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.     

Interferon Regulatory Factor 1 (IRF-1) induces p21(WAF1/CIP1) dependent cell cycle arrest and p21(WAF1/CIP1) independent modulation of survivin in cancer cells

We have shown that the ectopic expression of Interferon Regulatory Factor 1 (IRF-1) results in human cancer cell death accompanied by the down-regulation of the Inhibitor of Apoptosis Protein (IAP) survivin and the induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). In this report, we investigated the direct role of p21 in the suppression of survivin. We show that IRF-1 down-regulates cyclin B1, cdc-2, cyclin E, E2F1, Cdk2, Cdk4, and results in p21-mediated G1 cell cycle arrest. Interestingly, while p21 directly mediates G1 cell cycle arrest, IRF-1 or other IRF-1 signaling pathways may directly regulate survivin in human cancer cells.     

Identification and characterization of survival-related gene, a novel cell survival gene controlling apoptosis and tumorigenesis

Apoptosis plays a critical role in cellular homeostasis during development, immune responses, and tumorigenesis. Recent studies have identified a number of genes that control this process. We report here our identification of a novel cell survival-related gene (SRG) from a human expression cDNA library by functional cloning. SRG shows no significant nucleotide sequence homology to any known genes in the Genbank. Our fluorescence in situ hybridization analysis has estimated that SRG is located at 1p36, agreeing with the location at 1p36.22 in the human genome sequence. SRG encodes a putative protein of 172 amino acids, which is mainly located in the perinuclear region. Northern blotting analysis indicates that SRG is highly expressed in many human cancer cell lines although it is low in most tissues except liver and placenta. To investigate the function of SRG in apoptosis, we transfected SRG cDNA into BAF/BO3 and B16/F0 cells and induced apoptosis by cytokine/serum deprivation. We found that SRG-transfected cells are resistant to apoptosis induced by cytokine/serum deprivation. In addition, mice bearing SRG-transfected melanoma had more tumor formation and larger tumor growth. Melanoma transfected with antisense SRG showed significantly less tumor formation and smaller tumor growth. Interestingly, mouse SRG gene was also identified on chromosome 4 and blocking SRG expression with small interfering RNA promoted serum deprivation-induced apoptosis of NIH3T3 cells. Our results show that SRG is a novel cell survival gene that critically controls apoptosis and tumor formation.     

Overexpression of edg-2/vzg-1 induces apoptosis and anoikis in ovarian cancer cells in a lysophosphatidic acid-independent manner.

Lysophosphatidic acid (LPA) is one of the major growth factors in ascites from ovarian cancer patients and appears to play an important role in proliferation, survival, and invasion of ovarian cancer cells. Recently, several groups have shown that Edg-2, which belongs to the G-protein coupled receptor family, is a functional LPA receptor. Northern blot analysis showed that most ovarian cancer cell lines express Edg-2. Edg-2 expression was especially high in the cisplatin-resistant and slowly proliferating 2780cp cell line and was almost absent from the cisplatin-sensitive and rapidly proliferating A2780 cell line. We thus assessed whether Edg-2 could contribute to changes in cell viability, cell proliferation, or cisplatin resistance. Stable overexpression of Edg-2 in A2780 cells induced an exogenous LPA-independent decrease in proliferation but did not alter cisplatin sensitivity. The LPA-independent decrease in growth rate induced by overexpression of Edg-2 could be explained, at least in part, by Edg-2-induced apoptosis rather than by effects on cell cycle progression. In agreement with the results in stably transfected A2780 cells, transient expression of Edg-2 in Jurkat T cells also induced apoptosis. When cells were separated from the extracellular matrix, they underwent a specialized form of apoptosis called anoikis, which is particularly important in survival of cells in the circulation during metastasis. A2780 cells engineered to overexpress Edg-2 were particularly sensitive to anoikis. These observations suggest that Edg-2 may be a negative regulator for ovarian epithelial cell growth and metastasis.     

Involvement of activator protein-1 (AP-1) in induction of apoptosis by vitamin E succinate in human breast cancer cells

The purpose of this study was to document induction of apoptosis by vitamin E succinate (VES; RRR-α-tocopheryl succinate) in human breast cancer cells in culture and to characterize potential c-jun involvement. VES at 18.8 μM (10 μg/mL) induced DNA synthesis arrest, reduced total cell numbers, and induced apoptosis in estrogen receptor–positive and estrogen-responsive MCF-7 human breast cancer cells. VES at 10 μg/mL induced apoptosis in greater than 60% of cells within 3 d of treatment. Apoptosis was documented by detection of fragmented or condensed nuclei in 4′,6-diamindino-2-phenylindole–stained cells, detection of terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeled DNA, and DNA laddering. Analyses of mRNA and protein levels of candidate molecules involved in apoptosis showed that MCF-7 cells treated with VES exhibited elevated and persistent expression of c-jun. MCF-7 cells stably transfected with a dominant-negative interfering mutant c-jun, TAM-67, and expressing high levels of mutant jun exhibited approximately 50% blockage of VES-mediated apoptosis. In addition to increased c-jun expression after VES treatment, VES-treated MCF-7 cells exhibited elevated activator protein-1 (AP-1) binding activity. Comparisons of AP-1 binding factors by super-shift analyses with jun-specific antibodies in cells sensitive to VES-induced apoptosis (empty-vector control 7-1 cells) and cells resistant to VES-induced apoptosis (TAM-67–containing TAM-9 cells) showed that the sensitive cells expressed c-jun and jun D and the resistant cells TAM-67 AP-1 binding proteins after VES treatment. These studies suggested that c-jun may be involved in the apoptotic process initiated by VES treatment of human MCF-7 breast cancer cells. Mol. Carcinog. 19:180–190, 1997. © 1997 Wiley-Liss, Inc.     

SV40 T/t-common polypeptide specifically induces apoptosis in human cancer cells that overexpress HER2/neu

Previously, we reported that SV40 T/t-common polypeptide, which contains the NH(2)-terminal common domain of SV40 large T and small t antigens, can repress HER2/neu (also known as erbB-2) expression and consequently suppress the tumorigenic potential of the HER2/neu-overexpressing ovarian carcinoma cells. Here we report that T/t-common could specifically induce apoptosis in HER2/neu-overexpressing human cancer cell lines but not in nontransformed cell lines and HER2/neu low-expressing human cancer cell lines. The ability of T/t-common to induce apoptosis in HER2/neu-overexpressing cancer cells was derived from its ability to inhibit HER2/neu because reexpression of a large amount of HER2/neu could block apoptosis induced by T/t-common. T/t-common expression in HER2/neu-overexpressing SK-OV-3 cancer cells led to down-regulation of Bcl-2 and Bcl-X(L), and overexpression of Bcl-2 could inhibit the ability of T/t-common to induce apoptosis in these cells. Therefore, the apoptosis-inducing activity of T/t-common is related to its ability to inhibit Bcl-2 expression in HER2/neu-overexpressing cancer cells. Consistent with the apoptosis-inducing activity of T/t-common, we found that T/t-common could specifically inhibit the soft-agarose colony-forming ability of the HER2/neu-overexpressing human cancer cell lines but not that of the HER2/neu low-expressing human cancer cell lines. Finally, we showed that T/t-common could specifically sensitize HER2/neu-overexpressing human cancer cell lines, but not HER2/neu low-expressing human cancer cell lines, to chemotherapeutic agent etoposide. Together, these data suggest that T/t-common alone or in combination with chemotherapy may provide a new approach for treatment of cancers that overexpress HER2/neu.     

Nuclear translocation of apoptosis inducing factor is associated with cisplatin induced apoptosis in LNCaP prostate cancer cells

Prostate cancer (PC) is considered resistant to cisplatin chemotherapy. In order to identify novel causes of resistance to cisplatin, we explored the role of Apoptosis Inducing Factor (AIF) that mediates caspase independent apoptosis in cisplatin induced cell death in PC. Similar to treatment with pancaspase inhibitor Z-VAD-fmk, cisplatin induced apoptosis in LNCaP cells was inhibited by AIF inhibitor N-acetyl-L-cysteine (NAC), treatment of LNCaP cells with NAC prevented AIF translocation to the nucleus and over-expression of recombinant AIF gene increased apoptosis. Our results suggest that AIF is associated with cisplatin induced apoptosis in PC.     

Enhanced apoptosis and radiosensitization by combined 13-cis-retinoic acid and interferon-alpha2a; role of RAR-beta gene.

PURPOSE: Combined use of 13-cis-retinoic acid (cRA) and interferon-alpha2a (IFNalpha) induced significant radiosensitization in human cervical cancer ME-180 cell line, whereas it failed to achieve similar radiation enhancement in HeLa cells. The differential radiosensitization could be from the difference of retinoic acid receptor (RAR) expression because RAR-beta was highly expressed in ME-180 cells in contrast to the HeLa cells where RAR-beta was not detectable. We examined the role of this gene in mediating radiosensitization by cRA and IFNalpha, and explored the mechanism of radiation-induced cell killing. METHODS AND MATERIALS: Human cervical cancer cell lines, ME-180 and HeLa, were treated with cRA and IFNalpha followed by radiation. Apoptosis and radiosensitization were quantitated by TUNEL assay (in situ DNA nick end labeling) and colony-forming ability of surviving cells. The cells were transfected with bcl-2 gene and RAR-beta gene to test the role of these genes in mediating radiosensitization and apoptosis. RESULTS: Synergistic radiosensitization and apoptosis was observed by combined use of cRA and IFNalpha with radiation in ME-180 cells which express high level of RAR-beta mRNA, whereas these were not seen in HeLa cells where RAR-beta mRNA is not detectable. Both radiosensitization and apoptosis were abolished by bcl-2 gene in ME-180 cells. RAR-beta gene transfection induced similar radiation enhancement and apoptosis in HeLa cells. CONCLUSION: Apoptosis and radiation response were enhanced in the cells with high level of RAR-beta mRNA expression. The RAR-beta gene appears to mediate the radiation-induced apoptosis by cRA and IFNalpha. These findings indicate that presence of RAR-beta in the cancer cells could be exploited for patient selection in using these drugs for apoptosis and radiosensitization.     

环氧化酶抑制剂诱导人膀胱癌细胞系T24凋亡作用研究

 目的 探讨环氧化酶抑制剂尼美舒利对膀胱癌细胞系T24的凋亡诱导作用和对环氧化酶-2(COX-2)的表达抑制作用。方法 以膀胱癌细胞系T24为研究对象，已知COX-2高表达的胃癌细胞系SGC-7901为阳性对照。分别经不同浓度尼美舒利作用25小时后，Western blot检测COX-2的表达；凋亡DNA琼脂凝胶电泳检测DNA凋亡梯度。Image Tool凝胶分析软件分析Western blot电泳图像。结果 DNA梯度随尼美舒利浓度的加大而加大，Western blot结果显示COX-2的表达随着尼美舒利的浓度加大而明显抑制，在浓度为100μmol／L时，无法检测到COX-2的表达。Image Tool软件分析Western blot灰度值差异有显著性(P<0．01)。结论 尼美舒利有良好的诱导凋亡作用，其机制可能与COX-2的表达抑制有关。     

FADD在人肺腺癌细胞A549诱导T淋巴细胞凋亡中的作用

背景与目的:细胞凋亡是肺癌免疫逃逸的机制之一.Fas相关死亡结构域蛋白(Fas associated death domain protein,FADD)在细胞凋亡过程中起着重要作用.本研究旨在探讨FADD在人肺腺癌细胞A549诱导人T淋巴细胞凋亡过程中的作用.方法:用人肺腺癌细胞株A549细胞诱导人淋巴瘤T细胞株Ju...     

