BACKGROUND
triptolide is a biological diterpenoid derived from the chinese herb tripterygium wilfordii hook f. triptolide has been shown to have anti-inflammatory and immunosuppressive activities and has been used in traditional chinese medicine to treat several diseases, such as, rheumatoid arthritis, immune complex diseases, and systemic lupus erythematosus  <cit> .
it has been shown to have influence on several anti-tumor target genes and inhibit tumors by altering multiple signaling pathways, such as, inhibition of nfκb and c-kit pathway  <cit> , inhibition of jak <dig> transcription  <cit> , inducing apoptotic signals by activation of pro-apoptotic proteins  <cit> , activation of mapk8/jnk  <cit> , and inhibition of heat shock response  <cit> .
triptolide has also been shown to influence epigenetic modulation of genes by interaction with histone methyltransferase and demethylase  <cit> .
in spite of the wide therapeutic properties of triptolide, poor water solubility has limited its clinical use in the past.
however, recently a water-soluble analog of triptolide–minnelide has shown promising results in pancreatic cancer cell lines, human xenograft models, as well as in mouse models of pancreatic cancer  <cit> .
minnelide has been shown to reduce tumor burden in preclinical models of osteosarcoma  <cit> .
taken together with the anti-tumor properties of triptolide and the recent development of triptolide analogs to overcome its water solubility, triptolide has emerged as a promising anti-tumor agent.
in the present study, we evaluated the impact of genetic variations and gene expression profiles predictive of triptolide cytotoxicity using epstein-barr-virus transformed lymphoblastoid cell lines  that are part of international hapmap project   <cit> .
hapmap lcls has been used as model to identify genetic markers associated with in vitro chemo-sensitivity to several drugs .
genotype data is publically available allowing for genome-wide association analyses for biomarker identification.
our results validated some of the known genes/pathways as well as identified novel candidate genes/pathways of relevance to triptolide.
we further validated the functional significance of cflar in pancreatic cell lines.
methods
in vitro cytotoxicity assays
hapmap lcls from subjects with european ancestry  were obtained from the coriell institute for medical research and were maintained as recommended.
in vitro cytotoxicity was determined by treating lcls with varying concentrations of triptolide  for 48 hr followed by cell viability measurements using mtt - <dig> -diphenyltetrazolium bromide assays  and synergy  <dig> multi-plate reader.
cytotoxicity assays were performed in duplicates and cell lines were randomly chosen to repeat on different dates to rule out any experimental variation.
panc- <dig>  a pancreatic cell line  was used for functional validation of the top gene identified in the gwas.
panc- <dig> was cultured in dmem medium supplemented with 2 mm glutamine and 10 % fetal bovine serum.
real-time quantitative pcr analysis
mrna expression levels of cflar spliced isoforms were quantitated in lcls and panc- <dig> cell lines using cflar isoform specific oligonucleotide primers and 2^-ΔΔct method as described in additional file  <dig> and fig.
<dig>
genotyping of panc- <dig> cell lines
genomic dna from panc- <dig> cell line was genotyped for cflar snp rs <dig>  using taqman snp genotyping assay .
genomic dnas from hapmap cell lines with known genotype  were used as controls.
western blotting
western plotting was performed using whole-cell lysates and cflar  or β-actin  primary and mouse igg secondary antibodies.
sirna mediated knockdown of cflar in cancer cell lines
mission esirna were procured from sigma aldrich.
esirna were synthesized through in vitro transcription of a 300–600 bp gene specific dsrna, which is further digested in to complex pool of sirna using rnases.
this digested pools of esirna are verified by dna sequensing and gel electrophoresis to ensure identity and high specificity .
to ensure high specificity and efficacy of the esirna, the algorithm deqor is utilized .
cflar specific  <dig> base pair sequence used in this study to create esirna pool for cflar knockdown is “tccatcaggttgaagaagcacttg atacagatgagaaggagatgctgctctttttgtgccgggatgttgctatagatgtggttccacctaatgtcagggaccttctggatattttacgggaaagaggtaagctgtctgtcggggacttggctgaactgctctacagagtgaggcgatttgacctgctcaaacgtatcttgaagatggacagaaaagctgtggagacccacctgctcaggaaccctcaccttgtttcggactatagagtgctgatggcagagattggtgaggatttggataaatctgatgtgtcctcattaattttcctcatgaaggattacatgggccgaggcaagataagcaaggagaagagtttcttggaccttgtggttgagttggagaaactaaatctggttgccccaga”.
panc- <dig> cells were transfected with  <dig> nm cflar-esirna and negative sirna using lipofectamine®  <dig>  as per manufacturer’s protocol.
twenty-four hours post-transfections cell were treated with varying concentrations of triptolide and cell viability was determined 48 hr post-treatment using mtt assays.
mrna levels of all three isoforms of cflar sirna were quantitated 24 hr post-transfection to check for the knockdown.
transfection of cflar-short and cflar-long plasmid in panc- <dig> cell line
panc- <dig> cells were transfected with control and cflar expression plasmids  using fugene hd reagent  followed by triptolide treatment and mtt assay as described above.
cell pellets were also collected for protein analysis.
statistical analysis
as these cell lines are part of several publically available genotyping databases, genotype data was retrieved on all cell lines from the hapmap project .
for  <dig> samples, data was also retrieved from the  <dig> genomes project .
mrna expression was retrieved for all of the individuals from a publically available source .
ic <dig>  concentration that kills 50 % of the cells, was calculated from a 4-parameter logistic model using the package drc v <dig> .
<dig> in r v <dig> .
<dig>  <cit> .
association analysis of triptolide cytotoxicity with genetic variation
snp genotype  data was retrieved from the hapmap  for all  <dig> samples .
snps were filtered using various quality control criteria as, build changes, call rate, compliance with hardy weinberg and minimum allele frequency  as described in additional file  <dig>  in total  <dig> snps in  <dig> individuals passed quality control measures.
for genotype data for individuals in the  <dig> genomes only a maf filter was used, dropping snps with maf <  <dig> .
several snps overlap between the  <dig> genomes and hapmap data, data obtained from the  <dig> genomes project was used preferentially over data obtained from the hapmap.
since not all of the samples used have been sequenced as part of the  <dig> genomes project as of current, snps in the  <dig> genomes project, but not in hapmap  were imputed for samples not included in the  <dig> genomes project.
beagle v <dig> .
