EFFECT OF RADIATION C O M B I N E D W I T H HYPERTHERMIA ON H U M A N PROSTATIC CARCINOMA CELL LINES IN CULTURE ISSAC KAVER, M.D. JOY L. WARE, PH.D. JOHN D. WILSON, Ph.D. WARREN W KOONTZ, JR., M.D.
From the Division of Urology, Departments of Surgery, Pathology, and Radiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
ABSTRACT--The effect of radiation combined with heat on three human prostatic carcinoma cell lines growing in vitro was investigated. Cells were exposed to different radiation doses/followed by heat treatment at 43 °C /for one hour. Heat treatment, given ten minutes after radiation, significantly enhanced the radiation response of all the cell lines studied. The combined effect of radiation and heat produced greater cytotoxicity than predicted/from the additive effects o/f the two individual treatment modalities alone. These results indicate that a combined treatment regimen of radiation plus hyperthermia (43 °, 1 hr) might be an important tool in maintaining a better local control of prostatic cancer.
Over the last decade, interest in the application of radiation therapy combined with heat (hyperthermia) for patients with localized advanced carcinoma has increased. 1-3 The reasons for this are threefold: (1) in a solid tumor, the presence of hypoxic clonogenic cells may render the tumor radioresistant; yet these cells may be heat sensitive4; (2) nutritionally deprived ceils and cells in an acidic environment are more heat sensitiveS; and (3) under certain conditions heat can act as a radiosensitizing agent. 6 Despite recent improvement in surgical techniques with the preservation of sexual potency in radical prostatectomy, 7 there is still controversy regarding the optimal m a n a g e m e n t for localized prostatic cancer. 8,9 Furthermore, in elderly, poor-risk surgical patients, it would be preferable to avoid radical surgery. Thus definitive radiation therapy remains an alternative 88
curative modality for local tumor such cases. 1° Recently, several report~, vocated the application of hyperther and in combination with radiation foI ment of localized prostatic cancer, n-I We have previously documented th sensitivity of various established hm tatic carcinoma cell lines in vitro. 14Tt of this study was to evaluate in vitrc bined effect of radiation and heat on lines. Material and Methods
Cell lines H u m a n prostatic carcinoma cell ] grown in monolayer cultures in RPM] dium supplemented with 10 percent tivated calf serum and gentamicin (~ Three established cell lines, DU-145, TTRNT N/-'-V
/
TTTTV 1QCil
/
V~gT TTTkAf'I~ Y Y Y V T T T
K['IT~JJJ~J
~N), were used in this inves[45 and PC-3 lines were reLstatic lesions in brain (DUPC-3) 16 from two different •ed from prostatic cancer. 1'C-3, was recovered from a I node metastasis occurring ~aring a PC-3 tumor.17 Cells rypsinization from exponenltures, c o u n t e d and their :d by try-pan blue dye exclu3ns in 5 m L aliquots of cell a concentration of 2 x 105 ,'ed in sterile 15 m L plastic r subsequent exposure to raalone, or radiation followed ~t
were done by immersion of containing 5 m L of cell suscells/mL) in a w a t e r bath ,- Group) maintained at conwithin + 0.1 °C accuracy. tion of the cell suspension l was achieved in about five ad this time differential was ring time. Cell suspensions at alone at 43 °C for time in:ty, ninety, and one h u n d r e d :radiated cells were exposed 3 heat t r e a t m e n t at 43 °C for s exposed to 37°C served as .f the cell suspension before g remained constant in the determination ~tion and/or h y p e r t h e r m i c ls were washed and counted teter. The cells were diluted , plated in 60 m m tissue cultaintained at 37°C in a huatmosphere for the assessr m i n g ability. At three-day
/
V O L U M E XXXVIII, N U M B E R 1
~"~U--
145
0 < n-" CD Z
>
1-LN
> re(/3
10
-~
ons, the 15 m L centrifuge e cells were exposed at room ~00 Ci cesium-137 irradiator epherd and Associates) at a /rain. For samples that were diation heating, the interval f the radiation exposure and ,~heat t r e a t m e n t was fixed at
P91
6-4 70
0
I
1
I
I
30
60
90
120
150
TIME AT 43°C, MIN FIGURE 1. Cell survival curves of three human prostate tumor cell lines heated at 43 °C for different periods of time as indicated. Survival curves were fitted to data as described in text (Material and Methods). Plotted points are average values from three repeated experiments. Vertical bars are standard errors. intervals, 1-2 m L of fresh culture medium w e r e added to all dishes. The colonies were fixed on day 10 with 50 % methanol and stained with 0.2% methylene blue for thirty minutes, colonies containing m o r e t h a n fifty ceils w e r e scored as positive. Analysis of dose-response data f r o m colony f o r m a t i o n assays For each cell line the surviving fraction for each t r e a t m e n t was calculated from the ratio of the n u m b e r of colonies formed by the treated and untreated (control) cells. In every experiment, three replicate dishes for each t r e a t m e n t were scored. Survival data from two to four replicate dose-response experiments involving radiation and/or heat were averaged and plotted in standard fashion on semi-log coordinate axes. Survival curves were fitted to the data using a one-hit, multitarget model with initial slope ~8,1° and nonlinear regression methods.
Results The cell survival curves for heat t r e a t m e n t alone at 43°C are shown in Figure 1 for the three prostate t u m o r cell lines studied. Exposure at 43°C for up to thirty minutes p r o d u c e d
89
z o
X> fldlO 2 ~ :::3 (/3
10 ~0
R+H
R+H
R+I"
A
B
10"0 2"00 300 ' 400 ' 5"00 600 700
160 260 360 460 560 660 700
0
1(30 260 360 460 560 600~--
RADIATION DOSE, cGy FIGURE 9.. Cell survival curves for (A) DU-145, (13) PC-3, and (C) 1-LN human prostate tumor cell which received radiation alone (solid symbols) or radiation followed ten minutes later by heating at 43 o, one hour (open symbols). Curves were fitted to data as described in text (Material and Methods) with c', for heat-treated cells being normalized to 1.0 to account for heat killing. Effect of one-hour heat treat: alone can be assessed from Figure 1. Vertical bars are standard errors.
a moderate eytotoxie response resulting in less than a 20 percent reduction in cell survival in all three cell lines. However, as the t r e a t m e n t time increased, survival c o n t i n u e d to decrease, with the effect being greater in PC-3 and 1-LN cells. After two hours of heating, t h e survival of DU-145 cells and both PC-3 and 1-LN cells h a d been reduced to approximately 50 percent a n d 20 percent of u n h e a t e d controls, respectively. Cell survival curves for radiation alone and radiation followed by exposure to 43 °C for one hour are shown in Figure 2 for each of the cell lines. The curves representing the c o m b i n e d modality treatments (R + H) were normalized to a surviving fraction of 1.0 by using the 43 °C, one-hour sample as the control. This eliminates the contribution to eytotoxieity of the heat exposure in the combined t r e a t m e n t and allows only the radiation responses under the two conditions to be analyzed. Thus, a comparison of each pair of curves obtained for a specific cell line illustrates the radiosensitizing effect of the thermal treatment for those cells. In all eases it can be seen that the addition of h e a t modified the radiation survival curves in t w o ways: it increased the terminal slopes and r e d u c e d the size of the initial shoulders. T h e latter modification was most p r o n o u n c e d in PC-3 cells w h e r e postirradiation exposure to 43°C for one hour totally eliminated the shoulder of the PC-3 radiation survival curve.
90
A standard m e t h o d of q u a n t i t a t i n g the radi tion sensitizing effect of h y p e r t h e r m i a illU trated in Figure 2 involves using the paired su v i v a l c u r v e s to c a l c u l a t e a therm e n h a n c e m e n t r a t i o (TER). T h e T E R is defim as the ratio of radiation doses required to pr duee a given level of d a m a g e w i t h o u t and wi the addition of heat. Table I shows the radiatk doses required to reduce the surviving fractic of prostate t u m o r cells to the arbitrarily ehos! levels of 0.5 and 0.1 in the presence and absen, of heat (43 °C, 1 hr) as well as the eorrespo~ ing T E R values. At the 0.1 survival level, Jt addition of heat has the effect of approximate doubling the effectiveness of the radiation dos i.e., the T E R value is about 2 for all three cg TABLE I. Thermal enhancement ratios (TE for human prostate tumor cell lines* Cell Line DU-145 PC-3 I-LN
Surviving Surviving 0 --Fraetion= 0.5 --Fraetion = R,cGyT R + H,cGy~ TER§ R,cGy R + H,cGy 358 273 289
178 79 126
2.0 3.5 2.3
710 534 510
370 240 271
*Values for radiation doses appearing in this table we tained from equations for fitted survival curves shown inJ 2. $R = dose of radiation alone resulting in 50 percent suJ *R + H = dose of radiation with subsequent heating (4! hr) resulting in 50 percent survival. §R/R + H.
UROLOGY
/
JULY1991
/
VOLUME XXXVII1, NUMBN,iI
ermal enhancement factor (TEF) an prostate tumor cell lines exposed f cesium-137 gamma radiation*
Surviving Fraction - ,y Radia- 200 cGy RadiaAlone tion With Heat~ TEF$ .79 .70 .69
0.42 0.16 0.23
1.9 4.4 3.0
ppearing in this table were obtained from the :ed survival curves s h o w n in Figure 2. ) cGy alone/survival after 200 cGy w i t h heat.
