Expression Levels Of Cyclin D1 Research Articles

Similarity of oncogenic protein expression in KRASG12D gene delivery-based rat pancreatic cancer model to that of human pancreatic cancer

BackgroundThe development of effective therapies and biomarkers for pancreatic cancer is an unmet clinical need. To address this, we have developed an easy-to-use pancreatic cancer rat animal model via pancreas-targeted hydrodynamic gene delivery of human pancreatic cancer-related genes. Our study aimed to determine the molecular similarity between the pancreatic tumor in the rat model and human pancreatic cancer. MethodsKRASG12D gene-expressing plasmid was delivered to the pancreas of wild type rats via pancreas-targeted hydrodynamic gene delivery as previously reported. Tissue samples were collected at 5 weeks after the first gene delivery. The tumors developed in the rats were assessed for the expression of oncogenic proteins that are involved in human pancreatic cancer development. ResultsThe development of a tumor mimicking pancreatic ductal adenocarcinoma was confirmed. The expression levels of Cyclin D1, c-Jun, IL-33, and Zip4 proteins in the tumor were immunohistochemically assessed and the correlation of the proteins was confirmed. The expression pattern showed similarity to that of surgically resected human pancreatic cancer tissues. ConclusionsOur study findings showing a similar pattern of oncogenic protein expression in novel KRASG12D gene-induced rat pancreatic cancer model and human pancreatic cancer will be useful for establishing novel tumor markers and therapeutic options for pancreatic cancer.

Expression and significance of cyclinD1, cyclin-dependent kinase4 and cyclin-dependent kinase inhibitor P27 in patients with non-neoplastic epithelial disorders of the vulva.

Non-neoplastic epithelial disorders of the vulva (NNEDV) are prevalent and refractory gynecological diseases. However, the underlying pathogenesis of these diseases remain unclear. The present study aimed to investigate the expression and significance of cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase inhibitor P27 (P27) in patients with NNEDV and provide a reference for clinical diagnosis and treatment. Normal vulvar skin samples from patients with perineum repair (control group, n=20) and skin samples from the vulvar lesions of patients with NNEDV (NNEDV group, n=36) were collected. Expression levels of cyclin D1, CDK4 and P27 were assessed in the samples using immunohistochemistry. The expression of each protein was evaluated based on the mean optical density (MOD). The MODs of cyclin D1 and CDK4 were significantly higher in samples of the three pathological types of NNEDV, namely squamous hyperplasia (SH), lichen sclerosus (LS) and mixed SH and LS lesions, compared with those of the control group. The MOD of P27 was lower in samples of the three pathological types of NNEDV than in the control group, although the difference was not statistically significant. No significant differences in the MOD of cyclin D1, CDK4 and P27 were detected among the three pathological types of NNEDV. The ratios of the MOD of cyclin D1 and CDK4 in the prickle cell layer to those in the basal cell layer were significantly higher in the NNEDV group than in the control group. However, the ratio of the MOD of P27 in the prickle cell layer to that in the basal cell layer exhibited no significant difference between the NNEDV and control groups. NNEDV has the potential for malignant transformation. The occurrence and development of NNEDV may be associated with the acceleration of cell proliferation, in which cyclin D1, CDK4 and P27 contribute to regulation of the cell cycle. Therefore, cyclin D1, CDK4 and P27 may be potential targets in the development of new clinical therapeutic drugs for patients with NNEDV.

LncRNA ENST00000434223 exerts regulatory effects on NSCLC cells via Wnt/β-catenin signaling pathway

Purpose: To identify the role and underlying mechanism of lncRNA ENST00000434223 (lncRNA ENST) in the occurrence of NSCLC. 
 Methods: Quantitative real time-polymerase chain reaction (qRT-PCR) was used to evaluate relative lncRNA ENST, β-catenin, Wnt1, and cyclin D1 expression levels in NSCLC tissues, cells, and sera. Proliferation of NSCLC cells was assessed using CCK-8 assay, while Transwell assay was utilized in evaluating changes in cell invasion/migration. Changes in Wnt1, cyclin D1, and β-catenin expressions were determined using qRT-PCR, while alterations in their protein expressions were determined by western blot (WB). In vivo experiments were conducted in nude mice to investigate the effects of lncRNA ENST on tumor growth. 
 Results: lncRNA ENST expression was low in sera, cells, and tissues of NSCLC (p <0.05). Tumor metastasis and size, lymphatic metastasis, and clinical stage were correlated with lncRNA ENST expression (p < 0.05). It was found that ENST could be applied as independent prognostic factor in NSCLC patients. Upregulated expression of lncRNA ENST restrained cell migration, proliferation, and invasion (p < 0.05). Compared with t NC group, lncRNA ENST expression decreased expressions of Wnt1, cyclin D1, and β-catenin in SPC-A1 cells and A549 cells (p < 0.05). 
 Conclusion: lncRNA ENST inhibits the proliferation and apoptosis of NSCLC cells via Wnt/β-catenin pathway. Thus, it is a possible biomarker for NSCLC, and provides a novel insight into the clinical management of NSCLC.

