H22 Cells Research Articles

Glabridin inhibits proliferation and migration in hepatocellular carcinoma by regulating multi-targets

Ethnopharmacological relevanceGlycyrrhiza uralensis Fisch. (GC) is widely utilized in traditional Chinese medicine (TCM) for its properties in Qi tonification, heat clearing, and detoxification. Within TCM theory, Qi is also implicated in tumor development. Numerous TCM formulas containing GC are used for their anti-tumor effects, and contemporary pharmacological research has demonstrated that ethyl acetate extracts (EAe) of GC, along with potential bioactive compounds like glabridin (Gla), possess anti-tumor properties. Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and a major challenge to global healthcare, with high incidence and poor prognosis. Nevertheless, the effects and mechanisms of action of Gla in inhibiting HCC have not been extensively studied. Aim of studyThis study aims to elucidate the effects and mechanisms of action of Gla against HCC by in vitro and in vivo experiments. MethodsThe inhibitory effects of ethyl acetate extract (EAe) of GC and its bioactive compounds on HCC were studied using a drug-cell interaction system equipped with UPLC-MS/MS and high-throughput screening methods in vitro. RNA sequencing (RNA-seq) and bioinformatics technologies were employed to detect the differentially expressed genes (DEGs) and pathways in HepG2 cells. The findings were further validated using quantitative real-time PCR (qPCR) and Western blot (WB) assays. Additionally, an in vivo tumor-bearing mouse model established with H22 cells was utilized to examine alterations in tumor tissues via hematoxylin-eosin (HE) staining. Immunohistochemistry was used to assess the protein expression levels of hub targets within each group. ResultsBoth in vitro and in vivo experiments demonstrated the effects of EAe against HCC, identifying Gla was one of its main bioactive compounds. Integration of RNA-seq data with clinical databases revealed that Gla inhibited HCC by up-regulating the expression levels of DUSP5, ZFP36, KLF10, and NR4A1, while down-regulating RMI2 expression. These findings were further validated by Gene Expression Omnibus (GEO), qPCR, WB and immunohistochemistry assays. ConclusionsGla regulates the expression levels of DUSP5, ZFP36, KLF10, NR4A1, and RMI2 to against HCC, providing valuable insights for the application of Gla in HCC treatment.

Mitochondria-Targeted Icaritin Nanoparticles Induce Immunogenic Cell Death in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that is resistant to chemotherapy and immunotherapy. Icaritin (ICT), a traditional Chinese medicine, has been reported as an immunoregulatory agent for treating advanced unresectable HCC. ICT induces mitophagy to cause immunogenic cell death (ICD); however, the poor bioavailability of ICT limits its therapeutic efficacy and clinical use. Therefore, this study aimed to assess the effect of using the poly(2-(N-oxide-N,N-diethylamino) ethyl methacrylate)-b-poly(ε-caprolactone) copolymer (OPDEA-PCL) to encapsulate ICT into nanoparticles (ICT NPs). OPDEA-PCL/ICT NPs colocalized with the mitochondria, promoting the ICD induction effect of ICT in mouse HCC H22 cells. In the H22 subcutaneous tumor model, intravenously injected OPDEA-PCL/ICT NPs quickly accumulated in the tumor and efficiently activated systemic anticancer immunogenicity through their effects on mitophagy. The resulting tumor suppression rate was 60%, which was significantly higher than that of free ICT and poly(ethylene glycol) (PEG)-PCL/ICT NPs. Furthermore, mouse survival was also prolonged by nearly 2-fold with OPDEA-PCL/ICT NPs compared with PBS. In summary, this approach provides valuable insights into improving the immunotherapeutic efficacy of ICT for HCC.

Co-delivery of SN38 and MEF2D-siRNA via tLyp-1-modified liposomes reverses PD-L1 expression induced by STING activation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) exhibits an immunosuppressive tumor microenvironment, leading to a low objective response rate when immune checkpoint inhibitors (ICIs) are utilized. The cGAS-STING pathway demonstrates a powerful immune stimulatory effect, nevertheless, activation of this pathway triggers an upregulation of PD-L1, which inhibits the anti-tumor function of immune cells. The present study discovered that knockdown of MEF2D by a siRNA in H22 cells decreases the expression of PD-L1. Subsequently, tLyp-1-modified liposomes were developed for the delivery of SN38 and MEF2D-siRNA. The outcomes indicated that the modification of tLyp-1 could enhance the uptake of liposomes by tumor cells. tLip/siMEF2D/SN38 liposomes can effectively knockdown the expression of MEF2D in HCC cells and reduce the expression of PD-L1 in vitro and in vivo, thereby enhancing proliferation inhibition and apoptosis induction, and effectively suppressing the growth of tumors. SN38 treatment elevated the expression of p-TBK1 and p-IRF3 in tumor tissue, signifying the activation of the cGAS-STING pathway and facilitating the maturation of dendritic cells in vitro and in vivo. At the same time, the co-delivery of MEF2D-siRNA reduced the expression of PD-L1, thereby decreasing the quantity of M2 macrophages and myeloid-derived suppressor cells (MDSCs) in tumors, increasing the number of CD4+ T cells within the tumor, and strengthening the anti-tumor immune efficacy. In conclusion, our results suggest that tLyP-1 modified, SN38- and MEF2D siRNA-loaded liposomes have the potential for the treatment of HCC and optimize the immunotherapy of HCC via STING activation.

