Therapeutic agents with multiple gene targeting potentials could be the right choice for the treatment of cancer as various genes are known to be dysregulated in cancers. NF-κB, a “rapid-acting” primary transcription factor is the important intracellular target in chemotherapy for cancer as it responds to various cellular stimuli that regulate the expression of various genes resulting in inflammation, cell survival, invasion, angiogenesis and metastasis [21, 22]. Our previous reports have shown that BDMC-A is a more effective inhibitor of NF-κB and related markers in a breast cancer cell line in comparison with curcumin . We have also reported that BDMC-A inhibits Hep-2 cells more efficiently than curcumin through apoptosis rather than necrosis. In the present study we showed that BDMC-A affects the NF-κB signaling pathway in Hep-2 cells.
We observed that BDMC-A downregulated NF-κB, p65 and its related oncogene c-Rel more efficiently than curcumin in Hep-2 cell lines. Curcumin was reported to inhibit NF-κB activation and thereby suppress cancer cell proliferation in head and neck cancers . Curcumin inhibits the stimulation of the upstream signal of NF-κB i.e. Akt. It downregulates NF-κB targets COX-2 and MMP-9 [24, 25]. Agents that activate NF-κB also stimulate the transcription factor AP-1. Activated AP-1 has been associated with cell proliferation and chemical carcinogenesis. Expression of genes regulated by AP-1 has been related to the transformation from preneoplastic to the neoplastic state of cancer cells in ex vivo and in vivo models  AP-1 also participates in tumor progression and metastasis. The results of our present study showed that treatment of Hep-2 cells with BDMC-A significantly downregulated the c-Jun and c-Fos expression in comparison with curcumin treatment. This effect of BDMC-A may in part be due to the suppression of NF-κB as the previous studies have reported a distinct and essential role of NF-κB in regulating the AP-1 transcription factor. Curcumin has been reported to suppress AP-1 activation . It interacts with AP-1 DNA binding motif and inhibits AP-1 activation.
STAT3 converges with NF-κB in various oncogenic signaling pathways . Constitutive activation of STAT3 has been observed in cancer and immune cells. It takes part in carcinogenesis, as well as in tumor immune evasion. STAT3 has been proved to control cell proliferation, survival and regulate the expression of c-myc, cyclin D and Bcl-2 in colon cancer . Mutation in EGFR has been attributed for constitutive STAT3 phosphorylation in lung adenocarcinomas . In our study the STAT3 and STAT5 expression levels were more significantly downregulated in BDMC-A treated cells compared to curcumin treated cells. Thus, decreased level of NF-κB in BDMC-A treated Hep-2 cells may be partially due to the effect of the drug on STATs. Suppression of STAT3 activation by curcumin has been observed in T-cell leukemia , lung , HNSCC , ovarian cancer . Curcumin has been reported previously to downregulate the activation of STAT5 in K562 leukemia cells .
β-catenin is an important transcription factor that is activated by the WNT signaling pathway. We investigated the effect of BDMC-A on the β-catenin levels in Hep- 2 cells and found that the β-catenin level was more significantly reduced in BDMC-A than curcumin-treated cells. Curcumin stimulates caspase-3-mediated β-catenin cleavage as well as suppress β-catenin/Tcf/LEF transactivation by c-myc and cyclin D1 . Another transcription factor that is emerging as a potential target for cancer therapy is PPAR-γ. In our study, BDMC-A significantly upregulated the PPAR-γ level in Hep-2 cell lines. This could have also aided the previously observed downregulation of NF-κB in BDMC-A treated cells. The anti-inflammatory role of curcumin via PPAR-γ has been shown previously and activation of PPAR-γ by curcumin inhibits MOSER cell growth by suppressing cyclin D1 and EGFR . The recent report proposes that PPAR-γ ligands exert their effects in HT-29 colon cancer by interacting with the p65 subunit of NF-κB. This prevents NF-κB pathway activation .
Multiple cellular pathways influence the growth and metastatic potential of tumors. In view of their high metastatic potential, chemotherapy has become one of the main treatments for HNSCC in recent years. It has been reported that cancer cell-matrix interaction will play an important role in promoting cell invasion and metastasis . Overexpression of various metalloproteinases (MMPs) is proved to markedly increase the invasive behavior of tumor cells and their ability to metastasize in experimental animal models. Increased expression of MMPs correlates with the invasion and metastasis in head and neck cancer . In our study, both BDMC-A and curcumin treatment reduced the levels of MMP-9 in Hep-2 cells. This may be due to the effect of BDMC-A on the upstream targets of MMPs such as NF-κB and AP-1 as the mRNA levels of MMP-9 in BDMC-A treated cells was also found to be reduced. Curcumin has been shown to affect both the transcriptional and post transcriptional levels of MMPs .
TIMP-2 has been found to block tumor cell invasion both in vitro and in vivo and may act as a metastasis suppressor gene. TIMP overexpression results in the decreased invasion of endothelial and tumor cells both in vitro and in vivo. TIMP is reduced in many cancer cells . In our study, Hep-2 cells treated with BDMC-A showed higher levels of TIMP-2 than curcumin. Previous studies have shown that curcumin enhances the expression of the anti-metastatic protein, TIMP-2 in melanoma cells . Curcumin was reported for its potential to reduce tumor cell invasion and metastasis in Hep2 cells . BDMC-A has been proved to regulate aberrant levels of MMPs and TIMPs in other diseases , highlighting the potential of BDMC-A as an antimetastatic agent.
Tumour angiogenesis is an essential process for incessant growth and the spreading of solid tumors. Angiogenesis occurs by the angiogenic factors including the growth factors VEGF, cytokines like IL-6, IL-8, and a number of small molecules secreted by the cancer cells . Highly malignant tumors are characterized by enriched vascularization, which is further correlated with increased VEGF expression. ECM is considered as a reservoir for VEGF. Degradation of the ECM releases VEGF which is a pro-angiogenic factor . An effective oncotherapy should address angiogenesis as well. In this respect too, BDMC-A holds potential as it downregulated the expression of VEGF. Curcumin has been shown to downregulate VEGF in HNSCC . This correlates with the present study where both curcumin and BDMC-A downregulated VEGF expression and BDMCA was more potent.
Tissue microenvironment has been identified to exert an intense effect on cell proliferation and differentiation. IL-6, the pleiotropic cytokine, exerts proangiogenic activities in the tumor microenvironment. There is growing evidence that reveals significant relationships between IL-6 levels and failure of treatment directed against VEGF . In the present study, the increased levels of IL-6 were reduced upon treatment with BDMC-A in Hep-2 cells. Thus BDMC-A reduces the VEGF expression through IL-6.
NF-κB positively regulates COX-2 in varied cell types. The 5’- promoter region of COX-2 contains 2 putative NF-κB binding sites. Activation of AP-1 is also implicated in the transcription of COX-2 . The previous report showed that curcumin reduced the invasive and metastatic properties of cells through the inhibition of COX-2 and MMP-9 expressions . In our study, we found that BDMC-A was more effective in reducing the COX-2 level compared to that of curcumin in Hep-2 cells. Thus, the observed reduction in the mRNA levels of COX-2 with BDMC-A treatment in the present study can be attributed to the suppressed levels of NF-κB, AP-1, VEGF and IL-6 levels.
Epithelial to mesenchymal transition (EMT) is known to be associated with the progression of cancer and impacts cell motility and invasiveness. EMT is mediated and maintained by chemokines/cytokines such as IL-8 and TGF-β. Since IL-8 expression is also regulated by NF-κB and AP-1, as expected, BDMC-A decreased the IL-8 expression in Hep-2 cells. Inhibiting TGF-β induced EMT is an ideal strategy for the treatment of invasion and metastasis of cancer . Treatment with BDMC-A produced a significant downregulation of TGF-β in Hep2 cells. We observed that in Hep-2 cells, BDMC-A treatment not only significantly lowered the levels of TGF-β but also reduced the invasiveness of these cells. Curcumin has been reported to downregulate the expression of various pro-inflammatory cytokines including TNF-α, IL-1, IL-2, IL-6, IL-8, IL-12, and chemokines, mainly through inactivation of the transcription factor NF-κB. Curcumin was found to prevent tumor induction via down-regulation of TGF-β in cancer cells . In hepatoma cells, curcumin has been reported to inhibit IL-6 production and AP-1 activation . The observed effect of BDMC-A on TGF-β, IL-6 and, IL-8 levels can be corroborated with these studies of curcumin. Nevertheless, BDMC-A was more effective than curcumin in bringing out these changes.