Novel Oleanane-Type Triterpene Glycosides from the Saponaria officinalis L. Seeds and Apoptosis-Inducing Activity via Mitochondria

Saponaria officinalis L., commonly known as “Soapwort”, is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1–17), including seven new glycosides (1–7). The structures of 1–7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G₂/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells.     

Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.     

The Antidiabetic Drug Metformin Inhibits the Proliferation of Bladder Cancer Cells in Vitro and in Vivo

Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4), E2F1 and an increase of p21^waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK) and suppressed mammalian target of rapamycin (mTOR), the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.     

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.     

Ziyuglycoside II Inhibits the Growth of Human Breast Carcinoma MDA-MB-435 Cells via Cell Cycle Arrest and Induction of Apoptosis through the Mitochondria Dependent Pathway

Ziyuglycoside II is one of the major active compounds of Sanguisorba officinalis L., which has a wide range of clinical applications including hemostasis, antibiosis, anti-inflammation and anti-oxidation. This study investigated the effect of ziyuglycoside II on the growth of human breast carcinoma MDA-MB-435 cells for the first time. The results showed that ziyuglycoside II could significantly inhibit the growth of MDA-MB-435 cells through blocking cell cycle progression at G0/G1 and S phase as well as via inducing cell apoptosis. Accumulation of reactive oxygen species (ROS) was observed in the progression of cell cycle arrest, which was associated with the increased expression of cell cycle regulating factors, p53 and p21. Subsequent apoptosis induced by ziyuglycoside II was accompanied with the activation of mitochondrial pathway, in particular a decreased mitochondrial membrane potential (MMP) as well as increased Bax/Bcl-2 ratio, cytochrome c release and the activity of caspase-3 and caspase-9. In conclusion, our study was the first to report that ziyuglycoside II has inhibitory effect on the growth of MDA-MB-435 cells, which might become a potential therapeutic approach of breast cancer in the future.     

Low-Molecular-Weight Fucoidan as Complementary Therapy of Fluoropyrimidine-Based Chemotherapy in Colorectal Cancer

This study investigated the roles of low-molecular-weight fucoidan (LMWF) in enhancing the anti-cancer effects of fluoropyrimidine-based chemotherapy. HCT116 and Caco-2 cells were treated with LMWF and 5-FU. Cell viability, cell cycle, apoptosis, and migration were analyzed in both cell types. Potential mechanisms underlying how LMWF enhances the anti-cancer effects of fluoropyrimidine-based chemotherapy were also explored. The cell viability of HCT116 and Caco-2 cells was significantly reduced after treatment with a LMWF-–5FU combination. In HCT116 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through the (1) induction of cell cycle arrest in the S phase and (2) late apoptosis mediated by the Jun-N-terminal kinase (JNK) signaling pathway. In Caco-2 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through both the c-mesenchymal–epithelial transition (MET)/Kirsten rat sarcoma virus (KRAS)/extracellular signal-regulated kinase (ERK) and the c-MET/phosphatidyl-inositol 3-kinases (PI3K)/protein kinase B (AKT) signaling pathways. Moreover, LMWF enhanced the suppressive effects of 5-FU on tumor cell migration through the c-MET/matrix metalloproteinase (MMP)-2 signaling pathway in both HCT116 and Caco-2 cells. Our results demonstrated that LMWF is a potential complementary therapy for enhancing the efficacies of fluoropyrimidine-based chemotherapy in colorectal cancers (CRCs) with the wild-type or mutated KRAS gene through different mechanisms. However, in vivo studies and in clinical trials are required in order to validate the results of the present study.     

Epithelial Cell-Derived Extracellular Vesicles Trigger the Differentiation of Two Epithelial Cell Lines

Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.     

Benzyl Isothiocyanate Inhibits Prostate Cancer Development in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) Model,Which Is Associated with the Induction of Cell Cycle G1 Arrest

Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has been shown to have anti-tumor properties. In the present study, we investigated whether BITC inhibits the development of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) mice. Five-week old, male TRAMP mice and their nontransgenic littermates were gavage-fed with 0, 5, or 10 mg/kg of BITC every day for 19 weeks. The weight of the genitourinary tract increased markedly in TRAMP mice and this increase was suppressed significantly by BITC feeding. H and E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the TRAMP mice. The number of lobes with WDC was reduced by BITC feeding while that of lobes with prostatic intraepithelial neoplasia was increased. BITC feeding reduced the number of cells expressing Ki67 (a proliferation marker), cyclin A, cyclin D1, and cyclin-dependent kinase (CDK)2 in the prostatic tissue. In vitro cell culture results revealed that BITC decreased DNA synthesis, as well as CDK2 and CDK4 activity in TRAMP-C2 mouse prostate cancer cells. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in TRAMP mice treated with BITC.     

