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Chalcone-imidazolone conjugates induce apoptosis through DNA damage pathway by affecting telomeres.

Ramaiah MJ, Pushpavalli S, Krishna GR, Sarma P, Mukhopadhyay D, Kamal A, Bhadra U, Bhadra MP - Cancer Cell Int. (2011)

Bottom Line: Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event.The apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds.In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Chemical Biology, Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500607, India. ahmedkamal@iict.res.in.

ABSTRACT

Background: Breast cancer is one of the most prevalent cancers in the world and more than one million women are diagnosed leading to 410,000 deaths every year. In our previous studies new chalcone-imidazolone conjugates were prepared and evaluated for their anticancer activity in a panel of 53 human tumor cell lines and the lead compounds identified were 6 and 8. This prompted us to investigate the mechanism of apoptotic event.

Results: Involvement of pro-apoptotic protein (Bax), active caspase-9 and cleavage of retinoblastoma protein was studied. Interestingly, the compounds caused upregulation of p21, check point proteins (Chk1, Chk2) and as well as their phosphorylated forms which are known to regulate the DNA damage pathway. Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event. The telomeric protein such as TRF2 which is an important target for anticancer therapy against human breast cancer was extensively studied along with proteins involved in proper functioning of telomeres.

Conclusions: The apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds. Down regulation of TRF2 and upregulation of the TRF1 as well as telomerase assay indicated the decrease in telomeric length revealing telomeric dysfunction and thereby controlling the rapid rate of cell proliferation. In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

No MeSH data available.


Related in: MedlinePlus

Determination of telomerase activity in chalcone-imidazolone conjugates treated MCF-7 cells. MCF-7 cells were treated with 30 μM concentration of chalcone imidazolone conjugates for 24 h and total cell lysates were subjected to telomerase specific PCR using fluorescently labeled primers of TRAPEZE XL telomerase detection kit. The telomerase activity was measured based on green fluorescence produced due to fluorescein (F). Internal control primers which are fluorescently labeled produces red colour which is represented by (R), due to sulforhodamine. Here TMAC is the starting material used for the synthesis of chalcone-imidazolone conjugates 6 and 8. CA-4, Combretastatin is positive control. Fluorescence reading due to fluorescein (F) was measured with excitation wave length of 495 nm and emission wave length of 516 nm. The fluorescence reading due to sulforhodamine (R) was measured at excitation wave length of 600 nm and emission wave length of 620 nm. The F/R ratio gives the telomerase activity. The p value for each compound was <0.001. The p value was generated by using student t-test by comparing the each compound vs control (untreated cells). Each experiment was conducted in triplicates.
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Figure 10: Determination of telomerase activity in chalcone-imidazolone conjugates treated MCF-7 cells. MCF-7 cells were treated with 30 μM concentration of chalcone imidazolone conjugates for 24 h and total cell lysates were subjected to telomerase specific PCR using fluorescently labeled primers of TRAPEZE XL telomerase detection kit. The telomerase activity was measured based on green fluorescence produced due to fluorescein (F). Internal control primers which are fluorescently labeled produces red colour which is represented by (R), due to sulforhodamine. Here TMAC is the starting material used for the synthesis of chalcone-imidazolone conjugates 6 and 8. CA-4, Combretastatin is positive control. Fluorescence reading due to fluorescein (F) was measured with excitation wave length of 495 nm and emission wave length of 516 nm. The fluorescence reading due to sulforhodamine (R) was measured at excitation wave length of 600 nm and emission wave length of 620 nm. The F/R ratio gives the telomerase activity. The p value for each compound was <0.001. The p value was generated by using student t-test by comparing the each compound vs control (untreated cells). Each experiment was conducted in triplicates.

Mentions: Generally actively proliferating cancerous cells have more telomerase activity. Thus we have investigated the telomerase activity and conducted fluorescence based telomerase assay using cell extracts after compound (TMAC, CA-4, 6 and 8) treatment. Control untreated cells which serve as positive control has highest level of telomerase activity. Telomerase activity of the control cells is lost when the extract was heated at 85°C for 10 min. Fluorescence reading was taken as a measure of telomerase activity. To our surprise down regulation of telomerase activity was observed in compound 6 and 8 treated cells. Hence our data strongly supports the effect of compounds on telomere and regulate the DNA stability (Figure 10).


