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Disulfiram Chelated with Copper Promotes Apoptosis in Osteosarcoma via ROS/Mitochondria Pathway.

Disulfiram (DSF) chelated with copper has been confirmed to have a strong anti-tumor ability. In this study, we determined that DSF-Cu induced mitochondria-dependent apoptosis in osteosarcoma (OS), reflecting in DSF-Cu induces mitochondrial membrane potential decline, the production of reactive oxygen species (ROS), and inhibiting cells migration and invasion along with decreasing the concentration of intracellular glutathione (GSH) and facilitating the opening of mitochondrial permeability transition pore (PT) in osteosarcoma cells. These anti-tumor activities can be reversed by Cyclosporine A (CsA, PT inhibitors) and N-acetyl-L-cysteine (NAC, antioxidants). Our results suggested that DSF-Cu exerts its anti-tumor effects in OS via regulation of the ROS/Mitochondria pathway. Our findings provide the basis for DSF-Cu to treat osteosarcoma, even might develop as a potential therapy for other tumors.


INTRODUCTION
Osteosarcoma (OS), a primary malignant bone tumor, is most likely to occur in adolescents with strong aggressiveness, high degree of malignancy and high metastasis rate. 1) With the advancement of surgical technology and the development of the new methods of adjuvant chemotherapy, the therapeutic outcome of patients with osteosarcoma and the 5-year survival rate have been obviously improved, but the recurrence rate of patients with postoperative is still high and the outcome is not very satisfactory. The removal of the cancer cells and avoidance of recurrence and metastasis are still difficult, which could bring patients heavy physical and mental pain and economic burden. 2) Therefore, there is an urgent need for new therapies to improve the patients' survival rate and inhibit postoperative recurrence and metastasis and to reduce patient sufferings and economic burden.
Mitochondria are the main sites for ATP metabolism in eukaryotic cells. In addition to providing energy for biological behaviors such as cell's growth, proliferation and differentiation, they are also widely involved in the pathogenesis and development of apoptosis and tumor drug resistance. 3) Once the mitochondrial function appeared disordered, it can rapidly lead to apoptosis. As a regulatory factor, reactive oxygen species (ROS) plays an important role in the progress of apoptosis of mitochondrial pathway. 4) Under normal circumstances, the level of ROS in the body is in a dynamic equilibrium state, normal cellular physiological activities can produce a certain amount of ROS. When the level of ROS is slightly higher in the cell, it will be eliminated by various reactive oxygen scavengers in the cell. When various factors induce cell to produce excessive ROS, in the process of ROS treatment, the non-specific permeability transition pore in the mitochondrial inner membrane is opened, and the ions in the mitochondrial inner membrane gap enter the cytoplasmic matrix through the permeability transition pore, resulting in the disappearance of ion concentration gradient on both sides of the mitochondrial membrane, and the decrease of mitochondrial membrane potential (MMP). 5) The release of apoptosis factors in the mitochondrial membrane triggers cell dysfunction, and ultimately leads to apoptosis and necrosis. Thus, ROS accumulation and MMP reduction play an important role in mitochondrial apoptotic pathway.
Disulfiram (DSF) is a member of the dithiocarbamates family. It can inhibit acetaldehyde dehydrogenase in liver, make ethanol oxidized into acetaldehyde in body and then can't continue to decompose and oxidize, resulting in alcoholics' aversion to alcohol, so it is widely used in clinical anti-alcoholism. 6) Therefore, DSF is widely used to resist to alcoholism in clinical practice. At the same time, it has a strong force to chelate metal ions and new potential therapeutic uses for human cancers. 7) Some studies have shown that the anti-tumor effect of DSF-Cu is related to the nuclear factor-kappaB (NF-κB) pathway and c-Jun N-terminal kinase (JNK) pathway. 8,9) Moreover, because of its cheapness and high safety it has been attached more importance to its researches in the fields of anti-tumor activities in recent years. However, the anti-tumor mechanism of DSF-Cu is still not completely clear, especially in the effect of osteosarcoma, it has rarely been reported. Cell Proliferation and Cytotoxicity Assays Cells were seeded into 96-well plates and treated with different drugs for 24 h. Then the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) was added to plate for 2 h, the optical density (OD450) was measured using a plate reader. For colony formation, cells were seeded into 6-well plates. The 6-well plates were shaken to maintain sufficient distance between the individual cells. The colonies were measured by a microscope and photographed.

Cell Invasion and Migration Assays
The activity of migration was tested by wound-healing assay, making a wound line using a pipette tip when cells proliferation reached about 80% confluence in the 6-well plate, the cells in different groups were visualized and photographed after 24 h. Cell invasion was investigated by the Trans well chambers (with 8 µm-pore-sized polyester membranes, Corning Life Science, U.S.A.).

