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. 2012;7(6):e38522.
doi: 10.1371/journal.pone.0038522. Epub 2012 Jun 4.

Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway

Qian Wang  1 , Jizheng Chen, Yun Wang, Xiao Han, Xinwen Chen
Affiliations

Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway

Qian Wang et al. PLoS One. 2012.

Abstract

Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HCV infection decreases MIN6 cell viability.
(A) The images of MIN6 cells infected with HCV at 24, 48, 72 and 96 hpi. MIN6 cells were mock-infected or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1. Scale bar, 10 µm. (B) MIN6 cells were infected with HCV at different MOIs. At indicated time points post-infection, the percentage of viable cells was assessed by MTT assay and plotted versus time. The activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, **P<0.01, compared with respective controls.
Figure 2
Figure 2. HCV played a direct role in the reduction of cell viability and induction of cell death of beta cells.
(A, B) MIN6 cells were mock-infected (a) or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1 (b), ultracentrifugation-purified HCV particles (1.0 MOI) (c), the supernatant of HCV-infected Huh7.5.1 after ultracentrifugation (d), CON1 cell culture medium (e), or the supernatant of HCV-infected Huh7.5.1 after UV radiation treatment (f) at 96 hpi. Light microscopy images (scale bar, 10 µm) (A) were obtained and cell viability (B) of MIN6 cells was determined by MTT assay. (C, D) MIN6 cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of indicated dose of RBV (C) or BILN 2061 (D) at 96 hpi, cell viability was assessed by MTT assay. (E) MIN6 cells were infected with HCV as in Figure 1B. At different time-points, cell number was determined by DNA content staining with the fluorescent dye PI. (F) MIN6 cells were treated as in (A). Cell death induced by the indicated treatment was measured by trypan blue exclusion. (A–F) All activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, **P<0.01, compared with respective controls.
Figure 3
Figure 3. HCV infection induces novel morphological changes of MIN6 cells.
(A–C) Confocal image of MIN6 cells stained with Hoechst 33258. Scale bar, 10 µm. Cells were mock infected or infected with 1.0 MOI HCV for 96 h. Cells treated with CHX for 48 h served as an apoptosis positive control. White arrows indicate nuclei (A). MIN6 cells were treated as in Figure 2A (B). Cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of RBV (50 µM) or BILN 2061 (10 µM) at 96 hpi (C). (D) Electron microscopic analysis as in (A). Scale bar, 2 µm. All measurements were done in triplicates.
Figure 4
Figure 4. HCV infection induces apoptosis-like cell death in MIN6 cells.
MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. (A) Confocal image of cells stained with TUNEL at 96 hpi. Scale bar, 10 µm. Quantitative summary of the TUNEL-positive staining is provided on the left. (B) Caspase 3 activity levels at 24 and 48 hpi. The caspase 3 activity of the control cells at 0 h after treatment was arbitrarily expressed as 1.0. (C) Immunoblot analysis of caspase 3 and PARP at 48, 72, 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Immunoblots are representative of at least three independent experiments. Amounts of actin were measured as an internal control to verify equivalent sample loading. (D) Diagrams of FITC-Annexin V/PI flow cytometry in a representative experiment at 48 hpi. Numbers in the quadrants indicate the proportions of cells in the corresponding areas (left panel). (A, B, D) All measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). **P<0.01, compared with respective controls.
Figure 5
Figure 5. Mitochondrial changes in HCV-infected MIN6 cells.
MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. (A) Mitochondrial transmembrane potential changes at 48 hpi. Representative images from JC-1 staining are shown. The aggregated form of JC-1, characteristic of mitochondrial integrity (red) and monomeric JC-1 (green) were examined under laser confocal scanning microscope. (B) Electron microscopy of mitochondrial morphologies at 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Black and white arrows indicate mitochondria and insulin granules, respectively. Scale bar, 1 µm.
Figure 6
Figure 6. HCV infection induces ER stress in MIN6 cells.
(A) mRNA levels of GRP78 and CHOP. MIN6 cells were mock-infected or infected with 1.0 MOI HCV for 24, 48, 72 h. mRNA was analyzed by real-time RT-PCR. The results were normalized with the values obtained from actin in the same sample. Data are expressed as fold-increase relative to the values observed in mock control. The measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, compared with respective controls. (B) Immunoblot analysis of GRP78, CHOP and p-PERK corresponding to (A) probed with the indicated antibodies. Amounts of actin were measured as an internal control to verify equivalent sample loading. Immunoblots are representative of at least three independent experiments.
Figure 7
Figure 7. Hepatitis C virus replicates in MIN6 cells.
(A) Kinetics of detection of positive-strand HCV RNA. MIN6 and Huh7.5.1 cells were mock-infected or infected with 1.0 MOI HCV. At different time points, cells were harvested and total RNA was isolated and reverse-transcribed to cDNA. Single-round PCR products (150 bp) were obtained by amplification with POSF/R primers. Actin was measured as an internal control. (B) Detection of the synthetic negative-strand RNA as in (A). The sample from HCV-infected Huh7.5.1 cells was used as the positive control. Negative controls included PCR amplification from non-infected cells (−) and water (H2O). (C–D) MIN6 cells were mock-infected or infected with 1.0 MOI of HCV at 24 hpi. HCV core (red) and NS5A (green) labeled with respective antibody (C) or HCV dsRNA labeled with J2 antibody (green) (D) were examined by immunofluorescence assay. Blue fluorescence represents DAPI-stained nuclei as observed. (E) Immunoprecipitation and blotting of NS5A purified from 96 h-infected MIN6 and Huh7.5.1 cells. Actin from lysis was used as the internal control. All measurements were done in triplicates. Immunoblots are representative of at least three independent experiments.
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