J Cancer Prev 2020; 25(2): 100-110
Published online June 30, 2020
© Korean Society of Cancer Prevention
1Department of Food Science and Biotechnology, College of Knowledge-Based Services Engineering, Sungshin Women’s University, 2Department of Future Applied Sciences, College of Natural Sciences, Sungshin Women’s University, 3Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, Korea
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15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand for PPARγ, has differential effects on cancer cell proliferation and survival depending on the dose and the type of cells. In the present study, we have investigated the effects of 15d-PGJ2 on apoptosis of the Ha-ras transformed human breast epithelial (MCF10A-ras) cells. When MCF10A-ras cells were treated with 15d-PGJ2 (10 μM) for 24 hours, they underwent apoptosis as evidenced by characteristic morphological features, an increased proportion of sub-G0/G1 cell population, a typical pattern of annexin V/propidium iodide staining, perturbation of mitochondrial transmembrane potential (Δψm), and cleavage of caspase-3 and its substrate PARP. A pan-caspase inhibitor, Z-Val-Ala-Asp (OCH3)-fluoromethyl ketone attenuated cytotoxicity and proteolytic cleavage of caspase-3 induced by 15d-PGJ2. The 15d-PGJ2-induced apoptosis was accompanied by enhanced intracellular accumulation of reactive oxygen species (ROS), which was abolished by the antioxidant N-acetyl-L-cysteine (NAC). 15d-PGJ2 inhibited the DNA binding activity of NF-κB which was associated with inhibition of expression and catalytic activity of IκB kinase β (IKKβ). 15d-PGJ2-mediated inhibition of IKKβ and nuclear translocation of phospho- p65 was blocked by NAC treatment. 9,10-Dihydro-PGJ2, a non-electrophilic analogue of 15d-PGJ2, failed to produce ROS, to inhibit NF-κB DNA binding, and to induce apoptosis, suggesting that the electrophilic α,b-unsaturated carbonyl group of 15d-PGJ2 is essential for its pro-apoptotic activity. 15d-PGJ2-induced inactivation of IKKβ was also attributable to its covalent thiol modification at the cysteine 179 residue of IKKβ. Based on these findings, we propose that 15d-PGJ2 inactivates IKKβ–ΝF-κB signaling through oxidative or covalent modification of IKKβ, thereby inducing apoptosis in Ha-ras transformed human breast epithelial cells.
Keywords: 15-Deoxy-Δ12,14-prostaglandin J2, Apoptosis, Reactive oxygen species, IKKβ–,NF-kB, MCF10A-ras cells
An endogenous ligand for PPARγ, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a cyclopentenone prostaglandin formed via two step dehydration of prostaglandin D2. 15d-PGJ2 has been known to exert diverse biological activities such as pro-inflammatory/anti-inflammatory, carcinogenic/anti-carcinogenic, and prooxidant/anti-oxidant effects, depending on the types of cells and the concentration used . It has been shown that synthetic PPARγ ligands as well as 15d-PGJ2 can induce growth inhibition, apoptosis, and terminal differentiation of several types of cancerous and transformed cells. 15d-PGJ2 attenuated the capability of the MDA-MB-231 cells to induce xenograft tumors in nude mice . In addition, 15d-PGJ2 inhibits migration of breast cancer MDA-MD-231 cells and osteolytic breast cancer bone metastasis in nude mice . 15d-PGJ2 inhibits phorbol ester-induced expression of matrix metallopeptidase-9 and invasion of MCF-7 cells . Moreover, 15d-PGJ2 synergistically enhanced the anti-tumor activity of the chemotherapeutic agent doxorubicin in renal cell carcinoma , and markedly reduced growth of murine colorectal carcinoma and HL-60 leukemia xenograft tumors .
The anti-proliferative effects of 15d-PGJ2 are associated with de novo synthesis of proteins involved in regulating the cell cycle and apoptosis. 15d-PGJ2 inhibited c-myc, cyclin D2, and cyclin D1 expression with concomitant induction of p21waf1 and p27kip1 in various type of cancer cells [6,7]. In addition, 15d-PGJ2 has been reported to induce apoptosis in diverse types of cancer cells, including gastric, colorectal, osteosarcoma, pancreatic, breast cancer [5,8-11]. 15d-PGJ2-induced apoptosis was associated with production of reactive oxygen species (ROS)  and mediated by regulating expression levels of the Bcl-2 family member proteins, such as Bax and Bcl-2 [8,12] and by downregulation of SIRT1 .
