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. 2021 May 13;10(3):531–541. doi: 10.1093/toxres/tfab030

Ganoderic acid T improves the radiosensitivity of HeLa cells via converting apoptosis to necroptosis

Chang-Sheng Shao 1,2,#, Na Feng 3,4,#, Shuai Zhou 5, Xin-Xin Zheng 6,7, Peng Wang 8, Jing-Song Zhang 9,10,✉, Qing Huang 11,12,✉
PMCID: PMC8201584  PMID: 34141167

Abstract

The use of natural substances derived from traditional Chinese medicine and natural plants as safe radiosensitizing adjuvants is a new trend for cancer radiotherapy. Ganoderma lucidum has been used as a traditional Chinese medicine with a history of more than 2000 years. Ganoderic acid T (GAT) is a typical triterpene of G. lucidum, which has strong cytotoxicity to cancer cells, but whether it has radiation sensitization effect has not been explored. In this work, we treated the HeLa cells with different concentrations of GAT before exposure to gamma-ray radiation and investigated its influence on the radiosensitivity. The cell viability, apoptosis rate, necoptosis rate, intracellular ATP level, cell cycle, the amount of H2AX and 53BP1, reactive oxygen species, and mitochondrial membrane potential were examined. Apoptotic, necroptotic, and autophagic biomarker proteins, including caspase 8, cytochrome c, caspase 3, RIPK, MLKL, P62, and LC3, were analyzed. As a result, we confirmed that with treatment of GAT, the gamma-ray radiation induced both apoptosis and necroptosis in HeLa cells, and with increase of GAT, the percentage ratio of necroptosis was increased. The involved pathways and mechanisms were also explored and discussed.

Keywords: ganoderic acid T (GAT), triterpene, apoptosis, necroptosis, radiosensitivity, ionizing radiation

Introduction

Ganoderma lucidum is one of the famous Chinese herbal medicines that have a history of more than 2000 years in China [1, 2]. Ganoderma lucidum has many medicinal values, such as lowering high blood pressure, protecting the liver, relieving asthma, stimulating the human immune system, and inhibiting tumor cell proliferation [3]. It has generally been accepted that G. lucidum contains two important medicinal ingredients; one is polysaccharides which can stimulate immunity, and the other is triterpenes which has cytotoxicity to various cancer cells [4]. According to previous reports, ganoderic acid T (GAT) is a representative highly oxidized lanostane-type triterpene in G. lucidum, which possesses pro-apoptotic effects and can inhibit cell proliferation in human cervical cancer cells by inducing G1 phase arrest [5] and can cause cytotoxicity in various tumor cells [6, 7]. In addition, other types of ganoderic acids, such as ganoderic acid D, have also been reported to have the function of inhibiting the growth of cancer cells, such as esophageal squamous cell carcinoma (ESCC) cells, through apoptosis and autophagic death [8].

At present, more and more natural substances are used in the radiation therapy of tumors [9]. It is known that many natural products can resist oxidation and protect normal human tissues and cells, and under certain conditions, they may also enhance radiation sensitivity [10]. It has been demonstrated that some natural compounds extracted from medicinal plants can be used as radiosensitizers to induce cytotoxicity to human tumor cells, such as blocking the cell cycle and enhancing cell apoptosis [11]. Radiosensitivity can be generally examined by the factors such as increasing DNA break damage/blocking DNA damage repair, arresting of cell cycle in the radiation-sensitive period, hypoxia, increasing reactive oxygen species (ROS), and enhancing cell death via apoptosis and necroptosis [12, 13]. It has been reported that celastrol, which is also a type of triterpene, can increase the radiosensitivity of lung cancer cells [14]. This fact suggests that triterpenes, including GAT, may also improve the radiosensitivity by promoting the death of tumor cells. GAT is also reported to have strong cytotoxicity [5], while the mechanism is still unclear.

Therefore, in this study, we treated the HeLa (a model cancer cell line derived from cervical cancer) cells with GAT and gamma-ray radiation and investigated the role of GAT in the radiosensitizing effect. As a result, we have not only found that GAT could induce apoptosis but also confirmed that, under certain radiation conditions, GAT could also induce necroptosis accompanied with apoptosis in a dose-dependent manner. The effect of radiosensitization of GAT was scrutinized in detail, and the possible signal pathways and mechanism were explored and discussed.

