In situ generated D-peptidic nanofibrils as multifaceted apoptotic inducers to target cancer cells

Nanofibrils of small molecules, as a new class of biofunctional entities, exhibit emergent properties for controlling cell fates, but the relevant mechanism remains to be elucidated and the in vivo effect has yet to be examined. Here, we show that D-peptide nanofibrils, generated by enzyme-instructed self-assembly (EISA), pleiotropically activate extrinsic death signaling for selectively killing cancer cells. Catalyzed by alkaline phosphatases and formed in situ on cancer cells, D-peptide nanofibrils present autocrine proapoptotic ligands to their cognate receptors in a juxtacrine manner, as well as directly cluster the death receptors. As multifaceted initiators, D-peptide nanofibrils induce apoptosis of cancer cells without harming normal cells in a co-culture, kill multidrug-resistant (MDR) cancer cells, boost the activities of anticancer drugs, and inhibit tumor growth in a murine model. Such a supramolecular cellular biochemical process (consisting of reaction, assembly, and binding) for multi-targeting or modulating protein–protein interaction networks ultimately may lead to new ways for combating cancer drug resistance.

Kinases and phosphatases regulate phosphorylation and dephosphorylation, 1,2 respectively, and control a variety of cellular processes. Kinase inhibitors have achieved relative successes in chemotherapy, but the development of phosphatases inhibitors remains to be a challenge. Since a number of phosphatases (e.g., PTEN 3 ) are important tumor suppressors, the lack of selectivity of phosphatase inhibitors is a major concern. The inherent difficulty for developing specific phosphatase inhibitors has earned phosphatases the reputation of undruggable. 4 Thus, new approaches to make phosphatases druggable are needed. Departing from the dogmatic approach of enzyme inhibition, we have been exploring the use of alkaline phosphatase (ALP) to catalyze the formation of molecular nanofibrils via self-assembly 5,6 for killing cancer cells. [7][8][9][10][11][12] The merit is that nanofibrils of small molecules, spatiotemporally defined by enzymatic catalysis and self-assembly (i.e., EISA), can interact with multiple cellular proteins and interrupt multiple cellular processes to kill cancer cells selectively, 13,14 even without inducing acquired drug resistance. 13 After we demonstrated that EISAgenerated nanofibrils selectively kill cancer cells, 15,16 several labs have already validated this concept of EISA in cell assays. 17,18 However, the relevant molecular and cellular mechanisms of cell death induced by the nanofibrils remain to be elucidated, and the in vivo effects of the nanofibrils generated by EISA have yet to be examined.
Here, we report the mechanistic study of using ALP catalysis, not ALP inhibition, to generate molecular nanofibrils for selectively killing cancer cells. Specifically, ALP, as an ectoenzyme, 19 catalyzes the formation of pericellular nanofibrils of D-tripeptides (DTPs) on cancer cells, which pleiotropically activate cell death signaling by presenting autocrine death ligands (e.g., TNF-α 20 and TRAIL 21 ) to their cognate receptors (e.g., TNFR1/2, DR3/4/5) 22 in a juxtacrine manner, 23 as well as directly clustering the receptors (e.g., CD95 24 ). As the localized multifaceted initiators, DTP nanofibrils induce apoptosis. 25 The ALP-generated DTP nanofibrils kill cancer cells without harming normal cells in a mimicked tumor microenvironment, 26 kill multidrug-resistant (MDR) cancer cells, boost the activities of anticancer drugs (e.g., cisplatin 27 and NF-ĸB inhibitors 28,29 ), and inhibit tumor growth in a murine model. These results demonstrate a paradigm shift from inhibition to reaction, that is, the use of a multiple stepreaction, assembly, and bindingprocess 30 to modulate protein-protein interaction networks for selectively killing cancer cells.

Results and Discussion
ALP-generated nanofibrils for killing HeLa cells in a coculture. Since certain ALP (e.g., placental alkaline phosphatase (PLAP) on HeLa cells 31 ) is overexpressed on the surface of certain cancer cells in the co-culture (mixture of cancer (HeLa) and normal (HS-5) cells), the precursor of DTP (i.e., a phosphorylated DTP, pDTP) is dephosphorylated locally on the surface of the cancer cells to generate DTP nanofibrils (Figure 1a). Acting as a pericellular nanonet just around the cancer cells, the DTP nanofibrils present the secreted, different proapoptotic ligands (e.g., TNF-α, CD95L, and TRAIL) from cancer cells to bind with different extrinsic cell death receptors (e.g., TNFR1/2, CD95, and DR3/4/5), or directly interact with the death receptors (e.g., CD95), which leads to the death of cancer cells only. Multiple evidences in the following sections support the mechanism illustrated in Figure 1a.
