Xanthatin suppresses proliferation and tumorigenicity of glioma cells through autophagy inhibition via activation of the PI3K-Akt-mTOR pathway.

Glioma is the most common and aggressive primary brain tumor in adults with high morbidity and mortality. Rapid proliferation and diffuse migration are the main obstacles to successful glioma treatment. Xanthatin, a sesquiterpene lactone purified from Xanthium strumarium L., possesses a significant antitumor role in several malignant tumors. In this study, we report that xanthatin suppressed glioma cells proliferation and induced apoptosis in a time- and concentration-dependent manner, and was accompanied by autophagy inhibition displaying a significantly reduced LC3 punctate fluorescence and LC3II/I ratio, decreased level of Beclin 1, while increased accumulation of p62. Notably, treating glioma cells with xanthatin resulted in obvious activation of the PI3K-Akt-mTOR signaling pathway, as indicated by increased mTOR and Akt phosphorylation, decreased ULK1 phosphorylation, which is important in modulating autophagy. Furthermore, xanthatin-mediated pro-apoptosis in glioma cells was significantly reversed by autophagy inducers (rapamycin or Torin1), or PI3K-mTOR inhibitor NVP-BEZ235. Taken together, these findings indicate that anti-proliferation and pro-apoptosis effects of xanthatin in glioma are most likely by inhibiting autophagy via activation of PI3K-Akt-mTOR pathway, suggesting a potential therapeutic strategy against glioma.

Xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating endoplasmic reticulum stress-dependent CHOP pathway.

Xanthatin is a natural sesquiterpene lactone purified from Xanthium strumarium L., which has shown prominent antitumor activity against a variety of cancer cells. In the current study, we investigated the effect of xanthatin on the growth of glioma cells in vitro and in vivo, and elucidated the underlying mechanisms. In both rat glioma C6 and human glioma U251 cell lines, xanthatin (1-15 µM) dose-dependently inhibited cell viability without apparent effect on the cell cycle. Furthermore, xanthatin treatment dose-dependently induced glioma cell apoptosis. In nude mice bearing C6 glioma tumor xenografts, administration of xanthatin (10, 20, 40 mg·kg-1·d-1, ip, for 2 weeks) dose-dependently inhibited the tumor growth, but did not affect the body weight. More importantly, xanthatin treatment markedly increased the expression levels of the endoplasmic reticulum (ER) stress-related markers in both the glioma cell lines as well as in C6 xenografts, including glucose-regulated protein 78, C/EBP-homologous protein (CHOP), activating factor 4, activating transcription factor 6, spliced X-box binding protein-1, phosphorylated protein kinase R-like endoplasmic reticulum kinase, and phosphorylated eukaryotic initiation factor 2a. Pretreatment of C6 glioma cells with the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 7 mM) or knockdown of CHOP using small interfering RNA significantly attenuated xanthatin-induced cell apoptosis and increase of proapoptotic caspase-3. These results demonstrate that xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating the ER stress-related unfolded protein response pathway involving CHOP induction. Xanthatin may serve as a promising agent in the treatment of human glioma.

Dynamic contrast-enhanced magnetic resonance imaging for monitoring the anti-angiogenesis efficacy in a C6 glioma rat model.

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is useful in predicting responses to angiogenic therapy of malignant tumors. PURPOSE: To observe the dynamics of DCE-MRI parameters in evaluating early effects of antiangiogenic therapy in a C6 glioma rat model. MATERIAL AND METHODS: The Bevacizumab or vehicle treatment was started from the 14th day after glioma model was established. The treated and control groups (n = 13 per group) underwent DCE-MRI scans on days 0, 1, 3, 5, and 7 after treatment. Tumor volume was calculated according to T2-weighted images. Hematoxylin and eosin, microvessel density (MVD), and proliferating cell nuclear antigen (PCNA) examination were performed on day 7. The MRI parameters between the two groups were compared and correlations with immunohistochemical scores were analyzed. RESULTS: The average tumor volume of treated group was significantly lower than that of control group on day 7 (81.764 ± 1.043 vs. 103.634 ± 3.868 mm3, P = 0.002). Ktrans and Kep decreased in the treated group while they increased in the control group. The differences were observed on day 5 (Ktrans: 0.045 ± 0.018 vs. 0.093 ± 0.014 min-1, P < 0.001; Kep: 0.062 ± 0.018 vs. 0.134 ± 0.047 min-1, P = 0.005) and day 7 (Ktrans: 0.032 ± 0.010 vs. 0.115 ± 0.025 min-1, P < 0.001; Kep: 0.045 ± 0.016 vs. 0.144 ± 0.042 min-1, P < 0.001). The difference of Ve was observed on day 5 (0.847 ± 0.248 vs. 0.397 ± 0.151, P = 0.009) and 7 (0.920 ± 0.154 vs. 0.364 ± 0.105, P = 0.006). Ktrans and Kep showed positive correlations with MVD and Ve showed negative correlation with PCNA. CONCLUSION: DCE-MRI can assess the changes of early effects of anti-angiogenic therapy in preclinical practice.

