Malat1 regulates PMN-MDSC expansion and immunosuppression through p-STAT3 ubiquitination in sepsis.

Myeloid-derived suppressor cells (MDSCs) expand during sepsis and contribute to the development of persistent inflammation-immunosuppression-catabolism syndrome. However, the underlying mechanism remains unclear. Exploring the mechanisms of MDSCs generation may provide therapeutic targets for improving immune status in sepsis. Here, a sepsis mouse model is established by cecal ligation and perforation. Bone marrow cells at different sepsis time points are harvested to detect the proportion of MDSCs and search for differentially expressed genes by RNA-sequence. In lethal models of sepsis, polymorphonuclear-MDSCs (PMN-MDSCs) decrease in early but increase and become activated in late sepsis, which is contrary to the expression of metastasis-associated lung adenocarcinoma transcript 1 (Malat1). In vivo, Malat1 inhibitor significantly increases the mortality in mice with late sepsis. And in vitro, Malat1 down-regulation increases the proportion of PMN-MDSCs and enhanced its immunosuppressive ability. Mechanistically, Malat1 limits the differentiation of PMN-MDSCs by accelerating the degradation of phosphorylated STAT3. Furthermore, Stattic, an inhibitor of STAT3 phosphorylation, improves the survival of septic mice by inhibiting PMN-MDSCs. Overall, the study identifies a novel insight into the mechanism of sepsis-induced MDSCs and provides more evidence for targeting MDSCs in the treatment of sepsis.

Diagnosis of mixed infection and a primary immunodeficiency disease using next-generation sequencing: a case report.

Major Histocompatibility Complex Class II (MHC II) deficiency is a rare primary immunodeficiency disorder (PID) with autosomal recessive inheritance pattern. The outcome is almost fatal owing to delayed diagnosis and lacking of effective therapy. Therefore, prompt diagnosis, timely and effective treatment are critical. Here, we report a 117-day-old boy with diarrhea, cough, cyanosis and tachypnea who was failed to be cured by empiric antimicrobial therapy initially and progressed to severe pneumonia and respiratory failure. The patient was admitted to the pediatric intensive care unit (PICU) immediately and underwent a series of tests. Blood examination revealed elevated levels of inflammatory markers and cytomegalovirus DNA. Imaging findings showed signs of severe infection of lungs. Finally, the diagnosis was obtained mainly through next-generation sequencing (NGS). We found out what pathogenic microorganism he was infected via repeated conventional detection methods and metagenomic next-generation sequencing (mNGS) of sputum and bronchoalveolar lavage fluid (BALF). And his whole exome sequencing (WES) examination suggested that CIITA gene was heterozygous mutation, a kind of MHC II deficiency diseases. After aggressive respiratory support and repeated adjustment of antimicrobial regimens, the patient was weaned from ventilator on the 56th day of admission and transferred to the immunology ward on the 60th day. The patient was successful discharged after hospitalizing for 91 days, taking antimicrobials orally to prevent infections post-discharge and waiting for stem cell transplantation. This case highlights the potential importance of NGS in providing better diagnostic testing for unexplained infection and illness. Furthermore, pathogens would be identified more accurately if conventional detection techniques were combined with mNGS.

Sulodexide improves vascular permeability via glycocalyx remodelling in endothelial cells during sepsis.

