Sarcoma is a heterogeneous group comprised of over 160 different bone and soft tissue neoplasms and categorized more than 60 malignancies in terms of diverse biological and clinical characteristics (Gage
The transcriptional gene silencing is regulated by epigenetic mechanisms such as the DNA methylation, the histone modification, and alterations of nucleosome positions at the DNA level. Unlike genetic events, epigenetic events are reversible, which makes epigenetic regulation extremely interesting from the point of view in developing new therapeutic technologies for cancer treatment. In recent decades, DNA hypomethylating drugs (decitabine and 5-aza-2ʹ-deoxycytidine, also known as 5-aza-dC) have been reported to have anticancer activities in patients with leukemia, myelodysplastic syndrome (MDS), and some other solid tumors (Kaminskas
Ionizing radiation is one of the conventional approaches for local control, and the effects of preoperative radiotherapy in sarcomas include reducing tumor size before surgery and decreasing the risk of local recurrence (Ta
In the present study, we investigated the combined effect of radiation with epigenetic inhibitors (particularly, DNA hypomethylating drugs) in sarcoma cell lines and explored the biological mechanisms of radiosensitization that is mediated by epigenetic regulation in human sarcoma cells.
Human sarcoma cell lines (SK-LMS-1, SK-UT-1, SK-UT-1B, and SK-ES-1) were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were grown in MEM (Welgene, Daegu, Korea) containing 10% fetal bovine serum (HyClone Laboratories Inc, Logan, UT, USA) and 1% antibiotic-antimycotic (Thermo Fisher, Waltham, MA, USA) and maintained at 37°C in an atmosphere of 20% O2 and 5% CO2 incubator. Cells were treated with 5-aza-dC (0.1, 0.5 or 1 μM) (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) or SGI-110 (0.5 or 1 μM) (APExBio, Houston, TX, USA) once daily for 3 days. Cells were incubated with 5-aza-dC or SGI-110 for 3 days before and 24 h after irradiation. Cells that were treated with 5-aza-dC or SGI-110 for 3 days without exposure to radiation or those cells that were solely exposed to irradiation were used as controls. Irradiation (gamma-rays) of cells was performed with a 137Cs ray source (Eckert & Ziegler, Berlin, Germany) at a dose rate of 2.6 Gy/min.
Cells were seeded in 6-well plates (5,000 cells/well) and treated with 5-aza-dC or SGI-110. After drug treatment, cells were irradiated at the specific doses. Twelve to fourteen days after seeding, colonies were fixed and stained with 1.25% crystal violet. The number of colonies containing at least 50 cells was determined.
Cell proliferation was determined by using a CCK-8 assay (Dojindo Molecular Technologies, Rockville, MD, USA). Cells (2×105 cells/well) were seeded in 6-well plates and incubated at 37°C. After 48 h, the cells were washed twice with PBS, and CCK-8 reagent and culture media (1:1 ratio) were added to each well for 1 h. After the reaction, the absorbance range from 450 nm was measured by using a Spectra Max (Molecular Devices, San Jose, CA, USA).
The evaluation of cell apoptosis was performed by using flow cytometry. DNA hypomethylating drugs or irradiated cells were assessed by using an Annexin V/FITC Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). The cells were stained with FITC, Annexin V, and 7-amino-actinomycin (7-AAD), after which they were incubated for 15 min in the dark.
Cells were lysed with cell lysis buffer. Equal amounts of total proteins were loaded onto 4-12% SDS–PAGE gels and transferred to PVDF membranes (GE Health care Life Sciences, Marlborough, MA, USA). The membranes were blocked with 5% skim milk in TBST buffer and incubated with primary antibodies - anti-DNMT1 (Abcam, Cambridge, UK), anti-cleaved caspase 3, anti-E-cadherin, anti-cleaved caspase 9, anti-cleaved PARP1, anti-ATG3, anti-ATG5, anti-ATG7, anti-ATG12, anti-ATG16L1 (Cell Signaling Technology, Denvers, MA, USA), anti-LC3A (Novus Biologicals, Littleton, CO, USA), anti-Beclin (BD Biosciences), and anti-β-actin (Protein Tech group, Rosemont, IL, USA) overnight at 4°C. The membranes were subsequently incubated with specific horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by using a AI600 system (GE Healthcare, Marlborough, MA, USA).
Measurements of caspase 3/7 activities in cells were performed using the commercially available Caspase-Glo 3/7 Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Cell migration was determined by using transwell plates (8 μm pore size, Corning Costar, Merck, Darmstadt, Germany), and invasion assays were performed by using Matrigel-coated invasion chambers (8 μm pore size, Corning Costar, Merck). The upper chamber contained osteosarcoma cells in serum-free medium, and the lower chamber contained MEM supplemented with 10% FBS. Both migration and invasion assays were performed following the manufacturer’s instructions. Photographs were taken using a QIcam image camera system mounted on a Nikon ECLIPSE 80i microscope (Nikon, Minato, Tokyo, Japan).
