CN117323317A - Application of benserazide hydrochloride in preparation of anti-colon cancer drugs - Google Patents
Application of benserazide hydrochloride in preparation of anti-colon cancer drugs Download PDFInfo
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- CN117323317A CN117323317A CN202311324784.9A CN202311324784A CN117323317A CN 117323317 A CN117323317 A CN 117323317A CN 202311324784 A CN202311324784 A CN 202311324784A CN 117323317 A CN117323317 A CN 117323317A
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- Prior art keywords
- benserazide hydrochloride
- colon cancer
- cells
- benserazide
- hydrochloride
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- BNQDCRGUHNALGH-UHFFFAOYSA-N benserazide Chemical compound OCC(N)C(=O)NNCC1=CC=C(O)C(O)=C1O BNQDCRGUHNALGH-UHFFFAOYSA-N 0.000 title claims abstract description 89
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an application of benserazide hydrochloride in preparing anti-colon cancer drugs. The invention has found that the concentration of the single use is 10 ‑9 ～10 ‑7 The benserazide hydrochloride of M has the inhibition effect on proliferation and migration of colon cancer cells. Benserazide hydrochloride can inhibit proliferation and migration of colon cancer cells, inhibit glucose uptake by cells, reduce lactic acid accumulation, and down regulate mRNA levels of glycolysis key enzymes GLUT-1, PKM2, HK2 and LDHA and protein expression levels of GLUT-1 and LDHA, thereby realizing the effect of resisting colon cancer. Therefore, the benserazide hydrochloride can be used for preparing anti-colon cancer drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of benserazide hydrochloride in preparation of anti-colon cancer medicines; in particular to an application of benserazide hydrochloride serving as a unique active ingredient in preparing a medicine for treating colon cancer.
Background
Benserazide hydrochloride is a peripheral decarboxylase inhibitor, has the effect similar to carbidopa, is often combined with levodopa to prepare a compound preparation of the dobby hydrazine, and can inhibit the conversion of the levodopa into dopamine in the outside, so that the amount of the levodopa entering the brain is increased, and the compound preparation is used for treating parkinsonism.
Cancer is a disease that severely jeopardizes human health and is primarily manifested by disruption of the homeostasis of cell growth and death, unlimited proliferation and division, loss of contact inhibition, and susceptibility to metastasis. In solid tumors, the tumor cells consume oxygen and nutrients rapidly, have the characteristics of high metabolism and high oxygen consumption, and finally form an anoxic environment. This anaerobic environment induces the expression of hypoxia responsive factors, which bind to hypoxia responsive elements of the target gene, thereby mediating the transcriptional expression of the downstream target gene. One of the important is to regulate glucose metabolism, up-regulate glycolytic enzyme expression under anaerobic conditions, promote cell glycolysis, generate energy to sustain cell survival, and ultimately accelerate tumor growth and promote its migration.
Colon cancer is third among all cancers in worldwide mortality. Currently, methods of treatment for colon cancer mainly include surgical excision, chemotherapy, radiation therapy, and a combination of immunomodulatory treatments. There are many methods for treating colon cancer, but these methods may bring toxic side effects to patients, and may generate drug resistance and recurrence, so new therapeutic strategies and drugs with low toxic side effects are required for treating colon cancer at present.
At present, there are considerable researches on benserazide hydrochloride in the aspect of treating parkinsonism, and meanwhile, researches report the effects of benserazide hydrochloride in treating atherosclerosis, melanin and antifungal. However, the use of benserazide hydrochloride as the sole active ingredient for the treatment of anti-colon cancer has not been seen.
Disclosure of Invention
The invention aims to: the invention aims to provide an application of benserazide hydrochloride in preparing a medicine for resisting colon cancer. The invention has found that the concentration of the single use is 10 -9 ～10 -7 The benserazide hydrochloride of M has the inhibition effect on proliferation and migration of colon cancer cells. Benserazide hydrochloride can inhibit proliferation and migration of colon cancer cells, inhibit glucose uptake by cells, reduce lactic acid accumulation, and down regulate mRNA levels of glycolysis key enzymes GLUT-1, PKM2, HK2 and LDHA and protein expression levels of GLUT-1 and LDHA, thereby realizing the effect of resisting colon cancer. Thus, benseryl hydrochlorideHydrazine can be used for preparing anti-colon cancer drugs.
