Abstract
Background/Aim: Radiation therapy is pivotal in cancer treatment; however, its efficacy is limited by challenges such as tumor recurrence. This study delves into the role of exosomes, which are molecular cargo-bearing vesicles, in influencing cell proliferation, radioresistance, and consequent post-irradiation tumor recurrence. Given the significance of exosomes from irradiated malignancies in diagnostics and therapy, it is vital to delineate their functional dynamics, especially in breast and cervical cancer cell lines, where the impact of irradiation on exosome behavior remains enigmatic. Materials and Methods: Using MDA-MB-231 and HeLa cell lines, exosomes were isolated from the culture supernatant via ultracentrifugation. The bicinchoninic acid assay was used to measure exosome quantities in irradiated and non-irradiated cells. Radiosensitivity was assessed using colony formation assays, while the role of the MAPK/Erk signaling pathway in recipient cell proliferation and radioresistance was probed using western blotting. Results: Irradiated cells, in both MDA-MB-231 and HeLa lines, produced significantly more exosomes than their non-irradiated counterparts. Co-culturing irradiated cells with exosomes led to increased cell survival post-irradiation and enhanced cell proliferation in both cell lines. Western blotting indicated elevated p-Erk expression in such cells, underscoring the influence of the MAPK/Erk pathway in radioresistance and proliferation. Conclusion: The study establishes a potential nexus between exosome secretion and tumor resurgence following radiotherapy. The spotlight falls on the MAPK/ERK signaling conduit as a key influencer. This new knowledge provides an innovative strategy for counteracting cancer recurrence after radiotherapy, emphasizing the importance of understanding the multifaceted roles of exosomes in this context.
Exosomes are extracellular vesicles with diameters ranging from 30 to 200 nm that serve as integral cellular communicators by harboring an assortment of biological entities including DNA, mRNAs, miRNAs, proteins and lipids (1). In addition to their structural composition, these vesicles are pivotal regulators of both pathogenesis and physiological processes that influence immune responses, inflammation, apoptosis and angiogenesis (2).
Exosome formation is intricately linked to the endosomal pathway. It commences with the biogenesis of early-sorting endosomes (ESEs) enriched with cell surface proteins. This is achieved by maturation of ESEs into late-sorting endosomes (LSEs), during which internal budding produces intraluminal vesicles (ILVs). The terminal phase of this biogenetic cascade culminates with multivesicular bodies (MVBs) – entities loaded with ILVs – fusing to the plasma membrane, leading to the release of ILVs as exosomes (3, 4).
These findings shed light on the significant influence of exosomes on cancer phenotypes, metastatic potential, and chemoresistance (5). Changes in the surrounding milieu of exosomes can induce alterations in their cargo, rendering them capable of fostering a tumor-promoting environment. Supporting this, Hoshino et al. delineated the pivotal roles of MVBs and exosomes in invadopodia formation, emphasizing that exosomes emanating from cells with maturing invadopodia can trigger invadopodia formation in recipient cells (6). Moreover, the multifaceted roles of exosomes include the modulation of immune responses, attenuation of chemotherapy outcomes, and championing angiogenesis (7). Compelling evidence indicates that following X-ray irradiation, exosomes released from squamous head and neck cancer cell lines provide a survival advantage to neighboring malignant cells (8).
To gain a deeper understanding of the effects of radiation exposure on the secretion of exosomes by cancer cells and how these radiation-induced exosomes influence the proliferation and radiation resistance of other cancer cells, we conducted a series of investigations.
Materials and Methods
Cell culture. MDA-MB-231 human breast cancer cells and HeLa human cervical cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 1% penicillin, streptomycin, and L-glutamine (Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO2. The cells were maintained at less than 90% confluency.
Irradiation. MDA-MB-231 and HeLa cells were irradiated with gamma rays. 137Cs gamma-ray (0.662 MeV) irradiation was conducted using a Gammacell 40 Exactor (MDS Nordion, Ottawa, Canada).
