CN101125140B - Application of dihydroartemisinin in reinforcing chemotherapy medicine antitumor curative effect - Google Patents
Application of dihydroartemisinin in reinforcing chemotherapy medicine antitumor curative effect Download PDFInfo
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Abstract
The present invention provides an application of dihydroartemisinin in the preparation of the drugs to enhance the anti-tumor efficacy of the chemotherapeutic drugs. The pharmaceutical preparation contains preparation allowable pharmaceutical excipients or carriers. The form of the preparation is solid preparation. The pharmaceutical preparation provided by the present invention can enhance the anti-tumor efficacy of the chemotherapeutic drugs and can be used in the adjuvant therapy during the tumor chemotherapy process. The present invention takes the theory that the angiogenesis inhibitor can change the micro-environment of tumor as the background, studies and clarifies the function and the mechanism that the effective monomer in the traditional Chinese medicine can enhance the anti-tumor efficacy of the chemotherapy drugs, provides a pharmacological basis for the development of the new usages of the artemisinin drugs and provides an important basis for the development of the new clinical values of the angiogenesis inhibitor, so the present invention is a new research direction for the development of the theory of traditional Chinese medicine.
Description
Technical field
The invention belongs to medicinal usage, relate to the medicinal usage of dihydroartemisinine aspect the enhancing chemotherapy medicine antitumor curative effect.
Background technology
Malignant tumor is to threaten one of the most serious disease of human health, and the main cause of its treatment failure is that tumor cell produces drug resistance to chemotherapeutics.Malignant tumor produces drug resistance except cancerous cell makes a variation by the light of nature to chemotherapeutics, and another major reason is that tumor tissues microenvironment 26S Proteasome Structure and Function is unusual.The mechanism that this unusual microenvironment forms is not illustrated as yet fully, it is generally acknowledged with tumor tissues in new vessels 26S Proteasome Structure and Function major defect, organize factors such as high and low oxygen environment of internal clearance liquid voltage rise and acidosis relevant.Recent years, angiogenesis inhibitor is as the sensitizer paid more and more attention of chemotherapeutics, and fruitful in the treatment kinds of tumors.Some clinical researches show, angiogenesis inhibitor can suppress endotheli ocytosis and obviously suppress growth of tumor and transfer by specificity, its Combination chemotherapy makes the treatment of nonsmall-cell lung cancer (NSCLC) be expected to break away from pining down of " bottle-neck zone ", might become the standard care scheme of advanced NSCLC.
Angiogenesis inhibitor is not only that as the reasonable part of chemotherapeutic sensitizer chemotherapeutics and angiogenesis inhibitor can damage the different ingredient of tumor tissues: tumor cell and endotheliocyte, and angiogenesis inhibitor can change unusual structure of tumor vessel and function, thereby strengthens the therapeutic effect of chemotherapeutics.At first, angiogenesis inhibitor can improve the state of tumor vessel structure disturbance, seepage, reduces interstitial liquid pressure in the tumor, increases drug conveying to inside tumor, and tumor remission histanoxia will make chemotherapy effect strengthen greatly like this.Secondly, angiogenesis inhibitor can stop tumor cell in chemotherapy quick recovery later.Although chemotherapy or radiotherapy can make most of tumor occur disappearing, owing to the serious adverse reaction of chemotherapeutics or radiotherapy has limited its prolonged application.Treatment finishes the back tumor cell can recover apace, breed, and tumor cell again growth rate do not reduce along with the increase of chemotherapy or radiotherapy number of times.Therefore tumor regression does not have the life that really prolongs patient, and Hudis has proposed to implement the feasible solution of angiogenesis inhibitor treatment between the chemotherapy withdrawal time, and the oxygen and the nutrition that are provided by adjacent blood vessel still are provided with the quick propagation again of blocking tumor cell.Once more, angiogenesis inhibitor can strengthen the lethal effect of chemotherapeutics to endotheliocyte, chemotherapeutics not only has direct lethal effect to endotheliocyte during new vascularization, and functional, the great-hearted endotheliocyte of those bone marrow deriveds also had lethal effect, this class cell comprises endothelial progenitor cells, CD54
+Mononuclear cell etc.Endothelial progenitor cells can be divided into sophisticated endotheliocyte, plays a significant role in angiogenesis; CD54
+The cradle that mononuclear cell and other bone marrow like cell groups are angiogenesiss.The low dose of rhythmicity therapeutic modality of chemotherapeutics can the direct killing endotheliocyte, suppresses the endothelial progenitor cells in the blood circulation.These most of endothelial cells can secretion of VEGF, and therefore chemotherapeutics and some are that the drug combination of target spot can be brought into play therapeutic effect collaborative or that add up with VEGF in therapeutic process.Clinical studies show, low dose of rhythmicity treatment of chemotherapeutics and angiogenesis inhibitor share for a long time can bring into play tangible antitumous effect.
Arteannuin (artemisinin) is the sesquiterpene lactones with new structure that China scientist proposed from feverfew Herba Artemisiae annuae (Artemisia annua Linn) first in 1971, and it has very good malaria effect.Dihydroartemisinine (characteristics such as dihydroartemisinin is the intravital main active metabolite of artemisinin-based drug DHA), compares with arteannuin, has good water solubility, and the malaria curative effect is stronger.This seminar early-stage Study shows that artemisinin-based drug can significantly suppress the expression of VEGF among human ovarian cancer transplanted tumor in nude mice tissue and pernicious blood tumor cell strain K562, the RPMI8226, has clear and definite antineoplastic vascular nucleus formation.
Summary of the invention
This dihydroartemisinine that provides that makes eye bright is preparing the application that strengthens in the chemotherapy medicine antitumor curative effect medicine.Promptly strengthen chemotherapeutics in the effect aspect the antitumor curative effect as a kind of target tumor angiogenesis inhibitor.The molecular formula of described dihydroartemisinine is C
15H
24O
5
Drug prepared of the present invention is an antineoplastic chemotherapy medicine.Contain preparation allowable pharmaceutical excipients or carrier.The dosage form of described medicine is a solid preparation.Administering mode is an oral administration.
The present invention has the following advantages:
(1) dihydroartemisinine is the most representative medicine in the traditional artemisinin-based antimalarial drug, be used for the malaria treatment, characteristics of the present invention are to propose dihydroartemisinine strengthens chemotherapy medicine antitumor as a kind of target tumor angiogenesis inhibitor therapeutic effect first all the time.Malignant tumor is to threaten one of the most serious disease of human health, and chemotherapy will be the main means of oncotherapy, and dihydroartemisinine can be by the blocking-up tumor-blood-vessel growth in oncotherapy, and it is indeterminate to overcome the chemotherapeutics targeting, the deficiency that side effect is bigger.Therefore, combined chemotherapy enhancing antineoplaston effect will obtain important application.
(2) the present invention provides pharmacodynamics and Its Mechanisms foundation thereof for the dihydroartemisinine exploitation becomes the chemotherapeutics sensitizer, has the value with artemisinin-based drug exploitation becoming neoplasm targeted therapy medicine.
(3) the present invention tentatively inquires into the dosage regimen research that dihydroartemisinine strengthens chemotherapy medicine antitumor curative effect, and rationalizing administration for clinical Combination chemotherapy provides the pharmacology foundation.
Description of drawings
Fig. 1 a is dihydroartemisinine (DHA) and cyclophosphamide (CTX) combined treatment I tumor growth curve.
Fig. 1 b is DHA and CTX combined treatment II tumor growth curve.
Fig. 2 a is DHA and CTX combined treatment I tumor stereogram.
Fig. 2 b is DHA and CTX combined treatment II tumor stereogram.
Fig. 3 is the influence of DHA combined with CT X to the tumor-bearing mice tumor weight
Fig. 4 a is that DHA and CTX combined treatment I mouse lung are organized the metastasis stereogram.
Fig. 4 b is that DHA and CTX combined treatment II mouse lung are organized the metastasis stereogram.
Fig. 5 a is DHA and CTX combined treatment I tumor tissues vegf expression.
Fig. 5 b is DHA and CTX combined treatment II tumor tissues vegf expression.
Fig. 6 is the influence of DHA to the LLC cell proliferation.
Fig. 7 induces the apoptotic morphological observation of LLC for DHA.
Fig. 8 a, 8b are that DHA induces the apoptotic flow cytometry quantitative analysis of LLC.
Fig. 9 is the influence of DHA to LLC cell KDR/flk-1 protein expression.
The specific embodiment
The present invention is further described with accompanying drawing in conjunction with specific embodiments.Should be understood that these embodiment only are used for illustration purpose, and be not used in the restriction scope of the invention.With regard to mechanism of action, dihydroartemisinine is applicable to that all rely on the solid tumor (as gastric cancer, hepatocarcinoma, intestinal cancer and breast carcinoma etc.) that blood supply could be grown, and the treatment of neoplastic hematologic disorder (leukemia, multiple myeloma etc.).
Embodiment 1: dihydroartemisinine strengthens the therapeutical effect of cyclophosphamide to Lewis lung cancer growth and transfer
Test material: dihydroartemisinine tablet (Zhejiang Holley South Lake pharmaceutical Co. Ltd), LLC (LewisLung Carcinoma) cell strain (Chinese Academy of Sciences's Shanghai RESEARCH ON CELL-BIOLOGY provides), 100 of C57BL/6 mices, male, body weight 20 ± 2g, 6～8 ages (Chinese Academy of Sciences's Shanghai Experimental Animal Center provides) in week, SABC Elivision
TMPlus wide spectrum test kit (KIT-9901/9902/9903) (Foochow steps neoplasm technology company limited), mouse-anti people VEGF monoclonal antibody (Santa Cruz company).
Method:
(1) Lewis lung cancer growth and metastasis model are set up and dosage regimen
Cultivate mice LLC cell with the DMEM culture fluid that contains 10% calf serum, in 37 ℃, 5%CO
2Hatch in the cell culture incubator, go down to posterity after waiting to cover with the 70-90% area.The trophophase cell of taking the logarithm is adjusted cell concentration to 1 * 10 with serum-free medium
7Ml
-1, 6-8 week male C57BL/6 mouse back subcutaneous vaccination in age, 0.2ml/, totally two.Treat that tumor grows to 500mm
3Size is put to death mice, and aseptic condition takes out tumor piece and cutting down into about 1mm
3Size is inoculated into new 6-8 male C57BL/6 mice right fore oxter in age in week, totally 100 with the trocar.
Be divided into 10 groups after the inoculation at random, 10 every group, begin the administration time difference by cyclophosphamide, use two dosage regimens:
Dosage regimen I: solvent control group normal saline every day is irritated stomach, and dihydroartemisinine begins administration, every day 1 time, continuous 25 times next day after inoculation; Cyclophosphamide after inoculation the 5th day the beginning administration, the next day 1 time, continuous 5 times.Referring to table 1.
Dosage regimen II, solvent control group normal saline every day irritate stomach, and dihydroartemisinine begins administration, every day 1 time, continuous 25 times next day after inoculation; Cyclophosphamide after inoculation the 7th day the beginning administration, the next day 1 time, continuous 5 times.Referring to table 2.
Table 1. dihydroartemisinine strengthens cyclophosphamide to Lewis lung cancer therapeutical effect zoopery dosage regimen I
| Group | Number of animals (only) | Dosage * administration number of times | |
| DHA(mg/kg，qd，ig)×25 | ?CTX(mg/kg，qod，?ip)?×5 | ||
| Solvent control DHA CTX (d5-13) DHA+CTX (d5-13) DHA+CTX (d5-13) DHA+CTX (d5-13) | 10 10 10 10 10 10 | - 100 - 50 100 200 | - - 50 50 50 50 |
Table 2. dihydroartemisinine strengthens cyclophosphamide to Lewis lung city cancer therapeutical effect zoopery dosage regimen II
| Group | Number of animals (only) | Dosage * administration number of times | |
| DHA(mg/kg，qd，ig)×25 | ?CTX(mg/kg，qod，?ip)?×5 | ||
| Solvent control DHACTX (d7-15) DHA+CTX (d7-15) DHA+CTX (d7-15) DHA+CTX (d7-15) | 10 10 10 10 10 10 | - 100 - 50 100 200 | - - 50 50 50 50 |
Two treatment sequences all behind last administration 24h, are put to death mice, take out the tumor piece, and it is liquid-solid fixed to get the Bouin of pulmonary ' s.
(2) toxic and side effects of gross tumor volume variation and medicine
Observe ordinary circumstance and the growth of xenografted situation of mice during the administration, gross tumor volume of per 3 days mensuration.Assay method is for using vernier caliper measurement tumor major axis (a), minor axis (b), according to formula V=0.5ab
2Calculate gross tumor volume, draw the gross tumor volume growth curve, measure the mice body weight simultaneously.After mice is put to death, cut open and get tumor tissues and weigh, and calculate tumour inhibiting rate: tumour inhibiting rate=1-(the average tumor weight of the average tumor weight/matched group of medication group) * 100%.
(3) therapeutical effect that the tumor lung is shifted
Treatment finishes the back and puts to death mice, cuts open to get lung tissue and be fixed in Bouin ' s liquid, detects lung surface tumours metastasis behind the 24h, and it is yellow that lung tissue is, the neoplasm metastasis kitchen range protuberance that is white in color.
(4) synergism evaluation criterion
Drug combination effect An Jinshi formula is estimated: q=Ea
+B/ (Ea+Eb-EaEb), wherein Ea
+B is the suppression ratio that two medicines share, and Ea and Eb are the suppression ratio of each prescription time spent, represents that two medicines have share antagonism when q＜1.15, and q＞1.15 o'clock represent that two medicines have synergism.
(5) immunohistochemistry
Above-mentioned tumor tissues takes out the back and fixes with paraformaldehyde, makes the thick paraffin section of 5 μ m after routine is handled, and back SABC, step step the instant second filial generation SABC Elivision of neoplasm technology company limited by Foochow
TMThe operating instruction that provides in the plus reagents for spectrometry box is carried out.The DAB colour developing, gradient ethanol dehydration, neutral gum mounting.The one anti-mouse-anti people VEGF monoclonal antibody (dilution in 1: 50) that adopts, negative control group adopts PBS to replace specificity one anti-.With the xanchromatic strong and weak degree of palm fibre is observation index.
The result
(1) suppresses tumor growth
In Mice Bearing Lewis Lung Cancer transplanted tumor animal model, tumor growth rate of each group of dihydroartemisinine commissural arch phosphamide medication slowly (Fig. 1 a, 1b), treatment finish the back tumor weight than the solvent control group obviously reduce (p＜0.05) (Fig. 2 a, b).Dihydroartemisinine commissural arch phosphamide has therapeutical effect preferably to Lewis lung cancer: by dosage regimen I, 50mg/kgDHA+50mg/kgCTX (d5-13) administration group, 100mg/kgDHA+50mg/kgCTX (d5-13) administration group, the average tumor of 200mg/kgDHA+50mg/kgCTX (d5-13) administration group heavily are respectively 1.33 ± 1.02g, 0.84 ± 0.48g, 0.65 ± 0.45g; The average tumor of three dosage groups heavy with the solvent control group (2.69 ± 1.62g), single with the dihydroartemisinine group (1.67 ± 1.22g), singly organize with cyclophosphamide (d5-13) that (2.16 ± 1.06g) compare and all are obvious inhibition (p＜0.05).By dosage regimen II, 50mg/kgDHA+50mg/kgCTX (d7-15) administration group, 100mg/kgDHA+50mg/kgCTX (d7-15) administration group, the average tumor of 200mg/kgDHA+50mg/kgCTX (d7-15) administration group heavily are respectively 1.13 ± 0.51g, 0.49 ± 0.28g, 0.45 ± 0.22g, the average tumor of three dosage groups heavy with the solvent control group (2.69 ± 1.62g), single with the dihydroartemisinine group (1.67 ± 1.22g), singly organize with CTX (d7-15) that (1.94 ± 0.69g) compare and all are more obvious inhibition (p＜0.05).Show that dihydroartemisinine can strengthen the therapeutical effect of cyclophosphamide to Lewis lung cancer really.While is in the dihydroartemisinine and the cyclophosphamide combination group of same dose, because the time point different table of administering drug combinations reveals the inhibiting difference of tumor growth, 100mg/kgDHA+50mg/kgCTX (d5-13, dosage regimen I) tumor weight and 100mg/kgDHA+50mg/kgCTX (d7-15, dosage regimen II) tumor weight is compared with the tumor-inhibiting action of dosage regimen II more obvious, same 200mg/kgDHA+50mg/kg CTX (d5-13, dosage regimen I) tumor weight and 200mg/kgDHA+50mg/kgCTX (d7-15, dosage regimen II) tumor weight is compared with the tumor-inhibiting action of dosage regimen II more obvious (p＜0.05) (Fig. 3, table 3).
Among Fig. 1 a: ● the solvent control group, zero DHA (100mg/kg) is single with group, ▲ CTX (50mg/kg, d5-13) single with group, △ DHA (50mg/kg)+CTX (50mg/kg, d5-13) drug combination group, ■ DHA (100mg/kg)+CTX (50mg/kg, d5-13) drug combination group, DHA (200mg/kg)+CTX (50mg/kg, d5-13) drug combination group.
Among Fig. 1 b: ● the solvent control group, zero DHA (100mg/kg) is single with group, ▲ CTX (50mg/kg, d7-15) single with group, △ DHA (50mg/kg)+CTX (50mg/kg, d7-15) drug combination group, ■ DHA (100mg/kg)+CTX (50mg/kg, d7-15) drug combination group, DHA (200mg/kg)+CTX (50mg/kg, d7-15) drug combination group.
Among Fig. 2 a: (A) solvent control group, (B) DHA (100mg/kg) is single with group, (C) CTX (50mg/kg, d5-13) single with group, (D) DHA (50mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (E) DHA (100mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (F) DHA (200mg/kg)+CTX (50mg/kg, d5-13) drug combination group.
Among Fig. 2 b: (A) solvent control group, (B) DHA (100mg/kg) is single with group, (C) CTX (50mg/kg, d7-15) single with group, (D) DHA (50mg/kg)+CTX (50mg/kg, d7-15) drug combination group, (E) DHA (100mg/kg)+CTX (50mg/kg, d7-15) drug combination group, (F) DHA (200mg/kg)+CTX (50mg/kg, d7-15) drug combination group.
Among Fig. 3: (A) solvent control group, (B) DHA (100mg/kg) is single with group, (C) CTX (50mg/kg, d5-13) single with group, (D) DHA (50mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (E) DHA (100mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (F) DHA (200mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (G) CTX (50mg/kg, d7-15) single with group, (H) DHA (50mg/kg)+CTX (50mg/kg, d7-15) drug combination group, (I) DHA (100mg/kg)+CTX (50mg/kg, d7-15) drug combination group, (J) DHA (200mg/kg)+CTX (50mg/kg, d7-15) drug combination group.
(2) synergistic therapeutic action
Estimate the therapeutic alliance effect that dihydroartemisinine and cyclophosphamide share according to the Jin Shi formula, single tumour inhibiting rate with 100mg/kg dihydroartemisinine group (DHA) is 37.75%, beginning in the 5th day single tumour inhibiting rate with 50mg/kg cyclophosphamide (d5-13) group in inoculation back is 19.76%, the tumour inhibiting rate of combination group 100mg/kgDHA+50mg/kgCTX (d5-13) is 68.89%, and getting the q value is 1.38; Beginning in the 7th day single tumour inhibiting rate with 50mg/kg cyclophosphamide (d7-15) group in inoculation back is 27.97%, the tumour inhibiting rate of combination group 100mg/kgDHA+50mg/kgCTX (d7-15) is 81.71%, getting the q value is 1.48, show and under two treatment sequences, share dihydroartemisinine and cyclophosphamide all has good synergism, and to begin to share the synergism of cyclophosphamide (dosage regimen II) on the 7th day better to inoculate the back.
(3) toxic and side effects of medicine
During the medication, it is movable good that each organizes tumor-bearing mice, do not see special untoward reaction, and none example is dead.Medication after 25 days solvent control group and single tumor body weight of going with dihydroartemisinine (100mg/kg) be respectively 22.05 ± 1.91g, 22.15 ± 1.59g, it is 22.55 ± 2.32g that the 5th day beginning list in inoculation back goes the tumor body weight with cyclophosphamide (50mg/kg) group, cyclophosphamide is beginning in the 5th day and 50mg/kg after inoculation, 100mg/kg, the mice of respectively organizing that the 200mg/kg dihydroartemisinine share goes the tumor body weight to be respectively 21.89 ± 1.47g, 20.72 ± 0.89g, 21.58 ± 1.17g, it is 22.82 ± 2.17g that the 7th day beginning list in inoculation back goes the tumor body weight with cyclophosphamide (50mg/kg) group, and cyclophosphamide is beginning in the 7th day and 50mg/kg after inoculation, 100mg/kg, the mice of respectively organizing that the 200mg/kg dihydroartemisinine share goes the tumor body weight to be respectively 22.03 ± 1.32g, 21.21 ± 0.58g, 21.80 ± 1.00g.Relatively go tumor mice body weight, each treatment group and solvent control group all do not have significant difference (p＞0.05, table 3).
Table 3, dihydroartemisinine commissural arch phosphamide go the influence of tumor body weight to tumor-bearing mice tumor weight and mice
| Group | Number of animals (only) | Dosage * administration number of times | Go tumor body weight (g) | Tumor heavy (g) | Tumour inhibiting rate (%) | |
| DHA( mg/kg， qd，ig) ×25 | CTX(mg/ kg，qod， ip) ×5 | |||||
| Solvent control DHA CTX (d5-13) DHA+CTX (d5-13) DHA+CTX (d5-1 3) DHA+CTX (d5-13) CTX (d7-15) DHA+CTX (d7-15) DHA+CTX (d7-15) DHA+CTX (d7-15) | 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 | -- 100 -- 50 100 200 -- 50 100 200 | -- -- 50 50 50 50 50 50 50 50 | 22.05±1.9122.15±1.5922.55±2.3221.89±1.4720.72±0.8921.58±1.1722.82±2.1722.03±1.3221.21±0.5821.80±1.00 | 2.69±1.62 1.67±1.22 ** 2.16±1.06 1.33±1.02 **▲▲▲ 0.84±0.48 ***#▲▲▲ 0.65±0.45 ***##▲▲▲ 1.94±0.69 1.13±0.51 **▲▲▲ 0.49±0.28 **##▲▲▲ 0.45±0.22 **##▲▲▲ | 37.75 19.76 50.54 68.89 75.93 27.97 58.00 81.71 84.60 |
*, p＜0.05;
*, p＜0.01 vs solvent control.
#, p＜0.05;
##, p＜0.01 vs DHA.
▲ ▲, p＜0.01;
▲ ▲ ▲, p＜0.001 vs CTX.
(4) influence that the tumor-bearing mice lung is shifted
In subcutaneous transplantation tumor model, find that solvent control group mice major part spontaneous lung occurs and shifts, single have decline in various degree with DHA, CTX group lung surface metastasis number than the solvent control group, the treatment of dihydroartemisinine commissural arch phosphamide then can suppress the tumor spontaneous lung fully and shift (Fig. 4 a, b, table 4).
The influence that table 4 dihydroartemisinine commissural arch phosphamide shifts tumor-bearing mice tumor spontaneous lung
| Group | Dosage * administration number of times | Shift the generation ratio | The mean transferred kitchen range | |
| DHA(mg/kg，qd，ig)×25 | ?CTX(mg/kg，qod，ip) ×5 | |||
| ControiDHACTX(d5-13)DHA+CTX(d5-13)DHA+CTX(d5-13)DHA+CTX(d5-13)CTX(d7-15)DHA+CTX(d7-15)DHA+CTX(d7-15)DHA+CTX(d7-15) | -- 100 -- 50 100 200 -- 50 100 200 | -- -- 50 50 50 50 50 50 50 50 | 8/10 4/10 4/10 0/10 0/10 0/10 4/10 0/10 0/10 0/10 | 10.1 1.1 1.3 0 0 0 1.0 0 0 0 |
Among Fig. 4 a: (A) solvent control group, (B) DHA (100mg/kg) is single with group, (C) CTX (50mg/kg, d5-13) single with group, (D) DHA (50mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (E) DHA (100mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (F) DHA (200mg/kg)+CTX (50mg/kg, d5-13) drug combination group.
Among Fig. 4 b: (A) solvent control group, (B) DHA (100mg/kg) is single with group, (C) CTX (50mg/kg, d7-15) single with group, (D) DHA (50mg/kg)+CTX (50mg/kg, d7-15) drug combination group, (E) DHA (100mg/kg)+CTX (50mg/kg, d7-15) drug combination group, (F) DHA (200mg/kg)+CTX (50mg/kg, d7-15) drug combination group.
(5) vegf protein is expressed in the dihydroartemisinine downward modulation tumor tissues
Immunohistochemistry detects the expression of tumor tissues vegf protein, judges the variation of vegf expression amount in the tumor cell with pale brown color power in the cell.The result shows, the solvent control group shows as to be compared with the solvent control group to medium pale brown color by force, the dihydroartemisinine list with group and dihydroartemisinine obviously weaken with the interior pale brown color of each tumor cell of organizing of cyclophosphamide combined treatment (Fig. 5 a, b).(A) solvent control group among Fig. 5 a, (B) DHA (100mg/kg) is single with group, (C) CTX (50mg/kg, d5-13) single with group, (D) DHA (50mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (E) DHA (100mg/kg)+CTX (50mg/kg, d5-13) drug combination group, (F) DHA (200mg/kg)+CTX (50mg/kg, d5-13) drug combination group.(A) solvent control group among Fig. 5 b, (B) DHA (100mg/kg) is single with group, (C) CTX (50mg/kg, d7-15) single with group, (D) DHA (50m/gkg)+CTX (50mg/kg, d7-15) drug combination group, (E) DHA (100mg/kg)+CTX (50mg/kg, d7-15) drug combination group, (F) DHA (200mg/kg)+CTX (50mg/kg, d7-15) drug combination group.
Embodiment 2: the external evoked Lewis lung cancer of dihydroartemisinine (LLC) apoptosis and downward modulation vegf receptor KDR/flk-1 express
Dihydroartemisinine: Guilin pharmaceutical factory is so kind as to give
Method:
(1) mtt assay detects cell proliferation
5 * 10
4Ml
-1The LLC cell inoculation is in 96 orifice plates, and as solvent control, behind variable concentrations dihydroartemisinine effect 48h, mtt assay detects, and measures absorbance (A with microplate reader under the 570nm wavelength with DMSO
570nm) value, calculate cell proliferation rate, cell proliferation rate (%)=experimental group A
570nm/ solvent control group A
570nm* 100%.Adopt IC
50Software for calculation calculates the IC that dihydroartemisinine suppresses the LLC cell proliferation
50Value is with the confidence interval of SPSS10.0 statistical software calculating 95%.
(2) morphologic detection apoptosis
The LLC cell inoculation behind dihydroartemisinine effect 48 h of adding variable concentrations, washes twice with serum-free medium in 24 orifice plates, adds acridine orange 100mgL
-1With bromination second pyridine 100mgL
-1The dyed blended 5min in the darkroom, observation of cell form under the fluorescence inverted microscope.
(3) cells were tested by flow cytometry apoptosis rate and cell cycle
Behind LLC cell process dihydroartemisinine effect 48 h, centrifugal collection is cleaned 2 times through PBS, adjusts born of the same parents' number to 1 * 10
6ML
-1, slowly splash into 70% ice-cold ethanol fixed cell then, to put 4 ℃ and spend the night, oneself fixed cell adds 50mgL through PBS centrifuge washing 2 times
-1Propidium iodide, 0.1%Triton-X100,37gL
-1EDTA, 50mgL
-1Rnase A, lucifuge is carried out DNA dyeing 30min under the room temperature, crosses 300 order nylon mesh screens, and the up flow type cell instrument is measured in the 24h.Adopt Cell Quest 3.1f software (U.S. Becton Dickinson) to obtain rectangular histogram, ModFit 3.0 softwares (U.S. Becton Dickinson) calculate apoptosis rate and the analysis of cells cycle distributes mutually.
(4) expression of Western-blot quantitative analysis KDR/flk-1
The LLC cell behind the dihydroartemisinine effect 48h of variable concentrations, centrifugal collection, cell lysis extracts albumen, measures protein concentration with the Bradford method, carries out electrophoresis on 12%SDS-PAGE, total protein 50 μ g on each swimming lane.Protein band is transferred on the pvdf membrane behind the electrophoresis, film seals 1h with the TPBS room temperature that contains 5% defatted milk powder, 4 ℃ of placements of mouse-anti people flk-1 polyclonal antibody (dilution in 1: 500) are spent the night, wash film through TPBS, two anti-room temperature effect 1h of horseradish peroxidase-labeled (dilution in 1: 5000), TPBS washes film, adopts the ECL method that target stripe is detected.Adopt the eluting of same film by antibody, the protein band to β-actin detects again, as confidential reference items.
The result:
(1) suppresses the LLC cell proliferation
The MTT experimental analysis shows, handle 48h with dihydroartemisinine after, the LLC cell proliferation is obviously suppressed, and increases its suppression ratio with drug level and be and increase progressively trend, dihydroartemisinine suppresses the IC of LLC cell proliferation
50Value is 53.14 μ molL
-1, 95% confidence interval is 48.69-58.18 μ molL
-1Above result shows that dihydroartemisinine has obvious inhibitory action to LLC propagation, and is concentration dependent, and referring to Fig. 6, DHA is to the half-inhibition concentration (IC of LLC cell proliferation among the figure
50)=53.14 μ molL
-1, 95% confidence interval is 48.69-58.18 μ molL
-1
(2) dihydroartemisinine is induced the apoptotic morphological observation of LLC
The LLC cell is after the two dyeing of acridine orange/bromination second pyridine, and as seen, solvent control group nucleus presents the uniform green fluorescence of density under the fluorescence microscope, show as Normocellular dyeing characteristic, and behind dihydroartemisinine effect 48h, cell presents typical apoptosis morphological change such as the coagulation of chromatin height, marginalisation etc., the visible fluorescent orange of Fluirescence observation strengthens, be compact massive and distribute, and apoptotic body occurs, referring to Fig. 7, among Fig. 7: (A) solvent control group, (B) DHA 20 μ molL
-1, (C) DHA 40 μ molL
-1, (D) DHA 80 μ molL
-1
(3) the apoptotic streaming quantitative analysis of the inductive LLC of dihydroartemisinine
Through flow cytometry analysis, show that according to PI fluorescence rectangular histogram the LLC cell is through 20 μ molL
-1Behind the dihydroartemisinine effect 48h, percentage of cell apoptosis is significantly increased, through 40,80 μ molL
-1Behind the dihydroartemisinine effect 48h, the hypodiploid peak of apoptotic cell has appearred before the G1 peak representing, apoptosis rate rise to 9.82% and 13.86% respectively (Fig. 8 a, b).Wherein among Fig. 8 a: (A) solvent control group, (B) DHA20 μ molL
-1, (C) DHA 40 μ molL
-1, (D) DHA 80 μ molL
-1
(4) dihydroartemisinine suppresses the expression of LLC cell KDR/flk-1
The LLC cell extracts albumen after the variable concentrations dihydroartemisinine is handled, the Western-blot method detects the proteic expression of KDR/flk-1 in the cell, and the result shows that along with the increase of drug level, the proteic expression of cell KDR/flk-1 descends successively.40,80 μ molL
-1The expressing quantity of dihydroartemisinine concentration group cell KDR/flk-1 is reduced to 33.8% and 12.6% of solvent control group respectively, compares with the solvent control group to have significant difference (p＜0.001), and the result is referring to Fig. 9.
Claims (1)
1. the application of dihydroartemisinine in the anti-pulmonary carcinoma curative effect of preparation enhancing chemotherapeutics medicine, the molecular formula of described dihydroartemisinine is C
15H
24O
5, it is characterized in that: dihydroartemisinine is as the application of a kind of target tumor angiogenesis inhibitor in the anti-pulmonary carcinoma curative effect of preparation enhancing chemotherapeutics medicine, and wherein chemotherapeutics is a cyclophosphamide.
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