Combination of multifunctional ursolic acid with kinase inhibitors for anti-cancer drug carrier vesicles.

A sterically stabilized unilamellar nanocarrier vesicle (SSV) system containing dipalmitoylphosphatidylcholine, cholesterol, ursolic acid and PEGylated phospholipid has been developed by exploiting the structural advantages of ursolic acid: by spontaneously attaching to the lipid head groups, it induces curvature at the outer side of the bilayers, allowing the preparation of size-limited vesicles without extrusion. Ursolic acid (UA) also interacts with the PEG chains, supporting steric stabilization even when the amount of PEGylated phospholipid is reduced. Using fluorescence immunohistochemistry, vesicles containing ursolic acid (UA-SSVs) were found to accumulate in the tumor in 3 h on xenografted mouse, suggesting the potential use of these vesicles for passive tumor targeting. Further on, mono- and combination therapy with UA and six different kinase inhibitors (crizotinib, erlotinib, foretinib, gefitinib, refametinib, trametinib) was tested on seven cancer cell-lines. In most combinations synergism was observed, in the case of trametinib even at very low concentration (0.001 µM), which targets the MAPK pathway most often activated in human cancers. The coupled intercalation of UA and trametinib (2:1 molar ratio) into vesicles causes further structural advantageous molecular interactions, promoting the formation of small vesicles. The high drug:lipid molar ratio (~0.5) in the novel type of co-delivery vesicles enables their direct medical application, possibly also overcoming the multidrug resistance effect.

Combining immunotherapy with an epidrug in squamous cell carcinomas of different locations: rationale and design of the PEVO basket trial.

Squamous cell carcinomas (SCCs) are among the most frequent solid tumors in humans. SCCs, related or not to the human papillomavirus, share common molecular features. Immunotherapies, and specifically immune checkpoint inhibitors, have been shown to improve overall survival in multiple cancer types, including SCCs. However, only a minority of patients experience a durable response with immunotherapy. Epigenetic modulation plays a major role in escaping tumor immunosurveillance and confers resistance to immune checkpoint inhibitors. Preclinical evidence suggests that modulating the epigenome might improve the efficacy of immunotherapy. We herein review the preclinical and the clinical rationale for combining immunotherapy with an epidrug, and detail the design of PEVOsq, a basket clinical trial combining pembrolizumab with vorinostat, a histone deacetylase inhibitor, in patients with SCCs of different locations. Sequential blood and tumor sampling will be collected in order to identify predictive and pharmacodynamics biomarkers of efficacy of the combination. We also present how clinical and biological data will be managed with the aim to enable the development of a prospective integrative platform to allow secure and controlled access to the project data as well as further exploitations.

Identification of a methylation hotspot in the death receptor Fas/CD95 in bladder cancer.

We characterized Fas immunoreactivity, functionality and its role in the response to mitomycin-C (MMC) chemotherapy in vitro in cell lines and in vivo in bladder washings from 23 transitional cell carcinoma of the bladder (TCCB) patients, harvested prior to and during MMC intravesical treatment. Having established the importance of functional Fas, we investigated the methylation and exon 9 mutation as mechanisms of Fas silencing in TCCB. For the first time, we report p53 up-regulation in 9/14 and Fas up-regulation in 7/9 TCCB patients during intravesical MMC treatment. Fas immunoreactivity was strong in the TCCB cell line T24 and in 17/20 (85%) tumor samples from patients with advanced TCCB. T24 and HT1376 cells were resistant to MMC and recombinant Fas ligand, whilst RT4 cells were responsive to Fas ligand and MMC. Using RT4 cells as a model, siRNA targeting p53 significantly reduced MMC-induced p53 and Fas up-regulation and stable DN-FADD transfection decreased MMC-induced apoptosis, suggesting that functional Fas enhances chemotherapy responses in a p53-dependent manner. In HT1376 cells, 5-aza-2-deoxycytidine (12 µM) induced Fas immunoreactivity and reversed methylation at CpG site -548 within the Fas promoter. This site was methylated in 13/24 (54%) TCCB patient samples assessed using Methylation-Specific Polymerase Chain Reaction. There was no methylation at either the p53 enhancer region within the first intron or at the SP-1 binding region in the promoter and no mutation within exon 9 in tumor DNA extracted from 38 patients. Methylation at CpG site -548 is a potential target for demethylating drugs.

