Possible anticancer effects of pmc-a through the mechanisms of mitochondrial apoptosis and cell cycle arrest in colon carcinoma cell lines; hct116 wt and bax (-/-)

Karslı, Gizem (2008) Possible anticancer effects of pmc-a through the mechanisms of mitochondrial apoptosis and cell cycle arrest in colon carcinoma cell lines; hct116 wt and bax (-/-). [Thesis]

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In previous studies reported from our lab, pramanicin (PMC) induced apoptosis in Jurkat leukemia cells in a dose- and time-dependent manner; and this effect has been shown to be caspase dependent. Therefore, in this study, possible anticancer effects of PMC and its 9 analogs were determined; and further insight into the mechanism of action is provided by focusing on Bcl-2 family proteins and cell cycle arrest in HCT-116 WT and Bax(-/-) colon cancer cell lines. Among 9 analogs screened PMC-A was the most effective chemical in inducing apoptosis as shown by M30 and time-dependent Annexin V assays. Dose and time kinetics indicated that PMC-A was the most effective in inducing apoptosis (25 μM, ≈30% in 24 hours), therefore, it was chosen for further experiments. To understand the role of Bax in apoptotic pathway, parallel experiments were carried out in HCT116 WT and Bax(-/-) cells. Compared to the WT cell lines, the apoptotic response of Bax(-/-) cells after the treatment with PMC-A at 25 μM was detected at 24th hour indicating a late apoptotic response. Caspase-9 activation and apaf-1 up-regulation were observed consistently in a time-dependent manner. Although caspase-3 cleavage was observed in WT cells, there was no caspase-3 cleavage in HCT116 Bax(-/-) cells indicating a caspase-3- independent apoptotic pathway. To provide further insight into the apoptotic response, regulation of Bcl-2 protein family members (among pro-survivals, Bcl-2, Mcl-1 and Bcl-xL; among pro-apoptotics Bax, Bak, Bim, Puma, Bad and Bid) were monitored by immunoblotting in a time dependent manner. As Bcl-2 was down-regulated, Mcl-1 was up-regulated and Bcl-xL levels remained the same. On the other hand, among pro-apoptotic proteins, Bim and Bax was up-regulated, Bak was down-regulated, Bad and Puma levels remained constant. Since Bcl-2 and Mcl-1 were the regulated proteins among pro-survivals, their modulation in Bax(-/-) cells were determined. Their pattern did not change in comparison to WT cells, which can be explained by the upstream effect of these proteins. Among other pro apoptotic proteins, Bid was truncated in WT cells, whereas was not truncated in Bax(-/-) cells. Since equilibrium between cell proliferation and death has been proposed to be a fundamental point in carcinogenesis, effect of PMC-A on cell cycle was identified. There was an increase in p21 level, a general inhibitor protein, in both cell types; WT and Bax(-/-). WT cells did not arrest at any phases of the cell cycle, however, Bax(-/-) cells arrested at G2-M phase, indicating a sensitive profile of Bax(-/-) cells to cell cycle arrest. Our data indicate that PMC-A induced intrinsic apoptosis pathway, triggering caspase-cleavage and Bcl-2 regulation in HCT116 WT cells, whereas Bax deficiency induced a different pathway in programmed cell death response. In summary, apoptosis is a dominant effect of PMC-A in HCT116 cells, on the other hand Bax deficiency made cells more sensitive to PMC-A induced cell cycle arrest indicating alternative response to apoptosis.
Item Type: Thesis
Uncontrolled Keywords: PMC-A. -- Apoptosis. -- Bcl-2 protein family. -- Cell cycle arrest. -- HCT116 cell lines. -- Cancer. -- Cell cycle. -- PMC-A. -- Apoptoz. -- Bcl-2 protein ailesi. -- Hücre döngüsünden kaçış. -- HCT116 hücre hattı. -- Kanser. -- Hücre siklusu.
Subjects: T Technology > TA Engineering (General). Civil engineering (General) > TA164 Bioengineering
Divisions: Faculty of Engineering and Natural Sciences > Academic programs > Biological Sciences & Bio Eng.
Faculty of Engineering and Natural Sciences
Depositing User: IC-Cataloging
Date Deposited: 25 Aug 2011 10:55
Last Modified: 26 Apr 2022 09:55
URI: https://research.sabanciuniv.edu/id/eprint/16760

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