Expression of IAP family proteins in myelodysplastic syndromes transforming to overt leukemia

Bone marrow cells of patients with myelodysplastic syndromes (MDS) frequently undergo apoptosis, though the apoptotic cell ratio decreases when overt leukemia (OL) develops. Thus, we compared the expression of the inhibitor of apoptosis protein (IAP) gene family proteins in bone marrow samples from control, MDS, OL transformed from MDS (MDS --> OL), and de novo acute myelogenous leukemia (AML) subjects by the quantitative real-time RT-PCR method and an immunohistochemical approach. Overexpression of mRNA for survivin, cIAP1, NAIP and XIAP was significant in MDS bone marrow cells compared with control samples. However, the expression of mRNA for survivin, cIAP1 and cIAP2 exhibited a remarkable decrease after the development of OL (MDS --> OL). By immunohistochemistry, survivin was found to localize to the nucleus of myeloid cells in the majority of MDS cases. Next, the chronological changes in the expression of IAPs were determined in cases of MDS with evolution of OL. Although the expression of cIAP1 and cIAP2 revealed a sudden or gradual decrease as OL developed, survivin in many cases and XIAP in the majority of cases exhibited a peak of expression before a decline, indicating that these IAPs could be associated with the early events in the development of OL.     

Apoptotic effects of thiazolobenzimidazole derivatives on sensitive and multidrug resistant leukaemic cells

We investigated the cytotoxic activity of eight thiazolobenzimidazole derivatives on sensitive HL60 and multidrug-resistant (MDR) (HL60R) leukaemia cell lines. The antitumour effects of these compounds were compared with those of RS-TBZ, a thiazolobenzimidazole derivative, previously described in our reports, that was able to induce apoptosis more markedly in MDR cells than in the parental sensitive cell lines. Only two compounds in this study proved to have interesting effects: (a) the S-enantiomer of TBZ, that was able to induce apoptosis in MDR cells in a slightly more selective manner than TBZ (racemic form); and (b) TBZ-4-OCH3 (TBZ-4-OCH3), that showed cytotoxic and apoptotic effects on sensitive and resistant leukaemia cells greater than TBZ, without cytotoxic effects on normal haemopoietic progenitor cells. Moreover, we observed that TBZ-4-OCH3 was also active in cells expressing Bcr-Abl, an oncogene that confers resistance to apoptosis induced by several stimuli, including cytotoxic agents. The inhibition of caspase-9 and caspase-3 by specific polypeptide inhibitors decreased the apoptotic effects of TBZ-4-OCH3 in HL60 cells indicating that apoptosis induced by this compound was, at least partly, caspase-mediated. On the contrary, the blocking of FL-associated cell surface antigen (Fas) using a specific Fas-blocking monoclonal antibody did not affect the level of apoptosis induced by TBZ-4-OCH3 suggesting that the Fas pathway was not involved. In addition, the caspase 8 inhibitor was unable to inhibit the apoptotic activity of TBZ-4-OCH3. The very low toxicity shown by TBZ-4-OCH3 in normal haemopoietic progenitor cells and its high activity in sensitive and MDR neoplastic cells suggest a possible clinical use for this new compound.     

Induction of chemoresistance in HL-60 cells concomitantly causes a resistance to apoptosis and the synthesis of P-glycoprotein.

The appearance of multidrug-resistant (MDR) proteins or the acquisition of a defective apoptotic programme are major drawbacks in the treatment of cancers since both induce a resistance to classical chemotherapy. However, a link between the two mechanisms has not, as yet, been clearly established. In this study, HL-60 cells cultured in the continual presence of a sub-lethal dose of doxorubicin (dox; HL-60/Dox) were used as a model to study acquired chemoresistance. During the induction of chemoresistance, the appearance of a functional P-glycoprotein (P-gp), in addition to the expression of anti-apoptotic Bcl-2, Bcl-XL and pro-apoptotic Bax proteins was assessed. Parental cells which are sensitive to dox, have no P-gp activity and express Bcl-2 and Bax. After 4 weeks of treatment, a functional P-gp was detected in HL-60/Dox cells. In addition, the synthesis of Bcl-2 appeared to be replaced by Bcl-XL while that of Bax remained unchanged. These cells were also resistant to apoptosis induced by both P-gp and non-P-gp substrates. This inability to induce apoptosis could have resulted from the induction of the expression of the inhibitor of apoptosis protein (XIAP). Our data show that acquired chemoresistance could involve a parallel induction of P-gp and an impairment of the apoptotic pathway.     

Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy

It is well known that dysfunction of the apoptotic pathway confers apoptosis resistance and results in a low sensitivity of human cancer cells to therapeutic agents. A novel strategy to overcome the resistance is to target the apoptotic pathway directly. To identify molecular targets in the apoptotic pathway that are differentially regulated in cancer and normal cells, we have examined the levels of apoptotic effectors and inhibitors in human tumor and normal cell lines as well as in cancer and normal tissues. These include three pancreatic cancer lines (BXPC-3, MIA PaCa-2, and Panc-1), four breast cancer cell lines (MDA-MB-231, MDA-MB-435, MDA-MB-361, and MCF-7), and colon carcinoma line (SW620). Additionally, breast carcinoma tissue specimens were examined. Compared with normal human fibroblast and mammary epithelial cell lines, we detected high basal levels of caspase-3 and caspase-8 activities and active caspase-3 fragments in the tumor cell lines and cancer tissues in the absence of apoptotic stimuli. Furthermore, the tumor cells expressed high levels of survivin and XIAP, two members of the inhibitor of apoptosis (IAP) protein family. When the activity of these IAPs was blocked by expression of dominant-negative mutant survivin (survivinT34A) and XIAP-associated factor 1, respectively, apoptosis was induced in tumor but not normal cell lines. Moreover, down-regulation of both survivin and XIAP significantly enhanced tumor-cell apoptosis as compared with inhibition of either survivin or XIAP alone. These results suggest that up-regulated IAP expression counteracts the high basal caspase-3 activity observed in these tumor cells and that apoptosis in tumor cells but not normal cells can be induced by blocking IAP activity. Therefore, IAPs are important molecular targets for the development of cancer-specific therapeutic approaches.     

Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1.

The multidrug resistance (MDR) phenotype, induced by the overexpression of several ABC transporters or by antiapoptotic mechanisms, has been identified as the major cause of drug resistance in the treatment of patients with acute myeloid leukemia (AML). In this study, we have shown that valproic acid (VPA) (a histone deacetylase inhibitor) can inhibit the proliferation of both P-glycoprotein (P-gp)- and MDR-associated protein 1 (MRP1)-positive and -negative cells. VPA also induced apoptosis of P-gp-positive cells. VPA induced apoptosis in K562 cells led to decrease in Flip (FLICE/caspase-8 inhibitory protein) expression with Flip cleavage, which could not be observed in HL60 cells. In HL60/MRP cell line, which proved to be resistant to apoptosis by VPA, we observed an abnormal expression of apoptotic regulatory proteins, overexpression of Bcl-2 and absence of Bax. Also, the Bcl-2 antagonist HA14-1 rapidly restored apoptosis in this cell line. Cotreatment with cytosine arabinoside induced very strong apoptosis in both K562/DOX and HL60/DNR cell lines. VPA also induced apoptosis in AML patient cells expressing P-gp and/or MRP1. Our findings show VPA as an interesting drug that should be tested in clinical trials for overcoming the MDR phenotype in AML patients.     

Overexpression of Survivin and XIAP in MDR cancer cells unrelated to P-glycoprotein

Cancer cells developing multidrug resistance (MDR) is one of the most serious clinical problems responsible for the failure of cancer chemotherapy. P-glycoprotein (P-gp) overexpression and inhibitor of apoptosis proteins (IAPs) overexpression in cancer cells are the two common mechanisms of MDR. However, the relationship between IAPs and P-gp in MDR cancer cells is unknown. We investigated the expression levels of two IAPs, Survivin and XIAP, and their interaction with P-gp in MDR cancer cells. We have found that the human epidermoid carcinoma cells KBv200 and breast cancer cells MCF-7/Adr overexpress not only P-gp but also XIAP and Survivin, and showed high resistance to chemotherapeutic drugs doxorubicin, docetaxel and vincristine, in contrast to their parental cells KB and MCF-7. Furthermore, upregulation of Survivin or XIAP through transfection with the plasmid pECFPN1-Survivin or pcDNA3-6myc-XIAP in these four cell sublines did not affect the P-gp expression. Downregulation of Survivin or XIAP through transfection with the Survivin or XIAP siRNA did not have an effect on the P-gp expression in these resistant cells. Additionally, our immunoprecipitation results showed that Survivin or XIAP did not directly bind to P-gp. In summary, our study suggested that the overexpression of Survivin and XIAP in MDR cancer cells does not directly interact with P-gp.     

