Zhongli Shi

Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells

Nano-hydroxyapatite (nano-HAP) may be a better candidate for an apatite substitute of bone in biomedical applications than micro-sized hydroxyapatite (m-HAP). However, size control is always difficult when synthesizing well-defined nano-HAP particles. In this study, nano-HAP particles with diameters of approximately 20nm (np20) and approximately 80nm (np80) were synthesized and characterized. The size effects of these nano-HAPs and m-HAP were studied on human osteoblast-like MG-63 cells in vitro. Our results demonstrate that both cell proliferation and cell apoptosis are related to the size of the HAP particles. Np20 has the best effect on promotion of cell growth and inhibition of cell apoptosis. This work provides an interesting view of the role of nano-HAPs as ideal biomedical materials in future clinical applications.

Matrine induces caspase-dependent apoptosis in human osteosarcoma cells in vitro and in vivo through the upregulation of Bax and Fas/FasL and downregulation of Bcl-2

PurposeMatrine, one of the main active components of extracts from the dry roots of Sophora flavescens, has potent anti-tumor activity in various cancer cell lines. However, the activity of matrine against osteosarcoma remains unclear. In the present study, we examined the effects of matrine on human osteosarcoma cells and explored the underlying mechanism.MethodsFour human osteosarcoma cell lines: MG-63, U-2OS, Saos-2, and MNNG/HOS were treated by matrine and subjected to MTT assay, annexin V-FITC/PI double staining, and TUNEL assay. The activation of caspases and the expression of pro-apoptotic and anti-apoptotic factors were examined by qRT-PCR and Western blot. In addition, MNNG/HOS xenograft tumors were established in female nude BALB/c mice, and matrine was intraperitoneally (i.p.) administered to evaluate the anti-cancer capacity of matrine in vivo.ResultsWe found that matrine inhibited the proliferation and induced apoptosis of the four osteosarcoma cell lines in vitro and induced the activation of caspase-3, -8, and -9 in a dose-dependent manner. Furthermore, the pro-apoptotic factors Bax and Fas/FasL were upregulated, and the anti-apoptotic Bcl-2 was downregulated. More importantly our in vivo, studies showed that administration of matrine decreased tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated the downregulation of Bcl-2 and upregulation of Bax and Fas/FasL in MNNG/HOS tumor tissues following matrine treatment, consistent with the in vitro results.ConclusionOur results demonstrate that matrine inhibits the proliferation and induces apoptosis of human osteosarcoma cells in vitro and in vivo. The induction of apoptosis appears to occur through the upregulation of Fas/FasL and Bax, downregulation of Bcl-2, and activation of caspase-3, -8, and -9, which then trigger major apoptotic cascades.

Celecoxib, a cyclooxygenase-2 inhibitor, induces apoptosis in human osteosarcoma cell line MG-63 via down-regulation of PI3K/Akt

Cyclooxygenase‐2 (COX‐2), involved in the inhibition of apoptosis and, the potentiation of cell growth, is frequently overexpressed in human malignancies including osteosarcoma (OS). We have attempted to identify the anti‐proliferation of celecoxib, a selective COX‐2 inhibitor, and the combination of celecoxib and cisplatin in MG‐63 cells, and to explore the potential molecular mechanisms involved. MG‐63 cells were treated with the combination of celecoxib and cisplatin or either agent alone for 48 h in serum‐supplemented medium. Celecoxib caused G1 phase arrest and significantly inhibited cell growth, as well as potentiating cisplatin‐induced apoptosis. The effect was dose‐dependent, and apoptotic changes such as DNA fragments and apoptotic bodies were observed. However, downregulation of COX‐2 did not occur in cells treated with celecoxib. Phosphoinositide‐3‐kinase (PI3K)/Akt, survivin, bcl‐2 were significantly downregulated in cells treated with the combination of celecoxib and cisplatin, and decreased survivin and bcl‐2 levels were found in cells with wortmannin, a specific PI3K inhibitor. Moreover, the decreased expressions of procaspase‐9, procaspase‐3 and cleaved PARP‐1 were detected by Western blot analysis. Therefore, celecoxib exerts its anti‐tumor activities through COX‐2‐independent mechanisms, which may be PI3K/Akt‐dependent, and survivin and bcl‐2‐related. PI3K may be at the center of the celecoxib effects, which play an essential role in the regulation of survivin and Bcl‐2.

