Cytotoxicity of Medicinal Mushrooms Oudemansiella canarii and Ganoderma lucidum (Agaricomycetes) Against Hematologic Malignant Cells via Activation of Apoptosis-Related Markers.

Cancer is the second leading cause of death worldwide, and despite of the of the availability of the advanced chemical treatments, development of effective and safe alternatives derived from natural resources are still of high interest. Mushroom is one of the important resources of pharmacologically active cytotoxic compounds. In this paper, we report the cytotoxicity of ethanolic extracts of Oudemansiella canarii (Jungh.) Höhn. and Ganoderma lucidum (W. Curt.: Fr.) P. Karst. against nine hematologic malignant cells and describe their molecular mechanisms. Cell lines were exposed to varying concentrations of mushroom extracts for 48 h and the cell proliferation and apoptosis parameters were determined. Western blot analysis was performed to determine the extract-induced changes in the level of apoptosis-related proteins in cancer cell lines and patient-derived mononuclear cells. Results revealed that O. canarii and G. lucidum extracts exhibited cytotoxicity with IC50 values of 26.8-66.0 ppm and 48.1-78.4 ppm, respectively, in all the cancer cell lines used. Mushroom extracts inhibited cell proliferation by 57.3-72.5% (O. canarii) and 44.2-67.4% (G. lucidum), which correlates to the activation of apoptosis as indicated by increased annexin V positivity, cells in sub G0/G1 phase and production of reactive oxygen species, and decreased mitochondrial membrane potential. Western blot analysis showed increase in the level of apoptotic markers (cleaved PARP1, cleaved caspase 3 and phosphorylation of histone 2AX) and activation of the stress-activated protein kinase (SAPK/JNK) signaling pathway. The extract-activated apoptosis was also observed in mononuclear cells isolated from the peripheral blood of leukemia and lymphoma patients. In conclusion, activation of pro-apoptotic markers is one of the major mechanisms of the cytotoxicity of O. canarii and G. lucidum extracts against hematologic malignant cells.

Cytotoxicity of Gymnopilus purpureosquamulosus extracts on hematologic malignant cells through activation of the SAPK/JNK signaling pathway.

Treatment of hematologic malignancies is a formidable challenge for hematologists and there is an urgent need to identify safe and efficacious agents either via synthesis in the laboratory or isolation from natural products. Here, we report the cytotoxicity of extracts from mushroom Gymnopilus purpureosquamulosus Høil (G. pps) and describe its molecular mechanisms. Using leukemia, lymphoma and multiple myeloma cell lines, 28-35 ppm G. pps extract inhibited cell proliferation by ~46-79%, which correlates with activation of apoptosis as indicated by increase in annexin V-positive cells (~5-8-fold), production of reactive oxygen species (~2-3-fold), cells in sub G0/G1 phase (~3-13-fold), caspase 3 enzymatic activity (~1.6-2.9-fold), DNA fragmentation, PARP1 cleavage and down-regulation of prosurvival proteins. Mitochondrial membrane potential decreased and leakage of pro-apoptotic factors to cytoplasm was observed, consistent with the activation of intrinsic apoptosis. Western blot analysis showed activation of the ASK1-MEK-SAPK/JNK and ASK1-P38 MAPK pathways possibly due to changes in the cellular redox status as suggested by decreased protein levels of peroxiredoxin, thioredoxin and thioredoxin reductase. Moreover, antioxidant N-acetylcysteine alleviated the cytotoxicity of G. pps. Pharmacological inhibition of SAPK/JNK and P38 alleviated the G. pps-mediated cytotoxicity. The extract activated apoptosis in leukemia and lymphoma patient cell samples but not in mononuclear cells from healthy donors further supporting the therapeutic values of G. pps for hematologic malignancies.

Quorum quenching activity of Andrographis paniculata (Burm f.) Nees andrographolide compounds on metallo-ß-lactamase-producing clinical isolates of Pseudomonas aeruginosa PA22 and PA247 and their effect on lasR gene expression.

