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Autonomous inhibition of apoptosis correlates with responsiveness of colon carcinoma cell lines to ciglitazone.

ABSTRACT: Colorectal cancer is a leading cause of mortality worldwide. Resistance to therapy is common and often results in patients succumbing to the disease. The mechanisms of resistance are poorly understood. Cells basically have two possibilities to survive a treatment with potentially apoptosis-inducing substances. They can make use of their existing proteins to counteract the induced reactions or quickly upregulate protective factors to evade the apoptotic signal. To identify protein patterns involved in resistance to apoptosis, we studied two colorectal adenocarcinoma cell lines with different growth responses to low-molar concentrations of the thiazolidinedione Ciglitazone: HT29 cells underwent apoptosis, whereas SW480 cells increased cell number. Fluorescence detection and autoradiography scans of 2D-PAGE gels were performed in both cell lines to assess protein synthesis and turnover, respectively. To verify the data we performed shotgun analysis using the same treatment procedure as in 2D-experiments. Biological functions of the identified proteins were mainly associated with apoptosis regulation, chaperoning, intrinsic inflammation, and DNA repair. The present study suggests that different growth response of two colorectal carcinoma cell lines after treatment with Ciglitazone results from cell-specific protein synthesis and differences in protein regulation.

SUBMITTER: Baron DM

PROVIDER: S-EPMC4263530 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Autonomous inhibition of apoptosis correlates with responsiveness of colon carcinoma cell lines to ciglitazone.

Baron David M DM Kaindl Ulrike U Haudek-Prinz Verena J VJ Bayer Editha E Röhrl Clemens C Gerner Christopher C Marian Brigitte B

PloS one 20141211 12

Colorectal cancer is a leading cause of mortality worldwide. Resistance to therapy is common and often results in patients succumbing to the disease. The mechanisms of resistance are poorly understood. Cells basically have two possibilities to survive a treatment with potentially apoptosis-inducing substances. They can make use of their existing proteins to counteract the induced reactions or quickly upregulate protective factors to evade the apoptotic signal. To identify protein patterns involv ...[more]

PMID: 25502518

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Project description:Extensive research has been done in the search for innovative treatments against colon adenocarcinomas; however, the incidence rate of patients remains a major cause of cancer-related deaths in Malaysia. Natural bioactive compounds such as curcumin have been substantially studied as an alternative to anticancer drug therapies and have been surmised as a potent agent but, nevertheless, remain deficient due to its poor cellular uptake. Therefore, efforts now have shifted toward mimicking curcumin to synthesize novel compounds sharing similar effects. A synthetic analog, (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2-ene-1-one (DK1), was recently synthesized and reported to confer improved bioavailability and selectivity toward human breast cancer cells. This study, therefore, aims to assess the anticancer mechanism of DK1 in relation to the induction of in vitro cell death in selected human colon cancer cell lines. Using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, the cytotoxicity of DK1 towards HT29 and SW620 cell lines were investigated. Acridine orange/propidium iodide (AO/PI) dual-staining assay and flow cytometry analyses (cell cycle analysis, Annexin/V-FITC and JC-1 assays) were incorporated to determine the mode of cell death. To further determine the mechanism of cell death, quantitative real-time polymerase chain reaction (qRT-PCR) and proteome profiling were conducted. Results from this study suggest that DK1 induced changes in cell morphology, leading to a decrease in cell viability and subsequent induction of apoptosis. DK1 treatment inhibited cell viability and proliferation 48 h post treatment with IC50 values of 7.5 ± 1.6 µM for HT29 cells and 14.5 ± 4.3 µM for SW620 cells, causing cell cycle arrest with increased accumulation of cell populations at the sub-G₀/G₁phaseof 74% and 23%, respectively. Flow cytometry analyses showed that DK1 treatment in cancer cells induced apoptosis, as indicated by DNA fragmentation and depolarization of the mitochondrial membrane. qRT-PCR results show significant upregulation in the expression of caspase-9 in both HT29 and SW620 cell lines, further supporting that cell death induction by DK1 is via an intrinsic pathway. These outcomes, therefore, demonstrate DK1 as a potential anticancer agent for colon adenocarcinoma due to its anti-apoptotic attributes.

Project description:BackgroundNew efficient therapies for urothelial carcinoma (UC) are urgently required. Small-molecule drugs targeting chromatin regulators are reasonable candidates because these regulators are frequently mutated or deregulated in UC. Indeed, in previous work, Romidepsin, which targets class I histone deacetylases (HDAC), efficiently killed UC cells, but did not elicit canonical apoptosis and affected benign urothelial cells indiscriminately. Combinations of HDAC inhibitors with JQ1, an inhibitor of bromodomain-containing acetylation reader proteins like BRD4, which promote especially the transcription of pro-tumorigenic genes, have shown efficacy in several tumor types. We therefore investigated the effects of combined Romidepsin and JQ1 treatment on UC and benign urothelial control cells.ResultsJQ1 alone induced cell cycle arrest, but only limited apoptosis in eight UC cell lines with strongly varying IC50 values between 0.18 and 10 μM. Comparable effects were achieved by siRNA-mediated knockdown of BRD4. Romidepsin and JQ1 acted in a synergistic manner across all UC cell lines, efficiently inhibiting cell cycle progression, suppressing clonogenic growth, and inducing caspase-dependent apoptosis. Benign control cells were growth-arrested without apoptosis induction, but retained long-term proliferation capacity. In UC cells, anti-apoptotic and oncogenic factors Survivin, BCL-2, BCL-XL, c-MYC, EZH2 and SKP2 were consistently downregulated by the drug combination and AKT phosphorylation was diminished. Around the transcriptional start sites of these genes, the drug combination enhanced H3K27 acetylation, but decreased H3K4 trimethylation. The cell cycle inhibitor CDKN1C/p57KIP2 was dramatically induced at mRNA and protein levels. However, Cas9-mediated CDKN1C/p57KIP2 knockout did not rescue UC cells from apoptosis.ConclusionOur results demonstrate significant synergistic effects on induction of apoptosis in UC cells by the combination treatment with JQ1 and Romidepsin, but only minor effects in benign cells. Thus, this study established a promising new small-molecule combination therapy approach for UC.

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Autonomous inhibition of apoptosis correlates with responsiveness of colon carcinoma cell lines to ciglitazone.

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Autonomous inhibition of apoptosis correlates with responsiveness of colon carcinoma cell lines to ciglitazone.

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