| Background:The mortality of cancer patients in China is relatively high,accounting for about54%of the global cancer mortality.Lung cancer is the most common and highly malignant disease.The incidence of lung cancer increases rapidly after 65 years of age,and the incidence gradually increases with age.According to the microscopic morphological characteristics,prognosis and therapeutic significance of lung cancer tissue cells,the World Health Organization(who)preliminarily divided lung cancer into two types:small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Among them,small cell lung cancer(SCLC)accounts for 20%of primary lung cancer and non-small cell lung cancer accounts for 80%of primary lung cancer.Non-small cell lung cancer can be further divided into adenocarcinoma,squamous cell carcinoma and large cell carcinoma.In recent years,with our understanding of disease biology and tumor progression mechanisms,the treatment of non-small cell lung cancer(NSCLC)has made important progress in clinical practice.Surgical resection is one of the most common treatment for early-stage NSCLC.However,even after complete resection,NSCLC still has the risk of recurrence and distant metastasis.In addition,many patients with NSCLC died of recurrent NSCLC after surgical resection,which indicates that these patients have a certain proportion of micro-metastasis during surgical resection.Due to the lack of specific diagnostic symptoms in the early stage of lung cancer,detection of NSCLC has great challenges,and most patients with lung cancer have been diagnosed as advanced disease.In order to improve the survival rate of NSCLC,many studies have proved the benefits of chemotherapy for NSCLC.Chemotherapy regimens have a certain effect on non-small cell lung cancer,but the side effects are great.At present,the clinical treatment of advanced NSCLC is mainly based on western medicine chemotherapy.Although new drugs are constantly being developed and marketed,the overall cure rate of advanced NSCLC is still low.Therefore,finding new treatment options is a new strategy for the treatment of non-small cell lung cancer.Chinese herbal medicine has been widely used in the treatment of advanced non-small cell lung cancer,but its efficacy and safety are still controversial.Recently,people have focused their research on traditional medicines that have been in clinical use for hundreds of years.Many drugs used to treat cancer in the clinic are derived from natural products or modified natural products;However,at present the treasure of natural medicine,especially traditional Chinese medicine,has not been fully utilized.The treatment of traditional Chinese medicine is still only in the treatment,and the specific mechanism still needs to be further explored.Some new chemical monomers extracted from various medicinal plants,fruits and vegetables have great anticancer potential.In addition,these monomers or active ingredients from plants regulate a variety of molecular signaling pathways,including inflammation,apoptosis and anti-apoptotic proteins,and the regulation of protein kinase activity.Therefore,the research methods of modern medicine are worth absorbing and learning from traditional medicine.Similarly,the use of drugs in traditional medicine has been tested by practice.Finding the role of drugs from traditional medicine can greatly reduce the drug cycle,the research and development cost,and thus decrease the medical expenses of patients.They can learn from each other.This is also beneficial to the treatment of diseases and the modernization of traditional Chinese medicine.Safflower yellow composition is composed of quinone chalcones,which is a mixture of many water-soluble components.It is found in the dried petals of Carthamus tinctorius.It has been widely used in folk medicine.Hydroxysafflor yellow A is the most representative of safflor yellow compounds.Since HSYA was discovered,people are more and more interested in its scientific application value,and more and more researchers have found its health effect.Previous studies have found that HSYA significantly inhibited the expression of angiogenesis markers such as CD105 and VEGF,So it can inhibit tumor angiogenesis and play an anti-tumor role.However,there are few studies on its role and mechanism in lung cancer.Therefore,based on the above research,this experiment intends to continue to use human non-small cell lung cancer