Knockdown of Antisense Noncoding Mitochondrial RNA Reduces Tumorigenicity of Patient-Derived Clear Cell Renal Carcinoma Cells in an Orthotopic Xenograft Mouse Model.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of renal cancer and its treatment is hindered by a resistance to targeted therapies, immunotherapies and combinations of both. We have reported that the knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) with chemically modified antisense oligonucleotides induces proliferative arrest and apoptotic death in tumor cells from many human and mouse cancer types. These studies have been mostly performed in vitro and in vivo on commercially available cancer cell lines and have shown that in mouse models tumor growth is stunted by the treatment. The present work was performed on cells derived from primary and metastatic ccRCC tumors. We established primary cultures from primary and metastatic ccRCC tumors, which were subjected to knockdown of ASncmtRNAs in vitro and in vivo in an orthotopic xenograft model in NOD/SCID mice. We found that these primary ccRCC cells are affected in the same way as tumor cell lines and in the orthotopic model tumor growth was significantly reduced by the treatment. This study on patient-derived ccRCC tumor cells represents a model closer to actual patient ccRCC tumors and shows that knockdown of ASncmtRNAs poses a potential treatment option for these patients.

Preclinical evaluation of chimeric antigen receptor T cells targeting the carcinoembryonic antigen as a potential immunotherapy for gallbladder cancer.

Gallbladder cancer (GBC) is commonly diagnosed at late stages when conventional treatments achieve only modest clinical benefit. Therefore, effective treatments for advanced GBC are needed. In this context, the administration of T cells genetically engineered with chimeric antigen receptors (CAR) has shown remarkable results in hematological cancers and is being extensively studied for solid tumors. Interestingly, GBC tumors express canonical tumor-associated antigens, including the carcinoembryonic antigen (CEA). However, the potential of CEA as a relevant antigen in GBC to be targeted by CAR-T cell-based immunotherapy has not been addressed. Here we show that CEA was expressed in 88% of GBC tumors, with higher levels associated with advanced disease stages. CAR-T cells specifically recognized plate-bound CEA as evidenced by up-regulation of 4-1BB, CD69 and PD-1, and production of effector cytokines IFN-γ and TNF-α. In addition, CD8+ CAR-T cells up-regulated the cytotoxic molecules granzyme B and perforin. Interestingly, CAR-T cell activation occurred even in the presence of PD-L1. Consistent with these results, CAR-T cells efficiently recognized GBC cell lines expressing CEA and PD-L1, but not a CEA-negative cell line. Furthermore, CAR-T cells exhibited in vitro cytotoxicity and reduced in vivo tumor growth of GB-d1 cells. In summary, we demonstrate that CEA represents a relevant antigen for GBC that can be targeted by CAR-T cells at the preclinical level. This study warrants further development of the adoptive transfer of CEA-specific CAR-T cells as a potential immunotherapy for GBC.

Targeting antisense mitochondrial noncoding RNAs induces bladder cancer cell death and inhibition of tumor growth through reduction of survival and invasion factors.

Knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, supporting a selective therapy against different types of cancer. In this work, we evaluated the effects of knockdown of ASncmtRNAs on bladder cancer (BCa). We transfected the BCa cell lines UMUC-3, RT4 and T24 with the specific antisense oligonucleotide Andes-1537S, targeted to the human ASncmtRNAs. Knockdown induced a strong inhibition of cell proliferation and increase in cell death in all three cell lines. As observed in UMUC-3 cells, the treatment triggered apoptosis, evidenced by loss of mitochondrial membrane potential and Annexin V staining, along with activation of procaspase-3 and downregulation of the anti-apoptotic factors survivin and Bcl-xL. Treatment also inhibited cell invasion and spheroid formation together with inhibition of N-cadherin and MMP 11. In vivo treatment of subcutaneous xenograft UMUC-3 tumors in NOD/SCID mice with Andes-1537S induced inhibition of tumor growth as compared to saline control. Similarly, treatment of a high-grade bladder cancer PDX with Andes-1537S resulted in a strong inhibition of tumor growth. Our results suggest that ASncmtRNAs could be potent targets for bladder cancer as adjuvant therapy.

