CN103446162B - 3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is preparing the purposes in multidrug resistance reversing agent - Google Patents

Abstract

The invention belongs to field of medicaments, relate to 3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is preparing the application in antitumor drug, especially 3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is preparing the purposes in tumour medicine reversal agent of drug resistance.The present invention 3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is natural product, confirms the effect with reversing multiple medicine resistance of tumor cells through test, can as multidrug-resistance reversal agent; Also have and increase multidrug resistance cell to the effect of antitumor drug sensitivity, can as sensitizer.Present invention also offers antitumor drug and 3 β, 20 (S), the pharmaceutical composition of 21-trihydroxy dammarane-24-alkene conbined usage.Micromolecular compound of the present invention is developed as new antitumor drug or its auxiliary element, and tumor killing effect is obvious, environmental protection, provides a kind of new approach and means by for treatment and healing tumor.

Description

3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is preparing the purposes in multidrug resistance reversing agent

Technical field

The invention belongs to the medicine of enhanced sensitivity antitumor drug and reverse multiple drug resistance of tumor, be specifically related to gypenoside unit 3 β, the novelty teabag of 20 (S), 21-trihydroxy dammarane-24-alkene, is namely preparing the application in multidrug resistance reversing agent.

Background technology

The life and health of the malignant tumor serious harm mankind, has become the one of the main reasons of current disease death.The essential therapeutic arsenals of malignant tumor has operation, radiation and chemotherapy.Large quantifier elimination shows, malignant tumor is a kind of disease of general and the pathological changes of non-locality, therefore, compares the topical therapeutic means of operation and radiotherapy, the method of tumor pharmacother and chemotherapy becomes treatment tumor, extends the important means of survival of patients time and raising prognosis quality of life.According to statistics, the multidrug resistance of tumor cell to chemotherapeutics is the main cause of chemotherapy failure, accounts for more than 90% of chemotherapy failure.Therefore find and the compound of reverse multiple drug resistance of tumor can become the serious problems that oncotherapy needs solution badly.The drug resistance of tumours of chemotherapeutic agents can be divided into primary resistance (primary drug resistance, PDR) and the large class of multidrug resistance (multidrug resistance, MDR) two according to its drug-resistant phenotype.The former only produces drug resistance to induced drug; Latter refers to that tumor cell is after a kind of chemotherapeutics of contact, not only produce drug resistance to this medicine, and the chemotherapeutics different with mechanism of action to other structure also produces cross resistance.And it is different with mechanism according to the time of drug resistance generation, initial drug-resistant (innate resistance) and secondary drug resistance (acquired resistance) [R.Perez-Tomas can be divided into again, Curr Med Chem, 13 (2006) 1859.].The MDR phenomenon of tumor produces has complicated the Molecular Biology Mechanism, mainly comprise medicine and take in minimizing, drug efflux increase (primarily of P-gp glycoprotein and MRP protein mediated), pharmaceutically-active target molecule changes, the increase of inaxtivation of drug expression of enzymes, drug metabolism obstacle, DNA repair mechanism obstacle or DNA polymerase activity change etc.In the formation of MDR, these resistance mechanisms may work alone or in combination [T.Ozben, FEBS Lett, 580 (2006) 2903.].In recent years, how to overcome MDR and become domestic and international study hotspot.For different resistance mechanisms, medicine scholar has developed inversion agent and the sensitizer of a series of tumor multi-medicine drug-resistant.First generation tumor multi-medicine drug-resistant sensitizer comprises verapamil, ciclosporin, d-Verapamil etc., they can with chemotherapeutics carrier competition binding, thus strengthen the active anticancer of cancer therapy drug.But under required enhanced sensitivity dosage, these sensitizers have very strong toxicity usually, can produce obvious toxic and side effects, therefore and be not suitable for using clinically.Attempt going the toxicity reducing them also not obtain Expected Results.Although the sensitizer of the second filial generation does not find obvious toxicity, the pharmacokinetics of their energy appreciable impact cancer therapy drugs.The current third generation is developed based on directly suppressing the multidrug-resistance reversal agent (tariquidar, cyclopropyldibenzosuberane, zosuquidar) of carrier.These compounds do not show toxicity, and discord cytochrome P 540 is reacted, and does not also affect the pharmacokinetics of medicine.These inversion agent, at present in clinical trial, are expected to the multidrug resistance problem solving Partial tumors.But these inversion agent mostly to there is toxicity large, active low problem, seriously constrain its range of application [M.Susa, E.Choy, C.Yang, J.Schwab, H.Mankin, F.Hornicek and Z.Duan, J Biomol Screen, 15287.S.S.Winter, D.M.Lovato, H.M.Khawaja, B.S.Edwards, I.D.Steele, S.M.Young, T.I.Oprea, L.A.Sklar and R.S.Larson, J Biomol Screen, 13 (2008) 185.A.Katsman, K.Umezawa and B.Bonavida, Drug Resist Updat, 10 (2007) 1.].Therefore, one of multidrug resistance reversing agent difficult problem remaining oncotherapy finding new high-efficiency low-toxicity.

