Sesquiterpene from Polygonum Barbatum Disrupt Mitochondrial Membrane Potential to Induce Apoptosis and Inhibits Metastasis by Downregulating Matrix Metalloproteinase and Osteopontin in NCI-H460 Cells

Background: Globally, lung cancer accounts for 18% of cancer-associated mortalities. Among the subtypes, non-small-cell lung cancer (NSCLC) is the most prevalent one. The increased resistance and poor survival rates, signifies disease aggressiveness and thus require a search for an alternative anticancer molecule. Hypothesis/Purpose: Earlier, the isolated sesquiterpene i.e. compound 1 ((E)-Methyl 6-acetoxy-7-methoxy-1-(2-methylpropylidene)-1H-indene-3-carboxylate) from P. barbatum was isolated, characterized by us and reported for preliminary anticancer activity. Considering its potent activity, this study was designed to explore the underlying molecular mechanism of apoptosis and metastasis against NCI-H460 cells. Method: The molecular mechanism of compound 1 inducing apoptosis and inhibiting metastasis was elucidated by analyzing mitochondrial membrane potential, DNA fragmentation, clonogenic assay, invasion assay and expression of apoptotic (Caspases 3, 6, 8, 9 and BAK) and metastatic markers (MMP 2, 9 and osteopontin). Results: Compound 1 significantly inhibited cell proliferation and induced apoptosis via intrinsic route i.e. the mitochondrial pathway by disrupting mitochondrial membrane potential. The enhanced expression of caspases 6, 9, BAK and HRK with downregulation of Bcl-2L1 and Ki67 further confirmed the involvement of the intrinsic apoptotic pathway. Moreover, compound 1 restricted the invasive nature of NCI-H460 cells evinced by reduced cell invasion in Boyden chamber invasion assay and downregulating the expression of metastatic markers i.e. matrix metalloproteinase 2 / 9 and VEGF. It was also found that it blocks osteopontin by negatively regulating its expression; a marker protein in cancer management. Conclusion: Conclusively, this sesquiterpene exhibited potent anticancer and anti-metastatic activity and can be explored further as possible pharmacophores

The m6A Methyltransferase METTL3 Promotes Cisplatin Resistance and Invasion in Testicular Seminoma via BCL2

Background: Methyltransferase-like 3 (METTL3) involves in promoting tumor progression through tumor-related genes N6-methyladenosine (m6A) modification. Our previous study found that METTL3 plays an important role in seminoma chemosensitivity. BCL-2 is a key cause of cisplatin resistance in many tumors. Therefore, we want to explore whether METTL3 affects cisplatin resistance of seminoma by regulating BCL-2.Methods: In this study, we downregulated and overexpressed METTL3 in TCam-2 cisplatin resistant cells (TCam-2/CDDP). Then, m6A RNA methylation quantification of BCL-2 and cell viability assay, cell apoptosis analysis and cell invasion assay were investigated under the condition of with or without cis‐dichlorodiammine platinum (CDDP) treatment. Results: Consistent with our previous results, METTL3 significantly affects the chemosensitivity of TCam-2/CDDP. After METTL3 downregulated, the proliferation and anti-apoptosis ability of TCam-2/CDDP cells significantly weakened. Correspondingly, overexpression of METTL3 could promote the chemoresistance. However, the phenotype could be partly reversed by decreasing the expression of BCL-2. Moreover, we found that the m6A modification of BCL-2 is more abundant in cisplatin-resistant strains. Knockdown and overexpression of METTL3 significantly affected the m6A modification and the protein level of BCL-2 in TCam-2/CDDP. Finally, we found that METTL3 also promoted the invasion ability of TCam-2/CDDP cells via BCL2.Conclusion: This study revealed that METTL3 promotes anti-apoptosis and invasion of TCam-2/CDDP through BCL-2. And it indicated that METTL3 and BCL-2 may be an effective treatment target for CDDP-resistance seminoma.