Biological Response of Osteosarcoma Cells to Size-Controlled Nanostructured Hydroxyapatite

Osteosarcoma is a primary malignant bone tumor, most prevalent in children and adolescents, and is usually highly aggressive and eventually lethal. Despite multimodal therapies, there is no effective approach to treat this malignant disease. In this study, we observed the biological response of osteosarcoma cells to two kinds of hydroxyapatite nanoparticles (Nano HA), NanoHA-S and NanoHA-L. These nanospheres have the same crystallinity (phase) and morphology, but they differ in size. Cells treated with two kinds of Nano HA were inhibited and mainly led to apoptotic cell death. Caspase-9-dependent intrinsic apoptotic pathway plays a role. It was interesting that the suppression and the apoptosis of osteosarcoma cells was directly related to the size of nanoparticles and that the larger-sized Nano HA exhibited more effectiveness than the smaller one. This in vitro study suggested the potential of size-controlled calcium phosphate nanoparticles for use in therapeutic replacement and reconstruction of bone merits after osteosarcoma extraction. *Authors to whom correspondence should be addressed. E-mail: hmtao-zr@163.com, Rtang@zju.edu.cn Figures 6–8 appear in color online: http://jba.sagepub.com JOURNAL OF BIOMATERIALS APPLICATIONS Volume 25 — July 2010 19 0885-3282/10/01 0019–19

Enhanced anticancer effect of gemcitabine by genistein in osteosarcoma: the role of Akt and nuclear factor-κB

10.00/0 DOI: 10.1177/0885328209339396 The Author(s), 2010. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.navOsteosarcoma is a primary malignant bone tumor, most prevalent in children and adolescents, and is usually highly aggressive and eventually lethal. Despite multimodal therapies, there is no effective approach to treat this malignant disease. In this study, we observed the biological response of osteosarcoma cells to two kinds of hydroxyapatite nanoparticles (Nano HA), NanoHA-S and NanoHA-L. These nanospheres have the same crystallinity (phase) and morphology, but they differ in size. Cells treated with two kinds of Nano HA were inhibited and mainly led to apoptotic cell death. Caspase-9-dependent intrinsic apoptotic pathway plays a role. It was interesting that the suppression and the apoptosis of osteosarcoma cells was directly related to the size of nanoparticles and that the larger-sized Nano HA exhibited more effectiveness than the smaller one. This in vitro study suggested the potential of size-controlled calcium phosphate nanoparticles for use in therapeutic replacement and reconstruction of bone merits after osteosarcoma extraction.

Gallic Acid Induces the Apoptosis of Human Osteosarcoma Cells In Vitro and In Vivo via the Regulation of Mitogen-Activated Protein Kinase Pathways

Genistein, a nontoxic flavonoid compound, has potent antitumor activity in various cancer cell lines. This study was designed to investigate whether combination therapy with gemcitabine and genistein enhances antitumor efficacy in osteosarcoma cell lines (MG-63 and U2OS). Our results show that significant reduction in cell viability and corresponding induction of apoptosis were observed with combination treatment in both cell lines. On the molecular level, we found that gemcitabine alone can activate nuclear factor κB (NF-κB) in osteosarcoma, suggesting the potential mechanism of acquired chemoresistance. In contrast, genistein reversed the cancers resistance to gemcitabine through the downregulation of NF-κB activity and the suppression of Akt. These findings suggest that the combination of gemcitabine and genistein enhanced the antitumor efficacy by abrogating the Akt/NF-κB pathway. The marked ability to induce apoptosis with a combination of gemcitabine and genistein suggests that this could be a rational and novel approach for osteosarcoma preclinical and clinical trials.

