A drug for treating non-small cell lung cancer

Abstract

The present invention provides a drug for treating non-small cell lung cancer, belonging to the field of medicine. The drug's raw materials include gefitinib and andrographolide, wherein the concentration of gefitinib is 12-20 μM and the concentration of andrographolide is 200-250 μM. The drug is in the form of an oral liquid preparation or an oral tablet. Experimental results show that the drug provided by the present invention has a significant inhibitory effect on the expression of apoptosis-related proteins and genes in PC9 / G cells, with a greater inhibitory effect than gefitinib or andrographolide alone, indicating a certain synergistic relationship between the two.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a medicine for treating non-small cell lung cancer. Background Art

[0002] Lung cancer is a common malignancy and a leading cause of cancer-related death. It is the third most common cancer after breast and prostate cancer, but accounts for the largest proportion of all cancer-related deaths. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases. Due to the atypical early symptoms of NSCLC, early diagnosis is difficult. By the time of diagnosis, 70%-80% of patients have already progressed to the advanced stage, missing the opportunity for radical treatment such as surgical resection.

[0003] At present, new chemotherapy drugs represented by glycosides, taxanes, pemetrexed, etc. are continuously put into clinical use. The efficacy of drugs has been improved compared with the first-generation chemotherapy drugs, but the overall survival benefit has not been significantly improved. Some patients even experience serious toxic side effects, which seriously endanger human health and life. Therefore, exploring new therapeutic drugs has become a hot topic in lung cancer research.

[0004] With in-depth research into the pathogenesis of NSCLC and the continuous development and application of novel molecular targeted drugs, clinical practice has gradually confirmed that epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, offer new opportunities for the treatment of advanced NSCLC with their relatively good therapeutic efficacy and significant improvement in quality of life. However, despite the high response rate to EGFR-TKIs in patients with non-small cell lung cancer, drug resistance is inevitable after 9-13 months of treatment, and 60% of this acquired resistance is caused by the EGFR T790M mutation. Therefore, new and effective strategies to enhance the sensitivity of EGFR-TKIs are urgently needed.

[0005] Andrographolide is a diterpene lactone isolated from Andrographis paniculata (the king of bitter herbs). Andrographis paniculata is an important herbal remedy used to treat a variety of conditions, such as respiratory infections, fever, bacillary dysentery, and diarrhea. Studies have shown that andrographolide can induce ROS production and cysteine-containing aspartate proteinase (caspase)-dependent apoptosis in lymphoma cell lines and primary tumor samples. Andrographolide-induced apoptosis is mediated through the mitochondrial pathway but is dependent on caspases in both cell lines and patient samples. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a drug for treating non-small cell lung cancer. The present invention obtains a drug that is non-cytotoxic and can effectively treat non-small cell lung cancer by combining andrographolide and gefitinib.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] A medicine for treating non-small cell lung cancer comprises gefitinib and andrographolide; the concentration of the gefitinib is 12-20 μM, and the concentration of the andrographolide is 200-250 μM.

[0009] Preferably, the concentration of gefitinib is 14-18 μM, and the concentration of andrographolide is 220-240 μM.

[0010] Preferably, the molar ratio of gefitinib to andrographolide is 1-5:1-5.

[0011] Preferably, the dosage form of the drug is an oral liquid preparation or an oral tablet.

[0012] The present invention also provides the use of andrographolide and gefitinib in preparing a reagent for promoting apoptosis of PC9 / G cells.

[0013] The present invention also provides the use of andrographolide and gefitinib in preparing a reagent for inhibiting PC9 / G cell proliferation.

[0014] The present invention also provides the use of andrographolide and gefitinib in preparing an inhibitor for inhibiting the expression of one or more genes among Bcl-2, K167 and PCNA.