Six-week-old male athymic nude mice obtained from OrientBio (Seongnam, Korea) were maintained with freely provided sterile food and water under specific pathogen-free conditions. After a week-long adaptation period to the new environment, the mice were randomized into nine different groups before tumor implementation (n=3 mice per group). All of the animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Dongnam Institute of Radiological and Medical Sciences (DIRAMS) (DI-2019–009). To establish the tumor xenograft models, SK-UT-1 or SK-LMS-1 cancer cells (which were treated as indicated) were subcutaneously injected (2×106 cells in 200 μL PBS) into the flank of each mouse. Tumor growths were measured 2 times a week. Tumor volume (mm3) was calculated via the “(W2×L)/2” formula (Faustino-Rocha
The results in this study are presented as the mean ± standard deviation. The significance of data was estimated using either two tailed paired or unpaired student’s t test and ANOVA.
First, we determined whether sarcoma cell lines affected the protein expression of well-known regulators of DNA methylation, such as DNA-methyltransferase 1 (DNMT1), via the classical DNA hypomethylating drug (5-aza-dC) or second-generation DNA hypomethylating prodrug (SGI-110). By using several doses (0.1, 0.5, and 1 μM) of these drugs, Western blot analyses showed that the protein levels of DNMT1 were significantly decreased in sarcoma cells after treatment with these two DNA hypomethylating drugs. Interestingly, we found that a low dose (0.1 μM) of these drugs was sufficient to induce enough epigenetic inhibition in sarcoma cell lines; accordingly, a low dose (0.1 μM) was chosen for subsequent experiments to examine the combined effects of DNA hypomethylating drugs (5-aza-dC or SGI-110) and IR (Fig. 1).
To characterize whether 5-aza-dC or SGI-110 increased the sensitivity of sarcoma cell lines to IR, we performed conventional clonogenic assays to compare the cytotoxic effects of 5-aza-dC or SGI-110, IR at different doses by itself, and combination treatments of each DNA hypomethylating drug and IR in four different sarcoma cell lines (SK-LMS-1, SK-UT-1, SK-UT-1B, and SK-ES-1). Sarcoma cells were pretreated with 5-aza-dC (0.1 μM) or SGI-110 (0.1 μM) for 72 h before being exposed to various doses of IR (0.1, 0.5, 2, 4, and 6 Gy), including a lower dose, without knowing the cytotoxicity level that was induced by IR, and the cells were plated to assess the colony-forming efficiency. In response to IR, there was a dose-dependent reduction in cell survival with or without DNA hypomethylating drugs (5-aza-dC or SGI-110). After pretreatment with 5-aza-dC or SGI-110, radiation-induced cell death was significantly increased, accompanied by the formation of few and small colonies in only SK-LMS-1 and SK-UT-1 [(Fig. 2A) but not in SK-UT-1B and SK-ES-1 (data not shown, Supplementary Fig. 1)] cell lines, compared to those in the IR control groups (
To evaluate whether 5-aza-dC or SGI-110 in combination with IR affects cell proliferation, we performed a CCK-8 assays in two sarcoma cell lines (SK-LMS-1 and SK-UT-1) because there was no significant change in the number of colonies that was formed after combination treatment with 5-aza-dC, SGI-110, and IR in the other two cell lines. In addition, low doses (0.1 and 0.5 Gy) of sole IR exposure to both sarcoma cell lines indicated no significant changes in cell growth compared to control cells, thus we excluded the combination treatment of 5-aza-dC or SGI-110 in subsequent experiments. For the SK-LMS-1 cells, there was a 1-39% decrease in proliferation after treatment with 5-aza-dC, SGI-110, and IR alone, as well as a 43-70% decrease in proliferation after combination treatment with 5-aza-dC or SGI-110 and IR (
To further determine the combined effect of DNA hypomethylating drugs and IR on the metastasis of sarcoma cell lines, the migratory and invasive potential of SK-LMS-1 and SK-UT-1 cells were assessed using migration and invasion assays. As shown in Fig. 4, the migrating and invading cells in the combination of DNA hypomethylating drugs and IR group were significantly decreased compared with those in the control, 5-aza-dC, SGI-110, or IR alone in both sarcoma cell lines. Our data indicated that the combination of DNA hypomethylating drugs and IR affects sarcoma cell migration and invasion.