<dig>  <cit>  was used to impute snps with the reference of the  <dig> genomes as described in additional file  <dig>
association analysis of triptolide cytotoxicity with gene expression variation
mrna expression from the illumina sentrix human- <dig> expression beadchip version  <dig> was normalized as per stranger et al., using  <dig> probes  <cit> .
for this analysis, only the caucasian samples were used and data was normalized using quantile normalization across replicates and median normalization across individuals.
original illumina annotation was retrieved from remoat  <cit> .
expression was not adjusted for gender .
integrative analyses
with the wealth of data produced by current genomic technologies, collection of multiple types of genomic data on a set of samples is becoming commonplace.
new methods explore a multifactor approach that combine different kinds of genomic data, sometimes referred to as “integrative genomics” or “genomic convergence”, in which a multistep procedure is used to identify potential key drivers of complex traits that integrate dna variation and gene expression data.
to integrate the genotype, expression and drug cytotoxicity data, we first identified markers associated with triptolide ic <dig> using a liberal significance threshold of  <dig> .
next, we determined which expression probe sets were associated with these ic <dig> associated snps  .
finally, to determine whether the expression probe sets associated with these snps were also associated with triptolide ic <dig> values, we identified which expression probe sets were associated with ic <dig> with a p-values ≤  <dig> ).
a similar integrative analysis approach has been used successfully to detect novel candidate genes  <cit> .
the association between ic <dig> and snp genotypes modeled as count of rare alleles , or log <dig> normalized mrna expression and cytotoxicity phenotypes, or snps was quantified using a spearman correlation coefficient, and p-value calculated for the null hypothesis of no association using an f-test.
RESULTS
genetic associations with triptolide ic <dig> in lcls
we evaluated  <dig> lcls derived from unrelated subjects with caucasian ancestry for cellular sensitivity to triptolide.
triptolide ic <dig> values ranged from  <dig> to  <dig> nm indicating wide inter-individual variation in chemo-sensitivity.
gwas analysis identified  <dig> snps in  <dig> genes that were associated with triptolide ic <dig> at p < 10− <dig> .
significant proportion of snps  clustered in ~293 kb region on chromosome  <dig> , which maps to multiple biologically interesting genes  including cflar, clk <dig>  fam126b, ndufb <dig>  nif3l <dig>  orc <dig> and ppil  <dig> .
other genes with significant snps included tp53bp <dig> , mtsu <dig> , znf <dig>  and fndc3b .
ingenuity pathway analysis of these genes mapped them to  <dig> networks .
these networks are involved in cellular movement, inflammatory response, cell-to-cell signaling and interaction; cell death and survival, cellular function and maintenance, molecular transport; cancer, dermatological diseases and conditions, developmental disorder; cellular movement, hematological system development and function, immune cell trafficking and carbohydrate metabolism.fig.
<dig> genome-wide association study of snps with triptolide cytotoxicity.
a manhattan plot showing association of snps with triptolide ic <dig> .
b genomic region on chr  <dig> with strongest association with triptolide cytotoxicity.
y-axis represents -log  <dig>  and x-axis presents chromosomal location
rs10190751
202006096
a
<dig>
− <dig>
<dig> e-06
2
cflar/intron
cflar  with maximum number of most significant snps  codes for protein c-flip.
c-flip regulates apoptosis and is structurally similar to caspase- <dig>  however, lacks the caspase activity.
it has been implicated as a crucial link between cell survival and cell death pathways in mammalian cells.
gene expression associations with triptolide ic <dig> in lcls
genome-wide gene expression analysis identified  <dig> probes that were associated with triptolide ic <dig> .
some of the biologically interesting genes with expression levels associated with triptolide sensitivity included: jak <dig> , dtx <dig> , agl , and muc <dig> .
pathway analysis using ingenunity pathway analysis tool mapped these genes to jak/stat, il, inos, egfsignalining pathways .table  <dig> top probe and genes with expression levels in lcls associated with triptolide lc50
integrated snp-mrna association analysis with ic50
we performed integrated analysis between snps-mrna expression-ic50; we selected top snps that were significantly  associated with cytotoxicity, and top gene expression signatures significantly  associated with cytotoxicity for association with each other i.e.
snp vs. gene expression.
this analysis basically identified eqtls associated with triptolide ic <dig> and at p < <dig>  we identified  <dig> unique snp-mrna pairs that were associated with triptolide cytotoxicity however these snp-mrna pairs mapped to only  <dig> genes , indicating association of multiple snps with one gene .
some of the biologically interesting snp-mrna pairs included: association of multiple snps in chromosome  <dig> spanning genes chst <dig>  tmem <dig>  gabarapl <dig> and adat <dig> with expression levels of agl; snps in asxl <dig> with ascl <dig> expression; camta <dig> snps and crygs expression; tiam <dig> snps with dtx <dig> expression; snps in gpatch <dig>  and multiple snps on chr  <dig> and  <dig> with jak <dig> expression.
multiple snps within cflar were associated with expression levels of mtvr <dig>  pip5k1b and c9orf19/glrip <dig> table  <dig> summary of  <dig> way integrated analysis of gene-expression-snp and triptolide cytotoxicity
tiam1
arrb1/ opcml
manba/ myo18b
nif3l1/ c7orf42/ cflar/ fam126b/nif3l1/orc2
capn2/cgnl1/tp53bp2
mpg/nprl3/ccdc46/kank1/usp34
erbb4/ fhad1/ gpatch2
nif3l1/ abcc4/ cflar/ fam126b/ gpatch2/ndufb3/nif3l1/orc2/ppil3/smyd3/tsga14
ldlrad3
ablim2/ manba/ ttll1
fhad1/ppcs/ smyd3/zmynd12
clk1/ fam126b/
snx6/ strn3/ rsu1
actn1/ arrb1/ grm7/ sez6l
functional significance of cflar splicing snp
we selected cflar for functional validation in our study on the basis of following observations; i) higher number of snps in cflar were associated with the triptolide cytotoxicity ; ii) cflar.
