Genome-wide loss-of-function genetic screen identifies INSIG2 as the vulnerability of hepatitis B virus-integrated hepatoma cells.

There are approximately 250 million people chronically infected with hepatitis B virus (HBV) worldwide. Although HBV is often integrated into the host genome and promotes hepatocarcinogenesis, vulnerability of HBV integration in liver cancer cells has not been clarified. The aim of our study is to identify vulnerability factors for HBV-associated hepatocarcinoma. Loss-of-function screening was undertaken in HepG2 and HBV-integrated HepG2.2.15 cells expressing SpCas9 using a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library. Genes whose guide RNA (gRNA) abundance significantly decreased in HepG2.2.15 cells but not in HepG2 cells were extracted using the MAGeCK algorithm. We identified four genes (BCL2L1, VPS37A, INSIG2, and CFLAR) that showed significant reductions of gRNA abundance and thus potentially involved in the vulnerability of HBV-integrated cancer cells. Among them, siRNA-mediated mRNA inhibition or CRISPR-mediated genetic deletion of INSIG2 significantly impaired cell proliferation in HepG2.2.15 cells but not in HepG2 cells. Its inhibitory effect was alleviated by cotransfection of siRNAs targeting HBV. INSIG2 inhibition suppressed the pathways related to cell cycle and DNA replication, downregulated cyclin-dependent kinase 2 (CDK2) levels, and delayed the G1 -to-S transition in HepG2.2.15 cells. CDK2 inhibitor suppressed cell cycle progression in HepG2.2.15 cells and INSIG2 inhibition did not suppress cell proliferation in the presence of CDK2 inhibitor. In conclusion, INSIG2 inhibition induced cell cycle arrest in HBV-integrated hepatoma cells in a CDK2-dependent manner, and thus INSIG2 might be a vulnerability factor for HBV-associated liver cancer.

Pentraxin 3 is an adipose tissue-related serum marker for pancreatic cancer cachexia predicting subsequent muscle mass and visceral fat loss.

Cancer cachexia, a paraneoplastic syndrome characterized by ongoing skeletal muscle mass loss, is accompanied by adipose tissue loss and strongly affects chemotherapy endurance. Our aim was to detect a serum marker reflecting pancreatic cancer cachexia and predicting subsequent loss of muscle mass and adipose tissue, focusing on adipose tissue-secreted proteins. Murine-derived pancreatic cancer cells were orthotopically injected into the mouse pancreatic tail. After 3 weeks, RNA sequencing of perigonadal fat and orthotopic tumors was carried out. We analyzed stocked sera and clinical data of metastatic pancreatic cancer patients who received chemotherapy. Perigonadal fat weight/body weight decreased in mice with orthotopic tumors compared to those without tumors. By RNA sequencing and real-time PCR validation, pentraxin 3 (PTX3) was identified as a secreted protein-encoded gene whose expression was significantly higher in the perigonadal fat of mice with orthotopic tumors than in that of mice without orthotopic tumors and was least expressed in orthotopic tumors. Serum PTX3 levels correlated with PTX3 mRNA levels in perigonadal fat and were higher in mice with orthotopic tumors than in those without tumors. In 84 patients diagnosed with metastatic pancreatic cancer, patients with high serum PTX3 levels showed a greater visceral fat loss/month and skeletal muscle mass index (SMI) decrease/month than those with low serum PTX3 levels. High serum PTX3 was an independent risk factor for visceral fat loss, decreased SMI, and poor prognosis. High serum PTX3 in pancreatic cancer patients predicts visceral fat and muscle mass loss and major clinical outcomes of cancer cachexia.

Inhibition of nonhomologous end joining-mediated DNA repair enhances anti-HBV CRISPR therapy.

