Transwell Chamber Assay Research Articles

Diacylglycerol kinase γ facilitates the proliferation and migration of neural stem cells in the developing neural tube.

In this study, we aim to investigate diacylglycerol kinase (DGK) γ expression in developing neural tubes (NTs) and its effects on neural stem cell (NSC) proliferation and migration. Whole-mount in situ hybridization (WMISH) and immunohistochemistry are performed to explore DGKγ localization in developing NTs in vivo. NSCs are treated with sh-DGKγ, R59949, or PMA in vitro. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and neurosphere formation assay are utilized to evaluate NSC proliferation. Neurosphere migration assay and a transwell chamber assay are used to assess NSC migration. The diacylglycerol (DAG) content is detected via enzyme-linked immunosorbent assay (ELISA). The mRNA expression of DGKγ is detected via quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of DGKγ, protein kinase C (PKC) and phosphorylated PKC (p-PKC) are detected via western blot analysis. The results show that DGKγ mRNA is expressed predominantly in developing NTs. The neuroepithelium in developing NTs is positive for NSC markers, including Nestin, glial fibrillary acidic protein (GFAP), and DGKγ. DGKγ is expressed in the cytoplasm and nucleus of the neuroepithelium and is coexpressed with p-PKCγ and p-PKCδ. The proliferation of NSCs, the number of EdU-positive NSCs, and the number of neurospheres are decreased by sh-DGKγ and R59949 but increased by PMA. There is a shorter migration distance of NSCs and fewer migrated NSCs in the sh-DGKγ, R59949 and PMA groups. DAG content and the p-PKCδ/PKCδ ratio are increased by sh-DGKγ, R59949 and PMA, whereas the p-PKCγ/PKCγ ratio is decreased by PMA. Taken together, our findings indicate that DGKγ facilitates NSC proliferation and migration, which is responsible for the participation of DGK in NT development. DGKγ facilitates NSC migration via the DAG/PKCδ pathway.

EZH2 mutation is associated with the development of visceral metastasis by enhancing proliferation and invasion and inhibiting apoptosis in breast cancer cells

BackgroundThe prognosis of breast cancer patients with visceral metastasis (VM) is significantly worse than that of patients without VM. We aimed to evaluate EZH2 (enhancer of zeste homolog 2) mutation as a biomarker associated with VM.MethodsData from forty-nine patients with metastatic breast cancer (MBC) pathologically confirmed at our hospital between March 2016 and September 2018 were collected. Metastatic tissue samples were obtained via ultrasound-guided needle biopsy, and paired peripheral blood samples were also collected. Tissue and blood samples were subjected to targeted next-generation sequencing via a 247-gene panel. Stably transfected MDA-MB-231 cells expressing wild-type EZH2 (EZH2WT) or a mutant form of EZH2 (EZH2K515R) were generated. Cell proliferation, colony formation ability, migration and invasion abilities and apoptosis were assessed using CCK-8 assays, plate colony formation assays, Transwell chamber assays and flow cytometry.ResultsThe incidence of EZH2 mutations in the VM subgroup was greater than that in the non-VM subgroup in the entire cohort (n = 49, 42.3% vs. 13.0%, p = 0.024) and in the triple-negative breast cancer (TNBC) subgroup (n = 20, 50.0% vs. 10.0%, p = 0.05). Patients carrying EZH2 mutations had a significantly greater risk of developing VM than did those in the non-EZH2 mutation group in the entire cohort (HR 2.9) and in the TNBC subgroup (HR 6.45). Multivariate analysis revealed that EZH2 mutation was an independent prognostic factor for VM (HR 2.99, p = 0.009) in the entire cohort and in the TNBC subgroup (HR 10.1, p = 0.006). Data from cBioPortal also showed that patients with EZH2 mutations had a significantly greater risk of developing VM (HR 3.1), and the time to develop VM was significantly earlier in the EZH2 mutation group (31.5 months vs. 109.7 months, p = 0.008). Multivariate analysis revealed that EZH2 mutation (HR 2.73, p = 0.026) was an independent factor for VM after breast cancer surgery. There was no correlation between EZH2 mutations and BRCA1/2 mutations. Most of the patients (81.8%) in our cohort who developed VM carried the “c.1544A > G (p.K515R)” mutation. Compared with EZH2WT MDA-MB-231 cells, EZH2K515R MDA-MB-231 cells had greater colony formation rates (p < 0.01), greater migration and invasion rates (p < 0.001), and lower apoptosis rates (p < 0.01). The proportion of S + G2/M phase cells in the EZH2K515R group was significantly greater than that in the EZH2WT group.ConclusionsEZH2 mutation is associated with VM development in breast cancer patients. The EZH2K515R mutation leads to VM and a poor prognosis by enhancing proliferation and invasion and inhibiting apoptosis in breast cancer cells.

