VIII. FIGURE AND LEGENDS
7. ΔΨm assay
ΔΨm was determined using MitoPT™ JC-1 Assay Kit
(ImmunoChemistry Technologies, Bloomington, USA). Briefly, the cells were cultured in medium containing different concentration of EMMQ and incubated at 37 °C for 3, 6 and 12 h. The collected cells were washed with 1× assay buffer. After centrifugation at 1,000 rpm for 5 min, cell pellets were stained with 250 μL mixture containing 5 μL of JC-1 with 995 μL 1× assay buffer for 25 min at 37 °C. The residual JC-1 was removed by centrifugation at 1,000 rpm for 5 min. The pellet was mixed with 1× assay buffer. JC-1 fluorescence was measured to assess the emission shift from green (530 nm) to red (590 nm) using 488 nm excitation wavelength. Data were given as the relative ratio of green to red fluorescence intensities, indicating the level of depolarization of the mitochondrial membrane potential. The flow cytometer FACS CaliburTM (BD Bioscience) was used for analysis. Data were quantified and the expressed as the percentage of mitochondrial membrane potential drop relative to those of untreated cells.
8. Release of cytochrome c release
The harvested cells after treatment were treated with 100 μL
digitonin (50 µg/mL PBS, 100 mM potassium iodide and 1 mM EDTA) for 5 min on ice until more than 95 % of cells were permeabilized. Cells were fixed and stained with 3.7% formaldehyde and DAPI (1:3,000)
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(Sigma, USA) in PBS for 20 min at room temperature. Cells were washed thrice in PBS, and incubated in blocking buffer (3% bovine serum
albumin (Themo, USA and 0.05% saponin in PBS) for 1 h. The cells were incubated overnight at 4°C with anti-cytochrome c mouse monoclonal antibody (BD PharMingen). Cells were washed thrice in PBS, and incubated for 1 h at room temperature with TRITC-conjugated goat
anti-mouse (Santa Cruz) in blocking buffer. The cells were counterstained with Mitotracker Green (Invitrogen Life Technologies). The samples were detected using a Leica TCS SP5 Confocal Spectral Microscope.
9. Western blot analysis
Cell lysates were collected and the concentrations quantitated using BCA assay (Pierce Biotechnology, Rockford, IL). A total of 20 µg of protein was resolved by electrophoresis through SDS-PAGE gel. The gel was transferred to nitrocellulose filters, blocked with 5% of Skim Milk (BD, Mansfield, MA) and incubated with primary and secondary antibodies. The emitted chemiluminescence signals were visualized by ECL detection kit (Millipore).
10. Transfection with p53 shRNA
The HepG2 hepatocellular carcinoma cells were seeded in 60-mm dishes at 5 × 105 cells/dish, incubated overnight. Cells were transfected with shRNA targeting exon 7 of p53 with NS as control prior to treatment.
After a 24 h transfection period, cells were treated with EMMQ for 48 h.
Cell lysates were collected for western blot analysis.
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11. Isolation of mitochondria and cytosol fractions
Cell lysates of cytosol and mitochondria fractions were separated according to the manufacturer’s instruction (BioVision, Milpitas, CA).
Cells (5× 107) were collected by centrifugation at 600×g for 5 minutes at 4°C. Cells were washed with 10 mL of ice-cold PBS and centrifuged at 600×g for 5 mins at 4°C. The supernatant was removed. Cells were resuspend with 1.0 mL of 1× cytosol extraction buffer mix containing DTT and protease inhibitors. Cells were incubated on ice for 10 minutes.
The homogenate was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 700×g for 10 mins at 4°C. The supernatant was collected carefully and the pellet discarded. The supernatant was collected
transferred to a fresh 1.5 mL tube, and centrifuged at 10,000×g for 30 mins at 4°C. The supernatant was collected and saved at -80°C (cytosol fraction). The pellet was suspended with 100 µL of 1× mitochondrial extraction buffer mix containing DTT and protease inhibitors. The mixture was saved at -80°C (mitochondrial fraction). The lysed cytosol and mitochondrial fractions as were used for western blot analysis.
12. Statistical Analysis
Experiments were performed for tree times. The differences between the treated and control cells were analyzed using the Student's t-test
between two groups, or one-way ANOVA was applied to compare more than two groups. The data were expressed as mean values ± SD of three independent experiments and p<0.05 were considered significant.