Materials and Methods

Reagents

GAT was provided by the Shanghai Academy of Agricultural Sciences. Necrostatin-1 (Nec-1), Z-VAD-FMK (Z-VAD), chloroquine (CQ), and rapamycin (Rap) were purchased from Tsbiochem (Shanghai, China). PI, DAPI were acquired from Beyotime (Shanghai, China). Tetramethylrhodamine ethyl ester (TMRE) was acquired from USeverbright. ROS probe CellRox Green was purchased from Invirogen (CA, USA). The antibodies of caspase 8, MLKL, RIPK, cytochrome c (Cyto C), caspase 3, and GAPDH were purchased from Bimake (MA, USA). The γH2AX(S139), 53BP1, LC3, and P62 were purchased from CST (MA, USA). Alexa Fluor® (488 Goat Anti-Mouse, 647 Goat Anti-Rat) IgG secondary antibodies were obtained from Invitrogen (CA, USA). Gamma irradiation was performed using Biobeam Cs137 irradiator (cat no. GM 2000; Gamma-Service Medical, Leipzig, Germany). Flow cytometry was performed using CytoFLEX (Beckman, USA). Fluorescence measurements were performed using EVOS FL Auto Cell Imaging System (Invitrogen, USA).

Cell culture and gamma-ray irradiation

HeLa and BHK-21 cell lines were obtained from American Type Cultures Collection (ATCC, Manassas, VA) and were cultured in DMEM and MEM medium (HyClone, USA) comprised of 10% fetal bovine serum (FBS, ExCell Bio). The cells were cultured at 37°C in 5% CO2.

The HeLa and BHK-21 cells were irradiated at 1, 2, and 4 Gy with 3.37 Gy/min dose rate, while in sham group (0 Gy), the cells were unexposed to radiation.

Cell viability

Cell viability experiments were performed using CCK-8 assay kit. In brief, 24 h before irradiation, ca. 6000 cells in 100 μl of the medium were placed in a 96-well plate and were then cultured for 24 h. Then, CCK-8 solution was added to the cells and the plate was further incubated for 1 h. The optical density was estimated at 450 nm using microplate reader (SpectraMax M5, Molecular Devices, USA).

Determination of ATP levels

Intracellular ATP was measured using a luciferin/luciferase assay kit (Invitrogen, USA). Cells were cultured in black 96-well plates at density of 1 × 105 cells per well. Following treatment with GAT for 24 h, the cells were washed with HBSS and were then lysed according to the manufacturer’s instructions. Luminescence (which is proportional to the amount of ATP) was measured with the SpectraMax M5 microplate reader.

Annexin V-FITC/PI staining and cell cycle analysis

The HeLa cells were seeded in a six-well plate at 2 × 105 cell/well for gamma-ray treatment. After 24 h, the cells were treated with chilled ethanol and were stained with PI to perform cell-cycle assay, and around 20 000 cells were analyzed using flow cytometry.

The cells were rinsed and suspended in annexin V binding buffer and were then stained with 5 μl of annexin V-FITC and PI. After 15 min, the HeLa cells were again analyzed with flow cytometry.

Analysis of mitochondrial membrane potential and ROS

Mitochondrial membrane potential (MMP) was estimated using the TMRE MMP assay kit (Useverbright, USA). For this purpose, the cells were cultured for 45 min at 37°C with a medium containing 200 nM TMRE, which was followed by rinsing with PBS for four times and further incubation with HBSS buffer containing 50 nM of TMRE. The TMRE staining was evaluated using the EVOS FL Auto Cell Imaging System (Invitrogen, USA).

ROS measurement was performed using CellRox Green probe (Invitrogen, USA). There were 4000 cells seeded into 96-well plates, and they were incubated overnight. Prior to radiation exposure, the cells were incubated for 30 min with HBSS solution with 5 μM of CellRox Green. After 30 min post-irradiation, the cells were rinsed thrice, and fluorescence and flow cytometry measurements were performed.

Western blot analysis and immunofluorescence staining

Total protein was extracted from cells using RIPA buffer and protease inhibitor cocktail (Selleck, USA) and was quantified using BCA Protein Assay (Thermo Scientific, USA). Western blot was used to assess the protein levels of caspase 8, RIPK, MLKL, caspase 3, Cyto C, LC3, P62, and loading control GAPDH.