NitraTh epitope-based neoantigen vaccines for effective tumor immunotherapy

Neoantigen vaccines represent an emerging and promising strategy in the field of tumor immunotherapy. Despite their potential, designing an effective neoantigen vaccine remains a challenge due to the current limitations in predicting CD4+ T cell epitopes with high accuracy. Here, we introduce a novel approach to neoantigen vaccine design that does not rely on computational prediction of CD4+ T cell epitopes. Utilizing nitrated helper T cell epitope containing p-nitrophenylalanine, termed “NitraTh epitope,” we have successfully engineered a series of tumor neoantigen vaccines capable of eliciting robust neoantigen-specific immune responses. With the help of NitraTh epitope, even mutations with low predicted affinity for MHC class I molecules were successfully induced to elicit neoantigen-specific responses. In H22 cell allograft and patient-derived xenograft (PDX) liver cancer mouse models, the NitraTh epitope-based neoantigen vaccines significantly suppressed tumor progression. More strikingly, through single-cell sequencing we found that the NitraTh epitope-based neoantigen vaccines regulate macrophage reprogramming and modulate macrophages to decrease the levels of the immunosuppressive molecule prostaglandin E2 (PGE2), which in turn reshapes the tumor immunosuppressive microenvironment. In summary, NitraTh epitope-based neoantigen vaccines possess the dual effects of potently activating neoantigen-specific immunity and alleviating immunosuppression, potentially providing a new paradigm for the design of tumor neoantigen vaccines.

Construction of DNA Vaccine of Extracellular Region of Vascular Endothelial Growth Factor Receptor 2 and Its Antitumor Activity in Vivo

Introduction: it constructed recombinant DNA vaccine of extracellular region of vascular endothelial growth factor receptor 2 (VEGFR2) and analyzed its in vivo activity against H22 transplanted hepatoma. Methods: the gene fragment of VEGFR2 extracellular region 1-3 was amplified, and the pcDNA3.1(+)/exVEGFR2 recombinant plasmid was constructed. The expression of exVEGFR2 was detected by Western blot. Subsequently, liposome (LP) pcDNA3.1(+)/exVEGFR2 complex (LP-pcDNA3.1(+)/exVEGFR2) was prepared and immunized to C57BL/6 mice. The cytotoxic activity mediated by cytotoxic T cells (CTLs) was analyzed by 51Cr release assay. H22 tumor bearing C57BL/6 mouse model was prepared. Quadriceps femoris muscle was injected with normal saline (saline group, AG), LP-pcDNA3.1(+) 100 μL (1 μg/μL, LP-pcDNA3.1(+) group, BG), and LP-pcDNA3.1(+)/exVEGFR2 100 μL (1 μg/μL, LP-pcDNA3.1(+)/exVEGFR2 group, CG). Tumor growth and death were recorded, and microvessel density (MVD) was evaluated by CD31 immunohistochemical staining. Results: a 1252 bp of exVEGFR2 sequence was amplified, and a specific band expressing 44 kDa molecular weight was constructed by transfecting pcDNA3.1(+)/exVEGFR2 into COS-7 cells. After immunizing C57BL/6 mice for 6 weeks, CTLs experiment suggested that LP-pcDNA3.1(+)/exVEGFR2 could effectively mediate the toxic effect of VEGFR2 positive H22 cells. The in vivo antitumor activity experiment found that as against AG and BG, the tumor volume, weight, and MVD were markedly reduced, the tumor latency time was markedly prolonged, and the average survival time was also markedly prolonged in CG (P < 0.05). Conclusion: LP-pcDNA3.1(+)/exVEGFR2 can effectively activate C57BL/6 mice to produce specific anti-VEGFR2 immune response, and then produce anti-tumor cell immunotoxicity in vitro and in vivo. Inhibiting angiogenesis can effectively prolong the survival time of H22 tumor bearing mice.

