CN105168241A - Application of hydrogen sulfide to increasing tumor inhibition rate of anti-tumor drug - Google Patents

Abstract

The invention discloses the application of hydrogen sulfide in improving the tumor inhibition rate of anti-tumor drugs and the application in preparing synergists of anti-tumor drugs. The invention also discloses a method for increasing the tumor inhibition rate of antitumor drugs, the method comprises administering antitumor drug synergists to cancer cells before or at the same time of treatment with antitumor drugs. The synergist in the present invention uses hydrogen sulfide, soluble hydrogen sulfides or soluble sulfides as active ingredients. After the synergist acts on tumor cells in the body, it can significantly improve the tumor-inhibiting effect of anti-tumor drugs. The side effects are very small and negligible, which greatly reduces the side effects on normal cells.

Description

Application of Hydrogen Sulfide in Improving the Inhibition Rate of Antitumor Drugs

technical field

The invention relates to the technical field of medicine, in particular to the application of hydrogen sulfide in improving the tumor inhibition rate of antitumor drugs and a method for increasing the tumor inhibition rate of antitumor drugs.

Background technique

For tumor/cancer patients, there are no more than three treatment methods: surgery, radiotherapy and chemotherapy. The scope of application of surgery is relatively narrow, and it is generally only suitable for patients with benign tumors, and only unnecessary organs can be removed; radiotherapy and chemotherapy are the preferred options for most tumor/cancer patients.

Radiation therapy is a method of killing cancer cells by using the radiation energy of radiation (such as X-rays, gamma rays, and electron rays). High doses of radiation can directly act on DNA, causing breakage or cross-linking of DNA molecules; low doses of radiation can cause ionization of interstitial fluid and generate free radicals, which in turn damage biological macromolecules and cause cell death. The dose of radiation used in radiation therapy is generally low dose, mainly to kill cancer cells in an indirect way.

Chemotherapy is a method of using chemical drugs to kill cancer cells. There are many types of chemotherapy drugs, and most chemotherapy drugs, such as doxorubicin, mitomycin, 5-fluorouracil, etc., will also form free radicals in the body after being applied to specific parts, and kill cancer cells through the action of free radicals (Free Radical Biol. Med. 1990, 8:567).

Free radicals include a variety of components, among which the most reactive is the hydroxyl radical (HO ), which mainly comes from the Fenton reaction:

Fe ²⁺ +H ₂ O ₂ +H ⁺ →HO·+H ₂ O+Fe ³⁺

Since there are many iron-containing proteins in cells, Fe ²⁺ will be released after being attacked by radiation or chemotherapy drugs, and then react with H ₂ O ₂ to generate HO·. Although HO· has a short lifetime, its chemical reactivity is extremely high, and it can react with almost all macromolecules (such as proteins, DNA, RNA, and lipids) in cells, and oxidize these biomacromolecules. For example, HO can react with amino acid residues to cause carbonylation of molecules. Carbonylation is an irreversible process that not only causes protein fragmentation to form small peptides, but also further derivates to form toxic proteins. HO· also oxidizes lipids, turning unsaturated lipids into polar peroxidized lipids, thereby increasing membrane fluidity, resulting in loss of cell contents and inactivation of membrane proteins. The DNA damage caused by intracellular H ₂ O ₂ is also related to the Fenton reaction. Since ferrous iron is easily combined with the negatively charged phosphate skeleton on DNA, when there is H ₂ O ₂ in the cell, it will bind to the Fe bound to DNA. ²⁺ undergoes a Fenton reaction to produce HO·, thereby destroying the structure of DNA, and even breaking DNA in severe cases.

In order to fight against hydrogen peroxide and free radicals imposed by the outside world or produced by its own metabolism, cells have evolved a variety of response pathways. One of the most important pathways is the production of hyperactive/excessive amounts of catalase. For example, the catalase and superoxide dismutase activities of colon cancer cells are 2-3 times higher than those of the surrounding mucosal cells (Mekhailetal, Cancer Res. 1989, 49:4866). To this end, many drug research and development institutions have been working on the development of anticancer drug synergists.

The invention patent application document with the publication number CN101583366A discloses a mixed preparation of hydrogen peroxide and hyaluronic acid, which can be used to enhance the curative effect of anticancer drugs. The principle is to use the oxidation synergy of hydrogen peroxide and anticancer drugs to kill cancer cell. Although such synergistic processing can improve the efficacy of anticancer drugs, its side effects will also be enhanced, so hyaluronic acid needs to be added to strengthen the protection of normal cells.

