From circadian rhythms to cancer chronotherapeutics

Mammalian circadian rhythms result from a complex organization involving molecular clocks within nearly all “normal” cells and a dedicated neuroanatomical system, which coordinates the so-called “peripheral oscillators.” The core of the central clock system is constituted by the suprachiasmatic nuclei that are located on the floor of the hypothalamus. Our understanding of the mechanisms of circadian rhythm generation and coordination processes has grown rapidly over the past few years. In parallel, we have learnt how to use the predictable changes in cellular metabolism or proliferation along the 24h time scale in order to improve treatment outcome for a variety of diseases, including cancer. The chronotherapeutics of malignant diseases has emerged as a result of a consistent development ranging from experimental, clinical, and technological prerequisites to multicenter clinical trials of chronomodulated delivery schedules. Indeed large dosing-time dependencies characterize the tolerability of anticancer agents in mice or rats, a better efficacy usually results from treatment administration near the least toxic circadian time in rodent tumor models. Programmable in time multichannel pumps have allowed to test the chronotherapy concepts in cancer patients and to implement chronomodulated delivery schedules in current practice. Clinical phase I and II trials have established the feasibility, the safety, and the activity of the chronotherapy schedules, so that this treatment method has undergone further evaluation in international multicenter phase III trials. Overall, more than 2000 patients with metastatic disease have been registered in chronotherapy trials. Improved tolerability and/or better antitumor activity have been demonstrated in randomized multicenter studies involving large patient cohorts. The relation between circadian rhythmicity and quality of life and even survival has also been a puzzling finding over the recent years. An essential step toward further developments of circadian-timed therapy has been the recent constitution of a Chronotherapy cooperative group within the European Organization for Research and Treatment of Cancer. This group now involves over 40 institutions in 12 countries. It is conducting currently six trials and preparing four new studies. The 19 contributions in this special issue reflect the current status and perspectives of the several components of cancer chronotherapeutics.     

Circadian chemotherapy for gynecological and genitourinary cancers

The circadian timing of surgery, anticancer drugs, radiation therapy, and biologic agents can result in improved toxicity profiles, tumor control, and host survival. Optimally timed cancer chemotherapy with doxorubicin or pirarubicin (06:00h) and cisplatin (18:00h) enhanced the control of advanced ovarian cancer while minimizing side effects, and increased the response rate in metastatic endometrial cancer. Therapy of metastatic bladder cancer with doxorubicin–cisplatin was made more tolerable by this same circadian approach resulting in a 57% objective response rate. This optimally timed therapy is also effective in the adjuvant setting, decreasing the expected frequency of metastasis from locally advanced bladder cancer. Circadian fluorodeoxyuridine (FUDR) continuous infusion (70% of the daily dose given between 15:00h and 21:00h) has been shown effective for metastatic renal cell carcinoma resulting in 29% objective response and stable disease of more than 1 yr duration in the majority of patients. Toxicity is reduced markedly when FUDR infusion is modulated to circadian rhythms. In a multicenter trial in patients with metastatic renal cell cancer, patients were randomized to a flat or a circadian-modified FUDR infusion. This study confirmed a significant difference in toxicity and dose intensity, favoring the circadian-modified group. Hormone refractory metastatic prostate cancer has been treated with circadian-timed FUDR chemotherapy; however, without objective response. Biological agents such as interferon-α and IL-2 have shown low but effective disease control in metastatic renal cell cancer, however, with much toxicity. Each of these cytokines shows circadian stage dependent toxicity and efficacy in model systems. In summary, the timing of anthracycline, platinum, and fluoropyrimidine-based drug therapies during the 24h is relevant to the toxic–therapeutic ratio of these agents in the treatment of gynecologic and genitourinary cancers.     

