La inflamación inducida por formalina favorece el desarrollo del melanoma B16 en ratones Balb/C / inflamation induced by formaline promotes the development of melanoma B16 in Balb/C mice

El cáncer es la segunda causa de muerte en el mundo representando el melanoma 1% de todos los tipos de cáncer. Se ha planteado que el cáncer es una enfermedad inflamatoria sistémica que genera radicales libres causando mutaciones y liberan factores tróficos que favorecen la iniciación tumoral y la proliferación celular, respectivamente. Con el objetivo de estudiar el efecto de la inflamación inducida por formalina sobre el desarrollo del melanoma B16, 22 ratones Balb/C fueron distribuidos en tres grupos: Control Melanoma, Melanoma-Formalina y Control Formalina. A los grupos CF y MF se les aplicó 20 µl de Formalina al 2% en el dorso de la pata trasera derecha a nivel subcutáneo; a los grupos CM y MF se le trasplantaron 100.000 células melanocíticas vía subcutánea en la superficie plantar de la pata derecha, 24 horas posteriores a la formalina. Los ratones del grupo CF desarrollaron una inflamación que fue máxima entre la primera y segunda semana y luego cedió progresivamente hasta desaparecer a la sexta semana. Los ratones del grupo CM desarrollaron máculas tumorales hasta 30 mm² que involucionaron espontáneamente. Los ratones del grupo MF desarrollaron masas tumorales que alcanzaron hasta 300 mm³ entre la 3-4 semanas post-trasplante y luego disminuyeron progresivamente de volumen. Los ratones de los grupos CF y MF disminuyeron significativamente de peso respecto al grupo CM. En conclusión, la inflamación inducida por formalina favorece el desarrollo tumoral en un modelo alogénico de melanoma maligno.

Cancer is the second cause of death around the world, representing melanoma 1% of all cancer. It has been suggested that cancer is a systemic inflammatory disease that generates free radicals causing mutations and releasing trophic factors that favors tumor initiation and cell proliferation. In order to study the effect of formalin-induced inflammation on the development of B16 melanoma, 22 Balb/C mice were divided into three groups: Control Melanoma (CM), Melanoma-Formalin (MF) and Control Formalin (CF). CF and MF groups were injected with 20 µl of 2% formalin on the back of the right paw at the subcutaneous level; CM and MF groups were transplanted with 100,000 melanocytic cells subcutaneously in the plantar surface of the right paw, 24 hours after formalin. CF group mice developed an inflammation that was maximal between the first and second week, then progressively diminished until disappearance by the sixth week. CM group mice developed tumoral macules up to 30 mm², which involute spontaneously. MF group mice developed tumor masses that reached up to 300 mm³ between 3-4 weeks post-transplant and then progressively decreased in volume. CF and MF mice significantly decreased in weight with respect to CM group. In conclusion, inflammation induced by formalin favors tumor development in an allogenic model of malignant melanoma, indicating that anti-inflammatory treatments may be useful in the management of melanoma.

In vitro anti-tumor effect of mGM-CSF-GnRH3 and mGM-CSF-GRP6 recombinant fusion protein and theirbioinformatics prediction / 中国肿瘤生物治疗杂志

@#Objective: To prepare the fusion protein mGM-CSF-GnRH3 (mGGn) of mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) combining with gonadotropin releasing hormone (GnRH) and the fusion protein mGM-CSF-GRP6 (mG6) of mGM-CSF combining with gastrin-releasing peptide (GRP), and to investigate the inhibitory effect of the above two fusion proteins on B16F10 melanoma in vitro as well as to preliminarily predict their isoelectric point, relative molecular weight, hydrophobicity, stability, subcellular localization, signal peptide, spatial structure and potential epitopes. Methods:After the successful preparation of mGGn and mG6, the effects of different concentrations of fusion proteins on tumor cell morphology, migration, proliferation and cell cycle were detected by microscopic observation, scratch test, CCK-8 method and flow cytometry, respectively. The protein online analysis systems EXPASY, GOR4, SWISS MODEL were used to predict the basic properties and secondary/tertiary structure of recombinant fusion proteins. The B cell epitopes were predicted by IEDB and ABCpred software, the CTL epitopes were comprehensively predicted by SYFPEITHI, BlMAS and NetCTL software, and the Th epitopes were predicted by NetMHCIIpan 3.1 Server and IEDB software. Results:Both mGGn and mG6 inhibited the migration and proliferation of tumor cells. mGGn could block B16F10 cell cycle at G1 phase while mG6 could block B16F10 cell cycle at S phase, all of which prevented cells entering into G2 phase to inhibit tumor cell growth. The mGGn and mG6 fusion proteins got diverse structures and had multiple potential B epitopes, CTL epitopes and Th epitopes. Conclusion: mGGn and mG6 have inhibitory effect on B16F10 melanoma in vitro, and bioinformatics predictions have laid a foundation for further study of the biological functions and immunological activities of these fusion proteins.

