CN114107426B

CN114107426B - Method for screening glutamine transport protein inhibitor, inhibitor screened by method and application of inhibitor

Info

Publication number: CN114107426B
Application number: CN202111328929.3A
Authority: CN
Inventors: 张洪建; 周志芸; 段泽琳
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2023-01-06
Anticipated expiration: 2041-11-10
Also published as: CN114107426A

Abstract

The invention relates to a method for screening a glutamine transport protein inhibitor, and screens a new glutamine transport protein inhibitor mianserin based on a human glutamine transport protein inhibitor screening system, thereby expanding a new application field for mianserin. The invention discloses a new application of mianserin, and the compound is beneficial to the development of medicaments for preventing and treating various tumors clinically.

Description

Method for screening glutamine transport protein inhibitor, inhibitor screened by method and application of inhibitor

Technical Field

The invention relates to the technical field of biological medicines, in particular to a method for screening a glutamine transporter inhibitor, an inhibitor screened by the method and application of the inhibitor.

Background

Tumor cells, in order to adapt to biosynthetic and redox requirements during proliferation and metastasis, readjust cell metabolism to meet the requirements of energy (glucose), amino acids, fatty acids, and other nutrients. Studies have shown that interfering with or blocking certain metabolic pathways (e.g., glutamine metabolism) can effectively reverse the resistance of tumor cells to anti-tumor therapies. Glutamine is the most abundant nonessential neutral amino acid in plasma, can be obtained from dietary breakdown or produced in muscle, liver, adipose tissue, lung and brain, and is a key intermediate in many metabolic processes, including energy formation, redox homeostasis, macromolecular synthesis and signaling, among others. During rapid growth or disease, the glutamine supply is exceeded by the cell and glutamine is changed from a non-essential amino acid to an essential amino acid, so that tumor cell glutamine addiction is a metabolic compensation that indirectly promotes oxidative glycolysis to provide nutrients and energy to tumor cells under hypoxic adverse conditions.

The key step in glutamine metabolism is SLC1A5 (ASCT 2) mediated cellular uptake: SLC1A5 is Na ⁺ The dependent transmembrane transporter, belonging to the SLC transporter superfamily, is one of the metabolic weaknesses of tumor cells in the Tumor Microenvironment (TME). It has been found that the inhibition or deletion of SLC1A5 results in a decrease in the glutamine content in the tumor cells, which results in a lack of energy source substances, and ultimately in an inhibition of tumor growth. Therefore, in the tumor cells, the absorption of glutamine is blocked or reduced, the effect of inhibiting the growth and the proliferation of the tumor cells can be achieved, and the sensitivity of the tumor cells to the antitumor drugs is improved. Based on this hypothesis, the possibility of cancer treatment by modulating the SLC1A5 transport function was explored, using the glutamine transporter SLC1A5 as a potential novel target.

SLC1A5 is a specific glutamine transporter, and a plurality of SLC1A5 uptake glutamine transport inhibitors have been developed at present, while the development of novel SLC1A5 inhibitors based on small molecular compounds is significant, and more medicaments and methods can be provided for treating tumors. The design and screening of the transporter inhibitor can be completed by some tumor cells expressing SLC1A 5. However, the screening method has the defects of poor specificity, low flux and high cost, so that the establishment of a reasonable inhibitor screening system has great significance.

Disclosure of Invention

In order to solve the technical problems, the invention provides a human SLC1A5 stable transfection cell strain, and a screening method is established by applying the cell strain to screen out a novel glutamine transporter inhibitor.

In the invention, the glutamine transporter inhibitor is screened by a human SLC1A5 stable transfection cell strain, and the construction method of the cell strain comprises the following steps: a cell line obtained by inserting a humanized SLC1A5 gene into HEK293 cells,

(1) Assembling the hSLC1A5 gene with a pcDNA5-TO (Hygromycin/Ampicillin) expression vector TO construct a pcDNA5-TO-hSLC1A5 plasmid; wherein, the hSLC1A5 gene sequence (NM _ 005628.3) is obtained from the gene bank, and the nucleotide sequence is shown as SEQ ID NO. 1;

(2) And (2) transfecting the pcDNA5-TO-hSLC1A5 plasmid constructed in the step (1) TO an HEK293 cell, screening out positive clones by using hygromycin B, and confirming that the cell strain stably expresses the human SLC1A5 gene and the protein thereof at the same time, so that the human SLC1A5 stably transfected cell strain is constructed.

According to the invention, the HEK293 cell is used as a transfected cell, the HEK293 cell is a human embryonic kidney cell, the inventor discovers that most metabolic enzymes, uptake transporters and efflux transporters are absent in the HEK293 cell through biochemical characteristic examination, the HEK293 cell has the advantages of short culture period, low background, easiness in culture and the like, the cell line is selected for construction of a screening system, and the HEK293 cell is verified to be the optimal selection through comparison with other various transfected cell strains.

Further, in step (1), the cleavage sites for constructing the recombinant plasmid are NotI and XbaI.

