CN117205201A - 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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- CN117205201A CN117205201A CN202311092590.0A CN202311092590A CN117205201A CN 117205201 A CN117205201 A CN 117205201A CN 202311092590 A CN202311092590 A CN 202311092590A CN 117205201 A CN117205201 A CN 117205201A
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- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 title claims abstract description 87
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- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 39
- 239000000178 monomer Substances 0.000 title claims abstract description 29
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims abstract description 22
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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.
The asiatic toddalia root (Toddalia asiatica (L.) Lam) is also named as thorn Mitong, sanbai stick, xuefei, and the like, and is a plant of the genus of the asiatic toddalia of the family Rutaceae, and is also the only plant in the genus of the asiatic toddalia. 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 a medicament for reversing lung cancer resistance. The research of Guan Feilong palmatine in medicines for reversing lung cancer resistance is not found, and the research of effective traditional Chinese medicine monomer components for reversing non-small cell lung cancer resistance from traditional Chinese medicines is found, and the action mechanism of the effective traditional Chinese medicine monomer components is clear, so that the research has great significance.
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. Selection of A549 and A549/DDP cells in log phase growth phase at 1X 10 with good growth status 4 The density of each hole is inoculated into a 96-well plate (6 compound holes 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, and the cells are plated and then placed at 37 ℃ and 5% CO 2 After 24h incubation in incubator, one group was added with cisplatin at drug concentrations of 0. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 80. Mu.M to A549 and A549/DDP cells, respectively, another group was added with palmatine at drug concentrations of 0. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 80. Mu.M to A549 and A549/DDP cells, respectively, and the third group was added with cisplatin and 10. Mu.M palmatine at drug concentrations of 0. Mu.M, 5. Mu.M, 10. Mu.M, 40. Mu.M, 80. Mu.M to A549 and A549/DDP cells, respectively, after 24h incubation was performed, the absorbance was measured at 450nm with a microplate reader at 37℃for 2h, and the 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 in logarithmic growth phase to 1X 10 with good growth state 4 The density of each hole is inoculated into a 96-well plate (6 compound holes 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, and the cells are plated and then placed at 37 ℃ and 5% CO 2 After 24h incubation in incubator, one group added cisplatin at drug concentration of 0. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 80. Mu.M to A549/DDP cells, the other group added cisplatin and 10. Mu.M toyophyllin to A549/DDP cells at the same drug concentration, after 24h of incubation, CCK-8 reagent (10. Mu.L/well) was added respectively, absorbance was measured with an enzyme-labeled instrument at 450nm wavelength after incubation for 2h at 37℃and the relative cell viability was calculated and statistically analyzed 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
Selecting A549/DDP cells in logarithmic growth phase to 1X 10 with good growth state 4 Density of each well was inoculated into 96-well plates (6 wells per group), and experimental control group (containing culture medium and cells), blank control group (normal culture only) was additionally setLiquid without cells), cells were plated and placed at 37℃with 5% CO 2 Culturing in incubator for 24h, treating with 10 μm of toddalia element or/and 10 μm of cisplatin (DMSO treatment control group) respectively, adding CCK-8 reagent (10 μl/hole) respectively after 24h, incubating at 37deg.C for 2h, measuring absorbance at 450nm with enzyme-labeled instrument, calculating relative cell activity value, and performing statistical analysis, wherein the calculation formula is: 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
Selecting good growth state, and dispersing A549/DDP cells in logarithmic growth phase into single cell state by digesting with 0.25% pancreatin to obtain 1×10 3 Density of individual/well was seeded in 6-well plates, cells plated at 37℃and 5% CO 2 After 24h of incubation in the incubator, the culture medium containing the corresponding drug was replaced every 3 days during treatment with 10. Mu.M of palmatine or/and 10. Mu.M of cisplatin, respectively (DMSO-treated control group). 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. Selecting A549/DDP cells in logarithmic growth phase to 1X 10 with good growth state 6 Density of individual/well was seeded in 6-well plates, cells plated at 37℃and 5% CO 2 After 24h of incubation in the incubator, 24h of incubation were performed with 10. Mu.M of palmatine or/and 10. Mu.M of cisplatin (DMSO-treated control group), 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 the instruction of a ChamQ SYBR qPCR MasterMix kit. The sequence of the realtem PCR primer is 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'; beta-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. Selecting 1×10A 549/DDP cells in logarithmic growth phase with good growth state 6 Density of individual/well was seeded in 6-well plates, cells plated at 37℃and 5% CO 2 After 24h of incubation in the incubator, 24h of incubation were performed with 10. Mu.M of palmatine or/and 10. Mu.M of cisplatin (DMSO-treated control group), respectively. 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 (5)
1. A traditional Chinese medicine monomer for reversing cisplatin resistance of lung cancer is characterized in that: the traditional Chinese medicine monomer is toddalia element, and the structural formula is:
2. the traditional Chinese medicine monomer for reversing cisplatin resistance of lung cancer as claimed in claim 1, which is characterized in that: the lung cancer is non-small cell lung cancer.
3. The use of a traditional Chinese medicine monomer for reversing cisplatin resistance in lung cancer as claimed in claim 1 in the preparation of a medicament for reducing cisplatin resistance in lung cancer.
4. The use of a traditional Chinese medicine monomer for reversing the cisplatin resistance of lung cancer as defined in claim 1 in the preparation of a medicament for improving the sensitivity of lung cancer cells to cisplatin.
5. The use of a combination of cisplatin with a traditional Chinese medicine monomer for reversing cisplatin resistance in lung cancer as claimed in claim 1 in the preparation of a drug for preventing and treating lung cancer.
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| 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, vol. 19, no. 8, 15 June 2012 (2012-06-15), pages 737 * |
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