LRRK2 G2019S promotes the development of colon cancer

Abstract LRRK2-G2019S is the most prevalent variant associated with Parkinson’s disease (PD), found in 1–3% of sporadic and 4–8% of familial PD cases. Intriguingly, emerging clinical studies have suggested that LRRK2 G2019S mutation carriers have an overall increased risk of cancer, including colon cancer. However, it remains to be experimentally tested whether and how this mutant directly affects cancer development. Here, by using the AOM/DSS-induced colitis-associated colon cancer model combined with LRRK2 G2019S knock-in (KI) mice, we found that LRRK2 G2019S promoted the pathogenesis of colon cancer, characterized by increased tumor loads and tumor-promoting cytokines compared with those in wild-type controls. Immunoblotting and immunofluorescence indicated that expression levels of Cyclin-D1, Ki-67, β-catenin and p-STAT3 were dramatically increased in the colon of KI mice, and these proteins are well known to promote the pathogenesis of colon cancer. As inflammation is a major factor contributing to the progression of colon cancer, we thus further tested if LRRK2 G2019S promotes colitis in the initiation of the disease. Indeed, we found KI mice were more susceptible to colitis induction, evident by rapid body weight loss and elevated clinical histology score. Mechanistically, LRRK2 G2019S promoted gut inflammasome activation and enhanced the activation of the NF-ƙB and MAPK pathways upon Dectin-1 stimulation. Together, our data for the first time provide direct evidence that gain-of-function of kinase activity in LRRK2 promotes colon cancer development, implicating LRRK2 as a potential target in colon cancer, especially in those LRRK2 G2019S carrier patients and cancer with hyper LRRK2 expression. Startup fund from the Department of pathology at the University of IOWA, NIH R21AG076895, R01 NS104164

HSDL2 overexpression promotes rectal cancer progression by regulating cancer cell cycle and promoting cell proliferation

To analyze the expression of hydroxysteroid dehydrogenase like 2 (HSDL2) in rectal cancer tissues and the effect of changes in HSDL2 expression level on proliferation of rectal cancer cells. Clinical data and tissue samples of 90 patients with rectal cancer admitted to our hospital from January 2020 to June 2022 were collected from the prospective clinical database and biological specimen database. The expression level of HSDL2 in rectal cancer and adjacent tissues was detected by immunohistochemistry, and based on the median level of HSDL2 expression, the patients were divided into high expression group (n=45) and low expression group (n=45) for analysis the correlation between HSDL2 expression level and the clinicopathological parameters. GO and KEGG enrichment analyses were performed to explore the role of HSDL2 in rectal cancer progression. The effects of changes in HSDL2 expression levels on rectal cancer cell proliferation, cell cycle and protein expressions were investigated in SW480 cells with lentivirus-mediated HSDL2 silencing or HSDL2 overexpression using CCK-8 assay, flow cytometry and Western blotting. The expressions of HSDL2 and Ki67 were significantly higher in rectal cancer tissues than in the adjacent tissues (P < 0.05). Spearman correlation analysis showed that the expression of HSDL2 protein was positively correlated with Ki67, CEA and CA19-9 expressions (P < 0.01). The rectal cancer patients with high HSDL2 expressions had significantly higher likelihood of having CEA ≥5 μg/L, CA19-9 ≥37 kU/L, T3-4 stage, and N2-3 stage than those with a low HSDL2 expression (P < 0.05). GO and KEGG analysis showed that HSDL2 was mainly enriched in DNA replication and cell cycle. In SW480 cells, HSDL2 overexpression significantly promoted cell proliferation, increased cell percentage in S phase, and enhanced the expression levels of CDK6 and cyclinD1 (P < 0.05), and HSDL2 silencing produced the opposite effects (P < 0.05). The high expression of HSDL2 in rectal cancer participates in malignant progression of the tumor by promoting the proliferation and cell cycle progress of the cancer cells.

The increased expression of cytokeratin 13 leads to an increase in radiosensitivity of nasopharyngeal carcinoma HNE-3 cells by upregulating ERRFI1.

