Application of Streptococcus sanguis extract in preparing medicine for preventing and treating cancer

Abstract

The present invention relates to the use of a Streptococcus sanguis extract in the preparation of a drug for the prevention and treatment of cancer, belonging to the field of pharmaceutical technology. To develop a new use of the Streptococcus sanguis extract (XLS) in cancer treatment, the present invention provides the use of a Streptococcus sanguis extract in the preparation of a drug for the prevention and treatment of cancer. The present invention demonstrates the inhibitory effect of the Streptococcus sanguis extract (XLS) on the ovarian cancer cell line SKOV3. CCK-8 assays demonstrate that the Streptococcus sanguis extract (XLS) can reduce SKOV3 cell viability and inhibit cell proliferation, invasion, and migration. Molecular biological experiments demonstrate that XLS inhibits activation of the PI3K / AKT signaling pathway by inhibiting AKT phosphorylation, thereby regulating the expression of apoptosis-related genes and inducing apoptosis in SKOV3 cells. Furthermore, XLS impairs tumor cell structure and function by disrupting mitochondrial membrane potential, increasing oxidative stress, and causing Ca2+ imbalance.

Description

Technical Field

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

[0002] Bacteriocin-like substances are a class of peptides, proteins, or protein complexes with antibacterial activity, primarily produced by bacteria through ribosomal synthesis during metabolic processes. Bacteriocin-like substances exert their antimicrobial effects primarily by disrupting bacterial cell membranes, inhibiting cell wall synthesis, or inhibiting nucleic acid and protein synthesis. These substances are widely present in nature and, due to their natural properties, non-toxicity, and resistance to drug resistance, hold broad application prospects in a variety of fields, including antimicrobial agents, food preservation, and animal feed additives.

[0003] Streptococcus sanguis extract (XLS) is a bacteriocin-like substance extracted from oral Streptococcus sanguis. It has a strong inhibitory effect on fungi, particularly against fungal infections such as Candida albicans and Candida tropicalis. It can also inhibit the growth of Porphyromonas gingivalis. Streptococcus sanguis extract (XLS) offers numerous advantages, including safety, efficacy, lack of toxic side effects, and resistance resistance. It is expected to replace antibiotics that have developed extensive drug resistance, providing a new option for clinical antibacterial therapy. However, beyond its significant antibacterial effects, the mechanisms of action of Streptococcus sanguis extract (XLS) as a natural active substance are not fully understood, which has hindered its further development and application. Summary of the Invention

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

[0005] The technical solution of the present invention:

[0006] Application of Streptococcus sanguis 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 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 12,000 r / min for 30 minutes, and the bacterial precipitate was collected. The obtained bacterial cells were resuspended in PBS buffer and centrifuged, and ultrasonically disrupted in an ice-water bath. The resulting system after ultrasonic disruption was placed at 4°C and 12,000 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 15,000 r / min for 45 minutes. PBS buffer was added to dissolve the precipitate, and then desalted by passing through a cross-linked dextran column. 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 contains the Streptococcus sanguis 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 0.1 wt% to 99 wt%.

[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 sanguis extract can inhibit the proliferation, invasion and migration of human ovarian cancer cell SKOV3.

[0014] Furthermore, the Streptococcus sanguis 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 sanguis 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 present invention provides a new use of a Streptococcus sanguis extract XLS for preventing and treating cancer, namely, use of the Streptococcus sanguis extract in preparing a drug for preventing and treating cancer, especially use of the extract in preparing a drug for preventing and treating ovarian cancer.

