JNK inhibition

c-Jun N-terminal kinase (JNK) is a member of mitogen-activated protein kinase (MAPK) that links inflammation to the development of various kinds of cancer, especially prostate cancer, breast cancer, leukemia, stomach cancer and hepatocellular carcinoma (HCC). JNK was reported for its role in cancer cell proliferation, angiogenesis and metastasis [1, 2].

A number of small compounds that can interfere and block JNK signaling pathway have been developed rapidly. SP600125, an anthrapyrazolone derivative, exhibited high efficacy in blocking the phosphorylation activity of JNK by interact with ATP-binding site on JNK. SP600125 is non-selective JNK inhibitor which can inhibit three isoforms of JNK with different activity (IC50 of 40 nM for JNK1 and JNK2, and 90 nM for JNK3 in vitro, respectively) [3]. There were many attempts trying to examine how SP600125 works in different kinds of cancer in the last few years in order to find an alternative way for cancer therapy. The ability of SP600125 in inhibit the 92-kDa type IV collagenase which causes cancer cell metastasis was studied in OVCAR-3 cell line derived from a malignant ascites of a patient with progressive adenocarcinoma of the ovary. SP600125 can diminish secretion of collagenase after blocking JNK and c-Jun phosphorylation [4]. Moreover, in another study, SP600125 effectively inhibits endothelial cell migration and proliferation which are angiogenesis processes in human microvascular endothelial cells (HMVEC). The administration of SP600125 also inhibited JNK in DU145 human prostate carcinoma xenografts and murine Lewis lung carcinoma [5]. For breast cancer, epidermal growth factor receptor 2 (HER2) is high expression. The HER2-mediated signaling negatively regulates CCNG2 gene which encodes for cyclin G2, an unconventional cyclin family, which relates in cell cycle arrest. The downstream JNK pathway of HER2 receptor regulates the expression of cyclin G2 which is reduced in breast cancer. Therefore, this study focused on the inhibition of JNK by SP600125 in order to increase the cyclin G2 expression level. SP600125 induced RNA expression of cyclin G2 in MCF7 cell (human breast cancer cell line) in a dosage-dependent manner. The ectopic expression of cyclin G2 inhibits cyclin-dependent kinase 2 activity (Cdk2), Rb phosphorylstion, cell cycle progression and cellular proliferation without affecting p27Kip1 leads to cell cycle arrest at G1-phase [6]. SP600125 also induces apoptosis and inhibits cell cycle progression in G2/M phase by reduces the level of Cdk2, cyclin B1 and cdc25c proteins in human leukemia U937 cells [7].

Doxorubicin (DOX) resistance breast and stomach cancer cell lines were screened and treated with SP600125. DOX is a chemotherapeutic drug used in various cancers for treatment of endocrine-resistant and metastatic cancers. SP600125 was a new target therapy that might be used for DOX-resistant cancers. Four breast cancer cell lines and one stomach cancer cell line viability were reduced significantly after treatment with SP600125. Moreover, the weight and volume of mouse xenograft tumors were also reduced significantly [8].

Nowadays the only way to cure for hepatocellular carcinoma (HCC) is the surgical resection. HCC patients normally have pathological symptom when they are in advanced stage. Surgical resection therefore can not remove the large size of tumors [9].

For HCC, SP600125 was also tested for its potential JNK inhibitor ability. WW-domain containing oxidoreductase (WWOX) gene has been implicated as p53-mediated apoptosis and functions as a tumor suppressor in breast, ovarian, prostate, oral, gastric, pancreatic, esophageal, cutaneous, thyroid and lung carcinomas and meningiomas. In 18 human HCC cell lines showed 61% of WWOX mRNA expression downregulation. JNK interacts and phosphorylates WWOX to inhibit its role of apoptosis. To study the inhibitory property of SP600125 against JNK, human HCC cell lines were treated with SP600125. SP600125 enhanced apoptosis and suppressed HCC cell proliferation [10]. Echography of rat HCC which were induced by diethylnitrosamine (DEN) was also studied to examine the lesion of tumors. After SP600125 injection directly to 2-4 mm diameter lesions, the size of HCC was reduced significantly visualized by echographic detection [11]. Another study that showed the activity of SP600125 in HCC was performed in 4 week-old male rats. The rats were treated with DEN that activated JNK in order to induce HCC development. From liver tissues of DEN treated rats, the control group which was not injected by SP600125 developed multiple tumors. Comparing to control group, SP600125 group had the smaller number of tumors that larger than 3 mm in diameter. The mechanism of SP600125 in growth inhibition was also identified in this study. DEN administration rats results in the production of inflammatory cytokines from kuffer cells that is a hallmark of inflammation-related hepacarcinogenesis. JNK is activated by the inflammatory cytokines and it can further phosphorylates pSmad3L (JNK substrate) leading to cell proliferation. DEN can also stimulate the production of TGF-b which then binds to TGF-b type I receptor (TbRI) triggering pSmad3C and cell growth inhibition. In cancer development, pSmad3L inhibits pSmad3C activation leading to cease p21WAF1 pathway. However, when JNK is inhibited by SP600125, pSmad3L is not activated and stop cancer cell growth (Fig. 1) [12].

Apart from SP600125, hesperidin, a flavonoid present in citrus fruits, was used to treat HCC metastasis by inhibit phosphorylation of JNK. Tumor invasion and metastasis relate in increasing of matrix metalloproteinase (MMP) expression level [13]. The MMP-9 gene is highly expressed particularly in invasive HCC. MMP-9 contains activator protein 1 (AP-1) element which can get activated by JNK signaling pathway. Hesperidin showed an effective ability to inhibit acetaldehyde-induced AP-1 in HepG2 cells by interfering JNK phosphorylation activity which then reduced the MMP-9 expression and HCC invasion. Unfortunately, hesperidin is not specific to JNK only. It also can inhibit p38 MAPK and IkB pathway [13]. Therefore, using hesperidin as JNK inhibitor in HCC should be depended on the stages of HCC.

