Hengyuan Li

Piperine inhibits proliferation of human osteosarcoma cells via G2/M phase arrest and metastasis by suppressing MMP-2/-9 expression

The piperidine alkaloid piperine, a major ingredient in black pepper, inhibits the growth and metastasis of cancer cells both in vivo and in vitro, although its mechanism of action is unclear. Furthermore, its anticancer activity against osteosarcoma cells has not been reported. In this study, we show that piperine inhibited the growth of HOS and U2OS cells in dose- and time-dependent manners but had a weaker effect on the growth of normal hFOB cells. Piperine inhibited osteosarcoma cell proliferation by causing G2/M phase cell cycle arrest associated with decreased expression of cyclin B1 and increased phosphorylation of Cyclin-dependent kinase-1(CDK1) and checkpoint kinase 2 (Chk2). In addition, piperine treatment inhibited phosphorylation of Akt and activated phosphorylation of c-Jun N-terminal kinase (c-JNK) and p38 mitogen-activated protein kinase (MAPK) in HOS and U2OS cells. Piperine induced colony formation in these two cell types. We proved that piperine could suppress the metastasis of osteosarcoma cells using scratch migration assays and Transwell chamber tests. Moreover, gelatin zymography showed that piperine inhibited the activity of matrix metalloproteinase (MMP)-2/-9 and increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1/-2. Taken together, our results indicate that piperine inhibits proliferation, by inducing G2/M cell cycle arrest, and the migration and invasion of HOS and U2OS cells, via increased expression of TIMP-1/-2 and down-regulation of MMP-2/-9. These findings support further study of piperine as a promising therapeutic agent in the treatment of osteosarcoma.

Prognostic value of pathologic fracture in patients with high grade localized osteosarcoma: a systemic review and meta-analysis of cohort studies.

Consensus has not been reached regarding the ability of pathologic fracture to predict local recurrence and survival in osteosarcoma. We aim to review the available evidence to examine the association between pathologic fracture and osteosarcoma prognosis. A comprehensive literature search for relevant studies published until March 2014 was performed using PubMed, Cochrane and Web of Science. The studies investigating pathologic fracture of osteosarcoma patients were systematically analyzed. The overall relative risk (RR) was estimated using a fixed‐effect model or random‐effect model according to heterogeneity between the trials. We included nine cohort studies involving 2,187 patients (311 with pathologic fracture and 1,876 without fracture) for the analysis of survival rate and local recurrence. Studies were assessed for quality using the Newcastle–Ottawa Assessment Scale. In the fixed‐effects model, the meta‐analysis showed that pathologic fracture in osteosarcoma patients predicted poor 3‐year overall survival (OS) (RR = 1.86, 95% CI: 1.37–2.53, p < 0.001) and 5‐year OS (RR = 1.34, 95% CI: 1.06–1.70, p = 0.016). Similarly, pathologic fracture was significantly correlated with worse 3‐year event free survival (EFS) (RR = 1.52, 95% CI: 1.21–1.92, p < 0.001) and 5‐year EFS (RR = 1.24, 95% CI: 1.03–1.49, p = 0.021), whereas no significant association was noted with local recurrence (RR = 1.30, 95% CI: 0.84–2.02, p = 0.233). The meta‐analysis confirmed that pathologic fracture in osteosarcoma was a prognostic marker for both OS and EFS but not for local recurrence.

SIRT1 promotes metastasis of human osteosarcoma cells.

Pulmonary metastasis is the leading cause of mortality in patients with osteosarcoma; however, the underlying mechanism remains unclear. The NAD+-dependent deacetylase, sirtuin 1 (SIRT1), has been reported to play a key role in carcinogenesis through deacetylation of important regulatory proteins. Here, we report that SIRT1 promotes osteosarcoma metastasis by regulating the expression of metastatic-associated genes. The SIRT1 protein was significantly upregulated in most primary osteosarcoma tumours, compared with normal tissues, and the SIRT1 expression level may be coupled with metastatic risk in patients with osteosarcoma. Moreover, the results of cell migration and wound-healing assays further suggested that higher expression of SIRT1 promoted invasive activity of osteosarcoma cells. Importantly, downregulating SIRT1 with shRNA inhibited the migration ability of osteosarcoma cells in vitro and suppressed tumour lung metastasis in mice. Finally, a gene expression analysis showed that knockdown of SIRT1 profoundly activated translation of its downstream pathway, particularly at migration and invasion. In summary, high levels of SIRT1 may be a biomarker for a high metastatic rate in osteosarcoma patients; inhibiting SIRT1 could be a potent therapeutic intervention for these patients.

