Total RNA was isolated using Trizol (Invitrogen) and cDNA was prepared using avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI). PCR was performed with 25-30 cycles of the reaction involving 95°C for 30 seconds, 53-58°C for 30 seconds, and 72°C for 70-90 seconds. The PCR primer sets were produced by Bioneer (Seoul, Korea) to detect all variants of Claudin-1 (5’-GAGCGAGTCATGGCCAACGCG-3’
and 5’-GCCTCTGTGTCACACGTAGTC-3’) and β-actin (5'-CCTTCCTGGGCATGGAGT CCTGT-3’ and 5'-GGAGCAATGATCTTGATCTTC-3’).
F. Measurement of intracellular and mitochondrial ROS level.
To determine intracellular and mitochondrial ROS levels, dichlorofluorescin diacetate (DCFH-DA) (Molecular probe, Eugene, OR) and mitochondrial specific MitoSOX® (Invitrogen) fluorogenic probes were used, respectively (Yu and Kim, 2011). Briefly, cells were incubated in media containing DCFH-DA (20 μM) and MitoSOX® (25 μM) for 20 min at 37oC. Stained cells were washed and resuspended in PBS, and analyzed by flow cytometry (FACS Vantage, Becton Dickinson Corp.). Mean values of arbitrary fluorescence units of 10,000 cells were used and expressed as percentage of negative control.
8
G. Construction of HSF1 cDNA plasmids and transfection of cDNA plasmids and siRNAs.
To generate a cDNA plasmid, pcDNA-HSF1-HA, conventional cloning procedures were applied. Briefly, pcDNA-HSF1-HA plasmid was constructed by TA cloning into pGEMT-easy (Promega) with HSF1 cDNA fragment amplified by PCR using total Chang cell cDNAs and the primer set (5’-AGAATTCATGGATCTGCCCG-3’ and 5’-TGAGCTCGGAGACAG TGGG-3’). The HSF1 cDNA was inserted into EcoRI and XhoI sites of the pcDNA3-HA vector previously constructed (Seo et al., 2008).
To introduce plasmids and small interfering RNAs (siRNAs) into cells, cells were transfected with the plasmids and siRNA duplexes using FuGENE HD (Promega) and OligofectamineTM Reagent (Invitrogen), respectively, according to the manufacturer’s instructions. HSF1 siRNAs (#1, 5'- ACUGUAGAUUGCUUCUGUA-3' and 5'- UACAGA AGCAAUCUACAGU-3') (#2, 5'- GAACUAAAGCCAAGGGUAU-3' and 5'- AUACCCU UGGCUUUAGUUC -3') were obtained from Bioneer Co. Negative control siRNAs (5’-CC UACGCCACCAAUUUCGU-3’ and 5’-ACGAAAUUGGUGGCGUAGG-3’) were obtained from Bioneer.
H. ChIP assay.
A chromatin immunoprecipitation (ChIP) assay was performed with a ChIP kit (Upstate Biotechnology, Lake Placid. NY), according to the manufacturer’s instructions, using antibodies against HSF1 (4356S) or control IgG (Santa Cruz). The precipitated DNA was subjected to PCR amplification with specific primers for the Claudin-1 promoter region containing HSF1-binding sites (Song et al., 2013). The following primers were used for PCR: HSF-1 (5’- TTTGTCTGAGGGTACATAGCAGA-3’ and 5’-GGCAGCACTGAGAC CAAGAA-3’).