Background: Degradation of the endothelial glycocalyx is critical for sepsis-associated lung injury and pulmonary vascular permeability. We investigated whether sulodexide, a precursor for the synthesis of glycosaminoglycans, plays a biological role in glycocalyx remodeling and improves endothelial barrier dysfunction in sepsis. Methods: The number of children with septic shock that were admitted to the PICU at Children's Hospital of Fudan University who enrolled in the study was 28. On days one and three after enrollment, venous blood samples were collected, and heparan sulfate, and syndecan-1 (SDC1) were assayed in the plasma. We established a cell model of glycocalyx shedding by heparinase III and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Sulodexide was administrated to prevent endothelial glycocalyx damage. Endothelial barrier function and expression of endothelial-related proteins were determined using permeability, western blot and immunofluorescent staining. The survival rate, histopathology evaluation of lungs and wet-to-dry lung weight ratio were also evaluated. Results: We found that circulating SDC1 levels were persistently upregulated in the non-alive group on days 1 and 3 and were positively correlated with IL-6 levels. Receiver operating characteristic curve analysis showed that SDC1 could distinguish patients with mortality. We showed that SDC1-shedding caused endothelial permeability in the presence of heparinase III and sepsis conditions. Mechanistically, sulodexide (30 LSU/mL) administration markedly inhibited SDC1 shedding and prevented endothelial permeability with zonula occludens-1 (ZO-1) upregulation via NF-κB/ZO-1 pathway. In mice with LPS and CLP-induced sepsis, sulodexide (40 mg/kg) administration decreased the plasma levels of SDC1 and increased survival rate. Additionally, sulodexide alleviated lung injury and restored endothelial glycocalyx damage. Conlusions: In conclusion, our data suggest that SDC1 predicts prognosis in children with septic shock and sulodexide may have therapeutic potential for the treatment of sepsis-associated endothelial dysfunction.

Metagenomic next-generation sequencing of bronchoalveolar lavage fluid from children with severe pneumonia in pediatric intensive care unit.

Background: Severe pneumonia due to lower respiratory tract infections (LRTIs) is a significant cause of morbidity and mortality in children. Noninfectious respiratory syndromes resembling LRTIs can complicate the diagnosis and may also make targeted therapy difficult because of the difficulty of identifying LRTI pathogens. In the present study, a highly sensitive metagenomic next-generation sequencing (mNGS) approach was used to characterize the microbiome of bronchoalveolar lavage fluid (BALF) in children with severe lower pneumonia and identify pathogenic microorganisms that may cause severe pneumonia. The purpose of this study was to use mNGS to explore the potential microbiomes of children with severe pneumonia in a PICU. Methods: We enrolled patients meeting diagnostic criteria for severe pneumonia admitted at PICU of the Children's Hospital of Fudan University, China, from February 2018 to February 2020. In total, 126 BALF samples were collected, and mNGS was performed at the DNA and/or RNA level. The pathogenic microorganisms in BALF were identified and correlated with serological inflammatory indicators, lymphocyte subtypes, and clinical symptoms. Results: mNGS of BALF identified potentially pathogenic bacteria in children with severe pneumonia in the PICU. An increased BALF bacterial diversity index was positively correlated with serum inflammatory indicators and lymphocyte subtypes. Children with severe pneumonia in the PICU had the potential for coinfection with viruses including Epstein-Barr virus, Cytomegalovirus, and Human betaherpesvirus 6B, the abundance of which was positively correlated with immunodeficiency and pneumonia severity, suggesting that the virus may be reactivated in children in the PICU. There was also the potential for coinfection with fungal pathogens including Pneumocystis jirovecii and Aspergillus fumigatus in children with severe pneumonia in the PICU, and an increase in potentially pathogenic eukaryotic diversity in BALF was positively associated with the occurrence of death and sepsis. Conclusions: mNGS can be used for clinical microbiological testing of BALF samples from children in the PICU. Bacterial combined with viral or fungal infections may be present in the BALF of patients with severe pneumonia in the PICU. Viral or fungal infections are associated with greater disease severity and death.

Effect of methomyl on water quality, growth performance and antioxidant system in liver of GIFT (Oreochromis niloticus) in the presence of peppermint (Mentha haplocalyx Briq.) as a floating bed.

This study was conducted to investigate the effect of methomyl (MET) on water quality, growth and antioxidant system of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) in the presence of peppermint as a floating bed. The concentration of NH3-N, NO2--N, NO3--N and TP in T3 (with 200 g wet peppermint) was significantly lower (P < 0.05) than that in T2 (100 g), T1 (50 g) and control, and the nutrient removal rates were 61.90%, 31.59%, 59.86% and 45.92% in 20 days, respectively. Juveniles GIFT (5.1 ± 0.2 g) were exposed to sub-lethal concentrations of 0.2, 2.0, 20 and 200 µg/L of MET for 45 days. After 6 weeks of a feeding trial, percentage weight gain (PWG), specific growth rate (SGR) and feed conversion ratio (FCR) were significantly decreased in 0.2, 2.0, 20 µg/L MET groups respectively and increased in the 200 µg/L MET group. Compared with the control, no significant changes in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were detected in the 0.2 µg/L group. The significant increase in activities of SOD, CAT and GPx was accompanied by a diminution in reduced glutathione (GSH) levels resulting with tilapia exposed to 2.0, 20, or 200 µg/L for 45 days. The highest rates observed in SOD, CAT, GPx were 157.63%, 164.05% and 167.46% of the control respectively, and the lowest inhibition rate in GSH was 66.42% of the control. Peppermint as a floating bed can alleviate the adverse effects of MET, such as growth retardation and oxidative stress.

BBOX1-AS1 Accelerates Nasopharyngeal Carcinoma Progression by Sponging miR-3940-3p and Enhancing KPNA2 Upregulation.

BACKGROUND: Upregulation of lncRNA BBOX1 antisense RNA 1 (BBOX1-AS1) has been examined in various tumors. However, its role in nasopharyngeal carcinoma (NPC) remains poorly understood. METHODS: RT-qPCR was performed to measure the expression of BBOX1-AS1, KPNA2, and miR-3940-3p. In vitro assays were performed to determine the alteration of cell phenotypes in NPC cells upon transfection or co-transfection with sh-BBOX1-AS1, sh-KPNA2, or miR-3940-3p inhibitor. The BBOX1-AS1-miR-3940-3p and miR-3940-3p-KPNA2 interplay was verified via luciferase reporter and RNA pull-down assays. RESULTS: High BBOX1-AS1 levels were detected in the nasopharyngeal carcinoma tissues. BBOX1-AS1 silencing considerably suppressed the proliferative, migratory, and invasive abilities of NPC cells in vitro. Interestingly, BBOX1-AS1 could specifically bind to miR-3940-3 and abrogate the inhibition of KPNA2 induced by miR-3940-3. Additionally, analysis of tissue samples showed that miR-3940-3 was inversely correlated with BBOX1-AS1 and KPNA2. CONCLUSION: Our findings revealed that the BBOX1-AS1/miR-3940-3/KPNA2 axis is pro-oncogenic in NPC progression, uncovering novel insights into targeted therapy for this disorder.

Incidence, persistence and clearance of cervical human papillomavirus among women in Guangdong, China 2007-2018: A retrospective cohort study.

BACKGROUND: Previous studies showed the incidence, persistence and clearance of cervical human papillomavirus (HPV) among women varies from regions. There is no study on dynamic changes of HPV infection among women in Guangdong. METHODS: It is a retrospective cohort study that included gynecological outpatients aged ≥15 years and retested for HPV within 24 months in Guangdong Women and Children Hospital to estimate HPV incidence, persistence and clearance. Outcomes were estimated through the proportion of HPV incidence, persistence and clearance in HPV-negative or HPV-positive women. Moreover, we examined HPV incidence, persistence and clearance among women who retested in four calendar periods: 0-6, 6-12, 12-18, 18-24 months after the first test. RESULTS: 33,328 gynecological outpatients were included in our study. Incidence rates of any HPV, high-risk (HR) HPV and low-risk (LR) HPV were 10.58%, 8.68% and 4.83%. The most common incident HR HPV were HPV52 (2.69%), HPV16 (1.23%) and HPV58 (1.23%). Persistence rates of any HPV, HR HPV and LR HPV were 47.55%, 42.77% and 33.88%. HPV52 (42.33%), HPV58 (40.74%) and HPV68 (32.36%) were commonly found persistent types. And clearance rates of any HPV, HR HPV and LR HPV were 52.44%, 57.23% and 66.12%.The lowest clearance rates were observed for HPV52 (57.67%), HPV68 (67.64%) and HPV39 (68.56%). HPV incidence and persistence were higher among women aged 15-19 years and ≥55 years. HPV incidence and persistence were found higher among women who retested within 6 months than others in other periods. CONCLUSIONS: HPV52, 58, 68, and 39 were the more likely to cause incident and persistent infection, and less likely to be cleared among women in Guangdong. HPV incidence and persistent infection were higher among women aged both younger and older women compared to middle aged women. HPV retesting period may impact the detection of HPV incidence, persistence and clearance.

