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Tumor Biol. DOI 10.1007/s13277-016-4972-7

ORIGINAL ARTICLE

Colchicine induces autophagy and senescence in lung cancer cells at clinically admissible concentration: potential use of colchicine in combination with autophagy inhibitor in cancer therapy Surela Bhattacharya 1 & Amlan Das 1 & Satabdi Datta 1 & Arnab Ganguli 1 & Gopal Chakrabarti 1

Received: 15 October 2015 / Accepted: 3 February 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract Colchicine is a well-known and potent microtubule targeting agent, but the therapeutic value of colchicine against cancer is limited by its toxicity against normal cells. But, there is no report of its cytotoxic potential against lung cancer cell, at clinically permissible or lower concentrations, minimally toxic to non-cancerous cells. Hence, in the present study, we investigated the possible mechanism by which the efficacy of colchicine against lung cancer cells at less toxic dose could be enhanced. Colchicine at clinically admissible concentration of 2.5 nM had no cytotoxic effect and caused no G2/M arrest in A549 cells. However, at this concentration, colchicine strongly hindered the reformation of cold depolymerised interphase and spindle microtubule. Colchicine induced senescence and reactive oxygen species mediated autophagy in A549 cells at this concentration. Autophagy inhibitor 3-methyladenine (3-MA) sensitised the cytotoxicity of colchicine in A549 cells by switching senescence to apoptotic death, and this combination had reduced cytotoxicity to normal lung fibroblast cells (WI38). Together, these findings indicated the possible use of colchicine at clinically relevant dose along with autophagy inhibitor in cancer therapy.

Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-4972-7) contains supplementary material, which is available to authorized users. * Gopal Chakrabarti [email protected]

1

Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700 019, India

Keywords Colchicine . ROS . Autophagy . Microtubule . Lung cancer

Introduction Colchicine has been used for centuries for treatment of gout attacks and familial Mediterranean fever (FMF), and it remains one of the oldest known drugs still currently in use [1–4]. In 2009, the FDA approved colchicine for treatment of gout and FMF [5]. The history of the use of colchicine unravels its efficacy but also indicates its toxicity especially at high doses that can also be lethal [6]. Extensive research studies with colchicine establish that it inhibits microtubule polymerization by binding to tubulin and thus acts as mitotic spindle poison in cells [7, 8]. Recent investigations have thrown insight into possibilities of treatment of various ailments including cancer, with colchicine thus unfolding new chapter in its long history of medication [6]. Chemotherapy has remained the most important approach in treatment of both early- and late-stage cancer treatment. The goal of chemotherapy is to successfully induce cell death in cancer cells with minimal toxicity to normal cells. However, the conventional drug regimens at Bmaximum tolerated doses^ (MTDs) of the cytotoxic agents, which have been designed to kill maximum number of tumour cells, inflict severe damage to normal cells. To allow the normal cells to recover from the chemotherapeutic insult, treatment-free period must be included in the drug administration regime of the patients. This treatment-free period is in turn utilised by the endothelial cells within the tumour which again initiate angiogenesis, thus causing resurgence of tumour growth. Thus, the new concept of low-dose chemotherapy emerges, wherein the toxicity caused by high dose is drastically reduced and multidrug resistance can be avoided [9–11]. Recent studies indicate that

Tumor Biol.

low-dose chemotherapy can induce premature senescence (also known as accelerated or stress-induced premature senescence) which has been identified recently as a tumour suppressor mechanism and a key determinant of cancer chemotherapy outcome [12–15]. However, stress-induced senescence could also reflect efforts by the cell to evade direct cell killing. The senescence-associated secretory phenotypes (SASP) components secreted by senescent tumour cells might stimulate the malignant phenotypes of nearby tumour cells [16]. Additionally, there is every possibility of resurgence of senescent cells into their active dividing state, unless they are not cleared by the system [17, 18]. Making the situation worst, this stress-induced premature senescence is often associated with relative inability to undergo apoptosis [19]. The chemotherapeutic agents unfavourably induce stress response-activated pathways that trigger cell survival and resistance to such chemotherapeutics. Autophagy is one such stress-regulated evolutionarily conserved pathway of largescale lysosomal degradation of long-lived proteins, macromolecules and cellular organelles which provides a means of recycling macromolecules as an alternative energy source and thus rendering the cell ability to survive and maintain homeostasis in stressful environment. Autophagy may act as a saviour from chemotherapy-mediated cancer cell death [20–22] or may act as programmed cell death type II or autophagic death [23, 24]. Thus, depending on the context such as cancer cell type, nature and duration of stress, autophagy plays various roles in maneuvering cancer cell fate. Recent evidences have suggested that autophagy and senescence are interconnected. An increase of autophagic vacuoles and senescenceassociated β-galactosidase (SA-β-gal) activity was observed in aging fibroblasts [25]. Markers of autophagy and senescence were collaterally observed in the bile duct cells of patients with primary biliary cirrhosis as well as in biliary epithelial cells isolated from mice and treated with either hydrogen peroxide or etoposide [26]. A recent study revealed the upregulation of autophagy-related genes during oncogeneinduced senescence and that inhibition of autophagy delayed the senescence phenotype [27]. There are reports of microtubule targeting agents inducing both senescence and autophagy [28, 29]. However, there is no report until date on the senescence or autophagy inducing property of clinically admissible concentration of colchicine. In spite of limited application of colchicine due to its toxicity to normal cells, oral colchicine is a safe medication when used in proper dose, and contraindications have been limited. Thus, the present study reports induction of both premature senescence and autophagy upon clinically admissible dose of colchicine treatment in human lung cancer cell line (A549 cells). The manipulation of this senescent phenotype by autophagy inhibition compels the cell to undergo apoptotic cell death.

