CN101797242A - Application of cysteamine in preparing medicine for treating cancer - Google Patents
Application of cysteamine in preparing medicine for treating cancer Download PDFInfo
- Publication number
- CN101797242A CN101797242A CN 201010143701 CN201010143701A CN101797242A CN 101797242 A CN101797242 A CN 101797242A CN 201010143701 CN201010143701 CN 201010143701 CN 201010143701 A CN201010143701 A CN 201010143701A CN 101797242 A CN101797242 A CN 101797242A
- Authority
- CN
- China
- Prior art keywords
- cell
- cysteamine
- cancer
- chemotherapeutics
- autophagy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses application of cysteamine in preparing a medicine for treating cancer. Sensitivity on cancer cells by chemotherapeutics is increased by the cysteamine with low dosage, thereby realizing that cancer cells are effectively killed by the chemotherapeutics with low dosage. The invention greatly improves the medicine effect of the chemotherapeutics and lowers the side effects of chemotherapy.
Description
Technical field
The present invention relates to the application of a kind of cysteamine in the medicine of preparation treatment cancer.
Background technology
Chemotherapy is a present clinical anticancer method very effective and commonly used.But the Drug resistance of serious adverse and cancerous cell tends to cause the interruption or the failure for the treatment of.Chemotherapeutics and corresponding side effect commonly used now have: cyclophosphamide (CTX) side effect is bone marrow depression, hemorrhagic cystitis; Ifosfamide (IFO) side effect is hemorrhagic cystitis, bone marrow depression; Cisplatin (DDP) side effect is Toxicity of Kidney, digestive tract reaction, ototoxicity, neurotoxicity; Carboplatin (CARBO) side effect is bone marrow depression; Mitoxantrone (Mx) side effect is cardiac toxicity, bone marrow depression; Methotrexate (MT) side effect is nephrotoxicity, pulmonary fibrosis, oral ulcer; Cytosine arabinoside (Ara-C) side effect is a hepatic injury; Bleomycin (BLM) side effect is pulmonary fibrosis; Bleomycin A5 (PYM) pulmonary fibrosis; Paclitaxel (TAXOL) side effect allergy, cardiac conduction obstacle, peripheral neuritis; Vincristine (VCR) peripheral neuritis, the skin lesion of exosmosing; 5-fluorouracil (5-FU) side effect is diarrhoea; Podophyllin (etoposide, VP-16) side effect is bone marrow depression; With U.S. new (TOPO) side effect be bone marrow depression; Amycin (ADR) side effect is cardiac toxicity, bone marrow depression, digestive tract and Toxicity of Kidney etc.
Therefore a large amount of compound cancer therapy drugs or combination anticarcinogen occur, wherein patent has CN101040859A, CN101495148A, CN101040959A, CN101273963A, CN101495148A etc.; The composition that these combinations are wanted relates to alkylating agent, antimetabolite class medicine, the antibiotics anticarcinogen, the vegetable alkaloids medicine, amerantrone class medicine, natural product, hormone, hormone antagonist and other medicines: radiosensitizer platinum coordination complex adrenal cortex inhibitor immunosuppressant functional therapeutic agent gene therapeutic agents antisense therapy agent tyrosine kinase inhibitor monoclonal antibody immunity toxin radioimmunoassay conjugate cancer vaccine interferon interleukin substituted urea class taxanes cox 2 inhibitor.
Cysteamine claims β one to dredge basic ethamine (being called for short CS) again, can from animal hair, extract, but also chemosynthesis, as the decarboxylate of cysteine, cysteamine is the constituent and the intravital bioactive substance of animal of coenzyme A molecule, has the important physical effect in vivo.The application of cysteamine in poultry production has regulate hormone level, promotes growth of animal; Improve the digestion and metabolism of nutrient substance; Improve animal body immunity.The application of cysteamine in clinical treatment now has treatment cataract and cystinosis, radiation sickness syndrome, acute and chronic metal poisoning.
Summary of the invention
The present invention be directed to chemotherapy effect and serious side effects, the application of cysteamine in the medicine of preparation treatment cancer is provided.
Utilize cysteamine to add the combination medicament that can be made into new treatment cancer in the chemotherapeutics of present use to.This combination medicament can reduce chemotherapeutics when using effective dose can reach therapeutic effect, thereby reduces the side effect that chemotherapeutics produces.
The present invention also provides a kind of combination medicament for the treatment of cancer, and its effective ingredient comprises cysteamine and chemotherapeutics.
Also comprise medically acceptable adjuvant in the combination medicament of described treatment cancer.
Two kinds of compositions of composition of medicine of the present invention are administration or separate administration together, and medicine type can be, but be not limited to: tablet, unguentum, capsule, powder, injection, solution etc.
The anticancer composite medicine agent administration in the following manner that the present invention prepares:
Topical (topical): direct drug injection comprises in the body part that will influence: epidermis administration, inhalation, coloclysis administration.
Digestive tract administration (enteral): oral administration, sensitizer in the present technique and chemotherapeutics can be made tablet, capsule, liquid medicine etc., other intubate, anum administration etc. in addition.
Parenteral administration (parenteral): intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, bone marrow injection, intradermal injection, transdermal administration, mucosa delivery, inhalation etc.
Other administering modes: lumbar injection, epidural space injection, spinal cord injection, vitreous body of eye injection etc.
The medicine of this treatment cancer can separate administration, the preferred administering mode of component cysteamine has: conventional administering modes such as intravenous injection, intramuscular injection, because treat acute metal poisoning (as tetraethyl lead poisoning) and prevent and treat in the clinical practice of radiation sickness at cysteamine, intravenous injection is considered to first-selected reliable administering mode.And treat the administering mode of chronic metal poisoning clinically, first-selected be intramuscular injection, thus these two kinds of injection systems by clinical verification, the safe and reliable administering mode of cysteamine, just, need select according to clinical example according to the difference of absorption rate etc.The high dose cysteamine may cause gastric ulcer in addition, so avoid oral administration under the situation of high dose administration as far as possible.
