Browsing by Author "van der Watt, Pauline"
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- ItemOpen AccessA tight balance of Karyopherin β1 expression is required in cervical cancer cells(BioMed Central, 2018-11-16) Carden, Sarah; van der Watt, Pauline; Chi, Alicia; Ajayi-Smith, Aderonke; Hadley, Katie; Leaner, Virna DBackground 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. Methods 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. Results 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. Conclusions 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.
- ItemOpen AccessInhibition of Kpnβ1 mediated nuclear import enhances cisplatin chemosensitivity in cervical cancer(2021-02-02) Chi, Ru-pin A; van der Watt, Pauline; Wei, Wei; Birrer, Michael J; Leaner, Virna DBackground Inhibition of nuclear import via Karyopherin beta 1 (Kpnβ1) shows potential as an anti-cancer approach. This study investigated the use of nuclear import inhibitor, INI-43, in combination with cisplatin. Methods Cervical cancer cells were pre-treated with INI-43 before treatment with cisplatin, and MTT cell viability and apoptosis assays performed. Activity and localisation of p53 and NFκB was determined after co-treatment of cells. Results Pre-treatment of cervical cancer cells with INI-43 at sublethal concentrations enhanced cisplatin sensitivity, evident through decreased cell viability and enhanced apoptosis. Kpnβ1 knock-down cells similarly displayed increased sensitivity to cisplatin. Combination index determination using the Chou-Talalay method revealed that INI-43 and cisplatin engaged in synergistic interactions. p53 was found to be involved in the cell death response to combination treatment as its inhibition abolished the enhanced cell death observed. INI-43 pre-treatment resulted in moderately stabilized p53 and induced p53 reporter activity, which translated to increased p21 and decreased Mcl-1 upon cisplatin combination treatment. Furthermore, cisplatin treatment led to nuclear import of NFκB, which was diminished upon pre-treatment with INI-43. NFκB reporter activity and expression of NFκB transcriptional targets, cyclin D1, c-Myc and XIAP, showed decreased levels after combination treatment compared to single cisplatin treatment and this associated with enhanced DNA damage. Conclusions Taken together, this study shows that INI-43 pre-treatment significantly enhances cisplatin sensitivity in cervical cancer cells, mediated through stabilization of p53 and decreased nuclear import of NFκB. Hence this study suggests the possible synergistic use of nuclear import inhibition and cisplatin to treat cervical cancer.
- ItemOpen AccessInvestigating nuclear transport proteins as secreted cancer biomarkers(2019) Okpara, Michael Obinna; Leaner, Virna; van der Watt, PaulinePrevious studies in our laboratory using microarray gene expression analysis identified members of the nuclear transport protein family as significantly upregulated in cervical cancer biopsies compared to normal cervical epithelial tissues. These results were validated at both mRNA and protein levels, and similar upregulation observed in oesophageal cancer. Recent mass spectrometry (MS) analysis of cancer cell secreted proteins identified elevated levels of 13 members of the nuclear transport protein family in the secretomes of transformed, cervical cancer and oesophageal cancer cell lines. The nuclear transport proteins have functions in many cellular processes including proliferation, mitosis, maturation of RNA, activation of the actin cytoskeleton and restructuring of the nuclear envelope. In addition, they are required for the nuclear import and export of numerous cargo proteins such as transcription factors, oncoproteins and kinases, which often display deregulated activity in cancer cells. The aims of this study were to 1) independently validate the MS data showing elevated levels of the nuclear transport proteins in the secretomes of cervical and oesophageal cancer cell lines, 2) investigate the diagnostic potential of members of the nuclear transport protein family using cervical and oesophageal cancer serum samples and 3) identify the potential binding partners of Kpnβ1, a key member of the nuclear transport protein family, in normal and cancer cells. This study investigated the levels of endogenous expression and secretion of 8 members of the nuclear transport protein family; Kpnβ1, IPO5, IPO7, TNPO1, CRM1, CAS, Kpnα2 and Ran in a normal epithelial cell line (hTERT-RPE1) in comparison to transformed (SVWI38 and CT-1), cervical cancer (HeLa and CaSki) and oesophageal cancer (WHCO5 and KYSE 30) cell lines using Western blot analysis. Our data revealed differential endogenous expression in the cell lines. An analysis of the secretomes of the cell lines showed that all 8 proteins assayed were secreted at elevated levels by the transformed, cervical cancer and oesophageal cancer cell lines compared to the normal cell line. These results validate previous MS data generated in our laboratory. To investigate whether members of this protein family can be detected in the serum of cancer patients, ELISA for Kpnβ1, CRM1, Kpnα2 and CAS proteins were performed using commercially available ELISA kits. The results showed significantly elevated levels of Kpnβ1, CRM1 and CAS in the serum of cervical cancer patients compared to the non-cancer controls. Serum levels of Kpnβ1, CRM1, Kpnα2 and CAS were elevated in the oesophageal cancer patients compared to the non-cancer controls. To investigate the diagnostic potential of these proteins, logistics regression analysis was performed. Our results showed that CAS was the best performing individual candidate biomarker in discriminating between cervical cancer cases and non-cancer controls. It had the highest AUC (0.85±0.03) and highest sensitivity (55%) at 95% specificity compared to those of Kpnβ1 (AUC=0.77±0.04 with 35% sensitivity at 95% specificity), CRM1 (AUC=0.64±0.05 with 20% sensitivity at 95% specificity) and Kpnα2 (AUC=0.51±0.05 with <10% sensitivity at 95% specificity). The combination of Kpnβ1, CRM1, Kpnα2 and CAS as a panel of biomarkers had an improved AUC of 0.89 with a sensitivity of 100% at 60% specificity. In discriminating oesophageal cancer cases from the non-cancer controls, CAS (AUC=0.86±0.03 with 56% sensitivity at 95% specificity) similarly performed better compared to Kpnβ1 (AUC=0.62±0.05 with 15% sensitivity at 95% specificity), CRM1 (AUC=0.75±0.04 with 32% sensitivity at 95% specificity) and Kpnα2 (AUC=0.73±0.04 with 21% sensitivity at 95% specificity). The combination of Kpnβ1, CRM1, Kpnα2 and CAS as a panel of biomarkers had the highest diagnostic capacity with an AUC of 0.90 and 84% sensitivity at 86% specificity. These results suggest that individual members of the nuclear transport protein family have potential as diagnostic biomarkers for both cervical and oesophageal cancers, with a combination of Kpnβ1, CRM1, Kpnα2 and CAS being the best predictor. Our investigation aimed at identifying the binding partners of Kpnβ1 in normal, cervical cancer and oesophageal cancer cell lines using immunoprecipitation coupled to mass spectrometry (IP-MS) identified 100 potential Kpnβ1 binding partners in hTERT-RPE1 normal cell extracts, 179 in HeLa cervical cancer cell extracts, 147 in WHCO5 cell extracts and 176 in KYSE30 oesophageal cancer cell extracts. Venn Dis JavaFX-based Venn and Euler diagram software was used to identify common and unique Kpnβ1 binding partners. 38 proteins were identified as common binding partners of Kpnβ1 in normal and cancer cells and 56 common binding partners of Kpnβ1 in the three cancer cell lines. Of these, 18 proteins were found to be unique to the three cancer cell lines and of these, 10 could be linked via protein-protein interaction mapping using STRING bioinformatic analysis. These include nucleoporin 214 (Nup214), Prem RNA 3'-end-processing factor FIP1 (FIP1L1), cell division cycle and apoptosis regulator 1 (CCAR1), cleavage and polyadenylation specific factor 7 (CPSF7), ribosomal protein L7 (RPL7), ribosomal protein L10 (RPL10), ribosomal protein L13A (RPL13A), ribosomal protein S6 (RPS6), ribosomal protein S4, X isoform (RPS4X) and Ras-related nuclear protein (Ran). Among these, FIP1L1, CCAR1 and CPSF7 have not been previously described as binding partners of Kpnβ1. In conclusion, elevated levels of nuclear transport proteins in the extracellular environment of cancer cells and in cancer patient serum samples suggest that they have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a combination of Kpnβ1, CRM1, Kpnα2 and CAS being the best predictor. In addition, this study shows that Kpnβ1 interacts with several different proteins in normal and cancer cells, with some of the interactions unique to cancer cells presenting as novel binding partners for further investigation.
- ItemOpen AccessThe role of Karyopherin β1 in the nuclear import of HIV-1 proteins(2014) Shaw, Tamlyn Marion; Leaner, Virna; van der Watt, PaulineIncludes abstract. Includes bibliographical references.