An increasing number of cyclic dipeptides (CDPs) have been shown to exhibit important biological activity including antifungal, antibacterial, anticonvulsant and immunomodulatory activity. Furthermore, some CDP derivatives have been shown to exhibit antitumour activity in vitro and in vivo. Several proline-based CDPs that exhibit biological activity have been detected in various processed foods and beverages. In the present study, the potential of seven proline-based CDPs to inhibit cancer cell growth was investigated in HT-29 (colon), HeLa (cervical), MCF-7 (breast) and WHCO3 (oesophageal) cancer cell lines. The CDPs used in this study were cyclo(Phe-Pro), cyclo(Tyr-Pro), cyclo(Gly-Pro), cyclo(Pro- Pro), cyclo(His-Pro), cyclo(Leu-Pro) and cyclo(Thr-Pro). The sulforhodamine B (SRB) cell growth assay was used in an initial screening phase to investigate the effects of the CDPs in HT-29, HeLa and MCF-7 cells. After exposing the cells to 10mM of the respective CDPs for 48 hours, the SRB assay results showed that only cyclo(Phe-Pro) exhibited more than 50% growth inhibition (p<0.01) in the three cell lines. The other CDPs showed comparatively marginal growth-inhibitory effects, except for cyclo(Tyr-Pro), which exhibited a pronounced effect in MCF-7 cells compared to HT-29 and HeLa cells. The MTT assay was used to confirm the SRB assay results for cyclo(Phe-Pro) and cyclo(Tyr-Pro), extending the investigation to the use of the fourth cell line WHCO3 and using a longer exposure time of 72 hours. The MTT assay demonstrated a dosedependent (0.008-10 mM) growth inhibition by cyclo(Phe-Pro) with an IC50 value of 4.04 ± 1.15 mM for HT-29 cells. Cyclo(Phe-Pro) was subsequently used to investigate whether the growth-inhibitory effects of this CDP were related to the induction of apoptosis in HT-29 cells. Hoechst 33342 staining showed that 5mM cyclo(Phe-Pro) induced characteristic chromatin condensation and nuclear fragmentation in 18.3 ± 2.8% (p<0.01) of HT-29 cells after 72 hours. Furthermore, annexin V binding revealed that HT-29 cells treated with 5 mM cyclo(Phe-Pro) displayed phosphatidylserine externalization after 48 hours. In addition, it was shown that 10 mM cyclo(Phe-Pro) induced poly(ADP-ribose)polymerase PARP cleavage, one of the hallmark events of apoptosis. The use of the broad-range caspase inhibitor Z-VAD-FMK, showed that this PARP cleavage was caspase-dependent, which in turn was confirmed by demonstrating an increase in caspase-3 activity (p<0.01) in cyclo(Phe- Pro)-treated HT-29 cells. In conclusion, these findings demonstrate that cyclo(Phe-Pro) inhibited the growth of HT- 29, MCF-7, HeLa and WHCO3 cells, and induced apoptosis in HT-29 colon cancer cells, suggesting the potential antitumour activity of cyclo(Phe-Pro)-related CDPs.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:11088 |
| Date | January 2004 |
| Creators | Brauns, Seth Clint Aron |
| Publisher | University of Port Elizabeth, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Doctoral, PhD |
| Format | 132 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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