In Vivo Evaluation of the Antitumor and Immunogenic Properties of Silver and Sodium Dichloroacetate Combination against Melanoma

Posgrado Conjunto de las Facultades de Agronomía y Medicina Veterinaria y Zootecnia, Universidad Autónoma de Nuevo León, Ave. Universidad S/N, Cd. Universitaria, San Nicolás de los Garza, N. L., CP 66455, Mexico Laboratorio de Inmunología y Virología, Unidad C, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Ave. Universidad S/N, Cd. Universitaria, San Nicolás de los Garza, N. L., CP 66455, Mexico


Introduction
Targeted therapies have increased the chances of survival for people with melanoma [1]; however, cancer cells present within the tumor favor different metabolic pathways [2]; as a consequence, the tumor eventually becomes resistant to targeted therapies, especially the ones designed against a single target [3].
The development of silver-based therapies is a promising tool in cancer treatment. Silver ions and silver nanoparticles induce oxidative stress, mitochondrial membrane dysfunction, DNA damage, and cytokines upregulation [4]. The exact action mechanism varies depending on the physical and chemical properties of the nanoparticle and the type of cancer [5]. Furthermore, the clinical use of colloidal silver for bactericidal and antiviral purposes proves that this treatment is safe [6,7].
Sodium dichloroacetate (DCA) is a pyruvate analog which interferes with tumor-associated glycolysis (Warburg effect), decreases cancer malignancy, and reduces lactate production by altering cancer cell metabolic pathways [8]. A decrease in lactate counteracts the acidic state of tumoral microenvironment, reducing tumor growth and metastasis [8]. WZB117, a bis-hydroxybenzoate, 2-deoxy-d-glucose, metformin, and DCA reduce glycolysis and block glucose uptake in cancer cells. Under low intracellular glucose levels, biosynthetic pathways, such as nucleotides and amino acids genesis, are interrupted due to a shortage of intermediate molecules, putting a break on cell proliferation. Despite of its use as monotherapy or combined with chemotherapy, few or none adverse effects have been reported [9].
Because of these activities, we evaluated immunogenic cell death as a possible action mechanism, owing to the increasing number of studies that demonstrate that cellular and mitochondrial danger-associated molecular patterns (DAMPs) can be actively released when exposed to external stimuli [10]. The release of alarmins (Hsp70, HSP90, calreticulin, HMGB1, ATP, DNA, and RNA) and tumor neoantigens induce a tumor-specific immune response that eliminates live cancer cells and residual tumor tissue, avoiding cancer recurrence [11].
The main focus of this study was to use silver and DCA as dual-function agents that affect the DNA integrity and mitochondria activity in order to increase the antitumor response in melanoma treatment. Furthermore, this study could serve as a starting point for the next level developmental stage of dichloroacetate-loaded silver nanoparticles targeted pharmacological formulation. 2.3. Cell Viability. Cells (5 × 10 3 cells/well) were plated on 96 flat-bottom well plates and incubated for 24 h at 37°C in 5% CO 2 atmosphere. After incubation, culture medium was removed, and Ag (0.8mM to 6:5 × 10 −5 mM) or DCA (75mM/ml to 750mM/ml) diluted in the same medium were added. The plates were then incubated for 4h at 37°C and 5% CO 2 atmosphere. Thereafter, the supernatant was removed, and cells were washed twice with DMEM/F-12 medium. Cell viability was determined by the resazurin (Alamar Blue) method, and cytotoxicity was expressed as the concentration of 50% cell growth inhibition (LD 50 ). Results were given as the mean ± standard deviation (SD) of three independent experiments. The LD 50 of each treatment was used in further experiments.
2.5. Animals. Female C57BL/6 mice aged 6 and 10 weeks with a body weight around 23 (±2) g were purchased from the Harlan Laboratories (Mexico City, Mexico). The mice were kept at 25-29°C and a 12h light to 12h dark cycle. Food and water were provided ad libitum. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the Biological Sciences Faculty, Autonomous University of Nuevo Leon (San Nicolas de los Garza, Mexico).

Tumor Implantation and Treatment Administration.
Tumors were induced subcutaneously by injecting 1 × 10 6 B16F10 cells in 200 μl of phosphate buffered saline (PBS) solution. Seven days after B16F10 cell transplantation, a noticeable tumor mass appeared, and mice were distributed randomly into four groups (five mice per group). The control group received only saline solution. The DCA group received 50mg/kg of DCA, whereas the Ag group received 28mg/kg of Ag. Saline solution, Ag, and DCA were administrated by peritumor route, daily, for 21 days. Finally, the Ag + DCA group received the same of doses Ag and DCA by peritumor route every other day, alternating between treatments. Tumor length and width were measured weekly, and tumor volume was determined using the equation: L × W 2 , where L is the longest side and W is the shortest side. Animals were euthanized at the study endpoint (21 days), and the tumors were excised for further experiments.
2.7. ELISA for Active NF-κB p65 Subunit. To measure NF-κB p65 subunit activation, nuclear extracts were prepared from 3 × 10 6 tumor cells, using a Nuclear Extract Kit according to the manufacturer's protocol. Levels of nuclear p65 concentrations were determined by a sensitive ELISA assay (TRANS-AM, Active Motif, Rixensart, Belgium).

