Effect of kappa elastin on melanogenesis in A375 human melanoma cells and its related mechanism.

BACKGROUND: Elastin derived peptides can regulate melanocyte precursor development. Ultraviolet irradiation, infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis. The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes. METHODS: A375 human melanoma cells were treated with different concentrations of kappa elastin for 24 hours. A375 human melanoma cells were randomly assigned to control, kappa elastin, and lactose pre-incubated groups. The cell viabilities were detected by the methyl thiazoleterazolium assay. Melanin content and tyrosinase activity in A375 melanoma cells were measured. The expressions of endothelin B receptor (ET(B)R) mRNA and c-kit mRNA in A375 melanoma cells were measured by quantative reverse transcription polymerase chain reaction. RESULTS: Fifty µg/ml of kappa elastin significantly increased the melanin content by 56.64% compared with the control (P < 0.05). Kappa elastin increased cellular tyrosinase activity by 46.73% compared with the control at 24 hours (P < 0.05). Kappa elastin increased the expressions of ET(B)R and c-kit mRNA levels by 2.13-fold and 2.47-fold compared with the controls, respectively. When pre-incubating cells with a lactose solution (10 mmol/L), the inhibition on melanin production was 34.96% compared with the kappa elastin group (P < 0.05), tyrosinase activity was inhibited by 29.93% compared with kappa elastin group (P < 0.05), and the expressions of ET(B)R mRNA and c-kit mRNA were decreased by 1.56-fold and 0.82-fold compared with kappa elastin group, respectively. CONCLUSION: Kappa elastin increased the melanogenesis in A375 melanoma cells via the stimulation of tyrosinase activity and the expression of ET(B)R and c-kit. The over expressed tropoelastin produced by keratinocytes might play a role in melanogenesis of epidermal melanocytes.

[Effect of integrin-linked kinase on the growth of prostate cancer in nude mice].

OBJECTIVE: To investigate the effect of the integrin-linked kinase (ILK) small interfering RNA (siRNA) on prostate cancer in nude mice by orthotopic injection of human cell line DU145. METHODS: The cultured human cell line DU145 was knocked down for ILK using a siRNA .Cellular ILK expression was quantified by RT-PCR and Western blot analysis. Moreover, cell attachment, invasiveness and microfilament dynamics assays were performed. Furthermore, the impact of the ILK siRNA on the prostate cancer was tested using a nude mice model in which prostate cancer was induced by orthotopic injection of human prostate cancer cell line DU145.Gross tumor volume of prostate in nude mice,cell differentiation,the state of apoptosis and proliferation were tested after 5 weeks of injection. RESULTS: The expression of ILK was suppressed significantly by siRNA, cellular mRNA and protein of ILK decreased 87% and 81% separately. The knockdown of ILK also induced the attachment and invasiveness of DU145 cell growing down. The tumor volume, cell differentiation, apoptosis index and proliferation index of prostate in nude mice of ILK siRNA orthotopic injection model were significantly smaller, better, increased and decreased separately than those in control group. CONCLUSION: Targeting inhibition of ILK not only decreases attachment and invasiveness of human DU145 cells, but also suppresses the growth and development of prostate cancer of orthotopic injection human DU145 cell line model in nude mice.

Quercetin in combating H2O2 induced early cell apoptosis and mitochondrial damage to normal human keratinocytes.

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of epidermal diseases. This study aimed to investigate the effects of quercetin on the anti-oxidative response and on mitochondrial protection in cultured normal human keratinocytes. METHODS: Cultured HaCaT cells were treated with different concentrations of H2O2 (0, 50, 100, 250, 500 micromol/L) for different periods of time (0.5, 1, 2, 4 hours) to establish an oxidative stress model. The cultured HaCaT cells were randomly assigned to control, H2O2, and quercetin + H2O2 groups. For the quercetin groups, the cells were treated with different concentrations of quercetin (0, 10, 25, 50 micromol/L) before exposure to H2O2. Morphological changes of the cells were observed under an inverted microscope and an electron microscope. The cell viability was detected by the MTT method. The cell apoptosis (AnnexinV/propidium iodide double stain) and mitochondrial membrane potential (DeltaPsim) changes were detected by flow cytometry. RESULTS: An oxidative stress model of HaCaT cells was established under a suitable concentration (250 micromol/L) and treated time of H2O2 (2 hours). The cell viability and DeltaPsim decreased in a concentration-dependent and time-dependent manner while the percentage of apoptotic cells significantly increased in the H2O2 groups compared with the control group (P < 0.05). The cell viability and DeltaPsim of the quercetin treated group increased (P < 0.05) and the percentage of apoptotic cells decreased at concentrations of 1-50 micromol/L quercetin (P < 0.01) compared with H2O2 treated group. CONCLUSION: Quercetin can relieve the cell damage and apoptosis from H2O2 induced injury to HaCaT cells by anti-oxidation and mitochondrial protection.

[Chemoattractive effects of chemokine-like factor 1 on human arterial smooth muscle cells].

OBJECTIVE: To investigate the chemotactic effect of chemokine-like factor 1 (CKLF1) on human arterial smooth muscle cells (ASMCs). METHODS: The recombinant eukaryotic expression vectors pEGFP-N1-CKLF1 (test group) and pEGFP-N1 (control group) were transiently transfected into 293T cells. The supernatants were harvested 72 h after transfection for bioactivity study. The chemotactic effect of CKLF1 on ASMCs were assayed by cellular chemotactic experiments. RESULTS: ASMCs number migrated from the test and control groups diluted by none and 10-fold supernatants had statistical significance (114+/-4 vs 41+/-4, P<0.05; 74+/-4 vs 34+/-3, P<0.01), but have no statistical significance (28+/-4 vs 25+/-5, P>0.05; 26+/-5 vs 23+/-5, P>0.05) between the two groups diluted by 100-fold and 1 000-fold supernatants. When ASMCs were treated at different concentrations of 0 and 2 microg/L of pertussis toxin (PTX), the cell number migrated from the test and control groups diluted by 10-fold supernatants, they had statistical significance (74+/-4 vs 34+/-3, P<0.01; 45+/-3 vs 34+/-3, P<0.01). And when ASMCs were treated at 10 microg/L of PTX, the cell number migrated from both groups diluted by 10-fold supernatants, they had no statistical significance (37+/-4 vs 34+/-3, P>0.05). CONCLUSION: CKLF1 has significant chemotactic effects on ASMCs and such a CKLF1-induced chemotaxis could be inhibited by PTX at concentration of 10 microg/L.

[Phosphodiesterase type 5 siRNA increases cGMP in the smooth muscle cells of human corpus cavernosum].

OBJECTIVE: To investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on the cGMP in the smooth muscle cells of human corpus cavernosum, and to provide an experimental groundwork for the gene therapy of erectile dysfunction (ED). METHODS: Small interfering RNAs targeting PDE5 gene were synthesized by using web design software provided by Ambion, three siRNAs and a control siRNA were synthesized by Ambion. siRNAs were transfected into the smooth muscle cells of human corpus cavernosum by using siPORT Lipid reagent. cGMP was detected by ELISA at different times (24, 48, 72 and 96 h) after transfection. RESULTS: The cGMP levels of the siRNA1, siRNA2 and siRNA3 groups were significantly higher than those of the siRNA control and blank control groups (P < 0.05), and so was it in the siRNA1 group than the siRNA2 and siRNA3 groups (P < 0.05), with significant difference between the siRNA control and the blank control group (P > 0.05). CONCLUSION: The synthesized siRNAs in vitro are capable of increasing the level of cGMP in the smooth muscle cells of human corpus cavernosum, different siRNAs with different capabilities. The siRNA technique could provide not only an extremely powerful tool for the functional analysis of genome but also a new approach to ED gene therapy.

[Effects of 1alpha,25-dihydroxyvitamin D(3) and UVB on cell proliferation and melanin synthesis of cultured human melanocyte].

OBJECTIVE: To study the effects of 1alpha,25-dihydroxyvitamin D(3) and UVB on cell proliferation and melanin synthesis of normal human melanocytes. METHODS: Melanocytes of foreskins of healthy men were cultured and treated with various concentration of 1alpha,25-dihydroxyvitamin D(3) or UVB (55 mJ/cm(2)),or both. Cell proliferation was measured with MTT assay .The synthesis of melanin was determined by chromatography. RESULTS: 1alpha,25-dihydroxyvitamin D(3) and UVB could promote the proliferation of cultured melanocyte,and 1alpha,25-dihydroxyvitamin D(3) could promote the melanin synthesis. The significant concentration of 1alpha,25-dihydroxyvitamin D(3) ranging from 10(-7) to 10(-10) mol/L. CONCLUSION: 1alpha,25-dihydroxyvitamin D(3) and UVB irradiation could promote the proliferation of melanocyte,which indicates that they might be effective in the treatment of vitiligo.

Gene transfer of kringle 5 of plasminogen by electroporation inhibits corneal neovascularization.

PURPOSE: To test the efficacy of naked plasmid that expresses human kringle 5 of plasminogen (K5) in suppressing experimental corneal neovascularization in a rat model. METHODS: A eukaryotic expression plasmid encoding human K5 (pSecK5) was constructed. COS cells were transiently transfected with pSecK5 using a lipid-based transfection reagent. K5 secretion was confirmed by Western blot analysis. The effect of the secreted K5 on the proliferation of human umbilical vein endothelial cells (HUVECs) was investigated colorimetrically. Forty-three Sprague-Dawley rats were used for a corneal neovascularization suppression experiment. Corneal injury was induced by placing a disk of filter paper (immersed in 1 mol/l NaOH, 3.0 mm in diameter) on the corneal surface for 2 min. The cornea was immediately washed with saline. pSecK5 and empty plasmids were injected subconjunctivally, and square-wave electric pulses were immediately applied to the eyes. The expression of K5 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The extent of corneal neovascularization was evaluated by scores. RESULTS: The constructed plasmid could express itself in COS cells. Conditioned medium from K5-transfected COS cells apparently inhibited HUVEC proliferation, compared with conditioned medium from COS cells transfected with empty plasmid or nontransfected cells. RT-PCR and immunohistochemistry confirmed the expression of K5 in the conjunctiva and cornea. Corneal neovascularization was significantly suppressed by K5 gene transfer in rats' eyes. CONCLUSION: In a rat model, K5 gene transfer by subconjunctival injection and electroporation can effectively inhibit corneal neovascularization induced by an alkali burn.

[The effects of antisenes oligodeoxynucleotide on the cyclic nucleotide monophosphates in smooth muscle cells of human corpus cavernosum].

OBJECTIVES: To investigate the effects of antisense oligodeoxynucleotide(ASON) on the cyclic nucleotide monophosphates (cNMP) in smooth muscle cells of human corpus cavernosum, and provide experimental groundwork for the gene therapy of erectile dysfunction. METHODS: PDE5 gene ASON(containing exon 1) was transfected into the corpus cavernosum smooth muscle cells with the presence of liposome DOTAP. Another sense oligodeoxynucleotide(SON) and 1% of bovine serum were also transducted into the cells as controls. Two of cNMP, cAMP and cGMP, were probed and measured by ELISA at 1, 2, 4, 6, 10, 24 and 48 h after transfection. RESULTS: After transfection, the level of cGMP(1-6 h) in human corpus cavernosum smooth muscle cells was significantly higher than that in controls(P < 0.01). CONCLUSIONS: The PDE5 gene ASON had been showed to manifest stimulative effect on the cGMP in smooth muscle cells of human corpus cavernosum in vitro, and it provides experimental groundwork for the gene therapy of erectile dysfunction.