Cell Colony Formation Assays Research Articles

SGPP2 is activated by SP1 and promotes lung adenocarcinoma progression.

The late diagnosis and easy metastasis of lung adenocarcinoma (LADC) remains a challenge. SGPP2 is reported to modulate cell processes in many cancers. However, the roles and molecular mechanisms of SGPP2 in LADC are unclear. Online bioinformatics tools GEPIA, CPTAC, and K-M plotter were used to analyze the expression of SGPP2 and the prognosis in LADC. JASPAR and PROMO were used to predict the transcription factors of SGPP2. Real-time quantitative reverse transcription PCR and western blot were used to detect the levels of SGPP2 in LADC cell lines and tissues. Cell counting kit-8, colony formation, flow cytometry, and transwell assay were used to detect cell proliferation, apoptosis, and invasion. The anti-cancer effect of SGPP2 silence was evaluated in the LADC xenograft model. It was found that SGPP2 was highly expressed and related to the poor prognosis of LADC patients. Elevated SGPP2 expression was detected in LADC cell lines and tissues. The chi-square test indicated that the expression of SGPP2 was positively related to tumor, node, metastasis grades and lymph node metastasis. Knocking down SGPP2 significantly inhibited LADC cell viability, and invasion, but induced apoptosis. The anti-tumor effects of SGPP2 were verified in vivo. The upstream transcription factor of SGPP2 was predicted to be SP1, which was highly expressed in LADC tissues and cell lines. Overexpression of SP1 partly rescued the inhibition of SGPP2-shRNA in cell growth, colony formation, and invasion capabilities, and decreased apoptotic cell number in LADC cells. This study demonstrated that SGPP2, activated by SP1, promotes LADC cell proliferation and invasion, and suppresses apoptosis in LADC.

Identification of phytoestrogens as sirtuin inhibitor against breast cancer: Multitargeted approach

Despite progress in diagnosis and treatment strategies, breast cancer remains a primary risk to female health as indicated by second most cancer-deaths globally caused by this cancer. High risk mutation is linked to prognosis of breast cancer. Due to high resistance of breast cancer against current therapies, there is necessity of novel treatment strategies. Sirtuins are signaling proteins belonging to histone deacetylase class III family, known to control several cellular processes. Therefore, targeting sirtuins could be one of the approaches to treat breast cancer. Several plants synthesize phytoestrogens which exhibit structural and physiological similarities to estrogens and have been recognized to possess anticancer activity. In our study, we investigated several phytoestrogens for sirtuin inhibition by conducting molecular docking studies, and in-vitro studies against breast cancer cell lines. In molecular docking studies, we identified coumestrol possessing high binding energy with sirtuin proteins 1–3 as compared to other phytoestrogens. The molecular dynamic studies showed stable interaction of ligand and protein with higher affinity at sirtuin proteins 1–3 binding sites. In cell proliferation assay and colony formation assay using breast cancer cell lines (MCF-7 and MDAMB-231) coumestrol caused significant reduction in cell proliferation and number of colonies formed. Further, the flow cytometric analysis showed that coumestrol induces intracellular reactive oxygen species and the western blot analysis revealed reduction in the level of SIRT-1 expression in breast cancer cell lines. In conclusion, in-silico data and in-vitro studies suggest that the phytoestrogen coumestrol has sirtuin inhibitory activity against breast cancer.

CBX4/miR-190 regulatory loop inhibits lung cancer metastasis.

Lung cancer is one of the major threats to human life worldwide. MiR-190 has been found to perform essential roles in multiple cancer progression; however, there have been no studies focused on its function and underlying regulatory mechanism in lung cancer. The miR-190 expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The cell functional experiments, including cell counting kit-8 (CCK-8), colony formation and transwell assay were conducted in vitro, as well as animal experiments performed in vivo. The regulation and potential binding sites of CBX4 on miR-190 were predicted by TCGA data set and JASPAR website and verified by ChIP assay and dual-luciferase reporter assay. The prospects binding site of miR-190-3p on CBX4 3'UTR region was predicted by StarBase and verified by dual-luciferase reporter assay. MiR-190 was decreased in lung cancer cells. The overexpression of miR-190 had no effects on cell proliferation, but significantly inhibited cancer metastasis both in vitro and in vivo. Moreover, miR-190 expression could be transcriptionally inhibited by CBX4, and CBX4 was the direct target of miR-190-3p. MiR-190 served as a cancer metastasis inhibitor in lung cancer and formed a regulatory loop with CBX4. These findings provided emerging insights into therapeutic targets and strategies for metastatic lung cancer.

Bazedoxifene Inhibits Cell Viability, Colony-Forming Activity, and Cell Migration in Human Non-Small Cell Lung Cancer Cells and Improves the Treatment Efficacy of Paclitaxel and Gemcitabine.

Bazedoxifene is a third-generation selective estrogen receptor modulator that inhibits the IL6/IL6R/GP130 signaling pathway by inhibiting IL6-induced homodimerization of GP130. Considering that the IL6/IL6R/GP130 signaling pathway is important in tumorigenesis and metastasis, bazedoxifene is thought to have an antitumor effect, which has been proven preliminarily in breast cancer and pancreatic cancer but has not yet been studied in non-small cell lung cancer (NSCLC). This study is aimed at evaluating the antitumor effect of bazedoxifene in NSCLC. A549 and H1299 NSCLC cell lines were employed and exposed to various concentrations of bazedoxifene, paclitaxel, gemcitabine, and their combinations for cell viability, colony formation, and wound healing assays to demonstrate the antitumor effect of bazedoxifene with or without paclitaxel or gemcitabine. MTT cell viability, colony formation, and wound healing assays showed that bazedoxifene was capable of inhibiting cell viability, colony formation, and cell migration in a dose-dependent manner. In addition, bazedoxifene was capable of working with paclitaxel or gemcitabine synergistically to inhibit cell viability, colony formation, and cell migration. This study demonstrated the potential antitumor effect of bazedoxifene and its ability to improve the treatment efficacy of paclitaxel and gemcitabine.

Design, Synthesis and Evaluation of a Novel Teoptinib Derivative as an Effective Anti-hepatocellular Carcinoma Agent.

Hepatocellular Carcinoma (HCC) is a type of liver cancer known for its poor prognosis and high mortality. Teoptinib is a highly selective MET inhibitor that has been used in the treatment of liver cancer. Although good progress has been made in clinical treatment, further improvement is still needed. In this study, a series of novel Teoptinib derivatives were synthesized and evaluated as anti-cancer agents for the treatment of liver cancer, and an oral nanodrug delivery system was also explored. A series of novel Teoptinib derivatives were synthesized, and an oral nanodrug delivery system was also explored. HPLC, high-resolution mass spectrometer and NMR were used to determine the structure and molecular formula of the synthesized compounds. Zeta potential assay was used to access the particle size distribution and zeta potential of the nanoparticles. MTT assay, cell colony formation assay, cell apoptosis inhibition assay, cell scratch assay, and the MHCC-97H xenograft model of nude mice assay were used to evaluate the in vitro and in vivo anti-tumor activity of the synthesized compounds. Compound (R)-10 showed the best antitumor activity with 0.010 μM of the IC50 value against MHCC-97H, a human liver cancer cell line with high c-Met expression. The MHCC-97H xenograft model of nude mice assay showed that nano-prodrug of compound (R)-10 exhibited good in vivo activity with 87.67% of the TGI at the dosage of 8 mg/kg. We designed and synthesized a series of c-Met inhibitors containing different side chains and chiral centers as anti-liver cancer agents. Among them, compound (R)-10 shows a promising effect as a lead molecule for further study in the treatment of liver cancer. The successful incorporation of (R)-10 into a novel oral nanodrug delivery system highlights the importance of effective drug delivery systems for enhanced therapeutic efficacy.

Lindqvist-type Polyoxometalates Act as Anti-breast Cancer Drugs via Mitophagy-induced Apoptosis.

Lindqvist-type polyoxometalates (POMs) exhibit potential antitumor activities. This study aimed to examine the effects of Lindqvist-type POMs against breast cancer and the underlying mechanism. Using different cancer cell lines, the present study evaluated the antitumor activities of POM analogues that were modified at the body skeleton based on molybdenum-vanadium-centered negative oxygen ion polycondensations with different side strains. Cell colony formation assay, autophagy detection, mitochondrial observation, qRT-PCR, Western blotting, and animal model were used to evaluate the antitumor activities of POMs against breast cancer cells and the related mechanism. MO-4, a Lindqvist-type POM linking a proline at its side strain, was selected for subsequent experiments due to its low half maximal inhibitory concentration in the inhibition of proliferation of breast cancer cells. It was found that MO-4 induced the apoptosis of multiple types of breast cancer cells. Mechanistically, MO-4 activated intracellular mitophagy by elevating mitochondrial reactive oxygen species (ROS) levels and resulting in apoptosis. In vivo, breast tumor growth and distant metastasis were significantly reduced following MO-4 treatment. Collectively, the results of the present study demonstrated that the novel Lindqvist-type POM MO-4 may exhibit potential in the treatment of breast cancer.

WTAP promotes laryngeal carcinoma cell progression by posttranscriptional activation of CTHRC1 in an m6A-YTHDF1-dependent way.

Laryngeal carcinoma is one of the malignancies in the head and neck region with high incidence and mortality. Despite advances in therapeutic modalities, the 5-year survival rate remains low. Wilms tumor 1-associated protein (WTAP) has been reported to regulate cancer progression, however, its role and mechanism in regulating laryngeal carcinoma development remain unclear. In this study, the expressions of WTAP, collagen triple helix repeat containing 1 (CTHRC1), and YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) and other molecules were detected by quantitative real-time polymerase chain reaction or western blotting. Cell viability and colony formation rate were determined by cell counting kit-8 assay and cell colony formation assay. Cell migration and invasion were investigated by transwell assay. The relationship between CTHRC1 and YTHDF1 was identified by RNA immunoprecipitation assay. The results showed that WTAP and CTHRC1 were upregulated in laryngeal carcinoma tissues and cells. WTAP or CTHRC1 silencing inhibited the proliferation, migration and invasion of laryngeal carcinoma cells. WTAP knockdown inhibited CTHRC1 mRNA stability by suppressing CTHRC1 m6A modification and YTHDF1 from recognizing CTHRC1 m6A sites. Moreover, CTHRC1 overexpression attenuated WTAP knockdown-mediated effects on laryngeal carcinoma cell phenotypes and the expression of β-catenin, C-myc and cyclinD1. Thus, WTAP facilitated CTHRC1 mRNA stability in an m6A-dependent manner to activate the Wnt/β-catenin pathway and promote laryngeal carcinoma cell malignant phenotypes.

4,5-Dimethoxycanthin-6-one Inhibits Glioblastoma Stem Cell and Tumor Growth by Inhibiting TSPAN1 Interaction with TM4SF1.

Glioblastoma stem cells (GSCs) have been implicated in the self-renewal and treatment resistance of glioblastoma (GBM). Our previous study found that 4,5-dimethoxycanthin-6-one has the potential to inhibit GBM cell proliferation. This current study aims to elucidate the molecular mechanism underlying the effects of 4,5-dimethoxycanthin-6-one in GBM development. The effect of 4,5-dimethoxycanthin-6-one on GSC formation and differentiation was explored in human GBM cell lines U251 and U87. Subsequently, 4,5-dimethoxycanthin-6-one binding to tetraspanin 1 (TSPAN1) / transmembrane 4L six family member 1 (TM4SF1) was analyzed by molecular simulation docking. Co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were used to assess the interactions between TSPAN1 and TM4SF1 in GSCs. Cell proliferation was detected by cell counting kit-8 (CCK-8) and colony formation assay. To evaluate cell migration, invasion and apoptosis, we employed wound healing assay, transwell and flow cytometry, respectively. Furthermore, subcutaneous xenograft tumor models in nude mice were constructed to evaluate the impact of 4,5-dimethoxycanthin-6-one on GSCs in vivo by examining tumor growth and histological characteristics. 4,5-Dimethoxycanthin-6-one inhibited GSC formation and promoted stem cell differentiation in a concentration-dependent manner. Molecular docking models of 4,5-dimethoxycanthin-6-one with TM4SF1 and TSPAN1 were constructed. Then, the interaction between TSPAN1 and TM4SF1 in GSC was clarified. Moreover, 4,5-dimethoxycanthin-6-one significantly inhibited the expressions of TM4SF1 and TSPAN1 in vitro and in vivo. Overexpression of TSPAN1 partially reversed the inhibitory effects of 4,5-dimethoxycanthin-6-one on GSC formation, proliferation, migration and invasion. 4,5-Dimethoxycanthin-6-one inhibited GBM progression by inhibiting TSPAN1/TM4SF1 axis. 4,5-Dimethoxycanthin-6-one might be a novel and effective drug for the treatment of GBM.

