CN101797242A - Application of cysteamine in preparing medicine for treating cancer - Google Patents

Abstract

The invention discloses the application of cysteamine in the preparation of medicine for treating cancer. The application of the cysteamine provided by the invention in the preparation of medicines for treating cancer improves the sensitivity of cancer cells to chemotherapeutic drugs through low-dose cysteamine, thereby realizing the effective killing of cancer cells through low-dose chemotherapeutic drugs. The invention greatly improves the efficacy of chemotherapy drugs and reduces the side effects of chemotherapy.

Description

Application of cysteamine in the preparation of medicines for treating cancer

technical field

The invention relates to the application of cysteamine in the preparation of medicines for treating cancer.

Background technique

Chemotherapy is a very effective and commonly used method for clinical treatment of cancer. However, severe side effects and drug resistance of cancer cells often lead to treatment interruption or failure. Now commonly used chemotherapeutic drugs and corresponding side effects are: the side effects of cyclophosphamide (CTX) are myelosuppression and hemorrhagic cystitis; the side effects of ifosfamide (IFO) are hemorrhagic cystitis and myelosuppression; Renal toxicity, gastrointestinal reactions, ototoxicity, neurotoxicity; side effects of carboplatin (CARBO) are myelosuppression; side effects of mitoxantrone (Mx) are cardiotoxicity, bone marrow suppression; side effects of methotrexate (MT) are nephrotoxicity, Pulmonary fibrosis, oral ulcers; side effect of cytarabine (Ara-C) is liver damage; side effect of bleomycin (BLM) is pulmonary fibrosis; side effect of pingyangmycin (PYM) pulmonary fibrosis; side effect of paclitaxel (TAXOL) allergy , heart conduction disorder, peripheral neuritis; vincristine (VCR) peripheral neuritis, extravasation skin damage; 5-fluorouracil (5-FU) side effect is diarrhea; podophyllin (etoposide, VP-16) side effect is bone marrow Inhibition; the side effect of Hemeixin (TOPO) is bone marrow suppression; the side effect of adriamycin (ADR) is cardiotoxicity, bone marrow suppression, gastrointestinal and renal toxicity, etc.

Therefore, a large number of composite anticancer drugs or combined anticancer drugs have appeared, among which patents include CN101040859A, CN101495148A, CN101040959A, CN101273963A, CN101495148A, etc.; the main components of these combinations involve alkylating agents, antimetabolite drugs, and antibiotic anticancer drugs , plant alkaloids, anthracenediones, natural products, hormones, hormone antagonists and other drugs: radiosensitizers, platinum coordination complexes, adrenal cortex inhibitors, immunosuppressants, functional therapeutic agents, gene therapy agents, antisense Therapeutics Tyrosine Kinase Inhibitors Monoclonal Antibodies Immunotoxins Radioimmunoconjugates Cancer Vaccines Interferons Interleukins Substituted Ureas Taxanes COX-2 Inhibitors.

Cysteamine, also known as β-mercaptoethylamine (CS for short), can be extracted from animal hair or chemically synthesized as a decarboxylation product of cysteine. Cysteamine is a component of coenzyme A molecule and biologically active substances that play important physiological roles in the body. The application of cysteamine in poultry production can regulate hormone levels, promote animal growth; improve digestion and metabolism of nutrients; improve animal immunity. The application of cysteamine in clinical medicine today includes the treatment of cataract and cystinosis, radiation sickness syndrome, acute and chronic metal poisoning.

Contents of the invention

The invention aims at chemotherapy effect and serious side effect, and provides application of cysteamine in preparation of medicine for treating cancer.

Adding cysteamine to currently used chemotherapeutic drugs can make a new combination agent for treating cancer. When the combination medicine is used, the effective dose of the chemotherapy medicine can be reduced to achieve the therapeutic effect, thereby reducing the side effects of the chemotherapy medicine.

The present invention also provides a combination medicine for treating cancer, the active ingredients of which include cysteamine and chemotherapeutic drugs.

The combination medicament for treating cancer also includes medically acceptable auxiliary materials.

The two components of the combination medicine of the present invention can be administered together or separately, and the dosage forms can be, but not limited to: tablets, ointments, capsules, powders, injections, solutions and the like.

The anticancer combination medicament prepared by the present invention can be administered in the following ways:

Topical: Administration directly to the body part to be affected, including: epidermal administration, inhalation administration, and enema administration.

Enteral administration: oral administration, the sensitizers and chemotherapeutic drugs in this technology can be made into tablets, capsules, liquid medicine, etc. Others include intubation, anal administration, etc.

Parenteral administration: intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, bone marrow injection, intradermal injection, transdermal administration, mucosal administration, inhalation administration, etc.

Other administration methods: intraperitoneal injection, epidural injection, spinal cord injection, eyeball vitreous injection, etc.

