Cell Colony Formation Research Articles

Anti-tumor mechanism of total saponins of Paridis Rhizoma on inducing ferroptosis of breast cancer MCF-7 cells

This study aims to investigate the mechanism of total saponins of Paridis Rhizoma in inducing the ferroptosis of MCF-7 cells and provide a theoretical basis for the clinical treatment of breast cancer with total saponins of Paridis Rhizoma. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the effects of different concentrations of total saponins of Paridis Rhizoma on the proliferation of MCF-7 cells. A phase contrast inverted microscope was used to observe the morphological changes of MCF-7 cells. The colony formation assay was employed to test the colony formation of MCF-7 cells. The lactate dehydrogenase(LDH) release test was conducted to determine the cell membrane integrity of MCF-7 cells. The cell scratch assay was employed to examine the migration of MCF-7 cells. After that, the level of reactive oxygen species(ROS) in MCF-7 cells was observed by an inverted fluorescence microscope, and the content of Fe~(2+) in MCF-7 cells was detected by the corresponding kit. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of MCF-7 cells. Western blot was employed to determine the expression of ferroptosis-related proteins, such as p53, solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long-chain family member 4(ACSL4), and transferrin receptor protein 1(TFR1) in MCF-7 cells. The results showed that 1.5, 3, 4.5, 6, 7.5, and 9 μg·mL~(-1) total saponins of Paridis Rhizoma significantly inhibited the proliferation of MCF-7 cells, with the IC_(50) of 4.12 μg·mL~(-1). Total saponins of Paridis Rhizoma significantly damaged the morphology of MCF-7 cells, leading to the formation of vacuoles and the gradual shrinkage and detachment of cells. Meanwhile, total saponins of Paridis Rhizoma inhibited the colony formation of MCF-7 cells, destroyed the cell membrane(leading to the release of LDH), and shortened the migration distance of MCF-7 cells. Total saponins of Paridis Rhizoma treatment significantly increased the content of ROS, induced oxidative damage, and led to the accumulation of Fe~(2+) in MCF-7 cells. Furthermore, total saponins of Paridis Rhizoma changed the mitochondrial structure, increased the mitochondrial membrane density, led to the decrease or even disappear of ridges, promoted the expression of p53 protein, down-regulated the expression of SLC7A11 and GPX4, and up-regulated the expression of ACSL4 and TFR1. In summary, total saponins of Paridis Rhizoma can significantly inhibit the proliferation and migration of MCF-7 cells and destroy the cell structure by inducing ferroptosis.

Synthesis and Selective Anticancer Activity Evaluation of 2-phenylacrylonitrile Derivatives as Tubulin Inhibitors.

This study aimed at synthesizing 13 series of novel derivatives with 2-phenylacrylonitrile, evaluating antitumor activity both in vivo and in vitro, and obtaining novel tubulin inhibitors. The 13 series of 2-phenylacrylonitrile derivatives were synthesized by Knoevenagel condensation and the anti-proliferative activities were determined by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometer. Quantitative cell migration was performed using 24-well Boyden chambers. The proteins were detected by western blotting. in vitro kinetics of microtubule assembly was measured using ELISA kit for Human β-tubulin (TUBB). Molecular docking was done by Discovery Studio (DS) 2017 Client online tool. Among the derivatives, compound 1g2a possessed strong inhibitory activity against HCT116 (IC50 = 5.9 nM) and BEL-7402 (IC50 = 7.8 nM) cells. Compound 1g2a exhibited better selective antiproliferative activities and specificities than all the positive control drugs, including taxol. Compound 1g2a inhibited proliferation of HCT116 and BEL-7402 cells by arresting them in the G2/M phase of the cell cycle, inhibited the migration of HCT116 and BEL-7402 cells and the formation of cell colonies. Compound 1g2a showed excellent tubulin polymerization inhibitory activity on HCT116 and BEL-7402 cells. The results of molecular docking analyses showed that 1g2a may inhibit tubulin to exert anticancer effects. Compound 1g2a shows outstanding antitumor activity both in vivo and in vitro and has the potential to be further developed into a highly effective antitumor agent with little toxicity to normal tissues.

