CN106166296A - A kind of pharmaceutical composition assisting rapamycin treatment kinds of tumors - Google Patents

Abstract

The invention discloses a pharmaceutical composition for assisting rapamycin in treating various tumors, belonging to the technical field of biomedicine. In the present invention, n-3PUFA is added to the tumor cells being cultured at different concentrations alone or in combination with rapamycin, and the cell growth, cell proliferation, cell death, Erk1/2 protein activity changes, Akt Changes in protein activity and P38 protein activity are used to evaluate the effect of substances on the apoptosis and/or proliferation of tumor cells, so as to determine the effect of the n-3PUFA on assisting rapamycin drugs in treating various tumors.

Description

A pharmaceutical composition for assisting rapamycin in treating various tumors

technical field

The invention relates to a pharmaceutical composition for assisting rapamycin in treating various tumors, belonging to the technical field of biomedicine.

Background technique

Cancer is a common and frequently-occurring disease that seriously threatens human life. At present, there are about 10 million new cancer patients and nearly 6 million cancer deaths in the world every year. In my country, there are about 1.8 million new cancer patients and 1.3 million deaths every year, and the incidence rate is increasing year by year. In my country, cancer death accounts for the second cause of death of various diseases, and it has already occupied the first place in the city. Cancer not only causes huge physical damage to patients, but also brings serious trauma to patients psychologically and spiritually. It has brought hardships to the lives of countless people, ruthlessly claimed countless lives and destroyed The happy and happy life of the family has caused a heavy burden on the family and society. But the treatment of cancer has always been a worldwide problem. Over the years, countries all over the world have been looking for and exploring various effective means to prevent or treat cancer. Drug therapy is one of the three major therapies for cancer treatment. Traditional chemical drugs in the treatment of malignant tumors are mainly aimed at directly or indirectly killing tumor cells, and have a certain degree of lethality to normal cells, with obvious toxic and side effects. In addition, the existence of drug resistance of tumor cells greatly limits the efficacy of this type of cytotoxic drugs and apply.

Rapamycin (rapamycin) is a macrolide immunosuppressant produced by Streptomyces hygroscopicus. It is clinically used for the anti-rejection of organ transplantation and the treatment of autoimmune diseases. Rapamycin mainly acts on cell cycle G1, inhibiting the DNA synthesis of cytokines and growth factors in immune and non-immune cells. It is clinically used for the treatment of anti-rejection and autoimmune diseases in organ transplantation. It has high activity, small dosage (2mg/day, human), and low toxicity. The United States has conducted 1295 cases of kidney transplantation trials in 80 centers around the world. The effect Significantly, with few side effects.

It has been found that rapamycin alone or in combination with chemotherapy drugs has anti-tumor activity. In vivo and in vitro studies have shown that rapamycin can induce a variety of tumors to stagnate in the G1 phase, reduce tumor cell somatolysis, and induce tumor cell apoptosis independent of p53. Rapamycin prevents VEGF from interacting with blood vessels by reducing the production of VEGF. The role of endothelial cells to significantly reduce tumor angiogenesis, reduce tumor blood vessel density, cause tumor necrosis, and prolong the survival time of tumor-bearing mice.

Although rapamycin has anti-tumor activity, its tumor effect is poor when rapamycin is used alone, and severe hyperlipidemia and drug resistance will occur after long-term use. In addition, in the existing literature on the combined use of rapamycin and other antineoplastic drugs, the components used in combination only involve some existing synthetic rather than natural antineoplastic drugs, and these antineoplastic drugs are relatively toxic , has bone marrow suppression, leukopenia, and also has the effect of causing vomiting, nausea and other gastrointestinal reactions. At present, there are no reports showing that natural food components can assist rapamycin in the treatment of tumors.

Contents of the invention

In order to solve the above problems, it is an important solution to select natural anti-tumor food drugs combined with rapamycin, enhance the activity of anti-tumor drugs and rapamycin, and reduce the toxic and side effects of rapamycin and its derivatives . Therefore, the present invention assists rapamycin to treat various cancers through n-3PUFA, and the combination of active functional food components and drugs can obtain satisfactory therapeutic effects. The present invention provides a food-assisted therapeutic pharmaceutical composition with antitumor activity. On the one hand, it enhances the antitumor effect of rapamycin, expands its antitumor range (antitumor spectrum), and/or reduces its dosage so as to reduce Medication time and/or reduce potential side effects on patients; another object of the present invention is to provide the application of the pharmaceutical composition in tumor treatment.

