Effects of MicroRNA-195-5p on Biological Behaviors and Radiosensitivity of Lung Adenocarcinoma Cells via Targeting HOXA10

Objective. To explore the effects of miR-195-5p and its target gene HOXA10 on the biological behaviors and radiosensitivity of lung adenocarcinoma (LUAD) cells. Methods. The effects of miR-195-5p on LUAD cell proliferation, migration, invasion, cycle arrest, apoptosis, and radiosensitivity were investigated by in vitro experiments. The bioinformatics analysis was used to assess its clinical value and predict target genes. Double-luciferase experiments were used to verify whether the miR-195-5p directly targeted HOXA10. A xenograft tumor-bearing mouse model was used to examine its effects on the radiosensitivity of LUAD in vivo. Results. Both gain- and loss-of-function assays demonstrated that miR-195-5p inhibited LUAD cell proliferation, invasion, and migration, induced G1 phase arrest and apoptosis, and enhanced radiosensitivity. Double-luciferase experiments confirmed that miR-195-5p directly targeted HOXA10. Downregulation of HOXA10 also inhibited LUAD cell proliferation, migration, and invasion, induced G1 phase arrest and apoptosis, and enhanced radiosensitivity. The protein levels of β-catenin, c-myc, and Wnt1 were decreased by miR-195-5p and increased by its inhibitor. Moreover, the effects of the miR-195-5p inhibitor could be eliminated by HOXA10-siRNA. Furthermore, miR-195-5p improved radiosensitivity of LUAD cells in vivo. Conclusion. miR-195-5p has excellent antitumor effects via inhibiting cancer cell growth, invasion, and migration, arresting the cell cycle, promoting apoptosis, and sensitizing LUAD cells to X-ray irradiation by targeting HOXA10. Thus, miR-195-5p may serve as a potential candidate for the treatment of LUAD.

Imperatorin induces autophagy and G0/G1 phase arrest via PTEN-PI3K-AKT-mTOR/p21 signaling pathway in human osteosarcoma cells in vitro and in vivo

Background Osteosarcoma is the third most common cancer in adolescence and the first common primary malignant tumor of bone. The long-term prognosis of osteosarcoma still remains unsatisfactory in the past decades. Therefore, development of novel therapeutic agents which are effective to osteosarcoma and are safe to normal tissue simultaneously is quite essential and urgent. Methods Firstly, MTT assay, cell colony formation assay, cell migration and invasion assays were conducted to evaluate the inhibitory effects of imperatorin towards human osteosarcoma cells. RNA-sequence assay and bioinformatic analysis were then performed to filtrate and assume the potential imperatorin-induced cell death route and signaling pathway. Moreover, quantitative real-time PCR assay, western blot assay and rescue experiments were conducted to confirm the assumptions of bioinformatic analysis. Finally, a subcutaneous tumor-transplanted nude mouse model was established and applied to evaluate the internal effect of imperatorin on osteosarcoma by HE and immunohistochemistry staining. Results Imperatorin triggered time-dependent and dose-dependent inhibition of tumor growth mainly by inducing autophagy promotion and G0/G1 phase arrest in vitro and in vivo. Besides, imperatorin treatment elevated the expression level of PTEN and p21, down-regulated the phosphorylation of AKT and mTOR. In contrast, the inhibition of PTEN using Bpv (HOpic), a potential and selective inhibitor of PTEN, concurrently rescued imperatorin-induced autophagy promotion, cell cycle arrest and inactivation of PTEN-PI3K-AKT-mTOR/p21 pathway. Conclusions This work firstly revealed that imperatorin induced autophagy and cell cycle arrest through PTEN-PI3K-AKT-mTOR/p21 signaling pathway by targeting and up-regulating PTEN in human osteosarcoma cells. Hence, imperatorin is a desirable candidate for clinical treatments of osteosarcoma.