MDR1/P-Glycoprotein Overexpression in Bladder Transitional Cell Carcinoma and its Correlation with Expression of Survivin and Fas

Bladder transitional cell carcinomas (BTCC) are the most com- mon urologic tumors in China and the sixth most common ma- lignancy in developed countries.[1] One characteristic feature of BTCC is the high recurrence incidence which is about 75-80% after op…     

The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer

Apoptosis is a crucial biological process that prevents uncontrolled cell proliferation and eliminates harmful cells. Resistance to apoptotic stimuli is a hallmark feature of various cancers. One of the mechanisms through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of inhibitor of apoptosis proteins (IAPs). IAPs are a group of structurally related proteins that were initially identified in baculoviruses. Mammalian IAPs block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms. This family of proteins has become increasingly prominent in the field of cancer biology. To date, overexpression of several IAPs has been detected in various cancers. This paper reviews the recent advances in the research of IAPs. The differential expression and the biological significance of each IAP in various cancer types will be discussed. Finally, we review the most recent advances in the research efforts aimed at using IAPs as potential targets for cancer therapy.     

Expression of the inhibitors of apoptosis proteins in cisplatin-resistant prostate cancer cells

Acquired multi-drug resistance remains a major obstacle in the management of prostate cancer. The objective of this study was to examine whether chemoresistance could be due in part to the expression of the inhibitors of apoptosis proteins (IAPs). We established cisplatin-resistant LNCaP sublines. We examined the effects of cisplatin on cell growth and apoptosis in LNCaP cells and LNCaP sublines by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) assay and Hoechst 33258 staining, and analyzed cross-resistance to adriamycin, 5-fluorouracil, taxol, taxotere, and etoposide. In addition, the expression of IAP-1, IAP-2, X-linked IAP (XIAP), neuronal apoptosis inhibitory protein, and survivin was investigated by immunoblot analysis in LNCaP sublines. Although the growth rates were reduced in a dose-dependent manner by cisplatin in LNCaP sublines, the anti-proliferative effects of cisplatin were significantly decreased in LNCaP sublines compared to LNCaP cells. Cisplatin-resistant sublines, LNCaP/C1, LNCaP/ C2, and LNCaP/C3 cells, were 6.3-, 9.1-, and 22.3-fold more resistant to cisplatin than LNCaP cells, respectively, and this resistance was paralleled with reduced induction of apoptosis. LNCaP/C3 cells showed cross-resistance to adriamycin, 5-fluorouracil, and etoposide whereas those cells exhibited no or only weak cross-resistance against taxol and taxotere. With the exception of survivin, all the IAPs were identified in LNCaP cells by immunoblot analysis. Interestingly, the expression of IAP-2, XIAP, and survivin gradually increased with the extent of cisplatin-resistance. Altered expression of IAP-2, XIAP, and survivin was involved in these phenotypes of cisplatin-resistant LNCaP sublines. IAPs may make an important contribution to the resistance to the apoptotic effect of cisplatin in prostate cancer.     

P-glycoprotein and survivin simultaneously regulate vincristine-induced apoptosis in chronic myeloid leukemia cells

Overexpression of P-glycoprotein (Pgp/ABCB1) in tumor cells is associated with a classic phenotype of multidrug resistance (MDR). Moreover, some members of the inhibitor of apoptosis protein (IAP) family, such as survivin, contribute to an apoptosis-resistant phenotype, by inhibiting chemotherapy-induced cell death and promoting MDR. By using Western blotting, qRT-PCR, Annexin V and immunofluorescence assays we have demonstrated a relationship between Pgp and survivin in a prior sensitive chronic myeloid leukemia (CML) cell line (K562). A high dose of vincristine induced a concomitant overexpression of Pgp and survivin, which was associated with a low apoptotic index in the K562 cell line. In addition, we observed a cytoplasmic co-localization of Pgp and survivin, suggesting a functional association between these two proteins in apoptosis control by a common mechanism. In summary, our data suggest that Pgp and survivin should be analyzed in aggregate because they may have significant impact on drug resistance in CML cells.     

