CN116687913B

CN116687913B - Application of mycophenolic acid in preparation of medicine for treating esophageal cancer

Info

Publication number: CN116687913B
Application number: CN202310920085.4A
Authority: CN
Inventors: 孔维嘉; 丁彤晶; 张佳慧; 王玥; 陈栋; 张泽涵; 商钰; 陈佳祺; 陈梦园
Original assignee: Beijing University of Chinese Medicine
Current assignee: Beijing University of Chinese Medicine
Priority date: 2023-07-25
Filing date: 2023-07-25
Publication date: 2024-01-26
Anticipated expiration: 2043-07-25
Also published as: CN116687913A

Abstract

The invention relates to the technical field of medicines, and in particular discloses application of mycophenolic acid in preparation of medicines for treating esophageal cancer. The invention provides application of mycophenolic acid in preparing a medicament for treating esophageal cancer for the first time, and researches show that the main action mechanism of mycophenolic acid in treating esophageal cancer is as follows: it can inhibit the glycolytic energy level of KYSE150 and KYSE170 of esophageal squamous cell carcinoma, thereby inhibiting the cell activity and proliferation capacity of KYSE150 and KYSE170, blocking the cell cycle in G0/G1 phase, inhibiting cell migration and inducing apoptosis. The application of mycophenolic acid in esophageal squamous cell carcinoma is expected to provide a very promising treatment scheme for esophageal squamous cell carcinoma patients, and has great clinical significance for breaking through the bottleneck of esophageal cancer treatment.

Description

Application of mycophenolic acid in preparation of medicine for treating esophageal cancer

Technical Field

The invention relates to the technical field of medicines, in particular to application of mycophenolic acid in preparing medicines for treating esophageal cancer.

Background

Esophageal cancer (Esophageal Cancer) is a common tumor of the upper digestive tract, and has high incidence and serious malignancy. According to Global Cancer2020 data, the new cases of the Global esophageal Cancer are up to 60.41 ten thousand, the Global tumor is at 7 th position, the death cases are up to 54.41 ten thousand, and the Global tumor is at 6 th position, so that the Cancer-related death is one of the main causes of Cancer-related death. Esophageal cancer progresses rapidly and most patients are found to be already in advanced stages, so the prognosis of the patient is poor. The survival rate of the esophageal cancer in 5 years is 30.3%, the survival rate of the esophageal cancer in 5 years is low, and the life and health of residents in a high-rise area are seriously endangered.

The pathological tissue types of esophageal cancer are Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC), and ESCC is the main part, accounting for more than 85% of all cases. The common treatment method for early stage esophageal cancer is surgical treatment, but most patients have advanced to middle and late stages of tumor at the time of treatment due to the lack of typical clinical symptoms and signs of early stage esophageal cancer patients, and at this time, the patients cannot tolerate surgical treatment, and a plurality of conservative treatments such as drug treatment are performed. Conventional therapeutic drugs for esophageal cancer include gemcitabine hydrochloride, an Luoti Ni hydrochloride capsules, paclitaxel and the like. Because of the poor therapeutic effect of these conventional drugs, there is an urgent need to find new drugs for esophageal cancer treatment.

Disclosure of Invention

Aiming at the problems existing in the existing esophageal cancer treatment, the invention provides application of mycophenolic acid in preparing medicines for treating esophageal cancer. Research shows that energy metabolism is one of the markers of cancer cells, and the metabolic phenotype is characterized in that normal cells are powered by oxidative phosphorylation under the condition of sufficient oxygen, and the cancer cells selectively tend to utilize glycolysis, which is a lower energy efficiency mode for energy metabolism and supply, even if the oxygen supply is sufficient, and the glycolysis energy supply of the cancer cells directly determines the characteristics of proliferation, migration, cell cycle, apoptosis and the like, so that the reduction of the glycolysis level is expected to become a new target point for cancer treatment. And mycophenolic acid can inhibit glycolytic capacity of esophageal squamous cell carcinoma KYSE150 and KYSE170 cells under low-dose condition, so as to inhibit cell activity and proliferation capacity of KYSE150 and KYSE170, block cell cycle in G0/G1 phase, inhibit cell migration and induce apoptosis.

In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:

the invention provides application of mycophenolic acid in preparing a medicament for treating esophageal cancer.

Further, the esophageal cancer is esophageal squamous cell carcinoma.

Mycophenolic acid, also known as mycophenolic acid, english name: mycophenolic acid, a class of drugs extracted from penicillin bacteria. The invention provides application of mycophenolic acid in preparing a medicament for treating esophageal cancer for the first time, and researches show that the main action mechanism of mycophenolic acid in treating esophageal cancer is as follows: mycophenolic acid inhibits the glycolytic and proliferative capacity of esophageal squamous cell carcinoma KYSE150 and KYSE170 cells, thereby inhibiting the cell activity and proliferation capacity, blocking the cell cycle to the G0/G1 phase, inhibiting the cell migration and inducing the apoptosis. The mycophenolic acid provided by the invention is applied to the treatment of esophageal squamous cell carcinoma, and is expected to provide a very promising treatment scheme for ESCC patients.

The mycophenolic acid has the structural formula:

mycophenolic acid, CAS number: 24280-93-1, molecular formula: c (C) ₁₇ H ₂₀ O ₆ Molecular weight 320.34.

Preferably, the medicament for treating esophageal cancer comprises a pharmaceutically acceptable excipient or carrier.

Preferably, the pharmaceutical preparation form for treating esophageal cancer comprises a liquid preparation or a solid preparation.

Preferably, the drug administration mode for treating esophageal cancer comprises oral administration or injection administration.

The invention also provides a medicine for treating esophageal cancer, which comprises mycophenolic acid and a pharmaceutically acceptable excipient or carrier.

The mycophenolic acid has good application prospect in the aspect of treating esophageal cancer, can be used for preparing medicines for treating esophageal squamous cell carcinoma, provides a more effective treatment scheme for treating clinical esophageal cancer patients, also provides a new direction for the subsequent medicine research and development of esophageal cancer, clinical treatment and the like, and has extremely high social value and market application prospect.

Drawings

FIG. 1 is a graph showing the effect of various concentrations of mycophenolic acid on the viability of KYSE150 and KYSE170 cell lines in example 2 according to the invention, wherein FIG. 1A is KYSE150 and FIG. 1B is KYSE170;

FIG. 2 is a graph showing the effect of mycophenolic acid on KYSE150 and KYSE170 cell clone formation in example 3 of the present invention, wherein FIG. 2A and FIG. 2C are photographs of clone formation, and FIG. 2B and FIG. 2D are histograms of clone formation rate (P < 0.05);

FIG. 3 is a graph showing migration of mycophenolic acid after treatment of KYSE150 and KYSE170 cell lines in example 4 of the present invention, wherein FIG. 3A and FIG. 3C are graphs showing scratch healing results, and FIG. 3B and FIG. 3D are cell mobility histograms;

FIG. 4 is a graph showing the cell cycle distribution of mycophenolic acid on KYSE150 and KYSE170 according to the invention in example 5, wherein FIG. 4A and FIG. 4C are graphs showing the results of the flow cell cycle, and FIG. 4B and FIG. 4D are bar charts showing the cell cycle distribution;

FIG. 5 is a graph showing the apoptosis rate of mycophenolic acid on KYSE150 and KYSE170 cell lines in example 6 of the present invention, wherein FIG. 5A and FIG. 5C are flow charts of apoptosis, and FIG. 5B and FIG. 5D are bar charts of apoptosis rate;

FIG. 6 is a graph showing the effect of mycophenolic acid on the glycolytic capacity of KYSR150 and KYSE170 cell lines in example 7 of the present invention, wherein FIG. 6A and FIG. 6B are the effect of mycophenolic acid on the extracellular acidification rate (ECAR) and glycolytic proton efflux rate (glycoPER) of KYSE150 cells, and FIG. 6C and FIG. 6D are the effect of mycophenolic acid on the extracellular acidification rate (ECAR) and glycolytic proton efflux rate (glycoPER) of KYSE170 cells.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

1. Material

1.1 major instrumentation and reagents

1.2 cell lines

Human esophageal squamous cell carcinoma cell lines KYSE150 and KYSE170: is provided by the fourth hospital tumor institute of the university of Hebei medical science.

1.3 kit

1.3 preparation of main reagents:

RPMI-l640 cell culture (containing 10% fetal bovine serum): 50mL of FBS fetal bovine serum and 5mL of penicillin-streptomycin are added into 450mL of RPMI-1640 culture medium in an ultra-clean workbench, blown and mixed uniformly, and stored in a refrigerator at 4 ℃.

