Carmustine

Cortical neurons from E21 Sprague–Dawley rats were cultured as described60 (link)61 (link) and experiments performed at 8–10 DIV. Puma-knockout neurons were prepared from E17 Puma-null founder mice obtained from Professor Andreas Strasser62 (link). To obtain astrocyte-free cultures the antimitotic agent cytosine arabinoside (Sigma) was added to the cultures on the day of plating (DIV0) rather than the usual DIV4. This results in <0.2% GFAP-positive astrocytes rather than the usual 5–10% (ref. 29 (link)). Cortical astrocyte cultures were prepared as previously described63 (link). Before stimulations, neurons were transferred to a trophically deprived medium (22 (link) (Tmo) containing 10% MEM (Invitrogen) and 90% Salt/Glucose/Glycine medium consisting of: 114 mM NaCl, 0.219% NaHCO₃, 5.292 KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, 1 mM glycine, 30 mM glucose, 0.5 mM sodium pyruvate, 0.1% phenol red; osmolarity 325 mOsm l⁻¹). Bursts of action potentials were induced through stimulation with 50 μM BiC and 250 μM 4-aminopyridine (BiC/4-AP), which in turn disinhibits the neuronal network and depolarizes the cells, generating high frequency action potential firing22 (link). The following reagents were used: buthionine sulfoximine (BSO), carmustine (BCNU), MK-801 were purchased from Tocris, BiC and H₂O₂ from Sigma, γ-glutamylcysteine-ethyl ester (GCEE) from Bachem, 4-aminopyridine from Calbiochem. To quantify cell death, neurons were fixed and subjected to nuclear DAPI (Vectorlabs) staining, then imaged using a Leica AF6000 LX imaging system with a DFC350 FX digital camera. Cell death was quantified by counting (blind) the number of pyknotic nuclei as a percentage of the total, with ∼1,500 cells counted per treatment.

We performed a single-center retrospective cohort study. Our study population included patients who were diagnosed with lymphoma and submitted to autologous HSCT at the Centro Hospitalar Universitário Lisboa Norte, EPE (CHULN) between January 2005 and December 2015. As exclusion criteria we defined: Patients under the age of 18 years, patients with CKD already on renal replacement therapy; patients who underwent renal replacement therapy one week before transplantation and those with previous HSCT.
The conditioning regimens used followed institutional protocols – Carmustine, Etoposide, Cytarabine and Melphalan (BEAM) or Thiotepa, Etoposide, Cytarabine and Melphalan (TEAM). Total body irradiation is not available in our institution, and it is not contemplated in any of our institutional protocols.
Our data collection was based on registers of daily medical records, 6-h period nurses’ records and diagnostic exams during hospital admission period for HSCT, as well as all routine medical records and laboratorial analysis before and after HSCT. We collected variables related to patient demographic characteristics (age, gender, race, body weight, and height), related to patient comorbidities (diabetes mellitus, hypertension, arrythmia, valvular heart disease, ischemic heart disease, cerebrovascular disease, chronic liver disease, intestinal inflammatory disease, peptic ulcer, connective tissue disease, chronic obstructive pulmonary disease, and solid-organ cancer, psychiatric disease), related to lymphoma and previous treatment approach (subtype of lymphoma, number of previous lines of therapy, and exposure to radiotherapy in the past); related to HSCT (conditioning regimen, cells source, period of aplasia, length of stay in hospital, blood results on hospital admission day for HSCT, sinusoidal obstructive syndrome, thrombotic microangiopathy, sepsis, nephrotoxic drugs, shock, cytomegalovirus infection, AKI, and AKI stage) and related to prognostic impact (time to relapse, time to all-cause mortality and eGFR 1 year after HSCT and 3 years after HSCT).
All patients were followed until death or censored at 36 months (3 years) after HSCT. This timeline was defined because patients are often transferred to other hospitals closer to their residence for continued follow-up after this 3-year period.

To test the induction of apoptosis in cancer cells, Annexin V/propidium iodide assay was performed [43 (link)]. Cells were seeded at 10⁶ cells/well density in 6-well plates and left to adhere for 24 h. The next day, cells were treated with either one of the steroid compounds 10a, 10d, 10e, 10f, or 10g at 20 μM concentration for 24 h or with one of the drugs (Bleomycin, Carmustine, Cisplatin, Doxorubicin, or Epirubicin) in individual pre-determined concentrations based on the MTT screening. According to the MTT screening, we could identify effective steroid + chemotherapy drug combinations; and from these positive hits, the ones with the lowest chemotherapy drug concentration were always regarded (see Supplementary Material Table S2 green colored labeling). In such cases, where a given chemotherapy drug that was applied together with different steroid derivatives and the lowest efficient drug concentration to reduce the viability of cancer cells was different, then always the higher drug concentration was chosen for the apoptosis experiments. The drug concentrations for single or combination treatments upon apoptosis experiments were the following: Bleomycin 215 µM, Cisplatin 45 µM, Epirubicin 19 µM, Carmustine 603 µM, Doxorubicin 252 µM.
To estimate the degree of membrane efflux inhibition exhibited by the synthetic steroids and how this would affect the degree of apoptosis induced in steroid+drug combination treatments, furthermore, to compare the efficiency of these steroid-induced actions with the performance of a well-known ABC transporter inhibitor (Verapamil [44 (link)]), we used Verapamil in parallel experiments. Verapamil was applied at 4 μM concentration for 24 h.
After each treatment, media were discarded and the cells were collected and stained with Annexin V-fluorescein isothiocyanate/propidium iodide (PI), according to the manufacturer’s recommendations. The fluorescence values of 10,000 cells/sample were measured with FACSCalibur™, and the data was analyzed with FlowJo V10.0.7 software. Since the fluorescence of PI might interfere with that of Doxorubicin and Epirubicin, dot plots of Annexin V vs. forward scatter were created, rather than Annexin V vs. PI, to present the results.