FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

FTY720 produces caspase-independent cell death of acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL), the most common form of childhood cancer, usually responds to chemotherapy but patients who develop disease relapse have a poor prognosis. New agents to treat ALL are urgently required. FTY720 is an immunosuppressive drug that has promising in vitro activity in a number of malignancies, with the proposed mechanism being the reactivation of the protein serine/threonine phosphatase, PP2A. FTY720 reduced the proliferation and viability of Ph⁺ and Ph^- ALL cell lines and patient samples with IC₅₀ values for viability between 5.3 to 7.9 μM. Cell death was caspase-independent with negligible activation of caspase-3 and no inhibition of FTY720-induced cell death by the caspase inhibitor Z-VAD-FMK. The cytotoxic effects of FTY720 were independent of PP2A reactivation as determined by the lack of effect of the PP2A inhibitor okadaic acid. Features of autophagy, including autophagosomes, LC3II expression and increased autophagic flux, were induced by FTY720. However the phosphorylated form of FTY720 (FTY720-P) similarly induced autophagy but not cell death. This suggests that autophagy is prosurvival in this setting, a finding supported by protection from cell death induced by the cytotoxic agent vincristine. FTY720 also induced reactive oxygen species (ROS) and the antioxidant N-acetyl-cysteine (NAC) partially reversed the cytotoxic effects, demonstrating a role for ROS generation in the cell death mechanism. FTY720 is an active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase-independent mechanism of cell death with the occurrence of autophagy and necrosis.     

Artesunate induces necrotic cell death in schwannoma cells

Established as a potent anti-malaria medicine, artemisinin-based drugs have been suggested to have anti-tumour activity in some cancers. Although the mechanism is poorly understood, it has been suggested that artemisinin induces apoptotic cell death. Here, we show that the artemisinin analogue artesunate (ART) effectively induces cell death in RT4 schwannoma cells and human primary schwannoma cells. Interestingly, our data indicate for first time that the cell death induced by ART is largely dependent on necroptosis. ART appears to inhibit autophagy, which may also contribute to the cell death. Our data in human schwannoma cells show that ART can be combined with the autophagy inhibitor chloroquine (CQ) to potentiate the cell death. Thus, this study suggests that artemisinin-based drugs may be used in certain tumours where cells are necroptosis competent, and the drugs may act in synergy with apoptosis inducers or autophagy inhibitors to enhance their anti-tumour activity.Artemisinin, a sesquiterpene lactone isolated from the Chinese herb Artemisia annua L., has profound activity against malaria.¹ Artemisinin contains an endoperoxide moiety that reacts with iron to produce toxic reactive oxygen species (ROS). When malaria parasite (Plasmodia) consumes iron-rich haemoglobin within its acidic food vacuole in erythrocytes, the exposure of artemisinin to haem-derived iron results in lethal ROS production that exerts fatal toxicity to the parasite.² Therefore, artemisinin, its water-soluble derivative artesunate (ART) and other analogues are potent in killing malarial parasites.^1,3Cancer cells contain substantial free iron, resulting from their higher-rate iron uptake via transferrin receptors compared with normal cells. Therefore, artemisinin-based drugs such as ART possess selective toxicity to cancer cells.^{4, 5, 6} Importantly, the pharmacokinetics and tolerance of ART as an anti-malarial drug have been well documented, with clinical studies showing excellent safety. Collectively, these properties make artemisinin-based compounds attractive drug candidates for cancer chemotherapy. Artemisinin and ART have been shown to induce cell death in multiple cancer cells, including colon, breast, ovarian, prostate,⁷ pancreatic⁸ and leukaemia⁹ cancer cells. Preliminary in vivo experiments also indicate the therapeutic potential for these drugs as anti-cancer treatments. In animal models, artemisinin or ART has shown promising results in Kaposi Sarcoma,¹⁰ pancreatic cancer¹¹ and hepatoma,¹² while compassionate use of ART in uveal melanoma patients fortifies standard chemotherapy potential for the patients.¹³ Currently, ART is on clinical trial for breast cancer treatment (ClinicalTrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT00764036","term_id":"NCT00764036"}}NCT00764036).Programmed cell death (PCD) is one of the critical terminal paths for the cells of metazoans. Among PCD, apoptosis has been well studied and it is known that caspase activation is essential in this process.¹⁴ In addition to apoptosis, necroptosis is another form of PCD. The RIP1-RIP3 complex highlights the signals that regulate necroptosis.^{15, 16, 17} Artemisinin derivatives, mostly ART, have been suggested to lead to apoptosis via ROS production in cancer cells. Efforts have been focused on ROS-mediated mitochondrial apoptosis,^9,18,19 and DNA damage²⁰ in cancer cells. Recent data suggest that artemisinin and its derivatives may induce cell death or inhibit proliferation through diverse mechanisms in different cell types. Artemisinin or its analogues were shown to inhibit cell proliferation in multiple cancer cells by regulating cell-cycle arrest^{21, 22, 23} or inducing apoptosis.^24,25 Nevertheless, the detailed molecular mechanisms underlying artemisinin or ART-induced cell death are poorly understood, thus need to be further addressed.Neurofibromatosis 2 (NF2) is caused by the loss of NF2 gene encoding Merlin protein. NF2 gene mutations cause the low grade tumour syndrome, composed of schwannomas, meningiomas and ependymomas.²⁶ All spontaneous schwannomas, the majority of meningiomas and a third of ependymomas are caused by NF2 gene mutations. Notably, approximately 10% of intracranial tumours are schwannomas.²⁷ Interestingly, NF2 gene mutations are also found in a variety of cancers, including breast cancer and mesothelioma.^{28, 29, 30} The low grade tumours caused by NF2 gene mutations do not respond well to current cancer drugs and therapy is restricted to surgery and radiosurgery.²⁶ Therefore, there is a need for drug treatment of the diseases. Here, we show that ART sufficiently induced schwannoma cell death in both RT4 cell line and human primary cells. Importantly, we show, for the first time, that ART-induced cell death is largely dependent on necroptosis. Our data suggest that ART has great potential in schwannoma chemotherapy, especially when used in synergy with an apoptosis-inducing drug and/or an autophagy-inhibitory drug.     