Adding ALP to the solution of pDTP ( Figure 1b) results in DTP nanofibrils (Figure 1c) (diameters of 7 ± 2 nm), indicating that ALP overexpressed on the cancer cells can generate DTP nanofibrils. Adding pDTP in the co-culture of HeLa and HS-5 (as a mimic of tumor microenvironment 26 Table 1). As shown in the fluorescent imaging (Figure 1e and Supplementary Figure 1b)  of the co-culture treated with pDTP, contrasting to the control,  almost no HeLa-GFP cells live, while the HS-5 cells remain viable. The addition of L-phenylalanine (L-Phe), an uncompetitive inhibitor of PLAP, 33 almost doubles the viability of HeLa-GFP cells, in comparison with only pDTP in culture medium (Figure 1f and Supplementary Figure 1c), excluding the possibility that pDTP acts as surfactants to lyse cells and confirming that PLAP on HeLa cells catalyzes the EISA of pDTP to selectively kill cancer cells. siRNA knock-down of PLAP on HeLa cells protects the cells from pDTP, confirming that PLAP is essential for killing HeLa cells  Figure 2d). Collectively, these results confirm that ALPs, as ectoenzymes overexpressed on cancer cells, generate DTP nanofibrils in situ for selectively killing cancer cells without harming normal cells.
ALP-generated nanofibrils pleiotropically activate extrinsic death signaling. We focus on extrinsic cell death signaling because previous results suggest the ALPgenerated DTP nanofibrils inducing mitochondrial independent cell death. 7 Co-incubating zVAD-fmk 34 (a pan-caspase inhibitor) with pDTP significantly reduces the cell death (Figure 2a), indicating that DTP nanofibrils mainly induce apoptosis. We co-incubate pDTP with extrinsic cell death ligands and monoclonal antibodies (mAbs) of key extrinsic cell death signaling molecules, respectively. While anti-TNF-α considerably protects cells, anti-TNFR1 or anti-TNFR2 leads to more cell death (Figure 2b and Supplementary Figures  3a-c). hTNF-α also causes more cell death without the use of cycloheximide 35 (Supplementary Figure 3d). These results indicate that DTP nanofibrils enhance interactions between the autocrine TNF-α 36 and its cognate receptors, likely by presenting TNF-α in a juxtacrine manner. 37 siRNA knockdown 38 of TNFR1 or TNFR2 in HeLa cells decreases the cytotoxicity of pDTP (Figure 2b and Supplementary  Figure 3l), agreeing with the juxtacrine presentation of TNF-α (as well as anti-TNFR1 or anti-TNFR2) by DTP nanofibrils (Figure 1a). While anti-DR5 exhibits little effect, anti-DR3, anti-DR4, or TRAIL slightly promotes cell death (Figure 2c and Supplementary Figures 3e, 3f, and 3h). Anti-TRAIL only slightly decreases the cytotoxicity of DTP nanofibrils (Supplementary Figure 3g), but knock-down of DR3 or DR5 considerably rescues the cells (Supplementary Figure 3l). These results suggest that DTP nanofibrils, besides interacting with TRAILRs via the juxtacrine presentation of TRAIL, directly interact with TRAILRs. Either anti-CD95 or CD95L improves the cell survival, anti-CD95L results in more cell death (Figure 2d   We compare the amounts of secreted extrinsic cell death ligands trapped in the pericellular DTP nanofibrils and those in the conditioned medium of HeLa cells and find that DTP nanofibrils retain little TRAIL, a moderate level of TNF-α, and CD95L significantly (Figure 3a). Although the cell lysate of HeLa contains TNF-α, TRAIL, and CD95L (Figure 3b), only CD95L exhibits the considerably high amount in the pericellular DTP nanofibrils, agreeing with that cancer cells, under stress, increase the secretion of CD95L. This result further supports that DTP nanofibrils present the autocrine death ligands (Figure 3b). In addition, the western blot reveals that pDTP exhibits little effect on the expression of TNF-α or TRAIL while moderately increases the expression of CD95L (Supplementary Figure 5h), suggesting that the pericellular DTP nanofibrils, besides increasing autocrine CD95L of HeLa cells, mainly retain the autocrine death ligands near the cell  In situ generated D-peptidic nanofibrils X Du et al surface to interact with the death receptors. However, it is also possible that DTP nanofibrils possess slightly higher affinity to CD95L and CD95 than to TNF-α and TRAIL. Nevertheless, the fluorescent imaging of HeLa cells treated with pDTP, primary mAbs of the ligands, and the fluorescent secondary mAb (Figure 3c), together with quantification of the fluorescence (Figure 3d), indicates that the pericellular DTP nanofibrils increase the clustering of death ligands (e.g., TNF-α, CD95L), which is consistent with that DTP nanofibrils present the autocrine proapoptotic ligands to their cognate receptors in a juxtacrine manner.