Cell culture medium containing mycophenolic acid: the mycophenolic acid is weighed and added into dimethyl sulfoxide (DMSO), and 20mM mycophenolic acid mother solution is prepared and stored in a refrigerator at the temperature of minus 20 ℃ in a dark place. In the experiment, the mother solution is diluted according to the multiple ratio of the cell culture solution of mycophenolic acid with different concentrations to obtain the cell culture medium (DMSO is less than 1 mill).

2. Test method

2.1 Cell treatment

2.1.1 cell culture methods

(1) Before the experiment, the ultra-clean workbench is wiped by 75% ethanol, an ultraviolet lamp is turned on to irradiate for 30min, a relatively sterile environment is ensured, and the cell culture medium, PBS buffer solution and pancreatin which need to be used are placed at room temperature.

(2) Human esophageal squamous cell carcinoma cell lines KYSE150 and KYSE170 were added into a flask (culture area 25 cm) containing RPMI-1640 culture solution (containing 10% fetal bovine serum) ² ) The culture is carried out at 37 ℃ under 5% CO ₂ The growth state of cells is monitored every day, when the fusion degree between cells reaches 80% -85%, the color of the culture solution is slightly yellow, the culture solution is discarded, and 2mL PBS buffer solution is used for gently flushing for 2-3 times;

(3) Adding 1mL of 0.25% pancreatin into each flask, slightly shaking the flask to enable the pancreatin to be fully contacted with cells, placing the flask in a 37 ℃ incubator for 2min, taking out the flask, placing the flask in a microscope for observation, when the cells lose original form, shrink into round spheres, but are not completely floating and fall off, indicating proper digestion degree, adding 2mL of RPMI-1640 culture solution (containing 10% fetal calf serum) to neutralize the pancreatin, stopping digestion, gently blowing with a 1mL gun head to form single cell suspension, and adding the single cell suspension into a 15mL centrifuge tube;

(4) Centrifuging the tube in a low speed centrifuge at 1000rpm for 5min, removing supernatant, adding new RPMI-1640 culture solution (containing 10% fetal calf serum), gently blowing with 1mL gun head to form single cell suspension, diluting the single cell suspension 2 times, inoculating into new culture flask containing RPMI-1640 culture solution (containing 10% fetal calf serum), gently shaking the culture flask according to "crisscross" method to spread cells on the bottom of the flask uniformly, placing at 37deg.C under 5% CO ₂ Is continuously cultured in a constant temperature incubator. Cells in logarithmic growth phase were taken at the time of the experiment.

2.1.2 cell cryopreservation

Selecting cells with good growth condition and in logarithmic phase during freezing, discarding culture solution, gently flushing 2-3 times with 2mL PBS buffer solution, adding 1mL 0.25% pancreatin into each bottle, putting into a 37 ℃ incubator for digestion for 2min, adding 2mL RPMI-1640 culture solution (containing 10% fetal calf serum) to stop digestion, gently blowing with 1mL gun head to make cells fall off, forming single cell suspension, adding into 15mL centrifuge tube, putting the centrifuge tube into a low-speed centrifuge, centrifuging at 1000rpm for 5min, discarding supernatant after centrifugation, adding cell freezing solution to resuspend cell precipitate, and gently blowing with gun head to make the cell precipitate uniformly mixed.

The cell suspension is divided into sterile frozen storage tubes according to the volume of 1.5 mL/tube, and the cell concentration is as high as 1X 10 ⁶ The cell/mL frozen stock solution is prepared by placing a cell frozen stock tube into a refrigerator at-80 ℃ for short-term storage, and placing the cell frozen stock tube into a liquid nitrogen tank at-190 ℃ for long-term frozen stock.

