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Background: Pulmonary inflammatory myofibroblastic tumor (IMT) accounts for 0.04-0.7% of all lung tumors, and endobronchial IMT accounts for only 10% of all pulmonary IMTs. Little is known about the therapeutic outcomes of rigid bronchoscopy for endobronchial IMT. Here, we report a case series of eight patients with endobronchial IMT underwent rigid bronchoscopy., Methods: We retrospectively analyzed eight patients with endobronchial IMT between January 2004 and December 2023., Results: The median age of our patients was 36 years, and 62.5% were male. Dyspnea was the predominant symptom in cases where the tumor was centrally located (n = 6), whereas hemoptysis was the predominant symptom in peripherally located tumors (n = 2). Most cases had high contrast enhancement and a tumor stalk without bronchial wall invasion on computed tomography (CT) and bronchoscopy. Complete endoscopic resection and laser cauterization via rigid bronchoscopy were possible in five patients. There were only two cases in which tumors remained after the procedure, requiring additional treatment (chemotherapy and surgical resection, respectively). In one patient, surgical resection was performed three weeks after the procedure, and the surgical specimen was free of residual tumor. There was no mortality during the median follow-up duration of 18.8 months., Conclusions: Endoscopic resection and laser cauterization using rigid bronchoscopy may serve as a safe and effective alternative treatment modality to surgery for patients with endobronchial IMT., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Institutional Review Board of Samsung Medical Center (IRB no. 2024-04-098). Informed consent was waived by the IRB because it is a retrospective study, many patients are not being followed up, and it does not contain personal information that can be used to infer individual patients. However, we want to emphasize that we strictly adhered to ethical principles and ensured patient confidentiality throughout the study. Consent for publication: Not applicable. Conflict of interest: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Clinical trial number: Not applicable., (© 2025. The Author(s).)

Background: Glomus tumors (GTs) are rare, comprising only 2% of all soft tissue tumors. Pulmonary GTs are exceptionally rare, with fewer than 80 cases reported to date. Little is known about the therapeutic outcomes of rigid bronchoscopy for endobronchial GT., Methods: This is a case series of four patients with endobronchial GT who underwent therapeutic rigid bronchoscopy between February 2021 and June 2024., Results: The ages of the patients in our series ranged from 32 to 75 years, and all patients were male. Cough and blood-tinged sputum were present in all patients with endobronchial GT. The tumor sizes ranged from 1 to 3 cm. Complete endoscopic resection and laser cauterization via rigid bronchoscopy were achieved in two patients. One patient had incomplete resection of a 3-cm tumor in the segmental bronchus that showed radiological evidence of bronchial wall invasion. This patient subsequently underwent lobectomy seven months after bronchoscopic resection. The fourth patient was lost to follow-up. There was no mortality throughout the follow-up periods that ranged from 2.8 to 42.5 months. Factors favoring successful rigid bronchoscopy resection for endobronchial GT include a benign tumor in the central airways without bronchial wall invasion., Conclusion: Endoscopic resection and laser cauterization using rigid bronchoscopy may be a viable option for patients with endobronchial GT when surgery is not practical., Clinical Trial Number: Not applicable., Competing Interests: Declarations. Ethics approval and consent to participate: This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Samsung Medical Center (IRB no. 2024-08-125). Informed consent was waived by the IRB. Consent for publication: Informed consent was waived by the IRB because it is a retrospective study, some patients are not being followed up, and it does not contain personal information that can be used to infer individual patients. However, we want to emphasize that we strictly adhered to ethical principles and ensured patient confidentiality throughout the study. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: In the absence of significant pleural effusion, conventional medical thoracoscopy (MT) is often not feasible due to the risk of lung injury. Dry MT mitigates these risks by inducing artificial pneumothorax through needle insufflation or blunt dissection. Although the Veress needle is commonly used by surgeons to create pneumoperitoneum before laparoscopic surgeries, its application in dry MT has not been widely reported in recent times., Methods: We report on a series of 31 patients who underwent dry MT with artificial pneumothorax induction using Veress needle under thoracic ultrasonography (TUS) guidance. A procedure was considered technically successful if it met all the following criteria: successful pneumothorax induction, allowing smooth insertion of the semi-rigid thoracoscope; absence of immediate significant procedural-related complications; and no delayed complications such as persistent air leaks, defined as leakage lasting more than 5 days necessitating extended chest tube placement., Results: Complete pneumothorax induction was achieved in 25 cases, resulting in an 80.6% technical success rate; however, biopsies were successfully performed in all cases. The most frequent histopathological diagnoses were malignancy (n=9, 29.0%), followed by inflammatory pleuritis (n=8, 25.8%) and tuberculosis (n=8, 25.8%). No procedural complications were reported., Conclusion: These results indicate that TUS-guided dry MT utilizing a Veress needle is technically feasible and secure when performed by experienced MT practitioners in TUS.

Introduction: Depression is prevalent after traumatic brain injury (TBI). However, there is a lack of understanding of the brain-based correlates of depression post-TBI. This systematic review aimed to synthesize findings of structural and functional magnetic resonance imaging (MRI) studies to identify consistently reported neural correlates of depression post-TBI., Methods: A search for relevant published studies was conducted through OVID (MEDLINE, APA PsycINFO, and Embase), with an end date of August 3rd, 2023. Fourteen published studies were included in this review., Results: TBI patients with depression exhibited distinct changes in diffusion- based white matter fractional anisotropy, with the direction of change depending on the acuteness or chronicity of TBI. Decreased functional connectivity (FC) of the salience and default mode networks was prominent alongside the decreased volume of gray matter within the insular, dorsomedial prefrontal, and ventromedial prefrontal cortices. Seven studies reported the correlation between observed neuroimaging and depression outcomes. Of these studies, 42% indicated that FC of the bilateral medial temporal lobe subregions was correlated with depression outcomes in TBI., Discussion: This systematic review summarizes existing neuroimaging evidence and reports brain regions that can be leveraged as potential treatment targets in future studies examining depression post-TBI., Competing Interests: VB is supported by an Academic Scholar Award from the Department of Psychiatry, University of Toronto, and has received research support from the Canadian Institutes of Health Research, Brain & Behavior Foundation, Ontario Ministry of Health Innovation Funds, Royal College of Physicians and Surgeons of Canada, Department of National Defence (Government of Canada), New Frontiers in Research Fund, Associated Medical Services Inc. Healthcare, American Foundation for Suicide Prevention, Roche Canada, Novartis, and Eisai. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Baltazar, Demchenko, Tassone, Sousa-Ho, Schweizer and Bhat.)

Competing Interests: Disclosure: There is no conflict of interest or other disclosures.

Competing Interests: Declaration of interests We declare no competing interests.

Docetaxel (DTX) is widely used for metastatic castrated resistant prostate cancer, but its efficacy is often compromised by drug resistance associated with low intracellular concentrations. Piperine (PIP) could enhance the bioavailability of other drugs via the inhibition of CYPs and P-gp activities. Thus, we hypothesize a positive effect with the DTX-PIP combination on the anti-tumor efficacy and intra-tumor DTX concentrations in taxane-resistant prostate cancer. ICR-NOD/SCID mice implanted with taxane-resistant human prostate cancer cells were administrated with saline as well as PIP and DTX separately or in combination. The tumor growth was monitored together with intra-tumor concentrations of DTX. The inhibitory effects on CYPs and P-gp were further assessed in mouse liver microsome and MDCK-MDR1 cells. Compared with DTX alone, DTX-PIP combination significantly inhibited the tumor growth (114% vs. 217%, p = 0.002) with corresponding significantly higher intra-tumor DTX concentrations (5.854 ± 5.510 ng/ml vs. 1.312 ± 0.754 ng/mg, p = 0.037). The percentage of DTX metabolism was significantly decreased from 28.94 ± 1.06% to 18.14 ± 2.22% in mouse liver microsome after administration of PIP for two weeks. DTX accumulation in MDCK-MDR1 cell was significantly enhanced in the presence of PIP. Further microarray analysis revealed that PIP inhibited P-gp as well as CYP1B1 gene expression and induced a significant gene expression change relating to inflammatory response, angiogenesis, cell proliferation, or cell migration. In conclusion, DTX-PIP combination significantly induces activity against taxane-resistant prostate tumor. Such effect appeared to be attributed to the inhibitory effect of PIP on CYPs and P-gp activity as well as gene expression changes relating to tumorigenesis and cellular responses., Competing Interests: CONFLICTS OF INTEREST None.

Although over 100 chemotherapeutic agents are currently available for the treatment of cancer patients, the overall long term clinical benefit is disappointing due to the lack of effectiveness or severe side effects from these agents. In order to improve the therapeutic outcome, a new approach called precision medicine or personalized medicine has been proposed and initiated by the U.S. National Institutes of Health. However, the limited availability of effective medications and the high cost are still the major barriers for many cancer patients. Thus alternative approaches such as herbal medicines could be a feasible and less costly option. Unfortunately, scientific evidence for the efficacy of a majority of herbal medicines is still lacking and their development to meet FDA approval or other regulatory agencies is a big challenge. However, herbal medicines may be able to play an important role in precision medicine or personalized medicine. This review will focus on the existing and future technologies that could speed the development of herbal products for treatment of resistant cancer in individual patients. Specifically, it will concentrate on reviewing the phenotypic (activity based) rather than genotypic (mechanism based) approach to develop herbal medicine useful for personalized cancer chemotherapy.

Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy.

Zinc-finger, MYND-type containing 10 (ZMYND10), or more commonly called BLU, expression is frequently downregulated in nasopharyngeal carcinoma (NPC) and many other tumors due to promoter hypermethylation. Functional evidence shows that the BLU gene inhibits tumor growth in animal assays, but the detailed molecular mechanism responsible for this is still not well understood. In current studies, we find that 93.5% of early-stage primary NPC tumors show downregulated BLU expression. Using a PCR array, overexpression of the BLU gene was correlated to the angiogenesis network in NPC cells. Moreover, expression changes of the MMP family, VEGF and TSP1, were often detected in different stages of NPC, suggesting the possibility that BLU may be directly involved in the microenvironment and anti-angiogenic activity in NPC development. Compared with vector-alone control cells, BLU stable transfectants, derived from poorly-differentiated NPC HONE1 cells, suppress VEGF165, VEGF189 and TSP1 expression at both the RNA and protein levels, and significantly reduce the secreted VEGF protein in these cells, reflecting an unknown regulatory mechanism mediated by the BLU gene in NPC. Cells expressing BLU inhibited cellular invasion, migration and tube formation. These in vitro results were further confirmed by in vivo tumor suppression and a matrigel plug angiogenesis assay in nude mice. Tube-forming ability was clearly inhibited, when the BLU gene is expressed in these cells. Up to 70-90% of injected tumor cells expressing increased exogenous BLU underwent cell death in animal assays. Overexpressed BLU only inhibited VEGF165 expression in differentiated squamous NPC HK1 cells, but also showed an anti-angiogenic effect in the animal assay, revealing a complicated mechanism regulating angiogenesis and the microenvironment in different NPC cell lines. Results of these studies indicate that alteration of BLU gene expression influences anti-angiogenesis pathways and is important for the development of NPC.

SNPs in human AFP promoter are associated with serum AFP levels in hepatocellular carcinoma (HCC), suggesting that AFP promoter variants may generate better transcriptional activities while retaining high specificity to AFP-producing cells. We sequenced human AFP promoters, cloned 15 different genotype promoters and tested their reporter activities in AFP-producing and non-producing cells. Among various AFP variant fragments tested, EA4D exhibited the highest reporter activity and thus was selected for the further study. EA4D was fused with tBid and coupled with nano-particle vector (H1) to form pGL3-EA4D-tBid/H1. pGL3-EA4D-tBid/H1 could express a high level of tBid while retain the specificity to AFP-producing cells. In a HCC tumor model, application of pGL3-EA4D-tBid/H1 significantly inhibited the growth of AFP-producing-implanted tumors with minimal side-effects, but had no effect on non-AFP-producing tumors. Furthermore, pGL3-EA4D-tBid/H1 could significantly sensitize HCC cells to sorafenib, an approved anti-HCC agent. Collectively, pGL3-EA4D-tBid/H1, a construct with the AFP promoter EA4D and the novel H1 delivery system, can specifically target and effectively suppress the AFP-producing HCC. This new therapeutic tool shows little toxicity in vitro and in vivo and it should thus be safe for further clinical tests., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Mutations in HBx gene are frequently found in HBV-associated hepatocellular carcinoma (HCC). Activation of hypoxia-inducible factor-1α (HIF-1α) contributes to HCC development and progression. Wild-type HBx has been demonstrated to activate HIF-1α, but the effect of HBx mutations on HIF-1α has not been elucidated., Methods: HBx mutations were identified by gene sequencing in 101 HCC tissues. Representative HBx mutants were cloned and transfected into HCC cells. Expression and activation of HIF-1α were analysed by western blot and luciferase assays, respectively. The relationship between HBx mutants and HIF-1α expression in HCC tissues was also evaluated., Results: The dual mutations K130M/V131I enhanced the functionality of HBx as they upregulated the expression and transcriptional activity of HIF-1α. The C-terminal truncations and deletion mutations, however, weakened the ability of HBx to upregulate HIF-1α. Meanwhile, the C-terminus was further found to be essential for the stability and transactivation of HBx. In the HCC tissues, there was a positive association between the HBx mutants and HIF-1α expression., Conclusion: Different mutations of HBx exert differentiated effects on the functionality of HIF-1α, however, the overall activity of HBx mutants appears to increase the expression and transcriptional activity of HIF-1α.

Purpose: The overexpression of hypoxia-inducible factor 1α (HIF-1α) is a common finding in hepatocellular carcinoma (HCC), and it leads to angiogenesis and poor prognosis. Sorafenib, a multikinase inhibitor, has shown significant improvement in survival in patients with advanced HCC in clinical trials. However, the mechanisms that account for the antiangiogenic efficiency of sorafenib have not been fully elucidated. The present study aims to explore the effect of sorafenib on HIF-1α expression and activation in HCC cells and xenografts., Experimental Design: HCC cells and xenografts were treated with sorafenib or vehicles. Western blotting and quantitative PCR array were used to determine protein and mRNA expression, respectively. HIF-1α activity, de novo protein synthesis, and VEGF secretions were determined using assay kits., Results: Sorafenib dose dependently decreased the hypoxia-induced accumulation and activation of HIF-1α protein. Further analysis revealed that such reduction of HIF-1α was associated with the inhibition of HIF-1α protein synthesis rather than the promotion of HIF-1α protein degradation or the reduction of HIF-1α mRNA. Moreover, the phosphorylation levels of mTOR, extracellular signal-regulated kinase (ERK), p70S6K, RP-S6, 4E-BP1, and eIF4E were significantly suppressed by sorafenib. In vivo studies further confirmed the inhibitory effect of sorafenib on the expression of HIF-1α and VEGF proteins, leading to a decrease in tumor vascularization and growth of the xenografts., Conclusions: Sorafenib-mediated inhibition of HIF-1α synthesis is associated with previously undefined pathways in which mTOR/p70S6K/4E-BP1 and ERK phosphorylation are downregulated. Our preclinical data expand our understanding of sorafenib's antiangiogenic mechanism of action by inhibiting HIF-1α and VEGF protein expression., (©2012 AACR)

Objective: Esthesioneuroblastoma is an uncommon tumor that is described widely among the Caucasians. In Singapore, we see predominantly Asian patients with esthesioneuroblastomas. From our experience, we note significant and interesting differences between our data on Asian patients and the published ones on the Caucasian patients., Methods: A retrospective review of all patients who underwent craniofacial resection for esthesioneuroblastomas was conducted from January 1997 to January 2010. Relevant data were collected and statistical analyses were carried out to determine factors that predicted mortality or complications., Results: Out of a total of 48 patients who underwent craniofacial resections, half had esthesioneuroblastomas (50%). There was a peak age distribution at the sixth decade of life and 62% of our patients were male. Both local and regional recurrence rate was 50%., Conclusion: Majority of our Asian patients who underwent craniofacial resections had esthesioneuroblastomas. There is a male predilection, and we do not see a bimodal age distribution that is commonly reported., (Copyright © 2012. Published by Elsevier B.V.)

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC., (Copyright © 2010 Elsevier B.V. All rights reserved.)

ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A self-administered network-based questionnaire survey was conducted on 91 HIV-positive Chinese men having sex with men (MSM). Affiliation matrices were created to assess their networking pattern. The individuals' preferential use of venues for sex partnership before HIV infection has changed over three time periods of 1997-2000, 2001-2003 and 2004-2006. Over time, there was a parallel increase in network density (density scores from 0.26, through 0.36, to 0.53) and degree centrality (from a median score of 9, through 12, to 16), suggesting that connectivity of MSM was becoming higher through sexual networks. The overall practice of unprotected sex has, however, remained the same. The study demonstrated how the application of social network analysis could enrich the epidemiologic description of HIV infection in the population., (2009 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd.)

Investments in systems biology approaches by the pharmaceutical industry have not yet yielded the payoffs envisaged by many. In most cases, a plethora of novel drug targets arising from genomics led to many more failed projects in the pipeline, suggesting that target-based drug discovery may not be an optimal strategy for the industry. Before high-throughput '-omics' technologies and computer analysis became commonplace, most drug candidates were laboriously screened in animal systems to identify compounds that produced useful responses. Interestingly, the targets of many of the compounds that became drugs are still uncertain to this day. It is likely that drugs act on multiple targets in concert over time, the identification of which will require not only system level cataloguing and measurements, but next generation multiscale systems modelling. The concept of a 'differentiated drug response'- elucidating and integrating responses composed of a range of effects on different tissues and, importantly, different time scales - may eventually prove to be the dominant paradigm of systems biology research. In this article, we explore key relevant concepts and technologies that we believe are critical for the future of systems biology and its place in pharmaceutical research.

Background: Variations of serum AFP levels in HCC patients and cell lines are likely due to the differential activity of enhancer/silencer elements that control AFP. To understand the potential mechanism underlying the differential expression of AFP, we have examined the sequence of the AFP promoter in HCC., Methods: Direct DNA sequencing was carried out to sequence 980 bp of AFP promoter of DNA samples isolated from 83 HCC patients., Results: Three novel SNPs in the promoter region of the AFP gene, which have not been previously reported, were found at positions -330, -401 and -692. The level of serum AFP was significantly higher in HCC patients with the CT genotype of 330 SNP or the AG genotype of the 401 SNP. The genotype of CG in 692 SNP was also associated with a significant elevated level of serum AFP, and further this genotype appeared to be associated with the high risk of HCC development. 401 SNP and 692 SNP were located at the positions of known binding sites for transcription factors that have a role in the production of AFP and the growth of tumors., Conclusions: The novel polymorphisms identified in the promoter region of the AFP gene may be pathologically significant in HCC.

BH3-only protein Bim is a critical regulator of apoptosis and plays an essential role in mammalian development, but the characterization of Bim and its isoforms in hepatocellular carcinoma (HCC) has not been studied before. Here we investigated the expression, distribution, regulation and role of Bim isoforms in HCC cells. Fifteen Bim isoforms were identified in HCC, with six newly identified isoforms and two newly identified exons. Among of them, Bim EL, L, S, a1, a2, a3, b2, b4 and b6 are abundant isoforms according to their mRNA levels. However only Bim EL, L and S proteins could be clearly detected. Bim mRNA and protein were strongly expressed in HCC tissues compared to relevant non-tumorous regions, but the ratio of variant isoforms showed no difference between tumorous and non-tumorous tissues. Bim isoforms were differentially regulated after chemotherapeutic drug 5-Fluorouracil (5-FU) treatment. Interestingly, Bim EL, L and S, the isoforms known to induce apoptosis strongly, are the least inducible isoforms at their mRNA levels when exposed to the stress, suggesting that post-transcriptional rather than transcriptional, modulations may play a role to enhance their functions. Finally, overexpression of Bim EL, L, S and all alpha isoforms induced apoptosis in HCC cells, while overexpression of Bim beta isoforms showed no effects on cell survival after 5-FU treatment. In conclusion, Bim alpha isoforms appears to have a role in the regulation of apoptosis in HCC cells, which may contribute to not only the growth of tumor cells but also the sensitivity of HCC cells to chemotherapy.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, gene therapy may represent an alternative for HCC management. Our earlier study has shown that Bid plays a role in the development of HCC. The aim of our study is to evaluate the possibility of using truncated Bid (tBid) as a novel therapy for HCC treatment. Two HCC cell lines, Hep3B and PLC/PRF/5, were used in the experiment. Hep3B was a p53-resistant while PLC/PRF/5 a p53-sensitive. A recombinant adenovirus-Ad/AFPtBid, which contained a tBid gene driven by an alpha-fetoprotein (AFP) promoter, was constructed. Both Hep3B and PLC/PRF/5 cells infected with Ad/AFPtBid showed a significant decrease in cell viability. The decrease in cell viability by Ad/AFPtBid resulted from apoptosis of HCC cells, evident by enhanced activity of caspases and increased release of cytochrome c. In vivo experiment was performed by the intratumor injection of Ad/AFPtBid in nude mice inoculated with Hep3B. Ad/AFPtBid injection significantly inhibited tumor growth, and tumor tissues showed a marked increase in TUNEL-positive cells. Our experiment also demonstrated that Ad/AFPtBid only targeted AFP-producing cells but not those non-AFP producing cells. In conclusion, these results indicate that the introduction of Ad/AFPtBid can not only significantly but specifically kill HCC cells that produce AFP. The cell death induced by Ad/AFPtBid in HCC cells is via an apoptotic pathway that can be independent of p53 status., (Copyright 2006 Wiley-Liss, Inc.)

Background: Chronic infection by hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) in man. The viral transactivator HBV X (HBx) gene plays a critical role in the molecular pathogenesis of HBV-related HCC., Objectives: The aim of this study was to investigate whether there were particular HBx mutations associated with the Chinese Hong Kong patients with HCC., Study Design: We have examined HBx in 113 tumor tissue samples from patients with HCC and 48 serum samples from the same group. In addition, we also examined the expression of HBx protein and the index of apoptotic cell death in tumor tissues of HCC. The entire coding region of HBx gene from the sample was sequenced and aligned with the published HBx gene sequence., Results and Conclusions: We have identified total 54 different types of mutations in HBx gene. HBx mutations occurred in a very high percentage of samples tested. Mutation of HBx was found in 95.2% and 95.3% of the tumor tissue and serum samples, respectively. Most of samples contained more than one type of the mutation. Relative risk analysis indicated that the mutations in 12 sites of tissue HBx and nine sites of serum HBx were highly associated with HCC, suggesting a potential role of these mutants in carcinogenesis. An insert mutation at position 204: Insert 204AGGCCC, was always found to co-exist with point mutations at 260 (G-->A) and 264 (G/C/T-->A). Furthermore, this particular pattern of HBx mutation was most frequently detected. Immunochemical staining of HBx protein revealed that the nuclear localization of HBx protein in hepatocytes of tumor tissues was highly associated with this particular pattern of HBx mutation. In conclusion, HBx mutation occurs frequently in HCC samples tested and a sample usually has multiple types of mutations. A special pattern of insert at 204 and point mutations at 260 and 264 was identified, and it appears to be associated with the nuclear localization of HBx protein. The development of multiple types of mutations in a given sample may contribute to the process of multiple steps in hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Asia. Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an interferon-induced, serine/threonine protein kinase. Recent studies have suggested that PKR is involved in the pathogenesis of HCC with hepatitis virus C infection by inhibiting viral and cellular proteins related to cell growth and proliferation. In the present study, PKR was examined in both tumor and non-tumor tissues from HCC livers infected with HBV. The expression of PKR was determined by TaqMan real-time PCR and immunohistochemical methods. The level of PKR was also analyzed in relation to pathological changes observed in HCC. The result showed that PKR was reduced in tumor tissues of HCC from HBV carriers with low serum viral load (<0.7 x 10(6) copies/ml) compared to those with higher serum viral load. However, the overall PKR level was much lower in tumor tissues than that in non-tumor tissues, irrespective of HBV carrier status or serum viral load. PKR level tended to be lower in HCC samples with alpha-fetoprotein (AFP) more than 500 ng/ml (mean: 4024.2 ng/ml) than those with AFP less than 500 ng/ml (mean: 50.6 ng/ml). There was no significant difference in the expression of PKR between tumor tissues with well differentiation and those with poor or moderate differentiation. In conclusion, the level of PKR was reduced in HCC tumor tissues, suggesting a possible role of PKR in promoting the growth of tumor. HBV may participate in altering the level of PKR, but factors other than HBV should play a more determining role in the regulation of PKR in HCC. The association between PKR and AFP levels may offer an alternative tumor marker for HCC., (Copyright 2004 Wiley-Liss, Inc.)

Biosimulation software is being used in pharmaceutical drug development to mimic diseases. Virtual clinical trials of new developing pharmaceutical drugs can be conducted on computers running disease simulations. Using virtual patients instead of clinical research patients can save both time and money for pharmaceutical companies in their search to discover new drugs and bring them to market. In the future, this type of research will be commonplace.

p53 has recently been identified as a downstream target of ZBP-89, a zinc finger transcription factor. ZBP-89 promotes growth arrest through stabilization of the p53 protein. The aim of this study is to determine the status of the p53 gene in recurrent human hepatocellular carcinoma (HCC) and test the link between the expression of ZBP-89 and the p53 gene. The results showed that mutations in the p53 gene were frequently detected in recurrent HCC. The interval between surgical resection and the recurrence of HCC was significantly longer in patients with the wild-type p53 gene than those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence. Among those positive for the p53 protein, nearly 85% (18 of 21) showed nuclear localization of the p53 protein while only about 14% (3 of 21) were positive for the p53 protein in the cytoplasm. ZBP-89 co-localized with p53 in the nucleus in about 67% (12 of 18) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, co-localization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment.

As a mitochondrial membrane death ligand, Bid oligomerises Bak to release cytochrome C and its deficiency renders hepatocytes resistant to apoptosis induced by Fas. The Bid level in hepatocellular carcinoma (HCC) is unknown. In this report, we examined the expression of Bid protein and mRNA in HCC cancerous tissues and their corresponding non-cancerous ones. The effect of the hepatitis B x protein (HBx) on the expression of Bid was also evaluated by transfecting hepatoma cells with the HBx gene. The results showed that the expression of Bid was significantly lower in cancerous tissues than that in their corresponding non-cancerous tissues. Immunohistochemical study revealed that Bid molecule was mainly localised in hepato-cytoplasm. Some nuclei were also positive for Bid antigen though to a lesser degree. In vitro experiments demonstrated that the expression of Bid in cells transfected with HBx was significantly lower than that in the cells without HBx transfection. This finding suggests that HBx may play a causative role in the reduction of Bid expression in HCC. This in vitro result is, to some degree, supported by clinical data that all the HCC examined are positive for hepatitis B virus (HBV). We conclude from this data that the expression of Bid in HCC is significantly decreased and the reduction of Bid may result from a mechanism associated with HBx, a major hepatocarcinogenic product from HBV. The imbalance of increased anti-apoptosis and decreased pro-apoptosis seen in HCC is a critical mechanism leading to the uncontrolled growth of tumour cells. Therefore, this study suggests that a deficiency in the expression of Bid may contribute to the development of such an imbalance in HCC.

Objective: To compare the systemic cytokine response in patients after laparoscopic-assisted resection with those after open resection of rectosigmoid carcinoma., Summary Background Data: Laparoscopic resection of colorectal carcinoma is technically feasible, but objective evidence of its benefit is scarce. Systemic cytokines are accepted as markers of postoperative tissue trauma and mediators of the host immune response., Methods: Thirty-four patients with rectosigmoid carcinoma, without evidence of metastatic disease and suitable for laparoscopic resection, were randomized to undergo either laparoscopic (n = 17) or conventional open (n = 17) resection of the tumor. Clinical parameters were recorded. Sera were collected before surgery and at appropriate time points afterward and assayed for interleukin-1beta, tumor necrosis factor-alpha, interleukin-6, and C-reactive protein. The primary end points were the cytokine and C-reactive protein levels. Data were analyzed by intention to treat., Results: The demographic data of the two groups were comparable. The clinical outcome of both groups was satisfactory, with no surgical deaths and a reasonable complication rate. Both interleukin-1beta and interleukin-6 levels peaked 2 hours after surgery, with the responses in the laparoscopic group significantly less than those in the open group. C-reactive protein levels peaked at 48 hours, and the difference was also statistically significant. Levels of tumor necrosis factor-alpha were not elevated after surgery, and there was no difference between the groups., Conclusions: Tissue trauma, as reflected by systemic cytokine response, was less after laparoscopic resection than after open resection of rectosigmoid carcinoma. The difference in the systemic cytokine response may have implications on the long-term survival.

Background: Aminoleban EN contains branched-chain amino acids (BCAA) and is known to be beneficial for the protein-energy malnutrition in cirrhotic patients. Patients suffering from hepatocellular carcinoma often have background cirrhosis, and the present study investigates the effect of Aminoleban EN on these patients after hepatic resection for the primary disease., Methods: A prospective randomized controlled clinical trial, to which 50 patients were recruited, was carried out. The study group received Aminoleban EN in addition to normal diet for 12 weeks and the control group received an isonitrogenous and isocaloric diet only., Results: After exclusions, there were 21 patients in the study group and 23 patients in the control. The study group had a shorter hospital stay, and had a significantly higher haemoglobin level, higher sodium level, higher albumin level and lower bilirubin during the postoperative course. There was no significant difference in terms of neuropsychiatric symptoms or Karnofsky performance score. There was no difference in gastrointestinal symptoms or other signs. No adverse reaction was associated with the administration of Aminoleban, and there was no significant difference in terms of morbidity and mortality between the two groups of patients., Conclusion: Aminoleban EN is safe to administer and does not have significant adverse effects. It contributes to a shorter hospital stay and quicker improvement of liver function in the early postoperative period. These beneficial results require only a 12-week period of administration of BCAA after operation.

This laboratory has reported the conditions for an effective, non-toxic, chemoimmunotherapy utilizing doxorubicin in combination with prolonged administration of interleukin-2 and the identification of the critical role of activated CD8+ T cells in the therapeutic effect. Mice (C57BL/6) cured in those studies have been followed for the remainder of their life spans. These mice, approximately 2 months of age when initially inoculated with syngeneic EL4 lymphoma, survived for more than 2 years, the normal life span of C57BL/6 mice. Mice 4 months old reinoculated with the EL4 cells all survived. At about 1 year of age mice were sacrificed and the ability of their thymocytes and splenocytes to develop specific CD8+ anti-EL4 activity was as high as it had been at the time of tumor rejection. At about 2 years of age EL4 was reimplanted into mice; all of them survived. These surviving mice, at 2 years 2 months of age, as well as a group of 2-year-old mice not rechallenged, were killed and functional antitumor activity and phenotype characteristics of various lymphocyte populations were determined in comparison to those of young and age-matched control mice. The phenotyping of the lymphocytes from the cured mice indicated very notable differences in subset distribution and increased CD44 expression. Functionally they developed high levels of anti-EL4 activity, which was ablated by combined treatment with monoclonal antibodies against CD8 and CD44, indicating the role of memory cells. Consistent with cells from aged mice, these same cell populations had a very reduced allogeneic responsiveness. It appears that cured mice have developed an immune memory specific for EL4.

Recently we have reported the development of a safe and effective chemoimmunotherapy protocol involving doxorubicin (Dox) in combination with interleukin 2 which, in C57BL/6 mice, boosts local T cell responses, and, in 50 to 80% of the cases, this resulted in the complete eradication of established syngeneic EL4 lymphoma or its Dox-resistant variant, EL4/A. Accumulation of host-derived leukocytes in the peritoneal cavity was increased up to 8-fold after tumor inoculation, but, in absolute numbers, did not increase further following Dox administration. The cellular pharmacokinetic studies undertaken to clarify the role of Dox following a single IV injection indicate that 4 h later, lymphocytes found in the peritoneal cavity have detectable levels of Dox; but the lymphoma cells (both EL4 and EL4/A) have, in proportion to their larger size, taken up more drug as judged by flow cytometry. The estimated drug "concentration" (i.e., intracellular amount divided by estimated cell size) at the 4-h time point, however, was found to be essentially equivalent in both the lymphoma cells and the lymphocytes. Thereafter, the drug content and intracellular "concentration" in the EL4/A cells rapidly declined while their numbers progressively increased. In contrast, the EL4 lymphoma cells and the lymphocytes found in the peritoneal cavity in the presence of either lymphoma consistently exhibited higher levels of drug 24-48 h than at 4 h. Splenic and tumor-infiltrating mature T (CD3+) cells were completely insensitive to Dox cytotoxicity and actually showed increased CTL activity when examined ex vivo. Although EL4 cells had identical Dox uptake patterns to those of CD3+ cells, they were sensitive to the drug and their numbers decreased, resulting in increased host/tumor cell ratios in these mice. The pharmacokinetic parameters of the drug and the insensitivity of the mature T cells to the drug determined in this study can explain, in part, the efficacy of a chemoimmunotherapy protocol boosting local T-cell responses.

A safe and effective therapeutic combination of moderate doses of Adriamycin (doxorubicin, 4 mg/kg, IV, Days 1 and 8 or only Day 8) plus prolonged administration of moderate doses of interleukin 2 (2 micrograms, b.i.d., Days 9-40) was developed in the syngeneic EL4 (5 x 10(4) cells, IP, Day 0) lymphoma--C57B1/6 mouse model and has been reported in the companion paper. The studies described herein demonstrate that the effectiveness of this combination treatment against EL4 lymphoma growing intraperitoneally in C57B1/6 mice was dependent upon the presence of CD8+ cells. Thus, the induction of long-term survivors (60-80%) by Adriamycin plus interleukin 2 was completely ablated by pretreatment of mice with anti-CD8 monoclonal antibody (MAb), whereas pretreatment with anti-CD4 MAb only partially inhibited the therapeutic effects and anti-NK1.1 MAb had no effect. A close association between survival, an increase in phenotypically identified CD8+ cells, and an increase in specific anti-EL4 cytolytic activity was demonstrated with cells from the tumor site (peritoneum) but not consistently with cells from the spleen. No association was observed between survival and modulations in natural killer (NK), lymphokine-activated killer (LAK), or tumoricidal macrophage activity of spleen or peritoneal cells. Taken together the results indicate that, in this model, the most relevant correlate of a therapeutically effective host antitumor response is the level of specific EL4 tumor killing by cells present at the tumor site. Based on the findings reported herein, it can be predicted that weakly immunogenic tumors may be eradicated by immunologic mechanisms elicited in conjunction with properly designed therapeutic regimens.

This laboratory has extensively studied Adriamycin (doxorubicin)-induced immunomodulation. Despite demonstration of favorable effects, little therapeutic advantage was seen, and it was decided to test Adriamycin in combination with interleukin 2 (IL2). Considerable toxicity was seen with either high-dose IL2 or high-dose Adriamycin alone, using the syngeneic C57B1/6-EL4 T cell lymphoma model. When the doses of either agent were reduced to decrease toxicity, little therapeutic effect was seen. In contrast, an effective protocol without apparent toxicity was developed by combining a moderate dose of Adriamycin (4 mg/kg, IV, Days 1 and 8 or only Day 8) with prolonged administration of a moderate dose of IL2 (2 micrograms, b.i.d., i.p., Days 9 to 40). This protocol resulted in up to 80% long-term survivors among mice inoculated on Day 0 with EL4 lymphoma (5 x 10(4) cells). It should be noted that under these conditions, neither agent, when administered singly, induced long term survivors, and that following the inoculation of only 10-100 EL4 tumor cells all animals died in the absence of treatment. The survivors developed protective immunity as demonstrated by their ability to resist reimplantation with EL4 tumor. Furthermore, this resistance to tumor reimplantation could be transferred into naive hosts with spleen cells from tumor-bearing mice receiving the combination protocol; exposure of mice to sublethal whole body irradiation prior to tumor implantation completely abrogated the efficacy of this combination treatment. Finally, it was shown that this combination protocol was equally effective against an Adriamycin-resistant subline of EL4 that expresses the multidrug resistance phenotype.

Using the C57BL/6/EL4 tumor model, studies were carried out to demonstrate the feasibility of administering Adriamycin (ADM) in therapeutic doses and schedules such that the host antitumor defenses would not be suppressed and in some cases might be stimulated by treatment. ADM treatment caused prolongation of survival and, in general, either stimulated host cytolytic activities above untreated control levels or had no effect. These effects by ADM were observed with the ADM-sensitive parent EL4 line as well as with an ADM-resistant subline, indicating that the effects did not result entirely from direct antitumor activity. The cytolytic activities examined were those of cytolytic T-lymphocytes, lymphokine-activated killer cells, and splenic and peritoneal macrophages. All activities were assessed against the syngeneic EL4 target line. The information obtained in this investigation provides a rational basis for the future development of curative protocols with ADM plus biological response modifiers, which would depend on a functional immune system for optimum efficacy and would also exploit synergistic immunomodulating effects of the agents used in combination.

A number of recent studies have implied that a relationship exists between cellular sensitivities to tumor necrosis factor (TNF) and expression of the classic multidrug resistance (MDR) phenotype. However, different conclusions have been reported concerning whether TNF sensitivity is positively or negatively correlated with MDR (Hong, W.-S.; Sijo, N.; Sasaki, Y.; Shinkai, T.; Eguchi, K.; Sakurai, M.; Takamashi, H.; Nakano, H.; Nakagawa, K.; Twentyman, P. R. Jpn. J. Can. Res. (Gann) 78:1274-1280; 1987 and Dollbaum, C.; Creasey, A. A.; Dairkee, S. H.; Hiller, A. J.; Rudolph, A. R.; Lin, L.; Vitt, C.; Smith, H. S. Proc. Natl. Acad. Sci. USA 85:4740-4755; 1988). An apparent relationship of TNF sensitivity to P-glycoprotein (P-170gp) mediated MDR was investigated in EL4 murine T-lymphoma cell lines sensitive and resistant to Adriamycin (ADM). No consistent association was found between MDR and TNF responses when the lines were subcloned. Whereas the MDR phenotype of subclones (as assessed by ADM resistance and P-170gp expression) reflected that of the cell line from which they were derived, the TNF sensitivity of subclones varied widely. Also consistent with independence of P-170gp mediated MDR and TNF response, the P388/ADM cell line (exhibiting P-170gp mediated MDR) remained as resistant to TNF as the P388 parental line. In addition, no evidence was found of modified recognition of MDR EL4 cell lines by host defense effector cells, and gamma-interferon failed to enhance the susceptibility of either parental or MDR cell line to TNF. These results may be of value in considering therapeutic studies using the ADM/TNF combination treatment.

Recombinant murine (rMu) tumor necrosis factor (TNF), in a standard comitogenic assay with phytohemagglutinin, induced murine thymocyte proliferation, while up to 10,000-fold higher concentrations of recombinant human TNF did not. The induction of thymocyte proliferation was dependent upon TNF concentration in a biphasic manner. Thus, 100 to 1,000 units/ml TNF were near optimal while concentrations greater than or equal to 1,000 units/ml caused apparent down regulation. The effect was abrogated by neutralizing antibody to rMu-TNF but not by neutralizing antibody to rMu-interleukin 1 alpha or beta. The rMu-TNF did not induce proliferation of the mature murine T-helper cell line, D10.G4.1, in the presence of mitogen. Taken together the results indicate that TNF, in a strictly species-specific manner, can regulate thymocyte proliferation independently of interleukin 1.