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Distribution of β -tubulin isotypes, total tubulin content and polymerized microtubule fraction in P388 vinorelbine-sensitive and -resistant cell lines by Western blotting. a Monoclonal antibodies to β -tubulin isotypes I, II, III and IVa + IVb were used to measure the percentage of each β -tubulin isotype in these cell lines by Western blotting. The sum of these isotypes was considered to be the total tubulin for analysis. Each error bar represents the standard deviation from biological and technical triplicates. b Anti β -tubulin antibody Tu27 was used to compare total tubulin levels by Western blotting and densitometry. Anti-GAPDH antibody was used to determine GAPDH
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The work presented here was initiated to explore the mechanisms underlying vinorelbine resistance in two previously established murine leukemia P388 cell lines (N.63 and N2.5). IC(50) measurements demonstrated that the vinorelbine-resistant cell line N.63 was sensitive to both vinblastine and vinflunine. In addition, vinorelbine-resistant cell line...
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... are seven β-tubulin isotype classes based upon their carboxyl terminal sequences [33]. We compared β-tubulin I, II, III and IVa + IVb isotype protein levels in P388 vinorelbine-sensitive and -resistant cell lines using quanti- tative immunoblotting (Fig. 1a). We found that class I was the major β-tubulin isotype in all three cell lines (60-72% of total β-tubulin) while β-tubulin classes IVa + IVb were the least abundant (1.2-1.7% of total β-tubulin). β-Tubulin class III was constant in all cell lines (11.3-11.7% of total β-tubulin), and there were no significant differences in β- tubulin ...
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... content can be a cause for antimitotic resistance [35], we measured the total tubulin content in these cell lines by Western blotting. The value for P388-S cells was set to 100. We found no statistically significant difference (p<0.05) in total tubulin content in either P388-N.63 (119.8+45.4) or P388-N2.5 (113.2+33.3) when compared to P388-S (Fig. ...
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... We used two different buffers containing either taxol or glycerol as cellular microtubule stabilizers. These buffers have been well studied for preserving cellular microtubules [27]. With the glycerol buffer, we measured 36.4+9.2% tubulin in the microtubule fraction (pellet) in P388-S compared to 30.7+ 7.4% in P388-N.63 and 31.7+8.7% in P388-N2.5 (Fig. 1c). Similarly, with the taxol buffer, we measured 31.3+7.6% cellular tubulin polymerized in the microtubule fraction in P388-S compared to 23.0+4.3% in P388-N.63 and 30.3+ 9.3% in P388-N2.5 (Fig. 3c). Analysis by Student t test demonstrated that the differences between polymerized microtubules in vinorelbine-resistant P388 cells and ...
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... Istomin ve ark yaptıkları bir çalışmada, vinorelbin uygulanmış olan HeLa hücrelerinin başlangıçtaki hücre sayısının yarısını öldürmek için gerekli olan dozun 48 saat süre için 7,4ng/ml (= 9,4μM) olduğu belirlenmiştir(30).Çalışmamızda ise hücrelerin yarısını öldürmek için gerekli olan dozun 24 saat için daha yüksek olduğunu bulduk. Bu farklılığın nedenleri ilacın uygulama sürelerinin aynı olmaması, değişik kültür koşulları ve hücre sayıları olabilir.İnsan T hücresi lenfoma hücre hatlarında ve fare lösemi hücrelerinde vinorelbinin mitokondri aracılı içsel apoptotik yolağı aktive ettiği belirlenmiştir(31,32).Apoptotik cevabın kontrolünde iş gören bir molekülü şifreleyen BCL2 geninin mRNA ifadesinin mikrotübül hedefleyen ajanlarla düzenlenerek kemoterapötik yanıtla ilişkilendirilmesi çeşitli çalışmalarla desteklenmiştir(33,34). BCL2'nin aşırı ifade edilmesinin apoptozu geciktirdiği, birçok hücre ve dokuda in vivo ve in vitro olarak gösterilmiştir(35)(36)(37)(38). ...
