Capn4 is a marker of poor clinical outcomes and promotes nasopharyngeal carcinoma metastasis via nuclear factor‐κB‐induced matrix metalloproteinase 2 expression

Calpain small subunit 1 (Capn4) plays a key role in tumor migration or invasion. In this study, expression and function of Capn4 was investigated in human nasopharyngeal carcinoma (NPC). Here we report that both mRNA and protein levels of Capn4 were elevated in NPC tissues when compared to normal NP tissues. Similarly, Capn4 was also highly expressed in multiple NPC cell lines, compared to immortalized human nasopharyngeal epithelial cell line NP69. Moreover, expression of Capn4 was significantly correlated with Epstein‐Barr virus infection, advanced stages, and lymph node or distant metastasis (P < 0.001). The patients with NPC displaying higher Capn4 had a significantly shorter overall survival (P = 0.002) and progression‐free survival (P = 0.003). Furthermore, siRNA knockdown of Capn4 suppressed cell migration and invasion in vitro and in vivo. These events resulted from Capn4 downregulation were associated with reduced expression of matrix metalloproteinase 2 (MMP2), Snail, and Vimentin. Finally, we demonstrated that Capn4 upregulated MMP2 via nuclear factor‐κB (NF‐κB) activation, manifested by increased phosphorylation of p65, a subunit of NF‐κB. Together, these findings argue a novel function of Capn4 in invasion and metastasis of NPC, and thereby suggest that Capn4 may represent an independent prognostic factor and a potential therapeutic target in NPC.     

Association between activation of atypical NF‐κB1 p105 signaling pathway and nuclear β‐catenin accumulation in colorectal carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1. © 2009 Wiley‐Liss, Inc.     

Significance of NF‐κB activation in immortalization of nasopharyngeal epithelial cells

NF‐κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF‐κB is common in NPC and may contribute to NPC development. The role of NF‐κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF‐κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF‐κB signaling is a common phenotype in telomerase‐immortalized NPE cell lines. Our results suggest that NF‐κB activation promotes the growth of telomerase‐immortalized NPE cells, and suppression of NF‐κB activity inhibits their proliferation. Furthermore, we observed upregulation of c‐Myc, IL‐6 and Bmi‐1 in our immortalized NPE cells. Inhibition of NF‐κB downregulated expression of c‐Myc, IL‐6 and Bmi‐1, suggesting that they are downstream events of NF‐κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF‐κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF‐κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.     

Clonidine,an alpha‐2 adrenoceptor agonist relieves mechanical allodynia in oxaliplatin‐induced neuropathic mice; potentiation by spinal p38 MAPK inhibition without motor dysfunction and hypotension

Cancer chemotherapy with platinum‐based antineoplastic agents including oxaliplatin frequently results in a debilitating and painful peripheral neuropathy. We evaluated the antinociceptive effects of the alpha‐2 adrenoceptor agonist, clonidine on oxaliplatin‐induced neuropathic pain. Specifically, we determined if (i) the intraperitoneal (i.p.) injection of clonidine reduces mechanical allodynia in mice with an oxaliplatin‐induced neuropathy and (ii) concurrent inhibition of p38 mitogen‐activated protein kinase (MAPK) activity by the p38 MAPK inhibitor SB203580 enhances clonidine's antiallodynic effect. Clonidine (0.01–0.1 mg kg^?1, i.p.), with or without SB203580(1‐10 nmol, intrathecal) was administered two weeks after oxaliplatin injection(10 mg kg^?1, i.p.) to mice. Mechanical withdrawal threshold, motor coordination and blood pressure were measured. Postmortem expression of p38 MAPK and ERK as well as their phosphorylated forms(p‐p38 and p‐ERK) were quantified 30 min or 4 hr after drug injection in the spinal cord dorsal horn of treated and control mice. Clonidine dose‐dependently reduced oxaliplatin‐induced mechanical allodynia and spinal p‐p38 MAPK expression, but not p‐ERK. At 0.1 mg  kg^?1, clonidine also impaired motor coordination and decreased blood pressure. A 10 nmol dose of SB203580 alone significantly reduced mechanical allodynia and p‐p38 MAPK expression, while a subeffective dose(3 nmol) potentiated the antiallodynic effect of 0.03 mg kg^?1 clonidine and reduced the increased p‐p38 MAPK. Coadministration of SB203580 and 0.03 mg kg^?1 clonidine decreased allodynia similar to that of 0.10 mg kg^?1 clonidine, but without significant motor or vascular effects. These findings demonstrate that clonidine treatment reduces oxaliplatin‐induced mechanical allodynia. The concurrent administration of SB203580 reduces the dosage requirements for clonidine, thereby alleviating allodynia without producing undesirable motor or cardiovascular effects.     

