Since the mechanisms of the developmental processes of tumors remain unclear, early detection and early treatment of the tumors is necessary to save patients with malignant tumors. Therapies currently available to patients are surgery, chemotherapy, and radiotherapy. However, there are many patients who cannot be saved by such therapies. In this study, we found the common features of various tumor tissues, and we demonstrated the effect of therapeutics that target them by using experimental rats with spontaneous tumors. 26 kinds of human tumors (epithelial or mesenchymal origin, and malignant or benign) and Sprague-Dawley rats with spontaneous mammary gland tumors were examined by light and electron microscope. To detect of mast cells and leukotriene receptor, toluidine blue stain and immunohistochemical stain were performed. The rats were orally administered one of leukotriene receptor antagonists. We found that the presence of numerous mast cells and expression of leukotriene receptors in various tumor (human tumors and rat spontaneous tumors). And the therapeutic effects, which suppressed not only tumor progress but also angiogenesis and nerve formation, for rat tumors by administration of leukotriene receptor antagonist could obtain. Our data suggest the possibility that allergic reactions, in which leukotriene and leukotriene receptors in particular appear to play an important role, are involved in the development and progression of tumors and that it may be possible to control tumor progression by regulating such reactions. Our results might be useful for clinical applications of oncotherapy.
At present, the therapeutic options for malignant tumors include surgery, chemotherapy, and radiotherapy. However, many patients cannot be saved by these therapies. In many cases of a suspected benign tumor, the approach is to simply observe the progress of the tumor, and the tumor is then removed surgically when considered necessary. The patient then undergoes regular medical examinations for early discovery of malignant alterations to the tumor. Since the mechanisms and developmental processes of tumors remain unclear, this approach seems to represent the most effective means for achieving early detection of malignancy. However, a more effective method of saving patients with malignant tumors would be the early detection and early treatment of such tumors.
We recently discovered that mast cells are strongly involved in the development of endometriosis [
In this study, we confirmed the expression of leukotriene receptors, which were expressed as part of an allergic reaction, in various human tumor tissues and we discovered that this pathological finding was common to a range of tumors. We further demonstrated the therapeutic effect of targeting leukotriene receptors by treating spontaneous tumors of experimental animals.
Sprague-Dawley (SD) rats (all rats are females, mean weight: 605.62 ± 38.38 g) that had spontaneous mammary gland tumors were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan).
Tissue samples from the tumor area and a non-tumor area (for example, a left mammary gland was obtained as the comparison (non-tumor area) if the mammary gland which a tumor existed in was a right mammary gland) of the anesthetized SD rats with and without anti-leukotriene therapy, were fixed with 10% buffered formalin, and, after routine dehydration, were embedded in paraffin. Sections 5-μm thick were stained with hematoxylin and eosin (HE) and examined under a light microscope.
To identify mast cells in each specimen, the paraffin sections were stained with toluidine blue. The granules within mast cells contain heparin and sulfated glycosaminoglycan that stain metachromatically with toluidine blue. The 5-μm tissue sections were stained for 30 minutes with a staining solution containing a 0.05% concentration of toluidine blue O (Kanto Chemical Co., Inc., Tokyo, Japan) in a citric acid phosphate buffer (pH 2.5), and were then examined by light microscopy.
Immunohistochemical staining for the cysteinyl leukotriene receptor (CysLT) 1 and 2 was performed to detect the expression of the leukotriene receptors in the tumors under a light microscope. The 5-μm tissue sections were stained immunohistochemically using the streptavidin-biotin method (Histofine SAB-PO Kit; Nichirei, Tokyo, Japan). The primary antibodies used were polyclonal antibody to CysLT1 and polyclonal antibody to CysLT2 (Acris Antibodies, Inc., San Diego, CA, USA).
The rats were orally administered one of the following leukotriene receptor antagonists: Zafirlukast (Astra Zeneca, London, UK), Monterukast sodium (Merck, NJ, USA), Pranlukast hydrate (Ono Pharmaceutical Co., Ltd., Osaka, Japan) or Zileuton (Abbott Laboratories; Chicago, IL, USA). The experiment dose of each medicine was decided referring to the amount of prescription (to the adult asthmatic) that it was mentioned in each attached document. The medication periods for each medicine were for 3 or 7 days (Zafirlukast (1.33 mg/kg); the one oral administration/day; total three days (n = 3) or seven days (n = 3), Monterukast sodium (0.16 mg/kg per day); the one oral administration/day; total three days (n = 3) or seven days (n = 3), Pranlukast hydrate (7.5 mg/kg per day); the one oral administration/day; total three days (n = 3) or seven days (n = 3), Zileuton (34.3 mg/kg per day); the one oral administration/day; total three days (n = 3) or seven days (n = 3)). Four non-treated female rats (distilled water (0.5 ml), the one oral administration/day, total three days (n = 2) or seven days (n = 2)) that had spontaneous tumors were used as comparative controls.
All experimental animals were handled in accordance with institutional and national guidelines for the care and use of laboratory animals.
Tumor tissues obtained from patients who were diagnosed with various tumors, which were confirmed based on the standard for diagnosis of the Japanese society of surgical pathology, are listed in
The tissue samples were immediately fixed with 1% glutaraldehyde and 4% formalin for 6 hours at 4˚C and were then rinsed in 0.1 M cacodylate buffer overnight. The tissues were postfixed with 1% osmium tetroxide and were embedded in Epon 812 resin. Ultrathin sections were prepared using an Ultramicrotome (Model MT-XL; RMC, Arizona, USA), were doublestained with uranyl acetate and lead citrate, and were then examined under an electron microscope (Model JEM1400; JEOL, Tokyo, Japan).
Apoptotic appearance was evaluated based on the characteristics of an ultrastructural process in the apoptotic body, as described in our previous report [
We first checked for the presence of mast cells in the tissue specimens prepared from 26 kinds of human tumors (epithelial or mesenchymal origin, and malignant or benign tumors such as gastric carcinoma, colon cancer, esophagus epidermoid carcinoma, breast cancer, prostatic cancer, liver cancer, gastrointestinal stromal tumor, leiomyosarcoma, rhabdomyosarcoma and myoma uteri). In all tumor samples analyzed numerous mast cells were detected. These mast cells were diffusely distributed within the tumor, and such a diffuse distribution was very similar to that of mast cells in the lesion of the endometriosis patient that we previously studied [
We then used Sprague-Dawley rats with spontaneous mammary gland tumors (which, as expected, displayed many mast cells within the tumors [
Malignant tumors | Number of specimen | |
---|---|---|
Epithelial origin | ||
Pharyngeal carcinoma (Squamous cell carcinoma) | Well | N = 2 |
Poorly | N = 2 | |
Esophageal carcinoma (Squamous cell carcinoma) | Moderately | N = 2 |
Gastric cancer | Poorly | N = 5 |
Colon cancer (Adenocarcinoma) | Well | N = 5 |
Moderately | N = 3 | |
Poorly | N = 3 | |
Liver cancer (Hepatoblastoma) | N = 3 | |
(Hepatocellular carcinoma) | N = 4 | |
Gallduct carcinoma (Adenocarcinoma) | Well | N = 3 |
Poorly | N = 3 | |
Renal cell carcinoma | N = 5 | |
Pancreatic carcinoma (Adenocarcinoma) | Well | N = 5 |
Moderately | N = 3 | |
Poorly | N = 3 | |
Breast cancer (Intraductal carcinoma; noninvasive) | N = 5 | |
(Scirrhus carcinoma; invasive) | N = 3 | |
Prosstatic carcinoma (Adenocarcinoma) | N = 5 | |
Thyroid gland carcinoma (Papillary carcinoma) | N = 3 | |
Mesenchymal orgin | ||
Rhabdomyosarcoma | N = 2 | |
Leiomyosarcoma | N = 2 | |
Hemangiosarcoma | N = 2 | |
Total number of malignant tumors: 22 | Total of specimen: 73 | |
Benign tumors | Number of specimen | |
Epithelial origin | ||
Schwannoma | N = 2 | |
Pituitary adenoma | N = 4 | |
Follicle adenoma (Thyroid gland) | N = 3 | |
Mesenchymal orgin | ||
Myoma uteri | N = 15 | |
