Scutellarin inhibits proliferation and invasion of hepatocellular carcinoma cells via down‐regulation of JAK2/STAT3 pathway

The prognosis of hepatocellular carcinoma (HCC) is poor because of high incidence of recurrence and metastasis. JAK/STAT signalling pathway regulates cell proliferation, apoptosis, differentiation and migration and epithelial‐mesenchymal transition (EMT) is also considered to contribute to invasion and metastasis of epithelial malignant tumours. Scutellarin is an active component found in many traditional Chinese herbs and has been regularly used in anti‐inflammatory and antitumour medicine. This study aimed to identify the effect of scutellarin and its possible mechanism of action in HCC cells. Proliferation, colony‐forming, apoptosis and cell migration assays were used to examine the effect of scutellarin on HCC cells. Quantitative real‐time PCR and Western blotting were performed to study the molecular mechanisms of action of scutellarin. Light and electron microscopy and immunofluorescence analysis were performed to study the effect of scutellarin on cellular mechanics. We show that scutellarin potentially suppresses invasiveness of HepG2 and MHCC97‐H cells in vitro by remodelling their cytoskeleton. The molecular mechanism behind it might be the inhibition of the EMT process, which could be attributed to the down‐regulation of the JAK2/STAT3 pathway. These findings may provide new clinical ideas for the treatment of liver cancer.     

Tilianin inhibits the human ovarian cancer (PA-1) cell proliferation via blocking cell cycle,inducing apoptosis and inhibiting JAK2/STAT3 signaling pathway

Ovarian cancer is one of the deadliest gynecologic malignancies and is the seventh leading cause of mortalities and morbidities globally. Although there are various therapeutic strategies, a major challenge for scientific community is to come up with effective strategy to treat ovarian cancer. Tilianin, a polyphenol flavonoid is well known for its extensive biological actions like cardioprotective, neuroprotective, anti-oxidant, anti-inflammatory, anti-diabetic and anti-tumor properties. The current study is designed to investigate the anti-cancer action of Tilianin in ovarian cancer (PA-1) cells. The findings of this study revealed that Tilianin treatment results in significant and concentration dependent decrease in cell viability. The growth inhibiting action of Tilianin is associated with apoptosis which was confirmed by DAPI and AO/EtBr staining. The Tilianin-triggered apoptosis in PA-1 cells was correlated with elevated generation of ROS, loss of mitochondrial membrane potential, alterations in pro-apoptotic (upregulated mRNA expression of Bax) and anti-apoptotic (downregulated mRNA expression of Bcl2) factors and activation of caspase-8, −9 and −3. Cell cycle analysis revealed that Tilianin treatment prevented G1/S transition through reduced mRNA expression of cyclin D1. Additionally, the findings of this study also showed Tilianin inhibited JAK2/STAT3 signaling (downregulated expression of pJAK2, JAK2, pSTAT3, and STAT3) with no change in mRNA expression level of ERK indicating its non-involvement in the apoptotic and/or growth inhibition of ovarian cancer cells. In conclusion, the findings of this exploration provided clear evidence of anti-cancer effects of Tilianin in PA-1 cells through its anti-proliferative action, ability to induce apoptosis both through extrinsic and intrinsic pathways, cell cycle (G1/S) arrest and JAK2/STAT3 signaling inhibition.     

Increased proliferation of a human breast carcinoma cell line by recombinant interleukin-2

Two adenocarcinoma cell lines, Breast M25-SF and Breast M, were established from tumor tissue resected surgically from a patient with breast cancer. One, Breast M25-SF, expresses interleukin-2 receptor (IL-2R) on the cell surface and the other, Breast M, does not. The effects of recombinant inteleukin-2 (rIL-2) on the proliferation of these cell lines were investigated. The growth of Breast M25-SF was significantly promoted by rIL-2 ranging from 1,25 U/ml to 640 U/ml. Anti-CD25 (Tac) antibody, significantly blocked the growth enhancement of Breast M25-SF by rIL-2. Breast M, however, did not respond to rIL-2. To confirm more directly the promotion of Breast M25-SF growth by rIL-2, cloning of IL-2 responders from parent Breast M25-SF cells was carried out by limiting dilution without feeder cells in 96-well microplates. No colony formation was found in 24 wells without rIL-2. Eleven, 13 and 6 clones were established from groups of 24 wells containing rIL-2 at 200, 20 and 2 U/ml respectively. All of the clones expressed IL-2R and respond to rIL-2. By using a sensitive polymerase chain reaction technique, we demonstrated that Breast M25-SF but not Breast M expressed IL-2 mRNA, and IL-2 secretion from Breast M25-SF but not Breast M was also confirmed by radioimmunoassay. These findings suggest a role for IL-2 in autocrine support of Breast M25-SF growth. IL-2 may play an important role in the growth control of breast carcinoma cells.     

