Apoptosis Of Small Cell Lung Cancer Cells Research Articles

Exosome inhibition improves response to first-line therapy in small cell lung cancer.

Exosomes are recognized as important mediators of cell-to-cell communication, facilitating carcinogenesis. Although there have been significant advancements in exosome research in recent decades, no drugs that target the inhibition of sEV secretion have been approved for human use. For this study, we employed GW4869 and Nexinhib20 as inhibitors of exosome synthesis and trafficking combined. First, we found that Nexinhib20 and GW4869 effectively inhibited RAB27A and neutral sphingomyelinase 2 (nSMase2) nsMase2. Interestingly, the inhibition of nsMase2 and RAB27A decreased expression of CD9, CD63 and Tsg101, both at RNA and protein levels. We used a combination treatment strategy of cisplatin/etoposide plus GW4869 or Nexinhib20 on small cell lung cancer (SCLC) cell lines. The combination treatment of GW4869 or Nexinhib20 effectively enhanced the inhibitory effects of first-line chemotherapy on the SCLC cells. Furthermore, we demonstrated that reducing exosome release through GW4869 and Nexinhib20 treatment effectively reduced cellular proliferation and significantly induced apoptosis in SCLC cells. Also, we showed that combining exosome inhibition with chemotherapy has a significant synergistic effect on cellular proliferation. We also found increased p53 and p21 expressions with western blot and significantly changing Bax, BCL2, caspase-3 and caspase-9 expressions. Inhibiting the exosome pathway offers opportunities for developing novel, effective treatment strategies for SCLC.

Synergistic effect of a nonsteroidal anti-inflammatory drug in combination with topotecan on small cell lung cancer cells.

The topoisomerase I inhibitor topotecan (TPT) is used in the treatment of recurrent small cell lung cancer (SCLC). However, the drug has a limited success rate and causes distress to patients due to its side effects, such as hematologic toxicities, including anemia and thrombocytopenia. Due to these pharmacokinetic limitations and undesirable side effects of chemotherapeutic drugs, the development of combination therapies has gained popularity in SCLC. Meclofenamic acid (MA), a nonsteroidal anti-inflammatory drug, has demonstrated anticancer effects on various types of cancers through different mechanisms. This study aims to investigate the potential synergistic effects of MA and TPT on the small cell lung cancer cell line DMS114. To assess the cytotoxic and apoptotic effects of the combined treatment of MA and TPT, trypan blue exclusion assay, Annexin V, acridine orange/propidium iodide staining, western blot, and cell cycle analysis were conducted. The results demonstrated that the combination of MA and TPT elicited synergistic effects by enhancing toxicity in DMS114 cells (P < 0.01) without causing toxicity in healthy epithelial lung cells MRC5. The strongest synergistic effect was observed when the cells were treated with 60 µM MA and 10 nM TPT for 48h (CI = 0,751; DRI = 10,871). This study, for the first time, furnishes compelling evidence that MA and TPT synergistically reduce cellular proliferation and induce apoptosis in SCLC cells. Combinations of these drugs holds promise as a potential therapeutic strategy to improve efficacy and reduce the side effects associated with TPT.

MDM2-mediated Inhibitory Effect of Arsenic Trioxide on Small Cell Lung Cancer Cell Line by Degrading Mutant p53.

Small cell lung cancer (SCLC) is featured by a high TP53 mutant rate. Our previous research found that arsenic trioxide (As2O3) could significantly inhibit the growth and metastasis of SCLC. Studies have shown that the degradation of mutant p53 mediated by murine double minute 2 (MDM2) can be induced by As2O3, which probably contributes to the inhibition of SCLC, but the detailed mechanism is still unclear. We aimed to testify that As2O3 can inhibit the growth of SCLC cells by degrading mutant p53 protein via binding to MDM2. CCK-8 assay, cell cycle analysis, and western blot of apoptosis markers were used to evaluate the inhibitory effect of As2O3 on NCI-H446 cells (containing mutant p53) and NCI-H1299 cells (p53 null). The effects of As2O3 on p53 and its downstream proteins were identified by western blot using mut-p53-knockdown and overexpressed cell models. MDM2-knockdown cell models were constructed, and western blot, co-IP of mut-p53, and ubiquitin were carried out to explore the mediating effect of MDM2 in As2O3 induced mut-p53 degradation. As2O3 inhibited proliferation and induced cell cycle arrest and apoptosis of SCLC cells in a dose- and timedependent manner. After mut-p53 knockdown or overexpressed, the inhibitory effect of As2O3 was dampened or enhanced. Additionally, As2O3-induced mut-p53 ubiquitination was significantly weakened after MDM2 knockdown. As2O3 could inhibit SCLC cells by inhibiting proliferation and inducing cell cycle arrest and apoptosis. These inhibitory effects were achieved at least in part by upregulating MDM2, which, in turn, promotes ubiquitination and degradation of mut-p53.