Stromelysin-3 is a potent negative regulator of adipogenesis participating to cancer cell-adipocyte interaction/crosstalk at the tumor invasive front

The initial invasive processes during cancer development remain largely unknown. Stromelysin-3/matrix metalloproteinase 11 (ST3/MMP11) is associated with tumor invasion and poor prognosis. We present novel evidence that adipocytes present at human breast tumor invasive front are induced by cancer cells to express ST3. Using mouse syngeneic model, light and electron microscopy showed that in ST3-deficient mice but not in wild-type mice, forced cancer cell-adipocyte interaction/crosstalk results in adipocyte membrane alteration, allowing cancer cell fat infiltration and death. Thus, adipocytes are involved in initial cancer cell survival into connective tissue, and this effect is ST3 mediated. This suggested that ST3 might play a role in adipocyte metabolism. Accordingly, ST3-deficient mice exhibited fat excess and increased mRNA levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and adipocyte protein 2 (aP2) adipogenic markers, indicating that, in vivo, ST3 negatively regulates fat homeostasis. Moreover, ST3-deficient mouse embryonic fibroblasts exhibited a dramatic enhanced potential to differentiate into adipocytes associated with increased PPARgamma and aP2 expression, and recombinant ST3 treatment reverted their differentiation. Thus, in vitro, ST3 reduces adipocyte differentiation in an autocrine manner. High fibroblasts/adipocytes ratio is a stroma feature, and peritumoral fibroblast origin remains debated. Our results support the concept that invading cancer cells aberrantly restore the negative ST3 function on adipogenesis into proximal adipocytes/preadipocytes, leading to the accumulation/maintenance of a particular peritumoral fibroblast subpopulation. Accordingly, in human breast tumors, we observed that ST3-expressing peritumoral fibroblasts are distinct from alpha-smooth muscle actin-expressing myofibroblasts. This constitutes the first report of implication of a MMP in cancer cell-adipocyte interaction/crosstalk during early steps of connective tissue invasion.     

Induction of apoptosis by AN-152, a cytotoxic analog of luteinizing hormone-releasing hormone (LHRH), in LHRH-R positive human breast cancer cells is independent of multidrug resistance-1 (MDR-1) system

Objective. More than 50% of human breast cancers express receptors for luteinizing hormone-releasing hormone (LHRH-R). These receptors can be used for targeted chemotherapy with agents like AN-152, in which doxorubicin is linked to analog [D-Lys⁶]LHRH. We compared the effects of AN-152 and doxorubicin in human breast cancer cells. Methods. MCF-7, T47D, HCC-70 and ZR-75-1 cells were analysed for expression of LHRH-R using RT-PCR, immunostaining and flow cytometry. Apoptosis and expression of MDR-1 gene product Pgp were measured by flow cytometry. Cleavage of doxorubicin from AN-152 by serum carboxylesterase (CE) was inhibited by DFP. Results. In MCF-7, T47D and HCC-70 cells we found cell surface expression of LHRH-R. In ZR-75-1 cells only sparse surface expression was found. In HCC-70 cells, induction of apoptosis by AN-152 was stronger than by doxorubicin in medium containing fetal calf serum (FCS). Pretreatment with DFP increased AN-152-induced apoptosis in LHRH-R positive MCF-7 and HCC-70 cells and reduced apoptosis in ZR-75-1 cells. In serum-free medium apoptosis induced by AN-152 was stronger than that induced by doxorubicin in HCC-70, T47D and MCF-7 cells, but weaker in ZR-75-1 cells. In medium containing FCS, both AN-152 and doxorubicin induced surface expression of MDR-1 gene product Pgp, but the effect of AN-152 was weaker. Pgp-expression induced by AN-152 was inhibited by pretreatment with DFP. AN-152 did not induce Pgp-expression in serum-free medium. Conclusion.The cytotoxic LHRH analog AN-152 induces apoptosis independent of MDR-1 in LHRH-R positive breast cancer cells. The efficacy and/or specificity of AN-152 is improved by suppression or absence of CE activity.     

