Regulated Necrotic Cell Death in Alternative Tumor Therapeutic Strategies.

The treatment of tumors requires the induction of cell death. Radiotherapy, chemotherapy, and immunotherapy are administered to kill cancer cells; however, some cancer cells are resistant to these therapies. Therefore, effective treatments require various strategies for the induction of cell death. Regulated cell death (RCD) is systematically controlled by intracellular signaling proteins. Apoptosis and autophagy are types of RCD that are morphologically different from necrosis, while necroptosis, pyroptosis, and ferroptosis are morphologically similar to necrosis. Unlike necrosis, regulated necrotic cell death (RNCD) is caused by disruption of the plasma membrane under the control of specific proteins and induces tissue inflammation. Various types of RNCD, such as necroptosis, pyroptosis, and ferroptosis, have been used as therapeutic strategies against various tumor types. In this review, the mechanisms of necroptosis, pyroptosis, and ferroptosis are described in detail, and a potential effective treatment strategy to increase the anticancer effects on apoptosis- or autophagy-resistant tumor types through the induction of RNCD is suggested.

Pasakbumin A controls the growth of Mycobacterium tuberculosis by enhancing the autophagy and production of antibacterial mediators in mouse macrophages.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a major health problem worldwide. Thus, identification of new and more effective drugs to treat emerging multidrug-resistant TB (MDR-TB) and to reduce the side effects of anti-TB drugs, such as liver toxicity and other detrimental changes, is urgently needed. In this study, to develop a novel candidate drug for effective TB treatment with few side effects in the host, we selected pasakbumin A isolated from Eurycoma longifolia (E. longifolia) Jack, which protected host cells against Mtb infection-induced death. Pasakbumin A significantly inhibited intracellular Mtb growth by inducing the autophagy via the ERK1/2-mediated signaling pathway in Mtb-infected macrophages. We further investigated whether pasakbumin A could be used as a potential adjuvant for TB treatment. Treatment with pasakbumin A and anti-TB drug rifampicin (RMP) potently suppressed intracellular Mtb killing by promoting autophagy as well as TNF-α production via the ERK1/2- and NF-κB-mediated signaling pathways in Mtb-infected cells. Our results suggest that pasakbumin A could be developed as a novel anti-TB drug or host-directed therapeutic (HDT) strategy to protect against host cell death and improve host defense mechanisms against Mtb infection in macrophages.

Antiviral Activity of Fermented Ginseng Extracts against a Broad Range of Influenza Viruses.

Ginseng products used as herb nutritional supplements are orally consumed and fermented to ginsenoside compounds by the intestinal microbes. In this study, we investigated antiviral protective effects of fermented ginseng extracts against different strains of influenza viruses in genetically diverse mouse models. Intranasal coinoculation of mice with fermented ginseng extract and influenza virus improved survival rates and conferred protection against H1N1, H3N2, H5N1, and H7N9 strains, with the efficacy dependent on the dose of ginseng samples. Antiviral protection by fermented ginseng extract was observed in different genetic backgrounds of mice and in the deficient conditions of key adaptive immune components (CD4, CD8, B cell, MHCII). The mice that survived primary virus inoculation with fermented ginseng extract developed immunity against the secondary infection with homologous and heterosubtypic viruses. In vitro cell culture experiments showed moderate virus neutralizing activity by fermented ginseng extract, probably by inhibiting hemagglutination and neuraminidase activity. This study suggests that fermented ginseng extracts might provide a means to treat influenza disease regardless of virus strains.

Combination Therapy Comprising a Static Magnetic Field with Contractility Improves Skin Wounds.

Cutaneous wounds can present significant clinical problems because of abnormal healing after deep dermal damage. Despite technical advances in wound care, there are still unmet needs that result from inefficient treatment. In this study, we aimed to improve skin wound healing using a contractibility band with static magnetic field (SMF), termed a magnetic band (Mb). To examine the effect of the Mb on wound healing, full-thickness 15 × 35 mm excision wounds were surgically created on the dorsum of rats. An elastic and contractile band (nontreatment), or one neodymium magnet (Nd-1) or two magnets with an elastic and contractile band (Nd-2) were topically applied to the wound daily and the wound size was measured from day 1 to 7 after surgery. Nd-2 showed a significant (95%) reduction in the wound size on day 3. Histological analysis showed that proinflammatory cytokine levels were diminished by Nd-2, and granulation tissue and microvessels were increased compared with those in the sham group. During Mb-induced wound healing, apoptosis was significantly reduced and matrix remodeling-related factors were initially regulated. The results suggest that combination therapy comprising an SMF and an elastic and contractile band could be a promising tool to heal cutaneous wounds rapidly.

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.

Heat-killed Lactobacillus casei confers broad protection against influenza A virus primary infection and develops heterosubtypic immunity against future secondary infection.

