CN111000836B - Application of thymoquinone and combination use of thymoquinone and autophagy inhibitor ATG7-siRNA in preparation of drugs for treating esophageal cancer - Google Patents
Application of thymoquinone and combination use of thymoquinone and autophagy inhibitor ATG7-siRNA in preparation of drugs for treating esophageal cancer Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention relates to application of thymoquinone TQ in preparation of a composition for treating esophageal cancer. This application includes thymoquinone TQ alone, and thymoquinone TQ in combination with siRNA. The inventor of the invention finds that the TQ can regulate the expression levels of several key factors related to autophagy including ATG7, Bectrin-1 and LC3B in esophageal cancer cells, thereby realizing the treatment of esophageal cancer. Further, the inventor of the invention also finds that the siRNA and the TQ have the characteristic of synergistically inhibiting the proliferation of the esophageal cancer cells based on the combined use of the esophageal cancer cell autophagy promoter.
Description
Technical Field
The application relates to thymoquinone and application of thymoquinone in preparation of a medicine for treating esophageal cancer in combination with an autophagy inhibitor ATG 7-siRNA.
Background
About 30 million people die worldwide each year from esophageal cancer. China is one of high-incidence regions of esophageal cancer, accounts for 22.34 percent of the total number of malignant tumor deaths in China, is second to gastric cancer and is the second place. The incidence of diseases is highest in northern China, and forest counties in Henan province account for the first place. The incidence rate of esophageal cancer in China is higher for men than for women, but the proportion of men and women in high incidence areas is close, and the incidence age is more than 40 years old. Esophageal cancer mostly occurs in the middle section of the esophagus, and accounts for more than 50 percent; esophageal cancer refers to a cancer that occurs in the esophagus. Is a malignant tumor of esophageal mucosa epithelium. Studies have shown that thymoquinone (TQ, as shown below) is a bioactive compound, is a major component of the seed of Negella sativa, and has multiple antibiotic activities. Inhibition of tumor growth by modulation of apoptotic activity, however, few studies have reported its effect on esophageal cancer; the common medicines for treating esophageal cancer have drug resistance and drug toxic and side effects, and are also the main reasons for the failure of the treatment of esophageal cancer, so the research on natural, low-toxicity and high-efficiency biological medicines is promoted.
The siRNA is usually a 20-24 bp double-stranded RNA (dsRNA), two strands of which respectively extend from two ends of the RNA to the other end by 2 nucleotides, and each strand has a 5 'phosphate end and a 3' hydroxyl end. This structure is obtained by treatment with an enzyme called dicer, which cleaves long double-stranded RNA or small hairpin RNA (small hairpin RNA) into siRNA. In addition, siRNA can also be introduced into cells via a variety of different transfection (transfection) techniques and produce specific knockdown effects on specific genes. Therefore, the complementarity of properly tailored siRNA can be used to target genes of known sequence, which makes siRNA an important tool for studying gene function and drug target.
For the treatment of tumors, inhibition of tumor cell proliferation is a common therapeutic approach. For inhibiting tumor cell proliferation, there are several main approaches.
Apoptosis (apoptosis) refers to an autonomous, ordered process of death of cells controlled by genes in order to maintain homeostasis. Apoptosis is strictly regulated by a plurality of genes, including Caspase family, Bcl2 family, oncogenes such as C-myc, cancer suppressor gene P53, and the like.
Autophagy (autophagy) is an evolutionarily conserved important process in eukaryotes for the turnover of intracellular material. In the process, some damaged proteins or organelles are wrapped by autophagy vesicles with double-layer membrane structures, and then are delivered into lysosomes (animals) or vacuoles (yeasts and plants) for degradation and recycling. There are currently three different views of autophagy in academia: autophagy can be used as an initial factor to induce apoptosis, and blocking autophagy can delay apoptosis, while a broad-spectrum Caspase inhibitor can effectively inhibit apoptosis, but does not influence autophagy; low degree autophagy can antagonize apoptosis, at this time, if the autophagy is blocked, the sensitivity of the cell to apoptosis signals can be increased; and autophagy and apoptosis exist independently of each other, and inhibition of either pathway pushes cells to another programmed death. In conclusion, autophagy is an important mechanism for maintaining cellular homeostasis and helping to maintain cellular return to normal.
