Article Figures & Data
Figures
- Figure 1.
Suppression of ICMT inhibits proliferation and induces apoptosis and autophagy in multiple pancreatic cancer cells. A, Top, Cell viability curve of MiaPaCa2, AsPC-1, PANC-1, BxPC-3, PANC-10.05, CAPAN-2, and HPAF-II pancreatic cancer cells treated with cysmethynil at concentrations range from 10 to 40 μmol/L with increments of 2.5 μmol/L for 48 hours. Bottom, Immunoblot study on MiaPaCa2 cell lysates for prelamin A and loading control GADPH. The calculated ratios of prelamin A and GAPDH are shown. B, Immunoblot analysis on lysates of MiaPaCa2, AsPC-1, CAPAN-2, and HPAF-II cells prepared after 48 hours of treatment with 0, 20, 22.5, and 25 μmol/L cysmethynil. C, Flow cytometric quantification of apoptotic (sub-G0) population of MiaPaCa2 cells after 24-hour cysmethynil treatment at 0 or 22.5 μmol/L. D, Immunoblot analysis of the lysates of MiaPaCa2 and HPAF-II cells expressing either control shRNA or that targeting ICMT, 96 hours after infection by shRNA expressing lentiviruses (top). The ICMT knockdown efficiency is assessed by qPCR analysis (bottom). E, Colony formation assay of MiaPaCa2 cells treated with the indicated concentration of cysmethynil for 14 days. F, Colony formation assay of MiaPaCa2 cells expressing either control shRNA or that targeting ICMT; qPCR analysis of ICMT expression levels are presented on the right. G, Growth of MiaPaCa2 xenograft tumors under every other day treatment by vehicle, 100 or 150 mg/kg cysmethynil. n = 6 for each group. ***, P < 0.001 between different groups. H, Image to demonstrate the contralateral growth of xenograft MiaPaCa2 tumors derived from cells expressing either control shRNA (right side of mouse, pointed by black arrow) or that targeting ICMT (left side of mouse, pointed by red arrow). I, Analysis of xenograft data on tumors expressing control shRNA or that targeting ICMT. n = 3 for each dosing group. ***, P < 0.001 between treatment groups and control. The qPCR analysis of ICMT expression for the 2 groups of cells used for the study was done before implantation and presented on the right side of I. A–F, Data shown are from a single experiment that has been repeated 3 times with similar results.
- Figure 2.
p21, induced by suppression of ICMT, promotes apoptosis of MiaPaCa2 cells in vitro and inhibits tumor formation in vivo. A, p21 levels were assessed in parental MiaPaCa2 cells (EV) and 2 derived clones expressing shRNA targeting p21 (sh-p21 B and Q), after 48 hours of treatment with concentrations of cysmethynil of 0, 17.5, 20, and 22.5 μmol/L. B, Flow cytometric analysis of parental and p21 shRNA expressing MiaPaCa2 cells, after 48-hour treatment with vehicle or 22.5 μmol/L of cysmethynil. C, Viability of MiaPaCa2 parental and p21-knockdown clones after 48-hour treatment of cysmethynil at concentrations of 0, 17.5, 20, and 22.5 μmol/L. D, Soft-agar colony formation was evaluated for parental MiaPaCa2 and a stable p21-knockdown clone (sh-p21), under the treatment by vehicle or 20 μmol/L cysmethynil for 14 days. Samples from technical repeats were analyzed and the data are presented on the right of D. E and F, In vivo efficacy study of cysmethynil treatment in xenograft tumor model of parental MiaPaCa2 cells (E) and stable p21-knockdown cells (sh-p21; F). Animals were dosed with vehicle or cysmethynil at 150 mg/kg every other day. ***, P < 0.001. A–D, Data shown are from a single experiment that has been repeated 3 times with similar results.
- Figure 3.
ICMT inhibition–induced p21 promotes transcription of autophagy and apoptosis genes. All panels show qPCR expression analysis of the indicated genes. RNA samples were prepared from (A) MiaPaCa2 cells after 48-hour treatment with 0, 20, or 22.5 μmol/L cysmethynil, (B) MiaPaCa2 cells 96 hours after infection with lentivirus expressing either control shRNA or that targeting ICMT, (C) xenograft tumor samples obtained from mice treated every other day with either vehicle or cysmethynil at 100 and 150 mg/kg, respectively, (D) xenograft tumor samples derived from MiaPaCa2 cells expressing control shRNA or that targeting ICMT, and (E) MiaPaCa2 cells selected to express either control shRNA (−) or shRNA targeting p21 (+) after 48-hour treatment with cysmethynil at 0, 17.5, 20, or 22.5 μmol/L. All studies have been repeated 3 times with similar results.
