Peptide-based therapeutics for melanoma have received increasing attention in medical research. However, the local delivery of such therapeutics poses unique challenges. Cell-penetrating peptides (CPPs) with the ability to selectively enter cancer cells, with sufficient stability and increased endosomal escape mechanisms, can provide a new and improved delivery strategy for therapeutic agents for treating cancer. We developed a new combination strategy for the synthesis of penetrating peptides functionalized with targeting of integrin α6. The linear peptide S5 was multimerized with 4 copies in linear sequential order spaced by GSG between each copy to yield the 4S5 peptide. The multimerized 4S5 peptide coupled with an intracellular delivery peptide (N) and endosomal escape peptide (G) was separated by a GGS spacer. This optimized peptide was called 4S5NG. The 4S5NG, EGFP or PE24 peptide-protein conjugates were purified via a C-terminal His-tag. The uptake efficacy, intracellular distribution and integrin α6-targeting ability of these 4S5NG peptides were systematically characterized via IncuCyte, flow cytometry and in vivo imaging using 4S5NG-Cy5 or 4S5NG-EGFP. Moreover, 4S5NG-incorporated Pseudomonas aeruginosa (PE24) exotoxin A generated therapeutic peptides. The antitumor efficacy and underlying mechanism were studied in cell lines and a mouse model. In addition, the effect of 4S5NG-PE24 on antitumor immunity of a healthy immune system was investigated via a mouse model. Images of living cells and mice indicated that 4S5NG accumulated at tumor sites in vitro and in vivo and was much more effective than the S5 and 4S5 peptides. 4S5NG-PE24 induced cell pyroptosis in integrin α6-expressing melanoma through the caspase 3/gasdermin E (GSDME) signaling pathway in the absence of histological alterations in other organs. 4S5NG-PE24 also promoted the response rate of programmed cell death protein-1 (PD-1) checkpoint blockade to increase antitumor efficacy. Collectively, these results highlight the potential use of 4S5NG to deliver the toxin PE24 to selectively eliminate integrin α6+ cells in melanoma, which may represent a novel treatment approach for melanoma patients.

PURPOSE:Profilin 1 (Pfn1) has been implicated in cytoskeletal regulation; however, its role in breast cancer progression and DNA replication remains unclear. This study investigated the functional significance of Pfn1 nuclear-cytoplasmic shuttling in breast cancer. METHODS:We analyzed Pfn1 expression and its correlation with DNA replication, repair, and oncogenic markers in breast cancer cell lines. Chromatin-bound and soluble Pfn1 levels were quantified by western blotting. The effects of nuclear (nuclear localization sequence-Pfn1) and cytoplasmic (nuclear export sequence-Pfn1) localization on cell growth, DNA replication, and stemness were assessed using colony formation, Alamar blue fluorescence, replication protein A 32-kDa foci staining, and DNA fiber assays. Mouse xenografts of breast cancer cells were used to determine the effect of Pfn1 localization on tumor growth in vivo. We identified the direct interactors of nuclear Pfn1 by immunoprecipitation, and their affinity was determined using bio-layer interferometry. RESULTS:Pfn1 expression was positively correlated with DNA replication, repair, p53, and MYC expression. Chromatin-bound Pfn1 was significantly degraded in breast cancer cell lines compared to that in non-cancerous MCF10a cells. Nuclear Pfn1 inhibited cell growth and DNA replication in SKBR3 cells, while cytoplasmic Pfn1 promoted cell survival and DNA replication in MCF10a cells. Loss of nuclear Pfn1 in SKBR3 cells inhibited their growth in vivo. Additionally, cytoplasmic Pfn1 upregulated stemness markers (c-Myc, B lymphoma Mo-MLV insertion region 1, and Nijmegen breakage syndrome 1). Pfn1 regulated cell stemness by binding to the nucleosome remodeler sucrose non-fermenting 2 homolog. CONCLUSION:Our findings revealed that nuclear Pfn1 acts as a tumor suppressor by inhibiting DNA replication and cell growth, while cytoplasmic Pfn1 promotes tumorigenesis by enhancing stemness and replication efficiency. These results highlight the dual role of Pfn1 in breast cancer progression, governed by its subcellular localization. They suggested that modulating Pfn1 nuclear-cytoplasmic shuttling may be a potential therapeutic strategy.

