CN116585320A - Reversing method and application of non-small cell lung cancer octenib acquired drug resistance - Google Patents
Reversing method and application of non-small cell lung cancer octenib acquired drug resistance Download PDFInfo
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- CN116585320A CN116585320A CN202310575911.6A CN202310575911A CN116585320A CN 116585320 A CN116585320 A CN 116585320A CN 202310575911 A CN202310575911 A CN 202310575911A CN 116585320 A CN116585320 A CN 116585320A
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Abstract
The invention relates to a reversing method and application of non-small cell lung cancer octenib acquired drug resistance. The method for reversing the acquired resistance of the non-small cell lung cancer of the Ornitanib is to use a reversing drug and the Ornitanib to realize the reversing of the acquired resistance of the non-small cell lung cancer of the Ornitanib, wherein the reversing drug is selected from panobinostat, bortezomib, carfilzomib, vorinostat, auranofin, romidepsin or homoharringtonine. The invention also provides application of the auranofin in reversing drug of non-small cell lung cancer of the acquired resistance of the Ornitanib. The invention provides a novel reversing method for NSCLC cell Organtinib acquired drug resistance, which has important significance for promoting the combination of auranofin and Organtinib in lung cancer clinical treatment.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a reversing method and application of non-small cell lung cancer octenib acquired drug resistance.
Background
Lung cancer is a malignant tumor with high morbidity and mortality worldwide. Of these, about 80 to 85% are Non-small cell lung cancer (Non-small cell cancer, NSCLC). Because the disease has no typical symptoms at early stage, nearly 70% of NSCLC patients have been diagnosed at middle and late stages, not only lose the opportunity of operation, but also are insensitive to conventional chemotherapy and radiotherapy, and the survival rate of 5 years is only about 15%. In recent years, molecular targeted drugs such as an epidermal growth factor receptor-tyrosine kinase inhibitor (Epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI) and the like are introduced, so that clinical treatment of NSCLC is breakthrough progress.
Ornitinib (Osi) is the first third generation EGFR-TKI approved by the FDA in the United states, and can selectively irreversibly bind to sensitive mutant EGFR and T790M drug-resistant mutant EGFR to block downstream signaling to achieve the purpose of resisting tumors. In view of its good clinical efficacy and safety, the drug is recommended for first-line treatment of EGFR mutation positive NSCLC patients. However, most patients with good responsiveness at the initial stage of administration have acquired drug resistance after 9-14 months, resulting in treatment failure and disease progression. The reasons for this phenomenon are very complex, and the molecular mechanisms that have been found at present mainly include two main classes: EGFR-dependent mechanisms, such as EGFR C797S mutations, etc.; EGFR independent mechanisms such as compensatory signaling pathway establishment of c-MET, HER2 and the like, pathological tissue type transformation and the like. In order to overcome the drug resistance of the Osi and enhance the curative effect of the Osi, the research of searching for a drug resistance reversing agent and exploring a combined treatment scheme is a hot spot of the current research besides the fourth generation EGFR-TKI aiming at EGFR C797S mutation which is currently developed. At present, researches show that panobinostat, aspirin and curcumin have a certain reversal effect on acquired drug resistance of NSCLC cell EGFR-TKI drugs, but the drug reversal agents with higher specificity and safety and better curative effect stability and effectiveness still need to be further explored and discovered.
Auranofin is a gold-containing compound, which was approved by FDA in 1985 for the treatment of rheumatoid arthritis, and many studies have shown that it can act alone as an anticancer agent, but at a greater concentration, and with certain side effects.
