Zhenlong Yu

Melatonin enhances the anti-tumor effect of fisetin by inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways

Melatonin is a hormone identified in plants and pineal glands of mammals and possesses diverse physiological functions. Fisetin is a bio-flavonoid widely found in plants and exerts antitumor activity in several types of human cancers. However, the combinational effect of melatonin and fisetin on antitumor activity, especially in melanoma treatment, remains unclear. Here, we tested the hypothesis that melatonin could enhance the antitumor activity of fisetin in melanoma cells and identified the underlying molecular mechanisms. The combinational treatment of melanoma cells with fisetin and melatonin significantly enhanced the inhibitions of cell viability, cell migration and clone formation, and the induction of apoptosis when compared with the treatment of fisetin alone. Moreover, such enhancement of antitumor effect by melatonin was found to be mediated through the modulation of the multiply signaling pathways in melanoma cells. The combinational treatment of fisetin with melatonin increased the cleavage of PARP proteins, triggered more release of cytochrome-c from the mitochondrial inter-membrane, enhanced the inhibition of COX-2 and iNOS expression, repressed the nuclear localization of p300 and NF-κB proteins, and abrogated the binding of NF-κB on COX-2 promoter. Thus, these results demonstrated that melatonin potentiated the anti-tumor effect of fisetin in melanoma cells by activating cytochrome-c-dependent apoptotic pathway and inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways, and our study suggests the potential of such a combinational treatment of natural products in melanoma therapy.

Melatonin inhibits AP-2β/hTERT, NF-κB/COX-2 and Akt/ERK and activates caspase/Cyto C signaling to enhance the antitumor activity of berberine in lung cancer cells

Melatonin, a molecule produced throughout the animal and plant kingdoms, and berberine, a plant derived agent, both exhibit antitumor and multiple biological and pharmacological effects, but they have never been combined altogether for the inhibition of human lung cancers. In this study, we investigated the role and underlying mechanisms of melatonin in the regulation of antitumor activity of berberine in lung cancer cells. Treatment with melatonin effectively increased the berberine-mediated inhibitions of cell proliferation, colony formation and cell migration, thereby enhancing the sensitivities of lung cancer cells to berberine. Melatonin also markedly increased apoptosis induced by berberine. Further mechanism study showed that melatonin promoted the cleavage of caspse-9 and PARP, enhanced the inhibition of Bcl2, and triggered the releasing of cytochrome C (Cyto C), thereby increasing the berberine-induced apoptosis. Melatonin also enhanced the berberine-mediated inhibition of telomerase reverses transcriptase (hTERT) by down-regulating the expression of AP-2β and its binding on hTERT promoter. Moreover, melatonin enhanced the berberine-mediated inhibition of cyclooxygenase 2 (COX-2) by inhibiting the nuclear translocation of NF-κB and its binding on COX-2 promoter. Melatonin also increased the berberine-mediated inhibition of the phosphorylated Akt and ERK. Collectively, our results demonstrated that melatonin enhanced the antitumor activity of berberine by activating caspase/Cyto C and inhibiting AP-2β/hTERT, NF-κB/COX-2 and Akt/ERK signaling pathways. Our findings provide new insights in exploring the potential therapeutic strategies and novel targets for lung cancer treatment.

Gamabufotalin, a bufadienolide compound from toad venom, suppresses COX-2 expression through targeting IKKβ/NF-κB signaling pathway in lung cancer cells

BackgroundGamabufotalin (CS-6), a major bufadienolide of Chansu, has been used for cancer therapy due to its desirable metabolic stability and less adverse effect. However, the underlying mechanism of CS-6 involved in anti-tumor activity remains poorly understood.MethodsThe biological functions of gamabufotalin (CS-6) were investigated by migration, colony formation and apoptosis assays in NSCLC cells. The nuclear localization and interaction between transcriptional co-activator p300 and NF-κB p50/p65 and their binding to COX-2 promoter were analyzed after treatment with CS-6. Molecular docking study was used to simulate the interaction of CS-6 with IKKβ. The in vivo anti-tumor efficacy of CS-6 was also analyzed in xenografts nude mice. Western blot was used to detect the protein expression level.ResultsGamabufotalin (CS-6) strongly suppressed COX-2 expression by inhibiting the phosphorylation of IKKβ via targeting the ATP-binding site, thereby abrogating NF-κB binding and p300 recruitment to COX-2 promoter. In addition, CS-6 induced apoptosis by activating the cytochrome c and caspase-dependent apoptotic pathway. Moreover, CS-6 markedly down-regulated the protein levels of COX-2 and phosphorylated p65 NF-κB in tumor tissues of the xenograft mice, and inhibited tumor weight and size.ConclusionsOur study provides pharmacological evidence that CS-6 exhibits potential use in the treatment of COX-2-mediated diseases such as lung cancer.

Human transporters, PEPT1/2, facilitate melatonin transportation into mitochondria of cancer cells: an implication of the therapeutic potential

Melatonin is present in virtually all organisms from bacteria to mammals, and it exhibits a broad spectrum of biological functions, including synchronization of circadian rhythms and oncostatic activity. Several functions of melatonin are mediated by its membrane receptors, but others are receptor‐independent. For the latter, melatonin is required to penetrate membrane and enters intracellular compartments. However, the mechanism by which melatonin enters cells remains debatable. In this study, it was identified that melatonin and its sulfation metabolites were the substrates of oligopeptide transporter (PEPT) 1/2 and organic anion transporter (OAT) 3, respectively. The docking analysis showed that the binding of melatonin to PEPT1/2 was attributed to their low binding energy and suitable binding conformation in which melatonin was embedded in the active site of PEPT1/2 and fitted well with the cavity in three‐dimensional space. PEPT1/2 transporters play a pivotal role in melatonin uptake in cells. Melatonins membrane transportation via PEPT1/2 renders its oncostatic effect in malignant cells. For the first time, PEPT1/2 were identified to localize in the mitochondrial membrane of human cancer cell lines of PC3 and U118. PEPT1/2 facilitated the transportation of melatonin into mitochondria. Melatonin accumulation in mitochondria induced apoptosis of PC3 and U118 cells. Thus, PEPT1/2 can potentially be used as a cancer cell‐targeted melatonin delivery system to improve the therapeutic effects of melatonin in cancer treatment.

Targeting PDK1 with dichloroacetophenone to inhibit acute myeloid leukemia (AML) cell growth.

Pyruvate dehydrogenase kinase-1 (PDK1), a key metabolic enzyme involved in aerobic glycolysis, is highly expressed in many solid tumors. Small molecule compound DAP (2,2-dichloroacetophenone) is a potent inhibitor of PDK1. Whether targeting PDK1 with DAP can inhibit acute myeloid leukemia (AML) and how it works remains unknown. In this study, we evaluated the effect of inhibition of PDK1 with DAP on cell growth, apoptosis and survival in AML cells and identified the underlying mechanisms. We found that treatment with DAP significantly inhibited cell proliferation, increased apoptosis induction and suppressed autophagy in AML cells in vitro, and inhibited tumor growth in an AML mouse model in vivo. We also showed that inhibition of PDK1 with DAP increased the cleavage of pro-apoptotic proteins (PARP and Caspase 3) and decreased the expression of the anti-apoptotic proteins (BCL-xL and BCL-2) and autophagy regulators (ULK1, Beclin-1 and Atg). In addition, we found that DAP inhibited the PI3K/Akt signaling pathway. Furthermore, we demonstrated that PDK1 interacted with ULK1, BCL-xL and E3 ligase CBL-b in AML cells, and DPA treatment could inhibit the interactions. Collectively, our results indicated that targeting PDK1 with DAP inhibited AML cell growth via multiple signaling pathways and suggest that targeting PDK1 may be a promising therapeutic strategy for AMLs.

Characterization of Phase I Metabolism of Resibufogenin and Evaluation of the Metabolic Effects on Its Antitumor Activity and Toxicity

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxin-like compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5β-hydroxylresibufogenin, 5-HRB) by liquid chromatography–mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5β-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5β-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5β-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.

Gamabufotalin, a major derivative of bufadienolide, inhibits VEGF-induced angiogenesis by suppressing VEGFR-2 signaling pathway

Gamabufotalin (CS-6), a main active compound isolated from Chinese medicine Chansu, has been shown to strongly inhibit cancer cell growth and inflammatory response. However, its effects on angiogenesis have not been known yet. Here, we sought to determine the biological effects of CS-6 on signaling mechanisms during angiogenesis. Our present results fully demonstrate that CS-6 could significantly inhibit VEGF triggered HUVECs proliferation, migration, invasion and tubulogenesis in vitro and blocked vascularization in Matrigel plugs impregnated in C57/BL6 mice as well as reduced vessel density in human lung tumor xenograft implanted in nude mice. Computer simulations revealed that CS-6 interacted with the ATP-binding sites of VEGFR-2 using molecular docking. Furthermore, western blot analysis indicated that CS-6 inhibited VEGF-induced phosphorylation of VEGFR-2 kinase and suppressed the activity of VEGFR-2-mediated signaling cascades. Therefore, our studies demonstrated that CS-6 inhibited angiogenesis by inhibiting the activation of VEGFR-2 signaling pathways and CS-6 could be a potential candidate in angiogenesis-related disease therapy.

