Studies On Related Mechanisms For The Development Of Diethylstilbestrol (DES)-induced Prolactinoma And Experimental Treatment Of Melatonin In Rats

Part 1 Study on dynamic morphological changes and expressions of angiogenesis-related factors during the development of diethylstilbestrol (DES)-induced prolactinoma in ratsBackground and purpose Morphological changes are one of the basic signs of the initiation, development and formation of pituitary tumor. However, the dynamic morphological changes of pituitary tissues during tumor development are rarely discussed. Angiogenesis is the constitutive base and vital mark of tumor enlargement and invasion. Based on experimental pituitary tumor induced by diethylstilbestrol (DES) in rats, we explored the dynamic morphological changes of pituitary tissues and expressions of angiogenesis-related factors during the formation of DES-induced pituitary tumor.Methods 51 female Wistar rats were randomly allocated into vehicle-controlled group and DES group. Rats in DES group received intraperitoneal injection of DES (5mg/kg, twice a week), while their controls were correspondingly administered with sun-flower seed oil. At 4 week (w), 8w and 12w, animals were intracardially perfused and pituitary tissues were harvested for morphological investigations. HE staining was used to confirm the occurance of tumor, and each section was scored based on HE observations. Immunohistochemical method was undertaken to identify the tumor phenotype, in which PRL, ACTH, GH, and LH reactivities were examined. Expressions of three angiogenesis-related factors: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and CD31 were investigated. Body weight and fur change were observed at each time point. Prior to perfusion, MRI scans were undertaken in 3 randomly selected rats from each group to study the in vivo growth profile of the pituitary gland.Results 1 Body weight and appearance changes A rapid body weight gaining was observed in vehicle-controlled rats, whose body weights were 189.6±15.6g, 256.4±27.0g, and 244.5±23.8g after 4, 8 and 12 weeks of vehicle treatment. In contrast, rats in DES-treated group were 164.0±11.4g, 209.2±13.5g and 208.4±21.0g, significantly decreased compared with their controls (p<0.01 at each time point). Rats in DES-treated group began losing hair at 2 week, which appeared more severely in costal region and backneck。MRI The mid-sagittal areas of DES-treated pituitary were significantly larger compared to their controls at each time point (p<0.01 at each time point) based on T2WI investigations. MRI scans also demonstrated gradually upward enlargement of the pituitary tissue with no apparent changes in its base. Histological observations and tumor phenotype identification Hyperproliferation and gland cavity narrowing initially occurred in DES-treated pituitary tissues, which were then rapidly occupied by tumor-like cells. Afterwards, nuclear condensation was observed within areas with tumor-like changes. Endothelial cell emergence, double-wall emergence and cavity extension were concurrently observed in the same section. At each time point, the incidence rates of tumor were 40% (4/10), 60% (6/10) and 80% (8/10). Immunohistochemical stainings confirmed the tumor phenotype as prolactinoma, with dotted distribution of ACTH positive cells within these areas. Expressions of GH and LH were negative. Expressions of angiogenesis-related factors Each stage of the development of angiogenesis saw expressions of EGF, VEGF, and CD31 in the endothelial cells, and /or vascular walls and peripheral cells in areas with tumor-like changes.Conclusions (1) The growth of the pituitary gland is characterized by upward extension during the development of pituitary tumor. (2) Histologically, the thorough process of tumor development was composed of hyperproliferation to tumorigenesis, local to whole, solid to mixture joined by newly-generated vessels. The self reconstruction of pituitary tissues represents a morphological base for the transformation of pituitary tumor from benign to malignant, and from non-invasion to invasion. (3) Body weight reduction and fur loss can be regarded as signs of the development of pituitary tumor under the induction of DES. (4) EGF, VEGF, and CD31 were expressed in the endothelial cells and pituitary cells in their respective manner, suggesting they may function and cooperate with each other at each stage of angiogenesis.Expressions and activities of two ubiquitous calpains (μ-and m-calpains) during the development of diethylstilbestrol (DES)-induced prolactinoma in ratsBackground and Purpose Frequently occurred, the pituitary tumors account for 15% of the intracranial tumors, with high frequency of the pituitary microadenoma among normal people. Studies have demonstrated that the development and formation are based on the mutual actions between intrapituitary and extrapituitary factors in different levels. Calpains represent a Ca2+ dependent protease family comprised of more than 14 members, among whichμ- and m-calpains are ubiquitous calpains widely distributed in a variety of tissues, where they exert extensive biological functions. Data available have demonstrated the involvement of calpains in the secretion process of several lines of endocrine cells. However, roles of calpains in the development of pituitary tumor are rarely discussed. Based on the rat model of diethylstilbestrol (DES)-induced prolactinoma, we focused our main research on the expressions, activities of calpains and their possible relationships to prolactin secretion during the development of prolactinoma.Material and Methods 93 female Wistar rats were randomly allocated into vehicle-controlled group and DES-treated group, which were used for morphological and biomolecular investigations. In DES group, rats received DES intraperitoneally (5mg/kg, twice a week), and in the vehicle-controlled group, rats were intraperitoneally injected sun-flower seed oil as vehicle (1ml/kg, twice a week). At 4 week (w), 8w and 12w, some animals were intracardially perfused and pituitary tissues were harvested for morphological investigations. HE staining was used to confirm the occurance of tumor. Immunohistochemical method was employed to study the expressions of prolactin (PRL) and ACTH in areas with tumor-like changes. Expressions and distributions of two ubiquitous calpains as well as their downstream effector caspase-3 were also visualized by immunohistochemial investigation. Double immunofluorescence-labeling were used to cofirm the potential relationship betweenμ-calpain and PRL, and that between PRL and m-calpain. At the same time point, some other animals were anaesthetized, and the abdomen aorta was exposed for blood collection, soon after which the pituitary tissues were harvested for future studies after fluid N2 freezing. ELISA was used to study the serum level of ACTH. Cytosol expressions of calpains at each time point were investigated by using Western Blotting. Zymography was undertaken to study the cytosol and membrane activities of both ubiquitous calpains.Results 1 induction rate The induction rates in DES-treated group at each time point were 40% (4/10), 60% (6/10) and 80%(8/10). 2 immunohistochemistry and immunofluorescence Pituitary tissues with tumor development demonstrated a strong reactivity to prolactin and dotted ACTH positivity. In tumor located regions, expressions of bothμand m-calpain were increased, although physiological expressions of two ubiquitous calpains were seen in both the anterior lobe and the intermediate lobe. Double immunofluorescence-labeling confirmed the co-localization ofμ-calpain and PRL, as well as that of m-calpain and PRL. The distributions of two calpains were similar to that of PRL, whose secretion is uniquely characterized by extravasation in prolactinoma. 3 Western Blotting At 4w, cytosol expression of m-calpain was significantly increased, followed by a sharp reduction at 8w and an apparent increase at 12w. However, cytosol expressions ofμ-capain in DES group remained low levels at both 4w and 8w, followed by a significant increase at 12w. 4 Casein Zymography (ⅰ) Low-leveled cytosol activities ofμ-calpain were seen in the veicle-controlled group at each time point. In contrast, cytosol activity ofμ-calpain in DES group was 0 at 4w, followed by its emergence at 8w and significant increase at 12w. (ⅱ) The membrane activity ofμ-calpain in vehicle-controlled was present only at 8w. Its activity remained 0 at both 4w and 8w in both DES groups, and was significantly increased at 12 week. (ⅲ) At 4w, a comparatively high-leveled cytosol m-calpain activity was present in the DES group, followed by a sharp reduction at 8w, and an apparent reincreasing at 12w. (ⅳ) The membrane m-calpain activities remained low levels in the DES group at both 4w and 8w, followed by a significant increase at 12w. 5 ELISA for serum ACTH At 4w, the serum ACTH level was significantly reduced, and was restored to levels similar to those in the vehicle-treated group at 8w and 12w.Conlusions (1) Intraperitoneal injection of DES can induce prolactinoma in rats, and prolonged injection increased the incidence rates of prolactinoma. (2) With the induction of the development of prolactinoma, DES promoted the expressions of two ubiquitous calpains in both AL and IL. The two calpains were colocalized with PRL intracellarly in the AL, suggesting they are highly related to the functions of prolactatrophs and may represent one of the substantial bases of the pathophysiological functions of pituitary tumor. Cleaved caspase-3 was selectively and weakly expressed in hypophyseal cells, excluding the possibility of its activation by highly activated calpains, leading to apoptosis-induced cellular injury. (3) The subsequent restoration of serum ACTH occurred, in concurrence with the enhanced immunoreactivity of ACTH in the IL, suggesting the formation of hyperactivity in this hypophyseal part, which may exert roles in prolactin expressin and secretion in the AL through secreting several paracrine factors. (4) To explore the roles of two ubiquitous calpains in hormone secretion may be of vital importance in prolactinoma treatment clinically.Mechanisms for the treatment of melatonin in DES-induced prolactinoma in female Wistar rats Background and Purpose Prolactinoma is a type of frequently-occurred pituitary tumor clinically, which is mainly characterized by high serum prolactin and angiogenesis. In vivo and in vitro studies have demonstrated the anti-tumor effect of melatonin, but the detailed mechanisms remain elucidation. In the present research, we studied the effects of melatonin on expressions of angiogenesis-related factors EGF, VEGF, and CD31, as well as the expressions of bothμ-and m-calpains and their Ca2+-dependent activities during the development of DES-induced prolactinoma in female Wistar rats.Materials and Methods 64 female Wistar rats were randomly allocated into 5 groups. In group 1, rats received intraperitoneal injection of sun flower seed oil for 16 weeks. In group 2, rats were intraperitoneally administered with DES (5mg/kg, twice a week) for 16 weeks, while rats in group 3 was administered with DES for 12 weeks, followed by its discontinuation for 4 weeks. In group 4 and 5, rats received DES for 16 weeks, with subcutaneous administration of melatonin at doses of 0.25mg/day and 1.0mg/day at the beginning of the time point of 13 week. Body weight and fur changes were observed before each injection. Some rats were subjected to perfusion to harvest the pituitary tissues, which were used for HE staining, immunohistochemical studies for expressions of EGF, VEGF and CD31. In the other rats, 3ml of blood were collected from the abdomen aorta, which was used for determining the serum ACTH level by using ELISA. The pituitary tissues were harvested for cytosol expressions of two ubiquitous calpains and their cytosol and membrane activities.Results 1 Body weitht and fur Long-term treatment of DES significantly reduced body weight and fur loss, which could be inhibited by DES withdrawal and melatonin administration at different doses. 2 HE score Long-term treatment of DES significantly increased the HE score, which could be reduced by DES withdrawal and melatonin at different doses. 3 Expressions of EGF, VEGF and CD31 Expressions of EGF, VEGF, and CD31 were greatly enhanced after long-term DES administration, which could be reduced by DES withdrawal and melatonin at different doses. 4 Electronic microscopy Tumor-like cells contained multi-shaped secretion granuales, sheet-like bodies of the rough endoreticulum (RER) and displayed misplaced exocytosis. After DES withdrawal and melatonin treatment, misplaced exocytosis disappeared, granule extrusion was arrested. 5 Cytosol expressions of two ubiquitous calpains and their in vitro activities The cytosol expressions of two ubiquitous calpains were increased due to continuous treatment of DES for 16 weeks, with their membrane activities increased. DES discontinuation or melatonin administration at 2 doses could significantly reduce their cytosol expressions and in vitro activities. 6 Serum ACTH level A slight reduction of serum ACTH level was observed in group 2, and DES discontinuation and melatonin treatment could restore its serum level in a certain degree.Conclusions 1 Long term intraperitoneal administration of DES could induce angiogenesis and hyperproliferation in the pituitary gland in female Wistar rats, which provides an idea model for experimental prolactinoma treatment. Body weight and fur changes could be used to evaluate reaction of rats to DES and medical treatment. Serum ACTH level could be referred for modulating drug dose. 2 Expressions of EGF,VEGF, and CD31 were DES dependent, and melatonin may exert its anti-tumor effect by downregulating expressions of EGF, VEGF and CD31. 3 Increased cytosol expressions and upregulated membrane activities of bothμ-and m-calpains may be involved in the development of prolactinoma. And melatonin may reduce granule extrusion by inhibiting activities of two ubiquitous calpains. 4 The concurrence of high expressions of angiogenesis-related factors and cytosol calpains as well as their increased membrane activities indicated their cooperation during the development of experimental prolactinoma, which may represent the treatment targets of melatonin. The detailed roles of two ubiquitous calpains need further investigations.