Lysosomal membrane protein TMEM192 deficiency triggers crosstalk between autophagy and apoptosis in HepG2 hepatoma cells

  • Authors:
    • Zhen Liu
    • Ya Juan Lv
    • Yi Peng Song
    • Xiao Hong Li
    • Yuan Na Du
    • Cui Hong Wang
    • Li Kuan Hu
  • View Affiliations

  • Published online on: June 20, 2012     https://doi.org/10.3892/or.2012.1881
  • Pages: 985-991
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Abstract

As constituents of lysosomes, lysosomal membrane proteins play important roles in lysosome-related autophagy and apoptosis. In a recent proteomic study of lysosomal proteins, we identified transmembrane protein 192 (TMEM192) as a novel lysosomal membrane protein candidate. Using specific anti-TMEM192 antibody and lysosomal markers, the lysosomal localization of TMEM192 was determined by immunofluorescence. TMEM192 shows a wide expression pattern in mouse tissues. Interestingly, TMEM192 was found to be highly expressed in tumor cell lines, while it was not expressed or was detected at low levels in normal cell lines. By knockdown of TMEM192 expression using specific siRNAs, we found that TMEM192-deficient HepG2 hepatoma cells show growth inhibition and increased apoptosis. Autophagy was shown to be activated through detection of LC3II expression. Increased apoptosis was inhibited by blocking the expression of the key autophagy gene Atg7 in TMEM192-deficient HepG2 cells. The results suggest that TMEM192 is important for tumor cell growth and proliferation. TMEM192 deficiency can induce autophagy in tumor cells, and can further activate apoptosis by the mitochondrial pathway through autophagy. TMEM192 promotion of autophagy may be a new route for tumor therapy.

Introduction

Autophagy refers to the highly regulated and evolutionarily conserved process of turnover and maintenance of cellular components that is required for cellular homeostasis (1). It is a multistep process where portions of the cytoplasm and organelles are engulfed in double-membrane vesicles called autophagosomes. These structures then fuse with lysosomes, resulting in the destruction of their contents by the acid hydrolases provided by the lysosome. There are three main autophagic pathways: macroautophagy, microautophagy, and chaperone-mediated autophagy (2). Autophagy is controlled by autophagy-related genes, many of which are involved in autophagosome formation. This process features conjugation systems that are well-conserved among eukaryotes: Atg12-Atg5 and Atg8 (microtubule-associated protein 1 light chain 3, LC3)-phosphatidylethanolamine systems (3).

Numerous studies have demonstrated a variety of physiological and pathophysiological roles in autophagy, such as adaptation to nutrient deprivation, intracellular clearance of protein and organelles, development, antiaging, elimination of microorganisms, cell death, tumor suppression, and antigen presentation (46). In tumor cells, autophagy has been recognized as an important regulator of the promotion of tumor cell survival and development and restriction of necrosis (79). However, recent studies suggest that the role of autophagy is more complicated, and may have diametrically opposite consequences for the tumor, depending on the circumstances (10).

Here, we demonstrate a contradictory role for autophagy in HepG2 hepatoma cells, which was induced by knockdown of the expression of transmembrane protein 192 (TMEM192), a lysosome membrane protein.

Materials and methods

Cell lines and cell culture

If not stated otherwise, LO2 and HepG2 cells were grown in DMEM (Dulbecco’s modified Eagle’s medium; Gibco Life Technologies) supplemented with 10% fetal calf serum (Gibco Life Technologies) and 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA) under 5% CO2 atmosphere at 37°C.

Western blotting

Cells were harvested in lysis buffer and subjected to western blot analysis. Signals were detected using HRP-conjugated secondary antibodies and an ECL detection reagent. Details are provided in a previous study (11). The primary antibodies included rabbit anti-human TMEM192 (Sigma-Aldrich), anti-LAMP1 (Santa Cruz Biotecnhology, Santa Cruz, CA, USA), rabbit anti-human caspase-12 (Biotime, Haimen, Zhejiang, China), rabbit anti-caspase-3, anti-P-p38 MAPK and β-actin, rat anti-human Bax and caspase-8 (Biotime). The LC3 antibody was a gift of Dr Yi Wang (Shanghai Jiao Tong University).

