Transport, metabolism, cytotoxicity and effects of novel taxanes on the cell cycle in MDA-MB-435 and NCI/ADR-RES cells

Resistance of tumours to taxanes causes chemotherapy failure in numerous patients. Resistance is partly due to the low tumour uptake of taxanes and their rapid metabolism. Structural modifications of taxanes can reduce their P-glycoprotein-related efflux or decrease metabolism and consequently increase taxane efficiency. This study compared cytotoxicity and effects of the cell cycle, transport and metabolism of novel taxanes SB-T-1102, SB-T-1103, SB-T-1214 and SB-T-1216, fluorinated SB-T-12851, SB-T-12852, SB-T-12853, SB-T-12854 and IDN5109 with paclitaxel in paclitaxel-sensitive (MDA-MB-435) and paclitaxel-resistant (NCI/ADR-RES) human cancer cells. We have shown before that NCI/ADR-RES cells were 1,000-fold less sensitive to paclitaxel than MDA-MB-435 cells in correspondence to P-glycoprotein overexpression and up to 20-fold lower uptake of the drug in the resistant cells. The uptake of novel taxanes was 1.2 to 3.8 times lower than that of paclitaxel in the MDA-MB-435 cells, but 1.5 to 6.5 times higher in NCI/ADR-RES cells. NCI/ADR-RES cells were correspondingly only 2- to 6.6-fold less sensitive than the MDA-MB-435 cells to novel taxanes. Both cell lines showed minimal metabolism of the novel taxanes which was therefore not responsible for their different sensitivity, the observed differences in their individual efficiency and higher effects than paclitaxel. All novel taxanes caused G(2)/M block of the cell cycle similar to paclitaxel, but lower at concentrations by order of magnitude. Thus, structural modifications of taxanes resulting in their decreased P-glycoprotein-related transport probably caused their higher efficiency than paclitaxel in multidrug-resistant NCI/ADR-RES tumour cells.     

Cell death induced by novel fluorinated taxanes in drug-sensitive and drug-resistant cancer cells

The aim of this study is to compare the effects of new fluorinated taxanes SB-T-12851, SB-T-12852, SB-T-12853, and SB-T-12854 with those of the classical taxane paclitaxel and novel non-fluorinated taxane SB-T-1216 on cancer cells. Paclitaxel-sensitive MDA-MB-435 and paclitaxel-resistant NCI/ADR-RES human cancer cell lines were used. Cell growth and survival evaluation, colorimetric assessment of caspases activities, flow cytometric analyses of the cell cycle and the assessment of mitochondrial membrane potential, reactive oxygen species (ROS) and the release of cytochrome c from mitochondria were employed. Fluorinated taxanes have similar effects on cell growth and survival. For MDA-MB-435 cells, the C₅₀ of SB-T-12851, SB-T-12852, SB-T-12853 and SB-T-12854 was 3 nM, 4 nM, 3 nM and 5 nM, respectively. For NCI/ADR-RES cells, the C₅₀ of SB-T-12851, SB-T-12852, SB-T-12853, and SB-T-12854 was 20 nM, 20 nM, 10 nM and 10 nM, respectively. Selected fluorinated taxanes, SB-T-12853 and SB-T-12854, at the death-inducing concentrations (30 nM for MDA-MB-435 and 300 nM for NCI/ADR-RES) were shown to activate significantly caspase-3, caspase-9, caspase-2 and also slightly caspase-8. Cell death was associated with significant accumulation of cells in the G₂/M phase. Cytochrome c was not released from mitochondria and other mitochondrial functions were not significantly impaired. The new fluorinated taxanes appear to use the same or similar mechanisms of cell death induction as compared with SB-T-1216 and paclitaxel. New fluorinated and non-fluorinated taxanes are more effective against drug-resistant cancer cells than paclitaxel. Therefore, new generation of taxanes, either non-fluorinated or fluorinated, are excellent candidates for further and detailed studies.     

Metabolism of new-generation taxanes in human, pig, minipig and rat liver microsomes

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.     

Metabolism of new-generation taxanes in human,pig, minipig and rat liver microsomes

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.     