[0015] The present invention also provides the use of andrographolide and gefitinib in preparing an inhibitor for inhibiting the expression of one or more proteins in Cleabed-casepase-3 and BAX.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention combines andrographolide and gefitinib to prepare a drug for treating non-small cell lung cancer. The drug significantly promotes apoptosis and inhibits proliferation of PC9 / G cells, without cytotoxicity. Compared to using gefitinib and andrographolide alone, the drug exhibits a superior therapeutic effect. Furthermore, experimental results show that andrographolide and gefitinib exhibit a synergistic effect, significantly inhibiting the expression of Bcl-2, K167, and PCNA genes, as well as the expression of Cleabed-casepase-3 and BAX proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Cell viability was measured after H1299 and PC9 cells were treated with gefitinib and andrographolide, respectively;

[0019] Figure 2 Figure 2 is the flow cytometry apoptosis result after PC9 cells were treated with andrographolide for 24 hours and 48 hours respectively;

[0020] Figure 3 The apoptosis histograms of PC9 cells were obtained by flow cytometry after treatment with andrographolide for 24 h and 48 h, respectively.

[0021] Figure 4 Cell viability measured after PC9 / G cells were treated with different drugs;

[0022] Figure 5 The dose-effect curve was obtained by using CalcuSyn2.0 software;

[0023] Figure 6 The morphology of apoptotic cells was observed using an inverted phase contrast microscope;

[0024] Figure 7 Figure 2 shows the flow cytometry results of apoptosis in PC9 / G cells treated with different drugs for 24h and 48h.

[0025] Figure 8 The apoptosis histograms of PC9 / G cells treated with different drugs for 24h and 48h are shown in flow cytometry;

[0026] Figure 9 The expression results of apoptosis-related proteins were measured after PC9 / G cells were treated with different drugs;

[0027] Figure 10 The results of P-Akt protein expression were measured after PC9 / G cells were treated with different drugs;

[0028] Figure 11 The imaging results of Example 5, wherein “-” represents that andrographolide or gefitinib was not added, and “+” represents that andrographolide or gefitinib was added;

[0029] Figure 12 The results of Bcl-2 gene expression were measured after PC9 / G cells were treated with different drugs;

[0030] Figure 13 The results of PCNA gene expression were measured after PC9 / G cells were treated with different drugs;

[0031] Figure 14 The results of K167 gene expression were measured after PC9 / G cells were treated with different drugs;

[0032] Figure 15 The results of Caspase-3 gene expression were measured after PC9 / G cells were treated with different drugs;

[0033] Figure 16 The results of Bax gene expression were measured after PC9 / G cells were treated with different drugs;

[0034] Figure 17 The results of P53 gene expression were measured after PC9 / G cells were treated with different drugs;

[0035] Figure 18 The JC-1 fluorescent probe staining results were measured after PC9 / G cells were treated with different drugs;

[0036] Figure 19 The flow cytometry results of apoptosis in PC9 / G cells treated with different drugs for 24 hours;

[0037] Figure 20 The flow cytometry results of apoptosis in PC9 / G cells treated with different drugs for 48 hours;

[0038] Figure 21 The apoptosis histograms of PC9 / G cells treated with different drugs for 24h and 48h were analyzed by flow cytometry. DETAILED DESCRIPTION

[0039] The invention provides a medicine for treating non-small cell lung cancer, comprising gefitinib and andrographolide; the concentration of the gefitinib is 12-20 μM, and the concentration of the andrographolide is 200-250 μM.

[0040] In the present invention, the concentration of gefitinib is preferably 14-18 μM, more preferably 15-17 μM, and further preferably 16 μM; the concentration of andrographolide is preferably 220-240 μM, more preferably 225-235 μM, and further preferably 230 μM; the molar ratio of gefitinib to andrographolide is preferably 1-5:1-5, more preferably 2-4:2-4, and further preferably 3:3; the dosage form of the drug is preferably an oral liquid preparation or an oral tablet.

