CN112569240A - Application of chlorpromazine hydrochloride in preparation of cyclin inhibitor - Google Patents
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- CN112569240A CN112569240A CN201910936440.0A CN201910936440A CN112569240A CN 112569240 A CN112569240 A CN 112569240A CN 201910936440 A CN201910936440 A CN 201910936440A CN 112569240 A CN112569240 A CN 112569240A
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
- A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
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Abstract
The invention provides an application of chlorpromazine hydrochloride in preparing a cyclin protein inhibitor. The invention proves that chlorpromazine hydrochloride can inhibit proliferation of colorectal cancer cells and promote apoptosis of the colorectal cancer cells, phosphorylation of AKT and p44/p42MAPK proteins related to cell proliferation can be obviously reduced in the process, expression of cycle D1, cycle B1, cdc2, cdc25c, cycle E, cycle A2 and c-Myc related to cell cycle in the colorectal cancer cells can be reduced, expression of p-cdc (Tyr15) and p21 can be increased, expression of pro-caspase 3 and Bcl2 related to apoptosis in CT26 and HCT116 cells can be reduced, and expression of clear caspase3, Bax and p53 related to apoptosis can be increased. The chlorpromazine hydrochloride can be used for preparing cyclin protein inhibitors and medicaments for treating cancers, particularly colorectal cancers, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of chlorpromazine hydrochloride in preparation of a cyclin protein inhibitor.
Background
Cyclin proteins, also known as cyclins, are cell cycle regulatory molecules that regulate the turnover and progression of various phases of the cell cycle through the activation of cyclin-dependent kinases. The major cyclins of the cell cycle are: cyclin A, cyclin B, cyclin D and cyclin E. The regulatory proteins in stage G1 → stage S are cyclin D1(cyclin D1) and cyclin E (cyclin E), the regulatory protein in stage S → stage G2 is cyclin A, and the regulatory protein in stage G2 → stage M is cyclin B (cyclin B).
Colorectal cancer is the leading cause of cancer death worldwide, causing approximately 83 million deaths each year. The main therapeutic approaches for treating colorectal cancer include surgical resection, chemotherapy, radiation therapy, targeted therapy, and immunotherapy. Despite the advances in colorectal cancer treatment in recent years, there is a great need for more effective methods of treating colorectal cancer.
Among the treatment modalities for colorectal cancer, radical surgical resection is the main treatment modality for early colorectal cancer without distant metastasis. The chemotherapy drugs mainly comprise 5-FU, leucovorin calcium, capecitabine, oxaliplatin, irinotecan and the like, but chemotherapy and radiotherapy have obvious toxic and side effects of bone marrow toxicity, digestive tract toxicity and the like, and many patients do not respond to the treatment or resist the drug and the like. In recent years, some targeted drugs are also beginning to be used for treating colorectal cancer, such as cetuximab and panitumumab targeting Epidermal Growth Factor Receptor (EGFR), bevacizumab and aflibercept targeting Vascular Endothelial Growth Factor Receptor (VEGFR), and protein kinase inhibitor regorafenib, which bring survival benefits to patients with advanced colorectal cancer and improve clinical efficacy. Immunotherapy is also being used in the treatment of colorectal cancer, such as the immune checkpoint inhibitors PD-1 mabs Pembrolizumab and Nivolumab are recommended for the end-line treatment of colorectal cancer with the dMMR (mismatch repair protein deletion)/MSI-H (high microsatellite instability) molecular phenotype. However, these targeted therapies and immunotherapies also have problems of high price, patient non-response to the therapy, and resistance to the therapy. Therefore, more drugs that may be clinically used for treating colorectal cancer need to be developed.
Chlorpromazine hydrochloride is a blocking agent of thiazine central dopamine receptors, can be used for treating schizophrenia, mania or other psychotic disorders, and can also be used for treating emesis caused by various reasons. However, few reports have been made on its use for the treatment of colorectal cancer.
Disclosure of Invention
The invention aims to provide application of chlorpromazine hydrochloride in preparing cyclin protein inhibitors.
The invention provides an application of chlorpromazine hydrochloride in preparing a cyclin protein inhibitor.
Further, the cyclin protein is cyclin D1, cyclin B1, cyclin E and cyclin A2.
