A GPC2 antibody-drug conjugate is efficacious against neuroblastoma and small-cell lung cancer via binding a conformational epitope.

Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2's immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2.

Roles of Simvastatin and Sildenafil in Modulation of Cranial Irradiation-Induced Bystander Multiple Organs Injury in Rats.

In radiobiology and radiation oncology fields, the observation of a phenomenon called radiation-induced bystander effect (RIBE) has introduced the prospect of remotely located tissues' affection. This phenomenon has been broadly developed to involve the concept of RIBE, which are relevant to the radiation-induced response of a distant tissue other than the irradiated one. The current study aimed at investigating each of the RIBE of cranial irradiation on oxidative and inflammatory status in different organs such as liver, kidney, heart, lung, and spleen. Being a vital target of the cholinergic anti-inflammatory response to an inflammatory stimulus, the splenic α-7-nicotinic acetylcholine receptor (α-7nAchR) was evaluated and the hepatic contents of thioredoxin, peroxisome proliferator-activated receptor-alpha and paraoxinase-1 (Trx/PPAR-α/PON) were also assessed as indicators for the liver oxidative stress and inflammatory responses. Being reported to act as antioxidant and anti-inflammatory agents, simvastatin (SV) and/or sildenafil (SD) were investigated for their effects against RIBE on these organs. These objectives were achieved via the biochemical assessments and the histopathological tissues examinations. Five experimental groups, one sham irradiated and four irradiated groups, were exposed to cranial irradiation at dose level of 25 Gy using an experimental irradiator with a Cobalt (Co60) source, RIBE, RIBE + SV (20 mg.(kg.bw)-1 day-1), RIBE + SD (75 mg.(kg.bw)-1 day-1), and RIBE + SV + SD. Cranial irradiation induced structural, biochemical, and functional dys-regulations in non-targeted organs. RIBE-induced organs' injuries have been significantly corrected by the administration of SV and/or SD. Our results suggest the possibility of a potentiated interaction between SV and SD in the modulation of the RIBE associated with head and neck radiotherapy.

DHES0815A, a novel antibody-drug conjugate targeting HER2/neu, is highly active against uterine serous carcinomas in vitro and in vivo.

OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2 overexpressing tumors at a distinct epitope from that bound by trastuzumab and pertuzumab after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A against primary USC cell lines and xenografts. METHODS: Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for C-erbB2 gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability and bystander killing in USC cell lines after exposure to DHES0815A, the non-targeted ADC, and the unconjugated antibody (i.e. MHES0488A) were evaluated using flow cytometry-based-assays. In vivo activity of DHES0815A was tested against HER2/neu overexpressing USC xenografts. RESULTS: High HER2/neu protein expression was seen in 25% (3/12) of the primary USC cell lines. USC cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (p < 0.001). DHES0815A did not induce significant bystander killing of HER2/neu negative tumors when admixed with HER2/neu positive tumors. DHES0815A caused growth-inhibition and increased survival in USC HER2/neu overexpressing xenografts when compared to controls (p < 0.01). CONCLUSIONS: DHES0815A is both highly selective and toxic to USC tumors overexpressing HER2/neu both in vitro and in vivo. HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy.

Spatially-resolved pharmacokinetic/pharmacodynamic modelling of bystander effects of a nitrochloromethylbenzindoline hypoxia-activated prodrug.

PURPOSE: Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates for any diffusion limitations of the prodrug in tumour tissue. METHODS: Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC-MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green's function modelling, respectively. RESULTS: NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green's function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug. CONCLUSIONS: The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue.

Uncialamycin-based antibody-drug conjugates: Unique enediyne ADCs exhibiting bystander killing effect.

Antibody-drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics with at least 11 approved therapies and over 80 advancing through clinical trials. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin [Formula: see text], have a proven success track record. However, they pose a significant synthetic challenge in the development and optimization of linker drugs. We have recently reported a streamlined total synthesis of uncialamycin, another representative of the enediyne class of compounds, with compelling synthetic accessibility. Here we report the synthesis and evaluation of uncialamycin ADCs featuring a variety of cleavable and noncleavable linkers. We have discovered that uncialamycin ADCs display a strong bystander killing effect and are highly selective and cytotoxic in vitro and in vivo.

Study of modifying effects of astaxanthin on cytogenetic manifestations of bystander response in human peripheral blood lymphocytes in vitro.

AIM: To study the possible impact of astaxanthin on the cytogenetic manifestations of the simultaneous development of radiation-induced (RIBE) and tumor-induced bystander effect (TIBE) in human peripheral blood lymphocytes. MATERIALS AND METHODS: Peripheral blood lymphocytes from healthy persons and patients with chronic lymphocytic leukemia were cultured separately or cocultured with or without previous irradiation in vitro by 137Cs at a dose of 0.5 Gy. The cells were cultured with and without 20 µg/ml astaxanthin. RESULTS: In the presence of astaxanthin, the decrease of chromosomal instability both in the variant with separate TIBE and with simultaneous development of TIBE and RIBE was observed as the reduction in the frequency of simple chromosome-type aberrations, namely, double fragments. The average level of chromatid-type aberrations did not change under the action of astaxanthin. Although the total chromosome instability in bystander cells diminished, this did not lead to the elimination of the RIBE and TIBE development in the presence of astaxanthin. CONCLUSION: In the setting of experiment, astaxanthin did not reduce the frequency of chromatid-type aberrations in bystander cells due to RIBE and TIBE but reduced the frequency of simple aberrations of chromosomal type, not associated with the development of bystander response phenomenon.

Role of MicroRNA-145 in DNA Damage Signalling and Senescence in Vascular Smooth Muscle Cells of Type 2 Diabetic Patients.

Increased cardiovascular morbidity and mortality in individuals with type 2 diabetes (T2DM) is a significant clinical problem. Despite advancements in achieving good glycaemic control, this patient population remains susceptible to macrovascular complications. We previously discovered that vascular smooth muscle cells (SMC) cultured from T2DM patients exhibit persistent phenotypic aberrancies distinct from those of individuals without a diagnosis of T2DM. Notably, persistently elevated expression levels of microRNA-145 co-exist with characteristics consistent with aging, DNA damage and senescence. We hypothesised that increased expression of microRNA-145 plays a functional role in DNA damage signalling and subsequent cellular senescence specifically in SMC cultured from the vasculature of T2DM patients. In this study, markers of DNA damage and senescence were unambiguously and permanently elevated in native T2DM versus non-diabetic (ND)-SMC. Exposure of ND cells to the DNA-damaging agent etoposide inflicted a senescent phenotype, increased expression of apical kinases of the DNA damage pathway and elevated expression levels of microRNA-145. Overexpression of microRNA-145 in ND-SMC revealed evidence of functional links between them; notably increased secretion of senescence-associated cytokines and chronic activation of stress-activated intracellular signalling pathways, particularly the mitogen-activated protein kinase, p38α. Exposure to conditioned media from microRNA-145 overexpressing cells resulted in chronic p38α signalling in naïve cells, evidencing a paracrine induction and reinforcement of cell senescence. We conclude that targeting of microRNA-145 may provide a route to novel interventions to eliminate DNA-damaged and senescent cells in the vasculature and to this end further detailed studies are warranted.

Radiation-induced bystander effects impair transplanted human hematopoietic stem cells via oxidative DNA damage.

Total body irradiation (TBI) is commonly used in host conditioning regimens for human hematopoietic stem cell (HSC) transplantation to treat various hematological disorders. Exposure to TBI not only induces acute myelosuppression and immunosuppression, but also injures the various components of the HSC niche in recipients. Our previous study demonstrated that radiation-induced bystander effects (RIBE) of irradiated recipients decreased the long-term repopulating ability of transplanted mouse HSCs. However, RIBE on transplanted human HSCs have not been studied. Here, we report that RIBE impaired the long-term hematopoietic reconstitution of human HSCs as well as the colony-forming ability of human hematopoietic progenitor cells (HPCs). Our further analyses revealed that the RIBE-affected human hematopoietic cells showed enhanced DNA damage responses, cell-cycle arrest, and p53-dependent apoptosis, mainly because of oxidative stress. Moreover, multiple antioxidants could mitigate these bystander effects, though at different efficacies in vitro and in vivo. Taken together, these findings suggest that RIBE impair human HSCs and HPCs by oxidative DNA damage. This study provides definitive evidence for RIBE on transplanted human HSCs and further justifies the necessity of conducting clinical trials to evaluate different antioxidants to improve the efficacy of HSC transplantation for the patients with hematological or nonhematological disorders.

Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10-100 kDa Fraction of Conditioned Medium.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Quantifying ADC bystander payload penetration with cellular resolution using pharmacodynamic mapping.

With the recent approval of 3 new antibody drug conjugates (ADCs) for solid tumors, this class of drugs is gaining momentum for the targeted treatment of cancer. Despite significant investment, there are still fundamental issues that are incompletely understood. Three of the recently approved ADCs contain payloads exhibiting bystander effects, where the payload can diffuse out of a targeted cell into adjacent cells. These effects are often studied using a mosaic of antigen positive and negative cells. However, the distance these payloads can diffuse in tumor tissue while maintaining a lethal concentration is unclear. Computational studies suggest bystander effects partially compensate for ADC heterogeneity in tumors in addition to targeting antigen negative cells. However, this type of study is challenging to conduct experimentally due to the low concentrations of extremely potent payloads. In this work, we use a series of 3-dimensional cell culture and primary human tumor xenograft studies to directly track fluorescently labeled ADCs and indirectly follow the payload via an established pharmacodynamic marker (γH2A. X). Using TAK-164, an anti-GCC ADC undergoing clinical evaluation, we show that the lipophilic DNA-alkylating payload, DGN549, penetrates beyond the cell targeted layer in GCC-positive tumor spheroids and primary human tumor xenograft models. The penetration distance is similar to model predictions, where the lipophilicity results in moderate tissue penetration, thereby balancing improved tissue penetration with sufficient cellular uptake to avoid significant washout. These results aid in mechanistic understanding of the interplay between antigen heterogeneity, bystander effects, and heterogeneous delivery of ADCs in the tumor microenvironment to design clinically effective therapeutics.

Tolerogenic nanoparticles suppress central nervous system inflammation.

Therapeutic approaches for the induction of immune tolerance remain an unmet clinical need for the treatment of autoimmune diseases, including multiple sclerosis (MS). Based on its role in the control of the immune response, the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is a candidate target for novel immunotherapies. Here, we report the development of AhR-activating nanoliposomes (NLPs) to induce antigen-specific tolerance. NLPs loaded with the AhR agonist ITE and a T cell epitope from myelin oligodendrocyte glycoprotein (MOG)35-55 induced tolerogenic dendritic cells and suppressed the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, in preventive and therapeutic setups. EAE suppression was associated with the expansion of MOG35-55-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells), concomitant with a reduction in central nervous system-infiltrating effector T cells (Teff cells). Notably, NLPs induced bystander suppression in the EAE model established in C57BL/6 × SJL F1 mice. Moreover, NLPs ameliorated chronic progressive EAE in nonobese diabetic mice, a model which resembles some aspects of secondary progressive MS. In summary, these studies describe a platform for the therapeutic induction of antigen-specific tolerance in autoimmune diseases.

Astragalus polysaccharide inhibits radiation-induced bystander effects by regulating apoptosis in Bone Mesenchymal Stem Cells (BMSCs).

The purpose of this study was to investigate the effects of astragalus polysaccharides (APS) on the proliferation and apoptosis of bone marrow mesenchymal stem cells (BMSCs) induced by X-ray radiation-induced A549 cells bystander effect (RIBE), and to explore their mechanisms. In this study, APS increased the reduced cell proliferation rate induced by RIBE and inhibiting the apoptosis of bystander cells. In terms of mechanism, APS up-regulates the proteins Bcl-2, Bcl-xl, and down-regulates the proteins Bax and Bak, which induces a decrease in mitochondrial membrane potential, which induces the release of Cyt-c and AIF, which leads to caspase-dependent and caspase-independent pathway to cause apoptosis. In addition, we believe that ROS may be the main cause of these protein changes. APS can inhibit the generation of ROS in bystander cells and thus inhibit the activation of the mitochondrial pathway, further preventing cellular damage caused by RIBE.

