Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin

Abstract Osteosarcoma (OS) is the most common malignant bone tumor and a leading cause of cancer‐related deaths in children and adolescents. Current standard treatments for OS are a combination of preoperative chemotherapy, surgical resection, and adjuvant chemotherapy. Cisplatin is used as the standard chemotherapeutic for OS treatment, but it induces various adverse effects, limiting its clinical application. Improving treatment efficacy without increasing the cisplatin dosage is desirable. In the present study, we assessed the combined effect of ascorbate on cisplatin treatment using cultured human OS cells. Co‐treatment with ascorbate induced greater suppression of OS cell but not nonmalignant cell proliferation. The chemosensitizing effect of ascorbate on cisplatin treatment was tightly linked to ROS production. Altered cellular redox state due to increased ROS production modified glycolysis and mitochondrial function in OS cells. In addition, OS cell sphere formation was markedly decreased, suggesting that ascorbate increased the treatment efficacy of cisplatin against stem‐like cells in the cancer cell population. We also found that enhanced MYC signaling, ribosomal biogenesis, glycolysis, and mitochondrial respiration are key signatures in OS cells with cisplatin resistance. Furthermore, cisplatin resistance was reversed by ascorbate. Taken together, our findings provide a rationale for combining cisplatin with ascorbate in therapeutic strategies against OS.


| INTRODUC TI ON
Osteosarcoma (OS) is the most common primary malignant bone tumor and affects adolescents and children. 1 In addition, OS is the most frequent cause of cancer-related deaths in children and adolescents. 2 The current standard treatment for OS is a combination of chemotherapy and surgical resection. 3 The overall survival for patients with localized OS is approximately 65%-70%. 4 In recent decades, improved chemotherapy regimens have contributed to treatment outcomes, 5 but more than 30% of patients have recurrence and metastasis. 6 The response to preoperative chemotherapy is an important predictive factor for the prognosis of OS. 7 Thus, several attempts have been made to improve the treatment efficacy of poor responders using modified chemotherapy protocols, but a unified strategy has not been agreed upon. 5 The use of high-dose chemotherapeutic drugs for primary chemotherapy could be a solution, but systemic toxicity 8 and a risk of secondary cancer are concerns. 9,10 Thus, there is a pressing need to develop new strategies or modify current chemotherapy regimens to treat OS patients refractory to chemotherapy.
Cisplatin is one of the most widely used platinum-based anticancer drugs for treating a variety of solid tumors, including OS. 11 Cisplatin interacts with various cellular components, including chromosomal DNA, proteins, small peptides, lipids, and RNA, [12][13][14][15] resulting in suppression of tumor cell proliferation via multiple pathways. 15 In addition, cisplatin treatment induces oxidative stress, which contributes to its cytotoxic effects. 16,17 The adverse effects of cisplatin place limits on its clinical application. 18,19 Thus, the development of strategies to improve cisplatin treatment efficacy without increasing dose is important.
The anticancer effects of ascorbate (vitamin C) were proposed in the 1950s, 20,21 but contradictory results were reported in subsequent studies. 22 When administered intravenously at high doses, ascorbate has exhibited clinical and preclinical potential in cancer treatment, especially in synergy with other chemotherapeutic agents. 23,24 Additional studies have shown that ascorbate has an antitumor effect in a number of cancer models, including pancreatic, ovarian, and breast. [25][26][27][28][29] Several molecular mechanisms have been proposed for the cytotoxic effects of ascorbate, including increased pro-oxidant damage by generation of reactive oxygen species (ROS), but little is known about the effect of ascorbate on the cisplatin response in human OS. Normally, ROS are produced mainly by intracellular aerobic respiration and metabolism, consequently influencing cell and tissue homeostasis. An altered redox balance due to increasing ROS, however, is known to have pathophysiological effects, including oxidative damage of surrounding lipids, proteins, and DNA. It is thus important to better understand the effects of combined cisplatin and ascorbate treatment on the oxidative stress response in OS, including consequential effects on mitochondrial function and metabolic shift.
In the present study, we show that ascorbate enhances the antitumor effect of cisplatin via increased ROS production, and that the treatment of OS cells with cisplatin and ascorbate alters glycolysis and mitochondrial function. In addition, the co-treatment of OS cells with cisplatin and ascorbate reduces sphere formation, suggesting a chemosensitizing activity of ascorbate on cancer cells that retain cancer stem cell (CSC) properties. Briefly, 1 × 10 6 U2OS cells were cultured on three 10-cm dishes in the presence or absence of cisplatin. The cisplatin concentration was increased from 1 to 30 μmol/L over 6 months. The medium was changed every other day with occasional passage to maintain appropriate cellular confluency. Cisplatin resistance was confirmed by a cell proliferation assay.