Shikonin suppresses small cell lung cancer growth via inducing ATF3-mediated ferroptosis to promote ROS accumulation

Small cell lung cancer (SCLC) is a subtype of lung cancer with a very poor overall survival rate due to its extremely high proliferation and metastasis predilection. Shikonin is an active ingredient extracted from the roots of Lithospermum erythrorhizon, and exerts multiple anti-tumor functions in many cancers. In the present study, the role and underlying mechanism of shikonin in SCLC were investigated for the first time. We found that shikonin effectively suppressed cell proliferation, apoptosis, migration, invasion, and colony formation and slightly induced apoptosis in SCLC cells. Further experiment indicated the shikonin could also induced ferroptosis in SCLC cells. Shikonin treatment effectively suppressed the activation of ERK, the expression of ferroptosis inhibitor GPX4, and elevated the level of 4-HNE, a biomarker of ferroptosis. Both total ROS and lipid ROS were increased, while the GSH levels were decreased in SCLC cells after shikonin treatment. More importantly, our data identified that the function of shikonin was dependent on the up-regulation of ATF3 by performing rescue experiments using shRNA to silence the expression of ATF3, especially in the total and lipid ROS accumulaiton. Xenograft model was established using SBC-2 cells, and the results revealed that shikonin also significantly inhibited tumor growth by inducing ferroptosis. Finally, our data further confirmed that shikonin activated ATF3 transcription by impairing the recruitment of HDAC1 mediated by c-myc on the ATF3 promoter, and subsequently elevating of histone acetylation. Our data documented that shikonin suppressed SCLC by inducing ferroptosis in a ATF3-dependent manner. Shikonin upregulated the expression of ATF3 expression via promoting the histone acetylation by inhibiting c-myc-mediated HDAC1 binding on ATF3 promoter.

Castasterone, a Plant Steroid Hormone, Affects Human Small-Cell Lung Cancer Cells and Reverses Multi-Drug Resistance.

Small-cell lung cancer (SCLC) has a dismal prognosis, in part because of the development of multi-drug resistance. Castasterone (CAS) is the metabolic precursor of the plant steroid hormone epibrassinolide (EB). In some plants, EB accounts for the total hormone activity, whereas in other plants, CAS is the active form. The effects of CAS, a BR present in most plants, on animal cells in general and cancer cells in particular have not been described. Here, we report the effects of CAS on drug-sensitive (H69) and drug-resistant (VPA17) SCLC cells. CAS was equally cytotoxic to both cell lines (IC50 = 1 μM), indicating a lack of cross-resistance. Pre-incubation of VPA17 cells with CAS for 96 h reversed drug resistance to etoposide and doxorubicin. Synergism between CAS and EB, as well as with chemotherapy drugs, was investigated by exposure of VPA17 cells to 1:1 ratios of CAS and the other drugs at the respective IC50 values, with dilutions at 0.25 to 2.0 × IC50 and determination of the combination index (CI). CAS and EB were additive, indicating that the two drugs act on the same pathway, whereas CAS-etoposide (CI = 0.77) and CAS-doxorubicin were synergistic, indicating that CAS and the two chemotherapeutic drugs act on different pathways. Apoptosis in SCLC cells was measured by immuno-detection of single-strand DNA breaks. Following 96 h incubation of SCLC H69 cells in CAS, the level of DNA breaks was similar to measurements made after incubation in EB and etoposide, indicating that CAS is pro-apoptotic. Incubation of SCLC cells in CAS led to a time-dependent reduction (by 80%) in the transcriptional activator β-catenin. These data indicate that CAS may act via Wnt signaling. Taken together, our study reveals that CAS is pharmacologically active in both drug-sensitive and drug-resistant SCLC cells.

