Novel regulation of miR-34a-5p and HOTAIR by the combination of berberine and gefitinib leading to inhibition of EMT in human lung cancer.

HOTAIR is an important carcinogenic lncRNA and involves in tumorigenesis, and invasion. MiR-34a-5p functions as a tumour suppressor. However, the underlying mechanism of HOTAIR regulation especially in association with miR-34a-5p in non-small-cell lung cancer (NSCLC) has not been explored. Herein, we performed series of in vitro experiments, including viability, migration, invasion, apoptosis and in vivo xenograft model, and identified that HOTAIR was remarkably elevated in NSCLC cells. Enforced HOTAIR expression promoted migration and invasion, while depleted HOTAIR diminished the ability of migration and invasion of NSCLC cells. We also observed that miR-34a-5p was dramatically inhibited in NSCLC cells and the binding correlation between HOTAIR and miR-34a-5p was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. We also showed that induction of miR-34a-5p and reduction of HOTAIR, and the interaction between miR-34a-5p and HOTAIR resulted in the suppression of epithelial-mesenchymal transition (EMT) as illustrated by induction of key epithelial markers E-cadherin expression, reduction of vimentin and EMT-inducing transcription factor snail. Excessive expression of snail resisted miR-34a-5p-inhibited cell growth. Snail binds to E-cadherin promoter and regulates E-cadherin expression. There was a synergy in combination of berberine and gefinitib in this process. Similar findings were also observed in a tumour xenograft model. Collectively, this is the first report demonstrating reciprocal interaction of miR-34a-5p- and HOTAIR-mediated regulation of snail resulting in inhibition of EMT process by the combination of berberine and gefitinib suggesting that regulation of miR-34a-5p- and HOTAIR-mediated inhibition of EMT may provide novel treatment paradigms for lung cancer.

The regulation and interaction of colon cancer-associated transcript-1 and miR7-5p contribute to the inhibition of SP1 expression by solamargine in human nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a common head and neck malignancy with higher incidence in Southern China and Southeast Asia. Solamargine (SM), a steroidal alkaloid glycoside, has been shown to have anticancer properties. However, the underlying mechanism involved remains undetermined. In this study, we showed that SM inhibited the growth of NPC cells. Mechanistically, we found that solamargine decreased lncRNA colon cancer-associated transcript-1 (CCAT1) and increased miR7-5p expression. There was a reciprocal interaction of CCAT1 and miR7-5p. In addition, SM inhibited the expression of SP1 protein and promoter activity, which was strengthened by miR7-5p mimics and inhibited by overexpressed CCAT1. MiR7-5p could bind to 3'-UTR of SP1 and attenuated SP1 gene expression. Exogenously expressed SP1 feedback resisted SM-increased miR7-5p expression and more importantly reversed SM-inhibited growth of NPC cells. Finally, SM inhibited NPC tumor growth in vivo. Collectively, our results show that SM inhibits the growth of NPC cells through reciprocal regulation of CCAT1 and miR7-5p, followed by inhibition of SP1 gene expression in vitro and in vivo. The interregulation and correlation among CCAT1, miR7-5p and SP1, and the feedback regulatory loop unveil the novel molecular mechanism underlying the overall responses of SM in anti-NPC.

Novel reciprocal interaction of lncRNA HOTAIR and miR-214-3p contribute to the solamargine-inhibited PDPK1 gene expression in human lung cancer.

Solamargine (SM) has been shown to have anti-cancer properties. However, the underlying mechanism involved remains undetermined. We showed that SM inhibited the growth of non-small cell lung cancer (NSCLC) cells, which was enhanced in cells with silencing of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR), while it overcame by overexpression of HOTAIR. In addition, SM increased the expression of miR-214-3p and inhibited 3-phosphoinositide-dependent protein kinase-1 (PDPK1) gene expression, which was strengthened by miR-214-3p mimics. Intriguingly, HOTAIR could directly bind to miR-214-3p and sequestered miR-214-3p from the target gene PDPK1. Intriguingly, overexpression of PDPK1 overcame the effects of SM on miR-214-3p expressions and neutralized the SM-inhibited cell growth. Similar results were observed in vivo. In summary, our results showed that SM-inhibited NSCLC cell growth through the reciprocal interaction between HOTAIR and miR-214-3p, which ultimately suppressed PDPK1 gene expression. HOTAIR effectively acted as a competing endogenous RNA (ceRNA) to stimulate the expression of target gene PDPK1. These complex interactions and feedback mechanisms contribute to the overall effect of SM. This unveils a novel molecular mechanism underlying the anti-cancer effect of SM in human lung cancer.

