Arsenic Trioxide Is Highly Cytotoxic to Small Cell Lung Carcinoma Cells

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Arsenic trioxide is highly cytotoxic to small cell lung carcinoma cells

1 1 Helen M. Pettersson, Alexander Pietras, effect of As2O3 on SCLC growth, as suggested by an Matilda Munksgaard Persson,1 Jenny Karlsson,1 increase in neuroendocrine markers in cultured cells. [Mol Leif Johansson,2 Maria C. Shoshan,3 Cancer Ther 2009;8(1):160–70] and Sven Pa˚hlman1

1Center for Molecular Pathology, CREATE Health and 2Division of Introduction Pathology, Department of Laboratory Medicine, Lund University, 3 Lung cancer is the most frequent cause of cancer deaths University Hospital MAS, Malmo¨, Sweden; and Department of f Oncology-Pathology, Cancer Center Karolinska, Karolinska worldwide and results in 1 million deaths each year (1). Institute and Hospital, Stockholm, Sweden Despite novel treatment strategies, the 5-year survival rate of lung cancer patients is only f15%. Small cell lung carcinoma (SCLC) accounts for 15% to 20% of all lung Abstract cancers diagnosed and is a very aggressive malignancy Small cell lung carcinoma (SCLC) is an extremely with early metastatic spread (2). Despite an initially high aggressive form of cancer and current treatment protocols rate of response to chemotherapy, which currently com- are insufficient. SCLC have neuroendocrine characteristics bines a platinum-based drug with another cytotoxic drug and show phenotypical similarities to the childhood tumor (3, 4), relapses occur in the absolute majority of SCLC neuroblastoma. As multidrug-resistant neuroblastoma patients. At relapse, the efficacy of further chemotherapy is cells are highly sensitive to arsenic trioxide (As2O3) poor and the need for alternative treatments is obvious. in vitro and in vivo, we here studied the cytotoxic effects Arsenic-containing compounds have been used in tradi- of As2O3 on SCLC cells. As2O3 induced pronounced cell tional Chinese medicine for thousands of years. In the last death in SCLC cells at clinically relevant concentrations, decade, clinical studies have shown that low doses of and also at hypoxia. SCLC cells were more sensitive than arsenic trioxide (As2O3) can induce complete remission non–SCLC cells to As2O3. Cell death was mainly due with minimal toxicity in patients with relapsed acute to necrosis, although apoptotic responses were also seen. promyelocytic leukemia (APL; refs. 5–8), and As2O3 is A significant in vivo effect of As2O3 on SCLC growth now used as first-line treatment modality in these patients was shown in a nude mice-xenograft model, although a with APL. At clinically tolerable concentrations, As2O3 also fraction of the treated tumor-bearing animals did not induces cell death in multidrug-resistant neuroblastoma respond. The nonresponding SCLC tumors differed in cells in vitro and in vivo (9, 10). The cytotoxic effect of As2O3 morphology and cell organization compared with treat- is retained in neuroblastoma cells at hypoxia (11), a ment-responsive tumors, which in turn, showed decreased property of potential clinical importance because hypoxic vascularization and higher expression of neuroendocrine areas are frequent in solid tumors and have been linked to markers compared with control tumors. Our results drug-resistant phenotypes (12). suggest a potential clinical application of As2O3 in SCLC The mechanisms of As2O3-induced cytotoxicity are therapy. In addition to cell death induction, antiangiogenic complex and differ depending on the cell type and tumor induction of differentiation may also be part of the in vivo form. In APL cells, the induction of apoptotic mechanisms, growth inhibition, as well as differentiation have been observed (13, 14). In other cells, caspase-independent pathways are also important (10, 15) and can involve the Received 6/24/08; revised 9/29/08; accepted 10/16/08. activation of the proapoptotic Bcl-2 family member Bax Grant support: Swedish Cancer Society, the Children’s Cancer Foundation (16, 17), as observed in As2O3-induced death of neuroblas- of Sweden, Swedish Foundation of Strategic Research, the SSF Strategic toma cells (10). In drug-sensitive as well as multidrug- Center for Translational Cancer Research-CREATE Health, Hans von resistant neuroblastoma cells, p21 Bax is proteolytically Kantzow’s Foundation, the Royal Physiographic Society in Lund, Franke and Margareta Bergqvist’s Cancer Foundation, Crafoordska Foundation, cleaved into the more proapoptotic p18 Bax form in a Gunnar Nilsson’s Cancer Foundation, Magn. Bergvall’s Foundation, calpain-dependent process. We have further shown that and the research funds of Malmo¨University Hospital. p18 Bax generated upon As2O3 treatment is exclusively The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked localized to the nucleus (18), thus indicating a novel, advertisement in accordance with 18 U.S.C. Section 1734 solely to nuclear rather than mitochondrial death-signaling pathway indicate this fact. induced by a Bcl-2 family member. Requests for reprints: Helen Pettersson, Center for Molecular Pathology, Neuroblastoma and SCLC have many characteristics in Department of Laboratory Medicine, Lund University, University Hospital MAS, Entrance 78, SE-205 02 Malmo¨, Sweden. Phone: 46-40-332285; common, e.g., both tumors have neuronal/neuroendocrine Fax: 46-40-337063. E-mail: [email protected] traits and usually respond well to initial treatment with Copyright C 2009 American Association for Cancer Research. similar drugs. Furthermore, amplification of MYC, MYCN, doi:10.1158/1535-7163.MCT-08-0595 and MYCL is observed in 20% to 30% of SCLC (19) and