Therefore, it is necessary to explore a new anticancer drug synergist to solve the above-mentioned problem of high side effects of anticancer drugs.

Contents of the invention

The present invention finds that hydrogen sulfide has a strong inhibitory effect on catalase, and applying anticancer drugs after treating cancer cells with a reagent containing hydrogen sulfide or capable of releasing hydrogen sulfide gas, or applying anticancer drugs while treating, can greatly Increase the death rate of cancer cells.

Based on the above findings, the present invention provides the application of hydrogen sulfide in improving the tumor inhibition rate of antitumor drugs.

Studies have found that hydrogen sulfide is a gaseous substance at room temperature, and can freely pass through the cell membrane into the cell, inactivating the catalase in the cell; and hydrogen sulfide is a normal gaseous signal molecule component of the human body, and the human blood contains about 60 μM Hydrogen sulfide can even be as high as 1mM in intestinal tissues. As long as hydrogen sulfide or hydrogen sulfide releasing reagents are selectively and directionally applied to tumor tissues, the side effects on normal cells in the body are almost negligible.

The hydrogen sulfide releasing reagent of the present invention refers to the antitumor drug synergist capable of releasing hydrogen sulfide gas; the anticancer drug or antitumor drug is a general term for antitumor/cancer.

The invention also provides the application of hydrogen sulfide in the preparation of antitumor drug synergists.

Specifically, the antitumor drugs include hydrogen peroxide, doxorubicin, mitomycin, 5-fluorouracil and pingyangmycin.

Specifically, the tumor is one of liver cancer, lung cancer, gastric cancer, skin cancer and esophageal cancer.

Preferably, the active ingredient of the antitumor drug synergist is at least one of hydrogen sulfide, soluble hydrogen sulfides, and soluble sulfides.

Preferably, the concentration of the active ingredient of the antitumor drug synergist is 0.01-5 mmol/L.

Preferably, the soluble hydrogen sulfide salts are NaHS or KHS.

Preferably, the soluble sulfide is Na ₂ S or K ₂ S.

The present invention also provides a method for increasing the tumor inhibition rate of antitumor drugs, comprising the following steps: administering synergists of antitumor drugs to cancer cells before or at the same time of treatment with antitumor drugs;

The active ingredient of the synergist is at least one of hydrogen sulfide, soluble hydrogen sulfides, and soluble sulfides. Preferably, the concentration of the active ingredient of the antitumor drug synergist is 0.01-5 mmol/L. More preferably, the concentration is 0.05-1.00 mmol/L.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention explores a new application for improving the tumor inhibition rate of antitumor drugs for the known compound hydrogen sulfide, and opens up a new application field.

(2) The synergist of the present invention uses hydrogen sulfide, soluble hydrogen sulfides or soluble sulfides as active ingredients to act on tumor cells in the body, which significantly improves the antitumor effect of antitumor drugs, and has very little side effects on normal cells in the body. Negligible, greatly reducing the side effects on normal cells.

(3) The synergist provided by the present invention can be produced industrially, and the agent can be used to prepare anti-tumor auxiliary drugs, reduce the dose of traditional chemotherapy or radiotherapy, and reduce the damage of normal cells in the patient's body.

Description of drawings

Fig. 1 is the synergistic inhibitory effect of potassium hydrosulfide and hydrogen peroxide on the growth of human lung cancer cell line A549 (relative inhibition rate, %);

A: 0.01 mM KHS; B: 0.2 mM KHS; C: 5 mM KHS; D: 0.2 mM H ₂ O ₂ ; E: 1 mM H ₂ O ₂ ; F: ₅ mM H ₂ O _{2 ;} mMKHS _{+ 1mMH2O2} ; I: 0.2mMKHS _{+ 0.2mMH2O2} ; J: 0.2mMKHS _{+ 1mMH2O2} .

