Adel Kardosh

HIV-1 Protease Inhibitors Nelfinavir and Atazanavir Induce Malignant Glioma Death by Triggering Endoplasmic Reticulum Stress

HIV type 1 (HIV-1) protease inhibitors (PI) have been shown to have anticancer activity in non-HIV-associated human cancer cells. The underlying mechanism of this effect is unclear. Here, we show that the PIs nelfinavir and atazanavir cause cell death in various malignant glioma cell lines in vitro. The underlying mechanism of this antitumor effect involves the potent stimulation of the endoplasmic reticulum (ER) stress response (ESR), as indicated by increased expression of two ESR markers, GRP78 and CHOP, and activation of ESR-associated caspase-4. Induction of ESR seems to play a central role in PI-induced cell death because small interfering RNA-mediated knockdown of the protective ER chaperone GRP78 sensitizes cells; whereas knockdown of proapoptotic caspase-4 protects cells from PI-induced cell death. Furthermore, the treatment of cells with PIs leads to aggresome formation and accumulation of polyubiquitinated proteins, implying proteasome inhibition. Thus, our results support a model whereby PIs cause tumor cell death via triggering of the ESR, inhibition of proteasome activity, and subsequent accumulation of misfolded proteins. Inhibition of glioma growth via ESR takes place in the in vivo setting as well, as nelfinavir inhibits the growth of xenografted human malignant glioma, with concomitant induction of the proapoptotic ER stress marker CHOP. Because ER stress has also been reported as the mechanism for insulin resistance and diabetes, our ER stress model of PI function may also explain why these drugs may induce insulin resistance as one of their most common side effects.

Aggravated Endoplasmic Reticulum Stress as a Basis for Enhanced Glioblastoma Cell Killing by Bortezomib in Combination with Celecoxib or Its Non-Coxib Analogue, 2,5-Dimethyl-Celecoxib

The proteasome inhibitor bortezomib (Velcade) is known to trigger endoplasmic reticulum (ER) stress via the accumulation of obsolete and damaged proteins. The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex) causes ER stress through a different mechanism (i.e., by causing leakage of calcium from the ER into the cytosol). Each of these two mechanisms has been implicated in the anticancer effects of the respective drug. We therefore investigated whether the combination of these two drugs would lead to further increased ER stress and would enhance their antitumor efficacy. With the use of human glioblastoma cell lines, we show that this is indeed the case. When combined, bortezomib and celecoxib triggered elevated expression of the ER stress markers GRP78/BiP and CHOP/GADD153, caused activation of c-Jun NH(2)-terminal kinase and ER stress-associated caspase-4, and greatly increased apoptotic cell death. Small interfering RNA-mediated knockdown of the protective ER chaperone GRP78/BiP further sensitized the tumor cells to killing by the drug combination. The contribution of celecoxib was independent of the inhibition of COX-2 because a non-coxib analogue of this drug, 2,5-dimethyl-celecoxib (DMC), faithfully and more potently mimicked these combination effects in vitro and in vivo. Taken together, our results show that combining bortezomib with celecoxib or DMC very potently triggers the ER stress response and results in greatly increased glioblastoma cytotoxicity. We propose that this novel drug combination should receive further evaluation as a potentially effective anticancer therapy.

Calcium-activated endoplasmic reticulum stress as a major component of tumor cell death induced by 2,5-dimethyl-celecoxib, a non-coxib analogue of celecoxib

A drawback of extensive coxib use for antitumor purposes is the risk of life-threatening side effects that are thought to be a class effect and probably due to the resulting imbalance of eicosanoid levels. 2,5-Dimethyl-celecoxib (DMC) is a close structural analogue of the selective cyclooxygenase-2 inhibitor celecoxib that lacks cyclooxygenase-2–inhibitory function but that nonetheless is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. To further establish the potential usefulness of DMC as an anticancer agent, we compared DMC and various coxibs and nonsteroidal anti-inflammatory drugs with regard to their ability to stimulate the endoplasmic reticulum (ER) stress response (ESR) and subsequent apoptotic cell death. We show that DMC increases intracellular free calcium levels and potently triggers the ESR in various tumor cell lines, as indicated by transient inhibition of protein synthesis, activation of ER stress–associated proteins GRP78/BiP, CHOP/GADD153, and caspase-4, and subsequent tumor cell death. Small interfering RNA–mediated knockdown of the protective chaperone GRP78 further sensitizes tumor cells to killing by DMC, whereas inhibition of caspase-4 prevents drug-induced apoptosis. In comparison, celecoxib less potently replicates these effects of DMC, whereas none of the other tested coxibs (rofecoxib and valdecoxib) or traditional nonsteroidal anti-inflammatory drugs (flurbiprofen, indomethacin, and sulindac) trigger the ESR or cause apoptosis at comparable concentrations. The effects of DMC are not restricted to in vitro conditions, as this drug also generates ER stress in xenografted tumor cells in vivo, concomitant with increased apoptosis and reduced tumor growth. We propose that it might be worthwhile to further evaluate the potential of DMC as a non-coxib alternative to celecoxib for anticancer purposes. [Mol Cancer Ther 2007;6(4):1262–75]

