C.H. Kang

Caspase-independent cell death by arsenic trioxide in human cervical cancer cells: reactive oxygen species-mediated poly(ADP-ribose) polymerase-1 activation signals apoptosis-inducing factor release from mitochondria.

Although mechanisms of arsenic trioxide (As2O3)-induced cell death have been studied extensively in hematologic cancers, those in solid cancers have yet to be clearly defined. In this study, we showed that the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for As2O3-induced cell death in human cervical cancer cells. We also showed that reactive oxygen species (ROS)-mediated poly(ADP-ribose) polymerase-1 (PARP-1) activation is necessary for AIF release from mitochondria. The treatment of human cervical cancer cells with As2O3 induces dissipation of mitochondrial membrane potential (Δψm), translocation of AIF from mitochondria to the nucleus, and subsequent cell death. Small interfering RNA targeting of AIF effectively protects cervical cancer cells against As2O3-induced cell death. As2O3 also induces an increase of intracellular ROS level and a marked activation of PARP-1. N-acetyl-l-cystein, a thiol-containing antioxidant, completely blocks As2O3-induced PARP-1 activation, Δψm loss, nuclear translocation of AIF from mitochondria, and the consequent cell death. Furthermore, pretreatment of 1,5-dihydroxyisoquinoline or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, PARP-1 inhibitors, effectively attenuates the loss of Δψm, AIF release, and cell death. These data support a notion that ROS-mediated PARP-1 activation signals AIF release from mitochondria, resulting in activation of a caspase-independent pathway of cell death in solid tumor cells by As2O3 treatment.

The Rac1/MKK7/JNK pathway signals upregulation of Atg5 and subsequent autophagic cell death in response to oncogenic Ras

To prevent the development of malignancies, mammalian cells activate disposal programs, such as programmed cell death, in response to deregulated oncogene expression. However, the molecular basis for regulation of cellular disposal machinery in response to activated oncogenes is unclear at present. In this study, we show that upregulation of the autophagy-related protein, Atg5, is critically required for the oncogenic H-ras-induced autophagic cell death and that Rac1/mitogen-activated kinase kinase (MKK) 7/c-Jun N-terminal kinase (JNK) signals upregulation of Atg5. Overexpression of H-ras(V12) induced marked autophagic vacuole formation and cell death in normal fibroblasts, which remained unaffected by a caspase inhibitor. Pretreatment with Bafilomycin A1, an autophagy inhibitor, completely attenuated H-ras(V12)-induced cell death as well as autophagic vacuole formation. Selective production of Atg5 was observed in cells overexpressing H-ras(V12), and small interfering RNA (siRNA) targeting of Atg5 clearly inhibited autophagic cell death. Interestingly, inhibition of JNK or c-Jun by specific siRNA suppressed Atg5 upregulation and autophagic cell death. Moreover, inhibition of MKK7, but not MKK4, effectively attenuated H-ras(V12)-induced JNK activation. In addition, ectopic expression of RacN17 or Rac1-siRNA effectively inhibited MKK7-JNK activation, Atg5 upregulation and autophagic cell death. These data support the notion that upregulation of Atg5 is required for the oncogenic H-ras-induced autophagic cell death in normal fibroblasts and that activation of Rac1/MKK7/JNK-signaling pathway leads to upregulation of Atg5 in response to oncogenic H-ras. Our findings suggest that in cells acquiring deregulated oncogene expression, oncogenic stress triggers autophagic cell death, which protects cells against malignant progression.

Activation of Bak and Bax through c-Abl-Protein Kinase Cδ-p38 MAPK Signaling in Response to Ionizing Radiation in Human Non-small Cell Lung Cancer Cells

Intracellular signaling molecules and apoptotic factors seem to play an important role in determining the radiation response of tumor cells. However, the basis for the link between signaling pathway and apoptotic cell death machinery after ionizing irradiation remains still largely unclear. In this study, we showed that c-Abl-PKCδ-Rac1-p38 MAPK signaling is required for the conformational changes of Bak and Bax during ionizing radiation-induced apoptotic cell death in human non-small cell lung cancer cells. Ionizing radiation induced conformational changes and subsequent oligomerizations of Bak and Bax, dissipation of mitochondrial membrane potential, and cytochrome c release from mitochondria. Small interference (siRNA) targeting of Bak and Bax effectively protected cells from radiation-induced mitochondrial membrane potential loss and apoptotic cell death. p38 MAPK was found to be selectively activated in response to radiation treatment. Inhibition of p38 MAPK completely suppressed radiation-induced Bak and Bax activations, dissipation of mitochondrial membrane potential, and cell death. Moreover, expression of a dominant negative form of protein kinase Cδ (PKCδ) or siRNA targeting of PKCδ attenuated p38 MAPK activation and conformational changes of Bak and Bax. In addition, ectopic expression of RacN17, a dominant negative form of Rac1, markedly inhibited p38 MAPK activation but did not affect PKCδ activation. Upon stimulation of cells with radiation, PKCδ was phosphorylated dramatically on tyrosine. c-Abl-PKCδ complex formation was also increased in response to radiation. Moreover, siRNA targeting of c-Abl attenuated radiation-induced PKCδ and p38 MAPK activations, and Bak and Bax modulations. These data support a notion that activation of the c-Abl-PKCδ-Rac1-p38 MAPK pathway in response to ionizing radiation signals conformational changes of Bak and Bax, resulting in mitochondrial activation-mediated apoptotic cell death in human non-small cell lung cancer cells.

