A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer

  1. Qiaomu Tian
  2. Peng Zhang
  3. Yihan Wang
  4. Youhui Si
  5. Dengping Yin
  6. Christopher R Weber
  7. Melissa L Fishel
  8. Karen E Pollok
  9. Bo Qiu
  10. Fei Xiao
  11. Anita S Chong  Is a corresponding author
  1. Department of Surgery, The University of Chicago, United States
  2. Cinkate Pharmaceutical Corp, ZhangJiang District, China
  3. Department of Pathology, The University of Chicago, United States
  4. Department of Pediatrics, Indiana University, United States
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
CK21 exhibits a stable release of triptolide in vivo.

(a) Synthesis of compound CK21 as white solid after recrystallization in a mixed organic solvent. Compound structure was characterized by H-NMR, C-NMR, and HR-MS. (b) Thermal ellipsoid model …

Figure 1—source data 1

Safety profile of CK21.

Acute maximum tolerated dose (MTD) studies, toxicity and toxicokinetic studies on rats and beagle dogs.

https://cdn.elifesciences.org/articles/85862/elife-85862-fig1-data1-v2.pptx
Figure 1—source data 2

IC50 (µM) of triptolide (TP) or CK21 for different cancer cell lines in an in vitro cell viability assay.

https://cdn.elifesciences.org/articles/85862/elife-85862-fig1-data2-v2.pptx
Figure 1—figure supplement 1
In vitro viability assay of primary human fibroblasts cocultured with CK21 at the indicated concentrations for 72 hours.
Figure 2 with 4 supplements
CK21 shows efficacy and minimal toxicity at 3 mg/kg in different in vivo animal models.

(a) Scheme of in vivo efficacy studies. Human pancreatic cancer cell line, AsPc-1, was implanted into nude mice and CK21 treatment was initiated ~14 days later and administered daily for 4 weeks. (b)…

Figure 2—figure supplement 1
Survival curve of mice receiving CK21 at 5 mg/kg or triptolide (TP) at 0.25 mg/kg.
Figure 2—figure supplement 2
AsPC-1 subcutaneous tumors showed no tumor relapse after treated with CK21 at 5 or 3 mg/kg.
Figure 2—figure supplement 3
CK21 inhibited growth of Panc-1 tumors in a subcutaneous xenograft model.

(a) Tumor growth with CK21 treatment at 3 mg/kg daily for 28 days. (b) Weight change of mice during Ck21 treatment.

Figure 2—figure supplement 4
Male mice with AsPC-1 tumors respond to CK21.

(a) Subcutaneous AsPC-1 tumor in male mice after CK21 treatment at 1.5 mg/kg. (b) Male mice weight during CK21 treatment. (N=5 for each experimental group).

CK21 of 3 mg/kg daily shows efficacy in delay therapy and rescues mice that failed in synergistic therapy.

(a) Scheme of delayed therapy. Mice received CK21 at 3 mg/kg daily for 4 weeks, starting on day 42 post-tumor innoculation. (b) Tumor volume during delayed CK21 therapy. (c) Scheme of combination …

Figure 4 with 5 supplements
Transcriptome analysis of patient-derived pancreatic tumor organoids and AsPC-1 after CK21 treatment.

(a) In vitro culture of different organoids with escalating concentrations of CK21 for 72 hours. Gemcitabine was included as a positive control. (b) U049MAI tumor size in nude mice during CK21 …

Figure 4—source data 1

Essential information on the pancreatic tumor organoids used in this study.

Details of organoids from Patient# 1, 2, 6, and 7 are provided in reference 39 (Romero-Calvo et al., 2019).

https://cdn.elifesciences.org/articles/85862/elife-85862-fig4-data1-v2.pptx
Figure 4—figure supplement 1
Volcano plots highlighting differentially expressed genes by U049MAI and U123m15-T respectively after 3 hours, 6 hours, 9 hours, and 12 hours of CK21 (50 nM) treatment.
Figure 4—figure supplement 2
DDIT4 expression was significantly reduced after CK21 treatment.