Chalcone-imidazolone conjugates induce apoptosis through DNA damage pathway by affecting telomeres.

Ramaiah MJ, Pushpavalli S, Krishna GR, Sarma P, Mukhopadhyay D, Kamal A, Bhadra U, Bhadra MP - Cancer Cell Int. (2011)

Determination of telomerase activity in chalcone-imidazolone conjugates treated MCF-7 cells. MCF-7 cells were treated with 30 μM concentration of chalcone imidazolone conjugates for 24 h and total cell lysates were subjected to telomerase specific PCR using fluorescently labeled primers of TRAPEZE XL telomerase detection kit. The telomerase activity was measured based on green fluorescence produced due to fluorescein (F). Internal control primers which are fluorescently labeled produces red colour which is represented by (R), due to sulforhodamine. Here TMAC is the starting material used for the synthesis of chalcone-imidazolone conjugates 6 and 8. CA-4, Combretastatin is positive control. Fluorescence reading due to fluorescein (F) was measured with excitation wave length of 495 nm and emission wave length of 516 nm. The fluorescence reading due to sulforhodamine (R) was measured at excitation wave length of 600 nm and emission wave length of 620 nm. The F/R ratio gives the telomerase activity. The p value for each compound was <0.001. The p value was generated by using student t-test by comparing the each compound vs control (untreated cells). Each experiment was conducted in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 10: Determination of telomerase activity in chalcone-imidazolone conjugates treated MCF-7 cells. MCF-7 cells were treated with 30 μM concentration of chalcone imidazolone conjugates for 24 h and total cell lysates were subjected to telomerase specific PCR using fluorescently labeled primers of TRAPEZE XL telomerase detection kit. The telomerase activity was measured based on green fluorescence produced due to fluorescein (F). Internal control primers which are fluorescently labeled produces red colour which is represented by (R), due to sulforhodamine. Here TMAC is the starting material used for the synthesis of chalcone-imidazolone conjugates 6 and 8. CA-4, Combretastatin is positive control. Fluorescence reading due to fluorescein (F) was measured with excitation wave length of 495 nm and emission wave length of 516 nm. The fluorescence reading due to sulforhodamine (R) was measured at excitation wave length of 600 nm and emission wave length of 620 nm. The F/R ratio gives the telomerase activity. The p value for each compound was <0.001. The p value was generated by using student t-test by comparing the each compound vs control (untreated cells). Each experiment was conducted in triplicates.
Mentions: Generally actively proliferating cancerous cells have more telomerase activity. Thus we have investigated the telomerase activity and conducted fluorescence based telomerase assay using cell extracts after compound (TMAC, CA-4, 6 and 8) treatment. Control untreated cells which serve as positive control has highest level of telomerase activity. Telomerase activity of the control cells is lost when the extract was heated at 85°C for 10 min. Fluorescence reading was taken as a measure of telomerase activity. To our surprise down regulation of telomerase activity was observed in compound 6 and 8 treated cells. Hence our data strongly supports the effect of compounds on telomere and regulate the DNA stability (Figure 10).

Bottom Line: Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event.The apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds.In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Chemical Biology, Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500607, India. ahmedkamal@iict.res.in.

ABSTRACT

Background: Breast cancer is one of the most prevalent cancers in the world and more than one million women are diagnosed leading to 410,000 deaths every year. In our previous studies new chalcone-imidazolone conjugates were prepared and evaluated for their anticancer activity in a panel of 53 human tumor cell lines and the lead compounds identified were 6 and 8. This prompted us to investigate the mechanism of apoptotic event.

Results: Involvement of pro-apoptotic protein (Bax), active caspase-9 and cleavage of retinoblastoma protein was studied. Interestingly, the compounds caused upregulation of p21, check point proteins (Chk1, Chk2) and as well as their phosphorylated forms which are known to regulate the DNA damage pathway. Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event. The telomeric protein such as TRF2 which is an important target for anticancer therapy against human breast cancer was extensively studied along with proteins involved in proper functioning of telomeres.

Conclusions: The apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds. Down regulation of TRF2 and upregulation of the TRF1 as well as telomerase assay indicated the decrease in telomeric length revealing telomeric dysfunction and thereby controlling the rapid rate of cell proliferation. In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

No MeSH data available.


Related in: MedlinePlus