Lactic acid bacteria (LAB) are the common probiotics. Here, we investigated the antiviral protective effects of heat-killed LAB strain Lactobacillus casei DK128 (DK128) on influenza viruses. Intranasal treatment of mice with DK128 conferred protection against different subtypes of influenza viruses by lessening weight loss and lowering viral loads. Protection via heat-killed DK128 was correlated with an increase in alveolar macrophage cells in the lungs and airways, early induction of virus specific antibodies, reduced levels of pro-inflammatory cytokines and innate immune cells. Importantly, the mice that were protected against primary viral infection as a result of heat-killed DK128 pretreatment developed subsequent heterosubtypic immunity against secondary virus infection. For protection against influenza virus via heat-killed DK128 pretreatment, B cells and partially CD4 T cells but not CD8 T cells were required as inferred from studies using knockout mouse models. Our study provides insight into how hosts can be equipped with innate and adaptive immunity via heat-killed DK128 treatment to protect against influenza virus, supporting that heat-killed LAB may be developed as anti-virus probiotics.

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.

Oldenlandia diffusa Promotes Antiproliferative and Apoptotic Effects in a Rat Hepatocellular Carcinoma with Liver Cirrhosis.

Oldenlandia diffusa (OD) is commonly used with various diseases such as cancer, arthritis, and autoimmune disease. Liver cirrhosis is a predominant risk factor for hepatocellular carcinoma (HCC). Here, we show that the therapeutic effect of OD, which was investigated both in vitro and chemically, induced HCC model. OD significantly enhanced apoptosis and antiproliferative activity and reduced migration ability of HCC cells. In vivo, OD was treated twice a day for 28 days after confirmed HCC model through 2-[(18)F]-fluoro-2-deoxy-D-glucose ((18)F-FDG) imaging. The survival in OD treated groups was shown to have a greater therapeutic effect than the control group. 28 days after OD treatment, OD treated groups resulted in a significant reduction in tumor number, size, (18)F-FDG uptake, and serum levels such as alanine transaminase, aspartate transaminase, and alkaline phosphate compared to the control group. Also, proliferated cells in tumor sites by OD were reduced compared to the control group. Furthermore, several rats in OD treated group survived over 60 days and liver morphology of these rats showed the difference between tumor mass and normal tissue. These results suggest that OD may have antiproliferative activity, inhibition of metastasis, and apoptotic effects in chemically induced HCC model and can have the potential use for clinical application as anticancer drug of the herbal extract.

Ginseng diminishes lung disease in mice immunized with formalin-inactivated respiratory syncytial virus after challenge by modulating host immune responses.

Formalin-inactivated respiratory syncytial virus (FI-RSV) immunization is known to cause severe pulmonary inflammatory disease after subsequent RSV infection. Ginseng has been used in humans for thousands of years due to its potential health benefits. We investigated whether ginseng would have immune modulating effects on RSV infection in mice previously immunized with FI-RSV. Oral administration of mice with ginseng increased IgG2a isotype antibody responses to FI-RSV immunization, indicating T-helper type 1 (Th1) immune responses. Ginseng-treated mice that were nonimmunized or previously immunized with FI-RSV showed improved protection against RSV challenge compared with control mice without ginseng treatment. Ginseng-mediated improved clinical outcomes after live RSV infection were evidenced by diminished weight losses, decreased interleukin-4 cytokine production but increased interferon-γ production, modulation of CD3 T-cell populations toward a Th1 response, and reduced inflammatory response. Ginseng-mediated protective host immune modulation against RSV pulmonary inflammation was observed in different strains of wild-type and mutant mice. These results indicate that ginseng can modulate host immune responses to FI-RSV immunization and RSV infection, resulting in protective effects against pulmonary inflammatory disease.

A Pilot Study for the Neuroprotective Effect of Gongjin-dan on Transient Middle Cerebral Artery Occlusion-Induced Ischemic Rat Brain.

In this study, we investigated whether gongjin-dan improves functional recovery and has neuroprotective effects on reducing the infarct volume after transient middle cerebral artery occlusion (MCAo). Infarct volume was measured using TTC staining and glucose utilization by F-18 FDG PET. Functional improvement was evaluated with the Rota-rod, treadmill, Garcia score test, and adhesive removal test. At 14 days after MCAo, neuronal cell survival, astrocytes expansion, and apoptosis were assessed by immunohistofluorescence staining in the peri-infarct region. Also, the expression of neurotrophic factors and inflammatory cytokines such as VEGF, BDNF, Cox-2, TNF-α, IL-1ß, and IL-1α was measured in ischemic hemisphere regions. The gongjin-dan-treated group showed both reduced infarct volume and increased glucose utilization. Behavior tests demonstrated a significant improvement compared to the control. Also in the gongjin-dan treated group, NeuN-positive cells were increased and number of astrocytes, microglia, and apoptotic cells was significantly decreased compared with the control group in the ischemic peri-infarct area. Furthermore, the expression of VEGF and BDNF was increased and level of Cox-2, TNF-α, IL-1ß, and IL-1α was decreased. These results suggest that gongjin-dan may improve functional outcome through the rapid restoration of metabolism and can be considered as a potential neuroprotective agent.

