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Concomitant decrease of resistance and modifications of the cytoskeleton after all-trans retinoic acid and phorbol
V Debal1, F Breillout, M Manfait
1Laboratoire de Spectroscopie Biomoleculaire, GIBSA, IFR 53, U.F.R. de Pharmacie, Reims, France.
Abstract:
The bladder carcinoma cell line J82-NVB was selected for resistance to the new vinca alkaloid Navelbine. These cells possessed a non-MDR phenotype and were cross-resistant to vinca alkaloids and taxoids. Some morphological differences between sensitive (J82) and resistant (J82-NVB) cells were observed J82 cells had a heterogeneous population morphology with both epithelial and spindle shaped cells, while J82-NVB cells were almost all of the epithelial type. Vimentin intermediate filaments were less organized in J82-NVB than in J82 cells. Moreover, desmosomes were present in the membranes of J82NVB cells but not in J82 cells. These findings suggest that J82 cells are poorly differentiated epithelial cells while J82-NVB cells possess some characteristics of a more differentiated epithelial cell line. After a two-week treatment with all-trans retinoic acid, all the cells became spindle shaped, vimentin filaments reappeared in the cytoplasm of J82-NVB cells and desmosomes disappeared from the membranes of these cells. These changes were accompanied by a decrease from 17 to 4.6 of the resistance factor of J82-NVB cells to Navelbine. This decrease in resistance was concomitant with modifications of microtubules assembly regulation mechanisms. After Navelbine treatment, microtubule reassembly occurred in resistant but not in sensitive nor in retinoic acid treated cells. Okadaic acid, a protein phosphatase inhibitor, inhibited microtubule reassembly in resistant cells, and 2-aminopurine, a protein kinase inhibitor, induced microtubule reassembly in sensitive cells after Navelbine treatment. These findings show that microtubule reassembly after depolymerization is regulated by the kinase/phosphatase systems. A treatment with phorbol myristate acetate (PMA), a protein kinase C (PKC) agonist, induced the same morphological modifications and resistance decrease as retinoic acid treatment. A specific PKC inhibitor (Bisindolymaleimide) prevented these PMA-induced morphological modifications and resistance decrease in J82-NVB cells, showing that these effects were mediated by PKC. This study suggests that, in part by acting on some properties of the cytoskeleton, the differentiation modulator, retinoic acid, and the signal transduction modulator, phorbol myristate acetate, can decrease the resistance of J82-NVB cells to microtubule poisons.
Insights
Drug resistance in bladder cancer cells can be reduced by targeting cytoskeleton properties. Retinoic acid and phorbol myristate acetate decrease Navelbine resistance by modulating microtubule assembly via kinase/phosphatase systems.
Area of Science:
- Cell Biology
- Cancer Research
- Pharmacology
Background:
- The bladder carcinoma cell line J82-NVB exhibits resistance to Navelbine, a vinca alkaloid.
- This resistance is associated with a non-multidrug resistance (MDR) phenotype and cross-resistance to taxoids.
- Morphological and cytoskeletal differences, including vimentin organization and desmosome presence, distinguish resistant J82-NVB cells from sensitive J82 cells, suggesting altered differentiation states.
Purpose of the Study:
- To investigate the mechanisms underlying Navelbine resistance in J82-NVB cells.
- To explore the effects of differentiation modulators, all-trans retinoic acid and phorbol myristate acetate (PMA), on drug resistance and cellular characteristics.
- To elucidate the role of microtubule assembly regulation and kinase/phosphatase systems in modulating drug resistance.
Main Methods:
- Comparative morphological analysis of J82 and J82-NVB cells.
- Treatment of J82-NVB cells with all-trans retinoic acid and PMA.
- Assessment of Navelbine resistance factor before and after treatment.
- Investigation of microtubule assembly dynamics following Navelbine treatment in sensitive and resistant cells.
- Utilizing protein phosphatase (Okadaic acid) and protein kinase (2-aminopurine, Bisindolymaleimide) inhibitors to probe regulatory pathways.
Main Results:
- All-trans retinoic acid treatment induced morphological changes in J82-NVB cells, decreased Navelbine resistance, and altered microtubule assembly regulation.
- Microtubule reassembly after depolymerization was regulated by kinase/phosphatase systems, with Okadaic acid inhibiting and 2-aminopurine inducing reassembly.
- PMA treatment mimicked retinoic acid effects, inducing similar morphological changes and resistance decrease, which were prevented by a protein kinase C (PKC) inhibitor, indicating PKC mediation.
- These findings suggest that differentiation modulators can decrease resistance to microtubule poisons by affecting cytoskeleton properties.
Conclusions:
- J82-NVB cells display altered differentiation and cytoskeletal characteristics compared to sensitive J82 cells.
- All-trans retinoic acid and PMA can reduce Navelbine resistance in J82-NVB cells by modulating microtubule dynamics.
- The kinase/phosphatase system plays a critical role in regulating microtubule reassembly and drug resistance, with PKC being a key mediator of PMA's effects.