ROS, Glutathione Peroxidase (GPx) and Glutathione (GSH) Determination
Using Oxidative conversion of 2′,7′-dichlorodihy drofluorescein diacetate (DCFH-DA) to test the levels of intracellular ROS, as per manufacturer's instructions, the cells were seeded in 6-well plates and exposed to DSF-Cu for 24 h, and then incubated with DCFH-DA for 15 min. Next, the cells from each well were detected by the fluorescence microscope. Respective cell samples were investigated for the GPx and GSH contents by the appropriate assay kits (Beyotime Biotech Inc., Jiangsu, China). According to the manufacturer's instructions, the optical density (OD) values were measured using the SpectraMax microplate reader (Molecular Devices). The concentration value was obtained from the acquired OD values that were fit to the standard curve.
MMP Assay The fluorescent probe JC-1 (Beyotime, Jiangsu, China) was used to test mitochondrial membrane potential (ΔΨm). According to the manufacturer's instructions, the cells were stained with JC-1 staining solution for 15 min at 37 °C. Thereafter, images were captured by a fluorescent microscope (Olympus, Tokyo, Japan).
Transmission Electron Microscope (TEM) Analysis Using Transmission Electron Microscopy (TEM) analysis to investigate the morphological of OS cells. The cells were harvested and fixed in 2.5% glutaraldehyde overnight and then fixed with 1% osmium tetroxide for 2 h at room temperature, before embed in the pellets spur resin, the sample was dehydrated in different concentrations of alcohol. The ultrastructural analysis was analysed by TEM (Hitachi H-7650, Japan).
Fluorescence Immunoassays The sample was blocked by bovine serum albumin (BSA) then incubated with anti-Active Animals and Model The athymic nude BALB/c male mice were procured from the Shanghai Laboratory Animal Center, of Chinese Academy of Science and maintained under specific pathogen-free conditions. The mice were inoculated with K7M2 cells (2 × 106/mouse) via marrow cavity of the right tibia to establish tumor xenograft. After 7 d, those mice were randomly divided into two groups. The mice received y DSF-CuCl 2 (100, 5 mg/kg per os (p.o.), qod) and PBS (equal volume). The tibial tumors were harvested 4weeks after treatment and each tumor was weighed. Tumor sizes were calculated as volume (cm 3 ) = [width2 (cm 2 ) × length (cm)]/2, all the experiments followed Institutional Animal Care and Use Committee (IACUC) of Wenzhou Medical University (Approval No. wydw2020-0392) for humane animal treatment and complied with NIH guidelines.
Histological Examination The tissue was harvested and fixed in 4% paraformaldehyde (PFA) for 72 h. The samples were mounted onto slides and stained with hematoxylin. Images were obtained by a Nikon ECLIPSE 80i (Nikon, A1 PLUS, Tokyo, Japan).
Statistical Analysis All data are presented as mean ± standard error of the mean (S.E.M.). One-way ANOVA was used to test the significance of multiple groups and Student's t-test was used to test differences between the two groups, and p-values <0.05 were defined as significance. All experiments were performed for at least three times.

DSF-Cu Inhibit the Activity of Osteosarcoma Cell
As shown in Figs. 1A-E, the survival rate of OS cells was gradually decreased after DSF-Cu treatment and was dose-dependent. According to this result (0.2 µM DSF), 1 µM Cu was selected for the subsequent cell experiments. The morphology of the DSF-Cu-treated cells shrink in shape and in number (Figs. 1F, G). The invasion and migration of the OS cells was restricted by DSF-Cu (Figs. 1H-K) and the tumorigenesis  ability of OS cells was restrained by DSF-Cu (Fig. 1L). The Western blotting analysis of cleaved-caspase3 showed a significant higher expression of cleaved-caspase3 proteins in the DSF-Cu treatment group compared with the untreated con-trols group (Figs. 1M-O).

DSF-Cu Induced Mitochondrial Dysfunction in Osteosarcoma Cell
When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of the mitochondria to form J-aggregates, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria. At this time, JC-1 is the monomer and can produce green fluorescence. The results of JC-1 staining of OS cells showed that in comparison with the untreated group, the green/red fluorescence ratio increased in the DSF-Cu group, (Figs. 2A-C). The mitochondrial probe (MitoTracker Red) showed that mitochondria becomes rounder after DSF-Cu treatment when compared with control group (Fig. 2D). Furthermore, immunostaining of Mff showed that the expression of Mff were higher in the DSF-Cu group compared with the control group (Figs. 2E, F). The expression of Bax protein showed a significant higher level than that in control group and the expression of BCL-2 and TOM20 proteins showed the adverse trend. Furthermore, we also found that DSF-Cu treatment promotes the release of Cytc from mitochondria into the cytoplasm of OS cells (Figs. 2H-K). To further investigate the effect of DSF on mitochondria in OS, we performed TEM analysis on OS cells, the TEM results showed that the mitochondria ridges of OS cells became blurred and edema after DSF-Cu treatment when compared with control group (Fig. 2G).