[0041] The present invention also provides the use of andrographolide and gefitinib in preparing a reagent for promoting apoptosis of PC9 / G cells.

[0042] The present invention also provides the use of andrographolide and gefitinib in preparing a reagent for inhibiting PC9 / G cell proliferation.

[0043] The present invention also provides the use of andrographolide and gefitinib in preparing an inhibitor for inhibiting the expression of one or more genes among Bcl-2, K167 and PCNA.

[0044] The present invention also provides the use of andrographolide and gefitinib in preparing an inhibitor for inhibiting the expression of one or more proteins in Cleabed-casepase-3 and BAX.

[0045] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1 Effects of Gefitinib and Andrographolide on Cell Proliferation Activity

[0047] The CCK-8 method was used for detection. The specific steps were as follows: PC9 and H1299 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and then 6×10 4 / mL cell concentration to prepare different cell suspensions for standby use; 100 μL of cell suspension was respectively plated in a 96-well plate, patted to mix, and placed in a 37°C incubator for static culture for 24 h. After the cells adhered, the culture medium in each well was discarded, and preheated culture medium containing different concentrations of andrographolide (Andro) (final concentrations of 320, 160, 80, 40, and 20 μM) and gefitinib (Gef) (final concentrations of 20, 40, 80, 160, and 320 μM) was added to each well. Each concentration was repeated for 5 wells, and a control group (no drug group) and a blank group were set up. The plates were placed in a 37°C, saturated humidity, 5% CO2 incubator for static culture for 24 h and 48 h; 10 μL / well of CCK-8 solution was added at 24 h and 48 h, respectively, and the plates were placed in a 37°C, saturated humidity, 5% CO2 incubator for static culture for 2 h. The OD value at 450 nm was measured on a microplate reader. The cell survival rate was calculated according to formula (1). Figure 1 shown.

[0048] Cell survival rate = [(A experimental - A blank) / (A control - A blank)] × 100% formula (1)

[0049] At the same time, the IC50 values ​​of different drugs on cells were obtained as shown in Table 1.

[0050] Table 1 IC50 values ​​of different drugs on cells

[0051]

[0052]

[0053] Depend on Figure 1As shown in Table 1, andrographolide significantly inhibited the proliferation of PC9 or H1299 cells, and the inhibitory effect of andrographolide on PC9 and H1299 cell proliferation increased with increasing drug concentration. Compared with andrographolide, gefitinib had a weaker inhibitory effect on PC9 or H1299 cell proliferation and a higher cell survival rate.

[0054] Based on the above results, andrographolide was selected as the drug and treated with the above method for HPAEpiC and 293T cells. The results showed that low concentrations of andrographolide (0-300 μM) had no significant cytotoxic effect on HPAEpiC and 293T cells. However, high concentrations of andrographolide (≥320 μM) had a significant cytotoxic effect on HPAEpiC and 293T cells. This result indicates that andrographolide has no significant cytotoxic effect on normal tissue cells at the dose administered in this study.

[0055] In summary, 240 μM was selected as the concentration of andrographolide; 16 μM was selected as the concentration of gefitinib.

[0056] Example 2 Effect of andrographolide on PC9 cells

[0057] Flow cytometry was used to detect the effect of andrographolide on apoptosis of PC9 cells. 5 / mL concentration was plated in a 96-well plate and cultured at 37℃ for 24h. A 70μM andrographolide solution was added to the wells and cultured for 24h and 48h. The liquid in the wells was aspirated, washed once with PBS, digested with trypsin and made into a cell suspension, centrifuged at 1000rpm for 4min, repeated twice and resuspended with 1mL of AnnexinV reagent. 100μL of cell suspension was transferred to different flow tubes, 5μL FITCAnnexinV and 5μL 7-AAD active staining solution were added respectively, and FITCAnnexinV and 7-AAD single staining tubes were set up, and the cells were gently vortexed. Incubated in the dark at 25℃ for 15min, 400μL AnnexinV Binding Buffer was added respectively; analyzed using a flow cytometer within 30min. A control group (no drug group) was set up. The results are as follows Figure 2 and Figure 3 shown.