To further confirm that DTP nanofibrils are able to present the death ligands, we test the effect of pDTP on CD95L-KO HeyA8 cells (Supplementary Figure 5c). pDTP only exhibits weak cytotoxicity to CD95L-KO HeyA8 cells at 48 h. But the addition of CD95L together with pDTP significantly increases the CD95L-KO HeyA8 cell death. This result supports the notion that DTP nanofibrils present CD95L to induce apoptosis.
For the pDTP-treated HeLa cells, Western blot reveals that, while the constitutive expression of TNFR1 increases initially and then decreases moderately, the inductive expression of TNFR2, especially the glycosylated form (75 kD), 41 significantly increases over time. While the expressions of CD95 and DR3 decrease slightly, expression of DR4 increases remarkably, and expression of DR5 increases moderately (Figure 3e). None of these changes of TNFR1 and TNFR2 occur in the controls ( Supplementary Figures 5d-g), confirming that the ALP-generated DTP nanofibrils result in those changes. Western blot of the down-stream proteins (e.g., caspase 8 or RIP1) shows that pDTP slightly decreases the expression of caspase 8 and the cleaved RIP1 while increases expression of RIP1 (Figure 3f), agreeing with that DTP nanofibrils mainly result in apoptosis. In the case of HeLa cells, we find that cleaved caspase 8 appears at 24 h, cleaved caspase 3 emerges around 36 h, and cleaved PARP also arises in small amount at 12 h and is significantly produced around 24 h. These results (Supplementary Figure 5i) confirm DTP nanofibrils activate caspases for apoptosis of HeLa cells. In addition, HeLa cells overexpress PLAP on membrane, while HS-5 and HepG2 cells barely express PLAP (Figure 3g). This fact, together with the cell viabilities of these three cells treated by pDTP (Supplementary Table 1), suggests that pDTP acts as the substrate of ALP (e.g., PLAP) on HeLa cells to form the nanofibrils that induce apoptosis. These results demonstrate DTP nanofibrils as multifaceted inducers of apoptosis by pleiotropically interacting with extrinsic cell death ligands and receptors.
ALP-generated nanofibrils selectively kill MDR cancer cells. As shown by western blot (Supplementary Figure 6a), several MDR cancer cells (e.g., MES-SA/Dx5, T98G, and A2780-cis) also overexpress ALPs. Besides overexpressing germ cell alkaline phosphatases, MES-SA/Dx5, T98G, and A2780cis overexpress tissue non-specific alkaline phosphatases. So we evaluate the responses of these three MDR cells to pDTP, including in their co-cultures with HS-5, and examine the corresponding cell death mechanisms. pDTP selectively induces apoptosis of those MDR cancer cells while remains innocuous to HS-5 cells in their co-cultures  Table 1). zVAD-fmk, co-incubated with pDTP, almost completely rescues MES-SA/Dx5, significantly protects T98G, and moderately reduces the death of A2780-cis (Figure 3i), indicating that DTP nanofibrils induce apoptosis of those cells, albeit at different extent. hTNF-α leads to more death of T98G than those of MES-SA/Dx5 and A2780-cis; anti-TNF-α significantly protects MES-SA/Dx5 and T98G, anti-TNFR1 rescues MES-SA/Dx5; anti-TNFR2 moderately protects MES-SA/Dx5, T98G, and A2780-cis (Figure 3j and Supplementary Figure 6c). TRAIL results in more death of T98G, but offers slight protection to MES-SA/Dx5 and A2780-cis; anti-TRAIL slightly promotes the death of MES-SA/Dx5 and A2780-cis; anti-DR3 slightly protects MES-SA/Dx5; anti-DR4 slightly increases cytotoxicity of all the three cells; anti-DR5 mainly protects MES-SA/Dx5 (Figure 3k and Supplementary Figure 6d). CD95L also causes more death of T98G, but offers considerable protection to MES-SA/Dx5 and A2780-cis; anti-CD95L only leads more death of T98G; anti-CD95 hardly affects MES-SA/Dx5, T98G, and A2780-cis cells (Figure 3l and Supplementary Figure 6e). Both anti-CD95L and CD95L increase cell death of T98G, indicating that the pericellular DTP nanofibrils may directly interact with CD95 receptors as well as present autocrine CD95L of T98G cells. These apparently paradoxical results reflect the complexity and versatility of the EISA process and molecular nanofibrils. These results indicate that DTP nanofibrils directly interact with CD95 and DR5 or present TNF-α to induce death of MES-SA/Dx5 and A2780-cis, but mainly present TNF-α, TRAIL, and CD95L to their cognate receptors inducing apoptosis of T98G cells. Table 1 summarizes the role of the receptor/ligand pairs in different cell lines contributed to the apoptosis induced by pDTP. Unlike HeLa cells that express the three extrinsic cell death receptors, the other cell lines lack the expression of at least one death receptor/ligand pair. The absence of the death receptors may contribute to the resistance of these tumor cells to the corresponding death ligands.
Western blot results show that the increasing inductive expression of TNFR2 and the up-and down-expression of TNFR1 over time (Figures 4a-c) are the common trends for Table 1 The summary of the receptor/ligand pairs required for pDTP toxicity in different cell lines (↑ indicates increasing the cytotoxicity;↓indicates decreasing the cytotoxicity; − indicates little effect)

Cells
HeLa  Figures. 7c and d). These results establish ALP-generated DTP nanofibrils pleiotropically activating extrinsic cell death signaling as a general mechanism for triggering apoptosis of ALP-overexpressing cancer cells.

by Student's t-test
In situ generated D-peptidic nanofibrils X Du et al HS-5 than to A2780-cis cells. pDTP turns the IC 50 value of CDDP against A2780-cis cells to an IC 95 value (Figure 4f and Supplementary Figures 7f and g), promising molecular nanofibrils for CDDP-based combination therapy. 15 The presence of NF-ĸB inhibitors (e.g., BTZ or BAY) considerably decreases the IC 50  with pDTP, agreeing with DTP as innocuous monomers. 7 In addition, pDTP hardly forms DTP nanofibrils in human serum ( Supplementary Figures 8d and e), suggesting low toxicity to in vivo. Thus, we conduct in vivo toxicity test in mice. The intravenous injection of pDTP has little adverse effect on mice, without changing body weight, spleen, thymus, and blood conditions (Figures 5b and c and Supplementary  Figures 9a-e). H and E staining indicates that major organs (e.g., heart, spleen, kidney, liver, and lung) of the injected mice are normal (Figure 5d and Supplementary Figure 9f), further confirming low in vivo toxicity of pDTP.
ALP generating nanofibrils as a multitargeting process for apoptosis. The goal of cancer therapy is to kill cancer cells without harming normal cells. Although the generic differences between cancer and normal cells are rare, recent tissue-based map of human proteome 42 indicates that overexpression of ALPs, one of the earliest tumor markers identified, 31 is a generic difference between cancer and normal cells. Moreover, it is common for late stage cancers overexpress ALPs. 43 However, ALP is 'undruggable'. Thus, the use of EISA as a process to generate nanofibrils as multifaceted, localized inducers for cell death signaling may be particularly valuable to address the problem of MDR in cancer therapy. DTP combined with anti-TNFR1/2 mAbs has no effect on HeLa cells (Supplementary Figure 5a) since the assembly of DTP hardly localizes on cell surface without ALP catalysis. Anti-TNFR1 or anti-TNFR2 makes little difference on the viability of HS-5 treated with pDTP ( Supplementary Figure 5b) because HS-5 expresses little ALP on cell surface (Figure 3g). In addition, unlike the case of HeLa cells treated with pDTP, western blot analysis of HeLa cells incubated with DTP itself, pDTP and L-Phe, or pDTP and ALP shows little change of the death receptors or down-stream proteins over time (i.e., little effect on the expression of TNFR1 and TNFR2). These results further confirm that the formation of DTP nanofibrils, a multiple step molecular process catalyzed by ALP in situ, is necessary for the selective inhibition of the cancer cells.
