Picropodophyllin inhibits proliferation and survival of diffuse large B-cell lymphoma cells
Thomas Stro¨mberg1 • Xiaoying Feng2,3 • Maryam Delforoush1 • Mattias Berglund1 •
Yingbo Lin2 • Magnus Axelson4 • Olle Larsson2 • Patrik Georgii-Hemming1 •
Johan Lennartsson5 • Gunilla Enblad1
Received: 28 April 2015 / Accepted: 3 May 2015 / Published online: 29 May 2015
© Springer Science+Business Media New York 2015
Abstract
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults. Although chemother- apy in combination with anti-CD20 antibodies results in a cure rate of 60–70 %, novel treatment approaches are warranted for the remaining patients. The insulin-like growth factor-1 receptor (IGF-1R) and its principal ligands IGF-1 and IGF-2 have been suggested to play pivotal roles in different cancers. However, in DLBCL the importance of this system is less well understood. To assess whether interference with IGF-1R-mediated signaling may repre- sent a therapeutic option for this malignancy, we used a panel of eight DLBCL cell lines together with primary tumor cells derived from lymph nodes in four DLBCL patients. The cells were treated with the cyclolignan pi- cropodophyllin (PPP), a small molecule compound initially described to selectively inhibit the IGF-1R. PPP dose-dependently inhibited proliferation/survival in all cell lines and primary cell preparations. In parallel experiments, the IGF-1R inhibitor NVP-AEW541 and the microtubule- destabilizing compounds podophyllotoxin (PPT) and col- chicine were demonstrated to also inhibit growth of the cell lines. Linear regression analysis showed that the responses of the cell lines to PPP correlated with their responses to the microtubule inhibitors PPT and colchicine, but not with the response to NVP-AEW541 or the expression level of surface IGF-1R. Analysis of cell cycle phase distribution revealed that treatment with PPP for only 1 h induced a clear accumulation of cells in the G2/M-phase with a corresponding depletion of the G0/G1-phase. Interestingly, these cell cycle effects could be closely mimicked by using PPT or colchicine. Treatment with PPP led to increased apoptotic cell death in the SU-DHL-6 and U-2932 cell li- nes, whereas the DB and U-2940 did not undergo apop- tosis. However, the DB cells were still killed by PPP, suggesting another mode of cell death for this cell line. The U-2940 cells responded to PPP mainly by inhibition of proliferation. Pretreatment of U-2932 or U-2940 cell lines with PPP at biologically active concentrations did not prevent ligand-induced phosphorylation of IGF-1R at Tyr1131/1136 or its downstream targets AKT and ERK1/2. In contrast, the IGF-1R inhibitor NVP-AEW541 clearly inhibited phosphorylation of IGF-1R and AKT, while ERK1/2 phosphorylation was less affected. Taken together, the inhibitory effects of PPP in DLBCL cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease. However, we suggest that the primary target of PPP in these cells is not related to inhibition of IGF-1R phosphorylation.
Keywords : Diffuse large B-cell lymphoma · DLBCL · Picropodophyllin · PPP · IGF-1R
Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid neoplasm and comprises a heteroge- nous group of tumors that can be subdivided into mor- phological variants, molecular and immunophenotypic subgroups and distinct disease entities. The introduction of anti-CD20 antibodies (rituximab) to standard chemother- apy has resulted in significant improvements in the overall survival rates of DLBCL patients [1]; however, for a sub- group of patients the prognosis remains poor.
The insulin-like growth factor-1 receptor (IGF-1R), a member of the transmembrane tyrosine kinase receptor family, has demonstrated increased expression in various tumors [2], where it facilitates anchorage-independent growth, migration and chemoresistance of the malignant cells [3]. Among the lymphohematopoietic cancers, the IGF-1R has been suggested to represent a therapeutic target in multiple myeloma [4–6], T cell ALK+ anaplastic large- cell lymphoma [7], T lymphoblastic leukemia/lymphoma [8], mantle cell lymphoma [9] and classical Hodgkin lymphoma [10].
