Proliferation inhibition and apoptosis promotion by dual silencing of VEGF and Survivin in human osteosarcoma.

Simultaneous silencing of multiple upregulated genes is an attractive and viable treatment strategy for many incurable diseases including cancer. Herein we used a dual gene-targeted siRNA conjugate composed of VEGF and Survivin siRNA sequences in the same backbone to inhibit proliferation and angiogenesis in two human osteosarcoma cell lines. We synthesized siRNA sequences targeting the VEGF and Survivin genes individually (VEGF siRNA and Survivin siRNA) or simultaneously (one-chain-double-target siRNA: dual siRNA). VEGF and Survivin mRNA and protein expression levels in human osteosarcoma MG-63 and Saos-2 cells were detected by qRT-PCR and western blot analysis. VEGF and Survivin protein location and expression were evaluated by immunohistochemistry and immunofluorescence staining. MG-63 and Saos-2 cell migration, proliferation, apoptosis, and angiogenesis were detected by scratch test, MTT assay, flow cytometry, and capillary tube assay respectively. The dual siRNA induced similar downregulation of VEGF and Survivin mRNA and protein levels, compared with VEGF siRNA or Survivin siRNA alone. The dual siRNA caused greater suppression of MG-63 and Saos-2 cell migration, proliferation and angiogenesis, and promoted more cell apoptosis than VEGF siRNA or Survivin siRNA alone, suggesting that the effects of the dual siRNA on inhibiting cell proliferation, migration, and angiogenesis and promoting apoptosis were superior to those of the single-target siRNAs. Simultaneous silencing of VEGF and Survivin using the dual siRNA may be an advantageous alternative for the development of therapeutic strategies against human osteosarcoma.

PRR14 acts a novel oncogene activating the PI3K signal pathway in human cutaneous squamous cell carcinoma.

Proline rich protein 14 (PRR14) is considered as a new component of the nuclear fiber layer, it may be a key molecule in mediating nuclear morphological changes and functional changes in tumorigenesis. But, it's still unclear in human cutaneous squamous cell carcinoma (cSCC). In the study, the expression profiles of PRR14 in patients with cSCC were investigated by immunohistochemistry (IHC), also the PRR14 expression in cSCC tissues were detected using the methods of real-time quantitative PCR (RT-qPCR) and Western blot; cell counting kit-8 (CCK-8) assay, wound healing assay, matrigel-based transwell assay and Annexin V-FITC and PI double-staining with flow cytometry assay were used to investigate the biological functions of PRR14 in A431 and HSC-1 cSCC cells. Overexpression of PRR14 in cSCC patients was reported firstly in this study and its high expression was related to differentiation, thickness and tumor node metastasis (TNM) stage of cSCC. PRR14 inhibition with RNA interfering (RNAi) method resulted in the suppression of cell proliferation, migration and invasion but promotion the apoptosis of cSCC cells, and upregulation of the protein phosphorylation levels of mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K) and Akt. The study shows PRR14 maybe an activator of cSCC carcinogenesis through PI3K/Akt/mTOR signal pathway, and it also maybe a prognostic factor and new therapeutical target for cSCC treatment.

LINC00467 induces melanoma deterioration by targeting miR-485-5p/p21 activated kinase 1.

Background: The purpose of the current research was to investigate the biological roles of LINC00467 in inducing melanoma deterioration. Methods: Differential level of LINC00467 in melanoma tissues and its prognostic value were analyzed in GEPIA, which were further confirmed in clinical samples we collected. Regulatory effects of LINC00467 on proliferation, migration and invasion capacities of A375 and SKMEL1 cell lines were examined by a series of functional experiments. Potential downstream targets of LINC00467 were identified through dual-luciferase reporter assay, and their synergistic role in melanoma process was finally explored by rescue experiments. Results: LINC00467 was up-regulated in melanoma samples, but it did not have a prognostic potential in melanoma. LINC00467 has the capacities to stimulate proliferation, migration and invasion of A375 and SKMEL1 cell lines. The feedback loop LINC00467/miR-485-5p/PAK1 was identified, which was responsible for inducing melanoma deterioration. Conclusions: LINC00467 stimulates proliferation, migration and invasion capacities of melanoma via targeting miR-485-5p to upregulate PAK1, which provides potential targets for treatment of melanoma.

Wogonin inhibits the growth of HT144 melanoma via regulating hedgehog signaling-mediated inflammation and glycolysis.