ZET Amaç: Hücre hareketi, hücre bölünmesi, hücre içi madde taşınması, hücresel yapının sağlanması gibi biyolojik işlevlerde rol alan mikrotübüller, antikanser ilaçların esas hedefi konumundadır. Vinorelbin'in (Navelbin) mikrotübüller üzerinden etki gösteren moleküllerden birisidir ve çeşitli kanserlerde kemoterapötik ajan olarak kullanıl-maktadır. Çalışmamızda, insan serviks kanser hücre hattı olan HeLa hücrelerine, 24 saat 5-100µM doz aralığında vinorelbin uygulaması sonunda hücrelerdeki olası apoptotik etkisini belirlemeyi amaçladık. Gereç ve yöntem: Vinorelbin uygulanan HeLa hücrelerinde hücre canlılığı ve apoptotik oranları belirlemek için sırasıyla XTT canlılık analizi ve floresan mikroskobunda hücrelerin karakteristik morfolojisi değerlendirildi. Vinorelbin uygulanan hücrelerde B hücresi lenfoma 2 (BCL2) ve Siklin D1 (CCND1) genlerinin mRNA ifade düzeyleri de araştırıldı. Bulgular: En yüksek vinorelbin konsantrasyonu olan 100μM'da hücre canlılığının 24 saat sonunda %60'ın altına düştüğü belirlendi. Apoptotik oranın %17,6 olduğu 40μM dozda aynı zamanda nekrotik oranın da 80 ve 100μM dozlara oranla oldukça az olması sebebiyle en etkili apoptotik doz olarak 40μM bulundu. Ayrıca 40μM vinorelbin uygulamasının anti-apoptotik özellikteki BCL2 ile proto-onkogen olan CCND1 genlerinin mRNA ifade edilme düzeylerinde azalmaya neden olduğu saptandı. Sonuç: Mikrotübül dinamiğini etkileyerek antikanser özellik gösteren vinorelbin'in moleküler düzeyde olası etki mekanizmalarının belirlenmesinin, kanser tedavisinde daha etkin yaklaşımların ortaya çıkmasında yardımcı olabileceği kanısındayız. Anahtar sözcükler: Apoptoz, BCL2, CCND1, HeLa Hücre Hattı, Vinorelbin SUMMARY Objective: Microtubules, having roles in many biological functions including cell motility, cell division, intracellular transport, cellular architecture, are major targets for anticancer drugs. Vinorelbine (Navelbin) is one of the molecules that affects microtubules and used as a chemotherapoetic agent in various cancers. In our study, we aimed to determine prospective apoptotic effect of the vinorelbine on human servical carcinoma cell line HeLa cells at 24 hours between 5-100µM dose incubation. Material and Method: For this purpose, vinorelbine treated HeLa cells, were assessed by XTT viability analysis and fluoresence microscopy to determine cell characteristic morphology. B cell lyphoma 2 (BCL2) and cyclin D1 (CCND1) genes' mRNA expression levels were also evaluated in vinorelbine applied cells. Bu çalışma, 9th International Meeting of the International Society for the Study of Xenobiotics (ISSX) Kongresi'nde poster bildirisi olarak sunulmuştur (04-08 Eylül 2010, İstanbul, Türkiye)
... 5,6 Vinflunine demonstrated pronounced activity in multiple cancer cell lines, including small cell lung cancer. [7][8][9][10] This early efficacy prompted clinical investigation of single agent vinflunine in multiple solid tumor settings, including non-small cell lung cancer and mesothelioma. [11][12][13][14][15][16] Herein, we report on a multicenter phase II trial where vinflunine was administered to patients with relapse-sensitive and refractory SCLC. ...
Vinflunine is a new microtubule inhibitor with preclinical activity in small cell lung cancer (SCLC). In this phase II trial, we evaluated vinflunine in patients with relapse-sensitive and refractory SCLC.
This trial aimed to achieve a 20% objective response rate (ORR) in platinum-sensitive patients. Patients with Eastern Cooperative Oncology Group performance status 0 to 2 and measurable disease received vinflunine (320 mg/m(2) IV) every 21 days (< or =6 cycles; response evaluation every 6 weeks).
Patient characteristics (N = 51): median age 63 years (range, 37-85 years); male, 55%; Eastern Cooperative Oncology Group performance status 2, 16%; relapse-sensitive SCLC, 53%. The overall ORR was 19.6% (95% confidence interval [CI] 10-33%). Twelve (23.5%) patients had stable disease; 18 (35.3%) patients had progressive disease. Among relapse-sensitive patients, ORR was 22.2% (95% CI 9-42%). Among relapse-refractory patients, ORR was 16.7% (95% CI 5-37%). Median follow-up was 15 months (range, 12-18 months); median progression-free survival (PFS) was 1.6 months (95% CI 1.3-3.9 months); median overall survival (OS) was 4.9 months (95% CI 3.2- 6.5 months). Among relapse-sensitive patients, PFS and OS were 1.6 and 4.9 months, respectively. Among relapsed-refractory patients, PFS and OS were 1.4 and 4.0 months, respectively. In general, vinflunine was well tolerated, although neutropenia was a notable toxicity. Grade 3/4 toxicities (>5%): neutropenia (32%), arthralgia/myalgia (16%), fatigue (16%), hyponatremia (12%), leukopenia (12%), nausea/vomiting (12%), constipation (6%), and thrombocytopenia (6%). The rates of toxicities were relatively well balanced among relapse-sensitive and refractory patients; one patient died of sepsis that was possibly treatment related.
Vinflunine has activity in relapsed SCLC, including refractory relapsed patients. Neutropenia was common but associated with rare febrile episodes. Additional study in relapse-refractory SCLC is indicated.
Cancer is the second most reason for huge mortality in human life which has attributed to a drastic drift in anticancer drug discovery and development. Small molecules as anticancer agents have contributed tremendously for the treatment of cancer however plants based natural products have the cardinal foundation for traditional medicine schemes and still offer relief to cancer patients. Medicinal plants are known for producing numerous bioactive secondary metabolites that have been used since antiquity due to their medicinal properties. Copious advantages of natural products over conventional anticancer drugs have expanded their use in the health care industry which has been witnessed by the extensive use of natural products in the treatment of multiple cancers. The pleiotropic effects of plant products on targets in multiple ways, phytochemicals are considered as suitable source candidates for anticancer drug design and development. In this review, we attempted to compile structural and functional attributes of natural products with an emphasis on their anticancer properties.
Purpose:
This open label phase II study evaluated the safety and efficacy of vinflunine in patients with breast cancer previously treated with a vinorelbine-based regimen and who progressed during or within 6 months of completing this chemotherapy.
Patients and methods:
Thirty eight patients received vinflunine 320 mg/m(2) once every 3 weeks. The primary efficacy endpoint was overall response rate (ORR). Secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety.
Results:
ORR was 8.3% (95% CI: 1.75-22.4) and DCR was 75% (95% CI: 57.8-87.9). PFS was 4.0 months (95% CI: 2.5-6.1) and OS was 13.6 months (95% CI: 8.7-18.9). Toxicities not hampering dose intensity were as expected neutropenia (75.6% of patients), fatigue (44.7%), constipation (28.9%) and abdominal pain (26.3%).
Conclusion:
Vinflunine demonstrated antitumour activity and can be safely administered in breast cancer patients refractory/resistant to vinorelbine.
Microtubules have been identified as a suitable target for anticancer therapy, primarily based on their biological importance in coordinating chromosomal segregation at mitosis. Two main classes of microtubule-targeted agents, the taxanes and vinca alkaloids, suppress the dynamic behavior of spindle microtubules, inducing mitotic arrest and subsequent apoptotic cell death. Clinical activity of taxanes and first-generation vinca alkaloids in the treatment of solid tumors and hematologic malignancies, respectively, has prompted further research for novel analogs with improved clinical efficacy and safety. Such efforts have led to the development of vinflunine, a bifluorinated vinca alkaloid endowed with unique antitumor properties. Highlighted in this review are the key features of vinflunine that lead to effective suppression of microtubule dynamics and induction of cell death in cancer cells.
Antimitotic drugs are key components of combination chemotherapy protocols for hematological and solid tumors. The taxanes (e.g., paclitaxel) bind to the β subunit of the tubulin heterodimer and reduce microtubule dynamics, leading to cell cycle arrest in G2/M. The effectiveness of combination chemotherapy is limited by tumor resistance to drugs initially or as a cumulative effect after several cycles of treatment. Because changes in the drug receptor may be linked to drug resistance, we investigated changes in β-tubulin isotypes in response to paclitaxel treatment in MCF7 breast cancer cells. We found that paclitaxel induced a 2-3 fold increase in mRNA for β-tubulin IIA and III genes, TUBB2A, and TUBB3. β-Tubulin class III protein increased; however, β-tubulin class II protein was not detected in these cells. Paclitaxel treatment following pretreatment with actinomycin D showed that the change in β-tubulin class III was due to increased transcription and linked to G2/M arrest. The increase in β-tubulin IIA mRNA was due to both enhanced stability and increased transcription, unassociated with G2/M arrest. We used micro-RNA superarrays to look for changes in families of micro-RNAs that might be linked to drug-induced changes in β-tubulin isotype mRNA and/or protein. We found a significant decrease in the tumor suppressor, miR-100, in MCF7 cells in response to paclitaxel treatment. Transfection of MCF7 cells with miR-100 significantly reduced β-tubulin I, IIA, IIB and V mRNA and prevented paclitaxel-induced increases in β-tubulin isotypes. This is the first report of a micro-RNA that regulates these specific β-tubulin isotype mRNAs.
Podophyllotoxin and its analogues have important therapeutic value in the treatment of cancer, due to their ability to induce apoptosis in cancer cells in a proliferation-independent manner. These ligands bind to colchicine binding site of tubulin near the alpha- and beta-tubulin interface and interfere with tubulin polymerization. The binding free energies of podophyllotoxin-based inhibitors of tubulin were computed using a linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model. A training set of 76 podophyllotoxin analogues was used to build a binding affinity model for estimating the free energy of binding for 36 inhibitors (test set) with diverse structural modifications. The average root mean square error (RMSE) between the experimental and predicted binding free energy values was 0.56kcal/mol which is comparable to the level of accuracy achieved by the most accurate methods, such as free energy perturbation (FEP) or thermodynamic integration (TI). The squared correlation coefficient between experimental and SGB-LIE estimates for the free energy for the test set compounds is also significant (R(2)=0.733). On the basis of the analysis of the binding energy, we propose that the three-dimensional conformation of the A, B, C and D rings is important for interaction with tubulin. On the basis of this insight, 12 analogues of varying ring modification were taken, tested with LIE methodology and then validated with their experimental potencies of tubulin polymerization inhibition. Low levels of RMSE for the majority of inhibitors establish the structure-based LIE method as an efficient tool for generating more potent and specific inhibitors of tubulin by testing rationally designed lead compounds based on podophyllotoxin derivatization.
