Xenograft Mouse Research Articles

RNAi Screen Identifies AXL Inhibition Combined with Cannabinoid WIN55212-2 as a Potential Strategy for Cancer Treatment.

Background and objective: Cannabinoids are commonly used as adjuvant cancer drugs to overcome numerous adverse side effects for patients. The aim of this study was to identify the target genes that show a synergistic anti-tumor role in combination with the cannabinoid WIN55212-2 in vitro and in vivo. Methods: A human kinome RNAi library was used to screen the targeted gene that silencing plus WIN55212-2 treatment synergistically inhibited cancer cell growth in an INCELL Analyzer 2000. Cell viability, cell phase arrest and apoptosis were evaluated by MTT and flow cytometry assay. In vivo combined anti-tumor effects and regulatory mechanisms were detected in immunocompromised and immunocompetent mice. Results: Using RNAi screening, we identified the tyrosine receptor kinase AXL as a potential gene whose silencing plus WIN55212-2 treatment synergistically inhibited the proliferation of cancer cells in an INCELL Analyzer 2000. Subsequently, we demonstrated that inhibition of AXL by TP-0903 potentiated the inhibitory role of WIN55212-2 on cellular viability, colony formation and 3D tumor sphere in HCT-8 cells. Meanwhile, TP-0903 plus WIN55212-2 treatment promoted the apoptosis of HCT-8 cells. We then investigated the synergistic anti-tumor effect of TP-0903 and WIN55212-2 using colon cancer cell xenografts in immunocompromised and immunocompetent mice. The in vivo study demonstrated that combined administration of TP-0903 plus WIN55212-2 effectively reduced tumor volume and microvessel density and promoted apoptotic cells of tumor tissues in HCT-8 exogenous mice compared to either TP-0903 or WIN55212-2 treatment alone. Moreover, in addition to tumor suppression, the combination therapy of TP-0903 and WIN55212-2 induced the infiltration of cytotoxic CD8+ T cells and significantly reduced mTOR and STAT3 activation in tumor tissues of C57BL/6J mice bearing MC-38 cells. Conclusions: This study demonstrated that targeting AXL could sensitize cannabinoids to cancer therapy by interfering with tumor cells and tumor-infiltrating CD8+ T cells.

An antibody against human interleukin-2 showing minimal binding to neonatal Fc receptor: potent anti-tumor activity and reduced systemic toxicity in mice

The anti-tumor efficacy of immune checkpoint inhibitors can be enhanced by combining them with interleukin-2 (IL-2), which promotes the clonal expansion of antigen-activated CD8+ T cells and natural killer cells. However, IL-2 can simultaneously bind to endothelial cells and regulatory T cells to induce adverse and immunosuppressive effects. Such off-target binding can be inhibited by co-administering IL-2 with anti-IL-2 antibodies, but these antibodies can interact with neonatal Fc receptor to protect the IL-2 from lysosomal degradation, leading to substantial toxicity. Here we developed and humanized a mouse monoclonal antibody against human IL-2 and introduced two mutations (H310A and H435Q) in the Fc segment in order to weaken binding to the neonatal Fc receptor. The humanized antibody bound tightly to IL-2 but minimally to neonatal Fc receptor. Hydrogen deuterium exchange mass spectrometry indicated that the antibody binds to IL-2 at the site where the cytokine binds to subunit α (CD25) of the trimeric IL-2 receptor. Co-administering the antibody with human IL-2 improved the cytokine's ability to slow growth of two types of colorectal tumor xenografts (CT26, MC38) in immunocompetent mice. Co-administration to mice with CT26 xenografts strongly expanded CD8+ T cells, without expanding regulatory T cells in spleen or blood. Co-administration to mice with MC38 xenografts downregulated PD-1 and CD44, while upregulating CD62L and CD69, on tumor-infiltrating CD8+ T cells. Notably, co-administration synergized with anti-PD-1 antibody to slow growth of MC38 xenografts. Our results suggest that the combination of human IL-2 and our novel antibody shows promise for providing more effective, less toxic cancer immunotherapy.

CRH upregulates supervillin through ERK and AKT pathways to promote bladder cancer cell migration.

