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Triptolide potentiates lung cancer cells to cisplatin-induced apoptosis by selectively inhibiting the NER activity.

Wang G, Wang X, Xu X - Biomark Res (2015)

Bottom Line: The results of our cell growth inhibition studies revealed that the presence of low-levels triptolide itself had little effect on cell growth but greatly enhanced cisplatin-induced cell growth inhibition in both A549 and HTB182 cells.The results of our reef coral-red protein reporter expression studies indicated that the presence of low-levels triptolide did not affect expression of the reef coral-red protein from pDsRed2-C1 plasmid but greatly inhibited expression of the reef coral-red protein from cisplatin-damaged pDsRed2-C1 plasmid DNA in A549 cells.In addition, the results of our protein phosphorylation studies indicated that the presence of low-levels triptolide caused a decrease for cisplatin-induced CHK1 phosphorylation at Ser(317/345) but an increase for cisplatin-induced ATM phosphorylation at Ser(1981) in both HTB182 and A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201 USA.

ABSTRACT

Background: Cisplatin and many other platinum-based compounds are important anticancer drugs that are used in treating many cancer types. The development of cisplatin-resistant cancer cells, however, quickly diminishes the effectiveness of these drugs and causes treatment failure. New strategies that reverse cancer cell drug resistance phenotype or sensitize cancer cells to these drugs, therefore, need to be explored in order to improve platinum drug-based cancer treatment. Triptolide is a bioactive ingredient isolated from Tripterygium wilfordii, a Chinese herbal medicine. Triptolide binds to the TFIIH basal transcription factor and is required for both transcription and nucleotide excision repair (NER), a DNA repair pathway involved in repairing DNA damage generated by the platinum-based anticancer drugs.

Methods: Caspase-3 activation and cell growth inhibition assays were used to determine the effect of triptolide on cisplatin-induced apoptosis and cell growth in lung cancer cells. Real time PCR, immunoblotting, and expression of reef coral red protein were used to determine a mechanism through which the presence of triptolide increased cisplatin-induced apoptosis of the lung cancer cells.

Results: Our caspase-3 activation studies demonstrated that the presence of low-levels of triptolide greatly increased the cisplatin-induced apoptosis of HTB182, A549, CRL5810, and CRL5922 lung cancer cells. The results of our cell growth inhibition studies revealed that the presence of low-levels triptolide itself had little effect on cell growth but greatly enhanced cisplatin-induced cell growth inhibition in both A549 and HTB182 cells. The results of our reef coral-red protein reporter expression studies indicated that the presence of low-levels triptolide did not affect expression of the reef coral-red protein from pDsRed2-C1 plasmid but greatly inhibited expression of the reef coral-red protein from cisplatin-damaged pDsRed2-C1 plasmid DNA in A549 cells. In addition, the results of our protein phosphorylation studies indicated that the presence of low-levels triptolide caused a decrease for cisplatin-induced CHK1 phosphorylation at Ser(317/345) but an increase for cisplatin-induced ATM phosphorylation at Ser(1981) in both HTB182 and A549 cells.

Conclusion: The results of our studies suggest that the presence of low-levels of triptolide potentiates lung cancer cells to cisplatin treatment by selectively inhibiting NER activity, resulting in an increase in apoptosis of the lung cancer cells.

No MeSH data available.


Related in: MedlinePlus

The effect of triptolide on reef coral red and GFP proteins expressions in A549 lung tumor cells. The undamaged or cisplatin-damaged pDsRed2-C1 plasmid (pDsRed2-C1-Cis*) was co-transfected with pmaxGFP plasmid DNA into A549 cells. The transfected cells were cultured in the presence or absence of triptolide (10 ng/ml) for 24 h and expressions of both reef coral red and GFP proteins were detected by fluorescence microscope using excitation/emission lights with wavelengths of 563 nm/582 nm and 475 nm/505 nm respectively for reef coral red and GFP proteins. The light image of the same view was also documented for visualization of the live cells
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Fig4: The effect of triptolide on reef coral red and GFP proteins expressions in A549 lung tumor cells. The undamaged or cisplatin-damaged pDsRed2-C1 plasmid (pDsRed2-C1-Cis*) was co-transfected with pmaxGFP plasmid DNA into A549 cells. The transfected cells were cultured in the presence or absence of triptolide (10 ng/ml) for 24 h and expressions of both reef coral red and GFP proteins were detected by fluorescence microscope using excitation/emission lights with wavelengths of 563 nm/582 nm and 475 nm/505 nm respectively for reef coral red and GFP proteins. The light image of the same view was also documented for visualization of the live cells

