Amy Zhang

Synthesis and anticancer potential of novel xanthone derivatives with 3,6-substituted chains.

In an effort to develop new drug candidates with enhanced anticancer activity, our team synthesized and assessed the cytotoxicity of a series of novel xanthone derivatives with two longer 3,6-disubstituted amine carbonyl methoxy side chains on either benzene ring in selected human cancer cell lines. An MTT assay revealed that a set of compounds with lower IC50 values than the positive control, 5-FU, exhibited greater anticancer effects. The most potent derivative (XD8) exhibited anticancer activity in MDA-MB-231, PC-3, A549, AsPC-1, and HCT116 cells lines with IC50 values of 8.06, 6.18, 4.59, 4.76, and 6.09μM, respectively. Cell cycle analysis and apoptosis activation suggested that the mechanism of action of these derivatives includes cell cycle regulation and apoptosis induction.

Transition pattern and mechanism of B-lymphocyte precursors in regenerated mouse bone marrow after subtotal body irradiation.

Little is known about the effects of ionizing radiation on the transition and the related signal transduction of progenitor B cells in the bone marrow. Thus, using an NIH Swiss mouse model, we explored the impact of ionizing radiation on the early stage of B-cell development via an examination of the transition of CLP to pro-B to pre-B cells within bone marrow as a function of radiation doses and times. Our results showed that while the total number of bone marrow lymphoid cells at different stages were greatly reduced by subtotal body irradiation (sub-TBI), the surviving cells continued to transition from common lymphoid progenitors to pro-B and then to pre-B in a reproducible temporal pattern. The rearrangement of the immunoglobulin heavy chain increased significantly 1–2 weeks after irradiation, but no change occurred after 3–4 weeks. The rearrangement of the immunoglobulin light chain decreased significantly 1–2 weeks after sub-TBI but increased dramatically after 3–4 weeks. In addition, several key transcription factors and signaling pathways were involved in B-precursor transitions after sub-TBI. The data indicate that week 2 after irradiation is a critical time for the transition from pro-B cells to pre-B cells, reflecting that the functional processes for different B-cell stages are well preserved even after high-dose irradiation.

Delayed Effects of Radiation on Mitochondrial DNA in Radiation-Sensitive Organs

Since Balb/c mice are sensitive to acute hematopoietic syndrome and C57BL/6 to late fibrovascular toxicity, we measured the effect of total body irradiation (TBI) on mitochondrial DNA (mtDNA) copy number in these two strains. Six months after TBI, we extracted total genomic DNA, including nuclear DNA (nDNA) and mtDNA, from various tissues. The findings included: (1) TBI had no significant effect on body weight for Balb/c or C57BL/6 when compared to aged controls. (2) For different organs in control mice, the ratio of mtDNA to nDNA ranged from 0.33 to 11.53 in Balb/c and 0.34 to 13.28 in C57BL/6. (3) Ratios of mtDNA to nDNA were generally similar to or higher in C57BL/6 compared to Balb/c. (4) In C57BL/6, TBI induced an increase in mitochondrial copy number only in the brain (p < 0.05). (5) In Balb/c, radiation induced a dose-dependent increase of mtDNA in the liver (p < 0.05 and p < 0.01) and a decrease in skeletal/muscle tissue (p < 0.01). Our findings indicate that radiation influences mtDNA copy number differently in various tissues. This may explain differences in late radiation toxicity to those organs and differences in radiation tolerance between murine strains.

Maternal Bias in Mouse Radiosensitivity: The Role of the Mitochondrial PTP

This study investigated, at the molecular level, mitochondrial responses to radiation. In three mouse strains, we found the following: (1) mitochondrial response to calcium stress was associated with a strains susceptibility to γ-radiation; (2) γ-radiation increased this calcium stress response in a dose-responsive manner; (3) the mitochondrial DNA (mtDNA) copy number in the liver of the radiosensitive mouse strain was significantly lower, as compared to that of the radioresistant strain; (4) adenine nucleotide translocase (ANT) mRNA copy numbers were significantly lower in the radiosensitive strain; (5) the F1 offspring (BC/C57M) of radiosensitive females mated with radioresistant males exhibited a significant difference in calcium stress response from that of the radiation-resistant strain, but the reverse cross did not exhibit this difference; and (6) only those mitochondria extracted from the livers of irradiated BC/C57M mice exhibited a heightened calcium stress response. We propose that a genetic change in ANT and a postirradiation change involving either mtDNA-encoded protein replacement or altered mtDNA association fit these data.

Mitochondrial DNA and functional investigations into the radiosensitivity of four mouse strains.

