Nude Mouse Xenograft Research Articles

Anticancer effect of covalent purine-containing EGFR TKI, ZZC4 and its mechanism of action through network pharmacology

AimsEpidermal growth factor receptor (EGFR) has been documented in many malignancies as participating in the progression of cancer cells. Here, we present a novel EGFR tyrosine kinase inhibitor, ZZC4, and examine its effect on cancer cell proliferation, migration, and tumor-bearing xenograft models. Main methodsThe antiproliferative effect of ZZC4 was assessed in vitro by MTT assay, colony formation, and wound healing assay and in vivo with tumor-bearing xenograft nude mice. Further, Western blotting analysis and computational network pharmacology were used to explore and understand the mechanism of ZZC4. Key findingsThe results showed that ZZC4 potently inhibited the proliferation of lung, breast, and melanoma cells, and was more sensitive to lung cancer cells HCC827, H1975, and breast cancer cell T47D. In vitro findings were corroborated in vivo as results showed the suppressive effect of ZZC4 on HCC827 and H1975 tumor growth. Western blotting analysis confirmed that ZZC4 is an effective inhibitor of the EGFR pathways as it down-regulated p-EGFR, p-Akt, and p-MAPK. Computational molecular docking confirmed the strong binding affinity between ZZC4 and EGFR. Moreover, network pharmacology suggested that ZZC4 might play a suppressive role in the progression of malignancies with EGFR/PI-3K/Akt axis dysregulation or in cancer-related drug resistance. SignificanceOur study showed that ZZC4 is an anticancer drug candidate.

Tumor-suppressive effect of Reg3A in COAD is mediated by T cell activation in nude mice

Regenerating family protein 3 A (Reg3A) is highly expressed in a variety of organs and inflammatory tissues, and is closely related to tumorigenesis and cancer progression. However, clinical statistics show that high expression of Reg3A is associated with better prognosis in colorectal cancer (CRC) patients, suggesting a tumor-suppressive effect. The precise action and underlying mechanism of Reg3A in CRC remain controversial. The present study sought to investigate the relationship among Reg3A expression, CRC development, and immune cell alteration in patients using the TCGA, GEPIA, PrognoScan, TIMER and TISIDB databases. Reg3A-overexpressing LoVo cell line (LoVo-Reg3A), a representative of colon adenocarcinoma (COAD), was constructed and the action of Reg3A was assessed in a xenograft nude mouse model. Our bioinformatical analyses revealed that Reg3A upregulation is highly associated with CRC, along with increased frequency of immune cell infiltration. In the xenograft nude mice, Reg3A overexpression offered a tumor-suppressive effect by inhibiting cell proliferation and promoting apoptosis. The result of RNA-seq suggested a positive regulation of leukocytes and an upregulation of T cells in LoVo-Reg3A tumor tissue. CD4+ and CD8+ T cells in tumors, splenic Reg3A-reactive IFN-γ+/CD4+ T cells, and serum TNF-α, IFN-γ and IL-17 were significantly increased by Reg3A overexpression. In the ex vivo co-culture experiment, elevated cytotoxic effect, increased proportion of CD3ε+ T cells, and upregulated expressions of TNF-α, IFN-γ and IL-17 were detected in the PBMCs isolated from LoVo-Reg3A cell-xenografted nude mice. In conclusion, high expression of Reg3A could activate and recruit T cells in COAD leading to the cytotoxic tumor-suppressive effect.

Silencing LY6D Expression Inhibits Colon Cancer in Xenograft Mice and Regulates Colon Cancer Stem Cells' Proliferation, Stemness, Invasion, and Apoptosis via the MAPK Pathway.

This study explored the role of lymphocyte antigen 6 family member D (LY6D) in colon cancer stem cells' (CCSCs) proliferation and invasion. LY6D was knocked down using siRNA, and the down-regulation of LY6D was verified using Western blotting. After LY6D knockdown, CCSCs' proliferation, stemness, and invasion were suppressed, whereas apoptosis was increased. Gene Ontology (GO) enrichment analysis revealed that the differentially expressed genes (DEGs) between siLY6D and the negative control groups were significantly enriched in the cell-substrate adherens junction, focal adhesion, and cell-substrate junction terms. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DEGs were significantly enriched in the MAPK pathway. In addition, Western blotting results showed that pBRAF and pERK1/2, cascade kinases of the MAPK pathway, were significantly down-regulated after LY6D knockdown. In addition, nude mice xenograft experiments showed that the siLY6D treatment decreased tumor sizes and weights and improved tumor-bearing mice survival rates compared with the control group. In conclusion, these findings indicate that LY6D, which is highly expressed in CCSCs, is a key factor involved in tumor growth and development and might be a potential cancer marker and therapeutic target for colon cancer.

