Effects of paclitaxel on cultured synovial cells from patients with rheumatoid arthritis

BACKGROUND: Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis (RA). Using paclitaxel, a unique antineoplastic agent known to suppress collagen-induced arthritis, we conducted an in vitro study of cell kinetics on cultured synovial cells from patients with RA. METHODS: Alterations of the cell cycle of cultured fibroblast-like synovial cells (FLSs) from patients with RA were studied using flow cytometry and laser scanning cytometry. Apoptosis and accumulation of cyclin concerning effects of paclitaxel were detected. RESULTS: Paclitaxel induced arrest of the cell cycle at G2/M phase and apoptosis in FLSs. The late stage of apoptosis was determined by the positivity of terminal deoxynucleotidyl transferase assay. Morphological observation by combined usage of both annexin V and propidium iodide on FLSs on a slide glass showed early apoptotic changes in detail. FLSs arrested at G2/M phase showed marked accumulation of cyclin B1. The effects of paclitaxel decreased on FLSs, which diminished proliferative activity. CONCLUSIONS: These data indicate that paclitaxel induces cell arrest at G2/M phase followed by apoptosis in human FLSs, which have high proliferative activity, and possible therapeutic effects of paclitaxel on RA.     

Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL-induced activation of NF-κB and ERK

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.     

Fluorescent microplate assay for cancer cell-associated cathepsin B.

Cathepsin B and in particular cell-surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.     

Coating of poly(p-xylylene) by PLA-PEO-PLA triblock copolymers with excellent polymer-polymer adhesion for stent applications

Poly(p-xylylene) (PPX) was deposited by chemical vapor deposition (CVD) on stainless steel substrates. These PPX films were coated by solution casting of poly(lactide)-poly(ethylene oxide)-poly(lactide) triblock copolymers (PLA-PEO-PLA) loaded with 14C-labeled paclitaxel. Adhesion of PLA-PEO-PLA on PPX substrate coatings was measured using the blister test method. Excellent adhesion of the block copolymers on PPX substrates was found. Stress behavior and film integrity of PLA-PEO-PLA was compared to pure PLA on unexpanded and expanded stent bodies and was found to be superior for the block copolymers. The release of paclitaxel from the biodegradable coatings was studied under physiological conditions using the scintillation counter method. Burst release of paclitaxel was observed from PLA-PEO-PLA layers regardless of composition, but an increase in paclitaxel loading was observed with increasing content of PEO.     

CTGF increases drug resistance to paclitaxel by upregulating survivin expression in human osteosarcoma cells

Osteosarcoma is the most common primary malignant tumor, and its treatments require more effective therapeutic approaches. Paclitaxel has a broad range of antitumor activities, including apoptosis-inducing effects. However, the majority of tumors in patients with advanced cancer eventually develop chemoresistance. Connective tissue growth factor (CTGF) is a secreted protein that modulates the invasiveness of certain human cancer cells by binding to integrins. However, the effect of CTGF in paclitaxel-mediated chemotherapy is unknown. Here, we report that the expression of CTGF in osteosarcoma patients was significantly higher than that of the CTGF expression in normal bone tissues. Overexpression of CTGF increased the resistance to paclitaxel-mediated cell apoptosis. In contrast, knockdown of CTGF expression by CTGF shRNA increased the chemotherapeutic effect of paclitaxel. In addition, CTGF increased resistance to paclitaxel-induced apoptosis through upregulation of survivin expression. Moreover, the AMP-activated protein kinase (AMPK)-dependent nuclear factor kappa B (NF-κB) pathway mediated paclitaxel-increased chemoresistance and survivin expression. In a mouse xenograft model, overexpression of CTGF promoted resistance to paclitaxel. In contrast, knockdown of CTGF expression increased the therapeutic effect of paclitaxel in this model. In conclusion, our data indicate that CTGF might be a critical oncogene of human osteosarcoma involved in resistance to paclitaxel treatment.     

紫杉醇载药体系及其抗肿瘤活性和临床应用

紫杉醇是临床上常用的抗肿瘤药物,主要作用机制为促进细胞微管聚合并抑制微管解聚,导致细胞纺锤体失去正常功能,抑制肿瘤细胞的有丝分裂,进而诱导细胞凋亡,目前已被用于治疗卵巢癌、乳腺癌、肺癌等恶性肿瘤。紫杉醇难溶于水,临床上常采用聚氧乙烯蓖麻油和乙醇助溶,而聚氧乙烯蓖麻油易引起过敏反应。为提高紫杉醇在水中的溶解度,减少毒副作用的发生,并提高紫杉醇的抗肿瘤活性,国内外学者对紫杉醇的不同载药体系、制剂剂型及临床用药方式等进行了广泛的研究。现在,纳米技术与生物医学结合产生的纳米载药体系已经用于改善紫杉醇的水溶性和临床疗效。     

