The Expression of Genes Involved in Hepatocellular Carcinoma

May 4, 2014 - University of Salamanca, Salamanca, Spain. ‡. National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Spai...
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The Expression of Genes Involved in Hepatocellular Carcinoma Chemoresistance Is Affected by Mitochondrial Genome Depletion Ester Gonzalez-Sanchez,† Jose J. G. Marin,†,‡ and Maria J. Perez*,†,‡,§ †

Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), Biomedical Research Institute of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain ‡ National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Spain § IECSCYL-IBSAL, University Hospital of Salamanca, 37007 Salamanca, Spain S Supporting Information *

ABSTRACT: Deletions and mutations in mitochondrial DNA (mtDNA), which are frequent in human tumors, such as hepatocellular carcinoma (HCC), may contribute to enhancing their malignant phenotype. Here we have investigated the effect of mtDNA depletion in the expression of genes accounting for mechanisms of chemoresistance (MOC) in HCC. Using human HCC SK-Hep-1 cells depleted of mtDNA (Rho), changes in gene expression in response to antitumor drugs previously assayed in HCC treatment were analyzed. In Rho cells, a decreased sensitivity to doxorubicin-, SN-38-, cisplatin (CDDP)-, and sorafenib-induced cell death was found. Both constitutive and drug-induced reactive oxygen species generation were decreased. Owing to activation of the NRF2-mediated pathway, MDR1, MRP1, and MRP2 expression was higher in Rho than in wild-type cells. This difference was maintained after further upregulation induced by treatment with doxorubicin, SN-38, or CDDP. Topoisomerase-IIa expression was also enhanced in Rho cells before and after treatment with these drugs. Moreover, the ability of doxorubicin, SN-38 and CDDP to induce proapoptotic signals was weaker in Rho cells, as evidenced by survivin upregulation and reductions in Bax/Bcl-2 expression ratios. Changes in these genes seem to play a minor role in the enhanced resistance of Rho cells to sorafenib, which may be related to an enhanced intracellular ATP content together with the loss of expression of the specific target of sorafenib, tyrosine kinase receptor Kit. In conclusion, these results suggest that mtDNA depletion may activate MOC able to hinder the efficacy of chemotherapy against HCC. KEYWORDS: chemoresistance, liver, mitochondria, retrograde regulation, Rho cells

1. INTRODUCTION Hepatocellular carcinoma (HCC) is the third cause of cancerrelated death worldwide.1 Although tumor resection is the therapy of choice, surgery is not always possible. Moreover, the efficacy of adjuvant chemotherapy is very low due to the marked refractoriness of this tumor to drugs. The major cause of this refractivity is the development of the multidrug resistance (MDR) phenotype in tumor cells, which confers resistance to many structurally unrelated cytostatic drugs. This characteristic depends in part on the aberrant expression of genes involved in the defense against chemical stress expressed in healthy tissues. These genes have been classified in a variety of mechanisms of chemoresistance (MOC),2 on the basis of whether they involve a reduction in drug uptake (MOC-1a) or enhanced drug export (MOC-1b), a reduction in metabolic prodrug activation or an increase in drug inactivation (MOC-2), changes in molecular targets (MOC-3), enhanced DNA repair (MOC-4), or a modification in the proapoptotic (MOC-5a) versus prosurvival (MOC-5b) balance. Despite the heterogeneity in the genetic signatures with respect to MOC among different patients with HCC, our group has recently shown that some common trends © 2014 American Chemical Society

toward the upregulation or downregulation of certain genes can be found in this type of tumor.3 Sometimes, tumors develop resistance during pharmacological treatment, whereas on many other occasions chemoresistance is present even before the treatment has begun. A possible link between the alteration of mitochondrial function and the development and progression of cancer has been investigated extensively.4−6 One of the mechanisms that may contribute to this process is the accumulation of mutations and deletions in the genome of this organelle, which may result in mitochondrial respiratory chain alterations. The proteins of this machinery are encoded by both nuclear and mitochondrial genomes.7 In fact, it has been reported that a wide range of human primary tumors, including HCC, exhibit mitochondrial DNA (mtDNA) abnormalities such as point mutations and deletions.4 Thus, dysfunctional mitochondria associated with mtDNA alterations Received: Revised: Accepted: Published: 1856

December 5, 2013 April 3, 2014 May 4, 2014 May 4, 2014 dx.doi.org/10.1021/mp400732p | Mol. Pharmaceutics 2014, 11, 1856−1868