In the immunofluorescence staining assay, the HeLa cells were seeded on confocal dishes. After irradiation, the cells were treated with chilled methanol for 10 min and were permeated with PBS containing 0.5% Triton X-100 for 20 min. Then, the cells were first incubated for overnight at 37°C with the first antibody and then labeled with second antibody with fluorescent label for 1 h. The resultant cells were observed and counted using a laser confocal microscopy (LEICA).

Statistical analysis

One-way analysis of variance (ANOVA) was performed in GraphPad Prism software (Prism version 8, San Diego, CA, USA) to assess the statistical significance of the data. P < 0.05 was considered for statistical significance, and the presented values are represented as mean ± standard deviation (SD).

Results

GAT inhibits proliferation of HeLa cells

Aiming to evaluate the effect of GAT on HeLa cell proliferation, CCK-8 analysis was performed on HeLa cells treated with different concentrations of GAT for 24 h. As shown in Fig. 1, we observed the dose-dependent decline in cell viability after GAT treatment, and the value of IC50 of GAT was 13 ± 1.4 μM.

Figure 1.

Figure 1

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GAD inhibits cell viability; after 24 h of GAT treatment of HeLa cells, the cell viability was detected and the IC50 value was calculated.

In order to evaluate the inhibition of proliferation by cell-cycle arrest, we examined the cell cycle profile of the cells treated with various concentrations of GAT by flow cytometry. The HeLa cells were treated with GAT at concentrations of 0, 2.5, 5, and 10 μM, and the observed proportions of the G1 phase were 46.6, 51.7, 55.1, and 58.4%, respectively (Supplementary Fig. S1). The proportion of G1 phase increased with the GAT concentration.

GAT increases radiation sensitivity

To investigate whether GAT could induce radiosensitizing effect, we then examined the cell viability of the HeLa cells irradiated by gamma-ray. As shown in Fig. 2, compared with the sham-irradiation (0 Gy) group, the irradiated group showed decrease of viability, with the fold change after 1, 2, and 4 Gy irradiation being 0.88 ± 0.14, 0.68 ± 0.21 and 0.48 ± 0.12, respectively. According to the experimental results, the irradiation dose that can reduce the viability of HeLa cells to 50% without GAT treatment was ca. 3.5 Gy. Next, the cells were treated with different concentrations of GAT and different doses of radiation. According to data analysis, when the amount of GAT at the respective concentration of 2.5, 5, and 10 μM, the irradiation dose that made cell viability reach 50% was 2, 1.5, and 0 Gy, respectively. These results indicated that the cell viability decreased significantly with the increase of GAT concentration, and with the increase of irradiation dose, the effect of GAT on decreasing cell viability became more and more significant. As for the study of radiosensitization, it is necessary to apply the drug dose as low as possible. Therefore, we used the low dose of GAT (2.5 μM) in the subsequent experiments.

Figure 2.

Figure 2

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effect of GAT on HeLa cell radiosensitivity; (A) cell viability under different irradiation doses; (B) cell viability of different doses of GAT under different irradiation doses; all the results are presented as mean ± SD; n = 3 (*P < 0.05, **P < 0.01, ***P < 0.001, compared to non-treated cells).

GAT induces both apoptosis and necroptosis in HeLa cells exposed to radiation

The irradiated cells may have various cell killing effects, such as apoptosis, necrosis (including necroptosis), autophagy, senescence, and mitotic catastrophe [15]. Among them, apoptosis and necrosis are the most important types of cell death induced by irradiation. Therefore, as the classic method of detecting apoptosis and necrosis, annexin V and PI double labeling method was employed to detect the changes in the proportion of apoptosis and necrosis after adding or not adding GAT under different radiation doses. Figure 3 shows the results from flow cytometry, where annexin V + PI− stands for apoptotic cells, and annexin V + PI+ stands for necrotic cells. As shown in Fig. 3A, after the HeLa cells were irradiated with 0, 1, 2, and 4 Gy, the proportion of apoptotic cells was 0, 2.92, 4.34, and 5.77%, respectively. After the HeLa cells were incubated with 2.5 μM GAT and then treated with different doses of radiation, the proportion of apoptotic cells increased remarkably, which was 2.42, 3.32, 4.46, and 6.86%, respectively (Fig. 3B). Similarly, the necrosis ratio (annexin V + P+) of HeLa cells only under different doses of gamma radiation was 0.07, 4.65, 4.42, and 9.11%, respectively. When the HeLa cells were co-treated with GAT and different doses of irradiation, the ratio of annexin V+P+ increased significantly, which was 3.38, 5.63, 7.89, and 11.8%, respectively (Fig. 3C). These results therefore showed that, compared with the cell group without GAT treatment, the proportion of apoptotic cells (annexin V + PI−) and necrotic cells (annexin V+PI+) increased with the combination of both GAT and gamma-ray radiation treatments.