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.

LncRNA LINC00847 Accelerates Melanoma Progression by Regulating MiR-133a-3p/TGFBR1 Axis.

Growing evidence has suggested that lncRNAs play a regulatory role in tumorigenesis. Dysregulation of a newly identified lncRNA (LINC00847) has been involved in several tumors. Nevertheless, the expression and roles of lncRNAs in skin melanoma remain unclear. Therefore, we attempted to investigate the expressions and roles of lncRNAs in this study. Expression levels of LINC00847 were quantified in tissue samples from the TCGA database and clinically recruited participants. LINC00847 was inhibited in cells by transfecting with si-LINC00847 or si-NC. Expressions of LINC00847 and miR-133a-3p were determined using RT-qPCR, and the TGFBR1 level was determined using Western blotting. Targeting sites of LINC00847 with miR-133a-3p and miR-133a-3p with TGFBR1 were predicted by bioinformatic tools and proved by dual-luciferase reporter system and RNA immunoprecipitation. Cell proliferation, invasion, and migration abilities were assessed using CCK8, cell colony formation, cell wound scratch, and transwell assay, respectively. In both TCGA and clinical cohorts, the expression of LINC00847 was abnormally upregulated in skin melanoma tissues than that of benign nevus. Besides, LINC00847 expression increased more markedly in A375 and SK-MEL-28 cells than in normal epidermal melanocytes (HEMa-LP cells). LINC00847 knockdown remarkably restrained skin melanoma cell proliferation, metastasis, and wound healing rate. Furthermore, miR-133a-3p/TGFBR1 was the downstream target for LINC00847. LINC00847 negatively regulated miR-133a-3p expression in skin melanoma cells. Both miR-133a-3p inhibitors and TGFBR1 vector transfection reversed the effect of LINC00847 silence in skin melanoma cells. LINC00847 was highly expressed in skin melanoma, and its overexpression accelerated the malignant tumor behavior of skin melanoma cells. The miR-133a-3p /TGFBR1 axis was involved in the roles of LINC00847 in skin melanoma.

USP3 inhibition is Active Against Chemo-resistant Hepatocellular Carcinoma Anchorage-independent Growth via Suppressing Wnt/β-catenin.

USPs are a family of enzymes that regulate protein degradation, and their dysregulation has been implicated in the development and progression of cancer. This study aimed to determine whether ubiquitin-specific proteases 3 (USP3) could be a potential target for therapy in hepatocellular carcinoma (HCC), particularly in resistant HCC. This study systematically investigated the role of USP3 in HCC, with a focus on chemo-resistant HCC cells. The level of USP3 from clinical samples was measured using an ELISA assay. Cell proliferation, apoptosis, migration, and anchorage-independent colony formation assays were performed. Transfection was performed to knock down USP3 expression and measure β-catenin activity, and real-time PCR was used to measure levels of MYC and CYCLIN D1 genes. USP3 protein was upregulated in HCC tissues, but its upregulation was not associated with clinicopathology. USP3 knockdown had a similar inhibitory effect on growth in both sensitive and resistant HCC cells, did not affect migration, and induced apoptosis in sensitive but not resistant HCC cells. Furthermore, USP3 knockdown was more effective in suppressing anchorage-independent colony formation in chemoresistant HCC cells compared to their chemo-sensitive counterparts. Pearson correlation coefficient analysis revealed a strong positive correlation between USP3 and CTNNB1, and consistently, USP3 knockdown reduced the levels and activities of β-catenin in HCC cells. Using a Wnt activator (lithium) in rescue studies significantly reversed the inhibitory effects of USP3 knockdown. The findings suggest that inhibiting USP3 is an effective strategy against cancer stem cells and chemo-resistant HCC cells.

Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

Chlorogenic Acid Inhibits Proliferation, Migration and Invasion of Pancreatic Cancer Cells via AKT/GSK-3β/β-catenin Signaling Pathway.

