Reduced phosphate transport in the renal proximal tubule cells in cystinosis is due to decreased expression of transporters rather than an energy defect

Nephropathic cystinosis is an autosomal recessive disorder caused by mutations in the CTNS gene [1], which encodes for a transporter (cystinosin) responsible for cystine efflux from lysosomes. In cystinotic renal proximal tubules (RPTs), the defect in cystinosin function results in reduced reabsorption of solutes by apical Na⁺/solute cotransport systems, including the Na⁺/phosphate (Pi) cotransport system [2]. However the underlying molecular mechanisms are unknown, given the lack of an appropriate cellular model. To obtain such a model system, we have knocked down cystinosin with siRNA in primary RPT cell cultures. An 80% reduction in cystinosin strongly inhibited Na⁺ dependent Pi uptake (70%). Although this finding could be explained by a direct effect on transporters as well as by altered energetics (the ATP level dropped by 52%), our results demonstrate a lack of involvement of Na, K-ATPase, and a reduction in the number of NaPi2a transporters.     

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G₂M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.     

Activation of the AMP-activated protein kinase–p38 MAP kinase pathway mediates apoptosis induced by conjugated linoleic acid in p53-mutant mouse mammary tumor cells

Conjugated linoleic acid (CLA) inhibits tumorigenesis and tumor growth in most model systems, an effect mediated in part by its pro-apoptotic activity. We previously showed that trans-10,cis-12 CLA induced apoptosis of p53-mutant TM4t mouse mammary tumor cells through both mitochondrial and endoplasmic reticulum stress pathways. In the current study, we investigated the role of AMP-activated protein kinase (AMPK), a key player in fatty acid metabolism, in CLA-induced apoptosis in TM4t cells. We found that t10,c12-CLA increased phosphorylation of AMPK, and that CLA-induced apoptosis was enhanced by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and inhibited by the AMPK inhibitor compound C. The increased AMPK activity was not due to nutrient/energy depletion since ATP levels did not change in CLA-treated cells, and knockdown of the upstream kinase LKB1 did not affect its activity. Furthermore, our data do not demonstrate a role for the AMPK-modulated mTOR pathway in CLA-induced apoptosis. Although CLA decreased mTOR levels, activity was only modestly decreased. Moreover, rapamycin, which completely blocked the activity of mTORC1 and mTORC2, did not induce apoptosis, and attenuated rather than enhanced CLA-induced apoptosis. Instead, the data suggest that CLA-induced apoptosis is mediated by the AMPK–p38 MAPK–Bim pathway: CLA-induced phosphorylation of AMPK and p38 MAPK, and increased expression of Bim, occurred with a similar time course as apoptosis; phosphorylation of p38 MAPK was blocked by compound C; the increased Bim expression was blocked by p38 MAPK siRNA; CLA-induced apoptosis was attenuated by the p38 inhibitor SB-203580 and by siRNAs directed against p38 MAPK or Bim.     

eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1

Reports on the role of AMP-activated protein kinase (AMPK) in thrombin-mediated activation of endothelial nitric-oxide synthase (eNOS) in endothelial cells have been conflicting. Previously, we have shown that under culture conditions that allow reduction of ATP-levels after stimulation, activation of AMPK contributes to eNOS phosphorylation and activation in endothelial cells after treatment with thrombin. In this paper we examined the signaling pathways mediating phosphorylation and activation of eNOS after stimulation of cultured human umbilical vein endothelial cells (HUVEC) with histamine and the role of LKB1-AMPK in the signaling. In Morgan's medium 199 intracellular ATP was lowered by treatment with histamine or the ionophore A23187 while in medium RMPI 1640 ATP was unchanged after identical treatment. In medium 199 inhibition of Ca^+ 2/CaM kinase kinase (CaMKK) by STO-609 only partially inhibited AMPK phosphorylation but after gene silencing of LKB1 with siRNA there was a total inhibition of AMPK phosphorylation by STO-609 after treatment with either histamine or thrombin, demonstrating phosphorylation of AMPK by both upstream kinases, LKB1 and CaMKK. Downregulation of AMPK with siRNA partially inhibited eNOS phosphorylation caused by histamine in cells maintained in medium 199. Downregulation of LKB1 by siRNA inhibited both phosphorylation and activity of eNOS and addition of the AMPK inhibitor Compound C had no further effect on eNOS phosphorylation. When experiments were carried out in medium 1640, STO-609 totally prevented the phosphorylation of AMPK without affecting eNOS phosphorylation. AMPKα2 downregulation resulted in a loss of the integrity of the endothelial monolayer and increased expression of GRP78, indicative of endoplasmic reticular (ER) stress. Downregulation of AMPKα1 had no such effect. The results show that culture conditions affect endothelial signal transduction pathways after histamine stimulation. Under conditions where intracellular ATP is lowered by histamine, AMPK is activated by both LKB1 and CaMKK and, in turn, mediates eNOS phosphorylation in an LKB1 dependent manner. Both AMPKα1 and − α2 are involved in the signaling. Under conditions where intracellular ATP is unchanged after histamine treatment, CaMKK alone activates AMPK and eNOS is phosphorylated and activated independent of AMPK.     

