Abstract
Background/Aim: To evaluate the anti-cancer mechanism of N-Farnesyl-norcantharimide (NC15). Materials and Methods: The viability of NC15-treated human leukemic Jurkat T (JKT) cells was assessed using the Kit-8 cell counting method. Flow cytometry analysis, human apoptosis antibody array assay, and whole genome sequencing were adopted to investigate the mechanism underlying the anti-cancer activity of NC15 in JKT cells. Results: The growth inhibition rates of NC15 in JKT cells were about 80% and 95% after treatment with 8 μmol/l NC15 for 24 and 48 h, respectively. The percentages of NC15-treated JKT cells in the sub-G1 phase at 24 and 48 h were 22.0% and 34.3%, respectively, in contrast to the 1.5% in the control. Next-generation sequencing showed that many tumor suppressor genes (TSG) were up-regulated, while many genes associated with steroid biosynthesis, metabolic pathways, and fatty acid metabolism were down-regulated. Conclusion: NC15 can reduce the cell viability and increase the percentage of JKT cells in the sub-G1 phase by up-regulating TSG and related genes, and down-regulating the genes for steroid biosynthesis, metabolic pathways and fatty acid metabolism, instead of through apoptosis.
- N-farnesyl-norcantharimide
- Jurkat T cells
- next-generation sequencing
- tumor suppressor gene
- biosynthesis
Acute T lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers with very poor prognosis (1). A quarter of childhood T-ALL patients have relapsed within 5 years of treatment with very poor prognosis (2). The survival rate of patients with T-ALL within 5 years is less than 25% (3). Therefore, it is necessary to search for more efficient yet less toxic anti-cancer drugs for leukemia.
The Jurkat T (JKT) cell line established from the peripheral blood of a 14 years old boy with T-ALL in the late 1970s was used in this study (4). Phorbol 12-myristate 13-acetate plus ionomycin (PMA + ION) are often used in the study of the underlying mechanism of anti-cancer drugs because PMA + ION can activate JKT cells to produce high levels of interleukin-2 (IL-2) (5-8), and activate the JKT cells via a PKC-Ras signaling pathway (7, 8).
Mylabris, a species of blister beetle (Mylabris phalerata Pall.), has been used in the treatment of many kinds of malignancies in traditional oriental medicine for two thousand years (9-12). Mylabris-derived Cantharidin is a potent serine/threonine protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) inhibitor (13-15). Though Cantharidin has anti-cancer properties (16, 17), its clinical applications are limited because of its toxicity towards the kidneys and the urinary system (18, 19).
Norcantharidin (NCTD) derived from demethylation of Cantharidin has been shown to be effective towards many kinds of cancers including hepatocellular cancer (20), gallbladder malignancy (21), leukemia (22), and colorectal cancer (23). NCTD is able to prolong the survival of nude mice transplanted with human HepG2 cells by inhibiting the growth of tumor cells (24). The NCTD has an anti-cancer activity similar to that of Cantharidin (25, 26) with a lower cytotoxicity toward normal cells as compared to Cantharidin (26-28), and has less nephrotoxicity and other side effects than Cantharidin in clinical practice (9).
N-Farnesyl-norcantharimide (C23H33NO3, denoted as NC15), is a newly synthesized NCTD derivative (29). Figure 1 shows the chemical structures of NCTD and NC15. NC15 has high anti-cancer activity in cell models, and can induce G2/M arrest and induce cell apoptosis on mouse leukemic L1210 cells (30). NC15 has been found to prolong the survival of mice and reduce the weight of the tumor mass in a mouse leukemia model (30). This study investigated the mechanism underlying the anti-cancer activity of NC15 in JKT cells.
Materials and Methods
Chemical reagents. Both NCTD and NC15 (Figure 1) were synthesized and characterized in the previous report (29). Anti-Caspase-9, Caspase-8, Caspase-3, Bcl-2, BAX, Cytochrome C and Actin antibodies were purchased from the Cell Signaling Technology (Danvers, MA, USA). The anti-PARP antibodies were purchased from the Thermo Fisher Scientific (Waltham, MA, USA).
Cytotoxicity assay. The IC50 values of the cytotoxic effects of NC15 on JKT cells were calculated using the dose–response curve. The JKT cells [Clone E6-1, Bioresource Collection and Research Center (BCRC) number: 60424] and human normal lymphoblast (HNL, BCRC number: 08C0058) were cultured in RPMI 1640 medium (Biological Industries, Kibbutz Beit Haemek, Israel) plus 10% heat-treated fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) in a humidified 5% CO2 incubator at 37°C. Both JKT and HNL cells were plated in 96-well plates at a density of 1×104 cells/well. The cells were treated with 0, 2, 4, 6, and 8 μmol/l NC15 for 24 and 48 h. After incubation, the viability of JKT and HNL cells was assessed by using CCK-8 (Sigma, St Louis, MO, USA). The detection wavelength was set at 450 nm.
Total proteins isolation. The untreated and NC15-treated JKT cells were harvested by centrifugation at 200 × g for 10 min at 4°C. The cell pellets were washed once by ice-cold phosphate-buffered saline, re-suspended in 100 μl lysis buffer [10X RIPA buffer, 200 mM sodium orthovanadate (Na2VO3), 7X protease inhibitor, and 10X phosphatase inhibitor], and then placed on ice for 30 min. The cells in the suspension were ruptured by vortexing and centrifuged at 13000×g for 10 min at 4°C. The supernatants of ruptured cells were collected for later analyses.
Flow cytometry analysis. Apoptosis array assay was performed using flow cytometry analysis to study whether or not the apoptosis is the main anti-cancer mechanism of NC15. JKT cells plated in 6-well plates at a density of 1×106 cells/well were treated with NC15 at the IC50 concentration for 24 and 48 h. To determine the populations of apoptotic and necrotic cells (sub-G1 and G0 cell fraction), the JKT cells were harvested, fixed with cold 70% ethanol, stored at −20°C overnight, and treated with with 0.2% Triton X-100, 0.1 mg/ml RNase A (Sigma, MO, USA) and stained with 20 μmol/l Propidium iodide (PI) at 37°C in the dark for 30 min. The NC15-treated cells were stained with Annexin V-FITC (eBioscience, San Diego, CA, USA) in Annexin V staining buffer at 25°C for 10 min, and counterstained with 20 mg/ml PI in binding buffer solution without phenol red, and then analyzed by a FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) to evaluate the JKT cells in the early stages of apoptosis.
Human apoptosis antibody array. To examine whether or not apoptosis is the main anti-cancer mechanism of NC15, human apoptosis antibody array assay was performed on JKT cells treated with or without NC15 for 1 day. After that, the protein extracts of JKT cells were harvested and 200 μg of each protein extract were analyzed by using the human apoptosis antibody array kit (RayBiotech, Norcross, GA, USA). To capture the chemiluminescence signals, the membranes were exposed to the autoradiography film for various periods of time. The AlphaImager HP System (ProteinSimple, San Jose, CA, USA) was employed to quantify the images with chemiluminescence signals within the linear ranges. The chemiluminescence signals of the negative control spots on each membrane were averaged and then subtracted from that of each experimental spot. If the value of the chemiluminescence signal exceeded both the averaged local background and the averaged valid negative control values, the signal was considered valid (31). The valid chemiluminescent signal was normalized by the values of the positive control spots. The fold changes in the chemiluminescence signals of each spot were divided by the value of the valid signals at the corresponding spot on the minus NC15 membrane. Finally, the averaged fold changes and the standard deviations of each protein were calculated.
Next-generation sequencing. Whole genome sequencing of NC15-treated JKT cells was performed using next-generation sequencing (NGS) (32-36) to determine which genes in the JKT cells were regulated by NC15 treatment.
RNA quantification and qualification. Both RNA degradation and contamination were monitored on 1% agarose gels. The purity of the RNA was assessed using NanoPhotometer® spectrophotometer (IMPLEN, Westlake Village, CA, USA) (37). The concentration of RNA was determined using Qubit® RNA Assay Kit and Qubit® 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA). The integrity of the RNA was assessed using RNA Nano 6000 Assay Kit and the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).
Library preparation for transcriptome sequencing. Three μg of RNA per sample were used as the input material for the preparation of RNA sample. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs (NEB), San Diego, CA, USA). The index codes were added to the attribute sequences to each sample. In short, the mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. The fragmentation was then carried out in the NEBNext first strand synthesis reaction buffer (5X) using divalent cations at 72°C. Random hexamer primer and M-MuLV reverse transcriptase (RNase H−) was used to synthesize the first strand cDNA. After that, the second strand cDNA was synthesized using DNA Polymerase I and RNase H. Through the help of exonuclease/polymerase activities, the remaining overhangs were converted into blunt ends. After the completion of adenylation at the 3’ ends of the DNA fragments, the NEBNext Adaptor with hairpin loop structure was ligated and then used for hybridization. In order to select appropriate cDNA fragments of about 150~200 bp, the library fragments were purified with the help of the AMPure XP system (Beckman Coulter, Beverly, MA, USA). Before PCR, 3 μl of USER enzyme (NEB) were used with the size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by incubation at 95°C for 5 min. The PCR was performed using the Phusion® High-Fidelity DNA polymerase (NEB), universal PCR primers, and Index (X) primer. The PCR products thus obtained were purified using the AMPure XP system. At last, the library quality of the products was assessed by the Agilent Bioanalyzer 2100 system (38).
Clustering and sequencing. By using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), the clustering of the index-coded samples was performed on a cBot Cluster Generation System following the manufacturer's instructions. The library preparations were sequenced on the Illumina HiSeq 2500 PE150 platform (Illumina) after cluster generation. The 125 bp/150 bp paired-end reads were then generated (38).
Quality control. Through in-house perl scripts, the raw data or raw reads of fastq format were processed. At this step, the clean data or clean reads were obtained when the reads containing adapter, the reads containing ploy-N, and the low quality reads from the raw data were removed. Meanwhile, the Q20, Q30 and GC containing the high quality clean data were calculated to perform all downstream analyses (39).
Reads mapping to reference genome. The gene model annotation files and reference genome were downloaded directly from the genome website. Bowtie v2.2.3 was used to build the index of the reference genome, and TopHat v2.0.12 was used to align the paired-end clean reads to the reference genome. Since the TopHat can generate a database of splice junctions based on the gene model annotation file and yield a better mapping result compared with other non-splice mapping tools (39), it was selected and used as the mapping tool.
Quantification of gene expression and differential expression analysis. The reads numbers mapped to each gene were counted using the HTSeq v0.6.1. Based on the length of the gene and reads count mapped to this gene, the fragments per kilobase of transcript per million mapped reads (FPKM) of each gene were calculated (40). For the estimation of gene expression levels, the FPKM is currently the most commonly used method because it takes into account the effects of both sequencing depth and gene length on the reads count simultaneously. The read counts of each sequenced library were adjusted using the edgeR program package through one scaling normalized factor before differential gene expression analysis. The DEGSeq R package (1.20.0) was employed to analyze the differential expression. The Benjamini & Hochberg method was used to adjust the p-values. A corrected p-value of 0.005 and a log2 (fold change) of 1 were adopted as the threshold of significantly different expression (41).
GO and Kegg enrichment analysis. To correct the bias caused by gene length, the GOseq R package was used to perform the Gene Ontology (GO) enrichment analysis of the differentially expressed genes. GO terms whose corrected p-Values were smaller than 0.05 were regarded as significantly enriched by differentially expressed genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was a resource constructed from molecular-level information for the understanding of high-level functions and utilities of the biological system, comprizing of the cell, organism and ecosystem (42). The KOBAS webserver software was employed to test the statistical enrichment of differentially expressed genes in the KEGG pathways (43, 44).