5 survival level, however, the elhe radiation t r e a t m e n t has been factor of 3.5 for the PC-3 cells omplete removal of the shoulder a survival curve by post-irradiathese cells. of examining the radiation senff the heat t r e a t m e n t revealed in c o m p a r e the levels of survival given radiation dose alone and o n of heat. T h e ratio of the t w o is called the t h e r m a l enhance?EF). In Table II, this type of m carried out at a radiation dose ose size representative of a single [ clinically in a standard multi:ion therapy regimen. Table II val levels achieved in each of the ed to 200 cGy of radiation alone ',43 °C, 1 hr) a n d the correspond;s. These data indicate that the ,t following exposure to 200 eGy [uction in survival to a level apse-half that obtained with 200 DU-145 cells ( T E F = 1.9). This :izing effect is i n t e r m e d i a t e for 1' = 3.0) and greatest for PC-3 their survival to approximately level achieved in the absence of 1.4). ~t to characterize the interaction id heat in bringing about the reth of prostate t u m o r cells in these ~pared the survival of each of the result of exposure to radiation ne, and the c o m b i n a t i o n of both u r e 3 shows the results of such an d out at the clinically relevant ~ls of 200 cGy a n d 43°C, one of the cell lines studied, the cyto)n of the c o m b i n e d treatments
LY 1991
/
V O L U M E XXXVIII, NUMBER 1
100-
[~ E~ lS23 mR
9080.
7060. 50.
X X X X X
r'-1 i i
LI
,0
5020. 10. 0
II
N ;4 N N
N DU-145
PC-3
43C, 1 HR 2 0 0 coy ADDITIVE COMBINED EFFECT OBSERVED COMBINED EFFECT
n I I
II II
i!
1- L N
FIGURE 3. Combined effect of radiation (200 cGy) and heat (43 °C, 1 hr) on survival of three human prostate tumor cell lines.. Survival following heat and radiation treatments alone are indicated by open and hatched bars, respectively. Additive combined effect calculated as products of separate heat and radiation effects assuming independent action and are indicated by cross-hatched bars. Actual responses of cells to combined treatments are indicated by solid bars. Standard errors are indicated. (solid bars) was significantly greater t h a n simple additivity based on the effects of the separate t r e a t m e n t s (eross-hatched bars). Comment Studies in vitro and in vivo have consistently shown t h a t applying h y p e r t h e r m i a before or after radiation resulted in a remarkable enhancem e n t of the radiation effect. Several i m p o r t a n t generalizations having application to the treatm e n t of cancers can be d r a w n from these studies4-a'20'21: (1) heat in the range of 4 1 - 4 5 ° C directly kills cells; (2) acidic and nutritionally deprived cells in general are m o r e heat sensitive t h a n their physiologically n o r m a l counterparts; (3) radioresistant cells are often heat sensitive; (4) heat can act as a radiosensitizing agent; and (5) the interaction of heat and radiation can p r o d u c e greater t h a n additive effects. To date, there has been little i n f o r m a t i o n concerning the use of h y p e r t h e r m i a in the t r e a t m e n t of urologic eancer. 1H3 T h e aim of this study was to explore a possible n e w therapeutic application of h y p e r t h e r m i a in the t r e a t m e n t of urologic cancer, w i t h the emphasis on examining the c o m b i n e d eytotoxic effect of radiation a n d heat on h u m a n prostatic cell lines. T h e results presented in this report clearly d e m o n s t r a t e a significant t h e r m o e n h a n c e m e n t of the radiation response of all three cell lines studied. H e a t - i n d u c e d modifications of radiation survival curves-involved the reduction of the shoulders as well as increases in the t e r m i n a l
91
slopes (Fig. 2). It can be seen from the TER analysis that the same degree of cell killing can be achieved with half the radiation dose when heat is added to the treatment protocol (Table
I). An important question relative to the clinical setting is what happens at a radiation dose representing a commonly used fraction size for conventional fractionated radiotherapy regimens, for example 200 cGy, when it is followed immediately by a one-hour heat treatment at 43°C. Figure 3 illustrates survival obtained with a single radiation dose of 200 cGy alone, hyperthermia alone (43 °C, 1 hr), and the combined treatments. By comparing the additive effect predicted from the individual treatments with the survival observed for the combined treatments, it is clear that radiation and heat interact in a synergistic way at these clinically relevant treatment levels. This is most pronounced in the PC-3 and 1-LN cell lines in which exposure to radiation and heat resulted in a level of survival one-third to one-fourth of that expected for simple additivity; a lesser effect was observed in the DU-145 cell line. Although the application of hyperthermia in urology is still in its infancy, 13,~2,23 in the last decade there has been considerable interest in combining hyperthermia with radiation