The cyclolignan picropodophyllin (PPP) is an epimer of podophyllotoxin (PPT), which occurs naturally and can be isolated from certain plant species. PPT inhibits micro- tubule assembly by binding to the colchicine-binding site of tubulin [11] and has previously been evaluated as an anticancer agent, however, with unacceptable side effects [12]. PPP, being at least 50 times less effective than PPT at inhibiting microtubule assembly [13], has been launched as a potent and selective inhibitor of the IGF-1R [14] by specifically blocking phosphorylation of the tyrosine resi- due Tyr1136 within the activation loop of the IGF-1R beta subunit [15]. Via inhibition of the IGF-1R, PPP has been demonstrated to reduce phosphorylation of the downstream targets AKT and/or ERK1/2 [5, 6, 14, 16, 17] and also to induce apoptosis of malignant cells as well as tumor re- gression in different tumor models [6, 14, 16–19].
NVP-AEW541 is a small molecule inhibitor of IGF-1R kinase belonging to the pyrrolo[2,3-d]pyrimidine class [20]. In intact cells, it was found to be 27-fold more potent toward the IGF-1R as compared to the structurally related insulin R. NVP-AEW541 has shown efficacy in preclinical tumor models of, e.g., myeloma [20] and Ewing’s sarcoma [21] but has, in contrast to PPP [22], not been tested in humans.In this study, PPP dose-dependently inhibited prolif- eration/survival in eight DLBCL cell lines as well as in primary tumor cells extracted from lymph nodes of four non-treated DLBCL patients. However, the inhibitory ac- tivity of PPP in the cell line panel correlated with the ac- tivity of the microtubule-destabilizing agents PPT and colchicine rather than with the activity of the IGF-1R in- hibitor NVP-AEW541 or the level of IGF-1R expression. Furthermore, the effects of PPP on the cell cycle could be closely mimicked by PPT and colchicine. These findings together with that PPP failed to prevent phosphorylation of IGF-1R at Tyr1135/Tyr1136 suggest that PPP in DLBCL cells primarily exerts its effect(s) via other, IGF-1R-inde- pendent mechanism(s).
Materials and methods
Cell culture
The human DLBCL cell lines DB, OCI-LY-1, OCI-LY-3, RC-K8, SU-DHL-6, SU-DHL-10, U-2932 and U-2940 were routinely cultured in RPMI 1640 supplemented with 10 % fetal bovine serum (FBS), glutamine and antibiotics (Sigma-Aldrich, St Louis, MO) at +37 °C in a humidified incubator containing 5 % CO2-in-air atmosphere. All cell lines were confirmed negative for mycoplasma infection by MycoAlert® (Lonza, Copenhagen, Denmark) as were their identities using single-tandem-repeat (STR) analysis.
Patient material
The study subjects comprised lymph nodes from four histopathologically confirmed non-treated DLBCL patients who underwent needle biopsies at the Uppsala University Hospital, Department of Oncology, Uppsala, Sweden. The study was approved by the local ethical committee ac- cording to the Declaration of Helsinki, and after informed consent the lymph nodes were surgically obtained and placed in liquid nitrogen within 20 min post-excision.
Reagents
PPP, synthesized as described [14] or purchased (Tocris Bioscience, Bristol, UK), NVP-AEW541 (Selleckchem. com, Houston, TX), PPT and colchicine (Sigma-Aldrich) were dissolved in desiccated DMSO (Sigma-Aldrich) at 16 mM and prediluted in the solvent before addition to cell cultures, where the final concentration of DMSO was al- ways ≤0.1 %, a concentration known not to affect cell growth or signaling (data not shown).