The history of TCM on Tumor is of long standing and well established, and as an importance of modern combined therapy of tumour, more and more receive publicity [W.C.Cho, Zhongguo Fei Ai Za Zhi, 13190.] in recent years.A lot of research shows, has the composition of a lot of active artitumor multi-medicine-resistant lower compared with high toxicity in natural product.【W.Xu,A.D.Towers,P.Li andJ.P.Collet,Eur J Cancer Care(Engl),15(2006)397.】。Cucurbitaceae Herb Gynostemmae Pentaphylli (Gynostemmapentaphyllum) is herbaceos perennial, is mainly distributed in Japan, Korea S, China and south east asia.Once be used as sweeting agent in Japan, also used by as folk medicine in China always.Extract from this plant and obtain the multiple dammarane type glycoside (gypenoside) similar to ginsenoside's structure, be separated from the various piece of Herb Gynostemmae Pentaphylli so far and obtained more than 100 kinds of gypenosides.Pharmaceutical research finds, the gypenoside of Herb Gynostemmae Pentaphylli and separation has antitumor, reduces cholesterol, Promote immunity, antioxidation, the various active such as blood sugar lowering and treatment ulcer, and the toxicity of this compounds of acute, the subacute and chronic toxicity research of Gynostemma pentaphyllum Makino total saponins display is very low.So gypenoside compounds is well suited for carrying out new drug development research as the lead compound of medicament research and development.

3 β, 20 (S), 21-trihydroxy dammarane-24-alkene; also gypenoside unit 3 β are called; 20 (S), 21-trihydroxy dammarane-24-alkene, English name 3 β; 20 (S); 21-trihydroxydammar-24-ene, in the present invention referred to as H6, is hydrolyzed in the basic conditions by gypenoside XLIX and obtains [Lin JJ; Hsu HY; Yang JS, Lu KW, Wu RS; Wu KC; LaiTY, Chen PY, Ma CY; Wood WG, Chung JG.Phytomedicine; 18:1075-1085.D.R.Cao SYX, P.H.Yu, and Y.M.Deng.CN200510100735.], its molecular weight is 460.73, and molecular formula is C ₃₀h ₅₂o ₃, see Fig. 1 for its chemical constitution of 17939-12-7. for No. CAS.

Summary of the invention

The object of this invention is to provide a kind of micromolecular compound H6 and prepare the novelty teabag in multidrug resistance reversing agent.

Another object of the present invention is to the antineoplastic pharmaceutical compositions of providing package containing the antitumor drug such as H6 and VCR for the preparation of the application in treatment multidrug resistance of tumor medicine.

The cytotoxicity of H6 is very faint, to the IC of kinds of tumor cells ₅₀all be greater than 50 μMs, relatively few about its research at present.The present invention, by random screening, finds that this natural product has the effect of reversion MDR.Because its low toxicity is efficient, there is good application prospect.Experiment shows, H6 can strengthen the inhibition that chemotherapeutics is bred drug-resistant cell strain, reversing drug resistance effect.Such as, show in embodiment, H6 can reverse the drug-resistant effect of KB/VCR cell to VCR, strengthens VCR to the apoptosis-promoting effect of KB/VCR cell, makes VCR again play apoptosis-promoting effect.H6 obviously can overcome the drug resistance of multidrug resistance cell KB/VCR and MCF-7/ADR, by affecting the function of P-gp glycoprotein, the resistant characterization of reverse multiple drug resistance of tumor cell, can strengthen chemotherapeutics to the therapeutic effect of cells of resistant tumors.