Effect of bisdemethoxycurcumin on the proliferation and apoptosis of melanoma B16-F10 cells / 实用医学杂志

Objective To investigate the effect of bisdemethoxycurcumin on the proliferation and apoptosis of melanoma B16-F10 cells. Methods The B16-F10 cells were incubated with bisdemethoxycurcumin for 24 h, and MTT assay was used to detect the proliferation of B16-F10 cell. Flow cytometry was used to detect cell cycle and cell apoptosis. A C57BL/6 mouse melanoma model was established to investigate the effect of bisdemethoxycurcumin on the proliferation of melanoma. Expression of BCL-1 in B16-F10 cells and tissues was detected by western blotting assay. Results bisdemethoxycurcumin could significantly inhibit B16-F10 cell proliferation, induce B16-F10 cell apoptosis and block the cell cycle at S phase. The intravenous dosing of bisdemethoxycurcumin could inhibit the growth of melanoma. Bisdemethoxycurcumin could inhibit the expression of BCL-1. Conclusion Bisdemethoxycurcumin can inhibit the proliferation of B16-F10 cell, resulting from its role in promoting cell apoptosis.

Inhibitory effect of p-hydroxylcinnamaldehyde from Momordicae Semen on growth of mouse melanoma xenograft in vivo andstudy on its mechanism / 中草药

Objective: To investigate the inhibitory effect of p-hydroxylcinnamaldehyde (PHD) from Momordicae Semen seeds on growth of mouse melanoma B16 cells in vivo. Methods: The inhibitory effect of PHD on growth of mouse melanoma B16 cells was measured by MTS method. Morphological changes of B16 cells were observed by phase contrast microscope. The xenograft tumor models of B16 cells in mice were established and divided into two groups: The mice in treatment group were treated with PHD (2 mg/kg) and in control group (treated with equal volume of normal saline). The growth of xenograft tumors in mice was observed and their sizes and weights were measured. The expression of Tyr, MMP-9, S-100B, p-P38, and p-ERK in tumor tissues was detected by immunohistochemical method. Morphological changes of lung and liver tissues in mice were observed by HE staining. Results: PHD had obvious inhibitory effect on the growth of B16 cells in vitro (P < 0.01). The morphological changes of B16 cells were typically differentiated after treated with 20 μmol/L PHD for 48 h. The average volume and weight of tumor tissues in mice of PHD treatment group were significantly decreased as compared with those of the control group (P < 0.01). Compared to the control group, the expression levels of Tyr and p-P38 in PHD treatment group were increased (P < 0.05), while the expression levels of MMP-9, S-100B, and p-ERK were decreased (P < 0.05). No obvious morphological changes were found in liver and lung tissues of mice in PHD treatment group and the control group. However, lung tumor metastasis was found in control group mice. Conclusion: PHD has inhibitory effect on the growth of xenograft tumor of mouse melanoma cells in mice.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished. .

Effect of p-hydroxylcinnamaldehyde on differentiation of melanoma cells and its mechanism / 中草药

Objective: To explore the monomer compounds in the seeds of Momrodica cochinchinensis and to study the differentiation of mouse melanoma B16 cells induced by p-hydroxylcinnamaldehyde (PHC). Methods: After being treated by five kinds of compounds [PHC, coniferylaldehyde, p-hydroxylbenzaldehyde (PHB), 3-O-methoxyaniline-p-hydroxylbenzaldehyde, and ligballinol] for 48 h, the inhibitory rate of B16 cell growth was measured by sulforhadamine B (SRB); Morphological changes of B16 cells induced by PHC for 24, 48, and 72 h were observed by Giemsa staining and phase contrast microscope; Melanin content and the activity of tyrosinase in B16 cells 48 h after the administration were assessed by colorimeter. The expression of tyrosinase mRNA was detected by RT-PCR. Results: All the five compounds had the inhibitory effect on the B16 cells. Among them, PHC showed the strongest effect in the dose-and time-dependent manner; PHC could induce B16 cells dendritic growth 48 h after the treatment, and the morphological changes were typically differentiated; PHC also increased the melanin production and the activity of tyrosinase. There was a significant difference compared to the control group (P < 0.05). After treated by PHC for 6, 12, and 24 h, the expression levels of tyrosinase mRNA, tyrosinase 1 mRNA, and tyrosinase 2 mRNA were significantly increased (P < 0.01) in a time-dependent manner. Conclusion: PHC could inhibit the proliferation of B16 cells and the mechanism is related to the differentiation of B16 cells.

Effect of granulysin (GLS) and IL-12 genes' expression products on proliferation and apoptosis of melanoma B16 cell / 重庆医科大学学报

Objective:To dectect the effect of granulysin and IL-12 genes'expression products on proliferation and apoptosis of melanoma B16 cell in vitro.Methods:Co-expression plasmid including granulysin peptide and murine interleukin 12(mIL-12)genes was transfected into melanoma B16 cell with Lipofectamin TM2000 and its expression products were detected by RT-PCR.Growth suppression was detected with MTT colorimetric assay,and cell apoptotic alterations were evaluated by Hoechst 33258 staining,AO/EB staining,and Annexin V-FITC flow cytometry(FCM).Results:GLS peptite and IL-12 genes could be expressed in B16 cells.Expression products inhibited the proliferation of melanoma cells under MTT observaton.Cells apoptosis with nuclear chromatin condensation,fragmentation and cell membrane change were observed under Hoechst 33258 staining and AO/EB staining.FCM analysis showed the apoptotic rates in test group was 21.02%,which was higher than that in control in control group(15.57%).Conclusion:Expression products of granulysin and mIL-12 genes can not only inhibit proliferation but also induce apoptosis of murine melanoma cell line B16 in vitro.