A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells.

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma.

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

Shared pathways: death receptors and cytotoxic drugs in cancer therapy.

Death ligands (TNF, FasL, TRAIL) and their respective death receptor signaling pathways can be used to induce tumor cells to undergo apoptosis. Chemotherapeutic drugs can induce apoptosis and the upregulation of death ligands or their receptors. Downstream events following cytotoxic stress-induced DNA damage and the signaling pathways that lead to the induction of apoptosis may be either dependent or independent of death receptor signaling. The involvement of the Fas signaling pathway in chemotherapy-induced apoptosis has been the most extensively studied, with the current emergence of information on the TRAIL signaling pathway. Fas-mediated and chemotherapy-induced apoptosis can converge at the level of the receptor, FasL, DISC formation, activation of the initiator caspase-8, at the level of the mitochondria, or at the level of downstream effector caspase activation. Convergence is influenced by the specific form of DNA damage, the cellular environment, and the specific pathway(s) by which death receptor-mediated or drug-mediated apoptosis are induced. This review discusses the different levels of interaction between signaling pathways in the different forms of cell death.

TGF beta 1 induces caspase-dependent but death-receptor independent apoptosis in lymphoid cells.

Transforming growth factor beta 1 (TGF beta 1) is an antiproliferative and proapoptotic cytokine for normal B-cells, however many B-cell lymphomas have lost their response to TGF beta 1. The aim of this study was to identify the sequence of events in apoptosis induced by TGF beta 1 in an EBV negative, human B-cell lymphoma line (HT58). The proportion of apoptotic cells increased gradually (up to 72 hr) at an optimal dose range of 0.5-1.0 ng/ml. The induced cell death required the action of downstream caspases. Caspase activation was accompanied by an increase in the permeability of mitochondrial membranes, but there was no change in the expression of certain members of Bcl-2 family (Bcl-2, Bax, Bcl-XL). Similarly, none of the death receptors or ligands were involved in apoptosis induction. Further study will include the participation of TGF beta 1 target genes in the pore formation of mitochondrial membranes and/or the elimination of a putative survival signal.

p53 dependence of Fas induction and acute apoptosis in response to 5-fluorouracil-leucovorin in human colon carcinoma cell lines.

We examined the patterns of induction of apoptosis, Fas expression, and the influence of the status of the p53 tumor suppressor gene, in response to treatment of human colon carcinoma cell lines to 5-fluorouracil (FUra) combined with leucovorin (LV) under conditions of both DNA-directed (HT29, VRC5/c1, and RKO) and RNA-directed (HCT8 and HCT116) cytotoxicity. Acute apoptosis was induced in cell lines expressing wtp53 (RKO, HCT8, and HCT116), independent of the mechanism of FUra action. In HT29 cells that expressed mp53, apoptosis was a delayed event. Cell lines undergoing DNA-directed FUra cytotoxicity demonstrated marked accumulation of cells in S-phase (HT29 and RKO), whereas those lines undergoing RNA-directed cytotoxicity (HCT8 and HCT116) demonstrated marked cell cycle phase arrest in G2-M, both reversible by dThd. dThd partially protected HCT8 and HCT116 cells from FUra-LV-induced apoptosis but had no influence on FUra-LV-induced loss in clonogenic survival. In cells expressing wtp53, the Fas death receptor was induced in response to FUra-LV treatment. FUra-LV sensitized RKO cells to the anti-Fas monoclonal antibody CH-11 that was completely reversed by dThd, demonstrating the involvement of DNA damage in FUra-LV-induced, Fas-dependent sensitization to CH-11. In contrast, FUra-LV sensitized HCT116 cells to CH-11-induced apoptosis, which was not dThd reversible. Transduction of HT29 cells with Ad-wtp53 induced elevated Fas expression and sensitized the cells to FUra-LV-induced apoptosis. Data indicate that the presence of a wtp53 gene determines FUra-LV-induced Fas expression, the kinetics of FUra-LV-induced apoptosis and not the extent of apoptosis induced, both being independent of the mechanism of FUra action. Therefore, in colon carcinomas that express wtp53, the approach to sensitize tumors to Fas-mediated apoptosis may be further enhanced from the effect of FUra-LV in elevating Fas expression in a p53-dependent manner.

