Healthy mitochondria: the brake of cell proliferation of malignant melanoma

Background: Melanoma has become the leading cause of death from skin disease, and male patients show higher mortality rate than female. Mitochondrial transplantation therapy is an active area of current research but the anti-melanoma activity and specific mechanism involved in the therapy remain to be fully characterized. Methods: In the study, we intravenously administrated mitochondria extracted from male and female mouse livers respectively to the mice bearing malignantly subcutaneous and metastatic melanoma, and identified the signal mechanism responsible for the mitochondrial treatment through transcriptomic analysis. Meanwhile, the efficiency of female mitochondria and male mitochondria was compared in the cultured melanoma cells and transplanted melanoma in mice. Results: The results suggested that the mitochondria significantly inhibited the tumor growth, and the transcriptomic analysis suggested that general chromosome silencing and local opening of UPR mt effect region were strongly associated with the mitochondria against melanoma., which represented cell cycle arrest, autophagy, and cell apoptosis in the mitochondrial therapy on the metastasis melanoma. Moreover, the anti-tumor activity of mitochondria from female animals was more efficient in comparison to the males, and the female mitochondria could probably induce more persuasive mitochondria-nuclear communication than the mitochondria from male mice. Conclusions: The study not only reveals the anti-tumor mechanism of the mitochondria but also provides a novel insight into the effect of mitochondria in different genders. cell cycle, cell division, chromosome sister mitotic nuclear and nuclear The subclasses of the chromosome, centromeric region,


Background
Mitochondria are the crucial organelle that is responsible for cell survival and apoptosis.
Healthy mitochondria are essential to maintain the normal function of cells. However, accumulating research evidence identifies that tumor mitochondria undergo adaptive changes to accelerate rapid proliferation of tumor cells in the acidic and hypoxic microenvironment.[1 , 2 Thus, introducing healthy mitochondria into tumor cells is proposed to have a high efficacy in preventing tumor growth. [3,4 Currently, exogenous healthy mitochondria have been utilized in treating several carcinoma, including breast cancer, pancreatic cancer, and glioma, and similar results exhibited the excellent antitumor activity of the healthy mitochondria. [5 , 6 , 7 In our recent studies, we have examined the anti-tumor potential of mitochondria derived from young and aged mouse liver, and found that mitochondria from young mice have stronger anti-melanoma effects in comparison with that of the aged mice. [8,9 The biochemical measurement suggested that healthy mitochondria can significantly decrease the oxidative phosphorylation (OXPHOS) capability and induce apoptosis in the tumor cells.
However, the molecular signal mechanism for the mitochondrial therapy is still unclear.
Moreover, mitochondria are the near-exclusive maternal inheritance in evolution, and there might be sex differences of mitochondria in therapy. The earlier findings reported that mitochondria from female animals (female mitochondria) are more sensitive to stress and are better equipped to deal with the harmful condition, [10,11 thereby the female mitochondria were assumed to have different activities in anti-tumor growth in comparison with the male mitochondria. In this sudy, we further investigated the influence of the gender differences of the mitochondria therapy. The study would not only clarify the molecular mechanism of the mitochondria on the tumor and also provide a new insight of the anti-tumor effect of mitochondria obtained from different genders.

Animals
Healthy BABL/c mice of 2 month-age-old (22  2 g) were used in the research. The mice were procured from the Chongqing Medical University, China. The mice were housed in SPF center and fed by standard mouse chow and water. The animal protocol was authenticated through the Animal Ethical Committee, Southwest University.

Mitochondrial isolation and activity measurement
Liver mitochondria from female and male mice were isolated according to the earlier reported protocol. [ 12 Further, the mice were euthanized quickly through cervical dislocation, and the mouse liver was isolated and homogenized at 4°C. The supernatant fraction was collected, and an isolated mitochondrial solution was kept for homogenization. The redox capacity of the isolated mitochondria was examined using resazurin, and membrane potential were obtained by JC-1 assay kits procured from Jiangsu Kaiji Biotech. Ltd. Co, Nanjing, China. The activity of pyruvate dehydrogenase (PDH) was measured according to the manufacturer's protocol (Nanjing Jiancheng Biotech. Institute, Nanjing, China). Each measurement was independently conducted about six times, respectively.

