SND1 protects the viability of the SKOV3 cells from cisplatin treatment.
The major treatment strategy for ovarian cancer is surgery and chemotherapy, and most chemotherapy agents are based on platinum. Therefore, here, we observed effects of SND1 on sensitivity to cisplatin in the ovarian cancer cell line, SKOV3. Previously, we have conducted the stable knockdown SND1 cells by shRNA lentivirus (sh1 and sh2) and stable over-expression SND1 cells with a Flag tag (Flag-SND1) in the SKOV3 cell line.
We firstly tested different concentrations of cisplatin to determine the cytotoxicity of the SKOV3 cells by MTT assay and found that the IC50 was approximately 10 µg/mL after 24 hours and 5 µg/mL after 72 hours treatment (Fig. 1A). We found that the viability of the SKOV3 cells was significantly inhibited by cisplatin in a time- and concentration-dependent manner.
To test whether SND1 increases the sensitivity of SKOV3 cells to cisplatin, we examined the SKOV3 cell viability through the clone formation assay. SKOV3 cells were seeded in 6-well plates at a density of 2000 cells/well and cultured with 5 µg/mL cisplatin for 72 hours and counted the clone number after cultured two weeks. We found that the viability of SKOV3 cells was slightly inhibited by knockdown SND1, especially under the cisplatin treatment (Fig. 1B and C). The cell viability was growing when overexpressed SND1 in SKOV3 cells even though with the cisplatin treatment (Fig. 1D and E). These data suggest SND1 may play a role in resistant chemotherapy of cisplatin.
SND1 suppresses cisplatin-induced apoptosis of SKOV3 cells.
Then we detected cell apoptosis using flow cytometry. The knockdown and over-expressed stable cells were treated with two concentrations of cisplatin (5 µg/ml and 10 µg/mL) for 72 hours and cell survival was determined by apoptosis assay. The apoptosis (UR quadrant) in high doses of cisplatin (10µg/ml) is more serious than in low doses (5 µg/mL) in all kinds of stable SKOV3 cells. SND1 deficient cells, both sh1and sh2, displayed more sensitivity to cisplatin than control cells (pLKO), especially at high concentrations; Only 1.97% of cells encountered apoptosis in normal pLKO, the values of apoptosis slightly increased to 4.53%, and 4.42%, respectively, in the treatment with cisplatin by 5 µg/mL and 10 µg/mL (Fig. 2A and B). And while over-expression of SND1 cells showed greater resistance to cisplatin than control cells (IRES) (Fig. 2D and E). The effect on apoptosis was confirmed by testing the expression status of apoptotic proteins such as caspase-3 and cleaved PARP. The results showed that cisplatin treatment alone slightly increased the level of apoptotic proteins in SKOV3 cells while knockdown of SND1 caused markedly increase expression of active caspase-3, and formation of cleaved PARP (Fig. 2C). At the same time, we determined the expression of apoptotic proteins in SND1 over-expression cells. Both cleaved PARP and cleaved caspase-3 were increased after treatment of cisplatin, while over-expression of SND1 could suppress cisplatin-induced apoptotic proteins’ expression (Fig. 2F). These data indicate the enhancement of cisplatin resistance in SKOV3 cells by SND1 may be mediated through the induction of apoptosis via a caspase-dependent mechanism and by suppression of the cisplatin-induced apoptotic protein expression.
GAS6/AKT signaling pathway was involved in the resistance of SND1 to cisplatin.
To understand the molecular mechanism of the chemo-resistance effect of SND1 in cisplatin-induced SKOV3 apoptosis. We screened our previous expression profile chip in SKOV3 cells of stably knockdown SND1. We found that knockdown of SND1 affected the expression of more than a dozen genes related to chemotherapy sensitivity, including AKT, GAS6, SFRP1, and BCL2. Real-time quantitative PCR was used to confirm the correlation between SND1 and the potential genes' expression in two shRNA knockdown cells and over-expression cells (Fig. 3A and B). The results showed that only the expression of GAS6 was the same as that of SND1 in both knockdown and over-expression cells. However, the expression trends of other genes such as AKT and BCL2 are not completely consistent with SND1.
Next, we examined SND1 and GAS6 expressions were both significantly decreased under cisplatin treatment (Fig. 4A and B). While cisplatin-induced GAS6 decreasing expression was much more serious in down-regulated SND1 cells, that was some degree of relief in SND1 over-expression cells. Furthermore, western blotting results showed the same protein expression pattern between SND1 and GAS6 under cisplatin treatment. As previous studies have proved GAS6 could activate the phosphorylation of AKT during chemotherapy, especially cisplatin. Therefore, we detected AKT and p-AKT in our model. Total AKT was a consistently stable expression upon treatment with cisplatin when SND1 expression in SKOV3 cells was down-regulated or up-regulated. However, p-AKT was severely decreased when SND1 was depleted (Fig. 4C, lanes 1, 4, and 7), and that was significantly increased in SND1 up-regulated cells (Fig. 4D, lanes 1 and 4). Cisplatin treatment drastically enhanced this expression type (Fig. 4C, lanes 2–3, 5–6, and 8–9; D, lanes 2–3, and 5–6). These data suggest SND1 may involve in resistance to cisplatin-induced apoptosis via regulating GAS6/AKT signaling pathway.