Regulation of p38 MAPK and glucocorticoid receptor activation by hydrocortisone in mono-and co-cultured pancreatic acinar and stellate cells.

BACKGROUND/OBJECTIVES: Acute pancreatitis develops as an inflammatory response to pancreatic tissue injury. Postoperative pancreatitis has recently been associated with increased occurrence of complications. Activation of the mitogen-activated protein kinase p38 (p38 MAPK) pathway occurs early in acute pancreatitis and its inhibition has been suggested to alleviate pancreatic inflammation. Glucocorticoids are potent anti-inflammatory steroids whose use in the management of acute pancreatitis remains controversial. Our aim was to examine the effect of crosstalk between pancreatic acinar cells (PACs) and stellate cells (PSCs) on p38 MAPK and glucocorticoid receptor (GR) activation and to assess the impact of hydrocortisone on these events. METHODS: The long-term co-culture setting for mouse PACs and PSCs developed in our laboratory was used. Parallel 4d mono- and co-cultures with or without 10 nM hydrocortisone were performed followed by immunocytochemical analysis of nuclear GR and phospho-p38 MAPK (pp38 MAPK). RESULTS: Hydrocortisone inhibited pp38 MAPK up-regulation evoked by co-culture in PACs and PSCs and increased nuclear translocation of GR in PAC monocultures and in co-cultured PACs and PSCs. In PSC monocultures and co-cultured PACs, ligand-independent expression of nuclear GR was observed. In the former no change in nuclear GR but a significant decrease in total GR as analyzed by Western blot was caused by hydrocortisone. CONCLUSIONS: Cellular microenvironment plays a significant role on p38 MAPK and GR activation in PACs and PSCs. Hydrocortisone is an effective means to inhibit p38 MAPK activation in PACs and PSCs. Both ligand-dependent and -independent regulatory roles for GR are suggested in the exocrine pancreas.

Wnt/ß-catenin signalling plays diverse functions during the process of fibrotic remodelling in the exocrine pancreas.

BACKGROUND/OBJECTIVES: Wnt/ß-catenin signalling plays vital roles in tissue homeostasis. Dysregulation of the pathway has been implicated in the pathogenesis of cancer and fibroses in numerous tissues, including the pancreas. We studied the effect of microenvironmental changes pertaining to fibrotic tissue remodelling on the expression of selected Wnt/ß-catenin pathway proteins in the human exocrine pancreas. The role of acinar/stellate cross-talk on the expression of the proteins was elucidated in a long-term mouse co-culture system. METHODS: Expression of ß-catenin, Wnt2, Wnt5a and SFRP4 was analysed immunohistochemically in normal and moderately or highly fibrotic human pancreata (n = 8). The effect of humoral interactions on the expression of the proteins was studied by immunocytochemical means in parallel mono- and co-cultures of mouse acinar and stellate cells (PSCs). RESULTS: In human pancreatic tissue, fibrotic microenvironment was associated with redistribution of the proteins in and between epithelial and stromal compartments, compared to acinar-rich tissue. In non-fibrotic and moderately fibrotic tissue the proteins appeared only in acinar cells whereas in highly fibrotic tissue stromal fibroblastoid/stellate cells and macrophages were their predominant locations. Subcellular changes in the expression of ß-catenin and Wnt5a were detected. Our in vitro data suggest potential involvement of acinar cell/PSC cross-talk in mediating the changes observed in tissue specimens. CONCLUSIONS: Wnt/ß-catenin pathway-associated proteins are abundantly expressed in the exocrine pancreas with prominent changes in their cellular and subcellular expression patterns along with increasing levels of fibrosis. Diverse functions for Wnt/ß-catenin signalling during the course of fibrotic remodelling in the exocrine pancreas are suggested.

Additive inhibitory effects of simvastatin and enzalutamide on androgen-sensitive LNCaP and VCaP prostate cancer cells.

We evaluated the effects of simvastatin and antiandrogen enzalutamide on growth and androgen signaling in androgen-sensitive LNCaP and VCaP prostate cancer cells. Simvastatin alone abolished androgen-induced growth in both cell lines but decreased androgen receptor (AR) and prostate-specific antigen protein expression only in LNCaP, indicating that statin-induced growth inhibition is beyond AR transcriptional activity in VCaP. Combination of simvastatin and enzalutamide exerted additive growth inhibition in both cell lines accompanied with strong induction of autophagy in LNCaP. The data provide new insight into statins' effects on androgen signaling and their proposed role in enhancing androgen deprivation therapy in prostate cancer.

Reciprocal stimulation of pancreatic acinar and stellate cells in a novel long-term in vitro co-culture model.

BACKGROUND/OBJECTIVES: Pancreatic stellate cells (PSCs) are the key fibrogenic cells in the pancreas. Acinar cell injury is known to trigger PSC activation. To facilitate the experimental analysis of the crosstalk between acinar cells and PSCs, an in vitro system for their long-term co-cultivation was developed. MATERIALS AND METHODS: PSCs and acinar cells capable of retaining their secretory phenotype in long-term in vitro culture were obtained from mouse pancreata. A dual-chamber co-culture model was built in 24-well format with acinar cells seeded in the wells and PSCs in tissue culture inserts. Acinar cell-3T3 fibroblast co-cultures served as controls. After 4-day maintenance, the acinar compartment was analyzed for cell morphology, secretory capability, necrosis (HMGB1), apoptosis (TUNEL) and inflammation (NFκB). PSCs were analyzed for migratory activity and extracellular matrix (ECM) protein expression. The results were compared to parallel monocultures. RESULTS: Acinar cells in monoculture and in co-culture with fibroblasts exhibited a healthy monolayer arrangement and an ability to respond to 0.1 nM caerulein stimulus by increased amylase release. Co-culture with PSCs caused marked changes in acinar cell morphology and rendered them insensitive to secretagogue stimulus. Activation of NFκB and necrotic changes, but not apoptosis, were identified in co-cultured acinar cells. Co-culture increased the migratory activity and ECM protein expression of PSCs. CONCLUSIONS: Humoral interactions between acinar and PSCs in co-culture were shown to reciprocally affect their cellular functions. With its two separable cell compartments the co-culture system provides a versatile culture setting that allows independent manipulation and analysis of both cell types.

Physiological and clinically attainable concentrations of 1,25-dihydroxyvitamin D3 suppress proliferation and extracellular matrix protein expression in mouse pancreatic stellate cells.

BACKGROUND/OBJECTIVES: Vitamin D is an antiproliferative and differentiation-promoting secosteroid hormone with pleiotropic homeostatic functions in bone and extraskeletal tissues. Signaling of vitamin D is mediated via its ubiquitously expressed nuclear receptor, the vitamin D receptor (VDR). Pancreatic stellate cells have recently been identified as targets of vitamin D action. Our aim was to elucidate the effectiveness of the most potent endogenous vitamin D metabolite, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the proliferation and extracellular matrix (ECM) protein expression in pancreatic stellate cells (PSCs) using concentrations of the compound from the physiological and clinically attainable range in humans. METHODS: Culture-activated mouse PSCs were exposed to 1,25(OH)2D3 concentrations ranging from 0.1 nM to 10 nM for 7 days and subjected to colorimetric crystal violet assay for cell growth assessment and to Western blot and immunohistochemical analyses of VDR, fibronectin and collagen I using protein-specific antibodies. Immunohistochemical localization of VDR was performed on mouse pancreatic tissue and on a set of human specimens obtained at pancreatic surgery. RESULTS: A low basal level of VDR was detected in PSCs that was strongly induced in the presence of ligand. Cell growth was suppressed dose-dependently by 1,25(OH)2D3, the mean percentages of inhibition ranging from 24% at the physiological 0.1 nM concentration to around 60% at 10 nM. Significant 48% and 40% reductions in fibronectin expression were seen at 0.5 nM and 1 nM 1,25(OH)2D3. A minor decrease in collagen I expression was detected at 5 nM. VDR was predominantly localized in the islets of Langerhans in mouse and human tissues. In the latter VDR was expressed also in the exocrine tissue showing individual variation in its cellular distribution. CONCLUSIONS: Mouse PSCs express VDR protein and are sensitive 1,25(OH)2D3 target cells with low levels of 1,25(OH)2D3 exerting antiproliferative and antifibrotic effects on activated PSCs in vitro.

Cryopreserved mouse pancreatic acinar cells from long-term explant outgrowth cultures maintain their secretory phenotype after thawing.

BACKGROUND/OBJECTIVES: We recently reported an explant outgrowth culture method for obtaining functionally competent mouse pancreatic acinar cells for long-term in vitro purposes. The aim of the present study was to explore the possibility of cryostoring these cells without loss of functional differentiation. METHODS: Acinar cells prepared by the explant outgrowth method were cryopreserved using a DMSO-based protocol and stored in liquid nitrogen for 4 weeks. The following characteristics were compared in cryopreserved and parallel non-frozen cell preparations: cell viability and recovery, amylase content in viable cells before culture, basal and stimulated amylase release in culture and the ability of the cells to form glandular structures in Matrigel. RESULTS: Immediate post-thaw viability of the cells was similar to that of freshly isolated cells. Approximately 53% of viable cells frozen were recovered after thawing. Intracellular amylase content was identical in frozen and non-frozen cells. Cryopreserved cells maintained their ability to secrete amylase and to respond to caerulein stimulation in 4-day secondary cultures. They also were observed to form amylase-expressing glandular structures in three-dimensional cultures in Matrigel in a similar manner as non-frozen cells. CONCLUSIONS: This study shows that pancreatic acinar cells can be cryopreserved for long-term storage in liquid nitrogen without dedifferentiation. Successful cryopreservation helps to refine the experimental use of primary acinar cells by enabling their banking for on-demand utilization.

Adaptive and non-adaptive gene expression responses in prostate cancer during androgen deprivation.

Androgen deprivation therapy is the cornerstone treatment of advanced prostate cancer. Eventually prostate cancer cells overcome androgen deprivation therapy, giving rise to castration resistant prostate cancer (CRPC) characterized by increased androgen receptor (AR) activity. Understanding the cellular mechanisms leading to CRPC is needed for development of novel treatments. We used long-term cell cultures to model CRPC; a testosterone-dependent cell line (VCaP-T) and cell line adapted to grow in low testosterone (VCaP-CT). These were used to uncover persistent and adaptive responses to testosterone level. RNA was sequenced to study AR-regulated genes. Expression level changed due to testosterone depletion in 418 genes in VCaP-T (AR-associated genes). To evaluate significance for CRPC growth, we compared which of them were adaptive i.e., restored expression level in VCaP-CT. Adaptive genes were enriched to steroid metabolism, immune response and lipid metabolism. The Cancer Genome Atlas Prostate Adenocarcinoma data were used to assess the association with cancer aggressiveness and progression-free survival. Expressions of 47 AR-associated or association gaining genes were statistically significant markers for progression-free survival. These included genes related to immune response, adhesion and transport. Taken together, we identified and clinically validated multiple genes being linked with progression of prostate cancer and propose several novel risk genes. Possible use as biomarkers or therapeutic targets should be studied further.

Effects of simvastatin, acetylsalicylic acid, and rosiglitazone on proliferation of normal and cancerous prostate epithelial cells at therapeutic concentrations.