Andrographis paniculata (Burm f.) Nees is a tropical plant native to Southeast Asia that has been used as an effective remedy for a wide variety of illnesses in traditional Chinese and Ayurvedic medicine. The antimicrobial activity of its crude extract had been shown to be due to its quorum quenching activity. The study determined the effect of purified extracted compounds from the leaf of A. paniculata, namely: andrographolide, 14-deoxyandrographolide, 14-deoxy-12-hydroxyandrographolide and neoandrographolide on quorum sensing-mediated virulence mechanisms in clinical isolates of metallo-ß-lactamase (MßL)-producing Pseudomonas aeruginosa. Their effect on the expression of the lasR gene, which codes for LasR, a transcription activator protein of the quorum sensing system in P. aeruginosa was also determined using RT-qPCR. All the pure compounds significantly decreased the biofilm formation, protease production and swarming motility of the P. aeruginosa isolates compared to the untreated controls (p < 0.05). Results of the RT-qPCR assay showed that all compounds significantly downregulated the expression of lasR compared to the untreated control (p < 0.05), supporting the position that the lower virulence activities of the treated group were due to quorum quenching activity of the pure compounds. Multiple comparisons using Tukey's HSD analysis revealed that the means of the relative expression of lasR of the isolates treated with the different compounds were not significantly different from each other (p > 0.05), suggesting equal potencies. Results show the potential of the isolated pure compounds from A. paniculata for use as antimicrobial agents as a result of their quorum quenching activities.

In-vitro study of monocytic THP-1 leukemia cell membrane elasticity with a single-cell microfluidic-assisted optical trapping system.

We studied the elastic profile of monocytic THP-1 leukemia cells using a microfluidic-assisted optical trap. A 2-µm fused silica bead was optically trapped to mechanically dent an immobilized single THP-1 monocyte sieved on a 15-µm microfluidic capture chamber. Cells treated with Zeocin and untreated cells underwent RT-qPCR analysis to determine cell apoptosis through gene expression in relation to each cell's deformation profile. Results showed that untreated cells with 43.05 ± 6.68 Pa are more elastic compared to the treated cells with 15.81 ± 2.94 Pa. THP-1 monocyte's elastic modulus is indicative of cell apoptosis shown by upregulated genes after Zeocin treatment. This study clearly showed that the developed technique can be used to distinguish between cells undergoing apoptosis and cells not undergoing apoptosis and which may apply to the study of other cells and other cell states as well.

Morphological Characterization of Emerging Cercariae among Lymnaeid Snails from Barangay Cawongan, Padre Garcia, Batangas, Philippines.

BACKGROUND AND OBJECTIVES: Lymnaeid snails are the known intermediate hosts of the liver fluke Fasciola spp. and therefore play an important role in the parasite's life cycle. The study is conducted to determine specificity of snail host-parasite interaction and to determine the snail-trematode infection rate by cercarial emergence, characterizing the emerging larvae using standardized key. MATERIALS AND METHODS: A total of 750 snails were collected from a rice field in Barangay Cawongan, Padre Garcia, Batangas, from November 2016 to March 2017 (n=150/month). Preliminary snail identification was based on morphological features of the shell. Each snail was acclimatized for 24 hours in a 50-ml capacity container before being exposed to strong artificial light. The 150 snails collected per month were grouped into 5 batches (n=30/batch) with each batch receiving different number of light exposures. Emerging cercariae were described and characterized using photo-referencing and standardized keys. All statistical tests were performed at p<0.05 level of significance using SPSS ver. 20. RESULTS: The total cercarial shedding rate of the snails studied, as a measure of the infected snails, was found to be 35.6% and was positively associated with the length of the snail shell [OR = 1.809; 95% CI: 1.471-2.225; p<0.001], but not with the weight [OR = 0.003; 95% CI: 0.00-0.275; p=0.012] and width of the shell [OR = 0.937; 95% CI: 0.672-1.305]. The rates varied from 29.3% to 38.0% based on the frequency of 6-hour light exposure. Appearance of encysted forms increased with increasing number of light exposures [OR = 10.27, 95% CI: 3.04-34.76, p<0.001]. Three distinct cercariae were identified, namely, echinostome, longifurcate-pharyngeate distome cercariae (Strigea cercariae), and the virgulate xiphidiocercaria, with 26.4%, 2.27%, and 0.67% infection monitored by cercarial emergence, respectively. CONCLUSION: Local lymnaeid snails were infected with a single type of trematode larvae and coinfection with multiple larvae was rare but was encountered.

Thin Layer Chromatography-Bioautography and Gas Chromatography-Mass Spectrometry of Antimicrobial Leaf Extracts from Philippine Piper betle L. against Multidrug-Resistant Bacteria.

