Echocardiographic manifestations of mitochondrial disease with GTPBP3 gene mutations: A case report.

RATIONALE: Mitochondrial diseases are a group of disorders in which mutations in mitochondrial DNA or nuclear DNA lead to dysfunctional oxidative phosphorylation of cells, with mutations in mitochondrial DNA being the most common cause of mitochondrial disease, and mutations in nuclear genes being rarely reported. The echocardiographic findings of mitochondrial diseases with nuclear gene mutations in children's hearts are even rarer. Even more valuable is that we followed up the patient for 4 years and dynamically observed the cardiac echocardiographic manifestations of mitochondrial disease. Provide ideas for the clinical diagnosis and prognosis of mitochondrial diseases. PATIENT CONCERNS: The patient was seen in the pediatric outpatient clinic for poor strength and mental retardation. echocardiography: mild left ventricular (LV) enlargement and LV wall thickening. Nuclear genetic testing: uanosine triphosphate binding protein 3 (GTPBP3) gene mutation. Diagnosis of mitochondrial disease. DIAGNOSES: Mitochondrial disease with GTPBP3 gene mutations. OUTCOMES: After receiving drug treatment, the patient exhibited a reduction in lactate levels, an enhanced physical condition compared to prior assessments, and demonstrated average intellectual development. LESSONS SUBSECTIONS: For echocardiographic indications of LV wall thickening and LV enlargement, one needs to be alert to the possibility of hereditary cardiomyopathy, especially in children.

Effects of Cavitation from Extracorporeal Shock Wave Combined with Sulfur Hexafluoride Microbubble on Myocardial Ultrastructure in Rats.

BACKGROUND: In the present study, the effects of extracorporeal cardiac shock waves combined with different concentrations of sulfur hexafluoride ultrasound microbubbles on myocardial ultrastructure in rats were observed. METHODS: Thirty-six rats were randomly divided into 6 groups: control group (N), extracorporeal cardiac shock wave group, and combined group, i.e., extracorporeal cardiac shock wave combined with different concentrations of sulfur hexafluoride microbubble (0.225 mL/kg/min, 0.45 mL/kg/min, 0.9 mL/kg/min, 1.8 mL/kg/min). The combination of extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles of different concentrations had no significant effect on hemodynamic indexes and left ventricular function in rats. RESULTS: There were significant differences in cardiac troponin I (cTnI) and nitricoxide among different groups. Histopathology showed that inflammatory cells infiltrated in the shock wave+microbubble 0.9 and shock wave+microbubble 1.8 groups. The myocardial ultrastructural injury score of shock wave+microbubble1.8 group was significantly higher than that of the N group, shock wave group, shock wave+microbubble 0.225 group, and shock wave+microbubble 0.45 group. The score of shock wave+microbubble 0.9 group was higher than that of the control group (P=.009). Western blot results showed that the expression of vascular endothelial growth factor and endothelial nitricoxide synthase (eNOS) protein in the rats treated with extracorporeal cardiac shock wave combined with sulfur hexafluoride microbubbles of different concentrations was higher than that in the N group and shock wave group, with shock wave+microbubble 0.45 group having the strongest expression. CONCLUSION: Myocardial ultrastructure damage occurs when high concentrations of sulfur hexafluoride microbubbles are present, but a proper concentration of sulfur hexafluoride microbubbles could promote the cavitation effect of extracorporeal cardiac shock waves. Thus combination therapy may become a new paradigm in coronary heart disease, especially contributing to the treatment of refractory angina. Combination therapy may change coronary heart disease treatment, especially for refractory angina.

IL-8 regulates the stemness properties of cancer stem cells in the small-cell lung cancer cell line H446.

PURPOSE: Cancer stem cells (CSCs) are a small population of cancer cells located within a tumor that are highly tumorigenic, capable of tumor initiation, and resistant to cancer therapies. We identified the potential genes involved in regulating stemness properties and investigated the mechanisms in small-cell lung cancer (SCLC). MATERIALS AND METHODS: Whole transcriptome sequencing technology was used to screen the potential genes involved in regulating stemness properties from SCLC-SCs (uPAR+) and differentiated cells (uPAR-) in the H446 cell line. The selected genes were validated by quantitative reverse transcription PCR and ELISAs. The effect of IL-8 on stemness of sphere-forming cells was determined through tumor sphere formation, wound healing migration, and in vivo tumorigenesis assays. RESULTS: In our study, uPAR+ and uPAR- cells showed different gene expression profiles. IL-8 was upregulated in SCLC sphere-forming cells. Blocking IL-8 expression with siRNA led to loss of stemness, including the self-renewal capability, migration, expression of stemness-related genes, and in vivo tumorigenicity, in sphere-forming cells. Consistently, exogenously added IL-8 enhanced stemness properties in parental cells. CONCLUSION: IL-8 was upregulated in SCLC sphere-forming cells, and critical for the acquisition and/or maintenance of the stemness features in the SCLC cell line H446. Our results suggest that blocking IL-8 signaling may provide a novel therapeutic approach for targeting SCLC-SCs and improve treatment and outcomes in SCLC.

Cancer Stem Cells in Small Cell Lung Cancer Cell Line H446: Higher Dependency on Oxidative Phosphorylation and Mitochondrial Substrate-Level Phosphorylation than Non-Stem Cancer Cells.

Recently, targeting cancer stem cells (CSCs) metabolism is becoming a promising therapeutic approach to improve cancer treatment outcomes. However, knowledge of the metabolic state of CSCs in small cell lung cancer is still lacking. In this study, we found that CSCs had significantly lower oxygen consumption rate and extracellular acidification rate than non-stem cancer cells. Meanwhile, this subpopulation of cells consumed less glucose, produced less lactate and maintained lower ATP levels. We also revealed that CSCs could produce more ATP through mitochondrial substrate-level phosphorylation during respiratory inhibition compared with non-stem cancer cells. Furthermore, they were more sensitive to suppression of oxidative phosphorylation. Therefore, oligomycin (inhibitor of oxidative phosphorylation) could severely impair sphere-forming and tumor-initiating abilities of CSCs. Our work suggests that CSCs represent metabolically inactive tumor subpopulations which sustain in a state showing low metabolic activity. However, mitochondrial substrate-level phosphorylation of CSCs may be more active than that of non-stem cancer cells. Moreover, CSCs showed preferential use of oxidative phosphorylation over glycolysis to meet their energy demand. These results extend our understanding of CSCs metabolism, potentially providing novel treatment strategies targeting metabolic pathways in small cell lung cancer.

Co-expression of uPAR and CXCR4 promotes tumor growth and metastasis in small cell lung cancer.

Urokinase-type plasminogen activator receptor (uPAR) and C-X-C-chemokine receptor-4 (CXCR4) are considered as key molecules in invasion and metastasis of several cancers via extracellular matrix degeneration and assist tumor metastasis to specific sites by chemotaxis. However, the combined effect of uPAR and CXCR4 on small cell lung cancer (SCLC), the most aggressive type of lung cancer, is not clear. In this study, we detected the expression of uPAR and CXCR4 in SCLC tissue samples (n = 50) by immunohistochemistry. The tumors with high expression of both uPAR and CXCR4 (12/50) had larger size, higher lymph node (LN) metastasis and worse prognosis of patients than those with low expression of uPAR and CXCR4 (38/50) (P < 0.05). We further identified and isolated the both uPAR and CXCR4 positive expression subpopulation cells (uPAR(+)CXCR4(+) cells) from the SCLC cell line H446 by flow cytometry. The uPAR(+)CXCR4(+) cancer cells showed a higher invasive and migrating capacity in the transwell and wound healing assays compared with other subpopulation cells (P < 0.05). uPAR(+)CXCR4(+) cells injected subcutaneously in nude mice markedly increased tumor growth and induced lung metastasis, while other subpopulation cells did not. In conclusion, these data suggest that uPAR and CXCR4 co-expression predicts worse prognosis of SCLC patients. uPAR(+)CXCR4(+) cells promote the tumor growth and play a potential role in metastasis of SCLC.

Clinicopathological and prognostic significance of HIF-1α and HIF-2α expression in small cell lung cancer.