The first object of the present invention is to provide a pharmaceutical composition, which contains A and B as main active components; wherein A is rapamycin, and B is n-3 polyunsaturated fatty acid.

In one embodiment of the present invention, the n-3PUFA is a polyunsaturated fatty acid, including α-linolenic acid (α-Linolenic acid, ALA) and/or eicosapentaenoic acid (Eicosapentaenoic acid, EPA, containing 5 unsaturated bonds) and/or docosahexaenoic acid (Docosahexaenoic acid, DHA, containing 6 unsaturated bonds).

In one embodiment of the present invention, the rapamycin drugs include rapamycin and rapamycin derivatives.

In one embodiment of the present invention, the n-3 polyunsaturated fatty acids include foods containing n-3 PUFA and/or nutritional supplements containing n-3 PUFA.

In one embodiment of the present invention, the food containing n-3 PUFA mainly includes fish oil.

In one embodiment of the present invention, the molar ratio of A and B in the pharmaceutical composition is 2:1˜1:20.

In one embodiment of the present invention, the molar ratio of A and B in the pharmaceutical composition is 1:10.

In one embodiment of the present invention, in the drug combination, A and B are alone or/and combined into a solid state and/or a solvate, that is, the forms of A and B can be the same or different, and the forms of A and B May be solid or solvated.

The second object of the present invention is to provide a pharmaceutical composition for inhibiting and/or preventing and/or treating tumors, said composition has A and B as main active components; wherein A is inhibiting and/or preventing And/or a drug for treating tumors, B is n-3 polyunsaturated fatty acid.

In one embodiment of the present invention, the A is a rapamycin drug.

In one embodiment of the present invention, the tumor comprises breast cancer, prostate cancer, leukemia, gastric cancer, liver cancer, ovarian cancer, rectal cancer and/or colon cancer.

The third object of the present invention is to provide a pharmaceutical composition for treating or/and inhibiting or/and preventing human breast cancer cells; said composition uses A and B as main active components; wherein A is inhibiting and /or prevent and/or treat the medicine of human breast cancer cell, B is n-3 polyunsaturated fatty acid; Described human breast cancer cell comprises human breast cancer MCF-7 cell, T47D, SUM185, BT474, BT-483, 600MPE , ZR-75, MDA-MB-468/453/231.

The fourth object of the present invention is to provide a pharmaceutical composition for enhancing and/or improving the activity of p38 protein in tumor cells, the pharmaceutical composition is mainly active components with A and B; wherein A is rapamycin Vegetarian drugs, B is n-3 polyunsaturated fatty acid.

The present invention also provides a pharmaceutical composition for reducing and/or inhibiting the activity of Erk1/2 protein. The pharmaceutical composition uses A and B as main active components; wherein A is a rapamycin drug, and B is n- 3 polyunsaturated fatty acids.

The present invention also provides a pharmaceutical composition for reducing and/or inhibiting the activity of Akt protein. The pharmaceutical composition uses A and B as main active components; wherein A is a rapamycin drug, and B is n-3 poly unsaturated fatty acid.

A fifth object of the present invention is to provide a method for detecting and/or testing and/or examining and/or experimenting the effect of a pharmaceutical composition for treatment and/or inhibition and/or prevention.

In one embodiment of the present invention, the method is to detect Erk1/2 protein activity, Akt protein activity and/or p38 protein activity; the method is to detect Erk1/2 protein phosphorylation by methods such as immunoblotting or Western Kinase activity, Akt protein phosphokinase activity and/or p38 protein phosphokinase activity.

In one embodiment of the present invention, in the method, when the analysis result is that Erk1/2 and/or Akt activity in human cancer cells cultured in vitro is significantly reduced and/or decreased, and/or p38 activity is significantly increased and/or or enhanced, then the adjuvant is beneficial; if the result is a significant increase and/or enhancement of Erk1/2 and/or Akt activity in human cancer cells cultured in vitro, and/or a reduction and/or reduction of p38 protein activity, then the Adjuvants are either useless or harmful.

In one embodiment of the present invention, the human cancer cells include human breast cancer, human prostate cancer, human leukemia, human gastric cancer, human liver cancer, human ovarian cancer, human rectal cancer and/or human colon cancer cells.

In one embodiment of the present invention, the benefit means that it is beneficial to cancer treatment, and means that when the substance is taken in combination, it can reduce the dosage of the drug and/or enhance the effect of the drug.

