Overexpression of ABCC1 and ABCG2 confers resistance to talazoparib, a poly (ADP-Ribose) polymerase inhibitor.

AIMS: The overexpression of ABC transporters on cancer cell membranes is one of the most common causes of multidrug resistance (MDR). This study investigates the impact of ABCC1 and ABCG2 on the resistance to talazoparib (BMN-673), a potent poly (ADP-ribose) polymerase (PARP) inhibitor, in ovarian cancer treatment. METHODS: The cell viability test was used to indicate the effect of talazoparib in different cell lines. Computational molecular docking analysis was conducted to simulate the interaction between talazoparib and ABCC1 or ABCG2. The mechanism of talazoparib resistance was investigated by constructing talazoparib-resistant subline A2780/T4 from A2780 through drug selection with gradually increasing talazoparib concentration. RESULTS: Talazoparib cytotoxicity decreased in drug-selected or gene-transfected cell lines overexpressing ABCC1 or ABCG2 but can be restored by ABCC1 or ABCG2 inhibitors. Talazoparib competitively inhibited substrate drug efflux activity of ABCC1 or ABCG2. Upregulated ABCC1 and ABCG2 protein expression on the plasma membrane of A2780/T4 cells enhances resistance to other substrate drugs, which could be overcome by the knockout of either gene. In vivo experiments confirmed the retention of drug-resistant characteristics in tumor xenograft mouse models. CONCLUSIONS: The therapeutic efficacy of talazoparib in cancer may be compromised by its susceptibility to MDR, which is attributed to its interactions with the ABCC1 or ABCG2 transporters. The overexpression of these transporters can potentially diminish the therapeutic impact of talazoparib in cancer treatment.

Hypoxia preconditioning of adipose stem cell-derived exosomes loaded in gelatin methacryloyl (GelMA) promote type H angiogenesis and osteoporotic fracture repair.

The challenges posed by delayed atrophic healing and nonunion stand as formidable obstacles in osteoporotic fracture treatment. The processes of type H angiogenesis and osteogenesis emerge as pivotal mechanisms during bone regeneration. Notably, the preconditioning of adipose-derived stem cell (ADSC) exosomes under hypoxic conditions has garnered attention for its potential to augment the secretion and functionality of these exosomes. In the present investigation, we embarked upon a comprehensive elucidation of the underlying mechanisms of hypo-ADSC-Exos within the milieu of osteoporotic bone regeneration. Our findings revealed that hypo-ADSC-Exos harboured a preeminent miRNA, namely, miR-21-5p, which emerged as the principal orchestrator of angiogenic effects. Through in vitro experiments, we demonstrated the capacity of hypo-ADSC-Exos to stimulate the proliferation, migration, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) via the mediation of miR-21-5p. The inhibition of miR-21-5p effectively attenuated the proangiogenic effects mediated by hypo-ADSC-Exos. Mechanistically, our investigation revealed that exosomal miR-21-5p emanating from hypo-ADSCs exerts its regulatory influence by targeting sprouly1 (SPRY1) within HUVECs, thereby facilitating the activation of the PI3K/AKT signalling pathway. Notably, knockdown of SPRY1 in HUVECs was found to potentiate PI3K/AKT activation and, concomitantly, HUVEC proliferation, migration, and angiogenesis. The culminating stage of our study involved a compelling in vivo demonstration wherein GelMA loaded with hypo-ADSC-Exos was validated to substantially enhance local type H angiogenesis and concomitant bone regeneration. This enhancement was unequivocally attributed to the exosomal modulation of SPRY1. In summary, our investigation offers a pioneering perspective on the potential utility of hypo-ADSC-Exos as readily available for osteoporotic fracture treatment.

scEnhancer: a single-cell enhancer resource with annotation across hundreds of tissue/cell types in three species.