Figure 3.

Figure 3

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GAT induced both apoptosis and necrosis; (A) apoptosis assessed by flow cytometer and the percentage of the apoptotic and necrotic cells calculated; the HeLa cells were treated with GAT and different doses of gamma-ray for 24 h, respectively; (B, C) quantitative analysis of apoptosis and necrosis cells in percentage; all the results are presented as mean ± SD; n = 3 (*P < 0.05, **P < 0.01, ***P < 0.001, compared to non-treated cells).

Furthermore, to verify that GAT induced necrosis or necroptosis, we tested sub-G1 of cells by flow cytometry. It is generally believed that when the cell cycle is blocked in the G1 phase (Supplementary Fig. S1), the sub-G1 peak that appears is more likely to be caused by necrosis. Meanwhile, the relevant inhibitors of cell death methods were used for verification. NEC1 is an inhibitor of necroptosis, while Z-VAD is a blocker of the caspase pathway for suppressing apoptosis. Figure 4A shows that with increase of GAT, sub-G1 content increased, but with co-treatment of both GAT and NEC1, sub-G1 was reduced; while when GAT and Z-VAD were both applied, sub-G1 was significantly increased (Fig. 4B). These results confirmed that GAT induced necroptosis.

Figure 4.

Figure 4

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analysis of HeLa cells sub-G1-level changes; the levels of sub-G1 were analyzed by flow cytometry; (A) the histograms obtained by flow cytometry show Sub-G1 portions under different treatments; (B) quantitative analysis data of the Sub-G1 portions are presented; all the results are presented as mean ± SD; n = 3 (*P < 0.05, **P < 0.01, ***P < 0.001, compared to non-treated cells).

Moreover, to confirm that necroptosis tended to occur with the condition of high concentration of GAT, we inspected the results with employments of Z-VAD (inhibitor of apoptosis) and NEC1 (inhibitor of programmed necrosis). Figure 5 shows the comparison of the viability of GAT-treated HeLa cells, again confirming the occurrence of both apoptosis and necroptosis. But as shown in Fig. 5A, the cells with low concentration of GAT did not show increase in cell viability after adding the inhibitor of programmed necrosis (NEC1), while after adding the inhibitor of apoptosis (Z-VAD), the cell viability increased remarkably, indicating that in this case most cells went to apoptosis. The cell survival rate could not be completely restored by adding Z-VAD, indicating that, except apoptosis, other cell death types such as necrosis also existed. As shown in Fig. 5B, with combined use of NEC1 after adding a high dose of GAT to the cells, the cell viability was significantly increased, while with combined use of Z-VAD, the cell viability decreased, suggesting that at high dose of GAT, the portion of apoptosis became smaller while necroptosis became dominant.

Figure 5.

Figure 5

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comparison of the effects of apoptosis and necroptosis on the viability of GAT-treated HeLa cells; HeLa cells were pretreated with 20 μM Z-VAD or 10 M NEC1 for 1 h; (A) cells treated with 2.5 μM GAT for 24 h; (B) cells treated with 10 μM GAT for 24 h; cell viability was measured using the CCK-8 assay; all the results are presented as mean ± SD; n = 3 (*P < 0.05, **P < 0.01, ***P < 0.001, compared to non-treated cells).

Verification of the signaling pathways of apoptosis and necroptosis

Furthermore, in order to verify the GAT-induced apoptosis and -necroptosis under irradiation, we also examined the signal pathways for the apoptosis/necroptosis by analyzing the expression-level changes of biomarkers responsible for apoptosis/necroptosis. As shown in Fig. 6, RIPK, MLKL, caspase 8, caspase 3, and Cyto C were measured using western blotting assay. The results showed that as the dose of GAT increased, the expression of caspase 8 gradually increased. While under radiation conditions, with the increase of the dose of GAT, the expression of caspase 8 decreased significantly. Because caspase 8 is a key protein that regulates the transition from apoptosis to necroptosis, the above result indicated that, under radiation conditions, GAT may induce more cells to go to death through the pathway of necroptosis as the applied dose of GAT increased.

Figure 6.