The main factors contributing to the unfavorable outcome in the clinical treatment of patients with nasopharyngeal carcinoma (NPC) patients are radiation resistance and recurrence. This study aimed to investigate the sensitivity and molecular foundation of cytokeratin 13 (CK13) in the radiotherapy of NPC. To achieve this, a human NPC cell line overexpressing CK13, HNE-3-CK13, was constructed. The effects of CK13 overexpression on cell viability and apoptosis under radiotherapy conditions were evaluated using the CCK-8 assay, immunofluorescence, and western blotting (WB). Next-generation sequencing was performed to identify the downstream genes and signaling pathways of CK13 that mediate radiotherapy response. The potential role of the candidate gene ERRFI1 in CK13-induced enhancement of radiosensitivity was investigated through rescue experiments using clone formation and WB. The effects of ERRFI1 on cell viability, cell apoptosis, cell cycle, and the related key genes were further evaluated using CCK-8, immunofluorescence, flow cytometry, quantitative polymerase chain reaction and WB. The results showed that CK13 overexpression in HNE-3 significantly inhibited cell survival under radiotherapy and promoted apoptosis marker γH2AX expression, leading to a significant increase of ERRFI1. Knockdown of ERRFI1 rescued the decreased cell viability and proliferation and the increased cell apoptosis that were caused by CK13 overexpression-mediated radiotherapy sensitization of NPC cells. In this process, EGFR, AKT, and GSK-3β were found involved. In the end, ERRFI1 was proven to inhibit expression levels of CDK1, CDK2, cyclin B1, and cyclin D1, resulting an increased G2/M cell ratio. Overexpression of CK13 enhances the radiosensitivity of NPC cells, which is characterized by decreased cell viability and proliferation and increased apoptosis. This regulation may affect the survival of HNE-3 cells by increasing the expression of ERRFI1 and activating the EGFR/Akt/GSK-3β signaling pathway, providing new potential therapeutic targets for the treatment of NPC.

5-Demethylnobiletin mediates cell cycle arrest and apoptosis via the ERK1/2/AKT/STAT3 signaling pathways in glioblastoma cells.

5-Demethylnobiletin is the active ingredient in citrus polymethoxyflavones that could inhibit the proliferation of several tumor cells. However, the anti-tumor effect of 5-Demethylnobiletin on glioblastoma and the underlying molecular mechanisms are remains unknown. In our study, 5-Demethylnobiletin markedly inhibited the viability, migration and invasion of glioblastoma U87-MG, A172 and U251 cells. Further research revealed that 5-Demethylnobiletin induces cell cycle arrest at the G0/G1 phase in glioblastoma cells by downregulating Cyclin D1 and CDK6 expression levels. Furthermore, 5-Demethylnobiletin significantly induced glioblastoma cells apoptosis by upregulating the protein levels of Bax and downregulating the protein level of Bcl-2, subsequently increasing the expression of cleaved caspase-3 and cleaved caspase-9. Mechanically, 5-Demethylnobiletin trigged G0/G1 phase arrest and apoptosis by inhibiting the ERK1/2, AKT and STAT3 signaling pathway. Furthermore, 5-Demethylnobiletin inhibition of U87-MG cell growth was reproducible in vivo model. Therefore, 5-Demethylnobiletin is a promising bioactive agent that might be used as glioblastoma treatment drug.

Targeting GPC3high cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in gastric cancer

Cancer-associated fibroblasts (CAFs) are an important part of tumour microenvironment, but its role in immunotherapy of gastric cancer (GC) is still needed to further study. In this study, we firstly distinguish the GC related CAFs via single cell sequencing dataset. CAFs in deep layers of GC tissues gain more developmental potential. Moreover, we found Glypican-3 (GPC3) is up-regulated in the CAFs subgroups of the advanced GC and correlated with poor prognosis in GC patients. In addition, higher GPC3 expression GC patients have higher TIDE (Tumour Immune Dysfunction and Exclusion) score, dysfunction and exclusion score. independent GC cohort also show GC patients with GPC3high CAFs have lower response rate to PD-1 therapy. GPC3 secreted from CAFs up-regulated PD-L1, TIM3, CD24, CYCLIN D1, cMYC and PDK mRNA expression level in HGC-27 cells. At last, in vivo model demonstrate that targeting GPC3high CAFs sensitizing the PD-1 blockage therapy in GC. In conclusion, GPC3 expression in CAFs is a critical prognostic biomarker, and targeting GPC3high cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in GC. Key messages Glypican-3 (GPC3) is up-regulated in the CAFs subgroups of the advanced gastric cancer. Gastric cancer patients with GPC3high CAFs have lower response rate to PD-1 therapy. Targeting GPC3high CAFs sensitizing the PD-1 blockage therapy in gastric cancer.

