Application of streptococcus sanguis extract in preparation of medicines for preventing and treating cancers

Abstract

The invention relates to application of streptococcus sanguis extract in preparation of drugs for preventing and treating cancers, and belongs to the technical field of medicines. In order to develop the new application of the streptococcus sanguis extract XLS in cancer treatment, the invention provides the application of the streptococcus sanguis extract in preparation of drugs for preventing and treating cancers. The invention demonstrates the inhibition effect of the streptococcus sanguis extract XLS on an ovarian cancer cell line SKOV3. CCK-8 experiments prove that the streptococcus sanguis extract XLS can reduce the cell activity of SKOV3 and has an inhibiting effect on cell proliferation, invasion and migration of the SKOV3. Molecular biology experiment research shows that XLS inhibits activation of a PI3K / AKT signal channel by inhibiting phosphorylation of AKT, regulates expression of apoptosis-related genes and induces SKOV3 cell apoptosis. In addition, XLS further damages the structure and function of tumor cells by means of destroying mitochondrial membrane potential, increasing oxidative stress, Ca < 2 + > imbalance and the like.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to the application of a Streptococcus sanguinis extract in preparing a medicine for preventing and treating cancer. Background Art

[0002] Bacteriocin-like substances are a class of peptides, proteins or protein complexes with antibacterial activity that are mainly produced by bacteria through ribosome synthesis during metabolism. Bacteriocin-like substances mainly exert their antibacterial effects by destroying bacterial cell membranes, inhibiting cell wall synthesis or inhibiting nucleic acid and protein synthesis. These substances are widely present in nature and have broad application prospects in many fields such as antimicrobial drugs, food preservation and animal feed additives due to their advantages such as being natural, non-toxic and not prone to drug resistance.

[0003] Streptococcus sanguinis extract XLS is a bacteriocin-like substance extracted from oral Streptococcus sanguinis. It has a strong inhibitory effect on fungi, especially Candida albicans, Candida tropicalis and other fungal infections. It can also inhibit the growth of Porphyromonas gingivalis. Streptococcus sanguinis extract XLS has many advantages such as safety, high efficiency, no toxic side effects and no drug resistance. It is expected to replace antibiotics that have developed extensive drug resistance and provide a new option for clinical antibacterial treatment. However, in addition to the significant antibacterial effect, the mechanism of action of other potential functions of Streptococcus sanguinis extract XLS as a natural active substance has not been fully understood, which restricts the further development and application of Streptococcus sanguinis extract XLS. Summary of the invention

[0004] In order to develop new uses of Streptococcus sanguinis extract XLS in cancer treatment, the present invention provides the use of Streptococcus sanguinis extract in preparing drugs for preventing and treating cancer.

[0005] The technical solution of the present invention:

[0006] Application of Streptococcus sanguinis extract in preparing medicine for preventing and treating cancer.

[0007] Furthermore, the preparation method of the Streptococcus sanguis extract is as follows:

[0008] The purified and identified Streptococcus sanguis was added to a BHI liquid culture medium, and cultured at a constant temperature of 37°C under anaerobic conditions for 48 hours. The Streptococcus sanguis suspension was placed at 4°C and 12000 r / min for 30 minutes, and the bacterial precipitate was collected. The obtained bacterial cells were resuspended with PBS buffer and centrifuged, and ultrasonically disrupted under ice-water bath conditions. The system obtained after ultrasonic disruption was placed at 4°C and 12000 r / min for 30 minutes, and the supernatant was collected; the obtained supernatant was subjected to gradient salting out with ammonium hydrochloride for 8 hours, and the precipitate was collected by low-temperature high-speed centrifugation at 4°C and 15000 r / min for 45 minutes. PBS buffer was added to dissolve the precipitate, and then desalted by passing through a cross-linked dextran column, and the liquid after the column was collected, dialyzed and concentrated, and freeze-dried to obtain a Streptococcus sanguis extract.

