MicroRNA‐379 suppresses osteosarcoma progression by targeting PDK1

Osteosarcoma is the most common primary bone tumour. Increasing evidence has demonstrated the pathogenic role of microRNA (miRNAs) dysregulation in tumour development. miR‐379 was previously reported to function as an oncogenic or tumour‐suppressing miRNA in a tissue‐dependent manner. However, its function in osteosarcoma remains unknown. In this study, we found that the expression of miR‐379 was downregulated in osteosarcoma tissues and cell lines. Further functional characterization revealed that miR‐379 suppressed osteosarcoma cell proliferation and invasion in vitro and retarded the growth of osteosarcoma xenografts in vivo. Mechanistically, PDK1 was identified as the direct target of miR‐379 in osteosarcoma, in which PDK1 expression was up‐regulated and showed inverse correlation with miR‐379. Enforced expression of PDK1 promoted osteosarcoma cell proliferation and rescued the anti‐proliferative effect of miR‐379. These data suggest that miR‐379 could function as a tumour‐suppressing miRNA via targeting PDK1 in osteosarcoma.     

The lncRNA HNF1A‐AS1 is a negative prognostic factor and promotes tumorigenesis in osteosarcoma

Recent studies have revealed that long noncoding RNA HNF1A‐antisense 1 (HNF1A‐AS1) plays an important role in the development of several human malignancy entities. However, the expression and function of HNF1A‐AS1 in the carcinogenesis and development of osteosarcoma remains unknown. In this study, we detected the HNF1A‐AS1 levels in human osteosarcoma tissues and cell lines by quantitative real‐time polymerase chain reaction (qRT‐PCR), and investigated its role in osteosarcoma by using in vitro assays. Our study showed that HNF1A‐AS1 expression was significantly up‐regulated in human osteosarcoma tissues and cell lines compared with their normal counterparts, and its expression level was positively correlated with the distance metastasis (P = 0.009) and tumour stage (P = 0.019). Moreover, Kaplan–Meier curves with the log‐rank test showed that higher expression of HNF1A‐AS1 conferred a significantly poorer survival and multivariate Cox proportional hazards analysis revealed that HNF1A‐AS1 was an independent risk factor of overall survival. In addition, the expression of HNF1A‐AS1 in serum is correlated with patients’ status and receiver operating characteristic (ROC) curve analysis demonstrated that HNF1A‐AS1 could distinguish patients with osteosarcoma from healthy individuals (the area under curve 0.849, P < 0.001). Furthermore, in vitro knockdown of HNF1A‐AS1 by siRNA significantly inhibited cell proliferation and G₁/S transition, and suppressed migration and invasion by reducing the epithelial‐mesenchymal transition (EMT) program in osteosarcoma cells. Taken together, our data suggested that HNF1A‐AS1 is a novel molecule involved in osteosarcoma progression, which may provide as a potential diagnostic, prognostic biomarker and therapeutic target.     

Knockdown of lncRNA HULC inhibits proliferation,migration, invasion,and promotes apoptosis by sponging miR‐122 in osteosarcoma

Osteosarcoma is a rare malignant bone tumor with high degree of malignancy. HULC (highly upregulated in liver cancer), a long noncoding RNA (lncRNA) was involved in hepatocellular carcinoma development and progression, but its underlying mechanism in osteosarcoma is unknown. The aim of this study was to explore the functional role of HULC in osteosarcoma. The study was conducted in human osteosarcoma cell lines and the expression of HULC in the cell lines was detected by qRT‐PCR. Furthermore, the effects of HULC on tumorigenicity of osteosarcoma cells were evaluated by in vitro assays. Results revealed that HULC was highly expressed in osteosarcoma MG63 and OS‐732 cells compared to osteoblast hFOB1.19 cells. Suppression of HULC in osteosarcoma cells inhibited cell viability, migration, invasion, and promoted apoptosis. HULC functioned as an endogenous sponge for miR‐122, and its silence functioned through upregulating miR‐122. HNF4G was a target of miR‐122, and the effect of HNF4G on OS‐732 cells was the same as HULC. Furthermore, overexpression of miR‐122 inactivated PI3K/AKT, JAK/STAT, and Notch pathways by downregulation of HNF4G. These findings suggest that knockdown of HULC inhibited proliferation, migration, and invasion by sponging miR‐122 in osteosarcoma cells. HULC may act as a novel therapeutic target for management of osteosarcoma.     

