In vitro and in vivo conditional sensitization of hepatocellular carcinoma cells to TNF-induced apoptosis by taxol.

High mortality among hepatocellular carcinoma (HCC) patients reflects both late diagnosis and low curability, due to pharmacoresistance. Taxol (TAX) is toxic for many human HCC-derived cell lines, yet its clinical efficacy on HCCs is poor. Combining TAX with other drugs appears a promising possibility to overcome such refractoriness. We analyzed whether combining tumor necrosis factor (TNF) with TAX would improve their toxicity. Human HCC-derived cell lines were treated with TAX or TNF, alone or combined. Apoptosis was assessed by morphology and flow-cytometry. Several pro- and anti-apoptotic molecules were evaluated by western blotting and/or enzymatic assay. After a 24 hour treatment, TNF was ineffective and TAX modestly cytotoxic, whereas HCC cells were conditionally sensitized to TNF by TAX. Indeed some relevant parameters were shifted to a prodeath setting: TNF-receptor 1 was increased, SOCS3, c-FLIP and pSTAT3 were markedly downregulated. These observations provide a significant clue to critically improve the drug susceptibility of HCC cells by combining 2 agents, TAX and TNF. The sequential application of TAX at a low dosage followed by TNF for only a short time triggered a strong apoptotic response. Of interest, prior TAX administration could also sensitize to TNF-induced apoptosis in the Yoshida AH-130 hepatoma transplanted in mice. Therefore, scrutinizing the possibility to develop similar combination drug regimens in suitable preclinical models seems highly advisable.

Intracellular free iron and acidic pathways mediate TNF-induced death of rat hepatoma cells.

Rat hepatoma HTC cells are intrinsically resistant to various apoptosis-inducing agents. Strategies to induce death in hepatoma cells are needed and the present experimental study was aimed to investigate the sensitivity of HTC cells to TNF and to clarify the mechanisms of action of this cytokine. Cells were treated with TNF and death mechanisms characterized employing an integration of morphological and biochemical techniques. HTC cells, sensitized to TNF toxicity with cycloheximide, died in a caspase-independent apoptosis-like manner. Although we found no evidence for a direct involvement of lysosomal cathepsins, bafilomycin A1 and ammonium chloride significantly attenuated TNF toxicity. Also desferrioxamine mesylate, an iron chelator, partly protected the cells from TNF, while a complete protection was afforded by combining ammonium chloride and iron chelator. Moreover, HTC were protected from TNF also by lipophylic antioxidants and diphenylene iodonium chloride, a NADPH oxidase inhibitor. These data depict a novel mechanism of TNF-mediated cytotoxicity in HTC cells, in which the endo-lysosomal compartment, NADPH oxidase and an iron-mediated pro-oxidant status contribute in determining a caspase-independent, apoptosis-like cell death.

Expression and posttranslational fate of cathepsin D in HT-29 tumor cells depend on their enterocytic differentiation state.

In the present work, we analyzed the variations in the expression and trafficking of cathepsin D (CD), a lysosomal endopeptidase, associated with the enterocytic differentiation of the human colon carcinoma HT-29 cell line. In spite of the fact that the abundance of CD mRNA was severalfold higher in undifferentiated HT-29 cells than in their enterocyte-like differentiated counterparts, the intracellular levels of CD activity and protein were found to be much higher in the latter. The kinetic of transport of newly synthesized proCD was different in the two cell populations: (a) full conversion of proCD into the lysosomal mature form required more than 24 h in differentiated cells, whereas it was almost complete within 8 h in undifferentiated HT-29 cells; and (b) the extracellular release of proCD was shown to occur more rapidly and to a higher degree in undifferentiated than in differentiated cells. Most of the secreted proCD contained phosphomannoses. Secretion of beta-hexosaminidase activity doubled, whereas that of CD activity was unchanged, upon vacuolar alkalinization with ammonium chloride or chloroquine. Inhibition of the lysosomal-autophagic degradative pathway resulted in the accumulation of proCD molecules in undifferentiated HT-29 cells. Altogether these data suggest that: (a) the expression and the posttranslational fate of CD in HT-29 colon cancer cells are largely affected by the state of their enterocytic differentiation; and (b) in this cell line the acid-dependent mannose 6-phosphate receptor pathway is, at best, little involved in the trafficking of CD.

Differentiation-induced changes in the content, secretion, and subcellular distribution of lysosomal cathepsins in the human colon cancer HT-29 cell line.

