Glucose restriction induces cell death in parental but not in homeodomain-interacting protein kinase 2-depleted RKO colon cancer cells: molecular mechanisms and implications for tumor therapy.

Tumor cell tolerance to nutrient deprivation can be an important factor for tumor progression, and may depend on deregulation of both oncogenes and oncosuppressor proteins. Homeodomain-interacting protein kinase 2 (HIPK2) is an oncosuppressor that, following its activation by several cellular stress, induces cancer cell death via p53-dependent or -independent pathways. Here, we used genetically matched human RKO colon cancer cells harboring wt-HIPK2 (HIPK2(+/+)) or stable HIPK2 siRNA interference (siHIPK2) to investigate in vitro whether HIPK2 influenced cell death in glucose restriction. We found that glucose starvation induced cell death, mainly due to c-Jun NH2-terminal kinase activation, in HIPK2(+/+)cells compared with siHIPK2 cells that did not die. (1)H-nuclear magnetic resonance quantitative metabolic analyses showed a marked glycolytic activation in siHIPK2 cells. However, treatment with glycolysis inhibitor 2-deoxy-D-glucose induced cell death only in HIPK2(+/+) cells but not in siHIPK2 cells. Similarly, siGlut-1 interference did not re-establish siHIPK2 cell death under glucose restriction, whereas marked cell death was reached only after zinc supplementation, a condition known to reactivate misfolded p53 and inhibit the pseudohypoxic phenotype in this setting. Further siHIPK2 cell death was reached with zinc in combination with autophagy inhibitor. We propose that the metabolic changes acquired by cells after HIPK2 silencing may contribute to induce resistance to cell death in glucose restriction condition, and therefore be directly relevant for tumor progression. Moreover, elimination of such a tolerance might serve as a new strategy for cancer therapy

P62/sqstm1/keap1/nrf2 axis reduces cancer cells death-sensitivity in response to zn(Ii)–curcumin complex

The hyperactivation of nuclear factor erythroid 2 p45-related factor 2 (NRF2), frequently found in many tumor types, can be responsible for cancer resistance to therapies and poor patient prognosis. Curcumin has been shown to activate NRF2 that has cytotprotective or protumorigenic roles according to tumor stage. The present study aimed at investigating whether the zinc–curcumin Zn(II)–curc compound, which we previously showed to display anticancer effects through multiple mechanisms, could induce NRF2 activation and to explore the underlying molecular mechanisms. Biochemical studies showed that Zn(II)–curc treatment increased the NRF2 protein levels along with its targets, heme oxygenase-1 (HO-1) and p62/SQSTM1, while markedly reduced the levels of Keap1 (Kelch-like ECH-associated protein 1), the NRF2 inhibitor, in the cancer cell lines analyzed. The silencing of either NRF2 or p62/SQSTM1 with specific siRNA demonstrated the crosstalk between the two molecules and that the knockdown of either molecule increased the cancer cell sensitivity to Zn(II)–curc-induced cell death. This suggests that the crosstalk between p62/SQSTM1 and NRF2 could be therapeutically exploited to increase cancer patient response to therapies

Targeting COX-2/PGE(2) pathway in HIPK2 knockdown cancer cells: impact on dendritic cell maturation.

production after HIPK2 depletion and how to modulate it./VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation./VEGF production; and 3) zinc treatment of HIPK2 depleted cells restored DCs maturation

Hipk2 cooperates with p53 to suppress γ-ray radiation-induced mouse thymic lymphoma

A genome-wide screen for genetic alterations in radiation-induced thymic lymphomas generated from p53+/− and p53−/− mice showed frequent loss of heterozygosity (LOH) on chromosome 6. Fine mapping of these LOH regions revealed three non-overlapping regions, one of which was refined to a 0.2 Mb interval that contained only the gene encoding homeobox-interacting protein kinase 2 (Hipk2). More than 30% of radiation-induced tumors from both p53+/− and p53−/− mice showed heterozygous loss of one Hipk2 allele. Mice carrying a single inactive allele of Hipk2 in the germline were susceptible to induction of tumors by γ-radiation, but most tumors retained and expressed the wild-type allele, suggesting that Hipk2 is a haploinsufficient tumor suppressor gene for mouse lymphoma development. Heterozygous loss of both Hipk2 and p53 confers strong sensitization to radiation-induced lymphoma. We conclude that Hipk2 is a haploinsufficient lymphoma suppressor gene

