Intelligent triggering of nanomicelles based on a ROS-activated anticancer prodrug and photodynamic therapy (PDT)-synergistic therapy for lung cancers.

The intelligent triggering of drug release at targeted sites is essential for the safety and efficacy of cancer therapies. This study aimed to design and synthesize a novel prodrug (DHA-S-CA) using a reactive oxygen species (ROS)-responsive moiety, thioacetal, to bridge cinnamaldehyde (CA) and dihydroartemisinin (DHA). As ROS are highly expressed in tumor tissues, the design uses the ROS-responsive moiety as an effective target for the nanodrug delivery system. Furthermore, the near-infrared dye IR808 and the prodrug were adopted to prepare co-loaded Soluplus®/TPGS nanomicelles (IR808/DHA-S-CA NMs). The photosensitized agent IR808 exhibited both tumor accumulation and cancer imaging properties while generating ROS during laser irradiation. Intracellular ROS detection indicated that the prodrug DHA-S-CA could degrade via the high concentration of ROS in cancer cells induced by laser irradiation, and the released CA stimulated mitochondria to regenerate additional ROS to further improve the antitumor effect of DHA. Combined with photodynamic therapy (PDT), IR808/DHA-S-CA (+) NMs outperformed free DHA, DHA NMs, and IR808/DHA-S-CA (-) in a comparison of their pharmacokinetic profiles because it had a longer circulation time and a greater area under the curve (AUC). Compared with other DHA groups, the ROS-responsive IR808/DHA-S-CA (+) micelles had comparable cytotoxic activity. Furthermore, the ROS-responsive IR808/DHA-S-CA (+) micelles exhibited markedly higher anticancer efficiency on lung cancer cells than the other DHA groups. Overall, these results indicated that the therapeutic strategy of our novel small-molecule prodrug combined with PDT has great potential for the treatment of tumors.

Development of Biomimetic Hepatic Lobule-Like Constructs on Silk-Collagen Composite Scaffolds for Liver Tissue Engineering.

Constructing an engineered hepatic lobule-mimetic model is challenging owing to complicated lobular architecture and crucial hepatic functionality. Our previous study has demonstrated the feasibility of using silk fibroin (SF) scaffolds as functional templates for engineering hepatic lobule-like constructs. But the unsatisfactory chemical and physical performances of the SF-only scaffold and the inherent defect in the functional activity of the carcinoma-derived seeding cells remain to be addressed to satisfy the downstream application demand. In this study, SF-collagen I (SFC) composite scaffolds with improved physical and chemical properties were fabricated, and their utilization for bioengineering a more hepatic lobule-like construct was explored using the immortalized human hepatocyte-derived liver progenitor-like cells (iHepLPCs) and endothelial cells incorporated in the dynamic culture system. The SFC scaffolds prepared through the directional lyophilization process showed radially aligned porous structures with increased swelling ratio and porosity, ameliorative mechanical stiffness that resembled the normal liver matrix more closely, and improved biocompatibility. The iHepLPCs displayed a hepatic plate-like distribution and differentiated into matured hepatocytes with improved hepatic function in vitro and in vivo. Moreover, hepatocyte-endothelial cell interphase arrangement was generated in the co-culture compartment with improved polarity, bile capillary formation, and enhanced liver functions compared with the monocultures. Thus, a more biomimetic hepatic lobule-like model was established and could provide a valuable and robust platform for various applications, including bioartificial liver and drug screening.

Honokiol Prodrug Nanoparticles Based on In Situ Albumin Binding for Long Circulation and High Tumor Uptake.

Honokiol (HK) has antiproliferation effects against numerous cancer cells, but its low solubility and bioavailability impede its application. In this study, a prodrug of HK (HP) featuring a maleimide group was synthesized and then mixed with tocopherol polyethylene glycol succinate to prepare prodrug nanoparticles (HP-NPs). In vitro albumin binding experiments showed that HP rapidly reacted with the cysteine thiols of albumin to form a covalent conjugate that released HK slowly in the LLC tumor cell line. In vitro cell apoptosis and uptake assays showed that the cellular uptake of the HK increased into the LLC cells as the albumin concentration increased. Strikingly, in vivo pharmacokinetics and pharmacodynamics measurements demonstrated that the HP-NPs significantly prolonged the circulation and increased tumor accumulation. Taken together, our study demonstrated, both in vitro and in vivo, that the albumin-based HP-NPs delivery system holds significant potential toward the treatment of lung cancer in clinical studies.

miR-206 as a prognostic and sensitivity biomarker for platinum chemotherapy in epithelial ovarian cancer.

BACKGROUND: Drug resistance is a major obstacle to successful chemotherapy for epithelial ovarian cancer (EOC). We found a subset of miRNAs associated with the response to first-line platinum-based chemotherapy in EOC by microarray, and miR-206 was one of the most significant miRNAs. The purposes of this study were to evaluate the prognostic and platinum-resistance predictive value of miR-206 in EOC patients and to investigate the functional roles of miR-206 in regulating the platinum resistance of EOC and the underlying mechanism. METHODS: MiRNA expression profiling in EOC specimens was performed using a TaqMan miRNA array. miR-206 expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. Overexpression of miR-206 in EOC cell lines was achieved by the stable transfection of a recombinant plasmid. In vitro assays of cisplatin cytotoxicity, cell cycle distribution, apoptosis, transwell invasion and cell scratching were employed. Connexin 43 (Cx43) expression was detected by Western blotting. Murine xenograft models were used to determine the effects of miR-206 on platinum resistance in vivo. RESULTS: miR-206 expression was increased in primary platinum-resistant EOC. High miR-206 expression was related to poor prognosis in EOC patients who received platinum-based chemotherapy and predicted chemoresistance to platinum treatment. Overexpression of miR-206 in cisplatin-sensitive EOC cell lines significantly increased cell viability, migration and invasion in the presence of cisplatin and decreased cisplatin-induced apoptosis. Cx43, a target gene of miR-206, was negatively regulated by miR-206 in EOC cell lines and significantly related to better prognosis in patients who received platinum-based chemotherapy (KmPlot). miR-206 had high expression and Cx43 had low expression in platinum-sensitive EOC cell lines compared with resistant ones. In vivo murine xenograft models showed that miR-206 profoundly promoted the chemoresistance of EOC to cisplatin treatment. CONCLUSION: miR-206 was highly expressed in primary platinum-resistant EOCs and functionally promoted platinum resistance in part by downregulating Cx43 expression, thereby providing a useful biomarker for prognostic and platinum-resistance prediction.

Fenretinide-polyethylene glycol (PEG) conjugate with improved solubility enhanced cytotoxicity to cancer cell and potent in vivo efficacy.

Fenretinide (4-HPR), a synthetic retinoid, has shown its antitumor activity in many tumor types with low cytotoxicity to normal cells and high clinical safety. However, the low water solubility limits its further biological applications. To increase solubility, 4-HPR was conjugated with methoxy polyethylene glycol carboxylic acid (mPEG2K-COOH) by an ester linkage between the phenol hydroxyl of 4-HPR and the carboxyl of mPEG2K-COOH. The 4-HPR-PEG2K conjugate micelles had mean size of 76.70 ± 1.248 nm with a narrow distribution and a low critical micelle concentration. In vitro cytotoxicity studies showed the micelles have higher cytotoxicity to A2780s and MCF-7 cells. Its IC50 was 4.7 and 4.1-fold lower than the free 4-HPR, respectively. Importantly, in vivo pharmacokinetic studies, the AUC of 4-HPR was found to be 2.3-fold higher in 4-HPR-PEG2K micelles compared to free 4-HPR. And the 4-HPR-PEG2K micelles had higher antitumor activity. Meanwhile, the histopathology analysis exhibited that the micellar treatment decreased the viability of A2780s cells and increased the level of induced apoptosis. Therefore, the enhanced activity of 4-HPR by the method of conjugation with mPEG2K-COOH could hopefully provide new insights into the matter of ovarian cancer and breast cancer treatment.

Mixed micelles of TPGS and Soluplus® for co-delivery of paclitaxel and fenretinide: in vitro and in vivo anticancer study.

Fenretinide (4-HPR), as a semi-synthetic retinoid, has apoptosis-promoting effects as a single agent and chemotherapy synergist in vitro. When a human ovarian cancer cells line (A2780s) was treated with both PTX and 4-HPR, there was a synergistic anti-cancer effect demonstrated with a average combination index of 0.44. In this research, a new TPGS-Soluplus® mixed micelles were developed which encapsulation efficiencies of paclitaxel (PTX) and fenretinide (4-HPR) were as high as 98%, and the average diameter of the micelles was 66.26 nm. Cytotoxicity of the mixed micelles co-delivered with PTX and 4-HPR reduced significantly 7.3 and 25.1 times compared with free drug respectively in A2780s cells. More importantly, in vivo pharmacokinetic study, the loaded drugs in mixed micelles exhibited higher AUC and t1/2 values than free drugs. Furthermore, in vivo antitumor efficacy experiments demonstrated that PF-TS exhibited superior in vivo antitumor activity on the inhibition rate of tumor growth than other treatment groups (77.8% corresponding tumor growth inhibition in PF-TS treated group vs 19.9, 12.5, and 26.0% of tumor growth inhibition rate in Taxol®, 4-HPR, and Taxol®+4-HPR, respectively). Therefore, the mixed micelles of co-deliver PTX and 4-HPR successfully constructed may hopefully be applied to the cancer combination treatment with less toxic effect and more antitumor activity.

Soluplus®/TPGS mixed micelles for co-delivery of docetaxel and piperine for combination cancer therapy.

In this study, mixed micelles of Soluplus® and TPGS were developed for co-administering docetaxel (DTX) and piperine (PIP) for exerting the synergistic effect, which increased the cytotoxicity and improved the anti-cancer activity in HepG2 cell lines compared to free DTX. These in vitro (MTT assay, intracellular uptake of micelles) and in vivo (pharmacokinetic study, immunostaining, TUNEL analysis) studies exhibited the advantages of co-delivery of anticancer drugs with Soluplus®/TPGS by mixed micelles and furthermore established that co-delivery of DTX and PIP via the mixed micelles of Soluplus®/TPGS could be a promising strategy for the treatment of liver cancer.

Dihydroartemisinin and doxorubicin co-loaded Soluplus®-TPGS mixed micelles: formulation characterization, cellular uptake, and pharmacodynamic studies.

Clinically, co-delivery of chemotherapeutics has been limited by poor water-solubility and severe systemic toxicity. This study was aimed at integrating the merits of combination chemotherapy and mixed micellar technology and demonstrating the anticancer potential of doxorubicin (DOX) and dihydroartemisinin (DHA) co-loaded Soluplus®-TPGS mixed micellar system. In this study, physiochemically stable multidrug loaded mixed micelles were successfully prepared, encapsulation efficiencies of DOX and DHA were as high as 90%, and the average diameter of the micelles was 64.27 nm. The cellular uptake of DOX from the mixed micelles increased by 1.3 and 1.2 times for MCF-7 and MCF-7/ADR cell lines, respectively. The micelles were more cytotoxic than free DHA-DOX. Surprisingly, the co-loaded mixed micelles exhibited higher antitumor activity, while the systemic toxicity was reduced during the treatment. Therefore, the DOX and DHA mixed micelle might be a potential, effective, and less toxic drug-delivery system for cancer therapy.

Three-dimensional tissue culture model of human breast cancer for the evaluation of multidrug resistance.