Materials and methods Reagents Nutrient mixture HAMS F12 medium (supplemented with 1 mM L-glutamine), foetal bovine serum, penicillin-streptomycin, amphotericin B and 0.25 % trypsin-EDTA were purchased from GIBCO (Invitrogen). Colchicine, monodansylcadaverine (MDC), 3-methyladenine (3-MA), chloroquine (CQ), N-acetylcysteine (NAC), monoclonal anti-α-tubulin FITC tagged antibody produced in mouse, DAPI and JC-1, and DCF-DA were purchased from Sigma. Mouse monoclonal anti-p53, mouse monoclonal anti-p21, mouse monoclonal anti-α-tubulin antibody, mouse monoclonal anti-Beclin1 antibody, rabbit polyclonal anti-LC3 antibody, rabbit polyclonal anti-Bcl-2 antibody, goat polyclonal anti-caspase-3 antibody, mouse monoclonal anti-bax antibody and annexin V-FITC apoptosis kit were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Bradford protein estimation kit, goat anti-mouse IgG-HRP, rabbit anti-goat IgG-HRP, goat anti-rabbit IgG-HRP and goat anti-mouse IgG-TRITC conjugate were purchased from Genei, India. All other chemicals and reagents were of analytical grade and were purchased from Sisco Research Laboratories. Cell culture Human non-small lung epithelium cell line A549 was obtained from cell repository of National Centre for Cell Science, (NCCS) Pune, India. A549 cells were cultured in nutrient mixture HAMS F12 medium supplemented with 1 mM Lglutamine, 10 % foetal bovine serum, 1.17 g/l NaHCO3, 50 μg/mL penicillin, 50 μg/mL streptomycin and 2.5 μg/mL amphotericin B. The morphology of normal and treated cells was kept under observation with an Olympus model CKX41 inverted microscope. Cell viability assay (MTT Assay) Cultured cells were seeded in 96-well plate at a density of 1 × 104cells/mL and grown overnight. Cells were treated accordingly. MTT (5 mg/mL) solution was prepared in PBS, filtered, and added to each well and incubated for 4 h at 37 °C. Subsequently, purple formazan crystal was dissolved in 150 μL of DMSO. The absorbance was measured on an ELISA reader (Multiskan EX, Lab systems, Helsinki, Finland) at a wavelength of 570 nm. Data were calculated as the percentage of inhibition by the following formula: %Inhibition ¼ ½100−ðAt‐AsÞ  100

ð1Þ

where At and As are the absorbance of the test substances and solvent control, respectively [30].

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Cultured A549 cells were grown at a density of 106 cells/mL and incubated in the presence of desired ligand for required time. After treatment, the cells were harvested, fixed in icecold methanol for at least 30 min in 4 °C, and incubated for 4 h at 37 °C in a PBS solution containing 1 mg/mL RNase A. Then, nuclear DNA was labelled with propidium iodide (PI). Cell cycle analysis was performed using a Becton Dickinson FACS Calibur flow cytometer, and the data were analysed using the Cell Quest program from Becton Dickinson [31].

000g) for 10 min. The 100-μL supernatants containing soluble (cytosolic) tubulin were separated from the pellets containing polymerised (cytoskeletal) tubulin. The pellets were resuspended in 100 μL of lysis buffer. The total concentrations of proteins in the soluble fraction and pellet fraction were estimated separately by the Bradford method. Equal amounts (40 μg) of each sample were added with sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis sample buffer and run in a 10 % SDS polyacrylamide gel. The sample was then analysed by western blotting and probed with the antibody against α-tubulin (1:1000 dilutions).