The sixth object of the present invention is to provide a cell method for analyzing the effects of adjuvants on cancer cells, which is to add n-3PUFA alone and in combination with rapamycin at different concentrations to human cancer cells being cultured in vitro , to analyze the effect of the adjuvant on the apoptosis and/or proliferation of the cancer cells cultured in vitro, so as to determine the effect of the adjuvant on the cancer cells cultured in vitro.

In one embodiment of the present invention, the individual n-3 polyunsaturated fatty acids (mainly including ALA, EPA and DHA) concentration (80 μ M), the single rapamycin drug (mainly including rapamycin and its derivatives) ) concentration (8μM), and rapamycin drug (4μM) combined with n-3 polyunsaturated fatty acid (40μM), respectively added to human cancer cells being cultured in vitro, at a certain time point (such as 6h, 12, 24h , 48h, 72h, etc.) to analyze the apoptosis and/or proliferation of auxiliary agents (food and/or nutritional supplements) on cancer cells cultured in vitro by detecting cell number, shape, DNA fragments, and nuclear rupture and concentration, thereby It is analyzed whether the adjuvant can assist the rapamycin drug to exert a synergistic effect.

In one embodiment of the present invention, cell apoptosis can be determined by TUNEL, nuclear fluorescent staining DAPI method, real-time quantitative PCR detection, the expression of anti-apoptosis gene Bcl-2, Bid after the combined action of fatty acid and rapamycin There was a significant decrease, and the pro-apoptotic genes Bax, Bak, Bim, Bim-EL, Puma, and Noxa had a significant increase after the combined action of fatty acids and rapamycin.

In one embodiment of the present invention, said cell proliferation and/or growth is detected by CCK8 method. The change of cell number can be directly counted by the microscope hemocytometer method, and the cell morphology can be observed by staining microscope.

The seventh object of the present invention is to provide a pharmaceutical auxiliary, which is n-3 polyunsaturated fatty acid or a substance containing n-3 polyunsaturated fatty acid.

In one embodiment of the present invention, the substance containing n-3 polyunsaturated fatty acid is a food containing n-3 PUFA and/or a nutritional supplement containing n-3 PUFA.

In one embodiment of the present invention, the drug adjuvant is an adjuvant of rapamycin drugs.

Beneficial effects of the present invention:

(1) The antitumor pharmaceutical composition of the present invention selects rapamycin or its derivatives and n-3 polyunsaturated fatty acids as main components. Through the synergistic effect of anti-tumor active ingredients, better therapeutic effect is achieved. The combined effect of the present invention for treating tumors has a guiding effect on treatment or prevention.

(2) In the present invention, n-3PUFA is added to human cancer cells being cultured in vitro at different concentrations alone or in combination with rapamycin, and the cell growth is detected at different time points, and the cells that significantly inhibit cell proliferation and promote cell death are screened out. The concentration of the individual n-3PUFA and rapamycin drug; and then the concentration of the two is halved as the synergistic concentration, and it can be clearly concluded that the synergy has better inhibitory effect on cells than adding substances and rapamycin drugs alone. proliferation and promote cell death.

Description of drawings

Figure 1: Effects of different concentrations of fatty acids on cell growth;

Figure 2: Effects of different concentrations of fatty acids on cell proliferation;

Figure 3: Effects of different concentrations of rapamycin on cell growth;

Figure 4: Effects of different concentrations of rapamycin on cell proliferation;

Figure 5: Effects of fatty acids and rapamycin on cell growth;

Figure 6: Effects of fatty acids and rapamycin on cell proliferation;

Figure 7: Effects of fatty acids and rapamycin on apoptosis;

Figure 8: Detection of fatty acids and rapamycin on the level of apoptosis proteins;

Figure 9: Detection of fatty acids and rapamycin on the level of apoptosis genes;

Figure 10: The expression of ERK, Akt and p38 after fatty acid and rapamycin treatment of cells.

detailed description

Edible oils derived from animals or plants contain a large amount of various fatty acids, including fatty acids with different carbon chain lengths and degrees of saturation, such as saturated fatty acids and unsaturated fatty acids. They provide a substantial source of energy and/or nutrition for humans and/or animals. Among them, n-3 polyunsaturated fatty acids (n-3 PUFAs) are found in large quantities in oils derived from fish, especially wild species native to cold seawater. Farmed fish contain considerably lower levels of n-3PUFAs than wild fish. The main component of deep-sea fish oil is n-3PUFA, which belongs to unsaturated fatty acids in medium and long-chain fatty acids, and has a wide range of physiological activities and functions. n-3PUFA mainly regulates the occurrence of metabolic diseases by activating downstream ERK1/2, p38MAPK and other signaling pathways through non-specific receptors such as GPR120 (GPCRs family). Studies have shown that n-3 polyunsaturated fatty acids are generally beneficial to human health. It has been found that it has good anti-cardiovascular disease and anti-fatty liver effects, and blood lipid-lowering drugs have been developed.