Quantifying the invasion and migration ability of cancer cells with a 3D matrigel drop invasion assay

Metastasis is the main cause of cancer associated morbidity which will account for approximately 600,000 deaths in the USA in 2021. Defining new mechanisms that drive cancer metastasis is vital for developing new therapeutic strategies and improving clinical outcomes for cancer patients. Herein, we describe a recently established three-dimensional Matrigel drop invasion assay to measure cancer cell invasion and migration capability in vitro. This assay is a versatile and simple tool to test the ability of cells to invade and migrate, test the functional role of genes of interest in cell invasion and migration, analyze the localization of the target proteins at the cell invasion edge in situ, and screen drug effects on cancer cell invasion and migration.

Effect of lncRNA PVT1/miR186/KLF5 Axis on the Occurrence and Progression of Cholangiocarcinoma

This study primarily focused on the effect of the long noncoding RNA (lncRNA) PVT1/miR186/KLF5 axis on the occurrence and progression of cholangiocarcinoma (CCA). miR186 was found both in the lncRNA PVT1 targeting miRNAs and KLF5 targeting miRNAs using bioinformatic analysis. The expression of lncRNA PVT1 and KLF5 in the TFK-1, QBC939, and HuCCT1 cell lines and normal biliary epithelial HIBEpiC cells was detected by RT-qPCR. The significance of lncRNA PVT1 and KLF5 on cell proliferation was analyzed using the MTT assay and clone formation assay in lncRNA PVT1 and KLF5 silencing HuCCT1 cell lines and lncRNA PVT1and KLF5 overexpressing TFK-1 and QBC939 cell lines, respectively. The potential role of lncRNA PVT1 and KLF5 in cell migration was detected using the transwell invasion assay in CCA cell lines and tumor formation assay. Additionally, lncRNA PVT1 and KLF5 were proved to be highly expressed in CCA tissues and cell lines. Silencing and overexpressing of lncRNA PVT1 or KLF5 markedly inhibited or increased the cell proliferation and cell invasion in CCA cell lines, respectively. Silencing and overexpressing of lncRNA PVT1 significantly inhibited and increased the expression of KLF5 in CCA cell lines, respectively. Silencing of lncRNA PVT1 increased the expression of miR186, and silencing of miR186 increased the expression of KLF5 in CCA cell lines. Cotransfection of lncRNA PVT1 and miR186 increased the expression of KLF5 compared with controls. Overall, these results demonstrated that the lncRNA PVT1/miR186/KLF5 axis might exert a key role in the occurrence and progression of CCA, and this axis might provide a new target for treating CCA.

Anti-PITPNM3 small molecular compounds reverse breast cancer metastasis by targeting PITPNM3.

e15005 Background: Recent studies highlight the fundamental roles of PITPNM3 in breast cancer metastasis. PITPNM3 is identified as the functional receptor of CCL18 and promotes breast cancer cell invasion and metastasis by binding with CCL18. Since anti-CCL18 neutralized antibodies shows medium binding affinity which restricts their clinical application, small molecular inhibitors targeting PITPNM3 are needed to be further investigated. Therefore, we identified several first in class small molecular inhibitors potentially targeting PITPNM3 and can inhibit breast cancer metastasis conducted by PITPNM3 activation. Methods: We performed computer-assisted drug design by constructing PITPNM3 homology model, characterizing potential binding pockets and docking preselected high diversity structured small molecule compounds into the static PITPNM3 model. Top 100 small molecules in silico scores were selected and screened through basic experiments. After screening, the anti-metastasis effects of selected compounds were tested through transwell migration and invasion assay. Immunofluorescence and qPCR were applied to confirm the expression of vimentin and CDH1. Western blot were used to clarify the inhibition effects of selected compounds on PITPNM3 signaling pathways. Results: By using homology remodeling, we successfully constructed the PITPNM3(680-920aa) protein model. The PITPNM3(680-920aa) domain is responsible for interacting with PYK2 and phosphorylating PYK2. The phosphorylation of PYK2 conducted by PITPNM3 signaling pathway will lead to metastasis and epithelial-mesenchymal transition (EMT) of breast cancer cells. We then characterized the potential binding pockets of this static model and a druggable site was founded. More than 50K molecules with high diversity were docked into this druggable site and scored through their docking performance. Finally, top 100 scored small molecules were selected. In addition, through 1 rounds of toxicity screening, 1 round of transwell migration assay screening and 1 round of transwell invasion assay screening, 4 small molecules with higher bioactivity is identified and 1 compound with the highest bioactivity as well as docking performance among 50K small molecules is chose. This compound can inhibit CCL18 treatment as well as tumor associated macrophage co-culture mediated migration and invasion. Besides, it can also inhibit the phophorylation of PYK2 and Src without inhibition the expression of PITPNM3. Conclusions: Our findings identify the first-in-class anti-PITPNM3 small molecule inhibitors. These compounds can inhibit PITPNM3 signaling pathway and reverse breast cancer metastasis.

