Indian J.ournal .of Experimental Bi.ol.ogy V.ol. 37, September 1999, pp. 865-870

Optimization of tumour radiotherapy: Part V-Radiosensitization by 2-deoxy-D-glucose and DNA ligand Hoechst-33342 in a murine tumour

B S Dwarakanath, Surinder Singh & Viney Jain*

Department.of Bi.ocybernetics, Institute.of Nuclear Medicine and Allied Sciences, Luckn.ow Marg, Delhi 110054

Received 2 December 1998; revised 26 May 1999

Radi.osenstitizing effects .of c.ombinati.on .of a min.or gro.ove DNA ligand, H.oechst-33342, with the glucose anal.ogue and inhibit.or.of glyc.olysis, 2-de.oxy-D-gluc.ose (2-DG) have been investigated in Ehrlich ascites tum.our (EAT) bearing mice f.oll.owing f.ocal irradiati.on .of the tum.our with 60C.o gamma-rays. Treatment-induced tumour gr.owth delay and tumour free animal survival were evaluated as parameters .of radiati.on resp.onse. F.ocal irradiati.on .of the tumour with a si ngle fraction .of 10 Gy induced a moderate delay in tum.our growth but did n.ot lead t.o complete regressi.on in any of the tumours. Intrave­n.ous administrati.on .of H-342 I hr bef.ore irradiati.on enhanced radiation-induced growth delay in a d.ose dependent manncr. Complete regressi.on .of the tum.our was .observed .only at a d.ose .of 10 mg/kg b.ody wt, leading t.o a cure (tumour free sur~ vival f.or m.ore than 100 days) rate .of 55%. Administrati.on .of 2-DG (2 g/kg b.ody wt; iv), immediately before irradiation sig­nificantly enhanced radiation-induced growth delay and resulted in a cure rate .of 45%. In c.ombinati.on with this dose of 2-DG (2 g/kg b.ody wt), H-342 at a l.ower d.ose (5 mg/kg bbdy wt) signiticantly enhanced the cure rate t.o 66%. H-342 or 2-DG given al.one.or in c.ombinati.on at the d.oses investigated here did n.ot sh.ow any significant effects .on the unirradiated tumour.

The success of conventional procedures presently employed in tumour radiotherapy is limited by (a) the presence of intrinsically radioresistant and repair pro­ficient subpopulations of cancer cells and (b) mor­bidity due to damage to the normal tissues at higher therapeutic doses1 ,2. Therefore, approaches that en­hance the induction of radiation damage and/or in­hibit repair processes differentially in cancer cells should improve the efficacy of tumour radiotherapy. Induction as well as repair of DNA lesions, consid­ered to be most important factors in cellular response to injury caused by ionizing radiation3.4, can be influ­enced by modulation of several physico-chemical as well as biological parameters such as DNA and h · S 6 . . 7

C romatm structure" , tissue oxygenatIOn, presence of antioxidants8 as well as optimal flow of metabolic

9- 11 energy . Our earlier work based on the energy linked modi­

fication of radiation damage, has shown that the glu­cose analogue and glycolytic inhibitor 2-deoxy-D­glucose (2-DG) when co-administered with radiation, selectively inhibits the post-irradiation repair proc­esses in cancer cells" ·12, thereby enhancing the radia-

*Present address .of corresp.ondent auth.or: Dr. B. R. Ambedkar Center f.or Biomedical Research, University of Delhi, Delhi 110007 Fax: 91-11-7257730

tion-induced cytogenetic damage arId cell death ,}·' }- :7.

Under similar conditions, 2-DG protects the normal cells I 8

.19, by reducing the fixation processes '. 2-DG

induced inhibition of strand break rejoining'4 :!o, un­scheduled DNA synthesis (repair synthesis)1.1 621 and the repair of potentially lethal damage (PLDR)11.21 have been demonstrated in cancer cells. Pre-clinical studies in mice have also shown that, administration of 2-DG just before irradiation leads to the sensitiza­tion of tumours, resulting in growth delay as well as enhancement of animal survivaI22.21 . Phase I clinical trials with 2-DG in human cerebral g lioma patients have shown that the combined treatment is well toler­ated with minimal late radiation damage to the brain24

.