Expression levels of Ki-67 and cyclin D1 in advanced nasopharyngeal carcinoma and its prognosis

Introduction and Aim: Nasopharyngeal cancer (NPC) prognosis has been associated with cyclin D1 and Ki-67. These tumor markers had never been tested in NPC patients in Indonesia before. Therefore, this study aims to evaluate the relationships between Ki-67 and cyclin D1 expression levels and the clinicopathological features of nasopharyngeal cancer (NPC) patients. Materials and Methods: During the years 2015 until 2018, thirty-one nasopharyngeal cancer patients were studied in a cohort study at RSUPN Dr. Cipto Mangunkusumo. Patients aged 18 years, diagnosed with NPC according to the histopathological results, metastasis NPC, Eastern Cooperative Oncology Group 0–1, and those who had completed first-line chemotherapy treatment with 5–fluorouracil were included in this study. The Ki-67 and cyclin D1 expression levels were evaluated with the h-score. The NLR values were collected prior to the treatment. The 2-year survival rate was analyzed based on the optimal cut-off value obtained. The optimal cut-off values of Ki-67 and cyclin D1 were categorized into high- and low-expression groups to observe the 2-year mortality. Results: The relationships between Ki-67 and cyclin D1, clinical, pathological, and prognosis of patients were examined. Patients with Ki-67 high expression had a worse 2-year survival rate than those with low expression (p0.05). Ki-67 expression was found to be significantly related to adverse outcomes (p0.05, OR: 41.107, 95% CI: 2.600–649.940), while the cyclin D1 expression was not significantly related to adverse outcomes (p0.05). Conclusion: Ki-67 expression was associated with poor prognosis in NPC patients. Nevertheless, further research is required to explore the specific mechanism of cyclin D1 expression in NPC prognosis.

Abstract 3073: Action of thyroid hormones in sorafenib treatment of thyroid cancer

Abstract Background: Thyroid carcinoma (TC) is the most common endocrine neoplasia. Its incidence has increased in the last 40 years worldwide. Sorafenib (Sor), a tyrosine kinase inhibitor (TKI), was approved for the treatment of TC, being hypothyroidism the most frequent consequence of Sor-induced endocrine dysfunction. We have described that thyroid hormones (TH) increase cell proliferation in TC cell lines. Thus, hormone replacement therapy to treat Sor-induced hypothyroidism could negatively affect its antitumor action. We have also shown that the selective inhibition of the TH membrane receptor, integrin αVβ3, diminishes proliferation. Objective: To study integrin αVβ3 inhibition to enhance Sor antineoplastic activity in TC and the molecular mechanisms involved. Experimental Procedures: 8505C human anaplastic TC cell viability was evaluated by MTS assay. Sor was the TKI used, and Cilengitide (Cile) was used to inhibit integrin αVβ3. Protein modulation was measured by Western blot. Apoptosis induction was determined by APC-Annexin V Propidium iodide staining, followed by flow cytometry. In silico analysis on several databases were performed using CBioPortal (https://cbioportal.org/; TCGA, Cell 2014, n=504) and R2 (http://r2.amc.nl; GSE126729, n=28). Results: We first performed a bioinformatic analysis by cBioPortal on the PanCancer Atlas data and found that thyroid tumors are those with the highest integrin αVβ3 subunits expression. We then studied the role of integrin αVβ3 in Sor inhibition of TH-induced proliferation in TC cells. Treatment of 8505C cells with integrin αVβ3 antagonist Cile inhibits TH-induced proliferation (p&amp;lt0.0001), confirming the participation of the integrin. As expected, Sor treatment decreases proliferation (p&amp;lt0.05) while Cile addition did not change the inhibition level. Also, we found that Sor and Cile treatment diminished PCNA and Cyclin D1 expression levels. Cells were preincubated with Sor and Cile and then treated or not with TH for 48h to analyze apoptosis. Sor treatment increases the number of apoptotic cells relative to untreated control (p&amp;lt0.0001) and the presence of TH reduces the rate of apoptosis (p&amp;lt0.01). We then studied Sor target genes expression by R2 on an anaplastic TC patients’ database. FGFR is highly expressed among Sor targets, and it is correlated with integrin αVβ3 expression. Thus, FGFR expression modulation was studied by Western blot on cells treated as previously described. Finally, we found that TH treatment increased FGFR expression, Sor diminishes expression and in presence of TH, FGFR expression was rescued. Interestingly, Cile addition resensitized cells to Sor in presence of TH. Conclusion: Our results establish that the effective dual Sor and Cile treatment can significantly drive tumor proliferation inhibition and apoptosis, and it could provide alternatives to the treatments currently used for this disease. Citation Format: Mateo N. Campos Haedo, Maria C. Diaz Flaque, Helena A. Sterle, Johanna A. Diaz Albuja, Maria F. Cayrol, Maria M. Debernardi, Marina Perona, Guillermo J. Juvenal, Graciela A. Cremaschi, Cinthia Rosemblit. Action of thyroid hormones in sorafenib treatment of thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3073.

