Sensitizing Effect And Mechanism Of D-Borneol Combined With Cisplatin On Cisplatin-Resistant H460 Non-Small Cell Lung Cancer

Objective:First,we selected the most sensitive borneol and cell line through comparing the cell viability of three borneol（natural borneol,levorotatory borneol and synthetic borneol）on non-small cell lung cancer（NSCLC）and cisplatin-resistant H460.And then detected the effects of cisplatin in combination with borneol on cell proliferation,migration,apoptosis,cell cycle and ferroptosis to elucidating the biological mechanism of borneol chemotherapy sensitization at multiple levels and revealing the modern scientific connotation of the effect of borneol"weak alone,while assisting envoys".Methods:The dosage of borneol and sensitive cell lines were optimized based on the cell level.The effects of cisplatin in combination with d-borneol on cell proliferation,migration,cell cycle,apoptosis,oxidative stress injury and P-gp efflux were investigated on cisplatin-resistant NSCLC（H460/CDDP）.The anti-tumor effect of d-borneol combined with cisplatin on H460/CDDP tumor bearing mice was investigated at the overall level.The target and possible mechanism of d-borneol underlying synergistic molecular mechanisms was explored through RNA-sequencing（RNA-seq）.And based on the optimal drug dose,a variety of molecular biology techniques were used to explore the relevant mechanism.（1）Effects of borneol combined cisplatin on NSCLC cells and cisplatin-resistant cells The cell viability of natural borneol,levorotatory borneol and synthetic borneol on two NSCLC cells（A549,H460）was investigated by MTT to select sensitive cell lines,borneol,exposure time,and suitable dose range.The effects of three borneol combined with cisplatin on the cell viability of H460/CDDP cells were further investigated to evaluate its sensitizing effect.Cell migration ability was investigated by scratch-wound healing recovery assays.Cell proliferation was investigated by colony formation assay.The P-gp function was detected by rhodamine 123 efflux assay.（2）Sensitizing effect of d-borneol on cisplatin-resistant NSCLC in vivo The H460/CDDP subcutaneous xenograft tumor model was constructed in 4-week-old male Balb/c nude mice.The mice were randomly divided into the following four groups:a control group,vehicle group,d-Borneol group,CDDP group,and a combination treatment group（d-Borneol plus CDDP）.The tumor volume,weight,body weight,organ index,tumor inhibition rate,alanine aminotransferase（ALT）,albumin（ALB）,aspartate aminotransferase（AST）,total protein（TP）,blood urea nitrogen（BUN）,creatinine（CRE）and lactate dehydrogenase（LDH）were detected in each group to investigate the anti-tumor effect of d-borneol combined with cisplatin.The morphology of liver and kidney was investigated by hematoxylin and eosin（HE）staining.Immunohistochemistry（IHC）method was used to observe the degree of cell proliferation and cell cycle related molecular in mouse tumor tissue.（3）The differentially expressed genes（DEGs）induced by d-borneol combined with cisplatin through RNA-seq RNA-seq was used to explore the DEGs of d-borneol combined with cisplatin in tumor tissues of H460/CDDP tumor-bearing mice.Then up-regulated and down-regulated DEGs were performed to explore the possible mechanism of d-borneol sensitizing cisplatin through GO function analysis and KEGG signaling pathway enrichment analysis.（4）Target verification and mechanism investigation Based on the optimal dose and combined with the results of RNA-seq,a variety of techniques were used to clarify the molecular biological mechanism of its sensitization.（1）Cell apoptosis and cycle arrest mechanism:The cycle distribution and apoptosis of d-borneol combined with cisplatin on H460/CDDP cells detected by Flow cytometry.Hoechst 33258 staining was used to observe the apoptosis cells.According to the results of RNA-seq,real-time PCR（RT-PCR）and Western Blot（Western Blot）methods were used to detect the effect of d-borneol combined with cisplatin on the expression of genes and proteins related to apoptosis and cycle arrest.