Ionizing Radiation: Down Regulation Ionizing Radiation: Down Regulation of ‘ATM’ by 2 Deoxy-D- Glucose. of ‘ATM’ by 2 Deoxy-D- Glucose.

A Time Dependent StudyA Time Dependent Study

S. S. LAHIRIS. S. LAHIRIInst. of Nuclear Medicine & Allied Sciences (INMAS)Inst. of Nuclear Medicine & Allied Sciences (INMAS)

Delhi, IndiaDelhi, India

NREP, Chicago, Mar.31.2010NREP, Chicago, Mar.31.2010

I am thankful to the Organizing committee, more so to Ms. Michele Skiermont and Mr. Prosanta Chowdhury, for giving me a chance to present our work to the august gathering of NREP at Chicago.

This is my first ever visit to the United States of America.

My first impressions :

(i) There is great commitment to the cause(ii) US means business(iii) People are positive, cordial, confident and

happy-go- lucky type

Chicago is the city where the great Indian monk

SWAMI VIVEKANADA

addressed the First ‘Parliament of Religions’ during 1893.

He addressed as “My Dear Brothers and Sisters in America”.

That was an epoch making speech, referred even today and the text as a small booklet is available abundantly.

Radiation: Down Regulation of

Institute of Nuclear Medicine & Allied Sciences (INMAS) Delhi India

S Hamborde, S S Lahiri, N. Saxena, S Chaturbedi and R P Tripathi

2 Deoxy-D-Glucose (2DG) :

(i) Radio-sensitizer for cancer cells

(ii) Calls for optimization of radio-therapy (low dose IR will be sufficient)

(iii) Radio-protector for healthy cells surrounding the cancerous cell

(iv) Kills cancer cells by metabolic inhibition

Ataxia Telangiectasia (ATM) : A protein which senses and repairs DNA breaks

2-DG differentially modifies energy status in normal and cancer cells

Can 2-DG differentially modulate cellular processes in cancer and normal cells?

G/ 2-DG

2-DG-6-PATP X

M G R

Normal cells

G-6-P

Cancer cells2-DG

2-DG-6-P

ATPX

M G R

/G

/G-6-P

2-DG is transported across Blood Brain Barrier and phosphorylated by same mechanisms as glucose

H

H

HH

CH2OH

H

OHOH OH

H2-DG

O

H

OH

HH

CH2OH

H

OHOH OH

HGlucose

O2-DG-6-Phosphate is not metabolized further and acts as a metabolic block

2-Deoxy-D-glucose (2-DG) as a modifier of therapeutic responses

BackgroundDNA and tissue damage mainly are responsible for tumor

causation Damage induced survival (repair) and death responses require

metabolic energy (ATP) Cancer cells show enhanced glucose usage & derive

considerable part of their energy from the glycolytic pathway 2-DG inhibits glycolytic ATP production and disturbs cell

signaling

2-DG may enhance treatment-induced damage selectively in cancer cells by inhibiting repair and inducing death

processes

H

H

HH

CH2OH

H

OHOH OH

H2-DG

O

H

OH

HH

CH2OH

H

OHOH OH

HGlucose

O

2-DG an ideal adjuvant to improve cancer therapy ?

Jain & Pohlit 1973

Modification of radiation responses by 2-DG

CellsHuman Tumor cell lines (>10)

Repair Mitotic death Apoptosis Clonogenicity

Tumor model Radiation Tumor control Animal survival

Sarcoma 180, EAT, DL

Low & High LET

Animal model Radiation Marrow damage

Animal survival

Balb/C and Strain “A”

Co-60 gamma rays

Experimental studies: Tumor

Experimental studies: Normal

Clinical studiesTumor Tolerance Toxicity Survival QOLTrials

Mal glioma Phase I, II & III Excellent Negligible

Organ cultures of brain tumors

- -

Phase III Trial Successful

Enhanced glycolysis in cancerMultiple mechanisms involved with functional

redistribution

Mit gene mut

Onco trans

Hypoxia (HIF)

Akt; myc, Src, Bcr-Abl…

12&16SrRNA; Cox I-III; Dloop; ATPase

Altered metabolic enzymes (HKII, FH, SDH)

Korn et al, Clin Cancer Res 2004

Ellstrom et al, Cancer Res 2004Dang 2006

Mechanisms of damage response modifications by 2-DG in vitro

Loss of clonogenicity

Glycolysis

Oxidative stress Repair inhibition

Energy depletionVEGF

X

2-DG

HMP Shunt

Apoptosis

Altered Trans regulation

2-DG-6-P

GLUT

X

NADPH depletion UPR

Cytogen. damage

Mitotic death

HK

Necrosis & death

ECSMetabolism

Growth inhibition Senescence

HK?

