The involvement of small heat shock protein in chemoresistance in ovarian cancer - in vitro study

Abstract

Ovarian cancer is the most deadly gynecologic malignancy worldwide. Although the primary response to chemotherapy is high, the majority of patients will develop resistance against applied treatment. In this study, we focused on resistance to cisplatin, a first-line drug used for the treatment of ovarian cancer. The mechanism of the resistance development process is widely described, but there is a lack of information about the involvement of members of small heat shock proteins (HSPs) and their transport via exosomes. In this study, we used two cell lines: A2780 and SKOV3, and their cisplatin-resistance variants: A2780 CDDP and SKOV3 CDDP. We have shown that the expression of three small HSPs (HSPB5, HSPB6, and HSPB8) in cisplatin-resistant cell lines differs from their sensitive counterparts. Further, we isolated exosomes and determined the small HSPs in their cargo. In A2780 WT we observed a low amount of HSPB5 and HSPB6. We did not observe the expression of small HSPs in the SKOV3 cell line in both sensitive and resistant variants. Our data suggest the involvement of small HSPs in drug resistance of ovarian cancer and their presence is not related to exosomal transport. Analysis of the biological consequences of the imbalance of small HSPs expression in cisplatin resistance needs further investigation.

Keywords: chemoresistance; exosomes; ovarian cancer; small HSPs.

Table 1. Detailed information about antibodies used in the analysis

Figure 1. The expression of small HSPs in cisplatin-resistant OvCa cell lines differs from their sensitive counterparts. (a, b) RT-qPCR analysis of mRNA levels of HSPB5, HSPB6, and HSPB8 in both A2780 and SKOV3 cell lines, and their sensitive and resistant variants. Relative expression levels of genes were normalized using GAPDH. The bars represent a mean ± SD expression of transcript (n=3). *: p < 0.05; **: p < 0.01; ***: p < 0.001; **** - p by Welch's t-test. (c, d) Western blot evaluation of expression of HSPB5, HSPB6 and HSPB8 in whole cell lysates derived from A2780 and SKOV3 cell lines and their sensitive and resistant variants with semiquantitative analysis of signals intensity, normalized to level signal of housekeeping gene - GAPDH. The bars represent a mean ± SD expression of proteins. *: p < 0.05; **: p < 0.01; ***: p < 0.001; **** - p by Welch's t-test. WT- Wild Type, CDDP - cisplatin-resistant variant, NS - non-significant

Figure 2. Characteristics of isolated exosomes from serum-deprived conditioned cell culture medium from sensitive and resistant to cisplatin OvCa cell lines and determination of small HSPs in their cargo. (a) The evaluation of cholinesterase activity in exosomes (5 µg) derived from cell culture of studied OvCa cell lines, PBS (diluent) was used as control. The bars represent mean ± SD from duplicate; (b) The representative Western blots describing phenotype of isolated exosomes from sensitive and resistant to cisplatin A2780 and SKOV3 cell lines conditioned culture medium. LAMP1 and Alix were used as positive control, which represents the endosomal origin of isolated particles and CNX served as a negative control, which is a protein specific for endoplasmic reticulum. The whole cell lysate was obtained from cells cultured in serum-free culture medium. The values below the detected bands determine the level of expression of tested proteins, which was normalized to the intensity of the total amount of protein of membranes stained with Ponceau; (c) Western blot analysis of isolated exosomes from sensitive and resistant to cisplatin A2780 and SKOV3 cell lines for HSPB5, HSPB6, and HSPB8. The whole cell lysate was obtained from cells cultured in serum-free culture medium.The values below the detected bands determine the level of expression of tested proteins, which was normalized to the intensity of the total amount of protein of membranes stained with Ponceau; (d) A representative picture of PVDF membranes stained with Ponceau, which indicated loading control of the same amount of protein. The band profile of cell lysates and exosomes is different. WT- Wild Type, CDDP - cisplatin-resistant variant, M - molecular protein marker, ND - not detected