Chemokine Receptor CXCR4 Radioligand Targeted Therapy Using 177Lutetium-pentixather for Pulmonary Neuroendocrine Cancers.

Intermediate to high-grade lung neuroendocrine tumors (NETs; i.e., atypical carcinoid tumors) and neuroendocrine carcinomas (NECs) are currently difficult to cure. These tumors were found to express the CXCR4 G-protein coupled receptor that can be targeted with radioligands. PCR and flow cytometric analysis of lung NET and NEC cell lines using an anti-CXCR4 antibody demonstrated that all cell lines tested expressed CXCR4. PET/CT imaging with 68Galium-pentixafor in mouse xenografts of NETs and NECs verified tumor targeting that was blocked by a CXCR4 agonist. Clonogenic survival analysis demonstrated a more than additive enhancement of killing when 1 μM auranofin (a thioredoxin reductase inhibitor) was used as a radiosensitizer in combination with 177Lu-pentixather (10 μCi). DMS273 small cell lung cancer xenografts in female nude mice treated with 25 μCi/g 177Lu-pentixather induced inhibition of tumor growth and resulted in an increase in overall survival without causing unacceptable normal tissue toxicities. Immunohistochemical staining of 95 retrospective human samples (containing 90 small cell lung carcinomas) demonstrated 84% CXCR4 positivity. In a multivariable analysis of this cohort that included age, gender, stage, primary site, SSTR2 status, and CXCR4 status, Cox regression models determined that only distant metastasis at presentation (P < 0.01) and a CXCR4 H-score >30 (P = 0.04) were significantly associated with reduced survival. Prospective clinical testing of patient tumors identified CXCR4-positivity in 76% of 21 NECs, 67% of 15 lung NETs (including 8 of 10 atypical carcinoids), and 0% of 25 non-lung NETs (including 5 NETS G3s). These data support the hypothesis that CXCR4-targeted theranostics can be utilized effectively for select NETs and NECs.

Epigenetic inhibition of CTCF by HN1 promotes dedifferentiation and stemness of anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is one of the deadliest cancers, whose important malignant feature is dedifferentiation. Chromatin remodeling is critical for tumorigenesis and progression, while its roles and regulator in facilitating dedifferentiation of ATC had been poorly understood. In our study, an emerging function of hematological and neurological expressed 1 (HN1) in promoting dedifferentiation of ATC cells was uncovered. HN1 expression was negatively correlated with the thyroid differentiation markers both at mRNA and protein level. Knockdown of HN1 in ATC cells effectively upregulated the thyroid differentiation markers and impeded the sphere formation capacity, accompanying with the loss of cancer stemness. In contrast, overexpression of HN1 drove the gain of stemness and the loss of thyroid differentiation markers. Nude mouse and zebrafish xenograft models showed that inhibition of HN1 in ATC cells effectively hindered tumor growth due to the loss of cancer stemness. Further study showed that HN1 was negatively correlated with CTCF in an independent thyroid-cancer cohort, and inhibition of HN1 enhanced the expression of CTCF in ATC cells. Overexpression of CTCF significantly reversed the dedifferentiation phenotypes of ATC cells, whereas simultaneously inhibiting HN1 and CTCF was unable to recover the level of thyroid differentiation markers. The combination of ATAC-seq and ChIP-seq analysis confirmed that CTCF regulated genes relating with thyroid gland development through influencing their chromatin accessibility. HN1 inhibited the acetylation of H3K27 at the promoter of CTCF by recruiting HDAC2, thereby inhibiting the transcriptional activation of CTCF. These findings demonstrated an essential role of HN1 in regulating the chromatin accessibility of thyroid differentiation genes during ATC dedifferentiation.

