Autophagy is a cellular catabolic degradation response to various stress whereby cellular proteins, organelles and cytoplasm are engulfed, digested and recycled to sustain cellular metabolism (Shintani and Klionsky, 2004; Mizushima and Klionsky, 2007; Rubinsztein et al., 2007). In recent years, the importance of autophagy in human diseases, including cancer, neurodegenerative diseases and immunity has been progressively appreciated based on the increasing understanding of the diverse biological function of autophagy (Mizushima et al., 2008). Especially for the cancer, one particular important aspect is its role on cell death and cell survival (Yue et al., 2003;
Kuma et al., 2004; Feng et al., 2005). Although autophagy is generally recognized as a defense mechanism in response to a number of cancer chemotherapeutics and therefore, inhibition of autophagy sensitizes cell death (Amaravadi et al., 2007; KO et al., 2009), there is emerging evidence supporting the notion that activation of autophagy sensitizes cancer cell death. Cell death by autophagy, or autophagic cell death is characterized by (i) independent of apoptosis, (ii) increased autophagic flux in the dying cells, and (iii) pro-death function of autophagy (Shen and Codogno, 2011). The autophagic flux is initiated with formation of phagophore and subsequent complete autophagosomes, and finally is finished by fusion with endosomes-lysosomes to form autolysosomes.
Moreover, even there is real increase of autophagic flux in the dying cells, the enhanced autophagy may indicate an attempt by the cell to save from cell death (Levine and Yuan, 2005; Kroemer and Levine, 2008; Scarlatti et al., 2009). Therefore, it is critical to demonstrate the pro-death function of autophagy in defining this form of cell death.
Hepatocellular carcinoma (HCC) is one of the most common solid tumor and the third leading cause of cancer death in the worldwide. One of the reasons for the high mortality rate in patients with HCC is the lack of effective treatment, especially for those with advanced disease. To date, systemic therapy with classical cytotoxic chemotherapy has been reported to be poorly tolerated and ineffective. For this reason,
new therapeutic options are eagerly needed for more effective treatment of HCC. To increase anticancer efficacy for HCC, several approaches that combination of autophagy modulation and conventional chemotherapeutics have been developed and suggested as a promising therapeutic strategy in treatment (Takimoto and Awada, 2008; Cabibbo et al., 2009). In these situations, autophagy modulation may be a good therapeutic strategy.
However, little is known about autophagy status and corresponding functions in HCCs after use of these therapies.
Ginsenoside Rg3 extracted from Panax ginseng C.A. Meyer is one of the diverse groups of steroidal saponins with high pharmacological activity. Several findings suggest that Rg3 may increase the efficacies of cancer chemotherapy, and the molecular mechanisms responsible for such anti-cancer function is related to its inhibitory effects on NF- B and AP-1 activity but it is still unclear that the mechanism of Rg3 in cancer chemotherapies (Keum et al., 2003). It has been reported that the effects and mechanisms of ginsenoside Rb1 on activation of autophagy in glutamate-injured neurons (Chen et al., 2010) and ginsenoside Rk1 has anti-tumor activity by modulating autophagy (KO et al., 2009). However, the mechanism of autophagy modulation by ginsenoside is unclear and there is no report that effects and mechanisms of ginsenoside Rg3 on activation of autophagy.
In the present study, we investigated the effect of the ginsenoside Rg3 on modulation of autophagy in hepatocellular carcinoma cell lines and evaluate whether such effect is relevant to the sensitization effect of Rg3 to apoptosis induced by DNA damage agents, doxorubicin which is commonly used in the treatment of a wide range of cancers. Our data show that Rg3 is capable of repressing autophagic flux via suppressing the late stage autophagosome maturation and degradation. Importantly, Rg3-induced inhibition of autophagy contributes to doxorubicin-induced cancer cell death. The synergistic effect of Rg3 on the therapeutic efficacy of doxorubicin was confirmed using the
II. Materials and Methods
A. Rg3 extraction. Sun ginseng (1.6 kg) was extracted with 70% MeOH (1.2 L) under reflux for 3 hr. The solvent was removed in vacuo to yield 320 g of 70% MeOH extract, which was suspended in water and extracted with n-BuOH. The n-BuOH fraction was concentrated in vacuo to yield 91.5 g of BuOH fraction. 40g of fractions was subjected to silica gel column chromatography. 9 fractions were obtained using stepwise gradient elution (EtOAc : MeOH : H20 =40 : 1 : 1 → 20 : 1 : 1 → 10 : 1 : 1). Fraction 8 was chromatographed over silica gel using CHCl3 : MeOH : H2O = 200 : 20 : 1 → 150 : 20 : 1 solvent. Rg3 rich fraction which contained Rg3 (R) and (S) forms was obtained solvent.
It was further purified over semi-preparative HPLC/ELSD analysis using a reverse-phase column (Phenomenex C18, 250 mm x 10 mm) with 40% ACN to isolate Rg3 (S) form (10 mg).
B. Reagents. Anti-p62, anti-PARP1 antibodies were purchased from Cell signaling.
Anti-LC3 antibody and Necrostatin-1 were purchased from Sigma. Doxorubicin, Chloroquine diphosphate (CQ), Bafilomycin, 3-methyladenine (3-MA), doxycyclin, and Cycloheximide were purchased from Calbiochem. zVAD was purchased from R&D system.
C. Cell culture. SK-Hep1, HepG2, Hep3B, A549, H322 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Huh-7 cells were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The Atg5-/- MEFs and the Tet-off Atg5 MEFs were provided by Dr. N Misushima (Tokyo Medical and Dental University). Normal liver cell line HL-7702 was purchased from Shanghai Institute of
Cell Biology (Shanghai, China) and cells were cultured in RPMI 1640 medium supplemented with 20 % fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin.
D. Western blot analysis. Upon treatment, cells were lysed in M2 buffer (20 mM Tris at pH 7, 0.5 % NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, 1 μg/ml leupeptin). Equal amounts of cell extracts were resolved by 12% or 15% SDS-PAGE and analyzed by western blot and visualized by enhanced chemiluminescence (ECL, Amersham).
E. Transfection. Transfection experiments in hepatoma cells were performed with Lipofectamine PLUS reagent by following the instruction provided by the manufacturer (GIBCO/BRL). Cells were transfected with the GFP-LC3 construct. The mRFP-GFP tandem fluorescent-tagged LC3 construct (tfLC3) was provided by Dr. T Yoshimori (Osaka University) (Kimura et al., 2007).
F. Confocal microscopy. Cells were seeded to a coverglass slide chamber (Lab-Tek®, NUNC), after designated treatments, the cells were examined under a confocal microscope (Carl Zeiss LSM710). The GFP-LC3 puncta were examined under a confocal microscope and tfLC3 were observed for the change of both green and red fluorescence using a confocal microscope. The data presented were from one representative experiment of at least 3 independent repeats.
G. Cytotoxicity assay. Cell death was determined using tetrazolium dye colorimetric test (MTT test). The MTT absorbance was then read at 570 nm. Representative images were taken by a phase-contrast microscope.
H. Reverse Transcription-PCR. RNA was extracted with the RNeasy Kit (Qiagen). 1 ug of total RNA from each sample was used as a template for cDNA synthesis with a reverse transcriptase kit (Invitrogen). An equal amounts of cDNA product was used in the PCR performed using the Taq DNA polymerase (Takara). PCR amplification was performed using the following primers: human p62 gene sense (5'-AATGTGATCTGCGATGGCTG-3'), p62 antisense (5'-CCAAGGCGATCTTCCTCATC T-3') and β-actin gene sense (5′-CAGGTCATCACCATTGGCAATGAGC-3′), β-actin gene antisense (5′-GATGTCCACGTCACACTTCATGA-3′ ). The final PCR products were resolved in 1.5 % agarose gel and stained with ethidium bromide.
I. Tumor xenograft study. Male nude mice were obtained from Central Lab. Animal Inc. (Seoul, Korea). The animals were fed standard rat chow and tap water ad libitum, and were maintained under 12 h dark/light cycle at 21 ºC. Male, 6-week-old nude mice were randomly divided into four groups (control, Rg3, Doxorubicin, Rg3+Doxorubicin, n=8/group). Huh-7 cells were harvested and mixed with PBS (200 μl/mouse) and then inoculated into one flank of each nude mouse (5x106 Huh-7 cells). When the tumors had reached a volume of about 50-70 mm3, mice were given a daily oral dose of 20 mg/kg Rg3 or the vehicle (200 μl PBS, control group), and i.p three times/week at dose of 1 mg/kg Doxorubicin, for 21 days, respectively. The tumor dimensions were measured twice a week using a digital caliper and the tumor volume was calculated using the formula: V = length x width2 x 0.5. At the end of the experiment, the mice were killed and the tumors were excised and weighed. Histopathological analysis for tumors was carried out by using hematoxylin and eosin (H&E) staining.
III. Results
A. Rg3 induces LC3-I to LC3-II conversion in dose- and time- dependent manner