Roberto Zuchini

Autophagy suppresses tumorigenesis of hepatitis B virus‐associated hepatocellular carcinoma through degradation of microRNA‐224

In hepatocellular carcinoma (HCC), dysregulated expression of microRNA‐224 (miR‐224) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. In this study we determined that autophagy is down‐regulated and inversely correlated with miR‐224 expression in hepatitis B virus (HBV)‐associated HCC patient specimens. These results were confirmed in liver tumors of HBV X gene transgenic mice. Furthermore, miR‐224 was preferentially recruited and degraded during autophagic progression demonstrated by real‐time polymerase chain reaction and miRNA in situ hybridization electron microscopy after extraction of autophagosomes. Our in vitro study demonstrated that miR‐224 played an oncogenic role in hepatoma cell migration and tumor formation through silencing its target gene Smad4. In HCC patients, the expression of low‐Atg5, high‐miR‐224, and low‐Smad4 showed significant correlation with HBV infection and a poor overall survival rate. Autophagy‐mediated miR‐224 degradation and liver tumor suppression were further confirmed by the autophagy inducer amiodarone and miR‐224 antagonist using an orthotopic SD rat model. Conclusion: A noncanonical pathway links autophagy, miR‐224, Smad4, and HBV‐associated HCC. These findings open a new avenue for the treatment of HCC. (Hepatology 2014;59:505–517)

Autophagy-preferential degradation of MIR224 participates in hepatocellular carcinoma tumorigenesis

Autophagy and microRNA (miRNA) are important regulators during cancer cell tumorigenesis. Impaired autophagy and high expression of the oncogenic microRNA MIR224 are prevalent in hepatocellular carcinoma (HCC); however, the relationship between the 2 phenomena remains elusive. In this study, we are the first to reveal that autophagy selectively regulates MIR224 expression through an autophagosome-mediated degradation system. Based on this finding, we further demonstrated that in hepatitis B virus (HBV)-related HCC, aberrant autophagy (low autophagic activity) results in accumulation of MIR224 and decreased expression of the target gene Smad4, which leads to increased cell migration and tumor formation. Preferential recruitment of MIR224 into the autophagosome was clearly demonstrated by a) miRNA in situ hybridization under confocal microscopy, and b) immunogold labeling of MIR224 under electron microscopy compared with a ubiquitously expressed microRNA MIRlet7e/let-7. Furthermore, we found that off-label use of amiodarone, an antiarrhythmic agent, effectively suppressed HCC tumorigenesis through autophagy-mediated MIR224 degradation both in vitro and in vivo. In summary, we identified amiodarone as a new autophagy inducer, which may provide an alternative approach in HCC therapy through a novel tumor suppression mechanism.

Electromagnetic thermotherapy using fine needles for hepatoma treatment

BACKGROUND AND AIMS Hepatocellular carcinoma can be treated with heat-based therapies, especially radiofrequency ablation (RFA). However, RFA has limited efficacy and is quite expensive. We designed a new system using fine needles combined with an alternating magnetic field to generate hyperthermia for the treatment of hepatocellular carcinoma in a rat hepatoma model. Our aims are to assess the efficacy of our method and determine survival up to 30 days. METHODS An N1-S1 cell line was inoculated into the livers of Sprague-Dawley rats, generating tumors after 14 days. The animals were randomized into 5 groups and treated after laparotomy either with normal saline (group I), iron oxide nanoparticles (group II), fine needles (group III), fine needles and iron oxide nanoparticles combined (group IV) or self-designed two-part needles placed under ultrasonographic guidance percutaneously (group V). Every rat was placed in an alternating magnetic field. The temperature in the treatment area was maintained between 55 and 60 °C. At day 30 after treatment, tumor volumes and mortality were assessed and histology samples were studied. RESULTS Tumor volumes were significantly reduced and survival rate was prolonged in groups III, IV and V versus groups I and II (P < 0.05). On pathological examination, groups III, IV and V presented obvious necrosis, apoptosis, calcifications and inflammatory changes in the treatment area. CONCLUSION Our study demonstrates that hyperthermia generated by fine stainless-steel needles combined with an alternating magnetic field effectively inhibits hepatoma growth in rats and prolongs their survival. Further, this method can be applied percutaneously under ultrasonographic guidance.

Bloodless Liver Resection Using Needle Arrays Under Alternating Electromagnetic Fields

Background/Aim: Hemostasis is a major difficulty associated with hepatectomies. The authors designed a new thermal surgery system to reduce blood loss. Methods: The newly designed system consists of an alternating magnetic field generator and stainless steel needle arrays with thermosensitive bands. Lanyu pigs were used: 4 for the Kelly crushing method and 4 for the newly designed method. The procedures used were S4-S5 segmentectomies or left lateral segmentectomies, after which the amount of blood loss and operation times were compared. The pigs were observed for 4 weeks, after which liver pathologies were studied. Results: The blood loss in the method proposed by the authors was almost 0 mL, whereas with the Kelly crushing method it was 116 ± 35 mL. The method proposed in this study can save 15 to 25 minutes of operation time. The resected liver margins exhibited prominent apoptosis and fibrotic change in the remnant livers. Conclusions: The method proposed is a novel new way of performing thermal surgery.

Electromagnetic thermoablation to treat thrombocytopenia in cirrhotic and hypersplenic rats

Background and Aim:  Thrombocytopenia due to hypersplenism is usually a serious condition in cirrhotic patients who have undergone invasive procedures. We designed a new treatment method using a high‐frequency alternating electromagnetic force to treat the disease condition in a rat model.

Electromagnetic thermal surgery system for liver resection: An animal study

Purpose: Electromagnetic thermal surgery is a new technique. It applies an electrical current through coils to generate a high frequency magnetic field to heat up magnetic materials in the targeted area. Using this technique, we aim to perform liver resection without bleeding in rats and rabbits. Materials and methods: The electromagnetic machine can produce a high frequency magnetic field, with an input of 220 V-55 A-60 Hz, an output frequency of 62.1 kHz, and a power of 2.2 kW. The magnetic materials used in this study were fine needles made of stainless steel. For ex vivo experiments, we used porcine liver explants; in the animal model, sixteen Sprague-Dawley rats and seven New Zealand White rabbits were used. We inserted one needle array along the attempted resection lines and then used the magnetic coils to heat up the needles for three min. After heating, we resected the designated liver portions using surgical scalpels. Results: In the ex vivo test, the fine needles were heated up effectively to achieve tissue coagulation (more than 90°C). In the animal model the liver resection was performed without bleeding and no bile peritonitis was observed after the surgery. All animals were alive after the surgery until the end of the experiment (30 days). Conclusions: The experiments showed that our thermal surgery system is very effective in performing bloodless liver resection without further ligation or embolisation needed. Our technique is new and the system has great potential to develop into clinical practice.

Partial splenectomy using an electromagnetic thermal surgery system in a porcine model

Purpose: Partial splenectomy is technically more complicated than total splenectomy due to difficulty in haemostasis, but it can preserve splenic function after operation. We evaluated the feasibility and safety of partial splenectomy performed by an electromagnetic thermal surgery system in a porcine model. Methods: Our system was comprised of an alternating electromagnetic field generator, an extensible coil applicator, comb-needle arrays, and a temperature feedback control component. Ten Lanyu pigs were anaesthetised to conduct partial splenectomy. Two rows of comb-like stainless-steel needle arrays were inserted into the tissue at 15 cm from the distal tip of the spleen. The temperature of the tissues around the needle arrays was raised to 150°C for 3 min and the spleen was transected directly between the needle arrays and then sent for histological examination. Two weeks later, the animals underwent a second celiotomy to remove the remaining spleen for histological examination. Results: The average duration of the partial splenectomy was 10 min as timed from insertion of the needle arrays to the transection of the spleen. There was no blood loss during the procedure. The cut surface of the spleen was well coagulated without any oozing sites. During the re-exploration, no intra-abdominal blood was found. There were dense adhesions between the spleen and the surrounding organs. Histological examination of the cut surface of the excised portion of the spleen showed coagulative necrosis with clot formation in the blood vessels. Conclusions: Partial splenectomy using our electromagnetic thermal system can achieve effective haemostasis and is safe and easy to perform.

Successfully seal pancreatic end after thermal distal pancreatectomy using needle arrays in alternating electromagnetic fields.

Background. Pancreatic fistula is still the major postoperative morbidity after distal pancreatectomy (DP). An inductive heat technology via needle arrays in a system of alternating magnetic fields (AMFs) was designed to seal off the pancreatic end. Methods. Twenty Lanyu pigs were divided into 2 groups for DP: the conventional group had hand-sewn closure of the pancreatic end (n = 10), and the AMF group received thermal DP by AMF (n = 10). Pathological examinations of the resected and remnant pancreas were studied immediately after resection and on the 14th postoperative day (POD), respectively. The severity and the incidence of pancreatic abscess were compared. Results. The incidence and severity of pancreatic abscess were significantly decreased in the AMF group than those in the conventional group (P = .009). In the immediate postoperative period, microscopic examination of the pancreatic resected end showed prominent coagulative necrosis, loss of NADPH-diaphorase activity, and significant apoptosis at the resected pancreas in the AMF group compared with the control group. Fourteen days after AMF ablation, the pancreatic stump end was covered with thick fibrosis, and histological study of the remnant pancreas showed that the parenchyma had well recovered with positive NADPH-diaphorase activity, and the pancreatic duct was sealed off successfully by prominent periductal fibrosis and intraductal plug. The body weight gain on the 14th POD was significantly increased in the AMF group (from 23.8 ± 1.8 kg to 25.4 ± 5.5 kg) compared with the conventional group (from 25.3 ± 2.1 to 25.4 ± 6.0 kg; P = .003). Conclusions. Inductive heats by the AMF system via needle array can be performed easily and can seal the pancreatic cut surface well during DP.

Liver Resection Using High Frequency Electromagnetic Thermotherapy

Background/Aim: Electromagnetic thermotherapy is a new modality for surgery. It applies a high-frequency electrical current passing through coils to generate a high-frequency magnetic field to heat up magnetic materials for treating targeted tissue. Using such a system, we aim to perform resection of rabbit liver without bleeding.