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. 2000 Nov;2(6):555–560. doi: 10.1038/sj.neo.7900123

Use of Arsenic Trioxide as an Antivascular and Thermosensitizing Agent in Solid Tumors1

Robert J Griffin *, Sang H Lee *, Kelly L Rood *, Michael J Stewart *, John C Lyons *, Young S Lew †, Heonjoo Park *, Chang W Song *
PMCID: PMC1508087  PMID: 11228548

Abstract

Arsenic trioxide, As2O3 (ATO), has been found to be an effective chemotherapy drug for acute promyelocytic leukemia but its effect on solid tumors has not been fully explored. In the present report, we describe our observation that ATO is a potent antivascular agent and that it markedly enhances the effect of hyperthermia on tumors. The tumor blood perfusion in SCK tumors of A/J mice and FSall tumors of C3H mice was significantly suppressed for up to 24 hours after an i.p. injection of 8 mg/kg ATO. ATO was also found to be able to increase the thermosensitivity of tumor cells in vitro. As a probable consequence of these effects, ATO treatment markedly increased the tumor growth delay caused by hyperthermia at 41.5–42.5°C. Immunohistochemical staining of tumor tissue revealed that the expression levels of several adhesion molecules and TNFα are noticeably increased in tumors 2–6 hours after systemic ATO treatment. It is concluded that ATO is potentially useful to enhance the effect of hyperthermia on tumors at a clinically relevant temperature.

Keywords: arsenic trioxide, tumor perfusion, adhesion molecules, thermosensitization

Introduction

Arsenic has been used since ancient times to treat a variety of diseases such as tooth marrow disease, psoriasis, and syphilis. In the early 1900s, chronic myelogenous leukemia was treated with Fowler's solution containing potassium arsenite. In the 1970s, arsenic trioxide (ATO) was found to be able to induce complete remission in patients with acute promyelocytic leukemia (APL) in China [1–4]. More recent clinical studies conducted in China and the US have also demonstrated that daily administration of low doses of ATO is strikingly effective in causing complete remission without serious side effects in patients with APL [5,6]. Amazingly, ATO was effective even against APL that had relapsed after standard treatment for APL using retinoic acid [7]. The mechanism of such remarkable clinical effectiveness of ATO against APL is unclear although induction of nonterminal cytodifferentiation and apoptosis of APL cells by the drug have been indicated [8]. In further investigating the anticancer effects of ATO we have recently observed that it induces vascular shutdown and massive central necrosis in intradermal and subcutaneous Meth-A fibrosarcomas of BALB/c mice [9]. The indication that ATO may be effective against various malignancies has led to the recent opening of several clinical trials using this agent such as in the treatment of hormone refractory prostate cancer (NCI sponsored protocol #NCI-T99-0077).

Although ATO may be used as a stand-alone treatment, the powerful antivascular effect may be exploited to enhance tumor treatment with hyperthermia, the effect of which is heavily dependent on the tumor blood perfusion. One of the major obstacles in giving effective hyperthermia doses is the dissipation of heat through blood perfusion, an effect that prevents raising the tumor temperature and causes heterogeneous temperature distributions [10,11]. In the present study, we report results of our investigation on the effect of ATO on experimental murine tumor perfusion and tumor response to heating in vivo and in vitro. In addition we describe the effect of ATO on the expression of adhesion molecules and cytokines in the tumor, which is a probable mechanism of the vascular shutdown caused by ATO.

Materials and Methods

Tumors

The SCK mammary carcinoma of A/J mice and the FSall fibrosarcoma of C3H mice were used. Stock cells are stored in liquid nitrogen and are thawed every 2 to 3 months, and cultured in RPMI 1640 medium supplemented with 10% bovine serum and antibiotics under a humidified 5% CO2 atmosphere. Tumors were induced by subcutaneous injection of about 2x105 tumor cells in 0.05 ml of serum-free medium into the right hind thigh of 20-–23-g male A/J mice for SCK tumors or female C3H mice for FSall tumors and used when they had grown to 7–9 mm in diameter. All animal procedures and care were performed using protocols approved by the University of Minnesota Institutional Animal Care and Use Committee in accordance with federally approved guidelines.

Arsenic Trioxide

A 1% w/v solution of As2O3 (Sigma, St. Louis, MO) in PBS was prepared by magnetic stirring for 4 days at room temperature in a glass bottle and then placing the bottle into boiling water for 1 h. After the solution became crystal clear, it was sterilized using a 0.2 µM bottle-top filter and stored in the dark at 4°C. The stock solution was then diluted to desired concentrations. For the animal injections the stock solution was diluted with PBS containing 5% dextrose and the required dose was injected i.p. at 20 ml/kg (i.e., 0.5 ml for a 25-g mouse). For the in vitro study the stock solution in PBS was diluted in culture media at the appropriate concentration.

Blood Perfusion

The blood perfusion in tumors and normal tissues were measured with the 86Rb uptake method [12]. Tumor-bearing mice were anesthetized by an i.p. injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). About 15 minutes after the injection of anesthetics, 5 µCi of 86RbCI in 0.1 ml PBS was injected through the lateral tail vein and the mice were sacrificed 60 seconds later by cervical dislocation. The tumor, tail, and several normal tissues from each mouse were removed and weighed, and the 86Rb activity in a reference aliquot of 5 µCi and the tissues was counted in a well-type gamma counter (1282 Compugamma, Pharmacia LKB Wallac, Turku, Finland). By comparing the activity in the tumor, skin, muscle, and kidney to that in the total amount injected, the percent uptake per gram of each tissue type was calculated. Animals in which the tail activity exceeded 5% of the total injected dose were excluded from the data analysis.

Blood Pressure Measurement

Mouse blood pressure was measured directly in anesthetized animals at 2, 4, and 6 hours after drug administration by catheterization of the right carotid artery with PE-10 flexible tubing under a dissecting microscope. A pressure transducer (Stoelting Instruments, Wheaton, IL), which was precalibrated using a manometer, and a digital pressure display monitor (Quintron, Menomonee Falls, WI) were used to record the mean blood pressure for a period of 60 minutes. An incandescent light was used to keep the animal warm during monitoring and a small dose of anesthesia was given when needed.

Thermosensitivity Studies

In vitro A known number of cells plated in 25-cm2 tissue culture flasks were incubated with medium containing various concentrations of ATO for 1 hour at 37°C. The flasks were then tightly capped and control flasks were further incubated for 1 hour at 37°C whereas those to be heated were immersed in a preheated water bath for 1 hour. Immediately afterwards, the cells in all flasks were gently rinsed with 4 ml of drug-free medium and cultured with fresh medium in a 5% CO2 incubator for 8 days. The colonies were stained with crystal violet and counted. A viable colony was defined as one containing more than 50 cells.

In vivo Tumors were heated by immersing the tumor-bearing legs of anesthetized animals into a preheated water bath. The animals were laid in a plastic jig with a hole allowing the tumor-bearing leg to be extended and taped to a vertical extension. Each jig was then placed on a Plexiglas board on the top of a water bath and the legs were immersed into the water through slots in the board. After treatment the tumor size was measured using a metric scale caliper every 2 to 3 days until the mean tumor volume of each group reached four times the volume on the day of treatment. The tumor volume was calculated using the formula a2b/2, where a and b are the shorter and longer diameters of the tumor, respectively.

Tumor temperature

A tumor-bearing mouse was anesthetized and the tumor was heated as described above with the following modifications. A needle-type (29-gauge) thermocouple connected to a digital display unit was vertically inserted approximately 5 mm into the tumor and taped in place on the jig holding the animal. This was estimated to be at or near the center of the tumor geometry as we used tumors that averaged 9 mm in length. The tumor temperature in five mice treated with vehicle alone (PBS containing 5% dextrose) and that in five mice treated with 8 mg/kg ATO 2 hours before heating was measured during the first 30 minutes of heating. These tumors were not used for further study.

Immunohistochemistry

FSall tumor-bearing animals were injected with ATO or vehicle. At 2 or 6 hours later the tumors were harvested, fixed in 10% formalin for at least 24 hours, paraffin-embedded, and sectioned at 6 µm. The staining for the protein in question was accomplished using the appropriate primary antibody and a commercially available immunohistochemical staining kit (Histostain basic kit, Zymed Laboratories, San Francisco, CA). This staining system uses a biotinylated secondary antibody and the addition of a streptavidin-horseradish peroxidase complex for detecting the antigen with diaminobenzidine (DAB) as a substrate for localization. The sections were treated for 30–60 minutes with primary antibody against ICAM-1, VCAM, E-selectin or TNFα (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution. After treating with the biotinylated secondary antibody and streptavidin-peroxidase, there was a 5-minute DAB exposure and the sections were counterstained with hematoxylin and mounted. Images of the staining were collected at x40 magnification using a CoolSNAP digital camera (Roper Scientific, Tucson, AZ) and a variety of image analysis software.

Statistical methods

Student's t test was used for all statistical comparisons. A two-tailed P value was calculated and is quoted throughout this communication at an arbitrary level of significance of P%0.05.

Results

Effect of ATO on Tumor Blood Perfusion and Blood Pressure

The 86Rb uptake in control SCK tumors was 2.1% of injected activity per gram. In control FSall tumors the 86Rb uptake was 3.5% of injected activity per gram. The effect of 8 mg/kg ATO on the 86Rb uptake in both tumor types is shown in Figure 1. In our previous report we tested 10 mg/kg ATO [9] but we used 8 mg/kg ATO in the present study to minimize any potential acute toxicity of ATO. The 86Rb uptake decreased to around 50% and 20% of the control value in SCK and FSall mice, respectively, at 2 hours after an i.p. injection of 8 mg/kg ATO (P<.05), remained suppressed 6 to 16 hours after the ATO injection (P<.05), and was still lower than control value in both tumors by 24 hours after drug injection. The 86Rb uptake in skin, muscle, and kidney was not significantly affected by 8 mg/kg ATO (data not shown). The average blood pressure in anesthetized control A/J mice was 69.9±0.9 mm Hg. When measured at 2, 4, or 6 hours after an injection of 8 mg/kg ATO the blood pressure was found to be 70.6±1.1, 67.6±1.2, and 69.5±0.8 mm Hg, respectively.

Figure 1.

Figure 1

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Uptake of 86Rb in SCK and FSall tumors after administration of 8 mg/kg of ATO. Data is expressed as the percent of control tumor uptake per gram of tumor. Each data point represents the mean of 12 to 17 individual tumors with 1 SE.

Effect of ATO on Thermosensitivity In Vitro

The effect of 5–20 µM ATO on the thermosensitivity of tumor cells in culture is shown in Figure 2. Heating the cells at 41.5°C for 1 hour without ATO decreased the clonogenicity by 10%. The clonogenicity of cells treated with ATO alone or ATO combined with heating is normalized to the clonogenicity of nontreated control cells or those treated with heat alone, respectively. An exposure to ATO at a concentration higher than 5 µM for 1 hour before and during a 1 hour heating significantly (P<.05) increased the sensitivity of SCK cells to 41.5°C. For example, ATO exposure at 15–20 µM alone for 2 hours reduced the clonogenicity of the cells to about 50% of control but when combined with heating reduced the survival to 10%–20% of the control value.

Figure 2.

Figure 2

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Viability of SCK cells assessed by clonogenic assay. Cells in culture were treated for 60 minutes with 8 to 20 µM of ATO before heating for 60 minutes at 41.5°C. The drug was rinsed away immediately after heating. Each data point represents the mean of five individual experiments ± 1 SE plated in duplicate and normalized to the control value at each timepoint.

Effect of ATO on Tumor Temperature During Heating

The average tumor temperature during 30 minutes of heating at 42.5°C is shown in Figure 3. In tumors heated without ATO treatment the tumor temperature was 0.3–0.5°C lower than the water temperature. However, in tumors of mice treated with 8 mg/kg ATO the tumor temperature was only 0.1°C lower than the water temperature. The difference in tumor temperature during heating between the ATO treated and non-ATO-treated groups was highly significant (P<.001).

Figure 3.

Figure 3

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Tumor temperature in FSall tumors measured during 30 minutes of heating in a 42.5°C water bath. Each data point is the average of five individually treated and measured tumors with the bars representing 1 SE.

