Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and ranks as the fourth leading cause of cancer-related deaths in both sexes, globally [1
]. In fact, a global estimate of 1,678,127 colorectal cancer cases, has been predicted for the year 2020 [2
]. With a 5-year relative survival rate is ≥90% in stage I disease, and ~10% in stage IV disease, the therapeutic mainstay of CRC therapy is surgery, neoadjuvant radiotherapy in rectal cases, and adjuvant chemotherapy for advanced stage and high-risk stage II colon cancer [3
Over the last decade, a decline in the incidence and mortality rates of CRC has been recorded in high-income countries; conversely, these rates continue to rise in low-resourced nations. This disparity in rates may be attributed to early screening, improved diagnostic techniques, and better therapeutic strategies in the former, and senilization, increased embrace of a Western lifestyle, late diagnosis, the excessive cost of conventional therapy in the latter [4
], thus necessitating the discovery and/or development of a relatively cheaper and more accessible therapeutic strategy. Similarly, the cumulative evidence of the presence and implication of a small subset of the CRC bulk-termed CSCs in the tumorigenicity, oncogenicity, tumor progression, and disease recurrence of CRC [6
], further begs the case for a novel therapeutic option with CSCs—targeting potentials, high curative efficacy, and low drug-associated toxicity.
In an earlier work by our team, we demonstrated that 4-acetylantroquinonol B (4-AAQB), a mycelial isolate of Antrodia camphorata
, a common Taiwanese camphor tree mushroom with a broad range of documented bioactivities, effectively disrupts essential oncogenic signaling pathways such as the Lgr5/Wnt/β-catenin, JAK-STAT, and non-transmembrane receptor tyrosine kinase signaling pathways, inhibits the acquisition of the CRC-stem cell (SC) phenotype, down-regulates the expression and/or activities of stemness-associated genes including ALDH1
, attenuates tumor aggression, and accentuates chemosensitivity in CRC cells [8
]. Consistent with the growing evidence that microRNAs (miRNAs) are deregulated in several human cancer types including breast cancer, lung cancer, and colon cancer, potentially serving as screening and diagnostic factors [9
], as well as Leufkens et al.’s prospective cohort-nested case-control study (1064 CRC cases, 1064 matched controls), which provided evidence for the involvement of biomarkers of oxidative stress in the development of CRC [11
], as a sequela to our previously published work, in this present study, we continued exploring the therapeutic potential of 4-AAQB in CRC, particularly in the context of the probable regulatory role of 4-AAQB on the epigenetic landscape and modulation of oxidative stress in CRC.
In humans, superoxide dismutases (SODs), with three subtypes, namely SOD1/ Cu,Zn-SOD, SOD2/Mn-SOD, and SOD3/extracellular SOD, constitute a principal component of the antioxidant defense system, and are also known as the antioxidases. SOD2 is a 96 kDa homotetramer nuclear-encoded mitochondrial manganese (Mn)-containing antioxidant enzyme. Through its interaction with, and binding to superoxide byproducts of oxidative phosphorylation, SOD2 is able to convert them into diatomic oxygen and hydrogen peroxide (H2
). The Mn at the SOD2 active site catalyzes this disproportionization of superoxide anion to oxygen and H2
, akin to SOD1 and SOD3 [12
]. Despite documented association between mutations in the SOD2 gene and several pathologies, including sporadic motor neuron disease, idiopathic dilated cardiomyopathy (IDC), premature aging (progeria), and cancer, the role of SOD2 in cancer cells, such as in CRC cells, is not fully understood. While there are divergent views on the actual role of SOD2 in cancer, there is growing evidence that the loss of SOD2 function may not facilitate metastatic disease progression as previously thought; rather, there is an indication from experimental and epidemiological studies that enhanced SOD2 expression levels correlate with metastatic malignantization and disease progression in various cancer types [13
]. It has been shown that the up-regulation of SOD2 protein expression is characteristic of certain cancers, including cervical and salivary adenoid cystic carcinoma [14
Deregulation of epigenetic factors and patterns, such as aberrant DNA methylation, histone tail modification, and non-coding RNA (ncRNA) regulation, are implicated in loss of cell identity and contribute to several human pathologies, including cancer. Recent advances in the field of epigenetics are coupled with accumulating evidence of the critical role of non-coding RNAs, particularly miRNA expression and/or activity in oncogenicity [16
]. miRNAs are approximately 22 nucleotide (nt) long evolutionarily conserved small non-coding RNAs (ncRNAs) with the ability to elicit endogenous post-transcriptional silencing of its target genes based on complementarity with target sites in the 3′-untranslated regions (3′-UTRs) of the target gene/messenger RNA (mRNA) [16
]. There is documented correlation or association between down-regulated miRNA expression, and tumor initiation or metastatic disease progression [17
], however, there is a dearth of information regarding SOD2-regulated miRNA(s) in CRC, and regarding how the epigenetic modulation of SOD2 contributes to the CSC-like and metastatic phenotype of CRC cells.
Against the background of the conflicting data on SOD2 in malignancies, in this present study, we investigated the role of SOD2 in CRC, as well as, if and how 4-AAQB affects SOD2 expression profile in CRC. Additionally, considering the suggested cancer-related bivalence of SOD2, we also probed for probable epigenetic undertone to SOD2-associated phenotype and likely SOD2-attenuating 4-AAQB therapeutic activity in CRC. Consequently, herein, we provide evidence that hsa-miR-324 interacts with SOD2, and that the aberrant expression of SOD2 enhances the oncogenicity and cancer stem cell-like phenotype of CRC cells and the therapeutic effect of 4-AAQB in these cells is mediated by inducing re-expression of SOD2-suppressed hsa-miR-324.
In our previous work [8
] on the role of 4-AAQB in CRC, we provided evidence that 4-AAQB exhibits potent antiproliferative and anti-CSC therapeutic effects in CRC. We showed that 4-AAQB effectively reverses or attenuates the resistance of cancer cells to 5-FU or FOLFOX anticancer therapy, thus, enhancing chemosensitivity both in vitro and in vivo. Furthermore, we demonstrated that the JAK/STAT signaling pathway plays a vital role in the 4-AAQB-mediated suppression of CRC-SCs, thus projecting it as a putative therapeutic target in CRC treatment. Thus, we concluded that the relatively novel phytoalexin, 4-AAQB, was a potent therapeutic agent for single-agent or components of standard combination chemotherapy. With increased understanding of the epigenetic landscape in cancer pathologies, there has been increased interest and investigation of an epigenetics-mediated multipronged targeted-therapy strategy in tackling the clinical challenge of primary or evolved required resistance [21
In this present study, consistent with current knowledge that the epigenetic modulation of genes is a critical mechanism underlying the CRC oncogenicity and disease progression, as well as the development of resistance to chemotherapeutics, we demonstrate a novel and previously undocumented epigenetic-based mechanism of 4-AAQB therapeutic activity in CRC, involving the down-regulation of tumor-promoting SOD2 by the suppressor ncRNA, hsa-miR-324-5p. This negative epigenetic modulation of SOD2 was shown to be associated with the converse enrichment of hsa-miR-324 and through the interaction of the 3′-UTR of SOD2 mRNA to the 5′-UTR of hsa-miR-324 (Figure 1
and Figure 2
). Located on chromosome 6 (6q25.3 region) and resident in the mitochondrial matrix, the human homotetrameric SOD2 consists of four Mn3+
-harboring sub-units, catalyzes the disproportionation of free radical superoxides to O2
, and lowers cell susceptibility to oxidative injury in a genotoxic condition [12
]. In most of the early documentations, SOD2 was implicated as a tumor suppressor [24
]; however, more recently, there is accruing evidence that of the oncogenic/tumor-promoting role of SOD2 [13
]. Consistent with the latter, in this study we demonstrated the oncogenic role of the aberrant expression of SOD2 in CRC, as evidenced by its ability to facilitate metastatic disease progression through the repression of hsa-miR-324 (Figure 3
). In addition, we also showed that 4-AAQB inhibits the viability and/or proliferation of human CRC SP cells in a hsa-miR-324-mediated manner, with associated attenuation of the SOD2-facilitated EMT and enhanced cell-death in the SP cells (Figure 4
and Figure 5
; Supplementary Figure S1
). These findings are consistent with the demonstrated suppression of the invasion and migration of hepatocellular carcinoma (HCC) cells by hsa-miR-324-5p-induced down-regulation of the specificity protein 1 (SP1) and E26 transformation-specific protein 1 (ETS1) [25
], where decreased SP1 binding to the SOD2 promoter inhibits the constitutive activation of SOD2 in breast cancer cells [13
], since the proximal promoter that mediates the transcription of SOD2 is TATA-less and contains binding sites for several transcription factors, including ETS1 and SP1. It is of therapeutic relevance that the hsa-miR-324-inducing 4-AAQB inhibits the SOD2-mediated motility, invasiveness, and clonogenicity of colorectal cancer SP cells (Figure 6
