Synergistic effects of Rapamycin and Fluorouracil to treat a gastric tumor in a PTEN conditional deletion mouse model.

The tumor suppressor gene phosphatase and tensin homolog (PTEN) in PI3K/Akt/mTOR pathway is essential in inhibiting tumor growth and metastasis. However, whether the mutation of PTEN gene could induce tumorigenesis and impact the treatment of gastric cancer is still unclear. The purpose of the study was to investigate the combined treatment of gastric tumorigenesis using Rapamycin and Fluorouracil (5-Fu) through interfering with the Akt/mTOR pathway in a mouse model with PTEN conditional deletion. Three groups of mice were exposed for 5 days to Rapamycin and 5-Fu separately and together. The gene expression of the Akt/mTOR pathway, the protein expression of caspase-3 and p-Akt, p-S6K and p-4EBP1, and the pathological changes in stomachs were analyzed. Our study demonstrates that the conditional PTEN deletion in the cells of glandular stomach induces hyperplastic gastric tumors in mice. The combined Rapamycin administration with 5-Fu resulted in better outcomes than their separate administration for the treatment of gastric cancer by inhibiting the mTOR signal pathway. Our study indicates that Rapamycin has a synergistic interaction with chemotherapeutic 5-Fu, and demonstrates a potential therapeutic combination treatment on glandular stomach tumor with PTEN functional absence or aberrantly activated Akt/mTOR pathway. It provides important insights into the inhibition of the Akt/mTOR pathway in gastric cancer clinical therapy.

Atorvastatin induces mitochondrial dysfunction and cell apoptosis in HepG2 cells via inhibition of the Nrf2 pathway.

Atorvastatin (ATO) is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor widely used to treat hypercholesterolemia. However, clinical application is limited by potential hepatotoxicity. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a master regulator of cellular antioxidants, and oxidative stress is implicated in statin-induced liver injury. This study investigated mechanisms of ATO-induced hepatotoxicity and potential mitigation by Nrf2 signaling. ATO reduced Nrf2 and antioxidant enzyme superoxide dismutase-2 (SOD2) expression in human hepatocarcinoma HepG2 cells. ATO also induced concentration-dependent HepG2 cell toxicity, reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential (MMP) and cellular adenosine triphosphate (ATP). Further, ATO induced mitochondria-dependent apoptosis as indicated by increased Bax/Bcl-2 ratio, cleaved caspase-3, mitochondrial cytochrome c release and Annexin V-fluorescein isothiocyanate/propidium iodide staining. Tert-butylhydroquinone enhanced Nrf2 and SOD2 expression, and partially reversed ATO-induced cytotoxicity, ROS accumulation, MMP reduction, ATP depletion and mitochondria-dependent apoptosis. In conclusion, the present study demonstrates that ATO induces mitochondrial dysfunction and cell apoptosis in HepG2 cells, at least in part, via inhibition of the Nrf2 pathway. Nrf2 pathway activation is a potential prevention for ATO-induced liver injury.

Subchronic toxicity study of yttrium nitrate by 90-day repeated oral exposure in rats.

Concerns regarding the adverse effects of long-term exposure to low levels of rare earth elements (REEs) from foods on human health have arisen in recent years. Nevertheless, no official acceptable daily intake (ADI) has yet been proposed for either total REEs or individual REE. In accordance with the Organization for Economic Co-operation and Development (OECD) testing guideline, the present study was undertaken to evaluate the subchronic toxicity of yttrium, a representative heavy REE with higher contaminated level in foods in China, to achieve a no observed adverse effect level (NOAEL) which is a critical basis for the establishment of an ADI. Yttrium nitrate was orally administered to rats at doses of 0, 10, 30 and 90 mg/kg/day for 90 days followed by a recovery period of 4 weeks. The following toxicity indices were measured: mortality, clinical signs, daily food consumption and weekly body weight; urinalysis, hematology, blood coagulation, clinical biochemistry and histopathology at the end of administration and recovery periods. No toxicologically significant changes were found in any yttrium-treated group as compared to the concurrent control group. Under the present experimental condition, the NOAEL in rats was thus set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium.

Metallothioneins attenuate paraquat-induced acute lung injury in mice through the mechanisms of anti-oxidation and anti-apoptosis.

Paraquat (PQ) is a widely used herbicide, and lung is the primary target of PQ poisoning. Metallothionein (MT) is a potent antioxidant and free radical scavenger, and has been shown to play a protective role in lung injury induced by different stressors. This study was undertaken to evaluate the protective potential of MT against PQ-induced acute lung injury using MT-I/II null (MT(-/-)) mice. Wild-type (MT(+/+)) mice and MT(-/-) mice were given one intragastric administration of 50mg/kg PQ for 24h, and it was revealed that MT(-/-) mice were more susceptible to PQ-induced acute lung injury than MT(+/+) mice evidenced by the following findings. As compared with MT(+/+) mice, MT(-/-) mice presented more severe histopathological lesions in the lung, higher pulmonary malondialdehyde content, and more reduced pulmonary antioxidative enzymes activities. PQ also induced more apoptosis in pneumocytes from MT(-/-) mice, and the expressions of apoptosis-related proteins Bax, Bcl-2, cleaved-caspase-3, and the ratio of Bax/Bcl-2 were all more significantly increased in PQ-treated MT(-/-) mice. Our results clearly demonstrate that endogenous MT can attenuate PQ-induced acute lung injury, possibly through the mechanisms of anti-oxidation and anti-apoptosis.

[The immunological responses induced by Mycobacterium tuberculosis heat shock protein 16.3 and its synthetic peptide in mice].

OBJECTIVE: To evaluate the immune responses and resistance against Mycobacterium tuberculosis (MTB) infection in the mice induced by HSP16.3 of MTB and its synthetic peptide. METHODS: BALB/c mice were immunized subcutaneously 3 times at 2 week interval at the base of tail. The doses of HSP16.3 protein and synthetic peptide were both 50 microg each time. A single dose of BCG (5 x 10(6) CFU/mouse) was used to immunize the mice. The concentrations of specific antibodies in serum obtained at 0, 2, 4, 6, 8 weeks after the first immunization and the titer of serum obtained at 8th week, were analyzed by enzyme linked immunosorbent assay (ELISA). Four weeks after the final immunization, 8 mice from each group were sacrificed and single-cell suspensions of splenocytes were prepared, some of which were used for lymphocyte proliferation by MTT colorimetry with HSP16.3 stimulation, and the remaining cells were used for IFN-gamma level assay by sandwich ELISA. The remaining mice in each group were challenged intravenously with 10(5) colony forming units (CFU) of MTB H(37)Rv and were sacrificed 4 weeks after infection, and the number of bacteria in the spleens and lungs were determined by plating serial dilutions of homogenized tissue on Middlebrook 7H10 agar. The statistical significance of differences among means was assessed by an LSD-t test. RESULTS: The level of specific antibody to HSP16.3 protein and the peptide increased rapidly in the former 4 weeks and moderately in the later weeks. The average antibody-specific titers of 3 experiment groups (HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA) were higher than the BCG group. The indexes of spleen lymphocyte proliferation (SI) of the 3 experiment groups (3.13 +/- 0.18, 3.21 +/- 0.21 and 2.40 +/- 0.15) were significantly higher than the BCG group (1.67 +/- 0.12) and the saline group (1.04 +/- 0.09) respectively. The SI of HSP16.3 protein + DDA + MPL group (3.13 +/- 0.18) and synthetic peptide + DDA + MPL group (3.21 +/- 0.21) were higher than the synthetic peptide + IFA group (2.40 +/- 0.15). The IFN-gamma levels induced among the 3 experiment groups [(182 +/- 6), (194 +/- 9) and (179 +/- 8) mg/L] were lower than the BCG group [(275 +/- 10) mg/L], but higher than the saline group [(71 +/- 3) mg/L]. The IFN-gamma level induced among the 3 experiment groups did not show any marked difference. Although the protection induced by HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA all showed resistance against MTB H(37)Rv infection in the spleens or lungs (the bacterial logarithmic loads of spleen: 6.74 +/- 0.14, 6.60 +/- 0.13 and 6.81 +/- 0.28; the bacterial logarithmic loads of lung: 5.81 +/- 0.21, 5.74 +/- 0.27 and 6.65 +/- 0.32), none of them was better than the conventional BCG (the bacterial logarithmic loads of spleen and lung: 5.95 +/- 0.17 and 5.62 +/- 0.23). CONCLUSIONS: Both HSP16.3 and its synthetic peptide can be considered as TB vaccine candidates or effective components in TB vaccines.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

OBJECTIVE: To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. METHODS: The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. RESULTS: The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. CONCLUSIONS: Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.