We propose a novel mechanism by which the combination of drugs that elevate ceramides by inhibiting their metabolism to pro-proliferative sphingolipids can be combined with inhibitors of BCL2-like proteins to eradicate human cancer cells

We propose a novel mechanism by which the combination of drugs that elevate ceramides by inhibiting their metabolism to pro-proliferative sphingolipids can be combined with inhibitors of BCL2-like proteins to eradicate human cancer cells. Materials and Methods Cell Culture Human leukemia cell lines, U937, K562, HL60 and RPMI-8226 were obtained from ATCC (Manassas, VA, USA) Solanesol and cultured in RMPI media containing 10% FBS, 1% L-glutamine and 1% Penicillin/Streptomycin. synergized with ABT-263 to kill leukemia cells and similarly increased ceramides and sphingosine. Data suggest that synergism with ABT-263 requires accumulation of ceramides and sphingosine, as AMP-deoxynojirimycin, (an inhibitor of the glycosphingolipid pathway) did not elevate ceramides or sphingosine and importantly did not sensitize cells to ABT-263 treatment. Taken together, our data suggest that combining inhibitors of anti-apoptotic BCL2-like proteins with drugs that alter the balance of bioactive sphingolipids will be a powerful combination for the treatment of human cancers. Introduction Cancer cells are a distorted version of their normal counterparts [1]. One of the important distortions that separate cancer cells from healthy cells is the inability to undergo programmed cell death, or apoptosis, triggered by homeostatic processes. However, a peculiar observation is that most cancer cells are capable of undergoing apoptosis following treatment with cytotoxic stimuli. The challenge lies in identifying the particular stimuli that will effectively kill a given cancer, while sparing the healthy cells of the patients body. Historically, high doses of toxic compounds have been used, and are still used, to Solanesol eradicate cancers; however, the unwarranted consequences of this type of regimen are the detrimental side effects that patients experience due to the death of normal cells of the body. By understanding the biochemical and molecular requirements of cancer cells, it may be possible to avoid these detrimental side effects by combining lower doses of drugs that trigger apoptosis preferentially in cancer cells. Two families of molecules have been actively studied that may fulfill these criteria and give therapeutic benefit to cancer patients. The first is the BCL2-family of proteins and the second are bioactive sphingolipids. Both families can be sub-divided into members that possess the ability to either cause or prevent apoptosis and modulators of these molecules are being explored as potential cancer therapeutics [2], [3], [4], [5]. Anti-apoptotic BCL2-like proteins regulate critical aspects of apoptosis by inhibiting mitochondria outer membranes permeabilization (MOMP), a requisite step in the initiation of the apoptotic pathway that results in the release of proteins from the mitochondrial intermembrane space to the cytosol where they activate caspases and DNases necessary for the execution of the cell [3], [6], [7]. It has been demonstrated that many cancer cells are critically dependent on the activity of anti-apoptotic BCL2-like proteins to maintain survival [8], [9], [10]. As such, multiple inhibitors of BCL2-like proteins are currently being explored in clinical trials as potential therapeutics. One such compound is ABT-263 (or first generation compound, ABT-737), is a small molecule designed to interact with three of the BCL2-like proteins, BCL2, BCLxL and BCLw [9], [11]. ABT-263 does not inhibit the activity of the other three BCL2-like proteins, BCLb, BFL1 and MCL1, and as a consequence expression of any of these three proteins can potentially lead to resistance to the drug [12], [13], [14]. Additional mechanisms have also been described that can cause cancer cells to become insensitive to ABT-263 treatment, such as loss of the pro-apoptotic BCL2 proteins, BAK or BIM [14], [15]. In addition, patients that receive ABT-263 display thrombocytopenia caused by the ability of ABT-263 to block the function of BCLxl [11], [16]. Like the family of BCL2-like proteins, sphingolipids are also known to regulate apoptosis [5]. Cellular levels of Rabbit polyclonal to DUSP7 ceramide, a central molecule in sphingolipid metabolism, are elevated following treatment of cells with cytotoxic stimuli and inhibiting its generation blocks or delays cell death [17]. Ceramide generation occurs upstream of the execution phase Solanesol of apoptosis and data suggest that ceramides plays an important role in MOMP [17], [18]. Ceramide generation has been shown to be regulated by BCL2-like proteins. For example, overexpression of anti-apoptotic BCL2-like proteins BCL2 and BCLxL have been shown to inhibit ceramide generation and/or its ability to induce MOMP and apoptosis [19], [20], [21], [22], [23], . In addition, our previously published data indicate the pro-apoptotic BCL2-like protein BAK is required for ceramide synthase-mediated long-chain ceramide generation during apoptosis [17]. Once generated ceramide can be metabolized via several pathways, resulting in the production of pro-proliferative users of the sphingolipid family. For example, ceramide glycosylation results in the production of glucosylceramide, a sphingolipid that promotes cellular proliferation. Similarly, ceramide can be broken.