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Comparative evaluation of different cell lysis and extraction methods for studying benzo(a)pyrene metabolism in HT-29 colon cancer cell cultures.

ABSTRACT: Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies.

SUBMITTER: Myers JN

PROVIDER: S-EPMC3709193 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Comparative evaluation of different cell lysis and extraction methods for studying benzo(a)pyrene metabolism in HT-29 colon cancer cell cultures.

Myers Jeremy N JN Rekhadevi Perumalla V PV Ramesh Aramandla A

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 20110816 2

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, be ...[more]

PMID: 21865728

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Project description:Functional foods, beyond basic nutrition, offer health benefits to consumers thanks to the presence of bioactive compounds such as some phytochemicals [1,2]. Today, these foods are of particular interest in biomedical research due to their chemopreventive potential, as they have been shown to induce various biological effects on tumor cells, including the ability to inhibit cell proliferation, induce apoptosis, arrest cell cycle progression, and increase reactive oxygen species [3,4]. Multiple studies have confirmed the relationship between diet and the onset and progression of colorectal cancer (CRC), a malignant neoplasm that arises in the lining of the colon and/or rectum. Therefore, finding foods that can intervene in the carcinogenesis process is an important avenue of research [5,6]. Chlorogenic acid (CGA) is one of the most abundant phenolic compounds in coffee and is also found in fruits and vegetables. Scientific evidence suggests that CGA has chemopreventive potential on CRC cells [7], [8], [9]. For example, in previous studies conducted by our research group, green and roasted coffee extracts were characterized, and this compound was identified as the most abundant [7]. In addition, it was found to significantly decrease cell viability, reduce migration capacity, cause DNA fragmentation, and induce the production of reactive oxygen species in colorectal adenocarcinoma cells cultured in monolayer and treated with different doses of CGA. Furthermore, the mechanism underlying this biological activity has been related to CGA's ability to modulate the Wnt- /β-catenin pathway, which is implicated in the development and progression of CRC [7,10,11]. This paper presents data on the cytotoxic response of CGA treatments on HT-29 cells cultured in a 3D model. To this end, morphological changes in cell spheroids, propidium iodide and DiOC6 uptake, quantification of reactive oxygen species (ROS) production, phosphatidylserine exposure, and cell cycle progression were evaluated by flow cytometry.

Project description:In the present study, we evaluated the antitumor effect of two synthetic analogs of vitamin D, namely PRI-2191 [(24R)-1,24-dihydroxyvitamin D3] and PRI-2205 (5,6-trans calcipotriol), in combined human colon HT-29 cancer treatment with 5-fluorouracil (5-FU). Mice bearing HT-29 tumors transplanted subcutaneously or orthotopically were injected with vitamin D analogs and 5-FU in various schedules. A statistically significant inhibition of subcutaneous or orthotopic tumor growth was observed as a result of combined therapy. In HT-29 tumors and in cells from in vitro culture, we observed increased vitamin D receptor (VDR) expression after treatment with either PRI-2205 or 5-FU alone, or in combination. Moreover, PRI-2205 decreased the percentage of cells from intestinal tumors in G2/M and S stages and increased sub-G1. Increased VDR expression was also observed after combined treatment of mice with 5-FU and PRI-2191. Moreover, our docking studies showed that PRI-2205 has stronger affinity for VDR, DBP and CAR/RXR ligand binding domains than PRI-2191. PRI-2191 analog, used with 5-FU, increased the percentage of subcutaneous tumor cells in G0/G1 and decreased the percentage in G2/M, S and sub-G1 populations as compared to 5-FU alone. In in vitro studies, we observed increased expression of p21 and p-ERK1/2 diminution via use of both analogs as compared to use of 5-FU alone. Simultaneously, PRI-2191 antagonizes some pro-apoptotic activities of 5-FU in vitro. However, in spite of these disadvantageous effects in terms of apoptosis, the therapeutic effect expressed as tumor growth retardation by PRI-2191 is significant. Our results suggest that the mechanism of potentiation of 5-FU antitumor action by both analogs is realized via increased p21 expression and decreased p-ERK1/2 level which may lead to diminution of thymidylate synthase expression. Higher binding affinity for VDR, DBP, but also for CAR\RXR ligand binding domain of PRI-2205 may, in part, explain its very low toxicity with sustained anticancer activity.

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Comparative evaluation of different cell lysis and extraction methods for studying benzo(a)pyrene metabolism in HT-29 colon cancer cell cultures.

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Comparative evaluation of different cell lysis and extraction methods for studying benzo(a)pyrene metabolism in HT-29 colon cancer cell cultures.

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