TNF-α and NO Production.
Tumors were macerated with RPMI and the supernatant collected and adjusted at a concentration of protein by BSA and stored at -20°C for evaluation. TNF-α was measured in the tumor supernatant by enzyme-linked immunosorbent assays (TNF alpha Mouse ELISA Kit; Invitrogen; Thermo Fisher Scientific; Viena, Austria). All assay procedures were performed according to the manufacturer's protocol.
Nitrate/Nitrite assay kit was used to measure the levels of NO in the tissue homogenates of the tumor followed the protocol established by the manufacturer (Nitrate/Nitrite colorimetric assay kit; Cayman Chemical, USA).

Journal of Nanomaterials
where V is the tumor volume, L is the tumor length, and W is the tumor width, same formula as previously used by Rodríguez-Salazar et al. [12].

Journal of Nanomaterials
Lower doses of Ag and DCA were required in the combinatorial setting to achieve DL 25 and DL 50 (Figure 1(c)).

Ag-DCA Induced Tumor
Regression. The administration of Ag, DCA, and Ag + DCA induced tumor volume regression (p < 0:05) in a time-dependent manner, observing a better effect in mice treated with Ag + DCA treatment (Figure 2(a)).

Calreticulin
Exposure in Ag, DCA, or Ag + DCA Treated Cells. Ag, DCA, and Ag + DCA treatments do not induce calreticulin surface exposure in B16F10 cells, as compared to the control (B16F10 untreated cells) (Figures 4(a) and 4(b)).

Discussion
The cytotoxic effect of colloidal silver (Ag), sodium dichloroacetate (DCA), and their combination was evaluated against B16F10 murine melanoma cells. Our results show that Ag has antiproliferative effects against B16F10 cells, as previously reported by our research group [13]. Further reports of the cytotoxic activity of silver against melanoma cells refer to silver nanoparticles, although the proposed toxicity 5 Journal of Nanomaterials mechanism remains the same [14]. DCA also exhibited an antiproliferative effect against melanoma cells. In a similar manner, Rivera-Lazarín et al. reported a dose-dependent viability decrease in B16F10 cells treated with DCA [15].
The cytotoxic activity of Ag and DCA increased when used as a combined treatment. This was expected since the combination of two or more agents is a cornerstone for cancer treatment; it allows to target key pathways simultaneously, achieving an efficacy increase [16].
After observing the increased cytotoxic effect, we evaluated whether our results correlated with an in vivo antitumor activity. At the tumor level, the generation of necrosis was noted; it is worth mentioning that the lesions completely healed in all cases of tumor elimination. Skin lesions can occur due to the overexpression of tumor necrosis factor alpha [17]. Our results revealed higher levels of TNF-α in untreated melanomas and a significant decrease of this factor in response to all of our treatments. TNF-α correlates with melanoma aggressiveness and metastatic potential in vivo [18], and its overexpression has been reported in advanced primary melanomas by Rossi et al. [19].
It is important to mention that TNF-α is a pleiotropic cytokine, and its proapoptotic effects against cancer cells have been widely described [20]; however, melanoma cells resist TNF-α-induced apoptosis through NF-κB and nitric oxide [21].
In this study, our results showed NF-κB and nitric oxide decrease in mice treated with Ag, DCA, or the combination of both, correlating with tumor regression. Wang et al. reported that NF-κB suppresses TNF-α-mediated apoptosis through the activation of the antiapoptotic proteins TRAF1, TRAF2, c-IAP1, and c-IAP2 [22]. On the    Journal of Nanomaterials other hand, Salvucci et al. reported nitric oxide production in human melanoma cells, and blocking this production induces cell death in human melanoma [23]. Specifically, nitric oxide inhibits at least seven caspases trough snitrosylation [24]. Despite our observations, it is important to mention that TNF-α, NF-κB, and NO have pleiotropic effects, and their role in melanoma is not well understood. However, we emphasize that our results indicate that these molecules decrease in correlation with tumor regression and wound healing.
Many anticancer therapies have the potential to induce cancer cell death, resulting in tumor elimination and a patient free of malignancy. However, only immunogenic cell death inducers can prevent cancer recurrence. Therefore, drugs that induce immunogenic cell death represent a recent innovation in the field of onco-immunotherapy [25], such is the case of the use of immunomodulator IMMUNEPOTENT CRP, that recently demonstrated this capacity [12]. We set out to evaluate whether our treatments were capable of inducing an immunogenic cell death.
The presence of alarmins in vitro indicates the potential to induce immunogenic cell death [26]. But, despite treatment-dependent increase of HMGB1, HSP70, and HSP90 (but not calreticulin), the vaccination of mice with B16F10 cells lysed with Ag, DCA, or the combination Ag + DCA did not induce immunogenic cell death as evidenced by the tumor appearance in all mice (vaccinated or unvaccinated) after challenge with viable B16F10 cells. Tumor growth indicates a specific immune response was not induced by the vaccines. A reason for this could be that DCA, Ag, and DCA + Ag do not induce the release of DAMPs in a coordinated spatiotemporal pattern; therefore, they lack the capacity to induce cytokines and efficient antigen presentation [27].
In conclusion, the combination of Ag and DCA has potential antitumor properties against melanoma cells; however, the in vivo antitumor mechanism is not immunogenic cell death. Further studies to elucidate the cell death mechanism are important in order to design strategies and combinations with clinical efficacy against melanoma.

Data Availability
Data associated with the manuscript is available upon reasonable request.

Conflicts of Interest
The authors declare that there is no conflict of interest.