Investigation on the mechanisms of scorpion venom in hepatocellular carcinoma model mice via untargeted metabolomics profiling

Metabolic reprogramming is frequently accompanied by hepatocellular carcinoma (HCC) progression. Disrupted metabolites act as potential biomarkers and drug therapeutic targets for HCC. Peptide extract of scorpion venom (PESV) induces cytotoxic anti-proliferative effects and apoptosis in tumors. However, the action mechanisms of PESV remain unknown. This study aimed to explore the serum metabolic profiles of tumor-bearing mouse model. We generated an orthotopic HCC xenograft mouse model by implanting H22 cells into the left hepatic lobe of male C57BL/6 mice. After surgery, the mice were assigned to two groups randomly: PESV (PESV-treated 40 mg/kg daily, i.g.; n = 6) and control (treated with the solvent equally for 14 d, n = 6) groups. Based on an untargeted metabolomics approach using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry, differential metabolites were screened via univariate and multivariate data analyses. A total of 48 differential metabolites in negative ion mode and 63 in positive ion mode were identified in the serum samples. Furthermore, metabolic pathway analysis revealed that aminoacyl-tRNA biosynthesis, amino acid pathway, glutathione metabolism, protein transports, protein digestion and absorption, and cAMP signaling pathways play vital roles in PESV-induced inhibition of tumors. These findings highlight the distinct changes in the metabolic profiles of HCC-bearing mice after PESV treatment, suggesting the potential of the identified metabolic molecules as therapeutic targets for HCC.

Nanoemulsion based lipid nanoparticles for effective demethylcantharidin delivery to cure liver cancer.

Demethylcantharidin (DEM) is a widely used antitumor drug; however, its poor tumor targeting and serious organotoxicity limit its application. The aim of this study was to develop a new drug delivery system for efficient delivery of DEM. Nanoemulsion based lipid nanoparticles containing demethylcantharidin (DNLNs) were prepared by loading nanoemulsions into lipid nanoparticles. The cells proliferation, apoptosis, cycle, and uptake were investigated by Cell counting kit-8 (CCK-8), flow cytometry, and insitu fluorescence assays, respectively. Then, we established the H22 tumor-bearing mouse model to evaluate the antitumor efficacy of DNLNs and further studied its organ toxicity and distribution. DNLNs significantly inhibited the proliferation and promoted apoptosis of H22 cells, and H22 cells could take up more DNLNs. Compared with DEM, DNLNs had certain tumor-targeting properties, and the tumor inhibition rate increased by 23.24%. Moreover, DNLNs can increase white blood cell count and reduce organ toxicity. This study paves the way for nanoemulsion-based lipid nanoparticle (NLNs)-efficient DEM delivery to treat liver cancer.

Combined Effects of Anti-PD-L1 and Nanosonodynamic Therapy on HCC Immune Activation in Mice: An Investigation.

Current therapeutic strategies, including immune checkpoint blockade (ICB), exhibit limited efficacy in treating hepatocellular carcinoma (HCC). Nanoparticles, particularly those that can accumulate specifically within tumors and be activated by sonodynamic therapy (SDT), can induce immunogenic cell death (ICD); however, ICD alone has not achieved satisfactory therapeutic effectiveness. This study investigates whether combining ICB with ICD induced by nanoparticle-mediated SDT could enhance anti-tumor immunity and inhibit HCC growth. We developed an iron-based micelle nanodelivery system encapsulating the Near-Infrared Dye IR-780, which was surface-modified with a cyclic tripeptide composed of arginine-glycine-aspartic acid (cRGD). This led to the synthesis of targeted IR780@FOM-cRGD nanoparticles. These nanoparticles were specifically engineered to kill tumor cells under sonication, activate immunogenic cell death (ICD), and be used in conjunction with immune checkpoint blockade (ICB) for the treatment of hepatocellular carcinoma (HCC). The synthesized IR780@FOM-cRGD nanoparticles had an average diameter of 28.23±1.750 nm and a Zeta potential of -23.95±1.926. Confocal microscopy demonstrated that IR780@FOM-cRGD could target HCC cells while minimizing toxicity to healthy cells. Upon sonodynamic activation, these nanoparticles consumed significant amounts of oxygen and generated substantial reactive oxygen species (ROS), effectively killing tumor cells and inhibiting the proliferation, invasion, and migration of H22 cells. Hemolysis assays confirmed the in vivo safety of the nanoparticles, and in vivo fluorescence imaging revealed significant accumulation in tumor tissues. Mouse model experiments showed that combining ICB(which induced by Anti-PD-L1) with ICD (which induced by IR780@FOM-cRGD), could effectively activated anti-tumor immunity and suppressed tumor growth. This study highlights the potential of IR780@FOM-cRGD nanoparticles to facilitate tumor eradication and immune activation when used in conjunction with Anti-PD-L1 therapy. This combination represents a non-invasive, efficient, and targeted approach for the treatment of hepatocellular carcinoma (HCC). By integrating sonodynamic therapy with immunotherapy, this strategy promises to substantially improve the effectiveness of traditional treatments in combating HCC, offering new avenues for clinical application and therapeutic innovation.