Chlorogenic acid (CA, United States Patent No. 10772340), a natural biologically active food ingredient, displays potent antitumor activity against a variety of cancer cells. However, the mechanism underlying its anticancer effect is not well elucidated. In the present study, we hope to dissect the mechanism underlying the anticancer effects of CA in pancreatic cancer cells. The cytotoxicity of CA in pancreatic cancer cells was determined by MTT assay. Flow cytometry was performed to evaluate the cells apoptosis, while a clonogenic assay was carried out to check the colony formation of cancer cells. Transwell assay was performed to assess the cells migration and invasion. The protein expression of AKT/GSK-3β/β-catenin signaling pathway was detected by Western Blot. Our data indicated that CA inhibited the proliferation of PANC-28 and PANC-1 cells in a dose and time-dependent manner. CA was able to inhibit colony formation, migration, and invasion ability and trigger apoptosis in PANC-28 and PANC-1 cells. Further study showed that CA down-regulated the expression of AKT, p-AKT(Thr308), p-GSK-3β(Ser9), β-catenin, N-cadherin, and vimentin while enhancing the expression of cleaved-caspase 3 and cleaved-caspase 7 in PANC-28 and PANC-1 cells. Our study provides significant evidence that CA is able to inhibit the growth of pancreatic cancer via the AKT/GSK-3β/β-catenin signaling pathway.

Chondroitin polymerizing factor promotes development and progression of colorectal cancer via facilitating transcription of VEGFB.

Colorectal cancer (CRC) is a highly prevalent malignancy affecting the digestive system on a global scale. This study aimed to explore the previously unexplored role of CHPF in the progression of CRC. Our results revealed a significant upregulation of CHPF expression in CRC tumour tissues compared to normal tissues, with its levels correlating with tumour malignancy. Invitro experiments using CRC cell lines demonstrated that inhibiting CHPF expression suppressed cell proliferation, colony formation and cell migration, while promoting apoptosis. Conversely, overexpressing CHPF had the opposite effect. Additionally, our xenograft models in mice confirmed the inhibitory impact of CHPF knockdown on CRC progression using various cell models. Mechanistic investigations unveiled that CHPF may enhance VEGFB expression through E2F1-mediated transcription. Functionally, suppressing VEGFB expression successfully mitigated the oncogenic effects induced by CHPF overexpression. Collectively, these findings suggest that CHPF may act as a tumour promoter in CRC, operating in a VEGFB-dependent manner and could be a potential target for therapeutic interventions in CRC treatment.

Gelsolin knockdown confers radiosensitivity to glioblastoma cells.

Radiotherapy (RT) is a cornerstone of the glioblastoma (GBM) treatment. However, the resistance of tumour cells to radiation results in early recurrence. The mechanisms underlying GBM radioresistance remain unclear. Screening for differentially expressed genes (DEGs) related to radiation might be a potential solution to this problem. RT-associated DEGs were screened based on the RNA sequencing of 15 paired primary and recurrent GBMs. The mRNA and protein expression of candidate genes were validated in RNA sequencing of The Chinese Genome Atlas (CGGA) dataset and 18 cases of GBM samples. The relationship between the candidate gene and radiation was confirmed in irradiated GBM cells. The association of candidate gene with clinical characteristics and survival was investigated in the CGGA and TCGA dataset. Biological function and pathway analysis were explored by gene ontology analysis. The association of the candidate gene with radiosensitivity was verified using cell counting Kit-8, comet, and colony formation assays invitro and subcutaneous tumour xenograft experiments invivo. Gelsolin (GSN) was selected for further study. GSN expression was significant elevated in recurrent GBM and up-regulated in irradiated GBM cell lines. High expression of GSN was enriched in malignant phenotype of glioma. Moreover, high expression of GSN was associated with poor prognosis. Further investigation demonstrated that GSN-knockdown (GSN-KD) combined with RT significantly inhibited cell proliferation and enhanced radiosensitivity invivo and invitro. Mechanistically, GSN-KD could lead to more serious DNA damage and promotes apoptosis after RT. Radiation induced up-regulated of GSN. GSN-KD could enhance the radiosensitivity of GBM.

