Peiheng He

Pristimerin Inhibits Prostate Cancer Bone Metastasis by Targeting PC-3 Stem Cell Characteristics and VEGF-Induced Vasculogenesis of BM-EPCs

Background/Aims: Prostate cancer (PCa) is one of the most common malignant cancers and a major leading cause of cancer deaths in men. Cancer stem-like cells are shown to be highly tumorigenic, pro-angiogenic and can significantly contribute to tumor new vessel formation and bone marrow derived-EPCs (BM-EPCs) are shown to recruit to the angiogenic switch in tumor growth and metastatic progression, suggesting the importance of targeting cancer stem cells (CSCs) and EPCs for novel tumor therapies. Pristimerin, an active component isolated from Celastraceae and Hippocrateaceae, has shown anti-tumor effects in some cell lines in previous studies. However, the effect and mechanism of Pristimerin on CSCs and EPCs in PCa bone metastasis are not well studied. Methods: The effect of Pristimerin on PC-3 stem cell characteristics and metastasis were detected by spheroid formation, CD133 and CD44 protein expression, matrix-gel invasive assay and colony-formation assay in vitro, VEGF and pro-inflammatory cytokines expression by ELISA assay, and tumor tumorigenicity by X-ray and MR in NOD-SCID mice model in vivo. In addition, we also detected the effect of Pristimerin on VEGF-induced vasculogenesis and protein expression of BM-EPCs. Results: Pristimerin could significantly inhibit spheroid formation and protein expression of CD133 and CD44, reduce VEGF and pro-inflammation cytokines expression of PC-3 cell, and prevent the xenografted PC-3 tumor growth in the bone of nude mice. The present data also showed that Pristimerin significantly inhibited VEGF-induced vasculogenesis of BM-EPCs by suppressing the EPCs functions including proliferation, adhesion, migration, tube formation and inactivation the phosphorylation of VEGFR-2, Akt and eNOS. Conclusion: These data provide evidence that Pristimerin has strong potential for development as a novel agent against prostate bone metastasis by suppressing PC-3 stem cell characteristics and VEGF-induced vasculogenesis of BM-EPCs.

Inhibitory action of pristimerin on hypoxia‑mediated metastasis involves stem cell characteristics and EMT in PC-3 prostate cancer cells

The aim of the present study was to investigate whether pristimerin affects the bone metastasis, stem cell characteristics and epithelial-mesenchymal transition (EMT) of prostate cancer (PCa) PC-3 cells subjected to hypoxia. The PC-3 cells were cultured under hypoxia or normoxia for 48 h and were then treated with increasing concentrations of pristimerin from 0 to 0.8 µmol/l, under normoxia. Hypoxia‑inducible factor-1α (HIF-1α) was detected by western blotting. Proliferation was assessed with the CCK-8 assay. Transwell invasion assay was used to analyze the potency of invasion. Stem cell characteristics were detected by sphere formation, colony formation assay and western blotting, including CD44, KLF4, OCT4 and AGO2, which are stem cell characteristic-related markers. EMT was confirmed by the expression changes of EMT-related markers, including N-cadherin, fibronectin, vimentin and ZEB1, which were evaluated by western blotting. The addition of pristimerin to the medium reduced the hypoxia-induced PC-3 cell proliferation in a dose-dependent manner. Pristimerin effectively inhibited hypoxia‑induced invasion of the PCa cells in vitro. Moreover, the treatment of cells with pristimerin induced the reversal of hypoxia-induced stem cell characteristics and EMT, which was confirmed by sphere formation, colony formation assay and the expression changes of CSC- and EMT-related markers. The reversal of hypoxia‑induced stem cell characteristics and EMT in the PCa cells by low-dose pristimerin was dose‑dependent. These results showed that treatment with pristimerin may be a potential strategy for the suppression of hypoxia-induced metastasis through the reversal of hypoxia-induced stem cell characteristics and EMT in cancer cells, which justifies the potential use of pristimerin as a practical chemopreventive approach for patients with PCa.

Coenzyme Q10 Prevents the Interleukin-1 Beta Induced Inflammatory Response via Inhibition of MAPK Signaling Pathways in Rat Articular Chondrocytes: COENZYME Q10 PREVENTS IN RAT ARTICULAR CHONDROCYTES

Preclinical Research

Downregulation of miR‑505‑3p predicts poor bone metastasis‑free survival in prostate cancer

The principal issue deriving from prostate cancer (PCa) is its propensity to metastasize to bone. To date, bone metastasis remains incurable, and therapeutic strategies are limited. Therefore, it is of paramount importance to explore predictive markers for bone metastasis of PCa. In the present study, we reported that miR-505-3p was significantly downregulated in bone metastatic PCa tissues compared with that in non-bone metastatic PCa tissues, but there was no significant difference in miR-505-3p expression between PCa and adjacent normal tissues. miR-505-3p expression was inversely associated with serum PSA levels, Gleason grade, N and M classification, and short bone metastasis-free survival in PCa patients, but had no effect on overall survival in PCa patients. Furthermore, upregulation of miR-505-3p suppressed the activity of TGF-β signaling by directly targeting downstream effectors of TGF-β signaling, SMAD2 and SMAD3, further inhibiting the invasion and migration abilities of PCa cells. Therefore, our findings unraveled a novel mechanism by which miR-505-3p inhibits bone metastasis of PCa, supporting the notion that miR-505-3p may serve as a predictive marker for bone metastasis of PCa.

Downregulation of miR-133a-3p promotes prostate cancer bone metastasis via activating PI3K/AKT signaling

BackgroundBone metastasis is a leading cause of morbidity and mortality in advanced prostate cancer (PCa). Downexpression of miR-133a-3p has been found to contribute to the progression, recurrence and distant metastasis in PCa. However, clinical significance of miR-133a-3p in bone metastasis of PCa, and the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa remain unclear.MethodsmiR-133a-3p expression was evaluated in 245 clinical PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-133a-3p expression and clinicopathological features, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-133a-3p in the bone metastasis of PCa were investigated both in vitro and in vivo. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to demonstrate the relationship between miR-133a-3p and its potential targets. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the anti-tumor role of miR-133a-3p. Clinical correlation of miR-133a-3p with its targets was verified in human PCa tissues.ResultsmiR-133a-3p expression is reduced in PCa tissues compared with the adjacent normal tissues and benign prostate lesion tissues, particularly in bone metastatic PCa tissues. Low expression of miR-133a-3p is significantly correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients by statistical analysis. Moreover, upregulating miR-133a-3p inhibits cancer stem cell-like phenotypes in vitro and in vivo, as well as attenuates anoikis resistance in vitro in PCa cells. Importantly, administration of agomir-133a-3p greatly suppresses the incidence of PCa bone metastasis in vivo. Our results further demonstrate that miR-133a-3p suppresses bone metastasis of PCa via inhibiting PI3K/AKT signaling by directly targeting multiple cytokine receptors, including EGFR, FGFR1, IGF1R and MET. The negative clinical correlation of miR-133a-3p with EGFR, FGFR1, IGF1R, MET and PI3K/AKT signaling activity is determined in clinical PCa tissues.ConclusionOur results unveil a novel mechanism by which miR-133a-3p inhibits bone metastasis of PCa, providing the evidence that miR-133a-3p may serve as a potential bone metastasis marker in PCa, and delivery of agomir-133a-3p may be an effective anti-bone metastasis therapeutic strategy in PCa.