Inhibitory Effects of Propolis Flavonoids on Migration and Invasion of Laryngeal Cancer Cell and Analysis of Related Signal Pathways

Laryngeal cancer (LGC) is a malignant tumor that occurs in the larynx, and it is mainly treated through chemotherapy, radiotherapy, and surgery. Nevertheless, the five-year survival rate for patients is poor. Bee propolis contains various bioactive compounds and abundant anti-tumor active ingredients. Nevertheless, research on the use of propolis extracts for the treatment of LGC is relatively limited. This research aimed to demonstrate the inhibitory effects of ethanol extracts of propolis on migration (Mig) and invasion (Inv ) of LGC cells, as well as the related signaling pathways. The effects of graded ethanol extraction of propolis on the proliferation (Pro), Inv, Mig, apoptosis (Apo), and related signaling pathways of Hep-2 cells were analyzed. Propolis was extracted using ethanol (0%, 25%, 50%, 75%, and 100%) for the graded extraction of crude propolis. The flavonoid content and yield of the extracts were determined. The effects of various concentrations of propolis flavonoids on the clearance of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, O2- radicals, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals were evaluated, as well as their effects on the Pro inhibition of normal human pancreatic ductal epithelial (hTERT-HPNE) cells. Hep-2 cells of LGC were cultured using media containing 0, 25, 50, and 100 μmol/L propolis flavonoids. The cell Pro activity, Inv, Mig, Apo, and expression of PI3K/Akt pathway-related proteins were evaluated using CCK-8 assay, Transwell chamber assay, acridine orange/ethidium bromide (AO/EB) double staining method, and Western blotting, respectively. It was revealed that extraction with 50% ethanol solution yielded a higher content and yield of flavonoids, which were 51.20% and 7.42%, respectively. As the concentration of propolis flavonoids increased, the clearance rates of DPPH, O2-, and ABTS radicals, as well as the inhibition of hTERT-HPNE Pro, gradually increased. The maximum clearance rates were 84.1%, 26.6%, and 92.3%, respectively, while the maximum cell Pro inhibition rate was only 8.6%. Relative to the 0 μmol/L propolis flavonoid treatment group, the Hep-2 cells treated with 25, 50, and 100 μmol/L propolis flavonoids exhibited decreased cell Pro activity, reduced number of invasive and migratory cells, increased Apo rate, decreased PI3K and p-Akt proteins, and demonstrated a concentration-dependent effect (P &lt; 0.05). In summary, the extraction with 50% ethanol solution resulted in a higher yield of flavonoids. Propolis flavonoids demonstrated marked antioxidant activity and did not cause damage to normal hTERT-HPNE cells. They exhibited inhibitory effects on the Pro, Inv, and Mig of Hep-2 cells in LGC, and promoted cell Apo. These effects may be associated with PI3K/Akt signaling inhibition.

Acidic Pepsin Affects Laryngeal Carcinoma Cell Growth and Invasion Through Glycolysis.

The pathogenic mechanism underlying the effects of acidic pepsin in laryngeal cancer remains unclear. This study investigated whether acidic pepsin influences Glut-1 expression and glycolytic activity in laryngeal carcinoma cells and whether it plays a role in the growth and migration of these cells through glycolysis. In vitro study. A university-affiliated hospital. Laryngeal carcinoma TU 212 and TU 686 cells were treated with acidic pepsin and 2-deoxy-d-glucose (2-DG), then transfected with Glut-1 small interfering RNA (siRNA). Glucose uptake was detected by a radioimmunoassay counter, lactate secretion was detected by a lactic acid kit, and Glut-1 expression was detected by western blotting. Cell viability, migration and invasion, and clonal formation were assessed using the Cell Counting Kit-8, Transwell chamber, and clonal formation assays, respectively. Acidic pepsin significantly increased Glut-1 expression in laryngeal carcinoma cells compared with the control group (P < .01). It also significantly enhanced 18F-fluorodeoxyglucose (Cin/Cout) uptake, lactate secretion, cell viability, migration, invasion, and clonal formation in laryngeal carcinoma cells compared with the control group (P < .01). The glycolytic inhibitor 2-DG and Glut-1 siRNA significantly reversed the effects of acidic pepsin on laryngeal carcinoma cells (P < .01). Acidic pepsin enhances the growth and migration of laryngeal carcinoma cells by upregulating Glut-1, thus promoting glycolysis.

