Discovery of novel trimethoxyphenylbenzo[d]oxazoles as dual tubulin/PDE4 inhibitors capable of inducing apoptosis at G2/M phase arrest in glioma and lung cancer cells.

To discover PDE4/tubulin dual inhibitors with novel skeleton structures, 7-trimethoxyphenylbenzo[d]oxazoles 4a-u and 4-trimethoxyphenylbenzo[d]oxazoles 5a-h were designed and synthesized by migrating the trimethoxyphenyl group of TH03 to the benzo[d]oxazole moiety. Among these compounds, approximately half of them displayed good antiproliferative activities against glioma (U251) and lung cancer (A549 and H460) cell lines. The structure-activity relationships of trimethoxyphenylbenzo[d]oxazoles led to the identification of 4r bearing indol-5-yl side-chain as a novel dual PDE4/tubulin inhibitor, which exhibited satisfactory antiproliferative activities against glioma (IC50 = 300 ± 50 nM) and lung cancer (average IC50 = 39.5 nM) cells. Further investigations revealed that 4r induced apoptosis at G2/M phase arrest and disrupted the microtubule network. The preliminary mechanism of action showed that 4r down-regulated the expression of cyclin B1 and its upstream regulator gene cdc25C in A549.

Ameliorative effect of acetylshikonin on cigarette smoke-induced lung inflammation in mice.

Cigarette smoke exposure is the major cause of chronic obstructive pulmonary disease (COPD). Acetylshikonin was the active principle component of Purple Gromwell that show anti-oxidative and anti-inflammatory effect. However, no data are available to elucidate the protective effect of acetylshikonin on COPD. Acetylshikonin could attenuate smoke-induced lung pathological changes, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and monocyte chemoattractant protein 1 (MCP-1) productions, and tissue damages caused by oxidative stress. Furthermore, acetylshikonin was found to enhance the expression of Nrf2 and Nur77-mediated COX-2 in vivo and in vitro.

Are estrogen-related drugs new alternatives for the management of osteoarthritis?

Osteoarthritis (OA) is a chronic degenerative disease involving multiple physiopathological mechanisms. The increased prevalence of OA after menopause and the presence of estrogen receptors in joint tissues suggest that estrogen could help prevent development of OA. This review summarizes OA research with a focus on the effects of estrogen and selective estrogen receptor modulators (SERMs). Preclinical studies and clinical trials of estrogen therapy have reported inconsistent results. However, almost all studies assessing SERM treatment have obtained more consistent and favorable effects in OA with a relatively safety and tolerability profiles. At present, some SERMs including raloxifene and bazedoxifene have been approved for the treatment of osteoporosis. In summary, estrogen-related agents may exert both a direct effect on subchondral bone and direct and/or indirect effects upon the surrounding tissues, including the articular cartilage, synovium, and muscle, to name a few. Estrogen and SERMs may be particularly favorable for postmenopausal patients with early-stage OA or osteoporotic OA, a phenotype defined by reduced bone mineral density related to high remodeling in subchondral bone. At present, no single drug exists that can prevent OA progression. Although estrogen-related drugs provide insight into the continued work in the field of OA drug administration, further research is required before SERMs can become therapeutic alternatives for OA treatment.

Protective effect of calcitonin on lumbar fusion-induced adjacent-segment disc degeneration in ovariectomized rat.

