The pomegranate fruit possesses therapeutically important constituents. Almost all parts of pomegranate serve as repository for biologically active constituents, which can cure wide variety of disease such as tissue inflammation, cancer, diabetes, skin diseases, bleeding disorders and cardiovascular diseases
16-24. The major food product made from pomegranate fruit is its juice, obtained either from its arils or from the whole fruit
25. Pomegranate fruit is a rich source of polyphenols such as the flavonoid and gallo- and ellagitannin classes. The ellagitannins represent a significant portion of PGJ polyphenols and coexist with the major product of hydrolysis of this class of tannins, ellagic acid. A number of health-beneficial effects manifested by PGJ consumption are attributed to the presence of ellagic acid
26,27. Yousef et al.
13 evaluated the inhibitor effect of ellagic acid in a concentration a range of 0 to 200% on cell proliferation of CaCo-2 and HCT-116 colorectal cancer cell lines and they demon-strated the IC50 of ellagic acid was 200 μg/ ml at 24h and 100 μg/ ml at 48 h for both of CaCo-2 and HCT-116 cells similarly. In the present study we determined 8,68 ± 0,168 mg/ml ellagic acid as the major component of PGJ and we evaluated the anti-proliferative effect of PGJ between a concentration range of 1 to 6% for 12 to 72h in SW480 cells. SW480 cells differ from Caco-2 and HCT-116 with their genetic background regarding TP53 and K-RAS mutation status (28). The p53 transcription factor regulates the expression of genes with central roles in cellular processes including DNA repair, cell cycle, and apoptosis. Thus, mutations in TP53 confer significant oncogenic functions and promote metastasis and resistance to anticancer therapy
29. In addition, KRAS activating mutations in exon 2 and exon 3 avoid the sufficient therapy with EGFR inhibitors (30−32). CaCo-2 cells have mutation in TP53 gene (E204X) and HCT-116 cells have mutations in K-RAS gene (G13D). SW480 cells have mutations in both TP53 (R273H; P309S) and KRAS (G12V) genes
28. Thus, SW480 cells are types of colorectal tumors which are more resistant to current medical therapies in compare to CaCo-2 and HCT-116 cells. According to present findings, we defied the IC50 of PGJ in concentration of 4% for SW480 cells in 24h incubation. 4% concentration of PGJ (~217 μg/mL ellagic acid) is similar to IC50 concentration of ellagic acid for CaCo-2 and HCT-116 in the study of Yousef et al.
13. In this aspect, similar to the effect of ellagic acid on Ca-Co-2 and HCT-116 cells, PGJ showed an anti-proliferative effect on SW480 cells independent from the TP53 or K-RAS mutation status.
The main purpose of cancer therapy is to target proliferating cells to induce cellular death pathways. The p53 protein is a transcription factor, which can induce apoptosis by regulating the proapoptotic and antiapoptotic genes. The ability of p53 to promote cell death could be directly linked to its tumor suppressive function. Development of certain tumors in p53 null mice was associated with decreased cell death rather than increased cell cycle progression 33,34. Bcl-2, an antiapoptotic protein, is localized to the outer membrane of mitochondria, where it plays an important role in promoting cellular survival and inhibiting the actions of proapoptotic proteins 35. Bcl-2 promotor contains a p53-negative response element, raising the possibility that Bcl-2 may be a direct target of p53-mediated transrepression 36. p53 may also directly impact Bcl-2 activity as part of a transcription-independent program of cell death. In this process, cytoplasmic p53 binds to proapoptotic Bcl-2-family proteins, leading to permeabilization of mitochondria and apoptosis 37-39. Structural studies have demonstrated that the DNA-binding domain of p53 is required for direct p53–Bcl-2 interaction 40,41. Thus, TP53 mutations causes’ impaired Bcl-2 interaction though impaired DNA binding 42. In the study of Bishayee et al. 43, PE dose-dependently suppressed cell proliferation and induced apoptosis in mammary tumors though increasing Bax and decreasing Bcl2 protein expressions. However, they did not evaluate the p53 mutation status of mammary tumors. In the present study, PGJ caused 3.8 fold decreases in the regulation of BCL2 mRNA expression in SW480 cells (p =0.736). Similar to SW480 cell line, HT-29 cell line is a colorectal cancer cell line with a mutation in TP53 and in an EGFR pathway gene, BRAF mutation 28. In the preliminary studies of Banerjee et al. 44 in HT-29 cells, PG induced apoptosis and increased the expression of microRNA-126. microRNAs (miRNAs) are small non-coding RNAs of 18–25 nucleotides in length that bind to complementary UTR regions of target mRNAs, regulating the transcriptional activity of the target gene 45. Variable dietary factors, including micronutrients and non-nutrient dietary components, have been shown to alter gene expression via modulating miRNA 46. According to findings of Bishayee et al. 43, PG involve in regulation of VCAM-1 and PI3K/AKT-mTOR path-ways via modulating miR-126 in HT-29 colon cancer cells. Besides, miR-126 not only targets these signaling pathways. According to microRNA and mirSNP databases, while BCL2 is also a directly target of miR-126, TP53 is not targeting by this miRNA (Figure 3).
