Wendan Yu

Melatonin enhances the anti-tumor effect of fisetin by inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways

Melatonin is a hormone identified in plants and pineal glands of mammals and possesses diverse physiological functions. Fisetin is a bio-flavonoid widely found in plants and exerts antitumor activity in several types of human cancers. However, the combinational effect of melatonin and fisetin on antitumor activity, especially in melanoma treatment, remains unclear. Here, we tested the hypothesis that melatonin could enhance the antitumor activity of fisetin in melanoma cells and identified the underlying molecular mechanisms. The combinational treatment of melanoma cells with fisetin and melatonin significantly enhanced the inhibitions of cell viability, cell migration and clone formation, and the induction of apoptosis when compared with the treatment of fisetin alone. Moreover, such enhancement of antitumor effect by melatonin was found to be mediated through the modulation of the multiply signaling pathways in melanoma cells. The combinational treatment of fisetin with melatonin increased the cleavage of PARP proteins, triggered more release of cytochrome-c from the mitochondrial inter-membrane, enhanced the inhibition of COX-2 and iNOS expression, repressed the nuclear localization of p300 and NF-κB proteins, and abrogated the binding of NF-κB on COX-2 promoter. Thus, these results demonstrated that melatonin potentiated the anti-tumor effect of fisetin in melanoma cells by activating cytochrome-c-dependent apoptotic pathway and inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways, and our study suggests the potential of such a combinational treatment of natural products in melanoma therapy.

Melatonin synergizes the chemotherapeutic effect of 5-fluorouracil in colon cancer by suppressing PI3K/AKT and NF-κB/iNOS signaling pathways.

5‐Fluorouracil (5‐FU) is one of the most commonly used chemotherapeutic agents in colon cancer treatment, but has a narrow therapeutic index limited by its toxicity. Melatonin exerts antitumor activity in various cancers, but it has never been combined with 5‐FU as an anticolon cancer treatment to improve the chemotherapeutic effect of 5‐FU. In this study, we assessed such combinational use in colon cancer and investigated whether melatonin could synergize the antitumor effect of 5‐FU. We found that melatonin significantly enhanced the 5‐FU‐mediated inhibition of cell proliferation, colony formation, cell migration and invasion in colon cancer cells. We also found that melatonin synergized with 5‐FU to promote the activation of the caspase/PARP‐dependent apoptosis pathway and induce cell cycle arrest. Further mechanism study demonstrated that melatonin synergized the antitumor effect of 5‐FU by targeting the PI3K/AKT and NF‐κB/inducible nitric oxide synthase (iNOS) signaling. Melatonin in combination with 5‐FU markedly suppressed the phosphorylation of PI3K, AKT, IKKα, IκBα, and p65 proteins, promoted the translocation of NF‐κB p50/p65 from the nuclei to cytoplasm, abrogated their binding to the iNOS promoter, and thereby enhanced the inhibition of iNOS signaling. In addition, pretreatment with a PI3K‐ or iNOS‐specific inhibitor synergized the antitumor effects of 5‐FU and melatonin. Finally, we verified in a xenograft mouse model that melatonin and 5‐FU exerted synergistic antitumor effect by inhibiting the AKT and iNOS signaling pathways. Collectively, our study demonstrated that melatonin synergized the chemotherapeutic effect of 5‐FU in colon cancer through simultaneous suppression of multiple signaling pathways.

Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556

The normal growth of mycobacteria attributes to the integrity of cell wall core which consists of peptidoglycan (PG), arabinogalactan (AG) and mycolic acids. N-acetyl glucosamine (GlcNAc) is an essential component in both PG and AG of mycobacterial cell wall. The biosynthetic pathway for UDP-N-acetylglucosamine (UDP-GlcNAc), as a sugar donor of GlcNAc, is different in prokaryotes and eukaryotes. The conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is catalyzed by phosphoglucosamine mutase (GlmM), is unique to prokaryotes. Bioinformatic analysis showed that Msm MSMEG_1556 and Mtb Rv3441c are homologous to Ec GlmM. In this study, soluble Msm MSMEG_1556 protein and Mtb Rv3441c protein were expressed in E. coli BL21(DE3) and their phosphoglucosamine mutase activity were detected. In order to further investigate the essentiality of MSMEG_1556 for the growth of M. smegmatis, we generated a conditional MSMEG_1556 knockout mutant, which harbored thermo-sensitive rescue plasmid carrying Mtb Rv3441c. As the rescue plasmid was unable to complement MSMEG_1556 deficiency at 42°C, MSMEG_1556 knockout mutant did not grow. The dramatic morphological changes of MSMEG_1556 knockout mutant after temperature shift from 30°C to 42°C have been observed by scanning electron microscope. These results demonstrated that MSMEG_1556 is essential for growth of M. smegmatis. This study provided evidence that GlmM enzyme could be as a potential target for developing anti-tuberculosis drugs.

