Haiyan Hu

MEK inhibitor effective against proliferation in breast cancer cell

The targeted small-molecule drug AZD6244 is an allosteric, ATP-noncompetitive inhibitor of MEK1/2 that has shown activity against several malignant tumors. Here, we report that AZD6244 repressed cell growth and induced apoptosis and G1-phase arrest in the breast cancer cell lines MDA-MB-231 and HCC1937. Using microRNA (miRNA) arrays and quantitative RT-PCR, we found that miR-203 was up-regulated after AZD6244 treatment. In accordance with bioinformatics and luciferase activity analyses, CUL1 was found to be the direct target of miR-203. Furthermore, miR-203 inhibition and CUL1 overexpression reversed the cytotoxicity of AZD6244 on the MDA-MB-231 and HCC1937 cells. Collectively, our data indicate that miR-203 mediates the AZD6244-induced cytotoxicity of breast cancer cells and that the MEK/ERK/miR-203/CUL1 signaling pathway may participate in this process.

Percutaneous Vertebroplasty Combined with Zoledronic Acid for the Treatment of Painful Osteolytic Spinal Metastases in Patients with Breast Cancer

PURPOSE To assess retrospectively the efficacy and safety of percutaneous vertebroplasty (PVP) combined with zoledronic acid (ZA) for the treatment of painful osteolytic spinal metastases from breast cancer. MATERIALS AND METHODS PVP was performed in 43 patients with breast cancer and painful osteolytic spinal metastases; 126 vertebrae were treated. The patients subsequently received 4 mg ZA via a 15-minute intravenous infusion every 4 weeks for 12 months. Pain and quality of life (QoL) were assessed using a visual analog scale (VAS) and Karnofsky performance scale (KPS), respectively, 24 hours before PVP and 24 hours, 1 month, 3 months, 6 months, and 12 months after PVP. Skeletal-related events (SREs) were assessed for 12 months following the intervention. RESULTS The mean VAS scores decreased significantly from 7.6 ± 1.9 at 24 hours before PVP to 3.6 ± 1.4 at 24 hours, 2.0 ± 1.5 at 1 month, 2.8 ± 1.6 at 3 months, 3.1 ± 0.8 at 6 months, and 2.5 ± 0.9 at 12 months after the intervention (P < .05). KPS scores increased significantly after the combination treatment (P < .05). Compared with previous studies without PVP or ZA treatment, this patient group had a lower incidence of SREs. No major complications were observed. CONCLUSIONS PVP combined with ZA was shown to be a highly effective and safe combination therapy to relieve pain and improve QoL in patients with osteolytic spinal metastases from breast cancer. The combination therapy also prevented the occurrence of SREs.

Modulation of miR-185-5p expression by EBV-miR-BART6 contributes to developmental differences in ABCG4 gene expression in human megakaryocytes.

Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by low platelet count and bleeding, and is usually triggered by viral infections. We previously reported that 14 viral microRNAs of megakaryocytes cultured with serum from patients with ITP, including ebv-miR-BART6, are up-regulated. Previous research has reported that ebv-miR-BART6 down-regulated the expression of miR-185-5p. We therefore predicted that the ABCG4 gene, which is highly expressed in megakaryocyte progenitor cells, is a direct target of miR-185-5p. We hypothesized that ebv-miR-BART6 may play a role in development and differentiation of megakaryocytes. First, we verified the negative regulation of ABCG4 by miR-185-5p through luciferase assay analysis. Second, after transfection of ebv-miR-BART6 into megakaryocytes developing from normal cord blood mononuclear cells (MNCs), we found that the level of miR-185-5p in the ebv-miR-BART6 group was reduced to almost a third of that in the control groups, accompanied by up-regulation of ABCG4 at both the mRNA and protein levels. Meanwhile, proliferation of megakaryocytes was significantly repressed in the ebv-miR-BART6 group compared with the blank and negative control groups (14.89%±3.13%, 34.15%±2.42% and 30.96%±4.37%, respectively; P<0.001). Our results further revealed that ebv-miR-BART6 inhibited megakaryocyte colony unit formation, decreased CD41 expression and inhibited megakaryocyte polyploidization. These data suggest a new paradigm to explain the mechanisms underlying ITP, involving the regulation of megakaryocytopoiesis by viral microRNAs through the intronic hsa-microRNA.