MiR-122-5p regulates erastin-induced ferroptosis via CS in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a tumor that occurs in the nasopharynx. Although advances in detection and treatment have improved the prognosis of NPC the treatment of advanced NPC remains challenging. Here, we explored the effect of microRNA (miR)-122-5p on erastin-induced ferroptosis in NPC cells and the role of ferroptosis in the development of NPC. The effect of miR-122-5p silencing and overexpression and the effect of citrate synthase on erastin-induced lipid peroxidation in NPC cells was analyzed by measuring the amounts of malondialdehyde, Fe2+, glutathione, and reactive oxygen species and the morphological alterations of mitochondria. The malignant biological behavior of NPC cells was examined by cell counting kit-8, EDU, colony formation, Transwell, and wound healing assays. The effects of miR-122-5p on cell proliferation and migration associated with ferroptosis were examined in vivo in a mouse model of NPC generated by subcutaneous injection of NPC cells. We found that erastin induced ferroptosis in NPC cells. miR-122-5p overexpression inhibited CS, thereby promoting erastin-induced ferroptosis in NPC cells and decreasing NPC cell proliferation, migration, and invasion.

H3K27ac-induced RHOXF2 activates Wnt2/β-catenin pathway by binding to HOXC13 to aggravate the malignant progression of triple negative breast cancer

Triple negative breast cancer (TNBC) is insensitive to conventional targeted therapy and endocrine therapy, and is characterized by high invasiveness and high recurrence rate. This study aimed to explore the role and mechanism of RHOXF2 and HOXC13 on the malignant progression of TNBC. RT-qPCR and western blot were used to detect RHOXF2 and HOXC13 expression in TNBC cells. The proliferation, colony formation, invasion, migration, apoptosis and cell cycle of TNBC cells after transfection were analyzed by CCK-8 assay, colony formation assay, transwell assay, wound healing assay and flow cytometry analysis. Co-Immunoprecipitation and GST pull-down assays were used to analyze the combination between RHOXF2 and HOXC13. ChIP-PCR and luciferase reporter gene assay were used to examine the regulation of H3K27ac on RHOXF2. Besides, the expression of Ki67 and cleaved Caspase3 in tumor tissues of nude mice was determined by immunofluorescence. Results revealed that RHOXF2 and HOXC13 expression was increased in TNBC cells. RHOXF2 knockdown suppressed the proliferation, invasion and migration, as well as induced G0/G1 cell cycle arrest and apoptosis of TNBC cells. Besides, RHOXF2 could bind to HOXC13 and RHOXF2 knockdown suppressed HOXC13 expression in TNBC cells. Furthermore, HOXC13 overexpression reversed the impacts of RHOXF2 downregulation on the proliferation, invasion, migration, G0/G1 cell cycle arrest and apoptosis of TNBC cells. In addition, RHOXF2 silencing limited the tumor volume in nude mice, which was reversed by HOXC13 overexpression. Moreover, RHOXF2 knockdown interfered with Wnt2/β-catenin pathway in vitro and in vivo by binding to HOXC13. Importantly, H3K27ac acetylation could activate the expression of RHOXF2 promoter region. In conclusion, RHOXF2 activated by H3K27ac functioned as a tumor promoter in TNBC via mediating Wnt2/β-catenin pathway by binding to HOXC13, which provided promising insight into exploration on TNBC therapy.

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

NLRX1: a key regulator in mitochondrial respiration and colorectal cancer progression.

Colorectal cancer (CRC) is a prevalent and aggressive malignancy with high mortality rates and significant risks to human well-being. Population-wide screening for tumor suppressor genes and oncogenes shows promise for reducing the incidence and fatality of CRC. Recent studies have suggested that NLRX1, an innate immunity suppressor, may play a role in regulating chronic inflammation and tumorigenesis. However, further investigation is needed to understand the specific role of NLRX1 in CRC. To evaluate the impact of NLRX1 on migration, invasion, and metastasis, two human colon cancer cell lines were studied in vitro. Additionally, a knockout mouse tumor-bearing model was used to validate the inhibitory effect of NLRX1 on tumor emergence and progression. The Seahorse XF96 technology was employed to assess mitochondrial function and glycolysis in colorectal cancer cells overexpressing NLRX1. Moreover, public databases were consulted to analyze gene and protein expression levels of NLRX1. Finally, the results were validated using a series of CRC patient samples. Our findings demonstrate that downregulation of NLRX1 enhances proliferation, colony formation, and tumor-forming capacity in HCT116 and LoVo cells. Conversely, overexpression of NLRX1 negatively impacts basal respiration and mitochondrial ATP-linked respiration in both cell lines, resulting in a notable decrease in maximal respiration during the standard mitochondrial stress test. Furthermore, analysis of data from the TCGA database reveals a significant reduction in NLRX1 expression in colon and rectal cancer tissues compared to normal tissues. This result was validated using clinical samples, where immunohistochemistry staining and western blotting demonstrated a notable reduction in NLRX1 protein levels in CRC compared to adjacent normal tissues. The decreased expression of NLRX1 may serve as a significant prognostic indicator and diagnostic biomarker for CRC patients.

Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO) are the two principals enzymes involved in the catabolization of tryptophan (Trp) into kynurenine (Kyn). Despite their well-established role in the immune escape, their involvement in angiogenesis remains uncertain. We aimed to characterize TDO and IDO1 in human umbilical venular endothelial cells (HUVECs) and human endothelial colony-forming cells (ECFCs). qRT-PCR and immunofluorescence were used for TDO and IDO1 expression while their activity was measured using ELISA assays. Cell proliferation was examined via MTT tests and in in vitro angiogenesis by capillary morphogenesis. HUVECs and ECFCs expressed TDO and IDO1. Treatment with the selective TDO inhibitor 680C91 significantly impaired HUVEC proliferation and 3D-tube formation in response to VEGF-A, while IDO1 inhibition showed no effect. VEGF-induced mTor phosphorylation and Kyn production were hindered by 680C91. ECFC morphogenesis was also inhibited by 680C91. Co-culturing HUVECs with A375 induced TDO up-regulation in both cell types, whose inhibition reduced MMP9 activity and prevented c-Myc and E2f1 upregulation. HUVECs and ECFCs express the key enzymes of the kynurenine pathway. Significantly, TDO emerges as a pivotal player in in vitro proliferation and capillary morphogenesis, suggesting a potential pathophysiological role in angiogenesis beyond its well-known immunomodulatory effects.

Circ-METTL15 stimulates the aggressive behaviors of papillary thyroid cancer cells by coordinating the miR-200c-3p/XIAP axis.

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer. The critical importance of circular RNA (circRNA) in a range of cancer types has been lately recognized. However, research on the functions of circRNAs in PTC has been limited thus far. Therefore, this research aimed at exploring the function and mechanism of circ-methyltransferase-like 15 (METTL15) in PTC cells. Quantitative measurements of circ-METTL15, miR-200c-3p, and X-linked inhibitor of apoptosis protein (XIAP) in PTC cells were conducted using reverse transcription-quantitative polymerase chain reaction or Western blot analysis. To investigate cell growth, cell counting kit-8 and colony formation tests were employed, apoptosis was analyzed using flow cytometry, and migration and invasion were studied through Transwell assays. The targeted binding sites between miR-200c-3p and circ-METTL15 or XIAP were predicted by starBase and then verified by dual luciferase reporter assay. circ-METTL15 and XIAP were upregulated in the PTC cells, while miR-200c-3p was downregulated. Downregulating circ-METTL15 or upregulating miR-200c-3p resulted in inhibited proliferation, migration, and invasion of PTC cells, while promoting apoptosis. miR-200c-3p was the downstream molecule of circ-METTL15, and XIAP was the direct target of miR-200c-3p. Forcing XIAP expression obstructed circ-METTL15 silencing to inhibit PTC cell activity. By coopting miR-200c-3p/XIAP, Circ-METTL15 stimulates aggressive behavior in PTC cells.

Transcriptomic analysis and experiments revealed that remimazolam promotes proliferation and G1/S transition in HCT8 cells.

