CN103990127A - Pharmaceutical composition treating tumors - Google Patents
Pharmaceutical composition treating tumors Download PDFInfo
- Publication number
- CN103990127A CN103990127A CN201310052018.1A CN201310052018A CN103990127A CN 103990127 A CN103990127 A CN 103990127A CN 201310052018 A CN201310052018 A CN 201310052018A CN 103990127 A CN103990127 A CN 103990127A
- Authority
- CN
- China
- Prior art keywords
- cell
- autophagy
- monoclonal antibody
- pharmaceutical composition
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to the technical field of medicines and biology, and concretely relates to a pharmaceutical composition treating tumors. The pharmaceutical composition consists of one or a plurality of autophagy adjusting medicines and a monoclonal antibody medicine. The autophagy adjusting medicine(s) and the monoclonal antibody medicine in the pharmaceutical composition are administrated in a combined administration manner or in a sequential administration manner. By adjusting cell autophagy, the curative effect of the monoclonal antibody medicine on various tumors is enhanced. By utilizing the cell autophagy adjusting medicines to activate/inhibit autophagy, the curative effects of a plurality of monoclonal antibody medicines on lymphoma, stomach cancer, breast cancer, lung cancer, ovarian cancer and brain tumor are enhanced, and especially the curative effects on lymphoma, stomach cancer and breast cancer are enhanced.
Description
Technical field
The present invention relates to medicine, biological technical field, relate to antitumor drug, be specifically related to a kind of pharmaceutical composition for the treatment of tumor, relate in particular to the pharmaceutical composition that is regulated class medicine and a kind of monoclonal antibody class ingredients by one or more autophagy.
Background technology
The method that is mainly used at present oncotherapy is still chemotherapy, but the side effect of chemotherapy is large, makes monoclonal medicine have huge market and demand.Monoclonal antibody (McAb, be called for short monoclonal antibody) medicine develops rapidly in recent years, and some have been applied to clinical or have made diagnostic kit and detectable etc.Wherein that comparatively outstanding is [the Bozhkov B. Monoclonal antibodies--their clinical and diagnostic importance. Vutr Boles. 1987 such as Rituximab (Rituximab), chLym-1 monoclonal antibody and Herceptin (Trastuzumab), 26 (6): 1-9.], if Rituximab is for clinical treatment non Hodgkin lymphoma (NHL), 8 course of therapy aggressive of associating CHOP scheme (filling the air large B cell) lymphoma, 8 the first-line treatment inertia course for the treatment of of associating CVP scheme (folliculus) lymphoma, inertia B cellularity non Hodgkin lymphoma [Clark J, the Longo D. Monoclonal antibody for the treatment of recurrence or chemotherapy resistance
?therapy of leukemias and lymphomas. J Exp Pathol. 1987, 3 (4): 319-27.], chlym-1 monoclonal antibody is used for the treatment of multiple B cell malignant lymphoma, and there are data to show that its drug effect is better than Rituximab, this monoclonal antibody to the cell strain of the specific HLA-DR positive as B-35M, Raji, Su-DHL-6 etc. have obvious growth inhibited effect, research shows that its Main Function mechanism is by direct cell death inducing, the cytotoxicity (ADCC) that antibody relies on and the cytotoxicity (CDC) of Complement Dependent thus killing tumor cell [Liu C, DeNardo G, Tobin E, et al. Antilymphoma Effects of Anti-HLA-DR and CD20 Monoclonal Antibodies (Lym-1 and Rituximab) on Human Lymphoma Cells. Cancer Biother Radiopharm. 2004, 19 (5): 545-561, Zhang N, Khawli LA, Hu P, et al. Lym-1-induced apoptosis of non-Hodgkin's lymphomas produces regression of transplanted tumors. Cancer Biother Radiopharm. 2007, 22 (3): 342-356.], Herceptin (Trastuzumab) is used for the treatment of breast carcinoma and gastric cancer [Tahover E, Sonnenblick A, Peretz T, et al. Update:adjuvant
?trastuzumab
?in HER2 positive breast cancer. Harefuah. 2012, 151 (1): 37-42,61.] etc., it is good that monoclonal antibodies medicine has specificity, and side effect is little, the mechanism of action advantage such as more clearly, and effective killing tumor cell, but the curative effect of most monoclonal antibody is all be not as obvious as chemotherapeutics.
Cell autophagy (autophagy) claims again II type programmed death (type II programmed cell death), it is the phenomenon of common " self-digestion " (cellular degradation) in most eukaryotes, conventionally be transported to the difference of lysosome intracavity mode according to substrate in cell, mammalian cell autophagy can be divided into three kinds of modes: the autophagy (chaperone-mediated autophagy, CMA) of large autophagy (macroautophagy), little autophagy (microautophagy) and molecular chaperones mediation.In recent years along with the development of molecular biology and gene technology and the deep understanding to cell autophagy, find that the development of itself and various diseases, especially tumor is in close relations.