The DNA ligands, bisbenzimidazole Hoechst 33258 (H-258) and its analogue Hoechst 33342 (H-342) bind selectively in AT rich regions of the DNA minor groove25 and have been shown to protect DNA against radiation damage in aqueous solutions26

.17 and

cells28.29

. H-342, has higher lipophilicity due to the ethoxy substitution on the 4-phenyl ring10 and is, therefore, more permeable across cell membrane as compared to H-258. Recently , we observed that, both H-342 and H-258 could provide significant protection against whole body irradiation in mice at non-toxic doses 31. Considerable reduction in the damage to the haemopoeitic system (cytogenetic damage in the bone

866 INDIAN J EXP BIOL, SEPTEMBER 1999

marrow cells) and longer animal survival were ob­served at sublethal as well as lethal doses of radia­tion' l. Furthermore, the combination of H-342 and 2-DG provided a significantly higher degree of protec­tion as compared to the effects of these agents ad­ministered alone' l.

In vivo studies in tumour bearing mice have shown that the intravenously admjnistered H-342 binds largely to the nuclear DNA in euoxic cells and iess to the hypoxic tumour cells" , probably due to diffusion limited accessibility of H-342 in hypox ic regions. Possibly therefore, the hypoxic tumour cells would not be protected to a significant extent by H-342. Hence, the present studies were undertaken to inves­tigate the radiomodifying effects of the combination of H-342 with 2-DG in tumour bearing mice. Radia­tion-induced growth delay, tumour regression and animal survival were investigated as end points.

Materials and Methods Chemicals-Hoechst-33342 (H-342) and 2-deoxy­

D-glucose (2-DG) were obtained from Sigma Chemi­cal Company (USA) and used without any further purification . All other chemicals were of analytical grade and obtained from E-Merck, India or Qualigens (Glaxo), India

Mice and tumour transplants-The inbred Swiss strain 'A' male mice (10-12 weeks) used in these

. studies were obtained from the Institutes' central animal facility and weighed 20-25 g at the time of tumour implantation. They were provided with water and standard mouse food (Liptin, India) ad libitum. The Ehrlich ascites tumour (EAT) cells (strain F-3) obtained from Institute for Biophysics, University of Frankfurt, Germany were maintained by serial pas­sage of tumour cell suspension in the peritoneal cav­ity of the mice l4. All experiments were conducted according to the guidelines for "Care and use of ani­mals in scientific research", established by Indian National Science Academy (INSA).

Subcutaneous tumours were grown by injecting ]5x106 cells (in 0.]-0.15 ml volume) into the flank region of right hind leg. Tumour volume was calcu­lated using the formula: V = Tt/6 (d l x d2 x d,), where d), d2 and d, are the three orthogonal diameters meas­ured with the help of a calipers. Experiments, were performed when the tumours had attained an average diameter of ] ± 0.] cm resulting in a volume of 0.5-0.6 cm' (6-7 days after implantation).

Administration of drugs-Tumour bearing mice were held in restrainers and H-342 as well as 2-DG was injected in the tail vein. The injection volumes of solutions of these chemicals prepared in normal saline were 0.1-0.15 ml. Unless menti oned otherwi se, H-342 (0.5-10.0 mg/kg body wt) was always admin­istered I hr before irradiation, while 2-DG ( 1-2 g/kg body wt) was administered immediately before irra­diation .

