Multiple resistance to carcinogens and xenobiotics: P-glycoproteins as universal detoxifiers.

The detoxification of toxic substances is of general relevance in all biological systems. The plethora of exogenous xenobiotic compounds and endogenous toxic metabolic products explains the evolutionary pressure of all organisms to develop molecular mechanisms to detoxify and excrete harmful substances from the body. P-glycoprotein and other members of the ATP-binding cassette (ABC) transporter family extrude innumerous chemical compounds out of cells. Their specific expression in diverse biological contexts cause different phenotypes: (1) multidrug resistance (MDR) and thus failure of cancer chemotherapy, (2) avoidance of accumulation of carcinogens and prevention of carcinogenesis in healthy tissues, (3) absorption, distribution, metabolization and excretion (ADME) of pharmacological drugs in human patients, (4) protection from environmental toxins in aquatic organisms (multi-xenobiotic resistance, MXR). Hence ABC-transporters may have opposing effects for organismic health reaching from harmful in MDR of tumors to beneficial for maintenance of health in MXR. While their inhibition by specific inhibitors may improve treatment success in oncology and avoid carcinogenesis, blocking of ABC-transporter-driven efflux by environmental pollutants leads to ecotoxicological consequences in marine biotopes. Poisoned seafood may enter the food-chain and cause intoxications in human beings. As exemplified with ABC-transporters, joining forces in interdisciplinary research may, therefore, be a wise strategy to fight problems in human medicine and environmental sciences.

Prediction of Cancer Drug Resistance and Implications for Personalized Medicine.

Drug resistance still impedes successful cancer chemotherapy. A major goal of early concepts in individualized therapy was to develop in vitro tests to predict tumors' drug responsiveness. We have developed an in vitro short-term test based on nucleic acid precursor incorporation to determine clinical drug resistance. This test detects inherent and acquired resistance in vitro and transplantable syngeneic and xenografted tumors in vivo. In several clinical trials, clinical resistance was predictable with more than 90% accuracy, while drug sensitivity was detected with less accuracy (~60%). Remarkably, clinical cross-resistance to numerous drugs (multidrug resistance, broad spectrum resistance) was detectable by a single compound, doxorubicin, due to its multifactorial modes of action. The results of this predictive test were in good agreement with predictive assays of other authors. As no predictive test has been established as yet for clinical diagnostics, the identification of sensitive drugs may not reach sufficiently high reliability for clinical routine. A meta-analysis of the literature published during the past four decades considering test results of more than 15,000 tumor patients unambiguously demonstrated that, in the majority of studies, resistance was correctly predicted with an accuracy between 80 and 100%, while drug sensitivity could only be predicted with an accuracy of 50-80%. This synopsis of the published literature impressively illustrates that prediction of drug resistance could be validated. The determination of drug resistance was reliable independent of tumor type, test assay, and drug used in these in vitro tests. By contrast, chemosensitivity could not be predicted with high reliability. Therefore, we propose a rethinking of the "chemosensitivity" concept. Instead, predictive in vitro tests may reliably identify drug-resistant tumors. The clinical consequence imply to subject resistant tumors not to chemotherapy, but to other new treatment options, such as antibody therapy, adoptive immune therapy, hyperthermia, gene therapy, etc. The high accuracy to predict resistant tumors may be exploited to develop new strategies for individualized cancer therapy. This new concept bears the potential of a revival of predictive tests for personalized medicine.

Could a revision of the current guidelines for cancer drug use improve the quality of cancer treatment?

Clinical practice guidelines are indispensable for such a variable disease as malignant solid tumors, with the complex possibilities of drug treatment. The current guidelines may be criticized on several points, however. First, there is a lack of information on the outcome of treatment, such as the expected success and failure rates. Treating not only drug responders but also nonresponders, that is, patients with drug resistance, must result in failures. There is no mention of the possibility of excluding the drug nonresponders, identifiable by special laboratory tests and no consideration is given to the different side effects of the recommended drug regimens. Nor are there any instructions concerning tumor cases for which anticancer drug treatment is futile. In such cases, early palliative care may lead to significant improvements in both life quality and life expectancy. Not least, there is no transparency concerning the preparation of the guidelines: persons cannot be identified who could give a statement of conflicts of interest, and responsibility is assumed only by anonymous medical associations. A revision of the current guidelines could considerably improve cancer treatment.