Molecular Pharmaceutics

Article

Figure 1. Effect of doxorubicin (A, E), SN-38 (B, F), cisplatin (CDDP) (C, G), and sorafenib (D, H) on the viability of wild-type (WT) and mtDNAdepleted (Rho) SK-Hep-1 human hepatocarcinoma cells as measured with the Neutral Red test, after 48 h of incubation. The IC50 was defined as the drug concentration required to reduce cell viability to 50% of control cells. Values are expressed as means ± SEM from 3 cultures in triplicate. *, p < 0.05, significantly different from untreated WT cells; †, p < 0.05, significant difference between Rho and WT cells.

from mitochondrial to nuclear genomes triggered by mitochondrial alterations and that results in cellular responses. The aim of the present study was to investigate whether mtDNA depletion may be a contributing factor in the expression of genes accounting for the failure of antitumor drugs, such as doxorubicin, cisplatin (CDDP), sorafenib, and the active metabolite of irinotecan (SN-38), which have been previously assayed for the treatment of HCC.2

may trigger retrograde signaling pathways from these organelles to the nucleus, which can modify the expression of genes involved in diverse cellular processes, including metabolism, nutrient sensing, stress responses, tumor progression, life span, and drug sensitivity.5 In previous studies, using in vitro models of partial and complete mtDNA depletion, we reported that retrograde regulation may be involved in the defense response of mouse and human liver cells against the chemical stress induced by potentially toxic compounds such as paracetamol8 and bile acids.8,9 Moreover, several lines of evidence obtained from mtDNA-depleted HCC cells have revealed that mitochondrial stress resulting from the reduction of mtDNA causes substantial resistance to apoptosis10,11 as well as the upregulation of the efflux pump ABCB1.12,13 These results suggest that depletion of mtDNA could induce resistance to cancer chemotherapy by retrograde regulation, a signaling pathway of communication

2. MATERIALS AND METHODS 2.1. Chemicals. CDDP, doxorubicin, and SN-38 were obtained from Sigma-Aldrich (Madrid, Spain). Sorafenib tosylate was obtained through the Pharmacy Department, University Hospital of Salamanca (Salamanca, Spain). The purity of all these compounds was ≥97%. All other chemicals were of analytical grade. 2.2. Cell Lines and Culture Conditions. The wild-type (WT) human hepatocarcinoma SK-Hep-1 cell line (HTB-52) 1857

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(DCFH-DA) (Sigma-Aldrich) for 30 min after the corresponding treatment. Then, they were trypsinized and resuspended in FCS-free medium. Propidium iodide (Sigma-Aldrich) (5 μg/mL) was added 10 min before fluorescence measurement, as described previously,16 to exclude dead cells from the analysis. ATP levels were determined with an ATP colorimetric assay kit (BioVision, Deltaclon, Madrid, Spain) as previously described.9 2.4. Determination of Gene Expression Levels. Total RNA isolation from cultured cells was carried out using the illustra RNAspin mini RNA isolation kit (GE, Healthcare, Barcelona, Spain). The SuperScript VILO cDNA Synthesis Kit (Invitrogen) was used to synthesize cDNA from total RNA. Real-Time QPCR was performed using Amplitaq Gold polymerase (Applied Biosystems, Madrid, Spain) in an ABI Prism 7300 Sequence Detection System (Applied Biosystems) for single reactions to determine mRNA levels by conventional RT-QPCR with SYBR Green I detection, or using TLDAs (TaqMan Low Density Arrays) in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) with TaqMan Mix detection. In all cases, the following thermal conditions were used: a single cycle of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Total RNA from human liver was used in all determinations as an external calibrator to compensate for differences between separate PCR runs (interassay variations). The results were normalized by the determination of GAPDH in each sample (including the calibrator) to compensate for sampleto-sample variation. The MOC genes included in the microfluidic cards (Supporting Information Table 1) were amplified using primer oligonucleotides together with their appropriate TaqMan probes designed and validated by our group or by Applied Biosystems (TLDAs) (data not shown). The primer oligonucleotide sequences and the conditions to carry out QPCR of human MRP1, MRP2, MRP4, MRP5, MDR1, NRF2, FXR, PXR, CAR, Bax, Bcl-2, and GAPDH have been described previously.9,17,18 The primer oligonucleotide sequences to carry out QPCR of human survivin, p21, CYP1A2, VEGFR, KEAP1, Kit, PDGFRA, p27, OCT1, OCTN1, OCTN2, ENT2, OATP1B1, TOPO-IIA, TYMP, TYMS, and UPP1 are listed in Supporting Information Table 2. All primers (obtained from Sigma-Genosys, Madrid, Spain) were designed with the assistance of Primer Express software (Applied Biosystems), and their specificity was checked using BLAST. 2.5. Western Blot Analyses. After the corresponding treatments, cells were harvested by washing with cold PBS and scraping into lysis buffer containing PBS, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate, and 1% protease inhibitor cocktail (Sigma-Aldrich). After mixing for 30 min at 4 °C, the cell lysates were centrifuged at 20000g for 20 min at 4 °C and the supernatant fractions were used for Western blot analyses. Protein concentrations were determined14 using bovine serum albumin as standard. Proteins (25−50 μg) were loaded onto 8, 12.5, or 15% sodium dodecyl sulfate−polyacrylamide gels and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). The primary antibodies (working dilution) used were as follows: rabbit polyclonal antibodies against Bax (P-19) (1:100), Bcl-2 (N-19) (1:100), and KEAP1 (H-190) (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse polyclonal antibody against NRF2 (C-20) (1:500) (Santa Cruz); and mouse monoclonal antibodies against survivin (D-8) (1:500) and GAPDH (6C5) (1:1000) (Santa Cruz). Anti-rabbit and antimouse IgG horseradish peroxidase-linked secondary antibodies (Amersham Pharmacia Biotech, Freiburg, Germany) were used. Blots were visualized using enhanced chemiluminescence

Figure 2. ROS production in wild-type (WT) and mtDNA-depleted (Rho) SK-Hep-1 human hepatocarcinoma cells treated with the indicated concentrations of doxorubicin (A), SN-38 (B), cisplatin (CDDP) (C), and sorafenib (D) for 48 h. ROS production was determined by flow cytometry using dichlorofluorescein diacetate (DCFH-DA). Values are expressed as means ± SEM from 3 independent experiments performed in triplicate. *, p < 0.05, significantly different from untreated WT cells; †, p < 0.05, significant difference between Rho and WT cells.

was obtained from the American Type Culture Collection (Manassas, VA, USA). SK-Hep-1 cells were cultured in minimum essential medium Eagle (MEM) (Sigma-Aldrich) containing 10% fetal calf serum (FCS) (TDI S.A., Madrid, Spain) and 1% antibiotic−antimycotic solution (Invitrogen, Barcelona, Spain) and the supplements reported by the supplier in a humidified atmosphere in 5% CO2 at 37 °C. SK-Hep-1 Rho cells totally depleted of mtDNA were derived from WT SK-Hep-1 cells by culturing them in the presence of 100 ng/mL ethidium bromide for more than 20 generations, as previously described.9 To carry out the experiments, the cells were plated and incubated for 24 h in the absence of any of the compounds tested and then treated with different doses of doxorubicin, SN-38, CDDP, or sorafenib for 24 or 48 h. For flow cytometry and gene expression studies, cells were detached with trypsin−EDTA solution and then pelleted by centrifugation at 250g for 10 min and washed once with phosphate-buffered saline (PBS). 2.3. Cell Death, ROS Generation, and ATP Levels. The cell growth rate was determined by measuring total protein14 at different time points during 48 h of cell culture. Cell viability was evaluated using the Neutral Red (Sigma-Aldrich) test.15 To measure the generation of reactive oxygen species (ROS), flow cytometry was used. Cells were incubated with medium containing 5 μg/mL 2′,7′-dichlorofluorescein diacetate 1858

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Figure 3. Rate of cell growth measured by total protein content (A) and effect of doxorubicin, SN-38, cisplatin (CDDP), and sorafenib on the ATP levels (B) in wild-type (WT) and mtDNA-depleted (Rho) SKHep-1 human hepatocarcinoma cells treated with 0.05 μM doxorubicin (Dox), 0.05 μM SN-38, 5 μM CDDP, and 1.25 μM sorafenib for 48 h. Values are expressed as means ± SEM from 3 cultures in triplicate. *, p < 0.05, significantly different from untreated WT cells; †, p < 0.05, significant difference between Rho and WT cells.

Figure 4. Effect of doxorubicin, SN-38, cisplatin (CDDP), and sorafenib on MDR1 (A), MRP1 (B), MRP2 (C), MRP4 (D), and MRP5 (E) mRNA expression in wild-type (WT) and mtDNA-depleted (Rho) SKHep-1 human hepatocarcinoma cells. Cells were treated with 0.05 μM doxorubicin, 0.05 μM SN-38, 5 μM CDDP, and 1.25 μM sorafenib for 48 h before mRNA levels were measured by RT-QPCR. Values, expressed as percentage of untreated WT cells, are means ± SEM from 3 independent experiments performed in triplicate. *, p < 0.05, significantly different from untreated WT cells; †, p < 0.05, significant difference between Rho and WT cells.

reagents (Amersham Pharmacia Biotech) and a Fujifilm LAS4000 (TDI S.A.) luminescent image analyzer. 2.6. Statistical Methods. Values are expressed as means ± SEM. Statistical analyses were performed using the IBM SPSS Statistics 20 software (IBM Corp., Armonk, NY, USA) for Windows (Microsoft Co., Seattle, WA, USA). To calculate the statistical significance of the differences between groups, the Bonferroni method for multiple range testing and Student’s t test or the paired t test were used, as appropriate.