Pediatric rhabdomyosarcoma cell lines are resistant to Fas-induced apoptosis and highly sensitive to TRAIL-induced apoptosis.

Seven pediatric rhabdomyosarcoma (RMS) cell lines were resistant to the induction of apoptosis via the Fas death receptor. In contrast, four of seven lines (RD, Rh1, Rh18, and Rh30) were highly sensitive to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). TRAIL induced apoptosis within 4 h and also reduced clonogenic survival, both reversible by caspase inhibitors. DR5 (but not DR4) was expressed at high level in all cell lines. Expression of the decoy receptors DcR1 and DcR2 did not correlate with TRAIL sensitivity. All RMS lines expressed the adapter molecule FADD, and six of seven expressed procaspase-8. Expression of the inhibitory proteins c-FLIPL and c-FLIPs was high in three TRAIL-sensitive (RD, Rh1, and Rh30) and two TRAIL-resistant (Rh28 and Rh41) lines. All RMS lines expressed Bid and procaspases-3, -6, -7, and -9. Procaspases-8 and -10 were highest in TRAIL-sensitive RMS (RD, Rh1, and Rh30), and procaspase-10 was not expressed in Rh18, Rh36, or Rh41. TRAIL induced loss of mitochondrial membrane potential in TRAIL-sensitive Rh1 but not in TRAIL-resistant Rh41 cells. There was no correlation between expression of members of the Bcl-2 family (Bcl-2, Bcl-xL, Bax, and Bak) and TRAIL sensitivity. TRAIL-sensitive Rh18 expressed procaspase-8 in the absence of procaspase-10 and c-FLIP, and procaspase-10 was not detected in TRAIL-resistant Rh41 in the presence of procaspase-8 and c-FLIP. Data suggest that caspase-8 may be sufficient to deliver the TRAIL-induced apoptotic signal in the absence of both caspase-10 and c-FLIP (Rh18) but not in the presence of c-FLIP (Rh41). In RD, Rh1, and Rh30, the presence of c-FLIP may require amplification of the apoptotic signal via caspase-10.

Fas-dependent and -independent mechanisms of cell death following DNA damage in human colon carcinoma cells.

In thymidylate synthase-deficient (TS-) colon carcinoma cells, thymineless death is mediated via Fas/Fas ligand (FasL) interactions after thymidine deprivation and inhibited by the Fas-inhibitory monoclonal antibody NOK-1. The objective of the study was to elucidate whether other modes of DNA damage induced by doxorubicin, topotecan, and etoposide (VP-16) could elicit a similar cytotoxic response in TS- cells by signaling via the Fas death receptor. After a 72-h drug exposure, a loss in clonogenic survival that was not prevented by NOK-1 was induced by each agent in the absence of acute apoptosis, yielding IC50 values of 5 (doxorubicin), 10 (topotecan), and 150 nM (VP-16). Furthermore, TS- cell clones selected for resistance to Fas-mediated apoptosis (CH-11) were cross-resistant to the induction of thymineless death after thymidine deprivation but were not cross-resistant to doxorubicin, topotecan, or VP-16. A close correlation was found between acute induction of apoptosis (24 h) and up-regulated expression of FasL at high concentrations of each of the three agents (0.3-3 microM doxorubicin, 0.3-3 microM topotecan, and 10-90 microM VP-16), which was caspase dependent but Fas independent. At all drug concentrations, cell cycle distribution analyses demonstrated marked accumulation of cells in the G2-M phase. At nanomolar drug concentrations, prolonged arrest of TS- cells in G2-M phase resulted in the up-regulation of FasL expression and the delayed appearance of apoptotic cells (6 days), which could also be inhibited by the general caspase inhibitor Z-VAD-FMK, but not by NOK-1 or Fas-Fc. In clonogenic assays, Z-VAD-FMK did not rescue cells treated with VP-16 in contrast to treatment with CH-11 or thymineless stress, suggesting an irreversible commitment to cell death in G2-M phase. Expression of FasL at all drug concentrations appeared to be unrelated to the mechanism of drug-induced apoptosis. This was in contrast to the Fas-dependent regulation of thymineless death, which could be inhibited by blocking Fas/FasL interactions.

