Indocyanine-Green-Loaded Liposomes for Photodynamic and Photothermal Therapies: Inducing Apoptosis and Ferroptosis in Cancer Cells with Implications beyond Oral Cancer.

Oral cancer represents a global health burden, necessitating novel therapeutic strategies. Photodynamic and photothermal therapies using indocyanine green (ICG) have shown promise due to their distinctive near-infrared (NIR) light absorption characteristics and FDA-approved safety profiles. This study develops ICG-loaded liposomes (Lipo-ICGs) to further explore their potential in oral cancer treatments. We synthesized and characterized the Lipo-ICGs, conducted in vitro cell culture experiments to assess cellular uptake and photodynamic/photothermal effects, and performed in vivo animal studies to evaluate their therapeutic efficacy. Quantitative cell apoptosis and gene expression variation were further characterized using flow cytometry and RNA sequencing, respectively. Lipo-ICGs demonstrated a uniform molecular weight distribution among particles. The in vitro studies showed a successful internalization of Lipo-ICGs into the cells and a significant photodynamic treatment effect. The in vivo studies confirmed the efficient delivery of Lipo-ICGs to tumor sites and successful tumor growth inhibition following photodynamic therapy. Moreover, light exposure induced a time-sensitive photothermal effect, facilitating the further release of ICG, and enhancing the treatment efficacy. RNA sequencing data showed significant changes in gene expression patterns upon Lipo-ICG treatment, suggesting the activation of apoptosis and ferroptosis pathways. The findings demonstrate the potential of Lipo-ICGs as a therapeutic tool for oral cancer management, potentially extending to other cancer types.

Identification of Prognostic Biomarkers Originating From the Tumor Stroma of Betel Quid-Associated Oral Cancer Tissues.

BACKGROUND: Partial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging. METHODS: Gene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan-Meier curves of estimated overall survival were compared for statistical difference using the log-rank test. RESULTS: Compared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types. CONCLUSIONS: As the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.

Enhanced aerobic glycolysis of nasopharyngeal carcinoma cells by Epstein-Barr virus latent membrane protein 1.

Latent membrane protein 1 (LMP1) is a principal viral oncoprotein in Epstein-Barr virus (EBV)-associated malignancies, including nasopharyngeal carcinoma (NPC), which acts through regulating tumorigenesis and metabolic reprogramming of cancers. In the presence of oxygen, we demonstrated that glucose consumption, lactate production and lactate dehydrogenase (LDH) activity were significantly increased upon LMP1 expression in NPC cells and in a LMP1 variant derived from NPC patients-transformed BALB/c-3T3 cells. The amounts of the α subunit of hypoxia-inducible factor-1 (HIF-1α), a key regulator of aerobic glycolysis, and its targets, pyruvate dehydrogenase kinase 1 (PDK1) and the pyruvate kinase M2 (PKM2) isoform, were also consistently elevated by LMP1. Moreover, in parallel with reductions in the oxygen consumption rate and mitochondrial membrane potential in cells, an augmented extracellular lactate concentration was observed due to LMP1 induction. In conclusion, our results proved facilitation of the Warburg effect by LMP1 through alteration of mitochondrial function in NPC cells.

Positive regulation of HIF-1A expression by EBV oncoprotein LMP1 in nasopharyngeal carcinoma cells.

Latent membrane protein 1 (LMP1) is a pivotal viral oncoprotein that contributes to the carcinogenesis of Epstein-Barr virus (EBV)-associated malignancies, including nasopharyngeal carcinoma (NPC). We investigated the regulation of hypoxia-inducible factor 1-α (HIF-1α) by LMP1. In NPC cells, we found that LMP1 significantly enhanced the HIF-1α mRNA level, and not only the protein amount as described previously. Mechanistically, the stability of the HIF-1α transcript was remarkably prolonged by LMP1 via reduced expressions of RNA-destabilizing proteins tristetraprolin (TTP) and pumilio RNA-binding family member 2 (PUM2) through C-terminal activation region 1 (CTAR1) and CTAR3 interaction with the ERK1/2 and STAT3 signaling pathways, respectively, in parallel with hindrance of PUM2 binding to the HIF-1α mRNA 3'-untranslated region (3'-UTR). On the other hand, HIF-1A promoter activity was also obviously facilitated by the LMP1 CTAR1-recruited ERK1/2/NF-κB pathway. Intriguingly, in this scenario, augmented HIF-1α further exhibited positive auto-regulation of its own gene transcription. Our results showed the first time that LMP1 directly up-regulates HIF-1A transcription and post-transcription in NPC cells, in addition to providing evidence of an increase in the HIF-1α mRNA level caused by a tumor-associated virus under normoxic conditions.

