Extracellular Tissue Transglutaminase Activates Noncanonical NF-κB Signaling and Promotes Metastasis in Ovarian Cancer

Tissue transglutaminase (TG2) is a multifunctional protein that binds to fibronectin and exerts protein transamidating activity in the presence of Ca²⁺. We previously reported that TG2 is upregulated in ovarian tumors and enhances intraperitoneal (i.p.) metastasis. TG2 is secreted abundantly in ovarian cancer (OC) ascites as an active enzyme, yet its function in the extracellular compartment remains unknown. To study the distinct functions of secreted TG2, we used recombinant His₆-tagged TG2 and catalytically inactive enzyme in vitro and in vivo. By using i.p. and orthotopic ovarian xenografts, we show that extracellular transglutaminase promoted OC peritoneal metastasis. The main pathway activated by extracellular TG2 was noncanonical nuclear factor-kappa B (NF-κB) signaling, and the enzymatic function of the protein was required to induce phosphorylation of IκB kinase α and processing of the precursor protein p100 into the active p52 subunit. A specific target of TG2-activated p52/RelB complex is the hyaluronan receptor, CD44. Noncanonical NF-κB activation by extracellular TG2 induced CD44 up-regulation and epithelial-to-mesenchymal transition, contributing to increased cancer cell invasiveness and OC peritoneal dissemination. Taken together, our data support that noncanonical NF-κB activation is the pathway through which extracellular TG2 promotes OC metastasis.     

Stabilisation of p53 enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation

Background:

Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored.

Methods:

Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit.

Results:

We show that despite similar reovirus replication in p53^+/+ and p53^−/− cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53^−/− or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors.

Conclusion:

Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.     

Id-1 regulates Bcl-2 and Bax expression through p53 and NF-κB in MCF-7 breast cancer cells

Although increasing evidence supports the protective role of inhibitor of differentiation and DNA binding-1 (Id-1) against anticancer drug-induced apoptosis, the underlying molecular mechanisms seem to vary depending on the tumor system. Here, we examined the direct role of Id-1 in MCF-7 breast cancer cells by ectopically overexpressing Id-1 under serum-free condition, where the endogenous Id-1 expression was suppressed. Id-1 expression resulted in increased number of viable cells, reduced Bax expression, enhanced Bcl-2 expression, but no change in Bcl-xL expression. The expression of nuclear factor-κB (NF-κB) was augmented, while those of p53 and IκB were reduced. Such changes in p53 and NF-κB pathways were also functional, as assessed by real-time polymerase chain reactions and reporter assays of their known downstream targets, p21 and Il-6, as well as Bax and Bcl-2 genes. Finally, Id-1 played a protective role against taxol-induced apoptosis in breast cancer cells as assessed by MTT assay and apoptotic cell count upon taxol treatment (0–200 nM). Reduced Bax expression and enhanced Bcl-2 expression by Id-1 were also noted in the presence of taxol. Taken together, we present a molecular mechanism where Id-1 regulates p53 and NF-κB pathways, which in turn regulates Bax and Bcl-2 genes, thus providing a survival advantage under exogenous stress such as serum-free or taxol treatment in MCF-7 breast cancer cells. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer progression and anti-cancer drug resistance. Hwan Kim and Heekyoung Chung equally contributed to this work.     

CCN1 promotes tumorigenicity through Rac1/Akt/NF-κB signaling pathway in pancreatic cancer

Aberrant CCN1 expression has been reported to play an important role in the tumor development. However, the pattern and the role of CCN1 in pancreatic cancer remain largely unknown. Therefore, we further deciphered the role CCN1 played in pancreatic cancer. We first evaluated the CCN1 expression in human pancreatic cancer tissues and pancreatic cancer cells. Then we forced expression and silenced CCN1 expression in pancreatic cancer cell lines MIA PaCa2 and PANC-1 respectively, using lentivirus vectors. We characterized the stable cells in vitro and in vivo using a nude mouse xenograft model. In this study, we found that CCN1 expression was significantly higher in cancer specimens which positively correlated with the expression level of phosphorylated Akt and p65. and poorer outcome. Moreover, our results demonstrated that CCN1 positively regulated pancreatic cell growth in vitro and in vivo and helped cancer cells resist to tumor necrosis factor alpha-induced apoptosis. Furthermore, we disclosed that activation of CCN1/ras-related c3 botulinum toxin substrate 1 (Rac1)/V-akt murine thymoma viral oncogene homolog (Akt)/nuclear factor-kappa B pathway inhibited apoptosis in pancreatic cancer cells. CCN1 is upregulated in pancreatic cancer and promotes the survival of pancreatic cancer cells. Taken together, these results indicate that CCN1 may be a potential target for pancreatic cancer therapy.     

Musca domestica pupae lectin induces apoptosis in HepG2 cells through a NF-κB/p65-mediated caspase pathway

A new lectin (42 kDa) from Musca domestica pupae (MPL) has been known to inhibit proliferation in tumor cells. In this study, flow cytometry analysis showed that MPL induced HepG2 cells apoptosis significantly and the cells were arrested at S phase. MPL inhibited IκB-α degradation and NF-κB/p65 translocation from cytoplasm into nucleus. Simultaneously, the expressions of FLIP, which is a target gene of NF-κB/p65 were down-regulated and the caspase-8 and caspase-3 were then activated to induce apoptosis. Taken together, these results showed that MPL induced a caspase-dependent apoptosis via NF-κB/p65 pathway in HepG2 cells.     

D-Pinitol Promotes Apoptosis in MCF-7 Cells via Induction of p53 and Bax and Inhibition of Bcl-2 and NF-κB

Development of drugs from natural products has been undergoing a gradual evoluation. Many plant derivedcompounds have excellent therapeutic potential against various human ailments. They are important sourcesespecially for anticancer agents. A number of promising new agents are in clinical development based on theirselective molecular targets in the field of oncology. D-pinitol is a naturally occurring compound derived from soywhich has significant pharmacological activitites. Therefore we selected D-pinitol in order to evaluate apoptoticpotential in the MCF-7 cell line. Human breast cancer cells were treated with different concentrations of D-pinitoland cytotoxicity was measured by MTT and LDH assays. The mechanism of apoptosis was studied with referenceto expression of p53, Bcl-2, Bax and NF-kB proteins. The results revealed that D-pinitol significantly inhibitedthe proliferation of MCF-7 cells in a concentration-dependent manner, while upregulating the expression ofp53, Bax and down regulating Bcl-2 and NF-kB. Thus the results obtained in this study clearly vindicated thatD-pinitol induces apotosis in MCF-7 cells through regulation of proteins of pro- and anti-apoptotic cascades.     

HMGB1 promotes the synthesis of pro-IL-1β and pro-IL-18 by activation of p38 MAPK and NF-κB through receptors for advanced glycation end-products in macrophages

The high mobility group box-1 (HMGB1) protein and NALP3 inflammasome have been identified to play important roles in inflammation and cancer pathogenesis, but the relationships between the two and cancer remain unclear. The current study investigated the relationship between HMGB1 and the NALP3 inflammasome in THP-1 macrophages. HMGB1 was found unable to activate the NALP3 inflammasome and failed to induce the release of the IL-1β and IL-18 in THP-1 macrophages. HMGB1 was also found significantly enhanced the activity of ATP to induce IL-1β and IL-18 by the induction of increased expression of pro-IL-1β and pro-IL-18. This process was dependent on activation of RAGE, MAPK p38 and NF-κB signaling pathway. These results demonstrate that HMGB1 promotes the synthesis of pro-IL-1β and pro-IL-18 in THP-1 macrophages by the activation of p38 MAPK and NF-κB through RAGE. HMGB1 likely plays an important role in the first step of the release of the IL-1β and IL-18, preparing for other cytokines to induce excessive release of IL-1β and IL-18 which promote inflammation and cancer progression.     

Heptaphylline Induces Apoptosis in Human Colon Adenocarcinoma Cells through Bid and Akt/NF-κB (p65) Pathways

Heptaphylline derivatives are carbazoles in Clausena harmandiana, a medicinal plant that is utilized forheadache, stomach ache, and other treatments of illness. The present study examined the effects of heptaphyllineand 7-methoxyheptaphylline on apoptosis of human colon adenocarcinoma cells (HT-29 cell line). Quantificationof cell viability was performed using cell proliferation assay (MTT assay) and of protein expression throughimmunoblotting. The results showed that only heptaphylline, but not 7-methoxyheptaphylline, significantlysignificantly activated cleaved of caspase-3 and poly (ADP-ribose) polymerase (PARP-1) which resulted inHT-29 cell death. We found that heptaphylline activated BH3 interacting-domain death agonist (Bid) and Bak,proapoptotic proteins. In contrast, it suppressed X-linked inhibitor-of-apoptosis protein (XIAP), Bcl-xL andsurvivin, inhibitors of apoptosis. In addition, heptaphylline inhibited activation of NF-κB/p65 (rel), a regulatorof apoptotic regulating proteins by suppressing the activation of Akt and IKKα, upstream regulators of p65.The findings suggested that heptaphylline induces apoptosis in human colon adenocarcinoma cells .     