Effect of ATO on Heat-Induced Tumor Growth Delay

The FSall tumor growth delay caused by heating at 42.5°C for 60 minutes alone or combined with 8 mg/kg ATO treatment 2 or 6 hours before heating is shown in Figure 4. Control tumors grew to four times the starting volume in about 9 days. After a single injection of 8 mg/kg ATO the tumor volume increased by four times in 10.5 days, a growth delay of 1.5 days. Tumors that were treated with heating alone had a growth delay of 2.5 days. In animals treated with ATO 2 or 6 hours before heating, the tumor growth delay was extended to 17.0 and 9.5 days, respectively.

Figure 4.

Figure 4

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Effect of ATO injection on heat-induced tumor growth delay. FSall tumor-bearing animals were given an i.p. injection of 8 mg/kg ATO 2 or 6 hours before heating the tumor at 42.5°C for 60 minutes. Each data point is the average tumor volume±1 SE measured in 8 to 15 animals per treatment group.

In the studies with the SCK tumor we opted to heat the tumors at 41.5°C, a more clinically relevant temperature, 2 hours after administration of ATO. As Figure 5 shows, control SCK tumors quadruple in volume in 6 days. The growth time was lengthened by 1 day after a single dose of 8 mg/kg ATO and by 1.5 days after heating the tumor at 41.5°C for 60 minutes. When ATO treatment and heating were combined the growth of this fast growing tumor was delayed by 7 days.

Figure 5.

Figure 5

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SCK tumor growth delay after 8 mg/kg ATO and 41.5°C alone or combined. Tumor-bearing animals were given an i.p. injection of 8 mg/kg ATO 2 hours before heating the tumor at 41°5 C for 60 minutes. Each data point is the average tumor volume±1 SE measured in 10 to 19 animals per treatment group.

Expression of ICAM-1, VCAM, E-Selectin, and TNFα

The effects of an injection of 8 mg/kg ATO on the expression of the adhesion molecules ICAM-1, VCAM, E-selectin, and the cytokine TNFα in tumors were examined using immunohistochemistry. Figure 6 shows representative images of three to four repeated studies. Control tumors expressed a small amount of all proteins studied. In sections from tumors harvested 2 hours after ATO treatment there was a clear increase in expression of ICAM-1, VCAM, E-selectin, and TNFα. This expression pattern was even further indicated at 6 hours after the drug treatment with abundant cytoplasmic and cell-surface staining evident throughout each tumor section analyzed.

Figure 6.

Figure 6

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Immunohistochemistry on FSall tumor sections. The substrate in all cases was DAB (brown color). Images are representative of results obtained in sections prepared from three to four individually treated tumors for each condition.

Discussion

The results obtained in the present study confirm our previous observation that ATO causes a drastic vascular shutdown in solid tumors [9] and illustrate how this shutdown may be exploited for therapeutic gain in treating tumors with hyperthermia. Our results also indicated that ATO causes little change in normal tissue perfusion, which is in agreement with that observed in our previous study. In recent years, the importance of angiogenesis in tumor growth and use of antiangiogenic agents have been widely discussed [13,14]. Agents that inhibit angiogenesis, however, may exert little effect on tumors with already established vascular beds. Conversely, the destruction of the existing vasculature or inhibition of blood flow in established tumors with antivascular agents like ATO would deprive the tumor cells from nutrient supply, including oxygen, leading to cell death.

Antivascular agents have the potential to allow higher and uniform temperature distributions in the tumor during hyperthermic treatment by reducing heat dissipation through blood perfusion. We observed that the tumor temperature in the animals treated with ATO averaged only 0.1°C lower than the temperature of the water bath (Figure 3). This was distinctly less than in control tumors where the tumor temperature was 0.3 to 0.5°C lower than the water bath temperature. These data suggest that, as we and others have previously observed [15], heating at relatively mild temperatures increases tumor perfusion and heat dissipation. Treatment with ATO is able to reduce tumor perfusion and inhibit the heat dissipation. As shown in Figures 4 and 5, ATO significantly enhanced the heat-induced growth delay of tumors treated at 41.5–42.5°C, which may be attributed to an improvement in tumor heating caused by ATO. However, a temperature of 41.5 or 42.5°C is only mildly cytotoxic, which suggests that ATO may directly affect tumor cell thermosensitivity in addition to causing vascular shutdown (Figure 2). ATO is known to deplete cellular glutathione [2,16,17] and therefore may cause oxidative stress, which is thought to be involved in the cellular response to hyperthermia [18–20]. It is therefore possible that the increases in cellular thermosensitivity caused by ATO resulted from cellular oxidative stress. Such direct cellular actions of ATO leading to a significant increase in cellular sensitivity to heat deserve further study. Taken together, our data appear to indicate that both the antivascular effects as well as direct cellular thermosensitization caused by this compound contributed to the increase in heat-induced tumor growth delay observed in these studies.

