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International Journal of Hyperthermia

ISSN: 0265-6736 (Print) 1464-5157 (Online) Journal homepage: https://www.tandfonline.com/loi/ihyt20

Long-duration, mild whole body hyperthermia

with cisplatin: Tumour response and kinetics

of apoptosis and necrosis in a metastatic rat

mammary adenocarcinoma

N. Toyota, F. R. Strebel, L. C. Stephens, H. Matsuda & J. M. C. Bull

To cite this article: N. Toyota, F. R. Strebel, L. C. Stephens, H. Matsuda & J. M. C. Bull (1997) Long-duration, mild whole body hyperthermia with cisplatin: Tumour response and kinetics of apoptosis and necrosis in a metastatic rat mammary adenocarcinoma, International Journal of Hyperthermia, 13:5, 497-506, DOI: 10.3109/02656739709023548

To link to this article: https://doi.org/10.3109/02656739709023548

Published online: 09 Jul 2009.

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INT. J . HYPERTHERMIA, 1997, VOL. 13, NO. 5, 497-506

Long-duration, mild whole body hyperthermia with cisplatin: tumour

response and kinetics of apoptosis and necrosis in a metastatic rat

mammary adenocarcinoma

N. TOYOTAT, F. R. STREBELT, L. C . STEPHENS$, H. MATSUDAI.4, and J. M. C . BULL?*

?Division of Oncology, Department of Internal Medicine, The University of Texas Medical School at Houston, and $Section of Veterinary Pathology, M D Anderson Cancer Center, Houston, TX 77030, USA

(Received 30 September 1996; revised 24 January 1997; accepted 2 February 1997) This study examines antitumour effect and induction of apoptosis and necrosis after treatment with long-duration, mild whole body hyperthermia (LL-WBH, 4O.O0C, for 6 h) in simultaneous combination with cisplatin (CDDP) on primary and metastatic tumour growth in a rat mammary adenocarcinoma. A signifi- cantly greater delay in primary mammary tumour growth was observed after treatment with LL-WBH

+

CDDP, compared to either modality alone ( p < 0.05). LL-WBH alone caused a significant delay in spontaneous metastasis to the axillary lymph node (ALN) and LL-WBH

+

CDDP tended to further increase the delay in ALN metastasis. Survival was longest in rats receiving LL- WBH

+

CDDP, compared to other groups ( p < 0.05). CDDP induced a peak of tumour apoptosis at 24 h after treatment beginning that was significantly greater than LL-WBH alone ( p

<

0.05). The peak of tumour apoptosis induced by LL- WBH

+

CDDP from 12 to 24 h was significantly greater than any other group ( p < 0.01). These results suggest that the extent of treatment-induced apoptosis seems to correlate positively with antitumour response and the combination of LL-WBH with CDDP may lead to a promising adjuvant therapy for breast cancer.

Key words: Long duration, mild whole body hyperthermia, cisplatin, apoptosis, necrosis, primary and metastatic mammary tumour.

1. Introduction

Recent clinical studies suggest that hyperthermia in combination with radiation and/or chemotherapy is a very useful modality in the treatment of advanced cancer (Overgaard et al. 1995, Van der Zee et al. 1996). Mechanisms that explain how hyperthermia acts as an antitumour agent are being actively investigated, however, it is well accepted that hyperthermia can potentiate the cytotoxicity of many chemotherapeutic agents (Hahn 1982). CDDP is widely used in treating a variety of tumours and its cytotoxicity is enhanced by hyperthermia both in vitro and in vivo

(Barlogie et al. 1980, Baba et al. 1989). Previously we reported on the antitumour and normal tissue effects of short-duration, high-temperature whole body

*Correspondence to J. M. C. Bull, Division of Oncology, Department of Internal Medicine, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225, USA.

$Present address: Department of Surgery 11, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan.

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hyperthermia (SH-WBH, at 41.5"C for 2 h) combined simultaneously with a number of cytotoxic drugs, including CDDP and carboplatin (CBDCA) (Wondergem et al. 1988a, Ohno et al. 1991, 1992). Therapeutic gain was limited by SH-WBH enhance- ment of normal tissue toxicity. Recently the use of long-duration, mild hyperthermia has demonstrated efficacy in hyperthermia research. Armour et al. (199 1, 1992, 1993) reported that the cytotoxic effect of low dose rate irradiation was enhanced by long duration mild hyperthermia at 41.0°C, using rat 9L gliosarcoma cells, and that human tumour cells were more sensitive to long duration mild hyperthermia than rodent cells. We have also reported that LL-WBH (40.0"C for 6 h) combined with tumour necrosis factor Q (TNF) and CBDCA provided a greater therapeutic efficacy than SH-WBH (41-5°C for 2 h) in combination with TNF and CBDCA (Sakaguchi et al. 1994).