Furthermore, compared with a physical transfection method, the method is time-consuming and labor-consuming and low in efficiency, an artificial liposome method in a chemical transfection method has high transfection efficiency, a cell line which is difficult to transfect by other methods can be transfected, and DNA plasmids, small interfering RNAs (microRNAs, shRNAs and the like) and even proteins can be transfected by the liposome method. In addition, the liposome transfection method is simultaneously suitable for transient expression and stable expression, wherein the former has the characteristics of rapidness and flexibility, and the latter has the advantages of stability, strong reproducibility and the like.

Further, in step (2), cells in the logarithmic growth phase were taken for cell transfection at a plasmid concentration of 0.8 to 1.2 ng/. Mu.L.

Further, in step (2), plasmid transfection was performed using lipo2000 transfection reagent in an amount of 4. Mu.L per 100. Mu.L of the medium.

Further, in step (2), the medium used for plasmid transfection is Opti-MEM reduced serum medium.

Further, in step (2), the concentration of hygromycin B is 180-220. Mu.g/mL. At this concentration, cells without the corresponding resistance die by 60% in about 2 days, while cells with hygromycin B resistance do not die in principle; too high a concentration will kill resistant cells and the morphology of cells cultured for a long period of time will be subject to change.

Further, in the step (2), when the hygromycin B is screened and the cell fusion degree exceeds 25%, immediately carrying out passage, replacing a culture solution containing hygromycin B every 3-4 days for culture, repeating the steps until the passage reaches 3-4 generations, selecting positive monoclones, and continuously culturing until the monoclones grow into cell strains.

Further, in step (2), verification is performed with respect to mRNA and protein expression to select a positive clone cell line stably expressing the hSLC1A5 gene.

The screening of the glutamine transfer protein inhibitor by adopting the human SLC1A5 stable transfection cell strain comprises the following specific steps:

(1) Culturing a human SLC1A5 stable transfection cell strain (HEK 293-SLC1A5 cell) and an HEK293 wild type cell (HEK 293-Mock cell) to a logarithmic growth period, paving the cells, changing culture solution into culture solution without glutamine to remove the influence of the glutamine in a culture environment, adding a compound to be detected for pre-incubation, removing the culture solution, adding deuterated glutamine prepared by the culture solution without the glutamine and the compound to be detected for incubation, removing the incubation solution after the incubation is ended, and cleaning a pore plate to remove the influence of the deuterated glutamine in the culture environment;

(2) Detecting the content and protein concentration of the deuterated glutamine in each hole of the cell, normalizing the content of the deuterated glutamine in each hole of the cell by the protein concentration, and screening out a glutamine transporter inhibitor in the compound to be detected according to the uptake of the deuterated glutamine by the cell.

In the screening method, because the cells contain a certain amount of inherent glutamine to cause higher matrix influence, the deuterated glutamine (L-glutamine-2,3,3,4,4-D5) is used as the SLC1A5 substrate to avoid the interference of endogenous glutamine.

Further, in step (1), the human SLC1A5 stably transfected cell line and the HEK293 wild type cell line were passaged every 3 days in a high-glucose DMEM medium containing 10% fetal bovine serum and placed in an incubator maintained at 37 ℃ with 5% carbon dioxide, 95% humidity.

Further, in step (1), the density of the cells when plated is 2X 10^ 5/well, and the cell culture solution when plated is high-sugar DMEM containing 10% fetal calf serum.

Further, in step (1), HEK293-Mock cells and HEK293-SLC1A5 cells were plated in polylysine coated well plates. The specific operation steps are that Polylysine (PDL) solution is added into the pore plate to soak the bottom surface, and the bottom surface is sucked off and washed after being placed for 3-10 minutes.

Further, the next day of cell plating was subject to culture medium change.

Further, in step (1), to remove the interference of glutamine in the culture environment, the high-sugar culture solution needs to be aspirated and washed once with Phosphate Buffered Saline (PBS) and aspirated to dry. Then, glutamine-free DMEM was added, and preincubation was performed with the test compound prepared from glutamine-free DMEM.

Further, in step (1), the pre-incubation time of glutamine-free high-glucose DMEM is 12-20h, and the pre-incubation time of the test compound-containing DMEM is 30-60min.

Further, in the step (1), during the pre-incubation, the final concentration of the compound to be tested is 100 μ M; the final concentration of the substrate deuterated glutamine was 50-150 μ M upon incubation.

Further, in step (1), a 1mM deuterated glutamine solution prepared with glutamine-free high-glucose DMEM was added to each well on the basis of the pre-incubation so that the final concentration of deuterated glutamine was 50. Mu.M, as the start of the incubation.

Further, in step (1), after adding the substrate deuterated glutamine and the test compound, incubation is carried out at 35-40 ℃ for 2-6 minutes, preferably 3 minutes.

Further, in step (1), at the termination of incubation, the incubation solution was decanted, washed with pre-ice HBSS solution and blotted dry.