Multidrug resistance (MDR) is one of the major obstacles to improving outcomes of chemotherapy in tumour patients. However, progress has been slow to overcome this phenomenon due to the limitations of current cell/tissue models in recapitulating MDR behaviour of tumour cells in vitro. To address this issue, a more pathologically relevant, three-dimensional (3D) culture of human breast cancer cells was developed by seeding the adriamycin-resistant cells MCF-7R in silk-collagen scaffolds. The cultures of the parental cell line MCF-7 served as controls. Distinct growth profiles of MCF-7R and MCF-7 cells were observed when they were cultured in the scaffolds in comparison with those in the monolayer culture, including cell proliferation, cellular aggregate formation, and expression of drug resistance-related genes/proteins. Moreover, the 3D cultures of these cell lines especially the cultures of MCF-7R exhibited a significantly enhanced drug resistance evidenced by their increased IC50 values to the anticancer drugs and improved drug efflux capability. An altered cell cycle distribution and improved percentage of breast cancer stem cell (BCSC)-like cells was also found in the present study. This might play an important role in promoting the drug-resistance production in those 3D cultures. Thus, we established improved 3D cultures of MDR human breast cancer. It would provide a robust tissue model for use to evaluate the efficacy of anticancer drugs, explore mechanisms of MDR, and enrich BCSCs in vitro.

Development and evaluation of a novel drug delivery: Soluplus®/TPGS mixed micelles loaded with piperine in vitro and in vivo.

BACKGROUND: Although piperine can inhibit cells of tumors, the poor water solubility restricted its clinical application. This paper aimed to develop mixed micelles based on Soluplus® and D-α-tocopherol polyethylene glycol succinate (TPGS) to improve the aqueous solubility and anti-cancer effect. METHODS: Piperine-loaded mixed micelles were prepared using a thin-film hydration method, and their physicochemical properties were characterized. The cellular uptake of the micelles was confirmed by confocal laser scanning microscopy in A549 lung cancer cells and HepG2 liver cancer cells. In addition, cytotoxicity of the piperine mixed micelles was studied in A549 lung cancer cells and HepG2 liver cancer cells. Free piperine or piperine-loaded Soluplus®/TPGS mixed micelles were administered at an equivalent dose of piperine at 3.2 mg/kg via a single intravenous injection in the tail vain for the pharmacokinetic study in vivo. RESULTS: The diameter of piperine-loaded Soluplus®/TPGS (4:1) mixed micelles was about 61.9 nm and the zeta potential -1.16 ± 1.06 mV with 90.9% of drug encapsulation efficiency and 4.67% of drug-loading efficiency. Differential scanning calorimetry (DSC) studies confirmed that piperine is encapsulated by the Soluplus®/TPGS. The release results in vitro showed that the piperine-loaded Soluplus®/TPGS mixed micelles presented sustained release behavior compared to the free piperine. The mixed micelles exhibited better antitumor efficacy compared to free piperine and physical mixture against in A549 and HepG2 cells by MTT assay. The pharmacokinetic study revealed that the AUC of piperine-loaded mixed micelles was 2.56 times higher than that of piperine and the MRT for piperine-loaded mixed micelles was 1.2-fold higher than piperine (p < .05). CONCLUSION: The results of the study suggested that the piperine-loaded mixed micelles developed might be a potential nano-drug delivery system for cancer chemotherapy. These results demonstrated that piperine-loaded Soluplus®/TPGS mixed micelles are an effective strategy to deliver piperine for cancer therapy.

Soluplus/TPGS mixed micelles for dioscin delivery in cancer therapy.