Peiminine Induces G0/G1-Phase Arrest, Apoptosis, and Autophagy via the ROS/JNK Signaling Pathway in Human Osteosarcoma Cells in Vitro and in Vivo

Aims: Peiminine has been reported to have various pharmacological properties, including anticancer activity. In this study, we investigated the effect of this alkaloid on osteosarcoma and explored the underlying mechanisms.Methods: To evaluate the antiosteosarcoma effects of peiminine in vitro, cell viability was assessed by CCK-8 and live/dead assays; the effects of the drug on apoptosis and the cell cycle were examined by flow cytometry; the effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively, while its effects on autophagy were observed by transmission electron microscopy and an LC3 fluorescent puncta formation assay. The role of autophagy in the peiminine-mediated effects in osteosarcoma cells was evaluated by CCK-8 assay and western blotting after the application of the autophagy inhibitor chloroquine. The effect of peiminine on reactive oxygen species (ROS) production was analyzed using fluorescence confocal microscopy and spectrophotometry. Additionally, peiminine-treated osteosarcoma cells were exposed to SP600125, a JNK inhibitor, and N-acetylcysteine, a ROS scavenger, after which the contribution of the ROS/JNK signaling pathway to osteosarcoma was assessed using cell viability and LC3 fluorescent puncta formation assays, flow cytometry, and western blotting. A xenograft mouse model of osteosarcoma was generated to determine the antitumor effects of peiminine in vivo.Results: Peiminine suppressed proliferation and metastasis and induced cell cycle arrest, apoptosis, and autophagy in osteosarcoma cells. These anticancer effects of peiminine were found to be dependent on intracellular ROS generation and activation of the JNK pathway. In line with these results, peiminine significantly inhibited xenograft tumor growth in vivo.Conclusions: Peiminine induced G0/G1-phase arrest, apoptosis, and autophagy in human osteosarcoma cells via the ROS/JNK signaling pathway both in vitro and in vivo. Our study may provide an experimental basis for the evaluation of peiminine as an alternative drug for the treatment of osteosarcoma.

Nucleolar and spindle associated protein 1 enhances chemoresistance through DNA damage repair pathway in chronic lymphocytic leukemia by binding with RAD51

Nucleolar and spindle-associated protein 1 (NUSAP1) is an essential regulator of mitotic progression, spindle assembly, and chromosome attachment. Although NUSAP1 acts as an oncogene involved in the progression of several cancers, the exact role of chronic lymphocytic leukemia (CLL) remains elusive. Herein, we first discovered obvious overexpression of NUSAP1 in CLL associated with poor prognosis. Next, the NUSAP1 level was modulated by transfecting CLL cells with lentivirus. Silencing NUSAP1 inhibited the cell proliferation, promoted cell apoptosis and G0/G1 phase arrest. Mechanistically, high expression of NUSAP1 strengthened DNA damage repairing with RAD51 engagement. Our results also indicated that NUSAP1 knockdown suppressed the growth CLL cells in vivo. We further confirmed that NUSAP1 reduction enhanced the sensitivity of CLL cells to fludarabine or ibrutinib. Overall, our research investigates the mechanism by which NUSAP1 enhances chemoresistance via DNA damage repair (DDR) signaling by stabilizing RAD51 in CLL cells. Hence, NUSAP1 may be expected to be a perspective target for the treatment of CLL with chemotherapy resistance.

Cell Cycle Regulation of Hippocampal Progenitor Cells in Experimental Models of Depression and after Treatment with Fluoxetine

Changes in adult hippocampal cell proliferation and genesis have been largely implicated in depression and antidepressant action, though surprisingly, the underlying cell cycle mechanisms are largely undisclosed. Using both an in vivo unpredictable chronic mild stress (uCMS) rat model of depression and in vitro rat hippocampal-derived neurosphere culture approaches, we aimed to unravel the cell cycle mechanisms regulating hippocampal cell proliferation and genesis in depression and after antidepressant treatment. We show that the hippocampal dentate gyrus (hDG) of uCMS animals have less proliferating cells and a decreased proportion of cells in the G2/M phase, suggesting a G1 phase arrest; this is accompanied by decreased levels of cyclin D1, E, and A expression. Chronic fluoxetine treatment reversed the G1 phase arrest and promoted an up-regulation of cyclin E. In vitro, dexamethasone (DEX) decreased cell proliferation, whereas the administration of serotonin (5-HT) reversed it. DEX also induced a G1-phase arrest and decreased cyclin D1 and D2 expression levels while increasing p27. Additionally, 5-HT treatment could partly reverse the G1-phase arrest and restored cyclin D1 expression. We suggest that the anti-proliferative actions of chronic stress in the hDG result from a glucocorticoid-mediated G1-phase arrest in the progenitor cells that is partly mediated by decreased cyclin D1 expression which may be overcome by antidepressant treatment.