Fluorometric microculture cytotoxicity assay (FMCA)
The FMCA is a semi-automated microculture viability assay that has been used extensively for screening of chemotherapeutic drugs [23]. Cells were harvested in log phase, and seeded (45 lL/well) into 384-well microtiter plates (Corning Life Sciences, Manassas, VA) at a con- centration of 100,000 cells/mL using Biomek® 2000 (Beckman Coulter, Brea, CA). The plates were incubated at 37 °C in a humidified atmosphere containing 95 % air and 5 % CO2 for 24 h before addition of DMSO-dissolved drugs at ten different concentrations in duplicates using Echo 550 Liquid Handler (Labcyte, Sunnyvale, CA). After incubation for 72 h, the cells were washed with PBS,centrifuged and aspirated leaving a volume of 10 lL in each well. Finally, 50 lL fluorescein diacetate (Sigma- Aldrich) at the concentration 10 lg/mL was added to each well. After incubation in the dark for 60 min at room temperature, fluorescence was analyzed at 485/538 nm (emission/excitation) using a FLUOstar Omega (BMG Labtech, Ortenberg, Germany).
Analysis of proliferation/survival in primary DLBCL cultures
After preparation from lymph nodes from DLBCL patients, the primary tumor cells were seeded in complete medium in tissue culture-treated 96-well plates (Corning Life Sci- ences) at the concentration 500,000 cells/mL and treated with PPP for 72 h. Cell growth (i.e., the net result of cell division minus cell death) was analyzed by using resazurin (Sigma-Aldrich) as described [24].
Analysis of cell cycle phase distribution and apoptosis by flow cytometry
Cell cycle phase distribution was analyzed according to Vindelov [25] using non-fixed, propidium iodide (PI)-stained nuclei followed by quantification using ModFit LT 3.0 Analysis Software (Verity Software House, Topsham, ME).The amount of apoptosis was analyzed using Annexin V-Alexa Fluor 647® (Molecular Probes, Eugene, OR) and PI- staining, where only Annexin V-positive/PI-negative cells were considered as truly apoptotic.
Analysis of IGF-1R and insulin R surface expression
Surface IGF-1R and insulin R expression was quantified flow cytometrically by incubation with anti-IGF-1Ra, in- sulin Ra and isotype control antibodies (BD Biosciences, San Jose´, CA) followed by staining with phycoerythrin- conjugated anti-mouse IgG antibodies (DAKO, Glostrup, Denmark). Fluorescence resulting from specific staining was divided with fluorescence from staining with isotype control, resulting in relative mean fluorescence intensity (RFI).
Western blotting
The lymphoma cell lines were seeded at 800,000 cells/mL (10 × 106 cells/experimental condition) in medium with- out FBS and incubated for 24 h. The cells were then ex- posed to PPP or NVP-AEW541 at indicated concentrations for 1 h, treated with IGF-1 for 5 min before addition of ice-cold PBS and collected by centrifugation at +4 °C. Following one wash in ice-cold PBS and centrifugation, the cells were lysed on ice in a volume of 250 lL RIPA buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 % Triton-X, 0.5 % sodium deoxycholate, 0.1 % SDS) supplemented with 1 mM Na3VO4 and 1 mM Pefabloc® (Sigma- Aldrich). The lysates were precleared by centrifugation at 13,000×g for 15 min at 4 °C and the supernatants then subjected to immunoblotting. Primary antibodies were specific for phospho (p)-IGF-1R (Tyr1135/Tyr1136), total IGF-1Rb, p-AKT (Ser473), p-ERK1/2 (Thr202/Tyr204), total ERK1/2, GAPDH (Cell Signaling Technology, Dan- vers, MA) and total AKT (Santa Cruz Biotechnology, Santa Cruz, CA). The antibodies were diluted in PBS 0.1 % Tween (PBS-T) with 5 % protease-free bovine serum albumin fraction V (Saveen Werner AB, Limhamn, Sweden) and used at the concentrations 1:200 (total AKT), 1:1000 (p-IGF-1R, total IGF-1Rb, p-AKT, p-ERK1/2, total ERK1/2) and 1:2000 (GAPDH). The membranes were developed using SuperSignal® (Thermo Scientific, Rock-ford, IL) or WesternBrightTM ECL (Advansta, Menlo Park, CA).