Circadian chemotherapy for gynecological and genitourinary cancers

The circadian timing of surgery, anticancer drugs, radiation therapy, and biologic agents can result in improved toxicity profiles, tumor control, and host survival. Optimally timed cancer chemotherapy with doxorubicin or pirarubicin (06:00h) and cisplatin (18:00h) enhanced the control of advanced ovarian cancer while minimizing side effects, and increased the response rate in metastatic endometrial cancer. Therapy of metastatic bladder cancer with doxorubicin-cisplatin was made more tolerable by this same circadian approach resulting in a 57% objective response rate. This optimally timed therapy is also effective in the adjuvant setting, decreasing the expected frequency of metastasis from locally advanced bladder cancer. Circadian fluorodeoxyuridine (FUDR) continuous infusion (70% of the daily dose given between 15:00h and 21:00h) has been shown effective for metastatic renal cell carcinoma resulting in 29% objective response and stable disease of more than 1 yr duration in the majority of patients. Toxicity is reduced markedly when FUDR infusion is modulated to circadian rhythms. In a multicenter trial in patients with metastatic renal cell cancer, patients were randomized to a flat or a circadian-modified FUDR infusion. This study confirmed a significant difference in toxicity and dose intensity, favoring the circadian-modified group. Hormone refractory metastatic prostate cancer has been treated with circadian-timed FUDR chemotherapy; however, without objective response. Biological agents such as interferon-alpha and IL-2 have shown low but effective disease control in metastatic renal cell cancer, however, with much toxicity. Each of these cytokines shows circadian stage dependent toxicity and efficacy in model systems. In summary, the timing of anthracycline, platinum, and fluoropyrimidine-based drug therapies during the 24h is relevant to the toxic therapeutic ratio of these agents in the treatment of gynecologic and genitourinary cancers.     

Targeted therapy for cancer using pH-responsive nanocarrier systems

Most of the conventional chemotherapeutic agents used against cancer have poor efficacy. An approach to improve the efficacy of cancer chemotherapy is the development of carrier systems that can be triggered to release the anticancer drug in response to extracellular or intracellular chemical stimuli. To this end, pH-responsive nanocarriers have been developed to target drugs either to the slightly acidic extracellular fluids of tumor tissue or, after endocytosis, to the endosomes or lysosomes within cancer cells. These systems can release the drug by specific processes after accumulation in tumor tissues via the enhanced permeability and retention (EPR) effect or they can release the drugs in endosomes or lysosomes by pH-controlled hydrolysis after they are taken up by the cell via the endocytic pathway. This strategy facilitates the specific delivery of the drug while reducing systemic side-effects with high potential for improving the efficacy of cancer chemotherapy.     

Chronotherapy of colorectal cancer

Chronotherapy consists of chemotherapy delivery according to circadian rhythms. These genetically based rhythms modulate cellular metabolism and cell proliferation in normal tissues. As a result, both the host tolerance and antitumor efficacy of 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), like 30 other anticancer drugs, vary largely according to the dosing time in laboratory rodents. The transfer of this concept to the clinic is aimed primarily at increasing the dose-intensity of the therapy through adjustment of drug-delivery to 24h rhythms in host tolerance. A specific technology (programmable-in-time infusion pumps) enables administration of chronotherapy to fully ambulatory patients. Phase I-III clinical trials show chronotherapy significantly increases tolerance to high doses of cancer drugs and improves antitumor activity in patients with metastatic colorectal cancer. These safe conditions of drug-delivery led to the first demonstration of the high activity of the 5-FU-leucovorin-L-OHP protocol. Chronotherapy with these three drugs also allows surgical removal of previously unresectable liver and lung metastases. This novel medico-surgical management provides hope for the cure of metastatic disease in patients with unresectable colorectal cancer metastases.     

Mitochondrial function and energy metabolism in cancer cells: Past overview and future perspectives

The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.     

Tumor Growth Rate Determines the Timing of Optimal Chronomodulated Treatment Schedules

In host and cancer tissues, drug metabolism and susceptibility to drugs vary in a circadian (24 h) manner. In particular, the efficacy of a cell cycle specific (CCS) cytotoxic agent is affected by the daily modulation of cell cycle activity in the target tissues. Anti-cancer chronotherapy, in which treatments are administered at a particular time each day, aims at exploiting these biological rhythms to reduce toxicity and improve efficacy of the treatment. The circadian status, which is the timing of physiological and behavioral activity relative to daily environmental cues, largely determines the best timing of treatments. However, the influence of variations in tumor kinetics has not been considered in determining appropriate treatment schedules. We used a simple model for cell populations under chronomodulated treatment to identify which biological parameters are important for the successful design of a chronotherapy strategy. We show that the duration of the phase of the cell cycle targeted by the treatment and the cell proliferation rate are crucial in determining the best times to administer CCS drugs. Thus, optimal treatment times depend not only on the circadian status of the patient but also on the cell cycle kinetics of the tumor. Then, we developed a theoretical analysis of treatment outcome (TATO) to relate the circadian status and cell cycle kinetic parameters to the treatment outcomes. We show that the best and the worst CCS drug administration schedules are those with 24 h intervals, implying that 24 h chronomodulated treatments can be ineffective or even harmful if administered at wrong circadian times. We show that for certain tumors, administration times at intervals different from 24 h may reduce these risks without compromising overall efficacy.     