Cistectomía radical abierta versus laparoscópica: comparación de resultados perioperatorios y oncológicos a mediano plazo / Open vs Laparoscopic Radical Cystectomy: Comparison of perioperative and Mid-term oncological outcomes

OBJETIVO: Comparar los resultados perioperatorios y oncológicos a mediano plazo entre Cistectomía Radical Abierta (CRA) versus Cistectomía Radical Laparoscópica (CRL). MÉTODOS: Se realizó una cohorte retrospectiva, en la cual se incluyeron 182 pacientes sometidos de forma consecutiva a Cistectomía radical (CR) como tratamiento de Cáncer Vesical Músculo-Invasor entre el 2000 y el 2010 en un solo centro. La serie se dividió en dos grupos: CRA (n = 83) y CRL (n = 99). Todas las CR fueron realizadas por el mismo cirujano. Las complicaciones perioperatorias fueron registradas según la clasificación Clavien-Dindo. Se evaluó sobrevida libre de recurrencia, sobrevida cáncer-específica y asociación entre la técnica quirúrgica y recurrencia de enfermedad ajustando co-variables. RESULTADOS: Las características clínicas y patológicas fueron similares entre los dos grupos. Se observaron diferencias significativas en pérdida sanguínea estimada, tiempo operatorio y estadía hospitalaria entre los grupos (p < 0,05). Se presentaron 27 (32,5%) complicaciones Clavien I-II en el grupo abierto y 11 (11,1%) en el grupo laparoscópico. Cuatro complicaciones Clavien ≥ III (4,8%) se presentaron en el grupo CRA versus 7 (7%) en el grupo CRL (ns). La mediana de seguimiento para pacientes sin recurrencia fue de 23 meses (12-48). Un total de 60 pacientes (72,3%) presentaron recurrencia de algún tipo en el grupo de CRA y 59 pacientes (59,6%) en el de CRL. La incidencia acumulada de mortalidad cáncer-específica, estratificada por abordaje quirúrgico, fue similar entre ambos grupos (p.0,9). CONCLUSIONES: En base a nuestra experiencia, la CRL mostró ventajas en sangrado intraoperatorio y estadía hospitalaria, sin encontrar diferencias en complicaciones mayores entre ambos grupos. El control oncológico a mediano plazo en relación a recurrencia como a sobrevida cáncer-especifica no presenta diferencias significativas entre la CRL y CRA en el manejo del cáncer vesical músculo-invasor

OBJECTIVE: To compare peri-operative and mid-term oncological outcomes between Open radical cystectomy (ORC) and Laparoscopic radical cystectomy (LRC). METHODS: A retrospective cohort was assembled, in which 182 patients had been subjected consecutively to Radical Cystectomy (RC) for treatment of muscle-invasive bladder cancer (MIBC) between 2000 and 2010 in a single center. Two cohorts were included: ORC (n = 83) and LRC (n = 99). All the RCs were performed by the same surgeon. Perioperatory complications were registered according to Clavien-Dindo classification. We evaluated recurrence-free survival, cancer-specific survival and association between the surgical technique performed and disease recurrence, with co-variable adjustment. RESULTS: Clinical and pathologic characteristics were similar for both groups. Significant differences were observed between the two groups, regarding blood loss, operative time and hospitalization days (p < 0.04). The ORC group displayed 27 (32.5%) Clavien I-II cases, vs. 11 (11.1%) in the LRC group. Four Clavien≥III (4.8%) complications were reported in the ORC, vs. 7 (7%) in the LRC group (NS). Mean follow-up time for patients without recurrence was 23 months (12-48). A total of 60 patients (72.3%) showed recurrence in the ORC group, compared to 59 (59.6%) in the LRC group. Cumulative cancer-specific mortality index, stratified by surgical technique, was similar between both groups (p.-0.9) CONCLUSIONS: Based on our experience, LRC showed advantages in intraoperative bleeding and length of hospital stay with no difference in major complications between both groups. Mid-term oncological control, regarding local recurrence and cancer-specific survival, showed no significant difference between LRC and ORC in the management of MIBC

[Open vs Laparoscopic Radical Cystectomy: Comparison of perioperative and Mid-term oncological outcomes.] / Cistectomía radical abierta versus laparoscópica: comparación de resultados perioperatorios y oncológicos a mediano plazo.

