[Study on the regulation of autophagy against anticancer drugs' toxicity].

Autophagy is a crucial biological process in eukaryotes, which is involved in cell growth, survival and energy metabolism. It has been confirmed that autophagy mediates toxicity of anticancer drugs, especially in heart, liver and neuron. It is important to understand the function and mechanism of autophagy in anticancer drugs-induced toxicity. Given that autophagy is a double-edged sword in the maintenance of the function of heart, liver and neuron, the autophagy-mediated toxicity are very complicated in the body. We provide a review on the concept of autophagy and current status about autophagy-mediated toxicity of anticancer drugs. The knowledge is crucial in the basic study of anticancer drugs-induced toxicity, and provides some strategies for the development of alleviating the toxicity of anticancer drugs.

[Research progress in toxicology of molecular targeted anticancer drugs].

OBJECTIVE: Drug therapy is essential for cancer treatment. The molecular targeted anticancer drugs develop rapidly in recent years, since the effectiveness of traditional chemotherapy is unsatisfactory and the adverse reactions are high. However, molecular targeted anticancer drugs would damage the function of heart, liver or lung, and may cause adverse effects such as hand-foot syndrome, which restrains their clinical application. Therefore, it is critical for pharmaceutical toxicology to study the toxicity, the related mechanisms and the preventive measures of molecular targeted anticancer drugs.

p8 attenuates the apoptosis induced by dihydroartemisinin in cancer cells through promoting autophagy.

Dihydroartemisinin (DHA) exhibits anticancer activities in a variety of cancer cells, but DHA alone are not effective enough for cancer therapy. In this study we found the stress-regulated protein p8 was obviously increased after DHA treatment in several cancer cells, which further to induce autophagy by the upregulation of endoplasmic reticulum stress-related protein ATF4 and CHOP. Furthermore, when we silenced p8 by siRNA in cancer cells, the apoptosis induced by DHA were notably increased, whereas the overexpression of p8 in cancer cells leaded to the resistance to DHA-induced apoptosis. Moreover, we found the inhibition of autophagy with chloroquine (CQ) can enhance the anticancer effect of DHA both in vitro and in vivo. In conclusion, we found that p8-mediated autophagy attenuates DHA-induced apoptosis in cancer cells, which provides evidence to support the use p8 as a cancer therapeutic target, and suggests that the combination treatment with DHA and autophagy inhibitor might be an effective cancer therapeutic strategy.

Dihydroartemisinin induces autophagy by suppressing NF-κB activation.

Nuclear factor-kappa B (NF-κB) and autophagy are two major regulators involved in both tumor initiation and progression. However, the association between these two signaling pathways still remains obscure. In this work, we demonstrate that dihydroartemisinin (DHA) stimulates the induction of autophagy in several cancer cell lines through repression of NF-κB activity. We also show that inhibiting NF-κB results in an accumulation of reactive oxygen species (ROS), which participate in the stimulation of autophagy. These findings present a pathway by which DHA promotes autophagy in cancer cells and provide evidence for the DHA-induced sensitization effect of some chemotherapeutics.

Reducing the oxidative stress mediates the cardioprotection of bicyclol against ischemia-reperfusion injury in rats.

OBJECTIVE: To investigate the beneficial effect of bicyclol on rat hearts subjected to ischemia-reperfusion (IR) injuries and its possible mechanism. METHODS: Male Sprague-Dawley rats were intragastrically administered with bicyclol (25, 50 or 100 mg/(kg∙d)) for 3 d. Myocardial IR was produced by occlusion of the coronary artery for 1 h and reperfusion for 3 h. Left ventricular hemodynamics was continuously monitored. At the end of reperfusion, myocardial infarct was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and serum lactate dehydrogenase (LDH) level and myocardial superoxide dismutase (SOD) activity were determined by spectrophotometry. Isolated ventricular myocytes from adult rats were exposed to 60 min anoxia and 30 min reoxygenation to simulate IR injuries. After reperfusion, cell viability was determined with trypan blue; reactive oxygen species (ROS) and mitochondrial membrane potential of the cardiomyocytes were measured with the fluorescent probe. The mitochondrial permeability transition pore (mPTP) opening induced by Ca(2+) (200 µmol/L) was measured with the absorbance at 520 nm in the isolated myocardial mitochondria. RESULTS: Low dose of bicyclol (25 mg/(kg∙d)) had no significant improving effect on all cardiac parameters, whereas pretreatment with high bicyclol markedly reduced the myocardial infarct and improved the left ventricular contractility in the myocardium exposed to IR (P<0.05). Medium dose of bicyclol (50 mg/(kg∙d)) markedly improved the myocardial contractility, left ventricular myocyte viability, and SOD activity, as well decreased infarct size, serum LDH level, ROS production, and mitochondrial membrane potential in rat myocardium exposed to IR. The reduction of ventricular myocyte viability in IR group was inhibited by pretreatment with 50 and 100 mg/(kg∙d) bicyclol (P<0.05 vs. IR), but not by 25 mg/(kg∙d) bicyclol. The opening of mPTP evoked by Ca(2+) was significantly inhibited by medium bicyclol. CONCLUSIONS: Bicyclol exerts cardioprotection against IR injury, at least, via reducing oxidative stress and its subsequent mPTP opening.

DHA regulates angiogenesis and improves the efficiency of CDDP for the treatment of lung carcinoma.

Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has been shown to exhibit anti-angiogenic and anti-tumor effects apart from its antimalarial activity. In this study, we demonstrate that the combined treatment of cisplatin (CDDP) and DHA exerts a strong, synergistic anti-proliferative effect in human lung carcinoma cells, including A549 and A549/DDP cells, with an average combination index below 0.7. Moreover, the in vivo anti-tumor efficacy of CDDP treatment was increased by DHA. The enhanced anti-cancer activities were also accompanied by reduced tumor microvessel density, increased CDDP concentration within A549 and A549/DDP xenograft BALB/c athymic mice models and suppressed expression of the vascularization-related proteins HIF-1α and VEGF both in vivo and in vitro. Furthermore, the level of apoptosis in the tumor cells increased with the combined treatment of DHA and CDDP. Taken together, our results indicate that a combination of DHA and CDDP treatments synergistically affects tumor angiogenesis, and these results provide a clear rationale for the investigation of these drugs in future clinical trials.

Bicyclol attenuates pro-inflammatory cytokine and chemokine productions in CpG-DNA-stimulated L02 hepatocytes by inhibiting p65-NF-kappaB and p38-MAPK activation.

Bicyclol, a novel synthetic anti-hepatitis drug, has a potent hepatocyte-protective effect and a mild anti-hepatitis virus function. However, its pharmaceutical effects and mechanism are still unclear. In the present study, we found that bicyclol pre-treatment could attenuate the production of the inflammatory cytokines (TNF-alpha and IL-18) and chemokines (MCP-1, MIP-1 alpha and Rantes) and inhibit the activation of the p65-NF-kappaB and MAPK signaling pathways in CpG-ODN 2006-stimulated L02 hepatocytes in a dose-dependent manner. Collectively, these results suggest that bicyclol could exert its hepatocyte-protective effect through attenuating CpG-ODN 2006-stimulated inflammatory responses in L02 hepatocytes, which might be associated with the activations of NF-kappaB and MAPK pathways.

Dihydroartemisinin improves the efficiency of chemotherapeutics in lung carcinomas in vivo and inhibits murine Lewis lung carcinoma cell line growth in vitro.

PURPOSE: Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has exhibited the strongest antimalarial activity among the derivatives of artemisinin. There is growing evidence that DHA has some impact against tumors. Our purpose was to evaluate in vitro antitumoral properties of DHA in the murine Lewis lung carcinoma (LLC) cell line. At the same time, we observed the therapeutic effect of DHA combined with cyclophosphamide (CTX) in the LLC and combined with cisplatin (CDDP) in the human non-small cell lung cancer A549 xenotransplanted carcinoma in vivo. METHODS: Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, apoptosis was measured by AO/EB double staining and flow cytometry. The expression of vascular endothelial growth factor (VEGF) receptor KDR/flk-1 was analyzed by western blotting and RT-PCR. In vivo activity of DHA combined with CTX or CDDP was assayed through tumor growth and metastasis. RESULTS: Dihydroartemisinin exhibited high anti-cancer activity in LLC cell line. DHA also induced apoptosis of LLC cells and influenced the expression of VEGF receptor KDR/flk-1. Furthermore, in both tumor xenografts, a greater degree of growth inhibition was achieved when DHA and chemotherapeutics were used in combination. The affection by DHA combined CTX on LLC tumor metastasis was significant. CONCLUSIONS: Dihydroartemisinin is a potent compound against LLC cell line in vitro. In vivo, the combination strategy of DHA and chemotherapeutics holds promise for the treatment of relatively large and rapidly growing lung cancers.

Bicyclol attenuates oxidative stress and neuronal damage following transient forebrain ischemia in mouse cortex and hippocampus.

To assess its potential neuroprotective effect against ischemia/reperfusion (IR) injury in mice, bicyclol was administered intragastrically once a day for 3 days. After 6h of bicyclol pretreatment on the third day, forebrain ischemia was induced for 1h by bilateral occlusion of the carotid arteries. After different times of reperfusion, the histopathological changes and the levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the cortex and hippocampus were measured. We found that extensive neuronal death occurred in the cortex and the CA1 area of the hippocampus at day 7 after IR and that bicyclol significantly attenuated IR-induced neuronal death in a dose-dependent manner. We also found that pretreatment with bicyclol dose dependently decreased the generation of ROS and the MDA content and reduced the compensatory increase in SOD activity in the cortex and hippocampus at 4h of reperfusion. These results suggest that bicyclol protects the mouse brain against cerebral IR injury by attenuating oxidative stress and lipid peroxidation.

Inhibitory effects of artesunate on angiogenesis and on expressions of vascular endothelial growth factor and VEGF receptor KDR/flk-1.

Artesunate (ART) is a semi-synthetic derivative of artemisinin extracted from the plant Artemisia annua is a safe and effective antimalarial drug. In the present investigation, ART was found also to inhibit angiogenesis in vivo and in vitro. The anti-angiogenic effect in vivo was evaluated in nude mice by means of human ovarian cancer HO-8910 implantation and immunohistochemical stainings for microvessel (CD(31)), vascular endothelial growth factor (VEGF) and VEGF receptor KDR/flk-1. Tumor growth was decreased and microvessel density was reduced following drug treatment with no apparent toxicity to the animals. ART also remarkably lowered VEGF expression on tumor cells and KDR/flk-1 expression on endothelial cells as well as tumor cells. The in vitro effect of ART was tested on models of angiogenesis, namely, proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC). The results showed that ART significantly inhibited angiogenesis in a dose-dependent form in the range of 0.5 approximately 50 micromol/l. Additionally, the inhibitory effect of ART on HVUEC proliferation was stronger than that on Hela, JAR, HO-8910 cancer cells, NIH-3T3 fibroblast cells and human endometrial cells, indicating that ART was selectively against HUVEC. These findings and the known low toxicity of ART are clues that ART may be a promising angiogenesis inhibitor.

[Lipid-peroxidantion damage of embryo and placenta induced by artesunate in rats].

OBJECTIVE: To examine the effect and mechanism of artesunate (Art) on embryo development. METHODS: Rat embryo and placental glutathione peroxidase (GSH-Px)and malondialdehyde (MDA) were identified by using DTNB (dithionitrobenzene) direct method and TBA (thiobarbituric acid). We investigated the damage of decidual cells caused by Art using cell culture techniques. RESULTS: Subcutaneous administration of Art in rats on d 6 approximate, equals d 10 of gestation induced developmental toxicity. Absorption increased when progressively increased doses were given (r=0.996,P<0.01). Twenty four hours post injection, GSH-Px in embryo decreased significantly while MDA content was significantly higher than that in the control group (P<0.05). GSH Px: study group was(43.7+/-10.7)micromol/min.mg(-1)Hb, control group was(54.5+/-10.1)micromol/min.mg(-1)Hb; MDA:study group was(230.2+/-19.8)nmol/g tissue, control group was(150.4+/-44.1)nmol/g tissue. Placental GSH-Px activity was significantly higher than that in the control group(P<0.01). After cultured human decidual cells were exposed to Art for 24 h, the LC50 was (25.2+/-3.5)mg/L. CONCLUSION: Art may induce developmental toxicity in rat embryo and placenta by neutralizing the antioxidant defense mechanism.