Jinhua Zheng

Low-intensity Ultrasound Combined with 5-aminolevulinic Acid Administration in the Treatment of Human Tongue Squamous Carcinoma

We investigated the anti-tumor efficiency of sonodynamic therapy (SDT) on human tongue squamous carcinoma SAS cell line using low intensity ultrasound (LIU) of 0.6 and 0.8 W/cm2, plus 5-aminolevulinic acid (ALA). Xenograft in vivo experiments using Balb/ca nude mice and MTT assays in vitro showed that ALA-LIU therapy significantly suppressed the proliferation of SAS cells. ALA-LIU therapy markedly enhanced SAS cell apoptosis rate compared to LIU alone. Based on TEM and fluorescence microscopy observations, there are notably morphology changes and seriously swollen mitochondria in xenograft tissues, and ALA-induced PpIX bond strongly to mitochondria of SAS cells. Immunohistochemical staining and western blotting demonstrated upregulation of Bax, cytochrome c and caspase-3, and downregulation of Bcl-2 for both in vivo and in vitro cases after ALA-LIU treatment. Increase of reactive oxygen species (ROS) in the ALA-LIU treatment groups were found using 2, 7-dichlorofluorescin diacetate (DCFH-DA) staining. Administration of the ROS scavenger, N-acetylcysteine (NAC), suppressed ALA-LIU-induced apoptosis and the expression of mitochondria apoptosis-related proteins, which confirmed that the ALA-LIU induced SAS cell apoptosis is through the generation of ROS. The process initially damaged mitochondria, activated pro-apoptotic factors Bax and cytochrome c and supressed the anti-apoptotic factor Bcl-2, activated caspase-3 to executed apoptosis through mitochondrial signaling pathway.

Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarins effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarins angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and mRNA expression in cultured HUVECs in a concentration-dependent manner. Taken together, these results suggest that scutellarin promotes angiogenesis and may form a basis for angiogenic therapy.

Scutellarin inhibits the growth and invasion of human tongue squamous carcinoma through the inhibition of matrix metalloproteinase-2 and -9 and αvβ6 integrin

Scutellarin can inhibit the growth and migration of tongue cancer cells in vitro and can regulate cell adhesion; such agents will be the next generation anticancer therapeutics. In this study, we evaluated the antitumor effects of the scutellarin on human tongue squamous carcinoma (SAS) cell line and investigated its molecular mechanism. To explore the possible mechanisms underlying the antitumor effect of scutellarin, we studied its impact on the growth and invasion of SAS cells xenografted into nude mice, on the expression of MMP-2, MMP-9, integrin αvβ6, and c-JUN, and also on changes in collagen fibers. We observed that the growth of xenograft SAS tumors in nude mice was significantly inhibited by the administration of scutellarin without major adverse effects. Results showed that scutellarin mediated inhibition of tumor cell proliferation, induced apoptosis, and regulated expression of matrix metallo-proteinase (MMP)-2 and -9, and integrin αvβ6 at the mRNA and protein levels in vivo. Moreover, scutellarin regulated the expression of collagen fibers in the tumor microenvironment (surrounding the tumor), thereby inhibiting cell invasion and metastasis. Our in vitro and in vivo studies have shown that scutellarin reduces the expression of MMP-2 and -9, and integrin αvβ6 in SAS cells, possibly through the regulation of expression of transcription factor AP-1. These results suggest that scutellarin can have an anti-tumor therapeutic effect by inhibition of the ability of SAS cells to invade and metastasize.

5-Aminolevulinic acid-mediated sonodynamic therapy induces anti-tumor effects in malignant melanoma via p53-miR-34a-Sirt1 axis

BACKGROUD Malignant melanoma is a very refractory skin tumor due to its high metastasis, poor prognosis, and insensitivity to chemotherapy. Sonodynamic therapy has recently evolved as a potential method to treat cancers. In this study, 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) was used to treat malignant melanoma in vivo. OBJECTIVE To investigate whether ALA-SDT induces anti-tumor effects in malignant melanoma and to see if miRNAs are involved in this process. METHODS Tumor transplantation experiments in BALB/c nude mice were used to assess anti-tumor effects after ALA-SDT treatment. Cell apoptosis was evaluated by TUNEL assays and cell proliferation was measured using immunohistochemisty with anti-PCNA antibody. Microarray analysis was performed to measure miRNAs expressions. Endogenous miR-34a and its upstream and downstream genes were assayed by real-time PCR. Western blottings were used to determine these protein expressions. Intracellular ROS levels were detected by measuring the fluorescence intensity of DCF. RESULTS Tumor transplantation experiments revealed that ALA-SDT could inhibit mouse melanoma cell proliferation and tumor growth. Compared with the control group, TUNEL assays revealed that apoptosis was increased and proliferation was inhibited in the SDT group. Real-time PCR analysis showed 14-fold increase of miR-34a expression in the SDT group compared to the control group. In addition, ALA-SDT significantly increased intracellular ROS levels in vitro, which were almost inhibited by the ROS scavenger NAC. Also, the mRNA, total protein, and acetylation levels of p53 were increased, whereas some downstream anti-apoptotic or pro-proliferative factors of miR-34a such as BCL2, CCND1, CDK6, and SIRT1 were decreased in the SDT group compared with the control, ALA alone, and ultrasound alone groups. When miR-34a was inhibited in vitro, the protein expressions of BCL2, CCND1, CDK6, and SIRT1 recovered. By targeting SIRT1, which inhibits p53 acetylation, miR-34a promoted the transcriptional activity of p53, and finally led to increased expression of miR-34a itself. Therefore, the p53, miR-34a, and SIRT1 constituted a positive feedback loop. CONCLUSION ALA-SDT showed synergistic anti-tumor effects in malignant melanoma by constituting a positive feedback loop of p53-miR-34a-Sirt1 axis.