Improve efficacy of topical ALA-PDT by calcipotriol through up-regulation of coproporphyrinogen oxidase.

BACKGROUND: Topical 5-aminolevulinic acid-mediated photodynamic therapy (topical ALA-PDT) is effective for treating oral precancerous lesions. The aim of this in vivo and in vitro study was to examine whether the efficacy of topical ALA-PDT could be further improved by calcipotriol (CAL). METHODS: Precancerous lesions in the buccal pouch of hamsters were induced by dimethylbenz(a)anthracene (DMBA). Lesions were treated with multiple topical ALA-PDT with or without CAL pretreatment. ALA-induced protoporphyrine IX (PpIX) was monitored by in situ fluorescence measurement. The effect of CAL on heme-related enzymes (CPOX, PPOX, and FECH) were examined in an in vitro model using human squamous cell carcinoma (SCC) cells (SCC4, SAS) using Western blots. RESULTS: Fluorescence spectroscopy revealed that PpIX reached its peak level in precancerous epithelial cells of buccal pouch at 2.5 or 3.5h without or with CAL pretreatment, respectively. Both treatment regimens showed similar response rates, but the complete response was achieved after 5 times of ALA-PDT and 3 times of CAL-ALA-PDT (p<0.001). Pretreatment of SCC cells with 10(-8) or 10(-7)M CAL could result in a significant cell death (p<0.05) and an elevation of CPOX protein level. CONCLUSION: Topical CAL can improve the efficacy of ALA-PDT in treating precancerous lesions, likely through the increase in CPOX level and in PpIX production.

Topical methotrexate pretreatment enhances the therapeutic effect of topical 5-aminolevulinic acid-mediated photodynamic therapy on hamster buccal pouch precancers.

BACKGROUND/PURPOSE: Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is effective for treatment of human oral precancerous lesions. This animal study aimed to assess whether topical methotrexate (MTX) pretreatment could enhance the therapeutic effect of topical ALA-PDT on hamster buccal pouch precancerous lesions. METHODS: Twenty hamster buccal pouch precancerous lesions were treated with either topical ALA-PDT with topical MTX pretreatment (topical MTX-ALA-PDT group, n = 10) or topical ALA-PDT alone (topical ALA-PDT group, n = 10). The intracellular protoporphyrin IX (PpIX) level in another 12 precancerous lesions (n = 6 for either the topical MTX-ALA or topical ALA group) was monitored by fluorescence spectroscopy. RESULTS: The intracellular PpIX reached its peak level in precancerous lesions 6.5 hours and 2.5 hours after topical ALA application for the topical MTX-ALA group (5.63-fold higher in the lesion than in the normal mucosa) and topical ALA group (2.42-fold higher in the lesion than in the normal mucosa), respectively. The complete response rate of precancerous lesions was 80% for the topical MTX-ALA-PDT group and 70% for the topical ALA-PDT group. In addition, the topical MTX-ALA-PDT group required a significantly lower mean treatment number (2.1 ± 0.6) to achieve complete response than the topical ALA-PDT group (4.4 ± 1.3, p < 0.001)). Moreover, the topical MTX-ALA-PDT group had a lower recurrence rate (12.5%) than the topical ALA-PDT group (28.6%). CONCLUSION: We conclude that topical MTX-pretreatment can increase intracellular PpIX production in hamster buccal pouch precancerous lesions and significantly improves the outcomes of the precancerous lesions treated with topical ALA-PDT.

Monocyte chemotactic protein 1 (MCP-1) modulates pro-survival signaling to promote progression of head and neck squamous cell carcinoma.

BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) recruits monocytes and macrophages to inflammation sites, and inflammatory infiltration correlates with the progression of head and neck squamous cell carcinoma (HNSCC). This study aims to determine whether MCP-1 expression is related to HNSCC malignancy and patient survival. We also investigated the relationship between MCP-1 expression and the phosphorylation state of the pro-survival pathway factors Akt, ERK, and STAT3. METHODS: Expression of MCP-1 and related proteins in HNSCC cell lines was investigated using western blotting. HNSCC patients (34) without distant metastasis at diagnosis were recruited for tissue specimen evaluation of MCP-1 expression and clinical outcomes. The relationship between MCP-1 expression and survival was evaluated using the Cox proportional hazard model with stepwise selection. RESULTS: High-grade HNSCC cell lines were found to have higher levels of active Akt, ERK, and/or STAT3 than did lower grade cell lines under serum-free condition. OCSL, the most malignant cell line, had the highest level of endogenous MCP-1. Administration of exogenous recombinant MCP-1 increased phosphorylation of Akt, ERK, and STAT3 in a dose- and time-dependent manner and increased cellular resistance to serum starvation. Inhibition of Akt, ERK, or STAT3 reduced cell growth and caused cell death. Long-term survival of HNSCC patients was negatively associated with the histological intensity of MCP-1, implicating MCP-1 as a potential prognostic marker for HNSCC. CONCLUSIONS: These results suggest that overexpressed MCP-1 in cancer cells may promote HNSCC progression through upregulating pro-survival signaling pathways. High cellular MCP-1 expression is related to poor overall survival rate in HNSCC patients.