Remimazolam is a new ultrashort-acting benzodiazepine for sedation and anesthesia. The effects of remimazolam and the mechanism by which it functions in cancer cells have not been determined. This research aimed to explore the mechanism of remimazolam action in colon cancer treatment, using bioinformatics analysis and in vitro experiments. Cell cycle progression, colony formation, self-renewal capacity, and apoptosis detection were performed in HCT8 cells treated with or without remimazolam. Transcriptome sequencing, Gene Ontology, Kyoto Encyclopedia of Genes and Genome, Protein-Protein Interaction, Gene Set Enrichment Analysis, Western blotting, and qPCR were performed to investigate the mechanism of action of remimazolam in HCT8 colon cancer cells. Remimazolam promoted proliferation and cell-cycle progression of HCT8 cells. After remimazolam treatment, a total of 1,096 differentially expressed genes (DEGs) were identified: 673 genes were downregulated, and 423 genes were upregulated. The DEGs were enriched mainly in "DNA replication", "cell cycle", and "G1/S transition" related pathways. There were 15 DEGs verified by qPCR, and representative biomarkers were detected by Western Bloting. The remimazolam-mediated promotion of cell proliferation and cell cycle was reversed by G1T28, a CDK4/6 inhibitor. Remimazolam promoted cell-cycle progression and proliferation in HCT8 colon cancer cells, indicating that the long-term use of remimazolam has potential adverse effects in the anesthesia of patients with colon cancer.

Norcantharidin Enhances the Antitumor Effect of 5-Fluorouracil by Inducing Apoptosis of Cervical Cancer Cells: Network Pharmacology, Molecular Docking, and Experimental Validation.

The high recurrence rate of cervical cancer is a leading cause of cancer deaths in women. 5-Fluorouracil (5-FU) is an antitumor drug used to treat many types of cancer, but its diminishing effectiveness and side effects limit its use. Norcantharidin (NCTD), a demethylated derivative of cantharidin, exhibits various biological activities. Here, we investigated whether NCTD could potentiate 5-FU to induce cervical cancer cell death. To assess the cell viability and synergistic effects of the drugs, cell counting kit-8 and colony formation assays were performed using HR-HPV-positive cervical cancer cell lines. Annexin V-FITC/PI staining and TUNEL assays were performed to confirm the induction of apoptosis. The synergistic effect of NCTD on the antitumor activity of 5-FU was analyzed using network pharmacology, molecular docking, and molecular dynamics simulations. Apoptosis-related proteins were examined using immunoblotting. The combination of NCTD and 5-FU was synergistic in cervical cancer cell lines. Network pharmacological analysis identified 10 common targets of NCTD and 5-FU for cervical cancer treatment. Molecular docking showed the strong binding affinity of both compounds with CA12, CASP9, and PTGS1. Molecular dynamics simulations showed that the complex system of both drugs with caspase-9 could be in a stable state. NCTD enhanced 5-FU-mediated cytotoxicity by activating apoptosis-related proteins. NCTD acts synergistically with 5-FU to inhibit cervical cancer cell proliferation. NCTD enhances 5-FU-induced apoptosis in cervical cancer cell lines via the caspase-dependent pathway.

THAP3 recruits SMYD3 to OXPHOS genes and epigenetically promotes mitochondrial respiration in hepatocellular carcinoma.

Mitochondria harbor the oxidative phosphorylation (OXPHOS) system to sustain cellular respiration. However, the transcriptional regulation of OXPHOS remains largely unexplored. Through the cancer genome atlas (TCGA) transcriptome analysis, transcription factor THAP domain-containing 3 (THAP3) was found to be strongly associated with OXPHOS gene expression. Mechanistically, THAP3 recruited the histone methyltransferase SET and MYND domain-containing protein 3 (SMYD3) to upregulate H3K4me3 and promote OXPHOS gene expression. The levels of THAP3 and SMYD3 were altered by metabolic cues. They collaboratively supported liver cancer cell proliferation and colony formation. In clinical human liver cancer, both of them were overexpressed. THAP3 positively correlated with OXPHOS gene expression. Together, THAP3 cooperates with SMYD3 to epigenetically upregulate cellular respiration and liver cancer cell proliferation.

