CN105030788A - Application of natural compound Homoharringtonine in preparation of tumor treatment medicine - Google Patents
Application of natural compound Homoharringtonine in preparation of tumor treatment medicine Download PDFInfo
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Abstract
The invention discloses application of a natural compound Homoharringtonine in preparation of a tumor treatment medicine. Homoharringtonine of the invention has following characteristics of: inhibiting transcriptional activity of a transcription factor STAT3, inhibiting expression of a downstream gene MCL1 and survivin of STAT3, and inhibiting an anti-apoptosis effect of the MCL1 and surviving. Homoharringtonine has an action of effectively resisting tumor proliferation and resisting STAT3 phosphorylation in non-small cell lung cancer subcutaneous tumor bearing migration model of a nude mice. As a natural compound, Homoharringtonine can overcome the promotion action of interleukin IL-6 to tumors. The application achieves a new level in tumor targeted therapy aspect of natural compounds, and has well application prospect.
Description
Technical field
The present invention relates to the application of native compound Homoharringtonine in preparation tumor in biomedical sector.
Background technology
Pulmonary carcinoma is one of modal tumor, about has the cancer patient of 1/4th to die from pulmonary carcinoma every year.According to statistics, in the world, newly-increased 224,210 routine patients with lung cancer (wherein male patient 116 in 2014,000 example, female patient 108,210 examples), 159,260 routine Died Patients (wherein male patient 86,930 examples, female patient 72,330 examples).Pulmonary carcinoma is divided into two classes usually: small cell lung cancer (smallcelllungcancer, and nonsmall-cell lung cancer (non-smallcelllungcancer SCLC), NSCLC), wherein common with nonsmall-cell lung cancer, account for 80% of cases of lung cancer sum.According to an advancing of disease stage pathologic condition, the method for the treatment of pulmonary carcinoma mainly contains operation chemotherapy, targeted therapy and composite treatment.At present, some active drugs are developed, the tyrosinase inhibitor (tyrosinekinaseinhibitors, TKIs) being wherein representative with gefitinib (Gefitinib) and Erlotinib (Erlotinib) shows good effect in oncotherapy.But in use for some time, most patients because the sudden change of growth factor receptors (epithelialgrowthfactorreceptor, the EGFR) T790M of Cuticle of cell is developed immunity to drugs, thus causes Endodontic failure.Although have some such as Ah methods for the medicine such as Buddhist nun (Afatinib) and Dacomitinib to overcome Drug resistance, effect is still limited.Therefore develop new non-small cell lung cancer cell of can inducing dead, the new drug overcoming EGFRTKI drug resistance is instant to extend the life cycle of lung cancer patient.
The acquisition of antitumor drug is except de novo synthesis micromolecular compound, and the effective active composition be separated from plant extract is also an important sources.Have report to point out, the many monomer micromolecular compounds extracted from natural Chinese medicinal herb have new construction features and unique pharmaceutically active.Clinically, there is compared with the chemotherapeutics of routine the feature that side effect is little, and in many tolerances existed in oncotherapy, recurrence waits also to have in the exploitation of medicine also there is certain mitigation.Therefore, from plant, extracting and developing, screening and identification bioactive ingredients are important sources in tumour medicine exploitation.
Natural small molecule compounds Homoharringtonine (HHT) is a kind of extraction one of isolated alkaloid in cephalotaxus plant, structural formula is as formula I, and chemical name is (2R)-2-hydroxyl-2-(4-hydroxy-4-methyl amyl group) succinic acid (methyl) (cephalotaxin) ester.
Summary of the invention
Technical problem to be solved by this invention how to utilize Homoharringtonine to treat tumor.
For solving the problems of the technologies described above, the invention provides following arbitrary application:
1, Homoharringtonine (HHT) is preparing the application in inducing apoptosis of tumour cell and/or inhibition tumor cell propagation product (as medicine or pharmaceutical preparation);
2, the application of Homoharringtonine in preparation treatment tumor product (as medicine or pharmaceutical preparation);
3, Homoharringtonine is preparing the application in the tumor cell proliferation medicine (as medicine or pharmaceutical preparation) suppressing interleukin-6 (interleukin-6, IL-6) to promote;
4, Homoharringtonine promotes the application in tumor cell Mitochondria apoptosis product (as medicine or pharmaceutical preparation) in preparation;
5, Homoharringtonine suppresses transcription factor STAT3 to the application in the transcriptional activity product (as medicine or pharmaceutical preparation) of anti-apoptotic genes expression in preparation;
6, Homoharringtonine suppresses the application in the transcriptional activity product (as medicine or pharmaceutical preparation) of STAT3, MCL1 and/or Survivin in preparation;
7, Homoharringtonine suppresses the application in STAT3, MCL1 and/or Survivin gene expression products (as medicine or pharmaceutical preparation) in preparation;
8, Homoharringtonine suppresses the application in the enzymatic activity product (as medicine or pharmaceutical preparation) of STAT3, MCL1 and/or Survivin in preparation;
9, Homoharringtonine suppresses the application in the STAT3 phosphorylation product (as medicine or pharmaceutical preparation) of interleukin-6 induction in preparation.
In above-mentioned application, described STAT3 can be MCL1 or Survivin to anti-apoptotic genes expression.
In above-mentioned application, described tumor can be nonsmall-cell lung cancer, pulmonary carcinoma, hepatocarcinoma, esophageal carcinoma, gastric cancer, colon cancer, cancer of pancreas, leukemia, rectal cancer, cerebroma or skin carcinoma.
Described tumor can be resistant tumors further; Described resistant tumors can be resistance to gefitinib (Gefitinib) tumor, as the nonsmall-cell lung cancer of resistance to gefitinib (Gefitinib).
In an embodiment of the invention, described tumor is nonsmall-cell lung cancer, and described tumor cell is gefitinib drug resistant non-small cell lung cell, as Lines A549 and NCI-H1975.
In above-mentioned application, the concentration of HHT described in described product can be 0.125 ~ 6 μM, 0.25 ~ 6 μM, 0.5 ~ 6 μM, 0.5 ~ 4 μM, 1 ~ 6 μM, 2 ~ 6 μMs, 3 ~ 6 μMs, 4 ~ 6 μMs or 5 ~ 6 μMs.The concentration of described HHT specifically can be 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs.
Above, described MCL1 and Survivin can be MCL1 and Survivin in people source, and transcription factor STAT3 can be human transcriptionfactor hTF STAT3.