H1299 and A549 as the research target to preliminarily explore the effect and possible mechanism of HSYA on the proliferation and migration of lung cancer cell lines,so as to provide some reference for enriching the clinical chemotherapy treatment of non-small cell lung cancer.Part 1:The effect of HSYA on lung cancer cell proliferationObjective:To observe the effect of HSYA on the proliferation of human non-small cell lung cancer cell lines A549 and H1299 in vitro,and calculate the IC50 value to measure the toxicity of the drug to cells.Methods:1.Cell culture:A549 and H1299 cells were cultured in DMEM complete medium containing 10%fetal bovine serum with 1%penicillin and streptomycin,placed at37°C,5%CO2.The cells were fused to about 80%of the bottom on the flask and passaged once.2.CCK8 method to detect the effect of HSYA on the proliferation of A549 and H1299 cells at different concentrations:the cells were digested and resuspended in the logarithmic growth phase to make cell suspension,and then inoculated it on a96-well culture plate at a cell density of 6×103.A549 and H1299 cells in the experimental group were added with different concentrations of HSYA(0?M,10?M,20?M,30?M,40?M,50?M,100?M).The control group was added with drug-free DMEM medium.After the cells was cultured for 48 hours in the incubator,the OD value of each well was measured by CCK-8 assays,and the cell proliferation inhibition rate and the half inhibitory concentration IC50 of each concentration group were calculated.3.The effect of HSYA on the cell proliferation of A549 and H1299 cells was conducted by CCK-8 at different times:after the cells were digested by 0.25%trypsin in logarithmic growth phase,the cells were resuspended in complete medium to make a single cell suspension.The cells were inoculated on a 96-well culture plate with a cell density of 6×103 cells.After the cells adhered to the wall,the culture medium was removed and a specific concentration of HSYA was added to co-incubate with A549and H1299 cells.The cells in each group cultured for 24h,48h,72h respectively,and a control group was set up.According to the measured absorbance(OD)value,the growth inhibitory effect of HSYA was calculated under the different times.4.Immunofluorescence method was used to detect the cell proliferation of HSYA on A549 and H1299:the cells were put into a six-well plate with tweezers carefully,the cells were inoculated into the six-well plate,and the cells were adhered to the wall.the experiment was divided into two groups,one was treated with 20?M HSYA for 48h,the other group was added with PBS as a control,then the culture medium was discarded,paraformaldehyde was added for fixation for 15min and then blocked,the Ki67 primary antibody was incubated,and the corresponding secondary antibody was added.Take pictures with an fluorescent microscope.Results:1.The results of CCK8 showed that HSYA inhibited the proliferation of A549and H1299 cells at different concentrations(10?M,20?M,30?M,40?M,50?M,100?M),and this inhibitory effect increased significantly with the increase of the concentration.2.The results of CCK8 showed that the inhibitory effect of HSYA on A549 and H1299 increased with the extension of the time at 20?M(24h,48h,72h).The cell proliferation inhibition rate of A549 cells after 24h was 19.42±1.23%;after 48h,the proliferation inhibition rate was 52.47±3.32%.The difference between different concentrations of HSYA was statistically significant(P<0.05).After 72 hours of treatment,the proliferation inhibition rate was 59.61±2.23%.In order to facilitate subsequent experiments,we choose the IC50 value closer to 20?mol/LHSYA for 48h.Similarly,the proliferation inhibition results of HSYA on H1299 cells are consistent with the above.3.The immunofluorescence results showed that compared with the control group,the fluorescence intensity of A549 and H1299 treated with HSYA(20?mol/L)decreased significantly,which was statistically significant.Conclusion:Different concentrations of HSYA inhibited the proliferation of A549and H1299 cells in a time-dose-dependent manner.Part 2:HSYA inhibits the proliferation of lung cancer cells by regulating the FAK and AKT pathways induced by EGFObjective:To explore the possible mechanism of HSYA on the proliferation of non-small cell lung cancerMethods:1.Determination of the expression and activation level of FAK and AKT protein by HSYA on A549 and H1299 cells:After treating