CN114921520A - Method for measuring induction effect of niclosamide on cell cycle arrest and apoptosis - Google Patents

Method for measuring induction effect of niclosamide on cell cycle arrest and apoptosis Download PDF

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CN114921520A
CN114921520A CN202210645854.XA CN202210645854A CN114921520A CN 114921520 A CN114921520 A CN 114921520A CN 202210645854 A CN202210645854 A CN 202210645854A CN 114921520 A CN114921520 A CN 114921520A
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colon cancer
niclosamide
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apoptosis
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蒋传命
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Shaoyang University
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Abstract

The invention belongs to the technical field of medical treatment, and discloses a method for determining induction effects of PI3K-AKT signal pathway mediated niclosamide on colorectal cancer cell cycle arrest and apoptosis. MTT experimental results show that the niclosamide has obvious proliferation inhibition effect on different colon cancer cells, the inhibition effect shows time and dosage effects to a certain extent, and the IC50 values of different colon cancer cells are slightly different. Flow cytometry results show that niclosamide can block colon cancer HCT116 cells in the G2/M stage, and DAPI fluorescent staining shows that niclosamide induces colon cancer HCT116 cells to be apoptotic. Niclosamide can block cell proliferation and induce cell apoptosis through a PI3K-AKT signal channel, thereby inhibiting tumor cell proliferation and playing an anti-tumor role; meanwhile, the colon cancer cells obtained by the method for separating and culturing the colon cancer cells provided by the invention are as follows: high yield, high purity, high activity, high cell survival rate up to 95% or more, and high cell purity up to 96% or more.

Description

Method for measuring cell cycle retardation and apoptosis induction effect of niclosamide
Technical Field
The invention belongs to the technical field of medical treatment, and particularly relates to a method for determining induction effects of PI3K-AKT signal pathway mediated niclosamide on colorectal cancer cell cycle retardation and apoptosis.
Background
Niclosamide has long been used as an insect repellent for the treatment of cestode infections for over 50 years. It is found that niclosamide has wider pharmacological activity, and can regulate diabetes and diabetic nephropathy, improve dendritic cell function, inhibit replication of Zika virus, improve arthritis, prevent systemic sclerosis and other pharmacological effects. Niclosamide is also a potent mammalian target of rapamycin (mTOR) inhibitor and is a potential antidiabetic agent. In recent years, a series of researches indicate that niclosamide shows anticancer activity, such as glioblastoma, ovarian cancer, acute myelocytic leukemia, lung cancer and breast cancer, but the anticancer activity of niclosamide on colon cancer is not reported in related documents. A plurality of researches show that niclosamide can play an anti-tumor role through a plurality of signal pathways, such as Wnt/beta-catenin, STAT3, Notch [13] and the like. The research shows that niclosamide is a potential anticancer drug. Niclosamide as a traditional old medicine may have multiple new uses, and may have good development prospects as an antitumor drug. The PI3K-AKT signal pathway plays an important role as one of upstream pathways of mTOR in pathogenesis of colon cancer, and the research explores the role of the PI3K-AKT signal pathway in China with niclosamide anti-colon cancer effect.
In summary, the problems of the prior art are as follows: the anticancer activity of niclosamide on colon cancer is not reported in related documents; meanwhile, most of the established methods for separating and culturing human colon cancer cells mainly adopt a tissue mass culture method, and the separated cells are low in purity, small in quantity and long in culture time.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for determining the induction effect of PI3K-AKT signal pathway mediated niclosamide on colorectal cancer cell cycle retardation and apoptosis.
The invention is realized in such a way that a method for determining the effects of mediating niclosamide on the cell cycle arrest and apoptosis induction of colon cancer by a PI3K-AKT signal pathway comprises the following steps:
step one, separating and culturing colon cancer cells;
step two, detecting the proliferation inhibition effect of niclosamide on colon cancer cells by using an MTT (methyl thiazolyl tetrazolium) experimental method;
step three, detecting the inhibition effect of niclosamide on the colon cancer cell cycle through flow cytometry;
observing the induction effect of niclosamide on colorectal cancer cell apoptosis by a DAPI fluorescent staining method;
step five, detecting the expression change of niclosamide on mRNA of the PI3K/AKT pathway related gene of the colon cancer cells by using a real-time quantitative PCR technology;
and step six, analyzing the expression change condition of niclosamide on the PI3K/AKT pathway related proteins of the colon cancer cells by using a Western Blot technology.
Further, the method for separating and culturing colon cancer cells comprises the following steps:
(1) stirring and mixing 1 part of dimethyl sulfoxide, 2 parts of acetamide, 5 parts of glucose, 3 parts of sodium dihydrogen phosphate, 2 parts of sodium citrate, 3 parts of potassium citrate, 4 parts of magnesium sulfate, 1 part of adenosine and 2 parts of L-arginine according to the weight part data to obtain a preservation solution, cutting fresh colon cancer tissues under aseptic condition, placing the fresh colon cancer tissues into the preservation solution for preservation, and sealing and transporting the fresh colon cancer tissues back to a laboratory at 5 ℃; soaking the tissue processing apparatus in 76% ethanol water solution, placing in a sterile operating table, performing ultraviolet sterilization for 30min, taking out the apparatus, and air drying in the sterile operating table;
(2) placing the tissue into a precooled sterile PBS solution containing double antibiotics, placing a culture dish on ice to wash and remove the mesentery, blood vessels, fat and necrotic tissues on the surface, and shearing the colon cancer tissue into fragments of 1mm multiplied by 1mm by using an ophthalmic scissors in the precooled PBS solution under the sterile condition;
(3) digesting with trypsin-EDTA digestive juice and collagenase respectively, which are preheated at 38 ℃; terminating digestion with mixed culture medium containing fetal calf serum, double antibody and insulin (mixed culture medium for short), centrifuging the obtained filtrate 356g for 6min, removing supernatant, retaining precipitate, and repeating twice; taking cell filtrate, and detecting the cell activity by trypan blue staining;
(4) adding complete culture medium for re-suspension, and counting by a cell counting plate; after counting, the cells were inoculated into a culture flask coated with polylysine and placed at 38 ℃ in 6% CO 2 Continuously culturing in the environment, and changing the culture solution every 4 days;
(5) cell purification and enzyme digestion; subculturing cells; and (5) observing the cell morphology and growth condition.
Further, the concentration of the pre-cooled PBS was 0.02M and the pH was 7.5.
Further, the digestion environment is 38 ℃ and 6% CO 2 Incubator, digestion with 0.4% trypsin for 16min, and then 0.3% collagenase iv for 5 h.
Further, the 0.26% trypsin digest was digested for 11min and then digested for 3.6min with collagenase iv containing 0.2%.
Further, the detection method of the MTT experimental method comprises the following steps:
succinate dehydrogenase in mitochondria of living cells can reduce exogenous 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) into insoluble formazan, and dead cells do not have the function;
the dimethyl sulfoxide (DMSO) can dissolve formazan in cells, 570nm) is used for determining the light absorption value of the formazan, and the light absorption value is in direct proportion to the number of living cells, so that the cell survival rate after the drug treatment can be determined;
different colon cancer cells (5X 10) in logarithmic growth phase 3 One) were inoculated in 96-well plates, and HCT116, SW480, SW620 cells were treated with different concentrations of niclosamide (0, 2, 4, 6, 8 μmol/L, respectively; HT29 cells treated with 0, 5, 10, 15, 20 μmol/L) were treated, each set was provided with 5 replicate wells;
after 72h incubation, 150. mu.L MTT (5. mu.g/. mu.L) was added to each well and incubated for 4 h; after dissolving in 200. mu.L DMSO, the absorbance of each well was measured at a wavelength of 570nm using a microplate reader;
and (4) carrying out three biological repetitions, calculating the average value of absorbance, calculating the cell survival rate, and calculating the half effective inhibition concentration (IC50 value) of niclosamide on different colon cancer cells.
Further, the flow cytometry detection method comprises the following steps:
selecting HCT116 cells with lower IC50 value for subsequent experiments; taking 1X 10 5 2mL of cell suspension was seeded in a six-well plate, and after treatment with various concentrations of niclosamide (0, 0.5, 1, 2. mu. mol/L) for 48 hours, the cells were harvested and incubated overnight at 4 ℃ with 75% ice-cold ethanol;
washing with PBS, removing ethanol, and incubating in a dark room for 30min at room temperature with a cell cycle detection kit (containing 1mg/mL PI and 10mg/mL RNase A); cell cycle distribution and apoptosis rate were determined by flow cytometry, and experimental results were analyzed by ModFit software.
Further, the DAPI fluorescent staining method is as follows:
4', 6-diamidino-2-phenylindole (DAPI) can be strongly combined with DNA, fluorescence is emitted under the excitation of ultraviolet light, and the apoptosis condition can be observed according to the staining condition of cell nucleus; in the experiment, colon cancer HCT116 cells are inoculated in a 6-well plate with a cover glass and cultured for 24 hours until the cells adhere to the wall; adding niclosamide with different concentrations (0, 0.5, 1, 2 μmol/L) and continuing culturing for 48 hours, taking out and fixing with 4% paraformaldehyde for 10 minutes, washing with PBS, staining with DAPI staining working solution, and observing apoptosis under a fluorescence microscope.
Further, the detection method of the quantitative PCR technology comprises the following steps:
extracting total mRNA of colon cancer cells treated by niclosamide with different concentrations (0, 0.5, 1.0 and 2.0 mu mol/L) by a TRIzol reagent method, and synthesizing cDNA (Hangzhou Watson Biotech Co.) by a reverse transcription kit method; according to the method provided by the kit operating instruction, SYBRmix is used for carrying out real-time fluorescence quantitative PCR on a cDNA template, and RT-PCR reaction is carried out according to the following system; performing PCR amplification by using the synthesized cDNA as a template;
the PCR amplification system is as follows: 1 μ L of cDNA template, 1 μ L of dNTP Mixture (10mmol/L), 1 μ L of upstream primer (20 μmol/L), 1 μ L of downstream primer (20 μmol/L), 8 μ L of 2 μ M ultra-SYBR Mixture (With ROX), 8 μ L of double distilled water, and 20 μ L of total volume;
the amplification reaction procedure was: pre-denaturing at 95 ℃ for 10min, then circulating for 35 times by a program of 94 ℃ for 30s, 60 ℃ for 45s and 72 ℃ for 1min, and extending at 72 ℃ for 10 min; designing specific primers for real-time fluorescent quantitative PCR amplification cDNA by Premier Premier 5.0 primer design software, confirming amplification curve and dissolution curve after amplification reaction is finished, and adopting 2 for amplification result -△△Ct The method shows that the expression change of each gene is calculated by comparing with the reference gene GAPDH.
Further, the Western Blot analysis method comprises the following steps:
treating colon cancer HCT116 cells with niclosamide with different concentrations (0, 0.5, 1.0, 2.0 μmol/L) for 48 hours, collecting cells, washing twice with PBS, lysing at 4 deg.C for 30 minutes, centrifuging at 14000g for 15 minutes to remove insoluble precipitate, and extracting total cell protein;
the BCA method is used for measuring the concentration of the total protein, and then 10 percent SDS-polyacrylamide gel electrophoresis is carried out for separating the protein, wherein the sample loading amount of each sample loading hole is 30 mu g of the total protein;
transferring the protein self-gel to a PVDF membrane after electrophoresis, sealing with 5% skimmed milk powder, washing with TTBS, adding primary antibody (diluted according to a ratio of 1: 1000), and incubating for 2 hours or overnight at 4 ℃;
then adding horseradish peroxidase-labeled secondary antibody IgG working solution (a mouse-anti-rabbit secondary antibody diluted according to the proportion of 1: 5000), and incubating for 2 hours at room temperature or overnight at 4 ℃; the expression change of each related protein is compared with the internal reference GAPDH by developing on a gel imager and analyzing by Image Lab software, and the ratio of the expression change of the related protein is expressed.