Previous studies on enhancers and their target genes were largely based on bulk samples that represent 'average' regulatory activities from a large population of millions of cells, masking the heterogeneity and important effects from the sub-populations. In recent years, single-cell sequencing technology has enabled the profiling of open chromatin accessibility at the single-cell level (scATAC-seq), which can be used to annotate the enhancers and promoters in specific cell types. A comprehensive resource is highly desirable for exploring how the enhancers regulate the target genes at the single-cell level. Hence, we designed a single-cell database scEnhancer (http://enhanceratlas.net/scenhancer/), covering 14 527 776 enhancers and 63 658 600 enhancer-gene interactions from 1 196 906 single cells across 775 tissue/cell types in three species. An unsupervised learning method was employed to sort and combine tens or hundreds of single cells in each tissue/cell type to obtain the consensus enhancers. In addition, we utilized a cis-regulatory network algorithm to identify the enhancer-gene connections. Finally, we provided a user-friendly platform with seven useful modules to search, visualize, and browse the enhancers/genes. This database will facilitate the research community towards a functional analysis of enhancers at the single-cell level.

Cell membrane-camouflaged bufalin targets NOD2 and overcomes multidrug resistance in pancreatic cancer.

AIMS: Multidrug resistance in pancreatic cancer poses a significant challenge in clinical treatment. Bufalin (BA), a compound found in secretions from the glands of toads, may help overcome this problem. However, severe cardiotoxicity thus far has hindered its clinical application. Hence, the present study aimed to develop a cell membrane-camouflaged and BA-loaded polylactic-co-glycolic acid nanoparticle (CBAP) and assess its potential to counter chemoresistance in pancreatic cancer. METHODS: The toxicity of CBAP was evaluated by electrocardiogram, body weight, distress score, and nesting behavior of mice. In addition, the anticarcinoma activity and underlying mechanism were investigated both in vitro and in vivo. RESULTS: CBAP significantly mitigated BA-mediated acute cardiotoxicity and enhanced the sensitivity of pancreatic cancer to several clinical drugs, such as gemcitabine, 5-fluorouracil, and FOLFIRINOX. Mechanistically, CBAP directly bound to nucleotide-binding and oligomerization domain containing protein 2 (NOD2) and inhibited the expression of nuclear factor kappa-light-chain-enhancer of activated B cells. This inhibits the expression of ATP-binding cassette transporters, which are responsible for chemoresistance in cancer cells. CONCLUSIONS: Our findings indicate that CBAP directly inhibits NOD2. Combining CBAP with standard-of-care chemotherapeutics represents a safe and efficient strategy for the treatment of pancreatic cancer.

A hepatocyte differentiation model reveals two subtypes of liver cancer with different oncofetal properties and therapeutic targets.

Clinical observation of the association between cancer aggressiveness and embryonic development stage implies the importance of developmental signals in cancer initiation and therapeutic resistance. However, the dynamic gene expression during organogenesis and the master oncofetal drivers are still unclear, which impeded the efficient elimination of poor prognostic tumors, including human hepatocellular carcinoma (HCC). In this study, human embryonic stem cells were induced to differentiate into adult hepatocytes along hepatic lineages to mimic liver development in vitro. Combining transcriptomic data from liver cancer patients with the hepatocyte differentiation model, the active genes derived from different hepatic developmental stages and the tumor tissues were selected. Bioinformatic analysis followed by experimental assays was used to validate the tumor subtype-specific oncofetal signatures and potential therapeutic values. Hierarchical clustering analysis revealed the existence of two subtypes of liver cancer with different oncofetal properties. The gene signatures and their clinical significance were further validated in an independent clinical cohort and The Cancer Genome Atlas database. Upstream activator analysis and functional screening further identified E2F1 and SMAD3 as master transcriptional regulators. Small-molecule inhibitors specifically targeting the oncofetal drivers extensively down-regulated subtype-specific developmental signaling and inhibited tumorigenicity. Liver cancer cells and primary HCC tumors with different oncofetal properties also showed selective vulnerability to their specific inhibitors. Further precise targeting of the tumor initiating steps and driving events according to subtype-specific biomarkers might eliminate tumor progression and provide novel therapeutic strategy.

Genome-Wide Analysis of Long Non-Coding RNAs Related to UV-B Radiation in the Antarctic Moss Pohlia nutans.