BACKGROUND: Intervertebral disc (IVD) degeneration and pathological changes in the spinal cord are major causes of back pain. In addition to its well-established anti-resorptive effect on bone, calcitonin (CT) potentially exerts protective effects on IVD degeneration in ovariectomized rats. However, possible therapeutic effects of CT on lumbar fusion-induced adjacent-segment disc degeneration (ASDD) have not been investigated yet. In this study, we examined the effects of CT on IVD degeneration adjacent to a lumbar fusion in ovariectomized rats. METHODS: Posterolateral lumbar fusion (PLF) at L4-5 was performed 4 weeks after ovariectomy (OVX) or sham surgery in female Sprague-Dawley rats. Following PLF + OVX, rats received either salmon CT (OVX + PLF + sCT, 16 IU/Kg/2d) or vehicle (OVX + PLF + V) treatment for 12 weeks; the remaining rats were divided into Sham + V, OVX + V, and PLF + V groups. Fusion status was analyzed by manual palpation and radiography. Adjacent segment disc was assessed by histological, histomorphometric, immunohistochemical analysis. L6 vertebrae microstructures were evaluated by micro-computed tomography. RESULTS: Histological analysis showed more severe ASDD occurred in OVX + PLF + V rats compared with the OVX + V or PLF + V groups. CT treatment suppressed the score for ASDD, increased disc height, and decreased the area of endplate calcification. Immunohistochemical staining demonstrated that CT decreased the expression of collagen type-I, matrix metalloproteinase-13, and a disintegrin and metalloproteinase with thrombospondin motifs-4, whereas it increased the expression of collagen type-II and aggrecan in the disc. Micro-computed tomography indicated that CT increased bone mass and improved the microstructure of the L6 vertebrae. CONCLUSIONS: These results suggest that CT can prevent ASDD, induce beneficial changes in IVD metabolism, and inhibit deterioration of the trabecular microarchitecture of vertebrae in osteoporotic rats with lumbar fusion.

Age dependent changes in cartilage matrix, subchondral bone mass, and estradiol levels in blood serum, in naturally occurring osteoarthritis in Guinea pigs.

The Dunkin Hartley (DH) guinea pig is a widely used naturally occurring osteoarthritis model. The aim of this study was to provide detailed evidence of age-related changes in articular cartilage, subchondral bone mineral density, and estradiol levels. We studied the female Dunkin Hartley guinea pigs at 1, 3, 6, 9, and 12 months of age (eight animals in each group). Histological analysis were used to identify degenerative cartilage and electron microscopy was performed to further observe the ultrastructure. Estradiol expression levels in serum were assessed, and matrix metalloproteinase 3 and glycosaminoglycan expression in cartilage was performed by immunohistochemistry. Bone mineral density of the tibia subchondral bone was measured using dual X-ray absorptiometry. Histological analysis showed that the degeneration of articular cartilage grew more severe with increasing age starting at 3 months, coupled with the loss of normal cells and an increase in degenerated cells. Serum estradiol levels increased with age from 1 to 6 months and thereafter remained stable from 6 to 12 months. Matrix metalloproteinase 3 expression in cartilage increased with age, but no significant difference was found in glycosaminoglycan expression between 1- and 3-month old animals. The bone mineral density of the tibia subchondral bone increased with age before reaching a stable value at 9 months of age. Age-related articular cartilage degeneration occurred in Dunkin Hartley guinea pigs beginning at 3 months of age, while no directly positive or negative correlation between osteoarthritis progression and estradiol serum level or subchondral bone mineral density was discovered.

Purification, crystallization and preliminary crystallographic analysis of the 23S rRNA methyltransferase RlmM (Cm2498) from Escherichia coli.

RlmM is an AdoMet-dependent methyltransferase that is responsible for 2'-O-methylation of C2498 in the peptidyl-transferase loop of bacterial 23S rRNA. This modification occurs before assembly of the 50S ribosomal subunit, and lack of C2498 methylation can cause a slight reduction in bacterial fitness. Here, the purification and crystallization of RlmM from Escherichia coli as well as its preliminary crystallographic analysis are presented. Cocrystallization of RlmM with AdoMet was carried out and X-ray diffraction data were collected to a resolution of 2.30 Å on beamline BL17U at the SSRF. However, electron density for AdoMet cannot be observed by comprehensive crystallographic analysis, indicating that it is not bound by RlmM during the cocrystallization process. The structure was solved by molecular replacement and refinement is in progress. The crystal contained one molecule in the asymmetric unit and belonged to space group P2(1), with unit-cell parameters a = 56.07, b = 59.38, c = 54.35 Å, ß = 94.84°, which differs from the P3(1) or P3(1)21 space groups of previously reported RlmM structures (PDB entries 4auk, 4atn and 4b17).

Alendronate retards the progression of lumbar intervertebral disc degeneration in ovariectomized rats.

OBJECTIVE: Increasing evidence has revealed a positive correlation between postmenopausal osteoporosis and intervertebral disc degeneration, the underlying mechanism of which might be associated with changes in the vertebral bone and endplate. Alendronate (ALN) can increase bone mass and improve the microstructure of osteoporotic vertebrae, which might be helpful in preserving disc morphology and mechanical properties. This study aims to investigate the effects of ALN on lumbar intervertebral disc degeneration related to osteoporosis using an ovariectomized (OVX) rat model. METHODS: Thirty female Sprague-Dawley rats aged 3 months were randomly divided into three groups (with 10 rats each) as follows: the Sham group underwent sham surgery; the OVX + ALN group had twice-a-week subcutaneous injections of ALN (15 µg/kg) for 6 months. The OVX + V group received an equivalent volume of saline solution as placebo post-OVX. After animals were sacrificed at 6 months post-OVX, the L3-6 spinal segments were harvested. Bone mineral density (BMD), micro-CT analysis and biomechanical testing were performed to evaluate the bone quality and microstructural changes in the lumbar vertebral bodies. Histological analysis with van Gieson stain and the histological score were used to identify the characteristics of the degenerative discs. The disc height and the thickness of the cartilage endplate were measured and compared. Immunohistochemistry and real-time PCR measurements for aggrecan, type I collagen, type II collagen, and matrix metalloprotease (MMP)-1, MMP-3 and MMP-13 expressions on the disc were performed to assess the underlying molecular signaling changes in matrix metabolism during intervertebral disc degeneration. RESULTS: The OVX + ALN group significantly maintained vertebrae BMD, percent bone volume and biomechanical strength, when compared with the OVX + V group. Histological evaluation suggests that there was no significant difference in disc height between the OVX + ALN and Sham groups, and ALN significantly prevented cartilage endplate thickening and the development of abnormal bony tissues within the cartilage endplate. The histological score in the OVX + ALN group was significantly lower than the OVX + V group, suggesting that ALN treatment was effective in delaying the process of the disc degeneration. The results of molecular analysis revealed a significant increase in aggrecan and type II collagen expressions, but marked reductions in MMP-1, MMP-3 and MMP-13 expressions at both the protein and mRNA levels in the OVX + ALN group. CONCLUSIONS: ALN can retard the progression of lumbar intervertebral disc degeneration in OVX rats. The underlying mechanisms might be related to preservation of the structural integrity and function of the adjacent structures, including the vertebrae and endplates, which further links with modulations in extracellular matrix metabolism to protect the disc from degeneration. These results suggest that ALN might be a promising drug agent for preventing lumbar intervertebral disc degeneration related to osteoporosis.

[Feasibility study of preserving intercostobrachial nerve during dissection for breast carcinoma: evidences from a preliminary pathologic exploration].

OBJECTIVE: To determine the feasibility of preservation of intercostobrachial nerve (ICBN) in breast cancer. METHODS: During June 2004 to June 2006, 99 patients with operable breast cancer receiving an axillary lymph node dissection at our department were analyzed. The extirpated ICBN and ambient tissues were tested by HE staining to observe the pathological changes. RESULTS: In 96 (96.97%) cases with ICBN sacrificing, the nerves were not violated microscopically and the nerve cells remained intact. Of 28 patients with axillary lymphadenectasis, only 3 cases (10.71%) were found to have tumor emboli in the peri-neural vessels. CONCLUSION: The preservation of ICBN is a feasible and safe technique. The operative approach should be advocated. If at all possible, a surgeon should identify ICBN and preserve it.

[Relevance of tumor angiogenesis in occurrence and development of breast cancer].

