Studies On The Mechanism Of Endoplasmic Reticulum Stress-mediated Apoptosis Induced By Dihydroartemisinin In Human Hepatocarcinoma HEPG2 Cell Lines

PART ONE STUDIES ON THE PROLIFERATION INHIBITORY EFFECTS OF DHA ON HUMAN HepG2, PC-3, SGC7901 AND Caco2 CELL LINESOBJECTIVE:To compare the inhibitory effects of dihydroartemisinin (DHA) on four human carcinoma cell lines in vitro, and select the most sensitive cell lines for further investigation.METHODS: The proliferation inhibition of DHA on human HepG2, PC-3, SGC7901 and Caco2 cell lines was evaluated using MTT chromatometry; Flow cytometry analysis was used to detect the apoptosis of the four cell lines.RESULTS: There was no significant difference in proliferation of control and DMSO group of four cell lines (P>0.05). Proliferation inhibitory rate of each cell lines was positively correlated to DHA concentration and treatment time(P<0.05), and inhibitory rates of various DHA concentration groups of HepG2 had significant difference compared with that of other three cell lines at the same treatment time(P<0.01, except for 25μmol/L DHA 24h treatment time P<0.05). The apoptotic rates of each DHA concentration groups after 24h treatment were markedly higher than that of the control groups in four cell lines (P<0.01). DHA had the stongest effect on HepG2 among the four cell lines, with the highest inhibitory rate of (86.46±19.65) % at DHA 200μmol/L for 48 h, and the highest apoptotic rate of (31.74±2.80) % at DHA 100μmol/L for 24 h.CONCLUSION: DHA can efficiently inhibit proliferation and induce apoptosis of HepG2, PC-3, SGC7901 and Caco2 cell lines; HepG2 is the most sensitive to DHA among the four cell lines.PART TWO STUDIES ON THE INITIATORS OF APOPTOTIC INDUCTION OF DHA ON HUMAN HepG2 CELL LINESOBJECTIVE:To investigate the initiators of apoptotic induction of DHA on HepG2 cell lines.METHODS: HepG2 cells were treated with 0, 50,100 and 200μmol/L DHA for 6, 24 h. The fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and Fluo-3AM were employed to detecte the levels of reactive oxygen species(ROS) and intracellular calcium concentration, respectively; Flow cytometer was used to measure the mitochondrial membrane potential (ΔΨm), and the ultrastructural changes of HepG2 cells treated with DHA were observed using transmission electron microscope (TEM).RESULTS:Compared with control group, intracellular fluorescent intensity markedly increased (P<0.01) after DHA treatment in a time- and concentration-dependent manne; However, the increase of [Ca2+]i and the reduction ofΔΨm, which were concentration-dependent, occurred only in 24h treatment group(P<0.01). After 24h treatment of DHA 100μmol/L, a lot of apoptotic cells as well as significant distention of endoplasmic reticulum lumina were obserbed using TEM. The above effects of DHA could be markedly attenuated by antioxidant N-acetylcysteine (NAC) 5mmol/L.CONCLUSION: DHA significantly increases ROS generation and [Ca²⁺]_i, reduces the level ofΔΨm. ROS generation is probably upstream event.PART THREE STUDIES ON ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOTIC SIGNAL TRANSDUCTION MEDIATED BY DHA IN HUMAN HepG2 CELL LINESOBJECTIVE : To study the molecular mechanism of apoptosis induced by DHA in HepG2 cell linesMETHODS: Human HepG2 cells were treated with DHA, DHA+NAC and DHA+SP600125. The inhibitory effect on cell proliferation was assayed by MTT; Apoptotic induction was measured by Flow cytometry; Ultrastructural changes of HepG2 cells treated with DHA were observed with scanning electron microscope (SEM); The expression of CHOP, XBP1 and ATF4 mRNA were detected using reverse transcription-polymerase chain reaction (RT-PCR); Levels of CHOP, p-JNK, Bax and Bcl-2 protein expression were determined using Western Blot and immunocytochemistry.RESULTS: Proliferation inhibitory rate of HepG2 cells treated with DHA correlated with DHA treatment time and concentration (P<0.05). NAC blocked the effect of DHA treatment for both 24h and 48h, which could also be partially attenuated by JNK specific inhibitor SP600125 except for DHA at high concentration ( 50, 100, 200μmol/L) 48h groups; Compared to the control group, the apoptotic rates of HepG2 cells in DHA group were significantly higher and had a positive correlation with DHA concentration(P<0.05). Both NAC and SP600125 could attenuate the DHA effect. SEM showed decreasing and even deminish of microvilli on the cellular surface after DHA treatment, varying sized of ball-shaped membrane blebbing on the cellular surface of apoptotic cells, apoptosis body, broken plasma membranes and cytoplasma outflow in necrotic cells; RT-PCR showed up-regulation of mRNA expression of CHOP, XBP1u, XBP1s and ATF4 by DHA while Western-Blot and immunocytochemistry showed up-regulation of protein expression of CHOP and Bax, and down-regulation of Bcl-2, NAC inhibited the effects of DHA on these genes and protein expression; Phosphorylation of JNK was detected at 6h DHA treatment time by Western-Blot, both NAC and SP600125 blocked the phosphorylation of JNK induced by DHA.CONCLUSION: DHA could activate the three signal transduction pathways of UPR (PERK,ATF6,IRE1), and induce the endoplasmic reticulum stress-mediated apoptotic pathway via CHOP and JNK activation. ROS plays an important role in this mechanism.PART FOUR STUDIES ON INHIBITION OF HepG2 XENOGRAFT TUMOR IN NUDE MICEOBJECTIVE:To investigate the inhibitory effect of DHA on growth of HepG2 xenograft tumor in nude mice.METHODS: The nude mice model of human hepatocarcinoma was established by subcutaneous injection with HepG2 cell suspension, and growth inhibition of the xenograft tumor induced by DHA was observed in vivo; Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were employed to evaluate CHOP, Bax, Bcl-2 and p-JNK expression; Ultrastructural changes of xenograft tumor tissues were observed by transmission electron microscope (TEM).RESULTS: Compared to control group, the volume and weight of tumor of DHA group were significantly decreased (P<0.05), and the inhibitory rate was 50.91%. No significant difference in body weight between control and DHA group was recorded; RT-PCR and immunohistochemistry showed the expression of CHOP and Bax were up-regulated, while that of Bcl-2 was down-regulated, and the differences were statistically significant (P<0.05), and no expression of p-JNK in both groups; TEM indicated apoptosis morphological changes including significant distension of endoplasmic reticulum lumina and swelling of mitochondria in xenograft tumor histocytes after DHA treatment.CONCLUSION: DHA can inhibit the growth of HepG2 xenograft tumor in nude mice, and endoplasmic reticulum stress-induced apoptosis pathway may be involved in this action of DHA.