Further, the inhibitor is used to reduce expression of cdc2, cdc25c, c-Myc; and/or, increased expression of p-cdc (Tyr15), p 21.
Further, the inhibitor is used for preparing a medicament for treating cancer; preferably, the cancer is colorectal cancer.
Further, the medicament is a medicament for inhibiting proliferation of colorectal cancer cells; and/or, the drug is a drug that induces apoptosis in colorectal cancer cells.
Further, the drug is a drug for reducing phosphorylation of AKT protein, p44/p42MAPK protein related to colorectal cancer cell proliferation.
Further, the drug is a drug causing G2/M cycle arrest of colorectal cancer cells.
Further, the medicament is a medicament for reducing the expression of pro-caspase 3, Bcl2 related to apoptosis and increasing the expression of clear caspase3, Bax and P53 related to apoptosis.
Furthermore, the medicine is a preparation prepared by taking chlorpromazine hydrochloride as an active ingredient and adding pharmaceutically acceptable auxiliary materials and/or auxiliary ingredients.
Further, the preparation is an injection preparation.
In vitro experiments show that chlorpromazine hydrochloride can inhibit the proliferation of mouse colorectal cancer cells (CT26) and human colorectal cancer cells (HCT116 and SW620) in a concentration and time-dependent manner, can also inhibit the capability of HCT116 and SW620 cells to form clones in a 6-well plate, and can reduce the phosphorylation of AKT and p44/p42MAPK proteins related to cell proliferation. Chlorpromazine hydrochloride can also induce colorectal cancer cells CT26 and HCT116 to generate G2/M cell cycle block in a concentration and time-dependent mode, reduce the expression of cell cycle-related cyclin D1, cyclin B1, cdc2, cdc25c, cyclin E, cyclin A2 and c-Myc, and increase the expression of p-cdc (Tyr15) and p 21. In addition, chlorpromazine hydrochloride can also induce colorectal cancer cells to undergo apoptosis, reduce the expression of pro-caspase 3 and Bcl2 related to apoptosis and increase the expression of clear caspase3, Bax and P53 related to apoptosis. Chlorpromazine hydrochloride also lowers mitochondrial membrane potential of CT26 and HCT116 and increases intracellular reactive oxygen species. Animal experiments further prove that chlorpromazine hydrochloride can obviously inhibit the growth of human HCT116 colorectal cancer subcutaneous tumors in nude mice and CT26 subcutaneous tumors in Balb/c mice, reduce the proliferation of tumor cells in the subcutaneous tumors of the mice (the proportion of Ki67 positive cells is reduced), and increase the apoptosis of the tumor cells in the subcutaneous tumors of the mice (the proportion of clear caspase3 positive staining cells is increased). Compared with colorectal cancer cells, chlorpromazine hydrochloride has low toxicity to human normal intestinal epithelial cells and mouse embryonic fibroblasts, and the in vivo administration of chlorpromazine hydrochloride does not obviously reduce the body weight of mice, so that the safety is good.
In conclusion, the chlorpromazine hydrochloride has potential effects of inhibiting cyclin protein expression and treating colorectal cancer, and the chlorpromazine hydrochloride can be used for preparing a cyclin protein inhibitor and a medicine for treating the colorectal cancer, and has obvious clinical and social meanings.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a graph showing the inhibition rate of cell proliferation of human colorectal cancer cells SW620 by chlorpromazine hydrochloride of example 1.
FIG. 2 is a graph showing the inhibition rate of chlorpromazine hydrochloride on cell proliferation of human colorectal cancer cells HCT116 in example 1.
FIG. 3 is a graph showing the cell proliferation inhibition rate of chlorpromazine hydrochloride on mouse colorectal cancer cells CT26 in example 1.
FIG. 4 is a graph showing the cell proliferation inhibition rate of chlorpromazine hydrochloride in example 1 on human colonic epithelial cells HCoEpiC.
FIG. 5 is a graph showing the inhibition rate of chlorpromazine hydrochloride on cell proliferation of mouse embryonic fibroblasts NIH-3T3 in example 1.
FIG. 6 is a graph showing the inhibition of clonality of chlorpromazine hydrochloride on human colorectal cancer cells SW620 and HCT116 in example 1.