Further, in the step (2), the method for detecting the content and the protein concentration of the deuterated glutamine in each hole of the cell comprises the following steps:

taking a cell disruption solution from each hole, adding acetonitrile with 0.1 mu g/mL of cytosine arabinoside (Ara-C) containing an internal standard, carrying out vortex for 2-5min, then precipitating protein, centrifuging at the rotating speed of 10000-15000rpm at 3-5 ℃, transferring supernatant to a sample bottle, and absorbing a small amount of the supernatant to carry out LC-MS/MS analysis so as to detect the content of deuterated glutamine in cells in each hole; in addition, a cell disruption solution was taken from each well, and the BCA kit by TaKara was used to detect the protein concentration in the cells of each well.

Further, when LC-MS/MS analysis is performed, the chromatographic column is Venusil HLPC185 μm

(4X 150 mm); mobile phase a phase: water with 0.1% formic acid in 10mM ammonium acetate, phase B: acetonitrile, sample injection running time of 5min, isocratic elution mode, mobile phase A: the proportion of phase B is 9:1 (v/v), the flow rate is 0.5mL/min, the temperature of the column incubator is 50 ℃, and the sample injection amount is 10 mu L. The detected ion information of the deuterated glutamine is 152.1 → 135.2. The ion source is an ESI source, the positive ion scanning mode is a multi-reaction detection (MRM) scanning mode, and the scanning time is 100ms.

Through the screening method, a new glutamine transporter inhibitor, namely mianserin, is screened out, the new glutamine transporter inhibitor inhibits the ingestion of glutamine transporters by organisms, and the structure of the new glutamine transporter inhibitor is shown as follows:

based on the new application of the mianserin, the mianserin can be used for preparing anti-tumor drugs, and the administration mode can be oral administration or intravenous injection. Through experiments, the IC of mianserin on colorectal cancer SW480 cells is found ₅₀ The value was 32.45 μ M, the IC50 of fluorouracil (5-FU) alone inhibited SW480 cells was 22.8 μ M, and the IC50 of combined administration of 20 μ M mianserin inhibited SW480 cells was 7.6 μ M, showing that mianserin can increase the sensitivity of SW480 to fluorouracil, enhancing the anti-tumor effect of fluorouracil on SW480 cells. The tumor-bearing experiment in a nude mouse shows that the mianserin can inhibit the growth of SW480 tumor, and the mianserin can enhance the anti-tumor effect of fluorouracil after being combined with fluorouracil group. In addition, mianserin has an anti-tumor effect on various cells such as colorectal cancer SW620 cells, HEK293 cells, PANC-1 cells, hep-G2 cells, HCT-8 cells and the like, so that mianserin has great potential in developing novel medicines for various cancers such as colorectal cancer, pancreatic cancer, liver cancer, ileocecal cancer and the like.

By the scheme, the invention at least has the following advantages:

the invention discloses construction of a human-derived glutamine transporter inhibitor screening system and application of a compound mianserin screened by applying the system in preparation of a human-derived glutamine transporter inhibitor-related anti-tumor drug, in particular application in preparation of a colorectal cancer treatment drug.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a schematic diagram of the pcDNA5-TO (Hygromycin/Ampicillin) plasmid of the present invention;

FIG. 2 shows the result of checking the mRNA expression level of SLC1A5 gene in hSLC1A5 monoclonal cells of the present invention;

FIG. 3 shows the result of verifying the protein expression level of SLC1A5 gene in hSLC1A5 monoclonal cells according to the present invention;

FIG. 4 shows the effect of SiRNA transfection on SLC2A5 gene level and protein level in HEK293-hSLC1A5 monoclonal cell strain (27 #);

FIG. 5 is a typical chromatogram of deuterated glutamine (L-glutamine-2,3,3,4,4-D5, 10 μ M) of the present invention;

FIG. 6 shows the result of taking in deuterated glutamine from HEK293-hSLC1A5 monoclonal cell strain (No. 27) during pre-incubation time in glutamine-free medium according to the present invention;

FIG. 7 shows the results of concentration-dependent assay of HEK293-hSLC1A5 monoclonal cell strain (27 #) with deuterated glutamine as substrate;

FIG. 8 shows the uptake results of the HEK293-hSLC1A5 monoclonal cell strain (27 #) with deuterated glutamine as the substrate;

FIG. 9 shows the result of screening the inhibitor of SLC1A5 uptake/transport deuterated glutamine by using the HEK293-hSLC1A5 monoclonal cell strain (27 #) deuterated glutamine inhibitor screening experimental system successfully constructed in the invention;

FIG. 10 is a graph of the concentration-dependent experimental results of the SLC1A 5-mediated uptake of deuterated glutamine by mianserin of the present invention;

FIG. 11 shows the results of the relative expression levels of SLC1A5 gene in different tumor cells according to the present invention;

FIG. 12 shows the relative expression levels of SLC1A5 protein in various tumor cells of the present invention;

FIG. 13 is a graph showing experimental results of the effect of mianserin of the present invention on SW480 activity;

FIG. 14 is a graph showing the experimental results of the effect of the combination of mianserin of the present invention and fluorouracil (5-FU) on SW480 activity;

FIG. 15 shows the experimental results of the growth curve of the SW480 cell transplantation tumor of colorectal cancer in nude mice;

FIG. 16 shows the results of body weight changes of nude mice with colorectal cancer SW480 cell transplantation tumor.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

EXAMPLE 1 construction of plasmid

Obtaining hSLC1A5 gene sequence (NM-005628.3) from a gene bank, and obtaining hSLC1A5 target gene by gene synthesis by using a synthetic biology method, wherein the nucleotide sequence of the hSLC1A5 target gene is shown as SEQ ID No. 1. The genes were cloned into vector pcDNA5-TO (Hygromycin/Ampicillin) via 5'NotI and 3' XbaI (see FIG. 1), mini-scale recombinant plasmid DNA and a puncturing bacterium containing the recombinant plasmid were prepared, and pcDNA5-TO-hSLC1A5 plasmid was constructed for subsequent experiments.

Example 2 construction and validation of stably transfected cell lines

(1) Source and culture of cells:

the transfected cells were selected as human embryonic kidney cells (HEK 293 cells), and the HEK293 cells were purchased from ATCC (American type culture Collection). Cells were grown in 100mm cell culture dishes every three days at a rate of 1:5 passages once, the medium was DMEM containing 10% FBS, and was cultured in an incubator containing 5% carbon dioxide, 95% humidity, 37 ℃.

(2) And (3) screening antibiotic concentration:

HEK293 cells were seeded in 6-well plates, and 50mg/mL hygromycin B in PBS at 0, 4, 8, 16, and 32. Mu.L, respectively, was added to the plates to give final concentrations of 0, 100, 200, 400, 800, and 1000. Mu.g/mL, and the cell status was observed, and the final selection concentration was selected to be 200. Mu.g/mL, and the maintenance concentration was selected to be 100. Mu.g/mL.

(3) Plasmid transfection:

HEK293 cells in the logarithmic growth phase are taken and paved on a 6-well plate according to 10 ten thousand per well of HEK293 cells, and the transfection experiment is carried out after 24 hours.

Mu.g of pcDNA5-TO-hSLC1A5 plasmid was added TO 100. Mu.l of Opti-MEM reduced serum medium, then 100. Mu.l of Opti-MEM reduced serum medium containing 4. Mu.l of lipofectin 2000 was added, mixed well, left TO stand for 15min and dropped into 6 well plates of the corresponding cell line for transfection.

24hr after cell transfection, DMEM cell culture medium containing a certain concentration of selective antibiotic (hygromycin B at 200. Mu.g/mL) was replaced for selection. Once the cell fusion degree exceeds 25%, the cells are immediately passaged, the culture solution containing the antibiotics with specific concentration is replaced every 3-4 days, and after the cycle is repeated for 2-3 weeks until the passage reaches 3-4 generations, the cells are passaged to a 96-well plate, monoclonals are picked, diluted to 1 cell per well, and the plate is paved. After 5-10 days, screening out the hole with only one cell colony in each hole, and performing amplification culture to obtain the screened monoclonal strain for mRNA expression verification, protein expression verification and seed conservation. And selecting the cell strain with the highest mRNA expression level and the highest protein expression level according to the result, namely constructing the human SLC1A5 stable transfection cell strain, and taking the cell strain as a stable over-expression cell model.

(4) Verification of stably transfected cell lines:

in step (3), pcDNA5-TO-hSLC1A5 is transfected into HEK293 cells, a plurality of sets of parallel experiments are carried out, the numbers of the sets are respectively 1-48, and a plurality of HEK293-hSLC1A5 positive monoclonals are screened out. The positive monoclonals obtained in parallel experiments were subjected to mRNA identification (fluorescent quantitative PCR) and protein expression (Westernblot analysis).

Besides multiple groups of parallel experiments, HEK293 cells are replaced by other cells in the experimental process to construct stable transfected cell strains (such as CHO cells and the like), but the cells die in a large amount in the antibiotic screening process, and only the HEK293 cells successfully construct stable transfected cell strains.

(a) And (3) constructing a cell line for stably expressing hSLC1A5 and verifying the gene level:

total RNA from selected cell clones was extracted using RNAiosolplus and the concentration and purity of the RNA was determined using a Q5000UV-Vis spectrophotometer (Quawell). RNA was reverse transcribed into cDNA according to the PrimerScriptTM (Takara) kit method, GAPDH was selected as an internal reference gene, and hSLC1A5 was quantified using iQTM SYBRGreenSupermix (Bio-Rad) reagent and CFX96Real-Time fluorescent quantitative PCR instrument (Bio-Rad). The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 20s, circulation for 35 times, and extension at 72 ℃ for 10min. SLC1A5 expression at mRNA level was examined. The results are shown in FIG. 2, wherein Mock refers to HEK293 wild type cells, and the numbers in the figure represent the numbers of HEK293-hSLC1A5 positive monoclonals, compared with Mock cells, the expression levels of SLC1A5mRNA of cell strains 1#, 2#, 4#, 6#, 13#, 14# and 27# are higher. The primers used therein are shown in table 1.

