CN113116917A

CN113116917A - Application of 8-Br-cGMP as PKG I activator in preparation of medicine for preventing or treating ovarian epithelial cancer

Info

Publication number: CN113116917A
Application number: CN202110270575.5A
Authority: CN
Inventors: 蓝婷; 李颖; 罗兰; 王忠诚
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2021-07-16
Anticipated expiration: 2041-03-12
Also published as: CN113116917B

Abstract

The invention discloses application of 8-Br-cGMP as a PKG I activator in preparation of a medicine for preventing or treating ovarian epithelial cancer, and belongs to the field of biomedicine. Experiments show that 8-Br-cGMP can inhibit EGF-induced EOC cell proliferation, migration and invasion, and inhibit the growth of ovarian epithelial cancer by promoting the phosphorylation of threonine 693 site of EGFR protein, inhibiting the phosphorylation of tyrosine 1068 site of EGFR protein and activating downstream MAPK/ERK signal pathways, thereby indicating that 8-Br-cGMP can be used as a PKGI specific activator for treating or preventing the ovarian epithelial cancer.

Description

Application of 8-Br-cGMP as PKG I activator in preparation of medicine for preventing or treating ovarian epithelial cancer

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of 8-Br-cGMP as a PKG I activator in preparation of a medicine for preventing or treating ovarian epithelial cancer.

Background

Ovarian epithelial cancer (EOC) is the most common type of ovarian cancer, accounting for 75-100% of primary ovarian cancers. Only about 30% of ovarian epithelial cancer patients have tumors confined to the ovaries, with the remainder spreading to the uterus, bilateral appendages, greater omentum, and pelvic cavity. Meanwhile, statistical data at home and abroad show that 90% of patients with early-stage diagnosed ovarian cancer can survive for more than 5 years after operation and interventional therapy, and the survival rate of the patients with late-stage ovarian cancer who have metastasized is less than 30% in 5 years.

PKG I is a type I cyclic guanosine monophosphate dependent protein kinase, belonging to the serine/threonine protein kinase family, widely present in eukaryotic cells. The PKG-I is in an activated state after being combined with cyclic guanosine monophosphate (cGMP), and the cGMP is formed by combining and activating soluble guanylate cyclase GC and NO and catalyzing by taking guanosine triphosphate GTP as a substrate, so the cascade activation signal path is also called as a NO-GC-cGMP-PKG-I signal path. Wherein, the 8-Br-cGMP is a hydrolysis-resistant cGMP derivative which has stable performance and is difficult to be hydrolyzed by phosphodiesterase, and is also a specific activator of PKG-I. PKG-I activation can cause a range of physiological changes, such as vasodilation of vascular smooth muscle, inhibition of platelet aggregation, cytoskeletal remodeling, and the like.

Current studies indicate that PKGI affects cell proliferation, migration, and invasion in prostate and colon cancers. In the prostatic cancer, a PDE5-cGMP-PKG signal pathway controls the stem cell characteristics and differentiation of prostatic cancer cells, and the intervention of PDE5 can effectively inhibit the occurrence, metastasis and recurrence of prostatic cancer. In addition, activated PKG-I can also be regulated by Ca²⁺And (3) internal flow, and ERK, beta-catenin/TCF and AKT signal transduction are inhibited, so that colon cancer cell proliferation is inhibited. However, the role of PKG I in the pathogenesis of ovarian cancer is not well defined, and only studies have reported that PKGI activity in ovarian cancer cells is associated with DNA synthesis and can inhibit spontaneous apoptosis of the cells. Therefore, the intensive research on the role of PKG-I activity in EOC and the specific mechanism thereof is helpful for defining the role of PKG-I in the pathogenesis of EOC and providing a new strategy and thought for the treatment of the ovarian epithelial cancer.

Disclosure of Invention

The invention aims to provide a new medical application of 8-Br-cGMP, and the 8-Br-cGMP can inhibit the proliferation, migration and invasion of EOC cells induced by Epidermal Growth Factor (EGF) and inhibit the growth of ovarian epithelial cancer; in addition, the invention discusses the activity level and action mechanism of PKG I in the ovarian epithelial cancer cells, and provides important data for further developing drugs aiming at the ovarian epithelial cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

provides the application of 8-Br-cGMP as a PKG-I activator in preparing medicaments for preventing or treating the epithelial ovarian cancer.