Antarctic organisms are consistently suffering from multiple environmental pressures, especially the strong UV radiation caused by the loss of the ozone layer. The mosses and lichens dominate the vegetation of the Antarctic continent, which grow and propagate in these harsh environments. However, the molecular mechanisms and related regulatory networks of these Antarctic plants against UV-B radiation are largely unknown. Here, we used an integrated multi-omics approach to study the regulatory mechanism of long non-coding RNAs (lncRNAs) of an Antarctic moss (Pohlia nutans) in response to UV-B radiation. We identified a total of 5729 lncRNA sequences by transcriptome sequencing, including 1459 differentially expressed lncRNAs (DELs). Through functional annotation, we found that the target genes of DELs were significantly enriched in plant-pathogen interaction and the flavonoid synthesis pathway. In addition, a total of 451 metabolites were detected by metabonomic analysis, and 97 differentially change metabolites (DCMs) were found. Flavonoids account for 20% of the total significantly up-regulated metabolites. In addition, the comprehensive transcriptome and metabolome analyses revealed the co-expression pattern of DELs and DCMs of flavonoids. Our results provide insights into the regulatory network of lncRNA under UV-B radiation and the adaptation of Antarctic moss to the polar environments.

CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in cancer cells.

BACKGROUND: Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown. METHODS: Based on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis. RESULTS: Deficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110ß, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110ß deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110ß. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110ß level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice. CONCLUSIONS: CDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

Syntaxin of plants31 (SYP31) and SYP32 is essential for Golgi morphology maintenance and pollen development.

Pollen development is a key process for the sexual reproduction of angiosperms. The Golgi plays a critical role in pollen development via the synthesis and transport of cell wall materials. However, little is known about the molecular mechanisms underlying the maintenance of Golgi integrity in plants. In Arabidopsis thaliana, syntaxin of plants (SYP) 3 family proteins SYP31 and SYP32 are the only two Golgi-localized Qa-soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) with unknown endogenous functions. Here, we demonstrate the roles of SYP31 and SYP32 in modulating Golgi morphology and pollen development. Two independent lines of syp31/+ syp32/+ double mutants were male gametophytic lethal; the zero transmission rate of syp31 syp32 mutations was restored to largely normal levels by pSYP32:SYP32 but not pSYP32:SYP31 transgenes, indicating their functional differences in pollen development. The initial arrest of syp31 syp32 pollen occurred during the transition from the microspore to the bicellular stage, where cell plate formation in pollen mitosis I (PMI) and deposition of intine were abnormal. In syp31 syp32 pollen, the number and length of Golgi cisterna were significantly reduced, accompanied by many surrounding vesicles, which could be largely attributed to defects in anterograde and retrograde trafficking routes. SYP31 and SYP32 directly interacted with COG3, a subunit of the conserved oligomeric Golgi (COG) complex and were responsible for its Golgi localization, providing an underlying mechanism for SYP31/32 function in intra-Golgi trafficking. We propose that SYP31 and SYP32 play partially redundant roles in pollen development by modulating protein trafficking and Golgi structure.

A case study of combined neoadjuvant chemotherapy and neoadjuvant immunotherapy in resectable locally advanced esophageal cancer.

BACKGROUND: The prognosis of patients under existing neoadjuvant chemotherapy or neoadjuvant chemoradiotherapy requires improvement. Whereas programmed cell death 1 (PD-1) inhibitors have shown promising response in advanced esophageal cancer, they have not been used in the perioperative treatment of resectable locally advanced esophageal cancer. Whether immunotherapy can be incorporated into neoadjuvant therapy has became a challenging question for researchers. CASE PRESENTATION: We present a case of a 65-year-old male who had a history of progressive dysphagia for approximately 1 month. He underwent pertinent studies including computed tomography (CT)，gastroscopy，and pathological biopsy resulting in a diagnosis of medium-low differentiated squamous carcinoma of the thoracic segment of the esophagus (cT2N2M0 stage III). After 4 cycles of neoadjuvant chemotherapy combined with immunotherapy, gastroscopy showed the lesion in the esophagus was no longer present. Subsequently, the patient received thoracoscopic radical resection of esophageal cancer and achieved a pathological complete response (pCR) in postoperative pathological evaluation. During the whole treatment, no adverse effect was recorded and to date no evidence of recurrence has been recorded. CONCLUSION: Our report suggest that neoadjuvant chemotherapy combined with immunotherapy not only improve the R0 resection and pCR rate in patients with resectable locally advanced esophageal cancer, but also the adverse effects are within the control range. However, the selection of therapeutic strategy, predictors of response to treatment, and interval time between neoadjuvant treatment and surgery still await more reliable evidence-based studies with large prospective samples.

No Associations Between Regular Use of Proton Pump Inhibitors and Risk of All-Cause and Cause-Specific Mortality: A Population-Based Cohort of 0.44 Million Participants.

INTRODUCTION: The association between proton pump inhibitors' (PPIs) use and mortality remains unclear. METHODS: This was a prospective analysis of 440,840 UK residents and 13,154 deaths. We evaluated the associations with multivariate Cox regression. RESULTS: After adjusting for confounders, such as over health status and longstanding diseases, the regular use of PPIs was not associated with an increased risk of all-cause mortality and mortality due to neoplasms, circulatory system diseases, respiratory system diseases, digestive system diseases, external causes, and other causes. DISCUSSION: Regular use of PPIs was not associated with an increased risk of all-cause and cause-specific mortality.

Proscillaridin A induces apoptosis and inhibits the metastasis of osteosarcoma in vitro and in vivo.

The side effects of chemotherapy, drug resistance, and tumor metastasis hinder the development of treatment for osteosarcoma, leading to poor prognosis of patients with the disease. Proscillaridin A, a kind of cardiac glycoside, has been proven to have anti-proliferative properties in many malignant tumors, but the efficacy of the drug in treating osteosarcoma is unclear. In the present study, we assessed the effects of Proscillaridin A on osteosarcoma and investigated its underlying action mechanism. The cell cytotoxicity assay showed that Proscillaridin A significantly inhibited the proliferation of 143B cells in a dose- and time-dependent manner. Also, flow cytometry and invasion assay revealed that Proscillaridin A induced apoptosis and reduced 143B cell motility. Western blotting and PCR were used to detect the expressions of Bcl-xl and MMP2 and showed that mRNA/protein expression levels decreased significantly in Proscillaridin A-treated osteosarcoma cells. Using a mouse xenograft model, we found that Proscillaridin A treatment significantly inhibited tumor growth and lung metastasis in vivo and decreased the expression levels of Bcl-xl and MMP2. No noticeable side effect was observed in the liver, kidney, and hematological functions. Conclusively, Proscillaridin A suppressed proliferation, induced apoptosis, and inhibited 143B cell metastasis in vitro and in vivo, and these effects could be mediated by downregulating the expressions of Bcl-xl and MMP2.

The use of modified keystone flap in periarticular or large superficial tumor resection surgery.

BACKGROUND AND OBJECTIVES: The keystone design perforator island flap (KDPIF) is often used to cover defects in reliable blood supply and similar skin patterns, but its mobility is limited, especially when the wound is large or occurs around joints. Here, we describe a modified KDPIF, boat-shaped flap. We added a V shape along the lateral arc, forming a V-Y flap on KDPIF's outer arc shapes like a sail. This paper also describes a clinical study to evaluate this method. METHOD: From September 2014 to March 2017, 31 patients were operated on using the boat-shaped flap in our department and were followed up annually with clinical evaluation. The wound locations included joints (n = 11), trunk (n = 18), and face (n = 2). Fifteen defects were ≥5 × 5 cm2 . RESULTS: After 6 to 24 months of follow-up, 29 patients had first-intention healing and were satisfied with the morphology and function. Secondary healing was observed in two patients, and the wounds were closed after dressing treatment for 2 weeks. CONCLUSION: The boat-shaped flap enhances the mobility and achieves strong resistance to tension. The modified curvilinear shape prevents the joint activity from being restricted, with visually concealed scars. It is particularly applicable for repairing large wounds and defects around joints.

Adsorption Study of Lignin Removal from Recycled Alkali Black Liquor by Adsorption Resins for Improved Cellulase Hydrolysis of Corn Straw.