snp-mrna pairs associated with triptolide cytotoxicity included three cflar mrna probes additionally multiple snps within cflar were associated with each  <dig> mrna probes.
majority of the significantly associated snps within cflar gene were intronic and occurred in high ld .
however, one cflar snp, rs <dig> g > a, was present at the splice junction of exon  <dig>  and was significantly associated with triptolide cytotoxicity .
we screened hapmap cell lines with aa, ag and gg genotype  for long , short  and raji  forms of cflar splice variants.
cflar-l form was present in all cell lines irrespective of the genotype whereas cflar-s form was only expressed in cell lines with at least one g allele .
real-time quantification of cflar splice variants showed significant association of aa genotype with low levels of cflar-l; complete absence of cflar-s and higher levels of cflar-r form .
western blot analysis confirmed the association of c-flip protein isoform levels corresponding to rs <dig> genotype and cflar mrna isoforms .fig.
<dig> schematic representation of cflar gene and its splice variants.
a the schematic of cflar gene shows all the exons and the location of rs <dig> snp at the 3’splice site of intron  <dig>  presence or absence of this snp regulates production of cflar-short form .
difference splice variants of cflar are also shown along with the isoform specific primer pair’s used in this study.
b box plot showing association of rs <dig> splicing snp with triptolide lc <dig> in hapmap lcls.
y-axis is log  <dig> triptolide ic <dig> and x-axis represents rs <dig> genotype.
box plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding outliers.
the outliers are defined as data points that fall outside of the first and third quartiles by more than  <dig> -times the interquartile range.
circles falling outside the whiskers represent outliers
fig.
<dig> correlation of rs <dig> genotype with cflar splice variants.
a isoform specific amplification of cflar splice-variants  in lcls with aa, gg and ag genotype for rs <dig>  b real-time-mrna quantification showing relative levels of cflar-l, cflar-s and cflar-r isoforms in different genotype groups.
box plots details are same as in figure 2b.
c western blotting showing protein levels of cflar-l and cflar-s/r forms in hapmap lcls with aa, ag or gg genotype for rs10190751
sirna mediated functional studies on cflar
we selected panc- <dig> for further investigation for impact of sirna mediated transient knockdown of cflar on cellular sensitivity to triptolide.
we selected panc- <dig> for further investigation for impact of sirna mediated transient knockdown of cflar on cellular sensitivity to triptolide.
panc- <dig> was selected based on the literature evidence of efficient use of triptolide in pancreatic cancer at pre-clinical and clinical level  as well as cflar being reported as a therapeutic target for triptolide in pancreatic cancer .
genotype of panc- <dig> for rs <dig>  was identified as gg, therefore all isoform of cflar expressed in this cell line and it makes this cell line a perfect model to do functional validation for different isoform.
in a pancreatic cancer cell line, panc- <dig>  sirna mediated knockdown resulted in significant reduction in the cflar-l, cflar-s and cflar-r isoforms and significant increase in sensitivity to, triptolide .fig.
<dig> impact of sirna mediated knockdown or overexpression of cflar isoforms on triptolide sensitivity in panc- <dig> cancer cell lines.
a impact of sirna medicated knockdown of cflar on mrna expression levels of cflar-l, cflar-s and cflar-r isoforms b impact of sirna medicated knockdown of cflar on cellular cytotoxicity to triptolide.
c western blot showing over expression of cflar-l and clfar-s isoforms as compared to cells transfected to control plasmids.
d impact of overexpression of cflar-l and cflar-s isoforms in cellular sensitivity to triptolide
over expression of cflar-long and short isoform
since panc- <dig> demonstrated change in chemo-sensitivity in sirna mediated knockdown of cflar, we further overexpressed cflar-l and cflar-s forms in panc- <dig> cell lines.
transient transfection of most abundant isoforms of cflar was done in panc- <dig> cell line using pefa-cflar-l and pef6-v5-cflar-l plasmid for long and short form of cflar respectively.
compared to cells transfected with control plasmids the level of cflar protein isoforms were significantly increased .
over-expression cflar-l or cflar-s isoforms resulted in significant decrease in sensitivity for triptolide, .
discussion
triptolide is a diterpenoid triepoxide and has been used in traditional chinese medicine for years.
poor water solubility and toxicity has limited its use in clinics, however recent advances focused on developing triptolide derivatives with better solubility such as mc <dig>  <cit> , omtritolide  <cit> , minnelide  <cit>  etc.
are showing promising advancements especially in pancreatic cancer.
the anticancer activity of triptolide has been associated with its ability to inhibit various pro-proliferative or anti-apoptotic factors thereby inducing apoptosis  <cit> .
triptolide has been implicated in activation of both intrinsic and extrinsic apoptotic pathways by inducing caspase- <dig>  − <dig> and  <dig> as well as by inducing cleavage of parp  <cit> .
given the fact that triptolide has a promising potential as a therapeutic agent, we designed this study to identify the genomic markers associated with triptolide cytotoxicity using lcls from international hapmap project.
our results identified a qtl on chromosome  <dig> consisting of several snps with significant association with triptolide ic <dig>  this region on chromosome  <dig> included biologically interesting genes such as clk <dig>  ppl <dig>  nif3l <dig>  cflar, ndufb <dig>  casp <dig>  casp <dig> etc.
with important roles in apoptosis and cell cycle regulation pathways.
of particular interest was significant over-representation the top most significant snps in cflar gene .
cflar gene encodes for protein c-flip, best-known for its anti-apoptotic regulatory role by inhibiting tnf-alpha, fas-l and trail induced apoptosis  <cit> .
although most of the snps in cflar were intronic, one splicing snp, rs <dig>  was of particular interest  <cit> .