9
I. Total genomic DNA isolation.Total genomic DNA was isolated as described previously with slight modification (Yoon et al., 2006). Briefly, cell lysates were incubated at 37 °C for 1 h with 0.1 mg/ml RNase A, and then at 55 °C for 3 h with 0.1 mg/ml Proteinase K and 1 % SDS. Phenol/chloroform/iso amyl alchol were treated for several times. Genomic DNA (gDNA) was precipitated by addition of a 2.5 volume of absolute ethanol and 1/10 volume of 3 M sodium acetate (pH 5.2), pelleted by centrifugation at 13,000 rpm for 20 min, and dissolved in 100 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
J. Promoter assay.
For transfection of reporter plasmids, Chang cells were plated on 6-well plates at a density of 1×104 cells/well. Plasmid DNA was mixed with FuGENE HD transfection reagent (Promega) and transfected into the cells following the manufacturer’s protocol. After 48 hours of transfection, the cells were washed twice with PBS and then lysed in reporter lysis buffer (Promega). Luciferase activity was measured with a Dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Luciferase activity was measured in triplicate, averaged, and relative promoter activity was computed by normalizing the Firefly luciferase activity against that of the Renilla luciferase (Song et al., 2013).
10 RESULTS
Cln-1 is upregulated in hepatoma cell with mitochondrial dysfunction and high invasion activity.
First, I have investigated the relationship between mitochondrial dysfunction and Cln-1 expression in hepatoma cells, by employing four types of SNU hepatoma cells (SNU-354, SNU-387, SNU-423, and SNU449) derived from human hepatocellular carcinomas (Park et al., 1995; Ku and Park 2005), and compared these levels to those of Chang-L clone, which was derived from Chang cell (immortalized human hepatocyte) and were previously characterized as having certain liver-characteristics and active mitochondrial respiration (Kim et al, 2011). Interestingly, SNU354, SNU423, and SNU449 cells, which have defective mitochondrial respiration and increased cell invasion activity, showed increased Cln-1 expression (Fig. 1). Cellular morphology of these three cells with high Cln-1 expression displayed more flattened and extended like fibroblast whereas Chang and SNU387 look like typical epithelial octagonal shape (Fig. 2A). Moreover, increased Cln-1 expression was also observed in 80% of HCC tumor samples with mitochondrial defects (Fig. 2B). These results imply that Cln-1 is associated with mitochondrial dysfunction of hepatoma cells with high invasion activity.
11
Fig. 1. Cln-1 is highly expressed in invasive SNU hepatoma cells with mitochondrial dysfunction. Chang cell clone (Ch-L) and four different SNU hepatoma cell lines (SNU354, SNU387, SNU423, and SNU449) were cultured for 2 days to maintain exponentially growing state. A) Cellular oxygen consumption rate (OCR) was measured using XF analyzer as described in ‘Materials and methods.’ B) Cell invasion activity was assessed using MatrigelTM-coated traswell as described in ‘Materials and methods.’ Invaded cell numbers were counted. C) Western blot analysis for Cln1 expression. **, p<0.01 vs. Chang clone (Ch) by student t-test.
12
Fig. 2. Cln-1 is associated with mitochondrial dysfunction of hepatoma cell. A) Cellular morphology of Chang cell clone (Ch-L) and four different SNU hepatoma cell lines (SNU354, SNU387, SNU423, and SNU449). B) human HCC tumor sample (T) and their surrounding tissues (S) were applied to western blot analysis.
13
Complex I inhibition by rotenone effectively induce Cln-1 expression at transcriptional level.
Next, I have examined whether mitochondrial respiratory defects may directly induce Cln-1 expression. When Chang cell was treated with subcytotoxic doses of respiratory inhibitors, rotenone (complex I inhibitor), TTFA (complex II inhibitor), antimycin A (complex III inhibitor), KCN (complex IV inhibitor), and oligomycin (complex V inhibitor) for 12h as previously reported (Byun et al., 2008), Cln-1 protein expression was increased significantly in the cells treated with rotenone, TTFA and oligomycin (Fig. 3A and 3B). The effect of rotenone was the most effective. Similar increases were observed at mRNA levels as evidenced by RT-PCR (Fig. 3C and 3D). These results were not accompanied with any alteration of respiratory protein expressions (Fig. 3E).
I have further examined the effect of rotenone in detail. Complex I inhibition by rotenone increased Cln-1 expression in a dose-dependent manner and the mRNA induction started 3h after treatment (Fig. 4). These results indicate that direct inhibition of respiratory activity effectively induce Cln-1 expression at transcriptional level.
14
Fig. 3. Respiratory dysfunction induces Cln-1 expression at transcriptional level. A-D) Chang clone was challenged with 5 μM Rotenone (R), 200 μM TTFA (T), 5 μM antimycin A (A), 5 mM KCN (K) or 5 μM oligomycin (O) for 12h. A) Expression levels of Cln-1 protein (A) and mRNA (C) were monitored by Western blot and RT-PCR analysis, respectively. Quantitative analyses of the expression levels are shown (B,D). E) Western blot analysis for mitochondrial respiratory complex expression. *, p<0.05; **, p<0.01 vs. control by student t-test.
15
Fig. 4. Respiratory dysfunction by complex I inhibition induces effectively Cln-1 expression at transcriptional level. A) Chang clone was treated with the indicated concentration of rotenone for 12h. Western blot analysis for Cln-1 expression. B) Endogenous cellular oxygen consumption rate was measured and its specificity for mitochondrial respiration was confirmed by adding rotenone. C-D) Chang clone was challenged with 5 μM rotenone for the indicated time periods. Western blot (C) and RT-PCR (D) analyses for Cln-1 expression are shown. **, p<0.01 vs. control by student t-test.
16
Mitochondrial dysfunction by rotenone induce Cln-1 expression through mitochondrial ROS generation.
I further aimed to investigate the molecular mechanism involved in the mitochondrial defect-mediated Cln-1 induction. It is well known that mitochondrial respiratory defect is closely linked with mitochondrial ROS generation. As expected, mitochondrial ROS level was significantly increased in a dose dependent manner after cells were exposed to rotenone, (Fig. 5A). In the same condition, Cln-1 mRNA level was also increased (Fig. 5B). These results implied that mitochondrial ROS may be involved in the Cln-1 mRNA expression.
Pretreatment of N-acetyl cysteine blocked the rotenone-induced Cln-1 expression (Fig.
6A). In addition, treatment with exogenous H2O2 induced Cln-1 mRNA expression in a biphasic pattern, well corresponded with the intracellular ROS profile (Fig. 6B and 6C).
These results clearly indicated that mitochondrial ROS induced by rotenone was one of the important factors involved in Cln-1 expression.
17
Fig. 5. Mitochondrial ROS induced by complex I defect is the mediator to induce Cln-1 expression. A-B) Chang clone was treated with the indicated concentrations of rotenone for the indicated periods. A) Mitochondrial ROS levels were monitored by flow cytometric analysis after staining cells with MitoSOX fluorescence dye. B) mRNA expression levels for Cln-1 were examined by RT-PCR analysis. **, p<0.01 vs. control by student t-test.
18
Fig. 6. Intracellular ROS is the mediator to induce Cln-1 expression. A) Chang clone was challenged with 5 μM rotenone for 12h with or without pretreatment of NAC for 6h.
mRNA expression levels for Cln-1 were examined by RT-PCR analysis. B-C) Chang clone was treated with 300 μM H2O2 for the indicated time periods. Intracellular ROS levels (B) and mRNA expression levels (C) are shown. *, p<0.05; **, p<0.01 vs. control by student t-test.
19
ROS-mediated HSF1 phosphorylation is an important event involved in Cln-1 expression.