Corticotropin-releasing hormone (CRH) has been well documented playing a role in the regulation of cellular processes, immune responses, and inflammatory processes that can influence the occurrence and development of tumors. Supervillin (SVIL) is a membrane-associated and actin-binding protein, which is actively involved in the proliferation, spread, and migration of cancer cells. This work investigated CRH's influence on bladder cancer cells'migration and relevant mechanisms. By using human bladder cancer cells T24 and RT4 in wound healing experiments and transwell assay, we found that the migration ability of the T24 cells was significantly increased after CRH treatment. In vivo experiments showed that CRH significantly promoted the metastases of T24 cells in cell line-derived xenograft (CDX) mouse model. Interestingly, downregulation of SVIL by SVIL-specifc small hairpin RNAs significantly reduced the promoting effect of CRH on bladder cancer cell migration. Furthermore, CRH significantly increased SVILmessenger RNA and protein expression in T24 cells, accompanied with AKT andERK phosphorylation in T24 cells. Pretreatment with AKT inhibitor (MK2206) blocked the CRH-induced SVIL expression and ERK phosphorylation. Also, inhibition of ERK signaling pathway by U0126 significantly reduced the CRH-induced SVIL expression and AKT phosphorylation. It suggested that cross-talking between AKT and ERK pathways was involved in the effect of CRH on SVIL. Taken together, we demonstrated that CRH induced migration of bladder cancer cells, in which AKT andERK pathways -SVIL played a key role.

EZH2 Promotes Glioma Cell Proliferation, Invasion, and Migration via Mir-142-3p/KCNQ1OT1/HMGB3 Axis : Running Title: EZH2 Promotes Glioma cell Malignant Behaviors.

This study investigates the role and molecular mechanism of EZH2 in glioma cell proliferation, invasion, and migration. EZH2, miR-142-3p, lncRNA KCNQ1OT1, LIN28B, and HMGB3 expressions in glioma tissues and cells were determined using qRT-PCR or Western blot, followed by CCK-8 assay detection of cell viability, Transwell detection of invasion and migration, ChIP analysis of the enrichment of EZH2 and H3K27me3 on miR-142-3p promoter, dual-luciferase reporter assay and RIP validation of the binding of miR-142-3p-KCNQ1OT1 and KCNQ1OT1-LIN28B, and actinomycin D detection of KCNQ1OT1 and HMGB3 mRNA stability. A nude mouse xenograft model and a lung metastasis model were established. EZH2, KCNQ1OT1, LIN28B, and HMGB3 were highly expressed while miR-142-3p was poorly expressed in gliomas. EZH2 silencing restrained glioma cell proliferation, invasion, and migration. EZH2 repressed miR-142-3p expression by elevating the H3K27me3 level. miR-142-3p targeted KCNQ1OT1 expression, and KCNQ1OT1 bound to LIN28B to stabilize HMGB3 mRNA, thereby promoting its protein expression. EZH2 silencing depressed tumor growth and metastasis in nude mice via the miR-142-3p/KCNQ1OT1/HMGB3 axis. In conclusion, EZH2 curbed miR-142-3p expression, thereby relieving the inhibition of KCNQ1OT1 expression by miR-142-3p, enhancing the binding of KCNQ1OT1 to LIN28B, elevating HMGB3 expression, and ultimately accelerating glioma cell proliferation, invasion, and migration.

2′-Hydroxycinnamaldehyde induces ROS-mediated apoptosis in cancer cells by targeting PRX1 and PRX2

2′-Hydroxycinnamaldehyde (HCA) is a component of the commonly used spice cinnamon, which has beneficial effects on cancer, allergies, bacterial/viral infections, and Alzheimer’s disease. Our previous study showed that HCA induced reactive oxygen species (ROS) and apoptosis in cancer cells, and pretreatment of cancer cells with antioxidants abolished HCA-mediated ROS production and apoptosis. This indicates that ROS are critical effector for HCA activity. However, the molecular target of HCA for ROS induction has not been identified. In the present study, we identified peroxiredoxin 1 (PRX1) and peroxiredoxin 2 (PRX2) as target proteins of HCA using affinity chromatography, and further confirmed these association using a cellular thermal shift assay (CETSA). In addition, we used mutagenesis to identify important cysteine residues in PRX1 for HCA binding. PRX1 has four cysteines (Cys52, Cys71, Cys83, and Cys173), and when Cys173 (but not the other cysteine sites) was mutated to serine, it was unable to bind biotin-conjugated HCA, suggesting that Cys173 is important for HCA binding. Treatment of SW620 cancer cells transfected by control vector with 20 μM HCA increased ROS levels by 5.2-fold compared to DMSO-treated cells. However, downregulation of target proteins PRX1 and PRX2 using shRNAs (short hairpin RNA) significantly reduced HCA-mediated ROS induction (1.6-fold), supporting that PRX1 and PRX2 are targets of HCA for ROS elevation. Additionally, intraperitoneal injection of 50 mg/kg HCA inhibited SW620 tumor growth, resulting in a 59.9 % reduction in tumor volume. CETSA analysis of tumor tissues showed that PRX1 and PRX2 were bound and thus inactivated by HCA in a mouse xenograft model. These findings demonstrate that PRX1 and PRX2 are molecular target proteins responsible for HCA-induced ROS elevation and cancer cell death.