[0009] Furthermore, the drug for preventing and treating cancer uses the Streptococcus sanguinis extract as the only active ingredient or one of the active ingredients.

[0010] Furthermore, the content of the Streptococcus sanguis extract in the drug for preventing and treating cancer is 0.1wt% to 99wt%.

[0011] Furthermore, the drug for preventing and treating cancer also includes pharmaceutically acceptable excipients and / or carriers.

[0012] Furthermore, the cancer is ovarian cancer.

[0013] Furthermore, the Streptococcus sanguinis extract can inhibit the proliferation, invasion and migration of human ovarian cancer cell SKOV3.

[0014] Furthermore, the Streptococcus sanguinis extract inhibits the phosphorylation of AKT and thus the activation of the PI3K / AKT signaling pathway, thereby inducing apoptosis of human ovarian cancer cell SKOV3 cells.

[0015] Furthermore, the Streptococcus sanguinis extract destroys mitochondrial membrane potential, increases oxidative stress and Ca 2+ Imbalance impairs the structure and function of human ovarian cancer cell SKOV3 cells.

[0016] Beneficial effects of the present invention:

[0017] The invention provides a new use of a Streptococcus sanguinis extract XLS for preventing and treating cancer, namely, use of the Streptococcus sanguinis extract in preparing a drug for preventing and treating cancer, especially use of the Streptococcus sanguinis extract in preparing a drug for preventing and treating ovarian cancer.

[0018] The present invention demonstrates the inhibitory effect of Streptococcus sanguinis extract XLS on ovarian cancer cell line SKOV3. CCK-8 experiments have shown that Streptococcus sanguinis extract XLS can reduce the cell activity of SKOV3 and has an inhibitory effect on the proliferation, invasion and migration of SKOV3 cells. Molecular biological experimental studies have shown that XLS inhibits the activation of PI3K / AKT signaling pathway by inhibiting the phosphorylation of AKT, regulates the expression of apoptosis-related genes, and induces apoptosis of SKOV3 cells. In addition, XLS also destroys mitochondrial membrane potential, increases oxidative stress and Ca 2+ Imbalance in these pathways can damage tumor cell structure and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the cell viability of SKOV3 after treatment with different concentrations of XLS detected by CCK-8 method;

[0020] Figure 2 This is a comparison chart of the cell viability of SKOV3 cells after being treated with different concentrations of paclitaxel detected by CCK-8 method;

[0021] Figure 3 Comparison photos of the cell proliferation colonies of SKOV3 after treatment with different concentrations of XLS detected by plate colony formation method;

[0022] Figure 4 This is a comparison chart of the number of SKOV3 cells proliferating after being treated with different concentrations of XLS using the plate clone formation method;

[0023] Figure 5 Comparison photos of SKOV3 cell membrane permeability after treatment with different concentrations of XLS and paclitaxel in Transwell chamber experiment;

[0024] Figure 6 This is a comparison chart of the number of SKOV3 cells that passed through the membrane after being treated with different concentrations of XLS and paclitaxel using the Transwell chamber experiment;

[0025] Figure 7 Comparative photos of the scratch healing of SKOV3 after being treated with different concentrations of XLS in the scratch experiment;

[0026] Figure 8 This is a comparison of the scratch areas of SKOV3 after being treated with different concentrations of XLS in the scratch experiment;

[0027] Fig. 9 The scatter plot is a flow cytometry analysis of SKOV3 cells after treatment with different concentrations of XLS;

[0028] Fig.10 This is a comparison of the early and late apoptosis rates of SKOVS after treatment with different concentrations of XLS detected by flow cytometry;

[0029] Fig.11 This is a color-developed photograph of Western Blot detection of proteins related to the PI3K / AKT signaling pathway in SKOVS after treatment with different concentrations of XLS;

[0030] Fig.12 This is a comparison of the mRNA levels of proteins related to the PI3K / AKT signaling pathway in SKOVS after qPCR detection of different concentrations of XLS treatment;

[0031] Fig.13 Fluorescence micrographs of SKOVS treated with different concentrations of XLS detected by JC-1 fluorescent probe;