This study isolated and identified the antimicrobial compounds of Philippine Piper betle L. leaf ethanol extracts by thin layer chromatography- (TLC-) bioautography and gas chromatography-mass spectrometry (GC-MS). Initially, TLC separation of the leaf ethanol extracts provided a maximum of eight compounds with R f values of 0.92, 0.86, 0.76, 0.53, 0.40, 0.25, 0.13, and 0.013, best visualized when inspected under UV 366 nm. Agar-overlay bioautography of the isolated compounds demonstrated two spots with R f values of 0.86 and 0.13 showing inhibitory activities against two Gram-positive multidrug-resistant (MDR) bacteria, namely, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. The compound with an R f value of 0.86 also possessed inhibitory activity against Gram-negative MDR bacteria, namely, carbapenem-resistant Enterobacteriaceae-Klebsiella pneumoniae and metallo-ß-lactamase-producing Acinetobacter baumannii. GC-MS was performed to identify the semivolatile and volatile compounds present in the leaf ethanol extracts. Six compounds were identified, four of which are new compounds that have not been mentioned in the medical literature. The chemical compounds isolated include ethyl diazoacetate, tris(trifluoromethyl)phosphine, heptafluorobutyrate, 3-fluoro-2-propynenitrite, 4-(2-propenyl)phenol, and eugenol. The results of this study could lead to the development of novel therapeutic agents capable of dealing with specific diseases that either have weakened reaction or are currently not responsive to existing drugs.

Molecular and phenotypic characterization of methicillin-resistant Staphylococcus aureus isolates from a tertiary hospital in the Philippines.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to public health worldwide. There are relatively few studies addressing the molecular epidemiology of MRSA in the Philippines. METHODS: This study characterized MRSA isolates in terms of their antimicrobial susceptibility profile, the SCCmec type, and the presence of lukF-lukS genes for Panton-Valentine leukocidin (PVL) and determined the relatedness of the isolates by random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR). RESULTS: A total of 236 S. aureus were isolated from clinical specimens of the Makati Medical Center in Makati City, Philippines, between January 2013 and June 2013, and 108 or 45.76 % were found to be MRSA. Results showed that the MRSA strains were resistant to trimethoprim-sulfamethoxazole (20.37 %), azithromycin (10.19 %), gentamicin (5.56 %), and linezolid (4.63 %), while all were susceptible to vancomycin, nitrofurantoin, levofloxacin, minocycline, rifampin, and tetracycline. One isolate was found positive for inducible clindamycin resistance. All of the 108 MRSA strains were confirmed to carry the mecA and SCCmec genes, while the PVL genes were detected in 41 (38 %) of the isolates. Ninety-six isolates (89 %) carried SCCmec type IV, while the remaining isolates carried SCCmec type I (11 isolates) or type III (one isolate). CONCLUSION: This study is the first to present a comprehensive MRSA surveillance data with molecular characterization in a tertiary hospital in the Philippines.

Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance.

Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum ß-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-ß-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19µg/ml to 1250 µg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 µg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria.

Chemical constituents and bioactivities of Glinus oppositifolius.

OBJECTIVES: To isolate the secondary metabolites from the dichloromethane (DCM) extracts of Glinus oppositifolius; to test for the cytotoxicity of a new triterpene, oppositifolone (1); and to test for the hypoglycemic, analgesic, and antimicrobial potentials of 1, DCM and aqueous leaf extracts of G. oppositifolius. METHODS: The compounds were isolated by silica gel chromatography and identified by nuclear magnetic resonance spectroscopy. The cytotoxicity potential of 1 was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Triterpene 1, DCM, and aqueous leaf extracts were tested for hypoglycemic potential using the oral glucose tolerance test; analgesic potential using the tail-flick assay, and antimicrobial potential using the disc diffusion method. RESULTS: The DCM extracts of G. oppositifolius afforded 1, squalene, spinasterol, oleanolic acid, phytol, and lutein from the leaves; squalene and spergulagenin A from the stems; and spinasterol from the roots. Triterpene 1 was cytotoxic against human colon carcinoma 116 with an IC50 value of 28.7 but did not exhibit cytotoxicity against A549. The aqueous leaf extract at 200 mg/kg body weight (BW) exhibited hypoglycemic activity with a pronounced % blood glucose reduction of 70.76% ±17.4% within 0.5 h after introduction. The DCM leaf extract showed a lower % blood glucose reduction of 18.52% ±13.5% at 200 mg/kg BW within 1.5 h after introduction, while 1 did not exhibit hypoglycemic activity. The samples did not exhibit analgesic property and were inactive against multiple drug resistant bacterial pathogens. CONCLUSION: The compounds responsible for the hypoglycemic activity of G. oppositifolius which are fast acting (0.5 h) are found in the aqueous leaf extract.

Cytotoxicity of probiotics from Philippine commercial dairy products on cancer cells and the effect on expression of cfos and cjun early apoptotic-promoting genes and Interleukin-1 ß and Tumor Necrosis Factor-α proinflammatory cytokine genes.