The enhancement of combination of berberine and metformin in inhibition of DNMT1 gene expression through interplay of SP1 and PDPK1.

Berberine (BBR), one of active alkaloid found in the rhizome, exhibited anti-cancer properties. We have showed that BBR inhibited growth of non-small cell lung cancer (NSCLC) cells through mitogen-activated protein kinase (MAPK)-mediated increase in forkhead box O3a (FOXO3a). However, the in-depth mechanism underlying the anti-tumor effects still remained to be elucidated. Herein, we further confirmed that BBR not only induced cell cycle arrest, but also reduced migration and invasion of NSCLC cells. Mechanistically, we observed that BBR reduced 3-phosphoinositide-dependent protein kinase-1 (PDPK1) and transcription factor SP1 protein expressions. Exogenously expressed SP1 overcame BBR-inhibited PDPK1 expression. Moreover, BBR inhibited DNA methyltransferase 1 (DNMT1) gene expression and overexpressed DNMT1 resisted BBR-inhibited cell growth. Intriguingly, overexpressed PDPK1 antagonized BBR-inhibited SP1 and DNMT1 expressions. Finally, metformin enhanced the effects of BBR both in vitro and in vivo. Collectively, we observe that BBR inhibits proliferation of NSCLC cells through inhibition of SP1 and PDPK1; this results in a reduction of DNMT1 expression. The interplay of PDPK1 and SP1 contributes to the inhibition of DNMT1 in response to BBR. In addition, there is a synergy of BBR and metformin. This study uncovers a new mechanism of BBR in combination with metformin for NSCLC-associated therapy.

HOTAIR: An Oncogenic Long Non-Coding RNA in Human Cancer.

Long non-coding RNAs (LncRNAs) represent a novel class of noncoding RNAs that are longer than 200 nucleotides without protein-coding potential and function as novel master regulators in various human diseases, including cancer. Accumulating evidence shows that lncRNAs are dysregulated and implicated in various aspects of cellular homeostasis, such as proliferation, apoptosis, mobility, invasion, metastasis, chromatin remodeling, gene transcription, and post-transcriptional processing. However, the mechanisms by which lncRNAs regulate various biological functions in human diseases have yet to be determined. HOX antisense intergenic RNA (HOTAIR) is a recently discovered lncRNA and plays a critical role in various areas of cancer, such as proliferation, survival, migration, drug resistance, and genomic stability. In this review, we briefly introduce the concept, identification, and biological functions of HOTAIR. We then describe the involvement of HOTAIR that has been associated with tumorigenesis, growth, invasion, cancer stem cell differentiation, metastasis, and drug resistance in cancer. We also discuss emerging insights into the role of HOTAIR as potential biomarkers and therapeutic targets for novel treatment paradigms in cancer.

Functions and Roles of Long-Non-Coding RNAs in Human Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common cancers originating in the nasopharynx and occurring at high frequency in South-eastern Asia and North Africa. Long non-coding RNAs (lncRNAs) are a class of non-protein-coding RNA molecules and key regulators of developmental, physiological, and pathological processes in humans. Emerging studies have shown that lncRNAs play critical roles in tumorgenicity and cancer prognosis. With the development of deep sequencing analyses, an extensive amount of functional lncRNAs have been discovered in nasopharyngeal carcinoma tissues and cell lines. However, the roles and mechanisms of aberrantly expressed lncRNAs in the pathogenesis of NPC are not fully understood. In this review, we briefly illustrate the concept, identification, functional characterization, and summarize recent advancements of biological functions of lncRNAs with heterogeneous mechanistic characterization and their involvement in NPC. Then, we describe individual lncRNAs that have been associated with tumorgenesis, growth, invasion, cancer stem cell differentiation, metastasis, drug resistance and discuss the strategies of their therapeutic manipulation in NPC. We also review the emerging insights into the role of lncRNAs and their potential as biomarkers and therapeutic targets for novel treatment paradigms. Finally, we highlight the up-to-date of clinical information involving lncRNAs and future directions in the linking lncRNAs to potential gene therapies, and how modifications of lncRNAs can be exploited for prevention and treatment of NPC.