Methotrexate enhances 5-aminolevulinic acid-mediated photodynamic therapy-induced killing of human SCC4 cells by upregulation of coproporphyrinogen oxidase.

BACKGROUND/PURPOSE: Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is effective for treatment of oral precancerous and cancerous lesions. This in vitro study tried to examine whether the SCC4 cell killing by ALA-PDT was enhanced by pretreatment of methotrexate (MTX). METHODS: To measure the SCC4 cell killing abilities by MTX-pretreated ALA-PDT (MTX-ALA-PDT), the SCC4 cells were pretreated with 0 mg/L, 0.001 mg/L, 0.01 mg/L, 0.1 mg/L, or 1 mg/L of MTX for 72 hours, then incubated with 0 mM, 0.0625 mM, 0.125 mM, 0.187 mM, 0.25 mM, or 0.375 mM ALA for 4 hours, and subsequently illuminated with a 640-nm light-emitting diode array at a light dose of 10 J/cm(2). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted at 24 hours to quantify SCC4 cell survival rates after MTX-ALA-PDT treatment. Western blot analyses were used to examine the MTX-mediated enhancement in the expressions of the heme production-related enzymes, coproporphyrinogen oxidase (CPOX), protoporphyrinogen oxidase (PPOX), and ferrochelatase, in the MTX-preconditioned SCC4 cells. RESULTS: Pretreatment of SCC4 cells by 0.001 mg/L MTX for 72 hours resulted in a significant augmentation in MTX-ALA-PDT-induced killing of SCC4 cells (p < 0.05). The SCC4 cells treated with 0.001 mg/L MTX for 72 hours showed a significant and 1.65-fold increase in CPOX expression compared with the control SCC4 cells without MTX treatment (p < 0.05). However, no significant changes in the expressions of PPOX and ferrochelatase were observed in the SCC4 cells pretreated with different concentrations of MTX. CONCLUSION: MTX enhances ALA-PDT-induced SCC4 cell killing through upregulation of CPOX expression and subsequent increase in intracellular protoporphyrin IX production in SCC4 cells.

Concomitant induction of apoptosis and autophagy by prostate apoptosis response-4 in hypopharyngeal carcinoma cells.

The tumor-suppressive activity of prostate apoptosis response-4 (Par-4) has been demonstrated in a variety of human cancers. In this study, for the first time to our knowledge, we demonstrated that a higher intensity of Par-4 was significantly correlated with a better response in patients with hypopharyngeal carcinoma undergoing radiotherapy alone or concurrent chemoradiotherapy. Mechanistically, an elevated expression of Par-4 induced apoptosis of hypopharyngeal carcinoma cells and sensitized cells toward chemotherapeutic agents or X-ray irradiation. Along with apoptotic incitation, intriguingly, autophagic flux also increased on Par-4 stimulation and contributed to cell death. Moreover, the expressions of multiple common regulators involved in apoptosis and autophagy were regulated by Par-4. Taken together, our results suggested a prognostic role of Par-4 in hypopharyngeal carcinoma and showed novel activity of Par-4 in apoptosis and autophagy induction.

Differential miRNA expression in repeated recurrence of nasopharyngeal carcinoma.

Deregulated microRNAs (miRNAs) are known to be involved in the tumorigenesis of nasopharyngeal carcinoma (NPC). However, the role of miRNA expression in tumor recurrence is not yet understood. We found distinctive miRNA expression in repeated recurrent tumors using miRNA microarray, and verified this using quantitative real-time RT-PCR and miRNA in situ hybridization analysis. Computational analysis and immunohistochemistry further revealed that differentially expressed miRNAs may work in concert to modulate a multitude of biological pathways. The results not only indicate differential miRNA expression during tumor relapse, but imply the potential use of miRNAs to classify repeated recurrence of NPC beyond the histological approach.

Relevance of ultrastructural alterations of intercellular junction morphology in inflamed human esophagus.

