In vitro synergy of azole antifungals and methotrexate against Candida albicans.

OBJECTIVE: This study aims to evaluate the effective of azoles and MTX for patients with invasive candidiasis. METHODS: We used the disk diffusion assay and the checkerboard assay to evaluate the in vitro interactions between MTX and antifungals. In addition, we used the transmission electron microscopy to observe the ultrastructure of the effect of MTX and fluconazole on Candida albicans. RESULTS: The rates of synergy for the combination of MTX with fluconazole (FLC), itraconazole (ITC), and voriconazole (VRZ) were 91.3%, 65.2%, and 87% in checkerboard testing. No antagonism was found between methotrexate and azole antifungals in any of the strains. Furthermore, MTX treated C. albicans showed extensive cell wall vacuolations and the inhibition of blastospores growth, as observed using transmission electron microscopy. There was an apparent destruction of the cell membrane and cell wall resulting in the destruction of cytoplasm, a phenomenon observed when MTX was combined with azoles. CONCLUSION: This study provides evidence that the combination of azoles and MTX is effective for patients with invasive candidiasis, which on the other hand, will reduce the side effects of the drugs.

CDK5RAP1 targeting NF-κB signaling pathway in human malignant melanoma A375 cell apoptosis.

Malignant melanoma is characterized by rapid deterioration, early metastasis and high mortality. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1), which catalyzes 2-methylthio (ms2) modification of mitochondrial transfer RNAs, has been reported to induce cancer cell apoptosis, by a phospho-c-Jun N-terminal kinase (p-JNK) signaling pathway. The present study was the first to report on the association between CDK5RAP1 deficiency and nuclear factor-κB (NF-κB) signaling pathway during the apoptosis process in human malignant melanoma (A375) cells. CDK5RAP1 small interfering RNA (siRNA) and control siRNA were transfected into A375 cells. CDK5RAP1 deficiency inhibited Ca2+ influx in A375 cells. CDK5RAP1 deficiency also suppressed the proliferation of A375 cells, induced A375 cells apoptosis, and increased the generation of reactive oxygen species (ROS). In addition, CDK5RAP1 deficiency induced the phosphorylation of NF-κB and Bcl-2/Bcl-xl-associated death promoter (Bad). Notably, the phosphorylation of B-cell lymphoma-xl (Bcl-xl) and B-cell lymphoma-2 (Bcl-2) was downregulated by CDK5RAP1 deficiency. Pretreatment with pyrrolidine dithiocarbamate (PDTC), the inhibitor of NF-κB, prevented the decrease in cell proliferation and apoptosis induced by CDK5RAP1 deficiency in A375 cells. However, pretreatment with PDTC did not affect the generation of ROS in A375 cells, indicating that ROS is an upstream target of NF-κB signaling pathway during the apoptosis process. Taken together, CDK5RAP1 deficiency induces cell apoptosis in malignant melanoma A375 cells via the NF-κB signaling pathway. The results from the present study indicated a potential novel candidate for the treatment of skin cancer.

Low-dose UVB irradiation prevents MMP2-induced skin hyperplasia by inhibiting inflammation and ROS.

Skin cancer is one of the most common types of malignancy in the world. UV radiation is known as the primary environmental carcinogen responsible for skin cancer development. However, UV radiation is a ubiquitous substance existing in the environment and the physiological effect of UV radiation is consistently ignored. Therefore, in the present study, the physiological effect of UV radiation on inhibition of skin cancer was investigated. Normal mouse skin was processing by no pre-radiation or pre-radiation of low-dose UV before a medium or high dose of UV radiation. We found that the low-dose pre-radiated mouse skin tissue exhibited low skin inflammation, skin ROS production and consequently low skin epithelial hyperplasia after the medium-dose UV radiation compared with the no pre-radiated mouse. However, this inhibition was not indicated in the high-dose UV radiation group after low-dose pre-radiation. Furthermore, western blot analysis and gelatin zymography showed low expression and activation of MMP2 in the skin tissues processed following medium-dose radiation, but not in tissues treated with high-dose radiation after a low-dose pre-radiation. Further investigation of MMP2 inhibitors of TIMP2/TIMP4 showed an upregulated TIMP2 expression, but not TIMP4. Collectively, these data indicate that low-dose pre-radiation attenuates the skin inflammation and ROS production induced by medium-dose UV radiation and also elevates TIMP2 to withstand MMP2, therefore suppressing skin hyperplasia. The present study indicates a novel concept or prophylactic function of moderate UV radiation as a preventative strategy.

Role of the complement anaphylatoxin C5a-receptor pathway in atopic dermatitis in mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a genetic background. The C5a­receptor (C5aR) pathway has been reported to be involved in AD; however, the precise pathogenesis remains to be elucidated. In the present study, the contribution of the C5aR pathway to AD in mice was investigated. A BALB/c mouse model of AD was induced by application of 2,4­dinitrochlorobenzene (DNCB) onto hairless dorsal skin. Following DNCB application for 2 weeks, C5aR expression in skin tissue was assessed by reverse transcription quantitative polymerase chain reaction. C5aR expression in skin tissue was significantly increased in mice with AD. In an additional experiment, C5aR antagonist (C5aRA) intracutaneously injected in combination with DNCB treatment. The skin­fold thickness, number of total infiltrating leukocytes and mast cells infiltrating in skin tissue were measured. Interleukin­4 (IL­4) and interferon­Î³ (IFN­Î³) levels in skin tissue and IL­4, IFN­Î³, histamine and immunoglobulin E (IgE) levels in serum were measured using ELISA. The skin­fold thickness, numbers of total infiltrating leukocytes and mast cells in skin tissue, as well as levels of IL­4, IFN­Î³, histamine and IgE were significantly increased in mice with AD. However, simultaneous treatment with C5aRA significantly attenuated increases in skin fold thickness and the numbers of total infiltrating leukocytes and mast cells in skin tissue. Treatment with C5aRA also decreased IL­4 and IFN­Î³ levels in skin tissue, as well as the levels of IL­4, IFN­Î³, histamine and IgE in the serum. In conclusion, C5aRA inhibited AD in mice, possibly through suppression of the C5aR­mediated cascade action of mast cells.