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

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.


Introduction
Renal cell carcinoma (RCC) is among the top 10 most common cancers in the world; about 20%-30% of patients with RCC have metastasis to the first diagnosis, and over 95% of patients have multiple metastases [1]. Renal cell carcinoma resistance to conventional medical therapy remains the main obstacle to survival, and it is therefore important to develop new therapeutic strategies [2,3]. Indeed, many chemotherapeutic agents have been experienced in the treatment of RCC; among them, the cisplatin-based drugs are among the most common and effective chemotherapeutic agents for Biomolecules 2019, 9,92 2 of 14 many types of human cancer [4]. However, patients that receive conventional platinum-based drugs also develop side effects and resistance to treatment, decreasing their effectiveness.
The development of drugs targeting mammalian target of rapamycin (mTOR) (rapamycin analogs, i.e., temsirolimus and everolimus) has led to significant improvement in RCC prognosis. Although these new agents improve progression-free survival, none have shown a statistically significant improvement in overall survival. In effect, none are curative, and the duration of response is often limited [5]. In addition, based on clinical data, it has been argued that chronic drug exposure triggers the development of resistance, ultimately limiting the utility of mTOR inhibitor. Particularly, the mTOR-related proteins, AKT and S6K1, have been shown to be reactivated under long-term everolimus exposure [6].
In the search for new Pt complexes with better antitumor profiles that are less likely develop resistance, [Pt(O,O -acac)(γ-acac)(dimethyl sulfide (DMS))] has been synthesized, a Pt(II) complex containing two acetyl groups (acac) and a sulfide ligand in the Pt coordination sphere [7,8]. Previous studies have shown that this new complex has a high and rapid cytotoxic activity, being able to induce apoptotic cell death in HeLa human endometrial carcinoma cells [9], in MCF-7 human breast cancer cells [10], in SH-SY5Y human neuroblastoma cells [11], and in ZL55 [12] and ZL34 [13] mesothelioma cells. Quite recently, we also showed that [Pt(O,O -acac)(γ-acac)(DMS)] also has high and rapid citotoxicity on renal Caki-1 cells both in vitro and in vivo [14]. In these xenograft mice, [Pt(O,O -acac)(γ-acac)(DMS)] also displayed potent antiangiogenic activity though the inhibition of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1) expression in tumor tissues [14]. This is of interest since Caki-1 cells are intrinsically able to resist and inhibit apoptosis [15], whilst [Pt(O,O -acac)(γ-acac)(DMS)] kills cells through a potent induction of apoptosis. Resistance to apoptosis may be due to various factors. During nephrogenesis, the transcription factor paired box protein 2 (PAX2) is differentially expressed depending upon the kidney development phase, and it is barely quantifiable in mature kidney. In Caki-1 cells, PAX2 is overexpressed and contributes to cisplatin resistance [16]. Furthermore, Caki-1 cells resist apoptosis through the secretion of IL-6 that suppresses the induction of apoptosis acting in an autocrine way [17]. Although [Pt(O,O -acac)(γ-acac)(DMS)] most often activates the mitochondrial apoptotic pathway, another recent in vitro investigation of neuroblastoma cells has revealed that [Pt(O,O -acac)(γ-acac)(DMS)] leads to both apoptosis and activation of autophagy [18].
Thus, the aim of the present paper is to investigate apoptotic and/or autophagic pathways caused by cisplatin and [Pt(O,O -acac)(γ-acac)(DMS)] treatment in Caki-1 cells.

Cell Culture
Caki-1 cells (human renal carcinoma cell line) were maintained in McCoy's medium supplemented with 10% fetal bovine serum, penicillin/streptomycin, and 1% L-glutamine. All cell cultures were routinely grown in 75 cm 2 cell culture flasks and were sustained at 37 • C, 5% CO 2 , and 95% relative humidity. Cells were grown to 70%-80% confluence and then treated with cisplatin and [Pt(O,O -acac)(γ-acac)(DMS)] at various concentrations and for different incubation periods.