Platelet‐type 12‐lipoxygenase induces MMP9 expression and cellular invasion via activation of PI3K/Akt/NF‐κB

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of death in males in the United States. Using human prostate cancer specimens, the authors have previously shown that elevated expression levels of 12‐lipoxygenase (12‐LOX) occurred more frequently in advanced stage, high‐grade prostate cancer, suggesting that 12‐LOX expression is associated with carcinoma progression and invasion. Previous reports from their group and others have shown that 12‐LOX is a positive modulator of invasion and metastasis; however, the mechanism remains unclear. In this work, a new link between 12‐LOX and the matrix metalloproteinase 9 (MMP9) in prostate cancer angiogenesis is reported. This study demonstrated that overexpression of 12‐LOX in prostate cancer PC‐3 cells resulted in elevated expression of MMP9 mRNA, protein and secretion. Exogenous addition of 12(S)‐hydroxy eicosatetraenoic acid, the sole and stable end product of arachidonic acid metabolism by 12‐LOX, is able to increase MMP9 expression in wild‐type PC‐3 cells. Furthermore, using pharmacological and genetic inhibition approaches, it was found that 12‐LOX activates phosphoinositol 3 kinase (PI3K)/Akt, which results in nuclear factor‐kappa B (NF‐κB)‐driven MMP9 expression, ensuing in enhanced chemoattraction of endothelial cells. Specific inhibitors of 12‐LOX, PI3K or NF‐κB inhibited MMP9 expression in 12‐LOX‐expressing PC‐3 cells and resulted in the blockade of the migratory ability of endothelial cells. In summary, the authors have identified a new pathway by which overexpression of 12‐LOX in prostate cancer cells leads to augmented production of MMP9 via activation of PI3K/Akt/NF‐κB signaling. The role of 12‐LOX‐mediated MMP9 secretion in endothelial cell migration may account for the proangiogenic function of 12‐LOX in prostate cancer.     

Pak1 and Pak2 are activated in recurrent respiratory papillomas,contributing to one pathway of Rac1‐mediated COX‐2 expression

Recurrent respiratory papillomas are premalignant tumors of the airway caused by human papillomaviruses (HPVs), primarily Types 6 and 11. We had reported that respiratory papillomas overexpress the epidermal growth factor receptor (EGFR), the small GTPase Rac1 and cyclooxygenase‐2 (COX‐2), and have enhanced nuclear factor‐κB (NFκB) activation with decreased levels of IκB‐β but not IκB‐α. We also showed that EGFR‐activated Rac1 mediates expression of COX‐2 through activation of p38 mitogen‐activated protein kinase. We have now asked whether the p21‐activated kinases Pak1 or Pak2 mediate activation of p38 by Rac1 in papilloma cells. Pak1 and Pak2 were constitutively activated in vivo in papilloma tissue compared with normal epithelium, and Rac1 siRNA reduced the level of both phospho‐Pak1 and phospho‐Pak2 in cultured papilloma cells. Reduction in Pak1 and Pak2 with siRNA decreased the COX‐2 expression in papilloma cells, increased the levels of IκB‐β and reduced the nuclear localization of NF‐κB, but had no effect on p38 phosphorylation. Our studies suggest that Rac1 → Pak1/Pak2 → NFκB is a separate pathway that contributes to the expression of COX‐2 in HPV‐induced papillomas, independently of the previously described Rac1 → p38 → COX‐2 pathway.     

Polycyclic aromatic hydrocarbons induce migration in human hepatocellular carcinoma cells (HepG2) through reactive oxygen species-mediated p38 MAPK signal transduction

Although polycyclic aromatic hydrocarbons (PAHs) are carcinogenic and have been extensively studied with regard to tumor formation, few studies have investigated the involvement of these environmental chemicals in tumor migration and invasion. Polycyclic aromatic hydrocarbons induce reactive oxygen species (ROS) and activate MAPK signal transduction. The p38 signaling transduction pathway, one of the most typical MAPK pathways, plays an essential role in regulating cell migration. Therefore, we investigated whether three PAHs, benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F), and indeno[1,2,3-c,d]pyrene (IND), induce migration in human hepatocellular carcinoma cell line HepG2 through ROS-mediated p38 MAPK signal transduction. Reactive oxygen species generation and p38 MAPK activity both increased in a dose-dependent manner and were prevented by SB203580, an inhibitor of p38 MAPK, and N-acetylcysteine (NAC), a ROS scavenger. Expression of migration-related genes was also increased by B[a]A, B[k]F, and IND in a dose-dependent manner and was inhibited by SB203580 and NAC. The migration of HepG2 cells, observed using the Transwell migration assay, also increased in a dose-dependent manner and was prevented by SB203580 and NAC. Our results indicate that the ROS-mediated p38 MAPK signaling pathway plays an essential role in the PAH-induced migration of HepG2 cells.     

p38MAPK抑制剂SB203580抑制人绒癌JAR细胞的体外侵袭作用

目的：研究p38MAPK通路在人绒癌JAR细胞体外侵袭中的作用。方法：用细胞ELISA法测定JAR细胞中p38MAPK的活性变化，用Transwell细胞侵入系统检测细胞的侵袭作用。结果：PMA呈浓度依赖性地激活JAR细胞中p38MAPK,PMA能促进人绒癌JAR细胞的体外侵袭作用，而p38特异性抑制剂SB203580抑制了JAR细胞的侵袭能力。结论：p38MAPK通路在人滋养细胞的侵袭行为以及人绒癌的形成中具有重要作用，P38抑制剂可能会为人绒癌的防治提供新的途径。     

p38 mitogen-activated protein kinase pathway is involved in protein kinase Calpha-regulated invasion in human hepatocellular carcinoma cells

Protein kinase Calpha (PKCalpha) has been suggested to play an important role in tumorigenesis, invasion, and metastasis. In this study, we investigated the signal pathways selectively activated by PKCalpha in human hepatocellular carcinoma (HCC) cells to determine the role of mitogen-activated protein kinases (MAPK) in PKCalpha-mediated HCC migration and invasion. A stable SK-Hep-1 cell clone (siPKCalpha-SK) expressing DNA-based small interfering RNA (siRNA) PKCalpha was established and was then characterized by cell growth, migration, and invasion. The expression of PKCalpha was decreased in siPKCalpha-SK, and cell growth, migration, and invasion were reduced. These changes were associated with the decrease in p38 MAPK phosphorylation level, but not in c-jun-NH(2)-kinase-1/2 (JNK-1/2) and extracellular signal-regulated kinase-1/2 (ERK-1/2). This phenomenon was confirmed in the SK-Hep-1 cells treated with antisense PKCalpha olignucleotide. The p38 MAPK inhibitor SB203580 or dominant negative p38 mutant plasmid (DN-p38) was used to evaluate the dependency of p38 MAPK in PKCalpha-regulated migration and invasion. Attenuation of cell migration and invasion was revealed in the SK-Hep-1 cells treated with the SB203580 or DN-p38, but not with ERK-1/2 inhibitor PD98059 or JNK-1/2 inhibitor SP600125. Overexpression of constitutively active MKK6 or PKCalpha may restore the inactivation of p38 and the attenuation of cell migration and invasion in siPKCalpha-SK. Similar findings were observed in the stable HA22T/VGH cell clone expressing siRNA PKCalpha. This study provides new insight into the role of p38 MAPK in PKCalpha-mediated malignant phenotypes, especially in PKCalpha-mediated cancer cell invasion, which may have valuable implications for developing new therapies for some PKCalpha-overexpressing cancers.     

Epstein‐Barr virus‐encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan‐2 and synaptotagmin‐like‐4 in nasopharyngeal carcinoma cells

Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell‐to‐cell communication. The Epstein‐Barr virus (EBV)‐encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan‐2 (SDC2) and synaptotagmin‐like‐4 (SYTL4) through nuclear factor (NF)‐κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF‐κB signaling to promote EV secretion, and further enhance cancer progression of NPC.     

Glutathione S‐transferase M1 inhibits dexamethasone‐induced apoptosis in association with the suppression of Bim through dual mechanisms in a lymphoblastic leukemia cell line

(Cancer Sci 2010; 101: 767–773) Glutathione S‐transferase μ (GSTM1) is mainly known as a detoxification enzyme but it has also been shown to be a negative regulator of apoptosis‐related signaling cascades. Recently GSTM1 has been reported to be a significant risk factor for hematological relapse in childhood acute lymphoblastic leukemia, although the underlying mechanism remains largely unknown. Glucocorticoids play a crucial role in the treatment of childhood acute lymphoblastic leukemia, therefore we hypothesized that GSTM1 plays important roles in glucocorticoid‐induced apoptotic pathways. To clarify the relationship between GSTM1 and drug resistance, GSTM1 was transfected into a T‐acute lymphoblastic leukemia cell line, CCRF‐CEM (CEM), and we established the GSTM1‐expressing cell lines CEM/M1‐4 and CEM/M1‐9. Transduction of GSTM1 into CEM selectively decreased cellular sensitivity to dexamethasone in a manner that was independent of glutathione conjugation, but was due to apoptosis inhibition. Dexamethasone‐induced p38‐MAPK and Bim activation were concomitantly suppressed. Interestingly, nuclear factor kappa b (NF‐κB) p50 activity was upregulated in GSTM1‐expressing CEM. Inhibition of NF‐κB by the pharmacological agent BAY11‐7082 greatly enhanced the sensitivity of the GSTM1‐expressing CEM to dexamethasone and was accompanied by an increase in Bim expression. Thus, we propose that GSTM1, a novel regulator of dexamethasone‐induced apoptosis, causes dexamethasone resistance by suppression of Bim through dual mechanisms of both downregulation of p38‐MAPK and upregulation of NF‐κB p50.     

TM‐233, a novel analog of 1′‐acetoxychavicol acetate,induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities

Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well‐tolerated agents for myeloma, we have previously reported that 1′‐acetoxychavicol acetate (ACA), a natural condiment from South‐East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF‐κB‐related functions. Searching for more potent NF‐κB inhibitors, we developed several ACA analogs based on quantitative structure–activity relationship analysis. TM‐233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC₅₀ than ACA. Treatment with TM‐233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti‐apoptotic Mcl‐1 protein, but not Bcl‐2 and Bcl‐xL proteins. In addition, TM‐233 rapidly decreased the nuclear expression of NF‐κB and also decreased the accumulation of cytosolic NF‐κB. We also examined the effects of TM‐233 on bortezomib‐resistant myeloma cells that we recently established, KMS‐11/BTZ and OPM‐2/BTZ. TM‐233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS‐11/BTZ and OPM‐2/BTZ cells. Interestingly, the combination of TM‐233 and bortezomib significantly induced cell death in these bortezomib‐resistant myeloma cells through inhibition of NF‐κB activity. These results indicate that TM‐233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM‐233 might be a more potent NF‐κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells.     

Silibinin inhibits the invasion and migration of renal carcinoma 786-O cells in vitro, inhibits the growth of xenografts in vivo and enhances chemosensitivity to 5-fluorouracil and paclitaxel

Silibinin is a flavonoid antioxidant that is widely used for its anti‐hepatotoxic properties. It exerts a dose‐dependent inhibition on the invasion and migration of 786‐O renal cell carcinoma (RCC) cells in the absence of cytotoxicity. 786‐O cells were treated with silibinin at various concentrations, up to 50 µM, for a defined period and then subjected to gelatin zymography, casein zymography, and Western blot to investigate the impacts of silibinin on metalloproteinase (MMP) ‐2, ‐9, urokinase plasminogen activator (u‐PA), and MAPK pathway signaling proteins, respectively. The results showed that silibinin decreased MMP‐2, MMP‐9, u‐PA, p‐p38, and p‐Erk1/2 expressions in a concentration‐dependent manner. The reduced expressions of MMP‐2 and u‐PA, as well as inhibition of cell invasion were obtained in the cultures pre‐treated with PD98059 (Erk1/2 inhibitor) and SB203580 (p38 inhibitor). An in vivo anti‐tumor study with a nude mice xenograft model by a subcutaneous inoculation of 786‐O cells demonstrated small solid tumors after eight days following cell inoculation. There was a 70.1% reduction in tumor volume and 69.7% reduction in tumor weight by silibinin feeding on day 44, compared to those of controls. Moreover, combination treatment with silibinin and 5‐fluorouracil, paclitaxel, vinblastine, or RAD‐001 enhanced the chemosensitivity of 5‐fluorouracil and paclitaxel. In conclusion, silibinin inhibits the invasion and migration of 786‐O cells in vitro, inhibits the growth of xenografts in vivo, and enhances chemosensitivity to 5‐fluorouracil and paclitaxel. © 2011 Wiley‐Liss, Inc.     

蛋白酶体抑制剂诱导甲状腺癌细胞凋亡机制的探讨

目的:探讨p38MAPK信号转导通路参与蛋白酶体抑制剂诱导甲状腺癌细胞凋亡的作用机制。方法:选取甲状腺癌细胞系,利用丝裂原活化蛋白激酶(MAPK)的三种抑制剂抑制磷酸化途径,利用微小RNA干扰技术转染细胞,封闭p38MAPK。用蛋白质印迹法检测红系衍生的核因子2相关因子2(Nrf2)及p38MAPK的表达,实时定量PCR检测细胞内GCLC mRNA的表达,测定还原型谷胱甘肽的含量,流式细胞仪法检测甲状腺癌细胞凋亡率。结果:在甲状腺癌细胞系8305C细胞中,与对照组相比,p38MAPK的抑制剂SB203580和sip38MAPK均能够抑制蛋白酶体抑制剂诱导的Nrf2细胞核易位(P<0.01),并使随后的GCLC mRNA表达减少、GSH含量减少(P<0.01),显著提高蛋白酶体抑制剂诱导的甲状腺癌细胞凋亡率(P<0.01),而其他2种抑制剂和随机序列核酸siRNA无上述作用。结论:蛋白酶体抑制剂通过p38MAPK途径激活Nrf2的细胞核易位,进而导致GCLC的诱导作用和谷胱甘肽生成的连锁反应,成为蛋白酶体抑制剂在甲状腺癌细胞中的一种抗凋亡机制。