Like most malignancies, CRC is a SC-driven pathology, characterized by the presence of a sub- or side-population within the tumor bulk which is characteristically quiescent, phenotypically ALDH
+, functionally capable of self-renewal, and underlies innate or acquired insensitivity to chemotherapy, metastasis, and disease recurrence [26
]. The targeting and killing of these CSC-like SP cells by 4-AAQB is posited as a rational and efficacious therapeutic approach, as it eliminates the quiescent, slowly-dividing, and characteristically therapy-resistant tumor-initiating (and maintaining-) cells, alongside the sensitive rapidly-dividing non-SP cells. Consistent with this, we provided evidence that 4-AAQB, in a dose-dependent manner, effectively targets the primary colonospheres and subsequent generations of colonospheres, recapitulating the pharmacological inhibition of CRC-SC-related self-renewal or propagation, with associated attenuation of the expression of critical pluripotency transcription factors (Figure 7
Understanding that self-renewal, a vital property of the CSC-like SP cells, is associated with the facilitation and maintenance of the proliferative capacity of cancerous cells, makes the clinical implication of our data apparent, as it highlights the putative ability of 4-AAQB to negatively modulate oncogenic self-renewal signaling, thus enhancing the sensitivity of malignant cells to chemotherapeutics, altering their survival strategies, limiting tumorigenesis and/or oncogenicity, and apparently impeding disease recurrence [28
]. From in vivo validation of our in vitro findings, we confirmed that 4-AAQB alone or in synergism with FOLFOX reduces the tumorigenicity of CRC cells by suppressing SOD and up-regulating hsa-miR-324 expression, in vivo (Figure 8
). This is clinically relevant and important, not only because it is, to the best of our knowledge, the first demonstration of an hsa-miR-324-5p-mediated anticancer activity of 4-AAQB in CRC, but also because despite advances in anticancer therapy, resistance to chemotherapy remains a great challenge in the long-term management of incurable metastatic disease, and it subsequently contributes to cancer-related mortality. FOLFOX is the therapeutic mainstay in postoperative CRC patients, having been shown in several trials to contribute to increased progression-free (PFS) and overall (OS) survival, with response rates as high as 50%; however, resistance to FOLFOX does occur because of its reduced intracellular entry or enhanced efflux from the CRC cells by ABC-type multi-drug resistance (MDR) transporters or copper-transporting p-type ATPases (reviewed in [29
]). In the light of this, we showed in addition that 4-AAQB enhances the sensitivity of CRC cells to the standard of care FOLFOX, and significantly potentiates the anticancer effect of FOLFOX in the murine CRC xenograft models (Figure 8
; Supplementary Figure S2
Ranking as the third most common cause of cancer and the fourth most common cause of cancer-related deaths worldwide, CRC remains a major health problem. FOLFOX, comprising of folinate (leucovorin), fluorouracil (5FU), and oxaliplatin, is currently the drug of choice for patients with CRC; however, some patients are non-responsive or develop insensitivity to FOLFOX, resulting in treatment failure and consequently, tumor progression, thus, necessitating a therapeutic approach with enhanced efficacy. This present study provides evidence that 4-AAQB alone or by synergistically interacting with FOLFOX, exhibits an enhanced ability to kill CRC cells, inducing marked apoptosis via an epigenetically modulated pathway. Mechanistically, hsa-miR-324-mediated inhibition of the SOD2-activated oncogenicity and the cancer stem cell-like phenotype of CRC cells is a probable critical component of our observed 4-AAQB—FOLFOX anticancer synergism.
Interestingly, corroborating the critical role of epigenetic alterations in the unrestrained growth, invasion, colonization and acquired resistance of cancerous cells [16
], we validated the reciprocal suppression of the pro-tumorigenic and pro-metastatic SOD2 and suppressor miR, hsa-miR-324 ex-vivo, showing that the expression of hsa-miR-324 levels in the resected tumor tissue from 4-AAQB- and/or FOLFOX- treated mice was significantly up-regulated, while conversely, that of SOD2 was reduced markedly, compared to the untreated group (Figure 8
). Overall, the data demonstrates that the hsa-miR-324/SOD2 signaling axis is a putative novel therapeutic target, and a potential prognostic marker in patients with CRC.
Put together, this present study uncovers a novel mechanistic underlining the therapeutic effect of 4-AAQB alone or in combination with FOLFOX in the activation of cell death signals, attenuation of CSC-like phenotypes, and induction of apoptosis in the CRC cells. Akin to the single-agent therapy, the pharmacological synergism involves multiple mechanisms as summarized in the Scheme 1
. The enhanced cytotoxicity of 4-AAQB—FOLFOX dual-agent therapy is clinically relevant. We propose that SOD2-enrichment in the CRC cells accentuates mitochondrial biogenesis, activates the intracellular anti-oxidative machinery, alleviates oxidative stress, and enhances the maintenance of the CSC-like phenotype, as well as acquisition of resistance to chemotherapeutic agents, by functional suppression of hsa-miR-324-5p expression and/or activity; however, exposure to 4-AAQB induces the re-expression of hsa-miR-324-5p, attenuates SOD2 expression, and sensitizes the CSC-like SP cells in CRC to FOLFOX therapy. Thus, we propound a novel perspective on the putative roles of 4-AAQB as a hsa-miR-324-mediated CSC-targeting small molecule inhibitor of SOD2 in CRC, in vitro and in vivo.
4. Materials and Methods
4.1. Reagents and Drugs
The compound 4-acetylantroquinonol B (4-AAQB, >99% purity) was obtained from New Bellus Enterprises Co., Ltd. (Tainan, Taiwan), while Leucovorin calcium (PHR1541 SIGMA-ALDRICH, 5-FU (F6627 SIGMA, ≥99% HPLC) and oxaliplatin (PHR1528 SIGMA-ALDRICH) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Stock solutions of 10 mM in dimethyl sulfoxide (DMSO, Sigma-Aldrich) or sterile ddH2O for 4-AAQB or FOLFOX, were stored at −20 °C or 4 °C, respectively, until use. Anti-SOD2 (ab13533 Rabbit pAb) antibody was purchased from Abcam plc. (Biochiefdom International Co., Ltd., New Taipei City, Taiwan), antibodies against E-cadherin (24E10: #3195, Rabbit mAb), N-cadherin (D4R1H: #13116, Rabbit mAb), and vimentin (D21H3: #5741, Rabbit mAb) were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Anti-c-Myc (9E10: sc-40), Oct4 (C-10: sc-5279), Sox2 (E-4: sc-365823), Bax (B-9: sc-7480), Bcl-xL (7B2.5: sc-56021), and β-actin (C4: sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 647 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-rabbit IgG were purchased from Invitrogen (Grand Island, NY, USA).
4.2. Cells and Cell Culture
The human non-tumor colon epithelial cell line FHC (ATCC-CRL-1831), as well as the CRC cell lines DLD-1(ATCC-CCL-221) and HCT116 (ATCC-CCL-247) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in DMEM/F-12 (GibcoTM, 12-634-010; Thermo Fisher Scientific Inc., Waltham, MA, USA), RPMI-1640 (GibcoTM, 11-875-119) and McCoy’s 5A modified (GibcoTM, 16-600-082) medium, respectively. Both media were supplemented with 10% fetal bovine serum (FBS, Sigma, F7524) and 1% penicillin-streptomycin (GibcoTM, 15140) at 37 °C, in a 5% humidified CO2 incubator (Shel Lab, Sheldon Manufacturing Inc, Cornelius, OR, USA). Cells were sub-cultured at 90% confluence and the media changed every 72 h.