Since autophagy is an important mechanism for maintaining cellular homeostasis, it helps to maintain cellular return to normal. Therefore, if new therapeutic compositions could be provided, by inducing autophagy in cancer cells, they could be used as an alternative to cancer therapy, particularly esophageal cancer therapy.
Disclosure of Invention
Technical problem to be solved
At present, few reports of esophageal cancer treatment medicines exist, and the invention discovers that the TQ can inhibit the proliferation of esophageal cancer cells and simultaneously has a promoting effect on two esophageal cancer cell proliferation inhibition pathways of apoptosis and autophagy of the esophageal cancer cells. Therefore, the purpose can be achieved by adding the components of TQ into the medicine for preventing or treating esophageal cancer.
Means for solving the problems
One mode of the invention is the application of thymoquinone TQ in the preparation of a pharmaceutical composition for treating cancer. The thymoquinone TQ of the invention has obvious effect on cancer cells
In another embodiment of the present invention, the cancer is esophageal cancer. Surprisingly, the inventor of the present invention found that thymoquinone TQ has a promoting effect on both apoptosis and autophagy of cancer cells in inhibiting the proliferation pathway of esophageal cancer cells. Therefore, the purpose can be achieved by adding the components of TQ into the medicine for preventing or treating esophageal cancer.
In another mode of the invention, the thymoquinone TQ is applied to the induction of esophageal cancer cell apoptosis and/or the promotion of esophageal cancer cell autophagy.
In another embodiment of the present invention, the thymoquinone TQ is used in combination with a cancer cell autophagy promoting agent in order to further promote the therapeutic effect of esophageal cancer.
In another embodiment of the present invention, the cancer cell autophagy promoting agent is selected from the group consisting of siRNA. The inventor finds that the thymoquinone TQ has promotion effects on inhibiting the proliferation pathways of esophageal cancer cells simultaneously in apoptosis and autophagy of the cancer cells.
In another embodiment of the present invention, the ratio of the cancer cell autophagy promoter to the thymoquinone TQ is: 1: 1-5.
In the invention, the thymoquinone TQ and the autophagy inhibitor ATG7-siRNA are combined for use in inducing the apoptosis of esophageal cancer cells and/or promoting the autophagy phase of the esophageal cancer cells.
In another embodiment of the present invention, the composition is an oral preparation or an injectable preparation.
The concentration of TQ or a combination of TQ and siRNA may be set to 10 to 500mg/kg body weight. The dose may be administered in a single dose or divided doses.
The composition for treating esophageal cancer according to the present invention may also contain one or more excipients. The excipients are not limited, and examples thereof include solvents, isotonic agents, excipients, pH adjusters, antioxidants, disintegrants, flavors, fragrances, preservatives, and the like, which are generally used in the art.
Examples of the solvent include: distilled water for injection, physiological saline, vegetable oil, alcohols such as propylene glycol, polyethylene glycol, ethanol, and glycerin, and the like.
Examples of isotonic agents include: sorbitol, sodium chloride, glucose, and the like, which are isotonic agents commonly used in the art.
Examples of excipients include: lactose, mannitol, glucose, microcrystalline cellulose, starch, and the like.
Examples of the pH adjuster include: hydrochloric acid, citric acid, sodium hydroxide, strong potassium oxide, sodium bicarbonate, disodium hydrogen phosphate and the like.
Examples of antioxidants include: sodium sulfite, sodium bisulfite, ascorbic acid, and the like.
Examples of the disintegrant include: potato starch.
Examples of flavoring agents include: sucrose, simple syrup, and the like.
Examples of the perfume include: peppermint oil, orange peel oil, and the like.
