Scratch Wound Healing Assay Research Articles

The role of autophagy in the treatment of colon cancer by chlorin e6 photodynamic therapy combined with oxaliplatin

BackgroundPhotodynamic therapy is a tumour treatment method. Its mechanism mainly induces apoptosis, autophagy, and other ways to cause cell death. Therefore, this study aims to evaluate the therapeutic effect of chlorine e6 photodynamic therapy (Ce6-PDT) combined with oxaliplatin (L-OHP) in colon cancer and to investigate the role of autophagy in L-OHP treatment and Ce6-PDT combined with L-OHP in colon cancer. MethodsCCK-8 assay, Scratch wound healing assay, and Western Blot (WB) were used to identify drug-resistant colon cancer cell line SW620/L-OHP. Annexin V/FITC assay, laser confocal double immunofluorescence staining method and WB were employed to investigate the apoptosis and autophagy changes in Ce6-PDT combined with L-OHP. ResultsDrug resistance cells SW620/L-OHP were developed under the continuous multi-generation of L-OHP treatment, and the expression of ATP-binding cassette subfamily B member 1 (ABCB1) and ATG5 proteins were increased. The results of immunofluorescence showed that LC3B accumulated in SW620 cells and SW620/L-OHP cells under the treatment of L-OHP. The WB results indicated that LC3B and ATG5 protein expression was increasing in SW620 cells and SW620/L-OHP cells. Inhibition of L-OHP-induced autophagy reduces SW620 cells and SW620/L-OHP cells’ viability while increasing apoptosis and the Pro Caspase-3 protein expression. The combination of Ce6-PDT and L-OHP decreased the cell viability, the cell migration ability, the Bcl-2 protein expression, and increased the apoptosis rate, Pro Caspase-3 protein expression in SW620 cells. ConclusionsL-OHP can cause SW620 cells drug resistance. Autophagy plays a protective role in the L-OHP treatment of SW620 cells and SW620/L-OHP cells, and inhibition of autophagy can increase the efficacy of L-OHP. Ce6-PDT combined with L-OHP can further improve the tumor's therapeutic effect, and autophagy inhibition can improve the efficacy of combined therapy.

ANTI-CANCER PROPERTIES OF CEPHAELINE IN ORAL SQUAMOUS CELL CARCINOMA CELL LINE

Objectives To evaluate the anticancer properties of cephaeline in an oral squamous cell carcinoma (OSCC) cell line, including in cancer stem cells (CSC) regulation. Study Design The SCC-9 cell line was used as an in vitro OSCC model. Briefly, cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and cell migration by wound healing scratch assay. Gistone acetylation (H3K9ac) status was determined by immunofluorescence staining. CSC was evaluated by clonogenic assay. Results A single-dose administration of cephaeline reduced viability of OSCC cells and delayed cell migration. Moreover, cephaeline halted CSC colony formation. Additionally, cephaeline affected histone acetylation status. Conclusions Our findings suggest that cephaeline has anti-cancer properties in OSCC. Further analysis with other cell lines and in vivo studies are needed to validate these findings. To evaluate the anticancer properties of cephaeline in an oral squamous cell carcinoma (OSCC) cell line, including in cancer stem cells (CSC) regulation. The SCC-9 cell line was used as an in vitro OSCC model. Briefly, cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and cell migration by wound healing scratch assay. Gistone acetylation (H3K9ac) status was determined by immunofluorescence staining. CSC was evaluated by clonogenic assay. A single-dose administration of cephaeline reduced viability of OSCC cells and delayed cell migration. Moreover, cephaeline halted CSC colony formation. Additionally, cephaeline affected histone acetylation status. Our findings suggest that cephaeline has anti-cancer properties in OSCC. Further analysis with other cell lines and in vivo studies are needed to validate these findings.

Isolation and characterization of plasma-derived exosomes from olive flounder (Paralichthys olivaceus) and their wound healing and regeneration activities

Exosomes have garnered enormous interest for their role in physiological and pathological processes and their potential for therapeutic and diagnostic applications. In this study, exosomes were isolated from plasma of olive flounder (Paralichthys olivaceus) and their physiochemical and morphological characteristics, as well as wound healing and regeneration activities were determined. Isolated exosomes had typical characteristics, including average particle diameter (151.82 ± 9.17 nm), concentration (6.31 × 1010 particles/mL) with a membrane-bound, cup-shaped morphology. Exosome marker proteins, tetraspanins (CD63, CD9, and CD81), and acetylcholinesterase were detected, indicating the presence of exosomes in olive flounder plasma. Exosomes exhibited no toxicity in in vitro and in vivo studies, even at the highest treatment concentrations (100 and 400 μg/mL, respectively), confirming their suitability for further functional studies. Following exosome treatment (50 and 100 μg/mL), substantial cell migration with rapid closure of the open wound area in in vitro scratch wound healing assay and faster zebrafish larvae fin regeneration rate was observed compared to that of the vehicle. Moreover, exosomes exhibited immunomodulatory properties associated with wound healing, based on mRNA expression patterns in fathead minnow (FHM) cells. In conclusion, exosomes isolated from olive flounder plasma using ultracentrifugation exhibited minimal toxicity and enhanced wound healing and tissue regeneration activities. Identification and in-depth investigation of olive flounder plasma-derived exosome constituents will support the development of exosomes as an efficient therapeutic carrier system for fish medicine in the future.

