ROS as a Novel Indicator to Predict Anticancer Drug Efficacy
KEYWORDS Reactive oxygen species, cisplatin, dequalinium chloride hydrate, drug sensitivity, cancer biomarker 2 Abstract Background Mitochondria are considered a primary intracellular site of reactive oxygen species (ROS) generation. Generally, cancer cells with mitochondrial genetic abnormalities (copy number change and mutations) have escalated ROS levels compared to normal cells. Since high levels of ROS can trigger apoptosis, treating cancer cells with low doses of mitochondria-targeting /
... ondria-targeting / ROS-stimulating agents may offer cancer-specific therapy. This study aimed to investigate how baseline ROS levels might influence cancer cells' response to ROS-stimulating therapy. Methods Four cancer and one normal cell lines were treated with a conventional drug (cisplatin) and a mitochondria-targeting agent (dequalinium chloride hydrate) separately and jointly. Cell viability was assessed and drug combination synergisms were indicated by the combination index (CI). Mitochondrial DNA copy number (MtDNAcn), ROS and mitochondrial membrane potential (MMP) were measured, and the relative expression levels of the genes and proteins involved in ROS-mediated apoptosis pathways were also investigated. Results Our data showed a correlation between the baseline ROS level, mtDNAcn and drug sensitivity in the tested cells. Synergistic effect of both drugs was also observed with ROS being the key contributor in cell death. Conclusions Our findings suggest that mitochondriatargeting therapy could be more effective compared to conventional treatments. In addition, cancer cells with low levels of ROS may be more sensitive to the treatment, while cells with high levels of ROS may be more resistant. Doubtlessly, further studies employing a wider range of cell lines and in vivo experiments are needed to validate our results. However, this study provides an insight into understanding the influence of intracellular ROS on drug sensitivity, and may lead 3 to the development of new therapeutic strategies to improve efficacy of anticancer therapy. Background Mitochondria are implicated in many cellular processes such as cellular energy metabolism, cell communication, differentiation and apoptosis . Mitochondrial dysfunction leads to alterations in mitochondrial structure, disruption of mitochondrial membrane potential, instability of electron transport reactions resulting in reactive oxygen species (ROS) overproduction, activation of caspase cascades and initiation of apoptosis pathway. Hence, any mitochondrial abnormality can lead to the development of several human diseases, including cancers . One unique feature of mitochondria is that they contain their own genome, mitochondrial DNA (mtDNA; a small circular DNA of approximately 16569 bp), independent of nuclear DNA . Mitochondria are the primary source of intracellular ROS, a group of chemically reactive molecules containing oxygen, hydroxyl radical (•OH), superoxide anion (O 2 -), singlet oxygen (O₂) and hydrogen peroxide (H 2 O 2 ), as side-products of the mitochondrial electron transport chain reaction during cellular respiration . ROS play important roles in cell signalling pathways such as growth, differentiation, metabolism and apoptosis [4, 5] . They are also regarded as a double-edged sword in cancer cells since low doses of ROS can promote cell proliferation and invasion, whereas excessive levels of ROS cause oxidative damage to proteins, lipids, RNA and DNA which consequently induce cell death [6, 7] . Therefore, a slight increase of ROS is associated with the initiation and progression of cancer [4, 8] , but high levels of ROS can induce cell death by activating several signalling pathways resulting in 4 cell apoptosis [6, 7] . For example, in cancer cells with wild type p53, DNA damage by ROS induces apoptosis in a mitochondria-dependent manner via activation of the p53/BAX signalling pathway  . In healthy tissues, the intracellular ROS are preserved at a steady and low level by the equilibrium between ROS production and elimination by enzymatic antioxidants such as cytoplasmic superoxide (SOD1), mitochondrial superoxide (SOD2), catalase (CAT) and glutathione (GSH)  . Tumour cells express lower antioxidants than normal cells, and therefore have higher ROS levels. Furthermore, defective mitochondrial oxidative metabolism in tumour cells also render higher ROS levels  , and therefore ROS induction is a promising approach to cancer therapy [4, 8] . Despite its strong side effects, chemotherapy is still widely used in clinical practice. Many chemotherapy drugs cause cell death by a direct damage to the nucleic acids while others disrupt the redox balance within the cell. Some chemotherapeutic agents can cause an excessive accumulation of ROS either via an overproduction of ROS or by supressing their elimination in tumour cells by the antioxidant systems . Cisplatin [cisplatinum or cis-diamminedichloroplatinum (II)] is one of the most commonly used chemotherapeutic agents employed in the treatment of various human cancers. It is a highly reactive molecule which forms various types of adducts by binding to DNA, RNA and proteins, and the cytotoxic effect of cisplatin is mainly due to the lesions formed within the nuclear DNA . Moreover, previous studies have demonstrated that cisplatin accumulates in mitochondria and causes significant changes in mitochondrial structure and metabolic function [11,12]. Recent reports evinced that cisplatin-induced apoptosis could be inhibited