[0058] Depend on Figure 2-3 It can be seen that andrographolide can significantly promote the apoptosis of PC9 cells, thereby inhibiting the proliferation of PC9 cells.

[0059] Example 3 Effects of different drugs on PC9 / G cells

[0060] The CCK-8 method was used to detect the effects of gefitinib, andrographolide, and the complex of andrographolide and gefitinib on the proliferation rate of PC9 / G cells. The specific method is the same as that in Example 1. The concentrations of andrographolide were 1, 2, 4, 8, 16, and 32 μM, the concentrations of gefitinib were 20, 40, 80, 160, and 320 μM, and the concentrations of the complex of andrographolide and gefitinib were 20, 40, 80, 160, and 320 μM, respectively. The andrographolide and gefitinib complex were added in equal proportions according to their respective IC50 values ​​during preparation. The results are shown in FIG. Figure 4 shown.

[0061] At the same time, CalcuSyn2.0 software was used to calculate the synergistic effect of andrographolide and gefitinib. Figure 5 shown.

[0062] Depend on Figure 4-5 As shown, when the gefitinib concentration was 8 μM, there was no significant difference in apoptosis rate between the combination and andrographolide alone. However, at a gefitinib concentration of 32 μM, the apoptosis rate was too high after the combination. Therefore, a gefitinib concentration of 16 μM was selected. At a gefitinib concentration of 16 μM, apoptosis began to be detected by flow cytometry. At a gefitinib concentration of 32 μM, the apoptosis rate exceeded 50%.

[0063] Example 4 Effects of different drugs on nuclear morphology

[0064] The cells treated with different drugs were stained with Hoechst33342 staining solution to observe the morphology of the cell nucleus. The specific method is: take out 1×10 5 2mL of PC9 / G cells in the logarithmic growth phase were placed in complete culture medium containing 240μM andrographolide, 16μM gefitinib, and a mixture of 240μM andrographolide and 16μM gefitinib for 24h; the three groups of cells treated with different drugs were placed in an incubator at 37℃, saturated humidity, and 5% CO2 for 24h and 48h, and the cells were collected; the 6-well plate was taken out, 1mL of Hoechst33342 staining solution was added to each well, and the staining solution was fully covered with cells, and the cells were placed in an incubator for reaction for 15min, the staining solution was discarded, and the cell apoptosis morphology was observed using an inverted phase contrast microscope after washing with PBS. The results are as follows Figure 6 shown.

[0065] At the same time, flow cytometry was used to detect the apoptosis rate of PC9 / G cells by different drugs. The specific method was the same as that in Example 2. Among them, the concentration of andrographolide was 240 μM, the concentration of gefitinib was 16 μM, and the concentrations of andrographolide and gefitinib were 240 μM and 16 μM respectively. The measurement results are shown in Figure 2. Figure 7 and Figure 8 shown.

[0066] Depend on Figure 7 and Figure 8 It can be seen that compared with the use of andrographolide and gefitinib alone, the inhibitory effect of andrographolide and gefitinib on PC9 / G cells was significantly enhanced after combined use, that is, andrographolide and gefitinib have a synergistic effect.

[0067] Example 5 Effects of different drugs on the expression of apoptosis-related proteins in PC9 / G cells

[0068] The number of cells was 1×10 5 PC9 / G cells in logarithmic growth phase (100 cells / ml) were digested with 0.25% trypsin and centrifuged to prepare a cell suspension, which was then inoculated into culture dishes. When the cells reached 80% growth, they were cultured for 24 hours in complete medium containing 240 μM andrographolide, 16 μM gefitinib, or a mixture of 240 μM andrographolide and 16 μM gefitinib. Protein was then extracted after further incubation for 24 and 48 hours. A blank control group was also established.