Modulation of POPDC1 Expression by Phenothiazine and Trifluoperazine Suppress Colon Cancer Growth and Migration.

The aim of this study was to investigate the effects of CaM antagonist, PTZ, and TFP on cell proliferation and migration of colon cancer cells and its impact on POPDC protein expression. The 50% inhibitory concentration (IC50) of PTZ and TFP in SW1116, SW480, HCT-15, and COLO205 colon cancer cell lines are measured using MTT. Western blot and immunocytochemistry were used to determine the expression of PCNA, cyclin D1 (CD1), and POPDC proteins. Cell migration was observed using a scratch wound-healing assay. Treatment with PTZ and TFP inhibited colon cancer cells growth in a dose-dependent manner. PTZ and TFP significantly inhibited the activation of proliferation markers, PCNA and CD1, and the migration of colon cancer cells. Furthermore, POPDC protein was significantly suppressed in all cell types of colon cancer, particularly in SW480. Finally, the CaM antagonist upregulates the POPDC1 expression in colon cancer cells. These findings suggest that CaM antagonists suppress colon cancer cells proliferation via downregulation of CD1 and PCNA. In addition, POPDC protein could be used as a biomarker in colon cancer, and CaM antagonist could be used to regulate POPDC1 expression. This study suggests that targeting POPDC1 with CaM inhibition could be a potential therapeutic strategy for colon cancer treatment.<br />.

Transforming growth factor β1-enriched secretome up-regulate osteogenic differentiation of dental pulp stem cells, and a potential therapeutic for gingival wound healing: A comparative proteomics study

Objectives: Current study aimed at comparing the human dental pulp-derived stem cell (hDPSC) secretome (Control secretome) and transforming growth factor beta1 (TGF-β1)-transfected hDPSC secretome (TGF-β1 Secretome), which have the potential to be therapeutic in terms of regenerative dentistry, in terms of osteogenesis, adipogenesis and gingival wound healing with proteomic analyses. Materials and Methods: pCMV-TGF-β1 plasmid was transfected into hDPSCs by electroporation. hDPSC and TGF-β1 transfected hDPSC secretomes were collected for LC-MS/MS. Protein contents in control secretome and TGF-β1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations for canonical pathways, upstream regulators and networks were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. Surface marker expressions between groups, treated secretome were measured by flow cytometry. To support the proteomic data morphologically, we performed osteogenic-adipogenic differentiation in hDPSCs treated with control secretome and TGF-β1 secretome, and scratch wound healing assay in gingival fibroblasts. Statistical analyses were performed by GraphPad Prism 8.02. Results: Venn diagram classification showed us 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-β1 secretome. TGF-β1 secretome was found to have therapeutic effect on MSC-specific immunophenotypes. TGF-β1 secretome was determined to up-regulate osteogenesis-related molecules and pathways while down-regulating adipogenesis-related pathways. Analysis of canonical pathways showed that TGF-β1 secretome is associated with the wound healing pathway. Conclusion: Our study provided the first evidence that proteins identified in TGF-β1-transfected hDPSC secretomes are potential regulators of osteogenic/adipogenic differentiation and fibroblast wound healing. Clinical Significance: Based on these results, TGF-β1 secretome may have a therapeutic effect in repairing osteoporosis-related bone injuries, wound healing of oral mucosa and gingival tissue. TGF-β1 secretome may be a potential cell-free therapeutic in orthopedics and regenerative dentistry.

The Compatibility of Alisma and Atractylodes Affects the Biological Behaviours of VSMCs by Inhibiting the miR-128-5p/p21 Gene.

