CN117205201B - Traditional Chinese medicine monomer for reversing cisplatin resistance of lung cancer and application thereof - Google Patents
Traditional Chinese medicine monomer for reversing cisplatin resistance of lung cancer and application thereof Download PDFInfo
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention provides a traditional Chinese medicine monomer for reversing cisplatin resistance of lung cancer and application thereof. The traditional Chinese medicine monomer of the invention, which is the pterocarpus santalin, reduces the cisplatin resistance index from 4.46 to 1.53, has remarkable reversing effect, and can be used as a traditional Chinese medicine monomer for reversing the cisplatin resistance of non-small cell lung cancer.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a traditional Chinese medicine monomer for reversing cisplatin resistance of lung cancer and application thereof.
Background
Based on statistics of the international cancer research center (IARC) 2020, the number of new lung cancer patients worldwide is about 220 ten thousand, accounting for 11.4% of the total number of new cancers, which is the most common cancer type next to breast cancer. Lung cancer is also currently the leading cause of cancer death in china, accounting for nearly 20% of all cases of cancer death. Despite the continual advances in medical technology, the five-year survival rate of lung cancer patients is still very low, only about 15%. Therefore, the prevention and control of lung cancer is a great challenge facing the prevention and control of malignant tumors in China.
Lung cancer is generally classified into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC) according to histopathological and immunohistochemical characteristics. NSCLC has a much higher incidence than SCLC, accounting for about 85% of the total lung cancer. The current clinical treatment of NSCLC is increasingly individualized. For NSCLC patients with different genetic alterations, treatment with different tyrosine kinase inhibitors can result in higher response rates, longer progression free survival and lower toxicity than chemotherapy. However, combination chemotherapy based on platins, particularly cisplatin (DDP), is still an indispensable treatment for NSCLC patients without sensitive genetic mutations. The acquired resistance to drugs that they develop during the course of treatment remains a significant problem that is currently facing clinically. Therefore, the search for new drugs that reverse the resistance of non-small cell lung cancer is critical for the clinical treatment of non-small cell lung cancer.
Toddalina volvulus (Toddalia asiatica (L.) Lam) is also known as Jumi Tong, sanbai ban, xuefei, is a plant of the genus Toddalina of the family Rutaceae, and is also the only plant in the genus Toddalina. The asiatic toddalia root blood is a traditional folk medicinal material of minority nationality in southwest regions of China, contains chemical components such as coumarin, alkaloid, terpenes, flavone, phenolic acid and the like, has pharmacological effects of anti-inflammatory, analgesic, antioxidant, antibacterial, cardiovascular protection, anti-tumor and the like, and has wide clinical application. The invention patent with the patent number of CN109172571A discloses application of alkaloid in reversing lung cancer cisplatin resistance, and the invention patent with the patent number of CN114917219A discloses application of isoorientin combined with cisplatin in preparing medicines for reversing lung cancer resistance. There is no research on Guan Feilong palmatine in medicines for reversing lung cancer drug resistance, and the research on effective traditional Chinese medicine monomer components for reversing non-small cell lung cancer drug resistance are found from traditional Chinese medicines, and the research has significant meaning in defining the action mechanism of the effective traditional Chinese medicine monomer components.
Disclosure of Invention
In view of the above, the invention provides a traditional Chinese medicine monomer for reversing the cisplatin resistance of lung cancer and application thereof, wherein the traditional Chinese medicine monomer can overcome the cisplatin resistance effect of non-small cell lung cancer, and the combination of the traditional Chinese medicine monomer and cisplatin can remarkably improve the sensitivity of the non-small cell lung cancer to cisplatin and inhibit the proliferation of cancer cells.
The technical scheme of the invention is realized as follows: first, the invention provides a traditional Chinese medicine monomer for reversing cisplatin resistance of lung cancer, which is characterized in that: the traditional Chinese medicine monomer is toddalia element, and the structural formula is:
On the basis of the above technical solution, preferably, the lung cancer is non-small cell lung cancer.
Second, the invention provides an application of a traditional Chinese medicine monomer for reversing lung cancer cisplatin resistance in preparing a medicine for reducing lung cancer cisplatin resistance.
Third, the invention provides an application of a traditional Chinese medicine monomer for reversing the cisplatin resistance of lung cancer in preparing a medicine for improving the sensitivity of lung cancer cells to cisplatin.
Fourth, the invention provides an application of a traditional Chinese medicine monomer combined cisplatin for reversing the cisplatin resistance of lung cancer in preparing a lung cancer prevention and treatment medicine.
Compared with the prior art, the traditional Chinese medicine monomer for reversing the cisplatin resistance of the lung cancer and the application thereof have the following beneficial effects:
(1) The invention discloses that the toddalin has remarkable effect on reversing cisplatin resistance of non-small cell lung cancer, and has definite and reliable effect.
(2) The traditional Chinese medicine monomer has single and definite components, and can obviously improve the sensitivity of non-small cell lung cancer to cisplatin and inhibit the proliferation of cancer cells by combined use of the traditional Chinese medicine monomer and cisplatin.
(3) The traditional Chinese medicine monomer has good curative effect, convenient use, safety and no toxic or side effect, and is suitable for preventing and treating cis-platinum resistance of non-small cell lung cancer.
(4) The traditional Chinese medicine monomer toddalin reverses the cisplatin resistance action mechanism of the non-small cell lung cancer, and the traditional Chinese medicine monomer toddalin has the effects of overcoming the cisplatin resistance of the non-small cell lung cancer by inhibiting the Nrf2 signal path and down regulating the expression of related drug resistance genes, so that the cisplatin resistance index is reduced from 4.46 to 1.53, and the traditional Chinese medicine monomer toddalin has remarkable reversing effect.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of the results of comparison of the non-small cell lung cancer parent cell strain A549 and its cisplatin resistant strain A549/DDP cisplatin and toddalin resistance index;
FIG. 2 is a graph showing the effect of toddalin on cisplatin sensitivity of non-small cell lung carcinoma cisplatin resistant cell line A549/DDP;
FIG. 3 is a graph showing the effect of combination of metacarposin and cisplatin on proliferation of non-small cell lung cancer cisplatin-resistant cell line A549/DDP cells;
FIG. 4 is a graph showing the effect of metacarposin and cisplatin in combination on A549/DDP cell clone formation;
FIG. 5 is a graph of results of the effect of metacarposin and cisplatin in combination on Nrf2 signaling pathway (mRNA levels) in A549/DDP cells;
FIG. 6 is a graph of results of the effect (protein levels) of metacarposin and cisplatin and combinations thereof on Nrf2 signaling pathways in A549/DDP cells.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical aspects of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
The toddalin used in the invention is directly purchased from Cheng Doude Site biotechnology Co., ltd., purity: HPLC is more than or equal to 98 percent, and the product codes: DF0089, the toddalia volvata hems molecular structural formula is:
EXAMPLE 1 Toddalin enhances cisplatin drug sensitivity in lung cancer cisplatin-resistant cell lines
1. Selecting A549 and A549/DDP cells in logarithmic growth phase, inoculating into 96-well plate (6 compound wells are arranged in each group) at1×10 4 cells/well, adding experimental control group (containing culture solution and cells), blank control group (only normal culture solution and no cells), plating cells, culturing in 5% CO 2 incubator at 37deg.C for 24h, adding cisplatin into A549 and A549/DDP cells according to drug concentration of 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, another group was supplemented with palmatine at drug concentrations of 0. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 80. Mu.M, 160. Mu.M in A549 and A549/DDP cells, respectively, and the third group was supplemented with cisplatin and 10. Mu.M palmatine at drug concentrations of 0. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 80. Mu.M in A549 and A549/DDP cells, respectively, after 24h of action with CCK-8 reagent (10. Mu.L/well), absorbance was measured with an enzyme-labeled instrument at 450nm wavelength after incubation for 2h at 37℃and relative cell viability was calculated and statistically analyzed according to the formula: cell relative viability (%) = (experimental group absorbance-blank group absorbance)/(control group absorbance-blank group absorbance) ×100%.
