Pharmacological Ascorbate Induces Osteosarcoma Cells Death via H2O2 Mediated- Oxidative Stress and Enhances the E cacy of Cisplatin in Osteosarcoma


 Background: Finding emerging strategies for new use of the “old drug-vitamin C” on cancer treatment-combined with chemotherapy drugs.Methods: This study investigates the mechanisms that pharmacological ascorbate (vitamin C, AA) mediated toxicity in osteosarcoma cells and the effects of pharmacological ascorbate combined with cisplatin (DDP) on osteosarcoma cells in vitro and in vivo.Results: Pharmacological ascorbate not only promoted the production of hydrogen peroxide (H2O2) to kill osteosarcoma cells, but also improved its lethality to tumor cells by reducing the activity of catalase (CAT). DNA damage was the primary reason leading to osteosarcoma cell death, and depletion of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP), activated apoptosis-inducing factor (AIF) all contribute to ascorbate-induced toxicity. The combination of pharmacological ascorbate with cisplatin showed synergistic effect on osteosarcoma cells in vitro. Pharmacological ascorbate enhanced the efficacy of cisplatin on inhibiting osteosarcoma tumor growth in orthotopic intra-tibial mouse model.Conclusion: These results suggest that pharmacological ascorbate causes DNA damage and induces osteosarcoma cells death via H2O2 mediated-oxidative stress. The combination of pharmacological ascorbate and cisplatin has synergistic effects on osteosarcoma cells and orthotopic intra-tibial mouse model. Pharmacological ascorbate could be a safe and effective adjuvant agent against osteosarcoma tumor.

demonstrate any effects on cancer therapy with pharmacological oral administration [6,7]. Later pharmacokinetic studies found that blood levels of ascorbate are tightly controlled by oral use, and intravenous administration can produce high plasma concentrations [8]. This study provided a possible explanation for the discrepancy between the ndings of earlier trials. Additional clinical trials showed that patients show good tolerability to pharmacological intravenous ascorbate [9]. Recent researches also suggested that pharmacological intravenous ascorbate could be applied to cancer treatment alone or in combination with the chemotherapies or radiation therapy [10][11][12].
Chen et al. reported that the anti-cancer effect of pharmacological ascorbate is dependent on the action of ascorbate as a pro-drug for hydrogen peroxide (H 2 O 2 ) generation which involves in the catalytic metals [13]. H 2 O 2 and catalytic metals promote the production of reactive oxygen species (ROS) through the Fenton reaction which causes oxidative stress and induces cancer cell death [14,15]. Although the cytotoxic effect of ascorbate has been demonstrated on many cancer cell lines, its clearly molecular mechanism has not been identi ed yet [16]. Many previous studies have showed oxidative stress, DNA damage and ATP depletion play critical roles in pharmacological ascorbate-induced cancer cell death [14,17,18]. Apoptosis, autophagy and other forms of cell death have been proposed as potential cell death mechanisms [19][20][21].
In this study, we investigated the mechanisms that pharmacological ascorbate mediated toxicity in osteosarcoma cells. Pharmacological ascorbate mediated toxicity and DNA damage in osteosarcoma cells was found to be dependent upon H 2 O 2 mediated oxidative stress, and apoptosis-inducing factor activation was involved in this process. Furthermore, synergistic effects of pharmacological ascorbate combined with cisplatin were also observed in vitro and in vivo.

Materials And Methods
Reagents and cell culture Ascorbate acid, Cisplatin, N-acetyl-L-cysteine (NAC), bovine catalase (CAT), Deferoxamine (DFO), Chloroquine (CQ) and Ferrostatin-1 were purchased from Sigma-Aldrich. Ascorbate solution was prepared as 1 M stock solution in tri-distilled water. Then the solution was adjusted the pH to 7

Measurement of cellular ATP and NAD + levels
After treated with ascorbate, cells were washed with PBS and lysed by lysis buffer. The lysates were centrifuged at 12,000 g for 5 min at 4 °C.
The supernatants were collected for the ATP assay with ATP Assay Kit (Beyotime Biotechnology, China). Chemiluminescence mode of microplate reader and black 96-well plates were used for measuring ATP, and cellular ATP content was calculated by ATP standard curve line.
Determination of the total amount of NAD + / NADH and the amount of NADH in the supernatants by using NAD + /NADH Assay Kit with WST-8 (Beyotime Biotechnology, China). The amount of NAD + was derived by subtracting NADH from total NAD + /NADH. All values were normalized by BCA protein quanti cation assay.

