Role of Bim and p53 in transcription- independent apoptosis induced by Combretastatin A-4 in human non-small lung cancer cells

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(1)Facoltà di Scienze Matematiche, Fisiche e Naturali Dipartimento di Biologia Scuola Dottorale in Biologia Sezione di Biologia Applicata alla Salute dell’Uomo XXV Ciclo. "Role of p53 and Bim in transcription-independent apoptosis induced by Combretastatin A-4 in human non-small lung cancer cells". "Ruolo di p53 e Bim nell'apoptosi trascrizione-indipendente indotta da Combretastatina A-4 in cellule umane di carcinoma del polmone". _____________ Ph.D student Dr. GM. Mendez Callejas. _____________ Supervisor Dr. A. Antoccia. ____________________ PhD School Director: Prof. Paolo Ascenzi.

(2) INDEX. SUMMARY English. 4. Italiano. 7. INTRODUCTION. 10. 1. Microtubules as target for anticancer drugs. 10. 1.1 Microtubules Damaging Agents. 10. 1.2 Combretastatin A-4. 11. 2. MDAs induce cell cycle arrest and apoptosis. 13. 2.1 Extrinsec pathway. 14. 2.2 Intrinsec pathway. 15. 2.2.1 Transcription dependent pathway. 16. 2.2.2 Transcription independent pathway. 16. 3. The Bcl-2 family members and its regulation by MDAs. 18. 4. The BH3-only protein Bim. 20. 5. P53 status. 21. 6. Mitocondrial Inhibition of p53 functions. 23. RATIONALE OF THE PROJECT. 24. OBJECTIVES. 25.

(3) RESULTS 1. Microtubules depolymerisation, mitotic arrest and apoptosis in H1299, wt-p53 H460 cells and PFTµ-pre-treated H460 cells. 2. Cytochrome c release from mitochondria and changes in Mitochondrial Membrane Potential ( m) induced by CA-4 in H1299, wt-p53 H460 cells and PFTµ-pre-treated H460 cells 3. Role of CA-4 treatment on p53 and Bim expression and relocalization in wt-p53 H460 cells, p53-null H1299, and PFTµpre-treated H460 cells 4. Inhibition of Bim and caspase-3 activation in wt-p53 H460 cells 5. Interaction of mitochondrial p53 protein with the antiapoptotic proteins Bcl-2 and Bcl-XL and with the pro-apoptotic proteins Bim and Bax after CA-4 treatment in wt-p53 H460 cells 6. Interaction of cytosolic p53 protein with the pro-apoptotic protein Bim after CA-4 treatment in wt-p53 H460 cells and PFTµ-pre-treated H460 cells. 7, Interaction of mitochondrial pro-apoptotic proteins Bim and Bax with antiapoptotic proteins under CA-4 treatment in wtp53 H460, H1299 and PFTµ-pre-treated H460 cells. 26. 26. 29. 31 37. 40. 43. 44. DISCUSSION. 48. CONCLUSIONS. 53. REFERENCES. 54. SUPPLEMENTARY MATERIAL. 63 63 71. Material and methods Publications.

(4) Summary. Microtubules play an important role in many cellular processes including cell division, motility, intracellular trafficking and cell shape maintenance, therefore, these are an interesting target for cancer treatments. Molecules that suppress microtubule dynamics and functions are called Microtubule Damaging Agents (MDAs). Some MDAs are very remarkable because they are not being recognized by multidrug-resistance pumps, and therefore particularly appealing in cancer treatments. In this group, the Combretastatin A-4 (CA-4) is one of the most effective MDAs of natural origin used in chemotherapy. This molecule, has been demonstrated to be able to arrest cells in mitosis, causing successively apoptosis in several cellular types. To committing tumor cell to apoptosis is a pursued strategy in chemotherapy. In this respect, molecules taking part in the regulation of apoptosis may represent clinically relevant targets for chemical intervention The mechanisms upstream of mitochondria membrane permeability, by which regulatory proteins as p53 and Bim are involved in the CA-4-induced apoptosis in non-small lung cancer cells (NSCLC), remains unresolved, as is, more specifically, for the involvement of these proteins in the activation of the mitochondrial pathway by a transcription-independent mechanism. P53 is functionally impaired in a high percentage of human tumors, indeed its activation or reactivation in tumor cells has been recognized as a promising strategy for cancer treatment. As p53, Bim, a member of Bcl-2 (B-cell leukaemia-2) family, may represent a promising target for anticancer drugs however it is unclear how is involved in the apoptotic process induced by MDAs. Based on the observation that in wild-type p53 NSCLC, p53 and Bim are sequestered on the microtubule-array, it was investigated upon depolymerisation, whether these proteins could be involved in the commitment of apoptosis, and to investigate whether p53 status can affect the CA-4 mechanism of action in NSCLC. With this purpose p53-null H1299 and wt-p53 H460 NSCLC cells, in the absence and presence of phitirin-µ (PFTµ), an inhibitor of p53 mitochondrial-translocation, were treated with 7.5 nM CA-4 for 8, 24 and 48 h, and cellular endpoints related.

(5) to cell cycle arrest, apoptosis, protein levels, relocalization and proteinprotein interactions were analyzed. The effect of CA-4 on the microtubular network was not so marked in p53null cells H1299 whereas extensive microtubular disorganization was observed in wt-p53 H460 cells. It was found that CA-4 failed to activate an apoptotic response in H1299 cells, contrastingly to wt-p53 H460 cells, indicating an involvement of p53 in cell death as induced by CA-4. Upon treatment, cells were arrest at the G2/M-phase, and died through a caspase3-dependent mitotic catastrophe, but transient knock down of Bim in wtp53 H460 cells drastically reduced apoptosis, indicating that as p53, Bim is involved in cell death induced by microtubules disorganisation. Cytochrome c was released from mitochondrial inter membrane space in a timedependent manner, independent of p53 status and not always accompanied by the decrease in mitochondrial membrane potential ( m) In response to increasing conditions of microtubules depolymerisation, the protein levels of p53 and Bim were strongly upregulated in a timedependent manner in wt-p53 H460 cells. CA-4 led to p53 and Bim cellular relocalization; in particular, these proteins were released from the microtubular network and accumulated at mitochondria. Bim was translocated to mitochondria, even in a condition of protein synthesis inhibition, however, interestingly, cytosolic and the mitochondrial accumulation of protein Bim was strictly dependent on p53 status. P53 and Bim interacted each other physically at both, cytosolic and mitochondrial level by treatment in wt-p53 H460 cells, and once in the mitochondria, these proteins interacted with other mitochondrial proteins of Bcl-2 family. P53 and Bim showed a markedly increased interaction with Bcl-2 but not with the pro-survival protein Bcl-XL. In turn, the fraction of Bax bound to Bcl-2 decreases in response to treatment, thereby indicating that p53 and Bim possibly promote Bax release from the pro-survival protein Bcl-2 in these cells, although the p53/Bax interaction, though increased, did not reach a significant level. The levels of Bim/Bcl-2 immunocomplexes strongly increased after 24 h of PFTµ-pre-treatment and co-treatment with CA-4 and Bax/Bcl-2 interaction decreased in a timedependent manner, as in wt-p53 H460 cells. On the contrary, CA-4 treatment on H1299 cells was not followed by an increased protein-protein interaction between Bim/Bcl-2, while Bax/Bcl-2 interaction significantly decreased after 48 h treatment..

(6) CA-4 failed to activate an apoptotic response in H1299 cells, contrastingly to what observed in wt-p53 H460 cells, thus indicating an involvement of p53 in cell death as induced by CA-4, moreover, the extent of cell death was not reduced in H460 after combined treatment of CA-4 with PFTµ. Overall, the data support a model of CA-4-induced cells cycle arrest at the G2/Mphase and apoptosis in NSCLC, for which the expression of p53 and Bim proteins are essential, but where the mitochondrial function of p53, linked to its p53-transcription independent mechanism of apoptosis-induction is negligible. Defining the contribution of p53 and Bim in the mechanism of apoptosis may offer some different clues in view of developing new strategies for chemotherapy with CA-4, underlining the relevance of the cytoskeleton integrity in the apoptotic response..

(7) Riassunto. I microtubuli giocano un ruolo importante in molti processi cellulari, inclusi la divisione cellulare, la motilità, il ‘’trafficking’’ e il mantenimento della forma cellulare, inoltre, i microtubuli rappresentano un target interessante nel trattamento farmacologico delle neoplasie. Le molecole che sopprimono le dinamiche dei microtuboli e le loro funzioni sono chiamate agenti che danneggiano i microtuboli (MDA). Alcuni MDA sono molto interessanti perché essi non vengono riconosciuti dalle pompe multi-resistenti alle droghe, cosa che li rende molto promettenti per la chemioterapia. In questo gruppo, la Combretastatina A-4 (CA-4) è una dei più efficaci MDA di origine naturale usata in chemioterapia. Questa molecola ha dimostrato di essere capace di arrestare le cellule nella mitosi, causando successivamente apoptosi in molti tipi cellulari. Coinvolgere le cellule tumorali nell’apoptosi è una strategia richiesta nella chemioterapia. A questo proposito, le molecole partecipanti nella regolazione dell’apoptosi potrebbe rappresentare target clinicamente rilevanti per l’intervento chimico. I meccanismi a monte della permeabilità della membrana mitocondriale, nei quali proteine regolatorie come p53 e Bim sono coinvolte nell’apoptosi indotta da CA-4 in cellule umane di carcinoma del polmone (NSCLC), resta irrisolta, come accade, più specificamente, per il coinvolgimento di queste proteine nell'attivazione della via mitocondriale da un meccanismo di trascrizione indipendente. P53 è funzionalmente alterato in una grande percentuale di tumori umani, inoltre la sua attivazione o riattivazione nelle cellule tumorali è stata riconosciuta come una strategia promettente per il trattamento del cancro. Come p53, Bim, un membro della famiglia di Bcl-2 ( B-cell leukaemia-2), potrebbe rappresentare un target promettente per le droghe anticancerogene, comunque non è chiaro come sia coinvolto nel processo apoptotico indotto dagli MDA. In base all’ osservazione che in cellule p53 wild-type NSCLC, p53 e Bim sono sequestrati dai microtubuli è stato investigato come in seguito alla loro depolimerizzazionequeste proteine potessero essere coinvolte nei meccanismi apoptotici ed il ruolo giocato in queti processi dallo stato di p53..

(8) Con questo scopo, le cellule p53-mutate H1299 e wt-p53 H460 NSCLC, in assenza e presenza di phitirin-µ (PFTµ), un inibitore della traslocazione mitocondriale di p53, sono state trattate con 7.5 nM di CA-4 per 8, 24 e 48 ore, e processi cellulari quali l'arresto del ciclo cellulare, apoptosi, livelli proteici, rilocalizzazione ed interazioni proteina-proteina sono stati analizzati. L'effetto del CA-4 sui microtubuli non era così marcato nelle cellule p53mutate H1299 mentre una marcata disorganizzazione microtubulare è stata osservata nelle cellule wt-p53 H460. E' stato notato che CA-4 non è in grado di attivare una risposta apoptotica nelle cellule H1299, contrariamente a quanto avviene in wt-p53 H460, indicando un coinvolgimento di p53 nella morte cellulare provocata da CA-4. In seguito al trattamento, le cellule si arrestano nella fase G2/M, e muoiono attraverso un processo particolare di apoptosi definito catastrofe mitotica dipendente dalla caspase-3. Il transitorio knock-down di Bim nelle cellule wt-p53 H460, ha drasticamente ridotto l’apoptosi, indicando che come p53, Bim è coinvolto nella morte cellulare provocata dalla disorganizzazione dei microtubuli. In seguito al trattamento si assiste al rilascio di citocromo c dallo spazio inter membrana mitocondriale, in una maniera dipendente dal tempo, ma indipendente dallo status di p53 e non sempre accompagnato da una diminuzione del potenziale membrana mitocondriale. In risposta alla depolimerizzazione dei microtubuli, il livello proteico di p53 e Bim è fortemente aumentato nelle cellule wt-p53 H460. CA-4 induce la rilocalizzazione cellulare di p53 e Bim; in particolare, queste proteine rilasciate dalla rete microtubulare si sono accumulate nel mitocondrio. La rilocalizzazione mitocondriale di Bim avviena anche in condizione di inibizione della sintesi proteica; inoltre è sato osservato che l’accumulo citosolico e mitocondriale della proteina Bim era strettamente dipendente dallo status di p53. P53 e Bim interagiscono tra loro tanto a livello citosolico che mitocondriale in cellile H460 wt-p53,.Inoltre, una volta traslocate nel mitocondrio sia p53 che Bim interagiscono con altre proteine mitocondiali della famiglia Bcl-2. P53 e Bim hanno mostrato una marcata interazione con Bcl-2, ma non con la proteina Bcl-XL. La frazione di Bax legata a Bcl-2 diminuisce in risposta al trattamento, ciò indica che p53 e Bim promuovano il rilascio di Bax dalla proteina Bcl-2. I livelli degli immunocomplessi Bim/Bcl-2 sono.

(9) fortememente incrementati dopo il cotrattamento con PFTµ e CA-4, mentre l’interazione Bax/Bcl-2 è diminuita in una maniera dipendente dal tempo, come nelle cellule wt-p53 H460. Contrariamente, il trattamento di cellule H1299 con CA-4 non determinava un aumento dell’interazione Bim/Bcl-2, mentre l’ interazione Bax/Bcl-2 era significativamente diminuita. CA-4 non era in grado di attivare una risposta apoptotica nelle cellule H1299, contrariamente a quanto osservato per le cellule wt-p53 H460, indicando perciò un coinvolgimento di p53 nella morte cellulare indotta da CA-4., Inoltre, il trattamento di PFTµ non riduceva quantitativamente la morte cellulare indotta da CA-4 in cellule H460. Complessivamente, i dati supportano un modello di arresto del ciclo cellulare, nella fase di G2/M e apoptosi indotta da CA-4 in NSCLC, per cui l’espressione delle proteine p53 e Bim è essenziale, ma dove la funzione mitocondriale di p53, collegata al suo meccanismo di induzione dell’apoptosi indipendente di trascrizione di p53 è trascurabile. Definire il contributo di p53 e Bim nel meccanismo dell’apoptosi potrebbe aprire interessanti scenari per lo sviluppo di strategie chemioterapiche basate sulla CA-4..

(10) INTRODUCTION. 1.. Microtubules as target for anticancer drugs. 1.1 Microtubules Damaging Agents Microtubules are cytoskeletal polymers formed by globular protein subunits of - tubulin and -tubulin that are tightly bound by non-covalent bonds (Fig 1). Those heterodimers play an important role in many cellular processes including cell division, motility, intracellular trafficking of vesicles, organelles and proteins, and cell shape maintenance [1]. Fig 1. Structural organization of microtubules from - and - tubulin subunits. The figure shows the colchicine binding site at - tubulin from Teleomechanist: Memetic Algorithms [2]..

(11) Molecules that suppress microtubule dynamics and functions so-called Microtubule Damaging Agents (MDAs). The MDAs also named “spindle poisons” are often classified into two major groups, the microtubuledestabilizing agents and the microtubule-stabilizing agents, according to their effects at high concentrations on microtubule polymer mass [3, 1] The “microtubule-stabilizing” agents enhance microtubule polymerization at high drug concentrations and include taxol (paclitaxel, Taxol ™), docetaxel (Taxotere), the epothilones, ixabepilone (Ixempra™) and patupilone, discodermolide, eleutherobins, sarcodictyins, cyclostreptin, dictyostatin, laulimalide, rhazinilam, peloruside A, certain steroids and polyisoprenyl benzophenones. Most of the stabilizing agents bind to the same, or an overlapping, taxoid binding site on beta tubulin which is located on the inside surface of the microtubule [3] The “microtubule-destabilizing” agents inhibit microtubule polymerization when they are presents at high concentrations. Most of these agents bind in one of two domains on tubulin, the “vinca” domain and the “colchicine” domain. Vinca site binders include the vinca alkaloids, the cryptophycins, the dolastatins, eribulin, spongistatin, rhizoxin, maytansinoids, and tasidotin [3]. Colchicine-site binders include colchicine and its analogs, podophyllotoxin, 2-methoxyestradiol, phenylahistins (diketopiperazine), steganacins, curacins and combretastatins [3] Combretastatin A4 (CA-4) is one of the most potent among these compounds [4].. 1.2 Combretastatin A-4 The microtubule destabilizing agent, CA-4 was isolated from the South African Willow Combretum caffrum. Structurally, CA-4 is composed by two substituted benzene rings linked by a saturated, hydroxy- substituted two carbon bridge, that interacts with the colchicine binding site of βtubulin and strongly inhibits tubulin polymerization [5, 4, 1] (Fig 2). The IUPAC Name is 2-Methoxy-5-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl] phenol. This compound shows an IC50 of 7nM [1]..

(12) Fig 2. Combretastatin A-4 structure. Because cancer cells need oxygen and nutritional requairments, blocking of blood flow using vascular damage agents (VDAs) is a strategy to stop their growing. The mechanism of action of the VDAs is to prevent angiogenesis by targeting the already established tumor blood vessels of larger, solid tumors with a major effect on the central part of the tumor, causing vessel occlusion and necrosis, having a preventive effect. [6]. CA-4 is one of the most powerful anti-cancer drugs, that causes disruption of the tumour blood flow and subsequent tumour necrosis [4, 7, 8, 9]. Previously, the CA-4 ability to interfere with endothelial cells was demonstrated, and non-cytotoxic concentrations of CA-4 were found to cause rapid disruption of Human Vein Umbelical Endothelial Cells (HUVEC) cultures networks and to inhibit migration through type I collagen [10]. In addition, CA-4 was demonstrated to play an important role in the mechanism of disruption and formation of selective tumour neovessels in colon cancer, with minimal toxicity and without affecting normal stabilized vasculature [11] by that suggesting a vascular mechanism of action. The multidrug resistance (MDR) is the principal mechanism by which many cancers develop resistance to chemotherapy drugs and it has been correlated to the presence of at least two molecular "pumps" in tumor-cell membranes, which avoid the toxic effects of the drug within the cells. The two pumps most common are P-glycoprotein and the so-called multidrug resistance– associated protein (MRP) [12]. In this respect CA-4 has been proven to induce classical apoptosis or mitotic catastrophe in different cells types,.

(13) primary and tumour ones, and has the peculiarity of not being recognized by the MRP [13]. There is evidence showing that CA-4 has a low toxicity [14, 15, 3] and is well tolerated in a high percentage of patients with NSCLC, ovarian, prostate adenocarcinoma, and squamous cell carcinoma of the head and neck [16, 17]. Furthermore, this agent induced apoptosis in human chronic myeloid leukemia cells and in “ex vivo” patient samples including those displaying multidrug resistance [4].. 2.. MDAs induce cell cycle arrest and apoptosis. Targeting the cell-cycle checkpoints, responsible for the control of cellcycle phase progression, is one of the strategies used for tumor treatment. It is known that the cell-cycle checkpoints can regulate the quality and rate of cell division; agents are now under development that either increase or decrease the degree of checkpoint arrest. [18]. In general, in the G1 phase, cells commit to enter the cell-cycle and prepare to duplicate their DNA in S phase. After S phase, cells enter the G2 phase, where repair might occur along with preparation for mitosis in M phase. In the M phase, chromatids and daughter cells are separated [18]. MDAs have been used in cancer chemotherapy for a long time because of their ability to block cell cycle progression at the transition from prometaphase/metaphase to anaphase, and ultimately led to apoptosis [19, 1]. Apoptosis is a form of cell death that permits the removal of damaged, senescent or unwanted cells in multicellular organisms, without damage to the cellular microenvironment [20], and is associated with biochemical and physical changes involving the cytoplasm, the nucleus, and the plasma membrane [21]. The majority of chemotherapeutic agents use the apoptosis pathways to induce cancer cell death by targeting of those proteins acting in the extrinsic/intrinsic pathway that control the apoptosis machinery [20] (Fig 3).

(14) Fig 3. Intrinsec and extrinsec pathways of Apoptosis. 2.1 Extrinsic pathway The extrinsic pathway involves the transmembrane death receptors of the tumor necrosis factor (TNF) receptor gene superfamily that binds to extrinsic ligands and transduces intracellular signals that ultimately result in the destruction of the cell. This pathway does not seem to be activated by MDAs, to the contrary of the intrinsic pathway. [21].

(15) 2.2 Intrinsic pathway The intrinsic pathway is characterized by non-receptors–mediated intracellular signals that lead to the mitochondrial permeabilization and the release of proapoptotic factors into the cytoplasm [22]. Among the pro-apoptotic factors, cytochrome c was the first mitochondrial factor shown to be released from the mitochondrial inter-membrane space and much effort has been directed toward elucidating its mechanism of release. A massive cytochrome-c release has been largely associated with the mitochondrial membrane potential ( m) dissipation [23]. Once in the cytosol, cytochrome c binds to the apoptotic protease-activating factor 1 (Apaf-1) in the presence of ATP. This complex form a heptamer of seven molecules Apaf-1 and seven molecules cytochrome c binds and activates the initiator caspase-9, which in turn ignites the downstream caspase cascade. [22]. Smac/Diablo and Omi/Htra2 are also released from mitochondria and enhance the caspase activity by preventing action of the inhibitor of apoptosis proteins (IAPs) [24]. The caspases are mediators of apoptosis induced by MDAs. Procaspase-9 is the caspase initiator of the apoptosome, which induces the activation of caspase-3, the most important effector of caspases, leading to cleavage of substrates such as the DNA repair enzyme poly(ADP-ribose)polymerase (PARP), DFF45 /inhibitor of caspase-activated DNAse, topoisomerase I, or Cdc6 [25, 26]. Evidences have been gained showing that upon MDA treatment p53 acts upstream of Bax, a proapoptotic member of the Bcl-2 (B-cell leukaemia 2) family, to promote both transcription-dependent and transcriptionindependent apoptosis in several types of human tumor cell lines, including NSCLC. [27, 28, 29]. In both mechanisms of p53-induced apoptosis, the mitochondria play a critical role in the regulation of apoptosis because mitochondrial membrane permeabilization (MMP) permits the release of proapoptotic factors activating appropriate downstream signaling [30, 31]..

(16) 2.2.1 Transcription dependent pathway In cells, p53 associates with microtubules and uses the motor complex dynein/dynactin for nuclear targeting [32]. Once in the nucleus, p53 act as a transcription factor that binds to DNA in a sequence-specific manner to activate transcription of target genes. Some of these that represent potential downstream mediators of p53-dependent apoptosis include: Bax, CD95 (Fas/APO-1), Killer/DR5, Ei24/PIG8, Noxa, PERP, Pidd, p53AIP1, and PUMA [33].. 2.2.2 Transcription independent pathway It has been shown that the impairment of microtubules dynamics alter p53 translocation to the nucleus, thus affecting its function as a transcriptional regulator of pro-apoptotic target genes [34]. Alternatively, p53 can act in the cytosol and mitochondria to promote apoptosis trough a transcriptionindependent mechanism [35] . Such mechanism includes a direct signalling after p53 translocation from the cytosol to mitochondria [27]. Translocation occurs very rapidly in the cells, preceding a series of events, as the interaction with pro- and anti-apoptotic Bcl-2 family proteins, inducing Bax and Bak activation [36, 24, 37, 31]. However, different models of how these interactions promote apoptosis have been proposed; among them: •. After cellular stress, p53 interacts with the proapoptotic mitochondrial membrane protein Bak and the Bak/Mcl-1 complex disruption, leading to oligomerization and activation of Bak [38].. •. P53 mediates mitochondrial permeabilization through direct physical interactions with the pro-apoptotic Bcl-2 family members, Bax and Bak [39].. •. Binding of stress-induced mitochondrial p53 to Bcl-xL and Bcl-2 antagonizes their function of keeping Bax and Bak inactive. Consequently, the formation of p53–Bcl-2 and p53–Bcl-xL complexes leads to oligomerization and activation of Bax and Bak [35].. •. A physical interaction of p53 with the pro-apoptotic Bcl-2 protein Bad was proposed to enhance trafficking of p53 to the cytosol and mitochondria, thus, the accumulation of large amounts of non-nuclear.

(17) p53 can also be the result of specific localization signals, such as posttranslational p53 modifications or the interaction with nuclear export. [35]. •. Bim releasing from sequestrating complexes with Mcl-1, Bcl-2 and Bcl-XL seems to mediate some mechanisms of mitochondrial p53 transcription-independent apoptosis through the formation of new complexes between p53 and pro-survival proteins [40] (Fig 4).. Fig 4. A schematic model for p53-mediated de-repression of Bim. Under quiescent conditions (left panels), cells express low levels of p53; Bim is sequestered in complexes with Mcl-1, Bcl-2 and Bcl-XL; and Bax and Bak are present as unactivated monomers. Upon cellular activation by cytotoxic drugs or radiation (right panels), upregulated p53 generates de novo complexes with Mcl-1, Bcl-2 and Bcl-XL through Bim displacement. In turn, the unsequestered Bim is engaged in Bax/Bak activation.From (Jie Han, et al., 2010) [40].