Cell culture and viability tests
Murine melanoma B16 cells were cultured with RPMI 1640 media (containing 10% FBS) in a hypoxic incubator chamber with 37°C, 5% CO2. When the tumor cell mass reached 60% in 96-well plates, the isolated mitochondria were respectively added into the wells. The cells were observed using a confocal microscope (Zeiss. Germany) after mitochondrial addition. After 24 h incubation with the mitochondria, cell viability was measured by Alamar blue according to the manufacturer protocol. The levels of lactate, pyrvate, and ATP in cells were respectively determined using the commercial kits obtained from Nanjing Jiancheng Biotech. Institute, Nanjing, China. The mitochondrial morphology in tumor cells was examined using the transmission electron microscope (TEM). Further, cell cycle and apoptosis were respectively measured by using flow cytometery (BD FACS Calibur, USA).

Preparation of subcutaneous melanoma mouse model
In order to prepare the mouse model of subcutaneous melanoma, the cell suspension (cell concentration 10 7 /mL) with a volume of 0.2 mL was subcutaneously injected into the subaxillary of the right forelimb. After 6 days of cell transplantion, the longest diameter (L) and the largest transverse diameter (W) of the vertical direction of tumor mass were measured by using the vernier caliper, respectively. The volume of the tumor (V) was calculated according to the formula: V = LW 2 /2.

Animal assignment and mitochondrial administration
After 6 days of cell transplantation, the mice were randomly classified into 4 groups.
The groups were assigned as tumor model group, female mitochondria group (F-mito), and male mitochondria group (M-mito). In the mitochondria treating group, the isolated exogenous mitochondria (10 6 ) were injected intravenously once in two days, while the mice in the model group were injected with the same amount of saline. Every group contained 8 mice (n = 8 in each group) for the mitochondrial therapy on the subcutaneous melanoma. The subcutaneous tumors in each group were observed and recorded until the mice in the model group were euthanized through the administration of overdosage of sodium pentobarbital. Then the tumor tissues were isolated and weighed, and stained with HE. Meanwhile, the tissues were homogenated to respectively measure the levels of pyruvate kinase (PK), lactate, pyruvate, and ATP according to the commercial kits (Nanjing Jiancheng Biotech. Institute, Nanjing, China).

Preparation of mouse pulmonary metastatic melanoma and mitochondrial treatment
The tumor cells in the logarithmic growth period were digested by 0.25% trysin/EDTA and adjusted the concentration to 10 6 /mL. Then the cell suspension (0.2 mL) was administered into the tail vein. The mice were given mitochondrial therapy once every two days at the sixth day after cell transplantation. On the 24 th day, the mice were euthanized, and the numbers of metastatic tumor colonies per lung were examined.
Subsequently, the melanoma mass was respectively removed out for further measurement.

Western blot
Protein extraction of melanoma was exposed to SDS-PAGE and transmitted onto PVDF membrane, further investigated with antibodies versus BCL6, LC3, parkin, Ccnb, Cadherin, Mcam and -actin (Beijing boason Biotech. Co., Ltd., Beijing, China), then with HRP-conjugated secondary antibodies (1:5000; Beijing Dingguo Biotech. Co., Ltd., Beijing, China) as the secondary antibody. Further, the membranes were thoroughly washed twice for about 15 min for each wash, and the signals were respectively recognized by the ECL system (Pierce Co., USA).

Immunohistochemistry for cell apoptosis
TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) staining was conducted on the tumor tissues obtained from the mice. Apoptotic cells were identified by using DAB in-situ cell apoptosis kit according to the manufacturer's protocol (Beijing Beyotime Biotec. Co. Beijing, China). Tissues sections were observed under an optical microscope (Olympus, Japan).

Transcriptomic analysis
In order to elucidate the molecular mechanism of mitotherapy on the tumor, the metastatic lung melanomas of each group were dissected for transcriptomic analysis. In short, the total RNA of the melanomas was extracted based on the manufacturer's procedure, and then NanoDrop ND-2000 (Thermo Scientific, USA) was used to examine the purity and quantification of the total RNA. The library construction and sequencing were carried out by Shanghai Personalbio Biotechnology Co., Ltd.
(Shanghai, China). Differential expression genes (DEGs) between model and mitotherapy groups were identified through the calculation of the gene expression level of each transcript according to the fragments per kilobase of exon per million mapped reads (FRKM) method. Then SEM and edgeR software were respectively used to measure the concentration of genes/isoform and differential expression analysis. For the functional annotations, the collected transcripts were searched using String, KEGG databases, and NCBI protein nonredundant (NR) databases. For Gene Ontology (GO) annotations, the BLAST2GO program and KEGG database were used to analyze the pathways.

