Cell Colony Formation Ability Research Articles

RUNX3 exerts tumor-suppressive role through inhibiting EXOSC4 expression.

Breast cancer severely affects women health. 70% of breast cancer are estrogen receptor positive. Breast cancer stem cells are a group of tumor with plasticity, causing tumor relapse and metastasis. RUNX3 is a tumor suppressor frequently inactivated in estrogen receptor positive breast cancer. However, the mechanism of how RUNX3 is involved in the regualation of cancer stem cell traits in estrogen receptor positive breast cancer remains elusive. In this study, we utilized cut-tag assay to investigate the binding profile RUNX3 in BT474 and T47D cell, and confirmed EXOSC4 as the bona-fide target of RUNX3; RUNX3 could bind to the promoter are of EXOSC4 to suppress its expression. Furthermore, EXOSC4 could increase the colony formation, cell invasion and mammosphere formation ability of breast cancer cells and upregulate the the expression of SOX2 and ALDH1. Consistent with these findings, EXOSC4 was associated with poorer survival for Luminal B/Her2 breast cancer patiens. At last, we confirmed that EXOSC4 mediated the tumor suppressive role of RUNX3 in breast cancer cells. In conclusion, we demonstrate that RUNX3 directly binds to the promoter region of EXOSC4, leading to the suppression of EXOSC4 expression and exerting a tumor-suppressive effect in estrogen receptor postivive breast cancer cells.

Bioinformatics approach combined with experimental verification reveals OAS3 gene implicated in paclitaxel resistance in head and neck cancer.

This study aimed to identify a candidate gene associated with paclitaxel (PTX) resistance and to evaluate functionally its biological role in the PTX-resistant head and neck squamous cell carcinoma (HNSCC) cell lines and clinical specimens. Microarray data series containing samples of different types of cancers resistant to PTX were analyzed and then a candidate gene associated with PTX resistance was identified using various bioinformatics tools. After the suppression of the target gene expression, changes in cell viability and colony-forming ability were evaluated in PTX-resistant FaDu and SCC-9 cell lines. Bioinformatics analyses of upregulated genes in PTX-resistant cancer cells indicated that OAS3 was associated with PTX resistance. The downregulation of OAS3 expression significantly reduced the viability and colony-forming capacity of PTX-resistant SCC-9 cells by inducing apoptosis and cell cycle arrest at G0/G1 phase. The therapeutic targeting of OAS3 may resensitize PTX-resistant HNSCC cells with high OAS3 expression to PTX treatment.

Blocking SLC7A11 attenuates the proliferation of esophageal squamous cell carcinoma cells

ABSTRACT The role of ferroptosis-associated gene SLC7A11 in esophageal cancer progression is largely unknown, therefore, the effects of blocking SLC7A11 on esophageal squamous cell carcinoma (ESCC) cells are evaluated. Results showed that SLC7A11 was overexpressed in ESCC tissues both in mRNA and protein levels. Blocking SLC7A11 using Erastin suppressed the proliferation and colony formation of ESCC cells, decreased cellular ATP levels, and improved ROS production. Sixty-three SLC7A11-binding proteins were identified using the IP-MS method, and these proteins were enriched in four signaling pathways, including spliceosome, ribosome, huntington disease, and diabetic cardiomyopathy. The deubiquitinase inhibitors PR-619, GRL0617, and P 22077 could reduce at least 40% protein expression level of SLC7A11 in ESCC cells, and PR-619 and GRL0617 exhibited suppressive effects on the cell viability and colony formation ability of KYSE30 cells, respectively. Erastin downregulated GPX4 and DHODH and also reduced the levels of β-catenin, p-STAT3, and IL-6 in ESCC cells. In conclusion, SLC7A11 was overexpressed in ESCC, and blocking SLC7A11 using Erastin mitigated malignant phenotypes of ESCC cells and downregulated key ferroptosis-associated molecules GPX4 and DHODH. The therapeutic potential of targeting SLC7A11 should be further evaluated in the future.

Chalcomoracin promotes apoptosis and endoplasmic reticulum stress in hepatocellular carcinoma cells.

