Regorefenib induces extrinsic/intrinsic apoptosis and inhibits MAPK/NF‐κB‐modulated tumor progression in bladder cancer in vitro and in vivo

The aim of the present study is to investigate anticancer effect and mechanism of regorafenib in bladder cancer in vitro and in vivo. Human bladder cancer TSGH 8301 cells were treated with regorafenib, NF‐κB, AKT, or mitogen‐activated protein kinase (MAPK) inhibitors for different time. The changes of cell viability, NF‐κB activation, apoptotic signaling transduction, and expression of tumor progression‐associated proteins were evaluated with MTT, NF‐κB reporter gene assay, flow cytometry, and Western blotting assay. TSGH 8301 tumor bearing mice were established and treated with vehicle (140 μL of 0.1% DMSO) or regorafenib (10 mg/kg/day by gavage) for 15 days. The changes of tumor volume, body weight, NF‐κB activation, MAPK activation, and tumor progression‐associated proteins (MMP‐9, XIAP, VEGF, and Cyclin‐D1) after regorafenib treatment were evaluated with digital caliper, digital weight, and ex vivo Western blotting assay. Our results demonstrated NF‐κB activation and protein levels of MMP‐9, XIAP, VEGF, and Cyclin‐D1 were significantly reduced by NF‐κB (QNZ), ERK (PD98059), and P38 (SB203580) inhibitors. Regorafenib also significantly induced extrinsic and intrinsic apoptotic signaling transduction in bladder cancer in vitro. In addition, regorafenib significantly inhibited tumor growth, NF‐κB, p38, ERK activation and expression of tumor progression‐associated proteins in bladder cancer in vitro and in vivo. Taken together, these results proved that regorafenib not only induced apoptosis through extrinsic and intrinsic pathways and but suppressed MAPK/ NF‐κB‐modulated tumor progression in bladder cancer.     

MiR‐146a regulates PM1‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells

Exposure to particulate matter (PM) leads to kinds of cardiopulmonary diseases, such as asthma, COPD, arrhythmias, lung cancer, etc., which are related to PM‐induced inflammation. We have found that PM_2.5 (aerodynamics diameter <2.5 µm) exposure induces inflammatory response both in vivo and in vitro. Since the toxicity of PM is tightly associated with its size and components, PM₁ (aerodynamics diameter <1.0 µm) is supposed to be more toxic than PM_2.5. However, the mechanism of PM₁‐induced inflammation is not clear. Recently, emerging evidences prove that microRNAs play a vital role in regulating inflammation. Therefore, we studied the regulation of miR‐146a in PM₁‐induced inflammation in human lung bronchial epithelial BEAS‐2B cells. The results show that PM₁ induces the increase of IL‐6 and IL‐8 in BEAS‐2B cells and up‐regulates the miR‐146a expression by activating NF‐κB signaling pathway. Overexpressed miR‐146a prevents the nuclear translocation of p65 through inhibiting the IRAK1/TRAF6 expression, and downregulates the expression of IL‐6 and IL‐8. Taken together, these results demonstrate that miR‐146a can negatively feedback regulate PM₁‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells.     

Nonylphenol regulates cyclooxygenase‐2 expression via Ros‐activated NF‐κB pathway in sertoli TM4 cells

The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)‐2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX‐2 protein expression and interleukin‐6 (IL)‐6 and prostaglandin E2 (PGE2) secretion in a dose‐dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N‐acetylcysteine (NAC), attenuated NP‐induced ROS production, COX‐2 expression, and IL‐6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF‐κB, whereas the NF‐κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP‐enhanced COX‐2 expression and IL‐6 and PGE2 release in TM4 cells in a dose‐dependent manner. Furthermore, NAC blocked NP‐induced activation of NF‐κB. In addition, inhibition of COX‐2 mitigated NP‐induced IL‐6 release. In conclusion, NP induced ROS generation, activation of NF‐κB pathway, COX‐2 upregulation, and IL‐6 and PGE2 secretion in TM4 cells. NP may regulate COX‐2 expression via ROS‐activated NF‐κB pathway in Sertoli TM4 cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1144–1152, 2015.     