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Figure 3: miR-126 binding sites in 3’UTR of BCL2 gene (www.microRNA.org). |
This data imply that, PGJ may cause reduction in BCL2 mRNA level though modulating miR-126 level, independent from TP53 status.
Survivin, which encoded by BIRC5 gene, is also a target of p53 for its action and downregulation and p53 may induce apoptosis by antagonizing the antiapoptotic activity of survivin. Survivin inhibits caspases and blocks apoptosis and is expressed highly at G2/M phase and declines rapidly in G1 phase of cell cycle 47,48. G1 phase of cell cycle requires Cyclin D1 protein which encoded by CCND1 gene to dimerize with CDK4/6 and regulate the G1/S phase transition (49). Rocha et al. 50 demonstrated that, p53 indirectly involve in regulation of Cyclin D1. Kasimsetty et al. 51 demonstrated the inhibitor effect of PG on HT-29 cell line, mediated through cell cycle arrest in the G0/G1 and G2/M stages of the cell cycle followed by induction of apoptosis. However, in the present study, although we determined a reduction in CCND1 and BIRC5 expression level after PGJ treatment, the fold differences were negligible (1.84 fold; p =0.542 and 1.17 fold; p =0.718; respectively). HT-29 cells consist of a TP53 mutation (R273H) and a BRAF (V600E) mutation instead of a KRAS mutation, which is another down-stream gene of EGFR signaling pathway 28. According to study of Pek et al. 52, in CRC tumors with KRAS or BRAF mutations, CDK4/6 and MAPK coregulated gene set is highly enriched and targeting this KRAS-associated gene signature with Cdk4/6 and MEK inhibitors efficiently inhibited CRC growth and elicited apoptosis in KRAS-dependent and BRAF-mutant CRC. Kasimsetty et al. 51, demonstrated the cell cycle arrest in the G0/G1 and G2/M stages after PG treatment using a flow cytometric cell cycle analysis, but they didn’t evaluated the molecular background of this effect. Whereby we did not demonstrate any significant alteration in the expression level of CCND1 and BIRC5 genes, PGJ may target Cdk4 or Cdk6 but not Cyclin D1. In addition, PGJ may not be directly effect on BIRC5 expression since it is a direct target of TP53, which is mutated in these cell lines.
In conclusion, our observations and previous studies suggest that modulation of gene expressions may be an important mechanism underlying the biological effects of PGJ. PGJ may target specific genes though modulating miRNA expressions. Advance studies are required to define the effect of PGJ on these miRNAs. Moreover; to elucidate the molecular mechanism of this effect; beside ellagic acid; the role of other phenolic compounds of PGJ on regulation on these miRNA expressions need to be analyzed. Our results provide evidence that PGJ can induce apoptosis with reducing BCL2 gene expression independently from TP53 mutation status, suggesting a new mechanism of action for this extract. To the best of our knowledge, this is the first time that the proapoptotic capability of PGJ has been demonstrated in a both KRAS and TP53 mutated CRC cell line which may contribute to the development of a treatment for drug resistant CRC due to KRAS and TP53 mutations.
Conflicts of Interest: The authors declare that they have no conflict of interest.
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