The structural formula of the 8-Br-cGMP is shown as follows:

preferably, the medicament is used for regulating a cGMP-PKG I signal pathway.

Preferably, the medicament is used for promoting phosphorylation of threonine 693 site of EGFR protein, inhibiting phosphorylation of tyrosine 1068 site of EGFR protein and activating downstream MAPK/ERK signaling pathway.

Further, the ovarian epithelial cancer is ovarian epithelial cancer cell proliferation, migration and invasion induced by epidermal growth factor EGF.

Preferably, the dosage of the 8-Br-cGMP is 20 mg/kg.

In another aspect, the present invention provides a pharmaceutical formulation for treating ovarian epithelial cancer, comprising (1) an effective amount of 8-Br-cGMP as an active ingredient, and (2) optionally a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical dosage form is capsule, tablet, powder, oral liquid, pill, tincture, syrup or injection.

Cell experiments and animal experiments show that 8-Br-cGMP can inhibit the proliferation, migration and invasion of EOC cells induced by Epidermal Growth Factor (EGF), and inhibit the growth of ovarian epithelial cancer by promoting the phosphorylation of threonine 693 site of EGFR protein, inhibiting the phosphorylation of tyrosine 1068 site of EGFR protein and activating downstream MAPK/ERK signal pathways, thereby indicating that 8-Br-cGMP can be used as a PKG-I activator for preparing a medicament for preventing or treating the ovarian epithelial cancer. The PKG I in the normal ovarian epithelial cells and the ovarian epithelial cancer cells provided by the invention has activity difference, the PKG I specific activator 8-Br-cGMP can inhibit the proliferation, migration and invasion activity of the ovarian epithelial cancer, and the growth of the ovarian epithelial cancer transplanted on a mouse has an obvious inhibition effect, so that a new strategy and a new treatment mode are provided for clinically treating the ovarian epithelial cancer.

Drawings

FIG. 1 is a graph showing the results of Western blot analysis of normal ovarian epithelial (IOSE80) and ovarian epithelial cancer cells (SKOV3, A2780);

FIG. 2 is a graph showing the results of Western blot analysis of ovarian epithelial cancer cells SKOV3 and A2780 after addition of PKG I activator 8-Br-cGMP at various concentrations: (A) SKOV 3; (B) a2780;

FIG. 3 is a graph showing the results of the phosphorylation levels of the p-EGFR (Y1068) site and downstream MAPK/ERK pathway in cells after various concentrations of the PKG I activator 8-Br-cGMP were added to the ovarian epithelial cancer cell SKOV3 and the ovarian epithelial cancer cell A2780: (A) SKOV 3; (B) a2780;

FIG. 4 is a graph showing the results of the phosphorylation level analysis of threonine 693(T693) site of EGFR caused by the addition of 8-Br-cGMP-activated PKG I to ovarian epithelial cancer cell A2780;

FIG. 5 is a comparison graph of cell numbers after addition of different concentrations of PKG I activator 8-Br-cGMP to ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780: (A) SKOV 3; (B) a2780;

FIG. 6 is a comparison graph of cell number statistics at different times after addition of 250. mu.M PKG I activator 8-Br-cGMP to ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780: (A) SKOV 3; (B) a2780;

FIG. 7 is a graph showing the result of staining the clone cells after adding PKG I activator 8-Br-cGMP to ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780 and a statistical graph showing the number of colonies formed by the flat cells; (A) a plot of clonal cell staining results for SKOV 3; (B) the plate cell clone formation number of SKOV3 is shown as a statistical chart; (C) a graph showing the results of staining the clonal cells of A2780; (D) a2780 plate cell clone forming a number statistical chart;

FIG. 8 is an appearance map and a statistical map of scratch area of a scratch board after adding PKG I activator 8-Br-cGMP into ovarian epithelial cancer cells SKOV3 and ovarian epithelial cancer cells A2780; (A) a scratch board appearance map of SKOV 3; (B) a scratch area statistical plot of SKOV 3; (C) a2780 scratch board appearance; (D) a2780, a scratch area statistical chart;

