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A bis-sulphamoylated estradiol derivative induces ROS-dependent cell cycle abnormalities and subsequent apoptosis

View Article: PubMed Central - PubMed

ABSTRACT

Clinical trials have revealed that the potential anticancer agent, 2-methoxyestradiol (2ME2) has limitations due to its low bioavailability. Subsequently, 2ME2 derivatives including (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) have shown improved efficacies in inducing apoptosis. However, no conclusive data exist to explain the mode of action exerted by these drugs. This study investigated the mode of action used by EMBS as a representative of the sulphamoylated 2ME2 derivatives. Hydrogen peroxide and superoxide production was quantified using dichlorofluorescein diacetate and hydroethidine. Cell proliferation and mitochondrial metabolism were investigated using crystal violet and Alamar Blue. Apoptosis was assessed using Annexin V-FITC while mitochondrial integrity was assessed using Mitocapture. Autophagy was visualised using LC3B II antibodies. The effects of EMBS on H2A phosphorylation and nuclei were visualised using phospho H2A antibody and 4',6-diamidino-2-phenylindole, dihydrochloride. Data showed that EMBS exposure leads to increased reactive oxygen species (ROS) production which is correlated with loss of cell proliferation, mitochondrial membrane damage, decreased metabolic activity, G2/M arrest, endoreduplication, DNA double stranded breaks, micronuclei and apoptosis induction. Treatment of EMBS-exposed cells with the ROS scavenger, N-acetyl cysteine, abrogated ROS production, cell cycle arrest and apoptosis implying an essential role for ROS production in EMBS signaling. The inhibition of c-Jun N-terminal kinase (JNK) activity also inhibited EMBS-induced apoptosis suggesting that EMBS triggers apoptosis via the JNK pathway. Lastly, evaluation of LC3IIB protein levels indicated that autophagy is not activated in EMBS-exposed cells. Our data shows that EMBS targets a pathway that leads to increased ROS production as an early event that culminates in G2/M arrest and apoptosis by means of JNK-signaling in cancer cells. This study suggests a novel oxidative stress-dependent mode of action for sulphamoylated derivatives.

No MeSH data available.


Related in: MedlinePlus

Antiproliferative and apoptotic effects exerted by EMBS are ROS-dependent.(A) Hydrogen peroxide production of EMBS-treated cells in the presence of absence of 20 mM NAC. The graph represents the average of 3 independent experiments with error bars representing s.e.m. Values represent fold-change compared to vehicle-treated cells. A * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (B) Crystal violet staining demonstrating the dose-dependent inhibition of NAC on the antiproliferative activity of 0.4 μM EMBS measured after 24 h. Cell growth is expressed as a percentage compared to cells propagated in control growth medium. The graph represents the average of 3 independent experiments with error bars representing s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (C) Mitochondrial membrane potential of EMBS-treated cells in the presence or absence of 20 mM NAC. Cells treated with EMBS alone (dark grey bar) or EMBS and NAC (light grey bar) were analysed using Mitotracker. The graph represents three independent experiments with error bars of s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to cells exposed to 0.4 μM EMBS and 20 mM NAC. (D) MDA-MB-231 cells were either left unblocked or were blocked in G1/S by thymidine and were exposed EMBS alone or together with 20 mM NAC. The graph represents the average results of crystal violet of three independent experiments with error bars representing s.e.m. * demonstrates a statistically significant P value of <0.05 when compared to EMBS-treated cells. An ** demonstrates a statistically significant P value <0.05 when compared to cells treated with EMBS, thymidine and NAC.
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pone.0176006.g003: Antiproliferative and apoptotic effects exerted by EMBS are ROS-dependent.(A) Hydrogen peroxide production of EMBS-treated cells in the presence of absence of 20 mM NAC. The graph represents the average of 3 independent experiments with error bars representing s.e.m. Values represent fold-change compared to vehicle-treated cells. A * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (B) Crystal violet staining demonstrating the dose-dependent inhibition of NAC on the antiproliferative activity of 0.4 μM EMBS measured after 24 h. Cell growth is expressed as a percentage compared to cells propagated in control growth medium. The graph represents the average of 3 independent experiments with error bars representing s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (C) Mitochondrial membrane potential of EMBS-treated cells in the presence or absence of 20 mM NAC. Cells treated with EMBS alone (dark grey bar) or EMBS and NAC (light grey bar) were analysed using Mitotracker. The graph represents three independent experiments with error bars of s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to cells exposed to 0.4 μM EMBS and 20 mM NAC. (D) MDA-MB-231 cells were either left unblocked or were blocked in G1/S by thymidine and were exposed EMBS alone or together with 20 mM NAC. The graph represents the average results of crystal violet of three independent experiments with error bars representing s.e.m. * demonstrates a statistically significant P value of <0.05 when compared to EMBS-treated cells. An ** demonstrates a statistically significant P value <0.05 when compared to cells treated with EMBS, thymidine and NAC.

