In vitro and in vivo antitumor effects of 50 to 100-KDa components from B16 melanoma culture supernatant.

The development of immunological therapies for melanoma has been of considerable concern in recent years. Whole tumor cell lysates have been used to develop antitumor vaccines, but the effective components of the lysates have not been identified. In the present study, protein elements were purified from the B16 supernatant to analyze the in vitro chemotaxis towards mouse spleen lymphocytes using a Boyden chamber. Prior to establishing a B16 melanoma model, C57BL/6 mice were vaccinated with these proteins, and melanoma growth, tumor appearance time and behavioral changes were observed. Next, the cytotoxicity and subsets of the tumor infiltrating lymphocytes, and the histological characteristics of the melanoma were analyzed. The isolated purified fragments of B16 melanoma culture supernatant had strong antitumor effects. The possible antitumor mechanism was delineated, and was identified to possibly be through the activation of cluster of differentiation 8-positive T cells and the promotion of B16 cell differentiation. These methods will provide a novel insight into understanding antitumor immunological mechanisms and provide a potential avenue for immunotherapy.

Difference in glycerol levels between leukemia and normal bone marrow stem cells.

Aquaglyceroporin 9 (AQP9) is considered to be involved in numerous types of carcinogenic processes, particularly in liver carcinoma. AQP9 expression is significantly decreased in the human hepatocellular carcinoma when compared with the non-tumourigenic liver, which leads to increased resistance to apoptosis. In addition, AQP9 is permeable to glycerol and urea. The involvement of AQP9 in leukemia has not been fully delineated. It is proposed that abnormal proliferation of hematopoietic stem cells (HSCs) contributes to leukemia carcinogenesis. Therefore, the present study aimed to investigate the possible roles of AQP9 in HSCs and its effect on the intracellular glycerol content. HSCs and non-HSCs (nHSCs) were isolated via magnetic-activated cell sorting and then subjected to flow cytometry for evaluation of purity. White blood cells (WBCs) were isolated from peripheral blood from healthy volunteers. Furthermore, AQP9 expression was examined at the mRNA and protein levels using western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The glycerol content of HSCs, nHSCs and WBCs was evaluated by ELISA. Finally, in order to observe the morphology of HSCs and nHSCs, a blood smear was conducted and the cells were observed with Wright-Giemsa staining. The results indicated that the glycerol content in the HSCs was markedly greater than that in the nHSCs. AQP9 mRNA and protein expression was not detected in the HSCs and nHSCs, but was identified in the WBCs. Moreover, the HSC morphological characteristics included round or oval cells with round, slightly oval or irregularly shaped nuclei. Additionally, the nuclei occupied almost the entire cell, were located in the middle or were biased toward one side, and were stained light purple or red. Overall, our results indicated that intracellular glycerol is involved in HSC proliferation, despite the fact that glycerol is not mediated by AQP9. Hence, our findings may be useful in further understanding the mechanism of leukemia carcinogenesis, and these data may be valuable in developing future therapeutic strategies.

[Effect of histamine on metastasis of melanoma B16 cell xenograft in C57BL/6 mice].

BACKGROUND & OBJECTIVE: In the biotherapy for tumors, microvessel permeability is the main barrier for large molecular antibody-coupled antitumor drugs to enter the tumor mass. Histamine may enrich these drugs in the tumor matrix through enhancing microvessel permeability and tissue-fluid formation. This study was to investigate whether the increased microvessel permeability and more tissue fluid formation could increase the probability of lymphatic or hemal metastasis of tumor cells. METHODS: Cultured melanoma B16 cells were inoculated into the left armpit of C57BL/6 mice to develop melanoma. Five days after inoculation, the test group were injected subcutaneously at the dorsal part with histamine (300 mg/kg) every other day for 5 times, while the control group were given normal saline. The metastasis statuses in the lymph node, liver, lung, spleen, and brain were examined by histochemistry. Student t-test and Fisher's exact test were used respectively to analyze the effects of histamine on tumor growth and metastasis. RESULTS: All the mice developed melanoma after inoculation. At the end of the experiment, the tumor weight was significantly lighter in test group than in control group [(5.26 +/- 1.55) g vs. (6.96 +/- 1.31) g, P < 0.01]. The lymphatic and hemal metastasis rates were significantly lower in test group than in control group (33.3% vs. 75.0%, P < 0.05; 25.0% vs. 75.0%, P < 0.05). CONCLUSION: Histamine can inhibit the metastasis of melanoma B16 cells in C57BL/6 mice either through lymphatic or hemal route, and this partly because of its inhibitory effect on tumor growth.

[Immuno-blot detection of hemangiopoietin in the human fetal liver].

AIM: To detect the expression of a novel protein, hemangiopoietin (HAPO), in the human fetal liver at the protein level. METHODS: Monoclonal antibodies (moAbs) against HAPO were produced by traditional hybridoma technique. Their affinities were calculated from the results of non-competitive ELISA and the antibodies were purified by protein G affinity chromatography. Expression of HAPO in the liver was detected by SDS-PAGE and Western blot. RESULTS: Five strains of moAbs were screened out in total, among which three were IgG1 and the other two were IgM. Their light trains were all belonged to kappa. The relative affinities of the three IgG1 form moAbs were 3.06 x 10(9) mol/L, 6.07 x 10(8) mol/L and 1.71 x 10(10) mol/L respectively. After purification, the purity of the moAbs could reach more than 99%. HAPO expression was detected at the protein level in the human fetal liver, and the apparent molecular weight of the nature HAPO was very close to but a little higher than our recombinant one. CONCLUSION: HAPO was expressed in the human fetal liver at the protein level.