Somatostatin increases mitogen-induced IL-2 secretion and proliferation of human jurkat T cells via sst3 receptor isotype

The neuropeptide somatostatin (SRIF) modulates normal and leukemia T cell proliferation. However, neither molecular isotypes of receptors nor mechanisms involved in these somatostatin actions have been elucidated as yet. Here we show by using RT-PCR approach that mitogen-activated leukemia T cells (Jurkat) express mRNA for a single somatostatin receptor, sst3. This mRNA is apparently translated into protein since specific somatostatin binding sites (K_I1 = 78 ± 3 pM) were detected in semipurified plasma membrane preparations by using ¹²⁵I-Tyr¹-SRIF14 as a radioligand. Moreover, somatostatin inhibits adenylyl cyclase activity with similar efficiency (IC₅₀ = 23 ± 4 pM) thus strongly suggesting a functional coupling of sst3 receptor to this transduction pathway. The involvement of sst3 receptor in immuno-modulatory actions of somatostatin was assessed by analysis of neuropeptide effects on IL-2 secretion and on proliferation of mitogen-activated Jurkat cells. Our data show that in the concentrations comprised between 10 pM and 10 nM, somatostatin potentiates IL-2 secretion. This effect is correlated with somatostatin-dependent increase of Jurkat cell proliferation since the EC₅₀ concentrations for both actions were almost identical (EC₅₀ = 22 ± 9 pM and EC₅₀ = 12 ± 1 pM for IL-2 secretion and proliferation, respectively). Altogether, these data strongly suggest that in mitogen-activated Jurkat cells, somatostatin increases cell proliferation through the increase of IL-2 secretion via a functional sst3 receptor negatively coupled to the adenylyl cyclase pathway. J. Cell. Biochem. 68:62–73, 1998. © 1998 Wiley-Liss, Inc.     

IL-12 p35 silenced dendritic cells modulate immune responses by blocking IL-12 signaling through JAK-STAT pathway in T lymphocytes

Dendritic cells (DC) constitute a complex system of uniquely specialized antigen-presenting cells (APC) that play crucial roles in the initiation and regulation of immune responses. Recent studies have demonstrated that DC silenced by siRNA IL-12 p35 showed tolerogenic capacity in vitro. However, their mechanism of action is not fully understood. In this study, IL-12p35 siRNA was chemically synthesized and transfected into DCs. A coculture of T cells and DCs was performed. After 30 min coculture, T cells were harvested and analyzed. We showed that the IL-12 p35 silenced DCs decreased IL-12-induced T cell responses through blocking tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4 proteins in T cells. These results demonstrate IL-12 p35 silenced DCs modulate immune responses by blocking IL-12 signaling through JAK-STAT pathway in T cells.     

Urokinase-type plasminogen activator receptor signaling is critical in nasopharyngeal carcinoma cell growth and metastasis

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in southern China and Southeast Asia, with the highest metastasis rate among head and neck cancers. The mechanisms underlying NPC progression remain poorly understood. Genome-wide expression profiling on 18 NPC vs. 18 noncancerous nasopharyngeal tissues together with GeneGo pathway analysis and expression verification in NPC cells and tissues revealed a potential role of urokinase-type plasminogen activator receptor (uPAR) in NPC progression, which has not been investigated in NPC. We then observed that uPAR expression is increased in poorly differentiated, highly metastatic NPC cells compared with lowly metastatic cells or differentiated NPC cells. In vitro studies demonstrated that uPAR regulates NPC cell growth, colony formation, migration, and invasion and promotes the epithelial–mesenchymal transition (EMT). Additional tumor xenograft and spontaneous metastasis experiments revealed that uPAR promotes NPC cell growth and metastasis in vivo. The JAK–STAT pathway is involved in uPAR-regulated signaling in NPC cells as determined by immunoblotting. Moreover, uPAR-mediated growth and motility is partially abolished upon treatment with the Jak1/Jak2 inhibitor INCB018424. We suppressed uPA expression in uPAR-overexpressing NPC cells and found that uPAR-mediated cellular growth and motility is not exclusively dependent on uPA. In summary, uPAR is a significant regulator of NPC progression and could serve as a promising therapeutic target.     

