Boning Liu

Isothiocyanates induce oxidative stress and suppress the metastasis potential of human non-small cell lung cancer cells

BackgroundIsothiocyanates are natural compounds found in consumable cruciferous vegetables. They have been shown to inhibit chemical carcinogenesis by a wide variety of chemical carcinogens in animal models. Recent studies have also shown that isothiocyanates have antitumor activity, inhibiting the growth of several types of cultured human cancer cells. Our previous study showed that PEITC inhibited human leukemia cells growth by inducing apoptosis. However, the effect of isothiocyanates on lung cancer cell metastasis has not been studied. In the present study, we investigated the inhibitory effects of BITC and PEITC on metastatic potential of highly metastatic human lung cancer L9981 cells.MethodsCell migration and invasion were measured by wound healing assay and transwell chemotaxis assay. Expression of metastasis-related genes was assessed by quantitative RT-PCR and Western blotting. The mechanisms of action were evaluated by flow cytometry, reporter assay and Western blotting.ResultsOur data showed that both BITC and PEITC inhibited L9981 cell growth in a dose-dependent manner, the IC50 values were 5.0 and 9.7 μM, respectively. Cell migrations were reduced to 8.1% and 16.5% of control, respectively; and cell invasions were reduced to 2.7% and 7.3% of control, respectively. Metastasis-related genes MMP-2, Twist and β-catenin were also modulated. BITC and PEITC inhibited cell survival signaling molecules Akt and NFκB activation. Moreover, BITC and PEITC increased ROS generation and caused GSH depletion. Pretreatment with NAC blocked BITC and PEITC induced ROS elevation and NFκB inhibition.ConclusionOur results indicated that BITC and PEITC suppress lung cancer cell metastasis potential by modulation of metastasis-related gene expression, inhibition of Akt/NFκB pathway. Induction of oxidative stress may play an important role.

Apoptosis Induced by Benzyl Isothiocyanate in Gefitinib-Resistant Lung Cancer Cells is Associated with Akt/MAPK Pathways and Generation of Reactive Oxygen Species

Gefitinib is the first targeted drug approved for non-small cell lung cancer (NSCLC) treatment. Clinical trails showed that patients with certain clinical and histologic characteristics (such as women, patients of East Asian descent, no history of smoking, and adenocarcinoma) had higher rates of response and overall survival. Despite excellent clinical response to gefinitib in certain NSCLC patients, nearly all patients who respond initially to gefitinib later develop drug resistance. Isothiocyanates have been shown to possess antitumor activity, inhibiting several types of cancer cells growth. However, there are limited studies on their effects on chemoresistance of cancer cells. In this report, we found that benzyl isothiocyanate (BITC) inhibited gefitinib-resistant human NSCLC cells growth by inducing apoptosis in a dose-dependent manner, and activated caspase-3. There were no effects of BITC on epidermal growth factor receptor and multidrug resistant proteins expression. BITC caused cell cycle arrest at G2/M phase, reactive oxygen species generation, and glutathione depletion. Akt activity and NFκB transcriptional activation were suppressed; mitogen-activated protein kinase and activator protein 1 (AP-1) were activated. Our results demonstrated that BITC overcame gefitinib resistance in lung cancer cells. The further understanding of the anti-resistance mechanism of BITC would contribute to establish it as a potent lead compound for the synthesis of novel anticancer drugs.

MicroRNA-449a inhibits cell growth in lung cancer and regulates long noncoding RNA nuclear enriched abundant transcript 1

OBJECTIVE Lung cancer has become the primary cause of cancer-related death now. New therapies targeting the molecular regulatory machinery were required imperatively. MicroRNAs and long noncoding RNAs can respectively or cooperatively function as oncogenes or tumor suppressor genes in human cancers. The present study identified that miR-449a was down-regulated in tissue of human lung cancer. In this study, we aimed to investigate the function of miR-449a in NL9980 and L9981 lung carcinoma cells lines and the relationship with lncRNA nuclear enriched abundant transcript 1 (NEAT1). MATERIALS AND METHODS miR-449a was profiled in several lung carcinoma cell lines by quantitative reverse transcription-polymerase chain reaction RT-PCR. We analyzed the effects of miR-449a overexpression on proliferation, apoptosis and cell cycle in L9981 cells. The regulatory relationship between miR-449a and NEAT1 was predicted in silico and further studied by miR-449a inhibitor and mimics assay. RESULTS miR-449a was expressed in four cell lines, which we selected, however miR-449a was in high level in NL9980 and in low level in L9981 (P < 0.05). When the miR-449a was the overexpression in L9981 cells, the cell growth was suppressed, and the apoptosis cells were promoted compared with the control group (P < 0.05). The G1/G0 became longer and S, G2/M became shorter (P < 0.05) by miR-449a overexpression. Further study of the interaction between miR-449a and NEAT1 show that NEAT1 was up-regulated when cells were transfected with miR-449a inhibitor, and NEAT1 was down-regulated when cells transfected with miR-449a mimics. CONCLUSIONS Our data indicate that miR-449a may function as a suppressor of lung cancer, and affects the expression of NEAT1 in lung cancer cells.