Chalcomoracin (CMR), a Diels-Alder adduct obtained from mulberry leaves, demonstrated wide-spectrum anti-cancer activity. Herein, we aimed to explore the function of CMR and how it works in hepatocellular carcinoma (HCC). Human HCC cell lines Hep3B and SNU-387 were cultured and treated with various concentrations of CMR (1.5, 3, and 6 µM). Subsequently, the effects of CMR on cell viability, colony formation, apoptosis, migration, and invasion abilities were studied in vitro. Furthermore, the levels of endoplasmic reticulum (ER) stress-related proteins and mitogen-activated protein kinase (MAPK) pathway-related proteins in cells under CMR exposure were detected using western blot. Experiments in vivo were conducted to examine the effects of CMR on tumor growth in HCC. CMR administration inhibited the viability and clonogenic, migration, and invasion abilities, as well as promoted cell apoptosis and ER stress in Hep3B and SNU-387 cells. In addition, CMR treatment reduced the phosphorylation levels of ERK, P38, and JNK in the MAPK pathway. Moreover, an in vivo study showed that CMR administration could inhibit tumorigenesis and MAPK pathway activity in HCC. Our data indicate that CMR has the potential to inhibit the development of HCC, potentially through the inhibition of the MAPK pathway. These findings suggest that CMR may have promising applications as an anticancer agent in future therapeutics for HCC.

Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2.

Purpurogallin carboxylic acid (PCA) is a natural phenol compound derived from Macleaya microcarpa (Maxim.) Fedde, which exerts particular antioxidant and anti-inflammatory capacities. However, the effects and mechanisms of PCA on liver cancer cells remain unknown. Therefore, network pharmacology and computer virtual docking were used to identify the target-proteins of PCA. In addition, surface plasmon resonance, protease activity and rhodamine excretion assays were carried out to evaluate the effects of PCA on the activity of ATP binding cassette subfamily G member 2 (ABCG2). The synergistic effects of PCA and 5-fluorouracil (5-FU) on liver cancer cell proliferation, cell cycle arrest, colony formation and spheroid formation abilities in vitro were determined by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, western blot analysis, colony formation and spheroid formation assays, respectively. ABCG2 was identified as a potential target of PCA, with a high docking score. The equilibrium dissociation constant of PCA for ABCG2 protein was 1.84 µM, while the median inhibitory concentration of this protein was 3.09 µM. In addition, the results demonstrated that PCA could significantly reduce the drug efflux capacity of liver cancer cells. CCK-8 assays revealed that liver cancer cell treatment with 10 µM PCA and 10 µM 5-FU exhibited the most potent synergistic effects on liver cancer cell proliferation at 48 h. Additionally, cell co-treatment with PCA and 5-FU also significantly attenuated the colony and spheroid formation abilities of liver cancer cells in vitro, while it promoted their arrest at the G1 phase of the cell cycle. Furthermore, ABCG2 silencing in liver cancer cells notably abrogated the synergistic effects of PCA and 5-FU. In conclusion, the present study demonstrated that PCA exhibited synergistic effects with 5-FU on liver cancer cells in vitro via targeting ABCG2. Therefore, PCA combined with 5-FU may be a potential strategy for liver cancer therapy.

Effects of 4-hydroxy-2(3H)-benzoxazolone on proliferation and apoptosis of pancreatic cancer L3.6 cells