Synthetic derivatives of the natural product 13-amino 2-desoxy-4-epi-pulchellin inhibit STAT3 signaling and induce G2/M arrest and death of colon cancer cells

2-Desoxy-4-epi-puchellin (PCL) is a sesquiterpene-lactone, which naturally occurs in many traditional Chinese medicinal plants, and has antitumor and anti-inflammatory activities. In this work, a series of 13-amino derivatives of PCL were synthesized through Michael addition reaction. Inhibition of IL-6-induced STAT3 signaling pathway and in vitro cytotoxicity of these compounds were evaluated, and their effect on the cell cycle was also studied. The methyl hydroxyethylamine derivatives showed higher potency than PCL, which could induce significant mitotic arrest via G2/M arrest in HCT116 cancer cells, indicating that these derivatives have the potential to be developed into anti-colon cancer drugs.     

Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis within the individual cell. Recent reports have suggested that leptin, an adipocyte-secreted hormone, phosphorylates AMPK in skeletal muscle directly. However, little is known about the interaction between leptin signaling and AMPK activation. Here, we report that the leptin-induced phosphorylation of AMPK was detected in Huh7 cells expressing long form leptin receptor (OBRb) as well as short form leptin receptor (OBRa). In addition, we demonstrate that AMPK activation does not require the phosphorylation of either Tyr-985 or Tyr-1138 within the OBRb and may occur via a STAT3-independent signaling pathway. We also show that Huh7 cells expressing OBRb and SOCS3 (inhibitor of JAK2) resulted in a marked reduction of AMPK activation in response to leptin. These findings suggest that the activation of JAK2, but not STAT3, may play a critical role in leptin-induced AMPK activation in Huh7 cells.     

Ryanodine receptor signaling is required for anti-CD3-induced T cell proliferation, interleukin-2 synthesis, and interleukin-2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

A cell-free Salmonella typhimurium extract modulates interleukin-2 (IL-2) receptor expression but not IL-2-stimulated responses of murine splenic lymphocytes

Abstract In a previous study, we observed that a cell-free Salmonella typhimurium extract induced suppression of mitogen-induced T-cell proliferation and this suppression involved non-responsiveness of T-cells to interleukin-2 (IL-2). In this study, we found that a cell-free S. typhimurium extract modulated IL-2 receptor (IL-2R) expression on phytohemagglutinin (PHA)-stimulated murine spleen cells and this was a mechanism of T-cell non-responsiveness to IL-2, but did not affect IL-2 binding to IL-2R and the consequent responses. Western blotting using anti-phosphotyrosine antibodies showed that IL-2R-mediated tyrosine phosphorylation of protein substrates in PHA-activated murine splenic T-cells, which express a high-affinity IL-2R (α- and β-chains), was not affected by treatment with the S. typhimurium cell-free extract. Furthermore, PHA-activated spleen T-cells responded to recombinant IL-2 and this was not inhibited by the extract. Surprisingly, IL-2R expression was augmented by treatment with the extract, although this was independent of IL-2 production. These results suggest that the suppression of T-cell proliferation induced by the Salmonella cell-free extract was associated with augmentation of IL-2R expression, rather than down-regulation of the IL-2 response. This may be a mechanism responsible for the Salmonella extract-evoked suppression of mitogen-induced T-cell proliferation.     

Preferential proliferation of T cell receptor V gamma 3-positive cells in IL-2-stimulated fetal thymocytes

Thymocyte cell suspensions, prepared from mice at different ages, were cultured in vitro with human rIL-2. This stimulation resulted in a cell population that contained almost 50% TCR-gamma delta-positive cells if thymocytes were taken from fetal day 17 until just after birth. Analysis of the variable (V gamma) region used by the TCR-gamma delta cells revealed that 90% of them expressed TCR-V gamma 3, and less than 5% expressed TCR-V gamma 2. Cells positive for TCR-alpha beta were barely detectable. If fetal day 18 organ cultured thymus lobes, instead of a cell suspension, were stimulated with IL-2, no rise in the number of TCR-V gamma 3+ or TCR-delta+ cells was observed, whereas a partial outgrowth of TCR-alpha beta+ cells occurred. From day 1 after birth, the number of TCR-gamma delta cells recovered from an IL-2-stimulated thymocyte cell suspension dropped to reach a plateau of 15% of the total cell number, whereas TCR-V gamma 3+ cells became undetectable in older animals. TCR-alpha beta+ cells, on the other hand, quickly rose in cell number after birth. Kinetic analysis showed that the preferential outgrowth of TCR-V gamma 3+ cells in IL-2-stimulated fetal day 18 thymocyte cell suspensions was present from the onset of the culture; a significant proliferation of CD4 or CD8 single positive TCR-alpha beta cells was never observed. This lack of proliferation of TCR-alpha beta cells was not due to inhibition by the activated TCR-V gamma 3+ cells. Throughout the IL-2 culture, one-fourth of the TCR-V gamma 3+ thymocytes was positive for CD8. Analysis of the DNA content and the IL-2 receptor (IL-2R) p55 expression showed that during the first days of culture the TCR-V gamma 3+ cells had a much higher proliferation rate than the TCR-V gamma 3- cells, although TCR-V gamma 3+ IL2R p55+ cells could not be detected. From day 3 to 4 of culture, the proliferation rate of TCR-V gamma 3+ cells equaled that of the rest of the cells and less than 20% of the TCR-V gamma 3+ cells expressed the IL-2R p55. The biologic significance of our findings is discussed.     

