Kyeong Sook Choi

Two distinct modes of cell death induced by doxorubicin: apoptosis and cell death through mitotic catastrophe accompanied by senescence-like phenotype

Chronic exposure of many human hepatoma cell lines to a low dose (LD) of doxorubicin induced a senescence-like phenotype (SLP) accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity. LD doxorubicin-induced SLP was preceded by multinucleation and downregulation of multiple proteins with mitotic checkpoint function, including CENP-A, Mad2, BubR1, and Chk1. LD doxorubicin-treated cells eventually underwent cell death through mitotic catastrophe. When we investigated whether LD doxorubicin-induced cell death shares biochemical characteristics with high dose (HD) doxorubicin-induced apoptosis in Huh-7 cells, we observed that externalization of phosphatidyl serine and release of mitochondrial cytochrome c into the cytosol was associated with both types of cell death. However, propidium iodide exclusion assays showed that membrane integrity was lost in the initial phase of LD doxorubicin-induced cell death through mitotic catastrophe, whereas it was lost during the late phase of HD doxorubicin-induced apoptosis. Furthermore, HD doxorubicin-induced apoptosis but not LD doxorubicin-induced mitotic catastrophe led to transient activation of NF-κB and strong, sustained activations of p38, c-Jun N-terminal kinase, and caspases. Collectively, these results indicate that different doses of doxorubicin activate different regulatory mechanisms to induce either apoptosis or cell death through mitotic catastrophe.

Sulforaphane Sensitizes Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL)–Resistant Hepatoma Cells to TRAIL-Induced Apoptosis through Reactive Oxygen Species–Mediated Up-regulation of DR5

Sulforaphane is a chemopreventive agent present in various cruciferous vegetables, including broccoli. Here, we show that treatment with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in combination with subtoxic doses of sulforaphane significantly induces rapid apoptosis in TRAIL-resistant hepatoma cells. Neither TNF-alpha- nor Fas-mediated apoptosis was sensitized in hepatoma cells by cotreatment with sulforaphane, suggesting that sulforaphane can selectively sensitize cells to TRAIL-induced apoptosis but not to apoptosis mediated by other death receptors. We found that sulforaphane treatment significantly up-regulated mRNA and protein levels of DR5, a death receptor of TRAIL. This was accompanied by an increase in the generation of reactive oxygen species (ROS). Pretreatment with N-acetyl-l-cysteine and overexpression of catalase inhibited sulforaphane-induced up-regulation of DR5 and almost completely blocked the cotreatment-induced apoptosis. Furthermore, the sulforaphane-mediated sensitization to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs for DR5. These results collectively indicate that sulforaphane-induced generation of ROS and the subsequent up-regulation of DR5 are critical for triggering and amplifying TRAIL-induced apoptotic signaling. We also found that sulforaphane can sensitize both Bcl-xL- and Bcl-2-overexpressing hepatoma cells to TRAIL-induced apoptosis, indicating that treatment with a combination of TRAIL and sulforaphane may be a safe strategy for treating resistant hepatomas.

Autophagy and cancer

Basal autophagy plays a critical role in maintaining cellular homeostasis and genomic integrity by degrading aged or malfunctioning organelles and damaged or misfolded proteins. However, autophagy also plays a complicated role in tumorigenesis and treatment responsiveness. It can be tumor-suppressing during the early stages of tumorigenesis (i.e., it is an anti-tumor mechanism), as reduced autophagy is found in tumor cells and may be associated with malignant transformation. In this case, induction of autophagy would seem to be beneficial for cancer prevention. In established tumors, however, autophagy can be tumor-promoting (i.e., it is a pro-tumor mechanism), and cancer cells can use enhanced autophagy to survive under metabolic and therapeutic stress. The pharmacological and/or genetic inhibition of autophagy was recently shown to sensitize cancer cells to the lethal effects of various cancer therapies, including chemotherapy, radiotherapy and targeted therapies, suggesting that suppression of the autophagic pathway may represent a valuable sensitizing strategy for cancer treatments. In contrast, excessive stimulation of autophagy may also provide a therapeutic strategy for treating resistant cancer cells having high apoptotic thresholds. In order for us to develop successful autophagy-modulating strategies against cancer, we need to better understand how the roles of autophagy differ depending on the tumor stage, cell type and/or genetic factors, and we need to determine how specific pathways of autophagy are activated or inhibited by the various anti-cancer therapies.

Roscovitine sensitizes glioma cells to TRAIL-mediated apoptosis by downregulation of survivin and XIAP

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in many glioma cell lines. However, treatment with TRAIL in combination with subtoxic doses of roscovitine, a specific inhibitor of Cdc2 and Cdk2, induced rapid apoptosis in TRAIL-resistant glioma cells. Roscovitine could sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells, but not human astrocytes, to TRAIL-induced apoptosis, offering an attractive strategy for safely treating resistant gliomas. Treatment with roscovitine significantly inhibited Cdc2 activity, and expression of a dominant-negative Cdc2 mutant sensitized glioma cells to TRAIL-induced apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in U87MG and T98 glioma cells, treatment with roscovitine recovered TRAIL-induced activation of caspases very efficiently in these cells. We found that treatment with roscovitine or expression of a dominant-negative Cdc2 mutant downregulated the protein levels of survivin and XIAP, two major caspase inhibitors. Overexpression of survivin or XIAP attenuated the apoptosis induced by roscovitine and TRAIL. Taken together, these results suggest that downregulation of survivin and XIAP by subtoxic doses of roscovitine contributes to the amplification of caspase cascades, thereby overcoming glioma cell resistance to TRAIL-mediated apoptosis.

Transforming Growth Factor β1 Induces Apoptosis through Cleavage of BAD in a Smad3-Dependent Mechanism in FaO Hepatoma Cells

ABSTRACT Transforming growth factor β (TGF-β) induces apoptosis in a variety of cells. We have previously shown that TGF-β1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-β1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-XL, and Bax. However, TGF-β1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-β1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-β1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-β1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-β1-induced apoptosis and BAD cleavage. These results suggest that TGF-β1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.

A Human scFv Antibody against TRAIL Receptor 2 Induces Autophagic Cell Death in Both TRAIL-Sensitive and TRAIL-Resistant Cancer Cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death in a variety of tumor cells without significant cytotoxicity on normal cells. However, many cancer cells with apoptotic defects are resistant to treatment with TRAIL alone, limiting its potential as an anticancer therapeutic. Here, we report on the tumoricidal activity of a human single-chain fragment variable, HW1, which specifically binds to TRAIL receptor 2 (TR2) without competing with TRAIL for the binding. HW1 treatment as a single agent induces autophagic cell death in a variety of both TRAIL-sensitive and TRAIL-resistant cancer cells, but exhibits much less cytotoxicity on normal cells. The HW1-induced autophagic cell death was inhibited by an autophagy inhibitor, 3-methyladenine, or by RNA interference knockdown of Beclin-1 and Atg7. We also show that the HW1-mediated autophagic cell death occurs predominantly via the c-Jun NH(2)-terminal kinase pathway in a caspase-independent manner. Analysis of the death-inducing signaling complex induced by HW1 binding to TR2 exhibits the recruitment of TNF receptor-associated death domain and TNF receptor-associated factor 2, but not Fas-associated death domain, caspase-8, or receptor-interacting protein, which is distinct from that induced by TRAIL. Our results reveal a novel TR2-mediated signaling pathway triggering autophagic cell death and provides a new strategy for the elimination of cancer cells, including TRAIL-resistant tumors, through nonapoptotic cell death.

TGF beta1 induces prolonged mitochondrial ROS generation through decreased complex IV activity with senescent arrest in Mv1Lu cells.

Transforming growth factor β1 (TGF β1) is a well-characterized cytokine that suppresses epithelial cell growth. We report here that TGF β1 arrested lung epithelial Mv1Lu cells at G1 phase of the cell cycle with acquisition of senescent phenotypes in the presence of 10% serum, whereas it gradually induced apoptosis with lower concentrations of serum. The senescent arrest was accompanied by prolonged generation of reactive oxygen species (ROS) and persistent disruption of mitochondrial membrane potential (ΔΨm). We demonstrated that the sustained ROS overproduction was derived from mitochondrial respiratory defect via decreased complex IV activity and was involved in the arrest. Moreover, we verified that hepatocyte growth factor released Mv1Lu cells from the arrest by protecting mitochondrial respiration, thereby preventing both the ΔΨm disruption and the ROS generation. Our present results suggest the TGF β1-induced senescent arrest as another plausible mechanism to suppress cellular growth in vivo and provide a new biochemical association between the mitochondrial functional defects and the cytokine-induced senescent arrest, emphasizing the importance of maintenance of mitochondrial function in cellular protection from the arrest.

Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation

ABSTRACT Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

Sodium butyrate sensitizes human glioma cells to TRAIL-mediated apoptosis through inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP

In TNF-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells, co-treatment with nontoxic doses of sodium butyrate and TRAIL resulted in a marked increase of TRAIL-induced apoptosis. This combined treatment was also cytotoxic to glioma cells overexpressing Bcl-2 or Bcl-xL, but not to normal human astrocytes, thus offering an attractive strategy for safely treating resistant gliomas. Cotreatment with sodium butyrate facilitated completion of proteolytic processing of procaspase-3 that was partially blocked by treatment with TRAIL alone. We also found that treatment with sodium butyrate significantly decreased the protein levels of survivin and X-linked inhibitor of apoptosis protein (XIAP), two major caspase inhibitors. Overexpression of survivin and XIAP attenuated sodium butyrate-stimulated TRAIL-induced apoptosis, suggesting its involvement in conferring TRAIL resistance to glioma cells. Furthermore, the kinase activities of Cdc2 and Cdk2 were significantly decreased following sodium butyrate treatment, accompanying downregulation of cyclin A and cyclin B, as well as upregulation of p21. Forced expression of Cdc2 plus cyclin B, but not Cdk2 plus cyclin A, attenuated sodium butyrate/TRAIL-induced apoptosis, overriding sodium butyrate-mediated downregulation of survivin and XIAP. Therefore, Cdc2-mediated downregulation of survivin and XIAP by sodium butyrate may contribute to the recovery of TRAIL sensitivity in glioma cells.

Involvement of c-Src kinase in the regulation of TGF-beta1-induced apoptosis.

Transforming growth factor-β1 (TGF-β1) is a potent inducer of apoptosis in normal hepatocytes, and acquiring resistance to TGF-β1 may be a critical step in the development of hepatocellular carcinoma (HCC). In this study, we investigated the possible involvement of c-Src in the regulation of TGF-β1-induced apoptosis. TGF-β1 induced transient activation of c-Src and its subsequent caspase-mediated degradation concomitant with cell death in FaO hepatoma cells, which are sensitive to TGF-β1. In response to TGF-β1, activated c-Src was translocated into the cytoplasmic membrane, then relocated to the nuclei of apoptotic cells during its cleavage. In TGF-β1-induced apoptotic cells, c-Src maintained its tight association with p85 FAK fragment cleaved by caspases, possibly contributing to focal adhesion disassembly. TGF-β1-induced apoptosis was enhanced by either inhibition of c-Src activity using PP1 or PP2, or by overexpression of dominant-negative c-Src. In contrast, overexpression of constitutively active c-Src inhibited apoptosis suppressing TGF-β1-induced activation of p38, JNK and caspases. In many HCC cell lines resistant to TGF-β1, enhanced c-Src activity was detected. We hypothesize that activated c-Src in HCC may contribute to resistance against the apoptotic and/ or antiproliferative properties of TGF-β1.