Background
NBDHEX (6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol) is the leading compound of a class of nitrobenzoxadiazole derivatives (NBDs) with promising anticancer properties. Indeed, by targeting glutathione transferases (GSTs) these compounds have been shown to impair growth and survival of cancer cells at multiple levels [
1‐
5]. First, NBDs act as strong inhibitors of GSTs catalytic activity; accordingly, they can hinder the GST-mediated conjugation of several electrophilic anticancer drugs to reduced glutathione (GSH), which in turn would result in drug detoxification and extrusion from the cell via specific export pumps [
1,
6‐
9]. Moreover, NBDHEX is not a substrate of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) transporters, so that it accumulates in tumor cells, overcoming another major mechanism of cancer cell chemoresistance [
10‐
12]. Secondly, NBDs are able to disrupt the interaction between the GST isoform GSTP1-1 and key signaling effectors involved in the regulation of cell survival and proliferation, namely the adaptor protein TNF-Receptor associated factor 2 (TRAF2) and the c-Jun N-terminal kinase (JNK) [
2,
3,
13,
14]. The NBDs-induced release of TRAF2 from the complex with GSTP1-1 leads to the activation of the apoptosis signal-regulating kinase (ASK1), which in turn activates both p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways [
3,
8]. As a result, p38 causes cell cycle arrest, while JNK promotes apoptosis, the activation of this MAPK pro-apoptotic pathway being further sustained by the NBDs-induced release of JNK from the complex with GSTP1-1 [
3,
9]. Therefore, NBDs can exert antitumor effects either per se or by potentiating the efficacy of conventional anticancer drugs whose action relies on the activation of these MAPK pathways [
9].
In fact, NBDHEX has shown a broad-spectrum of activity against cancer cells of different origins, including osteosarcoma, Ewing’s sarcoma, melanoma, mesothelioma, lung and hepatic carcinoma and different types of leukemia, either alone or in combination with antitumor drugs such as cisplatin, doxorubicin, vincristine, and temozolomide [
2,
3,
10,
15‐
18]. Moreover, in in vivo studies performed on various tumor types xenografted in mice, NBDHEX proved to be effective in reducing both cancer growth and metastatic spread and was well tolerated [
17,
19,
20].
Besides NBDHEX, the most promising compound among the NBDs is the recently developed water-soluble analogue MC3181 (2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol) [
5,
21]. This compound bears two oxygen atoms within the alkyl chain bound at the C4 position of the NBD scaffold, resulting in a more than 50-fold increase in aqueous solubility compared to NBDHEX [
5].
Given the encouraging results obtained with NBDHEX on osteosarcoma models [
3,
15,
20], we performed experiments initially aimed at evaluating the activity of the new compound MC3181 on U-2OS human osteosarcoma cells cultured in vitro. Treatment with either MC3181 or the parent compound NBDHEX caused the accumulation of cytoplasmic vacuoles in U-2OS cells and prompted us to investigate the possible effects of these NBDs on autophagy. We here demonstrate that NBDs cause late-stage autophagy inhibition via the activation of JNK.
Discussion
Autophagy is an intracellular catabolic pathway by which superfluous or damaged intracellular proteins and organelles are first engulfed by autophagosomes and then delivered to autolysosomes for degradation and recycling of their molecular constituents [
39,
40]. Therefore, autophagy is deeply involved in cell homeostasis and adaptation to stress; by promoting the survival of transformed cells under hypoxic, nutritional and therapeutic stress conditions, it plays a major role in the progression of established neoplasms [
40,
41]. Cancer cells live under a strong metabolic pressure due to their high energy demand and inefficient energy production; they often reside in a microenvironment that provides an inadequate supply of oxygen, nutrients and growth factors, so that they are predicted to be more susceptible to the suppression of autophagy than non-cancerous cells [
29,
39,
40]. On the other hand, prolonged stress and sustained autophagy activation may eventually lead to cell death in certain cellular contexts such as in apoptosis-defective cells [
41‐
43]. Based on such evidence, modulators able to induce autophagic cell death or inhibit protective autophagy are currently being investigated in order to manipulate autophagy for clinical benefit in cancer patients [
41‐
44]. In this context, autophagy inhibition is emerging as a promising therapeutic strategy against cancer [
41‐
44]. Moreover, the potential of autophagy inhibitors such as CQ to increase tumor cell death upon therapeutic stress conditions induced by radiation, genotoxic agents, or tumor-targeted agents is documented by several studies [
41‐
45].
We here demonstrate that NBD compounds, already known to inhibit both the catalytic and TRAF2/JNK1-sequestering activity of GSTs and to trigger cell cycle arrest and apoptosis in cancer cells [
4,
5,
14], share the ability to act as late-phase autophagy inhibitors. In fact, we provide evidence that NBDs induce the accumulation of autophagic vesicles and LC3-II while reducing both basal and nutritional stress-induced autophagic flux in U-2OS cells. Furthermore, we show that the effects of NBDs on autophagy are general rather than cell type-specific. Indeed, these compounds produce a variable increase of both LC3-II and the autophagy selective substrate p62 in a panel of tumor cell lines of different origins, the concurrent increase of these markers being consistent with an impairment of autophagosome clearance [
27,
28]. At present we do not know whether this effect is due to the inhibition of autophagosome-lysosome fusion or to the defective degradation of the autophagic material in the autophagolysosome [
27].
The tumor cell lines used in this study display different degrees of sensitivity to the cytostatic/cytotoxic effects of NBDHEX and MC3181, their IC
50s for the two compounds ranging from about 1 μM for U-2OS to about 11–16 μM for HT-29 cells. No evident correlation emerges from the comparison of the cell lines’ sensitivity to the cytostatic/cytotoxic effects of NBDs and the degree of autophagy inhibition induced by the compounds. For instance, based on autophagic markers accumulation levels, NBDs induce comparably high levels of autophagy impairment in U-2OS and HT-29 cells, which however display the lowest and highest IC
50 values among the cell lines included in the study. Conversely, the IC
50s of U-2OS, MM-B1 and MCF-7 cells are quite similar, while the extent of autophagy inhibition observed in the latter two cell lines appears much lower as compared to that of U-2OS. On the other hand, this apparent lack of correlation is not conclusive since different cell lines can be characterized by different degrees of autophagy addiction and, accordingly, can display marked differences in how they respond to autophagy inhibition [
29,
44,
46,
47]. Therefore, experiments performed in a given cell line after silencing of essential autophagy genes [
27] will be necessary to investigate whether and to what extent the effects of NBDs on tumor growth and survival actually depend on the impairment of the autophagic pathway. In any case, our results suggest that the therapeutic potential of NBDs may not rely solely on their effectiveness in inducing cell cycle arrest and apoptosis, but also on their ability to weaken the capacity of tumor cells to endure stress conditions via autophagy. These findings may bear relevance for future studies specifically aimed at evaluating the efficacy of NBDs on autophagy-dependent tumor types as well as for the rational design of combined approaches based on the association of NBDs with antitumor drugs known to induce pro-survival autophagy [
43‐
45].
A second interesting finding of this study regards the role played by JNK in the autophagy-inhibitory effect of NBDs. In fact, treatment with NBDs triggers the activation of JNK, along with other signaling pathways [
3,
5,
14,
21], and leads to autophagic flux impairment. Moreover, by silencing JNK1 expression in U-2OS cells we here provide evidence that autophagy impairment by NBDs requires JNK activity: as compared to the untreated cells, in JNK1-silenced cells treated with NBDHEX we found an increase of LC3-II compatible with an increased formation of autophagosomes, but autophagic flux inhibition was no longer observed. These results implying a role for JNK in mediating autophagy impairment are unexpected given that the literature extensively supports the pro-autophagic function of this MAPK [
35,
36]. In particular, JNK has been reported to promote autophagy in response to different types of stress signals by two main mechanisms. First, by phosphorylating Bcl-2, JNK induces its dissociation from the autophagy-regulatory protein Beclin-1 which, in turn, interacts with multiple partners to promote the formation and maturation of autophagosomes [
36]. Second, JNK activation drives the upregulation of damage-regulated autophagy modulator (DRAM), a lysosomal protein whose stimulatory role in autophagy is thought to rely on the ability to regulate the fusion of autophagosomes and lysosomes [
48]. By contrast, two recent papers report that suppression of JNK signaling induces autophagy in neurons [
37], lens fiber cells and MCF-7 cells [
38] by decreasing FoxO-dependent expression of Bnip3, which in turn promotes autophagy via the dissociation of Beclin1 from Bcl-xl [
37], and by acting as a positive regulator of the autophagy inhibitor MTORC1 [
38]. Therefore, it appears that the autophagy inhibitory action of JNK can be mediated by different mechanisms. Besides, a factor that may explain the involvement of JNK in the inhibition of autophagy caused by NBDs is the JNK-dependent activation of caspases, since it has been reported that these proteases can inhibit autophagy through the cleavage of essential autophagy proteins including Beclin-1 and different Atg proteins [
49]. The finding that NBDs caused autophagy impairment and caspase-3 activation in JNK-positive U-2OS, but no autophagic flux inhibition or caspase-3 activation in JNK-silenced cells supports this hypothesis.
What emerges from this complex scenario is that JNK can act at multiple levels in the dynamic multistep process of autophagy to generate context-specific responses, probably depending on the mode and kinetics of its activation as well as on the cooperation with different signal transduction pathways [
37]. In this respect, due to their ability to induce the dissociation of both the TRAF2-GSTP1-1 and JNK-GSTP1-1 complexes, NBDs act as multi-target compounds able to activate not only JNK but also the TRAF2 downstream target p38 [
3,
14,
21], which has been reported to act, through mechanisms still poorly defined, both as a positive and negative regulator of autophagy [
35,
36]. Accordingly, the concurrent activation of different signaling pathways by NBDs may participate in modulating JNK-dependent responses leading to autophagic flux impairment.
Authors’ contributions
CP conceived of the study, designed and performed most of the experiments, drafted the manuscript. ADL carried out PLA and FACS analysis, participated in data analysis/interpretation. NR participated in data analysis/interpretation and in critical revision of the manuscript. MF and DR synthesized NBDHEX and MC3181. AMC conceived of the study, participated in its design and coordination, provided the funding, helped to draft the manuscript. All authors read and approved the final manuscript.