Disclosure of Invention
The invention aims to provide a reversing method and application of non-small cell lung cancer octreotide acquired drug resistance, so as to provide a brand new reversing method of NSCLC cell octreotide acquired drug resistance, and also provide a new application of auranofin.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention is largely researched and explored to screen the reversing agent of the obtained drug resistance of the oritinib from the 1470 drug molecular library approved by the FDA, namely, the reversing agent is selected from panobinostat, bortezomib, carfilzomib, vorinostat, auranofin, romidepsin or homoharringtonine, in particular, the reversing agent is selected from the group consisting of the palbociclib, the bortezomib, the carfilzomib and the homoharringtonine. The research result shows that the auranofin can realize the reversion of the acquired resistance of the non-small cell lung cancer cell Organtinib, and the combination of the auranofin and the Organtinib can evaluate the acquired resistance of the Organtinib to the growth proliferation, the apoptosis, the migration and the invasion of the Organtinib through CCK-8, edU cell proliferation, flow cytometry and Transwell detection, and the synergy is determined through CI values. Meanwhile, mRNA with different changes in drug-resistant strains after combined drug administration and single drug administration is detected through RNA-seq, and target gene and protein expression quantity are verified through qRT-PCR and Western blot, so that the growth proliferation and invasion and migration capacity of drug-resistant cell strains are obviously inhibited through drug combination, and the apoptosis rate is improved; upregulation of HSPB8 and LC3-II/LC3-I resulted in increased autophagy following combination. Thus, it is proved that the combination of auranofin and octreotide enhances the sensitivity of the octreotide to NSCLC drug-resistant cells, the mechanism of the combination is related to the increase of autophagy caused by the up-regulation of HSPB8, and the synergistic effect of the combination and the octreotide is verified in vitro and in vivo.
Therefore, the invention provides a method for reversing the acquired resistance of the non-small cell lung cancer of the Ornitanib, which adopts the combination of a reversing drug and the Ornitanib to realize the reversing of the acquired resistance of the non-small cell lung cancer of the Ornitanib;
the reversing drug is selected from the group consisting of panobinostat, bortezomib, carfilzomib, vorinostat, auranofin, romidepsin, and homoharringtonine.
Preferably, the reversing drug is selected from the group consisting of auranofin.
Preferably, the ratio of the auranofin to the octreotide is 0.8-2 mu mol.L -1 ：2～8μmol·L -1 。
Preferably, the ratio of the auranofin to the octreotide is 1 mu mol.L -1 ：2μmol·L -1 。
Preferably, the NSCLC cells in the non-small cell lung cancer are H1975/OR cells and/OR PC9/OR cells.
Preferably, the combination drug of the auranofin and the octreotide can inhibit the growth proliferation, migration and invasion of NSCLC drug-resistant cells and can improve the apoptosis rate of the cells.
Preferably, the combination of auranofin and octreotide is used for reversing the acquired resistance of non-small cell lung cancer octreotide by regulating the expression of HSPB8 and the level of LC3-II/LC3-I in cells.
Preferably, the combination of auranofin and octreotide is used for reversing the acquired resistance of non-small cell lung cancer octreotide by regulating the expression of HSPB8 and the level of LC3-II/LC3-I in up-regulated cells.
The invention also provides application of the auranofin in reversing drug of non-small cell lung cancer of the acquired resistance of the Ornitanib.
The invention has the beneficial effects that:
the double-emission carbon dot provided by the invention has a pH recognition function, is used for B, G-CDs of Trp sensing, and has good water solubility, salt resistance stability and excellent optical stability;
according to the reversing method of the non-small cell lung cancer of the invention, through high-throughput screening of the FDA approved drug molecular library, the method discovers that panobinostat, bortezomib, carfilzomib, vorinostat, auranofin, romidepsin or homoharringtonine, especially Auranofin (ANF), has a remarkable reversing effect on the NSCLC cell of the acquired drug resistance of the Ornitanib, and provides a new thought for clinically overcoming the NSCLC cell of the acquired drug resistance of the Ornitanib.
Drawings
FIG. 1 is a graph showing the results of the validation of the Aoditinib-derived drug-resistant cell lines H1975/OR and PC 9/OR; wherein A is a CCK-8 detection result diagram; b is a flow type apoptosis detection result graph;
FIG. 2 is a graph showing the results of screening NSCLC cells for an obtained drug resistance reversal drug; wherein A is a screening flow chart of a reverse drug of NSCLC cell Ornitinib acquired drug resistance; b is the inhibition rate result of the cell viability; c is the distribution result of 91 medicines with cell viability inhibition rate more than 50%; d is the drug distribution with 14 cell viability inhibition rate more than 50%; e is the inhibition rate result of the cell viability of the 7 drugs;
FIG. 3 is a graph showing the results of a combination of auranofin and oxximtinib; wherein A is the IC50 results of auranofin on H1975/OR and PC 9/OR; b is the inhibition rate result of the cell growth activity after the combination of the auranofin and the oxmeitinib; c is the CI value of H1975/OR and PC9/OR for the combination of auranofin and oxmeitinib;
FIG. 4 is a graph showing the results of growth proliferation and apoptosis of an Aoshiib-acquired drug resistant NSCLC cell by combining auranofin with Aoshiib; wherein A is an EdU cell proliferation experimental result graph; b is a flow apoptosis detection result graph;
FIG. 5 is a graph showing the results of the combination of auranofin and Ornitinib on the migration and invasion of Ornitinib-acquired drug resistant NSCLC cells; wherein A is a cell migration experimental result graph; b is a cell invasion experimental result graph;
FIG. 6 is a graph showing the results of induction of HSPB8 expression following a combination of auranofin and octreotide; wherein A and B are differentially expressed genes of the combination of the auranofin and the octreotide alone; c is the RNA-seq level of HSPB8 in H1975 and H1975/OR;
FIG. 7 is a graph of Western blot detection results.