Regulation of FasL by NF-kappaB and AP-1 in Fas-dependent thymineless death of human colon carcinoma cells.

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.

Interaction of cytocidal drugs and the inhibition of caspase-3 by 3-nitrosobenzamide.

The effect of 3-nitrosobenzamide (NOBA) on the etoposide, staurosporine and dexamethason induced rapid (4-6 hr), caspase-dependent apoptosis was investigated in thymocytes and lymphoma cells by flow cytometric assay of DNA fragmentation. When NOBA (ED(50) = 4 microM) was added to these cell systems, the rapid onset of apoptosis was prevented. Such apparent protection by NOBA was related to the inactivation of caspase-3, by s-nitrosylation of 1.3 mol -SH per enzyme molecule out of 7 -SH groups. Since NOBA by itself induces DNA fragmentation within 18 hr in lymphoma cells, our results indicate that at least two active cell death pathways exist with apparent dissimilar kinetics and molecular mechanisms.

A specifically radiolabeled somatostatin analog with strong antitumor activity induces apoptosis and accumulates in the cytosol and the nucleus of HT29 human colon carcinoma cells.

The new heptapeptide somatostatin analog TT-232 decreases proliferation of HT-29 human colon carcinoma cells in vitro by reducing mitotic and increasing apoptotic activity. We have synthesized and characterized a specifically tritium labeled 3H-Tyr3-TT-232 (30 Ci/mmol) to investigate the effect and the fate of this antitumor peptide on human colon tumor cells. 3H-labeled TT-232 could be detected on the cell surface, on cytoplasmic membranes and also in the nucleus of HT-29 cells, 1-6 h after the administration of 0.5 and 50 microg/mL [3H]TT-232. Binding and internalization of TT-232 to human colon tumor cells at a relatively high dose provide further evidence for the existence of low-affinity somatostatin receptors in such cells, which might mediate the apoptosis-inducing effect. Our data suggest the possible use of TT-232 in the treatment of human colon tumors.

A Fas-dependent component in 5-fluorouracil/leucovorin-induced cytotoxicity in colon carcinoma cells.

We have shown previously (J. A. Houghton et al., Proc. Natl. Acad. Sci. USA, 94: 8144-8149, 1997) that thymineless death in thymidylate synthase-deficient (TS-) colon carcinoma cells is mediated via Fas/FasL interactions after deoxythymidine (dThd) deprivation, and that Fas-dependent sensitivity of human colon carcinoma cell lines may be dependent upon the level of Fas expressed. The objective of this study was to elucidate whether a Fas-dependent component exists in 5-fluorouracil (FUra)/leucovorin (LV)-induced cytotoxicity of colon carcinoma cells, and whether this may be potentiated by IFN-gamma-induced elevation in Fas expression, using the HT29 cell line as a model. The cytotoxic activity of FUra/LV was inhibited by dThd in HT29 cells and also, in part, by NOK-1+NOK-2 MoAbs that prevent Fas/FasL interactions. FUra/LV-induced cytotoxicity was significantly potentiated by IFN-gamma, reversed by exposure to NOK-1+NOK-2 antibodies, and correlated with a 4-fold induction of Fas expression in the presence of IFN-gamma and significant elevation in expression of FasL. Using five additional human colon carcinoma cell lines, FUra/LV-induced cytotoxicity was dThd-dependent in GC3/c1, VRC5/c1, and Caco2 but not in HCT8 or HCT116 cells. Like HT29 cells, this cytotoxicity was potentiated by IFN-gamma in GC3/c1 and VRC5/c1 but not in Caco2, which fails to express Fas, nor in HCT8 and HCT116, in which no dThd-dependent FUra-induced cytotoxicity was demonstrated. Data suggest that a Fas-dependent component, potentiated by IFN-gamma, exists in FUra/LV-induced cytotoxicity but requires FUra/LV-induced DNA damage for IFN-gamma-induced potentiation to occur.

