Antifibrogenic effects of vitamin D derivatives on mouse pancreatic stellate cells

AIM To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells(PSCs).METHODS Quiescent PSCs were isolated from mouse pancreas and activated in vitro by seeding on plastic surfaces. The cells were exposed to D-vitamins as primary cultures(early-activated PSCs) and upon re-culturing(fullyactivated cells). Exhibition of vitamin A-containing lipid droplets was visualized by oil-red staining. Expression of α-smooth muscle actin(α-SMA), a marker of PSC activation, was monitored by immunofluorescence and immunoblot analysis. The rate of DNA synthesis was quantified by 5-bromo-2'-deoxyuridine(Brd U) incorporation assays. Real-time PCR was employed to monitor gene expression, and protein levels of interleukin-6(IL-6) were measured by ELISA. Uptake of proline was determined using 18 F-proline.RESULTS Sustained culture of originally quiescent PSCs inducedcell proliferation, loss of lipid droplets and exhibition of stress fibers, indicating cell activation. When added to PSCs in primary culture, all three D-vitamins diminished expression of α-SMA(to 32%-39% of the level of control cells; P < 0.05) and increased the storage of lipids(scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; P < 0.05). No such effects were observed when D-vitamins were added to fully-activated cells, while incorporation of Brd U remained unaffected under both experimental conditions. Treatment of re-cultured PSCs with D-vitamins was associated with lower expression of IL-6(-42% to-49%; P < 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene(209%-321% vs controls; P < 0.05). There was no effect of D-vitamins on the expression of transforming growth factor-β1 and collagen type 1(chain α1). The lowest uptake of proline, a main component of collagen, was observed in calcipotriol-treated PSCs.CONCLUSION The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.

Distinct antifibrogenic effects of erlotinib,sunitinib and sorafenib on rat pancreatic stellate cells

AIM: To study if three clinically available small molecule kinase inhibitors (SMI), erlotinib, sunitinib and sorafenib, exert antifibrogenic effects on pancreatic stellate cells (PSC) and analyze the basis of their action.

Insights into erlotinib action in pancreatic cancer cells using a combined experimental and mathematical approach

AIM:To gain insights into the molecular action of erlotinib in pancreatic cancer (PC) cells. METHODS:Two PC cell lines, BxPC-3 and Capan-1, were treated with various concentrations of erlotinib, the specific mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and protein kinase B (AKT) inhibitor XIV. DNA synthesis was measured by 5-bromo-2'-deoxyuridine (BrdU) assays. Expression and phosphorylation of the epidermal growth factor receptor (EGFR) and downstream signaling molecules were quantified by Western blot analysis. The data were processed to calibrate a mathematical model, based on ordinary differential equations, describing the EGFRmediated signal transduction. RESULTS:Erlotinib significantly inhibited BrdU incorporation in BxPC-3 cells at a concentration of 1 mol/L, whereas Capan-1 cells were much more resistant. In both cell lines, MEK inhibitor U0126 and erlotinib attenuated DNA synthesis in a cumulative manner, whereas the AKT pathway-specific inhibitor did not enhance the effects of erlotinib. While basal phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) did not differ much between the two cell lines, BxPC-3 cells displayed a more than five-times higher basal phospho-AKT level than Capan-1 cells. Epidermal growth factor (EGF) at 10 ng/mL induced the phosphorylation of EGFR, AKT and ERK in both cell lines with similar kinetics. In BxPC-3 cells, higher levels of phospho-AKT and phospho-ERK (normalized to the total protein levels) were observed. Independent of the cell line, erlotinib efficiently inhibited phosphorylation of EGFR, AKT and ERK. The mathematical model successfully simulated the experimental findings and provided predictions regarding phosphoprotein levels that could be verified experimentally. CONCLUSION:Our data suggest basal AKT phosphorylation and the degree of EGF-induced activation of AKT and ERK as molecular determinants of erlotinib efficiency in PC cells.

Establishment,functional and genetic characterization of three novel patient-derived rectal cancer cell lines

AIM To establish patient-individual tumor models of rectal cancer for analyses of novel biomarkers, individual response prediction and individual therapy regimens.METHODS Establishment of cell lines was conducted by direct in vitro culturing and in vivo xenografting with subsequent in vitro culturing. Cell lines were in-depth characterized concerning morphological features, invasive and migratory behavior, phenotype, molecular profile including mutational analysis, protein expression, and confirmation of origin by DNA fingerprint. Assessment of chemosensitivity towards an extensive range of current chemotherapeutic drugs and of radiosensitivity was performed including analysis of a combined radioand chemotherapeutic treatment. In addition, glucose metabolism was assessed with 18 F-fluorodeoxyglucose(FDG) and proliferation with 18 F-fluorothymidine.RESULTS We describe the establishment of ultra-low passage rectal cancer cell lines of three patients suffering from rectal cancer. Two cell lines(HROC126, HROC284 Met) were established directly from tumor specimens while HROC239 T0 M1 was established subsequent to xenografting of the tumor. Molecular analysis classified all three cell lines as CIMP-0/non-MSI-H(sporadic standard) type. Mutational analysis revealed following mutational profiles: HROC126: APC^(wt), TP53^(wt), KRAS^(wt), BRAF^(wt), PTEN^(wt); HROC239 T0 M1: APC^(mut), P53^(wt), KRAS^(mut), BRAF^(wt), PTEN^(mut) and HROC284 Met: APC^(wt), P53^(mut), KRAS^(mut), BRAF^(wt), PTEN^(mut). All cell lines could be characterized as epithelial(EpCAM+) tumor cells with equivalent morphologic features and comparable growth kinetics. The cell lines displayed a heterogeneous response toward chemotherapy, radiotherapy and their combined application. HROC126 showed a highly radio-resistant phenotype and HROC284 Met was more susceptible to a combined radiochemotherapy than HROC126 and HROC239 T0 M1. Analysis of 18 F-FDG uptake displayed a markedly reduced FDG uptake of all three cell lines after combined radiochemotherapy. CONCLUSION These newly established and in-depth characterized ultra-low passage rectal cancer cell lines provide a useful instrument for analysis of biological characteristics of rectal cancer.