Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress

Abstract

Unfolded protein response (UPR) is a cytoprotective mechanism that alleviates the protein-folding burden in eukaryotic organisms. Moderate activation of UPR is required for maintaining endoplasmic reticulum (ER) homeostasis and profoundly contributes to tumorigenesis. Defects in UPR signaling are implicated in the attenuation of various malignant phenotypes including cell proliferation, migration, and invasion, as well as angiogenesis. This suggests UPR as a promising target in cancer therapy. The pharmacological effects of the plant Scindapsus cf. hederaceus on human cancer cell lines is not understood. In this study, we identified an ethyl acetate extract from Scindapsus cf. hederaceus (SH-EAE), which markedly altered the protein expression of UPR-related genes in human non-small cell lung cancer (NSCLC) cells. Treatment with the SH-EAE led to the dose-dependent suppression of colony forming ability of both H1299 and H460 cells, but not markedly in normal bronchial epithelial BEAS-2B cells. SH-EAE treatment also attenuated the migration and invasion ability of H1299 and H460 cells. Moreover, SH-EAE strikingly suppressed the protein expression of two ER stress sensors, including inositol requiring enzyme-1α (IRE-1α) and protein kinase R-like ER kinase (PERK), and antagonized the induction of C/EBP homologous protein (CHOP) expression by thapsigargin, an ER stress inducer. SH-EAE induced the formation of massive vacuoles which are probably derived from ER. Importantly, SH-EAE impaired the formation of intersegmental vessels (ISV) in zebrafish larvae, an index of angiogenesis, but had no apparent effect on the rate of larval development. Together, our findings demonstrate, for the first time, that the ability of SH-EAE specifically targets the two sensors of UPR, with significant anti-proliferation and anti-migration activities as a crude extract in human NSCLC cells. Our finding also indicates potential applications of SH-EAE in preventing UPR activation in response to Tg-induced ER stress. We suggest that SH-EAE attenuates UPR adaptive pathways for rendering the NSCLC cells intolerant to ER stress.

Keywords: ER stress; NSCLC; Scindapsus cf. hederaceus; UPR; ethyl acetate extract; non-small cell lung cancer cell; selective anti-cancer therapeutics; unfolded protein response.

Figure 1

Identification of ethyl acetate extract of Scindapsus cf. hederaceus as a novel UPR modulator. The 12 samples of 10 plant species, labeled as 1197 (ethyl acetate), 4643 (ethyl acetate), 2278a (ethyl acetate), 2278b (water), 8106a (water), 8106b (butanol), 1349 (methanol), 1009 (ethyl acetate), 1339 (ethyl acetate), 3872 (ethyl acetate), 4634 (hexane), and 7265 (ethyl acetate), were collected from Dr. Cecilia Koo Botanic Conservation Center, Kaohsiung County, Taiwan. Ethyl acetate extract of Scindapsus cf. hederaceus (SH-EAE) was labeled as 1339 and stored at −20 °C for the screening of biological activity. H1299 cells were exposed to a single dose (20 μg/mL) of 12 extracts from a family Araceae for 48 h followed by immunoblot assay. The protein levels of Grp78, IRE-1α, SQSTM1, LC3, SOD1, and SOD2 were evaluated. Dimethyl sulfoxide (DMSO) as vehicle control. Dox, Doxorubicin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control.