Although 15d-PGJ2 was first identified as an endogenous PPARγ ligand, its biological effects are mainly achieved by PPARγ-independent mechanisms through direct interaction with diverse signaling molecules and their regulators. 15d-PGJ2 has a reactive a,b-unsaturated carbonyl group in the cyclopentane ring which reacts with nucleophilic cellular moiety such as cysteine and hence covalently modifies and regulates the activation of the redox proteins . For instance, 15d-PGJ2 directly binds to c-Jun at a specific cysteine residue located in the DNA binding domain of AP-1, thereby inactivating this transcription factor . Furthermore, it has been known that 15d-PGJ2 induces expression of phase II detoxification or antioxidant enzymes through Nrf2 activation, which may confer cellular defense against or adaptation to carcinogenic insult or oxidative stress . Also, 15d-PGJ2 directly inhibits NF-κB-dependent gene expression through covalent modifications of critical cysteine residues in IκB kinase (IKK) [17,18] and the DNA-binding domains of NF-κB subunits [18,19]. The inhibitory effect of 15d-PGJ2 on NF-κB signaling through thiol modification of NF-κB may contribute to anti-inflammatory and anti-proliferative effects through inhibition of target gene expression such as Bcl-2. In this study, we investigated pro-apoptotic activity of 15d-PGJ2 in Ha-
15d-PGJ2 and 9,10-dihydro15d-PGJ2 (H2-15d-PGJ2) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Dulbecco’s modified Eagle’s medium (DMEM)/F-12, heat-inactivated horse serum, L-glutamine, penicillin/streptomycin/fungizone mixture were products of Gico BRL (Grand Island, NY, USA). MTT, insulin, cholera toxin, hydrocortisone, recombinant epidermal growth factor,
To quantify the percentage of cell population that are actively undergoing apoptosis, Annexin V-FITC was used according to manufacturer’s protocol. Briefly, MCF10A-
To measure the mitochondrial transmembrane potential (Δψm), the lipophilic cationic probe TMRE was used. MCF10A-
To monitor net intracellular accumulation of ROS, the fluorescence generating probe DCF-DA was used. MCF10A-
Values were expressed as the mean ± SE of at least three independent experiments. Statistical significance was determined by Student’s t-test. The criterion for statistical significance was
First, we determined the effect of 15d-PGJ2 on viability of MCF10A-
ROS is an universal entity mediating apoptosis in cancer cells . To determine whether 15d-PGJ2-induced apoptosis was attributable to generation of ROS, we utilized a fluorescent dye DCF-DA, capable of detecting peroxides including H2O2. 15d-PGJ2 treatment led to an enhanced accumulation of ROS, which was attenuated by the general antioxidant NAC (Fig. 2A). NAC treatment attenuated 15d-PGJ2-induced cytotoxicity (Fig. 2B), the proportion of sub-G0/G1 population (Fig. 2C) and apoptotic cells stained with Annexin-V/FITC (Fig. 2D). ROS generation is mediated by perturbation of mitochondrial membrane potential . When MCF10A-
NF-κB has been known to be associated with resistance to apoptosis in various cancer cells . 15d-PGJ2 has been known to inhibit the NF-κB signaling . The predominant form of NF-κB consists of p50 and p65 subunits that is sequestered in the cytoplasm by its inhibitory counterpart IκB-a. Signal dependent activation of IKK complex leads to the inducible phosphorylation of IκB proteins at two conserved serine residues located within their
IKK is a multi-subunit complex that contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO (NF-κB essential modulator) . Downregulation of IKKβ is associated with suppression of tumor development . Therefore, targeting NF-κB and its activating kinase, IKK have become an appealing therapeutic strategy in the progression of many diseases including chronic inflammation and cancer .
To determine whether the 15d-PGJ2-induced apoptosis was mediated by targeting the NF-κB pathway, we examined the effect of 15d-PGJ2 on DNA binding activity of NF-κB. As shown in Figure 3A, 15d-PGJ2 inhibited the NF-κB DNA binding activity. Phosphorylation of the p65 subunit has been considered to facilitate the translocation of NF-κB into nucleus. 15d-PGJ2 treatment suppressed the localization of phospho-p65 into the nucleus which was blunted by co-treatment with NAC (Fig. 3B).
In addition, 15d-PGJ2 inhibited the expression and catalytic activity of IKKβ, which were attenuated by NAC and DTT, respectively (Fig. 4A and 4B). IKKβ has a cysteine residue at position 179 within its activation loop . To determine whether this cysteine of IKKβ is critical for loss of its catalytic activity and subsequent induction of apoptosis by 15d-PGJ2, we utilized a mutant IKKβ construct in which cysteine 179 is replaced by alanine. 15d-PGJ2-induced suppression of IKKβ catalytic activity and proteolytic cleavage of PARP were less prominent in MCF10A-
In another experiment, a pharmacological inhibitor of IKKβ (Bay11-7082) increased the apoptotic cell population positive for Annexin V and proteolytic cleavage of caspase-3, which were suppressed by NAC treatment (Fig. 5A and 5B). Moreover, the pharmacologic inhibition of IKKβ suppressed the cell viability (Fig. 5C) as well as its catalytic activity (Fig. 5D), which were attenuated by NAC (Fig. 5C and 5D).
The α,β-unsaturated carbonyl group in the cyclopentane ring of 15d-PGJ2 renders it reactive towards cellular nucleophilics to form covalently bound Michael adducts. To determine whether the α,β-unsaturated carbonyl moiety of 15d-PGJ2 plays an important role in induction of apoptosis in MCF10A-
We observed that 15d-PGJ2 (10 μM) induced apoptosis in ras oncogene transformed human breast epithelial MCF10A cells, which was attributable to the ROS-mediated inhibition of catalytic activity of IKKβ and subsequently NF-κB signaling. 15d-PGJ2 is characterized by the presence of a reactive a,β-unsaturated carbonyl group in the cyclopentenone ring. This moiety has been known to bind to sulfhydryl groups of cysteine residues of proteins by Michael addition, resulting in alteration of the protein structure and function . 15d-PGJ2 has been reported to direct modify the Cys 179 of IKKβ within their activation loop, thereby suppressing catalytic activity of IKK and transcriptional activity of NF-κB . Consistent with this result, we observed that 15d-PGJ2-induced suppression of catalytic activity of IKKβ and proteolytic cleavage of caspase 3 were attenuated in the MCF10A-
However, phosphorylation is critical mechanism to regulate NF-κB activation. Phosphorylation of two sites at the activation loop of IKKβ was essential for activation of IKK by tumor necrosis factor and interleukin-1 . Oxidation or chemical modification of the thiols in the IKKβ and NF-κB can result in a conformational change and thus affection the phosphorylation. Thiol modification of IKKβ may induce its conformational change, which may prevent upstream kinase from phosphorylating the serine residues, thereby preventing activation of the complex . Therefore, thiol modification of Cys179 of IKKβ can indirectly inhibit its kinase activity, resulting in suppression of NF-κB signaling.
We observed that ROS plays an important role in induction of apoptosis and nuclear translocation of p-p65 as well as suppression of IKKβ. Intracellular events associated with generation of ROS by 15d-PGJ2 are likely to be a consequence of depletion of reduced glutathione and glutathione peroxidase and increased production of protein–bound lipid peroxidation products (e.g., 4-hydroxy-2-nonenal and acrolein) [31,32]. In addition, 15d-PGJ2-inducd ROS generation is associated with reduction of thioredoxin (Trx) which plays an important role in the redox regulation of signal transduction and in cytoprotection against oxidative stress . The overexpression of Trx protected against 15d-PGJ2-induced apoptosis in human neuroblastoma SH-SY5Y cells. Further, 15d-PGJ2 directly binds and modifies the Cys residues of Trx . Consistent with these results, 9,10-dihydro-15d-PGJ2 did not affect modification of Cys in Trx and inhibit the expression of epidermal growth factor receptor in oral squamous cell carcinoma . Based on the previous studies, 15d-PGJ2-induced disruption of mitochondrial membrane potential is attributed to disruption of redox homeostasis in MCF10A-
In conclusion, 15d-PGJ2 inhibited IKKβ activity, thereby inactivating NF-κB signaling in MCF10A-
This work was supported by grants from Sungshin Women’s University (2018-1-29-016).
No potential conflicts of interest were disclosed.
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