Mathematical Model for Evaluation of Tumor Response in Targeted Radionuclide Therapy with 211At Using Implanted Mouse Tumor.

Recent introduction of alpha-emitting radionuclides in targeted radionuclide therapy has stimulated the development of new radiopharmaceuticals. Preclinical evaluation using an animal experiment with an implanted tumor model is frequently used to examine the efficiency of the treatment method and to predict the treatment response before clinical trials. Here, we propose a mathematical model for evaluation of the tumor response in an implanted tumor model and apply it to the data obtained from the previous experiment of 211At treatment in a thyroid cancer mouse model. The proposed model is based on the set of differential equations, describing the kinetics of radiopharmaceuticals, the tumor growth, and the treatment response. First, the tumor growth rate was estimated from the control data without injection of 211At. The kinetic behavior of the injected radionuclide was used to estimate the radiation dose profile to the target tumor, which can suppress the tumor growth in a dose-dependent manner. An additional two factors, including the time delay for the reduction of tumor volume and the impaired recovery of tumor regrowth after the treatment, were needed to simulate the temporal changes of tumor size after treatment. Finally, the parameters obtained from the simulated tumor growth curve were able to predict the tumor response in other experimental settings. The model can provide valuable information for planning the administration dose of radiopharmaceuticals in clinical trials, especially to determine the starting dose at which efficacy can be expected with a sufficient safety margin.

4-O-methylascochlorin stabilizes hypoxia-inducible factor-1 in a manner different from hydroxylase inhibition by iron chelating or substrate competition.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays essential roles in human diseases including cancer. The synthetic ascochlorin derivative 4-O-methylascochlorin stabilizes HIF-1α protein, and activates its transcriptional activity, resulting to induce gene expression of its downstream targets such as VEGF and GLUT-1. Here, we quantified protein level of HIF-1α in human osteosarcoma U2OS cells treated with ascochlorin-related compounds and typical HIF-1α stabilizers to characterize properties of HIF-1α stabilization by 4-O-methylascochlorin. Structure-activity relationship studies suggested that the aromatic moiety and hydrophobic substitution of the 4'-hydroxyl group are important for HIF-1α stabilization by ascochlorin-related compounds. 4-O-Methylascochlorin-induced HIF-1α stabilization was suppressed by ascorbic acid and compound C, but not by Fe(II), whereas ascorbic acid only suppressed HIF-1α stabilization by dimethyloxaloylglycine, an analog of the HIF-1 hydroxylase substrate. Fe(II) completely suppressed iron chelator-induced stabilization. These results suggest that ascochlorin-related compounds stabilize HIF-1α in a manner distinct from iron chelating or substrate competition.

Study of mutation from DNA to biological evolution.

Purpose: This is a paper based on a talk given in the BER2018 conference by M. Bando. We first emphasize the importance of collaborations among scientists in various fields for the low dose/dose-rate effects on biological body. We make comparisons of quantitative estimations of mutation caused by the radiation exposure on various animals and plants using one mathematical model. We derive the importance of the spontaneous mutation at the DNA level, which provides the key to understand the biological evolution. We try to make a guide map to solve this problem and find that the mutation is an important stage of the pathway from the DNA damage to the macroscopic biological evolution. Materials and methods: We construct a mathematical model for the mutation, named as 'WAM' model, which takes into account the recovery effect. The model setting is regarded as an extension of the survival and the hazard functions. The WAM model is used to reproduce accumulated data of mutation frequency of animals and plants. Especially the model analysis shows that the dose-rate dependence is important to understand various mutation data. Results and conclusions: The WAM model is successful in reproducing various mutation data of animals and plants. We find that the inclusion of the dose rate is important to understand all the mutation data. Hence, we are able to develop the 'scaling law' to make the cross-species comparison of mutation frequency data. With this finding, we can extract the dominant effect on the mutation to be caused by the spontaneous mutation, and quantify this amount. We are able to write then the artificial radiation frequency by subtracting the spontaneous mutation. With this success, we estimate the origin of the spontaneous mutation as due to ROS, the order of which agrees to the spontaneous mutation.

[Vinorelbine, a Microtubule Toxin, Induces Apoptosis and Polyploidy in MX-1, a Human Triple-Negative Breast Cancer Cell Line].