Inactivation of endopolyphosphatase gene PPN1 results in inhibition of expression of exopolyphosphatase PPX1 and high-molecular-mass exopolyphosphatase not encoded by PPX1 in Saccharomyces cerevisiae

Saccharomyces cerevisiae possesses multiple forms of exopolyphosphatases, the enzymes involved in the metabolism of inorganic polyphosphates, which are important regulatory compounds. In S. cerevisiae, inactivation of endopolyphosphatase gene PPN1 leads to the inhibition of expression of both exopolyphosphatase PPX1 and high-molecular-mass exopolyphosphatase of approximately 1000 kDa not encoded by PPX1. In the single endopolyphosphatase mutant CRN, the expression of exopolyphosphatase PPX1 decreases 6.5-fold and 2.5-fold at the stationary and exponential growth phases, respectively, as compared with the parent strain CRY. In this mutant, the activity of the high-molecular-mass exopolyphosphatase of approximately 1000 kDa decreases approximately 10-fold as compared with that in strains with the PPN1 gene. In a double mutant of PPX1 and PPN1, no exopolyphosphatase activity is detected in the cytosol at the stationary growth phase. Thus, the exopolyPase activity in cell cytosol depends on the endopolyPase gene PPN1.     

Chemotherapy of breast cancer: focus on paclitaxel

Paclitaxel is among the most effective agents in the treatment of breast cancer. Both as a single agent and in combinations, paclitaxel is effective as first-line therapy and as a salvage therapy in patients with locally advanced or metastatic disease. Paclitaxel also demonstrated efficacy in patients who received prior anthracyclin therapy and those with anthracyclin-resistant disease. In the adjuvant setting, data from randomized study have supported the sequential use of paclitaxel after therapy with doxorubicin / cyclophosphamide for patients with node-positive disease. The drug may be used in combination with other chemotherapeutical agents and immune stymulatory agents. Therapy on weekly and every-three-week schedules has been effective.     

Inhibition of the cathepsin cysteine proteases B and K by square-planar cycloaurated gold(III) compounds and investigation of their anti-cancer activity

Gold(III) compounds have been examined for potential anti-cancer activity. It is proposed that the molecular targets of these compounds are thiol-containing biological molecules such as the cathepsin cysteine proteases. These enzymes have been implicated in many diseases including cancer. The catalytic mechanism of the cathepsin cysteine proteases is dependent upon a cysteine at the active site which is accessible to the interaction of thiophilic metals such as gold. The synthesis and biological activity of square-planar six-membered cycloaurated Au(III) compounds with a pyridinyl-phenyl linked backbone and two monodentate or one bidentate leaving group is described. Gold(III) cycloaurated compounds were able to inhibit both cathepsins B and K. Structure/activity was investigated by modifications to the pyridinyl-phenyl backbone, and leaving groups. Optimal activity was seen with substitution at the 6 position of the pyridine ring. The reversibility of inhibition was tested by reactivation in the presence of cysteine with a bidentate thiosalicylate compound being an irreversible inhibitor. Five compounds were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. The thiosalicylate compound was tested in vivo against the HT29 human colon tumor xenograft model. A modest decrease in tumor growth was observed compared with the untreated control tumor.     

Cisplatin Induces Resistance by Triggering Differentiation of Testicular Embryonal Carcinoma Cells

Although testicular germ cell tumors are generally quite responsive to treatment with cisplatin, a small fraction of them acquire resistance during therapy. Even when cisplatin treatment is successful the patient is often left with a residual teratoma at the site of the primary tumor suggesting that cisplatin may trigger differentiation in some tumors. Using the human embryonal carcinoma cell line NTera2/D1, we confirmed that exposure to the differentiating agent retinoic acid produced a reduction in pluripotency markers NANOG and POU5F1 (Oct3/4) and an acute concentration-dependent increase in resistance to both cisplatin and paclitaxel that reached as high as 18-fold for cisplatin and 61-fold for paclitaxel within four days. A two day exposure to cisplatin also produced a concentration-dependent decrease in the expression of the NANOG and POU5F1 and increased expression of three markers whose levels increase with differentiation including Nestin, SCG10 and Fibronectin. In parallel, exposure to cisplatin induced up to 6.2-fold resistance to itself and 104-fold resistance to paclitaxel. Paclitaxel did not induce differentiation or resistance to either itself or cisplatin. Neither retinoic acid nor cisplatin induced resistance in cervical or prostate cancer cell lines or other germ cell tumor lines in which they failed to alter the expression of NANOG and POU5F1. Forced expression of NANOG prevented the induction of resistance to cisplatin by retinoic acid. We conclude that cisplatin can acutely induce resistance to itself and paclitaxel by triggering a differentiation response in pluripotent germ cell tumor cells.     

Synthesis and antitumor activity of peptide-paclitaxel conjugates.

Paclitaxel (Pac) is the most important anticancer drug used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. In this work, we present the synthesis of five 2'-paclitaxel-substituted analogs in which paclitaxel was covalently bound to peptides or as multiple copies to synthetic carriers. Ac-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Arg-NH(2), Folyl-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Ser-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](2)-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](3)-NH(2) and Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) were synthesized using 2'-halogeno-acetylated paclitaxel derivatives. Paclitaxel conjugates showed greater solubility in water than paclitaxel and inhibited the proliferation of human breast, prostate, and cervical cancer cell lines. Although all synthesized compounds had an antiproliferative activity, the Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) derivative showed improved biological activity in comparison with paclitaxel in cervical and prostate human cancer cells.     

Resistance to paclitaxel in hepatoma cells is related to static JNK activation and prohibition into entry of mitosis

Hepatocellular carcinoma (HCC) generally shows chemoresistant features to anticancer agents. Paclitaxel has been clinically used in the treatment of various cancers. However, effect of paclitaxel on HCC has not been adequately addressed. Here, we found two categories of hepatoma cells in response to paclitaxel. Paclitaxel effectively decreased the cell viability of SNU475, Hep3B, and SNU387 HCC cells and Chang liver cells (death prone). In contrast, the other five hepatoma cell lines (SNU449, SNU398, SUN368, SNU354, and HepG2 cells) were resistant to paclitaxel (death reluctant). In response to paclitaxel, Bcl-2 was highly phosphorylated in death-prone cells, whereas much less Bcl-2 was phosphorylated in death-reluctant cells. Cotreatment with SP600125, an inhibitor JNK, significantly reduced the phosphorylated Bcl-2 in death-prone cells and caused a significant reduction in cell death. The reduced cell death was due to prohibition into mitotic entry as evidenced by low cyclin B(1)/Cdk1 kinase activity. In death-reluctant cells, inbuild-phospho-JNK levels were high but no longer activated in response to paclitaxel. We found that paclitaxel combined with caffeine or UCN-01, inhibitors of G(2) DNA damage checkpoint, was able to partially overcome resistance to paclitaxel in these cells. Thus our data provide the molecular basis of paclitaxel resistance in hepatoma cells, and appropriate combination therapy may increase treatment efficacy.     

Tumor necrosis factor induces the loss of sphingosine kinase-1 by a cathepsin B-dependent mechanism

Sphingosine kinase-1 (SK1) has emerged as a key component of cytokine responses, including roles in apoptosis, yet the specific mechanisms by which cytokines regulate SK1 in the apoptotic responses have not been studied. In this study, we show that prolonged treatment of MCF-7 cells with tumor necrosis factor (TNF) induces a dose- and time-dependent decrease in SK1 protein. Inhibition of the upstream caspase 8 by IETD significantly rescued TNF effects on SK1, yet the caspase 7 inhibitor DEVD failed to have any effect, suggesting that the decline in SK1 occurs downstream of the initiator caspase but upstream of the effector caspase. In addition to caspase activation, TNF caused disruption of lysosomes with relocation of the cysteine protease cathepsin B into the cytosol. Down-regulation of cathepsin B using small interfering RNA significantly restored SK1 levels following exposure to TNF, suggesting that SK1 loss was dependent on cathepsin B activity. The regulation of SK1 by the lysosomal protease was further supported by the colocalization of SK1 with the lysosome and cathepsin B in cells and the loss of the colocalization following exposure to TNF. The ability of cathepsin B to regulate SK1 was further corroborated by an in vitro approach where recombinant cathepsin B cleaved SK1 at multiple sites to produce several cleavage fragments. Therefore, these studies show that SK1 down-regulation by TNF is dependent on the "lysosomal pathway" of apoptosis and specifically on cathepsin B, which functions as an SK1 protease in cells.     

Short exposure to paclitaxel induces multipolar spindle formation and aneuploidy through promotion of acentrosomal pole assembly

Paclitaxel is a widely used microtubule drug and cancer medicine. Here we report that by short exposure to paclitaxel at a low dose, multipolar spindles were induced in mitotic cells without centrosome amplification. Both TPX2 depletion and Aurora-A overexpression antagonized the multipolarity. Live cell imaging showed that some paclitaxel-treated cells accomplished multipolar cell division and a portion of the daughter cells went on to the next round of mitosis. The surviving cells grew into clones with varied genome content. The results indicated that an aneuploidy population could be induced by short exposure to paclitaxel at a low dose, implicating potential side effects of paclitaxel.     