The invention has the advantages and positive effects that: the MTT experiment result shows that the niclosamide has obvious proliferation inhibition effect on different colon cancer cells, the inhibition effect has time and dosage effects to a certain extent, and the IC50 values of different colon cancer cells are slightly different. Flow cytometry results show that niclosamide can block colon cancer HCT116 cells in the G2/M stage, and DAPI fluorescent staining shows that niclosamide induces colon cancer HCT116 cells to be apoptotic. Further researching a PI3K/AKT passage, and a Western Blot experiment shows that niclosamide can down-regulate the expression of proteins such as PI3K, p-AKT, p-NF-kappa B, p-ERK, Survivin and the like of HCT116 cells, up-regulate the expression of TNF-alpha protein, and a real-time quantitative PCR result is consistent with a Western Blot result; niclosamide can block cell proliferation and induce cell apoptosis through a PI3K-AKT signal channel, thereby inhibiting tumor cell proliferation and playing an anti-tumor role; meanwhile, the colon cancer cells obtained by the method for separating and culturing the colon cancer cells provided by the invention are as follows: high yield, high purity, high activity, high cell survival rate up to 95% or more, and high cell purity up to 96% or more.
Drawings
FIG. 1 is a flow chart of a method for determining the cell cycle arrest and apoptosis induction effects of niclosamide on colon cancer mediated by PI3K-AKT signaling pathway.
FIG. 2 is a graph showing the effect of niclosamide on the inhibition of proliferation of colon cancer cells.
FIG. 3 is a graph showing the effect of niclosamide treatment for 48h on the cell cycle of colon cancer cells HCT116 according to the present invention.
(A) Control group: stage G1: 61.27 percent; stage G2, 12.31%; 26.12 percent of S phase; the apoptosis rate is 0.58%. (B) 59.58% in G1 stage of 0.5 μmol/L treatment group; 9.95% in stage G2; 28.63 percent of S period; the apoptosis rate was 0.23%. (C) 1.0. mu. mol/L treatment group, G1 group, 51.52%; stage G2, 5.83%; 42.61% in S phase; the apoptosis rate is 0.56%. (D) 53.78% of G1 stage in a treatment group of 2.0 mu mol/L; stage G2, 4.90%; 41.31% in S phase; the apoptosis rate is 27.05%.
FIG. 4 is a graph showing the effect of niclosamide treatment for 72h on the cell cycle of colon cancer cells HCT116 according to the present invention.
(A) 63.16% of G1 stage in a control group; stage G2: 1.28%; 35.65 percent of S period; apoptosis rate 0.02%, (B) 0.5. mu. mol/L treatment group, stage G1: 60.11%; stage G2, 6.38%; 33.56 percent of S phase; the apoptosis rate is 0.05%; (C) 1.0. mu. mol/L treatment group, G1 stage 41.12%; stage G2, 10.86%; 48.09% of S phase; the apoptosis rate is 0.8%; (D)2.0 μmol/L treatment group, G1 stage 14.13%; 29.85% in stage G2; 56.13% in S phase; the apoptosis rate is 21.61%.
FIG. 5 is a graph showing the effect of niclosamide on apoptosis (DAPI staining, 20X) of colon cancer cells HCT116 according to the present invention.
FIG. 6 is a graph showing the effect of niclosamide on cell signaling pathway of colon cancer cells HCT116 according to the present invention.
(A) (ii) an expression profile of a key molecule of the PI3K-AKT signaling pathway; (B) (ii) a change in expression of a molecule downstream of the PI3K-AKT signaling pathway; (C) expression of apoptosis-related protein molecules changes.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The application of the principles of the present invention will now be further described with reference to the accompanying drawings.
As shown in figure 1, the invention provides a method for determining the effect of the PI3K-AKT signal pathway mediated niclosamide on the cell cycle arrest and apoptosis induction of colon cancer, which comprises the following steps:
s101, separating and culturing colon cancer cells;
s102, detecting the proliferation inhibition effect of niclosamide on colon cancer cells by using an MTT (methyl thiazolyl tetrazolium) experimental method;
s103, detecting the inhibition effect of niclosamide on the colon cancer cell cycle through flow cytometry;
s104, observing the induction effect of niclosamide on colorectal cancer cell apoptosis by a DAPI fluorescent staining method;
s105, detecting the expression change of niclosamide on the mRNA of the gene related to the PI3K/AKT pathway of the colon cancer cells by using a real-time quantitative PCR technology;
s106, analyzing the expression change of the niclosamide on the PI3K/AKT pathway related protein of the colon cancer cells by using a Western Blot technology.
The method for separating and culturing colon cancer cells provided by the invention comprises the following steps:
(1) stirring and mixing 1 part of dimethyl sulfoxide, 2 parts of acetamide, 5 parts of glucose, 3 parts of sodium dihydrogen phosphate, 2 parts of sodium citrate, 3 parts of potassium citrate, 4 parts of magnesium sulfate, 1 part of adenosine and 2 parts of L-arginine according to the weight part data to obtain a preservation solution, cutting fresh colon cancer tissues under aseptic condition, placing the fresh colon cancer tissues into the preservation solution for preservation, and sealing and transporting the fresh colon cancer tissues back to a laboratory at 5 ℃; soaking the tissue processing apparatus in 76% ethanol water solution, placing in a sterile operating table, performing ultraviolet sterilization for 30min, taking out the apparatus, and air drying in the sterile operating table;
(2) putting the tissue into a precooled sterile PBS (phosphate buffer solution) containing double-antibody, putting a culture dish on ice to wash and remove a mesentery, a blood vessel, fat and necrotic tissues on the surface, and shearing the colon cancer tissue into fragments of 1mm multiplied by 1mm by using an ophthalmic scissors in the precooled PBS under the sterile condition;
(3) digesting with trypsin-EDTA digestive juice and collagenase respectively, which are preheated at 38 ℃; terminating digestion with mixed culture medium containing fetal calf serum, double antibody and insulin (mixed culture medium for short), centrifuging the obtained filtrate 356g for 6min, removing supernatant, retaining precipitate, and repeating twice; taking cell filtrate, and detecting the cell activity by trypan blue staining;
(4) adding complete culture medium for resuspension, and counting by a cell counting plate; after counting, the cells were inoculated into a culture flask coated with polylysine and placed at 38 ℃ in 6% CO 2 Continuously culturing in the environment, and changing the culture solution every 4 days;
(5) cell purification and enzyme digestion; subculturing cells; and (5) observing the cell morphology and growth condition.
The concentration of the precooled PBS provided by the invention is 0.02M, and the pH is 7.5.
The digestion environment provided by the invention is 38 ℃, and 6% CO 2 Incubator, digestion with 0.4% trypsin for 16min, and then 0.3% collagenase iv for 5 h.
The trypsin digestive juice with the concentration of 0.26% provided by the invention is digested for 11min, and then digested for 3.6min by collagenase with concentration of 0.2%.
The MTT experimental detection method provided by the invention comprises the following steps:
succinate dehydrogenase in mitochondria of living cells can reduce exogenous 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT) into insoluble formazan, and dead cells do not have the function;
the dimethyl sulfoxide (DMSO) can dissolve formazan in cells, 570nm) is used for determining the light absorption value of the formazan, and the light absorption value is in direct proportion to the number of living cells, so that the cell survival rate after the drug treatment can be determined;
different colon cancer cells (5X 10) in logarithmic growth phase 3 One) were inoculated in 96-well plates, and HCT116, SW480, SW620 cells were treated with different concentrations of niclosamide (0, 2, 4, 6, 8 μmol/L, respectively; HT29 cells were treated with 0, 5, 10, 15, 20 μmol/L) with 5 multiple wells per group;
after 72h incubation, 150. mu.L MTT (5. mu.g/. mu.L) was added to each well and incubated for 4 h; after dissolving in 200. mu.L DMSO, the absorbance of each well was measured at a wavelength of 570nm using a microplate reader;
and (4) performing three biological repetitions, calculating the average value of absorbance, calculating the cell survival rate, and calculating the half effective inhibition concentration (IC50 value) of niclosamide on different colon cancer cells.
The flow cytometry detection method provided by the invention comprises the following steps:
selecting HCT116 cells with lower IC50 values for subsequent experiments; taking 1X 10 5 2mL of cell suspension was seeded in a six-well plate, and after treatment with various concentrations of niclosamide (0, 0.5, 1, 2. mu. mol/L) for 48 hours, the cells were harvested and incubated overnight at 4 ℃ with 75% ice-cold ethanol;
washing with PBS, removing ethanol, and incubating in a dark room for 30min at room temperature with a cell cycle detection kit (containing 1mg/mL PI and 10mg/mL RNase A); cell cycle distribution and apoptosis rate were determined by flow cytometry, and experimental results were analyzed by ModFit software.
The DAPI fluorescent staining method provided by the invention comprises the following steps:
4', 6-diamidino-2-phenylindole (DAPI) can be strongly combined with DNA, fluorescence is emitted under the excitation of ultraviolet light, and the apoptosis condition can be observed according to the staining condition of cell nucleus; in the experiment, colon cancer HCT116 cells are inoculated in a 6-well plate with a cover glass and cultured for 24 hours until the cells adhere to the wall; adding niclosamide with different concentrations (0, 0.5, 1 and 2 mu mol/L) for continuous culture for 48 hours, taking out and fixing with 4% paraformaldehyde for 10 minutes, washing with PBS, dyeing with DAPI dyeing working solution, and observing apoptosis under a fluorescence microscope.
The quantitative PCR technology detection method provided by the invention comprises the following steps:
extracting total mRNA of colon cancer cells treated by niclosamide with different concentrations (0, 0.5, 1.0 and 2.0 mu mol/L) by a TRIzol reagent method, and synthesizing cDNA (Hangzhou Watson Biotech Co.) by a reverse transcription kit method; according to the method provided by the kit operating instruction, SYBRmix is used for carrying out real-time fluorescence quantitative PCR on a cDNA template, and RT-PCR reaction is carried out according to the following system; carrying out PCR amplification by taking the synthesized cDNA as a template;
the PCR amplification system is as follows: 1 μ L of cDNA template, 1 μ L of dNTP Mixture (10mmol/L), 1 μ L of upstream primer (20 μmol/L), 1 μ L of downstream primer (20 μmol/L), 8 μ L of 2 μ M ultra-SYBR Mixture (With ROX), 8 μ L of double distilled water, and 20 μ L of total volume;
the amplification reaction procedure was: pre-denaturation at 95 deg.C for 10min, and then repeating for 35 times with a program of 94 deg.C for 30s, 60 deg.C for 45s, and 72 deg.C for 1min, and extending at 72 deg.C for 10 min; the specific primer of the real-time fluorescence quantitative PCR amplified cDNA is designed by Premier Premier 5.0 primer design software, the amplification curve and the dissolution curve are confirmed after the amplification reaction is finished, and the amplification result adopts 2 -△△Ct The method shows that the expression change of each gene is calculated by comparing with the reference gene GAPDH.
The Western Blot technical analysis method provided by the invention comprises the following steps:
treating colon cancer HCT116 cells with niclosamide with different concentrations (0, 0.5, 1.0, 2.0 μmol/L) for 48 hours, collecting cells, washing twice with PBS, lysing at 4 deg.C for 30 minutes, centrifuging at 14000g for 15 minutes to remove insoluble precipitate, and extracting total cell protein;
the BCA method is used for measuring the concentration of the total protein, and then 10 percent SDS-polyacrylamide gel electrophoresis is carried out for separating the protein, wherein the sample loading amount of each sample loading hole is 30 mu g of the total protein;
transferring the protein self-gel to a PVDF membrane after electrophoresis, sealing with 5% skimmed milk powder, washing with TTBS, adding primary antibody (diluted according to a ratio of 1: 1000), and incubating for 2 hours or overnight at 4 ℃;
then adding horseradish peroxidase-labeled secondary antibody IgG working solution (a mouse-anti-rabbit secondary antibody diluted according to the proportion of 1: 5000), and incubating for 2 hours at room temperature or overnight at 4 ℃; the expression change of each related protein is compared with the internal reference GAPDH by developing on a gel imager and analyzing by Image Lab software, and the ratio of the expression change of the related protein is expressed.
