Mitochondrial ceramide increases in UV-irradiated HeLa cells and is mainly derived from hydrolysis of sphingomyelin

Sphingolipids are important signaling molecules in many biologic processes, but little is known about their organelle-specific roles. Using HeLa cells, we investigated the effects of UV and etoposide-induced apoptosis on the contents of sphingomyelin (SM) and ceramide in subcellular compartments. UV irradiation of HeLa cells increased the levels of SM in all subcellular fractions, but the change was most dramatic in mitochondria. Using diacylglycerol kinase assays to quantify ceramide, we found that the levels of ceramide in mitochondria increased as early as 2 h after UV irradiation and remained elevated at 6 h. The increase in mitochondrial SM and ceramide was inhibited by D609, an inhibitor of sphingomyelinase and SM synthase. The inhibition of sphingolipid production correlated with protection of the mitochondrial transmembrane potential and prevention of cytochrome c release following UV irradiation. In contrast, myriocin, an inhibitor of the de novo ceramide synthesis pathway, only partially suppressed the production of ceramides in mitochondria and cannot suppress UV-induced apoptosis. Fumonicin B1, an inhibitor of ceramide synthase, can only prevent mitochondrial ceramide synthesis and UV-induced apoptosis in a small degree. These results indicate that mitochondrial ceramide production in UV-irradiated HeLa cells is not mediated by the de novo synthesis pathway, but mainly through SM hydrolysis.     

Targeting the ceramide system in cancer

Sphingolipids, in particular ceramide, have been described as important components of cellular signalling pathways. Ceramide can be produced via multiple mechanisms including through the hydrolysis of sphingomyelin by acid and neutral sphingomyelinase or by a de novo synthesis pathway. Recent studies have identified sphingomyelinases and ceramide synthases as important targets for γ-irradiation and chemotherapeutic drugs. Likewise, common cancer treatment modalities, such as γ-irradiation and many chemotherapeutic agents, induce cell death via the generation of ceramide. This suggests that the manipulation of ceramide production and metabolism could offer promising means for the enhancement of anti-tumor therapies. The focus of this mini-review will be to discuss contemporary evidence suggesting that ceramide forming pathways and ceramide itself are important targets for the treatment of tumors and the development of novel tumor treatment strategies.     

Curcumin mediates ceramide generation via the de novo pathway in colon cancer cells

A wealth of evidence supports the notion that curcumin, a phytochemical present in turmeric, is a potent chemopreventive agent for colon cancer. Its mechanism of action remains incompletely understood. Here we report that curcumin's apoptosis-inducing effects in colon cancer cell lines are accompanied by robust ceramide generation. This occurs through de novo synthesis as the increase in ceramide could be attenuated by pre-incubation of the cells with myriocin, and no changes were observed in sphingomyelin levels, or in either acidic or neutral sphingomyelinase activities. Furthermore, cell death could in part be reversed by myriocin, indicating, for the first time, that endogenous ceramide generation by this agent contributes towards its biological activity. We then investigated the role of reactive oxygen species (ROS) in this phenomenon and demonstrated that curcumin induced robust oxidant generation in the cell lines tested, and its reversal by N-acetylcysteine, completely attenuated apoptosis. We next confirmed that curcumin could activate c-jun N-terminal kinase (JNK) and that its modulation could reverse cell death; however, this intervention could not block ceramide generation, or ROS production. Conversely, however, the inhibition of ROS using N-acetylcysteine led to an inhibition of JNK activation. Hence, we conclude that curcumin induces apoptosis via a ROS-associated mechanism that converges on JNK activation, and to a lesser extent via a parallel ceramide-associated pathway.     

N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138-1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [(3)H]palmitic acid, caused sequential formation of [(3)H]sphinganine (220% over control) and [(3)H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B(1), an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB(1) were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 microM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 microM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.     

Bortezomib-induced apoptosis in cultured pancreatic cancer cells is associated with ceramide production

Purpose

The proteasome inhibitor bortezomib (PS-341) has displayed significant efficiency against pancreatic cancer cells. However, the underlying mechanisms are not fully understood. Here, we tested if ceramide production was involved in the bortezomib’s effect.

Methods

Two transformed pancreatic cancer cell lines (PANC-1 and Mia) and the primary pancreatic cancer cells were used. Cell death was analyzed by MTT viability assay and trypan blue staining. Cell apoptosis was analyzed by Histone DNA-ELISA assay and Annexin V FACS. Western blots were used to test signal protein changes. The cellular ceramide level after bortezomib treatment was also determined.

