A herbal medicine for the treatment of lung cancer

Lung cancer is the leading cause of cancer-related deaths throughout the world. Extracts of medicinal plants are believed to contain different chemopreventive or chemotherapeutic compounds. In this study, we determined the anti-cancer property of one of the traditional Indian medicine Rasagenthi Lehyam (RL) for the treatment of lung cancer. Two lung cancer cell lines (A-549 and H-460) and one normal bronchial epithelial (BEAS-2B) cell line were used to test the chemotherapeutic effect of RL. Out of five fractions of RL, chloroform fraction of RL (cRL) demonstrated a significant inhibition of cell proliferation and induction of apoptosis in A-549 and H-460 cells but not in normal BEAS-2B cells. The cRL fraction up-regulated the pro-apoptotic genes p53 and Bax and induced caspase-3 activation, and down-regulated the pro-survival gene Bcl-2 in both the lung cancer cell lines. Also, nuclear export of p53 was seen in cRL-treated lung cancer cells. In addition, cRL induced G₂/M arrest of cell cycle and enhanced the radio-sensitivity of both the lung cancer cell lines. This study suggests that cRL may prove to be a potent anti-cancer agent that may be used for the treatment of lung cancer. However, further studies are required to bring cRL into the mainstream of medicine in the treatment of lung cancer. (Mol Cell Biochem xxx: 125–133, 2005)     

Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer

Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.     

Gefitinib loaded PLGA and chitosan coated PLGA nanoparticles with magnified cytotoxicity against A549 lung cancer cell lines

In the current study, gefitinib loaded PLGA nanoparticles (GFT-PLGA-NPs) and chitosan coated PLGA nanoparticles (GFT-CS-PLGA-NPs) were synthesized to investigate the role of surface charge of NPs for developing drug delivery system for non-small-cell lung cancer (NSCLC). The developed NPs were evaluated for their size, PDI, zeta potential (ZP), drug entrapment, drug loading, DSC, FTIR, XRD, in vitro release profile, and morphology. The anti-cancer activity of GFT loaded PLGA NPs and GFT loaded CS-PLGA-NPs were examined in human A549 lung cancer cell lines. In vitro release studies of GFT-CS-PLGA-NPs showed more sustained release in comparison to GFT-PLGA-NPs due surface charge attraction of chitosan. In addition, viability of A549 cells decreases significantly with the increasing concentration of GFT-PLGA NPs and GFT-CS-PLGA-NPs when compared to that of pure GFT and blank PLGA NPs. In addition, the microscopic analysis and counting of viable cells also validate the cytotoxicity of the developed NPs. This investigation proved that the developed NPs would be efficient carriers to deliver GFT with improved efficacy against NSCLC.     

Nicotine induces cell survival and chemoresistance by stimulating Mcl-1 phosphorylation and its interaction with Bak in lung cancer

Nicotine is a major carcinogen in cigarettes, which can enhance cell proliferation and metastasis and increase the chemoresistance of cancer cells. Our previous data found that nicotine promotes cell survival in lung cancer by affecting the expression of antiapoptotic protein Mcl-1, suggesting that the Mcl-1 may be a therapeutic target for patients with lung cancer. In this study, we found that the effects of drug resistance on nicotine-induced lung cancer cell lines were shown to influence the phosphorylation of Mcl-1. Moreover, nicotine induces Mcl-1 phosphorylation exclusively at the T163 site, which results in enhancement of the antiapoptotic activity of Mcl-1 and increased cell survival. Meanwhile, nicotine can reduce the sensitivity of H1299 cells to CDDP via enhancement of the binding of Mcl-1 to Bak, which inhibits the proapoptotic effect of Bak and ultimately leads to increased survival and drug resistance of lung cancer cells. Thus, nicotine-induced cell survival and chemoresistance may occur in a mechanism by stimulating Mcl-1 phosphorylation and its interaction with Bak, which may contribute to improving the efficacy of chemotherapy in the treatment of human lung cancer.     

Aldehyde dehydrogenase activity as a functional marker for lung cancer

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry, we sorted cells with bright (ALDH(br)) and dim (ALDH(lo)) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH(br) cells are indeed a different clone, but when left in normal culture conditions will give rise to ALDH(lo) cells. Furthermore, the ALDH(br) cells grow slower, have low clonal efficiency, and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH(br) and ALDH(lo) cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension, measure ALDH activity, and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH(br) cells grew much slower in primary recipient mice. Histologically, there was no significant difference in the expression of ALDH in primary tumors originating from ALDH(br) or ALDH(lo) cells. Secondary and tertiary xenografts originating from ALDH(br) grew faster and bigger than those formed by ALDH(lo) cells. In conclusion, ALDH(br) cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide, but require support of other cells (ALDH(lo)) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer.     

