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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

Silencing of S100A11 leads to increased formation of DNA strand breaksA. Immunostaining of γH2AX in A549 cells treated as indicated (Scale bar, 10 μm); B. Cleavage of PARP and γH2AX level in A549 cells treated as indicated. GAPDH was used as loading control. C. TUNEL staining of DNA strand breaks (top image) and Hoechst 33342 (bottom image) in A549 cells treated as indicated. (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (arbitrary units). P < 0.05. For details see “Materials and Methods” section. All data are representative of three independent experiments.
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Figure 7: Silencing of S100A11 leads to increased formation of DNA strand breaksA. Immunostaining of γH2AX in A549 cells treated as indicated (Scale bar, 10 μm); B. Cleavage of PARP and γH2AX level in A549 cells treated as indicated. GAPDH was used as loading control. C. TUNEL staining of DNA strand breaks (top image) and Hoechst 33342 (bottom image) in A549 cells treated as indicated. (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (arbitrary units). P < 0.05. For details see “Materials and Methods” section. All data are representative of three independent experiments.

Mentions: Superoxide, initially produced by mitochondria, is further dismutated to hydrogen peroxide, which, in turn, gives rise to highly reactive hydroxyl radicals [34], which can significantly affect cell signaling, causing damage to cellular proteins, lipids and DNA [35]. Here we observed enhanced formation of DNA double-strand breaks (DSB) in S100A11 knocked-down compared to S100A11-expressing NSCLC cells treated by chemotherapeutic agents. DNA DSB formation was assessed by the level of phosphorylated histone H2AX (γH2AX) using confocal microscopy (Fig. 7A) and western blotting (Fig. 7B). Immunostaining of paraformaldehyde-fixed A549 cells, exposed to cisplatin for 8 hours, revealed increased formation of γH2AX foci upon S100A11 silencing (Fig. 7A). In addition, the level of γH2AX was elevated in S100A11 knocked-down compared to S100A11-expressing cells treated with cisplatin or 5-FU for 12 hours (Fig. 7B). To ensure assessment of primary DNA damage (and not a result of DNA fragmentation due to induction of apoptosis), the pan-caspase inhibitor Z-VAD was added to the cells 1 hour before the chemotherapeutic agents. As shown in Fig. 7B, Z-VAD abolished the increased level of cleaved PARP in response to chemotherapeutic treatment, while the elevated level of γH2AX upon S100A11 silencing was preserved in the presence of this inhibitor. Formation of DNA strand breaks was further confirmed by TUNEL assay (Fig. 7C) in A549 cells treated with cisplatin for 12 hours upon inhibition of caspase activity by Z-VAD. These data indicate that chemosensitization of NSCLC cells upon S100A11 silencing (which leads to elevated mitochondrial superoxide formation) might, at least in part, result from enhanced DNA damage that potentiates DNA damage response signaling and 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)

Silencing of S100A11 leads to increased formation of DNA strand breaksA. Immunostaining of γH2AX in A549 cells treated as indicated (Scale bar, 10 μm); B. Cleavage of PARP and γH2AX level in A549 cells treated as indicated. GAPDH was used as loading control. C. TUNEL staining of DNA strand breaks (top image) and Hoechst 33342 (bottom image) in A549 cells treated as indicated. (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (arbitrary units). P < 0.05. For details see “Materials and Methods” section. All data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494929&req=5

Figure 7: Silencing of S100A11 leads to increased formation of DNA strand breaksA. Immunostaining of γH2AX in A549 cells treated as indicated (Scale bar, 10 μm); B. Cleavage of PARP and γH2AX level in A549 cells treated as indicated. GAPDH was used as loading control. C. TUNEL staining of DNA strand breaks (top image) and Hoechst 33342 (bottom image) in A549 cells treated as indicated. (Scale bar, 50 μm). The data were quantified using ImageJ software; the results are shown as the mean ± SEM of three independent experiments (arbitrary units). P < 0.05. For details see “Materials and Methods” section. All data are representative of three independent experiments.
Mentions: Superoxide, initially produced by mitochondria, is further dismutated to hydrogen peroxide, which, in turn, gives rise to highly reactive hydroxyl radicals [34], which can significantly affect cell signaling, causing damage to cellular proteins, lipids and DNA [35]. Here we observed enhanced formation of DNA double-strand breaks (DSB) in S100A11 knocked-down compared to S100A11-expressing NSCLC cells treated by chemotherapeutic agents. DNA DSB formation was assessed by the level of phosphorylated histone H2AX (γH2AX) using confocal microscopy (Fig. 7A) and western blotting (Fig. 7B). Immunostaining of paraformaldehyde-fixed A549 cells, exposed to cisplatin for 8 hours, revealed increased formation of γH2AX foci upon S100A11 silencing (Fig. 7A). In addition, the level of γH2AX was elevated in S100A11 knocked-down compared to S100A11-expressing cells treated with cisplatin or 5-FU for 12 hours (Fig. 7B). To ensure assessment of primary DNA damage (and not a result of DNA fragmentation due to induction of apoptosis), the pan-caspase inhibitor Z-VAD was added to the cells 1 hour before the chemotherapeutic agents. As shown in Fig. 7B, Z-VAD abolished the increased level of cleaved PARP in response to chemotherapeutic treatment, while the elevated level of γH2AX upon S100A11 silencing was preserved in the presence of this inhibitor. Formation of DNA strand breaks was further confirmed by TUNEL assay (Fig. 7C) in A549 cells treated with cisplatin for 12 hours upon inhibition of caspase activity by Z-VAD. These data indicate that chemosensitization of NSCLC cells upon S100A11 silencing (which leads to elevated mitochondrial superoxide formation) might, at least in part, result from enhanced DNA damage that potentiates DNA damage response signaling and 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