Preparation and Antiproliferative Activity of Liposomes Containing a Combination of Cisplatin and Procainamide Hydrochloride

We have previously reported the enhancement of the antiproliferative and apoptotic activities of cis-diamminedichloroplatinum­(II) (DDP) when it is coadministered with a class I antiarrhythmic drug procainamide hydrochloride (PA). Here, we determined the antiproliferative activity of DDP, either in solution or loaded in liposomes, in the presence of PA, in the bulk solution, or directly embedded in liposomes together with DDP. Our results show that PA potentiates the activity of DDP-liposomes and that this effect is maintained at least in some of the investigated cell types when both drugs were mixed and loaded together into liposomes

Supplementary Data from z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

Supplementary Figures S1-S6 and Supplementary Tables S1-S3.</p

Relationship between total CNAs and DNA ploidy in OPMDs/OSCCs.

<p>(A) shows the number of total CNAs detected in oral potentially malignant disorders (OPMDs) and oral squamous cell carcinomas (OSCCs); (B) shows the number of total CNAs detected in non-dysplastic OPMDs (ND-OPMDs); (C) shows the number of total CNAs detected in dysplastic OPMDs (D-OPMDs) and OSCCs. The bottom and the top of each box show the first and third quartile, respectively, while the line inside the box represents the median (second quartile). Please notice that when the median is not shown, its value = 0. The tips of the whiskers represent the minimum and the maximum data value. The boxes corresponding to the number of CNAs detected in DNA diploid sites of oral mucosa are shown in white; the boxes corresponding to the number of CNAs detected in DNA aneuploid sites of oral mucosa are shown in gray. CNAs are referred to as: total focal gains, TFG; total broad gains, TBG; total focal losses, TFL; total broad losses, TBL. Broad gains and broad losses correspond to gains or losses of more than half a chromosome arm, respectively. Significant MW P-values (P < 0.05) and their corresponding q-values are shown. The FDR q-value method was applied for multiple testing (n = 4) correction; q-values < 0.1 are indicated in bold. (A) N = 94 OPMDs/OSCCs; N = 58 DNA diploid; N = 36 DNA aneuploid. (B) N = 46 ND-OPMDs; 35 DNA diploid; 11 DNA aneuploid. (C) N = 48 D-OPMDs/OSCCs; 24 DNA diploid; 24 DNA aneuploid.</p

NAC, Tiron and Trolox Impair Survival of Cell Cultures Containing Glioblastoma Tumorigenic Initiating Cells by Inhibition of Cell Cycle Progression

<div><p>Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.</p></div

NAC, tiron and trolox determine only modest changes in the ROS levels in the PT4 cell culture containing GBM TICs.

<p>Representative experiment of FCM analysis of PT4 culture containing GBM TICs cells incubated with the indicated fluorescent probe after 48(containing NAC, tiron or trolox) replacement. DCFDA, MitoSOX Red and TMRE were used to evaluate global ROS, mitochondrial superoxide and mitochondrial proton gradient, respectively. This analysis showed that trolox reduced global cellular ROS levels but did not substantially modify mitochondrial superoxide levels. NAC and tiron, instead, slightly decreased mitochondrial superoxide levels while slightly enhancing global cellular ROS levels. This analysis also showed that the drugs used in this study did not induce changes of the mitochondrial proton gradient displayed by the PT4 cells in control conditions.</p

Cell cultures containing GBM TICs display higher endogenous ROS generation than normal astrocytes.

<p>Representative experiment of FCM analysis of normal human astrocytes (NA) and cell cultures containing GBM TICs obtained from four different patients (PT1, PT2, PT4, PT5) incubated with the indicated fluorescent probe. Mitotracker Green, TMRE, DCFDA and MitoSOX Red were used to evaluate mitochondrial mass, mitochondrial proton gradient, global ROS and mitochondrial superoxide, respectively.</p

Cell survival of the PT4, MM1 and FO-1 cell cultures containing TICs.

<p>Survival of PT4 cells (A, E); MM1 cells (B) and FO-1 cells (C, D) after 6 days of treatment with the indicated substances and solvent controls. The medium renewal schedule was identical to that used for the cultures containing GBM TICs (see Introduction). Cell survival is expressed in arbitrary units as evaluated by MTT analysis. Standard deviations are indicated as vertical bars (n = 3 independent assays). DMSO concentration in (D) was 0.1% vol/vol. Drug concentrations in (D) were: NAC 20 mM, PLX4032 10 µM. Gefitinib final concentration in (E) was 3.9 µM. #The unpaired t-test was significant at P<0.05. §The unpaired t-test was significant at P = 0.01 or less. *The unpaired t-test was significant at P<0.001. **The unpaired t-test was significant at P<0.0001.</p

Patients enrolled in the study, type and number of oral mucosa subsites that underwent biopsy and were analyzed by hr DNA-FCM and aCGH.

<p>¹ Non-dysplastic oral potentially malignant disorder.</p><p>² Dysplastic oral potentially malignant disorder</p><p>³ Oral squamous cell carcinoma, OSCC.</p><p>^a 155 females and 137 males; median age 66.9 (range 19.8–93.8) and 60.3 (range 18.5–86.3) years, respectively. Three hundred thirty one OPMDs/OSCCs oral subsites of 292 patients were sampled by single or multiple biopsies yielding 522 bioptic samples (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142294#pone.0142294.t001" target="_blank">Table 1</a>). The nuclei suspensions of a subset of 145 bioptic samples obtained from 99 oral mucosa subsites of 99 of these 292 patients were also processed for DNA extraction and subsequent aCGH analysis. Therefore, the DNA index (DI) was also measured for each of the 145 DNA samples processed for aCGH. A single OPMD or OSCC for each patient was examined by aCGH.</p><p>^b 52 females and 42 males; median age 70.5 (range 19.8–93.8) and 60.3 (range 18.5–86.3) years, respectively.</p><p>^C The bioptic samples of 5 patients were removed from the statistical analysis because they did not comply with the aCGH quality control criteria.</p><p>Patients enrolled in the study, type and number of oral mucosa subsites that underwent biopsy and were analyzed by hr DNA-FCM and aCGH.</p

Relationship between total CNAs and histological diagnosis in TNG and BM OPMDs/OSCCs.

<p>(A) shows the number of total CNAs detected in oral potentially malignant disorder (OPMD) and oral squamous cell carcinoma (OSCC) in tongue (TNG) and buccal mucosa (BM) sites; (B) shows the number of total CNAs detected in non-dysplastic OPMDs (ND-OPMDs) in TNG and BM sites. (C) shows the number of total CNAs detected in dysplastic OPMDs (D-OPMDs) and OSCC TNG and BM sites. The bottom and the top of each box show the first and third quartile while the line inside the box represents the median (second quartile). Please notice that when the median is not shown, its value = 0. The tips of the whiskers represent the minimum and the maximum data value. CNAs are referred to as: total focal gains, TFG; total broad gains, TBG; total focal losses, TFL; total broad losses, TBL. Broad gains and broad losses correspond to gains or losses of more than half a chromosome arm, respectively. The boxes corresponding to the number of CNAs detected in TNG OPMDs/OSCCs are shown in white bars, whereas those of CNAs detected in BM OPMDs/OSCCs are shown in gray bars.</p

NAC, tiron and trolox treatments of the PT4 cultures containing GBM TICs inhibit the phosphorylation of the retinoblastoma (RB) protein.

<p>Western blot analyses were performed with lysates of the PT4 cell culture containing GBM TICs treated for 48 h and 6 days with the indicated antioxidant drug. The membrane was challenged first with an anti phosphorylated RB (Ser 807/811) antibody, then with an anti total RB antibody and lastly with an anti tubulin antibody. Stripping and control of effective stripping were performed before re-challenging of the membrane.</p