Role of metabolism in cancer cell radioresistance and radiosensitization methods

GLUT family represents a class of 14 proteins essential for glucose metabolism and found in the membranes of various cells. GLUT1 is the most common and widely distributed member of this family [78, 81], involved in the glucose transport and its expression is upregulated under hypoxic conditions, and therefore, it is often used as a cellular hypoxia marker [82]. GLUT1 overexpression was shown to be associated with the radioresistance and poor prognosis in oral squamous cell carcinoma and head and neck squamous cell carcinoma patients, which suggests that GLUT1 may be used as an indicator of the sensitivity to and prognosis of cancer radiotherapy [83, 84]. Radioresistant tumor cells often have high GLUT1 levels, which was associated with oncogene activation, tumor suppressor inactivation, stimulation of hypoxia, and the regulation of different signaling pathways, such as MAPK and PI3K/AKT [78].

Targeting GLUT1 and related signaling pathways may represent an effective way to improve radiotherapy efficacy [85]. As a natural flavonoid, apigenin was shown to have anti-proliferative and anti-angiogenic effects, exerted through the downregulation of GLUT1, HIF1α, and vascular endothelial growth factor (VEGF) expression [86]. Apigenin was confirmed to inhibit the expression of GLUT1 by regulating PI3K/AKT pathway, and improving the radiosensitivity of laryngeal carcinoma, prostate cancer, and adenoid cystic carcinoma cells [87‐89]. Additionally, WZB117, a small molecule, acts as a specific inhibitor of GLUT1, overcoming the resistance of cancer cells to radiation [90]. WZB117 and radiation therapy combined can inhibit the growth of breast cancer cells and sensitize cancer cells to radiotherapy by increasing the level of intracellular ROS [91]. Furthermore, the antisense oligonucleotide chain (AS-ODNs) of GLUT1 can also induce the radiosensitivity of laryngeal carcinoma cells (Fig. 2) [92, 93]. Co-suppression of GLUT1 and the members of PI3K/AKT signaling pathway was shown to improve the radiosensitivity of laryngeal carcinoma xenograft cells in nude mice, suggesting that PI3K/AKT signaling pathway plays an important role in the development of radioresistance [94].

Lactic acid is one of the main products of glycolysis and one of the key factors in the development of malignant tumors. Alterations in glucose metabolism after radiotherapy can lead to the accumulation of large amounts of lactic acid, which is one of the unique malignant tumor phenotypes [26, 95]. The concentration of lactic acid in tumor tissues was shown to be significantly higher than that in the healthy tissues [96], and it can promote tumor metastasis, recurrence, and radioresistance, resulting in poor prognosis in many cancers [97]. Lactic content can predict metastasis rates, overall survival of patients, is a genotoxic stress biomarker, and it was shown to be associated with hypoxia-induced radioresistance [98]. In the nude mouse model of human squamous cell carcinoma, the accumulation of lactic acid was shown to correlate with radioresistance [80]. Michael et al. [99] demonstrated that the lactic acid accumulation induces many adverse effects in the cancer-associated stromal cells, which can help regulate angiogenesis by affecting proliferation, differentiation, and maturation of fibroblasts and endothelial cells. Lactic acid inhibits the activation and differentiation of many immune cells, such as dendritic and T-cells, by interfering with their metabolism and mediating immune escape [100, 101]. Additionally, it can induce the release of hyaluronic acid by tumor-associated fibroblasts, which promotes cell migration and clustering, VEGF secretion, and neovascularization [102]. All these represent potential mechanisms involved in the lactic acid-associated radioresistance (Fig. 2).

The monocarboxylate transporters (MCTs) can transport lactate through the cell membrane, and these molecules are encoded by the SLC16 gene family comprising 14 members, with only four subtypes (MCT1-MCT4) known to be proton transporters. Of these, MCT1 shows the highest affinity for the lactic acid [103]. Shiho et al. [97] showed that the levels of lactic acid in myeloma cells are associated with MCT1 expression. A competitive inhibitor of MCT1, CHC reduces the expression of lactic acid, inducing cell apoptosis (Fig. 2). The downregulation of MCT1 expression induces the expression of FAS protein in ovarian cancer cells, significantly inhibiting the activation of its downstream targets, such as FASL and FAP1, and promoting the expressions of apoptosis-related protein caspase-3, which indicates that MCT1 can induce resistance to cisplatin by antagonizing FAS (Fig. 2) [104], and that it may play the same role in radioresistance development.