Abstract 5794: Targeting CD133+/CD90+ liver cancer stem cells by piperlongumine: A novel strategy for liver cancer treatment

Abstract Liver cancer is an aggressive malignant disease. It’s the most common cancer worldwide and the leading cause of cancer death. Because of the atypical early symptoms of liver cancer, the majority of liver cancer patients are diagnosed with advanced-cancer stage, decreasing the chance of resection and resulting in a poor survival rate. Liver cancer stem cells (LCSCs) are a small subpopulation inside the tumor bulk, characterized by the maintenance of self-renewal and pluripotency. Thus, LCSCs are responsible for cancer initiation, metastasis, relapse, and chemoresistance. Elucidating LCSC characteristics and disclosing their regulatory mechanism might facilitate the development of prognostic, diagnostic, and therapeutic approaches to improve the clinical management of liver cancer. Piperlongumine (PL) an active biological pyridine alkaloid has anti-angiogenic, anti-diabetic, and anti-bacterial activities. The present study aimed to isolate highly tumorigenic and aggressive subpopulation of LCSCs (CD133+/CD90+) from primary tumors and different cell lines and investigate the effect of (PL), a novel targeting therapy, on LCSCs proliferation. The isolated LCSCs were treated with different concentrations of PL and its effect was examined by cell viability, cell morphological changes, western blot, caspase-3 activity, apoptotic induction, and clonogenic formation. By using Immunocytochemistry (ICC) analysis, the localization of nuclear and cytoplasmic Aldehyde dehydrogenase (ALDH6A1) was detected. PL inhibited the viability of CD133+/CD90+LCSC-sub-popupation and caused morphological changes after 48 hours while enhancing the pre-apoptotic signals and caspase-3 activity. In addition, PL decreased the colonies’ formation ability compared to control group. Furthermore, PL suppressed the protein expression levels of ALDH6A1, Notch-1, β-catenin, ERK1/2, and Cyclin-D1 in the dose and time-dependent manners. Besides, PL decreased the localization and intensity of nuclear and cytoplasmic ALDH6A1 compared to the control group, indicating that PL decreased the proliferation and stemness markers of LCSCs. Overall, Piperlongumine as a novel targeting therapy has an important role in targeting CD133+/CD90+ LCSCs sub-population proliferation. Citation Format: Basma A. Abdelaziz, Omayma M. Sadek, Ahmed S. Sultan. Targeting CD133+/CD90+ liver cancer stem cells by piperlongumine: A novel strategy for liver cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5794.

Kallistatin as an inhibitory protein against colorectal cancer cells through binding to LRP6

Kallistatin (KL) is a member of the serine proteinase inhibitor (serpin) family regulating oxidative stress, vascular relaxation, inflammation, angiogenesis, cell proliferation, and invasion. The heparin-binding site of Kallistatin has an important role in the interaction with LRP6 leading to the blockade of the Wnt signaling pathway. In this study, we aimed to explore the structural basis of the Kallistatin-LRP6E1E4 complex using in silico approaches and evaluating the anti-proliferative, apoptotic, and cell cycle arrest activities of Kallistatin in colon cancer lines. The molecular docking showed Kallistatin could bind to the LRP6E3E4 much stronger than LRP6E1E2. The Kallistatin-LRP6E1E2 and Kallistatin-LRP6E3E4 complexes were stable during Molecular Dynamics (MD) simulation. The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) showed that the Kallistatin-LRP6E3E4 has a higher binding affinity compared to Kallistatin-LRP6E1E2. Kallistatin induced higher cytotoxicity and apoptosis in HCT116 compared to the SW480 cell line. This protein-induced cell-cycle arrest in both cell lines at the G1 phase. The B-catenin, cyclin D1, and c-Myc expression levels were decreased in response to treatment with Kallistatin in both cell lines while the LRP6 expression level was decreased in the HCT116 cell line. Kallistatin has a greater effect on the HCT116 cell line compared to the SW480 cell line. Kallistatin can be used as a cytotoxic and apoptotic-inducing agent in colorectal cancer cell lines.

Adipose Tissue-Mesenchymal Stem Cells Caused to Change the Methylation Status of hTERT Gene Promoter CpG Islands of Molt-4 Leukemia Cells as Cell-based Therapy.

DNA methylation was considered as prognostic information in some hematological malignancies. Previous studies have reported the in vitro and in vivo biology role of mesenchymal stem cells (MSCs) on leukemic cells. The aim of this study was to investigate the effect of MSCs on the promoter methylation status of hTERT as a catalytic subunit of telomerase enzyme. In the experimental study, the Molt-4 leukemic cells were co-cultured with MSCs for 7 days. At the end of the co-culture period, the Molt-4 cells were collected, DNA and protein were extracted. Then methylation specific-PCR and western blotting were done for evaluating the hTERT gene promoter methylation status and cyclin D1 and hTERT protein expression, respectively. In the following, the flow cytometry was done for cell cycle distribution assay. It was found that MSCs resulted in a significant decrease in the cyclin D1 and hTERT protein expression levels. Also, MSCs caused changes in the methylation status of the CpG islands in the hTERT gene promoter region. The following results showed that MSCs caused a significant increase in the number of cells at G0/G1 phase and arrest the G0/G1 phase as well as decrease in the cell proliferation of Molt-4 cells. It is concluded that co-culture of MSCs with Molt-4 cells could be involved in changing the methylation status of hTERT gene promoter, cell cycle and hTERT protein expression; it could be potentially beneficial for further investigations regarding the cell transplantation and cell-based therapy.