- Figure 4.
BNIP3-stimulated apoptosis, but not autophagy, promotes cell death resulted from ICMT inhibition. A, qPCR analysis of BNIP3 expression in MiaPaCa2 cells expressing either control shRNA (−) or shRNA targeting BNIP3 (+) after 48-hour treatment with 0, 20, or 22.5 μmol/L cysmethynil. B, Flow cytometric analysis of MiaPaCa2 cells with or without BNIP3 knockdown after 48-hour treatment with either vehicle or 22.5 μmol/L cysmethynil. C, Immunoblot analysis of the autophagy and apoptosis markers on cell lysates from (A). D, Immunoblot analysis of autophagy and apoptosis markers on MiaPaCa2 cells treated with cysmethynil at 0, 20, and 22.5 μmol/L, with or without Atg5 knockdown as indicated. E, Viability of MiaPaCa2 cells treated with 0, 17.5, 20, and 22.5 μmol/L cysmethynil for 48 hours, with or without Atg5 knockdown as indicated. F, Flow cytometric analysis of MiaPaCa2 cells treated with vehicle or 22.5 μmol/L cysmethynil for 48 hours, with or without of Atg5 knockdown as indicated. For all studies, data shown are from a single experiment that has been repeated 3 times with similar results.
- Figure 5.
Suppression of ICMT inhibits cell-cycle progression and induces metabolic stress and cell death in sensitive pancreatic cancer cells. A, qPCR analysis of BNIP3 and p21 mRNA levels in MiaPaCa2 (top) and HPAF-II (bottom) cells after 48-hour treatment with DMSO control or 22.5 μmol/L cysmethynil. B and C, Immunoblot analysis of the indicated proteins in MiaPaCa2 and HPAF-II cells following 48-hour treatment with DMSO control (−) or 22.5 μmol/L (+) of cysmethynil (B), or MiaPaCa2 cells 96 hours after infection with lentivirus-expressing either control shRNA (−) or that targeting ICMT (+) (C).
- Figure 6.
Metabolic stress–induced p21 elevation inhibits cell proliferation and promotes autophagy and apoptosis. A and B, Cell respiration analysis of MiaPaCa2 (A) and HPAF-II (B) cells for intact cell, basal mitochondrial, and maximum mitochondrial OCR (O2 consumption) and non-mitochondrial oxygen consumption (ROX) following 24 hours of treatment with 0 μmol/L (DMSO), 20 μmol/L (low-dose cysm), or 22.5 μmol/L (high-dose cysm) cysmethynil. C, Immunoblot analysis of the indicated proteins in MiaPaCa2 cells after growth in normal (+) or glucose-deprived (−) media. D, qPCR analysis of mRNA levels of BNIP3 and p21 in the cells from (C). A–D, Data shown are from a single experiment that has been repeated 3 times with similar results. E, Schematic model summarizing the findings that energy depletion, induced by ICMT inhibition or nutrient deprivation, leads to transcriptional activation of p21 and p21-dependent inhibition of cell proliferation and induction of ULK1, LC3, and BNIP3, which results in further induction of autophagy and apoptosis in susceptible pancreatic cancer cells.
Additional Files
Supplementary Data
- Supplementary Data - Supplementary Fig. S1. ICMT inhibitor treatment leads to reduced viability in sensitive pancreatic cancer cell lines. (A) MTS viability assay that measures the total viable cells (OD measured at 490 nm) at the time of initiation (0 hr) and end (48 hr) of cysmethynil treatment for multiple pancreatic cell lines. (B) IC50 of different cell lines in response to cysmethynil treatment, determined by MTS assay shown in (A). Supplementary Fig. S2. ICMT inhibitor induces p21 and LC3 expression independent of p53. (A) Immunoblot analysis of the indicated proteins in Miapaca2 cells after treatment with 0, 20 or 22.5 μM cysmethynil for 48 h. (B) Immunoblot analysis of the indicated proteins in Miapaca2 cells with (si�p53) or without (si�control) p53 knockdown, and concurrent treatment with 0, 20, 22 and 24 μM Cysmethynil. Supplementary Fig. S3. Forced expression of BCL�XL rescues MiaPaca2 cells from apoptotic cell death induced by ICMT inhibition. (A) Light microscopy analysis of MiaPaca2 cells infected with either control retrovirus or that expressing BCL�XL, with (+) or without (�) treatment with 22.5 μM Cysmethynil for 48 h. (B) Flow cytometry assessment of the cell preparations from (A). Supplementary Fig. S4. Immunoblot study on the cell lysates prepared from AsPC1 and BxPC3 cells after 48 h treatment by either DMSO control or 22.5 μM of cysmethynil.
Volume 16, Issue 5
