Microtubule disrupting agent‐mediated inhibition of cancer cell growth is associated with blockade of autophagic flux and simultaneous induction of apoptosis

Abstract Objectives Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN‐16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. Materials and methods The effect of TN‐16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti‐tumour efficacy of TN‐16 was investigated in syngeneic mouse model of breast cancer. Results TN‐16 inhibited cancer cell proliferation by impairing late‐stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy‐specific substrate p62 and lack of additional LC3‐II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome‐lysosome fusion. Further studies revealed that TN‐16–mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN‐16–mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. Conclusions Our data signify that TN‐16 is a potent autophagy flux inhibitor and might be suitable for (pre‐) clinical use as standard inhibitor of autophagy with anti‐cancer activity.


| INTRODUC TI ON
Autophagy is an evolutionarily conserved process of salvaging cellular constituents. It serves as an alternative mechanism of energy production during metabolic stress. Autophagy is a genetically regulated homeostatic control mechanism for maintaining quality of intracellular protein and organelle. The relevance of autophagy in pathophysiology of human diseases is becoming increasingly evident in recent years. Defects in autophagy have been linked with various diseases such as Crohn's disease, 1 Paget disease of bone, 2 asthma, 3 cancer 4 and several other neurodegenerative, 5 cardiovascular 6 and autoimmune disorders. 7 Many of these diseases have been reproduced in animal models through systemic or tissue-specific alterations of autophagy regulatory genes. 8 With the growing evidences on fundamental roles of autophagy in pathogenesis of human diseases, continuous efforts are being made in therapeutic targeting of this cellular process for various human health problems. The rationale of inhibiting autophagy in cancer therapy emanated from the observations of frequently enhanced level of autophagy in cancer cells with oncogenic mutations. 9 In this endeavour, pharmacological inhibition of autophagy had shown some success in combination with other standard therapies to enhance chemosensitivity of solid tumours. 10 Direct inhibition of autophagy, by chemical inhibitors, is usually achieved by targeting either at early phase of phagophore formation (eg, 3-MA, LY294002, Wortmannin) or at late phase of autophagolysosomal proteolysis (eg, Bafilomycin A1, CQ, HCQ).
Autophagosomes, formed in cytosol, move along the microtubule track to transport cellular cargo towards lysosomes at perinuclear region, and disruption of tubulin network results in suppression of autophagic process. 11 By targeting this essential step of autophagic process, microtubule destabilizing agents indirectly inhibit autophagy by preventing autophagosome-lysosome fusion. Supported with the known pharmacologic and toxicologic profiles, chloroquine (CQ) and hydroxychloroquine (HCQ) have been commonly used in various clinical trials to inhibit autophagy for management of multiple malignancies. However, prolonged use of CQ is associated with various side effects among which retinal toxicity is the most important complication in patients. 12 On the other hand, relatively safer HCQ has been linked with various potential drawbacks such as higher medication dose to achieve sufficient autophagy inhibition, prolong retention in the system after completion of therapy and various other side effects including retinopathy and indigestion. 13 Thus, there is obvious room for other potent autophagy inhibitors, with better efficacy, for clinical use.

| Cell culture
The mammary carcinoma cell lines of human (MCF-7 and MDA-MB-231) and mouse (4T1) origin were obtained from American

| Cell proliferation assay
Proliferation of cells, before and after incubation with TN-16, was determined by sulforhodamine B (SRB) assay as previously described. 18 Briefly, cells were seeded in 96-well plates at a density of 10 4 cells/well and grown overnight in CO 2 incubator. The following day, TN-16 was added at various concentrations and cells were incubated further for 48 hours. Viable cells were then fixed with 50% trichloroacetic acid (TCA) and stained with SRB for 30 minutes.
Thereafter, excess dye was collected, and after brief washing, protein bound dye was dissolved in 10 mmol/L Tris base solution for colorimetric measurement at 510 nm.

| Cell cycle analysis
Effect of TN-16 on cell cycle distribution was determined by flowcytometric analysis, as described earlier. 19 Cells were grown overnight in 6-well culture plate and treated with TN-16 at different concentrations for 24 hours. The cells were fixed with ice-cold 70% ethanol (v/v), stained with propidium iodide (PI) solution containing TritonX100 (5%) and RNase (1 mg/mL) for 30 minutes at 37°C in dark and analysed on FACSCalibur™ flow cytometer (BD Biosciences) with inbuilt software.

| Immunoblot analysis
Protein extracts from cells, treated with vehicle or TN-16, were prepared by lysing on ice in a lysis buffer consisting 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X100, and 0.5% Tween-20 and supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich). T-PER reagent (Thermo) was used for lysis of tumour tissue samples. The protein concentrations in cell and tissue samples were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo).
Equal amount of proteins were electrophoresed on SDS-PAGE and transferred onto PVDF membranes. Membranes were then blocked with 5% non-fat milk in 0.1% Tween-20 in TBS for 1 hour and subsequently incubated with respective primary antibodies as indicated in the figures. Immune complexes were visualized in a Chemidoc XRS + Imaging system (Bio-Rad) after incubation with appropriate secondary antibodies (horseradish peroxidase conjugated) and subsequent addition of enhanced chemiluminescence (ECL) solution.

| Immunocytochemistry
Cells, after growing overnight on coverslips to required confluence, were treated with TN-16 for indicated time periods and fixed with 4% paraformaldehyde. Cells were subsequently permeabilized  Chemically synthesized shRNA sequences targeting Atg7, 20,22 Bak 23 and scrambled control (http://www.holli ngsca ncerc enter.

| Plasmids and RNA interference
org/resea rch/shared-resou rces/shRNA/ Vecto rs.pdf) were procured from Integrated DNA Technologies, Inc. The sequences were annealed and sub-cloned into the AgeI and EcoRI sites of the pLKO.1 puro vector. Standard protocol was followed for generation of lentivirus and transduction of cells.
Cells, transduced with retro/lentiviruses encoding desired genes/shRNAs, were cultured in growth media as mentioned above.
Puromycin was added into the media after 48 hours of transduction. After maintaining the cells for ~10 days in puromycin-containing media, viable stable clones were propagated and used for experiments.

| Scanning electron microscopy
The cells were cultured on glass coverslips in a 6-well culture plate before incubation with vehicle or TN 16. Cells were then fixed overnight with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at 4°C and thereafter subjected to osmication. Post-washing, the samples were dehydrated using ascending grades of ethanol followed by critical point drying (CPD). The samples were then sputter coated with ~15 nm thickness of Au:Pd with a Polaron E5000 sputter coater and imaged at 20 kV in FEI Quanta 250 scanning electron microscope with SE detector. About 200 cells from two stubs for each sample were analysed.

| Transmission electron microscopy (TEM)
The study was performed as described previously. 18,24 Vehicle control or TN16-treated cells were fixed overnight at 4°C using 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) followed by osmication and encapsulation in agarose by centrifugation. Approximately 1 mm 3 pieces of pellet were cut and subjected to dehydration in ascending series of ethanol and acetone, before finally being embedded into Spurr resin. Sixty to eighty nanometre thick sections were obtained Leica EM UC7 microtome which were collected over 200 mesh copper grids. The sections were double-stained with uranyl acetate and lead citrate and air-dried before viewing under JEOL JEM 1400 transmission electron microscope at 80kV using Gatan Orius SC200B CCD camera.

| LysoTracker staining
For fluorescence microscopic analysis, MCF-7 cells were grown overnight on coverslips in 6-well plates at 37°C and exposed to indi-

| In vivo experiments
Orthotopic 4T1 syngenic tumour model was used for this study.

| Statistical analysis
Data are represented as mean value ± standard error (SE) of at least 3 independent experiments. Statistical analyses were performed by GraphPad Prism or Microsoft Excel using Student's t test. A P < .05 or less was considered as statistically significant.

| TN-16 inhibits cell proliferation by promoting G2/M arrest and induction of apoptosis
In our study, we first validated the microtubule destabilizing activ- F I G U R E 1 TN-16 disrupts microtubular dynamics. MDA-MB-231 and MCF-7 cells were treated with vehicle (DMSO) or TN-16 (1.5 µmol/L) for 24 h. Cells were then probed with Alexa Fluor 488 phalloidin (for staining of actin filaments) and anti-βtubulin antibody followed by Alexa Fluor 594-conjugated secondary antibody (for staining of microtubules). Images were captured using confocal microscope at 63× magnification. Colchicine (1 µmol/L for 24 h) was used as positive control

| TN-16 triggers intracellular autophagosomes formation
Given the essential role of microtubules in regulation of autophagosome dynamics, 26 most if not all tubulin inhibitors modulate autophagic machinery. Since TN-16 was originally developed as microtubule destabilizing agent, here we investigated alterations of a microtubule-associated protein light chain 3 (LC3), which is an established biomarker for detection of autophagy, by biochemical and morphological assays. Lipidation of LC3, by conjugating with phosphatidylethanolamine during autophagy, allows it to be redistributed and associated with autophagosomes which gives a typical puncta appearance in fluorescence microscopy. In our study, we immunostained LC3 and monitored its redistribution from cytosol to autophagosomes by confocal microscopy. As can be seen in Figure 3A  In autophagy, p62 acts as an adaptor molecule for recruitment of ubiquitylated substrates into nascent autophagosomes through tethering with the membrane-bound LC3-II and subsequently degraded in autophagolysosomes together with cargo. 27,28 Therefore, reduction of p62 level is widely used as an indicator for autophagic flux. In our study, we assessed p62 expression in breast cancer cells before and after incubation with TN-16 to further confirm its effect on autophagic process. As shown in Figure 5A is probably due to enhanced lysosomal volume, because of microtubule destabilization, instead of induction of autophagolysosmes. 29

| TN-16 inhibits in vivo growth of orthotopic mouse model of breast cancer
In the present study, 4T1 cells were implanted into the mammary fat pad of nude mice to induce orthotropic model of breast cancer.
By day 9, a palpable mass of tumour was developed measuring approximately 100 mm 3 volume. The mice were then treated at every alternate day with either TN-16 (@ 1 mg/kg B.W) or vehicle (5% PEG400 + 5% Tritonx100 in PBS) through intraperitoneal injection for 11 days. As shown in the Figure 7A-C, TN-16 treatment led to the significant reduction in tumour growth in comparison with the control. Likewise, average weight of the harvested tumours in treatment group was much less compared with the control ( Figure 7D).
Finally, we analysed tumour tissue-derived protein lysates by immunoblotting to determine expression level of various apoptosis and autophagy markers. As can be seen in Figure 7E  that their anti-cancer efficacy may be partially attributable to their ability to modulate autophagy. In general, small molecules that destabilize microtubules disrupt autophagic flux and thereby cause accumulation of toxic protein aggregates and/or damaged organelles which in turn promote cancer cell death. 33 In this study, we investigated efficacy of TN-16, a colchicine site binding agent, for anti-cancer activity and its underlying mechanisms. We found that TN-16 induces apoptosis in human cancer cell lines which is in agreement with earlier observations. 34 We also demonstrated TN-16-mediated inhibition of in vivo tumour growth and blockade of late-stage autophagy which further facilitates its pro-apoptotic activity.

| D ISCUSS I ON
As mentioned above, dynamics of autophagosome formation as well as its ensuing maturation essentially rely on the integrity of cytoskeletal network and especially on microtubule assemblage. 35   was synthesized as tubulin targeting agent which interferes microtubule assembly through reversible binding to the colchicine-sensitive site of tubulin. 14 Thus, it is quite likely that TN-16, by virtue of its ability to destabilize tubulin network, may modulate autophagy. Keeping Summing up, this study demonstrates that TN-16 inhibits in vitro and in vivo cancer cell growth through blockade of autophagic flux with simultaneous induction of apoptosis. As a MT F I G U R E 7 TN-16 inhibits murine breast tumour growth in vivo. The 4T1 mouse mammary tumour cells were inoculated into the mammary fat pad of female nude mice. Once the tumour reached approximately 100 mm 3 in size, the animals were treated either with vehicle or TN-16 (1 mg/kg body weight) by i.p. daily for 11 days. A, Tumour volumes were calculated by measuring length and width on alternate days. Data, comparing vehicle versus TN-16-treated group, were analysed by two tail t test for each denoted day after treatment. *P < .05 compared with control group. Gross appearance of tumour bearing mice (B) and harvested tumours (C) at the end of the study. D, Average weight of tumours from mice in each group. E, Western blot analysis of tumour lysates to determine expression of indicated proteins destabilizing agent, TN-16 prevented trafficking of autophagosomes along the tubulin track and thereby inhibited autophagosome-lysosome fusion at later stage of autophagic process. We also found that TN-16-induced cancer cell death is mediated in part by activation of apoptosis and blockade of autophagic flux facilitated its pro-apoptotic activity. Taken together, this study concludes that TN-16 can be a potential autophagy inhibitor which can be used alone or in combination with standard therapeutic agents to induce cancer cell death.

ACK N OWLED G EM ENTS
We

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.