BACKGROUND: Dioscin has shown cytotoxicity against cancer cells, but its poor solubility and stability have limited its clinical application. In this study, we designed mixed micelles composed of TPGS and Soluplus® copolymers entrapping the poorly soluble anticancer drug dioscin. METHOD: In order to improve the aqueous solubility and bioactivity of dioscin, TPGS/Soluplus® mixed micelles with an optimal ratio were prepared using a thin-film hydration method, and their physicochemical properties were characterized. Cellular cytotoxicity and uptake of the dioscin-loaded TPGS/Soluplus® mixed micelles were studied in MCF-7 breast cancer cells and A2780s ovarian cancer cells. The pharmacokinetics of free dioscin and dioscin-loaded TPGS/Soluplus® mixed micelles was studied in vivo in male Sprague-Dawley rats via a single intravenous injection in the tail vein. RESULTS: The average size of the optimized mixed micelle was 67.15 nm, with 92.59% drug encapsulation efficiency and 4.63% drug loading efficiency. The in vitro release profile showed that the mixed micelles presented sustained release behavior compared to the anhydrous ethanol solution of dioscin. In vitro cytotoxicity assays were conducted on human cancer cell lines including A2780s ovarian cancer cells and MCF-7 breast cancer cells. The mixed micelles exhibited better antitumor activity compared to free dioscin against all cell lines, which may benefit from the significant increase in the cellular uptake of dioscin from mixed micelles compared to free dioscin. The pharmacokinetic study showed that the mixed micelle formulation achieved a 1.3 times longer mean residual time (MRT) in circulation and a 2.16 times larger area under the plasma concentration-time curve (AUC) than the free dioscin solution. CONCLUSION: Our results suggest that the dioscin-loaded mixed micelles developed in this study might be a potential nano drug-delivery system for cancer chemotherapy.

MiR-770-5p inhibits cisplatin chemoresistance in human ovarian cancer by targeting ERCC2.

In this study, we examined the role of the miRNA miR-770-5p in cisplatin chemotherapy resistance in ovarian cancer (OVC) patients. miR-770-5p expression was reduced in platinum-resistant patients. Using a 6.128-fold in expression as the cutoff value, miR-770-5p expression served as a prognostic biomarker and predicted the response to cisplatin treatment and survival among OVC patients. Overexpression of miR-770-5p in vitro reduced survival in chemoresistant cell lines after cisplatin treatment. ERCC2, a target gene of miR-770-5p that participates in the NER system, was negatively regulated by miR-770-5p. siRNA-mediated silencing of ERCC2 reversed the inhibition of apoptosis resulting from miR-770-5p downreglation in A2780S cells. A comet assay confirmed that this restoration of cisplatin chemosensitivity was due to the inhibition of DNA repair. These findings suggest that endogenous miR-770-5p may function as an anti-oncogene and promote chemosensitivity in OVC, at least in part by downregulating ERCC2. miR-770-5p may therefore be a useful biomarker for predicting chemosensitivity to cisplatin in OVC patients and improve the selection of effective, more personalized, treatment strategies.

Nanoparticles with Optimal Ratiometric Co-Delivery of Docetaxel with Gambogic Acid for Treatment of Multidrug-Resistant Breast Cancer.

It has been reported that gambogic acid (GA), the main active component of gamboge, could directly inhibit and reduce the expression level of P-gp by promoting protein degradation through post-translational proteasome pathway. In this study, the optimal molar ratio of GA/docetaxel (DTX) that could recover the sensitivity of MCF-7/ADR cells to DTX was firstly investigated. Then GA and DTX were loaded simultaneously in PLGA nanoparticles in terms of the optimal ratio. In vitro cell apoptosis and western-blot assays showed that co-delivery of anticancer drugs resulted in enhanced cell apoptosis through the downregulation of the expression level of P-gp. Interestingly, in vivo pharmacokinetic study demonstrated that GA and DTX are released synchronously in blood from the NPs. Finally, the most effective tumor growth inhibition in the MCF-7/ADR human breast tumor xenograft was observed in the co-delivery nanoparticle formulation group in comparison with saline control, free DTX solution and free DTX/GA solution. Taken together, our study demonstrated that DTX/GA PLGA NPs based combination therapy holds significant potential towards the treatment of multidrug-resistant breast cancer.

Profile of differentially expressed miRNAs in high-grade serous carcinoma and clear cell ovarian carcinoma, and the expression of miR-510 in ovarian carcinoma.

