|Year : 2009 | Volume
| Issue : 9 | Page : 48-52
Modification of 2-deoxy-D-glucose on radiation-and chemotherapeutic drug-induced chromosomal aberrations
Venkatachalam Perumal, Paul FD Solomon, Vikram R Jayanth
Department of Human Genetics, Sri Ramachandra Medical College & Research Institute (Deemed University), Porur, Chennai, India
|Date of Web Publication||21-Aug-2009|
Department of Human Genetics, Sri Ramachandra Medical College & Research Institute (Deemed University), Porur, Chennai - 600 116
Source of Support: None, Conflict of Interest: None
Background: Chemotherapy is the treatment of cancer with drugs, often used as either adjuvant or neoadjuvant or in conjunction with radiation and surgery. Unfortunately, majority of the drugs are toxic to normal tissues, the toxicity being resulting from multidrug protocol used to induce remissions and achieve tumor care. While it has been demonstrated for compounds like the 2-deoxy-glucose (2-DG) used as a modulator for radiation-induced damages, such studies were rarely reported for chemotherapeutic drugs.
Objective: To study the effect of 2-DG on radiation-and chemotherapeutic drug-induced chromosomal aberrations in normal and tumor cells exposed in vitro.
Materials and Methods: The peripheral blood lymphocytes (PBLs) and BMG-1 cells were exposed to radiation and chemotherapeutic drugs (bleomycin and mitomycin-C) in the presence and absence of 2-DG. The treated cells were cultured for various durations, arrested at either metaphase or cytokinesis stage of the cell cycle. The stable and unstable aberrations were recorded using Giemsa staining and FISH technique. The cell cycle kinetics was studied using fluorescence plus Giemsa (FPG) staining.
Results: The presence of 2-DG reduced stable and unstable chromosome aberrations (CA) significantly (P < 0.001), in PBLs induced by radiation, bleomycin and mitomycin-C, when compared to cells treated with radiation or the drugs and increased significantly in BMG cells (P < 0.001). Furthermore, the presence of 2-DG altered the cell cycle kinetics in the PBLs and BMG-1 cells. Thus the overall results showed protection effect on the normal cell damages induced by radiation and chemotherapeutic drugs, while sensitizes the tumor cell.
Conclusion: The obtained results suggest that 2-DG in combination with radiotherapy/chemotherapy could lead to an improvement in tumor therapy by sensitizing the tumor cells while protecting the normal cells.
Keywords: Bleomycin, chromosomal aberrations, mitomycin-C, radiation, 2-deoxy-D-glucose
|How to cite this article:|
Perumal V, Solomon PF, Jayanth VR. Modification of 2-deoxy-D-glucose on radiation-and chemotherapeutic drug-induced chromosomal aberrations. J Can Res Ther 2009;5, Suppl S1:48-52
|How to cite this URL:|
Perumal V, Solomon PF, Jayanth VR. Modification of 2-deoxy-D-glucose on radiation-and chemotherapeutic drug-induced chromosomal aberrations. J Can Res Ther [serial online] 2009 [cited 2019 May 19];5:48-52. Available from: http://www.cancerjournal.net/text.asp?2009/5/9/48/55142
| > Introduction|| |
Chemotherapy is the treatment of cancer with drugs which interfere with cell division. These drugs enter the bloodstream and circulate throughout the body and control the cell proliferation, making this treatment potentially useful for cancers that have spread (metastasized) to distant organs. While, the drug is carried in the blood, it can reach cancer cells anywhere in the body, the healthy cells also being exposed to the toxic effects. The drugs were used as either adjuvant or neoadjuvant or in conjunction with radiation and surgery. Often a combination of drugs is used, because of its different effects and multiple cycles to check the tumor growth more efficiently. An enhanced cytotoxic effect has been reported in animal models and in vitro in tumor cells  upon simultaneous exposure to radiation and bleomycin. Unfortunately, majority of the drugs are toxic to normal tissues, the toxicity being resulting from the multidrug protocol used to induce remissions and achieve tumor care.
To maximize the therapeutic effect of radiation, studies were reported on the modulatory effect of compounds like Hoechst,  cysteine,  caffeine,  calcium channel blockers,  and 2-deoxy-glucose (2-DG) on animal models,  glioma cells,  and breast tumor cells  followed by radiation, and of radionuclides on tumor cells.  Scrutiny of the literature did not show any evidence for the modulatory effect of chemotherapeutic drugs though their use is inevitable in combined modality treatment. Along with the support of DRDO (INM-280), we started to investigate the effect of 2-DG on bleomycin- and mitomycin-C- induced cytogenetic damages in peripheral blood lymphocytes (PBLs) and in BMG-1 cells. The results obtained with chemotherapeutic drugs were compared with our earlier studies obtained with radiation, as the modulatory effect of 2-DG on the radiation-induced damages was well demonstrated.