To investigate how mitochondrial ROS modulate Cln-1 expression, I have first analyzed potential transcription factor binding sites in the promoter region of Cln-1 gene by using TFSEARCH program. Several HSF1 binding sites with 100% matched score were found within -2730 base upstream from transcription start site, implying its potential involvement (Fig. 7). Upon exposure to rotenone, HSF1 protein band was shifted with delayed mobility on SDS-PAGE (Fig. 8A) and the delayed band mobility by rotenone was recovered when incubated with λ phosphatase, indicating that the delay in band mobility is due to phosphorylation of HSF1 (Fig. 8B). HSF1 has several phosphorylation sites and is activated by phosphorylation on S326 residue (Guettouche et al., 2005). Phosphorylation status of HSF1 was further confirmed to be increased in response to rotenone treatment by using phospho-specific antibody against serine 326 residue (S326) of HSF1 (Fig. 8C).
In addition, I have examined whether HSF1 phosphorylation is modulated by ROS. Both delayed migration of HSF1 and Cln-1 induction by rotenone was restored by NAC pretreatment (Fig. 9A). And treatment with exogenous H2O2 induced Cln-1 mRNA expression in a biphasic pattern (Fig. 9B). The rotenone-mediated HSF1 band shift was restored by siRNA-mediated HSF1 knockdown and overexpression of HSF1 increased Cln-1 expression (Fig. 9C and 9D). These results suggest that respiratory defects induced by rotenone activate HSF1 by phosphorylation through mitochondrial ROS, thereby inducing Cln-1 expression.
20
Fig. 7. Analysis of transcription factor binding elements of Cln-1 promoter sequence.
Identification of putative HSEs within the human Cln-1 gene promoter. TFSearch, a web-based search engine for transcription factor binding sites, was used to search for HSEs within the Cln-1 promoter.
21
Fig. 8. Respiratory dysfunction by complex I inhibition induces HSF phosphorylation (activation). A-C) Chang clone was treated with 5 μM rotenone for the indicated time periods. A) HSF1 protein expression by Western blot analyses. B) Western blot analyses after cell lysates were incubated with lambda phosphatase for 2h. C) HSF1 phosphorylation status on ser326 were monitored.
22
Fig. 9. ROS-mediated HSF phosphorylation (activation) is involved in Cln-1 expression.
Western blot analysis. A) Chang clone was challenged with 5 μM rotenone for 12h with or without pretreatment of NAC for 6h. B) Chang clone was treated with 300 μM H2O2 for the indicated time periods. C) Chang clone was treated with 5 μM rotenone 36 h after the cells were transfected with siRNAs for HSF1. D) Chang clone was transfected with pcDNA-HSF1-HA to overexpress HSF1.
23
Mitochondrial ROS enhance HSF1 binding to the promoter region of Cln-1 gene and enhances its activity.
Next, I being monitored phosphorylation status of HSF1 in SNU hepatoma cells. SNU hepatoma cells with high Cln-1 expression and mitochondrial defects (SNU354, SNU423 and SNU449) has delayed migration of HSF1 and increased phosphorylation status on S326 as shown in Fig. 10A. Among the three SNU cells, SNU449 showed highest expression of phosphorylated HSF1, well corresponded with Cln-1 expression level. These three SNU cells with mitochondrial defects showed increase in intracellular ROS level (Fig. 10B). When SNU449 cell was exposed to NAC, HSF1 protein band migrated faster and Cln-1 expression decreased (Fig. 11A). Moreover, HSF1 binding to the promoter region of Cln-1 was decreased (Fig. 11B). These results imply that mitochondrial ROS enhance HSF1 binding to the promoter region of Cln-1 gene and enhances its activity.
24
Fig. 10. HSF1 phosphorylation was involved Cln-1 expression through ROS. A-B) Chang cell clone (Ch-L) and four different SNU hepatoma cell lines (SNU354, SNU387, SNU423, and SNU449) were cultured for 2 days to maintain exponentially growing state.
Western blot analysis (A), and Intracellular ROS levels (B) were monitored. **, p<0.01 vs.
Chang clone (Ch) by student t-test.
25
Fig. 11. Mitochondrial ROS enhance HSF1 binding to the promoter region of Cln-1 gene and enhances its activity. A and B) SNU449 cell was treated with NAC for 6h.