The potential effect and mechanism of benserazide hydrochloride for inhibiting colon cancer development are researched, and the result shows that the anti-colon cancer effect of benserazide hydrochloride is achieved by regulating glycolysis of cancer cells. Specifically, a cell hypoxia model is constructed by using a hypoxia simulator cobalt chloride, so that the living state of tumor cells in solid tumors is simulated, and meanwhile, the influence of a drug on proliferation and migration of the tumor cells is amplified. Based on the results, the anti-colon cancer effect and mechanism of the benserazide hydrochloride are researched through Transwell migration, lactic acid accumulation and other experiments, and the results show that the benserazide hydrochloride can inhibit proliferation and migration of colon cancer cells, uptake of glucose and lactic acid accumulation, and the benserazide hydrochloride can down regulate mRNA level and protein level of glycolytic key enzymes. The research can provide a new choice for preparing the medicine for treating colon cancer.
The technical scheme is as follows: the aim of the invention is achieved by the following technical scheme:
the invention provides an application of benserazide hydrochloride in preparing anti-colon cancer drugs.
The benserazide hydrochloride is used as the only active ingredient for preparing the medicine for treating the colon cancer.
The adding amount of the benserazide hydrochloride is 10 -9 ～10 -7 M。
CN114259566a discloses that the cell proliferation rate of benserazide hydrochloride alone applied to colon cancer HCT-116 cells is not significantly different from that of a blank control group, indicating that benserazide hydrochloride alone applied to colon cancer HCT-116 cells is not inhibited by benserazide hydrochloride at 85 μm.
However, the present invention has been studied to find that the concentration of 10 alone -9 ～10 -7 The benserazide hydrochloride of M has the inhibition effect on proliferation and migration of colon cancer cells. The invention researches the mechanism of inhibiting proliferation and migration of colon cancer cells by using benserazide hydrochloride, adopts an anoxic simulator cobalt chloride to simulate the anoxic environment of solid tumors, and adopts benserazide hydrochloride with different concentrations to act on colon cancer cells HCT-116. As a result, it was found that benserazide hydrochloride can inhibit proliferation and migration of colon cancer cells, inhibit uptake of glucose and accumulation of lactic acid, andthe mRNA levels of the glycolytic key enzymes GLUT-1, PKM2, HK2 and LDHA are regulated down, and the protein expression levels of the GLUT-1 and the LDHA are regulated down, so that the benserazide hydrochloride plays a role in resisting proliferation and migration of colon cancer cells by inhibiting glucose metabolism of the colon cancer cells.
The medicine comprises benserazide hydrochloride and pharmaceutically acceptable carriers or auxiliary materials.
The auxiliary materials comprise one or more of antioxidant, emulsifier, wetting agent, diluent, preservative, disintegrating agent or adhesive.
The antioxidant is at least one selected from ascorbic acid, sulfite, bisulfite, gallic acid and lipid thereof.
The emulsifier is at least one selected from tween, span, glycerol fatty acid esters, pectin, agar, sodium alginate or silicon dioxide.
The wetting agent is selected from at least one of water or ethanol.
The diluent is at least one selected from starch, saccharide, cellulose or inorganic salt.
The preservative is at least one selected from benzoic acid and salts thereof, sorbic acid and salts thereof or parabens.
The disintegrating agent is at least one selected from starch, sodium carboxymethyl starch, crosslinked povidone, low-substituted hydroxypropyl cellulose or crosslinked polyvinylpyrrolidone.
The binder is at least one selected from starch slurry, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose or ethyl cellulose.
The dosage forms of the medicine are tablets, granules, capsules, pills, oral liquid, injection or infusion solutions.
The medicaments of the present invention may be administered in a variety of known ways, for example orally, by injection, by inhalation spray. The medicine of the invention can be used alone or in combination with other medicines. The oral composition may be any orally acceptable dosage form including, but not limited to, tablets, granules, capsules, pills, suspensions, and solutions.
Sterile injectable compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, and the like.
The medicine of the invention can be prepared into common preparations, and also can be prepared into sustained release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.
The actual dosage level of the active ingredient in the medicament of the present invention may be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for the particular patient, composition and mode of administration, which is non-toxic to the patient. The selected dosage level depends on a variety of factors including the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with benserazide hydrochloride, the age, sex, weight, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The benserazide hydrochloride inhibits the growth and/or metastasis of colon cancer cells.
The benserazide hydrochloride has obvious inhibition effect on glucose metabolism of colon cancer HCT-116 cancer cells.