Lactate dehydrogenase (LDHA) is found in almost all human tissues; it is a major enzyme catalyzing the conversion of pyruvate to lactic acid, and plays an important role in the glycolytic process [105]. Michael et al. [106‐108] demonstrated that LDH5 overexpression can indicate hypoxic conditions, which can be associated with local recurrence, distant metastases, lower overall survival, and radioresistance of head and neck squamous cell carcinoma, prostate, and bladder cancers. Furthermore, soluble adenylate cyclase (sAC) promotes the release of LDHA, accelerates cell proliferation, and induces the anti-irradiation effects in prostate cancer cells through the activation of BRAF/ERK1/2 signaling pathway [109]. FX-11, a specific inhibitor of LDHA, can promote the generation of DSBs and cell apoptosis by reducing the EMT, DNA repair capacity, hypoxia, and autophagy in prostate cancer cells, improving cell sensitivity to radiotherapy [105]. Acting as a tumor suppressor, the expression of miR-34 was shown to negatively correlate with radioresistance development and to induce the sensitivity of the hepatocellular carcinoma cells to radiotherapy by inhibiting the expression of LDHA (Fig. 2) [110, 111].

Hypoxic conditions in cells often have a negative impact on the radiotherapy outcomes [112]. Tumors rely more on anaerobic glycolysis for energy production than the normal tissues. Malignant tumor environments are often hypoxic, and they rely on exacerbated glycolysis to meet the increased demand for ATP and biosynthetic precursors [113]. Hypoxic environments promote the transformation of tumor cell metabolism from the oxidative metabolism to anaerobic glycolysis, which protects tumor cells and induces the development of radioresistance in tumor stem cells [114]. In human osteosarcoma cells, hypoxia was shown to confer anti-irradiation effects and induce the expression of autophagy-related proteins LC3 and LC3-II, suggesting that hypoxia can activate cell autophagy and accelerate the removal of ROS from the cells, leading to radioresistance development [115]. Moreover, hypoxia can activate EGFR and NRF2 expression in lung cancer cells, inducing radioresistance (Fig. 2) [116], together with the activation of HIF1 transcription, which regulates the adaptive cellular responses to hypoxia [112]. HIF1 has been identified as an important mediator of the carbohydrate metabolic pathway reprogramming from OxPhos to glycolysis [117], and it is composed of two subunits, HIF1α and HIF1β, and HIF1α expression is oxygen-dependent due to the presence of an oxygen-dependent degradation domain. HIF1β, known as aryl hydrocarbon receptor nuclear translocator (ARNT), is constitutively expressed, and therefore, not affected by environmental conditions [118]. The heterodimer formed by HIF1α/HIF1β constitutes a functional HIF1 molecule, characterized by the presence of hypoxia-responsive element within the promoter or enhancer region [119]. HIF1α is the main regulatory subunit of this molecule, which binds to the promoter and upregulates HIF1β expression [120]. A previous study confirmed that HIF1α can activate the transcription of target genes that regulate various biological processes, including cell proliferation, glucose metabolism, and pH regulation, playing a vital role in the adaptation of cancer cells to hypoxic conditions (Fig. 2) [121].

A number of studies demonstrated that HIF1 promotes tumor invasion, metastasis, and mediates the anti-irradiation effects [112]. The mechanisms underlying the development of radioresistance may include the following:

An increasing number of studies have shown that, by targeting HIF1 activity, tumor antioxidant capacity can be reduced, as it affects the TME and promotes the sensitivity of solid tumors to radiotherapy. By combining HIF1 targeting and radiotherapy, improved therapeutic effects can be achieved [142]. For example, the use of chetomin, a chemical HIF1α inhibitor, can disrupt the interactions between this molecule and p300, attenuate hypoxia-induced gene expression, and increase radiosensitivity of cancer cells under severe hypoxic conditions [143]. Additionally, HIF1α inhibition leads to the downregulation of stem cell markers and a decrease in radioresistance of cervical cancer cells [144]. Various HIF1 inhibitors function through different signaling pathways, thereby enhancing the efficacy of radiotherapy. KNK437 is a benzylidene lactam compound that inhibits the synthesis of heat shock proteins (HSPs), which increase DNA damage repair and inhibit cell death, stabilizing HIF1α expression and promoting radioresistance. KNK437 can abrogate hypoxia-induced anti-radiation effects by targeting both AKT and HIF1α [145]. Furthermore, 2-methoxyestradiol is an estrogen metabolite that suppresses HIF1α levels and its transcriptional activity. It depolymerizes microtubules and prevents HIF1α nuclear accumulation [146], increasing the radiosensitivity of NPC stem cells and melanoma cells by inactivating NF-κB/HIF1 or HIF1α/PDK1 signaling pathway [147, 148]. Berberine can inhibit tumor metastasis, tumorigenicity, and growth, and transforming growth factor-β (TGF-β)-induced tumor invasion and EMT [149]. Moreover, it enhances the radiosensitivity of NPC cells by inhibiting the expression of HIF1α and VEGF [150]. NVP-BEZ235, an inhibitor of PI3K/mTOR signaling pathway, can inhibit the activation of HIF1α/VEGF signaling pathway in endometrial cancer and suppress radioresistance development [151]. As STAT3 inhibitors, NSC74859 and Stattic can improve the radiosensitivity of esophageal cancer through the inhibition of hypoxia and radiation-induced STAT3 activation, as well as the expression of HIF1α and VEGF [152, 153]. Additionally, docetaxel is a semi synthetic paclitaxel derived from European yew, which has been widely applied in the clinical treatment of gastric, non-small cell lung (NSCL), ovarian, and breast cancers [154], and which was shown to induce the activation of JNK2 signaling pathway, mediate the phosphorylation of PHD1, and inhibit the expression of HIF1α, leading to the apoptosis of the tumor cells in hypoxic conditions [155]. Finally, paclitaxel pretreatment was shown to inhibit the radioresistance of HIF1α-induced hepatocellular carcinoma and lung adenocarcinoma, suggesting that it can be used as a sensitizer for radiotherapy (Fig. 2) [156].

In addition to the inhibitors of HIF1, many miRNAs can promote or inhibit hypoxia-induced radioresistance. MiR-210 was shown to be expressed in the different types of tumor and normal cells in hypoxic environments [157]. The expression of miR-210 promotes the DSB repair, increases the production of lactic acid, and HIF1α stability [158]. The downregulation of miR-210 significantly inhibits cell viability, inducing G0/G1 phase cell cycle arrest, and increasing apoptosis rates and the radiosensitivity of hypoxic hepatocarcinoma cells [157, 159]. Furthermore, miR-21 was shown to regulate the radiosensitivity of cervical cancer cells through the PTEN/AKT/HIF1α feedback loop and the AKT-mTOR signaling pathway [160]. Hypoxia-responsive miR-124 and miR-144 overexpression can inhibit hypoxia-induced autophagy and enhance the radiosensitivity of prostate cancer cells by downregulating the expression of the PIM1 oncogene [161], while miR-216a can enhance the radiosensitivity of pancreatic cancer cells by inhibiting BECN1-mediated autophagy (Fig. 2) [162]. Additionally, a decrease in miR-23a expression promotes the radioresistance of NPCs, determining their response to radiotherapy [163].

Pyruvate kinase (PK) can convert phosphoenolpyruvate and ADP into pyruvate and ATP, which makes it one of the major rate-limiting enzymes in glycolysis. A previous study demonstrated that the PK expression positively correlates with the radiotherapy resistance in tumor cells [164]. The M2 isoform (PKM2) is a key regulator of glycolysis, expressed only in cancer cells [165], and targeting this molecule can inhibit cell viability, induce G2/M arrest, and promote apoptosis. Additionally, this can increase the radiosensitivity of NSCLCs and the IR-induced apoptosis and autophagy rates, which are associated with the inhibition of AKT and PDK1 phosphorylation [166]. PKM2 is targeted by miR-133 as well, and this miRNA is downregulated in radioresistant lung cancer cells. MiR-133b resensitizes radioresistant lung cancer cells by inhibiting PKM2-mediated glycolysis [167]. Nitric oxide (NO) levels were shown to be significantly associated with cellular metabolism, and a decrease in the NO levels leads to a significant reduction in PDK1 expression, enhancing the radiosensitivity of hypoxic NSCLCs [168]. Furthermore, dichloroacetate, a PDK inhibitor, can effectively radiosensitize glioblastoma cells [164], while the treatment of esophageal squamous cell carcinoma cells with diisopropylamine dichloroacetate (DADA) can increase their sensitivity to radiation (Fig. 2) [169].