This study explored the effects of 4-hydroxy-2(3H)-benzoxazolone(HBOA) on the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. The L3.6 cells cultured in vitro were treated with HBOA of 0-1.0 mmol·L~(-1). The cell viability was detected by the cell counting kit-8(CCK-8) method, and the half inhibitory concentration(IC_(50)) was analyzed to determine the drug concentration and time. The cell morphology was observed under an inverted microscope and by acridine orange(AO) staining. The ability of proliferation and self-renewal were evaluated through live cell counting and colony formation experiments. The cell cycle progression and cell apoptosis rate were detected by flow cytometry. The morphology of cell apoptosis was observed by scanning electron microscopy. The mRNA expression of proliferating cell nuclear antigen(PCNA), cyclinA1, cyclinA2, cyclin dependent kinase 2(CDK2), and cyclin dependent kinase inhibitor 1A(P21) were determined by qPCR. The level of reactive oxygen species(ROS), lipid peroxide, and mitochondrial membrane potential were measured by flow cytometry. The activity of protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway was detected by Western blot. Compared with the control group, the cells treated with HBOA exhibited a significant decrease in viability. Then the optimal concentration and intervention time of HBOA were determined to be 0.4 mmol·L~(-1), 0.6 mmol·L~(-1), and 48 h. Compared with the control group, groups with HBOA of 0.4 mmol·L~(-1 )and 0.6 mmol·L~(-1) showed a significant suppression in cell proliferation and colony formation ability, down-regulated mRNA of PCNA, cyclinA1, cyclinA2, and CDK2, up-regulated P21 mRNA, S-phase cell cycle arrest, and increased cell apoptosis rate. There was an appearance of apoptotic bodies, increased ROS and lipid peroxide, decreased mitochondrial membrane potential(with a significant decrease in 0.6 mmol·L~(-1) group), and down-regulated p-Akt and p-mTOR proteins. The results show that HBOA inhibits the proliferation of pancreatic cancer L3.6 cells and induces cell apoptosis, which may be related to the increase in reactive oxygen species and the inhibition of the Akt/mTOR pathway.

Discovery of gefitinib-1,2,3-triazole derivatives against lung cancer via inducing apoptosis and inhibiting the colony formation

A series of 20 novel gefitinib derivatives incorporating the 1,2,3-triazole moiety were designed and synthesized. The synthesized compounds were evaluated for their potential anticancer activity against EGFR wild-type human non-small cell lung cancer cells (NCI-H1299, A549) and human lung adenocarcinoma cells (NCI-H1437) as non-small cell lung cancer. In comparison to gefitinib, Initial biological assessments revealed that several compounds exhibited potent anti-proliferative activity against these cancer cell lines. Notably, compounds 7a and 7j demonstrated the most pronounced effects, with an IC50 value of 3.94 ± 0.17 µmol L−1 (NCI-H1299), 3.16 ± 0.11 µmol L−1 (A549), and 1.83 ± 0.13 µmol L−1 (NCI-H1437) for 7a, and an IC50 value of 3.84 ± 0.22 µmol L−1 (NCI-H1299), 3.86 ± 0.38 µmol L−1 (A549), and 1.69 ± 0.25 µmol L−1 (NCI-H1437) for 7j. These two compounds could inhibit the colony formation and migration ability of H1299 cells, and induce apoptosis in H1299 cells. Acute toxicity experiments on mice demonstrated that compound 7a exhibited low toxicity in mice. Based on these results, it is proposed that 7a and 7j could potentially be developed as novel drugs for the treatment of lung cancer.

ALKBH5 Modulates Asthma Progression by Downregulating N6-methyladenosine Methylation.

Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma. We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay. Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5. Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis.

Abstract LB274: Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression

Abstract HIKESHI, under heat stress conditions, has been reported to transport molecular chaperone HSP70 into the nucleus to prevent cancer cell death from heat stress. The tumor-supporting roles of molecular chaperones in various solid tumors are explored extensively. Yet, the sole targeting of the molecular chaperones could not significantly impact anticancer treatment. Thus, we hypothesized that the specific transporter protein HIKESHI might improve this outcome. However, understanding of the clinical relevance of HIKESHI in pancreatic cancer remained unclear, and few studies addressed the HIKESHI role in cancer and whether targeting can inhibit pancreatic cancer cell viability and the other HSPs. Here, we first investigated the expression pattern of the HIKESHI protein in surgically resected pancreatic cancer samples (n=142) and analyzed the clinical relevance of the HIKESHI. We found that the HIKESHI protein was overexpressed in cancer compared to the adjacent normal tissue, and positive expression of cancer HIKESHI was related to poor overall survival. In addition, HIKESHI-positive expression in cancer was identified as an independent prognostic marker for overall survival. Next, using shRNA, we established the stable HIKESHI-suppressed pancreatic cancer cell line (PANC-1). HIKESHI suppression inhibited cell growth and colony formation ability in vitro. The expression and nuclear translocation of HSP70 and HSP110/105 were suppressed in HIKESHI-suppressed cells compared to the control shRNA cells despite no heat stress conditions. Furthermore, we found that the tumor growth, HIKESHI, and Ki67 expression in HIKESHI-suppressed tumors were inhibited compared to the control shRNA tumors. Next, we performed Cap Analysis of Gene Expression and IP-MS analysis to discover the potential downstream and novel interactive partner of the HIKESHI protein. The extensive mechanism of these analyses is under investigation. Our findings suggest that targeting HIKESHI may be a promising therapeutic strategy against refractory pancreatic cancer. Citation Format: Bilguun Erkhem-Ochir, Takehiko Yokobori, Gendensuren Dorjkhorloo, Haruka Okami, Takaomi Seki, Norifumi Harimoto, Ryo Muranushi, Kouki Hoshino, Kei Hagiwara, Norihiro Ishii, Mariko Tsukagoshi, Kenichiro Araki, Hiroshi Saeki, Ken Shirabe. Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB274.

