Full length article
Nuclear factor-κB plays an important role in Tamarixetin-mediated inhibition of matrix metalloproteinase-9 expression

https://doi.org/10.1016/j.ejphar.2020.173808Get rights and content

Highlights

  • Tamarixetin inhibits MMP-9 expression in HT1080 fibrosarcoma cells.

  • Tamarixetin attenuates NFĸB pathway, preventing nuclear translocation of p65 subunit.

  • Tamarixetin modulates endogenous regulators of MMP-9 such as TIMP-1 and MMP-14.

  • Tamarixetin inhibits migration, invasion and clonogenic potential of HT1080 cells.

  • Tamarixetin-mediated MMP-9 inhibition and invasion also observed in 3D cultures.

Abstract

Flavonoids possess a broad spectrum of pharmacological properties, including anti-cancer, anti-oxidant and immunomodulatory activities. The current study explored the potential of some less-studied flavonoids in inhibiting Matrix Metalloproteinase-9 (MMP-9), a prominent biomarker, upregulated in a variety of cancers and known to promote migration and invasion of cancer cells. Amongst these, Tamarixetin, a naturally occurring flavonoid derivative of Quercetin, demonstrated significant dose-dependent inhibition of MMP-9 expression. Furthermore, a substantial inhibition of migration, invasion and clonogenic potential of HT1080 cells was also observed in the presence of Tamarixetin, which further suggests its role as a potential anti-cancer agent. It is noteworthy that Tamarixetin inhibits nuclear translocation as well the activity of nuclear factor kappa B (NFκB), both of which are functions essential for the activation of MMP-9 in promoting tumorigenesis. Additionally, the endogenous regulators of MMP-9 that tightly control its activity were also modulated by Tamarixetin, as evident from the 1.9 fold increase in the expression of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), with a concomitant 2.2 fold decrease in Matrix Metalloproteinase-14 (MMP-14) expression. The results obtained were further corroborated in three dimensional (3D) tumor models, which showed significant inhibition of MMP-9 activity as well as reduced invasive potential in the presence of Tamarixetin. Taken together, our observations demonstrate for the first time, the anti-invasive potential of Tamarixetin in cancer cells, indicating its possible use as a template for novel therapeutic applications.

Introduction

Metastasis, which makes the treatment of cancer extremely difficult and ineffective, is one of the primary reasons for cancer related mortality. Of the numerous matrix-remodelling enzymes, MMPs are pivotal for the breakdown of extracellular matrix and in the progression of metastasis (Bonnans et al., 2014). Amongst the different types of MMPs, MMP-9 has been connected with increased tumor metastasis and poor prognosis, emphasising its importance in cancer progression (Luo et al., 2017). Several studies have correlated elevated levels of MMP-9 expression with metastasis in different types of tumors such as lung, breast, colon, brain and prostate cancer, including fibrosarcoma and melanoma (Chintala et al., 1998; Huang, 2018). These studies clearly indicate the crucial role of MMP-9 in inducing tumor invasion and malignancy, promoting its selection as a promising target for developing cancer therapeutics.

MMP-9 is regulated by a host of endogenous modulators {inhibitors such as Tissue Inhibitor of Metalloproteinase-1 (TIMP) and Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), along with activators like Matrix Metalloproteinase-14 (MMP-14) and Extracellular matrix metalloproteinase inducer (EMMPRIN)}, that play a prominent role in its expression and tightly control its activity. One of the mechanisms of regulatory inhibition is via the activity of Tissue Inhibitor of Matrix Metalloproteinase, especially TIMP-1, which has a binding site in the hemopexin domain of pro-MMP-9, thereby blocking its activation (Antonov et al., 2018; Vilela et al., 2015; Yuan et al., 2018). Silencing of MMP-14 by siRNA prevented Phorbol 12-myristate 13-acetate (PMA)-mediated MMP-9 induction in HT1080 fibrosarcoma cells (Sheehy and Annabi, 2017). Additionally, MMP-14 in association with MMP-2, is known to activate MMP-9 (Li et al., 2017). Yet another key regulator of MMP-9 is the nuclear factor kappa B (NFкB), the well-known transcriptional regulator, known for its prominent role in promoting tumorigenesis (Li et al., 2012). NFкB is modulated by Inhibitor of KappaB Kinase 2 (IKK2), an important enzyme involved in the induction of NFκB pathway, which phosphorylates and degrades Inhibitor of kappaB (IκB) of the IκB/p65/p50 complex, resulting in the release and subsequent nuclear translocation of the NFкB subunits (p50/p65) from the cytoplasm, leading to activation of NFκB target genes. Down-regulation of NFкB activation is linked to inhibition of MMP-9 expression and decrease in tumor migration and invasion. PMA is an activator of NFкB, which in turn enhances MMP-9 expression leading to increased cell migration (Shin et al., 2007). Suppression of transcription factors that are necessary for the expression of oncogenes are considered as therapeutic targets for anti-tumor therapy. Thus, inhibitors of NFкB could be therapeutically exploited for regulation of MMP-9 expression.

Natural products serve as a major repository for the discovery of many novel drug molecules for anti-cancer therapy (Cragg and Newman, 2013). A number of natural products have been shown to regulate the expression of MMP-9 (Moon et al., 2009; Sen et al., 2010; Yu and Lin, 2010). The flavonoid Quercetin, is known to inhibit cell migration and invasion in a wide variety of cancers by decreasing MMP-9 expression (Kong et al., 2008; Vijayababu et al., 2006). In this study, we have evaluated the modulation of PMA-induced MMP-9 expression of Quercetin analogues in HT1080 fibrosarcoma cells. Taken together, our results demonstrate, for the first time, the molecular basis by means of which Tamarexitin regulates MMP-9, a key player in a wide variety of cancers and suggests the potential candidature of Tamarixetin as a lead molecule to be investigated for further development of anti-metastatic therapeutics.

Section snippets

Cell culture and treatments

The Human fibrosarcoma cell line HT1080, melanoma cell line A375 and lung cancer cell line A549 were obtained from NCCS, Pune Maharashtra, India. Cells were cultured in DMEM media (Sigma Aldrich, USA), supplemented with 10% FBS (GIBCO, USA). Primary cells were obtained from surgical tissues of breast cancer patients as described earlier (Drishya et al., 2020). All media compositions contained 100 mg/ml Penicillin, 100 mg/ml Streptomycin and 0.5 μg/ml Amphotericin B (Sigma Aldrich, USA). All the

Induction of MMP-9 with PMA

PMA is reported to up-regulate the expression of MMP-9 in a wide variety of cancer cells. Since treatment of HT1080 fibrosarcoma cells with 5 nM PMA for 24 h resulted in a significant induction of expression of MMP-9 (Supplementary Fig. 1), all the experiments were carried out under PMA-induced conditions.

Quercetin derivatives inhibit MMP-9 activity

The effect of seven Quercetin derivatives (Supplementary Fig. 2) on PMA (5 nM) induced MMP-9 expression was studied in HT1080 cells using gelatin zymography. Upon treatment with 12.5 μM of

Discussion

Flavonoids such as Quercetin, have been widely studied and reported to possess various anti-cancer properties (Chahar et al., 2011). In the present study, Quercetin and its analogues were screened to identify potent inhibitors of MMP-9. Earlier studies have demonstrated a strong correlation between MMP-9 expression and metastasis in various cancer cells, in particular due to its ECM-degrading capacity, thereby making MMP-9 an important druggable target in anti-metastatic therapy (Huang, 2018).

Conclusions

In conclusion, the present study identified Tamarixetin as a potent inhibitor of MMP-9 expression as compared to Quercetin. This inhibition is mediated primarily by the regulation of NFκB activity, resulting in attenuation of cell migration as well as invasion in HT1080 cells (Fig. 10). Additionally, multiple modulators of MMP-9 expression, such as MMP-14 and TIMP-1 were also regulated by Tamarixetin. These results were also observed in 3D culture systems as well as primary cells obtained from

Funding

This work was supported by Institutional funding (Amrita Vishwa Vidyapeetham).

CRediT authorship contribution statement

Sanu K. Shaji: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Roles, Writing - original draft, Writing - review & editing. Drishya G: Conceptualization, Data curation, Formal analysis, Investigation, Methodology. Damu Sunilkumar: Investigation, Methodology. Nanjan Pandurangan: Resources, Methodology. Geetha B. Kumar: Conceptualization, Formal analysis, Supervision, Validation, Roles, Writing - original draft, Writing - review

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgements

We acknowledge Mata Amritanandamayi Devi, Chancellor, Amrita Vishwa Vidyapeetham for being the inspiration behind this study. We thank Dr. Jyotsna Nambiar for her critical suggestions and help with conducting experiments. We would like to thank the University Grants Commission, Government of India for providing financial assistance in the form of fellowship (Beneficiary Code: BININ00357398, ID: 374719) to Mr. Sanu K. Shaji.

References (41)

  • R.P. Thomas et al.

    Selective targeting of the nuclear factor-kappaB pathway enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated pancreatic cancer cell death

    Surgery

    (2002)
  • P. Vilela et al.

    In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human monocytes

    Arch. Oral Biol.

    (2015)
  • Y.M. Yu et al.

    Curcumin prevents human aortic smooth muscle cells migration by inhibiting of MMP-9 expression

    Nutr. Metabol. Cardiovasc. Dis.

    (2010)
  • I.B. Antonov et al.

    Matrix metalloproteinases MMP-1 and MMP-9 and their inhibitor TIMP-1 as markers of dilated cardiomyopathy in patients of different age

    Bull. Exp. Biol. Med.

    (2018)
  • J.W. Antoon et al.

    Targeting NFkB mediated breast cancer chemoresistance through selective inhibition of sphingosine kinase-2

    Canc. Biol. Ther.

    (2011)
  • C. Bonnans et al.

    Remodelling the extracellular matrix in development and disease

    Nat. Rev. Mol. Cell Biol.

    (2014)
  • M.K. Chahar et al.

    Flavonoids: a versatile source of anticancer drugs

    Phcog. Rev.

    (2011)
  • P.J. Chen et al.

    Honokiol suppresses TNF-α-induced neutrophil adhesion on cerebral endothelial cells by disrupting polyubiquitination and degradation of IκBα

    Sci. Rep.

    (2016)
  • G. Drishya et al.

    RECK and TIMP-2 mediate inhibition of MMP-2 and MMP-9 by Annona muricata

    J. Biosci.

    (2020)
  • Y.P. Han et al.

    TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP

    J. Cell Sci.

    (2001)
  • Cited by (0)

    View full text