Novel anticancer drug discovery.

There is at present, much optimism about the possibility of finding selective anticancer drugs that will eliminate the cytotoxic side effects associated with conventional cancer chemotherapy. This hope is based on uncovering many novel molecular targets that are 'cancer-specific', which will allow the targeting of cancer cells while normal cells are spared. Thus far, encouraging results have been obtained with several of these novel agents at the preclinical level, and clinical trials have begun. These targets are involved at one level or more in tumor biology, including tumor cell proliferation, angiogenesis and metastasis. Novel targets for which advances are being made include the following: growth factor receptor tyrosine kinases such as the epidermal growth factor receptor and HER-2/neu (proliferation); the vascular endothelial growth factor receptor and the basic fibroblast growth factor receptor (angiogenesis); the oncogenic GTP-binding protein Ras (especially agents targeting Ras farnesylation, farnesyltransferase inhibitors) (proliferation); protein kinase C (proliferation and drug resistance); cyclin-dependent kinases (proliferation); and matrix metalloproteinases and angiogenin (angiogenesis and metastasis). Less explored, but potentially useful targets include the receptor tyrosine kinase platelet-derived growth factor receptor, mitogen-activated protein kinase cascade oncogenes such as Raf-1 and mitogen-activated protein kinase kinase, cell adhesion molecules such as integrins, anti-apoptosis proteins such as Bcl-2, MDM2 and survivin, and the cell life-span target telomerase.     

Autophagy is a therapeutic target in anticancer drug resistance

Autophagy is a type of cellular catabolic degradation response to nutrient starvation or metabolic stress. The main function of autophagy is to maintain intracellular metabolic homeostasis through degradation of unfolded or aggregated proteins and organelles. Although autophagic regulation is a complicated process, solid evidence demonstrates that the PI3K-Akt-mTOR, LKB1-AMPK-mTOR and p53 are the main upstream regulators of the autophagic pathway. Currently, there is a bulk of data indicating the important function of autophagy in cancer. It is noteworthy that autophagy facilitates the cancer cells' resistance to chemotherapy and radiation treatment. The abrogation of autophagy potentiates the re-sensitization of therapeutic resistant cancer cells to the anticancer treatment via autophagy inhibitors, such as 3-MA, CQ and BA, or knockdown of the autophagy related molecules. In this review, we summarize the accumulation of evidence for autophagy's involvement in mediating resistance of cancer cells to anticancer therapy and suggest that autophagy might be a potential therapeutic target in anticancer drug resistance in the future.     

Apoptosis enzyme-linked immunosorbent assay distinguishes anticancer drugs from toxic chemicals and predicts drug synergism

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.     

Free radicals and anticancer drug resistance: Oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.     

Reversal of anticancer drug resistance by COTC based on intracellular glutathione and glyoxalase I

Suppression of resistance to anticancer drugs by COTC of glyoxalase I (GloI) inhibitor targeting intracellular glutathione (GSH) and GloI was studied. Depletion of the cellular GSH content and inhibition of GloI by COTC increased chemotherapy-mediated apoptosis in apoptosis-resistant pancreatic adenocarcinoma AsPC-1 cells.     

Apoptosis in non-small cell lung cancer as related to drug resistance and prognosis

The percentage of apoptotic cells among the tumor cells (apoptotic index) was determined in a series of 178 non-small cell lung carcinomas with a long term clinical follow-up by a terminal desoxynucleotidyl transferase mediated dUTP nick end labelling technique. In survival analysis, we found no statistically significant correlation between the apoptotic index and survival times. We estimated also the sensitivity of specimens to doxorubicin by a short term test. Tumors with a high apoptotic activity were more sensitive to doxorubicin than tumors with a less apoptotic index. Thus, our data indicate that apoptosis may be involved in drug response of lung tumors and it could be useful for the chemotherapeutic strategy to design drugs which trigger apoptosis.     

Kinetic resistance to anticancer agents

Adherent epithelial cancer cells, such as colon cancer cells, are much more resistant to anthracyclines and to many other major anticancer agents when the cell population reaches confluence. Our purpose is to analyze the mechanisms of this confluence dependent resistance (CDR) that is probably the major cause of the natural resistance of solid tumors to chemotherapy. Some drugs (anthracyclines, etoposide and vincristine) but not others (cisplatin, melphalan and 5-fluorouracil) accumulate less in confluent than in nonconfluent cells. A decrease of the passive transmembrane drug transport in confluent cells is associated to a reduced membrane fluidity. However, the predominant mechanism of CDR is an increase in the intrinsic resistance of the DNA to the drug-induced damage. This mechanism is now relatively well understood for anthracyclines and etoposide that act mainly through an inhibition of the topoisomerase II: as the enzyme level is low in slowly proliferating confluent cells, the number of drug-induced DNA strand breaks is lower than in rapidly growing nonconfluent cells which highly express the topoisomerase II gene. Mechanisms of CDR for the other drugs are less clear and could involve an increase in the ability to repair damaged DNA. Attempts to circumvent CDR could consist in the stimulation of the cell proliferation by hormones or growth factors, or in the recruitment of quiescent cells into the S and G2 phases by previous treatment of confluent cells with infratoxic concentration of DNA-damaging agents.     