by compounds that interfere with ROS generation. These observations elucidate that the killing effect is correlated to increased ROS generation  . However, the 5 clinical use of cisplatin is limited because of its severe irreversible side effects including neurotoxicity, ototoxicity and nephrotoxicity which has been reported as the main limitation of cisplatin  . Furthermore, the majority of current systemic cancer chemotherapeutic drugs exert their toxicity on mitochondria indirectly via different signalling pathways, and they do not localise at tumour sites efficiently and therefore can cause unwanted damage to normal tissues [2, 14] . Recently, due to their critical role in metabolism, ATP synthesis and redox status, and because of their involvements in many pathways related to the cell death, mitochondria have become one of the main interests in developing cancer treatments. Since cancer cells generally have higher levels of ROS compared to normal cells, and because of the differences in the mitochondrial membrane potential between cancer and normal cells, a direct targeting on mitochondrial functions could be an effective approach to triggering cancer-specific cell death. Delocalised lipophilic cations (DLCs), a group of small membrane permeable agents driven by negative potential across the mitochondrial membrane, accumulate in mitochondria and are more toxic to cancer cells compared to normal cells . This characteristic attracts researchers to evaluate DLCs for selective cancer cell elimination . Within a wide range of DLCs, dequalinium (DQA) has been reported to demonstrate a potent anticancer activity in vitro and in vivo in different malignancies . Several studies have suggested that the cytotoxicity mechanism of DQA is related to mitochondrial dysfunction due to the damage of mitochondrial DNA and the inhibition of mitochondrial complex I . It has also been reported that DQA causes cell death in the HeLa cells by selective depletion of mtDNA . Moreover, it has been postulated that DQA induces human leukaemia cell death by affecting the redox balance  , and another study showed that DQA caused 6 oxidative stress and apoptosis in a human prostate cancer cell line  . Due to the merit of mitochondria-targeting therapy, the combination of conventional chemotherapy drugs such as cisplatin with mitochondria-targeting agents may offer a promising strategy for enhanced anticancer therapy  . Furthermore, mitochondrial DNA copy number (mtDNAcn) per cell is preserved within a stable range to achieve the required energy of the cell and hence ensure normal physiological functions. It ranges from 10 3 to 10 4 according to the population and cell type. Such variations also reflect the imbalance between ROS production and the antioxidant capacity, so mtDNAcn has been considered as a potential diagnostic and prognostic biomarkers for several cancer types  . This study aimed to investigate the link between mtDNAcn and baseline intracellular ROS level in untreated cancer cells, as well as how baseline ROS level might influence cells' response to ROS-stimulating therapy. The potential synergistic effect of cisplatin and dequalinium chloride in killing cancer cells was also assessed. Methods Cell culture The four cancerous (Ishikawa/endometrium, MDA-MB-231/breast, Caco-2/colon, PC-3/prostate) and one normal (PNT-2/prostate) cell lines were obtained from the departmental cell bank at the University of Portsmouth. All cell lines were originally purchased from the European Collection of Authenticated Cell Cultures (Ishikawa, MDA-MB-231, Caco-2, PNT-2) or the American Type Culture Collection (PC-3). Cells were maintained in required media (suppl. Table 1 ) and harvested at 90% confluence for the downstream assays. All cell lines were authenticated using STR profiling and screened for mycoplasma contamination in our laboratories over the 7 period of the investigation. Drug treatment Stock solutions of cisplatin (CDDP) and dequalinium chloride hydrate (DQA) (Sigma, Dorset, UK) were prepared at 100 mM in DMSO and 2 mM in distilled water, respectively. Both drugs were added to the cells in various concentrations and incubated for 24 hours to determine their IC50s (IC50: the half maximal inhibitory concentration) which were used in all subsequent experiments. N-Acetyl-L-cysteine (NAC) (Sigma), a powerful antioxidant, was dissolved in distilled water at the concentration of 100 mM shortly before each experiment, and the pH was adjusted to 7.4 before diluting the solution to the working concentration (10 mM) with complete cell culture medium. Cells were pre-incubated with NAC (10 mM) for 1 hour prior to the CDDP and DQA treatments. All experiments related to NAC followed the same procedure as described in the sections below. Cell viability assay Cell viability was measured colorimetrically using 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (CellTiter 96 Aqueous) (Promega, Southampton, UK). Briefly, 90 µl of cell suspension containing 10000 cells was added in each well of the 96-well plate and incubated for 24 hours. Cells were then treated with the 10 × drug solution (10 µl) for the desired amount of time. At the end of the experiment, the medium was replaced with 100 µl of fresh medium containing MTS (final concentration -0.3 mg/ml) and incubated for 90-180 min according to the optimised protocol for each cell line. Absorbance was measured using the microplate reader (Multiskan® GO) at 490 nm. IC50 was calculated as the concentration of the drug that caused a 50% loss of metabolic activity.