HSP70 desensitizes osteosarcoma cells to baicalein and protects cells from undergoing apoptosis

Baicalein is a new drug that has shown promising anti-cancer effects against a broad spectrum of tumors. However, the potential effect on osteosarcoma cells and the mechanisms involved are still largely unknown. Resistance to chemotherapy remains a major obstacle in cancer therapy. Therefore, the aim of the present study was to investigate the anti-tumor effect of baicalein on human osteosarcoma cancer cells and the molecular mechanism involved, as well as identify possible mechanisms of drug resistance. Our results revealed that baicalein-induced apoptosis in osteosarcoma cells was via a mitochondrial pathway involving both caspase-dependent and independent mechanisms. Notably, baicalein treatment upregulated the expression of HSP70, which partially prevented human osteosarcoma cells from undergoing apoptosis. Moreover, it was revealed that HSP70 expression decreased the sensitivity of osteosarcoma cells to baicalein via activation of PI3K/AKT and MAPK/ERK pathways. These results suggest that targeting HSP70-mediated drug resistance, in combination with chemotherapy drugs, may provide novel therapeutic opportunities.     

Protein disulfide isomerase inhibits endoplasmic reticulum stress response and apoptosis via its oxidoreductase activity in colorectal cancer

Protein disulfide isomerase (PDI), a principal endoplasmic reticulum resident oxidoreductase chaperone, is known to play a role in malignancies. This study aims to explore the molecular mechanism by which PDI regulates endoplasmic reticulum stress and the apoptosis signaling pathway in colorectal cancer (CRC). We determined the expression of PDI in CRC tissues and adjacent normal tissues. Gain- and loss- of function assays were conducted to evaluate the effects of PDI on oxidative stress, endoplasmic reticulum stress, and apoptosis in CRC cells, as reflected by hydrogen peroxide (H₂O₂) level and the expression of related proteins. PDI protein expression was upregulated in CRC tissues. Small molecule inhibitor of PDI or PDI knockdown reduced CRC cell viability and induced apoptosis. Overexpression of wild-type PDI augmented the viability of CRC cells and inhibited endoplasmic reticulum stress response and apoptosis. Small molecule inhibitor of PDI or PDI knockdown increased intracellular H₂O₂ level and activated apoptosis signaling pathway, which could be reversed by wild-type PDI restoration. Moreover, the catalytic active site of C-terminal of PDI was found to be indispensable for the regulatory effects of PDI on H₂O₂ levels, apoptosis and cell viability in CRC cells. Collectively, PDI inhibits endoplasmic reticulum stress and apoptosis of CRC cells through its oxidoreductase activity, thereby promoting the malignancy of CRC.     

Theabrownin triggers DNA damage to suppress human osteosarcoma U2OS cells by activating p53 signalling pathway

Osteosarcoma becomes the second leading cause of cancer death in the younger population. Current outcomes of chemotherapy on osteosarcoma were unsatisfactory to date, demanding development of effective therapies. Tea is a commonly used beverage beneficial to human health. As a major component of tea, theabrownin has been reported to possess anti‐cancer activity. To evaluate its anti‐osteosarcoma effect, we established a xenograft model of zebrafish and employed U2OS cells for in vivo and in vitro assays. The animal data showed that TB significantly inhibited the tumour growth with stronger effect than that of chemotherapy. The cellular data confirmed that TB‐triggered DNA damage and induced apoptosis of U2OS cells by regulation of Mki67, PARP, caspase 3 and H2AX, and Western blot assay showed an activation of p53 signalling pathway. When P53 was knocked down by siRNA, the subsequent downstream signalling was blocked, indicating a p53‐dependent mechanism of TB on U2OS cells (p53 wt). Using osteosarcoma cell lines with p53 mutations (HOS, SAOS‐2 and MG63), we found that TB exerted stronger inhibitory effect on U2OS cells than that on p53‐mut cell lines, but it also exerted obvious effect on SAOS‐2 cells (p53 null), suggesting an activation of p53‐independent pathway in the p53‐null cells. Interestingly, theabrownin was found to have no toxicity on normal tissue in vivo and could even increase the viability of p53‐wt normal cells. In sum, theabrownin could trigger DNA damage and induce apoptosis on U2OS cells via a p53‐dependent mechanism, being a promising candidate for osteosarcoma therapy.     