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f amplification of MYCN occurs in 20% of all patients with To evaluate the efficacy of As2O3 on hypoxic SCLC neuroblastomas (20). In both cases, amplification strongly cells, U-1690 cells were seeded on day 0 under normoxic correlates with poor clinical outcome. This, together with conditions, As2O3 was added on day 1 and cells were the observation that As2O3 effectively kills multidrug- further cultured at normoxia or hypoxia for 72 h. When resistant neuroblastoma cells, prompted us to investigate indicated, cells were also preincubated at normoxia or the in vitro and in vivo effects of As2O3 on SCLC cells. We hypoxia for 72 h before the addition of As2O3. In these show here that SCLC cells are highly sensitive to As2O3 experiments, the cells were re-seeded 1 day before the in vitro, both at normoxia and hypoxia. As2O3-induced initiation of drug treatment. Cell viability was assessed SCLC cell death is mainly necrotic but to some extent also with trypan blue (Sigma-Aldrich). Approximately 200 apoptotic. Finally, As2O3 treatment results in a marked cells per culture were counted using a light microscope. reduction in xenograft tumor growth, suggesting that All treatments were done in triplicate and the experi- As2O3-induced cytotoxicity is a potential treatment strategy ments were repeated thrice. Untreated controls were set for patients with relapsed SCLC. to 100%. Immunoblot Analyses and Flow Cytometry Whole cell lysates were analyzed by Western blotting as Materials and Methods described previously (10). The following primary anti- Cell Culture, Drugs, and CellViabilityAssays bodies and antiserum were used: Bax antiserum (BD All cell culture media were supplemented with 10% FCS PharMingen) diluted 1:2,000, anti–caspase 3 antibody (EuroClone, Ltd.), 100 units/mL of penicillin, and 100 (Alexis Biochemicals) diluted 1:500, anti–glyceraldehyde- Ag/mL of streptomycin (Life Technologies). The SCLC cell 3-phosphate dehydrogenase antibody (Chemicon Interna- lines U-1285, grown in suspension, and U-1690, U-1906, tional, Inc.) diluted 1:2,000, anti–hypoxia-inducible and U-2020, all of which are semiadherent, were grown in factor-1a (HIF-1a) antibody (Abcam PLC) diluted 1:700, RPMI 1640 (Sigma-Aldrich), as were the APL cell line NB4, and anti–poly(ADP-ribose) polymerase (PARP) antibody grown in suspension, and the semiadherent cell line (Biomol Research Laboratories, Inc.) diluted 1:5,000. U-1568, which originates from a large-cell morphologic The fractions of apoptotic and necrotic SCLC cells were variant of SCLC (SCLC-mv). The other lung cancer cell analyzed by flow cytometry using the TACSAnnexin V- lines H-125 (adenocarcinoma), U-1752 (squamous cell FITC apoptosis detection kit (R&D Systems) according to carcinoma), and U-1810 (large cell carcinoma) were grown the manufacturer’s recommendation. Cells were seeded as monolayers in F10 medium (Life Technologies). 1 day before As2O3 was added, and were collected after MCF7 and T-47D breast carcinoma cells were cultured as 24 to 72 h of As2O3 treatment, stained with Annexin V-FITC monolayers in RPMI 1640 supplemented with 0.01 mg/mL and propidium iodide (PI) and thereafter analyzed in of insulin (Actrapid Penfill; Novo Nordisk), 1.0 mmol/L a FACSCalibur flow cytometer (Becton Dickinson Immu- of sodium pyruvate (PAA Laboratories), and 1.5 g/L of nocytometry Systems). SCLC cells treated with up to sodium bicarbonate (ICN Biomedicals). The rat cell lines 5 Amol/L of staurosporine for 24 h were used as positive PC12 and HiB5 of neuroendocrine and neural derivation, controls for the induction of apoptosis. respectively, were grown as monolayers, PC12 cells in Xenograft TumorTreatment RPMI 1640 supplemented with 10% horse serum and 5% Six- to 8-week-old female athymic mice (NMRI strain FCS, and HiB5 cells in DMEM containing 4.5 g/L of glucose nu/nu; Taconic), weighing 20 to 25 g at arrival, were housed (Life Technologies). All cells were cultured at 37jC in a 95% in a controlled environment. After 1 week of acclimatiza- air, 5% CO2 humidified incubator, with the exception of the tion, the mice were anesthetized with halothane and j Â 6 HiB5 cells, which were grown at 33 C. To achieve 1% O2, injected subcutaneously on the upper back with 10 10 cells were grown in a humidified oxygen-regulated U-1690 cells suspended in PBS. When U-1690 tumors with a chamber (Invivo2 Hypoxia Workstation 400; Ruskinn diameter of 5 mm were established, the animals were Technology). randomized into two groups and thereafter given daily i.v. A As2O3 (Sigma-Aldrich) was dissolved in 1.0 mol/L of injections of either PBSor 150 gofAs2O3 (5 mg/kg) in the NaOH, carboplatin (Sigma-Aldrich) in distilled H2O, and tail vein. Tumor growth and animal weight were moni- etoposide (Sigma-Aldrich) as well as staurosporine (Sigma- tored on a daily basis. When the tumors reached a size of Aldrich) in DMSO. 20 mm in diameter, the mice were sacrificed with CO2 gas The effects of cytotoxic drugs on cell viability were and the tumors were dissected and weighed. All proce- determined by the WST-1 assay (Roche Molecular Bio- dures were carried out in accordance with the regional chemicals), according to the manufacturer’s recommenda- ethical committee for animal research (approval no. tion. The cells were seeded in 96-well plates at day 0 and M66-05). varying concentrations of the drugs were added on day 1. Immunohistochemistry and Quantitative Real- Where indicated, the pan-caspase inhibitor zVAD-fmk time PCR (Enzyme System Products) was added at various concen- Sections (4 Am) of formalin-fixed, paraffin-embedded trations on day 1. All treatments were done in triplicate and U-1690 xenograft tumors described above were analyzed the experiments were repeated thrice. Untreated control by immunohistochemistry using the following antibodies: viability was set to 100%. anti-PCNA (Dako) diluted 1:4,000, anti-CD34 (Santa Cruz

