Abstract
PurposeThis work aimed to investigate the effect of costunolide, a sesquiterpene lactone isolated from Michelia compressa, on cell cycle distribution and radiosensitivity of human hepatocellular carcinoma (HCC) cells.MethodsThe assessment used in this study included: cell viability assay, cell cycle analysis by DNA histogram, expression of phosphorylated histone H3 (Ser 10) by flow cytometer, mitotic index by Liu's stain and morphological observation, mitotic spindle alignment by immunofluorescence of alpha-tubulin, expression of cell cycle-related proteins by Western blotting, and radiation survival by clonogenic assay.ResultsOur results show that costunolide reduced the viability of HA22T/VGH cells. It caused a rapid G2/M arrest at 4 hours shown by DNA histogram. The increase in phosphorylated histone H3 (Ser 10)-positive cells and mitotic index indicates costunolide-treated cells are arrested at mitosis, not G2, phase. Immunofluorescence of alpha-tubulin for spindle formation further demonstrated these cells are halted at metaphase. Costunolide up-regulated the expression of phosphorylated Chk2 (Thr 68), phosphorylated Cdc25c (Ser 216), phosphorylated Cdk1 (Tyr 15) and cyclin B1 in HA22T/VGH cells. At optimal condition causing mitotic arrest, costunolide sensitized HA22T/VGH HCC cells to ionizing radiation with sensitizer enhancement ratio up to 1.9.ConclusionsCostunolide could reduce the viability and arrest cell cycling at mitosis in hepatoma cells. Logical exploration of this mitosis-arresting activity for cancer therapeutics shows costunolide enhanced the killing effect of radiotherapy against human HCC cells.
Highlights
Costunolide is a sesquiterpene lactone isolated from Michelia compressa in our previous work [1]
Cell viability and estimation of IC50 As demonstrated in Figure 1A, costunolide inhibited the viability of HA22T/VGH hepatocellular carcinoma (HCC) cells in a concentrationand time-dependent manner
To sensitize tumor cell to radiation at a concentration range not extensively cytotoxic, costunolide at and below 5 μM was used for further cell cycle analysis and radiosensitizing experiments
Summary
Costunolide is a sesquiterpene lactone isolated from Michelia compressa in our previous work [1]. Michelia compressa is a common origin of wooden furniture used worldwide. Costunolide has been identified in several species of plants, including Saussurea lappa C.B. Clarke [2], Aucklandia lappa Decne [3], Laurus nobilis [4], Magnolia grandiflora [5] and Michelia floribunda [6]. Bocca et al reported that costunolide interfered with the microtubule proteins [7]. Whether this activity refers to mitosis arrest and subsequent
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