Jinghua Ren

Acquired cisplatin resistance in human lung adenocarcinoma cells is associated with enhanced autophagy.

Activation of autophagy is a hallmark in tumor cells treated with chemotherapy, but the role of autophagy in acquired resistance of lung adenocarcinoma to cisplatin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental lung adenocarcinoma cell line A549 and its 8-fold, more resistant subcell line, A549/DDP, which was obtained by treating cisplatin with increasing concentrations. A549/DDP and A549 cells were exposed to serum-free culture medium or ionizing radiation. To measure the stress-induced autophagy, LC3-II, as an autophagosome marker, was measured by immunofluorescence and Western blotting. To determine the effect of 3-MA, a known inhibitor of autophagy, on overcoming acquired cisplatin resistance, the MTT assay and flow cytometry were performed. Western blotting analysis demonstrated that LC3-II was increased in A549/DDP cells, compared with those of parental A549 cells, under stress conditions. Meanwhile, immunofluorescence staining showed that LC3-II protein was located mainly in the cytoplasm of A549/DDP. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of cisplatin in acquired resistant cells (A549/DDP). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in cisplatin resistance of A549/DDP cells.

Inhibitory action of Celastrol on hypoxia-mediated angiogenesis and metastasis via the HIF-1α pathway

Celastrol, a natural biologically active compound isolated from Tripterygium wilfordii Hook F root extracts, has been shown to possess antitumor properties and therefore, is an interesting candidate for the development of novel chemotherapeutic cancer agents. In this study, we have demonstrated that Celastrol is a potent inhibitor of hypoxia-induced angiogenic and metastatic activity as shown by a decrease in the proliferation of both endothelial and cancer cells, blocking of migration as well as of tube formation of endothelial cells, and by inhibition of cancer cell invasion under hypoxic conditions. Moreover, Celastrol decreased hypoxia-inducible factor-1α (HIF-1α) mRNA levels under both normoxia and hypoxia and inhibited hypoxia-induced accumulation of nuclear HIF-1α protein. Meanwhile, inhibition of nuclear HIF-1α protein levels were accompanied by a reduction in the transcriptional activity of HIF-1α target genes, including VEGF. In addition, the inhibitory effect of Celastrol on HIF-1α protein was partly due to its suppression of HSP90 activity. We conclude that Celastrol regulates HIF-1α at multiple levels that may together or individually contribute to its antitumor activity against hypoxia-induced angiogenesis and metastasis.

Rho kinase inhibitor fasudil suppresses migration and invasion though down-regulating the expression of VEGF in lung cancer cell line A549

Rho and Rho-associated kinase play an important role in focal adhesion, stress fiber formation and cell motility. Fasudil is a kind of Rho kinase inhibitor. The effect and precise molecular mechanism of fasudil on the biology behavior of lung cancer cell A549 remains unclear. The cytotoxic effect of fasudil on A549 cell was measured by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Wound-healing assay was used to evaluate the effect of fasudil on migration activity of A549 cells. The invasion activity of A549 cells was detected by transwell chamber assay. The expression of MMPs was measured by gelatin zymography. RT-PCR and western blot were used to investigate the molecular change of A549 cells after treated with fasudil. Fasudil-inhibited proliferation of A549 cells in a concentration-dependent manner, decreased the migration and invasion activity. After treated with fasudil, the expression of MMP-2 and MMP-9 was significantly inhibited compared with the control group. Furthermore, the expression of RhoA and VEGF of A549 cell treated with fasudil was significantly down-regulated. Our findings indicate that fasudil might have a therapeutic potential for lung cancer though inhibiting cell proliferation, migration, invasion, MMPs activity and down-regulating the expression of RhoA and VEGF.

VEGF-PKD1-HDAC7 signaling promotes endothelial progenitor cell migration and tube formation.

Histone acetylation/deacetylation is a key mechanism for regulating transcription, which plays an important role in the control of gene expression, tissue growth, and development. In particular, histone deacetylase 7 (HDAC7), a member of class IIa HDACs, is crucial in maintaining vascular integrity. Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, whether HDAC7 plays a role in the processes of EPCs angiogenesis remains unclear. Migration and tube formation were the two major components of EPC angiogenesis. In this study, we show for the first time that HDAC7 silencing weakened the migration and tube formation abilities of EPCs. VEGF-A induced an increase of phospho-HDAC7 and its nuclear export in a time-dependent manner, which could be partly inhibited by protein kinase D1 (PKD1) inhibitor, but not by the PI3K inhibitor or the MEK inhibitor. Our results showed that EPCs involved in the angiogenesis might be controlled by VEGF-PKD1-HDAC7 axis, which regulates the EPCs angiogenesis by PKD1, but not the ERK and PI3K pathway.

