Curcumin induces EGFR degradation in lung adenocarcinoma and modulates p38 activation in intestine: the versatile adjuvant for gefitinib therapy

Background

Non-small cell lung cancer (NSCLC) patients with L858R or exon 19 deletion mutations in epidermal growth factor receptor (EGFR) have good responses to the tyrosine kinase inhibitor (TKI), gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are resistant to gefitinib treatment. Here, we showed that curcumin can improve the efficiency of gefitinib in the resistant NSCLC cells both in vitro and in vivo models.

Methods/Principal Findings

After screening 598 herbal and natural compounds, we found curcumin could inhibit cell proliferation in different gefitinib-resistant NSCLC cell lines; concentration-dependently down-regulate EGFR phosphorylation through promoting EGFR degradation in NSCLC cell lines with wild-type EGFR or T790M EGFR. In addition, the anti-tumor activity of gefitinib was potentiated via curcumin through blocking EGFR activation and inducing apoptosis in gefitinib-resistant NSCLC cell lines; also the combined treatment with curcumin and gefitinib exhibited significant inhibition in the CL1-5, A549 and H1975 xenografts tumor growth in SCID mice through reducing EGFR, c-MET, cyclin D1 expression, and inducing apoptosis activation through caspases-8, 9 and PARP. Interestingly, we observed that the combined treatment group represented better survival rate and less intestinal mucosal damage compare to gefitinib-alone therapy. We showed that curcumin attenuated the gefitinib-induced cell proliferation inhibition and apoptosis through altering p38 mitogen-activated protein kinase (MAPK) activation in intestinal epithelia cell.

Conclusions/Significance

Curcumin potentiates antitumor activity of gefitinib in cell lines and xenograft mice model of NSCLC through inhibition of proliferation, EGFR phosphorylation, and induction EGFR ubiquitination and apoptosis. In addition, curcumin attenuates gefitinib-induced gastrointestinal adverse effects via altering p38 activation. These findings provide a novel treatment strategy that curcumin as an adjuvant to increase the spectrum of the usage of gefitinib and overcome the gefitinib inefficiency in NSCLC patients.     

Angio-associated migratory cell protein interacts with epidermal growth factor receptor and enhances proliferation and drug resistance in human non-small cell lung cancer cells

Angio-associated migratory cell protein (AAMP) is expressed in some human cancer cells. Previous studies have shown AAMP high expression predicted poor prognosis. But its biological role in non-small cell lung cancer (NSCLC) cells is still unknown. In our present study, we attempted to explore the functions of AAMP in NSCLC cells. According to our findings, AAMP knockdown inhibited lung cancer cell proliferation and inhibited lung cancer cell tumorigenesis in the mouse xenograft model. Epidermal growth factor receptor (EGFR) is a primary receptor tyrosine kinase (RTK) that promotes proliferation and plays an important role in cancer pathology. We found AAMP interacted with EGFR and enhanced its dimerization and phosphorylation at tyrosine 1173 which activated ERK1/2 in NSCLC cells. In addition, we showed AAMP conferred the lung cancer cells resistance to chemotherapeutic agents such as icotinib and doxorubicin. Taken together, our data indicate that loss of AAMP from NSCLC inhibits tumor growth and elevates drug sensitivity, and these findings have clinical implications to treat NSCLC cancers.     

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ET_A receptor (R) and ET_BR on cancer cells. High levels of tumor ET-1 and ET_AR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ET_AR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ET_AR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca²⁺ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.     

Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer

Blockade of epidermal growth factor receptor (EGFR) activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC). As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs), there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK) activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975) were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271) both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be particularly sensitive to this combination treatment. As such, further evaluation of this combination therapy is warranted and could prove to be an effective therapeutic approach for patients with inherent EGFR TKI-resistant NSCLC.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.     

Overexpression of CARMA3 in non-small-cell lung cancer is linked for tumor progression