Preclinical evaluation of the CD38-targeting engineered toxin body MT-0169 against multiple myeloma.

Despite significant progress in the treatment of multiple myeloma (MM), relapsed/refractory patients urgently require more effective therapies. We here describe the discovery, mechanism of action, and preclinical anti-MM activity of engineered toxin body MT-0169, a next-generation immunotoxin comprising a CD38-specific antibody fragment linked to a de-immunized Shiga-like toxin A subunit (SLTA) payload. We show that specific binding of MT-0169 to CD38 on MM cell lines triggers rapid internalization of SLTA, causing cell death via irreversible ribosome inhibition, protein synthesis blockade, and caspase 3/7 activation. In co-culture experiments, bone marrow mesenchymal stromal cells did not induce drug resistance against MT-0169. In the preclinical setting, MT-0169 effectively lysed primary MM cells from newly diagnosed and heavily pretreated MM patients, including those refractory to daratumumab, with minimal toxicity against nonmalignant hematopoietic cells. MM cell lysis showed a significant correlation with their CD38 expression levels but not with cytogenetic risk, tumor load, or number of prior lines of therapy. Finally, MT-0169 showed efficient in vivo anti-MM activity in various mouse xenograft models, including one in which MM cells are grown in a humanized bone marrow-like niche. These findings support clinical investigation of MT-0169 in relapsed/refractory MM patients, including those refractory to CD38-targeting immunotherapies.

Antitumor activities of anti‑CD44 monoclonal antibodies in mouse xenograft models of esophageal cancer.

CD44 is a type I transmembrane glycoprotein associated with poor prognosis in various solid tumors. Since CD44 plays a critical role in tumor development by regulating cell adhesion, survival, proliferation and stemness, it has been considered a target for tumor therapy. Anti‑CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody‑drug conjugates and chimeric antigen receptor‑T cell therapy. Anti-pan‑CD44 mAbs, C44Mab‑5 and C44Mab‑46, which recognize both CD44 standard (CD44s) and variant isoforms were previously developed. The present study generated a mouse IgG2a version of the anti‑pan‑CD44 mAbs (5‑mG2a and C44Mab‑46‑mG2a) to evaluate the antitumor activities against CD44‑positive cells. Both 5‑mG2a and C44Mab‑46‑mG2a recognized CD44s‑overexpressed CHO‑K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5‑mG2a and C44Mab‑46‑mG2a could activate effector cells in the presence of CHO/CD44s cells and exhibited complement-dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5‑mG2a and C44Mab‑46‑mG2a significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control mouse IgG2a. These results indicate that 5‑mG2a and C44Mab‑46‑mG2a could exert antitumor activities against CD44‑positive cancers and be a promising therapeutic regimen for tumors.

Schistosoma mansoni soluble egg antigen suppresses colorectal cancer growth in vitro and in vivo

BackgroundColorectal cancer (CRC) is the third most common malignant disease around the world. Because the hosts’ immunity plays a great part in regulating tumor cells' growth and progression, immunotherapies have therefore aroused great interest in treating cancers. Currently, scientists have investigated the use of Schistosoma-derived soluble egg antigens (SEA), which is known as a strong immune modulator, in treating a series of immune-related diseases. MethodsIn this study, we investigated the anti-tumor effect of SEA against CRC using in vitro cell lines, HCT-116 and DLD-1, as well as in vivo mouse xenograft model. Approaches such as migration assay, invasion assay, and western blotting were done to analyze the anti-tumor effect of SEA. Furthermore, qRT-PCR and ELISA were performed to identify the immune profile of SEA-treated cells as well as SEA-treated xenograft mice. ResultsIn vitro studies suggested that SEA can dose-dependently inhibit the growth and progression of HCT-116 and DLD-1 cells. This inhibition was accompanied by a reduction of epithelial-mesenchymal transition (EMT), inflammasome inactivation, and apoptosis. SEA also downregulated the expression of IL-4 and IL-10 in the CRC cells, which may be the reason why their growth and progression were suppressed. In vivo studies showed a similar beneficial effect of SEA, as local administration of 25 μg SEA significantly inhibits tumor cell growth. SEA treatment also shifts the host’s immunity from a pro-tumorigenic response to an anti-tumor response. ConclusionIn conclusion, SEA may provide a beneficial effect against CRC, and further investigation may give promise in CRC treatment.

A novel goldfish orthotopic xenograft model of hepatocellular carcinoma developed to evaluate antitumor drug efficacy

Tumor xenograft animal models play a crucial role in hepatocellular carcinoma (HCC) research. Mice xenograft models are time consuming, laborious and expensive while zebrafish tumor xenograft models are cost-effective and effortless. However, the development of orthotopic xenograft models for HCC in zebrafish embryos has been challenging due to the small size of zebrafish livers. In this study, we utilized 7-day-old goldfish embryos as hosts and successfully established an orthotopic xenograft model of HCC in goldfish livers. Through injecting fluorescence labeled HCC cells into the liver of goldfish, we could visualize the proliferation and migration of tumor cells in vivo. In addition, we found that the temperature of 36 °C was better for tumor cell survival in goldfish larvae compared to 28 °C, assessed by EdU and TUNEL assays. Moreover, macrophage infiltration in the goldfish liver could be evaluated by neutral red staining. Finally, we evaluated the efficacy of the targeted therapy drug Sorafenib and the traditional Chinese medicine, Huaier granules, alone or in combination in the goldfish HCC orthotopic xenograft model. We found that the combination therapy showed the best efficacy against HCC cells in terms of macrophage infiltration, polarization as well as tumor cells proliferation, metastasis and apoptosis. In conclusion, the proposed goldfish HCC orthotopic xenograft model opens new avenues for HCC related research, including evaluation of tumor progression, cell interactions in the immune microenvironment, drug efficacy, and screening of anti-tumor drugs.

A LANA peptide inhibits tumor growth by inducing CHD4 protein cleavage and triggers cell death

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

Telaglenastat as an alternative to cisplatin as a radiosensitizer in the treatment of head and neck squamous cell carcinoma

The efficacy of radiation treatment (RT) of head and neck squamous cell carcinoma (HNSCC) is limited by radioresistance and the toxicity of FDA approved radiosensitizers. In extension to our previous research where we demonstrated that telaglenastat (CB839) increased efficacy of RT in in vitro and in vivo HNSCC models, here, we examine the radiosensitizing effects of telaglenastat in comparison to cisplatin's, as cisplatin is currently the standard of care for concurrent therapy. Combination of telaglenastat with RT reduced tumor volume in a HNSCC patient derived xenograft mouse model. The efficacy of telaglenastat with RT in reducing cell survival and increasing apoptosis was similar if not greater than that of cisplatin with RT in Cal27 and HN5 HNSCC cells. The addition of telaglenastat increased reactive oxygen species and reduced the antioxidant glutathione in both Cal27 and HN5 cells. Reverse Phase Protein Array analyses revealed alterations in cell death and DNA damage response proteins. This study provides the scientific underpinnings for the use of telaglenastat as a radiosensitizer in the treatment of HNSCC either as an alternative to cisplatin or in cisplatin-ineligible patients.

Decorin-armed oncolytic adenovirus promotes natural killers (NKs) activation and infiltration to enhance NK therapy in CRC model

Colorectal cancer (CRC) is a prevalent malignant tumor of the gastrointestinal system, with the third and second highest incidence and mortality rates globally in 2020, respectively. Immunotherapy has developed rapidly in recent years. Natural killer (NK) cells have received increasing attention in the field of tumor immunotherapy due to their recognition and killing tumor cells without the limitations of major histocompatibility complexes. However, constraints within the tumor microenvironment that impede the infiltration and proliferation of NK cells result in poor efficacy of NK cell therapy for solid tumors. Oncolytic viral therapy is an immunogenic treatment with the potential to enhance anti-tumour immune responses and promote immune cell infiltration. In this study, we synergistically combine NK cells with an oncolytic adenovirus carrying Decorin (rAd.DCN) for the treatment of colorectal cancer (CRC) in a xenograft mouse model. By using Flow cytometry, real-time quantitative PCR and Calcein-AM release assay, we found that rAd.DCN could effectively promote proliferation, activation and degranulation of NK cells, up-regulate expression and secretion of NK cell killing activity-related factors, and enhance their killing activity. The efficacy is better than that of the blank control oncolytic virus rAd.Null. Combined treatment significantly inhibited tumor growth, increased the number of NK cells in peripheral blood, promoted the killing function of NK cells, and increased the expression levels of perforin and IFN-γ. At the same time, more NK cells were recruited to infiltrate tumor tissue. Our study established the feasibility of combination NK cells and oncolytic adenovirus application, thus expanding the scope of potentially curative treatments for NK cells in CRC.

Trichosanthin elicits antitumor activity via MICU3 mediated mitochondria calcium influx

IntroductionTrichosanthin (TK) is a glycoprotein extracted from the Chinese medicinal herb Trichosanthes kirilowi, which has anti-virus and anti-tumor activity. However, the target and detailed mechanism of TK remains elusive. ObjectivesWe aimed to identify novel antitumor targets of TK in lung adenocarcinoma and study its anti-tumor mechanism. MethodsWe utilized a Lewis lung carcinoma mouse model to evaluate the inhibition of TK on tumor growth. CCK8 assay was utilized to calculate IC50 of trichosanthin on A549 and H1299. In-vitro cellular assays and in-vivo xenograft mice studies were used to investigate MICU3 overexpression and TK treatment on tumor growth. Fluo-4 dye and JC-1 staining was used to measure the mitochondrial calcium levels and membrane potential. H&E and immunohistochemistry staining were applied the asses the effect of TK on tumor and microenvironment. RNA sequencing was applied to analyze transcriptome changes in TK-treated and MICU3-overexpressed tumor cells. The influence of trichosanthin on DNMT3B expression and MICU3 methylation were detected by qPCR and Western blotting. Transcriptional activity of the MICU3 gene was measured by ChIP-PCR and luciferase assays. ResultsTrichosanthin ihibited the tumor growth in vivo, resulting cancer cell growth inhibition and cell death, with almost no effect on normal cells. IC50 of trichosanthin in A549 and H1299 cells were 62.8 μg/ml and 39.7 μg/ml, respectively. Mitochondrial Calcium Uptake Family complex MICU3 was shown to associated with favorable prognosis and was upregulated upon trichosanthin treatment, along with reduces tumor cell growth and migration, and increased cell death both in vitro and in vivo. Increased mitochondrial calcium level was observed in MICU3 overexpression cells. Pathway analysis of RNA-seq data revealed that cytokine and receptor pathways were enriched in MICU3-overexpressing cells. Trichosanthin decreased DNMT3B expression and altered MICU3 methylation while increased FOSL2 expression and reduced methylation that correlated with increased transcription of the MICU3 gene. ConclusionTrichosanthin elicits antitumor activity in lung adenocarcinoma via repressing DNMT3B and increasing FOSL2, which in turn induces MICU3-mediated mitochondrial calcium influx and tumor cell death.

High expression of SLC34A2 contributes to chemoresistance of non-small cell lung cancer against gefitinib: The critical role of miR-124–3p

Gefitinib is a therapeutic agent used to treat lung carcinoma, including non-small cell lung cancer (NSCLC). However, mechanisms underlying NSCLC cell resistance to gefitinib remain largely uncharacterized. In this study, we explored the association between the miR-124–3p/SLC34A2 axis and gefitinib resistance using a series of in vivo and in vitro assays. Data indicated that miR-124–3p is downregulated, while SLC34A2 is upregulated, in gefitinib-resistant NSCLC cells. Overexpression of miR-124–3p reduced NSCLC cell resistance to gefitinib by suppressing cell viability, inducing apoptosis, and decreasing N-cadherin expression. Conversely, inhibiting miR-124–3p in NSCLC cells led to increased cell viability and reduced apoptosis. Overexpression of SLC34A2 in NSCLC cells further heightened gefitinib resistance. In a xenograft mouse model, SLC34A2 overexpression promoted solid tumor growth and metastasis, while miR-124–3p overexpression inhibited these effects. Our results highlight that the interaction between miR-124–3p and SLC34A2 plays an indispensable role in determining gefitinib resistance in NSCLC cells.

