Proteomic approaches for studying chemoresistance in cancer

The role of various proteins involved in drug resistance in tumor cells is discussed in this review. Two types of studies are covered: those performed in the preproteomics era and those carried out with modern proteomic tools, namely 2D (electrophoretic) maps and 2D chromatography. In the preproteomic studies, one protein had generally been held responsible for a given chemoresistance. However, analysis via proteomic tools may reveal entire sets of proteins that are up- or downregulated (or switched on/off) in chemoresistant tumor cell lines compared with parental tumor lines. Therefore, it appears more realistic to expect that exposure of cells to drugs results in the activation of different mechanisms of resistance. Such investigations have led to the broadly shared opinion that exposure of cells to drugs results in the activation of different mechanisms of resistance, and that a specific drug-resistant phenotype consists of several molecular mechanisms that are simultaneously active. The proteomic papers reviewed clearly support the hypothesis that many metabolic pathways are affected during the resistance process. Although the modulation of expression levels of such proteins is not clear proof of their role in drug resistance per se, at least some of the themes are very likely to be involved in the resistance phenotype, and thus may be potential targets for new drugs. It is hoped that this review will bring new insight in this field and will stimulate novel and deeper searches with proteomic tools (including prefractionation of subcellular organelles, such as nuclei, to bring to the fore low-abundance proteins that might be responsible for the onset of drug resistance).     

Mechanisms of altered sequestration and efflux of chemotherapeutic drugs by multidrug-resistant cells

This review considers the mechanisms associated with the pleiotropic resistance of cancer cells to chemotherapeutic drugs, and more particularly those related to intracellular pH (pHi). The multidrug resistance (MDR) phenomenon responsible for the decreased accumulation and increased efflux of cytotoxic drugs is generally associated with excess levels of P-glycoproteins (Pgps) encoded by MDR genes and/or the multidrug resistance-associated protein (MRP). MDR cell lines, derived from normal or tumor cells, frequently exhibit abnormally elevated pHi and changes in the production of various proteins. Recent studies have suggested that, in addition to the impact of the ATP-dependent membrane transporters Pgp and MRP on drug transport, other mechanisms linked to pHi changes in MDR cells may play an important role in drug resistance. We have shown that alkalinization of the acidic compartments (endosomes and lysosomes) by lysosomotropic agents could stimulate the efflux of vinblastine from drug-resistant mouse renal proximal tubule cells. The fact that weak base chemotherapeutic drugs can be sequestered within the acidic organelles of MDR cells sheds new light on the cellular mechanisms of drug resistance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modeling adaptive drug resistance of colorectal cancer and therapeutic interventions with tumor spheroids

Drug resistance is a major barrier against successful treatments of cancer patients. Various intrinsic mechanisms and adaptive responses of tumor cells to cancer drugs often lead to failure of treatments and tumor relapse. Understanding mechanisms of cancer drug resistance is critical to develop effective treatments with sustained anti-tumor effects. Three-dimensional cultures of cancer cells known as spheroids present a biologically relevant model of avascular tumors and have been increasingly incorporated in tumor biology and cancer drug discovery studies. In this review, we discuss several recent studies from our group that utilized colorectal tumor spheroids to investigate responses of cancer cells to cytotoxic and molecularly targeted drugs and uncover mechanisms of drug resistance. We highlight our findings from both short-term, one-time treatments and long-term, cyclic treatments of tumor spheroids and discuss mechanisms of adaptation of cancer cells to the treatments. Guided by mechanisms of resistance, we demonstrate the feasibility of designing specific drug combinations to effectively block growth and resistance of cancer cells in spheroid cultures. Finally, we conclude with our perspectives on the utility of three-dimensional tumor models and their shortcomings and advantages for phenotypic and mechanistic studies of cancer drug resistance.     

A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431

Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant subline, A431/Pt, were used as a model system. The experimental set-up involved not just a two-way comparison of the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat-shock proteins HSP60, HSP90, HSC71, heat-shock cognate 71 kDa protein), Ca2+-binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione-S-transferase, GST), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ion channels (such as VDAC-1, voltage-dependent anion-selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.     

Nano carriers that enable co-delivery of chemotherapy and RNAi agents for treatment of drug-resistant cancers

Tumor cells exhibit drug resistant phenotypes that decrease the efficacy of chemotherapeutic treatments. The drug resistance has a genetic basis that is caused by an abnormal gene expression. There are several types of drug resistance: efflux pumps reducing the cellular concentration of the drug, alterations in membrane lipids that reduce cellular uptake, increased or altered drug targets, metabolic alteration of the drug, inhibition of apoptosis, repair of the damaged DNA, and alteration of the cell cycle checkpoints ( and ). siRNA is used to silence the drug resistant phenotype and prevent this drug resistance response. Of the listed types of drug resistance, pump-type resistance (e.g., high expression of ATP-binding cassette transporter proteins such as P-glycoproteins (Pgp; also known as multi-drug resistance protein 1 or MDR1, encoded by the ATP-Binding Cassette Sub-Family B Member 1 (ABCB1) gene)) and apoptosis inhibition (e.g., expression of anti-apoptotic proteins such as Bcl-2) are the most frequently targeted for gene silencing. The co-delivery of siRNA and chemotherapeutic drugs has a synergistic effect, but many of the current projects do not control the drug release from the nanocarrier. This means that the drug payload is released before the drug resistance proteins have degraded and the drug resistance phenotype has been silenced. Current research focuses on cross-linking the carrier's polymers to prevent premature drug release, but these carriers still rely on environmental cues to release the drug payload, and the drug may be released too early. In this review, we studied the release kinetics of siRNA and chemotherapeutic drugs from a broad range of carriers. We also give examples of carriers used to co-deliver siRNA and drugs to drug-resistant tumor cells, and we examine how modifications to the carrier affect the delivery. Lastly, we give our recommendations for the future directions of the co-delivery of siRNA and chemotherapeutic drug treatments.     

Molecular mechanisms of drug resistance and its reversal in cancer

Chemotherapy is the main strategy for the treatment of cancer. However, the main problem limiting the success of chemotherapy is the development of multidrug resistance. The resistance can be intrinsic or acquired. The resistance phenotype is associated with the tumor cells that gain a cross-resistance to a large range of drugs that are structurally and functionally different. Multidrug resistance arises via many unrelated mechanisms, such as overexpression of energy-dependent efflux proteins, decrease in uptake of the agents, increase or alteration in drug targets, modification of cell cycle checkpoints, inactivation of the agents, compartmentalization of the agents, inhibition of apoptosis and aberrant bioactive sphingolipid metabolism. Exact elucidation of resistance mechanisms and molecular and biochemical approaches to overcome multidrug resistance have been a major goal in cancer research. This review comprises the mechanisms guiding multidrug resistance in cancer chemotherapy and also touches on approaches for reversing the resistance.     

Differential protein expression analysis of Leishmania major reveals novel roles for methionine adenosyltransferase and S-adenosylmethionine in methotrexate resistance

Leishmania is a trypanosomatid parasite causing serious disease and displaying resistance to various drugs. Here, we present comparative proteomic analyses of Leishmania major parasites that have been either shocked with or selected in vitro for high level resistance to the model antifolate drug methotrexate. Numerous differentially expressed proteins were identified by these experiments. Some were associated with the stress response, whereas others were found to be overexpressed due to genetic linkage to primary resistance mediators present on DNA amplicons. Several proteins not previously associated with resistance were also identified. The role of one of these, methionine adenosyltransferase, was confirmed by gene transfection and metabolite analysis. After a single exposure to low levels of methotrexate, L. major methionine adenosyltransferase transfectants could grow at high concentrations of the drug. Methotrexate resistance was also correlated to increased cellular S-adenosylmethionine levels. The folate and S-adenosylmethionine regeneration pathways are intimately connected, which may provide a basis for this novel resistance phenotype. This thorough comparative proteomic analysis highlights the variety of responses required for drug resistance to be achieved.     

Overexpression of interleukin-2 receptor alpha in a human squamous cell carcinoma of the head and neck cell line is associated with increased proliferation,drug resistance,and transforming ability

It has been previously demonstrated that human carcinomas express interleukin-2 receptor (IL-2R) alpha, beta, and gamma chains. The beta and gamma chains of IL-2R have intermediate binding affinity for IL-2 and are responsible for the intracellular signaling cascades after IL-2 stimulation. IL-2Ralpha lacks the cytoplasmic domain, but is essential for increasing the IL-2-binding affinity of other receptors. Overexpression of IL-2Ralpha in tumor cells is associated with tumor progression and a poor patient prognosis. To define molecular mechanisms responsible for the effects associated with IL-2Ralpha expression, ex vivo experiments were performed with the squamous cell carcinoma head-and-neck cancer line, PCI-13, which was genetically engineered to overexpress the IL-2Ralpha chain. While IL-2Ralpha-overexpressing PCI-13 cells were capable of forming colonies in soft agar, PCI-13 cells transfected with the control vector or those expressing IL-2Rgamma did not. Consistently, IL-2Ralpha-expressing tumor cells proliferated more rapidly than the control or IL-2Rgamma+ cells, associated with increased levels of cyclins A and D1 and cyclin-dependent kinase (cdk(s)) 2 and 4 proteins. In addition, IL-2Ralpha-expressing cells were significantly more resistant to apoptosis induction by a tripeptidyl proteasome inhibitor (ALLN) and two chemotherapeutic drugs (VP-16 and taxol) than the control or IL-2Rgamma+ cells. Accompanying the drug resistance, high levels of anti-apoptotic Bcl-X(L) and Bcl-2 proteins were found in the mitochondria-containing fraction of IL-2Ralpha-expressing tumor cells. Treatment of IL-2Ralpha-expressing cells with a specific Janus kinase 3 (Jak3) inhibitor decreased expression of cyclin A, cyclin D1, Bcl-X(L), and Bcl-2 proteins. Finally, high levels of ubiquitinated proteins were detected in the proliferating IL-2Ralpha-expressing cells. Our data suggest that increased proliferation rates and decreased drug sensitivity of IL-2Ralpha-expressing tumor cells are responsible for the enhanced tumor aggressiveness and poor clinical prognosis of patients whose tumors express IL-2Ralpha.     

Influence of human tumor suppressor PTEN on sensitivity of malignant cells to anticancer drugs

The influence of the human tumor suppressor PTEN on sensitivity of tumor cells to cytostatic drugs was studied. Rat ras-transformed (N-ras ^Asp12 ) fibroblasts were stably transfected with a full-size PTEN gene. Transfected clone was characterized by an enhanced expression of PTEN and a more normal phenotype in comparison with the parental cells. The effect of transient transfection with PTEN on the sensitivity of several malignant cell lines to the cytostatic drugs colchicine and adriablastine was studied. These drugs differ from each other in action mechanisms and intracellular targets. The tumor cell lines tested in this study included parental cell lines and stable sublines possessing drug resistance due to overexpression of P-glycoprotein. In all cell lines, introduction of exogenous PTEN caused a decrease in proliferation rates. This indicated that transgene was active. The chemosensitivity of some drug-resistant sublines was changed after PTEN transfection, but the drug sensitivity of parental cell lines remained unaffected. The effect of PTEN overexpression on chemosensitivity of malignant cells to cytostatic drugs was found to depend both on their mechanisms of action and on the origin of transfected cells. Our data suggest that PTEN is involved into the molecular mechanisms of drug resistance in cells studied.     

Autocrine production of interleukin-8 confers cisplatin and paclitaxel resistance in ovarian cancer cells

It has been widely reported that interleukin-8 (IL-8) is overexpressed in ovarian cyst fluid, ascites, serum, and tumor tissue from ovarian cancer (OVCA) patients, and elevated IL-8 expression correlates with a poor final outcome and chemosensitivity. However, the role of IL-8 expression in the acquisition of the chemoresistance phenotype and the underlining mechanisms of drug resistance in OVCA cells are not yet fully understood. Here we show that both exogenous (a relatively short period of treatment with recombination IL-8) and endogenous IL-8 (by transfecting with plasmid encoding for sense IL-8) induce cisplatin and paclitaxel resistance in non-IL-8-expressing A2780 cells, while deleting of endogenous IL-8 expression in IL-8-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-8) promotes the sensitivity of these cells to anticancer drugs. IL-8-mediated resistance of OVCA cells exhibits decreased proteolytic activation of caspase-3. Meanwhile, the further study demonstrates that the chemoresistance caused by IL-8 is associated with increased expression of both multidrug resistance-related genes (MDR1) and apoptosis inhibitory proteins (Bcl-2, Bcl-xL, and XIAP), as well as activation of PI3 K/Akt and Ras/MEK/ERK signaling. Therefore, modulation of IL-8 expression or its related signaling pathway may be a promising strategy of treatment for drug-resistant OVCA.     

Cellular mechanisms of multidrug resistance of tumor cells

Multidrug resistance (MDR) is the protection of a tumor cell population against numerous drugs differing in chemical structure and mechanisms of influence on the cells. MDR is one of the major causes of failures of chemotherapy of human malignancies. Recent studies show that the molecular mechanisms of MDR are numerous. Cellular drug resistance is mediated by different mechanisms operating at different steps of the cytotoxic action of the drug from a decrease of drug accumulation in the cell to the abrogation of apoptosis induced by the chemical substance. Often several different mechanisms are switched on in the cells, but usually one major mechanism is operating. The most investigated mechanisms with known clinical significance are: a) activation of transmembrane proteins effluxing different chemical substances from the cells (P-glycoprotein is the most known efflux pump); b) activation of the enzymes of the glutathione detoxification system; c) alterations of the genes and the proteins involved into the control of apoptosis (especially p53 and Bcl-2).     

Comparative 2D-DIGE proteomic analysis of ovarian carcinoma cells: toward a reorientation of biosynthesis pathways associated with acquired platinum resistance

Ovarian cancer is the fifth most frequent cause of cancer death in women. Emergence of chemoresistance in the course of treatments with platinum drugs is in part responsible for therapeutic failures. In order to improve the understanding of the complex mechanisms involved in acquired platinum chemoresistance, we decided to compare the basal protein expression profile of the platinum-sensitive cell line OAW42 and that of its resistant counterpart OAW42-R by a proteomic approach. Reversed-phase HPLC pre-fractionated extracts from both cell lines were subjected to 2D-DIGE coupled to mass spectrometry (MS). Forty eight differentially expressed proteins were identified, 39 being up-regulated and 19 down-regulated in OAW42-R versus OAW42 cells. From the current knowledge on biological activities of most differentially expressed proteins, it can be inferred that the acquisition of resistance was associated with a global reorganization of biochemical pathways favoring the production of precursors for biosynthesis, and with the mobilization of macromolecule quality control mechanisms, preserving RNA and protein integrity under damage-inducing conditions.     

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.     

Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

Verapamil hypersensitivity of vincristine resistant Chinese hamster ovary cell lines

Vincristine resistant CHO cell lines, obtained by prolonged selection in semi-inhibitory drug concentrations show considerable hypersensitivity to verapamil. Their D10 values are around 0.2 micrograms/ml compared to 23 micrograms/ml for unselected controls. Reversion of vincristine resistance during growth in vincristine free medium is correlated with reversal of verapamil sensitivity indicating that the two aspects of the cells' phenotype have a common underlying cause. The rate of uptake of calcium in the absence and presence of verapamil is similar in the vincristine resistant cells and the controls. The correlation of verapamil sensitivity with vincristine resistance is not a universal feature of CHO cell lines resistant to antimicrotubular drugs, since it was found that other resistant cell lines which have been selected by short term exposure to high drug concentrations were not verapamil hypersensitive.     

Proteomic analysis of gemcitabine-induced drug resistance in pancreatic cancer cells

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.     

Type I interferon-mediated pathway interacts with peroxisome proliferator activated receptor-γ (PPAR-γ): At the cross-road of pancreatic cancer cell proliferation

Pancreatic adenocarcinoma remains an unresolved therapeutic challenge because of its intrinsically refractoriness to both chemo- and radiotherapy due to the complexity of signaling and the activation of survival pathways in cancer cells. Recent studies have demonstrated that the combination of some drugs, targeting most of aberrant pathways crucial for the survival of pancreatic cancer cells may be a valid antitumor strategy for this cancer. Type I interferons (IFNs) may have a role in the pathogenesis and progression of pancreatic adenocarcinoma, but the limit of their clinical use is due to the activation of tumor resistance mechanisms, including JAK-2/STAT-3 pathway. Moreover, aberrant constitutive activation of STAT-3 proteins has been frequently detected in pancreatic adenocarcinoma. The selective targeting of these cell survival cascades could be a promising strategy in order to enhance the antitumor effects of type I IFNs. The activation of peroxisome proliferator-activated receptor γ (PPAR-γ), on the other hand, has a suppressive activity on STAT-3. In fact, PPAR-γ agonists negatively modulate STAT-3 through direct and/or indirect mechanisms in several normal and cancer models. This review provides an overview on the current knowledge about the molecular mechanisms and antitumor activity of these two promising classes of drugs for pancreatic cancer therapy. Finally, the synergistic antiproliferative activity of combined IFN-β and troglitazone treatment on pancreatic cancer cell lines, evaluated in vitro, and the consequent potential clinical applications will be discussed.     

Molecular mechanism of multidrug resistance in tumor cells

The ability of tumor cells to develop simultaneous resistance to multiple lipophilic cytotoxic compounds represents a major problem in cancer chemotherapy. This review describes recent molecular biological studies which resulted in the identification and cloning of the gene responsible for multidrug resistance in human tumor cells. This gene, designated mdr1, is overexpressed in all and amplified in many of the multidrug-resistant cell lines analyzed. Gene transfer and expression assays have indicated that the mdr1 gene is both necessary and sufficient for multidrug resistance. The product of the mdr1 gene is P-glycoprotein, a transmembrane protein which shares homology with several bacterial proteins involved in active membrane transport. P-glycoprotein appears to function as an energy-dependent efflux pump responsible for the removal of drugs from multidrug-resistant cells. The functions of the mdr system in normal cells and its potential clinical implications are discussed.