Examples of preservatives include: preservatives commonly used in the art, such as parabens, sorbic acid and salts thereof.
The composition for treating esophageal cancer of the present invention may be in any form, for example, oral liquid, patch, tablet, capsule, injection, etc., preferably injection, and most preferably skeletal muscle injection.
The inventor of the invention finds that the TQ can regulate the expression levels of several key factors related to autophagy including ATG7, Bectrin-1 and LC3B in esophageal cancer cells, thereby realizing the treatment of esophageal cancer. Further, the inventor of the invention also finds that the siRNA and the TQ have the characteristic of synergistically inhibiting the proliferation of the esophageal cancer cells based on the combined use of the esophageal cancer cell autophagy promoter.
Drawings
FIG. 1 is a graph showing the relationship between ECA-109 cell inhibition rate over time after TQ treatment for 24h and 48 h.
FIG. 2 is a graph showing that the rate of apoptosis of cells was promoted when the TQ concentration reached 36. mu.M in the drug-added treatment for 24 hours.
FIG. 3 is a graph showing the blocking effect of TQ on the cell division cycle of ECA-109.
FIG. 4 is a graph showing that the motility of esophageal cancer cells is inhibited as the concentration of TQ increases in the control group and the TQ-treated test group.
FIG. 5 is a graph showing that the main and shear bands of ATG7 and Lc3B show a tendency to increase with increasing TQ concentration for ECA-109 cells at 24h of TQ treatment.
FIG. 6 is a graph showing the inhibition of ECA-109 cells by TQ alone, siRNA alone, TQ and siRNA in combination, siRNA and ATG7 in combination, and siRNA-ATG7 and TQ in combination, respectively.
FIG. 7 is a graph showing that the main band and the cleavage band of the combination of TQ and ATG7-siRNA show a decreasing trend.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
First, the effect of TQ on the proliferation of esophageal cancer cells ECA-109
1. In vitro culture of esophageal cancer cell strain
1.1 sources of cells
Human esophageal cancer cell ECA-109;
1.2 cell culture and passage
ECA-109 uses 1640 medium (Gibco). 10% fetal bovine serum (Capricorn) was added to the medium at the time of cell culture. The cell lines are all at 37 ℃ and 5% CO2Culturing in an incubator, digesting by using 0.25% pancreatin-EDTA and carrying out passage when the cells grow to 80-90% of the bottom of the culture dish.
2. Effect of TQ on cell proliferative Capacity
Main test materials: thymoquinone (TQ) (#03416) was dissolved in absolute ethanol by Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) to make 10mM concentrate, stored at-20 ℃, diluted to 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60 and 66 μ M at the time of use, respectively, ready to use; CCK-8 (beyond); pancreatin (Gibco); PBS; 96-well plates (Nunc).
The test method comprises the following steps: the cells were digested, centrifuged and resuspended in the corresponding medium, seeded at a density of 5X 103/well in 96-well plates, placed at 37 ℃ in 5% CO2After 24h incubation in the incubator, the stock culture was removed, freshly prepared culture medium containing 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60 and 66. mu.M TQ was added and incubation continued for 24h and 48h, respectively, after which 10. mu.L of CCK-8 was added to each well and the incubation was again carried out at 37 ℃ with 5% CO2After 2h incubation in the incubator, the absorbance of each well was measured at microplate reader OD450 nm. Three replicate groups were set for each concentration.
And (3) test results:
TQ had no significant dose-dependent effect on ECA-109 cells, but the inhibition of cell proliferation tended to increase with treatment time: after 48h of TQ treatment, the inhibition rate gradually increased with time, wherein the inhibition rate of ECA-109 at treatment concentration of 30 μ M at 48h of TQ treatment can exceed 50%; after the ECA-109 is acted on the TQ for 48 hours, the inhibition rate of the test group with the treatment concentration of more than 40 mu M can exceed 80 percent (figure 1).