Fig. 2 is the synergistic inhibitory effect of sodium sulfide and doxorubicin on the growth of human gastric cancer cell line SGC7901 (relative inhibition rate, %);

A: 0.05mM Na ₂ S; B: 0.2mM Na ₂ S; C: 1mM Na ₂ S; D: 0.2mg/mL doxorubicin; E: 1mg/mL doxorubicin; F: 5mg/mL doxorubicin; G: 0.05mMNa ₂ S+0.2mg/mL doxorubicin; H:0.05mMNa ₂ S+1mg/mL doxorubicin; I:0.2mMNa ₂ S+0.2mg/mL doxorubicin; J:0.2mMNa ₂ S+1mg /mL doxorubicin.

Figure 3 is the effect of sodium hydrosulfide on the activity of catalase in human gastric cancer cell line SGC7901.

Figure 4 is the effect of potassium hydrosulfide on the activity of catalase in mouse liver cancer cell line H22.

Figure 5 is the tumor inhibition rate of mouse sarcoma cells after co-treatment with sodium hydrosulfide and pingyangmycin.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific implementation examples.

Embodiments 1 to 4 are processed according to the following method (methylazotetrazolium salt (MTT) analysis method):

Methylazotetrazole solution: 250 mg of methylazotetrazole salt was dissolved in 50 mL of phosphate buffer (PBS, 0.01 mol/L, pH 7.4), filtered and sterilized, and stored in a refrigerator at 4°C.

Other materials: DMEM medium containing 10% fetal bovine serum, 0.25% trypsin, etc.

Methylazotetrazolium salt (MTT) analysis method: After 0.25% trypsin digests monolayer cultured cells, prepare single cell suspension with DMEM culture medium containing 10% fetal bovine serum, count and dilute to 10000 cells/ mL. In a 96-well culture plate, add 100 μL of single cell suspension to each well, transfer it into a CO ₂ incubator, and incubate for 72 hours at 37° C., 5% CO ₂ and saturated humidity. Then, carefully suck off the culture solution, add 100 μL of H ₂ S release reagent, treat at room temperature for 10 minutes, carefully suck off the solution, wash with distilled water, add 100 μL of hydrogen peroxide or anticancer drug solution, and incubate for 5 hours, and measure the cultured cells after washing with distilled water cell viability. Each experiment was repeated 3 times.

Determination of cell viability: Add 20 μL of methylazotetrazole solution to each well, react at 37° C. for 4 hours, absorb the solution, then add 150 μL of dimethyl sulfoxide (DMSO) to the well, and shake for 10 minutes to lyse the cells. The absorbance of each well was measured on a microplate reader at a wavelength of 570 nm, and the relative inhibition rate was calculated.

Relative inhibition rate = (control group A _{570 -experimental} group A ₅₇₀ )/control group A ₅₇₀

Example 1 Synergistic inhibition of growth of human lung cancer cell line A549 by potassium hydrosulfide and hydrogen peroxide

The in vitro growth characteristics of human lung cancer cell line A549 were tested by methylazotetrazolium salt (MTT) assay. It was found that potassium hydrosulfide treatment alone had no inhibitory effect on the growth of cancer cells, while hydrogen peroxide treatment alone had an inhibitory effect on cancer cells, and this inhibition was dose-related; The inhibitory effect on cancer cells is enhanced, and the synergistic inhibitory effect of the two is obvious (as shown in Figure 1).

Example 2 Synergistic inhibition of growth of human gastric cancer cell line SGC7901 by sodium sulfide and doxorubicin

The in vitro growth characteristics of the human gastric cancer cell line SGC7901 were tested using the methylazotetrazolium (MTT) assay. It was found that sodium sulfide treatment alone had no inhibitory effect on the growth of cancer cells, but doxorubicin alone had an inhibitory effect on cancer cells, and this inhibitory effect was dose-related; and adding doxorubicin after sodium sulfide treatment could significantly enhance the growth of cancer cells. The inhibitory effect on cancer cells, the synergistic inhibitory effect of the two is obvious (as shown in Figure 2).

Example 3 Synergistic inhibition of growth of mouse melanin skin cancer cell line B16 by sodium hydrosulfide and mitomycin

The in vitro growth properties of the mouse melanotic skin cancer cell line B16 were tested using the methylazotetrazolium salt (MTT) assay. It was found that sodium hydrosulfide treatment alone had no inhibitory effect on the growth of cancer cells, and mitomycin treatment alone had an inhibitory effect on cancer cells, and this inhibition was dose-related; while treatment with sodium hydrosulfide followed by mitomycin, It can significantly enhance the inhibitory effect on cancer cells, and the synergistic inhibitory effect of the two is obvious (see Table 1).

