Youngjong Ko

Inflammatory cytokines are suppressed by light-emitting diode irradiation of P. gingivalis LPS-treated human gingival fibroblasts

Human gingival fibroblasts (hGFs) play an important role in the inflammatory reaction to lipopolysaccharide (LPS) from P. gingivalis, which infects periodontal connective tissue. In addition, although light-emitting diode (LED) irradiation has been reported to have biostimulatory effects, including anti-inflammatory activity, the pathological mechanisms of these effects are unclear. This study examined the effects of 635-nm irradiation of P. gingivalis LPS-treated human gingival fibroblasts on inflammatory cytokine profiles and the mitogen-activated protein kinase (MAPK) pathway, which is involved in cytokine production. Gingival fibroblasts treated or not treated with P. gingivalis LPS were irradiated with 635-nm LED light, and cytokine profiles in the supernatant were assessed using a human inflammation antibody array. Expression of cyclooxyginase-2 (COX-2) protein and phosphorylation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun-N-terminal kinase (JNK) were assessed by Western-blot analysis to determine the effects on the MAPK pathway, and prostaglandin E2 (PGE2) in the supernatant was measured using an enzyme-linked immunoassay. COX-2 protein expression and PGE2 production were significantly increased in the LPS-treated group and decreased by LED irradiation. LPS treatment of gingival fibroblasts led to the increased release of the pro-inflammatory-related cytokines interleukin-6 (IL-6) and IL-8, whereas LED irradiation inhibited their release. Analysis of MAPK signal transduction revealed a considerable decrease in p38 phosphorylation in response to 635-nm radiation either in the presence or absence of LPS. In addition, 635-nm LED irradiation significantly promoted JNK phosphorylation in the presence of LPS. LED irradiation can inhibit activation of pro-inflammatory cytokines, mediate the MAPK signaling pathway, and may be clinically useful as an anti-inflammatory tool.

The effects of cadmium on VEGF-mediated angiogenesis in HUVECs.

Cadmium (Cd) is a highly toxic element that causes morphologic alterations and dysfunction in blood vessels. The altered vascular function caused by cadmium has been implicated in a range of chronic diseases, including hypertension. The effects of cadmium are a multisystem phenomenon involving inflammation, hypertrophy, apoptosis, angiogenesis and important processes involved in vascular remodeling systems. Vascular endothelial growth factor (VEGF) plays a major role in cell growth and angiogenesis under pathologic conditions. VEGF secretion is related to anti‐apoptosis protein expression and attenuates apoptosis in endothelial cells. This study examined the VEGF‐dependent mechanisms of angiogenesis and apoptosis in cadmium‐treated endothelial cells (HUVECs). The effects and mechanisms of cadmium in endothelial cells (HUVECs) were examined by exposing the cells to different doses of cadmium chloride (2.5–40 μ m). After the cadmium treatment, the angiogenesis and apoptosis mechanisms related to VEGF in cadmium‐treated HUVECs were examined. As a result, the low concentration of cadmium increased the tube formation in HUVECs. In addition, cadmium at concentrations of 5 and 10 μ m increased VEGF secretion and VEGFR2 activity, which suggest that cadmium affects the growth of blood vessels. All three MAPK pathways, namely ERK, JNK and p38, were activated by cadmium in HUVECs. However, high concentrations of cadmium caused cell damage, disrupted tube formation and inhibited VEGF expression and the activities of VEGFR2 and MAPK in HUVECs. Cadmium has dual functions through VEGF‐dependent mechanisms in a dose‐dependent manner. In this study, the dual effects of cadmium might alter angiogenesis and induce apoptosis through VEGF pathways in HUVECs. Copyright

Inhibition of mitochondria-dependent apoptosis by 635-nm irradiation in sodium nitroprusside-treated SH-SY5Y cells

