ABSTRACT
To study the effect of small interfering RNA targeting metastasis-associated lung adenocarcinoma transcript1 (si-MALAT1) combining with curcumin on the invasion and migration abilities of human colon cancer SW480 cells, and to explore the involved molecular mechanism. The recombinant lentiviral vector expressing si-MALAT1 was constructed, and its titer was determined by gradient dilution method. The colon cancer SW480 cells with stable expression of si-MALAT1 was established, followed by treatment with curcumin at different concentrations. The effect of curcumin or si-MALAT1 alone and the combination of the two on the cell activity was detected by MTT assay. The cell invasion and migration abilities were detected by transwell and scratch-wound assay. The relative expression level of MALAT1 was detected by RT-qPCR. The protein expression was determined by Western blot analysis. The IC50 of curcumin alone was 77.69 mmol/L, which was 51.17 mol/L when combined with curcumin and random sequence. The IC50 of curcumin was 30.02 mmol/L when combined with si-MALAT1. The increased susceptibility multiples was 2.58. The wound healing rates were 30.9% and 67.5% after treatment with si-MALAT1 combined with curcumin for 24 hrs and 48 hrs, respectively. The numbers of invasion cells were 200±12, 162±13, 66±8, 53±4 and 16±3 after treatment with si-MALAT1 combined with curcumin for 48 hrs. The relative expression level of lncRNA-MALAT1 in the curcumin group was 68%, and the relative expression level of lncRNA-MALAT1 in si-MALAT1group was 56%, and that for the combination treatment group was about 21%. The protein expression levels of β- catenin, c-myc and cyclinD1 were significantly down-regulated upon treatment with certain concentration of si-MALAT1 alone or combined with curcumin.si-MALAT1 could significantly inhibit the invasion and migration of SW480 cells by enhancing the sensitivity of SW480 cells to curcumin. The mechanism involved mignt be related to the down-regulation of β-catenin, c-myc and cyclinD1 proteins.
Subject(s)
Cell Migration Inhibition/drug effects , Colonic Neoplasms , Curcumin/pharmacology , Neoplasms/prevention & control , RNA , RNA, Small Interfering/drug effectsABSTRACT
Background Interleukin-1β (IL-1β) is an important inflammation-related factor in the initial stage of proliferative vitreoretinopathy (PVR).The previous research showed that curcumin can inhibit IL-1 β-induced proliferation of rabbit retinal pigment epithelium (RPE) cells,but the anti-inflammatory mechanism and effect of curcumin are still undefined.Objective This study was to observe the migration of IL-1β-induced rabbit RPE cells,and evaluate the function and mechanism of inhibition of curcumin on IL-1β-induced inflammation of RPE cells.Methods Cultured rabbit RPE cells of generation 4 were used in this experiment.The cells were cultured in serum-free DMEM and 0,0.1,1.0 and 10.0 μg/L IL-1β were separately added in the medium for 24 hours.The expressions of cyclooxygenase-2 (COX-2) protein and mRNA in the cells were detected by Western blot and reverse transcription PCR to determine the optimal concentration of IL-1β.The cells were divided into IL-1β group and curcumin+IL-1β group,and 1.0 μg/L IL-1 or 1.0 μμg/L IL-1 β combined with 10 μg/ml curcumin was respectively added into the medium for 24,48 and 72 hours.The cells cultured by only serum-free medium served as the control group.Hematoxylin and eosin staining was conducted for the cells to count the number of cells migrating into the injured area under the optical microscope.The relative expression levels of COX-2 protein and mRNA in the cells were detected by Western blot and reverse transcription PCR,and the relative expression levels of nuclear factor (NF)-κBp65 and inhibitor of NF-κB-α (IκB-α) protein were also detected by Western blot assay.The expression intensity and location of NF-κBp65,IκB-α and COX-2 in the cells were detected by immunochemistry.Results RPE cells just isolated from the rabbit eyes were in round shape and abundant in melanin.The melanin significantly decreased in the fourth generations of RPE cells.The shape of cells became long and narrow,and net shaped distribution.Immunochemistry demonstrated the strong positive response of RPE cells for keratin (AE1/AE3).There were (31.93 ±1.21),(36.27±2.50) and (38.33±2.40) migratory cells in the control group after 24,48 and 72 hours respectively.The number of migratory cells increased to 45.73 ± 2.30,71.13 ± 1.92 and 80.60 ± 1.71 in the IL-13 group,but obviously decreased to 13.13 ± 2.20,14.93 ± 1.10 and 12.60 ± 1.51 in the curcumin + IL-1β group.A Significant increase in the migrating cell number was found in the IL-1 β group compared with the control group and the curcumin+IL-1β group in various time points (all at P＜0.05).The relative expression levels of COX-2 protein and mRNA peaked in the 1.0 μg/L IL-1β group,so 1.0 μg/L of IL-1β was determined as the optimal concentration in the experiment.In 24,48 and 72 hours after culture,the expression levels of COX-2 protein and mRNA in the cells were significantly lower in the curcumin + IL-1β group than those in the control group (all at P＜0.05).The relative expression level reached peak in NF-κBp65 protein and lowed bottom in IκB-α proteins at 48 hours after cultured in the IL-1β group,and the reverse trend was seen in the curcumin+IL-1β group,with the significant differences between the two groups (both at P＜0.05).Immunochemistry showed that NF-κBp65 was expressed strongly in the cell nuclei and cytoplasm in the IL-1 β group and presented the weaker expression in the control group and the curcumin+IL-1 β group.Compared with the control group,the expression was weaker in IκB-α and stronger in COX-2 in the IL-1β group.In addition,the expression of IκB-α was enhanced and that of COX-2 was attenuated in the curcumin+IL-1β group in comparison with the IL-1β group.Conclusions Curcumin inhibits the movement of rabbit RPE cells induced by IL-1β.IL-1β up-regulates the expression of COX-2 by activating NF-κB signal pathway,and curcumin plays an anti-inflammatory role by blocking this pathway.
ABSTRACT
Objective To discuss the effects of curcuma on different phases of the Hela cell proliferation in order to find the effective medicine in cervix cancer treatment.Methods MTT colorimetry and flow cytometry were used to measure the inhibitory rate of Hela cell proliferation and the changes of cell cycle, and transmission electron microscope(TEM) was used to observe the changes of the Hela subcells treated with the different concentrations of curcuma(0,10,20 and(40 mg?L~(-1))).Results Curcuma(0,10,20 and 40 mg?L~(-1))had obvious inhibitory effects on the Hela cell proliferation in a dose-dependant manner,the inhibitory rates were 3.0%,21.4%,32.8% and 49.2%,respectively.Furthermore,flow cytometry showed that the number of cells in G_1 phase increased and the number of cells in S phase decreased,the number of cells in G_2/M phases relatively increased.The changes of subcell structure could be seen,such as cavernous cells,cytoplasm agglutination,increasing apoptosis.(Conclusion Curcuma) can inhabit the Hela cell proliferation, prevent the cells in G_1 phase from entering into(S phase),and promote Hela cell apoptosis.
ABSTRACT
Objective To investigate the preventive effects of curcumin for status epilepticus(SE) induced by lithium chloride-pilocarpine.Methods Totally 45 Sprague-Dawley(SD) rats were randomly divided into three groups: preventive group(n=15),non-preventive group(n=15),and control group(n=15).The latency peroid and incidence of SE were recorded.The surviving neurons were stained by using nissl staining,and the programme death cells were detected by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) in hippocampal CA3.Results The SE incidence of preventive group was 66.7%,which was significantly lower than that of non-preventive group(P0.05).Conclusion Pretreatment of curcumin can prevent the SE induced by lithium chloride-pilocarpine and the pretreatment can not protect the neuron.
ABSTRACT
【Objective】To observe the effect of different active ingredients from Chinese herbal medicine on interleukin 8(IL-8) secretion and Toll-like receptor 4(TLR4) mRNA expression in human intestinal tumor cell(HT-29)line.【Methods】Reverse transcription polymerase chain reaction(RT-PCR) was used to detect TLR4 mRNA expression,and enzyme-linked immunosorbent assay(ELISA) was used to examine IL-8 secretion.【Results】Astragaloside and emodin inhibited TLR4 mRNA expression stimulated by interferon ?(IFN-?),and emodin decreased IL-8 secretion stimulated by IFN-? and lipopolysaccharide(LPS).However,curcumin,naringin and jasminoidin had no effect on TLR4 mRNA expression.【Conclusion】The anti-inflammatory cellular mechanism of emodin may be related to the inhibition of TLR4 mRNA expression stimulated by IFN-?,and to the inhibition of IL-8 secretion stimulated by IFN? and LPS in HT-29 cell line.