Serum soluble interleukin-2 (IL-2) receptor levels in women with breast carcinoma and its correlation with IL-2 receptor expression on blood lymphocytes and lymphocytic infiltration within the tumour

Summary This study describes serum levels of soluble interleukin-2 receptor (sIL-2R) in 13 healthy women, 10 women with breast dysplasia and 37 patients with breast carcinoma. A difference was found between sIL-2R levels in normal women and cancer patients. sIL-2R increased with the advance in stage of cancer but the extent of increase fell from stage III to stage IV as compared to the change from stage II to stage III. Of the 15 patients who were followed after surgery and/or therapy, 10 (67%) showed a fall in the serum sIL-2R levels. A negative correlation was found between sIL-2R levels and lymphocytic infiltration only within the malignant tissue. These findings probably indicate that sIL-2R exerts an immunomodulatory effect on blood lymphocytes by preventing their infiltration into the tumour tissue. To our knowledge, this is the first report of its kind in immunology of breast carcinoma.     

Specific signaling pathways triggered by IL-2 in human V gamma 9V delta 2 T cells: an amalgamation of NK and alpha beta T cell signaling

The global immune response can be simplified into two components: the innate and the acquired systems. The innate immune response comprises primarily macrophages and NK cells, while B and T cells orchestrate the acquired response. Human Vgamma9Vdelta2 T cells represent a minor T cell subpopulation in blood (1-5%) that is activated via the TCR by small nonpeptidic molecules. Their percentage dramatically increases during the early phase of infection by intracellular pathogens, and they display many characteristics of NK cells, which places them at a unique position within the immune system. Our aim was to explore the behavior of these cells when they are activated by a receptor that is common to NK and alphabeta T cells, and to determine signaling pathways and biological responses induced in these cells through this receptor. Thus, we investigated whether Vgamma9Vdelta2 T cells behave as NK cells or as alphabeta T cells. We demonstrated that IL-2 activates not only STAT3, STAT5, the phosphatidylinositol 3-kinase pathway, and extracellular signal-regulated kinase-2 pathway, but also STAT4 as in NK cells, and the p38 mitogen-activated protein kinase pathway as in alphabeta T cells. Moreover, IL-2 induces the production of IFN-gamma in Vgamma9Vdelta2 T cells as observed in NK cells. Due to their double profiles, Vgamma9Vdelta2 T cells are at the interface of the innate and the acquired immune response and may therefore not only modulate the activity of innate cells, but also influence Th1/Th2 differentiation.     

IGF2BP3 facilitates cell proliferation and tumorigenesis via modulation of JAK/STAT signalling pathway in human bladder cancer

Insulin‐like growth factor‐2 messenger RNA‐binding protein 3 (IGF2BP3) has been reported to contribute to tumorigenesis in several human cancers. However, the biological functions of IGF2BP3 in bladder cancer are poorly understood. We investigated the relation between IGF2BP3 expression and prognosis of bladder cancer patients. Cell proliferation, cell cycle and cell apoptosis assays were performed to assess IGF2BP3 functions. The results showed that IGF2BP3 was overexpressed in bladder cancer tissues compared with that in normal bladder tissues, and its higher expression was closely correlated with poor prognosis in bladder cancer patients. Overexpression of IGF2BP3 markedly promoted cell proliferation and cell cycle progression and inhibited cell apoptosis, while knockdown of IGF2BP3 notably suppressed the proliferation, promoted cell apoptosis and induced cell cycle arrest at the G0/G1 phase. Mechanistically, we revealed that IGF2BP3 promotes the activation of the JAK/STAT pathway in bladder cancer cells. Moreover, the JAK/STAT inhibitor dramatically blocked the tumour‐promoting activity of IGF2BP3. Tumour growth in vivo was also suppressed by knocking down of IGF2BP3. Hence, IGF2BP3 facilitated bladder cancer cell proliferation by activating the JAK/STAT signalling pathway. These findings suggest that IGF2BP3 exhibits an oncogenic effect in human bladder cancer progression.