Colorectal cancer (CRC) is the third most common cancer in both men and women [1
]. The standard treatment for CRC is chemotherapy following colon resection by surgery [2
]. For chemotherapy, either FOLFOX (5-fluorouracil and leucovorin with oxaliplatin) or FOLFIRI (5-fluorouracil, leucovorin with irrinotecan) combined with bevacizumab is mainly used [3
]. Despite advances in treatment strategies for CRC, the prognosis remains poor because of the high rates of metastasis [4
]. Oxaliplatin is the first platinum-based drug to demonstrate clinical effectiveness against CRC, and it remains one of the most effective chemotherapeutic drug for CRC treatment along with 5-fluorouracil and leucovorin [4
]. However, repeated and long-term administration induces drug resistance through the promotion of export from the cells and nucleotide excision repair by increasing expression of the multidrug resistance protein, glutathione, and excision repair cross-complementation group 1 [6
]. Therefore, there is a need to explore new strategies to improve the efficiency of CRC treatment by identifying molecules and mechanisms associated with oxaliplatin resistance.
Autophagy (macroautophagy) is a process involving the lysosomal degradation of cytosolic proteins, damaged organelles, and invasive microbes in autophagosomes, which are double-membrane vesicles generated by extension of phagophores [8
]. Chemotherapy acts as a stress in the cells and increases apoptosis inhibition, autophagy increase, and epithelial-mesenchymal transition (EMT)-competent phenotypes through Beclin-1, Bcl-2, mammalian target of rapamycin (mTOR), adenisine monophosphate (AMP)-activated protein kinase (AMPK), and select microRNAs. In CRC, autophagy increases EMT-competent CRC cells and acquires resistance to chemotherapeutic drugs by the TP53-dependent pathway [9
Nitric oxide synthases (NOSs) are enzymes that catalyze the production of nitric oxide (NO) from L-arginine. NO is important for maintaining vascular tone, insulin secretion, and angiogenesis. There are three mammalian NOS isoforms: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3) [10
]. Although NOS expression is associated with cancer progression and metastasis, recent studies have suggested that NOS3 may inhibit tumor growth, invasion, and angiogenesis, particularly in breast cancer, and CRC [11
Cannabidiol (CBD) is one of the major components of Cannabis sativa
]. It is non-psychoactive and widely used to treat diseases, such as neurological diseases and cancer [15
]. Many clinical trials for its use in glioblastoma treatment are also currently underway [16
]. CBD is known to exert its antitumor effects through Noxa activation, downregulation of protein kinase B (AKT)/mTOR, and mitogen-activated protein kinase signaling [14
]. However, CBD has not been studied for its potential to overcome resistance to chemotherapeutic drugs.
In this study, we investigated whether CBD overcomes oxaliplatin resistance in CRC cells, and the relationship between NOS3 downregulation and combined oxaliplatin and CBD treatment-induced autophagy. We demonstrate, for the first time, that CBD enhances oxaliplatin-mediated autophagy via NOS3-mediated mitochondrial dysfunction, suggesting that NOS3 is a potential therapeutic target for overcoming oxaliplatin resistance and that CBD may be a new therapeutic agent for CRC treatment.
Oxaliplatin, which is commonly used for CRC treatment, binds covalently to DNA nucleobases to form guanine–guanine and guanine–adenine DNA links [4
]. This abnormal binding interferes with the structure of DNA and inhibits DNA replication, repair, and transcription, triggering double-strand breaks and causing apoptosis [5
]. However, drug resistance occurs in ~40% of CRCs treated with oxaliplatin [21
]. Therefore, a new therapeutic strategy needs to be developed to overcome oxaliplatin resistance in CRCs. In this study, we investigated whether there is a synergistic effect when CBD is combined with oxaliplatin for CRC treatment. Additionally, we analyzed the mechanism whereby CBD overcomes oxaliplatin resistance in CRC cells and report, for the first time, that CBD downregulates NOS3 activity, resulting in mitochondrial dysfunction and finally, leading to autophagy.
Oxaliplatin resistance is acquired through a variety of mechanisms, including inefficient cellular accumulation of drugs [22
], promotion of DNA repair [23
], activation of anti-apoptotic pathways [24
], and changes in cellular metabolism. An important finding of this study is that NOS3 is closely related to oxaliplatin resistance. NOS is an enzyme that converts L-arginine to L-citrulline to produce NO in various cell types [25
]. It has been mainly studied for its function in endothelial cells. In addition, NOS has been reported to be associated with resistance to cisplatin and 5-fluorouracil through Wnt signaling and family with sequence similarity 171, member B in non-small cell lung cancer, breast cancer, and leukemia [7
]; but this is a report of drug resistance regulated by NOS2. Our results showed that the activity of NOS3 was increased in oxaliplatin-resistant cells compared to that in their parent cells and this was attenuated by CBD, resulting in decreased NO production. Moreover, studies on the effects of CBD on overcoming drug resistance have rarely been performed on cancers other than glioblastoma [26
]. Therefore, NOS3 plays an important role in acquiring resistance to oxaliplatin, and CBD can decrease NO production by reducing NOS3 activity and thus increase oxaliplatin sensitivity.