Hedgehog (Hh) signaling has been proved to be closely associated with the occurrence of melanoma. Wogonin is one of the active components of flavonoids that extracts from Scutellariae radix. Previous studies showed that wogonin could inhibit the invasion and migration of B16F10 cells, and suppress the synthesis of melanin in A375 melanoma cells. However, the regulatory effects of Hh signaling in wogonin against melanoma and its potential mechanisms remain largely unknown. The present study aimed to investigate the effect of wogonin on the growth of HT144 melanoma, and to elucidate the role of Hh signaling in wogonin-induced antitumor effects by focusing on inflammation and glycolysis regulation. Wogonin inhibited the proliferation, colony formation and tumor growth of HT144 melanoma cells. Wogonin showed strong anti-inflammatory effect in HT144 melanoma, as shown by the decreased levels of pro-inflammatory factors, the increased level of anti-inflammatory factor and the decreased expression of inflammatory cytokines. Wogonin decreased the glucose consumption and the production of lactic acid and ATP, and decreased the activities of hexokinase (HK), phosphofructokinase(PFK) and pyruvate kinase (PK), and further inhibited the expression of monocarboxylate transporter 1 (MCT-1), MCT-4 and glucosecotransporter-1 (GLUT1), showing potent anti-glycolysis effect against HT144 melanoma. Wogonin inhibited the patched and Smo expression while increased Hhip expression in HT144 cells, suggesting that wogonin blocked the Hh signaling in HT144 cells. The Hh signaling inhibitor cyclopamine, like wogonin, inhibited the colony formation of HT144 cells, however, the inhibitory effect of wogonin on colony formation of HT144 cells was abrogated by the Hh signaling agonist SAG. In addition, SAG abrogated the inhibitory effect of wogonin on the secretion of inflammatory factors and the expression of inflammatory cytokines. Furthermore, SAG abrogated the inhibitory effect of wogonin on several key molecules controlling glycolysis. Overall, these findings suggested that the anti-tumor effect of wogonin can be attributed to the inhibition of Hh signaling-mediated regulation of inflammation and glycolysis in HT144 melanoma.

Photodynamic therapy enhances skin cancer chemotherapy effects through autophagy regulation.

Non-melanotic cutaneous cancers and melanoma are the main common type skin cancers worldwide. Despite many therapeutic options, therapeutic efficacy is not satisfied in all patients with advanced skin cancers, especially in melanoma. Photodynamic therapy (PDT) is a technology for skin disease treatment because of its high effectiveness, has no drug resistance and is easy to use compared with traditional therapy. Our previous study explored that autophagy plays an important role in the formation and development of SCC. But there was no evidence about the association between PDT with autophagy in skin cancers and the mechanism is also still unclear. In the study, we want to explore the effects of 5-aminolevulinic acid-PDT (ALA-PDT) on the skin cancers through autophagy regulation. The result showed that autophagy was regulated by ALA-PDT combined with or without 3-Methyladenine (3-MA) or 5-Fluoracil (5-FU), the proliferation of skin cancer cells A431 and A375 were suppressed while the apoptosis were induced by ALA-PDT and the effects can be enhanced by 3-MA or 5-FU pretreated. The results suggest that autophagy regulation may be a key point of ALA-PDT therapy; ALA-PDT combined with the chemotherapy of 3-FU or 5-FU may be a new strategy for treatment of skin cancers including non-melanotic cutaneous cancers or melanoma.

Inhibition of skin squamous cell carcinoma proliferation and promote apoptosis by dual silencing of NET-1 and survivin.

The simultaneous silencing of multiple upregulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. In the present study, skin squamous cell carcinoma (SSCC) tissue microarray was constructed and the expression of NET-1 and survivin was identified. The high expression of NET-1 and survivin gene in SSCC was confirmed as an important event for the formation and development of the cancer. A total of 100 primary SSCC patients were included in the present study. Expression of NET-1 and survivin in cancer cells was evaluated immunohistochemically in tissue microarrays. The interaction between NET-1 and survivin in SSCC by co-immunoprecipitation was subsequently verified by producing the siRNA sequence targeting the single gene (siRNA-NET-1 and siRNA-survivin) as well as NET-1 and survivin gene (one-chain-double-target siRNA). The levels of NET-1 and survivin mRNA and protein expression in A431 cells were detected by RT-qPCR and western blotting, and the expression of related genes including vascular endothelial growth factor (VEGF), cortactin, Bcl-2, caspase-3 and -8 was identified using RT-qPCR. The protein localization and expression of NET-1 and survivin in A431 cells were documented by immunohistochemistry and immuno-fluorescence staining. The proliferation and apoptosis of A431 cells were detected by CCK-8 assay and flow cytometry (FCM). The tissue microarray showed that NET-1 and survivin were highly expressed in SSCC, while the correlation analysis showed NET-1 expression was positively associated with survivin. In addition, we reported that using the one-chain-double-target siRNA conjugate composed of NET-1 and survivin siRNA sequences in the same backbone inhibited proliferation and promoted apoptosis of SSCC. The one-chain-double-target siRNA showed further downregulation on NET-1 and survivin mRNA and protein levels compared with NET-1 siRNA or survivin siRNA. It also exhibited greater suppression on proliferation and triggering of apoptosis in A431 cells than NET-1 siRNA or survivin siRNA. This result may be explained by the significant downregulation of VEGF, cortactin and Bcl-2, and upregulation of caspase-3 and -8. NET-1 and survivin were overexpressed in SSCC and an interaction between NET-1 and survivin was identified. The one-chain-double-target siRNA appears to be superior in inhibiting cell proliferation and promoting apoptosis compared with the single target siRNA. NET-1 and survivin may have correlative signaling pathways with VEGF, cortactin, Bcl-2, caspase-3 and -8. Simultaneous silencing of NET-1 and survivin using one-chain-double-target siRNA thus provides an advantageous alternative in the development of therapeutics for SSCC.