Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line / Efeito apoptótico da natalensia bulbina e do clorofito comoso na linhagem celular mielogênea da leucemia K562

Abstract Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Resumo Bulbine natalensis Baker e Chorophytum comosum (Thunb.) Jacques são potenciais fontes medicinais para o tratamento de cânceres. A Leucemia Mieloide Crônica (LMC) é um distúrbio das células-tronco hematopoiéticas que é tratado com inibidores da tirosina quinase, mas frequentemente, causa recorrência da leucemia após a interrupção da terapia, portanto, requer um tratamento alternativo. Este estudo determinou o efeito anticancerígeno de extratos metanólicos e aquosos de folha, raiz e bulbo de B. natalensis e C. comosum na linhagem celular de leucemia mieloide humana crônica (K562) por ensaios de MTT, Hoechst bis-benzimida nuclear e anexina V. O extrato metanólico da raiz de B. natalensis e C. comosum apresentou alta citotoxidade de 8,6% e 16,7% respectivamente, na linhagem celular K562 com a concentração de 1,000 μg / ml. Perda morfológica da integridade da membrana celular causando degradação dos núcleos, citoplasma e encolhimento celular foi observada no extrato metanólico da raiz de ambas as plantas. Uma alta apoptose (p <0,0001) foi induzida nas células K562 por extratos de folhas e raízes de C. comosum em comparação com B. natalensis. Este estudo mostrou que ambas as plantas possuem efeito apoptótico contra leucemia mieloide in vitro que contribui para as propriedades anticâncer gerais de B. natalensis e C. comosum para justificar futuras aplicações terapêuticas contra câncer de sangue de LMC.

Inhibition of Proliferation, Migration, and Invasion of Keloid Fibroblasts By miR-183-5p Through Downregulating EGR1 / Inhibición de la Proliferación, Migración e Invasión de Fibroblastos Queloides por miR-183-5p Mediante la Regulación Negativa de EGR1

SUMMARY: Keloid scar is a unique benign fibroproliferative tumor of the human skin. Previously, it was reported that early growth response 1 (EGR1), a transcription factor, promotes keloid fibrosis; however, the mechanism by which EGR1 modulates keloid formation was not elaborated. In this research, the specific function and the microRNA (miRNA) regulatory network of EGR1 in keloids was examined. Keloid fibroblasts (KFs) were transfected with EGR1-small interfering RNA (siEGR1), EGR1-overexpression plasmid (pcDNA3.1-EGR1), and microRNA (miR-183-5p)-mimics to regulate the expression of EGR1 and miR-183-5p. The study employed dual-luciferase reporter assays to explore the targeting regulation of miR-183-5p on EGR1. Additionally, Western blotting, flow cytometry, qRT-PCR, cell count kit-8 (CCK-8), transwell, and wound healing assays, and RNA sequencing were conducted. EGR1 was upregulated in KFs, and EGR1 silencing diminished proliferation, fibrosis, migration, invasion, and apoptosis of cells. In KFs, the expression of miR- 183-5p was reduced, leading to the inhibition of cell proliferation, migration, and invasion. Conversely, it enhanced apoptosis. By targeting EGR1, miR-183-5p partially counteracted the impact of EGR1 on migration, invasion, and fibrosis in KFs. The findings imply that miR-183-5p suppresses keloid formation by targeting EGR1. As a result, EGR1 holds promise as a potential therapeutic target for preventing and treating keloids.

La cicatriz queloide es un tumor fibroproliferativo benigno único de la piel humana. Anteriormente, se informó que la respuesta de crecimiento temprano 1 (EGR1), un factor de transcripción, promueve la fibrosis queloide; sin embargo, no se explicó el mecanismo por el cual EGR1 modula la formación de queloides. En esta investigación, se examinó la función específica y la red reguladora de microARN (miARN) de EGR1 en queloides. Se transfectaron fibroblastos queloides (KF) con ARN de interferencia pequeño de EGR1 (siEGR1), plásmido de sobreexpresión de EGR1 (pcDNA3.1-EGR1) y miméticos de microARN (miR-183-5p) para regular la expresión de EGR1 y miR-183. -5p. El estudio empleó ensayos de indicador de luciferasa dual para explorar la regulación dirigida de miR-183-5p en EGR1. Además, se realizaron pruebas de transferencia Western, citometría de flujo, qRT-PCR, kit de recuento celular-8 (CCK-8), transwell y curación de heridas, y secuenciación de ARN. EGR1 estaba regulado positivamente en KF, y el silenciamiento de EGR1 disminuyó la proliferación, fibrosis, migración, invasión y apoptosis de las células. En KF, la expresión de miR- 183-5p se redujo, lo que llevó a la inhibición de la proliferación, migración e invasión celular. Por el contrario, mejoró la apoptosis. Al apuntar a EGR1, miR-183-5p contrarrestó parcialmente el impacto de EGR1 en la migración, invasión y fibrosis en KF. Los hallazgos implican que miR-183-5p suprime la formación de queloides al apuntar a EGR1. Como resultado, EGR1 es prometedor como objetivo terapéutico potencial para prevenir y tratar los queloides.