(18) 3.. The Bcl-2 family members and its regulation by MDAs. Overexpression of Bcl-2-like proteins has been largely shown to suppress apoptosis induced by several factors, including MDAs [21].. Table 1. The Bcl-2 family members Antiapoptotic proteins Bcl-2-like Bcl-2 Bcl-xL Bcl-w Mcl-1 A1/Bfl-I NR-13 Boo/Diva/Bcl-1-L-10 Bcl-B. Bcl-2 Family members Proapoptotic proteins Bax-like BH3-only Bax Bik/Nbk Bak Blk Bok/Mtd Hrk/DP5 Bcl-x BNIP3 BimL/Bod Bad Bid Noxa PUMA/Bbc3 Bmf. From Carré M, and Braguer D, 2008 [21].. The Bcl-2 family members are divided into anti-apoptotic and pro-apoptotic members; the pro-apoptotic proteins are subdivided into “Bax-like” and “BH3-only” members (Tabla 1). Bcl-2 proteins are characterized by the presence of one or more BH3 domains, (Bcl-2 homology domains) (Fig 5) which are required for their survival functions and mediate the interaction of Bcl-2-like proteins with other protein partners. The BH1–3 domains form a hydrophobic groove that constitutes the functional part of the protein. The N-terminal BH4 domain stabilizes this structure, but is not present in all Bcl-2-like members, while the C-terminal domain helps to insert these proteins at the cytoplasmic face of organelles such as the outer mitochondrial membrane [21, 41]..

(19) Fig 5. The Bcl-2 family of proteins and Bcl-2 homology domains. From Anvekar R et al., 2011 [41].. These proteins are recognized as essential initiators/mediators of programmed cell death in different kind of organisms, from worms to humans [42]. Bcl-2-like members maintain mitochondrial integrity in the absence of any apoptotic signal. However when they are activated, the proapoptotic Bcl-2 members relies on their capability to bind and inactivate the pro-survival Bcl-2-like relatives, thus allowing the permeability of the outer mithochondrial membrane by Bax and Bak and consequently a series of events leading to cell death [43, 44]. The anti-apoptotic function of the Bcl-2-like protein family is regulated by phosphorylation of residues in the loop domain. Once hyperphosphorylated during mitosis, Bcl-2-like members become ineffective in preventing apoptosis induced by anticancer drugs. [21]..

(20) 4.. The BH3-only protein Bim. Bim, a member of the BH3-only Bcl-2 subfamily, is a well known apoptosis promoting protein, and the three major isoforms, BimEL, BimL, and BimS [45], have been described in cells of the hematopoietic, neural and epithelial lineage [6]. BimS, the shortest isoform, is only transiently expressed in apoptotic cells [45], whereas BimEL and BimL have been detected in a variety of normal and tumoral cell types [46]. The apoptotic activity of BimEL and BimL is suppressed by the interaction with the light chain 1 (DLC1) LC8 of dynein motor complex, the latter keeping Bim sequestered on the cytoskeleton and therefore, away from Bcl-2 and its homologous [47]. Treatment with doxorubicin, staurosporin, UV light and Gadd45a overexpression causes the release of the BimEL- and BimL-LC8 from microtubules and their translocation to mitochondria membrane [48, 49]. In case of UV-stimulated apoptosis, it has been shown that the release of Bim from dynein motor complexes is mediated by c-Jun N-terminal protein kinase (JNK) phosphorylation of Bim within the conserved DLC binding domain [50, 51, 52]. In addition to post-translational mechanisms like phosphorylation and re-localisation, the regulation of Bim, after apoptotic stimuli, has been reported to occur also at a transcriptional level, as shown for the stress kinase JNK in UV-exposed cells [51, 52] and for the forkheadbox transcription factor FOXO3a (FKHRL1), in breast cancer cells exposed to paclitaxel [53]. In particular, the members of the FoxO family activate gene expression via interaction with a specific DNA sequence, and known targets include among others, the proapoptotic Bim [54]. Bim protein levels increase dramatically after paclitaxel treatment, and gene-silencing experiments show that the transcriptional upregulation of Bim can be a direct cause of apoptosis in cancer cells [53]. In this respect, apoptosis has been shown to occur in a Bim-dependent manner in lymphocytes, in NSCLC expressing high basal level of BimEL [55] as well as in a Bim-independent way in paclitaxel-treated breast cancer cells [56]. Recently the involvement of the pro-apoptotic BH3 protein Bim in the p53pathway responsible for cell death has been investigated. It was proposed that mitochondrial p53 functions as a Bim de-repressor by releasing Bim from sequestrating complexes with Mcl-1, Bcl-2 and Bcl-XL, and allowing its engagement in Bak/Bax activation [40]. In addition, our previous data have revealed that after CA-4-treatment of H460 NSCLC, Bim was strongly upregulated in a time-dependent manner, translocated to mitochondria,.

(21) where it was shown to markedly interact with the pro-survival protein Bcl2, contributing to cell death [57,. The BimEL form is predominant over BimL in H460 as well as in many investigated normal and tumor cell lines established from lung, breast and prostate [45, 55, 56], and for this reason, the data here presented are restricted to this isoform.. 5.. p53 status. The sensitivity to cancer therapies can be affected by several cellular conditions, including the status of the p53 tumor suppressor protein, which may interfere with critical aspects of cancer pharmacology as expression of drug targets, access of drugs to intracellular targets, response to DNA damage [58] and apoptosis induction [27]. The UMD_p53 web database [59] has been updated with new sections describing the p53 status in the majority of cell lines and a special section devoted to cell lines with controversial p53 status. In this database it is possible to find the NSCLC cell lines classified according with the p53 status in: cells wt-p53 as H460; cells with p53 gene deletion or rearrangement as H1299; cells with p53 splice mutation, and around of eighty cell lines with p53 mutations (missense or frameshift) [60] TP53 gene is mutated in more than half of all tumours, compromising the efficiency of the chemotherapy treatments [61, 62] and this is the most frequently mutated tumor supressor gene in human cancers [63]. Several evidences indicate that, p53 mutants can exert oncogenic functions beyond their negative domination over the wild-type p53 tumor suppressor functions, contributing actively to various stages of tumor progression and to increasing resistance to anticancer treatments. These activities are referred to as mutant p53 gain-of-function [64]. In fact, studies on various human cancer cell lines demonstrate that cells with mutant p53 are more resistant to drugs compared with those with wild-type p53 when treated with a wide variety of clinically used chemotherapeutic drugs [63]. NSCLC p53- null cell lines exposed to different drugs as andriamycin, paclitaxel, carboplatin, have shown a markedly drug resistance [62, 65]..

(22) Some studies have revealed that lack of p53 in cancer cells, can be seen as an advantage by selective therapies of p53-deficient cancer cells, protecting wt-p53 at normal proliferation rates [61]. In this respect, apoptosis can be induced in p53-deficient H1299 cells by combination of various antimitotic agents as isoerianin, taxol with nutlin-3 or 8-chloro-adenosine (8-Cl-Ado), that strongly disrupted microtubules, leading to G2/M phase cell-cycle arrest and cell death [66, 61, 67] .. 6.. Mitocondrial Inhibition of p53 functions. Temporary suppression of p53 has been suggested as a therapeutic strategy to prevent toxic side effects of anticancer treatment in tumours with deficient apoptotic functions of p53, to this purpose, different inhibitors have been isolated [68, 69]. Among such inhibitors, pifithrin-µ (PFTµ) has a high specificity for p53 [70] by competing with Bcl-xL for the DNA binding domain at loop 1 and helix 2 (Fig 6) [71] . In particular, PFTµ inserts its aromatic ring into p53-DBD (DNA Binding Domain) and causes a reduced affinity of p53 itself for the Bcl-2 antiapoptotic proteins, which directly inhibit p53 binding to mitochondria [71] .. Fig 6. PFTµ binds to p53 in the DNA binding domain at loop 1 and helix 2 From Franz Hagn, et al 2011 [71].