Benzyl isothiocyanate induces protective autophagy in human lung cancer cells through an endoplasmic reticulum stress-mediated mechanism

Isothiocyanates, such as allyl isothiocya¬nate (AITC), benzyl isothiocyanate (BITC), phenethyl isothio¬cyanate (PEITC) and sulforaphane (SFN), are natural compounds abundant in cruciferous vegetables, which have substantial chemopreventive activities against various human malignancies. However, the mechanisms underlying the inhibition of tumor cell growth by isothiocyanates are not fully understood. Since autophagy has dual functions in cancer, in the present study we investigated the effects of BITC on autophagy induction in human lung cancer cells in vitro and in vivo. BITC (1–100 μmol/L) dose-dependently inhibited the growth of 3 different human lung cancer cell lines A549 (adenocarcinoma), H661 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) with IC50 values of 30.7±0.14, 15.9±0.22 and 23.4±0.11 μmol/L, respectively. BITC (10–40 μmol/L) induced autophagy in the lung cancer cells, evidenced by the formation of acidic vesicular organelles (AVOs), the accumulation of LC3-II, the punctate pattern of LC3, and the expression of Atg5. Pretreatment with the autophagy inhibitor 3-MA (5 mmol/L) significantly enhanced the BITC-caused growth inhibition in the lung cancer cells. Furthermore, BITC (20–40 μmol/L) activated ER stress, as shown by the increased cytosolic Ca2+ level and the phosphorylation of the ER stress marker proteins PERK and eIF2α in the lung cancer cells. Pretreatment with the ER stress inhibitor 4-PBA (5 mmol/L) attenuated the autophagy induction and potentiated the BITC-induced cell growth inhibition. In nude mice bearing A549 xenografts, administration of BITC (100 mg·kg-1·d-1, ip) for 8 weeks markedly suppressed the lung tumor growth, and significantly enhanced both autophagy and ER stress in the tumor tissues. Our results demonstrate that BITC inhibits human lung cancer cell growth in vitro and in vivo. In addition, BITC induces autophagy in the lung cancer cells, which protects the cancer cells against the inhibitory action of BITC; the autophagy induction is mediated by the ER stress response.

MiR-26a enhances invasive capacity by suppressing GSK3β in human lung cancer cells

ABSTRACT Lung cancer is the common cause of death from cancer, and most lung cancer patients die of metastasis. MicroRNAs (miRNAs) function as either oncogenes or tumor suppressors, playing crucial role not only in tumorigenesis, but also in tumor invasion and metastasis. There are several studies showed that miR‐26a is involved in carcinogenesis, however, its role in tumor metastasis need to be elucidated. In this study, we showed that ectopic expression of miR‐26a enhanced migration and invasion of lung cancer cells. Glycogen synthase kinase‐3&bgr; (GSK3&bgr;) was identified as a direct target of miR‐26a. GSK3&bgr; expression negatively correlated with miR‐26a expression in lung cancer tissues. Silencing of GSK3&bgr; achieved similar effect as miR‐26a over‐expression; over‐expression of GSK3&bgr; reversed the enhanced effect of miR‐26a on lung cancer cell migration and invasion. Further study indicated that miR‐26a increased &bgr;‐catenin expression and nuclear translocation. C‐myc and cyclin D1, the downstream genes of &bgr;‐catenin, were also up‐regulated by miR‐26a. Furthermore, xenograft study showed that miR‐26a promoted lung cancer cell growth in vivo, and suppressed GSK3&bgr; expression. Collectively, our results demonstrated that miR‐26a enhanced metastatic potential of lung cancer cells via activation of &bgr;‐catenin pathway by targeting GSK3&bgr;, suggesting the potential applicability of miR‐26a as a target for cancer treatment. HighlightsmiR‐26a enhances migration and invasion of lung cancer cells.GSK3&bgr; is identified as a direct target of miR‐26a.miR‐26a activates &bgr;‐catenin pathway by targeting GSK3&bgr;.miR‐26a promotes lung cancer cell growth in vivo.

Cancer-associated fibroblasts enhance metastatic potential of lung cancer cells through IL-6/STAT3 signaling pathway

Recent studies indicate that cancer-associated fibroblasts (CAFs) are involved in tumor growth, invasion and metastasis, however, the underling mechanisms remain unclear. In the present study, we investigated the role of CAFs on the metastatic potential of lung cancer cells. The stromal fibroblasts we isolated from lung cancer tissues presented CAFs characteristics with high levels of α-smooth muscle actin (α-SMA) and fibroblast-activating protein (FAP). Our data showed that the conditioned medium from cultured CAFs (CAF-CM) dramatically enhanced migration and invasion of lung cancer cells. CAF-CM induced epithelial-mesenchymal transition (EMT) by regulating the expression of EMT-associated markers E-cadherin and vimentin, and also modulated metastasis-related genes MMP-2 and VEGF both in vitro and in vivo. Further mechanistic studies demonstrated that CAFs enhanced the metastatic potential of lung cancer cells by secreting IL-6, subsequently activating of JAK2/STAT3 signaling pathway. Additionally, the inhibition of IL-6/STAT3 signaling pathway by IL-6 neutralizing antibody or specific inhibitors of JAK2/STAT3 reversed CAF-CM induced EMT and migration of lung cancer cells. Taken together, these findings revealed a novel mechanism that CAFs induced EMT and promoted metastasis of lung cancer cells through the IL-6/STAT3 signaling pathway.

