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.

Morita-Baylis-Hillman Adducts Display Anti-Inflammatory Effects by Modulating Inflammatory Mediator Expression in RAW264.7 Cells.

Inflammatory response plays an important role not only in the normal physiology but also in pathologies such as cancers. The Morita-Baylis-Hillman adducts (MBHA) are a novel group of synthetic molecules that have demonstrated many biological activities against some parasitic cells such as Plasmodium falciparum, Leishmania amazonensis, and Leishmania chagasi, and antimitotic activity against sea urchin embryonic cells was also related. However, little is known about the mechanisms induced by MBHA in inflammatory process and its relation with anticancer activity. The present work investigated the cytotoxicity of three MBHA derivatives (A2CN, A3CN, and A4CN), on human colorectal adenocarcinoma, HT-29 cells, and their anti-inflammatory activities were examined in lipopolysaccharide- (LPS-) stimulated RAW264.7 macrophage cells, being these derivatives potentially cytotoxic to HT-29 cells. Coincubation with A2CN, A3CN, or A4CN and LPS in RAW264.7 cells inhibited NO production, as well as the production of reactive oxygen species (ROS) was also repressed. The mRNA expressions of IL-1ß and IL-6 were significantly downregulated by such MBHA compounds in RAW264.7 cells, but only A2CN was able to inhibit the COX-2 gene expression. We also showed that MBHA compounds decreased almost to zero the production of IL-1ß and IL-6. These findings display that such MBHA compounds exhibit anticancer and anti-inflammatory activities.

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.

Compound A398, a novel podophyllotoxin analogue: cytotoxicity and induction of apoptosis in human leukemia cells.

Despite advances in oncology research, cancer is one of the leading causes of death worldwide. Thus, there is a demand for the development of more selective and effective antitumor agents. This study showed that A398, a novel podophyllotoxin analogue, was cytotoxic to the HT-29, MCF-7, MOLT-4 and HL-60 tumor cell lines, being less active in human peripheral blood mononuclear cells and normal cell lines FGH and IEC-6. Tests using the HepG2 lineage indicated that its metabolites do not contribute to its cytotoxicity. In the HL-60 cells, A398 induced apoptosis in a time and concentration-dependent manner, promoting mitochondrial depolarization, inhibition of Bcl-2, phosphatidylserine exposure, activation of caspases -8, -9 and -3, and DNA fragmentation. The production of reactive oxygen species does not seem to be a crucial event for the apoptotic process. Pretreatment with specific inhibitors of kinases ERK1/2, JNK and p38 resulted in an increased percentage of death induced by A398. These results indicate that the compound induced apoptosis through activation of intrinsic and extrinsic death pathways with the mechanism involving the inhibition of the MAPKs and Bcl-2. Taken together, our findings suggest that A398 has an anticancer potential, proving itself to be a candidate for preclinical studies.

Antiproliferative effects of lectins from Canavalia ensiformis and Canavalia brasiliensis in human leukemia cell lines.

The antiproliferative activity of lectins Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) were studied using human leukemia MOLT-4 and HL-60 cell lines. It was revealed that both ConA and ConBr were markedly cytotoxic to cells using MTT and NAC assays. The IC(50) values were approximately 3 and 20 µg/mL for ConA and ConBr, respectively, for both MOLT-4 and HL-60 cells. However, in normal human peripheral blood lymphocytes, the lectins were not cytotoxic, even when tested at concentrations as high as 200 µg/ml. Using comet assay, the lectins produced a rate of DNA damage exceeding 80% in MOLT-4 and HL-60 cells. Fluorescence analysis revealed the morphology characteristic of apoptosis, with low concentrations of apoptotic bodies and fragmented DNA (5 µg/ml). Flow cytometric analysis demonstrated an accumulation of cells in the sub-G1 cell cycle that is characteristic of DNA fragmentation, and a decrease in membrane integrity at high concentrations. Lastly, we evaluated the alterations in mitochondrial potential that reduced after treatment with lectins. Our results indicate that ConA and ConBr inhibited cell proliferation selectively in tumor cells and that apoptosis was the main death mechanism. Therefore, lectins can be considered a class of molecules with a high antitumor activity potential.