Table 1. Synergistic inhibition of sodium hydrosulfide and mitomycin on the growth of mouse melanin skin cancer cell line B16 (relative inhibition rate, %)

deal with Relative inhibition rate, % 0.1mM NaHS 0 0.4mM NaHS 0 2mM NaHS 12±6 10 μg/mL mitomycin 26±8 50 μg/mL mitomycin 61±15 200 μg/mL mitomycin 92±5 0.1mM NaHS+10μg/mL mitomycin 45±6 0.1mM NaHS+50μg/mL mitomycin 90±7 0.4mM NaHS+10μg/mL mitomycin 51±11 0.4mM NaHS+50μg/mL mitomycin 98±5

Example 4 Synergistic inhibition of growth of mouse liver cancer cell line H22 by potassium sulfide and 5-fluorouracil

The in vitro growth characteristics of the mouse hepatoma cell line H22 were tested using the methylazotetrazolium (MTT) assay. It was found that the treatment of potassium sulfide alone had no inhibitory effect on the growth of cancer cells, and the treatment of 5-fluorouracil alone had an inhibitory effect on cancer cells. This inhibitory effect was dose-related; The inhibitory effect on cancer cells, the synergistic inhibitory effect of the two is obvious (see Table 2).

Table 2. Synergistic inhibition of potassium sulfide and 5-fluorouracil on the growth of mouse liver cancer cell line H22 (relative inhibition rate, %)

deal with Measured after 30min 0.1mM K ₂ S 0 0.5mM K ₂ S 0 1 mM K ₂ S 0

10 μg/mL 5-fluorouracil 21±6 50μg/mL 5-fluorouracil 52±9 200μg/mL 5-fluorouracil 85±11 0.1mM K ₂ S+10μg/mL 5-fluorouracil 42±8 0.1mM K ₂ S+50μg/mL 5-fluorouracil 84±4 0.5mM K ₂ S+10μg/mL 5-fluorouracil 48±7 0.5mM K ₂ S+50μg/mL 5-fluorouracil 93±8

Embodiments 5-8 are processed according to the following method (xylenol alcohol (FOX1) analysis method):

Xylenol alcohol solution: Dissolve 76mg of xylenol alcohol and 18.217g of sorbitol in 1000mL of distilled water, add 1.33mL of sulfuric acid, mix well, dissolve in 100mg of ferrous ammonium sulfate, and store in a refrigerator at 4°C.

Other materials: DMEM medium containing 10% fetal bovine serum, 0.25% trypsin, etc.

Method: Cells were cultured according to the method of Examples 1-4. After culturing, suspend the cancer cells with a small amount of phosphate buffer to make 1×10 ⁶ cells/mL, and divide them into two parts, one part is treated with a certain concentration of hydrogen sulfide release reagent, and the other part is replaced by buffer solution (as a control). After being treated at room temperature for 10 min, centrifuged and washed, the cell pellet was ground in an ice bath, and after centrifugation, the upper layer of cell lysate (cancer cell extract) was collected to measure its catalase activity.

Determination of catalase activity: Add 1mL of 0.4mM H ₂ O ₂ (final concentration 0.2mM) and 0.2mL of cell treatment solution to the 2mL reaction system, make up to 2mL with phosphate buffer, react at 30°C for 30min, and pipette 0.2mL of the reaction solution solution, add 1.8mL xylenol alcohol solution, and measure the absorbance at 562nm after incubating at 30°C for 30min. The standard curve was made in the same way, and the consumed H ₂ O ₂ was found according to the standard curve, and the relative enzyme activity inhibition rate was calculated.

Relative inhibition rate = (control group A _{562 -experimental} group A ₅₆₂ )/control group A ₅₆₂

Example 5 Sodium sulfide inhibits the catalase activity of human lung cancer cell line A549

The catalase activity of human lung cancer cell line A549 was tested by xylenol alcohol (FOX1) assay. Sodium sulfide treatment can significantly inhibit the catalase activity of human lung cancer cell line A549, and this inhibitory effect is dose-related (see Table 3).