This study determined cytotoxicity of probiotic Lactobacillus spp. from Philippine dairy products on cancer cells and normal fibroblasts and their effects on expression of early apoptotic-promoting cfos, cjun and proinflammatory cytokine IL-1ß, TNF-α genes. Cultures were from Yakult, Bear Brand Probiotic Drink, Nido3+ Powdered Milk. Filter-sterilized supernatants from cultures of Lactobacillus spp. were evaluated for cytotoxicity to colon cancer cells (HT-29 and HCT116), leukemia cells (THP-1), and normal human dermal fibroblasts (HDFn) using PrestoBlue. Bleomycin was the positive control. Absolute quantification of transcript levels was conducted using qRT-PCR. Cytotoxicity index profiles on HDFn, THP-1 of all probiotic supernatants and negative controls suggest nontoxicity to the cells when compared to bleomycin, whereas all probiotic supernatants were found to be cytotoxic to HT-29 and HCT-116 colon cancer cell lines. Expression of cfos, cjun transcripts was significantly upregulated in HT-29 and HCT116 cells treated with probiotic supernatants compared to untreated baseline levels (P < 0.05). Expression of IL-1ß and TNF-α by lipopolysaccharide-treated macrophages was significantly downregulated in cells with probiotic supernatants compared to those exposed to MRS medium (P < 0.05). Results provide strong support for the role of Lactobacillus spp. studied in anticancer therapy and in prevention of inflammation that may act as precursor to carcinogenesis.

Proximate composition and functionality of the culinary-medicinal tiger sawgill mushroom, Lentinus tigrinus (higher Basidiomycetes), from the Philippines.

The proximate composition and functionality of Lentinus tigrinus were evaluated to establish and popularize this mushroom as functional food source. The evaluation of functionality focused on the antibacterial and hypoglycemic activities of the mushroom extracts. An acute single oral dose toxicity test in mice was used for its biosafety analysis. The pileus contained higher amounts of protein (25.9%), fat (2.1%), and ash (7.4%) and a higher energetic value (142.1 kcal/100 g) than the corresponding stipe, whereas the stipe contained higher amounts of total carbohydrates (67.7%), which consist of dietary fiber (63.0%) and reducing sugar (4.7%), than the pileus. Biosafety analysis confirmed that L. tigrinus is an edible mushroom species; it was found to be toxicologically safe in imprinting control region mice. The administration of lyophilized hot water extract of the fruiting body (both 100 and 250 mg/ kg doses) to diabetic mice significantly lowered the glucose level by 26.9% in the third week, which was significantly comparable to the results of the antidiabetic agent glibenclamide, which was used as a positive control. In vitro antibacterial assay showed that the ethanolic extract of the fruiting body and the immobilized secondary mycelia had high antibacterial activities against Staphylococcus aureus but not on Escherichia coli. Combining its useful nutrients and significant biological properties, L. tigrinus can be considered a natural source of safe nutraceuticals.

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia.

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm(3) to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

First report on the occurrence of SHV-12 extended-spectrum beta-lactamase-producing Enterobacteriaceae in the Philippines.

BACKGROUND AND PURPOSE: Soon after the introduction of extended-spectrum beta-lactam antibiotics to treat infections caused by Gram-negative bacteria that were resistant to the existing beta-lactams, resistant strains quickly emerged. This study investigated the antimicrobial susceptibility patterns, conjugative transferability, and molecular characterization of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in the Philippines. METHODS: ESBL-producing Enterobacteriaceae were collected from patients at the Philippine General Hospital from June 2000 to August 2001. The antimicrobial susceptibility patterns were determined using the standard disk diffusion assay. ESBL production was confirmed using the double-disk synergy assay and the E-test, while the ESBLs were characterized for type using polymerase chain reaction, sequencing, and isoelectric focusing. Conjugation studies were conducted in vitro on solid medium. RESULTS: All of the ESBL-producing isolates were also resistant to most other classes of antimicrobials studied. The ESBL genes on conjugative and non-conjugative plasmids were bla(SHV-12). Fifteen isolates also carried bla(TEM-1). None of the isolates were positive for the CTX-M type of ESBL. Plasmids from cefoxitin-resistant isolates did not produce amplified products using CMY primers for the AmpC gene. CONCLUSIONS: SHV-12 is the dominating ESBL among the Enterobacteriaceae studied. The gene bla(SHV-12) is carried together with genes for multiple resistance to other classes of antimicrobials in conjugative and nonconjugative plasmids.