or chemotherapy in the treatment of other malignant disease, superficial 2 as well as deep-seated tumors. T M We believe that in vitro studies of the combined effect of radiation and hyperthermia on human prostatic carcinoma cells, such as described in this report, are necessary precursors for the application of these modalities in vivo. We are currently extending this investigation to an appropriate animal tumor model to verify the in vitro data obtained in the present study. In conclusion, our in vitro data on the cytotoxic effect of radiation and heat on human prostatic carcinoma cells indicate that a combined treatment regimen may have implications for the clinic by achieving a more effective therapeutic response and a better local control in the treatment of prostatic cancer. Richmond, Virginia 23298
(DR. KAVER)
92
References 1. Horhack NB, et ah Advanced stage IIIB cancer of the eerv treatment by hyperthermia and radiation, Gyneeol Onco123: (1986). 2. Marmour JB, and Hahn GM: Combined radiation and h perthermia in superficial human tumors, Cancer 46:1986 (198~ 3. Sapoznik MD, et ah Regional hyperthermia in the tre~ ment of clinically advanced deep-seated malignancy. Results of pilot study employing an annular array applicator,. Int J Radi Oncol Biol Phys 10:775 (1984). 4. Tannock IF: Oxygen diffusion and the distribution of cell: lar radiosensitivity in tumors, Br J Radiol 45:515 (1972). 5. Kim SH, Kim JH, and Hahn EW: Enhanced killing of h poxie tumor cells by hyperthermia, Br J Radiol 48:872 (1975i 6. Kim SH, Kim JH, and Hahn EW: The radiosensitization hypoxic tumor cells by hyperthermia, Radiology 114:727 (197~ 7. Walsh PC, Lepor H, and Eggleston JC: Radical prostate tomy with preservation of sexual function: anatomical and path logical consideration, Prostate 4:473 (1983). 8. Paulson DF, et ah Radical surgery versus radiotherapy f adenocarcinoma of the prostate, J Urol 128:502 (1982). 9. Whitmore WF Jr: Irradiation and/or surgery. Some areas confrontation in urologic oneology, Am J Clin Oncol (CCT) 606 (1984). 10. Bagshaw MA: Radiation therapy for prostatic earcmom in Crawford ED, and Borden TA (eds): Genitourinary Cane Surgery, Philadelphia, Lea & Febiger Inc, 1982, pp 406-411. 11. Yerushalmi A, et ah Local hyperthermia for treatment carcinoma of the prostate; a preliminary report, Prostate 6: g!
(1984). 12. Servadio C, Leib Z, and Lev A: Diseases of the prosta treated by local microwave hyperthermia, Urology 30:97 (198~ 13. Szmigielski S, et al: Local microwave hyperthermia treatment of advanced prostatic adenocarcinoma, Urol Res 16~ (1988). 14. Kaver I, Ware JI~, and Koontz W W Jr: The effect ofh perthermia on human prostatic carcinoma cell lines: evaluation vitro, J Urol 141:1025 (1989). 15. Stone LR, et ah Isolation of a human prostatic carcin0n cell line (DU-145), Int J Cancer 21:274 (1978). 16. Kaighn ME, et ah Establishment and characterization 0i human prostatic cell line (PC-3), Invest Urol 17:16 (1979). ii 17. Ware JL, et ah Spontaneous metastasis of cells of the ,i man prostate carcinoma cell line PC-3 in athymie nude micei Urol 128:1064 (1982). 18. Fertil B, et ah In vitro radiosensitivity of six human C~ lines. A comparative study with different statistical models;, ti diat Res 82:297 (1980). 19. Barendsen GW: In Harris RJC (Ed): The Initial EffectsI Ionizing Radiation on Cells, London/New York, Academic Pre 1961, pp 183-194. 20. Ben-Hut E, Elkind MM, and Bronk BV: Therma enhanced radioresponse of cultured Chinese hamster cell: inh!1 tion of repair of sublethal damage and enhancement of lett~ damage, Radiat Res 58:38 (1974). 21. Overgaard J: Fractionated radiation and hypertherm!l Experimental and clinical studies, Cancer 48:1116 (1981). 22. Hall RR, Schade ROK, and Swinney J: Effect o f perthermia on bladder cancer, Br Med J 2:593 (1974). 23. Servadio C, and Leib Z: Hyperthermia in the treatme!l prostate cancer, Prostate 5:205 (1984). 24. Sugimach K, et ah Long term effects of hypertheri combined with chemotherapy and irradiation for the treat~ of patients with carcinoma of the esophagus, Surg Gynecol Ohi 167:319 (1988). '~
UROLOGY
/ JULY 1991
/
VOLUME XXXVIII, NUMB :1~
From the Division of Urology, Departments of Surgery, Pathology, and Radiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
ABSTRACT--The effect of radiation combined with heat on three human prostatic carcinoma cell lines growing in vitro was investigated. Cells were exposed to different radiation doses/followed by heat treatment at 43 °C /for one hour. Heat treatment, given ten minutes after radiation, significantly enhanced the radiation response of all the cell lines studied. The combined effect of radiation and heat produced greater cytotoxicity than predicted/from the additive effects o/f the two individual treatment modalities alone. These results indicate that a combined treatment regimen of radiation plus hyperthermia (43 °, 1 hr) might be an important tool in maintaining a better local control of prostatic cancer.