Compared to other types of breast cancers, triple-negative breast cancer(TNBC)has poor prognosis. However, much work has been done towards establishing an effective therapeutic strategy. Vinorelbine(VNB)is an effective therapeutic agent for TNBC, however, the mechanism for its efficacy remains to be elucidated. We found that MX-1, a TNBC cell line, exhibits apoptosis and polyploidy upon VNB treatment. Neither apoptosis nor polyploidy were observed in other types of breast cancer cells upon VNB treatment. Furthermore, inhibitors of respiration, protein synthesis, and DNA synthesis suppressed apoptosis and polyploidy induced by VNB in MX-1 cells. Among microtubule toxins, clinically effective paclitaxel(PTX)and VNB had a greater effect than colchicine and nocodazole on polyploidy induction in MX-1 cells. These results suggest that VNB induces apoptosis in some types of TNBCs through the induction of polyploidy, which is, at least in part, the likely mechanism of its clinical efficacy.

Verification of a dose rate-responsive dynamic equilibrium model on radiation-induced mutation frequencies in mice.

Purpose: We have proposed a mathematical model (WAM model) expressing increment of the dose-rate dependent mutation frequency caused by artificial radiations. In this model, it is defined that the pool of mutant cells in dynamic equilibrium in organisms. We verified the accuracy of the WAM prediction of mutation frequency in mice. Materials and methods: The theoretical values calculated by the WAM model were compared with the experimental values obtained from the large mouse genetics program at the Oak Ridge National Laboratory (ORNL). Results: Most of all the theoretical values in acute and chronic irradiation conditions nearly coincided with the experimental values. However, the theoretical value of the chronic conditions at the dose-rate of 0.8 R/min was significantly higher than its experimental value. This discordance was able to be minimized in the WAM assumption, when the period from the end of exposure to start mating was two weeks longer. Conclusions: As a result of comparison between experimental and theoretical data, the certainty of the WAM model was confirmed in mice and it was shown that the genetic influence varies depending on the dose-rate.

Delayed Growth Suppression and Radioresistance Induced by Long-Term Continuous Gamma Irradiation.

Biological response to ionizing radiation depends not only on the type of radiation and dose, but also on the duration and dose rate of treatment. For a given radiation dose, the biological response may differ based on duration and dose rate. We studied the properties of two human cell lines, M059K glioma and U2OS osteosarcoma, continuously exposed to γ rays for long time periods of more than five months. Growth inhibition in both cell lines was dependent on total dose when exposed to acute radiation over several minutes, whereas prolonged growth inhibition was dependent on dose rate after continuous irradiation over several months. The minimum dose rate for growth inhibition was 53.6 mGy/h. Cell cycle analysis showed G1 phase accumulation in cell populations continuously exposed to γ rays, and G2 phase accumulation in cells acutely exposed to high-dose-rate γ rays. Cells continuously exposed to γ rays continued to exhibit delayed growth suppression even after one month in an environment of background radiation, and maintained a high-level expression of c-Jun and its phosphorylation forms, as well as resistance to apoptosis induced by staurosporine and chemotherapeutic agents. These delayed effects were not observed in cells acutely exposed to 5 Gy of radiation. These results suggest that optimization of the irradiation schedule is crucial for risk estimation, protection and therapeutic utilization of ionizing radiation.

Fukushima simulation experiment: assessing the effects of chronic low-dose-rate internal 137Cs radiation exposure on litter size, sex ratio, and biokinetics in mice.

To investigate the transgenerational effects of chronic low-dose-rate internal radiation exposure after the Fukushima Daiichi Nuclear Power Plant accident in Japan, 18 generations of mice were maintained in a radioisotope facility, with free access to drinking water containing (137)CsCl (0 and 100 Bq/ml). The (137)Cs distribution in the organs of the mice was measured after long-term ad libitum intake of the (137)CsCl water. The litter size and the sex ratio of the group ingesting the (137)Cs water were compared with those of the control group, for all 18 generations of mice. No significant difference was noted in the litter size or the sex ratio between the mice in the control group and those in the group ingesting the (137)Cs water. The fixed internal exposure doses were ∼160 Bq/g and 80 Bq/g in the muscles and other organs, respectively.

Imaging plant leaves to determine changes in radioactive contamination status in Fukushima, Japan.

The chemical composition of plant leaves often reflects environmental contamination. The authors analyzed images of plant leaves to investigate the regional radioactivity ecology resulting from the 2011 accident at the Fukushima No. 1 nuclear power plant, Japan. The present study is not an evaluation of the macro radiation dose per weight, which has been performed previously, but rather an image analysis of the radioactive dose per leaf, allowing the capture of various gradual changes in radioactive contamination as a function of elapsed time. In addition, the leaf analysis method has potential applications in the decontamination of food plants or other materials.

