Nude Mice Research Articles

Pyroptosis-related gene GSDMC indicates poor prognosis and promotes tumor progression by activating the AKT/mTOR pathway in lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is one of the most common malignant tumors of the respiratory. Pyroptosis plays an essential role in cancer, but there is limited research investigating pyroptosis in LUSC. In this study, pyroptosis-related genes were observed to have extensive multiomics alterations in LUSC through analysis of the TCGA database. Utilizing machine learning for selection and verifying expression levels, GSDMC was chosen as the critical gene for further experiments. Our research found that GSDMC is overexpressed in LUSC tissues and cells, and is associated with poor prognosis. Knockdown of GSDMC in LUSC inhibits cell proliferation, invasion, metastasis, chemotherapeutic sensitivity, and reduced tumor formation in nude mice, accompanied by downregulation of proliferative and EMT-related protein expression. However, these effects were counteracted in cells where GSDMC is overexpressed. Mechanistically, the oncogenic role of GSDMC is primarily achieved through the activation of the AKT/mTOR pathway, and this effect can be significantly reversed by rapamycin. Finally, SMAD4's interaction with the promoter region of GSDMC results in the suppression of GSDMC expression. In summary, our study through bioinformatics and experimental approaches not only proves that SMAD4 regulates the protumorigenic role of GSDMC through transcriptional targeting, but also indicates the possibility of developing the SMAD4/GSDMC/AKT/mTOR signaling axis as a potential biomarker and treatment target for LUSC.

Areca nut extract may be a potentially driving force for tumors to become autophagy addicted

We have previously described the mechanisms of action and composition of the autophagy-inducing ingredients found in areca nut (AN). These ingredients are 30–100 kDa molecules present in AN extract (ANE), referred to as ANE 30–100K. In other studies, we demonstrated that chronic stimulation with sub-lethal doses of ANE or ANE 30–100K resulted in increased tolerance to environmental challenges, including serum deprivation, hypoxic conditions, anti-cancer drugs, and accelerated tumor growth in nude mice, through upregulated autophagy activity. Such tumor cells were highly sensitive to the chemical inhibition of autophagy both in vitro and in vivo. Here, we further demonstrated similar inhibitory effects on the growth of stimulated CE81T/VGH cells in mice using atg5 shRNA. In contrast, the growth of non-stimulated control cells in mice was shown to be resistant to 3-methyladenine (3-MA). These findings suggest that autophagy blockade might be particularly effective for treating autophagy-dependent tumors in patients with AN-chewing habits.

MTHFD1 Regulates Autophagy to Promote Growth and Metastasis in Colorectal Cancer via the PI3K-AKT-mTOR Signaling Pathway.

Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) is the enzyme with the activities of methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase. Our aim was to elucidate the function of MTHFD1 in colorectal cancer (CRC). In vitro assessments of the proliferation, invasion, and migration abilities of CRC cells were conducted using Immunohistochemistry, Transwell invasion assays, Western blot (WB), and Cell counting Kit-8 assays. WB was also utilized to measure autophagy protein levels and PI3K-AKT-mTOR signaling pathway expression. Furthermore, the role of MTHFD1 was evaluated invivo by using subcutaneous xenograft tumor models and lateral tail vein metastasis models of human CRC in nude mice. Overexpression of MTHFD1 promoted the abilities of tumorigenesis and metastasis in CRC invitro and invivo and reduced autophagy, attributing to the PI3K-AKT-mTOR signaling pathway in CRC cells. In contrast, the down-regulation of MTHFD1 increased autophagy and suppressed their proliferation, migration, and invasion. MTHFD1 can modulate the PI3K-AKT-mTOR signaling pathway to suppress autophagy and stimulate tumorigenesis and metastasis.