Significance Statement
Chemoresistance is a major cause of cancer mortality.
We show that co-treatment with cisplatin and ascorbate induces higher ROS production and metabolic shift in osteosarcoma (OS) cells, leading to strong suppression of cell proliferation. In addition, acquired cisplatin resistance in OS cells is reversed by co-treatment with ascorbate, rationalizing a combination of cisplatin and ascorbate for OS treatment. and 10 μmol/L), or cisplatin plus ascorbate. Ninety-six hours after treatment, 10 µL of WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) was added to each well and incubated for 2 hours at 37°C, after which the absorbance at 450 nm was measured immediately on a microplate reader (iMark; Bio-Rad). The background readings were subtracted from each original reading. The cell viability assay was performed in triplicate and repeated at least three times. The IC 50 was calculated from the curves constructed by plotting cellular viability vs drug concentration. A drug combination effect between cisplatin and ascorbate was evaluated by Combination Index. 31  ROS measurements were performed in triplicate and the entire experiment was repeated at least three times.

| Sphere formation assay
For the sphere formation assay, U2OS cells were resuspended in 0.33% agar gel and plated onto the top of a 0.66% agar gel layer in a 6-well plate. McCoy's 5A medium containing cisplatin, ascorbate, or cisplatin plus ascorbate was added to cover the agar gel layers. Ninety-six hours later, the medium containing the regents was removed and changed to normal McCoy's 5A medium. The medium was changed every other day and the number of spheres  The raw reads were trimmed and quality-filtered using Trim Galore! (ver. 0.4.4), Trimmomatic (ver. 0.36), 32 and cutadapt (ver. 1.16) software. Clean reads were aligned using STAR (ver. 2.6.1a), 33 the count matrix generated using the featureCounts tool (ver. 1.6.1), 34 and differential expression analysis performed using DESeq (ver. 1.30.0) 35 with standard settings.
Genes with |log 2 FC| ≥ 2 and a P-value < .05 were subjected to Gene Ontology analysis using clusterProfiler (ver. 3.6.0). 36 We compared the gene expression levels from parental and cisplatin-resistant U2OS cells and picked the genes with significant expression in gene set enrichment analysis (GSEA). 37 The RNA sequence data have been deposited in the DDBJ database (accession number: DRA009407).

| Statistical analysis
Differences between groups were analyzed by an unpaired twotailed Student's t test. Multiple groups were analyzed by one-way analysis of variance. Results are presented as the mean ± standard deviation. P < .05 was considered significant.

| Ascorbate enhances the cytotoxicity of cisplatin in human OS cells
To assess the effect of ascorbate on cisplatin-induced cytotoxicity, we measured cellular viability after 96 hours of continuous cisplatin, ascorbate, or cisplatin plus ascorbate treatment. Cisplatin treatment decreased the viability of U2OS cells in a dose-dependent manner with an IC 50 of 15.5 μmol/L ( Figure 1A). In contrast, ascorbate treatment alone did not significantly affect the viability of U2OS cells at doses between 0.001 and 10 μmol/L. At 100 μmol/L, ascorbate treatment markedly reduced cellular viability ( Figure 1B). We next tested the chemosensitizing effect of ascorbate (1-30 μmol/L) on cisplatin. Although ascorbate treatment alone did not affect cellular viability at these doses, it enhanced the cytotoxic effect of cisplatin ( Figure 1C). The IC 50