Statistical analysis
The generated fluorescence from the FMCA and the re- sazurin assay was (after blank subtraction) used for cal- culation of 50 and 25 % inhibitory concentrations (IC50 and IC25, respectively) of the different compounds by nonlinear regression analysis using GraphPad Prism (GraphPad Software, San Diego, CA). This software was also used for linear regression analysis performed to assess correlations between activities of PPP, NVP-AEW541, PPT, colchicine and expression levels of IGF-1R and in- sulin R.
Results
PPP, NVP-AEW541, PPT and colchicine inhibit growth of DLBCL cell lines
The effect of PPP, NVP-AEW541, PPT and colchicine on cell proliferation/survival in the eight DLBCL cell lines were analyzed by FMCA. When treated with PPP, the DB, OCI- LY-1, OCI-LY-3, SU-DHL-6, SU-DHL-10 and U-2932 cell lines showed the highest sensitivity exhibiting IC50s be- tween 0.19 and 0.29 lM, whereas the RC-K8 and U-2940 was less responsive with IC50s approximately at 1.41 and 0.72 lM, respectively (Table 1A). NVP-AEW541 was less potent than PPP exhibiting IC50s between 2.0 and 8.7 lM, whereas PPT and colchicine were more potent showing IC50s in the lower nanomolar range (Table 1A). Isolated tumor cells from patient 1, 2 and 4 were highly sensitive to PPP exhibiting IC50s below what was detected in the cell lines (Table 1B). In contrast, tumor cells from patient 3 showed some degree of resistance to PPP, since more than half of the cell population remained alive despite treatment.
DLBCL cell lines express surface IGF-1R and insulin R at different levels
The IGF-1R and insulin R surface expression was quanti- fied flow cytometrically followed by calculation of RFI. OCI-LY-3 cells expressed the highest IGF-1R level, while RC-K8 and DB cells expressed no or very low levels of IGF-1R (Table 2). Low but clearly detectable IGF-1R levels were expressed in OCI-LY-1, SU-DHL-10 cells, whereas intermediate expression was found in the SU- DHL-6, U-2932 and U-2940 cell lines. The SU-DHL-6 cells expressed the highest insulin R level, and the DB, OCI-LY-1 and RC-K8 and U-2940 cells expressed no or very low levels of insulin R. Low but clearly detectable insulin R levels were expressed in OCI-LY-3, SU-DHL-10, whereas intermediate expression was found in the U-2932 cell line (Table 2).
The responses of DLBCL cell lines to PPP correlate with their responses to PPT and colchicine
Linear regression analysis comparing the IC50s for PPP, NVP-AEW541, PPT, colchicine and the levels of IGF-1R and insulin R expression in the eight DLBCL cell lines revealed that the activity of PPP showed moderate corre- lation to the activity of PPT (R2 = 0.72, Fig. 1a) and high correlation to colchicine activity (R2 = 0.97, Fig. 1b). However, no correlation was detected between the ac- tivities of PPP and NVP-AEW541 (R2 = 0.21, Fig. 1c) or between PPP activity and IGF-1R expression (R2 = 0.14, Fig. 1d). A moderate level of correlation could be demonstrated between the IC50s for NVP-AEW541 ac- tivity and IGF-1R expression (R2 = 0.70, Fig. 1e), whereas for d PPP and e NVP-AEW541 were also compared with the expression levels of IGF-1R, as were the IC50s for f NVP-AEW541 with the expression levels of insulin R no correlation was found for NVP-AEW541 activity and insulin R expression (R2 = 0.19, Fig. 1f).
Fig. 1 Correlation analyses including activities of PPP, NVP- AEW541, PPT, colchicine and expression levels of IGF-1R and insulin R. The IC50s for PPP in the different cell lines were compared with IC50s for a PPT, b colchicine and c NVP-AEW541. The IC50s.
PPP, PPT and colchicine induce rapid accumulation in the G2/M-phase
The effect of treatment with PPP, PPT and colchicine on cell cycle phase distribution in the DB, SU-DHL-6, U-2932 and U-2940 cell lines was analyzed by flow cytometry. To minimize potential artifacts emanating from cells disap- pearing from the cell population to be analyzed (due to early and/or extensive cell death), the cells were subjected to short-term treatment, i.e., 1, 2, 4, 6 and 8 h, during basal cell culture conditions after 40 h of preincubation. The different compounds PPP, PPT and colchicine all induced a clear accumulation of cells in the G2/M-phase with a corresponding depletion of cells in the G0/G1-phase (Fig. 2). This response was detected in all four cell lines tested and was evident already after 1 h treatment.
PPP decreases survival in 3/4 DLBCL cell lines
The amount of apoptosis/cell death resulting from treat- ment with PPP at indicated concentrations was analyzed at 48 h using Annexin V-Alexa Fluor 647/PI-staining and flow cytometry. Both the U-2932 and SU-DHL-6 cell lines responded to PPP with increased apoptosis (Fig. 3). How- ever, the amount of apoptosis was not increased in the DB and the U-2940 cell lines, although the former showed increased cell death when treated with PPP.
PPP at biologically active concentrations does not inhibits phosphorylation of the IGF-1R
Based on previous experiments demonstrating changes in of cell cycle phase distribution already at 1 h of incubation with 0.5 lM PPP, we decided to examine the short-term effects of PPP on phosphorylated and total forms of IGF- 1R, AKT and ERK1/2 by Western blotting. Thus, U-2932 and U-2940 cells were pretreated with PPP or NVP- AEW541 at different concentrations for 1 h before stimulation with IGF-1 for 5 min. Phosphorylation of the IGF-1R at Tyr1135/Tyr1136 in both cell lines was clearly upregulated by treatment with IGF-1 (Fig. 4). With ex- ception of the highest concentration, PPP failed to coun- teract this effect (Fig. 4, left panel). In contrast, NVP- AEW541 showed high efficacy in preventing IGF-1- induced increase in p-IGF-1R (Fig. 4, right panel). Similarly, IGF-1-stimulated increase in p-AKT and p-ERK1/2 in U-2932 cells was clearly inhibited by NVP- AEW541, while PPP was able to diminish these effects only at the highest concentration. In the U-2940 cells, high background levels of p-AKT and p-ERK1/2 were detected, and only p-AKT could be further increased by treatment with IGF-1. However, the discrete increase in p-AKT could be counteracted by PPP even at concentrations below 10 lM. On the other hand, NVP-AEW541 much more.
Fig. 2 Effects of PPP, PPT and colchicine on cell cycle phase distribution. The DB, SU-DHL- 6, U-2932 and U-2940 cell lines were incubated for 40 h during basal conditions before treatment with the compounds for indicated times. The analyses were performed by flow cytometry and show distribution of the cell cycle phases G0/G1 (green), S (blue) and G2/M (green) resulting from treatment with PPP (solid lines), PPT (dashed lines) or colchicine (dotted lines).
Fig. 3 Effects of PPP on cell death/apoptosis. The four different DLBCL cell lines were incubated for 48 h with PPP for at indicated concentrations followed by flow cytometric analysis of cell viability using the Annexin V/PI-staining, where Annexin V-positive/PI- negative cells were considered as truly apoptotic effectively downregulated p-AKT in this cell line, leading to levels even below background. The total protein levels of IGF-1R, AKT and ERK1/2 was not affected by treat- ment with PPP or NVP-AEW541 (Fig. 4).
Discussion
Due to its overexpression in many cancers, the IGF-1R has been suggested as a promising molecular target in treatment of cancer [2, 20]. However, the results from clinical trials using different modes of IGF-1R-targeted therapy have this far been disappointing, and in some cases even lead to unacceptable toxicity when combined with traditional cytotoxic drugs [26]. In contrast, the cyclolignan PPP, launched as a selective IGF-1R in- hibitor [14], has been well tolerated in a phase I/II clinical study and possibly contributed to disease stabi- lization in four squamous non-small cell lung cancer patients [22]. Although PPP has been suggested as a potential therapy for the B-cell neoplasm multiple myeloma [5, 6] and for different classes of lymphoma
Fig. 4 Effects of PPP or NVP-AEW541 on IGF-1-stimulated phos- phorylation of IGF-1R, AKT and ERK in the U-2932 and U-2940 cell lines. The two DLBCL cell lines were serum-starved for 24 h and then treated with PPP (left panel) or NVP-AEW541 (right panel) at indicated concentrations for 1 h followed by stimulation with 50 ng/ mL IGF-1 for 5 min before lysis. The expression levels of IGF-1R, AKT and ERK and their phosphorylated forms were then analyzed by Western blotting where GAPDH served as loading control [7–10], there are no reports investigating the potential benefits of using PPP against DLBCL.
Results from the studies with FMCA showed that the activity of PPP in the eight DLBCL cell lines did not to correlate with the activity of NVP-AEW541 [20], an IGF- 1R inhibitor chemically unrelated to PPP and included in this study as a control compound. Neither did the activity of PPP correlate with the expression level of IGF-1R in the different cell lines. Instead, the inhibitory effect of PPP correlated with the activities of the microtubule inhibitors PPT and colchicine, indicating that the highly dynamic microtubule network may constitute a target for PPP as recently suggested [27, 28]. Furthermore, treatment of four DLBCL cell lines with either PPT or colchicine at 0.5 lM closely mimicked the alterations in cell cycle phase dis- tribution resulting from incubation with PPP at the same concentration. These effects, i.e., accumulation of cells in G2/M with a concomitant decrease in cells in G0/G1, were evident already after 1 h of PPP treatment and could pos- sibly be explained by destabilization of microtubules, since microtubule inhibitors are known to block mitosis and thus arrest cells in G2/M [29].
Previous studies have shown that 1 h of incubation with PPP before stimulation for 5 min with IGF-1 inhibits phosphorylation of IGF-1R and the downstream AKT and ERK1/2 as analyzed by Western blotting [14–17]. Similar effects of PPP have been obtained by other investigators, but then by using incubation times exceeding 1 h, some- times up to 24 h [5–10, 30]. Due to the spontaneous iso- merization of PPP in water to its very potent and toxic epimer PPT [12, 31], prolonged incubation times with PPP may lead to difficulties in interpretation of the results. This together with the observed rapid effects on the cell cycle described above made us to use 1 h incubation with PPP or NVP-AEW541 in the experiments where we tried to elu- cidate whether the IGF-1R could represent the primary target of PPP. However, we failed to show any inhibitory effects of submicromolar concentrations of PPP on phos- phorylation of the IGF-1R at Tyr1135/Tyr1136. In contrast, NVP-AEW541 clearly inhibited IGF-1R phosphorylation at these sites. PPP was recently demonstrated to within minutes inhibit the microtubule of the mitotic spindle leading to mitotic arrest [28], thus providing evidence for an alternative target of PPP that possibly could be highly relevant for DLBCL cells as well. It is reasonable to pos- tulate that such a dramatic event as block of cell division potentially can lead to many different downstream conse- quences depending on which cell type that is affected, thus explaining some of the inconsistent findings resulting from treatment of malignant cells with PPP.
In summary, PPP decreases proliferation and survival of DLBCL cell lines and primary tumor cells of this disease.The observed early effects of PPP on cell cycle phase distribution together with the lack of decreased phospho- rylation of IGF-1R suggests that the anti-tumor effects of PPP do not primarily result from targeting the IGF-1R but could possibly be governed via inhibition of microtubules, in particular during assembly of the mitotic spindle [28]. However, further investigations are required to in detail unravel such an IGF-1R-independent mechanism(s).
Acknowledgments This work was supported by Swedish Cancer Foundation, Swedish Research Council, European Commission Marie Curie Fellowship (EA), Cancer Society in Stockholm, Children Cancer Society, Lundberg’s Research Foundation in Gothenburg, Stockholm County Council and Karolinska Institutet.
Conflict of interest The authors declare that there are no conflicts of interest.
References
1. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, Morel P, Van Den Neste E, Salles G, Gaulard P, Reyes F, Lederlin P, Gisselbrecht C. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large- B-cell lymphoma. N Engl J Med. 2002;346:235–42.
2. Pollak M. Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer. 2008;8:915–28.
3. Sachdev D, Yee D. Disrupting insulin-like growth factor sig- naling as a potential cancer therapy. Mol Cancer Ther. 2007;6:1–12.
4. Georgii-Hemming P, Wiklund HJ, Ljunggren O, Nilsson K. In- sulin-like growth factor I is a growth and survival factor in human multiple myeloma cell lines. Blood. 1996;88:2250–8.
5. Stromberg T, Ekman S, Girnita L, Dimberg LY, Larsson O, Axelson M, Lennartsson J, Hellman U, Carlson K, Osterborg A, Vanderkerken K, Nilsson K, Jernberg-Wiklund H. IGF-1 receptor tyrosine kinase inhibition by the cyclolignan PPP induces G2/M- phase accumulation and apoptosis in multiple myeloma cells. Blood. 2006;107:669–78.
6. Menu E, Jernberg-Wiklund H, Stromberg T, De Raeve H, Girnita L, Larsson O, Axelson M, Asosingh K, Nilsson K, Van Camp B, Vanderkerken K. Inhibiting the IGF-1 receptor tyrosine kinase with the cyclolignan PPP: an in vitro and in vivo study in the 5T33MM mouse model. Blood. 2006;107:655–60.
7. Shi P, Lai R, Lin Q, Iqbal AS, Young LC, Kwak LW, Ford RJ, Amin HM. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells. Blood. 2009;114:360–70.
8. Huang Z, Fang Z, Zhen H, Zhou L, Amin HM, Shi P. Inhibition of type I insulin-like growth factor receptor tyrosine kinase by picropodophyllin induces apoptosis and cell cycle arrest in T lymphoblastic leukemia/lymphoma. Leuk Lymphoma. 2014;55:1876–83.
9. Vishwamitra D, Shi P, Wilson D, Manshouri R, Vega F, Schlette EJ, Amin HM. Expression and effects of inhibition of type I insulin-like growth factor receptor tyrosine kinase in mantle cell lymphoma. Haematologica. 2011;96:871–80.
10. Liang Z, Diepstra A, Xu C, van Imhoff G, Plattel W, Van Den Berg A, Visser L. Insulin-like growth factor 1 receptor is a prognostic factor in classical Hodgkin lymphoma. PLoS One. 2014;9:e87474.
11. Cortese F, Bhattacharyya B, Wolff J. Podophyllotoxin as a probe for the colchicine binding site of tubulin. J Biol Chem. 1977;252:1134–40.
12. Imbert TF. Discovery of podophyllotoxins. Biochimie. 1998;80: 207–22.
13. Loike JD, Brewer CF, Sternlicht H, Gensler WJ, Horwitz SB. Structure-activity study of the inhibition of microtubule assembly in vitro by podophyllotoxin and its congeners. Cancer Res. 1978;38:2688–93.
14. Girnita A, Girnita L, del Prete F, Bartolazzi A, Larsson O, Ax- elson M. Cyclolignans as inhibitors of the insulin-like growth factor-1 receptor and malignant cell growth. Cancer Res. 2004; 64:236–42.
15. Vasilcanu D, Girnita A, Girnita L, Vasilcanu R, Axelson M, Larsson O. The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway. Oncogene. 2004;23:7854–62.
16. Yin S, Girnita A, Stromberg T, Khan Z, Andersson S, Zheng H, Ericsson C, Axelson M, Nister M, Larsson O, Ekstrom TJ, Girnita
L. Targeting the insulin-like growth factor-1 receptor by pi- cropodophyllin as a treatment option for glioblastoma. Neuro Oncol. 2010;12:19–27.
17. Yin SC, Guo W, Tao ZZ. Picropodophyllin inhibits tumor growth of human nasopharyngeal carcinoma in a mouse model. Biochem Biophys Res Commun. 2013;439:1–5.
18. Girnita A, All-Ericsson C, Economou MA, Astrom K, Axelson M, Seregard S, Larsson O, Girnita L. The insulin-like growth factor-I receptor inhibitor picropodophyllin causes tumor re- gression and attenuates mechanisms involved in invasion of uveal melanoma cells. Clin Cancer Res. 2006;12:1383–91.
19. Klinakis A, Szabolcs M, Chen G, Xuan S, Hibshoosh H, Efstratiadis A. Igf1r as a therapeutic target in a mouse model of basal-like breast cancer. Proc Natl Acad Sci USA. 2009;106: 2359–64.
20. Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Shrin- garpure R, Akiyama M, Hideshima T, Chauhan D, Joseph M, Libermann TA, Garcia-Echeverria C, Pearson MA, Hofmann F, Anderson KC, Kung AL. Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors. Cancer Cell. 2004;5:221–30.
21. Scotlandi K, Manara MC, Nicoletti G, Lollini PL, Lukas S, Benini S, Croci S, Perdichizzi S, Zambelli D, Serra M, Garcia- Echeverria C, Hofmann F, Picci P. Antitumor activity of the insulin-like growth factor-I receptor kinase inhibitor NVP- AEW541 in musculoskeletal tumors. Cancer Res. 2005;65: 3868–76.
22. Ekman S, Frodin JE, Harmenberg J, Bergman A, Hedlund A, Dahg P, Alvfors C, Stahl B, Bergstrom S, Bergqvist M. Clinical phase I study with an insulin-like growth factor-1 receptor in- hibitor: experiences in patients with squamous non-small cell lung carcinoma. Acta Oncol. 2011;50:441–7.
23. Lindhagen E, Nygren P, Larsson R. The fluorometric microcul- ture cytotoxicity assay. Nat Protoc. 2008;3:1364–9.
24. Stromberg T, Dimberg A, Hammarberg A, Carlson K, Osterborg A, Nilsson K, Jernberg-Wiklund H. Rapamycin sensitizes mul- tiple myeloma cells to apoptosis induced by dexamethasone. Blood. 2004;103:3138–47.
25. Vindelov LL. Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei. Virchows Arch B Cell Pathol. 1977;24:227–42.
26. Baserga R. The decline and fall of the IGF-I receptor. J Cell Physiol. 2013;228:675–9.
27. Wu X, Sooman L, Wickstrom M, Fryknas M, Dyrager C, Len- nartsson J, Gullbo J. Alternative cytotoxic effects of the postu- lated IGF-IR inhibitor picropodophyllin in vitro. Mol Cancer Ther. 2013;12:1526–36.
28. Waraky A, Akopyan K, Parrow V, Stro¨mberg T, Axelson M, Abrahmse´n L, Lindqvist A, Larsson O, Aleem E. Picrop- odophyllin causes mitotic arrest and catastrophe by depolymer- izing microtubules via Insulin-like growth factor-1 receptor- independent mechanism. Oncotarget. 2014;5:8379–92.
29. Yang JS, Hour MJ, Huang WW, Lin KL, Kuo SC, Chung JG. MJ- 29 inhibits tubulin polymerization, induces mitotic arrest, and
triggers apoptosis via cyclin-dependent kinase 1-mediated Bcl-2 phosphorylation in human leukemia U937 cells. J Pharmacol Exp Ther. 2010;334:477–88.
30. Feng X, Aleem E, Lin Y, Axelson M, Larsson O, Stromberg T. Multiple antitumor effects of picropodophyllin in colon carci- noma cell lines: clinical implications. Int J Oncol. 2012;40: 1251–8.
31. Gensler WJ, Gatsonis CD. The podophyllotoxin-picrop- odophyllin equilibrium. J Org Chem. 1966;31:3224–7.