Dwarakanath et al, 2006, 2009

N-Linked glycosylation of

proteins

Cellular responses to radiation damage

DNA & Non-DNA damageDeath inducing signals & processes

Pro-survival signals & processes

DNA repair

Division delay

Primary Apoptosis

GADD45BRCA1Nbs1

DNA PKKu70/80

RAD

TNFBax

PUMANOXA

CDKsChk1 & 2BRCA2Topo II

P2114-3-3

ATM-ATR-p53 EGR1; AKT; PI3K;RasProliferationSurvival

NFk

Bcl2Bcl2RbE2F

Residual DNA damage

Cytogenetic damage

Secondary apoptosis

Loss of clonogenicty

KinasesPhosphatase

sAcetylases

Energy

2-Deoxy-D-Glucose (2-DG)

ATM pathwayATM pathway

IntroductionIntroduction Ionizing radiation Ionizing radiation (IR) causes oxidative stress & damage DNA, (IR) causes oxidative stress & damage DNA,

primarily as Double Stand Breaks. Besides single stand breaks primarily as Double Stand Breaks. Besides single stand breaks & base modifications also occur.& base modifications also occur.

ATMATM or Ataxia Telangiectasia mutated protein is involved in DNA or Ataxia Telangiectasia mutated protein is involved in DNA strand break strand break sensory & repair sensory & repair pathwayspathways

AT causes increased sensitivity to IRAT causes increased sensitivity to IR Important role in Telomere maintenance & Neuronal Important role in Telomere maintenance & Neuronal

developmentdevelopment Autosomal recessive gene coding for 350 KD proteinAutosomal recessive gene coding for 350 KD protein ATM is a serine threonine kinase. ATM is a serine threonine kinase. Phosphorylation of serine & threonine moieties lead to cell cycle Phosphorylation of serine & threonine moieties lead to cell cycle

arrest, DNA repair & apoptosis.arrest, DNA repair & apoptosis. ATM promoters do not have TATA element. More than one ATM promoters do not have TATA element. More than one

mechanism of transcription regulation. An ubiquitous mechanism of transcription regulation. An ubiquitous transcription factor transcription factor “Sp1” “Sp1” regulate ‘atm’ transcription from regulate ‘atm’ transcription from TATA less promoter.TATA less promoter.

Transcription of ‘atm’ can also be controlled Transcription of ‘atm’ can also be controlled independent of Sp1.independent of Sp1.

Sp1 activation can be modulated by O-Sp1 activation can be modulated by O-GlcNAcylation at either serine or threonine GlcNAcylation at either serine or threonine moieties. moieties.

2DG or 2 deoxy-D-glucose, can modulate the 2DG or 2 deoxy-D-glucose, can modulate the process of glycosylation in the cell & can down process of glycosylation in the cell & can down regulate Sp1 activity. However the mechanism regulate Sp1 activity. However the mechanism of down regulation of Sp1 is different in of down regulation of Sp1 is different in hypoglycemia & 2DG treatment.hypoglycemia & 2DG treatment.

2DG block glycolysis, thus cause 2DG block glycolysis, thus cause ATP depletion & apoptosis. It alters protein glycosylation & apoptosis. It alters protein glycosylation causing endoplasmic reticulum stress, Unfolded causing endoplasmic reticulum stress, Unfolded Protein Response (UPR) leading to Cell DeathProtein Response (UPR) leading to Cell Death

• 2DG sensitizes cancerous cells to 2DG sensitizes cancerous cells to radiation & protect healthy cells.radiation & protect healthy cells.

• Presence of 2DG before & after Presence of 2DG before & after irradiation causes modulation of O- irradiation causes modulation of O- GlcNAcylation of Sp1 transcription GlcNAcylation of Sp1 transcription factor, leading to down regulation in factor, leading to down regulation in ‘atm’ expression.‘atm’ expression.