The combination medicament of this treatment cancer can be used for the treatment of the various solid tumors of human body: comprise former or the cancer of transfer or the medicine of sarcoma or carcinosarcoma originating from brain, central nervous system, kidney, liver, gallbladder, incidence, oral cavity, thyroid, skin, mucosa, body of gland, blood vessel, osseous tissue, lymph node, lungs, esophagus, stomach, mammary gland, pancreas, eyes, nasopharynx part, uterus, ovary, endometrium, cervix uteri, prostate, bladder, colon, rectum.
Though it is clinical that the combination medicament of this treatment cancer is not applied to, cell experiment proves that when using cysteamine and chemotherapeutic agents, the comparable single use chemotherapeutic agents of chemotherapeutic agents is a decrement.The doctor can determine dosage according to specifically to patient's state of an illness understanding and chemotherapy effect under safe dose.The component cysteamine is clinical to be used for the treatment of acute metal poisoning and to prevent and treat radiation sickness dosage, generally is intravenous injection, and dosage is 0.3g/ time, one day 1-2 time.During cysteamine in the clinical use combination medicament of the present invention, can use with reference to its clinical dosage safe in utilization, under safe dose, the high more curative effect of using dosage is good more.The dosage of suggestion clinical practice is with reference to existing clinical using dosage.Such as, separately but wherein the dosage of the clinical use of component amycin is generally at 30-100mg/m
2, high dose can reach 300mg/m
2, when using jointly, can take the circumstances into consideration decrement with cysteamine.
Experimental results show that chemotherapeutics that cysteamine only need work in coordination with low dosage just effectively kill cancer cell and chemotherapeutic resistance cancerous cell, thereby prove that promptly medicament of the present invention reduces cancer therapy drug greatly in the chemotherapy of cancerous cell and resistance cancerous cell use amount reduces the side effect of chemotherapy.The application of cysteamine provided by the invention in the medicine of preparation treatment cancer promoted the sensitivity of cancerous cell to chemotherapeutics by the cysteamine of low dosage, thereby realized the effective kill cancer cell of chemotherapeutics by low dosage.The present invention improves the drug effect of chemotherapeutics greatly, has reduced the side effect of chemotherapy.
The cancer therapy drug of the present invention that utilized this principle design, it comprises common chemotherapeutics and adjuvant cysteamine.Cysteamine is assisted low-dosage chemotherapy medicine killing tumor cell and chemotherapeutics resisting cell in the anticancer agent, thereby strengthens the side effect of the chemotherapy effect reduction chemotherapeutics of chemotherapeutics greatly.
And since cysteamine obtain easily, cheap, chemical constituent is single and bio-toxicity is low, more makes this clinical practice of cysteamine seem and means a great.Cysteamine is not found its side effect as the treatment that clinical medicine is used for cataract and cystinosis, and exploitativeness and success rate that this has also strengthened this composition of medicine make this combination cancer therapy drug have more practicality as clinical medicine.
Description of drawings
Fig. 1: cysteamine causes HeLa cell autophagy (the green point-like in the stably express eGFP-LC3 cell is assembled)
Fig. 2: cysteamine causes 293A cell autophagy (the green point-like in the stably express eGFP-LC3 cell is assembled)
Fig. 3: cysteamine causes B16, MCF cell autophagy (endogenic albumen LC3I transforms to the LC3II type)
Fig. 4: cysteamine causes the time effect of cell autophagy in the HeLa cell
Fig. 5: cysteamine causes the dosage effect of cell autophagy in the HeLa cell
Fig. 6: cysteamine causes increasing of autophagy associated protein P62 in the HeLa cell
Fig. 7: the high dose cysteamine can cause cell death (MTT detection method)
Fig. 8: cysteamine promotes low concentration chemotherapy medicine amycin (doxorubicin) to kill HeLa cell experiment (propidium iodide, PI dyeing).
Fig. 9: cysteamine promotes low concentration chemotherapy medicine amycin (doxorubicin) to kill the experiment of B16 cell (propidium iodide, PI dyeing).
Figure 10: cysteamine promotes low concentration chemotherapy medicine amycin (doxorubicin) to kill the experiment of MCF-7 cell (propidium iodide, PI dyeing).
Figure 11: cysteamine promotes low concentration chemotherapy medicine amycin (doxorubicin) to kill the experiment of amycin resisting cell (MCF-7/ADR cell) (propidium iodide, PI dyeing)
Figure 12: the HeLa transit cell dyes Atg5siRNA and reduces the autophagy level, reduces the cell killing (propidium iodide, PI dyeing) that autophagy is brought accordingly
Figure 13: the cysteamine of variable concentrations promotes concentration chemotherapeutics amycin (doxorubicin) kill cancer cell (propidium iodide, PI dyeing)
The specific embodiment
Experimental technique described in the following embodiment if no special instructions, is conventional method.
Experimental technique:
1, human cervical carcinoma cell (LC3-eGFP/HeLa) method of screening stably express LC3-eGFP is as follows:
(1) human cervical carcinoma cell HeLa cell (available from Shanghai cell institute of the Chinese Academy of Sciences) is seeded in (U.S. GIBCO company) 24 porocyte culture plates that the DMEM culture medium is housed, the about 3-5 of density * 10
4/ hole, at 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
(2) the every hole 1.5 μ l of Lipofectamine2000, being dissolved in 100 μ l serum-frees does not have in the plain DMEM culture medium of antibiosis, fully mix back incubated at room 5min, plasmid LC3-eGFP (U.S. invitrogen company) is every hole 2 μ g, be dissolved in the DMEM culture medium (U.S. GIBCO company) of 100 μ l DMEM serum-free antibiotic-frees, two kinds of dissolving mix homogeneously behind the incubated at room 5min after fully mixing, incubated at room 20-25min.