Differential effects of selective COX-2 inhibitors on cell cycle regulation and proliferation of glioblastoma cell lines.

It is well established that traditional NSAIDs, which inhibit cyclooxygenase (COX) 1 and COX-2, have the potential to reduce the risk of colorectal cancer. New generation COX inhibitors have been developed that selectively inhibit COX-2, which might cause less side effects while still retaining their therapeutic potential. As patients with brain tumors, such as glioblastoma, exhibit a very poor prognosis, we began to explore whether COX inhibitors could be useful for the treatment of this type of tumor. We found that celecoxib inhibited the proliferation of various glioblastoma cell lines in vitro much more potently than traditional NSAIDs. In addition, although several different selective COX-2 inhibitors potently reduced PGE2 levels in these cells, none of them exerted anti-proliferative effects that were comparable to celecoxib. The addition of external PGE2 to celecoxib-treated cells did not restore proliferation, indicating that growth inhibition by celecoxib was not mediated via the blockage of PGE2 production. In an effort to determine the underlying molecular processes that might mediate celecoxib’s potent anti-proliferative effects, we found a loss of the activity of cyclin-dependent kinases, the essential regulators of cell proliferation, which was due to the transcriptional down-regulation of cyclin A and cyclin B expression. Taken together, our results show that celecoxib exerts COX-2-independent anti-proliferative effects on glioblastoma cell growth, which are more potent than those of other selective COX-2 inhibitors or traditional NSAIDs, and which are mediated via the transcriptional inhibition of two essential components of the cell cycle machinery, cyclin A and cyclin B.

Dimethyl-celecoxib (DMC), a derivative of celecoxib that lacks cyclooxygenase-2-inhibitory function, potently mimics the anti-tumor effects of celecoxib on Burkitt's lymphoma in vitro and in vivo.

The non-steroidal anti-inflammatory drug (NSAID) celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has shown some promising results as an anti-cancer drug. However, the question arose as to whether or not its COX-2-inhibitory function is required for its anti-tumorigenic properties. We therefore employed dimethyl-celecoxib (DMC), which is a close structural analog of celecoxib that lacks COX-2-inhibitory function, to investigate this question. By performing a combination of in vitro and in vivo studies with Burkitt’s lymphoma cells, we found that DMC potently mimics all of the anti-proliferative and anti-tumorigenic effects of celecoxib. In cell culture, DMC effectively inhibits cell proliferation through the down-regulation of cyclins A and B and the ensuing loss of cyclin-dependent kinase activity. This effect appears to take place in vivo as well and results in significantly (p

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

BackgroundAn 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.ResultsWith the use of glioblastoma and pancreatic carcinoma cell lines, we comparatively analyzed the effects of celecoxib, UMC, and DMC in various short-term (≤48 hours) cellular and molecular studies, as well as in long-term (≤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.ConclusionThe antitumor activity of celecoxib in vitro did not involve the inhibition of COX-2. Rather, the drugs 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.

