Deoxypodophyllotoxin Mediates Autophagy Death through Inhibition of GRP78 in Human Osteosarcoma.

BACKGROUND: Glucose-regulated protein 78 (GRP78), as a chaperone protein, can protect the endoplasmic reticulum of cells and is expressed to influence chemoresistance and prognosis in cancer. Deoxypodophyllotoxin (DPT) is a compound with antitumor effects on cancers. DPT inhibits the proliferation of osteosarcoma by inducing apoptosis, necrosis, or cell cycle arrest. OBJECT: This study was performed to demonstrate the molecular mechanism by which DPT attenuates osteosarcoma progression through GRP78. METHODS: Natural compound libraries and western blot (WB) were used to screen the inhibitors of osteosarcoma GRP78. The expression of mitochondria-related genes in cancer cells of the treatment group was detected by quantitative real-time PCR (qPCR) and WB. 3-(4,5)- Dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and 5-ethynyl-2'- deoxyuridine (EDU) were used to discover the activity and proliferation of osteosarcoma cells treated with DPT. We constructed an in vivo mouse model of DPT drug therapy and carried out immunohistochemical detection of xenografts. The treated osteosarcoma cells were analyzed using bioinformatics and electron microscopy. The data were analyzed finally. RESULTS: DPT inhibited osteosarcoma cell survival and the growth of tumor xenografts. It promoted up-regulation of BCL2-associated X protein (Bax) and B-cell CLL/lymphoma 2 (Bcl-2), which serves to mediate and attenuate, respectively, the killing activities of DPT through mitochondria dysfunction. The effect of DPT against cancer cells could be attenuated by the overexpression of GRP78, characterized by the inactivation of the caspase cascade. The loss of GRP78 in osteosarcoma cells negatively mediated the basal level of autophagyassociated genes. DPT stimulated autophagy via the phosphoinositide 3-kinase (PI3K)-v-akt murine thymoma viral oncogene homolog (AKT), a mechanistic target of rapamycin (mTOR) axis. The autophagy caused by DPT played an active role in the osteosarcoma of humans and blocked the apoptotic cascade. CONCLUSION: Combination treatment with the GRP78 inhibitor DPT and pharmacological autophagy inhibitors will be a meaningful method of obviating osteosarcoma cells.

LPS-induced senescence of macrophages aggravates calcification and senescence of vascular smooth muscle cells via IFITM3.

BACKGROUND: Cellular senescence, macrophages infiltration, and vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation participate in the pathophysiology of vascular calcification in chronic kidney disease (CKD). Senescent macrophages are involved in the regulation of inflammation in pathological diseases. In addition, senescent cells spread senescence to neighboring cells via Interferon-induced transmembrane protein3 (IFITM3). However, the role of senescent macrophages and IFITM3 in VSMCs calcification remains unexplored. AIMS: To explore the hypothesis that senescent macrophages contribute to the calcification and senescence of VSMCs via IFITM3. METHODS: Here, the macrophage senescence model was established using Lipopolysaccharides (LPS). The VSMCs were subjected to supernatants from macrophages (MCFS) or LPS-induced macrophages (LPS-MCFS) in the presence or absence of calcifying media (CM). Senescence-associated ß-galactosidase (SA-ß-gal), Alizarin red (AR), immunofluorescent staining, and western blot were used to identify cell senescence and calcification. RESULTS: The expression of IFITM3 was significantly increased in LPS-induced macrophages and the supernatants. The VSMCs transdifferentiated into osteogenic phenotype, expressing higher osteogenic differentiation markers (RUNX2) and lower VSMCs constructive makers (SM22α) when cultured with senescent macrophages supernatants. Also, senescence markers (p16 and p21) in VSMCs were significantly increased by senescent macrophages supernatants treated. However, IFITM3 knockdown inhibited this process. CONCLUSIONS: Our study showed that LPS-induced senescence of macrophages accelerated the calcification of VSMCs via IFITM3. These data provide a new perspective linking VC and aging, which may provide clues for diagnosing and treating accelerated vascular aging in patients with CKD.

Utility of cooling patches to prevent hand-foot syndrome caused by pegylated liposomal doxorubicin in breast cancer patients.