Many companies have developed a number of JNK inhibitor compounds. However, most of them are poor water solubility and off-targeting effect on other kinases that limited their further study in pre-clinical and clinical trials [9]. JNK activation that involves in cancer from initial to advanced steps is so complicated in which JNK can have either tumor suppressor property or cancer cell proliferation function depended on the type of cancer and the opposite effects of JNK1 and JNK2 [14]. Non-specificity of SP600125 is therefore should be concerned. JNK1 was reported to be an important isoform that expresses highly in late stages of HCC to promote cancer cell proliferation [9]. JNK inhibited by SP600125 reduced only small size of tumors found in the early stage of HCC [11]. That might be because SP600125 inhibits JNK2 which was reported to promote apoptosis in HCC. Accordingly, JNK1 was suggested to be a new therapeutic target for HCC.

JNK-interaction protein (JIP1) is a scaffolding protein of JNK [15]. Nuclear localization of JNK is blocked when binds to JIP-1 resulting to c-Jun phosphorylation level. JIP1 then acts as regulator for JNK signaling pathway. JIP1 consists of 707 amino acids and contains an N-terminal JNK-binding domain and C-terminal MLK (mixed-lineage kinase) and MKK7 (MAPK kinase)-binding domain. The small region of JIP1 has been identified as the JNK-inhibitory property [16]. This peptide of JIP1 (pepJIP1) inhibited JNK activity in vitro significantly without any effect on Erk or p38 MAPKs. The docking sites of JNK have been identified for substrate transcription factor, MKKs and JIP. Interestingly, these docking sites share some common sequences among one another. However, JNK isoforms differ in their binding sites of transcription factors. The docking site of JIP1 binds at the same docking grooves of JNK1 as the upstream kinase and the transcription factor [15].

pepJIP1 which is an 11-mer peptide (residues 153-163 of JIP1 with the sequence of RPKRPTTLNLF) was studied for its JNK1 inhibitory property in vitro used myelin basic protein (MBP) as a substrate. It was concluded that pepJIP1 can inhibit JNK mainly due to the competition to substrates or the upstream kinases binding to the docking sites. JIP1 binding can also caused distortion of ATP-binding site at the catalytic cleft [16].

Peptide sequences are responsible for molecular recognition and biological activities. Inhibition of protein-protein interactions by peptides is a major goal of the peptide research field, with several successes. Peptides therefore seem to be ideal drug. However, there are limitations for peptides that they are metabolically unstable due to the protease cleavage of the peptide backbone and low membrane transport characteristics. To solve these problems, 10-amino-acid HIV Tat transporter sequence linked to the 20-amino-acid JNK-binding motif (JBD) of JIP1/IB1 (islet-brain 1) was synthesized in two forms which were L-form (L-JNKI-1) and protease-resistant D-form (D-JNKI-1). The inhibitory property against JNK, but not JNK1 to suppress excitotoxicity and cerebral ischemia was examined in adult mice. L-JNKI-1 and D-JNKI-1 were injected free-handedly into the lateral ventricle. The result clearly showed that the lesion volume was reduced more than 90% for at least 14 days, particularly by D-JNKI-1 [17].

Even though, the HIV Tat-derived peptide which is a small basic peptide has been reported to be able to deliver proteins, peptides and nucleic acids across cell membrane, the problems combined with poor cell permeability, peptide instability and short half-life in vivo still exist. A series of pepJIP1 mimics therefore were studied in vitro and in vivo. BI-78D3, MW 379.37, IC50 = 500 nM (DELFIA assay) was the most effective compound among various designed models. To test BI-78D3 ability, ConA was injected to induce liver failure into mice. The bio-analysis of the JNK-inhibitory property of BI-78D3 in blocking ConA-induced liver failure was measured for the level of alanine aminotransferase (ALT) released from liver. After BI-78D3 injection, the ALT level was reduced significantly comparing to DMSO-treated group [18]. Although, BI-78D3 was designed by mimic pepJIP1, there was no evident for JNK1 specific inhibition. Further and more studies should be done to open the way for development of JNK1 inhibition targeting on non-ATP binding site.

The compound which was not designed for ATP-binding site in order to inhibit JNK1 pathway is therefore a new strategy for HCC therapy [19]. 7-(6-N-phenylaminohexyl)amino-2H-anthra[1,9-cd]pyrazol-6-one (AV-7) was developed in-house using structure-based design that shows a selective inhibitory effect against JNK1. AV-7 was designed to specific the protein-protein interaction between JNK1 and its binding partners, not the ATP binding site as a target of SP600125. Mouse embryonic fibroblasts (MEFs) were used to test for the specificity of AV-7. JNKWT, JNK1-/- and JNK2-/- fibroblasts had been stimulated with ultraviolet B (UVB) before they were treated with and without AV-7. Western blot analysis showed no differences in c-Jun phosphorylation (p-c-Jun) levels before and after treated with AV-7 in JNKWT group. JNK2-/- MEFs that expresses only JNK1 isoform decreased p-c-Jun level dramatically comparing to JNK1-/- group. However, SP600125 can inhibit p-c-Jun in both JNK1-/- and JNK2-/- groups. Although, AV-7 has ability to inhibit JNK1, 10 �M of AV-7 can reduce the c-Jun phosphorylation level about 50% which is not so effective inhibitor [20]. Moreover, AV-7 has been developed recently. It is in the beginning step of the drug development process. Further studying to identify how AV-7 works in animal model, its toxicity, potential dose and adverse effects should be examined.

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