The expression of matrix metalloproteinase 9 and cathepsin B in gastric carcinoma is associated with lymph node metastasis, but not with postoperative survival

Degradation components of basement membrane could be crucial for tumor invasion. A key role in this process has been assigned to cysteine proteases, i.e. cathepsins and matrix metalloproteinases. The purpose of this study was to investigate the relationship of the expression of MMP-9 and cathepsin B with tumor aggressiveness expressed by lymph node metastases and survival rates in gastric carcinoma patients. Slides of 5 mum-thick serial sections from 91 patients with primary gastric carcinoma were prepared and analyzed for MMP-9 and cathepsin B expression using anti-human monoclonal antibody (NCL-MMP-9 clone; dilution 1:40 and NCL-CATH-B clone; dilution 1:40). The patients were clinically monitored for 84 months. We found no association between the expression of MMP-9 and cathepsin B in main mass of tumor and patients' gender, tumor location, Lauren's classification or histological differentiation. Also no correlation was observed between the expression of MMP-9 in main mass of tumor and depth of invasion. A strong statistically significant association was found between the expression of MMP-9 and cathepsin B in main mass of tumor and lymph node involvement (p<0.001; p<0.001, respectively). However, we observed no correlation between the expression of MMP-9 and cathepsin B in main mass of tumor and lymph node involvement or 5-year overall survival. Our results may suggest that the expression of matrix metalloproteinase-9 (MMP-9) and cathepsin B is correlated with lymph node metastasis in advanced gastric carcinoma, but not with patients' postoperative survival.     

Elicitation kinetics of taxane production in suspension cultures of Taxus baccata Pendula

Methyl jasmonate increased taxane production in suspension cultures of Taxus baccata Pendula. Time course changes of taxane production after methyl jasmonate addition were different from normal kinetics without elicitation. Baccatin III and 10-deacetyl baccatin III were detected first and paclitaxel, 10-deacetyl taxol and cephalomanine followed each other in sequence. Paclitaxel was not a dead-end metabolite.     

Prognostic significance of lysosomal cysteine proteases in the estimation of the effectiveness of the antitumor therapy

The influence of the antitumor drugs, cyclophosphamide (CPA) and nitrosomethylurea (NMU) on the activity of lysosomal cysteine proteases cathepsin B and L in tumor tissue has been investigated using CPA-sensitive (LS) and CPA-resistant mouse lymphosarcomas (RLS). (These drugs exhibit high and low antitumor efficiency towards LS and RLS mouse lymphosarcomas, respectively). Regression or reduction in the growth rate of LS and RLS lymphosarcomas caused by CPA or NMU administration was accompanied by the increase in the activity of cysteine proteases cathepsin B and L in the tumor tissue. The increase of cathepsin B and L activity in tumor tissue correlated with the therapeutic effect of these drugs. Data obtained suggest that activity of cathepsin B and L in tumor tissue has a prognostic significance for the estimation of the effectiveness of antitumor therapy.     

Exposure to paclitaxel or vinblastine down-regulates CD11a and CD54 expression by P815 mastocytoma cells and renders the tumor cells resistant to killing by nonspecific cytotoxic T lymphocytes induced with anti-CD3 antibody

Paclitaxel and vinblastine are drugs with anti-microtubule activity that are commonly used in the treatment of numerous types of cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel or vinblastine on the subsequent susceptibility of surviving P815 murine mastocytoma cells to cytolysis by major histocompatibility complex (MHC)-unrestricted mouse cytotoxic T lymphocytes that had been induced with anti-CD3 antibody. P815 cells that had survived culture for 24 h in the presence of paclitaxel (5 or 50 micro g/ml) or vinblastine (1.5 or 15 micro g/ml) were rendered resistant to anti-CD3-activated killer-T (AK-T) cell-mediated cytolysis in a standard (51)Cr-release assay. Resistance to killing was associated with a reduced ability of AK-T cells to form conjugates with drug-treated P815 target cells, suggesting a possible effect on adhesion molecules. Flow cytometric analysis of paclitaxel- or vinblastine-treated P815 cells revealed reduced cell-surface expression of the adhesion molecules LFA-1 (CD11a /CD18) and ICAM-1 (CD54). Similar results were obtained following paclitaxel or vinblastine treatment of Yac-1 lymphoma cells. RT-PCR analysis revealed reduced levels of mRNAs coding for CD11a and CD54 in paclitaxel- or vinblastine-pretreated P815 cells. Collectively, these data lead us to conclude that paclitaxel and vinblastine render P815 mastocytoma cells resistant to T cell-mediated cytotoxicity by interfering with CD11a and CD54 expression by the tumor cells. A similar effect by these drugs on tumor cells and/or leukocytes in cancer patients might compromise tumor-specific cell-mediated immune responses.     

Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor

Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion.