A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer
- Department of Surgery, The University of Chicago, United States
- Cinkate Pharmaceutical Corp, ZhangJiang District, China
- Department of Pathology, The University of Chicago, United States
- Department of Pediatrics, Indiana University, United States
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 …
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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
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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).
Figure 3
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 …
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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. …
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Figure 6—source data 1
Full unedited gels.
- https://cdn.elifesciences.org/articles/85862/elife-85862-fig6-data1-v2.zip
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 resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | CK21 | In house | NA | |
| Chemical compound, drug | Gemcitabine | Actavis | 45963-619-59 | |
| Cell line (Homo-sapiens) | AsPC-1 | ATCC | CRL-1682 | |
| Cell line (Homo-sapiens) | Luciferase transfected AsPC-1 | Indiana University | N/A | Luciferase transfected |
| Cell line (Homo-sapiens) | Panc-1 | ATCC | CRL-1469 | |
| Cell line (Mus) | KC-6141 | University of Nebraska | N/A | |
| Cell line (Mus) | KPC-960 | University of Nebraska | N/A | |
| Cell line (Mus) | KPC-961 | University of Nebraska | N/A | |
| Biological sample (Mus) | B6129SF1/J | Jackson Laboratory | 101043 | |
| Biological sample (Mus) | C57BL/6 J | Jackson Laboratory | 000664 | |
| Biological sample (Mus) | Athymic Nude-Foxn1nu | Envigo | ||
| Commercial assay or kit | DMEM | ATCC | 30–2002 | |
| Commercial assay or kit | RPMI | Quality Biological | 112-024-101 | |
| Commercial assay or kit | Fetal bovine serum | Atlanta Biologicals | S115OH | |
| Commercial assay or kit | Penicillin streptomycin | Gibco | 15140–122 | |
| Commercial assay or kit | L-Glutamine | Gibco | 25030–081 | |
| Commercial assay or kit | DMSO | Sigma | 276855 | |
| Commercial assay or kit | Trypsin-EDTA | Stemcell | 07901 | |
| Commercial assay or kit | TrypLE express | Gibco | 12605–010 | |
| Commercial assay or kit | Sodium pyruvate | Gibco | 11360–070 | |
| Commercial assay or kit | MEM nonessential amino acids | Cellgro | 25–025 CL | |
| Commercial assay or kit | 2-Mercaptoethanol | Gibco | 21985–023 | |
| Commercial assay or kit | IntestiCult organoid growth medium | Stemcell | 6005 | |
| Commercial assay or kit | A83-01 | Sigma | SML0788 | |
| Commercial assay or kit | FGF-10 | Sigma | SRP3262 | |
| Commercial assay or kit | Gastrin I | Sigma | G9145 | |
| Commercial assay or kit | N-acetylcysteine | Sigma | A9165 | |
| Commercial assay or kit | Nicotinamide | Sigma | N0636 | |
| Commercial assay or kit | B27 supplement | Gibco | 17504–044 | |
| Commercial assay or kit | Primocine | Invivogen | ant-pm-1 | |
| Commercial assay or kit | Y-27632 | Tocris | 1254 | |
| Commercial assay or kit | Matrigel | Corning | 356231 | |
| Commercial assay or kit | TrypLE | Gibco | 12605–010 | |
| Commercial assay or kit | CellTiter 96 AQueous one solution | Promega | G3580 | |
| Commercial assay or kit | Caspase-3/7 green detection | Thermo Fisher | C10427 | |
| Commercial assay or kit | SYTOX dead cell stain | Thermo Fisher | C10427 | |
| Commercial assay or kit | CFSE cell proliferation kit | Thermo Fisher | C34554 | |
| Commercial assay or kit | ACK lysing buffer | Quality Biological | 118-156-101 | |
| Commercial assay or kit | ROS-Glo H2O2 assay | Promega | G8820 | |
| Commercial assay or kit | NuPAGE 10% Bis-Tris gel | Invitrogen | NP0301BOX | |
| Commercial assay or kit | NuPAGE MES SDS running buffer | Novex | NP002 | |
| Commercial assay or kit | NuPAGE MOPS SDS running buffer | Novex | NP001 | |
| Commercial assay or kit | NuPAGE transfer buffer | Novex | NP0006-1 | |
| Commercial assay or kit | NuPAGE LDS sample reducing agent | Invitrogen | NP0007 | |
| Commercial assay or kit | NuPAGE sample buffer | Invitrogen | NP0009 | |
| Commercial assay or kit | NuPAGE antioxidant | Invitrogen | NP0005 | |
| Commercial assay or kit | TBS Tween-20 buffer | Thermo Scientific | 28360 | |
| Commercial assay or kit | Invitrolon PVDF filter paper | Novex | LC2005 | |
| Commercial assay or kit | PageRuler prestained protein ladder | Thermo Scientific | 26616 | |
| Commercial assay or kit | Methanol | Fisher Scientific | A452-4 | |
| Commercial assay or kit | Pierce protease&phosphatase inhibitor | Thermo Scientific | A32959 | |
| Commercial assay or kit | Bovine serum albumin | Sigma | A7906 | |
| Commercial assay or kit | SuperSignal west pico PLUS | Thermo Scientific | 34579 | |
| Commercial assay or kit | Pierce bradford assay kit | Thermo Scientific | 23246 | |
| Antibody | Anti-beta actin (Rabbit polyclonal) | Abcam | ab8227 | (1:2000) |
| Antibody | Recombinant anti-REDD-1/DDIT4 (Rabbit monoclonal) | Abcam | ab191871 | (1:1000) |
| Antibody | Anti-Caspase-3 (Rabbit polyclonal) | Abcam | ab13847 | (1:500) |
| Antibody | Recombinant anti- BCL2 (Rabbit monoclonal) | Abcam | ab182858 | (1:2000) |
| Antibody | Goat anti-rabbit IgG H&L (Goat polyclonal) | Abcam | ab205718 | (1:10000) |
| Antibody | Phospho-NFkB p65, PE, eBioscience(Mouse monoclonal) | Invitrogen | 12986342 | (1:100) |
| Commercial assay or kit | 4',6-Diamidino-2-Phenylindole, Dilactate | Biolegend | 422801 | (1:1000) |
| Commercial assay or kit | PowerUp SYBR green master mix | Applied Biosystem | A25742 | |
| Commercial assay or kit | High capacity cDNA reverse transcription | Applied Biosystem | 4368814 | |
| Commercial assay or kit | D-Luciferin potassium salt | Perkin Elmer | 122799 | |
| Commercial assay or kit | PBS | GenClone | 25–508 | |
| Commercial assay or kit | Cell recovery solution | Corning | 354253 | |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiangen | 74124 | |
| Commercial assay or kit | DNase I recombinant | Roche | 04536282001 |
