Studies on the Treatment of Malignant Tumors with Fibroblast-inhibiting Agent. I. Fibroblast-inhibiting Action of Chloroquine.

The effects of chloroquine on the growth and morphology of fibroblasts in tissue culture, and in vivo granulomas were investigated. As the result, the drug was shown to have a potent action to inhibit fibroblast growth, which has led to a possibility of its clinical use to patients with malignant tumors. ∗PMID: 14165348 [PubMed OLDMEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Med. Okayama 17, 231-238 (1963) STUDIES ON THE TREATMENT OF MALIGNANT TUMORS WITH FIBROBLAST-INHIBITING AGENT I. FIBROBLAST-INHIBITING ACTION OF CHLOROQUINE Kiyoshi HIRAKI and Ikuro KIMURA Department of Internal Medicine, Okayama University Medical School (Director: Prof. K. Hiraki) Received for publication, August 10, i963 Malignant tumors consist of the parenchyma and stroma whose intimate associated growth forms neoplasms. This association of the two elements are also seen in the metastatic tumors. Our attention has been directed to the role of the stromal tissue in supporting cancer cell as already pointed out by FISCHER and on the basis of our idea that damage of the stromal tissue might secondarily results in damage of the cancer cells we have been conducting experimental and clinical studies with animal and human tumors for the past several years. To our best knowledge, no attempt has yet been made to approach malignant tumors from this chemotherapeutic point of view. Chloroquine, a derivative of quinoline, was used as the agent having a fibroblast-inhibiting action (an action inhibiting growth of fibroblasts), as already been recognized by HABERLAND. We have employed this agent, because it was shown to exert a powerful fibroblast-inhibiting effect. It is the intent of this paper to report our experimental results indicating the fibroblast-inhibiting effect of chloroquine (Fig. 1). < C1H~ N~H-QiiCHa-C~N CH .CH. 1 5 7-chloro-4-{4'-diethylamino·l'-methylbutylamino)-quinoline Fig. 1 Structure of chloroquine MATERIALS AND METHODS To determine the degree of fibroblast-inhibiting effects of chloroquine, a study was made on the influences of this drug upon the growth and morphology of in vitro cultured chick embryo heart fibroblasts, in vivo agar granuloma, and granuloma pouch of the animal. Chick embryo heart fibroblasts were cultured on tissue culture slides No. 2 devised by HlRAKI ; cells taken from heart of 9-day·old chick embryos were cultured in a medium consisting of an equal amount of chick embryo juice, avian 231 1 Hiraki and Kimura: Studies on the treatment of malignant tumors with Produced by The Berkeley Electronic Press, 1963 232 K. HIRAKI and 1. KIMURA serum and Ringer's solution, with addition of 5Y96, 500Y96, and 50,000Y 96 of chloroquine diphosphate, respectively, and observation was performed at various time intervals of 12, 24, 36, 48 and 72 hours with respect to the relative growth rate and cell density index. The effects of other derivatives of chloroquine diorotate, chloroquine chondroitin sulfate and chloroquine polygaracturonate were compared at the chloroquine concentration of 2 96 with those of chloroquine diphosphate at the same concentration. For the observation of morphological changes of chick embryo heart fibroblasts, roller tubes implanted with rectangular coverslips were used; after 48 hours of culture, chloroquine diphosphate was added at the concentration of 200 Y % and phase contrast microscopy was performed after 72 hours in culture. Agar granulomas were produced in the back of male Wistar rats by injecting 2 C.c. of 4 % agar solution in 1/10M phosphate buffer at pH 7.5 and beginning after 24 hours, chloroquine diphosphate, chloroquine diorotate, chloroquine chondroitin sulfate and chloroquine polygaracturonate were injected daily into the tail vein in the net amount of chloroquine of 15 mg. per kg. of body weight. After 15 days the animals were sacrificed and the granulomas were weighed and examined histologically after being stained with hematoxylin and eosin and van Gieson stain. Similarly, granuloma pouches· were produced' in male Wistar rats by injecting 25 C.c. of air subcutaneously in the back and by injecting 1 C.c. of 0.5 % croton oil in corn oil into the air space. Daily intraperitoneal injection of 25 mg of chloroquine diphosphate per kg. of body weight was started after 24 hours and continued for 14 days. When the animals were killed, the total pouch weight, weight of the pouch wall, amount of the exudate, and cell count in the exudate were measured. In addition, the amount of chloroquine of the pouch wall was determined by the method of BRODIE. RESULTS 1. Effects of chloroquine on the growth of chick embryo heart fibroblasts (Fig. 2): A decrease in the relative growth rate and cell density index was observed in cultures grown in medium containing 50,000Y % and 500 Y% of chloroquine diphosphate in comparison with the control. Even at the concentration of 5Y%, a moderate decrease in the relative growth rate was seen. Cultures grown in the medium containing 2% of chloroquine in the form of chloroquine disphosphate, chloroquine diorotate, chloroquine chondroitin sulfate and chloroquine polygaracturonate likewise showed similar depressive effects of the same degree. 2. Morphological changes of chick embryo heart fibroblasts (Photos 1, 2): In the control culture the cells were slender with cytoplasmic processes. The 2 Acta Medica Okayama, Vol. 17 [1963], Iss. 5, Art. 3 http://escholarship.lib.okayama-u.ac.jp/amo/vol17/iss5/3 Treatment of Malignant Tumors with Chloroquine -Control .. Chloroquine diphosphate Chloroquine diorotate .. .. Chloroquine chondroitin sulfate . . -. Chloroquine polygaracturonate 233 10.10 ~ tI:l loo ..!: ; 1'· e b/) 1IJ > .~ ] time Fig. 2 Relative growth rate of fibroblasts of chick embryo heart in tissue culture added with various chloroquine derivatives (2'1 % expressed in chloroquine concentration) cell margin was regular and distinct. Many intracytoplasmic vacuoles were small and uniform in size. There were many mitochondrias around the nuclei and in the cytoplasmic process, and they were granular or rod-shaped. The nuclei were oval and had distinct nuclear membrane and nucleoli. The cells cultured in the chloroquine-containing medium, however, showed an increase in the number of intracytoplasmic vacuoles which varied in size and characteristically contained giant vacuoles apparently resulting from confluent smaller vacuoles. No other significant changes were observed. 3. Effects of chloroquine on the growth of agar granulomas (Fig. 3, Photos Weight of granuloma (gram) 2.0 2~5 ,.0 Chloroquine. diphosphate Chloroquine diorotate Chloroquine chondroitin sulfate Chloroquine polygaracturonate