Upregulation of ESPL1 is associated with poor prognostic outcomes in endometrial cancer

Background Extra spindle pole bodies-like 1 (ESPL1) is known to play a crucial role in the segregation of sister chromatids during mitosis. Overexpression of ESPL1 is considered to have oncogenic effects in various human cancers. However, the specific biological function of ESPL1 in endometrial cancer (EC) remains unclear. Methods The TCGA and GEO databases were utilized to assess the expression of ESPL1 in EC. Immunohistochemistry was utilized to detect separase expression in EC samples. Kaplan-Meier survival analysis and Cox regression analysis were performed to evaluate the diagnostic and prognostic significance of ESPL1 in EC. Gene Set Enrichment Analysis (GSEA) was employed to explore the potential signaling pathway of ESPL1 in EC. Cell proliferation and colony formation ability were analyzed using CCK-8 and colony formation assay. Results Our analysis revealed that ESPL1 is significantly upregulated in EC, and its overexpression is associated with advanced clinical characteristics and unfavourable prognostic outcomes. Suppression of ESPL1 attenuated proliferation of EC cell line. Conclusion The upregulation of ESPL1 is associated with advanced disease and poor prognosis in EC patients. These findings suggest that ESPL1 has the potential to serve as a diagnostic and prognostic biomarker in EC, highlighting its significance in the management of EC patients.

Abstract 3076: Unveiling the therapeutic potential of β-catenin inhibition in triple negative breast cancers: Targeting the Warburg effect

Abstract Introduction: Triple Negative Breast Cancer (TNBC) exhibits increased glycolysis, known as the Warburg effect. Recent research, including our own Nanostring nCounter studies, identify β-catenin as an oncoprotein linked to elevated glycolysis in TNBC tumors. Our current research explores the impact of β-catenin inhibitors on glycolysis in TNBC cell lines and an in vivo CDX model. Method: We employed immunohistochemistry to assess 98 TNBCs for the presence of β-catenin, PFKP, LDH-A, and MCT1 proteins, exploring their correlation with clinicopathological factors. In in-vitro experiments using MDA-MB-231, MDA-MB-468, and 4T1 cell lines, we investigated how β-catenin inhibitors (XAV939 & Axitinib) affected aerobic glycolysis components, and TNBC cell characteristics (proliferation, migration, invasion, and stemness). For in-vivo validation, 4T1 TNBC xenografts were employed to confirm our findings. Results: Among the 98 TNBC patients, 63 (64%) displayed cytoplasmic/or nuclear β-catenin staining. Additionally, cytoplasmic accumulation of PFKP was observed in 52/98 (53%), increased cytoplasmic LDH-A expression in 57/98 (58%), and membranous MCT-1 expression in 50/98 (51%) TNBC patients. Notably, significant positive correlations were found between nuclear/cytoplasmic β-catenin and cytoplasmic PFKP (p = 0.001) as well as membranous MCT1 (p = 0.014). Furthermore, β-catenin, PFKP, LDHA, and MCT1 proteins were overexpressed in all TNBC cell lines thereby supporting their glycolytic nature. Comparative analyses between XAV939 and Axitinib revealed that Axitinib significantly reduced various TNBC cell parameters, including migration, invasion, lactate production, colony-forming ability, and expression of aerobic glycolytic pathway proteins. In the in-vivo studies, 30mg/kg Axitinib in a female Balb/c-4T1 xenograft model resulted in a substantial reduction in tumor volume and weight. Conclusion: Our findings show that increased levels of β-catenin play an important role in regulating aerobic glycolysis in TNBCs. Furthermore, our findings also highlight Axitinib's potential as an effective therapeutic agent for the treatment of β-catenin-positive TNBCs. Citation Format: Sheikh Mohammad Umar, Shruti kahol, Arundhathi Dev JR, Akanksha Kashyap, Sandeep R. Mathur, Ajay Gogia, S.V.S Deo, Chandra Prasad. Unveiling the therapeutic potential of β-catenin inhibition in triple negative breast cancers: Targeting the Warburg effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3076.

