Wenshu Chen

A Critical Role of Luteolin-Induced Reactive Oxygen Species in Blockage of Tumor Necrosis Factor-Activated Nuclear Factor-κB Pathway and Sensitization of Apoptosis in Lung Cancer Cells

Nuclear factor κB (NF-κB) activated by tumor necrosis factor (TNF) attenuates the TNF-induced apoptosis pathway. Therefore, blockage of NF-κB should improve the anticancer activity of TNF. Luteolin, a naturally occurring polyphenol flavonoid, has been reported to sensitize colorectal cancer cells to TNF-induced apoptosis through suppression of NF-κB; however, the mechanisms of this effect have not been well elucidated. In this article, we provide evidence showing a critical role of reactive oxygen species (ROS) accumulation induced by luteolin in modulating TNF-activated pathways in lung cancer cells. Luteolin effectively suppressed NF-κB, whereas it potentiated the c-Jun N-terminal kinase (JNK) to increase apoptosis induced by TNF in lung cancer cells. Our results further demonstrate that luteolin induced an early phase ROS accumulation via suppression of the cellular superoxide dismutase activity. It is noteworthy that suppression of ROS accumulation by ROS scavengers butylated hydroxyanisole, and N-acetyl-l-cysteine prevented the luteolin-induced suppression of NF-κB and potentiation of JNK and significantly suppressed the synergistic cytotoxicity seen with cotreatment of luteolin and TNF. Taken together, these results suggest that the accumulation of ROS induced by luteolin plays a pivotal role in suppression of NF-κB and potentiation of JNK to sensitize lung cancer cells to undergo TNF-induced apoptosis.

Epidermal growth factor receptor-mediated tissue transglutaminase overexpression couples acquired tumor necrosis factor-related apoptosis-inducing ligand resistance and migration through c-FLIP and MMP-9 proteins in lung cancer cells.

Acquired chemoresistance not only blunts anticancer therapy but may also promote cancer cell migration and metastasis. Our previous studies have revealed that acquired tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in lung cancer cells is associated with Akt-mediated stabilization of cellular caspase 8 and Fas-associated death domain (FADD)-like apoptosis regulator-like inhibitory protein (c-FLIP) and myeloid cell leukemia 1 (Mcl-1). In this report, we show that cells with acquired TRAIL resistance have significantly increased capacities in migration and invasion. By gene expression screening, tissue transglutaminase (TGM2) was identified as one of the genes with the highest expression increase in TRAIL-resistant cells. Suppressing TGM2 dramatically alleviated TRAIL resistance and cell migration, suggesting that TGM2 contributes to these two phenotypes in TRAIL-resistant cells. TGM2-mediated TRAIL resistance is likely through c-FLIP because TGM2 suppression significantly reduced c-FLIP but not Mcl-1 expression. The expression of matrix metalloproteinase 9 (MMP-9) was suppressed when TGM2 was inhibited, suggesting that TGM2 potentiates cell migration through up-regulating MMP-9 expression. We found that EGF receptor (EGFR) was highly active in the TRAIL-resistant cells, and suppression of EGFR dramatically reduced TGM2 expression. We further determined JNK and ERK, but not Akt and NF-κB, are responsible for EGFR-mediated TGM2 expression. These results identify a novel pathway that involves EGFR, MAPK (JNK and ERK), and TGM2 for acquired TRAIL resistance and cell migration in lung cancer cells. Because TGM2 couples TRAIL resistance and cell migration, it could be a molecular target for circumventing acquired chemoresistance and metastasis in lung cancer.

