Additional file 3 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 3: Figure S3. Expression of extracellular signal-regulated kinase (ERK) upon treatments. Increasing concentrations of RTKi were used for the treatment, and levels of phospho-mTOR, phospho-PI3K, phospho-AKT and phospho-ERK (pERK) and total ERK were evaluated by western blot. GAPDH served as a loading control protein. D = DMSO; the numbers indicate the concentrations used in μM

Additional file 4 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 4: Figure S4. Autophagy activation in SH-SY5Y cell line upon treatment with RTKi. Autophagy activation upon treatment with Afatinib and Sorafenib was validated using immunofluorescence in order to visualize a creation of autophagosomes; 60X magnification. DMSO treatment was used as control. Green—autophagy vacuoles; Blue—Hoechst, nuclear staining

Additional file 1 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 1: Figure S1. Caspase-3 and PI measurement upon treatments. A) Apoptosis activation was evaluated by measuring the intensity of PARP cleavage (89 kDa proteolytic PARP fragment), and by evaluating a decrease in the pro-Caspase-3 (35 kDa protein) level. Active Caspase-3 forms are observed as cleaved proteins with molecular weight below 20 kDa. GAPDH served as a loading control protein. D = DMSO; Af—Afatinib (8 μM); Sor—Sorafenib (14 μM); TP—TP-0903 (0.15 μM). B) Percentage of PI positive cells with respect to total cell number was presented as mean ± SD out of triplicates. D—DMSO; Af—Afatinib; Sor—Sorafenib; TP—TP-0903. p value is marked as **p < 0.01; n.s.—non-significant. C) Changes in the proliferation rate upon treatment with the three RTKi were determined by means of PCNA expression. The numbers indicate relative expression (densitometry) obtained after normalization to GAPDH protein level, which was used a loading control, and with respect to DMSO control (DMSO = 1)

Additional file 2 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 2: Figure S2. Colony formation assay. Effects on the capacity of SH-SY5Y cells to form colonies upon treatment with RTKi alone or in combined treatment with CQ or SP1 was measured. Upper image is the representative out of triplicate experiment. Lower graph represents a colony number calculated as a percentage of control (DMSO; 100%), and presented as the mean ± SD out of triplicate measurement. p value is marked as **p < 0.01; n.s.—non significant (Dunnett’s test). CQ—Chloroquine; SP1—Spautin-1; Af—Afatinib

Impact of Interleukin-6 –174 G>C Gene Promoter Polymorphism on Neuroblastoma

<div><p>Background</p><p>Common variants in DNA may predispose to onset and progression of neuroblastoma (NB). The genotype GG of single nucleotide polymorphism (SNP) rs1800795 (−174 G>C) in interleukin (IL)-6 promoter has been associated with lower survival of high-risk NB.</p><p>Result</p><p>To evaluate the impact of <i>IL-6</i> SNP rs1800795 on disease risk and phenotype, we analyzed 326 Italian NB patients and 511 controls. Moreover, we performed <i>in silico</i> and quantitative Real Time (qRT)-PCR analyses to evaluate the influence of the SNP on gene expression in 198 lymphoblastoid cell lines (LCLs) and in 31 NB tumors, respectively. Kaplan-Meier analysis was used to verify the association between <i>IL-6</i> gene expression and patient survival. We found that <i>IL-6</i> SNP is not involved in susceptibility to NB development. However, our results show that a low frequency of genotype CC is significantly associated with a low overall survival, advanced stage, and high-risk phenotype. The <i>in silico</i> (<i>p</i> = 2.61×10⁻⁵) and qRT-PCR (<i>p</i> = 0.03) analyses showed similar trend indicating that the CC genotype is correlated with increased level of <i>IL-6</i> expression. In report gene assay, we showed that the −174 C variant had a significantly increased transcriptional activity compared with G allele (<i>p</i> = 0.0006). Moreover, Kaplan-Meier analysis demonstrated that high levels of <i>IL-6</i> are associated with poor outcome in children with NB in two independent gene expression array datasets.</p><p>Conclusions</p><p>The biological effect of SNP <i>IL-6</i>–174 G>C in relation to promotion of cancer progression is consistent with the observed decreased survival time. The present study suggests that SNP <i>IL-6</i>–174 G>C may be a useful marker for NB prognosis.</p></div

Case-Control study of <i>IL-6</i>–176 (G>C) SNP.

a<p>P-value and OR obtained by Armitage's trend test.</p

Test for independent statistical significance of −174 <i>IL-6</i> SNP after adjustment for NB risk factors.

<p>HR, hazard ratio; CI, confidence interval; C-index, concordance index.</p>a<p>C-index calculated including in the model only the NB risk factor (Age or MYCN or INSS stage 4).</p>b<p>C-index calculated including in the model the NB risk factor and <i>IL-6</i> SNP.</p

SNP genotype correlation to <i>IL-6</i> gene expression and NB outcome.

<p><i>In silico</i> and qRT-PCR analysis of <i>IL-6</i> mRNA expression in (A) 198 LCLs and (B) 31 NB tumors, respectively, stratified according to the SNP <i>IL-6</i>–174. (C) Luciferase report gene assay carried out in HEK293 cells. Data shown in percentage are the mean ± SD from three independent transfection experiments, each done in triplicate and compared with promoter less control. Kaplan-Meier analysis is shown, with individuals grouped by median of expression of <i>IL-6</i> for OS and Progression Free Survival (PFS) rates in (D and E) 88 NB patients and (F) only PFS for 102 INSS stage 4 patients with <i>MYCN</i> not amplified. OS data of Seeger dataset were not available.</p

Characteristics of NB patients stratified per <i>IL-6</i> -176 (G>C) SNP genotype.

<p>N.A.  =  not available.</p>a<p>p-values and ORs from comparison of allelic frequencies.</p>b<p>p-values and ORs from comparison of genotype frequencies (GG/GC vs CC).</p>c<p>p-values and ORs from comparison of stage 1+2 patients vs stage 3+4 patients.</p

Kaplan-Meier curves for OS rates.

<p>OS rates were compared between CC and any G (GC or GG) for the SNP <i>IL-6</i>–174 in (A) all patients, (B) in not <i>MYCN</i> amplified patients, (C) in high-risk patients and (D) not <i>MYCN</i> amplified high-risk patients.</p