Western blot analysis
Cells (3 × 10 5 /well) were seeded in 6-well plates and treated with different doses of ascorbate for the indicated times. Subsequently, cells were lysed on ice, and then centrifugation at 12,000 g for 20 min at 4 °C. The lysates were collected and determined using the BCA method and denatured by boiling in loading buffer, and then subjected to Western blot analysis. Blots were enhanced with electrochemiluminescence (ECL) and scanned with a T5200 The mice were randomized into the following 4 groups: (1) Ascorbate (AA) group, mice were injected with ascorbate via intraperitoneal injection (4 g /kg daily, IP); (2) DDP group, mice were injected with DDP (5 mg/kg once per week, IP) in 0.9% saline solution; (3) AA + DDP combinational group, both AA and DDP were administered, AA (4 g /kg daily, IP), DDP (5 mg/kg once per week, IP); (4) Control group, mice were injected with equivalent dose of saline (IP). Tumors were measured every other day with Vernier calipers (volume = length × width 2 × 0.5), and mouse weight were also recorded every other day. After 3 weeks, mice were euthanized and sacri ced. Tumors were dissected out, weighed, and stained with hematoxylin and eosin (H&E) and immunohistochemistry (IHC). The major organs were also analyzed by H&E staining.

Statistical analysis
The statistical analysis of the difference between two groups was performed using Student's t-test. All statistical analyses were performed using GraphPad Prism 7 Software. The data were presented as the mean ± standard deviation (SD), P-value < 0.05 was considered signi cant. * represents signi cant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001), n.s. represents no signi cant differences (p > 0.05).