The Circadian System of Crustaceans

Crustaceans exhibit a variety of overt circadian rhythms. Observations on intact animals suggest the existence of more than one circadian pacemaker in the nervous system. Ablation experiments so far have been inconclusive in pinpointing the location of putative pacemakers. However, various structures, most notably the optic peduncle, have been shown to sustain circadian rhythmicity in vitro. Retinal sensitivity and neurosecretory activity display circadian rhythms in the isolated optic peduncle, but they are also responsive to synchronizing influences from other regions of the central nervous system, most notably the supra-esophageal ganglion. A model based on a number of circadian pacemakers distributed in the central nervous system best fits the experimental results at present. Coupling of rhythmicity between independent circadian pacemakers is likely to occur, and a neuroendocrine stage of integration has been proposed for several rhythms. Various entraining agents have been identified, and more than one may play a part in the synchronization of a given rhythm.     

Daily rhythms are retained both in spontaneously developed sarcomas and in xenografts grown in immunocompromised SCID mice

The circadian clock generates and regulates many daily physiological, metabolic and behavioral rhythms as well as acute responses to various types of stresses including those induced by anticancer treatment. It has been proposed that modulatory function of the clock may be used for improving the therapeutic efficacy of established anti-cancer treatments. In order to rationally exploit this mechanism, more information is needed to fully characterize the functional status of the molecular clock in tumors of different cellular origin; however, the data describing tumor clocks are still inconsistent. Here we tested the status of clock in two models of tumors derived from connective tissue: sarcomas spontaneously developed in p53-deficient mice and human fibrosarcoma cells grown as xenografts in immunocompromised severe combined immunodeficient (SCID) mice. We show that both types of tumors retain a functional clock, which is synchronized in phase with normal tissues. We also show that spontaneously developed tumors are not only oscillating in the context of an organism where they receive hormonal and metabolic signals but continue oscillating ex vivo in tissue explants demonstrating that tumors have functional clocks capable of timing all their functions. We also provide evidence that similar to liver, tumors can be synchronized by food availability independent of the central pacemaker in the suprachiasmatic nuclei (SCN). These data provide the basis for the design of anticancer therapies that take into account the circadian metabolic and physiological patterns of both the tumor and normal tissues.     

Chronotherapy of colorectal cancer

Chronotherapy consists of chemotherapy delivery according to circadian rhythms. These genetically based rhythms modulate cellular metabolism and cell proliferation in normal tissues. As a result, both the host tolerance and antitumor efficacy of 5-fluorouracil (5-FU) and oxaliplatin (l-OHP), like 30 other anticancer drugs, vary largely according to the dosing time in laboratory rodents. The transfer of this concept to the clinic is aimed primarily at increasing the dose-intensity of the therapy through adjustment of drug-delivery to 24h rhythms in host tolerance. A specific technology (programmable-in-time infusion pumps) enables administration of chronotherapy to fully ambulatory patients. Phase I–III clinical trials show chronotherapy significantly increases tolerance to high doses of cancer drugs and improves antitumor activity in patients with metastatic colorectal cancer. These safe conditions of drug-delivery led to the first demonstration of the high activity of the 5-FU–leucovorin–l-OHP protocol. Chronotherapy with these three drugs also allows surgical removal of previously unresectable liver and lung metastases. This novel medico-surgical management provides hope for the cure of metastatic disease in patients with unresectable colorectal cancer metastases.     

Chronobiology of the mammalian response to ionizing radiation. Potential applications in oncology

Ionizing radiation from all sources under appropriate conditions leads to cell death and tissue damage. It is used in cancer treatment under the assumption of a higher radiosensitivity of the fast dividing tumor cells as compared with adjacent host tissues. The radiosensitivities of proliferating host tissues like bone marrow and gastrointestinal lining epithelium are dose limiting. Since these host tissues and many tumors show circadian and other periodicities in their cell proliferation, the timing of radiation treatment according to host and/or tumor rhythms is expected to improve the toxic/therapeutic ratio of the treatment. The experimental data on the chronobiology of radiation exposure show circadian rhythmicity in radiation response after whole body irradiation in mice and rats with highest toxicity in light-dark 12h:12h synchronized animals during their daily activity span. Bone marrow toxicity as well as gastrointestinal epithelial damage show circadian rhythms in part due to radiation damage to the stem cells involved and especially in the intestine also due to damage to the microvasculature. Chronoradiotherapy of malignant tumors seems promising, alone or in combination with response modifiers, provided the host and potential tumor rhythms can be monitored.     

Analysis of FDA Approved Anticancer Drugs Reveals the Future of Cancer Therapy

This review discusses 26 new anticancer drugs approved by the FDA in the past decade. Based on their targets, these anticancer agents can be divided into three groups. First group contains cancer-selective or semi-selective drugs that are effective in rare kinase-addictive cancers. For other malignancies, semi-selective drugs have to be judiciously combined with non-selective agents. The second group includes analogs of classic cytotoxic agents such as DNA alkylating agents, nucleoside analogs, and anti-microtubule agents. As expected, they have a marginal advantage over the existing cytotoxic drugs, nevertheless are more effective (in common cancers) than semi-selective agents. The third is a diverse group of tissue-selective agents that essentially attack the normal tissues of tumor origin and thus, exploit the tissue-specific similarities between normal and cancer cells. Our analysis predicts that monotherapy with semi-selective agents will be limited to rare cancers. In most cancers however, two anticancer strategies may be most fruitful: (a) combinations of cytotoxic drugs with semi-selective agents aimed at matching targets and (b) tissue-selective therapy aimed at normal and tumor cells of the same tissues.     

Development and validation of computational models for mammalian circadian oscillators

Circadian rhythms are endogenous rhythms with a cycle length of approximately 24 h. Rhythmic production of specific proteins within pacemaker structures is the basis for these physiological and behavioral rhythms. Prior work on mathematical modeling of molecular circadian oscillators has focused on the fruit fly, Drosophila melanogaster. Recently, great advances have been made in our understanding of the molecular basis of circadian rhythms in mammals. Mathematical models of the mammalian circadian oscillator are needed to piece together diverse data, predict experimental results, and help us understand the clock as a whole. Our objectives are to develop mathematical models of the mammalian circadian oscillator, generate and test predictions from these models, gather information on the parameters needed for model development, integrate the molecular model with an existing model of the influence of light and rhythmicity on human performance, and make models available in BioSpice so that they can be easily used by the general community. Two new mammalian models have been developed, and experimental data are summarized. These studies have the potential to lead to new strategies for resetting the circadian clock. Manipulations of the circadian clock can be used to optimize performance by promoting alertness and physiological synchronization.     

双硫仑抗癌作用研究进展

双硫仑作为一种治疗慢性酒精中毒的药物在临床中广泛使用。近几十年研究发现它除了戒酒作用还在治疗癌症中具有巨大潜力,针对它在体外和体内模型的研究结论已有部分在临床治疗中得到证实。双硫仑通过其代谢产物抑制乙醛脱氢酶活性导致体内乙醛含量积累,增加细胞毒性从而抑制肿瘤干细胞增殖分化;提高细胞内活性氧的浓度诱导细胞凋亡;抑制蛋白酶体活性,积累大量废弃蛋白质诱导细胞凋亡;通过抑制NF-κB下调来抑制上皮间质转化等。此外双硫仑与抗癌药物联合使用可提升抗癌药物药效。由于具有低毒、低成本且对肿瘤组织有趋向性等特点,双硫仑重新应用于临床作为抗癌药物具有广阔前景。简要回顾了双硫仑最新研究中阐明的双硫仑抗癌作用分子机制,展望了未来双硫仑用作新临床抗癌药物的前景,以期为双硫仑在抗癌药物中的应用研究提供参考。     

Activity of Pan-Class I Isoform PI3K/mTOR Inhibitor PF-05212384 in Combination with Crizotinib in Ovarian Cancer Xenografts and PDX

The Phosphatidyl inositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and c-Met signaling pathways are often deregulated in cancer. The two pathways are interconnected and at least c-Met has been implicated in drug resistance. The aim of the study was to assess in ovarian cancer preclinical models, the efficacy and tolerability of a dual PI3K mTOR inhibitor (PF-05212384 or gedatolisib) and a c-Met inhibitor (crizotinib) either as single agents or in combination. In vitro, both PF-05212384 and crizotinib showed a concentration dependent activity in the two ovarian cancer cell lines. The combination of the two did not result in synergistic activity. A subline resistant to gedatolisib was obtained and showed an increased expression of MDR-1 gene. In vivo results show that crizotinib alone did not display any activity in all the tumors investigated, while PF-05212384 alone had some marginal activity. The combination of the two resulted in all the experiments superior to single agents with a good tolerability. Considering that crizotinib did not show activity in the models used, the results indicate that crizotinib is able to potentiate the activity of PF-05212384. Although the activity of the combination was not striking in these three models of ovarian cancer, due to the good tolerability of the combination, the results would suggest the possibility to combine the two drugs in settings in which gedatolisib or crizotinib alone have already some significant activity.     