The benserazide hydrochloride down regulates mRNA levels and protein levels of key enzymes of glycolysis.
The beneficial effects are that:
(1) The invention researches the mechanism of inhibiting proliferation and migration of colon cancer cells by using benserazide hydrochloride, adopts an anoxic simulator cobalt chloride to simulate the anoxic environment of solid tumors, adopts benserazide hydrochloride with different concentrations to act on colon cancer cells HCT-116, and finds that the benserazide hydrochloride can inhibit proliferation and migration of colon cancer cells, inhibit ingestion of glucose and accumulation of lactic acid, and down regulate mRNA levels of glycolytic key enzymes GLUT-1, PKM2, HK2 and LDHA, and simultaneously down regulate protein expression levels of GLUT-1 and LDHA, so that the research shows that the benserazide hydrochloride plays a role of resisting proliferation and migration of colon cancer cells by inhibiting glucose metabolism of colon cancer cells, and can provide a new choice for preparing medicaments for treating colon cancer.
(2) The benserazide hydrochloride can be used as an active ingredient and applied to the treatment of colon cancer. Benserazide hydrochloride can inhibit proliferation and migration of colon cancer, inhibit glucose uptake by cells, reduce lactic acid accumulation, and down regulate mRNA levels of glycolysis key enzymes GLUT-1, PKM2, HK2 and LDHA and protein expression levels of GLUT-1 and LDHA, so that the benserazide hydrochloride has an effect of resisting colon cancer. Therefore, the benserazide hydrochloride can be used for preparing medicines for treating colon cancer.
Drawings
FIG. 1 is a graph of the results of statistical analysis of the data from MTT experiments to determine the effect of benserazide hydrochloride on HCT-116 cell proliferation;
FIG. 2 is a graph of the effect of Transwell migration experiments on HCT-116 cell migration determined by benserazide hydrochloride;
FIG. 3 is a graph of data statistical analysis results of a Transwell migration experiment;
FIG. 4 is a graph showing the results of a glucose assay to determine the effect of benserazide hydrochloride on glucose uptake by HCT-116 cells;
FIG. 5 is a graph showing the results of lactic acid assay to determine the effect of benserazide hydrochloride on accumulation of lactic acid in HCT-116 cells;
FIG. 6 is a graph of a statistical analysis of the data from qPCR experiments to determine the effect of benserazide hydrochloride on GLUT-1mRNA levels in HCT-116 cells;
FIG. 7 is a graph of a statistical analysis of the data from qPCR experiments to determine the effect of benserazide hydrochloride on PKM2mRNA levels in HCT-116 cells;
FIG. 8 is a graph of a statistical analysis of the data from qPCR experiments to determine the effect of benserazide hydrochloride on the levels of HK 2mRNA in HCT-116 cells;
FIG. 9 is a graph of a statistical analysis of the data from qPCR experiments to determine the effect of benserazide hydrochloride on LDHA mRNA levels of HCT-116 cells;
FIG. 10 is a graph showing the results of Western blotting experiments to determine the protein bands and gray scale analysis of the effect of benserazide hydrochloride on the GLUT-1 protein expression levels in HCT-116 cells;
FIG. 11 is a graph showing the results of Western blotting experiments to determine the protein bands and gray scale analysis of the effect of benserazide hydrochloride on the expression level of LDHA protein in HCT-116 cells.
Detailed Description
The technical scheme of the present invention is described in detail below through specific examples, but the scope of the present invention is not limited to the examples.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, are all commercially available products.
GLUT-1 as described in the examples below refers to glucose transporter-1;
the PKM2 referred to in the examples below is a type M2 pyruvate kinase;
HK2 described in the examples below refers to hexokinase 2;
the LDHA described in the examples below refers to lactate dehydrogenase;
example 1CCK-8 experiment to examine the effect of benserazide hydrochloride on HCT-116 cell proliferation
Step one: culturing colon cancer cells (HCT-cells, ATCC) to 90% confluence, digesting with 0.25% (w/v) pancreatin (Biosharp Co.) containing 0.02% (w/v) EDTA to prepare a cell suspension, and mixing thoroughly and then mixing thoroughly with 5X 10 3 The density of each/hole is inoculated in a 96-well plate, and the cells are cultured at 37 ℃ until the cells adhere to the wall and the density is proper.