Cancer inhibition mechanism of lung cancer mouse model based on dye trace method

To minimize the incidence and mortality of cancer, dye trace method was used to explore the mechanism of drug inhibition. 60 mice were selected as the research objects and randomly divided into five groups: model group, shikonin group, aconitine group, notoginsenoside R1 group, and compound group. When establishing the model, begin to administrate the medicine by gavage. The permeability of lung barrier was measured, and H.E staining, immunohistochemical staining, and Western blot test were carried out. The results showed that the mice in model group had decreased autonomic activity, increased permeability of the lung barrier, white nodules on the lung tissue, decreased protein expression related to cell proliferation and differentiation, and decreased protein expression associated with cell proliferation and differentiation, increased expression of related proteins in cancer stem cells, and low level of cell-linked communication. And the incidence of lung cancer in the model group mice was 100%. The histopathological changes in mice were improved to varying degrees after the intervention of the three drugs. Especially in the compound group, the incidence of lung cancer decreased to 8.3%. This study demonstrated that the combination of shikonin, aconitine and notoginsenoside R1 had a good anti-cancer effect, which provided a theoretical basis for clinical research.     

Establishment and characterization of a lung cancer cell line,SMC-L001, from a lung adenocarcinoma

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.     

Luteolin suppresses growth and migration of human lung cancer cells

Luteolin, 3′,4′,5,7-tetrahydroxyflavone, has been shown to possess antioxidant, anti-inflammation and anti-cancer properties. However, its role in lung cancer remains poorly understood. Here we examined the anti-tumorigenic role of luteolin in a commonly used lung cancer cell line. Luteolin inhibited the growth of A549 cells by inducing G1 phase cell cycle arrest and apoptosis. Furthermore, stress fiber assembly and cell migration in A549 cells was markedly suppressed by luteolin.     

Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification

The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.     

Viridiflorol induces anti-neoplastic effects on breast,lung, and brain cancer cells through apoptosis

All active natural molecules are not fully exploited as therapeutic agents, causing delays in the advancement of anticancer drug discovery. Viridiflorol is a natural volatile element that may work as anti-cancer compound. We tested the anticancer properties of viridiflorol at different concentrations ranging from 0.03 to 300 μM in vitro on three cancer cells including breast (MCF-7), lung (A549) and brain (Daoy). The cancer cells responses were documented after treatment using MTT and Annexin V assays. Viridiflorol showed cytotoxic effects against all tested cell lines, reducing cell viability in a concentration-dependent manner with variable IC₅₀ values. Daoy and A549 cell lines were more sensitive to viridiflorol when compared with temozolomide and doxorubicin, respectively. Viridiflorol demonstrated the highest anticancer activity against the Daoy cells with an estimated IC₅₀ of 0.1 µM followed by MCF-7 at 10 µM, and A549 at 30 µM. In addition, upon exposure to concentrations ranging from 30 µM to 300 µM of viridiflorol, early and late apoptotic cell death was induced in a concentration dependent manner in Daoy (55.8%-72.1%), MCF-7 (36.2%-72.7%) and A459 (35%-98.9%) cell lines, respectively. In conclusion, viridiflorol demonstrates cytotoxic and apoptotic ability in three different cancer cell lines (brain, breast and lung).     

Identification of bronchioalveolar stem cells in normal lung and lung cancer

Injury models have suggested that the lung contains anatomically and functionally distinct epithelial stem cell populations. We have isolated such a regional pulmonary stem cell population, termed bronchioalveolar stem cells (BASCs). Identified at the bronchioalveolar duct junction, BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties. Furthermore, BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo. These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma. Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma, this work points to BASCs as the putative cells of origin for this subtype of lung cancer.     

Regulatory roles of OASL in lung cancer cell sensitivity to <Emphasis Type="Italic">Actinidia chinensis</Emphasis> Planch root extract (acRoots)

Lung cancer is one of the most common malignancies worldwide. Actinidia chinensis Planch root extract (acRoots) was found to have the capacity of the anti-tumor, although the molecular mechanisms remain unclear. The present study aims to investigate the molecular mechanisms by which lung cancer cells sense to inhibitory effects of acRoots with a special focus on immune-associated gene profiles. We firstly provide a preclinical evidence that acRoots can significantly inhibit lung cancer cell proliferation and apoptosis via the PI3K-OASL signal pathway. The heterogeneous alterations of immune-associated gene profiles of lung cancer cell types were measured after treatment with various doses of acRoots. The OASL gene was identified as the key regulator in molecular networks of acRoots-treated lung cancer cells and validated. The OASL gene plays an important role in the regulation of lung cancer cell sensitivity to acRoots, which modulated by the PI3K signal pathway. Thus, our data indicate that OASL can be one of the decisive regulators to maintain lung cancer cell susceptibility to acRoots and may be associated with the development of drug resistance. The regulation of OASL can be an alternative strategy to improve drug efficacy during cancer therapies.     