markedly lower in Rho than in WT cells (Figure 2), in agreement with previous reports.9 In WT cells, treatment with doxorubicin (Figure 3A), SN-38 (Figure 2B), CDDP (Figure 2C), and sorafenib (Figure 2D) for 48 h increased ROS generation in a concentration-dependent manner. However, in Rho cells, although treatment with the antitumor drugs assayed also stimulated ROS production, this effect was much weaker than in WT cells (Figure 2A−D). Concentrations of anticancer drugs able to induce a significant increase in ROS generation but below their IC50 were used in further experiments. Under control conditions the growth rate of SK-Hep-1 WT and Rho cells in culture was similar (Figure 3A). In order to characterize the energy metabolism status, ATP levels were analyzed. Similar steady-state levels of ATP in WT and Rho cells were found (Figure 3B), which was consistent with previous reports.9 Intracellular ATP content was increased after treatment with doxorubicin and CDDP in WT and Rho cells in a similar manner while it was increased by treatment with sorafenib only in Rho cells (Figure 3B). 3.2. Basal Expression of Genes Involved in Chemoresistance in HCC. Despite the interindividual variability in the

3. RESULTS 3.1. Sensitivity of Rho Cells to Antitumor DrugInduced Cell Death, ROS Generation and Alterations in ATP Levels. Treatment with doxorubicin, SN-38, CDDP, and sorafenib for 48 h revealed different sensitivities to the toxicity induced by these drugs (Figure 1A−D). In the range of concentrations investigated here, these antitumor drugs reduced cell viability in WT and, to a lesser extent, also in Rho cells. Thus, the IC50 values were markedly higher in Rho than in WT cells (Figure 2E−H). This effect was more marked for doxorubicin (Figure 1A,E) followed by sorafenib (Figure 1D,H), but was modest for SN-38 (Figure 1B,F) and CDDP (Figure 1C,G) treatments. Determination of DCFH oxidation, used here as an index of ROS generation, revealed that the basal levels of ROS were 1859

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Molecular Pharmaceutics

values from PCR reactions have been included in order to reflect the basal levels of the target genes in SK-Hep-1 cells. The expression of MDR1, MRP1, and MRP2 was upregulated whereas MRP4 expression was reduced and that of MRP5 was unchanged in Rho cells when compared with WT cells (Table 1). Among them, the most significant change was that of MDR1 (a 20-fold increase in Rho cells). However, the expression of this protein in Rho cells was very poor (only 1.7% as compared with human liver). The mRNA levels of the uptake transporters OATP1B1 and OCT1, the enzyme CYP1A2, and the receptor PDGFRA were very low, while the mRNA expression of VEGFR was undetectable in both WT and Rho cells. As compared with human liver, the expression of TOPO-IIA and TYMS was markedly higher in WT and Rho cells. Although survivin was also 1860

60.8 ± 4.2* 88 ± 5.0 86 ± 6.9 83.8 ± 1.2 75.5 ± 1.3 91.4 ± 1.9 80.2 ± 1.7

Rho WT Rho

1680 ± 0.0† 159 ± 2.1† 235 ± 4.0 68.6 ± 2.0† 127 ± 3.5 175 ± 12.9† 116 ± 2.7

sorafenib WT Rho

2868. ± 147† 152 ± 25 292 ± 18† 40.9 ± 4.7† 167 ± 22 195 ± 10.8† 126 ± 8.2

97.3 ± 1.3 118 ± 9.8 160 ± 9.6* 91.3 ± 2.6 110 ± 2.7 107 ± 4.5 111 ± 3.0

CDDP WT

134.5 ± 4.8* 105 ± 3.6 179 ± 3.6* 50.3 ± 3.3* 143 ± 0.5* 100 ± 8.5 124 ± 5.6*

Cells were treated with 0.05 μM doxorubicin, 0.05 μM SN-38, 5 μM cisplatin (CDDP), and 1.25 μM sorafenib for 24 h before mRNA levels were measured by RT-QPCR. Values, expressed as percentage of untreated WT control cells, are means ± SEM from 3 independent experiments performed in triplicate. *p < 0.05, significantly different from untreated WT cells. †p < 0.05, significant difference between Rho and WT cells.

Values are expressed as percentages of the external calibrator (RNA from human liver) and normalized by the determination of GAPDH in each sample. *p < 0.05, as compared with WT cells. Ct, values of threshold cycle. ND, nondetected.

a

a

Rho

1.7 ± 0.3* 195 ± 9.4* 9.25 ± 0.8* 741 ± 98.5* 17.06 ± 0.8 28427 ± 4606*