Tumoricidal Activation of Macrophages using Jatropha curcas Leaf Extract: As a Proxy for the Treatment of Cancer

Background: Jatropha curcas (J. curcas) is a plant rich in methanolic. Macrophages can be activated to become tumoricidal through interactions with immunomodulators. The aim of this study was to investigate the effect of J. curcas leaf extracts on the phagocytic activity of macrophages. Methods: Peritoneal-derived macrophages from male BALB/c mice were obtained using the method of Colligan et al. with some modifications. Latex was added as an antigen to cultures of BALB/c mice macrophages treated with two different concentrations of J. curcas extract and the cultures were observed every 30 minutes for 2 hours. Results: Macrophage latex vacuoles were observed after treatment with 15 μg/mL J. curcas leaf extract, with a mean latex vacuole count of 7.2 vacuoles/cell and a phagocytic index (PI) of 369. Macrophage treatment with 250 μg/mL J. curcas leaf extract produced a mean latex vacuole count of 8.1 vacuoles/cell and a PI of 785. Conclusion: Treatment with J. curcas leaf extracts increased the phagocytic activity of mice macrophages.


Introduction
Biological activities of the methanolic was under study. Leaf extracts that contains methanolic acid of Jatropha curcas showed the highest antioxidant activity. Other research in cytotoxicity assay results indicated the anticancer therapeutic property of the root extract against human colon adenocarcinoma (HT-29) cell line but its cytotoxic effect on human hepatocyte (Chang cell) was high [1].
Macrophages can be activated to become neoplasticidal by interaction with a substance filled immune modulators. Neoplasticidal macrophages can recognize and destroy neoplastic cells in vitro and in vivo. Although the exact mechanism by which macrophages discriminate between neoplastic and normal cells is unknown, it is independent of neoplastic cell characteristics such as immunogenicity, metastatic potential, and sensitivity to drugs. Moreover, macrophage destruction of neoplastic cells apparently is not associated with the development of neoplastic cell resistance. Macrophages are found in association with neoplasm in a definable pattern, suggesting that the most direct way to achieve macrophage-mediated neoplasm regression is in situ macrophage activation [2][3][4].

Method
Peritoneal-derived macrophages from male BALB/c mice (Hayati Science Lab. GMU Yogyakarta, Indonesia) were obtained using the method of Colligan et al. with some modifications [5]. Macrophages obtained by using a 10 ml syringe and hypodermic needle 25G. The results collected in sizes 25 ml centrifuge tube and stored on ice. Macrophage cultures performed in culture dishes with a diameter of 20 mm. Macrophages were distributed into each well of the plate so that the culture wells containing an average of 1000 cells. For ease of painting it on the basis of prior pitting the cover glass is placed before the cell is inserted. Further culture medium Roswell Park Memorial Institute (RPMI) 1640 (Sigma Chem. Co. St. Louis, USA) inserted into the wells as much as 10 ml. Medium replacement done once every 24 hours and incubated at room temperature.
Prior to treatment, the culture medium was removed and the macrophages were washed extensively with PBS-10F (7 nM phosphate buffer, pH 7.4 with 137 nM NaCl and 3 nM KCl). The cultures were then incubated with fractionated R. communis extract for 10 min at 37ºC. The cells were washed again with PBS-10F to remove any remaining R. communis leaf extract. Latex was then added to the macrophage cultures as a test antigen, and the cultures were observed every 30 min over 2 hrs [6]. For time-lapse microscopy the samples were stained with Giemsa (Merck). Nikon phase contrast microscope images were collected with a 100X objective lens [7].

Results
An increase in the phagocytic activity of cultured macrophages was detected following treatment with J. curcas leaf extract [ Figure 1 (A and B) and Table 1]. Macrophages with latex-containing vacuoles were observed to form after treatment with 15 µg/mL J. curcas leaf extract.
The mean latex vacuole (i.e. mean phagocyte) count per cell was 7.2 and the phagocytic index (PI) was 369. After treatment with 250 µg/mL J. curcas leaf extract, the mean latex vacuole count per cell was 8.1 with a PI of 785.

Discussion
Terpenoid compounds are the major metabolites found in the Euphorbiaceae family. Among the terpenes, diterpenoids have dominated research in Jatropha species with respect to their novel chemical structures and medicinal values [8]. Recently, Oskoueian et al. [9] reported that extract of root and latex of J. curcas plant which contained phenolics, flavonoid and saponins showed notable antioxidant, anticancer and anti-inflammatory activities. These compounds have been reported to be involved in the biological activities of the plants [10]. Ricinus communis is a same species of plant that can vary greatly in growth patterns and appearance. This variability has been enhanced by breeders who have selectively bred a range of cultivars for different leaf and flower colors. Ricinus communis is the only plant whose seeds are rich in a hydroxy fatty acid, namely ricinoleic acid. This compound has known pharmacological activities (e.g., anti-microbial, anti-fungal, antioxidant and anti-Inflammatory) [11][12][13].
Tumor cells express tumor-specific antigens (TSA) which the immune system recognizes as foreign. Phagocytes identify these tumor cells and initiate phagocytosis via the nonspecific Fc-receptor. Macrophages are known to express receptors for the phagocytic process, namely IgG-R and Complement-R, which are also involved as intermediaries in the macrophage-mediated phagocytosis of tumor cells [14,15].
The triggering of phagocytosis by macrophages can induce inflammatory activity, which, if sufficiently severe, can affect the clinical outcome. Modulation of the environment of the inflammatory response using molecules or cells can enhance the healing process. The IL-1β and M1 macrophages were observed to be very important in the healing process following surgery through modulation of the inflammation environment [2].
The relationship between macrophages and tumor cells has been widely investigated. Macrophage activity was shown to improve significantly following intravenous delivery of liposomes containing immunomodulators. However, other studies have indicated that the activation of macrophages would be more useful if the immunomodulators were supplied in the form of an adjuvant as this would provide minimal side effects [16].
Research regarding the activity of macrophages has shown that the extension of pseudopodia is triggered by the cytokines IL-4 and IL-13, which also protect the type-1 macrophages from the influence of LPS and IFN-γ [17].
Sorafenib, which is used to fix macrophage polarization, triggers the interaction between tumor cells and natural killer (NK) cells. Sorafenib therefore affects the function of macrophages and NK cells in terms of the destruction of tumor cells. Sorafenib triggers the activity of inflammatory tumor cells connected with the activity of macrophages and triggers NK cells to fight tumor cells by expressing cytokines and NF-κB. The sorafenib compound can therefore be used in the clinic as an alternative polarization between macrophages and tumor cells [18].
Thiram is widely used as a dithiocarbamate pesticide and fungicide, but can also be used as a suppressor of inflammation; studies have shown that thiram can reduce the inflammation resulting from contact dermatitis. Explain when thiram interact with the agency and the intensity of the inflammation will decline.