The online version of this article (https://doi.org/10.1186/s12885-018-5044-8) contains supplementary material, which is available to authorized users.
Karyopherin β1 (Kpnβ1) is the main nuclear import protein involved in the transport of cargoes from the cytoplasm into the cell nucleus. Previous research has found Kpnβ1 to be significantly overexpressed in cervical cancer and other cancer tissues, and further studies showed that inhibition of Kpnβ1 expression by siRNA resulted in cancer cell death, while non-cancer cells were minimally affected. These results suggest that Kpnβ1 has potential as an anticancer therapeutic target, thus warranting further research into the association between Kpnβ1 expression and cancer progression. Here, the biological effects associated with Kpnβ1 overexpression were investigated in order to further elucidate the relationship between Kpnβ1 and the cancer phenotype.
To evaluate the effect of Kpnβ1 overexpression on cell biology, cell proliferation, cell cycle, cell morphology and cell adhesion assays were performed. To determine whether Kpnβ1 overexpression influences cell sensitivity to chemotherapeutic agents like Cisplatin, cell viability assays were performed. Expression levels of key proteins were analysed by Western blot analysis.
Our data revealed that Kpnβ1 overexpression, above that which was already detected in cancer cells, resulted in reduced proliferation of cervical cancer cells. Likewise, normal epithelial cells showed reduced proliferation after Kpnβ1 overxpression. Reduced cancer cell proliferation was associated with a delay in cell cycle progression, as well as changes in the morphology and adhesion properties of cells. Additionally, Kpnβ1 overexpressing HeLa cells exhibited increased sensitivity to cisplatin, as shown by decreased cell viability and increased apoptosis, where p53 and p21 inhibition reduced and enhanced cell sensitivity to Cisplatin, respectively.
Overall, our results suggest that a tight balance of Kpnβ1 expression is required for cellular function, and that perturbation of this balance results in negative effects associated with a variety of biological processes.
Additional file 1: Figure S1. Overexpression of Kpnβ1 results in changes in the morphology and adhesion properties of CaSki cervical cancer cells. A: Phase contrast images showing CaSki EGFP and Kpnβ1-EGFP cells, taken 48 h post plating. Cells were viewed at 20 x magnification using the Zeiss Primovert inverted phase microscope. B: Quantification of relative CaSki cell area ± SEM of forty cells from each condition was performed using the AxioVision 4.7 software (*p < 0.05). C: Fluorescent staining of polymeric F-actin using phalloidin (red) in EGFP and Kpnβ1-EGFP expressing CaSki cells. DAPI stain was used to visualize the cell nuclei (blue). D: Quantification of the number of cytoplasmic protrusions from the captured fluorescent images. Results shown represent the mean ± SEM over fifteen fields of view (*p < 0.05). E: Relative cell adhesion in CaSki EGFP and Kpnβ1-EGFP cells. Adherent cells were fixed (after removing non-adherent cells by washing) and stained with 0.5% crystal violet solution. Cells over ten fields of view, viewed at 10 x magnification, were counted using ImageJ and normalized to unwashed cells. Results shown represent the mean ± SEM (*p < 0.05). F: Western blot analysis was used to determine the expression levels of E-cadherin and Vimentin in CaSki Kpnβ1-overexpressing cells. GAPDH was used as a control for loading. G: An in vitro scratch wound healing assay was performed and showed no change in migration of CaSki EGFP and Kpnβ1-EGFP cells within a 24 h period. H: Quantification of the scratch wound healing assay in G. (PPTX 627 kb)12885_2018_5044_MOESM1_ESM.pptx
Additional file 2: Figure S2. Overexpression of Kpnβ1 results in increased sensitivity to Cisplatin. Dose-response curves are shown after treatment of HeLa EGFP- and HeLa Kpnβ1-EGF-expressing cells with Cisplatin. (PPTX 42 kb)12885_2018_5044_MOESM2_ESM.pptx
Additional file 3: Figure S3. p53 and p21 inhibition reduces and enhances CaSki EGFP and Kpnβ1-EGFP cell sensitivity to Cisplatin, respectively. A: CaSki EGFP (a) and Kpnβ1-EGFP (b) cells were co-treated with Cisplatin and the p53 inhibitor Pifithrin α, and cell proliferation monitored 24 h later using the MTT assay. B: CaSki EGFP (a) and Kpnβ1-EGFP (b) cells were transfected with control (ctl) or p21 siRNA, and 48 h later treated with Cisplatin for 24 h, whereafter cell proliferation was monitored using the MTT assay. Results shown represent the mean ± SEM of experiments (*p < 0.05). (PPTX 59 kb)12885_2018_5044_MOESM3_ESM.pptx
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- A tight balance of Karyopherin β1 expression is required in cervical cancer cells
Pauline van der Watt
Virna D. Leaner
- BioMed Central
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