Effect of Survivin Down-Regulation by Egr1-Survivin shRNA Combined with Radiotherapy on Radiosensitivity of Esophageal Squamous Carcinoma / Efecto de la Regulación Negativa de Survivina por shRNA de Egr1-Survivina Combinado con Radioterapia sobre la Radiosensibilidad del Carcinoma Escamoso de Esófago

SUMMARY: This study is to investigate the effect of survivin down-regulation by Egr1-survivin shRNA combined with radiotherapy on the apoptosis and radiosensitivity of esophageal squamous cell carcinoma ECA109 and KYSE150 cells. ECA109 and KYSE150 cells were transfected with Egr1-survivin shRNA, and then treated with radiotherapy. After 24 h, the mRNA and protein levels of Egr1-survivin were detected by qPCR and Western-Blot. Cell cycle and apoptosis were detected by flow cytometry. Western blot also detected levels of cleavaged Caspase 3 and Caspase 9. YM155 was used as a positive control to inhibit survivin expression. The levels of survivin mRNA and protein in ECA109 and KYSE150 cells treated with Egr1-survivin shRNA combined with radiotherapy were significantly lower than those of the blank control group, the empty vector control group, and, the YM155 + radiotherapy group (P<0.05). Meanwhile, after survivin down-regulation, the ratio of G2 to S phase of ECA109 and KYSE150 cells increased significantly, leading to significant G2 and S phase arrest. Additionally, apoptosis of ECA109 and KYSE150 cells increased significantly (P <0.01). Further, protein levels of cleavaged Caspase 3 and Caspase 9 significantly increased in Egr1-survivin shRNA combined with radiotherapy group. Egr1-survivin shRNA combined with radiotherapy can down-regulate survivin expression, which further increases the apoptosis, and enhances the radiosensitivity of ECA109 and KYSE150 cells.

Este estudio tuvo como objetivo investigar el efecto de la regulación negativa de survivina por el shRNA de Egr1-survivina combinado con radioterapia sobre la apoptosis y la radiosensibilidad del carcinoma de células escamosas de esófago Células ECA109 y KYSE150. Las células ECA109 y KYSE150 se transfectaron con shRNA de survivina Egr1 y luego se trataron con radioterapia. Después de 24 h, los niveles de ARNm y proteína de Egr1-survivina se detectaron mediante qPCR y Western-Blot. El ciclo celular y la apoptosis se detectaron mediante citometría de flujo. La transferencia Western también detectó niveles de Caspasa 3 y Caspasa 9 escindidas. Se usó YM155 como control positivo para inhibir la expresión de survivina. Los niveles de ARNm y proteína de survivina en células ECA109 y KYSE150 tratadas con shRNA de survivina Egr1 combinado con radioterapia fueron significativamente más bajos que los del grupo control en blanco, el grupo control de vector vacío y el grupo de radioterapia YM155 + (P <0,05). Mientras tanto, después de la regulación negativa de survivina, la proporción entre las fases G2 y S de las células ECA109 y KYSE150 aumentó significativamente, lo que llevó a una detención significativa de las fases G2 y S. Además, la apoptosis de las células ECA109 y KYSE150 aumentó significativamente (P <0,01). Además, los niveles de proteína de Caspasa 3 y Caspasa 9 escindidas aumentaron significativamente en el shRNA de Egr1- survivina combinado con el grupo de radioterapia. El shRNA de survivina de Egr1 combinado con radioterapia puede regular negativamente la expresión de survivina, lo que aumenta aún más la apoptosis y mejora la radiosensibilidad de las células ECA109 y KYSE150.

Genética y genómica de la longevidad y el envejecimiento / Genetics and genomics of longevity and aging

El envejecimiento y la longevidad son procesos que involucran una serie de factores genéticos, bioquímicos y ambientales. En esta revisión se tratan algunas cuestiones sobre estos dos procesos biológicos y epigenéticos. Se presentan los genes más importantes en estos procesos, así como se ejemplifican enfermedades que presentan un aceleramiento o falla en la longevidad y el envejecimiento. Se usa el análisis inteligente de datos para hallar interacciones de proteínas/genes que expliquen estos dos fenómenos biológicos.

Aging and longevity are processes that involve a series of genetic, biochemical and environmental factors. This review addresses some issues about these two biological and epigenetic processes. The most important genes in these processes are presented, as well as diseases that present an acceleration or failure in longevity and aging. Intelligent data analysis is used to find protein/gene interactions that explain these two biological phenomena.

Protective Effects and Mechanisms of Huangqi Decoction Against Radiation-Induced Bystander Effects / Efectos Protectores y Mecanismos de la Decocción de Huangqi Contra los Efectos Secundarios Inducidos por la Radiación

SUMMARY: The 12C6+ heavy ion beam irradiation can cause bystander effects. The inflammatory cytokines, endocrine hormones and apoptotic proteins may be involved in 12C6+ irradiation-induced bystander effects. This study characterized the protective effects and mechanisms of Huangqi decoction (HQD) against 12C6+ radiation induced bystander effects. Wistar rats were randomly divided into control, 12C6+ heavy ion irradiation model, and high-dose/medium-dose/low-dose HQD groups. HE staining assessed the pathological changes of brain and kidney. Peripheral blood chemical indicators as well as inflammatory factors and endocrine hormones were detected. Apoptosis was measured with TUNEL. Proliferating cell nuclear antigen (PCNA) expression was determined with real-time PCR and Western blot.Irradiation induced pathological damage to the brain and kidney tissues. After irradiation, the numbers of white blood cells (WBC) and monocyte, and the expression of interleukin (IL)-2, corticotropin-releasing hormone (CRH) and PCNA decreased. The damage was accompanied by increased expression of IL-1β, IL-6, corticosterone (CORT) and adrenocorticotropic hormone (ACTH) as well as increased neuronal apoptosis. These effects were indicative of radiation-induced bystander effects. Administration of HQD attenuated the pathological damage to brain and kidney tissues, and increased the numbers of WBC, neutrophils, lymphocyte and monocytes, as well as the expression of IL-2, CRH and PCNA. It also decreased the expression of IL-1β, IL-6, CORT and ACTH as well as neuronal apoptosis. HQD exhibits protective effects against 12C6+ radiation-induced bystander effects. The underlying mechanism may involve the promotion of the production of peripheral blood cells, inhibition of inflammatory factors and apoptosis, and regulation of endocrine hormones.