Sample preparation for confocal microscopy

SA-β-gal assay for the detection of senescence

Cultured A549 cells were grown at a density of 105 cells/mL and treated accordingly. Subsequently, cells were washed twice by PBS, fixed by 2 % paraformaldehyde and incubated with permeable solution (0.1 % Na-citrate, 0.1 % Triton) for 1 h. Non-specific binding sites were blocked by incubating the cells with 5 % BSA for 2 h at room temperature-controlled. Cells were then incubated with mouse monoclonal anti-αtubulin FITC tagged antibody (1:200 dilutions) followed by DAPI (1 μg/mL). After incubation, cells were washed with PBS and viewed under a Ziess LSM 510 Meta confocal microscope [31].

pH 6.0-dependent β-galactosidase expression was used as a marker for senescence along with senescent-related morphology [34]. At the appropriate times after treatment, cells were washed twice with PBS and fixed with 2 % formaldehyde, 0.2 % glutaraldehyde for 5 min. The cells were then washed again with PBS and stained with a solution of 1 mg/mL 5bromo-4-chloro-3-indolyl-β-D-galactosidase in dimethylformamide (20 mg/mL stock), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 40 mM citric acid/sodium phosphate, pH 6.0 and 2 mM MgCl2. Following overnight incubation at 37 °C, the cells were washed twice with PBS and photographed with a light microscope. The extent of senescence was quantified based on the mean number of cells displaying blue-green staining for three fields (containing at least 50 cells per field) for each experimental condition.

Cell cycle analysis by flow cytometry

Effect of colchicine on the reassembly of cold-depolymerised mitotic microtubules in A549 cells A549 cells were seeded on glass cover slips for 24 h and then incubated with 1.5 μM nocodazole for 20 h. Nocodazole was removed by washing with fresh medium. Cells were then incubated without or with 2.5 nM colchicine on ice for 30 min. Subsequently, cells were transferred to 37 °C, and the assembly of microtubules was followed by fixing the cells after 30 min. The microtubule network was visualised by staining the fixed cells with anti-α-tubulin antibody and subsequently by FITC-tagged secondary antibody. The DNA was stained with DAPI and observed under confocal microscope [32]. Western blot analysis of soluble and insoluble tubulin in cellular system The cellular tubulin polymerization was quantified by a modified method which was originally described by Minotti et al [33]. Cultured A549 cells were treated with 0, 2.5 and 50 nM of colchicine for 48 h. Then, the cells were washed twice with PBS and harvested by trypsinization. Cells were lysed at 37 °C for 5 min in the dark with 100 μL of hypotonic lysis buffer [1 mM MgCl2, 2 mM EGTA, 0.5 % NP-40, 20 μg/mL aprotinin, 20 μg/mL leupeptin, 1 mM orthovanadate, 2 mM PMSF, and 20 mM Tris-HCl (pH6.8)]. After a brief but vigorous vortex, the samples were centrifuged at 14000 rpm (21,

Labelling of autophagic vacuoles with MDC The auto-fluorescent agent MDC (Sigma) was recently introduced as a specific autophagolysosome marker to analyse the autophagic process [35]. Autophagic vacuoles were labelled with MDC by incubating cells with 0.05 mM MDC at 37 °C for 30 min. After incubation, cells were washed three times with PBS and immediately analysed with a fluorescence microscope (Nikon Eclipse TE 300, Japan) equipped with a filter system (V-2A excitation filter 380/420 nm, barrier filter 450 nm). Images were captured with a CCD camera and imported into Adobe Photoshop. The fluorescence intensity of cells was quantified by FACS Calibur flow cytometer (Becton-Dickinson, USA), and the data were analysed using Cell Quest program from Becton-Dickinson. Detection and quantification of AVOs To detect the development of acidic vesicular organelles (AVOs), A549 cells were treated with desired concentration of NMK-TD-100 for required time point. Then, the treated cells were stained with acridine orange (1 μg/mL) for

Tumor Biol.

