Saponin and Ribosome-Inactivating Protein Synergistically Trigger Lysosome-Dependent Apoptosis by Inhibiting Lysophagy: Potential to Become a New Antitumor Strategy.

The susceptibility of lysosomal membranes in tumor cells to cationic amphiphilic drugs (CADs) enables CADs to induce lysosomal membrane permeabilization (LMP) and trigger lysosome-dependent cell death (LDCD), suggesting a potential antitumor therapeutic approach. However, the existence of intrinsic lysosomal damage response mechanisms limits the display of the pharmacological activity of CADs. In this study, we report that low concentrations of QS-21, a saponin with cationic amphiphilicity extracted from Quillaja Saponaria tree, can induce LMP but has nontoxicity to tumor cells. QS-21 and MAP30, a type I ribosome-inactivating protein, synergistically induce apoptosis in tumor cells at low concentrations of both. Mechanistically, QS-21-induced LMP helps MAP30 escape from endosomes or lysosomes and subsequently enter the endoplasmic reticulum, where MAP30 downregulates the expression of autophagy-associated LC3 proteins, thereby inhibiting lysophagy. The inhibition of lysophagy results in the impaired clearance of damaged lysosomes, leading to the leakage of massive lysosomal contents such as cathepsins into the cytoplasm, ultimately triggering LDCD. In summary, our study showed that coadministration of QS-21 and MAP30 amplified the lysosomal disruption and can be a new synergistic LDCD-based antitumor therapy.

A high hydrophobic moment arginine-rich peptide screened by a machine learning algorithm enhanced ADC antitumor activity.

Cell-penetrating peptides (CPPs) with better biomolecule delivery properties will expand their clinical applications. Using the MLCPP2.0 machine algorithm, we screened multiple candidate sequences with potential cellular uptake ability from the nuclear localization signal/nuclear export signal database and verified them through cell-penetrating fluorescent tracing experiments. A peptide (NCR) derived from the Rev protein of the caprine arthritis-encephalitis virus exhibited efficient cell-penetrating activity, delivering over four times more EGFP than the classical CPP TAT, allowing it to accumulate in lysosomes. Structural and property analysis revealed that a high hydrophobic moment and an appropriate hydrophobic region contribute to the high delivery activity of NCR. Trastuzumab emtansine (T-DM1), a HER2-targeted antibody-drug conjugate, could improve its anti-tumor activity by enhancing targeted delivery efficiency and increasing lysosomal drug delivery. This study designed a new NCR vector to non-covalently bind T-DM1 by fusing domain Z, which can specifically bind to the Fc region of immunoglobulin G and effectively deliver T-DM1 to lysosomes. MTT results showed that the domain Z-NCR vector significantly enhanced the cytotoxicity of T-DM1 against HER2-positive tumor cells while maintaining drug specificity. Our results make a useful attempt to explore the potential application of CPP as a lysosome-targeted delivery tool.

Effects of motivational interviewing intervention on psychological status, compliance behavior and quality of life of patients with malignant tumor combined with diabetes mellitus.

Objective: To investigate the effects of motivational interview education on psychological status, compliance behavior and quality of life in patients with malignant tumors combined with diabetes mellitus. Methods: This is a retrospective study. Eighty patients with malignant tumors combined with diabetes mellitus admitted at The Fourth Hospital of Hebei Medical University from January 2021 to June 2022 were included as subjects and divided into observation group and control group according to the intervention measures. Patients in the control group were given routine health education intervention, while those in the observation group were given motivational interviewing intervention on the basis of the control group. We compared the prognosis, cognitive function, quality of life, relief of cancer pain before intervention and three months after the intervention of the two groups were compared. Results: At three months after the intervention, the total remission rate of cancer pain in the observation group was higher than that in the control group(p<0.05), while the levels of FBG and 2hPG in the observation group were significantly lower than those in the control group(p<0.05). Self-Rating Anxiety Scale(SAS) and Self-rating depression scale(SDS) scores decreased in both groups three months after the intervention, with the level of reduction in the observation group being higher than that in the control group(p<0.05). The overall compliance was higher in the observation group than in the control group(p<0.05). Conclusion: Motivational interviewing leads to alleviate negative emotions, improve the psychological status, enhance compliance behavior and improve quality of life in patients with malignant tumors combined with diabetes mellitus.