Divergent Modulation of Neuronal Differentiation by Caspase-2 and -9

Human Ntera2/cl.D1 (NT2) cells treated with retinoic acid (RA) differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2) or -9 (si-Casp9) was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM), microtubule associated protein-2 (MAP2) and tyrosine hydroxylase (TH) mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved) in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells

Targeting Hypoxia in Cancer Cells by Restoring Homeodomain Interacting Protein-Kinase 2 and p53 Activity and Suppressing HIF-1α

BACKGROUND:The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) by phosphorylating serine 46 (Ser46) is a crucial regulator of p53 apoptotic function. HIPK2 is also a transcriptional co-repressor of hypoxia-inducible factor-1alpha (HIF-1alpha) restraining tumor angiogenesis and chemoresistance. HIPK2 can be deregulated in tumors by several mechanisms including hypoxia. Here, we sought to target hypoxia by restoring HIPK2 function and suppressing HIF-1alpha, in order to provide evidence for the involvement of both HIPK2 and p53 in counteracting hypoxia-induced chemoresistance. METHODOLOGY/PRINCIPAL FINDINGS:Upon exposure of colon and lung cancer cells to hypoxia, by either low oxygen or cobalt, HIPK2 function was impaired allowing for increased HIF-1alpha expression and inhibiting the p53-apoptotic response to drug. Cobalt suppressed HIPK2 recruitment onto HIF-1alpha promoter. Hypoxia induced expression of the p53 target MDM2 that downregulates HIPK2, thus MDM2 inhibition by siRNA restored the HIPK2/p53Ser46 response to drug. Zinc supplementation to hypoxia-treated cells increased HIPK2 protein stability and nuclear accumulation, leading to restoration of HIPK2 binding to HIF-1alpha promoter, repression of MDR1, Bcl2, and VEGF genes, and activation of the p53 apoptotic response to drug. Combination of zinc and ADR strongly suppressed tumor growth in vivo by inhibiting HIF-1 pathway and upregulating p53 apoptotic target genes. CONCLUSIONS/SIGNIFICANCE:We show here for the first time that hypoxia-induced HIPK2 deregulation was counteracted by zinc that restored HIPK2 suppression of HIF-1 pathway and reactivated p53 apoptotic response to drug, underscoring the potential use of zinc supplementation in combination with chemotherapy to address hypoxia and improve tumor treatment

p53-Dependent PUMA to DRAM antagonistic interplay as a key molecular switch in cell-fate decision in normal/high glucose conditions

BACKGROUND: As an important cellular stress sensor phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The p53 activity mainly depends on its transactivating function, however, how p53 can select one or another biological outcome is still a matter of profound studies. Our previous findings indicate that switching cancer cells in high glucose (HG) impairs p53 apoptotic function and the transcription of target gene PUMA. METHODS AND RESULTS: Here we report that, in response to drug adriamycin (ADR) in HG, p53 efficiently induced the expression of DRAM (damage-regulated autophagy modulator), a p53 target gene and a stress-induced regulator of autophagy. We found that ADR treatment of cancer cells in HG increased autophagy, as displayed by greater LC3II accumulation and p62 degradation compared to ADR-treated cells in low glucose. The increased autophagy in HG was in part dependent on p53-induced DRAM; indeed DRAM knockdown with specific siRNA reversed the expression of the autophagic markers in HG. A similar outcome was achieved by inhibiting p53 transcriptional activity with pifithrin-α. DRAM knockdown restored the ADR-induced cell death in HG to the levels obtained in low glucose. A similar outcome was achieved by inhibition of autophagy with cloroquine (CQ) or with silencing of autophagy gene ATG5. DRAM knockdown or inhibition of autophagy were both able to re-induce PUMA transcription in response to ADR, underlining a reciprocal interplay between PUMA to DRAM to unbalance p53 apoptotic activity in HG. Xenograft tumors transplanted in normoglycemic mice displayed growth delay after ADR treatment compared to those transplanted in diabetics mice and such different in vivo response correlated with PUMA to DRAM gene expression. CONCLUSIONS: Altogether, these findings suggest that in normal/high glucose condition a mutual unbalance between p53-dependent apoptosis (PUMA) and autophagy (DRAM) gene occurred, modifying the ADR-induced cancer cell death in HG both in vitro and in vivo

