Isotretinoin and Thalidomide Down-Regulate c-MYC Gene Expression and Modify Proteins Associated with Cancer in Hepatic Cells.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer. The number of cases is increasing and the trend for the next few years is not encouraging. HCC is usually detected in the advanced stages of the disease, and pharmacological therapies are not entirely effective. For this reason, it is necessary to search for new therapeutic options. The objective of this work was to evaluate the effect of the drugs isotretinoin and thalidomide on c-MYC expression and cancer-related proteins in an HCC cellular model. The expression of c-MYC was measured using RT-qPCR and western blot assays. In addition, luciferase activity assays were performed for the c-MYC promoters P1 and P2 using recombinant plasmids. Dose-response-time analyses were performed for isotretinoin or thalidomide in cells transfected with the c-MYC promoters. Finally, a proteome profile analysis of cells exposed to these two drugs was performed and the results were validated by western blot. We demonstrated that in HepG2 cells, isotretinoin and thalidomide reduced c-MYC mRNA expression levels, but this decrease in expression was linked to the regulation of P1 and P1-P2 c-MYC promoter activity in isotretinoin only. Thalidomide did not exert any effect on c-MYC promoters. Also, isotretinoin and thalidomide were capable of inducing and repressing proteins associated with cancer. In conclusion, isotretinoin and thalidomide down-regulate c-MYC mRNA expression and this is partially due to P1 or P2 promoter activity, suggesting that these drugs could be promising options for modulating the expression of oncogenes and tumor suppressor genes in HCC.

Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

Trichomonas vaginalis and Giardia lamblia Growth Alterations by Low-Frequency Electromagnetic Fields.

BACKGROUND: There is an increasing interest in using physical factors such as magnetic fields as antimicrobial strategy, with variable results. The current study was aimed to evaluate the influence of extremely low-frequency electromagnetic fields (ELF-EMFs) on the axenically-cultured parasite protozoans Trichomonas vaginalis and Giardia lamblia growth. METHODS: Bioassays were developed using T. vaginalis, GT-13 and G. lamblia IMSS-0989 strains cultured at 37 ºC in TYI-S-33 medium. The following treatment regimens and controls were considered: (a) cells exposed to ELF-EMFs, (b) untreated cells, (c) cells treated with Metronidazole, used as positive controls, and (d) cells co-exposed to ELF-EMFs and Metronidazole. When cultures reached the end of logarithmic phase, they were exposed to ELF-EMFs for 72 h, in a standardized magnetic field exposure facility. For determining cytotoxic effects, trophozoite density was blindly evaluated in a Neubauer chamber. RESULTS: A significant decrease in trophozoite growth was observed for T. vaginalis, in magnetic field-treated cultures. On the other hand, cultures co-exposed to ELF-EMFs and Metronidazole showed no significant differences when compared with cultures treated with Metronidazole alone. On the contrary, an increased trophozoite density was observed in G. lamblia cultures after exposure to magnetic fields. An absence of a synergistic or antagonistic effect was observed. CONCLUSION: ELF-EMFs induced T. vaginalis and G. lamblia growth alterations, indicating a potential effect in cell cycle progression.

Antiprotozoal activity against Entamoeba histolytica of plants used in northeast Mexican traditional medicine. Bioactive compounds from Lippia graveolens and Ruta chalepensis.

Amoebiasis caused by Entamoeba histolytica is associated with high morbidity and mortality is becoming a major public health problem worldwide, especially in developing countries. Because of the side-effects and the resistance that pathogenic protozoa build against the standard antiparasitic drugs, e.g., metronidazole, much recent attention has been paid to plants used in traditional medicine around the world in order to find new antiprotozoal agents. We collected 32 plants used in Northeast Mexican traditional medicine and the methanolic extracts of these species were screened for antiprotozoal activity against E. histolytica trophozoites using in vitro tests. Only 18 extracts showed a significant inhibiting activity and among them six plant extracts showed more than 80% growth inhibition against E. histolytica at a concentration of 150 µg/mL and the IC50 values of these extracts were determined. Lippia graveolens Kunth and Ruta chalepensis Pers. showed the more significant antiprotozoal activity (91.54% and 90.50% growth inhibition at a concentration of 150 µg/mL with IC50 values of 59.14 and 60.07 µg/mL, respectively). Bioassay-guided fractionation of the methanolic extracts from these two plants afforded carvacrol (1) and chalepensin (2), respectively, as bioactive compounds with antiprotozoal activity.

Axenic cultivation and comparative phospholipase A2 activity of Giardia duodenalis in a serum-free medium.

Mammalian serum is essential for the growth of Giardia duodenalis cultivated under axenic conditions. Unfortunately, some factors present in bovine serum used as supplement in the culture medium may inhibit protozoal growth and activity. TYI-33-PACSR is a TYI medium supplemented with a serum replacement (PACSR) made up of Earle's amino acid solution, Diamond's vitamin-tween 80 mixtures and LCR (a lipid-cholesterol - rich mixture). PACSR was previously used in the culture media for axenic cultivation of Entamoeba histolytica and Trichomonas vaginalis. The main objective of this work was to demonstrate that TYI-33-PACSR is useful for axenic cultivation of G. duodenalis. Additionally, the activity of phospholipase A(2) (PLA A(2)) in the sub-cellular vesicular fraction (P30) of G. duodenalis grown in TYI-S-33 and TYI-33-PACSR was compared. All strains of Giardia grown in TYI-33-PACSR reached relative cellular densities of 91 to 95% compared to controls growing in serum-supplemented TYI-S-33 medium. Additionally, PLA A(2) activity was similar in the P30 sub-cellular fraction obtained from trophozoites growing in TYI-S-33 and TYI-33-PACSR. Thus, TYI-33-PACSR could be useful in analyzing the biological properties of G. duodenalis in the absence of serum.

Entamoeba histolytica: soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity.

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg(2+), Mn(2+), or Co(2+), and inhibited by Zn(2+), Hg(2+), Ca(2+), and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.