Exploring The Cisplatin-sensitizing Effect Of Curculigo Orchioides On Non-small Cell Lung Cancer Cells And Its Mechanism Based On P-g

Research background and aims:Lung cancer is one of the cancers with the highest incidence,and non-small cell lung cancer(NSCLC)is the major type of lung cancer.At present,chemotherapy remains an important part of the first-line treatment of NSCLC.Cisplatin,a classic chemotherapeutic drug,tends to be effective in the initial stages of chemotherapy,but easily induces tumor acquired resistance,which seriously affects the effect of chemotherapy and patient prognosis.Overexpression of multidrug resistance protein P-gp(P-glycoprotein)and inhibition of apoptosis are important reasons for reducing the therapeutic efficacy and promoting the development of drug resistance in cancer.Patients at advanced stages of lung cancer often present with deficiency of Qi and Yin,even deficiency of both Yin and Yang.Curculigo Orchioides functions to warm the kidney and remove dampness,and some studies have shown that Curculigo Orchioides has anti-tumor effects.Based on the previous studies on the pharmacological effects of Curculigo Orchioides and its active ingredients,this study is to observe the effects of Curculigo Orchioides decoction and orcinol glucoside on NSCLC cells from the perspective of cisplatin sensitivity,and to explore the possible mechanism based on P-gp and cell apoptosis.Research methods:1.First,using the network pharmacology approach,combined with the literature to analyze the relevant targets and pathways for the treatment of Curculigo Orchioides with NSCLC,and then using molecular docking to evaluate the binding ability of orcinol glucoside with P-gp,to find the possible targets and components for Curculigo Orchioides intervention on the chemosensitivity of NSCLC,and to make the way for subsequent cell experiments;2.Cell experimentsMouse NSCLC cell LLC,human NSCLC cell A549,and cisplatin resistant cell A549/cis were used as experimental models of NSCLC;Method and concentration range of administration:Curculigo Orchioides decoction,orcinol glucoside and cisplatin were applied to cells in graded concentrations alone and in combination at different concentrations for 48 h;Gradient concentration range:Curculigo Orchioides decoction:0-1600 μg/mL,orcinol glucoside:0,1×10-6-3×10-4 mol/L,cisplatin:0-18 μg/mL(range was determined based on the sensitivity of different cells to cisplatin);Concentrations of the combinations:2-3 selected concentrations ranging from low to high in the gradient concentrations of Curculigo Orchioides decoction and cisplatin,orcinol glucoside:1×10-5、5×10-5mol/L;Cell viability was assessed by CCK-8 assay;IC50 values were calculated.2.1 Curculigo Orchioides decoction and cisplatin were individually applied at gradient concentrations and in combination to LLC of mouse NSCLC cells.Then detect cell viability and observe the effect characteristics;2.2 Curculigo Orchioides decoction,orcinol glucoside and cisplatin were individually applied at gradient concentrations and in combination to human NSCLC cells A549.Then detect cell viability and observe the effect characteristics;The effect of Curculigo Orchioides decoction combined with cisplatin on the P-gp expression level in A549 were determined by Western blot analysis;2.3 A549/cis early drug-resistant cells were established at 2 months of time of cisplatin application using a concentration escalation,intermittent induction method.Cell viability was detected and the fold of drug resistance was calculated to examine the inhibitory effects of Curculigo Orchioides decoction,orcinol glucoside combined with cisplatin on A549/cis early drug-resistant cells;2.4 Curculigo Orchioides decoction,orcinol glucoside and cisplatin were individually applied at gradient concentrations and in combination to A549/cis mature drug-resistant cells.Then detect cell viability;P-gp expression levels in A549/cis cells were determined by Western blot analysis;2.5 A549/cis mature resistant cells were grouped according to the monotherapy and combination methods of administration of Curculigo Orchioides decoction,orcinol glucoside and cisplatin,and subjected to long-term drug administration lasting for 3 months.At the end of drug administration,the cell viability of A549/cis under the action of cisplatin in gradient concentration was detected to observe the change of its drug resistance.The cell apoptosis rate was detected by double staining flow cytometry.Research results:1.The results of network pharmacology study and further literature analysis found that Curculigo Orchioides may intervene NSCLC through caspase,Bcl-2,Bax,Jun,ESR and other apoptosis related targets and pathways.Molecular docking results indicated that the free binding energy of P-gp and orcinol glucoside was-7.6 kcal/mol,so