Second, induction of apoptosis of esophageal cancer cells by TQ
1. Cell culture and passage
The test uses human esophageal cancer cell line ECA-109, the culture medium is 1640 culture solution added with 10% FBS, and the temperature is 37 ℃ and the CO content is 5%2Culturing in an incubator, digesting by using 0.25% pancreatin-EDTA and carrying out passage when the cells grow to 80-90% of the bottom of the culture dish.
2. Effect of TQ on apoptosis of esophageal cancer cells
Main test materials: TQ is dissolved in absolute ethyl alcohol to prepare 10mM concentrated solution, and the concentrated solution is stored at minus 20 ℃ and is respectively diluted to 36 mu M when in use, thus the solution is ready to use; annexin V-kFluor647 cell apoptosis detection kit (Kayji organism); PBS; 30mm petri dish, flow-type special loading tube.
The test method comprises the following steps: after the cells were digested into a cell suspension, the cells were plated in a 30mm petri dish at 37 ℃ with 5% CO at an appropriate density2Incubating in incubator, removing original culture solution when cell growth is about 50% of the bottom of the culture dish, adding fresh culture solution containing 36 μ M TQ, and continuing culturing. After 24h of incubation, the supernatant was carefully collected and the cells were digested with trypsin without EDTA, the cell suspension was mixed with the supernatant and centrifuged, the collected cells were washed once with PBS, centrifuged and resuspended in 500. mu.LBinding Buffer, and the cell density was adjusted to 1-5X 105 cells/mL. And (3) sucking 100 mu L of cell suspension into an upper sample tube, adding 5 mu L of Annexin V-kFluor647 and 5 mu L of Propidium Iodide (PI), uniformly mixing, reacting for 15min at a dark room temperature, and detecting the apoptosis condition by using a flow cytometer.
And (3) test results: TQ has the ability to induce apoptosis in esophageal cancer cells. At 24h of the drug treatment, when the drug concentration reached 36. mu.M, the apoptosis rate of the cells increased with the drug concentration (FIG. 2).
| TQ | AnnexinV(％) | |
| Blank control group | - | 5.64 |
| Group | ||
| 1 | 36μM | 5.8％ |
| Group 2 | 36μM | 12.38％ |
| Group 3 | 36μM | 8.28％ |
Inhibition of esophageal cancer cell cycle by TQ
1. Cell culture and passage
The test uses human esophageal cancer cell line ECA-109, the culture medium is 1640 culture solution added with 10% FBS, and the temperature is 37 ℃ and the CO content is 5%2Culturing in an incubator, digesting by using 0.25% pancreatin EDTA and carrying out passage when the cells grow to 80-90% of the bottom of the culture dish.
2. Effect of TQ on the cell cycle of esophageal cancer
Main test materials: TQ is dissolved in absolute ethyl alcohol to prepare 10mM concentrated solution, and the concentrated solution is stored at minus 20 ℃ and is respectively diluted to 36 mu M when in use, thus the solution is ready to use; cell cycle detection kits (Kekiky organisms); PBS; 30mm petri dish, flow-type special loading tube.
The test method comprises the following steps: after the cells were digested into a cell suspension, the cells were plated in a 30mm petri dish at 37 ℃ with 5% CO at an appropriate density2Incubating in incubator, removing original culture solution when cell growth is about 50% of the bottom of the culture dish, adding fresh culture solution containing 36 μ M TQ respectively, and continuing culturing. After 24 hours of culture, the supernatant was carefully collected and the cells were digested with trypsin without EDTA, the cell suspension was mixed with the supernatant and centrifuged, and the collected cells were washed once with PBS to adjust the cell density to 1X 106 cells/mL. Sucking 1mL of cell suspension, adding the cell suspension into 500 mu L of 70% cold ethanol for fixation overnight, washing out the fixation solution by using PBS before dyeing, adding the fixation solution into 500 mu LRNase/PI dyeing working solution, uniformly mixing, reacting for 30min at room temperature in a dark place, and detecting the cell cycle condition by using a flow cytometer.
And (3) test results: with respect to the ability of TQ to induce the cell cycle of esophageal cancer, TQ has a function of blocking the cell division cycle of ECA-109, thereby preventing the cells from successfully dividing and proliferating. The cycle arrest occurs mainly in G0/G1 (FIG. 3).
Fourth, the influence of TQ on the motor function of esophageal cancer cells
1. Cell culture and passage
The test uses human esophageal cancer cell line ECA-109, the culture medium is 1640 culture solution added with 10% FBS, and the temperature is 37 ℃ and the CO content is 5%2Culturing in an incubator, digesting by using 0.25% pancreatin-EDTA and carrying out passage when the cells grow to 80-90% of the bottom of the culture dish.
2. Cell migration assay
Main test materials: TQ is dissolved in absolute ethyl alcohol to prepare 10mM concentrated solution, and the concentrated solution is stored at minus 20 ℃ and is respectively diluted to 36 mu M when in use, thus the solution is ready to use;
the test method comprises the following steps: cells were digested, centrifuged, resuspended and plated at a density of 1X 105 cells/well in 24-well platesAt 37 ℃ and 5% CO2Culturing for 24h in an incubator, scratching a six-hole plate with a gun head when the cells grow to a fusion state, carefully sucking out the culture solution after scratching, adding a proper amount of PBS (phosphate buffer solution) for cleaning for 3 times, adding serum-free 1640 into each hole, and adding drugs (0, 2.5,5,10 and 20 mu M). At 37 ℃ 5% CO2After further incubation in the incubator for 24 hours, the incubation was terminated, and the degree of healing of the scratch was observed under an inverted microscope and photographed.
And (3) test results: as can be seen from the cell scratch picture, it is not
In the drug-treated control group, a certain number of cells migrate to the scratch after scratching for 24 hours; the TQ-treated test group showed more and more significant inhibition of the motility of esophageal cancer cells as the drug concentration increased, and the cells migrated into the scratched area 24h after treatment were significantly less than the control group (FIG. 4).
Fifth, the Werstern Blotting detection of the TQ to the esophagus cancer cell autophagy related factor expression
1. Cell culture and passage
The test uses human esophageal cancer cell line ECA-109, the culture medium is 1640 culture solution added with 10% FBS, and the temperature is 37 ℃ and the CO content is 5%2Culturing in an incubator, digesting by using 0.25% pancreatin-EDTA and carrying out passage when the cells grow to 80-90% of the bottom of the culture dish.
2. Expression detection of autophagy-related factor
Main test materials: TQ is dissolved in absolute ethyl alcohol to prepare 10mM concentrated solution, and the concentrated solution is stored at the temperature of minus 20 ℃ and is respectively diluted to 10,20,30 and 40 mu M when in use, namely the mixture is ready to use; RIPA lysate; PMSF; BCA protein concentration assay kit, primary anti-PI 3K (#4292), ATG-7(#8558), GAPDH (#2118), ACTIN (#4970), Beclin-1(#3495), LC3B (#3868) purchased from Cell Signaling Technology (Beverly, MA), and immunoblotted secondary antibodies were peroxidase goat anti-mouse IgG (#7076) and peroxidase goat anti-rabbit IgG (#7074) from Cell Signaling Technology; acrylamide, SDS, ammonium sulfite, Tris-Base, PVDF membrane, ECL luminescence solution (GE Healthcare).
The test method comprises the following steps: after the cells are digested into a cell suspension, the method comprisesThe appropriate density was plated in 60mm petri dishes at 37 ℃ with 5% CO2Incubating in incubator for about 24h, removing original culture solution when cell growth is about 60% of the bottom of culture dish, adding fresh culture solution containing 10,20,30 and 40 μ M TQ respectively, and continuing culturing with culture solution without TQ as control group. The culture was terminated 24h after the addition of TQ, the culture was removed and washed twice with PBS, and the cells were lysed by adding RIPA lysate containing 1% PMSF. The lysate was centrifuged at 4 ℃ and 12000 Xg/min for 8min, the supernatant was collected, the protein concentration was determined, the protein amount was measured and loaded, the protein was separated by 10% SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% skim milk powder, washed with TBST (1% Tween 20) and incubated overnight at 4 ℃ with primary antibody (actin, ATG7, gapdh, beclin-1, lc3 b). TBST wash 3 times for 8min, secondary antibody incubation at room temperature for 1h, TBST wash 3 times for 8min, chemiluminescence using ECL luminescence and exposure to dark room, analysis of relevant protein expression trends.
And (3) test results:
when ECA-109 cells are treated for 24 hours by TQ, ATG7 and the main band and the shear band of Lc3B show enhancement tendency along with the increase of the concentration of the TQ; there was no significant change in Beclin-1 expression (FIG. 5).
Sixth, TQ combined application siRNA for evaluating proliferation activity rate
1. Cell culture and passage
The test uses human esophageal cancer cell line ECA-109, the culture medium is 1640 culture solution added with 10% FBS, and the temperature is 37 ℃ and the CO content is 5%2Culturing in an incubator, digesting by using 0.25% pancreatin-EDTA and carrying out passage when the cells grow to 80-90% of the bottom of the culture dish.
2. Effect of TQ in combination with ATG7-siRNA on cell proliferative Capacity
Main test materials: dissolving Thymoquinone (TQ) in anhydrous ethanol to obtain 10mM concentrate, storing at-20 deg.C, and diluting to 36 μ M for use; CCK-8 (beyond); pancreatin (Gibco); PBS; 6 well plates (Nunc). siRNA (Bio-Rad), 30mm petri dish;
the test method comprises the following steps: digesting the cells, centrifuging, resuspending in the corresponding culture medium, planting in 30mm culture dish, adding transfectionThe solution, ATG7, AGT7-siRNA, cultured for 24H and planted in 96-well plate at 5 × 103/well density, placed at 37 deg.C and 5% CO2After 24h incubation in the incubator, the stock culture was removed, freshly prepared culture medium containing 36. mu.M TQ was added and incubation continued, the incubation was stopped for 24h, respectively, 10. mu.L of serum-free CCK-8 was added to each well, and the wells were again incubated at 37 ℃ with 5% CO2After 2h incubation in the incubator, the absorbance of each well was measured at microplate reader OD450 nm.
And (3) test results: ATG7-siRNA can inhibit autophagy by interfering with ATG7, and the data show that combining 12 μ M siRNA increases the inhibitory effect of TQ on ECA-109 cells compared to TQ intervention alone, i.e. lower proliferative activity and higher apoptotic rate; the combination of ATG7-siRNA reduces the inhibition effect of TQ on ECA-109 cells, namely the proliferation activity is high and the apoptosis rate is low; (FIG. 6)
In order to further illustrate the synergistic effect of the TQ and the siRNA, the invention adopts a gold positive mean q value method. The q value is obtained by the following formula: q ═ PA+B/(PA+PB-PA*PB). In the formula, PA、PBAnd PA+BRespectively the treatment rates of the A medicine group, the B medicine group and the combination of the two medicines. q is less than 1, which indicates that the two medicines generate antagonism after being used together; q is more than 1, which indicates that the two medicines produce synergistic action after being used together; q is 1, which indicates that the two drugs produce additive action after being combined.
In particular, P of the inventionA+B＝92％、PA＝52％、PBThis yields a q of 92%/(52% + 4% -52% × 4%) of 1.7. Therefore, the TQ of the invention has very obvious synergistic effect with siRNA, and the effect is unexpected.
Seventhly, TQ combined application autophagy inhibitor ATG7-siRNA expression Werstern Blotting detection
1. Cell culture and passage
The test uses human esophageal cancer cell line ECA-109, the culture medium is 1640 culture solution added with 10% FBS, and the temperature is 37 ℃ and the CO content is 5%2Culturing in an incubator, digesting by using 0.25% pancreatin-EDTA and carrying out passage when the cells grow to 80-90% of the bottom of the culture dish.