[0018] This study demonstrates the inhibitory effect of Streptococcus sanguis extract (XLS) on the ovarian cancer cell line SKOV3. CCK-8 assays demonstrate that XLS can reduce SKOV3 cell viability and inhibit proliferation, invasion, and migration. Molecular biological studies have shown that XLS inhibits AKT phosphorylation, thereby inhibiting the activation of the PI3K / AKT signaling pathway, regulating the expression of apoptosis-related genes, and inducing apoptosis in SKOV3 cells. Furthermore, XLS also disrupts mitochondrial membrane potential, increases oxidative stress, and Ca 2+. 2+ Imbalance in these pathways can damage the structure and function of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0022] Figure 4 This is a comparison of the number of SKOV3 cell proliferation detected by plate colony formation assay after treatment with different concentrations of XLS;

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

[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 a Transwell chamber experiment;

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

[0026] Figure 8 This is a comparison of the scratch area of ​​SKOV3 after treatment with different concentrations of XLS in the scratch test;

[0027] Figure 9 The figure is a scatter plot of SKOV3 cells after being treated with different concentrations of XLS by flow cytometry;

[0028] Figure 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] Figure 11 This is a photo of Western Blot analysis of proteins related to the PI3K / AKT signaling pathway in SKOVS after treatment with different concentrations of XLS.

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

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

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

[0033] Figure 15 Flow cytometry was used to detect the Ca2+ level 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 with reference to the embodiments, but 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 scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0035] The purchase channels for the drugs, test 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 cytometry sorter V4-B5-R3 was purchased from Beckman Coulter, Inc., USA; and the q-PCR analyzer LIGHTCYCLER480II was purchased from Hoffmann-La Roche GmbH.

[0037] The following examples were statistically analyzed: Image data were processed using ImageJ software, and statistical analysis was performed using SPSS 25.0 software. All data are expressed as mean ± standard deviation, and all experiments were repeated three times. One-way analysis of variance was used to compare differences between multiple groups, and independent sample t-test was used to compare differences between two groups. P < 0.05 indicated that the difference was statistically significant. Statistical significance was indicated by * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001). 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 sanguis ATCC10556 was purchased from the Key Laboratory of Oral Biomedical Engineering of the Ministry of Health, 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 was prepared from Sephadex G-25 dextran gel, which absorbs 2.5 g of water per gram of gel when it swells. It has a separation range of 1000-5000 and 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 sanguis was added to BHI liquid culture medium and cultured at a constant temperature of 37°C under anaerobic conditions for 48 h. The Streptococcus sanguis suspension was immediately centrifuged at 4°C and 12,000 rpm 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 then resuspended in 100 mL of 1×PBS buffer and ultrasonically disrupted in an ice-water bath. The ultrasonic disruptor was operated in intermittent mode, specifically set to work for 4 seconds and stop for 8 seconds, with an ultrasonic power of 150 W and an ultrasonic time of 30 min.

[0045] The solution after ultrasonic disruption was centrifuged at 4°C and 12000 r / min for 30 minutes, and the supernatant was collected; the obtained supernatant was salted out with ammonium hydrochloride gradient at 4°C for 8 hours, and the precipitate was collected by low-temperature high-speed centrifugation at 4°C and 15000 r / min for 45 minutes, and an appropriate amount of PBS buffer was added to dissolve the precipitate, and then the precipitate was 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 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 sanguis 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 assay is as follows:

[0051] 96-well plates were cultured in a 37°C, 5% CO2 incubator until human ovarian cancer cell lines (SKOV3) reached 90% confluence. Complete medium containing 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM Streptococcus sanguis XLS was added. Simultaneously, SKOV3 cells were treated with complete medium containing 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM paclitaxel as a control. After 24 hours of treatment, 10 μL of CCK-8 solution was added to each well. The absorbance of the 96-well plates was measured using a microplate reader, and cell viability was calculated.

[0052] Figure 1 This is a comparison chart of SKOV3 cell viability after treatment with different concentrations of XLS detected by 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 SKOV3 cell viability after treatment with different concentrations of paclitaxel detected by 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-dependent effects on the activity of SKOV3 cells.