Objective The compatibility of Alisma and Atractylodes (AA) has been estimated to exhibit antiatherosclerotic effects, but the mechanism remains unclear. This study aimed to identify the role of AA in oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cell (VSMC) behaviours and to explore the effects of microRNAs (miRNAs). Methods A scratch wound-healing assay was used to detect the migration of VSMCs, and immunocytochemistry and western blotting for SM22ɑ were used to evaluate phenotypic transformation. Bromodeoxyuridine (BrdU) immunocytochemistry and flow cytometry were applied to detect the proliferation of VSMCs. miRNA microarray profiling was performed using Lianchuan biological small RNA sequencing analysis. VSMCs were transfected with the miR-128-5p mimic and inhibitor, and the migration, phenotypic modulation, and proliferation of VSMCs were investigated. The 3′UTR-binding sequence site of miR-128-5p on the p21 gene was predicted and assessed by luciferase assays. Result AA and the extracellular regulated protein kinase 1/2 (ERK1/2) blocker U0126 markedly inhibited migration, elevated smooth muscle 22α (SM22α) expression, repressed VSMC proliferation, elevated miR-466f-3p and miR-425-3p expression, and suppressed miR-27a-5p and miR-128-5p expression in ox-LDL-induced VSMCs. miR-128-5p targets the tissue inhibitor of metalloproteinases (TIMPs), silent information regulator 2 (SIRT2), peroxisome proliferator-activated receptor (PPAR), and p21 genes, which are linked to the behaviours of VSMCs. The miR-128-5p mimic promoted the migration and proliferation of VSMCs and suppressed p21, p27, and SM22ɑ expression. The inhibitor increased p21, p27, and SM22ɑ expression and repressed the migration, phenotypic transformation, and proliferation of VSMCs. miR-128-5p directly targeted the 3′UTR-binding sequences of the p21 gene, negatively regulated p21 expression, and supported the proliferation of VSMCs. Conclusion Our research showed that the migration, phenotypic transformation, and proliferation of ox-LDL-induced VSMCs were repressed by AA through inhibiting miR-128-5p by targeting the p21 gene, which may provide an effective option for the treatment of atherosclerosis.

Human antigen R affects the migration and invasion of human lung cancer A549 cells via regulating E-cadherin suppressor Snail.

Recent studies demonstrated that the progression and metastasis of lung cancer were associated with human antigen R (HuR), a post-transcriptional RNA-binding protein that stabilize and regulate the expression of many tumor-related genes. Although HuR was shown to affect the expressions of epithelial cadherin (E-cadherin), a tumor migration suppressor, in airway epithelial cells, esophageal squamous and colon cancer cells, direct evaluation for the effect and mechanism of HuR on the migration and invasion of lung cancer cells is not documented. In this study, HuR was knocked down via RNA interference and overexpressed using recombinant plasmid in adenocarcinomic human alveolar basal epithelial A549 cells. No apparent inhibition of cell viability was observed. HuR knocked down significantly suppressed A549 migration and invasion in scratch wound healing and transwell assays, with an increase in E-cadherin expression, while the overexpression of HuR notably facilitated A549 migration and invasion, with a decrease in E-cadherin level. In addition, immunoprecipitation study showed that HuR directly interacted with Snail, a repressor of E-cadherin, and upregulated the expression of Snail in A549 cells. These combined results suggested that the effect of HuR on A549 migration and invasion was realized by stabilizing and increasing the expression of Snail, which in-turn interfered with the expression of E-cadherin. The finding of this study revealed direct evidence that HuR affected the migration and invasion of lung cancer cells via regulating E-cadherin and Snail, providing an additional reference and mechanistic clue for further researches and therapeutic strategies in treating lung cancer.

A novel hypoxic lncRNA, HRL-SC, promotes the proliferation and migration of human dental pulp stem cells through the PI3K/AKT signaling pathway

BackgroundHuman dental pulp stem cells (hDPSCs) are critical for pulp generation. hDPSCs proliferate faster under hypoxia, but the mechanism by which long noncoding RNA (lncRNA) regulates this process is not fully understood.MethodsNovel lncRNAs were obtained by reanalysis of transcriptome datasets from RNA-Seq under hypoxia compared with normoxia, and a differential expression analysis of target genes was performed. Bioinformatics analyses, including gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis and gene set enrichment analysis, were used to understand the function of key novel lncRNAs. hDPSCs were isolated from dental pulp tissue. EdU and scratch wound healing assays were used to detect the proliferation and migration of hDPSCs. qRT-PCR was used to detect changes in the RNA expression of selected genes. RNA fluorescence in situ hybridization, small interfering RNA, qRT-PCR and Western blot analysis were used to explore the function of key novel lncRNAs.ResultsWe identified 496 novel lncRNAs in hDPSCs under hypoxia, including 45 differentially expressed novel lncRNAs. Of these, we focused on a key novel lncRNA, which we designated HRL-SC (hypoxia-responsive lncRNA in stem cells). Functional annotation revealed that HRL-SC was associated with hypoxic conditions and the PI3K/AKT signaling pathway. HRL-SC was mainly located in the cytoplasm of hDPSCs and had stable high expression under hypoxia. Knockdown of HRL-SC inhibited the proliferation and migration of hDPSCs and the expression levels of PI3K/AKT-related marker proteins. Furthermore, the AKT activator SC79 partially offset the inhibitory effect caused by the knockdown, indicating that HRL-SC promoted hDPSCs through the PI3K/AKT signaling pathway.ConclusionsHypoxia-responsive lncRNA HRL-SC promotes the proliferation and migration of hDPSCs through the PI3K/AKT signaling pathway, and this understanding may facilitate the regenerative application of hDPSCs.

