In vitro and in vivo effects of cisplatin and etoposide in combination on small cell lung cancer cell lines

The effects of cisplatin (CDDP) and etoposide (ETP) in combination were evaluated in vitro and in vivo using small cell lung cancer cell lines. The combination effects in vitro were investigated using isobologram analysis. Used together, CDDP and ETP showed a synergistic effect against cell growth on only 1 cell line (SBC-3), additive effects on 6 (SBC-2, SBC-5, Lu130, Lu134AH, Lu135T and H69) and an antagonistic effect on 1 (SBC-1). In the in vivo experiment, nude mice were inoculated with SBC-1, SBC-3 and SBC-5 cells. Two or 5 mg/kg CDDP and 10 or 30 mg/kg ETP were administered intraperitoneally alone and simultaneously in combination to nude mice. The in vivo effects of the combination were determined by comparing the observed growth ratio in mice treated with the combination with the expected value of this ratio calculated based on the assumption that the effects of the drugs were simply additive. According to this definition, synergistic effects were observed against all 3 tumors. Thus, the in vivo and in vitro effects differed. The toxicity of the combination therapy, which was analyzed by estimating the body weight change of mice, was no higher than that of CDDP or ETP alone. These results suggest that the excellent clinical effects of CDDP and ETP combination therapy may be attributable not to drug interaction at the cellular level but to the feasibility of combined use of them at full doses without overlapping side effects.     

Nerve growth factor abrogates the tumorigenicity of human small cell lung cancer cell lines

Nerve growth factor (NGF) has antiproliferative and differentiating effects on adenomas of neuroendocrine origin. Cell lines derived from small-cell lung carcinoma (SCLC), a very aggressive neuroendocrine tumor, express NGF receptors. The role of NGF in the control of proliferation and progression of this carcinoma, however, has never been investigated. Chronic exposure of NCI-N-592 and GLC8 SCLC cell lines to NGF remarkably inhibited their proliferation rate both in vitro and in vivo, prevented their anchorage-independent clonal growth in soft agar, impaired their invasive capacity in vitro, and abolished their tumorigenic potential in nude mice. The proliferative response of SCLC cell lines to nicotine was also remarkably impaired by in vitro NGF treatment. Furthermore, NGF treatment activates in SCLC cell lines the expression and secretion of NGF. NGF thus reverts SCLC cell lines to a noninvasive, nontumorigenic phenotype that does not respond to nicotine and produces NGF.     

Emergence of resistance to VP-16/cisplatin chemotherapy: study in a lymphoblast model system

Etoposide (VP-16) and cisplatin are widely used in the treatment of malignancy. It is a common clinical observation that patients may initially respond to this two drug combination but later become resistant to it. Data from the CCL-159 lymphoblast cell line suggests that the emergence of resistance may be by means other than multiple drug resistance gene expression. The data suggest that chemotherapeutic failure may be mediated in part by a relatively inefficient method of drug resistance, the unstable expression of low-level drug resistance by a minority of cells. These results may help explain how patients whose malignancies are initially sensitive to VP-16/cisplatin later develop drug resistance.     

Acquired resistance to cisplatin and doxorubicin in a small cell lung cancer cell line is correlated to elevated expression of glutathione-linked detoxification enzymes

