Proapoptotic Effects of triazol-1,4-Naphthoquinones Involve Intracellular ROS Production and MAPK/ERK Pathway in Human Leukemia Cells.

BACKGROUND: The natural products constitute an important source of antitumor and cytotoxic agents. Naphthoquinones are effectively quinones present in different plants, with demonstrated anticancer activities. A recent study conducted by our group demonstrated the antileukemic potential of two novel triazol-1,4- naphthoquinones derivatives, PTN (2-(4-Phenyl-1H-1,2,3-triazol-1-yl)-1,4-naphthoquinone) and MPTN (2-[4- (4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]-1,4-naphthoquinone). Although, the mechanisms underlying the proapoptotic effects of PTN and MPTN have not been fully elucidated so far. OBJECTIVE: The aim of this study was to evaluate the proapoptotic mechanism of PTN and MPTN in human acute leukemia cells. METHODS: We used fluorescence microscopy to observe acridine orange and annexin V staining cells. Flow cytometry assay has also been used for ROS quantification, BAX and cytochrome c proteins expression and apoptosis analysis. MTT assay and western blotting technique have been performed as well for MAPK pathway analysis. RESULTS: By using the acridine orange and annexin V staining with fluorescence microscopy, we have characterized the proapoptotic effects of PTN and MPTN in HL-60 cells involving the intrinsic mitochondrial pathway, since these compounds promoted an increase in the intracellular BAX and cytochrome c protein levels (p<0.05). We further demonstrated that apoptosis induction in HL-60 cells was mediated by increasing intracellular ROS levels via ERK but not p38 MAPKs pathway. CONCLUSION: Taken together, these results have demonstrated that PTN and MPTN are promising tools for the development of new anti-leukemic drugs.

Chemical Properties of Caffeic and Ferulic Acids in Biological System: Implications in Cancer Therapy. A Review.

The antioxidant properties of caffeic and ferulic acids in biological systems have been extensively demonstrated. As antioxidants, these compounds prevent the production of reactive oxygen species (ROS), which cause cell lesions that are associated with the development of several diseases, including cancer. Recent findings suggest that the chemoprotective action of these phenolic acids occurs through the following mechanisms: regulation of gene expression, chelation and / or reduction of transition metals, formation of covalent adducts and direct toxicity. The biological efficacy of these promising chemoprotective agents is strongly related with their chemical structure. Therefore, in this study, we discuss the structural characteristics of ferulic and caffeic acids that are responsible for their biological activities, as well as the mechanisms of action involved with the anti-cancer activity. Several reports indicated that the antioxidant effect of these phenylpropanoids results from reactions with free radicals with formation of stable products in the cells. The chelating effect of these compounds was also reported as an important protective mechanism against oxidative. Finally, the lipophilicity of these agents facilitates their entry into the cells, and thus, contributes to the anticancer activity.

Synthesis, Cytotoxic Activity on Leukemia Cell Lines and Quantitative Structure-Activity Relationships (QSAR) Studies of Morita-Baylis-Hillman Adducts.

BACKGROUND: The Morita-Baylis-Hillman reaction is an organocatalyzed chemical transformation that allows access to small poly-functionalized molecules and has considerable synthetic potential and promising biological profiles. The Morita-Baylis-Hillman adducts (MBHA) are a new class of bioactive compounds and highlight its potentialities to the discovery of new cheaper and efficient drugs, e.g. as anti-Leishmania chagasi and Leishmania amazonensis, anti- Trypanosoma cruzi, anti-Plasmodium falciparum and Plasmodium berghei, lethal against Biomphalaria glabrata, antibacterial, antifungal, herbicide and others. METHODS: The goal of this work is to describe the primary cytotoxic activities against strains of human leukemia HL-60 cell line for thirty-four Morita-Baylis- Hillman adducts (MBHA), followed by a Quantitative Structure-Activity Relationships study (QSAR). RESULTS: The conventional or microwave-assisted syntheses of MBHA, derived from substituted aromatics or Isatin, were performed in good to excellent yields (70-100%) in short reaction times, using protocols recently developed by us. Isatin derivatives, MBHA 31 and 32, were the most active in this congener series of compounds, with IC50 values of 10.8 µM and 7.8 µM, respectively. The primary cytotoxic activities against chronic leukemia cells (K562) were also evaluated to these two most active compounds (MBHA 31 and 32), presenting IC50 values of 53 µM and 43 µM respectively. QSAR study was performed considering 3D, 2D and constitutional molecular descriptors. These were selected from Ordered Predictor Selection algorithm and submitted to Partial Least Squares Modeling. CONCLUSION: We present an interesting investigation about cytotoxic activities on human leukemia cell line (HL-60) for 34 synthetic MBHA. In a good way we discovered that the most cytotoxic compounds (31-32, 10.8 µM and 7.8 µM respectively) were also prepared quantitatively (100% yields) in a short reaction time using microwave irradiation. We demonstrate that 31 and 32 induced apoptosis and not necrosis in HL-60 cells, observed by externalization of PS and increase Anexin-V positive cells. Quantitative Structure-Activity Relationships considering 3D, 2D and constitutional descriptors provided a robust and predictive PLS model, in accordance with SAR observations.

Distinct effects of novel naphtoquinone-based triazoles in human leukaemic cell lines.

OBJECTIVES: The aim of this study was to investigate the cytotoxic effect of new 1,4-naphthoquinone- 1,2,3-triazoles, named C2 to C8 triazole derivatives, towards human cancer cell lines. METHODS: The effect on cell viability was assessed by MTT and propidium iodide assays. The cytotoxic effect of C2 and C3 in K562 and HL-60 cells were analyzed by flow cytometry, DNA fragmentation and reactive oxygen species (ROS) production. Western blot and q-PCR procedures were also performed. KEY FINDINGS: C2 and C3 inhibited both K562 and HL-60 cells growth in a concentration-dependent manner. C2 presented the highest cytotoxic activity with an IC50 of approximately 14 µm and 41 µm for HL-60 and K562 cells, respectively, while being less toxic to normal peripheral blood monocyte cells. Both derivatives induced cellular changes in HL-60 cells, characteristic of apoptosis, such as mitochondrial membrane depolarization, phosphatidylserine externalization, increasing sub-G1 phase, DNA fragmentation, downregulating Bcl-2 protein and upregulating Bax protein. In K562 cells, C2 and C3 induced S-phase arrest of cell cycle, which was associated with upregulation of p21. The effect of these derivatives in HL-60 cells can be related to the ROS intracellular level. CONCLUSION: Taken together our results showed that C2 and C3 triazole derivatives presented the best potential for drug design.