The role of CTLA-4 and PD-1 in anti-tumor immune response and their potential efficacy against osteosarcoma

Immunotherapy is proved to be a promising therapeutic strategy against human malignancies. Evasion of immune surveillance is considered to be a major factor of malignant progression. Inhibitory receptors, especially CTLA-4 and PD-1, are found to play critical roles in the mediation of anti-tumor immune efficacy. Thus, antibodies targeting these immune checkpoints have emerged as the attractive treatment approaches to those patients with cancer. Osteosarcoma is highly malignant and current treatment remains a challenge, especially for those patients with metastasis. Despite some achievements, the effect of immunotherapy against osteosarcoma is still unsatisfactory. The present review attempts to show the role and mechanism of CTLA-4 and PD-1 in immune response and summarize the recent findings related to the effect of inhibitory receptor antibodies on the immune response against tumors, especially osteosarcoma, and the correlation between PD-1 or/and CTLA-4 expression and outcome of osteosarcoma patients. We further discuss the utilization of the combination therapy against osteosarcoma.

Trastuzumab enhanced the cytotoxicity of Vγ9Vδ2 T cells against zoledronate-sensitized osteosarcoma cells.

Preliminary studies of Vγ9Vδ2 T cells and zoledronate (ZOL) present promising reasons to exploit their immunotherapeutic potential for osteosarcoma treatment (OS). ZOL is a third-generation aminobisphosphonate (ABP) and is well established in the management of cancer-induced bone disease. However, ZOL is characterized by high tropism for bone matrix, and the efficacy of ZOL for sensitizing tumors remains to be optimized. Vγ9Vδ2 T cells are important effectors of antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we investigated whether Vγ9Vδ2 T cell-mediated killing of ZOL-pretreated OS cells could be increased by the anti-HER-2 monoclonal antibody trastuzumab (TTZ). The cytotoxic activity of Vγ9Vδ2 T cells against osteosarcoma was assessed by an MTS assay in the presence or absence of TTZ. A CD107a assay was used to measure degranulation in cytotoxic Vγ9Vδ2 T cells. Blocking studies were used to determine the effect of relative ligands on Vγ9Vδ2 T cell recognition. TTZ induced an ADCC response in the OS cell line, U2OS; however, it had no effect on another OS cell line HOS with low levels of surface HER2 expression. Although the OS cells pretreated with ZOL for clinically relevant time periods (2hours) stimulated a suboptimal immune response of Vγ9Vδ2 T cells, TTZ could further enhance the cytotoxicity of Vγ9Vδ2 T cells. These results demonstrate that combining TTZ and ZOL significantly increases the cytotoxic potential of Vγ9Vδ2 T cells. This study raises the possibility of utilizing ZOL and TTZ in Vγ9Vδ2 T cell-based immunotherapy for OS.

Prognostic role of hypoxia-inducible factor-1 alpha expression in osteosarcoma: a meta-analysis

Background Hypoxia-inducible factor-1α (HIF-1α) plays an important role in tumor progression and metastasis. A number of studies have investigated the association of HIF-1α with prognosis and clinicopathological characteristics of osteosarcoma but yielded inconsistent results. Method Systematic computerized searches were performed in PubMed, Embase, and Web of Science databases for relevant original articles. The pooled hazard ratios (HRs) and odds ratios (ORs) with corresponding confidence intervals (CIs) were calculated to assess the prognostic value of HIF-1α expression. The standard mean difference was used to analyze the continuous variable. Results Finally, nine studies comprising 486 patients were subjected to final analysis. Protein expression level of HIF-1α was found to be significantly related to overall survival (HR =3.0; 95% CI: 1.46–6.15), disease-free survival (HR =2.23; 95% CI: 1.26–3.92), pathologic grade (OR =21.33; 95% CI: 4.60–98.88), tumor stage (OR =10.29; 95% CI: 3.55–29.82), chemotherapy response (OR =9.68; 95% CI: 1.87–50.18), metastasis (OR =5.06; 95% CI: 2.87–8.92), and microvessel density (standard mean difference =2.83; 95% CI: 2.28–3.39). Conclusion This meta-analysis revealed that overexpression of HIF-1α is a predictive factor of poor outcomes for osteosarcoma. HIF-1α appeared to play an important role in prognostic evaluation and may be a potential target in antitumoral therapy.