Nitric oxide (NO) is a major factor contributing to the loss of neurons in ischemic stroke, demyelinating diseases, and other neurodegenerative disorders. NO not only functions as a direct neurotoxin, but also combines with superoxide (O(2)(-)) by a diffusion-controlled reaction to form peroxynitrite (ONOO(-)), a species that contributes to oxidative signaling and cellular apoptosis. However, the mechanism by which ONOO(-) induces apoptosis remains unclear, although subsequent formation of reactive oxygen species (ROS) has been suggested. The aim of this study was to further investigate the triggers of the apoptotic pathway using O(2)(-) scavenging with light irradiation to block ONOO(-) production. Antiapoptotic effects of light irradiation in sodium nitroprusside (SNP)-treated SH-SY5Y cells were assayed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation, flow cytometry, Western blot, and caspase activity assays. In addition, NO, total ROS, O(2)(-), and ONOO(-) levels were measured to observe changes in NO and its possible involvement in radical induction. Cell survival was reduced to approximately 40% of control levels by SNP treatment, and this reduction was increased to 60% by low-level light irradiation. Apoptotic cells were observed in the SNP-treated group, but the frequency of these was reduced in the irradiation group. NO, O(2)(-), total ROS, and ONOO(-) levels were increased after SNP treatment, but O(2)(-), total ROS, and ONOO(-) levels were decreased after irradiation, despite the high NO concentration induced by SNP treatment. Cytochrome c was released from mitochondria of SNP-treated SH-SY5Y cells, but not of irradiated cells, resulting in a decrease in caspase-3 and -9 activity in SNP-treated cells. Finally, these results show that 635-nm irradiation, by promoting the scavenging of O(2)(-), protected against neuronal death through blocking the mitochondrial apoptotic pathway induced by ONOO(-) synthesis.

Cell death and intracellular distribution of hematoporphyrin in a KB cell line.

OBJECTIVE The objective of this study is to investigate the effect of intracellular photosensitizer distribution on tumor cell death after photodynamic therapy (PDT). BACKGROUND DATA The photosensitizer accumulates in tumor tissue during PDT, and generates intracellular reactive oxygen species (ROS), resulting in tumor cell death. MATERIALS AND METHODS This study was carried out to elucidate the effects of PDT in a KB oral cancer cell line using hematoporphyrin with irradiation at 635 nm and 5 mW/cm(2). After irradiation, the MTT reduction method, agarose gel electrophoresis, flow cytometry, and Diff-Quick staining were performed. The intracellular ROS level was measured by DCF-DA. Intracellular hematoporphyrin was monitored with a confocal microscope, and Western blot and caspase activity assays were performed. RESULTS In our study, cell survival was reduced by about 50% after 3 h of hematoporphyrin incubation time. In DNA fragmentation, flow cytometry, and Diff-Quick assay, necrosis was identified within 12 h and apoptosis soon thereafter. Confocal microscopy revealed that hematoporphyrin was localized in the cell membrane, cytoplasm, and nucleus as time passed. The quantities of intracellular ROS correlated with the time of hematoporphyrin accumulation. Additionally, Western blot analysis of Bcl-2/Bax, the release of cytochrome C, and activity of caspase-3 and caspase-9 showed that apoptosis followed the mitochondria-dependent pathway. CONCLUSION PDT with hematoporphyrin in the KB cell line showed morphological changes of cell necrosis and apoptosis, which were associated with the time of distribution and localization of hematoporphyrin. Also, the apoptosis evoked followed the mitochondria-dependent pathway.

Down-regulation of heat-shock protein 27-induced resistance to photodynamic therapy in oral cancer cells.

Photodynamic therapy (PDT) of cells is a new treatment modality involving selective delivery of a photosensitive dye into target cells, followed by visible light irradiation. PDT induces cell death by excessive ROS generation. The effects of multiple photosensitizers were owing to the difference in cell types involving sensitizer-specific protein changes linked to resistance. HSP27 is regulated in response to stress and is associated with apoptotic process. The effects of HSP27 on PDT resistance are controversial and unclear. The purpose of this study was to investigate the role of HSP27 down-regulation in the PDT-induced cells and HSP27 regulation in the resistance to PDT. KB cells transfected with HSP27 siRNA were exposed to hematoporphyrin (HP) followed by irradiation at 635 nm at an energy density of 4.5 mW/cm(2). After irradiation, the effects on HSP27 down-regulation were assessed by MTT assay, flow cytometry, confocal analysis, Western blotting and caspase activity. The results of this study showed that down-regulation of HSP27 restored cell survival in HP-PDT-induced apoptotic KB cells. HSP27 down-regulation attenuated PDT-induced apoptosis through caspase-mediated pathway in KB cells. Also, HSP27 silencing regulated Bax, Bcl-2, and PARP protein expression in PDT-induced cells. Therefore, HSP27 down-regulation confers resistance to PDT through interruption of apoptotic protein activity in PDT-induced cell death. HSP27 might contribute to regulating PDT-induced apoptosis in PDT-resistant cells.

High-grade oncocytic mucoepidermoid carcinoma of the minor salivary gland origin: a case report with immunohistochemical study

An oncocytic mucoepidermoid carcinoma arising from the minor salivary gland origin is extremely rare. We report on a 44-year-old man with a high-grade oncocytic mucoepidermoid carcinoma originating in the minor salivary gland of the posterior mandible. All tumor cells showed the expected pattern of immunoreactivity, with positive results for the antimitochondrial antibody and p63, and negative results for the androgenic receptor antibody. Microscopically, the tumor was considered to be a high-grade carcinoma in the grading systems of the Armed Forces Institute of Pathology and Brandwein. The patient underwent a partial mandibulectomy, and the lesion was reconstructed with a right fibula osteofasciocutaneous flap under general anesthesia. The patient is currently under long-term follow-up.

Dichloromethane fraction from Gardenia jasminoides: DNA topoisomerase 1 inhibition and oral cancer cell death induction.

Context: A growing body of evidence shows that compounds of plant origin have the ability to prevent cancer. The fruit of gardenia, Gardenia jasminoides Ellis (Rubiaceae), has long been used as a food additive and herbal medicine, and its pharmacological actions, such as protective activity against oxidative damage, cytotoxic effect, and anti-inflammatory and anti-tumor activity, have already been reported. Objective: The purpose of the present study was to investigate the presence of DNA topoisomerase 1 inhibitor in various solvent fractions of Gardenia extract and examine the induction of oral cancer cell death upon treatment with Gardenia extract. Materials and methods: The methanol extract of Gardenia was partitioned with n-hexane, dichloromethane, ethyl acetate, n-butanol, and water. Results: In the DNA topoisomerase 1 assay, n-hexane and dichloromethane fractions inhibited topoisomerase 1 and led to a decrease in the cell viability of KB cells. The dichloromethane fraction (0.1 mg/mL) also showed 77% inhibition of cell viability in KB cells compared with HaCaT cells. Treatment with dichloromethane fraction led to apoptotic cell death as evidenced by flow cytometric analysis and morphological changes. In addition, treatment with Gardenia extract dichloromethane fraction led to the partial increase of caspase-3, caspase-8 and caspase-9 activities and the cleavage of poly (ADP-ribose) polymerase. Conclusion: Taken together, these results suggest that the dichloromethane fraction from Gardenia extract induces apoptotic cell death by DNA topoisomerase 1 inhibition in KB cells. These findings suggest the possibility that Gardenia extract could be developed as an anticancer modality.

Ectopic overexpression of CD133 in HNSCC makes it resistant to commonly used chemotherapeutics.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world. Resistance to cytotoxic chemotherapy is a major cause of mortality in patients with HNSCC. A small subset of cancer cells called cancer stem cells (CSCs) may be key contributors to drug resistance and tumor recurrence in HNSCC. The aim of this study was to determine whether CD133, which maintains properties of CSCs, promotes chemoresistance by arresting cell cycle transition and reducing apoptosis in HNSCC cells. CD133 overexpression was examined in KB cells, and colony forming and aldehyde dehydrogenase activity assays were performed. To investigate the role of CD133 in chemoresistance, cell death was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Diff-Quick, flow cytometry, and western blot of apoptosis-related protein expression in fluorouracil (5-FU)- or cisplatin-treated cells. In addition, microarray and related protein expression assessments were performed to investigate the mechanism of chemoresistance against 5-FU and cisplatin in KB cells. Moreover, chemoresistance against 5-FU or cisplatin in a KB-inoculated mouse model was analyzed by hematoxylin and eosin staining, immunohistochemical study of CD133, and immunofluorescence of tumor tissue. In this study, we demonstrate that ectopic overexpression of CD133 significantly promotes properties of stemness in KB cell lines. Furthermore, CD133 promotes chemoresistance by arresting transition of the cell cycle and reducing apoptosis, which results in inhibition of tumor growth in 5-FU- or cisplatin-injected mouse tumor model. Taken together, our findings show that elevated levels of CD133 lead to HNSCC chemoresistance through increased stemness and cell cycle arrest.

Real-time in vivo imaging of metastatic bone tumors with a targeted near-infrared fluorophore

Tumors of the prostate or breast are particularly likely to metastasize to the bone, and early diagnosis of metastatic bone tumors is important for designing an effective treatment strategy. Imaging modalities for the detection of bone metastasis are limited, and radiation-based techniques are commonly used. Here, we investigated the efficacy of selective near-infrared (NIR) fluorescence detection of metastatic bone tumors and its role in the detection of bone metastasis in prostate and breast cancer cell lines and in a xenograft mouse model. A targeted NIR fluorophore was used to monitor metastatic bone tumors using a NIR fluorescence imaging system in real time, enabling the diagnosis of bone metastasis in vivo by providing the location of the metastatic bone tumor. The NIR fluorescence imaging technique using targeted NIR contrast agents is a potential tool for the early diagnosis of bone tumors.