Figure 2

SH-EAE decreased both protein and mRNA expression of two ER stress sensors, PERK and IRE-1α. (A) H1299 and H460 cells treated with different concentrations of SH-EAE (10, 20, and 50 μg/mL) for 48 h. Protein lysates were collected and subjected to SDS-PAGE followed by immunoblotting using antibodies against Grp78, IRE-1α, PERK, ATF6, Calnexin, PDI, Ero-Lα, and GAPDH (loading control). (B) H1299 cells were treated with 20 μg/mL SH-EAE for 0, 2, 4, 6, 8, 10, 12, and 24 h. Protein lysates were subjected to SDS-PAGE followed by immunoblotting using antibodies against PERK, IRE-1α, CHOP, and Grp78. GAPDH served as the loading control. (C) Relative fold change of mRNA abundance of UPR-related genes including GRP78, PERK, IRE1α, and ATF6, was measured by qPCR in NSCLC H1299 cells treated with SH-EAE (20 and 50 μg/mL) or Tg (0.1 μΜ) for 24 h. Data are shown as fold change compared with vehicle-treated cells, and represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.001). (D) SH-EAE causes the accumulation of the Grp78 within the ER. Immunofluorescence staining of endogenous Grp78 proteins. H1299 cells were treated for 48 h with SH-EAE (20 and 50 μg/mL), Tg (0.1 μΜ), and Tm (0.5 μg/mL), respectively, followed by immunofluorescence staining using anti-Grp78 antibody and ER-Tracker Red. The nuclei were counterstained with 4’6-diamidino-2-phenylindole (DAPI). Scale bar indicates 50 μm. Representative images of three independent experiments are shown.

Figure 2

SH-EAE decreased both protein and mRNA expression of two ER stress sensors, PERK and IRE-1α. (A) H1299 and H460 cells treated with different concentrations of SH-EAE (10, 20, and 50 μg/mL) for 48 h. Protein lysates were collected and subjected to SDS-PAGE followed by immunoblotting using antibodies against Grp78, IRE-1α, PERK, ATF6, Calnexin, PDI, Ero-Lα, and GAPDH (loading control). (B) H1299 cells were treated with 20 μg/mL SH-EAE for 0, 2, 4, 6, 8, 10, 12, and 24 h. Protein lysates were subjected to SDS-PAGE followed by immunoblotting using antibodies against PERK, IRE-1α, CHOP, and Grp78. GAPDH served as the loading control. (C) Relative fold change of mRNA abundance of UPR-related genes including GRP78, PERK, IRE1α, and ATF6, was measured by qPCR in NSCLC H1299 cells treated with SH-EAE (20 and 50 μg/mL) or Tg (0.1 μΜ) for 24 h. Data are shown as fold change compared with vehicle-treated cells, and represent the mean ± SD of three independent experiments. (* p < 0.05; ** p < 0.001). (D) SH-EAE causes the accumulation of the Grp78 within the ER. Immunofluorescence staining of endogenous Grp78 proteins. H1299 cells were treated for 48 h with SH-EAE (20 and 50 μg/mL), Tg (0.1 μΜ), and Tm (0.5 μg/mL), respectively, followed by immunofluorescence staining using anti-Grp78 antibody and ER-Tracker Red. The nuclei were counterstained with 4’6-diamidino-2-phenylindole (DAPI). Scale bar indicates 50 μm. Representative images of three independent experiments are shown.

Figure 3

SH-EAE induced cytoplasmic vacuolization in NSCLC cells. Comparison of ER morphology affected by SH-EAE, Tg or Tm. (A) H1299 and (B) H460 cells expressing the fluorescent ER-Tracker were treated with SH-EAE (20 μg/mL), Tg (0.1 μM), or Tm (0.5 μg/mL) for 48 h and analyzed by Nikon Eclipse TE2000U inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan). The images on the left panel were taken with a bright-field objective. The second panel is that stained with ER-Tracker™ Red dye. The third panel is that stained with DAPI. The fourth panel is an overlay of the bright-field images with the fluorescence images. The last panel is a magnification of the boxed region. Scale bar indicates 50 μm. Representative images of H1299 and H460 cells for each condition over three independent experiments are shown. (C) Staining for various organelles in the SH-EAE-treated H1299 cells. Cells exhibiting SH-EAE-induced vacuolization were stained for trackers of ER (red), lysosomes (red), and mitochondria (green). Cell nuclei were stained with DAPI. Scale bar indicates 25 μm. None of the lysosome or mitochondria trackers overlapped with SH-EAE-induced vacuolization.

