Cisplatin

To generate cisplatin resistant BT-549 cell line, parental cell line BT-549 was given continuous exposure to cisplatin (Sigma-Aldrich) following IC₅₀ values which were obtained from initial dose-response studies of cisplatin (0.1 μM–100 μM) over 72 h. Initially, BT-549 was treated with cisplatin (IC₅₀) for 72 h and the medium was replaced with fresh media to allow cells to recover for further 72 h. This process of development was carried out for approximately 6 months, followed by re-assessing of IC₅₀ concentrations. Cells were then maintained at the new cisplatin IC₅₀ concentrations for further 6 months.

HK-2 cells (ATCC, Manassas, VA, USA), which were immortalized by transduction with human papillomavirus 16 E6/E7 genes, were maintained in a keratinocyte serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5 ng/mL human recombinant epidermal growth factor 1–53 and 50 μg/mL bovine pituitary extract at 37 °C in a humidified 5% CO₂.
For analyzing the protective role of SGLT2 inhibitors in cisplatin-induced cytotoxicity, HK-2 cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin (Sigma-Aldrich, St. Louis, MO, USA) in the absence or presence of 1–25 μM canagliflozin, dapagliflozin, or empagliflozin (APExBIO, Houston, TX, USA) for 24 h.
To test whether canagliflozin induces autophagy or modulates AMPK-mTOR pathway and whether the protective effect of canagliflozin on cisplatin-induced cytotoxicity occurs in autophagy or AMPK activation-dependent manner, the cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin in the absence or presence of 10 μM canagliflozin and/or 10 μM chloroquine, 5 nM bafilomycin A (Sigma-Aldrich), or 1 μM compound C (APExBIO) for 24 h.

Cisplatin (Sigma, USA) is dissolved in 1 mL of anhydrous methanol, and then the above Cisplatin solution and 3mL of PEG-PLA (Sigma, USA) solution dissolved in chloroform are uniformly mixed (the ratio of lipid material to Cisplatin is 5: 1). Then the mixed solution of Cisplatin and PEG-PLA were transferred into an eggplant bottle, and the organic solvent was removed by a vacuum rotary evaporator to form a dry drug lipid film at the bottom of the bottle. Subsequently, 2mL PBS buffer solution was added to dissolve the drug lipid membrane, and the drug lipid membrane was hydrated in a water bath at 60° C for 30 minutes. The hydrated solution was filtered through polycarbonate membrane (FormuMax Scientific, USA) with a pore diameter of 0.2 micron to obtain Cisplatin polylactic acid nanoparticles (CDDP-PLA NPs) solution [27 (link)]. CDDP/CQ-PLA NPs were prepared using the same method above using Cisplatin and chloroquine instead of Cisplatin. PLA NPs were prepared using the same method above using no drug instead of Cisplatin.

Three months old male Wistar albino rats (250–300 g, n = 32) were purchased from the Military Medical Academy, Serbia. All animals were kept in transparent cages (four animals per cage) under standard conditions (temperature 23 ± 1 °C, humidity 50 ± 5%) with a light/dark cycle (12/12h) and had free access to food and water.
The rats were randomly assigned into four equal groups: control, cisplatin (CIS), NAC, and CIS + NAC groups (n = 8). As shown in Table 1, at the start of the trial (day 1) the control and cisplatin groups received saline (approximately 2 mL i.p.), while the NAC and CIS + NAC groups were administered with NAC (500 mg/kg i.p., Sigma-Aldrich, Munich, Germany). On the fifth day, the control group received saline (in the same manner), the CIS group was treated with cisplatin (7.5 mg/kg i.p.) (Merck, Paris, France), the NAC group was administered with NAC again (500 mg/kg i.p.), and the CIS + NAC group was simultaneously treated with cisplatin and NAC (7.5 and 500 mg/kg i.p., respectively).
All research procedures were carried out in accordance with the European Directive for the welfare of laboratory animals No 86/609/EEC, the principles of Good Laboratory Practice (GLP), and in accordance with the ARRIVE guidelines. All experiments were approved by the Ethical Committee of the Faculty of Medical Sciences, University of Kragujevac, Serbia.