Experiment of the present invention shows on a molecular scale, HHT suppresses the phosphorylation of STAT3, and then affects the expression of its Downstream regulatory protein MCL1 and Survivin, and then suppresses the propagation of non-small cell lung cancer cell, inducing apoptosis of tumour cell, thus the object reaching treatment tumor.
Native compound HHT has following advantage in the application of preparation treatment nonsmall-cell lung cancer:
1.HHT is as a kind of natural small molecule compounds, comparatively strong to suppressing nonsmall-cell lung cancer propagation, apoptosis-induced effect, with the survival of hypotoxicity anticancer within shorter action time, especially can have the treatment of resistant tumors for gefitinib.
2. the Hyperphosphorylationof of STAT3 can be stimulated owing to there is autocrine (autocrine) and paracrine (paracrine) IL-6 in pulmonary carcinoma, and then excessive activation STAT3, and then promote the survival of tumor cell.HHT then can suppress IL-6 to the anti-apoptotic activities of non-small cell lung cancer cell, thus inducing apoptosis of tumour cell.Therefore the treatment that HHT is used for IL-6 inducing survival tumor should obtain better curative effect.
3., in the treatment of nonsmall-cell lung cancer, the tolerance for tyrosinase inhibitor TKIs is a treatment difficult problem.HHT can show good antiproliferative effect, the effect of cell death inducing in gefitinib drug-resistant cell strain A549 and NCI-H1975.Therefore in the treatment of drug resistance nonsmall-cell lung cancer, HHT shows good therapeutic effect.
4. the medicine prepared by HHT can be used for the treatment of multiple nonsmall-cell lung cancer.
Experiment proves, under the identical processing time, HHT raises with the increase of the concentration of HHT the suppression ratio of cancerous cell, and under same concentrations, HHT raises with the increase in HHT processing time the suppression ratio of cancerous cell.In the identical processing time with under identical concentration, HHT to the suppression ratio of cancerous cell higher than the suppression ratio of gefitinib to cancerous cell: constantly little in process 24, the suppression ratio of HHT to A549 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 1.18,1.35,1.24,1.48,2.34 and 4.47 times of respective concentration gefitinib, and the suppression ratio of HHT to NCI-H1975 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 1.08,0.57,1.41,1.70,2.12 and 2.91 times of respective concentration gefitinib; Constantly little in process 48, the suppression ratio of HHT to A549 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 2.17,2.59,2.68,2.60,2.73 and 4.38 times of respective concentration gefitinib, and the suppression ratio of HHT to NCI-H1975 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 1.51,2.14,2.57,2.50,2.65 and 4.02 times of respective concentration gefitinib.
Experiment proves, HHT can the propagation of anticancer, and this inhibitory action raises with the increase of HHT concentration: under 1 μM, 2 μMs and 4 μMs of HHT, A549 cell soft agar Clone formation number is respectively 73%, 44% and 26% of 0 μM of HHT; Under 0.5 μM, 1 μM and 2 μMs of HHT, NCI-H1975 cell soft agar Clone formation number is respectively 78%, 31% and 18% of 0 μM of HHT.
Experiment proves, HHT significantly can reduce gross tumor volume under the prerequisite not affecting the weight of animals, and at the 24th day, after HHT treatment group nude mice, the volume of tumor was respectively 0.32 times and 0.28 times of treated with gefitinib group and matched group.
In general, the invention relates to the clinical medicine purposes that the micromolecular compound HHT that extracts from natural Chinese medicinal herb is new.HHT can be nonsmall-cell lung cancer and other neoplasm metastasis patient provides new medicine and therapy approach, has dosage moderate, moderate cost, and medication is convenient, evident in efficacy, the advantages such as toxic and side effects is little.
Accompanying drawing explanation
Fig. 1 is that the HHT of variable concentrations is to the inhibitory action of A549 and NCI-H1975 cell.Wherein, A is HHT process 24 hours suppression ratio to A549 cell of variable concentrations; B is HHT process 48 hours suppression ratio to A549 cell of variable concentrations; C is HHT process 24 hours suppression ratio to NCI-H1975 cell of variable concentrations; D is HHT process 48 hours suppression ratio to NCI-H1975 cell of variable concentrations; E be under variable concentrations HHT HHT to the inhibitory action of A549 cell soft agar Clone formation, left figure is violet staining result, and right figure is the percentage ratio that under variable concentrations HHT, A549 cell soft agar Clone formation number accounts for A549 cell soft agar Clone formation number under 0 μM of HHT; F be under variable concentrations HHT HHT to the inhibitory action of NCI-H1975 cell soft agar Clone formation, left figure is violet staining result, and right figure is the percentage ratio that under variable concentrations HHT, NCI-H1975 cell soft agar Clone formation number accounts for NCI-H1975 cell soft agar Clone formation number under 0 μM of HHT.
Fig. 2 is the expression of the albumen (caspase9, caspase3, CytochromeC, PARP, BAX, BCL2, MCL1 and Survivin) that in A549 with the NCI-H1975 cell of variable concentrations HHT process, apoptosis is relevant.Wherein, A is the expression of CytochromeC, caspase9, caspase3 and PARP, and B is the expression of BAX, BCL2, MCL1 and Survivin, and H1975 represents NCI-H1975.
Fig. 3 is the testing result of STAT3, JAK1, AKT and ERK expression and phosphorylation state thereof in A549 and the NCI-H1975 cell of variable concentrations HHT process.Wherein, figure A is the testing result of STAT3 expression and phosphorylation state thereof, figure B is the testing result of JAK1, AKT and ERK expression and phosphorylation state thereof, H1975 represents NCI-H1975 cell, p-STAT3 (Y705) represents the STAT3 albumen that the 705th tyrosine is phosphorylated, p-STAT3 (S727) represents the STAT3 albumen that the 727th serine is phosphorylated, p-JAK1 represents the JAK1 of phosphorylation, p-AKT1/2/3 represents the AKT1/2/3 of phosphorylation, and p-ERK1/2 represents the ERK1/2 of phosphorylation.
Fig. 4 is the testing result of the STAT3 phosphorylation that HHT suppresses interleukin-6 to be induced in dose-dependent mode.Wherein, "+" represents the interleukin-6 adding 5ng/mL, and "-" expression does not add interleukin-6, and " * " represents non-specific band.
Fig. 5 is the testing result of the STAT3 phosphorylation that HHT suppresses interleukin-6 to be induced in time dependent mode."+" represents the interleukin-6 adding 5ng/mL, and "-" expression is not added interleukin-6 or do not add HHT, and " * " represents non-specific band.
Fig. 6 is the experimental result that HHT suppresses nonsmall-cell lung cancer.Wherein, A represents the gross tumor volume of nonsmall-cell lung cancer nude mice, B represents the appearance of tumors of nonsmall-cell lung cancer nude mice, C represents the body weight of nonsmall-cell lung cancer nude mice, D represents the western-blot testing result of STAT3 phosphorylation and MCL1 albumen in the tumor of nonsmall-cell lung cancer nude mice, and E represents the immunohistochemical experiment result of STAT3 phosphorylation and MCL1 albumen in the tumor of nonsmall-cell lung cancer nude mice; P-STAT3 (Y705) represents the STAT3 albumen that the 705th tyrosine is phosphorylated.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Lines A549 and NCI-H1975 in following embodiment is ATCC product.
Homoharringtonine (HHT) in following embodiment is Yuan Ye bio tech ltd, Shanghai product.
Hyclone in following embodiment is Hyclone Products.
DMEM culture medium powder in following embodiment and RPMI-1640 culture medium powder are Life Products.
MTT and DMSO in following embodiment is Sigma Products.
The research of the application shows that HHT is by inhibition tumor cell IL-6R/JAK1/STAT3 path, and then suppresses its downstream MCL1 and Survivin to express and activity, and then suppresses the propagation of non-small cell lung cancer cell, induces its apoptosis.HHT blocks the phosphorylation of STAT3 in gefitinib resistant Lines on a molecular scale, thus suppressor transposition activates.Employing concentration is the cell line A549 cell of the nonsmall-cell lung cancer of the HHT process Wild type EGFR of 1 ~ 4 μM, employing concentration is the cell line NCI-HA1975 cell of the nonsmall-cell lung cancer of the HHT process mutant egf R of 0.5 ~ 2 μM, can cause the propagation of gefitinib resistant non-small cell lung cancer cell, induces its apoptosis in 24 hours.The action effect of native compound HHT to non-small cell lung cancer cell depends on the activation of HHT to STAT3 transcription factor, and then makes anti-apoptotic associated protein MCL1, the expression of Survivin etc., thus T suppression cell survival.
Embodiment 1, HHT suppress the propagation of nonsmall-cell lung cancer, inducing apoptosis of tumour cell
1, HHT suppresses the propagation of nonsmall-cell lung cancer
1.1HHT suppresses A549 cell proliferation
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
A549 cell is divided into seven groups, often organizes 3 × 10
3individual cell, processes as follows these seven groups of A549 cells and obtains seven kinds of A549 cell culture fluids through the HHT process of variable concentrations:
One group in containing the DMEM culture medium of 10% hyclone containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process A549 cell culture fluid of 24 hours of 0 μM;
One group in 1 μM of HHT culture medium (concentration adding HHT to HHT in containing the DMEM culture medium of 10% hyclone is 1 μM), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process A549 cell culture fluid of 24 hours of 1 μM;
One group in 2 μMs of HHT culture medium (concentration adding HHT to HHT in containing the DMEM culture medium of 10% hyclone is 2 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process A549 cell culture fluid of 24 hours of 2 μMs;
One group in 3 μMs of HHT culture medium (concentration adding HHT to HHT in containing the DMEM culture medium of 10% hyclone is 3 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process A549 cell culture fluid of 24 hours of 3 μMs;
One group in 4 μMs of HHT culture medium (concentration adding HHT to HHT in containing the DMEM culture medium of 10% hyclone is 4 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process A549 cell culture fluid of 24 hours of 4 μMs;
One group in 5 μMs of HHT culture medium (concentration adding HHT to HHT in containing the DMEM culture medium of 10% hyclone is 5 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process A549 cell culture fluid of 24 hours of 5 μMs;
One group in 6 μMs of HHT culture medium (concentration adding HHT to HHT in containing the DMEM culture medium of 10% hyclone is 6 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process A549 cell culture fluid of 24 hours of 6 μMs.
According to the method described above, 44 hours are all replaced with by 20 hours, other steps are all constant, obtain the HHT process A549 cell culture fluid of 48 hours of the HHT process A549 cell culture fluid of 48 hours of 0M, the HHT process A549 cell culture fluid of 48 hours of 1 μM, the HHT process A549 cell culture fluid of 48 hours of 2 μMs, the HHT process A549 cell culture fluid of 48 hours of 3 μMs, the HHT process A549 cell culture fluid of 48 hours of 4 μMs, the HHT process A549 cell culture fluid of 48 hours of 5 μMs and 6 μMs respectively.
According to the method described above, HHT is replaced with gefitinib (Gefitinib), other steps are all constant, obtain the gefitinib process A549 cell culture fluid of 24 hours of 0 μM respectively, the gefitinib process A549 cell culture fluid of 24 hours of 1 μM, the gefitinib process A549 cell culture fluid of 24 hours of 2 μMs, the gefitinib process A549 cell culture fluid of 24 hours of 3 μMs, the gefitinib process A549 cell culture fluid of 24 hours of 4 μMs, the gefitinib process A549 cell culture fluid of 24 hours of 5 μMs and the gefitinib process A549 cell culture fluid of 24 hours of 6 μMs.
According to the method described above, HHT is replaced with gefitinib, 44 hours are all replaced with by 20 hours, other steps are all constant, obtain the gefitinib process A549 cell culture fluid of 48 hours of 0 μM respectively, the gefitinib process A549 cell culture fluid of 48 hours of 1 μM, the gefitinib process A549 cell culture fluid of 48 hours of 2 μMs, the gefitinib process A549 cell culture fluid of 48 hours of 3 μMs, the gefitinib process A549 cell culture fluid of 48 hours of 4 μMs, the gefitinib process A549 cell culture fluid of 48 hours of 5 μMs and the gefitinib process A549 cell culture fluid of 48 hours of 6 μMs.
Under 570nm, measure the light absorption value (table 1) of the A549 cell culture fluid of above-mentioned different disposal respectively, and the HHT calculating variable concentrations is to the suppression ratio of A549 cell, result is as shown in A and B in table 2 and Fig. 1.