Detailed Description
Further advantages and effects of the present invention will become readily apparent to those skilled in the art from the disclosure herein, by referring to the accompanying drawings and the preferred embodiments. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be understood that the preferred embodiments are presented by way of illustration only and not by way of limitation.
1. Material
Materials: RPMI1640 medium and fetal bovine serum were purchased from Gibco, inc., USA; FDA approved 1470 drug molecular library (L4200) and CCK-8 kit (C0005) were purchased from TargetMol, USA; matrigel (356234) and Transwell chamber (353097) are available from corning corporation of the united states; penicillin-streptomycin (C0222), crystal violet dye liquor (C0121), edU kit (C0071S) were purchased from shanghai bi yunshan biotechnology limited; 4% paraformaldehyde (DF 0135) is purchased from Beijing Lei Gen Biotechnology Co., ltd; orditinib (B1104) is available from APExBIO Biotechnology Inc. of America; jinnofin (HY-B1123) is purchased from Shanghai Haoyuan biological medicine technologies Co., ltd; reverse transcription kit (RR 073A) was purchased from TaKaRa company in japan; the PCR primer is synthesized by Shanghai bioengineering Co., ltd; anti-HSPB8 (DF 2481) was purchased from U.S. Pat. No. Affinity Biosciences, anti-GAPDH (bs-2188R) was purchased from Beijing Boaosen Biotechnology Co., ltd, anti-LC3B (3868T) was purchased from U.S. Pat. No. Cell Signaling Technology, and horseradish enzyme-labeled goat Anti-rabbit IgG (ZB-2301) was purchased from Beijing China fir gold bridge Biotechnology Co.
Example 1
Establishment and verification of NSCLC (non-volatile memory) octenib acquired drug resistant cell model
Cell culture
Human non-small cell lung cancer cell lines H1975 (carrying EGFR L858R and T790M mutations) and PC9 (carrying EGFR exon 19 deletion mutations) were purchased from the institute of basic medicine, national academy of sciences. H1975 and PC9 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37℃with 5% CO 2 Culturing in an incubator. H1975 and PC9 cells in logarithmic growth phase were collected and cultured in 0.about.0.5. Mu. Mol.L -1 Increasing concentrations of octenib were treated until the cells could be at a concentration of 0.5. Mu. Mol.L -1 The cells are H1975/OR and PC9/OR, which grow stably in the Ornitinib culture solution. The drug was removed 2 weeks prior to the experiment and routinely cultured for use.
Modeling
The method comprises the steps of establishing an Aoditinib-derived drug-resistant cell strain H1975/OR and PC9/OR by using the Aoditinib-sensitive H1975 and PC9 cells according to a drug concentration increasing method.
Verification
Cell viability was measured using CCK-8. The method comprises the following steps: H1975/OR and PC9/OR cells in logarithmic growth phase were plated in 96-well plates (5X 10) 3 Individual cells/well), after 24h, different concentrations of octenib (0, 1.25, 2.5, 5, 10. Mu. Mol.L) were added -1 ) At 37℃with 5% CO 2 After 72h of treatment under the condition, adding 10% of CCK-8 solution for further incubation for 1h, and calculating half-maximal inhibitory concentration (half maximal inhibitory concentration, IC 50) of the cells after detecting the absorbance at 450nm by using an enzyme-labeled instrument; cell viability (%) = (experimental group absorbance-blank group absorbance)/(control group absorbance-blank group absorbance), the result is shown in fig. 1A.