Previous work from our laboratory has demonstrated that urokinase plasminogen activator receptor (uPAR) may be a potential stem-like cell marker in SCLC. Hypoxia inducible factor (HIF) has been shown to transcriptionally regulate uPAR expression. Therefore, the aim of this study was to evaluate the relationship between HIF-1α/HIF-2α and uPAR expression, and to investigate the role of HIF-1α/HIF-2α in the clinical pathology and prognosis of patients with SCLC. Immunohistochemical analysis showed that HIF-1α/HIF-2α staining was mainly present in the nuclei of cancer cells. HIF-1α-positive cells were diffusely distributed in the nests of the tumor, while HIF-2α-positive cells were frequently distributed around necrotic regions. HIF-1α and HIF-2α were expressed in 22/45 (48.9%) and in 11/45 (24.4%) of SCLC patients, respectively; HIF-1α did not correlate with any of the clinicopathological parameters as evaluated in our study. In contrast, a significant association of HIF-2α with uPAR expression, tumor growth and distant metastasis (p=0.001, 0.010 and 0.008, respectively) was noted; Kaplan-Meier survival analysis demonstrated that HIF-1α and HIF-2α expressions were related to shortened overall survival (p=0.027 and 0.001, respectively). However, in multivariate analysis, only HIF-2α expression and distant metastasis were the independent prognostic indicators of SCLC (p=0.004 and 0.018, respectively). Our results suggest that HIF-2α may represent a more aggressive phenotype in SCLC. HIF-2α, in addition to HIF-1α, needs to be considered when developing drugs that target HIF pathway.

miR-27a regulates the self renewal of the H446 small cell lung cancer cell line in vitro.

Cancer growth is driven by cancer stem-like cells within a tumor, called cancer stem cells (CSCs). Since miRNAs can regulate cell-fate decisions, we compared miRNA expression in stem-like cells and differentiated cells from small cell lung cancer (SCLC) cell lines to develop further understanding of the molecular mechanisms involved in the pathogenesis of SCLC. First, SCLC stem-like cells were enriched by isolating sphere-forming cells using a defined serum-free medium. Further, microRNA microarrays were used to measure the expression of 1212 miRNAs in sphere-forming cells and parental cells. We found 86 miRNAs that were differentially expressed, including 48 upregulated miRNAs and 38 downregulated miRNAs between sphere-forming cells and parental cells. Among them, five downregulated miRNAs (let-7, miR-20, 21, 27a and 30b) and one upregulated miRNA (miR-149*) were selected for validation in 3 sets of SCLC cell lines by qRT-RCR. The qRT-PCR analysis confirmed that all six miRNAs were indeed differentially expressed. However, only miR-27a was consistently downregulated in sphere-forming cells of all 3 cell lines. Antagonizing miR-27a by inhibitor in parental cells enhanced proliferation, self renewal, and the proportion of undifferentiated cells in vitro. The candidate miRNA and some miRNAs with same seed sequence are predicted to have several target genes related to apoptosis, cell proliferation and cell cycle. Our results suggest that downregulation of miR-27a enhanced the stem-like properties of SCLC cells in vitro and may be critical to maintaining a stem cell function in SCLC.

Characterization of sphere-forming cells with stem-like properties from the small cell lung cancer cell line H446.

A relatively novel paradigm in tumor biology hypothesizes that cancer growth is driven by tumor cells with stem-like properties. However, direct proof of a population of stem cells in small cell lung cancer (SCLC) remains elusive. In this study, we enriched for stem-like cells from the SCLC cell line H446 by growing them as spheres in a defined serum-free medium. Sphere-derived cells have increased in vitro clonogenic and in vivo tumorigenic potentials as well as drug-resistant properties. After enrichment for stem-like cells, we used multiple candidate stem cell markers to examine the expression profile and found that the sphere-derived cells contained a higher proportion of cells expressing the stem cell surface markers uPAR and CD133 when compared with parental cells. To identify a selectable marker for the sphere-forming cells, we evaluated the sphere-forming abilities of uPAR(+) and uPAR(-) cells as well as the sphere-forming abilities of CD133(+) and CD133(-) cells. Both CD133(+) and CD133(-) cell fractions were capable of forming spheres, and no statistically significant difference was observed in the sphere-forming efficiency between these two populations. In contrast, cells derived from the uPAR(+) fraction were capable of forming spheres, whereas cells derived from the uPAR(-) fraction remained as single cells. Moreover, uPAR(+) cells efficiently formed transplantable tumors, whereas uPAR(-) cells were unable to initiate tumors when transplanted at equivalent cell numbers. In addition, uPAR(+) cells could differentiate into CD56(+)cells, CK(+) cells, and uPAR(-) cells. These data support the existence of a population of tumor sphere-forming cells with stem cell properties in the H446 SCLC cell line. Furthermore, the stem cell population may be enriched in cells expressing the uPAR cell surface marker.