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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

37386230
37796
10.1038/s41598-023-37796-5
Article
Downregulation of SODD mediates carnosol-induced reduction in cell proliferation in esophageal adenocarcinoma cells
Li Aihua 12
Cao Weibiao Weibiao_Cao@alumni.brown.edu

1
1 grid.40263.33 0000 0004 1936 9094 Department of Pathology and Laboratory Medicine, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, 593 Eddy St, APC12, Providence, RI 02903 USA
2 grid.190737.b 0000 0001 0154 0904 Department of Gastroenterology, Chongqing University Cancer Hospital, Chongqing, China
29 6 2023
29 6 2023
2023
13 1058014 12 2022
28 6 2023
© The Author(s) 2023
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Esophageal adenocarcinoma carries a poor prognosis associated with a 5-year survival rate of 12.5–20%. Therefore, a new therapeutic modality is needed for this lethal tumor. Carnosol is a phenolic diterpene purified from the herbs such as rosemary and Mountain desert sage and has been shown to have anticancer activities in multiple cancers. In this study we examined the effect of carnosol on cell proliferation in esophageal adenocarcinoma cells. We found that carnosol dose-dependently decreased cell proliferation in FLO-1 esophageal adenocarcinoma cells and significantly increased caspase-3 protein, indicating that carnosol decreases cell proliferation and increases cell apoptosis in FLO-1 cells. Carnosol significantly increased H2O2 production and N-acetyl cysteine, a reactive oxygen species (ROS) scavenger, significantly inhibited carnosol-induced decrease in cell proliferation, indicating that ROS may mediate carnosol-induced decrease in cell proliferation. Carnosol-induced decrease in cell proliferation was partially reversed by NADPH oxidase inhibitor apocynin, suggesting that NADPH oxidases may be partially involved in carnosol’s effect. In addition, carnosol significantly downregulated SODD protein and mRNA expression and knockdown of SODD significantly inhibited the carnosol-induced reduction in cell proliferation, suggesting that downregulation of SODD may contribute to carnosol-induced reduction in cell proliferation. We conclude that carnosol dose-dependently decreased cell proliferation and significantly increased caspase-3 protein. Carnosol’s effect may be through the overproduction of ROS and the downregulation of SODD. Carnosol might be useful for the treatment of esophageal adenocarcinoma.

Subject terms

Cancer
Gastroenterology
Chongqing Scientific Research Institute Performance Incentive Guidance Special Projectcstc2020jxjl130017 Li Aihua issue-copyright-statement© Springer Nature Limited 2023
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pmcIntroduction

Esophageal adenocarcinoma has increased in incidence over the past several decades1–3. The major risk factor for esophageal adenocarcinoma is Barrett’s esophagus4, which carries nearly a 30–125-fold increased risk for the development of esophageal adenocarcinoma. The cancer incidence in Barrett’s esophagus is about 0.5–1.0% per year. The mechanisms of the progression from Barrett’s esophagus to adenocarcinoma are not fully understood. Many genetic and epigenetic alterations, chromosomal gains, and losses, and hypermethylation of gene promoters may be involved in this progression5,6. Esophageal adenocarcinoma has a poor prognosis associated with a median survival of less than 1 year7,8 and a 5-year survival rate of 12.5–20%9,10. Therefore, a new therapeutic modality is needed for this lethal tumor.

Carnosol is a phenolic diterpene purified from the herbs such as rosemary and Mountain desert sage, and has been shown to have anticancer activities in multiple cancers, including colon11, breast12, stomach13 and prostate14. It may inactivate STAT3 through the production of reactive oxygen species (ROS), thus inhibiting tumor migration and growth in breast cancer cells12. Carnosol may selectively inhibit the p300 histone acetyl transferase15 and triggers a ROS-dependent ER-stress response through activation of the three ER stress sensor pathways in breast cancer cells16. ROS dependent inactivation of STAT3 also mediates carnosol-induced apoptosis in human colon cancer HCT116 cells11. In gastric cancer, carnosol suppresses gastric cancer growth via inhibiting the RSK-CREB signaling pathway13. Whether carnosol has anticancer activity in esophageal adenocarcinoma is not known. The aim of this study is to examine the effect of carnosol on cell proliferation in esophageal adenocarcinoma cells and whether its effect is mediated by the silencer of death domains (SODD).