RNA interference

Cells were transfected with a non-targeted control siRNA (small interfering RNA) or target-specific siRNAs directed to TMEM192 sequences (Shanghai Sangon, China). Briefly, 30 nM TMEM192 siRNA (5′-AATCTTCTGTGGTTTATTCUU-3′) was transfected into cells using Oligofectamine (Invitrogen). Transfected non-targeted siRNA (20 nM, 5′-AATATCTCGTTCGTTTGTTUU-3′) cells and untransfected cells were mocked as controls. After 24 h, cells were re-transfected with the indicated siRNAs for additional 24 h intervals. In the experiments of 1–5 days, cells were harvested for further analysis. The Atg7 gene was silenced by the same procedure. The siRNA targeting Atg7 mRNA comprised the 27-base RNA nucleotide sequence 5′-AACTTAGCCCAGTACCCTGGATGGCCT-3′. That of the control siRNA was 5′-AATAGCTCCACCTCCATGAGGGTCGCTUU-3′.

Cell proliferation assay

For growth curves, 106 cells were collected and transferred to a 24-well plate, and were seeded in 10% serum. Monolayers were washed and the medium was replaced by siRNA transfections as above, carried out on Days 1 and 2. Fresh medium was added on Day 4. Cell survival and growth were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) on different days, as previously described (12).

Apoptosis cell analysis by flow cytometry (FCM)

To measure the quantity and the ratio of apoptotic cells, the Annexin V-FITC Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) was utilized. The cells were treated as described above. Cells were harvested, fixed and stained with PI and Annexin V-FITC as described in the kit instructions. Finally, the stained cells were analyzed immediately on a FACScalibur flow cytometer (BD Biosciences).

Immunoflurorescence staining

For cells grown on coverslips in 24-well plates, the monolayer was washed twice with PBS (phosphate-buffered saline). Then the cells were fixed with 4% PFA (paraformaldehyde) for 10–30 min. After washing twice with PBS, cells were permeabilized/blocked by treatment with SS-PBS (0.2% saponin containing 10% bovine serum albumin in PBS) for 30 min. For double immunostaining, primary and secondary antibodies were overlaid on coverslips in SS-PBS for 1 h and 30 min separately, followed by 3 washes with PBS. Coverslips were mounted onto 1-mm glass slides using fluorescent mounting medium. All steps were performed at room temperature. Samples were analyzed using a Leica TCS SP2 confocal microscope. In the case of double staining with LysoTracker Red, cells were labeled with 50 nM LysoTracker Red for 2 h in serum-free DMEM and washed with PBS, then fixed with 4% PFA for 20 min at room temperature.

Quantitative real-time PCR and RT-PCR

Using 5 μg of total RNA as template, reverse transcription was performed with PrimeScript II first strand cDNA Synthesis kit (Takara). Measurement of the relative quantity of the cDNA of interest was carried out using SYBR® Premix Ex Taq™ (Takara), 300 nM of the appropriate forward and reverse primers, and 1 μl of the cDNA mixture. The sense and antisense primers used for TMEM192 were 5′-GCCATCTTGGCACTGGAAC-3′ and 5′-GCTTACTCAGCAGCGCATT-3′ (59°C, 245 bp), respectively. The primers for mouse homologous TMEM192 were 5′-GTATGGATGACGACCCGCTTCT-3′ and 5′-TGGGCACTTGTCTTCAGTTGGAT-3′ (63°C, 200 bp). Real-time PCR assays were performed in the Applied Biosystems 7500 Real-Time PCR System using thermal cycling parameters recommended by the manufacturer (40 cycles of 15 sec at 95°C and 1 min at 60°C). PCR product purity was determined by melting curve analysis. Within each plate, we included triplicates of each sample. Data were analyzed with SDS2.1 software (v.2.1.0.3, Applied Biosystems). At least three separate experiments per subject were performed. Values exceeding two standard deviations were excluded.

Statistical analysis

The data are expressed as means ± SD. Tests for significance of differences were performed by ANOVA or Student’s t-test as appropriate. P<0.05 was considered statistically significant.

Results

Lysosomal localization of TMEM192 by immunofluorescence

The lysosomal localization of TMEM192 was confirmed by immunofluorescence. Using specific anti-TMEM192 antibody, TMEM192 staining in HLF-I cells showed small punctuate patterns throughout the cytoplasm, predominantly in the perinuclear region. No signal was detected at the plasma membrane or in the nucleus. This suggested that TMEM192 protein was localized in an intracellular compartment (green fluorescence in Fig. 1A). Double staining with antibody against LAMP1, a marker protein for lysosomes (red fluorescence in Fig. 1A), showed colocalization with TMEM192. Double staining with the lysosome tracker red (LTR), a marker of the lysosome, also showed that it was colocalized with TMEM192 (Fig. 1B).

Tissue distribution of the TMEM192

Tissue expression analysis provides important information about the function of TMEM192. Therefore, we analyzed the expression of TMEM192 in mouse tissues and different cell lines. Using real-time PCR, we found that TMEM192 was a widely-expressed protein. Its mRNA was abundant in the kidney and liver while little or no expression was detected in other tissues in the mouse, such as the spleen and intestine (Fig. 2A and B). Interestingly and importantly, TMEM192 was highly expressed in tumor cell lines while little or low expression was detected in normal cell lines (Fig. 2C). The differential expression of TMEM192 was further confirmed by RT-PCR analysis (Fig. 2D).

TMEM192 siRNA induces apoptosis in HepG2 tumor cell lines by upregulating Bax, caspase-3 and p38 MAPK and activating autophagy

According to the experiment above, TMEM192 is highly expressed in tumor cells. To elucidate the potential molecular function of TMEM192, we analyzed the effect of TMEM192 deficiency in HepG2 hepatoma cells and normal LO2 cell lines, by silencing TMEM192 expression with specific siRNA targeting the TMEM192 gene. RT-PCR with specific primers demonstrated a marked reduction in the levels of TMEM192 mRNA in cells transfected with the target siRNA (data not shown). This was consistent with the protein expression data (Fig. 3C). Our results using MTT and FCM have shown that TMEM192 deficiency in HepG2 cells induced growth inhibition and increased apoptosis while LO2 cells were unaffected (Fig. 3A and B). Intracellular TMEM192 has been shown to localize in the lysosome, and may have a function in the lysosomes. In the present experiments, HepG2 cells transfected with TMEM192 siRNA were shown to activate autophagy by detecting the LC3 protein (Fig. 3C). LC3 exists in two forms: the free mature form (LC3-I) and the faster lapidated LC3 (LC3-II). The presence of LC3-II band confirms that the reaction of LC3 conjugation to phospholipids was activated. Caspase-12 is an ER stress-related protein. These cells also showed the presence of an activated degraded caspase-12 fragment (48 kDa, Fig. 3C). Caspase-3, normally present as a zymogen (32 kDa), was detected in its activated 17 kDa form, while caspase-8 was detected only as the zymogen. Synthesis of Bax protein was activated 48 h after transfection. In addition, we found that p38-MAPK was phosphorylated (Fig. 3C). These results indicate that TMEM192 deficiency induces autophagy and apoptosis of HEPG2 cells. It is likely that the apoptosis occurred by upregulating the expression of Bax and caspase-3, as well as the phosphorylation of p38-MAPK.

Blocking autophagy inhibits apoptosis of HEPG2 cells induced by TMEM192 deficiency and downregulates caspase-12, capase-3 and Bax

Atg7 is a crucial autophagy gene. We used Atg7 siRNA to investigate whether blocking the autophagy would affect the apoptosis of HepG2 cells induced by TMEM192 deficiency. In this experiment, the Atg7 mRNA was silenced successfully using Atg7 siRNA (Fig. 4A). In TMEM192-deficient HepG2 cells, autophagy and apoptosis were inhibited after transfection with Atg7 siRNA. Blocking of the activation of caspase-12, Bax and caspase-3 was also observed (Fig. 4B). These results indicate that TMEM192 siRNA induced apoptosis in HepG2 cells through autophagy and the ER pathway, and this effect was closely related to the mitochondrial pathway, all of which can be suppressed by downregulation of autophagy by silencing the Atg7 gene.

