HY5 functions as a systemic signal by integrating BRC1-dependent hormone signaling in tomato bud outgrowth.

Light plays an important role in determining plant architecture, which greatly influences crop yield. However, the precise mechanisms by which light signaling regulates bud outgrowth remain to be identified. Here, we show that light regulates bud outgrowth via both HY5 and brassinosteroid (BR)-dependent pathways in tomato. Inactivation of the red-light photoreceptor PHYB, or deficiencies in PHYB or the blue-light photoreceptor CRY1a, inhibits bud outgrowth and leads to decreased accumulation of HY5 protein and increased transcript level of BRANCHED1 (BRC1), a central integrator of branching signals. HY5, functioning as a mobile systemic signal from leaves, promotes bud outgrowth by directly suppressing BRC1 transcript and activating the transcript of BR biosynthesis genes within the lateral buds in tomato. Furthermore, BRC1 prevents the accumulation of cytokinin (CK) and gibberellin (GA) by directly inhibiting the transcript of CK synthesis gene LOG4, while increasing the transcript levels of CK and GA degradation genes (CKX7, GA2ox4, and GA2ox5), leading to an arrest of bud outgrowth. Moreover, bud outgrowth occurs predominantly in the day due to the suppression of BRC1 transcript by HY5. These findings demonstrate that light-inducible HY5 acts as a systemic signaling factor in fine-tuning the bud outgrowth of tomato.

The bZIP transcription factor SlAREB1 regulates anthocyanin biosynthesis in response to low temperature in tomato.

Low temperature and abscisic acid (ABA) are the two main factors that induce anthocyanin synthesis; however, their potential relationships in governing anthocyanin biosynthesis in Solanum lycopersicum (tomato) seedlings remains unclear. Our study revealed the involvement of the transcription factor SlAREB1 in the low-temperature response of tomato seedlings via the ABA-dependent pathway, for a specific temperature range. The overexpression of SlAREB1 enhanced the expression of anthocyanin-related genes and the accumulation of anthocyanins, especially under low-temperature conditions, whereas silencing SlAREB1 dramatically reduced gene expression and anthocyanin accumulation. There is a direct interaction between SlAREB1 and the promoters of SlDFR and SlF3'5'H, which are structural genes that impact anthocyanin biosynthesis. SlAREB1 can regulate anthocyanins through controlling SlDFR and SlF3'5'H expression. Accordingly, SlAREB1 takes charge of regulating anthocyanin biosynthesis in tomato seedlings via the ABA-dependent pathway at low temperatures.

The RALF2-FERONIA-MYB63 module orchestrates growth and defense in tomato roots.

Plant secreted peptides RAPID ALKALINISATION FACTORs (RALFs), which act through the receptor FERONIA (FER), play important roles in plant growth. However, it remains unclear whether and how RALF-FER contributes to the trade-off of plant growth-defense. Here, we used a variety of techniques such as CRISPR/Cas9, protein-protein interaction and transcriptional regulation methods to investigate the role of RALF2 and its receptor FER in regulating lignin deposition, root growth, and defense against Fusarium oxysporum f. sp. lycopersici (Fol) in tomato (Solanum lycopersicum). The ralf2 and fer mutants show reduced primary root length, elevated lignin accumulation, and enhanced resistance against Fol than the wild-type. FER interacts with and phosphorylates MYB63 to promote its degradation. MYB63 serves as an activator of lignin deposition by regulating the transcription of dirigent protein gene DIR19. Mutation of DIR19 suppresses lignin accumulation, and reverses the short root phenotype and Fol resistance in ralf2 or fer mutant. Collectively, our results demonstrate that the RALF2-FER-MYB63 module fine-tunes root growth and resistance against Fol through regulating the deposition of lignin in tomato roots. The study sheds new light on how plants maintain the growth-defense balance via RALF-FER.

