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SCIENCE CHINA Life Sciences, Volume 64 , Issue 8 : 1295-1310(2021) https://doi.org/10.1007/s11427-019-1753-3

Sodium cantharidinate, a novel anti-pancreatic cancer agent that activates functional p53

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  • ReceivedNov 7, 2019
  • AcceptedJun 9, 2020
  • PublishedNov 3, 2020

Abstract


Funded by

the National Key R&D Program of China(2019YFC1711000)

the National Natural Science Foundation of China(81772566,to,J.L.)

and in part by the Project Program of State Key Laboratory of Natural Medicines

China Pharmaceutical University(SKLNMZZCX201820,to,X.X.)

and the “Double First-Class” University Project(CPU2018GF04,to,X.X.)


Acknowledgment

This work was supported by the National Key R&D Program of China (2019YFC1711000 to P.L.), the National Natural Science Foundation of China (81772566 to J.L.), and in part by the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMZZCX201820 to X.X.), and the “Double First-Class” University Project (CPU2018GF04 to X.X.).


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

The supporting information is available online at https://doi.org/10.1007/s11427-019-1753-3. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    SC inhibited the cell viability and induced apoptosis and DNA-damage response of pancreatic cancer cells. A, Structure of SC. B, Pancreatic cancer cells were treated with vehicle or various concentrations of SC (0.3–100 μmol L–1), and after 72 h incubation MTT assay was performed. SC inhibited the viability of TP53 mutant pancreatic cancer cells, including PANC-1, AsPC-1, SW1990 and BXPC-3. C, Quantification of apoptosis cell ratio of SW1990 cells upon SC treatment (Annexin V+/PI as early apoptosis and Annexin V+/PI+ as late apoptosis). D, SW1990 cells were treated with SC (10, 20 and 40 μmol L–1) and stained with AnnexinV and PI, and cells were analyzed by using flow cytometry. E, Representative images of γ-H2AX immunofluorescence staining (400×). Insets (foci staining) were 64× magnified. Blue, DAPI staining of DNA; red, γ-H2AX. F, Representative images of comet assay. G, Quantification of tail moment after performing a comet assay.*P<0.05, **P<0.01, ***P<0.001.

  • Figure 2

    Proteome-wide sequencing analysis detected perturbation of p53 signaling pathway on PANC-1 cells by SC. A, Proteomic workflow for identifying upstream regulators of SC treatment. PANC-1 cells were incubated with control vehicle or SC for LC-MS (label free quantitation) and IPA analyses. B, Upstream regulator analysis of proteomic data under control and SC conditions. C, The top canonical pathways and representative molecules involved in the pathway induced by SC. D, HCT116 p53-WT cells and HCT116 p53-KO cells were treated with vehicle or various concentrations of SC (0.3–100 μmol L–1), and after 72 h incubation MTT assay was performed.

  • Figure 3

    SC modulated p53 signaling pathway on SW1990 cells in a dose-dependent manner. SW1990 cells were treated with SC (10, 20, and 40 μmol L–1), and total protein lysate was evaluated by Western blotting to detect the caspase-9, cleaved-caspase 9, caspase-3, cleaved-caspase-3, PARP, cleaved-PARP, BAX, Bcl-2, cytochrome-c protein expressions, and the phosphorylation levels of MDM2 and p53. Representative bands are shown in A, B, E, F, I, J, M, and N. Relative levels of phosphorylated MDM2 (O), cleaved caspase-3 (C), cleaved-caspase-9 (D), cleaved-PARP (G), BAX (H), Bcl-2 (K), and cytochrome-c (P, Q) normalized to those of β-actin. Relative levels of phosphorylated p53 normalized to those of total p53 and quantification of p53/β-actin (L). R, Cells were treated with vehicle or SC (20 μmol L–1) for 8 h, followed by treatment with 50 μmol L–1 cycloheximide (CHX) for the indicated periods of time. The levels of p53 expression were determined by Western blotting. S, Quantification of p53/β-actin ratio after normalization with 0 h data (without CHX). The results are presented as mean±SD (n=3). *P<0.05, **P<0.01 vs. the control group.

  • Figure 4

    SC caused the WT p53 activation, thus inducing the apoptosis of SW1990 cells. A–D, The mRNA expressions of MDM2, BAX, PMAIP1 (NOXA), and BBC3 (PUMA) after treatment of SW1990 cells with SC (10, 20 and 40 μmol L–1). MDM2, BAX, PMAIP1, and BBC3 mRNA expression levels were detected by RT-PCR and normalized to those of control GAPDH. E–J, The caspase-dependent apoptosis effect of SC was strongly abrogated by the TP53-targeting shRNA. Knockdown of total p53 level was obtained by silencing gene expression with a lentiviral shRNA construct targeting TP53 in SW1990 cells, and then the cells were treated with SC (20 µmol L–1). The representative bands of cleaved-caspase-3 and phosphorylated p53 are shown in G and E. The levels of phosphorylated p53 (F) and cleaved-caspase-3 (H) were normalized to those of total p53 and total caspase-3. I, The apoptosis was evaluated by Annexin V-FITC/PI staining. J, Quantification of apoptosis cell ratio (Annexin V+/PI as early apoptosis and Annexin V+/PI+ as late apoptosis). The results are presented as mean±SD (n=3). **P<0.01.

  • Figure 5

    SC suppressed PANC-1 cell proliferation via JAK2/STAT3 pathway. PANC-1 cells were treated with SC (10, 20, and 40 μmol L–1), and total protein lysate was evaluated by Western blotting to detect the levels of phosphorylated JAK2 and STAT3. Western blotting bands are shown in A. Relative expression of phosphorylated JAK2 (B) and phosphorylated STAT3 (C) were normalized to that of total JAK2 and STAT3 proteins. The reduction of activation of JAK2-STAT3 by SC was strongly abrogated by the TP53-targeting shRNA. Total p53 levels were knocked down with a lentiviral shRNA construct, which targeted TP53 in PANC-1 cells, followed by treatment with SC (20 µmol L–1). The representative bands showed the levels of phosphorylated JAK2 and STAT3 (D). Relative expression of phosphorylated JAK2 (E) and phosphorylated STAT3 (F) were normalized to that of total JAK2 and STAT3 protein.

  • Figure 6

    SC alone or in combination with gemcitabine reduced the tumor growth of PANC-1 xenografts. A and B, SC and gemcitabine treatments were detected by MTT, and the IC50 of growth inhibition of PANC-1 cells was calculated. C, Representative photos of excised tumors after 21 days of treatment. D, Weights of excised tumors in different groups after treatment. The results are presented as mean±SD (n=6). E and F, The excised tumor volumes and body weights of four different groups throughout the treatment. G, Representative images of H&E staining and Ki-67 immunohistochemical staining of excised tumor (200×). H, Quantification of Ki-67 positive cells was done by image J software.

  • Figure 7

    Combination of SC and GEM enhanced the p53-activating effect. A–D, SC and GEM synergistically upregulated cleaved-caspase-3 expression and p53 phosphorylation levels in human primary pancreatic cancer cells. E–I, MDM2, TP53, BAX, BBC3, and PMAIP1 mRNA expression levels of primary cells were detected by RT-PCR and normalized to those of the control. The results are presented as mean±SD (n=3). *P<0.05, **P<0.01. J–L, SC (10 µmol L–1) and GEM (10 µmol L–1) synergistically downregulated the activation of JAK2/STAT3 in PANC-1 cells. The representative bands of phosphorylated JAK2 and STAT3 proteins are shown in J. Relative expression levels of phosphorylated JAK2 (K) and phosphorylated STAT3 (L) were normalized to those of total JAK2 and STAT3 protein.