FIG. 9 is a graph showing the result of staining and the number statistics of migrating cells detected by the Trans-well method after adding PKG I activator 8-Br-cGMP to ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780; (A) migration cell staining results of SKOV 3; (B) a statistical plot of the number of migrating cells of SKOV 3; (C) a2780 migration cell staining result chart; (D) a2780 migration cell number histogram;

FIG. 10 is a graph showing the result of staining and the number statistics of invaded cells detected by the Trans-well method after adding PKG I activator 8-Br-cGMP to ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780; (A) a plot of invasive cell staining results for SKOV 3; (B) a statistical plot of the number of invading cells of SKOV 3; (C) A2780 invasion staining result graph; (D) a2780 statistical chart of the number of invading cells;

FIG. 11 is a graph showing the comparison between the tumor material (A) and the tumor weight data in the abdominal cavity of a mouse after the mouse is injected with 8-Br-cGMP with different concentrations into the abdominal cavity;

FIG. 12 is a real image of the liver in mice after injecting 8-Br-cGMP with different concentrations into the abdominal cavity of the mice.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.

Unless otherwise indicated, reagents, methods and equipment used in the present invention are conventional in the art, and materials of kits used in the following examples are commercially available.

Firstly, experimental steps

Experimental animals and experimental design:

normal human ovarian epithelial cells (IOSE80) and human ovarian epithelial cancer cells (SKOV3, A2780) were purchased from the cell resource center of Shanghai Life sciences institute of Chinese academy of sciences.

Female nude mice were purchased from Experimental animals technology, Inc. of Weitonglihua, Beijing under license number SCXK (Jing) 2007-0001. The mice are placed under the standard conditions of humidity of 50 +/-10% and temperature of 23 +/-2 ℃ for adapting to survival for 12 hours every day and night. Mice had free access to water and food. All animal management and treatment protocols were approved by the animal ethics committee of xu nationality medical university. All experiments were performed as recommended by the ethical guidelines for managing and using animal behavior.

Reagent:

8-Br-cGMP and Rp-8-Br-cGMP were purchased from BIOLOG, Germany.

Antibody: p-EGFR (Y1068), p-ERK/ERK, p-MEK/MEK were purchased from Cell Signal Technology; EGFR antibody, PKG I antibody, p-VASP (Ser239) antibody, and IgG antibody were purchased from Affinity; EGF is purchased from proteintech.

1. Western blot analysis:

preparing polyacrylamide gel with corresponding concentration according to the molecular weight of the target protein. And (3) putting the prepared gel into an electrophoresis tank, and pouring electrophoresis buffer solution. After loading, the protein was accumulated in the gel at a constant pressure of 80V, and when the protein entered the gel, the protein was separated at a constant pressure of 100V. After the electrophoresis is finished, the proteins on the gel are transferred to a PVDF membrane. The PVDF membrane should be soaked in methanol for 15s in advance and then put into a membrane transferring buffer solution for balancing for more than 5 min. The sequence of placing from the cathode to the anode during film transfer is as follows: sponge, filter paper, gel, PVDF membrane, filter paper, sponge, with care being taken to drive out air bubbles between the gel and the membrane. The film transfer conditions were 4 ℃, 90mA,12 h. After membrane transfer, the membrane was blocked with 5% skim milk at room temperature for 1 h. The main antibodies used were EGFR, PKG I and p-VASP (Ser239) (affinity), diluted in the proportions indicated for each antibody, and incubated for 12h at 4 ℃. The next morning, the membranes were removed and washed three times with TBS-T for 10min each. HRP-labeled IgG antibody was diluted with TBS-T in a ratio of 1: 10000, incubation at room temperature for 1 h. Three washes with TBS-T for 10min each. The luminophores were applied to the protein side of the film and the results were recorded using a berle chemiluminiscence imaging system.