Improved insight into the molecular and genetic profile of different types of epithelial ovarian cancer (EOC) is required for understanding the carcinogenesis of EOC and may potentially be exploited by future targeted therapies. The aim of the present study was to identify a unique microRNA (miRNA) patterns and key miRNAs, which may assist in predicting progression and prognosis in high­grade serous carcinoma (HGSC) and clear cell carcinoma (CCC). To identify unique miRNA patterns associated with HGSC and CCC, a miRNA microarray was performed using Chinese tumor bank specimens of patients with HGSC or CCC in a retrospective analysis. The expression levels of four deregulated miRNAs were further validated using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) in an external cohort of 42 cases of HGSC and 36 cases of CCC. Kaplan­Meier analysis was performed to analyze the correlation between the expression levels of the four miRNAs and patient prognosis. Among these validated miRNAs, miR­510 was further examined in another cohort of normal ovarian tissues, as well as the HGSC, low­grade serous carcinoma (LGSC) and CCC specimens using RT­qPCR and in situ hybridization. The results revealed that, of the 768 miRNAs analyzed in the microarray, 33 and 50 miRNAs were significantly upregulated and downregulated, respectively, with at least a 2­fold difference in HGSC, compared with CCC. The quantitative analysis demonstrated that miR­510 and miR­129­3p were significantly downregulated, and that miR­483­5p and miR­miR­449a were significantly upregulated in CCC, compared with HGSC (P<0.05), which was consistent with the microarray results. Kaplan­Meier analysis revealed low expression levels of miR­510 and low expression levels of miR­129­3p, advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymphatic metastasis and that HGSC was significantly associated with the poorer overall survival rates (P<0.05). The expression of miR­510 was significantly higher in the LGSC and CCC tissues, compared with the HGSC and normal ovarian tissues. The results of the present study suggested that different subtypes of EOC have specific miRNA signatures, and that miR­510 may be involved differently in HGSC and CCC. Thus, miR­510 and miR­129­3p may be considered as potential novel candidate clinical biomarkers for predicting the outcome of EOC.

Expression of miR-136 is associated with the primary cisplatin resistance of human epithelial ovarian cancer.

MicroRNAs (miRNAs) are involved in regulating the response of cancer cells to various therapeutic interventions, yet their involvement in the chemoresistance of human epithelial ovarian cancer is not fully understood. We found that miR-136 was significantly downregulated in specimens from patients with chemoresistant epithelial ovarian cancer. In the present study, we aimed to clarify the role of miR-136 in regulating the chemoresistance of ovarian cancer. Thirty-four tumor bank specimens and 2 well-established human ovarian cancer cell lines, C13 and OV2008, were used. We found that miR-136 expression was significantly reduced in primary platinum-resistant patients and the ovarian cancer OVC cell line. Enforced expression of miR-136 decreased the chemoresistance to cisplatin in OVC cells through inhibition of cell survival. In addition, we found no association between miR-136 and migration or invasion potential in the ovarian cancer cell lines. However, in the platinum-resistant C13 cell line, the overexpression of miR-136 markedly promoted an apoptotic response to cisplatin. Furthermore, the levels of adducts corrected with their extent of DNA damage/repair, in terms of the percentage of DNA in comet tails, tail length, tail moment (TM), and olive tail moment (OTM), revealed that miR-136 is essential for the repair of cisplatin-induced DNA damage. Our findings suggest that miR-136 may function as an anti-oncogene and deficiency of miR-136 expression in ovarian cancer can induce chemoresistance at least in part by downregulating apoptosis and promoting the repair of cisplatin-induced DNA damage. Thus, miR-136 may provide a biomarker for predicting the chemosensitivity to cisplatin in patients with epithelial ovarian cancer.

MiRNA expression signature for potentially predicting the prognosis of ovarian serous carcinoma.

