CN110606814A - Application of small molecule compound QW24 in preparation of medicine for treating colorectal cancer diseases - Google Patents
Application of small molecule compound QW24 in preparation of medicine for treating colorectal cancer diseases Download PDFInfo
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Abstract
The invention discloses a small molecular compound QW24 shown in formula (I) and application thereof in preparing a medicine for treating colorectal cancer, wherein the compound can remarkably reduce the protein level of BMI-1 through an autophagy-lysosome protein degradation way, reduce the self-renewal capacity of colorectal cancer tumor stem cells, and further inhibit the proliferation and migration of the tumor cells. In a mouse subcutaneous tumor-bearing model, the small molecular compound QW24 shown in the formula (I) can obviously inhibit the growth of tumors, and in a colorectal cancer liver metastasis animal model, can obviously inhibit the metastasis of the tumors, does not show obvious toxicity in the administration process, and has an application prospect in treating colorectal cancer.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of a small molecular compound QW24 in preparation of medicines for treating colorectal cancer diseases.
Background
Colorectal cancer (Colorectal cancer) is the cancer with the third grade of incidence and fatality worldwide, accounting for 10% of all cancer cases, and with the fatality reaching 50%. In 2012 140 million new cases and 694000 people died from colorectal cancer, with more common cases in developed countries, accounting for approximately 65% of the total number of cases. The incidence and mortality of colorectal cancer in China are increasing day by day, which seriously endangers the health of people and also becomes a hotspot for the research of colorectal cancer.
The main treatment method of the colorectal cancer is different along with the development of the colorectal cancer at different stages, the colorectal cancer is mainly treated by means of surgical excision when tumors at the I stage and the II stage of the colorectal cancer are only positioned on the intestinal wall, the tumors are widely spread and metastasized when the cancer is developed to the III stage and the IV stage, the surgical excision therapy is difficult to cure, and the radiotherapy or chemotherapy mode must be combined. The survival rate of colorectal cancer patients gradually decreases as the cancer progresses, and the five-year survival rate at stage iv is only lower than 5%. There is some difficulty in surgical resection at the late stage of cancer, and chemotherapy or radiotherapy is the main treatment means. However, patients with advanced colorectal cancer have serious recurrence problems, survivors after primary cure still have the risk of forming secondary primary tumors in the colon and rectum, and the survival rate of the patients is still low.
Tumor stem cells (CSCs) have been shown to be more resistant to traditional Cancer therapies than non-tumor stem cells, recurrence of colorectal Cancer is closely associated with colorectal Cancer tumor stem cells, and clinical data indicate that if chemotherapeutic drugs can target tumor stem cells, survival rates of patients can be significantly improved. Therefore, tumor stem cells have been the focus of research on cancer treatment. BMI-1 is a key regulatory factor for maintaining the self-renewal capacity of tumor stem cells, can maintain the pluripotency of the tumor stem cells, promote cell proliferation, inhibit cell aging and apoptosis, and further cause drug resistance and relapse of cancer. BMI-1 is highly expressed in colorectal cancer patients and is closely associated with low survival rates of patients. Thus, targeting BMI-1 is considered a new approach to the treatment of colorectal cancer.
Therefore, by combining the latest basic research results of colorectal cancer formation mechanism, the novel small molecular drugs for treating colorectal cancer, especially the drugs with the independent intellectual property rights in China, are developed, the molecular mechanism of the effect is clarified, the foundation is laid for the subsequent research and development of new drugs, and the novel small molecular drugs have important theoretical significance and wide application prospect.
Disclosure of Invention
The invention provides a QW24 small molecule compound or a hydrate or a pharmaceutically acceptable salt thereof, which has a structure shown in a formula (I):
QW24 shown in formula (I) is a small molecular compound with the molecular formula of C27H28N2O4And the molecular weight is 444.52.
The invention also provides a pharmaceutical composition, which comprises the QW24 small molecule compound shown as the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The invention also provides application of the QW24 small-molecule compound shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in preparing a medicament for treating colorectal cancer diseases.
In the application of the invention, QW24 shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition has the following functions in vitro and in vivo: protein levels of BMI-1 were significantly down-regulated by the autophagy-lysosomal protein degradation pathway without affecting their mRNA levels. Has obvious proliferation inhibiting effect on colorectal cancer cell strain and little damage to normal cells. In colorectal cancer cell strains with stem cell-like properties, the self-renewal capacity of colorectal cancer tumor stem cells is remarkably reduced, and further, the proliferation and migration of tumor cells are inhibited. In a mouse subcutaneous tumor-bearing model, the growth of tumors is remarkably inhibited, in a colorectal cancer liver metastasis animal model, the metastasis of the tumors is remarkably inhibited, and no obvious toxicity is shown in the administration process.