2. CCK-8 experiment, the procedure was as follows:

(1) plate paving and dosing: selecting SKOV3 and A2780 cells of ovarian epithelial cancer cells in logarithmic growth phase, digesting and counting, adjusting cell concentration to 4 × 104/mL, inoculating into 96-well plate, wherein each well is 100 μ L, and adding appropriate amount of PBS to the periphery of the 96-well plate to prevent water evaporation. After the cells are attached to the wall, starving the cells by using a serum-free culture medium overnight, respectively adding EGF, 8-Br-cGMP and Rp-8-Br-cGMP with corresponding amounts into each hole according to different groups, and putting the holes into a culture box for culture;

(2) termination and measurement: adding 10 μ L of CCK-8 reagent into each well in dark condition, mixing well, standing at 37 deg.C and 5% CO₂Reading at 450nm of an enzyme-labeling instrument in the incubator for 1-2 h.

3. Plate clone formation experiments, the procedure was as follows:

(1) plate paving and dosing: digesting and counting cells, adjusting cell concentration to 500/mL, inoculating into 6-well plate with 1mL per well, mixing, placing at 37 deg.C and 5% CO₂After the cells adhere to the wall, adding EGF and 8-Br-cGMP with corresponding amounts according to different groups respectively;

(2) culturing: culturing in a 37 deg.C cell culture box for 10-15 days, observing cell state during culture, timely replacing culture solution and re-administering, and stopping culture when the number of cells with single cell colony in the culture dish is more than or equal to 50;

(3) fixing and dyeing: removing the culture medium, washing with PBS 3 times, fixing with 4% paraformaldehyde for 15min, washing with PBS 3 times, dyeing with crystal violet dye solution for 15min, washing with deionized water, and air drying at room temperature;

(4) photographing and analyzing: the 6 well plates were placed upside down on white paper, photographed and statistically analyzed with Image J software.

4. Scratch test, the procedure was as follows:

(1) plate paving: one day before the experiment, a straight ruler, a marking pen and a 10 mu L high-pressure gun head are subjected to ultraviolet irradiation in a biological safety cabinet for 30min, and the marking pen is used for marking a straight line crossing the through hole on the back of the 6-hole plate; digesting the cells and inoculating the cells into a 6-well plate, wherein the inoculation density is about 90% of the fusion degree of the adherent cells on the next day;

(2) scratching and dosing: during the experiment, a sterile 10-mu-L gun head is used to mark than a ruler, the same angle and force are used for marking, and each hole is marked with 3 straight lines. Washing with PBS for 2 times, adding 1mL DMEM medium containing 1% FBS, adding EGF and 8-Br-cGMP with corresponding amounts into each well according to different groups, and placing the 6-well plate into an incubator for continuous culture;

(3) photographing and analyzing: photographs were taken at the same positions at 0h, 24h, and 48h, respectively, and the wound distances were measured at each time point, and the experimental results were analyzed by Image J software.

5. Trans-well migration and invasion experiments

The differences between the Trans-well migration and the invasion assay are that the invasion assay requires a basement membrane precoated on the upper layer of the chamber, and in addition, the incubation time of the invasion assay is 24h longer than that of the migration assay.

(1) Invasion assay coating basement membrane: the night before the experiment, Matrigel was removed from-20 ℃ and thawed overnight at 4 ℃. Diluting Matrigel matrix glue and a pre-cooled serum-free culture medium according to a ratio of 1: 5, adding 100 mu L of diluted glue into an upper chamber of a Trans-well chamber to avoid bubbles, putting the chamber into a 24-hole plate, and putting the chamber into an incubator for 4-6 hours;

(2) plate paving and dosing: the cells were digested and counted, the cell density was adjusted to 2X 105/mL using serum-free culture, 200. mu.L of cell suspension was aspirated into the upper chamber layer, 600. mu.L of complete medium into the lower chamber layer, and the chambers were placed in 24-well plates to avoid air bubbles. Adding EGF, 8-Br-cGMP and Rp-8-Br-cGMPS with corresponding amounts into each chamber according to different groups, placing the chambers into an incubator, performing migration experiment culture for 24 hours, and performing invasion experiment culture for 48 hours;

(3) fixing and dyeing: the chamber was removed and the medium was discarded. Fixing with 4% paraformaldehyde for 15min, washing with PBS for 3 times, dyeing with crystal violet dye for 10min, washing with PBS for 3 times, and slightly wiping off cells which do not pass through the upper chamber with cotton swab;

(4) photographing and analyzing: the chamber was removed, photographed under a 200-fold inverted microscope, and five field cells were counted at random.