15 min at room temperature [36]. The acidic autophagic vacuoles fluoresced bright red as visualised under a fluorescence microscope (Olympus, BX40F4, Japan). To quantify the development of AVOs, the fluorescence intensity of cells was quantified by FACS Calibur flow cytometer (BectonDickinson, USA), and the data were analysed using Cell Quest program from Becton-Dickinson. Western blot analysis Cultured A549 cells (3 × 106 cells/mL) were grown in the presence of colchicine and/or 3MA for 48 h. The respective cells were collected and extracted in cold lysis buffer (150 mM NaCl, 1 % NP-40, 20 mM Tris-HCl, 20 μg/mL aprotinin, 20 μg/mL leupeptin, 1 mM orthovanadate, 2 mM PMSF, pH 7.4). The protein content of the extracts was estimated by Bradford method. Total protein (30–50 μg) from each sample was loaded in 10–12 % SDS-PAGE. The proteins were electrophoretically transferred on to polyvinylidene difluoride membrane, blotted with different monoclonal and polyclonal antibodies according to the manufacturer’s mentioned dilution and subsequently with required secondary antibodies. Then, the membranes were exposed to Kodak X-ray films after chemiluminescent treatment [31].

assessed for red and green fluorescence with FACS Calibur flow cytometer (Becton-Dickinson, USA) [31]. Measurement of ROS Colchicine-treated A549 cells were labelled with 10 μM DCFDA for 30 min at 37 °C [37]. DCFDA is nonfluorescent that is oxidised by ROS to a fluorescent substrate DCF [38]. The green fluorescence of DCF was instantly assessed with FACS Calibur flow cytometer (BectonDickinson, USA). Statistical analysis All the experiments were repeated three times unless otherwise stated. All data are presented as mean ± SD and analysed using Student’s t test. The p values of the experimental data were calculated compared to the control data and considered significant if less than 0.05.

Results Lethality of colchicine diminished in A549 cells with lowering of its concentration

Apoptosis assay Colchicine and/or 3MA were added to cultured A549 cells and incubated for 48 h. Approximately 1 × 105 cells were then stained for 15 min at room temperature in the dark with fluorescein isothiocyanate (FITC)-conjugated annexin V (1 μg/ mL) and propidium iodide (PI) (0.5 μg/mL) in a Ca2+enriched binding buffer and analysed by a two colour flow cytometric assay. Annexin V and PI emissions were detected in the FL1 and FL2 channels of a FACS Calibur flow cytometer (Becton-Dickinson, USA), respectively. Flow cytometer data shows three distinct populations of cells. The normal healthy cells, early apoptosis, late apoptosis, and necrotic populations were represented by annexin V-negative/ PI-negative population, annexin V-positive/PI-negative, annexin V-positive/PI-positive and annexin-negative/PI-positive cells, respectively [31]. The data were analysed using Cell Quest program from Becton-Dickinson. Assessment of mitochondrial membrane potential To measure the mitochondrial membrane potential (ΔΨm), 5, 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), a sensitive fluorescent probe for ΔΨm was used. The A549 cells were treated with colchicine and/or 3MA for 48 h. Cells were then rinsed with PBS twice, stained with 5 μM JC-1 for 30 min at 37 °C. Cells were rinsed with PBS twice, resuspended in 1 mL PBS and instantly

Colchicine inhibited the proliferation of A549 cells in a concentration-dependent manner, and the IC50 value was 50 nM for 48-h treatment as revealed by MTT assay (Fig. 1a). However, at low concentration of 2.5 nM, colchicine was merely toxic and the viability of cancer cells remained almost similar to that of control cells not treated with colchicine. Although upon further incubation up to 96 h, around 17 % loss of cell viability was observed in the presence of 2.5 nM colchicine (Fig. 1b). Such behaviour of colchicine at low concentration even resonated in its effect on cell cycle phases of A549 cells (Fig. 1c). A549 cells treated with 2.5 nM had a normal distribution in cell cycle phases as that of control cells. In contrast, cells treated with 50 nM colchicine show prominent arrest at G2/M phase. However, there were certain structural changes in the interphase microtubule of A549 cells in the presence of 2.5 nM colchicine. As illustrated in Fig. 1e, the microtubule cytoskeleton of A549 cells treated with 2.5 nM colchicine was modified at the perinuclear region and at the edges in contrast to that of the control cells without colchicine (Fig. 1d). As expected, the microtubules in those cells treated with 50 nM colchicine were completely disrupted, and the cells gained a spherical structure typical of microtubule disassembly (Fig. 1f). Microtubules are known to control the characteristic length of a cell [39]. Therefore, the length of A549 cells in the absence and presence of colchicine was calculated from the confocal microscopic images of the cells. The length of A549

