Anja Berger

Sandra-Annika Quast

Michael Plötz

Martin Hein

Manfred Kunz

Langer

PhD Jürgen Eberle
email: juergen.eberle@charite.de

Sensitization of melanoma cells for death ligand-induced apoptosis by an 1 indirubin derivative -enhancement of both extrinsic and intrinsic apoptosis 2 pathways 3 4

Keywords: apoptosis, melanoma, death ligands, indirubin, p53 24 25

Until today effective therapies are lacking for metastatic melanoma. The death ligand TRAIL 2 appears as promising in cancer treatment; however, melanoma cells reveal both preexisting 3 and inducible TRAIL resistance. Here, we present evidence that the recently described 4 indirubin derivative 8-Rha-β enhances melanoma cell sensitivity for death ligands and 5 overcomes resistance to TRAIL and CD95 agonists. Indirubin is known from traditional 6 Chinese medicine and is a potent kinase inhibitor. Unraveling of apoptotic signaling pathways 7 revealed that TRAIL resulted in a quick (within 8 h) downregulation of both agonistic TRAIL 8 receptors DR4 and DR5, in a kind of negative feed-back loop. Treatment with indirubin, 9 however, mediated upregulation of both receptors, thus compensating this negative feed-back 10 loop by TRAIL. Furthermore, indirubin activated intrinsic apoptosis pathways, seen in loss of 11 mitochondrial membrane potential and release of cytochrome c. The mitochondrial response 12 appeared as related to upregulation of Bax and Bad and to downregulation of Mcl-1. 13 Remarkably, indirubin in combination with TRAIL was also able to overcome apoptosis 14 resistance due to ectopic Bcl-2 overexpression. The tumor suppressor p53 appeared as master 15 regulator of these propapoptotic changes and is the transactivator of proapoptotic proteins 16 which was upregulated by indirubin. Taking into account the physiological role of death 17 ligands in immune surveillance, sensitization of melanoma cells for death ligands may be 18 supportive for an anti-tumor immune response. Furthermore, combinations with kinase 19 inhibitors, such as indirubin 8-Rha-β may help for a breakthrough of TRAIL-mediated 20 strategies in melanoma.

The electric impedance corresponds to the cell density. 1,250 cells were seeded per microtiter 8 well, and treatment started after 24 h. The impedance was determined up to 120 h after 9 seeding.

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For monitoring cell viability and proliferation, a colorimetric assay (WST-1, Roche 11 diagnostics, Mannheim, Germany) was used. A number of 1,250 cells per well was seeded in 12 96-well plates and treated the next day for 24 h with 20 ng/ml TRAIL, 2.5 µM indirubin or 13 the combination. For 100 µl culture medium per well, 10 µl WST-1 reagent were added, 14 following by incubation of the cells at 37°C between 0.5 to 4 h, until color changes were seen.

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The optical density of triplicated samples was determined several times at 450 nm in an 16 ELISA reader. For background correction, the values of untreated controls were subtracted. For protein analysis, cells were lysed in 10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM 2 EDTA; 2 mM PMSF; 1 mM leupeptin; 1 mM pepstatin; 0.5% SDS and 0.5% Nonidet P-40.

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For cytochrome c, AIF and VDAC, cytosolic and mitochondrial cell fractions were separated 4 by a mitochondria/cytosol fractionation kit (Alexis, Gruenberg, Germany). Protocols for 5 protein extraction and Western blot analysis were described previously [23].

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The following primary antibodies were used: cleaved caspase-3 (Cell Signaling, rabbit, 7 1:10000), caspase-3 (Cell Signaling, rabbit, 1:1000), caspase-8 (Cell Signaling, mouse, 8 1:1000), caspase-9 (Cell Signaling, rabbit, 1:1000), PARP (Biomol, mouse, 1:5000), Bid (Cell 9 Signaling, rabbit, 1:1000), Mcl-1 (Santa Cruz, mouse, 1:200), Bcl-2 (Santa Cruz, mouse, 10 1:200), Bax (Santa Cruz, rabbit, 1:200), Bak (Dako, rabbit, 1:500), Bad (Cell Signaling, 11 rabbit, 1:1000), Puma (Epitomics, rabbit, 1:1000), Noxa (Pro Sci, rabbit, 1:500), cytochrome 12 c (BD Biosciences, mouse, 1:1000), AIF (Santa Cruz, goat, 1:200), anti-Porin 31 HL (VDAC) 13 (Calbiochem, mouse, 1:5000), XIAP (Cell Signaling, rabbit, 1:1000), Survivin (Santa Cruz, 14 mouse, 1:500), DR4 (abcam, rabbit, 1:500), DR5 (abcam, rabbit, 1:500), p53 (Santa Cruz, 15 rabbit, 1:500) and GAPDH (Santa Cruz, mouse, 1:200). In contrast, at combined treatment with indirubin stronger processing of caspase-8 was seen in 19 A-375-TS, as well as the 17 and 15 kDa cleavage products of caspase-3 representing its 20 completely processed large subunit. This coincided with significant processing of PARP.

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Characteristically, Bid and caspase-9 were now also activated (Fig 4A).

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In order to confirm caspase involvement, the pancaspase inhibitor zVAD-fmk was used.

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  Malignant melanoma revealed increased incidences over the past decades and is characterized 2 by an unbroken high mortality[1]. The high metastatic potential and its resistance to 3 conventional chemotherapy is of major importance, related to defects in proapoptotic 4 signaling [2;3]. Overcoming apoptosis resistance is therefore a promising target. Two main proapoptotic pathways (extrinsic and intrinsic) have been described. The extrinsic 6 pathway is initiated by the binding of death ligands to cell surface death receptors leading to 7 the formation of a death inducing signaling complex (DISC), where initiator caspases 8 and 8 10 are activated [4]. On the other hand, the intrinsic apoptosis pathway is triggered by 9 intracellular signals such as cellular and DNA damage, also when caused by chemotherapy. 10 Key events are the depolarization of the mitochondrial membrane potential and the release of 11 mitochondrial factors such as cytochrome c into the cytosol, which results in activation of 12 initiator caspase-9 [5;6]. Both pathways may meet at the mitochondria due to caspase-8-13 mediated activation of the proapoptotic Bcl-2 protein Bid [7]. Mitochondrial activation is 14 critically controlled by the family of Bcl-2 proteins, which consists of antiapoptotic proteins 15 (e.g. Bcl-2, Bcl-x L and Mcl-1), proapoptotic multidomain proteins (Bax and Bak) and 16 proapoptotic BH3-only proteins (e.g. Bid, Bad, Puma and Noxa) [8]. According to these 17 pathways, caspase cascades appear as central in apoptosis regulation. Initiator caspases cleave 18 and activate downstream effector caspases as caspase-3, which then target a number of death 19 substrates to set apoptosis into work [5;9]. 20 The death ligand TRAIL (TNF-related apoptosis-inducing ligand) appeared as a promising 21 strategy in cancer therapy due to selective killing of tumor cells, whereas normal cells were 22 largely spared [10]. It binds to two agonistic death receptors, DR4/TRAIL-R1 and 23 DR5/TRAIL-R2 [11]. The downstream signaling cascade includes receptor oligomerization, 24 formation of the DISC, activation of initiator caspases, cleavage of effector caspases and

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  c c e p t e d M a n u s c r i p t 3 finally DNA fragmentation [12]. In parallel, TRAIL may also trigger additional pathways 1 such as nuclear factor kappa B (NF-κB) and mitogen-activated protein (MAP) kinases [13]. In 2 a previous study, we have shown the significant role of DR4 in mediating TRAIL sensitivity 3 in melanoma cells, and as the majority of melanomas revealed immunoreactivity for DR4, 4 TRAIL and DR4-based strategies appeared as promising, also for melanoma [14]. However, 5 pre-existing TRAIL resistance was also seen and in addition, melanoma cell lines may acquire 6 induced resistance upon TRAIL treatment [15]. 7 Indirubin, the red isomer of indigo, is the active ingredient in a traditional Chinese medicinal 8 recipe [16]. It is a potent kinase inhibitor targeting GSK-3β, CDKs (cyclin dependent 9 kinases), c-Src and FGF-R1 (fibroblast growth factor receptor) [17-19]. Here, we show the 10 potential of a recently described indirubin derivative 8-Rha-β [20] to sensitize human 11 melanoma cells for death ligand-induced apoptosis. This effect is mediated by enhancing both 12 extrinsic and intrinsic apoptosis pathways.

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  Four human melanoma cell lines were investigated: death ligand sensitive Mel-HO and A-375 3 as well death ligand resistant Mel-2a and MeWo [15]. TRAIL-resistant subclones of Mel-HO 4 and A-375 (Mel-HO-TS; A-375-TS) derived from a selection with 5 ng/ml TRAIL [15]. 5 CD95-resistant subclones of A-375 (A-375-CS) were generated by selection with 20 ng/ml 6 CH-11 agonistic CD95 antibody. Selected cells were continuously cultured with the 7 respective death ligand. As previously described, other subclones of A-375 resulted from 8 stable transfection with a pIRES-Bcl-2 construct (A-375-Bcl-2) or the pIRES empty plasmid 9 (A-375-Mock) [21]. The pIRES plasmid originated from Clontech (Palo Alto, CA, USA). 10 Melanoma cells were cultivated at 37°C, 5% CO 2 in DMEM (4.5 g/l glucose; Gibco, 11 Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS and antibiotics (Biochrom, 12 Berlin, Germany). Experiments were performed in 6-well, 24-well or 96-well plates (200,000, 13 50,000 or 1,250 seeding density per well, respectively). For induction of apoptosis, soluble 14 human killerTRAIL (Alexis, Gruenberg, Germany ALX-201-073-C020; 20 ng/ml) or the 15 agonistic monoclonal CD95 antibody CH-11 (Beckman Coulter, Marseille Cedex, France PN 16 IM 1504; 50 ng/ml) were applied. As indicated, incubation with death ligands took place for 17 8-24 h. Treatment with indirubin 8-Rha-β, described previously [20], was set up in parallel 18 with death ligands, whereas control cells received only the solvent DMSO (Applichem, 19 Darmstadt, Germany). For inhibition of caspases, cells were preincubated for 1 h with 10 µM 20 of the pancaspase/panprotease inhibitor zVAD-fmk (R&D Systems, Wiesbaden, Germany), 21 which binds the active sites of caspase-like proteases. 22 In further experiments, the chemotherapeutic substances doxorubicin, paclitaxel and 23 vinblastine (Sigma, Taufkirchen, Germany) were applied in concentrations of 1, 10 and 100 A c c e p t e d M a n u s c r i p t 5 nM. Simultaneous treatments were used for combinations of chemotherapeutics and indirubin 1 8-Rha-β (24 h).

  -time cell analysis and cell viability assay 4 For obtaining growth curves, cell confluence was continuously monitored by real-time cell 5 analysis (RTCA, xCELLigence, Roche diagnostics; Penzberg, Germany). The technique is 6 based on microelectrodes integrated in the bottom of each well of special 96-well E-plates.

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 3 Quantification of apoptosis and cytotoxicity 19For quantification of apoptosis, cell cycle analyses were carried out after propidium iodide 20 staining, according to Nicoletti et al. [22]. Cells were harvested by trypsinisation, stained with 21 PBS buffer containing triton-X 100, sodium citrate and propidium iodide (Sigma, 22 Taufkirchen, Germany; 200 mg/ml), centrifuged, washed with PBS and analyzed by flow 23 cytometry in a FACS Calibur. Cytotoxicity was determined in parallel by measuring LDH 24 activity in culture fluids applying a cytotoxicity detection assay (Roche Diagnostics).

  mitochondrial membrane potential, the fluorescent dye TMRM 19 (Tetramethylrhodamine methyl ester perchlorate) was used. Cells were harvested by 20 trypsinisation, stained with TMRM (Sigma-Alderich, Taufkirchen, Germany; 1 nM, 15 min; 21 37°C), washed once with PBS buffer and analyzed in PBS by flow cytometry.

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  For measurement of intracellular ROS levels, the fluorescent dye H 2 DCFDA (20,70-2 dichlorodihydrofluoresceindiacetate) was used. Cells were harvested by trypsinisation, stained 3 with H 2 DCFDA (Molecular Probes, Invitrogen, Eugene, Oregon, USA; 15 mM, 30 min), 4 washed once with PBS buffer and analyzed in PBS by flow cytometry.

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  Cells were seeded in 6-well plates (2x10 5 cells/well) and treated for 8 h with indirubin or 8 TRAIL, alone or in combination. Then, cells were harvested with 500 µM EDTA, stained 9 with the primary mouse monoclonal antibodies for TRAIL-R1/DR4 (Alexis, clone HS101, 10 ALX-804-297-C100, 1:100), or TRAIL-R2/DR5 (Alexis, clone HS 201, ALX-804-298-100, 11 1:100), or for control with mouse IgG1 (Ancell, # 278-010). After 15 min of incubation at 12 4°C and 15 min at room temperature, cells were centrifuged, washed twice with PBS/1% BSA 13 and resuspended in 50 µl PBS/BSA containing the secondary antibody for mouse IgG1 14 (Alexis, ALX 211-201-050, 1:100). After 30 min incubation at room temperature, cells were 15 centrifuged, washed twice, resuspended in 200 µl of PBS and analyzed by flow cytometry. duplicate or triplicate values, and two or three independent experiments 19 were performed. Normal distribution of the samples was proven by the Kolmogorov-Smirnov 20 test. In case of normal distribution, which applied to most of our data sets, the Student's t-test 21 was used. In case that the Kolmogorov-Smirnov test did not prove normal distribution of the 22 data, the Wilcoxon test had to be applied for proving statistical significance. Mean values and 23 SDs were calculated by enclosing all individual values of the independent experiments (at 24 least 6 samples), and a p-value of < 0.05 was considered as statistically significant.

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  cell culture models of TRAIL resistance 2 We have previously described pre-existing and inducible TRAIL resistance in human 3 melanoma cell lines [15]. MeWo and Mel-2a (DR4-, DR5+) revealed resistance, whereas A-4 375 (DR4+, DR5+) and Mel-HO (DR4-, DR5+) were sensitive. In addition, A-375 and Mel-5 HO developed an inducible resistance upon continuous cultivation with TRAIL leading to 6 selected resistant cell populations (Mel-HO-TS, A-375-TS). The apoptotic response to TRAIL 7 of these six cell culture models is demonstrated by determination of sub-G1 cell populations 8 at 24 h of TRAIL treatment (20 ng/ml). At this time, A-375 revealed strongly increased 9 number (18%) and Mel-HO revealed moderately increased (4%) number of apoptotic cells, 10 whereas the other four cell lines remained resistant (Fig 1A). Cytotoxicity was unaffected at 11 this time, as determined by LDH in the cell culture supernatants (Fig 1B). 12 For targeting melanoma cells, we used the indirubin derivate 8-Rha-β. The synthesis as well 13 as induction of apoptosis and decreased cell proliferation in melanoma cell lines SK-Mel-29 14 and SK-Mel-147 have been recently described by our group [20]. Decreased cell proliferation 15 by indirubin 8-Rha-β treatment (2.5 µM) was also seen in A-375-TS cells (Fig 1C), which 16 was accompanied by a G2 arrest, seen in A-375, Mel-2a and A-375-TS (Fig 1D).

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 2 Indirubin sensitizes melanoma cells for death ligand-induced apoptosis 19Aiming at a sensitization of melanoma cells for death ligand induced apoptosis, we applied 20 different combinations of the new indiribin 8-Rha-β and death ligands in our cell culture 21 models. Significant induction of apoptosis by indirubin itself (10 µM, 24 h treatment) was 22 seen in Mel-2a (4%), A-375 (7%) and A-375-TS (4% apoptotic cells; Fig 2A). Most 23 strikingly, however, was the synergistic enhancement of apoptosis by the combination of 24 indirubin and TRAIL, resulting in increased sub-G1 cell populations in Mel-HO (15%), A-A c c e p t e d M a n u s c r i p t 9 375 (32%), Mel-2a (14%) and A-375-TS (12%). Also, visible effects such as reduced cell 1 numbers, rounded cells and cell detachment were clearly evident. In contrast, MeWo and 2 Mel-HO-TS remained resistant (Fig 2A). Enhanced apoptosis also exerted a strong impact on cell proliferation, monitored for A-375-4 TS by real-time cell analysis. In these selected cells, TRAIL treatment did not decrease cell 5 proliferation, cells rather showed an enhanced proliferation in response to TRAIL. The 6 combination of indirubin and TRAIL, however, appeared as highly efficient (Fig 2B). For investigation of the effects of indirubin on apoptosis sensitivity via the CD95/Fas system, 8 we selected A-375 cells for CD95 resistance by continuous incubation with the agonistic 9 CD95 antibody CH-11. The resultant CH-11-selected A-375-CS cells revealed a significantly 10 reduced apoptotic response to CD95 stimulation (5% vs. 12% in parental cells). Indirubin 8-11 Rha-β was able to enhance the sensitivity of A-375 parental cells to CH-11 as well as it 12 reduced the resistance of A-375-CS (Fig 2C; 2D). 13 Both selected A-375 cell populations revealed cross-resistance for the respective other death 14 ligand. Thus A-375-TS was largely resistant to CH-11, and A-375-CS was largely resistant to 15 TRAIL, suggesting parallel pathways to death ligand resistance. These cross-resistances were 16 also overcome by combinations with indirubin (Fig 2E). For understanding how general the 17 apoptosis sensitization by indirubin was, it was combined with the chemotherapeutics 18 vinblastine, paclitaxel and doxorubicin, respectively. Therefore, the cell line A-375 was 19 simultaneously treated with 8-Rha-β (10 µM) and chemotherapeutics in concentrations of 1, 20 10 and 100 nM (vinblastine, doxorubicin) or 1 and 10 nM (paclitaxel). For vinblastine, some 21 enhancement of apoptosis was seen in the combinations, however less than additive effects 22 (Fig 3A). Paclitaxel at 10 nM revealed a strong proapoptotic effect on A-375, which was 23 however diminished by the combination with 8-Rha-β (seen in two independent experiments, 24 p < 0.005). Only the combination with 100 nM doxorubicin showed a significant A c c e p t e d M a n u s c r i p t 10 enhancement of apoptosis by about two-fold, when compared to 8-Rha-β single treatment (p < 1 0.005). Cytotoxicity was unaffected for all treatments at this time (24 h) (Fig 3B). A complete 2 series of these combination experiments was also performed with A-375-TS. These cells 3 revealed essentially a similar response to chemotherapeutics as the parental cell line, namely 4 enhancement in the combination with 100 nM doxorubicin but antagonistic effects with 10 5 nM paclitaxel (data not shown).

  Activation of the full caspase cascade by combination with indirubin with TRAIL 8 For understanding the way of indirubin-mediated apoptosis sensitization, activation of 9 initiator and effector caspases was investigated in A-375-TS cells by Western blot analysis. 10 The extrinsic caspase cascade with typical cleavage products of the initiator caspase-8 and the 11 main effector caspase-3 was seen in A-375 parental cells upon TRAIL treatment. Further loss 12 of the proform of Bid and processing of caspase-9 were indicative for an amplification loop 13 through the mitochondrial/intrinsic pathway. As a result of caspase-3 activity, cleavage of its 14 characteristic substrate PARP (poly-ADP-ribose polymerase) was seen (Fig 4A). 15 Interestingly, A-375-TS revealed also visible processing of caspase-8 as well as a 20 kDa 16 cleavage product of caspase-3 upon TRAIL treatment. This 20 kDa form, however, appears as 17 not completely active, as PARP was not cleaved (Fig 4A).

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  Preincubation for 1 h with zVAD completely abolished apoptosis induction in parental A-375 24 as well as in A-375-TS cells treated with the combination (Fig 4B). Caspase inhibition also 25 A c c e p t e d M a n u s c r i p t 11 resulted in improved survival at 24 h of treatment, as determined by WST assay (Fig 4C) and 1 improved cell proliferation, as determined by RTCA (Fig 4D). However, the antiproliferative 2 effects of indirubin were not neutralized by zVAD (data not shown), and the inhibition of cell 3 proliferation by the combination with TRAIL was only partly reduced, thus indicating that 4 indirubin may engage also protease-independent effects (Fig 4D).

  of mitochondrial apoptosis pathways 8 A clear involvement of the mitochondrial apoptosis pathway became evident after measuring 9 the mitochondrial membrane potential Δψ, which is characteristically decreased in apoptotic 10 cells. This effect was completely blocked in A-375-TS, whereas TRAIL strongly decreased 11 Δψ in about 40% of parental A-375 cells. Indirubin, however, resulted in some decrease of 12 Δψ in A-375-TS, which was further strongly enhanced by the combination with TRAIL. 13 Thus, indirubin was able to restore also this TRAIL-mediated effect (Fig 5A; 5B). 14 Pre-incubation with zVAD only partly prevented the Δψ loss induced of indirubin but 15 completely abrogated any further TRAIL effect, both in parental and selected cells. Thus, the 16 mitochondrial response by TRAIL appeared as downstream of the early caspase cascade (Fig 17 5A; 5B).

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  time kinetic analysis revealed that the Δψ effect of indirubin and the combination came into 19 play at 8 h after start of treatment (Fig 5C), which was right in parallel with the induction of 20 apoptosis (Fig 5D). Thus, the decrease of mitochondrial membrane potential was going hand 21 in hand with apoptosis. Increased ROS levels are frequently associated with mitochondrial 22 activation as we have reported for melanoma cells [24]. However, no effects on ROS levels 23 were seen at 8 and 24 h after treatment with indirubin or with the combination (data not 24 shown).

  A c c e p t e d M a n u s c r i p t 12 Release of mitochondrial factors into the cytosol is a hallmark in the mitochondrial 1 proapoptotic pathway. Indeed, Western blot analysis of cytosolic extracts of A-375 treated 2 with TRAIL revealed at 8 h significant release of cytochrome c and apoptosis-inducing factor 3 (AIF),not seen in TRAIL treated A-375-TS. This deficiency of A-375-TS was completely 4 restored by the combination with indirubin (Fig 6).

  overrides apoptosis resistance by Bcl-2 8 Bcl-2 is a critical regulator in melanoma cells. For evaluating whether Bcl-2 or indirubin was 9 stronger in the A-375 cells, we applied A-375 cells stably transfected with a Bcl-2 construct 10 (A-375-Bcl-2) and compared to mock-transfected cells (A-375-Mock). The ectopic 11 overexpression of Bcl-2 resulted in complete CD95 and TRAIL resistance as previously 12 shown [14;21] and also seen here (Fig 7A; 7B). In contrast, the proapoptotic effects of 13 indirubin were not decreased in A-375-Bcl-2 cells as compared to A-375-Mock. 14 In particular, the TRAIL and CH-11 resistance of A-375-Bcl-2 cells was significantly 15 diminished by the combination with indirubin, resulting in 12% of apoptotic cells (TRAIL, 16 5% indirubin), 22% (TRAIL, 10% indirubin) and 8% (CH-11, 10% indirubin), respectively 17 (Fig 7A; B). Thus Bcl-2 was able to reduce but not to block apoptosis through indirubin and 18 death ligands. In contrast, the decrease of Δψ was completely blocked in A-375-Bcl-2, 19 proving the complete abrogation of the mitochondrial apoptosis pathway (Fig 7C). Thus, the 20 remaining apoptosis appeared as inpendent of mitochondria. Effects of indirubin on Bcl-2 proteins and cIAPs 23 Kinase-dependent survival pathways, which may be blocked by indirubin, are frequently 24 related to the expression of apoptosis regulators. Of the family of Bcl-2 proteins, antiapoptotic A c c e p t e d M a n u s c r i p t 13 (Bcl-2, Mcl-1), proapoptotic multidomain (Bax, Bak) and proapoptotic BH3-only proteins 1 (Bad, Noxa, Puma) were investigated by Western blot analysis. In addition, inhibitor of 2 apoptosis proteins (XIAP, Survivin) was enclosed. As concerning proapoptotic Bcl-2 proteins, 3 the central apoptosis regulator Bax was significantly upregulated in A-375-TS at 8 h after 4 indirubin treatment, whereas the functionally related Bak was unaffected. Of three BH3-only 5 proteins investigated, there was significant upregulation of Bad by indirubin. In contrast, the 6 combination treatment resulted in a down-modulation of these proteins in A-375-TS, as a kind 7 of counter-regulation (Fig 8). 8 As concerning antiapoptotic factors, indirubin alone remained without effect on the 9 expression of antiapoptotic Bcl-2 patterns (Bcl-2, Mcl-1) as well as on cIAPs (Survivin, 10 XIAP). However, in combination with TRAIL, in course of strong apoptosis, there was strong 11 downregulation of Mcl-1 and XIAP (Fig 8).

  Effects of indirubin on death receptors and p5315 Expression of the agonistic receptors DR4 and DR5 is elementary for TRAIL sensitivity.16Characteristically, A-375-TS cells revealed reduced surface expression of DR4 and DR5 than 17 parental cells, as reported before[15]. Importantly, we found here strongly decreased surface 18 expression of both death receptors in parental cells already at 8 h of TRAIL treatment. This 19 negative feed-back loop helps to understand the quick establishment of TRAIL resistance in 20 melanoma cells (Fig 9A). 21 Indirubin treatment alone restored a high death receptor expression in A-375-TS, seen for the 22 surface expression (Fig 9A) as well as by Western blot analysis for the total protein (Fig 9B). 23 Further addition of TRAIL in the combination treatment again resulted in downregulation of 24 the receptors, thus reflecting a most sensitive balance regulating TRAIL sensitivity.

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  c c e p t e d M a n u s c r i p t 14 As master regulator in apoptosis, the transcription factor and tumor suppressor p53 has been 1 described to trigger the expression of death receptors and of proapoptotic Bcl-2 proteins. In 2 clear agreement with upregulation of Bax, Bad, DR4 and DR5, there was strong upregulation 3 of p53 total protein levels that may in part explain the apoptotic response (Fig 9B).

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  An efficient therapy for metastatic melanoma is still outstanding. Thus, chemotherapeutic 8 regimens as well as small molecule inhibitors have been tested in numerous clinical trials, but 9 could not significantly increase overall survival rates of metastasized melanoma patients so 10 far [1;3]. New approaches are urgently needed to improve the prognoses of that dismal 11 disease.12 New hope may be set in the death ligand TRAIL, due to its induction of apoptosis in a variety 13 of human cancer cells while normal cells were largely spared [10]. In mouse and primate 14 models, suppression of tumor growth was reported, when applying TRAIL as monotherapy or 15 in combination with chemotherapy [25;26]. Recombinant TRAIL derivatives and agonistic 16 monoclonal antibodies have been developed enabling selective activation of DR4 or DR5 17 [27;28]. Clinical trials were also performed in patients with advanced colorectal carcinoma 18 and breast cancer, which revealed only little side effects, however, the response on TRAIL 19 monotherapy was also rather limited [29;30]. 20 Limited responsiveness appears as related to inducible TRAIL resistance, which has been 21 reported in different tumor models as in breast cancer and leukemia [31;32]. In melanoma 22 cells, we have previously reported both, pre-existing TRAIL resistance and inducible 23 resistance in course of continuous treatment with TRAIL. Resistance was correlated to 24 downregulation of the agonistic TRAIL receptors, the proapoptotic Bcl-2 protein Bid and the A c c e p t e d M a n u s c r i p t 15 initiator caspases 8 and 10 [14;15]. As shown here, the downregulation of the TRAIL 1 receptors, seen in total protein levels as well as on the cell surface, appeared as a direct and 2 very early effect, already seen at 8 h of treatment. This finding sheds new light on TRAIL 3 resistance in melanoma; which appears as a fast arising program that is induced by TRAIL 4 itself. As a second hallmark of TRAIL resistance in melanoma we have shown here the inhibition of 6 complete caspase-3 processing. This effector caspase is expressed as a 35 kDa proenzyme, 7 which is processed in a first step by initiator caspases leading to a 20 kDa intermediate form 8 corresponding to the large subunit. Final processing, suspected to be due to autocatalysis, then 9 results in the 17 kDa and the finally processed 15 kDa form [33]. This cascade was clearly 10 visible in parental A-375 melanoma cells after TRAIL treatment. In contrast, TRAIL-selected 11 A-375 cells did not show the 17 and 15 kDa cleavage products but high levels of the 20 kDa 12 intermediate product and efficient caspase-8 processing. Thus, resistant cells were 13 characterized by a block in caspase-3 autocatalytic activity, which may depend on insufficient 14 activation of the caspase cascade. 15 Aiming at a sensitization of tumor cells for TRAIL, multiple strategies have been tested. 16 Among these, compounds originating from herbal extracts appeared as promising. Thus, 17 sensitization for TRAIL-induced apoptosis has been reported with ingredients of traditional 18 Chinese medicine such as the flavonoid wogonin and triptolide, tested in leukemia and AML 19 cells, respectively [34;35]. 20 Indirubin, the red isomer of indigo, is the active ingredient of a traditional Chinese medicine. 21 Indirubin and its substituted derivatives are potent kinase inhibitors, and inhibition of GSK-22 3β, cyclin dependent kinases (CDKs), c-Src and FGF-R1 have been reported [17-19]. In 23 contrast, other kinases appear as activated, in particular a long-term activation of p38 MAP A c c e p t e d M a n u s c r i p t 16 kinase [19]. As a major appears a the cell cycle arrest in G2/M phase, that has been reported 1 by indirubin derivatives [36] and is also seen here in three of 6 human melanoma cell lines. 2 Another effect is the induction of apoptosis, that has been attributed to indirubins [37;38]. 3 Recently, we have reported proapoptotic effects also in human melanoma cell lines by the 4 new indirubin derivative 8-Rha-β [20]. Here, we present clear evidence that this substance is 5 able to efficiently enhance death ligand sensitivity of melanoma cells, and it overrides 6 preexisting as well as inducible resistance. Combinations of TRAIL and CD95 agonists with 7 indirubin, as used here, have not been reported, however in lung carcinoma cells the 8 combination of the derivative indirubin-3´-monoxime I3M has been shown to sensitize for 9 TNF--induced apoptosis. In these cells, the effects were correlated with decreased activity of 10 NF-κB [39]. 11 In melanoma cells, we present clear evidence that the tumor suppressor p53 was activated by 12 indirubin, seen in enhanced levels of p53, which is usually regulated by its stability [40]. 13 Furthermore, Bax, Bad and both agonistic TRAIL receptors were found upregulated upon 14 indirubin 8-Rha-βtreatment, which are characteristic targets of p53 [41;42]. Indeed, enhanced 15 activity of p38 could explain the activation of the p53 pathway in melanoma, as p38 16 activation has been reported as a major indirubin effect [19], and p53 can be activated by p38 17 [43;44]. Effects on p53 have also been stated for other indirubin derivatives, such as in 18 cervical cancer and lung cancer cells, which had been further related to upregulation of the 19 cell cycle inhibitor p21 [45;46]. Thus, activation of p53 may be a critical issue in indirubin-20 mediated cell cycle inhibition and induction of apoptosis. 21 Activation of the p53 pathway may also result from chemotherapy treatment [40]. The 22 combination of chemotherapeutic agents with indirubin however resulted in variable 23 responses. Whereas indirubin 8-Rha-β was able to enhance apoptosis induction by high dose 24 doxorubicin, it revealed antagonistic inhibition of apoptosis in combination with paclitaxel.

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  c c e p t e d M a n u s c r i p t 17 These results are indicative for the selectivity of this compound, which appears to particularly 1 affect death ligand sensitivity thus supporting extrinsic apoptosis pathways.2Of note, the role of p53 in melanoma cells is a non-dissolved question so far, namely despite a 3 lack of inactivating mutations, the p53 pathway appears as blocked in melanoma cells, also 4 underlined by the pronounced chemotherapy resistance [3;47]. Thus, indirubin appears to 5 overcome the block of p53 activation in melanoma cells at least to some extend, as 6 concerning death ligand sensitivity. The upregulation of Bax and Bad by indirubin 8-Rha-β correlated with parallel activation of 8 intrinsic mitochondrial pathways in A-375-TS cells, as demonstrated by decreased 9 mitochondrial membrane potential, release of cytochrome c and AIF as well as by caspase-9 10 processing. Expression of Bcl-2 is of major importance for apoptosis deficiency of melanoma 11 cells [48]. Thus, we used A-375 cells stably transfected with Bcl-2 [21], which completely 12 blocked apoptosis by TRAIL or CH-11 agonistic CD95 antibody. Indeed, the decrease of the 13 mitochondrial membrane potential upon indirubin treatment was also completely blocked in 14 these cells. However, apoptosis induction by the combination of indirubin and TRAIL 15 appeared as only partly reduced. This indicates that indirubin affects both the intrinsic and the 16 extrinsic apoptosis pathway, of which only the intrinsic part was blocked by Bcl-2. 17 Previously, we had shown that enhancement via the mitochondrial pathway was essential for 18 death ligand-induced apoptosis in melanoma cells [49]. Indirubin, however, was able to 19 override the Bcl-2-mediated block of apoptosis. 20 In addition, the combination with TRAIL resulted in downregulation of Mcl-1 and XIAP. 21 Simultaneous decrease of these two proteins was also reported for leukemia cells upon 22 induction of apoptosis [50], possibly indicating their parallel regulation. As XIAP is a potent 23 inhibitor of caspase-3 [51], its downregulation may partly explain the full caspase-3 24 processing observed in A-375-TS after combined treatment.

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  c c e p t e d M a n u s c r i p t 18 The present study presents clear evidence of an upregulation of both DR4 and DR5 by 1 indirubin in melanoma cells. The subsequent enhancement of the extrinsic apoptosis pathway 2 was evident by enhanced caspase-8 processing. Upregulation of death receptors has been 3 reported in HeLa cervix carcinoma cells by the indirubin derivative I3M [45]. Upregulation of 4 both death receptors in cancer cell lines was also seen for celastrol, another ingredient of 5 traditional Chinese medicine [52]. As TRAIL resistance in melanoma cells was associated 6 with a quick TRAIL-mediated downregulation of death receptors, their upregulation by 7 indirubin appears as a suitable compensating effect, preventing inducible death ligand 8 resistance. In summary, these results present an efficient strategy for overcoming and preventing death 10 ligand resistance in melanoma cells. Taking into account that death ligands are physiological 11 signals of T-lymphocytes in anti-tumor immune surveillance [53;54], indirubin may reveal a 12 therapeutic effect also by supporting the immune response against melanoma. Regarding 13 possible proapoptotic therapies, TRAIL monotherapy may not be sufficient in melanoma 14 cells, due to the quickly arising resistance. However, combinations with kinase inhibitors, 15 such as indirubin 8-Rha-β, may help for a breakthrough of TRAIL-mediated strategies. 16 Indirubin derivatives were largely well tolerated in animal tumor models as shown in rat, 17 mouse and fish [55-57]. Also in clinical trials for treatment of chronic myelogenous leukemia 18 (CML), indirubin and the derivative meisoindigo have already been tested in China [16].19Nevertheless, this derivative is an investigational compound, and further investigations are 20 needed to prove its applicability and its anti-tumor effects in the clinical situation.

24

  The study was supported by the Deutsche Krebshilfe (Melanomverbund, 10-8008, TP7) 25A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 21 29. Herbst RS, Eckhardt SG, Kurzrock R, Ebbinghaus S, O'Dwyer PJ, Gordon MS, Novotny W, 1 Goldwasser MA, Tohnya TM, Lum BL, AshkenaziA, Jubb AM, and Mendelson DS, Phase I 2 dose-escalation study of recombinant human Apo2L/TRAIL, a dual proapoptotic receptor 3 agonist, in patients with advanced cancer. J.Clin.Oncol. 28: 2839-2846, 2010. 4 30. Camidge DR, Herbst RS, Gordon MS, Eckhardt SG, Kurzrock R, Durbin B, Ing J, Tohnya 5 TM, Sager J, Ashkenazi A, Bray G, and Mendelson D, A phase I safety and pharmacokinetic 6 study of the death receptor 5 agonistic antibody PRO95780 in patients with advanced 7 malignancies. Clin.Cancer Res. 16: 1256-1263, 2010. 8 31. Park SJ, Kim MJ, Kim HB, Sohn HY, Bae JH, Kang CD, and Kim SH, Trichostatin A 9 sensitizes human ovarian cancer cells to TRAIL-induced apoptosis by down-regulation of c-10 FLIPL via inhibition of EGFR pathway. Biochem.Pharmacol. 77: 1328-1336, 2009. 11 32. Zhang Y and Zhang B, TRAIL resistance of breast cancer cells is associated with constitutive 12 endocytosis of death receptors 4 and 5. Mol.Cancer Res. 6: 1861-1871, 2008. 13 33. Roy S, Bayly CI, Gareau Y, Houtzager VM, Kargman S, Keen SL, Rowland K, Seiden IM, 14 Thornberry NA, and Nicholson DW, Maintenance of caspase-3 proenzyme dormancy by an 15 intrinsic "safety catch" regulatory tripeptide. Proc.Natl.Acad.Sci.U.S.A 98: 6132-6137, 2001. 16 34. Fas SC, Baumann S, Zhu JY, Giaisi M, Treiber MK, Mahlknecht U, Krammer PH, and Li-17 Weber M, Wogonin sensitizes resistant malignant cells to TNFalpha-and TRAIL-induced 18 apoptosis. Blood 108: 3700-3706, 2006. 19 35. Mak DH, Schober WD, Chen W, Konopleva M, Cortes J, Kantarjian HM, Andreeff M, and 20 Carter BZ, Triptolide induces cell death independent of cellular responses to imatinib in blast 21 crisis chronic myelogenous leukemia cells including quiescent CD34+ primitive progenitor 22 cells. Mol.Cancer Ther. 8: 2509-2516, 2009. 23A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 23 57. Kim SA, Kim YC, Kim SW, Lee SH, Min JJ, Ahn SG, and Yoon JH, Antitumor activity of 1 novel indirubin derivatives in rat tumor model. Clin.Cancer Res. 13: 253-259, 2007.

  Figure legends

  Figure 2: Sensitization for death ligand-induced apoptosis

A c c e p t e d M a n u s c r i p t 25 The3

 25 Figure 3: Combination of indirubin with chemotherapeutics

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  Figure 6: Release of mitochondrial factors

Figure 9 :

 9 Figure 9:Expression of death receptors and p53.