The Role Of ROS In Apoptosis Of Tumor Cells Induced By Hypocrellin B, New Topoisomerase Inhibitor GS-2

Reactive oxygen species (ROS) are the products of aerobic metabolism in vivo. Manyphysical and chemical factors such as ionizing radiation or cancer drugs can induce cellapoptosis and ROS (H2O2,·OH, O-2, NO) are generating meanwhile. Photosensitivecompounds and DNA topoisomerases inhibitors are valid anti-tumor drugs, which couldinduce ROS in cell apoptosis. In this paper, we investigated the important role of ROSradicals (including NO) in hypocrellin B (HB), a potent photosensitizer induced cellapoptosis. Meanwhile, considering strong cytotoxic activity of GS-2(a noveltopoisomerases inhibitor) against cancer cells, we investigated the role of ROS in thecytotoxic effect. The finding of this paper maked theoretical basis for the molecularmechanisms of anti-tumor activity of natural photosensitive drug and synthesizedtopoisomerases inhibitors, provided research methods to reveal the role of ROS ininduced-apoptosis.Part I The preliminary study of HB-induced phototoxicityWe initiated our study by examining the phototoxicity of HB using MTT assay onHepG2cells. The IC50(Inhibition concentration which reduces cell viability by50%) ofHB/light at15min,30min,1h,2h,3h are5.35,4.35,4.12,3.4,3.1μM, respectively. Thedata showed that HB caused signifcant inhibitory effects on HepG2cell growth in aconcentration-and time-dependent manner. To quantify the ratio of apoptotic cells,Annexin V-FITC/PI staining and DNA content were measured by flow cytometry. Fromthe data of Annexin V-FITC/PI staining, the ratio of HB-induced apoptosis was4times ofthe control group; from another method, apoptotic cells were judged from the appearanceof a sub-G1peak. HepG2cells treated with HB resulted in a dramatic increase of thesub-G1fraction from15.69%to31.52%. Moreover, HB could induce G2/M-phase cyclearrest in HepG2cells, which illustrates HB/light could induce cancer cell apoptosis.Part II The role of H2O2and NO in the HB/light induced cell apoptosisTo investigate whether ROS were involved in HB-mediated cell apoptosis, we used DCFH-DA for H2O2level and DAF-FMDA for NO level. A peak generation of H2O2wasobserved after10min of exposure to600lux light and5mM HB. At the same time,5μMHB/light promoted the increases in DAF fluorescence intensity with the illumination time.However, HB/light could not cause O-2generation when DHE was applied to detect it. Allabove illustrate that H2O2and NO were the main ROS induced by HB/light. Moreover, theadministration of ROS inhibitor N-acetyl-cysteine (NAC) abrogated the inducedcytotoxicity and cell apoptosis in HepG2cells. On the other hand, the increasingexpression if iNOS and eNOS indicated that the NOS was the source of NO, as a result, weapplied the NOS inhibitor–L-NMMA to pretreat with HepG2cells, it aggravate theinduced phototoxicity and cell apoptosis. To have a further research on NO effect, the NOscavenger cPTIO and NO donor SNP were applied to treat with HepG2cells for1h beforeHB, the results showed that cPTIO had the similar effect with L-NMMA while SNP hadthe contrary effect. At last, we found that NO also have the influence on caspase3and9ability. All these indicate that NO may be involved in the mitochondrial pathway of cellapoptosis.Part III GS-2, a new topoisomerase inhibitor, induces apoptosis in MDA-MB-231cellsGS-2caused a signifcant inhibitory effect on MDA-MB-231cell growth in aconcentration-and time-dependent manner. AO/EB double staining proved preliminarilythat GS-2could induce apoptosis in231cells. The single cell gel electrophoresis (cometassay) was used to test the DNA strand breakage degree, we found that the number oftrailing cells was increased with the GS-2concentration. There are more than50%cellsdamaged, that is to say, they experienced apoptosis. As signal factors of apoptosis, caspase3and9were activated by5μM GS-2at18h, and the activities were increased to2.3timescompared with control group, which indicated the apoptosis of231cells maybe via themitochondrial pathway.Part IV GS-2-induced cell apoptosis is related to ROS generation in MDA-MB-231cellsTo investigate whether ROS were involved in GS-2-mediated cell death, we usedDCFH-DA for ROS level. A significant generation of ROS was observed after2h ofexposure to GS-2at0-20μM. After24h treatment, mitochondrial membrane potential(MMP) level declined and the antioxidant enzyme activities increased significantly. Compared to the control group, GS-2at5,10,20μM promoted the increases in SODactivity by14.5，23.0and39.0folds and in CAT activity by1.52,5.01and21.31folds,respectively. While inhibit ROS generation by NAC, cell viability went up to105.39%,110.91%and105.25%, MMP were completely recovered, and the ratio of cell apoptosisdeclined from49.57%to6.00%. To investigate the apoptosis pathway, NAC also declinedcaspase3activity from2.3to1.8folds compared to control group. Moreover, the results ofRT-PCR and qPCR showed GS-2had a significant inhibitory effect to the expression ofTop I and Top IIα, and NAC attenuated the induced inhibition on expression of topos,indicating that topos might be the target of GS-2-induced ROS. All these indicated thatGS-2, the new topoisomerase inhibitor, could induce cell apoptosis via ROS pathway.Our results showed the first time that NO makes a cytoprotective role in HB/lightinduced cell apoptosis. We also first proved that ROS mediated cell apoptosis induced byGS-2. It was also proved that GS-2had the significant inhibitory effect to Topo I and TopoIIα in transcriptional level. The findings of paper maked theoretical basis for the molecularmechanisms of anti-tumor activity of natural photosensitive drugs and synthesizedtopoisomerases inhibitors, provided research methods to reveal the role of ROS in theinduced-apoptosis.