PLUNC inhibits invasion and metastasis in nasopharyngeal carcinoma by inhibiting NLRP3 inflammasome activation

Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the nasopharynx. Palate, lung, and nasal epithelium clone (PLUNC) has been identified as an early secreted protein that is specifically expressed in the nasopharynx. The aim of this study was to determine the role and mechanism of PLUNC in NPC. We used mRNA sequencing (seq) combined with ribosome-nascent chain complex (RNC)-seq to determine the biological role of PLUNC. The expression of epithelial-to-mesenchymal transition (EMT)-related molecules was detected by western blotting. Then, cell migration and invasion were detected by wound healing and Transwell chamber assays. NPC cells were injected into the tail vein of nude mice to explore the biological role of PLUNC in vivo. The sequencing results showed that PLUNC inhibited the progression of NPC and its expression was correlated with that of NOD-like receptors. Experiments confirmed that PLUNC inhibited the invasion and metastasis of NPC cells by promoting the ubiquitination degradation of NLRP3. PLUNC overexpression in combination with the treatment by MCC950, an inhibitor of NLRP3 inflammasome activation, was most effective in inhibiting NPC invasion and metastasis. In vivo experiments also confirmed that the combination of PLUNC overexpression and MCC950 treatment effectively inhibited the lung metastasis of NPC cells. In summary, our research suggested that PLUNC inhibited the invasion and metastasis of NPC by inhibiting NLRP3 inflammasome activation, and targeting the PLUNC-NLRP3 inflammasome axis could provide a new strategy for the diagnosis and treatment of NPC patients.

The flavonoids from the fruits of Psoralea corylifolia and their potential in inhibiting metastasis of human non-small cell lung cancers

Nineteen flavonoids were isolated from the fruits of Psoralea corylifolia L., including a novel flavanol (3) and three novel isoflavones (12–14). Their chemical structures were unequivocally determined through comprehensive spectral data analysis. The anti-proliferative effect of the isolated flavonoids was assessed in vitro using the MTT assay. Molecular docking and ELISA were employed to determine the inhibitory effects of the active compounds on ALK5. Isobavachalcone was found to inhibit TGF-β1 induced EMT in A549 cells by Wound healing assay and Transwell chamber assay. Immunofluorescence assay and Western blot assay showed that IBC could inhibit cytoskeleton rearrangement, reduce the phosphorylation of ALK5, ERK, and Smad, down-regulate Snail expression, and up-regulate E-cadherin expression in TGF-β1 induced A549 cells, thereby exerting the potential inhibitory effects on epithelial-mesenchymal transition (EMT) process in A549 cells. The findings presented herein establish a fundamental basis for investigating the anti-proliferative and anti-metastatic properties of psoralen flavonoids in human non-small cell lung cancer.

USP31 Activates the Wnt/β-catenin Signaling Pathway and Promotes Gastric Cancer Cell Proliferation, Invasion and Migration.

Gastric cancer (GC) has a poor prognosis because it is highly aggressive, yet there are currently few effective therapies available. Although protein ubiquitination has been shown to play a complex role in the development of gastric cancer, to date, no efficient ubiquitinating enzymes have been identified as treatment targets for GC. The TCGA database was used for bioinformatic investigation of ubiquitin-specific protease 31 (USP31) expression in GC, and experimental techniques, including Western blotting, qRT-PCR, and immunohistochemistry, were used to confirm the findings. We also analyzed the relationship between USP31 expression and clinical prognosis in patients with GC. We further investigated the effects of USP31 on the proliferation, invasion, migration, and glycolysis of GC cells in vitro and in vivo by using colony formation, CCK-8 assays, Transwell chamber assays, cell scratch assays, and cell-derived xenograft. Furthermore, we examined the molecular processes by which USP31 influences the biological development of GC. Patients with high USP31 expression have a poor prognosis because USP31 is abundantly expressed in GC. Therefore, USP31 reduces the level of ubiquitination of the Wnt/β-catenin pathway by binding to β-catenin, thereby activating glycolysis, which ultimately promotes GC proliferation and aggressive metastasis. USP31 inhibits ubiquitination of β-catenin by binding to it, stimulates the Wnt/β-- catenin pathway, activates glycolysis, and accelerates the biology of GCs, which are all demonstrated in this work.