Photoactivatable CRISPR/Cas12a Strategy for One-Pot DETECTR Molecular Diagnosis.

As a golden partner of recombinase polymerase amplification (RPA), CRISPR/Cas12a has been proven to solve the false-positive problem caused by nonspecific amplification perfectly; meanwhile, its trans-cleave activity has further enhanced the sensitivity. However, the solution transfer operation after tube cap opening greatly increases the risk of aerosol contamination of amplicon, which is inconsistent with point-of-care (POC) diagnostics requirements. This study proposes a photoactivated CRISPR/Cas12a strategy to achieve one-pot high-sensitivity nucleic acid detection. Using photocleavable complementary ssDNA to block crRNA, RPA amplification can smoothly pass through the exponential interval without being affected by activated Cas12a in the critical early stage. After enough amplicons were produced, the Cas12a test was activated by short bursts of ultraviolet radiation at 365 nm. This one-pot method achieved a sensitivity of 2.5 copies within 40 min. This simple and sensitive one-pot method can effectively avoid amplicon contamination and lower the threshold for molecular diagnostics in POC.

High-Selectivity Single-Nucleotide Variant Capture Technology Based on the DNA Reaction Network.

The extremely low abundance of circulating tumor DNA in blood samples has limited the development of liquid biopsy techniques for the early diagnosis of major diseases. In this study, we demonstrate a DRN-based screening technique, SCREEN, which achieves the specific capture and enrichment of low abundance SNV nucleic acid samples without selective amplification. The SCREEN technique achieved a 108-fold increase in the abundance of single-nucleotide variant (SNV) nucleic acids from highly homologous mixtures (from 0.01% to 1.08%) and has been shown to significantly increase the abundance of SNV nucleic acids from 0.1% to 51% further through two rounds of capture. As a highly effective pre-enrichment technique, SCREEN has demonstrated the ability to enhance NGS in detecting an ultralow abundance SNV nucleic acid powerfully and has high compatibility with existing molecular diagnostic methods.

A safety assessment of a 300-meter saturation dive at sea by mental ability and performance efficacy.

High pressure is an environmental characteristic of the deep sea that may exert critical effects on the physiology and mental abilities of divers. In this study we evaluated the performance efficacy and mental ability of four divers during a 300-meter helium-oxygen saturation dive at sea. Spatial memory, 2D/3D mental rotation functioning, grip strength, and hand-eye coordination ability were examined for four divers during the pre-dive, compression, decompression, and post-dive phases. The results showed that both the reaction time and the correct responses for the mental rotation and hand-eye coordination were slightly fluctuated. In addition, there was a significant decline in the grip strength of the left hand. It is concluded that the performance efficacy and mental ability of divers were virtually unaffected during 300-meter helium-oxygen saturation diving at sea.

ELONGATED HYPOCOTYL 5 mediates blue light-induced starch degradation in tomato.

Starch is the major storage carbohydrate in plants, and its metabolism in chloroplasts depends mainly on light. However, the mechanism through which photoreceptors regulate starch metabolism in chloroplasts is unclear. In this study, we found that the cryptochrome 1a (CRY1a)-mediated blue light signal is critical for regulating starch accumulation by inducing starch degradation through the transcription factor HY5 in chloroplasts in tomato. cry1a mutants and HY5-RNAi plants accumulated more starch and presented lower transcript levels of starch degradation-related genes in their leaves than wild-type plants. Blue light significantly induced the transcription of starch degradation-related genes in wild-type and CRY1a- or HY5-overexpressing plants but had little effect in cry1a and HY5-RNAi plants. Dual-luciferase assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation-qPCR revealed that HY5 could activate the starch degradation-related genes PWD, BAM1, BAM3, BAM8, MEX1, and DPE1 by directly binding to their promoters. Silencing of HY5 and these starch degradation-related genes in CRY1a-overexpressing plants led to increased accumulation of starch and decreased accumulation of soluble sugars. The findings presented here not only deepen our understanding of how light controls starch degradation and sugar accumulation but also allow us to explore potential targets for improving crop quality.