IntroductionCytotoxic agents have shown limited benefits in hepatocellular carcinoma (HCC), mediated in part by the lack of targeting. As cell-penetrating peptides (CPPs) are capable of delivering various biologically active molecules into cells, including protein, peptides, small chemo-drugs, and nucleic acid with or without targeting, we developed T22-PE24, a CXCR4-targeted self-assembling cytotoxic nanotoxin, to effectively induce HCC pyroptosis.MethodsT22 incorporating enhanced green fluorescent protein (EGFP) or PE24 was purified from DE3 bacterial cells and characterized using transmission electron microscopy, the Zetasizer Nano®, and SEC-HPLC. The internalization effect of T22-EGFP was detected by flow cytometry system (FCS) in CXCR4+/LM3(CXCR4−) HCC cells. The CCK8, lactate dehydrogenase (LDH) release, Western blot, and nude mice HCC models were used to estimate the cell viability of T22-PE24. The complete-immunity HCC tumor-bearing mice model was used to assess the immune response of T22-PE24.ResultsThe round shape under transmission electron microscopy, 49.4 nm hydrodynamic diameter, and −33.33 mV zeta potential indicated that T22-PE24 self-assembled into nanoparticles. T22 incorporating EGFP selectively internalized in CXCR4+ HCC cells and showed no accumulation in CXCR4-knockout HCC cells. The T22-PE24 nanotoxin induced HCC pyroptosis via the caspase-3/GSDME signaling pathway and suppressed tumor growth in the absence of histological alterations in normal organs. Using the complete-immunity HCC tumor-bearing mice model, we found that T22-PE24 nanotoxin effectively induces the global reprogramming of cell components of the immune tumor microenvironment, leading to enhanced antitumor effects compared to those observed in immunodeficient mice.ConclusionOur findings demonstrate the activation of the innate immune response in HCC by inducing pyroptosis with T22-PE24 nanotoxin treatment and support an implementation of this strategy for HCC treatment.

Background: Recent researches have demonstrate that cuproptosis, a copper -dependent cell death mechanism, is related to tumorigenesis, progression, clinical prognosis, tumor microenvironment, and drug sensitivity. Nevertheless, the function and impact of cuproptosis in cholangiocarcinoma (CCA), remain elusive. Methods: Utilizing data obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA-CHOL) datasets, we conducted subgroup typing of CCA according to cuproptosis-related genes (CRGs) and explored functional differences and prognostic value between groups. A CRG score was established considering clinical prognosis and gene expression. Furthermore, differences in the immune microenvironment, response to immunotherapy, metabolic patterns, and cancer progression characteristics between high- and low -risk groups were examined on the basis of these scores. In vitro experiments validated the function of the key gene glycine cleavage system protein H (GCSH) in cellular and tissues, respectively. Results: Prognostic models established on the basis of subgroup genetic differences achieved satisfactory results in validation. Metabolic -related gene expression levels and tumor microenvironment distribution were significantly different between the high and low CRG groups. GCSH was revealed as the singular prognostic CRG in CCA (HR =6.04; 95% CI: 1.15-31.80). Moreover, inhibition of the cupcoptosis key gene GCSH attenuated the malignant ability of CCA cell lines in vitro, including cell proliferation, migration and invasion, and this function of GCSH may be achieved via JAK-STAT signaling in CCA. Conclusion: The CRG scoring system accurately predicts prognosis and opens up new possibilities for cuproptosis-related therapy for CCA. The cuproptosis key gene GCSH has been preliminarily confirmed as a reliable therapeutic target or prognostic marker for CCA patients.