TABLE 1 drug resistance index of cisplatin and palmatine
The results of fig. 1 show that: as shown in fig. 1A, the proliferation activity of cisplatin-resistant cells was higher than that of the parent cells, regardless of the concentration of cisplatin treatment, indicating that cisplatin-resistant cells had reduced sensitivity to cisplatin compared to the parent strain. Both were statistically analyzed for IC50 values, and the A549/DDP Resistance Index (RI) was 4.46 (Table 1) as compared to the parental strain. And the proliferation activity between the A549 parent strain and the cisplatin resistant strain treated by the toddalin with different concentrations is not significantly different. That is, no matter what concentration of the pterocarpus santalinus is treated, the proliferation activities of the two cell lines are similar, which indicates that cisplatin resistance does not make cells resistant to the pterocarpus santalinus, namely: the molecular mechanism of the metacarpophaga in inhibiting lung cancer cells may be different from cisplatin (fig. 1B), and both were statistically analyzed for IC50 values, with a Resistance Index (RI) of a549/DDP of 1.02 (table 1) compared to the parental strain. After cisplatin and toddalin are combined, the drug resistance index is reduced from 4.46 to 1.53, which shows that toddalin has the effect of reversing cisplatin drug resistance.
2. Selecting A549/DDP cells with good growth state in logarithmic growth phase, inoculating the cells into 96-well plates at the density of 1X 10 4 cells/well (6 compound wells are arranged in each group), additionally arranging an experimental control group (containing culture solution and cells) and a blank control group (only normal culture solution and no cells are added), plating the cells, culturing the cells in a culture box with 5% CO 2 at 37 ℃ for 24 hours, adding cisplatin into the A549/DDP cells according to the drug concentration of 0 mu M,5 mu M, 10 mu M, 20 mu M, 40 mu M and 80 mu M in one group after 24 hours, adding cisplatin and 10 mu M of femtoxaemia into the A549/DDP cells according to the same drug concentration, respectively adding CCK-8 reagent (10 mu L/well) after 24 hours of action, measuring the absorbance value at 450nm by an enzyme-labeled instrument after incubation for 2 hours, and calculating the relative activity value of the cells and carrying out statistical analysis, wherein the calculation formula is as follows: cell relative viability (%) = (experimental group absorbance-blank group absorbance)/(control group absorbance-blank group absorbance) ×100%.
The results of fig. 2 show that: as the concentration of cisplatin increases, the viability of cisplatin-resistant cell lines A549/DDP decreases gradually. Compared with cisplatin group, the cell viability of drug-resistant cell strain treated by cisplatin and metacarpophyllin is obviously reduced.
Example 2 synergistic effects of Toddalin and cisplatin combination
A549/DDP cells in a good growth state and in a logarithmic growth phase are selected and inoculated into a 96-well plate at a density of 1×10 4 cells/well (6 compound wells are arranged in each group), an experimental control group (containing culture solution and cells) and a blank control group (only normal culture solution and no cells are added) are additionally arranged, the cells are plated and then are cultured for 24 hours in A5% CO 2 incubator at 37 ℃, 10 mu M of toddalia blood or/and 10 mu M of cisplatin are respectively treated (DMSO treatment control group) after 24 hours, CCK-8 reagent (10 mu L/well) is respectively added after 24 hours of action, an enzyme-labeled instrument is used for measuring absorbance value at a wavelength of 450nm after incubation for 2 hours at 37 ℃, and the relative activity value of the cells is calculated and statistical analysis is carried out, and the calculation formula is as follows: cell relative viability (%) = (experimental group absorbance-blank group absorbance)/(control group absorbance-blank group absorbance) ×100%.
The results of fig. 3 show that: compared with the control group, the cisplatin group and the metacarposin group, the metacarposin and cisplatin combined group can obviously inhibit the activity of A549/DDP cells.