the cells with 30?M HSYA for 0?2?4?6?8?10h,the protein was extracted on ice.The expression of FAK and Akt protein in A549 and H1299 cells were compared under HSYA treatment at different time.2.To detect the effect of HSYA on phosphorylation of FAK and Akt induced by EGF:the experiment was divided into four groups:the first group:A549 and H1299cells were treated with 30?M HSYA alone,and the protein was extracted 24 hours later;the second group was treated with EGF alone,the third group was treated with EGF and HSYA at the same time,and the fourth group was the control group.All groups were treated with 24h and the expression of FAK and AKT was detected.The effect of FAK inhibitor PF-562271 on the phosphorylation of FAK and Akt in A549and H1299 cells were consistent with the above.3.HSYA inhibits the proliferation of lung cancer cells by regulating the FAK and AKT pathways induced by EGF:The A549 and H1299 cells were pretreated with HSYA or 10?M PF-562271 for 60 minutes,and then incubated with 1ng/ml EGF and the pretreated cells for 48 hours.CCK8 was used to detect cell proliferation.Results:1.Western blot results showed that the protein expression levels of p-FAK and p-Akt in A549 cells were significantly decreased after 8 h and 10 h of HSYA treatment compared with the control group(P<0.05),and p-Akt was significantly decreased after 4 h of HSYA treatment,especially after 10 h.However,p-FAK changed significantly 8 h after HSYA treatment.Similarly,the protein expression levels of p-FAK and p-Akt in H1299 treated with HSYA for 6 h were significantly decreased(P<0.05),and with the extension of time,the protein expression levels of p-FAK and p-Akt decreased more significantly.2.EGF could significantly increase the expression of p-FAK and p-Akt in A549 cell line compared with the control group.HSYA can reverse the expression level of p-FAK and p-Akt caused by EGF.Similarly,the expression of p-FAK and p-Akt induced by EGF can be reversed by FAK inhibitor PF-562271.In H1299 cell lines,Western blot results were consistent with those in A549 cells3.EGF can promote the proliferation of A549 and H1299 cells.When HSYA and EGF were treated at the same time,cell proliferation could be inhibited.Meanwhile,FAK specific inhibitor and EGF could also inhibit cell proliferation.Conclusion:HSYA inhibits its proliferation ability by inhibiting the expression of EGF protein in A549 and H1299 cells.This inhibition is related to the regulation of FAK and AKT signaling pathways.Part 3:the effect of HSYA on the migration of lung cancer cellsObjective:To investigate the effect of HSYA on the migration of lung cancer cell lines and its possible molecular mechanism.Methods:1.The effect of different concentrations of HSYA on the migration of lung cancer cells:the cells in logarithmic growth phase were treated with 0,20 and 30?M HSYA,respectively.The A549 and H1299 cells were observed under the microscope after 24 h and 48 h.2.Fluorescence quantitative PCR experiment to detect the effect of different concentrations of HSYA on the expression levels of VEGF,MMP-2mRNA and MMP-9 mRNA in A549 and H1299 cells for 0,24,and 48 hours.To understand the mechanism of HSYA on lung cancer cell metastasis.Result:1.Compared with the control group,H1299 has weaker cell migration ability after being treated with 20?M and 30?M HSYA for 24h and 48h,and the difference is statistically significant(P?0.05).After A549 cells were treated with 20?M HSYA for48h,the cell migration ability was significantly weakened,while after 20?M HSYA treated the cells for 24h,the migration of the cells was not significantly changed,and there was no statistical difference.The results of the two cells'ability to significantly inhibit cell migration after 30?M treatment of the cells were consistent.2.A549 and H1299 cells were treated with different concentrations of HSYA(20?M,30?M)and then cultured for 24 and 48h.Compared with the control group,the expression levels of VEGF,MMP-2 mRNA and MMP-9 mRNA in the cells treated with 20?M or 30?M HSYA decreased significantly.Compared with the blank control group,the HSYA group had a statistically significant difference(P?0.05).Conclusion:HSYA inhibits the migration of A549 and H1299 cells and down-regulates the expression of MMP-2,MMP-9,and VEGF mRNA.This may be part of the molecular mechanism of HSYA inhibiting tumor cell migration. |
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