Current anti-hepatitis B virus (HBV) therapies have little effect on covalently closed circular DNA (cccDNA) and fail to eliminate HBV. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been reported to directly target cccDNA and exert antiviral effects. In this study, we hypothesized that the inhibition of the DNA repair machinery, which is important for the repair of CRISPR-induced double-strand breaks, may enhance the effect of CRISPR targeting cccDNA, and we investigated the antiviral effect of potential combination therapy. The antiviral effect of CRISPR targeting cccDNA (HBV-CRISPR) was evaluated in HBV-susceptible HepG2-hNTCP-C4 cells expressing Cas9 (HepG2-hNTCP-C4-iCas9) or primary human hepatocytes (PHHs) expressing Cas9. Following HBV infection, HBV-CRISPR reduced cccDNA levels, accompanied by decreases in pregenomic RNA (pgRNA) levels and supernatant HBV DNA, hepatitis B surface antigen and hepatitis B e antigen levels in HepG2-hNTCP-C4-iCas9 cells, and PHHs. HBV-CRISPR induced indel formation in cccDNA and up-regulated poly(adenosine diphosphate ribose) polymerase (PARP) activity in HBV-infected HepG2-hNTCP-C4-iCas9 cells. The suppression of PARP2-Histone PARylation factor 1 (HPF1) (involved in the initial step of DNA repair) with small interfering RNA (siRNA) targeting either PARP2 or HPF1 increased the reduction in pgRNA and cccDNA by HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells. The suppression of DNA Ligase 4 (LIG4) (essential for nonhomologous end joining [NHEJ]) but not breast cancer susceptibility gene (BRCA) (essential for homologous recombination) enhanced the antiviral effect of HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells. Finally, the clinically available PARP inhibitor olaparib increased the reductions in pgRNA and cccDNA levels induced by HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells and PHHs. Conclusion: The suppression of the NHEJ-mediated DNA repair machinery enhances the effect of CRISPR targeting cccDNA. The combination of CRISPR and olaparib may represent a therapy for HBV elimination.

Capsid Allosteric Modulators Enhance the Innate Immune Response in Hepatitis B Virus-Infected Hepatocytes During Interferon Administration.

Capsid allosteric modulators (CAMs) inhibit the encapsidation of hepatitis B virus (HBV) pregenomic RNA (pgRNA), which contains a pathogen-associated molecular pattern motif. However, the effect of CAMs on the innate immune response of HBV-infected hepatocytes remains unclear, and we examined this effect in this study. Administration of a CAM compound, BAY41-4109 (BAY41), to HBV-infected primary human hepatocytes (PHHs) did not change the total cytoplasmic pgRNA levels but significantly reduced intracapsid pgRNA levels, suggesting that BAY41 increased extracapsid pgRNA levels in the cytoplasm. BAY41 alone did not change the intracellular interferon (IFN)-stimulated gene (ISG) expression levels. However, BAY41 enhanced antiviral ISG induction by IFN-α in HBV-infected PHHs but did not change ISG induction by IFN-α in uninfected PHHs. Compared with BAY41 or IFN-α alone, coadministration of BAY41 and IFN-α significantly suppressed extracellular HBV-DNA levels. HBV-infected human liver-chimeric mice were treated with vehicle, BAY41, pegylated IFN-α (pegIFN-α), or BAY41 and pegIFN-α together. Compared with the vehicle control, pegIFN-α highly up-regulated intrahepatic ISG expression levels, but BAY41 alone did not change these levels. The combination of BAY41 and pegIFN-α further enhanced intrahepatic antiviral ISG expression, which was up-regulated by pegIFNα. The serum HBV-DNA levels in mice treated with the combination of BAY41 and pegIFN-α were the lowest observed in all the groups. Conclusion: CAMs enhance the host IFN response when combined with exogenous IFN-α, likely due to increased cytoplasmic extracapsid pgRNA.

SIRT1 enhances hepatitis virus B transcription independent of hepatic autophagy.

Autophagy is an intracellular process that can lead to the degradation of malfunctioned proteins and damaged organelles to maintain homeostasis during cellular stress. Here, we evaluated the change in hepatitis B virus (HBV) production by regulating hepatic autophagy in HBV-producing cells. We examined focusing on a relation with a positive autophagy regulator, sirtuin1 (SIRT1). Starvation and rapamycin treatment induced autophagy with increasing SIRT1 protein, HBc protein and pregenomic RNA (pgRNA) levels in HBV- producing cells. Knockdown of Atg7 or Atg13 suppressed hepatic autophagy, and it did not change SIRT1 protein, HBc protein or pgRNA levels in HBV- producing cells. Resveratrol, which increases SIRT1 expression and activity, promoted autophagy and increased HBc protein and pgRNA levels. siRNA-mediated knockdown of SIRT1 inhibited autophagy and decreased HBc protein and pgRNA levels. In SIRT1-knockdown cells, starvation promoted autophagy but did not increase HBc protein and pgRNA levels. In conclusion, HBc protein and pgRNA levels are upregulated not by the autophagic process itself but by the SIRT1 expression level.