BACKGROUND: Non-steroidal anti-inflammatory drugs and cholesterol-lowering statins have been reported to inhibit prostate cancer cell growth suggesting their chemopreventive potential within the prostate. However, the effect has been demonstrated only with advanced prostate cancer cell lines and with drug concentrations above the clinical therapeutic range. In this study we compared the effect of therapeutic concentrations of acetylsalicylic acid, simvastatin and rosiglitazone on the growth of a set of prostatic primary cultures and various prostate epithelial cell lines. METHODS: Two primary epithelial cell lines isolated from surgical resecates of normal prostate tissue (P96E, P97E), a primary cell line isolated from untreated prostate carcinoma (ESTO1), two transformed prostate epithelial cell lines (PWR1-E, RWPE-1) and advanced cancer cell lines LNCaP and VCaP were used in the study. Cells were treated for seven days with therapeutic concentrations of acetylsalisylic acid, simvastatin, rosiglitazone or their combination. Cellular growth rate was measured by crystal violet staining method. RESULTS: Acetylsalicylic acid (0.5 mM) and simvastatin (10 nM) inhibited the growth of prostate epithelial cells of normal and primary cancer origin, whereas advanced cancer cell lines were resistant to the effect. Rosiglitazone at the therapeutic level of 1 microM did not reduce the growth of any cell type studied. CONCLUSIONS: Our results demonstrate that acetylsalicylic acid and simvastatin inhibit prostate epithelial cell growth at clinically relevant doses. This should be acknowledged when designing possible prostate cancer chemopreventive trials.

Effects of tamoxifen and raloxifene on normal human endometrial cells in an organotypic in vitro model.

The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy though its use is associated with an elevated risk of endometrial carcinoma. An organotypic culture model was employed here to examine the effects of tamoxifen and raloxifene, a related compound with no known adverse uterine effects, on epithelial cells of the premenopausal human endometrium. Changes in the expression levels of the proliferation marker Ki67, and estrogen and progesterone receptors were evaluated. No change in the Ki67 index compared to untreated controls was detected in cultures exposed to tamoxifen or tamoxifen+estradiol. In response to tamoxifen, the level of progesterone receptor-expressing organoids was shown to vary markedly between individual samples, whereas no change in estrogen receptor expression could be demonstrated. A significant decrease in Ki67 expression was observed in raloxifene-exposed cultures. Raloxifene or raloxifene+estradiol had no effect on progesterone receptor expression. The expression of estrogen receptor was markedly inhibited in response to raloxifene or raloxifene+estradiol in all but two samples displaying an intense estrogen receptor labelling. The present observations add to current clinical data on the respective estrogen receptor agonist and antagonist activities of tamoxifen and raloxifene on the human uterus by providing novel insights into the interindividual variation in cellular responses. Our organotypic model may have uses as an alternative to animal experimentation in preclinical screening of the endometrial effects of selective estrogen receptor modulators and may serve as a tool in personalized medicine by identifying patients with an increased risk of developing endometrial pathologies.

The effects of metformin and simvastatin on the growth of LNCaP and RWPE-1 prostate epithelial cell lines.

The anti-diabetic drug metformin and cholesterol-lowering statins inhibit prostate cancer cell growth in vitro and have been linked with lowered risk of prostate cancer in epidemiological studies. We evaluated the effects of these drugs on cancerous and non-cancerous prostate epithelial cell lines. Cancer (LNCaP) and normal (RWPE-1) prostate epithelial cell lines were treated with pharmacologic concentrations of metformin and simvastatin alone and in combinations. Relative changes in cell number were measured with crystal violet staining method. Drug effects on apoptosis and cell cycle were measured with flow cytometry. We also measured changes in the activation and expression of a set of reported target proteins of metformin and statins with Western blotting. Metformin decreased the relative cell number of LNCaP cells by inducing G1 cell cycle block, autophagy and apoptosis, and slightly increased cytosolic ATP levels, whereas RWPE-1 cells were resistant to metformin. However, RWPE-1 cells were sensitive to simvastatin, which induced G2 cell cycle block, autophagy and apoptosis, and increased cytosolic ATP levels in these cells. Combination of metformin and simvastatin synergistically decreased cytosolic ATP levels, increased autophagy and instead of apoptosis, induced necrosis in LNCaP cells. Synergistic effects were not observed in RWPE-1 cells. These results suggest, that prostate cancer cells may be more vulnerable to combined growth-inhibiting effects of metformin and simvastatin compared to normal cells. The data presented here provide evidence for the potency of combined metformin and statin, also at pharmacologic concentrations, as a chemotherapeutic option for prostate cancer.

Calcitriol inhibits growth response to Platelet-Derived Growth Factor-BB in human prostate cells.

Calcitriol, a hormonal form of Vitamin D, regulates growth of normal and cancer cells of various origins by modulation of peptide growth factors signaling. Platelet-Derived Growth Factor (PDGF) signaling pathway is involved in prostate cancer progression. We studied the expression of PDGF receptors in human prostate primary stromal cells and cancer epithelial cell lines and growth response to PDGF-BB isoform. We found that the expression of PDGF receptors and PDGF-BB-mediated cell growth are regulated by calcitriol in prostate cells. Quantitative RT-PCR analysis revealed a lower level of mRNA for PDGF receptors in LNCaP and PC-3 cells than in primary stromal cells. Western blotting showed a high amount of PDGFRalpha and beta proteins in primary stromal cells that could not be detected in LNCaP, which may explain the resistance of LNCaP cells to growth-promoting effect of PDGF-BB. Addition of Epidermal Growth Factor (EGF) to the culture medium induces the expression of PDGFRbeta and restores responsiveness of LNCaP to PDGF-BB to some extent. Calcitriol down-regulates PDGFRbeta expression and negatively regulates PDGF-mediated cell growth. Calcitriol does not affect PDGFRalpha and PDGF-B mRNA expression. We suggest that inhibition of PDGFRbeta expression by calcitriol might reduce responsiveness of prostate cells to mitogenic action of PDGF-BB.

25-hydroxyvitamin D3 is an active hormone in human primary prostatic stromal cells.

According to the present paradigm, 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] is a biologically active hormone; whereas 25-hydroxyvitamin D3 (25OHD3) is regarded as a prohormone activated through the action of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase). Although the role of vitamin D3 in the regulation of growth and differentiation of prostatic epithelial cells has been well studied, its action and metabolism in prostatic stroma are still largely unknown. We investigated the effects of 25OHD3 and 1alpha,25-(OH)2D3 on two human stromal primary cultures termed P29SN and P32S. In a cell proliferation assay, 25OHD3 was found at physiological concentrations of 100-250 nM to inhibit the growth of both primary cultures, whereas 1alpha,25-(OH)2D3 at a pharmacological concentration of 10 nM exhibited the growth-inhibitory effects on P29SN cells but not on P32S cells. Quantitative real-time RT-PCR analysis revealed that both 25OHD3 and 1alpha,25-(OH)2D3 induced 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) mRNA in a dose- and time-dependent manner. By inhibiting 1alpha-hydroxylase and/or 24-hydroxylase enzyme activities, the induction of 24-hydroxylase mRNA by 250 nM 25OHD3 was clearly enhanced, suggesting that 1alpha-hydroxylation is not a prerequisite for the hormonal activity of 25OHD3. Altogether our results suggest that 25OHD3 at a high but physiological concentration acts as an active hormone with respect to vitamin D3 responsive gene regulation and suppression of cell proliferation.

Expression of nuclear receptors and cofactors in human endometrium and myometrium.

OBJECTIVE: To study the expression of nuclear receptors and cofactors in human endometrium and myometrium in proliferative and secretory phases of the menstrual cycle. METHODS: Multiprobe ribonuclease protection assay and real-time reverse transcriptase polymerase chain reaction were used to quantitate mRNA levels of steroid receptors, vitamin D receptor (VDR), retinoic acid receptors (RAR), and cofactors AIB1 (amplified in breast cancer-1), CBP (cyclic adenosine monophosphate response element binding protein), pCAF (p300/CBP-associated factor), TIF2 (transcription intermediary factor-2), N-CoR (nuclear receptor corepressor), and SMRT (silencing mediator of repressed transcription). Cyclin A expression was analyzed to determine the proliferation status of the tissues. RESULTS: The expression of androgen receptor, estrogen receptors alpha and beta, progesterone receptor, and RARalpha followed cyclin A expression. There was more abundant expression in the proliferative phase endometrium than in the secretory phase endometrium. Glucocorticoid receptor, VDR, RARbeta, and RARgamma were stably expressed during the menstrual cycle in both endometrium and myometrium. Cofactors N-CoR, SMRT, pCAF, CBP, TIF2, AIB1, and p300 mRNAs were expressed in all samples in both endometrium and myometrium. N-CoR, pCAF, AIB1, and p300 appeared not to be regulated when comparing proliferative and secretory phases of the cycle. Individual differences were found in the expression levels of both nuclear receptors and cofactors. CONCLUSION: The menstrual cycle-dependent regulation of nuclear receptor expression was more apparent in the endometrium than in the myometrium, whereas cofactor expression was not cycle dependent. There were individual differences in the expression levels of different receptors and cofactors. In hormonal therapy these differences might result in different responses, depending on the patient as well as the ligand used.

A novel 2-step culture model for long-term in vitro maintenance of human pancreatic acinar cells.

OBJECTIVES: Because of rapid loss of functional differentiation that regularly occurs in vitro, culture systems permitting long-term studies on pancreatic acinar cells pose a major technical challenge. We recently described a method for long-term cultivation of mouse acinar cells. Here, we introduce a novel 2-step culture system for human pancreatic acinar cells. METHODS: The system involves 2 successive culture phases, which are as follows: primary organotypic culture of isolated acinar clusters under soft Matrigel (BD Biosciences, Bedford, Mass; range, 2-3 days) followed by dissociation and secondary monolayer culture of acinar cells (4 days). Basal and agonist-induced amylase secretion was used to assess the secretory capability. RESULTS: Acinar clusters showed excellent morphology and stable basal amylase secretion throughout primary culture. Carbachol (0.1 mM/L) increased amylase secretion 1.4-fold (P = 0.021) versus basal in 3 independent 4-day secondary cultures. Despite the controversy about the presence and roles of cholecystokinin receptors in human acinar cells, one of them also responded to 0.1 and 10 nM/L concentrations of caerulein with 1.9- and 1.4-fold increases in the rate of amylase secretion, respectively. CONCLUSIONS: Our technique allows cultured human acinar cells to maintain secretory differentiation for a minimum of 7 days. The technique provides novel prospects for in vitro modeling of the human exocrine pancreas.

Difference in Early Activation of NF-κB and MCP-1 in Acinar-Cell-Rich versus Fibrotic Human Pancreas Exposed to Surgical Trauma and Hypoxia.

Objectives. Previously we have shown that a pancreas with over 40% acinar cells is exposed to postoperative pancreatitis and other complications after pancreaticoduodenectomy (PD). Our aim was to analyze the expression of NF-κB and MCP-1 in the cut edge of human pancreas after PD in both acinar-cell-rich and fibrotic pancreata. Methods. Several pancreatic samples from six patients, three with acinar-cell-rich and three with fibrotic pancreata, were exposed to surgical trauma in PD, and thereafter to hypoxemia for 15 minutes, 2-2.5 hours, 4 hours, or 6 hours, to mimic postoperative conditions of the pancreatic remnant in a patient. Immunohistochemical analysis of inflammation markers (NF-κB, MCP-1) was performed. Results. In the acinar-cell-rich pancreata, intra-acinar NF-κB and MCP-1 expression increased from mild at 15 minutes to high during the first 4 hours, whereas in ductal cells MCP-1 staining was highly intense at both time points. Acinar cell NF-κB and MCP-1 expression and ductal cell MCP-1 expression were also observed in the fibrotic pancreata, but the activation remained low throughout the 6 hours. Conclusions. In acinar-cell-rich pancreas, an extensive inflammatory cascade begins almost immediately after surgical trauma. Fibrosis may limit the progression of inflammatory process in pancreas.

Autophagy is needed for the growth of pancreatic adenocarcinoma and has a cytoprotective effect against anticancer drugs.

BACKGROUND AND AIM: Autophagy is a regulated process of degradation and recycling of cellular constituents. The role of autophagy in pancreatic cancer is still not clear. Some studies indicate that in pancreatic cancer autophagy exerts cytoprotective effects, whereas others suggest that autophagy positively contributes to cell death by enhancing cytotoxicity of anticancer drugs. The aim of this study was to investigate the role of autophagy in pancreatic cancer, and to provide insights into new strategies for treatment. MATERIALS AND METHODS: Pancreatic cancer cell lines PANC-1 and BxPC-3 were treated with anticancer drugs (5-fluorouracil or gemcitabine) alone and in combination with autophagy inhibitors (chloroquine or wortmannin). Biopsy samples were retrieved from patients from pancreatic normal tissue and adenocarcinoma. Western blot of microtubule-associated protein 1 light chain 3 (LC3)-II was performed to investigate the degree of autophagy and cell proliferation was assessed by a crystal violet assay. RESULTS: Autophagy was active in PANC-1 cells under basal conditions. Autophagy was significantly induced in pancreatic ductal adenocarcinoma compared to healthy pancreatic tissue in patients. Inhibition of autophagy by chloroquine suppressed the growth of PANC-1 and BxPC-3. Autophagy was markedly increased after treatment with 5-fluorouracil or gemcitabine. Inhibition of autophagy by chloroquine potentiated the inhibition of cell proliferation of PANC-1 and BxPC-3 by 5-fluorouracil and gemcitabine. CONCLUSIONS: Our results with pancreatic cancer cell lines and human pancreatic adenocarcinoma suggest that autophagy contributes to pancreatic cancer cell growth. Autophagy has a cytoprotective effect against 5-fluorouracil and gemcitabine in pancreatic cancer cells. Combination therapy of these anticancer drugs and chloroquine should be investigated.

The risk for immediate postoperative complications after pancreaticoduodenectomy is increased by high frequency of acinar cells and decreased by prevalent fibrosis of the cut edge of pancreas.

OBJECTIVES: Soft pancreas is considered as a factor for pancreatitis after pancreaticoduodenectomy, which in turn constitutes a high risk for local complications. The aim was to analyze the proportion of different cell types in the cut edge of pancreas (CEP) in relation to postoperative pancreatitis and other complications after pancreaticoduodenectomy. METHODS: Data from postoperative follow-up was collected on 40 patients who had undergone pancreaticoduodenectomy. Positive urine trypsinogen-2, an early detector of pancreatitis, was checked on days 1 to 6 after operation. Drain amylase was measured on postoperative day 3. Anastomotic leakages, delayed gastric emptying, and other complications were registered. The areas of different cell types were calculated from the entire hematoxylin-eosin-stained section of CEP. RESULTS: High frequency of acinar cells in the CEP significantly increased positive urine trypsinogen-2 days, drain amylase values, and delayed gastric emptying. In a subgroup of patients with more than 40% acini in the CEP, there were significantly more postoperative complications. Increased fibrosis correlated with a small number of positive urine trypsinogen-2 days and postoperative complications. CONCLUSIONS: A large number of acinar cells in the CEP increases, whereas extensive fibrosis in the CEP decreases, the risk for postoperative complications after pancreaticoduodenectomy. These results emphasize the importance of acini in the development of postoperative complications.

The importance of LDL and cholesterol metabolism for prostate epithelial cell growth.

Cholesterol-lowering treatment has been suggested to delay progression of prostate cancer by decreasing serum LDL. We studied in vitro the effect of extracellular LDL-cholesterol on the number of prostate epithelial cells and on the expression of key regulators of cholesterol metabolism. Two normal prostatic epithelial cell lines (P96E, P97E), two in vitro immortalized epithelial cell lines (PWR-1E, RWPE-1) and two cancer cell lines (LNCaP and VCaP) were grown in cholesterol-deficient conditions. Cells were treated with 1-50 µg/ml LDL-cholesterol and/or 100 nM simvastatin for seven days. Cell number relative to control was measured with crystal violet staining. Changes in mRNA and protein expression of key effectors in cholesterol metabolism (HMGCR, LDLR, SREBP2 and ABCA1) were measured with RT-PCR and immunoblotting, respectively. LDL increased the relative cell number of prostate cancer cell lines, but reduced the number of normal epithelial cells at high concentrations. Treatment with cholesterol-lowering simvastatin induced up to 90% reduction in relative cell number of normal cell lines but a 15-20% reduction in relative number of cancer cells, an effect accompanied by sharp upregulation of HMGCR and LDLR. These effects were prevented by LDL. Compared to the normal cells, prostate cancer cells showed high expression of cholesterol-producing HMGCR but failed to express the major cholesterol exporter ABCA1. LDL increased relative cell number of cancer cell lines, and these cells were less vulnerable than normal cells to cholesterol-lowering simvastatin treatment. Our study supports the importance of LDL for prostate cancer cells, and suggests that cholesterol metabolism in prostate cancer has been reprogrammed to increased production in order to support rapid cell growth.

A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.

The development of in vitro models able to support the long-term viability and function of acinar cells is critical for exploring pancreatic pathophysiology. Despite considerable efforts, no long-term culture models for non-transformed pancreatic acini exist. Our aim was to develop and validate culture conditions for this purpose. An explant outgrowth culture design was established in which mouse pancreatic explants were cultured at the gas-liquid interphase. An enriched culture medium, pH 7.8, was employed to promote the selective outgrowth of acinar cells and to support their differentiated phenotype. After 7 days, the outgrown primary acinar cells were subcultured and maintained up to an additional 7 days as secondary monolayers on tissue culture plastic. Measurements of basal and caerulein-induced amylase secretion, phase-contrast microscopy and immunohistochemical analyses were used to characterize the cultures. Explants retained their pancreatic cytoarchitecture for 2 days in vitro. A triphasic dose response to caerulein was detected in 7-day primary cultures. The maximal rate of secretion was 1.2-fold versus basal (p=0.009) and 1.7-fold versus 1 pM caerulein (p=0.014). In secondary cultures the response was biphasic with maximal rates of secretion being 1.9-fold in 3- to 4-day cultures at 0.01 nM (p=0.049) and 2-fold in 6- to 7-day cultures at 0.1 nM (p=0.003). The present culture model provides a means to obtain functionally competent normal mouse acinar cells for long-term in vitro experimentation.

Comparative effects of high and low-dose simvastatin on prostate epithelial cells: the role of LDL.

Epidemiological studies have linked statin use with a decreased risk of advanced prostate cancer and an improved recurrence-free survival after radical therapy. It is unclear, however, whether statins could have direct effects against prostate cancer in a clinical setting, as their growth-inhibiting effects on prostate cancer cells have been demonstrated at drug concentrations which exceed the level in plasma during standard clinical dosing. We compared responses to high-dose and therapeutic-dose simvastatin in normal and cancerous prostate epithelial cells. Simvastatin was more effective at inhibiting the growth of normal prostate epithelial cells than of cancer cells. At therapeutic 100 nM concentration simvastatin had a cytostatic effect on normal cells: apoptosis was only slightly induced, but a decrease in cell cycle activity and an increase in senescence were observed. At therapeutic concentrations, lipophilic simvastatin caused a stronger growth inhibition than did hydrophilic rosuvastatin. In contrast, 10 µM simvastatin had a cytotoxic effect both on normal and cancer cells. Addition of LDL-cholesterol effectively reversed the cytostatic effect in all cell lines, but overcoming the cytotoxicity of 10 µM simvastatin required a combination of LDL-cholesterol and mevalonate. As LDL-cholesterol completely prevented the growth-inhibiting effect of therapeutic-dose simvastatin already at low, subphysiological concentrations it is unlikely that statins have direct effects on growth of prostate epithelial cells in vivo. Statins' possible benefits against prostate cancer could be due to systemic cholesterol-lowering, as suggested by epidemiological studies. Future clinical studies evaluating the effects of statins on prostate cancer prevention should monitor serum LDL and should probably administer statins at higher concentrations than those currently used in the treatment of hypercholesterolemia.