Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy.
OBJECTIVE
To study the antitumor effect of matrine in human hepatoma G2 (HepG2) cells and its molecular mechanism involved in antineoplastic activities.


METHODS
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect viability of HepG2 cells. The effect of matrine on cell cycle was detected by fl ow cytometry. Annexin-V-FITC/PI double staining assay was used to detect cellular apoptosis. Cellular morphological changes were observed under an inverted phase contrast microscope. Transmission electron microscopy was performed to further examine ultrastructural structure of the cells treated with matrine. Monodansylcadaverine (MDC) staining was used to detect autophagy. Whether autophagy is blocked by 3-methyladenine (3-MA), an autophagy inhibitor, was evaluated. Expression levels of Bax and Beclin 1 in HepG2 cells were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
Matrine significantly inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner, and induced G1-phase cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner. The total apoptosis rate was 0.14% for HepG2 cells not treated with matrine. In contrast, the apoptosis rate was 28.91%, 34.36% and 38.80%, respectively, for HepG2 cells treated with matrine at the concentration of 0.5, 1.0 and 2.0 mg/mL. The remarkable morphological changes were observed under an inverted phase contrast microscope. Abundant cytoplasmic vacuoles with varying sizes were observed in HepG2 cells treated with matrine. Furthermore, vacuolization in cytoplasm progressively became larger and denser when the concentration of matrine was increased. Electron microscopy demonstrated formation of abundant autophagic vacuoles in HepG2 cells after matrine treatment. When the specific autophagic inhibitor, 3-MA, was applied, the number of autophagic vacuoles greatly decreased. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in HepG2 cells was greater in matrine treatment group than in control group. Fewer autophagic vacuoles were observed in the combined 3-MA and matrine treatment group when 3-MA was added before matrine treatment, indicating that both autophagy and apoptosis are activated when matrine-induced death of hepatoma G2 cells occurs. Real-time quantitative RT-PCR revealed that the expression levels of Bax gene, an apoptosis-related molecule, and Beclin 1 gene which plays a key role in autophagy were higher in matrine treatment group than in control group, indicating that Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanism of matrine may be related to its upregulation of Bax expression.


CONCLUSIONS
Matrine has potent antitumor activities in HepG2 cells and may be used as a novel effective reagent in treatment of hepatocellular carcinoma.
Zhang
Jun-Qiang
JQ
Second Hospital of Lanzhou University, 82 Cuiyingmen, Lanzhou 730030, Gansu Province, China.
Li
Yu-Min
YM
Liu
Tao
T
He
Wen-Ting
WT
Chen
Ying-Tai
YT
Chen
Xiao-Hui
XH
Li
Xun
X
Zhou
Wen-Ce
WC
Yi
Jian-Feng
JF
Ren
Zhi-Jian
ZJ
D000470:Alkaloids / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000473:pathology / Q000648:ultrastructure; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000473:pathology / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics; D008858:Microscopy, Phase-Contrast; D011807:Quinolizines / Q000494:pharmacology*; D014617:Vacuoles / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000235:genetics; D000093842:Matrines
D000470:Alkaloids; D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D011807:Quinolizines; D051028:bcl-2-Associated X Protein; D000093842:Matrines
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