Preliminary Investigation of the Molecular Mechanism of Empagliflozin Suppressing Gastric Cancer Through Mammalian Target of Rapamycin

To predict the intervention targets of empagliflozin (EMPA), a specific inhibitor of sodium-glucose cotransporter 2 (SGLT2), in gastric adenocarcinoma through comprehensive network pharmacology, and to validate the effects and the molecular mechanisms of EMPA through cellular and molecular biology experiments. Bioinformatics analysis of gastric adenocarcinoma was conducted to assess the correlation between gastric adenocarcinoma prognosis and SGLT2 expression. Network pharmacology was utilized to identify shared targets of EMPA and gastric adenocarcinoma. AGS cells, a human gastric adenocarcinoma cells line, were incubated with EMPA at different concentrations for 24 h and, then, cell proliferation was assessed using the CCK8 assay. After AGS cells were incubated with EMPA at the doses of 0, 3, and 6 mmol/L, real-time cell analysis (RTCA) and 5-ethynyl-2-deoxyuridine (EdU) incorporation were used to evaluate EMPA's inhibitory effects on the proliferation of the AGS cells. In addition, wound healing and Transwell assays were performed to assess the inhibitory effect of EMPA on the migration and invasion of the APC cells and Western blot analysis was conducted to examine the expression of mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR). BALB/c (nu/nu) nude mice were implanted with 5×106 AGS cells in the axilla. The mice were divided into three groups, a control group, a low-dose group, and a high-dose group, each consisting of 7 mice. After one week, the control group received daily intraperitoneal injections of normal saline, while the low-dose group and high-dose group received daily intraperitoneal injections of EMPA at the doses of 3 mg/kg and 5 mg/kg, respectively. The tumor volume was measured one week after the drug intervention started. Gastric adenocarcinoma patients with low expression of SGLT2 exhibited longer survival time and higher survival rate than those with high expression of SGLT2 did. A total of 104 EMPA-related potential targets and 2028 targets associated with gastric adenocarcinoma were identified. Among these, 45 targets associated with gastric adenocarcinoma overlapped with potential targets of EMPA. Further analysis revealed 10 relevant pathways and 4 core genes. The core genes were cyclin-dependent kinase 4 (CDK4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mTOR, and cyclin E1 (CCNE1). CCK-8 assay revealed that EMPA at concentrations ranging from 0.39 to 50 mmol/L effectively inhibited the proliferation of AGS cells. RTCA results indicated a downward shift in the cell growth curve. In comparison to the findings for the control group, EdU assay demonstrated that EMPA at the concentrations of 3 mmol/L and 6 mmol/L significantly inhibited AGS cell proliferation (P<0.05). Results from wound healing and Transwell assays indicated a decrease in the levels of cell migration and invasion (P<0.05) and, notably, there was a significant difference between the high and low-dose EMPA groups (P<0.05). Western blot showed no statistically significant difference in the expression of total mTOR protein between the groups. However, the expression of p-mTOR in the 3 mmol/L and 6 mmol/L EMPA groups decreased compared to that of the control group (P<0.05), with the 6 mmol/L EMPA group exhibiting a more pronounced reduction (P<0.05). Nude mice xenograft tumor experiment demonstrated that, compared to that of the control group, the tumor volumes in the EMPA-treatment groups were significantly reduced (P<0.05), with the high-dose group showing a more pronounced reduction (P<0.05). EMPA inhibits the abnormal proliferation and migration of gastric adenocarcinoma cells, potentially through the modulation of mTOR protein activation. This study provides new potential medication and intervention targets for gastric adenocarcinoma treatment.

Remodeling of intestinal epithelium derived extracellular vesicles by nanoparticles and its bioeffect on tumor cell migration

Extracellular vesicles (EVs) are an effective tool to elucidate the bioeffect of nanomedicines. To clarify the interaction between oral nanomedicines and intestinal epithelial cells, and their bioeffects on downstream cells, polystyrene nanoparticles (PS-NPs) with different sizes were used as the model nanomedicines for EVs induction. Caco-2 monolayers were selected as the model of the intestinal epithelium and DLD-1 cells as the colorectal cancer model proximal to the gastrointestinal tract. It is found that compared with small-sized (25, 50, 100 nm) PS-NPs, the large-sized (200 and 500 nm) exhibited higher co-localization with multivesicular bodies and lysosomes, and more significant reduction of lysosomal acidification in Caco-2 cells. Proteomic and western-blotting analysis showed that the EVs remodeled by large-sized PS-NPs exhibited a higher extent of protein expression changes. The in vitro and in vivo signaling pathway detection in DLD-1 cells and DLD-1 cell xenograft nude mice showed that the remodeled EVs by large-sized PS-NPs inhibited the activation of multiple signaling pathways including Notch3, EGF/EGFR, and PI3K/Akt pathways, which resulted in the inhibition of tumor cell migration. These results primarily clarify the regulation mechanisms of nanomedicines-EVs-receptor cells chain. It provides a new perspective for the rational design and bioeffect evaluation of oral drug nanomaterials and sets up the fundamental knowledge for novel tumor therapeutics in the future.