A human small cell lung cancer cell line, U-1906, developed altered functional properties upon continuous in vitro cultivation. Cells obtained at late (U-1906 L) and early (U-1906 E) passages of cultivation differ in drug resistance to the cytostatic therapeutic agents cisplatin and doxorubicin. The U-1906 L cells are 1.6-fold and 1.3-fold more resistant to cisplatin and doxorubicin respectively, than are the U-1906 E cells. In the more resistant U-1906 L cells, the total glutathione (GSH plus GSSG) level is 40% lower, whereas the activities of GSH-linked enzymes such as GSH peroxidase and GSH transferases are significantly higher. Quantitative analysis with isoenzyme-specific ELISAs demonstrated increased concentrations of all three of the measurable GSTs, M1-1, M3-3 and P1-1, in the more resistant cells. The intracellular protein expression patterns of the U-1906 E and the U-1906 L cells are very similar as revealed by two-dimensional denaturing electrophoresis, but show significant alterations in the concentrations of some components. Two 35 kDa proteins of different pI values, the concentrations of which are increased in the U-1906 L cells, were both identified as glyceraldehyde-3-phosphate dehydrogenase, either by N-terminal or by internal amino acid sequence analysis. The present study demonstrates that the increased resistance of the U-1906 L cells may involve multiple detoxification mechanisms and that the contribution of the GSH-linked detoxification can be ascribed to the elevation of cytosolic GST isoenzymes, GSH peroxidase and glutathione reductase, rather than to the intracellular GSH concentrations.     

Rapid polymerase chain reaction assay to detect variation in the extent of gene-specific damage between cisplatin- or VP-16-resistant and sensitive lung cancer cell lines

We previously established a rapid and facile polymerase chain reaction (PCR)-stop assay for quantitation of specific gene damage in very small numbers of cells. The present study investigated whether the PCR-stop assay was able to detect variation in the extent of DNA damage in transcribed active genes between cisplatin- or VP-16-resistant and sensitive cells. The assay demonstrated that about twice as much genetic damage occurs in PC-9 cells than in cisplatin-resistant PC-9/CDDP cells following cisplatin exposure and about 4.6 times more damage occurs in H69 than in VP-16-resistant H69/VP cells following VP-16 exposure. These results show that DNA damage, as detected by PCR-stop assay, correlates with cytotoxicity. In conclusion, the PCR-stop assay could be useful in detecting variation in DNA damage in specific genes.     

Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer

Female gender is a significant independent favorable prognostic factor in lung cancer. To study the possible role of sex hormones in lung cancer, the expression of sex-steroid receptors and the glucocorticoid receptor was investigated in 29 lung-cancer cell lines stemming from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) by means of immunocytochemistry, ligand-binding assays and RNA expression via polymerase chain reaction. In at least 2 methods of investigation, NSCLC cell lines showed a low expression of estrogen receptor in 6, progesterone receptor in 13 and androgen receptor in 12 out of 17 cases examined; sex-steroid-receptor expression was virtually absent in SCLC cell lines. The glucocorticoid receptor was expressed in all 29 cell lines studied. Additionally, 52 tumor samples from primary lung cancer were investigated for their receptor expression by means of immunohistochemistry. Among patients with primary lung-cancer sex-steroid-receptor expression in tumor biopsies was detected most frequently in female patients (in 69% of 16 cases, vs. 42% of 36 tumors from men) and in patients with adenocarcinoma. Further research will focus on these subgroups. Immunohistology is a feasible method of studying steroid-receptor expression in lung cancer.     

Neuropathies in small cell lung cancer

A routine neurological examination, electromyography studies and conductance in sensory and motoric fibres of upper and lower extremity peripheral nerves, was carried out in 65 subjects with small cell lung cancer prior instituting chemotherapy. None of the patients demonstrated metabolic changes nor toxic injury to the neurological system. The results of the neurological examination led to suspicion of neuropathy in 22 (34%) which was later confirmed by the electromyographic studies. In 12 subjects only EMG abnormalities were found allowing to diagnose a subclinical phase of neuropathy. Altogether 52% of the subjects demonstrated injury of the peripheral nervous system. Sensory neuropathy was observed in 6 patients, motor-sensory in 7, motoric neuropathy in 12. In one of the subjects from the latter group a myasthenic syndrome of the Eaton-Lambert type was found. In 7 patients the EMG results suggested injury of the anterior horn cells, in two further patients the clinical and EMG data suggested injury of the peripheral and spinal column.     

The small heat shock protein hsp27 is correlated with growth and drug resistance in human breast cancer cell lines

An emerging body of evidence suggests that the heat shock proteins (hsp) may be involved in drug resistance. When hsp are induced by elevated temperatures, resistance to doxorubicin (Dox), but not to other commonly used chemotherapeutic agents, is induced in breast cancer cells. To evaluate the role of hsp27 in this phenomenon, we have transfected MDA-MB-231 breast cancer cells, which normally express low levels of hsp27, with a full-length hsp27 construct. These hsp27-overexpressing cells now display a 3-fold elevated resistance to Dox. Anchorage-dependent proliferation and anchorage-independent growth were also increased 2-4-fold in these transfectants. We have also derived a MCF-7 breast cancer cell line with amplified endogenous hsp27 which is highly resistant to Dox. When these cells are transfected with an antisense hsp27 construct, they are rendered sensitive to Dox (3-fold) with anchorage-dependent as well as anchorage-independent growth, similarly decreased. These results suggest that hsp27 specifically confers Dox resistance in human breast cancer cells and, furthermore, that hsp27 may be involved in the regulation of cell growth.     

Carboplatin plus epirubicin plus VP-16, concurrent 'split course' radiotherapy and adjuvant surgery for limited small cell lung cancer. Gruppo Oncologico Centro-Sud-Isole (GOCSI)

Thirty-three patients with limited small cell lung cancer (SCLC) received carboplatin, epirubicin and VP-16 chemotherapy, concurrent 'split course' thoracic radiotherapy, followed by surgery for patients achieving an objective response (OR). High-risk patients and those staged T4-N3 (IIIB) at diagnosis, were excluded from surgery. After induction chemoradiotherapy we obtained 90.9% OR, with 63.3% obtaining complete response (CR). Ten patients (30.3%) were eligible for surgery after induction therapy. Five patients (15.1%) were subjected to surgery and five additional patients refused. Of the five patients who were subjected to surgery, four had a complete response (CR), (three pathological confirmations), and one had a partial response (PR), (unresectable). The median survival time for all patients was 16 months with 12.1% of the long-term survivors still living after 2 years and 9% still living after 3 and 4 years. Toxicity consisted mainly of myelosuppression. This study shows a high activity of the chemotherapy and the chemoradiotherapeutic regimen employed but a low feasibility for adjuvant surgery in SCLC.     

Oral combination antiemetics in patients with small cell lung cancer receiving cisplatin or cyclophosphamide plus doxorubicin

BACKGROUND: Intravenous antiemetic combinations containing a 5-HT3 receptor antagonist (like metoclopramide, ondansetron, or granisetron) with dexamethasone have become the standard therapy for the treatment of acute chemotherapy-induced vomiting. Intravenous antiemetics, however, can be more costly and take more time to prepare and deliver, and therefore are not preferred for home, outpatient, or office use. The objective of this study was to determine the antiemetic activity and safety of the oral combination antiemetic regimen of metoclopramide, dexamethasone, and diphenhydramine in patients with small cell lung cancer receiving standard outpatient chemotherapy programs. METHODS: Fifty-two patients receiving initial cisplatin (60 mg/m2) or cyclophosphamide (600-1500 mg/m2) plus doxorubicin (30-45 mg/m2) received an oral regimen of metoclopramide (3 mg/kg x 2 then 2 mg/kg x 2 or 4 doses), dexamethasone (20 mg) and diphenhydramine (50 mg x 2 or 3 doses) (oral MDD), beginning 30 minutes before chemotherapy. RESULTS: Vomiting was prevented in 15 of 21 (76%) patients (95% confidence interval [CI], 53%-92%) receiving cisplatin and 21 of 31 (71%) individuals (95% CI, 52%-86%) given cyclophosphamide plus doxorubicin. Adverse effects were mild and transient and included sedation, loose stools, akathisia, and hiccoughs. CONCLUSIONS: The oral MDD antiemetic regimen prevented acute emesis in 73% of the patients entered and was well tolerated in this population of patients with small cell lung cancer.     