Exploiting tumor metabolism for non-invasive imaging of the therapeutic activity of molecularly targeted anticancer agents

Rational drug discovery and development requires biomarkers to inform on target modulation and treatment efficacy. Many aspects of metabolism are altered in cancer, compared to normal tissues, and are often regulated by oncogene activation. Non-invasive imaging of spatio-temporal effects of molecularly targeted anticancer agents on tumor metabolism has considerable potential in the development and use of personalized molecular medicine approaches to cancer treatment. Here we describe how non-invasive monitoring of metabolism, using primarily magnetic resonance spectroscopy (MRS), can be used to follow treatment with novel molecularly targeted anticancer agents. We discuss how the regulation of metabolic pathways by oncogenic signaling can affect MRS-detectable metabolic signals together with the physiological factors that can influence the measured changes. Finally, the translation of these metabolic measurements from pre-clinical models to patients will be discussed.     

Antiangiogenic cancer therapy: why do mouse and human patients respond in a different way to the same drug?

The tumor vasculature is an increasingly attractive target for development of anticancer drugs. The fundamental principle for antiangiogenic cancer therapy is based on the inhibitory effect of chemical compounds, proteins or nucleotides on tumor angiogenesis. Indeed, in almost all preclinical tumor models, antiangiogenic monotherapy with different agents shows potent effects on suppression of tumor growth. However, antiangiogenic monotherapy has barely produced any clinical benefits in cancer patients. Although in combination with chemotherapy some antiangiogenic drugs demonstrate survival improvement in patients with certain types of cancers, the overall benefits by addition of antiangiogenic drugs (ADs) to chemotherapy remain modest. The disparity of AD responses between preclinical models and clinical cancer patients has raised important issues, which include: 1) Are current animal tumor models appropriate for assessing the therapeutic efficacy of ADs for clinical development? 2) What are the key differences between mouse tumor models and human cancer patients? 3) Are anti-VEGF drugs off target in cancer patients? 4) What are alternative options for improvement of the clinical benefits of ADs? In this short review, I discuss these critical issues in relation to the clinical practice of ADs.     

Effects of paclitaxel and doxorubicin in histocultures of hepatocelular carcinomas

Summary Cancer has been the leading cause of death in Taiwan over the past two decades and liver cancer is the leading cause of all cancer deaths in Taiwan with a trend of increase in incidence. Therapeutic options and efficacy for liver cancer have been limited and the 5-year survival rate is less than 7% in the Unite States. The study was conducted to establish a histoculture system of human hepatocellular carcinomas (HCC) for biological and pharmacological studies and to determine the efficacy of anticancer drugs with the established HCC histocultures. Patient HCC tissues freshly obtained after surgeries were prepared and histocultured. The histocultured HCC were treated with doxorubicin and paclitaxel of various concentrations for 96-h. Upon drug treatments, the activity of tumor cell proliferation and extent of cell death induction were measured and changes of the α-fetoprotein levels in the culture medium were determined. We demonstrated that human HCC can be successfully cultured in a 3-dimensional histoculture system and used for pharmacological studies. Doxorubicin and paclitaxel showed concentration-dependent activities in anti-proliferation and cell death induction against the human HCC. Inhibitory effects of both drugs on α-fetoprotein production of the cultured HCC were in agreement with their anti-proliferative effects. Exposure time-dependent antitumoral effects of paclitaxel treatments at 3-, 24-, and 96-h against the histocultured HCC PLC/PRF/5 xenograft tumors were also observed. In conclusion, we have demonstrated a histoculture system for patient HCC and it can be utilized in selection of active drugs prior to treatments in patients and in evaluation of new agents against HCC, for which therapeutic agents are in desperate needs worldwide.     

In-vitro circadian rhythm of murine bone marrow progenitor production

Hematopoietic processes display 24h rhythms both in rodents and in human beings. We hypothesized these rhythms to be in part generated by a circadian oscillator within the bone marrow. The ability of murine bone marrow granulo-monocytic (GM) precursors to form colonies following colony-stimulating factor (rm GM-CSF) exposure was investigated in liquid culture samples obtained every 3 h for a span of up to 198 h. The CFU-GM count varied rhythmically over the first 4 d of culture, with a reproducible maximum in the early morning hours, similar to that observed in vivo. These experiments provide the first evidence that bone marrow progenitors sustain in vitro circadian rhythmicity, and they demonstrate the presence of a circadian time-keeping system within these cells. The results support the potential usefulness of bone marrow cultures for investigating chronopharmacologic effects of anticancer drugs and cytokines on this target system.