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  • br Recently pharmacological inhibitors of the APC C called

    2021-03-03


    Recently, pharmacological inhibitors of the APC/C called proTAME and apcin [19,20] have been developed. Within living cells, the small molecule inhibitor proTAME, which is a cell-permeable prodrug, is converted to TAME (Tosyl-L-Arginine Methyl Ester) by intracellular esterases. The three-dimensional conformation of TAME resembles the isoleucine/arginine tail of the activator proteins CDH1 and CDC20 and has therefore the ability to bind to the APC/C, preventing the association of CDH1 or CDC20 with the APC/C [20].
    Polo-like kinase 1 (PLK1) regulates multiple steps of the cell-cycle progression [21–23]. The inhibition of PLK1, like the deregulation of microtubule dynamics by paclitaxel, activates the SAC. PLK1 is overexpressed in multiple cancer types including ovarian cancer [24–30]. Interestingly, PLK1 overexpression correlates with bad prognosis in various types of cancer patients [24,31–33]. Remarkably, high PLK1 levels have also been associated with a high risk of metastases, indicating a role for PLK1 in more malignant tumors. Numerous studies using xenograft models revealed PLK1 as a very attractive cancer target [34–37]. Volasertib (BI6727) is the most advanced clinical PLK1 inhibitor. A significant correlation between PLK1-positive cells and the histological grade of ovarian cancer was observed [38]. An investigation on benign and tumor tissues derived from human ovaries revealed that the frequency of PLK1 expression was low in normal ovarian epithelium and borderline tumors, but a high frequency was observed in 26% of ovarian cancer tissues [30]. In a recent study, a similar antitumor activity in patients with ovarian cancer was shown comparing Volasertib (BI6727) targeting PLK1 and a single-agent chemotherapy, e.g., paclitaxel [39]. However, the precise roles of PLK1 as prognostic marker and as therapeutic target in early stage ovarian cancer remain elusive.
    Cells arrested in mitosis by paclitaxel or by the PLK1 inhibitor BI6727 can enter an apoptotic pathway during mitosis or execute mitotic slippage, thereby entering G1 without undergoing GW311616 or cytokinesis to end up as single, tetraploid cell [40]. Following slippage, cells can arrest, cycle, or die after slippage [41]. The cellular factors that determine the GW311616 outcome of mitotic arrest currently remain unknown. A recent study has shown that preventing mitotic slippage by downregulating CDC20 via RNAi may increase the sensitivity of tumor cells to microtubule-targeting agents [42]. In EOC cells, little is known about the APC/C and its co-activator CDC20. In this study, we first studied the prognostic relevance of PLK1 in a cohort of 263 ovarian cancer patients (stages I/II). Moreover, we aimed to elucidate the importance and therapeutic potential of small molecule inhibitors of the APC/C in EOC cells mitotically arrested by paclitaxel and a clinical PLK inhibitor. 
    Materials and Methods
    Patients Characteristics (Immunohistochemistry)
    Following an institutional review board approval (Institutional Ethics Committee of Sichuan University, Ethics approval no. 20180928032) and after obtaining written informed consent, a total of 293 patients with ovarian carcinoma were included in this study. Our study conforms to the guidelines set by the Declaration of Helsinki. The median age was 51 years with a range of 34 years to 85 years. Seventy-one patients presented with stage I (24.2%) and 222 patients (75.8%) presented with stage II disease. A total of 285 patients (97.3%) were of serous and 8 patients (2.7%) of a mixed pathological subtype; 283 patients (96.6%) displayed a low- and 10 patients (3.4%) a moderate-grade differentiation. Median follow-up for all patients was 30 months (range: 1-109 months).
    Scoring for PLK1 Expression
    Production of tissue microarrays of ovarian cancer specimens was generated from West China Second Hospital, Sichuan University. All those patients included have not received chemotherapy prior to surgery, and at least two independent pathologists have examined the accuracy of the pathological diagnosis in a double-blinded manner. Slices were subject to a standardized horseradish peroxidase technique with primary anti-PLK1 antibodies (Santa Cruz Biotechnology) at a 1:100 dilution. Next, dextran polymer-conjugated horseradish peroxidase and 3,3-diaminobenzidine chromogen were utilized for visualization followed by counterstaining with hematoxylin solution. PLK1 immunoreactivity was evaluated semiquantitatively by two independent investigators (F.R. and M.S.) without knowledge of the patients’ clinicopathologic and clinical characteristics considering both the percentage of positive tumor cells [1 (0%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (N 75%)] and the intensity of staining scored as 1+ (weak), 2+ (moderate), and 3+ (intense). These parameters were multiplied to produce an individual weighted score (WS). To facilitate further statistical analysis, the histochemical sores were arbitrarily dichotomized: A WS ≤ 6 was classified as “low” and a WS of N6 was classified as “high” PLK1 expression. Overall survival was defined as the time of surgical tumor resection to death by ovarian cancer by any reasons or the day of the last follow-up. Survival curves were plotted according to the method of Kaplan-Meier. Statistical analysis was performed using IBM SPSS software version 25.