Int J Clin Exp Med 2018;11(7):7004-7017 www.ijcem.com /ISSN:1940-5901/IJCEM0069480 Original Article Identification of commonly regulated genes and biological pathways as potential targets in PC-3 and DU145 androgen-independent human prostate cancer cells treated with the curcumin analogue 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one Kamini Citalingam1, Faridah Abas2,3*, Nordin H Lajis2*, Iekhsan Othman1*, Rakesh Naidu1* 1Jeffery Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia; 2Laboratory of Natural Products, Faculty of Science, 3Department of Food Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. *Equal contributors. Received November 20, 2017; Accepted May 3, 2018; Epub July 15, 2018; Published July 30, 2018 Abstract: Diarylpentanoid [1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one] (MS17) demonstrated enhanced anti- cancer activity compared to curcumin but its effect on androgen-independent prostate cancer has not been well- studied. The present study was aimed to perform gene expression profiling on MS17 treated PC-3 and DU145 cells using microarray technology to identify mutually regulated genes as common targets in both androgen-independent prostate cancer cell lines as well as molecular pathways that contributes to the anticancer activity of MS17. The profiling data revealed a dose-dependent gene regulation, evident by higher fold change expression values when the treatment dose was increased by 1.5-fold in both cell lines. Gene ontology classification was performed on highly regulated differentially expressed genes (DEGs). Among these genes, the mutually regulated DEGs were identified as common targets in both cell lines. The mutually up-regulated DEGs included, CRYAB and DNAI2 associated with cytoskeletal organization, HSPA6 and HSPB8 with response to unfolded protein, MMP3 and MMP10 with proteoly- sis, CACNA1G with transporter activity, NGFR with apoptosis, CCL26 with immune response, DNAJA4 with protein folding and TRIML2 with protein ubiquitination. However, the down-regulated genes such as CTDSP1 were associ- ated with phosphatase activity, HIST1H2BF and HIST1H2AI with chromosome organization, MXD3 with regulation of transcription and TNFRSF6B with apoptosis. PC-3 and DU145 are androgen-independent prostate cancer cells with different biological properties, and identification of common targets in both cell lines could potentially be used as therapeutic targets. Pathway analysis of the DEGs in PC-3 cells demonstrated modulation of top pathways associ- ated with cell cycle checkpoint, DNA damage, and inflammatory response while in DU145 cells the pathways were associated with immune response and metabolism. Thus, the findings of the present study provide insight into the antitumor activity of MS17 and as a potential chemotherapeutic agent for androgen independent prostate cancer cells. Keywords: Androgen-independent prostate cancer, diarylpentanoid, microarray gene expression profiling, pathway analysis Introduction to prevent the progression of hormone sensi- tive prostate cancer cells to hormone refrac- Prostate cancer is the leading cause of cancer tory stage. Curcumin, a polyphenol active com- among men. Although in the initial stage pros- pound extracted from the rhizome of the tur- tate cancer cells responds well to androgen meric plant, Curcuma longa has known to dem- deprivation therapy, it often progresses to an onstrate potential anticancer effect against a androgen-independent phenotype that is resis- variety of carcinomas both in vitro and in vivo tant to treatment [1]. Despite available thera- [2]. Despite the success of curcumin in treating peutic options, much effort has been directed cancer, its poor pharmacokinetic characteris- towards developing novel treatment modalities tics stimulated chemists to synthesize variety Gene expression of prostate cancer cells of analogues with the hope to obtain com- the chemically purified diarylpentanoid, MS17 pounds that are more effective and with a wider [1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3- spectrum of anti-tumor activity while retaining one] was synthesized based on the method its safety profile [3-5]. Diarylpentanoids (DAPs), described previously [16], and was prepared by a group of curcumin-like analogues with 5-car- dissolving in 100% DMSO (Sigma-Aldrich, St. bon chain between its aryl rings was reported Louis, MO, USA) to a final concentration of 50 to display significant growth suppressive poten- mM. tial compared to curcumin [6]. Several studies have demonstrated anticancer activity of DAPs Treatment and total RNA isolation in a wide range of cancer cell lines [7-12]. EF- 24 [3,5-bis(2-fluorobenzylidene)-4-piperidone] Cells were seeded in triplicate wells containing a curcumin DAP exhibited potent growth inhibi- 4 × 105 cells/mL in 6-well tissue culture plates tory effects in a variety of cancer cells including and treated with 10 µM and 15 µM doses of prostate cancer at doses significantly lower MS17 for 24 hours. Control wells (untreated than curcumin [13]. Other investigators report- cells) containing media only with 0.5% DMSO ed the anti-tumorigenic effects of the structur- was also included. Following overnight incuba- ally identical DAP ca27 and Ca 37 in androgen- tion, cells were harvested and RNA was isolat- dependent and -independent prostate cancer ed using RNeasy® Mini Kit (Qiagen, Valencia, cells respectively [7, 14]. While many of these CA, USA) and purified using RNase-free DNA DAPs demonstrated an improved anticancer set according to the manufacturer’s protocol. effect compared to curcumin, the underlying Concentration and purity of the extracted RNA mechanisms that contributed to its anticancer was measured spectrophotometrically using activity in prostate cancer cells remains to be NanoDrop 1000 (Thermo Fisher Scientific, Wal- explored. tham, MA, USA). RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Tech- In our recent study, curcumin diarylpentanoid nologies, Santa Clara, CA, USA) and scored as 1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3- RNA integrity number (RIN). Samples with RIN one (MS17) demonstrated improved dose- and score exceeding 8 indicate high RNA quality time-dependent growth inhibitory effects on and were selected for microarray analysis. androgen-independent prostate cancer cell lin- es, PC-3 and DU145 respectively at EC50 values Microarray of approximately 5.0 µM compared to curcum- in. MS17 also demonstrated significant apop- Genome-wide expression profiles of the treated totic effects at 24 hours of treatment with early and untreated samples were analyzed using apoptosis noted at 2 × EC50 (10 µM) and 3 × Agilent one-color microarray-based gene expre- EC50 (15 µM) concentrations in the treated cells ssion analysis protocols (Agilent Technologies). [15]. Hence, in the present study we performed Briefly, 100 ng quality-checked total RNA was gene expression profiling on MS17 treated PC-3 reverse transcribed using a Low Input Quick and DU145 cells using microarray technology Amp labeling kit and then transcribed to Cy3- to identify mutually regulated genes as com- labeled cRNA according to the manufacturer’s mon targets in both androgen-independent protocol. Cy3-labeled cRNA samples were hy- prostate cancer cell lines as well as molecular bridized onto whole human genome SurePrint pathways that contributes to the anticancer G3 8X60K arrays (Agilent Technologies) using activity of MS17. Agilent’s Surehyb Chambers in an Agilent hy- Materials and methods bridization oven set at 65°C for 17 hours. Three independent replicates were performed for Cell culture and preparation of MS17 diaryl- each treatment. The slides were washed and pentanoid subsequently scanned by an Agilent G2600D SureScan Microarray Scanner (Agilent Techno- Androgen-independent metastatic human pro- logies). Images from the scanned array were state cancer cell line, PC-3 and DU145 cells analyzed using Agilent’s Feature Extraction were purchased from American Type Culture software, version 11.5.1.1 and further analyz- Collection (ATCC, Rockville, MD, USA) and cul- ed for statistical evaluations using GeneSpring tured as previously described [15]. Separately, GX version-13.0 software (Agilent Technologies). 7005 Int J Clin Exp Med 2018;11(7):7004-7017 Gene expression of prostate cancer cells Data normalization and statistical analysis gen.com/ingenuity) and DEG’s from the pros- tate cancer cells were mapped to canonical Data normalization and transformation steps pathways using reference genes from the Inge- recommended by Agilent Technologies for one- nuity Knowledge Base. Using the Fishers Exact color oligonucleotide microarrays were per- Test the p-value was set at p < 0.05, canonical formed using GeneSpring GX software. The raw pathways were considered as statistically sig- signals were log transformed and normalized nificant when it passed through a threshold of using the Percentile shift normalization meth- 1.3 [calculated by -log (p-value)]. A ratio value od. Quality control on the probe sets were fil- calculating the number of genes from the data- tered based on flag and expression values to set that matched components within the path- remove probes with low signal intensities (< way was also calculated. 20.0). Statistical comparisons were performed between the experimental groups by using Real-time PCR (qRT-PCR) validation Analysis of Variance (ANOVA) with Benjamini- Hochberg false discovery rate (FDR) set as