RNase A Domain-Swapped Dimers Produced Through Different Methods: Structure–Catalytic Properties and Antitumor Activity

Upon oligomerization, RNase A can acquire important properties, such as cytotoxicity against leukemic cells. When lyophilized from 40% acetic acid solutions, the enzyme self-associates through the so-called three-dimensional domain swapping (3D-DS) mechanism involving both N- and/or C-terminals. The same species are formed if the enzyme is subjected to thermal incubation in various solvents, especially in 40% ethanol. We evaluated here if significant structural modifications might occur in RNase A N- or C-swapped dimers and/or in the residual monomer(s), as a function of the oligomerization protocol applied. We detected that the monomer activity vs. ss-RNA was partly affected by both protocols, although the protein does not suffer spectroscopic alterations. Instead, the two N-swapped dimers showed differences in the fluorescence emission spectra but almost identical enzymatic activities, while the C-swapped dimers displayed slightly different activities vs. both ss- or ds-RNA substrates together with not negligible fluorescence emission alterations within each other. Besides these results, we also discuss the reasons justifying the different relative enzymatic activities displayed by the N-dimers and C-dimers. Last, similarly with data previously registered in a mouse model, we found that both dimeric species significantly decrease human melanoma A375 cell viability, while only N-dimers reduce human melanoma MeWo cell growth.

The Inhibition of Cell Growth Through the EGFR/ERK/MMP-2 Pathway Induced by Ampelopsin in the Human Malignant Melanoma A375 Cell Line

Malignant melanoma is one of the most aggressive skin cancers, having a very high mortality rate. However, its effective treatment is not clear. Ampelopsin, a plant flavonoid, has been reported to inhibit cell growth and/or induce apoptosis in various types of tumor. In this study, it was shown that ampelopsin significantly inhibits melanoma A375 cell line proliferation in a concentration-dependent/time-dependent manner. The flow cytometric data clearly demonstrated that ampelopsin causes cell cycle arrest in the G2/M phase. Moreover, it also confirmed that growth inhibition mediated by treatment with ampelopsin is related to the decreased expression of Cdc2, Cdc25c, cyclin B1, and activation of caspase-3 and Bax, purportedly by epidermal growth factor receptor (EGFR), extracellular regulated protein kinases, and matrix metalloproteinase-2 (MMP-2) downregulation. As a result of this work, these findings suggest that ampelopsin inhibits human malignant melanoma A375 cell line proliferation by suppressing the EGFR/ERK/MMP-2 pathway.

Optimization of Ultrasonic Flavonoid Extraction from Saussurea involucrate, and the Ability of Flavonoids to Block Melanin Deposition in Human Melanocytes

(1) Background: Flavonoids are the primary medicinal ingredient of Saussurea involucrate, which have significant antioxidant capacity. Optimizing the extraction of Saussurea involucrate flavonoids (SIFs) and exploring the ability to block melanin deposition caused by reactive oxygen can greatly promote the development of S. involucrate whitening products. (2) Methods: Ultrasonic extraction process was optimized using the Box–Behnken design (BBD) and response surface methodology (RSM). Then, the effect of SIFs on antioxidant activity and anti-deposition of melanin, and genes related to the melanin synthesis are studied. (3) Results: The optimal extraction procedures are as follows: the extraction time, ethanol content, and solvent ratio (v/w) are 64 min, 54%, and 54:1, respectively. The reducing activity and scavenging rates of 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical, and ABTS+ were promoted as more S. involucrate flavonoid extract was added. The SIFs extract induced a decrease in the melanin synthesis by inhibiting the human melanoma A375 cell tyrosinase activity. SIFs also depress expression of melanin synthesis related genes. (4) Conclusions: the highest SIFs content was obtained by using 54% ethanol and 54:1 solvent ratio (v/w) for 64 min. The extract of SIFs exhibited good ability of antioxidant and anti-deposition of melanin in human melanocytes.

INVESTIGATION ON THE DIFFERENCE EXPRESSION OF TYPE IV COLLAGEN 1(IV)- 6(IV) CHAIN MRNA IN NORMAL FIBROBLAST AND IN SKIN CELL MALIGNANT MELANOMA

Collagen IV is the major basement membrane protein that influences adhesion, proliferation, and migration of cells. The collagen composed of a network chains a1 to a6. The characterization of this collagen IV will correlates the relationship of collagen gene expression and cancer. This is important in order to provide more detailed understanding of the expression of collagen in tumor cells. . The aim of this study is to determine the a1 to a6 (IV) mRNA expression in the cell lines obtained from skin and melanoma cell. To investigate the mRNA expression, the RNA was extracted from the fibroblast and melanoma (A375) cell lines. The RNA was subjected to reverse transcription and then synthesized. The mRNA expression levels were measured using real time PCR with related to internal control, GAPDH. The study identified that a1, a2, a4, a5 and a6 of the a1-a6 (IV) were expressed in skin fibroblast. This corresponds to the a1a1a2 and a5a5a6 networks. However, in melanoma cell lines the collagen IV a2, a4, a5 and a6 mRNA was observed in low level compared to a1 and this suggested that the tumor has affected the expression of collagen and basement membrane of the cell.