Screening for Anticancer Activity of Leaf Ethanolic Extract of Alpinia elegans ("tagbak") on Human Cancer Cell Lines.

BACKGROUND: Lung, liver, and colorectal cancers are among the leading causes of cancer-related deaths in the Philippines. As chemotherapeutic treatments remain expensive, native plants are being studied as alternative treatments for use in primary care. In this study, Alpinia elegans leaf ethanolic extract ("tagbak," TGK) was screened for potential anti-cancer activity against lung (A549), colorectal (HCT116) and liver (HEPG2) cancer cells. METHODS: An ethanolic extract (TGK) was prepared from A. elegans leaves sampled from Infanta, Quezon. Its anti-proliferative activity on A549, HCT116 and HEPG2 was determined using MTS cell viability assay, with doxorubicin (DXR) as positive control and 0.1% DMSO in culture media as negative control. To differentiate cytostatic from cytotoxic effects, LDH cytotoxicity was performed, with 5-fluorouracil (5-FU) as positive control. In screening for metastatic potential, scratch wound assay was done, with percent gap closure as indicator of cell migration. To visualize the actin filaments and nuclei, the cells were stained with AlexaFluor488-tagged phalloidin and Hoechst 33342, respectively. RESULTS: Phytochemical analysis revealed traces of alkaloids, moderate amounts of sterols, and abundant triterpenes, flavonoids, saponins, glycosides and tannins in TGK. TGK exhibited anti-proliferative activity at high concentrations, with TGK being more effective against HEPG2 (IC50: 98.35 ppm) than A549 (IC50: 245.5 ppm) and HCT116 (IC50: 299.7 ppm). This can be attributed to the cytotoxic activity of TGK as seen in LDH release assay, with HEPG2 more affected than HCT116 or A549. TGK also attenuated cell migration, with significantly different gap closure from negative control at 500 ppm (p<0.05). Cytoskeleton and nuclei visualization via fluorescence microscopy showed cell shrinkage and pyknosis, as well as cellular debris, indicating both apoptotic and necrotic effects on cancer cells. CONCLUSION: The ethanolic leaf extract of Alpinia elegans significantly inhibits cellular proliferation and migration at high concentrations, with direct exposure-response relationship within concentrations.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human ß-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

Progesterone alters human cervical epithelial and stromal cell transition and migration: Implications in cervical remodeling during pregnancy and parturition.

The cervix undergoes extensive remodeling throughout pregnancy and parturition. This process involves both ECM collagen degradation and cellular remodeling, which includes cell proliferation, transition and migration. Progesterone (P4) has been used clinically to delay cervical ripening and prevent preterm birth (PTB). However, the mechanisms by which progesterone affects cell transition and the migration of cervical epithelial and stromal cells are not yet fully known. In this study, we documented the role of a gestational level of P4 in the cellular transition (epithelial-mesenchymal transition [EMT] and mesenchymal-epithelial transition [MET]), cell migration, and inflammatory responses of endocervical epithelial cells (EEC) and cervical stromal cells (CSC). EEC and CSC were treated with LPS and P4 for 6 days. The epithelial:mesenchymal ratio (regular microscopy and cell shape index analysis), shift in intermediate filaments (immunofluorescence microscopy and western blot analyses for cytokeratin [CK]-18 and vimentin), adhesion molecules and transcription factors (western blot analyses for E-cadherin, N-cadherin and SNAIL), were used to determine growth characteristics and EMT and MET changes in EEC and CSC under the indicated conditions. To test cell remodeling, scratch assays followed by cellular analyses as mentioned above were performed. Inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor α [TNFα]) and matrix metallopeptidase 9 (MMP9) were measured by ELISA. LPS promoted EMT (decreased cell shape index, decreased CK-18 and E-cadherin, increased vimentin, N-cadherin, and SNAIL), and increased IL-6 and MMP9 production by EEC. A gestational level of P4 prevented LPS-induced EMT in EEC and exhibited anti-inflammatory effect in both EEC and CSC. LPS slowed down wound healing in CSC but P4 treatment prevented the negative impact of LPS in CSC wound healing. These results may explain the cellular mechanisms by which P4 helps to stabilize the cervical epithelial barrier and preserve the mechanical and tensile strength of the cervical stromal layer, which are important in normal cervical remodeling processes during pregnancy.

Oxidative stress promotes cellular damages in the cervix: implications for normal and pathologic cervical function in human pregnancy†.

A physiologic increase in reactive oxygen species throughout pregnancy is required to remodel the cervix. Oxidative stress can cause cellular damage that contributes to dysfunctional tissue. This study determined the oxidative stress-induced cell fate of human cervical epithelial and cervical stromal cells. We treated the ectocervical and endocervical epithelial cells and cervical stromal cells with cigarette smoke extract, an oxidative stress inducer, for 48 h. Cell viability (crystal violet assay); cell cycle, apoptosis, and necrosis (flow cytometry); senescence (senescence-associated ß-galactosidase staining); autophagy (staining for autophagosome protein, microtubule-associated protein 1 light chain 3B); stress signaler p38 mitogen-activated protein kinases pathway activation (western blot analyses); and inflammation by measuring interleukin-6 (enzyme-linked immunosorbent assay) were conducted after 48 h of cigarette smoke extract treatment. Oxidative stress induced reactive oxygen species production in cervical cells, which was inhibited by N-acetylcysteine. Oxidative stress promoted cell cycle arrest and induced necrosis in cervical cells. High senescence and low autophagy were observed in cervical stromal cells under oxidative stress. Conversely, senescence was low and autophagy was high in endocervical epithelial cells. Oxidative stress induced p38 mitogen-activated protein kinases (p38MAPK) activation in all cervical cells but only increased interleukin-6 production by the ectocervical epithelial cells. Inhibition of interleukin-6 production by a p38 mitogen-activated protein kinases inhibitor confirmed the activation of an oxidative stress-induced pathway. In conclusion, oxidative stress can promote cell death and sterile inflammation that is mediated by p38 mitogen-activated protein kinases activation in the cellular components of the cervix. These cellular damages may contribute to the normal and premature cervical ripening, which can promote preterm birth.

Organ-on-chip of the cervical epithelial layer: A platform to study normal and pathological cellular remodeling of the cervix.

Damage to the cervical epithelial layer due to infection and inflammation is associated with preterm birth. However, the individual and/or collective roles of cervical epithelial layers in maintaining cervical integrity remain unclear during infection/inflammation. To determine the intercellular interactions, we developed an organ-on-chip of the cervical epithelial layer (CE-OOC) composed of two co-culture chambers connected by microchannels, recapitulating the ectocervical and endocervical epithelial layers. Further, we tested the interactions between cells from each distinct region and their contributions in maintaining cervical integrity in response to LPS and TNFα stimulations. The co-culture of ectocervical and endocervical cells facilitated cellular migration of both epithelial cells inside the microchannels. Compared to untreated controls, both LPS and TNFα increased apoptosis, necrosis, and senescence as well as increased pro-inflammatory cytokine productions by cervical epithelial cells. In summary, the CE-OOC established an in vitro model that can recapitulate the ectocervical and the endocervical epithelial regions of the cervix. The established CE-OOC may become a powerful tool in obstetrics and gynecology research such as in studying cervical remodeling during pregnancy and parturition and the dynamics of cervical epithelial cells in benign and malignant pathology in the cervix.

Revealing the anticancer potential of candidate drugs in vivo using Caenorhabditis elegans mutant strains.

Drug repurposing is used as a strategy for finding new drugs for cancer. The process is a faster and a more cost-effective way of providing new indications for drugs that can address emerging drug resistance and numerous side effects of chemotherapeutic drugs. In this study, the in vivo anticancer potential of itraconazole, disulfiram, etodolac, and ouabain were assessed using five different C. elegans mutant strains. Each strain contains mutations in genes involved in different signaling pathways such as Wnt (JK3476), Notch (JK1107 and BS3164), and Ras-ERK (SD939 and MT2124) that result to phenotypes of sterility, infertility, and multivulva formation. These same signaling pathways have been shown to be defective in several human cancer types. The four candidate drugs were tested on the C. elegans mutant strains to determine if they rescue the mutant phenotypes. Both ouabain and etodolac significantly reduced the sterile and infertile phenotypes of JK3476, JK1107, BS3164, and SD939 strains (p=0.0010). Finally, itraconazole and etodolac significantly reduced multivulva formation (p=0.0021). The degrees of significant phenotypic rescues of each mutant were significantly higher than vehicle only (1% DMSO). Therefore, this study demonstrated that the four candidate drugs have anticancer potential in vivo, and etodolac had the highest anticancer potential.

The dopamine receptor antagonist trifluoperazine prevents phenotype conversion and improves survival in mouse models of glioblastoma.

Glioblastoma (GBM) is the deadliest adult brain cancer, and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 mo over surgery alone, but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype, and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP causes loss of radiation-induced Nanog mRNA expression, and activation of GSK3 with consecutive posttranslational reduction in p-Akt, Sox2, and ß-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that the combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment-resistant, induced GICs (iGICs).

1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine treatment after brain irradiation preserves cognitive function in mice.

BACKGROUND: Normal tissue toxicity is an inevitable consequence of primary or secondary brain tumor radiotherapy. Cranial irradiation commonly leads to neurocognitive deficits that manifest months or years after treatment. Mechanistically, radiation-induced loss of neural stem/progenitor cells, neuroinflammation, and demyelination are contributing factors that lead to progressive cognitive decline. METHODS: The effects of 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine (NSPP) on irradiated murine neurospheres, microglia cells, and patient-derived gliomaspheres were assessed by sphere-formation assays, flow cytometry, and interleukin (IL)-6 enzyme-linked immunosorbent assay. Activation of the hedgehog pathway was studied by quantitative reverse transcription PCR. The in vivo effects of NSPP were analyzed using flow cytometry, sphere-formation assays, immunohistochemistry, behavioral testing, and an intracranial mouse model of glioblastoma. RESULTS: We report that NSPP mitigates radiation-induced normal tissue toxicity in the brains of mice. NSPP treatment significantly increased the number of neural stem/progenitor cells after brain irradiation in female animals, and inhibited radiation-induced microglia activation and expression of the pro-inflammatory cytokine IL-6. Behavioral testing revealed that treatment with NSPP after radiotherapy was able to successfully mitigate radiation-induced decline in memory function of the brain. In mouse models of glioblastoma, NSPP showed no toxicity and did not interfere with the growth-delaying effects of radiation. CONCLUSIONS: We conclude that NSPP has the potential to mitigate cognitive decline in patients undergoing partial or whole brain irradiation without promoting tumor growth and that the use of this compound as a radiation mitigator of radiation late effects on the central nervous system warrants further investigation.

Is the Philippines ready for HIV self-testing?

BACKGROUND: The Philippines is facing a rapidly rising HIV epidemic among young men who have sex with men (MSM). Testing rates among young populations is poor. HIV self-testing (HIVST) is a promising strategy to address this testing gap. The study's purpose was to explore the perceived acceptability, feasibility and programmatic challenges of HIVST among key informants and target users. METHOD: A qualitative study involving semi-structured interviews and focus group discussions (FGD). We interviewed 15 key informants involved with HIV testing programs or policies and 42 target users in six FGD in Metro Manila. We held separate discussions with high socio-economic MSM (n = 12), urban poor MSM (n = 15) and transgender women (TGW) (n = 15). Results were analysed using a thematic framework approach. RESULTS: MSM and TGW welcomed the convenience and privacy HIVST could provide. They preferred an inexpensive accurate blood-based kit attained from reputable sites. Key informants at national and local level equally welcomed HIVST but identified a number of policy and regulatory issues. Both groups articulated the challenge of enrolling those who test reactive using HIVST to further testing and treatment in an environment characterised by acute stigma around HIV. CONCLUSIONS: HIVST was found to be highly acceptable to target users and was welcomed as an additional testing approach at national level. Strategic alliances are now needed between stakeholders to proactively deliver a patient-centred HIVST program that could provide an effective, safe means of increasing testing coverage in this escalating context.

Radiation mitigation of the intestinal acute radiation injury in mice by 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine.

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS), identified in an unbiased high-throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays, reporter cells, and in silico docking. 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened, thereby expanding the ISC pool, increasing the number of regenerating crypts and preventing the GI-ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen.

1-(4-nitrobenzenesulfonyl)-4-penylpiperazine increases the number of Peyer's patch-associated regenerating crypts in the small intestines after radiation injury.

OBJECTIVE: Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches. METHODS: C3H mice were irradiated with 0 or 12 Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24 h later. 48 h after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96 h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry. RESULTS: Compound #5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24 h after drug treatment. A single dose of Compound #5 significantly increased the number of CD45+ cells after 12 Gy TBI. Importantly, 96 h after 12 Gy TBI Compound #5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts. CONCLUSION: In summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches.

Adaptation and validation of the gratitude questionnaire GQ-6 for the Ecuadorian context / Adaptação e validação do questionário de gratidão GQ-6 para o contexto equatoriano / Adaptación y validación del cuestionario de gratitud GQ-6 para el contexto ecuatoriano

This paper evaluates the factor structure, reliability and validity of the gratitude scale (GQ-6) of McCullough, Emmons and Tsang (2002) and the five-item version proposed by Chen et al. (2009). Results of a sample of 1112 adults show that the fiveitem version has excellent internal consistency (α=.926; ω=.891; GLB=.913); high and significant factor loadings (greater than .8; p<.01), and excellent goodness of fit indexes (χ²(5)=23.837, p<.001; CFI=.997; TLI=.995; RMSEA=.082, p=.045; SRMSR=.035; WRMSR=.837). The criterion validity was evaluated applying subscales of the PERMA-Profiler: positive emotions (ρ=.5702, p=.021) and negative emotions (ρ=-.1786, p=.0316). Finally, we find psychometric equivalence between the sex of the participants. In conclusion, the five-item questionnaire is valid and reliable in the Ecuadorian context. (AU)

Este estudo avalia a estrutura fatorial, a confiabilidade e a validade da Escala de Gratidão (GQ-6) de McCullough, Emmons e Tsang (2002) e a versão de cinco itens proposta por Chen et al. (2009). Resultados de uma amostra de 1.112 adultos mostram que a versão de cinco itens tem excelente consistência interna (α=0,926; ω=0,891; GLB=0,913); cargas fatoriais altas e significativas (maior que 0,8; p<0,01), e excelentes índices de qualidade de ajuste (χ²(5)=23,837, p<0,001; CFI=0,997; TLI=0,995; RMSEA=0,082, p=0,045; SRMSR=0,035; WRMSR=0,837). A validade de critério foi avaliada aplicando-se subescalas do PERMA-Profiler: emoções positivas (ρ=0,5702, p=0,021) e emoções negativas (ρ=-0,1786, p=0,0316). Finalmente, encontra-se equivalência psicométrica entre o sexo dos participantes. Em conclusão, o questionário de cinco itens é válido e confiável no contexto equatoriano. (AU)

Este estudio evalúa la estructura factorial, la confiabilidad y la validez de la escala de gratitud (GQ-6) de McCullough, Emmons y Tsang (2002) y la versión de cinco ítems propuesta por Chen et al. (2009). Resultados de una muestra de 1112 adultos indican que la versión de cinco ítems tiene excelente consistencia interna (α=.926; ω=.891; GLB=.913); cargas factoriales altas y significativas (mayores a .8; p < .01), y excelentes índices de calidad de ajuste (χ²(5)=23.837, p<.001; CFI=.997; TLI=.995; RMSEA=.082, p=.045; SRMSR=.035; WRMSR=.837). La validez de criterio se evaluó aplicando subescalas del PERMA-Profiler: emociones positivas (ρ=.5702, p=.021) y emociones negativas (ρ=-.1786, p=.0316). Finalmente, se ha encontrado equivalencia psicométrica entre el sexo de los participantes. En conclusión, el cuestionario de cinco ítems es válido y fiable en el contexto ecuatoriano. (AU)

Caffeine Protects Dopaminergic Neurons From Dopamine-Induced Neurodegeneration via Synergistic Adenosine-Dopamine D2-Like Receptor Interactions in Transgenic Caenorhabditis elegans.

Previous studies have suggested that caffeine reduces the risk of L-DOPA-induced dyskinesia. However, caffeine is also known to promote dopamine signaling, which seemingly contradicts this observed effect. To this end, the study aimed to clarify the mechanism of caffeine neuroprotection in vivo when excess dopamine is present. Transgenic Caenorhabditis elegans (UA57) overproducing dopamine was exposed to caffeine for 7 days and monitored by observing GFP-tagged dopaminergic (DA) neurons via fluorescence microscopy. Caffeine (10 mM) prevented neuronal cell loss in 96% of DA neurons, with a mean GFP intensity that is 40% higher than control (0.1% DMSO). To confirm if cAMP plays a role in the observed neuroprotection by caffeine, cAMP levels were elevated via forskolin (10 µM), an adenylyl cyclase activator. Forskolin (10 µM) exposure did not confer neuroprotection and was similar to control (0.1% DMSO) at the 7th day, suggesting that cAMP is not the sole secondary messenger utilized. Rotigotine (160 µM), a dopamine D2-like receptor (DOP2R) agonist, was not able to confer significant neuroprotection to the nematodes. This suggests that DOP2R activation is necessary but insufficient to mimic neuroprotection by caffeine. Lastly, co-administration of caffeine (10 mM) with olanzapine (160 µM), a DOP2R antagonist, eliminated neuroprotection. This suggests that the protective effect must involve both adenosine receptor antagonism and activation of DOP2Rs. Taken together, we show that caffeine protects DA neurons from dopamine-induced neurodegeneration and acts by modulating adenosine receptor-DOP2R interactions in C. elegans.

The learning curve of lateral access lumbar interbody fusion in an Asian population: a prospective study.

PURPOSE: Lateral access lumbar interbody fusion (LLIF) is a minimally invasive technique that has an increasing popularity. It offers unique advantages and circumvents risk of certain serious complications encountered in other conventional spinal approaches. This study provides a statistical analysis defining the lateral access learning curve in the Asian population. METHODS: This prospective study included 32 consecutive patients who underwent LLIF from April 2012 to August 2014. The surgeries were performed by two senior spine surgeons and follow-up was conducted at 6 weeks, 3, 6, 9 months and 1 year post-operation. RESULTS: The breakpoint in operating time occurred at the 22nd level operated, from a mean of 71 min in the early phase group to a mean of 42 min in the steady state group. LLIF at L4/5 level is technically more demanding but technically feasible as competency is achieved. CONCLUSIONS: During the learning process, there was no compromise of perioperative or clinical outcomes. It should be feasibly incorporated into a spine surgeon's repertoire of procedures for the lumbar spine.