The invention uses n-3PUFA to assist rapamycin to treat various cancers, and the combination of active functional food components and medicine can obtain satisfactory therapeutic effect.

Next, the in vitro cell experiment takes cancer cells as an example, which can prove that the pharmaceutical composition has a good synergistic effect: n-3PUFA is added to human cancer cells being cultured in vitro at different concentrations alone and in combination with rapamycin. Cell growth was detected at different time points, and the concentrations of individual n-3PUFA and rapamycin drugs that significantly inhibited cell proliferation and promoted cell death were screened out; Synergistically, it has a better effect of inhibiting cell proliferation and promoting cell death than adding substances and rapamycin alone.

Embodiment 1: The growth effect of n-3 polyunsaturated fatty acids on breast cancer cells

The inhibition of EPA and DHA on the growth and proliferation of cancer cells has been confirmed by many studies. However, there is no clear conclusion about the effect of ALA on breast cancer cells. The present invention is verified by two experimental methods from two aspects of growth and proliferation.

1. Take cancer cells in the logarithmic growth phase, inoculate them in 6-well culture plates (2×10 ⁵ cells/well), incubate at 37°C in 5% CO ₂ , and add concentration gradients (10 μM, 20 μM , 40 μM, 80 μM) of EPA, DHA and ALA, observed under a microscope after incubation for 24 hours, and took pictures.

2. Cell proliferation experiment: CCK-8 experiment, the specific method is as follows:

(1) Collect the logarithmic phase cells, adjust the concentration of the cell suspension, add 100ul to each well of a 96-well plate, and plate to adjust the density of the cells to be tested to 1000-10000 wells, (the edge wells are filled with sterile PBS).

(2) 5% CO ₂ , incubate at 37° C., add drug with concentration gradient after the cells adhere to the wall, 1 ul per well, and set 5 duplicate wells.

(3) After incubation for 24 hours, add 10 microliters of CCK-8 solution to each well. The wells added with the corresponding amount of cell culture medium, drug and CCK-8 solution but without cells were set as blank control.

(4) Continue to incubate for 1 hour in the cell culture incubator. Absorbance was measured at 450 nm and a dual wavelength measurement was performed using 650 nm as a reference wavelength.

Figure 1 is a comparison of the changes in the microscopic morphology of cells after different concentrations of fatty acids acted on the cells. Figure 2 is the change of cell proliferation after the action of fatty acids corresponding to different concentrations. It can be seen from the results in Figure 1 that EPA, DHA and ALA all have different degrees of damage to the cells, and the number of cells is greatly reduced. , the number of cells is only about 1/2 of the initial amount. Among the three fatty acids, EPA has the most obvious effect. Figure 2 reflects the changes in the proliferation level of cells after treatment with different concentrations of fatty acids. It can be seen from the results that the three fatty acids have strong inhibitory effects on cell proliferation, among which EPA has the most obvious effect, which is consistent with the above results.

Embodiment 2: The effect of rapamycin on the growth of cancer cells

Rapamycin is used as a breast cancer drug in clinical trials, and its apoptosis effect on breast cancer cells has been clearly confirmed. The present invention is verified by two experimental methods from two aspects of cell growth and proliferation.

1. Take cancer cells in logarithmic growth phase, inoculate in 6-well culture plate (2×10 ⁵ cells/well), incubate at 37°C in 5% CO ₂ , add concentration gradient (1 μM, 2 μM, 4 μM, 8 μM) of rapamycin, observed under a microscope after incubation for 24 hours, and photographed.