While immunotherapy has emerged as a promising strategy to treat melanoma, the limited availability of immunotherapeutic agents in tumors due to the immunosuppressive tumor microenvironment dampens its efficacy. Pyroptosis is a gasdermin-mediated programmed necrosis that triggers the inflammatory tumor microenvironment and enhances the efficacy of tumor immunotherapy. Here, we prove that the CXCR4 antagonist T22 peptide specially targeted and became internalized into CXCR4+ melanoma cells. Then we report a self-assembling nanotoxin that can be used to spatiotemporally target CXCR4-expression melanoma cells and enable tunable cellular pyroptosis. Specific activation of caspase 3 signal transduction triggers gasdermin-E-mediated pyroptosis. This nanotoxin induces pyroptotic cell death resulting in enhanced antitumor efficacy and minimized systemic side effects toward melanoma in vivo. This study offers new insights into how to engineer nanotoxins with tunable pyroptosis activity through specifically targeting CXCR4 for biomedical applications.

Objective:To establish donor liver quality related risk factors for the loss of function of transplanted liver.Methods:The data of donors and recipients of liver transplantation at the Organ Donation and Transplantation Center of the First Affiliated Hospital of Sun Yat-sen University from Nov 2011 to Dec 2018 were analyzed retrospectively. Propensity score matching (PSM) was performed to evaluate and screen the data of donors and recipients, in order to balance the covariates.Results:Of the organ donation, there were 70 males and 20 females , aging (40.6±16.3) years. Of the liver transplantation recipients, there were 70 males and 20 females , aging (41.8±20.3) years. Liver dysfunction after transplantation was significantly correlated with the following variables: the donor's CPR time( t=0.429, P=0.000), 15-minute retention rate of indocyanine green ( χ2=67.151, P=0.000), liver function grading ( χ2=54.154, P=0.000), bullae fatty liver grading ( χ2=8.120, P=0.017), vesicular fatty liver grading ( χ2=16.000, P=0.001), ICU stay time ( χ2=14.900, P=0.001)and serum creatinine level ( χ2=44.685, P=0.000). The donor scoring system was established in our studying. For the 90 organ donation cases, the donated liver quality were classified into four levels,which were of good correspondence to the prognosis of the recipients. Conclusion:This donor scoring system and grading standards established by analyzing the high-risk factors of liver dysfunction after transplantation helps evaluate the quality of donor liver in China.

Objective To study the expression of MREll-RAD50-NBS1 complex in normal gallbladder tissues,simple cholecystitis,calculous cholecystitis and gallbladder carcinoma.Methods The expression of MRN complex in different gallbladder lesion tissues were detected by immunohistochemistry.Western blot,Methyl thiazolyl tetrazolium(MTT) assay and flow cytometry were used to detect the influence of apoptosis and proliferation induced by NBS1.Results The differences between the expression of MRE11,RAD50 and different gallbladder lesion tissues were not statistically significant (respectively x2 =2.724,1.697,all P ＞ 0.05).The expression of NBS1 in GBC tissues [15.3％ (9/59)] was prominently lower than that in the normal gallbladder tissues [84.7％ (50/59)],simple cholecystitis [87.8％ (36/41)],calculous cholecystitis [61.2％ (30/49)] (x2 =87.388,P ＜ 0.01).The Western blot results reveal that NBS1 can mediate apoptosis by up-regulate Bax and down-regulate Bcl-2.The MTT assay results showed that the relative absorbance of treatment and control group after 24,48,72 h were[(1.120 ± 0.006)vs.(1.350±0.009),(1.600±0.004)vs.(1.99±0.01),(1.83±0.01)vs.(2.260±0.003)(F=7.659,P ＜0.01).The apoptosis rate in treatment group(17.23％ ± 0.56％)was higher than that in the control group (4.13％ ± 0.67％) (t =9.133,P ＜ 0.01).Conclusion NBS1 is closely related to gallbladder carcinoma.NBS1 can inhibit the proliferation and promote apoptosis of GBC-SD cells.