Relationship among human papillomavirus infection, p16(INK4a), p53 and NF-κB activation in penile cancer from northern Thailand

Human papillomavirus (HPV) E6 and E7 oncoproteins are essential factors for HPV oncogenesis. These E6 and E7 gene products play a central role in the induction of malignant transformation by interacting with several cellular regulatory proteins such as p16(INK4a), p53 and nuclear factor κB (NF-κB). In the present study, conducted in northern Thailand, HPV-DNA was detected in penile cancer cases using an in situ hybridization procedure and p16(INK4a), p53 and NF-κB were detected by immunohistochemistry. Using the cell cycle regulatory proteins p16(INK4a) (61.5%) and p53 (71.8%), it was found that of the 51 cases, 39 (76.5%) were HPV-DNA-positive in penile cancer. On the other hand, 25% p16(INK4a) and 75% p53, respectively, were found in HPV-negative cases. Prevalence of HPV infection (76.5%) was shown in penile cancer cases in northern Thailand. No difference was found between HPV-positive and HPV-negative cases with respect to the presence of the cell cycle regulatory protein p53. On the other hand, p16(INK4a) was found to be different between HPV-positive and HPV-negative cases. Inactivation of tumor suppressor genes, such as p16(INK4a) and p53, to genetic instability, cell immortalization, accumulation of mutations and cancer formation, with or without HPV and irrespective of HPV infection, is therefore suggested. Of the 39 HPV-positive cases, 35 (89.7%) were NF-κB-positive in the nucleus, 29 (74.4%) in the cytoplasm and 37 (94.9%) in the nucleus and/or cytoplasm. NF-κB was detected in 4 (33.3%) of the 12 HPV-negative cases. Therefore, we propose that penile cancer cases with HPV infection are more likely to activate NF-κB than those without HPV infection.     

IL-12 Regulates B7-H1 Expression in Ovarian Cancerassociated Macrophages by Effects on NF-κB Signalling

Background and Aim: B7-H1, a co-inhibitory molecule of the B7 family, is found aberrantly expressed inovarian cancer cells and infiltrating macrophage/dendritic-like cells, and plays a critical role in immune evasionby ovarian cancer. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects on ovariancancer. However, whether IL-12 regulates B7-H1 expression in human ovarian cancer associated-macrophageshas not been clarified. Therefore, we investigated the effects of IL-12 on the expression of B7-H1 in ovariancancer-associated macrophages and possible mechanisms. Methods: PMA induced THP-1-derived macrophagesor human monocyte-derived macrophages were treated with recombinant IL-12 (rIL-12) or infected withadenovirus carrying human IL-12 gene (Ad-IL-12-GFP) for 24 h, then cocultured with the SKOV3 ovariancancer cell line for another 24 h. Macrophages were collected for real-time PCR and Western blot to detect theexpression of B7-H1, and activation of the NF-κB signaling pathway. Moreover, supernatants were collected toassay for IL-12, IFN-γ and IL-10 by ELISA. In addition, monocyte-derived macrophages treated with IFN-γ werecocultured with SKOV3 and determined for the expression of B7-H1. Furthermore, the expression of B7-H1 inmonocyte-derived macrophages was also evaluated after blocking NF-κB signaling. Results: The expression ofB7-H1 was significantly upregulated in monocyte-derived macrophages treated with rIL-12 or Ad-IL-12-GFPcompared with the control groups (p<0.05), accompanied by a remarkable upregulation of IFN-γ (p<0.05), amarked downregulation of IL-10 (p<0.05) and activation of NF-κB signaling. However, the upregulation of B7-H1 was inhibited by blocking the NF-κB signaling pathway (p<0.05). Expression of B7-H1 was also increased(p<0.05) in monocyte-derived macrophages treated with IFN-γ and cocultured with SKOV3. By contrast, theexpression of B7-H1 in THP-1-derived macrophages was significantly decreased when treated in the same wayas monocyte-derived macrophages (p<0.05), and IL-10 was also significantly decreased but IFN-γ was almostabsent. Conclusions: IL-12 upregulates the expression of B7-H1 in monocyte-derived macrophages, which ispossible though inducing the secretion of IFN-γ and further activating the NF-κB signal pathway. However,IL-12 downregulates the expression of B7-H1 in THP-1-derived macrophages, associated with a lack of IFN-γand inhibition of expression of IL-10.     