Loss of inhibitor of apoptosis proteins as a determinant of polyamine analog-induced apoptosis in human melanoma cells

We have previously shown that the clinically relevant polyamine analog N1,N11-diethylnorspermine (DENSPM) causes rapid apoptosis in human melanoma SK-MEL-28 cells via a series of events that include mitochondrial release of cytochrome c and activation of the caspase cascade. Upstream to these events, DENSPM downregulates polyamine biosynthesis and potently upregulates polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT). In searching for downstream effectors that either contribute to or abrogate the apoptotic response, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs). Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP protein (but not XIAP) levels by 18 h via an apparently Smac/Diablo-independent pathway. Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis, suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome. We also observed that structural analogs of DENSPM which differentially induced SSAT and apoptosis lowered survivin and ML-IAP levels in a manner that correlated with enzyme activity. The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interfering RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment. Among the melanoma cell lines (SK-MEL-28, MALME-3M, A375 and LOX), survivin degradation correlated temporally with the onset of DENSPM induced apoptosis or growth inhibition. By contrast, ML-IAP degradation occurred only during rapid apoptosis seen in SK-MEL-28 cells. These data suggest a sequence of events whereby DENSPM induction of SSAT leads to loss of IAP proteins and a more fulminate apoptotic response. The findings implicate survivin and ML-IAP as important determinants of polyamine analog drug action in melanoma cells.     

Anti-apoptotic proteins, apoptotic and proliferative parameters and their prognostic significance in cervical carcinoma

The inhibitor of apoptosis proteins (IAP) suppress apoptosis induced by a variety of stimuli. The aims of this study were to: (a) compare the expression of X-linked IAP (Xiap) and Human IAP-2 (Hiap-2) in cervical carcinoma cells and normal cervix, (b) determine the correlation between IAP expression and tumour apoptosis or proliferation, and (c) assess their prognostic significance in cervical carcinomas. Paraffin-embedded tissue sections were retrieved from 77 patients with cervical squamous carcinomas prior to treatments and 47 normal subjects. Tumour apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuracil triphosphate (dUTP) nick-end labelling (TUNEL) and apoptotic index (AI), and the proliferative rate was measured by Ki-67 and mitotic (MI) indices. Immunoreactive Xiap and Hiap-2 were found in both cervical cancer cells and normal tissues. IAP expressions in cancers did not correlate with apoptotic and proliferative parameters, disease stage and patient survival. The lower AI and Ki-67 index were associated with a better survival. In conclusion, the basal expression levels of IAPs have no prognostic significance, but AI and Ki-67 expression are potential prognostic indicators in cervical carcinoma.     

Targeting P-glycoprotein expression and cancer cell energy metabolism: combination of metformin and 2-deoxyglucose reverses the multidrug resistance of K562/Dox cells to doxorubicin

P-glycoprotein (P-gp) is one of the major obstacles to efficiency of cancer chemotherapy. Here, we investigated whether combination of metformin and 2-deoxyglucose reverses the multidrug resistance (MDR) of K562/Dox cells and tried to elucidate the possible mechanisms. The combination of metformin and 2-deoxyglucose selectively enhanced the cytotoxicity of doxorubicin against K562/Dox cells. Metformin was not a substrate of P-gp but suppressed the elevated level of P-gp in K562/Dox cells. The downregulation of P-gp may be partly attributed to the inhibition of extracellular signal-regulated kinase pathway. The addition of 2-deoxyglucose to metformin initiated a strong metabolic stress in both K562 and K562/Dox cells. Combination of metformin and 2-deoxyglucose inhibited glucose uptake and lactate production in K562 and K562/Dox cells leading to a severe depletion in ATP and a enhanced autophagy. Above all, P-gp substrate selectively aggravated this ATP depletion effect and increased cell apoptosis in K562/Dox cells. In conclusion, metformin decreases P-gp expression in K562/Dox cells via blocking phosphorylation of extracellular signal-regulated kinase. P-gp substrate increases K562/Dox cell apoptosis via aggravating ATP depletion induced by combination of metformin and 2-deoxyglucose. Our observations highlight the importance of combination of metformin and 2-deoxyglucose in reversing multidrug resistance.     

Inhibitor of apoptosis proteins: new therapeutic targets in hematological cancer?

Apoptosis is an essential process for the selection and survival of lymphocytes. Resistance to apoptosis can promote malignant transformation of hematopoietic cells. Proteins that regulate apoptosis may therefore be critically involved in the development of hematological cancer. A delicate balance between pro- and antiapoptotic mechanisms determines whether a cell death signal can activate the execution of the apoptotic cell death program. The family of inhibitor of apoptosis (IAP) proteins is a recently identified, novel category of apoptosis-regulatory proteins. IAPs can inhibit the activation of caspases that are the executioners of apoptosis, activated by both the extrinsic and intrinsic pathway. IAPs may thereby set the threshold for apoptosis-activation and play a key role in the regulation of apoptotic cell death. IAPs themselves are also subject to strict regulation through feedback mechanisms. This paper focuses on the role of IAP family proteins in the regulation of apoptosis and discusses implications for their involvement in cancer and possible use for cancer therapy, especially in leukemias and lymphomas.