Liver regeneration (LR) is the result of a dynamic balance between the increased proliferation and decreased apoptosis of hepatocytes. However, the role of microRNA (miR)‑26a in regulating complex signalling networks involving E3 ubiquitin‑protein ligase Mdm2 (mdm2), p53, p21 and p27 in the process of LR is currently unclear. In the present study, it was hypothesized that miR‑26a may negatively regulate the mdm2/p53 signalling loop in response to LR. In vitro experiments were performed, whereby mouse liver cells were transfected with an miR‑26a vector or an anti/miR‑26a vector. Cell proliferation was analysed using an MTS assay and cell apoptosis, and cell cycle progression were analysed by flow cytometry. In addition, the expression of mdm2, p53, p21 and p27 were assessed using western blotting and reverse transcription‑quantitative polymerase chain reaction analyses. Dual‑luciferase reporter assays were also used to examine the association between mdm2 and miR‑26a. A 70% partial hepatectomy in C57BL/6J mice was then performed, which was followed by injection with an mdm2‑cDNA vector or an mdm2‑small interfering RNA vector. The liver‑to‑body weight ratio and liver function of mice were measured at 72 h following vector administration. The results demonstrated an increase in hepatocyte proliferation accompanied by decreased hepatocyte apoptosis levels. In addition, inhibition of miR‑26a expression was associated with a marked increase in mdm2 expression, while the expression of p53, p21 and p27 was decreased when compared with negative controls. The opposite effects were observed when miR‑26a was overexpressed. Notably, miR‑26a was demonstrated to target the 3'‑untranslated region of mdm2 directly. The results of the present study are the first to demonstrate as far as the authors are aware that the mdm2/p53 negative feedback loop may be targeted by miR‑26a directly in response to LR, and that mdm2 negatively regulates p53, p21 and p27 but not miR‑26a. miR‑26a may therefore function as an important factor that regulates the interaction between mdm2 and p53.

Objective Study on the mechanism of sliencing microRNA (miRNA,miR)-16 on chemoresistance in SMMC-7721.Methods The lentiviral vectors were used to construct stable cell lines.To determine whether constructed successfully,the expression of miR-16 was detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and inhibitor of kappaB kinase beta (IKBKB),the target gene of miR-16,was detected by Western blotting.The sensitivity and resistance mechanism of the stable cell lines to paclitaxel were detected by Western blotting and thiazole blue (MTT).Results The expression of miR-16 in 7721-miR16 group was significantly higher than SMMC-7721 and 7721-mock groups (4.63 ± 1.32 vs.1.00 ± 0.30 vs.0.93 ± 0.37,P =0.000).The expression of IKBKB in 7721-shmiR16 was 25.73 times than the controls (P =0.000).The expression of multidrug resistance protein 1 (MDR1) in 7721-shmiR16 was increased and the half maximal inhibitory concentration (IC50) to paclitaxel was 4.903 μmol/L,10.64 times than the control group (0.461 μmol/L) (P =0.000).The results of western showed that silencing miR-16 induced resistance mainly through activating nuclear factor-kappaB (NF-κB) pathway.Conclusion Silencing miR-16 in SMMC-7721 significantly increases IKBKB expression leadind paclitaxel resistance.

Hepatocellular carcinoma (HCC) is a common malignant tumor usually resistant to chemotherapy. MicroRNAs play important roles in modulation of carcinogenesis and chemoresistance, which miR-16 has been reported to mediate chemoresistance in many types of cancers. However, the role of miR-16 in HCC remains unknown. The aim of this study was to investigate whether miR-16 is participated in chemoresistance in HCC and shed light on the underlying molecular mechanisms. The findings of the current study discover that miR-16 is down-regulated in HCC tissue and cell lines. The results demonstrate that the inhibition of miR-16 renders resistance to paclitaxel in vitro and in vivo by targeting IKBKB via NF-κB signaling pathway, suggesting that miR-16 may be a meaningful therapeutic potential to overcome drug resistance in HCC.