ATF3 Suppresses Growth and Metastasis of Clear Cell Renal Cell Carcinoma by Deactivating EGFR/AKT/GSK3β/β-Catenin Signaling Pathway

BackgroundClear cell renal cell carcinoma (ccRCC) is one of the most common malignant cancers in East Asia, with high incidence and mortality. Accumulating evidence has shown that ATF3 is associated with tumor progression.MethodsUsing qPCR, the expression of ATF3 was detected in 93 patients with ccRCC, including 24 paired normal and tumor tissues, which were used to further compare ATF3 expression through western blotting and immunohistochemistry. Lentivirus was used for the overexpression or knockdown of ATF3, and the consequent alteration in function was analyzed through CCK8 assay, colony formation assay, wound healing assay, invasion assay, and flow cytometry. The potential mechanism affected by ATF3 was analyzed through gene set enrichment analysis (GSEA) and verified using western blotting, invasion assay, or immunofluorescence staining. Furthermore, a xenograft mouse model was used to assess the function of ATF3 in vivo.ResultsATF3 expression was significantly decreased in ccRCC compared to that in adjacent normal tissues. Through gain- and loss-of-function experiments performed in an in vitro assay, we found that ATF3 could regulate ccRCC cell proliferation, cycle progression, migration, and invasion. In the in vivo study, the xenograft mouse model revealed that ATF3 overexpression can inhibit the growth of ccRCC. Moreover, the mechanism analysis showed that suppression of ATF3 could lead to an increase the expression of β-catenin and promote β-catenin transfer to the nucleus, and might be affected by EGFR/AKT/GSK3β signaling.ConclusionATF3 could be utilized as an independent protective factor to inhibit the progression of ccRCC. Potential treatment strategies for ccRCC include targeting the ATF3/EGFR/AKT/GSK3β/β−catenin signaling pathway.

MicroRNA-16 inhibits the migration and invasion of glioma cell by targeting Bcl-2 gene

Purpose: To investigate the effect of microRNA-16 (miR-16) on glioma cell migration and invasiveness, and the mechanism involved.Methods: MicroRNA-16 mimic or inhibitor was transfected into human glioma (SHG44) cells. Cell migration, invasiveness and morphology were determined using scratch test, Transwell invasion assay, and immunohistochemical staining, respectively. Expressions of bcl-2, MMP-9 and MMP-2, and NF-κB1 proteins were measured using Western blotting.Results: Overexpression of MicroRNA-16 significantly down-regulated MMP-9 protein in SHG44 cells (p < 0.05), but MMP-2 protein expressions in the 2 groups were comparable (p > 0.05). Protein expressions of MMP-9 and NF-κB1 were significantly down-regulated in human glioma positive cells, relative to negative control.Conclusion: MiR-16 overexpression suppresses the migration and invasiveness of SHG44 cells via the regulation of NF-κB1/MMP-9 signaling pathway, and it directly targets bcl-2 gene by inhibiting its protein expression. This finding affords a new target for developing new anti-glioma drugs. Keywords: Bcl-2, Expression, Glioma, MicroRNA-16, NF-κB1signaling pathway

