Zsuzsanna MIHÁLY1, Gy. MUNKÁCSY1,
R. SCHÄFER2, R. ABDUL-GHANI2, B. GYÕRFFY1,3
1MTA-SE Research Group for Pediatrics and Nephrology;
1st Department of Pediatrics, Semmelweis University, Budapest, Hungary
2Charité Universitätsmedizin, Berlin, Germany
3Semmelweis University, 2nd Department of Pediatrics, Budapest, Hungary
Background: Doxorubicin and paclitaxel are first line drugs in the
chemotherapy of urinary bladder cancer. The development of
chemoresistance to these chemicals results in the reduction of their
efficiency. The product of the gene PSMB7 is a component of the
proteasome β subunit, so far its role has not been studied in resistance
to doxorubicin or paclitaxel. The gene has significantly overexpressed
in doxorubicin-resistant cell-lines therefore the option of
its role in the generation of resistance has been identified. Since
cell-lines indicated resistance to paclitaxel as well, tests have been
conducted with the use of both drugs. Our research aims were to examine whether RNAi silencing of the PSMB7 gene has an impact
on the chemoresistance of doxorubicin-resistant cell-lines.
Methods:We worked with MCF-7 cell-lines. The gene was silenced
by RNA interference, which was produced by self designed siRNA
oligos and siPORT NeoFX transfection reagent. The validation of
the RNA happened with RNeasy Mini kit, the gene expression was
measured by RT-PCR. The drug treatment took place 24 hours after
transfection. The cells were counted by CASY DT Cell Counter.
Experiments were carried out three times with both doxorubicin
and paclitaxel, and each well was measured three times in each
experiment.
Results: 79.8%±13.3% of resistance cells survived after doxorubicin
treatment. Combined with gene silencing, only
31.8%±6.4% of the MCF-7-RAdr cells survived. The significance
between siRNA-treated and siRNA-untreated MCF-7-RAdr cells
after doxorubicin treatment was p > 0.001. After doxorubicin
treatment, 48.3±8.1% of sensitive cells survived. 72.4% of negative
control siRNA-treated cells survived. After combination of
paclitaxel treatment and gene silencing, 22.6±4% of the MCF-7-
RAdr cells survived compared to siRNA-untreated cells, while
43.8±6% of sensitive cells survived. Relative cell vitality of siRNAtreated
and siRNA-untreated MCF-7-RAdr cells after paclitaxel
treatment differed significantly (p = 0.03).
Discussion: According to our findings, by reducing the overexpression
of the PSMB7 gene via gene silencing the resistant cell’s
chemoresistance significantly decreased. Consequently the doxorubicin-
and paclitaxel-therapy became more efficient. In order to
predict clinical significance gene expression should be measured in
tumor samples from therapy resistant patients.