The light absorption value of the A549 cell culture fluid of table 1, different disposal
The material of table 2, variable concentrations and processing time are to the suppression ratio (%) of A549 cell
Result shows, and constantly little with process 48 process 24 hours, HHT all raises with the increase of the concentration of HHT the suppression ratio of A549 cell, and under same concentrations, HHT raises with the increase in HHT processing time the suppression ratio of A549 cell.In the identical processing time with under identical concentration, HHT to the suppression ratio of A549 cell higher than the suppression ratio of gefitinib to A549 cell: constantly little in process 24, the suppression ratio of HHT to A549 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 1.18,1.35,1.24,1.48,2.34 and 4.47 times of respective concentration gefitinib; Constantly little in process 48, the suppression ratio of HHT to A549 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 2.17,2.59,2.68,2.60,2.73 and 4.38 times of respective concentration gefitinib.
1.2HHT suppresses NCI-H1975 cell proliferation
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
NCI-H1975 cell is divided into seven groups, often organizes 5 × 10
3individual cell, processes as follows these seven groups of NCI-H1975 cells and obtains seven kinds of NCI-H1975 cell culture fluids through the HHT process of variable concentrations:
One group in containing the RPMI-1640 culture medium of 10% hyclone containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process NCI-H1975 cell culture fluid of 24 hours of 0 μM;
One group in 0.125 μM of HHT culture medium (concentration adding HHT to HHT in containing the RPMI-1640 culture medium of 10% hyclone is 0.125 μM), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process NCI-H1975 cell culture fluid of 24 hours of 0.125 μM;
One group in 0.25 μM of HHT culture medium (concentration adding HHT to HHT in containing the RPMI-1640 culture medium of 10% hyclone is 0.25 μM), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process NCI-H1975 cell culture fluid of 24 hours of 0.25 μM;
One group in 0.5 μM of HHT culture medium (concentration adding HHT to HHT in containing the RPMI-1640 culture medium of 10% hyclone is 0.5 μM), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process NCI-H1975 cell culture fluid of 24 hours of 0.5 μM;
One group in 1.0 μMs of HHT culture medium (concentration adding HHT to HHT in containing the RPMI-1640 culture medium of 10% hyclone is 1.0 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process NCI-H1975 cell culture fluid of 24 hours of 1.0 μMs;
One group in 2.0 μMs of HHT culture medium (concentration adding HHT to HHT in containing the RPMI-1640 culture medium of 10% hyclone is 2.0 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process NCI-H1975 cell culture fluid of 24 hours of 2.0 μMs;
One group in 3.0 μMs of HHT culture medium (concentration adding HHT to HHT in containing the RPMI-1640 culture medium of 10% hyclone is 3.0 μMs), containing 5%CO
2incubator in 37 DEG C cultivate after 20 hours, the concentration adding 10 μ LMTT is the MTT aqueous solution of 5mg/mL, continue cultivation 4 hours, then the culture medium containing MTT is absorbed, add 150 μ LDMSO, shake 10 minutes on decolorization swinging table under lucifuge room temperature condition, obtain the HHT process NCI-H1975 cell culture fluid of 24 hours of 3.0 μMs.
According to the method described above, 44 hours are all replaced with by 20 hours, other steps are all constant, obtain the HHT process NCI-H1975 cell culture fluid of 48 hours of 0M respectively, the HHT process NCI-H1975 cell culture fluid of 48 hours of 0.125 μM, the HHT process NCI-H1975 cell culture fluid of 48 hours of 0.25 μM, the HHT process NCI-H1975 cell culture fluid of 48 hours of 0.5 μM, the HHT process NCI-H1975 cell culture fluid of 48 hours of 1.0 μMs, the HHT process NCI-H1975 cell culture fluid of 48 hours of 2.0 μMs and the HHT process NCI-H1975 cell culture fluid of 48 hours of 3.0 μMs.
According to the method described above, HHT is replaced with gefitinib, other steps are all constant, obtain the gefitinib process NCI-H1975 cell culture fluid of 24 hours of 0 μM respectively, the gefitinib process NCI-H1975 cell culture fluid of 24 hours of 0.125 μM, the gefitinib process NCI-H1975 cell culture fluid of 24 hours of 0.25 μM, the gefitinib process NCI-H1975 cell culture fluid of 24 hours of 0.5 μM, the gefitinib process NCI-H1975 cell culture fluid of 24 hours of 1.0 μMs, the gefitinib process NCI-H1975 cell culture fluid of 24 hours of 2.0 μMs and the gefitinib process NCI-H1975 cell culture fluid of 24 hours of 3.0 μMs.
According to the method described above, HHT is replaced with gefitinib, 44 hours are all replaced with by 20 hours, other steps are all constant, obtain the gefitinib process NCI-H1975 cell culture fluid of 48 hours of 0 μM respectively, the gefitinib process NCI-H1975 cell culture fluid of 48 hours of 0.125 μM, the gefitinib process NCI-H1975 cell culture fluid of 48 hours of 0.25 μM, the gefitinib process NCI-H1975 cell culture fluid of 48 hours of 0.5 μM, the gefitinib process NCI-H1975 cell culture fluid of 48 hours of 1.0 μMs, the gefitinib process NCI-H1975 cell culture fluid of 48 hours of 2.0 μMs and the gefitinib process NCI-H1975 cell culture fluid of 48 hours of 3.0 μMs.
Under 570nm, measure the light absorption value (table 3) of the NCI-H1975 cell culture fluid of above-mentioned different disposal respectively, and the HHT calculating variable concentrations is to the suppression ratio of NCI-H1975 cell, result is as shown in C and D in table 4 and Fig. 1.
The light absorption value of the NCI-H1975 cell culture fluid of table 3, different disposal
The material of table 4, variable concentrations and processing time are to the suppression ratio (%) of NCI-H1975 cell
Result shows, and constantly little with process 48 process 24 hours, HHT all raises with the increase of the concentration of HHT the suppression ratio of NCI-H1975 cell, and under same concentrations, HHT raises with the increase in HHT processing time the suppression ratio of NCI-H1975 cell.In the identical processing time with under identical concentration, HHT to the suppression ratio of NCI-H1975 cell higher than the suppression ratio of gefitinib to A549 cell: constantly little in process 24, the suppression ratio of HHT to NCI-H1975 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 1.08,0.57,1.41,1.70,2.12 and 2.91 times of respective concentration gefitinib; Constantly little in process 48, the suppression ratio of HHT to NCI-H1975 cell of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs and 6 μMs is respectively 1.51,2.14,2.57,2.50,2.65 and 4.02 times of respective concentration gefitinib.
2, HHT is to the inhibitory action of non-small cell lung cancer cell Clone formation
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
2.1HHT is to the inhibitory action of A549 cell clonal formation
DMEM culture medium containing 0.6% (mass percent concentration) soft agar is laid in 6 orifice plates, every hole 1mL, after culture medium solidifying, obtains the DMEM culture medium of solidifying; 1mL is contained the HHT of 4 μMs, 0.3% (mass percent concentration) soft agar and 10
3the DMEM culture medium of individual A549 cell is laid on the DMEM culture medium upper strata of solidifying, at 5%CO
2incubator in 37 DEG C cultivate after 15 days and take out, with A549 cell soft agar Clone formation number (in Fig. 1 E) under statistics 4 μMs of HHT after 0.1% violet staining.
According to the method described above, 4 μMs are replaced with respectively 0 μM, 1 μM and 2 μMs, other steps are all constant, obtain A549 cell soft agar Clone formation number (in Fig. 1 E) under 0 μM, 1 μM and 2 μMs of HHT respectively.
Result shows, under the HHT of 0 μM, 1 μM, 2 μMs and 4 μMs, A549 cell soft agar Clone formation number is respectively 289 ± 17,210 ± 13,127 ± 9 and 75 ± 11, and under 1 μM, 2 μMs and 4 μMs of HHT, A549 cell soft agar Clone formation number is respectively 73%, 44% and 26% of 0 μM of HHT.Result shows, the propagation of HHT to A549 cell is inhibited, and this inhibitory action raises with the increase of HHT concentration.
2.2HHT is to the inhibitory action of NCI-H1975 cell clonal formation
RPMI-1640 culture medium containing 0.6% (mass percent concentration) soft agar is laid in 6 orifice plates, every hole 1mL, after culture medium solidifying, obtains the RPMI-1640 culture medium of solidifying; 1mL is contained the HHT of 2 μMs, 0.3% (mass percent concentration) soft agar and 10
3the RPMI-1640 culture medium of individual NCI-H1975 cell is laid on the RPMI-1640 culture medium upper strata of solidifying, at 5%CO
2incubator in 37 DEG C cultivate after 15 days and take out, with NCI-H1975 cell soft agar Clone formation number (in Fig. 1 F) under statistics 2 μMs of HHT after 0.1% violet staining.
According to the method described above, 2 μMs are replaced with respectively 0 μM, 0.5 μM and 1 μM, other steps are all constant, obtain NCI-H1975 cell soft agar Clone formation number (in Fig. 1 F) under 0 μM, 0.5 μM and 1 μM of HHT respectively.
Result shows, under 0 μM, 0.5 μM, 1 μM and 2 μMs of HHT, NCI-H1975 cell soft agar Clone formation number is respectively 277 ± 9,216 ± 18,86 ± 10 and 51 ± 7, and under 0.5 μM, 1 μM and 2 μMs of HHT, NCI-H1975 cell soft agar Clone formation number is respectively 78%, 31% and 18% of 0 μM of HHT.Result shows, HHT raises with the increase of HHT concentration the inhibitory action of NCI-H1975 cell clonal formation.
Embodiment 2, HHT are by mitochondrial apoptotic pathway induction non-small cell lung cancer cell apoptosis
In triplicate, the concrete steps at every turn repeating to test are as follows in experiment:
A549 cell is seeded in 6 orifice plates containing DMEM culture medium, every hole 1mLDMEM culture medium, every hole 2 × 10
5individual A549 cell, at 5%CO
2incubator in 37 DEG C of overnight incubation after cell attachment, obtain A549 cell culture fluid; In this A549 cell, add HHT, obtain HHT-A549 cell culture fluid, in HHT-A549 cell culture fluid, the concentration of HHT is 4 μMs; By HHT-A549 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate 24 hours, discard culture medium, with 1 × PBS cleaning cell twice, (RIPA lysate is green skies Bioisystech Co., Ltd product then to use RIPA lysate; Article No. is P0013B) cell lysis (every hole 200 μ LRIPA lysate), then centrifugal at 1,2000rpm, 4 DEG C, abandon precipitation, obtain the supernatant containing A549 Cytoplasm total protein, by the A549 total protein of cell of the HHT process of its called after 4 μMs.
According to the method described above, 4 μMs are replaced with respectively 0 μM, 1 μM and 2 μMs, other steps are all constant, obtain the A549 total protein of cell of the HHT process of 0 μM, 1 μM and 2 μMs respectively.
NCI-H1975 cell is seeded in 6 orifice plates containing RPMI-1640 culture medium, every hole 1mLRPMI-1640 culture medium, every hole 2 × 10
5individual NCI-H1975 cell, at 5%CO
2incubator in 37 DEG C of overnight incubation after cell attachment, obtain NCI-H1975 cell culture fluid; In this NCI-H1975 cell, add HHT, obtain HHT-NCI-H1975 cell culture fluid, in HHT-NCI-H1975 cell culture fluid, the concentration of HHT is 2 μMs; By HHT-NCI-H1975 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate 24 hours, discard culture medium, cell is cleaned twice with 1 × PBS, then RIPA lysate cell lysis (every hole 200mLRIPA lysate) is used, then centrifugal at 1,2000rpm, 4 DEG C, abandon precipitation, obtain the supernatant containing NCI-H1975 Cytoplasm total protein, by the NCI-H1975 total protein of cell of the HHT process of its called after 2 μMs.
According to the method described above, 2 μMs are replaced with respectively 0 μM, 0.5 μM and 1 μM, other steps are all constant, obtain the NCI-H1975 total protein of cell of the HHT process of 0 μM, 0.5 μM and 1 μM respectively.
Utilize the method for westernblot to detect the expression of albumen (caspase9, caspase3, CytochromeC, PARP, BAX, BCL2, MCL1 and Survivin) relevant to apoptosis in above-mentioned total protein of cell respectively, result as shown in Figure 2.The primary antibodie detecting caspase9 expression is CleavedCaspase-9 (Asp353) Antibody (MouseSpecific) (CellSignalingTechnology product, article No. is #9509), the primary antibodie detecting caspase3 expression is CleavedCaspase-3 (Asp175) (5A1E) RabbitmAb (CellSignalingTechnology product, article No. is #9664), the primary antibodie detecting CytochromeC (cytochrome C) expression is Cytochromec (D18C7) RabbitmAb (CellSignalingTechnology product, article No. is #11940), the primary antibodie detecting PARP expression is CleavedPARP (Asp214) (19F4) MousemAb (HumanSpecific) (CellSignalingTechnology product, article No. is #9546), the primary antibodie detecting BAX expression is Bax (D2E11) RabbitmAb (CellSignalingTechnology product, article No. is #5023), the primary antibodie detecting BCL2 expression is Pro-ApoptosisBcl-2FamilyAntibodySamplerKit (CellSignalingTechnology product, article No. is #9942), the primary antibodie detecting MCL1 expression is Mcl-1 (D35A5) RabbitmAb (CellSignalingTechnology product, article No. is #5453), the primary antibodie detecting Survivin expression is Survivin (71G4B7) RabbitmAb (CellSignalingTechnology product, article No. is #2808), internal reference is the primary antibodie of GAPDH, GAPDH is Anti-GAPDHrabbitpolyclonalantibody (Sangon Biotech's product, article No. is D110016-0200).
