Pharmacodynamics and Toxicodynamics of Drug Action: Signaling in Cell Survival and Cell Death

In therapeutic response to drugs, the plasma concentration range leads to the establishment of a safe and effective dosage regimen. Our hypothesis is that by studying drug concentration-dependent effect on signal transduction mechanisms, a better understanding of the beneficial pharmacodynamic and adverse toxicodynamic responses elicited by the drug may be achieved. Using two classes of chemopreventive compounds (phenolic antioxidants and isothiocyanates), we illustrate the potential utility of two signal transduction pathways elicited by these agents to predict the pharmacodynamic effect (induction of Phase II drug metabolizing enzymes) and the potential toxicodynamic response (stimulation of caspase activity and cytotoxic cell death). At lower concentration, phenolic antioxidants and isothiocyanates activate mitogen-activated protein kinase (MAPK; extracellular signal-regulated protein kinase 2, ERK2; and c-Jun N-terminal kinase 1, JNK1) in a concentration-and time-dependent manner. The activation of MAPK by these compounds may lead to the induction of cell survival/protection genes such as c-jun, c-fos, or Phase II drug metabolizing enzymes. However, at higher concentrations, these agents activate another signaling molecule, ICE/Ced3 cysteine protease enzymes (caspases) leading to apoptotic cell death. The activation of these pathways may dictate the fate of the cells/tissues upon exposure to drugs or chemicals. At lower concentrations, these compounds activate MAPK leading to the induction of Phase II genes, which may protect the cells/tissues against toxic insults and therefore may enhance cell survival. On the other hand, at higher concentrations, these agents may activate the caspases, which may lead to apoptotic cell death, and have toxicity. Understanding the activation of these and other signal transduction events elicited by various drugs and chemicals may yield insights into the regulation of gene expression of drug metabolizing enzymes and cytotoxicity. Thus, the study of signaling events in cell survival (hemeostasis) and cell death (cytotoxicity) may have practical application during pharmaceutical drug development.     

Modulation of cell sensitivity to antitumor agents by targeting survival pathways

The advent of drugs targeting tumor-associated prosurvival alterations of cancer cells has changed the interest of antitumor drug development from cytotoxic drugs to target-specific agents. Although single-agent therapy with molecularly targeted agents has shown limited success in tumor growth control, a promising strategy is represented by the development of rational combinations of target-specific agents and conventional antitumor drugs. Activation of survival/antiapoptotic pathways is a common feature of cancer cells that converge in the development of cellular resistance to cytotoxic agents. The survival pathways implicated in cellular response to drug treatment are primarily PI3K/Akt and Ras/MAPK, which also mediate the signalling activated by growth factors and play a role in the regulation of critical processes including cell proliferation, metabolism, apoptosis and angiogenesis. Inhibitors of PI3K, Akt and mTOR have been shown to sensitize selected tumor cells to cytotoxic drugs through multiple downstream effects. Moreover, the MAPK pathway, also implicated in the regulation of gene expression in response to stress stimuli, can interfere with the chemotherapy-induced proapoptotic signals. Targeting Hsp90, which acts as a molecular chaperone for survival factors including Akt, may have the potential advantage to simultaneously block multiple oncogenic pathways. Overall, the available evidence supports the interest of rationally designed approaches to enhance the efficacy of conventional antitumor treatments through the inhibition of survival pathways and the notion that the concomitant targeting of multiple pathways may be a successful strategy to deal with tumor heterogeneity and to overcome drug resistance of tumor cells.     

Bcl-2 phosphorylation in a human breast carcinoma xenograft: a common event in response to effective DNA-damaging drugs