The medicine for the treatment of cancer can be administered separately, and the preferred administration methods of component cysteamine include: conventional administration methods such as intravenous injection and intramuscular injection, because cysteamine is used in the treatment of acute metal poisoning (such as tetraethyl lead poisoning) In the clinical application of prevention and treatment of radiation sickness, intravenous injection is considered to be the preferred and reliable way of administration. In clinical treatment of chronic metal poisoning, the first choice is intramuscular injection, so these two injection methods have been clinically verified. The safe and reliable administration method of cysteamine is only based on the difference in absorption rate. Choose according to clinical examples. In addition, high doses of cysteamine may cause gastric ulcers, so try to avoid oral administration in the case of high doses of cysteamine.

The combined drug for treating cancer can be used to treat various solid tumors in the human body: including those originating from the brain, central nervous system, kidney, liver, gallbladder, head and neck, oral cavity, thyroid, skin, mucous membrane, gland, blood vessel, bone tissue Primary or metastatic carcinoma or sarcoid or carcinosarcoma of , lymph node, lung, esophagus, stomach, breast, pancreas, eye, nasopharynx, uterus, ovary, endometrium, cervix, prostate, bladder, colon, rectum drug.

Although the combination medicine for treating cancer has not been applied clinically, cell experiments have proved that when cysteamine and chemotherapy medicine are used, the dose of chemotherapy medicine can be reduced compared with single use of chemotherapy medicine. Doctors can determine the dose at a safe dose according to the specific understanding of the patient's condition and the effect of chemotherapy. Component cysteamine is clinically used for the treatment of acute metal poisoning and the prevention and treatment of radiation sickness, usually by intravenous injection, the dose is 0.3g/time, 1-2 times a day. When clinically using the cysteamine in the combination medicament of the present invention, it can be used with reference to its clinically safe dosage. Under the safe dosage, the higher the dosage, the better the curative effect. The recommended dosage for clinical application refers to the existing clinical dosage. For example, the clinical dose of doxorubicin alone but its component is generally 30-100mg/m ² , and the high dose can reach 300mg/m ² , which can be reduced appropriately when it is used together with cysteamine.

Experiments have proved that cysteamine can effectively kill cancer cells and chemotherapeutic drug-resistant cancer cells only by cooperating with low-dose chemotherapy drugs, which proves that the medicament of the present invention greatly reduces the anti-cancer effect in the chemotherapy of cancer cells and resistant cancer cells. The amount of drugs used can reduce the side effects of chemotherapy. The application of the cysteamine provided by the invention in the preparation of medicines for treating cancer improves the sensitivity of cancer cells to chemotherapeutic drugs through low-dose cysteamine, thereby realizing the effective killing of cancer cells through low-dose chemotherapeutic drugs. The invention greatly improves the efficacy of chemotherapy drugs and reduces the side effects of chemotherapy.

Utilizing this principle, the anticancer drug of the present invention is designed, which contains common chemotherapeutic drugs and cysteamine as an auxiliary agent. Cysteamine in anticancer drugs assists low-dose chemotherapy drugs to kill tumor cells and chemotherapy drug-resistant cells, greatly enhances the chemotherapy effect of chemotherapy drugs and reduces the side effects of chemotherapy drugs.

The clinical application of cysteamine is of great significance because cysteamine is easy to obtain, low in price, single in chemical composition and low in biological toxicity. Cysteamine is used as a clinical drug for the treatment of cataract and cystinosis without any side effects, which also enhances the feasibility and success rate of this combined drug, and makes this combined anticancer drug more practical as a clinical drug.

Description of drawings

Figure 1: Cysteamine induces autophagy in HeLa cells (green punctate aggregation in cells stably expressing eGFP-LC3)

Figure 2: Cysteamine induces autophagy in 293A cells (green punctate aggregation in cells stably expressing eGFP-LC3)

Figure 3: Cysteamine induces autophagy in B16 and MCF cells (transformation of endogenous protein LC3I to LC3II)

Figure 4: Time effect of cysteamine-induced autophagy in HeLa cells

Figure 5: Dose effect of cysteamine on autophagy in HeLa cells

Figure 6: Cysteamine leads to an increase in autophagy-related protein P62 in HeLa cells

Figure 7: High dose cysteamine can cause cell death (MTT assay)

Figure 8: Cysteamine promotes low concentration of chemotherapy drug doxorubicin (doxorubicin) to kill HeLa cells (propidium iodide, PI staining).

Figure 9: Cysteamine promotes the killing of B16 cells by low concentration chemotherapy drug doxorubicin (propidium iodide, PI staining).

Figure 10: Cysteamine promotes the killing of MCF-7 cells by low concentration chemotherapy drug doxorubicin (propidium iodide, PI staining).

Figure 11: Cysteamine promotes low-concentration chemotherapeutic drug doxorubicin (doxorubicin) to kill doxorubicin-resistant cells (MCF-7/ADR cells) experiment (propidium iodide, PI staining)

Figure 12: Transfection of Atg5siRNA in HeLa cells reduces the level of autophagy, and correspondingly reduces the cell killing caused by autophagy (propidium iodide, PI staining)

Figure 13: Different concentrations of cysteamine promote the killing of cancer cells by the chemotherapy drug doxorubicin (propidium iodide, PI staining)

Detailed ways

The experimental methods described in the following examples are conventional methods unless otherwise specified.