Evaluation of serum midkine as a biomarker in differentiated thyroid cancer

AIMS Midkine is a multifunctional cytokine identified to be a promising cancer biomarker. We aimed to prospectively investigate serum midkine as a diagnostic and prognostic biomarker in differentiated thyroid cancer (DTC). MAIN METHODS 162 patients with thyroid nodules participated in the surgical cohort (post-surgical pathology proved 70 cases with DTC and 92 cases with benign thyroid nodules), 75 healthy subjects served as control. Diagnostic values of pre-surgical midkine and thyroglobulin for DTC were conducted by receiver operating characteristic (ROC) curves. 214 DTC patients participated in the (131)I treatment cohort. Prognostic values of pre-(131)I-ablative midkine and thyroglobulin to predict (131)I-avid metastases were performed by ROC curves. Metastasis-free survival was analyzed by the Kaplan-Meier method. KEY FINDINGS Much better diagnostic capability of midkine than thyroglobulin was shown to differentiate DTC from benign thyroid nodules, with cut-off midkine value of 323.12pg/ml and diagnostic accuracy of 75.31%. Nearly similar diagnostic capabilities of midkine and thyroglobulin were shown to distinguish DTC from normal participants. Pre-(131)I-ablative thyroglobulin demonstrated perfect ability to predict metastases, with cut-off value and diagnostic accuracy of 19.50ng/ml and 96.73%. Midkine also performed well with a cut-off value and diagnostic accuracy of 504.71pg/ml and 89.25%. DTC patients with midkine or thyroglobulin levels higher than those of thresholds (500pg/ml or 20ng/ml) showed a significantly worse (131)I-avid metastasis-free survival by the Kaplan-Meier method (P<0.01). SIGNIFICANCE Our results show that midkine is as good as or even better than thyroglobulin to screen patients with thyroid nodules for DTC before surgery, and to predict whether metastases exist before the first (131)I ablative therapy.

Activating transcription factor 3 promotes malignance of lung cancer cells in vitro

Lung cancer remains the most common cause of cancer‐related death, with high rates of recurrence and poor outcomes. An abnormally high expression of activating transcription factor 3 (ATF3) in various cancers suggests an oncogenic role; however, its function in lung cancer is largely unknown.

Abstract 1552: Isothiocyanates suppress human lung cancer cells metastasis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is one of the most aggressive cancer types, and is the leading cause of cancer-related deaths worldwide. Metastasis is of great importance to the clinical management of cancer since it causes 90% of deaths from solid tumours. Therefore the development of novel anti-metastasis drugs becomes one of the most active fields in cancer research. Isothiocyanates are natural compounds found in consumable cruciferous vegetables. They have been shown to have the ability to inhibit chemical carcinogenesis by a wide variety of chemical carcinogens in animal models, inhibiting tumorigenesis in the lung, stomach, colon, liver, etc. Recent studies have also shown that isothiocyanates have antitumor activity, inhibiting several types of cultured human cancer cells growth, such as leukemia, prostate cancer and breast cancer. Our previous study also showed that isothiocyanates inhibit human lung cancer A549 cell growth. In the present study, we investigated the inhibitory effects of isothiocyanates on human lung cancer cell metastasis. We established a high-metastatic human large cell lung cancer cell line L9981, and found that both benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) inhibited L9981 cells growth in a dose-dependent manner, the IC50 values were 5.0±0.22 μM and 9.7±0.39 μM, respectively. Apoptosis was induced at 20 μM after 24 h. Transwell migration assay showed that the metastatic abilities of L9981 cells were suppressed by 96%, and metastasis-correlated genes were modulated. Cell cycle was arrested at G2/M phase. BITC and PEITC treatment resulted in reactive oxygen species (ROS) accumulation and severe glutathione (GSH) depletion. Akt activation was diminished. At transcription level, both nuclear transcription factors NFκB and AP-1 activities were suppressed. NFκB activities were reduced to 28.8% and 68.5% by BITC and PEITC, respectively; AP-1 activities were reduced to 17.8% and 80.9% by BITC and PEITC, respectively. Our results showed that BITC and PEITC suppress human lung cancer cells metastasis by induction of apoptosis, modulation of metastasis correlated gene expression, and induction of oxidative stress. This study indicates the potential use of isothiocyanates in treatment of advanced lung cancer. The signal transduction pathways and validation of cellular therapeutic targets of these isothiocyanates are under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1552.