(3) cell of incubated overnight adds the mixture that step (2) was hatched after washing 2 times with DMEM (serum-free antibiotic-free) culture medium, at 37 ℃, and 5% (volumn concentration) CO
2Condition of culture is cultivated 30min, and every hole supplementing culture medium DMEM complete medium 300 μ l continue to cultivate.
(4) behind the transfection 48hr, cell is transferred to (U.S. GIBCO company) 24 porocyte culture plates that the DMEM culture medium is housed after by dilution in 1: 15, and adds the G418 antibiotic-screening cell (U.S. Sigma company) of 0.5mg/ml, changes a subculture in per three days.Under fluorescence microscope, choose the green positive colony LC3-eGFP/HeLa cell of stably express LC3-eGFP after ten days, stand-by.
2, the LC3-eGFP/HeLa cell inoculation that will obtain after step 1 screening is being equipped with in (U.S. GIBCO company) 24 porocyte culture plates of DMEM culture medium, and 24 orifice plate cell densities are about 3-5 * 10
4/ hole, at 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
3, adding final concentration in 24 holes of step 2 is the cysteamine (cysteamine among Fig. 1) of 1.5mM, Induces Autophagy, carrying out fluorescence microscope after cultivating 24 hours takes pictures, with without any the cell of handling as negative control (contrasting among Fig. 1), (final concentration is 200nM with autophagy promoter rapamycin, U.S. Sigma company) as the positive control (rapamycin among Fig. 1) that causes autophagy, use common processing of cysteamine of autophagy inhibitor 3-methyladenine (3-MA, final concentration are 2mM) and 1.5mM to prove further that cysteamine caused the ability (cysteamine among Fig. 1+3-methyl purine) of autophagy in 24 hours simultaneously.Behind the cell transfecting eGFP-LC3 plasmid, under normal circumstances even dispersion is in Cytoplasm for the eGFP-LC3 albumen of expression, but when cell generation autophagy, eGFP-LC3 will accumulate in and form point-like on the film of autophagic vacuole and assemble.
The result shows:
The result as shown in Figure 1, the result shows, after 1.5mM cysteamine is handled LC3-eGFP/HeLa, even dispersion GFP-LC3 protein aggregation in Cytoplasm forms green point-like on the film of autophagosome assembles, and the LC3 point-like assembles and can be suppressed (Fig. 1) by the special inhibitor 3-methyl purine of autophagy, illustrate cysteamine cause the LC3 point-like accumulative be the performance of cell autophagy.
Embodiment 2: cysteamine is induced 293A cell autophagy [Induces Autophagy in the HEKC 293A of stably express LC3-eGFP, with autophagy promoter rapmycin as positive control]
Experimental technique:
1, HEKC (LC3-eGFP/293A) method of screening stably express LC3-eGFP is as follows:
(1) 293A HEKC (available from Shanghai cell institute of the Chinese Academy of Sciences) is seeded in (U.S. GIBCO company) 24 porocyte culture plates that the DMEM culture medium is housed, the about 3-5 of density * 10
4/ hole, at 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
(2) preparation transfection composite, method is as follows: the every hole 1.5 μ l of Lipofectamine2000, be dissolved in 100 μ l DMEM (serum-free antibiotic-free) culture medium, fully mix back incubated at room 5min, LC3-eGFP is every hole 2 μ g, be dissolved in 100 μ l DMEM (serum-free antibiotic-free) culture medium two kinds of dissolving mix homogeneously behind the incubated at room 5min after fully mixing, incubated at room 20-25min.
(3) cell of incubated overnight washs with DMEM (serum-free antibiotic-free) culture medium and adds the transfection composite that step (2) obtains after 2 times, at 37 ℃, and 5% (volumn concentration) CO
2Condition of culture is cultivated 30min, and supplementing culture medium DMEM complete medium 300 μ l, continue to cultivate by 37 ℃.
(4) behind the transfection 48hr, cell is by (U.S. GIBCO company) 24 porocyte culture plates of 1: 15 dilution back switching DMEM culture medium, and adds the G418 screening cell of 0.5mg/ml, changes a subculture in per three days.Under fluorescence microscope, choose the green positive colony of stably express LC3-eGFP after ten days, stand-by.
2, the LC3-eGFP/293A cell inoculation after screening is in 24 porocyte culture plates, and cell density is about 3-5 * 10
4/ hole, incubated overnight, stand-by.
3, adding final concentration in 24 holes of step 2 is the cysteamine (cysteamine among Fig. 2) of 1.5mM, and Induces Autophagy is carried out fluorescence microscope and taken pictures after cultivating 24 hours.With without any the cell of handling as negative control (contrasting among Fig. 2), (final concentration is 200nM with autophagy promoter rapamycin, U.S. Sigma company) as the positive control (rapamycin among Fig. 1) that causes autophagy, use common processing of cysteamine of autophagy inhibitor 3-methyladenine (3-MA, final concentration are 2mM) and 1.5mM to prove further that cysteamine caused the ability (cysteamine among Fig. 1+3-methyladenine) of autophagy in 24 hours simultaneously.
The result shows:
1, the HeLa cell inoculation is in (U.S. GIBCO company) 24 porocyte culture plates of DMEM culture medium, and 24 orifice plate cell densities are about 3-5 * 10
4/ hole, at 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2, add final concentration respectively and be that the 1.5mM cysteamine handles, respectively at 37 ℃, 5% (volumn concentration) CO
2Collect after cultivating 0hr, 5hr, 12hr, 18hr, 24hr in the incubator,, add again after electrophoresis sample-loading buffer (available from green skies company) boiling water boils 10min, make the electrophoresis sample with cell pyrolysis liquid (available from green skies company) cell lysis.Wet commentaries on classics method is transferred to (available from U.S. Amersham company) on the nylon membrane with albumen after carrying out the 12%SDS-PAGE electrophoresis then.Anti-as one with anti-LC3 (Novus company), anti-rabbit (Promega company) is two anti-, carries out Western blot.Come the protein content (glyceraldehyde-3-phosphate dehydrogenase of quantitative matched group and experimental group as a reference as an anti-detection GAPDH (glyceraldehyde-3-phosphate dehydrogenase) with anti-GAPDH (U.S. Millipore Corp.), be intracellular composing type albumen, in each intracellular level unanimity).
The result shows:
The result as shown in Figure 4, the result shows, cysteamine causes that in the HeLa cell cell autophagy effect is with the prolongation of the time of processing enhanced (Fig. 4).
Embodiment 5: cysteamine causes the dosage effect [endogenic albumen LC3I transforms to autophagy marker protein LC3II type] of cell autophagy in the HeLa cell
Experimental technique:
1, the HeLa cell inoculation is in (U.S. GIBCO company) 24 porocyte culture plates of DMEM culture medium, and 24 orifice plate cell densities are about 3-5 * 10
4/ hole, at 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2, each hole adds the cysteamine that final concentration is 0.5mM, 1.0mM, 1.5mM or 2.0mM respectively, the cell that does not have any processing is matched group (contrast among Fig. 5), with autophagy promoter final concentration is that 200nM rapamycin treatment (U.S. Sigma company) is as the positive control that causes autophagy (rapamycin among Fig. 5), at 37 ℃, 5% (volumn concentration) CO
2Incubator, collecting cell behind the cell culture 24hr with cell pyrolysis liquid (available from green skies company) cell lysis, adds after electrophoresis sample-loading buffer (available from green skies company) boiling water boils 10min again, makes the electrophoresis sample.Wet commentaries on classics method is transferred to (available from U.S. Amersham company) on the nylon membrane with albumen after carrying out the 12%SDS-PAGE electrophoresis then.Anti-as one with anti-LC3 (Novus company), anti-rabbit (Promega company) is two anti-, carries out Western blot.Come the protein content (glyceraldehyde-3-phosphate dehydrogenase of quantitative matched group and experimental group as a reference as an anti-detection GAPDH (glyceraldehyde-3-phosphate dehydrogenase) with anti-GAPDH (U.S. Millipore Corp.), be intracellular composing type albumen, in each intracellular level unanimity)
The result as shown in Figure 2, the result shows, the 1.5mM cysteamine just can cause the green point-like gathering of eGFP-LC3 in LC3-eGFP/293A, the eGFP-LC3 point-like is assembled and can be suppressed (Fig. 2) by the special inhibitor 3-MA of autophagy, illustrate cysteamine cause the eGFP-LC3 point-like accumulative be the performance of cell autophagy.
Embodiment 3: cysteamine causes melanoma cell B16 and human breast carcinoma MCF-7 cell autophagy [endogenic albumen LC3I (microtubule-associated protein LC3I) transforms to autophagy marker protein LC3II type]
When melanoma cell B16 and human breast carcinoma MCF-7 cell autophagy, endogenic albumen LC3I transforms to autophagy marker protein LC3II type, therefore followingly is converted to autophagy marker protein LC3II type and determines whether melanoma cell B16 and human breast carcinoma MCF-7 cell autophagy takes place by detecting endogenic albumen LC3I.
Experimental technique:
1, respectively melanoma cell B16 (available from Shanghai cell institute of the Chinese Academy of Sciences) or human breast carcinoma MCF-7 cell are seeded in respectively in (U.S. GIBCO company) 24 porocyte culture plates of DMEM culture medium, 24 orifice plate cell densities are about 3-5 * 10
4/ hole, 37 ℃, incubated overnight, stand-by.
2, add final concentration respectively and handle for the 1.5mM cysteamine, at 37 ℃, 5% (volumn concentration) CO
2Cultivate in the incubator after 24 hours and collect,, add again after electrophoresis sample-loading buffer (available from green skies company) boiling water boils 10min, make the electrophoresis sample with cell pyrolysis liquid (available from green skies company) cell lysis.Carry out behind the 12%SDS-PAGE electrophoresis wet commentaries on classics method then albumen transferred to (available from U.S. Amersham company) on the nylon membrane, with the melanoma cell B16 that do not have any processing as matched group.Anti-as one with anti-LC3 (Novus company), anti-rabbit (Promega company) is two anti-, carries out Western blot.Come the protein content (glyceraldehyde-3-phosphate dehydrogenase of quantitative matched group and experimental group as a reference as an anti-detection GAPDH (glyceraldehyde-3-phosphate dehydrogenase) with anti-GAPDH (U.S. Millipore Corp.), be intracellular composing type albumen, in each intracellular level unanimity).
The result shows:
The result as shown in Figure 3, the result shows, in melanoma cell B16 and human breast carcinoma MCF-7, with respect to contrast, cysteamine is handled back LC3II protein content to be increased greatly, illustrating that cysteamine at melanoma cell B16, also can cause cell autophagy among the human breast carcinoma MCF-7, is the sign (Fig. 3) that cell autophagy takes place because strengthen endogenic albumen LC3I to the conversion of albumen LC3II type.
Embodiment 4: cysteamine causes the time effect [endogenic albumen LC3I transforms to autophagy marker protein LC3II type] of cell autophagy in the HeLa cell
Experimental technique:
The result shows:
The result as shown in Figure 5, the result shows that cysteamine causes that in the HeLa cell cell autophagy effect is with the increase of cysteamine concentration enhanced (Fig. 5).
Embodiment 6: cysteamine causes the increasing of autophagy substrate protein p62 [with autophagy inhibitor 3-MA as negative control] in the HeLa cell
The albumen of the false folding in the cell autophagy energy degradation of cell or the organelle of damaged, P62 albumen also is the substrate of cell autophagy, p62 albumen when complete autophagy takes place cell in the meeting degradation of cell, if the downstream of cell autophagy is blocked the degraded that will block p62, so the proteic non-integrity that increases explanation autophagy that can be indirect of p62.
Experimental technique:
1.HeLa cell inoculation is in being equipped with (U.S. GIBCO company) 24 porocyte culture plates of DMEM culture medium, 24 orifice plate cell densities are about 3-5 * 10
4/ hole, at 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2. each hole is handled and is respectively the combination that adds final concentration 1.5mM cysteamine, 2mM 3-methyladenine, 1.5mM cysteamine and 2mM 3-methyladenine, with the cell of not doing any processing as contrast, respectively at 37 ℃, 5% (volumn concentration) CO
2Cultivate collecting cell behind the 24hr in the incubator,, add again after electrophoresis sample-loading buffer (available from green skies company) boiling water boils 10min, make the electrophoresis sample with cell pyrolysis liquid (available from green skies company) cell lysis.Wet commentaries on classics method is transferred to (available from U.S. Amersham company) on the nylon membrane with albumen after carrying out the 12%SDS-PAGE electrophoresis then.With anti-p62 (BIOMOL company) is one anti-, anti-rabbit (Promega company) is two anti-, carry out Western blot, come the protein content (glyceraldehyde-3-phosphate dehydrogenase of quantitative matched group and experimental group as a reference as an anti-detection GAPDH (glyceraldehyde-3-phosphate dehydrogenase) with anti-GAPDH, be intracellular composing type albumen, in each intracellular level unanimity)
The result shows:
The result as shown in Figure 6, the result shows that cysteamine processed group and matched group comparison autophagy substrate p62 increase (see figure 6), illustrates that the downstream passages of cell autophagy may be blocked, the existence of this pair cell is deleterious.
Embodiment 7: the influence of cysteamine pair cell vigor [thiazole blue laws (MTT) method detects cell death]
Experimental technique:
1, the HeLa cell inoculation is in (U.S. GIBCO company) 96 porocyte culture plates of DMEM culture medium, the about 0.8-1 of 96 orifice plate cell densities * 10
4/ hole, 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2, add the cysteamine that final concentration is 0mM, 0.5mM, 1mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 4.0mM respectively in each hole of Tissue Culture Plate.37 ℃, 5% (volumn concentration) CO
2After cultivating 24hr in the incubator, according to the method detection cell mortality of step 3.
3,96 porocytes are after above-mentioned processing, and every hole adds 5mg/ml MTT (tetrazolium bromide) 10 μ l, at 37 ℃, and 5%CO
2After cultivation was hatched about 2-4 hour in the incubator, after purple crystals occurring in the cell, after supernatant was removed in careful suction, every hole added 100 μ l DMSO (dimethyl sulfoxide), is put in the dark place 2 hours.After shaking up, microplate reader detects the uv absorption at its 570nm place.Along with the many more absorption values at the 570nm place of cell death are just more little, the absorption value of the cell that the 0mM cysteamine is handled is made as 100% living cells, calculates cell viability then in proportion.
The result shows:
The result as shown in Figure 7, the result shows, low concentration cysteamine pair cell vigor is influence not, the cysteamine of high concentration can cause cell death (Fig. 7).
Embodiment 8: cysteamine promotes low concentration chemotherapy medicine amycin (doxorubicin) to kill the experiment of cancer HeLa cell [detecting with iodate third heavy stone used as an anchor (PI) staining]
Experimental technique:
1, the HeLa cell inoculation is in (U.S. GIBCO company) 96 porocyte culture plates of DMEM culture medium, the about 0.8-1 of 96 orifice plate cell densities * 10
4/ hole, 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2, to handle and be respectively final concentration be that 1.5mM cysteamine (CS among Fig. 8), 0.75 μ g/ml amycin (Dox among Fig. 8), final concentration are the combination (DOX+CS among Fig. 8) of 1.5mM cysteamine and 0.75 μ g/ml amycin each hole in the Tissue Culture Plate, is contrast not add low dosage amycin (CS among Fig. 8) and not add 1.5mM (final concentration) cysteamine (DOX among Fig. 8) wherein.After cultivating 24hr, according to the method detection cell mortality of step 3.
3, cell is taken pictures at microscopically then with 2 μ g/ml Hirsts (Hochest33342 is available from green skies company) and the dyeing of 5 μ g/ml iodate third heavy stones used as an anchor (PI) 15 minutes.Hochest33342 is used for transfect cell nuclear specially, be used for counting total cell quantity, PI dyes dead cell, and it can penetrate cell membrane dead or dead cell and insert in the double-stranded DNA, can not enter living cells, the percentage ratio that dead cell accounts for all cells is the mortality rate of cell.
The result shows:
The result as shown in Figure 8, the result shows that the cysteamine of low concentration and the amycin of low dosage cause cancer cell death hardly, the two synergism then causes dead 65.5 ± 2.6% (Fig. 8) that the HeLa cell is a large amount of.
Embodiment 9: cysteamine promotes the experiment that low concentration chemotherapy medicine amycin (doxorubicin) kills melanoma cancerous cell B16
Experimental technique:
1, melanoma cancerous cell B16 cell inoculation is in (U.S. GIBCO company) 96 porocyte culture plates of DMEM culture medium, the about 0.8-1 of 96 orifice plate cell densities * 10
4/ hole, 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2, to handle and be respectively final concentration be that 1.5mM cysteamine (CS among Fig. 9), 0.8 μ g/ml amycin (Dox among Fig. 9), final concentration are the combination (DOX+CS among Fig. 9) of 1.5mM cysteamine and 0.8 μ g/ml amycin each hole in the Tissue Culture Plate, is contrast not add low dosage amycin (CS among Fig. 9) and not add 1.5mM (final concentration) cysteamine (DOX among Fig. 9) wherein.After cultivating 24hr, according to the method detection cell mortality of embodiment 8
The result shows:
The result as shown in Figure 9, the result shows that the cysteamine of low concentration and the amycin of low dosage cause cancer cell death hardly, the two synergism then strengthens dead 21.892 ± 1.053% (Fig. 9) of B16 cell.
Embodiment 10: cysteamine promotes the experiment that low concentration chemotherapy medicine amycin (doxorubicin) kills human breast carcinoma MCF-7
Experimental technique:
1, human breast carcinoma MCF-7 cell inoculation is in (U.S. GIBCO company) 96 porocyte culture plates of DMEM culture medium, the about 0.8-1 of 96 orifice plate cell densities * 10
4/ hole, 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2, to handle and be respectively final concentration be that 1.5mM cysteamine (CS among Figure 10), 0.8 μ g/ml amycin (Dox among Figure 10), final concentration are the combination (DOX+CS among Fig. 9) of 1.5mM cysteamine and 1.0 μ g/ml amycin each hole in the Tissue Culture Plate, is contrast not add low dosage amycin (CS among Figure 10) and not add 1.5mM (final concentration) cysteamine (DOX among Figure 10) wherein.After cultivating 24hr, according to the method detection cell mortality of embodiment 8
The result shows:
The result as shown in figure 10, the result shows that the cysteamine of low concentration and the amycin of low dosage cause cancer cell death hardly, the two synergism then strengthens dead 54.23 ± 2.146% (Figure 10) of MCF-7 cell.
Embodiment 11: cysteamine promotes the experiment that low concentration chemotherapy medicine amycin (doxorubicin) kills drug resistance MCF-7 MCF-7/ADR
Experimental technique:
1, screening amycin drug resistance MCF-7 MCF-7/ADR cell method is as follows: MCF-7 MCF-7 is seeded in (U.S. GIBCO company) 24 porocyte culture plates of DMEM culture medium, the amycin that adds final concentration 0.01 μ M, 37 ℃, 5% (volumn concentration) CO
2Incubator is cultivated after two months, adds the amycin of 0.1 μ M again, takes turns screening and culturing after two months through second, improves drug level to 1 μ M, and screening and culturing obtains stable amycin drug resistance MCF-7 MCF-7/ADR cell after 3 months.
2, with drug resistance MCF-7 MCF-7/ADR cell inoculation in (U.S. GIBCO company) 96 porocyte culture plates of DMEM culture medium, the about 0.8-1 of 96 orifice plate cell densities * 10
4/ hole, 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
3, to handle and be respectively final concentration be that 1.5mM cysteamine (CS among Figure 11), 20 μ g/ml amycin (Dox among Figure 11), final concentration are the combination (DOX+CS among Figure 11) of 1.5mM cysteamine and 20 μ g/ml amycin each hole in the Tissue Culture Plate, is contrast not add low dosage amycin (CS among Figure 11) and not add 1.5mM (final concentration) cysteamine (DOX among Figure 11) wherein.After cultivating 24hr, detect the cell mortality result according to the method for embodiment 8 and show:
The result as shown in figure 11, the result shows that low dosage cysteamine and amycin cause drug-resistant cell death hardly, the two synergism then cause cancerous cell a large amount of dead 44.23 ± 1.8%.
Embodiment 12:HeLa transit cell dyes Atg5siRNA and reduces the autophagy level, reduces the cell killing that autophagy is brought accordingly
Experimental technique:
1, the HeLa cell inoculation is in the Tissue Culture Plate of (U.S. GIBCO company) 2 60mm of DMEM culture medium, and cell density is about 2 * 10
5/ hole, 37 ℃, 5% (volumn concentration) CO
2Incubated overnight in the incubator, stand-by.
2, the every hole 1.5 μ l of siRNA transfection: Lipofectamine2000, be dissolved in 100 μ l DMEM (serum-free antibiotic-free) culture medium, fully mix back incubated at room 5min, negative control siRNA (purchasing in Shanghai JiMa pharmacy Technology Co., Ltd) and each 2 μ g of Atg5siRNA (purchasing in Shanghai JiMa pharmacy Technology Co., Ltd) of not having homology with mammalian cell are dissolved in respectively in 100 μ l DMEM (serum-free antibiotic-free) culture medium, two kinds of dissolving mix homogeneously behind the incubated at room 5min after fully mixing, incubated at room 20-25min.
3, after the cell of incubated overnight washs 2 times with DMEM (serum-free antibiotic-free) culture medium, add respectively and hatched contrast siRNA and Atg5siRNA, at 37 ℃, 5% (volumn concentration) CO
2Cultivate 30min in the incubator, supplementing culture medium DMEM complete medium 300 μ l continue to cultivate.
4, in the cell culture fluid of transfection contrast siRNA and add the 1.5mM cysteamine respectively in the cell culture fluid of Atg5siRNA and handle, the cell of not doing any processing in contrast.
5, cysteamine is handled collecting cell behind the 24hr, and with cell pyrolysis liquid (green the skies company) cell lysis, after boiling water boiled 10min, wet commentaries on classics method was transferred to (Amersham company) on the nylon membrane with albumen behind the 12%SDS-PAGE electrophoresis.With antibody anti-Atg5 (santa company) and anti-LC3 (Novus company) is that an anti-anti-rabbit (Promega company) is two anti-, carries out Western blot.With respect to the cell (consiRNA among the right figure of Figure 12) that contrast siRNA handles, the level of autophagy has reduced (Atg5siRNA among the right figure of Figure 12) in the cell of transfection Atg5siRNA.
