Background: Hypoxia-inducible factor-1-alpha (HIF-1 alpha) has the potential to predict the neoadjuvant chemotherapy (NAC) response in pancreatic ductal adenocarcinoma (PDAC). This study aimed to assess the relationship between the pathological response and intratumoral HIF-1 alpha expression in patients with PDAC receiving NAC, and investigate the predictive value of contrast-enhanced computed tomography (CECT) features in HIF-1 alpha expression. Methods: A total of 58 patients from three centers with pathologically confirmed PDAC who underwent NAC followed by surgery were retrospectively enrolled in this study. Immunohistochemistry was performed to detect intratumoral HIF-1 alpha expression. The Chi-square test was used to evaluate the differences in intratumoral HIF-1 alpha expression in PDAC responders and non-responders after NAC. Binary logistic regression and receiver operating characteristic (ROC) curves were used to determine the optimal correlation factors of different pathological responses in PDAC patients after NAC and to predict these factors using CECT features. Results: Among the PDAC patients, 27 (46.55%) responders and 31 (53.45%) non-responders were identified via histopathological examination. Nuclear and cytoplasmic HIF-1 alpha expression was significantly higher in the responders than the non-responders (P<0.001, P=0.036). However, HIF-1 alpha expression in the stroma was not statistically significant (P=0.864). The multivariate logistic regression revealed that the %Delta carbohydrate antigen 19-9 (CA19-9), tumor differentiation, and nuclear HIF-1 alpha were independent predictors of different pathological responses [odds ratio (OR) =9.005, P=0.037; OR =0.005, P=0.044; OR =0.352, P=0.018, respectively]. The ROC curve showed that nuclear HIF-1 alpha expression was the optimal associated predictor of the pathologic response (area under the curve =0.873, 95% confidence interval: 0.782-0.964). The multivariate logistic regression also showed that of the CECT characteristics, the (post-NAC - pre-NAC) arterial phase (AP) was an independent predictive indicator of nuclear HIF-1 alpha expression (OR =1.012, P=0.020). Conclusions: Nuclear HIF-1 alpha was the best predictor of the pathological response in patients with PDAC after NAC, and it can be predicted using CT feature of the (post-NAC - pre-NAC) AP.

Neoantigen peptides hold great potential as vaccine candidates for tumor immunotherapy. However, due to the limitation of antigen cellular uptake and cross-presentation, the progress with neoantigen peptide-based vaccines has obviously lagged in clinical trials. Here, a stapling peptide-based nano-vaccine is developed, comprising a self-assembly nanoparticle driven by the nucleic acid adjuvant-antigen conjugate. This nano-vaccine stimulates a strong tumor-specific T cell response by activating antigen presentation and toll-like receptor signaling pathways. By markedly improving the efficiency of antigen/adjuvant co-delivery to the draining lymph nodes, the nano-vaccine leads to 100% tumor prevention for up to 11 months and without tumor recurrence, heralding the generation of long-term anti-tumor memory. Moreover, the injection of nano-vaccine with signal neoantigen eliminates the established MC-38 tumor (a cell line of murine carcinoma of the colon without exogenous OVA protein expression) in 40% of the mice by inducing potent cytotoxic T lymphocyte infiltration in the tumor microenvironment without substantial systemic toxicity. These findings represent that stapling peptide-based nano-vaccine may serve as a facile, general, and safe strategy to stimulate a strong anti-tumor immune response for the neoantigen peptide-based personalized tumor immunotherapy.

Due to their strong nucleophilicities, nucleophilic lysine and cysteine residues can be easily recognized and modified by electrophilic groups, thus, acting as the targets for covalent ligands or drugs. There-fore, the development of site-specific protein-modification chemistry for various nucleophilic residues has been explored to label proteins selectively for many biological and therapeutic applications. In this study, we constructed a series of sulfonium-based small molecules to react with the amine group of lysine residues by utilizing the strong electrophilicity of sulfonium, resulting in lysine-selective labeling via the formation of classical amide bonds under alkaline conditions (pH 9.0-11.0). After systematic optimization of the labeling conditions, this strategy was utilized for protein labeling across various bacteria's lysates. Finally, combined with the activity-based protein profiling (ABPP)strategy, we successfully identified and analyzed hundreds of labeled lysine residues in the bacterial proteome.