PPI analysis of differentially expressed genes. The protein-protein interactions (PPI) analysis of differentially expressed genes was performed using the STRING database. For the species in the database, the networks were constructed by extracting the target gene list in the STRING database in accordance with the known interactions of selected reference species (45). Otherwise, the search protein database Blastx v2.2.28 was utilized to align the target gene sequences with the selected reference protein sequences. .
Statistical analysis. The experimental data of this study were expressed as means±standard deviation (SD) when appropriate. Student's t-test was employed to compare the study data with the control values. A p<0.05 was considered to indicate statistically significant differences compared to the control values. All statistical analyses were performed using the GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA).
Results
NC15 inhibited the growth of JKT cells. To determine the inhibitory effects of NC15 on the growth of JKT and HNL cells, their viability was assessed using Kit-8 cell counting method after treatment with 0, 2, 4, 6 or 8 μmol/l NC15 for 24 and 48 h. Figure 2a and b show that the growth of JKT cells was inhibited by NC15 in a dose- and time-dependent manner. The IC50 of NC15 inhibition on the growth of JKT cells at 24 and 48 h was found to be 2.51 and 2.54 μmol/l, respectively. When 8 μmol/l of NC15 was added to the JKT cells for 24 and 48 h, the inhibition rate of NC15 on JKT cells was increased to about 40%, as compared to the cells without NC15 treatment. Figure 2a and b also show that there were no significant differences in the growth of HNL cells treated with 0, 2, or 4 μmol/l NC15. Thus, the NC15 could inhibit the growth of JKT cells, but has mild inhibitory effects on the growth of HNL cells.
Cell cycle analysis. To determine whether apoptosis is the main mechanism underlying the growth inhibitory effects of NC15, JKT cells were treated with the IC50 concentrations of NC15 (2.51 and 2.54 μmol/l) for 24 and 48 h, and then the number of cells in the sub-G1 phase was assayed using flow cytometry with PI staining. Figure 3a show that only a small number of apoptotic cells was found in untreated cells. Figure 3 further shows that, compared to the untreated cells, the percentages of NC15-treated cells in the sub-G1 phase at 24 and 48 h were significantly increased, while the percentages of NC15-treated cells in the G1 phase at 24 h and 48 h were significantly decreased. However, the percentages of NC15-treated cells in both the S and G2/M phases were not significantly different from the untreated cells. Thus, NC15 could inhibit the growth of JKT cells through interference with their cell cycle.
Effect of NC15 on cell death. To assess the possibility of NC15-induced apoptosis in JKT cells, the number of apoptotic cells after NC15 treatment was determined using flow cytometry plus Annexin V/PI staining. Flow cytometry analysis demonstrated that the number of Annexin V-positive/PI-negative cells (early apoptosis) and Annexin V-positive/PI-positive cells (trace amount of late apoptosis) was slightly increased in parallel with the concentration of NC15. Figure 4 shows that the early/possibly late apoptosis ratios were 0.8/2.8, 1.2/1.7, 1.9/2.3, 1.8/3.6, 2.1/7.4 in the untreated cells versus the cells treated with 2, 4, 6, and 8 μmol/l NC15, respectively. The result of Annexin-V/PI analysis suggested that NC15 may induce trace amount of late apoptosis, but not early apoptosis, in JKT cells. Whether or not late apotosis is the mechanism of cytotoxicity of NC15 over JKT cells was investigated in the following apoptosis array analysis.
NC15 did not alter the levels of apoptosis-related proteins. To assess whether apoptosis is the main mechanism of cell death, the JKT cells were treated with NC15 (0, 2, 6, and 8 μmol/l) for 24 h, and then lysed. The apoptotic markers were screened using apoptosis array. Figure 5 shows that there were no significant differences between the treated and untreated cells regarding bad, bax, BID, bcl-2, Caspase-3, Caspase-8, Cytochrome C, Fas, Fas ligand, and HSP70. This result suggested that NC15 did not induce apoptosis in JKT cells.
NC15 affected metabolic and biosynthetic pathways in JKT cells. To understand the underlying mechanism of cell death in the NC15-treated JKT cells, the cDNA of the JKT cells exposed to NC15 for 24 h was amplified using RT-PCR, and whole genome sequencing was performed using next-generation sequencing (NGS, Illumina). The data suggested that the tumor suppressor genes (TSG) including cytochrome B-245 alpha chain (CYBA) for NADPH, cyclin-dependent kinase inhibitor 1B (CDKN1B) for P27, activating transcription factor 4 (ATF4) for CREB3, and the genes for serine metabolism and aminoacyl-tRNA biosynthesis were up-regulated, while the genes for steroid biosynthesis, metabolic pathways, and fatty acid metabolism were down-regulated (Tables I and II). This result suggested that NC15 might inhibit the viability and proliferation of JKT cells through the up-regulation of the above-mentioned TSG and the genes for serine metabolism and aminoacyl-tRNA biosynthesis, and the down-regulation of the genes responsible for steroid biosynthesis, metabolic pathways, and fatty acid metabolism.
Discussion
NC15 is a newly synthesized NCTD derivative with better anti-cancer activity than NCTD towards cell viability and proliferation in JKT cells. It has been shown that NC15 had higher cytotoxicity and anti-proliferative effects on human liver carcinoma HepG2 cell line, yet had lower cytotoxic effect on normal murine embryonic liver BNL CL.2 cells (29). In this study, NC15 was shown to decrease the viability of cells, increase the percentage of cells in the sub-G1 phase and induce trace amount of late apoptosis in JKT cells. Though the mechanism of cytotoxic effects of NC15 on JKT cells is not clear yet, apoptosis might not be the main mechanism of anti-cancer effects of NC15 on JKT cells, because there were no significant differences between treated and untreated JKT cells in the expressions of bad, bax, BID, bcl-2, Caspase-3, Caspase-8, Cytochrome C, Fas, Fas ligand, and HSP70 (Figure 5), and the progression of JKT cells could be inhibited by NC15 through its effects on the expression of TSG and genes for serine metabolism, aminoacyl-tRNA biosynthesis, steroid biosynthesis, metabolic pathways, and fatty acid metabolism in JKT cells (Table I and II).
High-throughput sequencing or next-generation sequencing (NGS) technology has dramatically improved our capability to characterize various kinds of cancers at the genomic level by cataloguing all mutations, aberrations in copy number and somatic rearrangements in the entire cancer genome (46, 47). NGS has significantly accelerated biological and biomedical research by facilitating inexpensive, routine, widespread, and comprehensive analyses of genomes, transcriptomes and interactomes, without requiring significant production-scale efforts (48). In the present study, gene expression of the JKT cells was analyzed using NGS technologies. The changes in gene expression of JKT cells represented the effects of NC15 treatment on the JKT cells. We speculate that altered protein levels in NC15-treated JKT cells due to up- and down-regulation of genes may result in the disturbance of many metabolic pathways of the JKT cells, leading to cell death. This might be the underlying mechanism of cell death in the JKT cells after treatment with NC15, instead of apoptosis.
The NGS data showed that the over-expressed TSG after treatment with NC15 in JKT cells included the CYBA (NADPH), CDKN1B (P27), and ATF4 (CREB3) genes that control the progression of cell cycle. The mechanisms controlling cell cycle progression are frequently lost in human cancer cells. The cell cycle is driven forward by cyclin-dependent kinases (CDK), and the inhibitors of CDK (CDKI) are important regulators of CDK. Thus, CDKIs negatively regulate cell cycle progression, and are responsible for cell cycle arrest at the G1 phase (49-52). The down-regulation of CDKI or TSG can lead to increased cell proliferation. Since NC15 could inhibit the viability of JKT cells by increasing the percentage of cells in the sub-G1 phase and G0-G1 arrest, CDKI might be involved in cell cycle arrest at the G1 phase in JKT cells.
Recently, the links between metabolic enzymes and oncogene-induced signal transduction have attracted increasing interest because the metabolic pathways are targets of cancer chemotherapy. The assessment of the metabolic pathways and their interplay in cancer cells should be important for the understanding of the transformation mechanisms, the mechanisms of neo-growth and cancer cell drug resistance, and for the identification of potential drugs specific for cancer cells (53, 54). In this study, we found that the expression of TSG and the genes responsible for serine metabolism and aminoacyl-tRNA biosynthesis was up-regulated (Table I), while the expression of the genes responsible for steroid biosynthesis, metabolic and fatty acid metabolism enzymes in JKT cells was down-regulated (Table II) following treatment with NC15. These changes in gene expressions might lead to instability and even breakdown of the metabolic functions of the cells and the disturbance of cell cycle, leading to cell death. This might be the main mechanism of the anti-cancer activity of NC15 on JKT cells. The destruction of cancer cells through the derangement of their metabolic pathways might be termed “metabolic killing”. Thus, the derangement and disturbance of metabolic pathways in cancer cells might be a target for developing potential anti-cancer drugs.
The accumulation of cells in the sub-G1 phase of the cell cycle may not be evidence of apoptosis, because the observed DNA fragmentation may also be caused by necrosis or necroptosis. Annexin-V/PI analysis indicated that NC15 may induce trace amount of late apoptosis in JKT cells (Figure 4), but an apoptosis array analysis showed that there was no up-regulation of proteins associated with apoptosis (Figure 5). Thus, necrosis or necroptosis might not be the mechanism underlying the accumulation of NC15-treated JKT cells in the sub-G1 phase.
Cell apoptosis can be divided into early and late apoptosis. When apoptosis is measured over time, the cells can undergo transformation from Annexin V negative/PI negative (no apoptosis) to Annexin V positive/PI negative (early apoptosis with intact membranes), and finally to Annexin V positive/ PI positive (end stage apoptosis and cell death). The presence of cells with these three phenotypes in a mixed cell population, or the transformation of a synchronized cell population through these three stages suggests apoptosis (55). However, the cells in the late apoptosis stage may not necessarily come from apoptosis, but also from necrosis and possibly other mechanism of cell death. When the plasma membrane of the cells become permeable, early apoptotic cells can become late apoptotic cells or secondary necrotic cells (56). After treatment with NC15, the late apoptotic cells might become necrotic cells, and be detected in the late apoptosis phase by the apoptosis array assay. The secondary necrotic cells were not true apoptotic cells, because the proteins associated with apoptosis such as bad, bax, BID, bcl-2, Caspase-3, Caspase-8, Cytochrome C, Fas, Fas ligand and HSP70 in the NC15-treated cells were not significantly different from those of untreated cells. To find out whether or not the cells in the late apoptotic stage were mainly derived apoptosis, we performed the apoptosis array assay and found that apoptosis might not be the main mechanism responsible for the death of JKT cells in the late apoptosis region in Figure 4. Instead, derangements in metabolic pathways may be the cause of cell death in JKT cells after NC15 treatment, because NGS data showed no changes in RNA levels and related protein expressions. Further studies are needed to elucidate the metabolic mechanism of cell death in NC15-treated JKT cells.
In conclusion, the newly derived compound NC15 can reduce cell viability and increase the percentage of cells in the sub-G1 phase in the JKT cells. The main mechanism of anti-cancer activity of NC15 against JKT cells may be through the up-regulation of TSG and the genes responsible for serine metabolism and aminoacyl-tRNA biosynthesis, and the down-regulation of the genes responsible for steroid biosynthesis, metabolic pathways, and fatty acid metabolism. The JKT cells may be killed by NC15 through the disturbances in their metabolism and cell cycle, rather than through apoptosis.
Acknowledgements
This work was supported by the grants V104C-153 and V104E5-001 of the Taipei Veterans General Hospital, Taipei, Taiwan.
Footnotes
* These Authors contributed equally to the present study.
Authors' Contributions
CDK designed the research, planned the research, revised and finalized the manuscript. ETT performed most of the experiments and drafted the initial manuscript. JYW synthesized the NC15 for research and improved the manuscript. FYY performed the experiment of Figure 3. JFL, HFL and YJC participated in the discussion and refined the manuscript. All Authors read and approved the manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
- Received March 15, 2020.
- Revision received March 24, 2020.
- Accepted March 26, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved