| BackgroundLung cancer is the leading cause of cancer death worldwide.Non-small-cell lung cancer(NSCLC)accounts for about 75-80%of lung cancer cases,and the 5-year survival rate for all stages is about 10%-15%.Chemotherapy is one of the common and irreplaceable treatment methods for the treatment of tumors and the prevention of postoperative recurrence and metastasis of tumors.However,drug resistance of tumor cells in the treatment process often leads to the failure of chemotherapy and ultimately leads to the death of patients.Recent studies have shown that autophagy can be used to help tumor cells escape chemoradiotherapy or other treatment-mediated cell death,leading to drug resistance.After tumor formation,the nutrition and energy shortage,tumor cells can use autophagy remove damaged organelles and recycling of normal cells degradation products supply for oneself,make tumor cell in tumor microenvironment,under the pressure of survival and autophagy can serve as a kind of adaptive response to tumor cells resistant to radiation resistant treatments such as chemotherapy.FLP ointment is a traditional Chinese medicine compound with effects of nourishing qi and nourishing Yin,clearing heat and detoxifying,eliminating phlegm and relieving cough.And previous studies have shown that FLP ointment can inhibit the growth and proliferation of lung adenocarcinoma mice by regulating autophagy.We found that the combination of FLP ointment and chemotherapy drugs can increase the sensitivity of patients to chemotherapy drugs in clinical practice,but there have been no study its action mechanisms.ObjectiveThe purpose was to explain the scientific connotation of FLP ointment reversing drug resistance,to reveal the molecular mechanism of FLP ointment reversing drug resistance to chemotherapy from the perspective of autophagy,and to observe the effect of FLP ointment on the expression of drug resistance proteins in tumor tissues in the animal model of tumor bearing chemotherapy resistance in lung cancer.At the same time,it provides scientific basis for the further application of traditional Chinese medicine in the treatment of NSCLC.Methods(1)Drug resistant cell lines of A549/cis lung adenocarcinoma were inoculated into the right axilla of BALB/c nude mice to establish the A549/cis chemotherapy resistant tumor-bearing animal model of lung cancer.After 21 days of intervention with normal saline,FLP ointment,DDP,FLP ointment combined with DDP,CQ,DDP combined with CQ and FLP ointment combined with DDP and CQ,tumor weight was measured and tumor inhibition rate was calculated.The expression level of ki-67 in tumor tissues was determined by Immunohistochemistry(IHC).(2)The expression levels of drug-resistant proteins MDR1 and MRP1 in tumor tissues were detected by Western Blotting(WB)and IHC after 21 days of intervention with normal saline,FLP ointment,DDP,FLP ointment combined with DDP,CQ,DDP combined with CQ and FLP ointment combined with DDP and CQ(3)To establish the A549/cis chemotherapy resistant tumor bearing animal model of lung cancer,with normal saline,FLP ointment,DDP,FLP ointment combined with DDP,CQ,DDP combined with CQ and FLP ointment combined with DDP and CQ after 21 days.WB and IHC were used to detect the expression levels of autophagy-related proteins LC3,p62,Beclin 1,Atg3,Atg5 and Atg7 in tumor tissues,and the expression levels of LC3 were detected by immunofluorescence(4)To establish the A549/cis chemotherapy resistant tumor bearing animal model of lung cancer,with normal saline,FLP ointment,DDP,FLP ointment combined with DDP,CQ,DDP combined with CQ and FLP ointment combined with DDP and CQ after 21 days.WB was used to detect the expression levels of signaling pathway related proteins AMPK,p-AMPK,mTOR and p-mTOR in tumor tissues.(5)Lung adenocarcinoma drug-resistant cell lines A549/cis were tested in vitro.The effect of lung adenocarcinoma resistant cell lines A549/cis was determined by cck-8 method.The effect of lung adenocarcinoma resistant cell lines A549/cis was determined by Annexin V/PI method.(6)Lung adenocarcinoma drug-resistant cell lines A549/cis were tested in vitro.After intervention with FLP ointment,DDP,FLP ointment combined with DDP,CQ,DDP combined with CQ and FLP ointment combined with DDP and CQ for 48h.WB method was used to detect the effect of FLP ointment on the expression of drug-resistant MDR1 and MRP1 in A549/cis drug-resistant lung adenocarcinoma cell lines.