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Tetrathiomolybdate mediates cisplatin-induced p38 signaling and EGFR degradation and enhances response to cisplatin therapy in gynecologic cancers.

Kim KK, Han A, Yano N, Ribeiro JR, Lokich E, Singh RK, Moore RG - Sci Rep (2015)

Bottom Line: However, TM treatment prior to cisplatin markedly improves cisplatin-induced cytotoxicity.These results are in contrast to what we saw when cells were co-treated with cisplatin plus an EGFR tyrosine kinase inhibitor, where receptor activation was inhibited but receptor degradation was also blocked.We furthermore provide evidence that TM may provide an additional benefit by potentiating p38 activation following cisplatin treatment, which may in turn promote receptor degradation by cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapeutics Laboratory, Program in Women's Oncology, Departments of Obstetrics and Gynecology, Women and Infants Hospital, Alpert Medical School, Brown University, Providence, RI, USA.

ABSTRACT
Cisplatin and its analogs are among the most widely used chemotherapeutic agents against various types of cancer. It is known that cisplatin can activate epidermal growth factor receptor (EGFR), which may provide a survival benefit in cancers. Tetrathiomolybdate (TM) is a potent anti-cancer and anti-angiogenic agent and has been investigated in a number of clinical trials for cancer. In this study, we explore the therapeutic potential of TM on cisplatin-mediated EGFR regulation. Our study shows that TM is not cytotoxic, but exerts an anti-proliferative effect in ECC-1 cells. However, TM treatment prior to cisplatin markedly improves cisplatin-induced cytotoxicity. TM suppressed cisplatin-induced activation of EGFR while potentiating activation of p38; the activation of p38 signaling appeared to promote cisplatin-induced EGFR degradation. These results are in contrast to what we saw when cells were co-treated with cisplatin plus an EGFR tyrosine kinase inhibitor, where receptor activation was inhibited but receptor degradation was also blocked. Our current study is in agreement with previous findings that TM may have a therapeutic benefit by inhibiting EGFR activation. We furthermore provide evidence that TM may provide an additional benefit by potentiating p38 activation following cisplatin treatment, which may in turn promote receptor degradation by cisplatin.

No MeSH data available.


Related in: MedlinePlus

TM inhibits cell proliferation and sensitizes cancer cells to cisplatin.(A) ECC-1 cells were treated with TM (0, 30, 60 μM) for the indicated times, after which the cells were fixed, and the cell proliferation was measured by SRB assay. (B) ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin (0, 60, 120 μM) for another 24 h. The cell viability was evaluated using MTS assay. (C) ECC-1 cells were incubated with or without TM (30 μM) for 18 h, after which the cells were incubated with or without cisplatin (30 μM) for another 24 h. The apoptotic cells were visualized by the TUNEL method that detects DNA fragmentations in apoptotic cells. (D) Top: ECC-1 cells were treated with or without TM for 24 h at the indicated concentrations, followed by cisplatin treatment (30 μM) for 24 h. Bottom: ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin at the indicated concentrations for 24 h. The levels of cleaved PARP, cleaved caspase-3, or GAPDH were determined by Western blot analysis. (E) ECC-1 cells were incubated with TM (30 μM) for 24 h, after which each cell line was treated with cisplatin (15 μM) for 24 h. Apoptotic cell death was determined by caspase-3/7 activity.
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f1: TM inhibits cell proliferation and sensitizes cancer cells to cisplatin.(A) ECC-1 cells were treated with TM (0, 30, 60 μM) for the indicated times, after which the cells were fixed, and the cell proliferation was measured by SRB assay. (B) ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin (0, 60, 120 μM) for another 24 h. The cell viability was evaluated using MTS assay. (C) ECC-1 cells were incubated with or without TM (30 μM) for 18 h, after which the cells were incubated with or without cisplatin (30 μM) for another 24 h. The apoptotic cells were visualized by the TUNEL method that detects DNA fragmentations in apoptotic cells. (D) Top: ECC-1 cells were treated with or without TM for 24 h at the indicated concentrations, followed by cisplatin treatment (30 μM) for 24 h. Bottom: ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin at the indicated concentrations for 24 h. The levels of cleaved PARP, cleaved caspase-3, or GAPDH were determined by Western blot analysis. (E) ECC-1 cells were incubated with TM (30 μM) for 24 h, after which each cell line was treated with cisplatin (15 μM) for 24 h. Apoptotic cell death was determined by caspase-3/7 activity.