Cervical human papillomavirus among women in Guangdong, China 2008-2017: Implication for screening and vaccination.

Our study aimed to analyze genotype-specific, age-specific prevalence, and year-on-year trend of cervical human papillomavirus (HPV) detection among women in Guangdong, China 2008 to 2017. A total of 199 963 women attending the gynecological department and 11 999 women attending the medical examination center at Guangdong Women and Children Hospital were included. Prevalent HPV detection significantly differed between these two groups of women (20.16% vs 17.25%; P < .001). HPV genotypes of these two populations have a large overlap, with HPV52, 16, 58, CP8304, and 53 being the dominant subtypes among gynecological outpatients and HPV52, CP8304, 58, 53, and 16 among women receiving physical examinations. The distribution of prevalent HPV detection showed a bimodal pattern across age groups among these two populations. However, prevalent HPV detection among these two populations exhibited different trends from 2008 to 2017. Our study demonstrated that prevalent HPV detection among women in Guangdong is associated with age and different from that among women from other regions of China. Our study may provide valuable data to inform cervical cancer screening and HPV vaccination programs for women in this province.

Ribonucleic acid interference knockdown of IL-6 enhances the efficacy of cisplatin in laryngeal cancer stem cells by down-regulating the IL-6/STAT3/HIF1 pathway.

BACKGROUND: Cisplatin has been used in the treatment of many cancers, including laryngeal cancer; however, its efficacy can be reduced due to the development of drug resistance. This study aimed to investigate whether interleukin-6 (IL-6) knockdown may enhance the efficacy of cisplatin in laryngeal cancer stem cells (CSC) and the potential involvement of the signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1 (HIF1) in this effect. METHODS: The ALDH+ and CD44+ CSC in Hep2 human laryngeal squamous cancer cells were identified by the fluorescence-activated cell sorting technique. IL-6, STAT3 and HIF1 mRNA and protein expressions were examined with quantitative real-time polymerase chain reaction and Western blot, respectively. Cell proliferation was measured by MTT assay. Tumorigenicity was measured by a colony formation assay and invasion was determined by a cell invasion assay. Apoptotic cells were counted by flow cytometry. Immunohistochemistry was performed to detect immunoreactive IL-6, STAT3 and HIF1 cells in xenografts. RESULTS: The mRNA and protein levels of IL-6, STAT3 and HIF1 were significantly increased in Hep2-CSC as compared with those from Hep2 cells. Application of siRNA-IL-6 to knockdown IL-6 resulted in significantly decreased IL-6, STAT3 and HIF1 mRNA and protein levels. IL-6 knockdown reduced cell proliferation, tumorigenicity and invasion and increased apoptosis within CSC. Enhanced degrees of suppression in these parameters were observed when IL-6 knockdown was combined with cisplatin in these CSC. Results from the xenograft study showed that the combination of IL-6 knockdown and cisplatin further inhibited the growth of xenografts as compared with that obtained in the cisplatin-injected group alone. Immunoreactive IL-6, STAT3 and HIF1 cell numbers were markedly reduced in IL-6 knockdown tumor tissues. IL-6, STAT3 and HIF1 immunoreactive cell counts were further reduced in tissue where IL-6 knockdown was combined with cisplatin treatment as compared with tissue receiving cisplatin alone. CONCLUSIONS: IL-6 knockdown can increase chemo-drug efficacy of cisplatin, inhibit tumor growth and reduce the potential for tumor recurrence and metastasis in laryngeal cancer. The IL-6/STAT3/HIF1 pathway may represent an important target for investigating therapeutic strategies for the treatment of laryngeal cancer.

The leukotriene B4-leukotriene B4 receptor axis promotes cisplatin-induced acute kidney injury by modulating neutrophil recruitment.