Cytotoxicity Assay
To assess the cytotoxicity of [Pt(O,O -acac)(γ-acac)(DMS)] and cisplatin, we used both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT) [19] and sulforhodamine B (SRB) [20] methods, as previously described. Viable cells were also counted using the trypan blue exclusion assay and light microscopy. The percentage of live cells was evaluated as the absorbance ratio of treated to untreated cells. The data presented are means ± standard deviation (SD) from eight replicate wells per microtiter plate.
In addition, cell death was then measured by quantifying the percentage of cells that exhibited annexin V-fluorescein isothiocyanate (FITC) and/or propidium iodide (PI) fluorescence using a flow cytometer. Total cell death was quantified by adding the percentage of cells detected in the upper left (PI), upper right (PI + annexin V-FITC), and lower right (annexin V-FITC) quadrants in the FACS dotplots (Becton-Dickinson, CA, USA).

Apoptosis Analysis
For the 4,6-diammine-2-phenylindol (DAPI) staining, cells treated with [Pt(O,O -acac)(γ-acac)(DMS)] or cisplatin were fixed with 4% paraformaldehyde and incubated with 1 mg mL −1 DAPI in phosphate buffeed saline (PBS) for 15-20 min. Cells were mounted on glass slides and analyzed using fluorescence microscopy. For statistical analysis of each experiment, 5-10 fields (magnification 20×) were analyzed using the image analysis software ImageJ so as to detect only the nuclei with chromatin condensation. The mean ± SD was calculated and displayed as a bar graph. For the DNA fragmentation assay, genomic DNA from Caki-1 cells was prepared using the Wizard Genomic DNA Purification Kit (Promega, Milan, Italy) according to the manufacturer's protocol. DNA was dissolved in Tris-ethylenediaminetetraacetic acid (EDTA) buffer, and 40 µg of DNA was separated on a 1.2% agarose gel containing 0.1 mg mL −1 ethidium bromide, visualized under ultraviolet light, and photographed.

Preparation of Subcellular Fraction
Preparation of subcellular fractions performed as previously reported [21].

Western Blotting Analysis
Western blotting analysis, immunodetection, and densitometric analysis of subcellular fraction were performed as previously described [21]. Briefly, lysates or subcellular samples were subjected to 8%-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose by semi-dry transfer at 25 V for 1 h using a Trans-Blot SD apparatus (BioRad, Hercules, CA, USA). Membranes were blocked with PBS containing 5% milk and 0.1% Tween-20 at room temperature, and incubated with primary antibodies overnight at 4 • C. Membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse antibody (1:10,000) for 1 h. The signals were visualized with Enhanced Chemiluminescence detection solution (Amersham Pharmacia Biotech, Milan, Italy). The purity of fractions was tested by immunoblotting with β-actin. Densitometric analysis was carried out on the Western blots using the National Institute of Health (NIH) Image v1.63 software (National Institutes of Health, Bethesda, MD, USA). The pixel intensity for each region was analyzed, the background was subtracted, and the protein expressions were normalized to β-actin loading control for each lane.

Cell Cycle Analysis
Cell cycle analysis was performed using a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA). After treatments, cells were washed with cold PBS and harvested by centrifugation. Then, cells were resuspended in cold absolute ethanol and stored at −20 • C overnight. RNase A (0.2 mg mL −1 ) and propidium iodide (20 µg mL −1 ) were added, followed by incubation for 40 min in the dark, and cell cycle distribution was analyzed by flow cytometry cell sorting. Cell cycle distribution (sub-G1, G0/G1, S, and G2/M phase fraction) was analyzed using FlowJo software (Ver. 7.6.5, TreeStar, Ashland, OR, USA).

Design and Preparation of Small Interfering RNAs
Small interfering RNAs (siRNAs) were prepared by an in vitro transcription method. Silencing of apoptosis inducing factor (AIF) by siRNA was performed as previously reported using sense (5 -CUUGUUCCAGCGAU GGCAUTT-3 ) and antisense (5 -AUGCCAUCGCUGGAACAAGTT-3 ) RNA oligos that target nucleotides 151-171 in human AIF [21]. All template oligonucleotides were chemically synthesized and purified by polyacrylamide gel electrophoresis. In vitro transcription, annealing, and purification of siRNA duplexes were performed using the protocol supplied with the T7 RiboMAX Express RNAi System (Promega, Milan, Italy), as previously described [21].