One of the best prognostic predictors for patients with epithelial ovarian cancer is the Federation of Obstetrics and Gynecology (FIGO) stage at diagnosis. Advanced-stage ovarian serous carcinoma (OSC) generally have poor prognosis. The goal of this study is to develop and validate a miRNA expression profile that can differentiate the OSC at early and advanced stages and study its correlation with the prognosis of OSC. To identify a unique microRNA (miRNA) pattern associated with the progression of OSC at early and advanced stages, a miRNA microarray was performed using Chinese tumor bank specimens of patients with OSC stage I or III in a retrospective analysis. The expression of four dysregulated miRNAs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in an external cohort of 51 cases of OSC samples at stages I and III. Kaplan-Meier analysis was performed to analyze the correlation between the expression of some miRNAs and prognosis. Of the 768 miRNAs analyzed in the microarray, 26 miRNAs were significantly either up- or downregulated, with at least a 2-fold difference, in OSC stage I compared with stage III. The qRT-PCR results showed that miR-510, miR-509-5p, and miR-508-3p were significantly downregulated and that miR-483-5p was upregulated in stage III OSC compared with stage I, which was consistent with the microarray results. Kaplan-Meier analysis showed low miR-510 expression, low miR-509-5p expression, and advanced FIGO stage, and chemotherapy resistance were significantly associated with poorer overall survival (P < 0.05). Our results suggest that miRNAs may play a role in the progression of OSC, and miR-510 and miR-509-5p may be considered novel-candidate clinical biomarkers for predicting OSC outcome.

miRNA expression pattern associated with prognosis in elderly patients with advanced OPSC and OCC.

The long-term survival for elderly patients with advanced ovarian papillary serous carcinoma (OPSC) does not exceed 30%, and the incidence and prognosis rise continuously after menopause. The aim of this study was to identify the differences in key miRNAs and their potential regulators through miRNA microarray analysis, functional target prediction, and clinical outcome between the elderly patients with advanced OPSC and ovarian clear cell carcinoma (OCC) who all suffered poor prognosis, to identify the pathogenetic basis, and to improve the understanding of the molecular basis of advanced OPCS in elderly patients. Through microarray analysis, we found 52 unique miRNAs with significant fold­change in expression levels, of which 9 were upregulated, whereas 43 were downregulated in OCC patients compared to elderly OPSC patients with advanced stage. Among these prediction miRNAs, miR-30a, miR-30e and miR-505 were found to have some common cancer-related targets. Lower expression of these three miRNAs of advanced OPSC in elderly patients, all associated with significantly poorer survival rate, strongly suggesting that they could be critical oncogenes and take important roles in OPSC etiology in elderly patients with advantaged stage. Functional analyses support the above hypothesis. Their targets, ATF3, STMN1 and MYC suggest that OPSC also has aggressive biological behavior when presented with advanced stage, and support the epidemiology results that incidence and mortality of advanced OPSC increases continuously. miR-30a, miR-30e and miR-505 may be important pathogenetic factors for OPSC in elderly patients with advanced stage. Age could be regarded as a continuous covariate in this process. This may improve the understanding of molecular underpinnings of advanced OPSC in elderly patients, and provide improved diagnostic, prognostic and therapeutic approaches.

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained.

MicroRNA 376c enhances ovarian cancer cell survival by targeting activin receptor-like kinase 7: implications for chemoresistance.

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in gene regulation. We have previously reported that activin receptor-like kinase 7 (ALK7) and its ligand, Nodal, induce apoptosis in human epithelial ovarian cancer cells. In this study, we examined the regulation of ALK7 by miRNAs and demonstrate that miR-376c targets ALK7. Ectopic expression of miR-376c significantly increased cell proliferation and survival, enhanced spheroid formation and blocked Nodal-induced apoptosis. Interestingly, overexpression of miR-376c blocked cisplatin-induced cell death, whereas anti-miR-376c enhanced the effect of cisplatin. These effects of miR-376c were partially compensated by the overexpression of ALK7. Moreover, in serous carcinoma samples taken from ovarian cancer patients who responded well to chemotherapy, strong ALK7 staining and low miR-376c expression was detected. By contrast, ALK7 expression was weak and miR-376c levels were high in samples from patients who responded poorly to chemotherapy. Finally, treatment with cisplatin led to an increase in expression of mRNA encoding Nodal and ALK7 but a decrease in miR-376c levels. Taken together, these results demonstrate that the Nodal-ALK7 pathway is involved in cisplatin-induced cell death in ovarian cancer cells and that miR-376c enhances proliferation, survival and chemoresistance by targeting, at least in part, ALK7.