Content of antioxidative caffeoylquinic acid derivatives in field-grown Ligularia fischeri (Ledeb.) Turcz and responses to sunlight.

Ligularia fischeri (Ledeb.) Turcz, a commercial leafy vegetable, contains caffeoylquinic acid derivatives (CQAs) as major phenolic constituents. The HPLC chromatograms of leaf extracts collected from different areas in Korea showed a significant variation in CQA amount, and two tri-O-caffeoylquinic acids (triCQAs) were purified and structurally identified by NMR and MS from this plant. Radical scavenging activities among CQAs were found to be increased in proportion to the number of caffeoyl groups. Since this plant prefers damp and shady growth conditions, the effects of sunlight were investigated by growing plantlets in sunlight and shade for four weeks. Greater leaf thickness and higher phenolic contents were found for leaves grown in sunlight than in shade. Four major CQAs-5-mono-O-caffeoylquinic acid (5-monoCQA), and 3,4-, 3,5-, and 4,5-di-O-caffeoylquinic acid (diCQA)-were induced by solar irradiation, whereas the content of these compounds decreased steadily in shade leaves. The leaves of L. fischeri clearly showed adaptation responses to sunlight, and these characteristics can be exploited for cultivation of this plant for potential use as a nutraceutical and functional food.

Rapid identification and evaluation of antioxidant compounds from extracts of Petasites japonicus by hyphenated-HPLC techniques.

Gradient HPLC coupled to Diode Array Detector (DAD), MS/MS and NMR was applied to the rapid structure determination of major compounds of methanol extracts from leaves and roots of Petasites japonicus. The relative antioxidant capacities of the compounds were evaluated by an HPLC system with post-column on-line antioxidant detection based on 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging. Six compounds were successfully separated on a reverse-phase C(18) column and were identified as 5-caffeoylquinic acid (5-CQA), fukinolic acid (FA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), quercetin-3-O-(6″-acetyl)-ß-glucopyranoside (QAG), 4,5-di-O-caffeoylquinic acid (4,5-DCQA) and kaempferol-3-O-(6″-acetyl)-ß-glucopyranoside (KAG) by MS/MS and (1)H NMR data. Among these compounds, those containing a caffeoyl moiety (5-CQA, FA, 3,5- and 4,5-DCQA) showed relatively strong radical scavenging capacity, with 3,5-DCQA having the greatest radical scavenging capacity in leaf (23.09% of total antioxidant capacity) and root (26.47%) extracts. The relative radical scavenging portion of QAG was only 3.41% in the leaves and KAG did not show any radical scavenging activity. These results demonstrate that the hyphenated HPLC techniques can be successfully applied to rapidly identify structures and evaluate antioxidant activities without prior purification of compounds from plant tissues of P. japonicus.

Therapeutic Effects of Acupuncture through Enhancement of Functional Angiogenesis and Granulogenesis in Rat Wound Healing.

Acupuncture regulates inflammation process and growth factors by increasing blood circulation in affected areas. In this study, we examined whether acupuncture has an effect on wound healing in injured rat. Rats were assigned randomly into two groups: control group and acupuncture group. Acupuncture treatment was carried out at 8 sites around the wounded area. We analyzed the wound area, inflammatory cytokines, proliferation of resident cells, and angiogenesis and induction of extracelluar matrix remodeling. At 7 days after-wounding the wound size in acupuncture-treat group was decreased more significantly compared to control group. In addition, the protein levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were significantly decreased compared to the control at 2 and 7 days post-wounding. Also, we analyzed newly generated cells by performing immunostaining for PCNA and using several phenotype markers such as CD-31, α-SMA, and collagen type I. In acupuncture-treated group, PCNA-positive cell was increased and PCNA labeled CD-31-positive vessels, α-SMA- and collagen type I-positive fibroblastic cells, were increased compared to the control group at 7 days post-wounding. These results suggest that acupuncture may improve wound healing through decreasing pro-inflammatory response, increasing cell proliferation and angiogenesis, and inducing extracellular matrix remodeling.

Preterm infants fed nutrient-enriched formula until 6 months show improved growth and development.

