[Pt(O,O'-acac)(γ-acac)(DMS)] Induces Autophagy in Caki-1 Renal Cancer Cells

Abstract

We have demonstrated the cytotoxic effects of [Pt(O,O'-acac)(γ-acac)(dimethyl sulfide (DMS))] on various immortalized cell lines, in primary cultures, and in murine xenograft models in vivo. Recently, we also showed that [Pt(O,O'-acac)(γ-acac)(DMS)] is able to kill Caki-1 renal cells both in vivo and in vitro. In the present paper, apoptotic and autophagic effects of [Pt(O,O'-acac)(γ-acac)(DMS)] and cisplatin were studied and compared using Caki-1 cancerous renal cells. The effects of cisplatin include activation of caspases, proteolysis of enzyme poly ADP ribose polymerase (PARP), control of apoptosis modulators B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and BH3-interacting domain death agonist (Bid), and cell cycle arrest in G2/M phase. Conversely, [Pt(O,O'-acac)(γ-acac)(DMS)] did not induce caspase activation, nor chromatin condensation or DNA fragmentation. The effects of [Pt(O,O'-acac)(γ-acac)(DMS)] include microtubule-associated proteins 1A/1B light chain 3B (LC3)-I to LC3-II conversion, Beclin-1 and Atg-3, -4, and -5 increase, Bcl-2 decrease, and monodansylcadaverine accumulation in autophagic vacuoles. [Pt(O,O'-acac)(γ-acac)(DMS)] also modulated various kinases involved in intracellular transduction regulating cell fate. [Pt(O,O'-acac)(γ-acac)(DMS)] inhibited the phosphorylation of mammalian target of rapmycin (mTOR), p70S6K, and AKT, and increased the phosphorylation of c-Jun N-terminal kinase (JNK1/2), a kinase activity pattern consistent with autophagy induction. In conclusion, while in past reports the high cytotoxicity of [Pt(O,O'-acac)(γ-acac)(DMS)] was always attributed to its ability to trigger an apoptotic process, in this paper we show that Caki-1 cells die as a result of the induction of a strong autophagic process.

Keywords: apoptosis; autophagy; cancerous renal cells; cisplatin.

Figure 1

Cytotoxic effects of [Pt(O,O′-acac)(γ-acac)(DMS) and cisplatin and effects on the cell cycle of Caki-1 cells. Caki-1 cells were treated with 10 µM [Pt(O,O′-acac)(γ-acac)(dimethyl sulfide (DMS))] or with 50 µM cisplatin. Cell viability was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT) assay (A) and cell death was quantified by fluorescence-activated cell sorter (FACS) after propidium iodide (PI)/annexin V-fluorescein isothiocyanate (FITC) staining (B), over a period of 48 h. Data are means ± standard deviation (SD) of five independent experiments with eight replicates in each, and are presented as percent of control. * p < 0.01 between treated and untreated cells (white bar), by Student’s t-test. (C) Cells were treated with [Pt(O,O′-acac)(γ-acac)(DMS)] or cisplatin for 24 h, and cell cycle distribution was analyzed by flow cytometry after staining the cells with PI. Representative FACS histogram from six separate experiments is shown.

Figure 2

[Pt(O,O′-acac)(γ-acac)(DMS)] does not induce apoptosis in Caki-1 cells. (A) Expression of cleaved caspase-9 and -3 in Caki-1 cells. Cell were treated with 10 µM of [Pt(O,O′-acac)(γ-acac)(DMS)] or 50 µM of cisplatin for the indicated time, and then subjected to Western blotting. Incubation with anti-β-actin confirmed the equal protein loading. The results shown are representative of three different experiments. (B) (Up) Caki-1 cells were treated, or not, with cisplatin, or with [Pt(O,O′-acac)(γ-acac)(DMS)] for 24 h, and then stained with 4,6-diammine-2-phenylindol (DAPI); the representative fields by confocal microscopy (magnification 40×) of one of four independent experiments are shown. (Down) Quantification of the percentage of apoptotic nuclei obtained from cells stained with DAPI (means ± SD; n = 4), after treatment for different times with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)] or 50 µM cisplatin. ** p < 0.01 between cisplatin-treated and untreated cells and * p < 0.05 between [Pt(O,O′-acac)(γ-acac)(DMS)]-treated and untreated cells, by Student’s t-test. (C) Visualization of DNA fragmentation in [Pt(O,O′-acac)(γ-acac)(DMS)]-treated Caki-1 cells. Total DNA was isolated and separated on a 1% agarose gel. A representative example of three independent experiments is shown. (D,E) Caki-1 cells were transfected with 30 nM small interfering RNA (siRNA) oligos for apoptosis inducing factor (AIF) and then treated with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)]. (D) Immunoblotting detection of AIF in cell extracts 48 h after siRNA transfection using polyclonal anti-AIF antibody. Controls were provided by untransfected cells and cells transfected with scrambled siRNA oligos (Scr). Incubation with anti-β-actin confirmed the equal protein loading. A representative example of three independent experiments is shown. (E) Cell death was quantified by FACS after propidium iodide (PI)/annexin V-FITC staining, in transfected Caki-1 cells treated with 10 µM of [Pt(O,O′-acac)(γ-acac)(DMS)] or 50 µM cispltin for 24 h. Data are means ± SD of five independent experiments.

Figure 3

[Pt(O,O′-acac)(γ-acac)(DMS)] induces autophagy in Caki-1 cells. (A) Caki-1 cells were treated with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)] for the indicated times or (B) Caki-1 cells were pretreated, or not, with 1 mM of 3-MA or 2 mM of 3-MA, then treated with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)] for 24 h; subsequently, Caki-1 cells were stained with monodansylcadaverine (MDC) and 4′,6-diamidino-2-phenylindole (DAPI). The cellular fluorescent changes were observed through confocal microscopy Zeiss LSM 700 (Carl Zeiss AG, Oberkochen, Germany). (C) Quantification of the percentage of autophagic vacuoles are presented as means ± SD. * p < 0.001 between treated and untreated cells; ^§ p < 0.001 between cells treated with 3-MA and [Pt(O,O′-acac)(γ-acac)(DMS)], and cells treated with [Pt(O,O′-acac)(γ-acac)(DMS)] alone, by Student’s t-test (n = 5). (D) Caki-1 cells were treated with increasing concentrations of [Pt(O,O′-acac)(γ-acac)(DMS)] and 2 mM of 3-MA for 24 h, and cell viability was monitored by MTT assay. * p < 0.001 between treated and untreated cells, by Student’s t-test (n = 5). ^§ p < 0.001 between cells treated with SP600125 and [Pt(O,O′-acac)(γ-acac)(DMS)], and cells treated with [Pt(O,O′-acac)(γ-acac)(DMS)] alone; ^§ p < 0.001 between cells treated with 3-MA and [Pt(O,O′-acac)(γ-acac)(DMS)], and cells treated with [Pt(O,O′-acac)(γ-acac)(DMS)] alone, by Student’s t-test (n = 5).

Figure 4

[Pt(O,O′-acac)(γ-acac)(DMS)] induces autophagy in Caki-1 cells. (A–C) Caki-1 cells were treated with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)] or with 50 µM cisplatin for different times. Cell lysates were analyzed by Western blotting using microtubule-associated proteins 1A/1B light chain 3B (LC3)I-II, autophagy-related (Atg)-3, Atg-4, Atg-5, JNK, and B-cell lymphoma 2 (Bcl-2) antibodies. Sequential incubation with anti-β-actin confirmed the equal protein loading. Representative immunoblots of three experiments are depicted. (A, down) Densitometric analysis of LC3I-II normalized to β-actin. (B, Right) Densitometric analysis of Atg-3, Atg-4, and Atg-5, normalized to β-actin. The data are means ± SD of three different experiments. * p < 0.001 between treated and untreated cells, by Student’s t-test (n = 3). (D) (Up) Cells, were incubated with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)] for the indicated time or were pretreated with 10 µM of JNK inhibitor SP600125 (SB) and then incubated with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)] (Pt(acaca)₂)_, for 24 h. Cell lysates were analyzed by western blotting using monoclonal antibody specific to Beclin-1. Sequential incubation with anti-β-actin confirmed the equal protein loading. These figures are representative of five independent experiments. (Down) Densitometric analysis of Beclin-1 normalized to β-actin.

Figure 5

[Pt(O,O′-acac)(γ-acac)(DMS)] induces autophagy through inhibition of the phosphatidine 3-kinase (PI3K)/AKT-mTOR pathway. (A) Cell lysates obtained from Caki-1 cells treated with 10 µM [Pt(O,O′-acac)(γ-acac)(DMS)], for the indicated times, were subjected to Western blotting analyses with monoclonal antibodies specific to p-p70S6K, p70S6K, p-mTOR, m-TOR, p-AKT, and AKT. (B) (Up) Cell lysates from cells treated with 50 µM cisplatin, for the indicated times, were subjected to Western blotting analyses with monoclonal antibodies specific to p-AKT and AKT. Sequential incubation with anti-β-actin confirmed the equal protein loading. These figures are representative of six independent experiments. (Down) Densitometric analysis of p-AKT (normalized respectively to total AKT). (C) Caki-1 cells were treated, or not, with 100 µM temsirolimus for the indicated times and cell lysates were analyzed by Western blotting using antibodies specific to p-p70S6K and total p70S6K. The figure is representative of three independent experiments. (D) Caki-1 cells were treated, or not, with different concentrations of [Pt(O,O′-acac)(γ-acac)(DMS)] or temsirolimus for 24 h, and cell viability was monitored by MTT assay. * p < 0.001 between treated and untreated cells by Student’s t-test (n = 3).