Overexpression of interleukin-2 receptor alpha in a human squamous cell carcinoma of the head and neck cell line is associated with increased proliferation,drug resistance,and transforming ability

It has been previously demonstrated that human carcinomas express interleukin-2 receptor (IL-2R) alpha, beta, and gamma chains. The beta and gamma chains of IL-2R have intermediate binding affinity for IL-2 and are responsible for the intracellular signaling cascades after IL-2 stimulation. IL-2Ralpha lacks the cytoplasmic domain, but is essential for increasing the IL-2-binding affinity of other receptors. Overexpression of IL-2Ralpha in tumor cells is associated with tumor progression and a poor patient prognosis. To define molecular mechanisms responsible for the effects associated with IL-2Ralpha expression, ex vivo experiments were performed with the squamous cell carcinoma head-and-neck cancer line, PCI-13, which was genetically engineered to overexpress the IL-2Ralpha chain. While IL-2Ralpha-overexpressing PCI-13 cells were capable of forming colonies in soft agar, PCI-13 cells transfected with the control vector or those expressing IL-2Rgamma did not. Consistently, IL-2Ralpha-expressing tumor cells proliferated more rapidly than the control or IL-2Rgamma+ cells, associated with increased levels of cyclins A and D1 and cyclin-dependent kinase (cdk(s)) 2 and 4 proteins. In addition, IL-2Ralpha-expressing cells were significantly more resistant to apoptosis induction by a tripeptidyl proteasome inhibitor (ALLN) and two chemotherapeutic drugs (VP-16 and taxol) than the control or IL-2Rgamma+ cells. Accompanying the drug resistance, high levels of anti-apoptotic Bcl-X(L) and Bcl-2 proteins were found in the mitochondria-containing fraction of IL-2Ralpha-expressing tumor cells. Treatment of IL-2Ralpha-expressing cells with a specific Janus kinase 3 (Jak3) inhibitor decreased expression of cyclin A, cyclin D1, Bcl-X(L), and Bcl-2 proteins. Finally, high levels of ubiquitinated proteins were detected in the proliferating IL-2Ralpha-expressing cells. Our data suggest that increased proliferation rates and decreased drug sensitivity of IL-2Ralpha-expressing tumor cells are responsible for the enhanced tumor aggressiveness and poor clinical prognosis of patients whose tumors express IL-2Ralpha.     

Bcl-X(L) and calyculin A prevent translocation of Bax to mitochondria during apoptosis

During many forms of apoptosis, Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the mitochondria and induces cytochrome c release, followed by caspase activation and DNA degradation. Both Bcl-X(L) and the protein phosphatase inhibitor calyculin A have been shown to prevent apoptosis, and here we investigated their impact on Bax translocation. ML-1 cells incubated with either anisomycin or staurosporine exhibited Bax translocation, cytochrome c release, caspase 8 activation, and Bid cleavage; only the latter two events were caspase-dependent, confirming that they are consequences in this apoptotic pathway. Both Bcl-X(L) and calyculin A prevented Bax translocation and cytochrome c release. Bcl-X(L) is generally thought to heterodimerize with Bax to prevent cytochrome c release and yet they remain in different cellular compartments, suggesting that their heterodimerization at the mitochondria is not the primary mechanism of Bcl-X(L)-mediated protection. Using chemical cross-linking agents, Bax appeared to exist as a monomer in undamaged cells. Upon induction of apoptosis, Bax formed homo-oligomers in the mitochondrial fraction with no evidence for cross-linking to Bcl-2 or Bcl-X(L). Considering that both Bcl-X(L) and calyculin A inhibit Bax translocation, we propose that Bcl-X(L) may regulate Bax translocation through modulation of protein phosphatase or kinase signaling.     

Wnt3a regulates proliferation,apoptosis and function of pancreatic NIT‐1 beta cells via activation of IRS2/PI3K signaling

Wnt‐signaling pathway is implicated in pancreatic development and functional regulation of mature beta‐cells. Wnt3a/Wnt pathway activation expands islet cell mass in vitro by increasing proliferation and decreasing apoptosis of beta‐cells, thereby enhancing its function. However, the signaling pathways that mediate these effects remain unknown. By using a clonal beta‐cell line (NIT‐1), we examined the role of IRS2/PI3K in the mediation of Wnt3a‐stimulated beta‐cell growth. Real‐time PCR and Western blot were employed to investigate the activity of Wnt/β‐catenin and IRS2/PI3K signaling. Proliferation of NIT‐1 cells was assessed by BrdU incorporation, and apoptosis was quantitatively determined by TUNEL and flow cytometry (FCM). Dkk1, an inhibitor of Wnt signaling, and wortmannin, an inhibitor of PI3K, were also used. Results showed that Wnt3a rapidly activated Wnt/β‐catenin signaling, promoted IRS2 expression and Akt phosphorylation in NIT‐1 cells. These effects were completely abrogated by Dkk1 or partially eliminated by wortmannin. Wnt3a also promoted NIT‐1 cell proliferation, inhibited cytokine‐induced beta‐cell apoptosis, and increased insulin secretion. Both of these effects were also eliminated by Dkk1 or wortmannin. Our results demonstrated that Wnt3a regulates proliferation, apoptosis and enhances function of pancreatic NIT‐1 beta cells via activation of Wnt/β‐catenin signaling, involving crosstalk with IRS2/PI3K signaling, with the effect of Wnt signaling on beta‐cells also being IRS2/PI3K/AKT dependent. J. Cell. Biochem. 114: 1488–1497, 2013. © 2013 Wiley Periodicals, Inc.     

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions

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Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Regulation of Survivin and Bcl‐2 in HepG2 Cell Apoptosis Induced by Quercetin

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in a variety of human cancer cells. However, the regulation of survivin and Bcl‐2 on the quercetin‐induced cell‐growth inhibition and apoptosis in cancer cells remains unclear. In the present study, we report that quercetin can inhibit proliferation and induce apoptosis in HepG2 cells in dose‐ and time‐dependent manner. Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) staining showed that HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to quercetin. Cell‐cycle analysis reveals a significant increase of the proportion of cells in G₀/G₁ phase. We also demonstrate that the levels of survivin and Bcl‐2 protein expression in HepG2 cells decreased concurrently, and the levels of p53 protein increased significantly after treatment with quercetin by immunocytochemistry analysis. Relative activity of caspase‐3 and caspase‐9 increased significantly. These data clearly indicate that quercetin‐induced apoptosis is associated with caspase activation, and the levels of survivin and Bcl‐2. Our results indicate that the expression of survivin may be associated with Bcl‐2 expression, and the inhibition expression of survivin, in conjunction with Bcl‐2, might cause more pronounced apoptotic effects. Together, concurrent down‐regulated survivin and Bcl‐2 play an important role in HepG2 cell apoptosis induced by quercetin.     

Oroxylin A induced apoptosis of human hepatocellular carcinoma cell line HepG2 was involved in its antitumor activity

We previously reported that wogonin, a flavonoid compound, was a potent apoptosis inducer of human hepatoma SMMC-7721 cells and murine sarcoma S180 cells. In the present study, the effect of oroxylin A, one wogonin structurally related flavonoid isolated from Scutellariae radix, on human hepatocellular carcinoma cell line HepG2 was examined and molecular mechanisms were also investigated. Oroxylin A inhibited HepG2 cell proliferation in a concentration- and time-dependent manner measured by MTT-assay. Treatment with an apoptosis-inducing concentration of oroxylin A caused typical morphological changes and apoptotic blebbing in HepG2 cells. DNA fragmentation assay was used to examine later apoptosis induced by oroxylin A. FACScan analysis revealed a dramatic increase in the number of apoptotic and G(2)/M phase arrest cells after oroxylin A treatment. The pro-apoptotic activity of oroxylin A was attributed to its ability to modulate the concerted expression of Bcl-2, Bax, and pro-caspase-3 proteins. The expression of Bcl-2 protein and pro-caspase-3 protein was dramatically decreased after treatment with oroxylin A. These results demonstrated that oroxylin A could effectively induce programmed cell death and suggested that it could be a promising antitumor drug.     

A natural phenylpropionate derivative from Mirabilis himalaica inhibits cell proliferation and induces apoptosis in HepG2 cells

Bioactivity-guided study led to the isolation of a natural phenylpropionate derivative, (E)-3-(4-hydroxy-2-methoxyphenyl)-propenoic acid 4-hydroxy-3-methoxyphenyl ester from the roots of Mirabilis himalaica. Cellular analysis showed that compound 1 specifically inhibited the cancer cell growth through the S phase arrest. Mechanistically, compound 1 was able to induce the apoptosis in HepG2 cells through mitochondrial apoptosis pathway in which Bcl-2 and p53 were required. Interestingly, the cellular phenotype of compound 1 were shown specifically in cancer cells originated from hepatocellular carcinoma (HepG2) while compromised influence by compound 1 were detected within the normal human liver cells (L-02). Consistently, the in vivo inhibitory effects of compound 1 on tumor growth were validated by the in xenograft administrated with HepG2 cells. Our results provided a novel compound which might serve as a promising candidate and shed light on the therapy of the hepatocellular carcinoma.     

Direct inhibition of interleukin-2 receptor alpha-mediated signaling pathway induces G1 arrest and apoptosis in human head-and-neck cancer cells

Overexpression of the interleukin-2 receptor (IL-2R) alpha chain in tumor cells is associated with tumor progression and a poor patient prognosis. IL-2Ralpha is responsible for the high affinity binding of the receptor to IL-2, leading to activation of several proliferative and anti-apoptotic intracellular signaling pathways. We have previously shown that human squamous cell carcinoma of a head-and-neck line (PCI-13) genetically engineered to overexpress IL-2Ralpha exhibit increased transforming activity, proliferation, and drug resistance, compared to the vector control cells (J Cell Biochem 2003;89:824-836). In this study, we report that IL-2Ralpha(+) cells express high levels of total and phosphorylated Jak3 protein and are more resistant to apoptosis induced by a Jak3 inhibitor than the control LacZ cells. Furthermore, we used daclizumab, a monoclonal antibody specific to IL-2Ralpha, and determined the effects of IL-2Ralpha inhibition on cell cycle and apoptosis as well as the involvement of potential cell cycle and apoptosis regulatory proteins. We found that daclizumab induces G(1) arrest, associated with down-regulation of cyclin A protein, preferentially in IL-2Ralpha(+) cells, but not in LacZ cells. In addition, daclizumab activates apoptotic death program via Bcl-2 down-regulation preferentially in IL-2Ralpha(+) cells. Finally, daclizumab also sensitizes IL-2Ralpha(+) cells to other apoptotic stimuli, although the effect is moderate. These results indicate that daclizumab inhibits the proliferative potential of IL-2Ralpha(+) cells via inhibition of cell cycle progression and induction of apoptosis.     

Regulation of A(2A) adenosine receptor expression and functioning following permanent focal ischemia in rat brain

Ischemia, through modulation of adenosine receptors (ARs), may influence adenosine-mediated-cellular responses. In the present study, we investigated the modulation of rat A_2A receptor expression and functioning, in rat cerebral cortex and striatum, following in vivo focal ischemia (24 h). In cortex, middle cerebral artery occlusion did not induce any alterations in A_2A receptor binding and functioning. On the contrary, in striatum, a significant decrease in A_2A ligand affinity, associated with an increase in receptor density, were detected. In striatum, ischemia also induced a significant reduction both in G protein pool and in A_2A receptor-G protein coupling. On the contrary, A_2A receptor functional responsiveness, measured as stimulation of adenylyl cyclise, was not affected by ischemia, suggesting receptor up-regulation may represent a compensatory mechanism to maintain receptor functioning during cerebral damage. Immunohistochemical study showed that following 24 h middle cerebral artery occlusion, A_2A ARs were definitely expressed both on neurons and activated microglia in ischemic striatum and cortex, but were not detected on astrocytes. In the non-ischemic hemisphere and in sham-operated rats A_2A ARs were barely detected. Modifications of ARs may play a significant role in determining adenosine effects during ischemia and therefore should be taken into account when evaluating time-dependent protective effects of specific A_2A active compounds.     

Up-regulation of p21CIP1 expression mediated by ERK-dependent and -independent pathways contributes to hepatocyte growth factor-induced inhibition of HepG2 hepatoma cell proliferation

Strong activation of the ERK signal is required for hepatocyte growth factor (HGF) to inhibit proliferation of the human hepatocellular carcinoma cell line HepG2. However, it is still to be elucidated whether the activation alone is sufficient to induce the inhibitory effect. In this study, we constructed HepG2 cell clones expressing a high level of epidermal growth factor receptor (EGFR), and examined the effect of the strong activation of ERK on the proliferation of the cell clones. EGF treatment of the cell clones induced strong activation of ERK similar to HGF treatment, but did not inhibit cell proliferation. HGF treatment of the cell clones up-regulated the expression of a Cdk inhibitor p16(INK4a), which has previously been shown to be required to inhibit the proliferation of HepG2 cells, but EGF treatment did not. Furthermore, EGF treatment of the cell clones did not induce the up-regulation of another Cdk inhibitor p21(CIP1), whereas HGF treatment did. Knockdown of p21 by siRNA restored the proliferation of HepG2 cells inhibited by HGF, and restored Cdk2 activity suppressed in HGF-treated HepG2 cells. These results suggest that strong activation of ERK alone is not sufficient, and some other pathway(s), which is activated through the HGF receptor but not through EGFR, is also required to induce the up-regulation of p16 and p21 expression, and also suggest that in addition to the up-regulated expression of p16, that of p21 contributes to the suppression of Cdk2 activity leading to the inhibition of proliferation of HGF-treated HepG2 cells.     

Neuroprotection by adenosine A2A receptor blockade in experimental models of Parkinson's disease

Adenosine A2A receptors are abundant in the caudate-putamen and involved in the motor control in several species. In MPTP-treated monkeys, A2A receptor-blockade with an antagonist alleviates parkinsonian symptoms without provoking dyskinesia, suggesting this receptor may offer a new target for the antisymptomatic therapy of Parkinson's disease. In the present study, a significant neuroprotective effect of A2A receptor antagonists is shown in experimental models of Parkinson's disease. Oral administration of A2A receptor antagonists protected against the loss of nigral dopaminergic neuronal cells induced by 6-hydroxydopamine in rats. A2A antagonists also prevented the functional loss of dopaminergic nerve terminals in the striatum and the ensuing gliosis caused by MPTP in mice. The neuroprotective property of A2A receptor antagonists may be exerted by altering the packaging of these neurotoxins into vesicles, thus reducing their effective intracellular concentration. We therefore conclude that the adenosine A2A receptor may provide a novel target for the long-term medication of Parkinson's disease, because blockade of this receptor exerts both acutely antisymptomatic and chronically neuroprotective activities.     

Neuroprotection afforded by adenosine A2A receptor blockade is modulated by corticotrophin‐releasing factor (CRF) in glutamate injured cortical neurons

In situations of hypoxia, glutamate excitotoxicity induces neuronal death. The release of extracellular adenosine is also triggered and is accompanied by an increase of the stress mediator, corticotrophin‐releasing factor (CRF). Adenosine A_2A receptors contribute to glutamate excitoxicity and their blockade is effective in stress‐induced neuronal deficits, but the involvement of CRF on this effect was never explored. We now evaluated the interaction between A_2A and CRF receptors (CRFR) function, upon glutamate insult. Primary rat cortical neuronal cultures (9 days in vitro) expressing both CRF₁R and CRF₂R were challenged with glutamate (20–1000 μM, 24 h). CRF₁R was found to co‐localize with neuronal markers and CRF₂R to be present in both neuronal and glial cells. The effects of the CRF and A_2A receptors ligands on cell viability were measured using propidium iodide and Syto‐13 fluorescence staining. Glutamate decreased cell viability in a concentration‐dependent manner. Urocortin (10 pM), an agonist of CRF receptors, increased cell survival in the presence of glutamate. This neuroprotective effect was abolished by blocking either CRF₁R or CRF₂R with antalarmin (10 nM) or anti‐Sauvagine‐30 (10 nM), respectively. The blockade of A_2A receptors with a selective antagonist SCH 58261 (50 nM) improved cell viability against the glutamate insult. This effect was dependent on CRF₂R, but not on CRF₁R activation. Overall, these data show a protective role of CRF in cortical neurons, against glutamate‐induced death. The neuroprotection achieved by A_2A receptors blockade requires CRF₂R activation. This interaction between the adenosine and CRF receptors can explain the beneficial effects of using A_2A receptor antagonists against stress‐induced noxious effects.