FTY720 produces caspase-independent cell death of acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL), the most common form of childhood cancer, usually responds to chemotherapy but patients who develop disease relapse have a poor prognosis. New agents to treat ALL are urgently required. FTY720 is an immunosuppressive drug that has promising in vitro activity in a number of malignancies, with the proposed mechanism being the reactivation of the protein serine/threonine phosphatase, PP2A. FTY720 reduced the proliferation and viability of Ph(+) and Ph(-) ALL cell lines and patient samples with IC 50 values for viability between 5.3 to 7.9 μM. Cell death was caspase-independent with negligible activation of caspase-3 and no inhibition of FTY720-induced cell death by the caspase inhibitor Z-VAD-FMK. The cytotoxic effects of FTY720 were independent of PP2A reactivation as determined by the lack of effect of the PP2A inhibitor okadaic acid. Features of autophagy, including autophagosomes, LC3II expression and increased autophagic flux, were induced by FTY720. However the phosphorylated form of FTY720 (FTY720-P) similarly induced autophagy but not cell death. This suggests that autophagy is prosurvival in this setting, a finding supported by protection from cell death induced by the cytotoxic agent vincristine. FTY720 also induced reactive oxygen species (ROS) and the antioxidant N-acetyl-cysteine (NAC) partially reversed the cytotoxic effects, demonstrating a role for ROS generation in the cell death mechanism. FTY720 is an active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase-independent mechanism of cell death with the occurrence of autophagy and necrosis.     

Abortive autophagy induces endoplasmic reticulum stress and cell death in cancer cells

Autophagic cell death or abortive autophagy has been proposed to eliminate damaged as well as cancer cells, but there remains a critical gap in our knowledge in how this process is regulated. The goal of this study was to identify modulators of the autophagic cell death pathway and elucidate their effects on cellular signaling and function. The result of our siRNA library screenings show that an intact coatomer complex I (COPI) is obligatory for productive autophagy. Depletion of COPI complex members decreased cell survival and impaired productive autophagy which preceded endoplasmic reticulum stress. Further, abortive autophagy provoked by COPI depletion significantly altered growth factor signaling in multiple cancer cell lines. Finally, we show that COPI complex members are overexpressed in an array of cancer cell lines and several types of cancer tissues as compared to normal cell lines or tissues. In cancer tissues, overexpression of COPI members is associated with poor prognosis. Our results demonstrate that the coatomer complex is essential for productive autophagy and cellular survival, and thus inhibition of COPI members may promote cell death of cancer cells when apoptosis is compromised.     

