Anti-tumor therapies based on anti-inflammatory effects have been considered in cancer treatment. Survival, proliferation and, resultantly, invasion and metastasis of tumor cells are regulated by local inflammatory mediators. Primary inflammatory cytokines, such as tumor necrosis factor TNF , are targets for anticancer therapy. Berg Myrtaceae. For gene expression, analysis was performed by total RNA extraction followed by synthesis of complementary DNA and analysis by quantitative polymerase chain reaction.
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Inflammation associated with cancer is a promising target for the development of anticancer therapies. Cytokines, chemokines and growth factors may have an important function in the interaction between tumor cells and infiltrating leukocytes from blood vessels. The existence of inflammatory components in the microenvironment of neoplastic tissues frequently leads to increased angiogenesis, resistance to hormones and inhibition of adaptive anti-tumor immunity.
The survival, proliferation and subsequent invasion and metastasis of tumor cells is regulated by inflammatory mediators present at the tumor site 1. Primary inflammatory cytokines, including interleukin-1 IL-1 and tumor necrosis factor TNF , expressed by leukocytes and tumor infiltrating cells, are targets for anticancer therapy. Anti-cytokine strategies against tumors have been investigated with TNF inhibitors in certain inflammatory diseases 2.
However, it also exhibits oncogenic activity associated with various processes, including invasion, inflammation and angiogenesis, which are essential in tumor development 8.
Plants are a major source of active substances that are used in therapeutic medicine as their metabolites have great structural diversity 9. Anti-inflammatory compounds have been extracted from natural products, including fruits, vegetables, spices and traditional medicinal herbs, and these compounds have been gaining importance as potential chemopreventive or therapeutic agents for cancer In the last 2 decades, herbal products have been important candidates in the discovery of novel drugs for cancer There were 12 natural products and 32 derivatives of natural products among the anticancer drugs released to the market between and They were obtained from various sources, including plants and microorganisms.
Between and , small molecules were released for the treatment of cancer and of those, molecules were developed from natural products The investigation of novel plants with anti-inflammatory, anti-tumor and anti-carcinogenic potential is important and may enable the development of novel drugs for cancer treatment.
Several members of Myrtaceae family have been previously investigated and various activities were observed, including antioxidant 14 , anti-tumor 15 — 21 and anti-inflammatory activities 22 — To the best of our knowledge, there are no existing studies investigating the activity of Calyptranthes grandifolia.
However, important activities have been demonstrated in other members of the Calyptranthes genus 28 — Thus, this genus demonstrates potential and may be beneficial in the treatment of inflammatory and tumor processes.
The objective of the present study was to investigate the expression of genes associated with proliferation and inflammation in cells of the Caco-2 cell line treated with extracts from Calyptranthes grandifolia O.
Leaves of Calyptranthes grandifolia O. From this material ethanol and hexane extracts were isolated according to the following methodology. Subsequently, leaves were reduced to small fragments to increase the contact surface with the extraction solution. The leaves of Calyptranthes grandifolia O. Berg were packed with hexane solvent in an amber bottle at room temperature for 72 h.
Vacuum filtration was performed and the filtrate was stored in an amber jar at room temperature until the solvent was removed. Maceration of the leaves occurred for 2 weeks and the solvent was changed twice. Subsequent to 24 h incubation at different concentrations and treatments, extraction of total RNA was performed on the Caco-2 cell line and the supernatant of RAW In addition to treatment with extracts, each culture plate had a positive control, in which cells were stimulated with LPS only, and a negative control with no stimulation or treatment.
A total of 5 different experiments were performed for gene expression analysis. As a negative control, cells were placed only in culture medium and DMSO. Correction factor was calculated by staining the culture medium with no cells. Following centrifugation, the supernatant was discarded and the pellet was resuspended in 1 ml DMEM in a dilution with Trypan Blue dye, which stains non-viable cells.
Finally, a total count of viable cells was performed in a Neubauer chamber for subsequent plating of viable Caco-2 and RAW Synthesis of cDNA was performed from 0. Gene expression analysis was performed by qPCR. The qPCR results were expressed as the relative quantification of amplified cDNA with respect to the normalizer gene Primers used for amplification of specific cDNA fragments were selected from the published sequence of each gene using online tool Primer3 v.
To confirm specificity of the reaction, a dissociation curve was performed for each primer pair with melting temperature analysis of each gene. Following 24 h incubation of RAW Data were tabulated and analyzed with descriptive statistics using SPSS software version Comparison between the controls was performed using an unpaired Student's t-test and the effects of extracts were analyzed by one-way analysis of variance followed by the Tukey test.