Ultracentrifugation. Exosomes were collected from the culture supernatants of MDA-MB-231 and HeLa cells by ultracentrifugation. Prior to exosome collection, the cell culture medium was replaced with DMEM without FBS. Cell debris and relatively large extracellular vesicles were removed following centrifugation at 2,000 × g for 20 min at 4°C and 12,000 × g for 30 min at 4°C. Next, ultracentrifugation at 175,000 × g for 84 min at 4°C using Optima XPN-80 (Beckman Coulter, Brea, CA, USA) was conducted, and each pellet was resuspended in PBS and ultracentrifuged at 175,000 × g for 84 min at 4°C. Finally, each exosome pellet was suspended in PBS, and filtered through a 0.22 μm syringe filter (SLGPR33RS, Merck, Darmstadt, Germany).
Bicinchoninic acid assay. The bicinchoninic acid (BCA) assay was performed to measure the quantity of exosomes collected by ultracentrifugation using a TaKaRa BCA Protein Assay Kit (T9300A, TaKaRa Bio, Shiga, Japan) according to the manufacturer’s instructions. A standard curve was constructed using BSA standard solutions at different concentrations. Then, standards and samples were combined with a working solution, and 200 μl of each standard and sample was prepared in triplicates. After incubation at 37°C for 1 h in the dark, the absorbance values were measured at 562 nm with Varioskan LUX (Thermo Fisher Scientific).
Colony formation assay. Colony formation assay was conducted to assess radiosensitivity. Cells were seeded into dishes, and these dishes were irradiated. Exosomes were then added to the culture medium at a concentration of 1 μg/ml, while PBS was added to the control group. After 14 d, the cells were fixed with formalin and stained with 0.1% crystal violet solution. After staining, the number of colonies was counted, and the survival fraction (SF) was calculated. At least three independent experiments were performed for each group.
Cell proliferation assay. Cells were seeded into six-well plates after which exosomes were added to the culture medium at a concentration of 1 μg/ml, while PBS was added to the control group. After 24, 48, and 72 h, we collected all cells in three wells and the number of cells was counted.
Protein extraction and western blotting. Protein extraction was performed using RIPA Buffer (J63306.AK, Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (78441, Thermo Fisher Scientific). Cells were disrupted by ultrasonication, were then centrifuged to remove cell debris at 12,000 × g for 15 min at 4°C, and the supernatants were collected. After the preparation of samples and gels, proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto fluorescent polyvinylidene difluoride membranes (LF PVDF, Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% skimmed milk for 1 h. Subsequently the primary antibodies were added: phospho-p44/42 MAPK (Erk1/2) (1:1,000, Thr202/Tyr204, D13. 14. 4E, XP®, Cat#4370, CST), p44/42 MAPK (Erk1/2) (1:1,000, 137F5, Cat#4695, CST), and GAPDH (1:1,000, Cat#60004-1-IG, Proteintech), and allowed to react with the proteins. The mixture was incubated overnight on a shaker at 4°C. After washing three times with Tris-buffered saline containing 0.1% Tween 20 (TBS-T), a secondary antibody (peroxidase-conjugated Affinipure sheep anti-mouse IgG, H+L, Cat#5151-035-003, Jackson ImmunoResearch) was added and incubated on a shaker for 1 h at room temperature. After washing with TBS-T thrice, the chemiluminescence of the bands was detected using Clarity Western ECL Substrate (Bio-Rad).
Statistics. Each experiment was independently repeated at least three times. The results are presented as mean±standard deviation (SD). Statistical analyses were performed using Student t-test to compare the means of two groups or Tukey’s honestly significant difference (HSD) test to compare the means of three groups. Differences were considered statistically significant at p<0.05. JMP 16 Pro (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis.
Results
Radiation enhances exosomal protein release. Non-irradiated MDA-MB-231 cells released exosomes with 0.16 μg of protein per 1×107 cells, while HeLa cells released 0.71 μg. After 4 Gy of irradiation, the protein content of MDA-MB-231 cells rose to 0.71 μg (a 3.28-fold increase), and that of HeLa cells increased to 1.67 μg (a 1.73-fold rise), as shown in Figure 1. Protein levels were notably higher in the irradiated groups than in the non-irradiated groups, suggesting that irradiation modulates exosomal release or content.
Cancer-derived exosome isolation and post-irradiation quantitative analysis. The quantity of exosomes from MDA-MB-231 and HeLa cells 48 h after irradiation was determined using the bicinchoninic acid (BCA) assay. The quantities were standardized per 1×107 cells. Data are expressed as mean±standard deviation (SD); n=3, **p<0.01 using two-way ANOVA followed by Tukey’s test.
Exosomes from irradiated cells show increased radioresistance in MDA-MB-231 and HeLa cultures. The survival fraction of MDA-MB-231 and HeLa cells was assessed following incubation under diverse conditions, including the exposure to exosomes from different origins. These conditions comprised a control group treated with PBS, exosomes derived from non-irradiated cells (Exosome-0Gy), and exosomes obtained from cells exposed to 4 Gy of radiation (Exosome-4Gy). It is important to note that the exosomes applied to the MDA-MB-231 and HeLa cell cultures were derived from their respective cell types. A pronounced increase in the SF was observed in both MDA-MB-231 and HeLa cells treated with Exosome-4Gy compared to the other groups (Figure 2A). This suggests that exosomes derived from irradiated cells may confer radioresistance to recipient cells.
Effect of irradiated exosomes on cell proliferation and resistance. (A) Survival fraction (SF) after gamma-ray irradiation at 4 Gy. The SF in MDA-MB-231 and HeLa cells was determined using a colony formation assay. (B) Effect of radiation-treated exosomes on cell proliferation. The concentration of exosomes added in both groups was 1 μg/ml. Data are represented as mean±SD; n=3, n.s.: p>0.05, *p<0.05, **p<0.01 using one-way ANOVA followed by Tukey’s test.
Effects of exosomes from irradiated cells on cell proliferation. We compared the proliferation rates of cells treated with PBS, Exosome-0Gy, and Exosome-4Gy. Our results clearly indicated that MDA-MB-231 and HeLa cells exhibited a markedly enhanced proliferation rate when treated with Exosome-4Gy compared to the control (Figure 2B). In contrast, no significant difference in proliferation was observed between PBS and cells treated with Exosome-0Gy. This suggests that irradiation imbues exosomes with properties that promote cell proliferation.
Exosomes from irradiated cells elevate p-Erk expression, augmenting cell proliferation via the MAPK/Erk pathway. Our findings demonstrate that exosomes enhance cell survival and proliferation. Therefore, we investigated the changes in protein expression associated with cell proliferation, focusing primarily on extracellular signal-regulated kinase (Erk) and its phosphorylated counterpart p-Erk. To further understand the role of exosomes in Erk signaling, we assessed Erk and p-Erk expression in MDA-MB-231 and HeLa cells incubated with exosomes. Western blot analysis revealed increased p-Erk expression in cells treated with Exosome-4Gy for 72 h compared to the control and cells treated with Exosome-0Gy (Figure 3). These results suggest that exosome uptake augments cell proliferation via the MAPK/Erk pathway.
Cellular protein expression. The protein levels of extracellular signal-regulated kinase (Erk) in MDA-MB-231 and HeLa cell lysates incubated with exosomes for 72 h after adding exosomes from non-irradiated cells (Exosome-0Gy) or cells irradiated with 4 Gy of gamma rays (Exosome-4Gy). Experiments were repeated three times.
Discussion
Exosomes released from cells have various effects on recipient cells (9, 10). Furthermore, the environment around donor cells, including drugs, changes in temperature, and hypoxia, can alter the exosome content. Moreover, exosomes released from irradiated cancer cells are advantageous for the survival of the recipient cells (14). Due to this, the possibility of recurrence and metastasis might increase after radiation therapy (15, 16). Thus, understanding the dynamic interplay between exosome release and environmental factors is crucial for comprehending the intricate mechanisms underlying cellular responses to radiation and its implications for cancer treatment outcomes.
