Drug resistance is a significant obstacle for successful chemotherapy of colorectal cancer. 5-Fluorouracil (5-FU) is widely used for the treatment of numerous cancers, including colon cancer, despite the low response rate of approximately 20% in this cancer (Kim
The endoplasmic reticulum (ER) synthesizes lipids involved in the production of the plasma membrane and provides a non-vascular pathway for lipid transport, as well as enzymes for lipid metabolism reactions (Salvador-Gallego
To resolve ER stress, a three-pronged signal transduction cascade is activated. The upstream components of the three branches are double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) (Kim
Shikonin and antibodies against GRP78, phospho-eIF2α, phospho-IRE1, and XBP-1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Thiazolyl blue tetrazolium bromide (MTT), ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), Hoechst 33342, propidium iodide (PI), tauroursodeoxycholic acid (TUDCA), and actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rhod-2 acetoxymethyl ester (Rhod-2, AM) and ER-Tracer Blue-White DPX were purchased from Molecular Probes (Eugene, OR, USA). Antibodies against phospho-PERK, caspase-12, and C/EBP-homologous protein (CHOP) were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals and reagents used were of analytical grade.
SNU-C5/5-FUR cells were obtained from the Resistant Cell Research Center of Chosun University (Gwangju, Korea). The cells were subcultured twice a week in presence of 140 μM 5-FU for over 6 months until they were stably drug resistant (Kang
The cells were seeded into a 24-well plate at a density of 0.8×105 cells/mL and incubated for 16 h. They were then treated with shikonin at a concentration of 1, 2, 3, 4, 5, 6, 8, 10, or 15 μM, or pretreated with an ER stress inhibitor (TUDCA) prior to treatment with shikonin for 30 min. The cells were subsequently incubated at 37°C for 48 h, and then 125 μL MTT stock solution (2 mg/mL) was added to each well. After 4 h, the formazan crystals were dissolved in 350 μL DMSO, and the absorbance at 540 nm was measured using a SpectraMax i3X multi-detection microplate reader (Molecular Devices, Sunnyvale, CA, USA) (Piao
Cells were seeded into a 60 mm culture dish at a density of 0.8×105 cells/mL and incubated for 16 h. After treatment with 3.3 μM shikonin, cells were observed for 1, 2, and 3 days, and changes in cell morphology were documented using a phase contrast inverted microscope (DP71 digital microscope camera, Olympus, Tokyo, Japan). All images were acquired at 20× magnification.
Approximately 100 cells were inoculated into a 35 mm culture dish and allowed to grow for 2 days. The cells were then treated with 3.3 μM shikonin and incubated for 10 days until colonies formed. To aid visualization, the colonies were stained using a Diff-Quik kit (Sysmex, Kobe, Japan) according to the manufacturer’s instructions.
Flow cytometry analysis was performed after cells were stained with propidium iodide (PI) to assess the proportion of sub-G1 cells with hypodiploid DNA content, which are considered to represent apoptotic cells. Briefly, cells were seeded in 6-well plates in triplicate for each of the control and shikonin-treated groups. Twenty hours later, cells were pretreated with 3.3 µM shikonin (treated). The cells were harvested after 48 h and fixed with 70% ethanol (1 mL) for 30 min at 4°C. Subsequently, cells were washed twice with cold PBS+2 mM EDTA, to prevent aggregation, and incubated in the dark for 30 min at 37°C in PBS/2 mM EDTA containing PI (final concentration, 100 µg/mL) and RNase A (final concentration, 100 µg/mL). The analysis was performed using a FACSCalibur instrument, and the percentage of sub-G1 hypodiploid cells was evaluated using CellQuest and ModFit Software (Becton Dickinson, San Jose, CA, USA).
Cellular DNA fragmentation was assessed by measuring DNA fragments released into the cell cytoplasm. To facilitate detection, DNA was labeled with the non-radioactive thymidine analog, BrdU. DNA fragments were detected immunologically using an ELISA kit from Roche Diagnostics (Mannheim, Germany), according to the manufacturer’s instructions.
Mitochondrial Ca2+ levels were monitored using Rhod-2 AM (Mészáros
The cells were seeded at 1.0×105 cells/mL in medium with or without EGTA. After 16 h incubation, the cells were treated with shikonin and incubated at 37°C for another 48 h. Alternatively, the cells were seeded in a 24-well plate at 1.0×105 cells/mL. After 16 h incubation, the cells were treated with TUDCA and incubated for 30 min, followed by treatment with shikonin and incubation at 37°C for 48 h. After staining with Hoechst 33342 cell-permeable nuclear counterstain dye for 10 min, nuclear fragmentation (indicating apoptosis) was determined as previously described (Piao
The harvested cells were washed once with PBS, lysed with RIPA buffer containing protease inhibitors on ice for 20 min, and centrifuged at 14,000×g for 10 min. The supernatant was collected, and the Quant-iT™ protein assay kit was used to determine protein concentrations (Thermo Fisher Scientific). After boiling an aliquot of the lysate (40 µg protein) for 5 min, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on their molecular weight. The separated proteins were transferred onto nitrocellulose membranes, which were subsequently incubated with primary antibodies against GRP78, phospho-PERK, phospho-eIF2α, phospho-IRE1, XBP-1, caspase-12, CHOP, and actin, followed by a horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA). The membranes were incubated with enhanced chemiluminescence detection reagents (Amersham, Little Chalfont, Buckinghamshire, UK) and exposed to X-ray film in the dark to visualize protein bands.
RT-PCR was performed as previously described (Han
Cells were seeded on a 4-well chamber glass slide at a density of 1.5×105 cells/mL and incubated for 16 h. Then, the cells were directly treated with 3.3 μM shikonin, or pretreated with 1 μM of the ER stress inhibitor TUDCA for 30 min, followed by treatment with 3.3 μM shikonin. Protein expression was detected using GRP78 and CHOP antibodies, and ER-Tracker™ Blue-White DPX reagent was used for localization. Processing of the slides for immunocytochemistry was performed as previously described (Piao
The transfection of SNU-C5/5-FUR cells with control (siControl, Santa Cruz Biotechnology) and CHOP (siCHOP, Bioneer, Seoul, Korea) siRNAs was performed according to a previously described method (Zhang
All experiments were repeated three times, and the values presented are the mean ± standard error of the mean. Analysis of variance (ANOVA) and Tukey’s post-hoc test were performed to analyze the differences between the means. Statistical significance was set at
To determine the effect of shikonin on 5-FU–resistant colorectal cancer cells, we treated SNU-C5/5-FUR cells, into a wide range of shikonin concentrations and determined the effects on cell viability. Our results indicated that shikonin exhibits concentration-dependent cytotoxicity in SNU-C5/5-FUR cells, with significant cytotoxicity evident at concentrations greater than 3 µM. The calculated IC50 value was 5.7 µM (Fig. 1A). Based on past experience, it is convenient to observe cell changes when the cell survival rate is approximately 60%-70%; therefore, a concentration of 3.3 μM, at which the cell survival rate was 65%, was selected as the optimum concentration for further investigations. After 24 h treatment with 3.3 μM shikonin, morphological changes were observed in the cells, including some dead cells, although the number of dead cells was not prominent. However, after two days of incubation, a significant number of dead cells was observed, and after three days of incubation, most of the cells appeared to be dead (Fig. 1B). To determine whether shikonin inhibits cell proliferation, a colony formation assay was performed. The results showed that compared with the control group, shikonin significantly inhibited colony formation of SNU-C5/5-FUR cells (CFCs) (Fig. 1C). To confirm that the cytotoxic effect of shikonin was due to its ability to promote apoptosis, we used flow cytometry to measure the proportion of sub-G1 DNA content (considered to represent apoptotic cells) after shikonin treatment. Shikonin significantly increased the rate of apoptosis relative to that in the control group (Fig. 1D). Moreover, shikonin-treated cells had a higher proportion of fragmented DNA content than the control group (Fig. 1E). These results suggested that shikonin effectively inhibits the proliferation of SNU-C5/5-FUR cells by inducing apoptosis.
In the mitochondrial matrix, although the accumulation of Ca2+ can stimulate oxidative phosphorylation, high concentrations of Ca2+ can also transmit and amplify apoptotic signals. We observed high levels of fluorescence in the shikonin-treated cells, compared to control cells, which was detected using flow cytometry after staining with Rhod-2 AM, a mitochondrial Ca2+-specific dye (Fig. 2A). Images obtained by confocal microscopy also confirmed this result, with shikonin-treated cells exhibiting strong red fluorescence, indicating that the accumulation of mitochondrial Ca2+ was higher than that of the control cells (Fig. 2B). To confirm the effect of Ca2+ on shikonin-induced cell apoptosis, we treated SNU-C5/5-FUR cells with shikonin in culture medium with or without EGTA (a Ca2+ chelator) and performed a nuclear fragmentation analysis using Hoechst 33342 staining reagent to detect changes in apoptosis. Compared with the control group, shikonin treatment resulted in a significant increase in apoptosis, but this increase was significantly inhibited in the shikonin+EGTA treatment group (Fig. 2C).
Next, we examined the effect of shikonin on the expression of ER stress-related factors using Western blotting. Compared with the control group, the expression of GRP78, phospho-PERK, phospho-eIF2α, phospho-IRE1, spliced XBP-1, cleaved caspase-12, and CHOP in the shikonin-treated groups increased in a time-dependent manner (Fig. 3A). The activation of phospho-IRE1 leads to unconventional splicing of the mRNA encoding
It has been reported that overexpression of CHOP induces apoptosis through the Bcl-2 pathway, and CHOP has also been shown to regulate apoptosis during ER stress (Hu
To further confirm that ER stress is involved in the apoptosis of SNU-C5/5-FUR cells induced by shikonin, we examined the effect of the ER stress inhibitor, TUDCA, in our experiments. As shown in Fig. 5A, TUDCA effectively restored the shikonin-induced decrease in cell viability. Moreover, pretreatment with TUDCA significantly inhibited the emergence of apoptotic bodies (visualized by Hoechst 33342 staining) in the shikonin-treated group (Fig. 5B). To confirm this result, we also analyzed the expression of the ER stress marker proteins, GRP78 and CHOP, via immunocytochemistry after TUDCA treatment. TUDCA significantly inhibited the expression of GRP78 and CHOP that were induced by shikonin (Fig. 5C, 5D).
Shikonin has a variety of biological activities, making it an attractive compound. We have previously reported that shikonin induces apoptosis in SNU-407 colon cancer cells by triggering mitochondrial dysfunction and activating the caspase cascade (Han
Generally, the ER stress response is a short-term, homeostasis-linked event that is critical for cell survival, although long-term and severe ER stress can trigger apoptosis through ER stress-specific cell death signals, such as CHOP and caspase-12 (Coker-Gürkan
We also studied specific markers of ER stress. Under stress-free conditions, the luminal domain of the ER stress sensor binds to the ER chaperone, binding immunoglobulin protein (BiP), keeping it inactive. When unfolded or misfolded proteins accumulate, BiP preferentially binds to these abnormal proteins and releases the inhibition of PERK, ATF6, and IRE1 (Mei
Although CHOP is expressed at very low levels under physiological conditions, it is strongly induced at the transcriptional level under conditions of ER stress (Gotoh
This research was supported by the 2021 scientific promotion program funded by Jeju National University.
The authors declare that there are no conflicts of interest.