BACKGROUND: Pegylated liposomal doxorubicin (PLD) uses the hydrophilic layer of liposomes to reach the sweat on the skin surface or accumulate in the sweat glands, producing toxic free radicals and oxidative damage, resulting in hand-foot syndrome (HFS). Regional cooling can induce vasoconstriction to reduce the release of drugs in the limbs and reduce the accumulation of drugs in sweat glands; thus, decreasing the incidence and severity of HFS. AIM: To study the efficacy of cooling patches to prevent HFS caused by PLD in the short-term. METHODS: This is a retrospective cohort study. Female breast cancer patients (n = 101) who were treated with PLD in two breast wards at our department from February 2020 to February 2021 were enrolled in the study and were randomly divided into the cooling group (51 patients) and the control group (50 patients). Patients in the control group only received routine care, while the patients in the cooling group applied cooling patches, based on routine care, to the palm and back of the hands 15 min before chemotherapy infusion for 10 h. All patients took a corresponding dose of dexamethasone orally one day before chemotherapy, on the day of chemotherapy, and one day after chemotherapy. SPSS23.0 version was used to analyze the data in this study. The occurrence and severity of HFS was analyzed by the Mann-Whitney U test, and scores were analyzed by the Student's t test or Wilcoxon rank-sum test. A P value < 0.05 was regarded as statistically significant. RESULTS: In this study, neither group of patients developed Grade 3 HFS. In the control group, the incidence of Grade 1 HFS and Grade 2 HFS was 38% and 2%, respectively. However, in the cooling group, only one person developed Grade 1 HFS (2%), and none of the patients developed Grade 2 HFS. These findings showed that cooling patches can effectively reduce the frequency and severity of HFS (P < 0.0001) in the short-term. Before the fourth chemotherapy cycle, although general self-efficacy scale scores in the cooling group were low, they were still significantly higher than those in the control group (17.22 ± 5.16 vs 19.63 ± 6.42, P = 0.041). Compared with the control group, the mean Hand-Foot Skin Reaction and Quality of Life Questionnaire score in the cooling group was significantly lower (18.08 ± 7.01 vs 14.20 ± 7.39, P = 0.008). CONCLUSION: Cooling patches can effectively reduce the frequency and severity of HFS caused by PLD in the short-term. In addition, it may help delay the decline in patients' self-efficacy.

Comparison of clinical outcomes between open and modified endoscopic release for carpal tunnel syndrome.

The aim of the present study was to investigate a novel technology, requiring only a single portal and no special equipment, to perform endoscopic treatment of carpal tunnel (CT) syndrome (CTS). This novel technique involves a surgical approach and standard operating procedures and is designed to minimize the potential for complications. Patients with CTS were randomly assigned using a computer-generated random allocation and stratified by site to either the modified endoscopic CT release (MECTR) group (n=48) or open CT release (OCTR) group (n=46). Various medical indexes were compared between the two groups, including operative time, hospitalization time, the time required to resume a normal life or work, intraoperative complications, incision infection rate, the amelioration of symptoms (Kelly grading), post-operative scar pain score, recovery of grip strength and pinch strength, two-point discrimination and the presence of sympathetic dystrophy. The results revealed that all patients had grade A wound healing and the symptoms were completely relieved. No significant differences were observed between the two groups with regards to the incision infection rate, intraoperative complications, grip strength, pinch strength, two-point discrimination, presence of sympathetic dystrophy and clinical symptom amelioration. In addition, compared with the OCTR group, the MECTR group had a decreased operative and hospitalization time, post-operative scar pain score and time required to resume a normal lifestyle. Post-operative electromyographic examination also revealed that the median nerve sensory conduction speed increased compared with that prior to surgery in both groups. In conclusion, the use of MECTR for the treatment of CTS achieved higher patient satisfaction, a shorter operative time and hospitalization time, an earlier return to work time or resumption of a normal life, as well as less post-operative scar pain compared with OCTR. Thus, these results suggested that MECTR may be an effective method for the treatment of idiopathic CTS. Trial registration no. ChiCTR2000041165, retrospectively registered 20th December 2020.

Joint effect of pre-operative anemia and perioperative blood transfusion on outcomes of colon-cancer patients undergoing colectomy.