Table 3. Effect of hydrogen sulfide on the activity of catalase in human lung cancer cell line A549

Na ₂ S concentration 0.01mM 0.05mM 0.5mM 2mM Relative activity of catalase (%) 75±6 32±10 15±6 8±2

Example 6 Sodium Hydrosulfide Inhibits Catalase Activity of Human Gastric Cancer Cell Line SGC7901

The catalase activity of human gastric cancer cell line SGC7901 was tested by xylenol alcohol (FOX1) assay. Sodium hydrosulfide treatment can significantly inhibit the catalase activity of the above-mentioned cancer cells, and this inhibitory effect is dose-related (see Figure 3).

Example 7 Potassium sulfide inhibits catalase activity of mouse melanin skin cancer cell line B16

The catalase activity of the mouse melanotic skin cancer cell line B16 was tested using the xylenol alcohol (FOX1) assay. It was found that potassium sulfide can significantly inhibit the catalase activity of the above-mentioned cancer cells, and this inhibitory effect is dose-related (see Table 4).

Table 4. Effect of potassium sulfide on catalase activity of mouse melanoma skin cancer cell line B16

K ₂ S concentration 0.05mM 0.5mM 1mM 5mM Relative activity of catalase (%) 68±10 44±9 28±7 3±3

Example 8 Potassium Hydrosulfide Inhibits Catalase Activity of Mouse Hepatoma Cell Line H22

The catalase activity of mouse hepatoma cell line H22 was tested using xylenol alcohol (FOX1) assay. It was found that potassium hydrosulfide can significantly inhibit the above-mentioned catalase activity on cancer cells, and this inhibitory effect is dose-related (see Figure 4).

Example 9 Treatment with sodium hydrosulfide followed by chemotherapy with pingyangmycin can significantly inhibit the growth of sarcoma in mice

Subcutaneously inoculate 0.2 mL of S180 sarcoma cells (density 1×10 ⁶ cells/mL, cell death rate less than 5%) into the right hindlimb groin of 5-week-old Kunming mice (weight 25 ± 3 g), and form a diameter after 1 week of routine feeding. Subcutaneous sarcoma about 6mm. Sarcoma mice were randomly divided into three groups with 6 replicates in each group. The first group (A) injected normal saline into each mouse; the second group (B) injected 0.15 mg Pingyangmycin (prepared with normal saline) into each mouse, and the third group (C) injected 0.1 mg NaHS and 0.15 mg Mixture of Pingyangmycin (prepared with normal saline). The mice were administered once every 5 days, and after the 4th administration, they were reared for 5 days. The mice were sacrificed and the tumors were stripped off, weighed, and the tumor inhibition rate was calculated.

Tumor inhibition rate=(tumor weight of group A-tumor weight of group B)/tumor weight of group A

It can be clearly seen from Figure 5 that the tumor inhibition rate of chemotherapeutic drugs can be significantly improved after treatment with sodium hydrosulfide, and the statistical analysis shows that the difference is significant.

Claims (10)

1. The application of hydrogen sulfide in improving the tumor inhibition rate of antitumor drugs. 2. Application of hydrogen sulfide in the preparation of antitumor drug synergists. 3. The application according to claim 1 or 2, characterized in that the antitumor drugs include hydrogen peroxide, doxorubicin, mitomycin, 5-fluorouracil and pingyangmycin. 4. The application according to claim 1 or 2, characterized in that said tumors include liver cancer, lung cancer, gastric cancer, skin cancer and esophageal cancer. 5. The application according to claim 2, wherein the active ingredient of the antineoplastic drug synergist is at least one of hydrogen sulfide, soluble hydrogen sulfides, and soluble sulfides. 6. The application according to claim 5, characterized in that the concentration of the active ingredient of the antineoplastic drug synergist is 0.01-5 mmol/L. 7. The application according to claim 5, characterized in that, the soluble hydrogen sulfide salts are NaHS or KHS. 8. The application according to claim 5, wherein the soluble sulfide is Na ₂ S or K ₂ S. 9. A method for increasing the tumor suppression rate of antitumor drugs, comprising the following steps: administering antitumor drug synergists to cancer cells before or during treatment with antitumor drugs; The active ingredient of the antitumor drug synergist is at least one of hydrogen sulfide, soluble hydrogen sulfide salts, and soluble sulfide. 10. The method according to claim 9, characterized in that the concentration of the active ingredient of the antineoplastic drug synergist is 0.01-5 mmol/L.