Example 3 inhibition of proliferation of lung cancer cisplatin-resistant cell lines by combination of metacarposin and cisplatin
A549/DDP cells in the logarithmic growth phase were selected for good growth status, were digested with 0.25% pancreatin and blown out to a single cell state, inoculated in 6-well plates at a density of 1×10 3 cells/well, plated and cultured in A5% co 2 incubator at 37 ℃ for 24 hours, and treated with 10 μm metacarpophyllin or/and 10 μm cisplatin, respectively (DMSO-treated control group), during which medium containing the corresponding drug was changed every 3 days. When the cell clone was visible to the naked eye and the size was appropriate, the original medium was discarded, 1 XPBS solution was added to wash 1 time per well, PBS was discarded, and 500. Mu.L of 4% paraformaldehyde was added to each well for 30min at room temperature. The paraformaldehyde is discarded, and 500 mu L of 1 XPBS solution is added to each well for 3 times, 3-5 min/time. The PBS was discarded and each well was stained with 500. Mu.L of 0.2% crystal violet at room temperature for 20min. The crystal violet staining solution is discarded, 1 XPBS solution is added into each hole for washing for 3 times, 3-5 min/time, PBS is discarded, and the image is collected and subjected to statistical analysis after the drying.
The results of fig. 4 show: compared with a control group, the cisplatin group, the metacarposin group and the metacarposin combined cisplatin treatment group can reduce the cell clone number, and the inhibition capacity of the combined group is larger than that of the cisplatin group and the metacarposin group, which indicates that the metacarposin combined cisplatin can obviously inhibit the clone formation of lung cancer cisplatin resistant cells and plays a role in inhibiting cell proliferation.
EXAMPLE 4 Toddalin overcomes lung cancer cisplatin resistance by inhibiting the Nrf2 signaling pathway
1. A549/DDP cells in the logarithmic growth phase were selected to grow well, inoculated into 6-well plates at a density of 1×10 6/well, plated, placed in A5% co 2 incubator at 37 ℃ for 24h, and treated with 10 μm of metacarposin or/and 10 μm of cisplatin (DMSO-treated control group) for 24h, respectively. After the drug treatment, the original culture medium was discarded, and 500. Mu.L of 1 XPBS solution was added to each well to wash 1 time, PBS was discarded, and 1mL of Trizol reagent was added to each well and the mixture was placed in a shaker at 4℃for 20 minutes. After 20min, the bottom of the well plate was repeatedly blown and Trizol was transferred to a 1.5mL EP tube without nuclease, 200. Mu.L chloroform was added as 1/5Trizol volume, and the mixture was gently inverted and mixed up and down, and placed on ice for 10min. Followed by centrifugation at 12000g for 7min at 4 ℃. After centrifugation, adding pre-cooled equal volume isopropanol into the aqueous phase supernatant, mixing the mixture upside down, and standing the mixture at-20 ℃ for 1h. After 1h 12000g centrifuge for 10min at 4℃white precipitate appeared at the bottom of the EP tube. The supernatant was discarded, 1mL of 75% ethanol was added, and the white precipitate was washed upside down and centrifuged at 12000g at 4℃for 10min. The supernatant was discarded, the EP tube was left to stand at room temperature for 5min to volatilize the residual ethanol, and then an appropriate amount of DEPC water was added to dissolve RNA precipitate. After determining the RNA content by a micro-scale and identifying the structural integrity of the RNA by electrophoresis, cDNA was synthesized by using a reverse transcription kit (Nanjinouzan Biotechnology Co., ltd.). The synthesized cDNA is used as a template, and real-time fluorescence quantitative PCR detection (beta-actin is used as an internal reference gene) is carried out according to ChamQ SYBR qPCR MasterMix kit instructions. REALTIME PCR primer sequences are as follows :Nrf2,F：5′-CGACGGAA AGAGTATGAG-3′,R：5′-GGCAACCTGGGAGTAG-3′;HO-1,F：5′-GCTGGCA GGAGGTCAT-3′,R：5′-TTTCTGGGCAATCTTTT-3′;NQO1,F：5`-AAAGGACA TCACAGGTAAA-3`,R：5`-CAGAATGGCAGGGACT-3`;β-actin,F：5`-CATG TACGTTGCTATCCAGGC-3`,R：5`-CTCCTTAATGTCACGCACGAT-3`.