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Fig. 1 Colchicine at low concentration was merely cytotoxic and did not cause G2/M arrest in A549 cells. a Cultured A549 cells were treated with varying concentrations of colchicine (0–100 nM) for 48 h. Cell viability was assessed by MTT assay and is expressed as a percentage of control. Data are represented as the mean ± SD. [*p < 0.05 vs. control, where n = 4]. b Cultured A549 cells were treated with 2.5 nM colchicine for 0–96 h. Cell viability was assessed by MTT assay and is expressed as a percentage of control. Data are represented as the mean ± SD. [*p < 0.05

vs. control, where n = 4]. c Effect of colchicine on cell cycle progression of A549 cells, treated with ligand for 48 h. The abbreviations M1, M2, M3 and M4 written on figure represent Sub G0/G1, G0/G1, S and G2/M phases of the cell cycle in A549 cells, respectively. Data are representative of three identical experiments. d–f Effect of colchicine on interphase microtubule of A549 cells. Cells were incubated with various concentration of colchicine (0–50 nM) for 48 h, fixed and stained using antibody against α-tubulin (green) and nucleus with DAPI (blue)

cell in the absence of colchicine was 72 ± 8.49 μm, whereas in the presence of 2.5 and 50 nM colchicine, the lengths were 55.13 ± 5.94 and 23 ± 3.79 μm, respectively. In order to confirm the effect of low concentrations of colchicine on microtubule of A549 cells, reformation of cold depolymerised interphase and spindle microtubules were studied in the absence and presence of colchicine. The results showed that colchicine inhibited the reassembly of interphase (Fig. S1) and spindle microtubules in A549 cells (Fig. S2). In order to show the effect of colchicine on soluble and insoluble fraction of tubulin in A549 cells, western blot was performed in order to visualise the change in soluble tubulin fraction and polymerised microtubule mass with respect to total tubulin in A549 cells after treatment with colchicine (figure S3a). After 48 h of treatment with 2.5 nM colchicine, there was a minor decrease in insoluble polymerised microtubule mass with slight increase in soluble fraction in A549 cells. However, the insoluble polymerised microtubule mass

underwent a major decrease with concomitant increase in soluble fraction in A549 cells treated with 50 nM colchicine. The total tubulin mass as detected by using anti-α-tubulin antibody in both untreated and treated cells remains unchanged. In order to show the effect of colchicine on the reassembly of cold depolymerised tubulin, western blot was performed. After incubation of the cells in cold condition, the soluble tubulin level increased which again returned to level as that of control after incubation in warm media. However, in the presence of 2.5 nM colchicine, the soluble tubulin level failed to return to the level as that of control, indicating inhibition in reassembly of cold depolymerised tubulin (Fig. S3b). Therefore, colchicine, at 2.5 nM, does not show its usual cytotoxicity or G2/M blocking activity in A549 cells at 48 h. However, the effect of 2.5 nM colchicine on both interphase and spindle microtubule, although subtle, was prominent in A549 cells.

Tumor Biol. Fig. 2 Colchicine induced senescence in A549 cells (a–d) The time-dependent effect of 2.5 nM colchicine on cellular senescence was observed in A549 cells by SA-β-gal staining. e Quantification of the percentage of senescent cells in A549 cells was shown. [*p < 0.05 vs. control, where n = 3]. f Western blot was performed to show the expression of p53 and p21 from A549 cell extract

Fig. 3 Colchicine induced autophagy in A549 cells. A549 cells, treated with colchicine (0–10 nM), were stained with MDC. Images were captured by fluorescence microscope (a–f). Fluorescence particles in the cytoplasm indicate autophagic vacuoles. Microphotographs were shown as representative results from three independent experiments.

Magnification ×400. g MDC fluorescence was quantified by flow cytometer. h A549 cells were incubated with colchicine and/or 3MA. The MDC stained autophagosomes were quantified by flow cytometer. Data are represented as the mean ± SD [*p < 0.05 vs. control, and **p < 0.05 vs. 2.5 nM colchicine-treated cells, where n = 3]

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Colchicine induced senescence in A549 cells at clinically admissible concentration Microtubule targeting agents are known to induce senescence in cancer cells. To investigate whether low-dose colchicine treatment induces senescence, SA-β-galactosidase assay was performed in colchicine-treated A549 cells. Upon colchicine treatment for various time periods, A549 cells were senescent, based on staining for beta-galactosidase, cell expansion, and flattening (Fig. 2a–d). The senescence was also quantified by counting the cells with blue-green colour (Fig. 2e). Both p53 and its downstream effector p21 are known to be involved in induction of senescence in cells [40]. The expression levels of p21 and its upstream regulator p53 were detected by western blot experiment. Both p53 and p21 increased in a time-dependent manner with low-dose colchicine treatment (Fig. 2f). Colchicine induced autophagy in A549 cells at clinically admissible concentration Colchicine has a strong affinity for tubulin which can be inferred from its reported dissociation constant of 0.5 μM and also from its effect on cellular microtubule at low concentration. However, 2.5 nM colchicine had no effect on the viability of A549 cells. Rather, it induced senescence in the cancer cells. A number of studies have suggested that autophagy and senescence induced simultaneously in response to various modes of stress are interlinked. However, the relationship between autophagy and senescence in cells exposed to low-dose