Anticancer activity of 4α-(cyclopropyl formylpiperazinyl)-4-deoxypodophyllotoxin and its mechanism of action

Podophyllotoxin (PPT) is a naturally occurring lignan extracted from the rhizomes of the Podophyllum plant. It exhibits toxicity towards a broad spectrum of cancer cells and can induce cell death by inhibiting the polymerization of microtubule proteins. In our pursuit of more potent anticancer drug candidates, 4α-(cyclopropyl formylpiperazinyl)-4-deoxypodophyllotoxin (GHGT-17) was successfully synthesized by introducing a piperazine group, formyl group, and cyclopropyl group at the C-4 position of PPT. The activity of GHGT-17 against various types of cancer cells was evaluated using the MTT assay. It exhibited significant potency against lung cancer cells, with IC50 values of 0.080 ± 0.020 μM (A549) and 0.086 ± 0.004 μM (H460), without obvious toxicity towards lung epithelial cells 16HBE (IC50 > 100 μM). Further investigations demonstrated that GHGT-17 induced morphological changes in A549 and H460 cells, inhibited proliferation in a time- and dose-dependent manner. Furthermore, GHGT-17 inhibited the formation of cell colonies in a dose-dependent manner. It was found to inhibit cell migration, arrest the cell cycle in the G2/M phase, induce nuclear wrinkling, decrease mitochondrial membrane potential, and inhibit microtubule protein polymerization. These effects ultimately contribute to the induction of endogenous apoptosis. In silico studies, ADMET analysis revealed that GHGT-17 exhibited a satisfactory bioavailability with good water solubility, easy absorption, and well-proportioned distribution in the body. Molecular docking was performed to predict the binding mode and interactions of GHGT-17 with tubulin protein (1SA0). Additionally, GHGT-17 was submitted to molecular dynamics simulation and RMSD, RMSF, Rg, hydrogen bonding was analyzed. The complex showed a rather low binding free energy (−36.37 kJ mol−1) and thus exhibited a superior binding affinity towards to tubulin protein. In summary, the combined results indicated that GHGT-17 exhibits promise as a prospective potential novel drug candidate worthy of further investigation for its potential application in lung cancer therapy.

Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation.

Objective: To investigate the immune mechanism of osteosarcoma (OS)-specific markers to mitigate bone destruction in the aggressive OS, prone to recurrence and metastasis. Methods: Gene expression patterns from the Gene Expression Omnibus (GEO) database (GSE126209) were analyzed using weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) analysis, least absolute shrinkage and selection operator (LASSO) modeling, and survival analysis to identify charged multivesicular body protein 4C (CHMP4C). Subsequently, its role in regulating the immune system and immune cell infiltration was explored. CHMP4C expression and signaling molecules in OS were assessed in osteosarcoma cell lines (MG63, U2OS, HOS) and hFOB1.19 cells using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The impact of CHMP4C upregulation and interference on OS-related signaling molecules in MG63 cells was studied. Functional validation of CHMP4C in MG63 OS cells was confirmed through cell counting Kit-8 (CCK-8), transwell, and colony formation assays. In vivo experiments were conducted using Specific Pathogen Free (SPF)-grade male BALB/C nude mice for OS xenograft studies. Results: Based on the gene expression profiles analysis of six osteosarcoma samples and six normal tissue samples, we identified 1,511 upregulated DEGs and 5,678 downregulated DEGs in normal tissue samples. A significant positive correlation between the "yellow-green" module and OS was found through WGCNA analysis. Expression levels of CHMP4C, phosphorylated Glycogen Synthase Kinase 3β (p-GSK3β), and β-catenin were notably higher in U2OS, HOS, and MG63 OS cells than in hFOB1.19 human osteoblasts. Overexpressing CHMP4C in MG63 OS cells upregulated CHMP4C, p-GSK3β, and β-catenin while downregulating GSK3β, leading to increased proliferation and migration of MG63 cells. Conversely, interrupting CHMP4C had the opposite effect. High expression of CHMP4C significantly accelerated the growth of OS in nude mice, resulting in substantial upregulation of CHMP4C, p-GSK3β, and β-catenin expression and suppression of Glycogen Synthase Kinase 3β (GSK3β) expression in OS tissues. Conclusion: CHMP4C may serve as a specific immunomodulatory gene for OS. Its activation of the Wnt/β-catenin signaling pathway, mainly by increasing the phosphorylation echelon of GSK3β, promotes the invasion and spread of OS.