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.

Xiao Q ，Zheng F ，Tang Q ，Wu JJ ，Xie J ，Huang HD ，Yang XB ，Hann SS ... -  《-》

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

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. 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. 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. 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.

Xiao Q ，Zheng F ，Wu J ，Tang Q ，Wang W ，Hann SS ... -  《-》

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.

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. 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. 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. 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.

Xiang S ，Zou P ，Tang Q ，Zheng F ，Wu J ，Chen Z ，Hann SS ... -  《BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS》

The repression and reciprocal interaction of DNA methyltransferase 1 and specificity protein 1 contributes to the inhibition of MET expression by the combination of Chinese herbal medicine FZKA decoction and erlotinib.

The Chinese herbal medicine Fuzheng Kang-Ai (FZKA) decoction obtained from Guangdong Kangmei Pharmaceutical Company, which contains 12 components with different types of constituents, has been used as part of the adjuvant treatment of lung cancer for decades. We previously showed that FZKA decoction enhances the growth inhibition of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant non-small cell lung cancer (NSCLC) cells by suppressing glycoprotein mucin 1 (MUC1) expression. However, the molecular mechanism underlying the therapeutic potential, particularly in sensitizing or/and enhancing the anti-lung cancer effect of EGFR-TKIs, remains unclear. Cell viability was measured using 3-(4, 5-diMEThylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 5-ethynyl -2'-deoxyuridine (EdU) assays. Western blot analysis was performed to examine the protein expressions of DNA methyltransferase 1 (DNMT1), specificity protein 1 (SP1), and MET, an oncogene encoding for a trans-membrane tyrosine kinase receptor activated by the hepatocyte growth factor (HGF). The expression of MET mRNA was measured by quantitative real-time PCR (qRT-PCR). Exogenous expression of DNMT1 and SP1, and MET were carried out by transient transfection assays. The promoter activity of MET was tested using Dual-luciferase reporter assays. A nude mouse xenografted tumor model further evaluated the effect of the combination of FZKA decoction and erlotinib in vivo. The combination of FZKA and erlotinib produced an even greater inhibition of NSCLC cell growth. FZKA decreased the expressions of DNMT1, SP1, and MET (c-MET) proteins, and the combination of FZKA and erlotinib demonstrated enhanced responses. Interestingly, there was a mutual regulation of DNMT1 and SP1. In addition, exogenously expressed DNMT1 and SP1 blocked the FZKA-inhibited c-MET expression. Moreover, excessive expressed MET neutralized FZKA-inhibited growth of NSCLC cells. FZKA decreased the mRNA and promoter activity of c-MET, which was not observed in cells with ectopic expressed DNMT1 gene. Similar findings were observed in vivo. FZKA decreases MET gene expression through the repression and mutual regulation of DNMT1 and SP1 in vitro and in vivo. This leads to inhibit the growth of human lung cancer cells. The combination of FZKA and EGFR-TKI erlotinib exhibits synergy in this process. The regulatory loops among the DNMT1, SP1 and MET converge in the overall effects of FZKA and EGFR-TKI erlotinib. This in vitro and in vivo study clarifies an additional novel molecular mechanism underlying the anti-lung cancer effects in response to the combination of FZKA and erlotinib in gefitinib-resistant NSCLC cells.

Zheng F ，Zhao Y ，Li X ，Tang Q ，Wu J ，Wu W ，Hann SS ... -  《-》

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.

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. 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. 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. 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.

Xiang S ，Zou P ，Wu J ，Zheng F ，Tang Q ，Zhou J ，Hann SS ... -  《-》