• To check the hypothesis, ‘atm’ levels To check the hypothesis, ‘atm’ levels were measured both at were measured both at Transcriptional Transcriptional and and Translational Translational levels levels and also for theand also for the Pre & Post-Pre & Post-irradiation administration of 2DG.irradiation administration of 2DG.

Fig. 1a. Glucose

Fig. 1b. Glucose

H

MaterialsMaterials & Methods& Methods Human Glioma cell line (BMG1) Human Glioma cell line (BMG1)

maintained as monolayer culture in maintained as monolayer culture in Dulbeco’s modified essential medium with Dulbeco’s modified essential medium with 10% fetal bovine serum & grown in 5% 10% fetal bovine serum & grown in 5% CoCo22 at 37 at 37C.C.

Irradiation at room temperature to a dose Irradiation at room temperature to a dose rate of rate of 0.0115 Gy/sec 0.0115 Gy/sec using Co 60 Gamma using Co 60 Gamma ray source (Eldorado 78, Teletherapy unit, ray source (Eldorado 78, Teletherapy unit, AECL, Canada).AECL, Canada).

2DG (M/s Sigma Chemicals, USA) was 2DG (M/s Sigma Chemicals, USA) was given at a final conc. of 5mM at different given at a final conc. of 5mM at different time points before or after irradiation.time points before or after irradiation.

A. Pre-treatmentA. Pre-treatment Group (2DG+IR)Group (2DG+IR)2DG Administered before irradiation at various time 2DG Administered before irradiation at various time points & RNA isolated 4h after irradiation (IR).points & RNA isolated 4h after irradiation (IR).For 2DG group 2DG administered at same intervals For 2DG group 2DG administered at same intervals & RNA isolated as before but without IR.& RNA isolated as before but without IR.

B. Post –treatment Group (IR+2DG)B. Post –treatment Group (IR+2DG)2DG administered at various time points after IR & 2DG administered at various time points after IR & RNA isolated 4h after IR.RNA isolated 4h after IR.For IR group, RNA was isolated at various time For IR group, RNA was isolated at various time points as abovepoints as above

Transcriptional StudiesTranscriptional Studies Cells were lysed directly on the plate using TRI reagent (Ambion USA) Cells were lysed directly on the plate using TRI reagent (Ambion USA)

& total RNA isolated.& total RNA isolated. RNA conc. & Integrity were checked at A260, A280 & also in agarose RNA conc. & Integrity were checked at A260, A280 & also in agarose gel.gel. Equal amount of RNA was blotted on activated charge modified nylon Equal amount of RNA was blotted on activated charge modified nylon

membrane Biobond, Sigma) & UV cross linked at 254nm for one min. membrane Biobond, Sigma) & UV cross linked at 254nm for one min. DNA Probe : Oligo probe sequence-5’AGC TGT CGC TGT GTT TGC TT3’DNA Probe : Oligo probe sequence-5’AGC TGT CGC TGT GTT TGC TT3’ Probe was labeled with Dig Oligonucleotide Tailing kit (Roche Applied Probe was labeled with Dig Oligonucleotide Tailing kit (Roche Applied

Science, Germany) Science, Germany)

Hybridization performed on 6 X SSC, 5 X Denhardt’s reagent, 0.5% Hybridization performed on 6 X SSC, 5 X Denhardt’s reagent, 0.5% SDS, 100 µg/ml of Sonicated Salmon sperm DNA at 50SDS, 100 µg/ml of Sonicated Salmon sperm DNA at 50C O/N. C O/N. Washing on 6 X SSC & 0.1% SDS at 50Washing on 6 X SSC & 0.1% SDS at 50C, 52C, 52C & 54C & 54C.C.

Detection of probe was performed by DNA Dig Detection kit (Roche Detection of probe was performed by DNA Dig Detection kit (Roche Applied Science, Germany) Applied Science, Germany)

Densitometry of the blot was done by Image Quant® 5.2 software.Densitometry of the blot was done by Image Quant® 5.2 software.

Translational StudiesTranslational Studies BMG-1 cells grown on DMEM in 10% FBS, 5% BMG-1 cells grown on DMEM in 10% FBS, 5%

CO2 at 37CO2 at 37C. C. Cells irradiated as before & harvested at Cells irradiated as before & harvested at

0,2,4,&6 h after IR.0,2,4,&6 h after IR. Lysis on plate with Nonidet P-4 lysis buffer.Lysis on plate with Nonidet P-4 lysis buffer. Equal quality (µg) of each lysate were Equal quality (µg) of each lysate were

separated by SDS-PAGE & transferred on Nc separated by SDS-PAGE & transferred on Nc membrane.membrane.