Tumour irradiation and fo llow-up-Mice were held on a Styrofoam platform and restrained by adhe-. T . d' d . flOC I slve tapes. umours were Irra tate uSing 0 te e-

therapy unit (Eldorado, AECL, Canada), by posi­tioning them in a field size of 2 x 2 em" ac hieved with the help of appropriate beam shapers at a tumour (sample) to source distance (ssd) of 80 cm. A total dose of 10 Gy was delivered at a dose rate of - 0.4 Gy/rnin. Ten to twenty animals were recruited into each group in these studies.

Tumour volumes were measured on alternate days. Animals with complete tumour regress ion were ob­served for their general condition including body weight till death.

Statistical analysis-Significance of differences in the mean values of tumour doubling time, td and per­centages of cure rates were evaluated by standard statistical methods (Student' s t 'test and Lawshe­Baker nomograph, respectively).

Results Modification of radiation response of Ehrlich ascites tumour by H-342

Tumour growth dela~The initia l growth of tu­mours in untreated control s was Gompertzian in na­ture. Administration of H-342 (0 .5-10.0 mg Ikg body wt) did not cause any significant effect on the tumour growth (Fig. I). The average initi al doubling times (td), were comparable to the values in untreated tu·· mours (4.5- 5.0 days) even at higher doses (5 and )I[)

mg/kg body wt) of H-342 Irradiation of the tumour with Co-60 gamma-rays

at an absorbed dose of lOGy (single fraction) in­duced only a transient delay in the growth (8-10 days; Fig. I) with the mean value of td increasing to nearly ]4 days. This absorbed dose, however, did not lead to a complete regression of the tumour in any of the irradiated mice (Table I). Administration of H-342 I hr before irradiation did not significantly alter the tumour growth up to a dose of I mg/kg body wt bur delayed the growth in a dose dependent manner at

OWARAKANATH et al.: OPTIMIZATION OF TUMOUR RADIOTHERAPY 867

higher doses (Fig. 2) . Complete tumour regression was observed only at the highest dose of H-342 used (10 mg/kg body wt) which induced nearly 55 % of the tumours to regress completely.

-Con ... Rad .

10 +H-342+2-DG+Rad. *H-342+Rad .

-C')

E u -Q.)

E ::J

~ ... 50.1 E ::J I-

+2-DG+Rad.

0.01 _ .......................... ________ ~~I-II

o 10 20 30 40 50 60

Post-irradiation time (days)

Fig I-Effects of 2-0G (2 glkg body wt) administered immedi­ately before irradiation and H-342 (5 mg/kg body wt) adminis­tered I h before irradiation on the growth of Ehrlich ascites tu­mour in mice, following focal irradiation of the tumour with (oIICo gamma-rays (lOGy). Symbols: (e ) control ; ("') gamma rays ; (* ) H-342 + gamma rays; (_ ) 2-DG + gamma rays and (+ ) H-342 + 2-0G + gamma rays. Mean values (± I SO) from 10-15 animals are presented. Initial tumour volumes were between 0.5 and 0.6 cmJ

.

Survival : Kaplan-Meier plots of actuarial survival are presented in Fig. 3. The median survival of un­treated or sham irradiated mice bearing the tumour was 32 days from the day of treatment ( I week after implantation) and all the animals died within 40 days. Focal irradiation of the tumour ( lOGy) enhanced the survival by only 10 days, the med ian survival bei ng 42 days (Table I ) . Administration of H-342 before irradiation did not alter the survi val up to a dose of 2 mg/kg body wt (data not shown). However, a dose of 5 mg/kg body wt marginally enhanced the survival (Fig. 3), the median survival being 50 days. Interest­ingly, increasing the dose to 10 mg/kg body wt re­sulted in a signific~nt enhancement of the survi val (median survival = 144 days) .

Radiosensitization of EAT by a combination of H-342 alld 2-DG

The growth of unirradiated tumours was not sig­nificantly altered by the administration of 2-DG (2 g/kg body wt) alone or in combination wi th H-342.

Administration of 2-DG (2 g/kg body wt) just be­fore irradiation delayed significant ly the growth of the tumour (Fig. 1) as compared to radiation alone, the mean value of td being 45 days. 2-DG in combi­nation with radiation also induced complete tumour regressions in nearly 45 % of the mice (P<O.OOI ;Table I). Administration of H-342 along with 2-DG before irradiation, did not significantly alter the growth de-

Table I--Effects of H-342 and 2-0G on the response of Ehrlich ascites tumour bearing mice foll owi ng focal irradiation «(oIICo gamma-rays, lOGy) of the tumour.

No. of animals Tumour doubling time Median survival Cure" Treatment (n) (days) (10) time (days) (%)

Gamma-rays H-33342 2-DG (Gy) (mglkg body (glkg body

wt) wt)

0 0 0 12 4.2±0.3 32 0 0 \0 0 \0 4.6±0.5 33 0 0 0 2 14 4.8±0.2 3 1 0 0 \0 2 II 4.6 +0.3 32 0

IO 0 0 14 14.0'; O.4h 42 0 \0 0 2 J3 45.0:; 2.5 h.c 142 45 \0 5 0 \0 23.0:; 1.0 h.c 50 0 IO 5 2 15 44.3 :; 3.2 h.c 14R 66d

IO IO 0 15 36.0:; 0.7 h,c 145 55 10 IO 2 15 43 .0 ± 4.0 h.c 144 55

• Cure is defined as tumour free survival for more than 100 days h Significantly different than unirradiated tumour (P<O.OOI) c Significantly different than irradiated tumour (P<O.OOI) d Significantly different than irradiated tumour treated with 2-DG (P<O.05)

868 INDIAN J EXP BIOL, SEPTEMBER 1999

U) 50 >. III :3. 40 Q)

:§ 30

Cl .!: ::c ::::I

20 a "0

10 :; a E 0 ::::I I- 0 :2 4 6 B 10

Hoechst-33342 dose (mg/kg b.w)

Fig 2-Effects of H-342 on gamma-ray (lOGy) induced growth delay of Ehrlich ascites tumour in mice. Mean values (± SO) from the measurements in 10-15 animals are presented.

lay or the median survival time of mice any further. Interestingly however, the combination at a H-342 dose of 5 mg/kg body weight enhanced the cure rate to 66% as against 45% (P<0.05) observed in mice treated with 2-DG plus radiation, whereas, at a higher dose of H-342 (10 mg/kg body wt) additi ve effects due to 2-DG were not observed (Table I) .

Discussion Contrary to expectations, the results of the present

studies clearly demonstrate the radiosensitizing ef­fects of the DNA ligand H-342 in the Ehrlich ascites tumour irradiated in vivo. Present studies have a.lso shown that the combinat ion of H-342 (5 mg/kg body wt) with the glucose analogue, 2-DG could be more effective in enhancing the tumour cure rate (Table I). Since the combination (H-342 + 2-DG) has been ear­lier shown to effectively protect normal tissues (like the bone marrow) against radiation damage31

, there­fore, this combination of radiomodifiers is expected to produce a large differential effect between the normal and tumour tissues. Present results could have important implications for optimizing tumour radio­therapy, since the doses at which these agents demon­strate the tumour radiosensitizing effects are neither toxic nor mutagenic42

. The two approaches used here for radiosensitization, namely modification of energy flow (using 2-DG) and information processing (using H-342) provided cure rates (tumor free survival of> 100 days) of more than 60% in combination with ra­diation. The fact, that the animals after therapy sur­vived for more than a year indicates the absence of any undesirable side effects.

The delay in tumour growth induced by H-342 (Figs. I & 2) and H-342+2-DG in combination with radiation, could arise on account of cytostat ic as well

liean. . &~.(10Gy) ""~342(5mG)+~ . • 2.oG(2g)+~ _H-342+2.aG+_

120

~ 100

(ij 80 LI~ ~ ~

.~ ~ ~ 80 en -----40

20

o ~ o 50 100 150 200 250 300

Post-irradiation time (days)

Fig 3-Effects of H-342 (5 mg/kg body w t) and 2-0G (2 g/kg body wt) on the survival of EAT bearing mice (n =10-15) fo l­lowing focal irradi ation (10 Gy) of the tumour. Symbols : (e )

control; (A ) gamma rays; (* ) H- 342 + gamma rays: (_ ) 2-DG + gamma rays and (+ ) H-342 + 2-0G + gamma rays.

as cytotoxic effects of these treatments. Both H-342 and 2-DG are known to induce concentration depend­ent division delay in un-irradiated as well as irradi­ated cells ill vitro3.33.34. However, both these agents (alone or in combination) did not induce any signifi­cant growth delay in unirradiated tumours (Table I ).

A combination of gamma irrad iation with H-342 at a higher dose (10 mg/kg body wt) resulted in a com­plete regression of the tumour in 55 % of the animals. However, together with 2-DG, the comb ination at thi s dose did not significantly increase e ither the median survival or the cure rate, although an increase in tel was observed . The data on tumour doubling time do not correlate strictly with survival or the cure rate (Table 1) . Since disease free survi al is the ultimate aim of any cancer treatment, careful and ethicall y permissible observations on animal survival in ex­perimental oncology studies are considered essential to develop and evaluate new therapeutic approaches. Therefore, animals showing complete tumour regres­sion are being followed till death .

The mechanisms underlying radiomodifying action of H-342 are complex and as yet 110t c learly under­stood. However, their ability to scavenge OH radicals as well as quench DNA radicals cou ld be important as shown in simple in vitro systems26.27 .3S.16 III vivo , H-342 and H-258 could further interfe re in the cata­lytic action of topoisomerases, important enzymes involved in a number of DNA tran !>actions including DNA repair37

.38

. Camptothecin and etoposide, in­hibitors of topoisomerase I and II, have been shown to potentiate the cytotoxic effect s of radiation 39

.

DWARAKANATH eta/.: OPTIMIZATION OF TUMOUR RADIOTHERAPY 869

Binding of these ligands could also alter the degree of chromatin condensation40, which may modify the re­pair as well as fixation of DNA lesions. Since these processes are highly dependent on the concentration of H-342, dose dependent modifications in tumour radioresponse are likely.

Further, it has also been demonstrated that the dis­sociation of DNA bound Hoechst and topoisomerase activities (particularly to po II) are energy depend­ent41

.42. Available data support the hypothesis that the effects of the glucose antimetabolite, 2-DG, could be mediated through the energy linked modification of the repair and fixation of radiation-induced le­sionsJ.II ·16. Therefore, it appears that the radiomodi­fying actions of H-342 and 2-DG could be inter­related and may involve some common sites and pathways.

An important consideration, while using agents that interact with DNA, is mutagenicity, besides any acute toxicity. Induction of 6-thioguanine resistant mutants by H-342 have been demonstrated in cell cultures (V 79 cells), albeit at high concentrations (> 50 f1M)33. The maximum dose of H-342 that demon­strated its radiosensitizing effects (10 mg/kg body wt) is considerably below the LD50 dose in mice (300 mg/kg body wt)43. Preliminary experiments on the induction of cytogenetic damage in bone marrow cells of mice indicate that, even the higher doses of H-342 (5 and 10 mg/kg body wt) used in the present work induced only a transient increase in the micro­nuc1ei formation from 0.2 % (controls) to 0.5% and 0.8%, respectively, which is not very alarming.

In conclusion, present studies suggest that the combination of H-342 and 2-DG could be useful in improving the efficacy of tumour radiotherapy. Therefore, further pre-clinical investigations and studies to understand the underlying mechanisms re­sponsible for the tumour radiosensitizing effects of the combination of H-342 with 2-DG are warranted.

Acknowledgement This work has been carried out under the project

INM-280.

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