Second-generation taxanes effectively suppress subcutaneous rat lymphoma: role of disposition, transport, metabolism, in vitro potency and expression of angiogenesis genes

The study investigated possible mechanisms by which second-generation taxanes, established as significantly more effective than paclitaxel in vitro, suppress a rat lymphoma model in vivo. The studied mechanisms included taxane pharmacokinetics, expression of genes dominating their metabolism (Cyp3a1/2) and transport (Abcb1) and genes controlling tumour angiogenesis (growth factors and receptors). SB-T-1214, SB-T-12854 and IDN5109 suppressed rat lymphoma more effectively than paclitaxel, SB-T-12851, SB-T-12852, SB-T-12853 or IDN5390 as well as P388D1 leukaemia cells in vitro. The greater anti-lymphoma effects of SB-T-1214 in rats corresponded to a higher bioavailability than with SB-T-12854, and lower systemic toxicity of SB-T-1214 for rats reflected its lower cytotoxicity for P388D1 cells in vitro. Suppression of Abcb1 and CYP3a1 expression by SB-T-1214 and IDN5109 could partly explain their anti-lymphoma effects, but not that of SB-T-12854. Growth factors genes Egf, Fgf, Pdgf, and Vegf associated with tumour angiogenesis had significantly lower expression following treatment with anti-lymphoma effective IDN5109 and their receptors were unaffected, whereas inefficient IDN5390 increased expression of the most important Vegf. The effective SB-T-12854 inhibited Egf, Egfr, Fgfr and Pdgfr expression, while the ineffective SB-T-12851, SB-T-12852 and SB-T-12853 inhibited only Egf or Egfr expression. Vegfr expression was inhibited significantly by SB-T-12851 and SB-T-12854, but effect of SB-T-12851 was compromised by induced Vegf expression. The very effective SB-T-1214 decreased the expression of Vegf, Egf and all receptors most prominently indicating the possible supporting role of these genes in anti-lymphoma effects. In conclusion, SB-T-1214, SB-T-12854 and IDN5109 are good candidates for further study.     

Cytotoxic effects of 27 anticancer drugs in HeLa and MDR1-overexpressing derivative cell lines

The cytotoxic effects of 27 anticancer drugs including amrubicin, vinorelbine, paclitaxel, docetaxel, gemcitabine, and irinotecan were evaluated in human cervical carcinoma HeLa cells, and drug-resistant HeLa-derived Hvrl-1, HvrlO-6, and Hvr100-6 cells, which were newly established by stepwise exposure to vinblastine. FACS and RT-PCR analysis indicated that MDR1 (P-glycoprotein) was induced without any alterations in expression of its related transporters. Hvrl00-6 cells showed 2- to 200-fold higher resistance to anthracyclines than HeLa cells, and unexpectedly showed slight resistance to idarubicin and amrubicin. The relative resistance to vinca-alkaloids was 300- to 600,000-fold, and HvrlOO-6 cells showed the highest relative resistance to vinorelbine. HvrlOO-6 cells also showed 4000- and 60000-fold resistance to the taxanes paclitaxel and docetaxel, respectively. Hvr100-6 cells were also resistant to 6-mercaptopurine, actinomycin D, etoposide, and mitomycin C, with relative resistance of 8-, 45000-, 12-, and 9-fold, respectively. In contrast, HvrlOO-6 cells showed no or slight resistance to platinum derivatives, pyrimidine analogues, and alkylating agents or to irinotecan and its active form, or tamoxifen. The cytotoxicity of anthracyclines, vinca-alkaloids, taxanes, actinomycin D, and etoposide was extensively reversed by cyclosporin A. Cyclosporin A had no effect on the cytotoxicity of 6-mercaptopurine or mitomycin C, suggesting that resistance to these drugs was not mediated via MDR1. The alterations in cytotoxicity by overexpression of MDR1 and effects of cyclosporin A could be also qualitatively explained by [3H]vinblastine uptake experiments. The 27 anticancer drugs analyzed here could be classified into substrates and nonsubstrates for MDR1. This will be useful for designing effective regimens for chemotherapy.     