(a) CK21 (50 mM) reduced the expression of DDIT4 in AsPC-1, Panc-1, U049MAI, and U123M15-T after 24 hoursr of culture. (b) Baseline expression of DDIT4 in different tumor cells (without CK21 …

Figure 4—figure supplement 3
DDIT4 knockdown or overexpression didn’t impact the sensitivty of tumor cell lines to CK21 treatment.

(a) knockdown of DDIT4 in Panc-1 did not alter response to CK21 (50 nM). (b) AsPC-1 overexpression of DDIT4 did not alter response to CK21 (50 nM).

Figure 4—figure supplement 4
Tumor size of KC-6141 and KPC-961 after subcutaneous implantation in B6 or B6 X129.

CK21 given at 4 mg/kg/day for KC-6141 and 3 mg/kg/day for KPC-961 resulted in modest inhibition of tumor growth.

Figure 4—figure supplement 5
RT-qPCR analysis of differentially expressed genes by two mice pancreatic tumor cell lines after CK21 treatment at 50 nM for 24 hours.
Figure 5 with 2 supplements
Bioinformatic analysis of the effect of CK21 on patient-derived pancreatic tumor organoids.

(a) Volcano plots of differentially expressed genes in (a) U049MAI and (b) U123M15-T after 12 hours CK21 treatment (50 nM). Significance cutoff was s P<0.05. Upregulation was colored as red, and …

Figure 5—figure supplement 1
Pathway enrichment of U049MAI and U123M15-T after treatment with CK21 (50 nM) for 9 hours.

Top pathways for (a) U049MAI and (b) U123M15-T.

Figure 5—figure supplement 2
Pathway enrichment of orthotopic AsPC-1 tumors after treatment with CK21 (3 mg/kg) for 3 days.
Figure 6 with 2 supplements
CK21 inhibits NF-κB activation and induces mitochondrial mediated apoptosis.

(a) Heatmap of the relative expression of genes in the NF-κB pathway in U049MAI and U123M15-T after CK21 treatment. Genes are color coded where red means upregulated, and blue means downregulated. …

Figure 6—figure supplement 1
Flow plots illustrating active Caspase 3/7 expression in AsPC-1 and Panc-1 treated with CK21 (50 and 400 mM) for 24 hoursr.
Figure 6—figure supplement 2
Key regulators in NF-kB canonical signaling pathway are significantly downregulated in (a) U049MAI and (b) U123M15-T after treatment with CK21 (50 nM) for 12 hoursr.

Green represents downregulation and red represent upregulation by IPA analysis.

Figure 7 with 1 supplement
CK21 does not exhibit significant immunosuppression in a spontaneous tumor rejection model.

(a) Scheme of a subcutaneous model of mouse pancreatic tumor, KPC-960, with CK21 treatment. CK21 was provided at 3 mg/kg daily starting on day 5 or day 7. During secondary challenge, CK21 was …

Figure 7—figure supplement 1
Tumor size of KPC-960 after subcutaneous implantation in nude mice.

