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. 2018 Nov;40(5):2445-2454.
doi: 10.3892/or.2018.6641. Epub 2018 Aug 10.

CIP2A Depletion Potentiates the Chemosensitivity of Cisplatin by Inducing Increased Apoptosis in Bladder Cancer Cells

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Free PMC article

CIP2A Depletion Potentiates the Chemosensitivity of Cisplatin by Inducing Increased Apoptosis in Bladder Cancer Cells

Fengbin Gao et al. Oncol Rep. .
Free PMC article

Abstract

Poor response and chemotherapy resistance to cisplatin (DDP)‑based therapy frequently lead to treatment failure in advanced bladder cancer; however the underlying mechanism is extremely complex and unclear. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A), a recently identified human oncoprotein, has been shown to play important regulatory roles in cancer cell survival. The present study aimed to investigate the correlation of CIP2A with sensitivity to DDP in bladder cancer cells. In the present study, knockdown of CIP2A was performed using short hairpin‑RNA. IC50 determination was used to estimate the chemosensitivity of cells to DDP. Apoptosis and DNA damage indicators were tested in vitro and in vivo to clarify the role of CIP2A in enhancing DDP sensitivity. We observed that CIP2A knockdown enhanced DDP sensitivity. CIP2A depletion accelerated the process of DNA damage caused by DDP treatment. Furthermore, DDP triggered inhibition of CIP2A by preventing AKT Ser473 phosphorylation. In vivo, CIP2A suppression increased the cytotoxicity of DDP, which resulted in a decrease in the subcutaneous tumor growth in a xenograft mouse model. Our findings revealed that the mechanism underlying the involvement of CIP2A in DDP sensitivity enhancement is that CIP2A mediates DDP‑induced cell apoptosis and DNA damage. CIP2A is a potential target to improve the response to DDP‑based therapy in bladder cancer patients.

Figures

Figure 1.
Figure 1.
CIP2A depletion increases drug sensitivity to DDP in bladder cancer cells. (A) CIP2A protein and (B) mRNA levels were examined in sh-Control and sh-CIP2A T24 and J82 cells. (C and D) IC50 of DDP was determined by MTT assay upon DDP treatment for 24 h in sh-Control and sh-CIP2A transfected T24 (C) and J82 (D) cells. (E) The response to DDP was tested by colony formation assay. Cell lines were treated continuously with 0 or 5 µg/ml of DDP for 14 days. Every experiment was conducted at least 3 times. **P<0.01 and ***P<0.001. DDP, cisplatin; sh, short hairpin; CIP2A, cancerous inhibitor of protein phosphatase 2A.
Figure 2.
Figure 2.
Knockdown of CIP2A induces increased apoptosis in bladder cancer cells upon DDP treatment. (A) Apoptosis of sh-Control and sh-CIP2A bladder cancer cells was demonstrated by flow cytometry with 20 µg/ml of DDP treatment for 24 h. (B) The expression of CIP2A, Bcl2, cleaved caspase-3, and cleaved PARP was detected by western blotting with 20 µg/ml of DDP treatment for 24 h. Every experiment was conducted at least 3 times. *P<0.05, **P<0.01. DDP, cisplatin; sh, short hairpin; CIP2A, cancerous inhibitor of protein phosphatase 2A.
Figure 3.
Figure 3.
Knockdown of CIP2A in bladder cancer cells enhances DDP-induced DNA damage. (A) Following 0 or 20 µg/ml DDP treatment for 24 h in sh-Control and sh-CIP2A T24 and J82 cells, a comet assay was performed to assess DNA damage. Scale bar, 50 µm. (B) Immunofluorescence was performed to demonstrate the formation of γ-H2AX foci. Scale bar, 100 µm. (C) Western blotting of CIP2A and γ-H2AX proteins was conducted. Every experiment was conducted at least 3 times. *P<0.05, **P<0.01, ***P<0.001. DDP, cisplatin; sh, short hairpin; CIP2A, cancerous inhibitor of protein phosphatase 2A.
Figure 4.
Figure 4.
DDP attenuates the expression of CIP2A via the AKT pathway in bladder cancer cells. Upon DDP treatment at different concentrations (0, 10, 20 µg/ml) in T24 and J82 cells, CIP2A mRNA levels were determined by (A) RT-qPCR, and (B) the protein levels of AKT, p-AKT, CIP2A, Bcl2, cleaved caspase-3, and cleaved PARP were determined by western blotting. (C) The protein levels of AKT, p-AKT and CIP2A in sh-Control and sh-CIP2A T24 and J82 cells were detected by western blotting. (D) Following 20 µmol/l LY294002 treatment for 24 h, western blotting was used to test the protein levels of AKT, p-AKT and CIP2A in T24 and J82 cells. Every experiment was conducted at least 3 times. **P<0.01, ***P<0.001. DDP, cisplatin; sh, short hairpin; CIP2A, cancerous inhibitor of protein phosphatase 2A.
Figure 5.
Figure 5.
Inhibiting CIP2A expression enhances DDP sensitivity in nude mouse xenografts. (A) Tumor growth curve of sh-CIP2A or sh-Control T24 cell bladder cancer subcutaneous xenograft tumors was built following DDP treatment. (B) Photographs of dissected xenograft tumors from nude mice after they were sacrificed. (C) The weight of dissected xenograft tumors in each group was assayed. (D) IHC staining was performed to detect the expression levels of CIP2A, p-AKT and γ-H2AX, and TUNEL staining was conducted to test the degree of apoptosis in harvested tumor tissues. Representative photomicrographs of 200-fold high power fields in each group are presented. Scale bar, 100 µm. *P<0.05. DDP, cisplatin; sh, short hairpin; CIP2A, cancerous inhibitor of protein phosphatase 2A; IHC, immunohistochemistry.

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References

    1. Trenta P, Calabro F, Cerbone L, Sternberg CN. Chemotherapy for Muscle-invasive bladder cancer. Curr Treat Options Oncol. 2016;17:6. doi: 10.1007/s11864-015-0376-y. - DOI - PubMed
    1. Massari F, Santoni M, Ciccarese C, Brunelli M, Conti A, Santini D, Montironi R, Cascinu S, Tortora G. Emerging concepts on drug resistance in bladder cancer: Implications for future strategies. Crit Rev Oncol Hematol. 2015;1:81–90. doi: 10.1016/j.critrevonc.2015.05.005. - DOI - PubMed
    1. Grossman HB, Natale RB, Tangen CM, Speights VO, Vogelzang NJ, Trump DL, deVere White RW, Sarosdy MF, Wood DJ, Raghavan D, Crawford ED. Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med. 2003;9:859–866. doi: 10.1056/NEJMoa022148. - DOI - PubMed
    1. International Collaboration of Trialists; Medical Research Council Advanced Bladder Cancer Working Party (now the National Cancer Research Institute Bladder Cancer Clinical Studies Group); European Organisation for Research and Treatment of Cancer Genito-Urinary Tract Cancer Group; Australian Bladder Cancer Study Group; National Cancer Institute of Canada Clinical Trials Group; Finnbladder; Norwegian Bladder Cancer Study Group; Club Urologico Espanol de Tratamiento Oncologico Group, corp-author. Griffiths G, Hall R, et al. International phase III trial assessing neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy for muscle-invasive bladder cancer: Long-term results of the BA06 30894 trial. J Clin Oncol. 2011;16:2171–2177. - PMC - PubMed
    1. Advanced Bladder Cancer Meta-analysis Collaboration. Neoadjuvant chemotherapy in invasive bladder cancer: A systematic review and meta-analysis. Lancet. 2003;361:1927–1934. doi: 10.1016/S0140-6736(03)13580-5. - DOI - PubMed

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