Cleavage of tropomodulin-3 by asparagine endopeptidase promotes cancer malignancy by actin remodeling and SND1/RhoA signaling

BackgroundAbnormal proliferation and migration of cells are hallmarks of cancer initiation and malignancy. Asparagine endopeptidase (AEP) has specific substrate cleavage ability and plays a pro-cancer role in a variety of cancers. However, the underlying mechanism of AEP in cancer proliferation and migration still remains unclear.MethodsCo-immunoprecipitation and following mass spectrometry were used to identify the substrate of AEP. Western blotting was applied to measure the expression of proteins. Single cell/nuclear-sequences were done to detect the heterogeneous expression of Tmod3 in tumor tissues. CCK-8 assay, flow cytometry assays, colony formation assay, Transwell assay and scratch wound-healing assay were performed as cellular functional experiments. Mouse intracranial xenograft tumors were studied in in vivo experiments.ResultsHere we showed that AEP cleaved a ubiquitous cytoskeleton regulatory protein, tropomodulin-3 (Tmod3) at asparagine 157 (N157) and produced two functional truncations (tTmod3-N and tTmod3-C). Truncated Tmod3 was detected in diverse tumors and was found to be associated with poor prognosis of high-grade glioma. Functional studies showed that tTmod3-N and tTmod3-C enhanced cancer cell migration and proliferation, respectively. Animal models further revealed the tumor-promoting effects of AEP truncated Tmod3 in vivo. Mechanistically, tTmod3-N was enriched in the cell cortex and competitively inhibited the pointed-end capping effect of wild-type Tmod3 on filamentous actin (F-actin), leading to actin remodeling. tTmod3-C translocated to the nucleus, where it interacted with Staphylococcal Nuclease And Tudor Domain Containing 1 (SND1), facilitating the transcription of Ras Homolog Family Member A/Cyclin Dependent Kinases (RhoA/CDKs).ConclusionThe newly identified AEP-Tmod3 protease signaling axis is a novel “dual-regulation” mechanism of tumor cell proliferation and migration. Our work provides new clues to the underlying mechanisms of cancer proliferation and invasive progression and evidence for targeting AEP or Tmod3 for therapy.

Anti-EMT and anti-fibrosis effects of protocatechuic aldehyde in renal proximal tubular cells and the unilateral ureteral obstruction animal model

Context Protocatechuic aldehyde (PCA) is a natural product that has various benefits for fibrosis. Objective This study evaluated the effects of PCA on renal fibrosis. Materials and methods Epithelial–mesenchymal transition (EMT) was induced by 20 ng/mL transforming growth factor-β1 (TGF-β1), followed by treatment with 1 and 5 μM PCA, in the rat renal proximal tubular cell line NRK-52E. Cell viability, protein expression, and scratch wound-healing assays were conducted. Sprague–Dawley (SD) rats underwent unilateral ureteral obstruction (UUO) surgery for renal fibrosis indication and were treated with 50 and 100 mg/kg PCA for 14 days. Results The IC50 of PCA was appropriately 13.75 ± 1.91 μM in NRK-52E cells, and no significant difference at concentrations less than 5 μM. PCA ameliorated TGF-β1-induced EMT, such as enhanced E-cadherin and decreased vimentin. Fibrotic markers collagen IV and α-smooth muscle actin (α-SMA) increased in TGF-β1-induced NRK-52E. Moreover, PCA reduced TGF-β1-induced migration in the wound-healing assay. Analysis of rat kidneys indicated that PCA reduced UUO-induced hydronephrosis (control: 15.11 ± 1.00%; UUO: 39.89 ± 1.91%; UUO + PCA50: 18.37 ± 1.61%; UUO + PCA100: 17.67 ± 1.39%). Protein level demonstrated that PCA not only decreased vimentin expression and enhanced E-cadherin expression, but inhibited UUO-induced collagen IV and α-SMA upregulation, indicating that it could mitigate EMT in a rat model of UUO-induced renal fibrosis. Discussion and conclusions This study suggested that PCA decreases TGF-β1-induced fibrosis and EMT in vitro and in vivo. These findings demonstrate pharmacological effects of PCA and might be a potential strategy for the prevention of organ fibrosis in clinics.

Tumor-Inhibitory Effects of Zerumbone Against HT-29 Human Colorectal Cancer Cells.

