MK2206 inhibits hepatocellular carcinoma cellular proliferation via induction of apoptosis and cell cycle arrest

Background

Hepatocellular carcinoma (HCC) is commonly diagnosed at an advanced stage and has limited effective treatment options. The aberrant regulation of the phosphoinositide 3-kinase/Akt pathway in HCC makes it an attractive therapeutic target. The effect of MK2206, a novel, allosteric Akt inhibitor, on HCC cells is not yet fully understood. We hypothesized that inhibition of Akt by MK2206 would impact cellular viability.

Materials and methods

Human Huh7, Hep3B, and HepG2 cell lines were treated with 0–2 μM of MK2206 for 96 h. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western blot analysis was used to examine the expression level of various protein markers to assess the mechanism of drug action and proliferation inhibition.

Results

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a reduction in cellular viability by ≥55% for all cell lines (control versus 2 μM MK2206; P <0.001). Western blot analysis revealed reduction in the level of phosphorylated AKT-Ser473 with no change in AKT-thr308 expression confirming the specificity of MK2206. There was an observed reduction in caspase-9 and survivin. Importantly, there were increases in p21 and p27 along with decreased cyclinD1 expression after treatment.

Conclusions

This study demonstrates the anti-tumor activity of MK2206 in HCC cells. The observed reduction in survivin and pro-caspase 9 suggests that MK2206 induces apoptosis. However, HCC proliferation is also halted via induction of cell cycle arrest as indicated by the increase in p21 and p27 expression and decrease in cyclinD1. Importantly, the concentration needed to achieve growth inhibition in HCC is lower than that needed for other cancer types.     

Expression and prognostic significance of p21-activated kinase 6 in hepatocellular carcinoma

Background

p21-activated protein kinase (PAK) 6 is a serine-threonine kinase belonging to the PAK family. Previous studies have indicated that abnormal expressions of PAK1, PAK2, and PAK5 played critical roles in hepatocellular carcinoma (HCC). Recent studies suggested that deregulation of PAK6 expression played an important role in oncogenesis. To explore the potential roles of PAK6 in HCC, expression of PAK6 was detected in human HCC specimens.

Methods

Immunohistochemistry and Western blot analysis were performed for PAK6 in 121 HCC samples. The data were correlated with clinicopathologic features. The univariate and multivariate survival analyses were also performed to determine their clinical prognostic significance.

Results

PAK6 was overexpressed in HCC as compared with the adjacent noncancerous liver tissues. High expression of PAK6 was associated with Edmondson–Steiner grade (P = 0.006) and number of tumor nodules (P < 0.001), and PAK6 was positively correlated with proliferation marker Ki-67 (P < 0.01). Univariate analysis suggested that PAK6 expression was associated with poor prognosis (P < 0.001). Multivariate analysis indicated that PAK6 and Ki-67 protein expressions were independent prognostic markers for HCC (P = 0.0245 and 0.0331, respectively).

Conclusions

Our results suggest that PAK6 overexpression is involved in the pathogenesis of HCC; it may be an independent poor prognostic factor for HCC.     

miR-503 regulates metastatic function through Rho guanine nucleotide exchanger factor 19 in hepatocellular carcinoma

Background

Our previous work described a metastasis-related microRNAs expression profiling and revealed miR-503 regulating metastatic function in hepatocellular carcinoma (HCC) cells. Here, we investigate to define the mechanism of miR-503 regulating metastasis in HCC.

Materials and methods

The expressions of miR-503 in HCC cell lines and clinical tissues with different metastatic potential were investigated. Meanwhile, a metastatic human HCC cell BALB/c nude mice model was used to investigate whether miR-503 regulates metastasis of HCC in vivo. Furthermore, luciferase activity of reporter gene, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluorescence-activated cell sorting analysis (FACS), and invasion assay were carried out to characterize the mechanism of miR-503 regulating metastasis in HCC.

Results

We confirmed the negative correlation between miR-503 expression and metastatic potential of HCC in cell lines and in clinical HCC tissues. We also showed that overexpression of miR-503 resulted in inhibition of proliferation and metastasis of HCC in vivo. Furthermore, we demonstrated that ARHGEF19 is a direct target gene of miR-503. Finally, our results indicated that ARHGEF19 overcomes the suppressive influence of miR-503 in HCC cells.

Conclusions

Our results suggest an important role of miR-503 in inhibiting metastasis of HCC through deregulating ARHGEF19.     

Prognostic significance of receptor for advanced glycation end products expression in hepatocellular carcinoma after hepatectomy

Background

The receptor for advanced glycation end products (RAGE) is recognized to be responsible for cancer progression in several human cancers. In this study, we investigated the clinical impact of RAGE expression in patients with hepatocellular carcinoma (HCC) after hepatectomy.

Materials and methods

Sixty-five consecutive patients who underwent initial hepatectomy for HCC were investigated. The relationships between immunohistochemical expression of RAGE and clinicopathologic features, clinical outcome (overall survival [OS], and disease-free survival [DFS]) were evaluated.

Results

The cytoplasmic expression of RAGE in HCC cells was observed in 46 patients (70.8%) and correlated with histologic grade (poorly differentiated versus moderately differentiated HCC, P = 0.021). Five-year OS in RAGE-positive and RAGE-negative groups were 72% and 94%, respectively, whereas 5-y DFS were 29% and 55%, respectively. There were significant differences between OS and DFS (P = 0.018 and 0.031, respectively). Multivariate analysis indicated that RAGE was an independent predictor for both OS and DFS (P = 0.048 and 0.032, respectively).

Conclusions

Our data suggest for the first time a positive correlation between RAGE expression and poor therapeutic outcome. Furthermore, RAGE downregulation may provide a novel therapeutic target for HCC.     

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation

Background

Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells).

Materials and methods

Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways.

Results

Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12).

Conclusions

LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation.     

The glypican 3 hepatocellular carcinoma marker regulates human hepatic stellate cells via Hedgehog signaling

Background

Hepatocellular carcinoma (HCC) frequently represents two diseases as it often arises in the setting of cirrhosis caused by the proliferation and activation of hepatic stellate cells (HSCs). Previously, we identified that Hedgehog (Hh) signaling regulates HSC viability and fibrinogenesis, as well as HCC tumorigenesis. Although it is increasingly recognized that HSCs and HCCs communicate via paracrine signaling, Hh's role in this process is just emerging. We hypothesized that a secreted HCC tumor marker and Hh mediator, glypican 3 (GPC3), may regulate HSC.

Methods

Using three human HCC lines (Hep3B, PLC/PRF/5 and SK-Hep-1) and one Hh-responsive human HSC line (LX-2), we developed two in vitro models of HCC-to-HSC paracrine signaling using a Transwell coculture system and HCC-conditioned media. We then evaluated the effects of these models, as well as GPC3, on HSC viability and gene expression.

Results

Using our coculture and conditioned media models, we demonstrate that the three HCC lines decrease HSC viability. Furthermore, we demonstrate that recombinant GPC3 dose-dependently decreases the LX-2 viability while inhibiting the expression of Hh target genes that regulate HSC viability. Finally, GPC3's inhibitory effects on cell viability and Hh target gene expression are partially abrogated by heparin, a competitor for GPC3 binding.

Conclusions

For the first time, we show that GPC3, an HCC biomarker and Hh mediator, regulates human HSC viability by regulating Hh signaling. This expands on existing data suggesting a role for tumor–stroma interactions in the liver and suggests that GPC3 plays a role in this process.     

Liver Resection in Transplanted Patients: A Single-Center Western Experience

Background

Liver resection (LR) in liver transplant (OLT) recipients, an extremely rare situation, who performed on 8 recipients.

Methods

This retrospective analysis of prospectively collected data concerned 8 (0.66%) 1198 LR cases among OLT performed from 1997 to 2011. We analyzed demographic data, surgical indications, and postoperative courses.

Results

The indications were resectable recurrent hepatocellular carcinomas (HCC, n = 3), persistent fistula from a posterior sectorial duct (n = 1), recurrent cholangitis due to anastomotic stricture on the posterior sectorial duct (n = l), hydatid cyst (n = l), left arterial hepatic thrombosis with secondary ischemic cholangitis (n = 1), and a large symptomatic biliary cyst (n = 1). The mean interval time to liver resection was 23.7 months (range, 5–47). LR included right hepatectomy (n = 1), right posterior hepatectomy (n = 1), left lobectomy (n = 4), pericystectomy (n = 1), or biliary fenestration (n = 1). Which there was no postoperative mortality, the global morbidity rate was 62% (5/8). The mean follow-up after LR was 92 months (range, 11–156). No patients required retransplantation. None of the 3 patients who underwent LR for HCC showed a recurrence.

Conclusions

LR in OLT recipients is safe, but associated with a high morbidity rate. This procedure can avoid retransplantation in highly selected patients, presenting a possible option particularly for transplanted patients with a resectable, recurrent HCC.     

Long-term Survival Outcomes for Living Donor Liver Transplant Recipients With Pathologically Nonviable Hepatocellular Carcinoma

Purpose

Complete necrosis of hepatocellular carcinoma (HCC) lesions has occasionally been found by explant pathology after pretransplant neoadjuvant treatment. This study sought to investigate the long-term prognostic effect of loss of tumor viability after HCC treatment in living donor liver transplant (LDLT) recipients.

Methods

We reviewed retrospectively the 5-year records of 37 patients who demonstrated nonviable HCC on explant pathology.

Results

The most common primary disease was hepatitis-B-virus-associated liver cirrhosis (n = 34). Single explant tumors were found in 29 patients; the mean maximal tumor size was 2.1 ± 0.9 cm (range: 0.8–4.0). No patients showed microvascular invasion. The median level of alpha-fetoprotein was 12 ng/mL (range: 1–1160). The 1 patient who showed a recurrence at 20 months remains alive more than 6 years after adrenalectomy and repeated pulmonary metastasectomy. The 5-year HCC recurrence rate was thus 2.1%. There were 2 late mortalities, each due to graft failure and recurrent gastric cancer. The overall patient survival rate was 97.3% at 5 and 92.7% at 10 years.

Conclusions

The results of this study revealed that the loss of tumor viability induced by pretransplant neoadjuvant treatment definitely decreased the risk of post-transplant HCC recurrence. Therefore, patients with nonviable HCC can be regarded as members of a superselect group with minimal risk for HCC recurrence, and may be exempted from routine HCC screening.     

Neutrophil to lymphocyte ratio changes predict small hepatocellular carcinoma survival

Background

There is limited information available concerning the delta neutrophil to lymphocyte ratio (ΔNLR) in hepatocellular carcinoma (HCC). The present study was designed to evaluate the predictive value of dynamic change of NLR in patients who undergo curative resection for small HCC.

Methods

A retrospective cohort study was performed to analyze 189 patients with small HCC who underwent curative resection between February 2007 and March 2012. Patient data were retrieved from our prospectively maintained database. Patients were divided into two groups: group A (NLR increased, n = 80) and group B (NLR decreased, n = 109). Demographic and clinical data, overall survival (OS), and recurrence-free survival (RFS) were statistically compared and a multivariate analysis was used to identify prognostic factors.

Results

The 1, 3, and 5-y OS in group A was 92.7, 70.0, and 53.0%, respectively, and 96.2, 87.5, and 75.9%, respectively, for group B (P = 0.003); The corresponding 1, 3, and 5-y RFS was 58.7, 37.9, 21.8, and 81.2%, 58.5% and 53.8% for groups A and B, respectively (P <0.001). Multivariate analysis suggested that ΔNLR was an independent prognostic factor for both OS (P = 0.004, Hazard Ratio (HR) = 2.637, 95% confidence interval (CI) 1.356–5.128) and RFS (P <0.001, HR = 2.372, 95% CI 1.563–3.601).

Conclusions

Increased NLR, but not high preoperative NLR or postoperative NLR, helps to predict worse OS and RFS in patients with small HCC who underwent curative resection.     

Expression Pattern Analysis of Hepatocellular Carcinoma Tumor Markers in Viral Hepatitis B and C Patients Undergoing Liver Transplantation and Resection

Background

This study was conducted to compare the expression patterns of serum alpha-fetoprotein (AFP) and proteins induced by vitamin K absence or antagonist-II (PIVKA-II) in hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) and resection at a high-volume single institution.

Methods

First, 663 liver transplant recipients with HCC were selected. They were divided into hepatitis B virus (HBV) (n = 628) and hepatitis C virus (HCV) groups (n = 35). Their medical records were retrospectively reviewed. Second, another cohort of 2709 patients who underwent HCC resection included 2258 HBV, 143 HCV, and 308 non-HBV non-HCV (NBNC) patients.

Results

In the transplantation group, pretransplantation AFP level >20 ng/mL was observed in 42.5% of HBV patients and 60% of HCV patients (P = .042). PIVKA-II level >40 mAU/mL was observed in 30.6% of HBV patients and 42.9% of HCV patients (P = .127). In the resection group, a preoperative AFP level >20 ng/mL was observed in 51.7% of HBV patients and 43.3% of HCV patients (P = .052). PIVKA-II level >40 mAU/mL was observed in 59.7% of HBV patients and 56.6% of HCV patients (P = .47). Preoperative AFP level >20 ng/mL and PIVKA-II level >40 mAU/mL were observed in 35.7% and 61% of NBNC patients, respectively. Receiver-operator characteristic curve analyses revealed that the expression pattern of PIVKA-II in patients with elevated AFP level was not predictable and vice versa, regardless of background liver diseases.

Conclusions

This study indicates that serum AFP and PIVKA-II may be expressed variably regardless of the types of background liver disease. Further large-volume multicenter studies are needed to evaluate the possibility of the etiology-dependent expression of tumor markers.     

Liver Transplantation for Hepatocellular Carcinoma Within Milan Criteria in Patients With Model For End-Stage Liver Disease Score Below 15: The Impact of the Etiology of Cirrhosis on Long-Term Survival

Background

Liver transplantation (OLT) is the gold standard therapy for patients with cirrhosis complicated by hepatocellular carcinoma (HCC) within Milan Criteria (MC). We evaluated the impact of the etiology of the underlying liver disease on long-term outcomes of patients undergoing OLT for HCC within MC having a Model for End-stage Liver Disease (MELD) score < 15.

Methods

From November 2002 to December 2009, we performed 203 primary OLTs from brain-dead donors in recipients with HCC and cirrhosis with biochemical MELD scores below 15. We excluded 31 patients outside MC on the explant pathology of the native liver. The remaining 172 were divided into 3 groups according to the etiology of the underlying cirrhosis: hepatitis C virus-positive (HCV+; n = 78; 45%), hepatitis B virus-positive (HBV+; n = 65; 38%) and other indications (n = 29; 17%). The groups were compared for donor and recipient features, donor-recipient match, and transplant variables. The study endpoint was long-term patient survival.

Results

The groups were similar, except for a greater prevalence of hepatitis B core antibody-positive grafts in the HBV+ group and less frequent HCC bridging procedures in the other indications group. After a median follow-up of 72 months, HCC recurrence was observed in 8 (4.7%) patients (6 HCV+, 2 other indications), 5 of whom died. Overall 5-year patient survival of 82%, revealed significant differences among groups: 98.3% in HBV+, 67.1% in HCV+, and 85.8% in other indications (HBV+ vs other indications: P = .01; HBV+ vs HCV+: P = .0001; HCV+ vs other indications: P = NS). In the HCV+ group, recurrent HCV hepatitis was the most frequent cause of death. Upon multivariate analysis, HBV positivity in the recipient was an independent predictor of better patient survival (hazard ratio = 0.10, 95% confidence interval 0.02–0.64, P = .013).

Conclusions

Etiology of the underlying cirrhosis significantly influenced the long-term survival after OLT of patients with HCC within MC and MELD < 15. It should be taken into account in estimation of survival benefit.     

Augmenter of liver regeneration attenuates inflammatory response in the postischemic mouse liver in vivo

Background

Augmenter of Liver Regeneration (ALR), a protein synthesized in the liver is suggested to be protective against oxidative stress–induced cell death. Hepatic ischemia–reperfusion (I/R) injury is triggered by reactive oxygen species. Here, we tested the hypothesis that ALR attenuates hepatic I/R injury in vivo.

Methods

C57BL6 mice were subjected to warm hepatic ischemia for 90 min. Either recombinant ALR (100 μg/kg) or vehicle were administered to mice prior ischemia. During reperfusion, neutrophil and CD4+ T cell migration and sinusoidal perfusion were analyzed using intravital microscopy. Alanine aminotransferase–aspartate aminotransferase (plasma) and caspase-3 (tissue) activities were determined as markers of hepatocellular necrotic and apoptotic injury.

Results

Hepatic I/R led to dramatic enhancement of neutrophil and CD4+ T cell recruitment in hepatic microvessels, sinusoidal perfusion failure, and strong elevation of aspartate aminotransferase–alanine aminotransferase and caspase-3 activities. During early reperfusion (60 min), the pretreatment with ALR improved postischemic perfusion failure (P < 0.05) and attenuated liver enzyme activities. Recruitment of CD4+ T cells, but not of neutrophils was attenuated. After 240 min of reperfusion, the protective effect of ALR was stronger, since the liver enzyme activity, perfusion failure, and leukocyte influx were significantly attenuated. As shown by the measurement of caspase-3 activity, postischemic apoptosis was reduced in the ALR-treated group.

Conclusions

Our in vivo data show that ALR has a therapeutic potential against postischemic liver injury. As a mechanism, we suggest a direct protective effect of ALR on apoptotic and necrotic death of hepatocytes and an attenuation of inflammatory cell influx into the postischemic tissue.     

Liver Transplantation for Unresectable Hepatocellular Carcinoma in Elderly Patients: What to Expect

Introduction

Elderly patients have orthotopic liver transplantation (OLT) outcomes comparable to younger individuals. However, it is undefined whether such results are also seen in those with unresectable hepatocellular carcinoma (HCC). The primary endpoints of this study were overall survival (OS), retransplantation rate, and disease-free survival (DFS) in OLT recipients with HCC and aged ≥65 years compared with those aged <65 years.

Patients and Methods

This was a single-center retrospective review of a cohort of adult deceased-donor OLT recipients due to unresectable HCC within the Milan criteria from May 2006 through March 2013. Demographic and clinical variables and outcomes of patients aged ≥65 years were compared with those aged <65 years.

Results

Thirty-seven individuals ≥65 years of age (group 1) and 141 individuals <65 years of age (group 2) were analyzed. OS (group 1 vs group 2) at 1 year (78% vs 81%), 3 years (64% vs 70%), and 5 years (64% vs 66%) (P = .49) was comparable. Retransplantation rates were also similar (group 1: 13.5%; group 2, 10.6% [P = .61]). DFS (group 1 vs group 2) at 1 year (100% vs 95%), 3 years (91% vs 92%), and 5 years (91% vs 92%) (P = .56) was also not significantly different between groups. On multivariate analysis, age >65 years was not an independent predictor of OS or DFS.

Conclusions

Patients aged ≥65 years with HCC presented with outcomes similar to their younger counterparts. Chronologic age is not a good predictor of outcome, and transplantation is feasible if overall clinical conditions and comorbidities allow.     

Effect of Autologous Adipose-Derived Stem Cells in Renal Cold Ischemia and Reperfusion Injury

Objective

The aim of this study was to investigate the effect of autologous adipose-derived stem cells (ADSCs) on renal cold ischemia and reperfusion (I/R) injury via intravenous infusion on rats.

Methods

A renal cold I/R injury rat model was established. Rats were equally randomized into Sham group, Cold I/R group (cold I/R plus culture medium only), and ADSC-treated group (cold I/R plus immediate intrarenal administration of 2 × 10⁶ autologous ADSCs, followed by intravenous autologous ADSCs 6 hours after reperfusion). All rats were killed 24 hours after the I/R procedure.

Results

Serum creatinine levels were significantly reduced in the ADSC-treated group compared with the Cold I/R group (P < .01). The renal tissue in the ADSC-treated group had well conserved renal architecture compared with the Cold I/R group. The mRNA expression of tumor necrosis factor α was significantly lower and Bcl-2 was higher in the ADSC-treated group than in the Cold I/R group (P < .05).

Conclusions

Autologous ADSC infusions ameliorated renal damage undergoing cold I/R injury and improved the renal function, partly through inhibiting inflammatory reactions and reducing apoptosis.     

Increased platelet storage time is associated with mitochondrial dysfunction and impaired platelet function

Background

Hemorrhagic shock is a leading cause of death following severe trauma, and platelet transfusions are frequently necessary to achieve hemostasis. Platelets, however, require special storage conditions, and storage time has been associated with loss of platelet quality. We hypothesized that standard storage conditions have a deleterious effect on platelet mitochondrial function and platelet activation.

Materials and methods

Platelet donations were collected from healthy donors (n = 5) and stored in gas-permeable collection bags according to American Association of Blood Bank recommendations. Platelet units were sampled from day of collection (day 0) until day 7. High-resolution respirometry was used to assess baseline mitochondrial respiration, maximal oxygen utilization, and individual mitochondrial complex-dependent respiration. Fluorescence-activated cell sorting was performed to analyze mitochondrial content, mitochondrial reactive oxygen species, the expression of P-selectin (both before and after challenge with thrombin receptor–activating peptide), and apoptosis. Data were analyzed using analysis of variance and Pearson correlation (P < 0.05 significant).

Results

Mitochondrial respiration decreased significantly in platelets stored longer than 2 d (P < 0.05). Platelets also demonstrated a persistent decrease in response to stimulation with thrombin receptor–activating peptide by the third day of storage (P < 0.05) as well as an increase in mitochondrial reactive oxygen species and apoptosis (P < 0.05). Mitochondrial respiration significantly correlated with platelet capacity to activate (r = 0.8, P < 0.05).

Conclusions

Platelet mitochondrial respiratory function and activation response decrease significantly in platelets stored for 3 d or more. Because platelet transfusions almost universally occur between the third and fifth day of storage, our findings may have significant clinical importance and warrant further in vivo analysis.     

Clinical Outcomes and Risk Factors of Hepatitis B Virus Recurrence in Patients Who Received Prophylaxis With Entecavir and Hepatitis B Immunoglobulin Following Liver Transplantation

Introduction

Although entecavir (ETV) and hepatitis B immunoglobulin (HBIG) have widely been used for prophylaxis of hepatitis B virus (HBV) recurrence following liver transplantation (OLT), there have been few studies about clinical outcomes and risk factors of HBV recurrence.

Materials and methods

This study retrospectively assessed clinical outcomes and identified risk factors of post-transplant HBV recurrence in 154 patients who received prophylaxis with both ETV and HBIG after OLT.

Results

The median follow-up duration was 28.0 months (range, 1.0–57.8). Post-transplant HBV recurrence occurred in 5 patients (3.2%) without any ETV-resistant mutants. The overall rates of HBV recurrence at 1, 2, and 4 years were 0.6%, 1.6%, and 6.2%, respectively. We found that recurrent hepatocellular carcinoma (HCC) was an independent risk factor of HBV recurrence (hazard ratio = 13.5, 95% confidence interval, 2.4–74.4; P = .006).

Conclusions

Prophylaxis with a combination of ETV and HBIG resulted in a low HBV recurrence rate following OLT without any emergence of ETV-resistant mutants. Recurrent HCC was an independent risk factor of HBV recurrence in patients who received prophylaxis with both ETV and HBIG for prophylaxis following OLT.     

Bacterial translocation induces proinflammatory responses and is associated with early death in experimental severe injury

Objective

An experimental model of severe injury with great lethality was studied to define the impact of bacterial translocation on survival and on inflammatory response.

Methods

Forty-one rabbits were divided into two groups: A, femur myotomy; and B, myotomy and fracture of the femoral bone. Vital signs and survival were recorded. Serum circulating endotoxins (lipopolysaccharides; LPS) were determined and tissue cultures were performed at necropsy. A subgroup of animals was sacrificed at 48 h post injury; LPS was determined in abdominal aorta and portal vein, apoptosis of spleen cells was assessed by flow cytometry, and ex vivo production of tumor necrosis factor alpha by splenocytes was measured.

Results

Tissue bacterial burden was increased in animals that died early (i.e., within 48 h after injury) versus rabbits that died later. Portal vein LPS at 48 h was increased in group B compared with group A, whereas circulating LPS did not differ. No difference in apoptosis of either lymphocytes or macrophages of the spleen was found in group B compared with group A. Following stimulation with LPS or phytohemagglutinin, tumor necrosis factor α production by splenocytes of group B was greater than that of group A.

Conclusions

Bacterial translocation primes enhanced proinflammatory responses and it is associated with early death in severe trauma.     

Dasatinib is synergistic with cetuximab and cisplatin in triple-negative breast cancer cells

Background

Patients presenting with triple-negative breast cancers (TNBCs) have a poorer prognosis compared with those with other subtypes of breast cancer. The majority of TNBCs overexpress epidermal growth factor receptor (EGFR). However, EGFR inhibition as a monotherapy, as with the monoclonal antibody cetuximab, is ineffective. Src family tyrosine kinases play a critical role in signal transduction downstream of growth factor receptors and are involved in the development of EGFR inhibitor resistance. We hypothesize that dasatinib, an Src family tyrosine kinase inhibitor, may help overcome EGFR resistance to cetuximab, and in combination with cisplatin may enhance growth inhibition and apoptosis and reduce metastatic potential.

Methods

Growth inhibition, apoptosis, cell migration and invasion, and effects on EGFR, Akt, and mitogen-activated protein kinase phosphorylation were examined in a panel of breast cancer cell lines, including seven TNBC cell lines.

Results

Six out of seven TNBC cell lines demonstrated a synergistic interaction using the triple-drug combination, compared with only two TNBC cell lines with the cisplatin and cetuximab combination. An induction of apoptosis and decrease in EGFR and mitogen-activated protein kinase phosphorylation, and thus resensitization to EGFR inhibition, was observed using the three-drug treatment regimen. A significant reduction (P < 0.001) in tumor cell migration and invasion was also found following dasatinib treatment alone or in combination.

Conclusions

These findings may have important clinical implications in treating TNBC patients whose tumors co-overexpress both EGFR and c-Src. Identification of this subset of patients may be beneficial in the design of a clinical trial using this treatment regimen.     

Incidental Hepatocellular Carcinoma: Risk Factors and Long-Term Outcome After Liver Transplantation

Background

Orthotopic liver transplantation (OLT) currently represents the treatment of choice for early hepatocellular carcinoma (HCC). Preoperatively known HCC (pkHCC) is diagnosed via imaging methods before OLT or before HCC is found postoperatively in the liver explant, denoted as incidental HCC (iHCC). The aim of this study was a comprehensive analysis of the post-transplantation survival of patients with iHCC and the identification of risk factors of iHCC occurrence in cirrhotic liver.

Methods

We retrospectively reviewed 33 adult cirrhotic patients with incidentally found HCC, comparing them with 606 tumor-free adult cirrhotic patients with end-stage liver disease (group Ci) who underwent OLT in our center from January 1995 to August 2012. Within the same period, a total of 84 patients underwent transplantation for pkHCC. We compared post-transplantation survivals of iHCC, Ci, and pkHCC patients. In the group of cirrhotic patients (Ci + iHCC), we searched for risk factors of iHCC occurrence.

Results

There was no difference in sex, Model for End-Stage Liver Disease score, and time spent on the waiting list in either group. In the multivariate analysis we identified age >57 years (odds ratio [OR], 3.37; 95% confidence interval [CI], 1.75–8.14; P < .001), hepatitis C virus or alcoholic liver disease (OR, 3.89; 95% CI, 1.42–10.7; P < .001), and alpha-fetoprotein level >6.4 μg/L (OR, 6.65; 95% CI, 2.82–15.7; P = .002) to be independent predictors of iHCC occurrence. Both the 1-, 3-, and 5-year overall survival (OS) and the 1-, 3- and 5-year recurrence-free survival (RFS) differed in iHCC patients compared with the Ci group (iHCC: OS 79%, 72%, and 68%, respectively; RFS 79%, 72%, and 63%, respectively; vs Ci: OS = RFS: 93%, 94%, and 87%, respectively; P < .001).

Conclusions

The survival of iHCC patients is worse than in tumor-free cirrhotic patients, but similar to pkHCC patients. The independent risk factors for iHCC occurrence in cirrhotic liver are age, hepatitis C virus, or alcoholic liver disease etiology of liver cirrhosis and alpha-fetoprotein level.     

Pretreatment of Transfused Donor Splenocytes and Allografts With Mitomycin C Attenuates Acute Rejection in Heart Transplantation in Mice

Objective

The aim of this study was to investigate the effect of pretreatment of donor splenocytes and grafts with mitomycin C (MMC) on heart allograft survival, as well as to demonstrate the mechanism of function.

Methods

Donor splenocytes from Balb/C mice were incubated with MMC (40 μg/mL) in vitro and then transfused into recipients (C57BL/6 mice). The heart allograft was perfused with MMC before harvest. Graft survival and histopathology were examined. Lymphocyte activation, regulatory T cells, and donor splenocyte apoptosis were examined with the use of flow cytometry.

Results

MMC incubation in vitro induced apoptosis of donor splenocytes (15.5 ± 2.3% vs 23.2 ± 4.2%; P < .01). Either intravenous injection of MMC-treated donor splenocytes or transplantation of allograft pretreated with MMC prolonged heart allograft mean survival time from 7 ± 0.8 days to 20.5 ± 1.9 days or 10 ± 0.9 days, respectively (both P < .01). A combination of MMC-pretreated donor splenocyte transfusion and allografts showed the best effect on prolongation of graft survival (28.5 ± 1.8 days). Activation of CD4⁺ T cells in spleen and peripheral lymph nodes of recipients was significantly inhibited by either MMC-splenocyte transfusion or the combination treatment. Meanwhile, the percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells in the spleen was increased in the MMC-splenocyte transfusion group (15.5 ± 1.1% vs 18.2 ± 0.9%; P < .05).

Conclusions

Both injection of MMC-conditioned donor splenocytes and MMC-conditioned allograft have effects on prolongation of heart allograft survival in mice, and MMC-conditioned donor splenocytes might play an essential role. MMC pretreatment induced regulatory T cells likely through induction of donor splenocyte apoptosis, and thus it inhibited T-cell activation.