A molecular clock regulates angiopoietin-like protein 2 expression.

Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

A rapid and easy method for the qualitative detection of intracellular deposition of inhaled nanoparticles.

In recent years, nanoparticle exposure risk has drawn increasing attention from the research community and the general public. However, analysis of nanoparticles is hindered by their small size, which prevents the development of methods for their detection in cells and tissues. For risk assessment of nanoparticle exposure, it is important to measure the exact amount of deposited material in pulmonary tissue. Using a nanoparticle exposure device, A/JJmsSlc mice were chronically exposed transtracheally to anatase-type titanium dioxide particles. A microscope-integrated laser Raman spectrometer was used to detect differentially stained macrophages in a pulmonary wash obtained from the mice exposed to the particles. This detection method allowed rapid and easy sample collection and qualitative analysis, and the method may be useful for conducting large-scale evaluations in workers exposed to environments heavily contaminated with nanoparticles. FROM THE CLINICAL EDITOR: This paper discusses a microscope-integrated laser Raman spectrometer method to measure the exact amount of nanoparticles deposited in pulmonary tissue. This method allows rapid sample collection, qualitative analysis, and may be useful for large-scale evaluations.

Enhancement of paclitaxel-induced apoptosis in HER2-overexpressing human breast cancer cells by a pertuzumab mimetic peptide, HRAP.

HRAP, a pentapeptide designed to bind with the pertuzumab interacting site in an extracellular domain of the HER2 molecule, enhanced the cytotoxicity of paclitaxel in HER2-overexpressing human breast cancer cell lines, BT474 and SKBR-3, but not in MDA-231 cells, which express lower levels of HER2. HRAP enhanced mitochondria-dependent apoptosis induced by paclitaxel in SKBR-3 and BT-474, but not in MDA-231. HRAP enhanced the inhibition of phosphorylation of serine 473 in Akt and Ser380/Thy382/The383 in PTEN. These results suggest that HRAP enhances paclitaxel-induced apoptosis in a manner dependent on the PTEN/Akt signal transduction pathway.

Clinical outcomes of video-assisted skin-sparing partial mastectomy for breast cancer and immediate reconstruction with latissimus dorsi muscle flap as breast-conserving therapy.

BACKGROUND: Skin-sparing partial mastectomy (SSPM) has yet to be investigated as a breast-conserving therapy for early-stage breast cancer. We report the clinical outcomes for video-assisted SSPM (VA-SSPM) with immediate breast reconstruction using autogenous tissue. METHODS: VA-SSPM is indicated for early-stage breast cancer arising in the upper-outer or lower-outer quadrant without skin involvement. An incision is placed along the midaxillary line, and SSPM is performed under endoscopic guidance using subcutaneous tunneling and lifting methods. Through the same incision, a latissimus dorsi muscle flap is harvested for breast reconstruction. From January 2000 to October 2007, 168 patients (Tis, n = 24; T1, n = 37; T2, n = 107) underwent VA-SSPM, and morbidity, curability, and postoperative patient satisfaction were investigated. RESULTS: Postoperative complications included skin necrosis (2.4%, n = 4) and muscle flap necrosis (0.6%, n = 1), but no severe complications were observed. After a mean follow-up of 58.6 months, eight patients (4.8%) experienced local recurrence. Sixty-month distant metastasis-free survival rates for Tis, T1, and T2 were 100%, 97%, and 83.3%, respectively, with an overall rate of 88.4%. Furthermore, overall survival rates for Tis, T1, and T2 were 100%, 94.1%, and 94.4%, respectively, with an overall survival rate of 95% for all patients. A patient satisfaction survey showed that 81.6% of patients evaluated the surgery as "good." CONCLUSIONS: VA-SSPM for early-stage breast cancer improves cosmetic results and achieves high patient satisfaction without increasing local or distant organ recurrence. This method offers a useful local therapy for early-stage breast cancer.

Effects of fission neutrons on human thyroid tissues maintained in SCID mice.