Role of MNK/eIF4E pathway in CD4+ Th2 cytokine-induced lung fibrosis in chronic allergic asthma

Abstract Despite therapeutic advances in allergic asthma, the persistent challenge of airway remodeling still exists, necessitating further exploration. We aimed to investigate the influence of CD4+ Th2 cytokines on fibrosis associated with airway remodeling, with a focus on mesenchymal cell (MC) transformation. Using both human and mouse lung MCs, we investigated the pivotal role of MNK/eIF4E pathway in regulating fibrous protein expression driven by CD4+ Th2 cytokines. To assess this, we employed western blot and flow cytometry studies, while Luminex assays measured cytokine and chemokine secretion from activated lung MCs responding to Th2 cytokines. Treatment with Th2 cytokines significantly increased ATX expression in lung MCs, a response mitigated by the specific MNK1/2 inhibitor (eFT-508). Furthermore, this inhibitor not only abolished the phosphorylation of eIF4E but also reduced collagen levels within activated MCs induced by Th2 cytokines. Luminex assay results demonstrated a significant decrease in IL-6, CCL-2, and GM-CSF in the activated MCs upon inhibiting the MNK1/2 pathway. Additionally, eFT-508 exhibited a marked reduction in lymphocyte migration, utilizing the Boyden chamber transwell assay. Our study reveals that targeting the MNK/eIF4E pathway holds promise in mitigating airway remodeling associated with MCs fibrosis and provides valuable molecular insights for future therapeutic interventions related to fibrosis in the airway remodeling of allergic asthma.

Contribution of sphingomyelin phosphodiesterase acid-like 3B to the proliferation, migration, and invasion of ovarian cancer cells.

Cancer has the highest mortality rate among gynecological cancers and poses a serious threat to women's lives. However, the treatment options for ovarian cancer are still limited, and exploring effective targeted biomarkers is particularly important for predicting and treating ovarian cancer. Therefore, it is necessary to explore the molecular mechanisms of the occurrence and development of ovarian cancer. This investigation encompassed the analysis of gene expression profiles, measurement of transcription levels of potential target genes in peripheral blood samples from ovarian cancer patients and characterization of the ovarian cancer-related secretory protein sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B). Through bioinformatics analysis, potential target genes were identified, and their association with overall survival (OS) and progression-free survival (PFS) in ovarian cancer patients was assessed utilizing relevant databases. Subsequently, differences in target gene expression in ovarian cancer tissue samples were validated through protein blotting and quantitative real-time PCR (qRT-qPCR). Cell proliferation assays using the cell count kit-8 (CCK-8) method, as well as transwell chamber assay and pre coated matrix gel chamber assay were employed to elucidate the role of SMPDL3B in ovarian cancer cell migration and invasion. This study revealed a substantial upregulation of SMPDL3B in the serum of ovarian cancer patients, correlating with an unfavorable prognosis. High SMPDL3B expression was linked not only to increased proliferation of ovarian cancer cells, but also enhanced migration and invasion. Remarkably, the knockdown the human alkaline ceramidase 2 (ACER2) gene in cancer cells with heightened SMPDL3B expression significantly inhibited cell proliferation, migration, and invasion induced by SMPDL3B activation (P<0.05), highlighting the functional interplay between ACER2 and SMPDL3B in ovarian cancer. In summary, this study proposes SMPDL3B as a prognostic marker for ovarian cancer, with implications for potential therapeutic intervention targeting the ACER2-SMPDL3B axis.

Abstract 4221: Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models

Abstract The monocarboxylic acid transporter 4 (Mct-4), a downstream biomarker of hypoxia inducing factor (HIF)-1α, is involved in the cellular response to hypoxia, as indicated by the hypoxic response element in its promoter region. Using a tumorsphere assay as an in vitro 3-dimensional (3D) model generated using 384-well ultra-low attachment (ULA) plates for cell proliferation analysis using a plate-based image cytometer, we identify a hypoxic response in the tumorsphere model that is distinct from that of cells grown under 2-dimensional (2D) normoxic conditions and demonstrate a key role for Mct-4 in enabling 3D growth. The tumorsphere model yields evidence of an essential role for Mct-4 in multiple cell lines which were genetically modified to underexpress and overexpress Mct-4, evidence not apparent in a standard 2D model of growth in the same cell lines. In addition, we identify the effects of overexpressing Mct-4 in cancer cell migration using a transwell chamber assay. We also show that the response to hypoxia may be circumvented by transfection with a CMV promoter driven Mct-4, which confers constitutive 3D growth, wherein tumorsphere growth inhibition by small molecule HIF-1α inhibitors is mitigated. Finally, we demonstrate quantifiable gene/protein expression differences between 2D and 3D cancer models based on the normoxic and hypoxic conditions. Therefore, the tumorsphere 3D model generated using 384-well ULA plates in combination with high-throughput image cytometer is demonstrated to provide a convenient, robust, and reproducible tool and method for the elucidation of mechanisms of action underlying tumor growth and migration in the hypoxic tumor microenvironment. Citation Format: James McDonald, Leo Li-Ying Chan, Sarah Kessel, Bo Lin, Paul Weingarten. Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4221.