Current status of methods to assess cancer drug resistance.

Drug resistance is the main cause of the failure of chemotherapy of malignant tumors, resistance being either preexisting (intrinsic resistance) or induced by the drugs (acquired resistance). At present, resistance is usually diagnosed during treatment after a long period of drug administration.In the present paper, methods for a rapid assessment of drug resistance are described. Three main classes of test procedures can be found in the literature, i.e. fresh tumor cell culture tests, cancer biomarker tests and positron emission tomography (PET) tests. The methods are based on the evaluation of molecular processes, i.e. metabolic activities of cancer cells. Drug resistance can be diagnosed before treatment in-vitro with fresh tumor cell culture tests, and after a short time of treatment in-vivo with PET tests. Cancer biomarker tests, for which great potential has been predicted, are largely still in the development stage. Individual resistance surveillance with tests delivering rapid results signifies progress in cancer therapy management, by providing the possibility to avoid drug therapies that are ineffective and only harmful.

CD133 is indicative for a resistance phenotype but does not represent a prognostic marker for survival of non-small cell lung cancer patients.

Despite advances in anticancer treatment, lung cancer still has poor prognosis. Recently, a cancer stem cell (CSC) hypothesis has emerged describing a small subset of tumor cells with stem cell properties. CSCs found in many solid tumors express CD133 antigen on the cell surface. The presence of CSC is correlated with poor survival of patients with glioblastomas, colon or prostate cancers. In this study, we evaluated whether CD133 expression in non-small cell lung cancer (NSCLC) has a prognostic value in patients' survival. We also analyzed whether CD133 positivity of NSCLC correlates with the expression of resistance-related proteins, angiogenic factors, oncogenes, proliferative activity or apoptosis. CD133 expression was retrospectively examined in a total of 88 cases of previously untreated NSCLC by immunohistochemistry. We found no correlation between CD133 positivity or the amount of CD133(+) cells with NSCLC patients' survival, expression of oncogenes c-myc, c-N-ras, c-jun, c-fos, c-erbB1, c-erbB2 or p53, angiogenic factors VEGF, VEGFR-1, FGF, FGFR-1, tissue factor and with proliferative activity or apoptosis in NSCLC tissues. However, there was a significant association between the expression of resistance-related proteins glutathione S-transferase, thymidylate synthase, catalase, O(6)-methylguanine-DNA methyltransferase and p170 and CD133. Because CD133 expression is linked to a resistant phenotype, detection of CD133(+) cells may be useful to predict efficacy of cytotoxic therapy but CD133 is not a strong prognostic parameter for survival of patients with NSCLC.

Intrinsic and acquired drug resistance in malignant tumors. The main reason for therapeutic failure.

Drug resistance is the major reason for failure in cancer chemotherapy. Resistance may be either pre-existent (intrinsic resistance), or induced by drugs (acquired resistance), So far, no strategy has been found to overcome resistance, which is based on highly complex and individually variable biological mechanisms. In present clinical practice, drug resistance can only be recognized during treatment, after long lag times. Thus diagnostic tests are re quired, indicating resistance at an earlier stage, in order to avoid unnecessary medication, frequently associated with toxic side-effects. A number of new anti-cancer drugs are now available. In contrast to the unspecifically acting cytostatic chemotherapy, these compounds have targeted actions. However, as recent studies have shown, resistances and severe side-effects can also be found with targeted drugs. With the increasing number of new treatment regimens, the early diagnosis of resistance will optimize therapy, and indeed will be indispensable for individual cancer therapy. The resistance assays available for use in clinical practice should be integrated into cancer therapy. Research into this neglected area needs to be intensified.

Prediction of broad spectrum resistance of tumors towards anticancer drugs.

PURPOSE: Drug resistance is a major obstacle in cancer chemotherapy. Although the statistical probability of therapeutic success is known for larger patient groups from clinical therapy trials, it is difficult to predict the individual response of tumors. The concept of individualized therapy aims to determine in vitro the drug response of tumors beforehand to choose effective treatment options for each individual patient. EXPERIMENTAL DESIGN: We analyzed the cross-resistance profiles of different tumor types (cancers of lung, breast, and colon, and leukemia) towards drugs from different classes (anthracyclines, antibiotics, Vinca alkaloids, epipodophyllotoxins, antimetabolites, and alkylating agents) by nucleotide incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Hierarchical cluster analysis and COMPARE analyses were applied. RESULTS: Tumors exert broad resistance profiles, e.g., tumors resistant to one drug tend to also be resistant to other drugs, whereas sensitive tumors reveal sensitivity towards many drugs. Interestingly, the broad spectrum resistance phenotype could reliably be predicted by doxorubicin alone. Expression of the ATP-binding cassette transporter P-glycoprotein (ABCB1, MDR1) and the proliferative activity of tumors were identified as underlying mechanisms of broad spectrum resistance. To find novel compounds with activity against drug-resistant tumors, a database with 2,420 natural products was screened for compounds acting independent of P-glycoprotein and the proliferative state of tumor cells. CONCLUSIONS: Tumors exert cross-resistance profiles much broader than the classical multidrug resistance phenotype. Broad spectrum resistance can be predicted by doxorubicin due to the multifactorial mode of action of this drug. Novel cytotoxic compounds from natural resources might be valuable tools for strategies to bypass broad spectrum resistance.

Resistance in malignant tumors: can resistance assays optimize cytostatic chemotherapy?

The frequent resistance of malignant tumors to chemotherapy documents the fact that numerous patients are uselessly traumatized by highly toxic drugs. Investigations into this resistance have shown that many different mechanisms exist which can abolish the antitumor action of chemotherapeutics. So far, no safe method has been found to counteract this resistance. To date, the only way to avoid harm by an ineffective chemotherapeutic is to refrain from its use when resistance to it is detected. The present survey reports on the available assays that diagnose resistance reliably; sensitivity tests are excluded as being unreliable. Resistance assays are still not fully recognized in hospitals, despite the fact that they are able to optimize chemotherapy by eliminating treatment that is ineffective and merely harmful. Since the number of chemotherapeutics will increase enormously in the future, pharmacological cancer treatment cannot dispense with resistance assays.

Pharmacogenetics for individualized cancer chemotherapy.

The same doses of medication cause considerable heterogeneity in efficacy and toxicity across human populations. Genetic factors are thought to represent important determinants of drug efficacy and toxicity. Pharmacogenetics focuses on the prediction of the response of tumor and normal tissue to standard therapy by genetic profiling and, thereby, to select the most appropriate medication at optimal doses for each individual patient. In the present review, we discuss the relevance of single nucleotide polymorphisms (SNP) in genes, whose gene products act upstream of the actual drug target sites, that is, drug transporters and drug metabolizing phase I and II enzymes, or downstream of them, that is, apoptosis-regulating genes and chemokines. SNPs in relevant genes, which encode for proteins that interact with anticancer drugs, were also considered, that is, enzymes of DNA biosynthesis and metabolism, DNA repair enzymes, and proteins of the mitotic spindle. A significant body of evidence supports the concept of predicting drug efficacy and toxicity by SNP genotyping. As the efficacy of cancer chemotherapy, as well as the drug-related toxicity in normal tissues is multifactorial in nature, sophisticated approaches such as genome-wide linkage analyses and integrate drug pathway profiling may improve the predictive power compared with genotyping of single genes. The implementation of pharmacogenetics into clinical routine diagnostics including genotype-based recommendations for treatment decisions and risk assessment for practitioners represents a challenge for the future.

Glutathione-related enzymes contribute to resistance of tumor cells and low toxicity in normal organs to artesunate.

The anti-malarial artesunate (ART) also inhibits the growth of cancer cells. The active moiety is an endoperoxide bridge whose cleavage generates reactive oxygen species and free radicals. We analyzed whether glutathione-related enzymes contribute to tumor resistance to ART and to the low toxicity of ART towards normal organs. The microarray-based mRNA expression of dihydrodiol dehydrogenase, gamma-glutamylcysteine synthase (gamma-GCS), glutathione S-transferases GSTM4, GSTT2, GSTZ1, and microsomal glutathione S-transferase MGST3 showed significant relationships (p <0.05) to cellular response to ART in 55 cell lines of the National Cancer Institute, USA. A tendency for correlation (0.05

Protein Expression Profiles Indicative for Drug Resistance of Kidney Carcinoma.