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Biotechnology) diluted 1:100, anti-chromogranin A (Dako) As2O3 Induces Proteolytic Activation of Caspase 3, diluted 1:3,000, anti-synaptophysin (Dako) diluted 1:100, PARP, and Bax in SCLC Cells a and anti–HIF-1 (Abcam PLC) diluted 1:400. After antigen Depending on the cell system investigated, As2O3- retrieval in Target retrieval solution high pH (Dako), induced cell death has been associated with caspase- immunoreactivities were detected using the Envision dependent apoptosis as well as caspase-independent death system and DAKO Techmate 500 (Dako; ref. 21). pathways (10, 14, 15). To characterize the mechanisms by For quantitative real-time PCR experiments, RNA extrac- which As2O3 kills SCLC cells, we studied the cleavage of tion, cDNA synthesis, and PCR reactions with SYBR Green caspase 3 and PARP, two classical apoptosis markers. PCR master mix (Applied Biosystems) were done as Caspase 3 was proteolytically activated in all tested SCLC previously described (22). The comparative Ct method cell lines in an As2O3 dose-dependent manner (Fig. 1C); was used for relative quantification of expression levels, however, the cleavage seemed modest. PARP is a target and data were normalized to the expression of three protein for caspases during apoptosis and As2O3 treatment housekeeping genes, HPRT1, TBP, and UBC, which were induced cleavage of PARP to an 89 kDa form (Fig. 1C). stable during hypoxia, using geNorm (23). Each reaction PARP can also be processed during necrosis, resulting in was done in triplicate and the experiments were repeated multiple cleavage products, one of which is of a similar four times. Primers were designed using Primer Express molecular size as the apoptosis-induced fragment, i.e., 89 (Applied Biosystems), and sequences are listed in Supple- kDa. In previous reports, the antibody used in this study mentary Table S1.4 (C-2-10) detected main fragments of markedly lower molecular weights (50 and 62 kDa) under necrotic Results conditions (24). Here, only weak bands corresponding to such fragments were detected in As2O3-treated U-1690 cells As2O3 in Low Doses Induces Cell Death in SCLC Cells We examined SCLC cell lines with regard to their (data not shown). In neuroblastoma cells, As2O3 induces a caspase-inde- responsiveness to As2O3 after 72 hours of treatment. Their sensitivities were comparable to that of the APL cell line pendent cell death involving proteolytic activation of the proapoptotic Bcl-2 family member Bax (10, 18). In the SCLC NB4 (Fig. 1A) as well as neuroblastoma cells (10). The IC50 values were in the 1 to 2 Amol/L range for four SCLC cell cell lines tested, As2O3 similarly induced dose-dependent lines and the NB4 cells, i.e., concentrations well below cleavage of full-length p21 Bax to a truncated p18 form clinically tolerable doses. By comparison, three non–small (Fig. 1C). However, compared with As2O3-treated neuro- cell lung carcinoma (non-SCLC) cell lines, and two breast blastoma cells, the induction was weak (Fig. 1C). A slight increase in total Bax levels was also noted in two of the cell cancer cell lines were less sensitive to As2O3, with IC50 values ranging from 2 to 5 Amol/L (Fig. 1A). The differ- lines (Fig. 1C). Calpains are a group of proteases known to be involved ences in sensitivity to As2O3 between the SCLC cells and the other cells could not be explained by differences in in the degradation of proteins in dying cells (25) and also in growth rates (data not shown). As SCLC and neuroblasto- proteolytic activation of the proapoptotic proteins Bax and ma cells have neuroendocrine and neuronal characteristics, Bid (10, 18, 26, 27). To investigate the involvement of respectively, we also tested two rat cell lines, PC-12 and caspases and calpains in As2O3-induced SCLC cell death, HiB5, of neuroendocrine and neural derivation. These cells cells were treated with zVAD-fmk, a pan-caspase inhibitor which also inhibits calpains at high concentrations (28, 29). were not as sensitive to As2O3 as human SCLC or The viability of As2O3-treated U-2020 cells, i.e., the SCLC neuroblastoma cells, suggesting that As2O3 sensitivity is not associated with a neuroendocrine or neural phenotype cells with the most pronounced As2O3-induced caspase 3 activation and Bax cleavage (Fig. 1C), was rescued to a per se (Fig. 1A; ref. 10). A We further examined the SCLC cells with regard to their limited extent by high concentrations (100 mol/L) of zVAD-fmk (Fig. 1D). The effect of zVAD-fmk was only sensitivity to etoposide and carboplatin, two drugs used in A observed upon treatment with 2 mol/L of As2O3,a first-line treatment of SCLC (3, 4). In contrast to their As2O3 sensitivity, the SCLC cells differed in their responsiveness suboptimal drug concentration upon which the involve- to etoposide and carboplatin (Fig. 1B). Of the tested cell ment of caspases and calpains in As2O3-induced SCLC cell lines, U-1690 was the most resistant to these two drugs, death may be the most pronounced. By contrast, the same particularly to etoposide, whereas both drugs markedly concentration of zVAD-fmk clearly prevented a classical reduced the viability of U-2020 and U-1285 cells after apoptotic cell death induced by staurosporin (Fig. 1D). As O Induces a Mixed Necrotic and Apoptotic Cell treatment for 72 hours at clinically relevant concentrations. 2 3 Death in SCLC Cells Compared with drug-sensitive neuroblastoma cells, all four SCLC cell lines were considerably more resistant to We further studied the relative contributions of apoptosis etoposide and carboplatin (data not shown; ref. 10). and necrosis to As2O3-induced SCLC cell death by flow cytometry using Annexin V-FITC and PI. In U-1690, U-1906, and U-2020 cells, this assay revealed different well-defined cell populations (Fig. 2A; data not shown). The 4 dot plots in Fig. 2 show viable cells (bottom left quadrants, Supplementary material for this article is available at Molecular Cancer À À Therapeutics Online (http://mct.aacrjournals.org/). Annexin V /PI ), necrotic cells (top left quadrants, Annexin

