Role of high-mobility group box 1 in cancer. / 高迁移率族蛋白B1在肿瘤中的作用.

High-mobility group box 1 (HMGB1) is a non-histone nuclear protein in most eukaryocytes. Inside the nucleus, HMGB1 plays an important role in several DNA events such as DNA repair, transcription, telomere maintenance, and genome stability. While outside the nucleus, it fulfils more complicated functions, including promoting cell proliferation, inflammation, angiogenesis, immune tolerance and immune escape, which may play a pro-tumoral role.Meanwhile, HMGB1 acts as an anti-tumoral protein by regulating immune cell recruitment and inducing immunogenic cell death (ICD) during the carcinogenesis process. Therefore, abnormal expression of HMGB1 is associated with oncogenesis, development, and metastasis of cancer, which may play a dual role of pro-tumor and anti-tumor.

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris.

BACKGROUND: Codon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias. RESULTS: In this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris. CONCLUSIONS: Codon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

CYP2J2-produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPARγ-dependent manner. / CYP2J2介导生成的EETs通过PPARÎ³ä¿ƒè¿›èƒ°è ºå¯¼ç®¡è ºç™Œé“æ­»äº¡æŠµæŠ—.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant digestive tract tumors with a poor prognosis and high recurrence rate. Recently, ferroptosis resistance has been found in PDAC. However, the underlying mechanism of ferroptosis resistance has not been fully elucidated. Cytochrome P450 2J2 (CYP2J2) is the main enzyme which mediates arachidonic acid to produce epoxyeicosatrienoic acids (EETs) in human tissues. It has been reported that EETs involve in the development of cancer, while the roles of EETs in PDAC and ferroptosis remain unclear.This study aims to explore the effect of CYP2J2/EETs on ferroptosis of human pancreatic ductal adenocarcinoma cells PANC-1 cells and the underlying mechanisms. METHODS: The tumor tissues and para-carcinoma tissues of 9 patients with PDAC were collected and the expression of CYP2J2 was detected with real-time PCR and Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of 8,9-dihydroxyeicosatrienoic acid (8,9-DHET), and the degradation product of 8,9-epoxyeicosa-trienoic acid (8,9-EET). PANC-1 cells were used in this study. The ferroptosis inducer erastin was used to induce ferroptosis. The intracellular long-chain acyl-CoA synthetase 4 (ACSL4) protein level, lactate dehydrogenase (LDH) activity, malondialdehyde (MDA) content, Fe2+ concentration, and cell survival were detected. The 8,9-EET was pretreated to observe its effect on erastin-induced ferroptosis in PANC-1 cells. Lentivirus was used to construct a CYP2J2 knockdown cell line to observe its effect on the ferroptosis of PANC-1 cells induced by erastin. A peroxisome proliferation-activated receptor γ (PPARγ) blocker was used to observe the effect of 8,9-EET on erastin-induced glutathione peroxidase 4 (GPX4) and MDA content in PANC-1 cells. RESULTS: High expression of CYP2J2 was found in PDAC, accompanied by an increased level of 8,9-DHET. The 8,9-EET pretreatment significantly attenuated the PANC-1 cell death induced by erastin. The 8,9-EET reduced the Fe2+ concentration, LDH activity and MDA content, and ACSL4 protein expression in erastin-treated PANC-1 cells. The 8,9-EET also restored the ferroportin (FPN) and ferroptosis suppressor protein 1 (FSP1) mRNA expressions in erastin-treated PANC-1 cells. But CYP2J2 knockdown exacerbated the erastin-induced ferroptosis in PANC-1 cells. Besides, CYP2J2 knockdown furtherly down-regulated the gene expression of FPN and FSP1. The 8,9-EET increased the expression of GPX4 in the erastin-treated PANC-1 cells, which was eliminated by a PPARγ blocker GW9662. And GW9662 abolished the anti-ferroptosis effects of 8,9-EET. CONCLUSIONS: CYP2J2/EETs are highly expressed in PDAC tissues. EETs inhibit the ferroptosis via up-regulation of GPX4 in a PPARγ-dependent manner, which contributes to the ferroptosis resistance of PDAC.

Light and Abscisic Acid Coordinately Regulate Greening of Seedlings.

The greening of etiolated seedlings is crucial for the growth and survival of plants. After reaching the soil surface and sunlight, etiolated seedlings integrate numerous environmental signals and internal cues to control the initiation and rate of greening thus to improve their survival and adaption. However, the underlying regulatory mechanisms by which light and phytohormones, such as abscisic acid (ABA), coordinately regulate greening of the etiolated seedlings is still unknown. In this study, we showed that Arabidopsis (Arabidopsis thaliana) DE-ETIOLATED1 (DET1), a key negative regulator of photomorphogenesis, positively regulated light-induced greening by repressing ABA responses. Upon irradiating etiolated seedlings with light, DET1 physically interacts with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and subsequently associates to the promoter region of the FHY3 direct downstream target ABA INSENSITIVE5 (ABI5). Further, DET1 recruits HISTONE DEACETYLASE6 to the locus of the ABI5 promoter and reduces the enrichments of H3K27ac and H3K4me3 modification, thus subsequently repressing ABI5 expression and promoting the greening of etiolated seedlings. This study reveals the physiological and molecular function of DET1 and FHY3 in the greening of seedlings and provides insights into the regulatory mechanism by which plants integrate light and ABA signals to fine-tune early seedling establishment.