Mentions: Anti-proliferative effects of TM have been reported282932. To verify these results, we treated ECC-1 human endometrial cancer cells with various concentrations of TM (0, 30, 60 μM) and evaluated the effect of TM on cell proliferation (Fig. 1A). After treatment for 24 or 48 h, the cell population was analyzed using SRB cell proliferation assay and compared to that of the untreated cells before treatment (0 h). We found that TM inhibits ECC-1 cell proliferation in a dose- and time-dependent manner. Cells incubated with TM for 24 h at 30 μM showed only moderate inhibition (10.9%) compared to the untreated. Anti-proliferative effects of TM (30 μM) were further increased (32.7%) after 48 h (Fig. 1A). Next, we interrogated if TM potentiates cisplatin sensitivity in ECC-1 cells using an MTS cell viability assay. This assay revealed that the viability of cells treated with both TM and cisplatin was reduced compared to cells exposed to cisplatin or TM alone (Fig. 1B). A common method for visualizing cellular apoptotic events is the TUNEL assay. We observed a clear appearance of TUNEL-positive cells (labeled FITC) after cisplatin treatment (Fig. 1C). The number of apoptotic events was substantially increased when cells were pretreated with TM followed by cisplatin treatment while TM treatment alone did not cause apoptosis in ECC-1 cells. Next, we performed immunoblotting to detect the activation of caspase-3 and the cleavage of PARP, characteristic markers for the induction of apoptosis. As shown in Fig. 1D (top panel), TM pretreatment (24 h) prior to cisplatin treatment (30 μM, 24 h) clearly increased cellular apoptosis in ECC-1 cells, compared to those only exposed to cisplatin. TM-mediated cisplatin sensitivity was found to be dose-dependent. Similarly, a fixed concentration of TM (30 μM) caused a dose-dependent increase in cisplatin-mediated cytotoxicity (0, 15, 30 μM) in ECC-1 cells (bottom panel). In order to measure the effects of drugs on apoptosis quantitatively we carried out the caspase-3/7 activity assay. This assay revealed that ECC-1 cells exposed to combinatorial treatment had a 1.9-fold increase in apoptosis over cells exposed to cisplatin alone (Fig. 1E).


Tetrathiomolybdate mediates cisplatin-induced p38 signaling and EGFR degradation and enhances response to cisplatin therapy in gynecologic cancers.

Kim KK, Han A, Yano N, Ribeiro JR, Lokich E, Singh RK, Moore RG - Sci Rep (2015)

TM inhibits cell proliferation and sensitizes cancer cells to cisplatin.(A) ECC-1 cells were treated with TM (0, 30, 60 μM) for the indicated times, after which the cells were fixed, and the cell proliferation was measured by SRB assay. (B) ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin (0, 60, 120 μM) for another 24 h. The cell viability was evaluated using MTS assay. (C) ECC-1 cells were incubated with or without TM (30 μM) for 18 h, after which the cells were incubated with or without cisplatin (30 μM) for another 24 h. The apoptotic cells were visualized by the TUNEL method that detects DNA fragmentations in apoptotic cells. (D) Top: ECC-1 cells were treated with or without TM for 24 h at the indicated concentrations, followed by cisplatin treatment (30 μM) for 24 h. Bottom: ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin at the indicated concentrations for 24 h. The levels of cleaved PARP, cleaved caspase-3, or GAPDH were determined by Western blot analysis. (E) ECC-1 cells were incubated with TM (30 μM) for 24 h, after which each cell line was treated with cisplatin (15 μM) for 24 h. Apoptotic cell death was determined by caspase-3/7 activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4644948&req=5