Intracellular NO produced by NOS3 directly affects mitochondrial function through oxidative stress [28
]. Consistent with this, we found that CBD reduced OCR, a direct marker of mitochondrial dysfunction, and significantly decreased MMP, mitochondrial complex I activity, the number of mitochondria, and the levels of the mitochondrial membrane protein, cardiolipin. Moreover, CBD increased the levels of intracellular ROS, especially mitochondrial ROS, and decreased the protein expression of the mitochondrial antioxidant enzyme, SOD2. NOS3 knockdown further reduced SOD2 levels in cells treated with CBD and oxaliplatin, indicating that the downregulation of NOS3 induced by CBD causes mitochondrial dysfunction and ROS overproduction by SOD2 reduction.
Autophagy is known to be involved in cell survival by regulating intracellular homeostasis in response to stress [31
]. However, in recent decades, autophagy has also been reported to be associated with cell death [32
]. Previous studies have suggested that autophagy promotes the resistance of cancer cells to chemotherapy [33
]. In contrast to those of previous studies, our results showed that the induction of autophagy by CBD increased the response of cells to oxaliplatin. The reason for this discrepancy is unclear, but it may be due to differences in the mechanism of action of CBD or different cellular responses to oxaliplatin compared with other chemotherapy drugs.
Moreover, one of the serious side effects of oxaliplatin is neurotoxicity induced by the pro-inflammatory cytokines, interleukin 6, tumor necrosis factor alpha, and cyclooxygenase 2 [35
]. CBD enhances antioxidant activity and inhibits the secretion of pro-inflammatory cytokines and thus, possibly acts as a neuroprotectant [37
]. Therefore, CBD may attenuate the neurotoxic side effects of oxaliplatin.
4. Materials and Methods
4.1. Cell Lines and Cell Culture
Human CRC DLD-1 and colo205 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Oxaliplatin-resistant DLD-1 (DLD-1 R) and colo205 (colo205 R) cells were developed by long-term exposure to oxaliplatin, with stepwise increases in concentration. All cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 100 mg/mL penicillin and streptomycin in a 5% CO2 atmosphere incubator at 37 °C.
4.2. Establishment of Oxaliplatin-Resistant Cell Lines
Resistance to oxaliplatin was induced by exposing cells to increased concentrations of oxaliplatin (1–20 µM) for 6 months. Initially, 1 µM oxaliplatin was added to the medium, and surviving cells were cultured every 4–5 days. Oxaliplatin dosage was gradually increased when the effect of the drug was considered insignificant. In both cell lines (colo205 R, DLD-1 R), we confirmed the resistance to oxaliplatin in comparison to the parent cells (colo205, DLD-1).
4.3. Reagents and Antibodies
CBD, oxaliplatin, 4-amino-5-methylamino-2’,7’-difluorescein (DAF-FM), SNAP, and NAC were purchased from Sigma Aldrich (St. Louis, MO, USA). 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA), mitochondrial superoxide indicator (MitoSOX), tetramethylrhodamine, ethyl ester, perchlorate (TMRE), 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), MitoTracker, and NAO were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Specific antibodies against LC3A/B, NOS3, SOD1, and VDAC were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SOD3, succinate dehydrogenase complex flavoprotein subunit A (SDHA), RieskeFeS, COX I, and ATP synthase F1 subunit alpha (ATP5A) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phospho-NOS3 (S1177), p62, NDUFA9 were purchased from Abcam (Cambridge, UK). The anti-SOD2 antibody was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and the anti-β-actin antibody was purchased from Sigma Aldrich. Horseradish peroxidase-conjugated anti-mouse IgG was purchased from Santa Cruz Biotechnology and anti-rabbit IgG was from Cell Signaling Technology.
4.4. Cell Proliferation Assay
Cells were seeded onto 96-well plates at a density of 1 × 104 cells/well and incubated overnight. They were then treated with CBD or oxaliplatin for 24 h. Subsequently, WST solution was added to each well for 2 h. The absorbance at 450 nm was then measured using a microplate reader (SPECTRA190; Molecular Devices, Sunnydale, CA, USA).
Immunoblotting was performed as previously described [40
4.6. Green Fluorescent Protein (GFP)-LC3 Puncta
Cells were infected with recombinant adenoviruses expressing GFP-LC3 (a gift from Professor Chang Kyu Lim, Chungnam National University, Daejeon, Korea). Infected cells were then incubated with CBD and oxaliplatin for 6 h and stained with 5 μM lysotracker dye for 30 min at 37 °C. The cells were observed under a confocal microscope (Carl Zeiss, Oberkochen, Germany).
