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Decitabine Reactivate Secretoglobin (SCGB3A1) and SAP/JNK genes in Cisplatin Resistant PC-3 and LNCaP Prostate cell lines

Parra E

Laboratorio de Biomedicina Experimental, Facultad de Medicina, Campus Saucache, Universidad de Tarapacá, Avenida Senador Luis Valente Rossi, Arica, Chile

E-mail : aa

Ferreira J

Programme of Molecular and Clinical Pharmacology, ICBM. Medical Faculty, University of Chile, Avenida Independencia 1027, Independencia, Santiago-Chile

Hecht P

Laboratorio de Biomedicina Experimental, Facultad de Medicina, Campus Saucache, Universidad de Tarapacá, Avenida Senador Luis Valente Rossi, Arica, Chile

Rojas V

Laboratorio de Biomedicina Experimental, Facultad de Medicina, Campus Saucache, Universidad de Tarapacá, Avenida Senador Luis Valente Rossi, Arica, Chile

Facultad de Ciencias Agrarias y Forestales, Programa de Doctorado en Biotecnología Traslacional, Universidad Católica del Maule, Ave. San Miguel 3605, Talca, Chile

Maturana JC

Laboratorio de Biomedicina Experimental, Facultad de Medicina, Campus Saucache, Universidad de Tarapacá, Avenida Senador Luis Valente Rossi, Arica, Chile

Inostroza J

Laboratorio de Biomedicina Experimental, Facultad de Medicina, Campus Saucache, Universidad de Tarapacá, Avenida Senador Luis Valente Rossi, Arica, Chile

DOI: 0.15761/TiM.1000289

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Abstract

Prostate cancer is one of the leading causes of cancer related deaths in men worldwide. Recently, it has been determined that epigenetics, a stage of regulation of gene expression consisting of modifications of chromatin and histone tails, could be playing a role in prostate cancer formation and progression. In this study, we have investigated the effect of decitabine on the control of Secretoglobin (SCGB3A1) and the SAP/JNK kinase that are critical for the pathogenesis of prostate cancer in PC-3 and LNCaP prostate cell lines. We demonstrated that decitabine reactivates the SCGB3A1 gene in both LNCaP and PC3 prostate cancer cell lines. The effect of decitabine on JNK-1 and JNK-2 expression in prostate resistant PC-3 cells p53 negative which contrasts to the p53 positive LNCaP cells showed that JNK-1 and JNK-2 mRNA levels were significantly increased in PC-3 cells following treatment with 1, 3, 6 and 9 μM decitabine. However, the increase in LNCaP cells was only marginal compared with PC-3. In addition, DNA methyltransferase (DNMT) activity was decreased after decitabine treatment in both, LNCaP and PC-3 cell lines. The sum of results obtained suggests that Decitabine treatments can induce regression of the features of prostatic adenocarcinoma and targeting epigenetic mechanisms represents a novel strategy in anticancer therapy.

Keywords

Decitabine, prostate cancer, SCGB3A1, JNK1, JNK-2, GAPDH

Introduction

Prostate Cancer is a result of alterations in genetic and epigenetic factors, as well as inter- and intracellular signaling pathways. Such alterations lead to the dysregulation of complex physiological pathways regulating normal cellular processes, growth, and development, resulting in carcinogenesis. Therefore, identification of molecular drivers and development of therapeutic approaches to target them is an important aspect of managing Prostate Cancer [1,2].

Primary prostate cancers are often multifocal, having topographically and morphologically distinct tumour foci [3,4]. Sequencing studies have revealed that individual tumour foci can arise as clonally distinct lesions with no shared driver gene alterations [5]. This finding demonstrates that multiple genomically and phenotypically distinct primary prostate cancers can be present in an individual patient. Lethal metastatic prostate cancer seems to arise from a single clone in the primary tumour but can exhibit subclonal heterogeneity at the genomic, epigenetic, and phenotypic levels [6].