Discussion

In previous proteomic analyses, TMEM192 was identified in lysosomal membranes (13). The exogenous and endogenous localization of TMEM192 was further verified (14), which suggest that TMEM192 is a lysosomal membrane protein. In the present study, using immunofluorescence staining approaches, we also demonstrated that TMEM192 colocalized with the lysosomal marker protein, LAMP1, and the lysosomal tracker, LTR. TMEM192 has tissue-specific expression. In mouse, its ortholog is highly expressed in the liver and brain but shows little or low expression in spleen and other tissues. Importantly, TMEM192 was highly expressed in tumor cell lines, whereas lower expression was detected in normal cell lines. This distribution may suggest the important function of TMEM192. In order to investigate the potential role of TMEM192 in tumor cell line proliferation and growth, we obtained TMEM192-deficient HepG2 cells using RNAi. HepG2 cells deficient in TMEM192 showed growth inhibition.

Autophagy refers to the highly regulated and evolutionarily conserved process of turnover and maintenance of cellular components that is required for cellular homeostasis (1). Autophagy and apoptosis are partners in cell life and death. They crosstalk, interact and share common regulatory pathways (15). In this study, our results show that TMEM192 siRNA activated autophagy accompanied by an increased expression of LC3-II. In addition, apoptosis was induced as evidenced by caspase-12 activation. Autophagy and apoptosis were inhibited after treatment with Atg7 siRNA, which indicates that the autophagy pathway plays a critical role in apotosis of HepG2 cells.

Studies have shown that caspase activation through the death receptor pathway, the mitochondrial pathway, and the ER pathway causes cell apoptosis. Caspase-8 is the primary initiator of the death receptor pathway, while caspase-3 is the main caspase effector in the mitochondrial pathway (16,17). Bax plays an important role in the mitochondrial apoptosis pathway (18,19). In HepG2 cells, we found that RNAi of TMEM192 increased the expression of Bax and caspase-3, as well as the phosphorylation of p38 MAPK, without affecting the levels of caspase-8. When Atg7 siRNA was used to block the autophagy, however, expression of Bax and caspase-3 was significantly downregulated. Since caspase-12 is a key factor in ER stress, we therefore, hypothesized that TMEM192 siRNA induces ER stress in HepG2 cells by activating caspase-12, and further activates Bax and the phosphorylation of p38 MAPK. In turn this induces a translocation of Bax from the cytosol into the mitochondria promoting the release of cytochrome c, which finally activates caspase-3 resulting in tumor cell apoptosis.

Autophagy is controlled by autophagy-related genes, many of which are involved in autophagosome formation. This process features conjugation systems that are well-conserved among eukaryotes: Atg5–12 and Atg8 (LC3)-phosphatidylethanolamine systems. In this study, we have shown that TMEM192 deficiency can induce autophagy concurrently with apoptosis in HepG2 hepatoma cells. Previous studies have shown that there is crosstalk between apoptosis and autophagy (15). Although the two processes are markedly different, several pathways regulate both the autophagic and the apoptotic machinery and autophagy can cooperate with apoptosis. For example, alterations in the mitochondrial membrane potential-induced Bax/Bak ratio can signal to both apoptosis and mitochondrial autophagy (20). Indeed, earlier studies have shown that mitochondria that have sustained stress-induced damage can be removed by selective mitochondrial autophagy (2124). There is a consensus that autophagy itself is usually a survival mechanism (25). However, it is possible that autophagy can contribute to the induction of some death responses, such as apoptosis, in light of the fact that the coordinate activation of both autophagic and apoptotic signals were induced by TMEM192 siRNA. By knockdown of the Atg7 expression, we found that autophagy was inhibited by the reduction of LC3-II expression. Interestingly, caspase-12 and Bax-independent apoptosis was reversed. The impact of autophagy on apoptosis is highly context-dependent, with compelling evidence that autophagy can either inhibit or promote apoptosis, depending on the system. In tumor cells, the relations between autophagy and apoptosis are more complicated (10,26).