Highly expressed RPLP2 inhibits ferroptosis to promote hepatocellular carcinoma progression and predicts poor prognosis

BackgroundRPLP2, an integral part of ribosomal stalk, plays an important role in the tumorigenesis of various cancers. However, its specific effect on HCC remains elusive.MethodsTCGA, GTEx, HCCDB, HPA, UALCAN, MethSurv, TISIDB, K–M plotter, FerrDb, RNAactDrug, STRING, Cytoscape and R studio were conducted for bioinformatics analysis. RPLP2 expression level in HCC was verified by IHC and western blot. IHC was used to demonstrate the immune cell infiltration. Functional experiments including CCK8, transwell and colony formation assays, and nude mice xenograft model were performed for in vitro and in vivo validation. Western blot, IHC, CCK8 assay and detection of GSH and lipid ROS were adopted to determine the effect of RPLP2 on the ferroptosis of HCC cells.ResultsHere, we demonstrate that elevated level of RPLP2 is strongly associated with advanced clinicopathologic features, and predicts poor prognosis of HCC patients. Additionally, DNA methylation level of RPLP2 decreases in HCC, and significantly correlates with patients outcome. Moreover, high RPLP2 expression level is linked closely to the unfavorable immune infiltration. Most importantly, RPLP2 positively associates with ferroptosis suppressor GPX4, and inhibition of RPLP2 could lead to the acceleration of ferroptosis to suppress tumor progression of HCC. Last, drug sensitivity analysis predicts many drugs that potentially target RPLP2.ConclusionTogether, our study reveals previous unrecognized role of RPLP2 in HCC, and provides new regulatory mechanism of ferroptosis, indicating RPLP2 may be a novel therapeutic target for HCC.

Yi-qi-hua-yu-jie-du decoction induces ferroptosis in cisplatin-resistant gastric cancer via the AKT/GSK3β/NRF2/GPX4 axis

BackgroundResistance to chemotherapy in gastric cancer (GC) is a ubiquitous challenge for its treatment. Yi-qi-hua-yu-jie-du decoction (YJD), an empirical formula in Traditional Chinese Medicine (TCM), demonstrated survival-prolonging functions in patients with GC. Previous research has shown that YJD could also inhibit drug resistance in GC. However, the precise mechanisms for how YJD accomplishes this remain incompletely explained. PurposeThe research aimed to identify differential metabolic characteristics in cisplatin-resistant GC and investigate whether YJD can target these differences to suppress GC drug resistance. MethodsMetabolomic analysis was conducted to identify metabolic disparities between cisplatin-resistant and parental GC cells, as well as metabolic modifications resulting from YJD intervention in cisplatin-resistant GC cells. The effect of YJD on ferroptosis stimulation was assessed by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA), iron ions, the reduced glutathione (GSH) to oxidised glutathione (GSSG) ratio, and alterations in mitochondrial morphology. Western blotting and quantitative real-time polymerase chain reaction (Q-PCR) were employed to verity the mechanisms of YJD-triggered ferroptosis through GPX4 and NRF2 overexpression models, alongside the AKT activator SC79. In vivo validation was conducted using nude mouse xenograft models. ResultsCisplatin-resistant GC exhibited altered GSH/GPX4 metabolism, and ferroptosis was a significantly enriched cell death pattern with YJD treatment in cisplatin-resistant GC cells. Ferroptosis biomarkers, including ROS, MDA, iron ions, the GSH/GSSG ratio, and mitochondrial morphology, were remarkably changed with the YJD intervention. Mechanistic experiments demonstrated that YJD inhibited the phosphorylation cascade activity of the AKT/GSK3β pathway, thereby reducing NRF2 expression. The level of GPX4, a crucial enzyme involved in glutathione metabolism, was attenuated, facilitating ferroptosis induction in cisplatin-resistant GC. ConclusionThe research reveals, for the first time, changes in GSH/GPX4 metabolism in cisplatin-resistant GC cells based on metabolomic analysis. YJD induced ferroptosis in cisplatin-resistant GC by inhibiting GPX4 through the AKT/GSK3β/NRF2 pathway, thus attenuating the cisplatin drug resistance in GC. Our findings identify metabolic changes in cisplatin-resistant GC and establish a theoretical framework for YJD on tackling drug resistance in GC through ferroptosis.