The invention provides the application of H6 in the reversal agent of drug resistance (Reversal agent) preparing antitumor drug.

Described tumor can be the various tumors such as oral cancer, breast carcinoma, hepatocarcinoma, pulmonary carcinoma, cervical cancer or cancer of pancreas to chemotherapeutics generation drug resistance.Described tumor cell can be the tumor cell of oral cancer, breast carcinoma, hepatocarcinoma or cervical cancer to chemotherapeutics generation drug resistance.Such as, multidrug resistance cell KB/VCR and MCF-7/ADR adopted in embodiment.H6 has remarkable result for the drug resistance accelerating to cause due to drug metabolism, is particularly useful for the tumor cell being carried out drug metabolism by P-gp glycoprotein.

Described antitumor drug can be medicines resistant to liver cancer, anti-oral cancer medicine or Antilung gland cancer medicine, can be cell cycle specific agents (CCSA), as vincristine, paclitaxel etc., it also can be the cell cycle nonspecific agent (CCNSA) such as daunorubicin, 5-fluorouracil (CCNSA).

Described antitumor drug reversal agent of drug resistance can be made into the pharmaceutically acceptable dosage form of any one, comprises tablet, capsule, granule, oral liquid, slow releasing preparation, controlled release preparation, nanometer formulation, injection etc.

Present invention also offers the application of H6 in the sensitizer (sensitizer) preparing antitumor drug.

H6 can overcome the drug resistance of drug-resistant cell strain, increases tumor cell line to the sensitivity of medicine, strengthens VCR to the apoptosis-promoting effect of KB/VCR cell.H6 can increase Rhodamine 123 in the intracellular accumulation of KB/VCR, stimulates the atpase activity of P-gp albumen.

The reversal agent of drug resistance of the antitumor drug described in the present invention or the active component of sensitizer are H6.

Present invention also offers a kind of antineoplastic pharmaceutical compositions, said composition comprises H6 and antitumor drug.Described antitumor drug can be the cell cycle specific agents such as vincristine, paclitaxel (CCSA), also can be the cell cycle nonspecific agent (CCNSA) such as daunorubicin, 5-fluorouracil (CCNSA).H6 also can with surgical operation conbined usage, with one or more Western medicine conbined usage, with Chinese herbal medicine conbined usage, with radiation treatment conbined usage.

In one embodiment of the present of invention, H6 can strengthen the apoptosis-promoting effect of VCR to KB/VCR cell, can reverse the drug-resistant effect of KB/VCR cell to VCR, make VCR again play apoptosis-promoting effect.Because VCR itself is a microtubule inhibitors, the depolymerization of microtubule can be caused, cause apoptosis, make the cytosis being in the sub-G1 phase.For this reason, the present invention Flow cytometry impact of H6 on the apoptosis of the persister cell that VCR induces.As shown in Figure 3, KB/VCR cell, after 10 μMs of H6 or 100nM VCR individual processing 48h, is in the ratio shared by cell that namely apoptosis occur its cell of sub-G1 and is about 1%.When KB/VCR cell is after the VCR of H6 and the 100nM of 10 μMs or 20 μMs cooperatively processes, occur to adjust the cell proportion of dying to be respectively 9.8% and 15.1%, 10 times when being 100nM VCR individual processing and 15 times.Therefore, H6 significantly can strengthen the apoptosis of the KB/VCR cell of VCR induction, and this effect strengthens along with the increase of H6 concentration.In the test to KB cell, do not find similar phenomenon.

On the other hand, the invention provides a kind of method of multidrug resistance tumor cells to chemotherapy drug susceptibility increasing In vitro culture, namely in the culture medium of multidrug resistance tumor cells, add H6.

Described tumor comprises oral cancer, breast carcinoma, hepatocarcinoma, pulmonary carcinoma, cervical cancer, adenocarcinoma of lung or cancer of pancreas.Described tumor cell can be the various tumor cells such as oral cancer, breast carcinoma, hepatocarcinoma, pulmonary carcinoma, cervical cancer or cancer of pancreas to chemotherapeutics generation drug resistance.Such as, multidrug resistance cell KB/VCR and MCF-7/ADR adopted in embodiment.

