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01.12.2018 | Primary Research | Ausgabe 1/2018 Open Access

Cancer Cell International 1/2018

Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Zeitschrift:
Cancer Cell International > Ausgabe 1/2018
Autoren:
Sanja Aveic, Marcella Pantile, Pierfrancesco Polo, Viktoryia Sidarovich, Marilena De Mariano, Alessandro Quattrone, Luca Longo, Gian Paolo Tonini
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12935-018-0557-4) contains supplementary material, which is available to authorized users.

Abstract

Background

A growing field of evidence suggests the involvement of oncogenic receptor tyrosine kinases (RTKs) in cell transformation. Deregulated activity of RTKs in tumors can determine disease progression and therapeutic responses in several types of cancer, including neuroblastoma (NB). Therefore, RTKs targeting is a worthwhile challenge for the oncologists. Nevertheless, acquired resistance to RTK inhibitors (RTKi) remains a serious problem. Autophagy activation is among the possible obstacles for good efficacy of the therapy with RTKi.

Methods

Under different treatment conditions we measured autophagic flux using immunoblot and immunofluorescence assays. Death induction was validated by trypan blue exclusion assay and FACS analysis (calcein-AM/propidium iodide). The NB cell lines SH-SY5Y and Kelly were used for the in vitro study.

Results

In order to define whether autophagy might be a limiting factor for the efficacy of RTKi in NB cells, we firstly checked its activation following the treatment with several RTKi. Next, we investigated the possibility to increase their therapeutic efficiency by combining RTKi with autophagy blocking agents in vitro. We exploited the effectiveness of three RTKi either alone or in combination with autophagy inhibitors (Chloroquine—CQ and Spautin-1). We demonstrated that autophagy induction was drug-dependent, and that its inhibition increased the anti-tumor activity of a single RTKi unevenly. We observed that the combined use of blocking agents which impair late autophagy events, such as CQ, and RTKi can be more effective with respect to the use of RTKi alone.

Conclusions

In the present report, we assessed the conditions under which autophagy is activated during the use of different RTKi currently in the pre-clinical evaluation for NB. We summarized the achievements of combined RTK/autophagy inhibitors treatment as a promising approach to enhance the efficacy of RTKi in impairing tumor cells viability.
Zusatzmaterial
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: 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.
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: 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.
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