Results

In cultured pancreatic cancer cells, bortezomib increased cellular ceramide production to promote cell apoptosis. The ceramide de novo synthase inhibitor fumonisin B1 (F-B1) suppressed bortezomib-induced ceramide production and apoptosis, while exogenously added C6-ceramide facilitated bortezomib-induced pancreatic cancer cell death. Meanwhile, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the inhibitor of glucosylceramide synthetase as well as the sphingosine kinase 1 inhibitors (SKI-II and SKI-IV), facilitated bortezomib-induced ceramide production and subsequent cell apoptosis. Further, bortezomib-induced pro-apoptotic c-Jun N-terminal kinase (JNK) activation was also associated with ceramide production. JNK activation by bortezomib was suppressed by F-B1, but was enhanced by SKI-II and PDMP in pancreatic cancer cells. Finally, C6-ceramide, SKI-II, and PDMP dramatically enhanced bortezomib-induced cytotoxicity in primary cultured pancreatic cancer cells.

Conclusions

We found that bortezomib-induced apoptosis was associated with ceramide production in primary and transformed pancreatic cancer cells.     

Fenretinide stimulates redox-sensitive ceramide production in breast cancer cells: potential role in drug-induced cytotoxicity

The synthetic retinoid N-(4-hydroxphenyl) retinamide (4HPR) has manifold actions, which may contribute to its chemopreventive effects on breast cancer cell growth and progression. A role for ceramide as a stress-response signal is investigated here during the cytotoxic action of 4HPR in MCF-7 cells. N-(4-hydroxphenyl) retinamide induced a dose-dependent decline in cell growth and survival associated with a maximal 10-fold increase in ceramide production at 10 microM. N-(4-hydroxphenyl) retinamide exhibited a greater potency than all-trans retinoic acid (ATRA) on growth inhibition and ceramide production. The synthetic peroxisome proliferator-activated receptors agonist troglitazone (TGZ), but not the native ligand 15-deoxy-delta 12,14-prostaglandin J2, abrogated both these actions of 4HPR but not that of ATRA. The antioxidant N-acetylcysteine mimicked the abrogative effect of TGZ on 4HPR action, while the exogenous oxidant H2O2 also stimulated ceramide production. The inhibitors of de novo ceramide synthesis, fumonisin B1 and myriocin, blocked the ceramide response to 4HPR and partially reversed the apoptotic response, but did not prevent the overall decline in cell survival. The pancaspase inhibitor Z-VAD fmk reduced the decrease in cell survival caused by 4HPR, but did not affect the ceramide response. These findings describe a novel redox-sensitive elevation of ceramide levels associated with the cytotoxic response of breast cancer cells to 4HPR. However, a major mediatory role for this sphingolipid in this context remains equivocal.     

Myristic acid potentiates palmitic acid-induced lipotoxicity and steatohepatitis associated with lipodystrophy by sustaning de novo ceramide synthesis

Palmitic acid (PA) induces hepatocyte apoptosis and fuels de novo ceramide synthesis in the endoplasmic reticulum (ER). Myristic acid (MA), a free fatty acid highly abundant in copra/palmist oils, is a predictor of nonalcoholic steatohepatitis (NASH) and stimulates ceramide synthesis. Here we investigated the synergism between MA and PA in ceramide synthesis, ER stress, lipotoxicity and NASH. Unlike PA, MA is not lipotoxic but potentiated PA-mediated lipoapoptosis, ER stress, caspase-3 activation and cytochrome c release in primary mouse hepatocytes (PMH). Moreover, MA kinetically sustained PA-induced total ceramide content by stimulating dehydroceramide desaturase and switched the ceramide profile from decreased to increased ceramide 14:0/ceramide16:0, without changing medium and long-chain ceramide species. PMH were more sensitive to equimolar ceramide14:0/ceramide16:0 exposure, which mimics the outcome of PA plus MA treatment on ceramide homeostasis, than to either ceramide alone. Treatment with myriocin to inhibit ceramide synthesis and tauroursodeoxycholic acid to prevent ER stress ameliorated PA plus MA induced apoptosis, similar to the protection afforded by the antioxidant BHA, the pan-caspase inhibitor z-VAD-Fmk and JNK inhibition. Moreover, ruthenium red protected PMH against PA and MA-induced cell death. Recapitulating in vitro findings, mice fed a diet enriched in PA plus MA exhibited lipodystrophy, hepatosplenomegaly, increased liver ceramide content and cholesterol levels, ER stress, liver damage, inflammation and fibrosis compared to mice fed diets enriched in PA or MA alone. The deleterious effects of PA plus MA-enriched diet were largely prevented by in vivo myriocin treatment. These findings indicate a causal link between ceramide synthesis and ER stress in lipotoxicity, and imply that the consumption of diets enriched in MA and PA can cause NASH associated with lipodystrophy.     

