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.

Comparison of the alendronate and irradiation with a light-emitting diode (LED) on murine osteoclastogenesis.

Photomodulation therapy (PBMT) using light-emitting diode (LED) has been proposed as an alternative to conventional osteoporosis therapies. Our aim was to determine the effect of irradiation with a light-emitting diode on receptor activator of NF-κB ligand (RANKL)-mediated differentiation of mouse bone marrow macrophages into osteoclasts and compare it to alendronate treatment. The cells were irradiated with LED at 635±10 nm, 9-cm spot size, 5 mW/cm2, and 18 J for 60 min/day in a CO2 incubator. The differentiation of irradiated and untreated RANKL-stimulated bone marrow macrophages into osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and by molecular methods. These included assessing messenger RNA (mRNA) expression of osteoclastic markers such as TRAP, c-Fos, Atp6v0d2, DC-STAMP, NFATc1, cathepsin K, MMP9 and OSCAR; phosphorylation of various MAPKs, including extracellular signal-regulated kinase ERK1/2, P38, and JNK; NF-κB translocation; and resorption pit formation. Results were compared to those obtained with sodium alendronate. Production of reactive oxygen species was measured by a 2',7'-dihydrodichlorofluorescein diacetate assay. LED irradiation and alendronate inhibited mRNA expression of osteoclast-related genes, such as TRAP, c-Fos, and NFATc1, and reduced the osteoclast activity of RANKL-stimulated bone marrow macrophages. LED irradiation, but not alendronate, also inhibited the production of reactive oxygen species (ROS); phosphorylation of ERK, P38, and IκB; and NF-κB translocation. These findings suggest that LED irradiation downregulates osteoclastogenesis by ROS production; this effect could lead to reduced bone loss and may offer a new therapeutic tool for managing osteoporosis.

Anti-inflammatory effect of 635 nm irradiations on in vitro direct/indirect irradiation model.

Low-level laser therapy (LLLT) has been promoted for its beneficial effects on tissue healing and pain relief. As during laser treatment it is possible to irradiate only a small area of the surface body or wound and, correspondingly, of a very small volume of the circulating blood, it is necessary to explain how its photomodification can lead to a wide spectrum of therapeutic effects. To establish the experimental model for indirect irradiation, irradiation with 635 nm was performed on immortalized human gingival fibroblasts (IGFs) in the presence of Porphyromonas gingivalis lipopolysaccharides (LPS). The irradiated medium was transferred to non-irradiated IGFs which were compared with direct irradiated IGFs. The protein expressions were assessed by Western blot, and prostaglandin E2 (PGE2 ) was measured using an enzyme-linked immunoassay. Reactive oxygen species (ROS) were measured by DCF-DA; cytokine profiles were assessed using a human inflammation antibody array. Cyclooxygenase-2 (COX-2) protein expression and PGE2 production were significantly increased in the LPS-treated group and decreased in both direct and indirect irradiated IGFs. Unlike direct irradiated IGFs, ROS level in indirect irradiated IGFs was decreased by time-dependent manners. There were significant differences of released granulocyte colony-stimulating factor (G-CSF), regulated on activated normal T-cell expressed and secreted (RANTES), and I-TAC level observed compared with direct and indirect irradiated IGFs. In addition, in the indirect irradiation group, phosphorylations of C-Raf and Erk1/2 increased significantly compared with the direct irradiation group. Thus, we suggest that not only direct exposure with 635 nm light, but also indirect exposure with 635 nm light can inhibit activation of pro-inflammatory mediators and may be clinically useful as an anti-inflammatory tool.

Effects of light-emitting diode irradiation on RANKL-induced osteoclastogenesis.

BACKGROUND AND OBJECTIVE: Bone homeostasis is maintained by a balance between osteoblastic bone formation and osteoclastic bone resorption, where intracellular reactive oxygen species (ROS) are crucial mediators of osteoclastogenesis. Recently, low-level light therapy (LLLT), a form of laser medicine used in various clinical fields, was shown to alleviate oxidative stress by scavenging intracellular ROS. The present study aimed to investigate the impact of 635 nm irradiation from a light-emitting diode (LED) on osteoclastogenesis from receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-stimulated mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN/MATERIALS AND METHODS: The effects of LED irradiation on osteoclastogenesis were assessed in tartrate-resistant acid phosphatase (TRAP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell viability, and resorption pit formation, respectively. Quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses were also performed to assess mRNA expression of osteoclastogenesis-related genes and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun-N-terminal kinase (JNK). NF-κB activity was assayed by luciferase reporter assay and Intracellular ROS generation was investigated by the 2',7'-dichlorodihydrofluorescein diacetate (H2 DCF-DA) detection method. RESULTS: LED irradiation significantly inhibited RANKL-mediated osteoclast differentiation from BMMs and mRNA expression of TRAP, osteoclast-associated immunoglobulin-like receptor (OSCAR), and dendrocyte-expressed seven-transmembrane protein (DC-STAMP). Exposure to LED light likewise significantly decreased RANKL-facilitated NF-κB activity, p38 and ERK phosphorylation and intracellular ROS generation, and increased gene expression of nuclear factor E2-related factor 2 (Nrf2). CONCLUSIONS: Taken together, the results presented herein show that LED irradiation downregulates osteoclastogenesis by reducing ROS production. Therefore, LED irradiation/LLLT might be useful as an alternative, conservative approach to osteoporosis management.