Raddeanin A synergistically enhances the anti-tumor effect of MAP30 in multiple ways, more than promoting endosomal escape.

Biomacromolecules such as proteins and nucleic acids are very attractive due to their high efficiency and specificity as cancer therapeutics. In fact, the endocytosed macromolecules are often trapped in the endosomes and cannot exhibit pharmacological effects well. Many strategies have been used to address this bottleneck, and one promising approach is to exploit the endosomal escape-promoting effect of triterpenoid saponins to aid in the release of biomacromolecules. Here, Raddeanin A (RA, an oleanane-type triterpenoid saponin) was proved to significantly promote endosomal escape as it recruited Galectin-9, an endosomal escape event reporter. As expected, RA effectively enhanced the anti-tumor effect of MAP30 (a type I ribosome-inactivating protein derived from Momordica charantia). However, based on the results of fluorescent colocalization, RA did not significantly promote MAP30 release from endosomes, suggesting that RA enhances MAP30 activity not only by promoting endosomal escape. Furthermore, it was found that the inhibitors of micropinocytosis and caveolae could almost completely inhibit the cytotoxicity of MAP30 combined with RA without affecting the cytotoxicity of MAP30 alone, indicating that RA may regulate the endocytic pathway of MAP30. Meanwhile, the effect of RA is related to the intra vesicular pH and cholesterol content on cell membrane, and is also cell-type dependent. Therefore, RA enhanced the anti-tumor effect of MAP30 in multiple ways, not just by promoting endosomal escape. Our findings will help to further decipher the possible mechanisms by which triterpenoid saponins enhance drug activity, and provide a new perspective for improving the activity of endocytosed drugs.

Liraglutide Attenuates Restenosis After Vascular Injury in Rabbits With Diabetes Via the TGF-ß/Smad3 Signaling Pathway.

Background: Lower limb ischemia due to arterial stenosis is a major complication in patients with diabetes mellitus (DM). Liraglutide is a long-acting analogue of a glucagon-like peptide 1 (GLP-1) receptor agonist used for lowering blood glucose in patients with DM, and is believed to possess cardiovascular protective effects. The aim of this study was to investigate whether liraglutide has a protective effect on blood vessels and alleviates vascular intimal hyperplasia in streptozotocin (STZ)-induced rabbits with DM and its molecular mechanism. Methods: Rabbits with DM were induced by STZ, and a lower limb ischemia model was established. The animals were divided into a control group, DM-injury group and liraglutide treatment group. Pathological staining was used to observe the intimal growth, analyze the oxidation levels of malondialdehyde (MDA), superoxide dismutase (SOD) and plasma glutathione peroxidase (GSH-Px), and analyze the changes in expression of marker proteins and signaling pathway proteins by Western blotting. A hyperglycemia (HG)-injured vascular smooth muscle cells (VSMCs) model was established to analyze reactive oxygen species (ROS) levels, Cell-Counting Kit-8 (CCK-8) was used to analyze cell proliferation, scratch assay and Transwell Migration Assay to analyze cell migration, flow cytometry to analyze apoptosis and Western blotting was used to analyze changes in the expression of marker and signaling pathway proteins. Results: The results of pathological staining showed that intimal hyperplasia was severe after diabetes-induced lower limb ischemia in rabbits at 4 weeks, and liraglutide treatment reduced symptoms. Liraglutide treatment significantly decreased MDA content, increased SOD, GSH-Px content, and augmented total antioxidant capacity levels in tissues. The results of Western blotting analysis showed that E-cadherin, mitochondrial membrane potential 9 (MMP-9), proliferating cell nuclear antigen (PCNA), and type I collagen protein expression levels were significantly decreased after liraglutide treatment compared with the DM injury group. The results indicated that liraglutide inhibited epithelial-mesenchymal transition (EMT) progression, vascular cell proliferation and migration and collagen production. Liraglutide inhibits transforming growth factor beta 1 (TGF-ß1)/Smad3 signaling pathway protein expression. In vitro assays have shown that liraglutide reduces cellular ROS levels, inhibits cell proliferation and migration and promotes apoptosis. Liraglutide down-regulated the expression of E-cadherin, MMP-9, PCNA, type I collagen protein as well as the TGF-ß1/Smad3 signaling pathway, but this effect could be reversed by tumor necrosis factor alpha (TNF-α). Conclusion: Liraglutide can significantly improve tissue antioxidant capacity, reduce vascular cell proliferation and migration via the TGF-ß1/Smad3 signaling pathway, inhibit the EMT and collagen production processes, and alleviate hyperglycemia(HG)-induced lower limb ischemia and intimal hyperplasia.