Biological and genetic characterization of a newly established human external auditory canal carcinoma cell line, SCEACono2

Squamous cell carcinoma of the external auditory canal (EACSCC) is an extraordinarily rare and aggressive malignant disease. Establishment of EACSCC cell line with robust molecular characteristics is essential for the basic and translational research of EACSCC. Here, we show the newly established EACSCC cell line SCEACono2, derived from a patient with well-to-moderately differentiated EACSCC. We analyzed histologic and genetic features of SCEACono2 hiring multiple experiments, including next-generation sequencing (NGS). Immunocytochemical staining of SCEACono2 showed positivity of p53 and SCC1/2. Furthermore, SCEACono2 exhibited a unique characteristic that cytokeratin, vimentin as well as cancer stem cell markers (CD44, CD133, ALP and Oct3/4) were positive. SCEACono2 had an ability to form tumors at the temporal lesion xenograft nude mice model. NGS revealed that SCEACono2 harbored the somatic mutations of TP53 (p.G245S) and NOTCH1 (p.A465T). RNA-seq and downstream bioinformatics analysis revealed significant enrichment of genes involved in inflammation and cell adhesion in SCEACono2 compared to SCC-9 and HSC-4. STR profiling indicated no evidence of cross-contamination. In conclusion, SCEACono2 could serves as a promising and robust research resource of EACSCC in vitro and in vivo.

Expression of ZNF281 in colorectal cancer correlates with response to radiotherapy and survival

Background The treatment of Colorectal cancer (CRC) is extremely complex and survival rates vary depending on the stage of the disease at the time of diagnosis. Neoadjuvant chemoradiotherapy (NACRT), is the conventional treatment for locally advanced rectal cancer (LARC); however, the resistance to chemoradiotherapy in LARC is difficult to predict. Materials and methods In this study, clinical data of 126 LARC patients were collected and analyzed, and relevant validation was performed using GEO database and in vitro and in vivo experiments, including Western blotting and Real-time quantitative PCR, immunohistochemistry, immunofluorescence, clonogenic cell survival assays, and nude-mouse xenograft models. Results In patients with LARC who were treated with neoadjuvant radiotherapy (NART), higher ZNF281 expression in malignant tissue was associated with a poorer prognosis and lesser degree of tumor regression. Cell and mouse experiments have shown that ZNF281 reduces the damage caused by X-rays to CRC cells and tumors grown in mice. Conclusion We found that the expression of ZNF281 predicted the radiation response of CRC cells and suggested the prognosis of patients with LARC who received neoadjuvant radiation therapy.

Construction of truncated PSMA as a PET reporter gene for CAR T cell trafficking.

In solid tumors, there are multiple barriers for a chimeric antigen receptor (CAR) T cell to surmount in order to reach the tumor site. For better understanding whether CAR T cells effectively infiltrate into tumor site, and simultaneously, whether there are off-target effects, real-time monitoring technologies need to be established. Cell-based positron emission tomography reporter genes have been developed to monitor engineered cells in living subjects. In this study, we reported the construction of a novel reporter gene truncated prostate-specific membrane antigen (ΔPSMA) pending for monitoring CAR T cells using 68Ga-PSMA-617 and a method for tracking the distribution of CAR T cells in vivo was developed. Data were provided to demonstrate that ΔPSMA was predominantly localized on the plasma membrane and could take up 68Ga-PSMA-617 in vitro in a time-dependent manner. And the expression of ΔPSMA did not affect CAR expression and cytolytic capacity of CAR T cells. CAR-ΔPSMA T cell xenografts in nude mice were clearly imaged by positron emission tomography 60 min after injection of 68Ga-PSMA-617. PSMA paired with 68Ga-PSMA-617 was capable of identifying approximately 1 × 104 engineered CAR T cells. The ability to image small numbers of CAR T cells in vivo would be helpful to accelerate the translation of cell-based therapies into the clinic, and it may reinforce our understanding of treatment success, failure, and toxicity.