Intratumor pharmacokinetics, flow resistance, and metabolism during gemcitabine infusion in ex vivo perfused human small cell lung cancer

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.     

Co-amplification of a novel cyclophilin-like gene (PPIE) with L-myc in small cell lung cancer cell lines

Specific genetic alterations affecting proto-oncogenes of the myc gene family are frequently detected in human lung cancer. Among 11 SCLC cell lines with L-myc gene amplification, four were found to have alteration of the RLF gene by Southern blot and RT-PCR analyses. One cell line, NCI-H378, contained aberrantly-sized L-myc-hybridizing bands by Southern and Northern blot hybridization but had no alteration of RLF. Some L-myc-hybridizing cDNAs from NCI-H378 contained a novel sequence with close homology to the cyclophilins joined to antisense L-myc exon 2 sequence. Full length cDNAs isolated from human skeletal muscle containing only the novel sequence identify open reading frames of 301 and 296 amino acids and differ in the C-terminal region by 22 and 17 amino acids. This gene, tentatively named PPIE (peptidyl-prolyl cis-trans isomerase E), has 83% amino acid identity with the central conserved region of cyclophilin A, is evolutionarily conserved by Southern blot, and exhibits differential tissue expression with highest levels found in muscle and brain. Co-amplification of PPIE was observed in seven of eleven L-myc amplified cell lines. Analysis of radiation hybrids suggests that the gene order is RLF-PPIE-L-myc on chromosome 1p and pulse-field gel electrophoresis localizes all three genes to an 800 megabase Mlu I fragment. The prognostic and functional consequences of PPIE gene amplification in SCLC can now be determined.     

Expression of genes involved in nucleotide excision repair and sensitivity to cisplatin and melphalan in human cancer cell lines

DNA repair has been proposed to be an important determinant of cancer cell sensitivity to alkylating agents and cisplatin (DDP). Nucleotide excision repair (NER), which represents one of the most important cellular DNA repair processes able to remove a broad spectrum of DNA lesions, is involved in the recognition and repair of the crosslinks caused by DDP and melphalan (L-PAM). In this study, the mRNA levels of the different genes involved in NER (ERCC1, XPA, XPB, XPC, XPD, XPF) were examined in a panel of eight different human cancer cell lines, together with the overall DNA repair capacity using a host cell reactivation assay of a damaged plasmid. A statistically significant correlation was observed between the relative expression of XPA/XPC (P < 0.05) and ERCC1/XPC (P < 0.05) mRNAs. No correlation was found between the DDP and L-PAM IC50S and the relative mRNA expression of the tested NER genes. When the overall cellular DNA repair capacity was studied, carcinomas seemed to have a higher repair activity than leukaemias; but this repair DNA activity correlated neither with the mRNA expression of the different NER genes nor with DDP and L-PAM IC50S. These data seem to suggest that even if the NER pathway is an important determinant for the cytotoxicity of alkylating agents, as demonstrated by the extremely high sensitivity to alkylating agents in cells lacking this repair system, other factors have to play a role in regulating the cellular sensitivity/resistance to these antitumour drugs.     

Cytokines modulate expression of cell-membrane complement inhibitory proteins in human lung cancer cell lines