LASS2基因抑制HCCLM3肝癌细胞的转移

目的：探讨LASS2基因对HCCLM3肝癌细胞转移的影响。方法：Northern blot分析高转移潜能肝癌细胞系HCCLM3和低转移潜能肝癌细胞系MHCC97-L中LASS2基因的mRNA水平。构建包含LASS2基因编码框的真核表达载体pCMV—HA2-LASS2，通过脂质体转染HCCLM3细胞，经G418筛选，建立稳定表达LASS2基因的HCCLM3细胞株。通过分析LASS2在HCCLM3中过表达后，HCCLM3细胞在迁移、侵袭等转移能力上的改变，Western blot、明胶酶谱及原位酶谱分析MMP-2合成、分泌、激活的变化。结果：Northern blot显示，HCCLM3细胞中LASS2基因的表达水平低于MHCC97-L细胞。当LASS2基因过表达后，HCCLM3细胞的迁移及侵袭能力受到显著抑制（P〈0．001），虽然细胞内MMP-2的合成未改变，但细胞MMP-2的分泌及MMP-2的激活均受抑制。结论：LASS2基因可下调MMP-2的分泌及激活，可使肝癌细胞HCCLM3的转移能力受到明显抑制，提示LASS2基因对肝癌细胞HCCLM3的转移具有抑制作用。     

Ceramide synthesis and metabolism as a target for cancer therapy

Sphingolipids, which include ceramides and sphingosine, are essential structural components of cell membranes that also have messenger functions that regulate the proliferation, survival, and death of cells. Exogenous application of ceramide is cytotoxic, and exposure of cells to radiation or chemotherapy is associated with increased ceramide levels due to enhanced de novo synthesis, catabolism of sphingomyelin, or both. Ceramide can be metabolized to less toxic forms by glycosylation, acylation, or by catabolism to sphingosine, which is then phosphorylated to the anti-apoptotic sphingosine 1-phosphate. Glucosylceramide synthase overexpression has been shown to enhance resistance to doxorubicin, suggesting that inhibition of ceramide metabolism or catabolism might enhance cancer chemotherapy. Several anticancer agents, including the cytotoxic retinoid, fenretinide (4-HPR), have been shown to act, at least in part, by increasing tumor cell ceramide via de novo synthesis. Combinations of 4-HPR and modulators of ceramide action and/or metabolism demonstrated increased anti-tumor activity in pre-clinical models with minimal toxicity for non-malignant cells, and were effective in a p53-independent manner against tumor cell lines resistant to standard cytotoxic agents. Phase I trials of ceramide metabolism inhibitors in combination with 4-HPR and with other cytotoxic agents are in development. Thus, pharmacological manipulation of sphingolipid metabolism to enhance tumor cell ceramide is being realized and offers a novel approach to cancer chemotherapy.     

Selective susceptibility of transformed T lymphocytes to induction of apoptosis by PSC 833, an inhibitor of P-glycoprotein

P-glycoprotein is a cellular efflux pump. The P-glycoprotein inhibitor PSC 833 causes apoptosis of cancer cells and induces a rise in the intracellular levels of ceramide. Our aims were to determine whether a cause and effect relationship exists between these two actions of PSC 833, and to assess whether the PSC 833-induced apoptosis is restricted to transformed cells. Apoptosis was determined by flow cytometry and radioactive quantitation of DNA fragmentation. PSC 833 induced apoptosis in the human T leukemia cell lines: Molt-4 and Jurkat. Analysis of the apoptosis in Molt-4 and Jurkat cells revealed that PSC 833 induced a rise in the cellular ceramide levels (as measured by the DG kinase assay). PSC 833-induced apoptosis was significantly reduced by specific inhibitors of ceramide de novo synthesis (i.e., fumonisin B1 and L-cycloserine). On the other hand, PSC 833 did not induce apoptosis in normal peripheral blood T cells regardless of whether these cells were quiescent, activated, or proliferating. Our results suggest that PSC 833 induces apoptotic death in human transformed T lymphocytes through an increase in ceramide de novo synthesis. In addition, normal lymphocytes are not susceptible to induction of apoptosis by PSC 833. This difference between normal lymphocytes and leukemia cells presents a potential target for chemotherapy.     

Signaling pathways required for matrix metalloproteinase-9 induction by betacellulin in head-and-neck squamous carcinoma cells

The mechanisms by which c-erbB-dependent signaling contribute to the invasive potential of HNSCC remain to be fully elucidated. We have previously shown that c-erbB autocrine and/or paracrine stimulation upregulates MMP-9 but has no effect on the related gelatinase, MMP-2. BTC, a major c-erbB ligand, has the ability to efficiently activate all c-erbB receptors and to bind directly to EGFR and c-erbB-4. BTC is commonly expressed in HNSCC cells and exerts the most potent effects in terms of MMP induction relative to other c-erbB ligands so far tested. In the present study, we explored the contribution of major downstream events triggered by BTC/c-erbB receptor signaling to the regulation of MMP-9 and in vitro invasiveness of HNSCC cells. In human HNSCC cell lines, SIHN-006 and Detroit-562, BTC treatment resulted in rapid tyrosine phosphorylation of all c-erbB receptors whereas both endogenous MMP-9 and BTC-stimulated MMP-9 were predominantly mediated via EGFR. BTC induced ERK1/2, JNK/SAPK and Akt phosphorylation with differing kinetics but not p38 kinase. The BTC-dependent activation of JNK and PI3K/Akt pathways occurred predominantly via EGFR, whereas activation of the MEK-1/ERK pathway occurred via all 4 c-erbB receptors, although again predominantly via EGFR. Selective inhibition of ERK/MAPK (by PD98059 or U0126) and PI3K (by LY294002 or wortmannin) led to marked reduction of both basal and BTC-induced MMP-9 activity and invasive ability of HNSCC cells. In contrast, inhibition of p38 kinase with SB203580 produced no such effects. A specific inhibitor of NF-kappa B, BAY 11-7085, also blocked the stimulatory effect of BTC. No remarkable inhibition of MMP-9 and invasion was observed on targeting other cellular activities, such as PKA, PKC and PLC-gamma. Taken together, our data show that BTC induces MMP-9 production and invasion primarily through activation of EGFR, MAPK and PI3K/Akt in HNSCC cells.     

Resveratrol impairs the formation of MDA-MB-231 multicellular tumor spheroids concomitant with ceramide accumulation

Resveratrol exerts a drastic growth inhibitory effect on human breast cancer MDA-MB-231 cells grown both in vitro and in vivo. Here we show that resveratrol affects the aggregation properties of MDA-MB-231 cells into multicellular tumor spheroids (MCTSs), in association with induction of de novo synthesis of ceramide. After 9 days of 3D growth in the presence of resveratrol (64 microM), MDA-MB-231 cells formed significantly smaller MCTSs. Further, cells from these aggregates failed to form colonies. Addition of resveratrol (64 microM) to preformed MDA-MB-231 MCTSs caused no significant size change, consistent with lack of ceramide induction. Only some apoptotic blebs were found on the MCTSs surface. Altogether these findings indicate that resveratrol might be effective for prevention of breast cancer cell growth.     

N-(4-hydroxyphenyl)retinamide increases ceramide and is cytotoxic to acute lymphoblastic leukemia cell lines, but not to non-malignant lymphocytes.

The retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR), mediates p53-independent cytotoxicity and can increase reactive oxygen species and ceramide in solid tumor cell lines. We determined changes in ceramide and cytotoxicity upon treatment with 4-HPR (3-12 microM) in six human acute lymphoblastic leukemia (ALL) cell lines: T cell (MOLT-3, MOLT-4, CEM), pre-B-cell (NALM-6, SMS-SB), and null cell (NALL-1). Exposure to 4-HPR (12 microM) for 96 h caused 4.7 (MOLT-3), 3.5 (MOLT-4), 3.9 (CEM), 2.9 (NALM-6), 4.7 (SMS-SB), AND 4.5 (NALL-1) logs of cell kill. The average 4-HPR concentration that killed 99% of cells (LC(99)) for all six lines was 4.8 microM (range: 1.5-8.9 microM). Treatment with 4-HPR (9 microM) for 24 h resulted in an 8.9 +/- 1.0-fold (range: 4.9-15.7-fold) increase of ceramide. Ceramide increase was time- and dose-dependent and abrogated by inhibitors of de novo ceramide synthesis. Concurrent inhibition of ceramide glycosylation/acylation by d,l-threo-(1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol) (PPMP) further increased ceramide levels, and synergistically increased 4-HPR cytotoxicity in four of six ALL cell lines. 4-HPR was minimally cytotoxic to peripheral blood mononuclear cells and a lymphoblastoid cell line, and increased ceramide <2-fold. Thus, 4-HPR was cytotoxic and increased ceramide in ALL cell lines, but not in non-malignant lymphoid cell types.     

