Increased expression of cdc2 inhibits transport function of RLIP76 and promotes apoptosis

Abstract

RLIP76 is a stress-responsive glutathione-electrophile-conjugates (GS-E) and drugs transporter which is over-expressed in different types of cancers. Cdc2 is a cell-cycle check point control kinase which has been shown to bind to RLIP76 during mitosis, such that endocytosis is inhibited. In present studies, we have purified cdc2 and examined its effect on the transport activity of RLIP76 reconstituted into artificial liposomes. Both doxorubicin (DOX) and dinitro-phenyl S-glutathione (DNP-SG) transport were inhibited by cdc2 in a concentration dependent manner. Liposomal delivery of cdc2 to H358 cells caused apoptosis, resulted in an increased intracellular doxorubicin-accumulation and decreased rate of efflux from the cells. In the present communication, we propose that the accumulation-deficient drug-resistance mediated by RLIP76 can be modulated by inhibition of RLIP76 transport activity by cdc2.

Figure 1. Purification of RLIP76 and cdc2 from transformed E. coli

DNPSG-affinity purified rec-RLIP76 (5 μg) (panels A and B) and Ni-NTA Super-flow resin (Qiagen) purified rec-cdc2 (5 μg) (panels C and D) were applied to SDS-PAGE (panels A and C) and subjected to Western-blot analyses (panels B and D) against anti-RLIP76 IgG and anti-cdc2 IgG as primary antibody. SDS-PAGE was stained with Coomassie Brilliant Blue-R250 and Western-blots were developed using horseradish peroxidase-conjugated goat-anti-rabbit-IgG as secondary antibody.

Figure 2. Inhibition of DOX and DNP-SG transport in RLIP76-proteoliposomes by cdc2

¹⁴C-DOX (panels A and B) and ³H-DNPSG (panels C and D) transport activity was measured as ATP-dependent DOX and DNP-SG uptake in artificial liposomes reconstituted with purified rec-RLIP76. Control included liposomes prepared without RLIP76. Purified cdc2 protein was added in varying ratio to the transport reaction mixture. Results presented are representative from three separate experiments, each with triplicate determinations. •, control-liposomes treatment; ■, cdc2-proteoliposomes treatment; * p < 0.005 compared with control-liposomes treatment

Figure 3. Effect of cdc2 supplementation on uptake, efflux and DOX-cytotoxicity

H358 NSCLC cells were treated with control-liposomes (green circles) and proteoliposomes containing 40 μg/ml cdc2 (red squares). Uptake of ¹⁴C-DOX (sp. act. 8.9 × 10⁴ cpm/nmol) by cells was determined by incubating the cells for varying periods of time with radio-labeled ¹⁴C-DOX, and measuring radio-activity in the cell pellet (panel A). For efflux studies, cells were loaded with ¹⁴C-DOX for 60 min, followed by rapid dilution in 1 ml buffer. Aliquots (50 μl) of the buffer were taken at 1 min intervals, and cellular drug content was calculated by back-addition from the residual DOX in cells at the end of the experiment (panel B). The cytotoxic effects of DOX were measured by MTT assay 96 h after drug-exposure (panel C). * p < 0.001 compared between control-liposomes vs. cdc2-proteoliposomes treatment.

Figure 4. Effect of cdc2 on apoptosis and colony-forming efficiency in H358 NSCLC

The effect of cdc2 overloading on apoptosis was examined by TUNEL (panel A) and FITC-labeled Annexin V assay (panels B). The H358 cells grown on cover-slips incubated for 24 h with control and cdc2-proteoliposomes (40 μg/ml) prior to Annexin V or TUNEL assays and examined using Zeiss LSM 510 META (Germany) laser scanning fluorescence microscope with filters 520 nm and >620 nm. Photographs taken at identical exposure at 400 × magnification are presented. Apoptotic cells showed green fluorescence and characteristic of cell shrinkage. Colony-forming assay was performed by staining the cells with methylene blue and the colonies were counted using Innotech Alpha Imager HP (panel C). Values are means ± S.D. of three separate experiments. * p < 0.005 compared with no liposomes and control-liposomes treatment.