Heavy‐ion‐induced bystander killing of human lung cancer cells: Role of gap junctional intercellular communication

Abstract

It is thought that heavy-ion radiation therapy is a promising modality of cancer therapy that can deliver higher biological effects on tumors than conventional X-ray radiation therapy, and may also conserve the function of normal organs because of its excellent dose distribution.(1) Recently, excellent clinical results were reported in the treatment of lung cancer, prostate cancer, cancer of the head and neck, and malignant tumors of bone and soft tissue.(2) Heavy-ion has high linear energy transfer (LET) with increased relative biological effectiveness (RBE). However, the possible mechanism of increasing cell killing in heavyion therapy is not fully understood. Previous studies have demonstrated that the induction of cell killing of non-irradiated cells by adjacent cells that are directly irradiated by heavy-ions is considered to play an important role in radiation-induced cell death, not only in tumors but also in normal organs. This phenomenon is termed the "bystander effect", and was reported by Nagasawa and Little in 1992.(3) They reported that increased sister chromatid exchanges were observed in more than 30% of cells when less than 1% of the nuclei of cultured cells were actually traversed by an α-particle in Chinese hamster ovary cells. It has been proposed that the mediating mechanism involves secreted soluble factors, (4)(5)(6) an increase in oxidative stress,plasma membrane-bound lipid rafts, (8) and gap junctional intracellular communication (GJIC). (4,7,(9)(10)(11)(12)(13) Gap junctions are membrane channels that connect the cytoplasm of cells and allow direct exchange of small molecules and ions between adjacent cells. GJIC is essential for the maintenance of tissue homeostasis and proliferation and the regulation of embryonic development and differentiation, and it plays a key role in induction of the bystander effect. (14)(15)(16)(17) Here, bystander killing of cells by heavy-ion irradiation was investigated, focusing on the involvement of GJIC on cell survival, to clarify the underlying mechanisms of the bystander effect in a human lung cancer cell line in vitro. Materials and MethodsCell cultures. A549 (a human lung cancer cell line with wild-type p53) was obtained from the Cell Resource Center for Biomedical Research at the Institute of Development, Aging and Cancer, Tohoku University, Japan. Cells were cultured in RPMI-1640 medium (Immuno-Biological Laboratories, Takasaki, Japan) supplemented with 10% fetal calf serum (PAA Laboratories, Pasching, Austria). Two days prior to irradiation experiments, 5 × 10 5 and 5 × 10 4 cells were inoculated into a specially designed 35-mm microbeam dish that was constructed as previously described, (18) to prepare confluent cultures and sparsely populated cultures, respectively. Unless otherwise described, all cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO 2 in air. The size of the cell nucleus and the whole cells was determined to be 14.6 ± 4.3 and 106 ± 24.9 μm 2 , respectively. Drug treatment. Prior to irradiation, cells were p...

Identification of stage-specific biomarkers in lung adenocarcinoma based on RNA-seq data

“…GJB6 has been reported to encode one of connexin proteins, connexin 30 (Cx30). Disruption of gap junction or connexin dysfunction has been indicated to be involved in carcinogenic processes in multiple cancers, including lung carcinogenesis [32][33][34]. Intriguingly, the expression of GJB6 was increased in the stage II of LAC, suggesting that GJB6 serves as an enhancer in the proliferation of tumor cells [35], which may be involved in the LAC pathogenesis by disrupting intercellular communication.…”

Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

“…Funayama et al (2005) reported that the number of non-irradiated CHO-K1 cells in colonies decreased at 60 h after exposure to a high-LET Ar-ion microbeam (11.5 MeV/n, 1260 keV/μm) via the bystander response, using heavy-ion microbeams at the TIARA. The bystander cell killing effect was also reported in A549 (human lung cancer cell line) cells and AG01522D cells after exposure to carbon ions (18.3 MeV/n, 103 keV/μm) or neon ions (13.0 MeV/n, 375 keV/μm) Harada et al, 2009). In these studies, clonogenic survival decreased by about 10%.…”

“…An additional excellent feature of the TIARA facility is that the same particles with similar energy in the broadbeams and microbeams can be used to expose biological materials, thereby contributing significantly to the understanding of heavy-ion-induced bystander responses (Shao et al, 2003;Funayama et al, 2005;Kanasugi et al, 2007;Hamada et al, 2008;Iwakawa et al, 2008;Harada et al, 2009;Fournier et al, 2009;Hino et al, 2010;Mutou-Yoshihara et al, 2012;Autsavapromporn et al, 2013 and so on). The significant role of NO in the bystander response was determined in studies using heavy-ion microbeams (Kanasugi et al, 2007;Mutou-Yoshihara et al, 2012).…”

Health Risks of Space Exploration: Targeted and Nontargeted Oxidative Injury by High-Charge and High-Energy Particles

“…Studies of bystander effects in cell cultures exposed to low fluences of HZE particles are only emerging, and conflicting data have been reported. In early experiments with a microbeam, stressful effects were shown to be propagated from HZE particle-irradiated cells to contiguous bystander cells (125,127,256). In addition, when HZE particle-irradiated cells were cocultured with bystander cells in a manner in which they shared only the growth medium, stressful responses were also induced in the bystander cells and were similar in nature to those generated in the targeted cells (95,278,(298)(299)(300) (Fig.…”

“…The lack of clear knowledge about nontargeted responses (94,127,299) has been singled out by the U.S. National Academies (1) as one of the important factors limiting the prediction of radiation health risks associated with space exploration. This is because during deep space travel, only a small fraction of cells in the human body experiences extremely dense ionizations created along the tracks of the traversing particles in a given day (62).…”