In the application of the invention, QW24 shown in formula (I) or hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition significantly reduces the expression of BMI-1 protein.
In the application, QW24 shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is used for inhibiting the proliferation, growth, metastasis and migration of colorectal cancer in vitro and/or in vivo and promoting the apoptosis of the colorectal cancer.
Wherein the colorectal cancer comprises colorectal cancer cell strains HCT116, HT29, HCT8, HCT15, LS174T, LoVo and CT 26.
In the application of the invention, QW24 shown in formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition can significantly reduce the self-renewal capacity of colorectal cancer tumor stem cells and reduce the number of rectal cancer tumor stem cells.
In the application of the invention, QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition inhibits the self-renewal capacity of colorectal cancer cell strain HCT116 with stem cell property, and reduces the number of HCT 116.
In the application of the invention, QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition can inhibit the migration of colorectal cancer cells in vitro and influence the cell morphology.
In the application of the invention, QW24 shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition can inhibit liver metastasis of colorectal cancer cell CT26 in vitro and/or in vivo.
In the application of the invention, QW24 shown in formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition can down-regulate the protein level of BMI-1 without influencing the mRNA level of the BMI-1 in vitro and/or in vivo and reduce the stability of the BMI-1 protein.
In the application of the invention, QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition degrades BMI-1 protein through an autophagy-lysosome pathway.
In the application of the invention, the hydrate, the pharmaceutically acceptable salt or the pharmaceutically acceptable carrier of the QW24 compound shown in the formula (I) and the like also have the same inhibiting effect as the QW24 shown in the formula (I).
In the application of the invention, the QW24 compound shown in the formula (I) can be used alone or in combination with other medicines.
The invention also provides a method for down regulating the expression of the BMI-1 in vitro and/or in vivo by the QW24 compound shown in the formula (I) or the hydrate or the pharmaceutically acceptable salt thereof or the pharmaceutical composition. QW24 as shown in formula (I) can down-regulate the expression of BMI-1 in a concentration-dependent manner.
The invention also provides a method for inhibiting the self-renewal of colorectal cancer tumor stem cells by the QW24 compound shown in the formula (I) or the hydrate or the pharmaceutically acceptable salt thereof or the pharmaceutical composition.
QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition significantly inhibits the frequency of colorectal cancer stem-like cell HCT116 sphere formation, i.e., inhibits the ability of tumor initiating cells (tumor stem cells) to self-renew.
The invention provides a method for inhibiting colorectal cancer cell metastasis in vitro and/or in vivo by QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition. QW24 shown in formula (I) or its hydrate or pharmaceutically acceptable salt, or pharmaceutical composition can inhibit in vitro migration of colorectal cancer cell lines HCT116, HCT8 and CT26 and liver metastasis of CT26 cells.
The invention provides a method for degrading a BMI-1 protein through an autophagy-lysosome pathway by QW24 shown in a formula (I) or a hydrate or pharmaceutically acceptable salt thereof or a pharmaceutical composition. When the QW24 shown in the formula (I) or the hydrate or the pharmaceutically acceptable salt thereof or the pharmaceutical composition is used for treating HCT116 cells, obvious autophagosome formation is realized, and the expression level of an LC3 II band is increased by Westernblotting detection.
The invention provides an application of a QW24 compound shown in formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition in preparing a medicament for preventing and/or treating malignant tumor diseases related to BMI-1 overexpression.
The malignant tumor is a BMI-1 over-expression malignant tumor, and comprises colon cancer, glioma, prostatic cancer, breast cancer, lung cancer, liver cancer, leukemia and the like.
The invention has the beneficial effects that: the invention obtains a small molecular compound QW24 shown in formula (I) through an SRB (Sulforhodamine B) colorimetric method and protein immunoblotting screening, wherein the compound can strongly inhibit the proliferation of colorectal cancer cells in vitro and in vivo, and can significantly reduce the protein level of BMI-1 through an autophagy-lysosome protein degradation pathway. In colorectal cancer cell strains with stem cell-like properties, the self-renewal capacity of colorectal cancer tumor stem cells is remarkably reduced, and further, the proliferation and migration of tumor cells are inhibited. In an animal model of colorectal cancer hepatic metastasis, tumor metastasis is remarkably inhibited, and no obvious toxicity is shown during administration. Colorectal cancer metastasis is an important cause of late death of patients, and the invention makes a new breakthrough in colorectal cancer treatment and provides a potential novel medicine for colorectal cancer treatment.
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FIG. 1 shows that QW24 of formula (I) inhibits proliferation of various colorectal cancer cell lines and down-regulates BMI-1 expression. FIG. 1(A) shows the inhibitory effect of QW24 of formula (I) at different concentrations (cell proliferation activity (%) in the ordinate) in colorectal cancer cell lines of HCT116, HT29, HCT8, HCT15, LS174T, LoVo and CT 26; FIG. 1(B) shows the IC50 of QW24 of formula (I) in different colorectal cancer cell lines; FIG. 1(C) shows the IC50 of QW24 of formula (I) in a normal cell line; fig. 1(D) shows that the QW24 shown in formula (I) has a significant difference in IC50 (half maximal inhibitory concentration (μ M) of QW24 on the ordinate) between colorectal and normal cells; FIG. 1(E) shows that QW24 shown in formula (I) has better inhibitory effect on HCT116 of colorectal cancer cells with dryness than positive compound PTC209, wherein PTC209 is a published BMI-1 inhibitor (cell proliferation activity (%) is ordinate); FIG. 1(F) shows that QW24 of formula (I) has a more pronounced down-regulation of BMI-1 than does positive compound PTC 209; FIG. 1(G) shows the effect of QW24 of formula (I) on the levels of BMI-1 and the protein Ub-H2A downstream of BMI-1 in different colorectal cancer cells.
FIG. 2 shows that QW24 of formula (I) inhibits the self-renewal ability of colorectal cancer tumor stem cells and reduces the number of colorectal cancer tumor stem cells. FIG. 2(A, B, C) shows that QW24 of formula (I) reduces the number and size of HCT116 cell sphere formation; FIG. 2(D) shows an in vitro limiting dilution assay; fig. 2(E) shows the ability of QW24 shown in formula (I) to inhibit self-renewal of colorectal cancer tumor stem cells.
FIG. 3 shows that QW24 of formula (I) inhibits migration of colorectal cancer cells in vitro and affects cell morphology. FIG. 3(A) shows that QW24 of formula (I) inhibits migration of HCT116, HCT8 and CT26 cells of colorectal cancer; FIG. 3 (B) shows that QW24 shown in formula (I) affects cell morphology.
FIG. 4 shows QW24 of formula (I) inhibiting tumor growth and down-regulating BMI-1 protein expression levels in vivo in a subcutaneous tumor-bearing model of colorectal cancer. Figure 4(a, B, C) shows the effect of QW24 and positive compound PTC209 shown in formula (I) in inhibiting HCT116 tumor growth in a colorectal carcinoma subcutaneous tumor-bearing model with QW24 effect more pronounced than PTC 209; FIG. 4(D) shows that QW24 and positive compound PTC209 of formula (I) have no significant effect on mouse body weight; FIG. 4(E) shows that QW24 and positive compound PTC209, represented by formula (I), down-regulate the protein expression level of BMI-1 in tumors; FIG. 4(F) shows the inhibitory effect of QW24 of formula (I) on tumor proliferation and intratumoral BMI-1 in tissue sections.
FIG. 5 shows QW24 of formula (I) inhibits liver metastasis from colorectal cancer and prolongs survival of mice in vivo. Fig. 5(a) shows that QW24 and positive compound PTC209 shown in formula (I) inhibited liver metastasis of colorectal cancer cell CT26 in a mouse model, and QW24 effect was more significant than PTC 209; FIG. 5(B) is a statistical plot showing that QW24 and positive compound PTC209 of formula (I) inhibit liver metastasis of colorectal cancer cell CT26 in a mouse model; FIG. 5(C) shows that QW24 and positive compound PTC209, represented by formula (I), have no significant effect on mouse body weight; FIG. 5(D) shows that QW24 of formula (I) is effective in extending survival of mice and is superior to PTC-209; FIG. 5(E) shows the inhibitory effect of QW24 and positive compound PTC209 of formula (I) on tumors metastasized to the liver and the corresponding statistical plot; FIG. 5(F) shows the effect of QW24 and positive compound PTC209 in inhibiting liver nodule formation shown in formula (I) and the corresponding statistical plot.
FIG. 6 shows that QW24 of formula (I) degrades BMI-1 protein mainly through the lysosomal-autophagy pathway. FIG. 6 (A, B) shows that QW24 of formula (I) has no significant effect on BMI-1mRNA levels; FIG. 6(C) shows that QW24 represented by formula (I) decreases the stability of BMI-1 protein; FIG. 6(D) shows that QW24 of formula (I) causes degradation of BMI-1 protein by inducing the autophagy-lysosomal pathway in cells; fig. 6(E, F, G, H) shows that QW24 shown in formula (I) significantly induced the onset of autophagy.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, but the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1: QW24 shown in formula (I) inhibits proliferation of multiple colorectal cancer cells in vitro
The technical method comprises the following steps:
1. culture of cells
Colorectal cancer cell strains HCT116, HT29, HCT8, HCT15, LS174T, LoVo and CT26 used in the invention are all from ATCC; human normal colon epithelial cells NCM460, human normal liver cells L02 and human epidermal fibroblasts HAF cells were derived from ATCC, and human umbilical vein endothelial cells HUVEC were purchased from ScienCell. HCT116, HT29, HCT8, HCT15, LoVo, CT26, NCM460, L02, HAF used RPMI-1640 medium, LS174T used high-sugar DMEM medium, HUVEC used ECM medium. The medium contained 10% FBS and 1% penicillin-streptomycin diabody, and the cells were cultured in a 37 ℃ incubator (humidity 95%, CO2 concentration 5%).
Determination of cell proliferation by the SRB (sulforhodamine) method
Different cell lines at a density of 5X 103-8×103One cell/well was inoculated into a 96-well plate (Corning), after 24 hours, QW24 compounds of different concentrations were added, DMSO was added in an equal amount to the control group, 3 wells were set for each group, and after further culturing for 72 hours, cells were fixed by adding pre-cooled TCA (trichloroacetic acid, 50%, w/V) at 4 ℃ for more than 60 min. After fixation, the mixture is flushed for 5 times and dried in the air. Mu.l of SRB stain (4%, w/V) was added to each well and incubated at room temperature for 10min for staining. Then the dye solution was aspirated, and 100. mu.l of 1% acetic acid was added to each well to wash 5 times to remove unbound dye. After air drying, 100. mu.l of 10mM Tris solution was added to each well and the bound SRB dye was dissolved by shaking. The 96-well plate was placed in a microplate reader (SPECTRAMAX 190) and OD was measured at a wavelength of 515 nm. The experiment was independently repeated 3 times. Cell survival (%) ═ drug-added OD value/control OD value × 100%.
3. Immunoblotting (western blot) experiment
After the cells are treated by the medicines with different concentrations, the cells are cracked to extract protein, and the protein is boiled in a water bath kettle with the constant temperature of 100 ℃ for 10min until the protein is completely denatured. Protein samples were separated using SDS-PAGE electrophoresis. Proteins were transferred to NC membranes, which were blocked with 5% skim milk or BSA on a shaker at room temperature for 2 hours. The corresponding antibody (primary antibody) was incubated at room temperature for two hours or at 4 ℃ overnight. And (3) incubating for one hour by using a secondary antibody with a fluorescent label, and finally detecting the expression of the target protein by using a membrane scanner Odyssey.
The experimental result is shown in fig. 1A, and the formula (I) QW24 has a significant inhibitory effect on each colorectal cancer cell, and has concentration dependence. The maximum half inhibitory concentration (IC50) of QW24 on proliferation of each colorectal cancer cell line was between 0.6. mu.M and 2. mu.M (FIG. 1B). QW24 was less toxic to normal cells, and shown in FIG. 1C as IC50 of QW24 in human normal liver cells L02, human skin fibroblasts HAF, human normal colon epithelial cells NCM460 and human umbilical vein endothelial cells HUVEC. QW24 showed significant selectivity for killing of colorectal and normal cells (fig. 1D).
The present invention compares the effect of QW24 with that of PTC-209, and using the same concentration and time for treating HCT116 cells, the results show that QW24 has the same good anti-proliferative activity as PTC-209, and QW24 has better anti-tumor cell proliferative activity at treatment concentrations of 2 μ M and 4 μ M, and has a differential significance compared to PTC-209 (FIG. 1E), and the down-regulation effect on BMI-1 protein level is more significant than PTC-209 (FIG. 1F). Therefore, QW24 has better anti-colorectal cancer cell proliferation capacity and the ability to down-regulate BMI-1 protein levels than PTC-209.
BMI-1 as one of the important elements of PRC1, PRC1 was able to monoubiquitinate lysine 119 of histone H2A (H2AK119Ub), so the present invention detected the expression level of ubiquitinated H2A (Ub-H2A) immediately downstream of BMI-1, and the result (FIG. 1G) showed that QW24 down-regulates the expression of Ub-H2A by down-regulating BMI-1, and has no effect on background H2A, further showing that QW24 has certain specificity and effectiveness for the action of BMI-1.
Example 2: QW24 inhibits self-renewal of colorectal cancer tumor stem cells
The technical method comprises the following steps:
1. experiment of sphere formation
The spheroplasty experiment was performed using colorectal cancer cell line HCT116 having stem cell properties, and first, HCT116 was pretreated with QW24 at different concentrations of 0, 1, 2, and 4 μ M for 12 hours, and then, the collected cells were washed with PBS to sufficiently remove residual serum, and seeded in a 96-Well plate (96 Well Clear Flat Bottom Ultra Low Attachment micro plate, Corning #3474) at a density of 1000 cells per Well to make the cells unable to adhere to each other, and after one week, the spheroplasty was observed and photographed using an inverted phase contrast microscope, and Scar bar was 100 μ M. The effect of QW24 on the self-renewal capacity of colorectal cancer tumor stem cells in vitro was examined using Limiting Dilution Analysis (LDA) in combination with sphere culture techniques, HCT116 cells were seeded in 96-well plates at different cell densities: 10000 (n-8), 1000 (n-8), 100 (n-8), 10 (n-12), 1 (n-12).
2. Flow cytometry
The influence of different concentrations of QW24 on the number of the colorectal cancer CSCs is detected by using flow cytometry and markers CD133+/CD44+ of the colorectal cancer CSCs, HCT116 cells are pretreated for 12 hours by QW24, collected and incubated for 10min in the dark by using CD133-APC (CD 133 antibody with APC fluorescent markers) and CD44-PE (CD 44 antibody with PE fluorescent markers), and then analyzed and detected on a flow cytometer, wherein the upper right quadrant represents CD133+/CD44+, namely the number area of tumor stem cells, and the percentage value represents the proportion of the CSCs to the whole cells.
The experimental results are shown in fig. 2A, wherein QW24 significantly inhibits the frequency of colorectal cancer stem-like cell HCT116 Sphere formation (Sphere-initiating cell frequency), i.e., the ability to inhibit self-renewal of tumor initiating cells (tumor stem cells). At 2 μ M and 4 μ M concentrations, there was a significant difference compared to the control group, with the tumor initiating cell initiation frequency from 1/5 decreasing to 1/26 and 1/149 (fig. 2D), and with increasing concentration of compound treatment, the number of spheres in each well was progressively reduced, and the diameter (i.e. size) of the spheres was progressively reduced (fig. 2A, B, C).
To further examine the effect of QW24 on tumor stem cell (CSC) numbers, we performed an analysis of tumor stem cells using flow cytometry, stained with the marker CD133+/CD44+ for colorectal cancer CSC, and showed that the number of tumor stem cells gradually decreased and was concentration dependent with increasing concentration of QW24 treatment (fig. 2E). It was therefore concluded that QW24 significantly reduced the number of colorectal cancer tumor stem cells, reducing their ability to self-renew.
Example 3: QW24 of formula (I) inhibits metastasis of colorectal cancer cells
The technical method comprises the following steps:
transwell cell migration experiment
The effect of QW24 on colorectal cancer cell metastasis was examined by the Transwell method at concentrations of 0, 1, 2, 4. mu.M for compound treatment, cells were starved with HCT116, HCT8, CT26 overnight first, and then cells were resuspended in basal medium without serum and with different concentrations of QW24 at 6X 104The density of each well (200. mu.l each) was seeded into the upper chamber of a Transwell chamber. The lower chamber is filled with 600 μ l of a liquidComplete medium at the corresponding concentration of QW 24. Culturing in cell culture box for 12-24 hr. The Transwell chamber was removed and the cells which did not migrate on the upper surface of the chamber were gently removed with a cotton swab, the cells on the lower surface of the chamber were fixed with 4% paraformaldehyde for 15 minutes, then the cells were treated with 2% o crystal violet stain for 3 minutes, the chamber was washed, the crystal violet which did not bind to the cells was removed, the upper side of the chamber was gently wiped with a cotton swab, and the dye which did not specifically bind to the upper surface of the chamber was wiped off for subsequent microscopic examination. The naturally dried chamber was photographed under a microscope and the number of cells in multiple fields was counted. Cell migration (%) -. drug-added cell migration number/control cell migration number × 100%.
2. Drug-affected cell morphology assay
Cells were treated with 2 μ M QW24 at time gradients of 0, 2, 4, 6, 8h and observed for morphological changes using an inverted phase contrast microscope.
As shown in fig. 3, QW24 significantly inhibited the migration of colorectal cancer cells and exhibited a concentration dependence: at a drug concentration of 1 μ M, the number of cells that migrated had decreased significantly; at 2 μ M, the cell mobility had decreased to around 20%; at 4. mu.M, only a very small number of cells migrated with less than 5% mobility (FIG. 3A). But also the morphology of the cells after QW24 treatment changed significantly (fig. 3B). Therefore, QW24 significantly inhibited colorectal cancer tumor cell migration, and exhibited significant inhibitory effects at low doses.