Crosstalk of NF-κB/P65 and LncRNA HOTAIR-Mediated Repression of MUC1 Expression Contribute to Synergistic Inhibition of Castration-Resistant Prostate Cancer by Polyphyllin 1-Enzalutamide Combination Treatment.

BACKGROUND/AIMS: Polyphyllin I (PPI), one of the steroidal saponins in Paris polyphylla, reportedly exhibits antitumor effects. However, the detailed mechanism underlying PPI, particularly in enhancing the effect of the androgen receptor inhibitor enzalutamide in controlling castration-resistant prostate cancer (CRPC) has not been explored. METHODS: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) expression was measured by quantitative real time-PCR (qRT-PCR). Western blot analysis was performed to determine the protein expression levels of MUC1, p65, and p50. Silencing of HOTAIR was evaluated using the siRNA procedure. The promoter activity of the MUC1 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. Exogenous expression of HOTAIR, p65, and MUC1 was conducted by transient transfection assay. A xenograft tumor model in nude mice was used to further evaluate the effect of the combination of PPI and enzalutamide in vivo. RESULTS: We showed that PPI significantly inhibited growth and induced cell cycle arrest in CRPC cells. PPI also decreased p65 and MUC1 protein expression and reduced HOTAIR expression. Exogenously expressed p65 resisted the PPI-inhibited expression of HOTAIR, whereas silenced HOTAIR reduced MUC1 protein but exerted no effect on the expression of p65 and p50 proteins. Conversely, exogenously expressed HOTAIR resisted the PPI-inhibited MUC1 protein expression, and excessive expression of MUC1 antagonized the PPI-inhibited cell growth. Notably, PPI combined with enzalutamide exerted a synergistic effect. Consistent with this finding, PPI inhibited tumor growth, HOTAIR expression, as well as p65 and MUC1 protein expressions in vivo. CONCLUSIONS: Our results indicate that PPI inhibits the growth of CRPC cells by inhibiting p65 protein and concomitantly reducing HOTAIR expression, thereby suppressing MUC1 gene expression. The novel regulatory interaction of p65 and HOTAIR converge in the inhibition of MUC1 expression and overall PPI response. The combination of PPI and enzalutamide exhibits synergy. This study reveals a novel mechanism underlying the synergistic inhibitory effect of PPI and enzalutamide on the growth of CRPC cells.

Repression of PDK1- and LncRNA HOTAIR-Mediated EZH2 Gene Expression Contributes to the Enhancement of Atractylenolide 1 and Erlotinib in the Inhibition of Human Lung Cancer Cells.

BACKGROUND/AIMS: We previously showed that the major bioactive compound of Atractylodes macrocephula Koidz atractylenolide 1 (ATL-1) inhibited human lung cancer cell growth by suppressing the gene expression of 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1). However, the potentially associated molecules and downstream effectors of PDK1 underlying this inhibition, particularly the mechanism for enhancing the anti-tumor effects of epidermal growth factor receptor-tyrosine-kinase inhibitors (EGFR-TKIs), remain unknown. METHODS: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Western blot analyses were performed to examine the protein expressions of PDK1 and of zeste homolog 2 (EZH2). The levels of long non-coding RNA (lncRNA) and HOX transcript antisense RNA (HOTAIR) were examined via qRT-PCR. RNA-binding protein immunoprecipitation assays were used to analyze HOTAIR interaction with EZH2. The promoter activity of the EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. Exogenous expressions of PDK1, HOTAIR, and EZH2 were conducted via transient transfection assays. A xenografted tumor model was used to further evaluate the effect of ATL-1 in the presence or absence of erlotinib in vivo. RESULTS: We showed that the combination of ATL-1 and EGFR-TKI erlotinib further inhibited growth and induced cell arrest of the human lung cancer cells, determined by both MTT and flow cytometry assays. ATL-1 inhibited the protein expression and the promoter activity of EZH2, which was reversed in cells with PDK1 overexpression. Interestingly, ATL-1 inhibited the expression levels of HOTAIR. While silencing HOTAIR inhibited the expressions of PDK1 and EZH2, overexpression of HOTAIR reduced the ATL-1-reduced PDK1 and EZH2 protein expressions and EZH2 promoter activity. In addition, ATL-1 reduced the HOTAIR binding to the EZH2 protein. Moreover, we found that exogenously expressed EZH2 antagonized the effect of ATL-1 on cell growth inhibition. Consistent with the in vitro results, ATL-1 inhibited tumor growth and the expression levels of HOTAIR, protein expressions of EZH2 and PDK1 in vivo. Importantly, there was synergy of the combination of ATL-1 and erlotinib in this process. CONCLUSION: Here, we provide the first evidence that ATL-1 inhibits lung cancer cell growth through inhibiting not only the PDK1 but also the lncRNA HOTAIR, which results in the reduction of one downstream effector EZH2 expression. The novel interplay between the HOTAIR and EZH2, as well as repressions of the PDK1 and HOTAIR coordinate the overall effects of ATL-1. Importantly, the combination of ATL-1 and EGFR-TKI erlotinib exhibits synergy. Thus, targeting the PDK1- and HOTAIR-mediated downstream molecule EZH2 by the combination of ATL-1 and erlotinib potentially facilitates the development of an additional novel strategy to combat lung cancer.