The cells were divided into a Control group, a Model group (Model, containing 50. Mu.M CoCl) 2 ) Benserazide hydrochloride groups (10) with different concentrations -9 、10 -8 、10 -7 M, each group contains the same concentration of CoCl as the model group 2 ) Each group was plated with 6 wells, and each group was replaced with a drug-containing medium prepared from DMEM medium (Gibco, high-sugar DMEM medium) containing no FBS, and cultured at 37℃for 24 hours.
50μM CoCl 2 The benserazide hydrochloride with different concentrations is prepared by adopting DMEM culture medium without FBS.
Step two: 10. Mu.L of CCK-8 solution (GLPBIO Co.) was added to each well and incubated at 37℃for 1 hour. After shaking and mixing, the absorbance of each well was measured at 450nm (Multiskan FC microplate reader, thermo Corp.). The data obtained were statistically analyzed using Graph Pad Prism5.0 software and the quantitative analysis results are shown in FIG. 1.
As can be seen from the results of fig. 1: HCT-116 cells in 50. Mu.M CoCl 2 Proliferation is promoted after stimulation, at 10 -9 ～10 -7 Proliferation of HCT-116 cells was inhibited and dose dependent compared to model group after action of benserazide hydrochloride at different concentrations in M range, at a dose of 10 -7 The inhibition effect of the M benserazide hydrochloride is optimal.
Example 2Transwell cell migration assay to examine the effect of benserazide hydrochloride on HCT-116 cell migration
The experiment was performed using a Transwell 24 well plate of 8 μm polycarbonate membrane.
Experimental grouping: blank (Control), model (Model, 50. Mu.M CoCl) 2 ) Benserazide hydrochloride groups (10) with different concentrations -9 、10 -8 、10 -7 M, each group contains the same concentration of CoCl as the model group 2 ) Each group was provided with 3 duplicate wells, and each group of medium was prepared with DMEM medium (Gibco, high-sugar DMEM medium) containing 0.2% (w/v) BSA (Source leaf organism).
50μM CoCl 2 Different concentrations of benserazide hydrochloride were prepared using FBS-free DMEM medium containing 0.2% (w/v) BSA (Source leaf Biotechnology).
Step one: HCT-116 cells (ATCC) were cultured, and after growing to 80% confluence and good condition, the medium was replaced with DMEM medium (Gibco, high sugar DMEM medium) containing no FBS, starved-cultured at 37 ℃ for 12 hours, and after conventional digestion and centrifugation, washed 2 times with 2mL PBS (bi cloud biosystems), and the density was adjusted to 1×10 with each group of medicated media 5 And each mL.
Step two: 500. Mu.L of each group of medium (prepared with DMEM medium containing 10% FBS (Gibco Co.), was added to the lower chamber of the Transwell chamber, and 200. Mu.L of the cell suspension of step one was added to the upper chamber, and the culture was continued for 24 hours.
Step three: the chamber was removed, medium was aspirated, cells that did not pass through the membrane were gently wiped off with a cotton swab, the chamber was washed 2 times with PBS, 600. Mu.L of 4% paraformaldehyde was added to a clean well of a 24-well plate, and the well was fixed in the chamber for 30min.
Step four: the fixative was discarded, stained with 0.1% crystal violet for 15min, washed 3 times with pbs, unbound crystal violet was wiped off with a cotton swab, observed under a microscope (AE 2000 inverted microscope, maculodi) and photographed as a result of fig. 2. After completion of photographing, 500. Mu.L of 10% acetic acid was added to the mixture to extract crystal violet by shaking, 200. Mu.L of the mixture was measured for absorbance at 600nm (Multiskan FC microplate reader, thermo Co.) and the analysis results were shown in FIG. 3.
The results of fig. 2 and 3 show that: at the addition of 50. Mu.M CoCl 2 After stimulation, migration of HCT-116 cells (ATCC) into the lower chamber was promoted at 10 -9 ～10 -7 In the M range, after the action of the benserazide hydrochloride with different concentrations, compared with a model group, the migration of HCT-116 cells is inhibited and has dose dependence, wherein the dosage is 10 -7 The inhibition effect of the M benserazide hydrochloride is optimal.