Pharmacological ascorbate decreases cell viability and inhibits cell proliferation
Previous studies have shown that pharmacological ascorbate presents cytotoxicity and selective sensitivity effects in many cancer cell lines [16]. Therefore, we rst detected the effects of pharmacological ascorbate on cell viability in osteosarcoma cell lines. The results showed that pharmacological ascorbate decreases osteosarcoma cell viability in a dose-dependent manner of four osteosarcoma cell lines (Fig. 1A). Then, we performed the colony formation assays to determine the cytotoxic effects of pharmacological ascorbate on cell proliferation. As shown in Fig. 1B [13]. In subsequent researches, the pro-oxidative effects of ascorbate have also been con rmed in many cancer cell lines [11,17] We detected the changes of catalase activity in four osteosarcoma cell lines after ascorbate treatment (Fig. 2D). With the increasing treating time, we found that pharmacological ascorbate can reduce catalase activity signi cantly (Fig. 2B). These results demonstrated that pharmacological ascorbate not only promotes the production of H 2 O 2 to kill tumor cells, but also improves its lethality to tumor cells by reducing the activity of catalase. Previous studies have shown that the free radicals generated during the oxidation of ascorbate and semidehydroascorbate are all responsible for the inhibition of catalase [24,25]. Moreover, we tested the effect of catalase on ascorbate, and excessive exogenous bovine catalase could completely abolish ascorbate-induced toxicity in osteosarcoma cells (Fig. 2C). Our results indicated that the role of H 2 O 2 is crucial in the process of ascorbate-induced cell death.
Pharmacological ascorbate-mediated oxidative stress breaks the redox balance and leads to DNA damage Considering that pharmacological ascorbate treatment improves intracellular H 2 O 2 levels and H 2 O 2 reacts with intracellular labile iron to generate more reactive hydroxyl radicals (HO•) by Fenton reaction [26], we measured the cellular ROS levels in osteosarcoma cells treated with ascorbate by DCFH-DA. As expected, ascorbate treatment signi cantly increased cellular ROS levels in osteosarcoma cells, and this effect was reversed by bovine catalase (Fig. 3A). Labile iron promotes the production of hydroxyl radicals, and the cytotoxic effect of hydroxyl radicals is stronger than H 2 O 2 [23]. As shown in Fig. 3B, both iron chelator DFO and ROS scavenger NAC could eliminate pharmacological ascorbate-induced toxicity in osteosarcoma cells (Fig. 3B). Excessive ROS can break DNA double-stand and cause cell death [27,28].
To observe the DNA damage induced by excessive ROS, we used transmission electron microscopy (TEM) to determine morphological alterations. As displayed in Fig. 3C, we observed that obvious disorganized chromatin condensation, vacuolation of the cytoplasm and breakdown of the plasma membrane. We also observed that swelling of mitochondria and disappearance of mitochondria cristae. TEM revealed ascorbate-induced cell death presents necrosis morphological features rather than apoptosis. Additionally, to con rm ascorbate treatment caused DNA double-strand damage in osteosarcoma cells, we detected markers of DNA double-strand damage. As shown in Fig. 3D, the expression of phosphorylation of histone 2AX (γ-H2AX) was signi cantly increased in cells after ascorbate treatment. While, both catalase and ROS scavenger NAC could prevent the phosphorylation of H2AX caused by ascorbate (Fig. 3E). Next, we tested the ability of ve small molecule cell death inhibitors to prevent ascorbate-induced cell death in osteosarcoma cells (Fig. 3F). we found that ascorbate-induced death was not consistently modulated by inhibitors of pan-caspase (Z-VAD-FMK), RIPK1 (necrostatin-1), lysosomal autophagy (3-MA, CQ), lipid ROS (ferrostatin-1) compounds known to inhibit forms of apoptosis, necrosis, autophagic and ferroptosis cell death. The results indicated that ascorbate-induced osteosarcoma cell death is independent of these forms of cell death. Collectively, our data indicated that pharmacological ascorbate breaks cellular redox balance, leads to DNA damage and induces osteosarcoma cell death.
DNA damage depletes intracellular ATP and NAD + and activates apoptosis-inducing factor Poly (ADP-ribose) polymerase (PARP) is an important nuclear enzyme that responds to DNA damage and is required for DNA repair [29]. Activated PARP format Poly (ADP-ribose) (PAR) for DNA damage repair, and the changes in PAR levels re ect the activation of PARP. Therefore, we detected the PAR polymer formation in ascorbate-treat cells. The results showed that the levels of PAR are signi cantly increased (Fig. 3D). During DNA damage repair process, NAD + , as a substrate, is cleaved into ADP-ribose and nicotinamide to format PAR, then cellular NAD + levels are restored by recycling nicotinamide with two ATP molecules [30]. Next, we measured the effect of ascorbate on cellular NAD + and ATP levels in osteosarcoma cells. As shown in Fig. 4A and 4B, ascorbate treatment for 1 h signi cantly decreased cellular NAD + and ATP levels. Also, both CAT and NAC could restore NAD + and ATP levels, indicating that ascorbate induced osteosarcoma cell death probably affected by NAD + and ATP depletion during damage repair. In order to further verify the role of activated PARP in osteosarcoma cells, we used olaparib, a PARP inhibitor, and ascorbate to co-treat osteosarcoma cells, and then detected cell viability (Fig. 4C). Interestingly, ascorbate-induced cytotoxicity could be effectively reversed by olaparib in MG63 cells, and olaparib had no effect on ascorbate-induced cytotoxicity in the other three osteosarcoma cell lines. Ma et al. have reported that exogenous ATP can protect neuroblastoma cells from ascorbateinduced cytotoxicity [17]. However, exogenous ATP could inhibit ascorbate-induced cytotoxicity in MG63 cells, and had no effect on the other three cell lines (Fig. 4D) [17]. Previous studies showed that once excessive PARP activation, the formatted PAR can also act as a pro-death signaling molecule [34,35]. PAR interacts with the mitochondrial outer surface and induces apoptosis-inducing factor (AIF) release [36]. Then, AIF is cleaved into a soluble form (tAIF) by calpains, and Bax regulates tAIF release from mitochondria [37]. To investigate the role of PAR in ascorbate-induced cell death, we tested the expression of the related proteins. As shown in Fig. 4E, the expression of Bax and the ratio of Bax/Bcl-2 were signi cantly increased in cells after ascorbate treatment. Moreover, the ratio of tAIF/AIF also signi cantly increased in cells after ascorbate treatment. The results indicated that ascorbate treatment provokes mitochondrial outer membrane permeability and promotes tAIF release from mitochondria. Considering that calpains regulates tAIF release from mitochondria, we also tested the effect of PD 151746, a calpain inhibitor, on ascorbate-induced cytotoxicity. However, PD 151746 cannot inhibit ascorbate-induced cytotoxicity (Fig. 4F). In summary, we believe that DNA damage is the primary reason leading to osteosarcoma cell death, and NAD + and ATP depletion enhances ascorbate-induced cytotoxicity. DNA damage promotes excessive activation of PARP leading to activation of AIF and release of tAIF, and tAIF induced nuclear condensation also contributes to ascorbate-induced cytotoxicity (Fig. 4G).
Pharmacological ascorbate synergizes with cisplatin in osteosarcoma cell lines As a rst-line chemotherapy for osteosarcoma treatment, cisplatin reacts with DNA to form intra and interstrand crosslinks, which induces DNA damage leading to cell death [38]. Our data have shown that DNA damage is the primary reason in ascorbate-induced cell death in osteosarcoma cells. First, we tested the effect of cisplatin on osteosarcoma cells. As shown in Fig. 5A and 5B, the osteosarcoma cell viability could be effectively inhibited by cisplatin. In addition, the IC50 of K7M2 cells was lower than the other three cell lines. To investigate the effect of ascorbate in combination with cisplatin. As shown in Fig. 5C, the combination of cisplatin and ascorbate signi cantly suppressed cell viability compared with cisplatin alone in the four osteosarcoma cell lines. We also used Chou-Talalay analysis to determine if ascorbate synergized with cisplatin in osteosarcoma cells, and the combination index (CI) less than 1 indicated a synergistic effect between cisplatin with ascorbate [39]. As displayed in Fig. 5C, ascorbate and cisplatin synergistically induced cytotoxicity in K7M2, MNNG/HOS and U2OS cells, but we did not observe synergistical effects of two agents in MG63 cells. To further examined the synergy of ascorbate and cisplatin, we detected the expression of γ-H2AX. The combination treatment resulted in a signi cant increase in γ-H2AX expression compared to cisplatin or ascorbate alone in the three cell lines (Fig. 5D).
Given that lower dose of cisplatin was used in combination with ascorbate to treat osteosarcoma cells, our results suggested pharmacological ascorbate can reduce the use of chemotherapeutic agents in vitro.
The combination of pharmacological ascorbate and cisplatin suppresses osteosarcoma tumor growth in orthotopic intra-tibial mouse model Based on the synergistic effects of ascorbate and cisplatin treatment on osteosarcoma cells in vitro, we tested the e ciency of ascorbate and cisplatin in orthotopic intra-tibial mouse model of osteosarcoma. We found that both pharmacological ascorbate and cisplatin could suppress tumor growth compared with saline group, and the tumor volume and weight were signi cantly reduced. Although cisplatin alone was better than ascorbate, the combination of two agents was more effective compared to either ascorbate or cisplatin alone ( Fig. 6A-C). Then, we tested the tumor tissues from mouse model by H&E staining and IHC analysis to further validate the inhibitory effects of on tumor growth. As shown in Fig. 6D and 6E, drugs treatment induced varying grades of necrosis and the percentage of Ki-67 positive cells was remarkably decreased. In addition, ascorbate alone had no effect on body weight, cisplatin alone and the combination of two agents reduced the body weight compared with saline group (Fig. 6F). Despite the body weight was reduced, we did not observe obvious toxicity in the liver, kidney and spleen in drugs treatment groups (Fig. 6G). Taken together, our results indicated that the treatment with ascorbate alone or in combination with cisplatin signi cantly suppressed osteosarcoma tumor growth in orthotopic intra-tibial mouse model. All of our ndings suggested that pharmacological ascorbate maybe a safe and effective adjuvant agent against osteosarcoma tumor.