Small Interfering RNA Transfection
Caki-1 cells (50%-70% confluence) were transfected with siRNA duplexes using the protocol supplied with the siRNA transfection reagent sc-29528 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Briefly, 5 µL of siRNA duplex (i.e., 0.6 µg siRNA) was diluted into 100 µL siRNA transfection medium: sc-36868 (Santa Cruz Biotechnology) without serum and antibiotics for about 20 min to form a lipid-siRNA complex. Transfection was initiated by adding the lipid-siRNA complex to six-well plates. Cells were incubated for 6 h at 37 • C in a CO 2 incubator. The final concentration of siRNA was 30 nM. Then, 1 mL of normal growth medium containing two times the normal serum and antibiotics concentration (2× normal growth medium) was added to the cells, without removing the transfection mixture, and incubated for an additional 24 h. Finally, the medium was aspirated and replaced with fresh 1× normal growth medium.

Visualization of Monodansylcadaverine
Autophagic vacuoles were labeled with monodansylcadaverine (MDC) by incubating Caki-1 cells grown on coverslips with 50 µM MDC in PBS at 37 • C for 30 min. Cells were fixed in 4% paraformaldehyde and washed with PBS. The cellular fluorescent changes were observed through confocal microscopy Zeiss LSM 700. For densitometric analysis, the NIH Image (v1.63) software (National Institutes of Health) was used.

Statistical Analysis
Data, presented as means ± SD, were analyzed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA) with unpaired Student's t-test or one-way analysis of variance (ANOVA), and when this returned p < 0.05, a post hoc analysis using Bonferroni test was performed; we used the Bonferroni-Dunn post hoc test in the ANOVA after a significant omnibus F-test. p < 0.05 was accepted as the level of statistical significance.

Cytotoxicity and Flow Cytometric Analyses of [Pt(O,O -acac)(γ-acac)(DMS)] and Cisplatin in Caki-1 Cells
The cytotoxicity data shown here were obtained by MTT metabolic assay and confirmed by SRB assay to rule out potential effects of   It is known that nuclear translocation of AIF is induced by several death stimuli [17]; therefore, we evaluate the involvement of AIF using small interfering RNAs (siRNAs) for AIF, in [Pt(O,O -acac)(γ-acac)(DMS)]-treated Caki-1 cells. As shown by immunoblotting detection of AIF in Caki-1 extracts, following treatment with AIF siRNA, AIF protein levels were significantly decreased ( Figure 2D). Controls were provided by untransfected cells (Con) and cells transfected with scrambled

