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. 2024 Nov 9;14(1):27309.
doi: 10.1038/s41598-024-76135-0.

Auxiliary effect of trolox on coenzyme Q10 restricts angiogenesis and proliferation of retinoblastoma cells via the ERK/Akt pathway

Shikha Upreti  1 , Prachi Sharma  2 , Seema Sen  3 , Subhrajit Biswas  2 , Madhumita P Ghosh  4   5
Affiliations

Auxiliary effect of trolox on coenzyme Q10 restricts angiogenesis and proliferation of retinoblastoma cells via the ERK/Akt pathway

Shikha Upreti et al. Sci Rep. .

Abstract

Reactive oxygen species (ROS) are essential for cancer signalling pathways and tumour maintenance, making ROS targeting a promising anti-cancer strategy. Coenzyme Q10 (CoQ10) has been shown to be effective against various cancers, but its impact on retinoblastoma, alone or with trolox, remains unreported. Cytotoxicity of CoQ10 alone and with trolox was evaluated in normal human retinal pigment epithelium cells (ARPE-19) and Y79 retinoblastoma cells using CCK-8. Flow cytometry was used to assess apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Anti-angiogenic potential was tested using human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM) assays. Mechanistic studies were conducted via RT-PCR and western blotting. CoQ10, alone and with trolox, reduced Y79 cell viability, induced apoptosis through excess ROS generation, and decreased MMP significantly. Both treatments caused G2/M phase cell arrest. The CAM assay showed a significant reduction in endothelial cell proliferation, evidenced by fewer number of co-cultured HUVECs when exposed to CoQ10 or CoQ10 with trolox. The combination of CoQ10 and trolox significantly reduced VEGF-A, ERK, and Akt receptor levels, while CoQ10 alone significantly inhibited ERK and Akt phosphorylation. Together, CoQ10 and trolox reduced protein expression of VEGFA. CoQ10 alone and with trolox, induces apoptosis in Y79 retinoblastoma cells by inhibiting the ERK/Akt pathway and downregulating VEGFA. This study is the first to report the in vitro and in-ovo anti-cancer potential of CoQ10 alone or when combined with trolox, on human retinoblastoma Y79 cells.