The nanomaterial-induced bystander effects reprogrammed macrophage immune function and metabolic profile.

Bystander effects in biological systems are the responses shown by nontargeted neighboring cells, and critical to the bio-nano interface interactions. In addition to direct effects, bystander effects also determine the design, applications and safety of nanomaterials, although the related information of nanomaterial-induced bystander effects remain largely unknown. A coculture system of A549 and THP-1 was established to mimic the lung microenvironment to study the bystander effects of WS2 nanosheets (representative transition-metal dichalcogenide nanosheets) on microenvironment macrophages during the inhalation exposure or the nanomaterial biomedical application in the lung. Lung cells exposed to WS2 nanosheet resulted in an increase in reactive oxygen species and the depolarization of mitochondrial membrane potential in neighboring macrophages. Bystander exposure also induced macrophage polarization toward the anti-inflammatory M2 phenotype, which is adverse to disease therapy. Metabolomics showed that WS2 nanosheets disturbed the energy metabolism and amino acid metabolism of macrophages, consistent with the metabolic characteristics of M2 macrophages. Nitric oxide-transforming growth factor-ß1 played an important mediator in the bystander effects. Importantly, WS2 nanosheet bystander exposure affected macrophage phagocytosis and migration and altered the macrophage immune response to endotoxin. This study improves the current understanding of bio-nano interactions and highlights the importance of neighboring cell responses, allowing us to use the maximum benefits of nanomaterials while limiting their adverse bystander effects.

Evolution of the Systems Pharmacokinetics-Pharmacodynamics Model for Antibody-Drug Conjugates to Characterize Tumor Heterogeneity and In Vivo Bystander Effect.

The objective of this work was to develop a systems pharmacokinetics-pharmacodynamics (PK-PD) model that can characterize in vivo bystander effect of antibody-drug conjugate (ADC) in a heterogeneous tumor. To accomplish this goal, a coculture xenograft tumor with 50% GFP-MCF7 (HER2-low) and 50% N87 (HER2-high) cells was developed. The relative composition of a heterogeneous tumor for each cell type was experimentally determined by immunohistochemistry analysis. Trastuzumab-vc-MMAE (T-vc-MMAE) was used as a tool ADC. Plasma and tumor PK of T-vc-MMAE was analyzed in N87, GFP-MCF7, and coculture tumor-bearing mice. In addition, tumor growth inhibition (TGI) studies were conducted in all three xenografts at different T-vc-MMAE dose levels. To characterize the PK of ADC in coculture tumors, our previously published tumor distribution model was evolved to account for different cell populations. The evolved tumor PK model was able to a priori predict the PK of all ADC analytes in the coculture tumors reasonably well. The tumor PK model was subsequently integrated with a PD model that used intracellular tubulin occupancy to drive ADC efficacy in each cell type. The final systems PK-PD model was able to simultaneously characterize all the TGI data reasonably well, with a common set of parameters for MMAE-induced cytotoxicity. The model was later used to simulate the effect of different dosing regimens and tumor compositions on the bystander effect of ADC. The model simulations suggested that dose-fractionation regimen may further improve overall efficacy and bystander effect of ADCs by prolonging the tubulin occupancy in each cell type. SIGNIFICANCE STATEMENT: A PK-PD analysis is presented to understand bystander effect of Trastuzumab-vc-MMAE ADC in antigen (Ag)-low, Ag-high, and coculture (i.e., Ag-high + Ag-low) xenograft mice. This study also describes a novel single cell-level systems PK-PD model to characterize in vivo bystander effect of ADCs. The proposed model can serve as a platform to mathematically characterize multiple cell populations and their interactions in tumor tissues. Our analysis also suggests that fractionated dosing regimen may help improve the bystander effect of ADCs.

Membrane-bound TNF mediates microtubule-targeting chemotherapeutics-induced cancer cytolysis via juxtacrine inter-cancer-cell death signaling.

Microtubule-targeting agents (MTAs) are a class of most widely used chemotherapeutics and their mechanism of action has long been assumed to be mitotic arrest of rapidly dividing tumor cells. In contrast to such notion, here we show-in many cancer cell types-MTAs function by triggering membrane TNF (memTNF)-mediated cancer-cell-to-cancer-cell killing, which differs greatly from other non-MTA cell-cycle-arresting agents. The killing is through programmed cell death (PCD), either in way of necroptosis when RIP3 kinase is expressed, or of apoptosis in its absence. Mechanistically, MTAs induce memTNF transcription via the JNK-cJun signaling pathway. With respect to chemotherapy regimens, our results establish that memTNF-mediated killing is significantly augmented by IAP antagonists (Smac mimetics) in a broad spectrum of cancer types, and with their effects most prominently manifested in patient-derived xenograft (PDX) models in which cell-cell contacts are highly reminiscent of human tumors. Therefore, our finding indicates that memTNF can serve as a marker for patient responsiveness, and Smac mimetics will be effective adjuvants for MTA chemotherapeutics. The present study reframes our fundamental biochemical understanding of how MTAs take advantage of the natural tight contact of tumor cells and utilize memTNF-mediated death signaling to induce the entire tumor regression.

A Systems Pharmacology Model for Drug Delivery to Solid Tumors by Antibody-Drug Conjugates: Implications for Bystander Effects.

Antibody-drug conjugates (ADCs) are cancer drugs composed of a humanized antibody linked to a cytotoxic payload, allowing preferential release of payload in cancer cells expressing the antibody-targeted antigen. Here, a systems pharmacology model is used to simulate ADC transport from blood to tumor tissue and ADC uptake by tumor cells. The model includes effects of spatial gradients in drug concentration in a three-dimensional network of tumor blood vessels with realistic geometry and accounts for diffusion of ADC in the tumor extracellular space, binding to antigen, internalization, intracellular processing, and payload efflux from cells. Cells that process an internalized ADC-antigen complex may release payload that can be taken up by other "bystander" cells. Such bystander effects are included in the model. The model is used to simulate conditions in previous experiments, showing good agreement with experimental results. Simulations are used to analyze the relationship of bystander effects to payload properties and single-dose administrations. The model indicates that exposure of payload to cells distant from vessels is sensitive to the free payload diffusivity in the extracellular space. When antigen expression is heterogeneous, the model indicates that the amount of payload accumulating in non-antigen-expressing cells increases linearly with dose but depends only weakly on the percentage of antigen-expressing cells. The model provides an integrated mechanistic framework for understanding the effects of spatial gradients on drug distribution using ADCs and for designing ADCs to achieve more effective payload distribution in solid tumors, thereby increasing the therapeutic index of the ADC.

Population Dynamics in Cell Death: Mechanisms of Propagation.

Cell death can occur through numerous regulated mechanisms that are categorized by their molecular machineries and differing effects on physiology. Apoptosis and necrosis, for example, have opposite effects on tissue inflammation due to their different modes of execution. Another feature that can distinguish different forms of cell death is that they have distinct intrinsic effects on the cell populations in which they occur. For example, a regulated mechanism of necrosis called ferroptosis has the unusual ability to spread between cells in a wave-like manner, thereby eliminating entire cell populations. Here we discuss the ways in which cell death can propagate between cells in normal physiology and disease, as well as the potential exploitation of cell death propagation for cancer therapy.

Reduced exosomal L-Plastin is responsible for radiation-induced bystander effect.

