Search

Hibernation/torpor enables certain mammals to survive under extreme environmental conditions. However, pharmacological induction of hibernation-like or torpor state in most mammals remains a huge challenge. Here we show that a natural product P57 promptly induces hypothermia and decreases energy expenditure in rodents. Mechanistically, P57 inhibits the kinase activity of pyridoxal kinase (PDXK), a key metabolic enzyme of vitamin B6 catalyzing phosphorylation of pyridoxal (PL), resulting in the accumulation of PL in hypothalamus to cause hypothermia. The hypothermia induced by P57 is significantly recuperated in the mice with knockout of PDXK in medial preoptic area (MPA). We further found that P57 and PL have consistent effects on gene expression regulation in hypothalamus, and both directly activate MPA neurons to induce hypothermia. Taken together, our findings demonstrate that P57 has a potential application in therapeutic hypothermia through regulation of vitamin B6 metabolism and PDXK serves as a previously unknown target of P57 in thermoregulation. In addition, P57 may serve as a chemical probe for exploring the neuron circuitry related to hibernation-like state in rodents.

No targeted therapies have been found to be effective against hepatocellular carcinoma (HCC), possibly due to the large degree of intratumor heterogeneity. We performed genetic analyses of different regions of HCCs to evaluate levels of intratumor heterogeneity and associate alterations with responses to different pharmacologic agents.We obtained samples of HCCs (associated with hepatitis B virus infection) from 10 patients undergoing curative resection, before adjuvant therapy, at hospitals in China. We collected 4-9 spatially distinct samples from each tumor (55 regions total), performed histologic analyses, isolated cancer cells, and carried them low-passage culture. We performed whole-exome sequencing, copy-number analysis, and high-throughput screening of the cultured primary cancer cells. We tested responses of an additional 105 liver cancer cell lines to a fibroblast growth factor receptor (FGFR) 4 inhibitor.We identified a total of 3670 non-silent mutations (3192 missense, 94 splice-site variants, and 222 insertions or deletions) in the tumor samples. We observed considerable intratumor heterogeneity and branched evolution in all 10 tumors; the mean percentage of heterogeneous mutations in each tumor was 39.7% (range, 12.9%-68.5%). We found significant mutation shifts toward CT and CG substitutions in branches of phylogenetic trees among samples from each tumor (P .0001). Of note, 14 of the 26 oncogenic alterations (53.8%) varied among subclones that mapped to different branches. Genetic alterations that can be targeted by existing pharmacologic agents (such as those in FGF19, DDR2, PDGFRA, and TOP1) were identified in intratumor subregions from 4 HCCs and were associated with sensitivity to these agents. However, cells from the remaining subregions, which did not have these alterations, were not sensitive to these drugs. High-throughput screening identified pharmacologic agents to which these cells were sensitive, however. Overexpression of FGF19 correlated with sensitivity of cells to an inhibitor of FGFR 4; this observation was validated in 105 liver cancer cell lines (P = .0024).By analyzing genetic alterations in different tumor regions of 10 HCCs, we observed extensive intratumor heterogeneity. Our patient-derived cell line-based model, integrating genetic and pharmacologic data from multiregional cancer samples, provides a platform to elucidate how intratumor heterogeneity affects sensitivity to different therapeutic agents.

The chemokine receptor CCR7 is highly expressed in dendritic cells (DCs), T cells, and other immune effector cells. The high-affinity ligand that can bind to CCR7 is the secondary lymphoid tissue c...

Aim: To investigate the antitumor immunity by a dendritic cell (DC) vaccine encoding secondary lymphoid chemokine gene and tumor lysate on murine prostate cancer. Methods: DC from bone marrow of C57BL/6 were transfected with a plasmid vector expressing secondary lymphoid chemokine (SLC) cDNA by Lipofectamine2000 liposome and tumor lysate. Total RNA extracted from SLC+lysate–DC was used to verify the expression of SLC by reverse transcriptase-polymerase chain reaction (RT-PCR). The immunotherapeutic effect of DC vaccine on murine prostate cancer was assessed. Results: We found that in the prostate tumor model of C57BL/6 mice, the adminstration of SLC+lysate–DC inhibited tumor growth most significantly when compared with SLC–DC, lysate–DC, DC or phosphate buffer solution (PBS) counterparts (P < 0.01). Immunohistochemical fluorescent staining analysis showed the infiltration of more CD4 + , CD8 + T cell and CD11c + DC within established tumor treated by SLC+lysate–DC vaccine than other DC vaccines (P < 0.01). Conclusion: DC vaccine encoding secondary lymphoid chemokine and tumor lysate can elicit significant antitumor immunity by infiltration of CD4 + , CD8 + T cell and DC, which might provide a potential immunotherapy method for prostate cancer. (Asian J Androl 2008 Nov; 10: 883–889)

We previously showed that introduction of a normal, neomycin-tagged human chromosome 8 reduced the metastatic capacity of C5F rat liver cancer cell line, which had high metastatic potential without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 8. We proceeded to define further the region harboring the metastasis suppressor gene(s) and to determine the random loss of human chromosome 8 by PCR amplification of sequence tag site (STS) markers.The national Center for Biotechnology Information (NCBI) databases were used as references of the relative genetic distances of the STS markers. C5F genomic DNA and A9/neo8 genomic DNA were used as negative and positive controls for chromosome 8 amplification, respectively. Genomic DNA was isolated and quantified from cultured hybrid clones (A9/C5F-1 and A9/C5F-2 microcell hybrid clones served as metastasis-unsuppressed groups; A9/C5F-4, A9/C5F-8 and A9/C5F-10 microcell hybrid clones served as metastasis suppressed groups). STS-PCR products were separated by electrophoresis through 2% agarose gel.Metastasis-suppressed microcell hybrid clones (A9/C5F-4, A9/C5F-8 and A9/C5F-10) conserved STS markers between D8S542 --D8S1973 (8p21.1-23.1). In contrast, metastasis-unsuppressed clones (A9/C5F-1 and A9/C5F-2) lacked several markers in this region. In attempts to refine the region retained in the microcell suppressed clones, more densely spaced STS markers in the human chromosome 8p21.1-23.1 were used. We found that the metastasis-suppressed clones retained 18cM region between D8S542 and D8S1973 (8P21.1-23.1), where as the metastasis-unsuppressed clones lacked the region.Our results suggest that a metastasis suppressor gene is located within the interval between D8S542 and D8S1973 on human chromosome 8p21.1-23.1.

To study the tumor cell killing function of T lymphocytes stimulated by dendritic cells (DC) and to analyze the differences of protein contents of exosomes in each type of cell.The exosomes of hepatic cell lines with high (P group) or low (F group) metastatic potentials were isolated by a process of four-step centrifugation and the collected exosomes were observed under an electron microscope (EM). The tumor cell killing experiment was performed by adding T lymphocytes activated by DC loaded with exosomes from corresponding P and F group cells and was studied using 3H-TdR experiments. The proteomic analysis was performed by surface-enhanced laser desorption/ ionization time of flight mass spectrometry (SELDI-TOF-MS ) on the exosomes of P and F group cells.The density distribution and content of exosomes in the P group were not equal to those in the F group observed by EM. The CD80, CD86, MHC-I and MHC-II in the P group were 64.27+5.00, 44.89+10.11, 84.35+19.89 and 59.03+19.37, and those in the F group were 71.53+4.85, 50.01+9.50, 80.68+29.87 and 58.86+21.11, respectively (P0.05, compared with the control group). The counts per minute value in the P group was 528.40+179.06 and 78.80+24.44 in the F group after being loaded with exosomes (P0.01, compared with the control group). There were significant differences between the proteins in the exosomes of hepatic cancer cell lines with high or low metastatic potentials.Exosomes have potential values of application in immunotherapy and in biotherapy for recurrences and metastases of hepatic carcinomas.

To study the relationship between lymphangiogenesis and lymphatic metastasis in mice bearing hepatic carcinoma and analyze the mechanism of the lymphatic metastasis.Hepatic carcinoma cell lines of high and low potentialities of lymphatic metastasis were injected into the footpads of Balb/c mice. Their metastases to lymph nodes were examined. The tumor tissues of each group were stained with 5'-nucleotidase-ALP to observe the lymphoangiogenesis. The total RNA of high and low metastatic potential cell lines were extracted for metastasis gene DNA array. The vascular endothelial cell growth factor C (VEGF-C) and VEGF-D of each cell line were detected using semi-quantitative RT-PCR and were further quantatively analyzed using real time PCR.The para-common iliac a. and renal hilar lymph nodes metastases of the high metastatic potential cells were significantly higher than in the controls (P0.05). The quantity of lymphatic vessels in the high metastasis group was significantly larger than that of the control group (P0.05). The expressions of CD44, E-cadherin, HER2/neu, H-Ras and VEGF-C in the high metastasis group were higher than those in the low metastasis group shown by the cDNA micro array experiment but the expressions of nm23A, nm23-E4, p16ink4a, CD61 were lower. The VEGF-C expression was higher and the VEGF-D was lower in the high metastasis group compared to those of the low metastasis group shown by semi-quantitative RT-PCR. The secretion of VEGF-D was significantly lower and the ratio of VEGF-C/VEGF-D was significantly higher in the high metastasis group than the low metastasis group (P0.05).The lymphatic metastasis of hepatic carcinoma is related to lymphoangiogenesis. The changes of VEGF-C and VEGF-D expressions might be a cause influencing the lymphoangiogenesis. VEGF-C/VEGF-D might be an effective parameter in affecting lymphatic metastases.