Results

Carnosol reduced cell proliferation and increased cell apoptosis in esophageal adenocarcinoma cells

We first examined whether carnosol decreases cell proliferation by using WST-1 cell proliferation assay. We found that carnosol dose-dependently decreased cell proliferation in FLO-1 esophageal adenocarcinoma cells (Fig. 1, ANOVA, P < 0.001). To further confirm this result, we performed Western blot analyses by using caspase-3 antibody. We found that carnosol significantly increased caspase-3 protein (Fig. 2, t test, P < 0.05), indicating that carnosol increases cell apoptosis in esophageal adenocarcinoma cells. These data suggest that carnosol decreases cell proliferation and increases cell apoptosis in FLO-1 cells.Figure 1 Effect of carnosol on cell proliferation. 24-h treatment with carnosol dose-dependently decreased cell proliferation in esophageal adenocarcinoma cells FLO-1, suggesting that carnosol decreases cell proliferation in FLO-1 cells. N = 3, ANOVA, P < 0.001.

Figure 2 Effect of carnosol on caspase 3. (A) A typical image of Western blot analysis and (B) summarized data showed that carnosol (10–4 M, 24 h) significantly increased caspase-3 protein, indicating that carnosol increases cell apoptosis in FLO-1 cells. N = 4, * t-test, P < 0.05. Original blots are presented in Supplementary Figure 1.

Reactive oxygen species (ROS) may mediate carnosol’s effect

Intracellular reactive oxygen species may play an important role in cell apoptosis. Therefore, we examined the effect of ROS on carnosol-induced decrease in cell proliferation by using N-acetyl cysteine (NAC), a ROS scavenger, on cell proliferation. As shown in Fig. 3A, pre-treatment with NAC for 1 h significantly inhibited carnosol-induced decrease in cell proliferation (ANOVA, P < 0.001), indicating that ROS may mediate carnosol-induced decrease in cell proliferation.Figure 3 Reactive oxygen species (ROS) may mediate carnosol’s effect. (A) Pre-treatment with N-acetyl cysteine (NAC, 10–6 M), a ROS scavenger, for 1 h significantly inhibited carnosol-induced decrease in cell proliferation, indicating that ROS may mediate carnosol (10–4 M, 24 h)-induced decrease in cell proliferation. N = 3, ANOVA, *P < 0.001, compared with vehicle control; **P < 0.001 compared with carnosol. (B) Hydrogen peroxide in the culture medium was measured. Carnosol (10–4 M, 3 h) significantly increased H2O2 production, suggesting that ROS may contribute at least in part to carnosol’s effect. N = 3, *t test, P < 0.05.

To confirm this result, we measured hydrogen peroxide in the culture medium. Cells were treated with vehicle or carnosol (10–4 M) for 3 h and then culture medium was collected for measurement. As shown in Fig. 3B, carnosol significantly increased H2O2 production (t test, P < 0.05), suggesting that ROS may contribute at least in part to carnosol’s effect.

NADPH oxidases may be involved in carnosol’s effect

We have shown that NADPH oxidases were present in FLO-1 cells17. Therefore, we examined the role of NADPH oxidases in carnosol-induced decrease in cell proliferation. Figure 4 showed that carnosol-induced decrease in cell proliferation was partially reversed by NADPH oxidase inhibitor apocynin (ANOVA, P < 0.0001), suggesting that NADPH oxidases may be partially involved in carnosol’s effect.Figure 4 NADPH oxidases may be involved in carnosol’s effect. Carnosol (10–4 M, 24 h)-induced decrease in cell proliferation was partially reversed by NADPH oxidase inhibitor apocynin (10–6 M), suggesting that NADPH oxidases may be partially involved in carnosol’s effect. N = 12, ANOVA, *P < 0.0001, compared with vehicle control; **P < 0.0001 compared with carnosol.

SODD is involved in carnosol-induced decrease in cell proliferation in FLO-1 Cells.