OBJECTIVE: To compare peri-operative and mid-term oncological outcomes between Open radical cystectomy (ORC) and Laparoscopic radical cystectomy (LRC). METHODS: A retrospective cohort was assembled, in which 182 patients had been subjected consecutively to Radical Cystectomy (RC) for treatment of muscle-invasive bladder cancer (MIBC) between 2000 and 2010 in a single center. Two cohorts were included: ORC (n=83) and LRC (n=99). All the RCs were performed by the same surgeon. Perioperatory complications were registered according to Clavien-Dindo classification. We evaluated recurrence-free survival, cancer-specific survival and association between the surgical technique performed and disease recurrence, with co-variable adjustment. RESULTS: Clinical and pathologic characteristics were similar for both groups. Significant differences were observed between the two groups, regarding blood loss, operative time and hospitalization days (p<0.04). The ORC group displayed 27 (32.5%) Clavien I-II cases, vs. 11 (11.1%) in the LRC group. Four Clavien≥III (4.8%) complications were reported in the ORC, vs. 7 (7%) in the LRC group (NS). Mean follow-up time for patients without recurrence was 23 months (12-48). A total of 60 patients (72.3%) showed recurrence in the ORC group, compared to 59 (59.6%) in the LRC group. Cumulative cancer-specific mortality index, stratified by surgical technique, was similar between both groups (p.-0.9). CONCLUSIONS: Based on our experience, LRC showed advantages in intraoperative bleeding and length of hospital stay with no difference in major complications between both groups. Mid-term oncological control, regarding local recurrence and cancer-specific survival, showed no significant difference between LRC and ORC in the management of MIBC.

OBJETIVO: Comparar los resultados perioperatorios y oncológicos a mediano plazo entre Cistectomía Radical Abierta (CRA) versus Cistectomía Radical Laparoscópica (CRL).MÉTODOS: Se realizó una cohorte retrospectiva, en la cual se incluyeron 182 pacientes sometidos de forma consecutiva a Cistectomía radical (CR) como tratamiento de Cáncer Vesical Músculo-Invasor entre el 2000 y el 2010 en un solo centro. La serie se dividió en dos grupos: CRA (n=83) y CRL (n=99). Todas las CR fueron realizadas por el mismo cirujano. Las complicaciones perioperatorias fueron registradas según la clasificación Clavien-Dindo. Se evaluó sobrevida libre de recurrencia, sobrevida cáncer-específica y asociación entre la técnica quirúrgica y recurrencia de enfermedad ajustando co-variables. RESULTADOS: Las características clínicas y patológicas fueron similares entre los dos grupos. Se observaron diferencias significativas en pérdida sanguínea estimada, tiempo operatorio y estadía hospitalaria entre los grupos (p<0,05). Se presentaron 27 (32,5%) complicaciones Clavien I-II en el grupo abierto y 11 (11,1%) en el grupo laparoscópico. Cuatro complicaciones Clavien ≥III (4,8%) se presentaron en el grupo CRA versus 7 (7%) en el grupo CRL (ns). La mediana de seguimiento para pacientes sin recurrencia fue de 23 meses (12-48). Un total de 60 pacientes (72,3%) presentaron recurrencia de algún tipo en el grupo de CRA y 59 pacientes (59,6%) en el de CRL. La incidencia acumulada de mortalidad cáncer-específica, estratificada por abordaje quirúrgico, fue similar entre ambos grupos (p.0,9). CONCLUSIONES: En base a nuestra experiencia, la CRL mostró ventajas en sangrado intraoperatorio y estadía hospitalaria, sin encontrar diferencias en complicaciones mayores entre ambos grupos. El control oncológico a mediano plazo en relación a recurrencia como a sobrevida cáncer-especifica no presenta diferencias significativas entre la CRL y CRA en el manejo del cáncer vesical músculo-invasor.

Mitochondrial ncRNA targeting induces cell cycle arrest and tumor growth inhibition of MDA-MB-231 breast cancer cells through reduction of key cell cycle progression factors.