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Figure 1. A and B, SCLC cells are as sensitive to As2O3 as the APL cells NB4, which is in contrast with non-SCLC, breast carcinoma, and neurally derived cells. Cells were treated with cytotoxic drugs for 72 h and the relative amounts of viable cells were evaluated using the WST-1 assay. Points, mean from three experiments presented as a percentage of viable cells as compared with untreated controls; bars, SD. A, the indicated cell lines treated with various concentrations of As2O3. B, dose-response curves of SCLC cells upon treatment with either etoposide or carboplatin. C and D, analysis of As2O3- induced cell death pathways in SCLC cells. C, immunoblot analyses of PARP, caspase 3, Bax, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)in SCLC cells treated with the indicated concentrations of As2O3 for 72 h. SK-N-BE2(c) neuroblastoma cells (NB) treated in the same way were included for comparison. D, U-2020 cells exposed to the indicated concentrations of ZVAD-fmk together with increasing concentrations of As2O3 or staurosporine for 72 and 24 h, respectively. After treatment, the fraction of viable cells was determined using the WST-1 assay. Columns, mean from three experiments presented as a percentage of viable cells as compared with untreated controls; bars, SD. **, P < 0.01; ***, P < 0.001, statistically significant differences determined by ANOVA followed by Duncan’s multiple range test.

À V /PI+), and early apoptotic cells (bottom right quadrants, because in either case, dead cells will be Annexin V+/PI+. À Annexin V+/PI ). In all our experiments, the top right Here, we have defined cells within the top left quadrant quadrant comprised two well-defined cell populations plus within gate G1 as lytic-necrotic cells, and cells within which were both Annexin V+/PI+:PIhigh (gate G1) and the bottom right quadrant plus within gate G2 as apoptotic PIlow cells (gate G2). However, this assay does not and late apoptotic. These assumptions are based on the distinguish, per se, between cells that are in late apoptosis following facts: in our experiments, the necrotic cells in the and those that have died/lysed as a result of necrosis top left quadrant together with the PIhigh cells in gate G1

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comprised a continuous horizontal cell population ranging ences in levels of necrosis and apoptosis were much less over the upper two quadrants (Fig. 2A). Their high uptake pronounced, and the frequency of necrotic cells never of PI, as compared with gate G2 cells, reflects a higher exceeded 35% in either cell line after 72 hours of treatment. membrane permeability, i.e., a more lytic condition. In Altogether, these results indicate that As2O3 induces both addition, it is known that when apoptosis is measured over necrosis and apoptosis in SCLC cells, and necrosis seems to À À time, cells can often proceed from Annexin V /PI (viable, be the main cell death mode. + À or no measurable apoptosis) to Annexin V /PI (early SCLC Cells Are Sensitive to As2O3 at Hypoxia apoptosis), and finally to Annexin V+/PI+ (end stage The oxygen tension in solid tumors is generally low apoptosis and death). The movement of cells through these compared with normal adjacent tissues (f1% versus 5–6% three stages, or quadrants, reflects cell death dynamics. O2) and tumor cells have adapted to growth under hypoxic In our experiments, the apoptotic cell population shifted conditions. The phenotypical changes induced by hypoxia from the bottom right quadrant toward gate G2 when cell are frequently associated with induced drug resistance death in As2O3-treated U-1690 cells was assessed over time (reviewed in ref. 12), and in vitro testing of drugs under (Fig. 2A and B). Exposure to As2O3 gave rise to a normoxic (21% O2) culture conditions might, for this simple concentration-dependent and time-dependent increase in reason, generate data of reduced clinical relevance. The necrotic as well as apoptotic cells. After 24 hours of most drug-resistant as well as the most drug-sensitive of treatment, the majority of the U-1690 cells was viable, and the SCLC cell lines tested, i.e., U-1690 and U-2020 cells the fraction of necrotic as well as apoptotic cells was <20% (Fig. 1B), were therefore grown and treated at 21% and 1% irrespective of the As2O3 concentration used. Cell death was O2, respectively. As shown in Fig. 3A and B, SCLC cells not substantial unless the cells were treated with 3 Amol/L grown under either normoxia or hypoxia were equally of As2O3 for 48 h, resulting in necrosis and apoptosis levels sensitive to As2O3. Furthermore, U-1690 cells pre-exposed of 35 and 30%, respectively. Cells undergoing apoptosis to hypoxic conditions for 72 hours before treatment at accounted for no more than 30% of the total U-1690 cell hypoxia or normoxia retained their sensitivity to As2O3 population at all time points and treatment concentrations (Fig. 3C). tested (Fig. 2B). In contrast, the frequency of necrotic cells To ensure that U-1690 cells cultured under hypoxic A was as high as 70% in cells treated with 3 mol/L of As2O3 conditions had adapted to hypoxia, we investigated the for 72 hours. accumulation of the hypoxia-induced transcription factor As2O3 treatment gave rise to a concentration-dependent HIF-1a (Fig. 3D). As expected, after a 4-hour incubation increase of necrosis as well as apoptosis in U-1906 and under normoxia or hypoxia, no HIF-1a protein could be U-2020 cells (Fig. 2C). However, in these cells, the differ- detected in normoxic cells (Fig. 3D, lane 1), whereas a

Figure 2. Flow cytometric analyses of Annexin V-FITC binding and PI uptake in SCLC cells after As2O3 treatment. A, Annexin V-FITC vs. PI density plots of U-1690 cells treated with various concentrations of As2O3 for the indicated time periods. Defined cell populations: viable cells (bottom left quadrant), necrotic cells (top left quadrant + gate G1), and apoptotic cells (bottom right quadrant + gate G2). One of three representative experiments. B, frequencies of necrotic, and apoptotic U-1690 cells after the indicated time periods of treatment with As2O3. Columns, mean from three experiments, one of which is presented in A; bars, SD. C, frequencies of necrotic, and apoptotic U-1906 and U-2020 cells when treated with As2O3 for 72 h. Columns, mean from three experiments; bars, SD.

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Figure 3. Hypoxic SCLC cells are sensitive to As2O3. After As2O3 treatment at hypoxia or normoxia for 72 h, the numbers of viable (trypan blue excluding) and dead cells were counted. Points, mean percentage of dead cells or viable cells as compared with untreated controls from three experiments; bars, SD. U-1690 cells (A) and U-2020 cells (B) exposed to the indicated concentrations of As2O3 at 1% and 21% oxygen, respectively, for 72 h. C, U-1690 cells cultured at normoxia or hypoxia for 72 h before addition of the indicated concentrations of As2O3. After another 72 h of incubation under normoxic or hypoxic conditions in the presence of As2O3, the cells were collected and counted. D, immunoblot analyses of HIF-1a and actin in untreated or As2O3-treated SCLC cells grown at normoxia or hypoxia for 72 h. SK-N-BE2(c) neuroblastoma cells (NB) cultured under normoxic or hypoxic conditions for 4 h were included for comparison. marked accumulation was observed in hypoxic cells inhibition of tumor growth was observed. In contrast, the (Fig. 3D, lane 2). A robust HIF-1a protein stabilization remaining two animals developed tumor burdens compa- was also detected in cells treated with As2O3 under hypoxia rable to those seen in the PBScontrol animals. Despite the (Fig. 3D, lane 3). Altogether, this indicates that the cells lack of response to As2O3 in these two animals, the mean adapted to the hypoxic environment. tumor growth in the treated cohort was significantly V SCLC Cells Are Responsive to As2O3 In vivo different from that of PBScontrol animals ( P 0.05; To test the in vivo cytotoxic efficacy of As2O3 on SCLC Fig. 4B). An eighth As2O3-treated animal in this experiment cells, we used a xenograft model of subcutaneously grown had to be sacrificed early during the experiment due to a U-1690–derived tumors in nude mice. We chose the U-1690 skin lesion in the tumor area and was excluded from the cells because they gave a high tumor take (f80%; 16 of 20 study. The growth-inhibiting effect of As2O3 was not injected animals in the experiment shown in Fig. 4), and of related to a general intoxication of treated animals as they the tested SCLC cell lines, U-1690 showed the highest appeared healthy and did not lose weight during treatment resistance to etoposide and carboplatin (Fig. 1B). Animals (Fig. 4C). were treated daily with i.v. injections of either PBS(control) Distinct Morphology of As2O3 Nonresponding SCLC A or 150 gofAs2O3 (5 mg/kg), a concentration chosen to Tu m o r s mimic clinically relevant plasma concentrations and com- In an attempt to understand the varying degrees of parable to those used in in vivo models to treat APL and As2O3-induced growth inhibition of SCLC xenograft neuroblastoma (9, 30). In the experiment presented in tumors, we did immunohistochemical investigations of Fig. 4, tumors in the PBScontrol animals seemed to follow tumor morphology, fractions of proliferating cells, differ- one fast and one slower growth pattern (Fig. 4A). In the entiation status, and levels of vascularization. H&E-stained As2O3-treated cohort of animals, the picture was slightly tumor sections revealed striking morphologic intertumor more complex. First, in three out of seven animals, As2O3 differences as exemplified by tumors from animal C4, As1, almost completely prevented tumor growth, and in another and As4 (Fig. 5). The PBS-treated tumors had large areas of two out of seven As2O3-treated animals, significant initial necrosis and a large fraction of dividing cells (PCNA

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positivity; Fig. 5). As expected, the drug-responding of tumors from 15 nontreated animals were analyzed. Two tumors had extensive areas of necrotic cells and a smaller of these showed the variant morphologic pattern (Fig. 6A), fraction of dividing cells (Fig. 5). Interestingly, the cells of clearly demonstrating that the development of this partic- the As2O3 nonresponding SCLC tumor As1 showed a ular morphology did not require As2O3 treatment, and distinctly different morphology in that they were larger, the suggesting that the U-1690 cell line contains different nuclei varied in size, and they had disintegrated chromatin subpopulations of cells, which both can give rise to tumors and prominent nucleoli. Furthermore, these variant tumor in nude mice. Of interest, one large cell morphologic cells showed a more organized growth pattern with a high variant of SCLC (SCLC-mv) cell line showed markedly number of PCNA-positive cells (Fig. 5), suggesting selec- reduced sensitivity to As2O3 (Fig. 6B) as compared with the tion for a different, nonresponding cell type in this tumor. classic SCLC cell lines presented in Fig. 1A. Regarding tumor vascularization, the number of blood Expression of Neuroendocrine Differentiation vessel endothelial (CD34 positive) cells was low in As2O3- Markers in As2O3-Treated SCLC Tumors responding tumors and considerably higher in nonres- As As2O3 has been reported to induce differentiation in ponding and PBS-treated tumors (Fig. 5). APL cells (14), we investigated neuroendocrine differenti- To test whether the variant, nonresponding tumor type ation status in the SCLC tumors based on the expression of developed as a result of As2O3 selection, the morphologies chromogranin A and synaptophysin, two neuroendocrine

Â 6 Figure 4. SCLC in vivo growth is reduced by As2O3 treatment. Human U-1690 cells (10 10 ) were xenotransplanted in nude mice. When tumors with a diameter of 5 mm were established, treatment with daily i.v. injections of either PBS or 150 AgofAs2O3 (corresponding to 5 mg/kg) was initiated. A, tumor burden in all animals (top) after the indicated number of days of daily treatment. PBS-treated animals are labeled C1 – C8 (middle), As2O3-treated animals As1 – As7 (bottom). B, mean tumor burden in PBS-treated and As2O3-treated animals from A. Statistically significant differences were determined by Mann-Whitney’s one-tailed U test (P V 0.05). Bars, SE. C, total weight of PBS-treated and As2O3-treated animals from A.

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Figure 5. Immunohistochemical characterization of xenotransplanted U-1690 tumors from the experiment presented in Fig. 4. Sections of formalin-fixed paraffin-embedded tumors treated with PBS (C4)orAs2O3 (nonresponding As1 and As2O3-responding As4) were stained with H&E and analyzed immunohistochemically for PCNA, CD34, chromogranin A, synaptophysin, and HIF-1a, respectively. Note the unspecific staining for PCNA, chromogranin A, and HIF-1a in the necrotic parts. Magnifications are shown as insets in the respective panels. Bars, 100 Am(bars in insets, 50 Am).

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marker genes known to be expressed in SCLC cells. As Discussion exemplified in Fig. 5, these two differentiation markers We show here that As2O3, at clinically tolerable concen- were high in the As2O3-responding tumors, fairly high in trations, is highly cytotoxic to SCLC cells in vitro. the PBScontrols, but virtually absent in the nonresponding Interestingly, non–SCLC cells were considerably more As1 tumor. resistant to As2O3. The sensitivity of SCLC cells to As2O3 To further evaluate the potential influence of As2O3 on was comparable to that of APL cells as the toxic effect is neuroendocrine differentiation, we studied the expression achieved within the same concentration range for both of chromogranin A and synaptophysin in As2O3-treated cell types. In addition, As2O3 treatment significantly U-1690 cells. After 6 days of treatment, both genes were impaired SCLC tumor growth in a mouse xenograft model. slightly down-regulated compared with untreated controls Our findings that As2O3 retains its cytotoxicity at in vitro (Fig. 6C). As vascularization seemed to be lower in the hypoxia as well as under in vivo conditions provide proof As2O3 responding versus nonresponding tumors, expres- of principle regarding the potential use of As2O3 for sion of differentiation marker genes was investigated in treatment of patients with SCLC. cells grown at hypoxia in the presence or absence of As2O3. As2O3-induced cytotoxicity is complex and has been Hypoxic cells up-regulated both chromogranin A and shown to involve several different cell death pathways. In synaptophysin significantly, whereas As2O3 in combination APL cells, As2O3 treatment results in apoptosis, growth with hypoxia did not induce any further increase in marker inhibition and differentiation (13, 14). Moreover, caspase- gene expression levels (Fig. 6C). Immunohistochemical independent necrotic cell death has also been shown upon a analysis of HIF-1 protein in xenograft tumor sections As2O3 treatment (31). The precise mechanisms of As2O3- revealed that treatment-responsive tumors accumulated induced SCLC cell death are yet to be determined. We HIF-1a, whereas less HIF-1a immunoreactivity was seen in observe what seemed to be a modest induction of caspase 3, PBS-treated or nonresponding tumors (Fig. 5). which only marginally contributed to As2O3-induced death

Figure 6. A, morphologic characterization of subcutaneously grown U-1690 – derived tumors in nude mice. Human U-1690 cells (10 Â 106) were xenotransplanted and tumors were collected after 15 d of in vivo growth. Sections of the formalin-fixed, paraffin-embedded tumors were stained with H&E. B, cell viability was determined by the WST-1 assay in a large-cell morphologic variant of SCLC (SCLC-mv, U-1568) cell line and in U-1690 cells after 72 h of As2O3 treatment. Points, mean from three experiments presented as a percentage of viable cells as compared with untreated controls; bars, SD. C, quantitative real-time PCR analyses of expression levels of neuroendocrine differentiation genes in U-1690 cells after As2O3 treatment for 3 and 6 d at hypoxia and normoxia, respectively. Columns, mean from four experiments; bars, SD. *, P < 0.05; **, P < 0.01, statistically significant differences determined by ANOVA followed by Duncan’s multiple range test.

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because treatment with the pan-caspase inhibitor zVAD- We observed high expression levels of neuroendocrine fmk did not prevent cell death. In line with these results, differentiation markers in SCLC tumors that responded fluorescence-activated cell sorting analyses showed low well to As2O3.AsAs2O3 alone did not induce a similar up- levels of apoptosis compared with the high frequency of regulation of differentiation markers in vitro at normoxia, necrotic cells. the in vivo result is most likely not a direct effect of As2O3 Caspase-independent cell death has, in some cell sys- on the tumor cells. Instead, it could be indirect, e.g., via an tems, been associated with increased expression and/or antiangiogenic effect of As2O3 leading to tumor hypoxia. proteolytic activation of the proapoptotic Bcl-2 family This hypothesis is supported by our observations that member Bax (16, 17). Although the proteolytic cleavage of hypoxia induced the expression of chromogranin A and p21 Bax to a p18 form by calpains could be linked to the cell synaptophysin in cultured SCLC cells and that treatment- a deathinAs2O3-treated neuroblastoma cells (10, 18), responsive tumors had the highest HIF-1 protein levels proteolytic activation of Bax did not seem to be of major (Fig. 5). As the level of tumor cell differentiation can importance for As2O3-induced SCLC cell death. However, influence clinical outcome, an indirect, hypoxia-driven the combined effect of the observed increase in total Bax differentiating effect of As2O3 could be clinically relevant and the formation of low levels of the more proapoptotic and might contribute to the effects of As2O3 seen in vivo. form, p18 Bax, might contribute substantially to As2O3- As2O3 has shown substantial efficacy in the treatment of induced SCLC cell death. Altogether, our results indicate patients with relapsed and refractory APL (5–8), and in that As2O3-induced SCLC cell death is mainly due to recent years, also in patients with untreated, newly caspase-independent necrotic cell death, whereas the diagnosed APL (36, 37). In addition, As2O3 induces cell involvement of apoptosis is more cell line–dependent. death at clinically relevant doses in a variety of cancer cells We have previously shown that As2O3 induces neuro- in vitro (38, 39), and a number of phase I and II clinical trials blastoma cell death independently of p53 status (10), a have been initiated to evaluate the potential effects of feature of great interest because loss of p53 is commonly As2O3 in various cancer types (40). The observed As2O3- seen in SCLC. The fact that As2O3 can activate several death induced cytotoxicity under hypoxic conditions is clinically pathways is likely important for its clinical efficacy and important because low tumor oxygenation has been linked may explain why As2O3 is able to kill SCLC cells such as to aggressive behavior in several tumor forms and hypoxic U-1690, which is less sensitive to etoposide and carboplatin areas are frequent in solid tumors. In conclusion, the but is as sensitive to As2O3 as the other tested SCLC findings presented in this study suggest a potential clinical cells. Thus, similar to neuroblastoma cells (9, 10), SCLC application of As2O3 in SCLC therapy. cells retain sensitivity to As2O3 despite acquired resistance to other drugs. The significant As2O3-induced reduction of SCLC tumor growth observed in vivo is therefore Disclosure of Potential Conflicts of Interest promising. No potential conflicts of interest were disclosed. The varying degree of drug response in the different Acknowledgments tumors arising from the same cell line may relate to subpopulation(s) of cells, as suggested by the distinct We thank Siv Beckman, Elisabet Johansson, Elise Nilsson, Caroline Bergenfelz, and Sofie Johnsson for skilful technical assistance, and Gerry cellular morphology observed in As2O3 nonresponding Jo¨nsson for his contribution in the in vivo experiments. SCLC tumors. This is an important issue that needs to be further evaluated. As tumors containing cells with this References morphology were also observed with some frequency in 1. Chua YJ, Steer C, Yip D. Recent advances in management of small-cell untreated animals, we propose that this particular pheno- lung cancer. Cancer Treat Rev 2004;30:521 – 43. type does not develop as a result of As O -induced 2. Chen LC, Travis WD, Krug LM. Pulmonary neuroendocrine tumors: 2 3 what (little) do we know? J Natl Compr Canc Netw 2006;4:623 – 30. selection. Previous reports have described SCLC cell lines 3. Freyer G, Tranchand B, Ligneau B, et al. Population pharmacokinetics with different morphologies and treatment responses of doxorubicin, etoposide and ifosfamide in small cell lung cancer patients: (32, 33). Thus, it is possible that there exist subpopulations results of a multicentre study. Br J Clin Pharmacol 2000;50:315 – 24. of tumor cells in established SCLC cell lines that are 4. Inoue A, Saijo Y, Kikuchi T, et al. Pharmacokinetic analysis of resistant to As O . A highly relevant question is whether combination chemotherapy with carboplatin and etoposide in small-cell 2 3 lung cancer patients undergoing hemodialysis. Ann Oncol 2004;15:51 – 4. corresponding cell populations can also be found in tumor 5. Shen ZX, Chen GQ, Ni JH, et al. Use of arsenic trioxide (As2O3) in the specimens from patients with SCLC. Patients with mixed treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and histology small cell/large cell carcinoma exist and this pharmacokinetics in relapsed patients. Blood 1997;89:3354 – 60. group of patients shows lower response rates and dies 6. Soignet SL, Maslak P, Wang ZG, et al. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J earlier than patients with archetypical small cell cancer (34). Med 1998;339:1341 – 8. The fact that cells of As2O3 nonresponding SCLC tumors 7. Niu C, Yan H, Yu T, et al. Studies on treatment of acute promyelocytic were larger and that the SCLC-mv cell line (derived from a leukemia with arsenic trioxide: remission induction, follow-up, and large cell variant of SCLC; ref. 35) was less sensitive to molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood 1999;94:3315 – 24. As2O3 treatment may indicate that the highest efficacy of 8. Soignet SL, Frankel SR, Douer D, et al. United States multicenter study As2O3 is to be expected in patients with classic small cell of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol type SCLC. 2001;19:3852 – 60.

