CN100443080C - Medicinal composition for preventing and treating cancer diseases - Google Patents
Medicinal composition for preventing and treating cancer diseases Download PDFInfo
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Abstract
A composite medicine for preventing and treating cancer contains the conventional anticancer chemical medicine for preventing and treating cancer, and the versulin as synergistic for decreasing the dosage of said anticancer chemical medicine.
Description
Technical field
The present invention relates to a kind of prevention and antitumor medicine composition.
Background technology
The celery flavin, have another name called apigenin (apigenin, 4 ', 5,7 ,-trihydroxyflavone), be the nontoxic food source chemical compound that comes from plant.The celery flavin is a kind of flavonoid of common food, its low toxicity, and no mutagenicity is distributed widely in many fruits and vegetables, comprises parsley, Bulbus Allii Cepae, Fructus Citri junoris, tea, Flos Chrysanthemi, Fructus Hordei Germinatus and some flavoring agent.The celery flavin is used as a kind of health food additive, and it has anticancer function recent findings.
Cancer is the disease of serious harm health and lives in the world, and in the U.S., cancer has become the main causes of death of crowd below 85 years old, does not still have good treatment means so far.The chemotherapy cancer (abbreviation chemotherapy) of utilizing cancer therapy drug to carry out is the important means of treatment cancer, the growth of the main anticancer of antitumor drug and diffusion, thus reach the purpose of treatment.But, thereby many tumor cells can produce the effect of drug resistance opposing medicine gradually, make cancer return and cause the treatment failure.Simultaneously, many cancer therapy drugs have certain toxicity, produce no small toxic and side effects, infringement patient's health in therapeutic process.Therefore, except developing new cancer therapy drug, also be the important goal of studying thereby overcome the existing cancer therapy drug curative effect of tumor cell drug resistance reduction dosage raising.The quick growth needs nutrition of tumor, providing of nutrient substance needs blood vessel, and the neovascularity of tumor is the tumor growth essential condition.Therefore, suppress tumor-blood-vessel growth and become a kind of new important treatment and the means of prophylaxis of cancer.VEGF VEGF (Vascular Endothelial Growth Factor) plays an important role in tumor-blood-vessel growth, and the synthetic adjusting that is subjected to hypoxia inducible factor HIF-1 (Hypoxia-Inducible Factor1) of VEGF.Therefore suppress the expression of HIF-1 and can play the effect that suppresses angiogenesis and tumor growth.
Descending appears in the five-year survival rate of most of cancers, but mechanism is still unclear.Although cisplatin, paclitaxel and other chemotherapeutic have been widely used in the treatment cancer, and the resistance that cancer cell produces these widely used medicines gradually finally causes patient's death.Therefore, the task of top priority, we must seek new cancer treatment drugs.
Summary of the invention
The purpose of this invention is to provide a kind of antineoplastic pharmaceutical compositions that better curative effect and toxic and side effects significantly reduce that has.The present invention is used in combination by two kinds of different cancer therapy drugs, reaches collaborative mutually, improves curative effect, reduces toxic and side effects and chemical sproof effect.
Because the celery flavin extensively is present in the plant food, has the effect that suppresses growth of tumour cell and angiogenesis again, thereby can be used for the prevention of tumor, as be used to produce health food.Can suppress the growth of the tumor cell line of some kinds although known the celery flavin, comprise colon cancer, breast carcinoma and carcinoma of prostate etc., it is still unknown that it unites the effect of use to the potentiation of chemotherapeutics and other chemotherapy agents and it.
Hypoxia inducible factor HIF-1 and VEGF VEGF play an important role in angiogenesis, and the expression that suppresses these two kinds of factors can cause growth of tumor to be suppressed.Discover that the celery flavin has the very strong inhibition kinds of tumor cells HIF-1 and the ability of vegf expression, show that with the experiment in vitro result celery flavin is a good angiogenesis inhibitor, has potential oncotherapy and prophylactic applications prospect in the body.The celery flavin suppresses the angiogenesis that tumor cell causes, can be used to suppress tumor-blood-vessel growth and tumor growth, also can suppress the stimulating factor of angiogenic growth: VEGF VEGF and hypoxia inducible factor HIF-1 α.Simultaneously, this material can improve the sensitivity of tumor cell to clinical cancer therapy drug commonly used, has reduced the drug resistance of tumor cell, has improved curative effect.Data show that the celery flavin has the potential of the medicine that becomes treatment and prophylaxis of cancer.
And during the medication combined use of celery flavin and conventional chemotherapy, just become the medicine of very effective prevention and treatment cancer.The celery flavin not only can suppress the growth of kinds of tumor cells separately, main is the curative effect that it can promote multiple cancer therapy drug, promote the curative effect of these medicine anticancer growths (especially some drug-resistant tumor cells) and tumor growth, reduce the IC of medicine
50(thereby making cell improve the minimizing medication) to the sensitivity of medicine.Therefore, it can share with some cancer therapy drug, overcomes the drug resistance of cancerous cell, reduces dosage, improves therapeutic effect.
The pharmaceutical composition of prevention of the present invention and treatment cancer, it comprises the chemotherapeutics of effective anticancer and the derivant and the salt thereof of celery flavin or celery flavin.
Described chemotherapeutics is meant the conventional treatment or the chemical synthetic drug of prophylaxis of cancer, as cisplatin (Cisplatin), paclitaxel (Paclitaxel), amycin (Doxorubicin), 5-fluorouracil (5-fluorouracil), etoposide (Etoposide), ametycin (Mytomycin C) etc.