4.7. Autophagic Activity
The Autophagy Detection Kit (Abcam) measures autophagic activity in living cells using a fluorescent detection reagent. Cells were seeded in culture dishes, incubated overnight. Following incubation, they were washed with phosphate-buffered saline (PBS) and pretreated with 1 μM rapamycin (an autophagy inducer) in serum-free RPMI medium for 1 h at 37 °C. Cells were then treated with CBD and oxaliplatin for 12 h and subsequently, incubated with 1 μL of Green Detection Reagent for 30 min at 37 °C in the dark. Cells were harvested using trypsin- ethylenediaminetetraacetic acid (EDTA) and resuspended with 500 μL of 1× assay buffer. Cells were analyzed on the FL-1 fluorescence channel of a flow cytometer.
4.8. Analysis of Cell Death
After treatment with oxaliplatin and CBD, cells were harvested using trypsin-EDTA and stained with Trypan blue. Triplicate wells of viable cells were counted using a hemocytometer.
4.9. Human Phospho-Kinase Array
Cell lysates were assayed using a Proteome Profiler Human Phospho-Kinase Array kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.
4.10. Immunofluorescence Staining
After CBD and oxaliplatin treatment, cells were fixed, permeabilized, blocked, and incubated with primary antibodies. Bound primary antibodies were visualized using an Alexa Fluor-594-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) and cells were stained with 4’,6-diamidino-2-phenylindole (Invitrogen, CA, USA). Finally, cells were mounted and imaged using a confocal microscope.
4.11. Measurement of ROS
DLD-1 R and colo205 R cells were treated with CBD and oxaliplatin for 24 h and subsequently, incubated with 10 μM DCF-DA and 5 μM MitoSOX dye for 30 min at 37 °C. Cells were then harvested and the fluorescence intensity, indicating the level of ROS, was quantitated by flow cytometry.
Small interfering RNAs (siRNAs) targeting NOS3 and SOD2 were purchased from Santa Cruz Biotechnology. pcDNA3-NOS3-GFP and pBI-EGFP-SOD2 plasmids were purchased from Addgene (Watertown, MA, USA). Cells were transfected with siRNAs or plasmids using Lipofectamine RNA iMAX or Lipofectamine 2000 reagent, respectively (Invitrogen, Carlsbad, CA, USA).
OCR was measured in DLD-1 R and colo205 R cell lines using the Seahorse XF-24 extracellular flux analyzer (Seahorse Biosciences, MA, USA). Cells were seeded in Seahorse XF-24 cell culture microplates at a density of 2.5 × 104 cells/well and cultured for 24 h. Cells were then washed with PBS and incubated in serum-free RPMI, with 4 μM CBD and 10 μM oxaliplatin, for 12 h. Finally, cells were treated with 2 μg/mL oligomycin (ATP synthase inhibitor), 2.5 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and 3 μM rotenone (mitochondrial complex I inhibitor).
4.14. Determination of Mitochondrial Function
Cells were treated with 500 nM TMRE, 5 μM MitoTracker, and 5 μM NAO reagent before treatment with CBD and oxaliplatin. Cells were then analyzed by flow cytometry. Cells were treated with 5 μM JC-1 the following day after CBD and oxaliplatin treatment. They were then mounted and visualized under a confocal microscope.
4.15. In Vivo Tumor Xenograft Model
Animal experiments were performed according to the Guidelines and Regulations for the Care and Use of the Korea University Institutional Animal Care and Use Committee (KOREA-2018-0083). Four-week-old female BALB/c nude mice were acclimated for 1 week prior to the study and were provided free access to food and water. colo205 R cells (1 × 107) in 100 μL of PBS were subcutaneously injected into 4-week-old female BALB/c nude mice. Tumor size and body weight were measured every 2 days. CBD and oxaliplatin were intraperitoneally injected at the same time. Tumor volume was calculated using the formula, 0.5 × length × (width)2. Six mice were included in each treatment group.
4.16. Immunohistochemistry (IHC)
IHC was carried out as previously described [41
]. The tissue was observed by confocal microscopy (ZEISS-LSM 700, ZEISS, Oberkochen, Germany).
4.17. Statistical Analysis
All experiments were performed in triplicate and each yielded similar result. Results are presented as an average of three independent experiments. Statistical analysis was performed using Prism 6 software (GraphPad, San Diego, CA, USA). The results are expressed as the mean of arbitrary values ± SEM. All results were evaluated using an unpaired Student’s t test, in which a p-value of less than 0.05 was considered significant (*, **, and *** indicates p < 0.05, p < 0.01, and p < 0.001, respectively).