EUS-guided verteporfin photodynamic therapy for pancreatic cancer

BACKGROUND AND AIMS: Locally advanced pancreatic cancer (LAPC) often causes obstruction. Verteporfin photodynamic therapy (PDT) can feasibly "debulk" tumor more safely than noncurative surgery and has multiple advantages over older PDT agents. We aimed to assess the feasibility of EUS-guided verteporfin PDT in ablating nonresectable LAPC. METHODS: Adults with LAPC with adequate biliary drainage were prospectively enrolled. Exclusion criteria included significant metastatic disease burden, disease involving >50% duodenal or major artery circumference, and recent treatment with curative intent. CT was obtained between day -28 to 0. On day 0, verteporfin 0.4 mg/kg was infused 60 to 90 minutes before EUS, during which a diffuser was positioned in the tumor and delivered light at 50 J/cm for 333 seconds. CT was obtained on day 2, with adverse event monitoring occurring on days 1, 2, and 14. Primary outcome was presence of necrosis. RESULTS: Of 8 patients (62.5% male, mean age 65±7.9 y) included in the study, 5 were staged at T3, 2 at T2, and 1 at T1. Most (4) had primary lesions in the pancreatic head. Mean pretrial tumor diameter was 33.3±13.4 mm. On day 2 CT, 5 lesions demonstrated a zone of necrosis measuring a mean diameter of 15.7±5.5 mm; 3 cases did not develop necrosis. No adverse events were noted during the procedure or postprocedure observation period (day 1-3), and no changes in patient reported outcomes were noted. CONCLUSIONS: In this pilot study, EUS-guided verteporfin PDT is feasible and shows promise as a minimally invasive ablative therapy for LAPC in select patients. Tumor necrosis is visible within 48 hours after treatment. Patient enrollment and data collection are ongoing

Expression profile of genes regulated by activity of the Na-H exchanger NHE1

BACKGROUND: In mammalian cells changes in intracellular pH (pH(i)), which are predominantly controlled by activity of plasma membrane ion exchangers, regulate a diverse range of normal and pathological cellular processes. How changes in pH(i )affect distinct cellular processes has primarily been determined by evaluating protein activities and we know little about how pH(i )regulates gene expression. RESULTS: A global profile of genes regulated in mammalian fibroblasts by decreased pH(i )induced by impaired activity of the plasma membrane Na-H exchanger NHE1 was characterized by using cDNA microarrays. Analysis of selected genes by quantitative RT-PCR, TaqMan, and immunoblot analyses confirmed results obtained from cDNA arrays. Consistent with established roles of pH(i )and NHE1 activity in cell proliferation and oncogenic transformation, grouping regulated genes into functional categories and biological pathways indicated a predominant number of genes with altered expression were associated with growth factor signaling, oncogenesis, and cell cycle progression. CONCLUSION: A comprehensive analysis of genes selectively regulated by pH(i )provides insight on candidate targets that might mediate established effects of pH(i )on a number of normal and pathological cell functions

Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells

Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) to drive production of an intracellular photosensitiser, protoporphyrin IX (PpIX), is a promising cancer treatment. However, ALA-PDT is still suboptimal for thick or refractory tumours. Searching for new approaches, we tested a known inducer of cellular differentiation, methotrexate (MTX), in combination with ALA-PDT in LNCaP cells. Methotrexate alone promoted growth arrest, differentiation, and apoptosis. Methotrexate pretreatment (1 mg l−1, 72 h) followed by ALA (0.3 mM, 4 h) resulted in a three-fold increase in intracellular PpIX, by biochemical and confocal analyses. After exposure to 512 nm light, killing was significantly enhanced in MTX-preconditioned cells. The reverse order of treatments, ALA-PDT followed by MTX, yielded no enhancement. Methotrexate caused a similar relative increase in PpIX, whether cells were incubated with ALA, methyl-ALA, or hexyl-ALA, arguing against a major effect upon ALA transport. Searching for an effect among porphyrin synthetic enzymes, we found that coproporphyrinogen oxidase (CPO) was increased three-fold by MTX at the mRNA and protein levels. Transfection of LNCaP cells with a CPO-expressing vector stimulated the accumulation of PpIX. Our data suggest that MTX, when used to modulate intracellular production of endogenous PpIX, may provide a new combination PDT approach for certain cancers

siRNA Silencing of Proteasome Maturation Protein (POMP) Activates the Unfolded Protein Response and Constitutes a Model for KLICK Genodermatosis

Keratosis linearis with ichthyosis congenita and keratoderma (KLICK) is an autosomal recessive skin disorder associated with a single-nucleotide deletion in the 5′untranslated region of the proteasome maturation protein (POMP) gene. The deletion causes a relative switch in transcription start sites for POMP, predicted to decrease levels of POMP protein in terminally differentiated keratinocytes. To investigate the pathophysiology behind KLICK we created an in vitro model of the disease using siRNA silencing of POMP in epidermal air-liquid cultures. Immunohistochemical analysis of the tissue constructs revealed aberrant staining of POMP, proteasome subunits and the skin differentiation marker filaggrin when compared to control tissue constructs. The staining patterns of POMP siRNA tissue constructs showed strong resemblance to those observed in skin biopsies from KLICK patients. Western blot analysis of lysates from the organotypic tissue constructs revealed an aberrant processing of profilaggrin to filaggrin in samples transfected with siRNA against POMP. Knock-down of POMP expression in regular cell cultures resulted in decreased amounts of proteasome subunits. Prolonged silencing of POMP in cultured cells induced C/EBP homologous protein (CHOP) expression consistent with an activation of the unfolded protein response and increased endoplasmic reticulum (ER) stress. The combined results indicate that KLICK is caused by reduced levels of POMP, leading to proteasome insufficiency in differentiating keratinocytes. Proteasome insufficiency disturbs terminal epidermal differentiation, presumably by increased ER stress, and leads to perturbed processing of profilaggrin. Our findings underline a critical role for the proteasome in human epidermal differentiation

CHOP Potentially Co-Operates with FOXO3a in Neuronal Cells to Regulate PUMA and BIM Expression in Response to ER Stress

Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in various neurodegenerative diseases including Parkinson Disease, Alzheimer Disease and Huntington Disease. PUMA (p53 upregulated modulator of apoptosis) and BIM (BCL2 interacting mediator of cell death), pro-apoptotic BH3 domain-only, BCL2 family members, have previously been shown to regulate ER stress-induced cell death, but the upstream signaling pathways that regulate this response in neuronal cells are incompletely defined. Consistent with previous studies, we show that both PUMA and BIM are induced in response to ER stress in neuronal cells and that transcriptional induction of PUMA regulates ER stress-induced cell death, independent of p53. CHOP (C/EBP homologous protein also known as GADD153; gene name Ddit3), a critical initiator of ER stress-induced apoptosis, was found to regulate both PUMA and BIM expression in response to ER stress. We further show that CHOP knockdown prevents perturbations in the AKT (protein kinase B)/FOXO3a (forkhead box, class O, 3a) pathway in response to ER stress. CHOP co-immunoprecipitated with FOXO3a in tunicamycin treated cells, suggesting that CHOP may also regulate other pro-apoptotic signaling cascades culminating in PUMA and BIM activation and cell death. In summary, CHOP regulates the expression of multiple pro-apoptotic BH3-only molecules through multiple mechanisms, making CHOP an important therapeutic target relevant to a number of neurodegenerative conditions

Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death

Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF

Distinct Cytoplasmic and Nuclear Functions of the Stress Induced Protein DDIT3/CHOP/GADD153

DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders

Free Cysteine Modulates the Conformation of Human C/EBP Homologous Protein

The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid l-cysteine. Addition of l-cysteine caused a significant dose-dependent decrease in the protein helicity – dropping from 69.1% to 23.8% in the presence of 1 mM of l-cysteine – and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 µM, an 8∶1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free l-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of l-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress