The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline,?
WP 631 in comparison to
doxorubicin (DOX), a first generation
anthracycline, currently the most widely used
pharmaceutical in clinical oncology. Experiments were performed in SKOV-3
ovarian cancer cells which are otherwise resistant to standard drugs such as
cis-platinum and
adriamycin. As attention was focused on the ability of
WP 631 to induce apoptosis, this was examined using a double staining method with
Annexin V and
propidium iodide probes, with measurement of the level of intracellular
calcium ions and cytosolic
cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of
caspase activation and
DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX
histones) in the development of apoptotic events.
WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic
drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of
phosphatidylserine and the elevated level of
cytochrome c. An extension of incubation time led to an increase in intracellular
calcium levels
after treatment with DOX. Lower changes in the
calcium content were associated with the influence of
WP 631. DOX led to the activation of all tested
caspases, 8, 9 and 3, whereas
WP 631 only induced an increase in
caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active
caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that
WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that
WP 631 generates early apoptosis and cell death independent of
caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of
WP631 and DOX suggest that this bisanthracycline can be an effective alternative in
ovarian cancer treatment.