Mentions: To further define the mechanism through which the presence of low-level triptolide caused an increase of cisplatin-induced apoptosis in these lung tumor cells, we investigated whether the presence of low-level triptolide inhibited both transcription and the NER process or only one of these events using A549 lung tumor cells. The cisplatin-damaged pDsRed2-C1 plasmid DNA was transfected into A549 cells for DNA repair and expression of reef coral red protein in the presence or absence of triptolide. As a positive control, the undamaged pDsRed2-C1 plasmid was also transfected into A549 cells in a parallel experiment for expression of the reef coral red protein in the presence or absence of triptolide. As an internal control, pmaxGFP plasmid DNA, which carried a CMV promoter-driven green fluorescent protein (GFP) gene, was co-transfected with pDsRed2-C1 plasmid DNA into A549 lung cancer cells for expression of the GFP protein in the presence or absence of triptolide. Expressions of both reef coral red and GFP proteins were visualized using fluorescence microscope (Fig. 4). When undamaged pDsRed2-C1 plasmid was transfected into A549 lung cancer cells, the reef coral red protein was highly expressed in the transfected cells with or without triptolide (Fig. 4f vs 4c). The reef coral red protein was also highly expressed in the cisplatin-damaged pDsRed2-C1 plasmid-transfected A549 cells in the absence of triptolide (Fig. 4i). In the presence of triptolide, however, expression of the reef coral red protein from the cisplatin-damaged pDsRed2-C1 plasmid was greatly diminished in A549 cells (Fig. 4l vs 4i). As an internal control, expression of the GFP protein from pmaxGFP plasmid was not affected by the presence of triptolide, even when expression of the reef coral red protein from cisplatin-damaged pDsRed2-C1 plasmid DNA was greatly inhibited in A549 cells by triptolide in the same dish as demonstrated in our study (Fig. 4k vs 4l). These results suggest that inhibiting NER process plays an important role in the mechanism through which the presence of low-level triptolide reduces reef coral red protein expression from the cisplatin-damaged pDsRed2-C1 plasmid in A549 lung cancer cells.Fig. 4


Triptolide potentiates lung cancer cells to cisplatin-induced apoptosis by selectively inhibiting the NER activity.

Wang G, Wang X, Xu X - Biomark Res (2015)

The effect of triptolide on reef coral red and GFP proteins expressions in A549 lung tumor cells. The undamaged or cisplatin-damaged pDsRed2-C1 plasmid (pDsRed2-C1-Cis*) was co-transfected with pmaxGFP plasmid DNA into A549 cells. The transfected cells were cultured in the presence or absence of triptolide (10 ng/ml) for 24 h and expressions of both reef coral red and GFP proteins were detected by fluorescence microscope using excitation/emission lights with wavelengths of 563 nm/582 nm and 475 nm/505 nm respectively for reef coral red and GFP proteins. The light image of the same view was also documented for visualization of the live cells
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496860&req=5

Fig4: The effect of triptolide on reef coral red and GFP proteins expressions in A549 lung tumor cells. The undamaged or cisplatin-damaged pDsRed2-C1 plasmid (pDsRed2-C1-Cis*) was co-transfected with pmaxGFP plasmid DNA into A549 cells. The transfected cells were cultured in the presence or absence of triptolide (10 ng/ml) for 24 h and expressions of both reef coral red and GFP proteins were detected by fluorescence microscope using excitation/emission lights with wavelengths of 563 nm/582 nm and 475 nm/505 nm respectively for reef coral red and GFP proteins. The light image of the same view was also documented for visualization of the live cells
Mentions: To further define the mechanism through which the presence of low-level triptolide caused an increase of cisplatin-induced apoptosis in these lung tumor cells, we investigated whether the presence of low-level triptolide inhibited both transcription and the NER process or only one of these events using A549 lung tumor cells. The cisplatin-damaged pDsRed2-C1 plasmid DNA was transfected into A549 cells for DNA repair and expression of reef coral red protein in the presence or absence of triptolide. As a positive control, the undamaged pDsRed2-C1 plasmid was also transfected into A549 cells in a parallel experiment for expression of the reef coral red protein in the presence or absence of triptolide. As an internal control, pmaxGFP plasmid DNA, which carried a CMV promoter-driven green fluorescent protein (GFP) gene, was co-transfected with pDsRed2-C1 plasmid DNA into A549 lung cancer cells for expression of the GFP protein in the presence or absence of triptolide. Expressions of both reef coral red and GFP proteins were visualized using fluorescence microscope (Fig. 4). When undamaged pDsRed2-C1 plasmid was transfected into A549 lung cancer cells, the reef coral red protein was highly expressed in the transfected cells with or without triptolide (Fig. 4f vs 4c). The reef coral red protein was also highly expressed in the cisplatin-damaged pDsRed2-C1 plasmid-transfected A549 cells in the absence of triptolide (Fig. 4i). In the presence of triptolide, however, expression of the reef coral red protein from the cisplatin-damaged pDsRed2-C1 plasmid was greatly diminished in A549 cells (Fig. 4l vs 4i). As an internal control, expression of the GFP protein from pmaxGFP plasmid was not affected by the presence of triptolide, even when expression of the reef coral red protein from cisplatin-damaged pDsRed2-C1 plasmid DNA was greatly inhibited in A549 cells by triptolide in the same dish as demonstrated in our study (Fig. 4k vs 4l). These results suggest that inhibiting NER process plays an important role in the mechanism through which the presence of low-level triptolide reduces reef coral red protein expression from the cisplatin-damaged pDsRed2-C1 plasmid in A549 lung cancer cells.Fig. 4