Lysosomal membrane proteins have been associated with autophagy. For example, deficiency of mice or cells in lysosomal protein, such as LAMP1 and LAMP2, resulted in increased autophagy, while other proteins, such as LAPTM4B, exhibited promotion of autophagy. In the tumor cells, TMEM192 deficiency can also activate the autophagy signaling pathway which can promote apoptosis independently of the caspase-12 and Bax pathway. None of these results were found in normal cells (data not shown). In summary, this study offers proof of crosstalk between autophagy and apoptosis. TMEM192 promotion of autophagy may be a new route for tumor therapy.

Acknowledgements

The authors would like to gratefully acknowledge the contributions of Professor Xu Ra and Professor Chen Xiao.

Abbreviations:

ER

endoplasmic reticulum

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

HRP

horseradish peroxidase

LAMP

lysosome-associated membrane protein

siRNA

small interfering RNA

TMEM192

transmembrane protein 192

References

1 

Mizushima N: Autophagy: process and function. Genes Dev. 21:2861–2873. 2007. View Article : Google Scholar

2 

Yang W, Monroe J, Zhang Y, George D, Bremer E and Li H: Proteasome inhibition induces both pro- and anti-cell death pathways in prostate cancer cells. Cancer Lett. 243:217–227. 2006. View Article : Google Scholar : PubMed/NCBI

3 

Yokoyama T, Miyazawa K, Naito M, et al: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells. Autophagy. 4:629–640. 2008. View Article : Google Scholar : PubMed/NCBI

4 

Shintani T and Klionsky DJ: Autophagy in health and disease: a double-edged sword. Science. 306:990–995. 2004. View Article : Google Scholar : PubMed/NCBI

5 

Shi JJ, Liu KY and Yang YP: Autophagy in the pathogenesis of Parkinson’s disease. Sheng Li Ke Xue Jin Zhan. 40:67–71. 2009.(In Chinese).

6 

Levine B and Kroemer G: Autophagy in the pathogenesis of disease. Cell. 132:27–42. 2008. View Article : Google Scholar : PubMed/NCBI

7 

Degenhardt K, Mathew R, Beaudoin B, et al: Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 10:51–64. 2006. View Article : Google Scholar : PubMed/NCBI

8 

Abedin MJ, Wang D, McDonnell MA, Lehmann U and Kelekar A: Autophagy delays apoptotic death in breast cancer cells following DNA damage. Cell Death Differ. 14:500–510. 2007. View Article : Google Scholar : PubMed/NCBI

9 

Bauvy C, Gane P, Arico S, Codogno P and Ogier-Denis E: Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29. Exp Cell Res. 268:139–149. 2001. View Article : Google Scholar : PubMed/NCBI

10 

Kondo Y, Kanzawa T, Sawaya R and Kondo S: The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 5:726–734. 2005. View Article : Google Scholar : PubMed/NCBI

11 

Chen T, Guo J, Yang M, et al: Cyclosporin A impairs dendritic cell migration by regulating chemokine receptor expression and inhibiting cyclooxygenase-2 expression. Blood. 103:413–421. 2004. View Article : Google Scholar : PubMed/NCBI

12 

Oka M, Maeda S, Koga N, Kato K and Saito T: A modified colorimetric MTT assay adapted for primary cultured hepatocytes: application to proliferation and cytotoxicity assays. Biosci Biotechnol Biochem. 56:1472–1473. 1992. View Article : Google Scholar : PubMed/NCBI

13 

Lubke T, Lobel P and Sleat DE: Proteomics of the lysosome. Biochim Biophys Acta. 1793:625–635. 2009. View Article : Google Scholar : PubMed/NCBI

14 

Schroder B, Wrocklage C, Hasilik A and Saftig P: Molecular characterisation of ‘transmembrane protein 192’ (TMEM192), a novel protein of the lysosomal membrane. Biol Chem. 391:695–704. 2010.