In general,, because cell autophagy is conducive to the survival of cell, therefore no matter in normal cell or tumor cell, autophagy all is generally retained, and is all maintaining basic autophagy in the ordinary course of things.Actually but or autophagy suppresses to promote the generation development of tumor cell still not come to a conclusion at present.Studies show that, the autophagy initial stage can be used as tumorigenic a kind of inhibition factor, the tumor-inhibiting factor that some are known, for example PTEN, TSC1 and TSC2 can activate autophagy, and can make protein degradation reduce to the inhibition of autophagy, anabolism increases, finally cause former cancerous cell to continue propagation.Most of tumor cells (as liver, pancreas, breast carcinoma etc.) are although autophagy ability is had nothing in common with each other before canceration, and after canceration, its autophagy ability all weakens.Because autophagy role in tumor occurs and develops is still not clear; therefore its role in antitumor drug killing tumor cell process is also not quite similar; roughly may be summarized to be two kinds: a kind of is protection to tumor cell, another kind of be killing and wounding tumor cell.Research shows chemotherapeutics 5-FU Induces Autophagy significantly; and the cell autophagy that suppresses to be produced by these 3 kinds of medicines can significantly increase sensitivity [the Li J of tumor cell to treatment; Hou N; Faried A; Tsutsumi S, et al. Inhibition of Autophagy by 3-MA Enhances the Effect of 5-FU-Induced Apoptosis in Colon Cancer Cells. Ann Surg Oncol .2009; 16 (3): 761 – 771.].At present the adjusting class medicine of cell autophagy can be divided into two classes by it to the effect of autophagy, a class be autophagy derivant as; Rapamycin (Rapamycin), lithium salts (lithium) and trehalose (trehalose) etc., to also have a class be autophagy inhibitor as: 3-MA(3-Methyladenine), wortmannin (wortmannin), LY294002 etc., and NH
4cl, chloroquine (Chloroquine) and hydroxychloroquine (hydroxychloroquine) etc.
Up to now, there is not yet and make pharmaceutical composition or compound medicine is combined or the report of sequential administration treatment tumor about cell autophagy class regulating drug and monoclonal antibody drug.
Summary of the invention
The object of this invention is to provide a kind of pharmaceutical composition for the treatment of tumor, relate in particular to the pharmaceutical composition that is regulated class medicine and a kind of monoclonal antibody class ingredients by one or more autophagy.This pharmaceutical composition can strengthen the lethal effect of medicine to tumor cell by the cell autophagy of the drug-induced tumor cell of regulation and control monoclonal antibodies, thereby significantly strengthens the curative effect of monoclonal antibodies medicine.
Concrete, the invention provides a kind of pharmaceutical composition for the treatment of tumor, it is characterized in that, this pharmaceutical composition regulates class medicine and a kind of monoclonal antibody class ingredients by one or more autophagy; This pharmaceutical composition is by regulating cell autophagy can strengthen the curative effect of monoclonal antibody class medicine in the various tumors for the treatment of; And can strengthen multiple monoclonal antibody class medicine to lymphoma, gastric cancer, breast carcinoma, pulmonary carcinoma, the curative effect of ovarian cancer and cerebroma, especially lymphoma, gastric cancer and breast carcinoma by using cell autophagy to regulate class medicine to activate/suppress autophagy.
In the present invention, described cell autophagy regulates class medicine, comprise cell autophagy inhibitor and cell autophagy derivant, its feature is, self has auxiliary lethal effect and can be further strengthens described monoclonal antibodies medicine to the killing and wounding of tumor cell by strengthening/suppress cell autophagy without obvious cytotoxicity or to tumor, strengthens its curative effect.
In the present invention, cell autophagy derivant includes, but is not limited to: rapamycin (Rapamycin), lithium salts (lithium) and trehalose (trehalose) etc.; Preferred rapamycin (Rapamycin) in the present invention.
In the present invention, cell autophagy inhibitor comprises (but being not limited to): 3-MA(3-Methyladenine), wortmannin (wortmannin), LY294002, cycloheximide, Ba Faluo mycin A1(Bafilomycin A1), NH
4cl, chloroquine (Chloroquine) and hydroxychloroquine (hydroxychloroquine), preferred 3-MA(3-Methyladenine in the present invention).
In the present invention, described monoclonal antibody drug includes, but is not limited to: chLym-1 monoclonal antibody, Rituximab (Rituximab) and Herceptin (Trastuzumab) etc.
In the present invention, one or more in autophagy derivant or autophagy inhibitor, as: lithium salts, trehalose and/or rapamycin, and/or the pharmaceutical composition of 3-MA, wortmannin and/or chloroquine and a kind of monoclonal antibody composition treatment tumor.
The pharmaceutical composition that is regulated class medicine and a kind of monoclonal antibody class ingredients by one or more autophagy of the present invention, regulate class medicine and monoclonal antibody class Drug therapy lymphoma, gastric cancer, breast carcinoma by associating or autophagy wherein of sequential use, pulmonary carcinoma, the tumors such as ovarian cancer and cerebroma; Especially lymphoma, gastric cancer and breast carcinoma; Described lymphoma comprises hodgkin's lymphoma (HL) and non Hodgkin lymphoma (NHL).
The present invention, by using one or several autophagy to regulate class medicine, comprises autophagy inhibitor and autophagy derivant, makes compound medicine or compositions with a kind of monoclonal antibody, administering drug combinations or sequential administration, thus strengthen the curative effect of this monoclonal antibody to tumor.
The present invention has carried out inhibition tumor cell experiment in vitro, and result confirms, described pharmaceutical composition can strengthen monoclonal antibodies medicine to the killing and wounding of tumor cell by strengthening/suppress cell autophagy, strengthens its curative effect.
