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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

Screening of antitumor activity of benzofuroxan compounds in vitro and in vivo against murine melanoma. a Cytotoxicity of 23 compounds in B16F10-Nex2 cells. Compounds were incubated with tumor cells at 100 μM for 16 h and viability was assessed by MTT method. b Antitumor activity of active benzofuroxan derivatives in vivo. Number of metastatic melanoma nodules in the lung of animals after treatment with 500 μM of compounds injected i.p. For each experiment, five animals were used per group (*p < 0.05). c N-Br and N-I melanoma treatment (300 μg/day) in NOD/SCID IL-2R-gamma  mice for seven consecutive days. No. metastatic nodules were quantified using a Stereo Microscope (Nikon) (*p < 0.05)
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Fig1: Screening of antitumor activity of benzofuroxan compounds in vitro and in vivo against murine melanoma. a Cytotoxicity of 23 compounds in B16F10-Nex2 cells. Compounds were incubated with tumor cells at 100 μM for 16 h and viability was assessed by MTT method. b Antitumor activity of active benzofuroxan derivatives in vivo. Number of metastatic melanoma nodules in the lung of animals after treatment with 500 μM of compounds injected i.p. For each experiment, five animals were used per group (*p < 0.05). c N-Br and N-I melanoma treatment (300 μg/day) in NOD/SCID IL-2R-gamma mice for seven consecutive days. No. metastatic nodules were quantified using a Stereo Microscope (Nikon) (*p < 0.05)

Mentions: The cytotoxicity of the compounds (Table 1) and particularly, the N-Br and N-I derivatives was investigated primarily on murine melanoma B16F10-Nex2 cells at 100 μM for 16 h. All tested compounds were active against tumor cells; compounds 1, 5, 10, 17, 18, 19, 21 and 23 were the most cytotoxic (Fig. 1a). The IC50 values (concentrations needed to kill 50 % of tumor cells) were determined on B16F10-Nex2, ranging from 6.9 to 25.4 μM (Table 2). They were evaluated with different murine and human tumor cell lines and non-tumorigenic cells (Table 3). Derivatives N-Br and N-I exhibited similar effects with IC50 values between 10 and 30 μM, except for U87 cells, which were resistant to these compounds (IC50 > 50 μM).Fig. 1


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)

Screening of antitumor activity of benzofuroxan compounds in vitro and in vivo against murine melanoma. a Cytotoxicity of 23 compounds in B16F10-Nex2 cells. Compounds were incubated with tumor cells at 100 μM for 16 h and viability was assessed by MTT method. b Antitumor activity of active benzofuroxan derivatives in vivo. Number of metastatic melanoma nodules in the lung of animals after treatment with 500 μM of compounds injected i.p. For each experiment, five animals were used per group (*p < 0.05). c N-Br and N-I melanoma treatment (300 μg/day) in NOD/SCID IL-2R-gamma  mice for seven consecutive days. No. metastatic nodules were quantified using a Stereo Microscope (Nikon) (*p < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Screening of antitumor activity of benzofuroxan compounds in vitro and in vivo against murine melanoma. a Cytotoxicity of 23 compounds in B16F10-Nex2 cells. Compounds were incubated with tumor cells at 100 μM for 16 h and viability was assessed by MTT method. b Antitumor activity of active benzofuroxan derivatives in vivo. Number of metastatic melanoma nodules in the lung of animals after treatment with 500 μM of compounds injected i.p. For each experiment, five animals were used per group (*p < 0.05). c N-Br and N-I melanoma treatment (300 μg/day) in NOD/SCID IL-2R-gamma mice for seven consecutive days. No. metastatic nodules were quantified using a Stereo Microscope (Nikon) (*p < 0.05)
Mentions: The cytotoxicity of the compounds (Table 1) and particularly, the N-Br and N-I derivatives was investigated primarily on murine melanoma B16F10-Nex2 cells at 100 μM for 16 h. All tested compounds were active against tumor cells; compounds 1, 5, 10, 17, 18, 19, 21 and 23 were the most cytotoxic (Fig. 1a). The IC50 values (concentrations needed to kill 50 % of tumor cells) were determined on B16F10-Nex2, ranging from 6.9 to 25.4 μM (Table 2). They were evaluated with different murine and human tumor cell lines and non-tumorigenic cells (Table 3). Derivatives N-Br and N-I exhibited similar effects with IC50 values between 10 and 30 μM, except for U87 cells, which were resistant to these compounds (IC50 > 50 μM).Fig. 1

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