Figure 3

SH-EAE induced cytoplasmic vacuolization in NSCLC cells. Comparison of ER morphology affected by SH-EAE, Tg or Tm. (A) H1299 and (B) H460 cells expressing the fluorescent ER-Tracker were treated with SH-EAE (20 μg/mL), Tg (0.1 μM), or Tm (0.5 μg/mL) for 48 h and analyzed by Nikon Eclipse TE2000U inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan). The images on the left panel were taken with a bright-field objective. The second panel is that stained with ER-Tracker™ Red dye. The third panel is that stained with DAPI. The fourth panel is an overlay of the bright-field images with the fluorescence images. The last panel is a magnification of the boxed region. Scale bar indicates 50 μm. Representative images of H1299 and H460 cells for each condition over three independent experiments are shown. (C) Staining for various organelles in the SH-EAE-treated H1299 cells. Cells exhibiting SH-EAE-induced vacuolization were stained for trackers of ER (red), lysosomes (red), and mitochondria (green). Cell nuclei were stained with DAPI. Scale bar indicates 25 μm. None of the lysosome or mitochondria trackers overlapped with SH-EAE-induced vacuolization.

Figure 4

SH-EAE treatment inhibited colony-forming ability in malignant NSCLC cells but not in normal human bronchial epithelial cells. (A) Colony formation assay in 6-well plates. Two NSCLC cell lines, H1299 and H460, and non-tumorigenic bronchial epithelial BEAS-2B cells were treated with different concentrations of SH-EAE (10, 20, and 50 μg/mL) for 14 days. Afterward, the cells were fixed in 4% paraformaldehyde and stained with Giemsa. (B) The quantification analysis of the colony area. Cells were treated as described above, bright-field images were obtained independently with the same objective lens and the areas covered by the cell colonies were measured using ImageJ software. Data represent the mean ± SD of three independent experiments (* p < 0.05; ** p < 0.001).

Figure 5

SH-EAE decreased H1299 and H460 cell migration and invasion in vitro. (A) Representative 4× magnification images at 16 h. H1299 and H460 cells in confluence were scratched and then treated with different concentration (5, 10, and 20 μg/mL) of SH-EAE for 16 h. The cells were fixed in 4% paraformaldehyde solution. The area between the two dotted lines indicates the wound width of SH-EAE (20 μg/mL)-treated cells at 16 h after scratching. (B) The quantifications of the regions of the cell during migration were analyzed using a software “TScratch”. Data represent the mean ± SD of three independent experiments. (* p < 0.05). (C) Cell invasion was determined using ThinCert™ cell culture transwell inserts, showing representative images of the bottom surface of the transwell membrane in H1299 and H460 cells. Scale bar indicates 1000 μm. (D) The number of invaded cells in four random microscopic fields (×200) was counted for each group. Data are shown as means ± SD of three independent experiments. (* p < 0.05).

Figure 6

SH-EAE reduces the EGFR and VEGF signaling in NSCLC cells. Expression of phospho-EGFR (Tyr845), EGFR, and VEGF were analyzed by western blot in NSCLC cell line H1299. GAPDH was used as the loading control. Data are representative of three independent experiments.

Figure 7

SH-EAE regulates UPR signaling but does not induce apoptosis. (A) Western blotting demonstrates relative protein levels of UPR components, including Grp78, CHOP, and proapoptotic proteins, such as caspase-9 and caspase-3, in NSCLC cells. Different from the Tg treatment. The increased levels of Grp78 in the SH-EAE treatment groups were obviously lower than the Tg group, and SH-EAE did not induce CHOP expression compared to Tg. The cleaved (active) form of caspase-3 and -9 were only induced in the Tg treatment. GAPDH serves as loading control. One of the two independent experiments is shown. (B) Annexin V-PI staining was performed to analyze apoptotic cell populations. 2 × 10⁵ cells were seeded into a six-well plate and treated with different concentrations (10, 20, and 50 μg/mL) and Dox (2 μM) for 48 h. Data are presented as dot plots (Annexin V-FITC on the x-axis; PI on the y-axis). The cell numbers in the four quadrants represent the percentage of viable (lower left), necrotic (upper left), early apoptotic (lower right), and late apoptotic (upper right) cells determined by using a BD Accuri™ C6 flow cytometer. Data are representative of three independent experiments. (C) A gallery of representative images of γ-H2AX foci was shown. Both H1299 and H460 cells were treated with SH-EAE (20 and 50 μg/mL) and Dox (2 μM) for 48 h. Cells were fixed and stained by immunofluorescence with anti-γ-H2AX (FITC, green). Nuclei were counterstained with DAPI. Representative images of H1299 and H460 cells obtained from at least three independent experiments for each condition are shown. ■ γ-H2AX; ■ DAPI. Scale bar indicates 50 μm. (D) Quantification of the numbers of γ-H2AX foci per nucleus after treatment with Dox or SH-EAE. Dox served as a positive control for DNA damage. The average numbers of γ-H2AX foci per cells treated with SH-EAE (20 and 50 μg/mL) were insignificant at 48 h as compared with untreated control cells. More than 50 cells were randomly analyzed using a software “ImageJ”. Data represent the mean ± SD of three independent experiments. (** p < 0.001).

Figure 8

SH-EAE abrogated the Tg-mediated induction of Grp78 and CHOP expression. H1299 cells were pretreated with SH-EAE for 10 h and further treated with Tg for another 12 h. Protein lysates were subjected to SDS-PAGE followed by immunoblotting using antibodies against Grp78, CHOP, cleaved-caspase-9 and caspase-3. H1299 cells pretreated with 20 and 50 μg/mL SH-EAE were significantly inhibited by Tg-induced CHOP expression. α-tubulin served as the loading control.

Figure 9

SH-EAE decreased angiogenesis in the zebrafish model. (A) The image is representative of 20 individual zebrafish larvae Tg(flk1:EGFP) under a fluorescence light microscope after exposure to SH-EAE (20 µg/mL) or vehicle control for 14 h. The upper image is untreated larvae as the control group, and the lower image is larva treated with SH-EAE (20 µg/mL). The yellow arrows depict the regions where fluorescent intersegmental vessels (ISVs) were obvious. Scale bars = 200 μm. (B) The formation of the ISVs in zebrafish larvae at 24 hpf. n = 20 embryos pooled from two independent experiments. Centerlines indicate median values. The p-value from the Mann–Whitney U test is less than 0.05 (* p < 0.05). (C) The effect of SH-EAE on the survival rate of zebrafish embryos. Data are expressed as percentages. n = 20 for each group.

Figure 10

(a) The buds of Scindapsus cf. hederaceus; (b) the phyllotaxy, the arrangement of leaves on the stem of Scindapsus cf. hederaceus. Photos taken by Dr. Cecilia Koo Botanic Conservation Center (KBCC), Pingtung, Taiwan.

Figure 11

A proposed model of SH-EAE on anti-lung cancer effects through UPR modulation. Unfolded protein response (UPR) is a cytoprotective mechanism that alleviates the protein-folding burden in eukaryotic organisms. Both tunicamycin (Tm) and thapsigargin (Tg) cause a large accumulation of GRP78 chaperones in the ER lumen and the activation of all three sensors of ER stress ATF6, IRE1α, and PERK. This results in the hyperactivated UPR and the consequent growth arrest and apoptosis in cells. In contrast, SH-EAE selectively inhibited the clonogenicity, migration, and invasion in lung non-small cell lung cancer (NSCLC) cells through the attenuation of UPR by the downregulation of IRE1α and PERK.