Example 1. Cysteamine can induce autophagy in HeLA cancer cells [Induce autophagy in human cervical cancer HeLa cells stably expressing LC3-eGFP, and use the autophagy promoter rapmycin as a positive control]

experimental method:

1. The method for screening human cervical cancer cells stably expressing LC3-eGFP (LC3-eGFP/HeLa) is as follows:

(1) Human cervical cancer cell HeLa cells (purchased from Shanghai Institute of Cells, Chinese Academy of Sciences) were inoculated in 24-well cell culture plates (GIBCO, USA) with DMEM medium at a density of about 3-5×10 ⁴ /well, at 37 Cultivate overnight in a 5% (volume percent) CO ₂ incubator until use.

(2) 1.5 μl of Lipofectamine2000 per well, dissolved in 100 μl of serum-free and antibiotic-free DMEM medium, mixed thoroughly and incubated at room temperature for 5 min, 2 μg of plasmid LC3-eGFP (Invitrogen, USA) per well, dissolved in 100 μl of serum-free DMEM In the antibiotic-free DMEM medium (GIBCO, USA), mix well, incubate at room temperature for 5 minutes, and then dissolve and mix evenly, and incubate at room temperature for 20-25 minutes.

(3) The cells cultured overnight were washed twice with DMEM (serum-free and antibiotic-free) medium, then added to the mixture incubated in step (2), at 37° C., 5% (volume percentage) _CO Culture conditions were cultivated for 30 min, Each well was supplemented with 300 μl of DMEM complete medium, and the culture was continued.

(4) After 48 hours of transfection, the cells were transferred to a 24-well cell culture plate equipped with DMEM medium (GIBCO, USA) after 1:15 dilution, and 0.5 mg/ml of G418 antibiotics were added to select the cells (Sigma, USA). ), and the culture medium was changed every three days. Ten days later, the green positive clone LC3-eGFP/HeLa cells stably expressing LC3-eGFP were picked out under a fluorescent microscope for use.

2. Inoculate the LC3-eGFP/HeLa cells obtained after screening in step 1 in a 24-well cell culture plate (GIBCO, USA) containing DMEM medium, and the cell density of the 24-well plate is about 3-5×10 ⁴ / The wells were cultured overnight in a 37°C, 5% (volume percent) CO ₂ incubator until use.

3. Add cysteamine with a final concentration of 1.5mM (cysteamine in Figure 1) to the 24 wells in step 2 to induce cell autophagy. After culturing for 24 hours, perform fluorescence microscope observation and take pictures, and use cells without any treatment as Negative control (control in Fig. 1), with autophagy promoter rapamycin (final concentration is 200nM, U.S. Sigma company) as the positive control (rapamycin in Fig. 1) that causes autophagy, simultaneously with autophagy inhibition The agent 3-methyladenine (3-MA, the final concentration is 2mM) and 1.5mM cysteamine were co-treated for 24 hours to further prove the ability of cysteamine to induce autophagy (cysteamine+3-methyladenine in Figure 1 base purine). After the cells are transfected with the eGFP-LC3 plasmid, the expressed eGFP-LC3 protein is uniformly diffused in the cytoplasm under normal conditions, but when the cells undergo autophagy, eGFP-LC3 will accumulate on the membrane of the autophagic vacuoles to form punctate aggregates .

The results show that:

The results are shown in Figure 1. The results showed that after LC3-eGFP/HeLa was treated with 1.5mM cysteamine, the GFP-LC3 protein evenly diffused in the cytoplasm and aggregated on the membrane of autophagosomes to form green dot-like aggregates, and LC3 The punctate aggregation can be inhibited by the autophagy-specific inhibitor 3-methylpurine (Figure 1), indicating that cysteamine causes LC3 punctate aggregation to be a manifestation of autophagy.

Example 2: Cysteamine induces autophagy in 293A cells [Induces autophagy in human embryonic kidney cells 293A stably expressing LC3-eGFP, using the autophagy promoter rapmycin as a positive control]

experimental method:

1. The method for screening human embryonic kidney cells stably expressing LC3-eGFP (LC3-eGFP/293A) is as follows:

(1) 293A human embryonic kidney cells (purchased from Shanghai Institute of Cells, Chinese Academy of Sciences) were seeded in 24-well cell culture plates (GIBCO, USA) with DMEM medium at a density of about 3-5×10 ⁴ /well, at 37°C , cultivated overnight in a 5% (volume percent) CO ₂ incubator, ready for use.

