Beclin-1-independent autophagy mediates programmed cancer cell death through interplays with endoplasmic reticulum and/or mitochondria in colbat chloride-induced hypoxia

Abstract

Autophagy has dual functions in cell survival and death. However, the effects of autophagy on cancer cell survival or death remain controversial. In this study, we show that Autophagy can mediate programmed cell death (PCD) of cancer cells in responding to cobalt chloride (CoCl2)-induced hypoxia in a Beclin-1-independent but autophagy protein 5 (ATG5)-dependent manner. Although ATG5 is not directly induced by CoCl2, its constitutive expression is essential for CoCl2-induced PCD. The ATG5-mediated autophagic PCD requires interplays with endoplasmic reticulum (ER) and/or mitochondria. In this process, ATG5 plays a central role in regulating ER stress protein CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and mitochondrial protein second mitochondria derived activator of caspases (Smac). Two pathways for autophagic PCD in cancer cells responding to hypoxia have been identified: ATG5/CHOP/Smac pathway and ATG5/Smac pathway, which are probably dependent on the context of cell lines. The former is more potent than the latter for the induction of PCD at the early stage of hypoxia, although the ultimate efficiency of both pathways is comparable. In addition, both pathways may require ATG5-mediated conversion of LC3-I into LC3-II. Therefore, we have defined two autophagy-mediated pathways for the PCD of cancer cells in hypoxia, which are dependent on ATG5, interplayed with ER and mitochondria and tightly regulated by hypoxic status. The findings provide a new evidence that autophagy may inhibit tumor cell proliferation through trigger of PCD, facilitating the development of novel anti-cancer drugs.

Keywords: Cobalt chloride; autophagy; endoplasmic reticulum stress; mitochondria; programmed cell death.

Figure 1

Beclin-1-independent conversion of LC3 I to LC3 II induced by CoCl₂. A, B. HeLa cells and MDA-MB-231 cells were treated with CoCl₂ (0 (PBS), 100, 200 or 400 μM) for 24 h, and the levels of protein expression were analyzed by western blot using antibodies to Beclin-1, ATG5, p62, LC3 and β-actin, respectively. The band intensity of LC3 II normalized by LC3 I was compared with that in the control. The intensity ratios of LC3 II/I display below LC3 bands. C, D. HeLa cells and MDA-MB-231 cells were treated with 400 μM CoCl₂ for 24 h. Relative mRNA expression levels of BECN1 and ATG5 were determined by RT-PCR. Curves and histograms represent the quantity of proteins or mRNA from relevant experiments.

Figure 2

The effects of knockdown of Beclin-1 and ATG5 on autophagy formation in hypoxic cancer cells. The HeLa cells and MDA-MB-231 cells were transfected with control siRNA Beclin 1 siRNA (A, B) or ATG5 siRNA (C, D) for 24 h and then treated with CoCl₂ (400 μM) for another 24 h. Levels of protein expression were measured by western blot analysis using antibodies to Beclin-1, ATG5, LC3 and β-actin. The band intensity of LC3 II normalized by LC3 I and the ratios of LC3 II to LC3 I were compared with that of the control. (A, B) The effects of knocking down Beclin-1 by Beclin-1-specific siRNA on autophagy formation; (C, D) The effects of knocking down ATG5 by ATG5-specific siRNA on autophagy formation.

Figure 3

ER stress marker expression induced by CoCl₂ was differentially expressed in the HeLa cells from MDA-MB-231 cells. Hela and MDA-MB-231 cells were treated with 400 μM CoCl₂ for 24 and 48 h. Relative mRNA expression levels of Bip, ATF4, DDIT3, ATF6, XBP1u and XBP1s were determined by RT-PCR. BNIP3 was used as a positive control (A, B). The proteins expression of CHOP and β-actin was analyzed by Western blot in HeLa cells and MDA-MB-231 cell lines exposed to CoCl₂ (100, 200 or 400 μM) 24 h after treatment (C, D).

Figure 4

CHOP is required for CoCl₂-induced autophagy formation in HeLa cells but not in MDA-MB-231 cells. A. CHOP siRNA suppressed CHOP expression: HeLa cells and MDA-MB-231 cells were transfected with CHOP siRNA or control siRNA (si-NC) for 48 hrs and CHOP protein expression was analyzed by Western blot. B, C. CHOP is required for autophagy formation in HeLa cells but not in MDA-MB-231 cells in responding to CoCl₂: HeLa cells and MDA-MB-231 cells were transfected with si-NC or CHOP siRNA for 24 h and then treated with CoCl₂ (400 μM) or vehicles (PBS) for another 24 h. The cells were harvested and the levels of protein expression were measured by western blot analysis using antibodies against CHOP, ATG5, LC3, p62 or β-actin. The band intensity of LC3 II was normalized by LC3 I and the ratios of LC3 II to LC3 I was compared with that of the control. D, E. ATG5 is required for CHOP expression in HeLa cells: HeLa cells and MDA-MB-231 cells were transfected with control siRNA or ATG5 siRNA for 24 h and then treated with CoCl₂ (400 μM) for another 24 h. Levels of protein expression were measured by western blot analysis using antibodies to CHOP and β-actin.

Figure 5

Mechanistic links among autophagy, ER and mitochondria in the hypoxic cancer cells. A, B. HIF-1α expression in cancer cells induced by CoCl₂: HeLa and MDA-MB-231 cells were treated with CoCl₂ (100, 200 or 400 μM) for 24 h. Levels of protein expression were analyzed by western blot using antibodies to HIF-1α, Smac, cleaved caspas-3 or β actin. C, D. Association of ER stress with mitochondrial stress: HeLa and MDA-MB-231 cells were transfected with control siRNA, CHOP siRNAs for 24 h and then treated with CoCl₂ (400 μM) for another 24 h. Levels of protein expression were measured by western blot analysis using antibodies to Smac, cleaved caspase-3 or β actin. E, F. Association of autophagy with mitochondrial stress: HeLa and MDA-MB-231 cells were transfected with control siRNA or ATG5 siRNA for 24 h and then treated with CoCl₂ (400 μM) for another 24 h. Levels of protein expression were measured by western blot analysis using antibodies to Smac, cleaved caspase-3 or β actin.

Figure 6

ATG5/CHOP/Smac pathway was more potent than ATG5/Smac pathway in the induction of programmed cancer cell death in hypoxia. HeLa and MDA-MB-231 cells were treated with PBS or indicated concentrations of CoCl₂ (100, 200, 400 or 800 μM) and OD values were measured at 12 (A), 24 (B) and 48 h (C) by a cck-8 assay, respectively. Representative results of three independent experiments are expressed as percentage of cck-8 reduction, relative to NT control. The viability of the CoCl₂-treated cells at 0 h is regarded as 100%; *P<0.05 and **P<0.01 when compared between the groups of HeLa cells and MDA-MB-231 cells.

Figure 7

Schematic Model for ATG5/CHOP/Smac pathway and ATG5/Smac pathway in cancer cells responding to hypoxia. A. Interactions between autophagy, ER stress and mitochondrial response in cancer cells responding to hypoxia induced by CoCl₂: Although Beclin-1 can be down or up-regulated in responding to the CoCl₂-induced hypoxia, it does not participate in autophagy formation. In contrast, ATG5 does not significantly respond to the hypoxia in transcription, it critically participate autophagy formation in the hypoxia. There are two pathways for ATG5 participating the induction of programmed cell death of cancer, each pathway have a subpathway by passing LC3II formation. Pathway A, autophagy, ER and mitochondrial responses: (a1) ATG5/CHOP/LC3II/Smac; (a2) ATG5/CHOP/SMAC; pathway B, autophagy and mitochondrial responses: (b1) ATG5/LC3II/SMAC; and (b2) ATG5/SMAC. B. Comparison of the ATG5/CHOP/SMAC pathway in the HeLa cells with the ATG5/SMAC pathway in the MDA-MB-231 cells: In the HeLa cells, ATG5 up-regulates CHOP, which subsequently up-regulates SMAC; while in the MDA-MB-231 cells, ATG5 induces LC3 II/I, leading to activation of SMAC. In addition, ATG5-induced CHOP in the HeLa cells also promotes LC3II, forming a positive loop to strengthen PCD through activation of SMAC/caspase-3 pathway, whereas CHOP inhibits ATG5 in the MDA-MB-231 cells in the absence of CoCl₂. ATG5: Autophagy protein 5; ATF4: activating transcription factor 4 (tax-responsive enhancer element B67); XBP1 u/s: X-box binding protein 1 unspliced/spliced; LC3/ MAP1LC3A: Microtubule-associated proteins 1A/1B light chain 3A; a light chain of the microtubule-associated protein 1; CHOP/DDIT3: DNA-damage inducible transcript 3; SMAC/ DIABLO: second mitochondria-derived activator of caspases; ER: endoplasmic reticulum; Mit. Stress: mitochondrial stress; PCD: programmed cell death.