Figure 6

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GAT could regulate apoptosis and necrosis signaling pathway-related proteins under radiation conditions; the protein level of RIPK, caspase 8, MLKL, caspase 3, Cyto C, and caspase 3 were evaluated by western blots at 24 h after radiation or GAT treatment, and GAPDH was used as an internal control

At the same time, we also tested the biomarkers (MLKL, RIPK) of necroptosis, and the protein expression levels were higher given that both the radiation and GAT were applied. As the biomarker of apoptosis, the expression levels of Cyto C and caspase 3 also significantly increased under radiation conditions, indicating that GAT did not merely enhance apoptosis but rather necroptosis with the co-interaction of radiation.

Verification of the radiosensitization effect of GAT

Generally, the factors affecting tumor cell radiation sensitivity mainly include the following: DNA damage and repair, cell cycle arrest, apoptosis, oncogene changes, autophagy, tumor microenvironment, tumor stem cells, and tumor metabolism [16]. In radiation biological research, interference with DNA break damage repair is the most direct mode of action of radiosensitizing drugs [16]. Therefore, we tried to verify whether GAT could enhance radiosensitivity by interfering with the repair of DNA double-strand breaks (DSBs). Indeed, Fig. 7A shows the co-localization of the fluorescence emitted by γ-H2AX and 53BP1 after the GAT treatment or radiation exposure or co-treatment with GAT and irradiation. Figure 7B shows that the number of γ-H2AX as DNA fragmentation biomarker in the radiation treatment group was significantly higher than that in the GAT treatment group. When GAT was co-treated with radiation, the number of γ-H2AX foci in this group increased significantly. These results confirmed that GAT indeed gave rise to the increase the radiation sensitivity. Figure 7C shows that compared with the control group, the number of 53BP1 foci was the largest at 2 Gy, but the number of 53BP1 foci in the GAT and irradiation co-treatment group was significantly reduced. This result implies that cells under a certain dose of GAT can be more severely damaged, along with weaker DNA repair capabilities.

Figure 7.

Figure 7

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analysis of γ-H2AX and 53BP1 foci produced in HeLa cells treated with irradiation and GAT separately or together; (A) immunofluorescence staining of γ-H2AX and 53BP1 foci in HeLa cells treated with gamma-ray (2Gy) or GAT (2.5 μM) or GAT + gamma-ray, blue DAPI labeled nuclei, green labeled γ-H2AX, and red labeled 53BP1; scale bar: 5 μm; (B) the histogram shows the mean numbers of γ-H2AX foci; (C) the histogram shows the mean numbers of 53BP1 foci; all the results are presented as mean ± SD; n = 3 (*P < 0.05, ***P < 0.001, compared to non-treated cells).

To further understand the radiosensitization effect of GAT, the mitochondrial membrane damage caused by GAT under radiation-induced conditions and the accompanying ROS levels were also examined. For this purpose, the changes in ROS were observed using a fluorescence microscope, and the MMP was labeled with TMRE (Fig. 8A and B). The results showed that the ROS level in the irradiated cells was significantly increased compared with the control group and the GAT treatment group, and the ROS level in the GAT and radiation treatment group was highest (Fig. 8C). In addition, we also applied GAT to the cells exposed to different doses of irradiation and detected the changes of ROS by flow cytometry. The results showed that with the increase of the radiation dose, the ROS level of the GAT-treated group was significantly increased compared with that of the drug-untreated group (Supplementary Fig. S2). At the same time, the change of MMP was opposite to ROS-level changes. The MMP of the 2.5 μM GAT and 2 Gy radiation group was almost equivalent, but the MMP of the co-treatment group was the lowest (Fig. 8D). Meanwhile, we also measured the cell viability of the cells with GAT under radiation conditions and observed the MMP damage, which further confirmed that GAT induced the radiosensitizing effect (Supplementary Fig. S3).

Figure 8.

Figure 8

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effects of ROS and MMP by GAT; (A) fluorescence microscope analysis of ROS changes in HeLa cells after treated by GAT (2.5 μM) or IR (2 Gy) or GAT+ IR for 6 h; NAC (50 μM) as negative control, TBHP (200 μM) as positive control; ROS assessment using 5 μM CellRox Green probe; (B) fluorescence microscope analysis of MMP changes in HeLa cells after treated by GAT (2.5 μM) or IR (2 Gy) or GAT+ IR for 24 h; FCCP (50 μM) as negative control; MMP assessment using 50 nM TMRE dye; scale bar: 200 μm; (C, D) quantitative data of the ROS and MMP MFI are presented; all the results are presented as mean ± SD; n = 3, ***P < .001, compared to non-treated cells (IR: gamma-ray irradiation; NAC: N-acetyl cysteine, is a ROS inhibitor; TBHP: ROS positive control; FCCP: A positive control for detecting mitochondrial membrane potential).