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V. RESULTS
1. EMMQ inhibited cell proliferation in human liver cancer cells To investigate the cytotoxic effect of EMMQ in human liver cancer cells, cell morphological and MTT assays were performed after treatment of human liver cancer cells and human hepatic cells with EMMQ. As shown in Fig. 16A, marked morphological changes of HepG2 cells based on cells showed they are smaller, round and blunt in size as EMMQ concentrations were increased after 48 h treatment; while there showed no changes in Huh7 and Hep3B cells. EMMQ reduced cell viability in HepG2 cells was dose-dependent with an IC50 of 5 μM as determined by MTT assay. The results showed no apparent growth inhibition shown in Hep3B cells and human hepatic cells L02 within the concentrations studied by MTT assay (Fig. 16B). There is no apparent IC50 for Huh7 cells. In addition, the colony formation capacity was significantly
decreased HepG2 cells by EMMQ treatment (Fig. 16C). The numbers of colonies in wild type p53 of HepG2 cells were reduced by more than 50%
when treated with 5 μM EMMQ, however it seemed no obvious inhibitory effect on mutant p53 Huh7 and null p53 Hep3B cells (Fig.
16C)
2. The EMMQ increased sub-G1 population cells, G1 arrest and apoptosis in HepG2 cells
To identify whether the growth inhibitory effect of EMMQ is caused by specific perturbation of cell cycle-related events, cell cycle phase distribution of cells treated with EMMQ (1, 5 or 10 μM) for 48 h. The
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cells were analyzed by flow cytometry after PI staining. The sub-G1 cell population significantly increased in HepG2 cells than in DMSO vehicle controls and the effects were dose-dependent, while no affect shown in Huh7 and Hep3B cells (Fig. 17A).
To examine whether EMMQ treatment induces apoptosis‚ apoptosis assay was carried out. We treated the human liver cancer cells with
various concentrations of EMMQ (0, 1, 5, or 10 µM) and then conducted the flow cytometry-based annexin V and PI double staining assay (Fig.
17B). The early and late apoptotic phase cell population rose to 26.0 % and 31.4 % for HepG2 cells with a concentration of 10 μM after 48 h treatment of EMMQ (Fig. 17C). The results implied that the EMMQ induced cell viability reduction in HCC cells carrying wild-type p53 was caused by apoptotic cell death following DNA damage.
To examine the effect of EMMQ treatment on cell cycle phase distribution. We treated HepG2 cells with various concentrations of EMMQ (0, 1, 5, or 10 µM) for 24 h and the cells were analyzed by flow cytometry after PI staining. As shown in Fig. 17D, treatment of HepG2 cells in a significant increase in the proportion of cells at the G1 phase and a reduction in the proportion of cells at the G2/M phase. The G1 phase percentage of HepG2 cells increased by approximately 30% compared with vehicle control. As shown in Fig. 17E, EMMQ could effectively increase PARP cleavage and p53 expression, while suppress cyclin D1 and CDK2 expression at 5 and 10 μM. These results demonstrated that the p53 increase down-regulation of cyclin D1 and CDK2 expression
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responsible for the G1 growth arrest induced by EMMQ for 24 h in HepG2 cells.
3. EMMQ induces HepG2 cell apoptosis by way of DNA damage Base on the observation that EMMQ induced p53 activation and a G1 arrest in HepG2, we evaluated the effect of EMMQ on DNA damage that play important roles in apoptosis by comet assay. The tails by comet assay demonstrating DNA lesions emerged after 3, 6 and 12 h treatment of EMMQ. EMMQ induced DNA lesions emerged in HepG2 with wild-type p53 status, while no apparent tails shown in Huh7 and Hep3B cells within the concentrations studied at 24 h (Fig. 18A). At 5 μM
concentrations of EMMQ led to HepG2 cells with a long DNA migration smear (comet tail), and effects occurred in a dose-dependent manner at 24 h (Fig. 18C). The comet tails indicating DNA lesions in HepG2 cells were detected after 3 h EMMQ treatment and the appearance of the excluded tail length was dose-dependent (Fig.18B). The DNA migration smear occurred in a time course and dose-dependent manner (Fig.18C) and greater significantly difference at 5 μM at 3, 6, 12 and 24 h.
Base on the observation that EMMQ induced DNA damage in HepG2, we evaluated the effect of EMMQ on DNA damage by western blot analysis. The advances showed that DNA damage could induce p53 and γ-H2AX activation. The γ-H2AX is a sensitive marker when DSBs damage in cells. As shown in Fig. 18D, EMMQ activated p53 and γ-H2AX expression and the effects occurred in a time course and
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dose-dependent manner. The results suggested that wild type p53 human liver cancer cells were more susceptible to DNA damage and the effects occurred in 3 h by EMMQ treatment.
4. EMMQ-induced HepG2 cell apoptosis through intrinsic pathway of mitochondrial membrane permeabilization, ΔΨm and cytochrome c release
Generation Mitochondria-related apoptotic pathway was linked to signal cascade following the ΔΨm disruption, signaling mitochondrial dysfunction involvement. It’s well know that the attenuated ΔΨm, outer membrane regulator Bcl-2 and release of downstream modulator
mitochondrial cytochrome c indicated mitochondria-mediated pathway leading to apoptosis. Using florescent dye JC-1 as a membrane-permeant dye, the loss of ΔΨm rose to 27 % in HepG2 cells after 10 μM of EMMQ treatment at 6 h, however it seemed no obvious significantly decrease at 3 h (Fig.19A). The data show the loss of ΔΨm in HepG2 cells suggested that the mitochondria-mediated apoptosis by low dosage of EMMQ was initiated starting at a concentration of 10 μM after 6 h treatment of EMMQ. The impaired mitochondrial functions were further accentuated by cytochrome c release in HepG2 cells with increasing drug
concentrations after 24 h treatment (Fig.19D). The effects of the drug on the cytochrome c release from mitochondria in HepG2 cells by western blot. The results showed that the increase of cytochrome c contents in the cytosolic fraction, decreases of cytochrome c in the mitochondrial
fraction after EMMQ treatment were observed (Fig.19E). These results
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suggested that EMMQ induced apoptosis through intrinsic pathway of mitochondrial membrane permeabilization, ΔΨm and cytochrome c release in HepG2 cells.
5. EMMQ induces HepG2 cell apoptosis through ROS production ROS is produced especially when cells undergo one of the causative factors (chemical or environmental stress) leading to cell cycle arrest or apoptosis. The assay investigated whether EMMQ could induce ROS production. Using florescent dye DCF-DA as a general ROS indicator, a significant fluorescent increase was observed by flow cytometry in
HepG2 cells ( p<0.05) treated with various concentrations of EMMQ (0, 1, 5, or 10 µM) for 24 h (Fig. 19B). Fig. 19C shows the intracellular ROS production was increased in HepG2 cells suggested that ROS-mediated apoptosis by low dosage of EMMQ was initiated starting at a
concentration of 1 μM after 24 h treatment of EMMQ, however it seemed no obvious inhibitory effect at 6 h. The intracellular ROS production rose to 45 % in HepG2 cells at 24 h (Fig.19C). These results indicated that EMMQ induced apoptosis in HepG2 cells in dose dependent of the intrinsic apoptotic pathway.
6. EMMQ-induced apoptosis through intrinsic pathway
The apoptosis attributed to DNA damage can proceed through intrinsic pathway or extrinsic pathway with escalated p53 levels. To clarify whether the intrinsic pathway is involved in EMMQ-induced apoptosis, we firstly examined the protein expression of p53,
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anti-apoptotic Bcl-2 family protein (Bcl-2), proapoptotic Bcl-2 proteins Bax, cytochrome c, caspase-3, cleavage caspase-3 and cleavage of poly(ADP ribose) polymerase (PARP) by western blot. Fig. 20A shows the increased concentrations of EMMQ activated p53 and reduced
p-AktS473, Bcl-2 and procaspase-3 levels, and increased Bax, cytochrome c, cleavage caspase-3 and cleavage of poly(ADP ribose) polymerase (PARP) in HepG2 cells after 48 h treatment (Fig. 20A). On the other hand, in the presence of 5 μM of EMMQ, activation of p53, reduction of Bcl-2 intensities as well as procaspase-3 dissipation and increased Bax, cytochrome c, cleavage caspase-3 and PARP cleavage in HepG2 cells were detected in time-dependent manners. No change was shown in Bcl-2, Bax levels and procaspase-3 as well as cleavage caspase-3 and PARP in p53-null Hep3B cells within the time intervals and drug concentration ranges as studied (Fig. 20B). The results of western blots implied that EMMQ induced apoptosis through p53 activation and diminished Bcl-2 and cleavage of caspase-3 and PARP is related to intrinsic pathway.
7. Down-regulated p53 abolished the onset of EMMQ-induced cell death in hepatocellular carcinoma cells
To ensure that p53 was indeed necessary in drug-mediated cell death, experiments by transfecting shRNA targeting exon 7 of p53 to cells prior to drug treatment were carried out along with those of NS control. The result of cell viability indicated that HepG2 cells were transfected with p53 shRNA led to the sensitivity toward EMMQ was eliminated as compared with cells transfected with NS control (Fig. 21A). The DNA
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lesions were diminished with EMMQ treatment for 12 h in HepG2 cells with p53 shRNA indicated that p53 was selectively knocked down might influence the DNA damage induced by EMMQ (Fig. 22A and 22B).
PI and annexin V double staining assay showed that HepG2 cells were decreased sub-G1 population and apoptosis ratio with EMMQ by knocking down p53. The results indicated knocking down p53 might affect EMMQ induced HepG2 cells cell death (Fig. 21B and 21C).
Western blot analysis of HepG2 cells showed that cells introduced with p53 shRNA exhibited significant reduction of p53 as compared with those transfected with NS control alone. In addition, the mitochondria modulator Bcl-2, Bax, cytochrome c, γ-H2AX and pro-survival gene Akt were unaffected by EMMQ by knocking down p53 (Fig. 22C). The results altogether suggested that p53 was needed during mitochondrial pathway activation that predates the effectiveness of EMMQ in
motivating apoptotic cell death of HCC cells.
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VI. DISCUSSION
HCC is one of the most common malignances and the second most frequent cause of cancer death. HCC is a highly aggressive tumor with a poor or no response to common therapies [89]. There have been several early trials evaluating targeted therapies for advanced HCC. Sorafenib is a kinase inhibitor drug approved for the treatment of advanced HCC.
Sorafenib treatment induces autophagy [90] , which may suppress tumor growth. However, autophagy can also cause drug resistance [91].
Sunitinib is a multi-targeted receptor tyrosine kinase inhibitor drug approved for the treatment of advanced HCC. Bevacizumab is an
angiogenesis inhibitor, a drug that slows the growth of new blood vessels [92]. The main side effects of bevacizumab treatment are hypertension and heightened risk of bleeding [93]. Sunitinib blocks the tyrosine kinase activities of KIT, PDGFR, VEGFR2 and other tyrosine kinases involved in the development of tumors [94], but the side effects associated with drug treatment [95]. Many lines of clinical investigation indicate that none of the adjuvant therapies is particularly effective in treating HCC after surgery and systemic traditional chemotherapy has a very low response rate for HCC. Therefore, new effective and well-tolerated therapy strategies are urgently needed.
The DNA-damage response is a number of cellular processes, including recognize damaged DNA; amplify the damage signal; control cell cycle progression, DNA repair, and apoptosis [96]. Many anticancer drugs induced cell apoptosis as a result of DNA damage involving p53
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activation [96]. When DNA DSBs, it is always followed by the
phosphorylation of histone, H2AX. H2AX play a key role in the repair process of damaged DNA [97]. The DSBs damage marker, γ-H2AX plays a key role in apoptosis by interact with the tumor suppressor p53 [98]. In study further validated that DNA repair system is maintained.in L02, Huh 7 and Hep3B cells, but no in HepG2 cells as indicated by comet assay (Fig 18A and 18C). The deficient DNA integrity accounts for the sensitivity and the effectiveness of EMMQ. In addition, our data
indicated that DNA lesions were increased in HepG2 cells and the effects significantly increase at 5 μM after 24 h treatment with EMMQ by comet assay (Fig. 19A and 19B). As shown in Fig. 19C and 19D, DNA lesion was start at 3 h and the effects were increased in time-dependent manners.
Western blotting analysis showed that p53 and γ-H2AX expression were activated after treatment of EMMQ and the effects occurred in a time course and dose-dependent manner (Fig. 19E). The result demonstrated that EMMQ induced DNA damage was start at 3 h and activated p53 expression and caused the formation of γ-H2AX in dose and
time-dependent manners. The tumor suppressor p53 contributes to the preservation of genetic stability through mediating either a G1 arrest or apoptosis in response to DNA damage [99]. The tumor suppressor p53 expression increased in response to DNA damage arrests G1 phase of the cell cycle through inhibiting the synthesis of cyclin-dependent kinases.