Cisplatin is an effective chemotherapeutic agent and widely used in treatment of various solid organ malignancies, including head and neck, ovarian, and testicular cancers. However, the induction of acute kidney injury (AKI) is one of its main side effects. Leukotriene B4 receptor 1 (BLT1) mediates the majority of physiological effects of leukotriene B4 (LTB4), a potent lipid chemoattractant generated at inflammation sites, but the role of the LTB4-BLT1 axis in cisplatin-induced AKI remains unknown. Here we found upregulated LTB4 synthesis and BLT1 expression in the kidney after cisplatin administration. Cisplatin was found to directly upregulate gene expression of leukotriene A4 hydrolase and stimulate LTB4 production in renal tubular epithelial cells. Reduced kidney structural/functional damage, inflammation, and apoptosis were observed in BLT1-/- mice, as well as in wild-type mice treated with the LTA4H inhibitor SC-57461A and the BLT1 antagonist U-75302. Neutrophils were likely the target of this pathway, as BLT1 absence induced a significant decrease in infiltrating neutrophils in the kidney. Adoptive transfer of neutrophils from wild-type mice restored kidney injury in BLT1-/- mice following cisplatin challenge. Thus, the LTB4-BLT1 axis contributes to cisplatin-induced AKI by mediating kidney recruitment of neutrophils, which induce inflammation and apoptosis in the kidney. Hence, the LTB4-BLT1 axis could be a potential therapeutic target in cisplatin-induced AKI.

Reduced expression of citrate synthase leads to excessive superoxide formation and cell apoptosis.

A/J mice are a mouse model of age-related hearing loss. It has been demonstrated that a mutation in gene of citrate synthase (CS) contributes to the early onset of hearing loss occurring at about one month of age. To understand the effects of a decreased CS activity that results from the mutation in Cs gene on hearing loss in A/J mice, human kidney cell line (293T) was transiently transfected with short hairpin RNA for Cs (shRNA-Cs) to reduce expression of CS. In comparison with those of cells transfected with a scrambled sequence (shRNA-NC), the oxygen consumption rate and adenosine trisphosphate (ATP) production level were decreased in 293T cells transfected with shRNA-Cs. Meanwhile, excessive superoxide production was induced as determined by mitochondrial superoxide formation assay (MitoSOX) and superoxide dismutase 2 (SOD2) detection. Moreover, the expression levels of BIP (binding immunoglobulin protein) and CHOP (CCAAT/enhancer-binding protein-homologous protein), markers of endoplasmic reticulum stress, were upregulated. Furthermore, apoptosis related molecule caspase-3 and the mitochondrial membrane potential were reduced. It is therefore concluded that downregulation of Cs expression in 293T cells leads to low level of ATP production, excessive superoxide formation and cell apoptosis, which implies a possible mechanism for hearing loss in A/J mice.

[Expression and significance of phosphated signal transducer and activator of transcription 3 and p53 protein in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the expression and clinical significance of phosphated signal transducer and activator of transcription 3 (p-STAT3) and p53 protein in laryngeal squamous cell carcinoma (LSCC), and the relationship between the two genes. METHOD: Formalin-fixed and paraffin-embedded tissues, which came from 60 cases of LSCC and 32 samples of normal mucosa over 2.0 cm away from tumor margin in 32 patients with total or subtotal laryngectomy were detected for the expression of p-STAT3 and p53 protein by in SP immunohistochemistry. Micro-image analysis system was used to determine the optical density,and the result was analyzed statistically. RESULT: There is over expression of p-STAT3 and p53 protein in LSCC. The expression of p-STAT3 and p53 protein in LSCC was associated with clinical stage and lymph nodal metastases (P < 0.05). There was a positive correlation between the expression of p-STAT3 and P53 protein. The correlation coefficient (r) was 0.6558 (P < 0.01). CONCLUSION: The expression of p-STAT3 and p53 protein may play an important role in the tumorigenesis,metastases and poor prognosis of LSCC. There was a positive correlation between the over expression of p-STAT3 and p53 protein in LSCC.

A novel orthotopic mouse model of head and neck cancer with molecular imaging.