Statistical analysis
Data were expressed in terms of mean  SD. Further, to analyze the difference between groups, one-way ANOVA method, and an unpaired two-tailed Student's t-test was carried out to compare statistical significance (p < 0.05). For transcriptomic analysis, the mean difference criteria of the transcription between groups are set as [log2FoldChange > 1 and p < 0.05.

Mitochondrial characteristics from male and female mice
So as to identify the difference between the mitochondria from male mice (M-mito) and female mice (F-mito) (Fig. 1A), mitochondrial membrane potential, redox ability, and PDH activity were measured respectively. Increasing trends were showed in F-mito compared with M-mito despite of without significant difference between the two groups ( Fig. 1B). Nevertheless, redox ability and PDT activity of F-mito were higher than those of M-mito ( Fig. 1C and 1D).

Mitochondria prevented melanoma cell proliferation
The cell internalization of mitochondria in melanoma cells was evaluated by using the confocal microscopy, where mitochondria were labeled by commercial MitoTracker Red CMXRos, and the cell skeleton was stained by FITC-phalloidin. After incubation with mitochondria for 2 h, a prominent yellow fluorescence was identified in the melanoma cells ( Fig. 2A), as shown by overlay of the fluorescence of MitoTracker red and FITC. Moreover, the morphology of mitochondria in the cells was observed by using TEM, and the images showed that the mitochondria in melanoma cells exhibited elongated and cavitating shape (Fig. 2B). In contrast, these in mitochondria-treated cells represented a spherical and rod-like structure. Also, autophagic bodies appeared in the mitochondria-treated cells. Under the TEM, the mitochondrial numbers significantly increased after mitochondrial addition, assessed by counting the mitochondrial numbers on each image (Fig. 2C).
To determine the capacity of the mitochondria on melanoma, we examined the effect of the mitochondria on cell viability in anoxic conditions using the anaerobic incubator. As illustrated in Fig. 2D, mitochondrial treatment for 24 h resulted in reduction of cell viability in a concentration-dependent manner, and F-mito-treated cells had lower viability than the cells treated by M-mito.
Since tumor cells produce energy mainly through glycolysis under hypoxia condition,[13 we examined the levels of pyruvate and lactate, the metabolites of glycolysis, as well as ATP content after the mitochondria were introduced into the melanoma cell media. The results exhibited that both pyruvate and lactate levels were reduced, accompanying with decreases of ATP content in the mitochondria-treated cells

Mitochondria inhibited the growth of subcutaneous melanoma
After the B16 cell transplantation, the volume of subcutaneous tumors in the model group increased continuously. When the mice were euthanized at 16th days after transplantation, the tumor volume was 3307.7 ± 395.2 mm 3 ( Fig. 3A and 3B), and the weight reached about 3.1 g (Fig. 3C). However, the growth rate of tumors slowed down after the mitochondrion administration. On the 5th day after intravenous injection of Fmito, the tumor volume began to shrink, and the growth rate slowed down. After the 11th day of F-mito injection, the tumor volume reduced to 1198.8 ± 408.5 mm 3, and the weight was about 1.5 g. Also, after intravenous injection of M-mito for 11 days, the tumor volume was 2042.1 ± 260.9 mm 3 , and the weight was about 2.0 g.
On the 16th day of cell transplantation, the subcutaneous melanomas were respectively removed out and stained by HE. The images showed that the tissues in the model group were full of tumor cells (Fig. 3D), whereas cell numbers reduced and gaps appeared in the mitochondria-treated groups. In addition, the results of biochemical measurement showed that the PK, one of the key enzymes of glycolysis, as well as the contents of lactate and pyruvate, significantly reduced. Accordingly, the ATP level was also significantly decreased. In addition, the levels of PK, lactate, pyruvate, and ATP in the F-mito-treated group were lower than those in the M-mito-treated group.
Moreover, TUNEL staining was carried out to determine apoptosis after mitochondrial injection since mitochondria were a key player in cell apoptosis. As shown in Fig 4I, numbers of TUNEL-positive cells in melanoma tissues were stained brown by DAB in the mitochondria-treated mice. Also, tumor cells in the F-mito-treated group had enormous TUNEL-positive cells than those of the M-mito-treated group.
To further identify the cell apoptosis induced by mitochondrial administration, Western blot was performed to examine the level of BCL2, an anti-apoptotic protein that increased in tumor cell proliferation (Fig 3J). The gray value showed that the mitochondria could significantly decrease the BCL2 level (Fig 3K), suggesting that the anti-apoptotic ability of tumor cells might be inhibited. In addition, the levels of autophagy-related proteins, LC3, and parkin, remarkably increased in melanoma cells after mitochondrial administration, especially in the F-mito-treated group (Fig3J, 3L, and 3M).