cflar-protein, c-filp exists in several isoforms due to alternate splicing, the most studied forms include long  and short  isoforms of  <dig> kd and  <dig> kd, respectively.
cflar gene has  <dig> exons and inclusion or exclusion of intron  <dig> or exon  <dig> regulates the expression of long, or short or raji forms.
cflar long form  skips exon  <dig> and is expressed as a full-length protein of  <dig> amino acids.
cflar short form  includes exon  <dig> thereby changing the reading frame, creating an early stop codon, and hence a shorter isoform with  <dig> amino acids.
c-flip-l is composed of two death effector domains  at the amino terminus and a caspase homologous domain, structurally similar to caspase  <dig> and caspase  <dig> at carboxy terminus.
in contrast c-flip-s has two deds but lacks caspase homology domain.
presence of rs <dig> regulates the splicing event with rs10190751-a allele resulting in lack of expression of the short form .
in addition to these isoforms recently cflip-r forms has been identified in the raji cells  <cit> .
due to intronic insertion; cflar-r isoform has a premature stop codon resulting in a protein with  <dig> amino acids and like the cflar-s isoform lacks caspase like domain.
although the characterization of the functional differences of these isoforms is still ongoing, cell type specific pro-apoptotic role of cflar-l has been reported.
cflar-l expression levels are considered critical factor in determining the balance between apoptotic and pro-survival signaling.
the cflar-l has also been shown to play critical role in autophagy, necroptosis and apoptosis in t-lymphocytes with cflar-l deficiency triggering severe cell death upon stimulation  <cit> .
in spite of its major role in regulating death receptor signaling, it has been shown to be involved in regulation of apoptosis by several other mechanism including; modulating the activity of ripoptosome  <cit>  regulation of nectroptosis by preventing caspase  <dig> activation , inhibiting autophagosome formation by interfering with conjugation of lc <dig> and in nfkb signaling with its ectopic expression resulting in nfkb activation .
given the important role of cflar  as a key inhibitor of processing and activation of caspase 8; its prognostic and therapeutic relevance in aml  <cit>  as well as in development of drug resistance  <cit>  we designed this study to further explore the clinical significance of the cflar and its genetic variation especially the splicing snp  as biomarker of risk of disease as well as with development drug resistance.
our results of sirna mediated knock down and overexpression of cflar in pancreatic cancer cell lines further provides evidence of its involvement in chemo-sensitivity to triptolide.
gene expression levels of jak <dig>  agl, and dtx <dig> genes, all involved in cell-to cell signaling  has been associated with triptolide cytotoxicity analysis.
jak <dig>  janus kinase  <dig> is involved in interferon-alpha/beta and -gamma signal transduction pathways and is a critical component of jak/stat pathway; agl is member of  <dig> alpha-glucanotransferase and is involved in glycogen degradation; dtx <dig>  deltex homolog  <dig> is involved in notch signaling pathway which is a critical for cell fate determination and has been implicated in several diseases as well as tumorogenesis  <cit> .
in our integrative exploratory analysis we identified several biologically interesting gene-snp-gene-expression pairs as tiam1-dtx <dig>  asxl3: ascl <dig>  gpatch2: jak <dig>  campta1-crygs, erbb4-nadsyn <dig> etc.
in recent years there has been significant evidence suggesting triptolide mediated inhibition of atpase activity of xpb, thereby by influencing transcription as well as nucleotide excision repair  <cit> .
xpb, also known as ercc <dig> is a subunit of transcription factor tfiih.
triptolide has been shown to influence gene expression by globally reducing gene expression although to not to same extent for all genes by blocking transcription initiation  <cit> .
antiproliferative effects of triptolide due to inhibition of xpb/tfiih has also been shown to phenocopy jnk-dependent apoptosis phenotype in dp <dig> deficient wing disc cells in drosophila  <cit> .
this global reduction of transcription caused by triptolide, correlates well with the phenotypes observed in tumour cells and in inflammation.
if we take in account these evidences, and if the treatment with triptolide, reduce global transcription, cells with reduction of the cflar mrna isoforms by the splicing snp will be even more sensitive, since this gene may negatively modulates apoptosis.
the kd and overexpression results using panc- <dig> cells incubated with triptolide may also be explained in part by taking in account a reduction in global transcription caused by triptolide.
in conclusion, our results identified cflar as an important predictor of triptolide cytotoxicity.
splicing snp-rs <dig> regulates production of cflar- long and short isoforms, which are associated with triptolide cytotoxicity.
the central role of anti-apoptotic protein c-flip  in regulating death receptor signaling points to the fact that this splicing snp might of importance to other chemotherapeutic agents.
up-regulation of c-flip has been associated with poor clinical outcome and thus could be reliable prognostic factor for several types of cancer, however the significance of cflar genetic variation as predictor of therapeutic efficacy has not been explored so far, thus opening up opportunities for future studies.
CONCLUSIONS
triptolide being an emerging drug, provides us a reason to do a genome wide association study to identify specific genetic polymorphism which may affect triptolide induced cytotoxicity.
we observed significant association of triptolide ic <dig> with snps located in biological important genes from apoptotic pathway, such as cflar, ppil <dig>  caspase 8/ <dig>  nfkb and stat <dig>  cflar is an upstream regulator of apoptotic pathway.
due to its important position as a regulator of apoptosis, we validated its functional role in triptolide induced cytotoxicity in pancreatic cancer cell line.
our finding shows that cflar polymorphism plays important role in cancer cell death induced by triptolide.
further studies are needed to predict the therapeutic response in patients.
availability of supporting data
gene expression data is publically avialble from gene expression omnibus under submission number series: gse <dig>  genotype data is avaiable at hapmap.org-release  <dig> .
additional files
additional file 1:
supplementary notes.
additional file 2: figure s <dig>  ld plot generated using haploview for chromosome  <dig> region flanking cflar gene in ceu population.
additional file 3: figure s <dig>  genome-wide association study of mrna expression with triptolide cytotoxicity.
y-axis represents -log  <dig>  and x-axis presents chromosomal location.
additional file 4: figure s <dig>  overlapping networks identified by pathway analysis of genes identified in gene expression analysis with triptolide ic <dig> in hapmap lcls using ingenuity pathway analysis  tool.
competing interests
the authors declare they have no competing interests.
authors contributions
jkl, conceived the study design; lc, nb, tf and tmg performed the experiments; blf and gdj performed statistical analysis; all authors contributed to manuscript writing.
all authors read and approved the final manuscript.

