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.     

Increased Spinal Cord Na+-K+-2Cl? Cotransporter-1 (NKCC1) Activity Contributes to Impairment of Synaptic Inhibition in Paclitaxel-induced Neuropathic Pain

Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na⁺-K⁺-2Cl⁻ cotransporter-1 (NKCC1) and K⁺-Cl⁻ cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with β-tubulin and β-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins.     

End-binding protein 1 stimulates paclitaxel sensitivity in breast cancer by promoting its actions toward microtubule assembly and stability

Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.     

Thermally sensitive micelles self-assembled from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) for controlled delivery of paclitaxel

Thermally sensitive micelles self-assembled from poly(N-isopropylacrylamide-co- N,N-dimethylacrylamide)-b-poly(d,l-lactide-co-glycolide)[P(NIPAAm-co-DMAAm)-b-PLGA] are fabricated and used as a carrier for the controlled delivery of paclitaxel. Paclitaxel is efficiently loaded into the micelles by a membrane dialysis method. The lower critical solution temperature (LCST) of the micelles is 39.0 degrees C in PBS. Encapsulation efficiency and loading level of paclitaxel are affected by the initial loading level of paclitaxel, fabrication temperature and polymer composition. The blank and paclitaxel-loaded micelles are characterized by particle size analysis (DLS), morphology (TEM and AFM) and paclitaxel distribution (NMR, DSC and WAXRD). The micelles are spherical in shape, having an average size less than 130 nm. Paclitaxel is molecularly distributed within the core of micelles. Sustained release of paclitaxel is achieved, which is much faster at a temperature above the LCST than at the normal body temperature (37 degrees C). Cytotoxicity of free paclitaxel and paclitaxel-loaded micelles against a human breast carcinoma cell line (MDA-MB-435S) is studied at different temperatures. The cytotoxicity of the paclitaxol-loaded micelles is greater as compared to free paclitaxel. Enhanced cytotoxicity is achieved by the paclitaxol-loaded micelles when the environmental temperature increases slightly above the LCST. Paclitaxel-loaded P(NIPAAm-co-DMAAm)-b-PLGA micelles may provide a good formulation for cancer therapy.     

长循环紫杉醇纳米脂质体的合成及其活性评估

目的：研制甲氧基聚乙二醇二硬脂酰磷脂酰乙醇胺（mPEG2000-DSPE）修饰的长循环紫杉醇纳米脂质体（PEG-PTX-LP）,减少市售紫杉醇制剂的不良反应并增强疗效。方法：采用薄膜超声分散法制备PEG-PTX-LP,采用激光散射粒度分析仪和透射电镜观察其物理性状,超滤法检测药物包封率,透析法检测药物缓释能力,通过细胞摄取试验观察人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells,HUVEC）、A549肺癌细胞对PEG-PTX-LP的摄取能力。结果：透射电镜显示长循环紫杉醇纳米脂质体呈圆形囊泡样结构,粒径检测其平均粒径为99.1 nm,制备后第2、7、14、21、30天的紫杉醇包封率均大于99%,在血清中的缓释能力优于泰素溶液,HUVEC、A549细胞对PEG-PTX-LP中紫杉醇的摄取量明显高于泰素溶液（Taxol）。结论：采用mPEG2000-DSPE修饰的PEG-PTX-LP具有更高的稳定性和缓释能力,对肿瘤细胞和血管内皮细胞有一定的特异性,是一种更有效的紫杉醇新剂型。     

Inhibition of ErbB2/neuregulin signaling augments paclitaxel-induced cardiotoxicity in adult ventricular myocytes

Paclitaxel (Taxol) has been successfully combined with the monoclonal antibody trastuzumab (Herceptin) in the treatment of ErbB2 overexpressing cancers. However, this combination therapy showed an unexpected synergistic increase in cardiac dysfunction. We have studied the mechanisms of paclitaxel/anti-ErbB2 cardiotoxicity in adult rat ventricular myocytes (ARVM). Myofibrillar organization was assessed by immunofluorescence microscopy and cell viability was tested by the TUNEL-, LDH- and MTT-assay. Oxidative stress was measured by DCF-fluorescence and myocyte contractile function by video edge-detection and fura-2 fluorescence. Treatment of ARVM with paclitaxel or antibodies to ErbB2 caused a significant increase in myofilament degradation, similarly as observed with an inhibitor of MAPK-signaling, but not apoptosis, necrosis or changes in mitochondrial activity. Paclitaxel-treatment and anti-ErbB2 reduced Erk1/2 phosphorylation. Paclitaxel increased diastolic calcium, shortened relaxation time and reduced fractional shortening in combination with anti-ErbB2. A minor increase in oxidative stress by paclitaxel or anti-ErbB2 was found. We conclude, that concomitant inhibition of ErbB2 receptors and paclitaxel treatment has an additive worsening effect on adult cardiomyocytes, mainly discernible in changes of myofibrillar structure and function, but in the absence of cell death. A potential mechanism is the modulation of the MAPK/Erk1/2 signaling by both drugs.     