Curcumin sensitized the antitumour effects of irradiation in promoting apoptosis of oesophageal squamous‐cell carcinoma through NF‐κB signalling pathway

Objectives

To investigate the potential synergistic effect of curcumin with irradiation (IR ) in oesophageal squamous‐cell carcinoma (ESCC ) and elucidate the underlying molecular mechanisms.

Methods

The ESCC cell lines were established from clinical samples. Cell apoptosis post‐treatment was stained by Annexin V /PI staining and analysed by flow cytometry. Cells survived IR was evaluated with clonogenic assay. Xenograft tumour model was established by subcutaneous inoculation, and tumour progression was monitored. The NF ‐κB pathway was characterized by immunoblotting.

Key findings

Curcumin enhanced the pro‐apoptotic effect of IR in ESCC cells. Pretreatment with curcumin significantly sensitized ESCC cells to IR in a dose‐dependent manner. Coadministration with curcumin remarkably extended the median survival time of ESCC xenograft mice while exposed to IR therapy. The xenograft tumour progression was significantly suppressed as well. Mechanistically, curcumin treatment was demonstrated to efficiently inhibited NF ‐κB signalling.

Conclusions

We have well‐recapitulated the pathological properties and therapeutic response of ESCC with established cell lines derived from clinical samples. We further demonstrated the significantly synergistic effect of curcumin on IR ‐elicited cell apoptosis in ESCC both in vitro and in vivo . Our data suggested the potential therapeutic values of curcumin for future clinical investigations.

     

Synthesis and SAR Studies of Dual AKT/NF‐κB Inhibitors Against Melanoma

The protein Kinase B alpha (AKT) and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathways are central regulators of cellular signaling events at the basis of tumor development and progression. Both pathways are often up‐regulated in different tumor types including melanoma. We recently reported the identification of compound 1 (BI‐69A11) as inhibitor of the AKT and the NF‐κB pathways. Here, we describe SAR studies that led to novel fluorinated derivatives with increased cellular potency, reflected in efficient inhibition of AKT and IKKs. Selected compounds demonstrated effective toxicity on melanoma, breast, and prostate cell lines. Finally, a representative derivative showed promising efficacy in an in vivo melanoma xenograft model.     

Role of NF‐κB activation and Th1/Th2 imbalance in pulmonary toxicity induced by nano NiO

With the progress of nanotechnology, nano nickel oxide (NiO) has been extensively used as sensors, battery electrodes, catalysts, and cosmetics. Previous researches verified that nano NiO could exert pulmonary toxicity, but its mechanism was unclear. To shed light upon this, the role of nuclear factor‐κ B (NF‐κ B) activation and Th1/Th2 imbalance were to explore in pulmonary damage induced by nano NiO. Male Wistar rats were randomized into control group, nano NiO groups (0.015, 0.06, and 0.24 mg kg^?1) and micro NiO group (0.024 mg kg^?1) and treated by intratracheal instillation twice a week for 6 weeks. The results showed that the abnormal changes induced by nano NiO were found on indicators of nitrative stress (NO, TNOS, and iNOS), inflammatory cytokines (TNF‐α , IL‐2, and IL‐10) and cytokine‐induced neutrophil chemoattractants (CINC‐1, CINC‐2αβ , and CINC‐3) in lung tissue. In addition, nano NiO instillation induced the upregulated mRNA and protein expression of NF‐κ B, inhibitor of κB kinase‐α (IKK‐α ) and nuclear factor‐inducing kinase (NIK). The protein content of GATA‐3 increased as well as T‐bet decreased in nano NiO groups, and the ratio of T‐bet/GATA‐3, as a key evaluation indicator of Th1/Th2 balance, was lower than the control group. The findings indicated that nano NiO could enhance the nitrative stress and inflammatory response in lung tissue, and its mechanism was related to the NF‐κ B activation and Th1/Th2 imbalance. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1354–1362, 2017.     

