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Ann Thorac Surg 2001;72:1173-1178
© 2001 The Society of Thoracic Surgeons

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Original article: general thoracic

Digitonin enhances the antitumor effect of cisplatin during isolated lung perfusion

^a First Department of Surgery, Yamaguchi University School of Medicine, Yamaguchi, Japan

Accepted for publication June 18, 2001.

Address reprint requests to Dr Tanaka, First Department of Surgery, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505, Japan
e-mail: toshik{at}po.cc.yamaguchi-u.ac.jp

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. The antitumor effect of isolated lung perfusion with cisplatin was limited because the intracellular platinum concentration did not increase sufficiently. To solve this problem, digitonin, a detergent, was chosen to increase cell permeability and enhance intracellular uptake and antitumor effect. This study was designed to investigate toxicity, pharmacokinetics, and efficacy of isolated lung perfusion with the combined use of digitonin and cisplatin in Fischer 344 rats.

Methods. Systemic and local toxicities of isolated lung perfusion treatment were evaluated on the basis of body weight change, survival rate, and histologic findings. The maximal tolerated dose of digitonin was determined by assessing survival on day 21 after contralateral pneumonectomy, body weight change, and histologic findings. Pharmacokinetics were observed in a solitary lung tumor nodule model by measuring platinum concentration in tumor and normal lung tissue. The antitumor effect was evaluated by the number of tumor nodules in the left lung 21 days after isolated lung perfusion. Isolated lung perfusion was performed 7 days after 1.0 x 10⁶ methylcholanthrene sarcoma cells were injected into the external jugular vein.

Results. The maximal tolerated dose of digitonin was 20 µmol/L. Platinum concentration of tumor nodules in the digitonin-cisplatin–treated rats was 20% higher than in the cisplatin-only group (5.48 ± 0.64 µg/g tissue versus 4.50 ± 1.09 µg/g tissue; p = 0.067). The number of pulmonary nodules decreased significantly by digitonin use (1.3 ± 1.5 versus 9.7 ± 2; p < 0.0001).

Conclusions. Isolated lung perfusion with digitonin and cisplatin in combination was performed safely and enhanced the antitumor effect. These drugs in combination show promise for enhancing the effect of clinical isolated lung perfusion.

	Introduction

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Isolated lung perfusion (ILP) is a theoretically ideal and effective means for regional chemotherapy of lung tumors, particularly in comparison to systemic chemotherapy, because a relatively high concentration of antitumor agent can reach the tumor tissue and because of the relatively low systemic toxicity. The safety and pharmacokinetics of ILP have been demonstrated recently in experimental animal models [1–3]. The efficacy of ILP with cisplatin in rat solitary lung tumor node models, however, was not studied in comparison with that in a rat micrometastases model [4]. Furthermore, our recent studies showed that the platinum concentrations in tumor tissue did not increase with either perfusion time or perfusate cisplatin concentration [5] and were significantly inversely related to the weight of tumor nodules after ILP with cisplatin [6]. It is likely that these issues are at the heart of clinical therapeutic failure.

Digitonin, a detergent, increases cell permeability by binding specifically to cholesterol in the plasma membrane, and it has been shown to enhance the intracellular uptake of platinum in a human ovarian carcinoma cell line in vitro [7]. In addition, digitonin increased platinum concentrations in rat hepatoma tissue in a carboplatin intraarterial administration study [8]. We selected digitonin in our experiment because we expected that the increase of cell permeability enhanced intracellular platinum concentration and antitumor effect. We investigated the toxicity, pharmacokinetics, and synergistic antitumor effect of digitonin-cisplatin combination ILP.

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
Cell line and animals
We used a methylcholanthrene (MCA) -induced rat sarcoma cell line, which was kindly supplied by Memorial Sloan-Kettering Cancer Center, New York, NY, USA. The cells were maintained in RPMI-1640 medium containing 10% bovine calf serum with 1% penicillin-streptomycin (Life Technologies, Grand Island, NY, USA). The cells were cultured in a 5% CO₂ atmosphere at 37°C.

Male Fischer 344 rats weighing 250 to 300 g were used for experiments and were treated in accordance with the Animal Welfare Act and the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985). The Institutional Animal Care and Use Committee of Yamaguchi University approved the experiments. Rats were bred in a standard laboratory and allowed free access to food and water in a temperature-controlled environment under a 12-hour light and dark cycle.

Chemicals
Digitonin (Sigma Chemical Company, St. Louis, MO, USA) was dissolved in dimethylsulfoxide and diluted with phosphate-buffered saline solution (PBS) immediately before use [9]. Cisplatin (Sigma) was dissolved in 6% buffered hetastarch solution immediately before use [9].

Evaluation of intracellular concentration and antitumor effect in vitro
For the pharmacokinetic study, 1.0 x 10⁷ MCA sarcoma cells were seeded into RPMI-1640 medium containing 50 µg/mL cisplatin (Cis group), 50 µg/mL cisplatin and 20 µmol/L digitonin (Cis-D20 group), or 50 µg/mL cisplatin and 50 µmol/L digitonin (Cis-D50 group). After a 30-minute treatment, cells were washed twice with PBS and extracted by centrifuge. Cell platinum levels were measured by flameless atomic spectrometry.

For the antitumor effect study, MCA sarcoma cells (1.0 x 10³) were seeded for 12 hours in 96-well cell culture dishes (Nunclon Surface, Nalge Nunc International, Roskilde, Denmark). After seeding, the cells were exposed to PBS with 50 mmol/L dimethylsulfoxide (PBS group), 5 µmol/L digitonin alone (D group), 5 µg/mL cisplatin alone (C group), or 5 µg/mL cisplatin and 5 µmol/L digitonin (D + C group) for 10 minutes. The cells were then washed twice with PBS. After 72 hours in culture, bromodeoxyuridine uptake was measured to determine viable cell counts with a cell proliferation enzyme-linked immunosorbent assay system (Biotrak, Amersham Life Science Ltd, Little Chalfont, UK) [10].

Isolated left lung perfusion
Isolated left lung perfusion was performed by a method described previously [6]. Briefly, animals were anesthetized with 50 mg/kg of pentobarbital administered intraperitoneally, and tracheal intubation was performed with a 16F intravenous catheter [11]. The animals were ventilated with a volume ventilator (Harvard rodent ventilator, model 683, Harvard Apparatus Inc, South Natick, MA) with room air at a tidal volume of 10 mL/kg and a rate of 80 strokes/min. A left thoracotomy was performed through the fourth intercostal space, and the left pulmonary artery and vein were exposed under a surgical microscope. After the left main pulmonary artery and vein were clamped, a polyethylene (PE-10) catheter was inserted into the pulmonary artery for infusion, pulmonary venotomy was performed, and a suction catheter placed in the proximity of the venotomy collected the effluent. All rats were perfused for 10 minutes and followed by a 5-minute washout with buffered hetastarch solution at a rate of 0.5 mL/min. A 0.2-mL bolus of digitonin was injected into the pulmonary artery just before ILP treatment in digitonin-treatment groups.

Toxicity
A total of 12 rats were randomized into the following two treatments on day 0 for study of the toxicity of digitonin: ILP with 400 µmol/L digitonin (D400 group; n = 6) and 200 µmol/L digitonin (D200 group; n = 6), both with buffered hetastarch solution only, was performed. Eighteen rats were randomly selected for injection of digitonin at 50 µmol/L (Cis-D50 group; n = 6), 20 µmol/L (Cis-D20 group; n = 6), or 10 µmol/L (Cis-D10 group; n = 6), each with 100 µg/mL cisplatin, just before ILP on day 0 to study the synergistic toxicity of digitonin and cisplatin. Toxicity was evaluated in the same manner in both toxicity studies; all rats were monitored and their body weights recorded every second day. On day 21, a right pneumonectomy to evaluate the effect of perfusion on left lung function was performed on all surviving rats. On day 35, all rats that survived were killed. Histologic changes were evaluated in each group.

Pharmacokinetics
Solitary tumor nodules were established in 12 rats by a method described previously [5]. Animals were anesthetized and intubated, and ventilation was maintained as described above. A small left thoracotomy was performed, and the thoracic cavity was opened. A 27-gauge needle attached to a 100-µL microsyringe was inserted into the left lower lung at an angle of approximately 15 degrees to a depth of 3 mm, and 1.0 x 10⁶ tumor cells in 20 µL of PBS were inoculated into the lung parenchyma. After the lung was expanded, the chest and thoracotomy incision were closed.