Effectiveness of liposomal paclitaxel against MCF-7 breast cancer cells

Paclitaxel is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including breast, ovarian, and non-small-cell lung cancer. Due to its high lipophilicity, paclitaxel is difficult to administer and requires solubilization with Cremophor EL (polyethoxylated castor oil) and ethanol, which often lead to adverse side effects, including life-threatening anaphylaxis. Incorporation of paclitaxel in dimyristoylphosphatidylcholine:dimyristoylphosphatidylglycerol (DPPC:DMPG) liposomes can facilitate its delivery to cancer cells and eliminate the adverse reactions associated with the Cremophor EL vehicle. Accordingly, the effectiveness of liposomal paclitaxel on MCF-7 breast cancer cells was examined. The results from this study showed that (i) the lipid components of the liposomal formulation were nontoxic, (ii) the cytotoxic effects of liposomal paclitaxel were improved when compared with those seen with conventional paclitaxel, and (iii) the intracellular paclitaxel levels were higher in MCF-7 cells treated with the liposomal paclitaxel formulation. The results of these studies showed that delivery of paclitaxel as a liposomal formulation could be a promising strategy for enhancing its chemotherapeutic effects.     

Docetaxel for the treatment of hormone-refractory prostate cancer

Chemotherapy has historically proven toxic and ineffective for the treatment of metastatic hormone-refractory prostate cancer (HRPC), a disease with substantial morbidity and mortality. Progress has been made in symptom relief, and the combination of mitoxantrone and prednisone is considered the palliative standard of care. The effects of a variety of chemotherapeutic agents, both alone and in combination, on prostate-specific antigen decline rates, measurable disease response, and survival have been examined in numerous phase I and II trials. Results suggest that combining vinblastine or paclitaxel with estramustine confers a survival advantage over either agent alone. In addition, docetaxel-based therapy has been found to be effective and well tolerated, and phase III trials will soon determine whether docetaxel-based therapy should replace mitoxantrone-based therapy as the standard of care for HRPC.     

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。     

Flow cytometric identification of Paclitaxel-accumulating subpopulations

An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC. Paclitaxel accumulation was significantly increased by elicitation with methyl jasmonate (100 microM) on day 7 post-transfer as compared to unelicited cultures. Maximum accumulation was observed by day 12 post-transfer in both total paclitaxel (approximately 0.25 mg/L) and the percentage of paclitaxel-accumulating cells (approximately 95%). A similar trend was observed with isolated protoplasts, although protoplasts accumulated only ca. 40-75% of the paclitaxel present in single cells. In unelicited cell cultures, a small subpopulation (ca. 3-5%) of single cells was shown to accumulate paclitaxel. Although nearly all cells were observed to accumulate paclitaxel in methyl jasmonate-elicited cell cultures, a high degree of cell-to-cell variation was observed in paclitaxel content. The identified subpopulations represent targets for cell sorting, which may be applied to develop higher-accumulating cell lines. The quantification of single cell paclitaxel content is useful for characterizing production variability in cell cultures and can be utilized to develop rational strategies to increase paclitaxel production.     

Mechanism of paclitaxel activity in Kaposi's sarcoma

Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferation of spindle-shaped cells predominantly of endothelial cell origin, neoangiogenesis, inflammatory cell infiltration, and edema. At least in early stage, KS behaves as a reactive lesion sustained by the action of inflammatory cytokines and growth factors, has a polyclonal nature, and can regress. However, in time it can become monoclonal, especially in the nodular stage, evolving into a true sarcoma, likely in association with the increased expression of antiapoptotic oncogenes. We have recently demonstrated by immunohistochemical analysis that Bcl-2, a proto-oncogene known to prolong cellular viability and to antagonize apoptosis, is highly expressed in spindle cells and vessels of both AIDS-KS and classical KS lesions and that its expression increases with lesion stage. Paclitaxel, a microtubule-stabilizing drug known to inhibit Bcl-2 antiapoptotic activity and to be highly effective in the treatment of certain neoplasms, has recently been found to be active also in patients with advanced HIV-associated KS. In this report we investigated the mechanism(s) of paclitaxel activity in KS. By using a model of experimental KS induced by the inoculation of KS-derived spindle cells in nude mice and primary cultures of KS spindle cells, we found that paclitaxel promotes regression of KS lesions in vivo and that it blocks the growth, migration, and invasion of KS cells in vitro. Furthermore, paclitaxel treatment promoted apoptosis and down-regulated Bcl-2 protein expression in KS cells in vitro and in KS-like lesions in mice. Our results suggest that paclitaxel interferes with KS by down-regulating Bcl-2 antiapoptotic effect.     

Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules

Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.     

A simple method for enhancing paclitaxel release from<Emphasis Type="Italic"> Taxus canadensis</Emphasis> cell suspension cultures utilizing cell wall digesting enzymes

Paclitaxel storage in Taxus suspension cell cultures was studied through the simple use of cell wall digesting enzymes. The application of cellulase (1%) and pectolyase (0.1%) to Taxus canadensis suspension cultures induced a significant increase in the paclitaxel present in the extracellular medium while maintaining membrane integrity, suggesting that paclitaxel is stored in the cell wall. The addition of cell wall digesting enzymes to a cell culture bioprocess may be an effective way of enhancing paclitaxel release to the extracellular medium and hence simplify product recovery.Communicated by K.K. Kamo     

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.     

Biosynthesis of paclitaxel using synthetic biology

Paclitaxel is a tricyclic diterpenoid first isolated from the bark of Taxus brevifolia. It is notably one of the most effective natural anticancer drugs discovered so far, and has been widely used in the treatment of breast, ovarian, lung cancer, as well as other diseases. However, the content of paclitaxel in the bark of yew trees is extremely low, thus alternative sources need to be developed urgently to meet the clinical demands. Using methods of synthetic biology to realize the heterologous expression of paclitaxel-related genes in microbial hosts could be a good choice. This paper reviews the advances in the researches on the biosynthesis of paclitaxel to provide new research strategies and insights.

     

Paclitaxel probably enhances cytotoxicity of natural killer cells against breast carcinoma cells by increasing perforin production

Paclitaxel, a semisynthetic taxane, is one of the most active chemotherapeutic agents for the treatment of patients with breast cancer. We focused on the effect of paclitaxel on the cytotoxicity of natural killer (NK) cells. NK cells were purified by negative selection with magnetic beads from peripheral blood mononuclear cells of healthy volunteers. A human breast carcinoma cell line BT-474 and an NK cell–sensitive erythroleukemia cell line K562 were used as targets. Cytotoxicity of NK cells was determined by ⁵¹Cr-release assay with labeled target cells. Paclitaxel (1–100 nM) did not affect cellular viability, and significantly enhanced cytotoxicity of NK cells in a dose-dependent manner. Although paclitaxel did not affect Fas-ligand expression of NK cells, paclitaxel induced mRNA and protein production of perforin, an effector molecule in NK cell–mediated cytotoxicity. Concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, inhibited paclitaxel-dependent NK cell–mediated cytotoxicity. Furthermore, paclitaxel induced activation of nuclear factor B (NF-B) in NK cells. NF-B inhibitor pyrrolidine dithiocarbamate significantly suppressed both paclitaxel-induced perforin expression and NK cell cytotoxicity. Our results show for the first time that paclitaxel enhances in vitro cytotoxicity of human NK cells. Moreover, our results suggest a significant association between enhanced NK cell cytotoxicity, increased perforin production, and NF-B activation.     

Effect of osmotic pressure on paclitaxel production in suspension cell cultures of Taxus chinensis

The effect of osmotic pressure on paclitaxel production was investigated in the suspension cell cultures of Taxus chinensis. Paclitaxel production was definitely influenced by the initial sucrose concentration and the highest production yield was achieved at the concentration of 60 g.l(-1) sucrose (300 mOsm.kg(-1)). High osmotic pressure conditions generated by non-metabolic sugar (mannitol and sorbitol) also enhanced paclitaxel production by about two-fold. Kinetic studies revealed that high initial osmotic pressure enhanced paclitaxel production and that high concentration of sucrose was effective for sustaining secondary metabolism after induction of paclitaxel biosynthesis. Stoichiometric analysis with different combinations of sucrose and mannitol confirmed that osmotic pressure was the more important factor for enhancing paclitaxel metabolism. The addition of non-sugar osmotic agent, PEG also enhanced paclitaxel production. In this paper, we showed that high osmotic pressure led to increases in paclitaxel production and proposed that regulation of osmotic pressure may be useful in controlling paclitaxel production.