Hiroaki Kuroda

Regression of hepatocellular carcinoma in vitro and in vivo by radiosensitizing suicide gene therapy under the inducible and spatial control of radiation

To improve the efficacy and selectivity of gene therapy for hepatocellular carcinoma (HCC), we designed a strategy for suicide gene therapy in conjunction with radiation therapy using an HVJ-liposome vector system. The radio-inducible suicide gene was constructed by insertion of the early growth response gene 1 (Egr-1) promoter upstream of the HSV-tk gene (EGF-tk). First, to test the tumor specificity of Egr-1, RT-PCR and immunohistochemistry were performed. The Egr-1 gene was highly expressed in HCC compared with normal liver, where expression was barely detectable. Next, radiation-inducible activity of the Egr-1 promoter was examined in primary cultured normal hepatocytes and human hepatoma cell lines Huh7, HepG2, and PLC/PRF/5 by luciferase assay as a reporter gene system. Egr-1 promoter activity was markedly increased in hepatoma cell lines in a radiation dose-dependent manner, with maximum activation (15- to 28-fold) 12 hr after irradiation. In contrast, only a twofold increase in activation was noted in normal hepatocytes. An in vitro gene therapy experiment showed that EGR-tk-transduced hepatoma cells became highly sensitive to ganciclovir (GCV) after irradiation, but not without irradiation. GCV with or without irradiation did not show any cytotoxic effects against control gene-transfected cells. In addition, a radiosensitization effect was also demonstrated by combination therapy with the HSV-tk/GCV system and irradiation. To examine the efficacy of this EGR-tk/GCV gene therapy in vivo, xenografted liver tumors in nude mice were targeted using the HVJ-liposome vector system. EGR-tk-transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n = 8), and almost disappeared in 3 weeks without any side effects. In comparison, tumors continued to grow in all mice (n = 8 in each group) treated by transfer of EGR-tk followed by either irradiation without GCV or GCV without irradiation. Our data indicate that HSV-tk gene therapy under the control of a radioinducible promoter is effective, and might be selective for hepatoma cells because of its inducible and radiosensitive capacity after radiation exposure as well as its tumor-specific activation.

Overexpression of Insulin-Like Growth Factor-1 (IGF-I) Receptor and the Invasiveness of Cultured Keloid Fibroblasts

Keloid is a dermal fibroproliferative tissue of unknown etiology. Protein tyrosine kinases (PTKs) play an important role in the regulation of cell growth and differentiation. Activation of PTK cascades in keloid fibroblasts is thought to be closely linked to abnormal cell proliferation and migration. We determined the expression profile of PTK genes in normal skin and keloid fibroblasts using the homology cloning method with a degenerated primer. Eight PTK genes were expressed among a total of 46 receptor-type clones. The most abundant type of PTK receptors was the platelet-derived growth factor receptor in both fibroblasts. However, insulin-like growth factor-I receptor (IGF-IR) was overexpressed only in keloid-derived fibroblasts (9 of 24). Immunohistochemical analysis confirmed the high expression of IGF-IR in keloid fibroblasts, but not in normal fibroblasts. To examine the functional properties of the IGF-I/IGF-IR pathway, we investigated cell proliferation and invasion activities of both types of fibroblasts. The mitogenic effect of IGF-I on both fibroblasts was very weak compared with serum stimulation. In contrast, the invasive activity of keloid fibroblasts was markedly increased in the presence of IGF-I, and inhibited by a neutralizing antibody against IGF-IR. Our results indicate the involvement of activated IGF-I/IGF-IR in the pathogenesis of keloid by enhancing the invasive activity of fibroblasts.