After tumor cell dosing, in culture medium, the concentration of H6 can be greater than 20 μMs, is even greater than 50 μMs.But, even if the concentration of H6 is lower in culture medium, such as 5-20 μM, even close to but when not comprising 0 μM, H6 still can play the multidrug-resistance reversal agent of antitumor drug or the effect of sensitizer.

Micromolecular compound H6 of the present invention can adopt the preparation method of various routine to prepare.Such as, the method can synthesized from separation and purification in plant (such as, Herb Gynostemmae Pentaphylli) or artificial chemistry.

Utilize micromolecular compound of the present invention, by various conventional screening assays, can filter out, with H6, interactional material occur, as receptor, inhibitor or antagonist etc.

The present invention and inhibitor, antagonist etc., when carrying out using (administration) on treating, can provide different effects.Usually, but these materials are formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can with being formulated the character of material and disease to be treated and changing to some extent.The pharmaceutical composition prepared can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, Intradermal or topical.

For H6 of the present invention, can by itself and suitable pharmaceutically acceptable carrier coupling.This kind of pharmaceutical composition contains the compound and pharmaceutically acceptable carrier or excipient for the treatment of effective dose.This kind of carrier comprises (but being not limited to): normal saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.H6 of the present invention can be made into injection form, such as, be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.The pharmaceutical composition of such as Tablet and Capsula and so on, is prepared by conventional method.Pharmaceutical composition such as injection, solution, Tablet and Capsula should aseptically manufacture.The dosage of active component is treatment effective dose, such as every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, H6 of the present invention also can use together with other treatment agent.

When H6 of the present invention is used as medicine, the H6 for the treatment of effective dose can be applied to mammal, wherein this treatment effective dose is usually at least about 10 micrograms/kg body weight, and be in most of the cases no more than about 8 mg/kg body weight, preferably dosage is about 10 micrograms/kg body weight ~ 1 mg/kg body weight.Certainly, concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.

The invention provides H6 and prepare the application in antitumor drug.H6 is natural product, can the propagation of obvious inhibition tumor cell when high dose.Experiment shows, H6 obviously can overcome the drug resistance of multidrug resistance cell KB/VCR and MCF-7/ADR, by affecting the function of P-gp glycoprotein, the resistant characterization of reverse multiple drug resistance of tumor cell, can strengthen chemotherapeutics to the therapeutic effect of cells of resistant tumors.Micromolecular compound of the present invention is developed as new antitumor drug or its auxiliary element, and tumor killing effect is obvious, environmental protection, provides a kind of new approach and means by for treatment and healing tumor.

Accompanying drawing explanation

Fig. 1 is the chemical constitution of H6.

Fig. 2 shows the sensitivity that H6 can make KB/VCR cell again recover VCR,

Wherein, A figure and C figure represents that the VCR conbined usage of H6 or 10 μM VPL and the variable concentrations of working as 10 μMs is on the impact of KB/VCR cell proliferation, and B figure and H6 or the 10 μM VPL of D figure expression 10 μMs and the VCR conbined usage of variable concentrations are on the impact of KB cell proliferation.In figure, abscissa represents the concentration of VCR, vertical coordinate display be the survival rate of cell.

Fig. 3 shows the apoptosis that H6 can strengthen the KB/VCR cell of VCR induction,

Wherein, the KB/VCR cell that to be flow cytomery H6 induce VCR of scheming A and B display and the apoptotic impact of KB.C figure and D figure is the statistical result to A figure and B figure respectively.

Fig. 4 shows H6 can increase Rhodamine 123 in intracellular accumulation,

Wherein, figure A and figure B display be H6 on the flow cytometer detection result of Rhodamine 123 in the impact of KB and KB/VCR intracellular accumulation, in figure, VPL is positive control.Figure C is the statistical result to figure A and figure B.D figure is that the H6 that observes under fluorescence microscope is on the impact of Rhodamine 123 at KB and KB/VCR intracellular accumulation.A and b is respectively KB and the KB/VCR cell without any process, c and d is respectively the KB/VCR cell after VPL and the H6 process 3h of 10 μMs.

