Drug-resistant Small Cell Lung Cancer Research Articles

Targeting TREX1 Induces Innate Immune Response in Drug-Resistant Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation sequencing revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLCs. Analyses of human SCLC tumors and patient-derived xenografts (PDX) also showed an increase in TREX1 expression in postchemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase stimulator of interferon gene pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug-resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs. Significance: In this study, we show that targeting TREX1 induces an innate immune response and resensitizes SCLC cells to chemotherapy, representing a promising novel target for "immunologically" cold tumors, such as SCLC.

HMOX1 regulates ferroptosis via mic14 and its impact on chemotherapy resistance in small-cell lung cancer.

This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed bioinformatics, qPCR, and Western Blot to assess HMOX1 levels in SCLC versus normal tissues and its prognostic relevance. CCK-8, flow cytometry, and thiobarbituric acid assays determined HMOX1's impact on SCLC chemosensitivity, ferroptosis markers, lipid peroxidation, and mic14's role in chemoresistance. In the GSE40275 and GSE60052 cohorts, HMOX1 expression was downregulated in SCLC tissues compared to normal tissues. Higher HMOX1 expression was associated with improved prognosis in the Sun Yat-sen University Cancer Hospital cohort and GSE60052 cohort. The RNA and protein levels of HMOX1 were reduced in drug-resistant SCLC cell lines compared to chemosensitive cell lines. Upregulation of HMOX1 increased chemosensitivity and reduced drug resistance in SCLC, while downregulation of HMOX1 decreased chemosensitivity and increased drug resistance. Upregulation of HMOX1 elevated the expression of ferroptosis-related proteins ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while decreasing the expression of GPX4 and xCT. Conversely, downregulation of HMOX1 decreased the expression of ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while increasing the expression of GPX4 and xCT. Upregulation of HMOX1 promoted cellular lipid peroxidation, whereas downregulation of HMOX1 inhibited cellular lipid peroxidation. Upregulation of HMOX1 reduced the RNA level of mic14, while downregulation of HMOX1 increased the RNA level of mic14. mic14 exhibited inhibitory effects on cellular lipid peroxidation in SCLC cells and contributed to reduced chemosensitivity and increased drug resistance in chemoresistant SCLC cell lines. HMOX1 plays a role in ferroptosis by regulating mic14 expression, thereby reversing chemoresistance in SCLC.

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.

Evaluating in vitro cytotoxic effects of Artemisia afra and Artemisia annua infusions against selected lung cancer cell lines

Lung cancer was the leading cause of cancer deaths in 2020. This can mainly be attributed to the poor outcomes of chemotherapy as a result of acquired multidrug resistance. Traditional medicinal plant species such as Artemisia afra and A. annua have been used for decades to treat various health-related conditions, including cancer. Both species have previously exhibited anti-cancer activity, although studies to confirm their efficacy and safety in the treatment of lung cancer are still lacking. Therefore, the aim of this study was to identify the major compounds in the traditionally prepared infusions and to evaluate the infusions’ in vitro cytotoxicity against the following lung cancer cell lines: H69V, a chemo-sensitive small cell lung cancer (SCLC) line; H69AR, a drug-resistant SCLC line that hyper-expresses the multidrug resistance-associated protein 1 efflux transporter; and A549, a non-small cell lung cancer line. Non-cancerous lung fibroblast and kidney cells (MRC-5 and Vero) were also included in the study to evaluate the cancer selectivity of the samples. A series of concentrations of each of the samples were screened to determine the relative cell viability inhibitory concentration and selectivity index by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The major phytochemicals in the infusions were identified as organic acids and coumarins and both A. annua and A. afra infusions showed a high degree of selectivity towards the chemo-sensitive H69V cells with IC 50 values of 6 and 70 µg/mL, respectively, and a SI value of 10 for A. afra . However, cytotoxicity towards both non-cancerous cell lines was also observed. Furthermore, the H69AR cells showed considerable resistance to the plant extracts’ cytotoxicity, suggesting that some of the active phytoconstituents in the extracts may potentially be substrates of the MRP1 efflux transporter. From the outcomes of this study, it became evident that both A. afra and A. annua extracts have the potential as a treatment for chemosensitive SCLC. However, possible non-selective cytotoxic effects may be a concern that warrants further investigation. • The cancer cytotoxicity of infusions from Artemisia annua and A. afra was evaluated. • The major compounds in the infusions were identified as organic acids and coumarins. • Infusions were found to be active at > 50 µg/mL with A. afra being most active. • While less active than paclitaxel, A . afra was more selective against H69V and A549.

