Abstract 3286: CLEFMA synergizes with celecoxib in suppression of lung cancer cell growth.
Authors: Sahoo Kaustuv K. , Yadav Vivek R. , Awasthi Vibhudutta 
Published: August 2014

Background: Cyclooxygenase-2 (COX-2) is frequently over-expressed in various malignancies, often associated with poor clinical outcome. Celecoxib, a selective COX-2 inhibitor induces apoptosis in cancer cells. However, at higher concentrations needed for chemoprevention or treatment, celecoxib targets COX-2 independent drug targets resulting in undesired toxicity. CLEFMA or 4-[3, 5-bis (2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid], a novel molecule synthesized in our laboratory potently inhibits the proliferation of various cancer cells while sparing normal cells. In this study we have tested a hypothesis that CLEFMA would reduce the celecoxib dose required for effective suppression of cancer cell proliferation.

Methods: We determined IC50 values of CLEFMA and celecoxib in lung adenocarcinoma (H441 and A549) and small cell lung cancer (H1650) cells. Further, we tested their toxicity in normal lung fibroblast CCL151 cells at 1x IC50 and 2x IC50 dose levels required for H441 cells. Drug concentrations at ratio 1:3 (Mixture 1), 1:1 (Mixture 2) and 3:1 (Mixture 3) of respective IC50 values of CLEFMA and celecoxib were examined for synergism. The data was analyzed with the Calcusyn software. Following synergy experiments, we determined the effect of CLEFMA treatment on total COX and COX-2 enzymatic activity in cultured A549 cells and in tumor tissue lysates obtained from H441 xenografts in mice. Expression of anti-apoptotic (BCL-2, Bcl-XL and surviving) and apoptotic proteins (PARP, caspase-3 and caspase-7) were investigated by immunobloting.

Results: We observed that CLEFMA was more effective in suppressing the proliferation of all three cancer cell lines than celecoxib. Celecoxib showed significant toxicity in normal lung fibroblasts at 2x IC50 dose, whereas CLEFMA was non-toxic to the normal cells. From the fractional effect-combination index (Fa-CI) plots, we found that Mixture 1 (1:3:: CLEFMA:celecoxib) was synergistic (CI &lt; 1.0) in all three cell lines; in H441 cells mixture 2 (1:1) was synergistic at lower concentration of the drugs, Mixture 3 (3:1) was synergistic only in A549 cells. CLEFMA does not reduce COX-2 activity/unit enzyme, but reduced the expression levels of COX-2 protein; the combination of CLEFMA with celecoxib synergistically suppressed COX-2 expression. CLEFMA also potentiated the activity of celecoxib in down-regulating Bcl-2, Bcl-XL and survivin (anti-apoptotic proteins) and up-regulating PARP, caspase-3 and caspase-7 (pro-apoptotic proteins).

Conclusions: From the results we conclude that CLEFMA synergistically augments the growth inhibitory effect of celecoxib in human lung cancer cell lines without being toxic to normal cells. It appears that the synergy depends on the different mode of actions of the two drugs- CLEFMA suppressing COX-2 protein expression, whereas celecoxib reducing COX-2 activity.

Citation Format: Kaustuv K. Sahoo, Vivek R. Yadav, Vibhudutta Awasthi. CLEFMA synergizes with celecoxib in suppression of lung cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3286. doi:10.1158/1538-7445.AM2013-3286