[0069] Meanwhile, prepare a lysis buffer by mixing RIPA and PMSF at a ratio of 99:1. Lyse the cells on ice for 30 minutes. Once lysed, centrifuge at 12,000 rpm and 4°C for 15 minutes. Transfer the supernatant to a 1.5 mL centrifuge tube and store at 4°C until needed. Mix Solution A and Solution B at a ratio of 50:1 to prepare the BCA working solution.

[0070] The protein standard diluted to 0.5 mg / mL was added to the corresponding 96-well plate at volumes of 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL, respectively. Each well was filled to 20 μL with PBS. 1 μL of protein sample was added to the sample well and the volume was filled to 20 μL with PBS. Two replicate wells were set up for calculating the average absorbance. Finally, 200 μL of BCA working solution was added to each well and the plates were placed in the dark at 37°C for 30 minutes. The absorbance of each well was monitored at 562 nm using a microplate reader, and the protein concentration in the sample was calculated according to formula (2). Based on the obtained protein concentration, protein samples were prepared with a final concentration of 2-4 μg / μL.

[0071] Protein concentration = protein concentration calculated from the standard curve × 20 (sample dilution factor) Formula (2)

[0072] Clean the glass plate and fix it. Prepare 15% separation gel solution and mix it. Pipette the prepared protein sample solution and slowly inject it into the glass plate interlayer, making sure not to generate bubbles. Seal it with anhydrous ethanol. After solidifying for 40 minutes, pour it out and dry it with anhydrous ethanol. Prepare concentrated gel, add it to the glass plate, insert a comb, and wait for 30 minutes until it is completely solidified. Fix the glass plate, use the newly prepared electrophoresis solution for the inner layer, observe for leakage, use the recycled electrophoresis solution for the outer layer, and remove the comb. After adding the sample, turn on the power supply and set the initial voltage to 80V. When the bromophenol blue indicator band runs out of the concentrated gel, adjust the voltage to 120V and stop the electrophoresis when the minimum marker runs to the bottom of the gel.

[0073] Prepare 1× transfer buffer (transfer buffer formula: Tris 3.0g, Glycine 14.4g, methanol: 200ml, add deionized water to 1000ml), soak thick filter paper in transfer buffer, activate PVDF membrane with a pore size of 0.2μm in methanol for 3-5min, then remove it. Place the activated PVDF membrane in transfer buffer, remove the glue on the glue plate, discard the concentrated glue, and soak it in transfer buffer. According to the principle of paper-membrane-glue-paper, take it out in sequence and place it on the semi-dry transfer mold instrument. Turn on the power and transfer the membrane at a constant voltage of 12V for 20min; weigh 0.5g BSA powder and dissolve it in 10mL 1×TBST to prepare 5% BSA blocking buffer; place the PVDF membrane in the blocking buffer, place it on the desktop to mix, and block it for 1.5h;

[0074] The antibody of the target protein was diluted at a volume ratio of 1:1000, and the internal reference antibody was diluted at a volume ratio of 1:1500, and then added to the 5% BSA blocking solution according to the ratio; the PVDF membrane was placed in the corresponding antibody and incubated at 4°C for 10 hours; the primary antibody was recovered, 10mL 1×TBST was added, and the membrane was washed for 5 minutes, and repeated 3 times; the secondary antibody was diluted with 1×TBST at a ratio of 1:10000 (volume ratio), incubated for 2 hours, and then washed with TBST on a mixer for 5 minutes, and repeated 4 times, and then developed; the A and B solutions were mixed at a ratio of 1:1 to prepare the ECL developer, and the developer was evenly distributed on the PVDF membrane and placed in the developer for exposure. The results are as follows Figure 9 、 Figure 10 、 Figure 11 shown.