HuR affects chemoresistance of small cell lung cancer by regulating FGFRL1 expression.

Human antigen R (HuR), an RNA-binding protein, has been demonstrated to serve an oncogenic role in various types of cancer. Fibroblast growth factor receptor-like 1 (FGFRL1) has been shown to regulate small cell lung cancer (SCLC) chemoresistance. In the present study, the role of HuR in chemoresistance of SCLC, as well as its possible molecular mechanism involving FGFRL1, was explored by reverse transcription-quantitative PCR, western blotting, Cell Counting Kit-8 assay, flow cytometry and RNA immunoprecipitation. The results revealed that HuR expression levels were markedly upregulated in drug-resistant SCLC cell lines (H69AR and H446DDP) compared with in the parental cell lines (H69 and H446). Knockdown of HuR in drug-resistant SCLC cells enhanced drug sensitivity, cell apoptosis and cell cycle arrest. Furthermore, molecular mechanism studies indicated that HuR could bind and regulate FGFRL1 expression levels to increase FGFRL1 mRNA stability. Taken together, the present study suggested that HuR may mediate chemoresistance of SCLC by regulating FGFRL1 expression. HuR may represent a prognostic predictor and a potential target for overcoming chemoresistance in SCLC.

Design, synthesis, and biological evaluation of novel 7-substituted 10,11-methylenedioxy-camptothecin derivatives against drug-resistant small-cell lung cancer in vitro and in vivo

A series of novel 7-substituted 10,11-methylenedioxy-camptothecin (FL118) derivatives were designed, synthesized, and biologically evaluated. All the FL118 analogues showed significant cytotoxic activities in vitro with IC50 values in the nanomolar range and were more potent than topotecan. The most active compound 9c exhibited more significant anti-tumor activity against small-cell lung cancer (NCI–H446, H69, drug-resistant H69AR cells, drug-resistant NCI–H446/Irinotecan cells and drug-resistant NCI–H446/EP cells) in vitro. Additionally, 9c could also induce the expression of apoptosis proteins such as caspase-3, caspase-9, and PARP in small-cell lung cancer. Further studies showed that 9c induced apoptosis by inhibiting the expression of Mcl-1, Bcl-2, XIAP and survivin in small-cell lung cancer. In vivo9c also showed better anti-tumor efficacy, with the tumor growth inhibition rates were 40.4% (0.75 mg/kg), 73.7% (1.5 mg/kg), and 95.5% (3 mg/kg). It is noteworthy that 9c also demonstrated potent inhibition of drug-resistant tumor growth in NCI–H446/Irinotecan and NCI–H446/EP xenograft models, the tumor growth inhibition rates were 93.42% and 84.46%, respectively. Taken together, these findings indicated that compound 9c displays outstanding antitumor activity and drug-resistance in small-cell lung cancer both in vivo and in vitro, which could be worth further research as a novel anti-tumor drug against small-cell lung cancer.

Clinical efficacy and safety of irinotecan and docetaxel combined with cisplatin in the treatment of recurrent and drug-resistant small cell lung cancer.

Clinical efficacy and safety of irinotecan and docetaxel combined with cisplatin in the treatment of recurrent and drug-resistant small cell lung cancer.

MRP1-targeted near infrared photoimmunotherapy for drug resistant small cell lung cancer

Small cell lung cancer (SCLC), one of the most aggressive cancers, has a high mortality rate and poor prognosis, and the clinical therapeutic outcomes of multidrug resistant SCLC are even worse. Multidrug resistance protein 1 (MRP1), one of the ATP-binding cassette (ABC) transporter proteins that cause decreased drug accumulation in cancer cells, is overexpressed in drug resistant SCLC cells and could be a promising target for treating the patients suffering from this illness. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed approach for targeted cancer treatment which uses a conjugate of a monoclonal antibody and photoabosorber IR700 followed by NIR light irradiation to induce rapid cancer cell death. In the present study, an anti-MRP1 antibody (Mab) -IR700 conjugate (Mab-IR700) was synthesized, purified and used to treat chemoresistant SCLC H69AR cells that overexpressed MRP1, while non-MRP1-expressing H69 cells were used as a control. Then, the photokilling and tumor suppression effect were separately evaluated in H69AR cells both in vitro and in vivo. Higher cellular delivery of Mab-IR700 was detected in H69AR cells, whereas there was little uptake of IgG-IR700 in both H69 and H69AR cells. Due to the targeting activity of Mab, stronger photokilling effect was found both in H69AR cells and spheroids treated with Mab-IR700, while superior tumor suppression effect was also observed in the mice treated with Mab-IR700 and light illumination. Photoacoustic imaging results proved that oxygen was involved in NIR-PIT treatment, and TUNEL staining images showed the occurrence of cell apoptosis, which was also testified by HE staining. This research provides MRP1 as a novel target for PIT and presents a prospective way for treating drug resistant SCLC and, thus, should be further studied.

H3K27me3 induces multidrug resistance in small cell lung cancer by affecting HOXA1 DNA methylation via regulation of the lncRNA HOTAIR.

The long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) serves as a powerful predictor of tumor progression and overall survival in patients. Our previous studies showed that HOTAIR modulated HOXA1 DNA methylation by reducing DNMT1 and DNMT3b expression in drug-resistant small cell lung cancer (SCLC). Moreover, H3 lysine 27 trimethylation (H3K27me3) is catalyzed by enhancer of zeste homolog 2 (EZH2) and plays a critical role in SCLC chemoresistance. However, it is not completely clear whether H3K27me3 affects HOXA1 DNA methylation or whether this effect is mediated by HOTAIR. The levels of EZH2 and H3K27me3 were identified in SCLC tissues by immunohistochemical (IHC) staining and in SCLC multidrug-resistant cells by Western blotting. Cell counting kit-8 (CCK-8) and flow cytometry were used to detect and analyze the biological function of H3K27me3. Then, we assessed the role of HOTAIR in the regulation of EZH2 and H3K27me3 by using lentivirus and small interfering RNA. Further, bisulfite sequencing PCR was conducted to detect the methylation levels of HOXA1 DNA. Finally, Western blotting was performed to examine the regulatory role of H3K27me3 in controlling HOTAIR expression in SCLC. In this study, we found that EZH2 and H3K27me3 levels were markedly higher in SCLC tissues and multidrug-resistant SCLC cells. The results indicated that H3K27me3 was related to multidrug resistance. HOTAIR overexpression and knockdown showed that EZH2 and H3K27me3 were regulated by HOTAIR. Moreover, H3K27me3 affected HOXA1 DNA methylation levels. Strikingly, we found that H3K27me3 acted as a negative feedback regulator of HOTAIR. Our study showed that H3K27me3 affects HOXA1 DNA methylation via HOTAIR regulation, indicating that H3K27me3 may be a potential therapy target for SCLC chemoresistance.