(2) Prepare the transfection complex, the method is as follows: 1.5 μl of Lipofectamine2000 per well, dissolved in 100 μl DMEM (serum-free and antibiotic-free) medium, mixed well and incubated at room temperature for 5 minutes, 2 μg of LC3-eGFP per well, dissolved in 100 μl In DMEM (serum-free and antibiotic-free) medium, mix well, incubate at room temperature for 5 minutes, and then dissolve and mix evenly, and incubate at room temperature for 20-25 minutes.

(3) The cells cultured overnight were washed twice with DMEM (serum-free and antibiotic-free) medium, and then the transfection complex obtained in step (2) was added, and cultured at 37° C., 5% (volume percentage) CO ₂ culture conditions After 30 minutes, the medium was supplemented with 300 μl of complete DMEM medium, and the culture was continued at 37°C.

(4) After 48 hours of transfection, the cells were diluted 1:15 and transferred to a 24-well cell culture plate in DMEM medium (GIBCO, USA), and 0.5 mg/ml of G418 was added to screen the cells, and the medium was changed every three days. . Ten days later, green positive clones stably expressing LC3-eGFP were picked out under a fluorescent microscope for use.

2. The screened LC3-eGFP/293A cells were seeded in a 24-well cell culture plate at a cell density of about 3-5×10 ⁴ /well, cultured overnight, and set aside.

3. Add cysteamine at a final concentration of 1.5 mM (cysteamine in Figure 2) to the 24 wells in step 2 to induce cell autophagy, and observe and take pictures with a fluorescence microscope after culturing for 24 hours. The cells without any treatment were used as negative control (control in Fig. 2), and the positive control (rapamycin in Fig. ), while co-treating with autophagy inhibitor 3-methyladenine (3-MA, the final concentration is 2mM) and 1.5mM cysteamine for 24 hours to further prove the ability of cysteamine to induce autophagy (Fig. 1 cysteamine + 3-methyladenine).

The results show that:

1. HeLa cells are seeded in DMEM medium (GIBCO, USA) 24-well cell culture plate, the cell density of the 24-well plate is about 3-5×10 ⁴ /well, at 37°C, 5% (volume percentage) Incubate overnight in a CO ₂ incubator until use.

2. Add cysteamine at a final concentration of 1.5mM to treat them, respectively culture them in 37°C, 5% (volume percent) CO ₂ incubators for 0hr, 5hr, 12hr, 18hr, and 24hr, and collect them with cell lysate (purchased from Biyuntian Company) to lyse the cells, and then add electrophoresis loading buffer (purchased from Biyuntian Company) to boiling water for 10 minutes to prepare electrophoresis samples. After 12% SDS-PAGE electrophoresis, the protein was transferred to a nylon membrane (purchased from Amersham, USA) by wet transfer. Western blot was performed using anti-LC3 (Novus) as the primary antibody and anti-rabbit (Promega) as the secondary antibody. Using anti-GAPDH (Millipore, USA) as the primary antibody to detect GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference to quantify the protein amount of the control group and the experimental group (glyceraldehyde-3-phosphate dehydrogenase, is Constitutive protein in cells, the level is the same in each cell).

The results show that:

The results are shown in Figure 4, and the results show that the autophagy effect of cysteamine in HeLa cells is enhanced with the prolongation of the treatment time (Figure 4).

Example 5: Dose effect of cysteamine on autophagy in HeLa cells [transformation of endogenous protein LC3I to autophagy marker protein LC3II]

experimental method:

1. HeLa cells are seeded in DMEM medium (GIBCO, USA) 24-well cell culture plate, the cell density of the 24-well plate is about 3-5×10 ⁴ /well, at 37°C, 5% (volume percentage) Incubate overnight in a CO ₂ incubator until use.

2. Cysteamine with a final concentration of 0.5mM, 1.0mM, 1.5mM or 2.0mM was added to each well, and the cells without any treatment were used as the control group (the control in Figure 5), and the final concentration of the autophagy promoter was 200nM Rapamycin treatment (Sigma, USA) was used as a positive control (rapamycin in Figure 5) to cause autophagy, at 37 ° C, 5% (volume percentage) CO ₂ incubator, collected after cell culture 24hr Cells were lysed with cell lysate (purchased from Beyond Company), and then electrophoresis loading buffer (purchased from Beyond Company) was added to boiling water for 10 minutes to prepare electrophoresis samples. After 12% SDS-PAGE electrophoresis, the protein was transferred to a nylon membrane (purchased from Amersham, USA) by wet transfer. Western blot was performed using anti-LC3 (Novus) as the primary antibody and anti-rabbit (Promega) as the secondary antibody. Using anti-GAPDH (Millipore, USA) as the primary antibody to detect GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference to quantify the protein amount of the control group and the experimental group (glyceraldehyde-3-phosphate dehydrogenase, is Intracellular constitutive protein, the level is consistent in each cell)

The results are shown in Figure 2. The results show that 1.5mM cysteamine in LC3-eGFP/293A can cause green dot-like aggregation of eGFP-LC3, and the dot-like aggregation of eGFP-LC3 can be inhibited by the autophagy-specific inhibitor 3-MA Inhibition (Figure 2), indicating that cysteamine causes eGFP-LC3 point-like aggregation is the expression of autophagy.

Example 3: Cysteamine induces autophagy in melanoma cells B16 and human breast cancer MCF-7 cells [transformation of endogenous protein LC3I (microtubule-associated protein LC3I) to autophagy marker protein LC3 II]

During the autophagy of melanoma cell B16 and human breast cancer MCF-7 cells, the endogenous protein LC3I is converted to the type II autophagy marker protein LC3, so the following method is used to detect the conversion of the endogenous protein LC3I to the type II autophagy marker protein LC3 To determine whether autophagy occurs in melanoma cell B16 and human breast cancer MCF-7 cells.

experimental method:

1. Inoculate melanoma cells B16 (purchased from Shanghai Institute of Cells, Chinese Academy of Sciences) or human breast cancer MCF-7 cells in 24-well cell culture plates in DMEM medium (GIBCO, USA), respectively, and the cell density in 24-well plates is about 3-5×10 ⁴ /well, culture overnight at 37°C, set aside.

2. Add cysteamine at a final concentration of 1.5mM to treat, culture in a 37°C, 5% (volume percent) CO ₂ incubator for 24 hours, and then collect them, and use cell lysate (purchased from Biyuntian Company) to lyse the cells , and then add electrophoresis loading buffer (purchased from Beyontian Company) and cook in boiling water for 10 minutes to prepare electrophoresis samples. After 12% SDS-PAGE electrophoresis, the protein was transferred to a nylon membrane (purchased from Amersham, USA) by wet transfer method, and melanoma cell B16 without any treatment was used as a control group. Western blot was performed using anti-LC3 (Novus) as the primary antibody and anti-rabbit (Promega) as the secondary antibody. Using anti-GAPDH (Millipore, USA) as the primary antibody to detect GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference to quantify the protein amount of the control group and the experimental group (glyceraldehyde-3-phosphate dehydrogenase, is Constitutive protein in cells, the level is the same in each cell).

The results show that:

The results are shown in Figure 3. The results showed that in melanoma cell B16 and human breast cancer MCF-7, compared with the control, the amount of LC3II protein increased greatly after cysteamine treatment, indicating that cysteamine had a significant effect on melanoma cell B16, human breast cancer MCF-7. Autophagy can also be induced in breast cancer MCF-7, because enhancing the conversion of endogenous protein LC3I to protein LC3II is a sign of autophagy (Figure 3).

Example 4: Time effect of cysteamine-induced autophagy in HeLa cells [conversion of endogenous protein LC3I to autophagy marker protein LC3II]

experimental method:

The results show that:

The results are shown in Figure 5, and the results show that the autophagy effect of cysteamine in HeLa cells is enhanced with the increase of cysteamine concentration (Figure 5).

Example 6: Cysteamine leads to an increase in the autophagy substrate protein p62 in HeLa cells [using the autophagy inhibitor 3-MA as a negative control]

Autophagy can degrade misfolded proteins or damaged organelles in cells. P62 protein is also the substrate of autophagy. When cells undergo complete autophagy, they will degrade intracellular p62 protein. If the downstream of autophagy is Blocking will block the degradation of p62, so the increase of p62 protein can indirectly explain the incompleteness of autophagy.

experimental method:

1. HeLa cells are seeded in 24-well cell culture plates (GIBCO, USA) with DMEM medium, the cell density of the 24-well plates is about 3-5×10 ⁴ /well, at 37°C, 5% (volume percentage content) in a CO ₂ incubator for overnight cultivation.

2. The treatment of each well was the combination of adding final concentrations of 1.5mM cysteamine, 2mM 3-methyladenine, 1.5mM cysteamine and 2mM 3-methyladenine, and the cells without any treatment were used as controls , respectively at 37° C., 5% (volume percentage) CO ₂ Cultured in the incubator for 24 hours, the cells were collected, and the cells were lysed with the cell lysate (purchased from Biyuntian Company), and then the electrophoresis loading buffer (purchased from Biyuntian Company) was added. Yuntian Company) was boiled in water for 10 minutes, and the electrophoresis samples were prepared. After 12% SDS-PAGE electrophoresis, the protein was transferred to a nylon membrane (purchased from Amersham, USA) by wet transfer. Use anti-p62 (BIOMOL Company) as the primary antibody, anti-rabbit (Promega Company) as the secondary antibody, perform Western blot, and use anti-GAPDH as the primary antibody to detect GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference Quantify the amount of protein in the control group and the experimental group (glyceraldehyde-3-phosphate dehydrogenase, which is a constitutive protein in cells, has the same level in each cell)

The results show that:

The results are shown in Figure 6, the results show that the cysteamine treatment group compared with the control group autophagy substrate p62 increased (see Figure 6), indicating that the downstream pathway of autophagy may be blocked, which is harmful to the survival of cells .

Example 7: Effect of Cysteamine on Cell Viability [Detection of Cell Death by Thiazolium Blue Method (MTT)]

experimental method:

1. HeLa cells are seeded in 96-well cell culture plates (GIBCO, USA) in DMEM medium, the cell density of the 96-well plates is about 0.8-1×10 ⁴ /well, 37°C, 5% (volume percentage) CO ₂ Incubate overnight in an incubator until use.

2. Add cysteamine at final concentrations of 0mM, 0.5mM, 1mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM and 4.0mM to each well of the cell culture plate. After culturing in a 5% (volume percent) CO ₂ incubator at 37° C. for 24 hours, the cell death rate was detected according to the method in step 3.

3. After the 96-well cells were treated as above, add 10 μl of 5 mg/ml MTT (thiazolium blue) to each well, and incubate for about 2-4 hours in a 37°C, 5% CO ₂ incubator, when purple crystals appear in the cells , After carefully aspirating the supernatant, add 100 μl DMSO (dimethyl sulfoxide) to each well and place in the dark for 2 hours. After shaking well, the microplate reader detects its ultraviolet absorption at 570nm. The absorbance at 570 nm decreases as more cells die, and the absorbance of cells treated with 0 mM cysteamine is set as 100% live cells, and then the cell viability is calculated proportionally.

The results show that:

The results are shown in Figure 7, and the results indicated that low concentration of cysteamine had no effect on cell viability, but high concentration of cysteamine could lead to cell death (Figure 7).

Example 8: Cysteamine promotes low-concentration chemotherapeutic drug doxorubicin (doxorubicin) to kill cancer HeLa cells [detected by propidium iodide (PI) staining]

experimental method:

1. HeLa cells are seeded in 96-well cell culture plates (GIBCO, USA) in DMEM medium, the cell density of the 96-well plates is about 0.8-1×10 ⁴ /well, 37°C, 5% (volume percentage) CO ₂ Incubate overnight in an incubator until use.

2. Each well in the cell culture plate was treated with a final concentration of 1.5mM cysteamine (CS in Figure 8), 0.75 μg/ml doxorubicin (Dox in Figure 8), and a final concentration of 1.5mM cysteamine. and 0.75 μg/ml doxorubicin (DOX+CS in Fig. 8), wherein without adding low dose doxorubicin (CS in Fig. DOX in 8) was used as a control. After culturing for 24 hours, the cell death rate was detected according to the method in step 3.

3. The cells were stained with 2 μg/ml Hochest (Hochest33342, purchased from Beyentian) and 5 μg/ml propidium iodide (PI) for 15 minutes, and then photographed under a microscope. Hochest33342 is specially used to stain the nucleus and count the total number of cells. PI stains dead cells. It can penetrate the cell membrane of dying or dead cells and insert double-stranded DNA. It cannot enter living cells. The percentage of dead cells in all cells is the cell death rate.

The results show that:

The results are shown in Figure 8. The results showed that low concentrations of cysteamine and low doses of doxorubicin hardly caused cancer cell death, while the synergistic effect of the two caused a large number of HeLa cells to die by 65.5 ± 2.6% (Figure 8) .

Example 9: Cysteamine promotes low-concentration chemotherapeutic drug doxorubicin (doxorubicin) to kill melanoma cancer cell B16 experiment

experimental method:

1. Melanoma cancer cell B16 cells were inoculated in a 96-well cell culture plate (GIBCO, USA) in DMEM medium, the cell density of the 96-well plate was about 0.8-1×10 ⁴ /well, 37°C, 5% (volume percentage) content) in a CO ₂ incubator for overnight cultivation.

2. Each well of the cell culture plate is treated with a final concentration of 1.5 mM cysteamine (CS in Figure 9), 0.8 μg/ml doxorubicin (Dox in Figure 9), and a final concentration of 1.5 mM cysteamine. and 0.8μg/ml doxorubicin (DOX+CS in Fig. 9), wherein without adding low dose doxorubicin (CS in Fig. DOX in 9) was used as a control. After cultivating for 24hr, detect cell death rate according to the method of embodiment 8

The results show that:

The results are shown in Figure 9. The results showed that low concentration of cysteamine and low dose of doxorubicin hardly caused cancer cell death, and the synergistic effect of the two increased the death of B16 cells by 21.892±1.053% (Figure 9).

Example 10: Cysteamine promotes low-concentration chemotherapeutic drug doxorubicin (doxorubicin) to kill human breast cancer MCF-7 experiment

experimental method:

1. Human breast cancer MCF-7 cells were inoculated in 96-well cell culture plate (GIBCO, USA) in DMEM medium, the cell density of the 96-well plate was about 0.8-1×10 ⁴ /well, 37° C., 5% (vol. content) overnight in a CO ₂ incubator until use.

2. Each well in the cell culture plate is treated with a final concentration of 1.5 mM cysteamine (CS in Figure 10), 0.8 μg/ml doxorubicin (Dox in Figure 10), and a final concentration of 1.5 mM cysteamine. and 1.0 μg/ml doxorubicin (DOX+CS in Fig. 9), wherein without adding low dose doxorubicin (CS in Fig. DOX in 10) was used as a control. After cultivating for 24hr, detect cell death rate according to the method of embodiment 8

The results show that:

The results are shown in Figure 10, and the results show that low-concentration cysteamine and low-dose doxorubicin hardly cause cancer cell death, and the synergistic effect of the two increases the death of MCF-7 cells by 54.23 ± 2.146% (Fig. 10).

Example 11: Cysteamine promotes low-concentration chemotherapeutic drug doxorubicin (doxorubicin) to kill drug-resistant human breast cancer cell line MCF-7/ADR experiment

experimental method:

1. The method for screening the doxorubicin-resistant human breast cancer cell line MCF-7/ADR cell is as follows: the human breast cancer cell line MCF-7 is inoculated in a 24-well cell culture plate of (GIBCO, USA) in DMEM medium, and added The doxorubicin of final concentration 0.01 μ M, 37 ℃, 5% (volume percentage content) CO After _incubator culture for two months, then add the doxorubicin of 0.1 μ M, after the second round of screening culture for two months, Increase the drug concentration to 1 μM, and obtain stable adriamycin-resistant human breast cancer cell line MCF-7/ADR cells after screening and culturing for 3 months.

2. Inoculate the drug-resistant human breast cancer cell line MCF-7/ADR in a 96-well cell culture plate (GIBCO, USA) in DMEM medium, the cell density of the 96-well plate is about 0.8-1×10 ⁴ /well, Cultivate overnight in a 5% (volume percent) CO ₂ incubator at 37° C. until use.

3. The treatment of each well in the cell culture plate is that the final concentration is 1.5mM cysteamine (CS in Figure 11), 20 μg/ml doxorubicin (Dox in Figure 11), the final concentration is 1.5mM cysteamine and The combination of 20 μg/ml doxorubicin (DOX+CS in Figure 11), wherein without adding low-dose doxorubicin (CS in Figure 11) and without adding 1.5mM (final concentration) cysteamine (in Figure 11 DOX) was used as a control. After cultivating for 24hr, the results of detecting the cell death rate according to the method of Example 8 showed:

The results are shown in Figure 11. The results showed that low doses of cysteamine and doxorubicin hardly caused the death of drug-resistant cells, and the synergistic effect of the two caused a large number of cancer cells to die by 44.23±1.8%.

Example 12: Transfection of Atg5siRNA in HeLa cells reduces the level of autophagy and correspondingly reduces the cell killing caused by autophagy

experimental method:

1. HeLa cells are seeded in two 60mm cell culture plates in DMEM medium (GIBCO, USA), the cell density is about 2×10 ⁵ /well, 37°C, 5% (volume percentage) CO ₂ incubator Incubate overnight in medium and set aside.

2. siRNA transfection: 1.5 μl of Lipofectamine2000 per well, dissolved in 100 μl of DMEM (serum-free and antibiotic-free) medium, mixed well, incubated at room temperature for 5 minutes, negative control siRNA with no homology with mammalian cells (purchased from Shanghai Gemma Pharmaceutical Technology Co., Ltd.) and 2 μg of Atg5siRNA (purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.) were respectively dissolved in 100 μl DMEM (serum-free and antibiotic-free) medium, mixed thoroughly, incubated at room temperature for 5 min, and the two dissolved and mixed evenly, incubated at room temperature 20-25min.

3. After the overnight cultured cells were washed twice with DMEM (serum-free and antibiotic-free) medium, the incubated control siRNA and Atg5 siRNA were added respectively, and incubated at 37° C. in a 5% (volume percent) CO ₂ incubator for 30 min. Supplement medium DMEM complete medium 300μl, continue culturing.

4. Add 1.5 mM cysteamine to the cell culture medium transfected with control siRNA and Atg5siRNA respectively, and the cells without any treatment are used as controls.

5. Cells were collected after cysteamine treatment for 24 hours, and cells were lysed with cell lysate (Beiyuntian Company), boiled in water for 10 minutes, and then transferred to a nylon membrane (Amersham Company) by wet transfer after 12% SDS-PAGE electrophoresis. Western blot was performed using antibodies anti-Atg5 (Santa Company) and anti-LC3 (Novus Company) as the primary antibody and anti-rabbit (Promega Company) as the secondary antibody. The level of autophagy was reduced in cells transfected with Atg5 siRNA (Atg5 siRNA in the right panel of FIG. 12 ) relative to control siRNA-treated cells (consiRNA in the right panel of FIG. 12 ).

6. Add final concentrations of 1.5 mM cysteamine (CS in the left figure of Figure 12) and 20 μg/ml doxorubicin (left in Figure 12) to the cell culture medium transfected with control siRNA and the cell culture medium transfected with Atg5siRNA, respectively. Dox in the figure), the final concentration is the combination of 1.5mM cysteamine and 20μg/ml doxorubicin (DOX+CS in the left figure of Fig. CS) and no addition of 1.5 mM (final concentration) cysteamine (DOX in the left figure of Figure 12) were used as controls. After culturing for 24 hours, the cell death rate was detected according to the method in Example 8.

