Neuroblastoma is the second most prominent type of solid tumor in childhood, with more than 50% of cases occurring within two years of birth [1
]. Despite advances in treatment modalities such as surgery, radiation therapy, and chemotherapy, neuroblastoma is generally progressive and very malignant, with long-term survival rates of <40% for children with advanced and metastatic disease [3
]. Presently, the development of alternative medicines derived from plants with low side effects is being studied extensively [6
]. Accumulating evidence reveals that these anticancer drugs induce various cell death mechanisms, including apoptosis and autophagy in several types of cancer cells [7
]. Autophagy and apoptosis play an important role in determining cancer cell fate and are essential metabolic pathways for organism homeostasis, organ development, and cancer [8
]. Apoptosis is a selective physiological process in which activation of caspases (cysteine aspartyl proteases) results in mitochondrial membrane permeability and morphological changes, including chromatin condensation and DNA fragmentation, thereby leading to programmed cell death [10
]. Failure of apoptosis is thought to contribute to tumorigenesis, because it regulates the balance between cell proliferation and death. Therefore, targeting of key apoptotic modulators has become a strategy for the development of cancer therapies [11
Similarly, autophagy is an evolutionarily conserved catabolic process of lysosomal degradation of cytoplasmic content in eukaryotes [12
]. This autophagic pathway includes vesicle elongation, autophagosome maturation (sequestration of cargo), autophagosome–lysosome fusion, and degradation [13
]. It provides energy for cell functions through degradation of molecules and cellular organs but also reduces cell damage by promoting the elimination of pathogens, toxic molecules, damaged cellular organelles, and misfolded proteins. However, excess autophagy can induce autophagic cell death by excessive degradation of mitochondria and damaged molecules required for cell survival [14
]. Autophagy acts like a “double-edged sword” that plays a positive or negative role in cancer cells during the tumorigenesis process. Whether autophagy increases or inhibits cell death in response to cellular stress remains questionable [17
]. Therefore, to balance cell survival and death, it is necessary to understand the complexity of the relationship between cancer cell apoptosis and autophagy.
C.A. Meyer has been used as a health product and natural remedy in traditional medicine in many Asian countries such as China, Korea, and Japan for thousands of years [18
]. Ginsenoside (ginseng saponins) is the major active component of ginseng, and more than 20 ginsenosides have been reported to possess various biological activities, including anti-inflammation, anti-carcinogenesis, anti-metastasis, and neuroprotection [19
]. Compound K (CK) is a major metabolite component of several protopanaxadiol type (PPD) ginsenosides (Rb1, Rb2, and Rc) that is secreted by intestinal bacteria in humans and rats through the multistage cleavage of sugar moieties [23
]. CK is a derivative of the PPD-ginsenoside, and its chemical formula is C36
with a molecular weight of 622.86 g/mol (Figure 1
A). The biological function of CK has been explored for its antitumor and anti-inflammatory effects in several disease models [18
]. CK blocks proliferation and migration of tumor cells and promotes apoptosis and autophagy [26
]. However, its mechanism of action in neuroblastoma cells is unknown. Therefore, in the present study, we aimed to investigate the anticancer effects of CK and its underlying mechanisms on crosstalk between apoptosis and autophagy in neuroblastoma cell lines.
In this study, we demonstrate that CK induced apoptosis and ROS generation and inhibited autophagy flux in neuroblastoma cells in vivo and in vitro. Interestingly, chloroquine, an autophagy flux inhibitor, potentiates CK-induced apoptosis. Apoptosis is a systemically well-organized suicide program that removes defective, damaged, or unwanted cells. Autophagy is also an intracellular system that removes cytosolic protein aggregates, damaged organelles, and infectious organisms and contributes to cell survival mechanisms [15
]. Apoptosis and autophagy are two distinct processes required to maintain homeostasis of cells. In addition, the crosstalk between autophagy and apoptotic cell death was reported in many cancer cell types [10
]. Hence, we investigated the molecular mechanisms of CK on neuroblastoma cell growth inhibition, cell death, and autophagy to understand the complexity of the relationship between cancer cell apoptosis and autophagy.