Knockdown of cullin 4A inhibits growth and increases chemosensitivity in lung cancer cells

Cullin 4A (Cul4A) has been observed to be overexpressed in various cancers. In this study, the role of Cul4A in the growth and chemosensitivity in lung cancer cells were studied. We showed that Cul4A is overexpressed in lung cancer cells and tissues. Knockdown of the Cul4A expression by shRNA in lung cancer cells resulted in decreased cellular proliferation and growth in lung cancer cells. Increased sensitivity to gemcitabine, a chemotherapy drug, was also noted in those Cul4A knockdown lung cancer cells. Moreover, increased expression of p21, transforming growth factor (TGF)‐β inducible early gene‐1 (TIEG1) and TGF beta‐induced (TGFBI) was observed in lung cancer cells after Cul4A knockdown, which may be partially related to increased chemosensitivity to gemcitabine. G0/G1 cell cycle arrest was also noted after Cul4A knockdown. Notably, decreased tumour growth and increased chemosensitivity to gemcitabine were also noted after Cul4A knockdown in lung cancer xenograft nude mice models. In summary, our study showed that targeting Cul4A with RNAi or other techniques may provide a possible insight to the development of lung cancer therapy in the future.     

Platinum‐based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS,but not autophagy

These days, cancer can still not be effectively cured because cancer cells readily develop resistance to anticancer drugs. Therefore, an effective combination of drugs with different mechanisms to prevent drug resistance has become a very important issue. Furthermore, the BH3‐only protein BNIP3 is involved in both apoptotic and autophagic cell death. In this study, lung cancer cells were treated with a chemotherapy drug alone or in combination to identify the role of BNIP3 and autophagy in combination chemotherapy for treating cancer. Our data revealed that various combinational treatments of two drugs could increase cancer cell death and cisplatin in combination with rapamycin or LBH589, which triggered the cell cycle arrest at the S phase. Cells with autophagosome and pEGFP‐LC3 puncta increased when treated with drugs. To confirm the role of autophagy, cancer cells were pre‐treated with the autophagy inhibitor 3‐methyladenine (3‐MA). 3‐MA sensitized cancer cells to chemotherapy drug treatments. These results suggest that autophagy may be responsible for cell survival in combination chemotherapy for lung cancer. Moreover, BNIP3 was induced and localized in mitochondria when cells were treated with drugs. The transfection of a dominant negative transmembrane deletion construct of BNIP3 (BNIP3ΔTM) and treatment of a reactive oxygen species (ROS) inhibitor suppressed chemo drug‐induced cell death. These results indicate that BNIP3 and ROS may be involved in combination chemo drug‐induced cell death. However, chemo drug‐induced autophagy may protect cancer cells from drug cytotoxicity. As a result, inhibiting autophagy may improve the effects of combination chemotherapy when treating lung cancer.     

The 1991 Merck Frosst Award. Multidrug resistance in small cell lung cancer.

The two-year survival rate of patients with small cell lung cancer is less than 10%. The major reason for this poor outcome is the development of drug resistance. Panels of small cell lung cancer cell lines have been established, providing models for the study of drug resistance in this tumour. One such model is the doxorubicin-selected H69AR cell line. H69AR displays the typical multidrug resistance phenotype in that it is cross-resistant to anthracyclines, Vinca alkaloids (e.g., vinblastine) and epipodophyllotoxins (e.g., VP-16). However, H69AR cells do not overexpress P-glycoprotein, the membrane drug efflux pump frequently found on multidrug resistant cells. Some alterations in glutathione levels and associated enzyme activities were found but the data do not support the notion that enhanced drug detoxication is involved in H69AR cell resistance. Fewer drug-induced DNA strand breaks, reduced levels of topoisomerase II, and reduced formation of drug-stabilized DNA/topoisomerase II complexes were observed in H69AR cells. These data implicate topoisomerase II in the resistance phenotype of H69AR cells, but cannot explain H69AR cell resistance to the Vinca alkaloids, which do not have topoisomerase II as a target. Monoclonal antibodies against antigens overexpressed on H69AR cells have been derived and four have been characterized. Immunoscreening of an H69AR cDNA expression library has allowed the identification of one of these antigens as p36 (annexin II), a Ca2+/phospholipid binding protein. Chemosensitizers and novel xenobiotics have been examined for their ability to circumvent the drug resistance of H69AR cells. The limited success of these investigations suggests that innovative approaches may be required. In conclusion, the data obtained with H69AR and other models of small cell lung cancer indicate that multiple mechanisms contribute to drug resistance in this disease.     