We have previously showed that acid-induced increase in SODD expression depends on the activation of NOX5-S and NF-κB1 p50 in FLO EA cells18. Therefore, we examined whether carnosol-induced decrease in cell proliferation is through the reduction of SODD. Figure 5 showed that 24-h treatment with carnosol (10–4 M) significantly decreased SODD protein (Fig. 5A,B, t test, P < 0.05) and mRNA expression (Fig. 5C, t test, P = 0.001), supporting our hypothesis that SODD may be involved in carnosol induced reduction in cell proliferation.Figure 5 SODD is involved in carnosol-induced decrease in cell proliferation in FLO-1 cells. (A) A typical image of Western blot analysis and (B) summarized data showed that carnosol (10–4 M, 24 h) significantly decreased SODD protein, indicating that carnosol downregulates SODD protein in FLO-1 cells. N = 4, * t-test, P < 0.05. Original blots are presented in Supplementary Figure 2. (C) 24-h treatment with carnosol (10–4 M) significantly decreased SODD mRNA expression in FLO-1 cells. #P = 0.001, t test, N = 8.

To further confirm this result, we used SODD siRNA to knock down SODD. We have shown that SODD siRNA successfully downregulated SODD protein expression18. Figure 6A showed that knockdown of SODD at basal condition significantly decreased cell proliferation (P < 0.02), indicating that SODD contributes to the cell proliferation at the basal condition in FLO-1 cells. In cells transfected with control siRNA, carnosol caused 68 ± 1% inhibition of cell proliferation (Fig. 6B). This inhibition was significantly reduced by knockdown of SODD (28.5 ± 4.8%, P < 0.02). The data suggest that downregulation of SODD may mediate carnosol-induced reduction in cell proliferation.Figure 6 Role of SODD in carnosol-induced decrease in cell proliferation. (A) Knockdown of SODD at basal condition significantly decreased cell proliferation, indicating that SODD contributes to the cell proliferation at the basal condition in FLO-1 cells. Carnosol significantly decreased cell proliferation in cells transfected with control siRNA, a decrease which was partially reversed by knockdown of SODD. N = 3, ANOVA, ***P < 0.001, compared with control transfected with control siRNA, **P < 0.02, compared with control transfected with control siRNA; *P < 0.05, compared with carnosol transfected with control siRNA. B. In cells transfected with control siRNA, carnosol caused 68 ± 1% inhibition of cell proliferation. This inhibition was significantly reduced by knockdown of SODD (28.5 ± 4.8%). The data suggest that downregulation of SODD may mediate carnosol-induced reduction in cell proliferation. N = 3, t test, #P < 0.02.

Discussion

We found that carnosol inhibited cell proliferation and increased cell apoptosis in esophageal adenocarcinoma cells since carnosol dose-dependently decreased cell proliferation and significantly increased caspase-3 protein, suggesting that carnosol has anticancer activity in esophageal adenocarcinoma. The carnosol’s effect may be mediated by reactive oxygen species as supported by our results that N-acetyl cysteine (NAC) blocked carnosol-induced decrease in cell proliferation and carnosol significantly increased H2O2 production. This result is consistent with the results in breast cancer cells16 and human colon cancer HCT11611. In breast cancer cells16, carnosol activates ROS-dependent ER-stress responses, whereas in human colon cancer cells carnosol-induced apoptosis is mediated by inactivation of STAT3 through the production of ROS. Reactive oxygen species have dual effects. Low doses of ROS activate cell survival signaling pathway, whereas high doses of ROS induce cell apoptosis19. Therefore, carnosol might induce ROS production high enough to cause cell apoptosis.

ROS may be generated in mitochondria through the respiratory chains or by enzymes such as NADPH oxidases. Apocynin is a naturally occurring methoxy-substituted catechol and is an inhibitor of NADPH-oxidase20–22. It reduces the production of superoxide from activated neutrophils and macrophages, but the exact mechanism of its inhibition is not fully understood23. It is thought that apocynin inhibits reactive oxygen species (ROS) by inhibition of the assembly of NADPH-oxidase23. We found that apocynin partially reversed carnosol-induced decrease in cell proliferation, suggesting that NADPH oxidases might be partially involved in carnosol’s effect.

SODD is an anti-apoptotic protein and belongs to the BAG family24. SODD has been reported to be increased in cancer tissues; e.g. pancreatic cancers25, an increase of SODD expression which may decrease cell death and increase cell proliferation in various cancer cell lines25,26. SODD is associated with the ATPase domain of Hsc70/Hsp7024, the cytoplasmic domain of the tumor necrosis factor receptor 1 (TNFR1) and death receptor-3. TNFR1 signaling complex is activated by the release of SODD from TNFR1, which permits the recruitment of TNFR-associated death domain and TNFR-associated factor 226. SODD inhibits cell apoptosis through its binding to TNFR1 and preventing the ligand-independent oligomerization and spontaneous activation of TNFR127. We found that carnosol significantly decreased SODD protein and mRNA expression, indicating that carnosol may decrease cell proliferation via the downregulation of SODD. This result was further confirmed by the knockdown study showing that knockdown of SODD significantly blocked carnosol-induced inhibition of cell proliferation.

Carnosol might also play its anticancer role via epigenetic changes. For example, carnosol has been shown to inhibit p300 histone acetyl transferase activity by blocking the acetyl-CoA binding to the histone acetyl transferase catalytic domain since carnosol occupies the region where the pantetheine arm of the acetyl-CoA is bound15. It is well known that p300 is a coactivator of multiple transcription factors related to many biological processes28. High levels of p300 are associated with breast cancer progression29 and poor prognosis30. The inhibition of p300 histone acetyl transferase will decrease p300 acetylation, thus increasing p300 degradation. Whether this epigenetic modification of p300 is involved in carnosol’s anticancer effect in esophageal adenocarcinoma needs to be further explored.

In conclusion, carnosol dose-dependently decreased cell proliferation and significantly increased caspase-3 protein. Carnosol’s effect may be through the overproduction of ROS and the downregulation of SODD. Carnosol may be useful for the treatment of esophageal adenocarcinoma.

Materials and methods

Cell culture and treatment

The human Barrett’s adenocarcinoma cell line FLO-131 was obtained from Dr. David Beer (University of Michigan). The FLO-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and antibiotics.

Cell proliferation assay

Cell proliferation was determined by using WST-1 Cell Proliferation Assay. Cell Proliferation Reagent WST-1 was purchased from MilliporeSigma. 2 × 104 FLO-1 cells were seeded in a 96-well plate. When cells reached 50–60% confluence, different concentrations of carnosol or vehicle controls were added to each well. After cells were cultured for 24 h, 10 μl WST-1 reagent was added to each well and cells were cultured for additional 1 h. Then the absorbance of each well was determined by using a BioTek Synergy H4 hybrid reader at 440 nm. Medium without cells was used as a blank. Carnosol was dissolved in ethanol and the same concentrations of ethanol were used in the control groups. The experiments were repeated three times for the experiments of siRNA, N-acetyl cysteine and carnosol’s effects on cell proliferation and twelve times for apocynin experiments.

Amplex® red hydrogen peroxide fluorescent assay

Levels of H2O2 in culture medium were measured by using Amplex® Red H2O2 Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA) as we previously described32. The assay was performed in triplicates.

Small interfering RNA(SiRNA) transfection

FLO-1 cells were transfected with control siRNA or SODD siRNA by using Lipofectamine 2000 (Invitrogen, Grand Island, New York, USA) according to the manufacturer’s instruction and as we previously described18. 24 h after transfection, FLO-1 cells were treated with vehicle or carnosol (10–4 M) for additional 24 h.

Reverse-transcription PCR and quantitative real-time PCR

Reverse-transcription PCR and quantitative real-time PCR were performed as we previously described18. The primers used were as follows: SODD forward, 5′-GGGGTACCCAATGGTGCGATCTCGGCTCACTG-3′; SODD reverse, 5′-GAAGATCTCTCGAGGGGATCCGCTGCCCTGAAGCGCT-3′; 18S forward, 5′-CGGACAGGATTGACAGATTGATAGC-3′; and 18S reverse, 5′-TGCCAGAGTCTCGTTCGTTATCG -3′. The experiments were repeated eight times.

Western blot analysis

FLO-1 cells were treated with carnosol (10−4 M) or vehicle for 24 h and then collected for Western blot analysis. Western blot analysis was performed as described previously33. Primary antibodies used were as follows: SODD antibody (1:1000, Santa Cruz biotechnologies), caspase-3 antibody (1:2000, Upstate Biotechnology, Waltham, MA) and GAPDH antibody (1:2000, Santa Cruz biotechnologies). The experiments were performed in quadruplets.

Materials

Carnosol and N-acetyl cysteine (NAC) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Apocynin was purchased from Millipore Sigma (Burlington, MA).

Statistical analysis

All statistical analyses were performed by using GraphPad Prism software version 6 (GraphPad Software, Inc., Boston, MA). Data were expressed as mean ± S.E. Statistical differences between two groups were determined by Student’s t test. Differences among multiple groups were tested using analysis of variance (ANOVA) and checked for significance using Fisher’s protected least significant difference test.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-023-37796-5.

Acknowledgements

The work was supported in part by Chongqing Scientific Research Institute Performance Incentive Guidance Special Project [cstc2020jxjl130017] awarded to Dr. Aihua Li.

Author contributions

A.L. performed the experiments, analyzed data and wrote the manuscript draft. W.C. designed the experiments, performed some experiments, analyzed data and wrote the final version of the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information file.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Blot WJ Devesa SS Kneller RW Fraumeni JF Jr Rising incidence of adenocarcinoma of the esophagus and gastric cardia JAMA 1991 265 1287 1289 10.1001/jama.1991.03460100089030 1995976
2. Pennathur A Gibson MK Jobe BA Luketich JD Oesophageal carcinoma Lancet 2013 381 400 412 10.1016/S0140-6736(12)60643-6 23374478
3. Thrift AP Whiteman DC The incidence of esophageal adenocarcinoma continues to rise: analysis of period and birth cohort effects on recent trends Ann. Oncol. 2012 23 3155 3162 10.1093/annonc/mds181 22847812
4. Lagergren J Bergstrom R Lindgren A Nyren O Symptomatic gastroesophageal reflux as a risk factor for esophageal adenocarcinoma N. Engl. J. Med. 1999 340 825 831 10.1056/NEJM199903183401101 10080844
5. Wild CP Hardie LJ Reflux, Barrett's oesophagus and adenocarcinoma: Burning questions Nat. Rev. Cancer 2003 3 676 684 10.1038/nrc1166 12951586
6. Souza RF Morales CP Spechler SJ Review article: a conceptual approach to understanding the molecular mechanisms of cancer development in Barrett's oesophagus Aliment. Pharmacol. Ther. 2001 15 1087 1100 10.1046/j.1365-2036.2001.01046.x 11472311
7. Falk GW Barrett's esophagus Gastroenterology 2002 122 1569 1591 10.1053/gast.2002.33427 12016424
8. Crane SJ Survival trends in patients with gastric and esophageal adenocarcinomas: a population-based study Mayo Clin. Proc. 2008 83 1087 1094 10.4065/83.10.1087 18828967
9. Sihvo EI Luostarinen ME Salo JA Fate of patients with adenocarcinoma of the esophagus and the esophagogastric junction: a population-based analysis Am. J. Gastroenterol. 2004 99 419 424 10.1111/j.1572-0241.2004.04094.x 15056079
10. Hur C Trends in esophageal adenocarcinoma incidence and mortality Cancer 2013 119 1149 1158 10.1002/cncr.27834 23303625
11. Park KW Carnosol induces apoptosis through generation of ROS and inactivation of STAT3 signaling in human colon cancer HCT116 cells Int. J. Oncol. 2014 44 1309 1315 10.3892/ijo.2014.2281 24481553
12. Alsamri H Carnosol, a natural polyphenol, inhibits migration, metastasis, and tumor growth of breast cancer via a ROS-dependent proteasome degradation of STAT3 Front. Oncol. 2019 9 743 10.3389/fonc.2019.00743 31456939