MAZ-mediated up-regulation of BCKDK reprograms glucose metabolism and promotes growth by regulating glucose-6-phosphate dehydrogenase stability in triple-negative breast cancer

Tumour metabolic reprogramming is pivotal for tumour survival and proliferation. Investigating potential molecular mechanisms within the heterogeneous and clinically aggressive triple-negative breast cancer (TNBC) subtype is essential to identifying novel therapeutic targets. Accordingly, we investigated the role of branched-chain α-keto acid dehydrogenase kinase (BCKDK) in promoting tumorigenesis in TNBC. We analysed The Cancer Genome Atlas dataset and immunohistochemically stained surgical specimens to investigate BCKDK expression and its prognostic implications in TNBC. The effects of BCKDK on tumorigenesis were assessed using cell viability, colony formation, apoptosis, and cell cycle assays, and subsequently validated in vivo. Metabolomic screening was performed via isotope tracer studies. The downstream target was confirmed using mass spectrometry and a co-immunoprecipitation experiment coupled with immunofluorescence analysis. Upstream transcription factors were also examined using chromatin immunoprecipitation and luciferase assays. BCKDK was upregulated in TNBC tumour tissues and associated with poor prognosis. BCKDK depletion led to reduced cell proliferation both in vitro and vivo. MYC-associated zinc finger protein (MAZ) was confirmed as the major transcription factor directly regulating BCKDK expression in TNBC. Mechanistically, BCKDK interacted with glucose-6-phosphate dehydrogenase (G6PD), leading to increased flux in the pentose phosphate pathway for macromolecule synthesis and detoxification of reactive oxygen species. Forced expression of G6PD rescued the growth defect in BCKDK-deficient cells. Notably, the small-molecule inhibitor of BCKDK, 3,6-dichlorobenzo(b)thiophene-2-carboxylic acid, exhibited anti-tumour effects in a patient-derived tumour xenograft model. Our findings hold significant promise for developing targeted therapies aimed at disrupting the MAZ/BCKDK/G6PD signalling pathway, offering potential advancements in treating TNBC through metabolic reprogramming.

UBQLN4 promotes the proliferation and invasion of non-small cell lung cancer cell by regulating PI3K/AKT pathway

BackgroundUbiquilin-4 (UBQLN4), a member of the ubiquilin family, has received limited attention in cancer research to date. Here, we investigated for the first time the functional role and mechanism of UBQLN4 in non-small cell lung cancer (NSCLC).MethodsThe Cancer Genome Atlas (TCGA) database was employed to validate UBQLN4 as a differentially expressed gene. Expression differences of UBQLN4 in NSCLC cells and tissues were assessed using immunohistochemistry (IHC) experiment and western blotting (WB) experiment. Kaplan-Meier analysis was conducted to examine the association between UBQLN4 expression and NSCLC prognosis. Functional analyses of UBQLN4 were performed through cell counting kit-8 (CCK-8), colony formation, and transwell invasion assays. The impact of UBQLN4 on tumor-associated signaling pathways was assessed using the path scan intracellular signaling array. In vivo tumorigenesis experiments were conducted to further investigate the influence of UBQLN4 on tumor formation.ResultsUBQLN4 exhibited up-regulation in both NSCLC tissues and cells. Additionally, over-expression of UBQLN4 was associated with an unfavorable prognosis in NSCLC patients. Functional loss analyses demonstrated that inhibiting UBQLN4 could suppress the proliferation and invasion of NSCLC cells in both in vitro and in vivo settings. Conversely, functional gain experiments yielded opposite results. Path scan intracellular signaling array results suggested that the role of UBQLN4 is associated with the PI3K/AKT pathway, a correlation substantiated by in vitro and in vivo tumorigenesis experiments.ConclusionWe validated that UBQLN4 promotes proliferation and invasion of NSCLC cells by activating the PI3K/AKT pathway, thereby facilitating the progression of NSCLC. These findings underscore the potential of targeting UBQLN4 as a therapeutic strategy for NSCLC.