Example 3 glucose test kit to detect the effect of benserazide hydrochloride on HCT-116 glucose uptake
Step one: after HCT-116 cells which have good growth state and have a fusion degree of 90% are digested conventionally, the density is adjusted to 5X 10 5 A6-well plate was plated at a volume of each mL, and when the cell density was appropriate and the condition was good, the cells were divided into a blank group (Control) and a Model group (Model, 50. Mu.M CoCl) 2 ) Benserazide hydrochloride groups (10) with different concentrations -9 、10 -8 、10 -7 M, each group contains the same concentration of CoCl as the model group 2 ) And each group of the culture media was replaced with DMEM medium (Gibco, high-sugar DMEM medium) containing no FBS, and cultured at 37℃for 24 hours.
Step two: the cell culture supernatant was transferred to an EP tube, centrifuged at 1000r/min for 10min, and the culture supernatant was assayed according to the instructions of the glucose test kit (Nanjing institute of biological engineering, model A154-1-1). The measurement conditions are shown in Table 1, and 3 wells are provided for each group.
TABLE 1 measurement conditions
Glucose content (mM) = (assay-a blank)/(a standard-a blank) ×5.55mM
Glucose uptake (mM) =cell-free wells-sample wells
After the completion of the sample addition, the well plate was gently shaken, incubated in an incubator at 37℃for 10 minutes, and the absorbance value A of each well was measured at a wavelength of 505 nm. Glucose uptake was calculated from each set of absorbance values using the above calculation formula. The data were statistically analyzed using Graph Pad prism5.0 software and the final analysis results are shown in FIG. 4.
As can be seen from the results of fig. 4: at the addition of 50. Mu.M CoCl 2 After stimulation, glucose uptake by HCT-116 cells (ATCC) increased significantly at 10 -9 ～10 -7 After the action of benserazide hydrochloride with different concentrations in the M range, glucose uptake was inhibited compared with the model group, wherein the glucose uptake was inhibited by 10 -7 The inhibition effect of the M benserazide hydrochloride is optimal.
Example 4 lactic acid test kit to examine the effect of benserazide hydrochloride on accumulation of HCT-116 lactic acid
Step one: after HCT-116 cells which have good growth state and have a fusion degree of 90% are digested conventionally, the density is adjusted to 5X 10 5 A6-well plate was plated at a volume of each mL, and when the cell density was appropriate and the condition was good, the cells were divided into a blank group (Control) and a Model group (Model, 50. Mu.M CoCl) 2 ) Benserazide hydrochloride groups (10) with different concentrations -9 、10 -8 、10 -7 M, each group contains the same concentration of CoCl as the model group 2 ) And each group of the culture media was replaced with DMEM medium (Gibco, high-sugar DMEM medium) containing no FBS, and cultured at 37℃for 24 hours.
Step two: the cell culture supernatant was transferred to an EP tube, centrifuged at 1000r/min for 10min, and the culture supernatant was measured according to the specification of a lactic acid test box (Nanjing institute of biological engineering, model A019-2-1). Assay conditions referring to table 2, 3 duplicate wells were set per group.
TABLE 2 measurement conditions
Lactic acid content (mM) = (assay-a blank)/(a standard-a blank) ×3mM
After the sample addition and mixing are completed, 250 mu L of the reaction solution is taken into a 96-well plate, and the absorbance value A of each well is measured at the wavelength of 530nm by using an enzyme-labeled instrument. The lactic acid accumulation amount was calculated from each group of absorbance values by using the above calculation formula. The data were statistically analyzed using Graph Pad Prism5.0 software and the final analysis results are shown in FIG. 5.
As can be seen from the results of fig. 5: after stimulation with 50. Mu.M CoCl2, the accumulation of lactic acid in HCT-116 cells (ATCC) increased significantly at 10 -9 ～10 -7 After the benserazide hydrochloride with different concentrations within the M range acts, compared with a model group, the benserazide hydrochloride can inhibit lactic acid accumulation to different degrees, wherein the concentration is 10 -7 The inhibition effect of the M benserazide hydrochloride is optimal.
Example 5qPCR detection of the Effect of benserazide hydrochloride on the expression of the glycolytic key enzymes GLUT-1, PKM2, HK2 and LDHA mRNA
Step one: after routine digestion of HCT-116 cells (ATCC), the cell density was adjusted to 5X 10 5 Each mL was plated with 6-well plates, and when cells grew to 80% confluence on the wall, the cells were divided into a Control group (Control), a Model group (Model, 50. Mu.M CoCl) 2 ) Benserazide hydrochloride groups (10) with different concentrations -9 、10 -8 、10 -7 M, each group contains the same concentration of CoCl as the model group 2 ) And each group of the culture media was replaced with DMEM medium (Gibco, high-sugar DMEM medium) containing no FBS, and cultured at 37℃for 24 hours.