All 12 animals were randomized into two groups (n = 6 in each group) on day 14 after tumor cell inoculation. Rats in the Cis group underwent ILP with 100 µg/mL cisplatin. Rats in the Cis-D20 group underwent ILP with 100 µg/mL cisplatin after 20 µmol/L digitonin was injected. After perfusion, a left pneumonectomy was performed. The tumor nodule was removed from samples of the left lung. Total platinum concentrations of the tumor and the left lung were measured by flameless atomic spectrometry.

Antitumor effect
A total of 18 rats were randomized into two groups 7 days after 1 x 10⁶ MCA sarcoma cells had been injected through the external jugular vein. A Cis group of 9 rats underwent ILP with 100 µg/mL cisplatin, and a Cis-D20 group of 9 rats were given ILP with 100 µg/mL cisplatin after 20 µmol/L digitonin was injected. All 18 rats were killed 14 days after ILP treatment, and their lungs were stained with India ink for identification of metastases according to the method of Wexler [12]. All tumor nodules were counted on the left lung.

Statistical analysis
All data are presented as mean ± standard deviation. In vitro data were analyzed by Fisher’s exact test, and in vivo data, by unpaired Student’s t test. Significance was determined at p less than 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Synergistic effect of digitonin in vitro
Total platinum levels in cells were significantly elevated in the Cis-D50 and Cis-D20 groups in comparison with levels in the Cis group (Fig 1). Platinum levels were significantly elevated with the increase in digitonin.

View larger version (28K): [in this window] [in a new window]   Fig 1. Total platinum level of methylcholanthrene-induced sarcoma cells. Total platinum levels are significantly increased in the 50 µg/mL cisplatin and 20 µmol/L digitonin group (Cis-D20 group) and the 50 µg/mL cisplatin and 20 µmol/L digitonin group (Cis-D50 group) in comparison with levels in the control 50 µg/mL cisplatin group (Cis group; *p = 0.023, ***p < 0.0001). Platinum levels were significantly elevated with increase in digitonin (**p = 0.0004). Reprinted, with permission, from Tanaka T, Kaneda Y, Li T-S, Matsuoka T, Zempo N, Esato K. Digitonin enhances the anti-tumor effect of cisplatin against methyl cholanthrene-induced rat sarcoma cells in vitro. Anticancer Research 21:313–6.

View larger version (25K): [in this window] [in a new window]   Fig 2. Number of viable methylcholanthrene-induced sarcoma cells. The proliferation of methylcholanthrene-induced sarcoma cells is significantly decreased in the 5 µmol/L digitonin + 5 µg/mL cisplatin group (D + C group; 1.056 ± 0.308 optical density [O.D.]) versus the phosphate-buffered saline group (PBS group; 1.849 ± 0.96 O.D.), the 5 µmol/L digitonin alone group (D group; 1.836 ± 0.176 O.D.), and the 5 µg/mL cisplatin alone group (C group; 1.801 ± 0.133 O.D.; all *p < 0.0001). Reprinted, with permission, from Tanaka T, Kaneda Y, Li T-S, Matsuoka T, Zempo N, Esato K. Digitonin enhances the anti-tumor effect of cisplatin against methyl cholanthrene-induced rat sarcoma cells in vitro. Anticancer Research 21:313–6.

According to histologic analysis, the left lung recovered to almost normal architecture within 35 days in the D200 group, but slight interstitial thickness was revealed in the D400 group (Fig 3A, 3B). Severe left lung damage developed in the Cis-D50 group (Fig 3D). Body weight changes between the D400 and the D200 groups, and among Cis-D50, Cis-D20, and Cis-D10 groups, were not statistically different.

View larger version (131K): [in this window] [in a new window]   Fig 3. Histologic findings of left lungs after isolated lung perfusion. Isolated lung perfusion was performed with (A) 200 µmol/L digitonin, (B) 400 µmol/L digitonin, (C) 20 µmol/L digitonin and 100 µg/mL cisplatin, and (D) 50 µmol/L digitonin and 100 µg/mL cisplatin. Interstitial thickness of lung tissue, which indicated lung toxicity histologically, was observed to be severe in isolated lung perfusion with either 400 µmol/L digitonin or 50 µmol/L digitonin with 100 µg/mL cisplatin, and slight in isolated lung perfusion with 20 µmol/L digitonin with 100 µg/mL cisplatin.