Using mRNA expression profiling to determine anticancer drug efficacy.

Pharmacogenomics is a fast-growing field of investigations that aims to further elucidate the inherited nature of interindividual differences in drug disposition and effects, with the ultimate goal of providing a stronger scientific basis for selecting the optimal drug therapy. Providing the right drug for the right patient is an important problem in the treatment of cancer. This is mainly due to the lack of information about the sensitivity of the tumor for a specific treatment modality, such as either chemotherapy or radiation treatment. This presentation highlights two approaches to identify responsiveness to treatment. Both approaches are based on the identification of expression profiles. The first approach concentrates on drug resistance and the second on the signaling pathways leading up to the death of the cell. Both approaches provide expression profiles; however, the more dynamic expression profiling as used to determine the signaling in damage cells promises to be a better determinant for the pharmacogenomic changes in expression profiles and, consequently, a potential better determinant for drug efficacy.     

Antimalarial drug resistance and combination chemotherapy.

Antimarial drug resistance develops when spontaneously occurring parasite mutants with reduced susceptibility are selected, and are then transmitted. Drugs for which a single point mutation confers a marked reduction in susceptibility are particularly vulnerable. Low clearance and a shallow concentration-effect relationship increase the chance of selection. Use of combinations of antimalarials that do not share the same resistance mechanisms will reduce the chance of selection because the chance of a resistant mutant surviving is the product of the per parasite mutation rates for the individual drugs, multiplied by the number of parasites in an infection that are exposed to the drugs. Artemisinin derivatives are particularly effective combination partners because (i) they are very active antimalarials, producing up to 10,000-fold reductions in parasite biomass per asexual cycle; (ii) they reduce malaria transmissibility; and (iii) no resistance to these drugs has been reported yet. There are good arguments for no longer using antimalarial drugs alone in treatment, and instead always using a combination with artemisinin or one of its derivatives.     

Microtubule-targeting anticancer drug eribulin induces drug efflux transporter P-glycoprotein

This study examined the effects of microtubule-targeting anticancer drugs (paclitaxel, cabazitaxel, and eribulin) on the expression of drug efflux transporter P-glycoprotein, which is encoded by MDR1. Paclitaxel and eribulin induced MDR1 promoter activity in a concentration-dependent manner, while cabazitaxel had little effect in human intestinal epithelial LS174T cells. Overexpression of the nuclear receptor pregnane X receptor (PXR) gene (NR1I2) enhanced paclitaxel- and eribulin-induced MDR1 activation, but expression of the nuclear receptor co-repressor silencing mediator for retinoid and thyroid receptors (SMRT) gene (NCOR2) repressed MDR1 activation. Eribulin increased the mRNA and protein expression of P-glycoprotein in LS174T cells. Cellular uptake of rhodamine 123 and calcein-acetoxymethyl ester (calcein-AM), P-glycoprotein substrates, decreased in paclitaxel- or eribulin-treated LS174T cells. Eribulin also increased MDR1 promoter activity in human breast cancer MCF7 cells. The results suggest that the microtubule-targeting anticancer drug eribulin can induce the drug efflux transporter P-glycoprotein via PXR in human intestinal and breast cancer cells and thus influence the efficacy of anticancer drugs.     

Free radicals in anticancer drug pharmacology

This review examines the formation of free radical intermediates from a number of clinically active antitumor agents including quinone-containing antibiotics and etoposide. An attempt is also made to relate the formation of these reactive intermediates to biochemical and pharmacological basis for tumor cell kill and resistance. The formation of these intermediates in some tumor cells has been detected by both direct ESR and spin-trapping technique. The detection of free radicals in biological systems, however, depends upon cellular bioenvironments, e.g. reducing conditions, and the presence and/or absence of activation and detoxification mechanisms. Evidence shows that certain antitumor drugs generate free radicals in vitro and in vivo and that these reactive species kill tumor cells by causing damage to DNA, membranes or enzymes.     

Kinetic simulation of anticancer drug interactions

A model is described that simulates the biochemical pathways of folate and nucleotide metabolism involved in DNA precursor biosynthesis. Examples are given of use of the model to study various aspects of the biochemical pharmacology of antitumour drugs. Modelling may be done in two ways: detailed simulation of all variables may be conducted for short time periods (<6 h of real time); alternatively, by assuming that concentrations of rapidly interconvertible metabolites remain close to steady-state proportions, time periods of several days may be modelled, facilitating simulation of cell growth in presence of drugs. Experiments designed to test predictions for these various types of study are described.     

Discovery and development of sesquiterpenoid derived hydroxymethylacylfulvene: a new anticancer drug.

Hydroxymethylacylfulvene (HMAF, MGI 114) is derived from the sesquiterpene illudin S by treatment with dilute sulfuric acid and excess paraformaldehyde. It is less cytotoxic than illudin S but exhibits much greater selectivity in toxicity to malignant cells compared to normal cells. HMAF is believed to undergo bioreductive activation in vivo. It is now being tested in human clinical phase II trials against solid tumors.     

Dependence on RAD52 and RAD1 for anticancer drug resistance mediated by inactivation of mismatch repair genes

Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3], [4]. We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins – in addition to their role in DNA-damage recognition – decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.