We aimed to investigate the clinical significance of the expression of novel scaffold protein CARMA3 in non-small-cell lung cancer (NSCLC) and the biological function of CARMA3 in NSCLC cell lines. We observed moderate to high CARMA3 staining in 68.8% of 141 NSCLC specimens compared to corresponding normal tissues. The overexpression of CARMA3 was significantly correlated with TNM stage (P = 0.022) and tumor status (P = 0.013). CARMA3 upregulation also correlated with a shorter survival rate of patients of nodal status N0 (P = 0.042)as well as the expression of epidermal growth factor receptor (EGFR) (P = 0.009). In EGFR mutation positive cases, CARMA3 expression was much higher (87.5%) compared to non-mutation cases (66.1%). In addition, we observed that knockdown of CARMA3 inhibits tumor cell proliferation and invasion, and induces cell cycle arrest at the boundary between the G1 and S phase. We further demonstrated a direct link between CARMA3 and NF-κB activation. The change of biological behavior in CARMA3 knockdown cells may be NF-κB-related. Our findings demonstrated, for the first time, that CARMA3 was overexpressed in NSCLC and correlated with lung cancer progression, EGFR expression, and EGFR mutation. CARMA3 could serve as a potential companion drug target, along with NF-kB and EGFR in EGFR-mutant lung cancers.     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.     

miR-146a Inhibits Cell Growth,Cell Migration and Induces Apoptosis in Non-Small Cell Lung Cancer Cells

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P<0.05). The patients with high miR-146a expression in their tumors showed longer progression-free survival (25.6 weeks in miR-146a high patients vs. 4.8 weeks in miR-146a low patients, P<0.05). miR-146a is therefore a strong candidate prognostic biomarker in NSCLC. Thus inducing miR-146a might be a therapeutic strategy for NSCLC.     

LINC00184 plays an oncogenic role in non-small cell lung cancer via regulation of the miR-524-5p/HMGB2 axis

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. We aimed to investigate the role of LINC00184 in NSCLC. Migration, proliferation and invasion of NSCLC cells were analysed using the wound healing assay, cell counting kit-8 assay and transwell assay, respectively. Apoptosis and cell cycle were assessed using flow cytometry. Online bioinformatics tools were utilized to predict downstream microRNAs (miRNA) or genes related to LINC00184 expression. The RNA pull-down experiment and luciferase reporter assay were performed to verify the predictions thereof. LINC00184, miR-524-5p, and high mobility group 2 protein (HMGB2) expression levels in NSCLC tissues and cell lines were detected using quantitative real-time polymerase chain reaction. An NSCLC mouse model was constructed for in vivo experiments. LINC00184 overexpression was observed in NSCLC tissues and cell lines and was found to be correlated with poor prognosis. LINC00184 knockdown inhibited cell proliferation, migration and invasion, induced cell cycle arrest and accelerated apoptosis in NSCLC cell lines. LINC00184 suppressed tumour growth and proliferation in NSCLC mouse models and directly targeted the miR-524-5p/HMGB2 axis. Moreover, the expression levels of LINC00184 and HMGB2 were negatively correlated with miR-524-5p expression, whereas LINC00184 expression was positively correlated with HMGB2 expression. LINC00184 affected the cell cycle, proliferation, apoptosis, migration and invasion in NSCLC via regulation of the miR-524-5p/HMGB2 axis.     

MicroRNA-133a Suppresses Multiple Oncogenic Membrane Receptors and Cell Invasion in Non-Small Cell Lung Carcinoma

Non-small cell lung cancers (NSCLCs) cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R), TGF-beta receptor type-1 (TGFBR1), and epidermal growth factor receptor (EGFR), are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.     

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.     

SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner

Aims

The mechanism by which SR48692 inhibits non-small cell lung cancer (NSCLC) proliferation was investigated.

Main methods

The ability of SR48692 to inhibit the proliferation of NSCLC cell lines NCI-H1299 and A549 was investigated in vitro in the presence or absence of neurotensin (NTS). The ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation was investigated by Western blot using NSCLC cells and various inhibitors. The growth effects and Western blot results were determined in cell lines treated with siRNA for NTSR1.

Key findings

Treatment of A549 or NCI-H1299 cells with siRNA for NTSR1 reduced significantly NTSR1 protein and the ability of SR48692 to inhibit the proliferation of A549 or NCI-H1299 NSCLC cells. Treatment of A549 and NCI-H1299 cells with siRNA for NTSR1 reduced the ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation. SR48692 or gefitinib (EGFR tyrosine kinase inhibitor) inhibited the ability of NTS to cause EGFR and ERK tyrosine phosphorylation. NTS transactivation of the EGFR was inhibited by GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) or U73122 (phospholipase C inhibitor) but not H89 (PKA inhibitor). NTS stimulates whereas SR48692 or gefitinib inhibits the clonal growth of NSCLC cells.

Significance

These results suggest that SR48692 may inhibit NSCLC proliferation in an EGFR-dependent mechanism.     

Knockdown of LncRNA PVT1 inhibits tumorigenesis in non-small-cell lung cancer by regulating miR-497 expression

Plasmacytoma variant translocation1 (PVT1) was reported to be upregulated in non-small-cell lung cancer (NSCLC) tissues, serve as a promising biomarker for diagnosis and prognosis of NSCLC, and promoted NSCLC cell proliferation. However, the detailed molecular mechanism of PVT1 involved in the pathogenesis and development of NSCLC remains largely unknown. In this study, the expression levels of PVT1 and miR-497 in NSCLC cells were determined by qRT-PCR. Cell viability, invasion and apoptosis were detected by MTT assay, cell invasion assay and flow cytometry analysis, respectively. RNA immunoprecipitation (RIP) and luciferase reporter assay were performed to confirm whether PVT1 directly interacts with miR-497. A xenograft mouse model was established to confirm the effect of PVT1 on tumor growth in vivo and the underlying molecular mechanism. Our findings indicated that PVT1 was significantly upregulated and miR-497 was markedly downregulated in NSCLC cell lines. si-PVT1 effectively decreased the expression of PVT1 and increased the expression of miR-497. PVT1 knockdown remarkably inhibited cell viability, invasion and promoted apoptosis in NSCLC cells. RIP and luciferase reporter assay demonstrated that PVT1 could directly interact with miR-497. Moreover, PVT1 overexpression reversed the inhibitory effect of miR-497 on cell viability, invasion and promotion effect on apoptosis of NSCLC cells. Furthermore, in vivo experiment showed that knockdown of PVT1 inhibited tumor growth in vivo and promoted miR-497 expression. In conclusion, knockdown of PVT1 inhibited cell viability, invasion and induced apoptosis in NSCLC by regulating miR-497 expression, elucidating the molecular mechanism of the oncogenic role of PVT1 in NSCLC and providing an lncRNA-directed target for NSCLC.     

MicroRNA-212 displays tumor-promoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1

Dysexpression of microRNAs has been found in many tumors, including lung cancer. The hedgehog (Hh) signaling pathway plays an important role during normal development, and the abnormal regulation of its members has also been related to many tumors. However, little is known about the relationship between microRNA and the Hh pathway. In this paper, we report microRNA-212 (miR-212) playing a role in non-small cell lung cancer (NSCLC) and targeting PTCH1, a receptor of the Hh pathway. We found that miR-212 was up-regulated when cells were treated with 4ß-12-O-tetradecanoylphorbol-13-acetate (TPA). We ectopically expressed miR-212 in NSCLC cell lines to examine the influence of miR-212 overexpression. The results showed that overexpression of miR-212 in NSCLC cells promoted cell cycle progression and cell proliferation, migration, and invasion. The promoting effects of miR-212 on cell proliferation, migration, and invasion were partially reversed by the miR-212 inhibitor anti-miR-212. These results suggested that miR-212 might have tumor-promoting properties. Potential targets of miR-212 were predicted, and we showed tumor suppressor PTCH1 was a functional target of miR-212. PTCH1 may be responsible for the effect of miR-212 on cell proliferation. Altogether, our results indicated that miR-212 was involved in tumorigenesis, and the oncogenic activity of miR-212 in NSCLC cells was due, in part, to suppression of PTCH1.     

Structure based designing of triazolopyrimidone-based reversible inhibitors for kinases involved in NSCLC

Secondary acquired mutant EGFR (L858R-T790M) overexpressed NSCLC forms one of the prevalent form of resistant NSCLC. Another subset of resistant NSCLC includes amplified cMET in mutant EGFR derived tumours. Thus, in continuation to our previous work on these two major targets of resistant NSCLC, i.e., EGFR (L858R-T790M) and cMET, we are hereby reporting reversible inhibitors of these kinases. Out of 11 lead molecules reported in our previous study, we selected triazolo-pyrimidone (BAS 09867482) scaffold for further development of small molecule dual and reversible inhibitors. Analogues of lead with different substituents on the side ring were sketched and docked in both the target kinases, followed by molecular dynamic simulations. Analogues maintaining hydrophobic interaction with M790 in secondary acquired mutant EGFR (L858R-T790M) were selected and duly synthesized. In vitro biochemical evaluation of these molecules against EGFR (L858R-T790M) and cMET kinase, along with EGFR (L858R) kinase disclosed that three molecules were having significant dual kinase inhibitory potential with IC₅₀ values well below 100 nM. Further, in vitro anti-proliferative assay against three cell lines (A549, A431 and H460) was performed. Out of all, two compounds were having significant potency against these cell lines.     

In Vitro and In Vivo Antitumor Activity of a Novel Semisynthetic Derivative of Cucurbitacin B

Lung cancer is the most deadly type of cancer in humans, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer showing high resistance to radiation and chemotherapy. Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task. Here we describe a new semisynthetic derivative of cucurbitacin B (DACE) as a potent inhibitor of NSCLC cell proliferation. DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3. Consistent with our in vitro studies, intraperitoneal application of DACE significantly suppressed the growth of mouse NSCLC that arises from type II alveolar pneumocytes due to constitutive expression of a human oncogenic c-RAF kinase (c-RAF-1-BxB) transgene in these cells. Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.     

Marsdenia tenacissima extract enhances gefitinib efficacy in non-small cell lung cancer xenografts

PurposeThe stem of Marsdenia tenacissima (Roxb.) Wight et Arn. has long been used as a medicine to treat cancer in China. Our previous in vitro results showed that Marsdenia tenacissima extract (MTE) overcomes gefitinib resistance in non-small cell lung cancer (NSCLC) cells. However, it is unknown whether MTE could enhance gefitinib efficacy in vivo. The present study was intended to investigate the in vivo anti-tumour activity of MTE combined with gefitinib.MethodsHuman NSCLC H460 (K-ras mutation) or H1975 cells (EGFR T790M mutation) were subcutaneously inoculated into nude mice. Tumour volume and body weight were measured regularly. Resected tumours were weighed after the animals were sacrificed. Immunoblotting or immunohistochemistry was used to assess the cellular proliferation and apoptosis in xenograft tumour tissue. Expression of the EGFR downstream pathways and c-Met were measured with western blot analysis to explore possible mechanisms.ResultsMTE (5, 10, 20 g/kg) dose-dependently reduced tumour growth and induced cell apoptosis. MTE suppressed EGFR related signals, and 20 g/kg was the most effective dose. Low-dose MTE (5 g/kg) significantly enhanced gefitinib efficacy in resistant H460 and H1975 xenografts. The combination inhibited tumour proliferation and induced cell apoptosis in both resistant NSCLC xenografts. Constitutive activation of the PI3K/Akt and MEK/ERK pathways is related to EGFR-TKI resistance. Accordingly, phosphorylation of PI3K/Akt/mTOR and ERK1/2 was suppressed after combination treatment. Simultaneously, cross-talked c-Met and EGFR were also prominently lowered in the presence of MTE combined with gefitinib.ConclusionThe present results suggest that the combination of MTE and gefitinib may be a promising therapeutic approach to enhance gefitinib efficacy in resistant NSCLC.     

Non‐canonical Raf‐1/p70S6K signalling in non–small‐cell lung cancer

Lung cancer is the leading cause of cancer‐related death globally, with non–small‐cell lung cancer (NSCLC) being the predominant subtype. Overall survival remains low for NSCLC patients, and novel targets are needed to improve outcome. Raf‐1 is a key component of the Ras/Raf/MEK signalling pathway, but its role and downstream targets in NSCLC are not completely understood. Our previous study indicated a possible correlation between Raf‐1 levels and ribosomal protein S6 kinase (p70S6K) function. In this study, we aimed to investigate whether p70S6K is a downstream target of Raf‐1 in NSCLC. Raf‐1 was silenced in NSCLC cell lines by using small hairpin RNA, and Raf‐1 and p70S6K protein levels were measured via Western blot. p70S6K was then overexpressed following Raf‐1 knock‐down; then, cell proliferation, apoptosis and the cell cycle in NSCLC cell lines were examined. Tumour xenografts with NSCLC cells were then transplanted for in vivo study. Tumours were measured and weighed, and Raf‐1 and p70S6K expression, cell proliferation and apoptosis were examined in tumour tissues by Western blot, Ki‐67 staining and TUNEL staining, respectively. When Raf‐1 was silenced, p70S6K protein levels were markedly decreased in the A549 and H1299 NSCLC cell lines. A significant decrease in NSCLC cell proliferation, a profound increase in apoptosis and cell cycle arrest were observed in vitro following Raf‐1 knock‐down. Overexpression of p70S6K after Raf‐1 depletion effectively reversed these effects. Xenograft studies confirmed these results in vivo. In conclusion, Raf‐1 targets p70S6K as its downstream effector to regulate NSCLC tumorigenicity, making Raf‐1/p70S6K signalling a promising target for NSCLC treatment.