Brief description of the drawings
Fig. 1: chLym-1 monoclonal antibody can significantly induce the LC3-II expression of Raji cell to raise,
Wherein, A: process the expression of the LC3-II albumen of the Raji cell after 24 hours through the chLym-1 monoclonal antibody of 10 μ g/ml; B:LC3-II albumen through/not through the Raji cells statistics of variables result of the chLym-1 monoclonal antibody processing of 0 μ g/ml.
Fig. 2: the process chlym-1 under the laser confocal microscope visual field processes Raji cell and the matched group Raji cell of 48 hours.
Fig. 3: chLym-1 monoclonal antibody can significantly induce the autophagosome in Raji cell to assemble.
Fig. 4: the expression of the each pathway associated protein of cell autophagy in Raji cell after chLym-1 intervenes different time.
Fig. 5: the expression of the each pathway associated protein of cell autophagy in Raji cell after variable concentrations chLym-1 intervenes.
Fig. 6: suppress the cell autophagy that MEK/Erk path can not suppress the induction of chLym-1 monoclonal antibody.
Fig. 7: suppress autophagy by 3-MA and can suppress the lethal effect of chLym-1 monoclonal antibody to Raji cell.
Fig. 8: pass through NH
4cl suppresses autophagy can suppress the lethal effect of chLym-1 monoclonal antibody to Raji cell.
Fig. 9: can further strengthen the lethal effect of chLym-1 to Raji cell by rapamycin Induces Autophagy.
Figure 10: 3-MA can significantly suppress the increase of autophagy associated protein LC3-II in the Raji cell of chLym-1 monoclonal antibody induction.
Figure 11: NH
4cl can significantly increase the expression of autophagy associated protein LC3-II in Raji cell.
Figure 12: rapamycin can significantly strengthen the expression of the autophagy associated protein LC3-II in Raji cell.
Figure 13: suppress the CDC effect to Raji cell that cell autophagy can suppress chLym-1 monoclonal antibody mediation.
Figure 14: 3-MA suppresses the ADCC effect to Raji cell that cell autophagy can suppress chLym-1 monoclonal antibody mediation.
Figure 15: inhibition cell autophagy can significantly weaken the apoptosis of the Raji cell of chLym-1 monoclonal antibody induction.
Figure 16: Rituximab can be induced Raji cell generation cell autophagy significantly.
Figure 17: autophagy inhibitor 3-MA and NH
4cl can strengthen the cell death of Rituximab induction.
Figure 18: Herceptin can be induced SKBR3 cell and N87 cell generation cell autophagy.
Figure 19: inhibition cell autophagy can significantly strengthen the growth inhibited of the SKBR3 cell of Herceptin induction.
Figure 20: inhibition cell autophagy can significantly strengthen the growth inhibited of the N87 cell of Herceptin induction.
Detailed description of the invention
the preparation of monoclonal antibody mother solution:take the lyophilized powder of 10mg chLym-1 monoclonal antibody, be dissolved in the PBS solution of 1ml 0.02M pH=7.4.Fully stir and use 0.1 μ m sterile filters to filter.Being distributed into 10 pipes preserves.When in vitro tests, be diluted to 10 μ g/ml.
the preparation of Rituximab mother solution:take the lyophilized powder of 10mg Rituximab, be dissolved in the PBS solution of 1ml 0.02M pH=7.4.Fully stir and use 0.1 μ m sterile filters to filter.Being distributed into 10 pipes preserves.When in vitro tests, be diluted to 10 μ g/ml.
the preparation of Herceptin mother solution:take the lyophilized powder of 10mg Herceptin, be dissolved in the PBS solution of 1ml 0.02M pH=7.4.Fully stir and use 0.1 μ m sterile filters to filter.Being distributed into 10 pipes preserves.When in vitro tests, be diluted to 10 μ g/ml.
the configuration of autophagy inhibitor medicine
(1) preparation of chloroquine solution: get appropriate chloroquine and be dissolved in pure water and be made into the storage liquid of 10mmol/L, be stored in 4 DEG C after the filter filtration sterilization with 0.1 μ m, experiment in vitro chamber dilution 500-1000 is doubly for suppressing cell autophagy
(2) preparation of ammonium chloride solution: get the storage liquid of the water-soluble 0.4mol/L of being made into of appropriate ammonium chloride, be stored in 4 DEG C after the filter filtration sterilization with 0.1 μ m.When experiment in vitro, dilute 50-80 doubly for suppressing cell autophagy.
(3) preparation of 3-MA solution: get appropriate 3-MA and be dissolved in sterilized water and be mixed with the storage liquid of 200mM, be stored in-20 DEG C after the filter filtration sterilization with 0.1 μ m.When experiment in vitro, be diluted to 2mM for suppressing cell autophagy.
the configuration of autophagy derivant medicine
(1) preparation of rapamycin solution: appropriate rapamycin is dissolved in DMSO, is made into the storage liquid of 1 μ mol/L, be stored in-20 DEG C, be diluted to 5nM-20nM when use for active cell autophagy.
(2) preparation of lithium salts: take appropriate LiCl and be dissolved in sterilized water and be mixed with the storage liquid of 400mM, after using the sterile filters filtration sterilization of 0.1 μ m and be stored in 4 DEG C.When use, be diluted to 5mM for active cell autophagy.