2. Cell proliferation test method: consistent with the above.

Figure 3 is a comparison of the changes in the microscopic morphology of the cells after different concentrations of rapamycin acted on the cells. It can be seen from the results in Figure 3 that rapamycin can inhibit the proliferation of cancer cells to a certain extent, and when the drug concentration reaches 8 μM, the proliferation inhibition is more obvious and the number of cells decreases. Figure 4 reflects the changes in cell proliferation levels after treatment with different concentrations of rapamycin. It can be seen from the results that rapamycin has a strong inhibitory effect on cell proliferation, and the concentration reaches 8 μM, which is significant, and this result is consistent with the above results.

Example 3: The effect of polyunsaturated fatty acid combined with rapamycin on the growth of cancer cells

As mentioned above, fatty acids and rapamycin alone have been clearly confirmed to inhibit the proliferation of breast cancer cells. The toxic side effects and drug resistance of rapamycin have become important factors affecting its anti-tumor effect in clinical practice. . Therefore, the present invention proposes a new point of view, whether the combination of the two can play a synergistic effect, thereby reducing the effective therapeutic concentration of fatty acids and rapamycin. The present invention is verified by two experimental methods from two aspects of cell growth and proliferation.

1. Take cancer cells in the logarithmic growth phase, inoculate them in 6-well culture plates (2×10 ⁵ cells/well), incubate at 37° C. in 5% CO ₂ , and add individual fatty acids (EPA, DHA, ALA) concentration (80 μM), rapamycin drug concentration alone (8 μM), and rapamycin drug (4 μM) combined with fatty acid (40 μM), were observed under a microscope after incubation for 24 hours, and photographs were taken.

2. Cell proliferation test method: consistent with the above.

Figure 5 is a comparison of the changes in the microscopic morphology of cells after fatty acids and rapamycin act alone and in combination. As can be seen from the results in Figure 5, fatty acids (80 μM) and rapamycin (8 μM) alone have a certain reduction in the number of cancer cells, and half-concentration fatty acids (40 μM) and rapamycin combine to reduce the number of cells more (4 μM ). Figure 6 reflects the changes in the proliferation level of cells after fatty acids and rapamycin acted on cells alone or in combination. It can be seen from the results that the combination of half-concentration fatty acid and rapamycin has a significantly stronger inhibitory effect on cell proliferation than that of fatty acid and rapamycin alone, which is consistent with the above results. The above data indicate that fatty acids and rapamycin can play a synergistic effect, which can reduce the effective therapeutic concentration of fatty acids and rapamycin.

Example 4: Apoptotic effect of fatty acid combined with rapamycin on cancer cells

When fatty acid and rapamycin acted on cells separately or in combination, cell growth and proliferation were inhibited, and the effect of the combination of the two on cell apoptosis was further verified. The present invention is verified by three experimental methods related to apoptosis.

1. DPAI staining method

a) The solution is prepared as follows:

(1) 0.01mol/L (pH7.0): Take 0.1mol/L NaH ₂ PO ₄ ·H ₂ O 34ml, 0.1mol/L Na ₂ HPO ₄ 66ml, NaCl 0.9g, dissolve in 900ml double distilled water ;

(2) DAPI storage solution: dissolve 0.5 mg DAPI in 5.0 ml, subpackage, and store at low temperature for a long time;

(3) DAPI working solution: dilute the DAPI stock solution with a final concentration of 0.1ug/ml.

b) The specific dyeing method is as follows:

(1) Inoculate breast cancer cells in the logarithmic growth phase, incubate at 37°C in 5% CO ₂ , add concentration-gradient drug concentration of fatty acids (EPA, DHA, ALA) (80 μM) alone, and rapamycin alone Drug concentration (10 μM), and rapamycin drug (5 μM) combined with fatty acid (40 μM), after incubation for 24 hours, the culture medium was aspirated and washed once with PBS.

(2) Fix with 4% paraformaldehyde at room temperature for 15 minutes, rinse with PBS three times, two minutes each time;

(3) DAPI staining solution was incubated at room temperature for 30 minutes, wrapped in tinfoil, rinsed with PBS three times, two minutes each time;

(4) Observation under a fluorescence microscope, and use UV band excitation to take pictures.

Figure 7 reflects the apoptotic DAPI staining of cells after fatty acids and rapamycin acted on cells alone or in combination. It can be seen from the results that after the combined action of half-concentration fatty acid and rapamycin, the fluorescence intensity of nuclear DAPI staining is significantly higher than that of fatty acid and rapamycin alone. and the role of rapamycin.

2. Western Blot (protein immunoblotting) to detect the level of PARP protein

a) Solution preparation:

(1) 10% (w/v) sodium dodecyl sulfate SDS solution: 0.1 g SDS, 1 ml H2O deionized water, stored at room temperature.