13. Wang L Carnosol suppresses patient-derived gastric tumor growth by targeting RSK2 Oncotarget 2018 9 34200 34212 10.18632/oncotarget.24409 30344937
14. Johnson JJ Carnosol: A promising anti-cancer and anti-inflammatory agent Cancer Lett. 2011 305 1 7 10.1016/j.canlet.2011.02.005 21382660
15. Alsamri H Carnosol is a novel inhibitor of p300 acetyltransferase in breast cancer Front. Oncol. 2021 11 664403 10.3389/fonc.2021.664403 34055630
16. Alsamri H Carnosol induces p38-mediated ER stress response and autophagy in human breast cancer cells Front. Oncol. 2022 12 911615 10.3389/fonc.2022.911615 35712465
17. Hong J Role of Rac1 in regulation of NOX5-S function in Barrett's esophageal adenocarcinoma cells Am. J. Physiol. Cell Physiol. 2011 301 413 420 10.1152/ajpcell.00027.2011
18. Li D Hong J Cao W Silencer-of-death domain mediates acid-induced decrease in cell apoptosis in Barrett's associated esophageal adenocarcinoma cells J. Pharmacol. Exp. Ther. 2017 360 14 22 10.1124/jpet.116.236620 27756772
19. Redza-Dutordoir M Averill-Bates DA Activation of apoptosis signalling pathways by reactive oxygen species Biochim. Biophys. Acta 1863 2977–2992 2016 10.1016/j.bbamcr.2016.09.012
20. Lafeber FP Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis Rheumatology (Oxford) 1999 38 1088 1093 10.1093/rheumatology/38.11.1088 10556260
21. Zhang Y Apocynin but not allopurinol prevents and reverses adrenocorticotropic hormone-induced hypertension in the rat Am. J. Hypertens. 2005 18 910 916 10.1016/j.amjhyper.2005.02.017 16053986
22. Stolk J Hiltermann TJ Dijkman JH Verhoeven AJ Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol Am. J. Respir. Cell Mol. Biol. 1994 11 95 102 10.1165/ajrcmb.11.1.8018341 8018341
23. Stefanska J Pawliczak R Apocynin: Molecular aptitudes Mediators Inflamm 2008 2008 106507 10.1155/2008/106507 19096513
24. Takayama S Xie Z Reed JC An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators J. Biol. Chem. 1999 274 781 786 10.1074/jbc.274.2.781 9873016
25. Ozawa F Friess H Zimmermann A Kleeff J Buchler MW Enhanced expression of Silencer of death domains (SODD/BAG-4) in pancreatic cancer Biochem. Biophys. Res. Commun. 2000 271 409 413 10.1006/bbrc.2000.2610 10799310
26. Jiang Y Woronicz JD Liu W Goeddel DV Prevention of constitutive TNF receptor 1 signaling by silencer of death domains Science 1999 283 543 546 10.1126/science.283.5401.543 9915703
27. Doong H Vrailas A Kohn EC What's in the 'BAG'?—A functional domain analysis of the BAG-family proteins Cancer Lett. 2002 188 25 32 10.1016/S0304-3835(02)00456-1 12406544
28. Chen J Li Q Life and death of transcriptional co-activator p300 Epigenetics 2011 6 957 961 10.4161/epi.6.8.16065 21730760
29. Fermento ME Inhibition of p300 suppresses growth of breast cancer. Role of p300 subcellular localization Exp. Mol. Pathol. 2014 97 411 424 10.1016/j.yexmp.2014.09.019 25240203
30. Xiao XS High expression of p300 in human breast cancer correlates with tumor recurrence and predicts adverse prognosis Chin. J. Cancer Res. 2011 23 201 207 10.1007/s11670-011-0201-5 23467396
31. Hughes SJ Fas/APO-1 (CD95) is not translocated to the cell membrane in esophageal adenocarcinoma Cancer Res. 1997 57 5571 5578 9407969
32. Si J NADPH oxidase NOX5-S mediates acid-induced cyclooxygenase-2 expression via activation of NF-kappaB in Barrett's esophageal adenocarcinoma cells J. Biol. Chem. 2007 282 16244 16255 10.1074/jbc.M700297200 17403674
33. Cao W MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle Am. J. Physiol. Gastrointest. Liver Physiol. 2003 285 G86 95 10.1152/ajpgi.00156.2002 12799309