HOTAIR-mediated reciprocal regulation of EZH2 and DNMT1 contribute to polyphyllin I-inhibited growth of castration-resistant prostate cancer cells in vitro and in vivo.

BACKGROUND: Polyphyllin I (PPI), one of the steroidal saponins in paris polyphylla, has been reported to exhibit antitumor effects. However, the detailed molecular mechanism underlying this has not been elucidated. METHODS: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Flow cytometry assays, respectively. Cell invasion and migration were examined by Transwell invasion and wound healing assays. Western blot analysis was performed to examine the protein expressions of zeste homolog 2 (EZH2), DNA methyltransferase 1 (DNMT1). QRT-PCR was used to examine the levels of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR). Small interfering RNAs (siRNAs) method was used to knockdown HOTAIR. Exogenously expressions of HOTAIR, DNMT1 and EZH2 were carried out by Transient transfection assays. EZH2 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro. RESULTS: We showed that PPI significantly inhibited growth, induced cell cycle arrest of castration-resistant prostate cancer (CRPC) cells. In addition, PPI also reduced the migration and invasion in CRPC cells. In mechanism, we found that PPI decreased the protein expressions of EZH2, DNMT1 and levels of HOTAIR. Interestingly, silenced HOTAIR reduced EZH2 and DNMT1 protein expressions. On the contrary, exogenously expressed HOTAIR resisted PPI-inhibited EZH2 and DNMT1 protein expressions, EZH2 promoter activity and cell growth. Moreover, excessive EZH2 antagonized PPI-suppressed DNMT1 protein expression or vice versa. Consistent with this, PPI inhibited tumor growth, HOTAIR, the protein expressions of DNMT1 and EZH2 in vivo. CONCLUSION: Our results show that PPI inhibits growth of CRPC cells through inhibition of HOTAIR expression, subsequently; this results in the repression of DNMT1 and EZH2 expressions. The interactions among HOTAIR, DNMT1 and EZH2, and reciprocal regulation of DNMT1 and EZH2 contribute to the overall responses of PPI. This study reveals a novel mechanism for HOTAIR-mediated regulating DNMT1 and EZH2 in response to PPI in inhibition of the growth of CRPC cells.

WITHDRAWN: Emodin increases expression of insulin-like growth factor binding protein 1 through activation of MEK/ERK/AMPKα and interaction of PPARγ and Sp1 in lung cancer.

Due to an error in the publishing process, this article has been withdrawn at the request of the editors. We wish to clarify that this is in no way related to the integrity of the authors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Emodin Increases Expression of Insulin-Like Growth Factor Binding Protein 1 through Activation of MEK/ERK/AMPKα and Interaction of PPARγ and Sp1 in Lung Cancer.

