Extraction,isolation and in vitro evaluation of affinisine from Tabernaemontana catharinensis in human melanoma cells

Plant compounds have been identified as new drug prototypes. In this line, this work aimed to isolate the indole alkaloid affinisine from Tabernaemontana catharinensis and test its antitumor activity. The alkaloid was isolated by silica gel open column chromatography from the ethanolic extract of the stem of T. catharinensis. Afterwards, this molecule was characterized by high-resolution mass spectrometry and nuclear magnetic resonance. In the next step, the cytotoxicity of the compound was tested against human melanoma cell lines (A375, WM1366 and SK-MEL-28) and a normal skin cell line (CCD-1059Sk) using a MTT (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells treated with affinisine were evaluated by flow cytometry to analyze apoptosis and the induction of cell cycle arrest, to evaluate the dead mechanism. The metabolite was isolated in a 0.2% yield relative to the extract. Cytotoxic activity of the molecule was observed at 48 h, resulting in considerable growth inhibition rates in melanoma cells, especially in WM1366, which had the lowest IC₅₀ (32.86 ± 2.54 µg/mL). The apoptosis rate was lower in A375 (56.66 and 86.71% with 57 and 65 µg/mL, respectively). Moreover, affinisine was able to significantly induce cell cycle arrest in different phases in the A375 and WM1366 cell lines. However, in SK-MEL-28 cells, cycle arrest was not observed. In summary, this compound significantly decreased the viability of tumor cells in a dose- and time-dependent manner for all evaluated lineages, reduced cell viability by the apoptosis mechanism and presented prominent activities of cell cycle arrest. In this way, the use of antineoplastic agents is among the most widely used therapeutic measures for the control and treatment of cancer. Affinisine is a promising prototype in the search for new drugs to treat cancer.     

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer

BackgroundAberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties.PurposeIn this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells.MethodsIn vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells.ResultsConvallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model.ConclusionThe result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.     

Procaspase 8 and Bax Are Up-regulated by Distinct Pathways in Streptococcal Pyrogenic Exotoxin B-induced Apoptosis

We have previously identified integrin α_vβ₃ and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B that binds to Fas only. In the current study we found that after binding to α_vβ₃, SPE B stimulated the tyrosine phosphorylation of JAK2 and STAT1. STAT1 tyrosine phosphorylation was inhibited by a JAK2 inhibitor, AG490, short interfering RNA (siRNA) silencing of JAK2, and anti-α_Vβ₃ antibody. AG490 also decreased the binding of tyrosine-phosphorylated STAT1 to the procaspase 8 promoter, decreasing procaspase 8 expression, suggesting that SPE B up-regulates procaspase 8 expression via the JAK2/STAT1 pathway. Alternatively, both SPE B and G308S increased STAT1 phosphorylation at serine 727, which was inhibited by anti-Fas antibody, a p38 inhibitor, SB203580, and siRNA silencing of p38. In addition, SPE B and G308S increased binding of serine-phosphorylated STAT1 to the Bax promoter and Bax expression, which was decreased by SB203580. SPE B and G308S-stimulated Bax expression was also inhibited by anti-Fas antibody. These findings suggest that Fas mediate SPE B-induced Bax expression through p38. Silencing of JAK2 or p38 by siRNA blocked procaspase 8 expression, whereas only p38 siRNA decreased Bax expression. Furthermore, JAK2 inhibition and p38 inhibition reduced SPE B-induced apoptosis, but only p38 inhibition blocked G308S-induced apoptosis.     

Virtual screening and synthesis of quinazolines as novel JAK2 inhibitors

JAK2 is an important target in multiple processes associated with tumor growth. In this study, virtual screening was employed for hit compound identification with chemical libraries using SurflexDock. Subsequently, hit optimization for potent and selective candidate JAK2 inhibitors was performed through synthesis of diverse C-1 substituted quinazoline derivatives. A novel compound 5p, (6,7-dimethoxyquinazolin-4-yl)naphthalen-1-ylamine, was thus obtained. JAK2 inhibitory activity of 5p was 43% at 20 ??M and this was comparable to AG490, a representative JAK2 inhibitor. Moreover, 5p showed a positive correlation between JAK2 inhibition and cytotoxicity; 5p treatment in HT-29 cells strongly inhibited JAK2 activation and subsequent STAT3 phosphorylation, reduced anti-apoptotic protein levels, and finally induced apoptosis. This suggests that compound 5p is a candidate inhibitor of JAK2 and its downstream STAT3 signaling pathway for antitumor therapy. In the docking model, the quinazoline template of 5k, the lead compound, occupied a hydrophobic region such as Leu856, Leu855, Ala880, Leu932 and Gly935, and the highly conserved hydrogen bond was created by 6-OMe of the ring template, which binds to the NH of Arg980. Moreover, hydrophobic interactions were identified between morpholine moiety and the hydrophobic region formed by Leu855, Ala880, Tyr931, Val911 and Met929. Also, compound 5k more strongly inhibited JAK2 phosphorylation in mouse embryonic stem cells than AG490. Our study shows the successful application of virtual screening for lead discovery and we propose that the novel compound 5p can be an effective JAK2 inhibitor candidate for further antitumor agent research.     

Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling

Background

The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML) cell line U937.

Methodology/Principal Findings

Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-x_L and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2), the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP) SH2 domain-containing phosphatase (SHP)-1, and the addition of sodium pervanadate (a PTP inhibitor) prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells.

Conclusions/Significance

Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.     

Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2

The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. We have characterized a novel small molecule JAK2 inhibitor, AZ960, and used it as a tool to investigate the consequences of JAK2 V617F inhibition in the SET-2 cell line. AZ960 inhibits JAK2 kinase with a K(i) of 0.00045 microm in vitro and treatment of TEL-JAK2 driven Ba/F3 cells with AZ960 blocked STAT5 phosphorylation and potently inhibited cell proliferation (GI(50)=0.025 microm). AZ960 demonstrated selectivity for TEL-JAK2-driven STAT5 phosphorylation and cell proliferation when compared with cell lines driven by similar fusions of the other JAK kinase family members. In the SET-2 human megakaryoblastic cell line, heterozygous for the JAK2 V617F allele, inhibition of JAK2 resulted in decreased STAT3/5 phosphorylation and inhibition of cell proliferation (GI(50)=0.033 microm) predominately through the induction of mitochondrial-mediated apoptosis. We provide evidence that JAK2 inhibition induces apoptosis by direct and indirect regulation of the anti-apoptotic protein BCL-xL. Inhibition of JAK2 blocked BCL-XL mRNA expression resulting in a reduction of BCL-xL protein levels. Additionally, inhibition of JAK2 resulted in decreased PIM1 and PIM2 mRNA expression. Decreased PIM1 mRNA corresponded with a decrease in Pim1 protein levels and inhibition of BAD phosphorylation at Ser(112). Finally, small interfering RNA-mediated suppression of BCL-xL resulted in apoptotic cell death similar to the phenotype observed following JAK2 inhibition. These results suggest a model in which JAK2 promotes cell survival by signaling through the Pim/BAD/BCL-xL pathway.     

Lidocaine inhibits melanoma cell proliferation by regulating ERK phosphorylation

The melanoma is responsible for the majority of all skin cancer–related deaths worldwide. Evidence suggests that local anesthetics provide some benefit in the treatment of cancer via inhibition of cellular proliferation, invasion and migration. However, the potential antiproliferative effects of local anesthetics in the treatment of melanoma remain to be elucidated. In this study, we investigated the antiproliferative effects and underlying mechanism of the commonly used local anesthetic (lidocaine) on melanoma cells. A375 melanoma cells were treated by lidocaine or vemurafenib. Cell Counting Kit-8, histological staining, flow cytometric analysis, immunohistochemical staining, and Western blot analyses were carried out to test the effects of lidocaine and vemurafenib on A375 cells. BALB/C-nu/nu mice intraperitoneally injected with A375 cells were treated by lidocaine, and then tumor volume and weight were calculated. Lidocaine exhibited vemurafenib-like effects totally. Lidocaine inhibited A375 melanoma cell proliferation in a dose- and time-dependent manner and colony formation also showed a dose-dependent inhibition. Lidocaine treatment resulted in the arrest of cell-cycle progression in the G1 phase and inhibited Ki-67 expression in a dose-dependent manner. This effect was associated with inhibited extracellular signal–regulated kinase (ERK) phosphorylation. In vivo experiments revealed that intravenous injections of lidocaine suppressed tumor volume and weight. Lidocaine inhibits melanoma cell proliferation in a dose- and time-dependent manner via a mechanism that may involve inhibition of the ERK signaling pathway. Thus, lidocaine may provide some benefit for the treatment of melanoma.     

A trisubstituted pyrazole derivative reduces DMBA-induced mammary tumor growth in rats by inhibiting estrogen receptor-α expression

P16 is the product of cyclin-dependent kinase 2 (CDKN2A) gene and plays multi-pronged roles in the cancer progression. Breast cancer (BC) is the most commonly diagnosed cancer type among females. In the current study, the potential function of P16 in the growth and metastasis of BC was investigated. Firstly, the expression statuses of P16 in different cancer types were investigated using Oncomine database and validated with corresponding cancer cell lines. Afterwards, the expression of P16 was knocked down in BC cell line BT-549 and the effect on the cell proliferation, sensitivity to paclitaxel (TAX), apoptosis, migration, and invasion abilities was assessed using CCK-8, Edu, flow cytometry, scratch, and transwell assays, respectively. The influence of P16 inhibition and P16 overexpression on the activity of IL-6/JAK/STAT3 signaling was explored. Additionally, the effect of P16 inhibition on the tumor growth was verified with a BC xenograft mice model. The abnormal expression of P16 was detected in BC cell line BT-549 as well as colorectal cancer and osteosarcoma cell lines. The inhibition of P16 suppressed the cell proliferation, invasion, and migration abilities while induced the apoptosis and sensitivity to TAX in BT-549 cells. At molecular level, P16 knockdown inhibited the expression of IL6ST and Survivin, and the phosphorylation of JAK2 and STAT3. However, the induced expression of P16 in P16-knockdown BT-549 cells restored the activity of IL-6/JAK2/STAT3 pathway. The results of in vitro assays were confirmed with BC xenograft models: the inhibition of P16 decreased the tumor growth rate. Findings outlined in the current study demonstrated that the inhibition of P16 decreased the growth and metastasis potential of BC cells by inhibiting IL-6/JAK2/STAT3 signaling.     

Sodium orthovanadate suppresses palmitate-induced cardiomyocyte apoptosis by regulation of the JAK2/STAT3 signaling pathway

Elevated circulatory free fatty acids (FFAs) especially saturated FFAs, such as palmitate (PA), are detrimental to the heart. However, mechanisms responsible for this phenomenon remain unknown. Here, the role of JAK2/STAT3 in PA-induced cytotoxicity was investigated in cardiomyocytes. We demonstrate that PA suppressed the JAK2/STAT3 pathway by dephosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705), and thus blocked the translocation of STAT3 into the nucleus. Conversely, phosphorylation of S727, another phosphorylated site of STAT3, was increased in response to PA treatment. Pretreatment of JNK inhibitor, but not p38 MAPK inhibitor, inhibited STAT3 (S727) activation induced by PA and rescued the phosphorylation of STAT3 (Y705). The data suggested that JNK may be another upstream factor regulating STAT3, and verified the important function of P-STAT3 (Y705) in PA-induced cardiomyocyte apoptosis. Sodium orthovanadate (SOV), a protein tyrosine phosphatase inhibitor, obviously inhibited PA-induced apoptosis by restoring JAK2/STAT3 pathways. This effect was diminished by STAT3 inhibitor Stattic. Collectively, our data suggested a novel mechanism that the inhibition of JAK2/STAT3 activation was responsible for palmitic lipotoxicity and SOV may act as a potential therapeutic agent by targeting JAK2/STAT3 in lipotoxic cardiomyopathy treatment.     

15-Deoxy-Delta(12,14)-prostaglandin J(2) suppresses IL-6-induced STAT3 phosphorylation via electrophilic reactivity in endothelial cells

In this study, the effects of 15d-PGJ(2) were investigated in IL-6-activated endothelial cells (ECs). 15d-PGJ(2) was found to abrogate phosphorylation on tyr705 of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner, but did not inhibit serine phosphorylation of STAT3 and the upperstream JAK2 phosphorylation. Other PPAR activators, such as WY1643 or ciglitazone, had no effect upon IL-6-induced STAT3 phosphorylation. Additionally, neither orthovanadate nor l-NAME treatment reverses the inhibition of STAT3 phosphorylation by 15d-PGJ(2). Otherwise, the effect of 15d-PGJ(2) requires the alpha,beta-unsaturated carbonyl group in the cyclopentane ring. A 15d-PGJ(2) analog, 9,10-Dihydro-15d-PGJ(2), which lack alpha,beta-unsaturated carbonyl group showed no increase in ROS production and no effect in inhibition of IL-6-induced STAT3 phosphorylation. The electrophilic compound, acrolein, mimics the inhibition effect of 15d-PGJ(2). Among the antioxidants, only NAC and glutathione reversed the effects of 15d-PGJ(2). NAC, glutathione and DTT all reversed the inhibition of STAT3 phosphorylation when preincubated with 15d-PGJ(2). The inhibition of ICAM-1 gene expression by 15d-PGJ(2) was abrogated by NAC and glutathione in IL-6-treated ECs. Taken together, these results suggest that 15d-PGJ(2) inhibits IL-6-stimulated phosphorylation on tyr705 of STAT3 dependent on its own electrophilic reactivity in ECs.     

Inhibition of JAK2/STAT3 signalling induces colorectal cancer cell apoptosis via mitochondrial pathway

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.     

Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling.     

Rapid inhibition of leptin signaling by glucocorticoids in vitro and in vivo

Elevated secretion of glucocorticoids (GCs) or hypersensitivity to GCs has a permissive effect on the development of obesity and leads to abnormalities of body fat distribution. Recent studies demonstrated GCs act as antagonists of leptin in rodents. However, little is known about the interaction between GCs and leptin signaling. In the present study, we investigated the effects of GCs on leptin action in vitro and in vivo. GCs rapidly inhibited the leptin-induced STAT3 phosphorylation in a dose- and time-dependent manner, as assayed by Western blotting using anti-phosphospecific-STAT3 in human hepatoma cell lines (Huh7) transiently expressing long form leptin receptor. GCs also inhibited the leptin-induced JAK2 tyrosine phosphorylation but unaltered the specific binding of (125)I-leptin to the cells. Parallel experiments, however, demonstrated that the inhibitory effects of GCs were not observed in either IL-6- or LIF-induced STAT3 phosphorylation. Furthermore, we examined the feeding behavior and hypothalamic leptin signaling following intracerebroventricular (icv) infusion of GCs prior to icv leptin infusion in Sprague-Dawley rats. The food intake after 24 h of icv leptin injection increased 3-fold in GCs-treated animals. In addition, central infusion of GCs resulted in a marked reduction of hypothalamic STAT3 phosphorylation in response to icv infusion of leptin. To clarify the molecular mechanism by which GCs rapidly reduce leptin-induced JAK/STAT signaling, we examined the intracellular signal transduction pathway potentially mediated by GCs. PD98059, a specific MEK inhibitor, blocked the inhibitory effects of GCs on leptin-induced JAK/STAT activation in Huh7 cells. These results suggest GCs antagonize leptin action by a rapid inhibition of the leptin-induced JAK/STAT pathway partly via MAPK cascade.