Chemotherapeutic approach which uses various natural (plant-derived) agents has become the focus of widespread attention for managing cancers in recent years; it is hoped that such studies will show a positive outcome to provide a scientific basis as to being efficient and useful in chemoprevention/chemotherapy of various cancers.
Reduction in metabolic activity of the cell is due to reduction in number of the cells for cell cycle block and/or cell death. Since the 30% induction of apoptosis in Baneh-treated T47D cells could not be enough to explain the reduction in cell viability observed with MTT assay, we decided to check the effect of Baneh extract on the cell cycle progression.
Uncontrolled cell cycle has a pivotal role in cancer incidence. Our results demonstrated that Baneh extract blocked the cell cycle in G0/G1 phase in a time dependent manner up to 48 h (Table 1) accompanied by down regulation of positive regulators of G1/S transition including cyclin D1 and cdk4, depending on the time of exposure (Figure 1).
With respect to the phytochemical evaluation, the extract possesses considerable amounts of polyphenolic compounds including flavonoids and anthocyanins. Dietary flavonoids inhibit the proliferation of various cancer cells and tumor growth in animal models. Recent studies have shown that polyphenols directly or indirectly can inhibit different cells at different cell cycle phases. Recently, S phase delay of Baneh- treated HT29 cells was reported by our group. Pc-3 (prostate cancer cells) treated with gum mastic of P. lentiscus showed cyclin D1 downregulation and G1 block of the cell cycle. Treatment of LNcap cells with Gum mastic led to downregulation of cyclin D1. HCT116-treated cells with chios mastic gum (CMG) were shown G1cell cycle arrest. It is established that a single polyphenol is capable of interacting with several protein kinases.
Polyphenolic compounds have phytoestrogenic activity. T47D cells are known as ER+ (Estrogen Receptor) cells. Estrogenic estradiols, act through the regulation of expression and function of the G1 phase regulatory proteins and promote transition through G1 phase. Cyclin D1, c-Myc, p21 and cyclin E-cdk2 are estrogen downstream targets. Estradiol can increase cyclin D1 expression by stimulating cyclin D1 transcription via PI3-Kinase/Akt pathway. The inhibitory effect of anti-estrogens on estrogen receptor activity leads to decreased cyclin D1 expression and dissociation of p21 and p27 from the cyclin D1-cdk4/6 complex then association with cyclinE-cdk2 complexes. Because of the promoting effects of estrogen on expression of the cycling D1, it is conceivable that Baneh extract involves component(s) which act as anti-estrogenic agent in T47D cells. It has been established that anti-estrogen treatment of MCF7 breast cancer cells induced reduction of c-Myc expression, resulting in cyclin D1 downregulation and eventually cell cycle arrest. In tamoxifen-treated T47D cells, G0/G1 delay of cell cycle was reported. Cyclin D1 expression and p53 status, both at the gene and protein level, revealed a highly significant association. Cyclin D1 may be one of the downstream effectors of p53. Loss of G1/S control and loss of p53 are two serious factors in tumor formation which directly affect cell-cycle checkpoints. The status of p53 and ER affect the response of breast cancer cells to exogenous agents. T47D cells express mutated and nonfunctional form of p53.
It is reported that NF-κB promotes G1-to-S phase transition in mouse embryonic fibroblasts and in T47D carcinoma cells. Inhibition of NF-κB induces pRb phosphorylation and inhibits G1-to-S phase transition. NF-κB can functionally interact with other transcription factors which regulate cyclin D1 promoter including; c-Fos/c-Jun, SP1, E2F1. According to the findings, inhibition of signal transduction proteins in the pathways linked to activation of NF-κB, in part is responsible for the cell cycle arrest and remarkable cyclin D1 reduction induced by Baneh extract in T47D cells.
Cyclin D1 is amplified and or overexpressed in a subset of human cancers including breast cancer. Due to the astonishing downregulation effect of Baneh extract on cyclin D1 abundance it could be a good candidate for malignancies with deregulated expression of this protein.
Doxorubicin is one of the most common anticancer drugs and is used for the treatment of human cancers. The main anticancer effect of Dox is due to DNA damage and resulting growth arrest or cell death. Dox treated cells shown G2/M type of cell cycle arrest associated with cyclin B1enhancment (Table 1 and Figure 1). Increased expression of cyclin B1 and G2/M delay of cell cycle in Dox-treated HT29 cells was reported by our group. Increased cyclin A expression, accumulation of cells at G2/M and number of apoptotic cells in Dox-treated k562 cells were described. Maximized level of cyclin B1 protein in T47D exposed cells with Dox (10 ng/ml) has been shown. Previously, Ling et al., demonstrated that in p388 murine leukemia cells, Dox caused G2/M cell cycle arrest, reduced p34 kinase activity, increased cyclin B1, alteration of cyclin B1/cdk1 complex function and/or DNA damage may trigger apoptosis. Recently, induction of apoptosis in Dox- treated T47D cells was shown by our group. This is the first report to show that Baneh extract can cease cell cycle progression of T47D cells.