The purpose of this investigation was to evaluate whether different protein patterns exist between drug-sensitive and drug-resistant kidney carcinomas. As a first step, expressions of drug resistance proteins (P-glycoprotein (P-gp), glutathione S-transferase-σ (GST-σ), DNA topoisomerase IIα (Topo IIα), alkaline phosphatase (AP), catalase, thymidylate synthetase, metallothionein), signal transducers (protein kinase Cα/ß (PKCα/ß)), proliferation-associated proteins (Ki-67) and proteins of proto-oncogenes and tumor suppressors (ErbB1, ErbB2, Fos, Jun, Myc, Ras and p53) of primary cell cultures of human kidney carcinomas of 18 patients were determined. The expression levels of the proteins were compared with the response to doxorubicin, vincristine or mafosfamide measured by growth inhibition and nucleotide incorporation assays. As a second step, those proteins showing a relationship to doxorubicin resistance (P-gp, GST-σ, Topo IIα, PKCα/ß, AP, ErbB1, ErbB2, Fos, K-Ras, p53, and Ki-67) were analyzed by hierarchical cluster analysis and clustered image mapping. The resulting clusters were correlated with the drug resistance data. The data shows that different protein expression profiles exist between drug-resistant and -sensitive kidney carcinoma cell cultures. Finally, the clustered image map (CIM) demonstrates a sensitive area that is characterized by a lower expression of proteins and a resistant area with a higher expression of proteins. These results may have important implications for the diagnosis and therapy of kidney cancer. The resistance proteins and the resistance-related factors found in the present analysis may be suitable parameters to predict treatment outcome of kidney cancer.

Lung Cancer Protein Expression Profiles of Smokers and Non-Smokers.

Epidemiological studies have established a causal relationship between cigarette smoking and respiratory tract cancer. The knowledge about the interaction of tobacco constituents with cellular systems is, however, still incomplete. Therefore, we analyzed 36 factors with known or assumed relevance and found that 8 proteins in lung cancer were associated with the smoking habits of 94 patients. These 8 factors belong to different functional classes including products of drug resistance-related proteins (P-glycoprotein, glutathione S-transferase-π, lung resistance protein, catalase), proto-oncogenes and transcription factors (FOS, JUN, HIF-1ß), and proliferative factors (cyclin D). By means of hierarchical cluster analysis, we were able to show that the 94 patients analyzed could be separated into three different clusters, of which one contained significantly more patients who smoked than the others (p=0.0026). This cluster also contained significantly more drug-resistant tumors than the others (p=0.0069), pointing to a close interrelationship between the smoking habits of patients and drug resistance of tumors.

Prediction of Drug Sensitivity and Resistance of Cancer by Protein Expression Profiling.

Although the statistical probability of therapeutic success is known for larger groups of cancer patients, the clinical response to chemotherapy of the individual patient remains uncertain. It would be of great value to know whether or not an individual tumor responds to the proposed therapy. The concept of sensitivity testing of tumors for individualized therapy traces back to the 1970s. Currently, an astonishing revival has taken place due to the thriving development of genomic and proteomic technologies. This review discusses our own results on protein expression profiles of non-small cell lung cancer, kidney carcinoma and acute lymphoblastic leukemia regarding the prediction of drug sensitivity or resistance. A great diversity of drug resistance mechanisms are operative in the clinical drug resistance of cancer e.g., resistance proteins, proliferative, apoptotic, angiogenic factors, proto-oncogenes and tumor suppressor-genes. Hierarchical cluster analyses and cluster image maps reveal different resistance profiles even within cancer types of homogeneous histology. Protein arrays may be appropriate to perform sensitivity or resistance tests for individual patients because thousands of proteins may be detected in a single experiment. On the other hand, results suggest that already a set of a limited number of factors may be sufficient to detect the sensitivity or resistance of a cancer.

Imbalance of cell proliferation and apoptosis during progression of lung carcinomas.

To evaluate the relationship between cell proliferation and apoptosis during progression of lung carcinomas, immunohistochemistry for proliferating cell nuclear antigen (PCNA) and the in situ end labelling (TUNEL) method for identifying apoptotic bodies were performed on paraffin sections from 135 lung carcinomas. These results were correlated with the corresponding tumor volumes as a model of disease progression in lung tumors. We found that, with increasing tumor volume, the proliferation rate decreased significantly, whereas the apoptotic rate increased. There was no relationship between apoptotic and proliferative indices except in carcinomas with a tumor volume between 51 and 100 cm3. These data suggest that progression of lung carcinomas, i.e. the increase in tumor volume, is accompanied by an increase in apoptosis rather than an increase in cell proliferation.

Molecular modes of action of artesunate in tumor cell lines.

A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S. National Cancer Institute (NCI). The 50% inhibition concentrations (IC50 values) for ART correlated significantly to the cell doubling times (P = 0.00132) and the portion of cells in the G0/G1 (P = 0.02244) or S cell cycle phases (P = 0.03567). We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base. These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes). The constitutive expression of 54 of 465 (=12%) genes correlated significantly to the IC50 values for ART. Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P = 0.00017). For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship. The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for gamma-glutamylcysteine synthetase increased resistance to ART. The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART. Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART. ART acts via p53-dependent and- independent pathways in isogenic p53+/+ p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/- colon carcinoma cells.

Modulation of multidrug resistance in human ovarian cancer cell lines by inhibition of P-glycoprotein 170 and PKC isoenzymes with antisense oligonucleotides.

Multidrug resistance in human ovarian carcinoma cell lines is caused by the expression of several related proteins, namely P-glycoprotein 170 (Pgp-170), glutathione S-transferase-pi GST-pi), and thymidylate synthase (TS). These proteins seem to be regulated by a common mechanism in which the expression of protein kinase C (PKC) is involved. Additionally, the function of Pgp-170 is dependent on PKC phosphorylation. However, in ovarian carcinoma cell lines the role of different PKC enzymes responsible for resistance is not quite clear. In the present study we circumvented resistance in taxol resistant human ovarian carcinoma cell lines with antisense oligonucleotides to PKC alpha and PKC beta mRNA and compared the effects with those obtained by Pgp-170 antisense oligonucleotides. We found a significant inhibition of cell number after treatment with Pgp-170 antisense oligonucleotides in combination with taxol. Additionally, resistance could be reversed by treatment with taxol and antisense oligomers to PKC alpha and PKC beta. This shows that regulatory correlations between these proteins exist and that inhibition of the mRNA of PKC alpha and PKC beta isoforms and Pgp-170 can reverse multidrug resistance.

Expression of drug resistance gene products during progression of lung carcinomas.

In order to study whether the presence of mechanisms of drug resistance is a characteristic unique to advanced lung cancer or occurs already early in the course of the disease, we investigated the expression of gluathione S-transferase-pi (GST-pi), P-glycoprotein (P-gp) and metallothionein (MT) in 80 human lung carcinomas and in 20 normal lung tissues using immunohistochemistry. We found that all three proteins were expressed in resected normal lung and lung carcinomas. Expression of GST-pi and MT was elevated in tumor tissues in comparison to normal lung tissues, whereas P-gp was highly expressed in normal lung. GST-pi and MT expression increased with increasing tumor volume and differentiation grade. These results suggest that the level of GST-pi and MT in lung cells increases as cells progress from the normal to the transformed state and that drug resistance gene products are already present in lung carcinomas at the time of surgical resection.

Protein expression profile of primary human squamous cell lung carcinomas indicative of the incidence of metastases.

The purpose of this investigation was to evaluate firstly whether different protein expression patterns exist in primary squamous cell lung carcinomas of patients with and without lymph node involvement and secondly, whether or not different patterns exist in tumours with positive lymph nodes. For this reason, formalin-fixed, paraffin-embedded specimens from 130 patients with squamous cell lung carcinomas were analyzed by immunohistochemistry. In a first step, proteins were selected which showed a relationship to lymph node involvement. The expression of JUN, ERBB2, MYC, cyclin D, PCNA, bFGF, VEGF and Hsp70 proteins revealed a positive correlation to lymph node involvement. In contrast, caspase-3, Fas ligand, Fas/CD95, and PAI showed an inverse correlation to lymph node involvement. In a second step, these parameters were further analyzed by hierarchical cluster analyses. The resulting clusters were correlated to patients with or without lymph node involvement. The data show that different protein expression patterns exist between primary squamous cell lung carcinomas with and without lymph node involvement and within carcinomas with lymph node involvement. The data suggest that various metastasis profiles exist.