(23) It has been demonstrated that treatment with the anticancer agent Nutlin-3 combined with PFTµ in wt-p53 cells, reduces apoptotic response, so underlining the significance of p53 mitochondrial program in cell death [72]. Nevertheless, recently have been reported another role of PFTµ. This small molecule, also named PES (2-phenylethylenesulfonamide), interferes with the carboxy-terminal substrate-binding domain of inducible molecular chaperone HSP70. In different cell lines, as SKBR3 and MDA-MB-468 human breast carcinomas, H1299 NSCLC, SKOV3 human ovarian cancer, and FaDu human head and neck cancer, have been demonstrated that PES affects the interaction with Hsp90, Hsp40, BAG and CHIP chaperones and co-chaperones, causing overexpression of Hsp70 and undergo readily cell death through caspase-independent mechanisms that involve increased protein aggregation, impairment of lysosomal functions, and inhibited autophagy [73, 74]. Has been report also that PES led to activation of caspase-3 and reduction of the intracellular concentrations of AKT and ERK1/2 in NALM-6 cells [75]. Thus, PFTµ induced reduction of the cell viability, cell cycle arrest and apoptosis in a dose-dependent fashion in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines. [75]. However no reports were found about cytotoxicity or apoptosis induction in H460 cells..

(24) RATIONALE OF THE PROJECT Since cancer is one of the leading causes of death worldwide, there is a need to find a pursued strategy in chemotherapy to committing tumor cell to apoptosis. In this respect, MDAs and the biochemical events that lead to apoptosis downstream of inhibition of microtubule dynamics are being explored. It is know that the alteration of the microtubule network, the block at mitosis, and the consequent apoptosis, may depend on the cell line and moreover to the differentiation status of the cell [20] Several molecules taking part in the regulation of apoptosis, among them Bim and p53 may represent clinically relevant targets for chemical intervention. However the molecular mechanisms and Bim involvement in the cascade of events leading to apoptotis following microtubule-array interference CA-4 induced, have been poorly investigated as well as the contribution offered by p53 transcription-independent CA-4 inducedapoptosis. In particular, no data are available in the literature on CA-4 apoptosis induction in NSCLC after combined treatment with PFTµ in conditions of impaired p53 translocation to mitochondria..

(25) OBJECTIVES. GENERAL Investigate the relationship between microtubule-array interference Combretastatin A-4-induced and the role of Bim and p53 in the activation the transcription- independent mechanism of apoptosis in non-small lung cancer cells with different p53 status. SPECIFIC •. Analyze the microtubules disruption induced by CA-4 in p53-null H1299, wt-p53 H460 and PFTµ-pre-treated H460 cells in a timedependent manner.. •. Evaluated the events carry out in the apoptosis induced by CA-4 as cell cycle arrest, DNA fragmentation, cytochrome c release from mitochondria and the relationship with changes in mitochondrial membrane potential ( m), and activity of caspase-3 in p53-null H1299, wt-p53 H460 and PFTµ-pre-treated H460 cells. •. Study the role of CA-4 treatment on p53 and Bim expression and relocalization in wt-p53 H460 cells, p53-null H1299, and PFTµ-pretreated H460 cells.. •. Examine the activity of caspase-3 after iInhibition of Bim in wt-p53 H460 cells. •. Analyze the interaction of mitochondrial p53 protein with the antiapoptotic proteins Bcl-2 and Bcl-XL and with the pro-apoptotic proteins Bim and Bax after CA-4 treatment in wt-p53 H460 cells and the interactions of mitochondrial pro-apoptotic proteins Bim and Bax with antiapoptotic proteins under CA-4 treatment in wt-p53 H460, H1299 and PFTµ-pre-treated H460 cells.

(26) RESULTS 1.. Microtubules depolymerisation, mitotic arrest and apoptosis in H1299, wt-p53 H460 cells and PFTµ-pre-treated H460 cells.. Cells were incubated for 8, 24 or 48 h with 7.5nM of CA-4, a dose selected on the basis of our previous papers for the study of the mechanistic effects of the drug [15, 57] In p53-null H1299 cells, the effect of CA-4 on the microtubular network was not so marked at 8 h, whereas extensive microtubular disorganization was observed at 24 and 48 h of treatment. On the contrary, in wt-p53 H460 cells, as well as in PFTµ-pre-treated cells, CA-4 effectively disrupted microtubules starting from 8 h (Figure 1).. Figure 1. CA-4 leads to depolymerization of microtubules in a timedependent manner. Images of H1299, wt-p53 H460 and PFTµ-pre-treated H460 cells were collected through immunofluorescent microscopy at 0, 8, 24 and 48 h after 7.5 µM CA-4 treatment. -Tubulin is shown in green, while the DNA is counterstained with DAPI in blue..

(27) In accordance with immunofluorescence analysis, DNA profiles of the cytofluorimetric assay showed that the drug strongly arrested cells in the G2/M-phase of the cell cycle in the 3 cell lines (Figure 2). The Sub-G1 DNA content of cells, a measure of apoptosis, was analyzed by flow cytometry at 24 and 48 h after CA-4 treatment. Whereas the percentage of apoptotic cells in H1299 cells was pretty low even after prolonged CA-4 treatment, the drug strongly arrested those cells in the G2/M-phase compartment (Figure 2a). On the contrary, the cytofluorimetric analysis showed that after treatment of p53 expressing cells, that is wt-p53 H460 and PFTµ-pre-treated H460 cells, the drug efficiently induced cells to death particularly at 48 h, whereas at 24 h prevailed G2/M-phase cell cycle arrest (Figures 2b and 2c), thus suggesting that attempt to exit from the G2/Mblock in the presence of the drug caused cell death as previously reported [15]..

(28) Figure 2. DNA cytometric analysis of untreated and treated cells with 7.5 µM CA-4 for 24 and 48 h, showing the percentage of cells in Sub-G1 stage and cell cycle distribution in H1299 (a), wt-p53-H460 (b) and PFTµ-pre-treated H460 cells (c)..

(29) 2.. Cytochrome c release from mitochondria and changes in Mitochondrial Membrane Potential ( m) induced by CA-4 in H1299, wt-p53 H460 cells and PFTµ-pre-treated H460 cells. Western blot analysis was used to analyze the translocation of cytochrome c after CA-4 treatment; in this respect, cytochrome c was decreased at the mitochondrial level with a consequent increase at the cytosolic level, irrespectively of the p53 status (Figures 3 a, b). Such a CA-4-induced mitochondrial cytochrome c release occurred without noteworthy changes in Ψm assessed by means of the JC-10 probe in both flow cytometry analysis and immunofluorescent microscopy (Figures 3 d, e) The combination of PFTµ inhibitor and CA-4 agent in H460 cells resulted in the release of cytochrome c from mitochondria, though the expected significant accumulation in the cytosol was not detected (Figure 3c). The inhibition of p53 accumulation at mitochondria, mediated by PFTµ incubation did not avoid completely the loss of Ψm, which was found to occur in a timedependent manner (Figure 3f).

(30) Figure 3. Cytochrome c releasing from mitochondria and accumulation into the cytosol in H1299 (a), wt-p53-H460 (b) and PFTµ-pre-treated H460 cells (c) untreated and treated with 7.5 µM CA-4. Cytofluorometric and immunofluorescent analyses of m changes in H1299 (d), wt-p53-H460 (e) and PFTµ-pre-treated H460 cells (f). Valynomicin was used as positive control. In the right panel is shown the densitometric analysis of cytochrome c. The presence of alpha-tubulin, a cystosolic protein, and Cox IV, a mitochondrial protein, were evaluated as an internal control to verify the purity of the extracts. Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(31) 3.. Role of CA-4 treatment on p53 and Bim expression and relocalization in wt-p53 H460 cells, p53-null H1299, and PFTµ-pretreated H460 cells. By means of co-immunoprecipitation assays and western blot analysis, CA4 was analyzed to know whether it could induce p53 and Bim to be released from microtubules as a response to tubulin depolymerisation. For this purpose, wt-p53 H460 whole cell lysates were prepared after treatment with CA-4 for up to 48 h. Immunofluorescent microscopy analysis (Figure 4a) showed that depolymerization of microtubules by CA-4 treatment was accompanied by induction of the expression of p53 protein and its level increased in a time dependent manner. P53 release from microtubules was confirmed by co-immunoprecipitation (Figure 4b).. Figure 4. CA-4-induced depolymerization of microtubules is accompanied by release of p53 from the microtubules network. Immunofluorescent microscopy (a) and immunoprecipitation (b) analyses. In the right panel is shown the densitometry analysis of p53. -Tubulin is shown in green, p53 in red, while the DNA is counterstained with DAPI in blue. Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(32) Western blot analysis show its accumulation at cytoplasmatic, nuclear, and mitochondrial level in these wt-p53 H460 cells (Figure 5). Figure 5. Relocalization of p53 at cytoplasm, nucleus and mitochondria after disruption of microtubules induced by CA-4 in wt-p53 H460 cells..