Mitochondria prevented the proliferation of metastatic lung melanoma
Since one of the leading causes of mortality in melanoma is lung metastasis, here the animal model of metastatic lung melanoma was prepared to examine the therapeutic effect of the mitochondria. After the cell transplantation, the numbers of melanoma nodules increased daily, and the lung color changed to dark in the model mice. The model mice at autopsy exhibited large and dense nodules on day 16 after cell transplantation, calculated through gross anatomy and HE staining technique ( Fig. 4A and 4B). However, tumor development was prevented after mitochondrial treatment, showing smaller and lower tumor nodules in the lungs (Fig. 4A-C). The suppressive effect of melanoma is more prominent in the F-mito-treated group. Then levels of PK, lactate, pyruvate, and ATP were respectively measured to identify the metabolic changes after mitochondrial administration. Similarly, with the results of mitochondrial action on subcutaneous melanoma, the mitochondria decreased the levels of PK, lactate, pyruvate, and ATP ( Fig. 4D-G), suggesting that energy production through glycolysis was inhibited in the metastatic melanoma cells.
Moreover, mitochondria in the melanoma were visualized under the TEM. Only a few numbers of mitochondria were visualized in the model group, whereas in the mitochondrial treatment group, the number of mitochondria increased predominantly.
Also, mitochondria appeared as vacuoles and with swelling, and there were also autophagosomes in the melanoma cells of the treatment group (Fig. 4H), which could be related to the increase of protein levels of LC3 and parkin (autophagy-related proteins) (Fig. 4J-L). Besides, the expression of BCL2 protein decreased after mitochondrial treatment ( Fig. 4J and 4M). Meanwhile, the images of TUNEL staining showed that a large number of TUNEL-positive cells appeared in the mitochondriatreated group (Fig. 4N), indicating that mitochondria could initiate cell apoptosis through reduction of the anti-apoptotic protein.

Transcriptomic analysis of metastatic melanoma after mitochondrial treatment
In order to elucidate the molecular mechanism of mitochondrial inhibition on melanoma, transcriptome analysis was used to identify the genes and molecular signals  (1), genetic information processing (4), human diseases (7), metabolism (11), and organismal systems (3). The majority of down-regulated KEGG pathways were closely associated with cancer cell proliferation, including cell cycle, DNA replication, and mitosis (Fig. 5B), while three up-regulated pathways were involved in glutathione metabolism, drug and xenobiotic metabolism (Fig. 5C).

Mitochondria inhibited gene transcription of the tumor cell proliferation
From the KEGG pathway, the cell cycle was significantly arrested by the mitochondria.
An extensive range of genes involved in the cell cycle and division were downregulated after mitochondrial treatment (Table S1). The result was consistent with the Western blot analysis of CCNB1 (one of representative cell cycle regulators) ( Fig. 5D and 5E), and with the melanoma cell cycle arrest after mitochondrial administration by using flow cytometry (Fig. 2B).
Meanwhile, mitochondrial therapy inhibited the cancer-related gene transcription in melanoma. As tumorigenesis and development, numbers of cancer-related genes are transcribed and expressed to maintain the unlimited proliferation of tumor cells.
However, a series of the genes were down-regulated after mitochondrial treatment,  (Fig. 5D, 5F, and 5G).

Mitochondria regulated the gene transcription of autophagy-and apoptosisrelated proteins in the melanoma
The result of DEG analysis exhibited that the mitochondria activated gene transcription of autophagy-related proteins (Gabarapl1, LC3, Atg, Prkn, Pink1) ( Table S3). The activation would lead to an increase of the autophagy protein expression (Fig. 2B and   4H) and the emergence of autophagy ( Fig. 3J and 4J). Moreover, mitochondria downregulated gene transcription of anti-apoptotic proteins, including BCL6, Naip6. Naip5, Ddias, Apaf1, and Stk17b, but increased the transcription of mitochondrion-associated apoptosis-inducing factor gene (Aifm3) ( Table S4). As a consequence, it appeared numbers of apoptotic cells in the melanoma after treated by the mitochondria, evaluated by flow cytometry and TUNEL staining (Fig. 2E, 3I, and 4N).