Over the last decade, interest in the application of radiation therapy combined with heat (hyperthermia) for patients with localized advanced carcinoma has increased. 1-3 The reasons for this are threefold: (1) in a solid tumor, the presence of hypoxic clonogenic cells may render the tumor radioresistant; yet these cells may be heat sensitive4; (2) nutritionally deprived ceils and cells in an acidic environment are more heat sensitiveS; and (3) under certain conditions heat can act as a radiosensitizing agent. 6 Despite recent improvement in surgical techniques with the preservation of sexual potency in radical prostatectomy, 7 there is still controversy regarding the optimal m a n a g e m e n t for localized prostatic cancer. 8,9 Furthermore, in elderly, poor-risk surgical patients, it would be preferable to avoid radical surgery. Thus definitive radiation therapy remains an alternative 88
curative modality for local tumor such cases. 1° Recently, several report~, vocated the application of hyperther and in combination with radiation foI ment of localized prostatic cancer, n-I We have previously documented th sensitivity of various established hm tatic carcinoma cell lines in vitro. 14Tt of this study was to evaluate in vitrc bined effect of radiation and heat on lines. Material and Methods
Cell lines H u m a n prostatic carcinoma cell ] grown in monolayer cultures in RPM] dium supplemented with 10 percent tivated calf serum and gentamicin (~ Three established cell lines, DU-145, TTRNT N/-'-V
/
TTTTV 1QCil
/
V~gT TTTkAf'I~ Y Y Y V T T T
K['IT~JJJ~J
~N), were used in this inves[45 and PC-3 lines were reLstatic lesions in brain (DUPC-3) 16 from two different •ed from prostatic cancer. 1'C-3, was recovered from a I node metastasis occurring ~aring a PC-3 tumor.17 Cells rypsinization from exponenltures, c o u n t e d and their :d by try-pan blue dye exclu3ns in 5 m L aliquots of cell a concentration of 2 x 105 ,'ed in sterile 15 m L plastic r subsequent exposure to raalone, or radiation followed ~t
were done by immersion of containing 5 m L of cell suscells/mL) in a w a t e r bath ,- Group) maintained at conwithin + 0.1 °C accuracy. tion of the cell suspension l was achieved in about five ad this time differential was ring time. Cell suspensions at alone at 43 °C for time in:ty, ninety, and one h u n d r e d :radiated cells were exposed 3 heat t r e a t m e n t at 43 °C for s exposed to 37°C served as .f the cell suspension before g remained constant in the determination ~tion and/or h y p e r t h e r m i c ls were washed and counted teter. The cells were diluted , plated in 60 m m tissue cultaintained at 37°C in a huatmosphere for the assessr m i n g ability. At three-day
/
V O L U M E XXXVIII, N U M B E R 1
~"~U--
145
0 < n-" CD Z
>
1-LN
> re(/3
10
-~
ons, the 15 m L centrifuge e cells were exposed at room ~00 Ci cesium-137 irradiator epherd and Associates) at a /rain. For samples that were diation heating, the interval f the radiation exposure and ,~heat t r e a t m e n t was fixed at
P91
6-4 70
0
I
1
I
I
30
60
90
120
150
TIME AT 43°C, MIN FIGURE 1. Cell survival curves of three human prostate tumor cell lines heated at 43 °C for different periods of time as indicated. Survival curves were fitted to data as described in text (Material and Methods). Plotted points are average values from three repeated experiments. Vertical bars are standard errors. intervals, 1-2 m L of fresh culture medium w e r e added to all dishes. The colonies were fixed on day 10 with 50 % methanol and stained with 0.2% methylene blue for thirty minutes, colonies containing m o r e t h a n fifty ceils w e r e scored as positive. Analysis of dose-response data f r o m colony f o r m a t i o n assays For each cell line the surviving fraction for each t r e a t m e n t was calculated from the ratio of the n u m b e r of colonies formed by the treated and untreated (control) cells. In every experiment, three replicate dishes for each t r e a t m e n t were scored. Survival data from two to four replicate dose-response experiments involving radiation and/or heat were averaged and plotted in standard fashion on semi-log coordinate axes. Survival curves were fitted to the data using a one-hit, multitarget model with initial slope ~8,1° and nonlinear regression methods.
Results The cell survival curves for heat t r e a t m e n t alone at 43°C are shown in Figure 1 for the three prostate t u m o r cell lines studied. Exposure at 43°C for up to thirty minutes p r o d u c e d
89
z o
X> fldlO 2 ~ :::3 (/3
10 ~0
R+H
R+H
R+I"
A
B
10"0 2"00 300 ' 400 ' 5"00 600 700
160 260 360 460 560 660 700
0
1(30 260 360 460 560 600~--
RADIATION DOSE, cGy FIGURE 9.. Cell survival curves for (A) DU-145, (13) PC-3, and (C) 1-LN human prostate tumor cell which received radiation alone (solid symbols) or radiation followed ten minutes later by heating at 43 o, one hour (open symbols). Curves were fitted to data as described in text (Material and Methods) with c', for heat-treated cells being normalized to 1.0 to account for heat killing. Effect of one-hour heat treat: alone can be assessed from Figure 1. Vertical bars are standard errors.
a moderate eytotoxie response resulting in less than a 20 percent reduction in cell survival in all three cell lines. However, as the t r e a t m e n t time increased, survival c o n t i n u e d to decrease, with the effect being greater in PC-3 and 1-LN cells. After two hours of heating, t h e survival of DU-145 cells and both PC-3 and 1-LN cells h a d been reduced to approximately 50 percent a n d 20 percent of u n h e a t e d controls, respectively. Cell survival curves for radiation alone and radiation followed by exposure to 43 °C for one hour are shown in Figure 2 for each of the cell lines. The curves representing the c o m b i n e d modality treatments (R + H) were normalized to a surviving fraction of 1.0 by using the 43 °C, one-hour sample as the control. This eliminates the contribution to eytotoxieity of the heat exposure in the combined t r e a t m e n t and allows only the radiation responses under the two conditions to be analyzed. Thus, a comparison of each pair of curves obtained for a specific cell line illustrates the radiosensitizing effect of the thermal treatment for those cells. In all eases it can be seen that the addition of h e a t modified the radiation survival curves in t w o ways: it increased the terminal slopes and r e d u c e d the size of the initial shoulders. T h e latter modification was most p r o n o u n c e d in PC-3 cells w h e r e postirradiation exposure to 43°C for one hour totally eliminated the shoulder of the PC-3 radiation survival curve.
90
A standard m e t h o d of q u a n t i t a t i n g the radi tion sensitizing effect of h y p e r t h e r m i a illU trated in Figure 2 involves using the paired su v i v a l c u r v e s to c a l c u l a t e a therm e n h a n c e m e n t r a t i o (TER). T h e T E R is defim as the ratio of radiation doses required to pr duee a given level of d a m a g e w i t h o u t and wi the addition of heat. Table I shows the radiatk doses required to reduce the surviving fractic of prostate t u m o r cells to the arbitrarily ehos! levels of 0.5 and 0.1 in the presence and absen, of heat (43 °C, 1 hr) as well as the eorrespo~ ing T E R values. At the 0.1 survival level, Jt addition of heat has the effect of approximate doubling the effectiveness of the radiation dos i.e., the T E R value is about 2 for all three cg TABLE I. Thermal enhancement ratios (TE for human prostate tumor cell lines* Cell Line DU-145 PC-3 I-LN
Surviving Surviving 0 --Fraetion= 0.5 --Fraetion = R,cGyT R + H,cGy~ TER§ R,cGy R + H,cGy 358 273 289
178 79 126
2.0 3.5 2.3
710 534 510
370 240 271
*Values for radiation doses appearing in this table we tained from equations for fitted survival curves shown inJ 2. $R = dose of radiation alone resulting in 50 percent suJ *R + H = dose of radiation with subsequent heating (4! hr) resulting in 50 percent survival. §R/R + H.
UROLOGY
/
JULY1991
/
VOLUME XXXVII1, NUMBN,iI
ermal enhancement factor (TEF) an prostate tumor cell lines exposed f cesium-137 gamma radiation*
Surviving Fraction - ,y Radia- 200 cGy RadiaAlone tion With Heat~ TEF$ .79 .70 .69
0.42 0.16 0.23
1.9 4.4 3.0
ppearing in this table were obtained from the :ed survival curves s h o w n in Figure 2. ) cGy alone/survival after 200 cGy w i t h heat.
5 survival level, however, the elhe radiation t r e a t m e n t has been factor of 3.5 for the PC-3 cells omplete removal of the shoulder a survival curve by post-irradiathese cells. of examining the radiation senff the heat t r e a t m e n t revealed in c o m p a r e the levels of survival given radiation dose alone and o n of heat. T h e ratio of the t w o is called the t h e r m a l enhance?EF). In Table II, this type of m carried out at a radiation dose ose size representative of a single [ clinically in a standard multi:ion therapy regimen. Table II val levels achieved in each of the ed to 200 cGy of radiation alone ',43 °C, 1 hr) a n d the correspond;s. These data indicate that the ,t following exposure to 200 eGy [uction in survival to a level apse-half that obtained with 200 DU-145 cells ( T E F = 1.9). This :izing effect is i n t e r m e d i a t e for 1' = 3.0) and greatest for PC-3 their survival to approximately level achieved in the absence of 1.4). ~t to characterize the interaction id heat in bringing about the reth of prostate t u m o r cells in these ~pared the survival of each of the result of exposure to radiation ne, and the c o m b i n a t i o n of both u r e 3 shows the results of such an d out at the clinically relevant ~ls of 200 cGy a n d 43°C, one of the cell lines studied, the cyto)n of the c o m b i n e d treatments
LY 1991
/
V O L U M E XXXVIII, NUMBER 1
100-
[~ E~ lS23 mR
9080.
7060. 50.
X X X X X
r'-1 i i
LI
,0
5020. 10. 0
II
N ;4 N N
N DU-145
PC-3
43C, 1 HR 2 0 0 coy ADDITIVE COMBINED EFFECT OBSERVED COMBINED EFFECT
n I I
II II
i!
1- L N
FIGURE 3. Combined effect of radiation (200 cGy) and heat (43 °C, 1 hr) on survival of three human prostate tumor cell lines.. Survival following heat and radiation treatments alone are indicated by open and hatched bars, respectively. Additive combined effect calculated as products of separate heat and radiation effects assuming independent action and are indicated by cross-hatched bars. Actual responses of cells to combined treatments are indicated by solid bars. Standard errors are indicated. (solid bars) was significantly greater t h a n simple additivity based on the effects of the separate t r e a t m e n t s (eross-hatched bars). Comment Studies in vitro and in vivo have consistently shown t h a t applying h y p e r t h e r m i a before or after radiation resulted in a remarkable enhancem e n t of the radiation effect. Several i m p o r t a n t generalizations having application to the treatm e n t of cancers can be d r a w n from these studies4-a'20'21: (1) heat in the range of 4 1 - 4 5 ° C directly kills cells; (2) acidic and nutritionally deprived cells in general are m o r e heat sensitive t h a n their physiologically n o r m a l counterparts; (3) radioresistant cells are often heat sensitive; (4) heat can act as a radiosensitizing agent; and (5) the interaction of heat and radiation can p r o d u c e greater t h a n additive effects. To date, there has been little i n f o r m a t i o n concerning the use of h y p e r t h e r m i a in the t r e a t m e n t of urologic eancer. 1H3 T h e aim of this study was to explore a possible n e w therapeutic application of h y p e r t h e r m i a in the t r e a t m e n t of urologic cancer, w i t h the emphasis on examining the c o m b i n e d eytotoxic effect of radiation a n d heat on h u m a n prostatic cell lines. T h e results presented in this report clearly d e m o n s t r a t e a significant t h e r m o e n h a n c e m e n t of the radiation response of all three cell lines studied. H e a t - i n d u c e d modifications of radiation survival curves-involved the reduction of the shoulders as well as increases in the t e r m i n a l
91
slopes (Fig. 2). It can be seen from the TER analysis that the same degree of cell killing can be achieved with half the radiation dose when heat is added to the treatment protocol (Table
I). An important question relative to the clinical setting is what happens at a radiation dose representing a commonly used fraction size for conventional fractionated radiotherapy regimens, for example 200 cGy, when it is followed immediately by a one-hour heat treatment at 43°C. Figure 3 illustrates survival obtained with a single radiation dose of 200 cGy alone, hyperthermia alone (43 °C, 1 hr), and the combined treatments. By comparing the additive effect predicted from the individual treatments with the survival observed for the combined treatments, it is clear that radiation and heat interact in a synergistic way at these clinically relevant treatment levels. This is most pronounced in the PC-3 and 1-LN cell lines in which exposure to radiation and heat resulted in a level of survival one-third to one-fourth of that expected for simple additivity; a lesser effect was observed in the DU-145 cell line. Although the application of hyperthermia in urology is still in its infancy, 13,~2,23 in the last decade there has been considerable interest in combining hyperthermia with radiation or chemotherapy in the treatment of other malignant disease, superficial 2 as well as deep-seated tumors. T M We believe that in vitro studies of the combined effect of radiation and hyperthermia on human prostatic carcinoma cells, such as described in this report, are necessary precursors for the application of these modalities in vivo. We are currently extending this investigation to an appropriate animal tumor model to verify the in vitro data obtained in the present study. In conclusion, our in vitro data on the cytotoxic effect of radiation and heat on human prostatic carcinoma cells indicate that a combined treatment regimen may have implications for the clinic by achieving a more effective therapeutic response and a better local control in the treatment of prostatic cancer. Richmond, Virginia 23298
(DR. KAVER)
92
References 1. Horhack NB, et ah Advanced stage IIIB cancer of the eerv treatment by hyperthermia and radiation, Gyneeol Onco123: (1986). 2. Marmour JB, and Hahn GM: Combined radiation and h perthermia in superficial human tumors, Cancer 46:1986 (198~ 3. Sapoznik MD, et ah Regional hyperthermia in the tre~ ment of clinically advanced deep-seated malignancy. Results of pilot study employing an annular array applicator,. Int J Radi Oncol Biol Phys 10:775 (1984). 4. Tannock IF: Oxygen diffusion and the distribution of cell: lar radiosensitivity in tumors, Br J Radiol 45:515 (1972). 5. Kim SH, Kim JH, and Hahn EW: Enhanced killing of h poxie tumor cells by hyperthermia, Br J Radiol 48:872 (1975i 6. Kim SH, Kim JH, and Hahn EW: The radiosensitization hypoxic tumor cells by hyperthermia, Radiology 114:727 (197~ 7. Walsh PC, Lepor H, and Eggleston JC: Radical prostate tomy with preservation of sexual function: anatomical and path logical consideration, Prostate 4:473 (1983). 8. Paulson DF, et ah Radical surgery versus radiotherapy f adenocarcinoma of the prostate, J Urol 128:502 (1982). 9. Whitmore WF Jr: Irradiation and/or surgery. Some areas confrontation in urologic oneology, Am J Clin Oncol (CCT) 606 (1984). 10. Bagshaw MA: Radiation therapy for prostatic earcmom in Crawford ED, and Borden TA (eds): Genitourinary Cane Surgery, Philadelphia, Lea & Febiger Inc, 1982, pp 406-411. 11. Yerushalmi A, et ah Local hyperthermia for treatment carcinoma of the prostate; a preliminary report, Prostate 6: g!
(1984). 12. Servadio C, Leib Z, and Lev A: Diseases of the prosta treated by local microwave hyperthermia, Urology 30:97 (198~ 13. Szmigielski S, et al: Local microwave hyperthermia treatment of advanced prostatic adenocarcinoma, Urol Res 16~ (1988). 14. Kaver I, Ware JI~, and Koontz W W Jr: The effect ofh perthermia on human prostatic carcinoma cell lines: evaluation vitro, J Urol 141:1025 (1989). 15. Stone LR, et ah Isolation of a human prostatic carcin0n cell line (DU-145), Int J Cancer 21:274 (1978). 16. Kaighn ME, et ah Establishment and characterization 0i human prostatic cell line (PC-3), Invest Urol 17:16 (1979). ii 17. Ware JL, et ah Spontaneous metastasis of cells of the ,i man prostate carcinoma cell line PC-3 in athymie nude micei Urol 128:1064 (1982). 18. Fertil B, et ah In vitro radiosensitivity of six human C~ lines. A comparative study with different statistical models;, ti diat Res 82:297 (1980). 19. Barendsen GW: In Harris RJC (Ed): The Initial EffectsI Ionizing Radiation on Cells, London/New York, Academic Pre 1961, pp 183-194. 20. Ben-Hut E, Elkind MM, and Bronk BV: Therma enhanced radioresponse of cultured Chinese hamster cell: inh!1 tion of repair of sublethal damage and enhancement of lett~ damage, Radiat Res 58:38 (1974). 21. Overgaard J: Fractionated radiation and hypertherm!l Experimental and clinical studies, Cancer 48:1116 (1981). 22. Hall RR, Schade ROK, and Swinney J: Effect o f perthermia on bladder cancer, Br Med J 2:593 (1974). 23. Servadio C, and Leib Z: Hyperthermia in the treatme!l prostate cancer, Prostate 5:205 (1984). 24. Sugimach K, et ah Long term effects of hypertheri combined with chemotherapy and irradiation for the treat~ of patients with carcinoma of the esophagus, Surg Gynecol Ohi 167:319 (1988). '~
UROLOGY
/ JULY 1991
/
VOLUME XXXVIII, NUMB :1~