(2) Separating gel buffer: 1.5mmol/LTris-HCL (pH8.8): Dissolve 18.15g Tris with 80ml of water, adjust the pH to 8.8 with HCL, and dilute with water to a final volume of 100ml.

(3) Stacking gel buffer: 0.5 mmol/LTris-HCL (pH6.8): 6.05 g Tris was dissolved in 80 ml of water, adjusted to pH 6.8 with about HCL, and diluted with water to a final volume of 100 ml.

(4) SDS-PAGE loading buffer: pH6.8 0.5mol/LTris buffer 8 ml, glycerol 6.4 ml, 10% SDS 12.8 ml, mercaptoethanol 3.2 ml, 0.05% bromophenol blue 1.6 ml, H2O 32 ml and mix well spare.

(5) Tris-glycine electrophoresis buffer: 30.3g Tris, 188g glycine, 10g SDS, dissolved in distilled water to 1000ml, diluted 10 times before use.

(6) Transfer buffer: Weigh 14.4g of glycine and 6.04g of Tris, add 200ml of methanol, and add water to a total of 1L.

(7) Tris buffered saline solution (TBS): 20mmol/LTris/HCL (pH7.5), 500mmol/LNaCl.

b) The specific steps are as follows:

The breast cancer cells were washed 3 times with PBS, added to the lysate, boiled for 5 minutes directly, cooled on ice, centrifuged at 12000 rpm for 2 minutes, the supernatant was taken, and stored at -20 for later use.

(1) SDS-PAGE gel electrophoresis

(1) After the cleaned glass plate is aligned, put it into the clamp and clamp it, and then vertically clamp it on the shelf to prepare for glue filling.

(2) Prepare 10% separating gel, add TEMED and shake well immediately to pour the glue, then seal it with ethanol liquid, pour off the ethanol on the top layer of the glue and dry it with absorbent paper after the glue is fully solidified.

(3) Add 5% stacking gel, shake well immediately after adding TEMED to fill the gel. Fill the remaining space with stacking gel and insert the comb into the stacking gel. After the concentrated gel has solidified, pull it out vertically and gently.

(5) Put it into the electrophoresis tank, add enough electrophoresis solution and start preparing for loading. After measuring the protein content of the protein sample, add 5×SDS loading buffer to a final concentration of 1×, cook in boiling water for 3 minutes, mix well and load the sample, the total protein amount is 35 μg.

(6) Electrophoresis run at constant voltage 80V. When the sample enters the lower gel, electrophoresis at constant voltage 120V until the bromophenol blue reaches the bottom of the gel, then transfer to the membrane.

(2) Transfer film:

(1) Gel cutting: Pry off the glass plate, remove the small glass plate, scrape off the concentrated gel, and cut the gel according to the molecular weight of the protein according to the experimental needs, using Marker as a control.

(2) Membrane preparation: cut PVDF membrane and filter paper, and place the cut PVDF in 80% methanol solution to activate for 30s.

(3) Film loading: Open the clamp for film transfer to keep the black side (negative electrode) horizontal. Put a sponge pad on it, add the transfer solution to soak it, put the soaked filter paper on the pad, and then stack it in sequence according to the order of gel-nitrocellulose membrane-filter paper-sponge pad. Finally, cover the white plate (positive electrode) and put it into the transfer film tank.

(4) Transfer film: Put the clip into the transfer tank, make the black side of the clip face the black side of the slot, and the white side of the clip face the red side of the slot. Transfer membrane at 4°C, constant current 400mA.

(5) Remove the membrane after the rotation, and mark a corner.

(3) Immune response

(1) Sealing: Rinse the membrane 3 times with TBST, 5 min each time. After rinsing, put the nitrocellulose membrane into 5% skimmed milk powder and shake it for 1 hour at room temperature.

(2) Add primary antibody: put the blocked nitrocellulose membrane into a washing tank containing TBST and rinse on a shaker for 3 times, 5 min each time. Incubate overnight at 4°C in a plate with primary antibody.

(3) Add secondary antibody: recover the primary antibody, rinse the nitrocellulose membrane in a TBST washing tank for 3 times on a shaker at room temperature, each time for 5 minutes, and then put the rinsed nitrocellulose membrane into the plate with the secondary antibody , and incubate at room temperature for 45 min in the dark. After incubation, the nitrocellulose membrane was washed 3 times in TBST tank, 5 min each time.

(4) Chemiluminescence

(1) Mix the two reagents A and B in a small centrifuge tube according to the instructions of the luminescence kit, add them to the nitrocellulose membrane, and develop the color with a chemiluminescence imager.

After the fatty acid and rapamycin acted on the cells alone or in combination, the total protein was extracted to detect the PARP protein level by Western Blot. Figure 8 shows that in different time periods (12h, 24h, 48h), the cleaved form of PARP has a significant increase when fatty acids and rapamycin are combined, and only EPA and rapamycin have cleaved forms of PARP at 24h However, at 48h, all three fatty acids combined with rapamycin showed obvious cleavage of PARP, which means that with the delay of time, it is verified that the combination of fatty acids and rapamycin has obvious apoptosis occur.

3. Detection of apoptosis-related gene levels by RT-PRC

a) Extraction of RNA

Add 1ml Trizol to a 35mm Petri dish and pipette repeatedly, then transfer the cell lysate to a 1.5ml Eppendorf tube and let it stand at room temperature for 5 minutes to fully lyse. Add 200 μl of chloroform per 1 ml of Trizol, vigorously shake and mix for 15 seconds, then place at room temperature for 3 minutes. This was followed by centrifugation at 12000 g for 15 minutes at 4°C. It can be seen that the upper layer is a colorless water phase, the middle mixed phase, and the lower layer is a red cool phase. Aspirate the upper aqueous phase and transfer to another new Eppendorf tube. Add 0.5 ml of isopropanol for every 1 ml of Trizol, mix it upside down, and let it stand at room temperature for 10 minutes. Then centrifuge at 12000g at 4°C for 10 minutes, discard the supernatant, and the RNA can be seen precipitated at the bottom of the tube. Add 1 ml of 75% ethanol for every 1 ml of Trizol and mix vigorously. Then centrifuge at 7500 g at 4°C for 5 minutes, and discard the supernatant. Dry the RNA pellet at room temperature or vacuum for 5-10 minutes. Finally, it was dissolved in 30 μl of DEPC-treated water and stored at -80°C until use. The purity and concentration of RNA were detected with a spectrophotometer at A260 and A260/280, respectively.

b) reverse transcription

The extracted RNA was reverse-transcribed into cDNA using TaKaRa's PrimescriptTM RT reagent kit. The reaction solution was prepared on ice.

Table 1 reaction system

The reverse transcription reaction conditions are: 37°C for 15 minutes (reverse transcription reaction); 85°C for 10 seconds (reverse transcriptase inactivation reaction)

c) Real-time quantitative PCR

Real-time quantitative PCR was performed with SYBR Green I fluorescent staining. The PCR reaction system is 20 μl, and the reaction solution is prepared on ice. When preparing the PCR reaction solution, avoid strong light and avoid contamination as much as possible.

Table 2 PCR reaction system

The amplification conditions were: pre-denaturation at 95°C for 30s, followed by denaturation at 95°C for 10 seconds, annealing/extension at 60°C for 30s, a total of 40 cycles, and the specificity of the amplified product was monitored by a melting curve. The mRNA expression of the target gene in each group was normalized by the mRNA expression level of β-actin, and then the ratio of the mRNA expression level of the target gene to the mRNA expression level of the control group was used to reflect the mRNA expression of the target gene. The experiment was repeated 3 times.

After fatty acids and rapamycin acted on the cells alone or in combination, RNA was extracted and RT-PRC was used to detect the levels of apoptosis-related genes. Figure 9 shows that the anti-apoptotic genes Bcl-2 and Bid were significantly decreased after the combination of fatty acid and rapamycin, and the There was a significant increase after the combination of pamycin. From the above results, it is further demonstrated that the combination of fatty acid and rapamycin can significantly promote cell apoptosis.

Example 5: The effect of fatty acid combined with rapamycin on AKT, ERK1/2, p38 signal

Akt, also known as PKB or Rac, plays an important role in cell survival and apoptosis. Growth and survival factors such as insulin can activate the Akt signaling pathway, and Ser473 of Akt can be phosphorylated by PDK1.

