Mutant p53 prevents GAPDH nuclear translocation in pancreatic cancer cells favoring glycolysis and 2-deoxyglucose sensitivity

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating human malignancies. In about 70% of PDACs the tumor suppressor gene TP53 is mutated generally resulting in conformational changes of mutant p53 (mutp53) proteins, which acquire oncogenic functions triggering aggressiveness of cancers and alteration of energetic metabolism. Here, we demonstrate that mutant p53 prevents the nuclear translocation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) stabilizing its cytoplasmic localization, thus supporting glycolysis of cancer cells and inhibiting cell death mechanisms mediated by nuclear GAPDH. We further show that the prevention of nuclear localization of GAPDH is mediated by both stimulation of AKT and repression of AMPK signaling, and is associated with the formation of the SIRT1:GAPDH complex. By using siRNA-GAPDH or an inhibitor of the enzyme, we functionally demonstrate that the maintenance of GAPDH in the cytosol has a critical impact on the anti-apoptotic and anti-autophagic effects driven by mutp53. Furthermore, the blockage of its mutp53-dependent cytoplasmic stabilization is able to restore the sensitivity of PDAC cells to the treatment with gemcitabine. Finally, our data suggest that mutp53-dependent enhanced glycolysis permits cancer cells to acquire sensitivity to anti-glycolytic drugs, such as 2-deoxyglucose, suggesting a potential personalized therapeutic approach in human cancers carrying mutant TP53 gene

Non-Agonistic Bivalent Antibodies That Promote c-MET Degradation and Inhibit Tumor Growth and Others Specific for Tumor Related c-MET

The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519–561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed β-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies

Alzheimer's disease: an update of the roles of receptors, astrocytes and primary cilia

Lavoro di rilevanza internazionale su rivista con refere

Photoexcited calphostin C selectively destroys nuclear lamin B1 in neoplastic human and rat cells - A novel mechanism of action of a photodynamic tumor therapy agent

Lamin B1, a major component of the nuclear lamina, anchors the nucleus to the cytoskeletal cage, and controls nuclear orientation, chromosome positioning and, alongside several enzymes, fundamental nuclear functions. Exposing polyomavirus-transformed rat pyF111 fibroblasts and human cervical carcinoma (HCC) C4-I cells for 30 min to photoexcited perylenequinone calphostin C, i.e. Cal C(phiE), an established reactive oxygen species (ROS)-generator and protein kinase C (PKC) inhibitor, caused the cells to selectively oxidize and then totally destroy their nuclear lamin B1 by only 60 min after starting the treatment, i.e. when apoptotic caspases' activities had not yet increased. However, while the oxidized lamin B1 was being destroyed, lamins A/C, the lamin A-associated nuclear envelope protein emerin, and the nucleoplasmic protein cyclin E were neither oxidized nor destroyed. The oxidized lamin B was ubiquitinated and demolished in the proteasome probably by an enhanced peptidyl-glutaminase-like activity. Hence, the Cal C(phiE)-induced rapid and selective lamin B1 oxidation and proteasomal destruction ahead of the activation of apoptotic caspases was by itself a most severe molecular lesion impairing vital nuclear functions. Conversely, Cal C directly added to the cells kept in the dark damaged neither nuclear lamin B1 nor cell viability. Thus, our findings reveal a novel cell-damaging mechanism of a photodynamic tumor therapeutic agent

Calphostin C, a remarkable multimodal photodynamic killer of neoplastic cells by selective nuclear lamin B1 destruction and apoptogenesis

Perylenequinones that generate reactive oxygen species (ROS) when illuminated with visible light have been recommended as photodynamic chemotherapeutic agents. One of these is calphostin C (CalC), the action of the photo-activated derivative of which, CalCphiE, has been ascribed to its ability to selectively and irreversibly inhibit protein kinase Cs (PKCs). But recent results of experiments with neoplastic rat fibroblasts and human breast and uterine cervix cancer cells have revealed that the action of CalCphiE involves more than PKC inhibition. Besides suppressing PKC activity, CalCphiE rapidly causes endoplasmic reticulum (ER) stress in breast cancer cells and the selective complete oxidation and proteasomal destruction of the functionally essential nuclear envelope protein lamin B1, in human cervical carcinoma (HCC) cells and neoplastic rat fibroblasts. When these lamin B1-lacking cells are placed in the dark, cytoplasmic membrane-linked PKC activities suddenly rebound and apoptogenesis is initiated as indicated by the immediate release of cytochrome c from mitochondria and later on the activation of caspases. Hence, CalCphiE is a photodynamic cytocidal agent attacking multiple targets in cancer cells and it would be worth determining, even for their best applicative use, whether other perylenequinones also share the so far unexpectedly complex deadly properties of the CalCphiE

Involvement of protein kinase C-zeta at the nuclear membrane level of VP-16-treated human cervical carcinoma C4-I cells

The involvement of protein kinase C-zeta in several protein complexes at the nuclear envelope level of VP-16-treated human cervical carcinoma C4-I cells is demonstrated

Role-Shifting PKC\u3b6 Fosters Its Own Proapoptotic Destruction by Complexing with Bcl10 at the Nuclear Envelope of Human Cervical Carcinoma Cells: A Proteomic and Biochemical Study

Many features of deadly human cervical cancers (HCCs) still require elucidation. Among HCC-derived cell lines, here we used the C4-I one since its quantitative gene expression pattern most closely mimics invasive HCCs, including protein kinase-Czeta (PKCzeta) overexpression. Via proteomic, bioinformatic, and biochemical approaches we identified 31 and 33 proteins co-immunoprecipitating with PKCzeta from nuclear membranes (NMs) of, respectively, untreated or VP-16-exposed C4-I cells. Such proteins belonged to eight functional groups, whose compositions and relative sizes changed with either context.Of the 56 proteins identified, only eight were shared between the two subproteomes, including Bcl10. Surprisingly, proteins known to associate with Bcl10, like Carma1/3 and Malt1,in so-called CBM signalosomes were absent. Notably, in VP-16-treated C4-I cells,PKCzeta\ub7Bcl10 complexes increasingly accrued at NMs, where PKCzeta phosphorylated Bcl10, as PKCzeta also did in vitro and in cell-free systems, both processes being thwarted by interfering RNA (iRNA) PKCz depletion. Caspase-3 wasassociated with PKCzeta\ub7Bcl10 complexes and proteolyzed PKCzeta leading to its inactivation/destruction; both events were prevented by Bcl10 iRNA suppression. Thus,PKCzeta\u2019s molecular interactions and functional roles changed strikingly according to the untreated or apoptogen-treated cells context, and by complexing with Bcl10, PKCzeta surprisingly favored its own demise, which suggests both proteins as HCCs therapeutic targets

Comano\u2019s (Trentino) thermal water interferes with the interleukin-6 production and secretion and with cytokeratin-16 expression by cultured human psoriatic keratinocytes: Further potential mechanisms of its anti-psoriatic action.

Thermal balneotherapy with Comano's spa water (CW; Trentino, Italy) is used for psoriasis and other skin disorders but its mechanisms of action are mostly unknown. Previously, we showed that CW can interfere with the expression and secretion of various VEGF-A isoforms by cultured human psoriatic epidermal keratinocytes. In this study, confluent cultures of IL-6-hypersecreting keratino-cytes isolated from 6 psoriatic patients were exposed for 11-15 days to DMEM, the chemicals of which had been dissolved in either deionised water (DW-DMEM, controls) or CW (CW-DMEM, treated cells). As detected by means of immunocytochemistry, Western immunoblotting, and ELISA assays, the intracellular levels and secretion rates of IL-6 were drastically curtailed in the CW-DMEM-incubated keratinocytes and in their cell-conditioned media. A nearly maximal inhibition of IL-6 release had already been induced by a CW fraction in the DMEM as low as 25%. CW exposure also promptly, intensely, and persistently down-regulated the expression of cytokeratin-16 (CK-16), a marker associated with keratinocyte psoriatic phenotype. Hence, CW balneotherapy may beneficially affect the clinical manifestations of psoriasis via an attenuation of the local deregulation of several cytokines/chemokines, including IL-6 and VEGF-A isoforms, and of a concurrent, abnormal cell differentiation program entailing the expression, amongst other proteins, of CK-16

Comano\u2019s (Trentino) thermal water interferes with tumour necrosis factor-\u3b1 expression and interleukin-8 production and secretion by cultured human psoriatic keratinocytes: Yet other mechanisms of its anti-psoriatic action.

Thermal balneotherapy with Comano's spa water (CW; Trentino, Italy) is beneficial for psoriasis and other skin disorders but its operative mechanisms are largely unknown. Previously, we showed that CW interferes with the production and secretion of IL-6 and various VEGF-A isoforms and with CK-16 expression by cultured human psoriatic keratinocytes. In this study, confluent cultures of epidermal keratinocytes isolated from the lesional areas of 9 psoriatic patients were exposed for 11-13 days to DMEM, whose chemicals had been dissolved in either deionised water (DW-DMEM, controls) or CW (CW-DMEM, treated cells), in order to assess the expression and secretion of TNF-alpha and IL-8 by such cells. The results gained by means of immunocytochemistry, Western immunoblotting (WB), and ELISA assays showed that CW exposure significantly down-regulated the intracellular levels of TNF-alpha, a key inducer of IL-8, IL-6, and other chemokines. However, no assayable TNF-alpha secretion occurred in keratinocyte-conditioned DW- and CW-DMEM samples. Moreover, the intracellular levels and secretion rates of IL-8 were also markedly reduced in the protein extracts and conditioned media of CW-DMEM-incubated keratinocytes. Notably, the most effective inhibition of IL-8 secretion was elicited by a 25% CW fraction in the DMEM. Altogether, our findings indicate that by attenuating at lesional skin sites the deregulated production and secretion of a cascade of several cytokines and chemokines (e.g. TNF- alpha, IL-8, IL-6, and various VEGF-A isoforms), and by offsetting the keratinocytes' abnormal differentiation program entailing CK-16 expression, CW balneotherapy may beneficially influence the clinical manifestations of psoriasis