Hepatitis C virus infection suppresses hepatitis B virus replication via the RIG-I-like helicase pathway.

Mechanisms of hepatitis B virus (HBV) reactivation after hepatitis C virus (HCV) elimination by direct-acting antiviral (DAA) treatment in HBV/HCV-co-infected patients remain unclear. We examined RIG-I-like helicase (RLH) pathway activation by HBV mono-infection, HCV mono-infection or HBV/HCV co-infection and interference between HBV and HCV in primary human hepatocytes. Interference between HBV and HCV and HBV reactivation after DAA treatment in humanized-liver mice were assessed. HCV infection activated RLH pathway, as evidenced by RIG-I, ISG15 and ISG56 expression induction; HBV caused only RIG-I induction in vitro. RLH activation was also found in HBV/HCV-co-infected cells, and HBV replication were suppressed in HBV/HCV-co-infected than in HBV-mono-infected cells. siRNA-mediated double knockdown of ISG15 and ISG56 increased HBV replication in HBV/HCV-co-infected cells. HCV infection activated RLH pathway and suppressed HBV replication in humanized-liver mice. Subsequent elimination of HCV by DAA administration downregulated RLH pathway and upregulated HBV replication in mice. RLH pathway was activated in livers of chronic hepatitis C patients compared to those of chronic hepatitis B or non-B, non-C patients. The RLH pathway activation was downregulated by HCV elimination. In conclusion, HCV infection activated RLH pathway and suppressed HBV replication in human hepatocytes. HCV elimination upregulated HBV replication, probably through RLH pathway downregulation.

Effects and safety of ¹³¹I-metaiodobenzylguanidine (MIBG) radiotherapy in malignant neuroendocrine tumors: results from a multicenter observational registry.

Effective treatments for malignant neuroendocrine tumors are under development. While iodine-131 metaiodobenzylguanidine (¹³¹I-MIBG) radiotherapy has been used in the treatment of malignant neuroendocrine tumors, there are few studies evaluating its therapeutic effects and safety in a multicenter cohort. In the current study, we sought to evaluate the effects and safety of ¹³¹I-MIBG therapy for conditions including malignant pheochromocytoma and paraganglioma within a multicenter cohort. Forty-eight malignant neuroendocrine tumors (37 pheochromocytoma and 11 paraganglioma) from four centers underwent clinical ¹³¹I-MIBG radiotherapy. The tumor responses were observed before and 3 to 6 months after the ¹³¹I-MIBG radiotherapy in accordance with RECIST criteria. We also evaluated the data for any adverse effects. The four centers performed a total of 87 ¹³¹I-MIBG treatments on 48 patients between January 2000 and March 2009. Of the treatments, 65 were evaluable using RECIST criteria. One partial response (PR), 40 stable disease (SD), and 9 progressive disease (PD) in malignant pheochromocytoma were observed after each treatment. Fourteen SD and one PD-were observed in paraganglioma. Patients with normal hypertension (systolic blood pressure (BP) > 130 mmHg) showed significantly reduced systolic BP after the initial follow-up (n=10, 138.1±8.2 to 129.5±13.5 mmHg, P=0.03). In adult neuroendocrine tumors with a treatment-basis analysis, there were side effects following 41 treatments (47.1%) and most of them (90.2%) were minor. In this multicenter registry, PR or SD was achieved in 84.6% of the treatment occasions in adult neuroendocrine tumors through ¹³¹I-MIBG radiotherapy. This indicated that most of the ¹³¹I-MIBG radiotherapy was performed safely without significant side effects.

Prognostic values of initial responses to low-dose (131)I-MIBG therapy in patients with malignant pheochromocytoma and paraganglioma.

