Isocorydine targets the drug-resistant cellular side population through PDCD4-related apoptosis in hepatocellular carcinoma

Abstract

Isocorydine (ICD), an anticancer agent under current evaluation, decreased the percentage of side population (SP) cells significantly in hepatocellular carcinoma (HCC) cell lines. ICD treatment sensitized cancer cells to doxorubicin (DXR), a conventional clinical chemotherapeutic drug for HCC. We found that ICD decreased the percentage of SP cells in HCC cell lines by preferentially killing SP cells. In the early stage of treatment, ICD inhibited SP cell growth by arresting cells in G2/M; later, it induced apoptosis. Our xenograft model confirmed that ICD selectively reduced the size and weight of SP-induced tumor masses in vivo. Furthermore, it was found that programmed cell death 4 (PDCD4), a tumor suppressor gene, was relatively low when expressed in SP cells compared with non-SP cells, and its expression level was remarkably elevated when cells were treated with ICD. Taken together, these data suggest that ICD is a drug that may target the SP cells of HCC.

Figure 1

Altered SP fraction and ABCG2 expression after treatment with ICD. (A) Representative flow cytometry histograms of SP analysis of PLC/PRF/5, MHCC-97L, SNU449, MHCC-97H and MHCC-LM3 cells after treatment with 250 μg/mL ICD for 24 h. (B) Bar graph of (A), showing statistical analysis (t test) of SP percentages of four HCC cell lines treated with 150 μg/mL and 250 μg/mL ICD for 24 h; for MHCC-97L and MHCC-LM3 cells, the effect of 10 μg/mL DXR on SP cell percentages was also used as a control. *p < 0.05, **p < 0.01. (C) Western blot of ABCG2 protein in MHCC-97L and MHCC-LM3 cells after being treated with ICD and DXR, respectively, for 24 h; β-actin is a loading control. (D) Immunofluorescence staining confirmed that ABCG2 is downregulated in MHCC-97L and MHCC-LM3 cells after treatment with 150 μg/mL ICD for 24 h. ABCG2 (green) was detected with a monoclonal antibody and labeled with fluorescein isothiocyanate (FITC)-conjugated secondary antibody; nuclei were counterstained with 6-diamidino-2-phenylindole (DAPI).

Figure 2

ICD induces apoptosis in SP cells. (A) Cell proliferation was analyzed by BrdU assay in MHCC-97L and PLC/PRF/5 SP cells after treatment with ICD and DXR. (B) Representative flow cytometry histograms of SP and non-SP cells, in which G2/M arrest was induced by ICD treatment for 12, 24 and 48 h. The level of G2/M arrest in SP cells was more significant than the arrest in non-SP cells. (C) FACS analysis revealed that exposing MHCC-97L SP cells to ICD for 48 h induced apoptosis. Early apoptotic cells refer to cells labeled only by annexin V; late apoptotic cells refer to cells that have both signals of 7-AAD and annexin V (defined according to the manual of PE Annexin V Apoptosis Detection Kit from BD Biosciences). (D) Western blot of caspases in MHCC-97L SP and non-SP cells after treatment with 150 μg/mL ICD for 24 h. (E) Western blot of apoptosis proteins, which localized on the cellular and mitochondrial membranes in MHCC-97L SP and non-SP cells treated with 150 μg/mL ICD for 24 h.

Figure 3

Inhibitory effect of ICD on xenografts formed by SP and non-SP cells from the MHCC-97L line. (A) Tumor masses formed by MHCC-97L-SP or non-SP cells and treated with ICD or PBS in nude mice. (B) The weights of tumor masses induced by MHCC-97L SP or non-SP cells and treated with ICD or PBS (n = 5 each group, *p < 0.05). (C) The weights of tumor-bearing mice treated with ICD or PBS. (D) TUNEL assay of apoptotic cells in SP and non-SP cells induced subcutaneous tumor tissues of control groups (PBS) and ICD treatment groups. (E) Liver orthotopic SP and non-SP cells induced tumors treated with PBS or ICD. (F) TUNEL assay of apoptotic cells in SP and non-SP cells induced liver orthotopic tumor tissues of control groups (PBS) and ICD treatment groups. (G) Western blot of caspase proteins from the tumor tissues of the four groups. (H) Western blot of apoptosis-related proteins from the tumor tissues of the four subcutaneous xenograft groups.

Figure 4

ICD increases the sensitivity of HCC cell lines to DXR. (A) Cell cytotoxity detection with MTT assay; pretreatment with 150 μg/mL ICD increased the sensitivity of MHCC-97L and MHCC-LM3 cells to DXR treatment, and pretreatment with 10 μg/mL DXR decreased their sensitivity to DXR. (B) DXR efflux and retention assays showed that pretreatment with ICD reduces DXR efflux and prolongs its retention in both MHCC-97L and MHCC-LM3 cells. The left panel (red) shows the autofluorescence of DXR, the middle panel is the bright field image and the right panel is the merge of the previous two images. *p < 0.05; **p < 0.01.

Figure 5

PDCD4 is a potential target of ICD. (A) Western blot of ABCG2 and PDCD4 in sorted SP and non-SP cells from the MHCC-97L cell line. (B) Western blot of PDCD4 in MHCC-97L and MHCC-LM3 cells exposed to ICD for 24 h. (C) Western blot of PDCD4 in sorted SP and non-SP cells from MHCC-97L and MHCC-LM3 lines treated with PBS or 150 μg/mL ICD for 24 h. (D) Western blot of PDCD4 in tumors induced by MHCC-97L SP or non-SP cells and treated with ICD or PBS, respectively. (E) Western blot of PDCD4 protein in MHCC-97L cells after RNA interference. NC, negative control. (F) Analysis of ICD-induced apoptosis in SP and non-SP cells after PDCD4 siRNA. After silencing of PDCD4, cells were treated with 150 μg/mL ICD for 48 h.