[0032] Fig.14 This is a comparison of the reactive oxygen species (ROS) levels of SKOV3 cells after treatment with different concentrations of XLS and paclitaxel detected by flow cytometry;

[0033] Fig.15 Flow cytometry was used to detect the Ca2+ expression of SKOV3 cells after treatment with different concentrations of XLS and paclitaxel. 2+ Horizontal comparison chart. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0035] The purchase channels of the drugs, kits and equipment used in the present invention are as follows:

[0036] Paclitaxel, apoptosis detection kit 50assays, CCK-8 cell viability detection kit and Ca 2+ The concentration kits were purchased from Biosharp Lanjieke Technology Co., Ltd.; the ROS reactive oxygen species kit was purchased from Biyuntian Biotechnology Co., Ltd.; the flow cytometer V4-B5-R3 was purchased from Beckman Coulter, Inc., USA; and the q-PCR analyzer LIGHTCYCLER480II was purchased from Hoffmann-La Roche LLC.

[0037] The following examples are statistically analyzed: Image data were processed using ImageJ software, and statistical analysis was performed using SPSS25.0 software. All data are expressed as mean ± standard deviation, and all experiments were repeated three times. One-way analysis of variance was used for comparison of differences between multiple groups, and independent sample t-test was used for comparison of differences between two groups. P<0.05 indicates that the difference is statistically significant. Statistical significance is indicated by *(P<0.05), **(P<0.01), ***(P<0.001), ****(P<0.0001), respectively. When P>0.05, the difference was not statistically significant.

[0038] Example 1

[0039] This embodiment provides a method for preparing a Streptococcus sanguis extract.

[0040] In this example, the standard strain of Streptococcus sanguinis ATCC10556 was purchased from the Key Laboratory of Oral Biomedical Engineering of the Ministry of Health of West China School of Stomatology, Sichuan Province. This standard strain is available to the public through normal commercial channels.

[0041] The BHI bovine heart and brain infusion liquid culture medium used in this example was purchased from OXOID, England.

[0042] The cross-linked dextran used in this example is prepared from Sephadex G-25 dextran gel, which absorbs 2.5 grams of water per gram of gel when it swells. The separation range is 1000-5000, and it is suitable for desalting and separation of peptides and other small molecules.

[0043] The specific preparation method of Streptococcus sanguis extract is as follows:

[0044] The purified and identified Streptococcus sanguinis was added to BHI liquid culture medium and cultured at a constant temperature of 37°C under anaerobic conditions for 48 h. The Streptococcus sanguinis suspension was immediately centrifuged at 4°C and 12000 r / min for 30 min. The bacterial precipitate was collected and resuspended in 1×PBS buffer and centrifuged again at a low temperature. The resuspension and centrifugation process was repeated three times. The bacterial precipitate was resuspended in 100 mL 1×PBS buffer and ultrasonically disrupted in an ice water bath. The working mode of the ultrasonic disruptor was intermittent, specifically set to work for 4 seconds and stop for 8 seconds, the ultrasonic power was 150 W, and the ultrasonic time was 30 min.

[0045] The solution after ultrasonic disruption was centrifuged at 4°C and 12000r / min for 30min, and the supernatant was collected; the obtained supernatant was salted out with ammonium hydrochloride gradient at 4°C for 8h, and the precipitate was collected by low-temperature high-speed centrifugation at 4°C and 15000r / min for 45min, an appropriate amount of PBS buffer was added to dissolve the precipitate, and then the precipitate was desalted by cross-linked dextran column, the liquid after the column was collected, dialyzed and concentrated, and freeze-dried to obtain the Streptococcus sanguinis extract, which was stored at -80°C for future use.

[0046] Example 2

[0047] In this example, the CCK-8 method was used to detect the effects of different concentrations of Streptococcus sanguinis extract XLS on the viability of human ovarian cancer cell SKOV3 cells, and different concentrations of paclitaxel were used as a control.