| > Materials and Methods|| |
The PBLs collected from healthy volunteers were used as normal cells. The sample collected was divided into 2-ml aliquots and exposed to gamma radiation (dose range from 0.1 to 4 Gy) at a dose rate of 2Gy/min. Simultaneously, blood samples also suspended in the RPMI-1640 medium treated with bleomycin (10-80 μg/ml) or mitomycin-C (2-12 μg/ml). The samples were exposed to either radiation or the chemotherapeutic drug in the presence and absence of 2-DG (5 mM, 30min before treatment) for 3h at 37°C. After 3 h, cells were washed with a buffer to remove the drugs and 2-DG before used for the culture setup. To 1.0ml of the blood sample, 10 ml culture medium (RPMI-1640), supplemented with 7.5% NaHCO 3 , 20% fetal calf serum, 200 mM l-glutamine, penicillin 100 units/ml, and streptomycin 100 mg/ml, was added. PHA-P, 200ml, was added to the culture to initiate cell division. At 46h, the cells were blocked at the metaphase stage by adding colcemid (0.1 mg/ml) and the culture was further incubated until 48h. The sample was harvested by given hypotonic treatment, fixed with Carnoy's fixative, and cast on clean precooled slides. Multiple slides were casted for each sample and used for unstable chromosomal aberration analysis using Giemsa staining and stable aberrations using fluorescence in situ hybridization.
To analyse unstable chromosomal aberrations, the slides were stained with 10% Giemsa, air dried and mounted with a cover slip using DPX. The slides were observed under a microscope to record various types of aberrations such as dicentric chromosome, ring chromosome, minutes, gaps, acentric fragments, and chromatid gaps. 
The slide with metaphase chromosomes prepared as mentioned above was denatured, dehydrated, and air dried. The whole chromosome probe (no. 2 labeled with TRITC) in the hybridization buffer was denatured and applied to denatured chromosomes and hybridization was carried out for 24 h at 37°C in a moistened hybridization chamber. After 24 h of hybridization, the cover slip was removed and the slides were rinsed in a formamide wash solution, air dried, counterstained with DAPI, and covered with a cover slip. The number of painted chromosomes with and without translocation was recorded for each metaphase. The genomic translocation frequency was estimated from the fraction of genome painted as suggested by Lucas et al . 
To study the cell cycle kinetics, the chromosome culture was set up, as mentioned above, except for the addition of BrdU (100 μl/ml), and the culture time was 72h. After the preparation of slides, metaphase chromosomes were stained with Hoechst (33258) for 10min, washed with Mcllvaine's buffer, mounted temporarily with a cover slip, and exposed to sunlight for 2h. The slides were stained with 2% Giemsa and mounted with DPX, and the stages of the cell cycle and the number of exchanges in the second cell cycle metaphases alone were recorded. 
The BMG-1 cells obtained from INMAS, New Delhi, and maintained as a monolayer culture in our laboratory were used as model tumor cells. BMG-1 cells grown for 24 h (Dulbecco's modified essential medium supplemented with 5% fetal calf serum and antibiotics) after subculture were exposed to bleomycin at different concentrations (10-80 μg/ml) in the presence and absence of 2-DG (5 mM, 30-min pretreatment with bleomycin) for 3 h at 37°C. At the end of 3 h, bleomycin and 2-DG were removed from the cells by washing with HBSS (Hanks' balanced salt solution) three times. The cells were resuspended in the medium, grown further for various another 20-28h. Colcemid (0.1 mg/ml) was added at 20 h to block the cells at the metaphase stage. At the end of 28 h, the cells were harvested and caste onto slides, stained with Giemsa, and different types of chromosomal aberrations were recorded as mentioned for peripheral blood. For the micronucleus assay, cytochalasin-B at a final concentration of 2 μg/ml culture was added at 24 h of culture. The cells were further incubated for 24 h at 37°C. The cells were harvested with brief hypotonic treatment and slides were prepared by fixing the cells with Carnoy's fixative. The cell suspensions were dropped onto a clear cooled slide and stained with Giemsa. Cells with two daughter nuclei surrounded by cytoplasm were scored for the presence of micronuclei (MN). 
The modulatory effect of 2 DG was calculated as given below: 
| > Results|| |
[Table 1] shows the comparison between the frequencies of chromosomal aberrations (CAs) obtained from the PBL exposed to radiation in the presence and absence of 2-DG. Cells exposed to radiation showed a dose-dependent increase in the aberration frequency, whereas treatment of cells with 2-DG upon prior exposure to radiation reduced the CAs significantly in all the doses ( P > 0.01). The dose modulatory factor varies between 0.33 and 0.75.
The frequencies of stable aberrations (translocations, TLs) and unstable aberrations (CAs) obtained from PBLs treated with bleomycin in the presence and absence of 2-DG are shown in [Table 2] and [Table 3] respectively. It was observed that bleomycin induced a concentration-dependent increase in the frequencies of both types of aberrations. However, the addition of 2-DG reduced the bleomycin-induced aberration frequency significantly ( P < 0.001). The dose modualtory factors varied between 0.38 and 0.72 for different types of aberrations.