Effect of intracellular ATP depletion by GAT and irradiation

Necroptosis is a programmable type of cell death independent of caspase [17, 18]. The general characteristics of necroptosis include severe damage to cell integrity, swelling of cells and subcellular organelles, leakage of cell contents, changes in chromatin morphology, and a large amount of ROS [19]. The consumption of intracellular ATP is an important indicator of the induction of necroptosis [20]. In order to verify whether necroptotic cell death was caused by GAT, we measured the level of intracellular ATP. The results showed that GAT was still maintained after different doses of radiation in the non-GAT group (Fig. 9). However, after adding a certain amount of GAT, it was found that the ATP level was significantly decreased. At the same time, with the increase of GAT dose, the intracellular ATP level decreased significantly until it was exhausted. This result suggests that the GAT-induced cell death may be necroptotic, which is related to intracellular ATP consumption.

Figure 9.

Figure 9

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effects of intracellular ATP depletion by GAT; HeLa cells treated with different dose of radiation (0, 1, 2, and 4Gy) and different doses of GAT (0, 2.5, 5, and 10 μM); quantitative analysis of intercellular ATP level in percentage; *P < 0.05, **P < 0.01, and ***P < 0.001 indicated significant differences compared to non-treated cells group; all the results are presented as mean ± SD; n = 3

Discussion

GAT is a highly oxidized lanostane-type triterpene in G. lucidum, which has significant anti-cancer effect and cytotoxicity in vivo or in vitro [5, 6, 21]. In this study, we clearly demonstrated that GAT could also induce the effect of increasing radiation sensitivity. We observed that GAT increased the DNA damage and interfered with DNA repair, blocked the cell cycle in G1 phase, increased ROS production, and reduced MMP. On the other hand, we also observed that GAT could induce ATP depletion under radiation conditions, which thus increased the cell death by enhancing necroptosis in addition to apoptosis.

To confirm the radiation sensitization effect induced by GAT, we have inspected the typical factors, including cell cycle distribution, the ability to repair DNA damage, hypoxia, altered mitochondrial and energy metabolism, and regeneration [13]. First, we confirmed that GAT could block HeLa cells in G1 phase. Although G1 phase is not the most radiosensitive phase, it is not the most radiosensitive phase like G2 phase [22]. However, some studies have shown that G1 phase is second only to G2 phase in radiosensitivity [23]. It was previously reported that caffeine enhances the radiosensitivity of PTEN-deficient glioma cells by enhancing G1 phase arrest that was induced by ionizing radiation [24]. Therefore, the phenomena of GAT arresting cells at G1 phase could be regarded as one of the factors to increase radiosensitivity. Second, we employed the marker protein of DNA damage as γ-H2AX and DNA damage repair protein as 53BP1, which were labeled by the immunofluorescence method, to verify whether GAT interfered with the DNA damage and repair. By counting the number of foci of γ-H2AX and 53BP1, it was found that GAT increased the DNA damage and blocked DNA repair, confirming the increased radiosensitivity caused by GAT. However, we observed that the number of 53BP1 foci in the GAT and radiation co-treatment group was lower than that in the radiation group. This might be due to that GAT blocked the cell cycle in G1 phase and also interfered with the repair of DNA break damage. To be noted, normally, in the radiation research, either γ-H2AX or 53BP1 is employed for verifying the radiation damage on DNA. However, in more general cases, γ-H2AX may be not concomitant with 53BP1. DSB signaling is initiated by the MRN complex (MRE11-RAD50-NBS1), which senses and binds to the break region and subsequently recruits and activates ATM [25]. Next, ATM leads to rapid phosphorylation and conversion of the serine 139 site of histone H2AX at the break site to γ-H2AX, and it is involved in subsequent chromatin remodeling after DSBs [26]. As an early DSB signal, γ-H2AX can be detected as early as 30 s after irradiation. Subsequently, mediator of DNA damage checkpoint protein 1 (MDC1) is recruited to form a complex binding to γ-H2AX after which ubiquitin E3 ligases (RNF8 and RNF168) are recruited to bind to the γH2AX/MDC1 complex [27]. When RNF168 reaches the DNA break region, it rapidly ubiquitinates histone H2A, leading to the recruitment of DNA repair factor 53BP1 to proceed [28]. The 53BP1 is an essential component of DSB signaling and repair, facilitating non-homologous end joining (NHEJ)-mediated DSB repair during G1 phase [29]. Therefore, in our work, both γ-H2AX, an early signaling marker of DSB breaks, and 53BP1, a late repair factor marker, were selected to examine the interference of GAT on radiation damage repair. Third, we also examined the increase of ROS related to the enhancement of radiosensitivity. It is well known that ROS could be the mediator of tumor radiosensitivity [30]. It has been reported that 1α, 25 (OH) 2D3, as a potential anticancer molecule, can increase the radiosensitivity of lung cancer by promoting a series of increases in NADPH, ROS, and apoptosis [31]. In addition, it has been reported that the increase of ROS could inhibit the telomerase activity, such as CDDO-Me, a triterpenoid, which could inhibit the telomerase activity by increasing ROS and mediate cell apoptosis [32].