FIG. 7 is a graph showing the inhibition of clonality of chlorpromazine hydrochloride in example 1 on human colonic epithelial cells HCoEpiC and mouse embryo fibroblast NIH-3T3 cells.
FIG. 8 is a graph of the effect of chlorpromazine hydrochloride on phosphorylation of AKT and p44/p42MAPK proteins associated with cell proliferation of human colorectal cancer cell HCT116 and mouse colorectal cancer cell CT26 of example 1.
FIG. 9 is a graph showing the effect of chlorpromazine hydrochloride on the cell cycle of CT26, a mouse colorectal cancer cell, in example 2.
FIG. 10 is a graph of the effect of chlorpromazine hydrochloride on the cell cycle of human colorectal cancer cells HCT116 of example 2.
FIG. 11 is a graph showing the effect of chlorpromazine hydrochloride on cell cycle-related protein expression of CT26 in mouse colorectal cancer cells in example 2.
FIG. 12 is a graph showing the effect of chlorpromazine hydrochloride on the expression of human colorectal cancer cell HCT116 cell cycle-associated protein in example 2.
FIG. 13 is a graph showing the flow results of chlorpromazine hydrochloride apoptosis on mouse colorectal cancer cell CT26 and human colorectal cancer cell HCT116 of example 3.
FIG. 14 is a graph showing the effect of chlorpromazine hydrochloride on apoptosis-related protein expression in mouse colorectal cancer cell CT26 and human colorectal cancer cell HCT116 of example 3.
FIG. 15 shows the effect of chlorpromazine hydrochloride of example 4 on mitochondrial membrane potential of mouse colorectal cancer cell CT26 and human colorectal cancer cell HCT116 (flow-through results).
FIG. 16 is the effect of chlorpromazine hydrochloride on intracellular reactive oxygen content of mouse colorectal cancer cell CT26 and human colorectal cancer cell HCT116 of example 4 (flow results).
FIG. 17 is a graph of the inhibitory effect of chlorpromazine hydrochloride of example 5 on mouse colorectal cancer cell CT26 and human colorectal cancer cell HCT116 on a mouse subcutaneous model.
FIG. 18 is a graph of the change in body weight of mice during the treatment of CT26 and HCT116, mouse colorectal cancer cells, on a mouse subcutaneous model, with chlorpromazine hydrochloride of example 5.
FIG. 19 is a graph of Ki67 immunohistochemical staining results in mouse tumor tissues 12 days after treatment of mouse subcutaneous human colorectal cancer cells HCT116 model with chlorpromazine hydrochloride in example 5.
FIG. 20 is a graph of the results of immunohistochemical staining of clear caspase3 in mouse tumor tissues 12 days after treatment of mouse subcutaneous human colorectal cancer cells HCT116 model with chlorpromazine hydrochloride in example 5.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
Among them, chlorpromazine hydrochloride (CPZ, english name), CAS number: 69-09-0, the structure is as follows:
example 1 cellular experiment of chlorpromazine hydrochloride inhibition of colorectal cancer
First, experiment principle
A large characteristic of tumors is uncontrolled growth, which, if it is possible to reduce the number of tumor cells, would produce an antitumor effect. Tumor cells have the ability to proliferate from one cell division to form a cell clone, and if the ability of tumor cells to form a clone can be inhibited, an antitumor effect can also be produced. This example will demonstrate the inhibition ability of chlorpromazine hydrochloride on tumor cells from the above two aspects.
Second, Experimental methods
1. Culture of cells
The human colorectal cancer cell lines SW620, HCT116 and the mouse colorectal cancer cell line CT26 are cultured by a 1640 culture medium containing 10% FBS, the human colorectal epithelial cell line HCoEpiC and the mouse embryonic fibroblast cell line NIH-3T3 are cultured by a DMEM high-sugar medium containing 10% FBS, 100U/mL penicillin and streptomycin are required to be added in the culture and experiment processes of the cell lines, and the culture is carried out under the environmental conditions of 37 ℃ and 5% CO2Incubator (normal culture).