Mitomycin-C-induced CA frequencies obtained from PBLs exposed in the presence and absence of 2-DG are shown in [Table 4]. Similar to radiation and bleomycin, exposures of cells to mitomycin-C induced a concentration-dependent increase in the frequencies of all types of aberrations. Though the exposures of PBL to mitomycin-C induced different types of CAs, the yield is significantly less when compared to those induced by either radiation or bleomycin. However, exposure to mitomycin-C in the presence of 2-DG showed a significant reduction in CA and SCE frequencies. The cell average generation time was increased upon exposure to the drug.
Frequencies of CAs and the MN obtained from BMG-1 cells treated with bleomycin in the presence and absence of 2-DG are given in [Table 5] and [Table 6] respectively. In untreated BMG-1 cells, both CA and MN were higher when compared to those obtained in PBLs ( P = 0.001). When compared to MN, the CA induced by bleomycin was twofold higher in both PBLs and BMG-1 cells. Further, when compared to bleomycin-induced CA and MN frequency, the presence of 2-DG increased in BMG-1 ( P < 0.0001) cells with extreme significance.
| > Discussion|| |
Wide varieties of antineoplastic agents are used routinely in clinical oncology. Unfortunately, majority of the drugs are toxic to normal tissues resulting, the toxicity being resulting from the multidrug protocol used to induce remissions and achieve tumor care. The enormous literature provides concrete evidence on the modulatory effect of 2-DG in radiation-induced damages; we were interested to study the effect of 2-DG on chemotherapeutic drugs as they are used in combinational therapy. We used bleomycin and mitomycin-C in addition to radiation, of which the radiomimetic bleomycin is a free radical-based DNA-damaging agent which induces strand breaks by highly specific, concerted free-radical attack on deoxyribose moieties in both DNA strands, whereas mitomycin-C is an alkylating agent that binds with DNA and interferes with cell replication.
Exposure of cells to therapeutic agents induces variety of DNA lesions,  of which a double-strand break is believed to be most significant because it can lead to exchange-type chromosomal aberrations, which are lethal to the cells upon cell division. Our obtained results showed that though radiation and bleomycin induce variety of chromosomal aberrations in both PBLs and BMG-1 cells, 60-70% of them are exchange-type chromosome aberrations (both stable and unstable), which could have resulted due to restitution of double-strand breaks from two different chromosomes; the remaining include simple chromosome- and chromatid-type lesions without any exchanges. The results are in agreement with earlier reports obtained for CHO cells  and PBLs obtained from patients treated with bleomycin , in which the dicentric chromosomes accounted for 90% of aberrations. The yield of chromosomal aberrations induced by mitomycin-C is less when compared to that obtained from bleomycin or radiation; however, it induced many exchanges among sister chromatids. The difference could be attributed to their mechanism of action as mitomycin-C predominantly interferes with replication than inducing strand breaks directly. The presence of 2-DG significantly reduced both simple exchanges and unstable chromosomal aberrations induced by mitomycin-C.
It has been demonstrated that 2-DG sensitized the radiation- induced DNA damages in tumor cells  while protecting normal cells.  Similar to radiation, the presence of 2-DG showed an extremely significant reduction in the frequencies of CA the PBLs (ρ < 1), in all the bleomycin and mitomycin-C concentrations studied, showed a very significant enhancement in both CA and MN frequencies induced by bleomycin in BMG-1 cells (ρ > 1). These results suggest that 2-DG protect the chemotherapeutic drug-induced cytogenetic damages, especially exchange-type chromosome aberrations, in normal cells while sensitizing the tumor cells. The significant observation made in the current study, is that the presence of 2DG reduced the stable aberration (translocations) frequency induced by bleomycin in the PBL. It has been reported that persistence of the stable aberration like translocations is a possible initiating event for genomic instability and therapeutic agent induced second malignancy.  Thus, from our observation, it is reasonable to speculate that pretreatment of 2-DG before therapy might reduce the probability of developing therapy induced second malignancy in addition to the chemo-modulatory effect. However, to confirm the same we need more studies in multiple cellular systems.
A general mechanism proposed for the reduction in damages in normal cells during radiation treatment is either protecting the DNA against the induction of damage or interference with the progression of cells in the cell cycle and modifications in repair of damages. Earlier we had shown that exposure of cells to 2-DG increases the average generation time in normal cells.  The drug-induced transient block in cell cycle progression presumably permits the error-free repair of DNA damages before the cells initiate the synthesis or mitosis in normal cells, whereas in tumor cells, the rate of fixation is higher and therefore increased number of DNA lesions caused by a decrease in DNA repair in the presence of 2-DG would immediately transformed into irreparable lesions, which are expressed as chromosomal aberrations. In addition, it was shown that ATP synthesis and oxidative metabolism were essential for the repair of damages induced by radiation.  In the present study also, the increased cell cycle doubling time along with a shift in the energetic status of the cellular environment would play a significant role in the modulation of chemotherapeutic drug-induced chromosomal aberrations. Therefore, it has been suggested that 2-DG in combination with radiotherapy/chemotherapy could lead to an improvement in tumor therapy by inhibiting the repair process and enhance the damage by fixing the lesions in tumor cells while protecting the normal cells.
| > Acknowledgements|| |
The authors wish to thank the DRDO (INM-280) and CSIR for providing the financial assistance to carry out the research work.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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