Apoptosis was detected using flow cytometry. The method comprises the following steps: the logarithmic phase H1975/OR and PC9/OR cells were seeded in 6-well plates (2X 10) 5 Individual cells/well), after 24h, 0.5. Mu. Mol.L was added -1 After 48h of treatment with octenib, all cells in the adherent and supernatant were collected, 5. Mu.L of Annexin V-FITC and 5. Mu.L of Propidium Iodide (PI) staining solution were added, incubated at 4℃for 15min in the absence of light, and apoptosis was detected by flow cytometry, as shown in FIG. 1B.
In FIG. 1A, the CCK-8 assay shows that the IC50 values of the Organidine pairs H1975 and H1975/OR are 0.074. Mu. Mol.L, respectively -1 And 5.204 mu mol.L -1 The drug resistance index is 70.31; IC50 values of the Ornitinib pair PC9 and PC9/OR are 0.042. Mu. Mol.L respectively -1 And 5.692 mu mol.L -1 The drug resistance index was 136.99.
In FIG. 1B, the results of flow apoptosis detection show that compared with the sensitive strain, 0.5. Mu. Mol.L -1 After 48H of Ornitinib treatment, the apoptosis rate of H1975/OR cells was reduced by 72.6% compared with H1975 cells (6.99% vs 25.51%, P)<0.05 The apoptosis rate of PC9/OR cells was reduced by 84.6% compared with PC9 cells (4.55% vs 29.53%, P)<0.01). The results show that the obtained drug-resistant NSCLC cell strain of the Ornitinib is successfully constructed.
Example 2
Screening of NSCLC cell Ornitinib acquired drug resistance reversal agent
In order to find a reversal agent of acquired resistance of NSCLC cells to octenib, as shown in FIG. 2A, the screening was performed by CCK-8 method with 0.5. Mu. Mol.L using FDA-approved 1470 drug molecular library in this example -1 The drug of H1975/OR and PC9/OR cells can be obviously killed after the combination of the Ornitinib.
The method comprises the following steps: first, H1975/OR and PC9/OR cells in the logarithmic growth phase were inoculated into 96-well plates (5X 10) 3 Cells/well), after 24h, 10. Mu. Mol.L was added -1 1470 medicines to be tested are added with 0.5 mu mol.L at the same time -1 Ornitinib CO-acts on H1975/OR cells and PC9/OR cells for preliminary screening at 37℃with 5% CO 2 After the treatment for 72 hours under the condition, adding 10% of CCK-8 solution for further incubation for 1 hour, detecting the absorbance at 450nm by using a microplate reader, calculating the inhibition rate of the cell viability, and screening out medicines with the inhibition rate of more than 50% on the cell viability as shown in figure 2B, wherein as shown in figure 2C, 30% is kinase inhibitor, 17% is RNA, DNA, protein synthesis inhibitor, histone deacetylase inhibitor, ion channel inhibitor, proteasome inhibitor and the like. Subsequently, the concentration of the 91 drugs was reduced to 1. Mu. Mol.L -1 With 0.5 mu mol.L -1 The operation steps of the combined method are the same as that of the primary screening, and as shown in figure 2D, the inhibition rate of 14 medicines to H1975/OR cells is more than 50%, the inhibition rate of 11 medicines to PC9/OR cells is more than 50%, wherein the inhibition rate of 7 medicines to two drug-resistant cells is more than 50%, namely panobinostat, bortezomib, carfilzomib, vorinostat, auranofin, romidepsin and homoharringtonine.
In this example, the 7 drugs (panobinostat, bortezomib, carfilzomib, vorinostat, auranofin, romidepsin, homoharringtonine) were compared to 0.5. Mu. Mol.L when administered alone -1 Effect of octenib on viability of both drug resistant cells when used in combination. The method comprises the following steps: taking H1975/OR and PC9/O in logarithmic growth phaseR cells were seeded in 96-well plates (5X 10) 3 Cells/well), 24h later, 1. Mu. Mol.L each was added -1 Concentration of 7 drugs, or 7 drugs and 0.5. Mu. Mol.L respectively -1 Oritinib was combined at 37℃with 5% CO 2 After 72h of treatment under the condition, 10% of CCK-8 solution is added for further incubation for 1h, and after the absorbance at 450nm is detected by an enzyme-labeled instrument, the inhibition rate of the cell viability is calculated, and the result is shown in FIG. 2E.
From the results in fig. 2E, it was found that the difference in killing of cells by auranofin was most pronounced in two cases: when taken alone, the inhibition rates of the auranofin on H1975/OR and PC9/OR cell activities are 46.07% and 40.32%, respectively; when the concentration is 0.5 mu mol.L -1 When the combination of the Ornitinib is used, the inhibition rates of H1975/OR and PC9/OR cell activities are respectively increased to 66.67% and 70.76%.
The above study results show that, unlike other screened medicines, although the auranofin has a certain killing effect on NSCLC cells with the acquired drug resistance of the Ornitinib when taken alone, the effect is more obvious when the auranofin is combined with the Ornitinib, and the action target point of the auranofin is highly likely to be closely related to certain biological characteristics of the NSCLC cells changed in the drug resistance process. On the basis, the reverse action of the drug resistance on the NSCLC cell of the obtained drug resistance of the Ornitanib is further verified, and the drug resistance reverse mechanism is explored, so that the novel safe and effective drug resistance reverse scheme is explored, the more comprehensive cause of the obtained drug resistance of the Ornitanib can be known from a brand new view, and a novel solution idea is provided for clinically overcoming the phenomenon.
Example 3
Combined drug experiments of auranofin and octreotide
To further verify the efficacy of the combination of auranofin and octreotide and to determine the optimal dose of both drugs, in this example, the IC50 values of the treatment of the obtained drug-resistant NSCLC cells with auranofin alone were first examined by the CCK-8 method to preliminarily determine the drug concentration range. The method comprises the following steps: H1975/OR and PC9/OR cells in logarithmic growth phase were plated in 96-well plates (5X 10) 3 Individual cells/well), after 24h, different concentrations (0.625, 1.25) were added、2.5、5μmol·L -1 ) Auranofin of (5%) at 37 ℃ C., 5% CO 2 After 72h of treatment, 10% CCK-8 solution was added for further incubation for 1h, and after detection of absorbance at 450nm by a microplate reader, the half-maximal inhibitory concentration (half maximal inhibitory concentration, IC 50) of the cells was calculated and the results are shown in FIG. 3A.
From the results in FIG. 3A, it was revealed that the IC50 value of auranofin for H1975/OR cells was 1.16. Mu. Mol.L -1 IC50 value for PC9/OR cells was 1.40. Mu. Mol.L -1 。
Subsequently, H1975/OR and PC9/OR cells were cultured to the logarithmic phase and inoculated into 96-well plates (5X 10) 3 Individual cells/well). After 24h of incubation, different concentrations of auranofin (0, 0.8, 1, 2. Mu. Mol.L were measured by orthogonal test design method with reference to IC50 value of single drug -1 ) Different from the concentration of the Ornitanib (0, 2, 4, 6, 8 mu mol.L) -1 ) Acting on cells at 37deg.C with 5% CO 2 And (3) continuously incubating for 72 hours under the condition, adding 10% of CCK-8 solution, continuously incubating for 1 hour, detecting the absorbance at 450nm by using an enzyme-labeled instrument, calculating the inhibition rate of the cell viability at each concentration, analyzing the combined effect of the two drugs by using Compusyn software, and determining the drug combination index (Combination index, CI) by using the principle of median effect. Ci=d 1 /D x1 +D 2 /D x2 Wherein D is 1 And D 2 Respectively represent the dosages required by the combination application of the Ornitinib He Jinnuo and the like, D x1 And D x2 The doses of octenib He Jinnuo-fin required to inhibit a certain level of cell viability are shown, respectively. CI (CI)<1 represents that the two drugs have a synergistic effect at this concentration combination, ci=1 represents that the two drugs have an additive effect at this concentration combination, CI>1 represents that both drugs have antagonism at this concentration combination.
FIG. 3B shows the inhibition of cell growth activity, and CI values were calculated by inputting inhibition data into CompuSyn software, and the results are shown in FIG. 3C. From the results in fig. 3C, it is known that the calculated CI value in both cells is less than 1 at the selected drug concentration formulation, thus demonstrating that both drugs combined produce a synergistic effect at the above concentrations. Wherein, the concentration is 1 mu mol.L -1 Jinnofin+2 mu mol.L -1 The method has the advantages that the CI value of the octenib scheme is minimum, the synergistic effect is strongest, and the scheme is used as the optimal proportion and is used as the concentration of the subsequent experiment.