It is known that the co-incubation of a NF-ĸB inhibitor (e.g., cycloheximide) is necessary for TNF-α-induced apoptosis, 35 an observation has led to the recognition of the difference between paracrine and juxtacrine signaling of TNF-α. 44 This work reports that hTNF-α combined with pDTP induces cell death without NF-ĸB inhibitors (Figure 2b), a strong evidence of DTP nanofibrils promoting the juxtacrine signaling. Because PLAP catalyzes the formation of DTP nanofibrils, the inclusion of exogenous hTNF-α in the nanofibrils for juxtacrine signaling can occur during the self-assembly of DTP, which agrees with that more cells are dead upon the addition of hTNF-α. Moreover, the addition of anti-TNFR1 and anti-TNFR2 causes more death of HeLa cells incubated with pDTP, not only agreeing with that antibodies to TNF receptor can have TNF-like activity, 45 but also suggesting that nanofibrils may crosslink/aggregate the antibody to convert an otherwise neutralizing antibody to an agonistic antibody. In addition, the combination of pDTP and NF-ĸB inhibitors against cancer cells shows that pDTP enhances the efficacy of NF-ĸB inhibitors (Figures 4g and h). NF-ĸB inhibitors also increase the inhibition ability of pDTP against cancer cells. These results further demonstrate DTP nanofibrils to bias NF-ĸB pathway for cell death, suggesting that it might be worthwhile to combine pDTP with other NF-ĸB inhibitors for further elucidating the detailed mechanisms depending on specificity of NF-ĸB transcription factors.
Interestingly, the knock-down of either TNF-α, TRAIL, or CD95L receptors all rescues cell death~50%, strongly suggesting crosstalk between all death receptor pathways at the level of receptor clustering and/or engagement. In addition, western blot analysis shows that DTP nanofibrils cause the reemergence of death receptors (especially TNFR1, TNFR2, and DR5) on siRNA knock-down cells (Figure 2g). This result indicates that, even if one kind of death receptors is knocked down, DTP nanofibrils can affect or interact with other receptors, regulate their down-stream signaling, and induce the re-emergence of silenced receptors, implying that the nanofibrils may counter cancer drug resistance 13 due to downregulation of a death receptor. As the first mechanistic study on multitargeting by enzyme-triggered nanofibrils to result in apoptosis of cancer cells, this work also implies that the use of locally generated nanofibrils to present proapoptotic ligands (e.g., TNF-α, CD95L, and/or TRAIL) in a juxtacrine mode may lead a way to bringing back the early promises of proapoptotic ligands for cancer therapy.

Materials and Methods
Transmission electron microscopy (TEM). Before placing sample solution/gels on the grid (3 μl, sufficient to cover the grid surface), we glow discharge the carbon-coated grids to increase their hydrophilicity. About 10 s later, we place three large drops of ddH 2 O on parafilm to let the grid touch the water drops with the sample-loaded surface facing the parafilm, and then tilt the grid and gently absorb water from the edge of the grid using a filter paper. Immediately after rinsing, we place three large drops of uranyl acetate (UA) staining solution on parafilm to let the grid touch the staining solution drops with the sample-loaded surface facing the parafilm, and then tilt the grid and gently absorb the stain solutions from the edge of the grid using a filter paper. After drying the grid in air and it is ready for EM imaging, we examine the grid as soon as possible. 46 Solutions and supplies: 2.0% (w/v) UA (prepare by dissolving 200 mg UA in 10 ml of ddH 2 O. Add water to the tube containing UA, cover tube with foil and rotate in cold room for several hours till fully dissolved. Filter through a 0.22-μm filter that has be pre-rinsed well with ddH 2 O. Filtered stain stored at 4°C in a foil-wrapped tube can be used for 41 year); filter strips (prepared by cutting Whatman 1 filter paper into small slivers); grids (400 mesh copper grids coated with continuous thick carbon film~35 nm in thickness, purchased from Pacific Grid Tech. Co). We prepare samples by adding ALP (5 U/ml) to the solution of pDTP at concentration of 362 μg/ml. Cell viability assay. Cells in exponential growth phase are seeded in a 96-well plate at a density of 1 × 10 4 cells/well. The cells are allowed to attach to the wells for 4 h at 37°C, 5% CO 2 . The culture medium is removed and 100 μl culture medium containing compounds (immediately diluted from fresh prepared stock solution of 10 mM) at gradient concentrations (0 μM as the control) is placed into each well. After culturing at 37°C, 5% CO 2 for 24, 48, 72 h, each well is added by 10 μl of 5 mg/ml MTT ((3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), 47 and the plated cells are incubate at dark for 4 h. One-hundred microliters 10% SDS with 0.01 M HCl is added to each well to stop the reduction reaction and to dissolve the purple. After incubation of the cells at 37°C for overnight, the OD at 595 nm of the solution is measured in a microplate reader (DTX 880 multimode detector). Data represent the mean ± standard deviation of three independent experiments.