Primary antibody to ‘atm’ (M/s Santacuruz), Primary antibody to ‘atm’ (M/s Santacuruz), HRP conjugated secondary antibody (M/S HRP conjugated secondary antibody (M/S Bangalore Genei, India) were used.Bangalore Genei, India) were used.

Actin was used a loading control.Actin was used a loading control.

Studies on Cell survivalStudies on Cell survival

BMG-1 cells were grown as triplicate on BMG-1 cells were grown as triplicate on petridishes (60mm). After 48h of plating (80% petridishes (60mm). After 48h of plating (80% confluence), cells were treated with 2DG 4h confluence), cells were treated with 2DG 4h before IR (2Gy) or 2DG adm. soon followed by before IR (2Gy) or 2DG adm. soon followed by IR (2 Gy) & incubated as before for seven days IR (2 Gy) & incubated as before for seven days for colony formation & counting.for colony formation & counting.

Results and DiscussionResults and Discussion

Transcriptional StudiesTranscriptional StudiesExpression of ‘atm’ in response to IR (2Gy) was Expression of ‘atm’ in response to IR (2Gy) was monitored till 6h after irradiation. monitored till 6h after irradiation.

Pre-irradiation TreatmentPre-irradiation TreatmentSignificant down regulation (30-40%) of ‘atm’ Significant down regulation (30-40%) of ‘atm’ when treated with 2DG and 2Gy IR (Fig.2). when treated with 2DG and 2Gy IR (Fig.2).No change in ‘atm’ expression at 3h & 6 h No change in ‘atm’ expression at 3h & 6 h after IR (2Gy) even if 2 DG was administered after IR (2Gy) even if 2 DG was administered just prior to exposure. just prior to exposure.

Fig. 2:‘‘atm’’ expression in BMG1 cells treated with 2-DG immediately before irradiation (2 Gy) and RNA isolated 2,3,4,5 and 6 Hr after irradiation. Two independent experiments were performed for each treatment and hybridization done in triplicate for each time point. RNA blotted on nylon membrane and hybridized with ‘atm’ probes. All the bars represent percentage increase and decrease from untreated control. Error bars represent St. error.

2 DG alone reduces ‘atm’ expression 2 DG alone reduces ‘atm’ expression by 20% to 40% at all time points by 20% to 40% at all time points (Fig.2).(Fig.2).

Reduction of ‘atm’ mRNA was seen Reduction of ‘atm’ mRNA was seen with 2DG + 2 Gy IR only at 4h & 5h with 2DG + 2 Gy IR only at 4h & 5h after IR (Fig.2), except in 10 Gy at 2h.after IR (Fig.2), except in 10 Gy at 2h.

Non significant reduction in ‘atm’ Non significant reduction in ‘atm’ expression in response to 2DG + 5 or expression in response to 2DG + 5 or 10Gy IR (Fig. 3).10Gy IR (Fig. 3).

2 DG adm. 4 h before IR is expected 2 DG adm. 4 h before IR is expected to decrease cell survival and slow to decrease cell survival and slow down DNA repair & further down down DNA repair & further down regulation of ‘atm’.regulation of ‘atm’.

Fig. 4.‘‘atm’’ expression in BMG1 cells treated with 2, 5 and 10 Gy irradiation both in presence and absence of 2DG. RNA was isolated 4 Hr after respective treatment.

Fig. 3. In IR+2DG series BMG1 cells were treated with 2DG after 1,2 and 3 Hr of irradiation (5Gy) and RNA harvested after 4 Hr of irradiation. In IR series RNA was harvested after 1, 2, 3 and 4 Hr after irradiation without 2DG treatment. The experiment was repeated twice and each RNA sample was blotted in triplicates. The mean of all six observations was plotted with time. The error bars represent St. error.

Fig 5: In IR+2DG series BMG1 cells were treated with 2DG after 1,2 and 3 Hr of irradiation (10Gy) and RNA harvested after 4 Hr of irradiation. In IR series RNA was harvested after 1, 2, 3 and 4 Hr after irradiation without 2DG treatment. The experiment was repeated twice and each RNA sample was blotted in triplicates. The mean of all six observations was plotted with time. The error bars represent St. error.