Darinaparsin (ZIO-101) enhances the sensitivity of small-cell lung cancer to PARP inhibitors.

Small-cell lung cancer (SCLC) is an aggressive high-grade neuroendocrine carcinoma of the lung associated with early metastasis and an exceptionally poor prognosis. Little progress has been made in developing efficacious targeted therapy for this recalcitrant disease. Herein, we showed that H3.3, encoded by two genes (H3F3A and H3F3B), was prominently overexpressed in SCLC. Darinaparsin (ZIO-101), a derivative of arsenic trioxide, dose- and time-dependently inhibited the viability of SCLC cells in an H3.3-dependent manner. More importantly, ZIO-101 treatment resulted in substantial accumulation of H3.3 and PARP1 besides induction of G2/M cell cycle arrest and apoptosis in SCLC cells. Through integrative analysis of the RNA-seq data from Cancer Cell Line Encyclopedia dataset, JNCI and Genomics of Drug Sensitivity in Cancer 2 datasets, we found that H3F3A expression was negatively correlated with the IC50 values of PARP inhibitors (PARPi). Furthermore, co-targeting H3.3 and PARP1 by ZIO-101 and BMN673/olaparib achieved synergistic growth inhibition against SCLC in vitro and in vivo. In conclusion, it is feasible to target H3.3 by ZIO-101 to potentiate the response rate of PARPi in SCLC patients.

Pazopanib restricts small cell lung cancer proliferation via reactive oxygen species-mediated endoplasmic reticulum stress.

BackgroundPazopanib is an approved multitarget anticancer agent for soft tissue sarcoma (STS) and renal cell carcinoma (RCC), which is also under clinical investigation for other malignancies, including small cell lung cancer (SCLC). However, the potential anti‐SCLC mechanisms of pazopanib remain unclear.MethodsCell viability was evaluated by CCK‐8, apoptotic cell detection was conducted using annexin V/PI staining followed by flow cytometry, and Western blot analysis was used to detect the apoptotic‐related molecules and ER‐stress pathway effectors. The intracellular reactive oxygen species (ROS) level was determined by DCFH‐HA staining followed by flow cytometry. An NCI‐H446 xenograft model was established to evaluate pazopanib on tumor suppression in vivo. Immunohistochemistry (IHC) was used to assess the proliferative activity of xenograft in NCI‐H446 cell‐bearing NOD‐SCID mice.ResultsPazopanib dose‐ and time‐dependently inhibited SCLC cell proliferation induced significant apoptosis in SCLC cell lines, increased cleaved‐caspase3 and Bax, and decreased Bcl‐2. Moreover, the PERK‐related ER‐stress pathway was potently activated by pazopanib treatment, inhibiting ER‐stress by salubrinal significantly reversing pazopanib‐mediated apoptosis in SCLC cell lines. Furthermore, pazopanib‐induced intracellular ROS levels increased, while inhibiting ROS by NAC significantly reversed pazopanib‐induced apoptosis in SCLC cells. In addition, pazopanib significantly suppressed NCI‐H446 xenograft growth and decreased Ki67 positive cells in the tumor.ConclusionOur findings indicate that pazopanib induces SCLC cell apoptosis through the ER‐stress process via upregulation of ROS levels. Further investigation of relevant biomarkers to accurately select patients for benefit from pazopanib should be further investigated.

Inhibition of lncRNA KCNQ1OT1 Improves Apoptosis and Chemotherapy Drug Response in Small Cell Lung Cancer by TGF-β1 Mediated Epithelial-to-Mesenchymal Transition.

PurposeDrug resistance is one of the main causes of chemotherapy failure in patients with small cell lung cancer (SCLC), and extensive biological studies into chemotherapy drug resistance are required.Materials and MethodsIn this study, we performed lncRNA microarray, in vitro functional assays, in vivo models and cDNA microarray to evaluate the impact of lncRNA in SCLC chemoresistance.ResultsThe results showed that KCNQ1OT1 expression was upregulated in SCLC tissues and was a poor prognostic factor for patients with SCLC. Knockdown of KCNQ1OT1 inhibited cell proliferation, migration, chemoresistance and promoted apoptosis of SCLC cells. Mechanistic investigation showed that KCNQ1OT1 can activate transforming growth factor-β1 mediated epithelial-to-mesenchymal transition in SCLC cells.ConclusionTaken together, our study revealed the role of KCNQ1OT1 in the progression and chemoresistance of SCLC, and suggested KCNQ1OT1 as a potential diagnostic and prognostic biomarker in SCLC clinical management.

Combinations of proteasome inhibitors with obatoclax are effective for small cell lung cancer.

Proteasome inhibitors, bortezomib (BTZ), and carfilzomib (CFZ) are approved drugs for hematological malignancies, but lack anticancer activities against most solid tumors. Small cell lung cancer (SCLC) is a very aggressive neuroendocrine carcinoma of the lungs demanding effective therapy. In this study we investigated whether BTZ or CFZ combined with obatoclax (OBX), an antagonist for MCL-1 and a pan-BCL family inhibitor, could cause synergistic growth inhibition of SCLC cells. We showed that combined application of BTZ or CFZ with OBX caused synergistic growth inhibition of human SCLC cell lines (H82, H526, DMS79, H196, H1963, and H69) than single agent alone. Both BTZ-OBX and CFZ-OBX combinations displayed marked synergism on inducing apoptosis (~50% increase vs BTZ or CFZ alone). A comprehensive proteomics analysis revealed that BTZ preferentially induced the expression of MCL-1, an antiapoptotic protein, in SCLC cells. Thus, proteasome inhibitor-OBX combinations could specifically induce massive growth inhibition and apoptosis in SCLC cells. Subsequent proteome-wide profiling analysis of activated transcription factors suggested that BTZ- or CFZ-induced MCL-1 upregulation was transcriptionally driven by FOXM1. In nude mice bearing in SCLC H82 xenografts, both BTZ-OBX, and CFZ-OBX combinations exhibited remarkable antitumor activities against SCLC tumors evidenced by significant reduction of tumor size and the proliferation marker Ki-67 signals in tumor tissues as compared with single agent alone. Thus, proteasome inhibitor-OBX combinations are worth immediate assessments for SCLC in clinical settings.

The anti-infection drug furazolidone inhibits NF-κB signaling and induces cell apoptosis in small cell lung cancer.

Targeting nuclear factor kappa B (NF-κB) signaling pathway has become a promising strategy for the development of new antitumor drugs. In this paper, we found that anti-infection drug furazolidone (FZD) could significantly inhibit NF-κB-driven luciferase activity, and FZD could markedly inhibit both of the constitutive and tumor necrosis factor-α (TNFα)-triggered phosphorylation of NF-κB p65 in small cell lung cancer (SCLC). Further studies revealed that FZD inhibited the expression of inhibitor of kappa B kinase β (IKKβ) in SCLC cells. In addition, we found that FZD had significant antitumor activities in SCLC cells. FZD could markedly suppress the cell viability of SCLC cells dose-dependently, and FZD could significantly induce the cleavages of poly ADP-ribose polymerase (PARP) and Caspase3, the biomarkers of cell apoptosis, in SCLC cells. The flow cytometry also revealed that FZD induced cell apoptosis in SCLC cells. Finally, we also found that overexpression of constitutively activated IKKβ could significantly abolish FZD-induced cell growth inhibition in SCLC cells, which further confirmed that FZD displayed its anti-SCLC activity through regulating NF-κB signaling pathway.

SIRT3 increases cisplatin sensitivity of small-cell lung cancer through apoptosis.

Small-cell lung cancer (SCLC) is the most invasive of all lung cancer subtypes, and is characterized by its rapid response to chemotherapy resistance. Overcoming chemotherapy resistance is therefore the key to treating SCLC. P53 is mutated in most SCLCs, which has an effect of enhancing chemotherapy resistance. Regulation of p53 proteins by a variety of post-translational modifications, such as acetylation, which affects their function. Acetylation and deacetylation of p53 may be potential targets for modulating chemosensitivity. Recent studies have shown that SIRT3 acts as a deacetylase that regulates acetylation of p53. However, whether SIRT3 can regulate the post-translational modification of mutant p53 has not been studied. In the present study, we found that SIRT3 can deacetylate mutant p53, thus reducing its expression, inducing apoptosis in SCLC cells, and increasing SCLC chemosensitivity. The relationship between SIRT3 and mutant p53 could be the basis of a new SCLC treatment approach.