[0048] The culture method of human ovarian cancer cell SKOV3 in this embodiment is as follows:

[0049] The frozen cells were thawed and inoculated into RPMI 1640 complete medium and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0050] The CCK-8 method is as follows:

[0051] The 96-well plate was placed in an incubator at 37°C and 5% CO2, and when the human ovarian cancer cell SKOV3 cells grew to 90% density, complete medium containing 0μM, 2.5μM, 5μM, 10μM, 20μM, 40μM and 80μM of Streptococcus sanguinis extract XLS was added, and at the same time, SKOV3 cells were added to complete medium containing 0μM, 5μM, 10μM, 20μM, 40μM, 80μM and 160μM paclitaxel as a control. After 24 hours of treatment, 10μL CCK-8 solution was added to each well. The absorbance of the 96-well plate was detected using an ELISA reader, and the cell viability was calculated.

[0052] Figure 1 This is a comparison chart of the cell viability of SKOV3 after being treated with different concentrations of XLS using the CCK-8 method. Figure 1 It showed that after treatment with Streptococcus sanguis extract XLS, the viability of SKOV3 cells decreased in a concentration-dependent manner as the concentration of Streptococcus sanguis extract XLS increased.

[0053] Figure 2 This is a comparison chart of the cell viability of SKOV3 cells after being treated with different concentrations of paclitaxel using the CCK-8 method. Figure 2 The results showed that, under the same conditions, after paclitaxel treatment of SKOV3 cells for 24 hours, cell viability was significantly reduced when the concentration exceeded 20 μM, while there was no significant difference when the concentration was lower than 10 μM.

[0054] The results showed that Streptococcus sanguis extract XLS and paclitaxel showed similar concentration dependence in affecting the activity of SKOV3 cells.

[0055] Example 3

[0056] In this example, a plate cloning assay was used to detect the effects of different concentrations of Streptococcus sanguinis extract XLS on the proliferation of human ovarian cancer cell SKOV3 cells.

[0057] The plate colony formation assay method is as follows:

[0058] Human ovarian cancer cell SKOV3 cells were seeded in two 6-well plates and 500 cells were counted. After the cells adhered, they were treated with 0μM, 10μM, 20μM and 40μM of Streptococcus sanguinis extract XLS for 24 hours. After incubation at 37°C for 24 hours, fresh complete medium was replaced and cultured until colonies containing more than 15 cells were formed. After fixing the cells, they were stained with 0.5% crystal violet for 30 minutes and the number of visible cell colonies was counted.

[0059] Figure 3 These are comparison photos of the cell proliferation colonies of SKOV3 cells after being treated with different concentrations of XLS using the plate cloning assay. Figure 4 This is a comparison chart of the number of SKOV3 cell proliferation detected by plate clone formation method after treatment with different concentrations of XLS; Figure 3 and Figure 4 The results showed that with the increase of the concentration of Streptococcus sanguis extract XLS, the formation of SKOV3 cell colonies was significantly reduced, indicating that the Streptococcus sanguis extract XLS can significantly inhibit the proliferation ability of SKOV3 cells.

[0060] Example 4

[0061] In this example, a Transwell chamber experiment was used to detect the effects of different concentrations of Streptococcus sanguinis extract XLS on the invasion of human ovarian cancer cell SKOV3 cells, and paclitaxel was used as a control.

[0062] The Transwell chamber experimental method is as follows:

[0063] Dilute Matrigel with serum-free medium or PBS at a ratio of 1:8. Take 60 μL and add it evenly to the upper chamber of the Transwell chamber membrane, and incubate it in a 37°C incubator for 3 hours to form a thin film. Digest the human ovarian cancer cell SKOV3 cells in the logarithmic growth phase for 2-3 minutes, centrifuge, discard the supernatant, wash with PBS and resuspend the cells, and adjust the cell density to 1-10×105 / mL. Add the culture medium containing chemokines to the lower chamber of the 24-well plate, place the Transwell chamber in the 24-well plate, add the cell suspension to the upper chamber, the content of Streptococcus sanguinis extract XLS is 0μM, 10μM and 20μM, the content of paclitaxel is 20μM, and incubate for 12 to 48 hours. Finally, detect the number of cells that pass through the membrane.