Firstly, we found that the growth inhibition by CK in three human neuroblastoma cell lines, such as SK-N-BE(2), SH-SY5Y, and SK-N-SH cells, were dose-dependent (Figure 1
B–D), and we also demonstrated that CK induced the accumulation of sub-G1 population (apoptotic cells) and apoptosis in a caspase-dependent manner. We also found that CK significantly inhibited autophagic flux by blocking of autophagosome and lysosome fusion, the step of late-stage autophagy. Similar to our study, CK exerts anticancer effect by triggering apoptosis and autophagy via ROS generation on colon cancer cells [28
]. In addition, there is ample evidence that demonstrated that CK has an antitumor effect on various cancers by triggering apoptosis, inhibiting proliferation, or inducing autophagy through multiple pathways [27
]. However, previous studies have limited results such as acting as an autophagy inducer in colon cancer or non-small lung cancer [28
] or inhibiting cell death by autophagy after brain injury [47
]. Thus, the anticancer effect of CK will be controversial whether it contributes to apoptosis or not. In this context, the present study is first to show that CK inhibits autophagy flux in neuroblastoma. It also suggests that that natural product CK can be used for not only inducing apoptosis but also inhibiting autophagic flux in neuroblastoma.
Another cell death type, autophagic cell death (ACD) is an evolutionarily conserved catabolic process that requires the formation of autophagosomes and various autophagy-related proteins, including Atgs, BECN, and LC3B [33
]. Autophagic flux is defined as a measure of autophagic degradation activity, and LC3-II and p62 are widely used as indicators for the measurement of autophagic flux [49
]. In this study, CK-induced early-stage autophagy in neuroblastoma cells was evidenced by an increased number of autophagosomes and increased expression of the autophagy markers LC3-II, Atg7, and BECN proteins (Figure 5
). Additionally, autophagic flux analysis showed that CK treatment induces an increase in p62 expression. Furthermore, CK inhibited autophagic flux by blocking the autophagosome–autolysosome fusion process in neuroblastoma cells, similar to CQ, a late-stage autophagy inhibitor (Figure 6
A). According to our results, p62 expression was upregulated by CK treatment in a dose-dependent manner in neuroblastoma cells (Figure 5
E,F). Consistent with our data, recent studies demonstrate that upregulation of p62 expression is associated with ACD in breast cancer [52
], hepatocellular carcinoma cells [53
], and esophageal cancer [54
The role of autophagy in the treatment of cancer remains still unclear. In many solid tumors, including NB, after chemotherapy, autophagy acts as a survival mechanism of cancer cells, by which it can reduce the effectiveness of chemotherapy [2
]. Extensive autophagy can lead to cell death, and although autophagy is thought to have a dual role in tumorigenesis, its function is yet to be fully understood [56
]. Thus, identification of a clear interaction between autophagy and apoptosis could be an effective approach for cancer treatment. Among the inhibitors of autophagy, chloroquine (CQ), a specific late-phase autophagy inhibitor, inhibits lysosome fusion to the autophagosome and impairs autophagosome maturation into degradative autolysosomes [37
]. Currently, CQ has been used in cancer clinical trials. Thus, the combination of chloroquine and CK might be a promising strategy in the treatment of neuroblastoma. In the present study, we identified the anti-NB cancer activity of CK through in vitro and in vivo experiments (Figure 1
, Figure 2
, Figure 3
, Figure 4
, Figure 7
and Figure 8
). Additionally, CK inhibited autophagic flux through blockade of autophagosome and autolysosome fusion in neuroblastoma. This suppression of autophagy with the autophagy inhibitor CQ could significantly enhance CK-induced apoptosis in neuroblastoma cells. More detailed studies found that CK not only stimulated the generation of intracellular ROS, which subsequently triggered mitochondria dysregulation, but also inhibited autophagic flux. However, the relationship between increased ROS level and autophagic flux inhibition by CK remains to be revealed through further studies. This observation has demonstrated the cytoprotective role of autophagy in CK-treated neuroblastoma cells, suggesting that abolition of autophagy can improve the anti-NB cancer effect of CK in vitro. We also noted that the SK-N-BE (2) cell xenograft model confirms the protective effect of autophagy on CK therapy in vivo. As a result, the combination therapy of CK and CQ enhanced the therapeutic effect of CK, which proved that autophagy blocking could further increase the therapeutic effect of NB cells.