Design and Synthesis of Novel Anti-proliferative Formononetin Derivatives

aims: aimed to synthesize and evaluate the potential antineoplastic activity of eight newly synthesized FMN derivatives background: Formononetin (FMN) is an isoflavone component of the natural product Astragalus membranaceus. Its well-documented anticancer activity led to the synthesis of new derivatives. objective: This study aimed to synthesize and evaluate the potential antineoplastic activity of eight newly synthesized FMN derivatives. method: Evaluation of the anti-tumor effects and potential mechanisms of action of derivatives using methods such as tetrazolium bromidesalt (MTT), cell cloning, EdU staining, Tunel staining, Transwell chamber , flow cytometry, and enzyme-linked immunosorbent assay (ELISA). result: As indicated by the results, all compounds exhibited toxicity to all three types of cancer cells. Most derivatives exhibited more significant antiproliferative activity than FMN. In comparison with other derivatives, compound 8 displayed the maximum antiproliferative activity, with IC50 value of 8.973 ± 0.296, 7.240 ± 0.208, and 4.378 ± 0.380 μ mol/L for HeLa, A549, and HepG2 cells, separately. In addition, further experiments demonstrated that compound 8 could suppress in vitro proliferation of HepG2 cells, promote HepG2 cell apoptosis, suppress HepG2 cell migration and invasion, and arrest HepG2 cell growth in S phase. The mechanism may be through the reduction of Bcl-2 and Mcl-1 protein expression, the increase of Bax, P53, Fas, Caspase-3 and Caspase-9 protein expression, the reduction of mitochondrial membrane potential and the reduction of ATP production, thus promoting the apoptosis of HepG2 cells. conclusion: Compound 8 may serve as a lead compound with high potential in discovering novel antitumor drugs. Furthermore, it can be explored in depth as an anticancer drug. other: none

Taraxasterol enhanced bladder cancer cells radiosensitivity via inhibiting the COX-2/PGE2/JAK2/STAT3/MMP pathway

Purpose Radiotherapy with bladder preservation is highly acceptable among patients bearing bladder cancer (BCa), but the occurrence of secondary tolerance (ARR) during treatment is one of the important reasons for the failure of clinical radiotherapy. COX-2 has been frequently reported to be highly expressed and associated with radio-resistance in various cancers. In this study, the feasibility of Taraxasterol (Tara) as a radiosensitizer was investigated, and the target effect of Tara on COX-2 and its underlying mechanism were explored. Methods and materials The toxicity of Tara toward BCa cells was detected with the MTT method and cells in response to IR or Tara + IR were compared by clone formation assay. Next, a small RNA interference system (siRNA) was employed to decrease endogenous COX-2 expression in BCa cells, and the stem cell-like features and motion abilities of BCa cells under different treatments were investigated using microsphere formation and transwell chamber assay, respectively. Meanwhile, the expression of a series of inflammation-related molecules and stem cell characteristic molecules was determined by qRT-PCR, western blot and ELISA method. In vivo studies, BCa cells were subcutaneously injected into the right flank of each male mouse. Those mice were then grouped and exposed to different treatment: Tara, IR, IR + Tara and untreated control. The volumes of each tumor were measured every two days and target proteins were detected with immunohistochemical (IHC) staining. Results The results show that COX-2 decline, due to COX-2 knocking-down or Tara treatment, could greatly enhance BCa cells’ radiosensitivity and significantly decrease their migration, invasion and microsphere formation abilities, companied with the reduce of JAK2, phos-STAT3, MMP2 and MMP9 expression. However, Tara could not further reduce the expression of an above molecule of cells in COX-2-deficient BCa cells. Correspondingly, Tara treatment could not further enhance those siCOX-2 BCa cells response to IR. Conclusions Our data support that Tara can improve the radiosensitivity of BCa cells by targeting COX-2/PGE2. The mechanism may involve regulating STAT3 phosphorylation, DNA damage response protein activation, and expression of MMP2/MMP9.

Integrated single-cell and bulk characterization of branched chain amino acid metabolism-related key gene BCAT1 and association with prognosis and immunogenicity of clear cell renal cell carcinoma.