Stephania delavayi Diels. inhibits breast carcinoma proliferation through the p38 MAPK/NF-κB/COX-2 pathway

The nuclear factor κB (NF-κB)/inhibitor of κ kinase-β (IKKβ) signaling pathway is important in tumor promotion and progression. MDA-MB-231 human breast carcinoma cells express COX-2 and show a constitutive phosphorylation of NF-κB. Many non-specific inhibitors of IKKβ and NF-κB are used to inhibit tumor promotion and progression. The Stephania delavayi Diels. (S. delavayi Diels.) extract has been reported to safely activate B cell immunity and there is evidence suggesting that it may be a promising new anticancer therapeutic agent. S. delavayi Diels. extract suppressed proliferation of the breast cancer cell lines MDA-MB-231 and MCF-7 by inducing cell death. To aid in the development of the S. delavayi Diels. extract as a therapeutic agent, its mechanisms of action were investigated, in particular its effects on p38 MAPK, NF-κB and COX-2, which play important roles in inflammation and cancer. S. delavayi Diels. stimulated p38 MAPK phosphorylation but reduced NF-κB phosphorylation and COX-2 expression in a dose- and time-dependent manner. Thus, S. delavayi Diels., which appears to act primarily through p38 MAPK/NF-κB/COX-2 signaling in breast carcinomas, may be a potent anticancer agent with target specificity and low toxicity.     

Combinatorial inhibition of Plk1 and PKCβ in cancer cells with different p53 status

PKCβ and Plk1 are fascinating targets in cancer therapy. Therefore, we combined Enzastaurin targeting PKCβ and SBE13 targeting Plk1 to test synergistic effects in cells with different p53 status. We analyzed cell proliferation and apoptosis induction, and did Western blot and FACScan analyses to examine the combined PKCβ and Plk1 inhibition. p53-wild-type cells are more resistant to the combinatorial treatment than p53-deficient cells, which displayed a synergistic reduction of cell proliferation after the combination. HeLa, MCF-7 and HCT116^p53wt and HCT116^p53-/- cells differed in their cell cycle distribution after combinatorial treatment in dependence on a functional p53-dependent G1/S checkpoint (p53-deficient cells showed an enrichment in S and G2/M, p53-wild-type cells in G0/G1 phase). hTERT-RPE1 cells did not show the synergistic effects of cancer cells.Thus, we demonstrate for the first time that Plk1 inhibition using SBE13 enhances the effects of Enzastaurin in cancer cells. HCT116^p53wt and HCT116^p53-/- cells confirmed the p53-dependence of different effects after Plk1 and PKCβ inhibition observed in HeLa and MCF-7 cells. Obviously, p53 protects cells from the cytotoxicity of Enzastaurin in combination with SBE13. For that reason this combination can be useful to treat p53-deficient cancers, without displaying toxicity to normal cells, which all have functional p53.     

Gambogic acid synergistically potentiates cisplatin-induced apoptosis in non-small-cell lung cancer through suppressing NF-κB and MAPK/HO-1 signalling

Background:

Gambogic acid (GA) has been reported to have potent anticancer activity and is authorised to be tested in phase II clinical trials for treatment of non-small-cell lung cancer (NSCLC). The present study aims to investigate whether GA would be synergistic with cisplatin (CDDP) against the NSCLC.

Methods:

1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI) isobologram, western blot, quantitative PCR, flow cytometry, electrophoretic mobility shift assay, xenograft tumour models and terminal deoxynucleotide transferase-mediated dUTP nick-end labelling analysis were used in this study.

Results:

The cell viability results showed that sequential CDDP-GA treatment resulted in a strong synergistic action in A549, NCI-H460, and NCI-H1299 cell lines, whereas the reverse sequence and simultaneous treatments led to a slight synergistic or additive action. Increased sub-G1 phase cells and enhanced PARP cleavage demonstrated that the sequence of CDDP-GA treatment markedly increased apoptosis in comparison with other treatments. Furthermore, the sequential combination could enhance the activation of caspase-3, -8, and 9, increase the expression of Fas and Bax, and decrease the expression of Bcl-2, survivin and X-inhibitor of apoptosis protein (X-IAP) in A549 and NCI-H460 cell lines. In addition, increased apoptosis was correlated with enhanced reactive oxygen species generation. Importantly, it was found that, followed by CDDP treatment, GA could inhibit NF-κB and mitogen-activated protein kinase (MAPK)/heme oxygenase-1 (HO-1) signalling pathways, which have been validated to reduce ROS release and confer CDDP resistance. The roles of NF-κB and MAPK pathways were further confirmed by using specific inhibitors, which significantly increased ROS release and apoptosis induced by the sequential combination of CDDP and GA. Moreover, our results indicated that the combination of CDDP and GA exerted increased antitumour effects on A549 xenograft models through inhibiting NF-κB, HO-1, and subsequently inducing apoptosis.

Conclusion:

Gambogic acid sensitises lung cancer cells to CDDP in vitro and in vivo in NSCLC through inactivation of NF-κB and MAPK/HO-1 signalling pathways, providing a rationale for the combined use of CDDP and GA in lung cancer chemotherapy.