[0055] Example 3

[0056] In this example, a plate colony formation assay was used to detect the effects of different concentrations of Streptococcus sanguis 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 cells (SKOV3) were seeded in two 6-well plates, and 500 cells were counted. After cell attachment, the cells were treated with 0 μM, 10 μM, 20 μM, and 40 μM Streptococcus sanguis XLS extract for 24 hours. After incubation at 37°C for 24 hours, the medium was replaced with fresh complete medium and cultured until colonies of at least 15 cells were formed. After fixation, the cells were stained with 0.5% crystal violet for 30 minutes, and the number of visible cell colonies was counted.

[0059] Figure 3 The comparison photos of SKOV3 cell proliferation colonies after treatment with different concentrations of XLS were detected by plate colony formation assay. Figure 4 This is a comparison of the number of SKOV3 cell proliferation detected by plate colony formation assay 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 assay was used to detect the effects of different concentrations of Streptococcus sanguis extract XLS on the invasion of human ovarian cancer cell SKOV3 cells, with paclitaxel used as a control.

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

[0063] Dilute Matrigel gel at a ratio of 1:8 with serum-free medium or PBS. Add 60 μL of the solution evenly to the upper chamber of a Transwell membrane and incubate in a 37°C incubator for 3 hours to form a thin film. Human ovarian cancer cells (SKOV3) in the logarithmic growth phase were digested for 2-3 minutes, centrifuged, and the supernatant discarded. The cells were washed with PBS and resuspended to adjust the cell density to 1-10 × 10⁵ / mL. Add culture medium containing chemokines to the lower chamber of a 24-well plate. Place a Transwell chamber in the 24-well plate and add the cell suspension to the upper chamber. The concentrations of Streptococcus sanguinis extract (XLS) at 0 μM, 10 μM, and 20 μM, and paclitaxel at 20 μM, were varied. The cells were incubated for 12-48 hours. Finally, the number of cells that had crossed the membrane was determined.

[0064] Figure 5 Comparison photos of SKOV3 cell membrane permeability after treatment with different concentrations of XLS and paclitaxel in Transwell chamber experiments. 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 Transwell chamber experiments; Figure 5 and Figure 6 Paclitaxel significantly reduced the invasive ability of SKOV3 cells compared to the untreated control group, consistent with its known anti-tumor effects. Streptococcus sanguis extract XLS exhibited a concentration-dependent inhibition of SKOV3 cell invasion. Treatment with 10 μM and 20 μM of Streptococcus sanguis extract XLS reduced the number of migrating cells by approximately 33% and 45%, respectively, compared to the control group.

[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 cells (SKOV3) were evenly seeded into 6-well plates and cultured until a monolayer of cells formed. A scratch was made on the monolayer using a sterile pipette tip. The cells were treated with 10 μM and 20 μM Streptococcus sanguis extract (XLS) for 24 hours, respectively, and a blank control group was established. Images were captured immediately at the start of the scratch (0 hours) and again after 18 and 26 hours. The progress of wound healing was monitored using an inverted microscope, and changes in scratch area at different time points were analyzed to assess cell migration ability.

[0069] Figure 7 Comparative photos of the wound healing of SKOV3 after treatment with different concentrations of XLS in the scratch test. Figure 8This is a comparison of the scratch area of ​​SKOV3 after treatment with different concentrations of XLS in the scratch test; Figure 7 and Figure 8 The results showed that compared with the control group, Streptococcus sanguis extract XLS could inhibit the migration ability of SKOV3 cells. With the increase of Streptococcus sanguis extract XLS concentration, the wound healing rate of SKOV3 cells was significantly reduced, showing a concentration-dependent manner.

[0070] Example 6

[0071] In this example, flow cytometry was used to detect the effects of different Streptococcus sanguis extracts XLS on the apoptosis of human ovarian cancer cells 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 trypsinized. The cells were harvested and the suspension transferred to an EP tube and centrifuged at 2500 rpm for 5 minutes at room temperature. The cells were washed twice with 4°C pre-cooled PBS and resuspended in 300μL of 1× Binding Buffer. Annexin V-FITC / PI double staining was performed by adding 5μL of Annexin V-FITC and incubating at room temperature for 15 minutes in the dark. Five minutes before loading, 5μL of PI was added for staining, and 200μL of 1× Binding Buffer was added.