OBJECTIVES/HYPOTHESIS: Our goal was to develop a noninvasive, dynamic imaging method that would further the understanding of head and neck cancer (HNC) tumor growth and local spreading. We developed a novel orthotopic mouse model of HNC with a stable cell line expressing a red fluorescent protein gene to compare a molecular imaging tumor quantification with traditional caliper measurement. METHODS: An HNC-tdT stable cell line expressing the tdTomato gene was established, which were injected into the floor of the mouth of nude mice. Tumor growth was constantly monitored using molecular imaging for up to 35 days. The tumors were further evaluated by histologic examination. RESULTS: Established tumors consistently expressed fluorescent signals that were successfully imaged by molecular imaging during the study. Initial tumor development was detected earlier than caliper measurement would allow. The fluorescent signal quantities of tumors detected by the imaging correlated with the tumor sizes measured by calipers. CONCLUSIONS: This novel animal model represents an orthotopic human HNC model. The tumor can be detected earlier with molecular imaging than by conventional external caliper measurement. Unlike surgical measurement, the tumor can be quantified without disturbing the tumor environment. This model has significant potential for HNC oncologic research.

[Expression and significance of signal transducer and activator of transcription 3 and c-myc in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the expression and clinical significance of signal transducer and activator of transcription 3(STAT3) and its target gene c-myc in laryngeal squamous cell carcinoma (LSCC), and the relationship between the two genes. METHOD: Formalin-fixed and paraffin-embedded tissues, which came from 56 cases of LSCC and 30 samples of normal mucosas from 30 patients with total or subtotal laryngectomy over 2.0 cm away from tumor margin, were detected for the expression of STATS, c-myc by in situ hybridization and SP immunohistochemistry. Micro-image analysis system was used to determine the optical density, and the result was analyzed statistically. RESULT: There is overexpression of STAT3, c-myc mRNA and protein in LSCC. The expression of STAT3 and c-myc mRNA in LSCC was associated with clinical stage, differentiation grade and lymph nodal metastases (P < 0.05 or 0.01). The expression of STAT3 and c-myc protein in LSCC was associated with clinical stage and lymph nodal metastases (P < 0.05 or 0.01). There was a positive correlation between the expression of STAT3 and c-myc genes. mRNA and protein, The correlation coefficient (r) was 0.6224 (P < 0.01) for the mRNA expression and 0.7012 (P < 0.01 )for the protein expression. CONCLUSION: The expression of STAT3 and c-myc may play an important role in the tumorigenesis, metastases and poor prognosis of LSCC. There was a positive correlation between the overexpression of STAT3 and c-myc genes in LSCC.

Cucurbitacin B, a small molecule inhibitor of the Stat3 signaling pathway, enhances the chemosensitivity of laryngeal squamous cell carcinoma cells to cisplatin.

We have previously shown that the simultaneous exposure of Hep-2 cells to cucurbitacin B and docetaxel significantly enhances anticancer activity of these cells by suppressing Stat3 activation and down-regulating the expression levels of key cell cycle and anti-apoptosis regulators. In order to determine whether cucurbitacin B can also enhance the sensitivity of Hep-2 laryngeal cells to cisplatin, we treated Hep-2 cells with either cucurbitacin B, cisplatin, or the combination and evaluated these cells for proliferation, cell cycle distribution, and apoptosis. Our results demonstrate that, in comparison to single agent cucurbitacin B or cisplatin treated cells, Hep-2 cells treated with a cucurbitacin B/cisplatin combination display synergistic effects on growth inhibition, cell cycle arrest, and apoptosis induction. Western blot analysis using protein extracts from Hep-2 cells treated with cucurbitacin B, cisplatin, or the combination largely recapitulated the observations made when treated with the cucurbitacin B/docetaxel combination. More specifically, Hep-2 cell lines treated with the cucurbitacin B/cisplatin combination demonstrated a significantly reduced level of p-Stat3 in comparison with single agent treated cells. In addition, cucurbitacin B/cisplatin treated Hep-2 cells also demonstrated a significant reduction in Bcl-2 and Cyclin B1 protein levels compared to single agent cucurbitacin B or cisplatin treated cells. Xenograft models containing Hep-2 cells in mice also demonstrated that this cucurbitacin B/cisplatin combination led to the synergistic inhibition of tumor growth. Taken together, these results suggest that the cucurbitacin B/cisplatin combination treatment may be a potentially useful therapeutic option for individuals diagnosed with laryngeal cancer.