Alismanoid A, an unprecedented 1,2-seco bisabolene from Alisma orientale, and its protective activity against H2O2-induced damage in SH-SY5Y cells

A pair of novel sesquiterpenoids, (8R)-alismanoid A (1a) and (8S)-alismanoid A (1b), possessing an unprecedented 1,2-seco bisabolene carbon skeleton, were isolated from rhizomes of Alisma orientale together with two new sesquiterpenoids: a 15-nor guaiane alismanoid B (2) and alismanoid C (3). Their structures were elucidated by 1D and 2D NMR, HRESIMS, electronic circular dichroism (ECD), and theoretical calculations, and a plausible biosynthetic pathway for compound 1 is discussed. Meanwhile, compounds 1–3 were investigated for their protective effects on H2O2-induced damage in human dopaminergic neuroblastoma cells (SH-SY5Y), and compounds 1b, 2, and 3 showed significantly protective activities at a certain concentration. Further, the action mechanism of compound 3 was proved to be through inhibition of H2O2-induced apoptosis in SH-SY5Y cells. Herein, these results suggest that sesquiterpenoids are potential candidate drugs for treating Parkinsons disease induced by reactive oxygen species.

Activatable Near-Infrared Fluorescent Probe for Dipeptidyl Peptidase IV and Its Bioimaging Applications in Living Cells and Animals

Visualization of endogenous disease-associated enzymes is of great clinical significance, as it could allow earlier clinical diagnosis and timely intervention. Herein, we first synthesized and characterized an enzyme-activatable near-infrared fluorescent probe, GP-DM, for determining the activity of dipeptidyl peptidase IV (DPP IV), which is associated with various pathological processes, especially in diabetes and malignant tumors. GP-DM emitted significant turn-on NIR fluorescent signals simultaneously in response to DPP IV, making it favorable for accurately and dynamically monitoring DPP IV activity in vitro and in vivo. GP-DM exhibited excellent specificity and sensitivity in DPP IV imaging, as indicated by its higher catalytic activity than other human serine hydrolases and by its strong anti-interference ability to a complex biological matrix, which was fully characterized in a series of phenotyping reactions and inhibition assays. Encouraged by the advantages mentioned above, we successfully used GP-DM to evaluate endogenous DPP IV activity in various biological samples (plasma and tissue preparations) and living tumor cells and performed real-time in vivo bioimaging of DPP IV in zebrafish and tumor-bearing nude mice. All of the results reflected and highlighted the potential application value of GP-DM in the early detection of pathologies, individual tailoring of drug therapy, and image-guided tumor resection. Furthermore, our results revealed that DPP IV, a key target enzyme, is closely associated with the migration and proliferation of cancer cells and regulating the biological activity of DPP IV may be a useful approach for cancer therapy.

YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma

Y-box binding protein 1 (YBX1) is involved in the multi-tumor occurrence and development. However, the regulation of YBX1 in lung tumorigenesis and the underlying mechanisms, especially its relationship with CDC25a, was remains unclear. In this study, we analyzed the expression and clinical significance of YBX1 and CDC25a in lung adenocarcinoma and identified their roles in the regulation of lung cancer growth. The retrospective analysis of 116 patients with lung adenocarcinoma indicated that YBX1 was positively correlated with CDC25a expression. The Cox-regression analysis showed only high-ranking TNM stage and low CDC25a expression were an independent risk factor of prognosis in enrolled patients. High expression of YBX1 or CDC25a protein was also observed in lung adenocarcinoma cells compared with HLF cells. ChIP assay demonstrated the binding of endogenous YBX1 to the CDC25a promoter region. Overexpression of exogenous YBX1 up-regulated the expression of the CDC25a promoter-driven luciferase. By contrast, inhibition of YBX1 by siRNA markedly decreased the capability of YBX1 binding to CDC25a promoter in A549 and H322 cells. Inhibition of YBX1 expression also blocked cell cycle progression, suppressed cell proliferation and induced apoptosis via the CDC25a pathway in vitro. Moreover, inhibition of YBX1 by siRNA suppressed tumorigenesis in a xenograft mouse model and down-regulated the expression of YBX1, CDC25a, Ki67 and cleaved caspase 3 in the tumor tissues of mice. Collectively, these results demonstrate inhibition of YBX1 suppressed lung cancer growth partly via the CDC25a pathway and high expression of YBX1/CDC25a predicts poor prognosis in human lung adenocarcinoma.