BACKGROUND: Emodin has anti-neoplastic activities on multiple tumors. However, the molecular mechanisms underlying this effect still remain to be fully understood. METHODS: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays and flow cytometry, respectively. Cell invasion and migration were examined by transwell invasion and wound healing assays. Western blot analysis was performed to examine the phosphorylation and protein expression of AMP-activated protein kinase alpha (AMPKα), extracellular signaling-regulated kinase 1/2 (ERK1/2), peroxisome proliferators-activated receptor gamma (PPARγ), insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and the transcription factor Sp1. QRT-PCR was used to examine the mRNA levels of the IGFBP1 gene. Small interfering RNAs (siRNAs) were used to knockdown PPARγ and IGFBP1 genes. Exogenously expression of IGFBP1 and Sp1 was determined by transient transfection assays. IGFBP1 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings. RESULTS: We showed that emodin induced cell cycle arrest of NSCLC cells. Emodin increased PPARγ protein and luciferase reporter activity, which were abolished by inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK)/ERK and AMPK. Silencing of PPARγ abrogated emodin-inhibited cell growth and cell cycle arrest. Furthermore, emodin elevated IGFBP1 mRNA, protein, and promoter activity through activation of PPARγ. Intriguingly, overexpressed Sp1 attenuated emodin-induced IGFBP1 expression, which was not observed in cells with silenced PPARγ gene. Moreover, silencing of IGFBP1 gene blunted emodin-induced inhibition of cell growth and cell cycle arrest. On the contrary, overexpressed IGFBP1 enhanced emodin-induced phosphorylation of AMPKα and ERK1/2, and restored emodin-inhibited growth in cells with silenced endogenous IGFBP1 gene. Emodin also inhibited growth of lung xenograft tumors and Sp1, and increased IGFBP1 and PPARγ protein expressions In vivo. CONCLUSION: Collectively, our results show that emodin inhibits growth of non-small-cell lung cancer (NSCLC) cells through ERK and AMPKα-mediated induction of PPARγ, followed by reduction of Sp1. This in turn induces IGFBP1 gene expression. Thus, the signaling cascades, positive feedback loop and cooperative interplay between transcription factors-induced the expression of IGFBP1 gene contribute to the overall responses of emodin. This study provides a novel mechanism by which emodin inhibits growth of human lung cancer cells.

Combination of Solamargine and Metformin Strengthens IGFBP1 Gene Expression Through Inactivation of Stat3 and Reciprocal Interaction Between FOXO3a and SP1.

BACKGROUND/AIMS: Solamargine, one natural photochemical component from traditional plants, has been shown to have anti-cancers properties. We previously showed that solamargine inhibited the growth of non-small-cell lung cancer (NSCLC) cells through suppression of prostaglandin E2 (PGE2) receptor EP4 gene and regulation of downstream signaling pathways. However, the detailed mechanism underlying this, especially in combination of metformin, a known AMPK activator, still remained to be determined. METHODS: Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colorimetric 5-bromo-2-deoxyuridine (BrdU) ELISA methods, respectively. Western blot analysis and immunohistochemistry were performed to examine the phosphorylation and protein expressions of signal transducer and activator of transcription 3 (Stat3), SP1, forkhead box O3a (FOXO3a), and insulin-like growth factor (IGF)-IGF binding protein 1 (IGFBP1). The expression of IGFBP1 mRNA was measured by quantitative real time PCR (qRT-PCR). Silencing of FOXO3a and IGFBP1 were examined by siRNA procedures. Exogenously expression of SP1, FOXO3a, and IGFBP1 were carried out by transient transfection assays. The promoter activity of IGFBP1 was tested using Secrete-PairTM Dual Luminescence Assay Kit. A xenografted tumor model was used to further test the effect of solamargine in combining with metformin in vivo. RESULTS: We further demonstrated that solamargine inhibited growth and induced cell cycle arrest in other NSCLC cell lines. Through mechanism-based approaches, we showed that solamargine decreased the phosphorylation of Stat3; In addition, solamargine induced FOXO3a, whereas reduced SP1 protein levels; all of which were abrogated in cells with overexpressed Stat3 gene. Interestingly, there is interaction between FOXO3a and SP1. Moreover, solamargine increased mRNA, protein expression and promoter activity of IGFBP1, which was not observed in cells with overexpressed SP1 or with silenced FOXO3a genes. Finally, ablation of IGFBP1 expression by siRNA blocked the effect of solamargine on cell growth inhibition. More importantly, there was a synergy of combination of solamargine and metformin. Similar findings were also observed in vivo. CONCLUSION: Our results show that solamargine increases IGFBP1 gene expression through inactivation of Stat3, followed by regulation and reciprocal interaction of FOXO3a and SP1 in vitro and in vivo. This ultimately leads to suppression of human lung cancer cell growth. Moreover, this is a synergy of solamargine in combination with metformin in this process. This study unravels a novel mechanism underlying the anti-lung cancer effects of solamargine in combination of metformin, and suggests a potential new lung cancer associated therapy.

Activation of ERK and Mutual Regulation of Stat3 and SP1 Contribute to Inhibition of PDK1 Expression by Atractylenolide-1 in Human Lung Cancer Cells.

BACKGROUND/AIMS: Atractylodes macrocephula Koidz is an important ingredient in traditional Chinese herbs. One major bioactive compound, atractylenolide-1 (ATL-1), was reported to have anti-inflammatory and anti-tumor activities. However, the underlying molecular mechanism associated to this has not been well elucidated. METHODS: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Western blot analysis was performed to examine the phosphorylation and protein expression of extracellular signaling-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (Stat3), 3-phosphoinositide dependent protein kinase-1 (PDK1) and transcription factor SP1. QRT-PCR was used to examine the mRNA levels of PDK1 gene. Exogenously expressions of Stat3, PDK1 and SP1 were carried out by transient transfection assays. PDK1 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro. RESULTS: We showed that ATL-1 inhibited human lung cancer cell growth and induced cell cycle arrest. Furthermore, we found that ATL-1 stimulated phosphorylation of ERK1/2, inhibited phosphorylation and protein expressions of Stat3 and SP1; the latter were abrogated in the presence of MEK/ERK inhibitor PD98059. Moreover, ATL-1 reduced the protein, mRNA expression and promoter activity of PDK1. Intriguingly, exogenously expressed Stat3 and SP1 overcame ATL-1-inhibited SP1 and Stat3, and PDK1 protein expressions, respectively. Moreover, overexpression of PDK1 resisted the ATL-1-inhibited lung cancer cell growth. In consistent with the results in vitro, ATL-1 inhibited tumor growth, protein expressions of Stat3, SP1 and PDK1, and induced phosphorylation of ERK1/2 in vivo. CONCLUSION: In summary, our results show that ATL-1 inhibits lung cancer cell growth through activation of ERK1/2, followed by suppressing SP1 protein expression. ATL-1 also reduces phosphorylation and protein levels of Stat3. These are mutual regulation between Stat3 and SP1 proteins affected by ATL-1. This ultimately suppresses PDK1 gene expression. This study reveals a novel mechanism by which ATL-1 inhibits growth of lung cancer cells. Thus, targeting PDK1 pinpoints a potential in the lung cancer treatment.

ß-elemene inhibited expression of DNA methyltransferase 1 through activation of ERK1/2 and AMPKα signalling pathways in human lung cancer cells: the role of Sp1.

ß-elemene, a compound derived from Rhizoma zedoariae, is a promising new plant-derived drug with broad-spectrum anticancer activity. However, the underlying mechanism by which this agent inhibits human lung cancer cell growth has not been well elucidated. In this study, we showed that ß-elemene inhibits human non-small cell lung carcinoma (NSCLC) cell growth, and increased phosphorylation of ERK1/2, Akt and AMPKα. Moreover, ß-elemene inhibited expression of DNA methyltransferase 1 (DNMT1), which was not observed in the presence of the specific inhibitors of ERK (PD98059) or AMPK (compound C). Overexpression of DNMT1 reversed the effect of ß-elemene on cell growth. Interestingly, metformin not only reversed the effect of ß-elemene on phosphorylation of Akt but also strengthened the ß-elemene-reduced DNMT1. In addition, ß-elemene suppressed Sp1 protein expression, which was eliminated by either ERK1/2 or AMPK inhibitor. Conversely, overexpression of Sp1 antagonized the effect of ß-elemene on DNMT1 protein expression and cell growth. Taken together, our results show that ß-elemene inhibits NSCLC cell growth via ERK1/2- and AMPKα-mediated inhibition of transcription factor Sp1, followed by reduction in DNMT1 protein expression. Metformin augments the effect of ß-elemene by blockade of Akt signalling and additively inhibition of DNMT1 protein expression. The reciprocal ERK1/2 and AMPKα signalling pathways contribute to the overall responses of ß-elemene. This study reveals a potential novel mechanism by which ß-elemene inhibits growth of NSCLC cells.

Ursolic acid inhibited growth of hepatocellular carcinoma HepG2 cells through AMPKα-mediated reduction of DNA methyltransferase 1.

Hepatocellular carcinoma (HCC), the major histological subtype of primary liver cancer, remains one of the most common malignancies worldwide. Due to the complicated pathogenesis of this malignancy, the outcome for comprehensive treatment is limited. Chinese herbal medicine (CHM) is emerging as a promising choice for its multi-targets and coordinated intervention effects against HCC. Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid found in CHM, exerts anti-tumor effects and is emerging as an effective compound for cancer prevention and therapy. However, the molecular mechanisms underlying the action of UA remain largely unknown. In this study, we showed that UA inhibited the growth of HCC cells and induced apoptosis in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of AMP-activated protein kinase alpha (AMPKα) and suppressed the protein expression of DNA methyltransferase 1 (DNMT1) in the dose-dependent manner. The inhibitor of AMPK, compound C blocked, while an activator of AMPK, metformin augmented the effect of UA on DNMT1 expression. In addition, UA suppressed the expression of transcription factor Sp1. Conversely, overexpression of Sp1 reversed the effect of UA on DNMT1 expression and cell growth. Collectively, our results show for the first time that UA inhibits growth of HCC through AMPKα-mediated inhibition of Sp1; this in turn results in inhibition of DNMT1. This study reveals a potential novel mechanism by which UA controls growth of HCC cells and suggests that DNMT1 could be novel target for HCC chemoprevention and treatment.

Repression of phosphoinositide-dependent protein kinase 1 expression by ciglitazone via Egr-1 represents a new approach for inhibition of lung cancer cell growth.

BACKGROUND: Peroxisome proliferator-activated receptors gamma (PPARγ) ligands have been shown to inhibit the growth of non-small cell lung cancer (NSCLC) cells. However, the mechanisms underlying this effect remain incompletely elucidated. METHODS: Cell proliferation and apoptosis were measured by cell viability, MTT and caspase3/7 activity assays. Phosphorylation/protein expression and gene silence/overexpression of AMPKα, phosphoinositide-dependent protein kinase 1 (PDK1), Egr-1 and PPARγ were performed by Western blot and siRNA/transfection assays. Dual-Luciferase Reporter Kit was used to measure the PPAR response elements (PPRE) reporter and PDK1 promoter activities, and ChIP assay was used to detect the Egr-1 protein binding to the DNA site in the PDK1 gene promoter. RESULTS: We found that ciglitazone, one synthetic PPARγ ligand, inhibited growth and induced apoptosis of NSCLC cells through decreased expression of PDK1, which was not blocked by GW9662 (a specific PPARγ antagonist). Overexpression of PDK1 overcame the effect of ciglitazone on cell growth and caspase 3/7 activity. Ciglitazone increased the phosphorylation of AMPKα and c-Jun N-terminal kinase (JNK), and the inhibitor of AMPK (compound C), but not JNK (SP600125), reversed the effect of ciglitazone on PDK1 protein expression. Ciglitazone reduced PDK1 gene promoter activity, which was not observed in cells exposed to compound C, but not silenced of PPARγ siRNA. Combination of ciglitazone and metformin further reduced PDK1 expression and promoter activity. Furthermore, we showed that ciglitazone induced the protein expression of Egr-1, which was not observed in cells silencing of AMPKα. Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth. On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity. ChIP assays demonstrated that ciglitazone induced Egr-1 protein bind to the specific DNA site in the PDK1 gene promoter. CONCLUSION: Collectively, our results demonstrate that ciglitazone inhibits PDK1 expression through AMPKα-mediated induction of Egr-1 and Egr-1 binding to the specific DNA site in the PDK1 gene promoter, which is independent of PPARγ. Activation of AMPKα by metformin enhances the effect of ciglitazone. In turn, this leads to inhibition of NSCLC cell proliferation.

[Corrigendum] Traditional Chinese medicine, Fuzheng Kang­Ai decoction, inhibits metastasis of lung cancer cells through the STAT3/MMP9 pathway.

Following the publication of the above article, an interested reader drew to the authors' attention that various of the data panels shown for the cell migration assay experiments in Figs. 2B and 3 appeared to show overlapping regions, such that they were not generated from discretely performed experiments.The authors have re-examined their original data, and realize that the figures in question were assembled incorrectly. In addition, the authors have also realized that certain of the western blotting data panels in Figs. 5 and 6 had likewise been assembled incorrectly. The corrected versions of Figs. 2, 3, 5 and 6 are shown on the next two pages. All these corrections were approved by all authors. The authors regret that these errors were included in the paper, and are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this corrigendum. They also wish to emphasize that the errors made during the compilation of the figures did not substantially alter any of the major conclusions reported in the study, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 16: 2461-2468, 2017; DOI: 10.3892/mmr.2017.6905].

Biological Roles and Mechanisms of Circular RNA in Human Cancers.