Salvianolic acid-A induces apoptosis, mitochondrial membrane potential loss and DNA damage in small cell lung cancer cell lines

Purpose: To examine the anticancer effect of salvianolic acid-A against human small cell lung cancer (SCLC) cells (H-69). Methods: In vitro antiproliferative effect of salvianolic acid against SCLC cell lines was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction by salvianolic acid-A in these cells was measured by videomicroscopy along with fluorescence microscopy using Hoechst 33258 staining. The effect of the compound on mitochondrial membrane potential loss was detected by flow cytometry using rhodamine-123 as fluorescent probe. Gel electrophoresis was used to analyze DNA fragmentation after salvianolic acid treatment. Results: The results revealed that salvianolic acid-A induces dose-dependent as well as timedependent growth inhibitory effects against SCLC cancer cells. Videomicroscopy analysis revealed that SCLC cells became rounded (dead) and refringent after drug treatment which increased with increase in salvianolic acid-A dose. Fluorescence microscopy demonstrated that salvianolic acid-A induced dosedependent chromatin condensation in SCLC cells which is characteristic of apoptosis. DNA fragmentation was induced by various doses of the compound again hinting at apoptosis. Flow cytometry analysis using rhodamine-123 showed that salvianolic acid-A also caused substantial loss of mitochondrial membrane potential. Conclusion: The results suggest that salvianolic acid-A is a potential anticancer and apoptotic agent against drug-resistant small cell lung cancer.

Synergistic Anticancer Activity of Topotecan— Cyclin-Dependent Kinase Inhibitor Combinations against Drug-Resistant Small Cell Lung Cancer (SCLC) Cell Lines

Extended-stage small cell lung cancer (SCLC) responds to platinum/vepeside-based first-line chemotherapy but relapses rapidly as drug-resistant tumor. Topotecan (TPT) is the single chemotherapeutic agent approved for second-line treatment of SCLC. However, the response to TPT is short-lived and novel treatment modalities need to be developed. Sequential treatment of cytotoxic drugs and inhibitors of cyclin-dependent kinases (CDKs) showed promising preclinical anticancer activity and, in the present work, combinations of TPT with CDK inhibitors olomoucine, roscovitine and CDK4I are shown to exhibit synergistic cytotoxic activity against SCLC cell lines. Highest activity was found against TPT-resistant NCI-H417 and DMS153 cell lines and moderate chemosensitizing effects against a primary SCLC cell line and sensitive GLC19 cells at levels of CDK inhibitors which exerted low toxicity. A combination of 0.6 μM TPT with 0.6 μM roscovitine, exhibiting no significant cytotoxicity as single agents, reduced viability of the TPT-resistant NCI-H417 line (IC50 > 10 μM) by 50%. In the TPT resistant cell lines olomoucine and roscovitine, targeting CDK1,2,5,7, were highly effective, whereas in the more sensitive cell lines CDK4I, inhibiting mainly CDK4/6, showed activity. In NCI-417 cells, preincubation with roscovitine for one day proved synergistic with TPT. Thus, in good accordance with previous findings, CDK inhibitors are able to convert SCLC cancer cells which are cell-cycle arrested by a blockade of topoisomerase I by TPT to apoptotic cells. Since nowadays several CDK inhibitors are at various phases of clinical testing their combination with TPT seems to constitute a promising approach to improve second-line chemotherapy in SCLC.

Organic Anion Transporting Polypeptide 5A1 (OATP5A1) in Small Cell Lung Cancer (SCLC) Cells: Possible Involvement in Chemoresistance to Satraplatin

BackgroundThe role of organic anion transporting polypeptide 5A1 (OATP5A1) a member of a family of drug transporters that mediate cellular uptake of drugs has not been characterized so far.MethodsGene expression levels of OATP5A1 in small cell lung cancer (SCLC) cell lines were determined by real-time qPCR and chemosensitivity of HEK-293-SLCO5A1-transfected cells to satraplatin in MTT assays.ResultsSignificant expression of this transporter was found at the mRNA level, primarily in drug-resistant SCLC cells, and SLCO5A1-transfected HEK-293 cells showed higher resistance to satraplatin. OATP5A1 is found preferentially in cytoplasmic membranes of tumor cells, including SCLC.ConclusionsOATP5A1 seems to effect intracellular transport of drugs and may participate in chemoresistance of SCLC by sequestration, rather than mediating cellular uptake. Since satraplatin failed to improve survival in SCLC patients, the relation of OATP5A1 expression to clinical drug resistance and its use as marker of chemoresistance should be further investigated.

Gene expression profiling of drug-resistant small cell lung cancer cells by combining microRNA and cDNA expression analysis

MicroRNAs (miRNAs) are now known to play important roles in the regulation of gene expression for developmental timing, cell proliferation and apoptosis. Therefore, it is likely that they also modulate sensitivity and resistance to anti-cancer drugs. To better understand the molecular mechanisms of multidrug resistance in SCLC and identify novel molecular markers, we evaluated the expression of 856 miRNAs and ∼22,000 genes using miRNA microarray and cDNA microarray in cellular models of SCLC which were widely used as sensitive (NCI-H69) and resistant cell lines (NCI-H69AR) to chemotherapy. We also analysed the correlations between miRNA and mRNA expression patterns. Further studies were tested to determine whether the differentially expressed miRNAs were involved in multidrug resistance in SCLC. Our results showed that 61 miRNAs are presented significantly (>3-fold) including up-regulation of 24 miRNAs and down-regulation of 37 miRNAs. Among these miRNAs, 48 of 61 differentially expressed miRNAs were firstly reported to be closely associated with drug resistance and 37.7% (24/61) of miRNA genes were organised as 10 clusters in total 61 significantly expressed miRNAs. We also found that only 27 of 69 miRNAs were significantly correlated with 604 of 21,522 70 mRNA transcripts by MAS database. The sensitivity to anti-cancer drugs Cisplatin, Etoposide and Doxorubicin greatly increased or reduced following transfection of the drug-resistant H69AR cells with the mimics or antagomirs of miR-134, miR-379 and miR-495, respectively. miR-134 increases the cell survival by inducing G1 arrest in H69AR cells. MRP1/ABCC1 is negatively regulated by miR-134 and down-regulation of MRP1/ABCC1 at the protein level largely correlates with elevated levels of miR-134 in H69AR cells. Our results support for the first time a substantial role for miRNAs in multidrug resistance in SCLC. miR-134 could be a causal factor of the down-regulation of MRP1/ABCC1 in H69AR cells. These findings provide valuable information for potential utility of these miRNAs as specific diagnostic biomarkers and novel therapeutic approaches for drug resistance of SCLC.

Effect of Ganoderma on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells

Multidrug resistance is a major problem in small-cell lung cancer (SCLC). Ganoderma lucidum is a widely used herb is traditional Chinese medicine. We tested the effects of Ganoderma on drug-sensitive (H69) and multi-drug resistant (VPA) human SCLC cells. Both cells showed equal cytotoxicity when incubated with extracts of mycelia of 9 species of Ganoderma, including G. lucidum. Cells treated with the IC 50 of cytotoxic Ganoderma and analyzed by flow cytometry-PI staining showed increases in S phase. When compared untreated controls or SCLC cells treated with extracts of non-cytotoxic Ganoderma species, cells treated with extracts of cytotoxic Ganoderma species responded with an induction of apoptosis similar to cells treated with the chemotherapeutic drugs etoposide and doxorubicin. This was shown by four criteria: increased DNA fragmentation within cells as measured by ELISA; increased TUNEL staining for DNA breaks; increased specific activities of caspases 3 and 9, but not caspase 8 by colorimetric assays, indicating the endogenous pathway; and similar patterns changes in the expressions of 9 genes involved in the cell cycle/apoptosis, as measured by RT-PCR and capillary electrophoresis. Pre-incubation of drug-resistant SCLC cells with cytotoxic Ganoderma reduced the IC 50 for etoposide (3.4–0.21 μM) and doxorubicin (0.19–0.04 μM). These results show that extracts of several species of Ganoderma are cytotoxic to both drug-sensitive and drug-resistant SCLC cells, are pro-apoptotic, induce gene expression patterns that are similar to SCLC cells treated with chemotherapeutic drugs, and can reverse resistance to chemotherapeutic drugs.