Lysyl Oxidase 473 G>A Polymorphism and Breast Cancer Susceptibility in Chinese Han Population

Lysyl oxidase (LOX) is an extracellular enzyme critical for the cross-linking of collagens and elastin. The LOX gene has also been shown to inhibit the transforming activity of Ras oncogene signaling. Recently, a single-nucleotide polymorphism (SNP) of LOX G473A (rs1800449) has been demonstrated to be associated with increased risk of breast cancer in African American women. In this hospital-based case-control study, the association of LOX polymorphism with breast cancer susceptibility in Chinese Han population was investigated. In total, 238 female patients with breast cancer and 234 age-matched healthy controls recruited were genotyped. We found a significant difference in the frequency of the LOX G473A genotype between the breast cancer and control groups. Individuals with GA genotype showed a 2.79-fold (95% confidence interval = 1.87-4.16) increased risk of breast cancer compared with subjects carrying GG genotype (p < 0.001). Further statistical analysis revealed that this polymorphism was an independent parameter with regard to other variables that are significantly associated with breast cancer, that is, age, menopausal status, estrogen exposure interval, expression status of estrogen receptor, and progesterone receptor. These findings suggest that the LOX 473 GA genotype is independently associated with increased risk of breast cancer in Chinese female population.

Modulation of radiation-induced tumour necrosis factor-α and transforming growth factor β1 expression in the lung tissue by Shengqi Fuzheng injection

Radiation-induced lung injury (RILI) is one of the most common and severe side effects of thoracic radiotherapy. Therefore, novel therapeutic approaches to improve the effectiveness of RILI treatment are required. The present study was designed to determine the effectiveness of a traditional Chinese medicine regimen, Shenqi Fuzheng injection (SFI), in the treatment of RILI. SFI is composed of extracts from codonopsis pilosula and radix astragali. Here, we determined the protective effects of SFI on RILI with a single-dose irradiation (RT) of 12 Gy in C57BL/6 8-week-old mice. The mice were divided into four groups treated with i) phosphate-buffered saline (PBS; pH 7.4, 20 ml/kg/day) alone as normal a control; ii) SFI only (20 ml/kg/day); iii) RT + PBS (20 ml/kg/day); and iv) RT + SFI (20 ml/kg/day). SFI and PBS were administered via intraperitoneal injection 1 week before and 2 weeks after RT. The pathology of RILI and any clinical signs of toxicity were monitored. The expression of tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 in the lungs was analyzed by RT-PCR and immunohistochemistry. TNF-α and TGF-β1 expression was increased by RT, but was reversed by SFI treatment during the radiation pneumonic and fibrotic phases (P<0.05). Lung histology at 24 weeks revealed a significant decrease in structural damage and collagen deposition in the RT + SFI group compared to the RT + PBS group. In conclusion, TNF-α and TGF-β1 are key mediators for the pathogenesis of RILI, and SFI reduces TNF-α and TGF-β1 expression after RT. This may be a key mechanism behind the preventive effects of SFI on lung injury after radiation.

Rho Kinase Inhibitor Fasudil Suppresses the Vasculogenic Mimicry of B16 Mouse Melanoma Cells Both In Vitro and In Vivo

The aim of this study was to investigate the biologic role of the Rho kinase inhibitor fasudil in the vasculogenic mimicry (VM) of B16 mouse melanoma cells. It was previously reported that RhoA plays a critical role in angiogenesis by coordinating endothelial cell cytoskeleton remodeling and promoting endothelial cell motility. Although RhoA has been implicated in the regulation of angiogenesis, little has been described regarding its control of these tumor cell–lined channels. In this study, we established an in vitro model of VM using 3-dimensional cell culturing of mouse B16 melanoma cells and studied VM in vivo by transplanting B16 cells into C57/BL mice. Next, we explored the effect of RhoA and Rho-associated, coiled-coil containing protein kinase (ROCK) on VM formation using the Rho kinase inhibitor fasudil. We provide direct evidence that fasudil leads to reduced vascular-like channels in Matrigel. Additional experiments suggested that fasudil prevents both initial cellular architecture changes and cell migration in vitro. Finally, we provide in-depth evidence for the underlying mechanisms of fasudil-induced VM destruction using the Rho-GTPase agonist lysophosphatidic acid. In vivo studies revealed that fasudil reduced B16 melanoma cell xenograft tumor growth without causing significant toxicity in mice. Fasudil-treated tumors also displayed fewer VM channels. These results suggest that fasudil may be an emerging therapeutic option for targeting cancer VM. Mol Cancer Ther; 14(7); 1582–90. ©2015 AACR.

