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COX-2 inhibition is neither necessary nor sufficient for celecoxib to suppress tumor cell proliferation and focus formation in vitro.

Chuang HC, Kardosh A, Gaffney KJ, Petasis NA, Schönthal AH - Mol. Cancer (2008)

Bottom Line: We found that DMC exhibited the most potent antitumor activity; celecoxib was somewhat less effective, and UMC clearly displayed the overall weakest antitumor potential in all aspects.The antitumor activity of celecoxib in vitro did not involve the inhibition of COX-2.In addition, the newly discovered ability of this drug to restore contact inhibition and block focus formation during chronic drug exposure, which involved neither ERS nor COX-2, suggests a novel, as yet unrecognized mechanism of celecoxib action.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Microbiology & Immunology, University of Southern California, Los Angeles, USA. huanchic@usc.edu

ABSTRACT

Background: An increasing number of reports is challenging the notion that the antitumor potential of the selective COX-2 inhibitor celecoxib (Celebrex) is mediated primarily via the inhibition of COX-2. We have investigated this issue by applying two different analogs of celecoxib that differentially display COX-2-inhibitory activity: the first analog, called unmethylated celecoxib (UMC), inhibits COX-2 slightly more potently than its parental compound, whereas the second analog, 2,5-dimethyl-celecoxib (DMC), has lost the ability to inhibit COX-2.

Results: With the use of glioblastoma and pancreatic carcinoma cell lines, we comparatively analyzed the effects of celecoxib, UMC, and DMC in various short-term (< or =48 hours) cellular and molecular studies, as well as in long-term (< or =3 months) focus formation assays. We found that DMC exhibited the most potent antitumor activity; celecoxib was somewhat less effective, and UMC clearly displayed the overall weakest antitumor potential in all aspects. The differential growth-inhibitory and apoptosis-stimulatory potency of these compounds in short-term assays did not at all correlate with their capacity to inhibit COX-2, but was closely aligned with their ability to trigger endoplasmic reticulum stress (ERS), as indicated by the induction of the ERS marker CHOP/GADD153 and activation of the ERS-associated caspase 7. In addition, we found that these compounds were able to restore contact inhibition and block focus formation during long-term, chronic drug exposure of tumor cells, and this was achieved at sub-toxic concentrations in the absence of ERS or inhibition of COX-2.

Conclusion: The antitumor activity of celecoxib in vitro did not involve the inhibition of COX-2. Rather, the drug's ability to trigger ERS, a known effector of cell death, might provide an alternative explanation for its acute cytotoxicity. In addition, the newly discovered ability of this drug to restore contact inhibition and block focus formation during chronic drug exposure, which involved neither ERS nor COX-2, suggests a novel, as yet unrecognized mechanism of celecoxib action.

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Increased expression of markers for ER stress and apoptosis in response to treatment with CXB, UMC, and DMC. U251 glioblastoma cells were treated with the indicated concentrations of CXB, UMC, and DMC and cell lysates were analyzed by Western blot with specific antibodies to CHOP (a pro-apoptotic ER stress indicator protein), cleaved (i.e., activated) caspase 7 (an ER stress-associated protein that participates in the execution of apoptosis), and PARP (proteolytic cleavage of PARP is executed by caspase 3 and indicates ongoing apoptosis). To verify equal loading in each case, the blots were also probed with an antibody to actin. The top panels represent lysates from cells treated with drugs for 18 hours (to reveal earlier events during ER stress). The bottom panels represent lysates from cells treated with drugs for 48 hours (to reveal later stages of ER stress-induced apoptosis). f.l.: full length PARP; cl.: cleaved PARP.
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Figure 3: Increased expression of markers for ER stress and apoptosis in response to treatment with CXB, UMC, and DMC. U251 glioblastoma cells were treated with the indicated concentrations of CXB, UMC, and DMC and cell lysates were analyzed by Western blot with specific antibodies to CHOP (a pro-apoptotic ER stress indicator protein), cleaved (i.e., activated) caspase 7 (an ER stress-associated protein that participates in the execution of apoptosis), and PARP (proteolytic cleavage of PARP is executed by caspase 3 and indicates ongoing apoptosis). To verify equal loading in each case, the blots were also probed with an antibody to actin. The top panels represent lysates from cells treated with drugs for 18 hours (to reveal earlier events during ER stress). The bottom panels represent lysates from cells treated with drugs for 48 hours (to reveal later stages of ER stress-induced apoptosis). f.l.: full length PARP; cl.: cleaved PARP.

Mentions: Because it was reported before that ER stress might be a critical mechanism by which celecoxib exerts its antitumor function, we investigated two established markers of ER stress, i.e., the pro-apoptotic protein CHOP and caspase 7. U251 glioblastoma cells were treated with different concentrations of CXB, UMC, and DMC, and cell lysates were analyzed by Western blot. As shown in Figure 3, all three drugs were able to stimulate CHOP expression levels and trigger activation of caspase 7. However, the respective effective concentrations differed substantially. In the case of CHOP induction, 40 μM DMC was as effective as 60 μM CXB, but UMC even at 100 μM displayed overall weaker effects. In the case of caspase 7 activation, 60 μM DMC was approximately as potent as 75 μM CXB, but UMC, again, even at 100 μM displayed weaker effects. In addition, we examined the cleavage of PARP (poly-ADP-ribose polymerase), which serves as an indicator of ongoing apoptosis. As before, DMC displayed the strongest effects on PARP cleavage; CXB was somewhat less potent and UMC exhibited only weak effects. Thus, overall, the effects of the three drugs on molecular markers of ER stress and apoptosis correlated with their cytotoxic potency (as shown in Figure 2), but not with their ability to inhibit COX-2.


COX-2 inhibition is neither necessary nor sufficient for celecoxib to suppress tumor cell proliferation and focus formation in vitro.

Chuang HC, Kardosh A, Gaffney KJ, Petasis NA, Schönthal AH - Mol. Cancer (2008)

Increased expression of markers for ER stress and apoptosis in response to treatment with CXB, UMC, and DMC. U251 glioblastoma cells were treated with the indicated concentrations of CXB, UMC, and DMC and cell lysates were analyzed by Western blot with specific antibodies to CHOP (a pro-apoptotic ER stress indicator protein), cleaved (i.e., activated) caspase 7 (an ER stress-associated protein that participates in the execution of apoptosis), and PARP (proteolytic cleavage of PARP is executed by caspase 3 and indicates ongoing apoptosis). To verify equal loading in each case, the blots were also probed with an antibody to actin. The top panels represent lysates from cells treated with drugs for 18 hours (to reveal earlier events during ER stress). The bottom panels represent lysates from cells treated with drugs for 48 hours (to reveal later stages of ER stress-induced apoptosis). f.l.: full length PARP; cl.: cleaved PARP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2396175&req=5

Figure 3: Increased expression of markers for ER stress and apoptosis in response to treatment with CXB, UMC, and DMC. U251 glioblastoma cells were treated with the indicated concentrations of CXB, UMC, and DMC and cell lysates were analyzed by Western blot with specific antibodies to CHOP (a pro-apoptotic ER stress indicator protein), cleaved (i.e., activated) caspase 7 (an ER stress-associated protein that participates in the execution of apoptosis), and PARP (proteolytic cleavage of PARP is executed by caspase 3 and indicates ongoing apoptosis). To verify equal loading in each case, the blots were also probed with an antibody to actin. The top panels represent lysates from cells treated with drugs for 18 hours (to reveal earlier events during ER stress). The bottom panels represent lysates from cells treated with drugs for 48 hours (to reveal later stages of ER stress-induced apoptosis). f.l.: full length PARP; cl.: cleaved PARP.
Mentions: Because it was reported before that ER stress might be a critical mechanism by which celecoxib exerts its antitumor function, we investigated two established markers of ER stress, i.e., the pro-apoptotic protein CHOP and caspase 7. U251 glioblastoma cells were treated with different concentrations of CXB, UMC, and DMC, and cell lysates were analyzed by Western blot. As shown in Figure 3, all three drugs were able to stimulate CHOP expression levels and trigger activation of caspase 7. However, the respective effective concentrations differed substantially. In the case of CHOP induction, 40 μM DMC was as effective as 60 μM CXB, but UMC even at 100 μM displayed overall weaker effects. In the case of caspase 7 activation, 60 μM DMC was approximately as potent as 75 μM CXB, but UMC, again, even at 100 μM displayed weaker effects. In addition, we examined the cleavage of PARP (poly-ADP-ribose polymerase), which serves as an indicator of ongoing apoptosis. As before, DMC displayed the strongest effects on PARP cleavage; CXB was somewhat less potent and UMC exhibited only weak effects. Thus, overall, the effects of the three drugs on molecular markers of ER stress and apoptosis correlated with their cytotoxic potency (as shown in Figure 2), but not with their ability to inhibit COX-2.

Bottom Line: We found that DMC exhibited the most potent antitumor activity; celecoxib was somewhat less effective, and UMC clearly displayed the overall weakest antitumor potential in all aspects.The antitumor activity of celecoxib in vitro did not involve the inhibition of COX-2.In addition, the newly discovered ability of this drug to restore contact inhibition and block focus formation during chronic drug exposure, which involved neither ERS nor COX-2, suggests a novel, as yet unrecognized mechanism of celecoxib action.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Microbiology & Immunology, University of Southern California, Los Angeles, USA. huanchic@usc.edu

ABSTRACT

Background: An increasing number of reports is challenging the notion that the antitumor potential of the selective COX-2 inhibitor celecoxib (Celebrex) is mediated primarily via the inhibition of COX-2. We have investigated this issue by applying two different analogs of celecoxib that differentially display COX-2-inhibitory activity: the first analog, called unmethylated celecoxib (UMC), inhibits COX-2 slightly more potently than its parental compound, whereas the second analog, 2,5-dimethyl-celecoxib (DMC), has lost the ability to inhibit COX-2.

Results: With the use of glioblastoma and pancreatic carcinoma cell lines, we comparatively analyzed the effects of celecoxib, UMC, and DMC in various short-term (< or =48 hours) cellular and molecular studies, as well as in long-term (< or =3 months) focus formation assays. We found that DMC exhibited the most potent antitumor activity; celecoxib was somewhat less effective, and UMC clearly displayed the overall weakest antitumor potential in all aspects. The differential growth-inhibitory and apoptosis-stimulatory potency of these compounds in short-term assays did not at all correlate with their capacity to inhibit COX-2, but was closely aligned with their ability to trigger endoplasmic reticulum stress (ERS), as indicated by the induction of the ERS marker CHOP/GADD153 and activation of the ERS-associated caspase 7. In addition, we found that these compounds were able to restore contact inhibition and block focus formation during long-term, chronic drug exposure of tumor cells, and this was achieved at sub-toxic concentrations in the absence of ERS or inhibition of COX-2.

Conclusion: The antitumor activity of celecoxib in vitro did not involve the inhibition of COX-2. Rather, the drug's ability to trigger ERS, a known effector of cell death, might provide an alternative explanation for its acute cytotoxicity. In addition, the newly discovered ability of this drug to restore contact inhibition and block focus formation during chronic drug exposure, which involved neither ERS nor COX-2, suggests a novel, as yet unrecognized mechanism of celecoxib action.

Show MeSH
Related in: MedlinePlus