Prognostic significance of VEGF-C, semaphorin 3F, and neuropilin-2 expression in oral squamous cell carcinomas and their relationship with lymphangiogenesis

Neuropilin‐2 (NRP2), a receptor for vascular endothelial growth factor C (VEGF‐C) and semaphorin 3 F (SEMA3F), is a possible regulator of tumor progression and angiogenesis. However, little evidence of a correlation between NRP2 expression and lymphangiogenesis has been reported. SEMA3F might suppress lymphangiogenesis by competing with VEGF‐C for binding to NRP2.

5-Aminolevulinic Acid-Based Sonodynamic Therapy Induces the Apoptosis of Osteosarcoma in Mice

Objective Sonodynamic therapy (SDT) is promising for treatment of cancer, but its effect on osteosarcoma is unclear. This study examined the effect of 5-Aminolevulinic Acid (5-ALA)-based SDT on the growth of implanted osteosarcoma and their potential mechanisms in vivo and in vitro. Methods The dose and metabolism of 5-ALA and ultrasound periods were optimized in a mouse model of induced osteosarcoma and in UMR-106 cells. The effects of ALA-SDT on the proliferation and apoptosis of UMR-106 cells and the growth of implanted osteosarcoma were examined. The levels of mitochondrial membrane potential (ΔψM), ROS production, BcL-2, Bax, p53 and caspase 3 expression in UMR-106 cells were determined. Results Treatment with 5-ALA for eight hours was optimal for ALA-SDT in the mouse tumor model and treatment with 2 mM 5-ALA for 6 hours and ultrasound (1.0 MHz 2.0 W/cm2) for 7 min were optimal for UMR-106 cells. SDT, but not 5-ALA, alone inhibited the growth of implanted osteosarcoma in mice (P<0.01) and reduced the viability of UMR-106 cells (p<0.05). ALA-SDT further reduced the tumor volumes and viability of UMR-106 cells (p<0.01 for both). Pre-treatment with 5-ALA significantly enhanced the SDT-mediated apoptosis (p<0.01) and morphological changes. Furthermore, ALA-SDT significantly reduced the levels of ΔψM, but increased levels of ROS in UMR-106 cells (p<0.05 or p<0.01 vs. the Control or the Ultrasound). Moreover, ALA-SDT inhibited the proliferation of osteosarcoma cells and BcL-2 expression, but increased levels of Bax, p53 and caspase 3 expression in the implanted osteosarcoma tissues (p<0.05 or p<0.01 vs. the Control or the Ultrasound). Conclusions The ALA-SDT significantly inhibited osteosarcoma growth in vivo and reduced UMR-106 cell survival by inducing osteosarcoma cell apoptosis through the ROS-related mitochondrial pathway.

Expression of αvβ6 integrin and collagen fibre in oral squamous cell carcinoma: association with clinical outcomes and prognostic implications

BACKGROUND This study aims to investigate the expression and significance of the αvβ6 integrin, collagen fibres and matrix metalloproteinases (MMP)-3 in oral squamous cell carcinoma (OSCC) and to analyse the possible regulatory relationships between αvβ6, collagen fibres and MMP-3. MATERIALS AND METHODS A series of 80 patients (mean age 56.4 years) diagnosed with OSCC were enrolled. Associations between αvβ6, MMP-3, collagen fibre expression levels and clinicopathological parameters were evaluated using the Fisher exact test. Survival analysis was performed with Kaplan-Meier curves. Spearman rank correlation was used to analyse interactions between αvβ6, MMP-3 and collagen fibres. RESULTS αvβ6 and MMP-3 were strongly expressed in human OSCC, especially at the peripheral borders of invasive tumour islands, and collagen fibres were generally disrupted and degraded in the same areas. The expression intensity of αvβ6 was associated with the differentiation state of cells. β6 mRNA was expressed in almost all cancer cells. In carcinomas, αvβ6 and MMP-3 expression were correlated with the distribution of collagen fibres. CONCLUSIONS Tumour cells highly expressing αvβ6 have a strong capability for invasion and migration, due to concomitant protease production and the destruction and remodelling of collagen fibres. Increased αvβ6 integrin and MMP-3 expression and collagen fibre changes in human OSCCs are related to unfavourable clinical prognostic factors and decreased survival.