BACKGROUND: The purpose of the present study was to determine the effect of feeding nutrient-enriched preterm formula to preterm infants until 6 months' corrected age (CA) on growth and development in the first 18 months of life. METHODS: Very low-birthweight preterm infants were fed preterm formula until term (40 weeks CA). Infants were then assigned to one of three groups and were fed term formula until 6 months' CA (group 1, n= 29); preterm formula to 3 months' CA and then term formula to 6 months' CA (group 2, n= 30); or preterm formula until 6 months' CA (group 3, n= 31). Anthropometry was performed at term, 3, 6, 9, 12, 15, and at s18 months' CA. Mental and psychomotor development were assessed using the Bayley Scales of Infant Development II at 18 months' CA. RESULTS: Although body weight, length, head circumference and z score for CA at term in group 3 were significantly lower than those of groups 1 and 2, growth rates of these parameters were significantly higher in group 3 up to 18 months CA', as compared to groups 1 and 2. The mental developmental index and psychomotor developmental index of the Bayley test were not significantly different between the three groups. CONCLUSIONS: Very low-birthweight preterm infants fed nutrient-enriched preterm formula until 6 months' CA demonstrated significantly improved growth rates for bodyweight, length and head circumference, and comparable mental and psychomotor development throughout the first 18 months of life.

Hepatoprotective effect of flavonoid glycosides from Lespedeza cuneata against oxidative stress induced by tert-butyl hyperoxide.

The aerial parts of Lespedeza cuneata G. Don, perennial legume native to Eastern Asia, have been used therapeutically in traditional Asian medicine to protect the function of liver, kidneys and lungs. However, little is known about the pharmaceutical effect of extracts from this plant. In the present study, the aerial parts of L. cuneata were used to prepare an ethanol extract, which was then tested for hepatoprotective effects against injury by tert-butyl hyperoxide (t-BHP). At a dose of 20 µg/mL, the ethanol extract significantly protected HepG2 cells against the cytotoxicity of t-BHP. Further fractionation of the extract with ethyl acetate allowed the isolation of five flavonoid compounds that were structurally identified by ¹H and ¹³C NMR spectroscopy as isovitexin, hirsutrin, trifolin, avicularin and quercetin. Hirsutrin, avicularin and quercetin (10 µM) showed clear hepatoprotective activity against injury by t-BHP in HepG2 cells, whereas isovitexin and trifolin showed no protective effects. The observed hepatoprotective effect of the investigated compounds showed a high correlation with radical scavenging activity, which followed the structure-activity relationships of the flavonoid aglycones.

Luteolin ameliorates cisplatin-induced acute kidney injury in mice by regulation of p53-dependent renal tubular apoptosis.

BACKGROUND: Cisplatin chemotherapy often causes acute kidney injury in cancer patients. The causative mechanisms of cisplatin-induced acute kidney injury include renal inflammation, activation of p53 tumour suppressor protein and tubular apoptosis. Luteolin, a flavone found in medicinal herbs and plants, has been reported to exhibit anti-inflammatory, antioxidant and anticarcinogenic activities. The purpose of this study was to investigate the anti-apoptotic effect of luteolin on cisplatin-induced acute kidney injury and the molecular mechanism. METHODS: C57BL/6 mice were treated with cisplatin (20 mg/kg) with or without treatment with luteolin (50 mg/kg for 3 days). Renal function, histological changes, degree of oxidative stress and tubular apoptosis were examined. The effects of luteolin on cisplatin-induced expression of renal p53, PUMA-α and Bcl-2 family proteins were evaluated. RESULTS: Treatment of mice with cisplatin resulted in renal damage, showing an increase in blood urea nitrogen and creatinine levels, tubular damage, oxidative stress and apoptosis. Treatment of cisplatin-treated mice with luteolin significantly improved renal dysfunction, reducing tubular cell damage, oxidative stress and apoptosis. Examination of molecules involving apoptosis of the kidney revealed that treatment of cisplatin increased the levels of p53 and its phosphorylation, PUMA-α, Bax and caspase-3 activity that were significantly decreased by treatment with luteolin. CONCLUSION: These results indicate that cisplatin induces acute kidney injury by regulation of p53-dependent renal tubular apoptosis and that luteolin ameliorates the cisplatin-mediated nephrotoxicity through down-regulation of p53-dependent apoptotic pathway in the kidney.

Overexpression of the pineapple fruit bromelain gene (BAA) in transgenic Chinese cabbage (Brassica rapa) results in enhanced resistance to bacterial soft rot

Bromelain is a crude protein extract obtained from pineapple stems, which comprises a variety of proteolytic enzymes. It exhibits potential therapeutic activities against trauma, inflammation, autoimmune diseases and malignant disorders. In this study, we cloned BAA1 (the gene encoding fruit bromelain) into a plant expression vector that was then used to transform Brassica rapa and overexpress BAA1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. We demonstrate that constitutive overexpression of BAA1 in B. rapa confers enhanced resistance to the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum. These results suggest that it could be utilized for protecting plants from attack by bacterial pathogens.