The mechanisms responsible for the marked decrease in tumor blood perfusion caused by ATO are unknown. A decline in blood pressure has been known to decrease tumor blood flow [21]. We observed little change in blood pressure in response to 8 mg/kg ATO, whereas there were significant and lasting decreases in tumor perfusion. Therefore, the contribution of hypotension to the observed effects appears to be minimal. Another possibility lies in the expression of adhesion molecules in the tumor vasculature that slow the circulation and eventually block the passage of blood as white blood cells adhere to the vascular wall. Following the blockage of the blood vessel the inflammatory cascade may ensue. As Figure 6 shows, we found massive increases in the expression of three major adhesion molecules as well as TNFα. Interestingly, others have found that hyperthermia alone increases ICAM-1 expression [22] and therefore our results (Figure 6) may underestimate adhesion molecule expression in response to combined ATO and heating. We are currently studying the combined effects of hyperthermia and ATO on tumor physiology. It seems reasonable to assume that with an increase in adhesion molecule expression, flow through the irregular tumor neovasculature could be easily blocked leading to inflammation, hemorrhage, and parenchymal damage. The expression of TNFα, as shown in Figure 6, may be in response to the microenvironmental stress induced by the drastic reduction in tumor perfusion caused by ATO. It is also known that hyperthermia in combination with TNFα produces a high degree of procoagulant activity in endothelial cells [23]. Therefore, in the present study, the hyperthermic treatment may have added to the ATO-induced adhesion molecule expression in the tumor vasculature and also increased the vascular effect of TNFα produced as a result of ATO treatment.

In recent years, a number of agents capable of reducing tumor blood flow and thus able to improve tumor heating have been identified. Among them TNF, interleukins, flavone acetic acid, serotonin analogs, and hydralazine have been demonstrated to decrease tumor blood flow, at least in rodent tumors, through various mechanisms [24–27]. Unfortunately, most of the drugs mentioned here are not applicable in the clinic due to unwanted normal tissue effects or species-dependent effects. Most recently, a naturally occurring tubulin binding agent named combretastatin-A4 has been shown to be effective in reducing tumor blood flow by selectively damaging the tumor vasculature by affecting endothelial cells [28–30]. The clinical effectiveness of this drug still remains to be seen.

It is important to note that ATO, now demonstrated to be a potent and selective antivascular agent for solid tumors, has already been found to be capable of inducing complete remission in more than 80% of patients with APL with minor side effects [6,7]. These results have prompted the FDA to very recently approve ATO for clinical use against leukemia. Data from clinical hyperthermia treatments shows that 41.5–42.5°C is a realistic temperature range that can be achieved in human tumors. Therefore, the results of the present study appear to be extremely promising in light of the fact that the antitumor effect of a temperature as low as 41.5°C can be markedly enhanced by ATO at a dose devoid of acute toxicity. A challenge that may remain is the negative connotation of a compound that contains arsenic, a fact that may unfairly bias the patient or clinician opinion of this drug.

In conclusion, ATO is a potent chemotherapeutic against leukemia and a novel antivascular agent. Increasing tumor thermosensitivity may be an effective use of this antivascular agent in cancer treatment. Further study of the mechanisms by which it exerts its effects on solid and blood-borne malignancies are warranted.

Acknowledgement

The authors thank LuAnn Anderson for helpful suggestions and discussion in the histologic study.

Abbreviations

ATO

arsenic Trioxide

ICAM-1

intercellular adhesion molecule 1

VCAM

vascular cell adhesion molecule

TNF

tumor necrosis factor

APL

acute promyelocytic leukemia

Footnotes

1

This work was supported by grant CA44114 from the National Cancer Institute.

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