Keywords: Anti-cancer; CoQ10; ERK/Akt inhibition; Rb cells; Trolox.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Representative data from in-vitro assay of cell viability showing effects of CoQ10 alone, trolox and combination of CoQ10 and trolox on cell lines. (a) CoQ10 at concentrations of 50µM and 30 µM trolox were safe on ARPE-19 cells and produced no toxicity. (b) The combination index value was found to be 0.9 which indicates towards the synergistic activity of CoQ10 with trolox. (c) Representative images of cell death induced in Y79 cells when cultured in serum-supplemented RPMI media (control), trolox, CoQ10 alone and combination of CoQ10 with trolox. A significant decrease in cell number by both CoQ10 alone as well as CoQ10 with trolox was observed in Y79 cells. (d) The dose response curves of CoQ10 and trolox indicated that CoQ10 reduced Y79 population by 50% at IC50 of 5.04 and trolox at IC50 of 3.19. (e) The combined treatment value of CoQ10 and trolox was determined to be 30 µM each for Y79 cells. Each bar represents mean ± SEM where n = 3. *p < 0.05 vs. control. DMSO = dimethylsulfoxide. Scale bar- 500 μm.
Fig. 2
Fig. 2
Representative images showing the results of colony formation assay in Y79 cells. A significant decrease in the descending order was observed in the groups of trolox (p < 0.05*), CoQ10 (p < 0.05*) and CoQ10 combined with trolox (p < 0.01**) when compared to control respectively. Each bar represents mean ± SEM where n = 3. Scale bar- 500 μm.
Fig. 3
Fig. 3
Representative images of estimation of Y79 cells by flow cytometry for generation of ROS. The percent ROS generating cells were 64.6% in control and which was marginally increased to 67% cells by trolox (a). However, the percentage increase in ROS generating cells was significant when treated with CoQ10 alone (71%, p < 0.05*) and by CoQ10 with trolox (88.4%, p < 0.01**); x axis = fluorescence intensity and y axis = count of cells (b). Histogram representing changes in MMP in Y79 cells (c). Both the doses of CoQ10 alone and CoQ10 with trolox increased ROS generation in Y79 cells by resulting in depolarization of MMP which was evident in the cell counts. Even though MMP lowered in trolox group, significantly lowered levels could be seen only in the groups of CoQ10 alone (p < 0.05*) and CoQ10 combined with trolox (p < 0.01**) vs. control respectively (d). Each bar represents mean ± SEM where n = 3. x axis = rhodamine intensity and y axis = count percent.
Fig. 4
Fig. 4
A representative flow cytometric data for the analysis of cell cycle in Y79 cells. The accumulation of cells in the G2/M phase by treating with trolox (16.1%) increased marginally from the control group (13.9%). This percentage increased when cells were subjected to CoQ10 alone (20.5%) and CoQ10 combined with trolox (23.7%) after 48 h (a). A graphical representation of percentage of cells in the three stages of cell cycle (b). Each bar represents mean ± SEM where n = 3. *p < 0.05 and **p < 0.01 vs. control. x axis = treatment groups; y axis = percent cell cycle distribution.
Fig. 5
Fig. 5
The apoptotic stages of cells as demonstrated by Annexin V/PI assay in Y79 cells. The division of cells in the four apoptotic stages when exposed to treatment of trolox alone, CoQ10 alone and combination of CoQ10 with trolox (a). Graphical representation of quantification of cells showing significant deaths by trolox (p < 0.05*), CoQ10 (p < 0.01**) and CoQ10 with trolox (p < 0.01**) (b). Each bar represents mean ± SEM where n = 3. *p < 0.05 and **p < 0.01 vs. control. x axis = Annexin V; y axis = PI.
Fig. 6
Fig. 6
Representative images of co-culture of HUVECs with Y79 cells. The cell number of HUVECs in control group of HUVECs + Y79 remained unaffected after incubation of 48 h (a). Quantification of cell numbers which were significantly reduced by CoQ10 alone and with trolox (b). The appearance of tumour grafts on the CAM after inoculation of Y79 cells (4.3 × 105 cells) onto the chick CAM (c). Tumour observation and excision was done on ED12. Each bar represents mean ± SEM where n = 3. p < 0.05*, p < 0.01** vs. control. Scale bar- 500 μm.
Fig. 7
Fig. 7
Quantitative real-time RT-PCR analysis of (a) VEGF receptor A, (b) Akt, and (c) ERK receptor from control, CoQ10, Trolox and CoQ10 + trolox groups (n = 4, each group). The mRNA levels were normalized to GAPDH housekeeping gene. Each bar represents mean ± SEM where n = 3. *p < 0.05 vs. control.
Fig. 8
Fig. 8
The expressions of proteins produced by Y79 cells were analysed by western blotting. The blot of targeted proteins (a) showed that expression of targeted protein Akt was downregulated by both CoQ10 alone (p < 0.05*) as well as when combined with trolox (p < 0.05*) (b). A similar pattern was observed while examining the pAkt downregulation by both CoQ10 alone (p < 0.01**) and CoQ10 with trolox (p < 0.05*) (c). Interestingly, the expression of ERK protein was brought down by only CoQ10 alone (p < 0.05*) (d), with the phosphorylated ERK expression being lowered by both CoQ10 alone (p < 0.05*) and CoQ10 with trolox (p < 0.05*) almost equally (e). Also, estimated were the variations in levels of pro-angiogenic factor VEGFA (f) and it was found that only the combination of CoQ10 with trolox (p < 0.05*) could downregulate its expressions (g). Data is represented as mean ± SE from three different experiments. *p < 0.05 and **p < 0.01 vs. control.
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