Inhibition of mitochondrial OMA1 ameliorates osteosarcoma tumorigenesis

OMA1 is an ATP-independent zinc metalloprotease essential for maintaining mitochondrial homeostasis and plays a vital role in tumorigenesis. Depending on the type of cancer, a decrease in OMA1 expression has been linked to a varying prognosis for patients. The role of OMA1 in human osteosarcoma (OS), one of the most prevalent malignant bone tumors, remains elusive. Here, we observed elevated OMA1 expression in OS tumor tissues from four patients with advanced OS. Knockout of OMA1 in OS cells significantly reduces OS tumor weight and size, and lung metastatic nodules in BALB/c nude mice. Immunohistochemistry analysis showed a significant decrease in Ki67 and an increase in Cleaved-caspase 3 in OMA1 knockout tumor samples. Mechanistically, we found that OMA1 deficiency increases the levels of PINK1 and Parkin and consequently induces excessive mitophagy, leading to increased apoptosis and reduced cell proliferation and invasion in OS cells. Specifically, OMA1 deficiency reduces the amount of cytosolic p53 and p53-associated cytosolic Parkin but increases mitochondrial p53, which may lead to enhanced apoptosis. Regarding the effect on cell proliferation and invasion, loss of OMA1 reduces mitochondrial ROS levels and increases cytosolic glycogen synthase kinase 3β (GSK3β) levels, thereby increasing interaction between GSK3β and β-catenin and then reducing cytosolic and nuclear β-catenin. This contributes to reduced cell proliferation and migration in OMA1-deficient cells. Moreover, we found that ciclopirox (CPX), an antifungal drug, induces OMA1 self-cleavage and L-OMA1 degradation in cultured OS cells. CPX also reduces tumor development of control OS cells but not OMA1-deficient OS cells in mice. These findings strongly support the important role of OMA1 in OS tumorigenesis and suggest that OMA1 may be a valuable prognostic marker and a promising therapeutic target for OS.

Resveratrol promotes mitophagy via the MALAT1/miR-143-3p/RRM2 axis and suppresses cancer progression in hepatocellular carcinoma

ObjectiveResveratrol (Res) is a promising anticancer drug against hepatocellular carcinoma (HCC), but whether its anti-HCC effects implicate mitophagy remains unclear. Therefore, we aimed to explore the specific role of Res in mitophagy and the related mechanisms during the treatment of HCC. MethodsHepG2 cells and tumor-grafted nude mice were used to investigate the effects of low-, middle- and high-dose of Res on HCC progression and mitophagy in vitro and in vivo, respectively. A series of approaches including cell counting kit-8, flow cytometry, wound healing and transwell assays were used to evaluate tumor cell functions. Transmission electron microscopy, immunofluorescence and Western blotting analysis were used to assess mitophagy. Mitochondrial oxygen consumption rate, reactive oxygen species and membrane potential were used to reflect mitochondrial function. After disrupting the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), miR-143-3p, and ribonucleoside reductase M2 (RRM2), the effects of the MALAT1/miR-143-3p/RRM2 axis on cell function and mitophagy under Res treatment were explored in vitro. Additionally, dual-luciferase reporter and chromatin immunoprecipitation were used to confirm interactions between target genes. ResultsRes significantly inhibited the proliferation and promoted apoptosis of HCC cells in vitro, while significantly suppressing tumor growth in a dose-dependent manner and inducing mitophagy and mitochondrial dysfunction in vivo. Interestingly, MALAT1 was highly expressed in HCC cells and its knockdown upregulated miR-143-3p expression in HCC cells, which subsequently inhibited RRM2 expression. Furthermore, in nude mice grafted with HCC tumors and treated with Res, the expression of MALAT1, miR-143-3p and RRM2 were altered significantly. In vitro data further supported the targeted binding relationships between MALAT1 and miR-143-3p and between miR-143-3p and RRM2. Therefore, a series of cell-based experiments were carried out to study the mechanism of the MALAT1/miR-143-3p/RRM2 axis involved in mitophagy and HCC; these experiments revealed that MALAT1 knockdown, miR-143-3p mimic and RRM silencing potentiated the antitumor effects of Res and its activation of mitophagy. ConclusionRes facilitated mitophagy in HCC and exerted anti-cancer effects by targeting the MALAT1/miR-143-3p/RRM2 axis.

Toxicity and Oxidative Stress Biomarkers in the Organs of Mice Treated with Mesoporous Polydopamine Nanoparticles Modified with Iron and Coated with Cancer Cell Membrane.

Polydopamine nanoparticles (PDA NPs) have great potential in medicine. Their applications being widely investigated in cancer therapy, imaging, chemotherapy, photodynamic therapy (PDT), photothermal therapy (PTT), and tissue repair. The aim of our study was to assess the in vivo toxicity and changes in oxidative stress biomarkers in organs of animals treated with mesoporous PDA NPs modified with iron (MPDAFe NPs), coated with the cancer cell membrane and loaded with doxorubicin (DOX), and subsequently subjected to PTT. Liver and kidney homogenates were obtained from BALB/c nude mice with xenograft HepG2 human hepatoma cells, treated with iron modified mesoporous PDA nanoparticles, coated with the cancer cell membrane and loaded with doxorubicin (MPDAFe@DOX@Mem NPs), and subjected to PTT. These samples were used for histological evaluation and measurement of oxidative stress biomarkers, including total protein (TP), reduced glutathione (GSH), nitric oxide (NO), S-nitrosothiols (RSNO), thiobarbituric acid reactive substances (TBARS), trolox equivalent antioxidant capacity (TEAC), catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD). In the kidney, MPDAFe@DOX@Mem NPs in combination with PTT increased GSH (43%), TBARS (32%), and CAT (27%), while SOD decreased by 20% compared to the control group. Additionally, CAT activity in the liver increased by 79%. Significant differences in oxidative stress parameters and histological changes after administration with MPDAFe@DOX@Mem NPs and PTT were observed in the kidneys, showing more pronounced changes than the liver, indicating potential kidney toxicity. Our research provides insights into oxidative stress and possible toxic effects after in vivo administration of mesoporous PDA NPs combined with chemotherapy-photothermal therapy (CT-PTT), which is extremely important for their future applications in anticancer therapies.

The combination of FLCWK with 5-FU inhibits colon cancer and multidrug resistance by activating PXR to suppress the IL-6/STAT3 pathway.

5-fluorouracil (5-FU) is a preferred chemotherapeutic agent for the treatment of colon cancer. Nonetheless, its clinical effectiveness is frequently hampered by suboptimal therapeutic outcomes and the emergence of drug resistance. Therefore, there exists a pressing demand for novel therapeutic agents to circumvent chemoresistance. The pregnane X receptor (PXR) exerts a pivotal regulatory influence on the proliferation, invasion, and chemoresistance mechanisms in colon cancer. Activation of PXR drives up the transcription of the multidrug resistance gene (MDR1), thus prompting the expression of P-glycoprotein (P-gp) responsible for conferring tumour resistance. This study scrutinized the potential of Fengliao Changweikang (FLCWK) in augmenting the efficacy of 5-FU in the management of colon cancer. To this end, we engineered colon cancer cells with varied levels of PXR expression via lentiviral transfection, subsequently validating the findings in nude mice. By means of MTT assays, flow cytometry apoptosis analysis, Western blotting and immunofluorescence, we probed into the prospective impacts of FLCWK and 5-FU on cellular viability and resistance. Our results revealed that while upregulation of PXR amplified the therapeutic benefits in colon cancer treatment, it concurrently heightened resistance levels. FLCWK demonstrated a capacity to reduce P-gp expression, with the combined administration of FLCWK and 5-FU effectively reversing resistance mechanisms. Furthermore, activation of PXR was found to impede the IL-6/STAT3 signalling pathway. In an effort to mimic the development of colon cancer, we established an azomethane oxide (AOM)/ dextran sodium sulfate (DSS) mouse model, showing that FLCWK bolstered the inhibitory effects of 5-FU, impeding the progression of colon cancer. In summation, our findings point towards the potential of FLCWK in the treatment of colon cancer, particularly in strengthening the therapeutic efficacy of 5-FU in the prevention and control of the disease.

WITHDRAWN: Tumor cell extracellular vesicles derived long non-coding RNA X-inactive specific transcript regulates the blood–brain barrier and non-small cell lung cancer brain metastasis via miR-19b-3p/EPN2