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.

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.

Inflammatory cytokines are suppressed by light-emitting diode irradiation of P. gingivalis LPS-treated human gingival fibroblasts: inflammatory cytokine changes by LED irradiation.

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 E(2) (PGE(2)) in the supernatant was measured using an enzyme-linked immunoassay. COX-2 protein expression and PGE(2) 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.

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.

Regulation of Osteosclerosis by Inoculated Cd133+ PC3 Cells in Bone-marrow Microenvironmental Niches.

Bone is the most common site of prostate cancer (PC) metastasis. Studies suggest that cancer stem cells (CSCs) are associated with stemness characteristics, providing some support for the concept that CSCs act as osteosclerotic precursors in bone microenvironmental niches. Here, we asked whether ectopic overexpression of CD133 maintains stability of CSCs in human PC cell lines and induces the changes of molecular features in the bone microenvironment. Ectopic overexpression of CD133 in PC3 or DU145 cells led to increased expression of ALDHA1, OCT4, and NANOG, enhanced colony-forming ability, and increased ALDH activity. In addition, micro-CT imaging, confocal microscopy, and H&E staining of mouse tissue confirmed that CD133 overexpression in PC3 and DU145 led to marked osteolytic bone tumor. However, expression of osteoblastic markers such as collagen type I, bone sialoprotein, and osteocalcin (OC) at the tumor margin of CD133-overexpressing PC3 tumors in mouse tibiae was higher than that of CD133-overexpressing DU145 tumors with osteosclerotic molecular features. In addition, expression of osteopontin (OPN) mRNA/protein by CD133-overexpressing PC3 cells was higher than that by DU145 cells. Especially, conditioned medium (CM) from PC3CD133+ cells increased osterix (OSX) activity in bone marrow stromal cells (BMSCs), resulting in increased expression of OC mRNA/protein resulted in increased staining of mineralized matrix by Alizarin red. However, CM from OPN silenced PC3CD133+ cells led to a reduction of OC mRNA and protein expression through OSX activity resulted in reduced amount of mineralized matrix. In conclusion, these findings suggest that CD133 plays a functional role in regulating CSC characteristics in PCs and modulates their abilities in which induce the osteosclerosis of BMSCs. In addition, OPN from CSCs acts as a niche component that promotes osteosclerosis by supporting osteoblastic differentiation of BMSCs.

Regulation of RANKL-Induced Osteoclastogenesis by 635-nm Light-Emitting Diode Irradiation Via HSP27 in Bone Marrow-Derived Macrophages.

OBJECTIVE: This study was designed to investigate the effect of 635-nm irradiation from a light-emitting diode (LED) on osteoclastogenesis in receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-stimulated mouse bone marrow-derived macrophages (BMMs). We determined whether 635-nm irradiation modulated the RANKL-induced osteoclastic signaling pathway in heat shock protein-27 (HSP27)-silenced cells and analyzed the functional cross talk between these factors in osteoclastic differentiation and activation. BACKGROUND: HSP27, a member of the small HSP family, regulates oxidative stress. Clinical reports suggest that low-level laser therapy or LED therapy (LEDT) could be an effective alternative treatment for osteolytic bone disease. METHODS: In control or HSP27-siRNA-treated BMMs, the effects of LED irradiation with 635 nm and 5 mW/cm2 on RANKL-induced osteoclastic differentiation and activity were assessed by measuring tartrate-resistant acid phosphatase (TRAP) and resorption pit formation. Quantitative real-time polymerase chain reaction and western blot assays were carried out to assess the mRNA expression of osteoclastogenesis-related genes and phosphorylation of c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and p38, respectively. Intracellular reactive oxygen species (ROS) generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) detection method. RESULTS: The 635-nm irradiation treatment significantly increased HSP27 expression and decreased intracellular ROS generation, as well as p38 and AKT phosphorylation, leading to reductions in the expression of c-fos, NFATc1, and DC-STAMP and TRAP activation and osteoclastic bone resorption in RANKL-induced BMMs. However, in HSP27-silenced BMMs, no change was observed. CONCLUSIONS: Thus, 635-nm irradiation modulates RANKL-induced osteoclastogenesis via HSP27 in BMMs. Thus, HSP27 may play a role in regulating the osteoclastic response to LEDT.

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.

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.

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.

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.