Down regulation of fatty acid synthase via inhibition of PI3K/AKT/mTOR in ovarian cancer cell line by novel organoselenium pseudopeptide

Abstract

Ovarian cancer (OC) is the 7th most common cancer in women world-wide and the 3rd most common female cancer. For the treatment of OC, there is no successful therapeutic. The medications that are currently available have significant side effects and a low therapeutic index. This work aimed to evaluate the anticancer activity of organoselenium pseudopeptide compound against OC cell lines. After treatment with 50 ​μM of compound 4 (CPD 4), the viability was determined. The anticancer activity was further investigated by different methods including cell cycle and apoptosis analysis, colony formation assay, zymography, comet assay and Western blot. In comparison to a positive control, compound 4 showed cytotoxicity toward A2780CP cells rather than A2780 and SKOV-3 ​cells. Compound 4 was more selective to OC cells rather than HSF cells. Moreover, Compound 4 was able to inhibit cell migration and proliferation. The anticancer effect of compound 4 was found to be partially via cell cycle arrest, overexpression of p27 ​cell cycle inhibitor and induction of apoptosis through DNA fragmentation and activated production of ROS. Compound 4 had a differential effect on the modulation of PI3K/AKT/mTOR signaling pathway in the OC treated cell lines, also inhibited lipogenesis process via downregulation of FASN expression. Conclusion: This work highlights the unique role of Compound 4 against OC via modulation of oxidative stress, inhibition of survival PI3K/AKT/mTOR pathway. Compound 4 was found to be a promising alternative therapy for the treatment of OC in this investigation.

Keywords: DNA fragmentation Metalloproteniases; Lipogenesis; Ovarian cancer; PI3K/AKT/mTOR; Reactive oxygen species.

Graphical abstract

Scheme 1

Synthesis of ((diselanediylbis (4,1-phenylene)) bis (azanediyl)) bis(3-methyl-1-oxobutane-1,2-diyl) diacetate (compound 4).

Fig. 1

(A) Cytotoxicity of compound 4 on ovarian cancer cells, sensitive cells (A2780), resistant cells (A2780CP) and SKOV-3 ​cells were incubated with compound 4 for 48 ​h and as a negative control, DMSO was used. MTT assay was used to determine the cell viability. (B) Proliferation activity of compound 4 against ovarian cancer cells was determined by incubation of cells with compound 4 for 4 days. The cell viability was determined by MTT assay. Biotek plate reader (Gen5™) was used to measure the absorbance at λ_{570- 630}.

Fig. 2

(A) Effect of compound 4 on wound healing of ovarian cancer cells. The wound healing rate was monitored at zero time and 48 ​h. Image J software was used to measure the size of the wound. Results reveled that compound 4 inhibited cell migration and prevented cells from metastasis. (B): One-Way ANOVA was used to analyze data and significance was ∗∗∗∗P ​< ​0.0001.

Fig. 3

(A): The images showed the inhibitory effect of compound 4 on ovarian cancer cells through decreasing the number of colonies after treatment in comparison to negative control (DMSO). (B): One-Way ANOVA was used to analyze data and significance ∗∗∗∗P ​< ​0.0001.

Fig. 4

(A): Inhibition of MMP-2 and MMP-9 after compound 4 treatments. Serum starved SKOV-3 ​cells were seeded as described in the methods section, and then treated for 48 ​h with (½ IC₅₀ and IC₅₀) of compound 4. Media were collected following treatment and total protein content was analyzed by BCA assay then gelatin zymography was performed. (B): Statistical significance was indicated: ∗∗∗∗p ​< ​0.0001.

Fig. 5

(A): The effect of compound 4 treatment on cell cycle phases for cancerous cells. (B): Represent the number of cells in each phase of cell cycle.

Fig. 6

(A) Effectiveness of compound 4 treatment on the expression level of p27, cells were treated with IC₅₀ value of compound 4 as mentioned above for 48 ​h. As a loading control, glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used. (B): One-Way ANOVA was used to analyze the data, and the significance was ∗∗∗∗P ​< ​0.0001.

Fig. 7

(A): Induction of apoptosis in ovarian cancer cells as a result of compound 4 treatment. (B): Represent the number of the cells in each apoptotic phase.

Fig. 8

Effect of compound 4 treatment on DNA strand breakage. Ovarian cancer cells were treated with compound 4 for 48 ​h live cells were spread on a frosted microscope slide. After many steps mentioned in material and method section, Olympus PEN Lite E-PL3 camera connected with Olympus BX43 was used to take pictures.

Fig. 9

(A): Compound 4 causes intracellular ROS production in ovarian cancer cells. For 30 ​min, cells were loaded with 20 ​μM DCFDA stain and then treated with IC₅₀ value of compound 4 mentioned above and incubated for additional 4 ​h. ROS production was determined by gauging the level of H₂O₂ by flow cytometry BD Accuri C6 Plus DCF fluorescence intensity. (B): One-Way ANOVA was used to analyze the data and significance was ∗∗∗∗P ​< ​0.0001.

Fig. 10

(A): Western blot data showed decrease or increase in proteins SKOV-3, A2780 and A2780CP were treated with the IC₅₀ value of compound 4 for 48 ​h. The cells were lysed in RIPA buffer containing phosphatase and protease inhibitor. BCA assay was used to determine protein concentration. 30 ​μg of protein were loaded in the gel. Proteins were transferred onto nitrocellulose membrane and subjected to the primary antibodies overnight at 4 ​°C then incubated with HRP-conjugated secondary antibody. As a loading control GAPDH was used. The level of change in proteins expression was normalized to the amount of GAPDH. (B): Statistical analysis of data was calculated by One-way ANOVA and significance was ∗∗∗∗P ​< ​0.0001 and ∗∗∗P ​< ​0.0006.