Fig. 4 Colchicine caused formation of AVOs and change in expression of autophagic markers proteins. a A549 cells were treated with colchicine (0–10 nM) for 48 h, and acridine orangestained AVOs were analysed by flow cytometer. b Western blot was performed with cell lysate of A549 cells treated with colchicine (0–10 nM) for 48 h, using antiLC3 and anti-Beclin1 antibody. c Autophagic flux was analysed from cell lysate of A549 cells treated with 2.5 nM colchicine in the absence and presence of 10 μM CQ. A549 cells were cultured in the presence of 0 nM colchicine (lane 1), 10 μM CQ (lane 2), 2.5 nM colchicine (lane 3), and both CQ and colchicine (lane 4) for 48 h, and western blot was performed with LC3 antibody

chemotherapeutic agents is not well-established. Thus, it was next assessed whether colchicine induced autophagy in A549 cells. As per assumption, colchicine at a range of low concentrations induced autophagy in A549 cells, which was evident from the MDC-stained autophagosomes as observed under fluorescence microscope (Fig. 3a–f) and quantified by flow cytometry (Fig. 3g). Formation of acidic vesicular organelles (AVOs) is a marked feature of autophagy, and colchicine-mediated formation of AVOs was monitored by labelling cells with acridine orange (Fig. 4a). As seen in Fig. 4b, colchicine treatment led to the conversion of LC3І to membrane-bound LC3ІІ and an apparent increase in the level of Beclin1 in A549 cells in contrast to the control cells. To investigate whether increase in LC3II expression level was due to induction of autophagy by colchicine or rather due to blockade of autophagic pathway in A549 cells, autophagic flux was analysed by measuring the LC3-II protein levels in cells treated with 2.5 nM colchicine in the absence or presence of 10 μM chloroquine (CQ), a lysomotropic inhibitor that blocks the lysosomeautophagosome fusion and lowers autophagic flux [41]. In the presence of CQ, colchicine-induced increase in LC3-II protein was enhanced substantially (Fig. 4c) which indicated the prevention of LC3-II degradation in autophagosomes by lysosomal hydrolases after fusion with lysosome. Thus, a significant autophagic flux was evident in A549 cells, upon colchicine treatment. There was also a time-dependent increase in autophagosome formation in A549 cells upon 2.5 nM colchicine treatment as evident from fluorescence microscopic

Tumor Biol. Fig. 5 Time-dependent analysis of autophagy induction in colchicine (2.5 nM) treated A549 cells. a–e A549 cells were incubated with 2.5 nM of colchicine for various time points, labelled with MDC and observed under fluorescence microscope. f Western blot analysis of increase in LC3II expression in colchicine treated A549 cells for various time points

images of MDC-labelled autophagosomes (Fig. 5a–e) and western blot image of increase in LC3II protein expression over time (Fig. 5f). Colchicine-mediated autophagy was ROS-dependent ROS is known to play a major role in induction of autophagy, and low concentration of microtubule targeting agents is known to induce ROS generation [42]. To investigate whether ROS was an upstream event of autophagy, it was analysed whether colchicine at low concentration was capable of generating ROS in A549 cells. As illustrated in Fig. 6a, colchicine caused increase in ROS generation at low concentration as analysed by DCF-DA staining of A549 cells. To further study whether colchicine-induced ROS was responsible for autophagy induction, A549 cells were incubated with 2.5 nM colchicine in the absence and presence of 2 mM NAC, and formation of autophagosomal vacuoles was monitored. As seen in Fig. 6b, attenuation of ROS by NAC led to inhibition of autophagosome formation in colchicine-treated A549 cells. Thus, it can be inferred that ROS was an upstream player in autophagy induction in colchicine-treated A549 cells. Inhibition of autophagy by 3-MA attenuated senescence but enhanced apoptosis in colchicine-treated A549 cells As colchicine at 2.5-nM concentration caused depolymerization of microtubule but still is not lethal to cancer cells, it may be hypothesised that colchicine-induced autophagy is a survival

Fig. 6 Colchicine-mediated ROS generation at low concentration was responsible for autophagy induction in A549 cells. a A549 cells were incubated in the absence and presence of colchicine for 48 h. ROS generation was analysed by flow cytometer after staining the cells with DCF-DA. Data are represented as the mean ± SD [*p < 0.05 vs. control, where n = 3]. b A549 cells were incubated with or without colchicine in the presence of NAC. The MDC-stained autophagosomes were quantified by flow cytometer. Data are represented as the mean ± SD [*p < 0.05 vs. control, and **p < 0.05 vs. colchicine-treated cells, where n = 3]