The results of fig. 5 show that: compared with the control group, the mRNA levels of Nrf2 and downstream target proteins HO-1 and NQO1 in the metacarposin group, the cisplatin group and the metacarposin combined cisplatin treatment group are significantly reduced, and the expression of Nrf2, HO-1 and NQO1 in the metacarposin combined cisplatin treatment group is most significantly reduced, which indicates that the metacarposin overcomes lung cancer cisplatin resistance by inhibiting an Nrf2 signal path.
2. A549/DDP cells in good growth state and in logarithmic growth phase were selected and inoculated in 6-well plates at a density of 1×10 6 cells/well, and after cell plating, the cells were placed in A5% co 2 incubator at 37 ℃ for 24 hours, and after 24 hours, treated with 10 μm of metacarposin or/and 10 μm of cisplatin, respectively (DMSO-treated control group) for 24 hours. After the end of the drug treatment, the original medium was discarded, and 500. Mu.L of 1 XPBS solution was added to each well and washed 1 time, PBS was discarded, and 200. Mu.L of RIPA lysate containing protease inhibitor was added to each well and lysed on ice for 30min. Subsequently, the lysate was collected with a cell scraper, centrifuged at 12000g at 4℃for 10min, and the supernatant was taken and protein quantified using the BCA protein concentration kit. The supernatant was then added to 5 Xloading buffer and placed in a boiling water bath for 10min denaturation, each sample was loaded with 20. Mu.g/well of protein, proteins were separated by 10% or 12.5% SDS-PAGE gel electrophoresis and transferred to 0.45 μm or 0.22 μm PVDF membrane. The membrane was then blocked in 5% BSA (bovine serum albumin) solution for 1h at 37℃in an incubator. Primary antibodies were diluted according to antibody instructions for Nrf2, HO-1, NQO1 and β -actin and incubated overnight at 4 ℃. After 3 times (10 min/time) of membrane washing with 1 XTBE solution for a second time, incubating the HRP-labeled secondary antibody for 1h at room temperature, after the incubation, 3 times (10 min/time) of membrane washing with 1 XTBE solution, and then performing chemiluminescence imaging through ECL luminescent liquid and collecting images (beta-actin is taken as reference protein).
The results of fig. 6 show: compared with the control group, the protein levels of Nrf2 and target proteins HO-1 and NQO1 downstream of the Nrf2 in the Feibuxuridine group, the cisplatin group and the Feibuxuridine combined cisplatin treatment group are obviously reduced, and the expression reduction of the Nrf2, HO-1 and NQO1 in the Feibuxuridine combined cisplatin treatment group is most obvious, which indicates that the Feibuxuridine overcomes the cisplatin resistance of lung cancer by inhibiting an Nrf2 signal path.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (4)
1. The application of a traditional Chinese medicine monomer in preparing a medicine for reducing the drug resistance of lung cancer cisplatin resistant strain A549/DDP to cisplatin is characterized in that: the traditional Chinese medicine monomer is toddalia element, and the structural formula is:
。
2. the use of a traditional Chinese medicine monomer according to claim 1 in the preparation of a medicament for reducing cisplatin resistance of lung cancer cisplatin-resistant strain a549/DDP, characterized in that: the lung cancer is non-small cell lung cancer.
3. The application of a traditional Chinese medicine monomer in preparing a medicine for improving the sensitivity of lung cancer cisplatin resistant strain A549/DDP to cisplatin is characterized in that: the traditional Chinese medicine monomer is toddalia root hematoxylin, and the toddalia root hematoxylin has the structural formula:
。
4. The application of traditional Chinese medicine monomer combined cisplatin in preparing a medicine for preventing and treating lung cancer cisplatin resistant strain A549/DDP is characterized in that: the traditional Chinese medicine monomer is toddalia root hematoxylin, and the toddalia root hematoxylin has the structural formula:
。
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Non-Patent Citations (2)
| Title |
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| Ramiro Vázquez等.Toddaculin, a natural coumarin from Toddalia asiatica, induces differentiation and apoptosis in U-937 leukemic cells.Phytomedicine.2012,第19卷(第8-9期),737– 746. * |
| Toddaculin, a natural coumarin from Toddalia asiatica, induces differentiation and apoptosis in U-937 leukemic cells;Ramiro Vázquez等;Phytomedicine;20120615;第19卷(第8-9期);737– 746 * |
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