Shengqing Jiangzhuo decoction regulates the immunosuppressive microenvironment via the STAT3 signaling pathway for the treatment of malignant ascites in HCC

IntroductionMalignant ascites is a common complication of advanced hepatocellular carcinoma (HCC) that seriously affects the quality of life and survival of patients and has poor clinical efficacy. In the early stages, the authors found that the combination of the Shengqing Jiangzhuo decoction (SQJZD)and Endostar can effectively alleviate the clinical symptoms of malignant ascites in patients with HCC and reduce the amount of malignant ascites. The purpose of this study was to investigate the mechanism of action of SQJZD in HCC-related malignant ascites. MethodsA malignant ascites BALB/c mouse model was established by the intraperitoneal inoculation of H22 cells. The mice were randomly divided into model, Endostar, and high-, medium-, and low-dose traditional Chinese medicine (TCM) combined with Endostar groups. Normal mice from the same batch were used as the normal group. The Endostar and TCM + Endostar groups were intraperitoneally injected with Endostar, whereas the model and normal groups were intraperitoneally injected with equal amounts of normal saline. Different doses of SQJZD were administered to each TCM group, and equal amounts of distilled water were administered to the normal, model, and Endostar groups. ResultsThe malignant ascites volume and body weight were higher in the model group than in the normal group. After treatment, the volume and body weight of mice with malignant ascites decreased, especially in the SQJZD combined with Endostar group. Compared with that in the normal group, the number of regulatory T cells (Tregs) in the peripheral blood and spleen of mice in the model group was significantly decreased; the number of regulatory dendritic cells (DCregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) in the peripheral blood were not significantly changed; and the number of DCregs, MDSCs, and TAMs in the spleen was increased. Compared with those in the model group, the numbers of Tregs, DCregs, MDSCs, and TAMs in the peripheral blood increased in the Endostar or SQJZD combined with Endostar groups, whereas the numbers of Tregs, DCregs, MDSCs, and TAMs in the spleen decreased. Western blotting revealed that the percentage of p-STAT3-positive cells in the liver and spleen of mice increased in the model group, whereas the percentage of p-STAT3-positive cells in the liver and spleen decreased in mice treated with Endostar or SQJZD combined with Endostar. ConclusionsSQJZD combined with Endostar may attenuate immunosuppression and enhance the antitumor immune response by regulating the STAT3 pathway to alleviate malignant ascites in HCC.

4-Deoxy-ε-Pyrromycinone: A Promising Drug/Lead Compound to Treat Tumors.

Anthraquinone drugs are widely used in the treatment of tumors. However, multidrug resistance and severe cardiac toxicity limit its use, which have led to the discovery of new analogues. In this paper, 4-Deoxy-ε-pyrromycinone (4-Deo), belonging to anthraquinone compounds, was first been studied with the anti-tumor effects and the safety in vitro and in vivo as a new anti-tumor drug or lead compound. The quantitative analysis of 4-Deo was established by UV methodology. The anti-cancer effect of 4-Deo in vitro was evaluated by cytotoxicity experiments of H22, HepG2 and Caco2, and the anti-cancer mechanism was explored by cell apoptosis and cycle. The tumor-bearing mouse model was established by subcutaneous inoculation of H22 cells to evaluate the anti-tumor effect of 4-Deo in vivo. The safety of 4-Deo was verified by the in vitro safety experiments of healthy cells and the in vivo safety experiments of H22 tumor-bearing mice. Tumor tissue sections were labeled with CRT, HMGB1, IL-6 and CD115 to explore the preliminary anti-cancer mechanism by immunohistochemistry. In vitro experiments demonstrated that 4-Deo could inhibit the growth of H22 by inducing cell necrosis and blocking cells in S phase, and 4-Deo has less damage to healthy cells. In vivo experiments showed that 4-Deo increased the positive area of CRT and HMGB1, which may inhibit tumor growth by triggering immunogenic cell death (ICD). In addition, 4-Deo reduced the positive area of CSF1R, and the anti-tumor effect may be achieved by blocking the transformation of tumor-associated macrophages (TAMs) to M2 phenotype. In summary, this paper demonstrated the promise of 4-Deo for cancer treatment in vitro and in vivo. This paper lays the foundation for the study of 4-Deo, which is beneficial for the further development anti-tumor drugs based on the lead compound of 4-Deo.