CircRNA ATF6 suppresses bladder cancer cell proliferation and migration via miR-146a-5p/FLNA axis

BackgroundBladder cancer (BCa) is the most common malignancy with increasing morbidity and mortality. Circular RNA (circRNA) ATF6 level was downregulated in BCa after GSE92675 CircRNA microarray dataset was analyzed using GEO2R. However, its function and mechanism in BCa remain largely unknown. MethodsGEO2R and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to measure levels of circRNA ATF6, microRNA-146a-5p (miR-146a-5p), and filamin A (FLNA). CircRNA ATF6 stability was assessed by actinomycin D and RNase R assays, while circRNA ATF6 cellular localization was examined by FISH experiments in T24 cells. Cell counting kit-8 (CCK-8), colony formation, wound-healing, and transwell assays were used to study circRNA ATF6’s function in growth, motility, and invasion. By examining luciferase, starBase, RNA pull-down, and RNA immunoprecipitation (RIP) experiments, we anticipated and confirmed miR-146a-5p interactions with circRNA ATF6, as well as miR-146a-5p interactions with FLNA. On tumor-bearing mice, in vivo experiments were conducted. ResultsMiR-146a-5p expression in Bca was elevated, while circRNA ATF6 and FLNA were downregulated. CircRNA ATF6 showed better stability in BCa cells, with its expression primarily in the cytoplasm. Upregulating circRNA ATF6 lowered BCa cell viability, colony numbers, and invasion numbers, but broadened their migratory pattern. MiR-146a-5p was directly sponged up by circRNA ATF6, which also detrimentally affected miR-146a-5p levels in BCa. MiR-146a-5p reduced BCa FLNA expression by targeting FLNA. FLNA silencing abolished circRNA ATF6’s mitigating function in BCa cell proliferation, motility, and invasion. In vivo, overexpression of circRNA ATF6 significantly reduced tumor volume and weight. ConclusionCircRNA ATF6 suppresses BCa cell growth, migration and invasion through the miR-146a-5p/FLNA axis.

MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis.

Gastric cancer (GC) is the fourth leading cause of cancer-related death worldwide. Minichromsome maintenance proteins family member 8 (MCM8) assists DNA repair and DNA replication. MCM8 exerts tumor promotor function in multiple digestive system tumors. MCM8 is also considered as a potential cancer therapeutic target. Bioinformatics methods were used to analyze MCM8 expression and clinicopathological significance. MCM8 expression was detected by immunohistochemistry (IHC) staining and qRT-PCR. MCM8 functions in GC cell were explored by Celigo cell counting, colony formation, wound-healing, transwell, and annexin V-APC staining assays. The target of MCM8 was determined by human gene expression profile microarray. Human phospho-kinase array kit evaluated changes in key proteins after ribosomal protein S15A (RPS15A) knockdown. MCM8 functions were reassessed in xenograft mouse model. IHC detected related proteins expression in mouse tumor sections. MCM8 was significantly upregulated and predicted poor prognosis in GC. High expression of MCM8 was positively correlated with lymph node positive (p < 0.001), grade (p < 0.05), AJCC Stage (p < 0.001), pathologic T (p < 0.01), and pathologic N (p < 0.001). MCM8 knockdown inhibited proliferation, migration, and invasion while promoting apoptosis. RPS15A expression decreased significantly after MCM8 knockdown. It was also the only candidate target, which ranked among the top 10 downregulated differentially expressed genes (DEGs) in sh-MCM8 group. RPS15A was identified as the target of MCM8 in GC. MCM8/RPS15A promoted phosphorylation of P38α, LYN, and p70S6K. Moreover, MCM8 knockdown inhibited tumor growth, RPS15A expression, and phosphorylation of P38α, LYN, and p70S6K invivo. MCM8 is an oncogene and predicts poor prognosis in GC. MCM8/RPS15A facilitates GC progression.