α7-Nicotinic acetylcholine receptor antagonist QND7 suppresses non-small cell lung cancer cell proliferation and migration via inhibition of Akt/mTOR signaling

Lung cancer, one of the most commonly found carcinoma type, has the highest mortality rate in cancer patients worldwide. Therapeutic interventions targeting to lung cancer become remaining the world significant challenge. Recently, the α7-nicotinic acetylcholine receptor (α7-nAChR) was reported to play an important role in the mechanism underlying lung cancer progression, being intriguing drug target for lung cancer therapy. Hence, the top four α7-nAChR antagonists (QND7, PPRD10, PPRD11 and PPRD12) among our previously developed ligands were proceeded to the in vitro anti-cancer evaluations in non-small cell lung cancer (NSCLC) cell lines (H460 and A549). In this study, we found that QND7 exhibited the highest cytotoxic effect and induced cell apoptosis in both cell lines at a level comparable to cisplatin, whereas the PPRD compounds showed much lower cytotoxicity. Low doses of QND7 and PPRD11 were able to suppress H460 and A549 cell proliferation, whereas PPRD10 and PPRD12 were considered ineffective. In an in vitro wound healing assay, QND7-treatment showed the greatest suppression of H460 and A549 cell migration. The variations in the anti-cancer activities of PPRD compounds might be, at least in part of, their non-selective antagonisms to serotonin receptor (5-HT3) and α4β2-nAChR. Further investigation revealed that QND7 was able to minimize protein kinase B/mammalian target of rapamycin (Akt/mTOR) activity, in correlating to its anti-cancer effects. These findings warrant QND7 for further preclinical evaluation and demonstrate the potential of α7-nAChR as cancer drug target.     

Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways

Na(+)/K(+)-ATPase targeted cancer therapy has attracted increasing interests of oncologists in lung cancer field. Although multiple anti-cancer mechanisms of cardiac glycosides as Na(+)/K(+)-ATPase inhibitors are revealed, the role of autophagy and related molecular signaling pathway for the class of compounds in human non-small cell lung cancer (NSCLC) cells has not been systematically examined. We herein investigated the anti-cancer effects of two representative cardiac glycosides, digoxin and ouabain, in A549 and H460 cell lines. Both agents caused significant growth inhibition at nanomolar level. The cardiac glycosides were found to induce moderate G(2)/M arrest but not apoptosis at IC(50) level in the NSCLC cell lines. Moreover, autophagy was markedly induced by both agents, as evidenced by the time- and dose-dependent increase of LC3-II, up-regulation of Atg5 and Beclin1, as well as by the observations through acridine orange staining, transmission electron microscopy and quantification of GFP-LC3 fluorescence. Importantly, AMP-activated protein kinase (AMPK) pathway was activated, resulting in mammalian target of rapamycin (mTOR) deactivation during autophagy induction. Moreover, extracellular-signal-regulated kinase 1/2 (ERK1/2) activation was simultaneously found to be involved in the autophagy regulation. Co-treatment with respective inhibitors or siRNAs could either block the autophagic phenotypes and signals, or significantly increase the cellular viability, indicating the drugs-induced autophagy plays tumor-suppressing role. This work provides first evidence showing that the cardiac glycosides induce autophagy in human NSCLC cells through regulation of both mTOR and ERK1/2 signaling pathways. The autophagy may at least partially account for the growth inhibitory effects of the compounds in human NSCLC cells.     

Coxsackievirus and adenovirus receptor expression in non-malignant lung tissues and clinical lung cancers

Adenoviral vector mediated gene delivery has been applied in clinical trials and mechanistic studies to explore new treatment approaches for lung cancers. The expression of coxsackievirus adenovirus receptor (CAR), the primary receptor for the most commonly used adenovirus serotype 5 (Ad5)-based vectors, predominantly determines the permissiveness of lung cancer cells. CAR expression is also suggested to modulate tumor cell proliferation capacity. Here, we studied CAR expression in archival lung cancer specimens by using well-characterized CAR 72 antibodies. High levels of CAR expression were observed in most of the 32 cases of squamous cell carcinoma lung cancers and in all the five cases of small cell lung cancers investigated. In contrast, high levels of CAR expression were detected only in 6 of 22 adenocarcinoma lung cancers. The relative levels of CAR expression did not correlate with the pathologic grade in lung cancers, and was thus inconsistent with a role of modulating cancer cell proliferation. Of note, CAR expression was not detected in non-malignant alveolar cells. Our data suggest a preferred utility of Ad5 vector mediated gene delivery to squamous cell carcinoma lung cancers, small cell lung cancers, but not to the majority of adenocarcinoma lung cancers.