PDCL3 is a prognostic biomarker associated with immune infiltration in hepatocellular carcinoma

BackgroundPhosducin-like 3 (PDCL3) is a member of the photoreceptor family, characterized by a thioredoxin-like structural domain and evolutionary conservation. It plays roles in angiogenesis and apoptosis. Despite its significance, research on the biological role of PDCL3 in liver hepatocellular carcinoma (LIHC) remains limited. This study aims to explore the prognostic value and potential mechanisms of PDCL3 in cancer, particularly in LIHC, through bioinformatics analysis.MethodsRNA-seq data and corresponding clinical information for pan-cancer and LIHC were extracted from the TCGA database to analyze PDCL3 expression and survival prognosis. Differential expression of PDCL3 was analyzed using the HPA database. GO and KEGG enrichment analysis were performed for PDCL3-associated genes. The relationship between PDCL3 expression and various immune cell types was examined using the TIMER website. Clinical samples were collected, and immunohistochemistry and immunofluorescence experiments were conducted to validate the differential expression of PDCL3 in LIHC and normal tissues. In vitro assays, including CCK-8, wound healing, Transwell, and colony formation experiments, were employed to determine the biological functions of PDCL3 in LIHC cells.ResultsAnalysis from TIMER, GEPIA, UALCAN, and HPA databases revealed differential expression of PDCL3 in various tumors. Prognostic analysis from GEPIA and TCGA databases indicated that high PDCL3 expression was associated with poorer clinical staging and prognosis in LIHC. Enrichment analysis of PDCL3-associated genes revealed its involvement in various immune responses. TCGA and TIMER databases showed that high PDCL3 expression in LIHC decreased tumor immune activity by reducing macrophage infiltration. PDCL3 exhibited positive correlations with multiple immune checkpoint genes. Immunohistochemistry (IHC) and immunofluorescence (IF) experiments confirmed elevated PDCL3 expression in LIHC tissues compared to adjacent normal tissues. In vitro experiments demonstrated that PDCL3 promoted LIHC cell proliferation, migration, invasion, and colony-forming ability.ConclusionPDCL3 is highly expressed in various cancer types. Our study suggests that elevated PDCL3 expression in hepatocellular carcinoma is associated with poorer prognosis and may serve as a potential diagnostic biomarker for LIHC. PDCL3 may regulate the biological functions of LIHC by modulating immune infiltration. However, the precise regulatory mechanisms of PDCL3 in cancer warrant further investigation.

Polyphyllin Ⅲ regulates EMT of lung cancer cells through GSK-3β/β-catenin pathway.

Some studies have found that the application of traditional Chinese medicine in the treatment of lung cancer has achieved satisfying results. Polyphyllin Ⅲ (PP Ⅲ) is a natural steroid saponin from P. polyphylla var. yunnanensis, and its analogs have played a wide role in anticancer research. This study aimed to investigate the effect of PP Ⅲ on the development of lung cancer and its molecular mechanism. A549 and NCI-H1299 cell lines were treated with PP Ⅲ in gradient concentration to detect the IC50 of the cells, and the optimal concentration was selected for subsequent experiments. The effects of PP III treatment on lung cancer were investigated in vitro and in vivo. In vitro experiments, it was found that the proliferation, invasion, migration, and colony formation ability of cancer cells were significantly reduced after PP III treatment, while accompanied by a large number of cell apoptosis. Further detection showed that N-cadherin was significantly decreased, E-cadherin was increased, and Snail and Twist were decreased in A549 cells and NCI-H1299 cells, respectively. In addition, GSK-3β expression was increased, while β-catenin expression was reduced with PP III treatment. In the mouse model, it was demonstrated that the volume of transplanted tumors was significantly reduced after PP Ⅲ treatment. PP Ⅲ has the capacity to inhibit the progression of lung cancer and regulate epithelial-mesenchymal transition through the GSK-3β/β-catenin pathway to suppress the malignant behavior of cancer cells. The application of PP Ⅲ is expected to be an effective method for the treatment of lung cancer.