CCNE1 and E2F1 Partially Suppress G1 Phase Arrest Caused by Spliceostatin A Treatment

The potent splicing inhibitor spliceostatin A (SSA) inhibits cell cycle progression at the G1 and G2/M phases. We previously reported that upregulation of the p27 cyclin-dependent kinase inhibitor encoded by CDKN1B and its C-terminal truncated form, namely p27*, which is translated from CDKN1B pre-mRNA, is one of the causes of G1 phase arrest caused by SSA treatment. However, the detailed molecular mechanism underlying G1 phase arrest caused by SSA treatment remains to be elucidated. In this study, we found that SSA treatment caused the downregulation of cell cycle regulators, including CCNE1, CCNE2, and E2F1, at both the mRNA and protein levels. We also found that transcription elongation of the genes was deficient in SSA-treated cells. The overexpression of CCNE1 and E2F1 in combination with CDKN1B knockout partially suppressed G1 phase arrest caused by SSA treatment. These results suggest that the downregulation of CCNE1 and E2F1 contribute to the G1 phase arrest induced by SSA treatment, although they do not exclude the involvement of other factors in SSA-induced G1 phase arrest.

Novel chemotherapeutic agent FX-9 activates NF-κB signaling and induces G1 phase arrest by activating CDKN1A in a human prostate cancer cell line

Background The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. Methods In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. Results Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. Conclusions FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling.

Deciphering the Molecular Machinery—Influence of sE-Cadherin on Tumorigenic Traits of Prostate Cancer Cells

The serum level of soluble (s)E-cadherin is elevated in several malignancies, including prostate cancer (PCa). This study was designed to investigate the effects of sE-cadherin on the behavior of PCa cells in vitro, with the aim of identifying a potential therapeutic target. Growth as well as adhesive and motile behavior were evaluated in PC3, DU-145, and LNCaP cells. Flow cytometry was used to assess cell cycle phases and the surface expression of CD44 variants as well as α and β integrins. Confocal microscopy was utilized to visualize the distribution of CD44 variants within the cells. Western blot was applied to investigate expression of α3 and β1 integrins as well as cytoskeletal and adhesion proteins. Cell growth was significantly inhibited after exposure to 5 µg/mL sE-cadherin and was accompanied by a G0/G1-phase arrest. Adhesion of cells to collagen and fibronectin was mitigated, while motility was augmented. CD44v4, v5, and v7 expression was elevated while α3 and β1 integrins were attenuated. Blocking integrin α3 reduced cell growth and adhesion to collagen but increased motility. sE-cadherin therefore appears to foster invasive tumor cell behavior, and targeting it might serve as a novel and innovative concept to treat advanced PCa.

A Dual PI3K/HDAC Inhibitor Downregulates Oncogenic Pathways in Hematologic Tumors In Vitro and In Vivo

Purpose: To investigate the efficacy and mechanism of compound 23, a PI3K/HDAC dual-target inhibitor, on hematologic tumor cells in vitro and in vivo. Methods: The MTS Kit was used to study the antiproliferative effects in vitro. Western blot was used to analyze the involved signaling pathways. Flow cytometry was used to analyze apoptosis and the cell cycle. The antiproliferative effects were evaluated in vivo using EL4 and A20 xenograft models. The CCLE database was used to analyze gene expression. Results: Compound 23 significantly inhibited the proliferation of hematologic tumors; it simultaneously regulated PI3K/HDAC pathways and induced apoptosis and G1-phase arrest in EL4, NB4, and A20 cells in vitro. When tested in vivo, compound 23 significantly inhibited the proliferation of EL4 and A20. The expression levels of ErbB2 and ErbB3 decreased in hematologic tumors compared with it in solid tumors. Conclusion: Compound 23 modulates the PI3K/HDAC pathway, which results in significant inhibition of hematologic tumor proliferation in vivo and in vitro. The differential levels of ERBB2 and ERBB3 might be related to the difference in the effect of compound 23 on hematologic tumors and solid tumors.