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Tudor staphylococcal nuclease drives chemoresistance of non-small cell lung carcinoma cells by regulating S100A11.

Zagryazhskaya A, Surova O, Akbar NS, Allavena G, Gyuraszova K, Zborovskaya IB, Tchevkina EM, Zhivotovsky B - Oncotarget (2015)

Bottom Line: Lung cancer is the leading cause of cancer-related deaths worldwide.Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level.Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF(3), as well as N-acetyl-L-cysteine, which also mimicked the effect of PLA(2) inhibitor on NSCLC chemosensitization upon S100A11 silencing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Medicine, Division of Toxicology, Stockholm, Sweden.

ABSTRACT
Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC), the major lung cancer subtype, is characterized by high resistance to chemotherapy. Here we demonstrate that Tudor staphylococcal nuclease (SND1 or TSN) is overexpressed in NSCLC cell lines and tissues, and is important for maintaining NSCLC chemoresistance. Downregulation of TSN by RNAi in NSCLC cells led to strong potentiation of cell death in response to cisplatin. Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level. Downregulation of S100A11 by RNAi resulted in enhanced sensitivity of NSCLC cells to cisplatin, oxaliplatin and 5-fluouracil. AACOCF(3), a phospholipase A(2) (PLA(2)) inhibitor, strongly abrogated chemosensitization upon silencing of S100A11 suggesting that PLA(2) inhibition by S100A11 governs the chemoresistance of NSCLC. Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF(3), as well as N-acetyl-L-cysteine, which also mimicked the effect of PLA(2) inhibitor on NSCLC chemosensitization upon S100A11 silencing. Thus, we present the novel TSN-S100A11-PLA(2) axis regulating superoxide-dependent apoptosis, triggered by platinum-based chemotherapeutic agents in NSCLC that may be targeted by innovative cancer therapies.

No MeSH data available.


Related in: MedlinePlus

Chemosensitizing effect of S100A11 in NSCLC cells involves mitochondrial superoxide formation, which is abrogated by PLA2 inhibitorA. Cleavage of PARP and processing of caspase-9 and -3 in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent) (upper panel). GAPDH was used as loading control. Cytochrome c (cyt c) release into the cytoplasm of A549 cells, treated as indicated (24 hours with or without chemotherapeutic agent) (cytoplasmic fraction, lower panel). GAPDH was used as loading control (panel showing corresponding GAPDH bands is presented below the panel demonstrating the protein of interest). The data are representative of three independent experiments. B, C, D. Representative images displaying staining of A549 cells, treated as indicated (12 hours with or without chemotherapeutic agent), with MitoSox Red mitochondrial superoxide indicator (top picture) and Hoechst 33342 (bottom picture) (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (the values are presented above each image). P < 0.05. For details see “Materials and Methods” section.
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Figure 6: Chemosensitizing effect of S100A11 in NSCLC cells involves mitochondrial superoxide formation, which is abrogated by PLA2 inhibitorA. Cleavage of PARP and processing of caspase-9 and -3 in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent) (upper panel). GAPDH was used as loading control. Cytochrome c (cyt c) release into the cytoplasm of A549 cells, treated as indicated (24 hours with or without chemotherapeutic agent) (cytoplasmic fraction, lower panel). GAPDH was used as loading control (panel showing corresponding GAPDH bands is presented below the panel demonstrating the protein of interest). The data are representative of three independent experiments. B, C, D. Representative images displaying staining of A549 cells, treated as indicated (12 hours with or without chemotherapeutic agent), with MitoSox Red mitochondrial superoxide indicator (top picture) and Hoechst 33342 (bottom picture) (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (the values are presented above each image). P < 0.05. For details see “Materials and Methods” section.