| Synergistic ROS induction and DNA damage upon combined treatment with cisplatin and ascorbate
To gain insight into the potential mechanisms underlying the chemosensitizing effect of ascorbate on cisplatin treatment, we measured ROS production by DHE-based flow cytometry. U2OS cells were continuously exposed to cisplatin or cisplatin plus ascorbate at the indicated doses for 96 hours and intracellular ROS levels were measured. Cisplatin treatment increased intracellular ROS levels in a dosedependent manner (Figure 2A). In addition, ROS levels significantly increased in the cells treated with cisplatin plus ascorbate compared to cisplatin treatment alone. To evaluate the kinetics of intracellular ROS production in response to treatment with cisplatin and ascorbate, we measured ROS levels after 24-, 48-, and 96-hour exposure. Although ascorbate treatment alone did not increase intracellular ROS levels, the combined treatment results in an increase after 24 hours exposure, with further increase over time ( Figure 2B). Hence, cisplatin and ascorbate together enhance intracellular ROS production in U2OS cells.  Figure 2C,D). Less than 10% of cisplatin has been reported to bind DNA, 38 but it is possible that the increase in γH2AX foci is due to this direct effect, enhanced by ascorbate. Thus, to confirm that ROS production significantly contributes to the observed DNA damage, we examined the effects of a ROS scavenger, NAC, on γH2AX foci formation. The addition of NAC markedly suppressed γH2AX foci formation ( Figure 2D). We can conclude, therefore, that cisplatin-induced ROS production leads to an increase in DNA damage, and that this effect is exacerbated upon addition of ascorbate.

| Enhanced mitochondrial respiration altered the metabolism of U2OS cells
An altered cellular redox state due to increased ROS can shift the balance of metabolic processes in the cell. In particular, ROS can

| Cisplatin and ascorbate treatment influences gene expression in the glycolytic and pentose phosphate pathways
Since combined treatment with cisplatin and ascorbate increases basal glycolysis, we sought to confirm and explain this increase by examining the expression of several genes involved in glucose metabolism. Firstly, we examined the levels of mRNA for HK2, the first enzyme in the glycolytic pathway, which phosphorylates glucose to produce glucose-6-phosphate. Cisplatin treatment increases HK2 mRNA levels ( Figure 4). Subsequently, we examined MCT4 (required for extracellular excretion of lactic acid produced by glycolysis), LDHA (converts pyruvate to lactic acid in the glycolytic pathway), and G6PD. G6PD is the first enzyme in the PPP, a pathway which provides NADPH for the synthesis of fatty acids, steroids, and ribose (for nucleotide and nucleic acid formation) and for maintaining reduced glutathione (an antioxidant). MCT4 and G6PD were upregulated in U2OS cells treated by cisplatin, and the expression levels were further F I G U R E 2 Ascorbate enhances ROS production in osteosarcoma cells. A, ROS levels in U2OS cells treated with cisplatin (0-100 µmol/L) and ascorbate (10 µmol/L) for 96 h as measured by flow cytometry. Intracellular ROS levels were determined by measuring the mean fluorescence intensity (MFI) of DHE-positive cells. MFI in the treated cells was expressed relative to MFI of the untreated cells (set at 1). B, ROS levels in U2OS cells measured by flow cytometry, 24, 48, and 96 h after treatment with cisplatin (0-30 µmol/L) and ascorbate (10 µmol/L). MFI in the treated cells was expressed relative to MFI of the untreated cells (set at 1). C, U2OS cells were treated with cisplatin (10 µmol/L) and/or ascorbate (10 µmol/L) in the presence or absence of ROS scavenger NAC (2 mmol/L), 96 h after treatment and the number of γH2AX dots was counted. D, Immunostaining of γH2AX (green) and DAPI (blue). The data represent the mean ± SD of triplicate samples from three independent experiments. *P < .05; **P < .01 LDHA was upregulated in U2OS cells treated with cisplatin but not significantly enhanced by the combined treatment ( Figure 4).
Since increased ROS are known to affect metabolic pathways, we examined whether the enhanced gene expression observed here on treatment with cisplatin (and/or combined cisplatin and ascorbate) is linked directly to ROS production. RNA levels for the genes were, therefore, assessed in the presence of NAC, a ROS scavenger. NAC treatment significantly suppressed the induction of HK2, MCT4, LDHA, and G6PD ( Figure 4). These data further support our findings that cisplatin and ascorbate synergistically modify mitochondrial function and glycolytic metabolism in U2OS cells, and that this is achieved, at least in part, by increased ROS production.