AICAR potentiates ROS production induced by chronic high glucose: roles of AMPK in pancreatic beta-cell apoptosis

We previously demonstrated that chronic high glucose (33.3 mM) induced beta-cell dysfunction and apoptosis through glucokinase (GCK) downregulation, but the exact mechanisms involved remain unclear. Here, we show that prolonged exposure of 5-aminoimidazole-4-carboxamide (AICA)-riboside potentiated apoptosis induced by high glucose in MIN6N8 pancreatic beta-cells, correlating with enhanced GCK downregulation and decreased production of ATP and insulin. These events are potentiated in AMPK-overexpressing cells, but are prevented in cells transfected with mutant dominant-negative AMPK (AMPK-K45R). Furthermore, AMPK activation increases production of reactive oxygen species (ROS) and loss of mitochondria membrane potential induced by high glucose, which is significantly inhibited by treatment with compound C or by AMPK-K45R overexpression. Overexpression of GCK prevents apoptosis; decreased cellular ATP and insulin secretion, and ROS production enhanced by AICAR, but does not affect AMPK activation. Similar results are obtained using isolated primary islet cells. Collectively, these data demonstrate that AMPK activation potentiates beta-cell apoptosis induced by chronic high glucose through augmented GCK downregulation mediated by enhanced ROS production.     

Effects of short‐chain acyl‐CoA dehydrogenase on cardiomyocyte apoptosis

Short‐chain acyl‐CoA dehydrogenase (SCAD), a key enzyme of fatty acid β‐oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert‐butyl hydroperoxide (tBHP)‐induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down‐regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA‐1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl‐2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP‐activated protein kinase α (p‐AMPKα) and Peroxisome proliferator‐activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways.     

Activation of AMP-activated protein kinase is involved in vincristine-induced cell apoptosis in B16 melanoma cell

The molecular basis for induction of apoptosis in melanoma cells by vincristine remains unknown. Here we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found for the first time that vincristine induces AMPK activation (AMPKα, Thr 172) and Acetyl-CoA carboxylase (ACC, Ser 79) (a downstream molecular target of AMPK) phosphorylation in cultured melanoma cells in vitro. Reactive oxygen species (ROS) dependent LKB1 activation serves as the upstream signal for AMPK activation. AMPK inhibitor (compound C) or AMPKα siRNA knockdown inhibits vincristine induced B16 melanoma cell apoptosis, while AMPK activator 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAR) enhances it. AMPK activation is involved in vincristine induced p53 phosphorylation and stabilization, the latter is known to mediate melanoma cell apoptosis. Further, activation of AMPK by vincristine inhibits mTOR Complex 1 (mTORC1) in B16 melanoma cells, which serves as another important mechanism to induce melanoma cell apoptosis. Our study provides new insights into understanding the cellular and molecular mechanisms of vincristine induced cancer cell death/apoptosis. We suggest that combining AMPK activator AICAR with vincristine may have potential to be used as a new therapeutic intervention against melanoma.     

Activation of AMP-activated protein kinase is essential for lysophosphatidic acid-induced cell migration in ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-β3 (PLC-β3) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-β3, or (CaMKKβ) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.     

Requirement for ERK activation in cisplatin-induced apoptosis

Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal.     

Role of Autophagy in Cisplatin Resistance in Ovarian Cancer Cells

Cisplatin-based treatment is the first line chemotherapy for several cancers including ovarian cancer. The development of cisplatin resistance results in treatment failure, but the underlying mechanisms are not fully understood. Here we show that the induction of autophagy plays an important role in cisplatin resistance in ovarian cancer cells. Specifically, we show that cisplatin resistance is correlated with autophagy induction in a panel of ovarian cancer cells but not in immortalized human ovarian surface epithelial cells. Mechanistically, cisplatin treatment activates ERK and subsequently promotes autophagy. The inhibition of ERK activation with MEK inhibitors or knockdown of ERK expression with siRNA decreases cisplatin-induced autophagy and subsequently sensitizes ovarian cancer cells to cisplatin-induced apoptosis. In ovarian cancer cells that have developed acquired cisplatin resistance, both ERK activation and autophagy induction are increased. Importantly, knockdown of ERK or inhibition of autophagy promotes cisplatin-induced apoptosis in acquired cisplatin-resistant cells. Collectively, our data indicate that ERK-mediated autophagy can lead to cisplatin resistance and suggest that cisplatin resistance can be overcome by inhibition of autophagy in ovarian cancer cells.     