(3) preparation of aqueous trehalose: take trehalose and be dissolved in deionized water and be mixed with the storage liquid of 400mM, after using the sterile filters filtration sterilization of 0.1 μ m and be stored in 4 DEG C.When use, be diluted to 20mM-40mM for active cell autophagy.
cell culture:growth of Cells is to logarithmic growth after date, with 5*10
5the concentration of cells/ml proceeds in Tissue Culture Plate and cultivates, each group cell gives respectively chLym-1 monoclonal antibody, Rituximab or the Herceptin of 0-10 μ g/ml, and 1h adds the chloroquine of 20 μ mol/L before administration, the NH4Cl of 5mmol/L or the rapamycin of 10nM are for suppressing or activate the cell autophagy of Raji cell.Continuous culture 48h, with 1000r/min centrifugal collecting cell.
The LC3-II expression of embodiment 1 chLym-1 monoclonal antibody induction Raji cell raises
Collect Raji cell and matched group Raji Western Blot and the IP lysate cracking extraction total protein of cell of having processed 24h through the chLym-1 monoclonal antibody of 10 μ g/ml, carry out protein quantification, carry out protein electrophoresis and transferring film is carried out Western blot by every hole 50 μ g protein content loadings, carry out chemiluminescence with ECL chemical luminescence reagent kit, result is as shown in Figure 1: processed the expression of Raji cell autophagy associated protein LC3 II of 24h through 10 μ g/ml chLym-1 monoclonal antibodies higher than matched group, and through gray scale process software IQuentTL analyze find both possess extremely significant difference (
p<0.01), due to the expression of LC3 II and the existing positive correlation of the quantity of autophagosome, the expression of LC3 II is higher, and cell autophagy is also just more active, result shows, chLym-1 monoclonal antibody can be induced Raji cell generation cell autophagy significantly,
Raji cell and matched group Raji cell by 10 μ g/ml chLym-1 monoclonal antibodies being processed to 48h with after Cyto-ID autophagosome associated protein LC3-II protein staining reagent dyeing by observing under the laser confocal microscope at 560nm in excitation wavelength, result demonstration: processed through 10 μ g/ml chLym-1 monoclonal antibodies in the Cytoplasm of Raji cell of 48h and had more green fluorescence speckle (as shown in Figure 2); Green fluorescence speckle number depend on the expression of autophagosome associated protein LC3-II, compared with matched group, chLym-1 monoclonal antibody has induced the LC3-II expression of Raji cell to raise significantly.
Embodiment 2
autophagosome in chLym-1 monoclonal antibody induction Raji cell is assembled
Collect through the Raji cell of chLym-1 monoclonal antibody intervention 24h and be fixed, embedding, section, after dyeing in its ultrastructure of observed under electron microscope, result shows: the Raji nucleus of matched group is complete, be normal circle, and the Raji cell core gross distortion of having intervened 24h through 10 μ g/ml chLym-1 monoclonal antibodies, 8000 × amplification under, find a lot of cavity spline structures at core exterior domain, 50000 × multiple under, can clearly see a large amount of significantly double membrane structures (as shown in Figure 3), according to bibliographical information, the diameter of autophagosome is generally between nanometer more than 300 is to several microns, coincide with the size of the double membrane structure cavity shown in the present embodiment figure, the cavity of confirming by analysis the duplicature sample of arrow indication is autophagosome, compare cellular control unit, there is obvious cell autophagy at 10 μ g/ml chLym-1 induction Raji cells.
Embodiment 3
chLym-1 regulates and controls Akt/mTOR, MEK/Erk path Induces Autophagy
Extract total protein of cell with the Raji cell of the chLym-1 monoclonal antibody processing of 10 μ g/ml and matched group Raji Western Blot and the cracking of IP lysate, carry out protein quantification, carry out protein electrophoresis and transferring film is carried out Western blot by every hole 60 μ g protein content loadings, carry out chemiluminescence with ECL chemical luminescence reagent kit, result shows (as Fig. 4, shown in 5): the p-mTOR after chLym-1 monoclonal antibody different time and variable concentrations is processed in Raji cell, the expression of p-Akt weakens, in addition the expression of p-TSC2 and p-4E-BP1 strengthens, the interaction energy of prompting chLym-1 monoclonal antibody is lowered the activity of mTOR signal path, in addition the expression of p-Erk also strengthens to some extent, show that chLym-1 monoclonal antibody can raise the activity of MEK/Erk path, after the chLym-1 of different time and variable concentrations monoclonal antibody is processed, the expression of LC3-II all strengthens to some extent in Raji cell, shows that chLym-1 monoclonal antibody induced cell autophagy, after MEK/Erk signal path being suppressed with U0126, detect and suppress after MEK/Erk path the expression of LC3-II in Raji cell, result shows: the activation that suppresses MEK/Erk signal path can not weaken the increase (as shown in Figure 6) of the LC3-II expression in the Raji cell of chLym-1 monoclonal antibody induction.Prove that MEK/Erk signal path does not participate in the regulation and control of the cell autophagy of chLym-1 induction.