Example (b):
1. the material and the method are as follows:
1.1 materials
Niclosamide (2 ', 5 ' -dichloro-4 ' -nitrosalicylic acid anilide, Shanghai Yan Biotech Co., Ltd.; batch No. 0560000) was dissolved in dimethyl sulfoxide to prepare a mother liquor of 10mmol/L, which was stored in a refrigerator at-20 ℃ for further use. PI3K antibody, AKT antibody, p-AKT antibody (S473), ERK-1/2 antibody, p-ERK-1/2, NF-. kappa.B antibody, p-NF-. kappa.B antibody, Survivin antibody, GAPDH antibody, etc. were purchased from Abcam corporation (Louis Park, MN, USA). Cell cycle detection kit (Abcam company), reverse transcription reagent, TRIzol kit (Hangzhou Watson Biotechnology company), 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT), 4', 6-diamidino-2-phenylindole (DAPI), BCA protein concentration determination kit, DMEM medium and Fetal Bovine Serum (FBS) are purchased from Hangzhou Sijiqing company.
1.2 methods
1.2.1 cell culture
Human colon cancer cell lines HCT116, SW620, SW480, HT29 were purchased from the chinese typical culture storage center (china, wuhan). The colon cancer cells are cultured in DMEM medium containing 10% fetal calf serum at 37 deg.C and 5% CO 2 Culturing in a constant temperature incubator under the condition. And (4) carrying out passage when the cells grow over the culture flask, and taking the cells in the logarithmic growth phase for subsequent experiments.
1.2.2 MTT assay for cell viability
Succinate dehydrogenase in mitochondria of living cells can further convert exogenous 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT)It was originally insoluble formazan, and dead cells did not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, 570nm) is used for measuring the light absorption value of the formazan, and the light absorption value is in direct proportion to the number of living cells, so that the cell survival rate after the drug treatment can be measured. Different colon cancer cells (5X 10) in logarithmic growth phase 3 One) were inoculated in 96-well plates, and HCT116, SW480, SW620 cells were treated with different concentrations of niclosamide (0, 2, 4, 6, 8 μmol/L, respectively; HT29 cells were treated with 0, 5, 10, 15, 20 μmol/L) and 5 duplicate wells were set for each group. After 72h incubation, 150. mu.L MTT (5. mu.g/. mu.L) was added to each well and incubated for 4 h. After dissolving in 200. mu.L DMSO, the absorbance of each well was measured at a wavelength of 570nm using a microplate reader. And (4) carrying out three biological repetitions, calculating the average value of absorbance, calculating the cell survival rate, and further calculating the half effective inhibition concentration (IC50 value) of niclosamide on different colon cancer cells.
1.2.3 flow cytometry analysis of the cell cycle
HCT116 cells with lower IC50 values were selected for subsequent experiments. Take 1X 10 5 2mL of the cell suspension was inoculated into a six-well plate, treated with niclosamide (0, 0.5, 1, 2. mu. mol/L) at various concentrations for 48 hours, the cells were harvested and incubated overnight with 75% ice-cold ethanol at 4 ℃. PBS was washed, ethanol removed, and incubated with cell cycle assay kit (containing 1mg/mL PI and 10mg/mL RNase A) for 30min at room temperature in the dark. Cell cycle distribution and apoptosis rate were determined by flow cytometry, and experimental results were analyzed by ModFit software.
1.2.4 DAPI staining for apoptosis
4', 6-diamidino-2-phenylindole (DAPI) can be strongly combined with DNA, and can emit fluorescence under the excitation of ultraviolet light, and the apoptosis condition can be observed according to the staining condition of cell nucleus. In the experiment, colon cancer HCT116 cells are inoculated in a 6-well plate with a cover glass and cultured for 24 hours until the cells adhere to the wall. Adding niclosamide with different concentrations (0, 0.5, 1, 2 μmol/L) and continuing culturing for 48 hours, taking out and fixing with 4% paraformaldehyde for 10 minutes, washing with PBS, staining with DAPI staining working solution, and observing apoptosis under a fluorescence microscope.
1.2.5Western Blot technique for detecting expression of PI3K-AKT signal channel-related protein
The colon cancer HCT116 cells were treated with niclosamide at different concentrations (0, 0.5, 1.0, 2.0. mu. mol/L) for 48 hours, the cells were collected, washed twice with PBS, lysed at 4 ℃ for 30 minutes, centrifuged at 14000g for 15 minutes to remove insoluble precipitates, and total cell proteins were extracted. The total protein concentration was determined by the BCA method, followed by 10% SDS-polyacrylamide gel electrophoresis to separate the proteins in an amount of 30. mu.g total protein per well. After electrophoresis, the proteins were transferred from the gel to PVDF membrane, blocked with 5% skim milk, washed with TTBS, and then primary antibody was added (diluted 1: 1000) and incubated for 2 hours or overnight at 4 ℃. Then, horseradish peroxidase-labeled secondary antibody IgG working solution (mouse-anti-rabbit secondary antibody, diluted at a ratio of 1: 5000) was added thereto, and the mixture was incubated at room temperature for 2 hours or at 4 ℃ overnight. The expression changes of the related proteins are compared with the internal reference GAPDH by developing on a gel imager and analyzing by Image Lab software, and the ratio of the expression changes of the related proteins is expressed.
