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.
- Full Text:
- Date Issued: 2004
- 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.
- Full Text:
- Date Issued: 2004
An investigation into the induction of oxidative stress and apoptosis by microcystin-LR in the CaCo2 cell line and intestinal tract of Balb/c mice
- Authors: Botha, Nicolette
- Date: 2003
- Subjects: Microcystis aeruginosa -- Toxicology , Apoptosis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11066 , http://hdl.handle.net/10948/349 , Microcystis aeruginosa -- Toxicology , Apoptosis
- Description: This study reports the findings on the effect of Microcystin-LR (MCLR) on the gastrointestinal tract cells of mice and on two different cell lines, Caco2 and MCF-7. The cyanobacterium Microcystis aeruginosa produces the potent toxin, MCLR. This toxin has been implicated in a number of cases of ill-health. It was decided to investigate whether microcystin-LR induced apoptosis in the gastrointestinal tract of mice and also which possible mechanisms were involved in the induction in vitro. Balb/c mice were given a 75% LD50 intraperitoneal dose of pure microcystin -LR and sacrificed at 8, 16, 24 and 32 hours post-exposure. The small intestinal sections were stained with haematoxylin and eosin and examined for apoptotic cells. There was a time-dependent increase in the number of apoptotic cells with most in the duodenum and the jejunum. No change in glycogen content was evident at 24 hours post exposure when PAS-stained sections were examined. To determine that microcystin was the agent responsible for the changes, fluoroscein isothiocyanate (FITC) immunostaining for the toxin was done on the sections. Apoptosis in vitro was investigated in Caco2, a cell line that behaves like normal enterocytes when the cells are differentiated at confluency, and MCF-7, a breast cancer cell line deficient in pro-caspase-3, cells by 3-[dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and by staining with DAPI and Rhodamine 123. MCLR exposure induced apoptosis, as seen in decreased cell viability and increased leakage of LDH, as well as mitochondrial damage shown by Rhodamine staining. The MCF-7 cells, deficient in pro-caspase-3, and Caco2 cells did not show cleavage of poly(ADP)ribose polymerase (PARP) after exposure to 50μM MCLR after 72 hours exposure. Both micro- and milli-calpain activity was however significantly increased in both cell lines exposed to the toxin. There was a significant increase in H2O2, one of the key reactive oxygen species, production during the first 30 minutes that the cells were exposed to 50 mM MCLR.
- Full Text:
- Date Issued: 2003
- Authors: Botha, Nicolette
- Date: 2003
- Subjects: Microcystis aeruginosa -- Toxicology , Apoptosis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11066 , http://hdl.handle.net/10948/349 , Microcystis aeruginosa -- Toxicology , Apoptosis
- Description: This study reports the findings on the effect of Microcystin-LR (MCLR) on the gastrointestinal tract cells of mice and on two different cell lines, Caco2 and MCF-7. The cyanobacterium Microcystis aeruginosa produces the potent toxin, MCLR. This toxin has been implicated in a number of cases of ill-health. It was decided to investigate whether microcystin-LR induced apoptosis in the gastrointestinal tract of mice and also which possible mechanisms were involved in the induction in vitro. Balb/c mice were given a 75% LD50 intraperitoneal dose of pure microcystin -LR and sacrificed at 8, 16, 24 and 32 hours post-exposure. The small intestinal sections were stained with haematoxylin and eosin and examined for apoptotic cells. There was a time-dependent increase in the number of apoptotic cells with most in the duodenum and the jejunum. No change in glycogen content was evident at 24 hours post exposure when PAS-stained sections were examined. To determine that microcystin was the agent responsible for the changes, fluoroscein isothiocyanate (FITC) immunostaining for the toxin was done on the sections. Apoptosis in vitro was investigated in Caco2, a cell line that behaves like normal enterocytes when the cells are differentiated at confluency, and MCF-7, a breast cancer cell line deficient in pro-caspase-3, cells by 3-[dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and by staining with DAPI and Rhodamine 123. MCLR exposure induced apoptosis, as seen in decreased cell viability and increased leakage of LDH, as well as mitochondrial damage shown by Rhodamine staining. The MCF-7 cells, deficient in pro-caspase-3, and Caco2 cells did not show cleavage of poly(ADP)ribose polymerase (PARP) after exposure to 50μM MCLR after 72 hours exposure. Both micro- and milli-calpain activity was however significantly increased in both cell lines exposed to the toxin. There was a significant increase in H2O2, one of the key reactive oxygen species, production during the first 30 minutes that the cells were exposed to 50 mM MCLR.
- Full Text:
- Date Issued: 2003
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