[0074] Figure 9 This is a scatter plot of SKOV3 cells after being treated with different concentrations of XLS by flow cytometry. Figure 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; Figure 9 and Figure 10 The results showed that compared with the control group, 5μM and 10μM Streptococcus sanguis extract XLS had no significant effect on early and late cell apoptosis. However, when the concentration of Streptococcus sanguis extract XLS was 20μM, the early and late apoptosis rates of the cells increased to 4.65% and 66.82%, respectively, significantly higher than the control group, indicating that 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 protein immunoblotting experiment (Western Blot) method is as follows:

[0078] Human ovarian cancer cells (SKOV3) were treated with 0μM, 5μM, 10μM, and 20μM Streptococcus sanguinis extract XLS. Cellular proteins were extracted using the RIPA lysis solution according to the manufacturer's instructions. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was then blocked with 5% skim milk for 2 hours. After blocking, the primary antibody was added and incubated overnight at 4°C. Following 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 applied to the PVDF membrane. The results were exposed and recorded in the dark using an automated imaging system.

[0079] Figure 11 Western blot analysis of proteins involved in the PI3K / AKT signaling pathway in SKOVS cells after treatment with different concentrations of XLS. The images show that compared with the control group, treatment with Streptococcus sanguis extract XLS did not significantly affect PI3K and AKT protein levels, but significantly decreased p-AKT protein levels in a concentration-dependent manner. This suggests that Streptococcus sanguis extract XLS may inhibit PI3K / AKT signaling pathway activation by inhibiting AKT phosphorylation. Furthermore, treatment with Streptococcus sanguis extract XLS significantly upregulated the apoptotic protein Bax and downregulated the anti-apoptotic protein Bcl-2.

[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 were analyzed after treatment with 0 μM, 5 μM, 10 μM, and 20 μM of Streptococcus sanguis extract XLS. Cells were lysed with Trizol reagent. Cellular RNA was extracted according to the Trizol manufacturer's instructions. RNA concentration was measured using a Nanodrop spectrophotometer, and reverse transcription was performed using a cDNA reverse transcription kit. After preparing the q-PCR reaction system, fluorescence quantitative PCR was performed. 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. Figure 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 sanguis extract XLS promoted the apoptosis of ovarian cancer SKOV3 cells by upregulating the mRNA levels of pro-apoptotic proteins Bax, p53 and mTOR, while 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 primarily present on the mitochondrial membrane, while Bax translocates to the mitochondrial surface in response to apoptotic signals, forming transmembrane pores. This leads to a decrease in membrane potential and an increase in membrane permeability, releasing apoptotic factors (such as cytochrome c), thereby affecting cellular status. Abnormalities in mitochondrial membrane potential are closely associated with the development of cancer. To further explore the effects of Streptococcus sanguis extract (XLS) on apoptosis mechanisms, this example examined characteristic parameters and expression levels of mitochondrial-related substances. The JC-1 fluorescent probe was used to examine the effects of Streptococcus sanguis extract (XLS) on mitochondrial membrane potential in SKOV3 cells.

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

[0088] Cells were seeded at a density of 6×105 cells / well in 6-well plates and cultured for 24 hours. Subsequently, cells were treated with RPMI1640 complete medium containing 0μM, 10μM, or 20μM Streptococcus sanguis XLS extract (2mL) per well and cultured for an additional 24 hours. Following treatment, 1mL of RPMI1640 and 1μL of JC-1 were added to each well and incubated in the dark for 20 minutes. Mitochondrial membrane potential was measured by flow cytometry, with untreated cells serving as a negative control.

[0089] Figure 13 Fluorescence micrographs of SKOV3 cells treated with different concentrations of XLS using the JC-1 fluorescent probe. The images show that in the control group, JC-1 exists as multimers, exhibits red fluorescence, and has a high mitochondrial membrane potential. However, after treatment with the Streptococcus sanguis extract XLS, green fluorescence significantly increases, and the mitochondrial membrane potential decreases significantly. These results indicate that treatment with the Streptococcus sanguis extract XLS significantly decreases the mitochondrial membrane potential of SKOV3 cells and promotes apoptosis.

[0090] Example 9

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

[0092] ROS play a key role in cell signaling and metabolic homeostasis. Under environmental stress, ROS levels increase significantly, leading to oxidative stress damage to cellular structures. To investigate the effects of Streptococcus sanguis extract (XLS) on oxidative stress in SKOV3 cells, flow cytometry was used to measure changes in ROS levels in SKOV3 cells following treatment with S. sanguis extract (XLS).

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

[0094] Ovarian cancer SKOV3 cells were seeded in 6-well plates and incubated for 24 hours. The cells were treated with RPMI1640 complete medium containing 0 μM, 10 μM, or 20 μM Streptococcus sanguis XLS, or RPMI1640 complete medium containing 20 μM paclitaxel, respectively, at a 2 mL dose per well for 24 hours. After trypsinization, the cells were stained using a ROS detection kit, and ROS levels were measured by flow cytometry using the FITC channel.

[0095] Figure 14 This flow cytometric comparison shows reactive oxygen species (ROS) levels in SKOV3 cells after treatment with different concentrations of XLS and paclitaxel. The image shows that treatment with XLS, a Streptococcus sanguinis extract, significantly increased ROS levels in SKOV3 cells in a concentration-dependent manner. This trend is consistent with changes in cell membrane potential. These results suggest that the mechanism by which XLS, a Streptococcus sanguinis extract, inhibits ovarian cancer cells may be similar to that of paclitaxel, both by increasing ROS levels and damaging cancer cell structure.

[0096] Example 10

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

[0098] Ca 2+ Ca channels play an important role in tumorigenesis and progression, including regulating the proliferation, migration, invasion and apoptosis of cancer cells. The above studies have shown that Streptococcus sanguis 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. Cells were treated with RPMI1640 complete medium containing 0 μM, 10 μM, and 20 μM Streptococcus sanguis XLS, or RPMI1640 complete medium containing 20 μM paclitaxel, and incubated in the dark at 37°C for 20 minutes in the presence of 1 μg Oregon Green 488BAPTA-1. Ca quantification was performed by fluorescence microscopy and flow cytometry. 2+ level.

[0101] Figure 15 Flow cytometry was used to detect the Ca2+ level 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 sanguis extract XLS significantly increased the Ca of SKOV3 cells 2+ levels, similar to the positive drug paclitaxel, both of which lead to Ca 2+ Imbalance.

Claims

1. Use of a Streptococcus sanguis extract in preparing a drug for preventing and treating cancer, characterized in that: The cancer is ovarian cancer, and the preparation method of the Streptococcus sanguis extract is as follows: The purified and identified Streptococcus sanguis was added to 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 12,000 r / min for 30 minutes, and the bacterial precipitate was collected. The obtained bacterial cells were resuspended in PBS buffer and centrifuged, and ultrasonically disrupted in an ice-water bath. The resulting system after ultrasonic disruption was placed at 4°C and 12,000 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 15,000 r / min for 45 minutes. PBS buffer was added to dissolve the precipitate, and then desalted by passing through a cross-linked dextran column. The liquid after the column was collected, dialyzed and concentrated, and freeze-dried to obtain a Streptococcus sanguis extract.

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

3. The use of the Streptococcus sanguis extract in preparing a drug for preventing and treating cancer according to claim 2, characterized in that: The content of the Streptococcus sanguis extract in the cancer preventing and treating medicine is 0.1 wt% to 99 wt%.

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 drug for preventing and treating cancer further comprises pharmaceutically acceptable excipients and / or carriers.

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