Discussion
Osteosarcoma is the most common primary malignant tumor of bone in children and young adults. Before the 1970s, amputation surgery was the main treatment for osteosarcoma. Subsequently, surgery combined with adjuvant systemic chemotherapy signi cantly improved outcome for patients. However, the treatment and outcome have not changed since the 1980s [40]. Currently, surgery and systemic chemotherapy is still the standard care for osteosarcoma patients. The use of chemotherapy agents has a toxic effect on the main organs, such as cardiotoxic effects of doxorubicin, nephrotoxic effects of methotrexate and cisplatin [3]. Therefore, it is necessary to study safe and effective adjuvant therapeutic agents for patients.
Although the effect of pharmacological ascorbate on cancer treatment is full of controversy, with the deepening of researches, the toxicity effect of ascorbate has been con rmed in a variety of cancer cell lines [12]. Different cancer cells are selective sensitive to ascorbate, and the clearly molecular mechanism has not been identi ed yet. Some studies showed that the expression of sodium-dependent vitamin C transporter 2 (SVCT-2) and glucose transporter 1 (GLUT1) in cancer cells, which transport vitamin C and dehydroascorbate (DHA) into the cells, respectively, affected the sensitivities of cancer cells to ascorbate [14,33]. The abilities of cancer cells to metabolize H 2 O 2 might also cause different sensitives to ascorbate [22]. Our data revealed that ascorbate not only promotes the production of H 2 O 2 to kill osteosarcoma cells, but also improves its lethality to tumor cells by reducing the activity of catalase. A recent study reported that the O 2 •− and H 2 O 2 were able to disrupt cellular iron metabolism, thereby selectively sensitizing cancer cells to ascorbate [11]. In our recent research, similar conclusions were obtained, the basal levels of intracellular labile iron and effects of ascorbate on ferritin expression are related to the sensitivity of human osteosarcoma cell lines to ascorbate [23].
Intracellular labile iron reacts with H 2 O 2 to generate more reactive hydroxyl radicals (HO•) by Fenton reaction, and leads to DNA damage. TEM and the expression of γ-H2AX have veri ed that ascorbateinduced signi cant DNA damage in osteosarcoma cells, which was consistent with results in neuroblastoma cells [17]. Although several forms of cell death have been proposed as potential ascorbate-induced cell death mechanisms, our data showed several small molecule cell death inhibitors cannot prevent ascorbate-induced cell death in osteosarcoma cells. These four osteosarcoma cell lines have different sensitivities to ascorbate, and the factors that affect their death are not the same. During DNA damage repair process, PARP is activated and NAD + and ATP are consumed. Over-activated PARP produces a large amount of PAR which in turn activates AIF, then tAIF induces nuclear condensation [36].
Xia et al. also showed that AIF was activated by ascorbate in multiple myeloma cells [41]. We found that NAD + and ATP depletion and tAIF also contributes to ascorbate-induced cytotoxicity. Exogenous ATP and PARP inhibitor olaparib can inhibit ascorbate-induced cytotoxicity in MG63 cells, but have no effect on the other three cell lines. Perhaps multiple factors involved in the death of MG63 cells that make it more sensitive to ascorbate compared with the other three cell lines.
Pharmacokinetic studies found that plasma concentrations of ascorbate are tightly controlled by oral use due to the limitation of intestinal absorption, and intravenous injection can produce high plasma concentrations [8]. Therefore, high plasma concentrations of ascorbate in vivo are su cient to kill tumor cells. Subsequently, many preclinical and clinical studies have shown that intravenous administration of ascorbate can be used as a safe and effective adjuvant to combine with chemotherapies or radiation therapy for cancer treatment [9,[42][43][44][45]. Furthermore, ascorbate could also sensitize cancer cells to chemotherapy and protect normal tissues [10,46]. Our data showed that ascorbate and cisplatin synergistically induce cytotoxicity in different osteosarcoma cell lines, and ascorbate can reduce the use of chemotherapeutic agents in vitro. In vivo experiments, intravenous administration of ascorbate could enhance the e cacy of cisplatin in orthotopic mouse model of osteosarcoma. While, clinical trials will be needed to evaluate the safety and e cacy of ascorbate in osteosarcoma tumor therapy.

Conclusions
In this study, we found that pharmacological ascorbate promotes the production of H 2 O 2 and reduces the activity of catalase in osteosarcoma cells. H 2 O 2 -mediated oxidative stress leads to DNA damage, which induced cell to death. In addition, depletion NAD + and ATP and activation of PARP both contributed to ascorbate-mediated cytotoxicity. We also observed the synergistic effects of pharmacological ascorbate combined with cisplatin in osteosarcoma cells and orthotopic intra-tibial mouse model. In summary, our ndings suggested that pharmacological ascorbate could be a safe and effective adjuvant agent against osteosarcoma tumor.

Consent for publication
Not applicable.

Availability of data and material
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.