The mechanism of action and experimental verification of Gan-song Yin on renal clear cell carcinoma based on network pharmacology and bioinformatics

BackgroundGan-song Yin (GSY) is originated from the scripture “Gan-song Pills”, a medical work of the Ningxia ethnic minorities, and its treatment of kidney diseases has good results. Its method of treating Renal clear cell carcinoma (KIRC) is still unknown, nevertheless.MethodsFirstly, utilizing a network pharmacology strategy to screen GSY for active components and targets and looking up KIRC-related targets in GeneCards and GEO databases. Secondly, protein interaction networks were constructed and analyzed for GO and KEGG enrichment. Molecular docking was then performed and clinical and other correlations of the network pharmacology results were analyzed using bioinformatic analysis methods. Finally, we performed in vitro cellular experiments with 786-O cells and ACHN cells to validate the results of network pharmacology and bioinformatic analysis.ResultsWith the help of network pharmacological analysis, six hub targets were eliminated. Bioinformatics study revealed that the hub targets has clinically significant clinical guiding importance. The results showed that GSY inhibited the proliferation of 786-O cells and ACHN cells, induced cell apoptosis, blocked cell cycle, and reduced cell colony formation ability. qRT-PCR results showed that GSY promoted the expression of ALB and CASP3 genes, and inhibited the expression of EGFR, JUN, MYC and VEGFA genes. Western blot results showed that GSY could promote the expression of ALB and CASP3 protein, and inhibit the expression of EGFR, JUN, MYC and VEGFA protein.ConclusionsNetwork pharmacology and bioinformatics analysis showed that GSY could act on multiple targets through a variety of components to achieve the effect of treating KIRC. In this study, we confirmed that GSY inhibits KIRC by regulating the expression of core targets through in vitro cellular experiments, thus providing a reference for subsequent related studies.

Role of deubiquitinase JOSD2 in the pathogenesis of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly malignancy with limited treatment options. Deubiquitinases (DUBs) have been confirmed to play a crucial role in the development of malignant tumors. JOSD2 is a DUB involved in controlling protein deubiquitination and influencing critical cellular processes in cancer. To investigate the impact of JOSD2 on the progression of ESCC. Bioinformatic analyses were employed to explore the expression, prognosis, and enriched pathways associated with JOSD2 in ESCC. Lentiviral transduction was utilized to manipulate JOSD2 expression in ESCC cell lines (KYSE30 and KYSE150). Functional assays, including cell proliferation, colony formation, drug sensitivity, migration, and invasion, were performed, revealing the impact of JOSD2 on ESCC cell lines. JOSD2's role in xenograft tumor growth and drug sensitivity in vivo was also assessed. The proteins that interacted with JOSD2 were identified using mass spectrometry. Preliminary research indicated that JOSD2 was highly expressed in ESCC tissues, which was associated with poor prognosis. Further analysis demonstrated that JOSD2 was upregulated in ESCC cell lines compared to normal esophageal cells. JOSD2 knockdown inhibited ESCC cell activity, including proliferation and colony-forming ability. Moreover, JOSD2 knockdown decreased the drug resistance and migration of ESCC cells, while JOSD2 overexpression enhanced these phenotypes. In vivo xenograft assays further confirmed that JOSD2 promoted tumor proliferation and drug resistance in ESCC. Mechanistically, JOSD2 appears to activate the MAPK/ERK and PI3K/AKT signaling pathways. Mass spectrometry was used to identify crucial substrate proteins that interact with JOSD2, which identified the four primary proteins that bind to JOSD2, namely USP47, IGKV2D-29, HSP90AB1, and PRMT5. JOSD2 plays a crucial role in enhancing the proliferation, migration, and drug resistance of ESCC, suggesting that JOSD2 is a potential therapeutic target in ESCC.