Gambogenic acid inhibits the proliferation of small‑cell lung cancer cells by arresting the cell cycle and inducing apoptosis.

Gambogenic acid (GNA), which is an important active compound present in gamboge, exerts anticancer activity in various types of tumor cells. However, the effect of GNA on small-cell lung cancer (SCLC) cell lines and the underlying mechanism involved still remain unclear. In the present study, GNA inhibited the proliferation and cell cycle progression of SCLC cells. GNA also promoted the apoptosis of SCLC cells in a dose-dependent manner, which is associated with modulating the levels of proteins involved in apoptosis pathways in NCI-H446 and NCI-H1688 cells. The results demonstrated that GNA increased the level of cleaved caspase-3, −8 and −9, and Bax but decreased the expression of anti-apoptotic protein, Bcl-2. Furthermore, similar results were obtained in a mouse tumor xenograft model. Additionally, GNA exhibit low toxicity in tissues when administered to mice in the SCLC xenograft models. Collectively, our findings demonstrated that GNA significantly inhibited the proliferation of SCLC cells and promoted cell apoptosis via cell cycle arrest and induction of apoptosis.

Wnt inhibitor XAV939 suppresses the viability of small cell lung cancer NCI-H446 cells and induces apoptosis.

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer due to a fast tumor doubling time and early hematogenous spread. Advances in the treatment of non-small cell lung cancer using targeted therapies having been made, but no targeted drugs for SCLC have been approved. The Wnt signaling pathway is associated with tumor progression and metastasis; therefore, the inhibition of Wnt/β-catenin signaling is a strategy for anticancer drugs. Tankyrase 1 (TNKS1) is overexpressed in a number of types of cancer and XAV939 is a small molecule inhibitor of TNKS1 which may inhibit tumor growth. The present study aimed to investigate the potential molecular mechanisms underlying XAV939-induced suppression of the viability of SCLC cells. MTT assays were used to determine the viability-inhibition rate of cells and to identify the drug concentration which optimally inhibited cell viability. Flow cytometry was used to determine whether XAV939 induced apoptosis of SCLC cells, and to analyze the effect of the drug on the cell cycle. The results of the present study identified that XAV939 inhibited the viability of NCI-H446 cells in a dose-dependent manner, but cisplatin inhibited NCI-H446 cell viability in a time- and dose-dependent manner. The combination of XAV939 and cisplatin exhibited a slightly more pronounced inhibition of cell viability at an increased dose of XAV939. In addition, XAV939 markedly induced cell apoptosis of the SCLC cell line H446 by increasing the proportion of cells in the G0/G1 phase, leading to inhibition of the cell cycle. The results of the present study indicated that XAV939 inhibited the viability of the NCI-H446 SCLC cell line by inducing cell apoptosis through the Wnt signaling pathway. Therefore, XAV939 may be useful for the treatment of SCLC.

RBM5 reduces small cell lung cancer growth, increases cisplatin sensitivity and regulates key transformation-associated pathways.

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer, with almost 95% of patients succumbing to the disease. Although RBM5, a tumor suppressor gene, is downregulated in the majority of lung cancers, its role in SCLC is unknown. Using the GLC20 SCLC cell line, which has a homozygous deletion encompassing the RBM5 gene locus, we established stable RBM5 expressing sublines and investigated the effects of RBM5 re-expression. Transcriptome and target identification studies determined that RBM5 directly regulates the cell cycle and apoptosis in SCLC cells, as well as significantly downregulates other important transformation-associated pathways such as angiogenesis and cell adhesion. RNA sequencing of paired non-tumor and tumor SCLC patient specimens showed decreased RBM5 expression in the tumors, and expression alterations in the majority of the same pathways that were altered in the GLC20 cells and sublines. Functional studies confirmed RBM5 expression slows SCLC cell line growth, and increases sensitivity to the chemotherapy drug cisplatin. Overall, our work demonstrates the importance of RBM5 expression to the non-transformed state of lung cells and the consequences of its deletion to SCLC development and progression.