p21‐activated kinase 7 is an oncogene in human osteosarcoma

p21‐activated kinase 7 (PAK7), also named as PAK5, is a member of Rac/Cdc42‐associated Ser/Thr protein kinases. It is overexpressed in some types of cancer such as colorectal and pancreatic cancers. However, the expression status and biological function of PAK7 in osteosarcoma are still ambiguous. To evaluate the expression levels of PAK7 in osteosarcoma tissues and cell lines, immunohistochemistry was used. To investigate the role of PAK7 in cell proliferation, apoptosis and tumorigenicity in vitro and vivo, a recombinant lentivirus expressing PAK7 short hairpin RNA (Lv‐shPAK7) was developed and transfected into Saos‐2 cells. The silencing effect of PAK7 was confirmed by quantitative real‐time PCR (qRT‐PCR) and Western blot technique. PAK7 was overexpressed in osteosarcoma tissue and cell line. By knocking‐down of PAK7, the proliferation and colony formation of Saos‐2 cells were inhibited and apoptosis enhanced significantly. The in vivo tumorigenic ability in xenograft model of Saos‐2 cells was also notably inhibited when PAK7 was knocked down. Our results imply that PAK7 promotes cell proliferation and tumorigenesis and may be an attractive candidate for the therapeutic target of osteosarcoma.     

Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53‐dependent pathway

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53‐wild type U2OS cells (and not of p53‐null Saos and p53‐mutant MG63 cells) by slowing‐down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin‐induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub‐G1 population, Bcl‐2 downregulation, caspase‐3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination‐induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine‐alpha. Moreover, the doxorubicin‐induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53‐dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy. J. Cell. Physiol. 228: 198–206, 2013. © 2012 Wiley Periodicals, Inc.     

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR‐9‐5p/QKI‐5 axis

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT‐PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR‐9‐5p and QKI‐5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK‐8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR‐9‐5p, whereas miR‐9‐5p down‐regulated QKI‐5 expression. Overexpressed MEG3 and QKI‐5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR‐9‐5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up‐regulated expression of QKI‐5 in vivo. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR‐9‐5p and its targeting gene QKI‐5.     

Mechanisms of prostate cancer cell survival after inhibition of AR expression

Recent reports have shown that the AR is the key determinant of the molecular changes required for driving prostate cancer cells from an androgen‐dependent to an androgen‐independent or androgen depletion‐independent (ADI) state. Several recent publications suggest that down‐regulation of AR expression should therefore be considered the principal strategy for the treatment of ADI prostate cancer. However, no valid data is available about how androgen‐dependent prostate cancer cells respond to apoptosis‐inducing drugs after knocking down AR expression and whether prostate cancer cells escape apoptosis after inhibition of AR expression. This review will focus on mechanisms of prostate cancer cell survival after inhibition of AR activity mediated either by androgen depletion or by targeting the expression of AR by siRNA. We have shown that knocking down AR expression by siRNA induced PI3K‐independent activation of Akt, which was mediated by calcium/calmodulin‐dependent kinase II (CaMKII). We also showed that the expression of CaMKII genes is under AR control: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in an elevated level of kinase activity and in enhanced expression of CaMKII genes. This in turn activates the anti‐apoptotic PI3K/Akt pathways. CaMKII also express anti‐apoptotic activity that is independent from the Akt pathway. This may therefore be an important mechanism by which prostate cancer cells escape apoptosis after androgen depletion or knocking down AR expression. In addition, we have found that there is another way to escape cell death after AR inhibition: DNA damaging agents cannot fully activate p53 in the absence of AR and as a result p53 down stream targets, for example, microRNA‐34, cannot be activated and induce apoptosis. This implies that there may be a need for re‐evaluation of the therapeutic approaches to human prostate cancer. J. Cell. Biochem. 106: 363–371, 2009. © 2008 Wiley‐Liss, Inc.     

Inhibition of phosphodiestrase 9 induces cGMP accumulation and apoptosis in human breast cancer cell lines, MCF-7 and MDA-MB-468

Objectives

Phosphodiesterase 9 (PDE9) is a major isoform of phosphodiesterase hydrolysing cGMP and plays a key role in proliferation of cells, their differentiation and apoptosis, via intracellular cGMP signalling. The study described here was designed to investigate expression, activity and apoptotic effect of PDE9 on human breast cancer cell lines, MCF‐7 and MDA‐MB‐468.

Materials and methods

Activity and expression of PDE9 were examined using colorimetric cyclic nucleotide phosphodiesterase assay and real‐time RT‐PCR methods respectively; cGMP concentration was also measured. MTT viability test, annexin V‐FITC staining, Hoechst 33258 staining and caspase3 activity assay were used to detect apoptosis.

Results

Treatment of both cell lines with BAY 73‐6691 lead to reduction in PDE9 mRNA expression, PDE9 cGMP‐hydrolytic activity and elevation of the intracellular cGMP response. BAY 73‐6691 significantly reduced cell proliferation in a dose‐ and time‐dependent manner and caused marked increase in apoptosis through caspase3 activation.

Conclusion

Our results revealed that BAY 73‐6691 induced apoptosis in these breast cancer cell lines through the cGMP pathway. These data suggest that BAY 73‐6691 could be utilized as an agent in treatment of breast cancer.     

Sodium Selenite-Induced Apoptosis Mediated by ROS Attack in Human Osteosarcoma U2OS Cells

Sodium selenite (Na₂SeO₃, SSE) is an inorganic Se compound that is widely used in cancer chemoprevention studies. SSE has been shown to have anti-proliferative effects on several types of human cancer cells, but its effect on osteosarcoma cells has thus far not been reported. In this study, the cytotoxic effect of SSE on osteosarcoma cells U2OS was investigated in vitro and found to be higher than on comparable non-cancer cell lines 293 and L6. Treatment with SSE decreased cell growth in a dose- and time-dependent manner and altered cellular morphology. SSE also inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and accumulation of cells during the advanced phase of apoptosis. SSE-induced apoptosis correlated with the activation of CASP 3, downregulation of BCL-2, and upregulation of P53 and PTEN in U2OS cells. These results indicated that SSE induces apoptosis in U2OS cells mainly through an ROS-mediated caspase pathway. This is the first report to show a possible mechanism of the anti-proliferative effect of SSE for the prevention of osteosarcoma in cell culture models.     

Artocarpin induces cell apoptosis in human osteosarcoma cells through endoplasmic reticulum stress and reactive oxygen species

Osteosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. However, because of side effects and drug resistance in chemotherapy and the insufficiency of an effective adjuvant therapy for osteosarcoma, it is necessary to research novel treatments. This study was the first to investigate the anticancer effects of the flavonoid derivative artocarpin in osteosarcoma. Artocarpin induced cell apoptosis in three human osteosarcoma cell lines—U2OS, MG63, and HOS. Artocarpin was also associated with increased intracellular reactive oxygen species (ROS). Mitochondrial dysfunction was followed by the release of cytochrome c from mitochondria and accompanied by decreased antiapoptotic Bcl-2 and Bcl-xL and increased proapoptotic protein Bak and Bax. Artocarpin triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels and increased glucose-regulated protein 78 and 94 expressions, and also increased calpains expression and activity. Animal studies revealed a dramatic 40% reduction in tumor volume after 18 days of treatment. This study demonstrated a novel anticancer activity of artocarpin against human osteosarcoma cells and in murine tumor models. In summary, artocarpin significantly induced cell apoptosis through ROS, ER stress, mitochondria, and the caspase pathway, and may thus be a novel anticancer treatment for osteosarcoma.     

Drug resistance‐related microRNAs in osteosarcoma: Translating basic evidence into therapeutic strategies

Although the application of multiple chemotherapy brought revolutionary changes to improve overall survival of osteosarcoma patients, the existence of multidrug resistance (MDR) has become a great challenge for successful osteosarcoma treatment in recent decades. Substantial studies have revealed various underlying mechanisms of MDR in cancers. As for osteosarcoma, evidence has highlighted that microRNAs (miRNAs) can mediate in the processes of DNA damage response, apoptosis avoidance, autophagy induction, activation of cancer stem cells, and signal transduction. Besides, these drug resistance‐related miRNAs showed much promise for serving as candidates for predictive biomarkers of poor outcomes and shorter survival time, and therapeutic targets to reverse drug resistance and overcome treatment refractoriness. This review aims to demonstrate the potential molecular mechanisms of miRNAs‐regulated drug resistance in osteosarcoma, and provide insight in translating basic evidence into therapeutic strategies.