Human lung cancers overexpress several cell-membrane complement inhibitory proteins (CIP). These complement inhibitory proteins are membrane cofactor protein (CD46), decay-accelerating factor (DAF; CD55), and CD59 (protectin). These cell-membrane proteins have a wide normal tissue distribution, are known to protect normal host cells from homologous complement-mediated lysis, and are thought to facilitate tumor escape from immunosurveillance. To study whether proinflammatory cytokines that are involved in cancer growth can modulate cell-membrane CIP expression in lung cancer cells, we studied the effect of interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma on two human lung cancer cell lines. ChaGo K-1 and NCI-H596 cell lines, undifferentiated carcinoma and lung adenosquamous carcinoma, respectively, were stimulated with different cytokines, and the effects of incubation time and cytokine concentration on cell-membrane CIP expression were studied. Cell-membrane CIP expression was evaluated using flow cytometry and cytokine effect was calculated as percent change in mean fluorescence intensity of each CIP molecule from its untreated control. We found that DAF was the lung cancer cell-membrane CIP molecule that was the most responsive to cytokine stimulation. Maximal stimulatory effect was usually noted 72 h after a cytokine was introduced. In ChaGo K-1 and NCI-H596 lung cancer cell lines, IL-1alpha and TNF-alpha increased DAF expression. IL-1alpha (100 U/ml/72 h) increased DAF expression up to a maximal mean of 45 and 48%, respectively, in comparison with untreated cells. TNF-alpha (1, 000 U/ml/72 h) increased DAF expression up to a mean of 131 and 46%, respectively. IFN-gamma (1 U/ml/72 h) increased DAF expression in NCI-H596 cells up to a mean of 100%, but had a slight inhibitory effect on DAF expression in ChaGo K-1 cells, decreasing expression by a mean of 17% in comparison with untreated cells. We conclude that cell-membrane DAF expression in the studied human lung cancer cell lines is modulated by IL-1alpha, TNF-alpha, and IFN-gamma, and speculate that cytokine-mediated modulation of cell-membrane DAF in human lung cancer cells might affect lung cancer cell biology.     

Proliferation of human non-small-cell lung cancer cell lines: role of interleukin-6

PURPOSE: The aim of this study was to describe the dysmorphogenetic process leading to esophageal atresia and tracheoesophageal fistula (EA + TEF) in the recently developed Adriamycin model of the malformation. METHODS:Time-mated pregnant rats were given either Adriamycin (1.75 mg/kg intraperitoneally) or saline on days 6 to 9 of gestation, and their embryos recovered on days 12, 12.5, and 13 were serially sectioned in the transversal plane and studied microscopically after H&E and PAS staining. The findings were compared with those of age-matched untreated embryos. RESULTS: All untreated and saline embryos were normal, whereas 49% of Adriamycin embryos had foregut malformations. Tracheoesophageal separation was complete on day 12 in control embryos, whereas 9 of 10 Adriamycin-exposed embryos had a common esophagotrachea with low emergence of the bronchi at that stage. This pattern had evolved into that of a regular EA + TEF in all nine malformed embryos by day 13. On day 12.5, esophagotrachea was found in 6 of 13 and EA + TEF in 5 of 13 embryos. Two had less well-defined malformations. CONCLUSIONS: Esophagotrachea equivalent to complete tracheoesophageal cleft is the first step leading to EA + TEF in this model. The full-blown malformation is finally acquired by partial loss of the posterior wall of the foregut, which tapers-off in the mediastinal mesenchyme and respiratory differentiation of the anterior wall down to the level of bronchial bifurcation, where it constitutes the fistula and the distal esophagus.     

Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines

Expression of Bcl-2 is life-sustaining for small-cell lung cancer cells and associated with drug resistance. In the present study, the interactions between the bcl-2 antisense oligodeoxynucleotide 2009 and the chemotherapeutic agents etoposide, doxorubicin and cisplatin were investigated on small-cell lung cancer cell lines to search for synergistic combinations. The cell lines NCI-H69, SW2 and NCI-H82 express high, intermediate-high and low basal levels of Bcl-2, respectively, which are inversely correlated with the sensitivities of the cell lines to treatment with oligodeoxynucleotide 2009 and the chemotherapeutic agents alone. Moreover, differences were found in the responsiveness of the cell lines to treatment with combinations of oligodeoxynucleotide 2009 and the chemotherapeutic agents. In the cell lines NCI-H69 and SW2, all combinations resulted in synergistic cytotoxicity. In NCI-H69 cells, maximum synergy with a combination index of 0.2 was achieved with the combination of oligodeoxynucleotide 2009 and etoposide. In SW2 cells, the combination of oligodeoxynucleotide 2009 and doxorubicin was the most effective (combination index = 0.5). In the cell line NCI-H82, which expresses a low basal level of Bcl-2, most of the combinations were slightly antagonistic. Our data suggest the use of oligodeoxynucleotide 2009 in combination with chemotherapy for the treatment of small-cell lung cancer that overexpresses Bcl-2.     

Basic science of small cell lung cancer

The Gilles de la Tourette syndrome is a neuropsychiatric condition characterized by waxing and waning tics, both vocal and motor, over a lifelong course with an onset under the age of 21 years. Once considered a rare curiosity, its prevalence is now agreed to be around 5/10,000, and is seen worldwide. Many cases are mild and never require medical attention. It is now thought to have a genetic basis, but linkage has not yet been achieved, owing possibly to the inadequacy of a single-gene hypothesis. There is a strong association with obsessive compulsive disorder, and some postulate that many other psychiatric conditions may be determined as variations of the same phenotype. Attention deficit hyperactivity disorder is also common among sufferers. Management is with a combination of psychosocial measures and drugs; in particular dopamine antagonists, the efficacy of which has led to the theory that the syndrome is caused by an abnormality of the dopaminergic system. Studies using functional imaging techniques have both supported and rejected this hypothesis. Future research is expected to concentrate on the further application of genetic and brain imaging techniques in addition to clinical trials to identify optimal treatments. Studies relating to clinical, genetic, etiological, and historical aspects are reviewed.     

Induction of broad drug resistance in small cell lung cancer cells and its reversal by paclitaxel

In the pulsed arterial spin labeling (ASL) techniques EPISTAR, PICORE, and FAIR, subtraction of two images in which inflowing blood is first tagged and then not tagged yields a qualitative map of perfusion. An important reason this map is not quantitative is that there is a spatially varying delay in the transit of blood from the tagging region to the imaging slice that cannot be measured from a single subtraction. We introduce here two modifications of pulsed ASL (QUIPSS and QUIPSS II) that avoid this problem by applying additional saturation pulses to control the time duration of the tagged bolus, rendering the technique relatively insensitive to transit delays and improving the quantitation of perfusion.     

Decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan

An in vitro model of acquired melphalan resistance was developed by serial incubation of an MCF-7 human breast cancer cell line in increasing concentrations of melphalan. The resulting derivative cell line, Me1R MCF-7, was 30-fold resistant to melphalan. Uptake studies demonstrated decreased initial melphalan accumulation in Me1R MCF-7 cells. Inverse-reciprocal plots of initial melphalan uptake revealed a 4-fold decrease in the apparent Vmax of Me1R MCF-7 compared with WT MCF-7 (516 amol cell-1 min-1 vs 2110 amol cell-1 min-1 respectively) as well as a decrease in the apparent Kt (36 microM vs 70 microM respectively). Two amino acid transporters have previously been identified as melphalan transporters: system L, which is sodium-independent and inhibited by 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH), and system ASC which is sodium dependent and unaffected by BCH. At low concentrations of melphalan (3-30 microM), 1mM BCH competition eliminated the differences between the two cell lines, thus implicating an alteration of the system L transporter in the transport defect in the resistant cells. Me1R MCF-7 cells were also evaluated for glutathione-mediated detoxification mechanisms associated with melphalan resistance. There was no difference between Me1R MCF-7 and WT MCF-7 in glutathione content, glutathione-S-transferase activity and expression of pi class glutathione S-transferase RNA. In addition, buthionine sulfoximine did not reverse melphalan resistance in Me1R MCF-7 cells. Therefore, Me1R MCF-7 cells provide an in vitro model of transport-mediated melphalan resistance in human breast cancer cells.