Photodynamic treatment increases the lifespan and oxidative stress resistance of Caenorhabditis elegans.

Photodynamic therapy is a noninvasive treatment in which specific photosensitizers and light are used to produce high amounts of reactive oxygen species (ROS), which can be employed for targeted tissue destruction in cancer treatment or antimicrobial therapy. However, it remains unknown whether lower amounts of ROS produced by mild photodynamic therapy increase lifespan and stress resistance at the organism level. Here, we introduce a novel photodynamic treatment (PDTr) that uses 20 µM hypericin, a photosensitizer that originates from Hypericum perforatum, and orange light (590 nm, 5.4 W/m2, 1 min) to induce intracellular ROS formation (ROS), thereby resulting in lifespan extension and improved stress resistance in C. elegans. The PDTr-induced increase in longevity was abrogated by N-acetyl cysteine, suggesting the hormetic response was driven by prooxidative mechanisms. PDTr activated the translocation of SKN-1/NRF-2 and DAF-16/FOXO, leading to elevated expression of downstream oxidative stress-responsive genes, including ctl-1, gst-4, and sod-3. In summary, our findings suggest a novel PDTr method that extends the lifespan of C. elegans under both normal and oxidative stress conditions through the activation of SKN-1 and DAF-16 via the involvement of many antioxidant genes.

Photodynamic Therapy.

In 1903, Von Tappeiner and Jesionek [...].

Schisandrin C improves leaky gut conditions in intestinal cell monolayer, organoid, and nematode models by increasing tight junction protein expression.

BACKGROUND: Leaky gut symptoms and inflammatory bowel disease (IBD) are associated with damaged intestinal mucosa, intestinal permeability dysfunction by epithelial cell cytoskeleton contraction, disrupted intercellular tight junction (TJ) protein expression, and abnormal immune responses and are intractable diseases. PURPOSE: We evaluated the effects of schisandrin C, a dibenzocyclooctadiene lignan from Schisandra chinensis, on intestinal inflammation and permeability dysfunction in gut mimetic systems: cultured intestinal cells, intestinal organoids, and a Caenorhabditis elegans model. METHODS: Schisandrin C was selected from 9 lignan compounds from S. chinensis based on its anti-inflammatory effects in HT-29 human intestinal cells. IL-1ß and Pseudomonas aeruginosa supernatants were used to disrupt intestinal barrier formation in vitro and in C. elegans, respectively. The effects of schisandrin C on transepithelial electrical resistance (TEER) and intestinal permeability were evaluated in intestinal cell monolayers, and its effect on intestinal permeability dysfunction was tested in mouse intestinal organoids and C. elegans by measuring fluorescein isothiocyanate (FITC)-dextran efflux. The effect of schisandrin C on TJ protein expression was investigated by western blotting and fluorescence microscopy. The signaling pathway underlying these effects was also elucidated. RESULTS: Schisandrin C ameliorated intestinal permeability dysfunction in three IBD model systems and enhanced epithelial barrier formation via upregulation of ZO-1 and occludin in intestinal cell monolayers and intestinal organoids. In Caco-2 cells, schisandrin C restored IL-1ß-mediated increases in MLCK and p-MLC expression, in turn blocking cytoskeletal contraction and subsequent intestinal permeabilization. Schisandrin C inhibited NF-ĸB and p38 MAPK signaling, which regulates MLCK expression and structural reorganization of the TJ complex in Caco-2 cells. Schisandrin C significantly improved abnormal FITC-dextran permeabilization in both intestinal organoids and C. elegans. CONCLUSION: Schisandrin C significantly improves abnormal intestinal permeability and regulates the expression of TJ proteins, long MLCK, p-MLC, and inflammation-related proteins, which are closely related to leaky gut symptoms and IBD development. Therefore, schisandrin C is a candidate to treat leaky gut symptoms and IBDs.

3,3'-Diindolylmethane Supplementation Maintains Oocyte Quality by Reducing Oxidative Stress and CEP-1/p53-Mediated Regulation of Germ Cells in a Reproductively Aged Caenorhabditis elegans Model.