TABLE 1 primers for SLC1A5, GAPDH used in the present invention

(b) And (3) constructing a stably expressed hSLC1A5 cell line protein level verification:

the monoclonal cell strain is subjected to amplification culture, protein is extracted, the protein level is verified, the expression of the protein is verified through a Westernblot experiment, and as shown in figure 3, compared with a Mock cell, the SLC1A5 cell strains of 1#, 2#, 4#, 6#, 13#, 14#, 26# and 27# have higher protein expression levels and are basically consistent with an RT-PCR result. Among them, SLC1A5mRNA and protein expression in 27# monoclonal cell strain is highest and cell state is good, so 27# cell strain is selected for subsequent experiment.

(c) Constructing a cell line for stably expressing hSLC1A5 and verifying gene silencing:

the screened positive clone cell HEK293-hSLC1A5 (27 #) and Mock cell are normally cultured. 7X 10^ a day before transfection ⁴ The density of each well was seeded in 24-well plates. During transfection, serum-free DMEM is used for respectively diluting siRNA and Lipo2000 reagents, and then the siRNA/Lipo2000 compound which is fully and evenly mixed is added into a cell plate. After transfection, the cells were placed in a cell incubator for 6 hours, and then cultured in normal high-glucose DMEM without a complex for 48 hours, total RNA of the cells was extracted, and Q-PCR quantification was performed after reverse transcription to examine the change of SLC1A5mRNA level by siRNA interference. Proteins in cells were extracted 72 hours after transfection and protein validation was performed using a Westernblot. As a result, theAs shown in figure 4, the mRNA level and the protein level of SLC1A5 in HEK293-hSLC1A5-27# are obviously reduced by silencing the expression level through siRNA, and the successful construction of the cell strain is proved, so that the 27# cell strain is selected for subsequent functional experiments. The sequences of the siRNAs in this experiment are shown in Table 2.

TABLE 2 SiSLC1A5 sequences

Example 3 Condition optimization of SLC1A5 inhibitor screening test System

(1) Establishment of deuterated glutamine (L-glutamine-2,3,3,4,4-D5) by LC-MS/MS method:

HEK293-hSLC1A5 positive monoclonal transport function using SLC1A5 transporter substrate deuterated glutamine (L-glutamine-2,3,3,4,4-D5) test, evaluation of monoclonal cell transporter function. Firstly, an LC-MS/MS method capable of effectively measuring the content of L-glutamine-2,3,3,4,4-D5 is developed, a chromatogram is shown in FIG. 5, and in FIG. 5, the standard concentration of deuterated glutamine is 10 μ M. The conditions for analyzing the concentration of deuterated glutamine by LC-MS/MS are as follows: the chromatographic column is Venusil HLP C185 mu m

(4X 150 mm); mobile phase a phase: water 10mM ammonium acetate with 0.1% formic acid, phase B: acetonitrile, sample injection running time of 5min, isocratic elution mode, mobile phase A: the proportion of phase B is 9:1 (v/v), the flow rate is 0.5mL/min, the temperature of the column incubator is 50 ℃, and the sample injection amount is 10 mu L. The detected ion information of the deuterated glutamine is 152.1 → 135.2. The ion source is an ESI source, the positive ion scanning mode is a multi-reaction detection (MRM) scanning mode, and the scanning time is 100ms.

(1) Pre-incubation experiments with uptake of deuterated glutamine:

mock cells and HEK293-hSLC1A5-27# cells were plated when grown to log-extended periods. 100 mu g/mLPDL solution 180 mu L/well is added into a 24-well plate, the bottom is infiltrated for improving the adhesiveness of cells, and after 5 minutes, PDL solution is sucked off, and each well is washed three times with 500 mu L of sterilized ultrapure water for standby. HEK293 cells are digested, counted, 2 × 10^ 5/hole and 500 mu L/hole are formed, the cells are paved evenly, after 24 hours, after the cells are attached to the wall, culture media without glutamine are replaced 0hr, 2hr and 12hr before the cells are taken, sterilized PBS is used for washing twice before the cells are replaced, old culture solution is sucked off when the cells are taken, HBSS solution in water bath at 37 ℃ is used for washing three times, 180 mu L/hole of HBSS solution is added, preincubation is carried out for 10 minutes, 20 mu L of deuterated glutamine solution prepared by HBSS is added, the taking time is 3 minutes, the final concentration is 50 mu M, the HBSS solution containing deuterated glutamine is quickly sucked off, the cells are washed three times by precooled HBSS solution, 200 mu L of ultrapure water is added into each hole after the cells are sucked dry, the cells are crushed by an ultrasonic crusher, and the cells are tested. After 2 hours of pre-incubation in culture solution without glutamine, the cell uptake of deuterated glutamine HEK293-hSLC1A5-27# is less than 2 times of the rate of uptake of the cell compared with HEK 293-Mock. The uptake window is larger when the culture solution without glutamine is preincubated for 12 hours, the uptake rate of No. 27 is 15.5 times of that of Mock cells, the cell state is not affected, and the result is shown in FIG. 6, so that deuterated glutamine is used as a substrate and the culture solution without glutamine is preincubated for 12 hours for subsequent inhibitor screening experiments.

(3) Deuterium-substituted glutamine uptake concentration dependent experiment:

after the cells are attached to the wall and overgrow, the old culture solution is sucked before the experiment, washed twice by PBS, and added with culture solution without glutamine for pre-incubation for 12 hours. When taking, the old culture solution is absorbed, washed three times by HBSS solution in water bath at 37 ℃, 180 mu L/hole of HBSS solution is added, preincubation is carried out for 10 minutes, 20 mu L of deuterated glutamine solution prepared by HBSS is respectively added, the final concentration of deuterated glutamine is 10 mu M, 20 mu M, 50 mu M, 100 mu M and 150 mu M, the taking time is 3min, then HBSS solution containing deuterated glutamine is rapidly absorbed, washed three times by precooled HBSS solution, after absorption, 200 mu L of ultrapure water is added into each hole, cells are crushed by an ultrasonic crusher, and to be tested, the absorbing results of substrates in HEK293-hSLC1A5-27# cells and HEK293-Mock cells are summarized as shown in figure 7: at the concentration of 10 mu M and 20 mu M, the response is low, the signal-to-noise ratio is lower than 10, the quantification is difficult, the uptake rate of the HEK293-hSLC1A5-27# to the glutamine at the concentration of 50 mu M, 100 mu M and 150 mu M is larger than that of the HEK293-Mock cell, and finally 50 mu M is selected as the final uptake concentration, the uptake window is large, and the error is small.

(4) Deuterated glutamine uptake time-dependent experiments:

after the cells are attached to the wall and overgrow, the old culture solution is sucked before the experiment, washed twice by PBS, and added with culture solution without glutamine for pre-incubation for 12 hours. Sucking out the old culture solution during intake, washing the old culture solution with HBSS solution in water bath at 37 ℃ for three times, adding the HBSS solution 180 mu L/hole, pre-incubating for 10 minutes, adding 500 mu M glutamine solution prepared by HBSS for 20 mu L, wherein the final concentration of glutamine is 50 mu M, the intake time is 0, 1, 3 and 5min, quickly sucking out the HBSS solution containing deuterated glutamine, washing the old culture solution with precooled HBSS solution for three times, sucking to dry, adding 200 mu L of ultrapure water into each hole, crushing the cells by an ultrasonic crusher, and detecting. The results are collated and summarized in FIG. 8: the window of the glutamine uptake rate of HEK293-hSLC1A5-27# is larger than that of HEK293-Mock cells, the window is the largest at 1min, the times are 22 times, but the uptake time is too short, the experimental operation error is large, and finally 3min with the uptake time of 9 is selected as the final uptake time. After the intake time is more than 5min, the metabolism of glutamine by the cell is obvious, and the concentration of deuterated glutamine in the cell is lower than the lower limit of quantification.

The concentration of deuterated glutamine was selected to be 50 μ M based on the concentration-dependent and time-dependent results, and the uptake time was 3 minutes for subsequent screening of inhibitors.

Example 4 application of SLC1A5 inhibitor screening test System

The screened positive clone cell HEK293-hSLC1A5 (27 #) and Mock cell are normally cultured. The effect of different known compounds on the rate of SLC1A5 transport of deuterated glutamine was examined according to the method of example 3, with a pre-incubation time of 12 hours for a glutamine-free medium, a deuterated glutamine concentration of 50. Mu.M, an uptake time of 3 minutes, a pre-incubation time of 30 minutes for an inhibitor, and a first round of inhibitor compound concentration of 100. Mu.M, for subsequent screening of the inhibitor. The results are shown in FIG. 9 and Table 3. The results show that the known compounds have an inhibitory effect on SLC1A5 transport of deuterated glutamine, and can be used as an inhibitor of SLC1A5 transport of glutamine. Selection of mianserin with the strongest inhibitory effect by concentration dependenceExperiment and calculation of IC ₅₀ ，IC ₅₀ The concentration was 18.3. + -. 6.1. Mu.M, and the results are shown in FIG. 10.

TABLE 3 screening results of inhibitors of SLC1A5 uptake transport of Glutamine

Example 5 Effect of SLC1A5 inhibitors on tumor cell proliferation

(1) Tumor cell selection:

the relative expression level of SLC1A5 of different cells at the gene level and the protein level is obtained by collecting RNA samples and protein samples of 2 normal tissue cells (human embryonic kidney cells HEK293 cells, chinese hamster ovary cells CHO cells) and 7 tumor cells (human colorectal cancer cells HCT8, HCT116, SW480, SW620, human liver cancer cells HepG-2, human pancreatic cancer cells PANC-1 and human breast cancer cells Mcf-7) and performing RT-PCR analysis and Westernblot analysis.

a) Gene level expression:

the results of RT-PCR detection of cDNA reverse transcribed from mRNA of different tumor cell lines and normalization of expression with internal reference are shown in FIG. 11: compared with human embryonic kidney cell HEK293 cells, SLC1A5 in the human colorectal cancer cell line SW480 is expressed at the highest mRNA level, and then the human colorectal cancer cell line SW620 is used; the SLC1A5 expression of the human liver cancer cell HepG2 and the human pancreatic cancer cell PANC-1 is slightly higher than that of the HEK293 cell; SLC1A5 expression of human colorectal cancer cells HCT8, HCT116 and human breast cancer cells Mcf-7 is lower than that of HEK293 cells. All tumor cells had significantly higher expression of SLC1A5 than Chinese hamster ovary CHO cells.

b) Protein level expression:

after western blot analysis of proteins from different tumor cell lines, the results are shown in fig. 12: compared with the HEK293 cells, SLC1A5 was most highly expressed at the protein level in the colorectal cancer cell line SW480, followed by the colorectal cancer cell line SW620, and SLC1A5 protein expression was lower in the remaining cells.

SW480 with the highest SLC1A5 expression was selected for subsequent cell proliferation studies, combining gene level and protein level expression.

(2) Effect of the SLC1A5 inhibitor mianserin on SW480 cell proliferation in vitro:

SW480 cells in the logarithmic growth phase were taken, counted by trypsinization, and sterilized 96-well plates were inoculated at a cell density of 3000/well and 100. Mu.L of 10% FBS-containing RPMI1640 medium/well. The incubator is placed for 24 hours and then taken out for administration treatment, the mianserin mother liquor is diluted by DMSO gradient, the working solution and the culture medium are diluted to the final administration concentration according to the proportion of 1. The incubator was left for 72hr, and then the 96-well plate was taken out for treatment. Adding the prepared MTT solution into a 96-well plate according to the amount of 10 mu L/well, taking out after an incubator is placed for 2 hours, sucking the MTT solution in the 96-well plate, adding DMSO into the 96-well plate according to the amount of 150 mu L/well to dissolve formazan, fully and uniformly oscillating at 800rpm and 10min, detecting the OD value of each well of the 96-well plate at the wavelength of 490nm by using a full-wavelength microplate reader, and inspecting the influence of mianserin on SW480 cell proliferation. The process of calculating the cell viability is detailed in equation 5-1.

Cell survival% = (OD) _{Experimental group} -OD _{Zero setting hole} )/(OD _{Control group} -OD _{Withering hole} ) X 100 formula 5-1

The results are shown in fig. 13, where mianserin can inhibit the proliferation of SW480 cells, and the activity of the cells decreases with increasing inhibitor concentration. The IC50 value for mianserin inhibition of SW480 cells was 32.45. Mu.M.

(3) Effect of the SLC1A5 inhibitor mianserin in combination with the antineoplastic fluorouracil (5-FU) on SW480 cell activity and proliferation:

SW480 cells in the logarithmic growth phase were counted by trypsinization, and sterilized 96-well plates were seeded at a cell density of 3000/well and 100. Mu.L of culture medium/well. Placing the incubator for 24hr, taking out, and performing administration treatment: 1. fluorouracil treatment alone (0, 0.1, 0.5, 1, 5, 10, 20, 50, 100 μ M); 2. fluorouracil (0, 0.1, 0.5, 1, 5, 10, 20, 50, 100 μ M) + mianserin (20 μ M) treatment, control groups with equal volume of DMSO. The incubator was left for 72hr, and then the 96-well plate was taken out and MTT-treated. The results are shown in FIG. 14: the IC50 of the 5-FU used alone for inhibiting SW480 cells is 22.8 mu M, and the IC50 of the combined mianserin administration for inhibiting SW480 cells is 7.6 mu M, which shows that the mianserin can increase the sensitivity of SW480 to fluorouracil and enhance the anti-tumor effect of the fluorouracil on SW480 cells.

(4) The anti-tumor activity of the SLC1A5 inhibitor mianserin in vivo:

the SW480 graft tumor model was first established. After SW480 reached logarithmic growth phase, the culture solution was discarded, washed twice with PBS, and then blown out uniformly using RPMI1640 medium containing 10% FBS, and the obtained cell suspension was collected in a 50ml centrifuge tube. Centrifuging at 500rpm for 5min, discarding the culture solution, and resuspending in serum-free RPMI1640 medium. Mixed with matrigel 1:1 on ice and injected subcutaneously into the back of the right hind limb of each nude mouse 100 μ L of a suspension containing 500 ten thousand cells.

After about 5 days of cell inoculation, tumors grew to 100-150mm ³ At this time, the inoculated nude mice were randomly grouped into 6 mice each, which were divided into Control group (blank Control group), 5-FU group (fluorouracil group), mianseirn group (mianserin group), and 5-FU + mianseirn group (combination group), and each group was administered by intraperitoneal injection, formulated as 5% pefg400+95% physiological saline, once every 3 days. The administration dose is as follows: the blank control group is a blank formula; the fluorouracil group is fluorouracil 30mg/kg, 10ml/kg; the mianserin group is 30mg/kg mianserin, 10ml/kg; the combined administration group comprises 30mg/kg of fluorouracil and 30mg/kg of mianserin, and the dosage is 10ml/kg. The body weight of nude mice was measured every three days, the tumor major diameter (a) and minor diameter (b) were measured, and the tumor volume V =1/2 × a × b was calculated ² 。

The administration was stopped after 5 times, the experiment was terminated 3 days after the 5 th administration, the nude mice were euthanized, tumor tissues were taken, tumor-outer adipose tissues were removed, and the results were photographed as shown in fig. 15. The growth rate of the transplanted tumors was the slowest in the combination group, followed by the mianserin group, and then the fluorouracil group. The combined group had the smallest tumor volume 20 days after inoculation, followed by the mianserin group, then the fluorouracil group. Thus, the mianserin can inhibit SW480 tumor growth and enhance the antitumor effect of fluorouracil after being combined with fluorouracil group. And the mianserin has no obvious toxic or side effect, as shown in figure 16 and table 4.

TABLE 4 tumor growth Rate

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Sequence listing

<110> Suzhou university

<120> a method for screening glutamine transporter inhibitors, inhibitors screened by the method and use thereof

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<170> SIPOSequenceListing 1.0

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tcccgggacc aggtgcgccg ctgccttcga gccaacctgc ttgtgctgct gacagtggtg 180

gccgtggtgg ccggcgtggc gctgggactg ggggtgtcgg gggccggggg tgcgctggcg 240

ttgggcccgg agcgcttgag cgccttcgtc ttcccgggcg agctgctgct gcgtctgctg 300

cggatgatca tcttgccgct ggtggtgtgc agcttgatcg gcggcgccgc cagcctggac 360

cccggcgcgc tcggccgtct gggcgcctgg gcgctgctct ttttcctggt caccacgctg 420

ctggcgtcgg cgctcggagt gggcttggcg ctggctctgc agccgggcgc cgcctccgcc 480

gccatcaacg cctccgtggg agccgcgggc agtgccgaaa atgcccccag caaggaggtg 540

ctcgattcgt tcctggatct tgcgagaaat atcttccctt ccaacctggt gtcagcagcc 600

tttcgctcat actctaccac ctatgaagag aggaatatca ccggaaccag ggtgaaggtg 660

cccgtggggc aggaggtgga ggggatgaac atcctgggct tggtagtgtt tgccatcgtc 720

tttggtgtgg cgctgcggaa gctggggcct gaaggggagc tgcttatccg cttcttcaac 780

tccttcaatg aggccaccat ggttctggtc tcctggatca tgtggtacgc ccctgtgggc 840

atcatgttcc tggtggctgg caagatcgtg gagatggagg atgtgggttt actctttgcc 900

cgccttggca agtacattct gtgctgcctg ctgggtcacg ccatccatgg gctcctggta 960

ctgcccctca tctacttcct cttcacccgc aaaaacccct accgcttcct gtggggcatc 1020

gtgacgccgc tggccactgc ctttgggacc tcttccagtt ccgccacgct gccgctgatg 1080

atgaagtgcg tggaggagaa taatggcgtg gccaagcaca tcagccgttt catcctgccc 1140

atcggcgcca ccgtcaacat ggacggtgcc gcgctcttcc agtgcgtggc cgcagtgttc 1200

attgcacagc tcagccagca gtccttggac ttcgtaaaga tcatcaccat cctggtcacg 1260

gccacagcgt ccagcgtggg ggcagcgggc atccctgctg gaggtgtcct cactctggcc 1320

atcatcctcg aagcagtcaa cctcccggtc gaccatatct ccttgatcct ggctgtggac 1380

tggctagtcg accggtcctg taccgtcctc aatgtagaag gtgacgctct gggggcagga 1440

ctcctccaaa attacgtgga ccgtacggag tcgagaagca cagagcctga gttgatacaa 1500

gtgaagagtg agctgcccct ggatccgctg ccagtcccca ctgaggaagg aaaccccctc 1560

ctcaaacact atcgggggcc cgcaggggat gccacggtcg cctctgagaa ggaatcagtc 1620

atgtaa 1626

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tgcgggtgaa gaggaagtag 20

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tgcaccacca actgcttagc 20

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ggcatggacc tgtggtcatg ag 22

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gguccuguac cguccucaau guaga 25

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ucuacauuga ggacgguaca ggacc 25

Claims

1. The application of mianserin in preparing the inhibitor of the glutamine transfer protein SLC1A5 is characterized in that: the inhibitor of the glutamine transporter SLC1A5 is used for inhibiting the overexpression of the glutamine transporter SLC1A5 in the colorectal cancer.

2. Use according to claim 1, characterized in that: the inhibitor of the glutamine transporter SLC1A5 also comprises a preparation containing fluorouracil.