Multi-omics sequencing revealed endostar combined with cisplatin treated non small cell lung cancer via anti-angiogenesis

BackgroundEndostar, an anti-angiogenic drug, has been approved for treating non-small cell lung cancer (NSCLC). At present, endostar combined with radiotherapy or chemotherapy has achieved ideal results in the treatment of some tumors, but there is a lack of application and study in NSCLC. This study investigated the therapeutic effect and potential mechanism of endostar combined with cisplatin (EC) in NSCLC.MethodsHE staining, TUNEL staining, immunofluorescence, colony formation ability, and cell migration ability were used to evaluate the anti-tumor activity of EC. The expressions of FMOD, VEGF, FGF-2, and PDGF-B were detected by western blotting and qPCR. The target of combination therapy was analyzed by m6A sequencing and RNA sequencing. METTL3 knockdown and overexpressed A549 cells were constructed and co-cultured with HUVECs to further evaluate the effect of METLL3 on combination therapy.ResultsCombination therapy significantly reduced the colony formation and migration ability of NSCLC cells, induced cell apoptosis, and inhibited the tube formation ability of HUVECs. The results of m6A sequencing and RNA sequencing showed that the EC could down-regulate the expression level of FMOD in tumor tissues, which might be related to the reduction of its m6A methylation modification regulatory enzyme METTL3. Restricting FMOD expression could reduce the expression of FGF2, TGF-β1, VEGF and PDGF-B. Moreover, overexpression of METTLE almost abolished the anti-tumor effect of EC and promoted angiogenesis.ConclusionsEndostar combined with cisplatin might exert anti-tumor effects by down-regulating the expression of METTL3 and FMOD.

Xianling Lianxia formula enhances the inhibitory effects of trastuzumab on HER2-positive breast cancer.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) is characterized by high invasiveness. Trastuzumab considerably improves the prognoses of HER2-positive BC, but some patients exhibit drug resistance. In this study, the effects of XLLXF combined with trastuzumab on the proliferation, apoptosis, invasion, and migration of HER2-positive BC cells are evaluated, and network pharmacology is performed. Then, we conduct an in vivo study using a xenograft mouse model of HER2-positive BC, and tumor growth is monitored. The expression levels of cytokines are measured by ELISA. Molecular docking is performed to observe the binding stability of IL2, JAK, STAT, and TNF with curcumenol, icariside-II, lobetyolin, and scutellarein. Finally, we observe changes in JAK1 and TNF-α in tumor tissues by immunohistochemistry. The results show that XLLXF enhances the inhibitory effects of trastuzumab on the proliferation, colony formation ability, migration, and invasion of HER2-positive BC cells and promotes apoptosis. Network pharmacology reveals that XLLXF may exert its effects on HER2-positive BC by modulating pathways such as the ErbB, JAK-STAT, and NF-κB pathways. Potential targets include cytokines closely related to immune function. In the in vivo study, XLLXF synergistically enhances the inhibitory effects of trastuzumab on tumor growth. ELISA reveals that XLLXF combined with trastuzumab increases the levels of IL-15, IL-2, TNF-α, and IFN-γ in tumor-bearing mice. Immunohistochemistry confirms that XLLXF can regulate the expressions of JAK1 and TNF-α. This study demonstrates that XLLXF can synergistically enhance the efficacy of trastuzumab in targeting HER2-positive BC. The mechanism may involve the modulation of inflammatory factors.

Novel LIPA-Targeted Therapy for Treating Ovarian Cancer.

Ovarian cancer (OCa) is the most lethal form of gynecologic cancer, and the tumor heterogeneities at the molecular, cellular, and tissue levels fuel tumor resistance to standard therapies and pose a substantial clinical challenge. Here, we tested the hypothesis that the heightened basal endoplasmic reticulum stress (ERS) observed in OCa represents an exploitable vulnerability and may overcome tumor heterogeneity. Our recent studies identified LIPA as a novel target to induce ERS in cancer cells using the small molecule ERX-41. However, the role of LIPA and theutility of ERX-41 to treat OCa remain unknown. Expression analysis using the TNMplot web tool, TCGA data sets, and immunohistochemistry analysis using a tumor tissue array showed that LIPA is highly expressed in OCa tissues, compared to normal tissues. ERX-41 treatment significantly reduced the cell viability and colony formation ability and promoted the apoptosis of OCa cells. Mechanistic studies revealed a robust and consistent induction of ERS markers, including CHOP, elF2α, PERK, and ATF4, upon ERX-41 treatment. In xenograft and PDX studies, ERX-41 treatment resulted in a significant reduction in tumor growth. Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of ERS induction, which could be exploited to treat heterogeneity in OCa.

