Reciprocal interaction of HOTAIR and SP1 together enhance the ability of Xiaoji decoction and gefitinib to inhibit EP4 expression.

The Chinese herbal prescription Xiaoji decoction (XJD) has long been used for cancer treatment. However, the molecular mechanisms underlying the effects of this medicine, particularly to enhance the efficiency of EGFR-TKI in the treatment of lung cancer have not been well elucidated. Cell viability and cell cycle distribution were detected by MTT assay and flow cytometry, respectively. The phosphorylation of ERK1/2 and protein levels of SP1 and EP4 were determined by Western blot. The expression of the HOX transcript antisense RNA (HOTAIR) was measured by qRT-PCR. Transient transfection experiments were used to overexpress the HOTAIR, SP1 and EP4 genes. The interaction between HOTAIR and SP1 were further examined via RNA immunoprecipitation (RIP) assay. A tumor xenograft model was used to confirm the in vitro findings. We showed that XJD inhibited growth and induced cell arrest of human non-small cell lung cancer (NSCLC) cells. We also found that XJD increased the phosphorylation of ERK1/2 and inhibited levels of HOTAIR and SP1, EP4 proteins, which were blocked by inhibitor of MEK/ERK. There was reciprocal interaction between HOTAIR and SP1. Silencing of HOTAIR reduced EP4 protein levels and repressed the growth of NSCLC cells, while overexpression of HOTAIR and SP1 overcame XJD-reduced EP4 protein expression. Additionally, excessive expressed EP4 reversed the effect of XJD on cell growth. Importantly, there was synergy of XJD with another cancer treatment drug, EGFR-TKI gefitinib, in this process. We also found that XJD inhibited tumor growth in a xenograft nude mice model. Our results show that XJD inhibits NSCLC cell growth via ERK1/2-mediated reciprocal repression of HOTAIR and SP1 protein expression, followed by reduced EP4 gene expression. XJD and gefitinib exhibit synergy in this process. The in vitro and in vivo study provides a novel mechanism by which XJD enhances the growth inhibitory effect of gefitinib in gefitinib-resistant NSCLC cells.

Wu J ，Tang Q ，Ren X ，Zheng F ，He C ，Chai X ，Li L ，Hann SS ... -  《-》

Chinese herbal medicine Xiaoji decoction inhibited growth of lung cancer cells through AMPKα-mediated inhibition of Sp1 and DNA methyltransferase 1.

Xiaoji decoction (XJD), which was considered as a Chinese herbal prescription, has been used for cancer treatment, especially lung cancer, for decades to improve quality of life and prolong the patient survival. However, the molecular mechanisms underlying the therapeutic potential have not been well elucidated. The cell viability was examined by MTT assays. The phosphorylation and expression of AMP-activated protein kinase alpha (AMPKα), DNA methyltransferase 1 (DNMT1) and transcription factor Sp1 proteins were assessed by Western Blot. Exogenous expression of Sp1 and DNMT1 were performed by transient transfection methods. The effects of XJD on the growth of xenograft tumors were evaluated by in vivo bioluminescence imaging. We showed that XJD inhibited growth of human non small cell lung cancer (NSCLC) cells in vitro. We also found that XJD increased phosphorylation of AMPKα and inhibited protein expression of DNTM1, the latter was not observed in the presence of the inhibitor of AMPK (compound C). Overexpression of DNTM1 reversed the effect of XJD on cell growth. In addition, XJD decreased Sp1 protein expression, which was eliminated by compound C. Conversely, exogenous expressed Sp1 abrogated XJD-inhibited DNTM1 protein expression. Interestingly, exogenous expression of DNMT1 feedback antagonized the XJD-induced phosphorylation of AMPKα. In in vivo studies, we found that XJD inhibited tumor growth in xenograft nude mice model, which was accompanied by induction of phosphorylation of AMPKα and suppression of DNMT1 protein from xenograft tumors. Our results show that XJD inhibits NSCLC cell growth via AMPKα-mediated inhibition of transcription of Sp1, followed by the reduction of DNMT1 expression both in vitro and in vivo. The negative feedback regulation loop of AMPKα further demonstrates the critical role of DNMT1 in mediating the overall effects of XJD in this process. This study unveils novel molecular mechanism by which XJD controls NSCLC cell growth.

Zhao S ，Wu J ，Tang Q ，Zheng F ，Yang L ，Chen Y ，Li L ，Hann SS ... -  《-》

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 ... -  《-》

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.

Wu J ，Ma C ，Tang X ，Shi Y ，Liu Z ，Chai X ，Tang Q ，Li L ，Hann SS ... -  《-》

Chinese herbal medicine Fuzheng Kang-Ai decoction sensitized the effect of gefitinib on inhibition of human lung cancer cells through inactivating PI3-K/Akt -mediated suppressing MUC1 expression.

Chinese herbal medicine (CHM) Fuzheng Kang-Ai (FZKA for short) decoction has been used as adjuvant treatment strategies in lung cancer patients for decades. However, the molecular mechanism underlying the therapeutic potential especially in sensitizing the effect of EGFR-TKI gefitinib has not been well elucidated. Cell viability was detected by MTT assay. Cell cycle distribution was detected by flow cytometry. Western blot were used to examine phosphorylation and protein levels of Akt, p65, p50 and MUC1. The mRNA level of MUC1 was measured by qRT-PCR. Transient transfection experiments were used to overexpression of Akt, p65 and MUC1. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings. Cell viability was inhibited by FZKA treatment and showed more significant when treated with FZKA and gefitinib in combine in lung cancer cells. FZKA induced the cell arrest at G0/G1 phase. Mechanistically, we showed that the phosphorylation of Akt, protein expressions of p65 and MUC1 were suppressed by FZKA and even more responses were observed in the FZKA and gefitinib combining. Overexpressed Akt overcame the effect of FZKA on p65 protein, and exogenously expressed p65 resisted the inhibitory effect of MUC1 protein expression by FZKA. On the contrary, while overexpressed MUC1 had no effect on p65 expression, it feedback increased phosphorylation of Akt, and more importantly, reversed the cell growth inhibition affected by FZKA. In line with the above, our results confirmed the synergistic effects of FZKA and gefitinib combination on tumor growth, the phosphorylation of Akt, and protein expression of p65 and MUC1 in vivo. This study shows that FZKA decoction inhibits the growth of NSCLC cells through Akt-mediated inhibition of p65, followed by reducing the expression of MUC1. More importantly, there is a synergistic effect of FZKA decoction and gefitinib combination with greater suppression. The positive feedback regulatory loop of MUC1 to Akt signaling pathway further added the important role of MUC1 in mediating the overall responses of FZKA decoction in this process. The in vitro and in vivo study provides an additional and a novel mechanism by which the FZKA decoction enhances the growth inhibition of gefitinib in gefitinib-resistant NSCLC cells.

Li L ，Wang S ，Zheng F ，Wu W ，Hann SS ... -  《-》