Blocking NF-κB and Akt by Hsp90 inhibition sensitizes Smac mimetic compound 3-induced extrinsic apoptosis pathway and results in synergistic cancer cell death

NF-κB and Akt are two main cell survival pathways that attenuate the anticancer efficacy of therapeutics. Our previous studies demonstrated that the Smac mimetic compound 3 (SMC3) specifically suppresses c-IAP1 and induces TNF-α autocrine to kill cancer cells. However, SMC3 also induces a cell survival signal through NF-κB activation. In this report, we further found that SMC3 potently activates Akt, which inhibits SMC3-induced cancer cell death. Strikingly, concurrent blocking NF-κB and Akt resulted in a significantly potentiated cytotoxicity. Because heat shock protein 90 (Hsp90) plays an important role in maintaining the integrity of both the NF-κB and Akt pathways in cancer cells, we examined if suppression of Hsp90 is able to potentiate SMC3-induced cancer cell death. The results show that targeting Hsp90 does not interfere with SMC3-induced c-IAP1 degradation and TNF-α autocrine, the key processes for SMC3-induced cancer cell apoptosis. However, Hsp90 inhibitors effectively blocked SMC3-induced NF-κB activation through degradation of RIP1 and IKKβ, two key components of the NF-κB activation pathway, and reduced both the constitutive and SMC3-induced Akt activity through degradation of the Akt protein. Consistently, with the co-treatment of SMC3 and Hsp90 inhibitors, apoptosis was markedly sensitized and a synergistic cytotoxicity was observed. The results suggest that concurrent targeting c-IAP1 and Hsp90 by combination of SMC3 and Hsp90 inhibitors is an effective approach for improving the anticancer value of SMC3.

Receptor-interacting Protein 1 Increases Chemoresistance by Maintaining Inhibitor of Apoptosis Protein Levels and Reducing Reactive Oxygen Species through a microRNA-146a-mediated Catalase Pathway

Background: Whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. Results: RIP1 knockdown resulted in miR-146a-mediated catalase reduction, ROS induction, IAP degradation, and increased cisplatin cytotoxicity. Conclusion: RIP1 blunts the anticancer activity of cisplatin by releasing miR-146a-mediated catalase suppression. Significance: Our results establish a chemoresistant role for RIP1, and intervention within the RIP1-mediated pathway may be exploited for chemosensitization. Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.

Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts that promotes bronchial epithelial cell transformation by cigarette-smoke carcinogen

Despite decades of research in defining the health effects of low-dose (<100 mGy) ionizing photon radiation (LDR), the relationship between LDR and human cancer risk remains elusive. Because chemical carcinogens modify the tumor microenvironment, which is critical for cancer development, we investigated the role and mechanism of LDR in modulating the response of stromal cells to chemical carcinogen-induced lung cancer development. Secretion of proinflammatory cytokines such as interleukin-6 (IL-6), CXCL1 and CXCL5 from human lung fibroblasts was induced by cigarette-smoke carcinogen benzo[a]pyrene diol epoxide (BPDE), which was inhibited by a single dose of LDR. The activation of NF-κB, which is important for BPDE-induced IL-6 secretion, was also effectively suppressed by LDR. In addition, conditioned media from BPDE-treated fibroblasts activated STAT3 in the immortalized normal human bronchial epithelial cell line Beas-2B, which was blocked with an IL-6 neutralizing antibody. Conditioned medium from LDR-primed and BPDE-treated fibroblast showed diminished capacity in activating STAT3. Furthermore, IL-6 enhanced BPDE-induced Beas-2B cell transformation in vitro. These results suggest that LDR inhibits cigarette smoke-induced lung carcinogenesis by suppressing secretion of cytokines such as IL-6 from fibroblasts in lung tumor-prone microenvironment.

RIP1 potentiates BPDE-induced transformation in human bronchial epithelial cells through catalase-mediated suppression of excessive reactive oxygen species.

Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer and mouse lung tumor tissues. RIP1 expression was remarkably increased in cigarette smoke-exposed mouse lung. In human bronchial epithelial cells (HBECs), RIP1 was significantly induced by cigarette smoke extract or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo(a)pyrene. In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs), including c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The ROS-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced ROS accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production.

MUC1 Contributes to BPDE-Induced Human Bronchial Epithelial Cell Transformation through Facilitating EGFR Activation

Although it is well known that epidermal growth factor receptor (EGFR) is involved in lung cancer progression, whether EGFR contributes to lung epithelial cell transformation is less clear. Mucin 1 (MUC1 in human and Muc1 in animals), a glycoprotein component of airway mucus, is overexpressed in lung tumors; however, its role and underlying mechanisms in early stage lung carcinogenesis is still elusive. This study provides strong evidence demonstrating that EGFR and MUC1 are involved in bronchial epithelial cell transformation. Knockdown of MUC1 expression significantly reduced transformation of immortalized human bronchial epithelial cells induced by benzo[a]pyrene diol epoxide (BPDE), the active form of the cigarette smoke (CS) carcinogen benzo(a)pyrene (BaP)s. BPDE exposure robustly activated a pathway consisting of EGFR, Akt and ERK, and blocking this pathway significantly increased BPDE-induced cell death and inhibited cell transformation. Suppression of MUC1 expression resulted in EGFR destabilization and inhibition of the BPDE-induced activation of Akt and ERK and increase of cytotoxicity. These results strongly suggest an important role for EGFR in BPDE-induced transformation, and substantiate that MUC1 is involved in lung cancer development, at least partly through mediating carcinogen-induced activation of the EGFR-mediated cell survival pathway that facilitates cell transformation.

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis.

Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.

IKKβ-mediated nuclear factor-κB activation attenuates smac mimetic–induced apoptosis in cancer cells

Smac mimetics (SM) have been recently reported to kill cancer cells through the extrinsic apoptosis pathway mediated by autocrine tumor necrosis factor (TNF). SM also activates nuclear factor-κB (NF-κB). However, how SM induces NF-κB and the role of NF-κB in SM-induced cancer cell death has not been well elucidated. We found that effective blockage of NF-κB had no detectable effect on SM compound 3 (SMC3)–induced TNF secretion, suggesting that the induction of TNF by SMC3 is independent of NF-κB. Conversely, SMC3-induced NF-κB activation was found to be mediated by autocrine TNF because this effect of SMC3 was effectively inhibited when TNF was blocked with either a TNF neutralizing antibody or TNF small interfering RNA. In addition, although SMC3 dramatically reduced c-IAP1 level, it had marginal effect on c-IAP2 expression, TNF-induced RIP modification, NF-κB activation, and downstream antiapoptosis NF-κB target expression. Furthermore, blocking NF-κB by targeting IKKβ or RelA substantially potentiated SMC3-induced cytotoxicity, suggesting that the NF-κB pathway inhibits SMC3-induced apoptosis in cancer cells. Our results show that through TNF autocrine, SM induces an IKKβ-mediated NF-κB activation pathway that protects cancer cells against SM-induced apoptosis, and thus, NF-κB blockage could be an effective approach for improving the anticancer value of SM. [Mol Cancer Ther 2009;8(6):1636–45]

A Superoxide-Mediated Mitogen-Activated Protein Kinase Phosphatase-1 Degradation and c-Jun NH2-Terminal Kinase Activation Pathway for Luteolin-Induced Lung Cancer Cytotoxicity

Although luteolin is identified as a potential cancer therapeutic and preventive agent because of its potent cancer cell-killing activity, the molecular mechanisms by which its cancer cell cytotoxicity is achieved have not been well elucidated. In this report, luteolin-induced cellular signaling was systematically investigated, and a novel pathway for luteolins lung cancer killing was identified. The results show that induction of superoxide is an early and crucial step for luteolin-induced apoptotic and nonapoptotic death in lung cancer cells. The c-Jun N-terminal kinase (JNK) was potently activated after superoxide accumulation. Suppression of superoxide completely blocked luteolin-induced JNK activation, which was well correlated to alleviation of luteolins cytotoxicity. Although luteolin slightly stimulated the JNK-activating kinase mitogen-activated protein kinase kinase 7, the latter was not dependent on superoxide. We further found that luteolin triggers a superoxide-dependent rapid degradation of the JNK-inactivating phosphatase mitogen-activated protein kinase phosphatase-1 (MKP-1). Introduction of a degradation-resistant MKP-1 mutant effectively attenuated luteolin-induced JNK activation and cytotoxicity, suggesting that inhibition of the JNK suppressor MKP-1 plays a major role in luteolin-induced lung cancer cell death. Taken together, our results unveil a novel pathway consisting of superoxide, MKP-1, and JNK for luteolins cytotoxicity in lung cancer cells, and manipulation of this pathway could be a useful approach for applying luteolin for lung cancer prevention and therapy.