In recent decades, maternal age at first birth has increased, as has the risk of infertility due to rapidly declining oocyte quality with age. Therefore, an understanding of female reproductive aging and the development of potential modulators to control oocyte quality are required. In this study, we investigated the effects of 3,3'-diindolylmethane (DIM), a natural metabolite of indole-3-cabinol found in cruciferous vegetables, on fertility in a Caenorhabditis elegans model. C. elegans fed DIM showed decreased mitochondrial dysfunction, oxidative stress, and chromosomal aberrations in aged oocytes, and thus reduced embryonic lethality, suggesting that DIM, a dietary natural antioxidant, improves oocyte quality. Furthermore, DIM supplementation maintained germ cell apoptosis (GCA) and germ cell proliferation (GCP) in a CEP-1/p53-dependent manner in a reproductively aged C. elegans germ line. DIM-induced GCA was mediated by the CEP-1-EGL-1 pathway without HUS-1 activation, suggesting that DIM-induced GCA is different from DNA damage-induced GCA in the C. elegans germ line. Taken together, we propose that DIM supplementation delays the onset of reproductive aging by maintaining the levels of GCP and GCA and oocyte quality in a reproductively aged C. elegans.

Tropolone-Bearing Sesquiterpenes from Juniperus chinensis: Structures, Photochemistry and Bioactivity.

The tropolone-bearing sesquiterpenes juniperone A (1) and norjuniperone A (2) were isolated from the folk medicinal plant Juniperus chinensis, and their structures were determined by a combination of spectroscopic and crystallographic methods. Photojuniperones A1 (3) and A2 (4), bearing bicyclo[3,2,0]heptadienones derived from tropolone, were photochemically produced and structurally identified by spectroscopic methods. Predicted by the machine learning-based assay, 1 significantly inhibited the action of tyrosinase. The new compounds also inhibited lipid accumulation and enhanced the extracellular glycerol excretion.

Natural Photosensitizers in Antimicrobial Photodynamic Therapy.

Health problems and reduced treatment effectiveness due to antimicrobial resistance have become important global problems and are important factors that negatively affect life expectancy. Antimicrobial photodynamic therapy (APDT) is constantly evolving and can minimize this antimicrobial resistance problem. Reactive oxygen species produced when nontoxic photosensitizers are exposed to light are the main functional components of APDT responsible for microbial destruction; therefore, APDT has a broad spectrum of target pathogens, such as bacteria, fungi, and viruses. Various photosensitizers, including natural extracts, compounds, and their synthetic derivatives, are being investigated. The main limitations, such as weak antimicrobial activity against Gram-negative bacteria, solubility, specificity, and cost, encourage the exploration of new photosensitizer candidates. Many additional methods, such as cell surface engineering, cotreatment with membrane-damaging agents, nanotechnology, computational simulation, and sonodynamic therapy, are also being investigated to develop novel APDT methods with improved properties. In this review, we summarize APDT research, focusing on natural photosensitizers used in in vitro and in vivo experimental models. In addition, we describe the limitations observed for natural photosensitizers and the methods developed to counter those limitations with emerging technologies.

Natural photosensitizers from Tripterygium wilfordii and their antimicrobial photodynamic therapeutic effects in a Caenorhabditis elegans model.

Tripterygium wilfordii Hook. f. is a traditional medicinal plant and has long been used in East Asia to treat many diseases. However, the extract and active components have never been investigated as potential photosensitizers for photodynamic treatment to kill pathogenic microorganisms. Here, the antimicrobial photodynamic treatment (APDT) effects of the extract, fractions, and compounds of T. wilfordii were evaluated in vitro and in vivo. Ethanolic extract (TWE) and the photosensitizer-enriched fraction (TW-F5) were prepared from dried T. wilfordii. Six active compounds were isolated from TW-F5 by semipreparative high-performance liquid chromatography, and their chemical structures were characterized through spectroscopic and spectrometric analysis. The singlet oxygen from extracts, fractions, and compounds was measured by using the imidazole-N,N-dimethyl-4-nitrosoaniline method. These extracts, fractions, and compounds were used as photosensitizers for the inactivation of bacteria and fungi by red light at 660 nm. The in vitro APDT effects were also evaluated in the model animal Caenorhabditis elegans. APDT with TWE showed effective antimicrobial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans. TW-F5, consisting of six pheophorbide compounds, also showed strong APDT activity. The photosensitizers were taken up into the bacterial cells and induced intracellular ROS production by APDT. TWE and TW-F5 also induced a strong APDT effect in vitro against skin pathogens, including Staphylococcus epidermidis and Streptococcus pyogenes. We evaluated the APDT effects of TWE and TW-F5 in C. elegans infected with various pathogens and found that PDT effectively controlled pathogenic bacteria without strong side effects. APDT reversed the growth retardation of worms induced by pathogen infection and decreased the viable pathogenic bacterial numbers associated with C. elegans. Finally, APDT with TWE increased the survivability of C. elegans infected with S. pyogenes. In summary, TWE and TW-F5 were found to be effective antimicrobial photosensitizers in PDT.