[0075] Depend on Figure 9-11 It can be seen that the inhibitory effect of andrographolide and gefitinib on Cleabed-casepase-3 and BAX protein was significantly enhanced after combined use.

[0076] Example 6 Effects of different drugs on the expression of apoptosis-related genes in PC9 / G cells

[0077] The PC9 / G cells obtained by culture in Example 4 and treated with different drugs were placed in a 37°C incubator and cultured for 24 h and 48 h. The culture medium was discarded, the cells were washed with PBS, and trypsin was used for 2 min to terminate the digestion. The cells were collected in a 1.5 mL centrifuge tube without RNase, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; 600 μL of lysis buffer containing β-mercaptoethanol (20 μL of β-mercaptoethanol per milliliter of lysis buffer) was added, vortexed to fully mix, and then 360 μL of anhydrous ethanol was added and blown up and down with a pipette; 700 μL of lysate was added to the nucleic acid purification column, centrifuged at 12000 rpm and 4°C for 1 min, the column fluid was discarded, the purification column was put back into the collection tube again, centrifuged again until all the column fluid was separated, the collection tube was discarded, and a new 2 mL collection tube was added. 700 μL of washing solution 1 (10 mM Tris-HCl (pH7.5, 80% ethanol), centrifuged at 12000 rpm and 4°C for 1 min, discarded the column liquid again, added 600 μL of washing solution 2 (10 mM Tris-HCl (pH7.5), 70% ethanol) to the purification column, centrifuged again and discarded the liquid, added 250 μL of flushing solution 2 (10 mM Tris-HCl (pH7.5), 70% ethanol) to the purification column, centrifuged at 12000 rpm and 4°C for 2 min, changed the purification column to a new 1.5 mL EP tube, added 50 μL of RNase-free water to the center of the membrane of the purification column, centrifuged at 12000 rpm and 1 min to wash off the RNA, and then discarded the purification column again; used Nanodrop TM RNA concentration and purity were tested using a One / One C micro-volume UV-Vis spectrophotometer. The measured OD260 / OD280 ratio of the sample should be between 1.8 and 2.0 before use in subsequent experiments.

[0078] Perform reverse transcription according to the instructions of the PrimeScript™ RT Master Mix kit and adjust the sample concentration to 1000 ng / 20 μL. To ensure consistent sample volume, reverse transcribe RNA into cDNA and use this as a template for quantitative amplification (see Table 2 for reverse transcription reaction conditions).

[0079] Primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd. 18s was used as an internal reference gene to detect the relative expression of related genes and plot histograms (see Table 3 for gene primer sequences). The operation was performed according to the operating procedures of the ABIQuantStudio 3 quantitative PCR instrument (see Table 4 for reaction system and Table 5 for reaction conditions). Three replicate wells were set for each sample, and the experiment was repeated three times using 2 -ΔΔct The relative expression levels of apoptosis-related mRNA were calculated by the method. Figure 12-17shown.

[0080] Table 2 Reverse transcription conditions

[0081]

[0082] Table 3 Primer sequences of related genes

[0083]

[0084] Table 4 qRT-PCR reaction system

[0085]

[0086] Table 5 qRT-PCR reaction conditions

[0087]

[0088] Depend on Figure 12-17 It can be seen that the combined treatment of andrographolide and gefitinib significantly inhibited the expression of Bcl-2, K167 and PCNA genes.

[0089] Example 7 Effects of different drugs on mitochondrial membrane potential of PC9 / G cells

[0090] When PC9 / G cells grew to 60-80% confluence, they were cultured with complete medium containing andrographolide, gefitinib, or a mixture of andrographolide and gefitinib for 24 h or 48 h. Simultaneously, 50 μL of JC-1 (200X) was added to 8 mL of ultrapure water for dilution, and the JC-1 was fully dissolved by vigorous shaking. 2 mL of JC-1 staining buffer (5X) was added and mixed thoroughly to obtain the JC-1 staining working solution (Note: 1 mL of staining solution was added to each well of a six-well plate).