Objective To study the expressions of Nodal in normal gallbladder,and gallbladders with cholelithiasis,cholecystitis and carcinoma;and to study the impact of inhibiting or promoting Nodal expressions in gallbladder carcinoma on the Smad2/4 pathway and epithelial-mesenchymal transition.Methods Immunohistochemistry was used to detect the expressions and distributions of Nodal protein in 30 normal gallbladders,96 simple cholecystitis/calculous cholecystitis specimens and 42 gallbladder carcinoma specimens.The mRNA and protein expressions of Nodal in normal and malignant gallbladdcr mucosal epithelium cells and breast cancer cells were detected by RT-PCR,western blotting and wound healing tests.The impact of activating agents and inhibitors on the expression levels of Nodal and its signaling pathway Smad2/4 and EMT-related proteins were analyzed.Results Immunohistochemistry showed that the positive rates of Nodal in the gallbladder cancer group was significantly higher than that in the gallbladder stone group and the normal gallbladder group.The results were 83.3％ (35/42),44.8％ (43/96),6.7％ (2/30) respectively (P＜ 0.01).RT-PCR and Western blotting showed the expressions of Nodal in gallbladder carcinoma cells were higher than normal gallbladder cells (P＜0.05).After using rhNodal to up regulate the Nodal expression,the Smad2 protein phosphorylation was promoted and the EMT associated proteins were up-regulated.After using the inhibitor SB431542 to suppress the Nodal expression,the Smad2 protein phosphorylation decreased and the EMT associated proteins were down-regulated.Conclusions The expression of Nodal was closely related to cell proliferation and metastasis in gallbladder carcinoma.Tumor progression was promoted via the smad2/4 pathway through epithelial-mesenchymal transition.

Objective To investigate the expressions of Yes-associated protein-1 (YAP1) in gallbladder mucosal epithelium of normal persons,in patients with simple/calculous cholecystitis,and in patients with gallbladder carcinoma;and to study the mechanism of YAP1 in gallbladder carcinoma development.Methods Immunohistochemistry was used to detect the expression and distribution of YAP1 protein in 50 persons with normal gallbladder,101 patients with simple cholecystitis/calculous cholecystitis and 100 patients with gallbladder carcinoma.RT-PCR and western-blot were used to detect the mRNA and protein levels of YAP1 in normal and malignant gallbladder mucosal epithelium cells.siRNA was used to shut down the expression of YAP1 in SGC996 cells.MTT was used to test cell vitality.Flow cytometry was used to measure cell cycle.Results Immunohistochemistry revealed the expression rates of YAP1 in the gallbladder carcinoma group,the cholecystitis/gallstone group and the control group to be 87.0％ (87/100),56.4％ (57/101) and 5.0％ (1/20),respectively (P ＜ 0.01).The YAP1 protein levels were higher in gallbladder carcinoma tissues and cells when compared to normal tissues and cells.RT-PCR showed the mRNA levels of gallbladder carcinoma cells to be 12.5 ± 1.2 times of normal gallbladder mucosal epithelial cells (P ＜ 0.05).After using siRNA to shut down the YAP1 expression,EMT associated proteins were down-regulated,cell vitality was decreased,and cell cycle was arrested in the S-phase.Conclusions YAP1 is closely related to cell proliferation and metastasis of gallbladder carcinoma.It may promote tumor progression through epithelial-mesenchymal transition.transition;Tumor progress

Myeloid derived suppressor cell (MDSC) is a kind of myogenic stem cells that are induced by tumor-derived cytokines.MDSCs not only have their own immunosuppressive effects,but also can mediate the immunosuppressive effects of T lymphocytes through different mechanisms.Recent studies have shown that MDSCs involved in tumor immune escape,immune tolerance as well as other pathological processes,and played an important role in promoting the generation and development of tumors.The advances of the generation,immunosuppressive effects of MDSCs and their relation with digestive system tumors were summarized in this paper,which would provide evidences for the clinical use of MDSCs in treating digestive system tumors.