PURPOSE: We retrospectively examined whether or not initial responses of first low-dose (131)I-meta-iodo-benzyl-guanidine radiotherapy ((131)I-MIBG therapy) in patients with malignant pheochromocytoma and paraganglioma had prognostic values. MATERIALS AND METHODS: This study included 26 patients with malignant pheochromocytoma (n = 18) and paraganglioma (n = 8) who underwent the first (131)I-MIBG therapy between October 2001 and September 2007. Based on the initial subjective, hormonal, scintigraphic, and objective responses to (131)I-MIBG therapy, the responses were divided into progression disease (PD) and non-PD. We examined the following factors for prognostic significance: sex, age, disease, initial diagnosis (benign or malignant pheochromocytoma), hypertension, diabetes mellitus, palpitations, symptoms related to bone metastases, and number of low-dose (131)I-MIBG therapy. Univariate Cox proportional regression analysis was used to identify prognostic factors for overall survival. Overall survival was analyzed by Kaplan-Meier method and the curves were compared using the log-rank test. RESULTS: The median survival time was 56 months. In the follow-up period, 16 patients died from exacerbation of their diseases. Univariate analysis showed that the hormonal PD [hazard ratio (HR) 3.20, P = 0.034, confidence interval (CI) 1.09-9.93], objective PD (HR 11.89, P = 0.0068, CI 2.14-65.85), single-time (131)I-MIBG therapy (HR 3.22, P = 0.020, CI 1.21-8.79), hypertension (HR 2.93, P = 0.044, CI 1.02-10.50), and symptoms related to bone metastases (HR 3.54, P = 0.023, CI 1.18-13.04) were bad prognostic factors for overall survival. Kaplan-Meier analysis demonstrated that the hormonal non-PD (P = 0.026), objective non-PD (P = 0.0002), multiple-time (131)I-MIBG therapy (P = 0.013), and no symptom related to bone metastases (P = 0.024) were significantly associated with good prognosis. Overall survival rate was 70 and 50 % at 5 years from the initial diagnosis and from the first (131)I-MIBG therapy, respectively. CONCLUSION: The hormonal and objective responses to the first low-dose (131)I-MIBG therapy as well as complication of hypertension and symptoms related to bone metastases may be prognostic factors in patients with malignant pheochromocytoma and paraganglioma.

Thyroid hormone replacement one day before (131)I therapy in patients with well-differentiated thyroid cancer.

OBJECTIVE: The current study aimed to determine the efficacy of radioiodine-131 ((131)I) ablation therapy with thyroid hormone replacement one day before (131)I administration in patients with well-differentiated thyroid cancer (DTC). METHODS: This retrospective study included 29 patients who underwent (131)I therapies twice for DTC during 6-12 months. Since all the patients obviously had residual lesions by their serum thyroglobulin levels or their scintigrams at the first therapies, they underwent the second (131)I therapies without diagnostic scintigraphy after the first therapies. After confirming the sufficient elevation of TSH concentration, thyroid hormone replacement was resumed one day before (131)I administration (3.7-7.4GBq). The ablation rate of thyroid remnant at the first (131)I therapy was evaluated by comparing (131)I post-therapeutic images of the two treatments. RESULTS: Three patients were administrated thyroid hormone after (131)I therapy because of insufficient TSH concentration under thyroid hormone withdrawal. In the remaining 26 patients, 41 thyroid remnant accumulations were detected in all 26 patients at the first (131)I therapy. Based on the second (131)I post-therapeutic images, successful ablation was confirmed in 24 of 26 patients (92.3%) and 38 of 41 sites (92.7%), which was comparable with historically reported ablation rates. CONCLUSION: Thyroid hormone replacement one day before (131)I therapy could provide a sufficiently high ablation rate in patients with DTC.

Sensitive immunodetection of radiotoxicity after iodine-131 therapy for thyroid cancer using γ-H2AX foci of DNA damage in lymphocytes.