Total number of benign tumors: 4 | Total of specimen: 24 |
was expressed in human and rat tumor tissues, and thus, which of these receptors react with the LT-R antagonists that were administered to the rats. CysLT-positive cells that were diffusely distributed within the tumor tissue were observed in all of the rat spontaneous tumors. Positive reactivity to the anti-CysLT antibodies was detected not only in tumor cells, but also in fibroblasts, mast cells, and endothelial cells [
Under light microscopy, apoptotic cells were detected in leukotriene receptor antagonist-treated rat tumors. These apoptotic cells were diffusely distributed throughout the tumor, and were not detected in the non-treated group [
Furthermore, alterations in the nervous tissue of tumor tissues were observed that varied according to each leukotriene receptor antagonist administered. In rat tumors that were treated with Zafirlukast or Monterukast sodium, destruction of the myelin sheath was observed, although no degeneration of the axon was seen [
In various human tumors and in the rat spontaneous tumors, the presence of numerous mast cells and the expression of leukotriene receptors were common features of tumors of epithelial or mesenchymal origin, whether they were malignant or benign tumors. These pathological findings show that anti-leukotriene therapy has a therapeutic effect against spontaneous tumors in rats. This is the first report of such findings.
Moreover, the results suggest a lack of side effects of this treatment, since no apoptotic effects or adverse effects on nervous tissue were evident in non-tumor areas. The effects of the leukotriene receptor antagonists were
diverse. Thus, leukotriene receptor antagonist treatment not only affected tumor cells by inducing apoptosis, but inhibited angiogenesis by inducing apoptosis of endothelial cells and smooth muscle cells of blood vessels.
Furthermore, this treatment also inhibited neurogenesis in the tumor, presumably because the leukotriene receptors are diffusely distributed in the tumor tissue from rats with spontaneous tumors. Therefore, anti-leuko- triene treatment may not only result in a decline in the amount of tumor tissue, but pain in terminal patients might also be eased. Although these data are very interesting, more data need to be accumulated in order to understand the mechanism of these effects since some differences were seen in the effect of each antagonist. In any case, since leukotriene receptors are also expressed in various human tumors, the present results obtained in rats suggest that similar effects in human patients might be expected.
The pathological findings regarding mast cells and leukotriene receptors that were obtained in this study of rat spontaneous tumors, also appeared to apply to human tumors, regardless of the kind of tumor. There is therefore a high possibility that allergic reactions including infiltration of mast cells are involved in the development and progression of tumors. In addition, as for the endometriosis lesion as well, we have already detected the existence of leukotriene receptor [
In particular, leukotrienes and leukotriene receptors seem to play an important role in these allergic reactions and their regulation might therefore control tumor progression. Because our findings were observed in common with the benign tumor and the malignant tumor both, it may have the effect, which prevents a benign tumor from becoming malignancy or the recurrence of the malignant tumor. And, effect on metastasis inhibition will be anticipated because this therapy prevents angiogenesis and nerve formation, too. Anti-leukotriene treatment may not only have therapeutic effects on, but also preventive effects against tumors. Our data might be expected to lead to clinical applications of anti-leukotriene treatment in oncotherapy.
MasaoSugamata,TomomiIhara,MihoSugamata, (2015) Mast Cell Infiltration and Leukotriene Receptor Expression in Various Tumors: Possible Clinical Application of Common Pathological Findings Concerned with Tumor Development. Journal of Cancer Therapy,06,606-612. doi: 10.4236/jct.2015.67066