BACKGROUND/AIMS: Detailed characterization of the ultrastructural morphology of intercellular space in gastroesophageal reflux disease has not been fully studied. We aimed to investigate whether subtle alteration in intercellular space structure and tight junction proteins might differ among patients with gastroesophageal reflux disease. METHODS: Esophageal biopsies at 5 cm above the gastroesophageal junction were obtained from 6 asymptomatic controls, 10 patients with reflux symptoms but without erosions, and 18 patients with erosions. The biopsies were morphologically evaluated by transmission electron microscopy, and by using immunohistochemistry for tight junction proteins (claudin-1 and claudin-2 proteins). RESULTS: The expressions of tight junction proteins did not differ between asymptomatic controls and gastroesophageal reflux disease patients. In patients with gastroesophageal reflux disease, altered desmosomal junction morphology was only found in upper stratified squamous epithelium. Dilated intercellular space occurred only in upper stratified squamous epithelium and in patients with erosive esophagitis. CONCLUSIONS: This study suggests that dilated intercellular space may not be uniformly present inside the esophageal mucosa and predominantly it is located in upper squamous epithelium. Presence of desmosomal junction alterations is associated with increased severity of gastroesophageal reflux disease. Besides dilated intercellular space, subtle changes in ultrastructural morphology of intercellular space allow better identification of inflamed esophageal mucosa relevant to acid reflux.

Successful treatment of 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch precancerous lesions by topical 5-aminolevulinic acid-mediated photodynamic therapy.

BACKGROUND: Our previous studies found that topical 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (ALA-PDT) with a light dose of 100 J/cm(2) is very effective for human oral precancerous lesions. METHODS: In this study, 20 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch precancerous lesions were treated by topical ALA-PDT with a light dose of either 75 J/cm(2) (n = 10) or 100 J/cm(2) (n = 10) using a 640-nm light-emitting diode (LED) light to test which light dose could achieve a better clinical outcome. RESULTS: The 10 precancerous lesions treated by 75-J ALA-PDT showed complete response in 8 after an average of 3.4 (range, 2-6) treatments and partial response in 2. The 10 precancerous lesions treated by 100-J ALA-PDT demonstrated complete response in 7 after an average of 4.4 (range, 3-6) treatments and partial response in 3. Fisher exact test showed no significant difference in clinical outcome between these two treatment modalities (p = 1.000). One complete-response precancerous lesion in the 75-J ALA-PDT group recurred at the end of 19-week follow-up and another complete response precancerous lesion in the 100-J ALA-PDT group recurred at the end of 16-week follow-up. Both recurrence lesions were treated by the original topical ALA-PDT regimen and demonstrated complete response after 3 PDT treatments. Furthermore, the 5 partial-response precancerous lesions developed into squamous cell carcinomas after 30-week follow-up. CONCLUSION: Our findings indicate that both the 75-J and 100-J topical ALA-PDT treatment modalities are very effective for DMBA-induced hamster buccal pouch precancerous lesions and no significant difference in clinical outcome between these two treatment modalities.

Monomeric L-amino acid oxidase-induced mitochondrial dysfunction in Rhizoctonia solani Reveals a novel antagonistic mechanism of Trichoderma harzianum ETS 323.

The monomeric L-amino acid oxidase (mTh-LAAO) of Trichoderma harzianum ETS 323 has been suggested to antagonize Rhizoctonia solani by an unknown mechanism. Here, the mTh-LAAO-treated R. solani exhibited hyphal lysis and apoptotic characteristics such as DNA fragmentation, reactive oxygen species (ROS) accumulation, lipid peroxidation, and mitochondrial membrane potential depolarization. This hyphal lysis was suppressed by the mitochondria-dependent apoptosis inhibitor oligomycin while accompanied by reduction of ROS accumulation. This result suggested that mitochondria-mediated apoptosis in R. solani was involved in mTh-LAAO-induced growth inhibition, which was supported by the evidence of cytocheome c release and activation of caspases 9 and 3. Furthermore, the data indicated that the mTh-LAAO-induced fungal cell death was also closely interrelated with the interaction of mTh-LAAO with R. solani hyphal cell wall proteins. These results illuminate the biological function and mechanism underlying the antagonistic action of T. harzianum mTh-LAAO against fungal pathogens.

Effective treatment of 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch precancerous lesions by topical photosan-mediated photodynamic therapy.

BACKGROUND: Previous studies found that topical photodynamic therapy (PDT) is very effective for human oral precancerous lesions. METHODS: This study evaluated whether topical photosan-mediated PDT (topical photosan-PDT), using a 640-nm light-emitting diode (LED) light, is an effective treatment modality for hamster buccal pouch precancerous lesions. Fourteen 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch precancerous lesions were treated with topical photosan-PDT using the 640-nm LED light twice a week. RESULTS: All 14 of the precancerous lesions showed a complete histologically confirmed response to the lesions after an average of 3.79 (range, 3-5) PDT treatments. Normal and precancerous pouch mucosae in the other 4 hamsters received 17 or 19 treatments of topical photosan-PDT showed no cumulative side effects. No recurrence of the lesions was found in these 18 PDT-treated hamsters after a follow-up period of 50 weeks. CONCLUSION: Our findings indicate that topical photosan-PDT is a very effective treatment modality for DMBA-induced hamster buccal pouch precancerous lesions.

Avian reovirus S1133-induced DNA damage signaling and subsequent apoptosis in cultured cells and in chickens.

In this study, intracellular signaling in ARV S1133-mediated apoptosis was investigated. A microarray was used to examine the gene expression profiles of cells upon ARV S1133 infection and ARV-encoded pro-apoptotic protein σC overexpression. The analysis indicated that in the set of DNA-damage-responsive genes, DDIT-3 and GADD45α were both upregulated by viral infection and σC overexpression. Further investigation demonstrated that both treatments caused DNA breaks, which increased the expression and/or phosphorylation of DNA damage response proteins. ROS and lipid peroxidation levels were increased, and ARV S1133 and σC caused apoptosis mediated by DNA damage signaling. ROS scavenger NAC, caffeine and an ATM-specific inhibitor significantly reduced ARV S1133- and σC-induced DNA breaks, DDIT-3 and GADD45α expression, H2AX phosphorylation, and apoptosis. Overexpression of DDIT-3 and GADD45α enhanced the oxidative stress and apoptosis induced by ARV S1133 and σC. In conclusion, our results demonstrate the involvement of the DNA-damage-signaling pathway in ARV S1133- and σC-induced apoptosis.

Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323.

Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.

A novel L-amino acid oxidase from Trichoderma harzianum ETS 323 associated with antagonism of Rhizoctonia solani.

Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.

Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells.

Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors gamma used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G(2)/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.

Activation of PI 3-kinase/Akt/NF-kappaB and Stat3 signaling by avian reovirus S1133 in the early stages of infection results in an inflammatory response and delayed apoptosis.

Avian reovirus (ARV) strain S1133 causes apoptosis in host cells in the middle to late stages of infection. This study investigated the early-stage biological response and intracellular signaling in ARV S1133-infected Vero and chicken cells. Treatment with conditioned medium from ARV S1133-infected cells increased the chemotactic activity of U937 cells. Neutralizing antibodies against IL-1beta and IL-6 showed that both cytokines contribute to viral-induced inflammation but neither affect cell survival. Inhibition of Akt, NF-kappaB, and Stat3 released the chemotactic activity and anti-apoptotic effect elicited by ARV S1133. ARV S1133 activated PI 3-kinase-dependent Akt/NF-kappaB and p70 S6 kinase, as well as Stat3; however, p70 S6 kinase was not involved in ARV S1133-mediated effects. DF1 cells over-expressing constitutively active PI 3-kinase and Stat3 showed association with enhancement of anti-apoptotic activity. In conclusion, in the early stages of ARV S1133 infection, activation of cell survival signals contributes to virus-induced inflammation and anti-apoptotic response.

Widespread expression of prostate apoptosis response-4 in nasopharyngeal carcinoma.

BACKGROUND: Prostate apoptosis response-4 (Par-4) augments apoptosis in various tumors, either during apoptotic insult or by ectopic overexpression. However, investigation of Par-4 expression in nasopharyngeal carcinoma (NPC) is lacking. METHODS: Specimens from patients with NPC, hypopharyngeal carcinoma (HPC), or oral cavity cancer were examined for Par-4 expression using immunohistochemistry. NPC cell proliferation and apoptosis were analyzed using immunohistochemical staining for Ki67, B-cell lymphoma 2 (Bcl-2), and in situ terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick end-labeling (TUNEL) assay, respectively. RESULTS: Par-4 was ubiquitously expressed in NPC biopsies (96.2%, 25/26) and was significantly higher than in HPC (47.6%, 50/105, p < .0001) and oral cavity cancers (38.7%, 12/31, p < .0001). Remarkably, apoptosis of NPC cells was absent and Par-4 expression was associated with obvious expression of Bcl-2 and Ki67 in all patients tested with NPC. CONCLUSIONS: Immunohistochemistry results showed widespread expression of Par-4 in NPC and revealed sustainable proliferation of NPC cells regardless of Par-4 expression.