The effects of selected proline-based cyclic dipeptides on growth and induction of apoptosis in cancer cells
- Authors: Brauns, Seth Clint Aron
- Date: 2004
- Subjects: Cyclic peptides , Antineoplastic agents -- Testing , Apoptosis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:11088 , http://hdl.handle.net/10948/396 , Cyclic peptides , Antineoplastic agents -- Testing , Apoptosis
- Description: 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.
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- Date Issued: 2004
Physiological and non-physiological induction of gastrointestinal differentiation
- Authors: Brauns, Seth Clint Aron
- Date: 1999
- Subjects: Gastrointestinal system -- Differentiation , Gastrointestinal system -- Physiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11089 , http://hdl.handle.net/10948/d1015521
- Description: The human colonic carcinoma cell lines HT-29 and Caco-2 both exhibit structural and functional differentiation under appropriate culture conditions. HT-29 can be induced to differentiate by treatment with short-chain fatty acids or acetoacetate. Caco-2 cells differentiate spontaneously upon contact inhibition. In this study HT-29 cells were treated with 5 mM acetate, propionate, butyrate and acetoacetate (physiological inducers) to assess their effects on the expression of carbonic anhydrase 1, sucrase-isomaltase and alkaline phosphatase which are reported to be markers of gastrointestinal differentiation. The maturation induction observed was compared to that of the spontaneous differentiation observed in Caco-2 cells. Assays were performed over an 18 day period. Results showed a close correlation (p < 0.05) between HT-29 and Caco-2 cell on days 4 and 12. These results indicate that differentiation reported in both cell lines is comparable and can be used as a basis for further comparative studies. In addition, parallel experiments to the above were conducted using a selection of nine rationally designed cyclic dipeptides (CDPs) potential drug entities which were chosen as non-physiological inducers. The results showed that the cyclic dipeptides were able to induce the gastrointestinal phenotype as observed in HT-29 cells treated with physiological inducers. Studies on the effects of energy-related metabolism in HT-29 and Caco-2 cells as induced by physiological and non-physiological inducers indicated that energy metabolism is a significant role player in gastrointestinal differentiation. The results reported show a decrease in ATP concentrations indicating that the cyclic dipeptides, like physiological inducers, affect the energy state of the HT-29 cells and thus may effect the differentiation of these cells. A positive correlation was found between histone phsophorylation and differentiation confirming that histone phsophorylation was partly responsible for the decrease in ATP concentrations. It is suggested that the induction of differentiation in HT- 29 cells could be either due to non-specific transcription of genes by activation of a chromatin switch or specific by the activation of signal transduction pathways based on the flux of ATP through the cells. Differential display RT-PCR is probably the most sensitive method that could be used to validate the suggestion of either a nonspecific transcription of genes or a specific differentiation reported for HT-29 cells.
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- Date Issued: 1999