Synthetic curcumin analog: inhibiting the invasion, angiogenesis, and metastasis in human laryngeal carcinoma cells via NF-kB pathway

Laryngeal carcinoma, the most common among head and neck squamous cell carcinoma (HNSCC), induces 1% of all cancer deaths. Curcumin the active constituent of turmeric, is shown to be effective in the treatment of various cancers. In the present study, we explored the mechanistic role of bis-demethoxy curcumin analog (BDMC-A) as a chemotherapeutic agent. We investigated its inhibitory effect on invasion, angiogenesis, and metastasis in human laryngeal carcinoma (Hep-2) cells in comparison with curcumin. The effect of curcumin and BDMC-A on transcription factors (NF-κB, p65, c-Jun, c-Fos, STAT3, 5, PPAR-γ, β-catenin, COX-2, MMP-9, VEGF, TIMP-2) involved in signal transduction cascade, invasion, and angiogenesis in Hep-2 cells were quantified using Western blotting and RT-PCR technique. ELISA was used to measure the pro-inflammatory markers in Hep-2 cells treated with curcumin and BDMC-A. The results showed that BDMC-A inhibits the transcription factors NF-κB, p65, c-Jun, c-Fos, STAT3, STAT5, PPAR-γ and β-catenin, which are responsible for tumor progression and malignancy. Moreover, BDMC-A treatment downregulated MMP-9, VEGF, TGF- β, IL-6 and IL-8 and upregulated TIMP-2 levels. The effects were more significant compared to curcumin. Our overall results revealed that BDMC-A more effectively inhibited the markers of invasion, angiogenesis and metastasis in comparison with curcumin.


Introduction
Laryngeal carcinoma, the most common type of head and neck squamous cell carcinoma (HNSCC), comprises 1% of all the cancers and reported to be responsible for 1% of all cancer-induced deaths [1,2]. Laryngeal cancer mainly results from the usage of tobacco [3]. In addition to this, alcohol use, nutritional deficiencies, and genetic predisposition may also play a role [4,5]. Besides surgery, laryngeal carcinoma could be most effectively treated by chemotherapy, and the widely used drug cisplatin significantly improves the survival rate [6,7]. However, the side effects such as leukopenia and kidney failure, are reported due to chemotherapy [8]. Identification of new drugs with low toxicity showing improved therapeutic potential is of great interest among the researchers.
Curcumin, a principle ingredient isolated from the rhizomes of Curcuma longa, is reported for its anti-inflammatory, antiviral, antibacterial, antifungal, anti-diabetic, anti-carcinogenic, and pharmacological properties [9]. In previous studies, curcumin was reported for its inhibitory effect on tumor proliferation, metastasis, invasion and angiogenesis [10]. Curcumin inhibits the proliferation of various cancer cells through the down-regulation of cell-signaling targets like vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), Akt/protein kinase B, activator protein 1 (AP-1), cyclin D1, and signal transducer and activator of transcription 3 (STAT-3) [11]. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation suppresses apoptosis and induces proliferation, invasion, metastasis and inflammation [12]. Curcumin 1 3 possesses anticancer effects on pancreatic [13], and breast cancer cells [14] due to its ability to suppress NF-κB activity. However, due to its low stability and poor bioavailability researchers are interested to synthesize analogs of curcumin to acquire improved potency.
Bis-demethoxy curcumin analog (BDMC-A), a synthetic analog of curcumin, possesses the structural backbone similar to curcumin and differs by the absence of methoxy group in the meta position of both the benzene rings. Though BDMC-A is only slightly better soluble in water than curcumin, it was reported as more effective anticancer and antidiabetic compound in comparison with curcumin [15,16]. The anti-tumor, anti-mutagenic as well as anti-oxidant activities of BDMC-A, was reported [17]. We have investigated and reported the therapeutic potential of BDMC-A on alcohol and thermally oxidised PUFA (ΔPUFA) induced oxidative stress [18], hyperlipidemia [19] and matrix metalloproteinase [20] in rat model. Previously BDMC-A was reported for its anticarcinogenic effects on 1, 2-dimethylhydrazine (DMH) induced-colon cancer rat model [21]. Moreover, the anti-proliferative, cell cycle arrest potential and the molecular mechanisms of BDMC-A inducing apoptosis in human laryngeal cancer (Hep-2) cells have been compared with curcumin [16,22]. In continuation of this, the present study is focused on the investigation of the anti-invasive, anti-angiogenic, and anti-metastatic potential of BDMC-A and curcumin using various markers.

Western blotting
Western blotting was done using the method described by Fido et al. [24]. Briefly, Hep-2 cells (5 × 10 5 cells per well) were grown in 6-well plates and treated with sub optimal doses of curcumin (25 μM) and BDMC-A (10 μM) (50% of the IC50 dose) of curcumin and BDMC-A. After 24 h of incubation with curcumin or BDMC-A, cells were collected and washed twice with ice cold PBS. The cell pellet was then resuspended in RIPA buffer and lysed by vortexing and freeze-thawing. The lysates were centrifuged at 12,000×g for 20 min to extract the protein. The protein concentration in the supernatant was determined by 'Bio-Rad Protein Assay' Kit. The protein was resolved on 10% SDS-PAGE and electro-transferred to nitrocellulose membrane. The membranes were then blocked using blocking buffer (5% skimmed milk powder in TBST) and probed with corresponding primary antibody followed by corresponding HRP-conjugated anti-IgG secondary antibody. β-actin was used as a control for protein loading. Protein bands were visualized by enhanced chemiluminescence method and the signals were detected by ECL, (Pierce, Rockford, IL, USA) and Chemi Doc Imaging System (Bio-Rad, Hercules, CA, USA). All the experiments were performed in triplicate. Normalization to β-actin was done using the automated Bio-Rad software.

Measurement of proinflammatory cytokines
Amounts of proinflammatory cytokines TGF-β, IL-6 and IL-8 in the supernatant of curcumin or BDMC-A treated Hep-2 cells were determined using commercial ELISA assay kits (eBiosciences, USA) following the manufacturer's instructions. The samples were run in triplicate and the data were averaged.

Invasion assay
Matrigel-coated transwell cell culture chambers (8 mm pore size) (BD Biosciences, USA) were used for cell invasion assay. Hep-2 cells were placed in the upper chamber of the transwell insert (5 × 10 4 cells/well) followed by the incubation in serum-free medium for 24 h and then treated with the selected concentration of curcumin or BDMC-A. The lower chamber was filled with medium with serum. Plates were then incubated for 24 h and 48 h [14]. Non-invasive cells were removed from the upper chamber by wiping with a cotton swab. The invasive cells were stained with 0.4% Trypan Blue. Cells that penetrated through the Matrigel appeared in the lower surface of the filter and were counted using haemocytometer under a light microscope.

Statistical analysis
SPSS 7.5-Windows Students version software (SPSS Inc., Chicago, IL, USA) was used for data analysis. One-way ANOVA followed by Tukey's test was used to assess the statistical significance between groups at the level of p ≤ 0.05.

Results
In a previous study, we have determined the cell viability of Hep-2 cells treated with BDMC-A and the IC 50 at 48 h was found to be 20 μM, which is lesser in comparison with curcumin (IC 50 at 48 h is 50 μM) [16]. The present study compares the BDMC-A-induced inhibitory effect by modulating the NF-κB signaling pathway and related markers in Hep-2 cells using the sub-optimal cytostatic dose of curcumin (25 μM) and BDMC-A (10 μM). As the changes in various parameters were significant even at 24 h, all the parameters were analyzed at the end of 24 h [16].

Effects of BDMC-A on putative invasion and angiogenesis -associated markers
Hep-2 cells treated with curcumin and BDMC-A were lysed and analyzed by Western blot and RT-PCR. RNA extracted from cells that were used for RT-PCR to assess the markers of invasiveness and angiogenesis. Western blot results in Hep-2 cells, showed significant down regulation (p ≤ 0.05) of COX-2 ( Fig. 3a(i), b(i)) when treated with BDMC-A in comparison with curcumin. Western blot results of Hep-2 cells treated with BDMC-A showed a significant down regulation of MMP-9 levels (Fig. 3a(ii), b(ii)) and significant upregulation (p ≤ 0.05) of TIMP-2 levels (Fig. 3a(iii), b(iii)) when compared to that of curcumin treatment.

Effects of BDMC-A on the invasion of Hep-2 cells
The effect of curcumin and BDMC-A on the invasive potential of Hep-2 cells was tested by its ability to migrate across a membrane. Compared to control only fewer cells treated with curcumin and BDMC-A entered into the lower chamber from the upper separated by EHS-coated filter. The number of Hep-2 cells migrated across the filter was significantly decreased up on BDMC-A treatment compared to curcumin treated and untreated Hep-2 cells (Fig. 5).

Effects of BDMC-A on cancer-related inflammatory markers
Curcumin and BDMC-A treated Hep-2 cell supernatant were tested using ELISA kit for pro-inflammatory markers such as TGF-β, IL-6, and IL-8 as they are considered the hallmark of cancer. Compared with control cells, BDMC-A treated 1 3 Hep-2 cells had significantly downregulated expression of TGF-β ( Fig. 6(i)), IL-6 ( Fig. 6(ii)) and IL-8 ( Fig. 6 (iii)). Overall, these are the common pathways of tumor development that were observed with some bladder, breast, head and neck and lung cancers, despite their diverse tissue origins.

Discussion
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 [25,26]. 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 [14]. We have also reported that BDMC-A inhibits Hep-2 cells more efficiently than curcumin through apoptosis rather than necrosis [16]. 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 [27]. Curcumin inhibits the stimulation of the upstream signal of NF-κB i.e. Akt. It downregulates NF-κB targets COX-2 and MMP-9 [28,29]. 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 [30] 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. Fig. 1 Expression of transcription factors from Hep-2 cells treated with curcumin or BDMC-A. Untreated cells were used as control. β-actin was used as the internal loading control 1 3 Curcumin has been reported to suppress AP-1 activation [31]. It interacts with AP-1 DNA binding motif and inhibits AP-1 activation.
STAT3 converges with NF-κB in various oncogenic signaling pathways [32]. 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 [33]. Mutation in EGFR has been attributed Fig. 2 Densitometry analysis of protein levels expressed in relative band intensity. Values represent the mean ± SD of three independent experiments. *p ≤ 0.05, significance compared to control. #p ≤ 0.05, significance compared to curcumin for constitutive STAT3 phosphorylation in lung adenocarcinomas [34]. 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 [35], lung [36], HNSCC [37], ovarian cancer [38]. Curcumin has been reported previously to downregulate the activation of STAT5 in K562 leukemia cells [39].
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 [40]. 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 [41]. 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 [42].
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 [43]. 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 [44]. 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 [45].
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 [46]. 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 [47]. Curcumin was reported for its potential to reduce tumor cell invasion and metastasis in Hep2 cells [48]. BDMC-A has been proved to regulate aberrant levels of MMPs and TIMPs in other diseases [20], 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 [49]. 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 [50]. 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 [51]. This correlates with the present study where both curcumin and BDMC-A downregulated VEGF expression and BDMC-A 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 [52]. 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 [53]. The previous report showed that curcumin reduced the invasive and metastatic properties of cells through the inhibition of COX-2 and MMP-9  [45]. 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 [54]. Treatment with BDMC-A produced a significant downregulation of TGF-β in Hep-2 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 [40]. In hepatoma cells, curcumin has been reported to inhibit IL-6 production and AP-1 activation [55]. 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.

Conclusion
We conclude that BDMC-A is a more potent cytotoxic agent against Hep-2 cells in comparison with curcumin. This was further confirmed by the significant downregulation of metastasis, invasiveness, and angiogenesis markers in Hep-2 cells treated with BDMC-A compared to curcumin. Overall, BDMC-A could be promoted as a potential therapeutic agent for head and neck cancer in the future.