BPTF promotes tumor growth and predicts poor prognosis in lung adenocarcinomas

BPTF, a subunit of NURF, is well known to be involved in the development of eukaryotic cell, but little is known about its roles in cancers, especially in non-small-cell lung cancer (NSCLC). Here we showed that BPTF was specifically overexpressed in NSCLC cell lines and lung adenocarcinoma tissues. Knockdown of BPTF by siRNA significantly inhibited cell proliferation, induced cell apoptosis and arrested cell cycle progress from G1 to S phase. We also found that BPTF knockdown downregulated the expression of the phosphorylated Erk1/2, PI3K and Akt proteins and induced the cleavage of caspase-8, caspase-7 and PARP proteins, thereby inhibiting the MAPK and PI3K/AKT signaling and activating apoptotic pathway. BPTF knockdown by siRNA also upregulated the cell cycle inhibitors such as p21 and p18 but inhibited the expression of cyclin D, phospho-Rb and phospho-cdc2 in lung cancer cells. Moreover, BPTF knockdown by its specific shRNA inhibited lung cancer growth in vivo in the xenografts of A549 cells accompanied by the suppression of VEGF, p-Erk and p-Akt expression. Immunohistochemical assay for tumor tissue microarrays of lung tumor tissues showed that BPTF overexpression predicted a poor prognosis in the patients with lung adenocarcinomas. Therefore, our data indicate that BPTF plays an essential role in cell growth and survival by targeting multiply signaling pathways in human lung cancers.

YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma

Y-box binding protein 1 (YBX1) is involved in the multi-tumor occurrence and development. However, the regulation of YBX1 in lung tumorigenesis and the underlying mechanisms, especially its relationship with CDC25a, was remains unclear. In this study, we analyzed the expression and clinical significance of YBX1 and CDC25a in lung adenocarcinoma and identified their roles in the regulation of lung cancer growth. The retrospective analysis of 116 patients with lung adenocarcinoma indicated that YBX1 was positively correlated with CDC25a expression. The Cox-regression analysis showed only high-ranking TNM stage and low CDC25a expression were an independent risk factor of prognosis in enrolled patients. High expression of YBX1 or CDC25a protein was also observed in lung adenocarcinoma cells compared with HLF cells. ChIP assay demonstrated the binding of endogenous YBX1 to the CDC25a promoter region. Overexpression of exogenous YBX1 up-regulated the expression of the CDC25a promoter-driven luciferase. By contrast, inhibition of YBX1 by siRNA markedly decreased the capability of YBX1 binding to CDC25a promoter in A549 and H322 cells. Inhibition of YBX1 expression also blocked cell cycle progression, suppressed cell proliferation and induced apoptosis via the CDC25a pathway in vitro. Moreover, inhibition of YBX1 by siRNA suppressed tumorigenesis in a xenograft mouse model and down-regulated the expression of YBX1, CDC25a, Ki67 and cleaved caspase 3 in the tumor tissues of mice. Collectively, these results demonstrate inhibition of YBX1 suppressed lung cancer growth partly via the CDC25a pathway and high expression of YBX1/CDC25a predicts poor prognosis in human lung adenocarcinoma.