Western blot analysis (A) and ChIP assay for HSF1 binding on Cln-1 promoter region (B) are shown. C) luciferase activity was assayed after 2 days in the presence of the indicated rotenone. Luciferase activity was normalized to Renilla activity as expressed by the cotransfected plasmid. **, p<0.01 vs. control by student t-test.
26
HSF1 is a key transcription factor to control hepatoma cell invasion activity through inducing Cln-1 expression.
Finally, I was tested whether HSF1 is involved in hepatoma cell invasion activity.
Previous reports indicated that the three hepatoma cells with mitochondrial defects (SNU354, SNU423, and SNU449 cells) has high invasion activity (Kim et al., 2011). When HSF1 expression was decreased by siRNA-mediated knockdown in SNU449 cell (highest HSF1 phosphorylation), cell invasion activity was significantly diminished, together with decrease in Cln-1 expression (Fig. 12A and 12B). Taken together, HSF1 phosphorylation was critically involved in hepatoma cell invasion activity through ROS-mediated Cln-1 expression.
27
Fig. 12. HSF1 phosphorylation was critically involved in hepatoma cell invasion activity through ROS-mediated Cln-1 expression. A-B) SNU449 cell was transfected with siRNA for HSF1 for 24h. Western blot analysis (A) and cell invasion activity (B) are shown.
*, p<0.05 vs. control by student t-test.
28
Fig. 13. Schematic model for mechanism of Cln-1. These results suggest that Cln-1 induced by HSF1 activation through respiratory defect mediated ROS in hepatoma cells.
29
DISCUSSION
Mitochondrial defects are considered to be hallmarks of cancer cells and importantly involved in cancer cell metastatic properties, including invasion activity (Chang et al., 2009;
Hung et al., 2012; He et al., 2013; Ma et al., 2013). It is unclear how mitochondrial respiratory defects control cancer cell invasion activity. However, according to previous study, it was reported that mitochndrial dysfunction is linked with the claudin-1 induction.
Claudins are a family of proteins that are the critical components of the tight junctions, which controls paracellular permeability of cells and the flow of molecules between cells.
According to several recent reports Cln-1 overexpression increases cancer cell invasion and metastasis (Dhawan et al., 2005; Leotlela et al., 2007; Dos Reis et al., 2008). For example, hepatocellular carcinoma with mitochondrial dysfunction display increased expression Cln-1 and high invasion activity, implying the close link between mitochondrial defects and Cln-1-mediated tumor invasion (Kim et al., 2011). However, it is not clearly understood how Cln-1 expression is increased in cancer and linked with the metabolic hallmark of cancer, mitochondrial dysfunction. So, it is aimed to elucidate “How does mitochondrial defects induce Cln-1 expression?” I am screened whether any specific mitochondrial respiratory defect induces Cln-1 induction. Among diverse respiratory inhibitors, complex I inhibition by rotenone most effectively induced Cln-1 expression at transcriptional level, accompanied by increase in mitochondrial ROS production.
The role of mitochondria in tumorigenesis has been linked to their ROS production. Under normal physiological conditions, about 1 to 3% of total mitochondrial oxygen consumption is incompletely reduced and leads to ROS production. As mitochondrial respiratory chain is a permanent source of ROS, cell has an effective antioxidant systems to nullify the toxic effects of the ROS and other free radicals (Gao et al., 2007). Excessive ROS production can cause mtDNA mutations, which may lead to mitochondrial respiratory chain dysfunction that could contribute to the onset of many diseases including neoplasia. When mitochondria function is affected, mitochondrial ROS production can be increased (He et al., 2013). So, it has been confirmed treatment of H2O2 directly induced 1 expression, implying that Cln-1 induction was mediated by mitochondrial ROS.
