells is reduced by 92.2%, and the migration rate of 25 cells is reduced by 90.6%; at 24h, the migration rate of 2 mug/mL of colicin to SCC9 cells is reduced by 32.3%, the migration rate of 25 cells is reduced by 60.4%, the migration rate of 4 mug/mL of colicin to SCC9 cells is reduced by 108.1%, the migration rate of 25 cells is reduced by 92.3%, the migration rate of 8 mug/mL of colicin to SCC9 cells is reduced by 95.5%, and the migration rate of 25 cells is reduced by 96.6%. At low concentrations, 2 μ g/mL effectively reduced the rate of tongue cancer cell migration, and SCC25 cells were more sensitive. With increasing concentration, the effect of SCC9 cells was greater 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 assay

And inoculating SCC9 cells and SCC25 cells in a 24-well plate, adding different concentrations of selaginellin after 12h till the cell density is about 70%, treating for 24h, and adding a positive control CCCP (ccc) for 20 min. The cells were collected, stained with JC-1, and incubated in a cell incubator at 37 ℃ for 20 minutes for detection by flow cytometry.

As a result: sporotysin decreases mitochondrial membrane potential.

JC-1 experiment detects the influence of Bostrycin on mitochondrial membrane potential of tongue cancer cells, and a flow cytometer is adopted for green fluorescence. The results are shown in FIGS. 6A-6D, where FIGS. 6A-6B are for SCC9 cells and FIGS. 6C-6D are for SCC25 cells. The spinosad with the concentration of 2 mug/mL increases the mitochondrial apoptosis rate of SCC9 by 0.75 percent, the spinosad with the concentration of 4 mug/mL increases the mitochondrial apoptosis rate of SCC9 by 7.8 percent, and the spinosad with the concentration of 8 mug/mL increases the mitochondrial apoptosis rate of SCC9 by 20.9 percent; the spinosad with the concentration of 2 mug/mL increases the mitochondrial apoptosis rate of SCC25 cells by 4.1%, the spinosad with the concentration of 4 mug/mL increases the mitochondrial apoptosis rate of SCC25 cells by 39.8%, and the spinosad with the concentration of 8 mug/mL increases the mitochondrial apoptosis rate of SCC25 cells by 14.6%. The result shows that the mitochondrial apoptosis of SCC25 cells is more sensitive to the spinosad, the apoptosis rate is far greater than that of SCC9 cells when the concentration is 4 mug/mL, and the detection value is lower due to most cell death when the concentration is 8 mug/mL for treating SCC25 cells.

Example 6 Western Blot measures the expression of proliferation and apoptosis related proteins

And respectively culturing SCC9 cells and SCC25 cells in a 35mm dish until the confluence degree is 70%, adding the spinosad, culturing for 24h, collecting the cells, extracting the total protein, and freezing and storing. After polyacrylamide gel electrophoresis, an NC membrane is transferred, and after the corresponding strip is cut off, the corresponding strip is incubated at 4 ℃ overnight. And (5) recovering the primary antibody, and developing after the secondary antibody is incubated for 1h after the membrane is cleaned.

As a result: sporotidine increases apoptosis of tongue cancer cells SCC9 and SCC25 through caspase signaling pathways.

Western Blot is adopted to detect the expression conditions of proliferation and apoptosis related proteins, the results are shown in FIGS. 7A-7J, the expression level of p-AKT is increased and the expression level of ERK is gradually reduced along with the increase of the concentration of Bostrycin, which indicates that Bostrycin can inhibit cell proliferation through AKT pathway, the expression level of BAX is increased, and that Bostrycin can promote cell apoptosis through mitochondrial apoptosis pathway.

The monomer bostrycin is used as a material, the inhibition effect of the monomer bostrycin on tongue squamous cell carcinoma SCC9 and SCC25 is researched, and the action mechanism of the monomer bostrycin is researched. The research shows that bostrycin can effectively inhibit the proliferation of tongue squamous cell carcinoma, and MTT experimental results show that the IC50 of bostrycin is 5.37 mu g/ml when the bostrycin acts on SCC9 cells for 24 hours, and the IC50 of bostrycin acts on SCC25 cells for 24 hours is 3.50 mu g/ml. Therefore, bostrycin has better in-vitro anti-cancer activity on squamous cell carcinoma cells of tongue and better inhibition effect on proliferation of SCC25 cells. Cell cycle experiments show that the spinosad inhibits tongue cancer cell proliferation by inducing tongue cancer cell G2/M phase retardation, and cycle detection also shows that small apoptosis peaks in sub-G1 phase are obviously increased along with the increase of the concentration of the spinosad, which indicates that the spinosad may induce tongue cancer cell apoptosis. The use of annexin V-Alexa flow 488/PI double stain and AO/EB experiments respectively show that bostrycin cells can promote tongue squamous cell carcinoma cell apoptosis, and the annexin V-Alexa flow 488/PI double stain experiment shows that the cell apoptosis of SCC9 is increased by 10.5% at 4 mu g/mL, the cell apoptosis of SCC9 is increased by 38.2% at 8 mu g/mL, the cell apoptosis of SCC25 is increased by 3.1% at 4 mu g/mL, and the cell apoptosis of SCC25 is increased by 24.6% at 8 mu g/mL. Therefore, bostrycin can obviously increase the apoptosis of tongue cancer cells, and the increase of the apoptosis rate of SCC9 cells is higher than that of SCC25 cells. The AO/EB experiment result shows that the proportion of the red fluorescence is increased along with the increase of the concentration of bostrycin, and the increase of the red fluorescence observed by a fluorescence microscope is consistent with the detection result of a flow cytometer. Shows that the cell apoptosis rate of tongue squamous cell carcinoma is gradually increased along with the increase of bostrycin concentration, and the cell apoptosis increase rate of SCC9 is higher than that of SCC25, which is consistent with the conclusion of annexin V-Alexa Flour 488/PI double staining experiment.

In conclusion, bostrycin has better tongue squamous cell carcinoma inhibiting effect in vitro, the inhibiting effect on the proliferation of SCC25 cells is better than that of SCC9 cells, and the increase on the apoptosis of SCC9 cells is better than that of SCC25 cells. Western Blot detects the expression condition of proliferation and apoptosis related proteins, and the result shows that bostrycin can obviously increase the expression of p-AKT and reduce the expression of ERK protein, which is probably a mode of the bostrycin for reducing the proliferation of squamous cell carcinoma cells of tongue. In order to investigate whether bostrycin affects apoptosis through a mitochondrial apoptosis pathway, the expression of BAX protein was detected, and the result showed that the BAX expression level was increased, which indicates that bostrycin is likely to affect apoptosis through a mitochondrial apoptosis pathway.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many 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

1. Use of Spirosporin in preparing tongue cancer cell proliferation inhibitor and/or tongue cancer cell apoptosis promoter is provided.

2. Use according to claim 1, wherein the tongue cancer cells are tongue squamous cell carcinoma cells.

3. Use according to claim 2, wherein the tongue squamous cell carcinoma cells are SCC9 cells and/or SCC25 cells.

4. The use of claim 1, wherein the tongue cancer cell apoptosis is via a caspase signaling pathway.

5. Application of Sporotrichum in preparing mitochondrial apoptosis inducer is provided.

6. The use of claim 5, wherein spinosyn induces mitochondrial apoptosis by inducing mitochondrial membrane potential changes.