Téma: Molekulární mechanismy a biomarkery I
Číslo abstraktu: 020
Taxanes are cytostatics routinely used in the anticancer therapy of solid tumors. Two classical taxanes, paclitaxel and docetaxel, are often employed in the treatment of breast, ovary, prostate and other types of cancers. Unfortunately, resistance of cancer cells to the effect of classical taxanes remains serious problem of taxane therapy.
Cell resistance to taxanes can be associated with ABC transporters overexpression, changes of taxane metabolism, mutations of tubulin subunits and defects in proper apoptotic course. In addition, the taxane resistance can be caused by changes of expression of β tubulin classes; the resistance associated with autophagy was also recently described.
We used breast cancer cell lines SK-BR-3, MCF-7 and T47D and derived sublines SK-/BR-3/PACRES and MCF-7/PAC-RES that proliferate in the cell death inducing concentration of paclitaxel (100 and 300 nM, respectively). For cell death induction we used classical taxane paclitaxel, and novel taxane SB-T- 12854 that effectively induced cell death in paclitaxel-sensitive as well as resistant sublines.
For assessment of autophagy level, we detected proteins associated with autophagy induction and course as p62, beclin, LC3, ATG3 and ATG16. Moreover, we observed autophagosomes formation in the cells by confocal microscopy. The relationship of level of autophagy and paclitaxel resistance was tested by taxane treatment of cells with activated (rapamycin treated) or suppressed (bafilomycin treated) autophagy.
Next, we assessed the expression and localization of β tubulin classes II and III in paclitaxel- sensitive and paclitaxel-resistant breast cancer cells by western blot and confocal microscopy.
Results and conclusions
Taxane application did not change the basal level of autophagy in SKBR- 3 or MCF-7 cells either but it increased slightly the basal level of autophagy in T47D cells.
There were not differences in the level of autophagy between sensitive and resistant SK-BR-3 cells. The MCF- 7/PAC-RES subline had somewhat lower level of autophagy than the sensitive subline. Suppressing of autophagy did not affect the resistance to paclitaxel in any tested cells. The activation of autophagy by rapamycin had no effect either except of increasing autophagy in SK-BR-3/PAC-RES cells that led to increased resistance to paclitaxel.
As for β tubulin-classes expression, we did not find any significant changes in expression of βII tubulin between sensitive and resistant SK-BR-3 and MCF-7 sublines. Application of taxanes did not change level of βII tubulin in the cells; however the application increased βII tubulin level in T47D cells.
The level of βIII tubulin was higher in SK-BR-3/PAC-RES cells compared with sensitive subline. Surprisingly, the level of βIII tubulin was lower in MCF-7/PAC-RES cells than in the sensitive subline. As previously, application of taxanes did not change βIII tubulin level in SK-BR-3 and MCF-7 cell lines but it increased level of βIII tubulin in T47D cells.
We can conclude there is no important role of autophagy in paclitaxel resistance of MCF-7 cells. On the contrary, it can be involved in the paclitaxel-resistance of SKBR- 3 and T47D cells probably by nonspecific suppressing of cell death induction.
Next, while the role of βII tubulin remains rather unclear; βIII tubulin can have important function in the taxane resistance, especially in T47D and SK-BR-3 cells.
This work was supported by grant LH14096, KONTAKT II, Ministry of Education, Youth and Sports, by research project PRVOUK P27 from the Charles University in Prague and by grant NT 13679-4 from the Internal Grant Agency, Ministry of Health of the Czech Republic
Datum přednesení příspěvku: 2. 12. 2015