Feng-Hou Gao

JAK2/STAT3 signaling pathway activation mediates tumor angiogenesis by upregulation of VEGF and bFGF in non-small-cell lung cancer

We investigated the clinical significance of Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) during angiogenesis in non-small-cell lung carcinoma. JAK2, phospho-JAK2 (pJAK2), STAT3, and phospho-STAT3 (pSATA3) were observed in 40/68 (58.8%), 39/68 (57.4%), 49/68 (72.1%) and 40/68 (58.8%) of the cases. The high expression levels of molecules involved in the JAK2/STAT3 signaling pathway were associated with a decreased survival rate. Of the total number of cases, 73.5% were positive for VEGF and 80.9% for bFGF. Microvessel density (MVD), as determined by CD34 staining and morphology, was higher in NSCLC samples with high pJAK2 and pSTAT3 expression, and the patients with high MVD had poor survival status. In addition, the expression of pSTAT3 correlated with VEGF (r=0.593) and bFGF (r=0.519) (p<0.05). Inhibiting JAK2 and knocking down STAT3 both suppressed STAT3 activation and reduced the expression of VEGF and bFGF in A549 and NCI-H292 cells, demonstrating that STAT3 activation was associated with VEGF and bFGF expression in the two human lung carcinoma cell lines. Therefore, STAT3 may be a critical molecular target for powerful intervention in NSCLC anti-angiogenesis therapy.

Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

BackgroundOridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer.MethodsEffects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice.ResultsOridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression.ConclusionOridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.

Enhancing chemotherapeutic drug inhibition on tumor growth by ultrasound: an in vivo experiment

An in vivo study on enhancing epirubicin hydrochloride (EPI) inhibition on tumor growth by ultrasound (US) was reported. Five-week-old male nude mice were used and HL-60 cells were s.c. (subcutaneous injection) inoculated in axilla of these mice. Six groups were designed and five consecutive treatments were applied to investigate the inhibition on tumor growth and body weight growth. US applied locally to the tumor resulted in a substantially increased drug uptake in tumor cells. The inhibition on tumor growth depended on the position of drug injection and phospholipid-based microbubble (PMB) application. Tumor growth rate under group 1 (PMB+US) was similar to that of blank control. The order of the inhibition on tumor volume growth was: group 4 (s.c. EPI+PMB+US) > group 5 intraperitoneal (i.p. EPI+PMB+US) > group 2 (i.p. EPI) > group 3 (s.c. EPI+US) > group 1 (PMB+US). Similar conclusion was obtained from experimental measurements of tumor weight change. The order of animal survival status for EPI administration groups was: group 4 > group 5 > group 2 > group 3. Chemotherapeutic drug inhibition on tumor growth could be enhanced by local US combined with PMB, which might provide a potential application for US-mediated chemotherapy.

Apoptosis inducing and differentiation enhancement effect of oridonin on the all‐trans‐retinoic acid‐sensitive and ‐resistant acute promyelocytic leukemia cells

We investigated the effects of oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, on apoptosis and differentiation of all‐trans‐retinoic acid (ATRA)‐sensitive (NB4) and ATRA‐resistant (NB4‐R1) cells. The results showed that reactive oxygen species initiates Ori‐induced apoptosis. In addition, we found that neither Ori nor ATRA (10 nm) alone induced marked cell differentiation, while co‐treatment of these two compounds can induce differentiation of NB4 and NB4‐R1 cells which was accompanied by increased RARα, C/EBPε or C/EBPβ. This is the first report to show that RARα could be accumulated by Ori which may be useful as a probe to investigate the mechanism of RARα catabolism. These results suggest that Ori is a potential candidate for acute promyelocytic leukemia cancer therapy.

Tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts by the inhibition of superoxide anion production and glutathione depletion.

Background/Objectives:  Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may induce an irreversible growth arrest similar to senescence. Tiron, 4,5‐dihydroxy‐1,3‐benzene disulfonic acid, is a widely used antioxidant to rescue ROS‐evoked cell death. The aim of the article was to explore the effects of tiron on skin photoaging and associated mechanisms.

Abnormal activation of the EGFR signaling pathway mediates the downregulation of miR‑145 through the ERK1/2 in non-small cell lung cancer.

The expression of miR-145 with tumor suppressor function is decreased in lung cancer cells. Epidermal growth factor receptor (EGFR) signaling pathway is abnormally activated in lung cancer cells. It is not clear whether the EGFR signaling pathway is involved in the regulation of miR-145 expression in lung cancer. In the present study, we found that the reduction of miR-145 was associated with EGFR abnormal activation in lung cancer cells. AG1478, an inhibitor of EGFR, may restore the expression of miR-145, indicating that EGFR activation is involved in the downregulation of miR-145 in lung cancer cells. Then, the application of STAT3, AKT and ERK1/2 inhibitors and siRNA against these signaling molecules indicated that ERK1/2 or AKT instead of STAT3 was involved in the process of miR-145 downregulation by EGFR. It was confirmed that AKT through activation of the ERK1/2 signaling molecules mediated the effect of EGFR on miR-145. Furthermore, we found that EGFR downregulated miR-145 through ERK1/2 in lung cancer cells. These findings establish EGFR and miR-145 links in lung cancer cells and therefore contribute to a better understanding of the role of EGFR in lung cancer cells, and provide clues for in-depth study of miR-145 expression and a possible direction for the further increase of miR-145 in lung cancer cells.

Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts

Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced.

USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells.

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer.

Oridonin enhances the anticancer activity of NVP-BEZ235 against neuroblastoma cells in vitro and in vivo through autophagy

The aberrant activation of PI3K/Akt/mTOR signaling pathway plays an important role in the oncogenesis, prognosis and chemotherapy resistance of neuroblastoma. However, NVP-BEZ235, a potent dual PI3K and mTOR inhibitor have not shown beneficial effects on neuroblastoma especially in terms of apoptosis induction as a single agent. We therefore attempted to explore an effective combination regimen to enhance the anticancer activity of NVP-BEZ235. Interestingly, we found that oridonin, a natural biologically active compound extracted from the Chinese medicinal herb Rabdosia rubescens, combined with NVP-BEZ235 markedly induced apoptosis of neuroblastoma cells. Notably, the synergistic activation of the apoptotic pathway was accompanied with enhanced autophagy as evidenced by significant decreased p62 expression as well as upregulated conversion of LC3-II. Suppression of the Beclin-1, a core component of the autophagy machinery, by means of shRNA resulted in diminished synergistic antitumor effect. Furthermore, the co-treatment with oridonin and NVP-BEZ235 was also much more effective than either agent alone in inhibiting the growth of neuroblastoma xenografts and in inducing tumor cells apoptosis. Taken together, our results suggest that the combination of NVP-BEZ235 and oridonin is a novel and potential strategy for neuroblastoma therapy.

GSK3β-dependent cyclin D1 and cyclin E1 degradation is indispensable for NVP-BEZ235 induced G0/G1 arrest in neuroblastoma cells

ABSTRACT Cyclin D1 and cyclin E1, as vital regulatory factors of G1-S phase cell cycle progression, are frequently constitutive expressed and associated with pathogenesis and tumorigenesis in most human cancers and they have been regarded as promising targets for cancer therapy. In this study, we established NVP-BEZ235, a potent dual kinase inhibitor, could induce neuroblastoma cells proliferation inhibition without apoptosis activation. Moreover, we showed NVP-BEZ235 could induce neuroblastoma cells arrested at G0/G1 phase accompanied with significant reduction of the cyclin D1 and E1 proteins in a dose dependent manner at nanomole concentration. Additionally we found that GSK3β was dephosphorylated and activated by NVP-BEZ235 and then triggered cyclin D1 and cyclin E1 degradation through ubiquitination proteasome pathway, based on the evidences that NVP-BEZ235 induced downregulation of cyclin D1 and cyclin E1 were obviously recovered by proteasome inhibitor and the blockade of GSK3β contributed to remarkable rescue of cyclin D1 and cyclin E1. Analogous results about its anti-proliferation effects and molecular mechanism were observed on neuroblastoma xenograft mouse model in vivo. Therefore, these results indicate that NVP-BEZ235-induced cyclin D1 and cyclin E1 degradation, which happened through activating GSK3β, and GSK3β-dependent down-regulation of cyclin D1 and cyclin E1 should be available for anticancer therapeutics.