Abstract
The aim of present study is to elucidate autophagic mechanism of tanshinone I (Tan I) in H28 and H2452 mesothelioma cells. Herein, Tan I exerted cytotoxicity with autophagic features of autophagy protein 5 (ATG5)/ microtubule-associated protein 1A/1B-light chain 3II (LC3 II) activation, p62/sequestosome 1 (SQSTM1) accumulation and increased number of LC3II punctae, acridine orange-stained cells and autophagic vacuoles. However, 3-methyladenine (3MA) and NH4Cl increased cytotoxicity in Tan I treated H28 cells. Furthermore, autophagy flux was enhanced in Tan I-treated H28 cells transfected by RFP-GFP-LC3 constructs, with colocalization of GFP-LC3 punctae with LAMP1 or Lysotracker. Interestingly, C-terminal UBA domain is required for Tan 1 induced aggregation of p62 in H28 cells. Notably, Tan I upregulated CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring protein-1 (IRE1) and p-c-Jun N-terminal kinase (p-JNK), but silencing of IRE1 or p62 and JNK inhibitor SP600125 blocked the LC3II accumulation in Tan I-treated H28 cells. Overall, these findings demonstrate that Tan I exerts antitumor activity through a compromise between apoptosis and p62/SQSTM1-dependent autophagy via activation of JNK and IRE 1 in malignant mesothelioma cells.
Highlights
Malignant pleural mesotheliomas are caused by asbestos exposure [1, 2]
To confirm whether tanshinone I (Tan I) induces late stage autophagy by fusion with lysosomes, H28 cells were stained with acridine orange (AO), which is used for staining acidic vacuoles, including lysosomes, endosomes, and autolysosomes [20, 21], one day after Tan I treatment
The expression of p62/sequestosome 1 (SQSTM1) was increased in a concentration dependent fashion in Tan I-treated H28 cells, while the expression of p62/SQSTM1 was upregulated at 5 μM of Tan 1, but tended to decrease from 10 μM
Summary
Malignant pleural mesotheliomas are caused by asbestos exposure [1, 2]. Though the recent incidence of malignant mesothelioma has been still increasing due to past exposure to asbestos worldwide, the survival rate of mesothelioma patients is merely 9 to 12 months, due to few effective treatments for mesotheliomas [3].Autophagy, so called macroautophagy, is a self-digestion process with the features of the formation of double membrane-bound vacuoles called autophagosomes that can be subsequently fused with the lysosome to form the autolysosome [4, 5]. Malignant pleural mesotheliomas are caused by asbestos exposure [1, 2]. Though the recent incidence of malignant mesothelioma has been still increasing due to past exposure to asbestos worldwide, the survival rate of mesothelioma patients is merely 9 to 12 months, due to few effective treatments for mesotheliomas [3]. SQSTM1, elicit direct autophagosome formation [6]. These autophagosomes contain several structural domains, such as the PB1, LIR and UBA domains [7]. The underlying autophagic mechanism of Tan I was never investigated in mesothelioma cells until now. In the current study, an autophagic molecular mechanism of Tan I was elucidated by assessing the essential effect of p62ΔUBA domain andprotein- protein interactions between p62/
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