| Background:Anaplastic thyroid carcinoma(ATC)has a rapid progression,high mortality,and poor therapeutic effect.Its resistance to doxorubicin,a commonly used chemotherapy drug,has been a clinical problem.Our previous research found that after treating ATC cell lines with doxorubicin,a small number of cells survived because if containing cancer stem cells(CSCs),this is an important factor in the development of drug resistance in ATC cells.Therefore,finding suitable anti-tumor and tnti-tumor stem cells drugs is a hot research topic.Recent studies have confirmed that andrographo lide compounds have obvious anti-tumor e ffects on lung cancer,liver cancer,gastric cancer and breast cancer,also found to have the effect of anti-multiple myeloma tumor stem cells.However,there is no relevant study on the effect and mechanism of andrographolide on ATC cells.The development of nanotechnology provides a new method for tumor research.Our previous research also found that mixing DOX and Dopamine-melanin nanoparticles(DMNPs)to form DOX-DMNPs,can improve DOX uptake ability of ATC resistant strain HTh74Rdox cells,and improve drug resistance.The effect of DMNPs on the loading capacity of andrographolide and the improvement of DMNPs combined with andrographolide on HTh74Rdox resistance is unclear and worth exploring.Objectives:(1)To evaluate the effect of andrographolide on inducing cell apoptosis,inhibiting cell proliferation and migration in ATC and to analyze its mechanism(2)To explore the value and mechanism of andrographolide in enhancing the effect of doxorubicin chemotherapy.(3)Observe the effect and mechanism of andrographolide on thyroid tumor stem cells.(4)Study the loading efficiency of DMNPs on andrographolide and observe the effect of combined andrographolide and DMNPs on ATC cells.Methods:(1)MTT and real-time cell analysis were used to observe the inhibitory effect of andrographolide on the activity of HTh74 and doxorubicin-resistant HTh74Rdox,and the inhibitory effect of andrographolide combined with doxorubicin on HTh74Rdox activity.(2)Cell cloning assay was used to detect the effect of andrographolide on the proliferation of HTh74Rdox cells.(3)Caspase-3 activity assay and flow cytometry were used to evaluate the effect of andrographolide on apoptosis of HTh74Rdox cells.(4)Cell scratch assay was used to detect the effect of andrographolide on the migration function of HTh74Rdox cells.(5)Tube formation assay was used to assess the effect of andrographolide on tumor angiogenesis.(6)qPCR and Western Blot were used to detect the mRNA and protein expression levels of multidrug resistance channel proteins MDR1 and ABCG2 in HTh74Rdox cells treated with different concentrations of andrographolide.(7)qPCR and Western Blot were used to detect the mRNA and protein expression levels of stem cell markers CD133 and O ct-4 in HTh74Rdox cells treated with different concentrations of andrographolide.(8)Western Blot was used to detect the expression levels of ERK and p-ERK in MAPK/ERK signaling pathway in HTh74Rdox cells treated with diffe rent concentrations of andrographolide.(9)UV-Vis spectrophotometer was used to detect the loading efficiency of dopamine-melanin nanoparticles on andrographolide.(10)Detect the activity of combined andrographolide and DMNPs on HTh74 and HTh74Rdox cells by MTT.Results:(1)Andrographolide inhibited the activity of HTh74 and doxorubicin-resistant HTh74Rdox cells.The half-inhibitory concentration(IC50)of andrographolide on HTh74 cells was 17.2±2.77 ?mol/L and the IC50 of HTh74Rdox cells was 25.9±2.43 ?mol/L.The inhibition of activity of HTh74 and HTh74Rdox cells was in time-and dose-dependent manners.The half-inhibitory concentration(IC50)of doxorubicin on HTh74Rdox cells after andrographolide combined with doxorubicin was 629.6±19.87 ng/ml.The drug resistance index of doxorubicin was 5.15±0.32.There was a significant improvement in drug resistance compared to single drug use.(2)The number of clones decreased with the increase of andrographolide concentration.When the concentration of andrographolide reached 10?mol/L,the clonal proliferation level of HTh74Rdox cell line decreased significantly,and the effective rate of cloning was 22.3%(P<0.01);When the concentration of andrographolide reached 20?mol/L,the efficiency of cloning was only 5%(P<0.01).(3)Caspase-3 was increased in HTh74Rdox cell line with the increase of andrographolide concentration.The late apoptotic cells of HTh74Rdox increased significantly compared with 7.35%of the control group.When the concentration of the ester was 20?mol/L,the late apoptosis of the cells was 21.56%,(4)The migration ability of cells after treating with different concentrations of andrographolide was significantly lower than that of the control group.When the concentration of andro grapholide reached 10 gmo l/L,the open area of the scratch was comparable to the control group.Up to 126.7%,when the concentration of andrographolide reached 20?mol/L,the open area of the scratch was 150.3%compared with the control group,and cell death also occurred.(5)There was no significant change in the number of tube branches after using different concentrations of andrographolide.(6)The mRNA and protein expression levels of multidrug resistance channel proteins MDR1 and ABCG2 in HTh74Rdox cells were significantly decreased after treating with different concentrations of andrographolide compared with the control group(P<0.05).(7)The expression of CD133 and Oct-4 mRNA and protein in HTh74Rdox cells were significantly decreased after treating with different concentrations of andrographolide compared with the control group(P<0.05).(8)The expression of p-ERK in HTh74Rdox cells decreased after treating with different concentrations of andrographolide compared with the control group(P<0.05).(9)The loading efficiency of andrographolide was maintained at a low level with the increase of the mass ratio of dopamine-me lan in nanoparticles and andrographolide.(10)The combination of andrographolide and DMNPs inhibited the cell viability of HTh74 and HTh74Rdox,and there was no significant difference in the inhibitory effect of andrographolide on ATC cells.Conclusions:(1)Andrographolide can inhibit the activity of HTh74 and HTh74Rdox in a dose-and time-dependent manner;Andrographolide inhibits the proliferation,migration,and apoptosis of HTh74Rdox cells,but has no significant effect on angiogenesis.(2)Combined andrographolide and doxorubicin has the effect of reducing drugs and increasing the effect on HTh74Rdox cells,and can also inhibit the mRNA and protein expression of multidrug resistance chanrel proteins MDR1 and ABCG2 level by down-regulate the expression of p-ERK protein in the MAPK/ERK signaling pathway.(3)Andrographolide can inhibit the formation of HTh74Rdox stem cell spheres and reduce the mRNA and protein expression levels of stem cell markers CD133 and Oct-4.(4)Dopamine-melanin nanoparticles have a low loading efficiency for andrographolide.There was no significant difference in the effect of the combination of dopamine-melanin nanoparticles and andrographolide on the inhibition of ATC cells by the use of andrographolide alone. |
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