Bioactive Hexapeptide Reduced the Resistance of Ovarian Cancer Cells to DDP by Affecting HSF1/HSP70 Signaling Pathway

Abstract

Ovarian cancer is the leading cause of death in gynecologic malignancies. Ovarian cancer as a metastatic malignant tumor is highly recurrent and prone to drug resistance. Bioactive peptides are an emerging area of biomedical research in reducing resistance of tumor cell to drugs. In this paper, we investigated the effects and mechanisms of bioactive hexapeptide (PGPIPN) derived in milk protein on the sensitivity of ovarian cancer cells to cis-dichlorodiammine platinum (DDP). Human ovarian cancer cell lines (SKOV3 and COC1), their DDP-resistant sublines (SKOV3/DDP and COC1/DDP) and human primary ovarian cancer cells were cultured in vitro under the combined treatment of DDP (close to IC50) and different concentrations of PGPIPN. The viabilities, apoptosis and cell cycle changes were respectively measured by WST-8 and flow cytometry. The mRNA and protein expression levels of HSF1, HSP70, MDR1, ERCC1 and β-actin gene were respectively assayed by RT-qPCR and western blotting. The results showed that PGPIPN significantly increased the sensitivity of human ovarian cancer cells to DDP in inhibiting viability and inducing apoptosis in vitro. But the effects in sensitive cells were lower than DDP-resistant cells. PGPIPN significantly changed the cell cycles in all human ovarian cancer cells, which leaded to a significant increase in the percentage of cells blocked at G2/M phase and decrease the percentage of cells at G1 phases in a dose-dependent manner. PGPIPN affected the expression levels of HSF1, HSP70, MDR1 and ERCC1 genes. Compared with cells in DDP treatment alone, the expression levels of HSF1 and HSP70 in human ovarian cancer cells treated with DDP and PGPIPN together significantly decreased in dose-dependent manner. PGPIPN significantly decreased MDR1 and ERCC1 of drug-resistant ovarian cancer cell lines and human primary ovarian cancer cell in a dose-dependent manner. Pifithrin-μ (PFTμ, HSP70 inhibitor) decreased or removed the effects of peptide in increasing the sensitivity of ovarian cancer cells to DDP. This suggests that PGPIPN enhanced the sensitivity of ovarian cancer cells to DDP partially via reducing the activity of HSF1/HSP70 signaling pathway, thus inducing cell apoptosis and decreasing repairment of DNA damage.

Keywords: apoptosis; bioactive hexapeptide; cell cycle; drug resistance; human ovarian cancer; signaling pathway.

Figure 1

The culture and identification of human primary ovarian cancer cells. (A) Pathological section of normal human ovarian tissue with benign pathology (H&E stained, ×40). (B) Pathological section of human ovarian cancer tissue (H&E stained, ×100) that was classified as serous ovarian adenocarcinoma (IV grade) according to WHO criteria. (C) Human primary ovarian cancer cells subcultured in medium (H&E stained, ×200). (D) Cultured human primary normal ovarian glandular epithelium cells (NOGECs) stained with nuclear dyes-Hochest 33258 (×100). (E) Cultured human primary NOGECs stained with anti-cytokeratin 19-FITC (×100). (F) The confocal of D and E pictures (×100). (G) Cultured human primary ovarian cancer cells stained with nuclear dyes-Hochest 33258 (×100). (H) Cultured human primary ovarian cancer cells stained with anti-cytokeratin 7-FITC (×100). (I) The confocal of G and H pictures (×100).

Figure 2

PGPIPN increased the sensitivity of human ovarian cancer cells to DDP in the inhibition of cell viability in vitro, of which the effect of resistant cells were better than that of sensitive cells. Data are mean ± SD, the experiment in cell lines were performed in triplicate, and the experiments were duplicated with primary ovarian cancer cells from six patients. ^*P<0.05, ^**P<0.01 compared with human ovarian cancer cell lines, human primary ovarian cancer cells or human primary normal ovarian cells intervened by DDP alone in the same time.

Figure 3

PGPIPN increased DDP-induced apoptosis in human ovarian cancer cells for 48 h in vitro. (A) Representative flow cytometry dot plot of human ovarian cancer cells stained with Annexin-V-FITC and PI. (B) Histogram of apoptosis rates of lines (SKOV3, COC1) and their DDP-resistant sublines (SKOV3/DDP, COC1/DDP). (C) Histogram of apoptosis rates of human primary ovarian cancer cell. The data are shown as means ± SD, the experiment in cell lines was performed in triplicate, and the experiments were duplicated with primary ovarian cancer cells from six patients, ^*P<0.05, ^**P<0.01 compared with cells in DDP treatment alone.

Figure 4

PGPIPN induced G2/M-phase accumulation of human ovarian cancer cells under combining drugs for 48 h. (A) Representative cell cycle profiles by flow cytometry. (B) The histograms were analyzed by Flowjo software to display the cell cycle distribution. The data are shown as means ± SD, the experiment in cell lines was performed in triplicate, and the experiments were duplicated with primary ovarian cancer cells from six patients, ^*P<0.05, ^**P<0.01 compared with cells in DDP treatment alone.

Figure 5

PGPIPN regulates the mRNA expression levels of HSF1, HSP70, MDR1 and ERCC1. β‑actin was used as the reference gene. Data are presented as the mean ± SD (cell line: n=6; primary ovarian cancer cell: n=12). ^*P<0.05 and ^**P<0.01 vs. human ovarian cancer cells in DDP treatment alone.

Figure 6

PGPIPN regulated the contents of proteins associated with HSF1/HSP70 signaling pathway in human ovarian cancer cell lines and primary ovarian cancer cells.SKOV3 and its DDP-resistant subline SKOV3/DDP. (A) The related proteins were detected with western blotting in SKOV3 and SKOV3/DDP, and β-actin was used to show the similar amount of protein loaded in different lanes. (D) The related proteins were detected with western blotting in COC1 and COC1/DDP, and β-actin was used as the reference protein. (G)The related proteins were detected with western blotting in human primary ovarian cancer cells, and β-actin was used as the reference protein. (B, C, E, F and H) Relative intensities of protein bands in A, D and G were determined using Quantity-One software and normalized using β-actin band intensity. Data in B, C, E, F and H are presented as mean ± SD (cell line: n=6; primary ovarian cancer cell: n=12). ^*P<0.05, ^**P<0.01 vs. cells in DDP treatment alone.

Figure 7

HSF1/HSP70 signaling pathway inhibitors reduced the effect of PGPIPN. (A) SKOV3 and DDP-resistant subline SKOV3/DDP. (B) COC1 and DDP-resistant subline COC1/DDP. Data are mean ± SD, the experiment was performed with sextuplicate in three independent sets, ^*P<0.05, ^**P<0.01 compared with DDP intervention alone in the same cell line; ^#P<0.05, ^##P<0.01 compared with DDP and PGPIPN synergistic intervention in the same cell line.