CN106806362A - Application of the dehydrogenation rush diphenol in antineoplastic sensitizer is prepared - Google Patents

Abstract

The invention belongs to the fields of cell biology and medicine, and relates to the application of dehydrorurgediol in the preparation of antitumor drug sensitizers. Dehydrorurgediol is a natural product, which has the function of reversing the multidrug resistance of tumor cells, and can be used as a reversal agent for tumor multidrug resistance; dehydrorurgediol also has the ability to increase the sensitivity of tumor multidrug resistant cells to antitumor drugs It can be used as a chemosensitizer. The invention also provides a method for inhibiting tumor multidrug-resistant cell proliferation with the pharmaceutical composition combined with antineoplastic drugs and dehydrorurgediol. The small-molecule compound dehydrojuicediol in the present invention is developed as a new anti-tumor drug or its auxiliary component, has obvious tumor-suppressing effect, is environmentally friendly, and will provide a new way and means for treating and curing tumors.

Description

Application of dehydrorurgediol in preparation of antitumor drug sensitizer

technical field

The invention belongs to the fields of cell biology and medicine, and specifically relates to a new application of dehydrorurgediol. The invention also relates to the corresponding pharmaceutical composition and its application method.

Background technique

Tumor is an abnormal lesion formed by the body under the action of various carcinogenic factors when a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in its clonal abnormal proliferation. The number of cancer cases in my country is quite large, accounting for 55% of the total number of cases in the world.

Benign tumors have less impact on the body, mainly showing symptoms of local compression and obstruction. Malignant tumors have a serious impact on the body due to their immature differentiation, rapid growth, infiltration and damage to the structure and function of organs, and the possibility of metastasis. In addition to local compression and obstruction symptoms similar to those of benign tumors mentioned above, malignant tumors may also cause fever and intractable pain, and severe emaciation, fatigue, anemia, and systemic exhaustion may appear in the advanced stage.

Traditional Chinese medicine believes that the cause of cancer is the balance of yin and yang in the human body, and the tissue cells are caused by cell mutations under the long-term action of different carcinogenic factors. In fact, cancer tissue is also a part of the human body. Only when the balance of yin and yang is out of balance and the five elements change, the immune monitoring system of the human body will lose its monitoring and let it develop. Chinese herbal medicine can preserve life by regulating qi and blood, adjusting the balance of yin and yang, and maintaining normal vital signs; Therefore, Chinese herbal medicine extracts have become an important direction for the treatment of tumors.

Dehydrorurgediol is extracted from the Chinese herbal medicine rush. Juncus effusus (scientific name: Juncus effusus L) is a perennial herbaceous aquatic plant, with short underground stems, prostrate, erect, cylindrical, solid, brown at the base of the stem, degenerated scale-like sheath leaves, spikes, terminal, in The stem is pseudolateral, the bracts at the base extend into a stem shape, there are 2 bracteoles under the flower, 6 perianth lobes, 3 stamens, and 3 divergent stigmas of pistil. Brown-yellow capsule, ovate or elliptic, yellow seed obovate. Rush rush is also a medicinal plant. Its stem marrow or whole herb is used as medicine to clear away heat, diuresis and dampness. It can be used for gonorrhea, edema, restlessness, insomnia, sore throat and trauma.

Dehydrorurgediol is a natural product with high bioavailability and relatively stable properties, and has clinical application value. At present, there are few reports on the chemical structure of dehydrorurgediol, and even fewer studies on the biological activity of the system. With the deepening of its chemical and biological research, its molecular mechanism of action will be gradually clarified, which will further promote the chemical structure modification and structure-activity relationship research of such compounds, and help to improve the medicinal value of rushes.

Contents of the invention

The purpose of the present invention is to provide a new medicinal application of dehydrorucorbiol.

The object of the present invention is to provide an antitumor drug sensitizer.

On the one hand, the present invention provides the application of dehydrorurgediol in the preparation of antitumor drug sensitizers. Among them, the full name of dehydroeffusol in English is dehydroeffusol, and its structure is shown in Figure 1.