expression of MMP-1/Par-1 and patterns of invasion in oral squamous cell carcinoma as potential prognostic markers

Background Matrix metalloproteinase (MMP)-1 degrades type I collagen of the extracellular matrix and also activates protease activated receptor (PAR)-1 to induce angiogenesis. The aims of this study were to evaluate microvessel density (MVD) and the expression of PAR-1 and MMP-1 in oral squamous cell carcinoma (SCC) specimens with different patterns of invasion (POI) and to evaluate their association with clinical outcomes. Methods Seventy-four surgically obtained oral SCC samples were classified by POI according to hematoxylin-eosin staining. MVD and the localization and intensity of PAR-1 and MMP-1 expression were detected by immunohistochemistry. Results Of the 74 oral SCC samples, 18, 5, 34, and 17 showed type I, II, III, and IV POI, respectively. MVD and expression levels of MMP-1 and PAR-1 differed between POI types I–II and POI types III–IV. Patients with low tumor expression of MMP-1 and PAR-1 and low MVD had a longer survival time than those with high tumor expression of MMP-1 and PAR-1. Moreover, the survival time of patients with POI types III–IV was shorter than that of patients with POI types I–II. Conclusion POI combined with expression levels of MMP-1 and PAR-1 may be a valuable tool for assessing the clinical prognosis of patients with oral SCC.

MMP-1/2 and TIMP-1/2 expression levels, and the levels of collagenous and elastic fibers correlate with disease progression in a hamster model of tongue cancer

In the present study, the presence of extracellular matrix components, including collagenous and elastic fibers, and the expression of their key regulating enzymes, were investigated in different stages of hamster tongue carcinoma development. Immunohistochemical and computer-assisted morphological analyses were performed to quantify the staining intensity and area (integral optical density) of matrix metalloproteinase (MMP)-1 and −2, tissue inhibitors of metalloproteinase (TIMP)-1 and −2, and the extent of elastic and collagenous fibers in histological sections. MMP-1, MMP-2, TIMP-1 and TIMP-2 expression levels gradually increased with tongue cancer progression, and were associated with disease pathology staging (r=0.705, 0.633, 0.759 and 0.751, respectively). By contrast, elastic fiber levels gradually decreased with cancer progression and were negatively correlated with disease staging (r=-0.881). The levels of collagenous fibers gradually increased with cancer progression and showed a positive correlation (r=0.619). In summary, the study demonstrated that MMP1/2 and TIMP1/2 expression levels, and collagenous and elastic fiber levels were significantly correlated with disease progression in a hamster model of tongue cancer.

Effects of low-intensity ultrasound combined with low-dose carboplatin in an orthotopic hamster model of tongue cancer: A preclinical study

Low-intensity ultrasound (LIUS) combined with chemotherapy is an innovative modality for cancer treatment, but its effect on orthotopic carcinoma remains unknown. Our previous study revealed that LIUS enhanced the growth inhibitory effects of several chemotherapeutic drugs in nude mice with transplanted tumors. In the present study, we used 7,12-dimethylbenz(alpha)anthracene to induce orthotopic tongue carcinogenesis in hamsters. We used the first-line chemotherapy drug for tongue cancer, carboplatin (CBP) in combination with LIUS to investigate the synergistic effect. The results revealed that LIUS combined with low-dose CBP enhanced the inhibitory effects of CBP on tumor growth, prolonged survival, and did not increase the incidence of side-effects. It also enhanced the inherent DNA damage caused by CBP, suppressed the expression of the DNA repair proteins O6-methylguanine DNA methyltransferase (MGMT) and Chk1, and increased the expression of DNA damage-inducible protein GADD45α. Furthermore, compared with CBP alone, LIUS combined with CBP reduced the expression of cyclin D1 and cyclin B1, induced the expression of caspase-3, cleaved caspase-3, caspase-8, Bax, and Bak, and inhibited the expression of Bcl-2. Examination of clinical samples revealed that MGMT, Chk1, and Gadd45α were higher in OTSCC than in adjacent normal tissue. Hence, our results indicated that LIUS enhanced the ability of low-dose CBP to damage DNA in an orthotopic hamster model of tongue cancer, induced apoptosis, inhibited tumor growth and progression, while it did not increase the toxic side-effects of the drug, suggesting additional clinical benefits for patients treated with the combination of CBP with LIUS.