Example 4: QW24 formula (I) inhibits growth and metastasis of colorectal cancer tumors in vivo
The technical method comprises the following steps:
1. mouse subcutaneous lotus tumor model
Human colorectal cancer cell line HCT116 (with stem cell properties) was selected for experiments using male BALB/c-nude mice (purchased from Shanghai Spikey Co.) with immunodeficiencies for 6-8 weeks. The method comprises the following specific steps:
(1) HCT116 cells were extensively expanded and cultured in quantities to ensure sufficient tumor-bearing experimental doses, 300X 10 per mouse4The cell amount is required to ensure that the cell state is optimal in order to ensure the success rate of tumor bearing.
(2) When the number of cells was sufficient, the cells were digested with pancreatin, centrifuged to remove the supernatant, and washed twice with PBS to remove serum from the medium to reduce immune rejection. Cells were suspended in PBS and placed on ice to reduce cell neometabolism.
(3) Will be 300X 104One colorectal cancer cell, HCT116 (100. mu.L), was injected subcutaneously into the back of 6-8 week immunodeficient male mice.
(4) The length and width of the tumor were measured after about one week according to the formula: length x width2X 0.52 tumor volume was calculated. When the tumor grows to about 100mm3, the mice were randomly divided into four groups: blank control group (DMSO), positive control group (PTC209-30mg/kg) and experimental drug group (QW24-15mg/kg and QW24-30mg/kg), 10 groups, the weight of mice and the length and width changes of tumors were measured and recorded every 4 days for subsequent statistics, the administration frequency was once a day, 50 μ L each, tumors were detached after the end of the experiment, photographed, and H&E staining, immunohistochemistry experiments and WB experiments.
2. Mouse colorectal cancer liver metastasis model
Tumor cells were transferred from the spleen to the liver through the blood circulation system by the spleen injection method using the luciferase-expressing colorectal cancer cell line CT26 and 6-8 week BALB/c male mice. The operation flow is as follows:
(1) a colorectal cancer cell line CT26-luc stably expressing Luciferase (Luciferase) is constructed. Transferring a plasmid (carrying a G418 resistance screening marker) for expressing firefly Luciferase (Luciferase) into a CT26 cell line, adding G418 to screen a stably-transformed cell line, wherein the stably-transformed cell can express the Luciferase, the Luciferase can react with a substrate luciferin thereof to emit fluorescence, and then detecting the number and the position (the intensity and the position of the fluorescence) of tumor cells by using an IVIS (in vivo imaging System for small animals).
(2) After the stable transgenic cell strain is constructed, the stable transgenic cell strain is amplified to the required number, and G418 still needs to be added to maintain and screen cells which stably express luciferase in the amplification period.
(3) After the cell amount is enough, a part of the cells can be taken to verify whether the cells are stably expressed or notLuciferase, if positive, can be obtained by digesting cells with pancreatin, centrifuging to remove supernatant, washing twice with PBS to obtain a cell density of 1000 × 104and/mL. The cells were suspended in PBS and placed on ice to reduce the metabolic effects of the cells.
(4) Injecting albofloxacin to anesthetize the mouse before dissecting the mouse, shaving hair of spleen part below left rib of the mouse by using a hair shaving machine, wiping the part to be dissected by using an alcohol cotton ball, cutting a small opening on skin outside the spleen, pulling out the spleen by using forceps to pull connective tissue around the spleen, injecting 100 x 10 ingredients containing flufenadine along the spleen4100 μ L of cell suspension of each cell, the spleen was inserted into the body, the wound was closed, and the opening was wiped with an alcohol cotton ball. Photographs were taken using a small animal live imager IVIS.
(5) After tumor metastasis, the spleens of mice were removed to avoid interference with follow-up results.
(6) The day after removal of the spleen, mice were initially dosed, and were also randomly grouped into 3 groups: blank control group (DMSO), positive control group (PTC209-30mg/kg) and experimental drug group (QW24-30mg/kg), and mice were imaged in vivo by IVIS every 3 days to observe tumor growth and metastasis.
(7) And finally, analyzing and processing the photographing data by using IVIS imaging system software.
The experimental results are shown in fig. 4, QW24 was effective in inhibiting tumor growth by 24 consecutive days of administration, and QW24 was significantly more effective than PTC-209 at the same dose and had a concentration dependent effect (fig. 4A, B, C). During dosing, tumors in the QW24 group, 30mg/kg, proliferated very slowly. During the continuous administration process, the body weight of the mice in the QW 24-treated group is not obviously changed compared with that in the blank control group (figure 4D), and abnormal behaviors and adverse reactions of the mice are not found, which indicates that the medicine has no obvious toxicity and low toxic and side effects. In vivo, QW24 still significantly down-regulated the protein level of BMI-1, and its effect was more pronounced than PTC-209 (FIG. 4E). Immunohistochemical methods also showed that in tumors, QW24 still exerted its down-regulation effect on BMI-1 protein and significantly reduced the expression of markers Ki67 and PCNA for tumor proliferation (fig. 4F), and therefore, it can be concluded that QW24 effectively inhibited tumor growth and significantly down-regulated BMI-1 protein levels in an in vivo animal model, with superior efficacy to PTC-209, and without exhibiting significant toxicity.
In the liver metastasis model, QW24 is effective in inhibiting liver metastasis of colorectal cancer, has a significantly better effect than PTC-209 (FIG. 5A, B), has no significant influence on the body weight of mice during administration (FIG. 5C), and has a good survival state. To test whether QW24 could prolong the survival of mice, we further performed a survival curve experiment, and the results showed that QW24 effectively prolonged the survival of mice and was superior to PTC-209 (fig. 5D). Finally, the liver of the mice was dissected out, and it was found that in the liver of the mice in the QW 24-administered group, the number of tumor nodules was very small, the effect was very significant compared to the blank control group, and the anti-tumor metastasis effect was better than that of PTC-209 (fig. 5E, F).
Example 5: formula (I) QW24 degrades BMI-1 protein primarily through the lysosomal-autophagy pathway.
The technical method comprises the following steps:
1. polymerase Chain Reaction (PCR) assay
After the cells are treated by drugs with different concentrations, TRIzol is used for separating and extracting RNA, cDNA is obtained after the RNA is reversely transcribed, and the expression level of the mRNA is detected by real-time quantitative PCR (Q-PCR) by using different BMI-1 specific primers.
2. Protein stability test for Cycloheximide (CHX)
Cycloheximide (CHX) inhibits ribosomes and blocks protein synthesis. Firstly, treating colorectal cancer cells by cycloheximide to block protein synthesis, simultaneously adding QW24 for treatment, and then detecting the BMI-1 protein expression level by an immunoblotting experiment; comparison of BMI-1 protein expression levels of cycloheximide and QW24 in the simultaneous treatment group versus cycloheximide alone was performed to examine the effect of the drug on protein stability.
3. Protein ubiquitination degradation experiment for detecting proteasome inhibitor MG132
MG132 is a reversible, aldehyde peptide-specific proteasome inhibitor that inhibits 26S proteasome degradation of ubiquitinated target proteins. Treating colorectal cancer cells by respectively adding QW24 and MG132 and simultaneously adding QW24 and MG132, and detecting the expression level of BMI-1 protein by an immunoblotting experiment; comparing the expression levels of BMI-1 protein in the MG132 and QW24 simultaneous treatment group with the QW24 and MG132 treatment group alone to determine whether the drug degrades BMI-1 protein through ubiquitination pathway.
4. Protein autophagy assay for Chloroquine (Chloroquine) detection
The lysosome inhibitor chloroquine can block autophagy, and chloroquine is added to detect whether the protein degrades the protein through an autophagy-lysosome pathway. Treating colorectal cancer cells by respectively adding QW24 and chloroquine and simultaneously adding QW24 and chloroquine, and detecting the expression level of BMI-1 protein by an immunoblotting experiment; comparing the expression levels of BMI-1 protein in the group treated with chloroquine and QW24 simultaneously with the group treated with QW24 and chloroquine alone to determine whether the drug degrades BMI-1 protein by autophagy-lysosome pathway.
As shown in FIG. 6, QW24 had no significant effect on BMI-1 transcript levels, i.e., mRNA levels, and no concentration and time dependence, and QW24 showed no significant change in BMI-1mRNA even when the compound treatment concentration was increased and the compound treatment time was prolonged (FIGS. 6A and B).
The invention adds Cycloheximide (CHX) to detect whether the stability of BMI-1 protein is affected. The experimental results show (FIG. 6C) that after the addition of CHX to inhibit protein synthesis, the protein level of BMI-1 can still continue to decrease under the action of QW24, that is, QW24 shortens the half-life of BMI-1 protein, reducing the stability of BMI-1 protein.
In order to further verify the conclusion, the lysosome inhibitor Chloroquinone is added to detect whether the protein is degraded through a lysosome-autophagy pathway, and surprisingly, when the Chloroquinone and QW24 are added simultaneously, the protein level of BMI-1 is obviously higher than that of the BMI-1 treated by QW24 alone (FIG. 6D), namely, the lysosome inhibitor Chloroquinone prevents the protein degradation process of BMI-1, and in order to further verify the conclusion, the autophagy marker LC3 is used for detecting whether autophagy occurs. First, the present invention transfected the GFP-LC3 plasmid into HCT116 cells, and the results showed that after 4 hours of 2. mu.M QW24 short treatment, many autophagosome formation was evident (FIG. 6H). Further, the results of Western blotting revealed that the expression level of LC3 II band was increased (FIG. 6E, F, G), thus indicating that QW24 induced autophagy occurred, and the present invention used PTC-209 as a control compound, PTC-209 did not induce autophagy, while the phenomenon of autophagy induced by QW24 was very significant (FIG. 6E). Thus, from the above results, it can be concluded that QW24 degrades BMI-1 protein mainly through the autophagy-lysosomal pathway.