To investigate the effectiveness of reconstruction of immunological functions of T cells on the degree of metastases of mouse hepatocarcinoma and the mechanisms of their functioning.The T cell model of immunological functions in Balb/c nu/nu mice was established and the effectiveness of the model was evaluated. The mice were divided into 4 groups. The immunological functions of T cells in experiment groups of Balb/c nu/nu mice were reconstructed. Metastases of the cancer in lymph nodes in each group were examined histologically. The formation time and growth rate of the tumors were calculated. The expression of MHCI and II of the tumor cell line and the difference of expression of immune associated gene were detected by Th1-Th2-Th3 gene array.The ratio of CD3, CD4, CD8 and CD4/CD8 in the reconstructed group was higher than that in the control group. The average formation time was 7.7+/-0.6 days in Balb/c nu/nu mice and 11.5+/-1.3 days in Balb/c mice. The extent of metastases of the experiment group was lower than that of the control group (P0.05). The expression of MHCI of the high metastasis cell line was lower than that of the low metastasis cell line (P0.05). The expressions of Th1/Th2 associated genes in lymphocytes of high metastasis mice were lower than those of the low metastasis mice.Reconstruction of the immunological function of T cells can influence the metastasis of mouse hepatocarcinoma. The alteration of MHC molecule and low expression of Th1/Th2 correlated genes in lymphocytes may be a factor influencing the metastasis of liver cancer.

To study the effects of hepatectomized rat serum and hepatocyte growth factor (HGF) on the transdifferentiation of adult rat bone marrow stem cells (ABMSCs) into hepatic parenchymal cells.The serum was collected from the rats 24 hours after being subjected to subtotal hepatectomy. ABMSCs were collected and cultured in DMEM/F12 (1:1) containing the hepaetectomized rat serum or HGF. The differentiated hepatocyte-like cells were labeled with CM-DiI and administrated by tail vein injection into the isogeneic rats. The cultured and injected cells were both identified by immunocytochemistry and cultured cells were assayed using RT-PCR and Western blot.Hepatectomized rat serum and HGF were demonstrated to have the effect of inducing transdifferentiation of ABMSCs into hepatocyte-like cells in vitro. The differentiated cells expressed albumin mRNA and albumin after 7 days++'s co-incubation. Albumin-expressing and CM-DiI positive hepatocyte-like cells were characterized in livers and spleens of the rats injected with the cultivated cells.ABMSCs could transdifferentiate into hepatic parenchymal cells by hepatectomized rat serum or HGF.

AIM: To determine whether the bone marrow derived dendritic cell(BMDC) vaccine sensitized by heat shocked hepal-6 cell proteins affects the infiltration of intratumoral CD25+Foxp3+ Tregs in a mouse hepatocellular carcinoma(HCC) model.METHODS: In the presence of GM-CSF andIL-4, BMDCs were induced in vitro. BMDCs were sensitized by heat shocked hepal-6 cell proteins to generate a vaccine for HCC. The expression of CD11c, CCR7, CD80 and CD86 on thesesensitized BMDCs were analyzed by FACS. Theantitumor effect of this vaccine was evaluatedusing a mouse HCC model established by subcutaneous injection of Hepal-6 cells. Eight dayslater, the tumorbearing mice were divided intofour groups, which underwent intratumoralinjection of BMDCs sensitized by heat shockedhepal-6 cell proteins, serumfree culture medium, BMDCs without sensitization and BMDCssensitized by unheated hepal-6 cell proteins(onceevery 7 days, 2 times altogether), respectively.Nine days after final administration, the micewere sacrificed and the tumor samples weretaken for immunofluorescence staining for CD8+cells and intratumoral CD25+Foxp3+ Tregs. RESULTS: Light microscopy and scanning electron microscopy showed that BMDCs propagated in the presence of GM-CSF and IL-4 displayed the typical morphological characteristics of dendritic cells. Immunocy to chemical stainingshowed that they expressed the dendritic cell marks including CD11c, CCR7, CD80 and CD86.Compared with the controls(BMDCs without sensitization or sensitized by unheated hepal-6cells proteins), the BMDCs sensitized by heat shocked hepal-6 cells proteins showed increased expression of CD11c(67.2 ± 4.49 vs 52.4 ± 5.20,58.4 ± 4.43), CCR7(65.4 ± 5.34 vs 45.9 ± 5.04, 57.0± 3.46), CD80(62.9 ± 4.69 vs 46.9 ± 4.75, 54.4 ± 3.47)and CD86(73.3 ± 3.58 vs 60.1 ± 2.98, 63.7 ± 3.10)(P < 0.01 for all). Compared with the controls,the mice administrated with the BMDC vaccinesensitized by heat shocked Hepal-6 cell proteinsshowed increased CD8+ T cells(55.0 ± 4.11 vs38.2 ± 3.34, 44.6 ± 4.29, 45.6 ± 4.92, P < 0.01 forall) and decreased intratumoral CD25+Foxp3+Tregs(0.37 ± 0.028 vs 1.31 ± 0.020, 0.77 ± 0.057,0.57 ± 0.062, P < 0.05 for all).CONCLUSION: Heat shocked hepal-6 cell protein sensitization can upregulate the expression of CD11c, CCR7, CD80 and CD86 on BMDCs in vitro. Administration with this BMDC vaccine can increase CD8+ T cells and decrease intratumoral CD25+Foxp3+ Tregs in HCC mice.