4.3. Access and Probe of Online Cancer Data Set
The Cancer Genome Atlas (TCGA) PANCANCER cohort dataset consisting of 14 cancer types were accessed, and analyses of the expression profile of SOD2 and hsa-miR-324 in normal–cancer tissue pairs was performed using the StarBase v2.0 software algorithms [30
]. The TCGA colon cancer (COAD) cohort was also analyzed for the influence of SOD2 or hsa-miR-324 expression on survival rates.
4.4. miRNA Profiling and Secondary Structure Prediction
4.5. Cell Viability and Drug Combination Assays
CRC cells were seeded in supplemented media at a density of 4 × 103 cells/well in triplicates in 96-well plates and incubated for 24 h in humidified 5% CO2 at 37 °C before exposure to different concentrations of 4-AAQB for 48 h. Cell viability and/or proliferation were assessed by a sulforhodamine B (SRB, Sigma; 59012-5G) colorimetric assay following the manufacturer’s instructions. Untreated wild type cells served as control. The assay was performed three times in triplicates. Optical density (OD) was measured at 495 nm wavelength, using a SpectraMax microplate reader (Molecular devices, Kim Forest Enterprises Co., Ltd., New Taipei City, Taiwan).
4.6. Flow Cytometry-Based Side Population Analysis
To identify the CRC side population (SP), the DLD-1 or HCT116 cells were washed in warm RPMI-1640 media supplemented with 2% FBS and 10 mmol/L HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer (Invitrogen-Life Technologies, Carlsbad, CA, USA), re-suspended at a density of 1 × 106 cells per mL of RPMI-1640 supplemented with 2% FBS and 10 mmol/L HEPES buffer containing 5 μg/mL Hoechst 33342 dye, and incubated at 37 °C for 1.5 h with gentle agitation. As control, SP cells were incubated with 50 μM verapamil (Sigma-Aldrich), an ABC transporter inhibitor, washed with ice-cold Hank’s balanced salt solution (HBSS) supplemented with 2% FBS and 10 mmol/L HEPES buffer, centrifuged, and re-suspended in ice-cold supplemented HBSS. Cells were exposed to 1 μg/mL 7-Aminoactinomicin D (7-AAD, #A1310, Molecular Probes, Thermo Fisher Scientific Inc., Eugene, OR, USA) to delineate only viable cells. To obtain a single cell suspension, cells were filtered through a 70-μm filter, sorted into SP and non-SP cell fractions, then analyzed on the BD FACSAria II System (BD Biosciences, San Jose, CA, USA). The purity ≥ 98%.
4.7. Western Blot Analysis
Cultured CRC cells were harvested and washed thrice with PBS and lysate prepared using ice-cold lysis buffer solution. After being heated at 95 °C for 5 min, immunoblotting of the protein lysate was performed. Blots were blocked for 1 h with 5% skimmed milk in Tris Buffered Saline with Tween 20 (TBST), incubated overnight at 4 °C with specific primary antibodies against SOD2 (1:1000), E-cadherin (1:2000), N-cadherin (1:2000), vimentin (1:1000), c-Myc (1:1000), BAX (1:1000), BCL-xL (1:1000), and β-actin (1:500). Thereafter, the polyvinylidene difluoride (PVDF) membranes were washed three times with TBST, then incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 1 h at room temperature and washed with TBST again before band detection using enhanced chemiluminescence (ECL) Western blotting reagents and imaging with the BioSpectrum Imaging System (UVP, Upland, CA, USA).
4.8. miRNA and Small Interfering RNA (siRNA) Transfection
For miRNA transfection, micrONTM hsa-miR-324-5p mimic (#miR10000761-1-5), micrOFFTM hsa-miR-324-5p inhibitor (#miR20000761-1-5), micrONTM mimic negative control #22 (# miR01101-1-5) and micrOFFTM inhibitor negative control #22 (#miR02101-1-5) were purchased from RiboBio (Guangzhou RiboBio Co. Ltd, Guangzhou, China). For transient silencing of SOD2, the SOD2-specific siRNA (si-h-SOD2_001, B000006648A-1-5) were also purchased from RiboBio (Guangzhou RiboBio Co. Ltd., Guangzhou, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for the transfection of the miRNA oligonucleotides and siRNAs following the manufacturer’s protocol. The total RNA or protein were extracted 48 h after transfection and used for the RT-PCR or Western blot analyses.
4.9. Scratch Wound Healing Assay
Cell migration potential was evaluated using the wound healing assay. Briefly, DLD-1 or HCT116 cells dissociated from SP-derived DLD-1 or HCT116 tumorspheres (Sp) were seeded onto 6-well plates (Corning Inc., Corning, NY, USA) with complete growth media containing 10% FBS, and cultured to 99–100% monolayer confluency. The cell monolayers were scratched with a sterile yellow pipette tip to denude the culture wells. The cell migration images were captured at the 0 and 24 h time points after denudation, under a microscope with a 10× objective lens, and analyzed with the NIH ImageJ software (https://imagej.nih.gov/ij/download.html
4.10. Transwell Matrigel Invasion Assay
Using the 24-well plate Transwell matrigel invasion system, 3 × 104 DLD-1 or HCT116 SP cells were seeded into the upper chambers of the inserts (BD Bioscience, 8 μm pore size) containing FBS-free media with different concentrations of 4-AAQB; media containing 10% FBS in the lower chamber served as a chemo-attractant. After 24 h cell incubation, media were discarded; cells on filter membrane were fixed with 3.7% formaldehyde for 1 h, and then stained with 0.2% (w/v) crystal violet solution, for 15 min, while cells on the upper side of the inserts were gently removed with a cotton swab. The invaded cells were visualized, and the invasive capacity was evaluated as the total number of cells on the lower surface of the membrane, as determined by microscopy.
4.11. Colony Formation Assay
2 × 104 DLD-1 or HCT116 cells were plated into 6-well cell culture plates (Corning Inc., Corning, NY, USA) and incubated for 2 weeks at 37 °C after treatment with different concentrations of 4-AAQB. Then, cells were washed thrice with PBS, fixed with ice-cold methanol, stained with 0.005% crystal violet, washed with PBS, and dried at room temperature. Formed colonies were then assessed and counted under microscope. In each well, the total number of colonies with diameter ≥ 100 μm was counted over six randomly selected fields in triplicate assays.
4.12. Tumorsphere and Clonal Tumorsphere Formation Assay
5 × 103 DLD-1 or HCT116 cells were plated into wells containing stem cell media with different concentrations of 4-AAQB in ultra-low attachment 6-well plates (Corning Inc., Corning, NY, USA) in quadruplicate. The stem cell media consisted of serum-free growth media, supplemented with 1 × B27 supplement, 10 ng/mL human basic fibroblast growth factor (bFGF; Invitrogen, Grand Island, NY, USA) and 20 ng/mL epidermal growth factor (EGF; Invitrogen, Grand Island, NY, USA), and was changed every 72 h. After 12 days of culture, primary colonospheres consisting of ≥20 cells were counted, and images acquired. Secondary colonospheres were generated by dissociating primary colonospheres by trypsinization,and pipetted through a 22G needle to obtain a single-cell suspension (Thermo Fisher Scientific Inc.). After dissociation of the primary tumorspheres, cell seeding and exposure to 4-AAQB was akin to that for primary tumorspheres.
4.13. Immunohistochemistry and Immunofluorescence Staining
Immunohistochemical (IHC) analyses of the Taipei Medical University-Shuang Ho Hospital CRC cohort consisting of different tumor stage colorectal cancer specimens with different clinical stages was performed. Recommendations of the Declaration of Helsinki for biomedical research involving human subjects were followed. Ethical approval for the study was obtained from Joint Institutional Review Board of the Taipei Medical University (approval number: N201602054). A review of patients’ clinical records to determine tumor stage at the time of diagnosis and outcome was carried out. Antibodies against SOD2 (1:200, ab13533 Rabbit pAb) were used in accordance to standard IHC procedure. SOD2 expression was evaluated and scored by two independent pathologists using the quick-score (Q-score) based on the staining intensity (I) and stained cells percentage (P). Staining intensity was defined as 0 (no staining), 1+ (weak), 2+ (moderate), and 3+ (strong). where the maximum score was 300. For the immunofluorescence (IFC) staining, untreated or 4-AAQB-treated DLD-1 or HCT116-derived colonospheres were plated onto poly-L-lysine-coated glass cover-slips, fixed with 4% paraformaldehyde, and carefully washed thrice with PBS. This was followed by cell permeabilization with 0.1% Triton X-100/PBS solution for 10 min, and incubation with primary antibodies against Oct-4 and Sox2 (Santa Cruz, CA, USA), then with goat anti–mouse Alexa Fluor488 secondary antibodies (Cat. #: R37120, Thermo Fisher Scientific Inc.). Cell nuclei were labeled with DAPI (4′,6-diamidino-2-phenylindole; Cat. #: D1306, (Molecular Probes, Thermo Fisher Scientific Inc.). Cell visualization and imaging was done using the Nikon E800 fluorescent microscope.
4.14. RNA Extraction, Real-Time Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from the CRC cell line DLD-1 and resected xenograft tumor sample, using the RNeasy kit (Qiagen Inc., Gaithersburg, MD, USA). The miRNEasy kit (Qiagen) was used for miRNA purification. Total RNA concentration was determined using NanoDrop ND1000 spectrophotometer (Nyxor Biotech, Paris, France). The PCR mixtures were prepared using the SYBR Green Master Mix (Applied Biosystems, Life Technologies, Grand Island, NY, USA). The PCR contained the primers, the fluorogenic probe mix, and the TaqMan Universal PCR Master mix (Applied Biosystems). Amplification reactions were performed in triplicate from 20 ng complementary DNA (cDNA) using the Bio-Rad C1000 real-time PCR system (Bio-Rad, Cambridge, MA, USA) using the following conditions: 95 °C for 3 min, 35 cycles at 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, and 72 °C for 10 min. Results were analyzed, and all values were normalized to the levels of the housekeeping gene β-actin, which served as the internal control. All procedures were in accordance to the manufacturers’ instructions. The PCR primer sequences were as follows: SOD2 (forward): 5′-GCCTCCCTGACCTGCCTTAC-3′, (reverse): 5′-GTGATTGATATGGCCCCCG-3′; hsa-miR-324-5p (forward): 5′-CGCGGATCCGGGTGGATGTAAGGGATGAG-3′; (reverse): 5′-CCGGAATTCTTGGGCTGATCCAGGAGAAG-3′; and β-actin (forward): 5′-CCCTAAGGCCAACCGTGAA-3′, (reverse): 5′-CCAGAGGCATACAGGGACAAC-3′
4.15. Oral Administration of 4-AAQB In Vivo and Primary Tumor Experiments
Severely immunocompromised NOD/SCID mice (6 weeks old) were purchased from BioLASCO (Taipei, Taiwan) and housed in Memorial Hospital Animal Center under SPF conditions. All the animal experiments were performed with strict adherence to the regulations set by MacKay Memorial Hospital Animal Center (Approved protocol number: MMH-104-024). The mice were bred and maintained in pathogen-free conditions, with sterile water and feed, in air-controlled rooms with a specifically designed 12 h light/dark cycle. 1 × 105 DLD-1 cells suspended in 100 μL PBS were injected subcutaneously into the right flank of 6–8-week-old, female NOD/SCID mice (n = 5/treatment group; average body weight, 22.5 ± 3.74 g). Tumor growth was monitored daily, and tumor size was measured every 48 h with calipers based on the formula: , where x = longest diameter, and y = diameter perpendicular to x. When tumors became palpable (~50–150 mm3) on day 7–8 after inoculation, the mice were randomly assigned to treatment groups, with oral gavage of vehicle (100 mL 50 mM PBS), 4-AAQB (n = 5; 5 mg/kg q48h), FOLFOX (n = 5; 15 mg/kg 5-FU q24h, 5 mg/kg folinate q24h and 5 mg/kg oxaliplatin q1wk), or 4-AAQB + FOLFOX (n = 5), for five weeks. Tumors were resected and weighed after humane sacrifice of the mice. The feeding habits, motor activity and body weights of the mice were all monitored as indicators of murine general health. Analysis of variance (ANOVA) was used to establish differences between groups, and significance levels were determined by a non-parametric Kruskal-Wallis test.
4.16. Statistical Analysis
All assays were performed at least thrice in triplicates. Values were expressed as mean ± standard error of mean (SEM). Comparison between two groups was estimated using a 2-sided Student’s t-test, while a one-way analysis of variance (ANOVA) was used for comparison between three or more groups. All statistical analyses were performed utilizing the GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). p-value < 0.05 was considered to be statistically significant.