| Combined treatment with cisplatin and ascorbate suppresses U2OS sphere formation
A small subset of stem-like cells present in tumors, known as CSCs, are proposed to be responsible for cancer initiation, and tumor recurrence, metastasis, and chemoresistance. 39 Here, the sphere formation assay was used to identify a cell population with CSC properties in OS. 40 The assay is based on the ability of CSCs to form a three-dimensional sphere when grown in a gel matrix. To test whether combined treatment with cisplatin and ascorbate affects the sphere formation capacity of U2OS cells, the cells enclosed in agar gel were treated with cisplatin, ascorbate, or cisplatin plus ascorbate for 96 hours. The medium was changed to complete normal medium, and the culture continued for 21 days. Cisplatin plus ascorbate treatment significantly reduced the number and the average size of spheres, compared to untreated control and cisplatin or ascorbate treatment alone, or to untreated control ( Figure 5A-C).
These findings suggest that cisplatin-induced cytotoxicity is enhanced in the cells with sphere-forming ability (CSCs) in the presence of ascorbate.

| Ascorbate increases the chemosensitivity of cisplatin-resistant U2OS cells
Acquired chemoresistance of cancer cells is both a huge concern and a limiting factor for chemotherapy programs. Our next objective, therefore, was to establish whether ascorbate treatment restores the cisplatin sensitivity of cisplatin-resistant cells. We first estab-  Figure 6D).

| D ISCUSS I ON
Chemoresistance is a major cause of cancer mortality. The response to preoperative chemotherapy is a particularly important predictive factor for the prognosis of OS patients. 7 Although the mechanisms underlying chemoresistance are complex and may vary among cancers, increasing evidence supports that heterogeneity is a driving force for chemoresistance in general, 44 and that the existing cell populations with chemoresistance can confer intrinsic resistance to chemotherapy. Supporting this view, cisplatin-resistant OS cells possess stem-like properties, 45 a small population of such CSCs having been recognized as drivers for tumor resistance and recurrence in a wide range of cancers.
In the present study, cisplatin treatment induced robust induction of γH2AX, which correlated with raised intracellular ROS levels.   genes. 73 The signaling pathways activated or inactivated in cisplatin-resistant cells are not fully understood, but a link between cisplatin and ribosome biogenesis has been observed in multiple cancer cell lines. 74 For example, ribosomal protein L36 contributes to establishing cisplatin resistance in human cancer cells. 75 Expression levels of ribosomal protein L37 affect cell cycle arrest and DNA damage response after cisplatin exposure. 76 These reports, together with our data, indicate an intimate mechanistic link between cisplatin resistance and the ribosomal stress response.
Although further studies are necessary, our RNA sequence data suggest that the modification of ribosome biogenesis is dominant in cisplatin resistance in U2OS cells, which is at least partly due to MYC activation. In support of our findings, neuroblastoma with N-Myc overexpression become resistant to cisplatin 77 and cisplatin exposure activates c-Myc in head and neck squamous carcinoma. 78 In summary, the current study has identified a convergence of phenotypic and detailed metabolic transitions during shortand long-term cisplatin treatment. We propose that combined treatment with cisplatin and ascorbate efficiently suppresses the proliferation and sphere formation of human OS cells via ROS production and a significant metabolic shift. Importantly, the effects of a combination of cisplatin and ascorbate extend to sphere formation and, therefore, the stem-like cells in OS, which are often drug-resistant. Effective suppression of CSC growth, in addition to other tumor cells, could improve early treatment and survival in OS patients. In addition, potential therapeutic targets to circumvent cisplatin resistance have been identified by RNA sequencing analysis, which provides a potential platform for testing novel combinatorial therapies. We anticipate that these approaches will provide new therapeutic options for managing OS patients in the future.

ACK N OWLED G M ENTS
We thank Shinji Kurashimo, Shoei Sakata, and the members of the

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The RNA sequence data have been deposited in the DDBJ database (accession number: DRA009407).