 triptolide is a biological diterpenoid derived from the chinese herb tripterygium wilfordii hook f. triptolide has been shown to have anti-inflammatory and immunosuppressive activities and has been used in traditional chinese medicine to treat several diseases, such as, rheumatoid arthritis, immune complex diseases, and systemic lupus erythematosus. it has been shown to have influence on several anti-tumor target genes and inhibit tumors by altering multiple signaling pathways, such as, inhibition of nfκb and c-kit pathway, inhibition of jak transcription, inducing apoptotic signals by activation of pro-apoptotic proteins, activation of mapk8/jnk, and inhibition of heat shock response. triptolide has also been shown to influence epigenetic modulation of genes by interaction with histone methyltransferase and demethylase. in spite of the wide therapeutic properties of triptolide, poor water solubility has limited its clinical use in the past. however, recently a water-soluble analog of triptolide–minnelide has shown promising results in pancreatic cancer cell lines, human xenograft models, as well as in mouse models of pancreatic cancer. minnelide has been shown to reduce tumor burden in preclinical models of osteosarcoma. taken together with the anti-tumor properties of triptolide and the recent development of triptolide analogs to overcome its water solubility, triptolide has emerged as a promising anti-tumor agent. in the present study, we evaluated the impact of genetic variations and gene expression profiles predictive of triptolide cytotoxicity using epstein-barr-virus transformed lymphoblastoid cell lines that are part of international hapmap project. hapmap lcls has been used as model to identify genetic markers associated with in vitro chemo-sensitivity to several drugs. genotype data is publically available allowing for genome-wide association analyses for biomarker identification. our results validated some of the known genes/pathways as well as identified novel candidate genes/pathways of relevance to triptolide. we further validated the functional significance of cflar in pancreatic cell lines. methods in vitro cytotoxicity assays hapmap lcls from subjects with european ancestry were obtained from the coriell institute for medical research and were maintained as recommended. in vitro cytotoxicity was determined by treating lcls with varying concentrations of triptolide for 48 hr followed by cell viability measurements using mtt - -diphenyltetrazolium bromide assays and synergy multi-plate reader. cytotoxicity assays were performed in duplicates and cell lines were randomly chosen to repeat on different dates to rule out any experimental variation. panc- a pancreatic cell line was used for functional validation of the top gene identified in the gwas. panc- was cultured in dmem medium supplemented with 2 mm glutamine and 10 % fetal bovine serum. real-time quantitative pcr analysis mrna expression levels of cflar spliced isoforms were quantitated in lcls and panc- cell lines using cflar isoform specific oligonucleotide primers and 2^-ΔΔct method as described in additional file and fig.  genotyping of panc- cell lines genomic dna from panc- cell line was genotyped for cflar snp rs using taqman snp genotyping assay. genomic dnas from hapmap cell lines with known genotype were used as controls. western blotting western plotting was performed using whole-cell lysates and cflar or β-actin primary and mouse igg secondary antibodies. sirna mediated knockdown of cflar in cancer cell lines mission esirna were procured from sigma aldrich. esirna were synthesized through in vitro transcription of a 300–600 bp gene specific dsrna, which is further digested in to complex pool of sirna using rnases. this digested pools of esirna are verified by dna sequensing and gel electrophoresis to ensure identity and high specificity. to ensure high specificity and efficacy of the esirna, the algorithm deqor is utilized. cflar specific base pair sequence used in this study to create esirna pool for cflar knockdown is “tccatcaggttgaagaagcacttg atacagatgagaaggagatgctgctctttttgtgccgggatgttgctatagatgtggttccacctaatgtcagggaccttctggatattttacgggaaagaggtaagctgtctgtcggggacttggctgaactgctctacagagtgaggcgatttgacctgctcaaacgtatcttgaagatggacagaaaagctgtggagacccacctgctcaggaaccctcaccttgtttcggactatagagtgctgatggcagagattggtgaggatttggataaatctgatgtgtcctcattaattttcctcatgaaggattacatgggccgaggcaagataagcaaggagaagagtttcttggaccttgtggttgagttggagaaactaaatctggttgccccaga”. panc- cells were transfected with nm cflar-esirna and negative sirna using lipofectamine® as per manufacturer’s protocol. twenty-four hours post-transfections cell were treated with varying concentrations of triptolide and cell viability was determined 48 hr post-treatment using mtt assays. mrna levels of all three isoforms of cflar sirna were quantitated 24 hr post-transfection to check for the knockdown. transfection of cflar-short and cflar-long plasmid in panc- cell line panc- cells were transfected with control and cflar expression plasmids using fugene hd reagent followed by triptolide treatment and mtt assay as described above. cell pellets were also collected for protein analysis. statistical analysis as these cell lines are part of several publically available genotyping databases, genotype data was retrieved on all cell lines from the hapmap project. for samples, data was also retrieved from the genomes project. mrna expression was retrieved for all of the individuals from a publically available source. ic concentration that kills 50 % of the cells, was calculated from a 4-parameter logistic model using the package drc v. in r v.. association analysis of triptolide cytotoxicity with genetic variation snp genotype data was retrieved from the hapmap for all samples. snps were filtered using various quality control criteria as, build changes, call rate, compliance with hardy weinberg and minimum allele frequency as described in additional file in total snps in individuals passed quality control measures. for genotype data for individuals in the genomes only a maf filter was used, dropping snps with maf < . several snps overlap between the genomes and hapmap data, data obtained from the genomes project was used preferentially over data obtained from the hapmap. since not all of the samples used have been sequenced as part of the genomes project as of current, snps in the genomes project, but not in hapmap were imputed for samples not included in the genomes project. beagle v. was used to impute snps with the reference of the genomes as described in additional file association analysis of triptolide cytotoxicity with gene expression variation mrna expression from the illumina sentrix human- expression beadchip version was normalized as per stranger et al., using probes. for this analysis, only the caucasian samples were used and data was normalized using quantile normalization across replicates and median normalization across individuals. original illumina annotation was retrieved from remoat. expression was not adjusted for