It was observed that the ethanolic and hexane extracts of Calyptranthes grandifolia O. Berg did not affect the cell proliferation when compared with the negative control. The negative control exhibited a viability of C-, negative control; EE, ethanolic extract; HX, hexanic extract. For the analysis of the genes of interest, the positive control consisted of Caco-2 cells that were stimulated only by LPS with no extract treatment, and the negative control consisted only of cells in culture.
The comparison between negative and positive controls was performed in order to verify whether there was a difference in gene expression when stimulated by LPS. Thus, the expression of these genes was stable in the negative and positive control groups Fig.
Effect of LPS on gene expression compared with the negative control. A macrophage lineage was used, characterized by the release of inflammatory cytokines. The negative control There were no significant differences when treated with hexanic extract data not shown. Herath et al 42 demonstrated that different serotypes of LPS produce different responses upon cell stimulation. However, LPS significantly activated the pathway. Other pro-inflammatory genes also exhibit modified activation by different LPS serotypes.
When evaluating the effect of Calyptranthes grandifolia O. Thus, the anti-inflammatory activity of the extract may be associated with the inhibition of pro-inflammatory cytokines and increased IL Li et al 44 evaluated the activity of flavonoids in RAW The nuclear factor is a central regulator of immune responses and has an important role in the expression of cytokine genes, including IL-2, IL-6, C-C motif chemokine 2 and CD40 ligand.
Furthermore, it is also involved in the expression of genes associated with cell survival and proliferation, including cyclin D1, cyclin D2, c-Myc, c-Myb, cyclooxygenase-2, Bcl-2 and Bcl-xl 47 , The activation of p38 isoforms is specifically controlled by different regulators and they are co-activated by several combinations 53 , Thus, p38 signaling may be associated with inflammation, the cell cycle, cell death, development, differentiation, senescence and tumorigenesis However, it is important to note that this kinase is associated with various responses, and may also be involved in resistance to chemotherapy in certain types of tumors The increase in mRNA levels is not directly proportional to the amount of protein translated, due to transcriptional and post-translational modifications.
The extracts included in the present study did not affect the cell viability when compared with the negative control. In this aspect, cytotoxic extracts maybe potential candidates for anti-carcinogenic studies However, it is important to note that these statements should be tested in other cell lines in order to observe potential selective cytotoxicity. The antioxidant activity was also determined in ethanolic and hexane extracts of Calyptranthes grandifolia by the research group data not shown.
This activity was evaluated by antioxidant activity testing, by capturing the free radical 2,2-diphenylpicryl-hidrazila The results demonstrated that the hexane extract exhibited no antioxidant activity, however, the ethanolic extract exhibited a dose dependent antioxidant activity. However, further studies are required to elucidate the signaling pathway that may be activated. Given the variation between experiments and potential post-transcriptional regulation, the analysis of other genes is required in order to assess the potential pathways implicated.
Furthermore, it is important to analyze protein expression in order to confirm that the expressed genes in Caco-2 cells are translated and determine the respective levels of translation. Cells treated with hexane extract exhibited no significant variations in the expression of the genes investigated at any of the concentrations. The extracts were not considered to be cytotoxic and are not candidates for anti-carcinogenic studies in this lineage.
However, other studies using different cell lines maybe performed to identify selective cytotoxicity. Thus, it is important to investigate its genotoxicity and to conduct in vivo analysis to confirm its anti-inflammatory potential. J ClinOncol. View Article : Google Scholar. Topics Curr Genet. Planta Med. Int J Phytomed. Nat Prod Rep. J Nat Prod. Kausel Myrtaceae leaves. RevBras Farmacogn. Appl Microbiol Biotechnol.
Curr Med Chem. Pak J Pharm Sci. Oncol Res. J Pharm Pharmacol. Rev Bras Farmacogn. J Ethnopharmacol. Potential for COPD therapy. Food Chem. BMC Immunol. Alston and isolated compounds on acne vulgaris. Mol Cancer Ther.
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Calyptranthes is a genus of flowering plants in the family Myrtaceae. They are known commonly as lidflowers , spicewoods , and mountainbays. They are evergreen shrubs or small trees , growing to 5 meters tall. They have simple, opposite, evergreen leaves and clusters of small flowers lacking petals. The sepals are capped in bud by a lidlike cover, which is shed later. The fruit is a dry, berry-like, reddish brown.