Embodiment 4
suppress the cell death that cell autophagy can suppress the induction of chLym-1 monoclonal antibody, active cell autophagy can strengthen the cell death of chLym-1 monoclonal antibody induction,
Each group of Raji cell directly added in 96 porocyte plates to 10 μ lMTT reactant liquors after continuous culture 48h, 37 DEG C of lucifuges are hatched 4h and add three liquid (aqueous solution of 50%DMF, 20%SDS) cell lysis after MTT are fully reacted generation first a ceremonial jade-ladle, used in libation with succinate dehydrogenase in cell, hatch 6h for 37 DEG C and dissolve first a ceremonial jade-ladle, used in libation, measure the O.D value of each group in 570nm place, carry out the conversion of cell viability percentage ratio taking the O.D. of matched group as 100%, and carry out statistical analysis with SPSS statistics 19; Adopt the chLym-1 monoclonal antibody of 10 μ g/ml to intervene Raji cell, and 1h add 3-MA or NH before administration
4cl suppresses cell autophagy or the rapamycin active cell autophagy of Raji cell, after continuous culture 48h, adopt mtt assay to measure cell viability, result shows, compared with matched group, through the cell viability of the Raji cell of 10 μ g/ml chLym-1 monoclonal antibody interventions have significance decline (
p<0.01), add autophagy inhibitor 3-MA and NH
4compared with the cell viability of the cell viability of the Raji cell of Cl and the Raji cell of simple chLym-1 monoclonal antibody intervention, have significance improve (
p<0.05,
p<0.01) (as Fig. 7,8 and Fig. 9 shown in), prove that the administering drug combinations of autophagy inhibitor and chLym-1 monoclonal antibody can weaken the drug effect of chLym-1 monoclonal antibody, and only inhibiting 3-MA and NH
4the cell viability of the group of Cl is compared with matched group, without significant difference, illustrate that the cell growth of inhibitor own is without obvious facilitation, result shows, the cytoactive of the Raji cell that the administering drug combinations of autophagy inhibitor and chLym-1 monoclonal antibody increases is not due to the synergism of inhibitor and medicine, but suppresses to have weakened chLym-1 monoclonal antibody killing and wounding Raji cell after autophagy; Add compared with the cell viability of Raji cell and the cell viability of the Raji cell of simple chLym-1 monoclonal antibody intervention of autophagy derivant rapamycin, be decreased significantly (
p<0.05), show to strengthen autophagy and can increase the lethal effect of chLym-1 to Raji cell.
embodiment 5 suppresses and induces the expression of autophagy associated protein LC3 II after autophagy to detect
Each group Raji cell extracts total protein of cell with the cracking of RIPA lysate after centrifugal collection, quantitatively, carry out protein electrophoresis by every hole 50 μ g protein content loadings and transferring film is carried out Western blot, carry out chemiluminescence with ECL chemical luminescence reagent kit, result 3-MA can significantly suppress the increase of autophagy associated protein LC3-II in the Raji cell of chLym-1 monoclonal antibody induction, NH
4cl can significantly increase the expression of autophagy associated protein LC3-II in Raji cell, and rapamycin can significantly strengthen the expression (as Figure 10, shown in Figure 11 and Figure 12) of the autophagy associated protein LC3-II in Raji cell; Through the expression of the LC3 II of the Raji cell of 10 μ g/ml chLym-1 monoclonal antibody processing all higher than matched group, through the expression of the LC3 II of 2mmol/L 3-MA or 1mmol/L 3-MA and 10 μ g/ml chLym-1 monoclonal antibody Raji cell after treatment lower than 10 μ g/ml chLym-1 monoclonal antibody Raji cell after treatment, and through 10 μ g/ml chLym-1 monoclonal antibody and 5mmol/L NH
4cl or 2.5mmol/L NH
4the expression of the LC3 II of Cl Raji cell after treatment is higher than 10 μ g/ml chLym-1 monoclonal antibody Raji cell after treatment, and the expression through 5nmol/L or 10nmol/L rapamycin and 10 μ g/ml chLym-1 monoclonal antibody Raji cell after treatment will be higher than 10 μ g/ml chLym-1 monoclonal antibody Raji cell after treatment, result shows that 3-MA and NH4Cl can effectively suppress the cell autophagy of the Raji cell of chLym-1 monoclonal antibody induction, and rapamycin can further strengthen the cell autophagy of the Raji cell of chLym-1 monoclonal antibody induction.
embodiment 6 suppresses the CDC effect to Raji cell that cell autophagy can suppress chLym-1 monoclonal antibody mediation
Each experimental group Raji cell adds after human serum and chLym-1 monoclonal antibody measures cell survival with mtt assay after continuous culture 2h, compared with matched group, there is significance to decline through the cell viability of the Raji cell of 10 μ g/ml chLym-1 monoclonal antibodies and 5 μ l serum interventions, and add 2mmol/L 3-MA or 5mmol/L NH
4cl suppresses the vigor of cell of the Raji cell of cell autophagy and compares with the Raji of 5 μ l serum interventions with simple 10 μ g/ml chLym-1 monoclonal antibodies, have significance improve (
p<0.05) (as shown in figure 13), result has proved to kill and wound in the process of Raji cell at chLym-1 monoclonal antibody mediation CDC, suppress the survival that cell autophagy can promote Raji cell, suppress cell autophagy and can weaken the effect at the CDC of chLym-1 monoclonal antibody mediation, induction autophagy can improve the CDC effect that chLym-1 mediates in lymphoma cell.
