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

ROS generation and N-acetyl cysteine protective effects. a ROS generation after treatment of melanoma cells with N-Br, N-I and NAC, as detected by DHE fluorescence microscopy. **p ≤ 0.001; *p ≤ 0.05 (b) Role of ROS on the induction of apoptosis in B16F10-Nex2 cells by the benzofuroxan derivatives and protection by NAC. Cellular viability was assessed by MTT assay. c Evaluation of ∆ψm as affected by N-Br and N-I treatment. ΔΨm was assessed using TMRE by flow cytometry
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: ROS generation and N-acetyl cysteine protective effects. a ROS generation after treatment of melanoma cells with N-Br, N-I and NAC, as detected by DHE fluorescence microscopy. **p ≤ 0.001; *p ≤ 0.05 (b) Role of ROS on the induction of apoptosis in B16F10-Nex2 cells by the benzofuroxan derivatives and protection by NAC. Cellular viability was assessed by MTT assay. c Evaluation of ∆ψm as affected by N-Br and N-I treatment. ΔΨm was assessed using TMRE by flow cytometry

Mentions: ROS may damage cellular components and trigger a cascade of events leading to cell death [15]. Previous studies with benxofuroxan compounds have shown that these molecules are able to elicit ROS dependent oxidative stress [16]. B16F10-Nex2 cells treated for 6 h with IC50 concentrations of the benzofuroxan compounds (16 μM for N-Br and 12 μM for N-I), in presence or absence of NAC, were examined for intracellular ROS revealed with DHE probe. Both compounds led to increased ROS formation evaluated by fluorescence microscopy, as compared to untreated cells, and this effect was attenuated by NAC (Fig. 4a). ROS dependent cell death was further confirmed in a cytotoxicity assay of melanoma cells and the cytotoxic effects of N-Br and N-I were significantly inhibited by NAC (Fig. 4b).Fig. 4


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)

ROS generation and N-acetyl cysteine protective effects. a ROS generation after treatment of melanoma cells with N-Br, N-I and NAC, as detected by DHE fluorescence microscopy. **p ≤ 0.001; *p ≤ 0.05 (b) Role of ROS on the induction of apoptosis in B16F10-Nex2 cells by the benzofuroxan derivatives and protection by NAC. Cellular viability was assessed by MTT assay. c Evaluation of ∆ψm as affected by N-Br and N-I treatment. ΔΨm was assessed using TMRE by flow cytometry
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: ROS generation and N-acetyl cysteine protective effects. a ROS generation after treatment of melanoma cells with N-Br, N-I and NAC, as detected by DHE fluorescence microscopy. **p ≤ 0.001; *p ≤ 0.05 (b) Role of ROS on the induction of apoptosis in B16F10-Nex2 cells by the benzofuroxan derivatives and protection by NAC. Cellular viability was assessed by MTT assay. c Evaluation of ∆ψm as affected by N-Br and N-I treatment. ΔΨm was assessed using TMRE by flow cytometry
Mentions: ROS may damage cellular components and trigger a cascade of events leading to cell death [15]. Previous studies with benxofuroxan compounds have shown that these molecules are able to elicit ROS dependent oxidative stress [16]. B16F10-Nex2 cells treated for 6 h with IC50 concentrations of the benzofuroxan compounds (16 μM for N-Br and 12 μM for N-I), in presence or absence of NAC, were examined for intracellular ROS revealed with DHE probe. Both compounds led to increased ROS formation evaluated by fluorescence microscopy, as compared to untreated cells, and this effect was attenuated by NAC (Fig. 4a). ROS dependent cell death was further confirmed in a cytotoxicity assay of melanoma cells and the cytotoxic effects of N-Br and N-I were significantly inhibited by NAC (Fig. 4b).Fig. 4

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