Apoptosis (programmed cell death) is a process distinguished from other modes of cell death (such as necrosis) on the basis of characteristic biochemical and mor- phological features. Apoptosis occurs in both physiological and pathological condi- tions and plays an important role in the regulation of tissue development (Alison and Sarraf 1992), and it is induced in normal tissue, cultured tumour cells, and solid tumours in response to therapeutic stimuli such as cytotoxic drugs and radiation (Dyson et al. 1986, Barry et al. 1990, Stephens et al. 1991, Myen et al. 1994). Hyperthermia has also been shown to activate the process of apoptosis in tumours (Takano et al. 1991, Moroi et al. 1996). However, the role of treatment-induced apoptosis and necrosis in relation to antitumour response is not yet fully understood. In this study we examined the primary and metastatic growth of a highly meta- static rat mammary adenocarcinoma (MTLn3) following treatment with long- duration, mild whole body hyperthermia (LL-WBH, 4O.O0C, for 6 h) alone, CDDP alone, and CDDP in simultaneous combination with LL-WBH. We also studied the kinetics of tumour apoptosis and necrosis induced by treatment in an attempt to investigate possible associations between these parameters of cell death and antitumour response.

2. Materials and methods 2.1. Animals

Experiments were performed using female Fischer 344 rats (Harlan Sprague- Dawley, Inc., Indianapolis, IN) with body weights ranging from 100 to 120 gm ( 6 7 weeks of age). Rats were fed standard laboratory chow, allowed free access to water, and housed under controlled conditions with a 12-h light/dark cycle. All rats were allowed a 1-week environmental adaptation period before their experimental use.

2.2. Tumour

Rats were inoculated with 5 x lo5 MTLn3 cells (tissue culture passage number 44) in 0.25 ml of alpha-modified minimum essential medium (aMEM, GibcoBRL Life Technologies, Inc., Grand Island,

NY),

subcutaneously under the left abdom- inal nipple in the region of mammary fat pad. Solid tumours were palpable at the inoculation site in 100% of the rats by day 6. Subsequently, the rats were treated when the tumour size reached 10&200 mm3.

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Efficacy and apoptosis of mild whole body hyperthermia 499 in 100% of animals by 24 days after implantation of tumour cells in the mammary fat pad (Matsuda et al. 1996).

2.3. Drug

CDDP (Platinol; Bristol-Myers Squibb Co., Princeton, N.J.) was dissolved in sterile H20 to a concentration of 1 mg/ml according to manufacturer's recommen- dation, and administered by i.v. bolus through the lateral tail vein at a dose of 4 mg/ kg [the maximum tolerated dose (MTD) in combination with LL-WBH]. Since circadian rhythm has been shown to affect CDDP activity (Hrushesky et al. 1982), CDDP was administered at set times (120CL1300 h) during the Iight/dark cycle. In animals receiving combined CDDP and LL-WBH, CDDP was administered simul- taneously with LL-WBH when the core rectal temperature first reached the target therapeutic temperature (40.0"C). In the control and LL-WBH alone groups, an equivalent volume of 0.9% NaCl (USP) was injected intravenously.

2.4. Whole body hyperthermia (WBH)

WBH was induced by immersing halothane anaesthetized rats into a thermo- statically controlled circulating water bath, with rectal temperatures being measured continuously and recorded every 5 min for all rats as previously described (Wondergem et al. 1988a, b), and the water bath temperature being maintained at 40.0 f 0.1"C. An average of 15 min was required for the rectal temperature to reach the therapeutic temperature of 40.0"C for LL-WBH. The rats were then maintained at 40.0"C for the 6 h duration of LL-WBH treatment. General inhalation anaesthesia of 1.5% halothane in pure oxygen as described previously (Wondergem et al. 1988) was used for all treatments. Similarly anaesthetized normothermic control rats were maintained at 37.0"C for 6 h, by placement on a thermostatically controlled water blanket (Blanketrol, Cincinnati Sub-Zero Products, Inc., Cincinnati, OH).

2.5. Treatment response

Tumour size was measured every 2 days by using a vernier caliper to determine three perpendicular dimensions (d), and the tumour volume (V) was calculated by using the formula V = (dl x d2 x d3)/2. At the same time, body weight was recorded and the left axillary region was palpated to determine the occurrence of metastasis to the axillary lymph node. In our previous study, histopathological examination showed MTLn3 metastasis in all axillary lymph nodes that were palpable (Matsuda et al. 1996). Primary tumour response, in terms of tumour growth delay (TGD), was determined by comparing the tumour growth time (TGT) required for the tumours in the various treatment groups to reach 10 x the treatment volume; TGD = TGTtreatme,, - TGTControl. Post-treatment survival time for each rat was also recorded.

2.6. Histopathological studies of apoptosis and necrosis

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1995, Sakaguchi et al. 1995, Matsuda et al. 1996). Briefly, five microscopic fields of non-necrotic areas were randomly selected in each specimen, and in each of these fields the number of apoptotic cells was recorded as the number per 100 nuclei and expressed as a percentage. Necrosis was scored on a graded scale of 0-+ 4 as follows: 0 = none, 1

+

= small areas of necrosis ( < 25%), 2

+

= confluent, central extensive necrosis (25-50%), 3

+

= as in 2

+

,

plus cystic necrosis (50-75%), and

4

+

= mostly necrotic tumour ( > 75%).

2.7. Statistics

differences in the data between each treatment group.

The Wilcoxon signed-rank test was used to determine statistical significance of

3. Results

3.1. Antitumour effect

Figure 1 shows the tumour growth curves for each treatment group. CDDP alone caused a significant antitumour response compared to control and LL-WBH alone from day 4 through 14 post-treatment ( p

<

0.05). A significant enhancement of antitumour activity from days 4 2 0 occurred when CDDP was combined with LL-WBH ( p

<

0.05 compared to any other group). This effect resulted in a signifi- cantly greater ( p

<

0.05) tumour growth delay (TGD) of 11.5

f

0.9 days with com- bined CDDP

+

LL-WBH, compared to 7.01k0.4 days for CDDP alone and

1.5 -f. 0.8 days for LL-WBH alone (Figure 2).

As a general measure of treatment-induced normal tissue toxicity, we examined body weight change every two days following treatment. As shown in Figure 3, the combined treatment of CDDP

+

LL-WBH caused significant body weight loss from days 2 to 18 after treatment compared to control and LL-WBH alone, and from days

100

I

0.1 I 1 I I I I I I I I

!

0 2 4 6 8 10 12 14 16 18 20

Time after treatment (days)

Figure 1 . Tumour growth curves following treatment: tumour size is presented as relative tumour volume compared to pretreatment size. 0; control, 0 ; LL-WBH alone,

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Efficacy and apoptosis of mild whole body hyperthermia 50 1

*

-+*

CDOP LL-WBH LL-WBH + cwp

Treatment groups

Figure 2. Tumour growth delay (TGD) induced by treatment: TGD was calculated from the tumour growth time (TGT) required for the tumours in the various treatment groups to reach 10 x the treatment volume. Data are mean f SEM of six rats per group. *; p < 0.01 compared to LL-WBH alone,

**;

p < 0.05 compared to CDDP alone.

0 c

P

0 >I w 0 m

Figure 3. Body weight change following treatment: Body weight is presented as % of pre- treatment weight.

0;

control, 0 ; LL-WBH alone,

A;

CDDP alone, V; LL-WBH

+

CDDP. Data are mean & SEM of six rats per group.

4

to 12 compared to CDDP alone ( p

<

0.05). Rats treated with CDDP alone showed a brief, early significant body weight loss from days 2 to

4,

compared to control and LL-WBH alone ( p

<

0.05).

3.2. Axillary lymph node ( A L N ) metastasis and survival

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Table 1. Axillary lymph node (ALN) metastasis and survival.

Group CDDP dose Mean time (days) to Mean survival (mg/kg) ALN metastasis? (days)

Control 0 9.0 f 1.8 20.0 k 2.3

CDDP 4 13.5 f 1.7 27.6 & 0.9$

LL-WBH 0 16.4 f 2.63 25.2 j~ 1.6$ LL-WBH

+

CDDP 4 21.6 f 1.7* 31.3 z t 1.88

?Time to ALN metastasis is the first day following treatment that a palpable axillary lymph node could be observed. Previous studies showed 100% of such palpable nodes to be invaded with metastatic MTLn3 cells.

$p

<

0.05 compared to control.

* p

<

0.01 compared to control and CDDP alone. $p < 0.05 compared to any other group.

Data are presented as mean f SEM of 6 rats per group.

ALN metastasis occurred with LL-WBH alone and LL-WBH

+

CDDP, compared to control and CDDP alone ( p

<

0.05). Although not statistically significant, the longest delay in development of ALN metastasis appeared to occur with LL-WBH

+

CDDP. Significantly increased survival was observed with CDDP alone and LL-WBH alone, compared to control. LL-WBH

+

CDDP caused the longest mean survival (31.3

f

1.8 days) compared to any other group ( p

<

0.05).

3.3. Tumour apoptosis and necrosis induced by treatment

The kinetics of tumour apoptosis and necrosis induced by treatment are shown in Figures 4 and 5, respectively. LL-WBH alone induced a mild peak of apoptosis

*O

I

0 12 24 36 48 60 7 2 04 96

Time after starting treatment (h)

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Efficacy and apoptosis of mild whole body hyperthermia 503

I I I 1 I I I I

0 12 24 36 48 60 72 84 96

Time after starting treatment

(h)

Figure 5. Kinetics of tumour necrosis induced by treatment: tumour necrosis was scored on a graded scale of 0 - 4 + (see Materials and methods). 0; control, 0 ; LL-WBH alone,

A;

CDDP alone,

7;

LL-WBH

+

CDDP. Data are mean f SEM of three rats per time point in each group.

(2.1

f

0.1%) at 12 h after the beginning of treatment that was significantly greater than control ( < 1 %) ( p

<

0.05) and CDDP alone included a peak of apoptosis (5.9

f

0.5%) at 24 h after the start of treatment that was significantly greater than the peak of apoptosis observed with control or LL-WBH alone ( p

<

0.05). The peak of tumour apoptosis induced by LL-WBH

+

CDDP (8.9 f 0.5 to 11.8 f 0.6%) was significantly greater than any other group from 12 to 24 h after treatment ( p

<

0.01). Extensive ( 3 + ) levels of tumour necrosis were induced by both LL- WBH and LL-WBH

+

CDDP earlier (at 8 h after the start of treatment) than with CDDP alone (at 24 h).

4. Discussion

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To further evaluate the efficacy of LL-WBH combined with CDDP, we examined ALN metastasis and survival. The combination of LL-WBH with CDDP tended to cause the longest delay in development of ALN metastasis (21.6

f

1.7 days) and resulted in a significantly longer mean survival (31.3 f 1.8 days) compared to either modality alone. In cancer therapy, surgical excision of the primary tumour is often the first treatment. Then, however, it is very important to inhibit local recurrence as well as metastasis and dissemination which would lead to death of the patient. These data suggest that LL-WBH combined with CDDP may eventually lead to a promis- ing adjuvant therapy for breast cancer as well as a therapy for advanced disease.

In our previous study using different tumours (fibrosarcoma and Ward colon carcinoma), SH-WBH alone induced considerable levels of apoptosjs (2&40%) (Sakaguchi et al. 1995). Although LL-WBH alone induced only a mild 2% peak of apoptosis in the present study, the combination of LL-WBH with CDDP induced a 12% peak of apoptosis at 24 h after treatment beginning that was significantly greater than the peak of either LL-WBH alone (2.1%) or CDDP alone (5.9%). These data suggest that the extent and duration of apoptosis seems to correlate with the tumour response to each treatment. With regards to treatment induction of necrosis, we observed that extensive (3 +) levels of tumour necrosis appeared to occur earlier with LL-WBH and LL-WBH

+

CDDP (at 8 h after treatment beginning) than with CDDP alone (at 24 h). Recently Moroi et al. (1996) reported that the gliosarcoma tumour showed a rapid increase in the number of apoptotic cells following hyperthermia treatment of 43.0"C for 30 min, and necrosis was markedly enhanced only in the centre of the tumour at 3 and 6 h after hyperthermia (Moroi et al. 1996). The combined responses of apoptosis and necrosis observed in our study could be caused by microenvironmental changes within the tumour after thermo- chemotherapy.

In conclusion, LL-WBH in combination with CDDP demonstrated improved efficacy (compared to either agent alone) in delaying the onset and reducing nodal metastases as well as the primary lesion of an aggressive, highly metastatic rat mammary adenocarcinoma. This antitumour response was associated with an increased level of tumour apoptosis, and an earlier onset of extensive necrosis (observed with both LL-WBH and LL-WBH

+

CDDP). Further studies of different LL-WBH

+

CDDP combinations and/or sequences which show correlations between tumour apoptosis and necrosis and tumour response may lead to both a better understanding of treatment induced tumour cell death and optimization of therapy.

Acknowledgements

This work was supported by NCI grant CA43090.

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