Possible biomarkers for ionizing radiation exposure in human peripheral blood lymphocytes.

Abstract Kang, C-M., Park, K-P., Song, J-E., Jeoung, D-I., Cho, C-K., Kim, T-H., Bae, S., Lee, S-J. and Lee, Y-S. Possible Biomarkers for Ionizing Radiation Exposure in Human Peripheral Blood Lymphocytes. Radiat. Res. 159, 312–319 (2003). Biomarkers to indicate past exposure to radiation have not been entirely satisfactory. Using cDNA microarray hybridization to find new potential biomarkers, we identified highly expressed genes in human peripheral blood lymphocytes (PBLs) after irradiation 1 Gy ex vivo. The present set of radiation markers in PBLs was identified 12 h after radiation. A total of 44 genes were identified. However, when RT-PCR was performed with mRNA from the PBLs of five individuals, only four genes, including TRAIL receptor 2, DRAL (now known as FHL2), cyclin G, and cyclin protein gene, showed greater than 50% agreement between gene induction as detected by microarray analysis and by RT-PCR. When more than 32 donors were tested for the above four genes, greater than 85% agreement was obtained between gene induction measured by microarray analysis and by RT-PCR. There was a linear dose–response relationship between 0.5 and 4 Gy 12 h after irradiation; however, there was less linearity at later times. These results suggested that the relative expression levels of genes such as TRAIL receptor 2, FHL2, cyclin G, and cyclin protein gene in PBLs may provide estimates of radiation exposures.

Hspa4 (HSP70) is involved in the radioadaptive response: results from mouse splenocytes.

Abstract Kang, C-M., Park, K-P., Cho, C-K., Seo, J-S., Park, W-Y., Lee, S-J. and Lee, Y-S. Hspa4 (HSP70) is Involved in the Radioadaptive Response: Results from Mouse Splenocytes. Radiat. Res. 157, 650–655 (2002). In a continuation of our earlier study on the involvement of HSP25 (now known as Hspb1) and HSP70 (now known as Hspa4) in the induction of an adaptive response, we examined the involvement of these proteins in the induction of the adaptive response using an animal model system. C57BL6 mice were irradiated with 5 cGy of γ radiation three times in 1 week (for a total of 15 cGy), and a high challenge dose (6 Gy) was given on the day after the last low-dose irradiation. The survival time of the low-dose preirradiated mice was increased to 30%. The induction of apoptosis induced by 6 Gy was also reduced by this low-dose preirradiation regimen. To elucidate any link existing between the HSPs and the induction of the adaptive response, reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis was performed using splenocytes. High-dose radiation up-regulated the expression of Hspb1 and especially Hspa4, while expression of other HSPs such as HSC70 (now know as Hspa8), Hsp90, and αB-crystalline (now known as Cryab) did not change. When splenocytes from Hspa4 transgenic mice were preirradiated with a low dose of radiation, a reduction in cell death after high-dose irradiation was observed. These results suggest that Hspa4 is a key molecule in the induction of the adaptive response.

Ionizing radiation can overcome resistance to TRAIL in TRAIL-resistant cancer cells

Although the majority of cancer cells are killed by TRAIL (tumor necrosis factor‐related apoptosis‐inducing ligand treatment), certain types show resistance to it. Ionizing radiation also induces cell death in cancer cells and may share common intracellular pathways with TRAIL leading to apoptosis. In the present study, we examined whether ionizing radiation could overcome TRAIL resistance in the variant Jurkat clones. We first selected TRAIL‐resistant or ‐sensitive Jurkat clones and examined cross‐responsiveness of the clones between TRAIL and radiation. Treatment with γ‐radiation induced significant apoptosis in all the clones, indicating that there seemed to be no cross‐resistance between TRAIL and radiation. Combined treatment of radiation with TRAIL synergistically enhanced killing of TRAIL‐resistant cells, compared to TRAIL or radiation alone. Apoptosis induced by combined treatment of TRAIL and radiation in TRAIL‐resistant cells was associated with cleavage of caspase‐8 and the proapoptotic Bid protein, resulting in the activation of caspase‐9 and caspase‐3. No changes in the expressions of TRAIL receptors (DR4 and DR5) and Bcl‐2 or Bax were found after treatment. The caspase inhibitor z‐VAD‐fmk completely counteracted the synergistic cell killing induced by combined treatment of TRAIL and γ‐radiation. These results demonstrated that ionizing radiation in combination with TRAIL could overcome resistance to TRAIL in TRAIL‐resistant cells through TRAIL receptor‐independent synergistic activation of the cascades of the caspase‐8 pathway, suggesting a potential clinical application of combination treatment of TRAIL and ionizing radiation to TRAIL‐resistant cancer cells.