LEPR/FOS/JUNB signaling pathway contributes to chronic restraint stress-induced tumor proliferation

Background & aimsPsychosocial stress has become an unavoidable part of life, which was reported to promote tumor development. Chronic stress significantly promotes the norepinephrine (NE) secretion and the expression of leptin receptor (LEPR), leading to tumor invasion, metastasis, and proliferation. However, the mechanism of chronic stress-induced tumor proliferation remains unclear. MethodsTo reveal the effect of chronic stress on tumor proliferation, subcutaneous tumor models combined with chronic restraint stress (CRS) were established. Combined with the transcript omics database of liver cancer patients, the target pathways were screened and further verified by in vitro experiments. ResultsThe results showed that the CRS with subcutaneous tumor transplantation (CRS + tumor) group exhibited significantly larger tumor sizes than the subcutaneous tumor transplantation (tumor) group. Compared with the tumor group, CRS obviously increased the mRNA levels of LEPR, FOS, and JUNB of tumor tissues in the CRS + tumor group. Furthermore, the treatment with norepinephrine (NE) significantly elevated the survival rate of H22 cells and enhanced the expression of LEPR, FOS, and JUNB in vitro. Silencing LEPR significantly reduced the expression of FOS and JUNB, accompanied by a decrease in H22 cell viability. ConclusionsOur study demonstrated that CRS activates the LEPR–FOS–JUNB signaling pathway by NE, aggravating tumor development. These findings might provide a scientific foundation for investigating the underlying pathological mechanisms of tumors in response to chronic stress.

A Synergistic Combination of Oleanolic Acid and Apatinib to Enhance Antitumor Effect on Liver Cancer Cells and Protect against Hepatic Injury.

As a pentacyclic triterpenoid, OA (oleanolic acid) has exhibited antiinflammatory, immunomodulatory and antitumor effects. VEGFR-2 (vascular endothelial cells receptor-2) tyrosine kinase activity could be inhibited by apatinib, a small-molecule antiangiogenic agent. Thus, this study sought to investigate the mechanism underlying the synergistic antitumor activity of combined OA and apatinib patent. Through CCK8 (Cell counting kit 8 assay), flow cytometric and western blotting techniques, we conducted in vitro studies on apatinib and OA effects on cell proliferation and apoptosis in H22 cell line. H22 tumor-burdened mice model was established in vivo, while the related signaling pathways were studied via pathological examination, western blotting and qPCR (quantitative polymerase chain reaction). Growth of H22 cells in vitro and in vivo could be inhibited effectively by apatinib and OA. Thus, OA repaired liver function and inhibited oxidative stress induced by apatinib. OA can treat apatinib induced liver injury in H22 Tumor-burdened mice by enhancing the suppresssive effect of apatinib on the growth of tumor.

Synergistic strategies for enhanced liver cancer therapy with sorafenib/resveratrol PEGylated liposomes in vitro and in vivo

This study investigates the therapeutic efficacy of sorafenib (Sr) and resveratrol (Rv) encapsulated in liposomal formulations for liver cancer treatment. In vitro release profiles demonstrated sustained drug release, with 70 % and 56.7 % at intestinal pH of 6.8 and 79.3 % and 74.2 % at gastric pH of 1.2 after 72 h for liposomal (Sr/Rv-Lip) and PEGylated liposomal (Sr/Rv-PEG-Lip) formulations. pH-dependent release patterns highlighted adaptability of these formulations to distinct physiological conditions, crucial for targeted drug delivery. Stability assessments over three months revealed a marginal 8.5 % decrease in Sr/Rv-PEG-Lip encapsulation efficiency, accompanied by nonsignificant alterations in size, zeta potential, and polydispersity index (PDI). These indicate the formulations' capability to preserve physicochemical properties, offering promising enhanced stability for Sr and Rv. In vitro studies demonstrated superior anticancer effects of Sr/Rv-PEG-Lip with an IC50 of 5.1 μM, showing 3.1-fold increased potency, compared to Sr/Rv in H22 cells, demonstrating enhanced cytotoxicity. Sr/Rv-PEG-Lip, compared to Sr/Rv, was 50.5 and 33.1 % more potent in inhibiting proliferation and migration of H22 cells, respectively. The reactive oxygen species (ROS) assay underscored the synergistic potential of Sr and Rv, with PEGylation amplifying oxidative stress induction by 1.4-fold. In vivo, Sr/Rv-PEG-Lip demonstrated superior efficacy in suppressing tumor growth by reducing the tumor volume to 380.4 mm³ and inhibiting the weight gain by 22.7 % with minimal toxicity, demonstrating its translational potential in liver cancer therapy. These findings contribute valuable insights into nanomedicine, suggesting Sr/Rv-loaded PEGylated liposomes as a promising avenue for liver cancer treatment.