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Fig. 7 Inhibition of colchicine-mediated autophagy by 3-MA-attenuated senescence but augmented apoptosis in A549 cells. a The effect of inhibition of autophagy by 3-MA was observed in colchicine-mediated senescence in A549 cells. b Quantification of the percentage of senescent cells in colchicine-treated A549 cells in the absence and presence of 3MA was shown [*p < 0.05 vs. control, and **p < 0.05 vs. colchicinetreated cells, where n = 3]. c The cytotoxicity assay using MTT was done in A549 cells treated with colchicine (2.5 nM) and/or 3-MA. Data are

represented as the mean ± SD [*p < 0.05 vs. colchicine alone, where n = 4]. d Colchicine induced apoptosis in A549 cells in the presence of 3-MA. A549 cells were incubated with 2.5 nM colchicine in the presence or absence of 3-MA for 48 h. Cells were labelled with Annexin-V FITC and PI, and cell death was analysed by flow cytometry. The percentage of early apoptotic cells in the lower right quadrant (annexin V-FITC positive/PI negative cells), as well as late apoptotic cells located in the upper right quadrant (annexin V-FITC positive/PI positive cells)

strategy for the cancer cells that renders death-defying ability to them and helps in survival of the cancer cells in their senescent form. This hypothesis was investigated by using early-stage autophagy inhibitor 3-MA (5 mM). The SA-β-Gal assay revealed that inhibition of autophagy by 3-MA in colchicine-treated A549 cells led to the attenuation of senescence (Fig. 7a–b). Next, the cell viability results showed that pretreatment with 3-MA caused significant cell death in A549 cells, treated with 2.5 nM colchicine, while 3-MA alone has negligible cytotoxicity (Fig. 7c). To investigate the mode of cell death as a consequence of autophagy inhibition in colchicine-treated A549 cells, apoptosis was measured by biparametric analysis with annexin V/PI. Five millimoles 3-MA or 2.5 nM colchicine alone had negligible proapoptotic effect on A549 cell for the time period tested (48 h). However, significant increase in the annexin V-positive apoptotic cells was detected when colchicine was administered to 3-MA pretreated A549 cells (Fig. 7d). In order to investigate whether apoptosis induced in A549 cells due to colchicine and 3-MA treatment was mediated by the mitochondrial pathway, the status of the mitochondrial membrane potential in A549 cells was studied using JC1, a sensitive fluorescent probe for ΔΨm. As shown in Fig. 8a, colchicine, in the presence of 3-MA, induced a

collapse of mitochondrial membrane potential in A549 cells, illustrated by the decrease in the ratio of red/green fluorescence. However, colchicine or 3-MA alone has no effect on the mitochondrial membrane integrity. Thus, the collapse of mitochondrial membrane potential may be an early event of colchicineinduced apoptosis in 3-MA pre-treated A549 cells. The effect of autophagy inhibition on the mitochondrial membrane potential of colchicine-treated A549 cells intrigued us to study the major protein component of the mitochondrial apoptotic pathway. As shown in Fig. 8b, colchicine treatment, indeed, resulted in the increase in Bax (proapoptotic)/Bcl-2 (antiapoptoic) ratio in the presence of 3-MA. Furthermore, we found that colchicine treatment led to decrease in the amount of procaspase-3 in the presence of 3-MA. Colchicine and 3-MA treatment had no effect on viability of human lung fibroblast cell line One of the major areas of concern in chemotherapy remains the toxicity to the normal tissue. To investigate whether the colchicine and 3-MA treatment had any cytotoxic effect on the normal tissue, human foetal lung fibroblast cell line WI 38 cell

Tumor Biol.

Fig. 8 Colchicine and 3-MA combination was less toxic to WI-38 cells than in A549 lung cancer cells. a Decline in mitochondrial membrane potential was assessed by staining colchicine and/or 3-MA-treated A549 cells for 48 h by JC-1. Red fluorescence emitted from the cells with normal mitochondria (lower quadrant in figure) gradually decreases with concomitant increase in green fluorescence emitted from that containing declined mitochondrial membrane potential (upper quadrant in figure). b Western Blot analysis of change in expression of pro and anti-apoptotic

proteins (bax, bcl2 and procaspase 3) of 48 h colchicine-treated A549 cells in the presence of 3-MA. Probing of α-tubulin was used as a loading control. The results represent the best of data collected from three experiments with similar results. c The cytotoxicity assay in WI-38 cells treated with colchicine (2.5 nM) in the presence of 3-MA was done using MTT reagent. Data are represented as the mean ± SD [*p < 0.05 vs. control, where n = 4]

line was used as model for normal cell. Treatment with colchicine or 3-MA alone had no notable toxicity to the WI38 cells as that in A549 cells (Fig. 8c). The proliferation inhibition was ∼17 % in WI 38 cells when treated with 2.5 nM colchicine in the presence of 5 mM 3-MA for 48 h, whereas it was ∼40 % in A549 cells in similar treatment condition. Thus, the cytotoxicity of 2.5 nM colchicine along with 5 mM 3-MA in WI38 was much lower than that in A549 cells.