PURPOSE: The purpose of our study was to evaluate the degree of radiotoxicity to lymphocytes in thyroid cancer after iodine-131(I-131) therapy using γ-H2AX foci immunodetection. METHODS: This study focused on 15 patients who underwent I-131 therapy for differentiated thyroid cancer after surgery. All patients received 3.7 GBq of I-131. Venous blood samples were collected from each patient before therapy and 4 days thereafter. Lymphocytes were isolated from the blood samples and subjected to γ-H2AX immunofluorescence staining. RESULTS: The number (mean ± SD) of foci per lymphocyte nucleus was 0.41 ± 0.51 before and 6.19 ± 1.80 after radioiodine therapy, and this difference was statistically significant (P = 0.001 < 0.05). Absorbed doses estimated for the 15 patients were 0.77 ± 0.31 Gy applying standard line in vitro external radiation doses. CONCLUSION: γ-H2AX foci immunodetection in lymphocytes may detect radiation-induced DNA damage associated with I-131 therapy for thyroid cancer, and may facilitate estimation of the radiation doses absorbed with this therapy.

I-131-Metaiodobenzylguanidine therapy with allogeneic cord blood stem cell transplantation for recurrent neuroblastoma.

Iodine-131-metaiodiobenzylguanidine (131I-MIBG) therapy combined with allogeneic cord blood stem cell transplantation (SCT) was used to treat a 4-year-old girl with recurrent neuroblastoma. The patient experienced relapse 2 years after receiving first-line therapies, which included chemotherapy, surgical resection, irradiation, and autologous peripheral SCT. Although 131I-MIBG treatment did not achieve complete remission, the size of the tumor was reduced after treatment. Based on our findings, we suggest that 131I-MIBG treatment with myeloablative allogeneic SCT should be considered as first-line therapy for high-risk neuroblastoma patients when possible.

Low-dose (123)I-metaiodobenzylguanidine diagnostic scan is inferior to (131)I-metaiodobenzylguanidine posttreatment scan in detection of malignant pheochromocytoma and paraganglioma.

OBJECTIVE: We assessed the lesion detectability of low-dose diagnostic (123)I-metaiodobenzylguanidine (MIBG) whole-body scans obtained at 6 and 24 h compared with posttreatment (131)I-MIBG whole-body scans in malignant pheochromocytoma and paraganglioma. METHODS: Scintigrams obtained in 15 patients with malignant pheochromocytoma and paraganglioma were retrospectively analyzed. Diagnostic scans were performed with 111 MBq of (123)I-MIBG. Therapeutic doses of (131)I-MIBG (5.55-7.40 GBq) were administrated and whole-body scans were obtained at 2-5 days after (131)I-MIBG administrations. We compared the number of lesions and the lesion-to-referent count ratios at 6 and 24 h of (123)I-MIBG and at 2-5 days of (131)I-MIBG. RESULTS: In comparison with the 6-h images of (123)I-MIBG, the 24-h images of (123)I-MIBG could detect more lesions in eight patients. Posttreatment (131)I-MIBG scans revealed new lesions in eight patients compared with the 24-h images of (123)I-MIBG. The lesion-to-referent count ratios at 6 and 24 h of (123)I-MIBG and at 3 days of (131)I-MIBG were increasing at later scanning time. There were significant differences in the lesion-to-referent count ratios between 6 and 24 h of (123)I-MIBG (P = 0.031), 6 h of (123)I-MIBG and 3 days of (131)I-MIBG (P = 0.020), and 24 h of (123)I-MIBG and 3 days of (131)I-MIBG (P = 0.018). CONCLUSION: Low-dose diagnostic (123)I-MIBG whole-body scan is inferior to posttreatment (131)I-MIBG whole-body scan in malignant pheochromocytoma and paraganglioma. Considering the scan timing of (123)I-MIBG, 6-h images might have no superiority compared with 24-h images.

Double-phase (131)I whole body scan and (131)I SPECT-CT images in patients with differentiated thyroid cancer: their effectiveness for accurate identification.