Nicotinamide Phosphoribosyltransferase Is Essential for Interleukin-1β-mediated Dedifferentiation of Articular Chondrocytes via SIRT1 and Extracellular Signal-regulated Kinase (ERK) Complex Signaling

Although much is known about interleukin (IL)-1β and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1β signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1β treatment. IL-1β or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1β-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1β. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1β-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1β and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1β induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.

Aberrant promoter CpG island hypermethylation of the adenomatosis polyposis coli gene can serve as a good prognostic factor by affecting lymph node metastasis in squamous cell carcinoma of the esophagus

There has been no clear evidence demonstrating whether DNA hypermethylation can affect the prognosis of esophageal cancer. We collected tissue from 50 cases of squamous cell carcinoma of the esophagus and tested them for DNA hypermethylation using methylation-specific polymerase chain reaction. CpG island hypermethylations were observed in 10% for p16, 34% for RARbetaP2, 46% for adenomatosis polyposis coli (APC), 14% for RASSF1A, 84% for FHIT, and 8% for hMLH1. APC promoter hypermethylation was frequently found in patients without lymph node metastasis compared with those with lymph node metastasis (62.5% : 30.8%, P = 0.025). The number of metastatic lymph nodes were lower in patients with APC promoter hypermethylation (0.87 +/- 0.30 : 3.07 +/- 0.72, P = 0.008). Excluding operative mortalities and incomplete resections, 42 patients were analyzed for long-term outcome. During the mean follow-up period of 35 months, 17 developed recurrence and 14 died of cancer. Ten patients died of other causes. In univariable analysis, unmethylation of APC (P = 0.0015) and FHIT (P = 0.0044), as well as presence of lymph node metastasis (P = 0.0038), were risk factors for recurrence. In multivariable analysis, lymph nodes metastasis (P = 0.050) and unmethylation of APC promoter (P = 0.023) remained as significant risk factors. In conclusion, promoter hypermethylation of the APC gene is related to a lower number of metastatic lymph nodes and to superior prognosis in terms of recurrence, which suggests it might be involved in the process of lymph node metastasis in esophageal cancer.

Identification of inositol polyphosphate 4-phosphatase type II as a novel tumor resistance biomarker in human laryngeal cancer HEp-2 cells

Although tumor resistance remains a significant impediment to successful radiotherapy, associated regulatory markers and detailed molecular mechanisms underlying this phenomenon are not well defined. In this study, we identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a novel marker of radioresistance by systematically analyzing Unigene libraries of laryngeal cancer. INPP4B was highly expressed in radioresistant laryngeal cancer cells and was induced by treatment with either radiation or anticancer drugs in various types of cancer cells. Ectopic INPP4B overexpression increased radioresistance and anticancer drug resistance by suppressing apoptosis in HEp-2 cells. Conversely, INPP4B depletion with small interfering RNA resensitized HEp-2 as well as A549 and H1299 cells to radiation- and anticancer drug-induced apoptosis. Furthermore, radiation-induced INPP4B expression was blocked by inhibition of extracellular signal-regulated kinase (ERK). INPP4B depletion significantly attenuated radiation-induced increases in Akt phosphorylation, indicating an association of INPP4B-mediated radioresistance with Akt survival signaling. Taken together, our data suggest that ERK-dependent induction of INPP4B triggers the development of a tumor-resistance phenotype via Akt signaling and identify INPP4B as a potentially important target molecule for resolving the radioresistance of cancer cells.

Differential Gene Signatures in Rat Mammary Tumors Induced by DMBA and Those Induced by Fractionated γ Radiation

Abstract Lee, H-J., Lee, Y-J., Kang, C-M., Bae, S., Jeoung, D., Jang, J-J., Lee, S-S., Cho, C-K. and Lee, Y-S. Differential Gene Signatures in Rat Mammary Tumors Induced by DMBA and Those Induced by Fractionated γ Radiation. Radiat. Res. 170, 579–590 (2008). The aim of this work was to identify specific genes involved in rat mammary tumors induced by dimethylbenz(a)anthracene (DMBA) or radiation. More TUNEL- and PCNA-positive cells were present in mammary tumors induced by radiation than in tumors induced by DMBA, whereas DNA damage responses like p53 accumulation and histone H2AX phosphorylation were higher in DMBA-induced tumors, even though the pathology was similar in both types of tumors. cDNA microarray and real-time RT-PCR analysis of radiation- or DMBA-induced tumor tissues, revealed that stanniocalcin 2 (Stc2), interferon regulatory factor 1 (Irf1), interleukin 18 binding protein (Il18bp), and chloride channel calcium activated 3 (Clca3) were expressed in both, and that arachidonate 5-lipoxygenase activating protein 1 (Alox5ap) and cathepsin S (Ctss) were expressed only in radiation-induced tumors. No DMBA-specific gene signatures were found. Soft agar growth assays were carried out to identify the carcinogenic features of these specific genes. Cells stably transfected with Alox5ap, Ctss, Stc2, Irf1, Il18bp and Clca3 showed morphological changes compared to controls. These findings indicate different gene alterations in carcinogen- or radiation-induced mammary tumors with similar pathological stages.