BACKGROUND: Both pre-operative anemia and perioperative (intra- and/or post-operative) blood transfusion have been reported to increase post-operative complications in patients with colon cancer undergoing colectomy. However, their joint effect has not been investigated. The purpose of this study was to evaluate the joint effect of pre-operative anemia and perioperative blood transfusion on the post-operative outcome of colon-cancer patients after colectomy. METHODS: We identified patients from the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) database 2006-2016 who underwent colectomy for colon cancer. Multivariate logistic regression analysis was employed to assess the independent and joint effects of anemia and blood transfusion on patient outcomes. RESULTS: A total of 35,863 patients-18,936 (52.8%) with left-side colon cancer (LCC) and 16,927 (47.2%) with right-side colon cancer (RCC)-were identified. RCC patients were more likely to have mild anemia (62.7%) and severe anemia (2.9%) than LCC patients (40.2% mild anemia and 1.4% severe anemia). A total of 2,661 (7.4%) of all patients (1,079 [5.7%] with LCC and 1,582 [9.3%] with RCC) received a perioperative blood transfusion. Overall, the occurrence rates of complications were comparable between LCC and RCC patients (odds ratio [OR] = 1.01; 95% confidence interval [CI] = 0.95-1.07; P = 0.750). There were significant joint effects of anemia and transfusion on complications and the 30-day death rate (P for interaction: 0.010). Patients without anemia who received a transfusion had a higher risk of any complications (LCC, OR = 3.51; 95% CI = 2.55-4.85; P < 0.001; RCC, OR = 3.74; 95% CI = 2.50-5.59; P < 0.001), minor complications (LCC, OR = 2.54; 95% CI = 1.63-3.97; P < 0.001; RCC, OR = 2.27; 95% CI = 1.24-4.15; P = 0.008), and major complications (LCC, OR = 5.31; 95% CI = 3.68-7.64; P < 0.001; RCC, OR = 5.64; 95% CI = 3.61-8.79; P < 0.001), and had an increased 30-day death rate (LCC, OR = 6.97; 95% CI = 3.07-15.80; P < 0.001; RCC, OR = 4.91; 95% CI = 1.88-12.85; P = 0.001) than patients without anemia who did not receive a transfusion. CONCLUSIONS: Pre-operative anemia and perioperative transfusion are associated with an increased risk of post-operative complications and increased death rate in colon-cancer patients undergoing colectomy.

Nickel-Catalyzed Enantioconvergent Reductive Hydroalkylation of Olefins with α-Heteroatom Phosphorus or Sulfur Alkyl Electrophiles.

Substantial advances in enantioconvergent C(sp3)-C(sp3) bond formation reactions have been made in recent years through the use of transition-metal-catalyzed cross-coupling reactions of racemic secondary alkyl electrophiles with organometallic reagents. Herein, we report a general process for the asymmetric construction of alkyl-alkyl bonds adjacent to heteroatoms, namely, a nickel-catalyzed enantioconvergent reductive hydroalkylation of olefins with α-heteroatom phosphorus or sulfur alkyl electrophiles. Including the use of readily available olefins, this reaction has considerable advantages, such as mild reaction conditions, a broad substrate scope, and good functional group compatibility, making it a desirable alternative to traditional electrophile-nucleophile cross-coupling reactions.

Nickel-catalyzed three-component olefin reductive dicarbofunctionalization to access alkylborates.

We report a three-component olefin reductive dicarbofunctionalization for constructing alkylborates, specifically, nickel-catalyzed reductive dialkylation and alkylarylation of vinyl boronates with a variety of alkyl bromides and aryl iodides. This reaction exhibits good coupling efficiency and excellent functional group compatibility, providing convenient access to the late-stage modification of complex natural products and drug molecules. Combined with alkylborate transformations, this reaction could also find applications in the modular and convergent synthesis of complex compounds.

[Advanced glycated albumin induces macrophage pyroptosis via upregulating nucleotide-binding oligomerization domain-like receptor protein 3].

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 µmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1ß (IL-1ß) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1ß and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1ß and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.