Analysis of Chemotherapy Dosage and Dosage Intensity and Survival Outcomes of High-Grade Osteosarcoma Patients Younger Than 40 Years

BACKGROUND Chemotherapy is essential for long-term survival of osteosarcoma patients. However, the impact of dosage and dosage intensity (DI) of chemotherapeutic agents on patients with high-grade osteosarcoma is largely unknown. OBJECTIVE The object of this study was to evaluate the influence of these dosage-related variables on treatment outcomes in terms of event-free survival (EFS). METHODS PubMed was searched for relevant English-language articles. Two reviewers extracted data independently. Sufficient data were presented to calculate the planned total dosage and DI for doxorubicin, cisplatin, ifosfamide, and methotrexate. Univariate analysis and partial regression analysis were performed to determine the association of 5-year EFS and the planned total dosage and DI of each drug. RESULTS Seventeen studies comprising 23 trial arms met the inclusion criteria. The analysis recruited a total of 2257 patients. The study period ranged from 1976 to 2006. Using univariate analysis, the planned dosage and DI of methotrexate and ifosfamide correlated with better 5-year EFS (P = 0.001 for methotrexate dosage; P = 0.030 for ifosfamide dosage; P < 0.001 for methotrexate DI; and P = 0.033 for ifosfamide DI). There was a trend toward worse 5-year EFS with increase of doxorubicin DI (P = 0.055). Based on the partial regression analysis, the association of doxorubicin DI and ifosfamide dosage and DI with EFS became no longer statistically significant, and the planned total dosage and DI of methotrexate remained significantly correlated with better 5-year EFS (P = 0.001 and P = 0.004, respectively). CONCLUSIONS We observed a correlation of higher planned methotrexate total dosage and DI with better treatment outcomes in osteosarcoma patients. The present study showed that methotrexate dosage and DI were important predictors of the clinical outcomes and provided the rationale of high-dosage methotrexate in the context of multi-agent chemotherapy.

Recurrence of Giant Cell Tumor of the Spine after Resection: A Report of 10 Cases: Recurrence of SGCT

To review the clinical details and further treatments for recurrent spinal giant cell tumors (SGCT), and to analyze the risk factors of recurrence and shed new light on the treatment options and prognosis of recurrent SGCT.

Decitabine Enhances Vγ9Vδ2 T Cell-Mediated Cytotoxic Effects on Osteosarcoma Cells via the NKG2DL–NKG2D Axis

γδ T cell-based immunotherapy for osteosarcoma (OS) has shown limited success thus far. DNA-demethylating agents not only induce tumor cell death but also have an immunomodulatory function. In this study, we have assessed the potential benefit of combining decitabine (DAC, a DNA demethylation drug) and γδ T cells for OS immunotherapy. DAC increased the expression of natural killer group 2D (NKG2D) ligands (NKG2DLs), including major histocompatibility complex class I-related chains B (MICB) and UL16-binding protein 1 (ULBP1), on the OS cell surface, making the cells more sensitive to recognition and destruction by cytotoxic γδ T cells. The upregulation of MICB and ULBP1 was due to promoter DNA demethylation. Importantly, the killing of OS cells by γδ T cells was partially reversed by blocking the NKG2D receptor, suggesting that the γδ T cell-mediated cytolysis of DAC-pretreated OS cells was mainly dependent on the NKG2D–NKG2DL axis. The in vivo results were consistent with the in vitro results. In summary, DAC could upregulate MICB and ULBP1 expression in OS cells, and combination treatment involving γδ T cell immunotherapy and DAC could be used to enhance the cytotoxic killing of OS cells by γδ T cells.

Valproic Acid Combined with Zoledronate Enhance γδ T Cell-Mediated Cytotoxicity against Osteosarcoma Cells via the Accumulation of Mevalonate Pathway Intermediates

The long-term survival of osteosarcoma has remained unchanged in the last several decades. Immunotherapy is proved to be a promising therapeutic strategy against osteosarcoma, especially for those with metastasis. Our previous study explored the sensibilization of zoledronate (ZOL) in γδ T cell-mediated cytotoxicity against osteosarcoma, but we have not yet elucidated the specific mechanism. Besides, high concentration is required to achieve these effects, whereas plasma ZOL concentration declines rapidly in the circulation. Valproic acid (VPA), a histone deacetylase inhibitor commonly used as the antiepileptic drug, has attracted much attention due to its synergistic antitumor efficacy with chemotherapy or immunotherapy. Here, we demonstrated that VPA combined with ZOL revealed the synergistic effect in enhancing antitumor efficacy of γδ T cells against osteosarcoma cells. This enhancement was mainly TCR-mediated and largely dependent on granule exocytose pathway. Of note, our findings indicated that ZOL sensitized osteosarcoma cells to γδ T cells by increasing the accumulation of the mevalonate pathway intermediates, which could be facilitated by VPA. We also found that this combination had similar effects on primary osteosarcoma cells. All the results suggested that VPA combined with ZOL could reduce the dose required to achieve a significant antitumor effect of γδ T cells, promoting it to be a novel therapy against osteosarcoma.