JNK Inhibitor AS-01 Causes Regression of Endometriosis and Suppression of Inflammatory Cytokines and Tissue Remodeling Enzymes.

Endometriosis is an estrogen dependent gynecological disease that can result in an array of problems from severe chronic pelvic pain to infertility. It is caused by the ectopic growth of the endometrial tissue at extrauterine sites, predominantly in the peritoneal cavity. Immune system deficiency is thought to be the underlying pathogenesis of this disease. Increased levels of cytokines, growth and angiogenic factors in the peritoneal fluid suggest tissue remodeling and angiogenesis might also play important parts in disease progression. In preclinical models, JNK inhibitors have been shown to be potential therapeutic agents for chronic inflammatory diseases such as rheumatoid arthritis and atherosclerosis. In this study, we used AS-01, a small molecule inhibitor of c-jun N-terminal kinase (JNK), to treat a mouse model of endometriosis. Experimental endometriosis was established in athymic ovariectomized mice implanted with a slow-release estradiol capsule. Proliferative phase human endometrium was obtained from patients with disease and injected subcutaneously into mice; mice were randomized into treatment groups 10 days later. Mice were treated daily with vehicle or inhibitor for 4 weeks and sacrificed and necropsied for signs of disease. Endometriotic-like lesions were photographed, measured and removed for further analysis. Our results indicate that treated mice showed a remarkable 50% decrease in lesion size compared to control mice, with no significant effect on uterine weight or size. Removed tissues were subjected to real-time RT-PCR assay of selected genes. Chemoattractant cytokines RANTES, IL8, GM-CSF, ICAM-1, MCP-1, TNFa, IL6 are highly expressed in the peritoneal cavity of diseased women and are important for the recruitment of the leukocytes to the peritoneum. Our investigation revealed that TNFa, IL8, GM-CSF, ICAM-1 and RANTES expression was remarkably decreased by about 50% by AS-01; haptoglobin, IL6 and MCP-1 mRNA levels were also much lower than control, showing an overall decrease between 20 and 35%. Matrix metalloproteases (MMPs), a group of enzymes that mediate normal and pathogenic tissue remodeling, are also upregulated in women with endometriosis. Our results demonstrated that MMP9 mRNA level was suppressed 60% by the JNK inhibitor AS-01, followed by MMP2 and MMP7 which were both decreased by about 20% each. Angiogenic factors could potentially contribute to the establishment of endometriotic lesions; ectopic endometrium secretes many angiogenic and pro-angiogenic factors, including VEGF, IL6, IL8, TNFa and HGF. In our studies, AS-01 suppressed the levels of VEGF by 20 %, and levels of HGF by 60%. The steroid hormones, estrogen and progesterone, are both regulators of endometrial tissue. In our study, we have investigated the expression levels of the steroid pathway receptors that regulate these two hormones, ER-a and PR. Our results indicate that both ER-a and PR expression level are not affected by compound treatment compared to control. In conclusion, this study shows that a small molecule inhibitor of JNK has lead to regression of endometriosis possibly by modulating the expression levels of many important inflammatory cytokines, angiogenic factors and tissue remodeling enzymes. These results are also in concert with the current literature for potential therapeutic application of JNK inhibitors for immune diseases.