Effect of paclitaxel octreotide conjugate on human ovarian paclitaxel-resistant cell xenograft tumor model and the mechanism underlying reversal of paclitaxel resistance

Purpose: To determine the effect of paclitaxel octreotide conjugate (POC) on human ovarian paclitaxelresistant cell xenograft tumor model and the mechanism underlying reversal of paclitaxel resistance.&#x0D; Methods: Forty female BALB/c-nu/nu mice were subcutaneously inoculated with 106 paclitaxel-resistant cells (a2780/taxol) per mouse during the logarithmic growth phase of ovarian cancer. They were randomly divided into four groups (control, octreotide, paclitaxel and POC). Immunohistochemical streptavidin-peroxidase (SP) method was used to determine expression of nuclear proliferation antigen (PCNA) while TUNEL method was used to assess apoptosis of human ovarian cancer metastasis. Realtime polymerase chain reaction (PCR) was used to assay mRNA expression levels of somatostatin receptor 2 (SSTR2), multidrug-resistant gene (MDR1), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and acetylated tubulin (α-tubulin and β-III-tubulin), while the corresponding protein expressions were assayed using western blotting.&#x0D; Results: Immunohistochemical SP showed significantly lower PCNA levels in octreotide, paclitaxel and POC groups than in control mice, but that of POC mice was significantly reduced, relative to those of octreotide and paclitaxel groups (p &lt; 0.05). There were significantly higher expression levels of SSTR2 mRNA and protein in octreotide, paclitaxel and POC groups than in control mice, but they were significantly higher in POC group than in octreotide and paclitaxel groups (p &lt; 0.05). The mRNA and protein expressions of other factors in POC mice were significantly lower than those in both octreotide and paclitaxel groups (p &lt; 0.05).&#x0D; Conclusion: Paclitaxel-octreotide conjugate effectively inhibits the growth of a2780/taxol xenografts in nude mice, induces tumor cell apoptosis, and suppresses tumor cell growth via mechanism involving enhancement of SSTR2 expression, and decreases in levels of acetylated tubulin, matrix metalloproteinase-9, and vascular endothelial growth factor.

Polyphyllin I attenuates the invasion and metastasis via downregulating GRP78 in drug-resistant hepatocellular carcinoma cells.

Drug resistance to chemotherapy agents presents a major obstacle to the effective treatment of hepatocellular carcinoma (HCC), a common type of liver cancer. Increasing evidence indicates a link between drug resistance and the recurrence of HCC. Polyphyllin I (PPI), a promising pharmaceutical candidate, has shown potential therapeutic advantages in the treatment of sorafenib-resistant hepatocellular carcinoma (SR-HCC cells). In this study, we sought to investigate the mechanism underlying the inhibitory effect of PPI on the invasion and metastasis of SR-HCC cells. Our in vitro studies included scratch wound-healing migration assays and transwell assays to examine PPI's effect on HCC cell migration and invasion. Flow cytometry was employed to analyze the accumulation or efflux of chemotherapy drugs. The results of these experiments demonstrated that PPI increased the susceptibility of HCC to sorafenib while inhibiting SR-HCC cell growth, migration, and invasion. Molecular docking analysis revealed that PPI exhibited a higher binding affinity with GRP78. Western blot analysis and immunofluorescence experiments showed that PPI reduced the expression of GRP78, E-cadherin, N-cadherin, Vimentin, and ABCG2 in SR-HCC cells. Interference with and overproduction of GRP78 in vitro impacted the proliferation, migration, invasion, and metastasis of HCC cells. Further examination revealed that PPI hindered the expression of GRP78 protein, resulting in a suppressive effect on SR-HCC cell migration and invasion. Histological examination of tumor tissue substantiated that administering PPI via gavage to HepG2/S xenograft nude mice inhibited tumor growth and significantly reduced tumor size, as evidenced by xenograft experiments involving nude mice. Hematoxylin and eosin (HE) staining of tumor tissue specimens, along with immunohistochemistry (IHC), were conducted to evaluate the expression levels of Ki67, GRP78, N-cadherin, Vimentin, and ABCG2. The results indicated that PPI administration decreased the levels of proteins associated with metastasis and markers of drug resistance in tumor tissues, impeding tumor growth and spread. Overall, our findings demonstrated that PPI effectively suppressed the viability, proliferation, invasion, and metastasis of SR-HCC cells both in vitro and in vivo by modulating GRP78 activity. These findings provide new insights into the mechanism of PPI inhibition of SR-HCC cell invasion and metastasis, highlighting PPI as a potential treatment option for sorafenib-resistant HCC.

REST-restrained lncRNA EPB41L4A-AS2 Modulates Laryngeal Squamous Cell Carcinoma Development via Regulating miR-1254/HIPK2 Pathway.

LncRNAs have been corroborated to exert crucial effects in malignancies, including laryngeal squamous cell carcinoma (LSCC). Nevertheless, the role and mechanism of EPB41L4A- AS2 in LSCC are inadequately investigated and warrant further exploration. Relevant database was adopted to analyze the relationship between EPB41L4A-AS2 expression level and tumors. The expressions and relationships of EPB41L4A-AS2, RE-1 silencing transcription factor (REST), miR-1254, and homeodomain interacting protein kinase 2 (HIPK2) in LSCC cells were evaluated by qRT-PCR, Pearson's correlation tests, RNA immunoprecipitation, RNA pull-down assay, chromatin immunoprecipitation, database, and dual-luciferase reporter assay. Following the required transfection, the biological behaviors of LSCC cells were examined using cell function experiments. Meanwhile, the levels of Ki-67 and apoptosis-, and epithelial-mesenchymal transition (EMT) pathway-related proteins were quantified with Western blot. Moreover, xenografts in nude mice were constructed, and the tumor volume and weight were measured. Ki-67 positivity was determined by immunohistochemical staining. EPB41L4A-AS2 and HIPK2 were lower-expressed, yet miR-1254 and REST were higher- expressed in LSCC cells. Pearson's correlation assay results exhibited a positive correlation between HIPK2 and EPB41L4A-AS2 and a negative correlation between HIPK2 and miR-1254. Overexpressed EPB41L4A-AS2 diminished the biological behavior, and repressed the levels of Ki-67 and EMT-related markers in LSCC cells whilst enhancing those of apoptosis-related markers. These aforementioned effects were counteracted by miR-1254 mimic. Moreover, EPB41L4A- AS2 overexpression suppressed the growth of tumors and reduced the positive expression of Ki-67 in nude mice. Besides, miR-1254 aggravated the biological behaviors and elevated the levels of Ki-67 and EMT-related proteins in LSCC cells while reducing the levels of apoptosis-related markers via targeting HIPK2. REST-restrained EPB41L4A-AS2 modulates LSCC development via regulating miR-1254/HIPK2 pathway.

Hsa_circ_0001687 Function as a ceRNA to Facilitate Hepatocellular Carcinoma Progression via miR-140- 3p/FOXQ1 Axis.

Increasingly convincing evidence has revealed that circular RNAs (circRNAs) are critical regulatory components of hepatocellular carcinoma (HCC) genesis. However, the expression of circRNAs in HCC and the relevance of circRNAs to HCC progression remain largely unexplained. qRT-PCR or western blotting was utilized to confirm circ_0001687, miR-140-3p, and Forkhead Box q1 (FOXQ1) levels in HCC tissues or cells. Cell proliferation ability was evaluated via CCK-8 and colony formation assay. The correlation of circ_0001687 or FOXQ1 and miR-140- 3p was determined using dual luciferase reporter assay. Nude mice xenograft tumor model was constructed to verify the effect of circ_0001687 on tumor growth. Circ_0001687 was elevated in HCC. Function assays and the nude mice xenograft tumor model indicated that circ_0001687 acts as a promoting gene in HCC to regulate the proliferation of the tumor cell and foster tumor growth. Further mechanistic exploration revealed that the tumor growth-promoting mechanism of circ_0001687 relied on blocking the inhibitory effect of miR-140- 3p on FOXQ1 and activating FOXQ1 expression. This research indicated the role of circ_0001687/miR-140-3p/FOXQ1 network in regulating HCC development. These may provide new insights into the treatment of HCC.