"Combination treatments of imatinib with astaxanthin and crocin efficiently ameliorate antioxidant status, inflammation and cell death progression in imatinib-resistant chronic myeloid leukemia cells".

Imatinib resistance remains a major obstacle in the treatment of chronic myelogenous leukemia (CML). Crocin (CRC) and astaxanthin (ATX) are phytochemicals with anti-cancer properties. This study aimed to explore the effects of combination treatment of Imatinib with CRC and ATX on Imatinib-resistant K562 (IR-K562) cells. After the establishment of IR-K562 cells, growth inhibitory activity was determined by the MTT assay. To test the regeneration potential, a colony formation assay was performed. Cell cycle analyses were examined by flow cytometry. Cell injury was evaluated by lactate dehydrogenase (LDH) leakage. Real-time PCR was applied to assess the expression of IL6, TNF-α, STAT3, BAD, CASP3, TP53, and Bcl-2 genes. Caspase-3 activity was determined by a colorimetric assay. Antioxidant activity was measured using a diphenylpicrylhydrazyl (DPPH) assay. After 48h of treatment, ATX (IC50 = 30µM) and CRC (IC50 = 190µM) significantly inhibited cell proliferation and colony formation ability, induced G1 cell cycle arrest and cell injury, upregulated the expression of apoptosis-associated genes, and downregulated the expression of anti-apoptotic and inflammatory genes. The combination of IM with ATX and/or CRC synergistically reduced cell viability (combination index [CI] < 1). Our data suggest that IM shows better therapeutic efficacy at lower doses when combined with ATX and/or CRC.

Semilicoisoflavone B induces oral cancer cell apoptosis by targeting claspin and ATR-Chk1 signaling pathways.

The prevalence of oral squamous cell carcinoma (OSCC) is increasing worldwide mainly due to poor oral hygiene and unrestricted lifestyle. Advanced-stage OSCC is associated with poor prognosis and a 5-year survival rate of only 30%-50%. The present study was designed to investigate the anticancer effect and mode of action of Glycyrrhiza-derived semilicoisoflavone B (SFB) in 5-fluorourasil (5FU)-resistant human OSCC cell lines. The study findings revealed that SFB significantly reduces OSCC cell viability and colony formation ability by arresting cell cycle at the G2/M and S phases and reducing the expressions of key cell cycle regulators including cyclin A, cyclin B, CDC2, and CDK2. The compound caused a significant induction in the percentage of nuclear condensation and apoptotic cells in OSCC. Regarding pro-apoptotic mode of action, SFB was found to increase Fas-associated death domain and death receptor 5 expressions and reduce decoy receptor 2 expression, indicating involvement of extrinsic pathway. Moreover, SFB was found to increase pro-apoptotic Bim expression and reduce anti-apoptotic Bcl-2 and Bcl-xL expressions, indicating involvement of intrinsic pathway. Moreover, SFB-mediated induction in cleaved caspases 3, 8, and 9 and cleaved poly(ADP-ribose) polymerase confirmed the induction of caspase-mediated apoptotic pathways. Regarding upstream signaling pathway, SFB was found to reduce extracellular signal regulated kinase 1/2 (ERK) phosphorylation to execute its pro-apoptotic activity. The Human Apoptotic Array findings revealed that SFB suppresses claspin expression, which in turn caused reduced phosphorylation of ATR, checkpoint kinase 1 (Chk1), Wee1, and CDC25C, indicating disruption of ATR-Chk1 signaling pathway by SFB. Taken together, these findings indicate that SFB acts as a potent anticancer compound against 5FU-resistant OSCC by modulating mitogen-activated protein kinase (MAPK) and ATR-Chk1 signaling pathways.