The results show that:

The results are shown in Figure 12. The results showed that, in the cells transfected with Atg5siRNA, the ability of cysteamine to induce autophagy was weakened (Fig. 12 left figure, control siRNA in the left figure of Figure 12 is the cell transfected with control siRNA, and Atg5siRNA is the cell transfected with Atg5siRNA).

Example 13: Cysteamine synergistically kills tumors with doxorubicin

experimental method:

1. B16 cells are inoculated in cell culture flasks with DMEM medium (GIBCO, USA), cultivated in a 37° C., 5% (volume percent) CO2 incubator, and the cells are digested and centrifuged when the culture flask is full.

2. Inoculate 1x106 cells into C57 mice (purchased from Shanghai Shrek Company), and administer 0.5 mm3 when the tumor grows for a week. The mice are randomly divided into four groups. Control group: give normal saline, cysteamine group: Dosage administration of 500mg/kg (relative to the body weight of mice), Adriamycin group: dosage administration of 5mg/kg (relative to body weight of mice), combined drug group: 500mg/kg cysteamine and 5mg/kg doxorubicin (relative to mouse body weight) co-administered every other day.

3. After two weeks, the mice were killed by neck segment, the tumor was stripped off and weighed, and the tumor size was counted to measure the effect of cysteamine and doxorubicin on killing the tumor (Figure 13)

The results show that:

The results are shown in Figure 13, and the results showed that cysteamine can greatly enhance the ability of doxorubicin to kill cancer cells (Figure 13).

The above experiments in Examples 1-13 show that the sensitivity of cancer cells to chemotherapeutic drugs is enhanced by low doses of cysteamine, thereby realizing the effective killing of cancer cells by low doses of chemotherapeutic drugs. Experiments have proved that cysteamine can effectively kill cancer cells and chemotherapeutic drug-resistant cancer cells only by cooperating with low-dose chemotherapy drugs, which proves that the medicament of the present invention greatly reduces the anti-cancer effect in the chemotherapy of cancer cells and resistant cancer cells. The amount of drugs used can reduce the side effects of chemotherapy. Therefore, this principle can be used to design the anticancer drug of the present invention, which contains common chemotherapeutic drugs and cysteamine as an auxiliary agent. Cysteamine in anticancer drugs assists low-dose chemotherapy drugs to kill tumor cells and chemotherapy drug-resistant cells, greatly enhances the chemotherapy effect of chemotherapy drugs and reduces the side effects of chemotherapy drugs.

Although the combination medicine for treating cancer has not been applied clinically, cell experiments have proved that when cysteamine and chemotherapy medicine are used, the dose of chemotherapy medicine can be reduced compared with single use of chemotherapy medicine. Doctors can determine the dose at a safe dose according to the specific understanding of the patient's condition and the effect of chemotherapy. Component cysteamine is clinically used for the treatment of acute metal poisoning and the prevention and treatment of radiation sickness, usually by intravenous injection, the dose is 0.3g/time, 1-2 times a day. When clinically using the cysteamine in the combination medicament of the present invention, it can be used with reference to its clinically safe dosage. Under the safe dosage, the higher the dosage, the better the curative effect. The recommended dosage for clinical application refers to the existing clinical dosage. For example, the clinical dose of doxorubicin alone but its component is generally 30-100mg/m ² , and the high dose can reach 300mg/m ² , which can be reduced appropriately when it is used together with cysteamine.

Claims (10)

1. The application of cysteamine in the preparation of medicaments for treating cancer. 2. A combined medicament for treating cancer, the active ingredients of which are cysteamine and chemotherapy drugs. 3. The combined medicament according to claim 2, characterized in that: the combined medicament for treating cancer also includes medically acceptable auxiliary materials. 4. The combined medicament according to claim 2 or 3, characterized in that: the chemotherapeutic drug is doxorubicin. 5. The combined medicament according to any one of claims 2-4, characterized in that: said cancers include cancers originating in the brain, central nervous system, kidney, liver, gallbladder, head and neck, oral cavity, thyroid, skin, Primary or Metastatic carcinoma or sarcoma or carcinosarcoma. 6. The combination medicine according to claim 5, characterized in that: the cancer is cervical cancer, breast cancer, melanoma or kidney cancer. 7. The application of cysteamine in improving the sensitivity of cancer cells to chemotherapeutic drugs. 8. The use of cysteamine in the preparation of tumor cell inhibitors. 9. Use of cysteamine and chemotherapeutic drugs in the preparation of tumor cell inhibitors. 10. The application according to claim 8 or 9, characterized in that: the tumor cells are human cervical cancer cell HeLa cells, human breast cancer cell line MCF-7 or melanoma cancer cell B16 cells; the chemotherapy drugs are Adriamycin.

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