AMPK protects proximal tubular cells from stress-induced apoptosis by an ATP-independent mechanism: potential role of Akt activation

We examined the role of AMP-activated protein kinase (AMPK) in modulating the viability of cultured kidney proximal tubular cells subjected to metabolic stress induced by either dextrose deprivation, inhibition of glycolysis, or inhibition of mitochondrial respiration. We used BU.MPT cells, a conditionally immortalized kidney epithelial cell line derived from the proximal tubules of transgenic mice bearing a temperature-sensitive mutation of the simian virus 40 large-tumor antigen. All three forms of metabolic stress increased the phosphorylation and activity of AMPK. Activation of AMPK led to changes in the phosphorylation of two downstream targets of AMPK, acetyl coenzyme A carboxylase and p70 S6 kinase. Inhibition of AMPK, either pharmacologically with compound C (CC) or by gene silencing, significantly increased the amount of apoptosis in response to all three forms of metabolic stress. Although the amount of apoptosis was directly related to the severity of ATP depletion, inhibition of AMPK had no effect on cellular ATP levels. Notably, metabolic stress increased the phosphorylation and activity of Akt. Furthermore, inhibition of AMPK, with CC or gene silencing, abrogated the ability of metabolic stress to activate Akt. The augmentation of apoptosis induced by inhibition of AMPK was comparable to that induced by inhibition of Akt. We conclude that activation of AMPK following acute metabolic stress plays a major role in promoting the viability of cultured proximal tubular cells. Protection by AMPK appears to be due not to AMPK-mediated conservation of cell energy stores, but rather, at least in part, to AMPK-mediated activation of Akt.     

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.     

Inhibition of AMP-activated protein kinase sensitizes cancer cells to cisplatin-induced apoptosis via hyper-induction of p53

Cisplatin is one of the most effective and widely used chemotherapeutic agents. However, one of the most salient limitations to the clinical application of cisplatin is the acquired or intrinsic drug resistance exhibited by some tumors. In the present study, we have assessed the potential of an intracellular energy balancing system as a target for augmentation of cisplatin sensitivity in tumors. AMP-activated protein kinase (AMPK) regulates the energy balance system by monitoring intracellular energy status. Here we demonstrate that AMPK is rapidly activated by cisplatin in AGS and HCT116 cancer cells. The inhibition of AMPK in those cells and in xenografts of HCT116 resulted in a remarkable increase in cisplatin-induced apoptosis, which was associated with hyper-induction of the tumor suppressor p53. We further showed that ERK, but not ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) kinases, was involved in the hyper-induction of p53 by the inhibition of cisplatin-induced AMPK. By way of contrast, cisplatin did not induce AMPK activation in HeLa cells, which appear to have a relatively high sensitivity to cisplatin-induced cytotoxicity, but expression of the constitutive active form of AMPK in HeLa cells resulted in a significant increase of cell viability after cisplatin treatment. Collectively, our data suggest that AMPK performs a pivotal function for protection against the cytotoxic effect of cisplatin, thereby implying that AMPK is one of the cellular factors determining the cellular sensitivity to cisplatin. On the basis of these observations, we propose that a strategy combining cisplatin and AMPK inhibition could be developed into a novel chemotherapeutic modality.     

ERK-mediated suppression of cilia in cisplatin-induced tubular cell apoptosis and acute kidney injury

In kidneys, each tubular epithelial cell contains a primary cilium that protrudes from the apical surface. Ciliary dysfunction was recently linked to acute kidney injury (AKI) following renal ischemia–reperfusion. Whether ciliary regulation is a general pathogenic mechanism in AKI remains unclear. Moreover, the ciliary change during AKI and its underlying mechanism are largely unknown. Here we examined the change of primary cilium and its role in tubular cell apoptosis and AKI induced by cisplatin, a chemotherapy agent with notable nephrotoxicity. In cultured human proximal tubular HK-2 epithelial cells, cilia became shorter during cisplatin treatment, followed by apoptosis. Knockdown of Kif3a or Polaris (cilia maintenance proteins) reduced cilia and increased apoptosis during cisplatin treatment. We further subcloned HK-2 cells and found that the clones with shorter cilia were more sensitive to cisplatin-induced apoptosis. Mechanistically, cilia-suppressed cells showed hyperphosphorylation or activation of ERK. Inhibition of ERK by U0126 preserved cilia during cisplatin treatment and protected against apoptosis in HK-2 cells. In C57BL/6 mice, U0126 prevented the loss of cilia from proximal tubules during cisplatin treatment and protected against AKI. U0126 up-regulated Polaris, but not Kif3a, in kidney tissues. It is suggested that ciliary regulation by ERK plays a role in cisplatin-induced tubular apoptosis and AKI.     

LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner.     

4-O-methylascochlorin, methylated derivative of ascochlorin, stabilizes HIF-1α via AMPK activation

Chemopreventive or anticancer agents induce cancer cells to apoptosis through the activation of adenosine AMP-activated protein kinase (AMPK), which plays a major role as energy sensors under ATP-deprived condition or ROS generation. In this study, we compared the effects of ascochlorin (ASC), from the fungus Ascochyta viciae, and its derivatives on AMPK activity. We also examined a regulatory mechanism for hypoxia-inducible factor-1α (HIF-1α) stabilization in response to 4-O-methylascochlorin (MAC). We found that AMPK activation was mainly involved with MAC, but not ASC and 4-O-carboxymethylascochlorin (AS-6), indicating that the substitution of 4-O-methyl group from 4-O-hydroxyl group of ASC is important in the activation of AMPK and the expression of HIF-1α. MAC-stabilized HIF-1α via AMPK activation triggered by lowering the intracellular ATP level, not by ROS generation, increases glucose uptake and the expression of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1), major target genes of HIF-1α. Moreover, MAC-induced AMPK activity suppressed survival factors, including mTOR and ERK1/2 or translational regulators, including p70S6K and 4E-BP1. Our data suggest that AMPK is a key determinant of MAC-induced HIF-1α expression in response to energy stress, further implying its involvement in MAC-induced apoptosis.     

Angiotensin II activates AMPK for execution of apoptosis through energy-dependent and -independent mechanisms

At the cellular level, 5'-AMP-activated protein kinase (AMPK) serves as a critical link between energy homeostasis and the regulation of fundamental biological activities, including apoptosis. Angiotensin (Ang) II plays a key role in fibrotic lung remodeling. We recently demonstrated that Ang II induces apoptosis in pulmonary artery endothelial cells (PAEC) through the Ang type 2 receptor (AT(2)). AT(2) activates Src-homology two-domain-containing phosphatase-2 (SHP-2) in a signaling cascade leading to Bcl-x(L) mRNA destabilization and initiation of intrinsic apoptosis. We investigated the requirement of AMPK and ATP generation for Ang II-induced apoptosis in PAEC. Ang II activated AMPK, which was required for ATP generation. Inhibition of ATP production by compound C, an AMPK inhibitor, or by oligomycin suppressed Ang II-induced apoptosis. Experiments in Chinese hamster ovary-K1 cells expressing ectopic AT(2) (wild-type, mutant D90A, or carboxy terminal truncated mutant tC319) demonstrated that AT(2) activation of AMPK required the active conformation of the receptor and the carboxy terminal 44 amino acids. AMPK associated with and activated SHP-2 and was required for Bcl-x(L) mRNA destabilization. These are the first findings demonstrating that AMPK is activated by Ang II to produce ATP required for apoptosis. Our data also indicate that AMPK plays an energy-independent role by mediating SHP-2 activation.     

Ursolic acid-induced AMP-activated protein kinase (AMPK) activation contributes to growth inhibition and apoptosis in human bladder cancer T24 cells

Ursolic acid (UA) has shown the anti-tumor properties against a number of human cancers both in vivo and in vitro, however, its effect in bladder cancer and the corresponding mechanisms of action remain largely unknown. Here we found that UA dose-dependently induced growth inhibition and apoptosis in human bladder cancer T24 cells, and activation of AMP-activated protein kinase (AMPK) may contribute to the process. Our Western-blot results demonstrated a significant AMPK activation after UA treatment in T24 cells. Notably, knockdown of AMPKα by the targeted shRNA largely inhibited UA-induced T24 cell growth inhibition and apoptosis, while an AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or a constitutively active form of AMPK mimic UA's effect. We found the ceramide level was increased after UA treatment in T24 cells, and UA-induced AMPK activation and T24 cell apoptosis were inhibited by ceramide synthase inhibitor fumonisin B1, and was enhanced by exogenously adding cell permeable short-chain ceramide (C6), suggesting that ceramide might serve as an upstream signal for AMPK activation. Further, activation of AMPK by UA promoted c-Jun N-terminal kinase (JNK) activation, but inhibited mTOR complex 1 (mTORC1) signaling to cause survivin down-regulation. Our study suggests that activation of AMPK by UA contributes to growth inhibition and apoptosis in human bladder cancer cells.