DSF-Cu Induced Mitochondria-Dependent Apoptosis in Osteosarcoma Cell
For the reduction-oxidation (REDOX) system of OS cells, we have investigated the intracellular GSH and the activity of GPx of OS cells. The results revealed that DSF-Cu restrain the amount of intracellular GSH and the activity of GPx in OS cells, and NAC (GSH precursor antioxidants) reversed the trend (Figs. 3A-D). The DCFH-DA staining showed that The level of ROS in OS cells increased when be exposed to DSF-Cu (Figs. 3E-G). The effects of DSF-Cu on tumorigenesis ability of OS cells was alleviated by NAC (Fig. 3H). The immunofluorescence analysis of cleaved-caspase3 showed a higher expression of cleaved-caspase3 proteins in the DSF-Cu treatment group compared with the DSF-Cu + CsA group (Figs. 3I-K). In order to further investigate the correlation between DSF-Cu-induced apoptosis and mitochondria in OS cells. We added Cyclosporine A or NAC to DSF-Cu treated OS cells and found that Cyclosporine A or NAC mitigate cell death caused by DSF-Cu (Figs. 3L-P).
The Effect of DSF-Cu on OS in Vivo As shown in Fig.  4A, we exerted the treatment process of tumor model in mice. The volume and weight charts of tumor (Figs. 4B-D) illustrated that the sizes of the tumor were increased in the untreated control group, compared with the DSF-Cu group. Hematoxylin and eosin (H&E) staining demonstrated that more tumor cell death in DSF-Cu group compared to untreated group (Fig. 4E) and no significant injure in multiple organs of mice after DSF-Cu treatment (Fig. 4G).

DISCUSSION
At present, there are some problems in the development of new molecular targeted drugs for osteosarcoma, such as high development cost and high price. It is undoubtedly a promising research direction to develop high-efficiency and lowtoxic anti-tumor drugs from low-cost and low toxic and side effects of traditional drugs. For example, thalidomide, which was originally used for the treatment of sleep disorders, was later found to have good efficacy in the treatment of multiple myeloma and leprosy. And sildenafil, which is used for sexual dysfunction, is now used to treat pulmonary hypertension. DSF is an acetaldehyde dehydrogenase inhibitor and effective anticancer drug approved by U.S. Food and Drug Administration (FDA). Compared with drugs of common chemotherapeutics, DSF has fewer side effects, 10) so it is expected to become a new anti-cancer method.
Copper, an essential trace element for human body, plays an important role in a variety of biological activities, 11,12) including mitochondrial respiration, redox reaction, and free radical scavenging. Some studies have shown that the chelation of DSF with divalent metal ions enhances the killing effects of DSF on tumor cells that is realized through the consumption of anti-oxidation factors in tumor cells. 13,14) Based on these studies, we tested the anti-osteosarcoma the performance of DSF-Cu and we also found that DSF-Cu indeed has the ability of resisting the growth of osteosarcoma and has no response to other normal tissues form human body.
Due to the strong ability of metabolism of tumor cells, the intracellular ROS content is significantly higher than that of normal cells, resulting in tumor cells are more vulnerable to oxidative stress than normal cells. 15) Therefore, ROS mediated anti-tumor strategy is undoubtedly a great anti-tumor scheme. This feature is related to the state of the reduction system of ROS and GSH. Previously we mentioned that a large number of non-specific permeability transition pores were formed in the mitochondrial inner membrane during the increase process of ROS levels in cells, resulting in the release of apoptotic factors in mitochondria and consequently causing apoptosis (Fig. 4F). Low molecular weight thiol (LMWT), Disulfiram, is strong chelators of transition metals and can generate cyto-toxic ROS. 16,17) Therefore, we infer that the anti-osteosarcoma activity of DSF-Cu may be achieved by increasing the level of ROS. As we expected, our studies show that DSF-Cu can promote the increase of the level of ROS, induce the decline of MMP, lead to changes in mitochondrial function, and then induce the apoptosis of osteosarcoma cells. GSH also plays a key role in the balance between apoptosis and survival of tumor cells. As an early event in the process of apoptosis, GSH depletion will promote the occurrence of apoptosis. 18) A study has shown that the apoptosis of gastric cancer accompanied by glutathione decrement via activating mitochondrial dependent pathway. 19) There are some studies which have also shown that intracellular GSH levels are associated with drug resistance in a variety of tumors, 18,[20][21][22] such as leukemia, colon cancer, lung cancer and so on. However, reducing the GSH levels in tumor cells by taking some measures can significantly increase the sensitivity of tumor cells to chemotherapeutic drugs and improve the effect of tumor chemotherapies. Our results suggested that DSF-Cu induces apoptosis of osteosarcoma cells accompanied by the decreasing the concentration of intracellular GSH and facilitating the opening of mitochondrial permeability transition pore (PT). These antitumor activities can be reversed by Cyclosporine A and NAC.

CONCLUSION
In Conclusion, this study mainly involves the specific molecular mechanism of oxygen free radicals and mitochondrial pathway in apoptosis of osteosarcoma cells which induced by DSF-Cu. DSF-Cu consumes the GSH in cells, promotes the generation of ROS, the openness of PT pores and releases cytochrome c into the cytoplasm, by inhibiting the activity of GPx and finally activates the mitochondrial pathways. Caspase activation can cause the apoptosis of osteosarcoma cells.