A variety of cytotoxic agents effective as antitumor drugs are known to kill tumor cells through induction of apoptosis as the most relevant modality of cell death. A specific role for the protein Bcl-2 in the cell death pathway induced by antimicrotubule agents has been proposed, because Bcl-2 phosphorylation occurs in response to microtubule damage. In this study, we compared efficacy, apoptosis, and Bcl-2 phosphorylation in the Bcl-2-overexpressing MX-1 human breast carcinoma xenograft after treatment with cytotoxic agents characterized by different mechanisms of action. We demonstrated that, in addition to antimicrotubule agents, effective DNA-damaging agents were also able to induce Bcl-2 phosphorylation irrespective of the type of genotoxic lesion. A comparison of effects of drugs belonging to the same class but endowed with a different antitumor activity (i.e. cisplatin versus a novel multinuclear platinum complex and doxorubicin versus a disaccharide analogue) showed a correlation between drug efficacy, apoptotic response, and Bcl-2 phosphorylation. In conclusion, overexpression of Bcl-2 did not counteract the apoptotic effects of a number of cytotoxic agents and could not be regarded as a mechanism of cellular resistance. Since Bcl-2 phosphorylation is a common event in response to different types of cytotoxic damage and is not only related to microtubule dysfunction, we suggest that many cell death pathways converge on Bcl-2 and protein phosphorylation is a step of the signaling cascade activated by diverse stimuli and likely related to the onset of drug-induced apoptosis.     

Signal transduction events elicited by natural products: Role of MAPK and caspase pathways in homeostatic response and induction of apoptosis

Many natural products elicit diverse pharmacological effects. Using two classes of potential chemopreventive compounds, the phenolic compounds and the isothiocyanates, we review the potential utility of two signaling events, the mitogen-activated protein kinases (MAPKs) and the ICE/Ced-3 proteases (caspases) stimulated by these agents in mammalian cell lines. Studies with phenolic antioxidants (BHA, tBHQ), and natural products (flavonoids; EGCG, ECG, and isothiocyanates; PEITC, sulforaphane), provided important insights into the signaling pathways induced by these compounds. At low concentrations, these chemicals may activate the MAPK (ERK2, JNK1, p38) leading to gene expression of survival genes (c-Fos, c-Jun) and defensive genes (Phase II detoxifying enzymes; GST, QR) resulting in survival and protective mechanisms (homeostasis response). Increasing the concentrations of these compounds will additionally activate the caspase pathway, leading to apoptosis (potential cytotoxicity). Further increment to suprapharmacological concentrations will lead to nonspecific necrotic cell death. The wider and narrow concentration ranges between the activation of MAPK/gene induction and caspases/cell death exhibited by phenolic compounds and isothiocyanates, respectively, in mammalian cells, may reflect their respective therapeutic windowsin vivo. Consequently, the studies of signaling pathways elicited by natural products will advance our understanding of their efficacy and safety, of which many may become important therapeutic drugs of the future.     

The JNK,ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine,doxorubicin, and etoposide

Assessment of specific apoptosis and survival pathways implicated in anticancer drug action is important for understanding drug mechanisms and modes of resistance in order to improve the benefits of chemotherapy. In order to better examine the role of mitogen-activated protein kinases, including JNK and ERK, as well as the tumor suppressor p53, in the response of tumor cells to chemotherapy, we compared the effects on these pathways of three structurally and functionally distinct antitumor agents. Drug concentrations equal to 50 times the concentration required to reduce cell proliferation by 50% were used. Vinblastine, doxorubicin, or etoposide (VP-16) induced apoptotic cell death in KB-3 carcinoma cells, with similar kinetic profiles of PARP cleavage, caspase 3 activation, and mitochondrial cytochrome c release. All three drugs strongly activated JNK, but only vinblastine induced c-Jun phosphorylation and AP-1 activation. Inhibition of JNK by SP600125 protected cells from drug-induced cytotoxicity. Vinblastine caused inactivation of ERK whereas ERK was unaffected in cells exposed to doxorubicin or VP-16. Inhibition of ERK signaling by the MEK inhibitor, U0126, potentiated the cytotoxic effects of vinblastine and doxorubicin, but not that of VP-16. Vinblastine induced p53 downregulation, and chemical inhibition of p53 potentiated vinblastine-induced cell death, suggesting a protective effect of p53. In contrast, doxorubicin and VP-16 induced p53, and inhibition of p53 decreased drug-induced cell death, suggesting a pro-apoptotic role for p53. These results highlight the differential roles played by several key signal transduction pathways in the mechanisms of action of key antitumor agents, and suggest ways to specifically potentiate their effects in a context-dependent manner. In addition, the novel finding that JNK activation can occur without c-Jun phosphorylation or AP-1 activation has important implications for our understanding of JNK function.     

Role of X-linked inhibitor of apoptosis protein in chemoresistance in ovarian cancer: possible involvement of the phosphoinositide-3 kinase/Akt pathway.

Although cisplatin derivatives are first-line chemotherapeutic agents for the treatment of epithelial ovarian cancer, chemoresistance remains a major hurdle to successful therapy and the molecular mechanisms involved are poorly understood. Apoptosis is the cellular underpinning of cisplatin-induced cell death, which is associated with expression of specific "death" genes and down-regulation of "survival" counterparts. The X-linked inhibitor of apoptosis proteins (Xiap), an intracellular anti-apoptotic protein, plays a key role in cell survival by modulating death signaling pathways and is a determinant of cisplatin resistance in ovarian cancer cells in vitro. This review focuses on the role of Xiap and its interactions with the phosphoinositide-3 kinase (PI3K)/Akt cell survival pathway in conferring resistance of ovarian cancer cells to chemotherapeutic agents and discusses potential therapeutic strategies in overcoming chemoresistant ovarian cancer.     

MAPK信号转导通路与肿瘤多药耐药关系的研究进展

丝裂原活化蛋白激酶(Mitogen-activated protein kinases,MAPK)信号途径存在于所有生物体内的大多数细胞中,是真核生物细胞重要的信号转导通路,调节不同的进程,包括增殖、分化以及凋亡。近年来很多研究认为MAPK信号转导通路可能参与肿瘤化疗耐药,其作用机制可能是调控耐药相关基因和蛋白的表达,通过对该通路的干预可以提高肿瘤的化疗敏感性,从而逆转化疗耐药。该文主要对MAPK信号转导通路与肿瘤多药耐药关系的研究进展综述如下。     

Involvement of tyrosine kinase p56/Lck in apoptosis induction by anticancer drugs

Induction of apoptosis is a hallmark of the cellular response of human lymphocytes and lymphoma cells to treatment with anticancer drugs and irradiation. Both treatment modalities trigger apoptosis through intrinsic, mitochondrial apoptosis pathways resulting in the activation of caspases. We and others have shown that the tyrosine kinase p56/Lck is involved in the regulation of apoptosis induced by irradiation or treatment with ceramide but dispensable for death receptor triggered cell death. However, the role of p56/Lck for apoptosis induction in response to anticancer drugs is unclear. To elucidate the putative requirement of p56/Lck for apoptosis signaling of cytotoxic drugs, activation of caspases and alteration of mitochondrial functions were determined in Jurkat T cells, the p56/Lck deficient JCaM1.6 cells and the p56/Lck retransfected JCaM1.6/Lck cells in response to chemotherapeutic drugs with different targets of their primary action. Treatment with Doxorubicin, Paclitaxel or 5-Fluorouracil induced a breakdown of the mitochondrial membrane potential and apoptotic cell death in p56/Lck expressing Jurkat and the retransfected JCaM1.6/Lck cells within 48 h of treatment. However, almost no mitochondrial alterations and no induction of apoptosis could be detected in the p56/Lck deficient JCaM1.6 cells. Correspondingly, activation of caspases-9, -8, and -3 and cleavage of the caspase-3 substrate PARP (poly-(ADP-ribose)-polymerase) were almost completely absent in JCaM1.6 cells while present in p56/Lck positive Jurkat and JCaM1.6/Lck cells. In contrast, retransfection of the cells with the p56/Lck-related tyrosine kinase Src could not restore sensitivity to the treatment with cytotoxic drugs indicating a specific role of the tyrosine kinase p56/Lck in apoptosis signaling. Importantly, kinase-activity of p56/Lck may be dispensable for its pro-apoptoptic action since preincubation with the Src-kinase inhibitor PP2 did not reduce apoptosis induced by cytotoxic drugs. In conclusion, the tyrosine kinase p56/Lck is essential for apoptosis induction by Doxorubicin, Paclitaxel and 5-Fluorouracil regulating early steps of the mitochondrial apoptosis signaling cascade, including alteration of mitochondrial functions and caspase-activation.     

HPMA copolymers for modulating cellular signaling and overcoming multidrug resistance

Unique properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer granted its wide use as a carrier for different nanotherapeutics. However, the role of HPMA in a drug delivery system is not limited solely to a carrier for an active payload. Detailed investigations revealed its deep influence on the molecular mechanisms of antitumor action of chemotherapeutic drugs bound to HPMA copolymer. Such influence involves changing the internalization and intracellular trafficking of an entire HPMA-drug complex, modifying the topography of the accumulation of delivered anticancer drug in the tumor and inside tumor cells, overcoming and suppression of existing drug resistance and preventing its de novo development, inhibition of cellular drug detoxification and intracellular repair mechanisms, affecting cell death signaling pathways and mechanisms of apoptosis and necrosis induction. The present review underlines the major mechanisms of the aforementioned effects leading to the substantial enhancement of cell death-inducing ability of conjugated anticancer drugs.     

Genetic variants in genes involved in mechanisms of chemoresistance to anticancer drugs

Refractoriness to the pharmacological treatment of cancer is dependent on the expression levels of genes involved in mechanisms of chemoresistance and on the existence of genetic variants that may affect their function. Thus, changes in genes encoding solute carriers may account for considerable inter-individual variability in drug uptake and the lack of sensitivity to the substrates of these transporters. Moreover, changes in proteins involved in drug export can affect their subcellular localization and transport ability and hence may also modify the bioavailability of antitumor agents. Regarding pro-drug activation or drug inactivation, genetic variants are responsible for changes in the activity of drug-metabolizing enzymes, which affect drug clearance and may determine the lack of response to anticancer chemotherapy. The presence of genetic variants may also decrease the sensitivity to pharmacological agents acting through molecular targets or signaling pathways. Recent investigations suggest that changes in genes involved in DNA repair may affect the response to platinum-based drugs. Since most anticancer agents activate cell death pathways, the evasion of apoptosis plays an important role in chemoresistance. Several genetic variants affecting death-receptor pathways, the mitochondrial pathway, downstream caspases and their natural modulators, and the p53 pathway, whose elements are mutated in more than half of tumors, and survival pathways, have been reported. The present review summarizes the available data regarding the role of genetic variants in the different mechanisms of chemoresistance and discusses their potential impact in clinical practice and in the development of tools to predict and overcome chemoresistance.     

Biological activities,mechanisms of action and biomedical prospect of the antitumor ether phospholipid ET-18-OCH(3) (edelfosine), a proapoptotic agent in tumor cells

The antitumor ether lipid ET-18-OCH(3) (edelfosine) is the prototype of a new class of antineoplastic agents, synthetic analogues of lysophosphatidylcholine, that shows a high metabolic stability, does not interact with DNA and shows a selective apoptotic response in tumor cells, sparing normal cells. Unlike currently used antitumor drugs, ET-18-OCH(3) does not act directly on the formation and function of the replication machinery, and thereby its effects are independent of the proliferative state of target cells. Because of its capacity to modulate cellular regulatory and signaling events, including those failing in cancer cells, like defective apoptosis, ET-18-OCH(3), beyond its putative clinical importance, is an interesting model compound for the development of more selective drugs for cancer therapy. Although ET-18-OCH(3) enhances host defense mechanisms against tumors, its major antitumor action lies in a direct effect on cancer cells, inhibiting phosphatidylcholine biosynthesis and inducing apoptosis in tumor cells. Recent progress has allowed unraveling the molecular mechanism underlying the apoptotic action of ET-18-OCH(3), leading to the notion that ET-18-OCH(3) is selectively incorporated into tumor cells and induces cell death by intracellular activation of the cell death receptor Fas/CD95. This intracellular Fas/CD95 activation is a novel mechanism of action for an antitumor drug and represents a new way to target tumor cells in cancer chemotherapy that can be of interest as a new framework in designing novel antitumor drugs. ET-18-OCH(3) and some analogues are pleiotropic agents that affect additional biomedical important diseases, including parasitic and autoimmune diseases, suggesting new therapeutic indications for these compounds.     