The relationship between clear cell renal cell carcinoma (ccRCC) and branched-chain amino acids (BCAA) metabolism has yet to be thoroughly explored. The BCAA metabolism-related clusters were constructed using non-negative matrix factorization (NMF). The features of BCAA metabolism in ccRCC were evaluated by building a prognostic model using least absolute shrinkage and selection operator (LASSO) regression algorithm. Real-time quantitative PCR (RT-qPCR) was employed to analyze differential expression of branched-chain amino acid transaminase 1 (BCAT1) between cancer and paracancer tissues and between different cell lines. Cell counting kit-8, wound healing and Transwell chamber assays were conducted to determine changes in proliferative and metastatic abilities of A498 and 786-O cells. Two BCAA metabolism-related clusters with distinct prognostic and immune infiltration characteristics were identified in ccRCC. The BCAA metabolic signature (BMS) was capable of distinguishing immune features, tumor mutation burden, responses to immunotherapy, and drug sensitivity among ccRCC patients. RT-qPCR revealed overexpression of BCAT1 in ccRCC tissues and cell lines. Additionally, single-gene RNA sequencing analysis demonstrated significant enrichment of BCAT1 in macrophages and tumor cells. BCAT1 played tumor-promoting role in ccRCC and was closely associated with immunosuppressive cells and checkpoints. BCAT1 promoted ccRCC cell proliferation and metastasis. The BMS played a crucial role in determining the prognosis, tumor mutation burden, responses to immunotherapy and drug sensitivity of ccRCC patients, as well as the immune cell infiltration features. BCAT1 was linked to immunosuppressive microenvironments and may offer new sights into ccRCC immunotherapeutic targets.

PW06 suppresses cancer cell metastasis in human pancreatic carcinoma MIA PaCa-2 cells via the inhibitions of p-Akt/mTOR/NF-κB and MMP2/MMP9 signaling pathways in vitro.

PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one], a kind of the carbazole derivative containing chalcone moiety, inducedcell apoptosisin human pancreatic carcinoma in vitro. There is no investigation to show that PW06 inhibits cancer cell metastasis in human pancreatic carcinoma in vitro. Herein, PW06 (0.1-0.8 μM) significantly exists in the antimetastatic activities of human pancreatic carcinoma MIA PaCa-2 cells in vitro. Wound healing assay shows PW06 at 0.2 μM suppressed cell mobility by 7.45 and 16.55% at 6 and 24 hours of treatments. PW06 at 0.1 and 0.2 μM reduced cell mobility by 14.72 and 21.8% for 48 hours of treatment. Transwell chamber assay indicated PW06 (0.1-0.2 μM) suppressed the cell migration (decreased 26.67-35.42%) and invasion (decreased 48.51-68.66%). Atomic force microscopy assay shows PW06 (0.2 μM) significantly changed the shape of cell morphology. The gelatin zymography assay indicates PW06 decreased MMP2's and MMP9's activitiesat 48 hours of treatment. Western blotting assay further confirms PW06 reduced levels of MMP2 and MMP9 and increased protein expressions of EGFR, SOS1, and Ras. PW06 also increased the p-JNK, p-ERK, and p-p38. PW06 increased the expression of PI3K, PTEN, Akt, GSK3α/β, and E-cadherin. Nevertheless, results also show PW06 decreased p-Akt, mTOR, NF-κB, p-GSK3β, β-catenin, Snail, N-cadherin, and vimentin in MIA PaCa-2 cells. The confocal laser microscopy examination shows PW06 increased E-cadherin but decreased vimentin in MIA PaCa-2 cells. Together, our findings strongly suggest that PW06 inhibited the p-Akt/mTOR/NF-κB/MMPs pathways, increased E-cadherin, and decreased N-cadherin/vimentin, suppressing the migration and invasion in MIA PaCa-2 cells in vitro.

Protective effect of tretinoin on cervical cancer growth and proliferation through downregulation of pFAK2 expression.

Cervical cancer, a life-threatening disease, is the seventh most commonly detected cancer among women throughout the world. The present study investigated the effect of tretinoin on cervical cancer growth and metastasis in vitro and in vivo in the mice model. Cell Counting Kit-8, clonogenic survival, and transwell chamber assays were used for determination cells proliferation, colony formation, and invasiveness. Western blotting assay was used for assessment of protein expression whereas AutoDock Vina and Discovery studio software for in silico studies. Tretinoin treatment significantly (p < .05) reduced the proliferation of HT-3 and Caski cells in concentration-based manner. Incubation with tretinoin caused a significant decrease in clonogenic survival of HT-3 and Caski cells compared with the control cultures. The invasive potential of HT-3 cells was decreased to 18%, whereas that of Caski cells to 21% on treatment with 8 μM concentration of tretinoin. In HT-3 cells, tretinoin treatment led to a prominent reduction in p-focal adhesion kinase (FAK), matrix metalloproteinases (MMP)-2, and MMP-9 expression in HT-3 cells. Treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. The metastasis of tumor in model cervical cancer mice group was effectively inhibited in spleen, intestines, and peritoneal cavity. In silico studies showed that tretinoin interacts with alanine, proline, isoleucine, and glycine amino acid residues of FAK protein to block its activation. The 2-dimensional diagram of interaction of tretinoin with FAK protein revealed that tretinoin binds to alanine and glycine amino acids through conventional hydrogen bonding. In summary, tretinoin suppressed the proliferation, colony formation, and invasiveness of cervical cancer cells in vitro. It decreased the expression of activated focal adhesion kinase, MMP-2, and MMP-9 in HT-3 cells in dose-dependent manner. In silico studies showed that tretinoin interacts with alanine and glycine amino acids through conventional hydrogen bonding. In vivo data demonstrated that treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. Therefore, tretinoin can be developed as an effective therapeutic agent for cervical cancer treatment.

Targeting ENPP1 depletion may be a promising therapeutic strategy for treating oral squamous cell carcinoma via cytotoxic autophagy-related apoptosis.

ENPP1 depletion closely related with modulation immunotherapy of several types of cancer. However, the role of ENPP1 correlation with autophagy in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. In this study, effects of ENPP1 on OSCC cells invitro were examined by cell proliferation assay, transwell chamber assay, flow cytometry analysis and shRNA technique. Cellular key proteins related to cell autophagy and apoptosis were evaluated by Western blot and immunofluorescent staining. Moreover, functions of ENPP1 on OSCC process were observed in nude mouse model. We reported that overexpression of ENPP1 promote the growth of OSCC cell xenografts in nude mouse model. In contrast, ENPP1 downregulation significantly inhibits OSCC cancer growth and induces apoptosis both invitro and invivo, which are preceded by cytotoxic autophagy. ENPP1downregulation induces a robust accumulation of autophagosomes, increases LC3B-II and decreases SQSTM1/p62 in ENPP1-shRNA-treated cells and xenografts. Mechanistic studies show that ENPP1 downregulation increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ENPP1 downregulation-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates it's effects. Collectively, these data uncover that ENPP1 downregulation induces autophagic cell death in OSCC cancer, which may provide a potential therapeutic target for the treatment of OSCC.

Molecular Mechanism Analysis of the Effect of Hederagenin Combined with L-OHP on Chemosensitivity of AGS/L-OHP Based on Network Pharmacology.

This study aimed to evaluate the pharmacological mechanism of Hederagenin (HD) combined with oxaliplatin (L-OHP) in treating gastric cancer (GC) through network pharmacology combined with experimental verification. Network pharmacology methods were used to screen potential targets for HD, L-OHP, and GC-related targets from public databases, and the intersection of the three gene sets was taken. Cross genes were analyzed through protein-protein interaction (PPI) networks to predict core targets, and related pathways were predicted through GO and KEGG enrichment analysis. The experimental results were verified by the in vitro experiments. HD was applied on AGS/L-OHP cells, and then cellular chemosensitivity and the expressions of P-gp, Survivin, Bcl-2, p-Akt, and p-PI3K genes were detected. Wound assay and Transwell Chamber assay were employed to detect the effect of HD on AGS/L-OHP cells. Nude mice xenograft models transfected using AGS/L-OHP cells were also treated with HD in order to verify the results. The size and weight of the tumor, as well as the expressions of P-gp, Survivin, Bcl-2, p- Akt and p-PI3K genes, were also measured. KEGG analysis showed that the anti-gastric cancer effect of HD was mediated mainly by PI3K-Akt signaling pathways. The PI3K-Akt signaling pathway containing more enriched genes may play a greater role in anti-gastric cancer. It was observed that for AGS/L-OHP cells jointly treated with HD and L-OHP, their activity, migration and invasion were significantly lower than those treated only using HD or L-OHP group. Moreover, expressions of p-Akt, p- PI3K, Bcl-2, P-gp, and Survivin for the HD+L-OHP group decreased significantly. Results of the in vivo experiments showed that the sizes and weights of tumors in the HD+L-OHP group were the lowest compared to the HD group and L-OHP group. Our findings suggest that HD may reduce the resistance of AGS/L-OHP cells to LOHP by regulating the PI3K/Akt signaling pathway.

Creatine kinase mitochondrial 2 promotes the growth and progression of colorectal cancer via enhancing Warburg effect through lactate dehydrogenase B.

Mitochondrial creatine kinase (MtCK) plays a pivotal role in cellular energy metabolism, exhibiting enhanced expression in various tumors, including colorectal cancer (CRC). Creatine kinase mitochondrial 2 (CKMT2) is a subtype of MtCK; however, its clinical significance, biological functions, and underlying molecular mechanisms in CRC remain elusive. We employed immunohistochemical staining to discern the expression of CKMT2 in CRC and adjacent nontumor tissues of patients. The correlation between CKMT2 levels and clinical pathological factors was assessed. Additionally, we evaluated the association between CKMT2 and the prognosis of CRC patients using Kaplan-Meier survival curves and Cox regression analysis. Meanwhile, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of CKMT2 in different CRC cell lines. Finally, we explored the biological functions and potential molecular mechanisms of CKMT2 in CRC cells through various techniques, including qRT-PCR, cell culture, cell transfection, western blot, Transwell chamber assays, flow cytometry, and co-immunoprecipitation. We found that CKMT2 was significantly overexpressed in CRC tissues compared with adjacent nontumor tissues. The expression of CKMT2 is correlated with pathological types, tumor size, distant metastasis, and survival in CRC patients. Importantly, CKMT2 emerged as an independent prognostic factor through Cox regression analysis. Experimental downregulation of CKMT2 expression in CRC cell lines inhibited the migration and promoted apoptosis of these cells. Furthermore, we identified a novel role for CKMT2 in promoting aerobic glycolysis in CRC cells through interaction with lactate dehydrogenase B (LDHB). In this study, we found the elevated expression of CKMT2 in CRC, and it was a robust prognostic indicator in CRC patients. CKMT2 regulates glucose metabolism via amplifying the Warburg effect through interaction with LDHB, which promotes the growth and progression of CRC. These insights unveil a novel regulatory mechanism by which CKMT2 influences CRC and provide promising targets for future CRC therapeutic interventions.

PTEN-Long inhibits the biological behaviors of glioma cells.

PTEN-Long is a translational variant of phosphatase and tensin homolog (PTEN). This study aimed to assess the effect of PTEN-Long on the biological characteristics of glioma cells and related mechanisms. A vector stably expressing PTEN-Long was established and transfected into cells, serving as the overexpression group, while a set of empty vectors served as the negative control group. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to detect the expression of PTEN-Long and phosphatidylinositol 3-kinase, Protein kinase B, andnuclear factor-κB (PI3K-AKT-NF-κB). Cell proliferation was assessed with the Cell Counting Kit 8 (CCK8) assay, migration through the scratch test, and invasion by the transwell chamber assay. Cell cycle analysis was performed using flow cytometry. The volume and weight of subcutaneous tumors in nude mice were also evaluated. PTEN-Long expression led to downregulation of p-Akt, NF-κB p65, p-NF-κB p65, and Bcl-xl, and up-regulation of IκBα. In addition, it inhibited glioma cell proliferation, induced cell cycle arrest in the G0/G1 phase, and reduced cell migration and invasion. Moreover, PTEN-Long inhibited the growth of subcutaneous glioma in nude mice. PTEN-Long inhibits the proliferation, migration, and invasion and induces apoptosis in glioma cells by inhibiting PI3K-AKT-NF-κb signaling, implying that PTEN-Long may be a new target for glioma treatment.

Up-regulated SLC25A39 promotes cell growth and metastasis via regulating ROS production in colorectal cancer.

Background: The mitochondrial transporter SLC25A39 has been implicated in the import of mitochondrial glutathione (mGSH) from the cytoplasm, crucial for mitigating oxidative stress and preserving mitochondrial function. Despite the well-established involvement of mitochondria in cancer, the functional impact of SLC25A39 on CRC progression remains elusive. Methods: The mRNA and protein expressions were detected by PCR, immunohistochemistry, and Western blot, respectively. Cell activity, cell proliferation, colony formation, and apoptosis were measured by CCK8 assay, EdU incorporation assay, plated colony formation assay, and flow cytometry, respectively. Cell migration was detected by wound healing and transwell chamber assay. The tumor microenvironment (TME), immune checkpoint molecules, and drug sensitivity of CRC patients were investigated using R language, GraphPad Prism 8 and online databases. Results: Here, we report a significant upregulation of SLC25A39 expression in CRC. Functional assays revealed that overexpression of SLC25A39 promoted CRC cell proliferation and migration while inhibiting apoptosis. Conversely, SLC25A39 knockdown suppressed cell growth and migration while enhancing apoptosis in vitro. Additionally, reduced SLC25A39 expression attenuated tumor growth in xenograft models. Mechanistically, elevated SLC25A39 levels correlated with reduced reactive oxygen species (ROS) accumulation in CRC. Furthermore, bioinformatic analyses unveiled the high SLC25A39 levels was associated with decreased expression of immune checkpoints and reduced responsiveness to immunotherapy. Single-cell transcriptomic profiling identified diverse cellular expression patterns of SLC25A39 and related immune regulators. Lastly, drug sensitivity analysis indicated potential therapeutic avenues targeting SLC25A39 in CRC. Conclusion Our findings underscore the pivotal role of SLC25A39 in CRC progression and suggest its candidacy as a therapeutic target in CRC management.