GINS2 promotes the progression of human HNSCC by altering RRM2 expression.

GINS2 exerts a carcinogenic effect in multiple human malignancies, while it is still unclear that the potential roles and underlying mechanisms of GINS2 in HNSCC. TCGA database was used to screen out genes with significant differences in expression in HNSCC. Immunohistochemistry and qRT-PCR were used to measure the expression of GINS2 in HNSCC tissues and cells. GINS2 was detected by qRT-PCR or western blot after knockdown or overexpression. Celigo cell counting, MTT, colony formation, and flow cytometric assay were used to check the ability of proliferation and apoptosis. Bioinformatics and microarray were used to screen out the downstream genes of GINS2. GINS2 in HNSCC tissues and cells was up-regulated, which was correlated with poor prognosis. GINS2 gene expression was successfully inhibited and overexpressed in HNSCC cells. Knockdown of GINS2 could inhibit proliferation and increase apoptosis of cells. Meanwhile, overexpression of GINS2 could enhance cell proliferation and colony formation. Knockdown of RRM2 may inhibit HNSCC cell proliferation, while overexpression of RRM2 rescued the effect of reducing GINS2 expression. Our study reported the role of GINS2 in HNSCC for the first time. The results demonstrated that in HNSCC cells, GINS2 promoted proliferation and inhibited apoptosis via altering RRM2 expression. Therefore, GINS2 might play a carcinogen in HNSCC, and become a specific promising therapeutic target.

Downregulation of B4GALNT1 inhibits proliferation and metastasis of osteosarcoma cells.

Osteosarcoma is the most common malignant bone tumor in children and adolescents, characterized by a high potential for proliferation and metastasis. Patients with osteosarcoma who have distant metastases generally have a poor prognosis. Challenges in treatment include incomplete resection of tumor and chemotherapy resistance, with no effective cure currently available. Recent studies suggest that β-1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT1) plays a role in the progression of various malignant tumors. However, the function of B4GALNT1 in osteosarcoma cells has not been reported. This study aims to investigate the expression of B4GALNT1 in osteosarcoma tissues compared to normal tissues and to explore its effects on the proliferation, migration, and invasion of osteosarcoma cells, thereby providing new theoretical foundations and directions for the treatment of osteosarcoma patients. Tumor tissues and corresponding normal tissue samples were collected from 16 osteosarcoma patients who underwent tumor resection at the Second Xiangya Hospital of Central South University. The patients' ages ranged from 8 to 17 years (median age 12 years). The expression of B4GALNT1 mRNA in osteosarcoma tissues, corresponding normal tissues, 3 osteosarcoma cell lines (MG63, Saos-2, and U2OS), and human fetal osteoblastic cells (hFOB) was detected using real-time reverse transcription PCR (real-time RT-PCR). The effects of B4GALNT1 knockdown on the proliferation of osteosarcoma cells Saos-2 and U2OS were analyzed using cell counting kit-8 (CCK-8) assays and colony formation assays. The effects of B4GALNT1 knockdown on the migration and invasion abilities of Saos-2 and U2OS cells were evaluated using Transwell migration and invasion assays. Western blotting analysis was performed to assess the impact of B4GALNT1 knockdown on the expression of epithelial-mesenchymal transition (EMT) and invasion-related proteins in Saos-2 and U2OS cells. Real-time RT-PCR results showed that B4GALNT1 mRNA expression levels were significantly higher in osteosarcoma tissues and the 3 osteosarcoma cell lines compared to normal tissues and hFOB cells (all P<0.01). CCK-8 and colony formation assays indicated that B4GALNT1 knockdown significantly reduced the proliferation rate of osteosarcoma cells compared to the control group (all P<0.05). Transwell migration and invasion assays demonstrated that B4GALNT1 knockdown significantly decreased the number of migrating and invading osteosarcoma cells (all P<0.01). Western blotting analysis revealed that B4GALNT1 knockdown inhibited the expression of N-cadherin, Snail, Vimentin, and matrix metalloproteinase 9 (MMP9) compared to the control group (all P<0.01). B4GALNT1 is upregulated in osteosarcoma tissues and cell lines, and its knockdown suppresses the malignant phenotype of osteosarcoma cells. B4GALNT1 may function as an oncogene in the proliferation and metastasis of osteosarcoma cells.