Effective Therapeutic Drug Delivery by GALA3, an Endosomal Escape Peptide with Reduced Hydrophobicity.

Endosomal escape is a rate-limiting step in the cytosolic delivery of therapeutic drugs. Overcoming this barrier is crucial to achieve an effective biological based therapy. In this work, we evaluated the ability of a synthetic biomimetic peptide derived from the GALA to facilitate endosomal escape of protein drugs. Our results showed that the cytoplasmic distribution of GALA fusion proteins changed according to the hydrophobicity of GALA. One of the synthetic peptides, GALA3, significantly enhanced the endosomal escape efficiency of protein drugs. The cytosolic delivery capacity of GALA3 was significantly higher than that of several previously reported endosomal escape peptides, including hemagglutinin 2 (HA2). Moreover, when GALA3 was fused to BLF1-HBP, a ribosome-inactivating protein with cell-penetrating peptide HBP, the cytotoxicity of the fusion protein was significantly increased in various cell lines, including H460, HeLa, A549, and SMCC-7721. The growth inhibition effect of GALA3-BLF1-HBP was at least 20 times greater than that of BLF1-HBP alone in different tumor cell lines. GALA3 effectively promoted the endosomal escape of BLF1-HBP in a pH-dependent manner and greatly enhanced the apoptotic activity of BLF1-HBP. Taken together, our data show that by adjusting the hydrophobicity of GALA we obtained a more effective endosomal escape peptide. Therefore, GALA3-fusions can improve the efficiency of therapeutic protein drugs.

Analysis of Triterpenoid Saponins Reveals Insights into Structural Features Associated with Potent Protein Drug Enhancement Effects.

Plant-based saponins are amphipathic glycosides composed of a hydrophobic aglycone backbone covalently bound to one or more hydrophilic sugar moieties. Recently, the endosomal escape activity of triterpenoid saponins has been investigated as a potentially powerful tool for improved cytosolic penetration of protein drugs internalized by endocytic uptake, thereby greatly enhancing their pharmacological effects. However, only a few saponins have been studied, and the paucity in understanding the structure-activity relationship of saponins imposes significant limitations on their applications. To address this knowledge gap, 12 triterpenoid saponins with diverse structural side chains were screened for their utility as endosomolytic agents. These compounds were used in combination with a toxin (MAP30-HBP) comprising a type I ribosome-inactivating protein fused to a cell-penetrating peptide. Suitability of saponins as endosomolytic agents was assessed on the basis of cytotoxicity, endosomal escape promotion, and synergistic effects on toxins. Five saponins showed strong endosomal escape activity, enhancing MAP30-HBP cytotoxicity by more than 106 to 109 folds. These saponins also enhanced the apoptotic effect of MAP30-HBP in a pH-dependent manner. Additionally, growth inhibition of MAP30-HBP-treated SMMC-7721 cells was greater than that of similarly treated HeLa cells, suggesting that saponin-mediated endosomolytic effect is likely to be cell-specific. Furthermore, the structural features and hydrophobicity of the sugar side chains were analyzed to draw correlations with endosomal escape activity and derive predictive rules, thus providing new insights into structure-activity relationships of saponins. This study revealed new saponins that can potentially be exploited as efficient cytosolic delivery reagents for improved therapeutic drug effects.

Screening and characterization of a novel high-efficiency tumor-homing cell-penetrating peptide from the buffalo cathelicidin family.

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell-penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo-derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell-penetrating antimicrobial peptides, including the well-known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC-7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC-5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia-derived type-I ribosome-inactivating protein MAP 30, and the cytotoxicity of the MAP 30-CAT fusion protein in the tumor cell line SMMC-7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30-CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC-5 was approximately twofold higher than the value for SMMC-7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38-fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor-homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.

Enhanced anticancer effect of MAP30-S3 by cyclosproin A through endosomal escape.

Cyclosporin A (CsA) is a calcium antagonist and can enhance the efficacy of some protein drugs, but its mechanism remains unknown. In this study, MAP30, a ribosome-inactivating protein reported to have apoptotic effects on cancer cells, was fused with S3, an epidermal growth factor receptor (EGFR)-targeting peptide. In addition, CsA was used to investigate whether it can further promote the apoptotic effects of S3 fused MAP30 (MAP30-S3). Our result showed that the internalization of FITC-labeled MAP30-S3 was increased significantly by S3 in HeLa cells. Unexpectedly, MAP30-S3 only showed a minor decrease in the viability of EGFR-overexpressing cancer cells, including HeLa, SMMC-7721, and MGC803 (IC50>5 µmol/l). However, 2 µmol/l CsA significantly increased the cytotoxicity of MAP30-S3, especially for HeLa cells (IC50=40.3 nmol/l). In comparison, CsA did not further decrease the cytotoxicity of MAP30-S3 on MRC-5, an EGFR low-expressing cell line from normal lung tissue, indicating that CsA did not affect the cancer-targeting specificity of MAP30-S3. Our results also showed that CsA further increased the apoptotic activity of MAP30-S3 in HeLa cells. CsA could promote the endosomal escape of FITC-MAP30-S3 with a diffused pattern in the cytoplasm. Five endocytic inhibitors were used to investigate the cellular uptake mechanism of MAP30-S3, and the results showed that the endosomal escape-enhancing effect of CsA on MAP30-S3 may be associated with the clathrin-dependent endocytic pathways. Our study suggested that CsA could be a novel endosomal escape enhancer to potentiate the intracellular release of anticancer protein drugs, resulting in their improved therapeutic efficacy.

Effectively enhancing cytotoxic and apoptotic effects of alpha-momorcharin by integrating a heparin-binding peptide.

Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein, has attracted a great deal of attention because of its antitumor activity. However, the cytotoxicity of α-MMC is limited due to insufficient cellular internalization in cancer cells. To enhance the cytotoxicity of α-MMC, a heparin-binding domain derived from heparin-binding epidermal growth factor (named heparin-binding peptide [HBP]) was used as a cell-penetrating peptide and fused to the C-terminus of α-MMC. This novel α-MMC-HBP fusion protein was expressed and purified with a Ni2+ -resin. The N-glycosidase activity and DNase activity assay indicated that the introduction of HBP did not interfere with the intrinsic bioactivities of α-MMC. HBP was able to efficiently carry α-MMC into the tested cancer cells and significantly enhance the cytotoxic effects of α-MMC in a dose-dependent manner. This enhanced cytotoxic ability occurred due to the higher level of cell apoptosis induced by α-MMC-HBP, which was demonstrated in western blot analysis in which α-MMC-HBP triggered caspase 8, caspase 9, casapase 3, and PARP more intensely than α-MMC alone. α-MMC-HBP led to an upregulation of cleaved PARP and an increase in the Bax/Bcl-2 ratio. Our study provided a new practical way to greatly improve the antitumor activity of α-MMC, which could significantly expand the pharmaceutical applications of α-MMC.

A novel trichosanthin fusion protein with increased cytotoxicity to tumor cells.

OBJECTIVE: To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF). RESULTS: The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells. CONCLUSION: HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Effects of Endovascular Interventions on vWF and Fb Levels in Type 2 Diabetic Patients with Peripheral Artery Disease.

BACKGROUND: von Willebrand Factor (vWF) and fibrinogen (Fb) are associated with hypercoagulability and thrombosis, which are the pathology and symptom of arterial disease. This research aims to study the effects of endovascular interventions on blood vWF and Fb levels in patients with diabetic peripheral artery disease (PAD). METHODS: Totally, 66 type 2 diabetic patients with PAD (intervention group) and 26 type 2 diabetic patients without PAD (control group) were enrolled. These patients are matched at gender, age, and diabetes duration. For PAD patients, percutaneous interventions (balloon dilation or stent implantation) were performed, and blood samples were collected before, during, and after interventions. Then, enzyme-linked immunosorbent assay and prothrombin time-derived method were used to detect the levels of vWF and Fb, respectively. RESULTS: For intervention group, vWF and Fb levels in distal ischemic regions (vWF: 231.3%, Fb: 4.97 g/L) were significantly higher than that in nonischemic regions (vWF: 147.6%, Fb: 3.91 g/L, P value < 0.01). After interventions, ischemia was improved, whereas vWF and Fb levels were significantly increased (vWF: 299.2%, Fb: 5.69 g/L, P value < 0.01). During the 2 weeks after interventions, vWF and Fb levels reached a peak (vWF: 199.3%, Fb: 4.96 g/L) and then decreased gradually to lower than the preinterventional levels (vWF: 148.3%, Fb: 3.88 g/L, P value < 0.05). CONCLUSIONS: Interventions caused increases of blood vWF and Fb in the first week postintervention, leading to endothelial dysfunction and blood hypercoagulability. It suggested endothelial function protection and anticoagulant therapies should be applied to diabetic PAD patients after interventions.

Enhancing tumor-specific intracellular delivering efficiency of cell-penetrating peptide by fusion with a peptide targeting to EGFR.

Cell-penetrating peptides (CPPs) are well known as intracellular delivery vectors. However, unsatisfactory delivery efficiency and poor specificity are challenging barriers to CPP applications at the clinical trial stage. Here, we showed that S3, an EGFR-binding domain derived from vaccinia virus growth factor, when fused to a CPP such as HBD or TAT can substantially enhance its internalization efficiency and tumor selectivity. The uptake of S3-HBD (S3H) recombinant molecule by tumor cells was nearly 80 folds increased compared to HBD alone. By contrast, the uptake of S3H by non-neoplastic cells still remained at a low level. The specific recognition between S3 and its receptor, EGFR, as well as between HBD and heparan sulfate proteoglycans on the cell surface was essential for these improvements, suggesting a syngeneic effect between the two functional domains in conjugation. This syngeneic effect is likely similar to that of the heparin-binding epidermal growth factor, which is highly abundant particularly in metastatic tumors. The process that S3H entered cells was dependent on time, dosage, and energy, via macropinocytosis pathway. With excellent cell-penetrating efficacy and a novel tumor-targeting ability, S3H appears as a promising candidate vector for targeted anti-cancer drug delivery.

Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

Molecular diversity and hypoglycemic polypeptide-P content of Momordica charantia in different accessions and different seasons.

BACKGROUND: Momordica charantia (MC) has been used for treating diabetes mellitus from ancient times in Asia, Africa and South America. There are many MC accessions in local markets. Polypeptide-P as a main hypoglycemic component in MC was first studied in this experiment to illustrate the different contents in MC of different accessions and different harvesting times. RESULTS: Nineteen MC accessions collected from different regions were clustered into three groups using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers. Content of polypeptide-P in the tested MC accessions was detected by western blot (WB) method. The WB results revealed that polypeptide-P was detected in MC accessions harvested in June and July but not in September and October. Furthermore, Polypeptide-P content corresponded well with the MC accessions. CONCLUSION: Our results suggest that the MC accessions and the harvesting times or the weather during harvest play significant roles in high content of polypeptide-P.

Improving combination cancer immunotherapy by manipulating dual immunomodulatory signals with enzyme-triggered, cell-penetrating peptide-mediated biomodulators.