Example 4
Experiment of growth proliferation and apoptosis of Ornithine-derived drug resistant NSCLC cells in combination with Ornitinib
The EdU cell proliferation assay specifically comprises: inoculating logarithmic growth phase cells into 24-well plate (4×10) 4 Individual cells/well), 24h later, solvent DMSO (blank) was added, 2. Mu. Mol.L alone -1 Oritinib, added alone at 1. Mu. Mol.L -1 Auranofin or 2 mu mol/L -1 Ornitinib+1. Mu. Mol.L -1 The treatment with the combination of auranofin was continued for 2h after 48h with addition of EdU. After fixing the cells with 4% paraformaldehyde, the cells were stained with Click reaction solution and Hoechst in the dark, observed under a normal fluorescence microscope and photographed, and the image was processed and counted by ImageJ software. EdU positive cell rate = Edu number of staining positive cells/number of Hoechst labeled cells. The results are shown in FIG. 4A.
As can be seen from the results of the EdU cell proliferation experiments in FIG. 4A, after 48H of the combined treatment of auranofin and Ornitanib, the ratio of EdU positive cells of H1975/OR and PC9/OR cells was decreased by 71.7% and 86.5%, respectively, whereas the ratio of auranofin alone was decreased by 39.9% and 30.4%, respectively, and the ratio of Ornitanib alone was decreased by 13.6% and 11.5%, respectively.
The apoptosis test by flow cytometry comprises the following steps: inoculating logarithmic growth phase cells into 6-well plate (2×10) 5 Individual cells/well), 24h later, solvent DMSO (blank) was added, 2. Mu. Mol.L alone -1 Oritinib, added alone at 1. Mu. Mol.L -1 Auranofin or 2 mu mol/L -1 Ornitinib+1. Mu. Mol.L -1 The combination treatment of auranofin was performed, all cells in the adherent and supernatant were collected after 48 hours, 5. Mu.L of Annexin V-FITC and 5. Mu.L of Propidium Iodide (PI) staining solution were added, and the mixture was incubated at 4℃for 15 minutes in the absence of light, and apoptosis was detected by flow cytometry, and the results are shown in FIG. 4B.
As can be seen from the flow apoptosis detection results in fig. 4B, compared with the single administration of both, the apoptosis rate of both drug-resistant cell lines was significantly increased after 48h of the combined treatment of auranofin and octreotide. The above results demonstrate that the combination of auranofin and octreotide significantly inhibits the growth and proliferation of H1975/OR and PC9/OR cells and induces apoptosis.
Example 5
Experiment of NSCLC cell migration and invasion of acquired resistance to Ornitinib by combining auranofin and Ornitinib
The cell migration and invasion capacities were tested by Transwell experiments, specifically: in the cell migration experiment, cells in logarithmic growth phase were inoculated in 6-well plates (2×10) 5 Individual cells/well), 24h later, solvent DMSO (blank) was added, 2. Mu. Mol.L alone -1 Oritinib, added alone at 1. Mu. Mol.L -1 Auranofin or 2 mu mol/L -1 Ornitinib+1. Mu. Mol.L -1 Infofene combination treatment, 48 hours later, cells were transferred to the upper chamber of a 24-well plate Transwell chamber (2X 10) 4 Individual cells/well) was subjected to serum-free culture, 500 μl of 1640 medium containing 20% FBS was added to the lower chamber, and after 24 hours of culture, 4% paraformaldehyde was added for fixation for 15min, 0.1% crystal violet was added for staining at room temperature for 15min, photographed under a microscope and counted with Image J software, and the results are shown in fig. 5A. In the cell invasion experiment, matrigel was spread in a Transwell chamber 24h in advance, and the other steps were the same as in the cell migration experiment, and the results are shown in fig. 5B.