Fig. 5 shows the atpase activity that H6 can promote P-gp albumen,

Abscissa represents the concentration of H6, and vertical coordinate is the cold light value that records after H6 process of recombinant human P-gp protein membrane components and Na ₃vO ₄the difference of the cold light value after process, the i.e. relative changing value (Δ RLU) of this sample cold light value, can show the Expenditure Levels of ATP in reaction system by calculating Δ RLU, thus reflection H6 is on the impact of the atpase activity of P-gp albumen.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this.Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Embodiment 1:CCK-8 test kit detects the cytotoxicity of H6

Experiment material:

H6 extracts from cucurbitaceous plant Herb Gynostemmae Pentaphylli, and purity is not less than 95%.HepG2 cell lines, human hepatoma cell strain SMMC-7721, human hepatoma cell strain Hep3B, human leukemia cell line THP-1, human leukemia cell line K562, undifferentiated human stomach cancer cell line HGC-27, human oophoroma cell line SKOV3, human pancreas cancer cell strain PANC-1, human colon cancer cell strain SW480, Human cervical carcinoma cell line HeLa, human A459 lung cancer cell line, Human breast cancer cell line MDA-MB-453 are all purchased from Shanghai OEG cell institute of Chinese Academy of Sciences cell bank.Human umbilical vein epithelial cell (HUVEC-2C) cell strain is purchased from Cascade Biologics biotech company.

Experimental technique:

Cell recovery

1) from liquid nitrogen container, take out cryopreservation tube, directly drop in 37 DEG C of warm water, and shake makes it melt as early as possible frequently.

2) from 37 DEG C of water-baths, take out cryopreservation tube, with suction pipe sucking-off cell suspension, inject centrifuge tube and add more than 10 times culture fluid, low-speed centrifugal after mixing, abandons supernatant, then repeats to wash once with culture fluid.

3), after suitably diluting with culture fluid, inoculated and cultured bottle, be placed on 37 DEG C of incubator quiescent culture, next day changes culture fluid, continues to cultivate.Go down to posterity when being cultured to finite concentration.

Passage is cultivated

HepG2 cell lines, human hepatoma cell strain SMMC-7721, human hepatoma cell strain Hep3B, human leukemia cell line THP-1, human leukemia cell line K562, undifferentiated human stomach cancer cell line HGC-27, human oophoroma cell line SKOV3, human pancreas cancer cell strain PANC-1, human colon cancer cell strain SW480, Human cervical carcinoma cell line HeLa, human A459 lung cancer cell line, Breast cancer lines MDA-MB-45, human breast cancer cell line Bcap-37 are all incubated at containing in the DMEM culture fluid of 10% hyclone or 1640 culture medium, cultivate in 5%CO2 incubator.HUVEC-2C cell culture is in the culture medium 200(Cascade Biologics biotech company containing low Serum Growth-Factors composition) in.

The situation of observation of cell growth every day, when cell grows to about 90% degree of converging (attached cell) in culture bottle, goes down to posterity according to the ratio of 1:3 to 1:5, about went down to posterity once every 2 ~ 4 days.Method is as follows:

1) with 1 × phosphate buffer wash cell once.

2) add the digestion of 1 ~ 2ml 0.25% tryptic digestive juice, be placed in 37 DEG C of incubator numbers minute.Pat Tissue Culture Flask with hands, make cell separation.

3) trypsinization is stopped with the suitable culture medium containing 10%Gibco hyclone.Cell is sub-packed in new culture bottle, continues to cultivate.

Cell cryopreservation

1) get the cell being cultured to exponential phase, trypsinization, to be collected in centrifuge tube and to count, centrifugal.

2) reject trypsin and old culture fluid, add the frozen culture fluid (containing 10%DMSO, 40%DMEM and 50%Gibico hyclone) configured, in cryopreserving liquid, the ultimate density of cell is 0.5-1 × 10 ⁷/ ml.Blowing and beating gently with suction pipe makes cell even, and be then distributed in aseptic cryopreservation tube, often pipe adds 1-1.5ml.

3) cryopreservation tube is put into freezing storing box and be placed in-80 DEG C, move in liquid nitrogen container after 5 hours and preserve.