Comparison of the components of fresh Panax notoginseng processed by different methods and their anti-anemia effects on cyclophosphamide-treated mice

Ethnopharmacological relevanceThe traditional Chinese herb Panax notoginseng (PN) tonifies blood, and its main active ingredient is saponin. PN is processed by different methods, resulting in different compositions and effects. Aim of the studyTo investigate changes in the microstructure and composition of fresh PN processed by different techniques and the anti-anemia effects on tumor-bearing BALB/c mice after chemotherapy with cyclophosphamide (CTX). Materials and methodsFresh PN was processed by hot-air drying (raw PN, RPN), steamed at 120 °C for 5 h (steamed PN, SPN), or fried at 130 °C, 160 °C, or 200 °C for 8 min (fried PN, FPN1, FPN2, or FPN3, respectively); then, the microstructures were compared with 3D optical microscopy, quasi-targeted metabolites were detected by liquid chromatography tandem mass spectrometry (LC‒MS/MS), and saponins were detected by high-performance liquid chromatography (HPLC). An anemic mouse model was established by subcutaneous H22 cell injection and treatment with CTX. The antianemia effects of PN after processing via three methods were investigated by measuring peripheral blood parameters, performing HE staining and measuring cell proliferation via immunofluorescence. Results3D optical profiling revealed that the surface roughness of the SPN and FPN was greater than that of the other materials. Quasi-targeted metabolomics revealed that SPN and FPN had more differentially abundant metabolites whose abundance increased, while SPN had greater amounts of terpenoids and flavones. Analysis of the composition and content of the targeted saponins revealed that the contents of rare saponins (ginsenoside Rh1, 20(S)-Rg3, 20(R)-Rg3, Rh4, Rk3, Rg5) were greater in the SPN. In animal experiments, the RBC, WBC, HGB and HCT levels in peripheral blood were increased by SPN and FPN. HE staining and immunofluorescence showed that H-SPN and M-FPN promoted bone marrow and spleen cell proliferation. ConclusionThe microstructure and components of fresh PN differed after processing via different methods. SPN and FPN ameliorated CTX-induced anemia in mice, but the effects of PN processed by these two methods did not differ.

Abstract 5629: MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas

Abstract Background: Immune escape remains a challenge in the treatment of hepatocellular carcinoma (HCC), one of the most common and deadly cancers in the world. The transcription factor, myocyte enhancer factor 2D (MEF2D), is important in the regulation of differentiation and adaptive responses in many cancers, such that we tested its role in HCC. Methods: We knocked down MEF2D in HCC cell lines and analyzed HCC tissues and cell lines by RNA sequencing, Western blot and immunohistochemistry. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation assay. Chromatin immunoprecipitation was used to analyze the regulation of CD70 transcription by MEF2D. H22 cells, with MEF2D knockout or without (controls), were injected into the livers of syngeneic BALB/c mice. Flow cytometry was used to analyze the function of T cells in tumors, spleens, and lymph nodes. Results: Through database searches, we noted that MEF2D and CD70 were upregulated in HCC and associated with shorter patient survival times. Compared to WT tumors, injection of MEF2D-knockdown HCC cells into immunocompetent mice led to smaller tumors with increased T cell infiltration, activation, and impaired T-regulatory (Treg) cell suppressive function. Mechanistically, we found that MEF2D binds to the promoter region of CD70 gene and activates its transcription. Moreover, acetylation of MEF2D enhances the binding of MEF2D to the CD70 promoter and further promotes its transcription. CD70 blocking antibody inhibited activation of the CD70-CD27 signaling axis in murine HCC tumors, leading to impaired immunosuppressive function of Tregs and enhanced T cell-mediated anti-tumor immune responses. Thus, regulation of the CD70-CD27 signaling axis by MEF2D affects the numbers and functions of Treg cells and thereby controls T cell infiltration. Conclusions: MEF2D in HCC cells increases the expression of CD70 and blocks T cell-mediated anti-tumor immunity via the CD70-CD27 signaling axis. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC. Citation Format: Fanhua Kong, Liqing Wang, Qifa Ye, Yan Xiong, Wayne W. Hancock. MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5629.

