Dataset Information

Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells.

ABSTRACT:

Purpose

Cisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC). Autophagy induction has been implied to contribute to cisplatin resistance in ovarian cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines.

Materials and methods

Human BC cells (5637 and T24) were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3)-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL) formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12) were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation.

Results

Cisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose-and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1) was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis.

Conclusion

Collectively, the study results indicated that cisplatin-induced autophagy is mediated by BECN1 in BC cells. Therefore, combinative treatment using cisplatin and autophagy inhibitors could potentially overcome cisplatin resistance related to autophagy induction.

SUBMITTER: Lin JF

PROVIDER: S-EPMC5439993 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.