Said tumors include oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer or pancreatic cancer.

The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

Dehydrojuicediol can be used as a tumor multidrug resistance reversal agent.

On the other hand, the present invention provides an anti-tumor pharmaceutical composition, the active ingredients of which are anti-tumor drugs and dehydrorurgediol.

The anti-tumor drugs are cell cycle specific drugs or cell cycle non-specific drugs.

The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

The present invention also provides a method for inhibiting the growth and proliferation of tumor cells in vitro, that is, adding dehydrourgeryl and antitumor drugs to the medium of tumor cells.

Wherein, the final concentration of adding dehydrorurgediol is 1-100 μ mol/L.

The tumor includes oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer or pancreatic cancer.

When implementing the above method, first add dehydrorurgediol, and then add antineoplastic drugs. It is also possible to add dehydrorurgediol and antineoplastic drugs at the same time.

The present invention provides a new application of dehydrorurgediol, that is, its application in the preparation of antitumor drug sensitizers. Dehydrorurgediol is a natural product, which has the function of reversing the multidrug resistance of tumor cells, and can be used as a reversal agent for tumor multidrug resistance; dehydrorurgediol also has the ability to increase the sensitivity of tumor multidrug resistant cells to antitumor drugs It can be used as a chemosensitizer. The small molecular compound dehydrorurgediol in the present invention is developed as a new anti-tumor drug or its auxiliary component, has obvious tumor-suppressing effect, is green and environment-friendly, and will provide a new approach and means for treating and curing tumors.

Description of drawings

Fig. 1 is the structure of dehydrorurgediol.

detailed description

The invention provides an antineoplastic drug, wherein the active ingredient of the antineoplastic drug is dehydrorurgediol. The tumor can be liver cancer cells, gastric cancer cells, cervical cancer cells or blood cancer cells.

The small molecule compounds of the present invention can be prepared by various conventional preparation methods. For example, the method of artificial chemical synthesis is adopted.

Utilizing the small molecular compound of the present invention, through various conventional screening methods, substances that interact with dehydrorurgediol, such as receptors, inhibitors or antagonists, can be screened out.

When the present invention and its inhibitors, antagonists, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, and the pH value can be formulated according to Depending on the nature of the substance and the condition being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the dehydrorurgediol of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The dehydrorurgediol of the present invention can be prepared in the form of injection, for example, by normal methods using physiological saline or aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the dehydrorurgediol of the present invention can also be used together with other therapeutic agents. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dose, which are within the skill of skilled physicians.

experimental method:

1. Cell Recovery

1) Take out the cryotube from the liquid nitrogen tank, put it directly into warm water at 37°C, and shake it from time to time to melt it as soon as possible.

2) Take out the cryotube from the 37°C water bath, suck out the cell suspension with a pipette, pour into the centrifuge tube and add more than 10 times of culture medium, mix and centrifuge at low speed, discard the supernatant, and wash again with culture medium again.

3) After properly diluting with the culture medium, inoculate the culture bottle, place it in a 37°C incubator for static culture, replace the culture medium the next day, and continue the culture. Subculture when cultured to a certain concentration. PANC-1 cells were cultured in DMEM high-glucose medium containing 10% Gibico fetal bovine serum; K562, HGC, QGY, MCF-7, PC-3 and other cells were cultured in 1640 medium containing 10% fetal bovine serum. SK-hep1, HeLa, HepG2, and other cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, and the medium contained 100U/ml penicillin and 100μg/ml streptomycin.

2. Subculture of cells

Observe the growth of the cells every day. When the cells grow to about 90% confluence in the culture flask, they should be subcultured every 2-4 days. Passage one bottle into three flasks, or one 25 cm ² into one 75 cm ² culture flask. method:

1) Wash the cells once with 1X phosphate buffer.

2) Add 2-3ml trypsin digestion solution for digestion, and place in a 37°C incubator for several minutes. Tap the cell culture flask with your hands to detach the cells.