6. Nude mouse abdominal cavity transplantation tumor model

(1) Subcutaneous tumorigenicity in nude mice: 2 female nude mice of 4 weeks old were selected, A2780 cells were digested and counted, and the cell density was adjusted to 5X 10 with PBS ⁶200 μ L of the strain, inoculated on the right back of the forelimb of nude miceUnder the skin;

(2) sacrifice and tumor removal: at about two weeks, the subcutaneous tumor volume is about 1cm long³Nude mice were sacrificed, immersed in 75% alcohol for 3min, following the work in a clean bench: placing a nude mouse in a sterile big dish, taking out the tumor with sterile scissors and tweezers, removing surrounding connective tissues, washing with sterile PBS, and placing the tumor in another sterile big dish;

(3) digestion and selection of cells: cutting tumor as much as possible, adding a proper amount of 0.125% pancreatin, putting into a 37 ℃ incubator, and digesting for 8-10 times by times, wherein each time is 5 min. The supernatant from each digestion was collected into a centrifuge tube containing complete medium, filtered through a 200 mesh cell filter screen, and centrifuged at 800rpm for 5 min. Washing with PBS for 3 times, completely culturing basic suspension cells, inoculating into a big dish, and culturing in an incubator;

(4) intraperitoneal injection of nude mice: 12 nude mice of 6 weeks old were randomly divided into 3 groups of 4 mice each, namely, a control group, a low concentration 8-Br-cGMP group (5mg/kg), and a high concentration 8-Br-cGMP group (20 mg/kg). Digesting and counting the A2780 cells when the cells are cultured in vitro for 4-5 passages, and adjusting the cell density to 5 multiplied by 10 by PBS⁶One of the cells was injected into the abdominal cavity of a nude mouse at a volume of 200. mu.L. The next day, 200. mu.L of sterile PBS was intraperitoneally injected into the control group, and the corresponding 8-Br-cGMP was intraperitoneally injected into the 5mg/kg group and the 20mg/kg group, respectively. Then, the abdominal cavity is injected with 8-Br-cGMP once every three days, and the injection is repeated for 5 times;

(5) sacrifice and dissection: feeding normally, observing the state of the nude mice every day, after about 2 weeks, killing and dissecting all the nude mice, taking out the liver and the tumor, weighing, photographing and recording.

Second, experimental results

1. Comparison of protein expression and phosphorylation levels of epidermal growth factor receptors EGFR, PKG I and p-VASP (Ser239) which expresses PKG-I activity in normal ovarian epithelial cells (IOSE80) and ovarian epithelial carcinoma cells (SKOV3, A2780)

As can be seen from FIG. 1, the expression levels of EGFR and PKG I were higher in the ovarian epithelial cancer cells compared to the normal ovarian epithelial cells, but the level of p-VASP (Ser239) was lower, indicating that the PKG I activity of the ovarian epithelial cancer cells was lower.

2. Phosphorylation levels of p-VASP (Ser239) which expresses PKG I activity after adding different concentrations of PKG I activator 8-Br-cGMP into ovarian epithelial cancer cells SKOV3 and ovarian epithelial cancer cells A2780

After adding various concentrations of the PKG I-specific activator 8-Br-cGMP, the results are shown in FIG. 2, from which it can be seen that the PKG I activity, namely the p-VASP (Ser239) level, is significantly increased, and as the concentration of 8-Br-cGMP is increased, the p-VASP (Ser239) level is also increased, the p-VASP (Ser239) level is significantly increased after the addition of 8-Br-cGMP at a concentration of 250. mu.M.

3. Phosphorylation levels of p-EGFR (Y1068) site and downstream MAPK/ERK pathway in cells after adding different concentrations of PKG I activator 8-Br-cGMP to ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780

After adding different concentrations of PKG I-specific activator 8-Br-cGMP, the result is shown in figure 3, the phosphorylation level of the tyrosine 1068 site of EGFR is obviously reduced compared with the EGF group, and the phosphorylation levels of other proteins c-raf, ERK and MEK of MAPK/ERK pathway are also obviously inhibited.

4. 8-Br-cGMP activated PKG I causes phosphorylation of threonine 693(T693) site of EGFR to inhibit phosphorylation of EGFR tyrosine 1068 site

The western immunoblotting results show that, as shown in fig. 4, the threonine 693 site of EGFR is phosphorylated after 8-Br-cGMP is added based on EGF, compared to the control group and the group with EGF alone, indicating that PKG I as a serine/threonine protein kinase causes serine/threonine phosphorylation of EGFR after 8-Br-cGMP activates PKG I. And simultaneously detecting the EGFR tyrosine 1068 site related to the activation of MAPK/ERK pathway, and displaying that compared with the EGF group, the phosphorylation of the EGFR tyrosine 1068 site is obviously reduced after simultaneously adding 500 mu M of 8-Br-cGMP.

5. Influence of different concentrations of PKG I activator 8-Br-cGMP in ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780 on cell proliferation capacity

After various concentrations of the PKG I-specific activator 8-Br-cGMP were added to the ovarian epithelial cancer cells SKOV3 and ovarian epithelial cancer cells A2780, the PKG I was activated by adding various concentrations of 8-Br-cGMP (50. mu.M, 250. mu.M, 500. mu.M) while treating the cells with EGF (200 ng/mL). As shown in FIG. 5, it can be seen that the cell proliferation of the EGF (200ng/mL) group was significantly increased as compared with the control group without any treatment; compared with EGF group, 8-Br-cGMP (250. mu.M and 500. mu.M) group showed significant inhibition of cell proliferation.

6. Influence on cell proliferation capacity at different times after adding certain concentration of PKG I activator 8-Br-cGMP into ovarian epithelial cancer cells SKOV3 and A2780

After adding different concentrations of PKG I specific activator 8-Br-cGMP into ovarian epithelial cancer cells SKOV3 and ovarian epithelial cancer cells A2780, EGF (200ng/mL) is used for treating the cells, 250 mu M of 8-Br-cGMP is added for different time, and cell proliferation conditions at 12h, 24h and 48h are detected. The results are shown in FIG. 6, from which it can be seen that the proliferation of the 8-Br-cGMP group of cells is significantly reduced at 24h and 48 h.

7. Influence on cell clone forming capability after adding PKG I activator 8-Br-cGMP into ovarian epithelial cancer cell SKOV3 and ovarian epithelial cancer cell A2780

The results of the cell clonogenic experiments are shown in FIG. 7, from which it can be seen that the clonogenic capacity of the EGF group is significantly enhanced compared to the control group without any treatment; particularly SKOV3 cell, the number and area size of its cell clone increased nearly one-fold compared to the control group, and the number and area size of cell clone formation decreased nearly 50% compared to the EGF group after addition of 8-Br-cGMP (500. mu.M).

The CCK-8 experiment and the plate cell clone formation experiment together show that the addition of 8-Br-cGMP can inhibit EGF-induced EOC cell proliferation.

8. After the PKG I activator 8-Br-cGMP is added into the ovarian epithelial cancer cells SKOV3 and A2780, the influence on the cell migration capacity is detected by a scratch test

The result of the scratch analysis is shown in fig. 8, when cultured for 24h after scratching, the area and distance of the scratch of the cells of the EGF group are significantly increased compared to those of the control group, indicating that EGF can promote cell migration. After 8-Br-cGMP is added, the cell migration distance is obviously shortened, and as seen from a statistical chart of the scratch area, the addition of 8-Br-cGMP is increased by nearly 50 percent compared with the EGF group, and the scratch area and distance are gradually increased along with the increase of concentration and time, which shows that the 8-Br-cGMP obviously inhibits EGF-stimulated cell migration.

9. After the PKG I activator 8-Br-cGMP is added into the ovarian epithelial cancer cell SKOV3 and the ovarian epithelial cancer cell A2780, the influence on the migration and invasion capacity of the cells is detected by a Trans-well method

The effect of PKG I on the ability of cells to migrate was confirmed by the Trans-well migration and invasion experiments. From fig. 9 and 10, it can be seen that the cells stained purple are cells that migrate and invade across membrane, and the number of cells migrating and invading SKOV3 and a2780 cells after EGF stimulation is significantly increased compared to the control group, and it can be seen from the statistical chart that the EGF group is increased by 50% compared to the control group, while the number of cells migrating and invading after the simultaneous addition of 8-Br-cGMP is significantly decreased compared to the EGF group. The data from the statistical plot show a 25% reduction in cells after addition of 8-Br-cGMP. The results show that EGF can remarkably stimulate migration and invasion of SKOV3 and A2780 cells, and activator PKG I can inhibit EGF-stimulated cell migration and invasion.

10. The total number and volume of tumor bodies of the mice and the number and position of tumor bodies transferred in the liver after injecting 8-Br-cGMP with different concentrations into the abdominal cavity of the mice

The results of the intraperitoneal tumorigenic model of nude mice are shown in fig. 11 and 12, and the effect of the injection of different concentrations of 8-Br-cGMP to activate PKG I on the transplanted tumor is clarified. The control group was a control group injected with PBS, and the treatment groups were injected with 5mg/kg and 20mg/kg of 8-Br-cGMP, respectively. FIG. 11(A) is a graph of the intraperitoneal tumor of different groups, and the tumor volume of the injection of 8-Br-cGMP is smaller than that of the control group, wherein 20mg/kg of the tumor volume is more obvious; as seen from FIG. 11(B), the tumor weights of the 5mg/kg and 20mg/kg groups were lower than that of the control, indicating that the 20mg/kg dose of 8-Br-cGMP had a significant tumor-inhibiting effect. FIG. 12 is a real image of liver in different groups, and the results show that the number of transfer points in liver in control group is about 3-5, and the number of transfer points in liver in 5mg/kg group and 20mg/kg group is less than that in control group.

The cell and animal experiments show that the 8-Br-cGMP can inhibit EGF-induced EOC cell proliferation, migration and invasion, and inhibit the growth of ovarian epithelial cancer by promoting the phosphorylation of threonine 693 site of EGFR protein, inhibiting the phosphorylation of tyrosine 1068 site of EGFR protein and activating downstream MAPK/ERK signaling pathway. Indicating that 8-Br-cGMP can be used as a PKGI specific activator for treating or preventing the ovarian epithelial cancer.

Claims

Application of 8-Br-cGMP as PKG I activator in preparing medicine for preventing and treating epithelial ovarian cancer.
2. Use of 8-Br-cGMP as a PKG I activator in the preparation of a medicament for the prevention or treatment of epithelial ovarian cancer according to claim 1, wherein said medicament is for modulating the cGMP-PKG I signaling pathway.
3. The use of 8-Br-cGMP as a PKG I activator in the preparation of a medicament for the prevention or treatment of epithelial ovarian cancer according to claim 2, wherein the medicament is for promoting phosphorylation at threonine 693 site of the EGFR protein, inhibiting phosphorylation at tyrosine 1068 site of the EGFR protein, and activating downstream MAPK/ERK signaling pathway.
4. Use of 8-Br-cGMP as a PKG I activator in the manufacture of a medicament for the prevention or treatment of epithelial ovarian cancer according to claim 1 or 2, wherein the epithelial ovarian cancer is proliferation, migration and invasion of epithelial ovarian cancer cells induced by epidermal growth factor EGF.
5. The use of 8-Br-cGMP as a PKG I activator in the manufacture of a medicament for the prevention or treatment of epithelial ovarian cancer according to claim 1 or 2, wherein the amount of 8-Br-cGMP is 20 mg/kg.
6. A pharmaceutical preparation for treating ovarian epithelial cancer, which comprises (1) an effective amount of 8-Br-cGMP as an active ingredient, and (2) an optional pharmaceutically acceptable carrier.
7. The pharmaceutical preparation for treating ovarian epithelial cancer according to claim 6, wherein the pharmaceutical dosage form is capsule, tablet, powder, oral liquid, pill, tincture, syrup or injection.