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.

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.

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.

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.

Evidencias de la presencia de una actividad tipo-quimotripsina en el espermatozoide humano y su rol en la reacción acrosómica / Evidence of the quimiotrypsin activity in the human spermatozoid and its role in the acrosomic reaction

Se evaluó el efecto de inhibidores de la quimotripsina sobre la reacción acrosómica del espermatozoide humano estimulada por la zona pelúcida o fluido folicular humano. Espermatozoides mótiles, seleccionados por un gradiente de Percol, fueron incubadas en una concentración de 1x 10 células/ml, a 37 grados centigrados, y 5 por ciento de CO2. Después de 4.5 hrs. Se añadió el inhibidor de quimotripsina TPCK (N- Tosyl-L-Phenylalanine-Chloromethyl Ketone) o el sustrato ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) por 30 minutos. Luego, se añadieron cuatro ovocitos humanos y se determinó el porcentaje de reacción acrosómica sobre la zona pelúcida. El TPCK y el ATEE inhibieron la reacción acrosómica producida por la zona pelúcida. Los inhibidores de quimotripsina TPCK y quimostatina y los sustratos para quimotripsina ATEE, BTEE (N-Benzoyl-Tyrosine-Ethyl Esther), Succinyl-Ala-Ala-Phe-7-Amido-4 Methyl-Coumarin (Suc-Ala-Ala-Phe-AMC), y Succynil-Leu-Leu-Val-Try-7-Amido-4 Methyl-Coumarin (Suc-Leu-leu-Val-Tyr-AMC) inhibieron la reacción acrosómica inducida por el fluido folicular humano. Los extractos espermáticos mostraron una actividad hidrolítica hacia los sustratos Suc-Ala-Ala-Phe-AMC y Suc-Leu-Leu-Val-Tyr-AMC. Esta actividad enzimática fue anulada por el TPCK y la quimostatina, en ausencia de Ca2+ y no fue modificada por la 1,10 fenantrolina. Además, esta actividad estuvo presente en el sobrenadante después de que la reacción acrosómica fue inducida por el ionóforo de calcio y en espermatozoides epidídimales recuperados de la región caudal. Observaciones con el microscopio electrónico indicaron que los inhibidores impidieron los eventos de membrana de la reacción acrosómica. Estos datos sugieren una asociación entre el espermatozoide humano y una actividad tipo quimotripsina con un posible rol en la reacción acrosómica