VIII. FIGURE AND LEGENDS
5. EMMQ induces HepG2 cell apoptosis through ROS
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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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 and L02 cells at 48 h. The cells were cultured in 96-well plate for 48 h, and then were treated with different concentrations of EMMQ as
indicated. After 48 h of treatment, MTT cell viability assay was
performed and the blue formazan dye produced by the viable cells was quantified by measuring the absorbance of 540 nm after elution by DMSO. The DMSO treated cells were served as 100% control. Data are represented as the mean values ± standard deviation (SD). The asterisk ( ) p< 0.05 and ( ) p < 0.01 indicated a significant difference against the vehicle. (C) Clonogenicity, a marker of senescence, was determined in HCC cell lines by treating cells for 48 h with EMMQ at different
concentrations or vehicle control and subsequently plating 500 cells/well, culturing for ten days and then staining cells with 0.005 % crystal violet in PBS. EMMQ decreased clonogenicity of HepG2 cells by 7-fold as compared to control. Colony formation was calculated as a percentage to untreated control cultures.Symbol (D) indicates DMSO.
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Figure 17 EMMQ treatment induces change in cell cycle distribution, G1 phase cell cycle arrest and apoptosis ratio on HepG2 cells. (A) Cells were treated with EMMQ for 48 h and stained with PI and detected the cell cycle distribution by flow cytometry. The populations of cells in sub-G1 phase increased on EMMQ treatment and the populations were at the expenses of G1 cells. (B) HCC cells were treated with EMMQ for 48 h and analyzed by flow cytometry after double staining with annexin V-FITC/PI. (C) The quantitative determination of apoptosis cell
population. Early (dark) and late (light) apoptotic cell population ratio are shown in different cell lines with respect to DMSO. (D) EMMQ induced cell cycle arrest in HepG2 cells for 24 h. The HepG2 cells were treated with various concentrations of EMMQ for 24 h and stained with PI and detected the cell cycle distribution by flow cytometry. The treatment of HepG2 cells with EMMQ resulted 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 ( p< 0.05). (E) Protein lysates were prepared from HepG2 cells after treatment with various concentrations of EMMQ for 24 h. PARP, p53, cyclin D1 and CDK2 protein expression levels were analyzed by western blot.Symbol (D) indicates DMSO.
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Figure 18 EMMQ treatment induces DNA damage in HepG2 cells.
EMMQ induced DNA damage in HCC cells were examined by comet assay. (A) The HCC cells were treated with EMMQ for 24h. Cells were collected and DNA damage was determined by comet assay. DNA tail formations were determination in HCC cells after EMMQ treatment. (B) The HepG2 cells were treated with EMMQ for 3, 6 and 12 h. Cells were collected and DNA tail formations were determined by comet assay with various concentrations of EMMQ treatment. (C) The DNA damage represented by the magnitude of the comet tail moment for HepG2 cells exposed to the various time. The tail lengths ratio is shown in different time with respect to DMSO. EMMQ caused HepG2 cells DNA damage at 5 μM with various time. The tail lengths were quantified by using
CometScore™ software. The asterisk ( ) p< 0.05 and ( ) p < 0.01 indicated a significant difference against the vehicle. The tail lengths ratio is shown in different cell lines with respect to DMSO. EMMQ caused HepG2 cells DNA damage at 5 μM for 3, 6, 12 h and 24 h. (D) Protein lysates were prepared from HepG2 cells after treatment with various concentrations and time of EMMQ. The tumor suppressor p53 and γ-H2AX protein expression levels were analyzed by western blot. The treatment of HepG2 cells with EMMQ resulted in a significant increase of p53, and γ-H2AX protein expression levels at 3 h.Symbol (D)
indicates DMSO.
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Figure 19 EMMQ induces ROS causes apoptosis through reduced ΔΨm and enhanced cytochrome c release in HepG2 cells. (A) HepG2 cells were treated EMMQ demonstrate increased level of ROS. Huh7, HepG2 and Hep3B cells were treated with 1, 5 or 10 μM of EMMQ for 24 h. The DMSO treated cells were served as 100% control. (B)
Evaluation of ΔΨm following EMMQ treatment. Cells treated with different concentrations of EMMQ were determined for changes in ΔΨm in HepG2 cells at 6 and 24 h. Data are represented as the mean values ± standard deviation (SD). The asterisk ( ) p< 0.05 and ( ) p < 0.01 indicated a significant difference against the vehicle. (C) The EMMQ induced release of cytochrome c from mitochondria to cytosol in HepG2 cells. Cells were treated with 1, 5 or 10 μM EMMQ or vehicle control DMSO for 24 h. The cells were stained with Mitotracker (green, mitochondrial staining), DAPI (blue, nuclear staining) and secondary antibody conjugated with TRITC for cytochrome c (red). The pointed arrow signified the co-localization of red color cytochrome c and green color mitochondria, while blue color stood for nucleus. (D) Cytochrome c levels in the cytosolic fractions and mitochondrial fractions of EMMQ treatment were analyzed by western blot analysis. M and C stand for mitochondrial and a cytosolic fractions, respectively.Symbol (D) indicates DMSO.
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Figure 20 EMMQ treatment induces apoptotic cell death in intrinsic pathway related proteins in HepG2 cells. (A) The protein lysates of the collected Huh7, HepG2 and Hep3B cells as being treated with 1, 5 and 10 μM of EMMQ for 48 h were analyzed by western blot as specified in Materials and Methods. (B) Treatment of EMMQ at different time points.
Proteins from lysates in cells treated with 5 μM of EMMQ at different time points (12, 24 and 48 h) were analyzed by western blot.Symbol (D) indicates DMSO.
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Figure 21 Effects of EMMQ treatment by p53 silencing in HepG2 cells. (A) HepG2 cells were transfected with either p53 shRNA or NS for 24 h and then treated with 10 μM EMMQ for 48 h. The cell numbers were determined by trypsin-EDTA and counted by trypan blue exclusion assay. ( ) p<0.05, compared with drug alone with NS RNA-transfected cells. Symbol (-) indicates no transfection. (B) Cells were treated with EMMQ for 48 h and stained with PI and detected the cell cycle
distribution by flow cytometry. (C) EMMQ treatment reduces change in cell cycle distribution. HepG2 cells transfected with p53 shRNA were treated with EMMQ (10 μM) for 48 h and analyzed by flow cytometry after double staining with annexin V-FITC/PI.Symbol (D) indicates DMSO.
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Figure 22 EMMQ treatment reduced DNA damage by p53 silencing in HepG2 cells. (A) The DNA lesions were reduced in HepG2 cells with p53 shRNA with EMMQ treatment for 12 h. (B) The tail lengths were quantified by using CometScore™ software. The asterisk ( ) p< 0.05 and ( ) p < 0.01 indicated a significant difference against the vehicle. (C) Expression of p53 and apoptosis markers in shRNA and NS transfected cells as monitored by western blot. Symbol (D) indicates DMSO.
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Figure 23 Proposed a model for EMMQ mediated apoptosis in HepG2 cells. EMMQ induced cell death through DNA damage through intrinsic and cell cycle arrest leading to apoptosis. First, the DNA lesions increased expression of p53 and γ-H2AX and decreased cyclin D1 and CDK 2 leading to G1 arrest of cell cycle. Second, the tumor suppressor p53 down-regulating Akt, Bcl-2, Bax, cytochrome c, caspase-3 and cleavage of PARP, in cells treated with EMMQ is the critical event leading to cell death in HepG2 cells.
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