thrown insight into its possibility of application as anti-cancer agent in a controlled way of dose administration. Colchicine has been long used in treatment of gout and FMF, and its pharmacokinetics is well-studied. The peak plasma concentrations after oral administration of 0.6 to 1 mg colchicine range from around 2 to 6 ng/mL [43–45]. The lowest reported lethal doses of oral colchicine are 7–26 mg, and acute ingestions of colchicine exceeding 0.5 mg/kg have a high fatality rate [2]. Colchicine has been reported to show anticancer role in low concentration in hepatocellular carcinoma [46]. Present study thus showed that colchicine at clinically admissible concentration can be an effective therapeutic strategy in lung cancer cells when pharmacological inhibition of reactive oxygen species-mediated autophagy changes the fate of cancer cells from senescence to apoptosis. The reason for choosing the concentration of 2.5 nM is due to the inclusion of this concentration in the clinically acceptable range [46]. Although, colchicine at 2.5-nM concentration did not show notable cytotoxicity, its effect on both interphase and spindle microtubules of A549 cells, though subtle, was prominent. It affected the interphase microtubule of A549 cells resulting in some degree of depolymerization of microtubule of those cells. More importantly,

Discussion One of the most well-defined characteristic of cancer cells is significantly increased rate of mitosis. This feature of the cancer cells makes them more vulnerable to the microtubule targeting agents, as microtubules constitute the essential element of mitosis and form the dynamic spindle apparatus. Colchicine is a wellknown microtubule depolymerizing agent, which possesses strong binding affinity for tubulin, resulting in perturbation of both interphase and spindle microtubule. However, toxicity to normal tissue limits its use as anticancer agent. Recent studies have

Tumor Biol.

colchicine at this low concentration inhibited the reformation of both cold depolymerised interphase and spindle microtubules. The notable observation of inability to cause G2/M arrest, but the ability to hinder reassembly of cold depolymerised spindle microtubule might be due to the capacity of colchicine to modulate the microtubule structure even at this low concentration due to its high affinity for tubulin. The dissociation constant for colchicine interaction with tubulin is reported to be 0.5 μM which indicates very robust binding with tubulin [47]. Thus, modulation of microtubule structure by colchicine at 2.5 nM might be the potential reason for induction of stress-induced senescence phenotype in cancer cells. Microtubule-targeting agents are also known to induce ROS generation at low concentration [48]. Thus, ROS-mediated autophagy here acted as the mean for cell survival in senescent form under the stress inflicted by colchicine on A549 cells. This senescence state of A549 cells probably hindered the apoptosis which generally follows after microtubule depolymerization. The above fact was demonstrated by the fact that pharmacological hindrance of autophagy pathway by 3-MA resulted in attenuation of senescence and induction of apoptosis in colchicine-treated A549 cells. Thus, in spite of the effect of low concentration colchicine on microtubule, autophagy here acted as saviour, rendering death-defying characteristic to the cancer cells. The senescent A549 cancer cells, upon inhibition of colchicinemediated autophagy by 3-MA followed mitochondrial pathwaymediated apoptosis. Thus, in view of betterment of low-dose chemotherapy, clinically admissible dose of colchicine along with autophagy inhibitor has a potential anticancer property which needs further investigation for better combination therapy.

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14. Acknowledgments The work was supported by grants from DST, Govt. of India (No. SR/SO/BB-14/2008) and DBT, Government of India (No. BT/ PR12889/AGR/36/624/2009) to GC. Confocal Microscope facility is from University-DBT-IPLS programme, Government of India (No. BT/ PR14552/INF/22/123/2010), and FACS instrument facility is developed by grants from National Common Minimum Project, Government of India. SB was a senior research fellow of University Grant Commission. SD is a Senior Research Fellow of CSIR, Government of India. AG was a fellow of DBT-CU-IPLS programmne, Government of India (No. BT/ PR14552/INF/22/123/2010). Compliance with ethical standards

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Conflicts of interest None 19.

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