Bottom Line: The results of our cell growth inhibition studies revealed that the presence of low-levels triptolide itself had little effect on cell growth but greatly enhanced cisplatin-induced cell growth inhibition in both A549 and HTB182 cells.The results of our reef coral-red protein reporter expression studies indicated that the presence of low-levels triptolide did not affect expression of the reef coral-red protein from pDsRed2-C1 plasmid but greatly inhibited expression of the reef coral-red protein from cisplatin-damaged pDsRed2-C1 plasmid DNA in A549 cells.In addition, the results of our protein phosphorylation studies indicated that the presence of low-levels triptolide caused a decrease for cisplatin-induced CHK1 phosphorylation at Ser(317/345) but an increase for cisplatin-induced ATM phosphorylation at Ser(1981) in both HTB182 and A549 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201 USA.

ABSTRACT

Background: Cisplatin and many other platinum-based compounds are important anticancer drugs that are used in treating many cancer types. The development of cisplatin-resistant cancer cells, however, quickly diminishes the effectiveness of these drugs and causes treatment failure. New strategies that reverse cancer cell drug resistance phenotype or sensitize cancer cells to these drugs, therefore, need to be explored in order to improve platinum drug-based cancer treatment. Triptolide is a bioactive ingredient isolated from Tripterygium wilfordii, a Chinese herbal medicine. Triptolide binds to the TFIIH basal transcription factor and is required for both transcription and nucleotide excision repair (NER), a DNA repair pathway involved in repairing DNA damage generated by the platinum-based anticancer drugs.

Methods: Caspase-3 activation and cell growth inhibition assays were used to determine the effect of triptolide on cisplatin-induced apoptosis and cell growth in lung cancer cells. Real time PCR, immunoblotting, and expression of reef coral red protein were used to determine a mechanism through which the presence of triptolide increased cisplatin-induced apoptosis of the lung cancer cells.

Results: Our caspase-3 activation studies demonstrated that the presence of low-levels of triptolide greatly increased the cisplatin-induced apoptosis of HTB182, A549, CRL5810, and CRL5922 lung cancer cells. The results of our cell growth inhibition studies revealed that the presence of low-levels triptolide itself had little effect on cell growth but greatly enhanced cisplatin-induced cell growth inhibition in both A549 and HTB182 cells. The results of our reef coral-red protein reporter expression studies indicated that the presence of low-levels triptolide did not affect expression of the reef coral-red protein from pDsRed2-C1 plasmid but greatly inhibited expression of the reef coral-red protein from cisplatin-damaged pDsRed2-C1 plasmid DNA in A549 cells. In addition, the results of our protein phosphorylation studies indicated that the presence of low-levels triptolide caused a decrease for cisplatin-induced CHK1 phosphorylation at Ser(317/345) but an increase for cisplatin-induced ATM phosphorylation at Ser(1981) in both HTB182 and A549 cells.

Conclusion: The results of our studies suggest that the presence of low-levels of triptolide potentiates lung cancer cells to cisplatin treatment by selectively inhibiting NER activity, resulting in an increase in apoptosis of the lung cancer cells.

No MeSH data available.


Related in: MedlinePlus