| Lung cancer is one of the most common cancers and a leading cause of cancer-related deaths,with an estimated global death toll of 1.5 million per year.About 90% of cancer deaths are the result of metastases,not primary tumors.Due to local invasion or distant metastasis,most lung cancer patients are diagnosed at a late stage of the possibility of surgery or radiotherapy.More and more studies have found that natural products are an important source of anti-tumor chemicals.The anticancer properties of boswellic acid have been well established by many in vitro and in vivo studies.a pentacyclic triterpenic acid group containing(?,?,?)boswellic acid,acetyl-?-boswellic acid,11-keto-?-boswellic acid,acetyl-11-keto-?-boswellic acid,etc.The enormous anti-cancer effect is through the properties of different molecular targets.There has been little research on the anticancer effect of 11-carbonyl-?-boswellic acid.The purpose of this study was to investigate its in vitro effects on human lung cancer A549 and H446 cells.This study focused on the following three aspects of the effects of 11-carbonyl-?-boswellic acid on human lung cancer cells A549 and H446.1 To observe the effects of 11-carbonyl-?-boswellic acid on cell proliferation,apoptosis and cell cycle.2 To explore the mechanism of 11-carbonyl-?-boswellic acid on apoptosis.3 To study the effect of 11-carbonyl-?-boswellic acid on invasion and metastasis of lung cancer cells.The results may provide a theoretical basis for the subsequent development of low toxicity,high efficiency new anticancer drugs with 11-carbonyl-?-boswellic acid as the main component and better application in clinical treatment.Part ?: Effect of 11-carbonyl-?-boswellic acid on cell proliferation,apoptosis and cell cycleObjective:To observe the effects of 11-carbonyl-?-boswellic acid on proliferation,apoptosis and cell cycle of lung cancer cells.Methods: In this study,MTT assay,Edu staining and flow cytometry were used to detect the effects of 11-carbonyl-?-boswellic acid on the growth and proliferation of A549 ? H446 cells as well as changes in cell morphology and intracellular ROS levels.The effects of 11-carbonyl-?-boswellic acid on apoptosis index,apoptosis rate and mitochondrial membrane potential of A549 and H446 cells were detected by Hochest 33258 staining,Annexin V-FITC/PI double staining and JC-1 staining method.All data were analyzed using SPSS software(version 19.0,SPSS Inc.,Chicago)with P< 0.05 being a significant difference.Result:1.Cell morphology observationCompared with the control group,the morphology of A549 and H446 cells treated with 11-carbonyl-?-boswellic acid was similar,the distribution was sparse,the cell gap became large,the morphology was extremely irregular,and a large number of cells contracted and became round.The refractive index of the cell membrane decreases,and the nuclear chromatin condenses together.Cell adhesion decreased and cell colonies were smaller than the control group.And as time goes on,the cells fall off one by one,and many cells are in a floating state.Residual cells detach from the characteristics of colony growth and turn into a single growth.2.Effect of 11-carbonyl-?-boswellic acid on cell proliferation and cell cycle of A549 and H446 cellsCells were treated with different concentrations of 11-carbonyl-?-boswellic acid for 24 hours,and cell viability was measured by MTT assay.There is a significant negative correlation between the concentration of 11-carbonyl-?-boswellic acid and the proliferation of A549 and H446 cells.Flow cytometry analysis showed that 11-carbonyl-?-boswellic acid induced A549 and H446 cells to block in G2/M phase.3.Effect of 11-carbonyl-?-boswellic acid on apoptosis of A549 and H446 cellsHochest 33258 staining showed that high dose(23.5 and 44.8 ?M)group 11-carbonyl-?-boswellic acid promoted the formation of apoptotic bodies,showing apoptotic bodies,nuclear chromatin condensation,nuclear fragmentation,marginalization and uneven distribution.Except for the low concentration of 4.2 ?M group,the apoptotic index was significantly higher than that of the control group.Annexin V-FITC/PI double staining method detects the dose of 11-carbonyl-?-boswellic acid proportional to the apoptotic rate4.Fluorescence probe DCFH-DA showed that different doses of 11-carbonyl-?-boswellic acid(4.2-44.8 ?M)acted on the cells for 48 hours,and the intracellular ROS levels increased significantly,reflecting the dose-response relationship.Among them,the DCF fluorescence intensity values in the 23.5 ?M and 44.8 ?M treatment groups were the largest.The difference was statistically significant compared with the blank control group(P< 0.05).5.Detection of potential changes in mitochondrial membrane of H446 and A549 cells by JC-1 staining showed that 11-carbonyl-?-boswellic acid increases the mitochondrial membrane potential depolarization of these two types of cells with a dose-dependent effect.When the drug concentration reached 44.8 ?M,A549 and H446 cells decreased(51.173.26)% and(53.082.11)% ??m,respectively.Summary:11-carbonyl-?