IGF2BP3/CTCF Axis-Dependent NT5DC2 Promotes M2 Macrophage Polarization to Enhance the Malignant Progression of Lung Squamous Cell Carcinomas.

Lung squamous cell carcinoma (LUSC) is a type of lung cancer that develops in the squamous cells. It is known to be promoted by the activation of various signaling pathways and the dysregulation of key regulatory molecules. One such molecule, 5'-nucleotidase domain containing 2 (NT5DC2), has been identified as a critical regulator in various cancers including lung cancer. However, there are no data regarding its role in LUSC. The mRNA expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), CCCTC-binding factor (CTCF), and NT5DC2 was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR), whereas their protein expression was assessed using a western blotting assay. Cell proliferation was determined using a cell counting kit-8 (CCK-8) assay. Cell apoptosis, CD11b expression, and CD206 expression were analyzed using flow cytometry. Tube formation was assessed through a tube formation assay. Glucose consumption, lactate production, and ATP levels were measured using colorimetric methods. The effect of NT5DC2 on the malignant progression of LUSC cells was analyzed using a xenograft mouse model assay. The levels of transforming growth factor-beta 1 (TGF-β1) and interleukin-10 (IL-10) were detected using enzyme-linked immunosorbent assays. The associations among IGF2BP3, CTCF and NT5DC2 were identified using dual-luciferase reporter assay, RNA immunoprecipitation assay and m6A RNA immunoprecipitation assay. The expression of NT5DC2 was found to be upregulated in LUSC tissues and cells when compared with normal lung tissues and normal human bronchial epithelial cells. Silencing of NT5DC2 inhibited LUSC cell proliferation, tube formation, glycolysis, M2 macrophage polarization, and tumor formation while inducing cell apoptosis. In addition, CTCF was found to transcriptionally activate NT5DC2 in LUSC cells. IGF2BP3 stabilized the mRNA expression of CTCF through m6A methylation. Further, overexpression of CTCF or NT5DC2 attenuated the effects of IGF2BP3 silencing in both NCI-520 and SK-MES-1 cells. The IGF2BP3/CTCF axis-dependent NT5DC2 promotes M2 macrophage polarization, thereby enhancing the malignant progression of LUSC. This study was the first to reveal the role of NT5DC2 in LUSC and the underlying mechanism. The result suggests that targeting the IGF2BP3/CTCF/NT5DC2 axis may have clinical significance in the treatment of LUSC.

Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Chemotherapy is crucial in NSCLC treatment, and targeting Wee1 kinase, a key regulator of the G2/M cell cycle checkpoint, may enhance the efficacy of cytotoxic agents. This study investigates the potential of the Wee1 inhibitor MK-1775 in combination with gemcitabine and pemetrexed to enhance cytotoxicity in NSCLC cell lines. MethodsHuman NSCLC cell lines H1975, HCC827, A549, and H460 were treated with MK-1775 and chemotherapeutic agents, both alone and in combination. Growth inhibitory effects were assessed using the CCK8 assay. Apoptotic markers were evaluated via Western blotting, and cell cycle distribution was analyzed using FACS. In vivo efficacy was assessed using xenograft mouse models with H1975 and H460 cells, monitoring tumor growth and treatment toxicity. ResultsMK-1775 combined with gemcitabine or pemetrexed significantly decreased cell survival rates and IC50 values in A549 and HCC827 cell lines. Increased levels of phosphorylated cdc2, γ-H2AX, and PARP indicated enhanced apoptosis. Cell cycle analysis revealed G2/M phase arrest in p53-mutant HCC827 and H1975 cells treated with MK-1775 and gemcitabine. In xenograft models, the combination significantly inhibited tumor growth without significant toxicity. ConclusionsMK-1775 enhances the cytotoxic effects of gemcitabine and pemetrexed in NSCLC cell lines and effectively inhibits tumor growth in vivo. These findings suggest that Wee1 inhibition by MK-1775, combined with chemotherapy, represents a promising therapeutic strategy for NSCLC treatment.