The family of long noncoding mitochondrial RNAs (ncmtRNAs), comprising sense (SncmtRNA), and antisense (ASncmtRNA-1 and ASncmtRNA-2) members, are differentially expressed according to cell proliferative status; SncmtRNA is expressed in all proliferating cells, while ASncmtRNAs are expressed in normal proliferating cells, but is downregulated in tumor cells. ASncmtRNA knockdown with an antisense oligonucleotide induces massive apoptosis in tumor cell lines, without affecting healthy cells. Apoptotic death is preceded by proliferation blockage, suggesting that these transcripts are involved in cell cycle regulation. Here, we show that ASncmtRNA knockdown induces cell death preceded by proliferative blockage in three different human breast cancer cell lines. This effect is mediated by downregulation of the key cell cycle progression factors cyclin B1, cyclin D1, CDK1, CDK4, and survivin, the latter also constituting an essential inhibitor of apoptosis, underlying additionally the onset of apoptosis. The treatment also induces an increase in the microRNA hsa-miR-4485-3p, whose sequence maps to ASncmtRNA-2 and transfection of MDA-MB-231 cells with a mimic of this miRNA induces cyclin B1 and D1 downregulation. Other miRNAs that are upregulated include nuclear-encoded hsa-miR-5096 and hsa-miR-3609, whose mimics downregulate CDK1. Our results suggest that ASncmtRNA targeting blocks tumor cell proliferation through reduction of essential cell cycle proteins, mediated by mitochondrial and nuclear miRNAs. This work adds to the elucidation of the molecular mechanisms behind cell cycle arrest preceding tumor cell apoptosis induced by ASncmtRNA knockdown. As proof-of-concept, we show that in vivo knockdown of ASncmtRNAs results in drastic inhibition of tumor growth in a xenograft model of MDA-MB-231 subcutaneous tumors, further supporting this approach for the development of new therapeutic strategies against breast cancer.

Vaccination-induced skin-resident memory CD8+ T cells mediate strong protection against cutaneous melanoma.

Memory CD8+ T cell responses have the potential to mediate long-lasting protection against cancers. Resident memory CD8+ T (Trm) cells stably reside in non-lymphoid tissues and mediate superior innate and adaptive immunity against pathogens. Emerging evidence indicates that Trm cells develop in human solid cancers and play a key role in controlling tumor growth. However, the specific contribution of Trm cells to anti-tumor immunity is incompletely understood. Moreover, clinically applicable vaccination strategies that efficiently establish Trm cell responses remain largely unexplored and are expected to strongly protect against tumors. Here we demonstrated that a single intradermal administration of gene- or protein-based vaccines efficiently induces specific Trm cell responses against models of tumor-specific and self-antigens, which accumulated in vaccinated and distant non-vaccinated skin. Vaccination-induced Trm cells were largely resistant to in vivo intravascular staining and antibody-dependent depletion. Intradermal, but not intraperitoneal vaccination, generated memory precursors expressing skin-homing molecules in circulation and Trm cells in skin. Interestingly, vaccination-induced Trm cell responses strongly suppressed the growth of B16F10 melanoma, independently of circulating memory CD8+ T cells, and were able to infiltrate tumors. This work highlights the therapeutic potential of vaccination-induced Trm cell responses to achieve potent protection against skin malignancies.

The cluster [Re6Se8I6]3- penetrates biological membranes: drug-like properties for CNS tumor treatment and diagnosis.

Tumorigenic cell lines are more susceptible to [Re6Se8I6]3- cluster-induced death than normal cells, becoming a novel candidate for cancer treatment. Still, the feasibility of using this type of molecules in human patients remains unclear and further pharmacokinetics analysis is needed. Using coupled plasma optical emission spectroscopy, we determined the Re-cluster tissue content in injected mice, as a biodistribution measurement. Our results show that the Re-cluster successfully reaches different tissues, accumulating mainly in heart and liver. In order to dissect the mechanism underlying cluster biodistribution, we used three different experimental approaches. First, we evaluate the degree of lipophilicity by determining the octanol/water partition coefficient. The cluster mostly remained in the octanol fraction, with a coefficient of 1.86 ± 0.02, which indicates it could potentially cross cell membranes. Then, we measured the biological membrane penetration through a parallel artificial membrane permeability assays (PAMPA) assay. The Re-cluster crosses the artificial membrane, with a coefficient of 122 nm/s that is considered highly permeable. To evaluate a potential application of the Re-cluster in central nervous system (CNS) tumors, we analyzed the cluster's brain penetration by exposing cultured blood-brain-barrier (BBB) cells to increasing concentrations of the cluster. The Re-cluster effectively penetrates the BBB, reaching nearly 30% of the brain side after 24 h. Thus, our results indicate that the Re-cluster penetrates biological membranes reaching different target organs-most probably due to its lipophilic properties-becoming a promising anti-cancer drug with high potential for CNS cancer's diagnosis and treatment.