-boswellic acid may effectively inhibit the proliferation of A549 and H446 cells,inhibit tumor cell growth by inducing apoptosis and blocking the cell cycle to G2-M phase.11-carbonyl-?-boswellic acid can induce the increase of ROS level in lung cancer cells and decrease the membrane potential(??m)of mitochondria in lung cancer cells.Part ?: Mechanism of 11-carbonyl-?-boswellic acid on apoptosisObjective:To explore the mechanism of 11-carbonyl-?-boswellic acid on apoptosis.Methods:RT-PCR was used to detect the expression of apoptosis-related genes in lung cancer cell line A549 induced by 11-carbonyl-?-boswellic acid and to detect NF-?B activity.Western blot analysis was used to detect the expression of Pro-apoptotic proteins and anti-apoptotic proteins(Bax and Bcl-2)under different concentrations of 11-carbonyl-?-boswellic acid.Finally,the effects of 11-carbonyl-b-boswellic acid on the expression of PARP,p-JNK and survivin were detected by WB.Result:1.Transcriptional level detection of expression changes of apoptosisrelated genesAfter 12 hours of 11-carbonyl-?-boswellic acid treatment,JNK1 and FOXO1 genes were significantly down-regulated(P< 0.05),and P53 and c-jun were significantly up-regulated(P< 0.05).And the trend of change is proportional to the duration of drug action.2.Effect of 11-carbonyl-?-boswellic acid on NF-?B activityNF-?B activity is inhibited by 11-carbonyl-?-boswellic acid and is concentration dependent.3.Effect of 11-carbonyl-?-boswellic acid on the expression of apoptosisrelated proteinsUnder the action of 11-carbonyl-?-boswellic acid,the pro-apoptotic protein Bax expression was significantly up-regulated with the time and concentration increasing(P< 0.05).The anti-apoptotic protein Bcl-2 was expressed as a significant down-regulation of the negative correlation between time and concentration(P< 0.05).4.Effect of 11-carbonyl-?-boswellic acid on PARP,apoptosis inhibitory protein survivin and JNK kinaseWestern Blot assay showed that 11-carbonyl-?-boswellic acid activated PARP activity,down-regulated the expression of survivin,and activated JNK kinase activity.Summary:This part of the study showed that 11-carbonyl-?-boswellic acid induced down-regulation of apoptosis-related genes JNK1 and FOXO1,and upregulation of P53 and c-jun in human lung cancer cell line A549.The activity of NF-?B in the cell transcription factor family was inhibited,the expression of Bax was up-regulated,and the expression of Bcl-2 was down-regulated.At the same time,PARP activity was activated,survivin was inhibited and JNK kinase was activated.Part ?: Effect of 11-carbonyl-?-boswellic acid on cell invasion andmetastasisObjective:To study the effects of 11-carbonyl-?-boswellic acid on migration and invasion of lung cancer cells A549 and H446.Methods:Effects of different doses(4.2-44.8 ?M)of 11-carbonyl-?-boswellic acid on adhesion of A549 and H446 cells were detected by cell matrix adhesion assay.Transwell migration assay and cell scratch assay were used to detect the effects of 11-carbonyl-?-boswellic acid on the migration of lung cancer cells.Transwell matrigel invasion assay were used to detect the effects of 11-carbonyl-?-boswellic acid on invasiveness of lung cancer cells.Result:1.Effect of 11-carbonyl-?-boswellic acid on cell adhesionAfter application of different doses(4.2-44.8?M)of 11-carbonyl-?-boswellic acid,the adhesion ability of A549 and H446 cells showed a significant downward trend.2.Effect of 11-carbonyl-?-boswellic acid on cell migrationBoth the Transwell chamber experiment and the cell scratch test showed a significant decrease in the migration ability of A549 and H446 cells under the influence of 11-carbonyl-?-boswellic acid.3.Effect of 11-carbonyl-?-boswellic acid on cell invasionTranswell matrigel invasion assay showed that the invasive ability of A549 and H446 cells was generally decreased after 24 hours of exposure to different concentrations of 11-carbonyl-?-boswellic acid.The 0 ?M,4.2 ?M,8.6 ?M,23.5 ?M,and 44.8 ?M dose groups were much less than the blank control group(P< 0.05)in terms of the number of trans-basement membranes.Summary:In this part,Cell scratch assay,Cell matrix adhesion assay,Transwell migration assay and Matrigel invasion assay have demonstrated that 11-carbonyl-?-boswellic acid may inhibit cell migration and invasion of human lung cancer cells A549 and H446.It may provide a theoretical basis for the study of natural pharmaceutical ingredients for inhibiting the metastasis of advanced lung cancer.Conclusions: 11-Carbonyl-?-boswellic acid has a significant inhibitory effect on the proliferation of human lung cancer A549 and H446 cells.11-carbonyl-?-boswellic acid may promote the induction of apoptosis by way of regulating the expression of apoptosis-related genes,apoptosis-related proteins,inhibiting the activity of NF-?B,activating PARP activity,downregulating the survivin and activating the JNK kinase.It may inhibit tumor cell growth by blocking the cell cycle to the G2-M phase.11-carbonyl-?-boswellic acid may significantly inhibit the adhesion and migration of A549 and H446 cells as well as invasive ability in vitro. |
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