30
More recent studies have revealed that mitochondria are also engaged in retrograde regulation, in which cells respond to changes in the functional state of the organelle via changes in nuclear gene expression. Retrograde regulation encompasses a wide assortment of cellular activities, including nutrient sensing, growth control, aging, and other signaling processes that function in metabolic and organelle homeostasis (Liu and Butow, 2006;
Barbour and Turner, 2014). Interestingly, Cln-1 promoter region (-2700bp ~ +300bp) contained HSF1 binding site, This transcription factor is known to be regulated by intracellular redox status (Jacquier-Sarlin and Polla, 1996; Christians et al., 2002; Yan et al., 2002). Also HSF1 promoted cancer migration and invasion (Fang et al., 2012; Xi et al., 2012). So, it has been confirmed that activation of HSF1 through mitochondrial ROS induces Cln-1 expression and HSF1 knockdown using siRNA decreased Cln-1 expression and invasive activity in SNU449.
A prominent feature of HSF1 is conversion into a transcriptionally active trimer occurs concurrently with extensive hyperphosphorylation of serine residues, most of which reside within the regulatory domain. In previously study, the analysis of HSF1 phosphorylation sites was performed by Guettouche and colleagues, who purified exogenously expressed human HSF1 from heat-shocked HeLa cells and identified phosphorylation target sites by means of mass spectrometry. The analysis, covering >90% of the HSF1 amino acid sequence, demonstrated the phosphorylation of HSF1 on at least 12 serin residues (S121, S230, S292, S303, S307, S314, S319, S326, S344, S363, S419, and S444), with no detectable amount of threonine or tyrosine phosphorylation (Guettouche et al., 2005; Anckar and Sistonen, 2011).
Among these residue, S230, S326 and S419 is associated with regulation of HSF1 activity (Holmberg et al., 2001; Guettouche et al., 2005; Kim et al., 2005; Neef et al., 2011). The phosphorylation of S326, previously had been shown to facilitate the association between HSF1 and the coactivator Daxx (Kim et al., 2005). In addition, stress-inducible phosphorylation of S230 by the calcium/calmodulin-dependent kinase CaMKII contribute to HSF1 activation as both mutation of S230 and pharmacological inhibition of CaMKII lead to a reduced heat shock response (Holmberg et al., 2001). Phosphorylation of HSF1 on S419 by polo-like kinase 1 (PLK1) is an essential step for HSF1 nuclear translocation by heat stress (Guettouche et al., 2005).
31
In this study, it is aimed to elucidate how mitochondrial dysfunction is linked with Cln-1 induction and if it does, what the underlying mechanisms are. These data lead to three major findings. (ⅰ) Mitochondrial defects can affect cancer invasion activity by Cln-1 induction.
(ⅱ) Complex I deficiency-induced ROS generation may play a critical role in these processes by regulating HSF1 activation. (ⅲ) Mitochondrial ROS enhances HSF1 binding activity to the promoter of Cln-1 gene and enhances its activity. This study demonstrated that mitochondrial ROS activates HSF1 through phosphorylation of S326 residue and enhances its binding to Cln-1 promoter.
32
CONCLUSION
Mitochondrial dysfunction is hallmarks of cancer cells and importantly involved in cancer cell metastatic properties, including invasion activity. However, it is unclear how mitochondrial respiratory defects control cancer cell invasion activity. This study clearly demonstrate that mitochondrial dysfunction increase mitochondrial ROS and activates (phosphorylates) HSF1, thereby inducing Cln-1 expression is a key molecule to enhance hepatoma cell invasion activity. This study emphasize that mitochondrial ROS activates HSF1 through phosphorylation of S326 residue and enhances its binding to Cln-1 promoter.
33
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미토콘드리아 기능 손상을 가지고 있는 간암치료에 대한 새로운 제어타겟을 제시하게 될 것으로 기대한다.
핵심어 : 간암세포, 미토콘드리아 기능손상, Claudin-1, 활성산소, Heat Shock Factor1