The G Protein Estrogen Receptor (GPER) is involved in the resistance to the CDK4/6 inhibitor palbociclib in breast cancer

BackgroundThe cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients.MethodsBC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored.ResultsDissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs.ConclusionsOverall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.

Sevoflurane and Propofol Co-affect the Development of Colorectal Cancer by Regulating TM2D1.

Sevoflurane and propofol are the most commonly used anesthetics in surgery. In this study, we aim to explore and clarify the function of sevoflurane and propofol in colorectal cancer. Cell counting kit-8, colony formation, western blot, and transwell assays were performed to determine cell proliferation, apoptosis, ferroptosis, invasion, and migration. We performed overexpression experiments to detect the underlying molecular mechanism of sevoflurane and propofol. The genes related to epithelial-mesenchymal transition were measured by western blot. We discovered that sevoflurane and propofol co-treatment exerted more anti-tumor activities than just sevoflurane or propofol treatment in colorectal cancer cells in vitro. Mechanistically, our data showed that sevoflurane and propofol-induced apoptosis and ferroptosis and inhibited cell proliferation, invasion, and migration. Additionally, TM2D1 was considered a target of sevoflurane and propofol, and TM2D1 overexpression reversed the effect of sevoflurane and propofol on colorectal cancer cell biology behaviors. Our results showed a novel anti-tumor mechanism of sevoflurane and propofol in colorectal cancer cells, and TM2D1 might be an underlying therapeutic target for treating colorectal cancer patients.

Exploration of the sensitization effect of Chaihu Shugan powder on chemotherapy for triple-negative breast cancer and its active ingredients.

Chemotherapy plays a crucial role in the clinical treatment of triple-negative breast cancer (TNBC), but drug resistance limits its clinical application. The active ingredients of Chaihu Shugan Powder (CSP; Bupleurum Liver-Coursing Powder), quercetin and luteolin, both belong to flavonoid compounds and have significant anti-tumor potential, which can promote chemotherapy sensitivity. However, the correlation between the two and TNBC paclitaxel (PTX) chemotherapy sensitivity is unknown. We collected herbal components of CSP from the TCMSP database, and screened effective molecules and corresponding targets. STRING database was utilized to construct a protein-protein interaction network combining effective molecules and target genes. The top 50 nodes ranked by affinity were chosen for subsequent functional analysis, and the drug-active ingredient-gene interaction network was established using Cytoscape software. Molecular docking was used to determine the small molecules that target TNBC PTX resistance. The "clusterProfiler" package was utilized for GO and KEGG enrichment analyses on the top 50 genes to determine the pathways affected by CSP. Cell counting and colony formation assays evaluated cell viability, IC50 values, and proliferation capacity. Flow cytometry tested PTX intracellular accumulation. Western blot assayed the expression of TNF pathway-related proteins. Active ingredients of CSP, quercetin and luteolin, could inhibit TNBC cell proliferation and promote PTX chemotherapy sensitization. Quercetin and luteolin repressed the TNF signaling pathway and promoted PTX chemotherapy sensitization. Quercetin and luteolin could inhibit TNBC cell proliferation and promote PTX chemotherapy sensitization through the TNF signaling pathway. Therefore, the use of quercetin and luteolin plus PTX treatment provides a prospective strategy for TNBC treatment.

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells.

Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells

Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies &gt;90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.

UCHL3 induces radiation resistance and acquisition of mesenchymal phenotypes by deubiquitinating POLD4 in glioma stem cells

BackgroundThe high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive.MethodExpression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS.ResultWe determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model.ConclusionThese findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.