Antiangiogenic Activities of 2,5-Dimethyl-Celecoxib on the Tumor Vasculature

Our laboratory has previously shown that a novel compound, 2,5-dimethyl-celecoxib (DMC), which is structurally similar to the cyclooxygenase-2 (COX-2) inhibitor celecoxib but lacks the COX-2–inhibitory function, mimics the antitumor effects of celecoxib. Most studies on DMC, however, focused on its effects on tumor cells. Here, we investigated the activities of DMC as an antiangiogenic agent in both in vitro and in vivo systems. Using primary cultures of human glioma specimens, we found that DMC treatment was cytotoxic to tumor-associated brain endothelial cells (TuBEC), which was mediated through the endoplasmic reticulum stress pathway. In contrast, confluent cultures of quiescent human BEC did not undergo cell death. DMC potently suppressed the proliferation and migration of the TuBEC. DMC caused no apparent effects on the secretion of vascular endothelial growth factor and interleukin-8 but inhibited the secretion of endothelin-1 in tumor-associated EC. DMC treatment of glioma xenografts in mice resulted in smaller tumors with a pronounced reduction in microvessel density compared with untreated mice. In vitro and in vivo analyses confirmed that DMC has antivascular activity. Considering that DMC targets both tumor cells and tumor-associated ECs, this agent is a promising anticancer drug. Mol Cancer Ther; 9(3); 631–41

Potential misidentification of cyclooxygenase-2 by western blot analysis and prevention through the inclusion of appropriate controls

Cyclooxygenase (COX)-2 plays an important role in the development of cancer and has been recognized as a potential therapeutic target. Because nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the activity of this enzyme, the potential efficacy of such drugs for purposes of cancer prevention or therapy is an area of intense research. Therefore, it is of critical importance to unequivocally determine the expression levels of COX-2 protein in tumor cells. In this regard, there are several conflicting reports in the literature where the same type of tumor cell lines were reported as COX-2 positive and as COX-2 negative. We found that during Western blot analysis of COX-2 positive and COX-2 negative cells, different antibodies to COX-2 protein are able to generate strong signals, which are false-positives and can be confused with COX-2. Thus, we believe that some of the conflicting reports on COX-2 expression in tumor cell lines could be the result of improper interpretation of the Western blot signals. Here, we present some of these pitfalls and suggest the inclusion of appropriate controls to unequivocally identify COX-2 protein levels.

Outcome disparities among ethnic subgroups of Waldenström's macroglobulinemia: a population-based study.

Background: Ethnic disparities in cancers are associated with variability in clinical outcomes. We present a Surveillance Epidemiology and End Results (SEER)-based outcome analysis of multiethnic Waldenströms macroglobulinemia (WM) patients. Methods: Adult WM patients diagnosed in 1992 or later (n = 3,175) were analyzed. Median overall survival (OS) was compared across different ethnicities stratified by year of diagnosis, registry identification, age at diagnosis, sex, and marital status. Results: African-Americans (AA) had the youngest median age at diagnosis (63 years) and Whites had the oldest (73 years) (p < 0.001). Female gender, a younger age at diagnosis, and a recent year of diagnosis were associated with an improved OS. Hispanics had the worst (5.6 years) while Whites had the best (6.8 years) median OS. A significant interaction existed between median OS, gender, and race (p = 0.007). Among males, AA had the worst (4.3 years) and Asians had the best (7.3 years) median OS. A significant interaction was also noted between median OS, age at diagnosis, and race (p = 0.033). The worst median OS was seen in Hispanics among patients aged >75 years, and in AA among those aged <65 years. Conclusions: These disparities among WM patients may be multifactorial but need to be explored systematically to better understand the disease biology and for optimal triaging of health care resources.

Suppression of the transformed phenotype and induction of differentiation-like characteristics in cultured ovarian tumor cells by chronic treatment with progesterone

Epidemiological evidence suggests that elevated levels of the pregnancy hormone progesterone might play a role in the reduced risk of women to develop ovarian cancer. In vitro studies have supported this hypothesis by demonstrating negative effects of this hormone on the growth and proliferation of cultured ovarian carcinoma cells. However, little is known about the underlying molecular processes and how progesterone might decrease the risk for ovarian tumors. Therefore, we investigated the effects of chronic hormone treatment on the cell‐cycle and transformed phenotype of ovarian carcinoma cell lines in vitro. We found that long‐term treatment of these cells with progesterone caused a concomitant reduction of cyclin‐dependent kinase (CDK) activity. In parallel, these cells lost their transformed phenotype as indicated by the acquisition of contact inhibition and the loss of anchorage‐independence, as well as the reduced expression of tumor markers such as heat shock protein (HSP) 72 and carcinoma antigen (CA) 125. In addition, progesterone‐treated cells exhibited characteristics that resembled a more differentiated phenotype. Taken together, our data indicated that progesterone was able to suppress the transformed phenotype of ovarian tumor cells. This observation could serve to explain progesterones alleged protective effect in ovarian carcinogenesis.