Colorectal cancer (CRC) is the second cause of cancer-associated death globally. Recently, herbal medicinal products and, in particular, zerumbone have been widely studied and used for cancer treatment as they induce significant anti-cancer effects. However, there is limited information about the anti-cancer effects of zerumbone in CRC. Therefore, we aimed to investigate the in vitro anti-cancer effects of the zerumbone in CRC, focusing on cell apoptosis and migration. Anti-proliferative and anti-migratory effects of zerumbone on HT-29 cells were evaluated using MTT and scratch wound healing assay, respectively. Quantitative real-time PCR (qRT-PCR) was performed to determine the mRNA expression levels of migration and apoptosis-related genes. Apoptosis and cell cycle distribution were evaluated by flow cytometry. The intracellular level of reactive oxygen species (ROS) was measured using a ROS assay kit. Additionally, matrix metalloproteinase-2/-9 (MMP-2/-9) activity was determined using gelatin zymography. Zerumbone suppressed the viability of the HT-29 cells dose-dependently while having less cytotoxicity on normal NIH/3T3 cells. Zerumbone induced apoptosis in HT-29 cells and arrested the cell cycle in the G2/M phase. These effects were associated with alteration in the expression of apoptosis-related genes (up-regulation of Bax and down-regulation of Bcl-2 genes). Zerumbone also enhanced the generation of ROS in HT-29 cells. Furthermore, zerumbone significantly inhibited the migration of HT-29 cells and decreased MMP-2/-9 mRNA expression and activity. Our findings provide a potential use for zerumbone to induce apoptosis and suppress metastasis in HT-29 cells; thus, it could be developed as a promising natural agent for future CRC therapy.

Abstract 5998: Peroxiredoxin IV promotes progression of colorectal cancer

Abstract Peroxiredoxin IV (Prx4) is a multifunction enzyme with a primary antioxidant role of reducing free radicals such as hydrogen peroxide and peroxynitrite. Literature review suggests that Prx4 upregulation in colorectal cancer is associated with higher tumor metastasis and shorter survival of patients. However, the function and mechanisms of Prx4 in cancer development are not well understood. The goal of this study is to validate the oncogenic role of Prx4 and to identify potential mechanisms of invasion and metastasis. Western Blot was performed to compare the expression of Prx4 protein in a normal colon cell line and seven colorectal cancer cell lines. shRNA mediated knockdown of Prx4 was performed in cancer cell lines with higher expression of Prx4. Stable vector control and Prx4 knockdown cells were used to perform Scratch Wound healing assay to compare the rate of migration. Transwell Matrigel invasion assay was performed using the same cell lines with 10% FBS containing-medium as chemoattractant. Vector control and Prx4 knockdown HCT116 cells were orthotopically implanted into the cecum wall of immunodeficient mice and humanely euthanized four weeks later. Prx4 expression is upregulated in colorectal cancer cell lines compared to normal colon cell line NCM356. Stable knockdown of Prx4 in HCT116 and RKO cell lines resulted in a significant decrease in migration and invasion rates of cancer cell lines in vitro compared to vector control. Orthotopic implantation of HCT116 vector control and Prx4 knockdown cells into cecum wall resulted in a lower metastasis rate to the liver and the lungs. We are currently conducting mechanistic studies to determine how the loss of Prx4 leads to a decrease in malignancy of colorectal cancer cell lines. Prx4 promotes colorectal cancer cell invasion and metastasis. Our study suggests Prx4 is a critical oncogenic protein that can be used as a therapeutic target for patients. This work was partially supported by the National Institutes of Health R01CA222596, Molecular Mechanisms of Toxicity Training Grant in Toxicology (T32ES07266), and American Cancer Society RSG-16-213-01-TBE. Citation Format: Pratik Thapa, Hong Jiang, Yanning Hao, Na Ding, Aziza Alshahrani, Yekaterina Zaytseva, Qiou Wei. Peroxiredoxin IV promotes progression of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5998.

Stress-induced epinephrine promotes epithelial-to-mesenchymal transition and stemness of CRC through the CEBPB/TRIM2/P53 axis

BackgroundPrevious studies have indicated that chronic emotional stressors likely participate in the occurrence of cancers. However, direct evidence connecting stress and colorectal cancer development remains almost completely unexplored.MethodsChronic stress mouse model was used to investigate the influence of stress on tumorigenesis. Several major agonists and antagonists of adrenergic receptors were applied to investigate the effects of β-adrenergic signaling on the development of CRC. Chromatin immunoprecipitation assays (CHIP) were used to investigate the binding of p53 and CEBPB to TRIM2 promoter. Mammosphere cultures, Cell Counting Kit-8 (CCK-8) assay, colony-formation assay, scratch wound healing assays, qPCR, immunofluorescence, coimmunoprecipitation and western blotting were used to explore the effect of stress-induced epinephrine on the CEBPB/TRIM2/P53 axis and the progress of CRC cells.ResultsIn this study, we found that stress-induced epinephrine (EPI) promotes the proliferation, metastasis and CSC generation of CRC primarily through the β2-adrenergic receptor. Furthermore, our studies also confirmed that chronic stress decreased the stability of p53 protein by promoting p53 ubiquitination. Results of transcriptome sequencing indicated that TRIM2 was overexpressed in cells treated with EPI. Further studies indicated that TRIM2 could regulate the stability of p53 protein by promoting p53 ubiquitination. Finally, we further proved that CEBPB was regulated by EPI and acts as the upstream transcription factor of TRIM2.ConclusionsOur studies proved that stress-induced EPI promotes the development and stemness of CRC through the CEBPB/TRIM2/P53 axis.