HPV-18 E2 protein downregulates antisense noncoding mitochondrial RNA-2, delaying replicative senescence of human keratinocytes.

Human and mouse cells display a differential expression pattern of a family of mitochondrial noncoding RNAs (ncmtRNAs), according to proliferative status. Normal proliferating and cancer cells express a sense ncmtRNA (SncmtRNA), which seems to be required for cell proliferation, and two antisense transcripts referred to as ASncmtRNA-1 and -2. Remarkably however, the ASncmtRNAs are downregulated in human and mouse cancer cells, including HeLa and SiHa cells, transformed with HPV-18 and HPV-16, respectively. HPV E2 protein is considered a tumor suppressor in the context of high-risk HPV-induced transformation and therefore, to explore the mechanisms involved in the downregulation of ASncmtRNAs during tumorigenesis, we studied human foreskin keratinocytes (HFK) transduced with lentiviral-encoded HPV-18 E2. Transduced cells displayed a significantly extended replicative lifespan of up to 23 population doublings, compared to 8 in control cells, together with downregulation of the ASncmtRNAs. At 26 population doublings, cells transduced with E2 were arrested at G2/M, together with downregulation of E2 and SncmtRNA and upregulation of ASncmtRNA-2. Our results suggest a role for high-risk HPV E2 protein in cellular immortalization. Additionally, we propose a new cellular phenotype according to the expression of the SncmtRNA and the ASncmtRNAs.

Mitochondrial ASncmtRNA-1 and ASncmtRNA-2 as potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model.

Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma.

Corrigendum to "Modified urethrovesical anastomosis during robot-assisted simple prostatectomy: Technique and results" [Prostate Int 4 (2016) 61-64].

[This corrects the article DOI: 10.1016/j.prnil.2016.04.001.].

Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

The characterization of anti-T. cruzi activity relationships between ferrocenyl, cyrhetrenyl complexes and ROS release.

Trypanosoma cruzi (T. cruzi) is the parasite that causes Chagas disease. Nifurtimox is the most used drug against the T. cruzi, this drug increases intermediaries nitro group, being mainly responsible for the high toxicity component, for this reason it is important to study new organic compounds and thus improve therapeutic strategies against Chagas disease. The electronic effects of ferrocenyl and cyrhetrenyl fragments were investigated by DFT calculation. A close correlation was found between HOMO-LUMO gap of nitro radical NO 2 (-) with the experimental reduction potential found for nitro group and IC50 of two forms the T. cruzi (epimastigote and trypomastigote). The IC50 on human hepatoma cells is higher for both compounds compared to IC50 demonstrated in the two forms the T. cruzi, and additionally show reactive oxygen species release. The information obtained in this paper could generate two new drugs with anti-T. cruzi activity, but additional studies are needed.

Modified urethrovesical anastomosis during robot-assisted simple prostatectomy: Technique and results.

BACKGROUND: Despite significant developments in transurethral surgery for benign prostatic hyperplasia, simple prostatectomy remains an excellent option for patients with severely enlarged glands. The objective is to describe our results of robot-assisted simple prostatectomy (RASP) with a modified urethrovesical anastomosis (UVA). METHODS: From May 2011 to February 2014, RASP with UVA was performed in 34 patients by a single surgeon (O.C.) using the da Vinci S-HD surgical system. The UVA was performed between the bladder neck and urethral margin using the Van Velthoven technique. Demographic, perioperative, and outcome data were recorded. Complications were recorded with the Clavien-Dindo system. RESULTS: The mean (standard deviation) age was 68 years (62-74 years). The median preoperative prostate volume (interquartile range) was 117 cc (99-146 cc). Operative time was 96 minutes (78-126 minutes), estimate blood loss was 200 mL (100-300 mL), and two (5.8%) patients required a blood transfusion. No conversion to open surgery was needed. The median specimen weight on pathological examination was 76 g (58-100 g). The average hospital stay was 2.2 days (1-4 days) and average Foley catheter time was 4.6 days (4-6 days). No intraoperative complications were recorded. There were seven (20.5%) postoperative complications, most of them Clavien less than or equal to Grade II. CONCLUSION: The results of our study show that RASP with UVA is a feasible, secure, and reproducible procedure with low morbidity. Additional series with larger patient cohorts are needed to validate this approach.

Prevention of Crush Syndrome through Aggressive Early Resuscitation: Clinical Case in a Buried Worker.