Radiation-induced bystander effects (RIBE) are discussed as relevant processes during radiotherapy. Irradiated cells are suggested to release growth-inhibitory/DNA-damaging factors transported to non-irradiated cells. However, the molecular nature of this phenomenon has not yet been resolved. We aimed at identifying the growth-inhibitory factor(s) transmitted to non-irradiated cells. RIBE-competent PC3 cells were used to produce conditioned medium (CM) after exposure to ionizing radiation. Indicator cells were incubated with CM and clonogenic survival as well as cell proliferation were determined as endpoints. A549 indicator cells exhibited a bystander effect upon incubation with CM from irradiated PC3 cells. This bystander effect was not due to DNA-damaging factors, but a radiation-triggered reduction of mitogenic/clonogenic activity present in CM. Several tumor cells, but not normal fibroblasts secrete this factor, whose release is reduced by irradiation. We identified L-Plastin to be responsible for the mitogenic/clonogenic activity. Removal of L-Plastin from CM by immunoprecipitation or siRNA-mediated knockdown of L-Plastin expression resulted in loss or reduction of mitogenic/clonogenic activity transmitted via CM, respectively. Exosome-transported L-Plastin was constitutively Ser5-phosphorylated, indicative of its bioactive conformation. In summary, we observed production and exosomal secretion of L-Plastin by cancer cells. Via exosome-transmitted L-Plastin, tumors induce clonogenic and mitogenic activity in cancer and normal cells of the tumor microenvironment. Irradiation inhibits L-Plastin production targeting both cancer cells and the tumor niche and may explain the high impact of radiotherapy in tumor control.

Characterization of Radioprotective, Radiomitigative and Bystander Signaling Modulating Effects of Endogenous Metabolites - Phenylacetate, Ursodeoxycholate and Tauroursodeoxycholate - on HCT116 Human Colon Carcinoma Cell Line.

Exposures to ionizing radiation can cause depletion in stem cell reservoirs and lead to chronic injury processes that exacerbate carcinogenic and inflammatory responses. Therefore, radioprotective measures, against both acute and chronic biological effects of radiation, require frequent intake of nontoxic natural products, which have practical oral administration. The goal of this study was to characterize the radioprotective, radiomitigative and radiation-induced bystander effect-inhibiting properties of endogenous metabolites: phenylacetate, ursodeoxycholate and tauroursodeoxycholate. Compounds were administered pre- and postirradiation as well as in donor and recipient bystander flasks to analyze whether these might adequately protect against radiation injury as well as facilitate recovery from the exposures. The clonogenic HCT116 p53 wild-type cancer cell line in this study shares characteristics of stem cells, such as high reproductive viability, which is an effective marker to demonstrate compound effectiveness. Clonogenic assays were therefore used to characterize radioprotective, radiomitigative and bystander inhibiting properties of treatment compounds whereby cellular responses to radiation were quantified with macroscopic colony counts to measure cell survival in flasks. The results were statistically significant for phenylacetate and tauroursodeoxycholate when administered preirradiation, conferring radioprotection up to 2 Gy, whereas administration postirradiation and in bystander experiments did not confer radioprotection in vitro. These findings suggest that phenylacetate and tauroursodeoxycholate might be effective radioprotectors, although they possess no radiomitigative properties.

Astragalus Polysaccharide Eases G1 Phase-Correlative Bystander Effects through Mediation of TGF- ß R/MAPK/ROS Signal Pathway After Carbon Ion Irradiation in BMSCs.

Although Astragalus polysaccharide (APS) has been shown to have various pharmacological effects, there have been no studies concerning the inhibitory effects of APS on the radiation-induced bystander effects (RIBE). The aim of this study was to investigate whether APS could suppress RIBE damage by inhibiting cell growth, micronucleus (MN) formation and 53BP1 foci number increased in bone marrow mesenchymal stem cells (BMSCs), named bystander cells, as well as to explore its mechanism. In this study, APS decreased proliferation and colony rate of bystander cells by inducing cell cycle arrest at G1 phase via extrinsic and intrinsic DNA damage. Regarding mechanism, APS inhibited mitogen-activated protein kinase (MAPK) signal pathway by down-regulating the expression of the key proteins, phosphorylated JNK (p-JNK), phosphorylated ERK (p-ERK) but not phosphorylated P38 (p-P38), and down-regulating their downstream function protein and molecule, cyclooxygenase-2 (COX-2) and reactive oxygen species (ROS). Moreover, in bystander cells, APS inhibits expression of transforming growth factor ß receptor II (TGF- ß R II), a cell membrane receptor, resulting in lower ROS production and secretion via TGF- ß R-JNK/ERK-COX-2/ROS not P38 signaling. They gave a hint that the decreased RIBE damage induced by APS treatment involved TGF- ß R-JNK/ERK-COX-2/ROS down-regulation.