3) Terminate trypsinization with appropriate medium containing 10-15% Gibico fetal bovine serum. Aliquot the cells into new culture flasks and continue culturing.

When the suspension cells are subcultured, they are directly collected in a centrifuge tube and centrifuged, and the old medium is discarded. Generally, the ratio of one bottle to three bottles is divided into new culture bottles, and fresh medium is added to continue the culture.

3. Cell cryopreservation

1) Digest the cells cultured to the logarithmic growth phase with trypsin, collect them in a centrifuge tube, count them, and centrifuge.

2) Discard the trypsin and the old culture medium, add the prepared frozen culture medium (containing 10% DMSO, 40% DMEM and 50% Gibico fetal bovine serum), the final concentration of cells in the frozen medium is 0.5-1 ×10 ⁷ /ml. Gently pipette with a pipette to make the cells uniform, then divide into sterile cryopreservation tubes, add 1 -1.5ml to each tube.

3) Put the cryopreservation tube into the freezer box and place it at -80°C for quick freezing, and transfer it to a liquid nitrogen tank for storage after 5 hours.

4. Experimental materials

Drug preparation:

Dehydrorurgediol was dissolved in DMSO (dimethyl sulfoxide), and prepared into 100mM or 50mM mother solution for later use.

Dehydrorurgediol can be purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. (CAS No.: 137319-34-7).

The structure of dehydrojuicediol is shown in Figure 1.

Verapamil (VPL), vincristine (VCR) and doxorubicin (ADR) were purchased from Roche Chemical Company with a purity greater than 99%.

Cell source:

Human oral cancer cell line KB and its drug-resistant cell line KB/VCR were provided by the Chinese Academy of Sciences, human breast cancer cell line MCF-7 and its drug-resistant cell line MCF-7/ADR, human colon cancer HCT-8 and its drug-resistant cell line Drug cell line HCT-8/VCR were purchased from Nanjing Kaiji Biological Company.

Reagent test kit:

CCK-8 kit was purchased from Tongren Company.

CCK-8 experiment

KB and KB/VCR cells as well as MCF-7 and MCF-7/ADR cells were inoculated into 96-well plates at a density of 3500/well, and after 24 h, different concentrations of ADR, VCR, dehydrorurgediol, VCR and Hydrogen-rurgediol was added to each well after being prepared together in α-MEM containing 10% fetal bovine serum. After culturing for 48 h, the culture medium was discarded, and 90 μL of serum-free medium and 10 μL of CCK-8 reagent were added to each well. After reacting at 37°C for 2 h, the absorbance value (OD450) at a wavelength of 450 nm was read with a microplate reader. The cell proliferation ratio of the medication group relative to the control group was obtained by calculation. In the blank group, only the medium was added without cells, and in the control group, DMSO with the same volume as the drug was added, and the cell survival rate = (OD450 of the experimental group-OD450 of the blank group)/(OD450 of the control group-OD450 of the blank group). The resistance fold (Resistance Fold, RF) was calculated based on the IC ₅₀ value.

RF = IC ₅₀ value of drug-resistant cell line/IC ₅₀ value of parental cell line. Three replicate wells were set up for each concentration, and the experiment was repeated 3 times.

All drug-resistant cell lines were grown in drug-free medium for 3 days prior to growth inhibition experiments. Each value is the result of 3 independent experiments, and IC ₅₀ is expressed in the form of "mean ± standard deviation". VCR, vincristine; ADR, daunorubicin; RF, resistance fold.

Example 1

Experimental results:

KB/VCR and MCF-7/ADR are two commonly used multidrug-resistant cell lines, and in this example, these two kinds of cells also exhibit the characteristic of multidrug resistance. As shown in Table 1, compared with KB cells, the multiples of drug resistance of KB/VCR cells to VCR and ADR were 81.9 times and 94.4 times, respectively, while MCF-7/ADR cells were more resistant to VCR and ADR than MCF-7 cells. The drug multiples were 38.1 times and 20.9 times respectively, showing that the drug-resistant cells used in the experiment had multidrug resistance, and the MDR activity was similar to the results reported in the literature.