OBJECTIVE: This study aims to determine whether a (131)I double-phase whole body scan (WBS) and SPECT-CT images have added value over a single-phase WBS image in identifying benign and malignant lesions in patients with well-differentiated thyroid cancer (DTC) at their first radioactive iodine (RAI) treatment. METHODS: This study included 42 DTC patients who underwent their first radioablation. Post-therapeutic WBS images were acquired after 3 days (early phase) and 7 days (delayed phase). Following early-phase WBS, SPECT-CT images were obtained. The images were reviewed independently of the clinical data by 2 board-certified observers with a 6-point scoring system (benign to malignant -3 to +3). RESULTS: The double-phase WBS and SPECT-CT images showed 115 radioiodine-avid localizations (81 benign and 34 malignant accumulations). Confidence levels of benign accumulations were significantly higher with SPECT-CT (average score -2.40 ± 1.06) compared to those of the early-phase WBS (average score -1.39 ± 1.88) (p < 0.0001) and delayed-phase WBS images (average score -1.49 ± 1.19) (p < 0.0001). When the analysis was restricted to accumulations with a low confidence score in the early-phase WBS image, the confidence level of the delayed-phase WBS was higher compared to that of the early-phase WBS images (p = 0.0012). The confidence levels of malignant accumulations were significantly higher with SPECT-CT images (average score 2.37 ± 0.96) compared to the early-phase WBS (average score 1.44 ± 1.21) (p < 0.0001) and the delayed-phase WBS images (average score 1.50 ± 1.13) (p < 0.0001). CONCLUSION: Post-therapeutic SPECT-CT image was superior to the early-phase WBS image in enhancing the confidence level and accurately localizing the lesions. The delayed-phase WBS image contributed to the accurate diagnosis of benign lesions with a low confidence level in the early-phase WBS image.

Comparison of diagnostic value of I-123 MIBG and high-dose I-131 MIBG scintigraphy including incremental value of SPECT/CT over planar image in patients with malignant pheochromocytoma/paraganglioma and neuroblastoma.

PURPOSE: to compare lesion detectability of I-123 MIBG scintigraphy with that of high-dose I-131 MIBG and to evaluate incremental benefit of SPECT/CT over planar image for the detection and localization of the lesions in patients with I-131 MIBG therapy for malignant pheochromocytoma/paraganglioma and neuroblastoma. MATERIALS AND METHODS: we retrospectively investigated 16 patients with malignant pheochromocytoma/paraganglioma and neuroblastoma, who were referred for I-131 MIBG therapy. We investigated the lesion detectability in 10 pairs of I-123 and high-dose I-131 MIBG studies of the same patient, obtained within 2 weeks. In 31 studies of I-123 MIBG scintigraphy in 16 patients and 17 studies of high-dose I-131 MIBG scintigraphy in 12 patients, we compared planar and SPECT/CT images for the lesion detectability and localization. RESULTS: the number of lesions detected by I-123 MIBG planer image and SPECT/CT and high-dose planer I-131 MIBG and SPECT/CT were 3.0 and 3.7, 7.3 and 7.7 per study, respectively. SPECT/CT images provided additional diagnostic information over planar images in 25 studies (81%) of 12 patients (75%) in I-123 MIBG scintigraphy and in 9 studies (53%) of 9 patients (75%) in high-dose I-131 MIBG scintigraphy. CONCLUSION: post-therapy high-dose I-131 MIBG scintigraphy is superior to I-123 MIBG scintigraphy in lesion detectability even in comparison with I-123 MIBG SPECT/CT images and high-dose I-131 MIBG planar images in patients with malignant neuroendocrine tumors. SPECT/CT images are helpful for accurate identification of anatomic localization compared with planar images.

Effect of postconditioning on myocardial 99mTc-annexin-V uptake: comparison with ischemic preconditioning and caspase inhibitor treatment.

