| ObjectivesOsteosarcoma is the most common primary bone tumor in children and adolescents, with a5-year disease-free survival rate of70%. Neoadjuvant chemotherapy accompanied by large doses of doxorubicin (DXR) has greatly improved the survival rate, yet20-40%of the patients still die of metastasis and recurrence. The primary cause of treatment failure is the resistance of tumor cells to chemotherapeutic medicine. Multidrug resistance (MDR) is mainly attributed to the increased drug efflux mediated by the P-glycoprotein (P-gp) product of the multidrug resistance protein1(MDR1) gene. P-gp is a ATP-dependent transmembrane transporter that acts as a drug efflux pump to decrease intracellular drug accumulation and consequently reduce intracellular drug efficacy. Compounds such as verapamil have been reported to overcome MDR in vitro. However, these compounds have side effects that hinder their clinical applications. Recently, several researchers have focused on screening for natural product-derived drugs to reverse MDR Diallyl trisulfide (DATS) is the main sulfur-containing compound in garlic. Engdal et al proposed that garlic compounds can inhibit the expression of P-gp in vitroand in vivo. The capacity of DATS to reverse the drug resistance of osteosarcoma cells is still unknown. In the present study, we aimed to determine the effect of DATS on the reversal of drug resistance in human osteosarcoma cells in vitroand to investigate its potential mechanisms, of action. The study also explored whether the suppression of the nuclear factor κ light-chain enhancer of activated B cells (NF-κB) is involved in the DATS-induced inhibition of osteosarcoma cell proliferation. Content and MethodsIn vitro drug sensitivity assay.U2-OS cells were seeded at1×104cells/ml per well into a96-well plate. After24h, the medium was replaced with DMEM supplemented with DATS at a dose of10,50and100μM. After treatment for20,44and68h,20μl3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added into each well, and the cells were further incubated for4h. The medium was then removed, and150μl DMSO was added to each well to dissolve the formazan crystals. The absorbance (A) of each sample was measured using a spectrophotometer at a wavelength of570nm. Inhibition of cell viability=[1-(average A value of the experimental group/average A value of the control group)] x100%.Group:①U2OS+ADM (lug/ml)②U2OS+ADM (lug/ml)+DATS (10umol/L) After incubated24,48,72hours, MTT was used to test the survival rate.Expression of P-gp by flow cytometry (FCM)The U2-OS cells were cocultured with10,50and100μM DATS for48h. The cell suspensions were incubated with phycoerythrin-conjugated UIC2, mouse anti-human P-gp monoclonal antibody (P-gp-PE), and the homotype control IgG2a-PE, and then detected by FCM. The results were analyzed using Cell Quest software (BD Pharmingen Co., USA).Morphological changes by light microscopyU2-OS cells were grown in complete DMEM for24h on24-well plates that had a coverslip at the bottom of each well. When a cell density of1×104cells/ml was reached, the cells were treated with either50μM DATS alone or10μM DATS combined with1μg/ml ADM and incubated for another48h. Treatment with1μg/ml ADM alone was applied to the control group. The coverslips were removed from each well, and stained with hematoxylin and eosin (H&E), and observed under a light microscope.Apoptosis assay by statistical FCMAfter incubation at37℃for24h in DMEM with the different drug doses (i.e.,10,50and100μM DATS or10μM DATS with1μg/ml ADM or1μg/ml ADM alone), the cell labeling was performed using annexin V conjugated to FITC. The fluorescence intensities of the samples were measured by a flow cytometer with the FACS software (BD Pharmingen Co.).Semi-quantitative RT-PCR assayAfter treatment with the drugs (50μM DATS)for48hours, the total mRNA was extracted from the cells with the TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) according to the manufacturer’s instructions. The following cycling conditions were used for each PCR run:initial denaturation at94℃for1min, followed by26cycles of58℃for1min and72℃for1min, and then the final extension at72℃for7min. The products were further analyzed using the UVP bioimaging system (UVP, Upland, CA, USA), with β-actin as the internal reference.Protein expression by western blot analysisAfter treatment with the drugs (50μM DATS) for48hours, the total protein content was isolated and gel electrophoresis by SDS-PAGE.After transfer membrane, the blots were incubated with the rabbit anti-human NF-κB (p65) or the mouse anti-human IκBα primary antibodies, according to the corresponding level of β-actin expression, the protein expression levels were determined by densitometry scans and calculated using Quantity One software (Bio-Rad Co., USA).Statistical analysisThe results are expressed as the means±standard deviation (SD) of three independent experiments. The Student’s t-test was used for the statistical analyses, and P<0.05was considered to be significant. Statistical calculations were carried out using the Student’s t-test with SPSS13.0for Windows software package.ResultsDrug sensitivity.Treatment of U2-OS cells with DATS resulted in the inhibition of cell viability in a dose-and time-dependent manner. The survival rate of the U2-OS cells significantly decreased to68.19,51.9and42.44%at24,48and72h, respectively, after treatment using10μM DATS with1μg/ml ADM as compared with treatment using ADM alone (85.21,76.04and59.37%at24,48and72h, respectively; P<0.05).Alteration of P-gp expressionAfter treatment with different concentrations of DATS (10,50and100μM), the P-gp expression in the U2-OS cells was significantly decreased to4.91,4.28and3.94, as compared with the untreated group (6.4; P<0.01)Apoptosis as observed by light microscopyAfter treatment with50μM DATS, proliferation of the U2-OS cells was reduced, and the cells shrank into rounded shapes with abundant cytoplasm and vacuoles, chromatin condensation and margination, nuclear fragmentation, apoptotic bodies, as well as other typical apoptotic features. However, the proliferation of the U2-OS cells treated with ADM (1μg/ml) alone was slightly inhibited and apoptotic cells were rare. The apoptotic cytomorphological features appeared at24h after simultaneous treatment with ADM (1μg/ml) and DATS (10μM).Apoptosis assay by statistical flow cytometryThe percentages of early apoptotic cells were significantly increased at24h after treatment with10,50and100μM of DATS as compared with that of the controls. Moreover, the percentage of apoptotic U2-OS cells treated with both ADM and DATS was much higher than the percentage of apoptotic cells treated with ADM alone.Detection of NF-κB and IκB expressionSemi-quantitative RT-PCR revealed the gray value of NF-KB/p65expression to be100±9.21. After treatment with50μM DATS, an evident decrease in the NF-κB/p65levels was observed (86±5.57, P<0.05). Conversely, the IκBα mRNA expression in the U2-OS cells treatment with50μM DATS was increased as compared with that of the control group (27±4.80vs.41±5.94, P<0.05).Western blot analysis revealed that the NF-KB/p65protein expression was decreased by DATS (50μM) in the U2-OS cells from89.41±6.98to58.40±5.03, whereas the IκBα protein expression was increased from28.14±2.58to61.43±4.22(P<0.05).ConclusionThe present study demonstrated that DATS can serve as a novel modulator of MDR in vitro by downregulating P-gp expression and inducing the apoptosis of U2-OS cells. For the first time, we demonstrated that DATS blocks NF-κB activation, which subsequently produces downstream inhibitory effects on the sensitivity to chemotherapy and apoptosis of U2-OS cells. Thus, DATS could be a highly feasible candidate for the development of a new MDR reversal agent. |
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