Ruiguang Zhang

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.

Anti-Inflammatory Effects of Tanshinone IIA on Radiation-Induced Microglia BV-2 Cells Inflammatory Response

AIM The aim of this study was to explore the inhibitory effects of Tanshinone II(A) on the production of proinflammation cytokines in radiation-stimulated microglia. METHODS Microglia cells were treated with 2, 4, 8, 16, and 32 Gy of irradiation or sham-irradiated in the presence or absence of 1.0 microg/mL of Tanshinone II(A). The effects of Tanshinone II(A) on radiation-induced proinflammatory cytokines were evaluated by real-time polymerase chain reaction; the expression level of nuclear factor (NF-kappabeta) p65 in cytoplasm and nucleus was measured by Western blot. Immunofluorescence staining and confocal microscopy analysis were applied to detect the expression of gamma-H2AX and p65 postirradiation. RESULTS Radiation-induced release of proinflammatory cytokines in BV-2 cells was detectable after irradiation. Tanshinone II(A) decreased the radiation-induced release of proinflammatory cytokines. Further, Western blotting showed that Tanshinone II(A) could attenuate the nuclear translocation of (NF-kappabeta) p65 submit postirradiation. Immunofluorescence staining showed gamma-H2AX foci formation with p65 translocation into the nucleus postirradiation. CONCLUSIONS Our data indicated that Tanshinone II(A) exerts anti-inflammatory properties by suppressing the transcription of proinflammatory cytokine genes that might be associated with the NF-kappabeta signaling pathway. It is postulated that irradiation causes immediate cellular reaction, and that double-strand breaks trigger the molecular response that leads to NF-kappabeta pathway activation.

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.

MiR-363 sensitizes cisplatin-induced apoptosis targeting in Mcl-1 in breast cancer

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic Bcl-2 family member that is often overexpressed in breast tumors, and has been reported to have an important role in regulating drug resistance in various types of cancer including breast cancer. However, the mechanisms underlying the aberrant expression of Mcl-1 are still unclear. In this study, we used bioinformatics, cellular, and molecular methods to predict and prove that miR-363 directly targeted Mcl-1 3′-UTR (3′-untranslated regions) and caused downregulation of Mcl-1 in breast cancer. Resistance to chemotherapy is a major barrier for the effective treatment for advanced breast cancer, but our study indicated that miR-363 reversed the resistance of the breast cancer cell line MDA-MB-231 to the chemotherapeutic agent cisplatin (CDDP). In addition, transfection of breast cancer cells with Mcl-1 expression plasmid abolished the sensitization effect of miR-363 to cisplatin-inducing cytotoxicity. In summary, our study showed that miR-363 was a negative regulator of Mcl-1 expression, and the combination of miR-363 and cisplatin may be a novel approach in the treatment for breast cancer.

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.

Anticancer activity of SAHA, a potent histone deacetylase inhibitor, in NCI-H460 human large-cell lung carcinoma cells in vitro and in vivo

Suberoylanilide hydroxamic acid (SAHA), a potent pan-histone deacetylase (HDAC) inhibitor, has been clinically approved for the treatment of cutaneous T-cell lymphoma (CTCL). SAHA has also been shown to exert a variety of anticancer activities in many other types of tumors, however, few studies have been reported in large-cell lung carcinoma (LCC). Our study aimed to investigate the potential antitumor effects of SAHA on LCC cells. Here, we report that SAHA was able to inhibit the proliferation of the LCC cell line NCI-H460 in a dose- and time-dependent manner, induced cell apoptosis and G2/M cell cycle arrest, decreased AKT and ERK phosphorylation, inhibited the expression of pro-angiogenic factors (VEGF, HIF-1α) in vitro, and suppressed tumor progression in an NCI-H460 cell nude mouse xenograft model in vivo. These results indicate that SAHA can exert its strong antitumor effects in LCC patient.

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;.

NEMO modulates radiation-induced endothelial senescence of human umbilical veins through NF-κB signal pathway.

Recently several laboratories have reported that radiation induces senescence in endothelial cells. Senescent cells can secrete multiple growth-regulatory proteins, some of which affect tumor growth, survival, invasion or angiogenesis. The purpose of this study was to explore the mechanisms of radiation-induced senescence and its effects on angiogenesis in human umbilical vein endothelial cells (HUVECs). HUVECs were either pretreated with or without PS1145 prior to irradiation with 0–8 Gy. PS1145 is a novel, highly specific small-molecule inhibitor of nuclear factor kappa B essential modulator (NEMO). MTT assays showed that in HUVECs untreated with PS1145, there was an increase in the number of radiation-induced senescence-like endothelial cells 5 days after 8 Gy irradiation, while pretreatment with PS1145 significantly ameliorated the induction in senescence of HUVECs compared to the control group. Electrophoretic mobility shift assay (EMSA) showed that pretreatment with PS1145 inhibited the radiation-induced NF-κB activation, which regulates cell fate in response to genotoxic stress. In addition, Western blotting demonstrated less translocation of p65 from cytoplasm to nucleus. Furthermore, real-time polymerase chain reaction (PCR) showed that pretreatment with PS1145 inhibited the increase of mRNA expressions of interleukin-6 (IL-6) and p53-induced death domain (PIDD) protein, which have been show to play crucial roles in both senescence and apoptosis (P < 0.05). TUNEL staining revealed an increase in apoptotic HUVECs in the group pretreated with PS1145 after irradiation. The series of functional assays further showed that radiation-induced senescence-like HUVECs had malfunctions in migration, invasion and formation of capillary-like structures, compared with the sham-irradiated and untreated, irradiated groups. Taken together, these findings indicate that the angiogenic capacity of radiation-induced senescence-like HUVECs decreased, and that irradiation caused vascular endothelial cells to gain a senescence-like phenotype through the DSB/NEMO/NF-κB signal pathway. The data suggests that NEMO may be a critical switch that regulates cellular senescence and apoptosis caused by exposure to radiation, and provides new clues for the clinical potential of the combination of radiotherapy and angiogenesis inhibitors.

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.