TERAPIE AVANZATE: LE SPERANZE DEL DOMANI

La mappatura del genoma umano e lo sviluppo di tecnologie per il sequenziamento dei geni hanno portato rapidamente ad una maggiore comprensione delle basi genetiche di molte malattie. I notevoli progressi tecnologici nel campo delle manipolazioni in vitro di cellule (isolamento, coltivazione, espansione, diffe-renziamento) e del trasferimento di materiale genetico da una cellula ad un’altra hanno consentito di sviluppare tecniche di ingegneria cellulare tramite le quali manipolare sia cellule staminali che cellule ad uno stadio di differenziamento terminale. Queste metodiche, applicate a cellule umane, lasciano presagire nuove opzioni terapeutiche per un’ampia gamma di patologie, trasformando e talora mettendo in discussione la nozione convenzionale di “medicina”. Il termine “terapie avanzate” fa riferimento a nuovi prodotti medicinali (Advanced Therapeutic Medicinal Products/ATMPs) che utilizzano geni (terapia genica), cellule (terapia cellulare) e tessuti (ingegneria tissutale) quali preparazioni farmaceutiche a scopo terapeutico o preventivo (ad esempio, se un difetto genetico evidenziato dall’analisi molecolare viene corretto prima che la patologia si manifesti in modo conclamato). Le distinzioni fra le categorie sono sottili ed occasionalmente alcuni prodotti potrebbero rientrare in più di una categoria. La produzione e sperimentazione di questi prodotti è strettamente regolata a livello internazionale. Tale regolamentazione impone criteri specifici atti a garantire in primo luogo la sicurezza del prodotto finale, poiché destinato alla somministrazione a pazienti, ma non secondariamente anche l’efficacia, poiché non è ritenuto etico sottoporre dei pazienti ad un trattamento che non abbia dato sufficiente prova di efficacia terapeutica

A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells

Nickel, a known occupational/environmental hazard, may cross the placenta and reach appreciable concentrations in various fetal organs, including the brain. The aim of this study was to investigate whether nickel interferes with the process of neuronal differentiation. Following a 4 week treatment with retinoic acid (10 μM), the human teratocarcinoma-derived NTera2/D1 cell line (NT2 cells) terminally differentiate into neurons which recapitulate many features of human fetal neurons. The continuous exposure of the differentiating NT2 cells to a not cytotoxic nickel concentration (10 μM) increased the expression of specific neuronal differentiation markers such as Neural Cell Adhesion Molecule (NCAM) and Microtubule Associated Protein 2 (MAP2). Furthermore, nickel exposure increased the expression of Hypoxia-Inducible-Factor-1α (HIF-1α) and induced the activation of the AKT/PKB kinase pathway, as shown by the increase of P(Ser-9)-GSK-3β, the inactive form of glycogen synthase kinase-3β (GSK-3β). Intriguingly, by the end of the fourth week the expression of tyrosine hydroxylase (TH) protein, a marker of dopaminergic neurons, was lower in nickel-treated than in control cultures. Thus, likely by partially mimicking hypoxic conditions, a not-cytotoxic nickel concentration appears to alter the process of neuronal differentiation and hinder the expression of the dopaminergic neuronal phenotype. Taken together, these results suggest that nickel, by altering normal brain development, may increase susceptibility to neuro-psychopathology later in life