The above embodiments are only for illustrating the technical idea and features of the present invention, and the purpose of the embodiments is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made in the spirit of the present disclosure are intended to be covered by the scope of the present disclosure.
Claims (12)
1. A QW24 small molecule compound or a hydrate or a pharmaceutically acceptable salt thereof, which is characterized in that the structure is shown as formula (I);
2. a pharmaceutical composition comprising a QW24 compound represented by formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
3. Use of the QW24 small molecule compound or a hydrate or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 2, in the preparation of a medicament for the prevention and/or treatment of colorectal cancer diseases.
4. The use according to claim 3, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is for inhibiting the proliferation, growth, metastasis, migration and promoting apoptosis of colorectal cancer in vitro and/or in vivo.
5. The use of claim 4, wherein the colorectal cancer comprises colorectal cancer cell lines HCT116, HT29, HCT8, HCT15, LS174T, LoVo and CT 26.
6. The use according to claim 3, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition significantly reduces the self-renewal capacity of colorectal cancer tumor stem cells, reduces the number of rectal cancer tumor stem cells.
7. The use according to claim 6, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition inhibits the self-renewal ability of colorectal cancer cell line HCT116 with stem cell properties, reducing the number of HCT 116.
8. The use according to claim 4, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is for inhibiting liver metastasis of CT26 in colorectal cancer cells in vitro and/or in vivo.
9. The use of claim 3, wherein the QW24 compound of formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition down-regulates the protein level of BMI-1.
10. The use of claim 9, wherein the QW24 compound of formula (I), or a hydrate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition degrades BMI-1 protein via the autophagy-lysosomal pathway.
11. Use of the QW24 compound or a hydrate or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 2, for the preparation of a medicament for the prevention and/or treatment of a malignant tumor disease associated with BMI-1 overexpression.
12. The use of claim 11, wherein the malignancy comprises colon cancer, glioma, prostate cancer, breast cancer, lung cancer, liver cancer, leukemia.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104557602A (en) * | 2015-01-07 | 2015-04-29 | 华东师范大学 | Tricyclic diterpene derivative, as well as preparation method and application thereof in preparation of anti-tumour drug |
| CN106928147A (en) * | 2017-03-14 | 2017-07-07 | 华东师范大学 | Tricyclic diterpene analog and preparation method thereof and its application in antiprostate cancer is prepared |
| CN106928095A (en) * | 2017-03-14 | 2017-07-07 | 华东师范大学 | Cyano group ketene tricyclic diterpene analog and its preparation method and application |
| CN107954930A (en) * | 2017-11-14 | 2018-04-24 | 华东师范大学 | Cyano group ketene tricyclic diterpene analog and preparation method thereof and the application in anti-drug resistance breast cancer |
| CN109528700A (en) * | 2017-09-21 | 2019-03-29 | 华东师范大学 | Application of the small molecule compound QW07 in preparation treatment prostatic disorders drug |
-
2019
- 2019-08-30 CN CN201910815668.4A patent/CN110606814A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104557602A (en) * | 2015-01-07 | 2015-04-29 | 华东师范大学 | Tricyclic diterpene derivative, as well as preparation method and application thereof in preparation of anti-tumour drug |
| CN106928147A (en) * | 2017-03-14 | 2017-07-07 | 华东师范大学 | Tricyclic diterpene analog and preparation method thereof and its application in antiprostate cancer is prepared |
| CN106928095A (en) * | 2017-03-14 | 2017-07-07 | 华东师范大学 | Cyano group ketene tricyclic diterpene analog and its preparation method and application |
| CN109528700A (en) * | 2017-09-21 | 2019-03-29 | 华东师范大学 | Application of the small molecule compound QW07 in preparation treatment prostatic disorders drug |
| CN107954930A (en) * | 2017-11-14 | 2018-04-24 | 华东师范大学 | Cyano group ketene tricyclic diterpene analog and preparation method thereof and the application in anti-drug resistance breast cancer |
Non-Patent Citations (1)
| Title |
|---|
| LIAN-FANG YANG等: "Synthesis of Cyanoenone-Modified Diterpenoid Analogs as Novel Bmi-1-Mediated Antitumor Agents", 《ACS MED. CHEM. LETT.》 * |
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