Ligustilide from Radix Angelica Sinensis prevents the migration of vascular smooth muscle A7r5 cells based on network pharmacology and experimental verification

ObjectiveLigustilide (Lig), a major component of Radix Angelica Sinensis, exhibits a protective effect on many cardiovascular diseases. Our previous study showed that Lig inhibited the proliferation of vascular smooth muscle cells. In this study, we aimed to investigate the effect of Lig on the migration of vascular smooth muscle A7r5 cells induced by Angiotensin II (Ang II) and explore the mechanism underlying based on network pharmacology. MethodsIn this study, scratch-wound healing assay and transwell migration assay were used to assess the migration activity of Lig on Ang II-induced vascular smooth muscle A7r5 cells, and network pharmacology method was used to predict the possible target molecules. Western blot analysis, Gelatin zymography assay, and small interfering RNA (siRNA) were used to determine the underlying mechanism of Lig on the migration in Ang II-induced A7r5 cells. ResultsWe found that Lig could prevented the migration induced by Ang II in A7r5 cells, which could be associated with c-Myc and MMPs from the results of network pharmacology. Our results revealed that Lig could significantly reduce the increase in the protein expression levels of c-Myc and MMP-2 in Ang II-induced A7r5 cells, but not affect the protein expression levels of MMP-9. Our data further showed that c-Myc siRNA, just as Lig, could obviously inhibit the cell migration accompanied by decreased MMP-2 expression. ConclusionThese findings suggested the anti-migratory effect of Lig could be associated with the c-Myc/MMP-2 pathway, and Lig administration had a promising potential for preventing cardiovascular diseases.

CircSRSF4 Enhances Proliferation, Invasion, and Migration to Promote the Progression of Osteosarcoma via Rac1.

(1) Background: At present, cancer cell metastasis is the main cause of death in patients with malignant tumors, and up to 23% of osteosarcoma patients have died due to lung and lymph node metastasis. Therefore, finding new molecules involved in tumor development can provide new strategies for the diagnosis and treatment of osteosarcoma patients. Circular RNAs (circRNAs) are a type of RNA molecule that are connected head-to-tail to form a closed ring. There is increasing evidence that circRNAs are RNA molecules with many biological functions in various diseases. However, the role and mechanism of circRNAs in osteosarcoma have rarely been reported. (2) Methods: The expression of circSRSF4 in osteosarcoma tissues and cell lines was detected by quantitative real-time PCR (RT-qPCR), and the result of high-throughput sequencing was verified. In order to explore the effect of circSRSF4 on tumor proliferation, invasion, and migration, a dual-luciferase reporter assay, RNA binding protein immunoprecipitation assay, cell counting kit-8 (CCK-8), transwell assay, scratch wound healing assay, Western blot analysis, and other experiments were carried out in vitro. Rescue experiments and a xenograft model confirmed that circSRSF4 directly acted on miR-224 to regulate Rac1 expression. (3) Results: The expression of circSRSF4 was significantly higher in osteosarcoma tissues and cell lines. Down-regulating the expression of circSRSF4 in vitro significantly inhibited the proliferation, invasion, and migration of cells, and also reduced the expression of Rac1, while the overexpression of Rac1 and miR-224 inhibition could reverse these effects. The inhibition of circSRSF4 expression in vivo also attenuated tumor growth. A mechanistic study showed that circSRSF4 can be used as an miR-224 sponge to up-regulate the expression of Rac1, thereby promoting the development of osteosarcoma. (4) Conclusions: CircSRSF4 acting as a ceRNA promotes the malignant behavior of osteosarcoma through the circSRSF4/miR-224/Rac1 axis, which provides a new theoretical basis for the clinical prevention and treatment of osteosarcoma and the study of related markers and intervention targets.