[0064] Figure 5 This is a comparison photo of the cell membrane of SKOV3 after being treated with different concentrations of XLS and paclitaxel in the Transwell chamber experiment. Figure 6 This is a comparison chart of the number of SKOV3 cells that passed through the membrane after being treated with different concentrations of XLS and paclitaxel using the Transwell chamber experiment; Figure 5 and Figure 6 The results showed that the paclitaxel positive control group significantly reduced the invasive ability of SKOV3 cells compared with the untreated control group, which is consistent with its known effects as an anti-tumor drug. Streptococcus sanguinis extract XLS showed a concentration-dependent inhibition of the invasive ability of SKOV3 cells. After treatment with 10μM and 20μM Streptococcus sanguinis extract XLS, the number of cell migration decreased by approximately 33% and 45% compared with the control group, respectively.

[0065] Example 5

[0066] In this example, a scratch test was used to detect the effects of different concentrations of Streptococcus sanguis extract XLS on the migration of human ovarian cancer cell SKOV3 cells.

[0067] The scratch test method is as follows:

[0068] Human ovarian cancer cell SKOV3 cells were evenly seeded into 6-well plates and cultured to form a monolayer of cells. A scratch was made on the monolayer of cells using a sterile pipette tip. The cells were treated with 10 μM and 20 μM of Streptococcus sanguinis extract XLS for 24 hours, respectively, and a blank control group was set up. Images at the beginning of the scratch were captured immediately (0 hours), and images were taken again after 18 hours and 26 hours. The progress of scratch healing was monitored using an inverted microscope, and the changes in scratch area at different time points were analyzed to evaluate cell migration ability.

[0069] Figure 7 Comparison photos of the scratch healing of SKOV3 after being treated with different concentrations of XLS in the scratch experiment. Figure 8This is a comparison of the scratch areas of SKOV3 after being treated with different concentrations of XLS in the scratch experiment; Figure 7 and Figure 8 The results showed that compared with the control group, the Streptococcus sanguinis extract XLS could inhibit the migration ability of SKOV3 cells. With the increase of the concentration of Streptococcus sanguinis extract XLS, the wound healing rate of SKOV3 cells decreased significantly, showing a concentration-dependency.

[0070] Example 6

[0071] In this example, flow cytometry was used to detect the effects of different Streptococcus sanguis extracts XLS on apoptosis of human ovarian cancer cell SKOV3.

[0072] The flow cytometry method is as follows:

[0073] Human ovarian cancer cells SKOV3 were treated with 0μM, 10μM and 20μM Streptococcus sanguinis extract XLS, and the treated cells were digested with trypsin. The digested cells were collected, the suspension was transferred to an EP tube, and centrifuged at 2500rpm for 5 minutes at room temperature. The cells were washed twice with PBS precooled at 4°C, and 300μL 1×Binding Buffer was added to resuspend the cells. AnnexinV-FITC / PI double staining method was used, 5μL AnnexinV-FITC was added, and incubated at room temperature for 15 minutes in the dark. 5μLPI was added for staining 5 minutes before the machine, and 200μL 1×Binding Buffer was added.

[0074] Fig. 9 This is a scatter plot of SKOV3 cells treated with different concentrations of XLS detected by flow cytometry. Fig.10 This is a comparison of the early and late apoptosis rates of SKOVS after treatment with different concentrations of XLS detected by flow cytometry; Fig. 9 and Fig.10 The results showed that compared with the control group, 5μM and 10μM of Streptococcus sanguis extract XLS had no significant effect on early and late apoptosis of cells. However, when the concentration of Streptococcus sanguis extract XLS was 20μM, the early and late apoptosis rates of cells increased to 4.65% and 66.82%, respectively, which were significantly higher than the control group. Streptococcus sanguis extract XLS can promote apoptosis of SKOV3 cells.

