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  • br Gelatin zymography assays indicated that


    Gelatin zymography assays indicated that HC9 reduced the secre-tion of MMP-2 and MMP-9 proteins in both MCF-7 (Fig. 5G) and MDA MB-231 (Fig. 5H) compared to the untreated control cells. The ex-pression of MMP-2 was further confirmed by western blotting, which 
    3.4. HC9 altered the expression of inflammatory markers and chromatin modulators
    MAR (matrix attachment region) binding proteins SMAR1 and CDP/ Cux are chromatin modulating proteins that are known to regulate breast cancer. We tested the expression of these proteins in response to HC9 treatment. In MCF-7, at 40 μg/ml of HC9, there was ∼2.99-fold
    Fig. 3. HC9 induced S phase arrest in MCF-7 cells and and altered the expression of Y-27632 proteins. MCF-7 cells were treated with HC9 (0–40 μg/ml) for 24 h. (A) HC9 induced accumulation of cells in S phase with simultaneous decrease in G2/M phase population. (B) Bar graph of cell cycle analysis of three independent experiments. (C) Densitometric analysis and western blotting analysis shows expression of p53, p21 and p16 proteins upon HC9 treatment in MCF-7. Tubulin was used as a loading control. The bands were quantified by densitometry scanning by using ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA).
    (p = 0.010) increase in expression of SMAR1 (Fig. 6C) with ∼4.15-fold decrease in CDP/Cux expression, respectively, (Fig. 6E). Similarly, in MDA-MB-231, at 80 μg/ml concentration of HC9, there was ∼6.8-fold (p = 0.010) increase in expression of SMAR1 (Fig. 6D) with ∼2.91-fold decrease in CDP/Cux expression, respectively, (Fig. 6F). These findings strongly suggest that HC9 effectively altered the expression of in-flammatory markers and chromatin modulators in breast cancer cells.
    4. Discussion
    Herbal remedies in the form of single herb or polyherbal formula-tions are increasingly playing prime role in the healthcare system be-cause of their wide biological activities, easy accessibility, cost effec-tiveness and safe usage (Kumar and Kumar, 2009). Due to complex interaction between different phytochemicals present in polyherbal formulations, they may target multiple dysregulated pathways in cancer cells and provide an extremely promising way to block the de-velopment of cancer (Dandawate et al., 2016). However, these remedies have not yet been integrated into modern clinical practice due to lack of experimental and clinical evidences on their safety, efficacy, and pharmacological mechanisms (Calixto, 2000). Thus, it is necessary to evaluate the polyherbal formulations for their safety and therapeutic efficacy before they could be taken up for clinical trials. 
    Estrogen (ER) and progesterone (PR) receptors are important prognostic markers of aggressive breast cancer (Baumgarten and Frasor,
    2012; Thakkar and Mehta, 2011). Therefore, two representative breast cancer cell lines, namely MCF-7ER(+)/PR(+) and MDA MB-231ER(−)/ PR(−) were selected to investigate the anti-proliferative effect of HC9 in these cell lines. HC9 caused S phase arrest in MCF-7 and simultaneously increased the expression of p53, p21 and p16 proteins. p53 and its downstream effectors p21/p16 are known tumor suppressor proteins and have important roles in cell growth arrest and apoptosis under abnormal stress conditions (Li et al., 2012; Rayess et al., 2012). p53/ p21 and p16/Rb pathways are also believed to have important con-tributions in the regulation of both replicative and premature senes-cence, characterized by an irreversible cell cycle arrest, which has be-come an attractive cancer therapeutic strategy (Qian and Chen, 2010; Ninomiya et al., 2014; Rayess et al., 2012; Akakura et al., 2010). Most of the chemopreventive drugs regulate the growth of cancer cells by targeting cell cycle regulatory proteins (Neergheen et al., 2010; Heng, 2010). Thus, in MCF-7, activation of p53 by HC9, resulted into acti-vation of p21/p16, which blocked the cell cycle progression of MCF-7 in S phase. On the other hand, in MDA MB-231, there was an increase in the expression of p53, p21 and pRb and down regulation of ppRb. Hence, the decrease in phopshorylation of Rb and increase in the ex-pression of p21 and p53 might have led to G1 phase arrest in MDA MB-