The half maximal inhibitory concentration IC ₅₀ of dehydrorurgediol to the parental cell lines KB and MCF-7 were 223.1 μM and 189.6 μM, respectively, and the IC ₅₀ to the drug-resistant cell lines KB/VCR and MCF-7/ADR were 232.7 μM and 232.7 μM, respectively. 196.4 μΜ, there was no significant difference between the two, and the multidrug-resistant cell lines KB/VCR and MCF-7/ADR did not show cross-resistance to the compound dehydrorurgediol, indicating that dehydrorurgediol can escape Multidrug resistance in resistant cells. At a concentration below 20 μM, dehydrorurgediol has no obvious inhibition on cell growth, and at a concentration higher than 100 μM, it can inhibit cell proliferation.

The experimental results show that: dehydrorurgediol can overcome the drug resistance of drug-resistant cell lines, and the sensitivity of drug-resistant cell lines to dehydrorurgediol is basically the same.

Example 2: CCK-8 method detects the reversal effect of dehydrorurgediol on the multidrug resistance activity of tumor cells

KB/VCR, MCF-7/ADR and HCT-8/VCR cells were inoculated into 96-well plates at a density of 3500/well, and after 24 hours, different concentrations of VCR, and different concentrations of VCR and dehydrorurgediol were used together α-MEM containing 10% fetal bovine serum was prepared and added to each well. After culturing for 48 h, the culture medium was discarded, and the CCK-8 kit was used to measure the activity of the drug-resistant cell line and its parental cells to VCR and VCR+dehydrorurgediol in the same manner as in Example 1, and a curve was drawn. Three replicate wells were set for each concentration, and the experiment was repeated 3 times.

Experimental results:

The multiplicity of drug resistance of KB/VCR cells to VCR was 81.9 times, and that of MCF-7/ADR cells to ADR was 20.9 times. After adding 10 μM dehydrorurgediol, the sensitivity of KB/VCR cells to VCR was increased by 6.82 times, while the sensitivity of ADR to drug-resistant cells MCF-7/ADR was increased by 7.10 times. Dehydrorurgediol can reverse the drug resistance of tumor multidrug resistant cells to chemotherapy drugs. However, this effect was not obvious in the parental cells.

The experimental results show that dehydrorurgediol can reverse the drug resistance of tumor multi-drug resistant cells KB/VCR and MCF-7/ADR, and can be used as a sensitizer for anti-tumor drugs, or it can be used as a drug combination with anti-tumor drugs thing.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. Application of dehydrorurgediol in the preparation of antitumor drug sensitizers. 2. The application according to claim 1, characterized in that, dehydrorurgediol is a tumor multidrug resistance reversal agent. 3. The application according to claim 1, wherein the tumor comprises oral cavity cancer, nasopharyngeal cancer, breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer or pancreatic cancer. 4. The application according to claim 1, wherein the antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil. 5. An anti-tumor pharmaceutical composition, characterized in that the active ingredients of the anti-tumor pharmaceutical composition are anti-tumor drugs and dehydrorurgediol. 6. The antitumor drug composition according to claim 5, wherein the antitumor drug is a cell cycle-specific drug or a cell cycle non-specific drug. 7. The antitumor drug composition according to claim 5, wherein the antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil. 8. A method for inhibiting the growth and proliferation of tumor cells in vitro, characterized in that dehydrorurgediol and antitumor drugs are added to the medium of tumor cells. 9. The method according to claim 8, wherein the tumor comprises oral cancer, breast cancer, nasopharyngeal cancer, liver cancer, lung cancer, cervical cancer or pancreatic cancer. 10. The method as claimed in claim 8, characterized in that first adding dehydrorurgediol, and then adding antineoplastic drugs.