1.2.6 real-time fluorescent quantitative PCR technology for detecting expression of mRNA of PI3K-AKT signal pathway related gene
Total mRNA of colon cancer cells treated with niclosamide at different concentrations (0, 0.5, 1.0, 2.0. mu. mol/L) was extracted by TRIzol reagent method, and cDNA (Hangzhou Watson Biotech) was synthesized by reverse transcription kit method. According to the method provided by the kit operating instructions, SYBRmix is used to perform real-time fluorescence quantitative PCR on the cDNA template, and the RT-PCR reaction is performed according to the following system. PCR amplification was performed using the synthesized cDNA as a template. The PCR amplification system is as follows: 1. mu.L of cDNA template, 1. mu.L of dNTP Mixture (10mmol/L), 1. mu.L of upstream primer (20. mu. mol/L), 1. mu.L of downstream primer (20. mu. mol/L), 8. mu.L of 2 XM ultra-SYBR Mixture (With ROX), 8. mu.L of double distilled water, and 20. mu.L of total volume. The amplification reaction procedure was: pre-denaturation at 95 ℃ for 10min, followed by 35 cycles with a program of 94 ℃ for 30s, 60 ℃ for 45s, 72 ℃ for 1min, and extension at 72 ℃ for 10 min. The specific primers for real-time fluorescence quantitative PCR amplification of cDNA were designed by Premier Premier 5.0 primer design software (Table 1.), the amplification curve and the dissolution curve were confirmed after the amplification reaction was completed, the amplification results were expressed by 2- Δ Ct method, and the expression changes of the genes were calculated by comparing with the reference gene GAPDH.
TABLE 1 RT-PCR primers
Figure BDA0003684057450000131
1.2.7 statistical analysis
Statistical analysis was performed using SPSS 17.0 and the experimental data are presented as mean. + -. standard deviation (S. + -. D). Statistical significance test Using Student's t test, significant statistical differences were indicated by P < 0.05.
2 results
2.1 proliferation inhibitory Effect of niclosamide on colonic cancer cells
The method comprises the steps of treating colon cancer cells by niclosamide with different concentrations, and detecting the cell survival rate by an MTT (methyl thiazolyl tetrazolium) experiment. The results show that the niclosamide has a certain inhibition effect on various colon cancer cells. Survival curves for various colon cancer cells (HCT116, SW480, SW620, HT29) are shown (fig. 2). The half inhibitory concentrations (IC50) were measured as: HCT116 is 0.98 +/-0.105 mu mol/L; SW 480: 1.13 +/-0.103 mu mol/L; SW 620: 1.85. + -. 0.213 μm OL/L and HT 29: 2.78. + -. 0.308. mu. mol/L.
2.2 Effect of niclosamide on the cell cycle of colon cancer
Subject groups selected colon cancer HCT116 cells with relatively significant inhibition (lower IC50) for follow-up experiments. HCT116 cells were treated with niclosamide at concentrations of 0, 0.5, 1.0 and 2.0 μ M for 48h and 72h, respectively, then collected, fixed and stained, and the effect of niclosamide on the HCT116 cell cycle was analyzed by flow cytometry. The results show that with increasing concentrations of niclosamide, the proportion of cells in the G1 phase decreased, and the proportion of cells in the S phase and G2/M phase increased (Table one). The percentage of cells in the G1 phase decreased significantly with time. The inhibition of HCT116 cells in S phase and G2/M phase by niclosamide is somewhat time and dose dependent. Niclosamide inhibits cell proliferation and induces apoptosis by blocking HCT116 cell cycle, and the apoptosis rate shows a trend of obviously increasing with the increase of drug concentration. (FIGS. 3 and 4).
TABLE 2 Effect of niclosamide on the cell cycle of colon cancer cells HCT116
Table 2 Influence of niclosamide on HCT116 cell cycle distribution(
Figure BDA0003684057450000141
n＝3)
Figure BDA0003684057450000142
Comparison with control group: p <0.05, compared to control: p <0.01 vs control
Tab.1 Comparison of cell cycles between four groups
Table 14 cell cycle comparisons of the group of cells (n ═ 6,%,
Figure BDA0003684057450000143
)
Figure BDA0003684057450000144
＊＊P＜0.01； a compared with the control group, the compound is added, a p <0.05 compared to the 20. mu. mol/L T5450996 treated group
2.3 Effect of niclosamide on apoptosis of HCT116 cells
The flow cytometry result shows that the niclosamide obviously inhibits apoptosis, and the influence of the niclosamide on the apoptosis is further observed and verified by a DAPI staining method. The results show that the control group has clear visible cell nucleuses, large cell nucleuses volume and uneven distribution state. The nucleus of the niclosamide treated group has the phenomenon of nucleus condensation, the shape of the nucleus is irregular, and even the nucleus is broken into fragments to form apoptotic bodies. And the apoptosis phenomenon becomes more obvious with the increase of niclosamide concentration (figure 5).
2.4 Effect of niclosamide on PI3K/AKT Signaling pathway-related proteins in HCT116 cells
Treating HCT116 cells with niclosamide at 0, 0.5, 1.0 and 2.0 μ M for 48h, respectively, extracting total cell protein, and detecting PI3K/AKT pathway related factor by western blotting method. The results showed that niclosamide dose-dependently down-regulated PI3K expression, but had no significant effect on AKT expression, while dose-dependently down-regulating p-AKT expression (fig. 6A).
The downstream molecules of the PI3K/AKT signaling pathway, which are associated with cell cycle and apoptosis, were further examined using the methods described above. The results show that niclosamide can significantly reduce the expression of p-NF-kappa B, p-ERK1/2 and ERK1/2, but has no obvious effect on the expression of NF-kappa B (FIG. 6B). In addition, niclosamide also up-regulated the expression of tumor necrosis factor alpha (TNF- α), down-regulated the expression of the apoptotic protein survivin (fig. 6C).
2.5 Effect of niclosamide on mRNA levels of genes associated with PI3K/AKT signalling pathway in HCT116 cells
The method is used for detecting the expression change of the PI3K/AKT signal pathway related gene mRNA by adopting a real-time quantitative PCR technology, and further verifying the western blotting experiment result. After the real-time quantitative PCR reaction was completed, the amplification curve and the dissolution curve were confirmed. The results were calculated as 2-DELTA. Ct, and the change in the mRNA expression level of each relevant gene was expressed as the ratio of the objective gene to the reference gene, GAPDH (Table II).