Inhibition of NF‐κB and metastasis in irinotecan (CPT‐11)‐resistant LoVo colon cancer cells by thymoquinone via JNK and p38

Clinically used chemotherapeutics can effectively eliminate most tumor cells. However, they cause unwanted side effects and result in chemoresistance. To overcome such problems, phytochemicals are now used to treat cancers by means of targeted therapy. Thymoquinone (TQ) is used to treat different cancers (including colon cancer) and is an NF‐κB inhibitor. Irinotecan resistant (CPT‐11‐R) LoVo colon cancer cell line was previous constructed by step‐wise CPT‐11 challenges to un‐treated parental LoVo cells and expresses EGFR/IKKα/β/NF‐κB pathway. TQ resulted in reduced total and phosphorylation of IKKα/β and NF‐κB and decreased metastasis in CPT‐11‐R cells. TQ not only reduced activity of ERK1/2 and PI3K but also activated JNK and p38. Furthermore, TQ was also found to suppress metastasis through activation of JNK and p38. Therefore, TQ suppressed metastasis through NF‐κB inhibition and activation of JNK and p38 in CPT‐11‐R LoVo colon cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 669–678, 2017.     

The role of GSH in microcystin‐induced apoptosis in rat liver: Involvement of oxidative stress and NF‐κB

Microcystins (MCs) are potent and specific hepatotoxins produced by cyanobacteria in eutrophic waters, representing a health hazard to animals and humans. The objectives of this study are to determine the relationship between oxidative stress and NF‐κB activity in MC‐induced apoptosis in rat liver and the role of glutathione (GSH). Sprague‐Dawley rats were intraperitoneally (i.p.) injected with microcystin‐LR (MC‐LR) at 0.25 and 0.5 LD₅₀ with or without pretreatment of buthionine‐(S,R)‐sulfoximine (BSO), a specific GSH synthesis inhibitor. MC‐LR induced time‐dependent alterations of GSH levels in rat liver. Increased malondialdehyde (MDA) and significant changes of antioxidant enzymes including GSH peroxidase (GPX) and GSH reductase (GR) were also observed, particularly at 24 h post‐exposure. The results indicated that acute exposure to MC‐LR induced oxidative stress, and GSH depletion (BSO pretreatment) enhanced the level of oxidative stress. Furthermore, the modulation of pro‐apoptotic gene p53 and Bax and anti‐apoptotic gene Bcl‐2 was observed in 0.5 LD₅₀ group at 24 h, and the alteration was more pronounced by BSO injection before MC‐LR treatment, suggesting that GSH played a protective role against MC‐induced toxicity. Additionally, electrophoretic mobility shift assay (EMSA) showed that NF‐κB was induced at 0.25 LD₅₀ but inhibited at 0.5 LD₅₀. The above results indicated that the possible crosstalk of oxidative stress and NF‐κB activity was associated with MC‐LR‐induced hepatocytes apoptosis in vivo. Our data will provide a new perspective for understanding the mechanisms of MC‐induced liver injury. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 552–560, 2016.     

Apoptosis induction and ERK/NF‐κB inactivation are associated with magnolol‐inhibited tumor progression in hepatocellular carcinoma in vivo

Although hepatitis B and/or hepatitis C virus were recognized as major risk factor for the development of hepatocellular carcinoma (HCC), certain occupational, environmental, and lifestyle factors also play key roles in HCC tumorigenesis. Moreover, in molecular signaling route, extracellular signal‐regulated kinase (ERK)/nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) signaling was found to be overexpressed and linked to poor prognosis in HCC. Thus, to identify possible nature compound that can suppress ERK/NF‐κB may be benefit to HCC patient. Magnolol, a natural compound derived from herbal plant Magnolia officinalis, has been recognized as a liver protection and antitumor reagent. However, whether magnolol‐inhibited HCC progression correlates with disruption of ERK/NF‐κB signaling is remained unclear. In this studies, we performed SK‐Hep1/luc2 HCC bearing animal model to investigate the anticancer efficacy and mechanism of magnolol on tumor progression. Tumor size and tumor growth rate were dramatically suppressed after treatment of magnolol. In addition, expression of phospho‐ERK (p‐ERK), NF‐κB p65 (Ser536), and tumor progression‐associated proteins, such as matrix metallopeptidase 9 (MMP‐9), vascular endothelial growth factor (VEGF), X‐linked inhibitor of apoptosis protein (XIAP), and CyclinD1 were all significantly decreased by magnolol. Most important, major extrinsic and intrinsic apoptosis signaling factors, including active caspase‐8 and caspase‐9 were both enhanced by magnolol. This study indicated that apoptosis induction through extrinsic/intrinsic pathways and blockage of ERK/NF‐κB activation were associated with magnolol‐inhibited tumor progression in HCC in vivo.     