Mentions: As discussed above there are opposing data regarding the role of ROS in 2ME2-induced apoptosis. Therefore, in this study ROS production induced by EMBS was inhibited by co-treating cells with the antioxidant, NAC. Cleavage of NAC at the acetyl group liberates reduced cysteine resulting in sustained production of glutathione, an essential antioxidant. Glutathione neutralizes ROS by providing a sulfyhydryl group which serves as a reducing equivalent. Glutathione also functions as a substrate in order to promote the antioxidant activity of glutathione peroxidases [34]. To assess whether the antioxidant NAC inhibits EMBS-induced ROS, cells were co-treated with NAC and EMBS and hydrogen peroxide levels were measured (Fig 3A). Hydrogen peroxide levels were increased within 4 h after EMBS treatment, but co-treatment with NAC abrogated this increase. To determine whether there is a causal link between ROS production by EMBS and cell death, cells were treated with a concentration range of NAC in the presence of EMBS and the effects on proliferation were measured using crystal violet (Fig 3B). Data revealed an increase in cell viability that was correlated with increased concentrations of NAC. Furthermore, NAC abolished mitochondrial membrane potential depolarisation induced by EMBS after 4 h and 24 h (Fig 3C).


A bis-sulphamoylated estradiol derivative induces ROS-dependent cell cycle abnormalities and subsequent apoptosis
Antiproliferative and apoptotic effects exerted by EMBS are ROS-dependent.(A) Hydrogen peroxide production of EMBS-treated cells in the presence of absence of 20 mM NAC. The graph represents the average of 3 independent experiments with error bars representing s.e.m. Values represent fold-change compared to vehicle-treated cells. A * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (B) Crystal violet staining demonstrating the dose-dependent inhibition of NAC on the antiproliferative activity of 0.4 μM EMBS measured after 24 h. Cell growth is expressed as a percentage compared to cells propagated in control growth medium. The graph represents the average of 3 independent experiments with error bars representing s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (C) Mitochondrial membrane potential of EMBS-treated cells in the presence or absence of 20 mM NAC. Cells treated with EMBS alone (dark grey bar) or EMBS and NAC (light grey bar) were analysed using Mitotracker. The graph represents three independent experiments with error bars of s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to cells exposed to 0.4 μM EMBS and 20 mM NAC. (D) MDA-MB-231 cells were either left unblocked or were blocked in G1/S by thymidine and were exposed EMBS alone or together with 20 mM NAC. The graph represents the average results of crystal violet of three independent experiments with error bars representing s.e.m. * demonstrates a statistically significant P value of <0.05 when compared to EMBS-treated cells. An ** demonstrates a statistically significant P value <0.05 when compared to cells treated with EMBS, thymidine and NAC.
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pone.0176006.g003: Antiproliferative and apoptotic effects exerted by EMBS are ROS-dependent.(A) Hydrogen peroxide production of EMBS-treated cells in the presence of absence of 20 mM NAC. The graph represents the average of 3 independent experiments with error bars representing s.e.m. Values represent fold-change compared to vehicle-treated cells. A * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (B) Crystal violet staining demonstrating the dose-dependent inhibition of NAC on the antiproliferative activity of 0.4 μM EMBS measured after 24 h. Cell growth is expressed as a percentage compared to cells propagated in control growth medium. The graph represents the average of 3 independent experiments with error bars representing s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (C) Mitochondrial membrane potential of EMBS-treated cells in the presence or absence of 20 mM NAC. Cells treated with EMBS alone (dark grey bar) or EMBS and NAC (light grey bar) were analysed using Mitotracker. The graph represents three independent experiments with error bars of s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to cells exposed to 0.4 μM EMBS and 20 mM NAC. (D) MDA-MB-231 cells were either left unblocked or were blocked in G1/S by thymidine and were exposed EMBS alone or together with 20 mM NAC. The graph represents the average results of crystal violet of three independent experiments with error bars representing s.e.m. * demonstrates a statistically significant P value of <0.05 when compared to EMBS-treated cells. An ** demonstrates a statistically significant P value <0.05 when compared to cells treated with EMBS, thymidine and NAC.
Mentions: As discussed above there are opposing data regarding the role of ROS in 2ME2-induced apoptosis. Therefore, in this study ROS production induced by EMBS was inhibited by co-treating cells with the antioxidant, NAC. Cleavage of NAC at the acetyl group liberates reduced cysteine resulting in sustained production of glutathione, an essential antioxidant. Glutathione neutralizes ROS by providing a sulfyhydryl group which serves as a reducing equivalent. Glutathione also functions as a substrate in order to promote the antioxidant activity of glutathione peroxidases [34]. To assess whether the antioxidant NAC inhibits EMBS-induced ROS, cells were co-treated with NAC and EMBS and hydrogen peroxide levels were measured (Fig 3A). Hydrogen peroxide levels were increased within 4 h after EMBS treatment, but co-treatment with NAC abrogated this increase. To determine whether there is a causal link between ROS production by EMBS and cell death, cells were treated with a concentration range of NAC in the presence of EMBS and the effects on proliferation were measured using crystal violet (Fig 3B). Data revealed an increase in cell viability that was correlated with increased concentrations of NAC. Furthermore, NAC abolished mitochondrial membrane potential depolarisation induced by EMBS after 4 h and 24 h (Fig 3C).

View Article: PubMed Central - PubMed

ABSTRACT

Clinical trials have revealed that the potential anticancer agent, 2-methoxyestradiol (2ME2) has limitations due to its low bioavailability. Subsequently, 2ME2 derivatives including (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) have shown improved efficacies in inducing apoptosis. However, no conclusive data exist to explain the mode of action exerted by these drugs. This study investigated the mode of action used by EMBS as a representative of the sulphamoylated 2ME2 derivatives. Hydrogen peroxide and superoxide production was quantified using dichlorofluorescein diacetate and hydroethidine. Cell proliferation and mitochondrial metabolism were investigated using crystal violet and Alamar Blue. Apoptosis was assessed using Annexin V-FITC while mitochondrial integrity was assessed using Mitocapture. Autophagy was visualised using LC3B II antibodies. The effects of EMBS on H2A phosphorylation and nuclei were visualised using phospho H2A antibody and 4',6-diamidino-2-phenylindole, dihydrochloride. Data showed that EMBS exposure leads to increased reactive oxygen species (ROS) production which is correlated with loss of cell proliferation, mitochondrial membrane damage, decreased metabolic activity, G2/M arrest, endoreduplication, DNA double stranded breaks, micronuclei and apoptosis induction. Treatment of EMBS-exposed cells with the ROS scavenger, N-acetyl cysteine, abrogated ROS production, cell cycle arrest and apoptosis implying an essential role for ROS production in EMBS signaling. The inhibition of c-Jun N-terminal kinase (JNK) activity also inhibited EMBS-induced apoptosis suggesting that EMBS triggers apoptosis via the JNK pathway. Lastly, evaluation of LC3IIB protein levels indicated that autophagy is not activated in EMBS-exposed cells. Our data shows that EMBS targets a pathway that leads to increased ROS production as an early event that culminates in G2/M arrest and apoptosis by means of JNK-signaling in cancer cells. This study suggests a novel oxidative stress-dependent mode of action for sulphamoylated derivatives.

No MeSH data available.


Related in: MedlinePlus