(7)To construct a double fluorescent mRFP-GFP-LC3 system A549/cis lung adenocarcinoma resistant cell line,with FLP ointment,DDP,FLP ointment combined with DDP,CQ,DDP combined with CQ and FLP ointment combined with DDP and CQ after 48 h,high intension testing A549/cis cell lines autophagy protein expression of LC3.WB method to detect lung tumor ping paste of A549/cis lung adenocarcinoma cell lines resistant autophagy protein LC3,p62,Beclinl,Atg3 and Atg7 expression level;(8)A549/cis drug-resistant lung adenocarcinoma cell lines were used in vitro.After intervention with FLP ointment,DDP,FLP ointment combined with DDP,CQ,DDP combined with CQ and FLP ointment combined with DDP and CQ after 48 h,WB method was used to detect the expression levels of lung neoplasms ointment on signaling pathway related proteins AMPK,p-AMPK,mTOR and p-TOR of A549/cis drug-resistant lung adenocarcinoma cell lines.Results(1)FLP group,F+D group,D+C group and F+D+C group were all able to effectively inhibit tumor,with the tumor inhibition rates of 26.07%,44.01%,39.60%and 53.01%,respectively,among which,F+D+C group had the best tumor inhibition effect.IHC detected the expression of ki-67 in tumor tissues after 21 days of different drug interventions.It was found that the expression of ki-67 was effectively inhibited in the FLP group,F+D group,D+C group and F+D+C group,among which the expression of ki-67 was most significantly inhibited in the D+C group and F+D+C group.(2)DDP can increase the expression of drug-resistant related proteins MDR1 and MRP1 to a certain extent;FLP ointment and DDP combination,DDP and CQ combination and FLP and DDP combined with CQ group can obviously decrease the expression of MDR1,statistically significant differences,IHC also suggests that this 3 groups can significantly reduce the expression of MRP 1,statistically significant differences,above all,FLP ointment can increase the sensitivity of the DDP and DDP together.Its effect is similar to that of DDP combined with CQ.(3)Indirect immunofluorescence method was used to detect the expression of LC3.It was found that the specific spot distribution increased after DDP intervention,By analyzing the results of WB and IHC,it is found that DDP can increase the expression of Atg3,Atg5 and Atg7 to a certain extent,and reduce the expression of p62 at the same time,indicating that DDP could induce autophagy to a certain extent.The expression of autophagy related proteins was detected by WB,IHC and immunofluorescence.It was found that combined DDP and FLP can inhibit autophagy marker of the expression of LC3,enhance the expression of P62,at the same time inhibit autophagy related proteins in different degrees Atg3,Atg7,and the expression of Beclinl,DDP combined CQ group also can efifectively inhibit autophagy marker expression of LC3,enhance the expression of P62,at the same time inhibit autophagy related proteins in different degrees Atg3,Atg5,Atg7,and the expression of Beclinl,visible DDP to a certain degree of inducing autophagy related protein expression;FLP ointment combined with DDP can inhibit the expression of autophagy-related proteins,and its effect is similar to that of DDP combined with autophagy inhibitor.(4)WB was used to detect the expression of AMPK and mTOR related proteins.It was found that there was no obvious abnormality in the total protein expression of AMPK and mTOR.FLP ointment combined with DDP could reduce the expression of p-AMPK and increase the expression of p-mTOR.(5)FLP combined with chemotherapy DDP can effectively inhibit the growth of A549/cis resistant strains in vitro,and the effect is time-dependent,and the effect is similar to that of chemotherapy DDP combined with autophagy inhibitor CQ.Flow cytometry was used to detect cell apoptosis.It was found that FLP combined with DDP could effectively promote the apoptosis of A549/cis-resistant strains in vitro.(6)FLP combined with DDP,DDP combined with CQ and combination of three drugs can effectively inhibit the expression of MDR1 in vitro,but the effect on the expression of MRP1 is not obvious(7)Dual fluorescent mRFP-GFP-LC3 system was used to detect the intervention of FLP ointment,DDP and CQ alone or in combination with A549/cis human lung adenocarcinoma drug-resistant cell lines after 48h.It was found that the combination of FLP ointment with DDP and the combination of three drugs with CQ could significantly block autophagy flow and inhibit autophagy.Meanwhile,WB showed that FLP combined with DDP and DDP combined with CQ can inhibit the expression of autophagy marker LC3 Ⅱ/LC3 Ⅰ,enhance the expression of P62,and inhibit the expression of autophagy related proteins Atg3 and Atg7 to different degrees.The combination of three drugs can not only reduce the expression of LC3,Atg3 and Atg7,enhance the expression of P62,but also reduce the expression of Beclin1.(8)the combination of FLP ointment serum with DDP,DDP with autophagy inhibitor CQ and combination of three drugs can significantly inhibit the relative optical density value of p-AMPK/AMPK related proteins in the signaling pathway,and at the same time increase the relative optical density value of p-mTOR/mTOR.Conclusions(1)FLP ointment combined with DDP can effectively inhibit the growth and proliferation of tumor in A549/cis-resistant tumor-bearing mice,effectively inhibit the growth of A549/cis-bearing mice,and promote their apoptosis.(2)The combination of FLP ointment and DDP can inhibit the expression of drug resistance related proteins by inhibiting autophagy,increase the sensitivity of DDP and reverse drug resistance.(3)FLP ointment combined with DDP to reverse drug resistance by inhibiting autophagy may be related to AMPK/mTOR signaling pathway. |
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