But this did not occur. But this did not occur. There was drop in cell There was drop in cell survival but no change in survival but no change in ‘atm’ expression (Fig.9,7)‘atm’ expression (Fig.9,7)

Fig. 9. Clonogenic cell survival assay in BMGI cells treated with 2-DG, 4 h before irradiation at 2 Gy and cells treated with 2 DG simultaneously with irradiation of 2 Gy.

No change in 2DG pretreated & No change in 2DG pretreated & simultaneously treated Gps in simultaneously treated Gps in ‘atm’ expression at any time point. ‘atm’ expression at any time point. However atm levels immediately However atm levels immediately after 1R went higher (approx 2 after 1R went higher (approx 2 times) than control (Fig.7).times) than control (Fig.7).

Fig No.7: Comparison of four-hour-2DG-pretreated-cells and cells treated with 2DG simultaneous to IR treatment. Total RNA was harvested after 0, 2 and 4 Hr after respective treatment.

Post-irradiation TreatmentPost-irradiation Treatment IR alone IR alone No change in No change in

‘atm’ expression (Fig.6).‘atm’ expression (Fig.6). IR 2Gy IR 2Gy 2DG 1 & 3h after IR 2DG 1 & 3h after IR

40% to 50% in ‘atm’ mRNA 40% to 50% in ‘atm’ mRNA (Fig.6). (Fig.6).

At 2h, much increase in At 2h, much increase in ‘atm’ expression ‘atm’ expression

IR 5Gy or 10Gy IR 5Gy or 10Gy 2DG 1,2 & 2DG 1,2 & 3h after IR. No change seen 3h after IR. No change seen except at 2h in 10Gy (Fig. 4 except at 2h in 10Gy (Fig. 4 &5).&5).

To study if post IR expression To study if post IR expression is dependent on pre IR is dependent on pre IR expression level.expression level.

2Gy IR with & without 2DG & 2Gy IR with & without 2DG & before or after IR expression. before or after IR expression.

Atm levels (Fig. 6) checked Atm levels (Fig. 6) checked 4h after IR.4h after IR.

Fig. 6. Data before 0 Hr is of pre-treatment and after 0 Hr is post-treatment group. Pre-treatment group includes two series (1) “2DG+IR” and (2) “2DG”. Cells were treated with 2DG; 1, 2 and 3 Hr before irradiation for “2DG+IR” series. For “2DG” series, cells were treated with 2DG at the same time as “2DG+IR” series, but were not irradiated. Total RNA was harvested from all the cells after 4 Hr of irradiation. Post-treatment group includes two series (1) “IR+2DG” and (2) “IR”. For “IR+2DG” series, cells were treated with 2DG at 1, 2 and 3 Hr after irradiation and total RNA was isolated at four hour after irradiation. For “IR” series total RNA was isolated after 1, 2, 3 and 4 Hr of irradiation without 2DG treatment.

In pre-treatment Gp ‘atm’ expression In pre-treatment Gp ‘atm’ expression reduced by 40% to 50% at -1 &0h (Fig.6).reduced by 40% to 50% at -1 &0h (Fig.6).

No diff in “2DG +IR” & “2DG” without IR No diff in “2DG +IR” & “2DG” without IR Gps at any time (Fig.6).Gps at any time (Fig.6).

Extent of down regulation in the Extent of down regulation in the pretreatment Gp is greater than post-pretreatment Gp is greater than post-treatment Gp. (Fig.6).treatment Gp. (Fig.6).

In post treatment Gp, ‘atm’ level was In post treatment Gp, ‘atm’ level was more than normal level in IR +2DG at 2h more than normal level in IR +2DG at 2h post IR (Fig.6).post IR (Fig.6).

TRANSLATIONAL STUDIES:TRANSLATIONAL STUDIES: At different time points At different time points

and at 2, 5 and 10 Gy and at 2, 5 and 10 Gy doses of radiation, there doses of radiation, there was no effect of 2-DG on was no effect of 2-DG on basal level of ‘atm’.basal level of ‘atm’.

1(a) At 2 Gy1(a) At 2 Gy At 2 Gy, there was 20% At 2 Gy, there was 20%

up regulation of ‘atm’ at up regulation of ‘atm’ at protein level. This was protein level. This was followed by down followed by down regulation till 6 h. In regulation till 6 h. In presence of radiation, 2-presence of radiation, 2-DG suppresses ‘atm’ DG suppresses ‘atm’ expression up to 10% expression up to 10% (Fig. 8).(Fig. 8).

Fig. 8. Effect of 2-DG on radiation induced ‘atm’ expression

1(b) At 5 Gy 1(b) At 5 Gy Similar to 2 Gy, 20% up regulation of ‘atm’ was Similar to 2 Gy, 20% up regulation of ‘atm’ was

found following radiation treatment. Level was found following radiation treatment. Level was consistent till 6 h. At all time point 2-DG consistent till 6 h. At all time point 2-DG suppresses the radiation induced ‘atm’ level up to suppresses the radiation induced ‘atm’ level up to basal level (Fig. 8).basal level (Fig. 8).

1(c) At 10 Gy 1(c) At 10 Gy After irradiation of 10 Gy, ‘atm’ up regulation was After irradiation of 10 Gy, ‘atm’ up regulation was

found starting from 0 h till 6 h with maximum at 2 found starting from 0 h till 6 h with maximum at 2 h that is 28%. 2 DG suppresses the ‘atm’ level h that is 28%. 2 DG suppresses the ‘atm’ level effectively up to basal level (Fig.8).effectively up to basal level (Fig.8).

CELL SURVIVAL STUDIESCELL SURVIVAL STUDIES Percent Survival was almost same for IR (2Gy) & 2DG + 2Gy Percent Survival was almost same for IR (2Gy) & 2DG + 2Gy

(Fig.9)(Fig.9)

Pretreated 2DG led to better survivality than simultaneous Pretreated 2DG led to better survivality than simultaneous treatmenttreatment

4h pretreatment of 2DG resulted in greater cell death due to IR4h pretreatment of 2DG resulted in greater cell death due to IR

Radiation damage are repaired through a sensitive & efficient Radiation damage are repaired through a sensitive & efficient mechanism of sensing, signal transduction, DNA repair and protein mechanism of sensing, signal transduction, DNA repair and protein trafficking.trafficking.

The Promoter of ‘atm’ gene is present 700bp upstream & is shared The Promoter of ‘atm’ gene is present 700bp upstream & is shared bi-directionally by ‘atm’ & ‘NPAT’ gene. bi-directionally by ‘atm’ & ‘NPAT’ gene.

("The cyclin E/Cdk2 substrate p220(("The cyclin E/Cdk2 substrate p220(NPATNPAT) is required for S-phase entry, ) is required for S-phase entry, histone histone genegeneexpression, and Cajal body maintenance in human somatic cells.". Mol. expression, and Cajal body maintenance in human somatic cells.". Mol. ...)...)

ATM sense break & guide repair, auto phosphorylate & up regulate ATM sense break & guide repair, auto phosphorylate & up regulate transcription. 2DG down regulate Sp1 transcription factor by hyper transcription. 2DG down regulate Sp1 transcription factor by hyper GlcNAcylation.GlcNAcylation.

Our findings support the report that 2DG down regulate ‘atm’.Our findings support the report that 2DG down regulate ‘atm’.

Mutation in putative binding site of Sp1, Mutation in putative binding site of Sp1, cre, ire z, Fse, GCF & Xre caused reduction cre, ire z, Fse, GCF & Xre caused reduction in promoter activity to less than basal in promoter activity to less than basal level.level.

‘‘Atm’ promoter can also be regulated Atm’ promoter can also be regulated independent of Sp1. This could explain independent of Sp1. This could explain the unexpected observation (Fig. 6)the unexpected observation (Fig. 6)

Strongest response to 4Gy IR is observed Strongest response to 4Gy IR is observed in peritoneal membrane, skin & spleen in peritoneal membrane, skin & spleen Maximum promoter activity was seen at Maximum promoter activity was seen at 8h after IR (ref.A)8h after IR (ref.A)

Our in vitro data reveal no change in ‘atm’ level to IR. Our in vitro data reveal no change in ‘atm’ level to IR. However 2DG causes down regulation to 30% to 40% with However 2DG causes down regulation to 30% to 40% with or without IR. This down regulate occur 4 to 5h after IR.or without IR. This down regulate occur 4 to 5h after IR.