Enterocyte-like differentiated HT-29 colon carcinoma cells were shown to contain far higher intracellular levels of activity of lysosomal cathepsins B, D, and L than their undifferentiated counterparts. In the latter, inhibition of lysosomal functions by leupeptin or ammonium chloride led to a marked increase in the cell-associated activity of the three cathepsins. High levels of pro-cathepsins B, D, and L were found in the culture media of both HT-29 cell populations. Ammonium chloride and chloroquine, which are known to impair the mannose-6-phosphate-dependent trafficking of lysosomal-targeted proteins, did not increase the secretion of the three cathepsins in either undifferentiated or differentiated cultures of HT-29 cells. Analyses by cell fractionation revealed heterogeneities with regard to the density and the content of lysosomal cathepsins between the two cell populations. Leupeptin induced the accumulation of mature lysosomal cathepsins B and L in light density organelles in undifferentiated HT-29 cells. Altogether, these data demonstrate that (1) the expression and subcellular distribution of cathepsins B, D, and L in HT-29 cells are influenced by their state of enterocytic differentiation, (2) the segregation of lysosomal cathepsins is largely inefficient in this tumor cell line and does not increase upon differentiation, and (3) the mannose-6-phosphate-receptor-dependent pathway plays a minor role in the sorting of the three cathepsins, both in undifferentiated and enterocytic-differentiated HT-29 cells.

Differential targeting and processing of procathepsin D in normal and transformed murine 3T3 fibroblasts.

The kinetics of transport and the processing of procathepsin D (proCD), the precursor of a lysosomal aspartyl protease involved in tumor-cell proliferation and metastasis, were compared in normal and SV-40- or benzo[a]pyrene-transformed 3T3 mouse fibroblasts. Sorting of newly synthesized proCD in normal cells was almost complete within 3 hr, while in transformed cells a fraction of the precursor survives a long time. In both normal and transformed 3T3 cultures, secretion of proCD started at 3 hr of chase. However, in normal cells secretion of proCD remained constant between 3 and 24 hr of chase, while in transformed cells it increased along with the chase incubation. The efficiency of formation of the mannose-6-phosphate group on proCD varied among the 3 cell types, being minimal in benzo[a]pyrene-transformed 3T3 cells. Ammonium chloride, a drug known to disrupt the segregation and to enhance the secretion of lysosomal proenzymes, was 2-fold more effective in normal than in transformed 3T3 cells. Despite vacuolar alkalinization, about one third of proCD was segregated into the endosomal-lysosomal pathway in normal and in transformed 3T3 fibroblasts, indicating the existence in these cells of alternative, mannose-6-phosphate receptor-independent mechanisms for targeting proCD. Thus, while hypersecretion of proCD and reduced sensitivity to vacuolar alkalinization are common features of both transformed cell types, the mechanisms responsible for inefficient segregation of proCD may differ between virally and chemically transformed 3T3 cells.

Synthesis, maturation and extracellular release of procathepsin D as influenced by cell proliferation or transformation.

The relationship between cell growth and intra- and extracellular accumulation of cathepsin D (CD), a lysosomal endopeptidase involved in cell protein breakdown, was examined in cultures of normal and transformed BALB/c mouse 3T3 fibroblasts grown at various cell densities. In crowded cultures of normal 3T3 cells (doubling time, Td, 53 hr) intracellular CD activity was 2-fold higher than in sparse, rapidly-growing (Td, 27 hr) cultures. In uncrowded (Td, 18 hr) and crowded (Td, 32 hr) cultures of benzo[a]pyrene-transformed cells intracellular CD levels were one third and two thirds, respectively, of those measured in hyperconfluent 3T3 cultures. Regardless of cell density, SV-40-virus-transformed cells (Td, 12 hr) contained one third of CD levels found in hyperconfluent 3T3 cells. Both transformed cell lines released into the medium a higher proportion of CD, compared with their untransformed counterpart, yet the amount secreted was not sufficient to account for the reduced intracellular level of the enzyme. Serum withdrawal induced a marked increase of both intra- and extracellular levels of CD activity. In both normal and virally or chemically transformed 3T3 cells CD comprised a precursor (52 kDa) and processed mature polypeptides; the latter were mostly represented by a 48-kDa peptide, but a minor part was in a double-chain form (31 and 16 kDa respectively). The proportion of mature enzyme vs. precursor was much higher in confluent, slowly-growing cells than in fast-growing cells, whether normal or transformed. In the latter, conversion of mature 48-kDa peptide into the double-chain form occurred more efficiently.

Differentiation-dependent autophagy controls the fate of newly synthesized N-linked glycoproteins in the colon adenocarcinoma HT-29 cell line.

Our previous results have demonstrated that, in undifferentiated human colon cancer HT-29 cells, a pool of glycoproteins bearing high-mannose oligosaccharides rapidly escapes the exocytic pathway to be degraded in the lysosomal compartment [Trugnan, Ogier-Denis, Sapin, Darmoul, Bauvy, Aubery and Codogno (1991) J. Biol. Chem. 266, 20849-20855]. We report here on the mechanism that governs this degradative pathway. Using pulse-chase experiments in combination with subcellular fractionation, we have observed that the sequestration of high-mannose glycoproteins in lysosomes was impaired by drugs which interfere with the autophagic-lysosomal pathway. The accumulation of high-mannose glycoproteins in the lysosomal fraction was shown to be part of the general autophagic pathway constitutively expressed in undifferentiated cells, as independently measured by the sequestration of the cytosolic enzyme lactate dehydrogenase and electroloaded raffinose. Furthermore, when HT-29 cells were cultured under differentiation-permissive conditions, the decreased accumulation of high-mannose glycoproteins in the lysosomal compartment was correlated with the decrease in autophagy.

Localization and processing of glycosylphosphatidylinositol anchored cathepsin D.

We have investigated the effect of a glycosylphosphatidylinositol anchor on the distribution of the soluble lysosomal enzyme cathepsin D. Only 10% of the chimeric protein (CD-GPI) could be detected on the plasma membrane after transfection in CHO cells. Similarly to endogenous cathepsin D, intracellular CD-GPI was detected in vesicular structures, suggesting that CD-GPI is targeted to lysosomes. CD-GPI is present as three forms with M(r) 55, 50 and 37 kD which could correspond to the precursor, intermediate and mature forms of cathepsin D, respectively. CD-GPI was shown to be GPI anchored by differential extractability with Triton X-114 before and after phosphatidylinositol phospholipase C hydrolysis. Intracellular CD-GPI is mainly substituted with oligosaccharides containing uncovered mannose 6-phosphate residues whereas these residues are covered in the cell surface precursor form of CD-GPI. Ammonium chloride treatment reduces the lysosomal delivery of CD-GPI and increases the cell surface expression of its precursor form.

A heterotrimeric Gi3-protein controls autophagic sequestration in the human colon cancer cell line HT-29.

Human colon cancer HT-29 cells exhibit a differentiation-dependent autophagic-lysosomal pathway that is responsible for the degradation of a pool of newly synthesized N-linked glycoproteins in undifferentiated cells. In the present study, we have investigated the molecular control of this degradative pathway in undifferentiated HT-29 cells. For this purpose, we have modulated the function and expression of the heterotrimeric G-proteins (Gs and Gi) in these cells. After pertussis toxin treatment which ADP-ribosylates heterotrimeric Gi-proteins, we observed an inhibition of autophagic sequestration and the complete restoration of the passage of N-linked glycoproteins through the Golgi complex. In contrast, autophagic sequestration was not reduced by cholera toxin, which acts on heterotrimeric Gs-proteins. Further insights on the nature of the pertussis toxin-sensitive alpha subunit controlling autophagic sequestration were obtained by cDNA transfections of alpha i subunits. Overexpression of the alpha i3 subunit increased autophagic sequestration and degradation in undifferentiated cells, whereas overexpression of the alpha i2 subunit, the only other pertussis toxin-sensitive alpha subunit expressed in HT-29 cells, did not alter the rate of autophagy.

Altered intracellular processing and enhanced secretion of procathepsin D in a highly deviated rat hepatoma.

Both freshly-isolated rat hepatocytes and Morris hepatoma 7777 cells synthesized cathepsin D as a precursor that was either processed intracellular to smaller mature forms or secreted into the medium. The pattern of mature enzyme forms was different in the 2 cell types. In addition, the relative amount of precursor secreted was much higher for hepatoma cells. Monensin strongly enhanced the secretion and also impaired the intracellular transport-linked maturation of procathepsin D in hepatocytes, while it markedly inhibited intracellular maturation and only slightly increased secretion of the pro-enzyme in hepatoma cells. Ammonium chloride influenced the intralysosomal segregation and maturation of procathepsin D in hepatocytes but not in hepatoma cells. Our observations indicate that (i) the lysosomal segregation of cathepsin D was less efficient and its fractional secretion higher in hepatoma cells than in hepatocytes; (ii) in the 2 cell types, delivery to lysosomes and processing of procathepsin D were differently sensitive to increases in the vacuolar pH.

High levels of proteolytic enzymes in the ascitic fluid and plasma of rats bearing the Yoshida AH-130 hepatoma.

Plasma and ascitic fluid of rats bearing the Yoshida ascites hepatoma AH-130 were shown to contain high levels of proteolytic enzymes belonging to different classes active at neutral and acidic pH. Relative to those measured in control rat plasma, in tumor-bearing animals, the activity levels of lysosomal cathepsins B and L, in their latent, acidic-activatable form, were approximately 5-fold higher in plasma and 9-fold higher in ascitic fluid, and cathepsin D activity was about 5-fold higher in both plasma and ascitic fluid. Plasma and ascitic fluid of tumor-bearing rats also contained novel neutral and acidic gelatinolytic activities. The latter, as revealed by zymographic analysis conducted at pH 6.0, in the presence of dithiothreitol and in the absence of divalent metal ions, was sensitive to iodoacetamide inhibition but not to EDTA, showed a molecular mass of approximately 90 kD on SDS-PAGE, and was lost upon limited proteolysis with pepsin. Therefore, this enzyme is not identifiable as cathepsin B or L or their related latent forms and may represent a novel, so far undescribed, gelatinase. Its presence exclusively in the body fluids of AH-130-bearing rats suggests its possible use as a tumor marker.