EGFR mutations in non-small-cell lung cancer among smokers and non-smokers: A meta-analysis

Mounting evidence has suggested somatic mutations in the EGFR gene are associated with better responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in patients with non‐small‐cell lung cancer (NSCLC). Some, but not all, studies have reported that the mutations were more frequently observed in patients without a smoking history. To comprehensively address this issue, we performed a meta‐analysis to evaluate the association between cigarette‐smoking history and mutation of the EGFR gene in NSCLC. Twenty‐six studies, involving 3,688 patients with NSCLC were included in the analysis. The pooled analysis shows that the incidence of EGFR mutations in NSCLC differs according to cigarette‐smoking history. The odds ratio (OR) for the EGFR mutation in non‐smokers relative to smokers was 4.829 (95% confidence interval [CI]: 3.598–6.482; P < 0.001). These data may assist clinicians in assessing the likelihood of EGFR mutations in patients with NSCLC when mutational analysis is not feasible. Environ. Mol. Mutagen. 2012.

Combinational therapy of interferon-α and chemotherapy normalizes tumor vasculature by regulating pericytes including the novel marker RGS5 in melanoma.

Several large randomized clinical trials show that chemotherapy in combination with interferon-&agr; (IFN-&agr;) seems superior to single-agent chemotherapy, such as dacarbazine (DTIC) in melanoma, but the molecular mechanism of this better efficacy is unclear. IFN-&agr; has antiangiogenic activity and could downregulate expression of regulator of G-protein signalling-5 (RGS5) recognized as a novel pericyte marker and a master gene loss of which results in pericyte maturation, vascular normalization, and consequent marked reductions in tumor hypoxia and vessel leakiness in pancreatic carcinoma. Here, we investigated the molecular mechanism of the effects of this combination therapy on melanoma tumor growth. In B16 tumor-bearing mice, the addition of IFN-&agr; to DTIC treatment significantly reduced tumor volume, compared with control or DTIC alone. Consistently, Digital Radiography data showed less chaotic vessel morphology and a decrease in microvessel density and mean vessel diameter in the combinational treatment. Furthermore, the combination therapy showed a remarkable reduction in tumor hypoxia, downregulated RGS5 expression, and increased mature pericyte coverage. Our data suggest that the combination of IFN-&agr; and DTIC therapy more efficiently inhibits tumor growth by normalizing tumor vasculature. This study shows a previously unrecognized role of IFN-&agr; in melanoma vascular normalization and suggests that pericyte including its novel marker RGS5 is an important target of IFN-&agr;.

Beclin1/PI3K-Mediated Autophagy Prevents Hypoxia-Induced Apoptosis in EAhy926 Cell Line

Although hypoxia can induce cell death, the cancer cells and endothelial cells within a solid tumor that remain active in the hypoxia microenvironment often possess an enhanced survival potential. Developing approaches aimed at increasing the sensitivity of endothelial cells to hypoxia-induced cell death represents a potentially important avenue for antiangiogenesis treatment. This study investigated approaches to increase the sensitivity of endothelial cells to hypoxia-induced apoptosis. Autophagy and apoptosis of endothelial cells induced by hypoxia were investigated by transmission electron microscopy, confocallaser microscopy, and western blotting. Moreover, cell invasion was observed by a transwell assay and F-actin quantitative analysis. In this study, it was found that hypoxia could induce both autophagy and apoptosis in hypoxia-inducible factor-1- and Beclin1-dependent endothelial cells. Hypoxia-induced autophagy was prohibited by phosphatidylinositol 3-kinase/Akt inhibitor but not mitogen-activated protein kinase inhibitor. Inhibition of autophagy promoted the rate of apoptosis. Further, the reversal of hypoxia-induced autophagy increased cell migration compared with the normoxia condition. This study concludes that hypoxia triggers a feedback mechanism that delays apoptosis of endothelial cells and that is driven by hypoxia-induced autophagy. Thus, approaches aimed at the disruption of this mechanism can be expected to enhance the susceptibility of endothelial cells to hypoxia-induced apoptosis.