UNLABELLED: 99mTc-annexin-V imaging has been proved to be feasible to detect phosphatidylserine, which externalizes on the outer cell membrane early in the process of apoptosis. To determine whether postconditioning suppresses myocardial cell damage or apoptosis, we evaluated the intensity and distribution of 99mTc-annexin-V uptake after postconditioning in a rat model of ischemia and reperfusion and compared the effect to that of ischemic preconditioning and pretreatment with caspase inhibitor. METHODS: In control rats (n = 13), after thoracotomy the left coronary artery was occluded for 20 min followed by reperfusion for 30 or 90 min and injection of 99mTc-annexin-V (80-150 MBq). One hour later, to verify the area at risk, 201Tl (0.74 MBq) was injected intravenously just beyond the left coronary artery reocclusion, and the rats were sacrificed 1 min later. In the groups of rats with various interventions, postconditioning (n = 11) was performed just after the reperfusion, and preconditioning (n = 11) and caspase inhibitor treatment (n = 11) were performed before ischemia. Dual-tracer autoradiography was performed to assess 99mTc-annexin-V uptake and area at risk. RESULTS: In all control rats, intense 99mTc-annexin-V uptake was observed in the area at risk (uptake ratios at 30 or 90 min after reperfusion, 4.15 +/- 1.89 and 3.70 +/- 1.41, respectively). Postconditioning suppressed 99mTc-annexin-V uptake (uptake ratios at 30 or 90 min after reperfusion, 2.09 +/- 0.56, P < 0.05, and 1.88 +/- 0.69, P < 0.05, respectively). Preconditioning also suppressed uptake (uptake ratios at 30 and 90 min after reperfusion, 1.17 +/- 0.29, P < 0.005, and 1.33 +/- 0.74, P < 0.01, respectively), as did caspase inhibitor (uptake ratios at 30 and 90 min after reperfusion, 2.08 +/- 0.50, P < 0.05, and 1.27 +/- 0.24, P < 0.005, respectively). In all interventions, the percentage of cells positive on deoxyuride-5'-triphosphate biotin nick end labeling and histologic changes with myocardial cell degeneration and cell infiltrations were suppressed markedly. CONCLUSION: These data indicate that 99mTc-annexin-V imaging may be a way to monitor myocardial injury and its response to novel therapeutic interventions including postconditioning, preconditioning, and antiapoptotic therapy.

Intraperitoneal radioimmunotherapy to treat the early phase of peritoneal dissemination of human colon cancer cells in a murine model.

BACKGROUND AND AIM: In patients with a high risk of peritoneal dissemination of colon cancer, a treatment adjuvant to surgical resection would improve their prognosis. We aimed to determine whether radioimmunotherapy employing radiolabelled monoclonal antibody would work in this situation. METHODS: A murine model of peritoneal dissemination was established in female Balb/c nu/nu mice by intraperitoneal injection of LS180 human colon cancer cells. Radioimmunotherapy with 7.4 MBq of a murine IgG1, anti-colorectal A7 monoclonal antibody, radiolabelled with (131)I by the chloramine-T method was conducted intraperitoneally on days 0, 3, 7 and 14 after cell inoculation, respectively. RESULTS: Radioimmunotherapy at any timing improved survival of mice as compared with those of non-treated mice and mice treated with a daily dose of 30 mg x kg(-1) of 5-fluorouracil for 4 consecutive days. The best improvement was obtained when radioimmunotherapy was conducted on day 0. CONCLUSION: These results indicate that intraperitoneal radioimmunotherapy may effectively kill colon cancer cells disseminated in the peritoneal cavity before formation of tumours and, therefore, may work as an adjuvant treatment to prevent peritoneal metastasis of colon cancer.

Fluctuation of adenosine concentration by modes of intravenous infusion based on mathematical simulation and experiments.

OBJECTIVE: Adenosine, which has been used for a myocardial perfusion scan, shows rapid clearance from blood because of its short half-life of <10 seconds. This simulation study evaluates influences of modes of radionuclide injection on ventricular adenosine concentration when one intravenous injection line is used. METHODS: Assuming that radionuclide injection is a unit impulse, time-activity curves were measured in the left ventricle (LV) and fitted by a gamma function. Typical patterns of concentration fluctuation when adenosine infusion was temporarily modified were calculated by the convolution integral of input function and unit impulse response. Variation of concentration was measured by experiments using continuous 99mTc injection and co-infusion of water via a three-way stopcock. Modes of co-infusion with various infusion speeds and volumes were examined. RESULTS: Intermission of adenosine infusion and rapid displacement by radionuclide co-injection significantly influenced the adenosine concentration in LV. Intermission of adenosine infusion for 2 seconds caused a 15% decrease in the adenosine concentration in the left ventricle. When a square-shaped input was assumed, a three-fold higher concentration of adenosine for 3 seconds created by radionuclide injection resulted in a +42% increase in the LV concentration. Based on a measured input function, radionuclide injection using three-way stopcock through one route caused a two- to three-fold increase in the steady concentration in the vein just after injection. When 0.5 ml of radionuclide was slowly co-injected, with three ways opened, it caused a relatively low fluctuation, creating a +34% to -47% change in concentration of LV. A flush of radionuclide with physiological saline significantly increased the adenosine concentration in LV, when short half-lives were assumed. CONCLUSION: An intravenous adenosine and radiopharmaceutical injection in the same line is feasible. However, the fluctuation of concentration depends significantly on the mode of injection. To minimize the fluctuation, a slow injection of a small volume of a myocardial imaging agent via a co-injection route, with three ways opened, is recommended.

99mTc-sestamibi to monitor treatment with antisense oligodeoxynucleotide complementary to MRP mRNA in human breast cancer cells.

OBJECTIVE: Technetium-99m sestamibi (MIBI) has been utilized to evaluate multi-drug resistance (MDR) phenomenon of malignant tumors and to predict chemotherapeutic effects on them. The current investigation examined the possibility of monitoring changes with respect to mRNA expression of multi-drug resistance associated protein (MRP) following antisense oligodeoxynucleotide (AS-ODN) treatment involving 99mTc-MIBI. METHODS: The human breast cancer MCF-7 cell line and its MDR-induced MCF-7/VP cell line were employed. Cell suspensions of the two cell lines at 1 x 10(4) cells/ml were inoculated in 24-well plates (0.2 ml/well) and incubated for one day. Antisense (AS) 20-mer phosphorothioate ODN complementary to the coding region of MRP mRNA and its sense (S) ODN were administered at final concentrations up to 25 microM, followed by a 5-day incubation. 99mTc-MIBI solution was added to each well and incubated for 30 min. Cellular 99mTc-MIBI uptake was corrected for protein concentration. MRP mRNA expression levels were analyzed via the reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Cellular uptake of 99mTc-MIBI in MCF-7/VP cells was only 15% of that of MCF-7 cells. Following AS-ODN treatment at 25 microM for five days, 99mTc-MIBI uptake in MCF-7/VP cells increased 2.4-fold in comparison with non-treated control cells. 99mTc-MIBI uptake in MCF-7 cells was unaffected by AS-ODN administration. Sense ODN did not alter uptake in either cell line. RT-PCR confirmed reduction of MRP mRNA in MCF-7/VP cells following AS-ODN treatment. CONCLUSION: Effects of AS-ODN administration on MRP function can be monitored via assessment of cellular uptake of 99mTc-MIBI.

Anti-angiogenic therapy and chemotherapy affect 99mTc sestamibi and 99mTc-HL91 accumulation differently in tumour xenografts.

BACKGROUND: Favourable effects of cytotoxic chemotherapy for tumours are characterized by the reduced accumulation of radiotracers such as 99mTc sestamibi (MIBI). Anti-angiogenic therapy is primarily cytostatic; consequently, its influence on tracer accumulation may differ from that of cytotoxic treatments. METHODS: Anti-angiogenic therapy employing 2-methoxyestradiol was administered in mice bearing subcutaneous xenografts of LS180 colon cancer cells. The effects of chemotherapy with 5-fluorouracil were examined as a cytotoxic counterpart. Treatments were conducted for 4 days from day 8. Distribution of 99mTc-MIBI and Tc-HL91, a hypoxic marker, was observed on days 8 and 12. Oxygen tension (PO2) in tumours was measured by a microelectrode. Cellular uptake of tracers was examined in vitro in normoxic and hypoxic conditions. RESULTS: 99mTc-MIBI accumulation decreased with increasing tumour weight when no treatment was conducted. Tumour growth was suppressed by anti-angiogenic therapy and chemotherapy. 99mTc-MIBI accumulation in tumours decreased after chemotherapy as compared to pre-therapeutic values, whereas accumulation of 99mTc-HL91 increased. In contrast, accumulation of tracers did not significantly change after anti-angiogenic therapy as compared to that observed pre-therapeutically. Tumour PO2 decreased with increasing tumour volume when no treatment was conducted. Chemotherapy reduced PO2 in tumours. PO2 in tumours treated with anti-angiogenic therapy was as high as that observed before treatment. 2-Methoxyestradiol or 5-fluorouracil did not significantly affect tracer accumulation in cells under both normoxic and hypoxic conditions in vitro. CONCLUSION: These findings indicate that scintigraphic assessment of therapeutic efficacy of anti-angiogenic therapy should be performed from a perspective distinct from that of cytotoxic treatment.