Identification of uPAR-positive Chemoresistant Cells in Small Cell Lung Cancer

BackgroundThe urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype.Methods and FindingsWe screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers.ConclusionsThese data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC.

Transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells

Multiple drug resistance is a significant problem in small-cell lung cancer (SCLC). Artemisinin (ART) is a natural product used to treat drug-resistant malaria. The drug is effective because the Fe 2+ present in infected erythrocytes acts non-enzymatically to convert ART to toxic products. We tested the effects of ART on drug-sensitive (H69) and multi-drug-resistant (H69VP) SCLC cells, pretreated with transferrin (TF) to increase the intracellular Fe 2+ level. Antibody staining followed by flow cytometry analysis showed twice the level of TF receptors on the H69VP as compared to the H69 cells. Low doses of ART were cytotoxic to SCLC cells. The cytotoxicity of ART for H69VP cells (IC 50=24 nM) was ten-fold lower than for H69 cells (IC 50=2.3 nM), indicating that ART is part of the drug resistance phenotype. Pretreatment of H69 cells with 220–880 nM TF did not alter the IC 50 for ART. However, in the ART-resistant H69VP cells, pretreatment with TF lowered the ART IC 50 to near drug-sensitive levels (IC 50=5.4 nM after 4 h pretreatment with 880 nM TF). Desferrioxamine (5 μM) inhibited the effect of TF on the IC 50 for ART in drug-resistant cells but did not have an effect on ART cytotoxicity in drug-sensitive cells. DNA fragmentation as measured by ELISA occurred within ART-treated cells, with kinetics indicating apoptosis rather than necrosis. This was confirmed by TUNEL staining. These data indicate the potential use of ART and TF in drug-resistant SCLC.

Reduction of expression of the multidrug resistance protein (MRP) in human tumor cells by antisense phosphorothioate oligonucleotides

Multidrug resistance protein (MRP) is a member of the ATP-binding cassette superfamily of transport proteins which has been demonstrated to cause multidrug resistance when transfected into previously sensitive cells. Sixteen eicosomeric oligonucleotides complementary to different regions along the entire length of the MRP mRNA reduced MRP mRNA and protein levels in drug-resistant small cell lung cancer cells that highly overexpress this protein. In MRP-transfected HeLa cells that express intermediate levels of MRP, one oligonucleotide, ISIS 7597, targeted to the coding region of the MRP mRNA, decreased the levels of MRP mRNA to < 10% of control levels in a concentration-dependent manner. This effect was rapid but transient with a return to control levels of MRP mRNA 72 hr after treatment. A double treatment with ISIS 7597 produced a sustained inhibition, resulting in a greater than 90% reduction in MRP mRNA for 72 hr and a comparable decrease in protein levels. Increased sensitivity to doxorubicin was observed under these conditions. Northern blotting analyses using two DNA probes corresponding to sequences 5′ and 3′ of the ISIS 7597 target sequence, respectively, revealed the presence of low levels of two smaller sized RNA fragments as expected from an RNase H-mediated mechanism of action of the antisense oligonucleotide. These studies indicate that a specific reduction in MRP expression can be achieved with antisense oligonucleotides, a finding that has potential implications for the treatment of drug-resistant tumors.

Altered topoisomerase IIα in a drug-resistant small cell lung cancer cell line selected in VP-16 : Mirski SEL, Evans CD, Almquist KC, Slovak ML, Cole SPC. Cancer Research Laboratories, Queen's University, Kingston, Ont. K7L 3N6. Cancer Res 1993;53:4866–73

The H209/V6 cell line was derived from the H209 small cell lung cancer cell line by selection in etoposide (VP-16). Cytogenetic analysis indicates that the sensitive and resistant cell lines share 20 marker chromosomes and thus are clearly related. However, the H209/V6 cell line has four additional structurally altered chromosomes and a 2 N-modal chromosome number, while the H209 cell line is hypotetraploid (4 N-). H209/V6 cells are cross-resistant to some drugs that interact with topoisomerase II but not mitoxantrone. H209/V6 cells are also not cross-resistant to vincristine, trimetrexate, or cisplatin. The rates of VP-16 efflux are the same in the resistant and sensitive cell lines, which is consistent with the observation that P-glycoprotein mRNA is not detectable in either cell line. Fewer VP-16-induced DNA-protein complexes are observed in H209/V6 cells, and immunoblot analysis shows that levels of topoisomerase II alpha are reduced in H209/V6 cells compared to the sensitive H209 cells. Furthermore, the topoisomerase II alpha-related protein in H209/V6 cells has an increased electrophoretic mobility, with an apparent M(r) of 160,000. The levels of the topoisomerase II alpha 6.1-kilobase mRNA in H209/V6 cells are reduced > 10-fold. In addition, a second topoisomerase II alpha-related mRNA of approximately 4.8 kilobases is observed in H209/V6 cells but not in H209 cells. The quantity and electrophoretic mobility of the M(r) 180,000 topoisomerase II beta protein and its 6.1-kilobase mRNA are the same in the sensitive and resistant cell lines. The topoisomerase II strand-passing activity in H209/V6 nuclear extracts is reduced about 2-fold, but this activity is not more resistant to inhibition by VP-16 than the activity in H209 cells. However, band depletion immunoblot experiments show that the topoisomerase II alpha-related M(r) 160,000 protein in H209/V6 cells is not bound to DNA in the presence of concentrations of VP-16 that deplete the M(r) 170,000 topoisomerase II alpha in H209 cells and the M(r) 180,000 topoisomerase II beta in both the resistant and sensitive cells. We conclude that quantitative and qualitative alterations in topoisomerase II alpha have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.

Do glutathione and related enzymes play a role in drug resistance in small cell lung cancer cell lines?

Small cell lung cancer (SCLC) is treated primarily with combination chemotherapy. Despite high initial response rates, most patients eventually die with drug resistant disease. In some tumours, resistance to multiple chemotherapeutic agents is attributed to overexpression of P-glycoprotein (P-gp). However, this does not appear to be a frequent occurrence in drug resistant SCLC. Increased levels of glutathione (GSH) and related enzymes may play a role in resistance to alkylating agents as well as natural product drugs. We measured levels of GSH, glutathione S-transferase (GST), glutathione reductase (GSH Red), glutathione peroxidase (GSH Px), and gamma-glutamyl transpeptidase (gamma-GT) in a panel of 20 SCLC cell lines. Most of these lines were established from patients treated at this centre. Each cell line had a characteristic and reproducible profile of GSH and related enzyme levels. Immunoblot analysis indicated that the predominant GST in the cell lines was the anionic pi isoenzyme. The relative sensitivity of each of these cell lines to 16 different chemotherapeutic agents was measured using a modified MTT assay. Spearman rank correlation analysis was used to determine the relationships between the relative chemosensitivity of these cell lines and the levels of GSH and related enzymes. The number of positive correlations was no greater than expected by chance alone. Furthermore, there was no correlation with the treatment history of the patients from whom the cell lines were derived. These data suggest that alterations in glutathione metabolism do not play a major role in resistance to chemotherapeutic agents in these human SCLC cell lines.

Drug-resistant small cell lung cancer (SCLC) cells are more sensitive to lonidamine (LND), a new cytotoxic agent, than the parent cells

Drug-resistant small cell lung cancer (SCLC) cells are more sensitive to lonidamine (LND), a new cytotoxic agent, than the parent cells