ObjectiveTo investigate the function of tumor cell extracellular vesicles (EVs) derived long non-coding RNA X-inactive specific transcript (XIST) in non-small cell lung cancer (NSCLC) brain metastasis and to clarify its specific mechanism. MethodsEVs were isolated from A549 cells, and transmission electron microscopy, nanoparticle tracking, and western blotting were used to detect morphology and particle size of EVs. Human brain microvascular endothelial cells (HBMEC) and astrocytes were used to construct an in vitro blood–brain barrier (BBB) model. The BBB model was treated with EVs for 24 h. PKH67 staining was performed to examine EV engulfment by HBMEC and astrocytes. RT-qPCR was used to examine XIST, miR-19b-3p, and epsin 2 (EPN2) mRNA expression. Expression of EPN2 and tight junction (TJ) proteins such as claudin-5, occludin, and ZO-1 were measured using western blotting. Transepithelial electrical resistance (TEER), rhodamine-dextran, and immunofluorescence staining were performed to explore the effects of EVs and XIST on the BBB. Transwell assay was used to evaluate the invasive ability of A549 and H1299 cells. Dual-luciferase and immunoprecipitation assays were used to verify binding between miR-19b-3p, XIST, and EPN2. The Interactive Video Information System was used to observe brain metastasis of xenografts in BALB/c nude mice. ResultsA549 cell-derived EVs exhibited typical characteristics of EVs. HBMEC ingested EVs in the BBB model. EVs treatment decreased TEER and TJ protein level and increased the permeability to rhodamine-dextran. XIST was overexpressed in A549 and H1299 cells. Knockdown of XIST in A549 cell-derived EVs alleviated BBB damage and reduced NSCLC cell invasion. In vivo, XIST inhibition repressed NSCLC brain metastasis by ameliorating the BBB. Mechanistically, XIST sponged miR-19b-3p and positively regulated EPN2 expression. miR-19b-3p inhibition weakened the protective effect of XIST inhibition on the BBB. ConclusionA549 cells EVs-derived XIST inhibition ameliorates BBB injury and inhibits NSCLC brain metastasis by regulating the miR-19b-3p/EPN2 axis.

Alkannin Induces G2/M-Phase Arrest, Apoptosis, and Inhibition of Invasion by Targeting GSK3β in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most common malignant tumor of the upper gastrointestinal tract, characterized by high mortality and poor prognosis. There is an urgent need for the development of more effective drugs. Alkannin has been shown to inhibit the progression of various cancers, but its inhibitory effects on ESCC remain unclear. This study aims to investigate the therapeutic effects of Alkannin on ESCC and elucidate its potential targets and molecular mechanisms. Cell Counting Kit-8 (CCK-8) assays, colony formation assays, Hoechst 33342 staining, wound healing assays, Transwell migration assays, flow cytometry, and Western blotting were used to investigate the therapeutic effects of Alkannin on ESCC in vitro. Transcriptome sequencing and network pharmacology were employed to analyze the potential targets and pathways affected by Alkannin treatment. The anticancer effects of Alkannin in vivo were assessed in a nude mouse model. Alkannin suppressed cell proliferation, invasion, migration, and induced ESCC cell apoptosis. Mechanistic studies indicated that Alkannin inhibits ESCC by inducing G2/M-phase cell cycle arrest by targeting Glycogen Synthase Kinase 3β (GSK3β). Consistently, in vivo administration of Alkannin significantly reduced the growth of ESCC tumors in nude mice. This study is the first to demonstrate that Alkannin, by targeting GSK3β, induces G2/M-phase arrest in ESCC cells, thereby inhibiting migration, invasion, and inducing apoptosis, suggesting that Alkannin may be a promising antitumor agent for treating ESCC.

Sterile Alpha Motif Domain-Containing 5 Suppresses Malignant Phenotypes and Tumor Growth in Breast Cancer: Regulation of Polo-Like Kinase 1 and c-Myc Signaling in a Xenograft Model.

Background Breast cancer, particularly the triple-negative breast cancer (TNBC) subtype, remains a significant clinical challenge due to its resistance to standard chemotherapy and high recurrence rate. In this study, we explored the role of Sterile Alpha Motif Domain-Containing 5 (SAMD5) as a potential regulatory partner with the c-Myc oncogenic signaling pathway in breast cancer. Materials and methods Functional assays were conducted to investigate the effects of SAMD5 overexpression on cell viability, colony formation, and invasive behavior in TNBC cell lines. This study further assessed the expression levels of proliferation and invasion markers, including Ki67 (a marker for cell proliferation), Matrix Metalloproteinase-2 (MMP2), and Matrix Metalloproteinase-9 (MMP9). Mechanistic analyses identified a negative correlation between SAMD5 and Polo-like Kinase 1 (PLK1), a gene frequently overexpressed in breast cancer, particularly in TNBC. The effects of PLK1 knockdown on cell viability, colony formation, and invasion were observed, along with the impact of PLK1 overexpression on SAMD5's inhibitory activity. In vivo studies were performed using a xenograft tumor model in nude mice to evaluate the impact of SAMD5 overexpression on tumor weight and volume. Results SAMD5 overexpression significantly reduced cell viability, colony formation, and invasion in TNBC cells, and downregulated key proteins in the c-Myc signaling pathway, including c-Myc itself, β-catenin, Cyclin-Dependent Kinase 4 (CDK4), Cyclin-Dependent Kinase 6 (CDK6), and Cyclin D1. PLK1 overexpression was found to counteract SAMD5's inhibitory effects. In vivo experiments demonstrated that SAMD5 overexpression led to a marked reduction in tumor weight and volume, effects that were partially reversed by PLK1 overexpression. Conclusions SAMD5 acts as a tumor suppressor in breast cancer, particularly in TNBC, by inhibiting critical cellular processes and downregulating the c-Myc signaling pathway. This effect appears to be mediated, in part, through its negative association with PLK1. Targeting the SAMD5/PLK1 axis offers a promising therapeutic strategy for addressing aggressive breast cancers.

CEP70 in Prostate Cancer: A Novel Mechanism of Angiogenesis and Metastasis through Upregulation of Vascular Endothelial Growth Factor A Expression.

This study aims to investigate centrosomal protein 70 (CEP70) in prostate cancer and its effects on angiogenesis and tumour metastasis and elucidate its molecular mechanisms. We evaluated CEP70 and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in tissue samples from patients with prostate cancer by immunohistochemistry. In vitro experiments included overexpressing CEP70 through transfection and assessing its impact on human umbilical vein endothelial cells (HUVECs). Intervention experiments with an NF-κB pathway inhibitor were conducted to verify the mechanism. Finally, the effects of CEP70 on tumour growth, angiogenesis and metastasis were examined in a nude mouse model. CEP70 was significantly overexpressed in prostate cancer tissues compared with that in adjacent normal tissues (p < 0.001). In vitro experiments demonstrated that CEP70 overexpression promoted HUVEC migration (p < 0.001), invasion (p < 0.001) and tube formation (p < 0.05). CEP70 significantly upregulated VEGFA expression in prostate cancer cells at messenger RNA (mRNA) (p < 0.001) and protein levels (p < 0.05). VEGFA knockdown experiments confirmed CEP70 as an essential cytokine for CEP70-induced angiogenesis (p < 0.01). Mechanistically, CEP70 promoted VEGFA expression by activating the NF-κB signalling pathway, as evidenced by the reversal of CEP70-induced effects upon treatment with the NF-κB inhibitor BAY11-7082 (p < 0.01). CEP70 promotes tumour angiogenesis and metastasis by upregulating VEGFA through NF-κB pathway activation.

DEAD/H-box helicase 11 is transcriptionally activated by Yin Yang-1 and accelerates oral squamous cell carcinoma progression.

Oral squamous cell carcinoma (OSCC) is the most common oral malignancy. DEAD/H-box helicase 11 (DDX11), a DNA helicase, has been implicated in the progression of several cancers. Yet, the precise function of DDX11 in OSCC is poorly understood. The DDX11 expression in OSCC cells and normal oral keratinocytes was evaluated in the Gene Expression Omnibus database (GSE146483 and GSE31853). SCC-4 and CAL-27 cells expressing doxycycline-inducible DDX11 or DDX11 shRNA were generated by lentiviral infection. The role of DDX11 in OSCC cells was determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry assay, TUNEL staining, and western blot. The effects of DDX11 on tumor growth were explored in a xenograft nude mouse model. The relationship between DDX11 and transcription factor Yin Yang-1 (YY1) was researched using the dual luciferase report assay and chromatin immunoprecipitation assay. DDX11 expression was significantly upregulated in OSCC cells. Knockdown of DDX11 inhibited cell proliferation, induced cell cycle arrest, and suppressed PI3K-AKT pathway, while DDX11 overexpression showed opposite effects. The number of apoptotic cells was increased in DDX11 silenced cells. DDX11 upregulation or knockdown accelerated or suppressed tumor growth in vivo, respectively. Moreover, the YY1 bound and activated the DDX11 promoter, resulting in increasing DDX11 expression. Forced expression DDX11 reversed the anticancer effects of YY1 silencing on OSCC cells. DDX11 has tumor-promoting function in OSCC and is transcriptionally regulated by YY1, indicating that DDX11 may serve as a potential target for the OSCC treatment.

Ultrasound/Magnetic Resonance Bimodal Imaging-Guided CD20-Targeted Multifunctional Nanoplatform for Photothermal/Chemo Synergistic Therapy of B-Cell Lymphoma

B-cell lymphoma has a poor prognosis due to difficulties in early diagnosis and the negative effects of systemic chemotherapy. Therefore, there is an urgent need to develop highly accurate and effective theranostic strategies for B-cell lymphoma. In this study, we designed a poly (lactic-co-glycolic acid) (PLGA)-based theranostic nanoplatform (denoted as TscNPs) to achieve ultrasound (US)/magnetic resonance (MR) bimodal imaging-guided photothermal (PTT)/chemo synergistic therapy of B-cell lymphoma. The nanoplatform was conjugated with a CD20 monoclonal antibody specifically targeting B-cell lymphoma to promote tumor accumulation. Encapsulated superparamagnetic iron oxide nanoparticles (SPIONs) as photothermal and MR imaging agents enabled thermal ablation of tumors and imaging-guided tumor therapy. When exposed to near-infrared (NIR) laser, TscNPs generate heat that induces optical droplet vaporization (ODV) of perfluoropentane (PFP), which transforms into microbubbles. This process not only enhanced ultrasound imaging, but also facilitated the release of celastrol (CST) from the nanoplatform, ultimately achieving a PTT/chemo synergistic therapy effect. In the tumor-bearing nude mice model, TscNPs were effectively accumulated in the tumor region. Furthermore, the combined treatment mode of TscNPs and NIR laser irradiation demonstrated a tumor inhibition rate of approximately 96.57%, which was significantly superior to the rates observed with PTT or chemotherapy alone. These results suggest that the multifunctional theranostic nanoplatform represents a promising new strategy for the therapy of B-cell lymphoma.

Microspheres of stem cells from human exfoliated deciduous teeth exhibit superior pulp regeneration capacity

ObjectivesEngineering spheroids to create three-dimensional (3D) cell cultures has gained increasing attention in recent years due to their potential advantages over traditional two-dimensional (2D) tissue culture methods. Stem cells derived from human exfoliated deciduous teeth (SHEDs) demonstrate significant potential for pulpal regeneration applications. Nevertheless, the feasibility of microsphere formation of SHEDs and its impact on pulpal regeneration remain unclear. MethodsIn this study, SHEDs were isolated, identified, and cultured in ultra-low attachment six-well plates to produce SHED microspheres. The biological properties of SHED microspheres were compared to those of traditional 2D culture using live-dead staining, Alizarin red staining, Oil-red O staining, scratch experiments, Immunofluorescence, Transmission electron microscopy scan, Western blotting, RNA sequencing, and a nude mice subcutaneous transplantation model. ResultsWe found SHED cells can form microspheres with a dense internal structure. SHED microspheres exhibited notable advantages over SHED cells in terms of biological properties, maintaining cell activity and enhancing cell differentiation, migration, and stemness in vitro. RNA-seq revealed that the SHED microspheres potentially influenced cell development, regulation of neurogenesis, skeletal system development, tissue morphogenesis singling pathway. In vivo, SHED microspheres promoted the generation of pulp tissue in dental pulp compared to traditional 2D culture. ConclusionsMicrosphereization of SHED through 3D cell culture enhances its pulp regeneration capacity, presenting a novel strategy for dental pulp regeneration and the clinical treatment of dental pulp diseases.

Sonopermeation With Size-sorted Microbubbles Synergistically Increases Survival and Enhances Tumor Apoptosis With L-DOX by Increasing Vascular Permeability and Perfusion in Neuroblastoma Xenografts

ObjectiveDespite aggressive therapy, approximately 50% of patients with neuroblastoma (NB) fail to respond, and survivors endure lifelong toxicities. Sonopermeation increases drug uptake via cell bilayer disruption through focused ultrasound and microbubbles (MBs)—gas-filled, sound sensitive lipid spheres. MB response to a given ultrasound pulse (cavitation) varies according to MB size. We asked whether size-sorted MBs (SSMB) 4 to 5 µm in diameter will more consistently and predictably enhance doxorubicin uptake, compared with polydisperse MBs (PMB, 0.5–10 µm in diameter), thereby increasing drug delivery to NB xenografts. MethodsHuman NB cells were implanted into the left kidney of nude mice and grown for 5 to 6 wk. Mice received sonopermeation alongside either PMB or SSMB at low (0.6 MPa) or high (2 MPa) negative pressures. Some mice also received different chemotherapy agents (doxorubicin, etoposide or cyclophosphamide). Circulating tumor cells were assessed by flow cytometry 1 h after treatment. Survival was assessed for up to 21 d, a subset of mice was euthanized 24 h after treatment for histological assessment of apoptosis, vascular lumen size and tight junctions. ResultsTumors treated with SSMB and high pressure showed synergy with liposomal doxorubicin (L-DOX) owing to increased vascular lumen and disruption of tight junctions, resulting in drug uptake, apoptosis, lack of tumor growth and increased survival. We found no difference in the numbers of circulating tumor cells. ConclusionSonopermeation with SSMB at 2 MPa synergizes with L-DOX delivery, increasing apoptosis, perfusion and vascular permeability, suggesting that SSMB sonopermeation at high pressure is promising for NB-targeted treatment, especially in combination with L-DOX.

Enhanced in vivo Stability and Antitumor Efficacy of PEGylated Liposomes of Paclitaxel Palmitate Prodrug.

The clinical use of paclitaxel (PTX) in cancer treatment is limited by its poor water solubility, significant toxicity, and adverse effects. This study aimed to propose a straightforward and efficient approach to enhance PTX loading and stability, thereby offering insights for targeted therapy against tumors. We synthesized a paclitaxel palmitate (PTX-PA) prodrug by conjugating palmitic acid (PA) to PTX and encapsulating it into liposomal vehicles using a nano delivery system. Subsequently, we investigated the in vitro and in vivo performance as well as the underlying mechanisms of PTX-PA liposomes (PTX-PA-L). PTX had a remarkable antitumor effect in vivo and significantly decreased the myelosuppressive toxicity of PTX. Moreover, the introduction of PA increased the lipid solubility of PTX, forming a phospholipid bilayer as a membrane stabilizer, prolonging the circulation time of the drug and indirectly increasing the accumulation of liposomes at the tumor site. Our in vivo imaging experiments demonstrated that PTX-PA-L labeled with DiR has greater stability in vivo than blank liposomes and that PTX-PA-L can target drugs to the tumor site and efficiently release PTX to exert antitumor effects. In a mouse model, the concentration of PTX at the tumor site in the PTX-PA-L group was approximately twofold greater than that of Taxol. However, in a nude mouse model, the concentration of PTX at the tumor site in the PTX-PA-L group was only approximately 0.8-fold greater than that of Taxol. Furthermore, the originally observed favorable pharmacodynamics in normal mice were reversed following immunosuppression. This may be caused by differences in esterase distribution and immunity. This prodrug technology combined with liposomes is a simple and effective therapeutic strategy with promising developmental prospects in tumor-targeted therapy owing to its ability to convert PTX into a long-circulating nano drug with low toxicity, high pharmacodynamics, and good stability in vivo.

Qingrexiaoji Recipe Regulates the Differentiation of M2 TAM via miR-29 in GC.

Gastric cancer, one of the most familiar adenocarcinomas of the gastrointestinal tract, ranks third in the world in cancer-related deaths. Traditional Chinese medicine can suppress the growth of tumors, and the underlying mechanism may be associated with the tumor microenvironment. Here, we investigated the anti-cancer effects of the Qingrexiaoji recipe on gastric cancer and the underlying molecular mechanism. An in vivo nude mouse model was established, and the expression of CD206, CD80, and M2 phenotype-related proteins (Arg-1, Fizz1) was obtained by flow cytometry and western blotting. The expressions of the M2 phenotype-related cytokines were examined by ELISA. Qingrexiaoji recipe inhibited gastric tumor growth and downregulated the expression of CD206, IFN-γ, IL-13, IL-4, and TNF-α in vivo. Qingrexiaoji recipe deceased M2 phenotypic polarization by upregulating microRNA (miR)-29a-3p level. Luciferase activity assays showed that HDAC4 is a potential target of miR-29a-3p. In cells co-transfected with HDAC4 siRNA and miR-29a-3p inhibitor and treated with IL-4 and Qingrexiaoji recipe, the miR-29a-3p inhibitorinduced increase of M2 phenotypic polarization was reversed. In summary, these results suggested that the Qingrexiaoji recipe regulated M2 macrophage polarization by regulating miR-29a-3p/HDAC4, providing a different and innovative treatment for gastric cancer.

Preclinical ex vivo IL2RG gene therapy using autologous hematopoietic stem cells as an effective and safe treatment for X-linked severe combined immunodeficiency disease

X-linked severe combined immunodeficiency disease (X-SCID) is a rare inherited disease caused by mutations in the interleukin 2 receptor subunit gamma gene (IL2RG), which encodes the common γ chain protein, a subunit of the receptor for lymphocytes. X-SCID is characterized by profound defects in T-cell, B-cell, and natural killer cell function. Here, we report a Chinese cohort of nine X-SCID patients with six novel IL2RG mutations. Among those, the two adolescent patients carrying missense mutation with an atypical immunotype were confirmed by further analyzing IL-2-JAK-STAT5 signaling, T cell proliferation, and T cell receptor excision circles (Trecs). Interestingly, Bacillus Calmette-Guérin (BCG) disease occurred commonly in this cohort. Although allogeneic hematopoietic stem-cell transplantation is curative for the disease, it is not available to all patients due to the lack of suitable matched donors. Autologous gene therapy using a self-inactivating lentiviral vector (SIN-LV) technology, developed in recent decades has provided an alternative therapy for treating such mono-genetic diseases. Here, we performed the pre-clinical studies to assess our SIN-LV carrying IL2RG on human ED7R cells deficient in IL2RG and CD34+ stem cells derived from the bone marrow of a healthy donor and a patient with X-SCID. This work is done complied with the established “Good Manufacturing Practice” (GMP) used in the clinical trials. In addition, a safety study is performed using the transduced CD34+ cells implanted into the axilla of nude mice in vivo. Overall, our studies have demonstrated the efficiency and safety of SIN-IL2RG-LV, which paves the way for conducting X-SCID gene therapy clinical trials in China in the near future.

(-)-Epicatechin regulates endoplasmic reticulum stress and promotes ferroptosis in lung cancer cells via the PERK/eIF2α/ATF4 signaling pathway.

(-)-Epicatechin (EC) is an active ingredient of Fagopyrum dibtrys (D. Don) Hara and can regulate lung cancer progression. However, the specific regulatory mechanism is poorly understood. This study explored the specific mechanism of EC in the treatment of lung cancer. H460 cells were injected subcutaneously into the left dorsal sides of nude mice to establish an animal model of lung cancer. H460 and H1299 cells and nude mice were treated with different concentrations of EC. The expression levels of related proteins were detected by Western blotting. Cell proliferation, migration, and invasion were detected by CCK-8, colony formation, and Transwell assays. Flow cytometry was used to detect the Ca2+ level in lung cancer cells. Immunohistochemistry was used to detect the expression of Ki-67 in tumor tissues. This study revealed that ferroptosis in lung cancer cells was inhibited during lung cancer development. EC treatment promotes ferroptosis, inhibits the proliferation, migration and invasion of lung cancer cells, and inhibits the formation of tumors in vivo. Ferroptosis inhibitors (Fer-1) weaken the effects of EC on lung cancer cells, whereas a ferroptosis inducer (erastin) further promotes the effects of EC. In addition, endoplasmic reticulum (ER) stress is involved in the EC-induced ferroptosis of lung cancer cells, and treatment with GSK, an inhibitor of the ER stress protein PERK, can reverse the effect of EC. EC therapy activates the PERK-eIF2α-ATF4 signaling pathway to increase ER stress, thereby promoting ferroptosis in lung cancer cells and inhibiting the occurrence and development of lung cancer. Our research suggests that EC may become a drug candidate for treating lung cancer.

Gilteritinib reverses ABCB1-mediated multidrug resistance: Preclinical in vitro and animal investigations

Multi-drug resistance (MDR) poses a significant challenge to cancer treatment. Targeting ATP-binding cassette subfamily B member 1 (ABCB1) is a viable strategy for overcoming MDR. This study examined the preclinical in vitro and animal studies that used gilteritinib, a FLT3 inhibitor that reverses ABCB1-mediated MDR. At nontoxic levels, gilteritinib significantly increased the susceptibility of cancer cells overexpressing ABCB1 to chemotherapeutic drugs. Furthermore, it impaired the development of drug-resistant cell colonies and 3D spheroids. Studies on the reversal mechanism have shown that gilteritinib can directly bind to the drug-binding site of ABCB1, inhibiting drug efflux activity. Consequently, the substrate’s drug cytotoxicity increases in MDR cells. Furthermore, gilteritinib increased ATPase activity while leaving ABCB1 expression and subcellular distribution unchanged and inhibited AKT or ERK activation. Docking analysis indicated that Gilteritinib could interact with the drug-binding site of the ABCB1 transporter. In vivo studies have shown that gilteritinib improves the antitumor efficacy of paclitaxel in nude mice without obvious toxic effects. In conclusion, our preclinical investigations show that gilteritinib has the potential to successfully overcome ABCB1-mediated MDR in a clinical environment when combined with substrate medicines.