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Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo.
Figueiredo CR
,
Matsuo AL
,
Pereira FV
,
Rabaça AN
,
Farias CF
,
Girola N
,
Massaoka MH
,
Azevedo RA
,
Scutti JA
,
Arruda DC
,
Silva LP
,
Rodrigues EG
,
Lago JH
,
Travassos LR
,
Silva RM
.
Abstract
BACKGROUND: Pyrostegia venusta (Ker. Gawl.) Miers (Bignoniacea) is a medicinal plant from the Brazilian Cerrado used to treat leucoderma and common diseases of the respiratory system.
OBJECTIVE: To investigate the antitumor activity of P.venusta extracts against melanoma.
MATERIALS AND METHODS: The cytotoxic activity and tumor induced cell death of heptane extract (HE) from P. venusta flowers was evaluated against murine melanoma B16F10-Nex2 cells in vitro and in a syngeneic model in vivo.
RESULTS: We found that HE induced apoptosis in melanoma cells by disruption of the mitochondrial membrane potential, induction of reactive oxygen species and late apoptosis evidenced by plasma membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation, exposure of phosphatidylserine on the cell surface and activation of caspase-2,-3,-8,-9. HE was also protective against singeneyc subcutaneous melanoma HE compounds were also able to induce cell cycle arrest at G2/M phases on tumor cells. On fractionation of HE in silica gel we isolated a cytotoxic fraction that contained a mixture of saturated hydrocarbons identified by (1)H NMR and GC-MS analyses. Predominant species were octacosane (C28H58-36%) and triacontane (C30H62-13%), which individually showed significant cytotoxic activity against murine melanoma B16F10-Nex2 cells in vitro and a very promising antitumor protection against subcutaneous melanoma in vivo.
CONCLUSION: The results suggest that the components of the heptane extract, mainly octasane and triacontane, which showed antitumor properties in experimental melanoma upon regional administration, might also be therapeutic in human cancer, such as in the mostly epidermal and slowly invasive melanomas, such as acral lentiginous melanoma, as an adjuvant treatment to surgical excision.
Figure 1. Cytotoxicity of HE in B16F10-Nex2 murine melanoma and nontumorigenic HUVEC, HF and 3T3 cells. Dose dependent activity of HE over 104 tumor cells in 18 h
Figure 2. Morphological evidence and DNA degradation in HE-treated apoptotic melanoma cells. (a) B16F10-Nex2 cell morphology after treatment with 50 μg/ml HE for 18 h. Apoptotic bodies’ formation is indicated by white arrows; (b) Evaluation of chromatin condensation in HE treated cells. B16F10-Nex2 cells (104) were treated with 50μg/ml of HE for 18 h, labeled with Hoechst dye, and analyzed by fluorescence microscopy. Arrows indicate pronounced chromatin condensation in treated cells (Magnification 60x) Scale bar: 20μm; (c) Agarose gel electrophoresis of DNA fragmentation in B16F10-Nex2 cells induced by 50 μg/ml HE treatment for 24 h; (d) Fluorescence microscopy for DNA fragmentation. Melanoma cells (5 × 104) were treated with 50 μg/ml of HE and 150 μM of combretastatin A4 as positive apoptotic control for 24 h, and DNA fragmentation was detected using a TUNEL assay (green fluorescence). DAPI (blue) were used for total cell nuclei (Scale bar: 20 μm)
Figure 3. Phosphatidyl serine surface expression and caspase activation. (a) Annexin V and propidium iodide labeling of B16F10- Nex2 (3 × 105) cells grown for 24 h in 12-well plate and incubated with 12 and 25 μg/ml of HE or RPMI medium for 18 h at 37 °C. Positive annexin V (AV) and propidium iodide cells were detected with an inverted fluorescence microscope at 10X magnification; (b) Activation of caspase-2, 3, 8 and 9 in HE treated melanoma cells. B16F10-Nex2 cells (107) were treated with 50 μg/ml of HE for 24 h, and the HE induced enzymatic activity of caspase-2, 3, 8 and 9 was evaluated by colorimetric assay
Figure 4. Mitochondrial effects of HE treatment. (a) Representative images of superoxide anions’ negative and positive cells treated with 12.5 and 25 μg/ml of HE for 18 h, and 5 mm of hydrogen peroxide (positive control) for 30 min. B16F10-Nex2 cells were stained with 5 μM of DHE (dihydroethidium). Scale bar: 20 μm; (b) Representative images of mitochondrial ÄѰm following HE treatment. B16F10-Nex2 cells were treated with negative control and with 12 and 25 μg/ml of HE for 18 h (original magnification, 20×). Positive apoptotic cells were stained with annexin V (original magnification, 10X). (c) Percentage of TMRE (tetramethylrhodamine ethyl ester) and DHE positive cells; (d) Protective effect of N-acetyl cysteine (NAC) on HE-treated cells. B16F10-Nex2 cells (104) were pretreated for 2 h with 10 mM NAC, washed and incubated with 50 μg/ml of HE at 37°C for 18 h
Figure 5. Effect of HE on the melanoma cell cycle. (a) Cell cycle analysis of B16F10-Nex2 tumor cells treated with 25 μg/ml HE and 75 μM of CA4 for 24 h. (b) Representative images of tumor cell morphology following HE treatment
Figure 8. (a) 1H NMR spectrum of fraction HEF2 (~ CDCl3, 200 MHz)-the signal at ~ 7.24 ppm corresponding to hydrogens of residual CHCl3 in deuterated solvent; (b) GC-MS of HEF2 components with 4 main alkane species with the retention times of (1) tetracosane (C24H50), (2) hexacosane (C26H54), (3) octacosane (C28H58), (4) triacontane (C30H62), and 3 other minor species as listed in Table 3; (c) Mass spectrum of octacosane (GC component 3) and (d) triacontane (GC component 4), further identified by co-injection with standard n-octacosane, Rt 37.7 min, MM 620 Da, and triacontane, Rt 39.57 min, MM 422 Da
Figure 9. Cytotoxicity of HE individual alkanes. (a) Cytotoxicity of octacosane and triacontane in B16F10-Nex2 cells after 18 h. (b) Tumor cell morphology after incubation with different concentrations of octacosane and triacontane
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