embodiment 7 suppresses the ADCC effect to Raji cell that cell autophagy can suppress chLym-1 mediation
Each experimental group Raji cell (target cell) adds after people's mononuclearcell (effector lymphocyte) and chLym-1 monoclonal antibody the lactic acid dehydrogenase (LDH) with CytoTox 96 kit measurement cells after continuous culture 5h to discharge, result is as shown in figure 14: respectively imitate target and be significantly higher than matched group than lactic acid dehydrogenase (LDH) burst size of the chLym-1 monoclonal antibody group of group, and present the dependency of good effector lymphocyte's quantity ratio, prove chLym-1 monoclonal antibody in Raji cell, induced significant ADCC effect (
p, and adding PI3K inhibitor 2mmol/L 3-MA to suppress after cell autophagy <0.05), with chLym-1 comparison, the burst size of lactic acid dehydrogenase (LDH) obviously reduce (
p<0.05,
p<0.01), prove that autophagy inhibitor 3-MA has suppressed the ADCC effect that chLym-1 monoclonal antibody mediates in Raji cell, prompting strengthens autophagy can strengthen the ADCC effect that chLym-1 monoclonal antibody mediates in lymphoma cell.
embodiment 8 suppresses cell autophagy can weaken the apoptosis of the Raji cell of chLym-1 monoclonal antibody induction
Each group Raji cell gives respectively 0 μ g/ml and 10 μ g/ml chLym-1 monoclonal antibodies, and 1h adds the 3-MA of 2mmol/L and the NH of 5mmol/L before administration
4cl suppresses the cell autophagy of Raji cell, after continuous culture 48h, use cell apoptosis detection kit to carry out passing through Flow cytometry Raji cell after treatment after Annexin V and PI dyeing, result shows, suppress the apoptosis (as shown in figure 15) that cell autophagy can significantly weaken the Raji cell of chLym-1 monoclonal antibody induction, through the Anexin V of the Raji cell of 10 μ g/ml chLym-1 monoclonal antibody interventions+cell proportion be significantly higher than matched group, the NH of 10 μ g/ml chLym-1 monoclonal antibody+2mmol/L 3-MA groups and 10 μ g/ml chLym-1 monoclonal antibody+5mmol/L
4the Raji cell that the Anexin V positive cell ratio of the Raji cell of Cl group is significantly intervened lower than simple 10 μ g/ml chLym-1 monoclonal antibodies, the NH of 10 μ g/ml chLym-1 monoclonal antibody+2mmol/L 3-MA groups and 10 μ g/ml chLym-1 monoclonal antibody+5mmol/L
4compared with the Raji cell that the Raji cell PI+ cell proportion of Cl group is intervened with 10 μ g/ml chLym-1, no significant difference, shows to suppress Raji cell autophagy and can weaken the apoptosis that chLym-1 monoclonal antibody is induced.
embodiment 9 Rituximabs can be induced Raji cell generation cell autophagy
Collect Raji cell and matched group Raji cell Western Blot and the IP lysate cracking extraction total protein of cell of processing 24h through the Rituximab of 10 μ g/ml, carry out protein quantification, carry out protein electrophoresis and transferring film is carried out Western blot by every hole 50 μ g protein content loadings, carry out chemiluminescence with ECL chemical luminescence reagent kit, result shows that Rituximab can induce Raji cell generation cell autophagy (as shown in figure 16) significantly: the expression of Raji cell autophagy associated protein LC3 II of processing 24h through 10 μ g/ml Rituximabs is higher than matched group, due to the expression of LC3 II and the existing positive correlation of the quantity of autophagosome, the expression of LC3 II is higher, cell autophagy is just more active, show that Rituximab can induce Raji cell generation cell autophagy significantly.
embodiment 10 suppresses cell autophagy can strengthen the cell death of the Raji cell of Rituximab induction
Each group of Raji cell directly added in 96 porocyte plates to 10 μ lMTT reactant liquors after continuous culture 48h, 37 DEG C of lucifuges are hatched 4h and add three liquid (aqueous solution of 50%DMF, 20%SDS) cell lysis after MTT are fully reacted generation first a ceremonial jade-ladle, used in libation with succinate dehydrogenase in cell, hatch 6h for 37 DEG C and dissolve first a ceremonial jade-ladle, used in libation, measure the O.D value of each group in 570nm place, result shows autophagy inhibitor 3-MA and NH
4cl can strengthen the cell death (as shown in figure 17) of Rituximab induction: compared with matched group, through the cell viability of the Raji cell of 10 μ g/ml Rituximab interventions have significance decline (
p<0.01), add autophagy inhibitor 3-MA and NH
4compared with the cell viability of the cell viability of the Raji cell of Cl and the Raji cell of simple Rituximab intervention, have significance reduce (
p<0.05,
p<0.01), prove that the administering drug combinations of autophagy inhibitor and Rituximab can strengthen the drug effect of Rituximab, and only inhibiting 3-MA and NH
4the cell viability of the group of Cl is compared with matched group, without significant difference, illustrate that the cell growth of inhibitor own is without obvious inhibitory action, what the administering drug combinations of autophagy inhibitor and Rituximab increased is not due to the synergism of inhibitor and medicine to the lethal effect of Raji cell, but suppresses to have strengthened Rituximab killing and wounding Raji cell after autophagy.