Our data demonstrated that G1 phase of cell cycle increased in HepG2 cells and the effects significantly increase at 10 μM after 24 h treatment of EMMQ (Fig. 17D). Fig.17E showed that the tumor suppressor p53 and
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PARP cleavage were increased, which cyclin D1 and CDK2 that by treating EMMQ at 5 and 10 μM for 24 h. These data suggest that EMMQ induced G1 arrest due to p53, cyclin D1 and CDK2 expression
downstream of DNA damage and caused the cleavage of PARP to apoptosis at 24 h in HepG2 cells.
Previous reports have shown that ROS induce apoptosis by activating of MAPKs to instrumental in p53 activation and phosphorylation [100]. The evidence show that the increase in intracellular ROS associated with the magnitude of p53 expression correlated with apoptosis in cancer cells [101]. The p53 plays a pivotal role in cell survival and induction of ROS. The evidence showed that production of ROS and disruption of mitochondria was associated with p53 activation [102]. The study demonstrated that ΔΨm control ROS production. Previous describe showed that EMMQ induced p53 activation through DNA damage at 3 h. In current study showed that interference of ΔΨm in HepG2 cells at 6 h, which production of ROS at 24 h by EMMQ treatment (Fig. 20B and 20C). The data suggested that EMMQ induced apoptosis through DNA lesion lead to attenuate ΔΨm and ROS production in HepG2 cells. Our previous report showed that EMMQ induced dysfunction of mitochondrial lead to cytosolic
cytochrome c release in NSCLC cells. As shown Fig. 20D and 20E, cytosolic cytochrome c release from mitochondria as a result of
impairment of mitochondrial functions after 24 h treatment of EMMQ in HepG2 cells. These data demonstrated that EMMQ induced apoptosis
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through DNA damage lead to attenuate ΔΨm, ROS production and release of cytosolic cytochrome c in HepG2 cells.
The tumor suppressor p53 plays an important role in regulating apoptosis. Our previous report showed that EMMQ induced apoptosis through activation of p53 and corresponding down-stream Akt, Bcl-2, Bax, cytochrome c and caspase-3 in NSCLC cells. Several reports
showed that Akt, Bcl-2 and Bax were associated with p53 expression [74, 103, 104]. In the current study we showed that p53 was activated and down-regulating Akt, Bcl-2, Bax, cytochrome c, caspase-3 and cleavage of PARP after treatment of EMMQ in HepG2 cells (Fig. 21A). Our study also demonstrated that knocking-down p53 can be offset cell sensibility, DNA lesions and final apoptotic cell death after EMMQ treated in HepG2 cells (Fig.22 and 23). Thus, EMMQ-mediated apoptosis through Akt, Bcl-2 and Bax expression, cytochrome c release and cleavage of PARP were associated with p53 status.
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VII. CONCLUSION
Previously, we reported the compound attenuated ΔΨm, DNA damage and hastened apoptosis in NSCLC cells carrying wild-type p53.
In current study showed that EMMQ damaged DNA firstly and then attenuated ΔΨm and accelerated apoptosis in HepG2 cells carrying
wild-type p53. In Figure 23, this proposed model of EMMQ-induced cell death through DNA damage increased expression of p53 and γ-H2AX after treatment EMMQ 3 hours. And the compound attenuated ΔΨm after treatment for 6 hours. After treatment EMMQ for 24 hours, data showed that ROS production, the expression of Bax and cytochrome c levels increased but the expression of Bcl-2 was decreased. After treatment 48 hours, EMMQ down-regulated Akt and induced the expression of
caspase-3 and cleavage of PARP leading to cell death in HepG2 cells.
This is an event for indolylquinoline derivative mediated cell death in human hepatocellular carcinoma HepG2 cells. Based on these results, EMMQ would be a potential compound for treating human hepatocellular carcinoma.
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VIII. FIGURE AND LEGENDS (A)
(B)
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(C)
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Figure 16 Effect of EMMQ on cell growth in HCC cell lines and human hepatic cells (A) Cells were treated with various concentrations of EMMQ for 48 h and used microscope (100X) to observe the
morphology of cells. EMMQ induced HepG2 cells morphological
changes and cell death at 5μM; while no morphological changes shown in Huh7 and Hep3B cells (B) EMMQ inhibits cell viability of HCC cells
changes and cell death at 5μM; while no morphological changes shown in Huh7 and Hep3B cells (B) EMMQ inhibits cell viability of HCC cells