Ensnaring membrane type 1-matrix metalloproteinase (MT1-MMP) with tissue inhibitor of metalloproteinase (TIMP)-2 using the haemopexin domain of the protease as a carrier: a targeted approach in cancer inhibition.

Metastatic cancer cells express Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) to degrade the extracellular matrix in order to facilitate migration and proliferation. Tissue Inhibitor of Metalloproteinase (TIMP)-2 is the endogenous inhibitor of the MMP. Here, we describe a novel and highly effective fusion strategy to enhance the delivery of TIMP-2 to MT1-MMP. We can reveal that TIMP-2 fused to the haemopexin +/- transmembrane domains of MT1-MMP (two chimeras named T2PEX+TM and T2PEX) are able to interact with MT1-MMP on the cell surface as well as intracellularly. In the case of T2PEX+TM, there is even a clear sign of MT1-MMP:T2PEX+TM aggregation by the side of the nucleus to form aggresomes. In vitro, T2PEX+TM and T2PEX suppress the gelatinolytic and invasive abilities of cervical carcinoma (HeLa) and HT1080 fibrosarcoma cancer cells significantly better than wild type TIMP-2. In mouse xenograft, we further demonstrate that T2PEX diminishes cervical carcinoma growth by 85% relative to the control. Collectively, our findings indicate the effectiveness of the fusion strategy as a potential targeted approach in cancer inhibition.

ADAMTS13 and 15 are not regulated by the full length and N-terminal domain forms of TIMP-1, -2, -3 and -4.

A disintegrin and metalloproteinase with thombospondin motifs (ADAMTS) 13 and 15 are secreted zinc proteinases involved in the turnover of von Willebrand factor and cancer suppression. In the present study, ADAMTS13 and 15 were subjected to inhibition studies with the full-length and N-terminal domain forms of tissue inhibitor of metalloproteinases (TIMPs)-1 to -4. TIMPs have no ability to inhibit the ADAMTS proteinases in the full-length or N-terminal domain form. While ADAMTS13 is also not sensitive to the hydroxamate inhibitors, batimastat and ilomastat, ADAMTS15 can be effectively inhibited by batimastat (Kiapp 299 nM). In conclusion, the present results indicate that TIMPs are not the regulators of these two ADAMTS proteinases.

Expanding the Activity of Tissue Inhibitors of Metalloproteinase (TIMP)-1 against Surface-Anchored Metalloproteinases by the Replacement of Its C-Terminal Domain: Implications for Anti-Cancer Effects.

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). TIMP molecules are made up of two domains: an N-terminal domain that associates with the catalytic cleft of the metalloproteinases (MP) and a smaller C-terminal domain whose role in MP association is still poorly understood. This work is aimed at investigating the role of the C-terminal domain in MP selectivity. In this study, we replaced the C-terminal domain of TIMP-1 with those of TIMP-2, -3 and -4 to create a series of "T1:TX" chimeras. The affinity of the chimeras against ADAM10, ADAM17, MMP14 and MMP19 was investigated. We can show that replacement of the C-terminal domain by those of other TIMPs dramatically increased the affinity of TIMP-1 for some MPs. Furthermore, the chimeras were able to suppress TNF-α and HB-EGF shedding in cell-based setting. Unlike TIMP-1, T1:TX chimeras had no growth-promoting activity. Instead, the chimeras were able to inhibit cell migration and development in several cancer cell lines. Our findings have broadened the prospect of TIMPs as cancer therapeutics. The approach could form the basis of a new strategy for future TIMP engineering.

Expression Levels of Myostatin and Matrix Metalloproteinase 14 mRNAs in Uterine Leiomyoma are Correlated With Dysmenorrhea.

Uterine leiomyoma is the most common benign neoplasm of female reproductive system, found in about 50% of women in reproductive age. The mechanisms of leiomyoma growth include cell proliferation, which is modulated by growth factors, and deposition of extracellular matrix (ECM). Activin A and myostatin are growth factors that play a role in proliferation of leiomyoma cells. Matrix metalloproteinases (MMPs) are known for their ability to remodel the ECM in different biological systems. The aim of this study was to evaluate the expression levels of activin ßA-subunit, myostatin, and MMP14 messenger RNAs (mRNAs) in uterine leiomyomas and the possible correlation of these factors with clinical features of the disease. Matrix metalloproteinase 14 was highly expressed in uterine leiomyoma and correlated with myostatin and activin A mRNA expression. Moreover, MMP14 and myostatin mRNA expression correlated significantly and directly with the intensity of dysmenorrhea. Overall, the present findings showed that MMP14 mRNA is highly expressed in uterine leiomyoma, where it correlates with the molecular expression of growth factors and is further increased in cases of intense dysmenorrhea.

Increased progesterone receptor expression in uterine leiomyoma: correlation with age, number of leiomyomas, and clinical symptoms.

OBJECTIVE: To investigate the possible correlation between progesterone receptor (PR) expression in uterine leiomyoma or adjacent myometrium and patient's age, size/number of leiomyomas, or clinical symptoms such as dysmenorrhea, acyclic pelvic pain, or menstrual and intermenstrual uterine bleeding. DESIGN: Cross-sectional study. SETTING: Referral center. PATIENT(S): Sixty-two Chinese women undergoing elective hysterectomy for uterine leiomyomata. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Evaluation of PR-total and PR-B mRNA with real-time polymerase chain reaction; PR-A and PR-B proteins quantified by Western blot in leiomyoma tissue and myometrium; symptoms rated by the patients using visual analog scores. RESULT(S): The PR-B mRNA and PR-A and PR-B proteins were more concentrated in leiomyomas than in matched myometrium. A direct correlation between PR-B mRNA levels in leiomyoma and age (r = 0.347) and number of tumors (r = 0.295) was found. Conversely, there was an inverse correlation between PR-B mRNA levels in leiomyoma and dysmenorrhea (r = -0.260) and intermenstrual bleeding (r = -0.266). Multiple regression analysis indicated that age (ß = 0.363) and the number of myomas (ß = 0.296) were independently associated with PR-B mRNA levels in leiomyoma tissue. CONCLUSION(S): The levels of PR-B mRNA in leiomyoma tissue are directly associated with the number of tumors and inversely correlated with the intensity of intermenstrual bleeding and dysmenorrhea, suggesting that PR signaling may favor leiomyoma growth while attenuating clinical symptoms. This duality should be taken into account in the clinical management of patients with symptomatic uterine leiomyoma.

Inhibins as diagnostic markers in human reproduction.

Over the past 75 years, many publications have focused on measurement of inhibin concentration and/or activity in biological samples in order to understand its role in physiology and disease. This chapter highlights the accomplishments within this area of research over the past decade including development of specific inhibin assays. Inhibin A is a marker of dominant follicle and corpus luteum activity and decreases in polycystic ovary syndrome (PCOS). Inhibin A increases in gestational diseases such as pre-eclampsia and fetal Down's syndrome, and this increase in inhibin A improves early diagnosis of both conditions. The measurement of inhibin A in women with threatened abortion provides useful information about the likelihood of pregnancy loss. Inhibin B increases markedly in women with granulosa cell tumor and appears closely related to gametogenesis in men, that is, reflecting Sertoli cell activity. On the contrary, Inhibin B decreases in women with declining ovarian function and correlates with female response to ovulation induction. This review evaluates the biochemical significance ofinhibins including their use in clinical practice.

High serum concentration of total inhibin in polycystic ovary syndrome.

OBJECTIVE: To evaluate serum total inhibin (the sum of precursors, subunits, and mature molecules of inhibin) along with inhibin A and inhibin B in a large sample of women with polycystic ovary syndrome (PCOS) and to investigate whether these parameters differ between lean and overweight PCOS patients. DESIGN: Cross-sectional, controlled study. SETTING: Academic health centers in Siena and Bologna, Italy. PATIENT(S): A group of women with PCOS (n = 145) was divided according to body mass index (BMI) into "lean PCOS" (BMI 19-25 kg/m(2), n = 52) and "overweight PCOS" (BMI 26-51 kg/m(2), n = 93). A group of healthy women (n = 90) with BMI 19-24 kg/m(2) and a history of regular menstrual cycles and normal results on physical examination served as controls. INTERVENTION(S): Blood samples were drawn between cycle days 3 and 5 in PCOS patients and controls. MAIN OUTCOME MEASURE(S): Serum concentrations of total inhibin, inhibin A, inhibin B, FSH, and LH were measured using commercially available immunoassays. RESULT(S): Women with PCOS had serum total inhibin levels twice as high as in the control group (median = 115 vs. 47 pg/mL). Dimeric inhibin A concentration was lower in the PCOS group compared with controls (10 vs. 25 pg/mL), whereas inhibin B concentration did not differ significantly between PCOS and control groups. There was no difference of either total inhibin or inhibin A levels between lean and overweight women with PCOS, whereas the inhibin B level was higher in the lean PCOS subgroup compared with the overweight PCOS subgroup. Total inhibin did not show any linear correlation with inhibin B, inhibin A, or serum gonadotropins. CONCLUSION(S): Women with PCOS have high serum concentration of total inhibin but not of inhibin A or inhibin B, thus suggesting that PCOS women have an impaired processing of alpha-inhibin precursor proteins.

High serum inhibin concentration discriminates autoimmune oophoritis from other forms of primary ovarian insufficiency.

CONTEXT: Primary ovarian insufficiency (POI) is defined by hypergonadotropic amenorrhea occurring before the age of 40 yr. In 4-5% of women with POI, an ovarian autoimmune process can be demonstrated. DESIGN: We have determined the serum concentrations of total inhibin and inhibin B by sensitive ELISAs in 22 women with autoimmune POI (aPOI), 71 women with non-autoimmune idiopathic POI (iPOI), 77 postmenopausal women, and 90 healthy, fertile women (HW). Diagnosis of aPOI was made according to the presence of steroid cell autoantibodies and/or 17alpha-hydroxylase autoantibodies and/or cytochrome P450 side-chain cleavage autoantibodies. All aPOI patients were also positive for adrenal autoantibodies. RESULTS: Total inhibin levels were significantly higher in women with aPOI (median, 281 pg/ml) than in women with iPOI (median, 74 pg/ml) or HW (median, 133.5 pg/ml) (P < 0.001). Levels of inhibin B were also significantly higher in women with aPOI (median, 109 pg/ml) than in women with iPOI (median, 18 pg/ml) (P < 0.001) or HW (median, 39 pg/ml) (P < 0.05). Serum concentrations of total inhibin and inhibin B were significantly higher in women with POI than in postmenopausal women (P < 0.001), irrespective of the presence/absence of autoantibodies. At receiver-operating characteristic analysis, cutoff values of 133 pg/ml for total inhibin and 60.5 pg/ml for inhibin B ensured 86.4% sensitivity and 81-84.5% specificity for aPOI vs. iPOI. CONCLUSIONS: We conclude that a variable degree of ovarian function is preserved in women with POI and that aPOI is characterized by increased inhibin production resulting from a selective theca cell destruction, with initial preservation of granulosa cells.

Activin A and follistatin in menstrual blood: low concentrations in women with dysfunctional uterine bleeding.

Activin A and follistatin are growth factors produced by several organs, comprising the endometrium, where they modulate cell and tissue differentiation. In this study, the authors tested whether activin A and follistatin are measurable in menstrual blood and whether their concentrations change in women with dysfunctional uterine bleeding (DUB). The authors evaluated healthy women with regular menstrual cycles (n = 15) and women with DUB (n = 12). Activin A and follistatin were measured in both menstrual and peripheral blood samples using highly sensitive enzyme immunoassays, whereas their respective mRNAs were quantified by real-time polymerase chain reaction in endometrial samples collected during the perimenstrual period. Activin A concentrations were 4-fold higher in menstrual than in peripheral serum of healthy women (mean +/- SE, 4.24 +/- 0.18 vs 1.00 +/- 0.15 ng/mL, P < .001) and were significantly lower in women with DUB compared to healthy subjects (P < .001). Follistatin concentration was 8-fold higher in menstrual than in peripheral serum of healthy women (3.94 +/- 0.49 vs 0.49 +/- 0.04 ng/mL, P < .001) and was significantly lower in the menstrual serum of women with DUB compared to controls (P < .001). There was no correlation between menstrual and peripheral serum concentrations of both proteins. The endometrial expression of activin A and follistatin mRNA was lower in women with DUB compared to controls (P < .05). Both activin A and follistatin are measurable in high concentrations in human menstrual blood and are relatively lower in women with DUB. The quantitative assessment of activin A and follistatin in menstrual serum might be a putative clinical marker of endometrial function.

Total inhibin is a potential serum marker for epithelial ovarian cancer.

CONTEXT: Total inhibin is the sum of precursors, subunits, and mature molecules of inhibin, which the normal ovary nearly stops to produce after menopause, whereas ovarian tumors still release. OBJECTIVE: The aim of the present study was to evaluate whether the serum concentration of total inhibin has the sensitivity/specificity characteristics to become a diagnostic test for epithelial ovarian cancer in postmenopausal women. DESIGN: This was a controlled, cross-sectional study. SETTING: The study was conducted at the University of Siena. PATIENTS: Blood specimens were collected from postmenopausal women with: 1) epithelial ovarian cancer, stage II-III (n = 89); 2) benign ovarian tumors (n = 25); 3) breast (n = 10), colon (n = 10), and stomach (n = 10) cancers; and 4) controls (n = 95). In the group of women with epithelial ovarian cancer, blood specimens were also collected after surgical removal of the tumor. In four cases of women with stage IIC mucinous tumor, blood specimens were collected during the follow-up time. INTERVENTION: Total inhibin was measured by a new double-antibody ELISA. RESULTS: Women with epithelial ovarian cancers showed serum total inhibin levels significantly higher than those with benign tumor or with nonovarian tumors or controls (P < 0.001). Patients with serous (n = 40) or mucinous tumors (n = 17) showed the highest total inhibin levels (P < 0.001). At 95% specificity, the total inhibin assay detected 37 of 40 (93%) serous tumors and 16 of 17 (94%) mucinous tumors. When total inhibin was combined with CA-125, all cases of serous and mucinous tumors were detected, and the overall sensitivity for epithelial ovarian cancers was 99% at 95% specificity. A significant decrease of total inhibin levels was shown in women with serous and mucinous carcinoma as result of surgery (P < 0.001). In the four women who were followed up, recurrence was associated to an increase of total inhibin levels. CONCLUSIONS: The present data show that total inhibin is a sensitive and specific marker of epithelial ovarian cancers in postmenopausal women. Total inhibin may therefore be combined with CA-125 for noninvasive diagnosis of epithelial ovarian cancer and may also be a useful serum marker to monitor disease-free intervals.