Massimo Fini

Kaempferol induces apoptosis in two different cell lines via Akt inactivation, Bax and SIRT3 activation, and mitochondrial dysfunction†

Kaempferol (3,4′,5,7‐tetrahydroxyflavone) is a flavonoid with anti‐ and pro‐oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 µM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl‐2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase‐3, and ‐9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD‐dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de‐phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl‐2, release of cytochrome c, caspase‐3 activation, and cell death. J. Cell. Biochem. 106: 643–650, 2009.

Hypoxia-increased RAGE and P2X7R expression regulates tumor cell invasion through phosphorylation of Erk1/2 and Akt and nuclear translocation of NF-κB

The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.

Role of hypoxia and autophagy in mda-mb-231 invasiveness

Survival strategies adopted by tumor cells in response to a hypoxic stress include activation of hypoxia‐inducible factor 1 (HIF‐1) and autophagy. However, the importance and the function of each molecular response is not well defined. In the present study, we investigated invasiveness, migration, matrix metalloproteinases (MMPs) activity, and cell survival of MDA‐MB‐231 cells under normoxia, hypoxia, and hypoxia/reoxygenation (H/R). Moreover, to assess the importance of hypoxia and autophagy on the parameters studied, cells were either left untreated or treated with Chetomin (a selective inhibitor of HIF‐1α) or trifluoperazine (TFP, an activator of autophagy). We found that hypoxia and H/R stimulated invasiveness and migration of MDA‐MB‐231 cells with an increased MMP‐2 activity. Chetomin and TFP differently regulated the cellular behavior under the oxygenation conditions studied. In fact, Chetomin was most effective in inhibiting cell invasion, MMPs activity, and cell survival under hypoxia but not normoxia or H/R. By contrast, TFP inhibition of cell invasion, migration, and cell survival was independent from oxygenation conditions. TFP‐induced autophagy was inhibited by light chain protein 3 (LC3) silencing or 3‐methyladenine (3MA) treatment. In fact, LC3‐silenced cells were able to invade in the presence of TFP without any GATE16 processing and p62 degradation. Immunofluorescence assay showed that LC3 silencing inhibited TFP‐induced autophagosome formation. However, we also showed that both TPF treatment and LC3 silencing caused cytoskeleton impairments suggesting a possible interaction between LC3 and cytoskeleton components. In conclusion, our study shows that hypoxia and autophagy by acting on common (HIF‐1α) or separate (MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival. J. Cell. Physiol. 223: 359–368, 2010.

SIRT1 silencing confers neuroprotection through IGF-1 pathway activation

The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF‐1 protein expression and secretion and of IGF‐1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF‐1 and IGF‐1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF‐1 and IGF‐1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF‐1 survival pathway. J. Cell. Physiol. 228: 1754–1761, 2013.

Unilateral deep brain stimulation of the pedunculopontine tegmental nucleus in idiopathic Parkinson’s disease: Effects on gait initiation and performance

The pedunculopontine tegmental nucleus (PPTg) is a component of the locomotor mesencephalic area. In recent years it has been considered a new surgical site for deep brain stimulation (DBS) in movement disorders. Here, using objective kinematic and spatio-temporal gait analysis, we report the impact of low frequency (40 Hz) unilateral PPTg DBS in ten patients suffering from idiopathic Parkinsons disease with drug-resistant gait and axial disabilities. Patients were studied for gait initiation (GI) and steady-state level walking (LW) under residual drug therapy. In the LW study, a straight walking task was employed. Patients were compared with healthy age-matched controls. The analysis revealed that GI, cadence, stride length and left pelvic tilt range of motion (ROM) improved under stimulation. The duration of the S1 and S2 sub-phases of the anticipatory postural adjustment phase of GI was not affected by stimulation, however a significant improvement was observed in the S1 sub-phase in both the backward shift of centre of pressure and peak velocity. Speed during the swing phase, step width, stance duration, right pelvic tilt ROM phase, right and left hip flexion-extension ROM, and right and left knee ROM were not modified. Overall, the results show that unilateral PPTg DBS may affect GI and specific spatio-temporal and kinematic parameters during unconstrained walking on a straight trajectory, thus providing further support to the importance of the PPTg in the modulation of gait in neurodegenerative disorders.