Mentions: Arachidonic acid and its oxidized metabolites, as well as radicals generated as by-products during AA oxidation, may affect intracellular ROS production and subsequent signaling events regulating apoptosis [30–33]. To investigate whether S100A11 silencing chemosensitizes NSCLC cells by affecting intracellular ROS production, we exploited antioxidant N-acetyl-L-cysteine (NAC). A549 cells were transfected with scrambled nontargeting or S100A11-specific siRNA pools, 24 hours later the transfection mixture was replaced with complete growth medium containing 5 mM of NAC, then 24 hours later cisplatin was added for another 24 hours. As shown in Fig. 6A, NAC abolished the potentiation of apoptosis caused by S100A11 silencing. In addition, NAC abolished cytochrome c release stimulated by S100A11 silencing (Fig. 6A, lower panel). Confirming that the chemosensitizing effect of S100A11 silencing in NSCLC cells is mediated to a large extent by ROS production, we next examined the level of ROS formation in A549 cells. Mitochondria, being crucial regulators of apoptosis, also represent a major source of ROS in mammalian cells. Superoxide (superoxide anion radical, O2•−) is recognized as “primary” ROS; O2•− is generated within mitochondria, further giving rise to other, “secondary” forms of ROS [34]. To assess ROS production we used MitoSox Red, a mitochondrial superoxide indicator, which is oxidized by superoxide in mitochondria. As shown in Fig. 6B, S100A11 silencing resulted in ~twofold increase in superoxide production upon treatment with cisplatin. Treatment with NAC did not induce a statistically significant difference in superoxide production in control cells (transfected with scrambled nontargeting siRNA pool), while it completely abrogated the rise in superoxide level in S100A11 knocked-down cells (Fig. 6C). In the presence of PLA2 inhibitor AACOCF3, the superoxide level was slightly decreased in control cells, while the rise in O2•− production was diminished for ~80% (Fig. 6D). Our results indicate that in NSCLC cells treated with platinum-based chemotherapeutic agents, S100A11 silencing leads to higher PLA2 activity that drives enhanced mitochondrial superoxide production, which results in potentiation of apoptosis.


Tudor staphylococcal nuclease drives chemoresistance of non-small cell lung carcinoma cells by regulating S100A11.

Zagryazhskaya A, Surova O, Akbar NS, Allavena G, Gyuraszova K, Zborovskaya IB, Tchevkina EM, Zhivotovsky B - Oncotarget (2015)

Chemosensitizing effect of S100A11 in NSCLC cells involves mitochondrial superoxide formation, which is abrogated by PLA2 inhibitorA. Cleavage of PARP and processing of caspase-9 and -3 in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent) (upper panel). GAPDH was used as loading control. Cytochrome c (cyt c) release into the cytoplasm of A549 cells, treated as indicated (24 hours with or without chemotherapeutic agent) (cytoplasmic fraction, lower panel). GAPDH was used as loading control (panel showing corresponding GAPDH bands is presented below the panel demonstrating the protein of interest). The data are representative of three independent experiments. B, C, D. Representative images displaying staining of A549 cells, treated as indicated (12 hours with or without chemotherapeutic agent), with MitoSox Red mitochondrial superoxide indicator (top picture) and Hoechst 33342 (bottom picture) (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (the values are presented above each image). P < 0.05. For details see “Materials and Methods” section.
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Figure 6: Chemosensitizing effect of S100A11 in NSCLC cells involves mitochondrial superoxide formation, which is abrogated by PLA2 inhibitorA. Cleavage of PARP and processing of caspase-9 and -3 in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent) (upper panel). GAPDH was used as loading control. Cytochrome c (cyt c) release into the cytoplasm of A549 cells, treated as indicated (24 hours with or without chemotherapeutic agent) (cytoplasmic fraction, lower panel). GAPDH was used as loading control (panel showing corresponding GAPDH bands is presented below the panel demonstrating the protein of interest). The data are representative of three independent experiments. B, C, D. Representative images displaying staining of A549 cells, treated as indicated (12 hours with or without chemotherapeutic agent), with MitoSox Red mitochondrial superoxide indicator (top picture) and Hoechst 33342 (bottom picture) (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (the values are presented above each image). P < 0.05. For details see “Materials and Methods” section.
Mentions: Arachidonic acid and its oxidized metabolites, as well as radicals generated as by-products during AA oxidation, may affect intracellular ROS production and subsequent signaling events regulating apoptosis [30–33]. To investigate whether S100A11 silencing chemosensitizes NSCLC cells by affecting intracellular ROS production, we exploited antioxidant N-acetyl-L-cysteine (NAC). A549 cells were transfected with scrambled nontargeting or S100A11-specific siRNA pools, 24 hours later the transfection mixture was replaced with complete growth medium containing 5 mM of NAC, then 24 hours later cisplatin was added for another 24 hours. As shown in Fig. 6A, NAC abolished the potentiation of apoptosis caused by S100A11 silencing. In addition, NAC abolished cytochrome c release stimulated by S100A11 silencing (Fig. 6A, lower panel). Confirming that the chemosensitizing effect of S100A11 silencing in NSCLC cells is mediated to a large extent by ROS production, we next examined the level of ROS formation in A549 cells. Mitochondria, being crucial regulators of apoptosis, also represent a major source of ROS in mammalian cells. Superoxide (superoxide anion radical, O2•−) is recognized as “primary” ROS; O2•− is generated within mitochondria, further giving rise to other, “secondary” forms of ROS [34]. To assess ROS production we used MitoSox Red, a mitochondrial superoxide indicator, which is oxidized by superoxide in mitochondria. As shown in Fig. 6B, S100A11 silencing resulted in ~twofold increase in superoxide production upon treatment with cisplatin. Treatment with NAC did not induce a statistically significant difference in superoxide production in control cells (transfected with scrambled nontargeting siRNA pool), while it completely abrogated the rise in superoxide level in S100A11 knocked-down cells (Fig. 6C). In the presence of PLA2 inhibitor AACOCF3, the superoxide level was slightly decreased in control cells, while the rise in O2•− production was diminished for ~80% (Fig. 6D). Our results indicate that in NSCLC cells treated with platinum-based chemotherapeutic agents, S100A11 silencing leads to higher PLA2 activity that drives enhanced mitochondrial superoxide production, which results in potentiation of apoptosis.

Bottom Line: Lung cancer is the leading cause of cancer-related deaths worldwide.Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level.Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF(3), as well as N-acetyl-L-cysteine, which also mimicked the effect of PLA(2) inhibitor on NSCLC chemosensitization upon S100A11 silencing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Medicine, Division of Toxicology, Stockholm, Sweden.

ABSTRACT
Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC), the major lung cancer subtype, is characterized by high resistance to chemotherapy. Here we demonstrate that Tudor staphylococcal nuclease (SND1 or TSN) is overexpressed in NSCLC cell lines and tissues, and is important for maintaining NSCLC chemoresistance. Downregulation of TSN by RNAi in NSCLC cells led to strong potentiation of cell death in response to cisplatin. Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level. Downregulation of S100A11 by RNAi resulted in enhanced sensitivity of NSCLC cells to cisplatin, oxaliplatin and 5-fluouracil. AACOCF(3), a phospholipase A(2) (PLA(2)) inhibitor, strongly abrogated chemosensitization upon silencing of S100A11 suggesting that PLA(2) inhibition by S100A11 governs the chemoresistance of NSCLC. Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF(3), as well as N-acetyl-L-cysteine, which also mimicked the effect of PLA(2) inhibitor on NSCLC chemosensitization upon S100A11 silencing. Thus, we present the novel TSN-S100A11-PLA(2) axis regulating superoxide-dependent apoptosis, triggered by platinum-based chemotherapeutic agents in NSCLC that may be targeted by innovative cancer therapies.

No MeSH data available.


Related in: MedlinePlus