The oncogenic PIM kinase family regulates drug resistance through multiple mechanisms

Resistance to chemotherapeutic drugs is a significant clinical problem for the treatment of cancer patients and has been linked to the activation of survival pathways and expression of multidrug efflux transporters. Thus inhibition of these survival pathways or efflux transporter expression may increase the efficacy of drug treatment. Here we review the role of the oncogenic PIM kinase family in regulating important proliferation and survival pathways in cancer cells and the involvement of PIM kinases in the expression and activity of MDR-1 and BCRP, two of the most important drug efflux transporters. PIM kinases are over expressed in various types of tumors and regulate the activation of signaling pathways that are important for tumor cell proliferation, survival and expression of drug efflux proteins. This makes PIM kinases attractive targets for the development of anti-cancer chemotherapeutic drugs. Focussing mainly on solid tumors, we provide an update on the literature describing the tumorigenic functions of PIM kinases. Also we provide an overview of the development of selective small molecule PIM kinase inhibitors. Because of the intense effort by pharmaceutical companies and academia it is reasonable to expect that PIM kinase inhibitors will enter the clinic in the foreseeable future. We therefore finish this review with a discussion on the most efficient application of these PIM inhibitors. This includes a consideration of which tumor type is the most appropriate target for treatment, how to select the patient population that stands to gain the most from treatment with PIM inhibitors, which molecular markers are suitable to follow the course of treatment and whether PIM kinase inhibitors should be used as monotherapy or in combination with other cytotoxic agents.     

Targeting cell death in tumors by activating caspases

Cytotoxic approaches to killing tumor cells, such as chemotherapeutic agents, gamma-irradiation, suicide genes or immunotherapy, have been shown to induce cell death through apoptosis. The intrinsic apoptotic pathway is activated following treatment with cytotoxic drugs, and these reactions ultimately lead to the activation of caspases, which promote cell death in tumor cells. In addition, activation of the extrinsic apoptotic pathway with death-inducing ligands leads to an increased sensitivity of tumor cells toward cytotoxic stimuli, illustrating the interplay between the two cell death pathways. In contrast, tumor resistance to cytotoxic stimuli may be due to defects in apoptotic signaling. As a result of their importance in killing cancer cells, a number of apoptotic molecules are implicated in cancer therapy. The knowledge gleaned from basic research into apoptotic pathways from cell biological, structural, biochemical, and biophysical approaches can be used in strategies to develop novel compounds that eradicate tumor cells. In addition to current drug targets, research into molecules that activate procaspase-3 directly may show the direct activation of the executioner caspase to be a powerful therapeutic strategy in the treatment of many cancers.     

Gemcitabine-induced activation of checkpoint signaling pathways that affect tumor cell survival

Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.     

Synergistic combinations of signaling pathway inhibitors: Mechanisms for improved cancer therapy

Cancer cells contain multiple signal transduction pathways whose activities are frequently elevated due to their transformation, and that are often activated following exposure to established cytotoxic therapies including ionizing radiation and chemical DNA damaging agents. Many pathways activated in response to transformation or toxic stresses promote cell growth and invasion and counteract the processes of cell death. As a result of these findings many drugs, predominantly protein and lipid kinase inhibitors, of varying specificities, have been developed to block signaling by cell survival pathways in the hope of killing tumor cells and sensitizing them to toxic therapies. Unfortunately, due to the plasticity of signaling processes within a tumor cell, inhibition of any one growth factor receptor or signaling pathway frequently has only modest long-term effects on cancer cell viability, tumor growth, and patient survival. As a result of this realization, a greater emphasis has begun to be placed on rational combinations of drugs that simultaneously inhibit multiple inter-linked signal transduction/survival pathways. This, it is hoped, will limit the ability of tumor cells to adapt and survive because the activity within multiple parallel survival signaling pathways has been reduced. This review will discuss some of the approaches that have been taken to combine signal transduction modulatory agents to achieve enhanced tumor cell killing.     

Role of early plasma membrane events in chemotherapy-induced cell death.

Most current anticancer therapies induce tumor cell death through the induction of apoptosis. However, the pathways leading to cell death are not always understood. For example, for several DNA-damaging agents the specific biochemical lesions (DNA damage) have been associated with the induction of apoptosis. However, several of these DNA-damaging agents (cisplatin, 1-beta-arabinofuranosylcytosine, daunorubicin or doxorubicin) as well as other antitumor agents, such as edelfosine or resveratrol, have been recently shown to induce apoptosis via signaling through plasma membrane lipid rafts involving the death receptor pathway. In this review we focus on the role of early plasma membrane events in chemotherapy-induced cell death. Special attention is given to changes in plasma membrane fluidity, activation of the acid sphingomyelinase and the Fas death pathway in response to chemotherapy as well as their possible interrelationships.