View larger version (12K): [in this window] [in a new window]   Fig 4. Total platinum concentration in tumor tissue and normal lung tissue after isolated lung perfusion. Isolated lung perfusion was performed with 20 µmol/L digitonin and 100 µg/mL cisplatin in combination (Cis-D20 group) or with 100 µg/mL cisplatin alone (Cis group). Total platinum concentration of tumor tissue tended to increase with the combination treatment (*p = 0.068), but there was no significant difference in tumor tissue and normal lung tissue between the two groups.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
The lung is one of the organs most frequently involved in cases of both primary and metastatic malignant tumors. Although complete surgical resection is the most effective therapy, the majority of patients with primary lung cancer [13] and metastatic lung cancer [14] are not suitable candidates for surgical resection because of multiple lesions in the lung or other organ metastases. Systemic chemotherapy in unresectable cases results in a poor outcome owing to dose-limiting systemic toxicities. Isolated lung perfusion can be used to deliver high-dose antitumor agents and prevent systemic toxicity [1]. The antitumor potency expected in some clinical series was not achieved [15, 16]; these clinical trials indicated a need for improvement in ILP treatment.

Cisplatin is widely used in the treatment of various malignant neoplasms including sarcomas. Cisplatin causes cytotoxicity by DNA injury by means of DNA platination. The intracellular concentration of platinum plays a very important role in cytotoxicity, but platinum-containing drugs enter cells relatively slowly in comparison with many other cytotoxic agents [17]. Although the mechanism by which cisplatin enters cells is not understood completely, many investigators report that uptake of cisplatin occurs primarily by passive diffusion. This slow uptake may relate to the clinical failures and our previous experimental findings [5].

Digitonin increases permeability of the plasma membrane by binding to cholesterol in the bilayer membrane in proportion to its cholesterol content [18]. This permeabilization enhances intracellular uptake of cisplatin [19]. Our in vitro experiment showed that the combined use of digitonin and cisplatin increases the cellular uptake of platinum in MCA sarcoma cells significantly. Furthermore, a significant synergistic antitumor effect was observed with the combined use of digitonin and cisplatin at very low doses, each of which, separately, had not shown an antitumor effect. Cisplatin with dimethylsulfoxide did not enhance the intracellular platinum concentration compared with cisplatin only, and dimethylsulfoxide only did not show an antitumor effect (data not shown). Our in vitro results indicate the possibility of a synergistic effect in ILP treatment.

Isolated lung perfusion with digitonin only did not result in toxicity in normal lung tissue. The toxicity resulting from digitonin and cisplatin in combination was quite severe. We obtained a 67% survival rate after right pneumonectomy when the dose of digitonin was decreased to 20 µmol/L. Thus, this was the dose that was studied.

Regarding the significant increase in intracellular platinum concentration in vitro, we expected that a very high platinum concentration would be seen in tumor tissue in the Cis-D20 group. The total platinum concentration of tumor tissue in the Cis-D20 group increased only slightly, however, in comparison with that in the control group. Although the reason for this was not clear, a possible factor is the different blood supply from the pulmonary and bronchial circulations to the tumor tissue. According to investigations conducted by Milne and associates [20] in humans and rats, the source of blood in pulmonary metastatic deposits may be the pulmonary circulation, bronchial circulation, or a combination of both. In fact, tumor nodules were stained after trypan blue infusion from both pulmonary artery and the aorta in our previous study [5]. This suggests that blood supply was derived from both pulmonary and bronchial systems in the rat solitary tumor model.

An antitumor effect was significantly evident in ILP with digitonin and cisplatin combined, which agreed well with results of the in vitro study. This may be related, in part, to the slight increase in total platinum in tumor tissue, but the synergistic effect was important also. Digitonin increases cell permeability and deformity by corrugations of hemitubules in the cell bilayer membrane [18]; thus, the antitumor effect of cisplatin is by the fragility of tumor cells after digitonin exposure. In fact, the combined use of low-dose cisplatin and digitonin caused an excellent antitumor effect in our in vitro study.

In summary, we observed that the combined use of digitonin and cisplatin increased intracellular platinum concentration and enhanced the antitumor effect in vitro. Furthermore, ILP to sarcomatous pulmonary metastases in the rat with digitonin and cisplatin in combination was performed safely and enhanced the antitumor effect in comparison with ILP with only cisplatin. A trial in large animals closer to humans will be needed before phase I human trials because we are aware of no published reports that describe the information in humans regarding potential toxicity of digitonin. If the toxicity to humans is solved, our findings suggest the possibility of enhancing the antitumor effects of clinical ILP treatment.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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