[0075] Example 7

[0076] In this example, Western Blot and q-PCR were used to detect the effects of different concentrations of Streptococcus sanguis extract XLS on the expression of proteins and genes related to the PI3K / AKT signaling pathway in human ovarian cancer cells SKOV3.

[0077] The method of Western Blot is as follows:

[0078] Human ovarian cancer cells SKOV3 were treated with 0μM, 5μM, 10μM and 20μM of Streptococcus sanguinis extract XLS, and cell proteins were extracted according to the RIPA lysis solution instructions. Proteins were separated using SDS-PAGE and transferred to a PVDF membrane. Subsequently, the membrane was blocked in a 5% skim milk solution for 2 hours. After blocking, the primary antibody was added and incubated overnight at 4°C. After the incubation, the secondary antibody was added and incubated for 1-2 hours. After the secondary antibody incubation, the chemiluminescent working solution was mixed in equal proportions and evenly dropped onto the PVDF membrane. The experimental results were exposed and recorded using a fully automatic imaging system in a dark room.

[0079] Fig.11 The color photos of Western Blot detection of proteins related to the PI3K / AKT signaling pathway in SKOVS after treatment with different concentrations of XLS show that compared with the control group, there was no significant difference in the levels of PI3K and AKT proteins after treatment with Streptococcus sanguinis extract XLS, but the level of p-AKT protein was significantly reduced in a concentration-dependent manner, indicating that Streptococcus sanguinis extract XLS may inhibit the activation of the PI3K / AKT signaling pathway by inhibiting AKT phosphorylation. In addition, treatment with Streptococcus sanguinis extract XLS significantly upregulated the level of apoptotic protein Bax protein and downregulated the level of anti-apoptotic Bcl-2 protein.

[0080] The polymerase chain reaction (q-PCR) method is as follows:

[0081] The mRNA expression levels of AKT, Bax, Bcl-2, p53 and mTOR in SKOV3 cells after treatment with 0μM, 5μM, 10μM and 20μM Streptococcus sanguis extract XLS were analyzed. Trizol reagent was added to lyse the cells. Cell RNA was extracted according to the Trizol user guide, the RNA concentration was determined using a Nanodrop ultra-micro spectrophotometer, and reverse transcription was performed using a cDNA reverse transcription kit. After the q-PCR reaction system was prepared, a fluorescent quantitative PCR instrument was used for detection. GAPDH was used as an internal reference gene for quantitative analysis.

[0082] As a tumor suppressor gene, p53 induces cell cycle arrest, metabolic changes and apoptosis by regulating the transcription of downstream target genes; mTOR is an important regulator of cell growth and proliferation, promoting autophagy and apoptosis by regulating cell metabolism. Fig.12 This is a comparison of the mRNA levels of proteins related to the PI3K / AKT signaling pathway in SKOVS after qPCR detection of different concentrations of XLS treatment. The picture shows that XLS, an extract of Streptococcus sanguinis, significantly upregulated the mRNA levels of Bax, p53 and mTOR, while the mRNA level of Bcl-2 was significantly decreased, which is consistent with the Western blot results.

[0083] Molecular experiments showed that Streptococcus sanguinis extract XLS promoted the apoptosis of ovarian cancer SKOV3 cells by upregulating the mRNA levels of pro-apoptotic proteins Bax, p53 and mTOR, and downregulating the level of anti-apoptotic Bcl-2 protein.

[0084] Example 8

[0085] In this example, the JC-1 fluorescent probe was used to detect the effects of different concentrations of Streptococcus sanguis extract XLS on the mitochondrial membrane potential of human ovarian cancer cell SKOV3.

[0086] Bcl-2 is mainly present on the mitochondrial membrane, while Bax is transferred to the mitochondrial surface under the action of apoptosis signals to form transmembrane pores, resulting in decreased membrane potential and increased membrane permeability, releasing apoptotic factors (such as cytochrome c), thereby affecting the cell state. Abnormal mitochondrial membrane potential is closely related to the occurrence of cancer. In order to further explore the effect of Streptococcus sanguis extract XLS on the apoptosis mechanism, this example detects the characteristic parameters and substance expression levels related to mitochondria. The effect of Streptococcus sanguis extract XLS on the mitochondrial membrane potential of SKOV3 cells was detected using JC-1 fluorescent probe.

[0087] The JC-1 fluorescent probe detection method is as follows:

[0088] The cells were seeded in 6-well plates at a density of 6×105 cells / well and cultured for 24 hours. Subsequently, the cells were treated with RPMI1640 complete medium containing 0μM, 10μM and 20μM Streptococcus sanguis extract XLS, 2mL was added to each well, and the culture was continued for 24 hours. After treatment, 1mL RPMI1640 and 1μL JC-1 were added to each well and incubated in the dark for 20 minutes. Mitochondrial membrane potential was detected by flow cytometry, and untreated cells were used as negative controls.

[0089] Fig.13 The fluorescence micrographs of SKOVS cells treated with different concentrations of XLS detected by the JC-1 fluorescent probe show that in the cells of the Control group, JC-1 exists in the form of polymers, exhibits red fluorescence, and has a high mitochondrial membrane potential; however, after treatment with the Streptococcus sanguinis extract XLS, the green fluorescence increased significantly and the mitochondrial membrane potential decreased significantly. The results show that the treatment with the Streptococcus sanguinis extract XLS significantly reduced the mitochondrial membrane potential of SKOV3 cells and promoted cell apoptosis.

[0090] Example 9

[0091] In this example, flow cytometry was used to detect the effects of different concentrations of Streptococcus sanguinis extract XLS on the level of reactive oxygen species (ROS) in human ovarian cancer cell SKOV3.

[0092] ROS plays a key role in cell signaling and metabolic balance. Under environmental stress, ROS levels increase significantly, leading to oxidative stress damage to cell structures. To explore the effect of Streptococcus sanguis extract XLS on oxidative stress in SKOV3 cells, flow cytometry was used to detect the changes in ROS levels in SKOV3 cells after treatment with Streptococcus sanguis extract XLS.

[0093] The flow cytometry detection method is as follows:

[0094] Ovarian cancer SKOV3 cells were inoculated in 6-well plates and incubated for 24 hours. The cells were treated with RPMI1640 complete medium containing 0μM, 10μM and 20μM Streptococcus sanguinis extract XLS and RPMI1640 complete medium containing 20μM paclitaxel, respectively, adding 2mL to each well and exposed for 24 hours. After trypsin digestion, the cells were stained with a ROS reactive oxygen species detection kit, and the ROS level was detected by flow cytometry in the FITC channel.

[0095] Fig.14 This is a comparison of the ROS levels of SKOV3 cells after treatment with different concentrations of XLS and paclitaxel using flow cytometry. The picture shows that treatment with Streptococcus sanguinis extract XLS significantly increased the ROS level of SKOV3 cells in a concentration-dependent manner. This trend is consistent with the change in cell membrane potential. The results indicate that the mechanism by which Streptococcus sanguinis extract XLS inhibits ovarian cancer cells may be similar to that of paclitaxel, both of which damage the structure of cancer cells by increasing ROS levels.

[0096] Example 10

[0097] In this example, flow cytometry was used to detect the effects of different concentrations of Streptococcus sanguinis extract XLS on the calcium ion level of human ovarian cancer cell SKOV3.

[0098] Ca 2+ Ca channels play an important role in tumorigenesis and development, including regulating cancer cell proliferation, migration, invasion and apoptosis. The above studies have shown that Streptococcus sanguinis extract XLS can inhibit the proliferation and migration of SKOV3 cells. 2+ Flow cytometry was used to detect the effect of Ca on the level of Streptococcus sanguis extract XLS. 2+ Concentration changes.

[0099] The flow cytometry detection method is as follows:

[0100] Ovarian cancer cells were seeded in 6-well plates at a density of 6×105 cells / well and cultured for 24 hours. The cells were treated with RPMI1640 complete medium containing 0μM, 10μM and 20μM of Streptococcus sanguinis extract XLS and RPMI1640 complete medium containing 20μM paclitaxel, respectively, and incubated at 37°C in the dark for 20 minutes in the presence of 1μg Oregon Green 488BAPTA-1. Ca was quantified by fluorescence microscopy and flow cytometry. 2+ level.

[0101] Fig.15 Flow cytometry was used to detect the Ca2+ expression of SKOV3 cells after treatment with different concentrations of XLS and paclitaxel. 2+ Horizontal comparison chart, the picture shows that compared with the untreated group, Streptococcus sanguinis extract XLS significantly increased the Ca of SKOV3 cells 2+ level, similar to the positive drug paclitaxel, both of which lead to Ca 2+ Imbalance.

Claims

1. Application of Streptococcus sanguinis extract in the preparation of drugs for preventing and treating cancer.

2. The use of the Streptococcus sanguis extract according to claim 1 in preparing a drug for preventing and treating cancer, characterized in that: The preparation method of the Streptococcus sanguis extract is as follows: The purified and identified Streptococcus sanguis was added to a BHI liquid culture medium, and cultured at a constant temperature of 37°C under anaerobic conditions for 48 hours. The Streptococcus sanguis suspension was placed at 4°C and 12000 r / min for 30 minutes, and the bacterial precipitate was collected. The obtained bacterial cells were resuspended with PBS buffer and centrifuged, and ultrasonically disrupted under ice-water bath conditions. The system obtained after ultrasonic disruption was placed at 4°C and 12000 r / min for 30 minutes, and the supernatant was collected; the obtained supernatant was subjected to gradient salting out with ammonium hydrochloride for 8 hours, and the precipitate was collected by low-temperature high-speed centrifugation at 4°C and 15000 r / min for 45 minutes. PBS buffer was added to dissolve the precipitate, and then desalted by passing through a cross-linked dextran column, and the liquid after the column was collected, dialyzed and concentrated, and freeze-dried to obtain a Streptococcus sanguis extract.

3. The use of the Streptococcus sanguis extract according to claim 1 or 2 in the preparation of a drug for preventing and treating cancer, characterized in that: The drug for preventing and treating cancer uses the Streptococcus sanguinis extract as the only active ingredient or one of the active ingredients.

4. The use of the Streptococcus sanguis extract in the preparation of a drug for preventing and treating cancer according to claim 3, characterized in that: The content of the Streptococcus sanguinis extract in the drug for preventing and treating cancer is 0.1wt% to 99wt%.

5. The use of the Streptococcus sanguis extract in the preparation of a drug for preventing and treating cancer according to claim 4, characterized in that: The drug for preventing and treating cancer also includes pharmaceutically acceptable adjuvants and / or carriers.

6. The use of the Streptococcus sanguinis extract in the preparation of a drug for preventing and treating cancer according to claim 5, characterized in that: The cancer is ovarian cancer.

7. The use of the Streptococcus sanguinis extract according to claim 6 in the preparation of a drug for preventing and treating cancer, characterized in that: The Streptococcus sanguinis extract can inhibit the proliferation, invasion and migration of human ovarian cancer cell SKOV3.

8. The use of the Streptococcus sanguinis extract in the preparation of a drug for preventing and treating cancer according to claim 7, characterized in that: The Streptococcus sanguinis extract inhibits the activation of the PI3K / AKT signaling pathway by inhibiting the phosphorylation of AKT, thereby inducing apoptosis of human ovarian cancer cell SKOV3 cells.

9. The use of the Streptococcus sanguinis extract in the preparation of a drug for preventing and treating cancer according to claim 8, characterized in that: The Streptococcus sanguinis extract disrupts mitochondrial membrane potential, increases oxidative stress and Ca 2+ Imbalance impairs the structure and function of human ovarian cancer cell SKOV3 cells.

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