OBJECTIVE: To detect the differential expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) count in benign and malignant breast lesions to clarify the correlation of VEGF expression with the occurrence and progression of breast cancer and angiogenesis. METHODS: Immunohistochemistry (SP method) was used to examine the expression of VEGF and MVD count in 88 intra-operatively harvested samples of invasive ductal breast carcinoma, 25 samples of breast carcinoma in situ, 15 samples of atypical breast hyperplasia and 100 samples of benign breast lesions obtained. RESULTS: The positive rate of VEGF in invasive ductal breast carcinoma group was 70.5% and it was significantly higher than those of benign breast lesions, atypical breast hyperplasia and breast carcinoma in situ groups ( 22.0%, 33.3% and 56.0% respectively, P = 0.000). The positive rate of VEGF with lymph node metastasis was higher than that without lymph node metastasis (P = 0.015). The positive rate of VEGF in Stages II b- III was higher than that in Stages I - II a (P = 0.006). The positive rate of VEGF in C-erbB-2 positive group was higher than that in C-erbB-2 negative group (P = 0.016). An elevated expression of VEGF was observed as the tissue differentiation degree increased (P = 0.017). The MVD value of invasive ductal breast carcinoma group was 23 +/- 15 and it was significantly higher than those of benign breast lesions, atypical breast hyperplasia and breast carcinoma in situ groups (14 +/- 4, 18 +/- 4 and 20 +/- 6 respectively, P = 0.000). In invasive ductal breast carcinoma group, a significant higher MVD value was observed as the tissue differentiation degree increased (P = 0.006). The MVD value in VEGF positive group was higher than that in VEGF negative group (P = 0.000). In invasive ductal breast carcinoma, the MVD count increased significantly with the elevated expression of VEGF (P = 0.000). CONCLUSION: In invasive ductal breast carcinoma, angiogenesis and metastasis are mediated mainly by VEGF. The expressions of VEGF and MVD may be two of reference predictors for biological behaviors of breast carcinoma The occurrence and progression of breast cancer might be correlated with the expression of VEGF. The VEGF-targeting antiangiogenic therapy is expected to become a new therapeutic modality for C-erbB-2 positive patients.

Laparoscopic hepatojejunostomy for biliary atresia.

PURPOSE: The aim of this study was to evaluate the feasibility of laparoscopic hepatojejunostomy for types I and II biliary atresia (BA). MATERIALS AND METHODS: Between April 2003 and July 2007, 10 children with "correctable" types I and II BA were enrolled for the study. They presented with progressive jaundice, pale stools, and elevated aspartate transferase and alanine transferase levels. There were 6 girls and 4 boys, with ages ranging from 23 to 160 days (median,53). All BA had cysts of extrahaptic bile ducts. There were 6 type I and 4 were type II BA. The median diameter of the cysts was 1.5 cm (range, 1.0-1.8). All 10 children underwent laparoscopic cyst excision with Roux-en-Y hepatojejunostomy. Four trocars were inserted. The distal end of the cyst was resected.and a Rouxen-Y hepatojejunostomy was fashioned. The patients were followed up on median for 26 months (range, 4-51). RESULTS: The median duration of the operation was 3.0 hours (range, 2.4 - 3.2). There were no intraoperative complications. The blood loss was between 5 to 10 mL. Postoperatively, patients passed flatus after 18 hours(range, 16-28), and resumed oral intake in 20 hours (range, 16-30). Normal colored stools were passed after 3 days (range, 2-4). Jaundice started to disappear on postoperative day 10 (range, 7-16). In 6 cases, the total and the direct bilirubin were normalized on postoperative day 14-3 in 3 weeks. One patient had a persistent elevation of bilirubin. The postoperative course was uneventful in all patients. The median postoperable hospital stay was 7 days (range, 5-9). No postoperative complication was found at the follow-up visits. CONCLUSIONS: The laparoscopic Kasai' operation for children with type I or II biliary atresia is feasible, safe, and effective.

Total extrapleura sternal elevation for the correction of pectus excavatum in children.

PURPOSE: The aim of this study was to evaluate the efficacy and safety of the thoracoscopic total extrapleural approach of the Nuss procedure for the correction of pectus excavatum in children. MATERIALS AND METHODS: Under thoracoscopic guidance, an extrapleural tunnel was created by using a blunt dissector via a right thoracic incision. A steel bar was inserted in the entirely extrapleural tunnel. The bar was turned and fixed as in the standard Nuss procedure. RESULTS: The operations were completed successfully in all patients. The operating time ranged from 35 to 50 minutes (median, 45). The intraoperative blood loss was 2 to 3 mL. There was no pneumothorax or hydrothoraxin our series. All patients were followed up for 2-6 months, and the surgical outcomes were excellent. CONCLUSIONS: The extrapleura Nuss procedure under thoracoscopic guidance is a safe and less traumatic procedure for the correction of pectus excavatum.

[Expression of vascular endothelial growth factor in different breast tissues and clinical significance thereof].

OBJECTIVE: To investigate the differences in the expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) count in breast benign affection, breast atypical hyperplasia, and breast carcinoma in situ and to clarify the association of VEGF expression and MVD with the clinicopathological features of these diseases. METHODS: Immunohistochemistry (SP-method) was used to examine the expression of VEGF and MVD count in 100 samples of breast benign affection (including 35 cases of breast fibroid tumor, 35 cases of breast cystic hyperplasia, and 30 cases of intraductal papilloma), and 15 samples of breast atypical hyperplasia, and 25 samples of breast carcinoma in situ, obtained during operation. RESULTS: The positive rate of VEGF of the breast carcinoma in situ group was 56% , significantly higher than hose of the breast benign affection and breast atypical hyperplasia groups (22% and 33% respectively, P < 0.05). However, there was no significant differences in positive rate of VEGF among breast fibroid tumor, breast cystic hyperplasia, and intraductal papilloma (all P > 0.05). The MVD value of the breast carcinoma in situ group was 20.1 +/- 6.1, significantly higher than those of the breast benign affection group and breast atypical hyperplasia groups (14.3 +/- 3. 5 and 18.5 +/- 3.6 respectively, both P < 0.05). There was no significant differences in MVD value among breast fibroid tumor, breast cystic hyperplasia, and intraductal papilloma (all P > 0.05). CONCLUSION: In breast tumors, angiogenesis is probably mediated mainly by VEGF. The occurrence and progression of breast cancer may be related with the expression of VEGF.

[Investigation on the alteration of hepatitis B virus (HBV) markers in liver allograft of HBV related recipients in perioperative period].

OBJECTIVE: To investigate the alteration of HBV markers in liver allograft of HBV related recipients pre and post liver transplantation under Lamivudine or combination of Lamivudine with HBIG prophylaxis and explore the mechanism of HBV de nova infection in liver allograft after orthotopic liver transplantation, as well as seek to establish a optimal prophylactic protocol. METHODS: The serial liver biopsy specimens of 90 liver allograft and sera of 78 liver transplant recipients during operation and after 1 week, 1 month, 3 months, 6 months, 12 months, 24 months post transplantation have been collected and detected for HBV markers with enzyme-linked radioimmunoassay, fluorescent quantitative assay for HBV-DNA in serology and with immunohistochemistry stain, HBV-DNA in situ hybridization in histology for detection of HBV markers in liver allograft samples. RESULTS: Whether recipients with active replicative or inactive replicative HBV preoperatively, none of positive HBV-DNA, HBsAg and HBcAg in 100% liver biopsy specimens with HBV-DNA hybridization in situ and immunohistochemistry stains in histology within 2 hours after reperfusion. CONCLUSION: Whatever HBV replicative status the recipients have before surgery, no evidence of HBV particles direct invasion to the liver allograft from HBV related cirrhotics during operation under current prophylactic measures. However, the further supposed mechanism and its significance in HBV de nova infection of liver allograft remained to be disclosed further.