To determine the mechanisms that are associated with DNA hypomethylating drug (5-aza-dC and SGI-110)-induced radiosensitivity of sarcoma cells, as well as the inhibition of cell growth both
Annexin V analysis (Fig. 5) showed a significant increase in apoptotic cells (3.9 fold), as well as an increase in caspase 3/7 activities (3.1-fold), in SK-UT-1 cells after treatment with either DNA hypomethylating drugs, IR (2 and 4 Gy), or their combination, compared with control cells (
Interestingly, our Western blot analysis demonstrated that the protein levels of caspases 3 and 9 were also higher in cells treated with the combination of 5-aza-dC and IR than in cells treated with either 5-aza-dC or IR alone, as well as in control cells. Cleaved PARP1, a well known molecule inducing apoptosis (Kondo and Kondo, 2006; Faustino-Rocha
To examine whether the combination treatment of DNA hypomethylating drugs and IR induced autophagy in SK-LMS-1 cells, we analyzed the levels of autophagic characteristic markers, including ATG3, ATG5, ATG7, ATG12, AGT16L1, LC3, and Beclin by using Western blotting. As shown in Fig. 6, the expression levels of most of the ATG3, ATG5, ATG7, ATG12, ATG16L1, and Beclin markers were increased by the combination of both DNA hypomethylating drugs and IR, rather than by either treatment with DNA hypomethylating drugs or IR alone. More interestingly, although similar effects were observed at the LC3 protein level, the combination of SGI-110 and IR induced a significant increase in LC3 protein expression, compared with the combination of 5-aza-dC and IR, thus suggesting that SGI-110 is more associated with inducing the autophagy pathway to cancer cell death. LC3-II expression levels were significantly increased in the combination treatment of SGI-110 and IR, compared to the control or single agent treatments with either DNA hypomethylating drugs or IR alone (Fig. 6). It has been known that LC3-I converted into LC3-II via lipidation by an ubiquitin-like system during the autophagy process. Afterward, LC3-II stays with autophagosomes until the fusion with lysosomes is completed; therefore, this phenomenon can be used as an autophagy marker (Kondo and Kondo, 2006). Consequently, these results suggested that the combination treatment of DNA hypomethylating drugs and IR in SK-LMS-1 cells can also contribute to autophagic death.
IR is a well-known genotoxic agent that cause to key molecular damage through both direct and indirect biological mechanisms. Exposure to IR leads to both activation or inactivation of multiple signaling pathways that play key roles in cell survival or death (Goodhead, 1994). In the clinical setting, radiotherapy is most frequently used as the primary or adjuvant therapy in combination with surgery, chemotherapy, or both treatments and widely used as a standard treatment for many types of cancer. Most recently, there has been growing evidence that epigenetic mechanisms, such as DNA methylation and histone modification, are associated with transcriptional gene silencing and may be involved in control of radiosensitivity in cancer cells (De Schutter
Regarding the reversibility of epigenetic modification changes occurring in cancer, it was hypothesized that DNA hypermethylation at promoter CpG islands was reversed to re-express silenced genes and to reprogram cancer cells to a more normal-like state. Decitabine (5-aza-dC) is a well-known DNA hypomethylating drug that has been successful in the clinic for the treatment of various cancers, such as myelodysplastic syndrome (MDS) (Taby and Issa, 2010) or other leukemia (Issa
In the present study, a low dose of 5-aza-dC or SGI-110 (0.1 μM) was used in combination with irradiation, although numerous reports have indicated that 5-aza-dC (5 μM) was required to reactivate the expression of most silenced genes (Schuebel
Here, we used two different DNA hypomethylating drugs. Specifically, 5-aza-dC (decitabine) is a well-known demethylating drug and FDA approved agent for MDS, and the other drug is a next generation DNA hypomethylating drug that has been recently developed and is currently being investigated in clinical trials. Unlike previous studies, although we used low doses (0.1 μM) of both DNA hypomethylating drugs in sarcoma cell lines, the global level of DNMT1 protein was significantly decreased (Fig. 1). The data presented in this study indicates that 5-aza-dC and SGI-110 increased radiosensitivity in two sarcoma cell lines (SK-LMS-1 and SK-UT-1), which is consistent with reports on zeburaline, another DNA hypomethylating drug (Dote
Autophagy has been classified as a second form of programmed cell death. First, it was observed in cells that were exposed to starvation, which implicating that it presumably a cell protective mechanism to survive during starvation or when they were exposed to environmental toxicity. Therefore, it thought to be that autophagy enhances both cancer cell death and survival (Levine, 2007; Dalby
Little is known about the cellular mechanisms of the radiosensitivity that is induced by epigenetic inhibitors. Recently, we and other researchers have reported that the combinations of epigenetic inhibitors with radiotherapy are capable of increasing apoptosis in several solid tumors and is regarded as a potential mechanism for radiosensitization (Qiu
In the beginning of this study, we started analyzing the radiation response in four different sarcomas that originated from different tissues, such as bone, uterine, and vulva tissues; however, we ended up studying the use of two different cell lines, including uterine leiomyosarcomas (ULMSs). ULMS is a rare gynecologic malignancy with a low survival rate. Although it is a rare tumor that accounts for less than 1% of all uterine malignancies, over two thirds of patients with ULMS that has extended among the uterus experience tumor recurrence after initial chemotherapy (Dinh
This work was supported by grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (NRF-2020M2C8A2069356).
The author declares no conflict of interest.