A thermally targeted elastin-like polypeptide-doxorubicin conjugate overcomes drug resistance

The ability of cancer cells to become simultaneously resistant to different drugs, a trait known as multidrug resistance, remains a major obstacle for successful anticancer therapy. One major mechanism of resistance involves cellular drug efflux by expression of P-glycoprotein (P-gp), a membrane transporter with a wide variety of substrates. Anthracyclines are especially prone to induction of resistance by the P-gp mechanism. P-gp mediated resistance is often confronted by use of P-gp inhibitors, synthesis of novel analogs, or conjugating drugs to macromolecular carriers in order to circumvent the efflux mechanism. In this report, the effect of free and Elastin-like polypeptide (ELP) bound doxorubicin (Dox) on the viability of sensitive (MES-SA and MCF-7) and multidrug resistant (MES-SA/Dx5 and NCI/ADR-RES) human carcinoma cells was studied in vitro. The resistant MES-SA/Dx5 cells demonstrated about 70 times higher resistance to free Dox than the sensitive MES-SA cells, and the NCI/ADR-RES cells were about 30 fold more resistant than the MCF-7 cells. However, the ELP-bound Dox was equally cytotoxic in both sensitive and resistant cell lines. The ELP-bound Dox was shown to accumulate in MES-SA/Dx5 cells, as opposed to free Dox, which was rapidly pumped out by the P-gp transporter. Since ELP is a thermally responsive carrier, the effect of hyperthermia on the cytotoxicity of the ELP-Dox conjugate was investigated. Both cytotoxicity and apoptosis were enhanced by hyperthermia in the Dox resistant cells. The results suggest that ELP-Dox conjugates may provide a means to thermally target solid tumors and to overcome drug resistance in cancer cells.     

A cytotoxic ribonuclease reduces the expression level of P-glycoprotein in multidrug-resistant cell lines

We have previously described a cytotoxic human pancreatic-ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells. We have investigated whether PE5 could specifically inhibit the accumulation of P-glycoprotein in multidrug-resistant cells, since P-glycoprotein overexpression is one of the most important mechanisms contributing to the multiple drug resistance phenotype. We show that PE5 is able to reduce the amount of P-glycoprotein in two different multidrug-resistant cell lines, NCI/H460-R and NCI/ADR-RES, while glutathione S-transferase-л is not affected. We also show that onconase, an amphibian ribonuclease that is undergoing phase II/III clinical trials as an antitumor drug, does not affect the expression of these proteins. The reduction of P-glycoprotein accumulation, which has been functionally confirmed by flow cytometry analysis, may be caused by the previously reported underphosphorylation of JNK induced by PE5. We also show that PE5 has synergistic cytotoxicity with doxorubicin on the NCI/ADR-RES multidrug-resistant cell line. In conclusion, PE5 is a cytotoxic ribonuclease that cleaves nuclear RNA and decreases the expression of P-glycoprotein, showing anticancer activity in multidrug-resistant cell lines.     

Menadione serves as a substrate for P-glycoprotein: implication in chemosensitizing activity

Based on its chemosensitizing effect, we questioned whether menadione is an inhibitor or a substrate of P-glycoprotein (P-gp). To test this hypothesis, we assessed the effect of menadione on P-gp activity and examined the P-gp-dependency of cellular accumulation and cytotoxicity of menadione as well. Treatment with menadione resulted in the concentration-dependent increase of rhodamine 123 (Rh123) accumulation in P-gp-overexpressing MDCKII/MDR1 and NCI/ADR-RES cells, suggesting that menadione inhibits Rh123 extrusion by P-gp. Compared with MDCKII or MCF-7, intracellular distribution of [³H]-menadione was significantly lower in MDCKII/MDR1 or NCI/ADR-RES cells, which could be restored by the P-gp inhibitors, verapamil and quinidine. Consistent with these results, MDCKII/MDR1 or NCI/ADR-RES cells were more resistant to the cytotoxicity of menadione than MDCKII or MCF-7 cells, respectively. Such resistance was abolished by the combined treatment of verapamil and quinidine in NCI/ADR-RES cells. Our study identified menadione as a substrate of P-gp, which presumably, acts as the mechanism for the chemosensitizing effect. Menadione may be a promising chemotherapeutic enhancer by its ability of circumventing drug resistance, in addition to its own anti-cancer activity.     

Improved effectiveness of nanoparticle albumin-bound (nab) paclitaxel versus polysorbate-based docetaxel in multiple xenografts as a function of HER2 and SPARC status

Nanoparticle albumin-bound (nab)-paclitaxel (Abraxane) is an albumin-bound 130-nm particle form of paclitaxel that demonstrated higher efficacy and was well tolerated compared with solvent-based paclitaxel (Taxol) and docetaxel (Taxotere) in clinical trials for metastatic breast cancer. Nab-paclitaxel enhances tumor targeting through gp60 and caveolae-mediated endothelial transcytosis and the association with the albumin-binding protein SPARC (secreted protein, acidic and rich in cysteine) in the tumor microenvironment. The overexpression of human epidermal growth factor receptor-2 (HER2) in breast cancer has been shown to correlate with resistance to paclitaxel. To evaluate the importance of HER2 and SPARC status in determining the relative efficacy of nab-paclitaxel compared with polysorbate-based docetaxel, nude mice bearing six different human tumor xenografts were treated with nab-paclitaxel (MX-1: 15 mg/kg, once a week for 3 weeks; LX-1, MDA-MB-231/HER2+, PC3, and HT29: 50 and 120 mg/kg, every 4 days three times ; MDA-MB-231: 120 and 180 mg/kg, every 4 days three times) and polysorbate-based docetaxel (15 mg/kg). HER2 and SPARC status were analyzed by RT-PCR and immunohistochemical staining. MDA-MB-231 and MX-1 breast and LX-1 lung cancers were HER2 negative and low in SPARC expression. Nab-paclitaxel at submaximum-tolerated dosage was significantly more effective than polysorbate-based docetaxel at its maximum-tolerated dosage in these three HER2-negative tumors. The HER2-positive tumors had variable SPARC expression, with MDA-MB-231/HER2+ 相似文献   

Overcoming multidrug resistance in human cancer cells by natural compounds

Multidrug resistance is a phenomenon whereby tumors become resistant to structurally unrelated anticancer drugs. P-glycoprotein belongs to the large ATP-binding cassette (ABC) transporter superfamily of membrane transport proteins. P-glycoprotein mediates resistance to various classes of anticancer drugs including vinblastine, daunorubicin, and paclitaxel, by actively extruding the drugs from the cells. The quest for inhibitors of anticancer drug efflux transporters has uncovered natural compounds, including (-)-epigallocatechin gallate, curcumin, capsaicin, and guggulsterone, as promising candidates. In this review, studies on the effects of natural compounds on P-glycoprotein and anticancer drug efflux transporters are summarized.     

Oral Delivery of Taxanes

Oral treatment with cytotoxic agents is tobe preferred as this administration routeis convenient to patients, reducesadministration costs and facilitates theuse of more chronic treatment regimens. Forthe taxanes paclitaxel and docetaxel,however, low oral bioavailability haslimited development of treatment by theoral route. Preclinical studies with mdr1aP-glycoprotein knock-out mice, which lackfunctional P-glycoprotein activity in thegut, have shown significant bioavailabilityof orally administered paclitaxel.Additional studies in wild-type micerevealed good bioavailability after oraladministration when paclitaxel was combinedwith P-glycoprotein blockers such ascyclosporin A or the structurally relatedcompound SDZ PSC 833. Based on theextensive preclinical research, thefeasibility of oral administration ofpaclitaxel and docetaxel in cancer patientswas recently demonstrated in our Institute.Co-administration of cyclosporin A stronglyenhanced the oral bioavailability of bothpaclitaxel and docetaxel. For docetaxel incombination with cyclosporin A an oralbioavailability of 90% was achieved withan interpatient variability similar to thatafter intravenous drug administration; forpaclitaxel the oral bioavailability isestimated at approximately 50%. The safety of the oralroute for both taxanes is good. A phase IIstudy of weekly oral docetaxel incombination with cyclosporin A is currentlyongoing.     

Nanoparticle albumin-bound paclitaxel for treatment of metastatic breast cancer

Two taxanes, paclitaxel and docetaxel, are among the most widely used chemotherapeutic agents in solid tumor oncology, with efficacy against tumors of the breast, lung, head and neck, ovary, prostate, stomach and urothelium. The taxanes have been studied extensively and have been proven effective for treating early and advanced breast cancer. However, paclitaxel and docetaxel are both highly hydrophobic compounds, requiring synthetic solvents for parenteral administration. The solvents in commercially available preparations cause life-threatening toxic effects and decreased efficacy, and they are inconvenient to administer. Nanoparticle albumin-bound paclitaxel (nabP) is a novel, solvent-free formulation of paclitaxel. With nabP, in contrast to standard paclitaxel, life-threatening hypersensitivity reactions have not been observed, and it can be administered safely without steroid and antihistamine premedication. Furthermore, nabP exploits cellular and tumor transport mechanisms to preferentially target tumor cells. Data from phase III studies of metastatic breast cancer demonstrated higher response rates, longer time to progression and an improved toxicity profile for nabP compared with standard paclitaxel. The U.S. Food and Drug Administration approved nabP in late 2004 for treatment of metastatic breast cancer after failure of an anthracycline-based regimen.     

Detection of apoptosis and drug resistance of human breast cancer cells to taxane treatments using quartz crystal microbalance biosensor technology

Taxanes are used for the treatment of many human cancers, as first- and second-line chemotherapeutics. In the course of treatment many patients develop resistance or hypersensitivity to one form of taxane and require a different taxane to rescue the therapeutic benefit of the drug. There is currently no method to reliably predict tumor responses to taxanes prior to therapy or when resistance or hypersensitivity develops. We adapted the quartz crystal microbalance (QCM) biosensor technique to study responses of human mammary epithelial tumor cells to taxanes. Studies indicate that stable frequency and resistance levels are reached at 24 h. Cells in the QCM can then be treated with taxanes and responses monitored in real time via frequency and resistance changes reflecting alterations of cell mass distribution and viscoelastic properties. Distinct shifts in frequency and resistance accurately predicted apoptosis or resistance to treatment, as determined in parallel convention assays. QCM analysis accurately predicted docetaxel was more effective than paclitaxel and MCF-7 cells were more resistant to taxanes compared to MDA-MB-231 cells. These studies suggest "signature" patterns for taxane responsivity could be compared to those of patient biopsy samples to predict therapy outcome prior to treatment for initial therapy or to rescue therapy efficacy.     

Dual approach utilizing self microemulsifying technique and novel P-gp inhibitor for effective delivery of taxanes

In the present work, concomitant use of self-microemulsifying drug delivery systems (SMEDDS) and a novel third-generation P-gp inhibitor, GF120918 (elacridar), for the effective transport of taxanes (paclitaxel and docetaxel) across an in?vitro model of the intestinal epithelium and uptake into tumor cells were investigated. On the basis of solubility studies and ternary phase diagrams, different SMEDDS formulations of taxanes were prepared and characterized. In caco-2 cell permeation study, paclitaxel-loaded SMEDDS along with GF120918 showed a four-fold increase in apparent permeability, while docetaxel-loaded SMEDDS in combination with GF120918 showed a nine-fold increase in permeability, as compared to plain drug solution. Cell uptake studies on A549 cells were performed with microemulsions formed from both SMEDDS formulations loaded with rhodamine 123 dye and showed good uptake than plain dye solution. Confocal laser scanning microscopic images further confirmed the higher uptake of both SMEDDS formulations in the presence of GF120918.     

Use of P-glycoprotein and BCRP inhibitors to improve oral bioavailability and CNS penetration of anticancer drugs

P-glycoprotein (ABCB1) and breast cancer resistance protein [BCRP (also known as ABCG2)] are drug efflux transporters of the ATP binding cassette (ABC) family of proteins. Both P-glycoprotein and BCRP are located in the apical membrane of epithelial cells (e.g. in the intestinal wall and blood-brain barrier), where they can actively extrude a variety of structurally diverse drugs and drug metabolites. Consequently, the oral uptake and CNS penetration of substrate drugs can be low and variable. Inhibition of P-glycoprotein and/or BCRP is therefore a logical strategy to improve oral absorption, CNS penetration and delivery of anticancer agents to brain tumors or CNS metastases. As outlined in this review, this concept of improved oral pharmacokinetics has been demonstrated extensively for the anticancer drugs paclitaxel and topotecan both in preclinical models and in patients, and improved CNS penetration has been shown for paclitaxel, docetaxel and imatinib in preclinical models.     

Synthesis of Novel Biodegradable Methoxy Poly(ethylene glycol)-Zein Micelles for Effective Delivery of Curcumin

Novel biodegradable micelles were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG) to zein, a biodegradable hydrophobic plant protein. The mPEG-zein micelles were in the size range of 95-125 nm with a low CMC (5.5 × 10(-2) g/L). The micelles were nonimmunogenic and were stable upon dilution with buffer as well as 10% serum. Curcumin, an anticancer agent with multiple delivery challenges, was encapsulated in mPEG-zein micelles. The micelles significantly enhanced the aqueous solubility (by 1000-2000-fold) and stability (by 6-fold) of curcumin. PEG-zein micelles sustained the release of curcumin up to 24 h in vitro. Curcumin-loaded mPEG-zein micelles showed significantly higher cell uptake than free curcumin in drug-resistant NCI/ADR-RES cancer cells in vitro. Micellar curcumin formulation was more potent than free curcumin in NCI/ADR-RES cancer cells, as evidenced from the 3-fold reduction in IC(50) value of curcumin. Overall, this study for the first time reports a natural protein core based polymeric micelle and demonstrates its application for the delivery of hydrophobic anticancer drugs such as curcumin.     

Bcl-2 down-regulation is a novel mechanism of paclitaxel resistance

Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of deltapsim after stimulation of the respiratory state. To evaluate the binding of [14C]paclitaxel to isolated mitochondria, mitochondrial proteins were precipitated yielding 18.6 +/- 2.1 cpm/microg of protein. After stimulation of the respiratory state, binding of [14C]paclitaxel increased up to 163.2 +/- 46.7 cpm/microg of protein. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A (A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.     

Differential impact of Raf-1 kinase activity on tumor cell resistance to paclitaxel and docetaxel

Docetaxel (Taxotere) is becoming increasingly important in the treatment of many tumor sites and is unusually active in tumors that are resistant to the structurally similar taxane, paclitaxel. These data suggest that the processes that confer cellular paclitaxel resistance may have a substantially lower impact upon the cytotoxicity induced by docetaxel. We have recently reported that there is a marked Raf-1 kinase dependency of paclitaxel resistance in human cervical and ovarian tumor cell lines. We therefore characterized the impact that inherent and genetically induced variations in Raf-1 kinase activity have on the docetaxel cytotoxicity in human ovarian and cervical cancer cell lines. Our data suggest that docetaxel cytotoxicity is independent of Raf-1 kinase activity in the cell lines studied and that the lack of cross-resistance between these two taxane compounds may be due to the differential impact that Raf-1 kinase activity has on their cytotoxicity. Should these relationships pertain to the clinical situation, these findings could form the basis for a molecular-based triage of patients to receive docetaxel when response to paclitaxel may be unlikely due to high Raf-1 kinase activity.     

Enhanced efficiency of thermally targeted taxanes delivery in a human xenograft model of gastric cancer

Since successful chemotherapy with taxanes requires an improvement in their therapeutic index, especially by the reduction in unwanted systemic toxicity of either drug or adjuvants, we have investigated and are reporting results from an investigation of the use of a novel polymeric thermosensitive micellar delivery system for docetaxel and paclitaxel. Here we reported a novel metastable thermosensitive polymeric micelle for docetaxel and paclitaxel delivery [poly(N-isopropylacrylamide-co-acrylamide)-b-poly(DL-lactide), Poly(IPAAm-co-AAm)-b-PDLLA]. Previous in vitro efficacy studies indicated that, with hyperthermia, docetaxel-loaded micelles showed stronger cytotoxicity to different tumor cell lines than conventional docetaxel formulation while exhibiting slighter toxicity to normal cells. Present in vivo studies indicated that at the same dose level of docetaxel (paclitaxel), hyperthermia greatly enhanced the antitumor effect of micellar docetaxel (paclitaxel) in human gastric BGC mouse xenograft model by showing an extraordinary tumor volume and weight growth percentage inhibition of more than 80%. Meanwhile, acute toxicity tests features the lower LD(50) of the combination of hyperthermia and micellar docetaxel (paclitaxel) compared to that of the control group. The present results suggest that poly(IPAAm-co-AAm)-b-PDLLA micelles could be a clinically useful chemotherapeutic formulation and merit further research to evaluate the feasibility of clinical application.