The effects of TUG-891 on MG-63 osteosarcoma cell viability

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posted on 2022-10-19, 13:48 authored by Cynthia SitholeCynthia Sithole, Abe KasongaAbe Kasonga, Iman van den Bout

Cell viability using resazurin assay

Figure 1: TUG-891 showed no significant effect on cell viability at the tested concentrations. However, Triton X-100, as expected significantly decreased (P < 0.0001) cell viability compared to medium containing DMSO (V+).The medium only cells(V-) also showed a significant decrease (P < 0.05) in cell viability relative to V+. ANOVA analysis was done with Bonferroni correction.

Osteoblast differentiation using ALP activity

Figure 2: TUG-891 significantly increased ALP activity when compared to osteogenic medium-contaning (OM+) cells at day 7 (P<0.01) and day 21 (P<0.0001) for 100 µM concentration. At day 14, there was no significant changes in ALP activity in all the tested TUG-891 concentrations compared to the OM+ cells. OM- = negative control cells without osteogenic media. ANOVA analysis was done with Bonferroni correction.

Osteoblast mineralisation using Alizarin red staining

Figure 3: Photomicrographs of alizarin red stained MG-63 cells. The cells were stained with 2% (w/v) of alizarin red S stain and left to dry overnight, and photographs were taken (scale bar: 200µm). There were not many discrepancies between the baseline (OM+) and negative control (OM-) on days 7-21. In the cells exposed to TUG-891 however, a smaller number of stained granules were visible compared to the OM+ cells. Staining appeared to be more intense on day 7 with the stain appearing to diminish on days 14 and 21.

Figure 4: There were no significant differences in the amount of staining between TUG-891 and OM+ cells at day 7 of exposure. However, after 14 and 21 days, there was a significant decrease (P<0.0001) and (P<0.001) respectively, in staining for the cells exposed to 100 µM concentration of TUG-891 compared to the OM+ cells. The amount of staining was measured relative to OM+ at 405 nm wavelength. ANOVA analysis was performed with Bonferroni correction.

Osteoblastic gene expression using qPCR

Figure 5: After 7 or 14 days, no significant differences were seen between the expressions of Runx2, Osx, ALP, BSP, OPG and RANKL in OM+ cells compared to the OM- cells. Furthermore, TUG-891 did not show any significant differences in gene expression in any of the genes tested when compared to the OM+ cells. However, TUG-891 showed an increasing trend in gene expression of Runx2, Osx, ALP, BSP and RANKL when compared to OM- except for OPG expression after day 7. TUG-891 revealed a decreasing trend in gene expression for all the tested genes compared to OM+ cells after 14 days. Data was analysed using 2-ΔΔCT method with β-actin used as a housekeeping gene.

ERK and AKT protein expression using western blotting

Figure 6: TUG-891 decreased the relative fold change after 4 hours in the first repeat but increased the change in the second repeat. In the first repeat, only TUG-891 revealed the relative fold change over the total ERK signal after 60 minutes. At 24 hours, OM- showed higher fold change compared to OM+, and OM+ compared to TUG-891 (Figure 6aii). In the second repeat, the relative fold change over the ERK signal could not be observed after 60 minutes or 24 hours except for OM+ at 24 hours.GAPDH was used to normalise the data. R1= repeat 1, R2= repeat 2.

Figure 7: Both repeats revealed similar trends in the relative fold change over total AKT signal across the different time points. A peak was seen after 4 hours. OM+ showed higher relative fold change compared to the OM- cells in both repeats across the different time intervals except after 60 minutes in the second repeat. TUG-891 increased the fold change across the different time points in both repeats compared to OM+ except after 4 hours in the second repeat.

Silencing GPR120 using western blotting

Figure 8: The relative fold change over the total ERK in the OM+ cells decreased compared to OM- cells in the first repeat for the control and GPR120 siRNA cells whereas it increased in the second repeat. The GPR120 siRNA did not appear to inhibit TUG-891 in the GPR120 siRNA cells compared to the control siRNA cells in both repeats. GAPDH was used to normalise the data. PD= PD184352 an ERK inhibitor.

Figure 9: Again, similar to ERK, the relative fold change over the total AKT showed different trends for the two repeats. In both repeats, the relative fold change over AKT in OM+ cells appeared lower than the OM- cells. TUG-891 showed an increased trend in the relative fold change over the total AKT in the control siRNA cells compared to GPR120 siRNA cells in both repeats but the GPR120 siRNA appeared not to be effective.