In this study, irradiation increased the quantity of exosomes secreted by MM231 and HeLa cells. Jabbari et al. reported that exosome biogenesis was activated and secretion of exosomes from MCF-7 breast cancer cells was increased by X-ray irradiation in a dose-dependent manner, which could explain the therapeutic resistance of cancer cells to radiation therapy (17). Furthermore, Abramowicz et al. demonstrated a dose-dependent increase in proteins released from irradiated UM-SCC6 human head-and-neck cancer cells (18). Our results suggest that irradiated cancer cells increase the secretion of exosomes to prevent cancer cell death (Figure 4).
Schematic representation of exosome secretion and its effects after radiation exposure. Following radiation exposure, cancer cells exhibit an enhanced formation of multivesicular bodies (MVBs), leading to increased exosome secretion. These exosomes promote intercellular communication between cancer cells, resulting in cell proliferation and radioresistance. Images were obtained using BioRender.com.
We showed that the SF of MM231 and HeLa cells incubated with Exosome-4Gy was significantly higher than that of cells incubated with PBS and Exosome-0Gy. Mutschelknaus et al. indicated that exosomes from irradiated cells increase DNA repair in head and neck squamous carcinoma cells (8). Consequently, irradiation alters the cargo of exosomes, which contribute to communication between irradiated and non-irradiated cells.
Enhanced proliferation was observed in cells incubated with exosomes originating from 4 Gy-irradiated cells. Compared with the non-irradiated group, the expression of p-Erk protein was elevated in irradiated cells. Moreover, p-Erk expression in the Exosome-4Gy group was elevated. Yang et al. demonstrated that bladder cancer cell-derived exosomes inhibited the apoptosis of cancer cells, and these effects were triggered by the activation of the PI3K/Akt and MAPK/Erk pathways (19). Erk1/2 is a protein kinase expressed in a wide range of cell types and regulates cell proliferation, apoptosis, and treatment resistance (20–22). We hypothesized that an undefined factor was translocated from irradiated cells to other cells, resulting in enhanced cell proliferation via the ERK pathway.
Irradiation increases exosome release and alters their content, potentially promoting tumor growth, recurrence, or metastasis post-radiotherapy. Enhanced radioresistance and proliferation are linked to Erk1/2 in exosomes from irradiated cells. Targeting the MAPK/Erk pathway might improve the radiotherapeutic effect by disrupting communication between irradiated and non-irradiated cancer cells. Further research is required to understand how these exosomes influence the MAPK/Erk pathway and their roles in radiation therapy.
Conclusion
Exosomes are recognized as orchestrators of cellular dynamics and critically influence cell proliferation and radioresistance. Our investigation revealed a heightened effect of exosomes sourced from irradiated cells on target cells. Intriguingly, this augmented influence is potentially mediated via the MAPK/Erk signaling cascade, highlighting a pivotal nexus that warrants deeper exploration to comprehend radiation-mediated cellular responses.
Acknowledgements
This study was supported by the Center for Medical Research and Education of the Graduate School of Medicine, Osaka University, Japan (JSPS KAKENHI, Grant Number 23K07178, JSPS KAKENHI Grant Number JP22H03025, and JST SPRING, Grant Number JPMJSP2138).
Footnotes
Conflicts of Interest
The Authors declare no conflicts of interest related to this study.
Authors’ Contributions
Y.D. and K. T. designed the experiment and wrote the paper. Y. D., M. K., and S. K. performed the experiments. K. M., S. T., S. S., and M. K. discussed the results and reviewed the manuscript. K. O. supervised the study.
- Received October 12, 2023.
- Revision received November 20, 2023.
- Accepted November 23, 2023.
- Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