Effect of Epichlorosamide on mRNA of PI3K/AKT signal pathway-related gene of HCT116 cells with colon cancer
Table 3 Effect of niclosamide on the PI3K/AKT signal pathway related gene mRNA level in HCT116 cells
Figure BDA0003684057450000151
Comparison with control group: p < 0.05; comparing to control group: p <0.01
Discussion 3
Colon cancer is a malignant tumor of the digestive tract which is well developed in the colon part, the incidence rate of the malignant tumor is higher in the digestive system tumor, and long-term colon polyp can also be developed into colon cancer. At present, the operation treatment is the best treatment method for colon cancer, and the postoperative chemotherapy has better treatment effect on colon cancer. In the chemotherapy of colon cancer, the most important drug is the Aureoxaplatin, the long-term chemotherapy of tumor patients can generate drug resistance, and the wide-range multidrug resistance (MDR) seriously influences the chemotherapy effect of tumors [15-16 ]. Molecularly targeted drugs such as erbitux (cetuximab) and avastin (bevacizumab) are important choices for treating colon cancer, and bring new hopes for patients with advanced colon cancer, but the curative effects of the drugs are still far from satisfactory. The search for new antitumor drugs still has important significance for the treatment of colon cancer.
Niclosamide is used as a traditional anthelmintic and veterinary drug and has a good inhibition effect on tumor cells in recent years, and the experiment takes colon cancer cells as objects and explores the proliferation inhibition effect and the molecular mechanism of niclosamide on the colon cancer cells.
The experiment firstly verifies that niclosamide has proliferation inhibition effect on colon cancer cells by adopting an MTT (methyl thiazolyl tetrazolium) experiment, then observes the influence of niclosamide on the cycle and apoptosis of the colon cancer cells by adopting flow cytometry and DAPI (fluorescent staining of deoxyribonucleic acid) fluorescence, and finally researches the signal channel of the niclosamide for inhibiting the tumor cells by adopting a Western technology and a real-time quantitative PCR (polymerase chain reaction) technology. Studies show that niclosamide blocks the action of colon cancer cells in S phase and G2/M phase, thereby inhibiting the proliferation of HCT116 cells and inducing apoptosis. Niclosamide can reduce the expression of PI3K when treating colon cancer cells, PI3K is an intracellular phosphatidylinositol kinase, has serine/threonine kinase activity, reduces the expression, reduces the phosphorylation of AKT, further reduces the phosphorylation of downstream molecules ERK and NF-kB by p-AKT, and reduces the phosphorylation of NF-kB which is a transcription factor, so that the expression of TNF-alpha protein is increased, the expression of Survivin is reduced, the cell division is hindered, and the cell apoptosis is started.
In conclusion, the PI3K/AKT signaling pathway may play an important role in the inhibition of colon cancer cell cycle and the induction of apoptosis by niclosamide.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. A method for determining the effects of mediating niclosamide on cell cycle arrest and apoptosis induction of colon cancer by a PI3K-AKT signal pathway is characterized in that the method for determining the effects of mediating niclosamide on cell cycle arrest and apoptosis induction of colon cancer by the PI3K-AKT signal pathway comprises the following steps:
step one, separating and culturing colon cancer cells;
step two, detecting the proliferation inhibition effect of niclosamide on colon cancer cells by using an MTT (methyl thiazolyl tetrazolium) experimental method;
step three, detecting the inhibition effect of niclosamide on the colon cancer cell cycle through flow cytometry;
observing the induction effect of niclosamide on the colorectal cancer cell apoptosis by a DAPI fluorescent staining method;
step five, detecting the expression change of niclosamide on the mRNA of the gene related to the PI3K/AKT pathway of the colon cancer cells by using a real-time quantitative PCR technology;
sixthly, analyzing the expression change of the niclosamide on the PI3K/AKT pathway related protein of the colon cancer cells by using a Western Blot technology.
2. The method for measuring the effects of PI3K-AKT signaling pathway mediated by niclosamide on the cycle arrest and apoptosis induction of colon cancer cells as claimed in claim 1, wherein the method for isolating and culturing colon cancer cells comprises the following steps:
(1) stirring and mixing 1 part of dimethyl sulfoxide, 2 parts of acetamide, 5 parts of glucose, 3 parts of sodium dihydrogen phosphate, 2 parts of sodium citrate, 3 parts of potassium citrate, 4 parts of magnesium sulfate, 1 part of adenosine and 2 parts of L-arginine according to the weight part data to obtain a preservation solution, cutting fresh colon cancer tissues under aseptic condition, placing the fresh colon cancer tissues into the preservation solution for preservation, and sealing and transporting the fresh colon cancer tissues back to a laboratory at 5 ℃; soaking the tissue processing apparatus in 76% ethanol water solution, placing in a sterile operating table, performing ultraviolet sterilization for 30min, taking out the apparatus, and air drying in the sterile operating table;
(2) placing the tissue into a precooled sterile PBS solution containing double antibiotics, placing a culture dish on ice to wash and remove the mesentery, blood vessels, fat and necrotic tissues on the surface, and shearing the colon cancer tissue into fragments of 1mm multiplied by 1mm by using an ophthalmic scissors in the precooled PBS solution under the sterile condition;
(3) digesting with trypsin-EDTA digestive juice and collagenase respectively, which are preheated at 38 ℃; terminating digestion with mixed culture medium containing fetal calf serum, double antibody and insulin, centrifuging the obtained filtrate 356g for 6min, removing supernatant, retaining precipitate, and repeating twice; taking cell filtrate, and detecting the cell activity by trypan blue staining;
(4) adding complete culture medium for re-suspension, and counting by a cell counting plate; after counting the cells, the cells were inoculated into a culture flask coated with polylysine and placed at 38 ℃ in 6% CO 2 Continuously culturing in the environment, and changing the culture solution every 4 days;
(5) purifying cells and performing an enzyme digestion method; subculturing the cells; and (5) observing the cell morphology and growth condition.
3. The method of claim 1, wherein the pre-chilled PBS concentration is 0.02M and the PH is 7.5, and the PI3K-AKT signaling pathway mediates niclosamide cell cycle arrest and induction of apoptosis in colon cancer.
4. The method for determining the cell cycle arrest and apoptosis induction of colon cancer mediated by PI3K-AKT signaling pathway as claimed in claim 1, wherein said digestion environment is 38 deg.C and 6% CO 2 Incubators, digested with 0.4% trypsin for 16min and then 0.3% collagenase iv for 5 h.