Shikonin Inhibits Cholangiocarcinoma Cell Line QBC939 by Regulating Apoptosis, Proliferation, and Invasion

This study was designed to clarify whether Shikonin causes proliferation, apoptosis, and invasion in cholangiocarcinoma cells and to investigate the mechanism of action. QBC939 cells were cultured with different doses of Shikonin, and then 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium assay was used to detect cell viability. Apoptosis of cells was detected using flow cytometry with Annexin V/propidium iodide (PI) assay after being stained with Hoechst 33242. The role of Shikonin on the invasive and metastasis ability was detected using Transwell invasion assay. Real-time polymerase chain reaction and Western blotting were used to detect the expression of caspase-3, caspase-8, epidermal growth factor receptor (EGFR), and matrix metalloproteinase (MMP)-9. Shikonin inhibited proliferation and invasive ability of QBC939 cells in a dose-dependent manner; at the same time, apoptosis of cells was also observed in a concentration-dependent fashion. Moreover, Annexin V/PI assay and Transwell invasion assay results indicated that Shikonin induced apoptosis and invasion inhibitory probably due to upregulation of caspase-3 and caspase-8 expression and downregulation of MMP-9 and EGFR expression in a concentration-dependent fashion. Shikonin could enhance apoptosis and inhibit proliferation and invasion of QBC939 cells; such biological behaviors mainly occurred via upregulating the expression of caspase-3 and caspase-8 and downregulating the expression of MMP-9 and EGFR.

The inhibitory effect of curcumin via fascin suppression through JAK/STAT3 pathway on metastasis and recurrence of ovary cancer cells

Background Fascin is an actin-binding protein and highly expressed in ovarian cancer cells. It is associated with metastasis of cancer and may be a useful prognostic factor. Anticancer activity of curcumin is related to its effect on several signaling mechanisms. Although there have been many reports regarding the anticancer properties of curcumin, its inhibitory effects on migration and invasion of ovarian cancer cells, particularly in the context of fascin expression, have not been reported. The purpose of this study was to investigate the effect of curcumin on fascin expression in ovarian cancer cells and to propose a possible mechanism for the anticancer activity of curcumin through reduced fascin expression. Methods SKOV3, human epithelial ovary cancer cell line, was cultured with curcumin at various dose and duration. The fascin was quantified using cell viability test and Western blot. To determine the effect of curcumin on the upstream pathway of fascin expression, the signal transducer and activator of transcription 3 (STAT3) was analyzed by sandwich-ELISA. Attachment assay, migration assay and invasion assay were analyzed to approve the change of cellular invasiveness of ovary cancer after curcumin. To determine the morphological changes of ovarian cancer cells by curcumin, immunofluorescence was performed. Results MTS assays showed that cell viability was different at various concentration of curcumin, and as concentration increased, cell viability tended to decrease. Curcumin appears to suppress fascin expression, even with a minimal concentration and short exposure time. Also, curcumin may suppress fascin expression in ovarian cancer cells through STAT3 downregulation. The attachment assay, migration assay and invasion assay of the ovarian cancer cells exhibited a statistically significant decrease. Immunofluorescence revealed a change of cell shape from a typical form of uninfluenced cells to a more polygonal appearance, with a significant reduction in filopodia formation. Conclusions Curcumin reduces fascin expression through JAK/STAT3 pathway inhibition, which interferes with the cellular interactions essential for the metastasis and recurrence of ovarian cancer cells. Higher curcumin concentrations and longer exposure times concomitantly decreased fascin expression.