Molecular signal pathway of mitochondrial therapy on melanoma
In order to figure out all the relevant cell signaling pathways produced by the mitochondria, we outlined the signaling regulatory network through the existing DNAprotein interaction database and protein-protein interplay (Cytoscape 3.5.0 software).
Gene regulation by chromosome silencing and mitochondrial unfold protein reaction (UPR mt ) induced by mitochondrial stress was considered to be the most relevant mechanism in the series of biological activities of the mitochondrial therapy. The overall gene silencing of tumor cell proliferation caused by the mitochondria would be related to methylation modification of histone. Meanwhile, the mitochondrial stress would promote the local opening of UPR mt regulatory region to induce gene transcription of tumor suppressor proteins and autophagy-related proteins. The signal pathways could explain the signal mechanism of the inhibitory effect of mitochondria on melanoma.

Discussion
Currently, melanoma has become the leading cause of death from skin disease. [15 Many efforts have been made to find ways to treat the terrible disease. Here we suggest that the tumor growth of both subcutaneous and metastatic malignant melanoma was significantly inhibited after mitochondrial administration. The signal mechanism was closely associated with the chromosome silencing and the local UPR mt effect caused by mitochondrial stress, eventually leading to cell cycle arrest, energy deficiency, autophagy, and cell apoptosis. The tumor inhibition effect of F-mito in cell cycle arrest and energy depletion was more potent than that of M-mito. In order to determine which mitochondria from different genders have a stronger effect on the future application in animals, we tested the sex differences of mitochondria on treating melanoma. Although numbers of reports described tissue-specific sex differences in mitochondrial morphology and oxidative capacities [29 , 30 , scarcely studies showed the functional differences of mitochondria in therapy. From the limited data available, female mitochondria have more favorable mitochondria-nuclear communication in response to stress compared to male mitochondria.[31 In this study, we firstly assessed mitochondrial activities isolated from female and male mice, and the results showed that female mitochondria exhibited higher activity and ATP production ability than those of male mitochondria. Subsequently, the anti-tumor experiments both in vitro and in vivo proved that female mitochondria have higher efficacy to inhibit tumor cell proliferation than male mitochondria. The study also revealed that female mitochondria could induce more robust stress response to silence gene transcription than male mitochondria in tumor cells, suggesting that female mitochondria were more sensitive under the hypoxic microenvironment of the tumor than male mitochondria, and eventually led to the stronger anti-tumor effect.
Here we used "naked" mitochondria to study their anti-tumor activity through the intravenous administration. After the mitochondria are injected into blood vessels, they would increase the content of mitochondria in blood. It is known that blood contains intact cell-free mitochondria in both healthy people and cancer patients, but the level of circular mitochondrial DNA in plasma of the cancer patients is proportionally lower than that of normal individals, [32,33 implying [35,36 Thus, we assumed that the mitochondria in blood could be internalized by melanoma cells because of the high permeability of local tumor blood vessels (enhanced permeability and retention effect) [37 , which was evidenced by TEM of melanoma tissues (Fig. 4). Further, mitochondria in blood can activate immune system through increasing the activities of phagocytes and T cells, [38,39 which might increase the anti-tumor effect of the mitochondria to a certain extent. The mitochondria on tumor immunity will be further invstigated.

Conclusions
In summary, the results exhibit a novel insight into mitochondrial function in malignant melanoma, and propose that healthy mitochondria inhibit tumor cell proliferation by regulating tumor gene transcription. The overall down-regulation of genes results in cell cycle arrest and cell proliferation stagnation, as well as the activation of autophagy and apoptosis, which eventually leads to the apparent inhibition of melanoma growth in mitochondrial treatment. The effect of female mitochondria on anti-melanoma is stronger than that of male mitochondria, which may explain why incidence rate and mortality rate of melanoma in female are lower than those in male.
The study not only reveals the anti-tumor mechanism of the mitochondria but also suggests female mitochondria have more potent than male mitochondria on antimelanoma.

Conflict of interest
None of the authors have any competing financial interests.

Authors' contributions
ZY did the animal study and transcriptome analysis. ZZ performed the cell culture and data analysis. CF did the data analysis and interpretation the data. WZ and JW did the animal study. AF designed the study and wrote the manuscript.

Funding
The work is supported by Chongqing Research Program of Basic Research and Frontier

Technology (No.cstc2018jcyjAX0612) and Chongqing Innovation Project for
Returnees from Overseas Scholars in 2018 (cx2018086).

Ethical Approval and Consent to participate
The animal experiment have been approved by the Animal Ethical Committee, Southwest University, China.

Consent for publication
All authors agree to publish the paper.

Competing interests
The authors declare that no competing interests.

Availability of supporting data
The data supporting the results of this article are included within the article and its additional Tables.