Clinical heterogeneity of prostate cancer arises from complex genome rearrangements, somatic mutations, and epigenetic alterations, all of these increased by genomic instability [1-4]. In addition, gene amplification in chromosomes 1, 4, 5, 7, 8, 11, 16, and 19 might harbor genes that predispose individuals to prostate cancer and may affect tumour growth rate and aggressiveness. Accordingly, altered expression of regulatory proteins, involved in signal transduction pathways that control the apoptosis, necrosis, or cell defense mechanisms, can additionally make certain types of cancer rather insensitive to cytotoxic effects of antitumor drugs such as cisplatin [5-7]. Cisplatin-resistant cells often fail to activate proapoptotic signal transducers such as MAPK1 (p38MAPK) and c-Jun N-terminal kinase, thereby can survive chemotherapy [8-10]. Indeed, several studies show that JNK/SAPK pathway is activated by DNA-damaging chemotherapeutic agents such as cisplatin and that this pathway is required for the repair of cisplatin-DNA adducts [10,11]. This would limit signaling through the inducer of extrinsic apoptosis, FAS/FASL system, and hence promote cell survival [12].

Recently, it has been determined that epigenetics, a stage of regulation of gene expression consisting of modifications of chromatin and histone tails, could be playing a role in prostate cancer formation and progression [13,14]. In prostate cancer cells occurs both the hyper methylation of promoter regions that could silence the expression of tumor suppressor genes and global hypomethylation of the genome, which could result in the activation of oncogenes [15]. Most remarkably, these epigenetic alterations are clonally amplified within the tumor and have been observed in prostate premalignant lesions. The enzymes responsible for these chromatin modifications, such as histone acetylases and DNA methylases, are also altered in their activity and expression in tumor tissues [16-19]. DNA methyltransferase 1 (DNMT1) is the predominant enzyme that is responsible for maintaining DNA methylation. Upregulation of the DNMT1 gene indicates poor prognosis in malignant cancers including renal cell carcinoma, lymphoma, and pancreatic and bladder cancer. It has been shown that DNA methyltransferase inhibitors, such as Decitabine, can block the activity of DNMT1, enabling expression of repressed genes, and thus have been approved by the FDA for use in the treatment of myelodysplastic syndromes [20].

Among these activated genes is the tumor suppressor-like gene SCGB3A1, a growth-inhibitory cytokine that is down regulated in the majority of prostate, breast, lung, pancreatic, and nasopharyngeal cancers [21,22]. Interestingly, in these cancer tissues, the human SCGB3A1 gene promoter has been found to be extensively methylated, and thus, a connection was established, for this gene, between the extent of promoter hypermethylation, the loss of gene expression, and disease [23,24]. The AKT signaling pathway is responsible for the SCGB3A1 tumor suppressor function as characterized by the inhibition of cell growth, cell migration, and invasion [25].  In this connection, it was shown that EGF and TGFγ increase SCGB3A1 expression through activation of the ERK-MAPK and phosphoinositide-3 kinase-AKT pathways [26]. Further, the expression of SCGB3A1 is restricted to terminally differentiated airway epithelial cells and is upregulated during retinoic acid-induced differentiation of bronchial epithelial cells, suggesting that SCGB3A1 may be involved in the acquisition or maintenance of the terminally differentiated epithelial phenotype [27]. The epigenetic changes in prostate cancer cells may eventually lead to Cisplatin resistance due to altered patterns of gene expression [28].  These observations have important clinical consequences as they provide an opportunity for epigenetic drugs to prevent or reverse non responsiveness to this anti-cancer drug. In this work, we have studied the inhibitor effect of Decitabine on the expression of SCGB3A1 and JNK-1/JNK-2, GAPDH genes in prostate cancer cell lines. This information could be used to determine whether Decitabine treatment of a Cisplatin- resistant prostate cancer cell line restores the antiproliferative and apoptotic effects of cisplatin; Multiple genes would be targeted simultaneously, blocking diverse signaling pathways utilized by tumor cells. We hypothesize that de-methylation of these genes will result in their re-expression; thus, preventing tumor progression in a novel in vivo model that utilizes human prostate cell lines.

Materials and Methods

Cell culture.  The human prostate cancer cell lines, LNCaP cells (ATCC-CRL-1740), DU145 cells (ATCC HTB 81), BPH-1 (atcc-scc256) and PC3   PC-3 (ATCC-CRL-1435) were obtained from the American Type Culture Collection (ATCC-Manassas, VA).  PC-3 and LNCaP were grown and made resistant to cisplatin as described previously [29]. Cells were cultured and maintained in RPMI-1640 medium including L-glutamine, supplemented with 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin (100 000 U/l penicillin, 100 mg/l streptomycin), at 37º C in a humidified atmosphere containing 5% carbon dioxide. Drug preparation and treatment Decitabine (Sigma Aldrich, Poole, UK) stock solutions of 500 mM were prepared using cell growth media and stored at -80 ºC.

RNA extraction/reverse transcription

To extract RNA, cells were grown to 80% confluency and TRIzol reagent was used according to the manufacturer's instructions (Invitrogen, Paisley, UK). A total volume of 1 ml TRIzol reagent was added per flask and then processed further according to the protocol. RNA quality (A260/A280 ratio) and quantity was determined by Nanodrop spectrophotometry (Nanodrop 1000 Spectrophotometer, Thermo Scientific, Loughborough, UK). For further downstream applications, 2 mg RNA was reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol, and 150 ng random hexamers (GE Healthcare, Buckinghamshire, UK).

Quantitative real-time PCR

Quantitative PCR

SCGB3A1 (UGRP2) expression was measured in a PCR reaction containing iQ™ Supermix (Bio-Rad, Hercules, CA), 500 nM of the forward primer 5’- CGGAATTCCCCCGCGCCATGAAGCTC-3’, 500 nM of the reverse primer 5’ ACATCTAGAGCCAAACACTGTCAGG-3’, and 2 μL of cDNA. TBP was used as a reference gene. Each TBP PCR reaction contained iQ™ Supermix (BioRad), 500 nM of the forward primer (5′-GAGCTGTGATGTGAA-GTTTCC), 500 nM of the reverse primer (5′-TCTGGGTTTGATCATTCTGTAG) and 2 μL of cDNA. Primers for amplification of Jnk-1 were 5′-GAACAGCTTG-GAACACCATGTCCTG-3′ (Forward), and 5′-GTGGAGCTTCTGCTTCAGAAGG-ATC-3′ (Reverse); for Jnk-2 were: 5′- GAGCAGCTGGGAACACC-ATCAGCAGAG-3′ (Forward), and 5′- GTGGGGCTT-CTGCTTCGGCGGGGT -3′ (Reverse). For the GAPDH gene the primers were   3’ -GAGTCAACGGATTTG GTCGTA3’ (Forward) and   5’- GCAGAGATGATGACC-CTTTTG3’ (Reverse). PCR condition used was 94°C, 2 min, followed by 30 cycles of 94°C, 15 sec, 55°C, 15 sec, and 68°C, 30 sec. Each primer and probe sets were optimized to be efficient (ranged from 93.1% to 100.9%). Samples were run in triplicate on an iQ5 thermocycler (Bio-Rad, Hercules, CA) and analyzed using the ΔΔCt method. All experiments were repeated in triplicate using two independent cDNA extractions with RNA isolated from three independent RNA extractions. Fresh stock solutions were prepared every 3 or 4 months, and fresh aliquots were used for each experiment. Cisplatin-resistant cells were treated with varying concentrations of decitabine (Sigma Aldrich) including 1, 3, 6, and 9 mM, for 24 h.

DNA methyltransferase activity

DNA methylation is a key regulator of gene expression in mammals (30) DNA methyltransferase activity was measured with a modification of Sodium bisulfite and Methylight. 5x106 cells were harvested from culture, homogenized with a Mini Beadbeater-1 (BioSpec Products) and subject to DNA/RNA extraction with AllPrep DNA/RNA Kit (Qiagen). Methylight probes and primers for SCGB3A1; GenBank C122714 region at 80825 – 80912 within the Exon1/Intron1 boundaries. The forward primer sequence was 5′-GGCGTAGCGGGCGTC, the reverse primer sequence was 5′- TACGTAACCCTATCCTACAACTCCG, and the probe sequence was 6FAM- CGAACTCCTAACGCGCACGATAAAACCTAA. All primers were designed with Beacon Designer (Premier Biosoft). Sodium bisulfite conversion was performed according to manufacturer’s recommendations using the EZ DNA Methylation Kit (Zymo Research). Methylight PCR was performed as previously described with COL2A1 as the internal control gene. Percentage of methylated reference (PMR, i.e., degree of methylation) was calculated for each sample using M.SssI-treated, CpGenome universal methylated DNA (Millipore) as the positive control.

Cell viability assay

In order to measure cell viability after treatment with decitabine, the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenylte- trazolium bromide) assay was used as described previously [30]. Briefly, 5x104 cells were seeded onto a 96-well plate in 100 ml growth media and incubated for 24 h prior to decitabine treatment. For 24 h decitabine treatment, 100 ml of the appropriate decitabine concentration (1, 3, 6, 9 mM) diluted in growth media was added to each well. Growth media only served as control. MTT (12 mM) (Sigma-Aldrich) was added to each well and cell viability was measured as described previously [30]. Cell viability assays were repeated in triplicate with six replicates per experiment.

Results

Decitabine reactivates Secretoglobin (SCGB3A1) gene in both, LNCaP and PC-3 Prostate cancer cell lines

To understand the role and mechanism of the human Secretoglobin (SCGB3A) gene expression, in the induction of prostate cancer we compared the expression of this gene in two cell lines, LNCaP and PC-3 cisplatin-resistant prostate cancer cells. The cells were treated with 9 mM decitabine and kept in culture for 3, 6, 9, and 12 days. After this time, the presence of the protein was measured using real-time PCR (Figure 1A, 1B). We showed that Decitabine reactivates Secretoglobin (SCGB3A1) gene in both, LNCaP (Fig 1A) and PC-3 (Fig 1B) Prostate cancer cell lines.  Interestingly, SCGB3A1 was highly expressed in PC-3 cells than in LNCaP cells. These results correlate with SCGB3A1 expression in prostate cancer. This protein is usually altered in prostate cancer cells. The results showed that both, LNCaP (p53 positive, with androgen receptor expression) and PC-3 (p53 negative, androgen unresponsive) cell lines were affected for decitabine suggesting That the expression of SCGB3A1 could be a p53 independent process.

Figure 1: Effect of Decitabine 9 mM on expression of Secretoglobin (SCGB3A1) gene in prostate cancer cell lines PC-3 and LNCaP. Cells were incubated for 3, 6 9 and 12 days in presence of decitabine 9mM. Human prostate and untreated LNCaP cells served as control.  Results are representative of three independent experiments.

Decitabine decreases DNMT activity in human prostate cancer cells

Since decitabine appeared not to be cytotoxic, at any concentration, in either LNCaP, DU145, or PC-3 cells we investigated the functional effectiveness of decitabine in our cell model system. As control cell, we used the Human Benign Prostatic Hyperplasia cell line (BPH-1), a well-established model for human prostate biology and widely used for investigation into the causes and potential treatments for prostatic disease. We tested DNMT activity in all Prostate cancer immortalized cells BPH-1, LNCaP, DU145, and PC-3 Cisplatin-resistant prostate cancer cells with and without 9 μM decitabine treatment (Figure 2). This was the highest concentration of decitabine used and was selected to determine whether decitabine was inhibiting DNA methylation in our cell model system. DNMT is an enzyme that catalyzed DNA methylation, and we observed that DNMT enzyme activity was decreased by 18.1% (±3.6) in LNCaP cells, 32% (±5.6) in DU145 cells, and 57.3% (±7.6) in PC-3 cells after decitabine treatment (Figure 2). The BPH-1 cells were not affected by decitabine treatment. Our results confirm that decitabine, which is a DNA methylation inhibitor, was functionally active in the prostate cancer cells that were tested.

Figure 2:  Effect of decitabine on DNA methyltransferase (DNMT). The activity of DNMT was measured in LNCaP, DU145 and PC-3 cisplatin - resistant prostate cancer cells with and without 9 mM decitabine for 24 hrs. DNMT activity is represented as percentage of control where untreated resistant cells served as control (100%).  The data is represented as mean SEM.  Results are representative of three independent experiments.

Figure 3: Legend to figure. Effect of decitabine on the expression of GAPDH, JNK-1 and JNK-2 gene products. Gene expression was measure using quantitative real-time PCR to compare differences in dose response. The cells were treated with, 1, 3 6 and 9 mM of decitabine for 24 h. The untreated cells were used as control cells. The data is presented as mean +/- SEM. (A) PC-3 cisplatin resistant cells, (B) LNCaP cisplatin resistant cells.  All experiments were repeated in triplicate using two independent cDNA extraction with RNA isolated from two independent RNA extraction. Results are representative of two independent experiments.

Effect of decitabine on expression of JNK-1 and JNK-2 signaling pathway in prostate cell lines LNCaP and PC-3 cells.

The expression levels of JNK-1 and JNK-2 (Figure 3A and 3B respectively and Table 1). MAPK signaling is an important signaling system that utilizes a step-by-step phosphorylation process to amplify signals into the nucleus, regulate the activity of transcription factors and the expression of corresponding genes, and then cause cellular responses [24]. It was determined that decitabine affected the expression of GAPDH JNK-1 and JNK-1 in the MAPK pathway. GAPDH was used as a control of endogenous gene expression. Compared with the normal control group, the increase in activity of JNK-1 and JNK-2 expression in the model group was significantly increased after decitabine intervention.  It should be of interest to study if the effect of decitabine in prostate tissue can induce a similar response on endogenous expression of JNK-1 and JNK-2 gene products. 

Table 1: Effect of decitabine on gene expression of JNK-1 and JNK-2.

 The results are presented as a percentage of control ± (cell untreated with decitabine) SEM.

Cell Line

Decitabine Concentration

(μM)

GAPDH

JNK-1

JNK-2

 

 

PC-3

0

100 %

100 %

100 %

1

146 % ± 31

124 % ± 17

135 % ± 21

3

135 % ± 34

156 % ± 20

168 % ± 36

6

124 % ± 33

150 % ± 29

179 % ± 31

9

112 % ± 16

122 % ± 38

185 % ± 19

 

 

LNCaP

0

100 %

100 %

100 %

1

155 % ± 40

166 % ± 31

124 % ± 42

3

174 % ± 24

171 % ± 25

112 % ± 44

6

135 % ± 28

149 % ± 42

118 % ± 47

9

138 % ± 33

122 % ± 20

133 % ± 38

Discussion

Epigenetic events are critical contributors to the pathogenesis of cancer, and targeting epigenetic mechanisms represents a novel strategy in anticancer therapy. Demethylating agents, such as Decitabine, hold the potential for reprogramming somatic cancer cells demonstrating high therapeutic efficacy in hematological malignancies.

The N-terminal Jun Kinase/Stress-Activated Protein Kinase (JNK/SAPK) signal transduction is rapidly activated by a large variety of toxic stimuli and inflammatory cytokines suggesting roles it is mediating inflammatory responses, stress responses, and apoptosis and is involved in the resistance of prostate cancer to cisplatin treatment [31-32]. In addition, previous studies have shown that this pathway participates in the transformation of animal model cells including primary murine fibroblasts [33]. Another molecule of interest in this study is the SCGB3A1, a gene that has been frequently considered as a constitutive control gene and used to normalize changes in specific gene expression. This protein has been shown to be altered in several cancers and downregulated by chemotherapic drugs [34-39]. However, an appropriate delivery system of decitabine can improve and reduce the incidence of toxicity in healthy tissues. In this work, we have evaluated SCGB3A1 and JNK1/JNK-2 gene expression using several prostate cell lines such as DU145, PC3, and LNCaP cancer cell lines treated with various concentrations of decitabine. The reactivation of Secretoglobin or SCGB3A1 by decitabine treatment in both LNCaP and PC-3 prostate cell lines confirmed that decitabine has multiple effects in cells independent of the p53 gene expression. On the other hand, DNMT-DNA methyltransferase activity was reduced in all three prostate cells used in the experiment. The most affected cells were the PC-3 cells. These cells are androgen unresponsive and are a cell line from grade IV adenocarcinoma with high metastatic potential. Multiple genes would be targeted simultaneously, blocking diverse signaling pathways utilized by tumor cells.

Conclusion

We hypothesize that de-methylation of these genes will result in their re-expression; thus, preventing tumor progression in a novel in vivo model that utilizes human prostate cell lines. It should be of interest to study if the effect of decitabine in prostate tissue can induce a similar response on endogenous expression of JNK-1, JNK-2 and SCGB3A1 gene products. This information could be used to determine whether Decitabine treatment of a Cisplatin-resistant prostate cancer cell line restores the antiproliferative and apoptotic effects of cisplatin. Overall, these results suggest that decitabine treatments can induce regression of the features of prostatic adenocarcinoma.

Funding

This work was supported by grants from Universidad of Tarapacá. Project-UTA-Mayor-7706-22.

Conflict of Interest

The authors declare that they have no competing interests.

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Editorial Information

Editor-in-Chief

Ying-Fu Chen
Kaohsiung Medical University, Taiwan

Article Type

Research article

Publication history

Received date: November 12, 2022
Accepted date: November 22, 2022
Published date: November 26, 2022

Copyright

©2022 Parra E. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Parra E, Ferreira J, Hecht P, Rojas V, Maturana JC, et al. (2022) Decitabine Reactivate Secretoglobin (SCGB3A1) and SAP/JNK genes in Cisplatin Resistant PC-3 and LNCaP Prostate cell lines. Trends Med 22: DOI: 10.15761/TiM.1000289

Corresponding author

Dr. Eduardo Parra

School of Medicine, University of Tarapacá, Arica. Avenida Senador Luis Valente Rossi, Campus Saucache, Arica, Chile

Table 1: Effect of decitabine on gene expression of JNK-1 and JNK-2.

 The results are presented as a percentage of control ± (cell untreated with decitabine) SEM.

Cell Line

Decitabine Concentration

(μM)

GAPDH

JNK-1

JNK-2

 

 

PC-3

0

100 %

100 %

100 %

1

146 % ± 31

124 % ± 17

135 % ± 21

3

135 % ± 34

156 % ± 20

168 % ± 36

6

124 % ± 33

150 % ± 29

179 % ± 31

9

112 % ± 16

122 % ± 38

185 % ± 19

 

 

LNCaP

0

100 %

100 %

100 %

1

155 % ± 40

166 % ± 31

124 % ± 42

3

174 % ± 24

171 % ± 25

112 % ± 44

6

135 % ± 28

149 % ± 42

118 % ± 47

9

138 % ± 33

122 % ± 20

133 % ± 38

Figure 1: Effect of Decitabine 9 mM on expression of Secretoglobin (SCGB3A1) gene in prostate cancer cell lines PC-3 and LNCaP. Cells were incubated for 3, 6 9 and 12 days in presence of decitabine 9mM. Human prostate and untreated LNCaP cells served as control.  Results are representative of three independent experiments.

Figure 2:  Effect of decitabine on DNA methyltransferase (DNMT). The activity of DNMT was measured in LNCaP, DU145 and PC-3 cisplatin - resistant prostate cancer cells with and without 9 mM decitabine for 24 hrs. DNMT activity is represented as percentage of control where untreated resistant cells served as control (100%).  The data is represented as mean SEM.  Results are representative of three independent experiments.

Figure 3: Legend to figure. Effect of decitabine on the expression of GAPDH, JNK-1 and JNK-2 gene products. Gene expression was measure using quantitative real-time PCR to compare differences in dose response. The cells were treated with, 1, 3 6 and 9 mM of decitabine for 24 h. The untreated cells were used as control cells. The data is presented as mean +/- SEM. (A) PC-3 cisplatin resistant cells, (B) LNCaP cisplatin resistant cells.  All experiments were repeated in triplicate using two independent cDNA extraction with RNA isolated from two independent RNA extraction. Results are representative of two independent experiments.