Result shows, in the non-small cell lung cancer cell of HHT process, cytochrome C is discharged into kytoplasm (important symbol of mitochondrial apoptotic pathway) from mitochondrion, caspase9 and caspase3 occurs to shear and activates, PARP shears, and shows the non-small cell lung cancer cell apoptosis of HHT induction of mitochondrial apoptotic pathway.Meanwhile, through HHT process, the expression of anti-apoptotic proteins MCL1 and Survivin also decreases.
Embodiment 3, HHT by suppressing STAT3 phosphorylation, and then suppress STAT3 to activate, and survive from T suppression cell
The method of westernblot is utilized to detect 0 μM of embodiment 2 respectively, 1 μM, the A549 total protein of cell of the HHT process of 2 μMs and 4 μMs and 0 μM, 0.5 μM, STAT3 expression in the NCI-H1975 total protein of cell of the HHT process of 1 μM and 2 μMs and the state of phosphorylation thereof, the primary antibodie detecting STAT3 expression is Anti-STAT3antibody [EPR787Y] (Abcam product, article No. is ab68153), the primary antibodie detecting the state of STAT3 phosphorylation is Anti-STAT3 (phosphoS727) antibody [E121-31] (Abcam product, article No. is ab32143) and Anti-STAT3 (phosphoY705) antibody [EP2147Y] (Abcam product, article No. is ab76315), internal reference is GAPDH, the primary antibodie of GAPDH is Anti-GAPDHrabbitpolyclonalantibody (Sangon Biotech's product, article No. is D110016-0200), result is as shown in A in Fig. 3.
Result shows, HHT inhibits the phosphorylation of STAT3Thr705, but does not affect the phosphorylation of STAT3Ser727 and the expression of STAT3 total protein.
In order to be clearly that HHT have impact on the phosphorylation of STAT3 by which signal paths affecting cell further, inventor have detected the JAK1 in the typical three bars approach of its upstream, the state of AKT and ERK and corresponding phosphorylation thereof.Concrete grammar is as follows: utilize the method for westernblot to detect 0 μM of embodiment 2 respectively, 1 μM, the A549 total protein of cell of the HHT process of 2 μMs and 4 μMs and 0 μM, 0.5 μM, JAK1 in the NCI-H1975 total protein of cell of the HHT process of 1 μM and 2 μMs, the state of AKT and ERK and corresponding phosphorylation thereof, the primary antibodie detecting JAK1 and phosphorylation thereof is respectively Jak1 (6G4) RabbitmAb (CellSignalingTechnology product, article No. is #3344 and Phospho-Jak1 (Tyr1022/1023) Antibody (CellSignalingTechnology product, article No. is #3331), the primary antibodie detecting AKT and phosphorylation thereof is respectively Anti-AKT1/2/3antibody [EPR16798] (Abcam product, article No. is ab179463) and Anti-AKT1/2/3 (phosphoY315+Y316+Y312) antibody (Abcam product, article No. is ab131443), the primary antibodie detecting ERK and phosphorylation thereof is respectively Anti-ERK1+ERK2antibody [ERK-7D8] (Abcam product, article No. is ab54230) and Anti-Erk1 (pT202/pY204)+Erk2 (pT185/pY187) antibody [MAPK-YT] (Abcam product, article No. is ab50011), internal reference is GAPDH, the primary antibodie of GAPDH is Anti-GAPDHrabbitpolyclonalantibody (Sangon Biotech's product, article No. is D110016-0200), result is as shown in B in Fig. 3.
Found that, the phosphorylation of JAK1 is suppressed because of HHT process, and JAK1 protein expression level does not change; HHT process does not affect AKT and ERK protein expression level and corresponding phosphorylation state thereof.Above-mentioned experimental result show HHT inhibit JAK/STAT3 signal path thus have impact on STAT3 transcribe combination.
Embodiment 4, HHT suppress the STAT3 phosphorylation of interleukin-6 induction, thus cell death inducing
1, the HHT STAT3 phosphorylation that suppresses interleukin-6 to be induced in dose-dependent mode
By A549 cell containing 10% hyclone DMEM culture medium at 5%CO
2incubator in 37 DEG C cultivate 12 hours, then culture medium is changed to DMEM culture medium and continues cultivation 12 hours, obtain the hungry A549 culture fluid cultivated; In the A549 culture fluid that hunger is cultivated, add HHT, obtain HHT-A549 cell culture fluid, in HHT-A549 cell culture fluid, the concentration of HHT is 2 μMs; By HHT-A549 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate 4 hours, obtain the A549 cell culture fluid of HHT process; In the A549 cell culture fluid of HHT process, add interleukin-6 (IL-6), obtain IL-6-HHT-A549 cell culture fluid, in IL-6-HHT-A549 cell culture fluid, the concentration of IL-6 is 5ng/mL; By IL-6-HHT-A549 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate after 30 minutes, abandon culture medium, collecting cell, utilize RIPA lysate cell lysis, 1,2000rpm is centrifugal under 4 DEG C of conditions, abandons precipitation, obtains the A549 total protein of cell of IL-6 and 2 μM of-HHT process.
According to the method described above, 2 μMs are replaced with respectively 4 μMs, 1 μM and 0 μM, other steps are all constant, obtain the A549 total protein of cell that the A549 total protein of cell of IL-6 and 4 μM of-HHT process, the A549 total protein of cell of IL-6 and 1 μM-HHT process and IL-6 and 0 μM-HHT process respectively.
According to the method described above, replace with 0 μM by 2 μMs, and be the concentration that 5ng/mL replaces with IL-6 by the concentration of IL-6 be 0ng/mL, other steps are all constant, obtain untreated A549 total protein of cell.
