In Vitro Collagen Clearance of Ovarian Fibrosis Tissue With Purified Collagenase I

Abstract

economic negative impact. Current treatment options are primarily surgical and no US Food and Drug Administration approved medical treatment is available so far. Natural compounds may be beneficial for UFs patients as safe non-hormonal therapeutic option. Honokiol (HKL) is a natural compound that showed promising anti-fibrotic effects in several diseases via activation of Sirtuin3 (SIRT3), yet to be explored in UF.DESIGN: Cell culture study of immortalized human leiomyoma and patient-matched myometrium cells treated with honokiol and hexafluoro honokiol.MATERIALS AND METHODS: Human uterine leiomyoma (HuLM) and normal uterine smooth muscle (UTSM) cells were treated with (5mM-100mM) of HKL or its synthetic agonist hexafluoro honokiol (HEX), and cell proliferation was assessed by MTT assay after 24, 48 and 72 hr. Protein expression of several UF phenotype related markers were measured in both HKL or HEX treated HuLM (24 hr) and compared or untreated cells using western blot (WB) analysis, including fibrosis related markers {collagen type1 (COLL1A) and fibronectin (FN)}, proliferation markers {Cyclin D1 (CCND1) and (PCNA)}, tumor related marker (P21). Protein expression of Sirt3 was measured in matched UF and myometrium patients' tissues (n¼6) from African American (AA) and Caucasian (CC) patients. Studentt test was used for statistical analysis and p<0.05 was considered significant.RESULTS: HKL and HEX showed a potent anti-proliferative effect on HuLM cells in a concentration and time dependent manner (p<0.05), HKL inhibit HuLM cell growth significantly starting at 5 mM, while HEX showed inhibitory effect at higher dose (50 mM). Interestingly, normal UTSM cells showed resistant effects to both treatment as compared to HuLM at all used doses suggesting selective growth-inhibitory effect on HuLM not UTSM. HKL treatment at 20-50 mM for 24 hr. significantly decreased protein levels of COLL1A, CCND1 and PCNA while increased protein levels of FN1 in HuLM cells compared to untreated control in dose dependent manner (p<0.05). p21, Bax, and cleaved caspase-3 protein expression did not change in response to HKL treatment. Moreover, WB analysis showed that normal myometrial tissues from both AA and CC patients expressed higher Sirt3 protein expression as compared to its matched UF tissue (p<0.05), suggesting that Sirt3 activation using HKL might exert anti-UF effects.CONCLUSIONS: Our studies provide a novel link between Sirt3 and UF phenotype. HKL exhibited a promising anti-fibrotic effect via activation of this link. HKL might offer a promising therapeutic option as safer non-hormonal long term and cost-effective treatment against UFs with potential clinical utility, yet pending further research.