Pyrotinib induces cell death in HER2-positive breast cancer via triggering HSP90-dependent HER2 degradation and ROS/HSF-1-dependent oxidative DNA damage.

HER2-positive breast cancer (HER2+ BC) is distinguished by its poor prognosis, propensity for early onset, and high risk of recurrence and metastasis. Consequently, anti-HER2-targeted therapy has emerged as a principal strategy in the treatment of this form of breast cancer. Pyrotinib, a novel irreversible pan-HER2 tyrosine kinase inhibitor, has brought fresh hope to patients with advanced HER2+ breast cancer. In this study, we conducted a comprehensive exploration of pyrotinib's anti-tumor mechanism. The in vitro results showed that pyrotinib significantly inhibited SKBR3 cells viability and induced apoptosis by promoting HER2 endocytosis and ubiquitylation, leading to HER2 degradation through the displacement of HSP90 from HER2. Beyond targeting the HER2 signaling pathway, pyrotinib also induced DNA damage, which was mediated by the activation of the ROS/HSF-1 signaling pathway and the downregulation of PCNA expression. Furthermore, the in vivo results demonstrated a pronounced anticancer effect of pyrotinib in the SKBR3 xenograft mouse model, concomitant with a reduction in HER2 expression. In summary, our findings provide novel insights into the mechanism of pyrotinib in the treatment of HER2+ BC.

Codelivery of metformin and methotrexate with optimized chitosan nanoparticles for synergistic triple-negative breast cancer therapy in vivo

The development of effective therapeutic strategies for triple-negative breast cancer (TNBC), an aggressive subtype with limited treatment options, remains a critical challenge. This study aimed to design and evaluate a combination therapy using chitosan nanoparticles (Cs NPs) loaded with metformin (Met) and methotrexate (MTX) as a promising approach for TNBC management.The Cs NPs exhibited an average size of 78.8 ± 25.84 nm for blank Cs NPs, 84.50 ± 22.54 nm for Met-Cs NPs, and 86.70 ± 30.90 nm for MTX-Cs NPs, with positive surface charges of 26.40 ± 1.40 mV, 28.20 ± 1.60 mV, and 14.30 ± 2.40 mV, respectively. The drug encapsulation efficiency was 88.56 ± 2.26 % for Met-Cs NPs and 97.03 ± 0.52 % for MTX-Cs NPs.The cellular uptake studies demonstrated a time-dependent increase in the accumulation of Shikonin-labeled Cs NPs in 4T1 cells. The cytotoxicity assays revealed that Met-Cs NPs and MTX-Cs NPs exhibited significantly lower IC50 values (19.85 μg/mL and 103.2 ng/mL, respectively) compared to the plain drugs at 48 h. The combination of Met-/MTX-Cs NPs showed a synergistic cytotoxic effect, inducing 50 % cell death at 15.233 μg/mL of Met and 0.166 μg/mL of MTX. In vivo studies using a 4T1 xenograft mouse model demonstrated that the combination of Met-/MTX-Cs NPs resulted in a 100 % reduction in initial tumor volume, compared to a 40 % decrease with the free drug combination. The tumor growth inhibition was 70.45 % for the Met-/MTX-Cs NPs group, significantly higher than the 33.86 % observed in the free drug combination group. The findings of this study highlight the potential of the Met-/MTX-Cs NPs combination as a novel and effective therapeutic approach for TNBC management. The enhanced therapeutic efficacy, improved safety profile, and the ability to modulate key signaling pathways make this nanoparticle-based combination therapy a promising candidate for further clinical investigation.

Parecoxib inhibits tumorigenesis and angiogenesis in hepatocellular carcinoma through ERK-VEGF/MMPs signaling pathway.