（2）Ferroptosis and epithelial-mesenchymal transition（EMT）mechanism:Flow cytometry and DCFH-DA probe method were used to detect the effect of d-borneol combined with cisplatin on the level of reactive oxygen species（ROS）in H460/CDDP cells.The levels of malondialdehyde（MDA）,glutathione（GSH）and superoxide dismutase（SOD）were detected according to the manufactures’instructions.The enzyme-linked immunosorbent assay（ELISA）was used to detect the level of human thioredoxin（Trx）in H460/CDDP cells.According to the results of RNA-seq,RT-PCR and Western Blot were used to detect the effect of d-borneol combined with cisplatin on the expression of genes and proteins related to ferroptosis,autophagy and EMT.Results（1）Comparison of the cell viability of three borneol in A549,H460 and H460/CDDP cells Both levorotatory borneol and synthetic borneol（1,2,4,8,16,32μg/ml）showed no obvious effect on A549 and H460 cells,and natural borneol（1,2,4μg/ml）could significantly inhibit the proliferation of H460 cells（p<0.01）.Natural borneol showed a strong cell viability inhibition of H460/CDDP cells than other borneol,especially at concentrations ranging from 0.5μg/ml to 4μg/ml.d-Borneol（0.5,1,2,and4 g/ml）and 24 h were chosen as the best concentration and duration for further research.（2）d-Borneol combined with cisplatin inhibited the proliferation of H460/CDDP cells and the effect of P-gp（1）Cell proliferation:Compared with the cisplatin group,d-borneol（0.5,1,2,4μg/ml）combined with cisplatin inhibited proliferation of H460/CDDP cells,which inhibition rate were 72.27%,80.61%,78.08%,and 70.08%,respectively（p<0.01）.（2）Colony formation and migration:d-Borneol combined with cisplatin could reduce the number of colonies and inhibit the migration index,especially at 2μg/ml（p<0.01）.（3）P-gp function and expression:d-Borneol combined with cisplatin increased the enrichment of rhodamine 123,decreased the m RNA and protein expression of P-gp（p<0.05,p<0.01）.（3）d-Borneol combined with CDDP suppressed tumor growth in vivo（1）Tumor volume and weight:The cisplatin group showed a tendency to inhibit tumor growth,but there was no statistical difference,while the combination of d-borneol and cisplatin treatment led to a significant decrease in tumor volume and tumor weight from10d（p<0.01）.（2）Tumor inhibition rate:The tumor inhibition rate of the cisplatin group,d-borneol group and the combined treatment group were 47.52%,18.81%and82.35%,respectively（p<0.01）.（3）Ki-67 expression:The cisplatin group showed no obvious change of Ki-67 expression,but the expression of Ki-67 was significantly decreased after d-borneol and cisplatin treatment.（4）Organ index and pathological morphology of liver and kidney:Results showed that cisplatin group decreased the liver and kidney organ index,while the combination of d-borneol and cisplatin treatment restored its significantly（p<0.01）.No histological differences in the liver or kidney were found in the control groups,but there were increased inflammatory cells and necrosis both in the liver and kidney tissue after intraperitoneal injection of cisplatin.In addition,renal tubular epithelial edema cell increased,and damaged casts appeared in the lumen.When combined with d-borneol,inflammatory and edema cells were significantly decreased,indicating that d-borneol could alleviated the cisplatin-caused toxicity.（5）Biochemical indicators:The ALB,TP and LDH in all treatments showed no significant difference.Compared with the control group,cisplatin group increased the ALT,AST,BUN and CRE significantly,and the combination of d-borneol and cisplatin treatment effectively decreased these indicators,suggesting that d-borneol combined with cisplatin improved liver and kidney function（p<0.01）.（4）RNA-seq analysis:There were totally 1510 DEGs were identified in CDDP alone group and cisplatin+d-borneol group,including 720 up-regulated and 790 down-regulated genes.The up-regulated DEGs were mainly enriched in the ferroptosis pathway,and the down-regulated DEGs were mainly enriched in the cell cycle.It is speculated that ferroptosis and cell cycle may be an important mechanism of d-borneol sensitizing cisplatin.（5）d-Borneol increased cisplatin sensitivity to H460/CDDP through cell cycle arrest and apoptosis（1）Cell cycle:d-Borneol combined with cisplatin decreased the percent of G0/G1 phase and elevated the percent of S phase,indicating that d-borneol arrested the cell cycle at S phase.The molecular mechanism of d-borneol cotreatment with cisplatin arrested the cell cycle at S phase through inhibiting the expression of cyclin-dependent kinases（CDKs）（CDK2,CDK6）,cyclins（Cyclin A2,Cyclin D3）,and increasing the expression of cyclin-dependent kinase inhibitors（CDKIs）（p21,p27）that regulate CDKs and cyclin（p<0.05,p<0.01）.