La irradiación con haz de iones pesados 12C6+ puede provocar efectos secundarios. Las citoquinas inflamatorias, las hormonas endocrinas y las proteínas apoptóticas pueden estar involucradas en los efectos secundarios inducidos por la irradiación 12C6+. Este estudio caracterizó los efectos y mecanismos protectores de la decocción de Huangqi (HQD) contra los efectos externos inducidos por la radiación 12C6+. Las ratas Wistar se dividieron aleatoriamente en grupos control, modelo de irradiación de iones pesados 12C6+ y grupos de dosis alta/media/baja de HQD. La tinción con HE evaluó los cambios patológicos del cerebro y el riñón. Se detectaron indicadores químicos de sangre periférica, así como factores inflamatorios y hormonas endocrinas. La apoptosis se midió con TUNEL. La expresión del antígeno nuclear de células en proliferación (PCNA) se determinó mediante PCR en tiempo real y transferencia Western blot. La irradiación indujo daños patológicos en los tejidos cerebrales y renales. Después de la irradiación, disminuyó el número de glóbulos blancos (WBC) y monocitos, y la expresión de interleucina (IL)-2, hormona liberadora de corticotropina (CRH) y PCNA. El daño estuvo acompañado por una mayor expresión de IL-1β, IL-6, corticosterona (CORT) y hormona adrenocorticotrópica (ACTH), así como un aumento de la apoptosis neuronal. Estas alteraciones fueron indicativas de efectos inducidos por la radiación. La administración de HQD atenuó el daño patológico a los tejidos cerebrales y renales, y aumentó el número de leucocitos y monocitos, así como la expresión de IL-2, CRH y PCNA. También disminuyó la expresión de IL-1β, IL-6, CORT y ACTH, así como la apoptosis neuronal. HQD exhibe mecanismos protectores contra los efectos externos inducidos por la radiación 12C6+. El mecanismo subyacente puede implicar la promoción de la producción de células sanguíneas periféricas, la inhibición de factores inflamatorios y la apoptosis y la regulación de hormonas endocrinas.

The Effects of White Tea (Camellia Sinensis) and Infliximab Against Cisplatin- Induced Testicular Damage via Oxidative Stress and Apoptosis / Efectos del Té Blanco (Camellia Sinensis) e Infliximab Contra el Daño Testicular Inducido por Cisplatino a Través del Estrés Oxidativo y la Apoptosis

SUMMARY: Cisplatin (Cis) is an important chemotherapeutic agent used in cancer treatment. Males exposed to Cis were reported to exhibit testicular toxicity. Cis-induced testicular toxicity is mediated by oxidative stress, inflammation, testosterone inhibition and apoptosis. Accordingly, this study was conducted to evaluate the potential protective roles of infliximab (IFX), which is an anti- TNF-a agent, and of white tea (Camellia sinensis), which is known to possess antioxidant, anti-apoptotic, and anti-inflammatory effects, against Cis-induced testicular toxicity in rats. Rats were randomly assigned into five groups as follows: control group, Cisplatin (7 mg/kg) treatment group, Cisplatin (7 mg/kg) + infliximab (7 mg/kg) treatment group, cisplatin + white tea (WT) treatment group, and Cisplatin+ WT+IFX combined treatment group. In the present study, Cis exposure reduced the sperm count. It also increased testicular oxidative stress as well as the levels of inflammatory and apoptotic markers. Histopathological assays supported the biochemical findings. Treatment with IFX and/or WT restored testicular histology, preserved spermatogenesis, suppressed oxidative stress and apoptosis, and significantly ameliorated Cis-induced damage. It was concluded that white tea and infliximab could potentially serve as therapeutic options for the protection of testicular tissue against the harmful effects of Cis.

El cisplatino (Cis) es un importante agente quimioterapéutico utilizado en el tratamiento del cáncer. Se informó que los hombres expuestos a Cis exhibieron toxicidad testicular. La toxicidad testicular inducida por Cis está mediada por el estrés oxidativo, la inflamación, la inhibición de la testosterona y la apoptosis. En consecuencia, este estudio se realizó para evaluar las posibles funciones protectoras de infliximab (IFX), un agente anti-TNF-α, y del té blanco (Camellia sinensis), conocido por sus propiedades antioxidantes, antiapoptóticas y anti-TNF-α -efectos inflamatorios, contra la toxicidad testicular inducida por Cis en ratas. Cinco grupos de ratas se asignaron al azar de la siguiente manera: grupo control, grupo de tratamiento con cisplatino (7 mg/ kg), grupo de tratamiento con cisplatino (7 mg/kg) + infliximab (7 mg/kg), grupo de tratamiento con cisplatino + té blanco (WT), y grupo de tratamiento combinado Cisplatino+ WT+IFX. En el presente estudio, la exposición a Cis redujo el conteo de espermatozoides. También aumentó el estrés oxidativo testicular, así como los niveles de marcadores inflamatorios y apoptóticos. Los ensayos histopatológicos respaldaron los hallazgos bioquímicos. El tratamiento con IFX y/o WT restauró la histología testicular, preservó la espermatogénesis, suprimió el estrés oxidativo y la apoptosis, y mejoró significativamente el daño inducido por Cis. Se concluyó que el té blanco y el infliximab podrían potencialmente servir como opciones terapéuticas para la protección del tejido testicular contra los efectos nocivos de Cis.

Diacerein mitigates renal ischemia/reperfusion injury via inhibition of renal inflammation, dendritic cells maturation and apoptosis: the role of TLR4/NF-kB/NLRP3/IL-1beta signaling pathway / La diacereína mitiga la lesión por isquemia/reperfusión renal a través de la inhibición de la inflamación renal, la maduración de las células dendríticas y la apoptosis: el papel de la vía de señalización TLR4/NF-kB/NLRP3/IL-1beta

SUMMARY: One of the reasons for acute kidney damage is renal ischemia. Nevertheless, there are limited protective and therapeutic approaches for this problem. Diacerein is an anti-inflammatory drug characterized by numerous biological activities. We aimed to determine the ameliorative impact of diacerein on renal ischemia/reperfusion injury (I/R) condition, exploring the underlying mechanisms. Twenty-four male rats were allotted into four groups (n= 6): sham group; Diacerein (DIA) group; I/R group, in which a non-crushing clamp occluded the left renal pedicle for 45 min, and the right kidney was nephrectomized for 5 min before the reperfusion process; I/R + diacerein group, injected intraperitoneally with 50 mg diacerein/kg i.m 30 minutes prior to I/R operation. Ischemia/ reperfusion was found to affect renal function and induce histopathological alterations. The flow cytometry analysis demonstrated an elevated expression of innate and mature dendritic cells in I/R renal tissues. Moreover, upregulation in the expression of the inflammatory genes (TLR4, Myd88, and NLRP3), and overexpression of the pro-inflammatory cytokines (IL-1β), apoptotic (caspase-3) and pyroptotic (caspase-1) markers were observed in I/R-experienced animals. The aforementioned deteriorations were mitigated by pre-I/R diacerein treatment. Diacerein alleviated I/R-induced inflammation and apoptosis. Thus, it could be a promising protective agent against I/R.

La isquemia renal es una de los motivos del daño renal agudo. Sin embargo, los enfoques protectores y terapéuticos para este problema son limitados. La diacereína es un fármaco antiinflamatorio caracterizado por numerosas actividades biológicas. Nuestro objetivo fue determinar el impacto de mejora de la diacereína en la condición de lesión por isquemia/ reperfusión renal (I/R), explorando los mecanismos subyacentes. Veinticuatro ratas macho se distribuyeron en cuatro grupos (n= 6): grupo simulado; grupo de diacereína (DIA); grupo I/R, en el que una pinza no aplastante ocluyó el pedículo renal izquierdo durante 45 min, y el riñón derecho fue nefrectomizado durante 5 min antes del proceso de reperfusión; Grupo I/R + diacereína, inyectado por vía intraperitoneal con 50 mg de diacereína/kg i.m. 30 min antes de la operación I/R. Se encontró que la isquemia/ reperfusión afecta la función renal e induce alteraciones histopatológicas. El análisis de citometría de flujo demostró una expresión elevada de células dendríticas innatas y maduras en tejidos renales I/R. Además, se observó una regulación positiva en la expresión de los genes inflamatorios (TLR4, Myd88 y NLRP3) y una sobreexpresión de las citoquinas proinflamatorias (IL-1β), marcadores apoptóticos (caspasa-3) y piroptóticos (caspasa-1) en animales con experiencia en I/R. Los deterioros antes mencionados fueron mitigados por el tratamiento previo a la diacereína I/R. La diacereína alivió la inflamación y la apoptosis inducidas por I/R. Por lo tanto, podría ser un agente protector prometedor contra I/R.

Análise imuno-histoquímica da Yes-Associated Protein (YAP) e sua influência sobre a proliferação celular e a apoptose em lesões odontogênicas epiteliais benignas / Immunohistochemical analysis of Yes-Associated Protein (YAP) and its influence on cell proliferation and apoptosis in benign epithelial odontogenic lesions

Introdução: Os cistos e tumores odontogênicos são lesões que apresentam comportamento biológico heterogêneo e patogênese ainda não totalmente esclarecida. A Yes-associated protein (YAP) atua como um regulador transcricional de genes envolvidos na proliferação celular e na apoptose, participando da ativação de vias associadas ao crescimento cístico e à progressão neoplásica. Objetivo: Analisar a expressão imuno-histoquímica da proteína YAP e correlacioná-la com marcadores envolvidos na proliferação celular e na apoptose em lesões odontogênicas epiteliais benignas. Metodologia: A amostra consistiu de 95 casos de lesões odontogênicas - 25 cistos dentígeros (CDs), 30 CO não sindrômicos (COs), 30 AMB convencionais (AMB-Cs) e 10 AMB unicísticos (AMB-Us) -, além de 10 espécimes de folículo dentários (FD). Foi realizada coleta dos dados clinico-demográficos dos casos, bem como análise morfológica para melhor caracterização da amostra. Os cortes histológicos foram submetidos à técnica imuno-histoquímica através da utilização dos anticorpos YAP, ciclina D1, Ki-67 e Bcl-2, e a análise da expressão destes foi realizada quali-quantitativamente, mediante metodologia adaptada. Os dados coletados seguiram para análise descritiva e estatística (p ≤ 0,05). Resultados: Houve discreta predileção por mulheres (n = 55; 57,6%) e por indivíduos na faixa etária dos 21 aos 40 anos (n = 50; 47,6%), sendo a região posterior de mandíbula mais afetada (64%). A análise da imunoexpressão de YAP revelou maiores níveis de expressão em COs, especialmente nas camadas basal e parabasal, seguido dos AMB-Us e AMB-Cs, que demonstraram moderada imunorreatividade, predominantemente nas células periféricas. Além disso, houve diferenças significativas quanto à imunoexpressão de YAP entre os grupos analisados, com existência de correlações positivas e estatisticamente significativas entre YAP e ciclina D1 em CDs e AMB-Us, e entre YAP e Ki-67 em AMB-Us (p < 0,05). Todavia, entre a imunoexpressão YAP e Bcl-2, foi verificada ausência de correlação estatisticamente significativa. Conclusões: A YAP pode exercer influência sobre a proliferação celular do epitélio de cistos e tumores odontogênicos, auxiliando, assim, na progressão das diferentes lesões odontogênicas (AU).

Background: Odontogenic cysts and tumors present heterogeneous biological behavior, and their etiopathogenesis is not fully understood yet. Yes-associated protein (YAP) acts as a transcriptional regulator of genes involved in cell proliferation and apoptosis, activating pathways associated with cystic growth and neoplastic progression. Objective: To analyze the immunohistochemical expression of YAP protein and correlate it with markers involved in cell proliferation and apoptosis in benign epithelial odontogenic lesions. Methods: The sample consisted of 95 cases of odontogenic lesions - 25 dentigerous cysts (DCs), 30 non-syndromic odontogenic keratocyst (OKCs), 30 conventional AMB (C-AMBs), and 10 unicystic AMB (UAMBs) -, in addition to 10 specimens of dental follicles (DF). Clinicodemographic data collection was carried out, as well as morphological analysis for better characterization of the sample. The histological sections were submitted to the immunohistochemical technique using YAP, cyclin D1, Ki-67, and Bcl-2 antibodies, and their immunoexpression analysis was performed qualitatively and quantitatively, through an adapted methodology. The collected data were submitted for descriptive and statistical analysis (p ≤ 0.05). Results: There was a slight predilection for women (n = 55; 57.6%) and individuals aged between 21 and 40 years (n = 50; 47.6%), with the posterior region of the mandible as the most affected site (64%). Analysis of YAP immunoexpression revealed higher expression levels in OKCs, especially in the basal and parabasal layers, followed by U-AMBs and C-AMBs, which showed moderate immunoreactivity, predominantly in peripheral cells. In addition, there were significant differences in YAP immunoexpression between the analyzed groups, with positive and statistically significant correlations between YAP and cyclin D1 in DCs and U-AMBs, and between YAP and Ki-67 in U-AMBs (p < 0.05). However, between YAP and Bcl-2 immunoexpression, there was no statistically significant correlation. Conclusions: YAP may influence on the cell proliferation of odontogenic cysts and tumors epithelium, thus helping with the progression of the different odontogenic lesions (AU) .

Over-expression of gastrin inhibits apoptosis of gastric cancer cells via reactive oxygen species and annexin A2-mediated mitochondrial dysfunction / La sobreexpresión de gastrina inhibe la apoptosis de las células de cáncer gástrico a través de especies reactivas de oxígeno y disfunción mitocondrial mediada por anexina A2

SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.

La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.

Immunohistochemical evidence of melatonin protection on lung tissue during chemotherapy / Evidencia inmunohistoquímica de la protección de la melatonina en el tejido pulmonar durante la quimioterapia

SUMMARY: The present study investigated the possible protective effects of melatonin on Bleomycin, Cisplatin and etoposide (BEP) chemotherapy regimens using immunohistochemistry. Forty male Wistar rats were divided into four groups of ten as; group 1 as untreated control; group 2 as BEP group which received the three cycles of 21 days' regimen each of 0.5¥ dose levels ofBEP (bleomycin 0.75 mg/kg, etoposide 7.5 mg/kg and cisplatin 1.5 mg/kg). Rats in the group 3 (MEL group) received 10 mg/kg/day melatonin once daily. Group 4 received the melatonin (30 min before the BEP injections) and BEP as in groups 2. Proliferating cell nuclear antigen (PCNA) staining was used to detect cell proliferation and caspase-3, caspase-9 and Caspase-8 were detected to investigate apoptosis. PCNA immunostaining in alveolar epithelium, alveolar macrophages and bronchus was weak to moderate in BEP group. However, diffuse and strong caspase immunoreactions for caspase-3, caspase 8- and caspase-9 were detected in the bronchioles epithelium, vascular endothelium, alveolar luminal macrophages in the BEP group. PCNA and caspase immunoreactivities in MEL and Mel + BEP groups were close to the control one. The surface are in the BEP group was significantly reduced as compared to the control one ((P0.05). It can be concluded that BEP regimen can affects negatively on lung tissue and melatonin inhibits lung tissue injuries during BEP chemotherapy.

El presente estudio investigó los posibles efectos protectores de la melatonina en los regímenes de quimioterapia con bleomicina, etopósido y cisplatino (BEP) mediante inmunohistoquímica. Cuarenta ratas Wistar macho se dividieron en cuatro grupos de diez: grupo 1, control sin tratar; grupo 2, quimioterapia con una dosis de 0,5x de BEP (0,75 mg/kg de bleomicina, 7,5 mg/ kg de etopósido y 1,5 mg/kg de cisplatino) con tres ciclos de 21 días cada uno. Las ratas del grupo 3 (grupo MEL) recibieron 10 mg/kg/día de melatonina una vez al día. El grupo 4 (Mel + BEP) recibió melatonina (30 minutos antes de las inyecciones de BEP) y BEP, como en los grupos 2. Se usó la tinción del antígeno nuclear de células en proliferación (PCNA) para detectar la proliferación celular y, caspasa- 3, caspasa-9 y caspasa-8 para investigar apoptosis. La inmunotinción de PCNA en el epitelio alveolar, los macrófagos alveolares y los bronquios varió de débil a moderada en el grupo BEP. Sin embargo, se detectaron inmunorreacciones difusas y fuertes para caspasa-3, caspasa 8- y caspasa-9 en el epitelio de los bronquiolos, endotelio vascular y macrófagos luminales alveolares. Las inmunorreactividades de PCNA y caspasa en los grupos MEL y Mel + BEP fueron similares a las del control. El área de superficie en el grupo BEP se redujo significativamente en comparación con el control (P0,05). Se puede concluir que la quimioterapia con BEP puede afectar negativamente al tejido pulmonar y la melatonina inhibe las lesiones durante la quimioterapia.

Maackiain inhibits proliferation and promotes apoptosis of nasopharyngeal carcinoma cells by inhibiting the MAPK/Ras signaling pathway / 中国天然药物

Nasopharyngeal carcinoma (NPC) is the third most common malignancy with a high recurrence and metastasis rate in South China. Natural compounds extracted from traditional Chinese herbal medicines have been developed and utilized for the treatment of a variety of cancers with modest properties and slight side effects. Maackiain (MA) is a type of flavonoid that was first isolated from leguminous plants, and it has been reported to relieve various nervous system disorders and exert anti-allergic as well as anti-inflammatory effects. In this study, we demonstrated that MA inhibited proliferation, arrested cell cycle and induced apoptosis in nasopharyngeal carcinoma CNE1 and CNE2 cells in vitro and in vivo. The expression of the related proteins associated with these processes were consistent with the above effects. Moreover, transcriptome sequencing and subsequent Western blot experiments revealed that inhibition of the MAPK/Ras pathway may be responsible to the anti-tumor effect of MA on NPC cells. Therefore, the effects of MA and an activator of this pathway, tertiary butylhydroquinone (TBHQ), alone or combination, were investigated. The results showed TBHQ neutralized the inhibitory effects of MA. These data suggest that MA exerts its anti-tumor effect by inhibiting the MAPK/Ras signaling pathway and it has the potential to become a treatment for patients with NPC.

Cyasterone inhibits IL-1β-mediated apoptosis and inflammation via the NF-κB and MAPK signaling pathways in rat chondrocytes and ameliorates osteoarthritisin vivo / 中国天然药物

Osteoarthritis is a prevalent global joint disease, which is characterized by inflammatory reaction and cartilage degradation. Cyasterone, a sterone derived from the roots of Cyathula officinalis Kuan, exerts protective effect against several inflammation-related diseases. However, its effect on osteoarthritis remains unclear. The current study was designed to investigate the potential anti-osteoarthritis activity of cyasterone. Primary chondrocytes isolated from rats induced by interleukin (IL)-1β and a rat model stimulated by monosodium iodoacetate (MIA) were used for in vitro and in vivo experiments, respectively. The results of in vitro experiments showed that cyasterone apparently counteracted chondrocyte apoptosis, increased the expression of collagen II and aggrecan, and restrained the production of the inflammatory factors inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), metalloproteinase-3 (MMP-3), and metalloproteinase-13 (MMP-13) induced by IL-1β in chondrocytes. Furthermore, cyasterone ameliorated the inflammation and degenerative progression of osteoarthritis potentially by regulating the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. For in vivo experiments, cyasterone significantly alleviated the inflammatory response and cartilage destruction of rats induced by monosodium iodoacetate, where dexamethasone was used as the positive control. Overall, this study laid a theoretical foundation for developing cyasterone as an effective agent for the alleviation of osteoarthritis.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3β/STAT3 signaling axis / 中国天然药物

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKTSer473 and p-GSK3βSer9, and decreased the expression of p-STAT3Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3β, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.

Bavachin induces apoptosis in colorectal cancer cells through Gadd45a via the MAPK signaling pathway / 中国天然药物

Bavachin is a dihydroflavonoid compound isolated from Psoralea corylifolia, and exhibits anti-bacterial, anti-inflammatory, anti-tumor and lipid-lowering activities. Recent attention has gradually drawn on bavachin-induced apoptosis in many human cancer cell lines. However, the anti-cancer effects and related mechanisms in colorectal cancer remain unknown. Here, we investigated the effects of bavachin on colorectal cancer in vivo and in vitro. The results showed that bavachin inhibited the proliferation of human colorectal cancer cells and induce apoptosis. These changes were mediated by activating the MAPK signaling pathway, which significantly up-regulated the expression of Gadd45a. Furthermore, Gadd45a silencing obviously attenuated bavachin-mediated cell apoptosis. Inhibition of the MAPK signaling pathway by JNK/ERK/p38 inhibitors also weakened the up-regulation of Gadd45a by bavachin. The anticancer effect of bavachin was also validated using a mouse xenograft model of human colorectal cancer. In conclusion, these findings suggest that bavachin induces the apoptosis of colorectal cancer cells through activating the MAPK signaling pathway.

In vitro and in vivo anticancer potential and molecular targets of the new colchicine analog IIIM-067 / 中西医结合学报

OBJECTIVE@#The current study evaluated various new colchicine analogs for their anticancer activity and to study the primary mechanism of apoptosis and in vivo antitumor activity of the analogs with selective anticancer properties and minimal toxicity to normal cells.@*METHODS@#Sulforhodamine B (SRB) assay was used to screen various colchicine analogs for their in vitro cytotoxicity. The effect of N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(pyrrolidine-1-yl)5,6,7,9-tetrahydrobenzo[a] heptalene-7-yl] acetamide (IIIM-067) on clonogenicity, apoptotic induction, and invasiveness of A549 cells was determined using a clonogenic assay, scratch assay, and staining with 4',6-diamidino-2-phenylindole (DAPI) and annexin V/propidium iodide. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were observed using fluorescence microscopy. Western blot analysis was used to quantify expression of proteins involved in apoptosis, cell cycle, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling. Pharmacokinetic and in vivo efficacy studies against Ehrlich ascites carcinoma (EAC) and Ehrlich solid tumor models were conducted using Swiss albino mice.@*RESULTS@#IIIM-067 showed potent cytotoxicity and better selectivity than all other colchicine analogs screened in this study. The selective activity of IIIM-067 toward A549 cells was higher among other cancer cell lines, with a selectivity index (SI) value of 2.28. IIIM-067 demonstrated concentration- and time-dependent cytotoxicity against A549 cells with half-maximal inhibitory concentration values of 0.207, 0.150 and 0.106 μmol/L at 24, 48 and 72 h, respectively. It also had reduced toxicity to normal cells (SI > 1) than the parent compound colchicine (SI = 1). IIIM-067 reduced the clonogenic ability of A549 cells in a dose-dependent manner. IIIM-067 enhanced ROS production from 24.6% at 0.05 μmol/L to 82.1% at 0.4 μmol/L and substantially decreased the MMP (100% in control to 5.6% at 0.4 μmol/L). The annexin V-FITC assay demonstrated 78% apoptosis at 0.4 μmol/L. IIIM-067 significantly (P < 0.5) induced the expression of various intrinsic apoptotic pathway proteins, and it differentially regulated the PI3K/AKT/mTOR signaling pathway. Furthermore, IIIM-067 exhibited remarkable in vivo anticancer activity against the murine EAC model, with tumor growth inhibition (TGI) of 67.0% at a dose of 6 mg/kg (i.p.) and a reduced mortality compared to colchicine. IIIM-067 also effectively inhibited the tumor growth in the murine solid tumor model with TGI rates of 48.10%, 55.68% and 44.00% at doses of 5 mg/kg (i.p.), 6 mg/kg (i.p.) and 7 mg/kg (p.o.), respectively.@*CONCLUSION@#IIIM-067 exhibited significant anticancer activity with reduced toxicity both in vitro and in vivo and is a promising anticancer candidate. However, further studies are required in clinical settings to fully understand its potential.