MED27 promotes melanoma growth by targeting AKT/MAPK and NF-κB/iNOS signaling pathways

The inhibitors of BRAF and MEK targeting MAPK signaling pathway provide a comparatively effective therapeutic strategy for melanoma caused by BRAF mutation. However, melanoma, especially metastatic melanoma, has become one of the most threatening malignancies. Thus, the identification of exact molecular mechanisms and the key components involved in such mechanisms is urgently needed in order to provide new therapeutic options for patients with melanoma. Here, we identified MED27 as a potential melanoma target and explored its role and the associated molecular mechanism involved in melanoma progression. MED27 was found to be highly expressed in melanoma cells and tumor tissues. Its silencing led to melanoma cell proliferation inhibition, cell cycle arrest and apoptosis induction accompanied by the inactivation of PI3K/AKT and MAPK/ERK signaling and the activation of Bax/Cyto-C/Caspase-dependent apoptotic pathway. In addition, silencing of MED27 led to the decrease of iNOS expression through inhibiting the activation of a serial of upstream key proteins of NF-κB signaling pathway and the translocation of p50/p65 from cytoplasm to nucleus. MED27 was also found to be able to interact with NF-κB and p300 and to be acetylated by p300. Furthermore, the results in a xenograft tumor model indicated that melanoma progression was effectively suppressed by MED27 knockdown accompanied by the down-regulation of p-AKT, p-ERK, p-MEK1/2, MMP-9, Bcl-2 and iNOS expressions in the tumor tissues. Taken together, our study not only demonstrated the new function of MED27 as an oncogenic protein and the associated molecular mechanisms involved in melanoma progression, but also provided a possibility for the development of MED27 as a new anticancer target in melanoma therapy.

Cleavage and polyadenylation specific factor 4 targets NF-κB/cyclooxygenase-2 signaling to promote lung cancer growth and progression

Overexpression of cyclooxygenase 2 (COX-2) is frequently found in early and advanced lung cancers. However, the precise regulatory mechanism of COX-2 in lung cancers remains unclear. Here we identified cleavage and polyadenylation specific factor 4 (CPSF4) as a new regulatory factor for COX-2 and demonstrated the role of the CPSF4/COX-2 signaling pathway in the regulation of lung cancer growth and progression. Overexpression or knockdown of CPSF4 up-regulated or suppressed the expression of COX-2 at mRNA and protein levels, and promoted or inhibited cell proliferation, migration and invasion in lung cancer cells. Inhibition or induction of COX-2 reversed the CPSF4-mediated regulation of lung cancer cell growth. Cancer cells with CPSF4 overexpression or knockdown exhibited increased or decreased expression of p-IKKα/β and p-IκBα, the translocation of p50/p65 from the cytoplasm to the nucleus, and the binding of p65 on COX-2 promoter region. In addition, CPSF4 was found to bind to COX-2 promoter sequences directly and activate the transcription of COX-2. Silencing of NF-κB expression or blockade of NF-κB activity abrogated the binding of CPSF4 on COX-2 promoter, and thereby attenuated the CPSF4-mediated up-regulation of COX-2. Moreover, CPSF4 was found to promote lung tumor growth and progression by up-regulating COX-2 expression in a xenograft lung cancer mouse model. CPSF4 overexpression or knockdown promoted or inhibited tumor growth in mice, while such regulation of tumor growth mediated by CPSF4 could be rescued through the inhibition or activation of COX-2 signaling. Correspondingly, CPSF4 overexpression or knockdown also elevated or attenuated COX-2 expression in tumor tissues of mice, while treatment with a COX-2 inducer LPS or a NF-κB inhibitor reversed this elevation or attenuation. Furthermore, we showed that CPSF4 was positively correlated with COX-2 levels in tumor tissues of lung cancer patients. Simultaneous high expression of CPSF4 and COX-2 proteins predicted poor prognosis of patients with lung cancers. Our results therefore demonstrated a novel mechanism for the transcriptional regulation of COX-2 by CPSF4 in lung cancer, and also offer a potential therapeutic target for lung cancers bearing aberrant activation of CPSF4/COX-2 signaling.

RFPL3 and CBP synergistically upregulate hTERT activity and promote lung cancer growth

hTERT is the key component of telomerase and its overactivation contributes to maintaining telomere length and cell immortalization. Previously, we identified RFPL3 as a new transcription activator of hTERT in lung cancers. However, the exact mechanism of RFPL3 in mediating hTERT activation and its associated signal regulatory network remain unclear. In this study, we found that RFPL3 colocalized and interacted directly with CBP in the nucleus of lung cancer cells. Immunohistochemical analysis of tissue microarrays of lung cancers revealed the simultaneous overexpression of both RFPL3 and CBP predicted relatively poor prognosis. Furthermore, we confirmed their synergistic stimulation on hTERT expression and tumor cell growth. The binding of RFPL3 to hTERT promoter was reduced markedly when CBP was knocked down by its specific siRNA or suppressed by its inhibitor in lung cancer cells with stable overexpression of RFPL3. When one of the two proteins RFPL3 and CBP was upregulated or downregulated, whereas the another remains unchanged, hTERT expression and telomerase activity were activated or repressed accordingly. In the meantime, the growth of lung cancer cells was also promoted or attenuated accordingly. Furthermore, we also found that RFPL3 coordinated with CBP to upregulate hTERT through the CBP-induced acetylation of RFPL3 protein and their co-anchoring at hTERT promoter region. Collectively, our results reveal a new mechanism of hTERT regulation in lung cancer cells and suggest the RFPL3/CBP/hTERT signaling pathway may be a new targets for lung cancer treatment.

High expression of Y-box-binding protein 1 correlates with poor prognosis and early recurrence in patients with small invasive lung adenocarcinoma

Background Prognosis of small (≤2 cm) invasive lung adenocarcinoma remains poor, and identification of high-risk individuals from the patients after complete surgical resection of lung adenocarcinoma has become an urgent problem. YBX1 has been reported to be able to predict prognosis in many cancers (except lung adenocarcinoma) that are independent of TNM (tumor, nodes, metastases) staging, especially small invasive lung adenocarcinoma. Therefore, we examined the significance of YBX1 expression on prognosis and recurrence in patients with small invasive lung adenocarcinoma. Material and methods A total of 75 patients with small invasive lung adenocarcinoma after complete resection were enrolled from January 2008 to December 2010. Immunohistochemical staining was used to detect the expression of YBX1, and receiver operating characteristic curve analysis was performed to precisely assess the overall expression of YBX1. Meanwhile, primary lesions were identified based on the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society’s classification of lung adenocarcinoma. The effect of different clinicopathological factors on patients’ survival was examined. Furthermore, Western blot analysis was used to show the expression of YBX1 in vitro. Results Sensitivity and specificity of YBX1 for detecting small invasive lung adenocarcinoma from normal surrounding tissue were 66.7% and 74.7% (area under the receiver operating characteristic curve =0.731; P<0.001), respectively. High YBX1 expression was detected in 31 (41.3%) patients, and in A549, H322, Hcc827, and H1299 lung adenocarcinoma cells but not in HLF cells. In addition to sex, age, tumor size, TNM staging, pleural invasion, and lymph node metastasis, the expression of YBX1 was associated with the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society pathological grade risk (P=0.026) and differentiation (P=0.009). The patients with low YBX1 expression lived longer than those with high expression (5-year overall survival: 52.3% vs 79.0%; P=0.039) and showed fewer recurrences (P=0.024). In multivariate analyses, high YBX1 expression (odds ratio =2.737; 95% confidence interval: 1.058–7.082; P=0.038) was shown as an independent risk factor of overall survival but not of disease-free survival (odds ratio =1.696; 95% confidence interval: 0.616–4.673; P=0.307). Conclusion YBX1 is an important predictor for the prognosis in patients with small invasive lung adenocarcinoma after complete resection.

CREB-binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

CBP (CREB‐binding protein) is a transcriptional co‐activator which possesses HAT (histone acetyltransferases) activity and participates in many biological processes, including embryonic development, growth control and homeostasis. However, its roles and the underlying mechanisms in the regulation of carcinogenesis and tumor development remain largely unknown. Here we investigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells. Elevated expression of CBP was detected in lung cancer cells and tumor tissues compared to the normal lung cells and tissues. Knockdown of CBP by siRNA or inhibition of its HAT activity using specific chemical inhibitor effectively suppressed cell proliferation, migration and colony formation and induced apoptosis in lung cancer cells by inhibiting MAPK and activating cytochrome C/caspase‐dependent signaling pathways. Co‐immunoprecipitation and immunofluorescence analyses revealed the co‐localization and interaction between CBP and CPSF4 (cleavage and polyadenylation specific factor 4) proteins in lung cancer cells. Knockdown of CPSF4 inhibited hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating the synergetic effect of CBP and CPSF4 in the regulation of lung cancer cell growth and survival. Moreover, we found that high expression of both CBP and CPSF4 predicted a poor prognosis in the patients with lung adenocarcinomas. Collectively, our results indicate that CBP regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathways.