Inhibitory Effect Of Low-intensity Ultrasound Combined With Curcumin On Glioma Cell Proliferation And Study On Its Mechanism

Objective:Gliomas are the most common primary malignant tumors of the central nervous system in adults.The morbidity and mortality of these patients with glioma are extremely high,with the median overall survival of only 10-14 months after diagnosis.Patients with glioma need chemotherapy after resection and radiotherapy,so chemotherapy is a reliable method to eradicate glioma cells.Previous evidence has shown that low-intensity ultrasound(LIUS)could enhance the sensitivity of glioma cells to chemotherapeutic agents.The aim of this study was to investigate the anti-tumor activity and mechanism of LIUS combined with curcumin in human glioma cells.Methods:In this study,human glioma U87 and U251 cells were selected as the research objects.Groups were divided into control,50.4(U50.4),83.4(U83.4),142(U142),290(U290),and 474(U474)m W/cm²according to different ultrasonic intensity and its duration for 60 s;based on various concentrations of curcumin,groups were set as follows:0(Control,DMSO),10(C10),20(C20),40(C40),and 60(C60)?mol/l,and glioma cells were treated with curcumin for 24 h,48 h,and 72 h,respectively.Since ultrasonic intensity of 142 m W/cm² is more effective in both glioma cells and has minimal damage to these cells,142 m W/cm² was used for further experiments.Subsequently,the ultrasonic intensity of 142 m W/cm² was combined with different concentrations of curcumin,and the groups were as follows:control,U142+C10,U142+C20,U142+C40,U142+C60;based on the statistical analysis results,IC₅₀ value and experimental purpose,the optimum parameters were selected from the aforementioned data,namely ultrasonic intensity of 142 m W/cm² and curcumin concentration of 20?mol/l.Then,they were classified into the control group(control),low-intensity ultrasound group(LIUS),curcumin group(curcumin),and low-intensity ultrasound combined with curcumin group(curcumin+LIUS).After that,CCK-8 and Ed U assays were performed to detect the proliferation of glioma cells following LIUS and curcumin treatment.And cell apoptosis was detected by flow cytometric analysis and western blot assay,respectively.Also,the autophagy activity of glioma cells was determined by acridine orange(AO)staining,dansylcadaverine(MDC)staining,and western blot assay,respectively.After adding autophagy inhibitor 3-MA and caspase inhibitor Z-VAD-FMK,cell proliferation,autophagy and apoptosis-related protein expression were detected by CCK-8 and western blot assays.In addition,intracellular reactive oxygen species(ROS)production in response to LIUS and curcumin treatment was evaluated using fluorescent probe DCFH-DA by multimode microplate reader and fluorescence microscopy.Finally,western blotting was used to detect the level changes of ROS/MAPK and AKT/m TOR pathway-associated proteins after treatment with LIUS and curcumin,and then NAC and AKT activator IGF-1 were added to detect whether co-treatment with LIUS-curcumin combination and NAC or IGF-1 could affect the levels of pathway-associated proteins and the proliferation ability of glioma cells compared with LIUS and curcumin in combination.Results:1.CCK-8 assay was performed to detect the proliferation ability after treatment with LIUS and curcumin in U87 and U251 cells.The results showed that there was a significant difference in cell viability between LIUS group and the control group when the ultrasonic intensity was more than 83.4 m W/cm².With the increase of ultrasonic intensity,glioma cell viability significantly decreased in an intensity-dependent manner.So ultrasonic intensity of 142 m W/cm² was used in the following experiments.Also,the results of CCK-8 assay revealed that curcumin inhibited the proliferative activity of glioma cells in a dose-and time-dependent manner,and at the same incubation time point,a significant difference was observed between curcumin group and the control group when the concentration of curcumin was greater than 10?mol/l.Under the same concentration of curcumin(>10?mol/l),there was a significant difference in cell viability of U87 and U251 cells incubated with curcumin for 24 h and 48 h,and a significant difference was found between 48 h and 72 h at the curcumin concentration of60?mol/l.So the incubation time was used for 48 h when treated with curcumin administration.Then,the effect of LIUS combined with curcumin on the proliferation of U87 and U251 cells was detected by CCK-8 and Ed U assays.The results showed that a significant difference was observed in the IC₅₀ value between cells treated with or without LIUS,indicating that LIUS could effectively increase the sensitivity of glioma cells to curcumin.And the optimum parameters for ultrasonic intensity of 142 m W/cm²and curcumin concentration of 20?mol/l were also obtained.Compared with curcumin alone group,the inhibitory cell viability of LIUS-curcumin combination was 2.26-fold and 2.53-fold that of the curcumin group in U87 and U251 cells,respectively.These results demonstrate that LIUS combined with curcumin inhibited the proliferation of glioma cells in a synergistic manner.2.The apoptotic ability of glioma cells was detected by flow cytometry.Compared with the control group,LIUS,and curcumin groups,LIUS combined with curcumin synergistically promoted the number of apoptotic glioma cells;The results of western blot analysis revealed that combined treatment with LIUS and curcumin could increase the markers of cell death and decrease the marker of cell survival relative to the other three groups.The expression levels of Bax and Cleaved caspase-3 in curcumin+LIUS group were significantly augmented,but the expression of Bcl-2 was attenuated(P<0.05).3.The results of acridine orange(AO)staining showed that LIUS plus curcumin significantly increased the number of orange red acidic vesicles in U87 and U251 cells,and 3-methyladenine(3-MA),an autophagy inhibitor,partially blocked the increase in the number of acidic vesicle organelles induced by LIUS combined with curcumin;The results of dansylcadaverine(MDC)staining revealed that the relative fluorescence intensity and the number of autophagic vacuoles(AVs)in MDC staining elevated following treatment with LIUS and curcumin,and the use of 3-MA attenuated the increase of AVs number and fluorescence intensity triggered by LIUS-curcumin combination;the results of western blot analysis revealed that combined treatment with LIUS and curcumin enhanced the expression levels of LC3B-II and Beclin-1 in U87 and U251 cells,but the expression of p62 was down-regulated in comparison to the control group,LIUS group,and curcumin group.4.CCK-8 assay and western blotting were used to detect the effects of autophagy inhibitor 3-MA and caspase inhibitor Z-VAD-FMK on the inhibition of LIUS-curcumin combination.The results showed that double application of 3-MA and Z-VAD-FMK significantly restored the inhibitory viability of glioma cells triggered by the combination of LIUS and curcumin(P<0.05).Pretreatment with Z-VAD-FMK significantly reduced the increase of Cleaved caspase-3 expression induced by LIUS-curcumin combination.Compared with combined treatment with LIUS and curcumin,LC3B-II expression decreased after co-treatment with LIUS,curcumin,and 3-MA(P<0.05).5.Contrasted to the other three groups,LIUS combined with curcumin significantly promoted the accumulation of ROS in both U87and U251 cells.On the contrary,the ROS production elicited by the combination of curcumin and LIUS was significantly reduced by using ROS scavenger NAC in both two cell lines;CCK-8 assay revealed that pretreatment with NAC could restore the inhibition of cell viability induced by LIUS-curcumin combination.6.The expression levels of ROS/MAPK and AKT/m TOR pathway-associated proteins were examined following different treatment in glioma cells.The results of western blotting showed that LIUS combined with curcumin significantly augmented the levels of p-JNK,p-ERK,and p-p38,without affecting the total JNK,ERK,and p38 expression.However,NAC treatment restrained the increased expression of p-JNK,p-ERK,p-p38,and LC3B-II induced by LIUS and curcumin in combination.Additionally,LIUS plus curcumin significantly inhibited the protein levels of p-AKT and p-m TOR in U87 and U251 cells in comparison to the other three groups while the total protein levels of AKT and m TOR were not changed.In contrast,pretreatment with IGF-1,an activator of AKT pathway,reversed the inhibition of p-AKT and p-m TOR mediated by LIUS-curcumin combination,and restrained the increase of LC3B-II expression induced by combined treatment with LIUS and curcumin;CCK-8 assay revealed that the inhibitory viability of glioma cells elicited by LIUS-curcumin combination was significantly elevated after pretreatment with NAC or co-treatment with IGF-1.Conclusion:In conclusion,LIUS combined with curcumin promotes the autophagic cell death by regulating ROS/MAPK and inhibiting AKT/m TOR pathway.As curcumin is easily accessible from the curcuma longa underground rhizome and mild for a long-term treatment,and the non-invasive LIUS could be modified to focus on a specific location,this combined treatment with LIUS and curcumin may function as a promising strategy for anti-cancer therapy.