(33) In the case of, Bim, this protein interacts in a physical manner with dynein. Such interaction was partly retained for the 8 h treatment, whereas at longer incubation times of 24 and 48 h, the physical interaction between Bim and dynein was markedly impaired (Figure 6a). The potential role of CA-4 treatment on Bim expression at both protein levels and mRNA was investigated. In this respect, the wenstern blot analysis, using an antibody able to recognize BimEL form, clearly showed that Bim was induced by CA-4 treatment in a time-dependent manner in H460 cells (Figure 6b). The data were also confirmed by Real Time analysis which Bim mRNA was induced by CA-4 treatment (Figure 6c). Figure 6. CA-4 induced release of Bim from dynein in wt-p53 H460 cells (a) The level of endogenus Bim was assessed both at protein level (b and mRNA (c). Bim mRNA was supressed in H460 cells transfected with Bim siRNA (+) and also treated with CA-4 for 8, 24 and 48 h. Non specific scrambled (S) were also assesed. Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(34) Once assessed that CA-4 caused Bim release from microtubules, we analyzed its re-localising on mitochondria. Western Blot analysis showed a detectable enrichment of Bim in the mitochondrial fraction of wt-p53 H460 cells treated with 7.5nM CA-4 for 0-48 h (Figure 7a). Interestingly, an increase of Bim was detectable even in the cytosolic extracts, (Figure 7a), indicating that CA-4 promoted a transcriptional up-regulation of Bim, as also suggested by means of RT-PCR experiment. In untreated cells, the amount of Bim on mitochondria was very low in contrast to what observed in T-cells [57]. Bim accumulation on mitochondria as a function of treatment was also confirmed by means of confocal analysis, using the specific mitotracker staining (Figure 7b). A treatment with 50nM of Taxol for 6 h was used as internal control.. Figure 7. Mithocondrial re-localisation of Bim after CA-4 treatment. Western blot analysis show that Bim re-localises on mitochondria (a). Cells were incubated for 1 h at 37 C with 1 mM Mitotracker green Fm, and a Texas Red-conjugated secondary antibody was directed against Bim primary antibody. The merge images obtained by confocal analysis showed that, after 24 h and 48 h CA-4 treatment, Bim localises on mitochondria as.

(35) can be inferred from the orange staining resulting from the merge (b). Significant values according to Student’s t-test, *p<0.05, **p<0.01. Experiments on wt-p53 H460 cells in which the translational activity had been inhibited using CHX, were carried out in order to assess whether the relocalisation of Bim on mitochondria simply reflected the CA-4-increased transcription of Bim rather than CA-4-induced Bim release from microtubules. As internal control, cells were treated with 25U/ml of IFNγ to induce the expression of IRF-1; the protein synthesis was blocked after 24 h of incubation with 10µg/mL of CHX (Figure 8a). Then, cells were treated with 7.5nM CA-4 for 8 and 24 h, and mitochondrial and cytosolic protein fractions were isolated. Western blot analysis showed an increased Bim expression in the mitochondrial but not in the cytosolic fraction as a function of time in response to CA-4 treatment (Figure 8b). The inhibition of the protein synthesis achieved by CHX treatment rule dout the possibility that increased Bim mitochondrial levels were simply due to the transcriptional upregulation induced by CA-4.. Figure 8. Bim mitochondrial relocalisation was evaluated in response to CA-4 in cells with inhibited translational activity. The expression of IRF-1 induced by IFNγ is blocked by CHX (a). Bim level increases at mitochondria after CA4 treatment in cells in which protein synthesis was blocked by CHX (b). Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(36) To investigate whether Bim induction and relocalization was affected by p53 expression or localization in the NSCLC, immunoblotting was performed in H1299 and PFTµ-pre-treated H460 cells using a specific antibody against BimEL. First, we verified the absence of p53 protein in H1299 cells, and then the effect of CA-4 on Bim expression. In this respect, western blot analysis showed that CA-4 determined in H1299 cells a significant reduction in the level of Bim protein in both cytosolic and mitochondrial compartments (Figure 9a). The pre-treatment of H460 cells with PFTµ effectively blocked the p53 translocation into the mitochondrial compartment, whereas the CA-4-induced cytosolic accumulation was not affected (Figure 9b). The co-treatment of CA-4 with PFTµ was accompanied by a slight reduction in the level of cytosolic Bim, while in mitochondrial extracts Bim was accumulated, particularly at 24 h of CA-4 treatment (Figure 9b).. Figure 9. Western blot analysis of cytosol and mitochondrial p53 and/or Bim protein levels after treatment with 7.5 µM CA-4 in H1299 (a), and PFTµ-pre-treated H460 cells (b). In the right panel are shown the relative densitometry analyses. Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(37) 4.. Inhibition of Bim and caspase-3 activation in wt-p53 H460 cells. Based on our previous results that showed the capability of CA-4 to induce apoptosis in H460 cells in a caspase-3 dependent manner [57], we evaluated whether the inhibition of endogenous Bim following CA-4 induction would suppress cell death. In this respect, cells were exposed to Bim siRNA or non-specific siRNA for 24 h and then treated with CA-4 for 0, 8, 24 and 48 h. Cells were collected 72 h post-transfection for preparation of whole cell lysates and for isolation of RNA. The level of cellular mRNA and Bim protein were substantially suppressed after 48 h treatment with Bim siRNA (Figure 10a) in H460 cells treated with CA-4. In contrast, non-specific scrambled siRNA had no significant effect on expression of endogenous Bim. The activation of caspase-3 was evaluated as a readout in an immunoblot assay, after inhibition of endogenous Bim expression by siRNA (Figure 10b) and in “in vivo” time-lapse analysis using the Magic red substrate (Figure 10c). In both assays, the activation of caspase-3 was strongly reduced when CA-4 treatment was performed in the presence of Bim interference, thus indicating an active role played by Bim in CA-4-induced apoptosis. Inhibition of Bim alone did not modify the extent of mitotic arrest obtained by incubating cells in the presence of CA-4 (not shown)..

(38)

(39) Figure 10. wt-p53 H460 cells were transfected with Bim siRNA (+) and non specific scrambled (S) and were also treated with CA-4 for 8, 24 and 48 h. Cells have been collected after transfection for preparation of whole cell lysates and for isolation of RNA. The levels of cellular Bim mRNA (a) and protein (b) were assessed. The cleavage, and the activation of caspase-3, was also assessed in all the samples by Western Blot analysis (b). In order to assess the presence of the caspase-3 in its activated form, time-lapse experiments were performed both on siRNA Bim transfected cells and control cells, by adding the MagicRed™ substrate to cell cultures before CA-4 treatment (c)..

(40) 5.. Interaction of mitochondrial p53 protein with the anti-apoptotic proteins Bcl-2 and Bcl-XL and with the pro-apoptotic proteins Bim and Bax after CA-4 treatment in wt-p53 H460 cells. The pro-apoptotic activity of p53 was evaluated by analysis of interaction of p53 with either pro-survival proteins, such as Bcl-2 and Bcl-XL, or proapoptotic proteins as Bim and Bax in mitochondrial extracts. First was evaluated the anti and pro-apoptotic proteins expression after CA-4 treatment, and we found that drug treatment up to 48 h did not modify the amount of cytosolic and mitochondrial amounts of Bcl-2, Bcl-XL as well as of Bax (Figure11).. Figure 11. Bcl-2, Bcl-XL and Bax protein levels in cytosol and mitochondrial protein extracts after treatment with CA-4..

(41) In the absence of CA-4 treatment, the interaction between p53 and proapoptotic proteins Bim and Bax was low compared with the increase observed after treatment in wt-p53 H460 cells. In particular, the p53/Bim physical interaction was strongly increased at mitochondrial level (Figure 12a), whereas the p53/Bax interaction, though increased, did not reach a significant level (Figure 12b). With respect to the pro-survival partners, the interaction between p53 and pro-survival protein Bcl-XL was not increased significantly by treatment (Figure 12c). On the contrary, p53/Bcl-2 interaction was notably increased up to 24 h (Figure 12d), but the binding was found to be decreased at longer times. As we thought, no interaction was observed between p53 and pro- and anti-apoptotic members of Bcl-2 family in the mitochondrial extracts pre-treated with PFTµ (data not showed).