[Pt(O,O -acac)(γ-acac)(DMS)] Induces Autophagy in Caki-1 Cells
The lack of [Pt(O,O -acac)(γ-acac)(DMS)]-induced apoptosis prompted us to study another mechanism of cell death, autophagy. Monodansylcadaverine may be used as a specific marker for autophagic vacuoles, since it accumulates in acidic compartments enriched in lipids, and staining intensity increases in cells induced to undergo autophagy. Caki-1 cells were then treated with It is known that nuclear translocation of AIF is induced by several death stimuli [17]; therefore, we evaluate the involvement of AIF using small interfering RNAs (siRNAs) for AIF, in [Pt(O,O'acac)(γ-acac)(DMS)]-treated Caki-1 cells. As shown by immunoblotting detection of AIF in Caki-1 extracts, following treatment with AIF siRNA, AIF protein levels were significantly decreased ( Figure  2D). Controls were provided by untransfected cells (Con) and cells transfected with scrambled siRNA oligos (Scr) ( Figure 2D). The silencing of AIF did not lead to a significant decrease in cell death after cisplatin (p = 0.01) or [Pt(O,O'-acac)(γ-acac)(DMS)] (p = 0.06) treatment in Caki-1 cells ( Figure 2E).
Furthermore, renal neoplasms are clinically resistant to Pt coordination complexes, not least to the cisplatin itself. Indeed, many chemotherapeutic agents have been used in the treatment of renal cell carcinoma in the advanced stage, but only floxuridine, 5-fluorouracil, and vinblastine have individually obtained results, though scarce [25]. More recently, mTOR and vascular endothelial growth factor receptor (VEGFR) inhibitors have been approved for the treatment of RCC [26][27][28][29]. Our recent results on Caki-1 cells [14] were confirmed here, with [Pt(O,O -acac)(γ-acac)(DMS)] inducing cytotoxicity faster and greater than that induced by cisplatin. The different and important observation in renal cells was that the high mortality rate associated with [Pt(O,O -acac)(γ-acac)(DMS)] was not due to apoptotic processes (caspases were not activated, poly ADP ribose polymerase (PARP) was not degraded, nor were DNA degradation or formation of condensed chromatin observed). Instead, the Caki-1 cells incubated with [Pt(O,O -acac)(γ-acac)(DMS)] underwent a remarkable autophagic process that is not seen with the use of cisplatin. This conclusion is based on evidence that several autophagic markers are activated in the presence of [Pt(O,O -acac)(γ-acac)(DMS)]. Autophagy does not always produce the same cellular effect, especially when it is triggered by antitumor drugs. Indeed, sodium selenite, [30] arsenic trioxide [31] and bortezomib are able to induce cell death through autophagy, whilst other studies showed that autophagy is significantly associated with cell survival and therapy resistance [32,33].
In our case, the inhibition of the autophagic process obtained with 3-MA showed an decrease in cell death due to [Pt(O,O -acac)(γ-acac)(DMS)]. This data suggests that autophagy triggered in Caki-1 cells is a process fostering cell death. The MAPK JNK1/2 is known to be involved in the regulation of autophagy of cancer cells in response to pharmacological stress [34,35]. We show here that JNK1/2 was phosphorylated in [Pt(O,O -acac)(γ-acac)(DMS)]-treated cells and that its inhibition blocked the [Pt(O,O -acac)(γ-acac)(DMS)]-induced Beclin-1 increase. Beclin-1, a key component of the autophagosome nucleation complex, can interact with Bcl-2 to form Beclin-1/Bcl-2 complex, which functions as an inhibitor of autophagy [36].
The phosphorylation of Bcl-2 by JNK promotes Bcl-2 degradation and dissociation from Beclin-1, leading to induction of autophagy [37,38]. Consistently, JNK activation is also essential for autophagic cell death induced by anticancer agents [39,40]. We also made clear in this study that the PI3K/AKT/mTOR/p70S6K pathway is part of the transduction mechanism used by [Pt(O,O -acac)(γ-acac)(DMS)] in inducing Caki-1 cell death. Several studies demonstrated that autophagy was often triggered by the inhibition of the PI3K/AKT/mTOR/p70S6K pathway concomitantly with the activation of the JNK pathway [41,42]. The PI3K/AKT/mTOR/p70S6K pathway has an important role in regulating cell survival and death, proliferation, and apoptosis.
In addition, various cellular signaling pathways, including AMPK and PI3K/AKT/ mTOR/p70S6K, play a vital role in the process of autophagy [43][44][45]. The PI3K/AKT pathway acts as a positive regulator of the mTOR pathway, which serves as a negative regulator of autophagy in cancer cells [24], so that disruption of the PI3K/AKT/mTOR/p70S6K pathway by anticancer agents induces autophagy. This is used in in advanced RCC patients with multiple adverse risk features [46] where the inhibition of mTOR (notably by using rapamycin analogues) displays an overall progression-free survival advantage. More precisely, p-mTOR in abundant nutrient conditions phosphorylates and inactivates the Unc-51 like autophagy activating kinases (ULK1/2) protein complex, while the deprivation of nutrients brings about the dephosphorylation and activation of ULK1 and ULK2 and their location near the phagophore. Finally, anticancer drugs that inhibited the PI3K/AKT/mTOR axis putatively stimulated autophagy [47].
In conclusion, we have shown a drastic difference in Caki-1 cell response to cisplatin and [Pt(O,O -acac)(γ-acac)(DMS)]. Cisplatin cytotoxicity was scarce, probably because Caki-1 cells are intrinsically able to resist and inhibit apoptosis [17,48]. The cytotoxic effect of [Pt(O,O -acac)(γ-acac)(DMS)] was strong compared to that observed in other cancer cell lines, due to autophagy through a mechanism mediated by JNK and PI3K/AKT/mTOR/p70S6K pathways.
[Pt(O,O -acac)(γ-acac)(DMS)] may therefore represent an alternative approach to reducing renal cancer mass. However, to assess the general applicability of these findings on renal cell cancer patients, further experiments performed on more renal cancer cell lines are required.

Conflicts of Interest:
The authors declare no conflict of interest.