The form of salt, hydroxide, nitrogen oxide or other chemical compound that described celery flavin and derivant thereof be the compd A of following formula or its ester, glucosides, pharmaceutically generate:
Wherein R1, R2, R3 site can be OH, H or OCH
2, can be the same or different.
Test shows, when the derivant of celery flavin or celery flavin and the weight ratio of officinal salt and cisplatin thereof are 12～18: in the time of 1, its result of use when prevention and treatment colon cancer is better; Weight ratio is 1.5: 1～3: 1 o'clock, and the result of use when prevention and treatment pulmonary carcinoma or ovarian cancer is better.
Test shows, when the derivant of celery flavin or celery flavin and the weight ratio of officinal salt and paclitaxel thereof are 100～300: in the time of 1, the result of use when prevention and treatment carcinoma of prostate is better; Weight ratio is 150～300: 1 o'clock, and the result of use when prevention and treatment ovarian cancer is better.
Test shows, when the derivant of celery flavin or celery flavin and the weight ratio of officinal salt and amycin thereof are 1.5～4: in the time of 1, its result of use when prevention and treatment carcinoma of prostate is better; Weight ratio is 8: 1～16: 1 o'clock, and its result of use when prevention and treatment colon cancer is better.
Test shows, when the weight ratio of the derivant of celery flavin or celery flavin and officinal salt and 5-fluorouracil was 10: 1～30: 1, its result of use during colon cancer before prevention and treatment was better; Weight ratio is 6: 1～15: 1 o'clock, and its effect when prevention and treatment carcinoma of prostate is better.
Test shows, when the weight ratio of the derivant of celery flavin or celery flavin and officinal salt and etoposide was 1: 1.5～1: 2, its result of use when prevention and treatment carcinoma of prostate was better.
Test shows, when the derivant of celery flavin or celery flavin and the weight ratio of officinal salt and ametycin thereof are 15～30: in the time of 1, its effect when prevention and treatment carcinoma of prostate is better; Weight ratio is 3～6: 1 o'clock, and its effect when prevention and treatment colon cancer is better; Weight ratio is 1～2: 1 o'clock, and its effect when prevention and treatment pulmonary carcinoma is better.
Two kinds of components are united when using, and the dosage range of the derivant of celery flavin or celery flavin, salt, hydroxide, nitrogen oxide, ester and the dosage range of other chemical medicine are clinical normal using dosage scope, get final product according to the state of an illness and physician guidance when specifically using.
The pharmaceutical composition of prevention of the present invention and treatment cancer can be used for the treatment of or reduce the malignant change of the early stage malignant change of incidence rate, minimizing and the inhibition carcinoma of prostate of the early stage malignant change of ovarian cancer, the frequency that suppresses the early stage malignant change of pulmonary carcinoma and generation, the early stage malignant change of inhibition colon cancer, inhibition or processing ovarian cancer etc.
Beneficial effect of the present invention:
1, significantly reduced the consumption of celery flavin and other cancer therapy drug under the situation of identical curative effect, toxic and side effects reduces simultaneously;
2, the celery flavin uses with other cancer therapy drug and has synergism, can improve the sensitivity of tumor cell to clinical cancer therapy drug commonly used, has reduced the drug resistance of tumor cell, has improved curative effect;
3, the celery flavin can with the cancer therapy drug compatibility of multiple routine, all can reduce the dosage of medicine in a large number, result of use is obvious.
Description of drawings
Fig. 1 shows that the growth of prostate gland cancer cell PC-3 and DU-145 is subjected to the inhibition of celery flavin
Fig. 2 shows that the growth of colon cancer cell Colo-205 and HCT-116 is subjected to the inhibition of celery flavin
Fig. 3 shows that the growth of lung cell A549 and H460 is subjected to the inhibition of celery flavin
Fig. 4 shows that the celery flavin strengthens the inhibitory action of anticancer drugs, doxorubicin to prostate gland cancer cell PC-3
Fig. 5 shows that the celery flavin strengthens the inhibitory action of anticancer drugs, doxorubicin to colon cancer cell Colo-205 and HCT-116
Fig. 6 shows the sensitivity of celery flavin enhancement lung carcinoma cell to amycin
Fig. 7 shows that the celery flavin improves prostate gland cancer cell PC-3 and the Du145 sensitivity to etoposide
Fig. 8 shows that the celery flavin strengthens the inhibitory action of cancer therapy drug ametycin to prostate gland cancer cell PC3 and DU145
Fig. 9 shows that the celery flavin strengthens the inhibitory action of cancer therapy drug ametycin to colon cancer cell Colo-205 and HCT-116
Figure 10 shows that the celery flavin strengthens the inhibitory action of cancer therapy drug ametycin to lung cell A549 and H460
Figure 11 shows that the celery flavin strengthens the inhibitory action of cancer therapy drug 5-fluorouracil to prostate gland cancer cell PC3 and DU145
Figure 12 shows that the celery flavin strengthens the inhibitory action of cancer therapy drug 5-fluorouracil to colon cancer cell HC116
Figure 13 shows the effect of celery flavin cisplatin to colon cancer cell HCT116 and Colo-205
Figure 14 shows that the celery flavin strengthens the inhibitory action of cancer therapy drug cisplatin to lung cell A549 and H460
Figure 15 shows that the celery flavin strengthens ovarian cancer cell A2780/CP70 (A) and PC-3 (B) sensitivity to cisplatin
Figure 16 shows that the celery flavin increases considerably the apoptosis induced effect of cisplatin to the A2780/CP70 cell
Figure 17 shows that the celery flavin increases the effect that paclitaxel (paclitaxel) kills prostate gland cancer cell PC-3 (A) and ovarian cancer A2780/CP70 (B) greatly.