We investigated whether genetic radiosensitivity-related changes in mtDNA/nDNA ratios are significant to mitochondrial function and if a material effect on mtDNA content and function exists. BALB/c (radiosensitive), C57BL/6 (radioresistant), and F1 hybrid mouse strains were exposed to total body irradiation. Hepatic genomic DNA was extracted, and mitochondria were isolated. Mitochondrial oxygen consumption, ROS, and calcium-induced mitochondrial swelling were measured. Radiation influenced strain-specific survival in vivo. F1 hybrid survival was influenced by maternal input. Changes in mitochondrial content corresponded to survival in vivo among the 4 strains. Calcium-induced mitochondrial swelling was strain dependent. Isolated mitochondria from BALB/c mice were significantly more sensitive to calcium overload than mitochondria from C57BL/6 mice. Maternal input partially influenced the recovery effect of radiation on calcium-induced mitochondrial swelling in F1 hybrids; the hybrid with a radiosensitive maternal lineage exhibited a lower rate of recovery. Hybrids had a survival rate that was biased toward maternal input. mtDNA content and mitochondrial permeability transition pores (MPTP) measured in these strains before irradiation reflected a dominant input from the parent. After irradiation, the MPTP opened sooner in radiosensitive and hybrid strains, likely triggering intrinsic apoptotic pathways. These findings have important implications for translation into predictors of radiation sensitivity/resistance.

The Murine Common Deletion: Mitochondrial DNA 3,860-bp Deletion after Irradiation

This study demonstrates that mice, similar to humans, have a common mitochondrial DNA deletion (3,860 bp) that encodes 5 transfer RNA genes and 5 polypeptide genes that is related to aging, tissue type and radiotoxicity. Our research indicates that the deletion ratio in the liver was significantly higher than in the brain and gut tissues of 8-month-old mice, as compared to 8-week-old mice. Our results also demonstrate that tissue type, oxidative metabolic capacity and radiosensitivity influence the 3,860-bp deletion level. Therefore, this 3,860-bp deletion content may serve as a biomarker of aging and oxidative damage in mice.

The role of mitochondrial proteomic analysis in radiological accidents and terrorism.

In the wake of the 9/11 terrorist attacks and the recent Level 7 nuclear event at the Fukushima Daiichi plant, there has been heightened awareness of the possibility of radiological terrorism and accidents and the need for techniques to estimate radiation levels after such events. A number of approaches to monitoring radiation using biological markers have been published, including physical techniques, cytogenetic approaches, and direct, DNA-analysis approaches. Each approach has the potential to provide information that may be applied to the triage of an exposed population, but problems with development and application of devices or lengthy analyses limit their potential for widespread application. We present a post-irradiation observation with the potential for development into a rapid point-of-care device. Using simple mitochondrial proteomic analysis, we investigated irradiated and nonirradiated murine mitochondria and identified a protein mobility shift occurring at 2-3 Gy. We discuss the implications of this finding both in terms of possible mechanisms and potential applications in bio-radiation monitoring.

Abstract 1563: Characterization of two new recombinant rabbit anti-PDL1 IHC staining in bladder cancer, NSCLC, and melanoma with immune cell markers

Published studies show tumor cells overexpress PD-L1 to escape the host immune defense by binding to PD-1 on surveilling T-cells causing the T-cells to shut down. Immunotherapies disrupting PD-1/PD-L1 signaling interactions have great success in treating PD-L1 positive tumors. Immunohistochemistry (IHC) is an important diagnostic tool currently used to determine the expression level of PD-L1 in tumor and immune cells. The FDA approved PD-L1 clones (SP142 and 28-8) for IHC present different staining patterns when evaluated on the same tumor tissue. In this study, we assessed both the immune cells and tumor cells IHC positive stain of PD-L1. To do this we evaluated multiple immune cell markers with multiple PD-L1 antibodies. IHC screens were done with 5 PD-L1 antibodies, 2 recombinant rabbit monoclonal antibodies (clone OR-5E3 and OR-5H8), the mouse monoclonal antibody (clone UMAB229), and the FDA approved clones (SP142 and 28-8). The immune cell markers used were CD3, CD8A, CD20, CD68, and FOXP3. NSCLC, Bladder Cancer, and Melanoma immune cells, as indicated by the CD3, CD8A, CD20, CD68 and FOXP3 staining, generated different distribution pattern in the three tumor types. The five PD-L1 antibodies did show variation in detection of both the immune and tumor cells. For example, PD-L1 clones OR-5E3 and OR-5H8 stained tumor cells stronger and picked up weak expression of PD-L1 better than clone SP142, suggesting the two antibodies have higher affinity. The mouse monoclonal anti-PDL1 clone UMAB229 picked up strong and weak staining similar or better than clones OR-5H8 and picked up the immune cells similar to SP-142. The five PD-L1 antibodies were made from different antigens which may contribute to their sensitivity and specificity to detect PD-L1 in tumor cells. This study suggests the new generation of PD-L1 antibodies may make a better tool for diagnostic screens. Citation Format: Rachel M. Gonzalez, Wei Fu, Evelin Logis, Emma Ding, Casey Chen, Xiaojie Li, Sonia Merritt, Mingjuan Liu, Amy Zhang, Yiran Wang, Guangli Wang, Donghui Ma. Characterization of two new recombinant rabbit anti-PDL1 IHC staining in bladder cancer, NSCLC, and melanoma with immune cell markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1563.