6, adding final concentration in the cell culture fluid of transfection contrast siRNA and in the cell culture fluid of transfection Atg5siRNA respectively is that 1.5mM cysteamine (CS among the left figure of Figure 12), 20 μ g/ml amycin (Dox among the left figure of Figure 12), final concentration are the combination (DOX+CS among the left figure of Figure 12) of 1.5mM cysteamine and 20 μ g/ml amycin, is contrast not add low dosage amycin (CS among the left figure of Figure 12) and not add 1.5mM (final concentration) cysteamine (DOX among the left figure of Figure 12) wherein.After cultivating 24hr, according to the method detection cell mortality of embodiment 8.
The result shows:
The result as shown in figure 12, the result shows, the cell of transfection Atg5siRNA, the ability of autophagy that causes cysteamine weakens (the right figure of Figure 12), corresponding cysteamine promotes that amycin causes that the ability of cell death also weakens (the left figure of Figure 12, control siRNA is the cell of transfection contrast siRNA among the left figure of Figure 12, and Atg5siRNA is the cell of transfection Atg5siRNA).
Embodiment 13: cysteamine is worked in coordination with the amycin killing tumor cells
Experimental technique:
1, the B16 cell inoculation is in having (U.S. GIBCO company) Tissue Culture Flask of DMEM culture medium, and 37 ℃, cultivate in 5% (volumn concentration) CO2 incubator, wait to cover with culture bottle with cell dissociation and centrifugally get off.
2, with 1x106 cell inoculation in C57 mice (available from Shanghai Shrek company), 0.5mm3 administration when treating that tumor is long and reaching in a week, mouse is divided into four groups at random, matched group: give normal saline, the dosed administration of cysteamine group: 500mg/kg (with respect to the mouse body weight), the dosed administration of amycin group: 5mg/kg (with respect to the mouse body weight), combination medicine group: 500mg/kg cysteamine and 5mg/kg amycin (with respect to the mouse body weight) co-administered every other day is administered once.
3, after two weeks mouse section neck is put to death, tumor is stripped down weigh, statistics tumor size is weighed the effect (Figure 13) of the collaborative amycin killing tumor cells of cysteamine
The result shows:
The result as shown in figure 13, the result shows that cysteamine can be strengthened the ability (Figure 13) that amycin kills and wounds cancerous cell greatly.
The description of test of the foregoing description 1-13 is promoted the sensitivity of cancerous cell to chemotherapeutics by the cysteamine of low dosage, thereby has realized the effective kill cancer cell of chemotherapeutics by low dosage.Experimental results show that chemotherapeutics that cysteamine only need work in coordination with low dosage just effectively kill cancer cell and chemotherapeutic resistance cancerous cell, thereby prove that promptly medicament of the present invention reduces cancer therapy drug greatly in the chemotherapy of cancerous cell and resistance cancerous cell use amount reduces the side effect of chemotherapy.Therefore can utilize this principle design cancer therapy drug of the present invention, it comprises common chemotherapeutics and adjuvant cysteamine.Cysteamine is assisted low-dosage chemotherapy medicine killing tumor cell and chemotherapeutics resisting cell in the anticancer agent, thereby strengthens the side effect of the chemotherapy effect reduction chemotherapeutics of chemotherapeutics greatly.
Though it is clinical that the combination medicament of this treatment cancer is not applied to, cell experiment proves that when using cysteamine and chemotherapeutic agents, the comparable single use chemotherapeutic agents of chemotherapeutic agents is a decrement.The doctor can determine dosage according to specifically to patient's state of an illness understanding and chemotherapy effect under safe dose.The component cysteamine is clinical to be used for the treatment of acute metal poisoning and to prevent and treat radiation sickness dosage, generally is intravenous injection, and dosage is 0.3g/ time, one day 1-2 time.During cysteamine in the clinical use combination medicament of the present invention, can use with reference to its clinical dosage safe in utilization, under safe dose, the high more curative effect of using dosage is good more.The dosage of suggestion clinical practice is with reference to existing clinical using dosage.Such as, separately but wherein the dosage of the clinical use of component amycin is generally at 30-100mg/m
2, high dose can reach 300mg/m
2, when using jointly, can take the circumstances into consideration decrement with cysteamine.
Claims (10)
1. the application of cysteamine in the medicine of preparation treatment cancer.
2. combination medicament for the treatment of cancer, its active component is cysteamine and chemotherapeutics.
3. combination medicament according to claim 2 is characterized in that: also comprise medically acceptable adjuvant in the combination medicament of described treatment cancer.
4. according to claim 2 or 3 described combination medicaments, it is characterized in that: described chemotherapeutics is an amycin.
5. according to any described combination medicament among the claim 2-4, it is characterized in that: described cancer comprises former of originating from brain, central nervous system, kidney, liver, gallbladder, incidence, oral cavity, thyroid, skin, mucosa, body of gland, blood vessel, osseous tissue, lymph node, lungs, esophagus, stomach, mammary gland, pancreas, eyes, nasopharynx part, uterus, ovary, endometrium, cervix uteri, prostate, bladder, colon or rectum or cancer or sarcoma or the carcinosarcoma that shifts.
6. combination medicament according to claim 5 is characterized in that: described cancer is cervical cancer, breast carcinoma, melanoma or renal carcinoma.
7. cysteamine is improving cancerous cell to the application in the sensitivity of chemotherapeutics.
8. the application of cysteamine in preparation tumor cell inhibitor.
9. cysteamine and the chemotherapeutics application in preparation tumor cell inhibitor.