[Isolation,Purification and Differentiation of Hematopoietic Stem Cells into Dendritic Cells in Mouse Lung Tissue].

OBJECTIVE: To explore the method of isolation, purification and differentiation of hematopoietic stem cells (HSCs) into dendritic cells (DC) in lung tissue of mouse, so as to provide theoretical basis and experimental methods for the study of hematopoietic stem cells in mouse lung tissue. METHODS: Lung tissues of 4 male C57 mice were digested, separated and purified into mononuclear cells by type I collagenase, type I DNA enzyme and lymphocyte isolation solution. Lin-Sca-1+c-Kit+ cells, which are hematopoietic stem cells (HSCs) were identified and sorted by flow cytometry. Stem cell factor (SCF) and interleukin 3 (IL-3) were added in the obtained HSCs to promote cell proliferation. After discontinuation of SCF and IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 were added to induce differentiation of HSCs into DCs, and lipopolysaccharide (LPS) was added to promote cell maturation. The morphology of DCs was observed under inverted microscope, the expression of CD80, CD86, CD11c and MII-II on the surface of DCs was analyzed by flow cytometry, and the expression level of IL-12 was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: 2419.67±247.59 HSCs were collected from lung tissue mononuclear cells of 4 mice identified by flow cytometry with purity: (7.16±0.43)%. HSCs were amplified 62.34±3.23 times by induction with SCF and IL-3 for 7 days. After induction culture for 15 days, mature dendritic cells were obtained with typical dendrites on the cell surface, the DC expressed dendritic cell-specific surface molecules CDllc (92.62±3.68)%,MHC-II (83.89±6.28)%， CD80 (75.96±5.13)%, CD86(72.07±4.38)%, and the expression level of IL-12 was 136.12±16.59 pg/ml detected by ELISA. CONCLUSION: There are HSCs in lung tissue, which can be transformed into DCs by cytokine induction and proliferation.

Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Nickel-catalyzed allylic defluorinative alkylation of trifluoromethyl alkenes with reductive decarboxylation of redox-active esters.

Herein, we report a nickel-catalyzed allylic defluorinative alkylation of trifluoromethyl alkenes through reductive decarboxylation of redox-active esters. The present reaction enables the preparation of functionalized gem-difluoroalkenes with the formation of sterically hindered C(sp3)-C(sp3) bonds under very mild reaction conditions, while tolerating many sensitive functional groups and requiring minimal substrate protection. Therefore, this method provides an efficient and convenient approach for late-stage modification of biologically interesting molecules.

Tissue microarray staining reveals PLD1 and Sp1 have a collaborative, pro-tumoral effect in patients with osteosarcomas.

It has been reported that phospholipase D1 (PLD1) - a key enzyme involved in lipid metabolism - is important for the initiation and progression of various human solid cancers; however, its biological significance and regulation in human osteosarcomas remain elusive. In this study, We found that PLD1 and Specificity Protein 1 (Sp1) expression were elevated in 137 osteosarcoma specimens with immunohistochemical staining. Our results showed that both PLD1 and Sp1 were expressed at much higher rates in the cancerous tissue compared to adjacent normal tissue. A correlation analyze also indicated that PLD1 was significantly associated with lactate dehydrogenase expression (p = 0.041) and the Enneking stage (p = 0.000), while Sp1 was significantly associated with the nuclear grade (p = 0.024). Furthermore, survival analyses showed that elevated PLD1 confers a poor prognosis on patients with osteosarcomas, acting as an independent prognostic factor. Of note, we showed a positive correlation between PLD1 and Sp1 expression in the cancer tissues (r = 0.357; p < 0.001). High co-expression of the two molecules results in the worst prognosis for the patients, and can also be regarded as independent prognostic factor (p = 0.001; HR = 2.71; 95% CI 1.53-4.80).

Nickel-Catalyzed Defluorinative Reductive Cross-Coupling of gem-Difluoroalkenes with Unactivated Secondary and Tertiary Alkyl Halides.