Sinomenine: A Protential Biomaterial for Its Antitumor Effect in H22 Hepatoma-Bearing Mice and Its Mechanisms

Sinomenine (SIN) is a natural isoquinoline alkaloid extracted from the Chinese medicinal plant Sinomenium acutum with promising antitumor properties. Although its antitumor effect and mechanism have been studied, its effects on the structure and function of tumor cell membranes remain unclear. This study aimed to evaluate the antitumor effect of SIN on H22 hepatoma-bearing mice and its mechanism of action on tumor cell membranes, and the tumor inhibition rate and life extension rate were examined. Flow cytometry was used to evaluate the dose-dependent induction of apoptosis in H22 cells. The main components of tumor cell membrane, including total protein, cholesterol and sialic acid (SA) were characterized. The ultrastructure of cell membrane was observed using transmission electron microscopy (TEM). Furthermore, SIN’s effect on the function of cell membranes was studied by analyzing fluidity, sealing, and ion pump activity. The results demonstrated that SIN effectively inhibited the growth of liver cancer cells in vivo and prolonged the lifespan of H22 hepatoma-bearing mice. SIN induced apoptosis of H22 cells in a dose-dependent manner, significantly reduced total protein, cholesterol, and SA contents in H22 cell membranes, and significantly decreased the fluidity, sealing, Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities of H22 cell membranes with increasing doses. Overall, these findings suggested that SIN reduced the main component contents of H22 cell membranes, leading to changes in structure and function of cell membrane, ultimately resulting in tumor cell apoptosis and inhibition of tumor growth. The findings indicated that SIN has great potential as a biomaterial for anti-liver cancer drugs, and provides new ideas and methods for developing antitumor drugs through membrane pharmacology.

Hybridization-based discovery of novel quinazoline-2-indolinone derivatives as potent and selective PI3Kα inhibitors

IntroductionPhosphatidylinositol 3-kinases (PI3Ks) overexpression can elicit cellular homeostatic dysregulation, which further contributes to tumorigenesis, with PI3Kα emerging as the most prevalent mutant isoform kinase among PI3Ks. Therefore, selective inhibitors targeting PI3Kα have attracted considerable interest in recent years. Molecular hybridization, with the advantage of simplified pharmacokinetics and drug-drug interactions, emerged as one of the important avenues for discovering potential drugs. ObjectivesThis study aimed to construct PI3Kα inhibitors by hybridization and investigate their antitumor activity and mechanism. Methods26 quinazoline-2-indolinone derivatives were obtained by molecular hybridization, and their structure–activity relationship was analyzed by MTT, in vitro kinase activity and molecular docking. The biological evaluation of compound 8 was performed by transwell, flow cytometry, laser scanning confocal microscopy, Western blot, CTESA and immunohistochemistry. ResultsHere, we employed molecular hybridization methods to construct a series of quinazoline-2-indolinone derivatives as PI3Kα selective inhibitors. Encouragingly, representative compound 8 exhibited a PI3Kα enzymatic IC50 value of 9.11 nM and 10.41/16.99/37.53-fold relative to the biochemical selectivity for PI3Kβ/γ/δ, respectively. Moreover, compound 8 effectively suppressed the viability of B16, HCT116, MCF-7, H22, PC-3, and A549 cells (IC50 values: 0.2 μM ∼ 0.98 μM), and dramatically inhibited the proliferation and migration of NSCLC cells, as well as induced mitochondrial apoptosis through the PI3K/Akt/mTOR pathway. Importantly, compound 8 demonstrated potent in vivo anti-tumor activity in non-small cell lung cancer mouse models without visible toxicity. ConclusionsThis study presented a new avenue for the development of PI3Kα inhibitors and provided a solid foundation for novel QHIDs as potential future therapies for the treatment of NSCLC.

Drug-Loaded Tumor-Derived Microparticles Elicit CD8+ T Cell-Mediated Anti-Tumor Response in Hepatocellular Carcinoma.

Hepatocellular Carcinoma (HCC) poses significant challenges due to limited effective treatments and high recurrence rates. Immunotherapy, a promising approach, faces obstacles in HCC patients due to T-cell exhaustion and immunosuppression within the tumor microenvironment. Using doxorubicin-loaded tumor-derived microparticles (Dox-TMPs), the mice with H22 ascites model and subcutaneous tumors model were treated. Following the treatment, mice were re-challenged with H22 cells to compare the therapeutic effects and recurrence among different groups of mice, alongside examining the changes in the proportions of immune cells within the tumor microenvironment. Furthermore, Dox-TMPs were combined with anti-PD-1 to further validate their anti-tumor efficacy. In vitro studies using various liver cancer cell lines were conducted to verify the tumor-killing effects of Dox-TMPs. Additionally, CD8+ T cells from the abdominal cavity of tumor-free mice were co-cultured with H22 cells to confirm their specific tumor-killing abilities. Dox-TMPs demonstrate effective anti-tumor effects both in vitro and in vivo. In vivo, their effectiveness primarily involves enhancing CD8+ T cell infiltration, alleviating T cell immunosuppression, and improving the immune microenvironment to combat tumors. When used in combination with anti-PD-1, their anti-tumor effects are further enhanced. Moreover, some mice treated with Dox-TMPs developed anti-tumor immunity, displaying a self-specific T-cell immune response upon re-challenged with tumor cells. This suggests that Dox-TMPs also have the potential to act as a long-term immune response against tumor recurrence, indicating their capability as a tumor vaccine. Dox-TMPs exhibit a dual role in liver cancer by regulating T cells within the tumor microenvironment, functioning both as an anti-tumor agent and a potential tumor vaccine.