f1: TM inhibits cell proliferation and sensitizes cancer cells to cisplatin.(A) ECC-1 cells were treated with TM (0, 30, 60 μM) for the indicated times, after which the cells were fixed, and the cell proliferation was measured by SRB assay. (B) ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin (0, 60, 120 μM) for another 24 h. The cell viability was evaluated using MTS assay. (C) ECC-1 cells were incubated with or without TM (30 μM) for 18 h, after which the cells were incubated with or without cisplatin (30 μM) for another 24 h. The apoptotic cells were visualized by the TUNEL method that detects DNA fragmentations in apoptotic cells. (D) Top: ECC-1 cells were treated with or without TM for 24 h at the indicated concentrations, followed by cisplatin treatment (30 μM) for 24 h. Bottom: ECC-1 cells were incubated with or without TM (30 μM) for 24 h, after which the cells were treated with cisplatin at the indicated concentrations for 24 h. The levels of cleaved PARP, cleaved caspase-3, or GAPDH were determined by Western blot analysis. (E) ECC-1 cells were incubated with TM (30 μM) for 24 h, after which each cell line was treated with cisplatin (15 μM) for 24 h. Apoptotic cell death was determined by caspase-3/7 activity.
Mentions: Anti-proliferative effects of TM have been reported282932. To verify these results, we treated ECC-1 human endometrial cancer cells with various concentrations of TM (0, 30, 60 μM) and evaluated the effect of TM on cell proliferation (Fig. 1A). After treatment for 24 or 48 h, the cell population was analyzed using SRB cell proliferation assay and compared to that of the untreated cells before treatment (0 h). We found that TM inhibits ECC-1 cell proliferation in a dose- and time-dependent manner. Cells incubated with TM for 24 h at 30 μM showed only moderate inhibition (10.9%) compared to the untreated. Anti-proliferative effects of TM (30 μM) were further increased (32.7%) after 48 h (Fig. 1A). Next, we interrogated if TM potentiates cisplatin sensitivity in ECC-1 cells using an MTS cell viability assay. This assay revealed that the viability of cells treated with both TM and cisplatin was reduced compared to cells exposed to cisplatin or TM alone (Fig. 1B). A common method for visualizing cellular apoptotic events is the TUNEL assay. We observed a clear appearance of TUNEL-positive cells (labeled FITC) after cisplatin treatment (Fig. 1C). The number of apoptotic events was substantially increased when cells were pretreated with TM followed by cisplatin treatment while TM treatment alone did not cause apoptosis in ECC-1 cells. Next, we performed immunoblotting to detect the activation of caspase-3 and the cleavage of PARP, characteristic markers for the induction of apoptosis. As shown in Fig. 1D (top panel), TM pretreatment (24 h) prior to cisplatin treatment (30 μM, 24 h) clearly increased cellular apoptosis in ECC-1 cells, compared to those only exposed to cisplatin. TM-mediated cisplatin sensitivity was found to be dose-dependent. Similarly, a fixed concentration of TM (30 μM) caused a dose-dependent increase in cisplatin-mediated cytotoxicity (0, 15, 30 μM) in ECC-1 cells (bottom panel). In order to measure the effects of drugs on apoptosis quantitatively we carried out the caspase-3/7 activity assay. This assay revealed that ECC-1 cells exposed to combinatorial treatment had a 1.9-fold increase in apoptosis over cells exposed to cisplatin alone (Fig. 1E).

Bottom Line: However, TM treatment prior to cisplatin markedly improves cisplatin-induced cytotoxicity.These results are in contrast to what we saw when cells were co-treated with cisplatin plus an EGFR tyrosine kinase inhibitor, where receptor activation was inhibited but receptor degradation was also blocked.We furthermore provide evidence that TM may provide an additional benefit by potentiating p38 activation following cisplatin treatment, which may in turn promote receptor degradation by cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapeutics Laboratory, Program in Women's Oncology, Departments of Obstetrics and Gynecology, Women and Infants Hospital, Alpert Medical School, Brown University, Providence, RI, USA.

ABSTRACT
Cisplatin and its analogs are among the most widely used chemotherapeutic agents against various types of cancer. It is known that cisplatin can activate epidermal growth factor receptor (EGFR), which may provide a survival benefit in cancers. Tetrathiomolybdate (TM) is a potent anti-cancer and anti-angiogenic agent and has been investigated in a number of clinical trials for cancer. In this study, we explore the therapeutic potential of TM on cisplatin-mediated EGFR regulation. Our study shows that TM is not cytotoxic, but exerts an anti-proliferative effect in ECC-1 cells. However, TM treatment prior to cisplatin markedly improves cisplatin-induced cytotoxicity. TM suppressed cisplatin-induced activation of EGFR while potentiating activation of p38; the activation of p38 signaling appeared to promote cisplatin-induced EGFR degradation. These results are in contrast to what we saw when cells were co-treated with cisplatin plus an EGFR tyrosine kinase inhibitor, where receptor activation was inhibited but receptor degradation was also blocked. Our current study is in agreement with previous findings that TM may have a therapeutic benefit by inhibiting EGFR activation. We furthermore provide evidence that TM may provide an additional benefit by potentiating p38 activation following cisplatin treatment, which may in turn promote receptor degradation by cisplatin.

No MeSH data available.


Related in: MedlinePlus