Clonogenic cell survival of 2DG & also 2DG + IR (2Gy) Clonogenic cell survival of 2DG & also 2DG + IR (2Gy) treated BMGI cells revealed down regulation of ‘atm’ treated BMGI cells revealed down regulation of ‘atm’ causing decrease in % survival (20-90%) in a dose causing decrease in % survival (20-90%) in a dose

dependent manner.dependent manner.

Cells treated with 6mM 2DG 16h before IR, radio Cells treated with 6mM 2DG 16h before IR, radio sensitizer enhancement of 1.4 at 10% iso-survival was sensitizer enhancement of 1.4 at 10% iso-survival was observed (28) 5 & 10Gy has not made significant change observed (28) 5 & 10Gy has not made significant change in ‘atm’ expression. Only 2Gy & 2DG causes down in ‘atm’ expression. Only 2Gy & 2DG causes down regulation of ‘atm’ 2DG reported to enhance radiation regulation of ‘atm’ 2DG reported to enhance radiation induced cell death (13) in normal & in tumor cells.induced cell death (13) in normal & in tumor cells.

2 DG after IR of 5 or 10 Gy 2 DG after IR of 5 or 10 Gy No No significant alternation in ‘atm’ level (Fig.4 significant alternation in ‘atm’ level (Fig.4 & 5) [only 10-20 % lower ‘atm’ levels & 5) [only 10-20 % lower ‘atm’ levels indicate cell death at higher dose of IR] indicate cell death at higher dose of IR] (Fig.3)(Fig.3)

Cells incubated for 1h with 2 DG before IR Cells incubated for 1h with 2 DG before IR show ‘atm’ down regulations which is not show ‘atm’ down regulations which is not observed at 2 & 3 h (Fig.6).observed at 2 & 3 h (Fig.6).

This is probably the difference in time This is probably the difference in time required for 2DG to act on Glc N Acylation required for 2DG to act on Glc N Acylation of Sp1 & time of transcription regulation of Sp1 & time of transcription regulation of ‘atm’ promoter by Sp1.of ‘atm’ promoter by Sp1.

Pretreatment with 2DG impairs Sp1 Pretreatment with 2DG impairs Sp1 activity by not allowing it to act on activity by not allowing it to act on ‘atm’ promoter thereby causing down ‘atm’ promoter thereby causing down regulation of atm.regulation of atm.

Simultaneous treatment of 2DG & IR Simultaneous treatment of 2DG & IR causes 2DG to bind to Sp1 instead of causes 2DG to bind to Sp1 instead of ‘atm’ promoter.‘atm’ promoter.

However higher expression in However higher expression in IR+2DG at 2h after IR (Fig 6) needs IR+2DG at 2h after IR (Fig 6) needs further investigation.further investigation.

ConclusionConclusion

IR does not up regulate ‘atm’ in BMG1 cells IR does not up regulate ‘atm’ in BMG1 cells but 2DG treatment in presence or in but 2DG treatment in presence or in absence of IR causes down regulation of absence of IR causes down regulation of ‘atm’ & affect cell survival. It will be useful ‘atm’ & affect cell survival. It will be useful in optimization of 2DG associated radio in optimization of 2DG associated radio therapy of cancer. therapy of cancer.

AcknowledgementAcknowledgement

Dr. B. S. DwarakanathDr. B. S. Dwarakanath Dr. S. ChandnaDr. S. Chandna Dr. R. P. TripathiDr. R. P. Tripathi

Cytosol

ECM

Kim & Dang TIBS 2005

Multiple roles of glycolytic enzymes

Certain enzymes in the glycolytic pathway function as transcriptional regulators (LDH; ENO1; GAPD etc)

Others functions in cytoplasm and organelle (for ex HK)

Glycolysis a target for altering therapeutic response

Targeting glucose metabolism for

therapeutic gain

Huang 2006

Jain & Pohlit; 1973; Dwarakanath et al 1999

Treatment of leukemia and pancreatic tumor patients with varying i.v doses of 2-DG as a chemotherapeutic agent; Landau et al, 1958; Thomas & Duthie, 1968

Clinical studies

Hyperglycemia Fatty acid mobilization from epinephrine releaseNeurohormonal disturbances & Marked diaphoresis

Enhanced glucose metabolism as a therapeutic target

Discontinuation of clinical trials with 2-DG as primary therapeutic agent

Prospects for 2-DG as an adjuvant ? ?

Experimental studies

Inhibition of proliferation and cell death in several tumor cell linesGrowth delay in murine and rodent tumors