embodiment 11 Herceptins can be induced SKBR3 and N87 cell generation cell autophagy
Collect SKBR3 cell and N87 cell and cellular control unit Western Blot and the IP lysate cracking extraction total protein of cell of processing 48h through the Herceptin of 10 μ g/ml, carry out protein quantification, carry out protein electrophoresis and transferring film is carried out Western blot by every hole 50 μ g protein content loadings, and carry out chemiluminescence with ECL chemical luminescence reagent kit, result shows, Herceptin can be induced SKBR3 cell and N87 cell generation cell autophagy (as shown in figure 18): the expression of having processed the SKBR3 cell of 24h and the cell autophagy associated protein LC3 II of N87 cell through 10 μ g/ml Herceptins is all higher than matched group, due to the expression of LC3 II and the existing positive correlation of the quantity of autophagosome, the expression of LC3 II is higher, cell autophagy is also just more active, Herceptin can be induced SKBR3 cell and N87 cell generation cell autophagy significantly.
embodiment 12 suppresses cell autophagy can strengthen Herceptin killing and wounding SKBR3 cell and N87 cell
Through 96h Herceptin and cell autophagy inhibitor 3-MA or NH
4after the processing of Cl, the cell viability of SKBR3 cell and N87 detects through mtt assay, result shows, suppress cell autophagy and can significantly strengthen the SKBR3 cell of Herceptin induction or the growth inhibited of N87 cell (as Figure 19, shown in 20): compared with matched group, Herceptin is acting on after N87 cell and SKBR3 cell 96h, all significantly inducing cell growth inhibited of energy, 3-MA or NH
4the administering drug combinations of Cl and Herceptin can further strengthen the fragmentation effect of Herceptin to N87 cell and SKBR3 cell (
p<0.05,
p<0.01), prove to suppress autophagy and can significantly strengthen the lethal effect of Herceptin to breast carcinoma SKBR3 cell and gastric cancer N87 cell.
Claims (11)
1. a pharmaceutical composition for the treatment of tumor, is characterized in that, this pharmaceutical composition regulates class medicine and a kind of monoclonal antibody class ingredients by one or more autophagy; Described cell autophagy regulates class medicine to comprise cell autophagy inhibitor and cell autophagy derivant.
2. by the pharmaceutical composition for the treatment of tumor claimed in claim 1, it is characterized in that, described cell autophagy derivant is selected from rapamycin (Rapamycin), lithium salts (lithium) or trehalose (trehalose).
3. by the pharmaceutical composition for the treatment of tumor claimed in claim 1, it is characterized in that, described cell autophagy derivant is rapamycin (Rapamycin).
4. by the pharmaceutical composition for the treatment of tumor claimed in claim 1, it is characterized in that, described cell autophagy inhibitor is selected from 3-MA(3-Methyladenine), wortmannin (wortmannin), LY294002, cycloheximide, Ba Faluo mycin A1(Bafilomycin A1), NH
4cl, chloroquine (Chloroquine) or hydroxychloroquine (hydroxychloroquine).
5. by the pharmaceutical composition for the treatment of tumor claimed in claim 1, it is characterized in that, described cell autophagy inhibitor is 3-MA(3-Methyladenine).
6. by the pharmaceutical composition for the treatment of tumor claimed in claim 1, it is characterized in that, described monoclonal antibody class medicine is selected from chLym-1 monoclonal antibody, Rituximab (Rituximab) or Herceptin (Trastuzumab).
7. by the pharmaceutical composition for the treatment of tumor claimed in claim 1, it is characterized in that, the autophagy in this pharmaceutical composition regulates class medicine and monoclonal antibody class medicine to use by associating or sequential mode.
8. by the pharmaceutical composition of the arbitrary treatment tumor of claim 1-7, it is characterized in that, described tumor is selected from lymphoma, gastric cancer, breast carcinoma, pulmonary carcinoma, ovarian cancer or cerebroma.
9. by the pharmaceutical composition of the arbitrary treatment tumor of claim 1-7, it is characterized in that, described tumor is selected from lymphoma, gastric cancer or breast carcinoma.
10. by the pharmaceutical composition of the treatment tumor described in claim 8 or 9, it is characterized in that, described lymphoma is hodgkin's lymphoma (HL) or non Hodgkin lymphoma (NHL).