ERK-1 MAP kinase prevents TNF-induced apoptosis through bad phosphorylation and inhibition of Bax translocation in HeLa Cells.

Extracellular signal‐regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl‐2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK‐1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase‐deficient form of ERK‐1 (K71R) were more sensitive to TNF and CHX. In the ERK‐1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK‐1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK‐1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK‐1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase‐8 inhibitor IETD‐FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c‐Jun N‐terminal kinases activator, increased TNF‐killing. The ERK‐1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK‐1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death. J. Cell. Biochem. 108: 1166–1174, 2009.

Segmental muscle vibration modifies muscle activation during reaching in chronic stroke: A pilot study

BACKGROUND Segmental muscle vibration (SMV) improves motor performances in neurological conditions, including stroke. OBJECTIVE To determine if SMV modifies upper limb muscular activity in chronic stroke patients performing a reaching movement. METHODS We randomized 22 chronic stroke patients to an experimental group (EG; n = 12), receiving 10 sessions of exercise + 120 Hz SMV over the biceps brachii (BB) and the flexor carpi ulnaris (FCU) muscles, or to a control group (CG; n = 10) receiving exercise only. All subjects performed a reaching movement with the affected side before and 4 weeks after therapy ended. We recorded surface EMG activity of the anterior deltoid (AD), posterior deltoid (PD), BB, triceps brachii (TB), FCU and extensor carpi radialis (ECR) muscles. We calculated muscular onset times, modulation ratio, co-contractions and degree of contraction. RESULTS After SMV, onset times of the PD (p = 0.03), BB (p = 0.02) and ECR (p = 0.04) in the EG were less anticipated than at baseline; the modulation ratio increased in AD (p = 0.003) and BB (p = 0.01); co-contractions decreased in the pairs BB/TB (p = 0.007), PD/BB (p = 0.004) and AD/BB (p = 0.01); and the degree of contraction decreased in BB (p = 0.01). CONCLUSIONS The modulation of muscular function induced by SMV may aid to explain its action on smoothness and coordination of movements.

Kinematic analysis of reaching movements of the upper limb after total or reverse shoulder arthroplasty

Studies have analyzed three-dimensional complex motion of the shoulder in healthy subjects or patients undergoing total shoulder arthroplasty (TSA) or reverse shoulder arthroplasty (RSA). No study to date has assessed the reaching movements in patients with TSA or RSA. Twelve patients with TSA (Group A) and 12 with RSA (Group B) underwent kinematic analysis of reaching movements directed at four targets. The results were compared to those of 12 healthy subjects (Group C). The assessed parameters were hand-to-target distance, target-approaching velocity, humeral-elevation angular velocity, normalized jerk (indicating motion fluidity), elbow extension and humeral elevation angles. Mean Constant score increased by 38 points in Group A and 47 in Group B after surgery. In three of the tasks, there were no significant differences between healthy subjects and patients in the study groups. Mean target-approaching velocity and humeral-elevation angular velocity were significantly greater in the control group than in study groups and, overall, greater in Group A than Group B. Movement fluidity was significantly greater in the controls, with patients in Group B showing greater fluidity than those in Group A. Reaching movements in the study groups were comparable, in three of the tasks, to those in the control group. However, the latter performed significantly better with regard to target-approaching velocity, humeral-elevation angular velocity and movement fluidity, which are the most representative characteristics of reaching motion. These differences, that may be related to deterioration of shoulder proprioception after prosthetic implant, might possibly be decreased with appropriate rehabilitation.

Insulin-like growth factor-1 inhibits STS-induced cell death and increases functional recovery of in vitro differentiated neurons

NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-xL content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and down-regulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17,1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude without giving rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to 100% after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.