10. it is characterized in that: described tumor cell behaviour cervical cancer cell HeLa cell, MCF-7 MCF-7 or melanoma cancerous cell B16 cell according to Claim 8 or 9 described application; Described chemotherapeutics is for being amycin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010143701 CN101797242A (en) | 2010-04-07 | 2010-04-07 | Application of cysteamine in preparing medicine for treating cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010143701 CN101797242A (en) | 2010-04-07 | 2010-04-07 | Application of cysteamine in preparing medicine for treating cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101797242A true CN101797242A (en) | 2010-08-11 |
Family
ID=42593183
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010143701 Pending CN101797242A (en) | 2010-04-07 | 2010-04-07 | Application of cysteamine in preparing medicine for treating cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101797242A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013120086A1 (en) * | 2012-02-10 | 2013-08-15 | Whitehead Institute For Biomedical Research | Inhibition of the glycine cleavage system for treatment of cancer |
| US20140314841A1 (en) * | 2013-04-19 | 2014-10-23 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv | Use of Cysteamine and Derivatives Thereof to Suppress Tumor Metastases |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060089313A1 (en) * | 2001-11-29 | 2006-04-27 | Sound Pharmaceuticals Inc. | Methods and compositions for ameliorating the undesirable effects of chemotherapy |
-
2010
- 2010-04-07 CN CN 201010143701 patent/CN101797242A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060089313A1 (en) * | 2001-11-29 | 2006-04-27 | Sound Pharmaceuticals Inc. | Methods and compositions for ameliorating the undesirable effects of chemotherapy |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013120086A1 (en) * | 2012-02-10 | 2013-08-15 | Whitehead Institute For Biomedical Research | Inhibition of the glycine cleavage system for treatment of cancer |
| JP2015509489A (en) * | 2012-02-10 | 2015-03-30 | ホワイトヘッド・インスティテュート・フォー・バイオメディカル・リサーチ | Inhibition of the glycine cleavage system for the treatment of cancer |
| EP2811991A4 (en) * | 2012-02-10 | 2016-03-16 | Whitehead Biomedical Inst | Inhibition of the glycine cleavage system for treatment of cancer |
| US9493775B2 (en) | 2012-02-10 | 2016-11-15 | Whitehead Institute For Biomedical Research | Inhibition of the glycine cleavage system for treatment of cancer |
| US20140314841A1 (en) * | 2013-04-19 | 2014-10-23 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv | Use of Cysteamine and Derivatives Thereof to Suppress Tumor Metastases |
| US20160184242A1 (en) * | 2013-04-19 | 2016-06-30 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | Use of Cysteamine and Derivatives Thereof to Suppress Tumor Metastases |
| US20190142769A1 (en) * | 2013-04-19 | 2019-05-16 | Meshaberase, LLC | Use of cysteamine and derivatives thereof to suppress tumor metastases |
| US20200147010A1 (en) * | 2013-04-19 | 2020-05-14 | Meshaberase, LLC | Use of cysteamine and derivatives thereof to suppress tumor metastases |
| US11052057B2 (en) | 2013-04-19 | 2021-07-06 | Meshaberase, LLC | Use of cysteamine and derivatives thereof to suppress tumor metastases |
| US11844769B2 (en) | 2013-04-19 | 2023-12-19 | Meshaberase, LLC | Use of cysteamine and derivatives thereof to suppress tumor metastases |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114848589A (en) | Medicinal solution with anti-tumor synergistic attenuation effects and medicinal composition containing medicinal solution | |
| CN104398526B (en) | The application of triptolide and Celastrol in antineoplastic is prepared | |
| CN102552908B (en) | Pharmaceutical composition containing artemisinin, artemisinin derivatives and Bcl-2 inhibitor and application thereof | |
| EP2263668B1 (en) | Use of tea polyphenols in preparing medicaments for prevention or treatment of tumors | |
| CN104434939B (en) | Antitumor medicament composition of panax notoginseng saponins R7 and oridonin with capabilities of reducing toxicity and enhancing efficacy and application of antitumor medicament composition | |
| CN105476996A (en) | Application of curcumin and afatinib for combined treatment of non-small cell lung cancer | |
| CN105380956B (en) | A kind of pharmaceutical composition of Dana Delany containing Chinese mugwort for treating leukaemia and application | |
| CN101797242A (en) | Application of cysteamine in preparing medicine for treating cancer | |
| CN102218140A (en) | Tumor resistance effect of medicine combination of salvianolic acid B and coxib medicaments | |
| US20190183893A1 (en) | Low dose of sildenafil as an antitumor drug | |
| US10441564B2 (en) | Fructose analogs and their combinations as anti-cancer agents | |
| CN110496129A (en) | The anti-tumor activity and its new application of BI 1356 | |
| JP7381115B2 (en) | Compositions and their application in the preparation of medicines for cancer treatment | |
| CN115300624A (en) | Application of ginsenoside and PD-1 blocker in preparation of head and neck squamous cell carcinoma resisting medicine | |
| WO2013012771A2 (en) | Clinical applications of a recombinant human endostatin adenovirus (e10a) injection | |
| CN102440987B (en) | Drug compound of apigenin, apigenin-like derivants, artemisinin and artemisinin-like derivants and application thereof | |
| CN102440994A (en) | Application of ganoderic acid G as immune synergist and super-antigen dependent therapeutic medicine in tumour treatments | |
| CN106491603A (en) | A kind of pharmaceutical composition and its application containing capsaicin and Sorafenib | |
| CN103127510A (en) | Medicine composition containing hepatic cell growth factor receptor inhibitor and Bcl-2 inhibitor and application thereof | |
| CN102688490A (en) | Pharmaceutical composition containing evodiamine, evodiamine derivative and Bc1-2 inhibitor, and the application | |
| CN101856393B (en) | Application of compound Yiganling composition in aspect of auxiliary treatment of chemotherapy of tumors | |
| CN115177620A (en) | Application of seolonide or pharmaceutically acceptable salt thereof in preparation of medicine for preventing or treating follicular lymphoma | |
| CN106668061B (en) | A kind of anticancer pharmaceutical composition containing cis-platinum | |
| Chang et al. | Experimental study on growth inhibition against anaplastic thyroid carcinoma of animal model by application of ginsenoside Rg1 | |
| CN116688001A (en) | Traditional Chinese medicine composition for preventing and treating tumor bone metastasis and reducing osteoporosis and/or toxic and side effects of chemotherapy, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20100811 |