Evaluation of Tumor Hypoxia in C6 Glioma Rat Model With Dynamic Contrast-Enhanced Magnetic Resonance Imaging.

RATIONALE AND OBJECTIVES: To investigate the feasibility of determining quantitative parameters for evaluating tumor hypoxia in the C6 glioma model by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were inoculated to establish C6 brain glioma models. DCE-MRI scans were performed 14, 21, and 28 days after transplantation. Quantitative parameters comprising Ktrans, Ve, Kep, and Vp were calculated and analyzed. At the end of each scan, 10 rats were randomly selected for immunohistochemical (IHC) staining of hypoxia-inducible factor-17αl (HIF-1α), proliferating cell nuclear antigen (PCNA), and CD34. Correlations between quantitative parameters and IHC scores were analyzed. RESULTS: The tumor volumes increased with time. The Ktrans values on days 14, 21, and 28 were 0.03 ± 0.009 min-1, 0.084 ± 0.010 min-1, and 0.050 ± 0.016 min-1, while the Ve values were 0.17 ± 0.070, 0.46 ± 0.159, and 0.51 ± 0.193, the Kep values were 0.18 ± 0.070%, 0.220 ± 0.049%, and 0.06 ± 0.035%, and these three parameters all differed significantly among the three time points. The Vp values on days 14, 21, and 28 were 0.09 ± 0.040%, 0.120 ± 0.034%, and 0.06 ± 0.010%, but the values did not differ among the three time points (P = 0.073). Ktrans had significant negative correlations with the HIF-1α scores on days 14 and 21 when there was also a positive correlation between Ktrans and CD34. Ve had negative correlations with the HIF-1α score on days 14 and 21, and there was a negative correlation between Ve and PCNA on day 21. Kep had a negative correlation with the HIF-1α score and a positive correlation with MVD on day 21. CONCLUSIONS: DCE-MRI may be a useful method for the noninvasive evaluation of the hypoxia status in a glioma model.

The antitumour growth and antiangiogenesis effects of xanthatin in murine glioma dynamically evaluated by dynamic contrast-enhanced magnetic resonance imaging.

To investigate the suppressive effects of xanthatin on glioma growth in a nude mouse xenograft model and rat orthotopic implantation model using magnetic resonance imaging (MRI) to dynamically monitor the antitumour growth and antiangiogenesis effects of xanthatin. The nude mouse xenograft tumour model and rat orthotopic implantation model were established to observe the antitumour effects of xanthatin in vivo. In the rat orthotopic implanted tumour model, MRI scanning was used to dynamically monitor the antitumour growth effect and evaluate the antiangiogenesis effect of xanthatin. We found that xanthatin at a dose of 0.4 mg/10 g dramatically decreased the growth of xenograft tumours in nude mice. The antiangiogenesis effect of xanthatin C6 glioma was evaluated by dynamic contrast-enhanced (DCE) MRI via comparison of the volume transfer constant (Ktrans ) value, a parameter that reflects vessel permeability. We found that xanthatin at the doses of 8 and 16 mg/kg significantly decreased the Ktrans value, which suggests that xanthatin has antiangiogenesis effects. These data demonstrate the suppressive effects of xanthatin on C6 glioma occur via antiangiogenesis. Meanwhile, this study also provides evidence for the application of quantitative parameters of DCE-MRI for dynamically evaluating the growth and angiogenesis of intracranial tumours and for experimental and clinical research.

MRI Dynamically Evaluates the Therapeutic Effect of Recombinant Human MANF on Ischemia/Reperfusion Injury in Rats.

As an endoplasmic reticulum (ER) stress-inducible protein, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been proven to protect dopaminergic neurons and nondopaminergic cells. Our previous studies had shown that MANF protected against ischemia/reperfusion injury. Here, we developed a magnetic resonance imaging (MRI) technology to dynamically evaluate the therapeutic effects of MANF on ischemia/reperfusion injury. We established a rat focal ischemic model by using middle cerebral artery occlusion (MCAO). MRI was performed to investigate the dynamics of lesion formation. MANF protein was injected into the right lateral ventricle at 3 h after reperfusion following MCAO for 90 min, when the obvious lesion firstly appeared according to MRI investigation. T2-weighted imaging for evaluating the therapeutic effects of MANF protein was performed in ischemia/reperfusion injury rats on Days 1, 2, 3, 5, and 7 post-reperfusion combined with histology methods. The results indicated that the administration of MANF protein at the early stage after ischemia/reperfusion injury decreased the mortality, improved the neurological function, reduced the cerebral infarct volume, and alleviated the brain tissue injury. The findings collected from MRI are consistent with the morphological and pathological changes, which suggest that MRI is a useful technology for evaluating the therapeutic effects of drugs.