  • Figure 8

    Interactions of the WT p53 protein or R273H mutant p53 protein with SC. A, The two-dimensional and three-dimensional ligand–receptor interaction diagram of SC and DNA-contact WT p53 (PBDID: 1tup). B, The two-dimensional and three-dimensional ligand–receptor interaction diagram of SC and R273H p53 (PBDID: 2BIM). C, Schematic model of the hypothesized molecular mechanism underlying the anti-pancreatic cancer activity of SC. SC induced p53-mediated apoptosis of pancreatic cancer cells and activated the p53 function to inhibit JAK2-STAT3 signaling. SC has different interaction models with DNA-binding pockets of WT and mutant p53 protein in nuclei.

  • Table 1   The IC50 (μmol L–1) of sodium cantharidinate in different cell lines after 72 h incubation

    Cancer types

    Cell lines

    IC50 (μmol L–1)

    Breast cancer

    MCF-7

    23.22±2.23

    MDA-MB-231

    32.07±2.19

    MDA-MB-468

    37.33±2.67

    MDA-MB-453

    42.39±3.07

    Lung cancer

    95D

    82.22±6.24

    NCI-H460

    49.92±3.67

    A549

    8.465±2.56

    NCI-H23

    27.08±1.63

    NCI-H520

    15.46±1.09

    Gastric cancer

    SGC7901

    29.18±1.84

    NCI-N87

    27.33±1.69

    Pancreatic cancer

    PANC-1

    36.71±2.33

    AsPC-1

    29.25±2.07

    SW1990

    12.62 ± 2.69

    BXPC-3

    21.79±1.24

    Prostate cancer

    DU145

    78.8±4.21

    PC-3

    77.1±3.29

    Ovarian cancer

    OVCAR-3

    4.45±0.25

    OV90

    32.09±2.19

    A2780

    51.35±2.79

    OVCAR8

    21.77±1.16

    Cervical cancer

    HeLa

    18.46±1.67

    HeLa 229

    3.57±0.19

    C-33A

    47.60±2.19

    Liver cancer

    HepG2

    24.44±1.34

    Bel7402

    28.55±2.05

    SMMC-7721

    29.34±1.97

    Endometrial cancer

    ECC-1

    24.01±1.27

    AN3CA

    17.34±1.12

    Ishikawa

    19.65±1.59

    Colorectal cancer

    Lovo

    25.37±1.67

    Caco-2

    27.67±2.64

    Leukemia

    K-562

    18.70±1.67

    Glioblastoma

    U87

    47.24±2.19

    U251

    51.34±3.18

    Vascular endothelial

    HUVEC

    82.26±6.67

    Liver

    HL-7702

    80.69±5.39

    Kidney

    HEK293

    91.81±7.68

  • Table 2   The IC50 (μmol L–1) of gemcitabine and sodium cantharidinate after 72 h incubation

    Cell lines

    IC50 (μmol L–1)

    Sodium cantharidinate

    Gemcitabine

    Human primary pancreatic cancer cells

    20.63±2.26

    13.90±1.35

    Human primary paracancerous cells

    85.47±5.32

    221.00±6.39

  • Table 3   CI values at various ratios of concentration of sodium cantharidinate and gemcitabine

    Cell lines

    SC/GEM

    IC50 (μmol L–1)

    CI

    Human primary pancreatic cancer cells

    2:1

    15.28±1.04

    0.860

    1:1

    10.26±0.93

    0.618

    1:2

    8.52±0.51

    0.480

  • Table 4   Cell death induced by the combination therapy of sodium cantharidinate and gemcitabine

    Cell death (%)

    GEM (μmol L–1)

    Sodium cantharidinate (μmol L–1)

    0

    5

    10

    15

    0

    0

    25.95±12.02

    46.27±2.61

    61.65±6.22

    0.1

    27.61±1.06

    47.47±2.53

    56.31±9.39

    68.21±2.87

    1.0

    29.15±2.26

    50.46±5.72

    49.15±14.12

    64.18±5.42

    10

    38.11±1.27

    64.97±3.14

    66.86±9.33

    80.84±3.55

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