Bid participates in genotoxic drug-induced apoptosis of HeLa cells and is essential for death receptor ligands' apoptotic and synergistic effects

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling

Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling

Motoneurons secrete angiogenin to induce RNA cleavage in astroglia.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in angiogenin, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. We previously demonstrated that angiogenin administration shows promise as a neuroprotective therapeutic in studies using transgenic ALS mice and primary motoneuron cultures. Its mechanism of action and target cells in the spinal cord, however, are largely unknown. Using mixed motoneuron cultures, motoneuron-like NSC34 cells, and primary astroglia cultures as model systems, we here demonstrate that angiogenin is a neuronally secreted factor that is endocytosed by astroglia and mediates neuroprotection in paracrine. We show that wild-type angiogenin acts unidirectionally to induce RNA cleavage in astroglia, while the ALS-associated K40I mutant is also secreted and endocytosed, but fails to induce RNA cleavage. Angiogenin uptake into astroglia requires heparan sulfate proteoglycans, and engages clathrin-mediated endocytosis. We show that this uptake mechanism exists for mouse and human angiogenin, and delivers a functional RNase output. Moreover, we identify syndecan 4 as the angiogenin receptor mediating the selective uptake of angiogenin into astroglia. Our data provide new insights into the paracrine activities of angiogenin in the nervous system, and further highlight the critical role of non-neuronal cells in the pathogenesis of ALS

Calnexin, an ER-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance. METHODS: The expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells. RESULTS: There were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy. CONCLUSION: Increased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies

Erratum to: Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer

Erratum to: Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer

Real time single cell analysis of Bid cleavage and Bid translocation during caspase-dependent and neuronal caspase-independent apoptosis.

Bcl-2 homology domain (BH) 3-only proteins couple stress signals to evolutionarily conserved mitochondrial apoptotic pathways. Caspase 8-mediated cleavage of the BH3-only protein Bid into a truncated protein (tBid) and subsequent translocation of tBid to mitochondria has been implicated in death receptor signaling. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage and tBid translocation during death receptor-induced apoptosis in caspase 3-deficient MCF-7 cells. Cells treated with tumor necrosis factor-alpha (200 ng/ml) showed a rapid cleavage of the Bid-FRET probe occurring 75.4 +/- 12.6 min after onset of the tumor necrosis factor-alpha exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (DeltaPsim). We next investigated the role of Bid cleavage in a model of caspase-independent, glutamate-induced excitotoxic apoptosis. Rat cerebellar granule neurons were transfected with the Bid-FRET probe and exposed to glutamate for 5 min. In contrast to death receptor-induced apoptosis, neurons showed a translocation of full-length Bid to the mitochondria. This translocation occurred 5.6 +/- 1.7 h after the termination of the glutamate exposure and was also paralleled with a collapse of the DeltaPsim. Proteolytic cleavage of the FRET probe also occurred, however, only 25.2 +/- 3.5 min after its translocation to the mitochondria. Subfractionation experiments confirmed a translocation of full-length Bid from the cytosolic to the mitochondrial fraction during excitotoxic apoptosis. Our data demonstrate that both tBid and full-length Bid have the capacity to translocate to mitochondria during apoptosis.</p

Knockdown of Bid improves clonogenic survival after treatment with Oxaliplatin.

<p>HeLa Control and HeLa Bid kd cells were treated with the indicated concentrations of Oxaliplatin (1 h) or vehicle. After incubation, 1000 cells were transferred to 60 mm dishes and cultured in fresh medium for 14 days. Then colonies were fixed, stained with methylene blue and counted. A) Colony formation in representative dishes is shown. B) Graphical representation of the percentage of colonies after treatment compared to control cells treated with vehicle (100%). Data are means+/−SD from at least two independent experiments performed in triplicate. # p<0.05: difference from control cells (Ctrl). ∗ p<0.05: difference from Bid kd cells treated with vehicle.</p

Generation of stable Bid deficient HeLa cells.

<p>A) Cells were stably transfected with plasmids encoding one of three different <i>bid</i>-specific shRNAs or a scrambled control shRNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002844#s4" target="_blank">Materials and Methods</a>. For analysis of the <i>bid</i> knockdown, cells were subjected to Western blotting with a polyclonal Bid antibody. α-Tubulin served as a loading control. B) Control cells expressing the scrambled (Ctrl 1, Ctrl 2), or the <i>bid</i> specific shRNA (Bid kd 4) and clone (Bid kd 1) were incubated with Cycloheximide (CHX) (1 µg/ml) and an agonistic Fas antibody or vehicle (clone CH11) at the indicated concentrations or vehicle for 4 h. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05: difference from control cells (Ctrl). C) Cell lysates were subjected to Western blotting with antibodies for the indicated proteins, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002844#s4" target="_blank">Materials and Methods</a>. D) HeLa Bid kd cells were stably transfected with a vector coding for YFP-<i>bid</i>-CFP (GFP-Bid); cell lysates were subjected to Western blotting with GFP and Bid antibodies. E) HeLa Bid kd cells expressing GFP-Bid were treated with CHX (1 µg/ml) and an activating Fas antibody (100 ng/ml). Controls were treated with vehicle for 4 h; caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).</p

The synergistic effects of ER Stress and TRAIL on apoptosis depend on Bid.

<p>A) HeLa Control and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk for 1 h where specified; cells were subsequently treated with Tunicamycin or vehicle for the indicated times. Cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. B, C) Control cells and HeLa Bid kd cells were pre-incubated for 16 h with the indicated concentrations of Tunicamycin, Thapsigargin or vehicle before treatment with TRAIL (10 ng/ml). Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin, Thapsigargin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl). D) Control cells and HeLa Bid kd cells were pre-incubated with Tunicamycin (0.3 µM) or vehicle for the indicated times followed by treatment with TRAIL (10 ng/ml) for the indicated times. Cell lysates were subjected to Western blotting with a polyclonal Bid, a polyclonal caspase-3, a polyclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E) Control cells and HeLa Bid kd cells were pre-incubated with Tunicamycin (0.3 µM) for 16 h followed by treatment with recombinant TRAIL (10 ng/ml) for the indicated times. Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl). F) Control cells and HeLa Bid kd cells were pre-incubated with a DR5 blocking peptide (50 ng/ml) for 1 h followed by pre-treatment with Tunicamycin (0.3 µM) or vehicle for 16 h. Subsequently, cells were treated with recombinant TRAIL for 3 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl).</p