FTY720 induces cell cycle arrest and apoptosis of rat glomerular mesangial cells

The present study aimed to examine the effect of FTY720, a new immunosuppressive agent, on the proliferation and apoptosis of glomerular mesangial cells (GMC), and investigate the underlying mechanisms. Cultured rat GMC were treated by FTY720, and the cell viability, apoptosis and cell cycle progression were examined. Furthermore, cell cycle related gene expression profile was analyzed by cDNA microarray, and the protein expression of cell cycle related genes as well as Bax and Bcl-2 were examined by Western blot. The results showed that FTY720 inhibited GMC proliferation and induced apoptosis of GMC in a dose- and time-dependent manner, and induced G(1) phase cell cycle arrest in GMC in a dose-dependent manner as well. cDNA microarray analysis revealed that FTY720 regulated the expression of cell cycle-related gene. Western blot analysis showed that FTY720 induced the downregulation of cyclin D1, cyclin E, CDK2, CDK4, Bcl-2 and E2F1 and the upregulation of Kip1/p27, Cip1/p21, Bax and Rb in GMC in a dose-dependent manner. These results demonstrated that FTY720 could inhibit the proliferation of GMC through inducing cell cycle arrest and apoptosis, probably via the regulation of the expression of cell cycle-related genes and Bax/Bcl-2.     

Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis

Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.     

Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells

Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03?±?3.12, 22.19?±?1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Guttiferone K induces autophagy and sensitizes cancer cells to nutrient stress-induced cell death

BackgroundMedicinal plants have long been an excellent source of pharmaceutical agents. Autophagy, a catabolic degradation process through lysosomes, plays an important role in tumorigenesis and cancer therapy.PurposeThrough a screen designed to identify autophagic regulators from a library of natural compounds, we found that Guttiferone K (GUTK) can activate autophagy in several cancer cell lines. The objective of this study is to investigate the mechanism by which GUTK sensitizes cancer cells to cell death in nutrient starvation condition.MethodsCell death analysis was performed by propidium iodide staining with flow cytometry or Annexin V-FITC/PI staining assay. DCFH-DA staining was used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Cell viability was measured by MTT assay.ResultsExposure to GUTK was observed to markedly induce GFP-LC3 puncta formation and activate the accumulation of LC3-II and the degradation of p62 in HeLa cells, suggesting that GUTK is an autophagy inducer. Importantly, hydroxychloroquine, an autophagy inhibitor, was found to significantly prevent GUTK-induced cell death in nutrient starvation conditions, suggesting that the cell death observed is largely dependent on autophagy. We further provide evidence that GUTK inhibits Akt phosphorylation, thereby inhibiting the mTOR pathway in cancer cells during nutrient starvation. In addition, GUTK causes the accumulation of reactive oxygen species (ROS) and the phosphorylation of JNK in EBSS, which may mediate both autophagy and apoptosis.ConclusionThese data indicate that GUTK sensitizes cancer cells to nutrient stress-induced cell death though Akt/mTOR dependent autophagy pathway.     

FTY720-induced blockage of autophagy enhances anticancer efficacy of milatuzumab in mantle cell lymphoma

Inhibition of the autophagic pathway has recently revealed promising results in increasing pro-death activity of multiple cancer therapeutics. Here, we discuss our findings regarding the autophagy-blocking and anti-neoplastic effects of a synthetic sphingosine analog, FTY720, in mantle cell lymphoma (MCL). We also emphasize how FTY720 enhances the pro-death activity of the fully humanized monoclonal antibody milatuzumab by inhibiting the autophagy-lysosome dependent degradation of its therapeutic target, CD74. Our results provide justification for further evaluation of FTY720 and milatuzumab as a combination therapy for this aggressive B-cell malignancy.     