gender. integrative analyses with the wealth of data produced by current genomic technologies, collection of multiple types of genomic data on a set of samples is becoming commonplace. new methods explore a multifactor approach that combine different kinds of genomic data, sometimes referred to as “integrative genomics” or “genomic convergence”, in which a multistep procedure is used to identify potential key drivers of complex traits that integrate dna variation and gene expression data. to integrate the genotype, expression and drug cytotoxicity data, we first identified markers associated with triptolide ic using a liberal significance threshold of. next, we determined which expression probe sets were associated with these ic associated snps. finally, to determine whether the expression probe sets associated with these snps were also associated with triptolide ic values, we identified which expression probe sets were associated with ic with a p-values ≤  ). a similar integrative analysis approach has been used successfully to detect novel candidate genes. the association between ic and snp genotypes modeled as count of rare alleles, or log normalized mrna expression and cytotoxicity phenotypes, or snps was quantified using a spearman correlation coefficient, and p-value calculated for the null hypothesis of no association using an f-test.  genetic associations with triptolide ic in lcls we evaluated lcls derived from unrelated subjects with caucasian ancestry for cellular sensitivity to triptolide. triptolide ic values ranged from to nm indicating wide inter-individual variation in chemo-sensitivity. gwas analysis identified snps in genes that were associated with triptolide ic at p < 10−. significant proportion of snps clustered in ~293 kb region on chromosome, which maps to multiple biologically interesting genes including cflar, clk fam126b, ndufb nif3l orc and ppil. other genes with significant snps included tp53bp, mtsu, znf and fndc3b. ingenuity pathway analysis of these genes mapped them to networks. these networks are involved in cellular movement, inflammatory response, cell-to-cell signaling and interaction; cell death and survival, cellular function and maintenance, molecular transport; cancer, dermatological diseases and conditions, developmental disorder; cellular movement, hematological system development and function, immune cell trafficking and carbohydrate metabolism.fig. genome-wide association study of snps with triptolide cytotoxicity. a manhattan plot showing association of snps with triptolide ic. b genomic region on chr with strongest association with triptolide cytotoxicity. y-axis represents -log and x-axis presents chromosomal location rs10190751 202006096 a − e-06 2 cflar/intron cflar with maximum number of most significant snps codes for protein c-flip. c-flip regulates apoptosis and is structurally similar to caspase- however, lacks the caspase activity. it has been implicated as a crucial link between cell survival and cell death pathways in mammalian cells. gene expression associations with triptolide ic in lcls genome-wide gene expression analysis identified probes that were associated with triptolide ic. some of the biologically interesting genes with expression levels associated with triptolide sensitivity included: jak, dtx, agl, and muc. pathway analysis using ingenunity pathway analysis tool mapped these genes to jak/stat, il, inos, egfsignalining pathways.table top probe and genes with expression levels in lcls associated with triptolide lc50 integrated snp-mrna association analysis with ic50 we performed integrated analysis between snps-mrna expression-ic50; we selected top snps that were significantly associated with cytotoxicity, and top gene expression signatures significantly associated with cytotoxicity for association with each other i.e. snp vs. gene expression. this analysis basically identified eqtls associated with triptolide ic and at p < we identified unique snp-mrna pairs that were associated with triptolide cytotoxicity however these snp-mrna pairs mapped to only genes, indicating association of multiple snps with one gene. some of the biologically interesting snp-mrna pairs included: association of multiple snps in chromosome spanning genes chst tmem gabarapl and adat with expression levels of agl; snps in asxl with ascl expression; camta snps and crygs expression; tiam snps with dtx expression; snps in gpatch and multiple snps on chr and with jak expression. multiple snps within cflar were associated with expression levels of mtvr pip5k1b and c9orf19/glrip table summary of way integrated analysis of gene-expression-snp and triptolide cytotoxicity tiam1 arrb1/ opcml manba/ myo18b nif3l1/ c7orf42/ cflar/ fam126b/nif3l1/orc2 capn2/cgnl1/tp53bp2 mpg/nprl3/ccdc46/kank1/usp34 erbb4/ fhad1/ gpatch2 nif3l1/ abcc4/ cflar/ fam126b/ gpatch2/ndufb3/nif3l1/orc2/ppil3/smyd3/tsga14 ldlrad3 ablim2/ manba/ ttll1 fhad1/ppcs/ smyd3/zmynd12 clk1/ fam126b/ snx6/ strn3/ rsu1 actn1/ arrb1/ grm7/ sez6l functional significance of cflar splicing snp we selected cflar for functional validation in our study on the basis of following observations; i) higher number of snps in cflar were associated with the triptolide cytotoxicity; ii) cflar. snp-mrna pairs associated with triptolide cytotoxicity included three cflar mrna probes additionally multiple snps within cflar were associated with each mrna probes. majority of the significantly associated snps within cflar gene were intronic and occurred in high ld. however, one cflar snp, rs g > a, was present at the splice junction of exon and was significantly associated with triptolide cytotoxicity. we screened hapmap cell lines with aa, ag and gg genotype for long, short and raji forms of cflar splice variants. cflar-l form was present in all cell lines irrespective of the genotype whereas cflar-s form was only expressed in cell lines with at least one g allele. real-time quantification of cflar splice variants showed significant association of aa genotype with low levels of cflar-l; complete absence of cflar-s and higher levels of cflar-r form. western blot analysis confirmed the association of c-flip protein isoform levels corresponding to rs genotype and cflar mrna isoforms.fig. schematic representation of cflar gene and its splice variants. a the schematic of cflar gene shows all the exons and the location of rs snp at the 3’splice site of intron presence or absence of this snp regulates production of cflar-short form. difference splice variants of cflar are also shown along with the isoform specific primer pair’s used in this study. b box plot showing association of rs splicing snp with triptolide lc in hapmap lcls. y-axis is log triptolide ic and x-axis represents rs genotype. box plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding outliers. the outliers are defined as data points that fall outside of the first and third quartiles by more than -times the interquartile range. circles falling outside the whiskers represent outliers fig. correlation of rs genotype with cflar