Radiation and ceramide-induced apoptosis

Ceramide is a sphingolipid that acts as a second messenger in ubiquitous, evolutionarily conserved, signaling systems. Emerging data suggest that radiation acts directly on the plasma membrane of several cell types, activating acid sphingomyelinase, which generates ceramide by enzymatic hydrolysis of sphingomyelin. Ceramide then acts as a second messenger in initiating an apoptotic response via the mitochondrial system. Radiation-induced DNA damage can also initiate ceramide generation by activation of mitochondrial ceramide synthase and de novo synthesis of ceramide. In some cells and tissues, BAX is activated downstream of ceramide, regulating commitment to the apoptotic process via release of mitochondrial cytochrome c. Genetic and pharmacologic studies in vivo showed that radiation targets the acid sphingomyelinase apoptotic system of microvascular endothelial cells in the lungs, intestines and brain, as well as in oocytes, to initiate the pathogenesis of tissue damage. Regulated ceramide metabolism may produce metabolites, such as sphingosine 1-phosphate, shown to signal antiapoptosis, thus controlling the intensity of the apoptotic response and constituting a mechanism for radiation sensitivity or resistance. An improved understanding of this signaling system may offer new opportunities for the modulation of radiation effects in the treatment of cancer.     

Taxol-induced ceramide generation and apoptosis in human breast cancer cells

PURPOSE: Taxol has emerged as a valuable antimitotic chemotherapeutic agent, particularly in advanced breast and ovarian cancers. Although much is known about cytotoxic mechanisms, the effectiveness of Taxol cannot be solely explained by microtubular interaction. This study was undertaken to determine whether ceramide generation plays a role in Taxol-induced apoptosis. METHODS: Hormone-independent MDA-MB-468 and hormone-dependent MCF-7 breast cancer cell lines were employed, and ceramide metabolism was characterized using [3H]palmitic acid as lipid precursor. RESULTS: Exposure of cells to Taxol resulted in enhanced formation of [3H]ceramide. Ceramide increased nearly 2-fold in MDA-MB-468 cells exposed to 50 nM Taxol, and more than 2.5-fold in MCF-7 cells exposed to 1.0 microM Taxol. These concentrations mirrored the EC50 (amount of drug eliciting 50% cell kill) for Taxol in the two cell lines. Use of cell-permeable C6-ceramide as a medium supplement revealed that MDA-MB-468 cells were 20-fold more sensitive to ceramide than MCF-7 cells (P < 0.001). Ceramide was generated as early as 6 h after exposure to Taxol in MDA-MB-468 cells, whereas the earliest signs of apoptosis were detected 12 h after treatment, and by 24 h the apoptotic index was six times that of untreated cells. Both fumonisin B1, a ceramide synthase inhibitor, and L-cycloserine, a serine palmitoyltransferase inhibitor, blocked Taxol-induced ceramide generation, whilst sphingomyelin levels remained unchanged, indicating a de novo pathway of ceramide formation. L-Cycloserine reduced Taxol-induced apoptosis by 30% in MDA-MB-468 cells and totally blocked Taxol-induced apoptosis in MCF-7 cells. CONCLUSIONS: These results suggest that Taxol-induced apoptosis is, in part, attributable to ceramide and sphingoid bases. This is of relevance to drug mechanism studies, as ceramide is a known messenger of apoptosis. Clinical use of Taxol with ceramide-enhancing agents may maximize cytotoxic potential.     

Ceramide involvement in homocamptothecin- and camptothecin-induced cytotoxicity and apoptosis in colon HT29 cells

Topoisomerase I inhibitors of the camptothecin (CPT) family have emerged as potent clinical chemotherapeutic agents in first-line treatment of solid colorectal cancer and in second-line for 5-fluorouracil resistant patients. CPT and homocamptothecin (hCPT), derivative with enhanced lactone stability, induced growth inhibition in HT29 cells via p53-independent apoptosis. hCPT- and CPT-induced apoptosis was dependent on caspase-3 but not caspase-1. We report here substantial evidence that ceramide, resulting from de novo pathway or catabolism modulation, acted as a second messenger of these antitumor drugs in HT29 cells and leads to the activation of caspase-3. In addition, hCPT and CPT may favor ceramide signaling by disturbing sites of synthesis (Golgi) and trafficking of glucosylceramide from Golgi to lipid droplets. This work contributes to the understanding of the mechanism of action of CPTs, and suggests that inhibitors of glycosylation or activators of de novo metabolism could be of clinical interest in enhancing the effects of CPTs.     

Combining nanoliposomal ceramide with sorafenib synergistically inhibits melanoma and breast cancer cell survival to decrease tumor development.

PURPOSE: Deregulation of phosphatidylinositol 3-kinase/Akt and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathways occurs in melanoma and breast cancer, deregulating normal cellular apoptosis and proliferation. Therapeutic cocktails simultaneously targeting these pathways could promote synergistically acting tumor inhibition. However, agents with manageable toxicity and mechanistic basis for synergy need identification. The purpose of this study is to evaluate the preclinical therapeutic efficacy and associated toxicity of combining sorafenib with nanoliposomal ceramide. EXPERIMENTAL DESIGN: Effects of sorafenib and nanoliposomal ceramide as single and combinatorial agents were examined on cultured cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assays and CalcuSyn software used to assess synergistic or additive inhibition. Western blotting measured cooperative effects on signaling pathways. Rates of proliferation, apoptosis, and angiogenesis were measured in size- and time-matched tumors to identify mechanistic basis for inhibition. Toxicity was evaluated measuring animal weight, blood toxicity parameters, and changes in liver histology. RESULTS: Sorafenib and nanoliposomal ceramide synergistically inhibited cultured cells by cooperatively targeting mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling. A 1- to 2-fold increase in cellular apoptosis and 3- to 4-fold decrease in cellular proliferation were observed following combination treatment compared with single agents, which caused synergistically acting inhibition. In vivo, an approximately 30% increase in tumor inhibition compared with sorafenib treatment alone and an approximately 58% reduction in tumor size compared with nanoliposomal ceramide monotherapy occurred by doubling apoptosis rates with negligible systemic toxicity. CONCLUSIONS: This study shows that nanoliposomal ceramide enhances effectiveness of sorafenib causing synergistic inhibition. Thus, a foundation is established for clinical trials evaluating the efficacy of combining sorafenib with nanoliposomal ceramide for treatment of cancers.     

Arsenic trioxide prevents radiation-enhanced tumor invasiveness and inhibits matrix metalloproteinase-9 through downregulation of nuclear factor kappaB

Arsenic trioxide (ATO) has been implicated as a promising anticancer agent by inhibiting cell growth and inducing apoptosis in certain types of cancer cells. This study explored the antimetastasis property of arsenic, drew potential link between arsenic use and radiotherapy, and uncovered the specific mechanisms underlying these remarkable responses. Using gelatin invasion assay and intravasation assay, we report the novel finding that low-dose ATO (1 muM) reduced the intrinsic migration ability of HeLa cells and significantly inhibited radiation-promoted tumor invasive potential of CaSki cells without inducing apoptotic cell death. Using the murine Lewis lung carcinoma model, the control animals and ATO treatment animals (1 mg/kg i.p., twice weekly) displayed similar in vivo growth kinetics, whereas the radiation (30 Gy in one fraction) and concurrent treatment groups showed considerable growth inhibition. Importantly, although concurrent treatment did not enhance the effectiveness of radiation therapy to the primary tumor, further examination of the lungs revealed that all animals succumbed to radiation-accelerated lung metastases could be effectively treated by combination of ATO and radiation. Radiation-induced matrix metalloproteinase-9 (MMP-9) expression was significantly inhibited by ATO using sequential analysis of the expression of MMPs in xenografts. Supporting this observation, ATO directly downregulates radiation-induced MMP-9 mRNA expression by inhibiting nuclear factor kappaB activity in human cervical cancer cells. In sum, concurrent arsenic-radiation therapy not only achieves local tumor control but also inhibits distant metastasis. Experimental results of this study highlight a novel strategy in cancer treatment.