CK21 given at 3 mg/kg/day resulted in limited efficacy on tumor growth. Each line represents an animal.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compound, drugCK21In houseNA
Chemical compound, drugGemcitabineActavis45963-619-59
Cell line (Homo-sapiens)AsPC-1ATCCCRL-1682
Cell line (Homo-sapiens)Luciferase transfected AsPC-1Indiana UniversityN/ALuciferase transfected
Cell line (Homo-sapiens)Panc-1ATCCCRL-1469
Cell line (Mus)KC-6141University of NebraskaN/A
Cell line (Mus)KPC-960University of NebraskaN/A
Cell line (Mus)KPC-961University of NebraskaN/A
Biological sample (Mus)B6129SF1/JJackson Laboratory101043
Biological sample (Mus)C57BL/6 JJackson Laboratory000664
Biological sample (Mus)Athymic Nude-Foxn1nuEnvigo
Commercial assay or kitDMEMATCC30–2002
Commercial assay or kitRPMIQuality Biological112-024-101
Commercial assay or kitFetal bovine serumAtlanta BiologicalsS115OH
Commercial assay or kitPenicillin streptomycinGibco15140–122
Commercial assay or kitL-GlutamineGibco25030–081
Commercial assay or kitDMSOSigma276855
Commercial assay or kitTrypsin-EDTAStemcell07901
Commercial assay or kitTrypLE expressGibco12605–010
Commercial assay or kitSodium pyruvateGibco11360–070
Commercial assay or kitMEM nonessential amino acidsCellgro25–025 CL
Commercial assay or kit2-MercaptoethanolGibco21985–023
Commercial assay or kitIntestiCult organoid growth mediumStemcell6005
Commercial assay or kitA83-01SigmaSML0788
Commercial assay or kitFGF-10SigmaSRP3262
Commercial assay or kitGastrin ISigmaG9145
Commercial assay or kitN-acetylcysteineSigmaA9165
Commercial assay or kitNicotinamideSigmaN0636
Commercial assay or kitB27 supplementGibco17504–044
Commercial assay or kitPrimocineInvivogenant-pm-1
Commercial assay or kitY-27632Tocris1254
Commercial assay or kitMatrigelCorning356231
Commercial assay or kitTrypLEGibco12605–010
Commercial assay or kitCellTiter 96 AQueous one solutionPromegaG3580
Commercial assay or kitCaspase-3/7 green detectionThermo FisherC10427
Commercial assay or kitSYTOX dead cell stainThermo FisherC10427
Commercial assay or kitCFSE cell proliferation kitThermo FisherC34554
Commercial assay or kitACK lysing bufferQuality Biological118-156-101
Commercial assay or kitROS-Glo H2O2 assayPromegaG8820
Commercial assay or kitNuPAGE 10% Bis-Tris gelInvitrogenNP0301BOX
Commercial assay or kitNuPAGE MES SDS running bufferNovexNP002
Commercial assay or kitNuPAGE MOPS SDS running bufferNovexNP001
Commercial assay or kitNuPAGE transfer bufferNovexNP0006-1
Commercial assay or kitNuPAGE LDS sample reducing agentInvitrogenNP0007
Commercial assay or kitNuPAGE sample bufferInvitrogenNP0009
Commercial assay or kitNuPAGE antioxidantInvitrogenNP0005
Commercial assay or kitTBS Tween-20 bufferThermo Scientific28360
Commercial assay or kitInvitrolon PVDF filter paperNovexLC2005
Commercial assay or kitPageRuler prestained protein ladderThermo Scientific26616
Commercial assay or kitMethanolFisher ScientificA452-4
Commercial assay or kitPierce protease&phosphatase inhibitorThermo ScientificA32959
Commercial assay or kitBovine serum albuminSigmaA7906
Commercial assay or kitSuperSignal west pico PLUSThermo Scientific34579
Commercial assay or kitPierce bradford assay kitThermo Scientific23246
AntibodyAnti-beta actin (Rabbit polyclonal)Abcamab8227(1:2000)
AntibodyRecombinant anti-REDD-1/DDIT4 (Rabbit monoclonal)Abcamab191871(1:1000)
AntibodyAnti-Caspase-3 (Rabbit polyclonal)Abcamab13847(1:500)
AntibodyRecombinant anti- BCL2 (Rabbit monoclonal)Abcamab182858(1:2000)
AntibodyGoat anti-rabbit IgG H&L (Goat polyclonal)Abcamab205718(1:10000)
AntibodyPhospho-NFkB p65, PE, eBioscience(Mouse monoclonal)Invitrogen12986342(1:100)
Commercial assay or kit4',6-Diamidino-2-Phenylindole, DilactateBiolegend422801(1:1000)
Commercial assay or kitPowerUp SYBR green master mixApplied BiosystemA25742
Commercial assay or kitHigh capacity cDNA reverse transcriptionApplied Biosystem4368814
Commercial assay or kitD-Luciferin potassium saltPerkin Elmer122799
Commercial assay or kitPBSGenClone25–508
Commercial assay or kitCell recovery solutionCorning354253
Commercial assay or kitRNeasy Plus Mini KitQiangen74124
Commercial assay or kitDNase I recombinantRoche04536282001

Additional files

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