2.1.3 cryopreserved cell resuscitation

And taking out the cell freezing tube to be revived from the refrigerator or the liquid nitrogen tank at the temperature of-80 ℃, putting the freezing tube into PE gloves, and immersing the cell freezing tube in a water bath at the temperature of 37 ℃ to shake so as to reduce the thawing time. After the frozen stock solution is completely melted, wiping a frozen stock pipe orifice by alcohol gauze, sucking cell suspension in the frozen stock pipe, slowly adding the cell suspension into a centrifuge pipe filled with 10mL of fresh RPMI-1640 culture solution (containing 10% of fetal calf serum), centrifuging at a low speed of 1000rpm for 5min, discarding supernatant after centrifugation, re-suspending cells by the RPMI-1640 culture solution (containing 10% of fetal calf serum), gently blowing and uniformly mixing, adding the cell suspension into a culture bottle containing the RPMI-1640 culture solution (containing 10% of fetal calf serum), gently shaking to uniformly spread the cells on the bottom of the bottle, and putting 37 ℃ and 5% CO ₂ Is cultured in a constant temperature incubator. Observing the cell adherence condition, the growth condition and the color change of the culture solution at any time, and carrying out liquid exchange or subculture at a proper time. Cells in logarithmic growth phase were taken at the time of the experiment.

2.1.4 cell counts

The cell counting plate and the cover plate are wiped clean by alcohol, and the cover plate is covered at the corresponding position of the counting plate. The cells to be tested are digested by 0.25% pancreatin, gently blown and evenly mixed to form single cell suspension, 10 mu L of the single cell suspension is taken from the middle part of the single cell suspension by a 10 mu L small gun head, the single cell suspension is slowly injected into the edges of a cell counting plate and a cover plate, the cell suspension is filled between the cover plate and the counting plate without generating bubbles, the counting plate is placed under an optical microscope for observation, the total number of four big cells of the cell counting plate is calculated, and the line pressing cells only mark the left side and the upper side. The calculation is carried out according to the following formula:

cell number/mL = total number of four large lattice cells/4 x 10 ⁴ ；

Cell suspension amount added per 1000 cells = 100/(four large cell count/4) μl.

2.1.5 statistical treatments

And (3) statistical treatment: all experiments in the above examples were repeated 3 more times and the results were expressed as mean ± standard deviation. Relevant data were plotted and statistically tested using GraphPad Prism 9, with significant differences at P < 0.05.

Example 2

Effects of mycophenolic acid on the proliferative capacity of esophageal cancer cells:

1. taking KYSE150 and KYSE170 cells which have good growth condition and are in logarithmic growth phase, digesting with 0.25% pancreatin, adding RPMI-1640 culture solution (containing 10% fetal calf serum) to stop digestion, centrifuging at 1200rpm for 3min, discarding supernatant, adding new RPMI-1640 culture solution (containing 10% fetal calf serum), gently blowing and mixing with gun head to form single cell suspension, counting with cell counting plate, inoculating 96-well plates with 2000 cells, respectively adding 100 μl sterile PBS buffer solution into a round of blank holes around the periphery of the inoculated cells, placing the 96-well plates at 37deg.C and 5% CO ₂ Is cultured in a constant temperature incubator.

2. Observing the cell state on every other day, if the cells are normally attached and the morphology is not abnormal, discarding the culture solution in each well, changing the culture solution to the culture medium containing mycophenolic acid with different concentrations (the culture medium containing mycophenolic acid in the example 1 is diluted to 1 mu M,3 mu M,5 mu M and 7 mu M, 4 concentration gradients are set), continuing culturing, setting at least 3 repeated wells with the same concentration, and setting a control group, namely changing the culture medium to the culture medium without mycophenolic acid.

3. After the cells were treated with mycophenolic acid at different concentrations for 0h,24h and 48h, 20. Mu.L of MTS working solution in MTS cell proliferation-toxicity detection kit was added to each well of original medium, the mixture was slightly shaken, incubated at 37℃for 2.5h in the absence of light, and after the incubation was completed, 96-well plates were placed in a microplate reader, and absorbance (OD value) at 492nm was detected. Cell viability was calculated and finally cell viability was plotted on the ordinate versus time as shown in fig. 1A and 1B. The cell viability was calculated as follows:

cell viability= [ (As-Ab)/(Ac-Ab) ]x100%;

wherein, as: experimental hole, ac: control wells, ab: blank holes.

As a result, it was found that after 48 hours of treatment with mycophenolic acid (MPA) at various concentrations, the cellular activities of both the esophageal cancer cells KYSE150 and KYSE170 were inhibited. As shown in FIG. 1A, in KYSE150 cells, the inhibition rates of MPA of 1. Mu. Mol/L, 3. Mu. Mol/L, 5. Mu. Mol/L and 7. Mu. Mol/L for 48h proliferation were 10.24%, 39.53%, 45.94% and 52.20%, respectively. As shown in FIG. 1B, in KYSE170 cells, the inhibition rates of MPA of 1 mu mol/L, 3 mu mol/L, 5 mu mol/L and 7 mu mol/L on 48h proliferation are respectively 9.61%, 25.43%, 35.70% and 43.76%, and the inhibition degrees are obviously positively correlated with the concentration of mycophenolic acid.

Example 3

Effects of mycophenolic acid on the clonogenic potential of esophageal cancer cells:

1. taking KYSE150 and KYSE170 cells which have good growth condition and are in logarithmic growth phase, digesting with 0.25% pancreatin, adding RPMI-1640 culture solution (containing 10% fetal calf serum), stopping digestion, centrifuging at 1000rpm for 5min, discarding supernatant, adding new RPMI-1640 culture solution (containing 10% fetal calf serum), gently blowing and mixing with gun head to form single cell suspension, counting by using cell counting plate, inoculating 1000 cells per hole of six-hole plate, adding RPMI-1640 culture solution (containing 10% fetal calf serum), shaking uniformly by crisscross method, ensuring that cells are not adhered to the bottom of dish to form clusters as much as possible, and keeping a certain distance between single cells, uniformly distributing, placing the inoculated 6-hole plate at 37 ℃ and 5% CO ₂ Is cultured in a constant temperature incubator.

2. Observing the cell state every other day, if the cell is adhered normally and the morphology is not abnormal, discarding the culture solution in each hole, changing the culture solution into a culture medium containing 3 mu M mycophenolic acid, continuing to culture, setting at least 3 repeated holes at the same concentration, and setting a control group, namely changing the culture medium into the culture medium without mycophenolic acid.

3. Periodically, the inoculated plates were observed for 7 days, white spots were visible in the plates, cell clones were visible under a microscope (the number of cells per clone was 50 or more), at which time the culture was stopped and the culture medium in the six well plates was discarded.

4. Slowly adding 1-2mL of PBS buffer solution into each culture hole, gently shaking, immersing for two times, sucking out the PBS buffer solution, adding 1 mL/hole of 4% paraformaldehyde solution, fixing at room temperature for 20min, sucking out the paraformaldehyde solution, standing at room temperature for a period of time to volatilize the residual paraformaldehyde solution, dyeing with 0.1% crystal violet at room temperature for 20min, recovering the crystal violet dye solution, slowly flushing the six-hole plate with distilled water, removing the residual crystal violet dye solution, and reversely buckling the six-hole plate on absorbent paper for airing after flushing.

5. Cell clones after staining were photographed in a cell phone, the number of clones was counted using Image J and Graphpad Prism software (generally set to be more than 50 cells colonies were regarded as one clone count), and the clone formation rate was counted as follows:

clone formation rate (%) =clone number/inoculated cell number×100%.

Cloning of KYSE150 and KYSE170 cells after treatment with 3. Mu.M mycophenolic acid is shown in FIGS. 2A and 2C, and cell cloning is plotted in bar graph form with the cloning efficiency on the ordinate and mycophenolic acid concentration on the abscissa, as shown in FIGS. 2B and 2D.

As a result, it was found that the KYSE150 control had a clone formation rate of 67.88%, and the clone formation rate was reduced to 32.43% after the treatment with mycophenolic acid at a concentration of 3. Mu.M. The KYSE170 control had a clone formation rate of 45.03% and a clone formation rate of 9.07% after treatment with 3. Mu.M mycophenolic acid.

The above results demonstrate that the clonogenic capacity of KYSE150 and KYSE170 cells can be inhibited to varying degrees after treatment with mycophenolic acid.

Example 4

Effects of mycophenolic acid on esophageal cancer cell migration ability:

1. taking KYSE150 and KYSE170 cells in logarithmic growth phase, digesting with 0.25% pancreatin, adding RPMI-1640 culture solution (containing 10% foetal calf serum), stopping digestion, centrifuging at 1000rpm for 5min, discardingAdding new RPMI-1640 culture solution (containing 10% fetal calf serum) into the supernatant, gently blowing with gun head, mixing to form single cell suspension, and adjusting cell concentration to 1×10 ⁶ Re-suspending cells in culture medium containing mycophenolic acid in 0 and 3. Mu.M each, adding 400. Mu.L cell suspension in each hole in six holes, supplementing culture medium containing mycophenolic acid in 0 and 3. Mu.M to corresponding holes, ensuring 2mL each hole, repeating three holes in each treatment group, shaking uniformly by crisscross method, and mixing at 37deg.C under 5% CO ₂ Is cultured for 24 hours in a constant temperature incubator until cells grow full.

2. The full six well plate was scored along a ruler, perpendicular to the transverse line, with a 10 μl gun head.

The cells were gently rinsed three times in PBS buffer and 2mL of RPMI-1640 medium (containing 10% fetal bovine serum) was added to each well. The scratch spacing was observed under an inverted microscope at 0h and 24h, respectively, and photographed, and the percentage of cell migration was calculated as follows:

cell mobility= (0 h scratch area-24 h scratch area)/0 h scratch area×100%

Migration ability results of KYSE150 and KYSE170 cells treated with 3. Mu.M mycophenolic acid are shown in FIGS. 3A and 3C. The cell mobility is plotted on the abscissa with mycophenolic acid concentration and on the ordinate as a bar graph of cell mobility, as shown in fig. 3B and 3D.

As shown in fig. 3A and 3C, the scratch areas of the KYSE150 and KYSE170 cells were further reduced with the increase of the observation time, the scratch healing ability of the mycophenolic acid-treated group was significantly inhibited compared to the control group, and as shown in fig. 3B and 3D, the cell mobility of the KYSE150 cells after the mycophenolic acid treatment was reduced from 93.85% to 75.47% at 24 hours compared to the control group, and the cell mobility of the KYSE170 cells after the mycophenolic acid treatment was reduced from 77.68% to 51.70%.

The above results demonstrate that mycophenolic acid treatment can inhibit the migratory capacity of esophageal squamous cell carcinoma KYSE150 and KYSE 170.

Example 5

Effects of mycophenolic acid on esophageal cancer cell cycle:

after treatment of KYSE150 and KYSE170 cell lines with mycophenolic acid of 0. Mu.M and 3. Mu.M, respectively, for 48h, each treatment group was fixed together with 75% ethanol solution, and the effect of mycophenolic acid on the cell cycle was examined by flow cytometry after PI staining, as described in example 4 above. And (4) drawing a flow chart of the influence of mycophenolic acid on the cell cycle by taking the DNA amount as an abscissa and the counted effective cell number as an ordinate, as shown in fig. 4A and 4C. The effect of mycophenolic acid on the cell cycle is plotted on the abscissa and the ratio of each cell cycle on the ordinate, as shown in FIGS. 4B and 4D.

As shown in fig. 4A and 4B, mycophenolic acid blocked the KYSE150 cell cycle in G0/G1 phase, increasing the G0/G1 cell fraction from 38.70% to 67.32% compared to the control group; as shown in FIGS. 4C and 4D, mycophenolic acid blocked the KYSE170 cell cycle at the G0/G1 phase, increasing the G0/G1 cell ratio from 38.57% to 65.40% compared to the control group.

The above results demonstrate that mycophenolic acid can arrest the cell cycle of esophageal squamous cell carcinoma KYSE150 and KYSE170 in the G0/G1 phase.

Example 6

Influence of mycophenolic acid on apoptosis ability of esophageal cancer cells:

1. cells were harvested after 48h treatment of KYSE150 and KYSE170 cell lines with 0. Mu.M and 3. Mu.M mycophenolic acid, respectively, according to the procedure described in example 4 above.

2. The collected cells of each group were centrifuged at 1000rpm for 10min, resuspended in 2mL of PBS buffer, centrifuged again, and the cells were repeatedly washed 3 times.

3. Removing the supernatant, sequentially adding 100 mu L of loading buffer solution, 10 mu L of PI dye solution and 5 mu L of Annexin V-FITC dye solution into the apoptosis detection kit, incubating for 15-30min in a dark place, supplementing 400 mu L of loading buffer solution, and loading.

4. The apoptosis rate of each treatment group was quantitatively detected by an Annexin V-FITC/PI double-dye flow cytometer.

Apoptosis results of KYSE150 and KYSE170 cells treated with mycophenolic acid are shown in flow charts, with Annexin V-FITC as abscissa and PI as ordinate, as shown in FIGS. 5A and 5C. And drawing a bar graph of the apoptosis rate by taking the concentration of mycophenolic acid as an abscissa and the apoptosis rate as an ordinate, as shown in fig. 5B and 5D.

As shown in FIG. 5, mycophenolic acid increased the apoptosis rate of KYSE150 from 1.82% to 10.88% and KYSE170 from 1.52% to 11.17% compared to the control group.

The results prove that after being treated by mycophenolic acid, the apoptosis rate of KYSE150 and KYSE170 cells can be improved to different degrees.

Example 7

Effects of mycophenolic acid on glycolytic capacity of esophageal cancer cells:

1. early preparation: esophageal cancer cells were divided into a dosing group and a control group, both groups being under the same conditions (37 ℃,5% CO) ₂ Incubator) for 48 hours.

2. The day before the experiment: esophageal cancer cells cultured for 48h were resuspended in the corresponding medium (RPMI-L640 cell culture medium containing 10% fetal bovine serum) after digestion with 0.25% pancreatin, and after cell concentration adjustment, inoculated into Agilent seahorse XFe/XF 96-well plates with 10000 cells in 80. Mu.L medium per well, and 6 multiplex wells were placed per group (labeled group order). The Agilent seahorse XFe/XF96 analyzer was opened and pre-heated overnight, and the assay solution Calibrant and the assay plate of the 96-well plate (200. Mu.L of sterile water was added to each well of the assay plate) were brought to 37℃without CO ₂ The incubator was preheated overnight.

3. Day of the experiment: observing the cell confluence under a microscope, sucking out sterile water in a 96-well detection plate when the cell confluence is 50% -90%, adding 200 mu L of Calibrant standard solution, and keeping the temperature at 37 ℃ without CO ₂ The incubator was set for 1 hour. Supplement additives (sodium pyruvate, glutamine and glucose) to Seahorse XF RPMI medium, pH 7.4. After the preparation of the detection liquid is completed, the detection liquid is heated to 37 ℃ for standby.

4. Sucking out 60. Mu.L of the medium in the 96-well plate with the cells (the remaining 20. Mu.L of the medium prevents the cells from contacting with air) and then adding 200. Mu.L of the detection solution to each well, sucking out 200. Mu.L of the liquid, adding 200. Mu.L of the detection solution to the well, and placing the 96-well plate at 37 ℃ in the absence of CO ₂ Incubating in an incubator for 45-60min.

5. 20. Mu.L of working fluid was added to each well of dosing well A and 22. Mu.L of working fluid was added to each well of dosing well B, and after addition, the wells were placed on the instrument for calibration for about half an hour. The 96-well cell plate was subjected to the last pipetting in half an hour of calibration, 200. Mu.L of liquid was aspirated, 160. Mu.L of assay liquid was added and the final volume was 180. Mu.L.

6. Analysis of experimental results: the experimental results were checked and analyzed using the official software Wave of Agilent company.

Results of glycolytic rate levels for KYSE150 cells and KYSE170 cells treated with mycophenolic acid are shown in FIGS. 6A-6D, with time on the abscissa and ECAR values or glycoPER on the ordinate.

As shown in FIGS. 6A and 6C, MPA reduced ECAR in both KYSE150 cells and KYSE170 cells. After subtracting mitochondrial acidification, the resulting value was called glycolytic proton efflux (glycoPER) and correlated with extracellular lactic acid accumulation rate of 1:1. As shown in FIGS. 6B and 6D, MPA treatment can also reduce the glycoPER of cells.

The results show that MPA can reduce glycolysis level of esophageal cancer cells.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

Claims

1. Application of mycophenolic acid in preparing medicine for treating esophageal squamous cell carcinoma is provided.

2. The use according to claim 1, wherein: the mycophenolic acid can inhibit glycolytic levels of esophageal squamous cell carcinoma KYSE150 and KYSE170, thereby inhibiting cell activity and proliferation capacity of KYSE150 and KYSE170, blocking cell cycle in G0/G1 phase, inhibiting cell migration and inducing apoptosis.

3. The use according to claim 1, wherein: the medicament further comprises a pharmaceutically acceptable excipient or carrier.

4. A use according to claim 3, wherein: the preparation form of the medicine comprises a liquid preparation or a solid preparation.

5. A use according to claim 3, wherein: the mode of administration of the drug includes oral administration or injection administration.