The authors have declared that no competing interests exist.
Pancreatic cancer is a fatal malignancy worldwide and urgently requires valid therapies. Previous research showed that the HDAC inhibitor chidamide is a promising anti-cancer agent in pancreatic cancer cell lines. In this study, we elucidate a probable underlying anti-cancer mechanism of chidamide involving the degradation of Mcl-1. Mcl-1 is frequently upregulated in human cancers, which has been demonstrated to participate in oxidative phosphorylation, in addition to its anti-apoptotic actions as a Bcl-2 family member. The pancreatic cancer cell lines BxPC-3 and PANC-1 were treated with chidamide, resulting in Mcl-1 degradation accompanied by induction of Mcl-1 ubiquitination. Treatment with MG132, a proteasome inhibitor reduced Mcl-1 degradation stimulated by chidamide. Chidamide decreased O2 consumption and ATP production to inhibit aerobic metabolism in both pancreatic cancer cell lines and primary cells, similar to knockdown of Mcl-1, while overexpression of Mcl-1 in pancreatic cancer cells could restore the aerobic metabolism inhibited by chidamide. Furthermore, chidamide treatment or Mcl-1 knockdown significantly induced cell growth arrest in pancreatic cancer cell lines and primary cells, and Mcl-1 overexpression could reduce this cell growth inhibition. In conclusion, our results suggest that chidamide promotes Mcl-1 degradation through the ubiquitin-proteasome pathway, suppressing the maintenance of mitochondrial aerobic respiration by Mcl-1, and resulting in inhibition of pancreatic cancer cell proliferation. Our work supports the claim that chidamide has therapeutic potential for pancreatic cancer treatment.
Pancreatic cancer, one of the most lethal cancers has an extremely poor prognosis due to its aggressive nature, the 5-year relative survival rate is less than 6%[
A recent study showed that the function of mitochondria in tumor cells was intact and that these mitochondria have no defects compared with normal cells. Mitochondrial oxygen metabolism is still an important source of ATP in tumor cells to maintain an energy supply for rapid growth[
Histone deacetylases (HDACs) modify the structure of histone and non-histone proteins through altering the acetylation status[
Chidamide was synthesized by Shenzhen Chipscreen Biosciences Ltd. (Shenzhen, China) and dissolved in dimethyl sulfoxide (DMSO). MG132 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibody anti-Mcl-1 was purchased from Abcam® (Cambridge, MA, USA), anti-Acetylated-Lysine and anti-β-actin were obtained from Cell Signaling Technology (Boston, MA, USA), and anti-ubiquitin was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA).
Human pancreatic cancer cell lines (BxPC-3 and PANC-1) were provided by Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Primary human pancreatic cancer cells (pdc0001 and pdc0015) were provided by the National Center for Nanoscale Science (Beijing, China). The cell lines were maintained at 37°C in 5% CO2 in DMEM supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 U/ml each) and NaHCO3 (2 g/L).
Cells were washed and lysed in RIPA lysis buffer, protein lysates concentrations were measured by BCA protein assay, and equal amounts of proteins were separated by 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk for 1 h, the PVDF membranes were probed with corresponding antibodies.
Total RNA was extracted with Trizol (Invitrogen, MA, USA) and reverse transcribed to cDNA using a GoScriptTM Reverse Transcription kit (Promega, WI, USA). qPCR for Mcl-1 was performed with primers specific for Mcl-1 (forward: 5’-CAGCGACGGCGTAACAAAC-3’, and reverse: 5’-ACAAACCCATCCCAGC- CTCTTT-3’) and β-actin (forward: 5’-CATCGAGCACGGCATVGTCA-3’, and reverse: 5’-TAGCACAGCCTGGATAGCAAC-3’). qPCR was carried out with a GoTaq® qPCR Master Mix kit (Promega, WI, USA).
Cells were lysed in RIPA buffer, and then centrifuged to collect the supernatant and the amount of protein was quantified. Equal amounts of protein were precipitated with primary antibody overnight at 4°C, and then protein A/G agarose beads were added at 4°C for 4 h. The immunoprecipitates were washed and collected for Western Blot analysis.
Mcl-1 siRNA (5’-CCAAGAAAGCUGCAUCGAAdTdT-3’) and non-silencing siRNA (5’-UUCUCCGAACGUGUCACGUTT-3’) were purchased from Sigma-Aldrich (St Louis, MO, USA). Cells were transfected with siRNA (final concentration of 60 nM) using Lipofectamine® RNAiMAX Reagent (Invitrogen, MA, USA). The efficiency of gene knockdown in cells was verified by real-time RT-PCR and Western blot after 48 h of transfection.
PANC-1 cells were seeded in six-well plates. Cells were transfected with 2 μg of a pCMV6-Entry expression plasmid encoding human Mcl-1 under the control of a CMV promoter by Lipofectamine® 3000 Reagent (Invitrogen, MA, USA). The efficiency of gene overexpression in cells was verified by real-time RT-PCR and Western blot after 48 h of transfection.
Cells (4×103 cells/well) were seeded in 96-well plates, and then incubated at 37°C with 5% CO2. To each well 100 μL CellTiter-Glo® reagent (Promega, WI, USA) was added and incubated with cells at room temperature for 10 min before measurement. The fluorescence signal was recorded by automatic biochemical analyzer.
Cells (9×103 cells/well) were seeded in 96-well seahorse microplate 24 h before measurement in normal DMEM medium. Cells were washed and pre-incubated in Seahorse Assay Medium for 1 h prior to analysis. Oxygen consumption rate (OCAR) was measured on a Seahorse XF96 analyzer(Seahorse Bioscience, MA, USA).