Knockdown of circZMIZ1 enhances the anti-tumor activity of CD8+ T cells to alleviate hepatocellular carcinoma.

ZMIZ1 acts as an oncogene in hepatocellular carcinoma (HCC). circZMIZ1 (hsa_circ_0018964) derives from ZMIZ1; its underlying mechanism in HCC has not been reported. Peripheral blood and peripheral blood mononuclear cells (PBMCs) were obtained from HCC patients and healthy volunteers. CD8+ T cells were sorted from PBMCs of HCC patients. Applying flow cytometry, cell apoptosis and the proportion of KCNJ2/CD8+ T cells were examined. The cytotoxicity of CD8+ T cells against HCC cells was evaluated. The interaction among circZMIZ1, miR-15a-5p, and KCNJ2 was investigated by dual luciferase assay, RNA immunoprecipitation, and RNA pull-down assay. An orthotopic mouse model of HCC was constructed by intrahepatic injection of H22 cells. Upregulation of circZMIZ1 and KCNJ2 and downregulation of miR-15a-5p were observed in peripheral blood and PBMCs of HCC patients. The proportion of KCNJ2/CD8+ T cells was also increased in HCC patients. circZMIZ1 knockdown restrained apoptosis of CD8+ T cells and elevated cytotoxicity of CD8+ T cells. Mechanically speaking, circZMIZ1 elevated KCNJ2 expression by sponging miR-15a-5p. miR-15a-5p inhibitor reversed circZMIZ1 silencing-mediated inhibition of apoptosis and promotion of cytotoxicity in CD8+ T cells. In vivo, orthotopic mice of HCC exhibited increased expression of circZMIZ1 and KCNJ2, elevated proportion of KCNJ2/CD8+ T cells, and decreased expression of miR-15a-5p. This work demonstrated that circZMIZ1 inhibited the anti-tumor activity of CD8+ T cells in HCC by regulating the miR-15a-5p/KCNJ2 axis. This provides a theoretical basis for the development of effective circZMIZ1 in tumor immunotherapy.

Preliminary Study on Pharmacokinetics and Antitumor Pharmacodynamics of Folic Acid Modified Crebanine Polyethyleneglycol-Polylactic Acid Hydroxyacetic Acid Copolymer Nanoparticles.

Liver cancer is associated significantly with morbidity and mortality. The combination of low-intensity ultrasound with nanomedicine delivery systems holds promise as an alternative for the treatment for liver cancer. This study focuses on the utilization of folic acid (FA) modified nanoparticles, which are loaded with fluorescent dye DiR and liquid fluorocarbon (PFP). These nanoparticles have the potential to enhance liver cancer targeting under ultrasound stimulation and future applications in vivo. The pharmacokinetics and tissue distribution of folic acid-modified Crebanine polyethylene glycol-polylactic acid copolymer nanoparticles (FA-Cre@PEG-PLGA NPs) were investigated. The pharmacokinetic parameters, liver targeting, and in vivo distribution were assessed. Additionally, the inhibitory impacts of FA-Cre@PEG-PLGA NPs in combination with ultrasonic irradiation on the proliferation and acute toxicity of H22 cells of mouse hepatoma were investigated in vitro. The tumor targeting and anti-tumor efficacy of FA-Cre@PEG-PLGA NPs were assessed utilizing a small animal in vivo imaging system and an in situ hepatocellular carcinoma transplantation model, respectively. The pharmacokinetic studies and tissue distribution tests demonstrated that FA-Cre@PEG-PLGA NPs conspicuously prolonged the half-life and retention time of the drug in rats, and the liver targeting effect was pronounced. Additionally, the in vivo acute toxicity test indicated that FA-Cre@PEG-PLGA NPs had minimal adverse reactions and could fulfill the aim of attenuating the drug. The outcomes of the animal experiments further substantiated that FA-Cre@PEG-PLGA NPs had a longer retention time at the tumor site, a superior anti-tumor effect, and less damage to liver and kidney tissue. The integration of FA-Cre@PEG-PLGA NPs with ultrasound irradiation demonstrated exceptional safety and potent anti-tumor efficacy in vivo, presenting a promising therapeutic strategy for the treatment of liver cancer through the combination of ultrasound technology with a nanomedicine delivery system.