Target Identification in Small Cell Lung Cancer via Integrated Phenotypic Screening and Activity-Based Protein Profiling

To overcome hurdles in identifying key kinases in small cell lung cancer (SCLC), we integrated a target-agnostic phenotypic screen of kinase inhibitors with target identification using activity-based protein profiling (ABPP) in which a desthiobiotin-ATP probe was used. We screened 21 SCLC cell lines with known c-MYC amplification status for alterations in viability using a chemical library of 235 small-molecule kinase inhibitors. One screen hit compound was interrogated with ABPP, and, through this approach, we reidentified Aurora kinase B as a critical kinase in MYC-amplified SCLC cells. We next extended the platform to a second compound that had activity in SCLC cell lines lacking c-MYC amplification and identified TANK-binding kinase 1, a kinase that affects cell viability, polo-like kinase-1 signaling, G2-M arrest, and apoptosis in SCLC cells lacking MYC amplification. These results demonstrate that phenotypic screening combined with ABPP can identify key disease drivers, suggesting that this approach, which combines new chemical probes and disease cell screens, has the potential to identify other important targets in other cancer types. Mol Cancer Ther; 15(2); 334-42. ©2016 AACR.

MicroRNA-134 regulates lung cancer cell H69 growth and apoptosis by targeting WWOX gene and suppressing the ERK1/2 signaling pathway

MicroRNAs have been shown to act as crucial modulators during carcinogenesis. Recent studies have implied that miR-134 expression associated with epithelial-to-mesenchymal transition phenotype and invasive potential of NSCLC cells. Our study investigated the pathogenic implications of miR-134 in small cell lung cancer (SCLC). Overexpression or inhibition MiR-134 expression by miR-134 mimics or miR-134 inhibitors (anti-miR-134) in SCLC cell lines was detected using qRT-PCR. Lactate dehydrogenase (LDH) assay, MTT assays and flow cytometry were performed in order to clarify the growth and apoptosis of SCLC cells which had been transfected with miR-134 mimics or anti-miR-134. WWOX expression in H69 cells was detected by qRT-PCR and western blot, respectively. The results showed that overexpression miR-134 was significantly promoting SCLC cells growth and inhibit its apoptosis. In addition, reduced miR-134 expression was significantly correlated with cell growth inhibition and apoptosis promotion. Furthermore, transfection of miR-134 mimics into the SCLC cells markedly down-regulated the level of WWOX, whereas, anti-miR-134 up-regulated WWOX expression. We also found that overexpression WWOX attenuate miR-134 induced H69 cells growth, and promote cell apoptosis. Moreover, miR-134 promoted cell proliferation and inhibit apoptosis via the activation of ERK1/2 pathway. These findings suggest that miR-134 may be an ideal diagnostic and prognostic marker, and may be attributed to the molecular therapy of SCLC.

Knocking down the expression of Aurora-A gene inhibits cell proliferation and induces G2/M phase arrest in human small cell lung cancer cells.

A hallmark of small cell lung cancer (SCLC) is frequent relapse characterized by newfound resistance to formerly efficacious chemotherapies. The prognosis for SCLC patients is particularly unfavorable. Aurora kinase A (AURKA), a member of the serine/threonine kinase family, is overexpressed across many types of human tumors. Recent studies have identified AURKA as an important factor in tumorigenesis, but little is known regarding its specific roles in SCLC. The aim of the present study was to establish the roles of AURKA in the molecular pathogenesis of human SCLC. In the present study, we constructed a lentiviral vector to express siRNA against AURKA (LV-AURKA siRNA). As we expected, the viral construct effectively suppressed the expression of the AURKA gene and protein in H446 and H1688 cell lines. Additionally, RNA interference of AURKA inhibited the colony formation and subsequent growth of H446 and H1688 cell lines by increasing the incidence of cell cycle arrest in the G2/M phase. Furthermore, suppression of AURKA by LV-AURKA siRNA also increased apoptosis of SCLC cells. A potential mechanism for the increase of apoptosis is the downregulation of Bcl-2 and upregulation of Bax. AURKA gene suppression may provide a novel, effective therapy for SCLC patients by inhibiting cell division and increasing the rate of apoptosis of SCLC cells.

Higher Expression of Caveolin-1 Inhibits Human Small Cell Lung Cancer (SCLC) Apoptosis In Vitro

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer, and its treatment is closely associated with apoptosis. Caveolin-1 plays an important role in the development of a variety of human cancers. This study sought to investigate the influence of Caveolin-1 on the apoptosis of SCLC in vitro. We demonstrate that higher expression of Caveolin-1 leads to inhibition of cisplatin and Ultraviolet Radiation (UVR)-induced apoptosis in SCLC cells; and also could decrease caspase-3 activity and increase the stability of Bcl-2 at the protein level. Our findings illuminate a potential molecular mechanism regarding CAV-1's role as anti-apoptosis protein.

Abstract 4891: Radiation enhances the efficacy of Bcl-2 and Bcl-XL inhibition in small cell lung cancer: A new concept of using radiation for targeted therapy through contextual oncogene addiction

Abstract Purpose: Radiotherapy has a critical role in the treatment of small cell lung cancer (SCLC). Effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. Experimental Design: We investigated whether using a new Bcl-2/Bcl-XL inhibitor, S44563, can enhance radiosensitivity of SCLC cells in vitro and in vivo. We also explored the mechanisms of the potential interaction. Results: In vitro studies confirmed that S44563 induce apoptosis in SCLC cells by inducing hallmarks of apoptosis (cleaved caspase-3, sub-G1 fraction induction and induction of the mitochondrial caspase activation pathway). S44563 markedly enhanced sensitivity of H146 and H69 cells to radiation in clonogenic assay. In vitro experiments demonstrated a greater induction of apoptosis (sub-G1 fraction and cleaved caspase-3) with the combination as well as in vivo caspase-3 immunostaining. This positive interaction between S44563 and radiation was greater when S44563 was given during and after the completion of the radiation, which was explained by the radiation-induced over expression of anti-apoptotic proteins (Bcl-2 and Bcl-XL). in vitro studies based on gene expression and immunoblotting indicated that radiation activates NF-κB pathways which results in anti-apoptotic expression. In addition, S44563 combined with cisplatin-based chemo-radiation showed a dramatic tumor growth delay and increased overall survival in mouse xenograft models.Conclusion: This study underlines the critical role of sequence administration of targeted therapies with conventional therapies such as radiation and chemotherapy. SCLC cells which survive to IR highly rely on the induction of anti apoptotic proteins for their survival therefore yielding to the concept of “contextual oncogene addiction” to the Bcl-2/Bcl-XL pathway. These results suggest that radioresistance results in part from resistance to apoptosis in SCLC mediated by up regulation of Bcl-2/Bcl-XL. The inhibition of Bcl-2 and Bcl-XL leads to the induction of radiation-induced apoptosis and enhances radiation therapy both in vitro and in SCLC xenograft models. Our data suggest that targeting radiation induced survival pathways may constitute an efficient strategy to potentiate IR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4891. doi:1538-7445.AM2012-4891

TRAIL-induced survival and proliferation of SCLC cells is mediated by ERK and dependent on TRAIL-R2/DR5 expression in the absence of caspase-8

Small cell lung cancer (SCLC) is characterized by an aggressive phenotype and acquired resistance to a broad spectrum of anticancer agents. TNF-related apoptosis-inducing ligand (TRAIL) has been considered as a promising candidate for safe and selective induction of tumor cell apoptosis without toxicity to normal tissues. Here we report that TRAIL failed to induce apoptosis in SCLC cells and instead resulted in an up to 40% increase in proliferation. TRAIL-induced SCLC cell proliferation was mediated by extracellular signal-regulated kinase 1 and 2, and dependent on the expression of surface TRAIL-receptor 2 (TRAIL-R2) and lack of caspase-8, which is frequent in SCLC. Treatment of SCLC cells with interferon-gamma (IFN-gamma) restored caspase-8 expression and facilitated TRAIL-induced apoptosis. The overall loss of cell proliferation/viability upon treatment with the IFN-gamma-TRAIL combination was 70% compared to TRAIL-only treated cells and more than 30% compared to untreated cells. Similar results were obtained by transfection of cells with a caspase-8 gene construct. Altogether, our data suggest that TRAIL-R2 expression in the absence of caspase-8 is a negative determinant for the outcome of TRAIL-based cancer therapy, and provides the rationale for using IFN-gamma or other strategies able to restore caspase-8 expression to convert TRAIL from a pro-survival into a death ligand.