We're currently studying the cytotoxic effects of a plant extract on HCT & HEK cells, and we want to incorporate a morphological analysis of our cells during the pre-introduction and post-introduction of the extract. Could anyone suggest literature or a method of analysis for this?
Any help would be very much appreciated.
Dear Jason,
The following publications cover the answer to your question:
1-Synthesis and biological evaluation of 6H-1-benzopyrano[4,3-b]
quinolin-6-one derivatives as inhibitors of colon cancer cell growth
Tie-Ling Li1
, Hai-Feng Guo2, Fu-Juan Li3, Zhi-Guo Sun2 and Heng-Chun Zhang2
Abstract:
A convenient synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-one derivatives
was reported using 4-chloro-2-oxo-2H-chromene-3-carbaldehyde with different
aromatic amines using silica sulfuric acid. The compounds were tested for
their anticancer activity against colon (HCT-116 and S1-MI-80), prostate (PC3
and DU-145), breast (MCF-7 and MDAMB-231) cancer cells. These compounds
showed more selectivity and potent cytotoxic activity against colon
cancer cells. 3c was tested against five other colon cancer cell lines (HT-29,
HCT-15, LS-180, LS-174, and LoVo), which had similar cytotoxicity and
selectivity. 3c did not induce PXR-regulated ABCB1 or ABCG2 transporters.
In fact, 3c induced cytotoxicity in HEK293 cells over expressing ABCB1 or
ABCG2 to the same extent as in normal HEK293 cells. It was cytotoxic
approximately 3- and 5-fold to resistant colon carcinoma S1-MI-80 cells. 3c
also produced concentration-dependent changes in HCT-116 colon cancer
cells, in mitochondrial membrane potential, leading to apoptosis, and submicromolar concentrations caused chromosomal DNA fragmentation.
Cell culture
Colon carcinoma cell lines [HCT-116, HCT-15, HT-29, Lovo, LS-180, LS-174, S1 (a clone of LS174T cells)] and prostatic cancer cell lines [DU-145 and PC -3] and breast carcinoma cells [MDA-MB-231 and MCF-7] were grown as adherent monolayers in flasks with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator containing 5% CO2 at 37ºC. A G482 mutant ABCG2-overexpressing, drug-resistant colon cancer cell line, S1-M1-80, was maintained in medium with 80 µM mitoxantrone (Miri et al., 2011; Robey et al., 2001). The assay media for PXR transactivation assays included phenol red-free DMEM (Lonza) supplemented with
5% charcoal/dextran-treated FBS (HyClone) and the other additives.
PXR transactivation assay HepG2 cells were transfected with pcDNA3-hPXR
and CYP3A4-luc plasmids using FuGENE 6 (Promega). After 24 hours of transfection in growth media, 10,000 cells were plated into wells of 96-well
culture plates (PerkinElmer), and treated with DMSO, RIF, or 3c for an additional 24 hours. At 48 hours of transfection, a luciferase assay was performed to measure luminescence using the Neolite Reporter Gene Assay System (PerkinElmer) and a
FLUOstar Optima microplate reader (BMG Labtech). Normalized CYP3A4 promoter activity was expressed as fold induction over the DMSO control. Cell viability was measured in parallel by CellTiter-Glo luminescent assays (Promega), which determine the number of metabolically active cells by quantifying the ATP present. Luminescence was measured with a FLUOstar Optima plate reader (BMG Labtech).
Cell cytotoxicity as determined by MTT assays and morphological analysis
The MTT assay (Carmichael et al., 1987) was used to determine cytotoxicity of the compounds to the following cells: HCT-116, HCT-15, HT-29, LS-180, LS-174,
Lovo, S1, DU-145, PC-3, MDA-MB-231 and MCF-7. Briefly, the cells were harvested with trypsin and suspended at a final concentration of 5 X 103 cells/well. Cells were seeded (180 µL/well) into 96-well multiplates. Different concentrations of 6H-1-benzopyrano [4,3-b]quinolin-6-one derivatives (20 µL/well) were added. After 72 hours of incubation, 20 µL of MTT solution (4 mg/mL) was added in each well, and the plates were incubated further 4 hours, allowing the viable cells to convert the yellow-colored MTT into dark -blue formazan crystals. Subsequently, the medium was removed, and DMSO (100 µL) was added in each well to dissolve the formazan crystals. The absorbance was determined at 570 nm with an OPSYS microplate Reader (DYNEX Technologies, Inc., Chantilly, VA, USA). The means ± SD concentrations were calculated from at least three experiments performed in triplicate. The IC50 values were calculated from survival curves using the Bliss method. At 68 hours, cells with or without treatment were photographed by use of an inverted microscope (Olympus, BX53F) with fluorescent lamps and digital cameras. The data were acquired and analyzed by CellSens software.
For more details, please see attached file.
2-Bioorg Med Chem. 2015 May 1;23(9):2148-58. doi: 10.1016/j.bmc.2015.03.002. Epub 2015 Mar 21.
Design, synthesis and in vitro cell-based evaluation of the anti-cancer activities of hispolon analogs.
Balaji NV1, Ramani MV1, Viana AG2, Sanglard LP2, White J2, Mulabagal V3, Lee C2, Gana TJ4, Egiebor NO5, Subbaraju GV1, Tiwari AK6.
Author information
Abstract
Phytochemicals play an important role in cancer therapy. Hispolon and 26 of its analogs (9 known and 17 new) were synthesized and evaluated for their antiproliferative activities in a panel of six independent human cancer cell lines using the in vitro cell-based MTT assay. Among the hispolon analogs tested, compound VA-2, the most potent overall, produced its most significant effect in the colon cancer cell lines HCT-116 (IC₅₀ 1.4 ± 1.3 μM) and S1 (IC₅₀ 1.8 ± 0.9 μM) compared to its activity in the normal HEK293/pcDNA3.1 cell line (IC₅₀ 15.8±3.7 μM; p
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