To confirm the GAT-induced apoptosis accompanied with necroptosis, we employed the inhibitor of apoptosis Z-VAD and the inhibitor of necroptosis NEC1 in the experiments. As for necroptosis, it was initially thought to be incompatible with apoptosis, in other words, a backup of apoptosis [33, 34]. The previous experiments showed that necroptosis of cells could occur by adding an inhibitor of apoptosis (Z-VAD) [35], while adding inhibitor of programmed necrosis (NEC1) could turn the cells from necroptosis to apoptosis [36, 37]. However, it is not as simple as adding apoptosis inhibitors to cause necroptosis, as apoptosis and necroptosis are not mutually exclusive. Rather, in our case, we found that GAT actually induced both apoptosis and programmed necroptosis under radiation conditions. In our experiment, we compared the treatments of low-dose GAT (2.5 μM) and the high-dose GAT (10 μM) on the cells, respectively. We found that the cell viability with Z-VAD (suppressing apoptosis) under low-dose GAT condition was higher than that with NEC1 (suppressing necroptosis), indicating that low-dose GAT led to mainly apoptotic cells. On the other hand, under high-dose GAT condition, the cell viability with NEC1 (suppressing necroptosis) was higher than that with Z-VAD (suppressing apoptosis), indicating that the proportion of necroptosis cells was predominant. These results therefore confirmed that with the increase of GAT, the radiation-induced apoptosis would turn to necroptosis.

But what is the mechanism for the apoptosis accompanied with necroptosis? Or, in other words, how did apoptosis convert to necroptosis in our case of radiation effect? There are several critical factors to be considered in this process. First, caspase 8 is the key molecular switch for apoptosis and necroptosis [38]. Apoptosis is a “cell suicide program” that does not cause tissue damage and is induced by caspase 8 [39]. Necroptosis is another way of regulating cell death. It causes cell damage and usually acts when caspase 8 is inhibited [40]. When caspase 8 is inhibited, it can induce necroptosis by activating the kinases RIPK and MLKL. So in our case, when the HeLa cells were exposed to radiation, the protein expression level of caspase 8 gradually decreased with the increase of GAT concentration, so GAT affected the inhibitory level of caspase 8, leading to the occurrence of programmed necrosis. Second, the depletion of ATP is also one vital factor for the cell’s transition from apoptosis to necroptosis. The level of ATP determines the direction of cell fate [41]. In our study, GAT can reduce the MMP and ATP level of cells under radiation conditions, further aggravate the damage to cells, and increase the radiosensitivity. Previous studies have shown that in the state of ROS increase, the cell death type can change from caspase 3-mediated apoptosis to necrosis because ATP depletion can prevent the activation of caspase [42]. Some studies have also shown that changes in ATP levels can also change the fate of cells because high levels of intracellular ATP often appear in apoptotic cells and low levels of ATP often appear in necrotic cells [43]. Third, increase of ROS in mitochondria is also an important reason for turning to necroptosis. Mitochondria, as the main subcellular organelles, have become important radiation targets, so it produced more ROS during irradiation, leading to mitochondrial membrane damage and mitochondrial dysfunction [44]. Of course, these factors are not working independently. On the contrary, they may intervene each other or work synergistically. For example, mitochondrial dysfunction could also lead to a decrease in the ATP synthesis capacity. As ATP was depleted, the cell fate then turned to necroptosis. Some studies revealed that during apoptosis, cells could use caspase to shut down some biochemical processes that consume more ATP, such as gene transcription and translation, and during necroptosis, caspase 8 would be inhibited and so ATP would be continuously depleted [45]. In addition, studies also showed that as the most important kinases (RIPK and MLKL) in necroptosis [46], the interaction between the two can transfer necrosome to the mitochondrial-related membrane, thereby triggering the increase of ROS production and cell necroptosis [47]. It is worth noting that although the necroptosis mediated by ROS is more complicated, they all contribute to the development of cell fate in the direction of necroptosis.

To be noted, in this work, we not only analyzed the apoptosis and necroptosis in the GAT-treated HeLa cells under radiation condition but also considered other pathways, such as whether GAT caused autophagy and blocked autophagy flux, and tested it. As shown in Supplementary Fig. S4, with the increase of GAT dose to 10 μM, the protein expression of LC3 and P62 only increased, indicating that the ability to activate autophagy was weaker at low doses of GAT. By adding the autophagy inducer (Rap) and the autophagosome inhibitor (CQ), it was found that GAT did not block the autophagy flux. These phenomena suggest that GAT did not produce autophagic cell death on the HeLa cells at least at low dose and so the detected cell death included mainly apoptosis and necroptosis.

In addition, we also applied GAT to treat normal cells (BHK-21 cells) to show that GAT has the special radiosensitivity effect on cancer cells instead of normal cells. As shown in Supplementary Fig.S5, the inhibition of cell proliferation of normal cells (BHK-21 cells) at different concentrations of GAT was examined, and it was found that GAT was cytotoxic to normal cells at higher doses (40, 80 μM). However, there was no significant effect on cell proliferation at a low dose less than 10 μM. Through the detection of cell death rate, we confirmed that 2.5-μM GAT and 2-Gy radiation did not affect the number of cell deaths of BHK-21 cells significantly. Furthermore, we examined the apoptosis of BHK-21 cells pretreated by GAT under different irradiation doses (0, 1, 2, and 4 Gy) and confirmed that the addition of 2.5 μM GAT did not significantly increase the proportion of apoptosis either. Moreover, we found that under 2-Gy irradiation condition, by increasing the drug concentration of GAT (0, 2.5, 5, and 10 μM), the expression level of intracellular ATP did not change significantly either.

Conclusion

In summary, we revealed that GAT could induce not only apoptosis but also necroptosis in the HeLa cells exposed to ionizing radiation. It was found that when the concentration of GAT increased, the death state of HeLa cells would change from apoptotic to necroptotic. In addition, we also explored the mechanism of enhancing radiation sensitivity of GAT by analyzing the cell cycle arrest, consumption of intracellular ATP, increase of ROS, decrease of MMP, and the transformation of necroptosis (Fig. 10). This study has therefore not only showed some insights into the pathways and mechanism of the radiation sensitization effect of GAT but may also provide a new direction for the research and development of traditional Chinese medicines or natural products as effective radiosensitizing adjuvants in tumor radiation therapy.

Figure 10.

Figure 10

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schematic diagram of the biochemical events of GAT and ionizing radiation on cells

Conflict of interest statement

None declared.

Supplementary Material

Supplementary_Information-final_tfab030
Click here for additional data file. (1.7MB, docx)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grants Nos. 11635013 and 11775272) and the Special Repair and Purchase Fund for Central-level scientific institutions (No. Y79XG13361).

Contributor Information

Chang-Sheng Shao, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences (CAS), Hefei 230031, China; Science Island Branch of Graduate School, University of Science and Technology of China, Hefei 230026, China.

Na Feng, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, P. R., China; National Engineering Research Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China.

Shuai Zhou, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, P. R., China.

Xin-Xin Zheng, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences (CAS), Hefei 230031, China; Science Island Branch of Graduate School, University of Science and Technology of China, Hefei 230026, China.

Peng Wang, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences (CAS), Hefei 230031, China.

Jing-Song Zhang, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, P. R., China; National Engineering Research Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China.

Qing Huang, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences (CAS), Hefei 230031, China; Science Island Branch of Graduate School, University of Science and Technology of China, Hefei 230026, China.

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Supplementary Materials

Supplementary_Information-final_tfab030
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