(42) Figure 12. Co-immunoprecipitation analysis performed on mitochondrial extracts of cells shows the interactions between p53 and pro-apoptotic Bim (a), Bax (b), and anti-apoptotic proteins Bcl-XL (c) and Bcl-2 (d) after CA4 treatment in wt-p53 H460 cells. Relative densitometry analyses (e) Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(43) 6.. Interaction of cytosolic p53 protein with the pro-apoptotic Bim after CA-4 treatment in wt-p53 H460 cells and PFTµ-pre-treated H460 cells. Previously was showed the strong complex formation between p53 and Bim at mitochondrial level (Figure 12a), moreover, it was evaluated the p53/Bim interaction at cytosolic level in H460 cells without and with the inhibitor PFTµ (Figure 13). The cytosolic interaction between p53 and Bim was increased significantly in wt-p53 H460 cells (Figure 13a), however, in presence of the inhibitor PFTµ, cytosolic p53 decreases the binding capacity with Bim (Figure 13b).. Figure 13. Co-immunoprecipitation analysis performed on cytosolic extracts shows the interactions between p53 and the pro-apoptotic Bim in wt-p53 H460 (a), and pre-treated PFTµ H460 cells (b) Relative densitometry analyses (c) Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(44) 7.. Interaction of mitochondrial pro-apoptotic proteins Bim and Bax with antiapoptotic proteins under CA-4 treatment in wt-p53 H460, H1299 and PFTµ-pre-treated H460 cells. We examined the physical association of Bim with prosurvival proteins such as Bcl-2 and Bcl-XL at mitochondrial level. After exposure to CA-4, mitochondrial extracts were prepared and incubated with anti Bcl-2 and Bcl-XL antibodies for co-immunoprecipitation experiments (Figures 14 a and c). The analysis of Bim/Bcl-2 interaction was also extended to the cytosolic protein fraction (Figure 14b). The precipitated complexes were immunoblotted with antibodies directed against BimEL. In absence of treatment, the levels of Bim/Bcl-2 immunocomplexes were rather low; however such physical interaction was strongly increased by treatment at mitochondrial (Figure 12a) as well as at cytoplasmic level (Figure 14b). No augmented interaction levels were detected between Bcl-XL and Bim (Figure 14c). In addition, it was evaluated whether Bim interaction with prosurvival members Bcl-2 would lead in turn to the release of Bax from either Bcl-XL (Figure 14e) or Bcl-2 (Figure 14f) or at mitochondrion. In this respect, we found that CA-4 treatment leads to a reduction of Bax/Bcl-2 interaction in a time-dependent manner and to an increased amount of mitochondrial Bax..

(45) Figure 14. CA-4 treatment leads to an increased interaction between Bim and Bcl-2 as shown by co-immunoprecipitation experiments performed on mitochondria of wt-p53 H460 cells (a) and cytoplasmatic extracts (b). On the contrary, the interaction between Bim and Bcl-XL was not modified by the treatment (c). In response to CA-4 treatment, Bcl-2 releases Bax(e). While Bcl-XL/Bax interaction on mitochondria remains unaltered after drug treatment (f). For immunoprecipitations relative densitometry analyses (d y g) Significant values according to Student’s ttest, *p<0.05, **p<0.01..

(46) To verify whether this process is likely to involve p53 expression and localization and the relationship with apoptosis, the interaction between Bcl-2 and the proapoptotic members Bax and Bim was evaluated in p53null H1299 and PFTµ CA-4-treated H460 cells. Mitochondrial extracts were prepared and incubated with anti Bcl-2 antibody for coimmunoprecipitation analysis and the precipitated complexes were immunoblotted with antibodies directed against Bax or Bim. CA-4 treatment on H1299 cells was not followed by an increased protein-protein interaction between Bim/Bcl-2, whereas Bax/Bcl-2 interaction significantly decreased after 48 h treatment (Figure 15a). Only in the PFTµ-pretreatment of H460 cells and up to 8 h of co-treatment with CA-4 the levels of Bim/Bcl-2 immunocomplexes were lower; however, such physical interaction was strongly increased after 24 h of the treatment, while for Bax/Bcl-2, the interaction was decreased in a time-dependent manner (Figure 15b), as we had found in wt-p53 H460 cells (Figure 14e).

(47) Figure 15. Co-immunoprecipitation analysis performed on mitochondrial extracts of cells shows the interactions between Bcl-2 and pro-apoptotic proteins Bax or Bim after CA-4 treatment in H1299 (a), in PFTµ-pre-treated H460 cells (b) In the right panel are shown the relative densitometry analyses. Significant values according to Student’s t-test, *p<0.05, **p<0.01..

(48) DISCUSSION. It is known that the suppression of microtubule dynamics by MDA leads to disruption of the mitotic spindle in dividing cancer cells, cell cycle arrest at G2/M phase, and cell death by apoptosis [76, 1]. In this study, we have assessed the role played by p53 and Bim and their cellular localization in CA-4-induced cell death in H460 cell line, with and without modifications, and in p53- null cells H1299. P53-null cell lines have been shown to display a marked drug resistance to apoptosis after drug exposures with different cellular targets as andriamycin, paclitaxel, carboplatin [62, 65]. Similarly, although CA-4 induced a microtubule depolymerization and G2/M accumulation in wt-p53 H460 cells as well as in wt-p53 H460 cells treated with the inhibitor PFT-µ, and in H1299, we found that the latter cells suffered a far lower rate of cell death after exposure to this depolymerising agent, thus suggesting a relevant role of p53 in this process. Contrastingly to H1299 cells, H460 cells and their counterparts treated with PFTµ attempted to exit from the G2/M-block in the presence of the drug incurring in cell death. Cells that enter in apoptosis in response to MDA have in common the release of pro-apoptotic factors as cytochrome c from the inter-membrane mitochondrial space into the cytosol [77]. This event is known to occur rapidly and it can proceed in a single step independently of changes in m and without loss of outer membrane integrity in several cell types [78]. Once into the cytosol, the cytochrome c triggers the formation of the apoptosome, resulting in the activation of caspase-9 which, in turn, activates caspase-3 [39, 77, 79]. Previously, the group of research reported that CA-4 affects the morphology and functionality of mitochondria, promoting MMP and cytochrome c release in H460 cells [15], but the effect of CA-4 on m was not so clear, nor was it in its relationship with cytochrome c release. We observed that CA-4 induced cytochrome c release from mitochondria in all types of cells analyzed in a time dependent manner, but only in those cells pre-treated with PFTµ the mitochondrial reduction of cytochrome c seemed to occur at a lower rate and not to be followed by its cytosolic accumulation. This effect was consistently observed in independent experiments and to explain it, we hypothesized that the accumulation of cytosolic PFTµ-modified p53 may be related in some way with the rapid action and degradation of cytosolic cytochrome c. Certainly, the cytochrome c released from mitochondria was associated with the m dissipation attended in apoptosis CA4-induced in PFTµ-pre-treated H460.

(49) cells. In these cells, the mitochondrial membrane depolarization occurred in a time-dependent manner and still persisted at 48 h, thus indicating that the combination of loss of mitochondrial p53 accumulation, together with its cytosolic accumulation, promotes a decrease in m even for prolonged treatments. However, a different behaviour was found in wt-p53 H460 and H1299 cells in which the cytochrome c release was not always accompanied by the decrease in m. In these cells, CA-4 caused only a temporary decrease of m at 8 h of treatment, whereas hyper-polarization was assessed after 24 h. In this respect, as previously shown, after exposure of hepatoma HepG2 cells to MDA as paclitaxel, mitochondrial membrane may be even hyperpolarized [80]. Such hyper-polarization was attributed to free tubulin, that regulates changes in m by voltage-dependent anion-selective channel (VDAC), in situ inhibition and regulation of the supply of respiratory substrates and/or ATP required for mitochondrial polarization. This event is followed by mitochondrial swelling, rupture of the outer membrane, and release of proapoptotic factors into the cytosol as cytochrome c [80]. According to our results, CA-4 might act as paclitaxel in H1299 and wt-p53 H460 cells. During the microtubule damage, several pro- and anti-apoptotic proteins were regulated as a consequence of CA-4 treatment; among them, p53 and Bim turned out to be essential proteins for apoptosis. p53 is functionally impaired in a high percentage of human tumors [81], indeed its activation or reactivation in tumor cells has been recognized as a promising strategy for cancer treatment [82]. Several findings suggest that p53 acts upstream of Bax to promote MDA-mediated cell death through both transcriptiondependent and -independent mechanisms in cancer cells [27]. Beside p53, Bim may represent a suitable target for anticancer drugs but its role in apoptosis engaged by agents known to affect the microtubular integrity, as the spindle poisons, has been only poorly investigated, so far, except for microtubular-stabilising agents as taxanes [55, 56], persins [83] and for PBOX-15, a novel microtubule targeting agent [84]. Both Bim-dependent apoptosis in lymphocytes [85], non-small-cell lung cancer lines [55], kidney epithelial cells [86], and Bim-independent apotosis in HeLa and breast cancer cells [56] have been reported for paclitaxel, and hence suggesting cell specificity in paclitaxel-induced cell death. In this respect, we found by targeting Bim with siRNA in wt-p53 cells, that drug-induced apoptosis in.

(50) wt-p53 H460 is strongly dependent on such pro-apoptotic protein as confirmed by inhibition of caspase-3 activation. We found that after exposure to CA-4, microtubular array disruption and cell death were accompanied by an up-regulation of the p53 and Bim with a consequent accumulation of these proteins, particularly after 24 h of treatment. p53 expression in wt-p53 H460 cells increased in mitochondria, nuclear and cytosol compartments, after releasing from the microtubule array in a time-dependent manner. Previously it was reported that Bim may be sequestered at the microtubular dynein motor complex, in different cell lines (human embryonic kidney, human breast carcinoma, mouse promyelocytic and mouse fibroblastoid lines) [47], according with this, we found that Bim accumulates in both mitochondria and cytosol after release from dynein motor complex. Transcriptional up-regulation of Bim occurred after exposure to CA-4, as it is for other stress stimuli. The downstream mediator of PI3K signaling, FoxO3a, is translocated to the nucleus and selectively binds FoxO binding site at the Bim promoter and hence, mediates Bim transcription and apoptosis in paclitaxel-treated breast cancer cell lines [53, 87]. Notably, it should be noted that FoxO3 gene is a direct target of p53 in response to the DNA damaging agent doxorubicin, in both mouse embryonic fibroblasts and in thymocytes, and appears to modulate p53-dependent apoptosis via trans-activation of pro-apoptotic downstream genes [88]. Therefore, these findings suggest that Bim expression can be regulated also by p53, at least for those agents able of inducing DNA lesions. In this respect, we have investigated whether Bim mechanism of action is affected in p53-null H1299 cells exposed to the non DNA damaging agent CA-4. Interestingly, we found that in the absence of p53, Bim was not up-regulated in cytoplasm and even decreased in mitochondria after CA-4, contrastingly to what detected in wt-p53 cells. In adition, previous reports have indicated that Bim can act as a tumor suppressor protein independently of p53 intervention. Moreover, an important role has been demonstrated for the p73–Bim axis in regulating cell death during mitosis in p53-null cells treated with taxol [89]. To understand better the correlation between Bim mechanism and p53 status a cross-talk between Bim and p53 may be approached by the following observations: CA-4 treatment did not modify the level of Bcl2, Bcl-XL or Bax in wt-p53 H460 cells. On the other hand, under CA-4 apototic.

(51) conditions, we found an augmented amount of both cytosolic and mitochondrial Bim co-immunoprecipitated with Bcl-2. By contrast, such an effect was not detected with regards to the interaction between Bim and Bcl-XL, nor between Bcl-XL and Bax. We hypothesised that as result of drug-mediated enhanced binding between Bim and Bcl-2, the proapoptotic effector Bax was released from Bcl-2 and operated then as an apoptogenic factor, though a certain delay was detected between Bim/Bcl-2 interaction and Bax releases from Bcl-2. On the other hand, no changes in the mitochondrial interaction of Bim with the anti-apoptotic protein Bcl-2 were observed with respect to the untreated p53-null cells, indicating that expression and function of Bim, both, depend on p53 expression in NSCLC treated with CA-4. Furthermore, it should be pointed out some sort of p53/Bim relationship which is also supported by the observation that in the presence of PFTµ inhibition, Bim accumulation does not occur at the cytoplasm level, while it appears to be even increased at the mitochondrial level compared to the sole CA-4 treatment. To our knowledge, this is one of the few reports investigating such p53 and Bim relationship, pointing to either a p53-mediated control of Bim expression after drug, or some form of increased Bim/p53 interaction conferred by the presence of wt-p53, but not by wt-p53 somehow modified by interaction by PFTµ. In the latter scenario, a higher amount of cytoplasmic Bim would be free to localize at mitochondria in a manner independent of the p53 mitochondrial localization. Interesting to note that only in recent times, another mechanism has been proposed for the mitochondrial function of p53, independently of its transcriptional activity, in which p53 can act as a regulator of a Bim function in different tumor cell types expressing wt-p53, including colon carcinoma, Hct116, multiple myeloma RPMI-8266, and breast cancer MDA-MB-231 and MCF7 cells [40]. According to this mechanism, Bim is released from sequestrating complexes with Mcl-1, Bcl2 and Bcl-XL and becomes free of forming new complexes with p53, thus promoting conformational changes of Bax and Bak mediated by Bim itself [40]. Additionally, we found that p53 and Bim interacted physically in both, cytosol and mitochondria in wt-p53 H460 cells, suggesting a new function for p53 that may allow Bim to be maintained active during CA-4 treatment, thereby favoring Bax releases from Bcl-2. However, p53 and Bim loss their binding capacity in presence of the inhibitor PFTµ, possibly by the p53conformational change PFTµ-induced, that reduce the affinity by the proapoptotic Bim as was reported for anti-apoptotic Bcl-2 protein [70]..

(52) Besides, Bax activation has also been reported to possibly occur, independently of functional p53, as observed after treatment with microtubule destabilizing agents in colon cancer cells carrying a deletion of the TP53 gene [27], and in wt-p53 colon, prostate, and breast cancer cells, treated with resveratrol, with such a Bax activation being accounted for to the localization to mitochondria of X-linked inhibitor of apoptosis protein (XIAP), t-Bid, or Bim proteins [90]. We found that Bax releasing from sequestrating protein Bcl-2 took place at 48 h after CA-4 treatment in H1299 cells, while in pre-treated-PFTµ H460 cells, it occurred at 24 hours, similarly to what observed in wt-p53 H460 cells. This result suggests that the deletion of the TP53 gene delayed, but not avoided Bax releasing; in a similar way, Bax release was not affected by the lack of p53 at mitochondria, thus confirming Bax activation to occur independently of p53 status and cellular localization. Furthermore, several works have suggested that p53, through direct and physical interactions with Bcl-2, Bcl-XL and Mcl-1 proteins, can neutralize their anti-apoptotic effects, by promoting release of pro-apoptotic Bcl-2 like proteins as Bak or Bax for mitochondria permeabilization, leading to caspase activation and apoptosis [37, 39]. In wt-p53 H460 cells, we observed a significant increase in the complex formation between p53 and Bcl-2 but not with Bcl-XL, thereby suggesting that p53 may participate in the releasing of Bax from Bcl-2 after 24 hours of CA-4 treatment, allowing for apoptosis to be activated in the same way as described for Bim in wtp53 H460 cells..

(53) CONCLUSIONS. CA-4 is a compound characterized by tubulin-depolymerising activity and a promising chemotherapeutic agent not subjected to the mechanism of MDR. P53 and the BH3-only protein Bim are involved in the initial events triggering CA-4-induced apoptosis in the wt-p53 H460 cells, however CA-4 failed to activate an apoptotic response in H1299 p53-null cells, indicating an involvement of p53 expression in cell death as induced by CA-4, moreover, the extent of cell death was not reduced in H460 after combined treatment of CA-4 with PFTµ. By affecting the microtubule array, CA-4 causes the release of p53 and Bim from microtubular network and their translocation to mitochondria, where these proteins engages pro-survival Bcl-2-like relatives, such as Bcl-2; then, p53 and Bim could promote the release of the proapoptotic protein Bax, which is in turn responsible for mitochondrial dysfunction and caspases activation. Overall our results support a model of CA-4-induced cells cycle arrest at the G2/M-phase and apoptosis in NSCLC, for which the expression of p53 and Bim proteins are essential, but where the mitochondrial function of p53, linked to its p53-transcription independent mechanism of apoptosisinduction is negligible. Defining the contribution of p53 and Bim to the mechanism of apoptosis may offer some different clues in view of developing new strategies for chemotherapy with CA-4, underlining the relevance of the cytoskeleton integrity in the apoptotic response..

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