15 

Eisenberg-Lerner A, Bialik S, Simon HU and Kimchi A: Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 16:966–975. 2009. View Article : Google Scholar : PubMed/NCBI

16 

Oubrahim H, Chock PB and Stadtman ER: Manganese(II) induces apoptotic cell death in NIH3T3 cells via a caspase-12-dependent pathway. J Biol Chem. 277:20135–20138. 2002. View Article : Google Scholar : PubMed/NCBI

17 

Vermeulen K, Van Bockstaele DR and Berneman ZN: Apoptosis: mechanisms and relevance in cancer. Ann Hematol. 84:627–639. 2005. View Article : Google Scholar : PubMed/NCBI

18 

Nakagawa T, Zhu H, Morishima N, et al: Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature. 403:98–103. 2000. View Article : Google Scholar : PubMed/NCBI

19 

Cao XX, Mohuiddin I, Chada S, et al: Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Mol Med. 8:869–876. 2002.PubMed/NCBI

20 

Yee KS, Wilkinson S, James J, Ryan KM and Vousden KH: PUMA- and Bax-induced autophagy contributes to apoptosis. Cell Death Differ. 16:1135–1145. 2009. View Article : Google Scholar : PubMed/NCBI

21 

Elmore SP, Qian T, Grissom SF and Lemasters JJ: The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J. 15:2286–2287. 2001.PubMed/NCBI

22 

Zhu JH, Horbinski C, Guo F, Watkins S, Uchiyama Y and Chu CT: Regulation of autophagy by extracellular signal-regulated protein kinases during 1-methyl-4-phenylpyridinium-induced cell death. Am J Pathol. 170:75–86. 2007. View Article : Google Scholar : PubMed/NCBI

23 

Colell A, Ricci JE, Tait S, et al: GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation. Cell. 129:983–997. 2007. View Article : Google Scholar : PubMed/NCBI

24 

Xue L, Fletcher GC and Tolkovsky AM: Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution. Mol Cell Neurosci. 14:180–198. 1999. View Article : Google Scholar : PubMed/NCBI

25 

Kroemer G and Levine B: Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 9:1004–1010. 2008. View Article : Google Scholar : PubMed/NCBI

26 

Kimmelman AC: The dynamic nature of autophagy in cancer. Genes Dev. 25:1999–2010. 2011. View Article : Google Scholar

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September 2012
Volume 28 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

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Spandidos Publications style
Liu Z, Lv YJ, Song YP, Li XH, Du YN, Wang CH and Hu LK: Lysosomal membrane protein TMEM192 deficiency triggers crosstalk between autophagy and apoptosis in HepG2 hepatoma cells. Oncol Rep 28: 985-991, 2012
APA
Liu, Z., Lv, Y.J., Song, Y.P., Li, X.H., Du, Y.N., Wang, C.H., & Hu, L.K. (2012). Lysosomal membrane protein TMEM192 deficiency triggers crosstalk between autophagy and apoptosis in HepG2 hepatoma cells. Oncology Reports, 28, 985-991. https://doi.org/10.3892/or.2012.1881
MLA
Liu, Z., Lv, Y. J., Song, Y. P., Li, X. H., Du, Y. N., Wang, C. H., Hu, L. K."Lysosomal membrane protein TMEM192 deficiency triggers crosstalk between autophagy and apoptosis in HepG2 hepatoma cells". Oncology Reports 28.3 (2012): 985-991.
Chicago
Liu, Z., Lv, Y. J., Song, Y. P., Li, X. H., Du, Y. N., Wang, C. H., Hu, L. K."Lysosomal membrane protein TMEM192 deficiency triggers crosstalk between autophagy and apoptosis in HepG2 hepatoma cells". Oncology Reports 28, no. 3 (2012): 985-991. https://doi.org/10.3892/or.2012.1881