2. MTT method for detecting influence of chlorpromazine hydrochloride on cell proliferation
The cells in exponential growth phase were digested and collected separately, plated on 96-well plates, and cultured normally. Selecting cells with good logarithmic phase growth state, inoculating to 96-well plate with 2000 cells per well and 100 μ L per well according to cell growth speed, and culturing at 37 deg.C and 5% CO2Culturing in an incubator. The next day of inoculation, 100. mu.L of chlorpromazine hydrochloride diluted in the corresponding medium was added to each well to give final concentrations of 0. mu.g/mL (0. mu.M for short), 1.88. mu.M, 3.75. mu.M, 7.5. mu.M, 15. mu.M, and 30. mu.M, 3 duplicate wells were set at the same concentration, and 5% CO was added at 37 ℃ to each well2After culturing in an incubator for 48 and 72 hours respectively, 20 mu L (5mg/mL) of MTT stock solution is added into each hole, then the mixture is put into the incubator for culturing for 3 hours, the cell supernatant is slightly sucked away, 150 mu L of DMSO is added into each hole, the mixture is placed on a horizontal shaking table and shaken for 5 minutes at 90r/min, and an OD value is measured at 570nm by a microplate reader. Repeating the experiment for three times, sorting data, obtaining growth inhibition rate according to absorbance value, and calculating IC50。
3. Detection of effects of chlorpromazine hydrochloride on SW620 and HCT116 cell proliferation by clone formation experiment
SW620 and HCT116 cells in logarithmic growth phase were seeded in 6-well plates at a density of 800 cells/well. After 24 hours of culture, the cell culture medium was changed to the same volume of medium containing chlorpromazine hydrochloride (3.75. mu.M, 7.5. mu.M and 15. mu.M) at different concentrations, and then placed at 37 ℃ with 5% CO2Culturing in incubator for 10 days. The medium was carefully aspirated off, washed 2 times with PBS solution, and fixed with 4% paraformaldehyde for 15 minutes. The gel was stained with a methanol solution containing 0.5% crystal violet for 15 minutes, and finally washed with a PBS solution and deionized water 1 time, respectively, and air-dried. Then, photographs were taken on a light box, and the number of clones (one clone was a colony of more than 50 cells) was counted under an inverted microscope, and the clone formation inhibition rate was calculated according to the following formula: clone formation inhibition rate ═ 1- (number of clones in administration group/number of clones in solvent control group)]X 100%. Each group was provided with three replicate wells and the experiment was replicated 3 times. The solvent control group was cultured only normally without adding drugs to the cells.
4. Detecting the effect of chlorpromazine hydrochloride on the expression of proliferation-related proteins of colorectal cancer cells CT26 and HCT116
CT26 and HCT116 cells in a logarithmic growth phase are taken and inoculated into a 10 cm-diameter culture dish at a density of 60 ten thousand/dish, after 24 hours of culture, the cells are treated with chlorpromazine hydrochloride at the same volume and different concentrations (0 mu M, 10 mu M and 20 mu M) for 48 hours, then the cells are washed 3 times with 4 ℃ precooled PBS, immediately all groups of cells are cracked on ice for 30 minutes by RIPA lysate, then all adherent cells are scraped off by cell scraping, the RIPA lysate is sucked into a 1.5mL EP tube, then the cells are cracked by an ultrasonic crusher (once every 1 second and every 1 second for 10 times), and then the cells are centrifuged at 4 ℃ for 15 minutes (13000 rpm). The supernatant was stored in a new 1.5mL EP tube for use. The resulting protein samples were quantitated according to BCA protein quantitation kit (cloudband day): adjusting protein concentration to the same concentration as the appropriate concentration (protein concentration can be detected), adding protein sample buffer (5X), boiling for 5min, subpackaging, and freezing at-20 deg.C. According to the procedure of SDS-PAGE gel kit, 10% or 12.5% gel is prepared, and the concentration of the gel is determined according to the size of the target protein. The loading of each well was 40. mu.g, and 120V electrophoresis was performed for 90 min. After electrophoresis is finished, placing the NC membrane with the size equivalent to that of the separation gel into a membrane transferring buffer solution. Gel and NC membrane are made into a membrane transfer sandwich structure according to the sequence of (red surface) sponge → 3 pieces of filter paper → PVDF membrane → gel → 3 pieces of filter paper → sponge (black surface), air bubbles between layers are eliminated, and the membrane transfer sandwich structure is rapidly inserted into a membrane transfer clamp, membrane transfer is carried out under the constant current of 300mA, and the membrane transfer time is determined by the molecular weight of the target protein. After the membrane transfer is finished, the membrane is placed into a blocking buffer solution and blocked for 1h at room temperature. The blocked membrane was washed with TBST membrane-washing solution for five minutes, then the corresponding primary antibody was diluted to an appropriate concentration with a primary antibody diluent according to the antibody specification, the membrane was put into the diluted primary antibody, and shaken overnight at 4 ℃. Then the membrane was taken out and placed on a horizontal shaker, washed 3 times with TBST membrane washing solution for 5min each time, diluted with blocking solution for secondary antibody, and incubated for 1h at room temperature. The membrane was removed, washed 3 times in TBST for 5 minutes each, and the developer was dropped onto the PVDF membrane uniformly and exposed in an exposure apparatus.
Third, experimental results
The results of experiments on the effects of chlorpromazine hydrochloride on the proliferation of human colorectal cancer cell lines SW620 and HCT116, a mouse colorectal cancer cell line CT26, a human colonic epithelial cell line HCoEpiC and a mouse embryo fibroblast cell line NIH-3T3 are shown in FIGS. 1-5. As can be seen from fig. 1, chlorpromazine hydrochloride inhibits SW620 cell proliferation in a concentration and time dependent manner. As can be seen from fig. 2, chlorpromazine hydrochloride inhibits HCT116 cell proliferation in a concentration and time dependent manner. As can be seen from fig. 3, chlorpromazine hydrochloride inhibited murine CT26 cell proliferation in a concentration and time dependent manner. As can be seen from FIGS. 4 and 5, the chlorpromazine hydrochloride at the same concentration has lower inhibitory effect on human colon epithelial cell line HCoEpiC and mouse embryo fibroblast cell line NIH-3T3 than SW620, HCT116 and CT26 cells.
Effects of chlorpromazine hydrochloride on the clonal formation of HCT116 and SW620 cells on six-well plates the experimental results are shown in fig. 6. As can be seen from FIG. 6, after 10 days of culture, HCT116 and SW620, which did not use the drug chlorpromazine hydrochloride, were able to form many distinct cell clones on six-well plates, while the use of chlorpromazine hydrochloride significantly reduced the number of cell clones. As can be seen from FIG. 7, the chlorpromazine hydrochloride of the same concentration has lower inhibition effect on the clone formation of the human colon epithelial cell line HCoEpiC and the mouse embryo fibroblast cell line NIH-3T3 than that of SW620, HCT116 and CT26 cells.
The effect of chlorpromazine hydrochloride on the expression of proliferation-related proteins in HCT116 and CT26 cells is shown in fig. 8. As can be seen from FIG. 8, chlorpromazine hydrochloride was able to significantly reduce phosphorylation of AKT and p44/p42MAPK proteins associated with cell proliferation after 2 days of treatment.
Example 2, the following: cell experiment for inducing colorectal cancer cell cycle arrest by chlorpromazine hydrochloride
First, experiment principle
Propidium Iodide (PI) is a fluorescent dye of double-stranded DNA, can be selectively inserted between bases of double-stranded helices of nucleic acid DNA and RNA to bind with the bases, the binding amount of the Propidium Iodide (PI) is in direct proportion to the content of the DNA, and the DNA distribution of each stage of the cell cycle can be obtained by analyzing with a flow cytometer, thereby calculating the percentage content of each stage. A large feature of tumors is uncontrolled growth, if the proliferation rate of tumor cells can be reduced, the cell cycle is blocked at a certain period, and the cells cannot enter the next division stage, so that the anti-tumor effect can be produced.
Second, Experimental methods
1. Effect of chlorpromazine hydrochloride on cell cycle distribution of colorectal cancer cells CT26 and HCT116
CT26 and HCT116 cells in logarithmic growth phase were seeded at a density of 10 ten thousand/well in 6-well plates and after 24 hours of culture, the cells were treated with the same volume of medium containing chlorpromazine hydrochloride at different concentrations (0. mu.M, 7.5. mu.M and 15. mu.M) for 12 and 24 hours. Cells were then trypsinized and harvested and fixed in 75% ethanol for 24 hours. After incubation with a solution containing 50. mu.g/mL Propidium Iodide (PI) and 0.1% Triton X-100 for 15 minutes in the absence of light, cell cycle distribution was measured and analyzed by flow cytometry.
2. Effect of chlorpromazine hydrochloride on expression of cell cycle related proteins CT26 and HCT116 of colorectal cancer cells
CT26 and HCT116 cells in logarithmic growth phase were seeded at a density of 60 ten thousand/dish in a 10cm diameter culture dish and after 24 hours of culture, the cells were treated with the same volume of medium containing chlorpromazine hydrochloride at different concentrations (0. mu.M, 7.5. mu.M and 15. mu.M) for 24 hours. The cells were then washed 3 times with 4 ℃ pre-chilled PBS, and each group of cells was immediately lysed with RIPA lysate for 30 minutes on ice, followed by scraping of all adherent cells using a cell scraper, following the procedure as in example 1, item 4 of the experimental protocol.
Third, experimental results
As can be seen from FIGS. 9 and 10, significant G2/M arrest was observed in both CT26 and HCT116 cells after 12 and 24 hours of chlorpromazine hydrochloride treatment, and a simultaneous decrease in G0/G1 and S phase cells was also observed. As can be seen from FIG. 11 and FIG. 12, chlorpromazine hydrochloride treatment for 24 hours can reduce the expression of cell cycle-associated cyclin D1, cyclin B1, cdc2, cdc25c, cyclin E, cyclin A2 and c-Myc and increase the expression of p-cdc (Tyr15) and p21 in both cells CT26 and HCT 116.
Example 3 cellular experiment of chlorpromazine hydrochloride Induction of colorectal cancer cell cycle apoptosis
First, experiment principle
Annexin V is a calcium ion-dependent phospholipid binding protein, phosphatidylserine is everted from the membrane when cells undergo apoptosis, and Annexin V has high affinity with everted phosphatidylserine. PI is a nucleic acid dye that cannot cross living cell membranes and when a rupture occurs in a cell membrane, PI can enter the cell and bind nucleic acids. When FITC (fluorescein isothiocyanate) is labeled on Annexin V, the combination of Annexin V and PI can be used for detecting apoptosis by using a flow cytometer. If the tumor cells can be subjected to apoptosis, the number of the tumor cells can be reduced, and therefore, the anti-tumor effect can be generated.
Second, Experimental methods
1. Effect of chlorpromazine hydrochloride on apoptosis of CT26 and HCT116 colorectal cancer cells
CT26 and HCT116 cells in the logarithmic growth phase were seeded at a density of 10 ten thousand/well in a 6-well plate, and after 24 hours of culture, cancer cells were treated with the same volume of medium containing chlorpromazine hydrochloride at different concentrations (0. mu.M, 7.5. mu.M, 15. mu.M, and 30. mu.M) for 24 hours. Then, cells were digested with pancreatin and collected, Annexin V-FITC and PI were added according to the kit, respectively, and then apoptosis was measured and analyzed by a flow cytometer.
2. Effect of chlorpromazine hydrochloride on expression of apoptosis-related proteins of CT26 and HCT116 colorectal cancer cells
CT26 and HCT116 cells in the logarithmic growth phase were seeded at a density of 60 ten thousand/dish in a 10cm diameter dish, and after 24 hours of culture, the cells were treated with the same volume of medium containing chlorpromazine hydrochloride at different concentrations (0 μ M, 10 μ M and 20 μ M) for 24 hours, and then washed 3 times with 4 ℃ pre-chilled PBS, and immediately groups of cells were lysed on ice with RIPA lysate for 30 minutes, and then all adherent cells were scraped off with cells, followed by the procedure as in example 1, item 4.
Third, experimental results
From the flow assay of fig. 13, it can be seen that after chlorpromazine hydrochloride treatment for 24 hours, concentration-dependent apoptosis occurred in both CT26 and HCT116 cells. As can be seen from FIG. 14, the expression of pro-caspase 3, Bcl2 related to apoptosis in CT26 and HCT116 cells can be reduced and the expression of clear caspase3, Bax and p53 related to apoptosis can be increased by 24 hours of chlorpromazine hydrochloride treatment.
Example 4 cellular experiment of chlorpromazine hydrochloride to lower mitochondrial membrane potential and increase reactive oxygen species in colorectal cancer cells
First, experiment principle
Apoptosis is divided into intrinsic and extrinsic apoptotic pathways. Maintenance of mitochondrial membrane integrity and mitochondrial membrane potential is essential for preventing intrinsic apoptosis. If integrity is compromised and mitochondrial membrane potential is reduced, cytochrome c is released from mitochondria into the cytoplasm and intrinsic apoptosis is initiated. Tumor cells are in an oxidative stress state due to continuous rapid division and the like, the reactive oxygen species in the cells are high, and if the reactive oxygen species in the cells are continuously increased, the cells can be subjected to apoptosis due to the oxidative stress which cannot be borne. Elevated levels of reactive oxygen species are also involved in the intrinsic apoptotic pathway of cells.
Second, Experimental methods
1. Measurement of cell mitochondrial membrane potential
CT26 and HCT116 cells in logarithmic growth phase were seeded in 6-well plates at a density of 10 ten thousand/well, and after 24 hours of culture, two kinds of CT26 and HCT116 cells were treated with the same volume of medium containing chlorpromazine hydrochloride at different concentrations (0. mu.M, 10. mu.M and 20. mu.M) for 24 hours, respectively, and then the cells were incubated with 5. mu.g/mL rhodamine-123 [ Rh123, Sigma-Aldrich (Shanghai) tracing Company ] at 37 ℃ for 30 minutes in the absence of light. The cells were then washed with cold PBS and the fluorescence signal from Rh123 was detected by flow cytometry.
2. Determination of intracellular reactive oxygen species levels
CT26 and HCT116 cells in the logarithmic growth phase are taken and inoculated into a 6-well plate at the density of 10 ten thousand cells/well, after 24 hours of culture, the CT26 and HCT116 cells are respectively treated by the same volume of medium containing chlorpromazine hydrochloride with different concentrations (0 mu M, 10 mu M and 20 mu M) for 24 hours, and then the cells are incubated with DCFH-DA (10 mu M) at 37 ℃ for 30 minutes in the dark. After washing with PBS, the fluorescence intensity of the cells was detected by flow cytometry to measure ROS levels in cancer cells.
Third, experimental results
As can be seen from fig. 15, after the chlorpromazine hydrochloride treatment for 24 hours, a concentration-dependent decrease in the mitochondrial membrane potential of the cells occurred in both CT26 and HCT 116. It can be seen from fig. 16 that reactive oxygen species in both CT26 and HCT116 cells showed concentration-dependent increase after 24 hours of chlorpromazine hydrochloride treatment, indicating that chlorpromazine hydrochloride may induce intrinsic apoptosis in both colorectal cancer cells.
Example 5 in vivo experiment of chlorpromazine hydrochloride inhibition of growth in mouse colorectal cancer model
First, experiment principle
Compared with the results on cell lines, the results of the in vivo anti-tumor experiments on animals can reflect the potential anti-tumor effect of the anti-tumor compound. The method adopts an international universal method, and comprises the steps of injecting CT26 mouse colorectal cancer cells into BALB/c mice subcutaneously, injecting HCT116 human colorectal cancer cells into BALB/c nude mice subcutaneously to establish a colorectal cancer model in mice, and when the average subcutaneous tumor volume of the mice reaches about 100mm3Starting to use chlorpromazine hydrochloride for treatment, judging the anti-colorectal cancer effect of chlorpromazine hydrochloride through the change of tumor volume, preliminarily judging the toxicity condition of the compound through the change condition of the body weight of a mouse after administration, and taking the compound when the experiment is finishedThe mouse tumor is subjected to immunohistochemical staining, and the in vivo anti-tumor action mode of the compound is preliminarily determined.
Second, Experimental methods
1. Establishing a colorectal cancer model of a mouse: 100 ten thousand CT26 mouse colorectal cancer cells are injected into BALB/c female mice at 8-10 weeks, 1000 ten thousand HCT116 human mouse colorectal cancer cells are injected into BALB/c female nude mice at 8-10 weeks, and after about 7-14 days, the mouse subcutaneous tumor grows to about 100mm3。
2. The formula of the solvent for dissolving chlorpromazine hydrochloride is as follows: 2.5% DMSO, 12.5% Cremophor EL and 85% physiological saline.
3. Dose, route and frequency of administration: injecting chlorpromazine hydrochloride (dissolved by solvent) once daily into abdominal cavity of mice in chlorpromazine hydrochloride treatment group at a dose of 10 mg/kg; the mice in the solvent control group were injected with the same amount of solvent intraperitoneally once a day.
4. Treatment method at the end of experiment: when the average subcutaneous tumor volume reaches about 2000mm3At that time, dosing was stopped and the mice were sacrificed using excess carbon dioxide. Mice tumors from the HCT116 model were taken and soaked in 4% paraformaldehyde solution for 48 hours, and then stained for both Ki67 and clear caspase3 protein in the tumors by immunohistochemical staining.
Third, experimental results
FIG. 17: the chlorpromazine hydrochloride inhibited the HCT116 and CT26 models by more than 60% after 12 and 13 days of administration.
FIG. 18, chlorpromazine hydrochloride treatment did not result in a significant decrease in body weight in BALB/c female and nude mice after 12 and 13 days of administration.
Fig. 19, immunohistochemical staining results in HCT116 model after completion of the experiment show: compared with a solvent control group, the chlorpromazine hydrochloride can reduce the proportion of Ki67 positive tumor cells, which indicates that the chlorpromazine hydrochloride can inhibit the proliferation of the tumor cells in a mouse body;
FIG. 20, chlorpromazine hydrochloride increased the proportion of clear caspase3 positive tumor cells compared to the solvent control, indicating that chlorpromazine hydrochloride can induce apoptosis of tumor cells in mice.
In conclusion, the chlorpromazine hydrochloride is proved to be capable of inhibiting the proliferation of colorectal cancer cells and promoting the apoptosis of the colorectal cancer cells, the phosphorylation of AKT and p44/p42MAPK proteins related to the cell proliferation can be obviously reduced in the process, the expression of cycle D1, cycle B1, cdc2, cdc25c, cycle E, cycle A2 and c-Myc related to cell cycle in the colorectal cancer cells can be reduced, the expression of p-cdc (Tyr15) and p21 can be increased, the expression of pro-caspase 3 and Bcl2 related to the apoptosis in CT26 and HCT116 cells can be reduced, and the expression of clear caspase3, Bax and p53 related to the apoptosis can be increased. The chlorpromazine hydrochloride can be used for preparing cyclin protein inhibitors and medicaments for treating cancers, particularly colorectal cancers, and has wide application prospect.
Claims (10)
1. Use of chlorpromazine hydrochloride in the preparation of cyclin inhibitor.
2. Use according to claim 1, characterized in that: the cyclin protein is cyclin D1, cyclin B1, cyclin E and cyclin A2.
3. Use according to claim 1, characterized in that: the inhibitors are useful for reducing expression of cdc2, cdc25c, c-Myc; and/or, increased expression of p-cdc (Tyr15), p 21.
4. Use according to claim 1, characterized in that: the inhibitor is used for preparing a medicament for treating cancer; preferably, the cancer is colorectal cancer.
5. Use according to claim 4, characterized in that: the drug is a drug for inhibiting the proliferation of colorectal cancer cells; and/or, the drug is a drug that induces apoptosis in colorectal cancer cells.
6. Use according to claim 5, characterized in that: the drug is a drug for reducing phosphorylation of AKT protein and p44/p42MAPK protein related to colorectal cancer cell proliferation.
7. Use according to claim 5, characterized in that: the drug is a drug causing G2/M cycle arrest of colorectal cancer cells.
8. Use according to claim 5, characterized in that: the medicine is a medicine for reducing the expression of pro-caspase 3 and Bcl2 related to apoptosis and increasing the expression of cleared caspase3, Bax and P53 related to apoptosis.
9. Use according to any one of claims 1 to 8, characterized in that: the medicine is a preparation prepared by taking chlorpromazine hydrochloride as an active ingredient and adding pharmaceutically acceptable auxiliary materials and/or auxiliary ingredients.
10. Use according to claim 9, characterized in that: the preparation is an injection preparation.
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