Mechanistic engineering of celastrol liposomes induces ferroptosis and apoptosis by directly targeting VDAC2 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of most common and deadliest malignancies. Celastrol (Cel), a natural product derived from the Tripterygium wilfordii plant, has been extensively researched for its potential effectiveness in fighting cancer. However, its clinical application has been hindered by the unclear mechanism of action. Here, we used chemical proteomics to identify the direct targets of Cel and enhanced its targetability and anti-tumor capacity by developing a Cel-based liposomes in HCC. We demonstrated that Cel selectively targets the voltage-dependent anion channel 2 (VDAC2). Cel directly binds to the cysteine residues of VDAC2, and induces cytochrome C release via dysregulating VDAC2-mediated mitochondrial permeability transition pore (mPTP) function. We further found that Cel induces ROS-mediated ferroptosis and apoptosis in HCC cells. Moreover, coencapsulation of Cel into alkyl glucoside-modified liposomes (AGCL) improved its antitumor efficacy and minimized its side effects. AGCL has been shown to effectively suppress the proliferation of tumor cells. In a xenograft nude mice experiment, AGCL significantly inhibited tumor growth and promoted apoptosis. Our findings reveal that Cel directly targets VDAC2 to induce mitochondria-dependent cell death, while the Cel liposomes enhance its targetability and reduces side effects. Overall, Cel shows promise as a therapeutic agent for HCC.

FASN promotes gallbladder cancer progression and reduces cancer cell sensitivity to gemcitabine through PI3K/AKT signaling.

Lipid metabolism plays an important role in the growth and development of tumors. However, the role of lipid metabolism in gallbladder cancer (GBC) has not been clearly clarified. Here, we demonstrated that fatty acid synthase (FASN), a key enzyme in de novo fatty acid biosynthesis, had upregulated expression in GBC samples both at protein and mRNA levels. Analysis of clinical data indicated the association between elevated FASN expression and poorer histology grades. Furthermore, FASN activity impairment through FASN knockdown or treatment with orlistat resulted in the inhibition of cell proliferation and migration, as well as increased sensitivity to gemcitabine. Both FASN knockdown and orlistat treatment induced cell apoptosis. Mechanistically, impairment of FASN activity suppressed the activation of the PI3K/AKT signaling pathway, which led to increased cell apoptosis and sensitivity to gemcitabine. These findings were also validated through nude mouse xenograft models, thus highlighting the potential of targeting FASN as a clinical treatment strategy. Collectively, the present study underscores the crucial role of FASN in the progression of gallbladder cancer via the PI3K/AKT pathway.

Induction of ferroptosis and apoptosis in endometrial cancer cells by dihydroisotanshinone I

Danshen, also known as Salvia miltiorrhiza, is a medicinal herb used in traditional Chinese medicine. Its potential impact on endometrial cancer has not been thoroughly investigated. This study aimed to examine the effect of dihydroisotanshinone I (DT), a compound found in Danshen, on the viability of ARK1 and ARK2 endometrial cancer cells and its mechanisms. The results showed that 10 μM DT inhibited cell viability of ARK1 and ARK2 cells by inducing apoptosis and ferroptosis, which was achieved by blocking the expression of GPX4. In vivo experiments using a xenograft nude mouse model indicated that DT treatment significantly reduced tumor volume without causing any adverse effects. These findings suggest that DT may be a potential therapeutic agent for inhibiting endometrial cancer cell viability, but further research is needed to confirm these results.

HIMB1636-MMAE, a Novel TROP2-Targeting Antibody-Drug Conjugate Exerting Potent Antitumor Efficacy in Pancreatic Cancer.

Herein, we first prepared a novel anti-TROP2 antibody-drug conjugate (ADC) hIMB1636-MMAE using hIMB1636 antibody chemically coupled to monomethyl auristatin E (MMAE) via a Valine-Citrulline linker and then reported its characteristics and antitumor activity. With a DAR of 3.92, it binds specifically to both recombinant antigen (KD ∼ 0.687 nM) and cancer cells and could be internalized by target cells and selectively kill them with IC50 values at nanomolar/subnanomolar levels by inducing apoptosis and G2/M phase arrest. hIMB1636-MMAE also inhibited cell migration, induced ADCC effects, and had bystander effects. It displayed significant tumor-targeting ability and excellent tumor-suppressive effects in vivo, resulting in 5/8 tumor elimination at 12 mg/kg in the T3M4 xenograft model or complete tumor disappearance at 10 mg/kg in BxPc-3 xenografts in nude mice. Its half-life in mice was about 87 h. These data suggested that hIMB1636-MMAE was a promising candidate for the treatment of pancreatic cancer with TROP2 overexpression.