Cells (3×103cells/well) were seeded onto 96-well plates, MTT was added to a final concentration of 1mM for 4 h, and formazan crystals were solubilized by addition of 10%SDS in 10mM HCL. The optical density of each well was measured with a visible microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm.
All data were normalized to control values of each assay and were presented as the mean ± standard deviation (SD). Data were analyzed by a one-way ANOVA or t-tests by using GraphPad Prism version 6.0. Significance was chosen as
We firstly investigated the effect of chidamide on Mcl-1 expression in BxPC-3 and PANC-1 pancreatic cancer cell lines. BxPC-3 and PANC-1 cells were treated with different concentrations of chidamide (0–50 μM) for 24 h. We found that chidamide treatment decreased the protein level of Mcl-1 in BxPC-3 and PANC-1 cell lines, as measured by Western blot assay (
(A) Level of Mcl-1 protein in pancreatic cancer cells treated with chidamide for 24 h, determined by Western blot analysis. β-actin served as a loading control. (B) Transcript level of Mcl-1 in pancreatic cancer cells treated with chidamide for 24 h, determined by quantitative real-time PCR. T-tests, BxPC-3:
Recent research showed that the HDAC inhibitors (HDACi) promote metabolic protein PEPCK1 degradation through the ubiquitin-proteasome pathway[
(A) Acetylated level of Mcl-1 in BxPC-3 and PANC-1 cells treated with chidamide for 24 h, determined by Western blot analysis. (B) Ubiquitinated level of Mcl-1 in BxPC-3 and PANC-1 cells treated with chidamide for 24 h, determined by Western blot analysis. (C) Ubiquitinated level of Mcl-1 in BxPC-3 cells treated with TSA for 24 h, determined by Western blot analysis. (D) Level of Mcl-1 in BxPC-3 cells treated with TSA for 24 h, determined by Western blot analysis. β-actin served as a loading control. T-tests, **
The above studies demonstrated the degradation effects on Mcl-1 by chidamide in pancreatic cancer cell lines. In view of the Mcl-1 function in mitochondrial respiration, we next investigated the effects of chidamide on aerobic metabolism. Pancreatic cancer cells were treated with Mcl-1 siRNA or 50 μM chidamide for 24 h, and then the ATP production and O2 consumption were measured. CellTiter-Glo® assay results in
(A) ATP production in pancreatic cancer cells treated with 50 μM chidamide or Mcl-1 siRNA for 24 h, determined by CellTiter-Glo® assay. Data are shown as the means ± SD of three independent experiments. **
Finally, we studied the significance of aerobic respiration inhibition in cancer cell proliferation and investigated the anti-cancer effects of chidamide in pancreatic cancer cell lines. Cells were treated with Mcl-1 siRNA or different concentrations of chidamide (0–50 μM), and cell survival was evaluated by MTT assay. The MTT results showed that chidamide causes cell growth arrest in a dose- and time-dependent manner in BxPC-3 and PANC-1 cancer cell lines (
(A) Pancreatic cancer cells were treated with various concentrations of chidamide for indicated time, proliferation inhibition was determined by MTT assay. (B) Pancreatic cancer cells were treated with Mcl-1 siRNA for 48 h, proliferation inhibition of pancreatic cancer cells was determined by MTT assay. Data are shown as the means ± SD of three independent experiments. **
In this study, we demonstrate that Mcl-1 plays an important role in both metabolic and survival processes. In addition, the anti-cancer effect of chidamide in pancreatic cancer may relate to regulation of Mcl-1 degradation. Chidamide induces Mcl-1 degradation through the ubiquitin-proteasome pathway, and consequently inhibits the aerobic metabolism and proliferation of pancreatic cancer cells.
The Western blot assay indicates that chidamide potently promotes the degradation of Mcl-1 proteins via the ubiquitin-proteasome pathway. This result is consistent with previous research demonstrating that ubiquitin-proteasome mediated degradation is a common process regulating Mcl-1 expression. In addition, Mcl-1 can be ubiquitinated at K5, K40, K136, K194 and K197[
Next we investigated the function of Mcl-1 downregulation by chidamide in pancreatic cancer cells. Experimental results verified that chidamide dose- dependently decreased ATP production by decreasing O2 consumption, which was consistent with the effect of Mcl-1 gene silencing. These results indicate that Mcl-1 plays an important role in the process of energy metabolism. In addition, we observed that the ATP production inhibition induced by chidamide was attenuated by Mcl-1 overexpression. Therefore, chidamide may inhibit aerobic metabolism via suppressing Mcl-1 expression.
In addition to chidamide, other HDACi drugs, such as TSA, have been reported to have anti-cancer activities. Our work agrees with and extends the previous work that chidamide can serve as a potential drug for pancreatic cancer. Previous studies confirmed that chidamide suppresses the PI3K/Akt and MAPK/Ras signaling pathways, arresting pancreatic cancer cells at the G1 phase of the cell cycle and promoting apoptosis[
Our data indicate another underlying anti-cancer molecular mechanism of chidamide in pancreatic cancer. Mcl-1 is highly expressed in pancreatic cancer cells[
In conclusion, we verified that Mcl-1 is a chidamide target protein. Chidamide exerts anti-tumor effects by inducing Mcl-1 degradation via the ubiquitin- proteasome pathway, promoting apoptosis and inhibiting aerobic respiration of pancreatic cancer cells. This is the first report of the effect of energy metabolism modulation on proliferation by chidamide through Mcl-1 degradation. We have elucidated an underlying anti-cancer pathway by chidamide. Furthermore, our work suggests that the novel HDACi chidamide has antitumor potential against pancreatic cancer.