GH suppresses TGF-β-mediated fibrosis and retains cardiac diastolic function

Abstract The aims of this study were to elucidate the molecular mechanism by which growth hormone (GH) excess is anti-fibrotic in vitro and in vivo model. The in vivo model GH excess showed a significant increase of relative wall thickness with no concomitant disturbance of cardiac diastolic function. Western blot for extracellular matrix (ECM) structural proteins showed minimal change in the GH treatment group, compared to an Angiotensin II (Ang II) subpressor dose group. In cultured cardiac fibroblasts, we investigated the abundance of ECM proteins, phosphorylation of p38 mitogen-activated protein kinase (MAPK), and transforming growth factor-β (TGF-β)-specific transcriptional activity. GH down-regulated the expression of PAI-1 and fibronectin proteins activated by TGF-β. In reporter assays, GH, but not insulin-like growth factor-1 (IGF-1), reduced TGF-β-specific transcriptional activity. Moreover, GH markedly down-regulated TGF-β-induced phosphorylation of p38 MAPK. These results demonstrated that a chronic excess of GH have an anti-fibrotic effect on cardiac remodeling, probably through a down-regulation of TGF-β signaling via de-phosphorylation of p38 MAPK.

Involvement of receptor-type tyrosine kinase gene families in cardiac hypertrophy.

OBJECTIVEnThe activation of protein tyrosine kinases (PTKs) has been postulated to be involved in cell differentiation and proliferation. To elucidate the involvement of tyrosine kinase genes in normal and pathological conditions, we analysed the expression patterns of receptor-type PTKs in the normal and hypertensive hypertrophied heart in rats.nnnMATERIALS AND METHODSnHypertrophied and normal rat hearts were obtained from hypertensive rats; deoxycorticosterone acetate (DOCA)-salt and 2 kidney-1 clip (2K-1C), and their sham-operated rats, respectively. A reverse transcription-polymerase chain reaction (RT-PCR) was performed using degenerated primers which were designed from highly conserved regions in the catalytic domains of receptor-type PTKs. The PCR products were ligated into a sequence vector, and subcloned by transforming bacteria. To compare the expression level of these PTK mRNAs in the normal and hypertrophied heart, we performed semi-competetive RT-PCR and immunohistochemical and Western blot analyses.nnnRESULTSnNucleotide sequencing of approximately 80 clones of PTKs revealed 10 receptor-type, five nonreceptor-type and two unknown types in the rat heart. Tie-2/Tek, Ryk, insulin-like growth factor-I receptor were abundantly expressed in the rat heart as members of receptor-type PTKs. Immunohistochemistry and RT-PCR demonstrated the presence of platelet-derived growth factor (PDGF)-alpha receptor, PDGF-beta receptor and fibroblast growth factor-3 receptor in both normal and hypertrophied hearts. We also confirmed the presence of Flt-1, KDR/FIk-1, and their ligand vascular endothelial growth factor, c-Met and its ligand hepatocyte growth factor (HGF), and Tie-1, Tie-2/Tek by immunohistochemistry and RT-PCR. The coexpression of cardiac HGF and c-Met in hypertrophied hearts, especially in 2K-1 C rats, was induced more intensively than that in DOCA-salt rats.nnnCONCLUSIONnThese findings suggest that HGF/c-Met interactions may play an important role in cardiac hypertrophy and remodeling, probably as a result of the activation of the local renin-angiotensin system.

Eradication of hepatocellular carcinoma xenografts by radiolabelled, lipiodol-inducible gene therapy

The promoter region of the early-growth response-1(Egr-1) gene has been shown to be activated by external radiation, thus making a selective tumoricidal effect possible. A previous experiment showed that the Egr-1 promoter can be activated by internal radiation using radioisotopes as well as external radiation. Internal radiation using I-131 lipiodol (I-131-Lip) has been established as one of the most useful therapeutic strategies against hepatoma. We herein linked the Egr-1 promoter to the herpes simplex virus-thymidine kinase (HSV-TK) gene, and investigated its efficacy in hepatoma gene therapy in combination with I-131-Lip. A luciferase assay showed the Egr-1-promoter activity to be markedly increased in hepatoma tissue specimens in an I-131-dose-dependent manner, whereas a less than two-fold increase in this activity was observed in other organs. In addition, the radioactivity derived from I-131 was selectively accumulated in the tumor tissue specimens. To examine the efficacy of EgrTK/ganciclovir (GCV) gene therapy in vivo, subcutaneous hepatoma xenografts in nude mice were transfected using a hemagglutinating virus of Japan (HVJ)-liposome vector. Complete tumor regression was observed in all the EgrTK-transfected tumors following combination treatment with I-131-Lip and GCV 42 days after treatment without any side effects (n=8). In contrast, the tumors continued to grow in all control mice (n=10). Furthermore, the serum α-fetoprotein levels decreased in the combination therapy group, while they increased in the controls. In conclusion, these data indicate that Egr-1 promoter-based gene therapy combined with internal radiation has a selective effect on hepatoma tumors while also showing an improved in vivo efficacy. This combination therapy might, therefore, be an effective human hepatoma gene therapy, even in advanced multiple cases.

Pathologic Diagnosis and Genetic Analysis of a Lung Tumor Needle Biopsy Specimen Obtained Immediately After Radiofrequency Ablation

PurposeTo evaluate the possibility of pathologic diagnosis and genetic analysis of percutaneous core-needle biopsy (CNB) lung tumor specimens obtained immediately after radiofrequency ablation (RFA).Materials and MethodsPatients who underwent CNB of lung tumors immediately after RFA from May 2013 to May 2016 were analyzed. There were 19 patients (8 men and 11 women; median age, 69xa0years; range, 52–88xa0years) and 19 lung tumors measuring 0.5–2.6xa0cm (median, 1.6xa0cm). Thirteen tumors were solid, and 6 were predominantly ground-glass opacity (GGO) on computed tomography. All specimens were pathologically examined using hematoxylin and eosin (H&E) staining and additional immunostaining, as necessary. The specimens were analyzed for EGFR and KRAS genetic mutations. The safety and technical success rate of the procedure and the possibility of pathologic diagnosis and genetic mutation analysis were evaluated.ResultsMajor and minor complication rates were 11% (2/19) and 53% (10/19), respectively. Tumor cells were successfully obtained in 16 cases (84%, 16/19), and technical success rate was significantly lower for GGO-dominant tumors (50%, 3/6) compared with solid lesions (100%, 13/13, pxa0=xa00.02). Pathologic diagnosis was possible in 79% (15/19) of cases based on H&E staining alone (nxa0=xa012) and with additional immunostaining (nxa0=xa03). Although atypical cells were obtained, pathologic diagnosis could not be achieved in 1 case (5%, 1/19). Both EGFR and KRAS mutations could be analyzed in 74% (14/19) of the specimens.ConclusionPathologic diagnosis and genetic analysis could be performed even for lung tumor specimens obtained immediately after RFA.

Fibrolamellar hepatocellular carcinoma with multiple lung metastases treated with multidisciplinary therapy

A 20-year old man was diagnosed with fibrolamellar hepatocellular carcinoma (FLHCC) with multiple lung metastases, and chemotherapy with FOLFOX was administered. Contrast enhanced CT after 3 cycles of FOLFOX showed no disease progression. We therefore performed surgical resection and radiofrequency ablation of the liver lesions and lung metastases, after obtaining the patients informed consent. The liver lesions and lung metastases tested positive for DNAJB1-PRKACA. The treatment for FLHCC with extrahepatic metastasis has not been established; however, in a few cases, good long-term prognoses were obtained with multidisciplinary therapy. We herein report a case of FLHCC with multiple lung metastases that was treated with multidisciplinary therapies.

Spontaneous regression of lung squamous cell carcinoma with synchronous mediastinal progression: A case report: Spontaneous regression of lung cancer

Spontaneous regression (SR) of cancer implies the partial or complete disappearance of malignant disease without or with adequate medical treatment. Typically, SR of cancer is a sporadic event, especially in non‐small cell lung cancer (NSCLC). Although the underlying mechanism of SR remains unknown, stimulation of an immunological response has been proposed. Herein, we report the case of a 56‐year‐old woman exhibiting SR of NSCLC with a mediastinal disease. Despite regression of the primary site after a lung biopsy, simultaneous progression of mediastinal lymph node metastasis occurred. Specimens obtained by surgical resection pathologically confirmed both primary and metastatic sites. Reportedly, primary and metastatic tumors shrink synchronously in SR of metastatic NSCLCs. Thus, the fact that the SR of NSCLC can present inconsistent development in primary and metastatic sites should be considered, and direct intervention is recommended if physicians diagnose this phenomenon.

Detection of abundant megakaryocytes in pulmonary artery blood in lung cancer patients using a microfluidic platform

OBJECTIVESnThe lung was recently re-discovered as a hematopoietic organ for platelet production in mice. However, evidence for the role of the lung in thrombopoiesis in humans is still limited. In this study, we examined megakaryocytes in the pulmonary and systemic circulation, specifically in pulmonary arterial blood (PAB), venous blood (PVB) and peripheral blood using a newly developed microfluidic platform for rare cell isolation.nnnMATERIALS AND METHODSnWe analyzed 23 lung cancer patients who underwent surgery in our institute. PAB and PVB were obtained from the resected lung immediately after surgery. Blood samples were size-selected using a filtration-based microfluidic device and enriched rare cells on glass slide specimens were stained with Papanicolaou (Pap), immunocytochemistry (ICC), and immunofluorescence (IF). Lung tissues were also analyzed by immunohistochemistry.nnnRESULTSnPap/ICC/IF showed the presence of abundant CD61+/cytokeratin- giant cells with a megakaryocyte lineage in PAB, but only a few in PVB. These megakaryocytes were found to consist of CD61+/CD41+ immature megakaryocytes and CD61+/CD41- mature megakaryocytes with the potential to produce platelets. These findings were confirmed by the conventional hematological analysis of blood smears stained with Giemsa. In analysis of lung cancer, CD61+ megakaryocytes were observed exclusively in the capillaries of non-cancerous tissue, whereas platelets were selectively observed in the tumor blood vessels of cancerous tissue.nnnCONCLUSIONSnThese results indicate that numerous megakaryocytes migrate from systemic bone marrows to accumulate in PAs and arrest of mature megakaryocytes in the capillaries of normal lung, suggesting the possibility that the lung plays a physiological role in the systemic thrombopoiesis in lung cancer patients.

The eighth TNM classification system for lung cancer: A consideration based on the degree of pleural invasion and involved neighboring structures

OBJECTIVESnThe eighth tumor-node-metastasis (TNM) classification system for lung cancer has been used since January 2017 and must be applied to an individual institutions database.nnnMETHODSnWe analyzed pathological stage data of 2756 patients who underwent resection of non-small-cell lung cancer, particularly in terms of the degree of visceral pleural invasion and involved neighboring structures.nnnRESULTSnFew patients had stage IIA disease (103, 4%); stratification between stages IB and IIA was insufficient (pu202f=u202f0.129). When T2a tumors were divided into PL1 and PL2 subgroups based on the degree of pleural invasion, there was a significant prognostic difference between the subgroups (pu202f<u202f0.001). By incorporating T2a tumors with PL2 (T2a-PL2) into the T2b category, modified stages IB, IIA (234, 8%), and IIB were well stratified (IB vs. IIA, pu202f<u202f0.001; IIA vs. IIB, pu202f=u202f0.011). Focusing on T3 tumors with PL3 (T3-PL3) invading neighboring structures, multivariate analysis for surveying pT3N0-2M0 tumors revealed that completeness of resection (pu202f=u202f0.002), implementation of any postoperative therapies (pu202f=u202f0.003), and subcategorization of whether only the pleura was infiltrated or other deeper structures were also invaded (pu202f=u202f0.024) were significant and crucial predictors. N2 disease showed worse outcome than N0-1 diseases, with marginal difference (pu202f=u202f0.054).nnnCONCLUSIONnT2a-PL2 tumors could be categorized into a worse prognostic T2b category. For T3-PL3 tumors involving resectable neighboring organs, subcategorization of whether there is only pleura infiltration (T3a) or other deeper structure invasion (T3b) could be a practical consideration.