| Background:The poor sensitivity and insensitivity of liver cancer to chemotherapy drugs represent main factors in hindering the effects of clinical treatments, whereas live cancer stem cells (LCSCs) are the sources for the occurrence, development, drug-resistance, metastasis and recurrence of liver cancer. Therefore, the exploration of effective drugs or measures for liver cancer treatment or LCSCs elimination still remains an advanced research hotspot. Arsenic trioxide (As2O3 or ATO) performs anti-tumor functions via inhibiting cell proliferation, inducing apoptosis and repressing neovascularization in tumors, and effectively plays role in killing cancer stem cells (CSCs), so ATO generates excellent efficacies in clinical treatments of leukemia and solid tumors with relative lower toxic and side effects. However, the therapeutic effect of ATO on solid tumors is poorer than that on leukemias, and the needed higher doses of ATO for treatment often generates greater side effects, which have restrained the clinical applications of ATO. Therefore, the combination of ATO with other drugs to lower its toxic and side effects and improve the sensitivity of tumor cells to ATO may stand for an effective measure to enhance efficacies of of ATO in clinical treatments. Parthenolide (PTL) possesses the anti-tumor and anti-CSC potential and exerts sensitizing effect to other chemotherapeutics. Therefore, it is of important and valuable to explore the combined effects of PTL and ATO in killing hepatoma cells and eliminating LCSCs.Objectives:To investigate the proliferation-inhibiting and apoptosis-inducing effects of PTL combined with ATO on hepatoma HepG2 cells and LCSCs, and to probe into the relevant molecular mechanisms of their actions.Methods:MTT colorimetric determination was adopted for the detection of cell proliferation activity. Optical microscope with Wright-Giemsa staining and electron microscope were employed to observe morphological characteristics and ultra-structures of cells, respectively. The cell self-renewal ability was detected by colony-formation assay. Cellular apoptosis was examined with AnnexinV/PI double staining. Cell cycle, expression of apoptosis-related proteins Bax and Bcl-2, Caspase-3 activity, active oxygen species(ROS), mitochondrial transmembrane potential(MMP) and LCSC proportion were determined by flow cytometry. The CD133+ phenotype was used as immunological marker of cancer stem cell to sort LCSCs from HepG2 cell population with flow cytometer. Expressions of apoptosis-related genes, P53, bcl-2, bax, NF-κB and caspase-3 were detected with real-time fluorescence quantitative RT-PCR and western blotting, respectively.Results:1. After treatment with different concentrations of PTL and ATO for 24h, 48h and 72h, the proliferation of HepG2 cells was inhibited at a concentration- and time-dependent manner. The half-inhibitory concentration (IC50) of ATO was 22.7μM,18.2μM and 6.6μM at 24h,48h and 72h, respectively, and 25.3μM,16.6μM and 8.3μM for PTL. Combination of PTL and ATO exerted much more significant inhibiting effects than that by PTL or ATO alone, and similar results were also acquired from the experiment of colony-formation of HepG2 cells. Therefore, it was suggested that PTL combined with ATO could synergistically enhance the sensitivity of HepG2 cells to ATO.2. After induction of PTL and ATO alone or combination, HepG2 cells exhibited typical morphological characteristics of apoptosis, such as shrunk in sizes, condensed cytoplasm, nuclear fragmentation, chromatin condensation and margination, etc. The distribution of cell-cycle was arrested at G2/M phase. Annexin V/PI staining showed that the apoptotic rate of PTL plus ATO-induced HepG2 cells was much higher than that of the PTL-or ATO-induced cells, and the former mainly manifested as late apoptosis, while the latter as early apoptosis. The above indicated that the main mechanism of synergistic action of ATO plus PTL was to induce HepG2 cells to apoptosis, and PTL could increase the sensitivity of HepG2 cells to ATO-induced apoptosis.3. Alone or combination treatment with ATO and PTL was able to decline MMP of HepG2 cells, and the increased expression of Bax gene and decreased expression of Bcl-2 gene lead to conspicuous increase of Bax/Bcl-2 ratios, and activity of Caspase-3 also augmented apparently. The effects exerted by co-treament of ATO and PTL were more significant than those by single use of PTL or ATO, and the results suggested that combined apoptosis-induction of ATO and PTL on HepG2 cells was mainly mediated by activation of Caspase-3 via mitochondrial apoptosis pathway.4. After alone or combined treatment with ATO and PTL, the expression and phosphorylation of P53 and ROS content in HepG2 cells significantly increased, and the expression of NF-kB was markedly suppressed. The combined effects of ATO and PTL were far greater than those of single use of PTL or ATO, and These mean that P53 was possibly involved in the regulation of apoptosis in mitochondrial pathway by enhancing Bax and repressing Bcl-2 expression, promoting the Mitochondrial membrane depolarization and enabling the release of apoptosis factors, whereas NF-κB directly promoted abnormal cell proliferation and maintained cell survival by suppressing P53-mediated apoptosis. Therefore, it was presumed that P53 signal and NF-κB pathway might be an additional regulating way for combined apoptosis-induction of ATO and PTL in HepG2 cells.5. Single or combined treatment of ATO and PTL might both kill LCSCs. Regardless of sorted LCSCs or LCSC subset in HepG2 cell population, combination of ATO and PTL was capable of playing a synergistical role in killing the LCSCs, and the possible mechanism was mainly relied on induction of LCSCs to apoptosis.Conclusions:Parthenolide combined with arsenic trioxide plays a synergistical action in proliferation-inhibition and apoptosis-induction of hepatic carcinoma HepG2 cells and effective killing of liver cancer stem cells, and that parthenolide and arsenic trioxide induce the apoptosis of HepG2 cells mainly via mitochondrial, P53 and NF-κB pathways. |
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