Caspase‐8 dependent osteosarcoma cell apoptosis induced by proteasome inhibitor MG132

To examine the antitumor effects of gallic acid (GA) on osteosarcoma, two human osteosarcoma cell lines U-2OS and MNNG/HOS were treated by GA and subjected to cell proliferation and apoptosis assays. In addition, MNNG/HOS xenograft tumors were established in nude BALB/c mice to evaluate the anticancer capacity of GA in vivo. The results showed that GA inhibited the proliferation and induced the apoptosis of osteosarcoma cells, accompanied by the upregulation of p-38 activation and the downregulation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK1/2) activation. Additionally, p38 MAPK inhibitor abrogated GA-induced growth inhibition of osteosarcoma cells, whereas JNK or ERK1/2 inhibitors sensitized osteosarcoma cells to GA-induced growth inhibition. In vivo studies further showed that GA administration decreased xenograft tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated the downregulation of PCNA and CD31 expression and upregulation of apoptosis in MNNG/HOS tumor tissues following GA treatment. This study demonstrates the antitumor efficacy of GA for osteosarcoma that is mediated by the modulation of cell proliferation, apoptosis, and angiogenesis. Our findings suggest that GA could be a potent agent for osteosarcoma intervention.

Genistein Potentiates the Anti-cancer Effects of Gemcitabine in Human Osteosarcoma via the Downregulation of Akt and Nuclear Factor-κB Pathway

Many researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells, such as breast cancer cell, lung cancer cell, and lymphoma cell. However, the effect of proteasome inhibitors on osteocsarcoma cells and the mechanisms are seldom studied. In this study, we found proteasome inhibitor MG132 was an effective inducer of apoptosis in human osteosarcoma MG‐63 cells. On normal human diploid fibroblast cells, MG132 did not show any apoptosis‐inducing effects. Apoptotic changes such as DNA fragment and apoptotic body were observed in MG132‐treated cells and MG132 mostly caused MG‐63 cell arrest at G2–M‐phase by cell cycle analysis. Increased activation of caspase‐8, accumulation of p27Kip1, and an increased ratio of Bax: Bcl‐2 were detected by RT—PCR and Western blot analysis. Activation of caspase‐3 and caspase‐9 were not observed. This suggests that the apoptosis induced by MG132 in MG63 cells is caspase‐8 dependent, p27 and bcl‐2 family related.

New hypothesis of chronic back pain: low pH promotes nerve ingrowth into damaged intervertebral disks

Genistein, a nontoxic flavonoid compound, has potent antitumor activity in various cancer cells. In the present study, we investigated whether genistein could be employed as a novel strategy to enhance the anti-tumor activity of gemcitabine using human osteosarcoma MNNG/HOS tumor model. In vitro, by MTT, electron microscopy, immunobloting and qRT-PCR assay, we found that the combination treatment of genistein and gemcitabine resulted in stronger growth inhibition and apoptosis induction through the downregulation of NF-κB activity and Akt activation in osteosarcoma cells. Moreover, the synergetic effects were observed when genistein was replaced by PI3K/Akt-pathway inhibitor (LY-294002) or NF-κB inhibitor (BAY11-7082). In vivo, the combination therapy augmented tumor growth inhibition through the down-regulation of NF-κB activity and Akt activation in xenografts. Taken together, these results provide in vitro and in vivo evidence that genistein abrogates gemcitabine-induced activation of NF-κB and increases the chemosensitization of osteosarcoma to gemcitabine. Combination therapy appears as a rational and novel approach for osteosarcoma treatment.

Osteochondral repair using the combination of fibroblast growth factor and amorphous calcium phosphate/poly(l-lactic acid) hybrid materials

The pathogenesis of low back pain is still elusive. Here, we proposed a new hypothesis that low pH is a possible cause of the development and progression of low back pain. We propose that low pH promotes the production of the inflammatory mediators and the depletion of proteoglycan in the damaged intervertebral disk. The inflammation response, evoked by the dorsal root ganglia, changes the delicate nutrient balance in the nucleus, resulting in a vicious cycle and leading to choronic back pain. Our hypothesis may explain many of the available clinical and experimental data on low back pain, thus it may help elucidate the pathogenesis of low back pain and improve clinical management.