Ziming Dong

Raf and MEK Protein Kinases Are Direct Molecular Targets for the Chemopreventive Effect of Quercetin, a Major Flavonol in Red Wine

Considerable attention has focused on the health-promoting effects of red wine and its nonflavonoid polyphenol compound resveratrol. However, the underlying molecular mechanisms and molecular target(s) of red wine or other potentially active ingredients in red wine remain unknown. Here, we report that red wine extract (RWE) or the red wine flavonoid quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells. The activation of activator protein-1 and nuclear factor-kappaB induced by TPA was dose dependently inhibited by RWE or quercetin treatment. Western blot and kinase assay data revealed that RWE or quercetin inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 and Raf1 kinase activities and subsequently attenuated TPA-induced phosphorylation of ERK/p90 ribosomal S6 kinase. Although either RWE or quercetin suppressed Raf1 kinase activity, they were more effective in inhibiting MEK1 activity. Importantly, quercetin exerted stronger inhibitory effects than PD098059, a well-known pharmacologic inhibitor of MEK. Resveratrol did not affect either MEK1 or Raf1 kinase activity. Pull-down assays revealed that RWE or quercetin (but not resveratrol) bound with either MEK1 or Raf1. RWE or quercetin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are involved in the activation of MEK/ERK signaling. Docking data suggested that quercetin, but not resveratrol, formed a hydrogen bond with the backbone amide group of Ser(212), which is the key interaction for stabilizing the inactive conformation of the activation loop of MEK1.

Involvement of the Acid Sphingomyelinase Pathway in UVA-induced Apoptosis

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10–40 μm), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20–80 μm), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.

Regulation of PUMA- α by p53 in cisplatin-induced renal cell apoptosis

Nephrotoxicity is a major side effect of cisplatin, a widely used cancer therapy drug. Depending on its concentration, cisplatin induces necrosis or apoptosis of tubular cells in the kidneys, whereas the underlying injury mechanism is unclear. Our recent work has suggested a critical role for p53 in cisplatin-induced tubular cell apoptosis; nevertheless, the apoptotic events triggered by p53 remain elusive. The current study has examined Bcl-2 family proteins, critical regulators of apoptosis that may be subjected to p53 regulation. Following cisplatin treatment, the expression of Bcl-xL, an antiapoptotic molecule, was suppressed, while the expression of Bak, a proapoptotic molecule, increased slightly. Of interest, PUMA-α, a newly identified p53-responsive proapoptotic Bcl-2 family protein, was drastically induced by cisplatin. PUMA-α induction preceded or paralleled the development of apoptosis. Induced PUMA-α was localized in mitochondria and appeared to antagonize Bcl-xL via molecular interaction. PUMA-α induction during cisplatin treatment was attenuated by pifithrin-α, a pharmacological inhibitor of p53, which was accompanied by the amelioration of Bax activation, cytochrome c release and apoptosis. Moreover, PUMA-α induction was suppressed by dominant-negative p53. Importantly, cisplatin-induced apoptosis was ameliorated in PUMA-α knockout cells. In vivo, cisplatin induced PUMA-α in the kidneys, and the inductive response was abrogated in p53-deficient animals. Together, this study has demonstrated the first compelling evidence for the involvement of PUMA-α in p53-mediated renal cell apoptosis during cisplatin nephrotoxicity.

Inhibition of ultraviolet B-induced activator protein-1 (AP-1) activity by aspirin in AP-1-luciferase transgenic mice

Aspirin is under consideration as a promising chemopreventative agent for human cancers. To study the usefulness of aspirin as a chemopreventative agent for UV-induced human skin cancer, we investigated the effect of aspirin on UVB-induced activator protein-1 (AP-1) activity. In the JB6 cell culture system, aspirin or sodium salicylate (SA) inhibited UVB-induced AP-1 activity in a dose-dependent manner; this inhibitory effect occurred only in cells pretreated with aspirin or SA before UVB irradiation but not cells treated with aspirin or SA after UVB irradiation. Furthermore, these inhibitory effects on UVB-induced AP-1 activity appeared to be mediated through blocking of activation of MAP kinase family members, including extracellular signal-regulated protein kinases, c-Jun N-terminal kinases, and p38. It was not due to absorption of UVB light by aspirin. In the skin of AP-1-luciferase transgenic mice, UVB irradiation induced a rapid increase in AP-1 activity, which reached the peak at 48 h post-UVB irradiation. The topical pretreatment of mouse skin with aspirin markedly blocked the UVB-induced AP-1 transactivation in vivo. These data provide the first evidence that aspirin and SA are inhibitors of UV-induced signal transduction and thus could be used as a chemopreventative agent for skin cancer.

Overactivated Neddylation Pathway as a Therapeutic Target in Lung Cancer

BACKGROUND A number of oncoproteins and tumor suppressors are known to be neddylated, but whether the neddylation pathway is entirely activated in human cancer remains unexplored. METHODS NEDD8-activating enzyme (NAE) (E1) and NEDD8-conjugating enzyme (E2) expression and global-protein neddylation were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction analysis. Cell proliferation, clonogenic survival, migration, and motility in vitro, as well as tumor formation and metastasis in vivo, were determined upon neddylation inhibition by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Survival was analyzed with Kaplan-Meier methods and compared by the log-rank test. All statistical tests were two-sided. RESULTS The entire neddylation pathway, including NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and global-protein neddylation, is overactivated in both lung adenocarcinoma and squamous-cell carcinoma. Compared with lung adenocarcinoma patients with low expression, those with high expression had worse overall survival (NEDD8-activating enzyme E1 subunit 1 [NAE1]: hazard ratio [HR] = 2.07, 95% confidence interval [CI] = 0.95 to 4.52, P = .07; ubiquitin-conjugating enzyme E2M (UBC12): HR = 13.26, 95% CI = 1.77 to 99.35, P = .01; global protein neddylation: HR = 3.74, 95% CI = 1.65 to 8.47, P = .002). Moreover, inhibition of neddylation by the NAE inhibitor MLN4924 statistically significantly suppressed proliferation, survival, migration, and motility of lung cancer cells in vitro and tumor formation and metastasis in vivo. At the molecular level, MLN4924 inactivated Cullin-RING E3 ligases, led to accumulation of tumor-suppressive Cullin-RING E3 ligase substrates and induced phorbol-12-myristate-13-acetate-induced protein 1 (NOXA)-dependent apoptosis or cellular senescence. CONCLUSIONS Our study highlights the overactivated neddylation pathway in lung cancer development and as a promising therapeutic target.

Phosphorylation of Caspase-7 by p21-activated Protein Kinase (PAK) 2 Inhibits Chemotherapeutic Drug-induced Apoptosis of Breast Cancer Cell Lines

p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.

As an independent prognostic factor, FAT10 promotes hepatitis B virus-related hepatocellular carcinoma progression via Akt/GSK3β pathway

FAT10 is an oncogene that is localized at 6q21.3, a region frequently amplified in hepatocellular carcinoma (HCC). Recently, growing attention has been paid to its effect in the initiation of various cancers. However, there has been little research into the influence of FAT10 on the progression and prognosis of HCC, especially in hepatitis B virus (HBV)-related HCC. Here, we aimed at investigating clincopathological significance of FAT10 in HBV-related HCC and its underlying mechanisms. Based on the analysis of FAT10 expression in a reliable and large number of cases with 5-year follow-up, we showed that FAT10 was significantly increased in 260 samples from HBV-related HCC patients, compared with 30 normal tissue, 50 cirrhosis and matched adjacent nontumor tissues. FAT10 expression is correlated with recurrence and poor prognosis in HBV-related HCC. In addition, ectopic expression of FAT10 enhanced cell proliferation, inhibited apoptosis and induced cell cycle progression, whereas silencing FAT10 expression suppressed cell proliferation and induced apoptosis. FAT10 also induced the epithelial–mesenchymal transition (EMT) and promoted invasion of HCC cells. Furthermore, we found Akt/GSK3β pathway contributed to the effects of FAT10 in HCC cells. Blocking the Akt pathway significantly inhibited the actions of FAT10. Taken together, the ubiquitin-like protein FAT10 has a central role in regulating diverse aspects of the pathogenesis of HCC, indicating that it might be a potential therapeutic target.

Abstract 2808: Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450

Esophageal squamous carcinoma (ESCC) may be developed through a progressive sequence from mild to severe dysplasia, carcinoma in situ, and finally, invasive carcinoma. Chemoprevention can block or weaken the influence of development. Recent study has shown luteolin, a bioflavonoid, possesses anti-inflammatory, antioxidant, and anti-proliferative effects, and it might have a preventive effect in this progress. In this study, we focused on the effect of luteolin on cell cycle regulation in human Esophageal Squamous Carcinoma Cell Line EC1 and KYSE450 Cells in vitro and its potential mechanisms. Observations by flow cytometer showed that luteolin inhibited cell cycle progression at G2/M phase in a dose- and time-dependent manner. We also found that luteolin could induce cell apoptosis via decreasing activation of caspase-3 and down-regulation of mitochondrial membrane potential. Western blot results showed the protein expression of cycle related protein CyclinD1 and apoptosis related proteins caspase-3, caspase-9, and Bak were also significantly decreased in luteolin treated cells compared with the non-luteolin treated cells. Our results suggest that the proper use of luteolin might be a practical approach to the prevention of esophageal carcinoma via the inhibition of cell proliferation and other potential high risk regions for this disease. Citation Format: Ping Chen, Tao Hu, Yane Ma, Xiaoyu Chen, Liping Dai, Ningjing Lei, Ziming Dong, Pei Li. Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2808. doi:10.1158/1538-7445.AM2015-2808

Eriodictyol inhibits RSK2-ATF1 signaling and suppresses EGF-induced neoplastic cell transformation

RSK2 is a widely expressed serine/threonine kinase, and its activation enhances cell proliferation. Here, we report that ATF1 is a novel substrate of RSK2 and that RSK2-ATF1 signaling plays an important role in EGF-induced neoplastic cell transformation. RSK2 phosphorylated ATF1 at Ser-63 and enhanced ATF1 transcriptional activity. Docking experiments using the crystal structure of the RSK2 N-terminal kinase domain combined with in vitro pulldown assays demonstrated that eriodictyol, a flavanone found in fruits, bound with the N-terminal kinase domain of RSK2 to inhibit RSK2 N-terminal kinase activity. In cells, eriodictyol inhibited phosphorylation of ATF1 but had no effect on the phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically suppresses RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation and tumor promoter-induced transformation of JB6 Cl41 cells. Eriodictyol or knockdown of RSK2 or ATF1 also suppressed Ras-mediated focus formation. Overall, these results indicate that RSK2-ATF1 signaling plays an important role in neoplastic cell transformation and that eriodictyol is a novel natural compound for suppressing RSK2 kinase activity.

Sunlight UV-Induced Skin Cancer Relies upon Activation of the p38α Signaling Pathway

The activation of cellular signal transduction pathways by solar ultraviolet (SUV) irradiation plays a vital role in skin tumorigenesis. Although many pathways have been studied using pure ultraviolet A (UVA) or ultraviolet B (UVB) irradiation, the signaling pathways induced by SUV (i.e., sunlight) are not understood well enough to permit improvements for prevention, prognosis, and treatment. Here, we report parallel protein kinase array studies aimed at determining the dominant signaling pathway involved in SUV irradiation. Our results indicated that the p38-related signal transduction pathway was dramatically affected by SUV irradiation. SUV (60 kJ UVA/m(2)/3.6 kJ UVB/m(2)) irradiation stimulates phosphorylation of p38α (MAPK14) by 5.78-fold, MSK2 (RPS6KA4) by 6.38-fold, and HSP27 (HSPB1) by 34.56-fold compared with untreated controls. By investigating the tumorigenic role of SUV-induced signal transduction in wild-type and p38 dominant-negative (p38 DN) mice, we found that p38 blockade yielded fewer and smaller tumors. These results establish that p38 signaling is critical for SUV-induced skin carcinogenesis.