Step two: the medium was discarded, transferred to an EP tube after regular digestion, and the discarded supernatant was centrifuged. Total RNA extraction was performed using TRIzol (Solarbio Co.), and after drying the RNA precipitate, ddH was added 2 O, vortex at room temperature to dissolve completely. RNA concentration was measured by Nanodrop 2000 (Thermo Co.) and stored at-80℃for further use.
Step three: the resulting cDNA was reverse transcribed using a reverse transcription kit (Abcam Co.) and stored at-20℃for further use. NCBI searches for the mRNA sequences of the relevant human sources, and the sequence is designed and synthesized by the on-line primer. qPCR analysis was performed using the ChamQ Universal SYBR qPCR Master Mix kit (Norflu).
The components were added to the tube on ice with reference to table 3, thoroughly mixed and centrifuged. The reaction tubes were placed on a qPCR instrument in sequence, and the reaction procedure was set as follows:
pre-denaturation: 95 ℃ for 30s;
and (3) cyclic reaction: 95℃10s,60℃60s,95℃15s.
Ct value is obtained after the reaction is finished, and 2 is adopted -ΔΔt mRNA levels were calculated by the method. The results obtained by processing the data with Graph Pad prism5.0 software are shown in fig. 6-9.
TABLE 3 qPCR reaction System
Primers were purchased from Shanghai organisms and the sequences are shown in Table 4:
TABLE 4 primer sequences
| Forward primer | Reverse primer | |
| PKM2 | CCTCCTTCAAGTGCTGCAGT | TCATGGCAAAGTTCACCCGG |
| HK2 | CGACAGCATCATTGTTAAGGAG | GCAGGAAAGACACATCACATTT |
| LDHA | ATGGCAACTCTAAAGGATCAGC | CCAACCCCAACAACTGTAATCT |
| GLUT-1 | ATTGGCTCCGGTATCGTCAAC | GCTCAGATAGGACATCCAGGGTA |
| β-actin | CATGTACGTTGCTATCCAGGC | CTCCTTAATGTCACGCACGAT |
As can be seen from the results of fig. 6 to 9: at the addition of 50. Mu.M CoCl 2 After stimulation, mRNA expression of GLUT-1 (FIG. 6), PKM2 (FIG. 7), HK2 (FIG. 8), LDHA (FIG. 9) was elevated in HCT-116 cells (ATCC), promoting gene transcription at 10 -9 ～10 -7 After the benserazide hydrochloride with different concentrations within the M range acts, compared with a model group, the gene transcription of GLUT-1, PKM2, HK2 and LDHA can be inhibited to different degrees, and in general, the concentration is 10 -7 The inhibition effect of the M benserazide hydrochloride is optimal.
EXAMPLE 6Western blotting detection of the Effect of benserazide hydrochloride on the expression of GLUT-1 and LDHA proteins in HCT-116 cells
Step one: after routine digestion of HCT-116 cells (ATCC), the cell density was adjusted to 5X 10 5 Each mL was plated with 6-well plates, and when cells grew to 80% confluence on the wall, the cells were divided into a Control group (Control), a Model group (Model, 50. Mu.M CoCl) 2 ) Benserazide hydrochloride groups (10) with different concentrations -9 、10 -8 、10 -7 M, each group contains the same concentration of CoCl as the model group 2 ) And each group of the culture media was replaced with DMEM medium (high-sugar DMEM medium, gibco Co.) containing no FBS, and cultured at 37℃for 24h。
Step two: cells were transferred to an EP tube after routine digestion, and the supernatant was discarded by centrifugation. The cell pellet was resuspended in pre-chilled PBS and transferred to a 1.5mL EP tube, centrifuged at 3000rpm at 4℃for 10min (Biofuge Stratos high speed cryocentrifuge, thermo Inc., USA), and repeated 1 time with pre-chilled PBS. After discarding the supernatant, carefully sucking the residual liquid, adding the pre-prepared RIPA lysis working solution (Biyun Tian), blowing and suspending, and then fully lysing on ice for at least 30min, and vortex oscillating once every 5min (XW-80A miniature vortex mixer, shanghai West analysis Co., ltd.). After the completion of the lysis, the mixture was centrifuged at 12000rpm and 4℃for 10min, and the white precipitate at the bottom of the EP tube was slowly removed to obtain the total cell protein.
