Here, we report the ability of rfhSP-D to induce apoptosis TNF-/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines. Translocation of NF-B from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed western blot at 48?h. The cell number following the rfhSP-D treatment was Saterinone hydrochloride reduced in the order of Panc-1 (~67%)?>?MiaPaCa-2 (~60%)?>?Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype. (SP-D gene) polymorphisms increase the susceptibility to chronic and infectious lung diseases (8), pneumococcal lung disease (9), emphysema (10), tuberculosis (11, Saterinone hydrochloride 12), Crohns disease, and ulcerative colitis (12). SP-D has been shown to be a potent innate immune molecule at pulmonary as well as extra-pulmonary mucosal surfaces by virtue of its ability to control inflammatory response and helper T cell polarization (3). The first clue came a murine model of allergic hypersensitivity, when therapeutic treatment with a recombinant fragment of human Saterinone hydrochloride SP-D (rfhSP-D) lowered peripheral and pulmonary eosinophilia, in addition to specific IgE levels and Th2 cytokines in the spleen (13, 14). It turned out that rfhSP-D selectively induced apoptosis in sensitized eosinophils derived from allergic patients (15). Using an eosinophilic cell line, AML14.3D10 (a model cell line for leukemia), it was established, proteomics analysis, that apoptosis induction by rfhSP-D involved upregulation of p53 (16, 17). Another crucial study by Pandit et al. (18) revealed that rfhSP-D was able to induce apoptosis in activated human PBMCs, but not in resting, nonactivated PBMCs. These studies, for the first time, raised the possibility that SP-D can have a function of immune surveillance against activated self and perhaps altered self. Recently, human lung adenocarcinoma cells (A549 cell line), when exogenously treated with SP-D, showed suppressed epidermal growth factor (EGF) signaling by reducing the EGF binding to EGFR, which subsequently reduced the cell proliferation, invasion, and migration of cancer cells (19). Here, we set out to examine a possible pro-apoptotic role of SP-D in pancreatic cancer. Pancreatic cancer is the fourth leading cause of cancer-related mortality in the western world (20, 21) and its 5-year survival rate is ~5% (22). The poor prognosis has been attributed to the silent nature of the tumor in early stages, aggressive phenotype, surgical complications, and lack of targeted efficacious therapies (23). In this study, we show that rfhSP-D, composed of 8 Gly-X-Y repeats, homotrimeric neck and carbohydrate recognition domains (CRDs) (1), induces cell growth arrest in G1 phase and subsequent apoptosis in human pancreatic adenocarcinoma cells using Panc-1, MiaPaCa-2, and Capan-2 cell lines. The apoptosis induction appears to involve TNF-, NF-B, and Fas axis, revealing a p53 independent route of apoptosis induction in the p53 mutated Panc-1 and MiaPaCa-2 cell lines and p53-dependent apoptosis in p53 wild type Capan-2 cell line by rfhSP-D. Materials and Methods Cell Culture and Treatments Saterinone hydrochloride Human pancreatic cancer cells lines, Panc-1 (CRL-1469), MiaPaCa-2 (CRL-1420), and Capan-2 (HTB-80), were obtained from ATCC and used as an model in this study. All cell lines were cultured at 37C under 5% v/v CO2 using DMEM-F12 media (Thermo Fisher) containing 10% v/v fetal calf serum with 2?mM l-glutamine, and penicillin (100?U/ml)/streptomycin (100?g/ml) (Thermo Fisher) until 80C90% confluency was reached. Expression and Purification of rfhSP-D Plasmid pUK-D1 (containing cDNA sequences for 8 Gly-X-Y repeats, neck, and CRD region of human SP-D), Saterinone hydrochloride transformed into BL21 (DE3) pLysS (Invitrogen), was used to express rfhSP-D, as described earlier (15, 16). The expression cassette included a short stretch of eight N-terminal Mouse monoclonal to Fibulin 5 GlyCXCY triplets with substitution of S for P in position 2 (residue 180), followed by the -helical coiled-coil neck region (residues 203C235) and the globular CRD region.
Here, we report the ability of rfhSP-D to induce apoptosis TNF-/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines
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a 67 kDa type I transmembrane glycoprotein present on myeloid progenitors
and differentiation. The protein kinase family is one of the largest families of proteins in eukaryotes
Apoptosis
bladder
brain
breast
cell cycle progression
cervix
CSP-B
Cyproterone acetate
EGFR) is the prototype member of the type 1 receptor tyrosine kinases. EGFR overexpression in tumors indicates poor prognosis and is observed in tumors of the head and neck
EM9
endometrium
erythrocytes
F3
Goat polyclonal to IgG H+L)
Goat polyclonal to IgG H+L)Biotin)
GRK4
GSK1904529A
Igf1
Mapkap1
monocytes andgranulocytes. CD33 is absent on lymphocytes
Mouse monoclonal to CD33.CT65 reacts with CD33 andtigen
Palomid 529
platelets
PTK) or serine/threonine
Rabbit Polyclonal to ARNT.
Rabbit polyclonal to BMPR2
Rabbit Polyclonal to CCBP2.
Rabbit Polyclonal to EDG4
Rabbit polyclonal to EIF4E.
Rabbit polyclonal to IL11RA
Rabbit polyclonal to LRRIQ3
Rabbit Polyclonal to MCM3 phospho-Thr722)
Rabbit Polyclonal to RBM34
SB 216763
SKI-606
SNX-5422
STK) kinase catalytic domains. Epidermal Growth factor receptor
stomach
stomach and in squamous cell carcinoma.
TNFSF8
TSHR
VEGFA
vulva