A novel copper complex induces paraptosis in colon cancer cells via the activation of ER stress signalling

Abstract

Platinum anticancer drugs have been used for three decades despite their serious side effects and the emerging of resistance phenomena. Recently, a phosphine copper(I) complex, [Cu(thp)(4)][PF(6)] (CP), gained special attention because of its strong antiproliferative effects. CP killed human colon cancer cells more efficiently than cisplatin and oxaliplatin and it overcame platinum drug resistance. CP preferentially reduced cancer cell viability whereas non-tumour cells were poorly affected. Colon cancer cells died via a programmed cell death whose transduction pathways were characterized by the absence of hallmarks of apoptosis. The inhibition of 26S proteasome activities induced by CP caused intracellular accumulation of polyubiquitinated proteins and the functional suppression of the ubiquitin-proteasome pathway thus triggering endoplasmic reticulum stress. These data, providing a mechanistic characterization of CP-induced cancer cell death, shed light on the signaling pathways involved in paraptosis thus offering a new tool to overcome apoptosis-resistance in colon cancer cells.

Fig 1

Chemical structure of CP.

Fig 2

Clonal growth. Fraction of surviving cells determined by clonogenic assay in LoVo and CCD-18Co cells after CP exposure.

Fig 3

Non-apoptotic cell death induction. (A) Comet assay. LoVo cells treated for 12 hrs with IC₅₀ of CP or O×Pt. (B) Nuclear DNA fragmentation. LoVo cells were treated for 12 or 24 hrs with IC₅₀ of CP or O×Pt. Quantitative estimation of DNA fragmentation was obtained with an ELISA test. *P < 0.05 compared to control. Insert (b) LoVo–O×Pt cells treated for 12 or 24 hrs with IC₅₀ of O×Pt. Data are the means of five independent experiments. Error bars indicate S.D. *P < 0.05 compared to control and °P < 0.05 compared to O×Pt treated LoVo cells. (C) Caspase activity. LoVo cells incubated for 12 and 24 hrs with CP or O×Pt and processed for caspase-3/-7, -6, -8, -9 activity. Insert (c): LoVo–O×Pt cells treated for 12 or 24 hrs with IC₅₀ of O×Pt. Data are the means of at least three independent experiments. Error bars indicate S.D. *P < 0.05 compared to control and °P < 0.05 compared to O×Pt treated LoVo cells. (D) Cytochrome c release. LoVo cells treated with IC₅₀ of tested compounds for 12 hrs and cytochrome c was estimated by Western blotting. (E) Flow cytometric profiles of LoVo cells untreated and treated with IC₅₀ of CP for 12 and 24 hrs and stained with TMRM.

Fig 4

Morphological changes in CP-treated LoVo cells. (A) Phase-contrast micropho-tographs (100×) of control (a) or treated for 24 hrs with IC₅₀ of CP (a′) LoVo cells. (B) TEM analysis of control (b), or CP-treated LoVo cells for 12 hrs (b′) or 24 hrs (b″ and b‴). (C) Calnexin staining of control LoVo cells (c) or exposed to CP for 12 hrs (c′).

Fig 5

Effect of CP on proteasome inhibition. Inhibition of human 26S proteasome purified from LoVo cells (A) and in intact LoVo cells (B) estimated by means of specific fluorogenic substrates. IC₅₀ values calculated by four-parameter logistic model (P < 0.05). In (B), LoVo cells following 24-hr treatment with increasing concentrations of CP. Detection of poly-ubiquitinated proteins in LoVo cells (C and D). In (C), cells treated for 6, 12 and 24 hrs with IC₅₀ of CP and ubiquitinated proteins estimated by Western blotting. In (D), cells treated for 24 hrs with IC₅₀ of CP and submitted to immunofluorescence analysis.

Fig 6

Effect of CP on UPR. (A) Western blot analysis of p-PERK and p-IRE1 in LoVo cells treated with IC₅₀ of CP for 12 and 24 hrs. *P < 0.05 compared to control. (B) Immunofluorescence staining of LoVo cells treated with IC₅₀ of CP for 24 hrs for p-PERK and p-IRE1 detection.