Tea is one of the most widely consumed beverages in the world. Black tea, obtained from the leaves of Camellia sinensis is the preferred beverage in India and in most western countries. Epidemiological studies on black tea and cancer are limited. However, preliminary studies indicate a positive correlation between black tea consumption and a lower incidence of breast and ovarian cancer. In the present communication , we wanted to see the effect of black tea extract and the polyphenol theaflavin on cell-ECM interaction, MMP activity etc. to strengthen the anti-cancer effect of black tea.
Tea is one of the most widely consumed beverages in the world. Black tea, obtained from the leaves of Camellia sinensis is the preferred beverage in India and in most western countries [
Epidemiological studies on black tea and cancer are limited. However, preliminary studies indicate a positive correlation between black tea consumption and a lower incidence of breast and ovarian cancer [
Tumour invasion and formation of secondary tumours require coordinated matrix degradation and modulation of cell-cell and cell matrix attachment. Matrix metalloproteinases (MMPs), a family of zinc dependant endopeptidases, degrade various extra cellular matrix (ECM) components, creating pathways through which tumour cells can invade [
Elevated expression and activity of MMPs (e.g. MMP-2) strongly correlate with increased metastatic potential, tumour spread and poor prognosis in a number of cancers including melanomas, breast, lung, thyroid, oral, stomach and colon carcinomas [
Theaflavin downregulates MMP-2 and MT1-MMP expression and activity and upregulate TIMP-2 levels in A375 human melanoma cells has been reported [
FAK acts as a transducer of integrin-growth factor stimulated signals to downstream targets like extracellular signal related kinase (ERK), phosphoinositol-3-kinase (PI-3K), c-Jun kinase (Jnk) and other mitogen activated protein kinase (MAPK) cascades [
The theaflavins, major components of black tea extract, exert multiple roles on intracellular signalling pathways. TF-2 has been reported to downregulate TNF-α and NF-κB, preventing inflammation and inducing apoptosis [
In the present communication we wanted to see the effect of black tea extract and the polyphenol theaflavin on cell-ECM interaction, MMP activity etc. to strengthen the anti-cancer effect of black tea.
Black Tea (Darjeeling variety), B16F10 cells, gelatin and other biochemicals from Sigma, USA, different antibodies are from Santa Cruz, USA
10 gms of Tea were added to a sterile tube adding 100 ml of distilled water and were percolated for 1 hr at 90˚C. The suspension was run at 10,000 rpm for 10 mins at room temp and the clear supernatant was saved and used as black tea extract.
B16F10 cells were grown in DMEM medium (10% FCS). 300,000 cells were collected, washed and grown in DMEM (without FCS) in presence of 20/40/80 ul of tea extract/ml or 20/40/80 μg of theaflavin/ml for 24 and 48 hrs. Cells were collected and cell adhesion assay was performed in a fibronectin coated (1 μg/ml) 96 well plate (in trplicate). Controls were run side by side [
B16 cells were treated with tea extract and or Theaflavin for 48 hrs in DMEM. The cells were collected, extracted and the protein concentration was measured. 100 μg of proteins were then run on SDS-PAGE and transferred onto nitrocellulose membrane. The immune blot was developed with respective primary antibodies (Nf-κB, ERK1/2, PI-3K) followed by 2nd antibody coupled to alkaline phosphatase. The colour was developed with NBT/BCIP [
The culture (DMEM without FCS) medium from cell adhesion assay was saved and MMPs of control and experimental dishes were concentrated using gelatin-sepharose beads. The MMPs adsorbed was released in Lammeli’s buffer without B-mercaptoethanol and subjected to zymography. Zymography was also done with human salivary MMPs incubating it with black tea extract and theaflavin separately (in vitro) for 30 mins at 37˚C [
B16F10 cells were grown on clean glass slides. The cells were then washed and incubated with 3% BSA blocking buffer for 1 hr at 37˚C followed by incubation with alfa5 monoclonal antibody for 1 and half hour. The cells were then washed ×3 with PBS and then incubated with 2nd antibody coupled to FITC for 1 hr at 37˚C. The cells were washed x 5 with PBS and observed under fluorescence microscope [
Cell adhesion assay (
Zymogram shows clearly the 72 and 64 kD pro and activated MMP-2 (
To see the presence of alfa5beta1 integrin on B16F10 cells the immunocytochemistry (
The B16F10 cells were treated with polyphenol theaflavin at a concentration of 40 μg/ml for 24 hrs in serum free culture medium (SFCM). The comparative immunoblots show that theaflavin downregulate the expression of ERK, PI-3K and NF-κB (
Cell-ECM interaction is one of the fundamental aspects of developmental as well as cancer biology. We wanted to see whether black tea extract and polyphenol theaflavin has any effect on cell-ECM (Fibronectin) interaction. The findings were very meaningful. The results clearly show that the binding of black tea extract and theaflavin treated B16F10 cells reduced drastically to fibronectin (ECM) most probably via integrin alfa5beat1. Culture of B16F10 cells over fibronectin coated culture dish upregulates MMP-2 is another evidence of cell-ECM interaction in regulation of MMP-2 and the role of alfa5beta1 integrin in MMP-2 regulation. Interestingly, when alfa5beta1 integrin was capped using alfa5 antibody, the expression and activity of MMP-2 was drastically reduced indicating strongly the alfa5beta1 integrin mediated signalling in modulation of MMP-2 activity [
We acknowledge Vice Chancellor, Ramakrishna Mission Vivekananda University, Natonal Tea Research Foundation for funding the project. Code No: NTRF 171/2014 started on June 6, 2014.
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Bhattacharyya, N., Mondal, S., Moulik, S., Paul, S., Bhattacharrya, S., Hazra, A.K., Ali, Md.N., Adhikari, A. and Chatterjee, A. (2017) Effect of Black Tea Polyphenol on Cell- ECM Interaction and MMP. American Journal of Plant Sciences, 8, 856-866. https://doi.org/10.4236/ajps.2017.84058