Previous studies showed that aromatic compounds such as lignin, phenols, and furans were main inhibitors of cellulase hydrolysis in recycled alkali black liquor (RBL), which should be removed to improve alkali utilization. In this study, three polymeric resins, XAD-4, XAD-16N, and XAD-7HP, were evaluated for their abilities to remove lignin from alkali black liquor recycled at the third time. Adsorption conditions of adsorbent dose and equilibrium time, isotherms, and kinetics were investigated. Of three tested adsorbents, XAD-16N was the most efficient, which can remove 89.84% of lignin after adsorption at an adsorbent-to-solution ratio of 1:4 for 2.5 h. Pseudo-second-order model was efficient to represent XAD-16N and XAD-7HP adsorption kinetics. Adsorption behavior of XAD-4 on RBL was fitted better to Langmuir model, while XAD-16N and XAD-7HP adsorption were more consistent with Freundlich model. The cellulase hydrolysis rate of corn straw treated with RBL after XAD-16N adsorption combined with ozone was 86.89%, which was only 0.89% lower than that of sodium hydroxide combined with ozone treatment. Structure characterization proved that the damage of XAD-16N adsorbed RBL to corn straw was similar to that of sodium hydroxide. It indicated that adsorption was effective in inhibitor removal from RBL to improve alkali utilization.

Effect of Alkaline Black Liquor Recycling on Alkali Combined with Ozone Pretreatment of Corn Stalk.

In the early stage, the best conditions for alkali-bound ozone pretreatment were studied. But after treatment, the alkaline black liquor was directly discarded due to the large amount of organic matter, resulting in environmental pollution and waste of resources. In this paper, the alkaline black liquor was recycled under the optimal pretreatment conditions. The results showed that the number of alkaline black liquor cycles had little effect on hemicellulose content, and had a great influence on cellulose content and lignin content. Through structural characterization of corn stover, it was found that the pretreatment caused structural changes of lignin in straw. However, when the alkaline black liquor was recycled for the fourth time, the ether bond in the side chain of lignin and the covalent bond between the components were not sufficiently destroyed, and the damage to the phenolic hydroxyl group was also weakened. It was indicated that when the alkaline black liquor was recycled for the fourth time, the destruction effect of the alkaline black liquor on the straw was significantly inhibited. Therefore, the optimal circulation time of alkaline black liquor was three times, and the cellulolytic conversion rate was 81.53%.

Calcium-binding protein 39 promotes hepatocellular carcinoma growth and metastasis by activating extracellular signal-regulated kinase signaling pathway.

Calcium-binding protein (CAB39) is a key regulator of a group of sterile 20 kinases. Here, we report that CAB39 was frequently up-regulated in hepatocellular carcinoma (HCC), which was significantly associated with tumor metastasis (P = 0.000), poorer disease-free survival rate (P = 0.027), and poor prognosis (P = 0.000). Ectopic expression of CAB39 in immortalized human liver cell line LO2 and HCC cell lines QGY-7703 and BEL-7402 could increase foci formation, colony formation in soft agar, tumor formation in nude mice, and cell motility. Silencing CAB39 expression in two HCC cell lines, Huh7 and MHCC97H, with short hairpin RNA could effectively abolish its oncogenic function. Further study found that CAB39 contributed to extracellular signal-regulated kinase (ERK) pathway activation, and mutations of the key sites of CAB39 markedly decrease the level of phosphorylated ERK. In addition, CAB39 could promote epithelial-mesenchymal transition by up-regulating N-cadherin and Fibronectin and down-regulating E-cadherin and α-E-catenin. As a result, ß-catenin nuclear translocation was increased and its downstream target gene, matrix metalloproteinase-9, was up-regulated. CONCLUSION: Taken together, our findings suggested that CAB39 played very important oncogenic roles in HCC pathogenesis and progression by activating the ERK signaling pathway. Better understanding of CAB39 may lead to its clinical application as a biomarker for a prognosis predictor and a novel therapeutic target. (Hepatology 2017;66:1529-1545).

Evaluation of the Effects of Isolated Lignin on Cellulose Enzymatic Hydrolysis of Corn Stover Pretreatment by NaOH Combined with Ozone.

In this experiment, corn stover was treated with optimal combined pretreatment conditions: 2% NaOH at 80 °C treated 2 h combined with initial pH 9 at the ozone concentration of 78 mg/mL treated 25 min. The effect of lignin removal rate on the enzymatic hydrolysis degree of cellulose during the treatment process was studied. At the same time, the lignin in the optimal pretreated corn stover was separated and extracted by enzymatic acidolysis, and its structure and connection were characterized. The results showed that the alkali combined with ozone pretreatment improved the enzymatic hydrolysis degree of the cellulose while exfoliating and degrading the macromolecular lignin into small molecules. The stable crosslink structure of the lignin-cellulose-hemicellulose was destroyed, and the lignocellulosic structure changed in favor of the enzymatic hydrolysis of the cellulose.

CHD1L promotes lineage reversion of hepatocellular carcinoma through opening chromatin for key developmental transcription factors.

UNLABELLED: High-grade tumors with poor differentiation usually show phenotypic resemblance to their developmental ancestral cells. Cancer cells that gain lineage precursor cell properties usually hijack developmental signaling pathways to promote tumor malignant progression. However, the molecular mechanisms underlying this process remain unclear. In this study, the chromatin remodeler chromodomain-helicase-DNA-binding-protein 1-like (CHD1L) was found closely associated with liver development and hepatocellular carcinoma (HCC) tumor differentiation. Expression of CHD1L decreased during hepatocyte maturation and increased progressively from well-differentiated HCCs to poorly differentiated HCCs. Chromatin immunoprecipitation followed by high-throughput deep sequencing found that CHD1L could bind to the genomic sequences of genes related to development. Bioinformatics-aided network analysis indicated that CHD1L-binding targets might form networks associated with developmental transcription factor activation and histone modification. Overexpression of CHD1L conferred ancestral precursor-like properties of HCC cells both in vitro and in vivo. Inhibition of CHD1L reversed tumor differentiation and sensitized HCC cells to sorafenib treatment. Mechanism studies revealed that overexpression of CHD1L could maintain an active "open chromatin" configuration at promoter regions of estrogen-related receptor-beta and transcription factor 4, both of which are important regulators of HCC self-renewal and differentiation. In addition, we found a significant correlation of CHD1L with developmental transcriptional factors and lineage differentiation markers in clinical HCC patients. CONCLUSION: Genomic amplification of chromatin remodeler CHD1L might drive dedifferentiation of HCC toward an ancestral lineage through opening chromatin for key developmental transcriptional factors; further inhibition of CHD1L might "downgrade" poorly differentiated HCCs and provide novel therapeutic strategies.

Construction of a plasmid for overexpression of human circadian gene period2 and its biological activity in osteosarcoma cells.

Beyond their established role in the mammalian circadian clock, recent studies have confirmed that the circadian genes have been implicated in tumor onset and progression. Currently, the biological effects of circadian genes on osteosarcoma cells' proliferation and migration are not well understood. Period2 (Per2) is one of the core circadian genes that act as master regulators of development and is frequently dysregulated in several cancers. However, the effects of human Per2 (hPer2) on the biological behavior of osteosarcoma cells are rarely reported. In the present study, to address the expression of hPer2 in osteosarcoma cells, the pEGFP-N1-hPer2 eukaryotic expression vector was constructed and transfected into cultured MG63 cells using Lipofectamine™ 2000. The overexpression of hPer2 in MG63 cells was verified by qRT-PCR and Western blotting, respectively. Finally, we investigated the effects of hPer2 protein overexpression on MG63 cells' viability, cycle, apoptosis, and invasive ability. In conclusion, the recombinant pEGFP-N1-hPer2 plasmid had been constructed successfully and expressed effectively in MG63 cells. Furthermore, results also showed that the viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in MG63 cells. This preliminary study provides ground work for further research on the roles of circadian gene hPer2 in osteosarcoma cells MG63 and would offer promise for the development of novel therapeutic strategies in the treatment of osteosarcoma.

Functional vesicles formed by anticancer drug assembly.

In this Letter, a new type of nitrogen mustard conjugate vesicles is developed to improve the stability and efficiency of anticancer drug. Benzoic acid nitrogen mustard-peptide (AAAK) conjugate was designed and synthesized, which was found to self-assemble into vesicles in water. The formation of the vesicles was confirmed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and circular dichroism (CD). The degradation data revealed that the benzoic acid nitrogen mustard peptide (AAAK) conjugate vesicles are more stable than the parent drug in aqueous solution. Furthermore, MTT assay revealed that the free drug conjugate has similar antitumor activity against MCF-7, Hela, HepG-2 cell lines compared with the parent drug. The benzoic acid nitrogen mustard-peptide conjugate vesicles may have potential in the treatment of cancers.