Step three: protein content in the samples was determined by BCA method. The loading volume was calculated as 40 μg protein loading according to the standard curve. Adding appropriate amount of 5 Xprotein loading buffer solution (Northenzan) into each sample protein, boiling in boiling water bath for 5min to denature protein, cooling to room temperature, and preserving at-20deg.C for use. Preparing gel according to the rapid gel preparation kit (Nuo Wei Zan), and standing at room temperature until gel is solid. Taking out the sample, swirling, blowing and mixing uniformly, sequentially loading samples according to the loading volume, performing constant-pressure electrophoresis at room temperature of 160V until bromophenol blue runs to the bottom of the gel, and stopping electrophoresis. After electrophoresis, the gel containing the target protein was taken as a reference protein standard (nuuzan) and placed in a1 Xtransfer buffer (glycine 2.9g,SDS 0.38g,Tris 5.8g, distilled water was dissolved and fixed to 800mL, supplemented with 200mL methanol). The size of the gel cut on the glass plate was measured and a 0.45 μm PVDF film (from bi yun day) of the same size was cut and put into methanol for activation for 60s.
The sponge, filter paper, adhesive tape and PVDF membrane were placed in order. Placing into an electrotransfer tank, filling with 1 Xtransfer buffer solution, and transferring the 200mA constant flow film in an ice-water bath for 90min. 5% skim milk powder is prepared in advance as a sealing liquid. And after the film transfer is finished, putting the film into a sealing liquid, and slowly shaking the film in the sealing liquid at room temperature for sealing for 2-3h. After the end of the blocking, the cells were placed in a primary Anti-dilution (Anti-GLUT-1-Anti-body/Anti-LDHa-Anti-body) corresponding to the target protein, and incubated overnight at 4 ℃. The PVDF membrane was removed the next day, and the membrane was washed with 1% TBST solution by shaking 3 times for 10min each. And (3) placing the primary antibody into a secondary antibody diluent (the coat anti-Rabbit IgG HRP label and the positive organism) corresponding to the primary antibody, and incubating for 60 minutes at room temperature. The PVDF membrane was removed and washed 3 times with 0.1% TBST solution for 10min each. And mixing the solution A and the solution B in the ECL luminous solution kit (Tiansheng) according to the volume ratio of 1:1 to prepare the working solution. And (3) placing the PVDF films in an exposure area after absorbing water, uniformly dripping 200 mu L of luminous working solution on the surface of each PVDF film, performing exposure photographing by a Tanon 5200 full-automatic luminous analysis system (tenability), and obtaining photographing results and gray level analysis results as shown in fig. 10 and 11.
As can be seen from fig. 10 and 11: at the addition of 50. Mu.M CoCl 2 After stimulation, GLUT-1, LDHA protein expression was elevated in HCT-116 cells (ATCC) at 10 -9 ～10 -7 In the M range, after the benserazide hydrochloride with different concentrations acts, compared with a model group, the GLUT-1 and LDHA protein expression can be inhibited to different degrees, wherein the concentration is 10 -7 The M benserazide hydrochloride has the best effect of inhibiting GLUT-1 protein expression and takes 10 percent -9 The M benserazide hydrochloride has the best effect of inhibiting the expression of LDHA protein.
As described above, although the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limiting the invention itself. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. Application of benserazide hydrochloride in preparing anti-colon cancer medicine.
2. The use according to claim 1, characterized in that the benserazide hydrochloride is used as the sole active ingredient for the preparation of a medicament for the treatment of colon cancer.
3. The use according to claim 1 or 2, characterized in that the added amount of benserazide hydrochloride is 10 -9 ～10 - 7 M。
4. The use according to claim 1 or 2, wherein the medicament comprises benserazide hydrochloride and a pharmaceutically acceptable carrier or adjuvant.
5. The use according to claim 1 or 2, wherein the medicament is in the form of a tablet, granule, capsule, pill, oral liquid, injection or infusion.
6. The use according to claim 1 or 2, wherein the benserazide hydrochloride inhibits the growth and/or metastasis of colon cancer cells.
7. The use according to claim 1 or 2, wherein benserazide hydrochloride has a significant inhibitory effect on glucose metabolism in colon cancer HCT-116 cancer cells.
8. The use according to claim 1 or 2, characterized in that the benserazide hydrochloride down regulates mRNA levels and protein levels of the key enzymes of glycolysis.
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