Parecoxib, a well-recognized nonsteroidal anti-inflammatory drug, has been reported to possess anticancer properties in various tumor types. In this work, we aimed to investigate the potential anticancer effects of parecoxib on hepatocellular carcinoma (HCC) cells. To assess the impact of parecoxib on HCC cell proliferation, we employed Cell Counting Kit-8, colony formation, and 5-ethynyl-2'-deoxyuridine assays. Hoechst/propidium iodide (PI) double staining and flow cytometry were performed to evaluate apoptosis and cell cycle analysis. Wound healing and transwell assays were utilized to assess cell migration and invasion. Tube formation assay was employed to analyze angiogenesis. Protein levels were determined using western blotting, and mRNA expression levels were assessed using quantitative real-time polymerase chain reaction (PCR). A xenograft mouse model was used to confirm the antitumor effects of parecoxib on HCC tumors in vivo. Our data demonstrated that parecoxib effectively inhibited the proliferation of HCC cells in a dose- and time-dependent manner. In addition, parecoxib induced cell cycle arrest in the G2 phase and promoted apoptosis. Moreover, parecoxib hindered tumor migration and invasion by impeding the epithelial-mesenchymal transition process. Further investigation showed that parecoxib could significantly suppress angiogenesis through the inhibition of extracellular signal-regulated kinase (ERK)-vascular endothelial growth factor (VEGF) axis. Notably, treatment with the ERK activator phorbol myristate acetate upregulated the expression of matrix metalloproteinase (MMP)-2, MMP-9, and VEGF and reversed the function of parecoxib in HCC cells. Besides, parecoxib displayed its antitumor efficacy in vivo. Collectively, our results suggest that parecoxib ameliorates HCC progression by regulating proliferation, cell cycle, apoptosis, migration, invasion, and angiogenesis through the ERK-VEGF/MMPs signaling pathway.

Cyclovirobuxine D inhibits triple-negative breast cancer via YAP/TAZ suppression and activation of the FOXO3a/PINK1-Parkin pathway-induced mitophagy

BackgroundTriple-negative breast cancer (TNBC) is characterized by its rapid progression and aggressive nature, with limited effective therapeutic interventions currently available. Cyclovirobuxine D (CVB-D), a natural alkaloid extracted from the traditional Chinese herb Buxus sinica, is renowned for its cardioprotective and anti-ischemic effects, demonstrating notable anti-cancer properties. Nevertheless, the anti-tumor effects of CVB-D on TNBC remain unverified. PurposeThis study seeks to investigate the effects of CVB-D on TNBC and to uncover the underlying mechanisms. Study DesignNetwork pharmacology, SPR, DSF, and cell-based functional assays were conducted on TNBC cells to assess the impact of CVB-D. Findings were further corroborated using xenograft mouse models. MethodsCell Counting Kit-8, 5-Ethynyl-2′-deoxyuridine, transwell assays, flow cytometry, wound healing assays, immunofluorescence, and immunoblotting were employed to evaluate CVB-D's influence on TNBC cell lines. SPR, DSF and molecular docking techniques were utilized to assess the binding affinity of CVB-D to Yes-associated protein (YAP). The interaction between CVB-D and autophagy/mitophagy was further analyzed through plasmid transient transfection, JC-1 assay, TUNEL assay, and the use of autophagy inhibitors. The anti-TNBC mechanism of CVB-D was elucidated by overexpressing YAP in MDA-MB-231 cells. Additionally, the in vivo efficacy and safety of CVB-D were assessed in a xenograft mouse model. ResultsIn vitro analyses revealed that CVB-D effectively suppressed G1 phase arrest and inhibited TNBC cell proliferation. Moreover, CVB-D induced mitochondrial-dependent apoptosis and reduced cell migration by antagonizing epithelial-mesenchymal transition. Mechanistically, CVB-D exerted its anti-cancer effects by directly binding to YAP, thereby inhibiting the nuclear translocation of YAP/TAZ and suppressing the transcription of downstream oncogenic target genes. Furthermore, CVB-D triggered excessive mitophagy by activating the FOXO3a/PINK1-Parkin axis, promoting apoptosis and leading to mitochondrial dysfunction in TNBC cells. Elevated YAP expression counteracted the effects of CVB-D on TNBC, including the suppression of mitophagy-related protein expression induced by CVB-D, suggesting that YAP modulates mitophagy through the FOXO3a/PINK1-Parkin axis. The anti-tumor efficacy of CVB-D and its underlying mechanisms were further substantiated using a subcutaneous xenograft model. ConclusionsThis study is the first to demonstrate that CVB-D can directly bind to the YAP target, proposing a novel therapeutic strategy for TNBC. CVB-D may serve both as a YAP/TAZ inhibitor and as an activator of the FOXO3a/PINK1-Parkin axis, leading to excessive mitophagy.