The central role of Sphingosine kinase 1 in the development of neuroendocrine prostate cancer (NEPC): A new targeted therapy of NEPC.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is often diagnosed as a sub-type from the castration-resistant prostate cancer (CRPC) recurred from the second generation of anti-androgen treatment and is a rapidly progressive fatal disease. The molecular mechanisms underlying the trans-differentiation from CRPC to NEPC are not fully characterized, which hampers the development of effective targeted therapy. METHODS: Bioinformatic analyses were conducted to determine the clinical correlation of sphingosine kinase 1 (SphK1) in CRPC progression. To investigate the transcriptional regulation SphK1 and neuroendocrine (NE) transcription factor genes, both chromosome immunoprecipitation and luciferase reporter gene assays were performed. To demonstrate the role of SphK1 in NEPC development, neurosphere assay was carried out along with several biomarkers determined by quantitative PCR and western blot. Furthermore, in vivo NEPC xenograft models and patient-derived xenograft (PDX) model were employed to determine the effect of SphK1 inhibitors and target validation. RESULTS: Significant prevalence of SphK1 in NEPC development is observed from clinical datasets. SphK1 is transcriptionally repressed by androgen receptor-RE1-silencing transcription factor (REST) complex. Furthermore, sphingosine 1-phosphate produced by SphK1 can modulate REST protein turnover via MAPK signaling pathway. Also, decreased REST protein levels enhance the expression of NE markers in CRPC, enabling the transition to NEPC. Finally, specific SphK1 inhibitors can effectively inhibit the growth of NEPC tumors and block the REST protein degradation in PDX. CONCLUSIONS: SphK1 plays a central role in NEPC development, which offers a new target for this lethal cancer using clinically approved SphK1 inhibitors.

Sphingosine kinases protect murine embryonic stem cells from sphingosine-induced cell cycle arrest.

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule regulating organogenesis, angiogenesis, cell proliferation, and apoptosis. S1P is generated by sphingosine kinases (SPHK1 and SPHK2) through the phosphorylation of ceramide-derived sphingosine. Phenotypes caused by manipulating S1P metabolic enzymes and receptors suggested several possible functions for S1P in embryonic stem cells (ESCs), yet the mechanisms by which S1P and related sphingolipids act in ESCs are controversial. We designed a rigorous test to evaluate the requirement of S1P in murine ESCs by knocking out both Sphk1 and Sphk2 to create cells incapable of generating S1P. To accomplish this, we created lines mutant for Sphk2 and conditionally mutant (floxed) for Sphk1, allowing evaluation of ESCs that transition to double-null state. The Sphk1/2-null ESCs lack S1P and accumulate the precursor sphingosine. The double-mutant cells fail to grow due to a marked cell cycle arrest at G2/M. Mutant cells activate expression of telomere elongation factor genes Zscan4, Tcstv1, and Tcstv3 and display longer telomeric repeats. Adding exogenous S1P to the medium had no impact, but the cell cycle arrest is partially alleviated by the expression of a ceramide synthase 2, which converts excess sphingosine into ceramide. The results indicate that sphingosine kinase activity is essential in mouse ESCs for limiting the accumulation of sphingosine that otherwise drives cell cycle arrest.

Adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase mutants combined with gemcitabine harbor a safe cancer treatment profile.

The purpose of this analysis was to investigate the enzyme activity and specificity of adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) mutants in combination with gemcitabine. Compared to herpes simplex type 1 thymidine kinases (HSV-TK) and other known dNKs, this Dm-dNK enzyme has a broader substrate specificity and a higher catalytic rate. We created the Dm-dNK mutants (dNKmut) by site-directed mutagenesis at the sites of 244E, 245S, 251S, and 252R, with the last 10 amino acids in the amino acid sequence randomly alternated. We subsequently evaluated the enzyme activity and substrate specificity. The engineered enzymes showed a relative increase in phosphorylation in the nucleoside analogs of gemcitabine (dFdC, 2',2'-difluoro-deoxycytidine) compared to the wild-type enzyme. The dNKmut enzymes were expressed in breast (Bcap37) and gastric (SGC-7901) cancer cell lines. In studying the sensitivity of the cell lines to dFdC, conditionally replicative adenovirus (CRAd) ZD55-dNKmut showed higher expression and enzymatic activity than the replication-defective adenovirus Ad-dNKmut in cancer cells, but with less cytotoxicity to cancer cells than that of Ad-dNKmut. Our data suggest that the triple phosphorylated dFdC catalyzed by dNKmut inhibited the replication of adenovirus with a simultaneous positive therapeutic effect on cancer cells. Therefore, concomitant use of the ZD55-dNKmut and dFdC could be a novel targeted strategy in suicide gene therapy with safe control of excessive virus replication.

[Primary investigation on metabolism of hpt gene in transgenic rice modified with cowpea trypsin inhibitor gene in rats].

OBJECTIVE: The metabolism characteristic in the gastrointestinal tract and possibility of transfer from gastrointestinal tract into liver and kidney was investigated. METHODS: 48 SD rats were allotted into 8 subgroups randomly, 6 groups rats of them were fed with transgenic rice diets and 2 groups rats were fed with non-transgenic rice diets. All rats were fed with transgenic and non-transgenic rice for two weeks. All rats were fast and sacrificed, the contents of stomach, jejunum, the lower part of ileum, cecum and rectum were taken out at 2, 4, 6, 8, 10 and 24 hours respectively after eating final diets. Fed two groups of SD rats with transgenic diet and non-transgenic rice diet respectively for 4 weeks, the liver and kidney were sampled and stored. All samples was examined by PCR. RESULTS: The fragment of hpt was found the was only detected in stomach and jejunum, but none of the five fragments was detected in the lower part of ileum, cecum and rectum. The degradation of hpt was time-relationship and long-length fragment relationship, the longer was the action time, the more serious was the degradation. Foreign hpt gene was not detected in liver and kidney. CONCLUSION: hpt gene was easy to degraded in gastrointestinal tract and was not detectable in hepatic tissue and nephridial tissues.

[Study on the food safety of the marker gene expressed protein HPT].

OBJECTIVE: This article studied on the food safety of HPT protein (Hygromycin B Phosphotransferase), a kind of plant selectable marker gene expression product, which mainly included the quantity and quality detection of HPT in the SCK transgenic rice plants, the possible dietary intakes and its stability in the simulative digestive system. METHODS: HPT cDNA fragment was inserted into the prokaryotic expressing vector pBV222 for the expression of 6His-HPT fusion protein. The inclusion bodies were denatured by 8 mol/L urea and purifiee with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT were used to immunize rabbit and BALB/C mice for the preparation of the polyclonal and monoclonal antibodies, and the double-Ab sandwich ELISA to detect HPT was established then. Concerning the sensitivity assessment of HPT protein, the amino acid sequences of HPT was compared with the other allergens and the simulative digestive experiment in vitro was carried out too. RESULTS: 6His-HPT protein was obtained with the purity up to 95%. The specificity of antisera and ascites was detected by Western blot, which showed specific binding reaction between the antibodies to the purified 6His-HPT proteins and their expressed product. The double-Ab sandwich ELISA system was established successfully with the sensitivity of 30 ng/ml. The HPT expression level in the leaves of transgenic rice was proved in the range of 80 - 150 ng/ml, yet we cannot detect HPT protein in the seeds of the same rice. There showed no comparability in the amino acid sequences between HPT and other allergens, and HPT protein was digested quickly in the simulated stomach and intestines liquids. CONCLUSION: All the results showed that the expression level of HPT in the SCK transgenic rice plants is very low and they are not stable in the simulative digestive system. So we inferred that under the current detection level, HPT protein might not be able to induce any safety problem. Yet further study on the acute toxicity and sensitivity experiments by the animals need to be carried out, and the results will be reported in the near future.