Immunosuppressive tumor microenvironments challenge the effectiveness of protein-based biopharmaceuticals in cancer immunotherapy. Reestablishing tumor cell immunogenicity by enhancing calreticulin (CRT) exposure is expected to improve tumor immunotherapy. Given that CRT translocation is inherently modulated by phosphorylated eIF2α, the selective inhibition of protein phosphatase 1 (PP1) emerges as an effective strategy to augment tumor immunogenicity. To harness the PP1-disrupting potential of GADD34-derived motifs and address their limited intracellular delivery, we integrated these sequences into an enzyme-triggered, cell-penetrating peptide-mediated chimeric protein scaffold. This design not only facilitates efficient cytoplasmic delivery of these immunostimulatory motifs to induce "eat-me" signaling, but also provides a versatile platform for combination immunotherapy. Fabrication of biomodulators with cytotoxic BLF1 provides additional "eat-me" signaling through phosphatidylserine exposure or that with an immunomodulatory designed ankyrin repeat protein disables "don't-find-me" signaling by antagonizing PD-L1. Notably, these bifunctional biomodulators exhibit remarkable ability to induce macrophage phagocytosis, dendritic cell maturation, and CD8+ T activation, ultimately substantially inhibiting tumor growth. This study presents a modular genetic coding strategy for PP1-centered therapies that enables seamless integration of immunostimulatory sequences into protein-based anti-tumor cocktail therapies, thereby offering novel alternatives for improving antitumor efficacy.

Evaluation of the feeding safety of Moringa (Moringa oleifera L.) in the Sprague Dawley rat.

This study aimed to evaluate the safety of Moringa by comparing the effects of different gavage doses of Moringa. The general behavior, body weight, food intake, blood indexes, serum biochemical indexes, and histopathology of rats were used to determine the safety threshold and to provide a reference for the further development and use of Moringa as animal feed. 40 Sprague Dawley rats were selected and given transoral gavage for 28 consecutive days. The T1, T2 and T3 groups were observed for general behavior, body weight, and food intake. Blood and serum biochemical indices were quantified, and histopathology was performed to evaluate the effect and safety of Moringa. The results of the toxicological test showed that (1) Only T1 groups experienced diarrhea. (2) The body weight and food intake of rats in each group were normal compared with the control group. (3) The hematological and serum biochemical indices of rats in the T1 group were significantly different from those of CK but were in the normal range; (4) The results of microscopic examination of the heart, liver, spleen, lung, and kidney of rats in each group were normal, but inflammation occurred in stomach and jejunum of rats in the T1 group, but not in the ileum. The gastrointestinal tract of rats in the T2 and T3 groups were normal. (5) No abnormal death occurred in any of the treatment groups.The results of this study revealed that gavage of Moringa homogenate at a dose of 6 g/kg BW can cause diarrhea in rats. Although there is no pathological effect on weight, food intake, blood and serum biochemical indicators in rats, there are pathological textures in the gastrointestinal tissue caused by diarrhea. Therefore, the safety threshold of Moringa homogenate should be ≤ 3 g/kg BW.

Expression level of serum miR-374a-5p in patients with acute pancreatitis and its effect on viability, apoptosis, and inflammatory factors of pancreatic acinar cells induced by cerulein.

Acute pancreatitis (AP) is one of the life-threatening diseases of the digestive system. MicroRNA has been asserted to be a regulator of AP. This paper explored the miR-374a-5p expression in AP patients and investigated the efficacy of AR42J cells. In this study, 60 healthy people, 58 MAP patients and 58 SAP patients were included, and the serum miR-374a-5p levels of the subjects were detected by RT-qPCR technology. The pancreatitis cell model was structured by stimulating AR42J cells with cerulein. Next, cell viability and apoptosis were detected by CCK-8 assay and flow cytometry. ELISA was used to measure the concentration of cytokines, such as TNF-α, IL-6, and IL-1ß. The data showed that miR-374a-5p was downregulated in samples from AP patients, while showing discriminative power for AP populations. Attenuated miR-374a-5p were negatively bound up with patients' Ranson score and APACHE II score. Besides, miR-374a-5p was declined in cerulein-treated AR42J cells and forced elevation of miR-374a-5p was beneficial to increase cell viability, and inhibit cell apoptosis and inflammation. The present study found that miR-374a-5p was reduced in AP serum samples, and up-regulated expression level of miR-374a-5p in cell models had a protective effect on cerulein-induced inhibition of cell function and inflammatory response.