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Cytotoxicity and cell death pathways invoked by two new rhodium-ferrocene complexes in benign and malignant prostatic
Brandon Weber1, Antonio Serafin, John Michie
1Department of Radiation Oncology, University of Stellenbosch, Tygerberg, SA.
Background:
Apoptotic propensity is currently viewed as an important parameter in drug-induced toxicity. But other cell death pathways exist e.g. micronucleation, intermitotic cell death, abnormal nuclear morphology and necrosis. This investigation explores the onset of apoptosis and abnormal morphology in response to 3 drugs i.e. Cisplatin, a novel Ferrocene (fctfa) and a novel Rhodium-Ferrocene [Rh(fctfa)(cod)] complex.
Materials And Methods:
A pair of prostate cell lines from normal human prostate epithelium (1542N) and malignant human prostate epithelium (1542T) were exposed to increasing concentrations of the drugs for 24 hours, double-stained with FITC-Annexin V and with Propidium Iodide and analysed by dual parameter flow cytometry to quantitate viable cells in quadrant I, early apoptotic cells in quadrant IV and late apoptotic/necrotic cells in quadrant III. Apoptosis was also scored by microscopy after Acridine Orange staining, by Western blots for caspase 3 induction and for caspase 8 induction using a colorimetric assay.
Results:
The toxicity of Cisplatin and the Ferrocene and Rhodium-Ferrocene complexes was found to be 0.9-1.3 microM; 4.1-4.5 microM and 10.1-13.2 microM, respectively. Apoptotic propensity scored after 24 hours was found to be dose-dependent and in the range of 7-19% for Cisplatin and 1-4.1% for the Ferrocene and Rhodium-Ferrocene complexes. Cisplatin produces a distinct apoptotic response followed by a necrotic response, whereas the Ferrocene and the Rhodium-Ferrocene complexes produce a massive necrotic reaction in the region of 3-19% and very little if any apoptosis. Absence of apoptosis was corroborated by lack of caspase 3 activation, absence of typical apoptotic morphology and by lack of caspase 8 activation.
Conclusion:
The 3 drugs Cisplatin, the novel Ferrocene and the novel Rhodium-Ferrocene complexes show similar toxicities in the 1-10 micro-molar range in prostate cell lines. However the drugs differ significantly in the activation of death pathways. While Cisplatin predominantly induces apoptosis documented by morphology, Annexin V staining and caspase 8 activation, the Ferrocene and Rhodium-Ferrocene complexes induce late necrosis and abnormal nuclear morphology. Unlike Cisplatin-treated cells which enter apoptosis and necrosis sequentially, the 2 Ferrocene drugs invoke direct entry of cells into late necrosis without first entering the early apoptotic compartment.
Insights
Cisplatin induces apoptosis and necrosis sequentially in prostate cells, while Ferrocene and Rhodium-Ferrocene complexes cause direct necrosis. These findings highlight distinct drug-induced cell death pathways.
Area of Science:
- * Pharmacology
- * Toxicology
- * Cancer Research
Background:
- * Drug-induced toxicity is often assessed by apoptotic propensity.
- * Other cell death pathways, including necrosis and abnormal nuclear morphology, also contribute to toxicity.
- * Understanding diverse cell death mechanisms is crucial for drug development.
Purpose of the Study:
- * To investigate the cell death pathways induced by Cisplatin, a novel Ferrocene complex, and a novel Rhodium-Ferrocene complex.
- * To compare the apoptotic and morphological responses of normal and malignant prostate cell lines to these drugs.
- * To elucidate the mechanisms of cell death triggered by these compounds.
Main Methods:
- * Prostate cell lines (1542N and 1542T) were treated with increasing drug concentrations for 24 hours.
- * Cells were analyzed using Annexin V/Propidium Iodide staining and flow cytometry to quantify cell viability and death.
- * Apoptosis was further assessed via microscopy, Western blot for caspase 3, and colorimetric assays for caspase 8.
Main Results:
- * Cisplatin, Ferrocene, and Rhodium-Ferrocene complexes exhibited toxicities in the 0.9-13.2 micro-molar range.
- * Cisplatin induced dose-dependent apoptosis (7-19%) followed by necrosis.
- * Ferrocene and Rhodium-Ferrocene complexes primarily induced necrosis (3-19%) with minimal apoptosis, confirmed by absent caspase activation.
Conclusions:
- * Cisplatin, Ferrocene, and Rhodium-Ferrocene complexes display similar toxicity profiles but distinct cell death pathway activation.
- * Cisplatin predominantly triggers apoptosis and subsequent necrosis, evidenced by caspase 8 activation and morphological changes.
- * Ferrocene and Rhodium-Ferrocene complexes induce direct necrosis, bypassing early apoptotic stages and lacking caspase activation.