Sufentanil combined with parecoxib sodium inhibits proliferation and metastasis of HER2-positive breast cancer cells and regulates epithelial-mesenchymal transition.

Sufentanil combined with parecoxib sodium is a commonly used postoperative medication for cancer patients. However, the effects of this combination therapy on human epidermal growth factor receptor-2 (HER2)-positive breast cancer cells have still remained elusive. This study aimed to investigate the effects and potential mechanisms of sufentanil combined with parecoxib sodium on HER2-positive breast cancer cells. The cell counting kit-8 (CCK-8), colony formation, flow cytometry, scratch, transwell invasion, and angiogenesis assays were used to assess the proliferation, cell cycling, migration, invasion, and angiogenesis of HER2-positive breast cancer BT474 cells. Western blot assay was employed for detecting the expression levels of proteins involved in the cell cycle, migration, invasion, angiogenesis, and epithelial-mesenchymal transition (EMT). The in vivo effects of tumor growth and metastasis were examined by establishing an orthotopic transplantation mouse model of HER2-positive breast cancer (MMTV-PyMT). Functional assays indicated that sufentanil combined with parecoxib sodium induced blockade of HER2-positive breast cancer BT474 cells in the G1 phase of the cell cycle and inhibited cell proliferation, migration, angiogenesis, and invasion in vitro. Western blot assay revealed that sufentanil combined with parecoxib sodium downregulated the expression levels of cyclin D1, matrix metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2), vascular endothelial growth factor A (VEGFA), and EMT-related proteins (N-cadherin, Vimentin, and Snail), while up-regulated the expression level of E-cadherin in BT474 cells. In addition, it was found that sufentanil combined with parecoxib sodium inhibited tumor growth and metastasis in the orthotopic transplantation mouse model of HER2-positive breast cancer. Sufentanil combined with parecoxib sodium inhibited HER2-positive breast cancer progression, including cell proliferation, cell cycle, migration, invasion, and angiogenesis, and regulated EMT.

Hyperoxia exposure upregulates Dvl-1 and activates Wnt/β-catenin signaling pathway in newborn rat lung

BackgroundBronchopulmonary dysplasia is a serious and lifelong pulmonary disease in premature neonates that influences around one-quarter of premature newborns. The wingless-related integration site /β-catenin signaling pathway, which is abnormally activated in the lungs with pulmonary fibrosis, affects cell differentiation and lung development.MethodsNewborn rats were subjected to hyperoxia exposure. Histopathological changes to the lungs were evaluated through immunohistochemistry, and the activation of disheveled and Wnt /β-catenin signaling pathway components was assessed by Western blotting and real-time PCR. The abilities of proliferation, apoptosis and migration were detected by Cell Counting Kit-8, flow cytometry and scratch wound assay, respectively.ResultsContrasting with normoxic lungs, hyperoxia-exposed lungs demonstrated larger alveoli, fewer alveoli and thicker alveolar septa. Superoxide dismutase activity was significantly decreased (7th day: P < 0.05; 14th day: P < 0.01) and malondialdehyde significantly increased (7th day: P < 0.05; 14th day: P < 0.01) after hyperoxia exposure. Protein and mRNA expression levels of β-catenin, Dvl-1, CTNNBL1 and cyclin D1 were significantly upregulated by hyperoxia exposure on 7th day (P < 0.01) and 14th day (P < 0.01). In hyperoxic conditions, Dvl-l downregulation and Dvl-l downregulation + MSAB treatment significantly increased the proliferation rates, decreased the apoptosis rates and improved the ability of cell migration. In hyperoxic conditions, Dvl-l downregulation could decrease the mRNA expression levels of GSK3β, β-catenin, CTNNBL1 and cyclin D1 and decrease the protein relative expression levels of GSK3β, p-GSK3β, β-catenin, CTNNBL1 and cyclin D1.ConclusionsWe confirmed the positive role of Dvl-1 and the Wnt/β-catenin signaling pathway in promoting BPD in hyperoxia conditions and provided a promising therapeutic target.