[0091] CCCP apoptosis inducer (10 mM) was taken out and added to the culture medium at a ratio of 1:1000 (volume ratio), diluted from 10 mM to 10 μM. After treating the cells with CCCP for 20 minutes, the cells were stained with JC-1 staining working solution (Note: For most cells, the mitochondrial membrane potential in the cells will be completely lost after treating the cells with 10 μM CCCP for 20 minutes. Normal cells should show red fluorescence after JC-1 staining, while cells should show green fluorescence after JC-1 staining).

[0092] The adhered cells were removed, the culture medium was discarded, and the cells were washed once with fresh PBS. Subsequently, 1 mL of culture medium was added, and 1 mL of JC-1 staining working solution was added to each well. After thorough mixing, the cells were incubated in a cell culture incubator for 20 min. During the incubation process, the required JC-1 staining buffer (1X) was prepared by adding 4 mL of distilled water to every 1 mL of JC-1 staining buffer (5X), and the cells were placed on ice. After the incubation, the supernatant was discarded, the cells were washed with JC-1 staining buffer (1X), 2 mL of cell culture medium was added, and the cells were observed and photographed under an inverted phase contrast microscope.

[0093] Take out the six-well plate, wash it once with PBS, add trypsin to digest for 2 minutes, centrifuge at 1000rpm for 4 minutes, aspirate the culture medium, add 1mL culture medium to resuspend, then add 0.5mL JC-1 staining working solution, keep inverting to mix, and place in the cell culture incubator for incubation for 20 minutes; after the incubation, centrifuge at 600×g and 4℃ for 3 minutes, discard the supernatant; wash twice with JC-1 staining buffer (1X), add 1mL JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 600×g and 4℃ for 3 minutes, precipitate the cells, and discard the supernatant. Repeat the above operation twice and analyze using flow cytometer. The results are as follows Figure 18-21 shown.

[0094] The results showed that there were more normal cells in the cells treated with andrographolide group and andrographolide and gefitinib combined treatment group, which had a better therapeutic effect on non-small cell lung cancer, while gefitinib could not achieve the effect of treating non-small cell lung cancer; and the percentage of apoptotic cells in the andrographolide group and andrographolide and gefitinib combined treatment group was 34.28% and 44.10% of the total cells, respectively, which was significantly different from the gefitinib group and the control group.

[0095] In addition, the cells used in Examples 1-7 were all purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A drug for treating non-small cell lung cancer, characterized in that: Comprising gefitinib and andrographolide; the concentration of gefitinib is 12-20 μM, and the concentration of andrographolide is 200-250 μM; The molar ratio of gefitinib to andrographolide is 1-5:1-5.

2. The drug according to claim 1, characterized in that The concentration of gefitinib is 14-18 μM, and the concentration of andrographolide is 220-240 μM.

3. The medicine according to claim 1 or 2, characterized in that The dosage form of the drug is an oral liquid preparation or an oral tablet.

4. Use of andrographolide and gefitinib in the preparation of a reagent for promoting apoptosis of PC9 / G cells, characterized in that: The concentration of gefitinib is 12-20 μM, and the concentration of andrographolide is 200-250 μM; The molar ratio of gefitinib to andrographolide is 1-5:1-5.

5. Use of andrographolide and gefitinib in the preparation of a reagent for inhibiting PC9 / G cell proliferation, characterized in that: The concentration of gefitinib is 12-20 μM, and the concentration of andrographolide is 200-250 μM; The molar ratio of gefitinib to andrographolide is 1-5:1-5.

6. Use of andrographolide and gefitinib in the preparation of a medicament for treating non-small cell lung cancer, characterized in that: The concentration of gefitinib is 12-20 μM, and the concentration of andrographolide is 200-250 μM; The molar ratio of gefitinib to andrographolide is 1-5:1-5.

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