splice variants. a isoform specific amplification of cflar splice-variants in lcls with aa, gg and ag genotype for rs b real-time-mrna quantification showing relative levels of cflar-l, cflar-s and cflar-r isoforms in different genotype groups. box plots details are same as in figure 2b. c western blotting showing protein levels of cflar-l and cflar-s/r forms in hapmap lcls with aa, ag or gg genotype for rs10190751 sirna mediated functional studies on cflar we selected panc- for further investigation for impact of sirna mediated transient knockdown of cflar on cellular sensitivity to triptolide. we selected panc- for further investigation for impact of sirna mediated transient knockdown of cflar on cellular sensitivity to triptolide. panc- was selected based on the literature evidence of efficient use of triptolide in pancreatic cancer at pre-clinical and clinical level as well as cflar being reported as a therapeutic target for triptolide in pancreatic cancer. genotype of panc- for rs was identified as gg, therefore all isoform of cflar expressed in this cell line and it makes this cell line a perfect model to do functional validation for different isoform. in a pancreatic cancer cell line, panc- sirna mediated knockdown resulted in significant reduction in the cflar-l, cflar-s and cflar-r isoforms and significant increase in sensitivity to, triptolide.fig. impact of sirna mediated knockdown or overexpression of cflar isoforms on triptolide sensitivity in panc- cancer cell lines. a impact of sirna medicated knockdown of cflar on mrna expression levels of cflar-l, cflar-s and cflar-r isoforms b impact of sirna medicated knockdown of cflar on cellular cytotoxicity to triptolide. c western blot showing over expression of cflar-l and clfar-s isoforms as compared to cells transfected to control plasmids. d impact of overexpression of cflar-l and cflar-s isoforms in cellular sensitivity to triptolide over expression of cflar-long and short isoform since panc- demonstrated change in chemo-sensitivity in sirna mediated knockdown of cflar, we further overexpressed cflar-l and cflar-s forms in panc- cell lines. transient transfection of most abundant isoforms of cflar was done in panc- cell line using pefa-cflar-l and pef6-v5-cflar-l plasmid for long and short form of cflar respectively. compared to cells transfected with control plasmids the level of cflar protein isoforms were significantly increased. over-expression cflar-l or cflar-s isoforms resulted in significant decrease in sensitivity for triptolide,. discussion triptolide is a diterpenoid triepoxide and has been used in traditional chinese medicine for years. poor water solubility and toxicity has limited its use in clinics, however recent advances focused on developing triptolide derivatives with better solubility such as mc, omtritolide, minnelide etc. are showing promising advancements especially in pancreatic cancer. the anticancer activity of triptolide has been associated with its ability to inhibit various pro-proliferative or anti-apoptotic factors thereby inducing apoptosis. triptolide has been implicated in activation of both intrinsic and extrinsic apoptotic pathways by inducing caspase- − and as well as by inducing cleavage of parp. given the fact that triptolide has a promising potential as a therapeutic agent, we designed this study to identify the genomic markers associated with triptolide cytotoxicity using lcls from international hapmap project. our results identified a qtl on chromosome consisting of several snps with significant association with triptolide ic this region on chromosome included biologically interesting genes such as clk ppl nif3l cflar, ndufb casp casp etc. with important roles in apoptosis and cell cycle regulation pathways. of particular interest was significant over-representation the top most significant snps in cflar gene. cflar gene encodes for protein c-flip, best-known for its anti-apoptotic regulatory role by inhibiting tnf-alpha, fas-l and trail induced apoptosis. although most of the snps in cflar were intronic, one splicing snp, rs was of particular interest. cflar-protein, c-filp exists in several isoforms due to alternate splicing, the most studied forms include long and short isoforms of kd and kd, respectively. cflar gene has exons and inclusion or exclusion of intron or exon regulates the expression of long, or short or raji forms. cflar long form skips exon and is expressed as a full-length protein of amino acids. cflar short form includes exon thereby changing the reading frame, creating an early stop codon, and hence a shorter isoform with amino acids. c-flip-l is composed of two death effector domains at the amino terminus and a caspase homologous domain, structurally similar to caspase and caspase at carboxy terminus. in contrast c-flip-s has two deds but lacks caspase homology domain. presence of rs regulates the splicing event with rs10190751-a allele resulting in lack of expression of the short form. in addition to these isoforms recently cflip-r forms has been identified in the raji cells. due to intronic insertion; cflar-r isoform has a premature stop codon resulting in a protein with amino acids and like the cflar-s isoform lacks caspase like domain. although the characterization of the functional differences of these isoforms is still ongoing, cell type specific pro-apoptotic role of cflar-l has been reported. cflar-l expression levels are considered critical factor in determining the balance between apoptotic and pro-survival signaling. the cflar-l has also been shown to play critical role in autophagy, necroptosis and apoptosis in t-lymphocytes with cflar-l deficiency triggering severe cell death upon stimulation. in spite of its major role in regulating death receptor signaling, it has been shown to be involved in regulation of apoptosis by several other mechanism including; modulating the activity of ripoptosome regulation of nectroptosis by preventing caspase activation, inhibiting autophagosome formation by interfering with conjugation of lc and in nfkb signaling with its ectopic expression resulting in nfkb activation. given the important role of cflar as a key inhibitor of processing and activation of caspase 8; its prognostic and therapeutic relevance in aml as well as in development of drug resistance we designed this study to further explore the clinical significance of the cflar and its genetic variation especially the splicing snp as biomarker of risk of disease as well as with development drug resistance. our results of sirna mediated knock down and overexpression of cflar in pancreatic cancer cell lines further provides evidence of its involvement in chemo-sensitivity to triptolide. gene expression levels of jak agl, and dtx genes, all involved in cell-to cell signaling has been associated with triptolide cytotoxicity analysis. jak janus kinase is involved in interferon-alpha/beta and -gamma signal transduction pathways and is a critical component of jak/stat pathway; agl is member of alpha-glucanotransferase and is involved