NF‐κB/p53‐activated inflammatory response involves in diquat‐induced mitochondrial dysfunction and apoptosis

Inflammation generated by environmental toxicants including pesticides could be one of the factors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of caspases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant N‐acetyl‐cystein attenuated the cytotoxicity of caspase‐3 pathways. These results demonstrate that diquat‐induced apoptosis is involved in mitochondrial dysfunction through production of ROS. Furthermore, diquat increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Diquat induced nuclear accumulation of NF‐κB and p53 proteins. Importantly, an inhibitor of NF‐κB nuclear translocation blocked the increase of p53. Both NF‐κB and p53 inhibitors also blocked the diquat‐induced inflammatory response. Pretreatment of cells with meloxicam, a COX‐2 inhibitor, also blocked apoptosis and mitochondrial dysfunction. These results represent a unique molecular characterization of diquat‐induced cytotoxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through inflammatory responses via NF‐κB‐mediated p53 signaling. This suggests the potential to generate mitochondrial damage via inflammatory responses and inflammatory stimulation‐related neurodegenerative disease.     

Diallyl sulfide alleviates cisplatin‐induced nephrotoxicity in rats via suppressing NF‐κB downstream inflammatory proteins and p53/Puma signalling pathway

Despite being a potent anticancer drug, nephrotoxicity is an adverse effect which renders the clinical use of cisplatin (Cis) limited. The protective role of diallyl sulfide (DAS); a naturally occurring organo‐sulfide, present in garlic, in cisplatin‐induced nephrotoxicity has been reported earlier. However, the mechanism through which DAS exerts its nephroprotective activity remains elusive. The aim of the current study was to elucidate the possible mechanisms underlying the reno‐protective effect of DAS in cisplatin‐induced nephrotoxicity in rats. DAS was given at 2 dose levels; 50 and 100 mg/kg, orally for 4 consecutive days, starting 1 hour after administration of single dose of cisplatin (3.5 mg/kg, intraperitoneally [i.p.]). The Cis‐induced elevation in serum urea and creatinine, degree of histopathological alterations was significantly ameliorated in cisplatin groups co‐treated with DAS. In addition, DAS significantly restored Cis‐depleted glutathione (GSH) content and superoxide dismutase (SOD) activity and attenuated Cis‐elevated Malondialdehyde (MDA) level. Also, DAS significantly reduced Cis‐increased renal expression of nuclear factor kappa B (NF‐κB) and subsequent pro‐inflammatory mediators; tumour necrosis factor alpha (TNF‐α), interleukin‐1β (IL‐1β), intercellular adhesion molecule‐1 (ICAM‐1) and inducible nitric oxide synthase (iNOS) in kidney tissues. Moreover, co‐treatment with DAS significantly inhibited Cis‐increased caspase‐8 and ‐9 levels. Additionally, DAS significantly mitigated Cis‐induced protein expression of p53, Puma, and Bax while, it significantly restored Cis‐reduced protein expression of Bcl‐xL compared to the Cis group. In conclusion, these results demonstrate that DAS ameliorates cisplatin‐induced nephrotoxicity in rats through enhancement of antioxidant defense, reduction of inflammatory cytokine tissue levels as well as inhibition of apoptosis via p53/Puma signalling pathway.     

para‐Phenylenediamine induces apoptosis through activation of reactive oxygen species‐mediated mitochondrial pathway,and inhibition of the NF‐κB,mTOR, and Wnt pathways in human urothelial cells

para‐Phenylenediamine (PPD) has long been used in two‐thirds of permanent oxidative hair dye formulations. Epidemiological studies and in vivo studies have shown that hair dye is a suspected carcinogen of bladder cancer. However, the toxicity effects of PPD to human bladder remains elusive. In this study, the effects of PPD and its involvement in the apoptosis pathways in human urothelial cells (UROtsa) was investigated. It was demonstrated that PPD decreased cell viability and increased the number of sub‐G₁ hypodiploid cells in UROtsa cells. Cell death due to apoptosis was detected using Annexin V binding assay. Further analysis showed PPD generated reactive oxygen species (ROS), induced mitochondrial dysfunction through the loss of mitochondrial membrane potential and increased caspase‐3 level in UROtsa cells. Western blot analysis of PPD‐treated UROtsa cells showed down‐regulation of phosphorylated proteins from NF‐κB, mTOR, and Wnt pathways. In conclusion, PPD induced apoptosis via activation of ROS‐mediated mitochondrial pathway, and possibly through inhibition of NF‐κB, mTOR, and Wnt pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 265–277, 2017.     

NF‐κB activation mediates LPS‐or zymosan‐induced hypotension and inflammation reversed by BAY61‐3606, a selective Syk inhibitor,in rat models of septic and non‐septic shock

We have previously demonstrated that the activation of the spleen tyrosine kinase (Syk)/inhibitory‐κB (IκB)‐α/nuclear factor‐κB (NF‐κB) p65 signalling pathway contributes to hypotension and inflammatory response in a rat models of zymosan (ZYM)‐induced non‐septic shock. The purpose of this study was to further examine the possible mechanism underlying the effect of inhibition of Syk by BAY61‐3606 via NF‐κB activity at the level of nuclear translocation regarding the production of vasodilator and proinflammatory mediators in lipopolysaccharide (LPS) (septic)‐ and ZYM (non‐septic)‐induced shock. Administration of LPS (10 mg/kg, ip) or ZYM (500 mg/kg, ip) to male Wistar rats decreased mean arterial pressure and increased heart rate that was associated with an increase in the activities of cyclooxygenase and nitric oxide synthase, tumour necrosis factor‐α, and interleukin‐8 levels, and NF‐κB activation and nuclear translocation in sera and/or cardiovascular and renal tissues. BAY61‐3606 (3 mg/kg, ip), the selective Syk inhibitor, given 1 hour after LPS‐ or ZYM injection reversed all the above‐mentioned effects. These results suggest that Syk contributes to the LPS‐ or ZYM‐induced hypotension and inflammation associated with transactivation of NF‐κB in septic and non‐septic shock.     

Involvement of NF‐κB in the upregulation of cystathionine‐γ‐lyase,a hydrogen sulfide‐forming enzyme,and bladder pain accompanying cystitis in mice

Hydrogen sulfide (H₂S) is generated from l ‐cysteine by multiple enzymes including cystathionine‐γ‐lyase (CSE), and promotes nociception by targeting multiple molecules such as Ca_v3.2 T‐type Ca²⁺ channels. Bladder pain accompanying cyclophosphamide (CPA)‐induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H₂S/Ca_v3.2 pathway. Therefore, we investigated whether NF‐κB, as an upstream signal of the CSE/H₂S system, contributes to bladder pain in mice with CPA‐induced cystitis. Bladder pain‐like nociceptive behaviour was observed in CPA‐treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β‐Cyano‐l ‐alanine, a reversible selective CSE inhibitor, prevented CPA‐induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF‐κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF‐κB inhibitor. PDTC and curcumin, which inhibits NF‐κB signals, abolished CPA‐induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA‐induced activation of NF‐κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H₂S/Ca_v3.2 signaling. Therefore, NF‐κB‐inhibiting compounds including curcumin may be useful for the treatment of cystitis‐related bladder pain.