Ursolic acid induces cell death and modulates autophagy through JNK pathway in apoptosis-resistant colorectal cancer cells

Colorectal carcinomas (CRCs) with P53 mutations have been shown to be resistant to chemotherapy with 5-fluorouracil (5-FU), the most widely used chemotherapeutic drug for CRC treatment. Autophagy is emerging as a promising therapeutic target for drug-resistant tumors. In the present study, we tested the effects of ursolic acid (UA), a natural triterpenoid, on cell death mechanisms and its effects in combination with 5-FU in the HCT15 p53 mutant apoptosis-resistant CRC cell line. The involvement of UA in autophagy and its in vivo efficacy were evaluated.Our data show that UA induces apoptosis independent of caspases in HCT15 cells and enhances 5-FU effects associated with an activation of c-jun N-terminal kinase (JNK). In this cell line, where this compound has a more pronounced effect on the induction of cell death compared to 5-FU, apoptosis corresponds only to a small percentage of the total cell death induced by UA. UA also modulated autophagy by inducing the accumulation of LC3 and p62 levels with involvement of JNK pathway, which indicates a contribution of autophagy on JNK-dependent induction of cell death by UA. By using nude mice xenografted with HCT15 cells, we verified that UA was also active in vivo decreasing tumor growth rate.In conclusion, this study shows UA's anticancer potential both in vitro and in vivo. Induction of cell death and modulation of autophagy in CRC-resistant cells were shown to involve JNK signaling.     

Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells

Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.     

A role of autophagy in Trypanosoma brucei cell death

The early branching eukaryote Trypanosoma brucei contains functional autophagy machinery that allows regulated degradation of its own cellular components. In this study, we examined the function of two Atg8 genes, TbAtg8.1 and TbAtg8.2, in starvation-induced autophagosome formation and cell death in procyclic T. brucei. Upon starvation, both TbAtg8.1 and TbAtg8.2 localize to punctate structures characteristic of autophagosomes as shown by fluorescence and electron microscopy, and wortmannin and chloroquine treatments. While TbAtg8.1 depletion has no detectable effects on TbAtg8.2 recruitment to autophagosomes, TbAtg8.2 depletion greatly reduced the autophagosome relocation of TbAtg8.1. Depletion of TbAtg8.1 and 8.2, individually or together, promote cell survival under starvation conditions. Taken together, these observations confirm the presence of an autophagy-related cell death pathway in T. brucei, where TbAtg8.1 and TbAtg8.2 play essential but distinct roles in autophagosome formation and cell death.     

A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells

Cancer develops when molecular pathways that control the fine balance between proliferation, differentiation, autophagy and cell death undergo genetic deregulation. The prospects for further substantial advances in the management of colorectal cancer reside in a systematic genetic and functional dissection of these pathways in tumor cells. In an effort to evaluate the impact of p38 signaling on colorectal cancer cell fate, we treated HT29, Caco2, Hct116, LS174T and SW480 cell lines with the inhibitor SB202190 specific for p38alpha/beta kinases. We report that p38alpha is required for colorectal cancer cell homeostasis as the inhibition of its kinase function by pharmacological blockade or genetic inactivation causes cell cycle arrest, autophagy and cell death in a cell type-specific manner. Deficiency of p38alpha activity induces a tissue-restricted upregulation of the GABARAP gene, an essential component of autophagic vacuoles and autophagosomes, whereas simultaneous inhibition of autophagy significantly increases cell death by triggering apoptosis. These data identify p38alpha as a central mediator of colorectal cancer cell homeostasis and establish a rationale for the evaluation of the pharmacological manipulation of the p38alpha pathway in the treatment of colorectal cancer.     

FTY720, a novel immunosuppressive agent, induces apoptosis in human glioma cells

FTY720, a metabolite from Isaria sinclairii, has been developed to be a potent immunosuppressive drug with induction of apoptosis in T cells and several cell lines. We investigated whether FTY720 induces apoptosis in human glioma cell lines, since they are relatively resistant to multiple apoptotic stimuli. In human glioma cells including T98G, FTY720 induced apoptosiswith ED50 between 1 to 10 microg/ml, while etoposidedid not induce apoptosis at the same doses. Among the caspase family proteases, mainly caspase-6 was activated during the apoptosis by FTY720 but not etoposide. In addition, FTY720 caused tyrosine dephosphorylation of FAK and did not activate a FAK-PI3-kinase survival pathway. This was confirmed also by the observation that orthovanadate prevented FTY720-induced dephosphorylation of FAK and inhibited FTY720-induced cell death. We assumed that FTY720 induced FAK dephosphorylation and cut off the FAK-PI3-kinase pathway resulting in the induction of apoptosis via caspase-6 activation in these glioma cells.     

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles’ heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.