Oleuropein Counteracts Both the Proliferation and Migration of Intra- and Extragonadal Seminoma Cells

Recent and growing literature has reported that oleuropein (OLE), the main polyphenol in olive leaf extract, inhibits tumor cell proliferation and reduces the invasiveness properties of cancer cells; therefore, OLE may play a significant role in the development of new drugs for cancer treatment. These antineoplastic properties have been reported in many experimental cancer models, but the effect of OLE on seminoma cells is yet to be evaluated. In the present study, we demonstrate, for the first time, that OLE reduces cell viability in both intra- and extragonadal TCAM-2 and SEM-1 seminoma cells, respectively, in a dose-dependent manner. As shown by Western-blot analysis, OLE exposure reduced cyclin-D1 expression and upregulated p21Cip/WAF1, concomitantly affecting the upstream pathway of NF-κB, leading to the reduction of its nuclear content, thereby suggesting that OLE could modulate cell-cycle regulators by inhibiting NF-κB. Moreover, Annexin V staining revealed that OLE induced apoptosis in cancer cells and upregulated the pro-apoptotic factor BAX. Through wound-healing scratch and transmigration assays, we also demonstrated that OLE significantly reduced the migration and motility of TCAM-2 and SEM-1 cells, and downregulated the expression of TGFβ-1, which is known to be the main pro-fibrotic factor involved in the acquisition of the migratory and invasive properties of cancer cells. Collectively, our results indicate that OLE reduces seminoma cell proliferation, promotes apoptosis, and counteracts cell migration and motility. Further studies are needed to explore the molecular mechanisms underlying these observed effects.

LINC00839 Promotes Neuroblastoma Progression by Sponging miR-454-3p to Up-Regulate NEUROD1.

Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Increasing long non-coding RNAs (lncRNAs) are reported to be associated with NB tumorigenesis and aggressiveness. Here, we attempted to investigate the biological functions of LINC00839 in NB progression as well as its possible pathogenic mechanisms. Public microarray datasets were applied to unearth the abnormally expressed lncRNAs in NB. RT-qPCR analysis was used to measure the expression of LINC00839, miR-454-3p, and neuronal differentiation 1 (NEUROD1) mRNA. The protein level was determined by awestern blot assay. CCK-8, plate clone formation, EdU, wound-healing scratch, and transwell assays were employed to evaluate cell proliferation, migration, and invasion. Xenografts were developed in nude mice to determine the effects of LINC00839 on NB tumor growth. Dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments were performed to identify the interaction between miR-454-3p and LINC00839 or NEUROD1. According to GSE datasets (GSE16237 and GSE16476), LINC00839 was found as a potential driver of NB progression. LINC00839 expression was higher in NB tumor tissues and cells. Also, LINC00839 expression was positively correlated with MYCN amplification, advanced INSS stages, and worse prognosis. Silencing of LINC00839 suppressed cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, LINC00839 could act as a sponge of miR-454-3p to facilitate the expression of its target NEUROD1. Moreover, miR-454-3p was demonstrated to exert an anti-cancer activity in NB. More importantly, the tumor-suppressive properties mediated by LINC00839 knockdown were significantly counteracted by the inhibition of miR-454-3p or overexpression of NEUROD1. Our study demonstrates that LINC00839 exerts an oncogenic role in NB through sponging miR-454-3p to up-regulate NEUROD1 expression, deepening our comprehension of lncRNA involved in NB and providing access to thepossibility of LINC00839 as a therapeutic target for NB.

LINC02154 promotes the proliferation and metastasis of hepatocellular carcinoma by enhancing SPC24 promoter activity and activating the PI3K-AKT signaling pathway.

Abnormal expression of long non-coding RNAs (lncRNAs) has been associated with the initiation and progression of hepatocellular carcinoma but, as yet, the clinicopathologic significance and potential role of Linc02154 in HCC remains to be determined. Here, we aimed to investigate the potential role and mode of action of Linc02154 in HCC. The expression of Linc02154 in 20 pairs of HCC/normal tissues and 7 HCC cell lines was detected by qRT-PCR. The localization of Linc02154 in HCC cells was detected using fluorescence in situ hybridization and nuclear-plasma separation assays. MTS, EdU incorporation, colony formation, flow cytometry, scratch wound-healing and transwell assays were performed to assess the role of Linc02154 in HCC cell proliferation, migration and invasion in vitro, and BALB/c nude mice xenografts were used to evaluate its role in vivo. RNA sequencing and Western blotting were used to evaluate the regulatory effect of Linc02154 on SPC24 gene expression. A dual-luciferase reporter assay was used to assess a putative interaction of Linc02154 with the SPC24 promoter. We identified a new lncRNA, Linc02154, that is highly expressed in HCC cells and tissues of patients with a poor overall survival. Functional experiments revealed that exogenous Linc02154 expression in MHCC-97H and SK-Hep1 cells promoted their proliferation, migration and invasion in vitro and their tumorigenesis in vivo. Using a dual luciferase reporter assay we found that Linc02154 can enhance SPC24 promoter (-500bp ~ -1000 region) activity. Exogenous over-expression of Linc02154 led to up-regulation of SPC24 by activating PI3K/AKT and its downstream signals, including cell cycle progression and EMT-associated gene expression. Our data suggest that Linc02154 may serve as a valuable biomarker of HCC and as a potential therapeutic target.