UNLABELLED: Introduction Crush syndrome, of which little is known, occurs as a result of compression injury to the muscles. This syndrome is characterized by systemic manifestations such as acute kidney injury (AKI), hypovolemic shock, and hydroelectrolytic variations. This pathology presents high morbidity and mortality if not managed aggressively by prehospital care. Clinical Case A 40-year-old worker was rescued after being buried underground in a ditch for 19 hours. The patient was administered early resuscitation with isotonic solutions and monitored during the entire rescue operation. Despite having increased plasma levels of total creatine kinase (CK), the patient did not develop AKI or hydroelectrolytic variations. CONCLUSION: Aggressive early management with isotonic solutions before hospital arrival is an effective option for nephron-protection and prevention of crush syndrome. Mardones A , Arellano P , Rojas C , Gutierrez R , Oliver N , Borgna V . Prevention of crush syndrome through aggressive early resuscitation: clinical case in a buried worker. Prehosp Disaster Med. 2016;31(3):340-342.

Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4'-trimethoxy-2'-hydroxy-chalcone (CH1) and 3'-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas.

Chylous ascites and high-output chylous fistula after extended pelvic lymph node dissection for urological cancer: a rare postoperative complication.

OBJECTIVES: Chylous ascites and high-output chylous fistula are rare complications following abdominal or pelvic surgery. We report a series of five cases that occurred after pelvic lymph node dissection for urological cancer, in addition to their clinical presentation, diagnosis, and treatment. METHODS: The series comprises five patients; four men in whom robotic radical prostatectomy and extended pelvic lymphadenectomy were performed, and one woman with an infiltrating bladder cancer that underwent robotic anterior pelvic exenteration and extended pelvic lymphadenectomy. The first four patients developed chylous ascites, and the female patient a high-output chylous fistula. RESULTS: In all cases, diagnosis of chylous ascites or chylous fistula was confirmed, and they were handled in varied ways, from observation to medical treatment, paracentesis, and surgery, according to their clinical presentation and evolution. We describe a simple treatment algorithm. CONCLUSION: This rare surgical complication requires a grade of suspicion and a defined treatment according to the probability of the medical compromise. Prevention is an important element. This series, according to our knowledge, is the first description in patients undergoing robotic extended pelvic lymphadenectomy.

Down-regulation of the antisense mitochondrial non-coding RNAs (ncRNAs) is a unique vulnerability of cancer cells and a potential target for cancer therapy.

Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3'-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy.

Determination of the differential expression of mitochondrial long non-coding RNAs as a noninvasive diagnosis of bladder cancer.

BACKGROUND: Bladder cancer is a significant cause of morbidity and mortality with a high recurrence rate. Early detection of bladder cancer is essential in order to remove the tumor, to preserve the organ and to avoid metastasis. The aim of this study was to analyze the differential expression of mitochondrial non-coding RNAs (sense and antisense) in cells isolated from voided urine of patients with bladder cancer as a noninvasive diagnostic assay. METHODS: The differential expression of the sense (SncmtRNA) and the antisense (ASncmtRNAs) transcripts in cells isolated from voided urine was determined by fluorescent in situ hybridization. The test uses a multiprobe mixture labeled with different fluorophores and takes about 1 hour to complete. We examined the expression of these transcripts in cells isolated from urine of 24 patients with bladder cancer and from 15 healthy donors. RESULTS: This study indicates that the SncmtRNA and the ASncmtRNAs are stable in cells present in urine. The test reveals that the expression pattern of the mitochondrial transcripts can discriminate between normal and tumor cells. The analysis of 24 urine samples from patients with bladder cancer revealed expression of the SncmtRNA and down-regulation of the ASncmtRNAs. Exfoliated cells recovered from the urine of healthy donors do not express these mitochondrial transcripts. This is the first report showing that the differential expression of these mitochondrial transcripts can detect tumor cells in the urine of patients with low and high grade bladder cancer. CONCLUSION: This pilot study indicates that fluorescent in situ hybridization of cells from urine of patients with different grades of bladder cancer confirmed the tumor origin of these cells. Samples from the 24 patients with bladder cancer contain cells that express the SncmtRNA and down-regulate the ASncmtRNAs. In contrast, the hybridization of the few exfoliated cells recovered from healthy donors revealed no expression of these mitochondrial transcripts. This assay can be explored as a non-invasive diagnostic tool for bladder cancer.