Fungal taxol and baccatin III induce changes in nuclear morphology in JR4 Jurkat and HeLa cells Changes in cell nuclear morphology, product information such as condensed and fragmented nuclei are considered late events of apop tosis. In order to identify the changes in cell nuclei in JR4 Jurkat and HeLa cells upon treatment with indicated con centrations of fungal taxol and baccatin III, cells were stained with Hoechst or AO EB and visualized by fluores cence microscopy. Our data reveal that both fungal taxol and baccatin III induce chromatin aggregation and nu clear condensation in JR4 Jurkat and HeLa cells. The control cells that stained evenly with Hoechst were also found to stain lightly and evenly with AO but stained negative for EB, suggesting the presence of live cells.
On the other hand, HeLa cells after 12 h of treatment, exhibited a condensed orange nucleus, whereas the necrotic cells display a structurally intact nucleus with an evenly distributed orange staining. Fungal taxol and baccatin III induce DNA fragmentation in both JR4 Jurkat and HeLa cells The fragmentation of nuclear DNA is one of the hall marks of apoptosis. It is known that DNA fragmentation is carried out by the caspase activated DNase. Acti vation of CAD leads to cleavage of nuclear DNA into mul tiples of 200 bp oligonucleosomal size fragments. To confirm the induction of apoptosis, JR4 Jurkat and HeLa cells were treated with fungal taxol or baccatin III. Low molecular weight DNA isolated from these cells was ana lyzed in 1. 2% agarose gels.
DNA ladder formation is ob served upon taxol or baccatin III treatment in JR4 Jurkat and HeLa cells, while there is no DNA fragmentation seen in untreated and DMSO treated cells. This con firmed that both fungal taxol and baccatin III can induce apoptosis in JR4 Jurkat or HeLa cells. Discussion Taxol is a potent chemotherapeutic agent that induces apoptosis in a variety of cancer cells including ovarian, endometrial, lung, prostate, colorectal, thyroid, acute myeloid leukemia and breast cancer cells. It was of interest to investigate whether baccatin III, the biosynthetic precursor of taxol, functions by the same mechanism as taxol. This is the first report on the apoptotic mechanisms involved in fungal baccatin III induced cytotoxicity in cancer cells. Comparison of cell cycle analysis of Jurkat cells treated with fungal taxol or baccatin III revealed similar time and concentration dependent induction of apoptosis. However, increased apoptotic sub G1 cells Entinostat after fungal baccatin III treatment occurred at higher concentration compared to fungal taxol. This might be either due to the high affinity of taxol to microtubules or involvement of non tubulin factors.