Mol Cancer Ther 2009;8(1). January 2009

9. Øra I, Bondesson L, Jonsson C, et al. Arsenic trioxide inhibits calpain/calpastatin network: an organelle transient. Bioessays 2006;28: neuroblastoma growth in vivo and promotes apoptotic cell death in vitro. 850 – 9. Biochem Biophys Res Commun 2000;277:179 – 85. 26. Wood DE, Newcomb EW. Cleavage of Bax enhances its cell death 10. Karlsson J, Øra I, Po¨rn-Ares I, Pa˚hlman S. Arsenic trioxide-induced function. Exp Cell Res 2000;256:375 – 82. death of neuroblastoma cells involves activation of Bax and does not 27. Mandic A, Viktorsson K, Strandberg L, et al. Calpain-mediated Bid require p53. Clin Cancer Res 2004;10:3179 – 88. cleavage and calpain-independent Bak modulation: two separate path- 11. Karlsson J, Edsjo A, Pa˚hlman S, Pettersson HM. Multidrug-resistant ways in cisplatin-induced apoptosis. Mol Cell Biol 2002;22:3003 – 13. neuroblastoma cells are responsive to arsenic trioxide at both normoxia 28. Blomgren K, Zhu C, Wang X, et al. Synergistic activation of caspase-3 and hypoxia. Mol Cancer Ther 2005;4:1128 – 35. by m-calpain after neonatal hypoxia-ischemia: a mechanism of ‘‘patho- 12. Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer logical apoptosis’’? J Biol Chem 2001;276:10191 – 8. treatment. Nat Rev Cancer 2004;4:437 – 47. 29. Waterhouse NJ, Finucane DM, Green DR, et al. Calpain activation is 13. Chen GQ, Shi XG, Tang W, et al. Use of arsenic trioxide (As2O3) in upstream of caspases in radiation-induced apoptosis. Cell Death Differ the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts 1998;5:1051 – 61. dose-dependent dual effects on APL cells. Blood 1997;89:3345 – 53. 30. Lallemand-Breitenbach V, Guillemin MC, Janin A, et al. Retinoic acid 14. Cai X, Shen YL, Zhu Q, et al. Arsenic trioxide-induced apoptosis and and arsenic synergize to eradicate leukemic cells in a mouse model of differentiation are associated respectively with mitochondrial transmem- acute promyelocytic leukemia. J Exp Med 1999;189:1043 – 52. brane potential collapse and retinoic acid signaling pathways in acute 31. Scholz C, Wieder T, Starck L, et al. Arsenic trioxide triggers a promyelocytic leukemia. Leukemia 2000;14:262 – 70. regulated form of caspase-independent necrotic cell death via the 15. McCafferty-Grad J, Bahlis NJ, Krett N, et al. Arsenic trioxide uses mitochondrial death pathway. Oncogene 2005;24:1904 – 13. caspase-dependent and caspase-independent death pathways in myeloma 32. Carney DN, Mitchell JB, Kinsella TJ. In vitro radiation and chemo- cells. Mol Cancer Ther 2003;2:1155 – 64. therapy sensitivity of established cell lines of human small cell lung cancer 16. Quignon F, De Bels F, Koken M, Feunteun J, Ameisen JC, de The H. and its large cell morphological variants. Cancer Res 1983;43:2806 – 11. PML induces a novel caspase-independent death process. Nat Genet 33. Gazdar AF, Carney DN, Nau MM, Minna JD. Characterization of 1998;20:259 – 65. variant subclasses of cell lines derived from small cell lung cancer having 17. Xiang J, Chao DT, Korsmeyer SJ. BAX-induced cell death may not distinctive biochemical, morphological, and growth properties. Cancer Res require interleukin 1h-converting enzyme-like proteases. Proc Natl Acad 1985;45:2924 – 30. Sci U S A 1996;93:14559 – 63. 34. Radice PA, Matthews MJ, Ihde DC, et al. The clinical behavior of 18. Karlsson J, Pietras A, Beckman S, Pettersson HM, Larsson C, Pa˚hlman ‘‘mixed’’ small cell/large cell bronchogenic carcinoma compared to ‘‘pure’’ S. Arsenic trioxide-induced neuroblastoma cell death is accompanied by small cell subtypes. Cancer 1982;50:2894 – 902. proteolytic activation of nuclear Bax. Oncogene 2007;26:6150 – 9. 35. Bergh J, Larsson E, Zech L, Nilsson K. Establishment and character- 19. Johnson BE, Russell E, Simmons AM, et al. MYC family DNA ization of two neoplastic cell lines (U-1285 and U-1568) derived from amplification in 126 tumor cell lines from patients with small cell lung small cell carcinoma of the lung. Acta Pathol Microbiol Immunol Scand [A] cancer. J Cell Biochem Suppl 1996;24:210 – 7. 1982;90:149 – 58. 20. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. 36. Mathews V, Balasubramanian P, Shaji RV, George B, Chandy M, Amplification of N-myc in untreated human neuroblastomas correlates Srivastava A. Arsenic trioxide in the treatment of newly diagnosed acute with advanced disease stage. Science 1984;224:1121 – 4. promyelocytic leukemia: a single center experience. Am J Hematol 2002; 21. Holmquist-Mengelbier L, Fredlund E, Lofstedt T, et al. Recruitment of 70:292 – 9. HIF-1a and HIF-2a to common target genes is differentially regulated in 37. Shen ZX, Shi ZZ, Fang J, et al. All-trans retinoic acid/As2O3 neuroblastoma: HIF-2a promotes an aggressive phenotype. Cancer Cell combination yields a high quality remission and survival in newly 2006;10:413 – 23. diagnosed acute promyelocytic leukemia. Proc Natl Acad Sci U S A 22. Lofstedt T, Jogi A, Sigvardsson M, et al. Induction of ID2 expression 2004;101:5328 – 35. by hypoxia-inducible factor-1: a role in dedifferentiation of hypoxic 38. Rojewski MT, Korper S, Schrezenmeier H. Arsenic trioxide therapy neuroblastoma cells. J Biol Chem 2004;279:39223 – 31. in acute promyelocytic leukemia and beyond: from bench to bedside. 23. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization Leuk Lymphoma 2004;45:2387 – 401. of real-time quantitative RT-PCR data by geometric averaging of multiple 39. Amadori S, Fenaux P, Ludwig H, O’Dwyer M, Sanz M. Use of arsenic internal control genes. Genome Biol 2002;3:RESEARCH0034. trioxide in haematological malignancies: insight into the clinical develop- 24. Gobeil S, Boucher CC, Nadeau D, Poirier GG. Characterization of the ment of a novel agent. Curr Med Res Opin 2005;21:403 – 11. necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of 40. Pettersson HM, Karlsson J, Pietras A, Øra I, Pa˚hlman S. Arsenic lysosomal proteases. Cell Death Differ 2001;8:588 – 94. trioxide and neuroblastoma cytotoxicity. J Bioenerg Biomembr 2007;39: 25. Hood JL, Brooks WH, Roszman TL. Subcellular mobility of the 35 – 41.

Mol Cancer Ther 2009;8(1). January 2009

Helen M. Pettersson, Alexander Pietras, Matilda Munksgaard Persson, et al.

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