CCK-8 tests

All cells, according to above-mentioned CMC model, in time growing to exponential phase, is inoculated in 96 orifice plates according to the density of 3500 cells/well, adds H6 after 24h.If 9 Concentraton gradient, each concentration establishes 3 multiple holes.Cell at 37 DEG C, 5%CO ₂cultivate after 48 hours under condition, outwell culture fluid, every hole adds 90 μ L not containing culture medium and the 10 μ L CCK-8 reagent of serum.After 37 DEG C of reaction 2h, microplate reader reads the light absorption value (OD of 450nm wavelength ₄₅₀).By calculating the cell proliferation ratio of medication group relative to matched group.Blank group only adds culture medium for not adding cell, and matched group is add the DMSO with medicine same volume, cell survival rate=(experimental group OD ₄₅₀-blank group OD ₄₅₀)/(matched group OD ₄₅₀-blank group OD ₄₅₀).

IC ₅₀refer to that the suppressed half of cell proliferation is used inhibitorconcentration.Here the concentration of H6 when above-mentioned cell survival rate is 50% is.IC ₅₀calculating generally need the dosage effect of mensuration more than 5, then obtain function by curve fitting and calculate and obtain.

Each experiment at least repeats 3 times.IC ₅₀value is averaged, and calculates its standard deviation.

Table 1H6 is to the cytotoxicity of normal cell and kinds of tumor cells

Experimental result:

Result is as shown in table 1, and H6 is to the IC of most of tumor cell ₅₀all be greater than 100 μMs, to the IC of all tumor cells ₅₀all be greater than 50 μMs.

Conclusion: H6 is a kind of safety low-poison natural product.

Embodiment 2:CCK-8 test kit detects drug-resistant cell strain to the drug resistance multiple of H6

Experiment material:

Verapamil (VPL), vincristine (VCR) and amycin (ADR) are purchased from Roche chemical company, and purity is all greater than 99%.CCK-8 test kit is purchased from colleague company.

Human oral cavity epithelial cancer KB cell and vincristine persister KB/VCR, MCF-7 Human Breast Cancer Cells and Adriamycin resistant strain MCF-7/ADR thereof are prepared by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences.

Experimental technique:

Cell recovery and cell freezing method are with embodiment 1.

Passage is cultivated: human oral cavity epithelial cancer KB cell and vincristine persister KB/VCR thereof are incubated in the α-MEM culture medium containing 10%FBS, 2mM glutamine and 1mM Sodium Pyruvate.Human breast cancer cell line Bcap-37/ADR the cell strain of adriamycin-resistant is incubated in the α-MEM culture medium containing 10%FBS, 1mM Sodium Pyruvate and 0.01mg/ml insulin, and its parental cell MCF-7 cultivates containing in the DMEM of 10% hyclone.All persisters started withdrawal in experiment before three days, when cell is in exponential phase, carry out CCK-8 experiment.

CCK-8 tests

KB and KB/VCR cell and MCF-7 and MCF-7/ADR cell are all inoculated in 96 orifice plates according to the density in 3500/ hole, the ADR of variable concentrations after 24h, are added in each hole after VCR, H6 and VCR and the H6 mono-α-MEM reinstated containing 10% hyclone prepares.Cultivate after 48h, discard culture fluid, with method in embodiment 1, survey drug-resistant cell strain and parental cell thereof to the IC of H6 with CCK-8 test kit ₅₀value.Pass through IC ₅₀value calculates drug resistance multiple (Resistance Fold, RF) again.

The IC of RF=drug-resistant cell strain ₅₀the IC of value/parental cell strain ₅₀value.Each concentration establishes 3 repeating holes, experiment repetition 3 times.

Table 2H6 can overcome the drug resistance of drug-resistant cell strain

All mdr cells grow 3 days before tying up to Cell suppression test in without pharmaceutical culture medium.Each numerical value is 3 independent experiment results, IC ₅₀represent with " means standard deviation " form.VCR, vincristine; ADR, daunorubicin; RF, drug resistance multiple.

Experimental result:

KB/VCR and MCF-7/ADR is two kinds of conventional multidrug resistance cell strains, and in the present embodiment, these two kinds of cells also show the characteristic of multidrug resistance.As shown in table 2, KB/VCR cell is respectively 81.9 times and 94.4 times relative to the drug resistance multiple of KB cell to VCR and ADR, and the drug resistance multiple that MCF-7/ADR cell compares VCR and ADR with MCF-7 cell is 38.1 times and 20.9 times respectively, display experiment mdr cell used has multidrug resistance, and MDR activity is similar to the result of bibliographical information.On this MDR in-vitro screening model, find that H6 shows very strong cytotoxicity to multiple medicine-resistant cell line, as shown in table 2, the drug resistance multiple of KB/VCR to H6 is 1.03 times, and the drug resistance multiple of MCF-7/ADR cell to H6 is 1.04 times.Show that H6 can overcome the multidrug resistance characteristic of KB/VCR and MCF-7/ADR two strain cell.

Conclusion: H6 can overcome the drug resistance of drug-resistant cell strain, and the sensitivity of drug-resistant cell strain to H6 is identical.

Embodiment 3:CCK-8 method detects H6 to the reverse effect of the multidrug-resisting activity of drug-resistant cell strain

Experiment material: with embodiment 2.

Experimental technique:

H6 enhanced sensitivity is tested: KB and KB/VCR cell is inoculated in 96 orifice plates according to the density in 3500/ hole, the VCR of variable concentrations after 24h, and VCR and H6 mono-of the variable concentrations α-MEM reinstated containing 10% hyclone prepare after be added in each hole.Cultivate after 48h, discard culture fluid, with method in embodiment 1, survey drug-resistant cell strain and parental cell thereof to the activity of VCR and VCR+H6 with CCK-8 test kit, plot curve.

Each concentration establishes 3 repeating holes, experiment repetition 3 times.

Experimental result:

The drug resistance multiple of KB/VCR cell to VCR is 81.9 times, and when after the H6 adding variable concentrations (5 μMs, 10 μMs, 20 μMs), make the sensitivity of KB/VCR cell to VCR add 2-10 doubly (table 3, Fig. 2 A), H6 can reverse the drug resistance of KB/VCR to VCR.And this effect shows and not obvious (Fig. 2 B) in parent KB cell.FR(Fold Reversal) represent reversal index.VPL is positive control (Fig. 2 C, 2D).

Table 3H6 reverses KB/VCR to the drug resistance of VCR

Conclusion: H6 can reverse the drug resistance of KB/VCR to VCR.

The apoptotic impact of KB/VCR that embodiment 4: Flow cytometry H6 induces VCR

Experiment material: the equal available from Sigma of RNase A and PI.

Experimental technique:

To take the logarithm KB/VCR and the KB cell of trophophase, by 5 × 10 ⁴the density of individual cells/well inoculates 12 orifice plates, treats that cell enters logarithmic growth after date and adds H6, the compositions of VCR and H6 and VCR after 24h.After 48h, trypsinization, centrifugal collecting cell, abandons supernatant, and cleans cell twice with pre-cooling PBS, is resuspended in the Triton X-100 containing 0.03%, in the PBS of 200mg/mlRNase A and 50 μ g/ml PI.After lucifuge incubated at room 15min, Flow cytometry.In experiment, VPL is positive control.The DNA fragment produced due to cell in apoptotic process can cause the DNA content of cell to reduce, therefore by there will be a sub-G1 peak during FCM analysis before G1 phase peak.The ratio that apoptotic cell accounts for all cells can be reacted by the percentage ratio calculating total cell shared by sub-G1 phase cell.

Experimental result:

As shown in Figure 3, KB/VCR cell is after 10 μMs, 20 μMs H6 or 100nM VCR individual processing 48h, and the ratio shared by apoptotic cell is about 1%, illustrates that the VCR individual processing of H6 or 100nM can not cause obvious natural death of cerebral cells.After the VCR co-treatment of H6 and the 100nM with 10 μMs or 20 μMs, the apoptosis ratio of KB/VCR cell rises to 9.8% and 15.1% respectively, 10 times when being about 100nM VCR individual processing and 15 times.Therefore, H6 significantly can strengthen the apoptotic effect of the KB/VCR cell of VCR induction, and this effect strengthens along with the increase of concentration.In the test to KB cell, do not find similar phenomenon.

Conclusion: H6 can reverse the drug-resistant effect of KB/VCR cell to VCR, strengthens the apoptosis of VCR induction.

Embodiment 5: fluorescence microscope and Flow cytometry H6 are on the impact of Rhodamine 123 accumulation in cell

Experiment material: Rhodamine 123 available from Sigma.

Experimental technique:

KB and KB/VCR cell is according to 1 × 10 ⁵the density in/hole is inoculated in 12 orifice plates, adds 10 μMs of H6 after 24h, and after continuing to cultivate 3h, adding Rhodamine 123 to final concentration is 5 μMs, after lucifuge hatches 30min, the Rhodamine 123 that PBS cleaning twice removing cell surface is residual.Fluorescence microscopy Microscopic observation is also taken pictures.Collected by trypsinisation cell, flow cytometry 488nm exciting light detects the accumulation situation of Rhodamine 123 in cell.

Experimental result:

Rhodamine 123 is the substrate of P-gp glycoprotein, by can green fluorescence be produced after the laser excitation of 488nm wavelength, and can by fluorescence microscope and Flow cytometry.Result as shown in Figure 4.When there is no H6 process, Rhodamine 123 is very low in KB/VCR intracellular accumulation degree, after the of short duration process of 3 hours 10 μMs of H6, fluorescence microscopy Microscopic observation, intracellular green fluorescence (Rhodamine 123) obviously strengthens, and flow cytometry display Rhodamine 123 obviously increases in the intracellular accumulation of KB/VCR.

Conclusion: H6 can affect the function of P-gp glycoprotein, increases Rhodamine 123 in the intracellular accumulation of KB/VCR.

Embodiment 6:P-gp GLO ^tMtest kit detects H6 to the impact of P-gp glycoprotein A TP enzymatic activity

Experiment material: P-gp GLO test kit is purchased from Promega company

Experimental technique:

In order to test the impact of H6 on P-gp activity, we adopt the P-gp-Glo of promega company ^tMpilot system measures the change of P-gp protein A TP enzymatic activity, operational approach reference reagent box description.Gallbladder sodium vanadate (Na ₃vO ₄) be the inhibitor of P-gp protein A TP enzymatic activity.First by H6 and the 5mM MgATP of variable concentrations, the mankind P-gp protein membrane components mixing that 25 μ g recombinate, after 37 DEG C of incubation 40min, adds ATP and detects buffer, incubated at room 20min.Mixed liquor to be measured is proceeded in 96 hole blanks, application multiple detection system measurement cold light reading.The cold light value recorded is deducted Na ₃vO ₄the cold light value of the sample of process, namely obtains the relative changing value (Δ RLU) of the cold light value of this sample.

Experimental result:

By calculating the value of Δ RLU, the Expenditure Levels of P-gp albumen to ATP can be reflected, indirectly the atpase activity of reflection P-gp albumen.In the result of this experiment, as shown in Figure 5, along with the rising of H6 concentration, the consumption of P-gp albumen to ATP constantly increases, and illustrates that H6 can promote the atpase activity of P-gp albumen.H6 stimulates the half-maximal effect concentration (EC of P-gp protein A TP enzymatic activity ₅₀) be 38 μMs.

Conclusion: H6 can promote the atpase activity of P-gp albumen.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (7)

1.3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is preparing the application in antitumor drug, it is characterized in that, described application is 3 β, 20 (S), the application of 21-trihydroxy dammarane-24-alkene in the reversal agent of drug resistance preparing antitumor drug.

2. apply as claimed in claim 1, it is characterized in that, 3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is multidrug-resistance reversal agent.

3. apply as claimed in claim 1, it is characterized in that, described tumor comprises oral cancer, breast carcinoma, hepatocarcinoma, cervical cancer, adenocarcinoma of lung, colon cancer or cancer of pancreas.

4. apply as claimed in claim 1, it is characterized in that, described antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

5. apply as claimed in claim 1, it is characterized in that, 3 β, 20 (S), 21-trihydroxy dammarane-24-alkene is the sensitizer of antitumor drug.

6. impel a method for external cells of resistant tumors enhanced sensitivity, it is characterized in that, in the culture medium of cells of resistant tumors, add 3 β, 20 (S), 21-trihydroxy dammarane-24-alkene.

7. method as claimed in claim 6, it is characterized in that, described tumor comprises oral cancer, breast carcinoma, hepatocarcinoma, cervical cancer, adenocarcinoma of lung or cancer of pancreas.