Synthesis and biological evaluation of chalcone derivatives as neuroprotective agents against glutamate-induced HT22 mouse hippocampal neuronal cell death.

Seventeen chalcone analogues were synthesized from 7-methoxy-3,4-dihydronaphthalen1(2H)-one and various aromatic aldehydes under basic conditions and their therapeutic properties were studied in mouse hippocampal cell line HT-22 against neuronal cell death induced by glutamate. From this study, we selected an analogue C01 as a active compound which showed significantly high neuroprotection. This compound inhibited Ca2+ influx and reactive oxygen species (ROS) accumulation inside cells. The glutamate-induced cell death was analyzed by flow cytometry and it showed that C01 significantly reduced apoptotic or dead cell induced by 5 mM glutamate. Western blot analysis indicates that glutamate-mediated activation of MAPKs were inhibited by compound C01 treatment. In addition, the C01enhanced Bcl-2 and decreased Bax, the anti and pro apoptotic proteins respectively. Further analysis showed that, C01 prevented the nuclear translocation of AIF (apoptosis inducing factor) and inhibited neuronal cell death. Taken together, compound C01 treatment resulted in decreased neurotoxicity induced by 5 mM of glutamate. Our finding confirmed that compound C01 has neuro-therapeutic potential against glutamate-mediated neurotoxicity.

Improvement in host metabolic homeostasis and alteration in gut microbiota in mice on the high-fat diet: A comparison of calcium supplements.

Despite the previously reported health benefits of calcium intake for the attenuation of metabolic disease, few studies have investigated the relationships among calcium intake, gut microbiota, and host metabolism. In this study, we assessed the effects of calcium supplementation on host microbial community composition and metabolic homeostasis. Mice were fed a high-fat diet with different calcium concentrations (4 and 12 g/kg) of 2 calcium supplements, calcium carbonate and calcium citrate. Supplementation with the higher concentration of calcium citrate significantly prevented body weight gain and decreased plasma biomarkers for metabolic disorder compared to calcium carbonate supplementation. Both calcium supplementation led to changes in microbial composition, increased propionate production and increased anorexigenic GLP-1 gene expression. The calcium citrate groups also experienced less metabolic endotoxemia. Our findings suggested that calcium supplementation could ameliorate host metabolic disorder caused by a high-fat diet, due to gut microbiota changes as well as decreased intestinal inflammation.

Synthesis and biological evaluation of isoliquiritigenin derivatives as a neuroprotective agent against glutamate mediated neurotoxicity in HT22 cells.

Glutamate-induced neurotoxicity is characterized by cellular Ca2+ uptake, which is upstream of reactive oxygen species (ROS)-induced apoptosis signaling and MAPKs activation. In the present study, we synthesized isoliquiritigenin analogs with electron-donating and electron-withdrawing functional groups. These analogs were evaluated for neuroprotective effect against glutamate-induced neurotoxicity in HT22 cells. Among these analogs, compound BS11 was selected as a potent neuroprotective agent. Cellular Ca2+ concentration, ROS level, MAPKs activation and AIF translocation to the nucleus were increased upon treatment with 5 mM glutamate. In contrast, we identified that compound BS11 reduced the cellular Ca2+ concentration and ROS level upon glutamate exposure. Western blot analysis showed that MAPK activation was decreased by treatment with compound BS11. We further identified that cotreatment of compound BS11 and glutamate inhibited translocation of AIF to the nucleus.

Protective effect of Lactobacillus casei HY2782 against particulate matter toxicity in human intestinal CCD-18Co cells and Caenorhabditis elegans.

OBJECTIVES: To investigate the preventive effect of Lactobacillus casei HY2782 on toxicity induced by particulate matter (PM, inhalable particles less than 10 µm in diameter) in human intestinal CCD-18Co cells and a model animal Caenorhabditis elegans. RESULTS: L. casei HY2782 treatment prevented PM-induced intestinal cell death via cellular reactive oxygen species production and membrane disruption attenuation. PM significantly decreased the total number of eggs laid and the body bending activity of C. elegans, demonstrating PM toxicity. L. casei HY2782 treatment restored the reproductive toxicity and decline in locomotion activity induced by PM in C. elegans. Overall, L. casei HY2782 attenuated PM toxicity in vitro in cultured intestinal cells and in vivo in the model nematode. CONCLUSION: Our study provides a potential clue for developing L. casei HY2782 probiotics that attenuate PM-induced cellular and physiological toxicity; however, further in-depth preclinical trials using mammalian animal models and clinical trials are required.

Antimicrobial Biophotonic Treatment of Ampicillin-Resistant Pseudomonas aeruginosa with Hypericin and Ampicillin Cotreatment Followed by Orange Light.

Bacterial antibiotic resistance is an alarming global issue that requires alternative antimicrobial methods to which there is no resistance. Antimicrobial photodynamic therapy (APDT) is a well-known method to combat this problem for many pathogens, especially Gram-positive bacteria and fungi. Hypericin and orange light APDT efficiently kill Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and the yeast Candida albicans. Although Gram-positive bacteria and many fungi are readily killed with APDT, Gram-negative bacteria are difficult to kill due to their different cell wall structures. Pseudomonas aeruginosa is one of the most important opportunistic, life-threatening Gram-negative pathogens. However, it cannot be killed successfully by hypericin and orange light APDT. P. aeruginosa is ampicillin resistant, but we hypothesized that ampicillin could still damage the cell wall, which can promote photosensitizer uptake into Gram-negative cells. Using hypericin and ampicillin cotreatment followed by orange light, a significant reduction (3.4 log) in P. aeruginosa PAO1 was achieved. P. aeruginosa PAO1 inactivation and gut permeability improvement by APDT were successfully shown in a Caenorhabditis elegans model.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

3,3'-Diindolylmethane Improves Intestinal Permeability Dysfunction in Cultured Human Intestinal Cells and the Model Animal Caenorhabditis elegans.

3,3'-Diindolylmethane (DIM), a digestive metabolite originating from cruciferous vegetables, has dietary potential for the treatment of various human intestinal diseases. Although intestinal permeability dysfunction is closely related to the initiation and progression of human intestinal inflammatory diseases (IBDs), the effect of DIM on intestinal permeability is unclear. We evaluated the effect of DIM on the intestinal permeability of human intestinal cell monolayers and the animal model Caenorhabditis elegans, which were treated with IL-1ß and Pseudomonas aeruginosa, respectively, to mimic IBD conditions. DIM substantially restored the intestinal permeability of differentiated Caco-2 cells by enhancing the expression of tight junction proteins (including occludin and ZO-1). Compared to the IL-1ß single treatment (551.0 ± 49.0 Ω·cm2), DIM (10 µM) significantly increased the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers (919.0 ± 66.4 Ω·cm2, p < 0.001). DIM also ameliorated the impaired intestinal permeability and extended the lifespan of C. elegans fed P. aeruginosa. The mean lifespan of DIM-treated worms (10.8 ± 1.3 days) was higher than that of control-treated worms (9.7 ± 1.1 days, p < 0.01). Thus, DIM is a potential nutraceutical candidate for the treatment of leaky gut syndrome by improving intestinal permeability.

Anti-Obesity Effect of Standardized Extract of Microalga Phaeodactylum tricornutum Containing Fucoxanthin.

Fucoxanthin (FX), a marine carotenoid found in macroalgae and microalgae, exhibits several beneficial effects to health. The anti-obesity activity of FX is well documented, but FX has not been mass-produced or applied extensively or commercially because of limited availability of raw materials and complex extraction techniques. In this study, we investigated the anti-obesity effect of standardized FX powder (Phaeodactylum extract (PE)) developed from microalga Phaeodactylum tricornutum as a commercial functional food. The effects of PE on adipogenesis inhibition in 3T3-L1 adipocytes and anti-obesity in high-fat diet (HFD)-fed C57BL/6J mice were evaluated. PE and FX dose-dependently decreased intracellular lipid contents in adipocytes without cytotoxicity. In HFD-fed obese mice, PE supplementation for six weeks decreased body weight, organ weight, and adipocyte size. In the serum parameter analysis, the PE-treated groups showed attenuation of lipid metabolism dysfunction and liver damage induced by HFD. In the liver, uncoupling protein-1 (UCP1) upregulation and peroxisome proliferator activated receptor γ (PPARγ) downregulation were detected in the PE-treated groups. Additionally, micro computed tomography revealed lower fat accumulation in PE-treated groups compared to that in the HFD group. These results indicate that PE exerts anti-obesity effects by inhibiting adipocytic lipogenesis, inducing fat mass reduction and decreasing intracellular lipid content, adipocyte size, and adipose weight.

Effects of fermented milk treatment on microbial population and metabolomic outcomes in a three-stage semi-continuous culture system.

We investigated the impact of a fermented milk product on gut microbiota and their metabolism in 3 different conditions of the colon with a systemic viewpoint. An in vitro semi-continuous anaerobic cultivation was used to assess the colon compartment-specific influence of fermented milk, followed by a multiomics approach combining 16S rDNA amplicon sequencing and nuclear magnetic resonance (NMR) spectroscopy. The microbiome profiling and metabolomic features were significantly different across three colon compartments and after fermented milk treatment. Integrative correlation analysis indicated that the alteration of butyrate-producing microbiota (Veillonella, Roseburia, Lachnospira, and Coprococcus) and some primary metabolites (butyrate, ethanol, lactate, and isobutyrate) in the treatment group had a strong association with the fermented milk microorganisms. Our findings suggested that fermented milk treatment significantly affected microbial population in an in vitro cultivation system as well as the colonic metabolome in different ways in each of colon compartment.

High-throughput and direct measurement of androgen levels using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS) to discover chemicals that modulate dihydrotestosterone production in human prostate cancer cells.

OBJECTIVES: To develop a high-throughput screening system to measure the conversion of testosterone to dihydrotestosterone (DHT) in cultured human prostate cancer cells using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS). RESULTS: After optimizing the cell reaction system, this method demonstrated a screening capability of 103 samples, including 78 single compounds and 25 extracts, in less than 12 h without manual sample preparation. Consequently, fucoxanthin, phenethyl caffeate, and Curcuma longa L. extract were validated as bioactive chemicals that inhibited DHT production in cultured DU145 cells. In addition, naringenin boosted DHT production in DU145 cells. CONCLUSION: The method can facilitate the discovery of bioactive chemicals that modulate the DHT production, and four phytochemicals are potential candidates of nutraceuticals to adjust DHT levels in male hormonal dysfunction.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Data on the anti-tumor effects of Selaginella tamariscina extract and amentoflavone combined with doxorubicin in mice.

Here, we report animal experimental data associated with the article entitled "AKR1B10-inhibitory Selaginella tamariscina extract and amentoflavone decrease the growth of A549 human lung cancer cells in vitro and in vivo" (Jung et al., 2017) [1]. We tested the synergistic anti-tumor effects of Selaginella tamariscina extract and amentoflavone combined with doxorubicin hydrochloride in a nude mouse xenograft model of A549 human lung cancer cells. In our experiment, Selaginella tamariscina extract and amentoflavone were administered orally; and doxorubicin hydrochloride was injected intraperitoneally. We expect our preliminary data will be helpful to the development of the anticancer agent using Selaginella tamariscina extract or amentoflavone.

AKR1B10-inhibitory Selaginella tamariscina extract and amentoflavone decrease the growth of A549 human lung cancer cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Selaginella tamariscina (P.Beauv.) Spring is a traditional medicinal plant used to treat various human diseases, including cancer, in Asia. The detailed molecular mechanism underlying the anti-cancer effects of this plant and the anti-cancer action of the combinatorial treatment of S. tamariscina and doxorubicin have not yet been investigated. AIM OF THE STUDY: We evaluated the inhibitory activity of S. tamariscina extract (STE) and its major compound, amentoflavone, on human aldo-keto reductase family 1B10 (AKR1B10), which is a detoxification enzyme involved in drug resistance, to evaluate their anti-cancer effects and their potential as adjuvant agents for doxorubicin cancer chemotherapy. MATERIALS AND METHODS: We tested the AKR1B10 inhibitory activity of STE and amentoflavone via an in vitro biochemical assay using recombinant human AKR1B10. We tested the anti-proliferative activity in A549, NCI-H460, SKOV-3, and MCF-7 human cancer cells, which contain different expression levels of AKR1B10, and determined the combination index to evaluate whether the addition of STE and amentoflavone is synergistic or antagonistic to the anti-cancer action of doxorubicin. We finally evaluated the in vivo anti-tumor effects of STE in a nude mouse xenograft model of A549 cells. RESULTS: STE and amentoflavone potently inhibited human AKR1B10 and synergistically increased the doxorubicin anti-proliferative effect in A549 and NCI-H460 human lung cancer cells that express a high level of AKR1B10 mRNA and protein. STE also significantly inhibited A549 tumor growth in animal experiments. CONCLUSION: Our results suggest that STE and amentoflavone could be potential anti-cancer agents that target AKR1B10 and might be candidate adjuvant agents to boost the anti-cancer effect of doxorubicin.