Antitumor Effects Of Xhh, A Novel Nitrogen Mustard Derivative, And Its Mechanisms

Objective:We investigated the anti-tumor effects of XHH, a novel nitrogen mustard derivative, and its apoptotic mechanisms in K562 and HepG2 cells.Methods:The anti-proliferation effects of XHH were assessed by MTT assay and trypan blue exclusion assay in K562 and HepG2 cells. Morphological change of K562 and HepG2 cells after XHH treatment was observed with AO/EB double staining by fluorescence microscope. The inducing apoptosis effects and mitochondrial membrane potential (MMP) were assessed with AnnexinV-FITC/Hoechst33342, PI/Hoecsht33342 and Rh123/Hoechst33342 double staining by high content screening (HCS). The activity of caspase-3, -8, -9 was evaluated by spectrophotometric method. The expression of caspase-3, -8, -9, Bcl-2, Bax and cytochrome c (Cyt c) was measured using immunofluorescence by HCS .Results:MTT assay demonstrated that XHH exerted potent anti-tumor effect in a dose-dependent manner and the IC50 values after 48 h treatment were (5.26±1.08)μM and (6.07±1.41)μM in K562 and HepG2 cells, respectively. The anti-tumor effect was slightly lower than chlorambucil (CHB) in K562 cells, however, more powerful in HepG2 cells. Furthermore, the anti-tumor effect of XHH was also authenticated by trypan blue exclusion assay, the results demonstrated that the growth of K562 and HepG2 cells was significantly degraded in a dose- and time-dependent manner.Typical morphological changes of apoptosis, including chromatin condensation, dense chartreuse nucleolus, nuclear fragmentation, and"apoptotic body"were observed in the XHH-treated K562 and HepG2 cells using AO/EB double staining, indicating XHH induced K562 cells and HepG2 cells apoptosis. Furthermore, those apoptotic effects were also authenticated using AnnexinV-FITC/ Hoechst33342 and PI/Hoechst33342 double staining by HCS.The disruption of mitochondrial integrity is one of the early events in cell apoptosis, so the mitochondrial membrane potential (MMP) was measured with Rh123/Hoechst33342 double staining by HCS. The results indicated that XHH could induce the MMP loss and destroy membrane integrity of K562 cells and HepG2 cells in a dose- and time-dependent manner.The mitochondrial pathway and membrane death receptor pathway are the two classical pathways in cell apoptosis. To explore the apoptotic mechanism of XHH, the activity of caspase-3, -8, -9 was assessed by spectrophotometric method, and the results indicated that the activity of caspase-3, -8, -9 significantly increased in a dose- and time-dependent manner, compared with control group (P<0.01), indicating XHH induced cell apoptosis via mitochondrial pathway and membrane death receptor pathway.Furtherly, the expression of caspase-3, -8, -9, Bcl-2, Bax and Cyt c in K562 and HepG2 cells was also detected using immunofluorescence by HCS, the results indicated that the activity of caspase-3, -8, -9, Bax and Cyt c was significantly higher than control and the activity of Bcl-2 was higher than control at 5μM and 10μM XHH, while Bcl-2/Bax significantly decreased in a dose- and time- dependent manner, the trend of XHH inducing cell apoptosis reinforced incessantly.Conclusions:XHH exerted potent anti-tumor activity in K562 and HepG2 cells. XHH induced apoptosis via mitochondrial pathway and membrane death receptor pathway in K562 cells and HepG2 cells.