Head and neck squamous cell carcinoma targeted chemosensitization.

OBJECTIVE: The current treatment for advanced head and neck squamous cell carcinoma continues to result in poor outcomes. The purpose of this study is to investigate the benefit of fibroblast growth factor 2-targeted adenovirus-mediated mutant-Rad50 (FGF2-Ad-Rad50) gene transfer in enhancing chemosensitization for head and neck squamous cell carcinoma and reducing chemotoxicity. STUDY DESIGN: Randomized controlled laboratory study. SETTING: University of Pennsylvania, Philadelphia, PA. SUBJECTS AND METHODS: Human head and neck squamous cell carcinoma tumor cells and a mouse model with human head and neck squamous cell carcinoma were used for this study. There were five mice in each study group. FGF2-fab' molecule was conjugated with an adenoviral mutant-Rad50 construct. FGF2-targeted transgene expression efficiency was evaluated in vitro. Tumor cytotoxicity and growth inhibition were examined after combined FGF2-Ad-Rad50 with cisplatin treatment in vitro and in vivo. Anti-tumor mechanisms were investigated. RESULTS: FGF2-targeted gene transfer approach significantly improved transgene expression in head and neck squamous cell carcinoma tumor cells over a nontargeted approach (207.51+/-33.62 vs 51.44+/-8.28, respectively). FGF2-Ad-Rad50 with cisplatin demonstrated a superior tumor suppression effect (264.5+/-124.1 mm3 vs 567.1+/-267.6 mm3), increased DNA double-strand breaks (1349+/-51.67 vs 774+/-28.56), and anti-angiogenesis (%ROI: 0.76%+/-0.38% vs 2.10%+/-1.66%) in tumor cells over nontargeted adenovirus. CONCLUSION: Combination of FGF2-Ad-Rad50 with cisplatin significantly improves anti-tumor effect by targeting DNA repair systems and tumor angiogenesis. The great benefit of this strategy supports clinical trial for novel treatment of head and neck squamous cell carcinoma.

[Effects of cucurbitacin B on cell proliferation and apoptosis in Hep-2 cells].

OBJECTIVE: To investigate the mechanism underlying the anticancer activity of cucurbitacin B on human laryngeal cancer. METHOD: Hep-2 cells were treated with different concentrations of cucurbitacin B for different time. MTT assay was used to evaluate cell proliferation. Flow cytometry with PI staining and fluorescent microscopy with Hoechst 33258 staining were used to estimate cell cycle distribution and cell apoptosis. Expression of p-STAT3, cyclin B1 and Bcl-2 proteins was evaluated by Western blot assay. In vivo inhibitory effects of cucurbitacin B on tumor growth was evaluated in a nude mouse xenograft model. RESULT: Cucurbitacin B inhibited cellular proliferation in a dose and time dependent manner (P <0.05 or 0.01). Flow cytometry analysis showed that treatment with cucurbitacin B resulted in accumulation of cells at the G2/M phase of the cell cycle and cell apoptosis in a dose and time dependent manner (P <0.05 or P <0.01). Marked morphological changes of cell apoptosis including condensation of chromatin, nuclear fragmentation and apoptotic bodies were observed clearly by Hoechst 33258 staining. Western blot analysis demonstrated that the expression of p-STAT3, cyclin B1 and Bcl-2 proteins was suppressed significantly. In vivo studies showed that the inhibitory rates on laryngeal squamous carcinoma xenograft model were 32.43%, 43.24% and 70.27% for lower, moderate and higher dosage group, respectively. CONCLUSION: Cucurbitacin B inhibited cell proliferation and induced apoptosis of Hep-2 cells by suppressing STAT3 signal pathway, down regulating the expression of cyclin B1 and Bcl-2 proteins.

Combined antitumor activity of cucurbitacin B and docetaxel in laryngeal cancer.

Combination therapy with multiple drugs is a common practice in the treatment of cancer. The promising clinical activity of docetaxel has promoted considerable interest in combining it with other antitumor agents. To determine whether cucurbitacin B can enhance chemosensitivity to docetaxel in laryngeal cancer, in the present study, we investigated the combined antitumor effect of cucurbitacin B with docetaxel on Hep-2, a human laryngeal cancer cell line. We treated Hep-2 cells with cucurbitacin B alone or in combination with docetaxel and evaluated cell growth, cell cycle distribution, and apoptosis using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay, flow cytometry, and fluorescent microscopy. Our results showed that, in comparison with single agent treatment, the combination of cucurbitacin B and docetaxel produced greater efficacy in growth inhibition, cell cycle arrest at G2/M phase, and apoptosis induction. Measuring the modulation of regulators in the cell cycle, apoptosis and signal transductions by Western blot analysis showed that the combination effect of cucurbitacin B and docetaxel was due to suppress the expression of p-STAT3 (signal transducers and activators of transcription 3), Bcl-2, and cyclin B1. Moreover, our in vivo studies were reproduced in a mouse xenograft model, where, the combination of cucurbitacin B with docetaxel synergestively inhibited tumor growth. Together, this investigation suggests that cucurbitacin B combined with docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with laryngeal cancer.

Inhibitory effects of cucurbitacin B on laryngeal squamous cell carcinoma.

Cucurbitacins are compounds isolated from various plant families, which have been used as folk medicines for centuries in countries such as India and China because of their wide spectrum of pharmacological activities such as cytotoxic, anti-inflammatory, and anticancer effects. Accumulated evidences have shown that cucurbitacin B inhibits the growth of numerous human cancer cell lines and tumor xenografts. To determine whether cucurbitacin B can inhibit the growth of laryngeal squamous cell carcinoma, in the present study we investigated the antitumor effect of cucurbitacin B on Hep-2 cells. Hep-2 cells were treated with different concentrations of cucurbitacin B for different time. Cell proliferation, cell cycle distribution, and cell apoptosis were evaluated using MTT assay, flow cytometry, and fluorescent microscopy. It was found that cucurbitacin B exhibited significant efficacy in growth inhibition, cell cycle arrest at G2/M phase, and apoptosis induction in a dose- and time-dependent manner. Measuring the modulation of regulators in the cell cycle, apoptosis and signal transductions by Western blot analysis showed that the effect of cucurbitacin B was due to suppression of the expression of p-STAT3, Bcl-2, and cyclin B1. Moreover, in vivo studies were performed in a mouse xenograft model, where cucurbitacin B inhibited tumor growth in a dose-dependent manner. In conclusion, the antitumor effect of cucurbitacin B on Hep-2 cells was due to the induction of cell cycle arrest as well as apoptosis. The possible mechanisms underlying the action might be attributed to the suppression of STAT3 phosphorylation. This investigation suggests a potential clinical application of cucurbitacin B for the treatment of laryngeal cancer patients.

[FTIR spectroscopic explorations of freshly resected laryngeal carcinoma tissues].

OBJECTIVE: To investigate the characteristics of freshly resected laryngeal carcinoma by Fourier transform infrared spectroscopy (FTIR). METHODS: FTIR was applied to the study of the cancerous tissues and adjacent normal tissues in 32 patients. RESULTS: Compared with pathological diagnosis results, one benign specimen was classified as a malignant, the accuracy was 98.4%. Significant differences were seen in the FTIR spectra between the normal and malignant laryngeal tissues. The peak at 1085 cm(-1) shift to 1114 cm(-1) showed that the relative contents of DNA in laryngeal carcinoma cells was increased. The peak at 1397 cm(-1) was stronger than 1451 cm(-1) in normal tissues, while it was not obvious in cancer tissues. I(2926)/I(2870) in carcinoma cells was lower than that in normal tissues. The wave numbers of the bands of amide I and amide II, symmetric and asymmetric stretching bands of CH(3), stretching vibration bands of C-OH and NH band were shifted to higher number in cancer tissues. CONCLUSION: The study shows that the malignant and normal laryngeal tissues have different FTIR spectra, which are mainly due to changes in protein, nucleic acid and phospholipids. FTIR may become a new method for the diagnosis of laryngeal carcinoma in clinical practice.