Bufalin Inhibits Proliferation and Induces Apoptosis in Osteosarcoma Cells by Downregulating MicroRNA-221

Bufalin, a major component of the Chinese medicine ChanSu, which is prepared from the skin and parotid venom glands of toads, has shown cytotoxicity in several malignant tumors. Here, we reported that bufalin inhibited proliferation and induced mitochondria-dependent apoptosis in U-2OS and Saos-2 osteosarcoma cells with intracellular reactive oxygen species (ROS) production. By microRNA (miR) array analysis and quantitative reverse transcription polymerase chain reaction, we found that miR-221 was downregulated after treatment with bufalin. In accordance with TargetScan prediction and luciferase reporter assay, Bcl2 binding component 3 (BBC3) was the direct target of miR-221. Furthermore, upregulating miR-221 by its MIMIC and suppressing BBC3 by small interfering RNA (siRNA) reversed the effects of bufalin on osteosarcoma cells. Collectively, our data indicate that bufalin inhibits cell proliferation and induces mitochondria-dependent apoptosis in osteosarcoma cells through downregulating miR-221 and triggering BBC3 expression.

Elevated expression of serine/threonine phosphatase type 5 correlates with malignant proliferation in human osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in adolescents and young adults. However, the involvement of serine/threonine phosphatase type 5 (PP5) in osteosarcoma remains unclear. The aim of this study was to evaluate the functional role of PP5 in osteosarcoma cells. Firstly, we found that PP5 is widely expressed in several human osteosarcoma cell lines. Then we used lentivirus-delivered siRNA to silence PP5 expression in Saos-2 and U2OS cell lines. Knockdown of endogenous PP5 expression by shRNA-expressing lentivirus significantly decreased the viability and proliferation of the osteosarcoma cells. Moreover, FACS analysis showed that knockdown of PP5 expression induced a significant arrest in the G0/G1 phase of the cell cycle, which was associated with the inhibition of cell proliferation. Therefore, knockdown of PP5 is likely to provide a novel alternative to targeted therapy of osteosarcoma and deserves further investigation.

Selumetinib suppresses cell proliferation, migration and trigger apoptosis, G1 arrest in triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC) has aggressive progression with poor prognosis and ineffective treatments. Selumetinib is an allosteric, ATP-noncompetitive inhibitor of MEK1/2, which has benn known as effective antineoplastic drugs for several malignant tumors. We hypothesized that Selumetinib might be potential drug for TNBC and explore the mechanism.MethodsAfter treated with Selumetinib, the viability and mobility of HCC1937 and MDA-MB-231 were detected by MTT, tunnel, wound-healing assay, transwell assay and FCM methods. MiR array was used to analysis the change of miRs. We predicted and verified CUL1 is the target of miR-302a using Luciferase reporter assay. We also silenced the CUL1 by siRNA, to clarify whether CUL1 take part in the cell proliferation, migration and regulated its substrate TIMP1 and TRAF2. Moreover, after transfection, the antagomir of miR-302a and CUL1 over-expressed plasmid into HCC1937 and MDA-MB-231 cell accompanied with the Selumetinib treatment, we detected the proliferation and migration again.ResultsSelumetinib reduce the proliferation, migration, triggered apoptosis and G1 arrest in TNBC cell lines. In this process, the miR-302a was up-regulated and inhibited the CUL1 expression. The later negatively regulated the TIMP1 and TRAF2. As soon as we knockdown miR-302a and over-expression CUL1 in TNBC cells, the cytotoxicity of Selumetinib was reversed.ConclusionsMiR-302a targeted regulated the CUL1 expression and mediated the Selumetinib-induced cytotoxicity of triple-negative breast cancer.

