Yulia Pollak

Inhibition of doxorubicin‐induced autophagy in hepatocellular carcinoma Hep3B cells by sorafenib – the role of extracellular signal‐regulated kinase counteraction

A multikinase inhibitor of the Raf/mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK/ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co‐treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1/2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl‐2 family. ERK1/2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX‐induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib‐induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK/ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX‐induced autophagy and thus promote cancer cell survival. The use of MEK/ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects.

Dietary L-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat

BackgroundArginine (ARG) and nitric oxide maintain the mucosal integrity of the intestine in various intestinal disorders. In the present study, we evaluated the effects of oral ARG supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat.MethodsMale rats were divided into four experimental groups: Control rats, CONTR-ARG rats, were treated with oral ARG given in drinking water 72 hours before and 72 hours following vehicle injection, MTX rats were treated with a single dose of methotrexate, and MTX-ARG rats were treated with oral ARG following injection of MTX. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. RT-PCR was used to determine bax and bcl-2 mRNA expression.ResultsMTX-ARG rats demonstrated greater jejunal and ileal bowel weight, greater ileal mucosal weight, greater ileal mucosal DNA and protein levels, greater villus height in jejunum and ileum and crypt depth in ileum, compared to MTX animals. A significant decrease in enterocyte apoptosis in the ileum of MTX-ARG rats (vs MTX) was accompanied by decreased bax mRNA and protein expression and increased bcl-2 protein levels.ConclusionsTreatment with oral ARG prevents mucosal injury and improves intestinal recovery following MTX- injury in the rat.

Parenteral omega-3 fatty acids (Omegaven) modulate intestinal recovery after intestinal ischemia-reperfusion in a rat model

BACKGROUND/PURPOSE Fatty acids from fish oil (omega-3 polyunsaturated fatty acids, 3PUFAs) are emerging as powerful yet safe disease-modifying nutrients and are protective in severe critical care conditions including ischemia-reperfusion (IR) injury. The purpose of the present study was to examine the effects of 3PUFAs on intestinal structural changes, enterocyte proliferation, and apoptosis after intestinal IR in a rat. METHODS Male rats were divided into three experimental groups: sham rats underwent laparotomy, IR rats underwent occlusion of both superior mesenteric artery and portal vein for 30 minutes followed by 48 hours of reperfusion, and 3PUFA-treated IR (IR-3PUFA) rats underwent IR and were treated with Omegaven (Fresenius Kabi, Bad Homburg, Germany) given intraperitoneally at a dose of 1 mL twice a day. Intestinal structural changes (Park injury score, overall bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, cell proliferation, and apoptosis) were determined 48 hours after IR. Real-time polymerase chain reaction (PCR) was used to determine the level of bax and bcl-2 messenger RNA. RESULTS A significant decrease in bowel and mucosal weight was observed in the ileum of untreated IR rats compared with sham animals. Forty-eight hours after IR, cell apoptosis remained increased in the jejunum and ileum, which coincided with increased bax/bcl-2 ratio. Cell proliferation was increased 48 hours after IR, suggesting tissue repair. Treatment with Omegaven resulted in a significant increase in bowel and mucosal weight in the jejunum and ileum, villus height in the jejunum and ileum, and crypt depth in the jejunum compared with untreated IR animals. IR-3PUFA rats also demonstrated a significantly lower Park injury score in the jejunum and ileum as well as a lower apoptotic index in the ileum compared with untreated IR animals. CONCLUSIONS Parenteral Omegaven administration decreases the intestinal mucosal injury and inhibits enterocyte apoptosis after intestinal IR in a rat.

Dietary transforming growth factor-beta 2 (TGF-β2) supplementation reduces methotrexate-induced intestinal mucosal injury in a rat.

Background/Aims Dietary supplementation with transforming growth factor-beta (TGF-β) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-β2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model. Methods Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-β2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-β rats were treated with diet enriched with TGF-β2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-β rats were treated with diet enriched with TGF-β2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, β-catenin, IL-1B and bax protein expression. Results Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-β2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-β2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-β2 also led to increased pERK, IL-1B and β-catenin protein levels in intestinal mucosa. Conclusions Treatment with TGF-β2 prevents mucosal-injury, enhances p-ERK and β-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.

Glutamine attenuates the inhibitory effect of methotrexate on TLR signaling during intestinal chemotherapy-induced mucositis in a rat

Toll-like receptor 4 (TLR-4) is crucial in maintaining intestinal epithelial homeostasis, participates in a vigorous signaling process and heightens inflammatory cytokine output. The objective of this study was to determine the effects of glutamine (GLN) on TLR-4 signaling in intestinal mucosa during methotrexate (MTX)-induced mucositis in a rat. Male Sprague–Dawley rats were randomly assigned to one of four experimental groups of 8 rats each: 1) control rats; 2) CONTR-GLN animals were treated with oral glutamine given in drinking water (2%) 48 hours before and 72 hours following vehicle injection; 3) MTX-rats were treated with a single IP injection of MTX (20 mg/kg); and 4) MTX-GLN rats were pre-treated with oral glutamine similar to group B, 48 hours before and 72 hours after MTX injection. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. The expression of TLR-4, MyD88 and TRAF6 in the intestinal mucosa was determined using real time PCR, Western blot and immunohistochemistry. MTX-GLN rats demonstrated a greater jejunal and ileal mucosal weight and mucosal DNA, greater villus height in ileum and crypt depth and index of proliferation in jejunum and ileum, compared to MTX animals. The expression of TLR-4 and MyD88 mRNA and protein in the mucosa was significantly lower in MTX rats versus controls animals. The administration of GLN increased significantly the expression of TLR-4 and MyD88 (vs the MTX group). In conclusion, treatment with glutamine was associated with up-regulation of TLR-4 and MyD88 expression and a concomitant decrease in intestinal mucosal injury caused by MTX-induced mucositis in a rat.

PDGF-α stimulates intestinal epithelial cell turnover after massive small bowel resection in a rat

Numerous cytokines have been shown to affect epithelial cell differentiation and proliferation through epithelial-mesenchymal interaction. Growing evidence suggests that platelet-derived growth factor (PDGF) signaling is an important mediator of these interactions. The purpose of this study was to evaluate the effect of PDGF-α on enterocyte turnover in a rat model of short bowel syndrome (SBS). Male rats were divided into four groups: Sham rats underwent bowel transection, Sham-PDGF-α rats underwent bowel transection and were treated with PDGF-α, SBS rats underwent a 75% bowel resection, and SBS-PDGF-α rats underwent bowel resection and were treated with PDGF-α. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined at euthanasia. Illuminas Digital Gene Expression analysis was used to determine PDGF-related gene expression profiling. PDGF-α and PDGF-α receptor (PDGFR-α) expression was determined by real-time PCR. Western blotting was used to determine p-ERK, Akt1/2/3, bax, and bcl-2 protein levels. SBS rats demonstrated a significant increase in PDGF-α and PDGFR-α expression in jejunum and ileum compared with sham animals. SBS-PDGF-α rats demonstrated a significant increase in bowel and mucosal weight, villus height, and crypt depth in jejunum and ileum compared with SBS animals. PDGF-α receptor expression in crypts increased in SBS rats (vs. sham) and was accompanied by an increased cell proliferation following PDGF-α administration. A significant decrease in cell apoptosis in this group was correlated with lower bax protein levels. In conclusion, in a rat model of SBS, PDGF-α stimulates enterocyte turnover, which is correlated with upregulated PDGF-α receptor expression in the remaining small intestine.

Effect of N-Acetylserotonin on Intestinal Recovery Following Intestinal Ischemia–Reperfusion Injury in a Rat

OBJECTIVE N-acetylserotonin (NAS) is a naturally occurring chemical intermediate in the biosynthesis of melatonin. Extensive studies in various experimental models have established that treatment with NAS significantly protects heart and kidney injury from ischemia-reperfusion (IR). The purpose of the present study was to examine the effect of NAS on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. METHODS Male Sprague-Dawley rats were divided into four experimental groups: (1) Sham rats underwent laparotomy, (2) sham-NAS rats underwent laparotomy and were treated with intraperitoneal (IP) NAS (20 mg/kg); (3) IR rats underwent occlusion of both superior mesenteric artery and portal vein for 30 minutes, followed by 48 hours of reperfusion, and (4) IR-NAS rats underwent IR and were treated with IP NAS (20 mg/kg) immediately before abdominal closure. Intestinal structural changes, Park injury score, enterocyte proliferation, and enterocyte apoptosis were determined 24 hours following IR. The expression of Bax, Bcl-2, p-ERK, and caspase-3 in the intestinal mucosa was determined using real-time polymerase chain reaction, Western blot, and immunohistochemistry. A nonparametric Kruskal-Wallis analysis of variance test was used for statistical analysis with p less than 0.05 considered statistically significant. RESULTS Treatment with NAS resulted in a significant increase in mucosal weight in jejunum and ileum, villus height in the ileum, and crypt depth in jejunum and ileum compared with IR animals. IR-NAS rats also had a significantly proliferation rates as well as a lower apoptotic index in jejunum and ileum which was accompanied by higher Bcl-2 levels compared with IR animals. CONCLUSIONS Treatment with NAS prevents gut mucosal damage and inhibits programmed cell death following intestinal IR in a rat.

The role of Wnt/β-catenin signaling in enterocyte turnover during methotrexate-induced intestinal mucositis in a rat

Background/Aims Intestinal mucositis is a common side-effect in patients who receive aggressive chemotherapy. The Wnt signaling pathway is critical for establishing and maintaining the proliferative compartment of the intestine. In the present study, we tested whether Wnt/β-catenin signaling is involved in methotrexate (MTX)-induced intestinal damage in a rat model. Methods Non-pretreated and pretreated with MTX Caco-2 cells were evaluated for cell proliferation and apoptosis using FACS analysis. Adult rats were divided into three experimental groups: Control rats; MTX-2 animals were treated with a single dose of MTX given IP and were sacrificed on day 2, and MTX-4 rats were treated with MTX similar to group B and were sacrificed on day 4. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were measured at sacrifice. Real Time PCR and Western blot was used to determine the level of Wnt/β-catenin related genes and protein expression. Results In the vitro experiment, treatment with MTX resulted in marked decrease in early cell proliferation rates following by a 17-fold increase in late cell proliferation rates compared to early proliferation. Treatment with MTX resulted in a significant increase in early and late apoptosis compared to Caco-2 untreated cells. In the vivo experiment, MTX-2 and MTX-4 rats demonstrated intestinal mucosal hypoplasia. MTX-2 rats demonstrated a significant decrease in FRZ-2, Wnt 3A Wnt 5A, β-catenin, c-myc mRNA expression and a significant decrease in β-catenin and Akt protein levels compared to control animals. Four days following MTX administration, rats demonstrated a trend toward a restoration of Wnt/β-catenin signaling especially in ileum. Conclusions Wnt/β-catenin signaling is involved in enterocyte turnover during MTX-induced intestinal mucositis in a rat.

TGF-β affects enterocyte turnover in correlation with TGF-β receptor expression after massive small bowel resection.

Objectives: In the present study, we evaluated the effect of transforming growth factor-beta 2 (TGF-&bgr;2)-enriched diet on enterocyte turnover and correlated it with TGF-&bgr;2 receptor expression along the villus–crypt axis in a rat model of short bowel syndrome (SBS). Methods: CaCo-2 cells were incubated with increasing concentrations of TGF-&bgr;2. Alamar Blue reduction test was used for investigation of cell viability and evaluation of cell apoptosis was assessed by flow cytometry. Male rats were divided into 4 groups: Sham rats underwent bowel transection, Sham TGF-&bgr; rats were treated with diet enriched with TGF-&bgr;2, SBS rats underwent a 75% bowel resection, and SBS TGF-&bgr; rats were fed a diet enriched with TGF-&bgr;2 after bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined at sacrifice. TGF-&bgr;2r expression in villus tips, lateral villi and crypts was assessed by immunohistochemistry. The effect of TGF-&bgr;2 on enterocyte turnover for each compartment was evaluated in correlation with TGF-&bgr;2r expression. Results: Incubation of CaCo-2 cells with TGF-&bgr;2 resulted in a significant decrease in cell viability and increased cell apoptosis. TGF-&bgr;2r expression in crypts increased in SBS rats (vs sham) and was accompanied by decreased cell proliferation and increased cell apoptosis following TGF-&bgr;2 administration. A significant decrease in TGF-&bgr;2r expression at villous tips in SBS rats was accompanied by a decreased cell apoptosis in this compartment following exposure to TGF-&bgr;2-enriched diet. Conclusions: In a rat model of SBS, the inhibiting effect of TGF-&bgr;2 on enterocyte turnover correlates with TGF-&bgr;2 receptor expression along the villus–crypt axis.

The Effect of Elevated Intra-Abdominal Pressure on TLR4 Signaling in Intestinal Mucosa and on Intestinal Bacterial Translocation in a Rat

BACKGROUND Recent evidence suggests that elevated intra-abdominal pressure (IAP) may adversely affect the intestinal barrier function. Toll-like receptor 4 (TLR-4) is responsible for the recognition of bacterial endotoxin or lipopolysaccharide and for initiation of the Gram-negative septic shock syndrome. The objective of the current study was to determine the effects of elevated IAP on intestinal bacterial translocation (BT) and TLR-4 signaling in intestinal mucosa in a rat model. METHODS Male Sprague-Dawley rats were randomly assigned to one of two experimental groups: sham animals (Sham) and IAP animals who were subjected to a 15 mmHg pressure pneumoperitoneum for 30 minutes. Rats were sacrificed 24 hours later. BT to mesenteric lymph nodes, liver, portal vein blood, and peripheral blood was determined at sacrifice. TLR4-related gene and protein expression (TLR-4; myeloid differentiation factor 88 [Myd88] and TNF-α receptor-associated factor 6 [TRAF6]) expression were determined using real-time PCR, western blotting, and immunohistochemistry. RESULTS Thirty percent of sham rats developed BT in the mesenteric lymph nodes (level I) and 20% of control rats developed BT in the liver and portal vein (level II). abdominal compartment syndrome (ACS) rats demonstrated an 80% BT in the lymph nodes (Level I) and 40% BT in the liver and portal vein (Level II). Elevated BT was accompanied by a significant increase in TLR-4 immunostaining in jejunum (51%) and ileum (35.9%), and in a number of TRAF6-positive cells in jejunum (2.1%) and ileum (24.01%) compared to control animals. ACS rats demonstrated a significant increase in TLR4 and MYD88 protein levels compared to control animals. CONCLUSIONS Twenty-four hours after the induction of elevated IAP in a rat model, increased BT rates were associated with increased TLR4 signaling in intestinal mucosa.