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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 TSN leads to downregulation of S100A11 and downregulation of S100A11 by RNAi chemosensitizes NSCLC cells similarly to the silencing of TSNA. Cleavage of PARP and S100A11 expression in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. B. Cleavage of PARP in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. C. Immunostaining of S100A11 in A549 cells transfected with nontargeting siRNA (si scr, upper panel) and S100A11-specific siRNA (si S100A11, lower panel) (72 hours post transfection). S100A11 is stained in green, while nuclei are stained in blue by Hoechst 33342 (Scale bar, 20 μm). D. Cleavage of PARP in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. E. Cleavage of PARP in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. F. Caspase-3-like activity (fold change versus control) in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). G. Cytochrome c (cyt c) level in membrane and cytoplasmic fraction of A549 (upper panel) and U1810 (lower panel) cells, treated as indicated (24 hours with or without chemotherapeutic agent). SDHA was used to verify the absence of mitochondria in the cytoplasmic fraction. GAPDH was used as loading control for cytoplasmic fraction. For details see “Materials and Methods” section. Different western blot images correspond to different exposure times of SuperRX X-ray films to the membranes treated with enhanced chemiluminescence reagents (to detect a noticeable signal from all protein bands under consideration within one membrane). Therefore, the band intensities are intended for comparison only within one membrane. All data are representative of three independent experiments.
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Figure 3: Silencing of TSN leads to downregulation of S100A11 and downregulation of S100A11 by RNAi chemosensitizes NSCLC cells similarly to the silencing of TSNA. Cleavage of PARP and S100A11 expression in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. B. Cleavage of PARP in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. C. Immunostaining of S100A11 in A549 cells transfected with nontargeting siRNA (si scr, upper panel) and S100A11-specific siRNA (si S100A11, lower panel) (72 hours post transfection). S100A11 is stained in green, while nuclei are stained in blue by Hoechst 33342 (Scale bar, 20 μm). D. Cleavage of PARP in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. E. Cleavage of PARP in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. F. Caspase-3-like activity (fold change versus control) in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). G. Cytochrome c (cyt c) level in membrane and cytoplasmic fraction of A549 (upper panel) and U1810 (lower panel) cells, treated as indicated (24 hours with or without chemotherapeutic agent). SDHA was used to verify the absence of mitochondria in the cytoplasmic fraction. GAPDH was used as loading control for cytoplasmic fraction. For details see “Materials and Methods” section. Different western blot images correspond to different exposure times of SuperRX X-ray films to the membranes treated with enhanced chemiluminescence reagents (to detect a noticeable signal from all protein bands under consideration within one membrane). Therefore, the band intensities are intended for comparison only within one membrane. All data are representative of three independent experiments.

Mentions: Next we investigated which of the TSN-regulated genes contributes to NSCLC chemoresistance. The S100A11 gene demonstrates the most drastic decrease upon TSN silencing (Supplementary Fig. S2) and was previously reported to be overexpressed in NSCLC [21, 22]. Therefore, we examined S100A11 protein expression upon TSN silencing in the A549 NSCLC cells, and found significant downregulation of S100A11 at the protein level in both cisplatin-treated and non-treated cells (Fig. 3A).


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 TSN leads to downregulation of S100A11 and downregulation of S100A11 by RNAi chemosensitizes NSCLC cells similarly to the silencing of TSNA. Cleavage of PARP and S100A11 expression in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. B. Cleavage of PARP in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. C. Immunostaining of S100A11 in A549 cells transfected with nontargeting siRNA (si scr, upper panel) and S100A11-specific siRNA (si S100A11, lower panel) (72 hours post transfection). S100A11 is stained in green, while nuclei are stained in blue by Hoechst 33342 (Scale bar, 20 μm). D. Cleavage of PARP in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. E. Cleavage of PARP in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. F. Caspase-3-like activity (fold change versus control) in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). G. Cytochrome c (cyt c) level in membrane and cytoplasmic fraction of A549 (upper panel) and U1810 (lower panel) cells, treated as indicated (24 hours with or without chemotherapeutic agent). SDHA was used to verify the absence of mitochondria in the cytoplasmic fraction. GAPDH was used as loading control for cytoplasmic fraction. For details see “Materials and Methods” section. Different western blot images correspond to different exposure times of SuperRX X-ray films to the membranes treated with enhanced chemiluminescence reagents (to detect a noticeable signal from all protein bands under consideration within one membrane). Therefore, the band intensities are intended for comparison only within one membrane. All data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Silencing of TSN leads to downregulation of S100A11 and downregulation of S100A11 by RNAi chemosensitizes NSCLC cells similarly to the silencing of TSNA. Cleavage of PARP and S100A11 expression in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. B. Cleavage of PARP in A549 cells treated as indicated (cispl, 5 μg/ml for 24 hours). GAPDH was used as loading control. C. Immunostaining of S100A11 in A549 cells transfected with nontargeting siRNA (si scr, upper panel) and S100A11-specific siRNA (si S100A11, lower panel) (72 hours post transfection). S100A11 is stained in green, while nuclei are stained in blue by Hoechst 33342 (Scale bar, 20 μm). D. Cleavage of PARP in A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. E. Cleavage of PARP in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). GAPDH was used as loading control. F. Caspase-3-like activity (fold change versus control) in U1810 and A549 cells treated as indicated (24 hours with or without chemotherapeutic agent). G. Cytochrome c (cyt c) level in membrane and cytoplasmic fraction of A549 (upper panel) and U1810 (lower panel) cells, treated as indicated (24 hours with or without chemotherapeutic agent). SDHA was used to verify the absence of mitochondria in the cytoplasmic fraction. GAPDH was used as loading control for cytoplasmic fraction. For details see “Materials and Methods” section. Different western blot images correspond to different exposure times of SuperRX X-ray films to the membranes treated with enhanced chemiluminescence reagents (to detect a noticeable signal from all protein bands under consideration within one membrane). Therefore, the band intensities are intended for comparison only within one membrane. All data are representative of three independent experiments.
Mentions: Next we investigated which of the TSN-regulated genes contributes to NSCLC chemoresistance. The S100A11 gene demonstrates the most drastic decrease upon TSN silencing (Supplementary Fig. S2) and was previously reported to be overexpressed in NSCLC [21, 22]. Therefore, we examined S100A11 protein expression upon TSN silencing in the A549 NSCLC cells, and found significant downregulation of S100A11 at the protein level in both cisplatin-treated and non-treated cells (Fig. 3A).

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