Radiation therapy is one of the most common treatments for cancer. However, enhancing tumors' radiation sensitivity and overcoming tolerance remain a challenge. Previous studies have shown that the Ras signaling pathway directly influences tumor radiation sensitivity. Herein, we designed a series of Ras-targeting stabilized peptides, with satisfactory binding affinity (KD = 0.13 μM with HRas) and good cellular uptake. Peptide H5 inhibited downstream phosphorylation of ERK and increased radio-sensitivity in HeLa cells, resulting in significantly reduced clonogenic survival. The stabilized peptides, designed with an N-terminal nucleation strategy, acted as potential radio-sensitizers and broadened the applications of this kind of molecule. This is the first report of using stabilized peptides as radio-sensitizers, broadening the applications of this kind of molecule.

AbstractHormone therapy resistance and the ensuing aggressive tumor progression present a significant clinical challenge. However, the mechanisms underlying the induction of tumor malignancy upon inhibition of steroid hormone signaling remain poorly understood. Here, we demonstrate that Drosophila malignant epithelial tumors show a similar reduction in ecdysone signaling, the main steroid hormone pathway. Our analysis of ecdysone-induced downstream targets reveals that overexpression of the nuclear receptor E75, particularly facilitates the malignant transformation of benign tumors. Genome-wide DNA binding profiles and biochemistry data reveal that E75 not only binds to the transcription factors of both Hippo and Notch pathways, but also exhibits widespread co-binding to their target genes, thus contributing to tumor malignancy. We further validated these findings by demonstrating that depletion of NR1D2, the mammalian homolog of E75, inhibits the activation of Hippo and Notch target genes, impeding glioblastoma progression. Together, our study unveils a novel mechanism by which hormone inhibition promotes tumor malignancy, and describes an evolutionarily conserved role of the oncogene E75/NR1D2 in integration of Hippo and Notch pathway activity during tumor progression.

Targeted protein degradation is becoming more and more important in the field of drug development. Compared with proteasomal-based degraders, lysosomal-based degraders have a broader target spectrum of targets, which have been demonstrated to have great potential, especially in degrading undruggable proteins. Recently, we developed a programmable and facile screening PROTAC development platform based on peptide self-assembly termed split-and-mix PROTAC (SM-PROTAC). In this study, we applied this technology for the development of lysosome-based degraders, named a split-and-mix chaperone-mediated autophagy-based degrader (SM-CMAD). We successfully demonstrated SM-CMAD as a universal platform by degrading several targets, including ERα, AR, MEK1/2, and BCR-ABL. Different from other lysosomal-based degraders, SM-CMAD was capable of facile screening with programmable ligand ratios. We believe that our work will promote the development of other multifunctional molecules and clinical translation for lysosomal-based degraders.

Lysine-specific demethylase 1 (LSD1) is a promising therapeutic target, especially in cancer treatment. Despite several LSD1 inhibitors being discovered for the cofactor pocket, none are FDA-approved. We aimed to develop stabilized peptides for irreversible LSD1 binding, focusing on unique cysteine residue Cys360 in LSD1 and SNAIL1. We created LSD1 C360-targeting peptides, like cyclic peptide S9-CMC1, using our Cysteine-Methionine cyclization strategy. S9-CMC1 effectively inhibited LSD1 at the protein level, as confirmed by MS analysis showing covalent bonding to Cys360. In cells, S9-CMC1 inhibited LSD1 activity, increasing H3K4me1 and H3K4me2 levels, leading to G1 cell cycle arrest and apoptosis and inhibiting cell proliferation. Remarkably, S9-CMC1 showed therapeutic potential in A549 xenograft animal models, regulating LSD1 activity and significantly inhibiting tumor growth with minimal organ damage. These findings suggest LSD1 C360 as a promising site for covalent LSD1 inhibitors' development.

Covalent proteolysis-targeting chimeras (PROTACs) offer enhanced selectivity, prolonged action, and increased efficacy against challenging target proteins.

Figure S1：HDAC distribution in HeLa cells. Figure S2：Enzyme activity of peptides in SIRT1. Figure S3: CD spectra of peptide 16cyc-HxA, 16lin-HxA, 13cyc-HxA and 13lin-HxA. Figure S4: Enzyme activity of peptides in HDAC1, 3, 6, 8. Figure S5: Cellular uptake in cancer stem-like cells treated with peptide inhibitors for 4 hours. Figure S6: The transfection efficiency of peptides in cancer cells concluded from cell flow cytometry assays Figure S7: Cellular uptake in HeLa cells treated with different peptides for 4 hours. Figure S8: Cellular uptake in A549 cells treated with different peptides for 4 hours Figure S9: Time tracking of peptide cell stability in PA-1 cells treated with 10 μM FITC-labeled peptides Figure S10: The comparison of cellular permeability and anti-proliferation effect in PA-1 cells treated with 16cyc-HxA and the scramble peptides Scr-1 and Scr-2. Figure S11: Anti-proliferation effect of different small inhibitors in cancer stem like cells and normal cells. Figure S12: LDH release from PA-1 cells for 4 hours and 12 hours. Figure S13: Hemolysis assays were performed to assess the erythrocyte toxicity of peptide inhibitors. Figure S14: The peptide binding affinity towards these endogenous HDAC isoforms using SA pull down assays. Figure S15: HDACs expression affected by peptide inhibitors in cancer stem-like cells and HDAC activity in A549 cells treated with peptide inhibitors. Figure S16: The irreversible inhibition of HDACs in PA-1 cell lines for 48 hours' treatment. Figure S17: The validation of HDAC inactivation using HDAC selective small inhibitors as positive controls. Figure S18: The quantifying of the different protein levels from Fig 4C. Figure S19: Peptide inhibitors could alter the recruitment of HDAC1 and LSD1 to Sox2 gene promoters by ChIP experiment in PA-1 cells. Figure S20: Apoptotic cells stained with FITC-Annexin V/PI (propidium iodide) were measured by flow cytometry. Figure S21：The western blot assays were performed to confirm the caspase-3 dependent apoptosis in PA-1 cells Figure S22：Cell cycle distribution of flow cytometry analysis treated with different peptides. Figure S23: The expression of cell cycle regulatory proteins in PA-1 cells treated with different peptide inhibitors. Figure S24: Transcriptome analysis of PA-1 cells treated with different inhibitors Figure S25: 2D diagram to show the number of genes with change of expression level and gene ontology analysis in PA-1 cells treated with different peptides. Figure S26: The relative genes were selected for RT-PCR assays to valid the results of RNA-microarray analysis. Figure S27：The protein levels of EGFR and WNT5A1 in PA-1 cells treatment with peptide inhibitors Figure S28: The cage-wheel exercise assay was performed to study the motor learning ability of mice. Figure S29: The antitumor activities of peptides and SAHA in a NTERA-2 xenograft animal model Figure S30: The body distribution of Cy-5 labeled peptides 16cyc-HxA, 16lin-HxA and PBS at different time points after intratumor injections. Figure S31: Hematoxylin and eosin (H&E) staining of organs and tumor sections collected from different groups of mice 3 weeks after treatment Figure S32: Immunohistochemistry analysis of HDAC1 and HDAC6 and their related protein level in tumor tissue slides. Figure S33: The proliferative index was tested by doing KI67 staining on PA-1 tumor tissues. Table S1: Peptides Characterization. Table S2: Primer sequence of genes for RT-PCR analysis Table S3: Sequences of primers used for ChIP assays Table S4: Cancer related genes affect by HDAC inhibitor 16cyc-HxA in PA-1 cells.

Through systematic optimization of halopyridinium compounds, we established a peptide coupling protocol utilizing 4-iodine N-methylpyridinium (4IMP) for solid-phase peptide synthesis (SPPS). The 4IMP coupling reagent is easily prepared, bench stable, and cost-effective. Employing 4IMP in the SPPS process has showcased remarkable chemoselectivity and efficiency, effectively eliminating racemization and epimerization. This achievement has been substantiated through the successful synthesis of a range of peptides via the direct utilization of commercially available amino acid substrates for SPPS.

为解决目前药学类专业分析化学实验教学中遇到的矛盾,提升教学质量.利用智能手机和校园网络,拍摄制作分析化学实验相关手机短视频,并用于实验教学中.提高了学生的学习兴趣,解决了基本实验操作技能不牢固,对相应实验原理理解不够透彻,和大型实验仪器在教学中无法充分发挥作用的矛盾.利用了智能手机及网络的优势,提高了学生的自主学习能力,丰富了实验教学手段,有效地解决了分析化学实验教学中的矛盾,促进了教学质量的提高.

研究开发同时测定黄连上清片中栀子苷和盐酸小檗碱两种有效成分的定量方法.采用ZORBAX Eclipse XDB-C18(4.6 × 250mm,5μm)色谱柱,流动相:A 泵为乙腈,B 泵为 0.2％磷酸,切换波长检测:0～10分钟检测栀子苷(λ=238nm)、10～50分钟检测盐酸小檗碱(λ=345nm),流速为1.0ml/min,梯度洗脱,柱温为30℃,进样10μL.可使各组分有效分离,栀子苷的回归方程:y=43651x-1027(r=0.9996)、加样回收率的平均值为98.95％(RSD=1.5％);盐酸小檗碱的回归方程:y=27519x-2836(r=0.9995)、加样回收率的平均值为99.07％(RSD=1.7％).结果说明此法快速、专属性高,可用于对黄连上清片的质量控制.

介绍分析化学理论课和实验课教学的实践与思考,包括讲好绪论、引入口诀、引入表格、巧设比喻、引入实验案例、加强巡视指导、优化实验课评价方式以及课程思政的应用,以期与同行交流.

以内蒙古医科大学药学专业学生为研究对象,探讨药学专业分析化学多元化线上线下混合教学模式的构建.教学过程中充分发挥线上、线下教学的优势,对课前、课中、课后等环节进行整合,构建教学质量评价指标体系,有效提高分析化学教学质量与教学效率.

Background Targeting ubiquitin-dependent proteolysis is one of the strategies in cancer therapy. CRL CDT2 and CRL DDB2 are two key E3 ubiquitin ligases involved in DNA replication and DNA damage repair. But CDT2 and DDB2 are opposite prognostic factors in kinds of cancers, and the underlining mechanism needs to be elucidated. Methods Small interfering RNAs were used to determine the function of target genes. Co-immunoprecipitation (Co-IP) was performed to detect the interaction between DDB2 and CDT2. Immunofluorescence assays and fluorescence activating cell sorting (FACS) were used to measure the change of DNA content. In vivo ubiquitination assay was carried out to clarify the ubiquitination of CDT2 mediated by DDB2. Cell synchronization was performed to arrest cells at G1/S and S phase. The mechanism involved in DDB2-mediated CDT2 degradation was investigated by constructing plasmids with mutant variants and measured by Western blot. Immunohistochemistry was performed to determine the relationship between DDB2 and CDT2. Paired two-side Student’s t-test was used to measure the significance of the difference between control group and experimental group. Results Knockdown of DDB2 stabilized CDT2, while over-expression of DDB2 enhanced ubiquitination of CDT2, and subsequentially degradation of CDT2. Although both DDB2 and CDT2 contain PIP (PCNA-interacting protein) box, PIP box is dispensable for DDB2-mediated CDT2 degradation. Knockdown of PCNA had negligible effects on the stability of CDT2, but promoted accumulation of CDT1, p21 and SET8. Silencing of DDB2 arrested cell cycle in G1 phase, destabilized CDT1 and reduced the chromatin loading of MCMs, thereby blocked the formation of polyploidy induced by ablation of CDT2. In breast cancer and ovarian teratoma tissues, high level of DDB2 was along with lower level of CDT2. Conclusions We found that CRL4 DDB2 is the novel E3 ubiquitin ligases of CDT2, and DDB2 regulates DNA replication through indirectly regulates CDT1 protein stability by degrading CDT2 and promotes the assembly of pre-replication complex. Our results broaden the horizon for understanding the opposite function of CDT2 and DDB2 in tumorigenesis, and may provide clues for drug discovery in cancer therapy.

Up to now, the chemotherapy approaches for glioblastoma were limited. 1-[2-Thiazolylazo]-2-naphthol (named as NSC139021) was shown to significantly inhibit the proliferation of prostate cancer cells by targeting the atypical protein kinase RIOK2. It is documented that RIOK2 overexpressed in glioblastoma. However, whether NSC139021 can inhibit the growth of glioblastoma cells and be a potential drug for glioblastoma treatment need to be clarified. In this study, we investigated the effects of NSC139021 on human U118MG, LN-18, and mouse GL261 glioblastoma cells and the mouse models of glioblastoma. We verified that NSC139021 effectively inhibited glioblastoma cells proliferation, but it is independent of RIOK2. Our data showed that NSC139021 induced cell cycle arrest at G0/G1 phase via the Skp2-p27/p21-Cyclin E/CDK2-pRb signaling pathway in G1/S checkpoint regulation. In addition, NSC139021 also increased the apoptosis of glioblastoma cells by activating the p53 signaling pathway and increasing the levels of Bax and cleaved caspase 3. Furthermore, intraperitoneal administration of 150 mg/kg NSC139021 significantly suppressed the growth of human and mouse glioblastoma in vivo. Our study suggests that NSC139021 may be a potential chemotherapy drug for the treatment of glioblastoma by targeting the Skp2-p27/p21-Cyclin E/CDK2-pRb signaling pathway.

The proto-oncoprotein MET is a receptor tyrosine kinase that plays a key role in cancer cell growth and invasion. We have used fluorescence-tagged antibodies to activate MET in live serum-starved glioblastoma cells and monitor the fate of antibody-bound MET receptor in single cell-based assays. We found that the antibodies induced rapid and transient formation of highly polarized MET clusters on the plasma membrane and promoted the activation of MET, resembling the initial effects of binding to its ligand, HGF. However, the antibody-induced clustering and activation of MET led to the rapid removal of the receptor from cell surface and altered its intracellular processing, resulted in rapid degradation of the receptor. Consequently, while cells pre-treated with HGF remain competent to respond to further HGF stimulation, cells pre-treated with antibodies are refractory to further HGF stimulation due to antibody-mediated MET depletion. Removal of MET by sustained treatment of antibodies blocked cancer cell migration and invasion. Our studies reveal a novel mechanism to alter the recycling process of MET in glioblastoma cancer cells by promoting the receptor degradation through a proteasome-sensitive and lysosome-dependent pathway through the ligand-independent activation of MET using anti-MET antibodies.

Abstract FDA-approved HDAC inhibitors exhibit dose-limiting adverse effects; thus, we sought to improve the therapeutic windows for this class of drugs. In this report, we describe a new class of peptide-based HDAC inhibitors derived from the HDAC1-specific substrate H3K56 with improved nonspecific toxicity compared with traditional small-molecular inhibitors. We showed that our designed peptides exerted superior antiproliferation effects on cancer stem–like cells with minimal toxicity to normal cells compared with the small-molecular inhibitor SAHA, which showed nonspecific toxicity to normal and cancer cells. These peptide inhibitors also inactivated cellular HDAC1 and HDAC6 and disrupted the formation of the HDAC1, LSD1, and CoREST complex. In ovarian teratocarcinoma (PA-1) and testicular embryonic carcinoma (NTERA-2) cell xenograft animal models (5 mice/group, 50 mg/kg, every other day, intraperitoneal injection), these peptides inhibited tumor growth by 80% to 90% with negligible organ (heart, liver, spleen, lung, kidney, brain) lesions. These results represent the first attempt to design chemically stabilized peptide inhibitors to investigate HDAC inhibition in cancer stem–like cells. These novel peptide inhibitors have significantly enhanced therapeutic window and offer promising opportunities for cancer therapy. Significance: Selective antiproliferative effects of stabilized peptide HDAC inhibitors toward cancer stem–like cells provide a therapeutic alternative that avoids high nonspecific toxicity of current drugs.

本文选择药学专业分析化学课程“沉淀滴定法”进行说课设计,从教材分析、学情分析、教法与学法、教学过程、教学反思进行了说课设计,结合自身教学体会,在分析教材和学生的基础上,精心安排教学过程,以提高教学效果和学生的学习能力和综合素质.

The MET proto-oncogene-encoded receptor tyrosine kinase (MET) and AXL receptor tyrosine kinase (AXL) are independently operating receptor tyrosine kinases (RTKs) that are functionally associated with aggressive and invasive cancer cell growth. However, how MET and AXL regulate the migratory properties of cancer cells remains largely unclear. We report here that the addition of hepatocyte growth factor (HGF), the natural ligand of MET, to serum-starved human glioblastoma cells induces the rapid activation of both MET and AXL and formation of highly polarized MET-AXL clusters on the plasma membrane. HGF also promoted the formation of the MET and AXL protein complexes and phosphorylation of AXL, independent of AXL's ligand, growth arrest-specific 6 (GAS6). The HGF-induced MET-AXL complex stimulated rapid and dynamic cytoskeleton reorganization by activating the small GTPase RAC1, a process requiring both MET and AXL kinase activities. We further found that HGF also promotes the recruitment of ELMO2 and DOCK180, a bipartite guanine nucleotide exchange factor for RAC1, to the MET-AXL complex and thereby stimulates the RAC1-dependent cytoskeleton reorganization. We also demonstrated that the MET-AXL-ELMO2-DOCK180 complex is critical for HGF-induced cell migration and invasion in glioblastoma or other cancer cells. Our findings uncover a critical HGF-dependent signaling pathway that involves the assembly of a large protein complex consisting of MET, AXL, ELMO2, and DOCK180 on the plasma membrane, leading to RAC1-dependent cell migration and invasion in various cancer cells.