Circular RNA (circRNA) is an intriguing class of RNA with covalently closed-loop structure and is highly stable and conservative. As new members of the ncRNAs, the function, mechanism, potential diagnostic biomarker, and therapeutic target have raised increased attention. Most circRNAs are presented with characteristics of abundance, stability, conservatism, and often exhibiting tissue/developmental-stage-specific manner. Over 30,000 circRNAs have been identified with their unique structures to maintain stability more easily than linear RNAs. An increased numbers of circRNAs are dysregulated and involved in several biological processes of malignance, such as tumorigenesis, growth, invasion, metastasis, apoptosis, and vascularization. Emerging evidence suggests that circRNAs play important roles by acting as miRNA sponge or protein scaffolding, autophagy regulators, and interacting with RNA-binding protein (RBP), which may potentially serve as a novel promising biomarker for prevention, diagnosis and therapeutic target for treatment of human cancer with great significance either in scientific research or clinic arena. This review introduces concept, major features of circRNAs, and mainly describes the major biological functions and clinical relevance of circRNAs, as well as expressions and regulatory mechanisms in various types of human cancer, including pathogenesis, mode of action, potential target, signaling regulatory pathways, drug resistance, and therapeutic biomarkers. All of which provide evidence for the potential utilities of circRNAs in the diagnosis and treatment of cancer.

Regulations of miR-183-5p and Snail-Mediated Shikonin-Reduced Epithelial-Mesenchymal Transition in Cervical Cancer Cells.

BACKGROUND: Shikonin, the main ingredient of Lithospermum erythrorhizon, has been reported to have antitumor effects via multiple targets and signaling pathways. However, the detailed mechanism underlying the effects in cervical cancer still remained unknown. METHODS: MTT, wound-healing, transwell assays and flow cytometry experiments were used to measure cell growth, migration, invasion, and cell cycle analysis. Western blot was used to examine protein levels of Snail, Vimentin and E-cadherin. The expression level of miR-183-5p was measured via qRT-PCR. The E-cadherin promoter activity was detected via Secrete-PairTM Dual Luminescence Assay Kit. The transient transfection experiments were used for silencing of E-cadherin and overexpression of Snail genes. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings. RESULTS: We showed that shikonin inhibited cell viability, migration and invasion, and induced cell cycle arrest in a dose-dependent manner in cervical cancer Hela and C33a cells. Mechanistically, we found that shikonin increased miR-183-5p expression and inhibited expression of transcription factor Snail protein. The mimics of miR-183-5p reduced, while the inhibitors of miR-183-5p reversed shikonin-inhibited Snail protein expression. In addition, shikonin decreased Vimentin, increased E-cadherin protein expressions and E-cadherin promoter activity, the latter was reversed in cells transfected with exogenous Snail overexpression vectors. Moreover, silencing of E-cadherin significantly abolished shikonin-inhibited cervical cancer cell growth. Similar findings were also observed in vivo using one xenograft mouse model. CONCLUSION: Our results show that shikonin inhibits EMT through inhibition of Snail and stimulation of miR-183-5p expressions, which resulted in induction of E-cadherin expression. Thus, blockade of EMT could be a novel mechanism underlying the anti-cervical cancer effects of shikonin.

The regulation and interaction of PVT1 and miR181a-5p contributes to the repression of SP1 expression by the combination of XJD decoction and cisplatin in human lung cancer cells.

The Chinese herbal prescription Xiaoji decoction (XJD) has been used as an adjuvant treatment of cancer for decades. However, the molecular mechanisms underlying XJD enhancement of the efficiency of chemotherapy were undetermined. In this study, we observed that combination of XJD and cisplatin (DDP) showed a greater inhibition on growth and induced a high magnitude of apoptosis in non-small cell lung cancer (NSCLC) cells. We also found that XJD decreased lncRNA PVT1 and increased miR181a-5p expressions. There was a reciprocal interaction between PVT1 and miR181a-5p. XJD decreased SP1 protein, which were overcame by overexpressed PVT1 and inhibitors of miR181a-5p. Overexpressed SP1 reversed the inhibitory effect of XJD on cell growth. Importantly, XJD and DDP exhibited synergy on regulation of PVT1, miR181a-5p, and SP1 expressions. The similar results were observed in one in vivo model. In conclusions, XJD inhibits NSCLC cell growth via reciprocal interaction of PVT1 and miR181a-5p followed by reducing SP1 expression. XJD and DDP exhibit synergy. This study provides a novel mechanism by which XJD enhances the anti-cancer effect of DDP in NSCLC cells.