in glycogen degradation; dtx deltex homolog is involved in notch signaling pathway which is a critical for cell fate determination and has been implicated in several diseases as well as tumorogenesis. in our integrative exploratory analysis we identified several biologically interesting gene-snp-gene-expression pairs as tiam1-dtx asxl3: ascl gpatch2: jak campta1-crygs, erbb4-nadsyn etc. in recent years there has been significant evidence suggesting triptolide mediated inhibition of atpase activity of xpb, thereby by influencing transcription as well as nucleotide excision repair. xpb, also known as ercc is a subunit of transcription factor tfiih. triptolide has been shown to influence gene expression by globally reducing gene expression although to not to same extent for all genes by blocking transcription initiation. antiproliferative effects of triptolide due to inhibition of xpb/tfiih has also been shown to phenocopy jnk-dependent apoptosis phenotype in dp deficient wing disc cells in drosophila. this global reduction of transcription caused by triptolide, correlates well with the phenotypes observed in tumour cells and in inflammation. if we take in account these evidences, and if the treatment with triptolide, reduce global transcription, cells with reduction of the cflar mrna isoforms by the splicing snp will be even more sensitive, since this gene may negatively modulates apoptosis. the kd and overexpression results using panc- cells incubated with triptolide may also be explained in part by taking in account a reduction in global transcription caused by triptolide. in conclusion, our results identified cflar as an important predictor of triptolide cytotoxicity. splicing snp-rs regulates production of cflar- long and short isoforms, which are associated with triptolide cytotoxicity. the central role of anti-apoptotic protein c-flip in regulating death receptor signaling points to the fact that this splicing snp might of importance to other chemotherapeutic agents. up-regulation of c-flip has been associated with poor clinical outcome and thus could be reliable prognostic factor for several types of cancer, however the significance of cflar genetic variation as predictor of therapeutic efficacy has not been explored so far, thus opening up opportunities for future studies.  triptolide being an emerging drug, provides us a reason to do a genome wide association study to identify specific genetic polymorphism which may affect triptolide induced cytotoxicity. we observed significant association of triptolide ic with snps located in biological important genes from apoptotic pathway, such as cflar, ppil caspase 8/ nfkb and stat cflar is an upstream regulator of apoptotic pathway. due to its important position as a regulator of apoptosis, we validated its functional role in triptolide induced cytotoxicity in pancreatic cancer cell line. our finding shows that cflar polymorphism plays important role in cancer cell death induced by triptolide. further studies are needed to predict the therapeutic response in patients. availability of supporting data gene expression data is publically avialble from gene expression omnibus under submission number series: gse genotype data is avaiable at hapmap.org-release. additional files additional file 1: supplementary notes. additional file 2: figure s ld plot generated using haploview for chromosome region flanking cflar gene in ceu population. additional file 3: figure s genome-wide association study of mrna expression with triptolide cytotoxicity. y-axis represents -log and x-axis presents chromosomal location. additional file 4: figure s overlapping networks identified by pathway analysis of genes identified in gene expression analysis with triptolide ic in hapmap lcls using ingenuity pathway analysis tool. competing interests the authors declare they have no competing interests. authors contributions jkl, conceived the study design; lc, nb, tf and tmg performed the experiments; blf and gdj performed statistical analysis; all authors contributed to manuscript writing. all authors read and approved the final manuscript. 
in the present study, we used lymphoblast cell lines derived from unrelated caucasian subjects to identify genetic markers predictive of cellular sensitivity to triptolide using genome wide association study. overall out results identified genetic factors associated with triptolide chemo-sensitivity thereby opening up opportunities to better understand its mechanism of action as well as utilize these biomarkers to predict therapeutic response in patients. triptolide is a therapeutic diterpenoid derived from the chinese herb tripterygium wilfordii hook f. triptolide has been shown to induce apoptosis by activation of pro-apoptotic proteins, inhibiting nfkb and c-kit pathways, suppressing the jak transcription, activating mapk8/jnk signaling and modulating the heat shock responses. our results identified snps on chromosome associated with triptolide ic. our results from functional studies in panc- cell lines further demonstrate potential role of cflar in triptolide toxicity. this region included biologically interesting genes as cflar, ppil caspase 8/ nfkb and stat identification of a splicing-snp rs which regulates cflar alternatively spliced isoforms predictive of the triptolide cytotoxicity suggests its role in triptolides action. analysis of gene-expression with cytotoxicity identified jak expression to be a significant predictor of triptolide sensitivity.
 triptolide is a therapeutic diterpenoid derived from the chinese herb tripterygium wilfordii hook f. triptolide has been shown to induce apoptosis by activation of pro-apoptotic proteins, inhibiting nfkb and c-kit pathways, suppressing the jak transcription, activating mapk8/jnk signaling and modulating the heat shock responses.  in the present study, we used lymphoblast cell lines derived from unrelated caucasian subjects to identify genetic markers predictive of cellular sensitivity to triptolide using genome wide association study. our results identified snps on chromosome associated with triptolide ic. this region included biologically interesting genes as cflar, ppil caspase 8/ nfkb and stat identification of a splicing-snp rs which regulates cflar alternatively spliced isoforms predictive of the triptolide cytotoxicity suggests its role in triptolides action. our results from functional studies in panc- cell lines further demonstrate potential role of cflar in triptolide toxicity. analysis of gene-expression with cytotoxicity identified jak expression to be a significant predictor of triptolide sensitivity.  overall out results identified genetic factors associated with triptolide chemo-sensitivity thereby opening up opportunities to better understand its mechanism of action as well as utilize these biomarkers to predict therapeutic response in patients. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords triptolidegenome-wide association studieshap-mapsingle nucleotide polymorphismscflarissue-copyright-statement© the author 2015 