The purposes of the pharmaceutical composition of 11. claim 1 in the monoclonal antibody synergism medicine of preparation treatment tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310052018.1A CN103990127A (en) | 2013-02-17 | 2013-02-17 | Pharmaceutical composition treating tumors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310052018.1A CN103990127A (en) | 2013-02-17 | 2013-02-17 | Pharmaceutical composition treating tumors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103990127A true CN103990127A (en) | 2014-08-20 |
Family
ID=51304622
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310052018.1A Pending CN103990127A (en) | 2013-02-17 | 2013-02-17 | Pharmaceutical composition treating tumors |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103990127A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104758934A (en) * | 2015-04-14 | 2015-07-08 | 中国科学院微生物研究所 | Pharmaceutical composition for synergistic inhibition virus induced cell transformation and leukemia and application of pharmaceutical composition |
| CN106635994A (en) * | 2017-01-10 | 2017-05-10 | 东南大学 | Culture medium additive for preparing autophagosomes-type tumor vaccine and preparing method thereof |
| WO2018161279A1 (en) * | 2017-03-08 | 2018-09-13 | Johnpro Biotech Inc. | Use of mtor inhibitor and chloroquine for treating cancer |
| CN117187181A (en) * | 2023-11-08 | 2023-12-08 | 普华赛尔生物医疗科技有限公司 | Method for coating a composition and use thereof |
-
2013
- 2013-02-17 CN CN201310052018.1A patent/CN103990127A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| ALEJANDRO VAZQUEZ-MARTIN ETAL: "Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab", 《PLOS ONE》 * |
| JULIE TURZANSKI ETAL: "Involvement of macroautophagy in the caspase-independent killing of Burkitt lymphoma cell lines by rituximab", 《BRITISH JOURNAL OF HAEMATOLOGY》 * |
| XINQUN LI ETAL: "Roles of autophagy in cetuximab-mediated cancer therapy against EGFR", 《AUTOPHAGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104758934A (en) * | 2015-04-14 | 2015-07-08 | 中国科学院微生物研究所 | Pharmaceutical composition for synergistic inhibition virus induced cell transformation and leukemia and application of pharmaceutical composition |
| CN104758934B (en) * | 2015-04-14 | 2017-12-26 | 中国科学院微生物研究所 | It is a kind of that there is collaboration to suppress virus induction cell transformation and drug regimen and its application of leukaemia |
| CN106635994A (en) * | 2017-01-10 | 2017-05-10 | 东南大学 | Culture medium additive for preparing autophagosomes-type tumor vaccine and preparing method thereof |
| WO2018161279A1 (en) * | 2017-03-08 | 2018-09-13 | Johnpro Biotech Inc. | Use of mtor inhibitor and chloroquine for treating cancer |
| CN117187181A (en) * | 2023-11-08 | 2023-12-08 | 普华赛尔生物医疗科技有限公司 | Method for coating a composition and use thereof |
| CN117187181B (en) * | 2023-11-08 | 2024-03-19 | 普华赛尔生物医疗科技有限公司 | Method for coating a composition and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shingu et al. | Inhibition of autophagy at a late stage enhances imatinib‐induced cytotoxicity in human malignant glioma cells | |
| De et al. | Doubling down on the PI3K-AKT-mTOR pathway enhances the antitumor efficacy of PARP inhibitor in triple negative breast cancer model beyond BRCA-ness | |
| Allen et al. | Dual inactivation of Akt and ERK by TIC10 signals Foxo3a nuclear translocation, TRAIL gene induction, and potent antitumor effects | |
| Shen et al. | A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor | |
| Liu et al. | Enhanced cytotoxic effect of low doses of metformin combined with ionizing radiation on hepatoma cells via ATP deprivation and inhibition of DNA repair | |
| CN105873592A (en) | Combination therapy for treating cancer | |
| Wu et al. | Phosphatidylinositol 3-kinase inhibitor LY294002 suppresses proliferation and sensitizes doxorubicin chemotherapy in bladder cancer cells | |
| Shome et al. | Tweaking EMT and MDR dynamics to constrain triple-negative breast cancer invasiveness by EGFR and Wnt/β-catenin signaling regulation | |
| Nam et al. | Identification of a novel S6K1 inhibitor, rosmarinic acid methyl ester, for treating cisplatin-resistant cervical cancer | |
| CN103990127A (en) | Pharmaceutical composition treating tumors | |
| CN101743005A (en) | Chemotherapy for cancer enhancement with 7-(2, 5-dihydro-4-imidazolo [1,2-a] pyridin-3-yl-2, 5-dioxo-1H-pyrrol-3-yl)-9-fluoro-1, 2,3, 4-tetrahydro-2-(1-piperidinylcarbonyl)-pyrrolo [3,2,1-jk] [1,4] benzodiaz [1,4] benzodiaz | |
| Li et al. | Curcumol Inhibits Lung Adenocarcinoma Growth and Metastasis via Inactivation of PI3K/AKT and Wnt/β-Catenin Pathway | |
| Rudnick et al. | Oral therapy for nonmelanoma skin cancer in patients with advanced disease and large tumor burden: a review of the literature with focus on a new generation of targeted therapies | |
| CN101332301A (en) | Antineoplastic composition and use thereof | |
| Xie et al. | PKI-587 enhances radiosensitization of hepatocellular carcinoma by inhibiting the PI3K/AKT/mTOR pathways and DNA damage repair | |
| Sha et al. | USP8 inhibitor–induced DNA damage activates cell cycle arrest, apoptosis, and autophagy in esophageal squamous cell carcinoma | |
| Sarkar et al. | A novel chemotherapeutic protocol for peritoneal metastasis and inhibition of relapse in drug resistant ovarian cancer | |
| Walter et al. | Inhibition of MDM2 via Nutlin‐3A: A Potential Therapeutic Approach for Pleural Mesotheliomas with MDM2‐Induced Inactivation of Wild‐Type P53 | |
| Xie et al. | Anwulignan is a novel JAK1 inhibitor that suppresses non‐small cell lung cancer growth | |
| Jasinghe et al. | ABT-869, a multi-targeted tyrosine kinase inhibitor, in combination with rapamycin is effective for subcutaneous hepatocellular carcinoma xenograft | |
| Fei et al. | Assessment of GSK1904529A as a promising anti-osteosarcoma agent | |
| Meng et al. | Ellagic acid inhibits cell proliferation, migration, and invasion of anaplastic thyroid cancer cells via the Wnt/β-catenin and PI3K/Akt pathways | |
| Xiao et al. | Curcumin/L‐OHP co‐loaded HAP for cGAS‐STING pathway activation to enhance the natural immune response in colorectal cancer | |
| CN109419803A (en) | Cell autophagy inhibitor and Afatinib pharmaceutical composition and its purposes in preparation tumour Synergistic preparations | |
| CN103505732A (en) | Tumor treatment synergism combination drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140820 |