| Objective:To study the effects of forskolin,luteolin and resveratrol on Cyclic Adenosine Monop Hosp Hate(c AMP)in triple negative breast cancer(TNBC),and to explore the inhibition for metastasis and stemness of triple-negative breast cancer cell through the combination of three drugs;reveals that the combination of three drugs inhibits the migration and stemness of triple-negative breast cancer cells by increasing intracellular c AMP content and provides a scientific preliminary basis for subsequent research and clinical application.Methods:1.Combination of forskolin,luteolin and resveratrol inhibited triple-negative breast cancer cell metastasis and its mechanismThe three negative breast cancer cells MDA-MB-231 and BT549 were used as the research object.The experimental group was divided into blank control group(DMSO),positive control group: Three chemical drugs(forskolin + rolipram + probenecid),natural product Group: Three traditional Chinese medicine(forskolin + luteolin + resveratrol).Application of modern cell molecular biology and pharmacology techniques: verification of the effect of luteolin on phosphodiesterase(PDE4)expression and resveratrol on multidrug resistance protein(MRP1)by q RT-PCR at the genetic level;the intracellular c AMP content of three groups of MDA-MB-231 and BT549 cells were detected by ELISA,and to investigate whether the natural product group was effective in maintaining the steady state of c AMP in TNBC cells;Through cell migration,invasion,scratch test and establishment of triple-negative breast cancer lung metastasis model,Observe the mental status of each group of animal models after administration.After 6 weeks of administration,lung tissue was taken.Counting the number of lung metastases in nude mice and Perform HE staining experiments on lung tissue to analysis the inhibition of cell growth and metastasis by combination of forskolin,luteolin and resveratrol.Western-blot was used to detect the expression of EMTrelated proteins in triple-negative breast cancer cells,and to explore the mechanism of the combination of three drugs in inhibiting the metastasis of triple-negative breast cancer.2.Research the mechanism of inhibition of stemness of triple negative breast cancer cells by combination of forskolin,luteolin and resveratrolEstablishment of a triple negative breast cancer orthotopic tumor model in the third pair of breast pads of nude mice.To observe the effects of mental status and body weight changes after administration and compare the tumor size of each group.Establishment of MDA-MB-231 cell cloned spheres experiment to observe the effects of three drugs on the formation of triple-negative breast cancer stem cell clones;Further detection of expression of intracellular stemness associated protein CD44 by flow cytometry;Western-Blot method was used to investigate the effects of three natural products on the expression of cell stemness and hedgehog signaling pathway downstream of c AMP,and to explore the mechanism of the combination of three traditional Chinese medicines to inhibit the stemness of triple negative breast cancer.Results1.Through q RT-PCR experiments,we found that luteolin can inhibit the activity of PDE4,can achieve or even exceed the inhibition of c AMP hydrolysis by western medicine rolipram,while resveratrol inhibits the expression of MRP1,which can also replace the western medicine probenecid to reduce the effect of c AMP efflux,to maintain the steady state of intracellular c AMP content together.Through ELISA technology,we found that the three Chinese medicine group can significantly increase the intracellular c AMP content compared with the blank control group,and the effect is equivalent to the chemical group;in the cell scratch,migration and invasion experiments,we found that The three Chinese medicine groups can also inhibit cell metastasis,and the effect is even better than the chemical group.To observe the mental status of each group of animal models after administration,after the administration of the three Chinese medicine groups,the adverse reactions of the nude mice were significantly reduced compared with the chemical group.After 6 weeks of administration,lung tissue was taken and the number of lung metastases in nude mice was counted.The number of lung nodules in the Chinese medicine group and the chemical group was significantly lower than that in the DMSO group.HE was stained in lung tissue,and metastatic lesions were almost invisible in the lung tissue sections of the three Chinese medicine groups.The expression of EMT-related protein was detected by westernblot after administration of triple-negative breast cancer cells.The expressions of Snail,Slug,TCF8,ZO-1 and Vimentin,which promoted transfer,all showed different degrees of reduction,further confirming the three Drug combination inhibits the metastasis of triplenegative breast cancer.2.Establishment of the nude mouse orthotopic tumor model,and observe the mental status of each group.The performance of nude mice was basically the same as that of the tail vein model.The side effects of the Chinese medicine group were significantly lower than those of the chemical group.The weight changes of nude mice were recorded.The weight of the Chinese medicine group was generally higher than that of the other two groups,and the survival status was better than that of the chemical group.The survival curve of nude mice was drawn,and the survival rate of DMSO group was significantly decreased.The survival rate of the Chinese medicine group was the highest,the survival time was the longest,and the mental state was the best compared with the former two groups.Comparing the size of each group of tumors,it can be seen that the combination of three Chinese medicines has a significant inhibitory effect on the growth of TNBC orthotopic tumors.Observe the formation of three groups of MDA-MB-231 stem cell cloned spheres,it was found that the combination of forskolin,luteolin and resveratrol significantly inhibited the formation of MDA-MB-231 stem cell cloning spheres in TNBC cells and reduced tumors.The size of the spheres;flow cytometry showed that the expression of tumor stem cell marker CD44 in the three Chinese medicine group was significantly lower than that in the blank group and the chemical group.Through the Western-Blot experiment,compared with the blank control group,the expressions of the cell stemness related proteins CD44 and Nanog were significantly decreased,and the hedgehog signaling pathway was inhibited.The experimental results were consistent with the expectation,and the chemical group with obvious side effects could be replaced.Conclusion:The natural product forskolin combined with resveratrol,which inhibits multidrug resistance protein,and luteolin,which inhibits phosphodiesterase,can significantly increase the concentration of c AMP in triple-negative breast cancer cells.Thus inhibition of metastasis and stemness of triple-negative breast cancer cells,the effect is even better than the positive control group(forskolin+rolipram+probenecid),which is known to be effective in previous experimental studies.Along with further research results,the attenuation of cancer stem cells,the decrease of the expression of tumor stem cell surface marker proteins CD44 and Nanog,the decrease of the expression level of metastasis-associated protein Vimentin,and the change of the expression of Hedgehog signaling pathway protein suggest us that the combination of forskolin,luteolin and resveratrol on the metastasis and stemness of triple-negative breast cancer is closely related to Hedgehog signaling pathway. |
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