Thoracic gamma irradiation-induced obesity in C57BL/6 female mice

Abstract Purpose: To investigate the late effects of thoracic region irradiation (TRI) on mouse body weight. Materials and methods: Female C57BL/6 mice were divided into nonirradiated, 5 Gy total body irradiation, 9 Gy sub-total body irradiation, and 12.5 Gy thoracic region irradiation (TRI) groups. Changes in mouse weight were monitored every other week at similar time points for 12 months. The anatomical characteristics of abdominal visceral fat distribution were recorded, and mitochondrial DNA copy number in the hearts and livers and lipid metabolic signaling in the liver were analyzed. Data were analyzed by one-way analysis of variance and a student’s t-test. Results: TRI led to a significant increase (p < .001) in body weight that was dependent on time and individuals [42.1% of mice were overweight (50% increase in body weight) 4 months post-TRI and 100% of mice were overweight at 10 months post-TRI]. Gross anatomical features of abdominal visceral fat distribution and storage in radiation-induced overweight/severely overweight mice were similar to those of high fat diet-induced overweight/severely overweight mice. The mitochondrial genome of heart and liver tissues from overweight/severely overweight mice had significantly (p < .05) decreased functional mitochondrial DNA copy number (ratios decreased from 1 to 0.71 or 0.49, respectively) and significantly (p < .05) increased mitochondrial DNA mutations (ratios increased from 1 to 3.21 or 1.83, respectively). CPT1 and IRS2 lipid metabolic signaling was significantly (p < .05–.01) decreased for both mRNA (fold decrease from 1 to 0.60 or 0.55, respectively) and protein (fold decrease from 1 to 0.62 or 0.19, respectively). Conclusions: TRI can cause mice to gain weight. These findings indicate that TRI can result in lipid metabolic abnormalities and provide a model to study the factors that result in these abnormalities.

Mo2028 A Functional Approach to Improve Barrier Function in Irradiated Mice

Tight junctions are formed by mature enterocytes and create an efficient barrier that has the necessary machinery for electrolyte and nutrient absorption. Radiotherapy causes epithelial barrier dysfunction leading to increased macromolecular translocation into the systemic compartment causing endotoxemia and inflammatory response. Recently, it was shown that glucose and some amino acids (AAs) activated active anion secretion and/ or increased paracellular permeability, whereas our mitigating amino acids mixture (MAAM) comprised of lysine, glycine, tryptophan, tyrosine, aspartic acid, isoleucine, threonine, valine, and serine increased electrolyte absorption and decreased paracellular permeability. The mechanism by which these amino acids tightened the mucosal barrier was not known. Therefore, studies were undertaken to determine the mechanism by which MAAM tightened the mucosal barrier. NIH Swiss mice were irradiated using a 137Cs source. Radiation dose-dependent and time-dependent (0, 6, 12, 24, 48, 72, and 144 hr after irradiation) changes in transepithelial electrical resistance (TEER) and dilution potential (DP) and the relative permeability of Cland Na+ (PCl/PNa) were measured. Transmission electron microscopy (TEM) images, Western blot analysis, and immunohistochemistry (IHC) for the cell junction protein complex (Claudin 1, 2, 5, Nectin, and E-cadherin) were performed at similar time points. Radiation resulted in a dose-dependent increase in conductance (43.3 ± 1.2, 29.5 ± 1.2, 48.2 ± 2.3, 48.9 ± 1.7, 38.7 ± 1.2 mS at 0, 1, 3, 5, 7 Gy, respectively). Similarly, time-dependent changes in conductance showed a maximal increase occurring in the first 6 hr (46.3 ± 1.5); there was no significant difference with increasing time after irradiation. Treatment with MAAM resulted in a significant decrease in conductance. DP studies showed that MAAM restored ion selectivity. TEM showed disruption of the cell junction complex and formation of clear space between the cells as early as 6 hr after irradiation. The changes persisted for the duration of the experiment (6 days). MAAM prevented cell junction disruption in mice; this was identifiable at 6 hr after irradiation. Western analysis and IHC showed a radiation dose-dependent increase in Claudin 1, 2, 5, Nectin, and E-cadherin except at 7 Gy. MAAM further increased Claudin 1, 2, 5, Nectin, and E-cadherin across all radiation doses. MAAM treatment decreased paracellular permeability. TEM showed disruption of the cell junction complex that peaked at 6 hours and was corrected using MAAM. Western analysis and IHC showed that radiation-induced alterations in the cell junction protein complex were further corrected in the MAAM group. We conclude that the disruption in the barrier results from changes in the cell junction complex and that MAAM restores the barrier function by correcting these changes.