By NCI-H1975 cell containing 10% hyclone RPMI-1640 culture medium at 5%CO
2incubator in 37 DEG C cultivate 12 hours, then culture medium is changed to RPMI-1640 culture medium and continues cultivation 12 hours, obtain the hungry NCI-H1975 culture fluid cultivated; In the NCI-H1975 culture fluid that hunger is cultivated, add HHT, obtain HHT-NCI-H1975 cell culture fluid, in HHT-NCI-H1975 cell culture fluid, the concentration of HHT is 1 μM; By HHT-NCI-H1975 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate 4 hours, obtain the NCI-H1975 cell culture fluid of HHT process; In the NCI-H1975 cell culture fluid of HHT process, add interleukin-6 (IL-6), obtain IL-6-HHT-NCI-H1975 cell culture fluid, in IL-6-HHT-NCI-H1975 cell culture fluid, the concentration of IL-6 is 5ng/mL; By IL-6-HHT-NCI-H1975 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate after 30 minutes, abandon culture medium, collecting cell, utilize RIPA lysate cell lysis, 1,2000rpm is centrifugal under 4 DEG C of conditions, abandons precipitation, obtains the NCI-H1975 total protein of cell of IL-6 and 1 μM of-HHT process.
According to the method described above, 2 μMs, 0.5 μM and 0 μM is replaced with respectively by 1 μM, other steps are all constant, obtain the NCI-H1975 total protein of cell that the NCI-H1975 total protein of cell of IL-6 and 2 μM of-HHT process, the NCI-H1975 total protein of cell of IL-6 and 0.5 μM-HHT process and IL-6 and 0 μM-HHT process respectively.
According to the method described above, replace with 0 μM by 1 μM, and be the concentration that 5ng/mL replaces with IL-6 by the concentration of IL-6 be 0ng/mL, other steps are all constant, obtain untreated NCI-H1975 total protein of cell.
The method of westernblot is utilized to detect the state of STAT3 expression in above-mentioned total protein of cell and phosphorylation thereof respectively, the primary antibodie detecting STAT3 expression is Anti-STAT3antibody [EPR787Y] (Abcam product, article No. is ab68153), the primary antibodie detecting the state of STAT3 phosphorylation is Anti-STAT3 (phosphoS727) antibody [E121-31] (Abcam product, article No. is ab32143) and Anti-STAT3 (phosphoY705) antibody [EP2147Y] (Abcam product, article No. is ab76315), internal reference is GAPDH, the primary antibodie of GAPDH is Anti-GAPDHrabbitpolyclonalantibody (Sangon Biotech's product, article No. is D110016-0200), result as shown in Figure 4.
Result shows, IL-6 is induction of the phosphorylation of STAT3Thr705, and HHT is inhibited to this inducing action, and the HHT of various dose is all inhibited to this inducing action.
2, the HHT STAT3 phosphorylation that suppresses interleukin-6 to be induced in time dependent mode
By A549 cell containing 10% hyclone DMEM culture medium at 5%CO
2incubator in 37 DEG C cultivate 12 hours, then culture medium is changed to DMEM culture medium and continues cultivation 12 hours, obtain the hungry A549 culture fluid cultivated; In the A549 culture fluid that hunger is cultivated, add HHT, obtain HHT-A549 cell culture fluid, in HHT-A549 cell culture fluid, the concentration of HHT is 4 μMs; By HHT-A549 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate 4 hours, obtain the A549 cell culture fluid of HHT process; In the A549 cell culture fluid of HHT process, add interleukin-6 (IL-6), obtain IL-6-HHT-A549 cell culture fluid, in IL-6-HHT-A549 cell culture fluid, the concentration of IL-6 is 5ng/mL; By IL-6-HHT-A549 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate after 30 minutes, abandon culture medium, collecting cell, utilize RIPA lysate cell lysis, 1,2000rpm is centrifugal under 4 DEG C of conditions, abandons precipitation, obtains the A549 total protein of cell of IL-6 and HHT-4 hour.
According to the method described above, 4 hours are replaced with respectively 0 hour, 1 hour and 2 hours, other steps are all constant, obtain the A549 total protein of cell of IL-6 and HHT-0 hour, the A549 total protein of cell of IL-6 and HHT-1 hour and the A549 total protein of cell of IL-6 and HHT-2 hour respectively.
By NCI-H1975 cell containing 10% hyclone RPMI-1640 culture medium at 5%CO
2incubator in 37 DEG C cultivate 12 hours, then culture medium is changed to RPMI-1640 culture medium and continues cultivation 12 hours, obtain the hungry NCI-H1975 culture fluid cultivated; In the NCI-H1975 culture fluid that hunger is cultivated, add HHT, obtain HHT-NCI-H1975 cell culture fluid, in HHT-NCI-H1975 cell culture fluid, the concentration of HHT is 2 μMs; By HHT-NCI-H1975 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate 4 hours, obtain the NCI-H1975 cell culture fluid of HHT process; In the NCI-H1975 cell culture fluid of HHT process, add interleukin-6 (IL-6), obtain IL-6-HHT-NCI-H1975 cell culture fluid, in IL-6-HHT-NCI-H1975 cell culture fluid, the concentration of IL-6 is 5ng/mL; By IL-6-HHT-NCI-H1975 cell culture fluid at 5%CO
2incubator in 37 DEG C cultivate after 30 minutes, abandon culture medium, collecting cell, utilize RIPA lysate cell lysis, 1,2000rpm is centrifugal under 4 DEG C of conditions, abandons precipitation, obtains the NCI-H1975 total protein of cell of IL-6 and HHT-4 hour.
According to the method described above, 4 hours are replaced with respectively 0 hour, 1 hour and 2 hours, other steps are all constant, obtain the NCI-H1975 total protein of cell of IL-6 and HHT-0 hour, the NCI-H1975 total protein of cell of IL-6 and HHT-1 hour and the NCI-H1975 total protein of cell of IL-6 and HHT-2 hour respectively.