Calcium acts as a central player in melatonin antitumor activity in sarcoma cells

PurposeChondrosarcoma and osteosarcoma are the most frequently occurring bone cancers. Although surgery and chemotherapy are currently clinically applied, improved treatment options are urgently needed. Melatonin is known to inhibit cell proliferation in both tumor types. Although the underlying mechanisms are not clear yet, calcium homeostasis has been reported to be a key factor in cancer biology. Here, we set out to investigate whether regulation of calcium by this indolamine may be involved in its antitumor effect.MethodsCell viability was measured using a MTT assay and flow cytometry was used to measure levels of cytosolic calcium, intracellular oxidants, mitochondrial membrane potential and cell cycle progression. Mitochondrial calcium was analyzed by fluorimetry. Cell migration was determined using a scratch wound-healing assay. Western blot analysis was used to assess the expression of proteins related to cell cycle progression, epithelial to mesenchymal transition (EMT), Ac-CoA synthesis and intracellular signaling pathways.ResultsWe found that melatonin decreases cytosolic and mitochondrial Ca2+ levels, intracellular oxidant levels, mitochondrial function and the expression of the E1 subunit of the pyruvate dehydrogenase complex. These changes were found to be accompanied by decreases in cell proliferation, cell migration and EMT marker expression. The addition of CaCl2 prevented the changes mentioned above, while co-treatment with the calcium chelator BAPTA enhanced the effects.ConclusionsOur data indicate that regulation of calcium homeostasis is a key factor in the inhibition of cell proliferation and migration by melatonin. This effect should be taken into consideration in combined therapies with traditional or new antitumor compounds, since it may circumvent therapy resistance.

Toll Like Receptor 4 Inhibition Improves Antitumor Responses to MBQ‐167 in Triple Negative Breast Cancer Cells in vitro

Triple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen, progesterone, and human epithelial growth factor receptors. Due to the scarce availability of receptor‐targeted or hormonal treatments, TNBC patients are submitted to taxane‐based chemotherapies like paclitaxel. Patients initially respond to paclitaxel, but 4 out of 10 patients may develop tumor recurrence along with high metastasis and mortality rates. Several studies demonstrate that paclitaxel may induce chemoresistance and metastasis through the activation of Toll‐Like Receptor 4 (TLR4)/NFKB signaling pathway, a known cancer prognostic marker in breast cancer. Hence, new treatments are needed to improve TNBC prognosis in patients. Recently, our laboratory and collaborators developed MBQ‐167, a potent Rac and Cdc42 inhibitor that decreases tumor burden, cell proliferation, and metastasis in TNBC mouse models. In preliminary studies with TNBC mice, we observed that MBQ‐167/Paclitaxel combination treatment prevented metastasis progression compared to paclitaxel treatment alone. This led us to hypothesize that MBQ‐167 chemosensitizes aggressive breast cancer cells and reduces metastasis by blocking Rac/Cdc4, hence decreasing their downstream target NfkB activity. This research project aims to evaluate cellular and molecular events that explain how MBQ‐167 prevents paclitaxel‐induced metastasis. Here, we generated TLR4‐knockdowns (TLR4‐KD) in MDA‐MB‐231 TNBC cell lines using lentiviral particles with shRNAs against the TLR4 mRNA. After knockdown validation with western blot analysis (WB), Wildtype (MDA‐MB‐231), Scramble control knockdowns (Sc‐KD), and TLR4‐KD cells were treated with different concentrations of MBQ‐167, paclitaxel, doxorubicin, or combinations. After treatments, we evaluated the effect on cell viability from MTT assays at 96 and 120 hours, cell apoptosis through Caspase‐Glo 3/7 Assays at 48 hours, and cellular migration through scratch wound healing assays. Our results demonstrate that partial inhibition of TLR4 decreases the cell viability of cells, increases apoptosis, and decreases cell migration after treatment with MBQ‐167 (250 nM and 500 nM), Paclitaxel (5 nM and 10 nM), or Doxorubicin (250 nM and 500 nM). We also observed an additive response upon combinatorial treatments with MBQ‐167/Paclitaxel and MBQ‐167/Doxorubicin when TLR4‐KD cells were compared to wild‐type and Sc‐KD cells. Altogether, these results suggest that TLR4‐KD chemosensitizes cells to Paclitaxel and Doxorubicin chemotherapies. In addition, TLR4 inhibition improves cellular response to Rac/Cdc42 inhibitor, MBQ‐167. Therefore, TLR4‐KD may reduce Rac and NfkB signaling, improving treatment response to MBQ‐167, MBQ‐167/Paclitaxel, and MBQ‐167/Doxorubicin. Other cellular studies, including Rac and NfkB activation assays, are being evaluated in our laboratory.