Morphology and function (secretion of thyroid hormone) of human thyroid tissues from Graves' disease patients are well maintained in C57BL/6J-scid mice. Serum level of thyroid hormone was reduced by fission neutrons from the nuclear reactor UTR-KINKI, and changes in thyroid hormone by fission neutrons were bigger than those by low LET radiations, X-rays and (137)Cs gamma-rays, suggesting high relative biological effectiveness (RBE; 6.5) of fission neutrons. Microarray analyses revealed that about 3% of genes showed more than 4-fold change in gene expression in the unexposed thyroid tissues against surgically resected thyroid tissues from the same patient, probably due to the difficult oxygen and nutrient supply shortly after transplantation. Dose-dependent changes in gene expression against unexposed concurrent controls were observed with increasing doses of fission neutrons (0.2-0.6Gy) and (137)Cs gamma-rays (1.0-3.0Gy) and showed high RBE (4.2). Furthermore, there were some specific genes which showed more than 4-fold change in gene expression in all the thyroid tissues exposed to higher doses of radiation, especially neutrons (0.4 and 0.6Gy), but none at lower doses (0.2Gy of neutrons and 1.0 and 2.0Gy of gamma-rays). These genes related to degeneration, regeneration, apoptosis, and transcription, respond specifically and very sensitively to neutron injury in human thyroid tissues. This is the first experimental report that fission neutrons can induce some morphological and functional disorders in human tissues, showing high RBE against gamma-ray exposure. These results are useful to evaluate the risks of fission neutrons and cosmic rays to humans.

Menin, a product of the MENI gene, binds to estrogen receptor to enhance its activity in breast cancer cells: possibility of a novel predictive factor for tamoxifen resistance.

Multiple coactivator and corepressor complexes play an important role in endocrine processes and breast cancer; in particular, estrogen and estrogen receptor-alpha (ERalpha) promote the proliferation of breast cancer cells. Menin is a tumor suppressor encoded by Men1 that is mutated in the human-inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1); it also serves as a critical link in the recruitment of nuclear receptor-mediated transcription. Here, we show that menin expressed in breast cancer cell line MCF-7 is colocalized with ERalpha and functions as a direct coactivator of ER-mediated transcription in breast cancer cells. In MCF-7 cells, coexpression of menin and estrogen-response element-luciferase induced the activity of the latter in a hormone-dependent manner. Cells knocked down for ERalpha exhibited impaired ERE-luciferase activity induced by menin. Mammalian two-hybrid assay and GST pull-down assays indicated that menin could interact with the AF-2 domain of ERalpha. These results indicate that menin is a direct activator of ERalpha function. Tamoxifen inhibited the binding of menin to AF-2 in mammalian two-hybrid assay, but in menin-overexpressing clones, tamoxifen suppressed ERE-luciferase activity only to the levels of nontreated wild-type MCF-7. In a clinical study with 65 ER-positive breast cancer samples-all of which had been treated with tamoxifen for 2-5 years as adjuvant therapies--menin-positive tumors had a worse outcome than menin-negative ones. These indicated that menin can function as a transcriptional regulator of ERalpha and is a possible predictive factor for tamoxifen resistance.

Video-assisted skin-sparing breast-conserving surgery for breast cancer and immediate reconstruction with autologous tissue: clinical outcomes.

BACKGROUND: This study analyzed clinical results of video-assisted breast-conserving surgery for breast cancer. METHODS: Video-assisted breast-conserving surgery is indicated for breast cancer that has not invaded the skin. A skin incision is made at an inconspicuous site. Skin-sparing partial mastectomy was performed endoscopically on 244 patients (stage I, n = 94; stage II, n = 150). Morbidity, curability, and patient satisfaction were analyzed. RESULTS: Skin necrosis was seen in nine patients. Local recurrence was seen in 13 patients (mean postoperative interval 65.3 months). Distant metastasis-free survival at 60 months was 93.6% for stage I and 90.5% for stage II. Overall survival was 95.7% for stage I and 96.9% for stage II. Satisfaction with surgery as investigated by questionnaire was "good" for 72.3% of patients. CONCLUSIONS: Video-assisted breast-conserving surgery showed no increases in local or distant recurrence and patient satisfaction was high. Video-assisted breast-conserving surgery appears useful for local treatment of breast cancer.

Ascochlorin activates p53 in a manner distinct from DNA damaging agents.

Ascochlorin, a prenylphenol antitumor antibiotic, profoundly increases the expression of endogenous p53 by increasing protein stability in the human osteosarcoma cells and human colon cancer cells. Ascochlorin also increases DNA binding activity to the p53 consensus sequence in nuclear extract and enhances transcription of p53 downstream targets. Ascochlorin specifically induces p53 phosphorylation at ser 392 without affecting ser 15 or 20, whereas DNA damaging agents typically phosphorylate these serines. Moreover, ascochlorin does not induce phosphorylation of ATM and CHK1, an established substrate of ATR that is activated by genotoxins, nor does it increase DNA strand break, as confirmed by comet assay. The structure-activity relationship suggests that p53 activation by ascochlorin is related to inhibition of mitochondrial respiration, which is further supported by the observation that respiratory inhibitors activate p53 in a manner similar to ascochlorin. These results suggest that ascochlorin, through the inhibition of mitochondrial respiration, activates p53 through a mechanism distinct from genotoxins.

Suppression of PAI-1 expression through inhibition of the EGFR-mediated signaling cascade in rat kidney fibroblast by ascofuranone.

Fibrosis in glomerulosclerosis causes progressive loss of renal function. Transforming growth factor (TGF)-beta, one of the major profibrotic cytokines, induces the synthesis of plasminogen activator inhibitor (PAI)-1, a factor that plays a crucial role in the development of fibrosis. Here, we found that an isoprenoid antibiotic, ascofuranone, suppresses expression of profibrotic factors including matrix proteins and PAI-1 induced by TGF-beta in renal fibroblasts. Ascofuranone selectively inhibits phosphorylation of epidermal growth factor receptor (EGFR), and downstream kinases such as Raf-1, MEK-1/2, and ERK-1/2. PAI-1 transcription also is suppressed by treatment with kinase inhibitors for MEK-1/2 or EGFR, and with small interfering RNA for EGFR. Ascofuranone inhibits cellular metalloproteinase activity, and an inhibitor of metalloproteinases suppresses EGFR phosphorylation and PAI-1 transcription. These results suggest that ascofuranone suppresses expression of profibrotic factors through the inhibition of an EGFR-dependent signal transduction pathway activated by metalloproteinases.

Video-assisted skin-sparing breast-conserving surgery for breast cancer and immediate reconstruction with autologous tissue.

OBJECTIVE: To analyze therapeutic results of video-assisted breast-conserving surgery (VA-BCS) for early stage breast cancer. BACKGROUND: VA-BCS for breast cancer has been developed in Japan, and is indicated for breast cancer unaccompanied by skin involvement. The surgical incision is made at an inconspicuous site, followed by skin-sparing partial mastectomy (SSPM) and immediate reconstruction of the breast. This technique affords good cosmetic results. The long-term results are reported herein. METHODS: VA-BCS was performed on 551 patients. The skin incision was made as a peri-areolar incision or at the midaxillary line. Skin-sparing partial mastectomy was performed using an endoscope and the lifting and tunneling method. Morbidity, curability, and degree of satisfaction with regard to cosmesis were analyzed. RESULTS: Skin necrosis in 22 patients (4.0%) and necrosis of fatty tissue-muscle flap in 17 patients (3.1%) were recorded as postoperative complications. No other serious complications were encountered. Local recurrence occurred in 23 patients (4.2%) after a mean follow-up of 38.4 months. Distant-metastasis-free survival rate at 66 months was 100% for Tis, 95.5% for T1, and 90.7% for T2. Overall survival rate was 100% for Tis, 97.3% for T1, and 95.7% for T2. Degree of satisfaction with surgery as investigated by questionnaire was "good" for 76.1% of patients. CONCLUSION: VA-BCS for early stage breast cancer showed no association with increases in local or distant organ recurrence. The technique yielded improved cosmesis and a high degree of patient satisfaction. Follow-up observation of patients for a longer period is necessary, but VA-BCS seems useful for local treatment of breast cancer.

Anxiolytic effect of aromatherapy massage in patients with breast cancer.

We examined how aromatherapy massage influenced psychologic and immunologic parameters in 12 breast cancer patients in an open semi-comparative trial. We compared the results 1 month before aromatherapy massage as a waiting control period with those during aromatherapy massage treatment and 1 month after the completion of aromatherapy sessions. The patients received a 30 min aromatherapy massage twice a week for 4 weeks (eight times in total). The results showed that anxiety was reduced in one 30 min aromatherapy massage in State-Trait Anxiety Inventory (STAI) test and also reduced in eight sequential aromatherapy massage sessions in the Hospital Anxiety and Depression Scale (HADS) test. Our results further suggested that aromatherapy massage ameliorated the immunologic state. Further investigations are required to confirm the anxiolytic effect of aromatherapy in breast cancer patients.