The method of westernblot is utilized to detect the A549 total protein of cell of IL-6 and 0 μM of-HHT process of above-mentioned total protein of cell and step 1 respectively, untreated A549 total protein of cell, STAT3 expression in the NCI-H1975 total protein of cell of IL-6 and 0 μM of-HHT process and untreated NCI-H1975 total protein of cell and the state of phosphorylation thereof, the primary antibodie detecting STAT3 expression is Anti-STAT3antibody [EPR787Y] (Abcam product, article No. is ab68153), the primary antibodie detecting the state of STAT3 phosphorylation is Anti-STAT3 (phosphoS727) antibody [E121-31] (Abcam product, article No. is ab32143) and Anti-STAT3 (phosphoY705) antibody [EP2147Y] (Abcam Products, article No. is ab76315), internal reference is GAPDH, the primary antibodie of GAPDH is Anti-GAPDHrabbitpolyclonalantibody (Sangon Biotech's product, article No. is D110016-0200), result as shown in Figure 5.Result shows, along with the increase in HHT processing time, the phosphorylation level of STAT3 reduces gradually.
Embodiment 5, HHT suppress the experiment of nonsmall-cell lung cancer
HHT is dissolved in 1 × PBS, to be dissolved completely after add normal saline and supplement volume, the concentration making HHT is 3g/L, obtains HHT solution.Gefitinib is dissolved in by volume ratio be the DMSO (Sigma) of 0.8:12:8, in the liquid that forms of polyoxyethylene castor oil (Cremophor) (Sigma) and ethanol (Shanghai Sheng Gong biological engineering company limited), to be dissolved completely after add normal saline and supplement volume, the concentration making gefitinib is 5.00g/L, obtains gefitinib solution.
By the right veutro subcutaneous injection 2.5 × 10 of every Mus of 30 female Mus of SPF level BALB/c-nu/nu in 6 week age (Guangdong Medical Lab Animal Center's product)
6individual NCI-H1975 cell, obtains nonsmall-cell lung cancer nude mice.On the same day of injection NCI-H1975 cell, 30 above-mentioned nonsmall-cell lung cancer nude mices are divided into three groups at random, and one group is matched group (Vehicle), and one group is treated with gefitinib group, last group is HHT treatment group, often organizes 10 nonsmall-cell lung cancer nude mices.HHT treatment group every Mus gives above-mentioned HHT solution according to real-time measurement body weight abdominal cavity makes dosage be every kg body weight 10mgHHT (every kg body weight administration volume is 3.3mL), and every day is administered once, altogether administration 24 days; Treated with gefitinib group every Mus gives above-mentioned gefitinib solution according to real-time measurement body weight abdominal cavity makes dosage be every kg body weight 30mg gefitinib (every kg body weight administration volume is 6mL), and every day is administered once, altogether administration 24 days; The isopyknic carrier of matched group (Vehicle) every intraperitoneal administration (i.e. 1 × PBS), every day is administered once, altogether administration 24 days.It is the 0th day with administration moment first time, the body weight of gross tumor volume and nude mice is measured in the 4th, 6,8,10,12,14,16,18,20,22 and 24 day, after within 24th day, measuring the body weight of gross tumor volume and Mus, nude mice is put to death, get tumor cell and detect STAT3 phosphorylation state and MCL1 protein expression situation in tumor tissues by westernblot and SABC.
The primary antibodie detecting STAT3 phosphorylation state in westernblot is Anti-STAT3 (phosphoY705) antibody [EP2147Y] (Abcam Products, article No. is ab76315), the primary antibodie detecting MCL1 albumen is Mcl-1 (D35A5) RabbitmAb (CellSignalingTechnology product, article No. is #5453), GAPDH is internal reference, the primary antibodie of internal reference is Anti-GAPDHrabbitpolyclonalantibody (Sangon Biotech's product, article No. is D110016-0200).
Primary antibodie in immunohistochemical experiment is anti-phospho-STAT3 (Epitomics product).Primary antibodie 4 DEG C of overnight incubation, after PBS washing, two anti-incubated at room 1 hour, two resist the donkey anti-rabbit IgG (SantaCruz product) for FITC labelling (green) labelling; DAPI (blue, Sigma product), for transfect cell core, contaminates core 8 minutes with DAPI after two anti-washings, and add mountant mounting after PBS washing, laser confocal microscope is taken pictures.
Result shows that HHT can significantly reduce gross tumor volume (in see Fig. 6 A, B and C) under the prerequisite not affecting the weight of animals, at the 24th day, the weight differences of three groups of nude mices is little, but the volume of HHT treatment group nude mouse tumor is respectively 0.32 times and 0.28 times of treated with gefitinib group and matched group.Detect STAT3 phosphorylation (p-STAT3) and MCL1 albumen in tumor tissues by westernblot and SABC simultaneously, find, in administration treated animal tumor tissues, p-STAT3 and MCL1 expresses and reduces, illustrate HHT to nonsmall-cell lung cancer in vivo level there is significant curative effect, as shown in D and E in Fig. 6.
Claims (10)
- The application of 1.Homoharringtonine in the product preparing inducing apoptosis of tumour cell and/or inhibition tumor cell propagation.
- The application of 2.Homoharringtonine in preparation treatment tumor product.
- 3.Homoharringtonine suppresses the application in the tumor cell proliferation medicine of interleukin-6 promotion in preparation.
- 4.Homoharringtonine promotes the application in tumor cell Mitochondria apoptosis product in preparation.
- 5., according to described application arbitrary in claim 1-4, it is characterized in that: described tumor is resistant tumors.
- 6.Homoharringtonine suppresses transcription factor STAT3 to the application in the transcriptional activity product of anti-apoptotic genes expression in preparation.
- 7.Homoharringtonine suppresses the application in the transcriptional activity product of STAT3, MCL1 and/or Survivin in preparation.
- 8.Homoharringtonine suppresses the application in STAT3, MCL1 and/or Survivin gene expression products in preparation.
- 9.Homoharringtonine suppresses the application in the enzymatic activity product of STAT3, MCL1 and/or Survivin in preparation.
- 10.Homoharringtonine suppresses the application in the STAT3 phosphorylation product of interleukin-6 induction in preparation.
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| US20030175365A1 (en) * | 2002-03-15 | 2003-09-18 | Yaguang Liu | Natural drug induced differentiation cancer cells to resemble normal cells |
| CN101732254A (en) * | 2008-11-13 | 2010-06-16 | 杭州民生药业有限公司 | Injection-use emulsion composition containing cephalotaxus plant alkaloid, and preparation method thereof |
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| US20030175365A1 (en) * | 2002-03-15 | 2003-09-18 | Yaguang Liu | Natural drug induced differentiation cancer cells to resemble normal cells |
| CN101732254A (en) * | 2008-11-13 | 2010-06-16 | 杭州民生药业有限公司 | Injection-use emulsion composition containing cephalotaxus plant alkaloid, and preparation method thereof |
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