From the results of the cell migration experiments in FIG. 5A, it was shown that the migration capacities of H1975/OR and PC9/OR cells were reduced by 86.5% and 81.8% respectively after 48 hours of the combination treatment of auranofin and octreotide, whereas the individual treatment of auranofin was reduced by 60.6% and 40.2% respectively, and the individual treatment of octreotide was reduced by 14.2% and 32.5% respectively, as compared with the blank. Also, from the results of the cell invasion experiments in fig. 5B, it was shown that the combined treatment of auranofin and octreotide reduced the invasiveness of H1975/OR and PC9/OR cells by 75.5% and 80.3%, respectively, whereas the treatment with auranofin alone reduced 49.6% and 44.5%, respectively, and the treatment with octreotide alone reduced 11.5% and 13.1%, respectively, compared to the blank. The above results demonstrate that the combination of gold and octreotide significantly inhibits the invasion and migration ability of H1975/OR and PC9/OR cells.
Example 6
Experiment to induce increased HSPB8 and autophagy following combination of auranofin and Ornitinib
Extraction of RNA and real-time fluorescent quantitative PCR detection of gene expression experiments. The method comprises the following steps: H1975/OR and PC9/OR cells in logarithmic growth phase were seeded in 6-well plates and after 24H 2. Mu. Mol.L were added alone -1 Oritinib, added alone at 1. Mu. Mol.L -1 Auranofin or 2 mu mol/L -1 Ornitinib+1. Mu. Mol.L -1 The method comprises the steps of carrying out combined treatment on the auranofin, extracting total RNA of cells by using an RNA extraction kit after 48 hours, extracting total RNA of two cells of H1975 and PC9, synthesizing cDNA by using a reverse transcription kit, and carrying out real-time fluorescence quantitative PCR reaction by using the cDNA as a template, wherein the reaction conditions are as follows: 95 ℃ 30s,95 ℃ 5s,60 ℃ 30s, 40 cycles total, 2 is calculated -ΔΔCt The relative expression levels of the genes of interest were calculated and three duplicate wells were set per group using the primers shown in Table 1.
Primer sequences (5 '-3') used in Table 1
Western blot detection experiments. The method comprises the following steps: H1975/OR and PC9/OR cells in logarithmic growth phase were seeded in 6-well plates and after 24H 2. Mu. Mol.L were added alone -1 Oritinib, added alone at 1. Mu. Mol.L -1 The method comprises the steps of carrying out combined treatment on auranofin or with Ornitinib and auranofin, collecting cells after 48 hours to extract proteins, simultaneously collecting proteins of two cells of H1975 and PC9, detecting different histone concentrations by using a BCA kit, carrying out SDS-PAGE electrophoresis, then electrically transferring to a PVDF membrane, blocking for 2 hours by using 5% nonfat milk powder, incubating a primary antibody overnight by using a 4-degree shaking table, washing for 3 times by using TBST, adding a secondary antibody to incubate for 1 hour at room temperature, washing for 3 times by using TBST, detecting protein expression level by using an ECL system, and repeating the experiment for 3 times.
As shown in fig. 6A and 6B, screening of differentially expressed genes of combination and octtinib alone by RNA-seq results in H1975/OR and PC9/OR, up-regulated in both cells for 36 genes in total, down-regulated in both cells for 34 genes, and further down-regulated after HSPB8 resistance in the differentially expressed genes found in RNA-seq results in H1975 and H1975/OR, as shown in fig. 6C, up-regulated after combination, and significantly down-regulated after drug resistance (P < 0.01) for HSPB8 was found after drug resistance by qRT-PCR validation of sensitive and resistant strains, and significantly up-regulated after combination (P < 0.01).
As shown by Western blot results in fig. 7, HSPB8 expression levels were significantly downregulated (P <0.05 or P < 0.01) in both cells compared to the sensitive strain, the expression levels of austitinib He Jinnuo-fin combined with HSPB8 were significantly upregulated (P < 0.01) compared to austitinib alone, and LC3-II/LC3-I levels were significantly upregulated (P < 0.01), indicating that the cellular drug resistance mechanism was by downregulating HSPB8, whereas combined administration may promote HSPB8 expression, leading to increased autophagy ultimately leading to tumor cell death.
Extraction of RNA and RNA-seq detection. Sequencing was performed by the method of Trizol (Invitrogen) for the extraction of H1975 and PC9 sensitive strains and 2. Mu. Mol.L for H1975/OR and PC9/OR, respectively -1 Oxitinib alone and 2. Mu. Mol.L -1 Oritinib and 1. Mu. Mol.L -1 Total RNA from cells treated with the combination of auranofin was tested for quality of RNA samples to ensure that acceptable samples were used for transcriptome sequencing. RNA library construction Using TruSeq TM RNA sample preparation Kit (Illumina, san diego, CA) kit, library was high throughput sequenced using Illumina HiSeq xten/NovaSeq 6000 sequencing platform, sequencing read length PE150.
Wherein HSPB8 belongs to the small molecule heat shock protein family (small heat shock proteins, sHSPs) in heat shock proteins, is expressed more under environmental and physiological stimulus, has a molecular weight of 22kDa, can degrade misfolded proteins through autophagy after being combined with BAG3, has been reported to have both a cancer promoting effect and a cancer inhibiting effect in tumors, and has different expression conditions in different tumors, such as that in melanoma, the combination of HSPB8 and TAK1 results in activation of p38-MAPK signaling pathway, finally apoptosis, and the activated TAK1 binds and phosphorylates beta-catenin at Ser552 site, resulting in reduced CDK2 transcription, and finally cell cycle arrest. HSPB8 and PI3K binding in hepatocellular carcinoma inhibits invasion and migration of cancer cells. Less research in lung cancer, the invention discovers that HSPB8 plays a role in inhibiting cancer in lung cancer and is related to autophagy.
In summary, the method for reversing the acquired resistance of the non-small cell lung cancer of the invention starts from high-throughput screening, and selects drugs for enhancing the sensitivity of the Organtinib from 1470 FDA approved drug libraries in H1975/OR and PC9/OR cells, finally obtains the effect of the Jinofen on inhibiting the growth of tumor cells and enhancing the curative effect of the Organtinib when the blood concentration of the Organtifen is obtained, and researches on the functions of two cell lines find that the combined effect of the two drugs is obvious, the expression level of HSPB8 after the drug resistance is reduced after the research, and the expression level of the HSPB8 and the autophagy related protein LC3-II/LC3-I after the combined use is up-regulated, so that the increased death of the tumor cells caused by autophagy indicates that the HSPB8 acts as an anti-cancer gene in the non-small cell lung cancer resistant Organtinib cell strain. The study is expected to provide a new treatment idea for the drug administration scheme of clinical drug-resistant patients.
The above embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention.
Claims (9)
1. The method for reversing the acquired resistance of the non-small cell lung cancer of the Ornitinib is characterized in that the reversing drug and the Ornitinib are combined to realize the reversing of the acquired resistance of the non-small cell lung cancer of the Ornitinib;
the reversing drug is selected from the group consisting of panobinostat, bortezomib, carfilzomib, vorinostat, auranofin, romidepsin, and homoharringtonine.
2. The method of reversing acquired resistance to non-small cell lung cancer of octenib according to claim 1, wherein the reversing drug is selected from the group consisting of auranofin.
3. The method for reversing the acquired resistance of non-small cell lung cancer of octreotide according to claim 2, wherein the ratio of the auranofin to the octreotide is 0.8-2 mu mol.L -1 ：2 ~8μmol·L -1 。
4. The method for reversing the acquired resistance of non-small cell lung cancer of octenib according to claim 3, wherein the ratio of the auranofin to the octenib is 1. Mu. Mol.L -1 ：2 μmol·L -1 。
5. The method of reversing acquired resistance to non-small cell lung cancer of octenib according to claim 1, wherein the NSCLC cells in the non-small cell lung cancer are H1975/OR cells and/OR PC9/OR cells.
6. The method for reversing the acquired resistance of non-small cell lung cancer of octreotide according to claim 1, wherein the combination of auranofin and octreotide can inhibit the growth proliferation, migration and invasion of NSCLC resistant cells and can increase the apoptosis rate of the cells.
7. The method of reversing the acquired resistance of non-small cell lung cancer of oriatinib according to claim 2, wherein said combination of auranofin and oriatinib is by modulating the expression of HSPB8 and LC3-/LC3-/>The level achieves reversal of non-small cell lung cancer of the acquired resistance to octenib.
8. The method of reversing the acquired resistance of non-small cell lung cancer of oriatinib of claim 7, wherein said combination of auranofin and oriatinib is by modulating up-regulation of HSPB8 in cellsExpression and LC3-/LC3-/>The level achieves reversal of non-small cell lung cancer of the acquired resistance to octenib.
9. The application of the auranofin is characterized in that the auranofin is used for reversing the acquired resistance of the non-small cell lung cancer of the Ornitanib.
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