Molecular mechanisms governing shade responses in maize.

Light is one of the most important environmental factors affecting plant growth and development. Plants use shade avoidance or tolerance strategies to adjust their growth and development thus increase their success in the competition for incoming light. To investigate the mechanism of shade responses in maize (Zea mays), we examined the anatomical and transcriptional dynamics of the early shade response in seedlings of the B73 inbred line. Transcriptome analysis identified 912 differentially expressed genes, including genes involved in light signaling, auxin responses, and cell elongation pathways. Grouping transcription factor family genes and performing enrichment analysis identified multiple types of transcription factors that are differentially regulated by shade and predicted putative core genes responsible for regulating shade avoidance syndrome. For functional analyses, we ectopically over-expressed ZmHB53, a type II HD-ZIP transcription factor gene significantly induced by shade, in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing ZmHB53 exhibited narrower leaves, earlier flowering, and enhanced expression of shade-responsive genes, suggesting that ZmHB53 might participates in the regulation of shade responses in maize. This study increases our understanding of the regulatory network of the shade response in maize and provides a useful resource for maize genetics and breeding.

Bafilomycin A1 increases the sensitivity of tongue squamous cell carcinoma cells to cisplatin by inhibiting the lysosomal uptake of platinum ions but not autophagy.

The role of autophagy in tongue squamous cell carcinoma (TSCC) cisplatin resistance is unclear. We aimed to identify a possible synergistic effect of autophagy inhibitors and cisplatin in TSCC cells and explore the underlying mechanism. Our results indicate that autophagic flux was high in TSCC cells; Autophagy inhibitor bafilomycin A1 increased cisplatin cytotoxicity in TSCC cells by inhibiting lysosomal uptake of platinum and enhancing intracellular platinum ion binding to DNA; Autophagy gene (Atg5) knockout in TSCC cells did not duplicate the above-mentioned sensitization of bafilomycin A1. Furthermore, we found that cisplatin resistance of TSCC cells was related to cisplatin inducing lysosome biogenesis in a TFEB-dependent manner, which was regulated by c-Abl. In summary, this is the first study to show that Bafilomycin A1 increases the sensitivity of TSCC cells to cisplatin by inhibiting lysosomal function but not autophagy. Lysosomes may be a potential target to increase cisplatin cytotoxicity toward TSCC cells.

Functional Characterization of the Maize Phytochrome-Interacting Factors PIF4 and PIF5.

Phytochrome-interacting factors (PIFs) play important roles in photomorphogenesis, the shade avoidance response, and other aspects of plant growth and development. PIF family proteins have been well-studied in Arabidopsis thaliana, but little is known about their physiological functions and molecular mechanisms in maize (Zea mays). In this study, we investigated the physiological functions of ZmPIF4 and ZmPIF5, two highly conserved members of the PIF gene family. RT-qPCR and western blot analyses revealed that ZmPIF4 and ZmPIF5 expression and ZmPIF4 and ZmPIF5 levels peak at night and remain low during the day. Overexpression of ZmPIF4 and ZmPIF5 in Arabidopsis partially rescued the reduced hypocotyl elongation and defective response to gravity in pif1 pif3 pif4 pif5 quadruple mutants (pifq). In addition, under high red: far-red light conditions, Arabidopsis lines overexpressing ZmPIF4 exhibited a constitutive shade avoidance response, including early flowering, slender leaves and inflorescences, plant lodging and precocious leaf senescence. Furthermore, ZmPIF4 physically interacted with the Arabidopsis DELLA protein REPRESSOR OF GA1-3 (RGA), indicating a potential interaction between ZmPIF4 and gibberellin signaling pathway on plant growth. Taken together, our results revealed that ZmPIF4 and ZmPIF5 are functionally conserved proteins that may play conserved roles in the response to phytochrome signaling in plants. HIGHLIGHTS: In this study, the functions of ZmPIF4 and ZmPIF5 were characterized by expression in Arabidopsis, revealing conserved roles of PIF family proteins in photomorphogenesis and the shade avoidance response in land plants.

Treatment and prognosis in sinonasal mucosal melanoma: A retrospective analysis of 65 patients from a single cancer center.

BACKGROUND: The purpose of this study was to evaluate the outcome in sinonasal mucosal melanoma (SMM). METHODS: A retrospective analysis of clinicopathological data from January 1976 to December 2005 was performed. Survival curve, univariate, and multivariate analyses were undertaken. RESULTS: Sixty-eight patients with SMM were enrolled; 3 patients refused treatment. The 3-year and 5-year overall survival (OS) rates in the remaining 65 cases of SMM were 36.5% and 29.7%, respectively. Patients who underwent surgery had better 3-year and 5-year OS rates than those treated without surgery (40.7% and 34.1% vs 21.4% and 14.3%, respectively), and the same was true for patients treated with and without biotherapy (58.2% and 50.9% vs 30.0% and 23.4%, respectively). Distant metastasis at presentation was associated with a worse prognosis. Those patients managed with multimodality treatment had better OS rates. CONCLUSION: The prognosis in SMM is poor, particularly for those with distant metastasis or without surgery. Multimodality treatment may improve survival.

Treatment and prognosis of oral mucosal melanoma.

To evaluate the treatment and prognosis of oral mucosal melanoma (OMM) and provide basic data for clinical treatment. Retrospective analysis of clinicopathological data on OMM from January 1976 to December 2005. Survival analysis was performed and Kaplan-Meier analysis was used to compare the effects of clinicopathological factors on survival using SPSS 18.0 software. A Cox model was applied for multivariate analysis. The 3-year and 5-year overall survival (OS) rates of 51 cases of OMM were 35.0% and 20.7%, respectively. Lymph node metastatic sites were predominantly at levels Ib-III (29/31, 93.5%). Patients of age ≥55 years and size ≥4 cm had a lower survival rate than those of aged <55 years and size <4 cm. The 3-year OS and 5-year OS of patients who underwent surgery combined with biotherapy or biochemotherapy (70.1% and 58.4%, respectively) were significantly higher than that of patients who underwent other therapeutic regimens (including surgery, chemotherapy, surgery combined with radiotherapy or surgery combined with chemotherapy) (25.0% and 12.5%, respectively). Multivariate analysis showed that surgery combined with biotherapy or biochemotherapy and neck dissection were effective treatments for OMM. Patients aged ≥55 years had a worse prognosis than those aged <55 years. OMM has a poor prognosis, but multimodality treatment including surgery combined with biotherapy may improve the prognosis. In patients aged ≥55 years with tumor size ≥4 cm, increasing the scope of resection may be effective. Elective levels I-III neck dissection should be considered in TanyNOMO cases.

[Clinical analysis of 51 cases of oral mucosal melanoma].

OBJECTIVE: To investigate the treatment and prognosis of the patients with oral mucosal melanoma (OMM). METHODS: The clinicopathological and follow-up data of patients with OMM in Sun Yat-sen University Cancer Center from January 1976 to December 2005 were analyzed retrospectively. RESULTS: Fifty-one cases were analyzed. The pathological lymph node metastasis rate was 61% (31/51) and the affected sites were confined to level I(b)-III (94%). The overall three year and five yearsurvival rates were 35% and 21% respectively. No significant difference of three year and five year survival rates were found between the group of incisional biopsy and the group of excisional biopsy. The prognosis was not affected by pigmentation. The survival rate of the patients receiving surgery combined with biotherapy or biochemotherapy was significantly higher than that of the patients treated by other modalities (P = 0.003). CONCLUSIONS: In patients with OMM, lymph node metastasis was mostly confined to level I(b)-III. Incisional biopsy and pigmentation were not associated with an unfavorable prognosis. The prognosis of the patients with OMM was poor and the patients may get a better prognosis by receiving surgery combined with biotherapy or biochemotherapy.

TRB3 overexpression due to endoplasmic reticulum stress inhibits AKT kinase activation of tongue squamous cell carcinoma.

Our investigation aims to evaluate the significance of TRB3, an endoplasmic reticulum stress (ERS)-inducible gene, and explore its relationship with AKT in oral tongue squamous cell carcinoma (OTSCC). Expression of TRB3 and phosphorylated AKT (p-AKT) in OTSCC tissues and adjacent normal tissues were assessed by RT-PCR, Western blot and immunohistochemistry assay. Correlation of TRB3 and AKT was validated by TRB3 adenovirus plasmid (Ad-TRB3) transfection and short hairpin RNA (shRNA) inhibition. The mRNA expression of TRB3 was significantly higher than adjacent noncancerous tissues by RT-PCR in 15 of 18 specimens of OTSCC (83.3%, P<0.01). Both of TRB3 and AKT were highly expressed in 13 of 18 (72.2%) specimens of OTSCC comparing with adjacent noncancerous tissues by Western blot assay (P<0.05). TRB3 was significantly elevated in 49.2% (63/128) of pathologically confirmed specimens and 13.3% (4/30) of adjacent noncancerous specimens by immunohistochemical analysis (P<0.01). TRB3 overexpression was closely correlated with tumor pathological T stage, lymph node metastasis and tumor recurrence. In addition, both mRNA and protein expression of TRB3 was increased under thapsigargin (TG) or tunicmycin (TU)-induced ERS in Tca8113 and CAL-27 cells. Moreover, expression of p-AKT protein decreased when Ad-TRB3 was transected with OTSCC Tca8113 cells. However, expression of p-AKT protein increased when TRB3 was inhibited by TRB3 shRNA inhibition. TRB3 expression was closely correlated with OTSCC prognosis. Under ERS, TRB3 was up-regulated, resulting in inhibiting the activation of AKT in OTSCC.