Figure 18 shows that the celery flavin improves the inhibitory action of cisplatin to ovarian tumor (A) and carcinoma of prostate (B) growth, improves the inhibitory action (C) of paclitaxel to carcinoma of prostate
Figure 19 shows that the celery flavin suppresses ovarian cancer cell propagation, and induces the apoptosis of ovarian cancer cell
Figure 20 shows that the celery flavin suppresses ovarian tumor growth
Figure 21 shows that the celery flavin suppresses the expression of ovarian cancer cell hypoxia inducible factor HIF-1 α
Figure 22 shows that the celery flavin suppresses the expression of ovarian cancer cell VEGF VEGF
Figure 23 shows that the celery flavin suppresses the generation of tumor neovascularity
Figure 24 shows that celery flavin raising ovarian tumor also significantly increases the inhibitory action of cisplatin to angiogenesis to the sensitivity of cisplatin
The specific embodiment
Test method
1, cell culture: prostate gland cancer cell PC-3 and DU-145, ovarian cancer cell A2780/CP70 cultivates in RPMI 1640 culture medium, includes 10% hyclone, 2mM L-glutamate, Glu, 1% penicillin/streptomycin.Colon cancer cell Colo-205 is grown in RPMI 1640 culture medium, includes 10% hyclone, 2mM L-glutamate, Glu, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10mM 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 1.0mM Sodium Pyruvate and 1% penicillin/streptomycin.Colon cancer cell HCT-116 is grown in McCoy ' the s 5a culture medium, includes 10% hyclone, 1.5mM L-glutamate, Glu and 1% penicillin/streptomycin.Lung cell A549 and H460 cells cultivate in the RPMI1640 culture medium, include 10% hyclone, 2mM L-glutamate, Glu and 1% penicillin/streptomycin.All cells is all cultivated at 37 degree and is contained in the incubator of 5% carbon dioxide.
2, used cancer therapy drug
Amycin, reverse transcription and RNA polymerase inhibitor; Cisplatin destroys DNA and suppresses the DNA reparation; Etoposide promotes the DNA cutting; Ametycin, the synthetic and karyokinesis inhibitor of DNA; 5-fluorouracil, the DNA synthetic inhibitor; Paclitaxel influences the cell micro-tubular structure.
3, cell proliferating determining
Cell proliferation carries out with conventional MTT method.Cell inoculation in 96 orifice plates, every porocyte 1 * 10
4Remove old culture medium after one day, change the fresh culture medium of reagent thing to some extent that contains, handled 24 hours.Every then hole adds 10 μ L of5mg/ml tetrazolium bromides (thiozolyl blue tetrazolium bromide) reagent.37 degree add 100 μ L solvent DMSO after 2 hours.Photometry absorption under the 470nm wavelength after 5 hours.The growth rate of cellular control unit is decided to be 1, and the numerical value of drug treating group and its compare, and represent relative growth rate.
Test example 1
Use above-mentioned test method, the growth of test prostate gland cancer cell PC-3 and DU-145 is subjected to the inhibition of celery flavin, the results are shown in Figure 1.
Test example 2
Use above-mentioned test method, test colon cancer cell Colo-205 and HCT-116 are subjected to the influence of celery flavin, the results are shown in Figure 2.
Test example 3
Use above-mentioned test method, test lung cell A549 and H460 are subjected to the influence of celery flavin, the results are shown in Figure 3.
Test example 4
Use above-mentioned test method, test celery flavin strengthens the inhibitory action of anticancer drugs, doxorubicin to prostate gland cancer cell PC-3, the results are shown in Figure 4.
Test example 5
Use above-mentioned test method, test celery flavin strengthens the inhibitory action (drug level all be micromole level) of anticancer drugs, doxorubicin to colon cancer cell Colo-205 and HCT-116, the results are shown in Figure 5.
Test example 6
Use above-mentioned test method, test celery flavin enhancement lung cell A549 and H460 the results are shown in Figure 6 to the sensitivity of amycin.
Test example 7
Use above-mentioned test method, test celery flavin raising prostate gland cancer cell PC-3 and DU145 the results are shown in Figure 7 to the sensitivity of etoposide.
Test example 8
Use above-mentioned test method, test celery flavin strengthens the inhibitory action (drug level all be micromole level) of cancer therapy drug ametycin to prostate gland cancer cell PC3 and DU145, the results are shown in Figure 8.
Test example 9
Use above-mentioned test method, test celery flavin strengthens the inhibitory action (drug level all be micromole level) of cancer therapy drug ametycin to colon cancer cell Colo-205 and HCT-116, the results are shown in Figure 9.
Test example 10
Use above-mentioned test method, test celery flavin strengthens the inhibitory action (drug level all be micromole level) of cancer therapy drug ametycin to lung cell A549 and H460, the results are shown in Figure 10.
Test example 11
Use above-mentioned test method, test celery flavin strengthens the inhibitory action (drug level all be micromole level) of cancer therapy drug 5-fluorouracil to prostate gland cancer cell PC3 and DU145, the results are shown in Figure 11.
Test example 12
Use above-mentioned test method, test celery flavin strengthens the inhibitory action (drug level all be micromole level) of cancer therapy drug 5-fluorouracil to colon cancer cell HC116, the results are shown in Figure 12.
Test example 13
Use above-mentioned test method, test celery flavin strengthens the press down effect (drug level all be micromole level) of cancer therapy drug cisplatin to colon cancer cell HC116 and Colo-205, the results are shown in Figure 13.
Test example 14
Use above-mentioned test method, test celery flavin strengthens the inhibitory action (drug level all be micromole level) of cancer therapy drug cisplatin to lung cell A549 and H460, the results are shown in Figure 14.
Pulmonary carcinoma, carcinoma of prostate and rectal cancer are common cancers, and current chemotherapy toxic side effect commonly used can cause such as nausea and vomiting and causes Normocellular death.The poly aldehydes matter belongs to native compound, is present in vegetable and fruit and the Chinese herbal medicine.Celery flavin (celery flavin) is the poly aldehydes matter, has antioxidation and immunization.By above test as seen, prostate gland cancer cell PC-3 and DU-145 when handling with 15 μ M celery flavin, to anticancer drugs, doxorubicin commonly used, etoposide, the sensitivity of ametycin and 5-fluorouracil obviously increases.For rectum cancer cell HCT-116 and colo-205, the celery flavin improves the sensitivity of these cells to amycin, ametycin, 5-fluorouracil and cisplatin greatly.And for lung cell A549 and H460, the celery flavin then improves the effect of amycin, ametycin and cisplatin.Cell is weighed by measuring cell proliferation or apoptosis the sensitivity of medicine.Make the IC of various medicines behind the adding celery flavin
50Reduce 2-3 doubly, many persons can reach more than 10 times.
Test example 15
The ovarian cancer cell A2780/CP70 (Figure 15 A) and the prostate gland cancer cell PC-3 (Figure 15 B) of cisplatin opposing are inoculated in 96 orifice plates every hole 1 * 10 of inoculum density
4Individual cell, overnight incubation.Then renew bright culture fluid to cell, the cisplatin of the various concentration shown in containing in the fresh medium, the cisplatin that perhaps contains various concentration adds celery flavin (15 μ M).After 24 hours, with MTT kit detection cell propagation.Result of the test is seen shown in Figure 15 A and the B.As seen from Figure 15, the celery flavin increases considerably the growth inhibited effect of cisplatin to ovarian cancer and prostate gland cancer cell.
The ovarian cancer cell of inoculation cisplatin opposing is to culture plate.Second day, with cisplatin (10 μ M), celery flavin (10 μ M), perhaps cisplatin (10 μ M) added celery flavin (10 μ M) processing cell 24 hours, handles the back collecting cell, uses the apoptosis situation of Annexin V test kit (Roche) analysis of cells then.See Figure 16, the celery flavin increases considerably the apoptosis induced effect of cisplatin to the A2780/CP70 cell.
Test example 16: the celery flavin increases the effect of paclitaxel (paclitaxel claims taxol again) kill cancer cell.
In inoculation prostate gland cancer cell PC-3 and A2780/CP70 cell to 96 orifice plate, overnight incubation.The culture fluid that sucking-off is old is changed the fresh medium that contains the variable concentrations paclitaxel then, and the paclitaxel that perhaps contains variable concentrations adds the fresh medium of 20 μ M celery flavin.After 48 hours, detect cell viability with MTT.The results are shown in Figure 17A and Figure 17 B, Figure 17 A shows that the celery flavin increases considerably the death of the PC-3 cell that paclitaxel causes, IC
50Be 50.33nM, than a IC with the taxol treatment group
50Low 11 times of value 577.19nM.Figure 17 B shows that the celery flavin increases considerably the death of the A2780/CP70 cell that paclitaxel causes, IC
50Be 225.02nM, than a IC with the taxol treatment group
50Value 503.44 low twices.
Test example 17: the celery flavin improves the inhibitory action of cisplatin to ovarian tumor growth
(A) ovarian cancer cell A2780/CP70 is inoculated into the female nude mice both sides abdominal part (300 ten thousand a cells/side joint kind) in 4 ages in week.After treating about 5 millimeters of diameter of tumor size in 12 days, nude mice is divided into 4 groups immediately, 5 every group, the beginning medication is handled, and medicine adopts lumbar injection.Using of cisplatin is 6mg/kg, a Wednesday time injection.Using of celery flavin is 5mg/kg, once a day injection.From first day measurement gross tumor volume of medication, its relative volume is decided to be 1.After the medication 9 days, put to death nude mice.(●) matched group is injected isopyknic solvent; (■) cisplatin treated group; (△) celery flavin processed group; () celery flavin (medication in a day once) cisplatin (Wednesday time medication) Combined Treatment group.Celery flavin raising cisplatin the results are shown in Figure 18A to the inhibitory action of ovarian tumor growth.The initial tumor volume settings is 1, and gross tumor volume changes to be represented with relative value.
(B) prostate gland cancer cell PC3 is inoculated into the back, male nude mouse both sides (300 ten thousand a cells/side joint kind) in 4 ages in week.After treating about 5 millimeters of diameter of tumor size, nude mice is divided into 4 groups immediately, 5 every group, the beginning medication is handled, and medicine adopts lumbar injection.Using of cisplatin is 6mg/kg, a Wednesday time injection.Using of celery flavin is 5mg/kg, once a day injection.From first day measurement gross tumor volume of medication, put to death mice after 20 days and take out tumor.(●) matched group is injected isopyknic solvent; (■) celery flavin processed group; (△) cisplatin treated group; () celery flavin (medication in a day once) cisplatin (Wednesday time medication) Combined Treatment group.Celery flavin raising cisplatin the results are shown in Figure 18B to the inhibitory action of tumor of prostate growth.Gross tumor volume is represented with cubic millimeter.
(C) prostate gland cancer cell PC3 is inoculated into the back, male nude mouse both sides (300 ten thousand a cells/side joint kind) in 4 ages in week.After treating about 5 millimeters of diameter of tumor size, nude mice is divided into 4 groups immediately, 5 every group, the beginning medication is handled, and medicine adopts lumbar injection.Using of paclitaxel is 5mg/kg, a week 2 injections.Using of celery flavin is 5mg/kg, once a day injection.From first day measurement gross tumor volume of medication, put to death mice after 20 days and take out tumor.(●) matched group is injected isopyknic solvent; (■) celery flavin processed group; (△) taxol treatment group; () celery flavin (medication in a day once) paclitaxel (medication biweekly) Combined Treatment group.Celery flavin raising paclitaxel the results are shown in Figure 18C to the inhibitory action of tumor of prostate growth.Gross tumor volume is represented with cubic millimeter.
By test example 15～17 as seen, the celery flavin can improve the sensitivity of tumor cell to cancer therapy drug commonly used, reduces the IC of these medicines
50(cell growth inhibiting 50% required drug level) (2-3 times at least, at most more than 10 times), thereby the therapeutic effect of enhancing cancer therapy drug.Interior animal experiment shows that the celery flavin promotes clinical cancer therapy drug cisplatin commonly used, the paclitaxel inhibitory action to ovary and tumor of prostate growth.
Test example 18
The celery flavin suppresses ovarian cancer cell propagation, and induces the apoptosis of ovarian cancer cell.A inoculates A2780/CP70 cell and the former GM847 cell of people's fiber (usefulness is very little to Normocellular effect in order to show the celery flavin in contrast, and big to the tumor cell effect) in 96 orifice plates, the every hole 4 * 10 of cell concentration
3Individual cell, overnight incubation.The culture fluid that sucking-off is old adds the fresh medium that 100 μ l contain the celery flavin or do not contain the celery flavin.Cell continues to cultivate 24 hours, surveys cell proliferation with mtt assay.The results are shown in Figure 19A.B when cultivating A2780/CP70 cell or GM847 cell to 70-80% culture dish saturation, handled cell 24 hours with the celery flavin then.Use trypsin digestion cell, collecting cell is washed cell once with PBS then, follows the explanation transfect cell according to the Annexin-V-Folus test kit.The results are shown in Figure 19B.C, the A2780/CP70 cell was handled 24 hours with the celery flavin, collecting cell, extracting DNA.1.8% agarose gel electrophoresis, applied sample amount 8 μ g DNA.Figure 19 c shows that the celery flavin promotes the cell DNA fracture of cisplatin induction.Swimming lane 1: molecular weight standard; Swimming lane 2: matched group; Swimming lane 3:10 μ M cisplatin; Swimming lane 4:20 μ M celery flavin; Swimming lane 5:20 μ M celery flavin adds 10 μ M cisplatin.D handles the A2780/CP70 cell with 20 μ M celery flavin, detects the shearing situation of caspase-3 (Guang winter enzyme) and PARP with western (the immune marking) method.The generation of the shearing table clear-cells apoptosis of caspase-3 and PARP.The results are shown in Figure 19D.
Test example 19: the celery flavin suppresses tumor growth.
Ovarian cancer cell A2780/CP70 cell is inoculated in 9 days the Embryo Gallus domesticus of fertilization, hatches 9 angel's tumor growths.Simultaneously, processed group is accepted the celery flavin of 7.5 μ M, and matched group is accepted the solvent of same amount.After 9 days, tumor is scaled off and weigh.A: matched group and the representational tumor B of celery flavin processed group: data are expressed as mean+SD from two different experiments, and each experiment has 20 embryos (10 belong to matched group, and 10 belong to processed group).The results are shown in Figure 20A and Figure 20 B.
By test example 18～19 as seen, the celery flavin suppresses the propagation of tumor cell, and inducing apoptosis of tumour cell suppresses tumor growth.
Test example 20: the celery flavin suppresses the expression of HIF-1 α in OVCAR3 cell and the A2780/CP70 cell
OVCAR3 cell and A2780/CP70 cell are cultured to 80-90% and merge, and use the celery flavin of each concentration as described in Figure to handle cell then.The cell that solubilizer is handled in contrast.Detect the protein level of HIF-1 α and HIF-1 β with the method (describing) of the immune marking as materials and methods.HIF-1 β is used as confidential reference items and monitors the accurate of applied sample amount and change membrane efficiency.The results are shown in Figure 21.
Test example 21: the celery flavin suppresses the expression of the VEGF among ovarian cancer cell OVCAR3 and the A2780/CP70
In inoculation OVCAR3 cell and A2780/CP70 cell to 12 orifice plate, be cultured to 90% and merge.Change the fresh medium that contains the celery flavin then, continue to cultivate 15 hours.Detect the concentration of VEGF in the culture fluid with ELISA method (described in material and method).Data come from three independent experiments, are expressed as meansigma methods ± standard error, and each independent experiment has three repetitions.The results are shown in Figure 22.
Test example 22: the celery flavin suppresses the generation of tumor neovascularity
Mixed ovarian cancer cell A2780/CP70 and matrigel (matrigel), be inoculated into then on the allantocherion of Embryo Gallus domesticus in 9 day age.After 100 hours, tumor is taken off, newly-generated blood vessel is counted at microscopically.Figure 23 A is to photograph and picture, and the ovarian tumor peripheral vessels that does not have to handle generates situation; Figure 23 B is the picture of having used behind the 15 μ M celery flavin, and the oophoroma tumor peripheral vessels that the celery flavin is handled generates situation.
Test example 23: celery flavin raising ovarian tumor also significantly increases the inhibitory action of cisplatin to angiogenesis to the sensitivity of cisplatin
The inoculation have the cisplatin repellence the A2780/CP70 cell to the plate of 100mm, cultured cell merges to 90-100%.Collecting cell, cell is mixed (cell and matrigel ratio are 2: 1) with matrigel (matrigel), contain 2.5 μ M cisplatin in this mixed liquor, 10 μ M celery flavin, perhaps contain 2.5 μ M cisplatin and 10 μ M celery flavin simultaneously, inoculated mixture is to the Embryo Gallus domesticus of being fertilized 9 days.After 10 days, collect tumor, weigh.In the microscopically of 100 times of amplifications number count to the tumor medium vessels.At least from five different visual field countings.Data are expressed as mean+SD (n=8) from two different experiments.Figure 24 A is representational tumor form, and Figure 24 B is a tumor weight; Figure 24 C is relative blood vessel quantity.This shows that celery flavin raising ovarian tumor also significantly increases the inhibitory action of cisplatin to angiogenesis to the sensitivity of cisplatin.
More than all tests show that the celery flavin suppresses the angiogenesis that tumor cell causes, can be used to suppress tumor-blood-vessel growth and tumor growth, also can suppress the stimulating factor of angiogenic growth: VEGF VEGF and hypoxia inducible factor HIF-1 α.Simultaneously, this material can improve the sensitivity of tumor cell to clinical cancer therapy drug commonly used, has reduced the drug resistance of tumor cell, has improved curative effect.Our data show, celery flavin have the potential of the medicine that becomes treatment and prophylaxis of cancer.And during the medication combined use of celery flavin and conventional chemotherapy, just become the medicine of very effective prevention and treatment cancer.
In the present invention's test, used ovarian cancer cell line A2780/CP70 and OVCAR-3, and prostate cancer cell line PC-3 is as study model.Found that the celery flavin under the physiological concentration can optionally suppress the propagation of these ovarian cancer cells, simultaneously the apoptosis (Figure 16) of inducing cell.Under same experiment condition, the celery flavin is to the effect of people's normal fiber archeocyte very little (Figure 16).The celery flavin may pass through to suppress PI3K (phosphatidylinositols-3-phosphokinase)-AKT path, and induces p53, and p21 and BAX and activation Guang winter enzyme (caspase) cascade reaction suppress the growth of ovarian cancer cell.In addition, find that also the celery flavin can suppress the tumor growth of ovarian cancer cell (Figure 15,17,21) in vivo, increase the sensitivity (Fig. 5-17) of some tumor cell of drug resistance drug treating.
According to the daily inleting appetite of flavonoid, do not have during the concentration of employed celery flavin toxicly in the research, be in the physiological range to human body.The result shows, the celery flavin is with the ability that suppresses tumor cell proliferation and tumor growth, and the celery flavin can increase the effect of other chemotherapeutic simultaneously, can be used as a kind of at ovarian cancer, carcinoma of prostate, and the chemotherapeutics of other cancer or prophylactic agent.
Find that in the present invention's research the celery flavin can suppress tumor-blood-vessel growth.Angiogenesis refers to the formation of new blood vessel.Tumor can not be grown under the new angiopoietic situation not having, because tumor needs the new blood vessel that forms that nutrition and oxygen are provided.Angiogenesis can be induced by many factors, wherein VEGF (VEGF) significantly.Many tumor cells can both synthesize VEGF, and these can induce forming of new blood vessel by the VEGF that tumor produces.The generation of VEGF mainly is subjected to hypoxia inducible factor-1 regulation and control (HIF-1).Hypoxia inducible factor-1 is a transcription factor, is made up of HIF-1 α and two subunits of HIF-1 β.HIF-1 α is the proprietary subunit of HIF-1, and when the lowering of concentration of oxygen in the cell or during some factors stimulated growth, HIF-1 α is induced.HIF-1 β is fragrant hydroxyl receptor nuclear translocon, and it and fragrant hydroxyl receptor form different aggressiveness, are not subjected to the adjusting of cell oxygen concentration or somatomedin.HIF-1 overexpression in many tumor cells, its activity level is relevant with angiogenesis with the one-tenth tumor.VEGF and its upstream element HIF-1 are the target spots of all treatments of cancer.Therefore, celery flavin and chemotherapy drugs in combination use can be used in all treatment for cancer.
To the ovarian cancer of cultivating, pulmonary carcinoma, colon cancer and prostate cell line, the celery flavin can suppress cell proliferation, cell death inducing (Fig. 1-4,19).
The associating result of use of research celery flavin and the cancer therapy drug cisplatin of using always.The ovarian cancer cell line A2780/CP70 of cisplatin opposing and prostate cancer cell line PC-3 are as study model.As shown in figure 15, the celery flavin is handled can increase the sensitivity of cell to cisplatin greatly.Suppress the required cisplatin concentration (IC of 50%A2780/CP70 cell proliferation
50) approximately be 20 μ M, add that the celery flavin of 15 μ M can reduce the IC of cisplatin
50To 5 μ M (reducing by 4 times).The PC-3 cell is had similar result, and cisplatin is to the IC of PC-3
50Near 22 μ M, add the celery flavin of 15 μ M, the IC of cisplatin
50Be reduced to 3 μ M (reducing by 7 times).These results show that cisplatin and celery flavin unite after the use, and cisplatin has strengthened greatly to the toxic action of tumor cell.
Apoptosis can be used as an index weighing tumor cell to celery flavin and cisplatin sensitivity.In the present invention's experiment, used the A2780/CP70 cell.As shown in figure 16, the cisplatin of 10 μ M can cause very a spot of A2780/CP70 apoptosis in 24 hours, and the celery flavin of 10 μ M 24 hours is internal energy to cause about 12% apoptosis, and two kinds of medicines use 25% the cell can apoptosis simultaneously.
The influence of the pair cell apoptosis that uses celery flavin and paclitaxel is united in research.Prostate cancer cell line PC-3 and ovarian cancer cell line A2780/CP70 are used as study model.As shown in figure 17, under the situation of taxol treatment, the celery flavin of 20 μ M can significantly be induced the apoptosis of PC-3.Paclitaxel is to the IC of PC-3
50Be 577.19nM, be reduced to 50.33nM after adding the celery flavin.The celery flavin of 20 μ M equally also can increase the apoptosis (Figure 17) of A2780/CP70 cell.After adding the celery flavin, paclitaxel is to the IC of A2780/CP70
50Be reduced to 225.02nM from 503.44nM.These results show, unite and use paclitaxel and celery flavin can increase the death of tumor cell greatly, have therapeutic effect.
In order to verify that further the celery flavin can improve the raising of drug-resistant tumor to drug susceptibility, the present invention has selected several frequently seen drug-resistant tumor cell again, such as ovarian cancer cell, prostate gland cancer cell, lung carcinoma cell, rectum cancer cell has used amycin, etoposide, clinical common cancer therapy drugs such as ametycin and 5-fluorouracil.The result shows, the celery flavin not only can be separately to the propagation of these tumor cells inhibitory action again, what is more important, it can improve the sensitivity of these tested cells to various cancer therapy drugs.Because the drug resistance of tumor cell is unusual universal phenomenon and be the major reason that causes final treatment to be failed in the current treatment of cancer, and the celery flavin is the natural materials of a food source and nontoxic abdomen side effect, therefore, its this characteristic has potential huge applications prospect to from now on cancer chemotherapy.
Experimentation shows (as Figure 18, shown in Figure 24) in the body, and the celery flavin can increase the inhibition that cisplatin, paclitaxel form the inductive tumor of A2780/CP70, PC3 greatly.The result shows that the cisplatin of 2.5 μ M can only slightly suppress the tumor that the A2780/CP70 cell causes and form (Figure 24) because A2780/CP70 be have the cisplatin repellence cell.The tumor that the celery flavin of 10 μ M causes the A2780/CP70 cell is formed with 40% inhibition, and uses cisplatin and celery flavin can reach 70% inhibition (Figure 24 B) simultaneously.This result is further confirmed (Figure 18 A) in the nude mice animal model.Angiopoietic result is consistent with the result of tumor growth.Only only can slightly suppress the vascularization (Figure 24 C) of tumor cell induction with cisplatin treated, the celery flavin can suppress 30% tumor-blood-vessel growth, and the inhibition to tumor-blood-vessel growth can reach 60% when cisplatin and celery flavin Combined Treatment.In sum, result of the present invention shows that the celery flavin can increase the sensitivity of ovarian cancer cell A2780/CP70 to cisplatin, increases the effect of anti-tumor medicine commonly used in the past simultaneously.These results show that celery flavin and other chemotherapeutics comprise that cisplatin combined use can be as a kind of effective treatment ovarian cancer and other method for cancer.
Ovarian cancer cell A2780/CP70 is inoculated into the back, female nude mice both sides (300 ten thousand a cells/side joint kind) in 4 ages in week.After treating about 5 millimeters of diameter of tumor size, nude mice is divided into 4 groups immediately, 5 every group, the beginning medication is handled, and medicine adopts lumbar injection.Using of cisplatin is 6mg/kg, a Wednesday time injection.Using of celery flavin is 5mg/kg, once a day injection.From first day measurement gross tumor volume of medication, put to death mice after 9 days and take out tumor (the results are shown in Figure 18A).Celery flavin and cisplatin combined use have improved the inhibitory action to ovarian tumor greatly.
Prostate gland cancer cell PC3 is inoculated into the back, male nude mouse both sides (300 ten thousand a cells/side joint kind) in 4 ages in week.After treating about 5 millimeters of diameter of tumor size, nude mice is divided into 4 groups immediately, 5 every group, the beginning medication is handled, and medicine adopts lumbar injection.Using of cisplatin is 6mg/kg, a Wednesday time injection.Using of celery flavin is 5mg/kg, once a day injection.From first day measurement gross tumor volume of medication, put to death mice after 20 days and take out tumor (the results are shown in Figure 18B).Celery flavin and cisplatin combined use have improved the inhibitory action to tumor of prostate greatly.
Prostate gland cancer cell PC3 is inoculated into the back, male nude mouse both sides (300 ten thousand a cells/side joint kind) in 4 ages in week.After treating about 5 millimeters of diameter of tumor size, nude mice is divided into 4 groups immediately, 5 every group, the beginning medication is handled, and medicine adopts lumbar injection.Using of paclitaxel is 5mg/kg, a week 2 injections.Using of celery flavin is 5mg/kg, once a day injection.From first day measurement gross tumor volume of medication, put to death mice after 20 days and take out tumor (the results are shown in Figure 18C).Celery flavin and paclitaxel are united use and have been improved inhibitory action to tumor of prostate greatly.
Claims (24)
1, the pharmaceutical composition of a kind of prevention and treatment cancer, it comprises the conventional therapy of effective anticancer or the chemical synthetic drug and the celery flavin of prophylaxis of cancer, wherein, the chemical synthetic drug of said conventional therapy or prophylaxis of cancer is meant paclitaxel, amycin, 5-fluorouracil, etoposide or ametycin.
2, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant paclitaxel, and the weight ratio of celery flavin and paclitaxel is 100～300: 1.
3, the application of the pharmaceutical composition of described prevention of claim 2 and treatment cancer in preparation prevention or treatment carcinoma of prostate medicine.
4, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant paclitaxel, and the weight ratio of celery flavin and paclitaxel is 150～300: 1.
5, the application of the pharmaceutical composition of described prevention of claim 4 and treatment cancer in preparation prevention or treatment ovarian cancer medicine.
6, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant amycin, and the weight ratio of celery flavin and amycin is 1.5～4: 1.
7, the application of the pharmaceutical composition of described prevention of claim 6 and treatment cancer in preparation prevention or treatment carcinoma of prostate medicine.
8, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant amycin, and the weight ratio of celery flavin and amycin is 8: 1～16: 1.
9, the application of the pharmaceutical composition of described prevention of claim 8 and treatment cancer in preparation prevention or treatment colon cancer medicine.
10, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant 5-fluorouracil, and the weight ratio of celery flavin and 5-fluorouracil is 10: 1～30: 1.
11, the application of the pharmaceutical composition of described prevention of claim 10 and treatment cancer in preparation prevention or treatment colon cancer medicine.
12, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant 5-fluorouracil, and the weight ratio of celery flavin and 5-fluorouracil is 6: 1～15: 1.
13, the application of the pharmaceutical composition of described prevention of claim 12 and treatment cancer in preparation prevention or treatment carcinoma of prostate medicine.
14, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant etoposide, and the weight ratio of celery flavin and etoposide is 1: 1.5～1: 2.
15, the application of the pharmaceutical composition of described prevention of claim 14 and treatment cancer in preparation prevention or treatment carcinoma of prostate medicine.
16, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant ametycin, and the weight ratio of celery flavin and ametycin is 15～30: 1.
17, the application of the pharmaceutical composition of described prevention of claim 16 and treatment cancer in preparation prevention or treatment carcinoma of prostate medicine.
18, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant ametycin, and the weight ratio of celery flavin and ametycin is 3～6: 1.
19, the application of the pharmaceutical composition of described prevention of claim 18 and treatment cancer in preparation prevention or treatment colon cancer medicine.
20, the pharmaceutical composition of prevention as claimed in claim 1 and treatment cancer, it is characterized in that: the treatment of said routine or the chemical synthetic drug of prophylaxis of cancer are meant ametycin, and the weight ratio of celery flavin and ametycin is 1～2: 1.
21, the application of the pharmaceutical composition of described prevention of claim 20 and treatment cancer in preparation prevention or treatment pulmonary carcinoma medicine.
22, the pharmaceutical composition of a kind of prevention and treatment cancer, it comprises the cisplatin and the celery flavin of effective anticancer, and the weight ratio of celery flavin and cisplatin is 12～18: 1 or 1.5: 1～3: 1.
23, the pharmaceutical composition of described prevention of claim 22 and treatment cancer is when the weight ratio of celery flavin and cisplatin in the compositions is 12～18: in the time of 1, in the preparation prevention or treat application in the colon cancer medicine.
24, the pharmaceutical composition of described prevention of claim 22 and treatment cancer, when the weight ratio of celery flavin and cisplatin in the compositions is 1.5: 1～3: 1, the application in preparation prevention or treatment pulmonary carcinoma medicine or ovarian cancer medicine.
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| CN102440987B (en) * | 2010-10-12 | 2013-10-30 | 鼎泓国际投资（香港）有限公司 | Drug compound of apigenin, apigenin-like derivants, artemisinin and artemisinin-like derivants and application thereof |
| CN102688228B (en) * | 2011-03-25 | 2014-05-07 | 鼎泓国际投资（香港）有限公司 | Pharmaceutical composition containing apigenin, apigenin derivative, rubescensin and rubescensin derivative, and application thereof |
| WO2014047780A1 (en) * | 2012-09-25 | 2014-04-03 | 鼎泓国际投资（香港）有限公司 | Pharmaceutical composition containing apigenin and apigenin derivative and oridonin and oridonin derivative and use thereof |
| CN103479649B (en) * | 2013-08-09 | 2016-01-27 | 大连理工大学 | A kind of pharmaceutical composition and application thereof |
| CN104188953A (en) * | 2014-08-01 | 2014-12-10 | 南京伊度医疗技术有限公司 | Application of apigenin to preparation of medicines for inhibiting scaffolding protein expression and cancer cell metastasis |
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| CN1640872A (en) * | 2004-01-14 | 2005-07-20 | 南京莱尔生物化工有限公司 | Novel semi-synthetic versulin preparing process |
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