MAGED4B Promotes Glioma Progression via Inactivation of the TNF-α-induced Apoptotic Pathway by Down-regulating TRIM27 Expression / 神经科学通报·英文版

MAGED4B belongs to the melanoma-associated antigen family; originally found in melanoma, it is expressed in various types of cancer, and is especially enriched in glioblastoma. However, the functional role and molecular mechanisms of MAGED4B in glioma are still unclear. In this study, we found that the MAGED4B level was higher in glioma tissue than that in non-cancer tissue, and the level was positively correlated with glioma grade, tumor diameter, Ki-67 level, and patient age. The patients with higher levels had a worse prognosis than those with lower MAGED4B levels. In glioma cells, MAGED4B overexpression promoted proliferation, invasion, and migration, as well as decreasing apoptosis and the chemosensitivity to cisplatin and temozolomide. On the contrary, MAGED4B knockdown in glioma cells inhibited proliferation, invasion, and migration, as well as increasing apoptosis and the chemosensitivity to cisplatin and temozolomide. MAGED4B knockdown also inhibited the growth of gliomas implanted into the rat brain. The interaction between MAGED4B and tripartite motif-containing 27 (TRIM27) in glioma cells was detected by co-immunoprecipitation assay, which showed that MAGED4B was co-localized with TRIM27. In addition, MAGED4B overexpression down-regulated the TRIM27 protein level, and this was blocked by carbobenzoxyl-L-leucyl-L-leucyl-L-leucine (MG132), an inhibitor of the proteasome. On the contrary, MAGED4B knockdown up-regulated the TRIM27 level. Furthermore, MAGED4B overexpression increased TRIM27 ubiquitination in the presence of MG132. Accordingly, MAGED4B down-regulated the protein levels of genes downstream of ubiquitin-specific protease 7 (USP7) involved in the tumor necrosis factor-alpha (TNF-α)-induced apoptotic pathway. These findings indicate that MAGED4B promotes glioma growth via a TRIM27/USP7/receptor-interacting serine/threonine-protein kinase 1 (RIP1)-dependent TNF-α-induced apoptotic pathway, which suggests that MAGED4B is a potential target for glioma diagnosis and treatment.

Apolipoprotein E enhances migration of endometrial cancer cells byactivating the ERK/MMP9 signaling pathway / 南方医科大学学报

OBJECTIVE@#To study the role of apolipoprotein E (APOE) in regulating endometrial cancer metastasis and explore the signaling pathway in the regulatory mechanism.@*METHODS@#Human endometrial cancer cell line HEC-1B was transfected with a control siRNA (siCtrl) or a specific siRNA targeting APOE (siAPOE) or with either pEGFP-N1 plasmid or an APOEoverexpressing plasmid. The changes in migration, proliferation, apoptosis and cell cycle of the transfected cells were examined using wound healing assay, Transwell migration assay, MTT assay, flow cytometry, and Hoechst staining. The activity of the ERK/MMP9 signaling pathway in the transfected cells was assessed using RT-qPCR and Western blotting. The expression level of APOE in clinical specimens of endometrial cancer tissues were detected using immunohistochemistry and its correlation with differentiation of endometrial cancer tissues was analyzed.@*RESULTS@#Wound healing assay and Transwell migration assay showed that compared with those in siCtrl group, HEC-1B cells transfected with siAPOE showed significantly reduced migration ability (P < 0.05), whereas APOE overexpression significantly promoted the migration of the cells (P < 0.05). Neither APOE knockdown nor overexpression produced significant effects on HEC-1B cell proliferation as shown by MTT assay and flow cytometry. Hoechst staining revealed that transfection with siAPOE did not significantly affect apoptosis of HEC-1B cells. APOE knockdown obviously reduced and APOE overexpression enhanced ERK phosphorylation and MMP9 expression in HEC-1B cells (P < 0.05). Treatment with U0126 partially reversed the effects of APOE overexpression on ERK phosphorylation, migration and MMP9 expression in HEC-1B cells (P < 0.05). APOE is highly expressed in clinical samples of endometrial cancer tissues as compared with the adjacent tissues.@*CONCLUSION@#APOE is highly expressed in endometrial cancer tissues to promote cancer cell migration by enhancing ERK phosphorylation and MMP9 expression.

Intermittent heat exposure induces thoracic aorta injury in spontaneously hypertensive rats by activating the AMPK/mTOR/ULK1 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effects of different manners of heat exposure on thoracic aorta injury in spontaneously hypertensive rats (SHRs) and explore the underlying mechanism.@*METHODS@#Normal 6 to 7-week-old male SHRs were randomized into control group (cage at room temperature), intermittent heat exposure group (SHR-8 group, exposed to 32 ℃ for 8 h daily for 7 days) and SHR-24 group (with continuous exposure to 32 ℃ for 7 days). After the treatments, the pathologies of the thoracic aorta of the rats were observed with HE staining, and the expressions of Beclin1, LC3B and p62 were detected with Western blotting and immunofluorescence assay; TUNEL staining was used to observe cell apoptosis in the thoracic aorta, and the expressions of caspase-3, Bax, and Bcl-2 were detected using Western blotting. The effects of intraperitoneal injections of 3-MA (an autophagy agonist), rapamycin (an autophagy inhibitor) or compound C 30 min before intermittent heat exposure on the expressions of proteins associated with autophagy, apoptosis and the AMPK/mTOR/ULK1 pathway in the aorta were examined with immunohistochemistry.@*RESULTS@#In SHR-8 group, the rats showed incomplete aortic intima with disordered cell distribution and significantly increased expressions of Beclin1, LC3II/LC3I and Bax, lowered expressions of p62 and Bcl-2, and increased apoptotic cells in the thoracic aorta (P < 0.05). Pretreatment with 3-MA obviously inhibited the expressions of autophagy- and apoptosis-related proteins, whereas rapamycin promoted their expressions. Compared with the control group, the rats in SHR-8 group had significantly down-regulated p-mTOR and up-regulated p-AMPK and p-ULK1 expression of in the aorta; Treatment with compound C obviously lowered the expressions of p-AMPK and p-ULK1 and those of LC3B and Beclin1 as well.@*CONCLUSION@#In SHRs, intermittent heat exposure causes significant pathologies and promotes autophagy and apoptosis in the thoracic aorta possibly by activating the AMPK/mTOR/ULK1 pathway.

LASS2/TMSG1 overexpression inhibits proliferation and promotes apoptosis of human lung cancer A549 cells possibly by upregulating ceramide and p38 MAPK to activate a signaling cascade / 南方医科大学学报

OBJECTIVE@#To investigate the effects of LASS2/TMSG1 gene overexpression on proliferation and apoptosis of human lung cancer A549 cells and explore the possible mechanism.@*METHODS@#We examined LASS2/TMSG1 expression level in a previously constructed A549 cell line overexpressing LASS2/TMSG1 using Western blotting. The proliferation and apoptosis of the cells were detected using colony-forming assay, CCK-8 assay, Hoechst/PI double staining and flow cytometry. Fourteen nude mice were randomized into 2 groups (n=7) to receive subcutaneous injection of A549 cells with or without LASS2/TMSG1 overexpression on the back of the neck, and the cell proliferation in vivo was observed. The expression levels of p38 MAPK protein and p-p38 MAPK protein in the xenografts were detected with Western blotting. ELISA was used to detect the levels of ceramide and p38 MAPK protein in cultured A549 cell supernatants and the xenografts in nude mice.@*RESULTS@#Compared with the negative control cells, A549 cells with LASS2/TMSG1 overexpression had significantly lowered proliferation ability in vitro with increased early apoptosis rate (P < 0.05), and showed obvious growth inhibition after inoculation in nude mice(P < 0.05). Western blotting showed that in both cultured A549 cells and the xenografts in nude mice, LASS2/TMSG1 gene overexpression significantly increased the expression levels of p38 MAPK protein and p-p38 MAPK protein (P < 0.05); the results of ELISA also revealed significantly increased levels of ceramide and p38 MAPK protein in the cell supernatant andxenografts as well (P < 0.05).@*CONCLUSION@#Overexpression of LASS2/TMSG1 gene can significantly inhibit the proliferation and promote early apoptosis of human lung cancer A549 cells both in vitro and in vivo possibly by upregulating the expressions of ceramide and p38 MAPK protein to activate a signal transduction cascade.

Shikonin induces hepatocellular carcinoma cell apoptosis by suppressing PKM2/PHD3/HIF-1α signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721 cells.@*METHODS@#Cultured SMMC-7721 cells and normal hepatocytes (L-02 cells) were treated with 4, 8, or 16 μmol/L shikonin, and the changes in cell viability was assessed using MTT assay. The levels of ATP and lactic acid in the cell cultures were detected using commercial kits. Co-immunoprecipitation and immunofluorescence staining were used to determine the relationship among pyruvate kinase M2 (PKM2), prolyl hydroxylase 3 (PHD3), and hypoxia-inducible factor-1α (HIF-1α). The expressions of PHD3, PKM2, HIF-1α, Bax, cleaved caspase-3, and Bcl-2 in SMMC-7721 cells were detected with Western blotting, and cell apoptosis was analyzed with annexin V-FITC/PI staining. The effects of RNA interference of PKM2 on PHD3 and HIF-1α expressions in SMMC-7721 cells were detected using Western blotting.@*RESULTS@#The IC50 of shikonin against SMMC-7721 and L-02 cells was 8.041 μmol/L and 31.75 μmol/L, respectively. Treatment with shikonin significantly inhibited the protein expressions of PKM2, HIF-1α and PHD3 and nuclear translocation of PKM2 and HIF-1α in SMMC-7721 cells. Coimmunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF-1α complex and significantly reduced the contents of lactic acid and ATP in SMMC-7721 cells (P < 0.05). The expressions of PHD3 and HIF-1α decreased significantly after PKM2 knockdown (P < 0.05). Shikonin treatment significantly increased the apoptosis rate, enhanced the expressions of Bax and cleaved caspase-3, and decreased Bcl-2 expression in SMMC-7721 cells (P < 0.05).@*CONCLUSIONS@#Shikonin induces apoptosis of SMMC-7721 cells possibly by inhibiting aerobic glycolysis through the PKM2/PHD3/HIF-1α signaling pathway to cause energy supply dysfunction in the cells.