Hexokinase 2 (HK2) is a key glycolytic enzyme in glucose metabolism, highly expressed in a variety of human solid tumors. Its upregulation can induce glycolysis, and it is essential for tumor progression and maintenance. By inhibiting HK2 signaling in cancer cells, their radiosensitivity may increase [170]. Additionally, 2-deoxy-D-glucose (2-DG) is an inhibitor of glucose metabolism and ATP production, which can help suppress the IR-induced radioresistance [171]. Following the phosphorylation of HK2 by 2-DG, this molecule can disrupt the radiation-induced DNA damage repair in tumor cells and promote their apoptosis by reducing intracellular energy levels [172]. The combination of 2-DG and histone deacetylase transferase inhibitors can induce apoptosis in glioblastoma cells [173], while 2-DG can also significantly inhibit the expression of HK2 and induce apoptosis (Fig. 2) [174].

The redox environment, representing a balance between the generation of ROS species and their removal by antioxidant enzymes, is a key regulator of oxidative stress in cells. Excessive superoxide levels, if not removed by endogenous antioxidants or related enzymes, can cause oxidative stress damage in mitochondria. Manganese superoxide dismutase (MnSOD) is the main antioxidant enzyme, which can catalyze the disproportionation of superoxide anion radicals, protecting the body from ROS-induced damage. Moreover, MnSOD regulates the response of cells, tissues, and organs to the IR, which is essential for the protection of mitochondria and cells from oxidative stress (Fig. 3). The increased activity of MnSOD was shown to increase significantly the viability of pancreatic cancer cells after γ-ray irradiation and activate G2 checkpoint block, ultimately inducing radioresistance in pancreatic cancer cells [177].

Many studies demonstrated that the protection of cells against the mitochondrial oxidative stress-induced cell death can promote radioresistance development. Maus et al. [178] showed that the glial cell antigen 2 (NG2) can protect oligodendrocyte precursor cells from oxidative stress by interacting with mitochondrial serine protease OMI/HtrA2. NG2 downregulation can increase the apoptosis and cell sensitivity to oxidative stress. This interaction between NG2 and OMI/HtrA2 may contribute to radioresistance development in gliomas (Fig. 3). Additionally, following treatment with Il6, rat glioma cells developed radioresistance by suppressing mitochondrial oxidative stress. Therefore, Il6 treatment induces radioresistance in tumor cells by inhibiting the increase in ROS levels (Fig. 3) [179].

In contrast, enhanced mitochondrial oxidative stress can promote tumor cell radiosensitivity. Sorafenib is a novel anti-cancer drug that can induce the apoptosis of drug- and radiation-resistant hepatoma cells through mitochondrion-dependent oxidative stress mechanisms. The mechanism of its action includes rapid formation of ROS in mitochondria, triggering of the mitochondrial calcium overload, and activating apoptotic processes by releasing cytochrome C and activating caspase 3/7 pathway [180]. Ceramides induce ROS accumulation as well, through the activation of mitochondrial/caspase apoptosis pathways, and inhibiting radioresistance [181]. Moreover, DADA regulates the switch from glycolysis to OxPhos and induces intracellular ROS level increase, thereby enhancing the radiosensitization of esophageal squamous cell carcinoma (Fig. 3) [169].

The mechanisms underlying cell irradiation are complex, and they are involved in the inhibition of cell proliferation and induction of cancer cell apoptosis. Mitochondrial proteins are involved in apoptosis as well, and therefore, they may play a key role in the radiation signal transduction. The mitochondrial proteomes derived from Burkitt lymphoma before and after irradiation were analyzed and 23 differentially expressed proteins were identified [182]. This suggests that radiotherapy can lead to the considerable alterations in the mitochondrial protein expression, and therefore induce radioresistance.

Currently, the mitochondrion-associated proteins that were shown to be associated with radioresistance include the following: (1) Adenosine monophosphate family protein 3A (ATAD3A), often expressed in cancer patients and shown to be related to the sensitivity of cancer patients to chemotherapeutics. Increased ATAD3A expression can inhibit the IR-induced apoptosis in glioblastoma cells. After silencing ATAD3A, the expression of ATM, histone H2AX, and H3 was shown to decrease, inhibiting the DNA damage repair and ultimately promoting tumor cell radiosensitivity [183]. (2) NAD⁺-dependent protein deacetylase SIRT3 is a member of the sirtuin family, present in mitochondria, that promotes metabolic homeostasis by modulating mitochondrial protein deacetylation. Following the SIRT3 transcription, cyclin B1/CDK1 further induce SIRT3 activity. Mutations in the SIRT3 Thr150Ala/Ser159Ala lead to a decrease in MnSOD activity and the production of mitochondrial ATP, increasing sensitivity to radiotherapy. Therefore, the targeting of CDK1-mediated phosphorylation of SIRT3 may represent an effective way to sensitize tumor cells to radiation therapy [28]. (3) Mitochondrial MAPK phosphatase (MKP1) represents a potential target for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancers. MKP1 is overexpressed in radioresistant breast cancer cells and it can translocate to mitochondria after irradiation, preventing the activation of apoptosis by inhibiting the accumulation of phosphorylated JNK. MKP1 is the major downstream effector of the HER2-activated RAF/MEK/ERK1/2 pathway. Mitochondrial MKP1 confers radioresistance to HER2 overexpressing breast cancer cells, and by co-suppressing the expression of MKP1 and HER2, breast cancer cell apoptosis can be induced, while inhibiting radioresistance (Fig. 3) [184].

During respiratory oxidative processes, the MMP is generated due to the asymmetric distribution of protons and other ions on both sides of the inner mitochondrial membrane. A physiological MMP is a prerequisite for the maintenance of the physiological cell functions. Studies have shown that some molecules and related signaling pathways induce radioresistance by increasing MMP or inhibiting its decrease. Therefore, targeting the MMP can be an effective way to increase radiosensitivity. The main molecules and processes underlying MMP effects on radiosensitivity include the following: (1) Growth differentiation factor-15 (GDF15), a member of the TGF-β superfamily, participates in homeostasis maintenance and regulates radiosensitivity. Li et al. [185] demonstrated that GDF15 contributes to radioresistance development in head and neck cancer (HNC) cells by activating MMP and inhibiting intracellular ROS generation. Therefore, GDF15 levels may indicate radioresistance levels and its expression may represent a potential therapeutic target for the treatment of HNC. (2) MEK/ERK-mediated signaling selectively inhibits IR-induced decrease in MMP and inhibits FAS-mediated cell death by inhibiting caspase-8 activity. MEK-specific inhibitor PD98059 was shown to prevent the observed effects of MEK/ERK on MMP and the development of radioresistance [186]. (3) Histone deacetylase inhibitors increase radiosensitivity by reducing MMP and promoting ROS production, G2/M phase arrest, and the IR-induced apoptosis of the esophageal cancer cells [187]. (4) In the radiation-resistant cells (CRR) treated with paclitaxel, ROS levels can increase through the decrease in MMP and OxPhos activation, thus overcoming the radioresistance of these cells (Fig. 3) [188].

Furthermore, mitochondrial ion channels are involved in radioresistance development as well. Different types of ion channels can be found on the inner and outer mitochondrial membranes, and they are involved in many important cellular processes, including ATP production, apoptosis, and cell proliferation [189]. Some ion channels as therapeutic targets have been widely used in clinic. Mitochondrial KATP channel (mtKATP channel) is an important member of this family. MtKATP overexpression was found to be closely related to the degree of malignancy of gliomas and the overall survival of patients. Importantly, mtKATP channel can regulate glioma radioresistance development by modulating the ROS-induced ERK activation (Fig. 3), suggesting that the mtKATP pathway is a key regulator of radiosensitivity in gliomas, and the blockers and inhibitors of mtKATP channel and MAPK/ERK kinase, respectively, may represent novel therapeutics of the treatment of gliomas [190].