5. The method of claim 1, wherein the PI3K-AKT signaling pathway mediated effect of niclosamide on cell cycle arrest and induction of apoptosis in colon cancer is determined by digesting with 0.26% trypsin digest for 11min and then with collagenase iv containing 0.2% for 3.6 min.
6. The method for determining the cell cycle arrest and apoptosis induction of colon cancer mediated by PI3K-AKT signaling pathway as claimed in claim 1, wherein the MTT assay comprises the following steps:
1) succinate dehydrogenase in mitochondria of living cells can reduce exogenous 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) into insoluble formazan, and dead cells do not have the function;
2) dimethyl sulfoxide (DMSO) can dissolve formazan in cells, 570nm) and the light absorption value is measured, and the light absorption value is in direct proportion to the number of living cells, so that the cell survival rate after the drug treatment can be measured;
3) inoculating colon cancer cells in different logarithmic growth phases into a 96-well plate, and respectively treating with niclosamide with different concentrations, wherein each group is provided with 5 multiple wells;
4) after culturing for 72h, adding 150 mu L MTT into each well, and incubating for 4 h; after dissolving in 200 μ L DMSO, measuring the absorbance of each well at a wavelength of 570nm by using a microplate reader;
5) and (4) carrying out biological repetition for three times, calculating the average value of absorbance, calculating the survival rate of the cells, and calculating the half effective inhibition concentration of niclosamide on different colon cancer cells.
7. The method for determining the effects of niclosamide on the cell cycle arrest and apoptosis induction of colon cancer cell cycle mediated by PI3K-AKT signaling pathway as claimed in claim 1, wherein the flow cytometry detection method comprises:
selecting HCT116 cells with lower IC50 values for subsequent experiments; taking 1X 10 5 2mL of the cell suspension was inoculated into a six-well plate, treated with niclosamide at different concentrations for 48 hours, the cells were collected and incubated overnight at 4 ℃ with 75% ice-cold ethanol;
washing with PBS, removing ethanol, and incubating in dark room for 30min at room temperature with cell cycle detection kit; and measuring cell cycle distribution and apoptosis rate by using a flow cytometer, and analyzing an experimental result by using ModFit software.
8. The method for determining the cell cycle arrest and apoptosis induction of colon cancer mediated by PI3K-AKT signaling pathway as claimed in claim 1, wherein the DAPI fluorescence staining method is as follows:
4', 6-diamidino-2-phenylindole (DAPI) can be strongly combined with DNA, fluorescence is emitted under the excitation of ultraviolet light, and the apoptosis condition of cells can be observed according to the staining condition of cell nuclei; in the experiment, colon cancer HCT116 cells are inoculated in a 6-well plate with a cover glass and cultured for 24 hours until the cells adhere to the wall; adding niclosamide with different concentrations, continuously culturing for 48 hours, taking out, fixing with 4% paraformaldehyde for 10 minutes, washing with PBS, adding DAPI staining working solution for staining, and observing apoptosis under a fluorescence microscope.
9. The method for detecting the cell cycle arrest and apoptosis induction of colon cancer mediated by PI3K-AKT signaling pathway as claimed in claim 1, wherein the quantitative PCR detection method comprises the following steps:
extracting colon cancer cell total mRNA treated by niclosamide with different concentrations by using a TRIzol reagent method, and synthesizing cDNA by using a reverse transcription kit method; according to the method provided by the kit operating instruction, SYBRmix is used for carrying out real-time fluorescence quantitative PCR on a cDNA template, and RT-PCR reaction is carried out according to the following system; performing PCR amplification by using the synthesized cDNA as a template;
the PCR amplification system is as follows: 1 mu L of cDNA template, 1 mu L of dNTP Mixture, 1 mu L of upstream primer, 1 mu L of downstream primer, 2 XMm ultra-SYBR Mixture8 mu L, 8 mu L of double distilled water and 20 mu L of total volume;
the amplification reaction procedure was: pre-denaturation at 95 deg.C for 10min, and then repeating for 35 times with a program of 94 deg.C for 30s, 60 deg.C for 45s, and 72 deg.C for 1min, and extending at 72 deg.C for 10 min; the specific primer of the real-time fluorescence quantitative PCR amplified cDNA is designed by Premier Premier 5.0 primer design software, the amplification curve and the dissolution curve are confirmed after the amplification reaction is finished, and the amplification result adopts 2 -△△Ct The method shows that the expression change of each gene is calculated by comparing with the reference gene GAPDH.
10. The method for determining the effects of PI3K-AKT signaling pathway mediated by niclosamide on the inhibition of colon cancer cell cycle and the induction of apoptosis as claimed in claim 1, wherein the Western Blot analysis method comprises the following steps:
treating colon cancer HCT116 cells with niclosamide with different concentrations (0, 0.5, 1.0, 2.0 μmol/L) for 48 hours, collecting cells, washing twice with PBS, lysing at 4 deg.C for 30 minutes, centrifuging at 14000g for 15 minutes to remove insoluble precipitate, and extracting total cell protein;
the total protein concentration was measured by BCA method, followed by 10% SDS-polyacrylamide gel electrophoresis to separate the proteins, with a loading of 30. mu.g total protein per well;
transferring the protein self-gel to a PVDF membrane after electrophoresis, sealing with 5% skimmed milk powder, washing with TTBS, adding primary antibodies, diluting according to a ratio of 1:1000, and incubating for 2 hours or overnight at 4 ℃;
then adding horseradish peroxidase-labeled secondary antibody IgG working solution (a mouse-anti-rabbit secondary antibody diluted according to the proportion of 1: 5000), and incubating for 2 hours at room temperature or overnight at 4 ℃; the expression change of each related protein is compared with the internal reference GAPDH by developing on a gel imager and analyzing by Image Lab software, and the ratio of the expression change of the related protein is expressed.
CN202210645854.XA 2022-06-08 2022-06-08 Method for measuring induction effect of niclosamide on cell cycle arrest and apoptosis Withdrawn CN114921520A (en)

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