Background
Ionizing radiation plays an important role in the management of a majority of malignancies [
1], although many tumors like glioma and several carcinomas are known to be refractory to radiotherapy with marginal benefits in survival. However, the molecular mechanisms underlying this radio-resistance of cancer cells remain poorly understood. One of the most common signatures of highly malignant tumors is their capacity to metabolize more glucose to lactic acid than normal tissues, which confers a selective growth advantage [
2]. Cells derived from hypoxic tumors typically maintain their metabolic phenotypes even under normoxic culture conditions (Warburg effect), indicating that aerobic glycolysis is constitutively upregulated through stable genetic or epigenetic changes [
2]. It is also reported that mitochondrial defect linked stabilization of HIF1α induces glycolytic phenotype in cancer cells and promotes aggressiveness of tumors [
2,
3]. On the other hand, efficient oxidative phosphorylation in cancer cells is required for execution of apoptosis through the generation of reactive oxygen species (ROS) [
4]. Therefore, metabolically reprogrammed and highly glycolytic cancer cells can easily escape the death processes, conferring resistance to therapeutic modalities [
5].
The phenotypic characteristics of enhanced glycolysis associated with tumors have been well exploited for the diagnosis of the disease using fluoro-deoxy glucose (FDG) based positron emission tomography (PET) imaging and the efficacy of glycolytic inhibitors as sensitizers to radiation and chemotherapeutic drugs has established in pre-clinical studies, while clinical trials are at different stages of evaluation [
6-
9]. Considerable amount of evidences suggest that inhibition of glycolysis leads to compromised DNA repair, which is accompanied by the depletion of energy [ATP and AXP (AMP and ADP)] in cells with high rates of glycolysis like the cancer cells, causing death [
10-
14]. However, the mechanisms underlying enhanced resistance to radiation-induced cell death in cells with high endogenous rates of glycolysis, like the cancer cells are not completely understood, although alterations in pH and lactate levels have been implicated [
2]. Therefore, present studies were undertaken to examine if transient stimulation of glycolysis (before irradiation) using MRMs is sufficient to confer radio-resistance and also to unravel the underlying mechanisms. Results obtained in human malignant and non-malignant cell lines clearly show that stimulation of glycolysis using MRMs reduces radiation-induced cell death by enhancing the repair of DNA damage leading to a reduction in the mitotic death linked to cytogenetic damage (micronuclei formation).
Discussion
Irradiation of cells causes macromolecular damage (viz DNA, protein etc) stimulating multiple signaling pathways viz DNA repair, cell cycle check points, apoptosis and senescence that collectively determine the fate of cells [
22]. It is well established that at moderate absorbed doses of low LET radiation (like gamma rays and X-rays), the main contributing factor responsible for loss of clonogenecity (cell survival) is the mitotic death linked to cytogenetic damage that arises from residual DNA damage [
22,
27]. Incomplete repair and/or mis-repair of DNA strand breaks results in chromosomal damage that can be observed as various chromosomal aberrations in the metaphase of the irradiated population and as micronuclei in the daughter cells [
27], although mitotic spindle dysfunction and other disturbances also lead to micronuclei formation and related nuclear damage [
28]. Therefore, alterations in the DNA repair processes that are operative for few hours following irradiation are expected to influence the cell survival as well as the level of cytogenetic damage. In the present study, we observed an increase in the clonogenic survival following transient stimulation of glycolysis (Figure
3) that correlated with the decrease in the level of micronuclei expression (Figure
5C) suggesting a reduction in the residual DNA damage under these conditions. While stimulation of glycolysis did not significantly alter the level of induction of DNA damage (Figure
5A), the rate of DNA strand break rejoining as well as the extent of damage removal were clearly higher that resulted in a decrease in the residual DNA damage at the end of 30 minutes after irradiation (Figure
5B).
It will be interesting to see whether the mechanisms underlying radio-resistance seen in cancer cells, where a stable phenotype with enhanced glycolysis gradually develops during tumorigenesis will be identical to the transient stimulation of glycolysis observed here. In this respect, it is pertinent to note that inhibition of mitochondrial respiration [
29] that leads to enhanced glycolysis (as seen here; Figure
1) often results in the stimulation of HIF1α expression of many genes in the glycolytic pathway and up-regulated in malignantly transformed cells and tumors [
30,
31]. Stimulation of glycolysis seen as a compensatory mechanism following a fall in the mitochondrial ATP production induced by respiratory inhibitors has been suggested to be due to the activation of AMP kinase triggered by an increase in the AMP level (and AMP/ATP ratio) due to ATP breakdown [
29]. Some recent evidences suggest that inhibition of mitochondrial respiration leads to accumulation of glycolytic end products like pyruvate and lactate which could dramatically increase HIF1α accumulation in cancer cells by inhibiting the prolyl hydroxylase enzyme activity. Moreover, other mediators like the expression of TKTL1 (Transketolase-like 1, an enzyme of pentose phosphate pathway) in head and neck carcinomas and gliomas leads to metabolic switch by stabilizing HIF1α for improving energy yield from glucose via glycolysis and enhancing antioxidant defence against ROS via pentose phosphate pathway [
32,
33]. In this respect it is pertinent to note that the increase in the protein levels of regulators of glycolysis stimulated by DNP in BMG-1 cells viz. Glut-1 and HK-II observed here (Figure
1D) are all regulated by HIF1α [
9,
27]. Therefore, radio-resistance following DNP induced transient elevation in glycolysis appears to partly involve similar mechanisms that are reported as activators of glycolysis in cancer cells generally associated with resistance to radiation and other drugs. Further, sensitization of BMG-1 cells to radiation in the presence of Antimycin A (Figure
4B), which inhibits mitochondrial respiration without compensatory increase in glycolysis and the glycolytic inhibitor 2-DG strongly suggests that MRMs (DNP, PS3 and MB) induced radio-resistance in cells is mainly due to increase in glycolysis and not because of inhibition of mitochondrial respiration.
DNA double strand breaks, the most lethal lesions widely considered to be responsible for radiation-induced cell death are repaired by both homologous recombination (HR) and non-homologous end joining (NHEJ) in a proliferating mammalian cell population. Rad51 is a critical component of DNA DSB repair pathway [
34], which is widely reported to redistribute within the nucleus following DNA damage suggesting the formation of repair foci involving this recombinase [
25,
26,
35], although radiation induced elevated level of this protein enhances radio-resistance has also been reported [
35]. Rapid increase in the level of Rad51 protein at 30 minutes following irradiation in glycolysis stimulated (DNP treated) cells observed here (Figure
6) is similar to the observations in the radio-resistant spheroids of DU145 carcinoma cell line [
36]; which also interestingly has a significant level of HIF1α with elevated glycolysis and increased resistance to radiation [
37]. Increased level of Rad51 following irradiation seen here, particularly in DNP treated cells is at variance with earlier reports [
25,
26,
35] and may arise due to many reasons viz. proteasomal degradation, altered metabolic status of the cells [
36,
38] or changes in the interactions with other members of the repair complex thereby altering the immune reactivity, which needs further investigations. A profound increase in the level of Ku-70 (Figure
6) in DNP treated cells also facilitates a faster repair by NHEJ conferring resistance against radiation in high glycolytic cells. Increased level of Ku-70 has been reported to increase cellular tolerance against ischemic stress [
39] and adaptation to ischemia provides hypoxia mediated
in-vivo tumor radio-resistance [
40]. Our results also suggest that increased Ku-70 level may facilitate NHEJ pathway of DSB repair in high glycolytic cells leading to reduced micronuclei and increased cell survival against radiation. Some recent evidences suggest that DNA damage induced by adriamycin enhances the TIGAR and TKTL1 expression and knocking down the TKTL1 or WRN complex both leads to reduced glycolytic metabolism and accumulation of DNA damage in cancer cells [
33,
41]. Although these observations strongly suggest enhanced repair of radiation-induced DNA DSBs by stimulated glycolysis, underlying mechanisms responsible for this can only be unraveled using studies with DNA repair deficient and efficient cell systems following the stimulation of glycolysis. Pending this insight to be unraveled, results of the present studies lend support to our hypothesis that enhanced glycolysis is a favorable metabolic change that facilitates DNA repair, which appears to be partly responsible for radio-resistance, which is often observed in cancer cells.
The transient increase in glycolysis conferring resistance against radiation-induced cell death in normal HEK293 cells (clonogenic survival; Figure
3C) has important implications in radiation countermeasure, as pharmacological agents that stimulate glycolysis at the systemic level may act as radio-protective agents. Indeed, administration of low amounts of, DNP used in this study for stimulating glycolysis has been shown to be safe for humans/canine [
42]. Facilitated DNA repair leading to reduced cytogenetic damage and mitotic linked cell death along with upregulated level of HK-II and Ku-70 which are generally elevated with the increase in glycolysis is known to inhibit intrinsic pathway of apoptosis [
43] and therefore can protect both the hematopoietic and Gastro Intestinal system.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BSD and ANB conceived the study, designed the experiments and wrote the manuscript. ANB performed most of the experiments with help of AC, SK, YR, SS, RS and NK. AC conducted western blotting and helped in editing the manuscript. All authors read and approved the final manuscript.