Prediction of proapoptotic anticancer therapeutic response in vivo based on cell death visualization and TRAIL death ligand-receptor interaction

Tumor growth is often associated with insufficient apoptosis. The Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) and its proapoptotic receptors death receptor 4 (DR4) and DR5 agonistic monoclonal antibodies are being developed as targeted therapeutics because they kill cancer cells while sparing normal cells. A challenge to targeted therapeutics is the selection of patients who are most likely to benefit from targeted drugs because of the heterogeneity of cancer. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and for predicting therapeutic response. We hypothesized that the cell surface expression level of DR4/5 may predict the proapoptotic targeted therapeutic response if the signaling pathway downstream is intact. The goal of this proof-of-concept study was to develop a molecular imaging strategy to predict proapoptotic anti-cancer therapy response at an early stage of treatment. TRAIL and the DR5 agonistic monoclonal antibody HGS-ETR2 (Lexatumumab, TRM-2) were labeled with a near-infrared dye and these were used to image the TRAIL receptors on cultured TRAIL sensitive and TRAIL resistant human tumor cells as well as tumor xenografts. Imaging of cells and tumor-bearing animals was conducted with near infrared fluorescence imagers and apoptosis in cells was assessed by western blots of PARP-cleavage and flow cytometry of sub-G1 content. Apoptosis in tumors was evaluated by imaging near-infrared dye-labeled Annexin V and tumor tissue activated caspase-3 staining. Both in vitro and in vivo studies showed that imaging of death inducing ligand-receptor interaction was consistent with the apoptosis readout. Thus TRAIL sensitive tumors that express TRAIL receptors underwent cell death following treatment whereas tumors lacking TRAIL receptor expression were shown to be TRAIL resistant. In vivo molecular imaging of TRAIL receptor expression correlated with response to TRAIL therapy and an apoptotic response in vivo.     

Xenotransplantation elicits salient tumorigenicity of adult T‐cell leukemia‐derived cells via aberrant AKT activation

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL‐2Rγc^null (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T‐cell leukemia‐derived cell lines, ST1‐N6 and TL‐Om1‐N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL‐Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1‐N6 and TL‐Om1‐N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK‐2206 attenuated the progression of tumors induced by ST1‐N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T‐cell leukemia‐derived cells.     

Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells

Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cancer treatments. This cellular suicide program is known to be regulated by many different signals from both intracellular and extracellular stimuli. Here we report that p53 suppressed expression of the cellular FLICE-inhibitory protein (FLIP) that potentially blocks apoptotic signaling in human colon cancer cell lines expressing mutated and wild-type p53. In contrast, the expression of the death receptor KILLER/DR5 (TRAIL-R2) had no effect on FLIP expression, although exogenous p53 is known to induce KILLER/DR5 expression. In line with these observations, FLIP-negative cancer cells were sensitive to both p53- and KILLER/DR5-mediated apoptosis, whereas cells containing high levels of FLIP underwent apoptotic cell death when triggered by ectopic p53 expression but not by KILLER/DR5 expression. Treating the cells with a specific inhibitor of the proteasome inhibited the decrease of FLIP by p53, suggesting that p53 enhances the degradation of FLIP via a ubiquitin-proteasome pathway. Thus, the data indicate that p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer.     

Human pancreatic cancer cells express non-functional Fas receptors and counterattack lymphocytes by expressing Fas ligand; a potential mechanism for immune escape

The aim of this study was to investigate the expression and functional status of Fas ligand (FasL) and its receptor (Fas) in human pancreatic cancers. Using RT-PCR and Western blotting, Fas and FasL were expressed in seven surgically resected pancreatic cancer specimens and five cell lines; Capan-1, AsPC-1, BxPC-3, PANC-1, and MIA PaCa-2. In the resected specimens, pancreatic cancer cells induced apoptosis in the surrounding lymphoid cells. In coculture experiments of pancreatic cancer and Jurkat T cells, 50% of Jurkat T cells underwent apoptosis after 2 days, however, almost all pancreatic cancer cells remained viable. In addition, by testing Fas function using anti-Fas antibody (CH11), all cell lines were resistant to Fas-mediated apoptosis except Capan-1 cells which showed sensitivity similar to that of Jurkat T cells. These results suggest that pancreatic cancer cells evade immune surveillance by expression of FasL and non-functioning Fas that allow them to activated T-cells. These tumor escape mechanisms may contribute to the rapid fatal course of pancreatic cancer.