Under normal circumstances, ROS formed by the mitochondrial respiration is removed by antioxidant enzymes. However, excessive ROS build-up due to the inhibition of antioxidant activity is toxic to cells [60
]. This abnormal ROS production leads to MMP loss, causing leakage of various apoptotic molecules from the mitochondria into the cytoplasm, leading to apoptosis. Thus, ROS is an important mediator of intracellular signaling pathways, including the mitochondrial-mediated apoptosis pathway [60
]. Recently, a change in ROS level has also been reported to be associated with autophagic death [62
]. Our present results also found that CK-treated neuroblastoma cells have an increased level of intracellular ROS compared to control cells (Figure 3
A–C); however, the ROS scavenger NAC significantly reduced ROS production and rescued cell death induced by CK treatment (Figure 3
A–D). Interestingly, the CQ and CK combination treatment induced a significant increase in mitochondrial ROS production and loss of MMP compared with CK only treatment, but not RAPA treatment (Figure 6
E–H). Additionally, inhibition of autophagy can exacerbate mitochondrial damage and promote mitochondrial-dependent apoptosis in neuroblastoma cells (Figure 6
A–D). These results indicate that autophagy can play a protective role against CK-induced apoptosis by eliminating damaged mitochondria. However, further studies on the underlying molecular mechanisms of ROS-mediated apoptosis and autophagy with CK treatment are needed.
One of the advantages in our study is that low concentrations of CK were used against neuroblastoma cells. There are many studies that have reported the inhibitory effect of CK on the growth of various cancer cells, and IC50
for HL-60, K562, PC-14, MKN-45, Du145, HCT116, and U373MG were at 11.7, 8.5, 25.9, 56.6, 58.6, 30–50, and 15 μM, respectively [41
]. In our study, IC50
values with CK treatment were 5 μM in SK-N-BE(2) cells, 7 μM in SH-SY5Y cells, and 15 μM in SK-N-SH cells, respectively (Figure 1
B–D). It showed a definitely low concentration of CK in neuroblastoma compared to other cell lines, and neuroblastoma cells are much more sensitive to CK than other cancer cells. More importantly, cytotoxicity was not found in normal cells including HUVEC, BJ, and CCD-1079SK cells (Figure 1
E–G). This suggests that CK may be a good candidate for NB therapy. Our data provide a preliminary insight into the potential use of CK as an anticancer drug for NB cancer treatment. In addition, further studies are needed to know the mechanism or regulation of cancer-specific effects of CK, and it will be interesting.
Overall, we demonstrate that ginsenoside compound K induced ROS generation-mediated apoptosis and inhibited autophagy flux in neuroblastoma cells (Figure 9
). Moreover, chloroquine, an autophagy flux inhibitor, potentiates CK-induced apoptosis in vitro and in vivo. To our knowledge, we first report the anticancer effect of CK combination with chloroquine against neuroblastoma cells. The findings suggest that a combination strategy of the ginsenoside CK with chloroquine (autophagy inhibitor) could be a novel therapeutic potential for the treatment of neuroblastoma.
4. Materials and Methods
4.1. Chemicals and Reagents
CK (purity > 97%) was prepared by a transformation of PPD-type ginsenosides extracted from Korean ginseng using acid-heat treatment. N-acetyl-L-cysteine (NAC), dimethyl sulfoxide (DMSO), 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA), Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), rapamycin (RAPA), acridine orange (AO), chloroquine (CQ), and an in situ cell death detection kit (TMR red; TUNEL) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), 0.05% Trypsin-EDTA and penicillin-streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). The following antibodies were used: LC3-I/II was purchased from Sigma; GAPDH, Bcl-2, cleaved caspase-3, caspase-8, PCNA, ATG7, Beclin 1, and p62 (Cell Signaling, Danvers, MA, USA), PARP and Bcl-xL (Santa Cruz Biotechnology, Dallas, TX, USA), Alexa 488/594 conjugated secondary antibodies (Abcam, Cambridge, UK), Annexin V-FITC apoptosis detection kit, propidium iodide (PI), Hoechst 33342 staining kit, and Z-VAD-FMK were purchased from BD Biosciences (SanJose, CA, USA).
4.2. Cells and Culture Conditions
Human neuroblastoma (SH-SY5Y and SK-N-BE(2)), human foreskin fibroblasts (CCD-1079SK, and BJ), and human umbilical vein endothelial cell (HUVEC) lines were obtained from the American Type Culture Collection (Manassas, VA, USA). SK-N-SH cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM supplement with 10% (v/v) heat-inactivated FBS and 1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO2 atmosphere and 37 °C. CK was prepared in 100% dimethyl sulfoxide, stored in small aliquots at −80 °C and diluted in the cell culture medium as needed.
4.3. Cell Viability Assay
Cell viability was assessed using the cell count kit-8 (Dojindo Molecular Technologies, Tokyo, Japan) assay. Cells were seeded in the 96-well plates (1 × 104/well) and exposed to various concentrations of CK (2, 5, 10, 15, 20 µM) for 24 h. Pretreated with NAC (5 mM), CQ (10 µM), RAPA (100 nM), and Z-VAD-FMK (20 µM) for 3 h and then exposed to either with or without 5 µM CK for 24 h. Cells without addition of CK were taken as control. The medium was then removed, and 10 μL of the CCK-8 solution was added to each well of the plate. After 3 h incubation, the absorbance was measured at 450 nm using a microplate reader Synergy TM (BioTek Instruments, Inc., Winooski, Vermont, USA). The cell viability was expressed as the percentage of the control, which was set to 100. All experiments were performed in triplicate and repeated at least three times. The 50% inhibitory concentration (IC50) was obtained from the dose-response curve of percent viability (Y) versus tested concentrations (X). IC50 was calculated using linear regression analysis in Microsoft Excel.
4.4. Colony Formation Assay
For colony formation assay, SK-N-BE(2) and SH-SY5Y cells were placed into a 6-well plate at a density of 1 × 103 cells/well and incubated with different concentrations of compound K at 37 °C for 24 h. The medium was then replaced freshly every day, and cells were grown for 14 days. After staining with 0.5% crystal violet for 30 min, the colonies were visualized and quantified.
4.5. Cell Cycle Analysis
SK-N-BE(2) cells were seeded onto 6-well plates at a density of 5 × 105 cells/well and treated with various concentrations of CK for 24 h. Floating and adherent cells were collected with trypsin-EDTA (Sigma-Aldrich) and fixed in 70% ethanol overnight at −20 °C. The cells were washed with cold phosphate buffered saline (PBS) and stained with PI (100 μg/mL) in the dark at 37 °C for 30 min. Then, 10,000 fluorescent events were measured and analyzed with an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).
4.6. Hoechst 33342 Staining
For the Hoechst 33342 staining, SK-N-BE(2) and SH-SY5Y cells were pretreated with 5 mM NAC for 3 h and treated with 5 µM CK for 24 h. The cells were washed with cold PBS and then fixed with cold methanol and stained with Hoechst 33342 (1 μg/mL) for 10 min. The morphology was examined by fluorescence microscopy (CELENA S, Logos Biosystems, Anyang, Korea).
4.7. Apoptosis Assay with Annexin V and PI Staining
Apoptotic cell death was determined using apoptosis detection kit (Annexin V-PI: BD Biosciences, San Jose, CA, USA). Briefly, 5 × 105 cells were treated with CK (0, 5, 10, and 20 μM) for 24 h. Cells were harvested and washed once with PBS, and stained with 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) for 30 min at 37 °C. The stained cells were analyzed by Accuri C6 flow cytometry. Apoptotic cells (Annexin V-FITC + /PI− and Annexin V-FITC + /PI +) were counted and presented as a percentage of the total cell count.
4.8. Mitochondrial Membrane Potential (∆ψm) Assay
Cells (2 × 105) were seeded on 6-well plates and treated with either with or without CK for 24 h. Mitochondrial membrane potential changes were monitored by rhodamine 123 using flow cytometry. In brief, cells were incubated with rhodamine 123 (0.1 μg/mL) for 30 min at 37 °C and collected. The fluorescent intensity was measured using flow cytometry. The positive control was treated with CCCP.
4.9. Hoechst 33342 and PI Staining
The levels of nuclear condensation and fragmentation were observed by Hoechst 33342 (1 μg/mL) and PI (1 mg/mL). Cells were grown on cover slips and treated with CK at various concentrations (0, 5, 10, and 20 µM) after 24 h. Cells were washed twice with cold phosphate buffered saline (PBS), and fixed in cold methanol for 10 min at room temperature. The fixed cells were washed twice with PBS. The stain solution was dropped on a glass slide and incubated for 20 min at 37 °C, and coverslips were placed on a glass slide prior to the observation by a fluorescence microscopy. Four visual fields were randomly selected from each sample and data were collected from three independent experiments. The cell death rate (%) was expressed as the ratio of PI positive stained cells in total Hoechst stained cell.
4.10. RNA Isolation and Reverse Transcription (RT)-PCR
Total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol, and 5 μg of RNA was used for complementary DNA (cDNA) synthesis using synthesized using Go Script reverse transcription system (Promega, Madison, WI, USA). PCR was carried out with specific primers. The PCR products were electrophoresed on 1.2% agarose gel and visualized on a UV transilluminator using Red Safe (iNtRON Biotechnology, Seongnam, Korea). The relative expression level of a target gene was quantified by normalization with the internal control GAPDH gene. Primers used in the experiment were as follows: (1) human Bax (forward) 5′-TCTGACGGCAACTTCAACTG-3′ and (reverse) 5′-TCCCGCCACAAAGATGGTCACG-3′; (2) human Bcl-2 (forward) 5′-GAGGATTGTGGCCTTCTTTG-3′ and (reverse) 5′-ACAGTTCCACAAAGGCATCC-3′; (3) human Caspase-3 (forward) 5′-TTTGT TTGTGTGCTTCTGAGCC-3′ and (reverse) 5′-ATTCTGTTGCCACCTTTCGG-3′; (4) human Caspase-9 (forward) 5′-AACAGGCAAGCAGCAAAGTT-3′ and (reverse) 5′-TCCATCTGTGCCGTAGACAG-3′; (5) human Puma (forward) 5′-AGTGTCCTGCGGCCTCTG-3′ and (reverse) 5′-GGAGTCCCATGATGAGATTGT-3′; (6) human Noxa (forward) 5′-CGGAGATGCCTGGGAAGAA-3′ and (reverse) 5′-AGGTTCCTGAGCAGAAGAGT-3′; (7) human GAPDH (forward) 5′-GAGTCAACGGATTTGGTCGT-3′ and (reverse) 5′-GACAAGCTTCCCGTTCTCAG-3′.
4.11. Western Blot Analysis
After treated with various stimuli, cells were lysed for 30 min in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher scientific, Waltham, MA, USA). The samples were heated to 100 °C for 5 min and placed on ice. Whole cell extracts (30 µg protein) were loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was blocked with 5% milk or 5% BSA for 1 h at room temperature (RT), and incubated overnight with primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. The blot bands were detected with an enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
4.12. Measurement of Intracellular ROS Generation
The generation of ROS was evaluated by fluorescence microscopy and flow cytometry. ROS production was measured using the dye DCFH-DA (Sigma, MO, USA), as previously described [31
]. For flow cytometry, cells treated with or without CK for 24 h were rinsed with cold PBS and incubated in 10 μM DCFH-DA for 30 min at 37 °C in the dark. DCF fluorescence was measured using Accuri C6 FACS (BD Biosciences, San Jose, CA, USA), and data was analyzed using Accuri C6 program (BD Biosciences, San Jose, CA, USA). For fluorescence microscopy, cells were seeded on coverslips and treated with or without CK for 24 h. Cells were incubated in 10 μM DCFH-DA for 30 min at 37 °C in the dark, rinsed with cold PBS, and imaged under a fluorescence microscopy. On average, three microscope fields were quantified in three separate cultures per treatment condition. Image J was used to quantify fluorescence intensity. The results were expressed as percentage change from the controls.
4.13. Immunofluorescence Staining
Cells were cultured on coverslips and pretreated with 10 μM chloroquine for 3 h prior to treating with 5 μM CK for another 24 h. Cells were then washed twice in PBS, fixed with cold methanol, and permeabilized with 0.2% Triton X-100 in PBS for 20 min. After blocking with 10 % normal goat serum for 1 h, and incubated overnight at 4 °C with primary antibodies (cleaved caspase-3 and PCNA), washed three times in PBS, and incubated in Alexa 488-conjugated anti-rabbit IgG or Alexa-594-conjugated anti-mouse IgG for 1 h. Nuclei were counterstained using Hoechst 33342 (BD Biosciences, San Jose, CA, USA), and the stained cells were observed under a fluorescence microscopy CELENA S. Images were analyzed using ImageJ software version 1.52a (NIH, Bethesda, MD, USA).
4.14. Detection of Autophagic Vacuoles by Acridine Orange Staining (AO)
A fluorescent compound, acridine orange (Sigma, St. Louis, MO, USA) is commonly used to measure DNA and RNA levels in cells and can also be used to detect the level of acidic granule compartments within cells undergoing autophagy [66
]. Formation of acidic vesicular organelles (AVOs) was quantitated by acridine orange staining [66
]. SK-N-BE(2) cells (5 × 105
/well) were cultured in 24-well culture plates and treated without or with CK (5 μM) for 24 h, then cells were stained with 5 µg/mL acridine orange (Sigma) for 15 min at 37 °C. After washed with PBS, cells were examined under a fluorescence microscope and analyzed by flow cytometry using the Accuri C6 program.
4.15. Plasmids Construction
For the generation of enhanced green fluorescent protein-LC3 (EGFP-LC3) transfection vector, the full length of human microtubule-associated protein 1 light chain 3 (LC3; GenBank accession no. NM_022818.5) was amplified by RT-PCR using forward (5′-GTTCTCGAGCTATGCCGTCGGAGAAGA-3′) and reverse (5′-AAGGATCCTTACACTGACAATTTCATCC-3′) primers, and the SK-N-BE(2) cDNA was used as template. The PCR fragment of LC3 protein was digested using with XhoI and BamHI (Enzynomics, Daejeon, Korea) and was cloned into pEGFP-C1 plasmid (Clontech, Palo Alto, CA, USA). All constructs were verified by sequencing. The mRFP-GFP-LC3 expression plasmid was a generous gift from Jaerak Chang (Ajou University, Suwon, Korea).
4.16. Cell Transfection and Stable Cell Screening
Transient transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. SK-N-BE(2) cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS) and antibiotics in 5% CO2 at 37 °C. When the confluence reached 70% in 6-well culture plates and then transfected with 5 μg plasmid per well (plasmid (μg): Lipofectamine 2000 (μL) = 1:3). Forty-eight hours after transfection, cells were digested with 0.25% trypsin and the cultures were transferred to the plates for further culture with complete DMEM containing 1000 mg/L G418 for 14 days. When the amount of resistant cell clones was observed, they were digested with 0.25% trypsin and then transferred to a new culture flask using an aseptic pipette for further culture. Selected cells were used for additional autophagy analysis.
4.17. Detection of GFP-LC3 or mRFP-GFP-LC3B Assay
After the pEGFP-LC3 or mRFP-GFP-LC3B stable SK-N-BE(2) cells were treated with CK or/and CQ for 24 h, the cells were fixed, and mounted. Images from slides were observed under a fluorescence microscope CELENA S.
4.18. Measurement of Mitochondrial ROS Generation
The formation of mitochondrial ROS was measured using MitoSOXTM
Red (Life technologies, Eugene, OR, USA) assay. MitoSOXTM
Red reagent is oxidized by superoxide once inside the mitochondria, and is converted to a fluorogenic oxidation product upon binding to nucleic acids [68
]. For microscopy, cells were treated with 5 μM CK for 24 h in the presence or absence of CQ and RAPA. After staining with MitoSOXTM
Red, images were collected using a fluorescence microscopy. Mean fluorescence intensity of images was analyzed using the Image J.
4.19. Tumor Xenograft Studies
Animal studies were performed as described previously [69
]. The mice were maintained in a specific pathogen free environment. SK-N-BE(2) cells (3 × 107
) in 100 µL (PBS: Matrigel = 1:1) were injected subcutaneously into the right flank of each mouse. A week after, tumor injected mice were randomly divided into vehicle, CK, CQ, and CK + CQ groups (n = 5 per group). The vehicle group was given DMSO, and the treatment groups were injected with CK (30 mg/kg), CQ (50 mg/kg) (Sigma-Aldrich) or both drugs, respectively. All drug injection was administered intraperitoneal three times per week. Body weight and tumor volume were also measured three times a week, and the tumor volume was calculated using the following formula: tumor size (mm3
) = π/6 × (length × width × height). Tumor inoculated mice were sacrificed after 60 days. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Chonbuk National University (permit number: CBNU-2018-013).
4.20. Histology and TdT-Mediated dUTP Nick end Labelling (TUNEL)
All immunohistochemistry experiments were performed as described previously [69
]. Paraffin-embedded samples were sliced into 4 µm sections. After antigen retrieval, tissue samples were blocked using blocking buffer (5% goat serum) for 30 min. After washed three times with PBS, samples were incubated in primary antibody (cleaved caspase 3, 1:250) at 4 °C overnight. After washed three times with PBS, samples were incubated for 1 h at RT with an Alexa-488 conjugated antibody. TdT-mediated dUTP nick end labelling (TUNEL) was performed with an In Situ Cell Death Detection (TMR red) Kit (Sigma; St. Louis, MO, USA) according to the manufactured instructions.
4.21. Statistical Analysis
All experiments were performed in triplicate and data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS (version 12.0, SPSS Inc., Chicago, IL, USA). Significant differences between treatment effects were determined by one-way analysis of variance or two-tailed Student’s t-test analysis, and p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) were considered statistically significant.