ERKs regulate the proliferation, differentiation and survival of cells, and are the downstream proteins of various growth factors (EGF, NGF, PDGF, etc.). It accepts a large number of signals from growth factors, mitogens, environmental stimuli, etc. On the other hand, it acts on nuclear transcription factors such as AP-1, NF-кB, etc. through the ERK signaling cascade reaction to regulate gene expression. Excessive activation of ERK can be found in many human cancers (such as breast cancer, prostate cancer, ovarian cancer, leukemia, oral cancer, melanoma, etc.).

The p38 signaling pathway is an important branch of the MAPK pathway, which plays an important role in various physiological and pathological processes such as inflammation, cell stress, apoptosis, cell cycle and growth. The 4 known isoforms of p38 include p38α, p38β, p38γ and p38δ. Over the years it has been found that the p38MAPK pathway can be activated by stress including hypertonicity, heat shock, radiation and other stress responses. Therefore, the p38MAPK pathway is involved in a variety of stimuli-induced signaling cascades, indicating that it plays an important role in eliciting a variety of cellular responses, and p38 also exhibits regulatory effects in apoptosis.

The present invention adopts the method of western blotting to verify the AKT, ERK1/2, p38 signal pathway.

Figure 10 shows the changes in the phosphorylation levels of AKT, ERK1/2, and p38 signals after fatty acids and rapamycin were combined to act on cells. It can be seen from Figure 9 that after fatty acid treatment, the activity of p38 was up-regulated, and the activities of AKT and ERK1/2 were decreased.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (14)

1. a pharmaceutical composition, it is characterised in that described pharmaceutical composition is with A and B as main active component；Wherein A is thunder Handkerchief mycin drug, B is n-3 polyunsaturated fatty acid (n-3PUFA).

Pharmaceutical composition the most according to claim 1, it is characterised in that described n-3 polyunsaturated fatty acid includes α-Asia Fiber crops acid ALA and/or eicosapentaenoic acid EPA and/or docosahexenoic acid DHA.

Pharmaceutical composition the most according to claim 1, it is characterised in that described n-3 polyunsaturated fatty acid include containing The food of n-3PUFA and/or the supplementary containing n-3PUFA.

4. according to the arbitrary described pharmaceutical composition of claim 1-3, it is characterised in that described rapamycin class medicine includes thunder Handkerchief mycin and the derivant of rapamycin.

Pharmaceutical composition the most according to claim 1, it is characterised in that in described pharmaceutical composition, the mol ratio of A and B is 2:1～1:20.

Pharmaceutical composition the most according to claim 3, it is characterised in that the described food containing n-3PUFA mainly includes Fish oil.

7. according to the arbitrary described pharmaceutical composition of claim 1 or 5, it is characterised in that A and B in described pharmaceutical composition Mol ratio is 1:10.

Pharmaceutical composition the most according to claim 1, it is characterised in that in described drug regimen, A, B are individually or/and tie Composite solid state and/or solvate.

9. one kind for suppression and/or is prevented and/or the pharmaceutical composition for the treatment of tumor, it is characterised in that described compositions is with A It is main active component with B；Wherein A is suppression and/or prevents and/or the medicine for the treatment of tumor, and B is n-3 polyunsaturated fat Acid.

10. according to the pharmaceutical composition described in claim 9, it is characterised in that described tumor include following any one or many Kind: breast carcinoma, carcinoma of prostate, leukemia, gastric cancer, hepatocarcinoma, ovarian cancer, colon and rectum carcinoma.

11. 1 kinds are used for treatment or/and suppression is or/and prevent the pharmaceutical composition of human breast cancer cell, it is characterised in that described Compositions is with A and B as main active component；Wherein A is suppression and/or prevents and/or the medicine for the treatment of human breast cancer cell, B For n-3 polyunsaturated fatty acid；Described human breast cancer cell include MCF-7 Human Breast Cancer Cells, T47D, SUM185, BT474, BT-483、600MPE、ZR-75、MDA-MB-468/453/231。

12. 1 kinds are used for strengthening and/or improve p38 protein active in tumor cell, reduce and/or suppress Erk1/2 protein Activity and/or the pharmaceutical composition of Akt protein active, it is characterised in that described pharmaceutical composition is with A and B as chief active Component；Wherein A is rapamycin class medicine, and B is n-3 polyunsaturated fatty acid.

13. 1 kinds of pharmaceutical auxiliaries, it is characterised in that described adjuvant be n-3 polyunsaturated fatty acid or containing n-3 many not The material of satisfied fatty acid.

14. adjuvant according to claim 13, it is characterised in that described adjuvant is the auxiliary of rapamycin class medicine Agent.