Cordyceps sinensis aqueous extract regulates the adaptive immunity of mice subjected to 60 Co γ irradiation.

Cordyceps sinensis (CS) is a traditional Chinese medicine that is known for treating various diseases, and particularly for exerting therapeutic effects in immune disorders. The adaptive immunoregulatory effects of CS aqueous extract (CSAE) on γ-irradiated mice have not been reported previously. The study aimed to evaluate the therapeutic effects of CSAE in mice immunosuppressed by irradiation. We observed that CSAE administration significantly increased body weight and spleen index, as well as the number of white blood cells, lymphocytes, and platelets in peripheral blood, T and B lymphocytes in spleen tissue, and total serum immunoglobulins in irradiated mice, whereas total serum pro-inflammatory cytokine levels were decreased. Collectively, CSAE maintained the structural integrity of spleen tissue and repaired its damage in irradiated mice as shown by hematoxylin and eosin staining, and decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive splenocytes. Mechanistically, CSAE upregulated Bcl-2, and downregulated Bax and cleaved caspase-3 in spleen of irradiated mice. However, there were no significant differences in red blood cells and neutrophils in different groups. The results revealed that CSAE had protective effects against irradiation-induced immunosuppression, which was likely associated with an antiapoptotic effect and the regulation of adaptive immunity.

Cordyceps sinensis Oral Liquid Inhibits Damage Induced by Oxygen and Glucose Deprivation in SH-SY5Y Cells.

CONTEXT: Cordyceps sinensis has been used in traditional Chinese medicine for thousands of years. It has been demonstrated to have a variety of biological activities, and an extract of it has been demonstrated to possess a protective effect in occlusion-induced focal cerebral ischemia of the middle cerebral artery in rats. It could be explored as an agent for treatment of ischemic stroke, and the mechanisms need to be studied further. OBJECTIVE: The study intended to investigate the protective effects of the Cordyceps sinensis oral liquid (CSOL) against damage induced by oxygen and glucose deprivation (OGD) in SH-SY5Y cells. DESIGN • The research team designed an in vitro study. SETTING: The study occurred at the Naval Medical Research Institute in Shanghai, China. INTERVENTION: SH-SY5Y cells were exposed to CSOL in doses of 0.01, 0.03, 0.10, 0.30, and 1.00 mg/mL, creating 5 intervention groups. The OGD condition was induced by transfer of the cells from high-glucose Dulbecco's Modified Eagle's medium (DMEM) in a box gassed with air containing 5% CO2 to glucose-free DMEM in a box gassed with 94% N2, 5% CO2, and 1% O2. Like the cells for the interventions groups, the cells for a model group were cultured with high-glucose DMEM and were transferred to the OGD, but they received no dose of COSL. Cells in a control group were cultured with high-glucose DMEM, were not transferred to the OGD condition, and did not receive any dose of COSL. OUTCOME MEASURES: Cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The apoptosis and the mitochondrial membrane potential (MMP) were detected by flow cytometry, and the protein expression of caspase-3 was observed by western blot. RESULTS: After exposure to OGD, the cell viability of cells treated with 0.01, 0.03, 0.10, 0.30, and 1.00 mg/mL of CSOL increased in a dose-effect relationship. Compared with the cells in the model group, the treatment of CSOL at all the experimental concentrations significantly inhibited both the cell apoptosis (P < .01) and the capase-3 activation (P < .01). The MMP dissipation in the cells of the model group increased significantly compared with those of the control group (P < .01). The treatment with all doses of CSOL significantly inhibited the MMP dissipation (P < .01). CONCLUSIONS: CSOL protects against the damage induced by OGD through inhibiting the mitochondrial apoptosis pathway in SH-SY5Y cells.

α-Lipoic acid protects against the oxidative stress and cytotoxicity induced by cadmium in HepG2 cells through regenerating glutathione regulated by glutamate-cysteine ligase.

Alpha-lipoic acid (α-LA) is an important antioxidant that is capable of regenerating other antioxidants, such as glutathione (GSH). In the present study, we examined the protective effects of α-LA against the oxidative stress and cytotoxicity induced by cadmium in human hepatoma cell lines (HepG2) and investigated if the process was mediated through regenerating GSH. Our results showed that after exposure to 25 µM cadmium for 16 h, there was a significant decrease in the cell viability and glutathione levels and a significant increase in lipid peroxidation (p<0.01) compared with untreated cells. The presence of α-LA significantly attenuated cadmium-induced cytotoxicity and lipid peroxidation, and reversed cellular GSH levels compared with cadmium-treated cells (p<0.05). Compared with the cells treated with cadmium, co-treatment with α-LA and cadmium significantly increased the activities of γ-glutamylcysteine ligase (γ-GCL), the rate limiting enzyme in GSH biosynthesis and the mRNA and the protein levels of γ-GCL catalytic subunit (GCLC) and a modifier subunit (GCLM). In conclusion, our results indicated that α-LA is an effective agent to reduce the oxidative stress and cytotoxicity induced by cadmium by regenerating GSH levels through increasing the activities and the expressions of γ-GCL.

Comparison of extracts and toxicities of organic compounds in drinking water concentrated by single and composite XAD resins.

We compared extracts and toxicities of organic compounds (OCs) in drinking water concentrated by composite XAD-2/8 resin (mixed with an equal volume of XAD-2 and XAD-8 resins) with those extracted by single XAD-2 (non-polar) and XAD-8 (polar) resins. Drinking water was processed from raw water of the Han River and the Yangtze River in Wuhan section, China. The extraction efficiency of all resins was controlled at 30%. The types of extracted OCs were detected by gas chromatography-mass spectrometry, and the cytotoxicity and genotoxicity were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and comet assays, respectively, in human hepatoma HepG2 cells. Our results showed that XAD-2/8 extracted a larger variety of OCs, compared with XAD-8 and XAD-2. The cytotoxicity and genotoxicity of extracted OCs were in the order of XAD-8> XAD-2/8> XAD-2 at almost all tested concentrations after 24 h treatment (P < 0.05). Our findings suggest that single XAD resin selectively extracts either polar or non-polar OCs, which would lead to over- or under-estimation of the toxicity of drinking water. Nevertheless, composite resin extracts both polar and non-polar OCs, and could be utilized as a useful extraction technique to evaluate the level and toxicity of OCs in drinking water.

Abscisic acid alleviates harmful effect of saline-alkaline stress on tomato seedlings.

Saline-alkaline stress inhibits plant growth and reduces yield. Abscisic acid (ABA) is an important plant hormone in response to plant stress. However, the role of ABA under saline-alkaline stress is poorly understood. Therefore, the mechanisms of ABA accumulation and resistance improvement in tomato seedlings were studied under saline-alkaline stress. We investigated whether ABA accumulation improved the saline-alkaline stress resistance ability of tomato. Here, wild-type (Solanum lycopersicum cv. Ailsa Craig) and ABA-deficient mutant (notabilis) seedlings were used to determine the membrane lipid peroxidation, osmotic substance and chlorophyll contents. ABA synthesis and signal transduction changes and ABA roles regulating the antioxidation in tomato seedlings subject to saline-alkaline stress were further explored. Results showed that ABA synthesis and signal transduction were induced by saline-alkaline stress. Under saline-alkaline stress, tomato seedlings had decreased relative water content, increased relative electrical conductivity and malondialdehyde content, and these changes were alleviated by exogenous ABA treatment. Exogenous ABA alleviated the degradation of chlorophyll in the leaves of tomato seedlings caused by saline-alkaline stress, further promoted the accumulation of proline and soluble sugar, reduced the content of ROS and improved the ability of the antioxidant enzyme system. Moreover, notabilis appeared to be sensitive to saline-alkaline stress. Overall, ABA is involved in the resistance of tomato seedlings to saline-alkaline stress, and exogenous ABA improves the saline-alkaline tolerance of tomato seedlings.

Design of Cyclodextrin-Based Functional Systems for Biomedical Applications.

Cyclodextrins (CDs) are a family of α-1,4-linked cyclic oligosaccharides that possess a hydrophobic cavity and a hydrophilic outer surface with abundant hydroxyl groups. This unique structural characteristic allows CDs to form inclusion complexes with various guest molecules and to functionalize with different substituents for the construction of novel sophisticated systems, ranging from derivatives to polymers, metal-organic frameworks, hydrogels, and other supramolecular assemblies. The excellent biocompatibility, selective recognition ability, and unique bioactive properties also make these CD-based functional systems especially attractive for biomedical applications. In this review, we highlight the characteristics and advantages of CDs as a starting point to design different functional materials and summarize the recent advances in the use of these materials for bioseparation, enzymatic catalysis, biochemical sensing, biomedical diagnosis and therapy.

Human urinary kallidinogenase in acute ischemic stroke: A single-arm, multicenter, phase IV study (RESK study).

AIMS: Human urinary kallidinogenase (HUK) has shown favorable efficacies in acute ischemic stroke (AIS) treatment. We sought confirmation of the safety and efficacy of HUK for AIS in a large population. METHODS: RESK study enrolled patients with AIS of anterior circulation to receive HUK infusion. The primary endpoint was the incidence of treatment-emergent adverse events (AEs). Secondary endpoints assessed neurological and functional improvements and stroke recurrent rate. RESULTS: Of 1206 eligible patients, 1202 patients received at least one dose of HUK infusion and 983 (81.5%) completed the study. The incidence of treatment-emergent AEs and serious AEs were 55.99% and 2.41%, respectively. Pre-specified AEs of special interest occurred in 21.71% of patients, but the majority were mild and unrelated to therapy. Hypertension, age, treatment time, and drug combination were identified to be associated with drug-related blood pressure reduction. Neurological and functional evaluations revealed favorable outcomes from baseline to post-treatment assessment. The cumulative recurrence rate of stroke was 2.50% during the 90-day assessment. CONCLUSION: HUK had an acceptable safety and tolerability profile in AIS patients. Besides, HUK demonstrated the neurological and functional improvements in AIS, further confirming its clinical efficacy in a real-world large population.

[Effect of chitosan on the level of ROS in HepG2 cell induced by cadmium chloride].

OBJECTIVE: To study the effect of chitosan on ROS levels in HepG2 cell induced by cadmium chloride. METHODS: The experiments were divided into 6 groups: normal control group, cadmium chloride group (20micromol/L), chitosan group (0.50mg/ml) and three combined treatment group (cadmium chloride 20micromol/L and chitosan 0.02mg/ml, 0.10mg/ml and 0.50mg/ml). The HepG2 cells were treated by chitosan and cadmium chloride simultaneously for 4h. The fluorescence of 2', 7'-dichlorofluorescin (DCF) was measured by floweytometry as a mean of estimating the formation of reactive oxygen species (ROS). RESULTS: In the comparison with normal control, the DCF intensity of chitosan group only exhibited somewhat decrease (P > 0.05) and significantly increased in cadmium chloride group (20micromol/L) (P < 0.01). The DCF intensity decreased in all three combined groups were more higher than those of cadmium chloride group, and when the concentration of chitosan improved from 0.01mg/ml to 0.50mg/ml, the DCF intensity decreased from 275.593 to 174.597 and the DCF intensity significant decrease was observed at concentration of 0.10mg/ml and 0.50mg/ml chitosan. CONCLUSION: It was suggested that chitosan could antagonize the increase of ROS in HepG2 cell induced by cadmium chloride.

Regeneration of glutathione by α-lipoic acid via Nrf2/ARE signaling pathway alleviates cadmium-induced HepG2 cell toxicity.

Alpha-lipoic acid (α-LA) is an important antioxidant that is capable of regenerating other antioxidants, such as glutathione (GSH). However, the underlying molecular mechanism by which α-LA regenerates GSH remains poorly understood. The current study aimed to investigate whether α-LA regenerates GSH by activation of Nrf2 to alleviate cadmium-induced cytotoxicity in HepG2 cells. In the present study, we found that cadmium induced cell death by depletion of GSH through inactivation of Nrf2. Addition of α-LA to cadmium-treated cells reactivated Nrf2 and regenerated GSH through elevating the Nrf2-downstream genes γ-glutamate-cysteine ligase (γ-GCL) and GR, both of which are key enzymes for GSH synthesis. However, blocking Nrf2 with brusatol in the cells co-treated with α-LA and cadmium reduced the mRNA and the protein levels of γ-GCL and GR, thus suppressed GSH regeneration by α-LA. Our results indicated that α-LA activated Nrf2 signaling pathway, which upregulated the transcription of the enzymes for GSH synthesis and therefore GSH contents to alleviate cadmium-induced cytotoxicity in HepG2 cells.

α-Lipoic acid protects against the cytotoxicity and oxidative stress induced by cadmium in HepG2 cells through regeneration of glutathione by glutathione reductase via Nrf2/ARE signaling pathway.

α-Lipoic acid (α-LA) is a potent natural antioxidant, which is capable of regenerating glutathione (GSH). However, the mechanisms by which α-LA regenerates reduced glutathione (rGSH) via the reduction of oxidized glutathione (GSSG) by glutathione reductase (GR) are still not well understood. In the present study, we investigated if α-LA replenished rGSH by GR via Nrf2/ARE signaling pathway in cadmium-treated HepG2 cells. We found that α-LA antagonized the oxidative damage and alleviated the cytotoxicity in cadmium-induced HepG2 cells by regeneration of rGSH. α-LA regenerated rGSH by activating Nrf2 signaling pathway via promoting the nuclear translocation of Nrf2, which upregulates the transcription of GR, and thus increased the activity of GR. Our results indicated that α-LA was an effective agent to antagonize the oxidative stress and alleviate the cytotoxicity in cadmium-treated HepG2 cells by regenerating rGSH through activating Nrf2 signaling pathway.

Medical swab touch spray-mass spectrometry for newborn screening of nicotine and cotinine in meconium.

Newborn screening is one of public health concerns designed to screen infants shortly after birth. Prenatal exposure to tobacco smoke such as nicotine has been reported to affect babies. Levels of nicotine and cotinine in meconium were widely used to evaluate the tobacco exposure of foetuses during pregnancy in a polluted environment. In this study, medical swabs were applied by using touch spray-mass spectrometry (TS-MS) to collect meconium from newborn infants for detection of nicotine and cotinine. Parameters such as choice of spray solvents, solvent volume and collision energy for screening of nicotine and cotinine were optimized. The limits of detection, reproducibility and matrix effect for analysis of meconium were also investigated. In this study, the levels of nicotine and cotinine in 54 puerpera volunteers were screened by TS-MS and were validated by using traditional liquid chromatography-mass spectrometry. These results showed that medical swab TS-MS would be useful for newborn screening of nicotine and cotinine in meconium with high reproducibility, speed, sensitivity and specificity. The use of disposable medical swabs involves no sample preparation and no chromatographic separation, significantly reducing the cost and time required for screening a large number of clinical sample. Copyright © 2016 John Wiley & Sons, Ltd.

Cordyceps sinensis oral liquid prolongs the lifespan of the fruit fly, Drosophila melanogaster, by inhibiting oxidative stress.

This study investigated the effect of Cordyceps sinensis oral liquid (CSOL) on the lifespan of Drosophila melanogaster (fruit fly). Following the lifelong treatment of fruit flies with CSOL, lifespan was examined. The activity of copper-zinc-containing superoxide dismutase 1 (SOD1), manganese-containing superoxide dismutase 2 (SOD2) and catalase (CAT), as well as the lipofuscin (LF) content were determined. The mRNA levels of SOD1, SOD2 and CAT were quantified by qPCR. Hydrogen peroxide (H2O2) and paraquat were used to mimic the effects of damage caused by acute oxidative stress. D-galactose was used to mimic chronic pathological aging. CSOL significantly prolonged the lifespan of the fruit flies under physiological conditions. The activity of SOD1 and CAT was upregulated, and LF accumulation was inhibited by CSOL. CSOL had no effect on the transcriptional levels (mRNA) of these enzymes. The survival time of the fruit flies which were negatively affected by exposure to H2O2 or paraquat was significantly prolonged by CSOL. In fruit flies pathologically aged by epxosure to D-galactose, CSOL also significantly prolonged their lifespan, upregulated the activity of SOD1 and CAT, and inhibited LF accumulation. The findings of our study indicate that CSOL prolongs the lifespan of fruit flies through an anti-oxidative stress pathway involving the upregulation of SOD1 and CAT activity and the inhibition of LF accumulation. CSOL may thus be explored as a novel agent for slowing the human aging process.

Extracellular Ca(2+) Selectively Enhances Adriamycin-induced Cell Death in Human Hepatoma Cells.

It has been shown that the regulatory mechanisms of Ca(2+) signaling between tumor and normal cells are different, providing new insight into the pharmacological modulation of anticancer drugs. However, it remains unclear whether there is any difference between hepatoma and normal hepatic cells in their response to extracellular Ca(2+) ([Ca(2+)]e). In the present study, the growth inhibition, apoptosis and necrosis rates of human normal embryo hepatic L02 and human hepatoma HepG2 cells exposed to additional calcium ([Ca(2+)]a)and adriamycin (ADM), a chemotherapeutic agent to treat hepatocellular carcinoma, were measured by MTT and annexin V/PI assays, respectively. The results showed that the growth inhibition, necrosis and apoptosis rates, as well as intracellular Ca(2+) concentrations ([Ca(2+)]i) induced by [Ca(2+)]e in HepG2 cells were higher than those in L02 cells. Moreover, [Ca(2+)]e was able to selectively enhance ADM-induced growth inhibition, apoptosis and necrosis in HepG2 cells, but not in L02 cells. ADM and [Ca(2+)]a co-treatment had a significant interaction effect to increase [Ca(2+)]i in both cell lines, although there was no significant difference in [Ca(2+)]i between the two cells. To further elucidate the mechanisms involved in the selective promotion of [Ca(2+)]e in HepG2 and L02 cells, the levels of these apoptosis regulatory proteins (bcl-2, bax and procaspase-3) and the caspase-3 activity following treatment of HepG2 and L02 cells with ADM or/and [Ca(2+)]a were investigated. The results showed that treating HepG2 cells with [Ca(2+)]a and ADM increased the level of bax protein and caspase-3 activity while decreasing the level of bcl-2 protein, compared with treatment with ADM alone. However, no significant change was noted in L02 cells. These results indicate that hepatoma HepG2 cells are more sensitive to [Ca2+]e than normal hepatic L02 cells and that [Ca(2+)]a can selectively enhance ADM-induced cell death in HepG2 cells. The mechanism of this intensive pro-apoptotic effect can be ascribed to up-regulation of bax and the simultaneous down-regulation of bcl-2, followed by the switch from procaspase-3 to caspase-3, which executed apoptosis. The present data suggest the potency of the calcium ion as an enhancer of ADM.