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Simultaneous Interference with HER1/EGFR and RAC1 Signaling Drives Cytostasis and Suppression of Survivin in Human
G Karpel-Massler1,2,3,4, M-A Westhoff5, R E Kast6
1Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany. georg.karpel@gmail.com.
Abstract:
We have previously reported that combined inhibition of the epidermal growth factor receptor by erlotinib and of RAC1 by NSC23766 yielded a synergistic antiproliferative effect on established and primary cultured glioblastoma cells. The current study aimed at identifying the molecular mechanism. Staining for annexin V/PI or carboxyfluorescein succinimidyl ester was performed in order to determine the induction of apoptosis, necrosis or cytostasis in established and primary cultured glioblastoma cells. Moreover, expression of Ki-67 was determined by immunofluorescence, and the expression of cell cycle proteins was analysed by Western blot. Our data show that combined treatment with erlotinib and NSC23766 resulted in a reduced number of cell divisions, a significantly decreased Ki-67 expression, increased apoptosis and autophagy when compared to single agent treatments. On the molecular level, concomitant treatment with both agents resulted in a pronounced downregulation of cyclin D1, cyclin-dependent kinases 2, 4 and 6, as well as of survivin when compared to treatments with either agent alone. In conclusion, we demonstrate that combined treatment of human glioma cell lines in vitro with erlotinib and NSC23766 markedly inhibits cell division, induces apoptosis independent of caspase-3 activation and induces autophagy concomitant with suppression of survivin.
Insights
Combined treatment with erlotinib and NSC23766 synergistically inhibits glioblastoma cell proliferation. This approach induces apoptosis and autophagy while downregulating key cell cycle proteins and survivin.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Glioblastoma multiforme (GBM) is an aggressive brain tumor with limited treatment options.
- Targeted therapies inhibiting epidermal growth factor receptor (EGFR) and RAC1 show promise.
- Previous studies indicated synergistic antiproliferative effects of combined erlotinib and NSC23766 on glioblastoma cells.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying the synergistic antiproliferative effect of combined erlotinib and NSC23766 treatment on glioblastoma cells.
- To investigate the induction of apoptosis, necrosis, and cytostasis.
- To analyze the impact on cell cycle progression and key protein expression.
Main Methods:
- Annexin V/PI and carboxyfluorescein succinimidyl ester staining to assess cell death.
- Immunofluorescence for Ki-67 expression analysis.
- Western blot analysis for cell cycle protein expression.
Main Results:
- Combined erlotinib and NSC23766 treatment significantly reduced cell divisions and Ki-67 expression.
- Enhanced apoptosis and autophagy were observed compared to single-agent treatments.
- Pronounced downregulation of cyclin D1, cyclin-dependent kinases (CDKs) 2, 4, and 6, and survivin was noted.
Conclusions:
- Combined erlotinib and NSC23766 treatment effectively inhibits glioblastoma cell division in vitro.
- The combination induces apoptosis independent of caspase-3 activation and promotes autophagy.
- Suppression of survivin and key cell cycle proteins contributes to the observed anti-cancer effects.
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