Herein, we described a nickel-catalyzed monofluoroalkenylation through defluorinative reductive cross-coupling of gem-difluoroalkenes with alkyl halides. Key to the success of this strategy is the combination of C-F cleavage with alkyl halides activation. This reaction enables the convenient synthesis of a large variety of functionalized monofluoroalkenes under mild reaction conditions with broad functional group compatibility and excellent Z-selectivity. The combination of Ni catalysis with (Bpin)2/K3PO4 as terminal reductant promoted the efficient C(sp2)-C(sp3) formation especially the generation of all-carbon quaternary centers with high chemoselectivity.

MicroRNA-638 inhibits cell proliferation by targeting suppress PIM1 expression in human osteosarcoma.

MicroRNAs (miRNAs) are a type of small noncoding RNAs that often play important roles in carcinogenesis, but the carcinogenic mechanism of miRNAs is still unclear. This study will investigate the functions and the mechanism of miR-638 in osteosarcoma (OS). The expression of miR-638 in OS and the DNA copy number of miR-638 were detected by real-time PCR. The effect of miR-638 on cell proliferation was measured by CCK8 assay. Different assays, including bioinformatics algorithms, luciferase report assay, and Western blotting, were used to identify the target gene proviral integration site for Moloney murine leukemia virus 1 (PIM1) of miR-638 in OS. The expression of PIM1 in clinical OS tissues was also validated by immunohistochemical assay. From this research, we found that miR-638 was downregulated in OS tissues compared with corresponding noncancerous tissues (NCTs), and the DNA copy number of miR-638 was lower in OS than in NCTs, which may induce the corresponding downregulation of miR-638 in OS. Ectopic expression of miR-638 inhibited OS cell growth in vitro. Subsequently, we identified that PIM1 is the downstream target gene of miR-638 in OS cells, and silencing PIM1 expression phenocopied the inhibitory effect of miR-638 on OS cell proliferation. Furthermore, we observed that PIM1 was overexpressed in OS tissues, and high expression of PIM1 in OS predicted poor overall survival. In summary, we revealed that miR-638 functions as a tumor suppressor through inhibiting PIM1 expression in OS.

Overexpression of pyruvate kinase M2 predicts a poor prognosis for patients with osteosarcoma.

It is stated that high expression of pyruvate kinase (PKM2) emerges as a significant player in the metabolism and progression of various human malignancies. However, the expression of PKM2 and its association with the prognosis of osteosarcoma had not yet been studied. In the present study, the expression and biological significance of PKM2 in osteosarcoma were investigated. We found that PKM2 expression was elevated in the cancerous tissues and it was more abundant than the adjacent normal tissues (60.2 vs 26.1 %, p < 0.001). Moreover, we showed that high PKM2 expression was positively correlated with Enneking stage (p = 0.006) and distant metastasis (p = 0.007) but not with the age, gender, tumor site, tumor size, histologic grade, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and local pain of the patients. Furthermore, Kaplan-Meier analysis revealed that the overall survival (OS) for patients with high PKM2 expression was significantly lower than those with low PKM2 expression (p < 0.001). Finally, multivariate analysis revealed that high PKM2 expression was an independent prognostic factor for osteosarcoma patients (p = 0.004). Collectively, these data indicated that elevated PKM2 might serve as a novel target for the treatment of osteosarcoma.

Tetraspanin CD9 in bovine oocytes and its role in fertilization.

This study was conducted in bovine to investigate whether CD9 (a member of the tetraspanin superfamily of proteins) is present on oocytes and whether it functions in sperm-oocyte binding and fusion. First, the presence of CD9 in bovine matured oocytes was examined by immunofluorescence with the anti-CD9 monoclonal antibody (mAb) and fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and the results showed that CD9 was expressed on the plasma membrane of matured oocytes. Sperm binding and fusion with oocytes was then examined by in vitro fertilization. When the zona pellucida-free matured oocytes were fertilized, both sperm binding to ooplasma and sperm penetrating into oocytes were significantly (P<0.01) reduced in anti-CD9 antibody-treated oocytes (6.3 +/- 0.7 per oocyte and 41.6%, respectively) compared with untreated control oocytes (19.0 +/- 0.7 per oocyte and 81.3%, respectively), indicating that the anti-CD9 mAb potentially inhibits sperm-oocyte binding and fusion. These results demonstrated that the CD9 present on bovine matured oocytes is involved in sperm-oocyte interaction during fertilization.