（2）Apoptosis:Flow cytometry results showed that d-borneol combined with cisplatin increased the proportion of apoptotic cells（p<0.05）.Hoechst 33258 staining results showed that d-borneol combined with cisplatin increased dense blue fluorescence nucleus and the number of apoptotic cells（p<0.05）.The molecular mechanism of d-borneol cotreatment with cisplatin promoted cell apoptosis through increasing the expression of B lymphoma-2 gene related protein（Bax）,decreasing B lymphoma-2 gene（Bcl-2）,increasing the ratio of Bax/Bcl-2,and activating the caspase-3（p<0.05,p<0.01）.（6）d-Borneol increased cisplatin sensitivity to H460/CDDP by promoting ferroptosis and inhibiting EMT（1）Oxidative damage:d-Borneol combined with cisplatin induces oxidative stress damage in tumor cells by increasing the green DCF fluorescence intensity and ROS fluorescence intensity,decreasing the content of antioxidants including GSH,SOD and Trx,and increasing the level of lipid peroxidation marker MDA（p<0.01）.（2）Ferroptosis:d-Borneol combined with cisplatin promoted the expression of NCOA4,autophagy-related regulators LC3II,ATG5 and Beclin-1,reduced the expression of HO-1,increased the expression of ACSL4,and activated the NCOA4-mediated autophagy pathway（ferritin phagocytosis）,which promoted ferroptosis.（3）Iron conversion:d-Borneol combined with cisplatin up-regulated the expression of PRNP,down-regulated the expression of PCBP2,and promoted the conversion of Fe³⁺to Fe²⁺,thereby inducing ferroptosis.（4）EMT:d-Borneol combined with cisplatin inhibited the EMT process by increasing the expression of epithelial marker E-cadherin and decreasing the expression of mesenchymal markers（N-cadherin,Vimentin）and transcription factors（SNAIL,ZEB1）.Conclusion（1）Compared with levorotatory borneol and synthetic borneol,natural borneol has a greater effect on the inhibition rate of H460 cells than A549 cells.Natural borneol combined with cisplatin showed stronger inhibitory effect on the viability of H460/CDDP cells than levorotatory borneol and synthetic borneol.（2）The main component of natural borneol,d-borneol combined with cisplatin significantly inhibited the cell proliferation,cell migration,colony formation and arrested cell cycle at S phase,induced oxidative damage,promoted apoptosis of H460/CDDP cells.d-Borneol combined with cisplatin significantly inhibited tumor volume,tumor weight,increased tumor inhibition rate,inhibited the expression of Ki-67,improved the weight loss of mice caused by cisplatin,protected the function of liver and kidney,reduced the damage of pathological morphology of liver and kidney.It not only expresses d-borneol can enhance the anti-tumor sensitivity of cisplatin,but also alleviates the toxic and side effects of cisplatin,reflecting the advantage of"enhancing the efficacy and reducing the toxicity"of the combination of traditional Chinese and western medicine.（3）Sensitization mechanism:d-Borneol combined with cisplatin could inhibit the expression and function of P-gp,reduce drug efflux.d-Borneol enhanced CDDP chemosensitivity involved in arresting the cell cycle at S phase via p27/p21-mediated cyclin A2/D3-CDK2/6 signaling pathway and promoting apoptosis via upregulating Bax and Caspase-3,downregulating of Bcl-2 expression.d-Borneol combined with cisplatin triggered ferroptosis through NCOA4-mediated autophagy-dependent ferritin degradation,decreased PCBP2 and HO-1 expression,increased PRNP expression,induced ROS accumulation,thereby promoting ferroptosis.Additionally,d-borneol combined with cisplatin inhibited EMT progression by increasing the expression of epithelial marker E-cadherin and decreasing the expression of mesenchymal markers（N-cadherin,Vimentin）and transcription factors（SNAIL,ZEB1）and increased cisplatin sensitivity.In summary,it is suggested that d-borneol can enhance cisplatin anti-tumor activity by regulating multiple signaling pathways,and thus play a synergistic anti-tumor role.This study is based on the drug resistance of lung cancer,according to the penetration and absorption of borneol,taking borneol combined with cisplatin as the starting point,using transcriptomics to explore chemotherapy targets and signaling pathways.And with the variety of modern technologies,from the cell-tissue-organ-overall level,to explain the biological mechanism of d-borneol and cisplatin against drug resistance,revealing the modern scientific connotation of the effect of borneol"weak alone,while assisting envoys",and providing a reference for expanding applications.It has both academic significance and application value.In addition,it has the characteristics of clinical Chinese pharmacy.