Ming Ping Wu

Anti-angiogenesis mediated by angiostatin K1-3, K1-4 and K1-4.5. Involvement of p53, FasL, AKT and mRNA deregulation

The molecular mechanism mediated by multiple forms of angiostatin via acting on proliferating vascular endothelium remains elusive. To address whether three forms of angiostatin, K1-3, K1-4 or K1-4.5, utilized similar or distinct pathways to mediate anti-angiogenesis, we adopted an adenoviral expression system to express secretable angiostatin molecules for CM collection. The anti-angiogenic activity of K1-3, K1-4 or K1-4.5 was confirmed by using proliferation, migration, tube formation and apoptotic assays of human endothelial cells. These angiostatin molecules at comparable expression level inhibited various in vitro angiogenesis assays with some variations. Furthermore, K1-3, K1-4 or K1-4.5 increased the expression of p53 protein and its downstream effectors, enhanced FasL-mediated signaling pathways, and decreased activation of AKT. At least three different receptors, Fas, integrin alpha(v)beta3 and ATP synthase, were involved in the anti-angiogenic action of angiostatin molecules. Besides, the expression of 189 genes at mRNA level was significantly altered by K1-3, K1-4 or K1-4.5. More than 70% of these genes participate in growth, inflammation, apoptosis, migration and extracellular matrix. Taken together, K1-3, K1-4 and K1-4.5, regardless of the number of kringles in the angiostatin molecules, mediated anti-angiogenesis via mostly similar pathways. We are the first to demonstrate the involvement of DAPK1 in the mediation of anti-angiogenesis by angiostatin.

Thrombospondin-1 acts as a fence to inhibit angiogenesis that occurs during cervical carcinogenesis.

PURPOSEThe acquisition of an angiogenic phenotype (angiogenic switch) is essential for cervical carcinogenesis. This study was aimed to examine the spatial and temporal relationship of thrombospondin-1 (TSP-1) expression in patients with precursor lesions and squamous cell carcinoma of uterine cervix and to correlate its expression with tumor angiogenesis. PATIENTS AND METHODSTSP-1 expression and microvessel density were assessed by immunohistochemistry in samples obtained from patients with pathological diagnoses of cervical intraepithelial neoplasm I, carcinoma in situ, invasive squamous cell carcinoma (SCC), and benign disease (N = 12 from each group). Two representative blocks that contained serial changes of cervical lesions from these 48 subjects were examined, and the pathological findings were categorized into the four groups of (1) normal cervical epithelia, (2) low-grade squamous intraepithelial lesions (LSILs), (3) high-grade SILs (HSILs), and (4) SCC. RESULTSA total of 120 foci with various cervical lesions from 98 slides were examined and classified into normal (48), LSIL (36), HSIL (24), and SCC epithelium (12). Immunohistochemical studies showed that TSP-1 was mainly localized at the basal epithelial cells, and we named it as the “TSP-1 fence.” The mean microvessel density counts and TSP-1 scores for normal, LSIL, HSIL, and SCC epithelium were 7.3 ± 2.9, 9.9 ± 3.4, 17.7 ± 5.1, and 22.8 ± 8.6, and 3.8 ± 0.4, 3.8 ± 0.4, 1.8 ± 0.4, and 1.5 ± 0.5, respectively. The TSP-1 intensities were significantly higher and the MVD counts lower in the groups of normal and LSIL epithelium than in those with HSIL and SCC epithelium. In addition, microvessel density count was negatively associated with the intensity of TSP-1. DISCUSSIONOur results indicate that the disruption of TSP-1 fence and the switch to angiogenic phenotype occurred during the transition from LSIL into HSIL. This concordance suggests that TSP-1 plays a role in the regulation of angiogenic switch. We conclude that the onset of angiogenesis is an early event in cervical carcinogenesis due, in part, to the down-regulation of TSP-1 by the dysplastic epithelium.

Multitarget therapy of malignant cancers by the head-to-tail tandem array multiple shRNAs expression system

Coexpression of multiple shRNAs can simultaneously inhibit multiple genes or target multiple sites on a single gene. These approaches can be used for dissecting complex signaling pathways and even be applied to targeting multiple genes in cancer therapy. Here we established a simple and efficient multiple shRNAs expression system based on pSUPER, the most popular expression vector in mammalian cells. A series of head-to-tail tandem array multiple shRNAs expression vectors were constructed containing different combinations of six shRNA expression cassettes targeting genes involved in cell proliferation and survival pathways: Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB. In HeLa and HEK293 cells, the multiple shRNAs expression constructs could efficiently and simultaneously induce inhibition of all six genes. We further evaluated the inhibition effects of the multiple shRNAs expression vectors on the human prostate cancer cell line PC3, which contains different cell variants with distinct oncogenic signaling alterations. The results revealed that the multiple shRNAs expression system could inhibit all six genes and was much more efficient in inducing apoptosis in the PC3 cells. Our results suggest that the multitarget shRNAs expression system could be an effective strategy in cancer therapy and be applied to any other DNA vector-based shRNA expression system.

A novel role of thrombospondin-1 in cervical carcinogenesis: Inhibit stroma reaction by inhibiting activated fibroblasts from invading cancer

Thrombospondin (TSP)-1, a potent angiogenesis inhibitor, has been shown to exert different biological functions on various cell types. Here, we investigate the role of TSP-1 in tumor-stroma reaction, which is mainly characterized by fibroblast activation to create a permissive microenvironment for tumor progression. Immunohistochemistry examinations in the human surgical specimens have shown that a downregulation of TSP-1 during the progression of cervical carcinogenesis was accompanied by an emergence in the upregulation of stroma markers, alpha-smooth muscle actin (alpha-SMA) and desmin. Transfection of SiHa cervical cancer cells with a plasmid expressing the TSP-1 protein exhibited antiangiogenic activity in vitro and resulted in reduced tumor growth in severe combined immunodeficiency (SCID) mice, which was accompanied by a decrease in tumor vascularization and lower expressions of alpha-SMA and desmin than those in the vector controls. Transfection with TSP-1 and purified TSP-1 added to NIH3T3 cells did not alter the protein levels of alpha-SMA and desmin but significantly inhibited matrix metalloprotease-2 activity. Transforming growth factor-beta (TGF-beta), a major factor in the activation of fibroblasts, increased alpha-SMA and desmin expression and the ability of cell migration and invasion in NIH3T3 cells. The increased migration ability and the invasive ability into tumor cluster of TGF-beta-treated NIH3T3 cells were dose dependently inhibited by TSP-1. In contrast, ectopic TSP-1 expression in SiHa cells has little effect on the invasive ability of the NIH3T3 cells. Together, our findings demonstrate a novel role of TSP-1 to inhibit tumor-stroma reaction that could be attributed to the blockage of activated fibroblasts from invading cancer cells.

The use of prostheses in pelvic reconstructive surgery: joy or toy?

The high recurrence rate of pelvic organ prolapse (POP) of up to 30% after pelvic reconstructive surgery makes a more refined surgery imperative, as well as the need for either biological or synthetic prostheses as adjuvant treatment. Patients with recurrence risks may benefit from the adjuvant treatment: (1) to substitute for the lack of supportive tissue; (2) to reinforce inadequate tissue; (3) to induce new supportive tissue; and (4) to consolidate and complement the insufficient surgical techniques. However, some debatable issues in use of the prosthetics remain. The use of prosthetics enables the simultaneous repair of all vaginal defects of POP and concomitant anti-incontinence surgery to be faster, easier and more precise. Nevertheless, great care should be devoted to the actual and theoretical short- and long-term risks, many of which have not been fully elucidated. Despite the lack of various ideal characteristics, the type I monofilament, macroporous polypropylene, has been suggested to have the lowest incidence of infection and erosion among the nonabsorbable prostheses. There is good evidence to support the use of nonabsorbable synthetic mesh for abdominal sacrocolpopexy, while the use of prostheses for repairing isolated anterior and posterior compartment defects remains controversial. There have been no long-term studies with sufficient patient numbers to prove whether synthetic or biological prostheses are superior during vaginal surgery. Tension-free vaginal mesh techniques with procedural kits are being adopted increasingly, despite the paucity of data. Although short-term follow-up studies have shown tension-free vaginal mesh to be a safe and effective technique to correct POP, anatomic and functional results of long-term follow-up studies, however, have not yet confirmed the effectiveness and safety. Mesh erosion remains a concern, with variable rates according to different materials and approaches. Newly developed prostheses offer an alternative option to pelvic reconstructive surgery. However, some questions remain: (1) Should prostheses be considered for primary repairs, secondary repairs, or solely in patients with risk factors for recurrence? (2) Which prosthetic material is better: synthetic or biological ones; absorbable or nonabsorbable ones? (3) Do the benefits of prosthetics in pelvic reconstructive surgery outweigh the risks of complications? These questions are explored and reports in the literature reviewed.

Concomitant trocar-guided transvaginal mesh surgery with a midurethral sling in treating advanced pelvic organ prolapse associated with stress or occult stress urinary incontinence

OBJECTIVE The purpose of this study was to evaluate the efficacy and feasibility of concomitant trocar-guided transvaginal mesh (TVM) surgery with a midurethral sling (MUS) for treating women with advanced pelvic organ prolapse (POP) and stress urinary incontinence (SUI) or occult SUI (OSUI). MATERIALS AND METHODS Eighty-nine women with advanced POP and SUI or OSUI were retrospectively enrolled. The Total Prolift and Tension-free Vaginal Tape-Obturator Systems were used for trocar-guided TVM surgery and MUS. Patients received regular follow-up at 1 week, and 1 month, 3 months, 6 months, and 12 months postoperatively, and then annually thereafter. The endpoints were the success rate for POP, and perioperative and postoperative complications. Functional outcomes were the presence of voiding difficulty, persistent or de novo overactive bladder symptoms, postoperative SUI, and paresthesia. RESULTS The median follow-up period was 35 months (range, 12-50 months). Within the follow-up period, 84 patients (94.4%) were objectively cured, five patients (5.6%) had vaginal apical mesh exposure, 29 individuals (32.6%) had persistent or de novo overactive bladder symptoms, six individuals (22.5%) had de novo SUI (two were found by urodynamics), and nine individuals (10.1%) had voiding difficulties (two were found by urodynamics). In addition, the vaginal hysterectomy group had greater blood loss, longer operation times, and a higher mesh erosion rate compared to the uterine suspension group. CONCLUSION Concomitant trocar-guided TVM surgery and MUS with the use of total Prolift and Tension-free Vaginal Tape-Obturator offer good efficacy in treating women with advanced POP and SUI or OSUI. The vaginal hysterectomy group had more perioperative complications.

Surgical trends for benign ovarian tumors among hospitals of different accreditation levels: An 11-year nationwide population-based descriptive study in Taiwan

OBJECTIVE Using a population-based nationwide database, we describe the changing surgical trends for laparoscopy or laparotomy for benign ovarian tumors among hospitals of different accreditation levels in Taiwan (medical centers, regional hospitals, and local hospitals). MATERIALS AND METHODS Women who had National Health Insurance and received either laparoscopy or laparotomy as the primary surgery for benign ovarian tumors in Taiwan during 1999-2009 were identified for analysis. RESULTS In total, 135,793 women who received either laparotomic (39,779) or laparoscopic surgery (96,014) for benign ovarian pathology were identified. The increase in annual laparoscopy number from 7176 in 1999 to 11,046 in 2009 was significant according to a log-linear regression test (p < 0.0001). The decrease in laparotomies from 3845 to 3567 was not significant (p = 0.190). Service volume shifts from local hospitals to regional hospitals were noted, with a concomitant decrease in the numbers of local hospitals. Laparoscopy was used more often than laparotomy among all three hospital accreditation levels. An increasing trend for choosing laparoscopy was observed for medical centers and local hospitals (p < 0.0001), but not regional hospitals (p = 0.0745). Laparoscopy was used more often in younger patients, by younger surgeons, and by male surgeons among hospitals at all three accreditation levels. CONCLUSION Laparoscopy was preferentially used over laparotomy at all three hospital levels. An increasing trend for choosing laparoscopy was observed for medical centers and local hospitals, but not regional hospitals. Service volume shifts from local hospitals to regional hospitals were noted. Use of laparoscopy differed according to patient age, surgeon age, and surgeon gender among different hospital levels.

Angiostatin K1-3 induces E-selectin via AP1 and Ets1: A mediator for anti-angiogenic action of K1-3

Summary.  Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1‐3 included. We previously showed that K1‐3 was the most potent angiostatin to induce E‐selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1‐3‐induced E‐selectin expression and investigate the role of E‐selectin in the anti‐angiogenic action of K1‐3. Methods and results: Quantitative real time RT‐PCR and Western blotting analyses confirmed a time‐dependent increase of E‐selectin mRNA and protein induced by K1‐3. Subcellular fractionation and immunofluorescence microscopy showed the co‐localization of K1‐3‐induced E‐selectin with caveolin 1 (Cav1) in lipid rafts in which E‐selectin may behave as a signaling receptor. Promoter‐driven reporter assays and site‐directed mutagenesis showed that K1‐3 induced E‐selectin expression via promoter activation and AP1 and Ets‐1 binding sites in the proximal E‐selectin promoter were required for E‐selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1‐3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E‐selectin by K1‐3. A modulatory role of E‐selectin in the anti‐angiogenic action of K1‐3 was manifested by both overexpression and knockdown of E‐selectin followed by cell proliferation assay. Conclusions: We show that K1‐3 induced E‐selectin expression via AP1 and Ets‐1 binding to the proximal E‐selectin promoter (−356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E‐selectin as a novel target for the anti‐angiogenic therapy.