PAK5 overexpression is associated with lung metastasis in osteosarcoma

p21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases, which are associated with cytoskeletal organization, cellular morphogenesis, migration and survival. PAKs are overactive in a number of tumor tissues and have attracted attention as a potential target for cancer therapy. In the present study, PAK5 levels were analyzed in primary osteosarcoma (OS) samples (n=65) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) methods. In the primary OS tissue, increased PAK5 expression (IHC score >2, n=37) was associated with significantly decreased overall survival (P=0.036) compared with decreased PAK5 expression (IHC score ≤2, n=28). PAK5 expression was identified to be significantly associated with metastasis (P=0.010). The lung is the most common metastasis site for OS. In addition, the level of PAK5 in lung metastasis tissue (n=13) was detected using RT-qPCR and IHC methods. PAK5 expression was increased in lung metastasis tissue compared with in primary OS samples. PAK5 was silenced using short hairpin RNA in OS cell lines. Wound healing, migration and nude mice model assay results consistently demonstrated that PAK5 knockdown was able to significantly inhibit OS migration. In PAK5-knockdown cells, the alteration in the expression of a number of metastasis-associated factors, including epithelial cadherin, vimentin, fibronectin and matrix metalloproteinase 2 (MMP2), was analyzed. Only MMP2 expression was decreased significantly (P<0.05). The expression level of MMP2 was analyzed in primary OS tissue and lung metastasis tissue using RT-qPCR and IHC methods. Expression of MMP2 was identified to be associated with expression of PAK5. The results of the present study suggest that PAK5 promotes OS cell migration and that PAK5 expression may be used to predict lung metastasis.

Safety and efficacy of multilevel vertebroplasty for painful osteolytic spinal metastases: a single-centre experience

ObjectiveTo retrospectively assess the safety and efficacy of percutaneous vertebroplasty (PVP) for painful osteolytic spinal metastases when treating more than three vertebrae per session.MethodsA total of 153 patients with painful osteolytic spinal metastases underwent PVP. Group A patients (n = 93) underwent PVP at up to three vertebral levels per session. Group B patients (n = 60) underwent PVP at more than three levels in one session. Pain, quality of life (QoL), and mobility were assessed before and after PVP. Minor and major complications were systematically assessed.ResultsBoth groups experienced significant pain relief and QoL improvement after the intervention (p < 0.001). Mobility improvement was observed in both groups, despite worse mobility status before PVP in group B compared with group A. There was no significant difference between the two groups throughout the follow-up period in overall pain relief and improvement in QoL and mobility. There was also no significant difference between groups in minor and major complications.ConclusionsMultilevel vertebroplasty is safe and effective for the treatment of multiple osteolytic spinal metastases. Multilevel PVP relieves pain and improves QoL and mobility.Key Points• Percutaneous vertebroplasty is safe and effective for painful osteolytic spinal metastases.• Multilevel vertebroplasty does not cause more complications than single-level vertebroplasty.• Multiple spinal metastases patients may regain functional independence after multilevel vertebroplasty.

Knockdown of ubiquitin-specific peptidase 39 inhibited the growth of osteosarcoma cells and induced apoptosis in vitro

BackgroundUbiquitin specific peptidase 39 (USP39), an essential factor in the assembly of the mature spliceosome complex, has an aberrant expression in several cancer. However, its function and the corresponding mechanism on human osteosarcoma has not been fully explored yet.MethodsThe mRNA and DNA copies of USP39 were increased in osteosarcoma cancer tissues compared with the one in human normal tissues according to datasets from the publicly available Oncomine database. A further western blot analysis also demonstrated an aberrant endogenous expression of USP39 in three different osteosarcoma cells. Then lentivirus-mediated short hairpin RNA (shRNA) was designed to silence USP39 in human osteosarcoma cell line U2OS, which is used to test the impact of USP39-silencing on cellular proliferation, colony formation, cell cycle distribution and apoptosis.ResultsKnockdown of USP39 expression in U2OS cell significantly decreased cell proliferation, impaired colony formation ability. A further analysis indicated suppression of USP39 arrested cell cycle progression at G2/M phase via p21 dependent way. In addition, the results of Annexin V/7-AAD staining suggested the knockdown of USP39 could promote U2OS cell apoptosis through PARP cleavage.ConclusionsThese results uncover the critical role of USP39 in regulating cancer cell mitosis and indicate USP39 is critical for osteosarcoma tumorigenesis.

Monitoring cancer stem cells: insights into clinical oncology.

Cancer stem cells (CSCs) are a small, characteristically distinctive subset of tumor cells responsible for tumor initiation and progression. Several treatment modalities, such as surgery, glycolytic inhibition, driving CSC proliferation, immunotherapy, and hypofractionated radiotherapy, may have the potential to eradicate CSCs. We propose that monitoring CSCs is important in clinical oncology as CSC populations may reflect true treatment response and assist with managing treatment strategies, such as defining optimal chemotherapy cycles, permitting pretreatment cancer surveillance, conducting a comprehensive treatment plan, modifying radiation treatment, and deploying rechallenge chemotherapy. Then, we describe methods for monitoring CSCs.