2. Expression detection of TQ in combination with ATG7-siRNA
Main test materials: dissolving TQ in anhydrous ethanol to obtain 10mM concentrated solution, storing at-20 deg.C, and diluting to 36 μ M when in use; RIPA lysate; PMSF; BCA protein concentration assay kit, primary anti-PI 3K (#4292), ATG-7(#8558), GAPDH (#2118), ACTIN (#4970), Beclin-1(#3495), LC3B (#3868) purchased from Cell Signaling Technology (Beverly, MA), and immunoblotted secondary antibodies were peroxidase goat anti-mouse IgG (#7076) and peroxidase goat anti-rabbit IgG (#7074) from Cell Signaling Technology; acrylamide, SDS, ammonium sulfite, Tris-Base, PVDF membrane, ECL luminescence solution (GE Healthcare), siRNA (Bio-Rad).
The test method comprises the following steps: after the cells were digested into a cell suspension, the cells were plated in a six-well plate at 37 ℃ with 5% CO at an appropriate density2And (3) incubating in an incubator, removing the original culture solution when the cells grow to about 30-40% of the bottom of the culture dish, adding the transfection solution, ATG7 and AGT7-siRNA respectively, culturing for 24H, adding a freshly prepared culture solution of 36 mu M TQ, and continuing culturing, wherein the culture solution without the TQ is used as a control group. The culture was terminated 24h after the addition of TQ, the culture was removed and washed twice with PBS, and the cells were lysed by adding RIPA lysate containing 1% PMSF. The lysate was centrifuged at 4 ℃ and 12000 Xg/min for 8min, the supernatant was collected, the protein concentration was determined, the protein amount was measured, the protein was separated by 10% SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% skim milk, washed with TBST (1% Tween 20) and incubated overnight at 4 ℃ with primary antibody (actin, ATG 7). TBST wash 3 times for 8min, secondary antibody incubation at room temperature for 1h, TBST wash 3 times for 8min, chemiluminescence using ECL luminescence and exposure to dark room, analysis of relevant protein expression trends.
And (3) test results:
siRNA interference ATG7 can obviously inhibit the occurrence of autophagy, and the combination of TQ and ATG7-siRNA shows a weakening trend; cancer cell autophagy was inhibited (fig. 7).
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (3)
1. The thymoquinone is combined with an autophagy inhibitor ATG7-siRNA to be used in the preparation of a pharmaceutical composition for treating cancer; the autophagy inhibitor ATG7-siRNA refers to small interfering RNA that specifically inhibits ATG7 expression; the administration molar ratio of the thymoquinone to the autophagy inhibitor ATG7-siRNA is 1-5: 1; the cancer is esophageal cancer.
2. The use of claim 1, wherein the thymoquinone is used to induce apoptosis and/or promote autophagy in esophageal cancer cells.
3. The use according to claim 1, wherein the composition is an oral liquid, a patch, a tablet, a capsule or an injection.
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Effective date of registration: 20230607 Address after: Room 302, Building 16, Lianhua Technology Small and Micro Enterprise Pioneer Park, Jiangkou Street, Huangyan District, Taizhou City, Zhejiang Province 318020 Patentee after: Taizhou Meika Medical Technology Co.,Ltd. Address before: 215334 Building 1, No.3 Gutan Avenue, economic development zone, Gaochun District, Nanjing City, Jiangsu Province Patentee before: Jiangsu Meika Medical Technology Development Co.,Ltd. |
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Address after: 318020 Floor 4, Building 16, Lianhua Technology Small and Micro Enterprise Pioneer Park, Jiangkou Street, Huangyan District, Taizhou City, Zhejiang Province Patentee after: Taizhou Meika Medical Technology Co.,Ltd. Address before: Room 302, Building 16, Lianhua Technology Small and Micro Enterprise Pioneer Park, Jiangkou Street, Huangyan District, Taizhou City, Zhejiang Province 318020 Patentee before: Taizhou Meika Medical Technology Co.,Ltd. |
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