Inhibition of Ferulic Acid on U937 Cell Growth and Its Related Mechanism

To investigate the effects of ferulic acid (FA) on proliferation, apoptosis and Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway of human acute myeloid leukemia (AML) U937 cells. Different concentrations of FA (0、10、25、50、100、200 μmol/L) was used to treat human AML cell lines Kasumi-1, HL-60, and U937 cells respectively for 24 h. The cell survival rate was detected by cell counting kit-8 method, and the 50% inhibitory concentration (IC50) of each cell line was calculated. The U937 cells were divided into control group, FA group (50 μmol/L FA), FA+pcDNA group (50 μmol/L FA+transfected with empty plasmid), FA+pcDNA-TLR4 group (50 μmol/L FA+transfected with TLR4 overexpression plasmid). Flow cytometry was used to detect U937 cell cycle and cell apoptosis; plate clone formation test was used to detect U937 cell clone formation ability; fluorescence quantitative PCR was used to detect the TLR4, NF-κB p65 mRNA levels in U937 cells; Western blot was used to detect the expression levels of CyclinD1, CyclinE, Bcl-2 related X protein (Bax), B cell lymphoma-2 (Bcl-2), Caspase-3, TLR4, and NF-κB p65 protein in U937 cells. With the increase of FA concentration, the survival rates of Kasumi-1, HL-60 and U937 cells gradually decreased(r=-0.919, r=-0.909, r=-0.900), the IC50 of U937 cells was 50.25±2.23 μmol/L. Compared with the control group, after drug treatment of U937 cells, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA group were significantly increased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and protein were significantly decreased (P<0.05); compared with FA group and FA+pcDNA group, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA+pcDNA-TLR4 group were significantly decreased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and proteins were significantly increased (P<0.05). FA inhibits U937 cell proliferation and promotes cell apoptosis by inhibiting the TLR4/NF-κB signaling pathway.

MiR-139 Affects Radioresistance in Esophageal Cancer by Targeting the PDK1/AKT/Cyclin D1 Signaling Pathway.

We explored the mechanism by which miR-139 modulates radioresistance of esophageal cancer (EC). The radioresistant cell line KYSE150R was obtained from the parental KYSE150 cell line by fractionated irradiation (15×2 Gy; total dose of 30 Gy). The cell cycle was assessed by flow cytometry. A gene profiling study was conducted to detect the expression of genes related to the radioresistance of EC. In the KYSE150R line, flow cytometry revealed increased number of G1-phase cells and decreased number of G2-phase cells; the expression of miR-139 increased. Knockdown of miR-139 decreased radioresistance and changed the distribution of cell cycle phases in KYSE150R cells. Western blotting showed that miR-139 knockdown increased the expression levels of cyclin D1, p-AKT, and PDK1. However, PDK1 inhibitor GSK2334470 reversed this effect for p-AKT and cyclin D1 expression. A luciferase reporter assay indicated that miR-139 directly bound to the PDK1 mRNA 3'-UTR. Analysis of the clinical data from 110 patients with EC showed an association of miR-139 expression with the TNM stage and the effect of therapy. MiR-139 expression significantly correlated with EC and progression-free survival. In conclusion, miR-139 enhances the radiosensitivity of EC by regulating the cell cycle through the PDK1/Akt/Cyclin D1 signaling pathway.

Sq-2, a biotinylated annonaceous acetogenin, induces apoptosis, autophagy and S-phase arrest by activating the MAPK pathway in breast cancer cells.

<p indent="0mm">Squamocin, an annonaceous acetogenin isolated from plants in the <italic>Annonaceae</italic> family, has antitumour activity. In this study, we report that Sq-2, a biotinylated squamocin monomer, has a favorable antitumour effect on MDA-MB-231 and SKBR3 breast cancer cells <italic>in vitro</italic>. MTT assays show that Sq-2 has a better antitumour effect on MDA-MB-231 cells than Sq-5 and Sq-6. Furthermore, RNA-Seq and KEGG enrichment analyses reveal that Sq-2 activates the MAPK signaling pathway, and results of western blot analysis demonstrate that Sq-2 activates the JNK and p38 pathways in MDA-MB-231 and SKBR3 cells. Flow cytometry and western blot analysis reveal that Sq-2 induces cell apoptosis by increasing the expressions of cleaved Caspase-3 and cleaved PARP as well as the ratio of Bax/Bcl-2. Inhibition of the Caspase family by Z-VAD-FMK attenuates the viability of MDA-MB-231 cells, indicating that Sq-2 induces apoptosis in a Caspase-dependent manner. Additionally, pretreatment with the p38 inhibitor SB203580 or JNK inhibitor SP600125 partially reverses the increase in the apoptosis rate and decrease in cell viability prompted by Sq-2. Furthermore, Sq-2 treatment decreases the expression level of CyclinD1 and increases the expression levels of p21, p27, CyclinA1, and CDK2, causing S-phase arrest in MDA-MB-231 and SKBR3 cells. Further study indicates that Sq-2 stimulates autophagy in MDA-MB-231 and SKBR3 cells, and inhibition of autophagy by bafilomycin A1 increases cell viability and promotes cell survival. Sq-2, a novel biotin-squamocin compound, shows a significant inhibitory effect on the propagation of SKBR3 and MDA-MB-231 breast cancer cells. Furthermore, Sq-2 treatment not only induces S-phase arrest and activates the JNK and p38 pathways to trigger apoptosis but also causes autophagy to promote apoptosis in MDA-MB-231 and SKBR3 cells. </p>.

N-Propargylic β-enaminones in breast cancer cells: Cytotoxicity, apoptosis, and cell cycle analyses.

Breast cancer is one of the most common cancers worldwide and the discovery of new cytotoxic agents is needed. Enaminones are regarded to be a significant structural motif that is found in a variety of pharmacologically active compounds however the number of studies investigating the anticancer activities of N-propargylic β-enaminones (NPEs) is limited. Herein we investigated the potential cytotoxic and apoptotic effects of 23 different NPEs (1-23) on human breast cancer cells. Cytotoxicity was evaluated via MTT assay. Apoptotic cell death and cell cycle distributions were investigated by flow cytometry. CM-H2DCFDA dye was used to evaluate cellular ROS levels. Expression levels of Bcl-2, Bax, p21, and Cyclin D1 were measured by quantitative real-time PCR. ADME properties were calculated using the ADMET 2.0 tool. NPEs 4, 9, 16, and 21 showed selective cytotoxic activity against breast cancer cells with SI values >2. NPEs induced apoptosis and caused significant changes in Bcl-2 and Bax mRNA levels. The cell cycle was arrested at the G0/G1 phase and levels of p21 and Cyclin D1 were upregulated in both breast cancer cells. ROS levels were significantly increased by NPEs, suggesting that the cytotoxic and apoptotic effects of NPEs were mediated by ROS. ADME analysis revealed that NPEs showed favorable distributions in both breast cancer cell lines, meaning good lipophilicity values, low unfractionated values, and high bioavailability. Therefore, these potential anticancer compounds should be further validated by in vivo studies for their appropriate function in human health with a safety profile, and a comprehensive drug interaction study should be performed.

Anticancer effects of the HDAC inhibitor, 3β,6β‑dihydroxyurs‑12‑en‑27‑oic acid, in MCF‑7 breast cancer cells via the inhibition of Akt/mTOR pathways.

Astilbechinensis (A.chinensis) is a perennial herb that is used to treat chronic bronchitis and pain. The anticancer activity of 3β,6β‑dihydroxyurs‑12‑en‑27‑oic acid (ACT‑3), a major component isolated from A.chinensis, has not yet been investigated in detail. The purpose of the present study was to investigate the histone deacetylase (HDAC) inhibitory and anticancer activities of ACT‑3 compared with suberoylanilide hydroxamic acid (SAHA) in MCF‑7 human breast cancer cells. The purity of ACT‑3 was determined using high‑performance liquid chromatography. In the present study, the effects of ACT‑3 on anticancer effects of MCF‑7 cells were determined by measuring the level of apoptotic cell death and cell cycle regulator using flow cytometry analysis and western blot analysis, respectively. The effects of ACT‑3 on HDAC enzyme activity were measured using assay kits. ACT‑3 and SAHA increased the levels of acetylated histone H3 and reduced the levels of HDAC1 and HDAC3 in MCF‑7 cells. ACT‑3 significantly decreased the cell viability in a concentration‑dependent manner and induced different morphological changes at high concentrations. ACT‑3 and SAHA significantly inhibited the colony formation in MCF‑7 cells. ACT‑3 inhibited total HDAC activity in a dose‑dependent manner. ACT‑3 significantly reduced the expression levels of cyclin D1 and cyclin‑dependent kinase 4, and upregulated the expression levels of p21WAF1 and p53. A significant increase in the G1 phase cell population was observed in MCF‑7 cells and ACT‑3 induced apoptosis by reducing the ratio of B‑cell lymphoma‑2 (Bcl‑2)/Bcl‑2‑associated X (Bax) and releasing cleaved caspase 9. Additionally, ACT‑3 significantly increased autophagic cell death by inhibiting the serine‑threonine kinase/mammalian target of the rapamycin pathway. Autophagy induction was confirmed via acridine orange staining. ACT‑3 significantly increased the pERK1/2 and p21 in MCF‑7 cells. Thus, the activated ERK pathway played an important role in cell cycle arrest and apoptosis via ERK‑dependent induction of p21 in MCF‑7 cells. These data indicated that ACT‑3 can be used as a promising anticancer agent to overcome the limitations and reduce the side effects of conventional anticancer drugs.