Novel method for evaluating the health condition of mice in space through a video downlink.

Clarification of the criteria for managing animal health is essential to increase the reliability of experiments and ensure transparency in animal welfare. For experiments performed in space, there is no consensus on how to care for animals owing to technical issues, launch mass limitation, and human resources. Some biological processes in mammals, such as musculoskeletal or immune processes, are altered in the space environment, and mice in space can be used to simulate morbid states, such as senescence acceleration. Thus, there is a need to establish a novel evaluation method and evaluation criteria to monitor animal health. Here, we report a novel method to evaluate the health of mice in space through a video downlink in a series of space experiments using the Multiple Artificial-gravity Research System (MARS). This method was found to be more useful in evaluating animal health in space than observations and body weight changes of the same live mice following their return to Earth. We also developed criteria to evaluate health status via a video downlink. These criteria, with "Fur condition" and "Respiratory" as key items, provided information on the daily changes in the health status of mice and helped to identify malfunctions at an early stage. Our method and criteria led to the success of our missions, and they will help establish appropriate rules for space experiments in the future.

Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in an HER2 transgenic model.

Aberrant sphingolipid metabolism has been reported to promote breast cancer progression. Sphingosine kinase 1 (SphK1) is a key metabolic enzyme for the formation of pro-survival S1P from pro-apoptotic ceramide. The role of SphK1 in breast cancer has been well studied in estrogen receptor (ER)-positive breast cancer; however, its role in human epidermal growth factor 2 (HER2)-positive breast cancer remains unclear. Here, we show that genetic deletion of SphK1 significantly reduced mammary tumor development with reduced tumor incidence and multiplicity in the MMTV-neu transgenic mouse model. Gene expression analysis revealed significant reduction of claudin-2 (CLDN2) expression in tumors from SphK1 deficient mice, suggesting that CLDN2 may mediate SphK1's function. It is remarkable that SphK1 deficiency in HER2-positive breast cancer model inhibited tumor formation by the different mechanism from ER-positive breast cancer. In vitro experiments demonstrated that overexpression of SphK1 in ER-/PR-/HER2+ human breast cancer cells enhanced cell proliferation, colony formation, migration and invasion. Furthermore, immunostaining of SphK1 and CLDN2 in HER2-positive human breast tumors revealed a correlation in high-grade disease. Taken together, these findings suggest that SphK1 may play a pivotal role in HER2-positive breast carcinogenesis. Targeting SphK1 may represent a novel approach for HER2-positive breast cancer chemoprevention and/or treatment.

Construction and expression of vectors encoding biologically active rodent gonadotropins.

The gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are important hormones in vertebrate reproduction. The isolation of gonadotropins from the pituitary gland is sub-optimal, as the cross-contamination of one hormone with another is common and often results in the variation in the measured activity of LH and FSH. The production of recombinant hormones is, therefore, a viable approach to solve this problem. This study aimed to express recombinant rat, mouse, and mastomys FSH and LH in Chinese hamster ovary (CHO) cells. Their common α-subunits along with their hormone-specific ß-subunits were encoded in a single mammalian expression vector. FSH from all three species was expressed, whereas expression was achieved only for the mouse LH. Immunohistochemistry for rat alpha subunit of glycoprotein hormone (αGSU) and LHß and FSHß subunits confirmed the production of the dimeric hormone in CHO cells. The recombinant rodent gonadotropins were confirmed to be biologically active; estradiol production was increased by recombinant FSH in granulosa cells, while recombinant LH increased testosterone production in Leydig cells.

Sphingosine Kinase 1 expression in peritoneal macrophages is required for colon carcinogenesis.

Accumulating evidence suggests that the sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) pathway plays a pivotal role in colon carcinogenesis. Our previous studies indicate that the SphK1/S1P pathway mediates colon carcinogenesis at least by regulating cyclooxygenase 2 (COX-2) expression and prostaglandin E2 (PGE2) production. However, the mechanisms by which this pathway regulates colon carcinogenesis are still unclear. First, we show that SphK1 deficient mice significantly attenuated azoxymethane (AOM)-induced colon carcinogenesis as measured by colon tumor incidence, multiplicity, and volume. We found that AOM activates peritoneal macrophages to induce SphK1, COX-2, and tumor necrosis factor (TNF)-α expression in WT mice. Interestingly, SphK1 knockout (KO) mice revealed significant reduction of COX-2 and TNF-α expression from AOM-activated peritoneal macrophages, suggesting that SphK1 regulates COX-2 and TNF-α expression in peritoneal macrophages. We found that inoculation of WT peritoneal macrophages restored the carcinogenic effect of AOM in Sphk1 KO mice as measured by aberrant crypt foci (ACF) formation, preneoplastic lesions of colon cancer. In addition, downregulation of SphK1 only in peritoneal macrophage by short hairpin RNA (shRNA) reduced the number of ACF per colon induced by AOM. Intraperitoneal injection of sphingolipids demonstrates that S1P enhanced AOM-induced ACF formation, while ceramide inhibited. Finally, we show that SphK inhibitor SKI-II significantly reduced the number of ACF per colon. These results suggest that SphK1 expression plays a pivotal role in the early stages of colon carcinogenesis through regulating COX-2 and TNF-α expression from activated peritoneal macrophages.

Sphingosine kinase 1 expression enhances colon tumor growth.

BACKGROUND: Accumulating evidence suggests that sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate pathway plays a pivotal role in colon carcinogenesis. METHODS: To further support the evidence, we investigated the effects of SphK1 using three separate animal models: SphK1 knockout mice, SphK1 overexpressing transgenic mice, and SphK1 overexpression in human colon cancer xenografts. Using azoxymethane (AOM, colon carcinogen), we analyzed colon tumor development in SphK1 KO and SphK1 overexpression in intestinal epithelial cells regulated by a tet-on system. Then, we analyzed subcutaneous tumor growth using xenografts of HT-29 human colon cancer cell. Finally, immunohistochemical analyses for SphK1 and COX-2 were performed on human colon cancer tissue microarray. RESULTS: SphK1 KO mice, compared to wild-type mice, demonstrated a significant inhibition in colon cancer development induced by AOM (58.6% vs. 96.4%, respectively, P < 0.005). Tumor multiplicity (1.00 vs. 1.64 per colon, respectively, P < 0.05) and tumor volume (14.82 mm3 vs. 29.10 mm3, P < 0.05) were both significantly reduced in SphK1 KO mice compared to wild-type mice. Next, SphK1 overexpression in HT-29 enhanced tumor growth as compared to GFP control in nude mice (229.5 mm3 vs. 90.9 mm3, respectively, P < 0.05). Furthermore, overexpression of SphK1 in intestinal epithelial cells significantly enhances AOM-induced colon tumor formation (P < 0.05). Lastly, SphK1 and COX-2 intensity tended to reduce overall survival of late stage colon cancer patients. CONCLUSIONS: SphK1 expression regulates the early stage of colon carcinogenesis and tumor growth, thus inhibition of SphK1 may be an effective strategy for colon cancer chemoprevention.

The impact of sphingosine kinase-1 in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) has a high reoccurrence rate and an extremely low survival rate. There is limited availability of effective therapies to reduce the rate of recurrence, resulting in high morbidity and mortality of advanced cases. Late presentation, delay in detection of lesions, and a high rate of metastasis make HNSCC a devastating disease. This review offers insight into the role of sphingosine kinase-1 (SphK1), a key enzyme in sphingolipid metabolism, in HNSCC. Sphingolipids not only play a structural role in cellular membranes, but also modulate cell signal transduction pathways to influence biological outcomes such as senescence, differentiation, apoptosis, migration, proliferation, and angiogenesis. SphK1 is a critical regulator of the delicate balance between proliferation and apoptosis. The highest expression of SphK1 is found in the advanced stage of disease, and there is a positive correlation between SphK1 expression and recurrent tumors. On the other hand, silencing SphK1 reduces HNSCC tumor growth and sensitizes tumors to radiation-induced death.  Thus, SphK1 plays an important and influential role in determining HNSCC proliferation and metastasis. We discuss roles of SphK1 and other sphingolipids in HNSCC development and therapeutic strategies against HNSCC.