Limits...
Benzofuroxan derivatives N-Br and N-I induce intrinsic apoptosis in melanoma cells by regulating AKT/BIM signaling and display anti metastatic activity in vivo.

Farias CF, Massaoka MH, Girola N, Azevedo RA, Ferreira AK, Jorge SD, Tavares LC, Figueiredo CR, Travassos LR - BMC Cancer (2015)

Bottom Line: The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP.The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM.We conclude that N-Br and N-I are promising agents aiming at cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, 8 andar, São Paulo, SP, 04023-062, Brazil. camyla.ff@gmail.com.

ABSTRACT

Background: Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model.

Methods: B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins.

Results: Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM.

Conclusion: We conclude that N-Br and N-I are promising agents aiming at cancer treatment. They may be useful in melanoma therapy as inducers of intrinsic apoptosis and by exerting significant antitumor activity against metastatic melanoma, as presently shown in syngeneic mice.

No MeSH data available.


Related in: MedlinePlus

Benzofuroxan derivative effects on melanoma cells (a) Morphological effects. Arrows point to surface membrane alterations. Original magnifications, 100× left panels; 200× right panels; 400x inserts. b DNA condensation induced by N-Br and N-I. Yellow arrows indicate positive cells for Hoechst staining. 100×, and 400× inserts. c Quantification of Hoechst 33342 relative fluorescence in treated and control cells, shown in B; **p ≤ 0.001; *p ≤ 0.05 (d) DNA fragmentation showing a ladder pattern; A: Control; B: N-Br (16 μM); C: N-I (12 μM); D: N-Br (100 μM); E: N-I (100 μM)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4621849&req=5

Fig2: Benzofuroxan derivative effects on melanoma cells (a) Morphological effects. Arrows point to surface membrane alterations. Original magnifications, 100× left panels; 200× right panels; 400x inserts. b DNA condensation induced by N-Br and N-I. Yellow arrows indicate positive cells for Hoechst staining. 100×, and 400× inserts. c Quantification of Hoechst 33342 relative fluorescence in treated and control cells, shown in B; **p ≤ 0.001; *p ≤ 0.05 (d) DNA fragmentation showing a ladder pattern; A: Control; B: N-Br (16 μM); C: N-I (12 μM); D: N-Br (100 μM); E: N-I (100 μM)

Mentions: Both N-Br and N-I caused morphological alterations in B16F10-Nex2 melanoma cells, with cytoplasm retraction, round cells and loss of tumor cell adhesion (Fig. 2a). DNA condensation and fragmentation were also observed in treated cells. Melanoma cells were incubated with both compounds at the IC50 concentration for 24 h and the density of nuclear chromatin previously stained with Hoechst was assessed by fluorescence microscopy (Fig. 2b). The relative intensity of fluorescence was quantified by processing images with ImageJ software (Fig. 2c). As to DNA fragmentation, the ladder pattern was observed using Agarose gel electrophoresis, upon treatment with both compounds at 100 μM during 24 h of incubation (Fig. 2d).Fig. 2


Benzofuroxan derivatives N-Br and N-I induce intrinsic apoptosis in melanoma cells by regulating AKT/BIM signaling and display anti metastatic activity in vivo.

Farias CF, Massaoka MH, Girola N, Azevedo RA, Ferreira AK, Jorge SD, Tavares LC, Figueiredo CR, Travassos LR - BMC Cancer (2015)

Benzofuroxan derivative effects on melanoma cells (a) Morphological effects. Arrows point to surface membrane alterations. Original magnifications, 100× left panels; 200× right panels; 400x inserts. b DNA condensation induced by N-Br and N-I. Yellow arrows indicate positive cells for Hoechst staining. 100×, and 400× inserts. c Quantification of Hoechst 33342 relative fluorescence in treated and control cells, shown in B; **p ≤ 0.001; *p ≤ 0.05 (d) DNA fragmentation showing a ladder pattern; A: Control; B: N-Br (16 μM); C: N-I (12 μM); D: N-Br (100 μM); E: N-I (100 μM)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4621849&req=5

Fig2: Benzofuroxan derivative effects on melanoma cells (a) Morphological effects. Arrows point to surface membrane alterations. Original magnifications, 100× left panels; 200× right panels; 400x inserts. b DNA condensation induced by N-Br and N-I. Yellow arrows indicate positive cells for Hoechst staining. 100×, and 400× inserts. c Quantification of Hoechst 33342 relative fluorescence in treated and control cells, shown in B; **p ≤ 0.001; *p ≤ 0.05 (d) DNA fragmentation showing a ladder pattern; A: Control; B: N-Br (16 μM); C: N-I (12 μM); D: N-Br (100 μM); E: N-I (100 μM)
Mentions: Both N-Br and N-I caused morphological alterations in B16F10-Nex2 melanoma cells, with cytoplasm retraction, round cells and loss of tumor cell adhesion (Fig. 2a). DNA condensation and fragmentation were also observed in treated cells. Melanoma cells were incubated with both compounds at the IC50 concentration for 24 h and the density of nuclear chromatin previously stained with Hoechst was assessed by fluorescence microscopy (Fig. 2b). The relative intensity of fluorescence was quantified by processing images with ImageJ software (Fig. 2c). As to DNA fragmentation, the ladder pattern was observed using Agarose gel electrophoresis, upon treatment with both compounds at 100 μM during 24 h of incubation (Fig. 2d).Fig. 2

Bottom Line: The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP.The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM.We conclude that N-Br and N-I are promising agents aiming at cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, 8 andar, São Paulo, SP, 04023-062, Brazil. camyla.ff@gmail.com.

ABSTRACT

Background: Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model.

Methods: B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins.

Results: Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM.

Conclusion: We conclude that N-Br and N-I are promising agents aiming at cancer treatment. They may be useful in melanoma therapy as inducers of intrinsic apoptosis and by exerting significant antitumor activity against metastatic melanoma, as presently shown in syngeneic mice.

No MeSH data available.


Related in: MedlinePlus