BCNU is a caspase-mediated inhibitor of drug-induced apoptosis.

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea], a bifunctional (alkylating/carbamoylating) anticancer agent, in noncytotoxic doses (12-50 microM) inhibited drug-induced apoptosis in HT58 human lymphoma cells exposed to etoposide (ETO; 50 microM) as well as in mouse thymocytes exposed to dexamethasone (5 microg/ml) in vitro in 4-h cultures. The cytoplasmic extracts of ETO-treated HT58 cells cleaved both purified poly(ADP-ribose)polymerase and Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin fluorogenic caspase substrate, indicating the presence of active caspases, and these effects were inhibited by BCNU concentration dependently. The carbamoylating decomposite, 2-chloroethyl-isocyanate (6-25 microM), also decreased ETO-induced apoptosis in HT58 cells in vitro and their caspase 3-like activity ex vivo, whereas N-(2-chloroethyl)-N-nitrosocarbamoyl-valinamide, an alkylating and mainly intramolecularly carbamoylating nitrosourea derivative (400 microM), did not influence these phenomena. Furthermore, the activity of recombinant caspase 3 was also strongly inhibited by BCNU and 2-chloroethyl-isocyanate. These results indicate that BCNU, via its carbamoylating capacity, can inactivate cysteine protease(s) essential for ETO-induced apoptosis. This apoptosis-modulating property of BCNU, in turn, may influence the efficacy of chemotherapeutic protocols in the treatment of cancer.

Modulation of apoptosis signaling in etoposide-treated lymphoma cells.

Signals of etoposide (ETO) induced apoptosis were studied in a human (B) lymphoma cell line, HT58. Morphology and DNA fragmentation assays proved the appearance of apoptosis after a short ETO treatment (4 hours). Modulation of signal components of this apoptotic pathway resulted the following a) phorbol ester (PMA) or heat shock inhibited apoptosis, which was prevented by staurosporine b) 3-amino-benzamide, a potent poly(ADP-ribose)polymerase inhibitor, had no significant effect; c) cysteine reactive compounds, such as iodoacetamide and phenylarsine oxide, as well as protease inhibitor TPCK were very active inhibitors of apoptosis; d) protein synthesis inhibitor, cycloheximide, potentiated cell death; e) the ETO-induced p53 protein overexpression had neither enhancing nor protecting effect on the apoptotic process. In conclusion, in the majority of HT58 lymphoma cells the apoptotic machinery is "primed" (the components are already expressed) and ETO-induced apoptosis is regulated by STA sensitive phosphorylation and proteolysis by cystein proteases, but not affected by ADP-ribozylation or p53.

Apoptotic cell death induced by inhibitors of energy conservation--Bcl-2 inhibits apoptosis downstream of a fall of ATP level.

Energy charge controls intermediary metabolism and cellular regulation. Here we show that inhibition of energy conservation at the level of glucose uptake, glycolysis, citric acid cycle, and oxidative phosphorylation induces cell death, leading to fragmentation of DNA into an oligonucleosomal ladder and morphological changes typical for apoptosis. Bcl-2, the prototype of oncogenes that suppress cell death, efficiently inhibits apoptosis induced by metabolic inhibitors. Bcl-2 does not antagonize the inhibitory potential of mitochondrial inhibitors, and cannot prevent or delay the decrease of the cellular ATP level subsequent to metabolic inhibition. Thus, we propose that Bcl-2 blocks apoptosis at a point downstream of the collapse of the cellular-energy homeostasis.