Transcriptional downregulation of miR-133b by REST promotes prostate cancer metastasis to bone via activating TGF-β signaling

High avidity of bone metastasis is an important characteristic in prostate cancer (PCa). Downexpression of miR-133b has been reported to be implicated in the development, progression and recurrence in PCa. However, clinical significance and biological roles of miR-133b in bone metastasis of PCa remain unclear. Here we report that miR-133b is downregulated in PCa tissues and further decreased in bone metastatic PCa tissues. Downexpression of miR-133b positively correlates with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-133b inhibits invasion, migration in vitro and bone metastasis in vivo in PCa cells. Mechanistically, we find that miR-133b suppresses activity of TGF-β signaling via directly targeting TGF-β receptor I and II, which further inhibits bone metastasis of PCa cells. Our results further reveal that overexpression of REST contributes to miR-133b downexpression via transcriptional repression in PCa tissues. Importantly, silencing miR-133b enhances invasion and migration abilities in vitro and bone metastasis ability in vivo in REST-silenced PCa cells. The clinical correlation of miR-133b with TGFBRI, TGFBRII, REST and TGF-β signaling activity is verified in PCa tissues. Therefore, our results uncover a novel mechanism of miR-133b downexpression that REST transcriptionally inhibits miR-133b expression in PCa cells, and meanwhile support the notion that administration of miR-133b may serve as a rational regimen in the treatment of PCa bone metastasis.

Downregulation of miR‑19a‑3p promotes invasion, migration and bone metastasis via activating TGF‑β signaling in prostate cancer

Constitutive activation of TGF-β signaling pathway is a well-documented mechanism responsible for the bone metastasis of prostate cancer (PCa). MicroRNAs (miRNAs) have been reported to be crucial for the activation of TGF-β signaling via targeting downstream components of TGF-β signaling pathway. Here, we report that miR-19a-3p is downregulated in bone metastatic PCa tissues and cells. Upregulation of miR-19a-3p suppresses invasion, migration in vitro and inhibits bone metastasis in vivo in PCa cells. Conversely, silencing miR-19a-3p yields the opposite effect. Our results further demonstrate that miR-19a-3p inhibits invasion and migration abilities of PCa cells via targeting downstream effectors of TGF-β signaling, SMAD2 and SMAD4, resulting in the inactivation of TGF-β signaling. Therefore, our results uncover a novel mechanistic understanding of miR-19a-3p-induced suppressive role in bone metastasis of PCa, which will facilitate the development of effective cancer therapy methods against PCa.

Acidic stress induces apoptosis and inhibits angiogenesis in human bone marrow‑derived endothelial progenitor cells

Bone marrow-derived endothelial progenitor cells (BM-EPCs) are exposed to acidotic environments in a variety of physiological and pathological conditions, including in tumors. However, the effect of acidosis on the function of BM-EPCs is still not fully understood. In the present study, BM-EPCs were isolated and cultured at an extracellular pH (pHe) of 6.5 or pHe 7.4 in vitro prior to various experiments being performed. Cellular proliferation, migration and tube formation ability were detected by DNA content quantification, Transwell assay and Matrigel-based angiogenesis assay. ELISA and western blot analysis measured protein secretion and expression, respectively. The results demonstrated that BM-EPCs cultured at pHe 6.5 compared with at pHe 7.4 demonstrated: Induced apoptosis; inhibited cellular proliferation, migration and adhesion; markedly reduced vascular endothelial growth factor (VEGF) expression; and the capacity to incorporate into vascular networks. Acidic pHe 6.5 induced ratio expression of B-cell lymphoma 2 (Bcl2)/Bcl2 associated X-protein (Bax), which in turn induced apoptosis, and inhibited cellular proliferation and other functional activities, with involvement of activation of VEGF receptor 2, protein kinase B and p38 mitogen activated protein kinase. These observations raise the possibility that the acidic extracellular environment may perform an important role in the vasculogenesis of BM-EPCs in tumor microenvironments. Therefore, culturing cancer cells at a lower pH that simulates endogenous tumor conditions may improve retention of the cellular heterogeneity identified in tumors.

miR-30b regulates chondrogenic differentiation of mouse embryo‑derived stem cells by targeting SOX9

The present study aimed to investigate the mechanisms underlying microRNA (miRNA)-mediated regulation of chondrogenic differentiation. Mouse embryo-derived stem cells C3H10T1/2 were cultured and chondrogenic differentiation was induced using transforming growth factor-β3 (TGF-β3). In addition, miRNA expression profiles were detected via miRNA array analysis, and quantitative polymerase chain reaction was performed to verify the differentially expressed miRNAs. Furthermore, bioinformatics software was used to predict the putative targets and the prediction was validated by dual-luciferase reporter assays and western blot analysis. In addition, cell proliferation and glycosaminoglycans were measured by a direct cell count method and alcian blue staining, respectively. Compared with the control group, 86 miRNAs were identified as differentially expressed in TGF-β3-induced cells and the expression levels of 28 miRNAs were increased while the remaining 58 miRNAs exhibited a decline in expression. Amongst the differentially expressed miRNAs, miR-30b expression was observed to have significantly decreased during chondrogenic differentiation. SOX9 is a target gene of miR-30b, and miR-30b inhibits SOX9 expression during chondrogenic differentiation. Furthermore, the alcian blue staining results demonstrated that miR-30b inhibited early chondrogenic differentiation. However, the data of the present study indicated that miR-30b had no influence on C3H10T1/2 cell line proliferation. In conclusion, miR-30b is a key negative regulator of TGF-β3-induced C3H10T1/2 cell chondrogenic differentiation, which functions by directly targeting SOX9.

Relationship between the tibial mechanical axis and bony anatomical landmarks of the calf and foot as measured on radiographs obtained with a new laser-calibrated position

OBJECTIVE To investigate relationship between the tibial mechanical axis and bony landmarks of the calf and foot by developing a new laser-calibrated position for radiography of the lower limb. MATERIALS AND METHODS A total of 120 volunteers were randomly divided into two groups. All subjects were marked with skin projection of the hypothetical axis of the calf on the frontal and sagittal planes. Radiographs of weight-bearing full-length lower-limb were obtained by the laser-calibrated positioning in the experimental group, and by the use of conventional technique in the control group. To consider the rotation of the calf, radiological features of the knee and ankle were investigated. The relationship between the tibial mechanical axis and the bony landmarks of the calf and foot were also measured. RESULTS Anteroposterior view depicted a tangential projection on the superior/inferior tibiofibular syndesmosis and between lateral malleolus and talus in ankle mortise in the experimental group. Bony overlap on the superior/inferior tibiofibular syndesmosis and between lateral malleolus and talus was seen in control group. On the tangential projection, it also presented a clear wheel-like contour of the medial femoral condyle, but a partial overlap between medial femoral condyle and tibial plateau. The femoral joint angle between the connecting line at the lowest point of the medial and lateral femoral condyles and the tibial mechanical axis was 83.6° ± 2.49° in the experimental group and 85.3° ± 2.18° in the control group (P < 0.001). The tibial tubercle-axis distance from the center of the medial and middle one-third of the tibial tubercle to the tibial mechanical axis was 1.5 mm in the experimental group and 3.7 mm in the control group (P < 0.05). The malleoli-axis distance from the midpoint of the bimalleolar line joining the tips of the medial and lateral malleoli to the tibial mechanical axis was 1.9 mm in the experimental group and 6.9 mm in the control group (P < 0.001). Lateral view showed no difference between the tibial mechanical axis and the fibular reference line within two groups. CONCLUSION In the new radiographic position, our data indicate that the hypothetical tibial mechanical axis and fibular reference line, obtained by marking the specified anatomic landmarks of the calf and foot, are located more closely to the tibial mechanical axis by correcting the rotation of the calf and foot during the radiography. On anteroposterior view, the tibial mechanical axis was approximately 2 mm medial to the center of the ankle. This indicates that the hypothetical tibial mechanical axis marked on the skin could be referred to guide the tibial osteotomy in total knee arthroplasty.