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.     

Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis

Melanoma is known to aggressively metastasize and is one of the prominent causes of skin cancer mortality. This study was designed to assess the molecular mechanism of decursinol angelate (DA) against murine melanoma cell line (B16F10 cells). Treatment of DA resulted in growth inhibition and cell cycle arrest at G0/G1 (p < 0.001) phase, evaluated through immunoblotting. Moreover, autophagy-related proteins such as ATG-5 (p < 0.0001), ATG-7 (p < 0.0001), beclin-1 (p < 0.0001) and transition of LC3-I to LC3-II (p < 0.0001) were markedly decreased, indicating autophagosome inhibition. Additionally, DA treatment triggered apoptotic events which were corroborated by the occurrence of distorted nuclei, elevated reactive oxygen species (ROS) levels and reduction in the mitochondrial membrane potential. Subsequently, there was an increase in the expression of pro-apoptotic protein Bax in a dose-dependent manner, with the corresponding downregulation of Bcl-2 expression and cytochrome C expression following 24 h DA treatment in A375.SM and B16F10 cells. We substantiated our results for apoptotic occurrence through flow cytometry in B16F10 cells. Furthermore, we treated B16F10 cells with N-acetyl-L-cysteine (NAC). NAC treatment upregulated ATG-5 (p < 0.0001), beclin-1 (p < 0.0001) and LC3-I to LC3-II (p < 0.0001) conversion, which was inhibited in the DA treatment group. We also noticed a systematic upregulation of important markers for progression of G1 cell phase such as CDK-2 (p < 0.029), CDK-4 (p < 0.036), cyclin D1 (p < 0.0003) and cyclin E (p < 0.020) upon NAC treatment. In addition, we also observed a significant fold reduction (p < 0.05) in ROS fluorescent intensity and the expression of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase-9 (p > 0.010) and cleaved caspase-3 (p < 0.0001). NAC treatment was able to ameliorate DA-induced apoptosis and cell cycle arrest to support our finding. Our in vivo xenograft model also revealed similar findings, such as downregulation of CDK-2 (p < 0.0001) and CDK-4 (p < 0.0142) and upregulation of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase 3 (p < 0.0001) and cleaved caspase 9 (p < 0.0001). In summary, our study revealed that DA is an effective treatment against B16F10 melanoma cells and xenograft mice model.     

Fargesin Inhibits EGF-Induced Cell Transformation and Colon Cancer Cell Growth by Suppression of CDK2/Cyclin E Signaling Pathway

Although the lignan compound fargesin is a major ingredient in Shin-Yi, the roles of fargesin in carcinogenesis and cancer cell growth have not been elucidated. In this study, we observed that fargesin inhibited cell proliferation and transformation by suppression of epidermal growth factor (EGF)-stimulated G₁/S-phase cell cycle transition in premalignant JB6 Cl41 and HaCaT cells. Unexpectedly, we found that signaling pathway analyses showed different regulation patterns in which fargesin inhibited phosphatidylinositol 3-kinase/AKT signaling without an alteration of or increase in mitogen activated protein kinase (MAPK) in JB6 Cl41 and HaCaT cells, while both signaling pathways were abrogated by fargesin treatment in colon cancer cells. We further found that fargesin-induced colony growth inhibition of colon cancer cells was mediated by suppression of the cyclin dependent kinase 2 (CDK2)/cyclin E signaling axis by upregulation of p21^WAF1/Cip1, resulting in G₁-phase cell cycle accumulation in a dose-dependent manner. Simultaneously, the suppression of CDK2/cyclin E and induction of p21^WAF1/Cip1 were correlated with Rb phosphorylation and c-Myc suppression. Taken together, we conclude that fargesin-mediated c-Myc suppression inhibits EGF-induced cell transformation and colon cancer cell colony growth by the suppression of retinoblastoma (Rb)-E2F and CDK/cyclin signaling pathways, which are mainly regulated by MAPK and PKB signaling pathways.     

PTP61F Mediates Cell Competition and Mitigates Tumorigenesis

Tissue homeostasis via the elimination of aberrant cells is fundamental for organism survival. Cell competition is a key homeostatic mechanism, contributing to the recognition and elimination of aberrant cells, preventing their malignant progression and the development of tumors. Here, using Drosophila as a model organism, we have defined a role for protein tyrosine phosphatase 61F (PTP61F) (orthologue of mammalian PTP1B and TCPTP) in the initiation and progression of epithelial cancers. We demonstrate that a Ptp61F null mutation confers cells with a competitive advantage relative to neighbouring wild-type cells, while elevating PTP61F levels has the opposite effect. Furthermore, we show that knockdown of Ptp61F affects the survival of clones with impaired cell polarity, and that this occurs through regulation of the JAK–STAT signalling pathway. Importantly, PTP61F plays a robust non-cell-autonomous role in influencing the elimination of adjacent polarity-impaired mutant cells. Moreover, in a neoplastic RAS-driven polarity-impaired tumor model, we show that PTP61F levels determine the aggressiveness of tumors, with Ptp61F knockdown or overexpression, respectively, increasing or reducing tumor size. These effects correlate with the regulation of the RAS–MAPK and JAK–STAT signalling by PTP61F. Thus, PTP61F acts as a tumor suppressor that can function in an autonomous and non-cell-autonomous manner to ensure cellular fitness and attenuate tumorigenesis.     

Anti-Proliferative Activities and Apoptosis Induction by Triterpenes Derived from Eriobotrya japonica in Human Leukemia Cell Lines

Eriobotrya japonica leaf is a traditional herbal medicine that contains numerous triterpenes, which have various pharmacological properties. In this study, we investigated the anti-proliferative activity of four triterpenes derived from E. japonica, including corosolic acid (CA), ursolic acid (UA), maslinic acid (MA) and oleanolic acid (OA), in human leukemia cell lines. CA showed the strongest anti-proliferative activity in all of the leukemia cell lines tested, but not in normal human skin fibroblast cell lines. To determine the mechanism underlying the anti-proliferative effect of CA, we examined the effect of CA on molecular events known as apoptosis induction. CA induced chromatin condensation, DNA fragmentation, sub-G₁ phase DNA, activation of caspase-3, -8 and -9 and the cleavage of PARP in HL-60. CA also activated Bid and Bax, leading to the loss of mitochondrial membrane potential (Δψ_m) and cytochrome c release into the cytosol, whereas Bcl-2 and Bcl-xL were unaffected by CA. These results suggest that CA has an anti-proliferative effect on leukemia cells via the induction of apoptosis mediated by mitochondrial dysfunction and caspase activation. CA may be a potential chemotherapeutic agent for the treatment of human leukemia.     

Kub3 Deficiency Causes Aberrant Late Embryonic Lung Development in Mice by the FGF Signaling Pathway

As a Ku70-binding protein of the KUB family, Kub3 has previously been reported to play a role in DNA double-strand break repair in human glioblastoma cells in glioblastoma patients. However, the physiological roles of Kub3 in normal mammalian cells remain unknown. In the present study, we generated Kub3 gene knockout mice and revealed that knockout (KO) mice died as embryos after E18.5 or as newborns immediately after birth. Compared with the lungs of wild-type (WT) mice, Kub3 KO lungs displayed abnormal lung morphogenesis and pulmonary atelectasis at E18.5. No difference in cell proliferation or cell apoptosis was detected between KO lungs and WT lungs. However, the differentiation of alveolar epithelial cells and the maturation of type II epithelial cells were impaired in KO lungs at E18.5. Further characterization displayed that Kub3 deficiency caused an abnormal FGF signaling pathway at E18.5. Taking all the data together, we revealed that Kub3 deletion leads to abnormal late lung development in mice, resulting from the aberrant differentiation of alveolar epithelial cells and the immaturation of type II epithelial cells due to the disturbed FGF signaling pathway. Therefore, this study has uncovered an essential role of Kub3 in the prenatal lung development of mice which advances our knowledge of regulatory factors in embryonic lung development and provides new concepts for exploring the mechanisms of disease related to perinatal lung development.