they have good binding activity.Thus the subsequent cell experiments focused on the stepwise unfolding of P-gp,orcinol glucoside and apoptotic effect;2.Cell experiments2.1 Gradient concentration of Curculigo Orchioides decoction had no significant effect on viability of mouse NSCLC cells LLC.The inhibition rate of LLC by cisplatin increased with the increasing concentration,and the IC50 value was 1.80 ± 0.03 μg/mL.The drug combination experiment showed that the inhibition rate of LLC cells was significantly increased in the combination of cisplatin and Curculigo Orchioides decoction group compared with the cisplatin alone group(P<0.05,P<0.01,and P<0.001,respectively);2.2 Gradient concentrations of Curculigo Orchioides decoction,orcinol glucoside had no significant effect on viability of human NSCLC cells A549.The inhibition rate of cisplatin on A549 increased with the concentration,and the IC50 value was 2.22 ± 0.03 μg/mL.Drug combination experiments showed that A549 cells were significantly inhibited in the combination of cisplatin and Curculigo Orchioides decoction、orcinol glucoside when compared with the cisplatin alone group(P<0.05,P<0.01);Western blot experiments showed that compared with cisplatin(1.5 μg/mL)alone group,the P-gp expression levels of A549 in cisplatin(1.5 μg/mL)and Curculigo Orchioides decoction(1000 μg/mL)combination group were significantly reduced(P<0.05);2.3 Using a concentration escalation,intermittent induction,after 2-month induction,the established A549 cell lines can maintain growing at medium with cisplatin(0.5 μg/mL).And the IC50 of cisplatin was 3.05 ± 0.02 μg/mL,which was significantly higher(P<0.001)than that of A549 cells.The drug-resistant fold was approximately 1.4-fold,indicating that it had developed preliminary drug resistance,named A549/cis early drug-resistant cells.The drug combination experiment showed that the inhibition rate of A549/cis early drug-resistant cells treated by the combination of cisplatin and Curculigo Orchioides decoction or orcinol glucoside was significantly increased compared with the cisplatin alone group(P<0.05,P<0.01);2.4 Gradient concentrations Curculigo Orchioides and orcinol glucoside alone did not significantly affect the viability of mature resistant A549/cis cells.The IC50 value of cisplatin on A549/cis mature resistant cells was 5.45 ± 0.21 μg/mL,and the resistance fold was about 2.45-fold.Drug combination experiments showed that the inhibition rate of A549/cis cells was significantly increased in the combination of cisplatin with Curculigo Orchioides decoction or orcinol glucoside compared with the cisplatin alone group(P<0.05,P<0.01);Western blot experiments showed that compared with cisplatin(1.5μg/mL)alone group,the P-gp expression levels of A549/cis cells in cisplatin(1.5 μg/mL)and Curculigo Orchioides decoction(1000 μg/mL)combination group were significantly reduced(P<0.05);2.5 After a long-term administration lasting for 3 months to A549/cis mature resistant cells,compared with the blank group,Curculigo Orchioides decoction(1000 μg/mL)and orcinol glucoside(5×10-5 mol/L)alone reduced the IC50 value of cisplatin by 16.6%versus 16.0%(P<0.001)and significantly increased the apoptosis rate(P<0.05)in A549/cis cells.In the combination group of cisplatin(4 μg/mL)and Curculigo Orchioides decoction(1000 μg/mL)or orcinol glucoside(5×10-5 mol/L),the IC50 of cisplatin was reduced by 11.3%and 6.9%respectively(P<0.001),and the apoptosis rate was significantly increased(P<0.05,P<0.01)compared with cisplatin(4 μg/mL)alone.In the combination group,the apoptosis rate was significantly higher in the combination group of cisplatin and Curculigo Orchioides decoction than that in the combination group of cisplatin and orcinol glucoside(P<0.05).Research conclusions:1.Curculigo Orchioides decoction or its active ingredient,orcinol glucoside alone had no significant inhibitory effect on NSCLC cells LLC,A549 and A549/cis;2.Curculigo Orchioides decoction and orcinol glucoside can synergize in combination with cisplatin in LLC,A549 and A549/cis early drug-resistant cells,A549/cis mature resistant cells,and can partially slow or reverse cisplatin resistance of A549/cis when administered chronically over a period of 3 months.Comprehensing the network pharmacology and cell experimental study the mechanisms of above cisplatin sensitizing effect of Curculigo Orchioides on NSCLC cells may be related to the inhibition of P-gp expression and the promotion of cell apoptosis;3.The trend of the effects of orcinol glucoside alone and in combination with cisplatin on viability,inhibition of P-gp expression and promotion of apoptosis in NSCLC cells was close to that of Curculigo Orchioides decoction.So orcinol glucoside might be one of the effective components responsible for the related effects of Curculigo Orchioides.