Live cell imaging. HeLa-GFP and HS-5 cells in exponential growth phase are seeded in glass bottomed culture chamber at 1 × 10 5 cells/well (5 × 10 4 HeLa-GFP and 5 × 10 4 HS-5 cells per well). The cells are allowed for attachment for 24 h at 37°C, 5% CO 2 . The culture medium is removed, and new culture medium containing pDTP at 216 or 362 μg/ml or f-pDTP at 493 μg/ml is added. After incubation for certain time (e.g., 24 h for pDTP or 4 h for f-pDTP), cells are stained with 1.0 μg/ml Hochst 33342 for 5 min at 37°C in dark. After that, cells are rinsed three times by PBS buffer, and then kept in PBS buffer for imaging.
Cell imaging with antibody staining. After incubating 1 × 10 5 HeLa cells per glass bottomed culture chamber for 24 h at 37°C, 5% CO 2 , the cells are treated with 216 or 362 μg/ml of pDTP for 24 h. After three times rinse by PBS buffer, cells are 4% formaldehyde fixed (10 min) and then incubated in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h to permeabilize the cells and block non-specific protein-protein interactions. The cells are then incubated with the primary antibodies (e.g., anti-CD95, anti-TNFR1, anti-TNFR2, anti-DR3, anti-DR5 In situ generated D-peptidic nanofibrils X Du et al mAbs) overnight at +4°C. The secondary antibody (green) is Alexa Fluor 488 goat anti-rabbit IgG (H+L) (abcam) used at 2 μg/ml for 1 h. Hochst 33342 is used to stain the cell nuclei (blue) at a concentration of 1.0 μg/ml. 48 The fluorescent intensity in each image is analyzed using ImageJ. 49 Knock-down HeLa cells. In a 3 cm petri dish, we seed 2 × 10 5 HeLa cells in 2 ml antibiotic-free normal growth medium supplemented with FBS and incubate the cells at 37°C in a CO 2 incubator for 24 h to make cells 60-80% confluent. We prepare two solutions, siRNA duplex solution (diluting 32 μl siRNA duplex (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) into 100 μl siRNA transfection medium (Santa Cruz Biotechnology, Inc.)) and the dilute transfection reagent solution (dilute 6 or 32 μl siRNA transfection reagent (Santa Cruz Biotechnology, Inc.) into 100 μl siRNA transfection medium), and then mix gently the two solutions by pipetting the solution up and down and incubate the mixture 45 min at room temperature. After washing the cells once with 2 ml of siRNA transfection medium, we immediately dilute the siRNA duplex mixture with 0.8 ml transfection medium and overlay the mixture onto the washed cells. After incubating the cells 7-12 h (depending on the cell conditions) at 37°C in a CO 2 incubator, 1 ml of normal growth medium containing two times the normal serum and antibiotics concentration (2 × normal growth medium) is added without removing the transfection mixture (if toxicity is still a problem, we would remove the transfection mixture and replace with 1 × normal growth medium). In all, 18-24 h later, we aspirate the medium and replace with fresh 1 × normal growth medium, and then the cells are ready for western blot or cell viability assay after 72 more hours. 50 Time-dependent western blot. Four of 10 cm petri dishes seeded 2 × 10 6 of HeLa, MES-SA/Dx5, T98G, A2780-cis, HS-5, or HepG2 cells onto each dish are prepared for the time-dependent western blotting experiment. After overnight incubation, we randomly choose three of them to be incubated with medium containing 300 μM of pDTP, or just fresh medium. The other dish of cells is washed with PBS buffer for three times and scrapped from petri dish after adding 500 μl cell lysis buffers, which represents the cell lysates at 0 h. Similarly, we collect one dish of treated cell lysates at 6 h, 12 h, 24 h, respectively, by using the same method. The collected cell lysates are mixed with 5 μl of 100 × proteinase inhibitor cocktail and then snap-frozen and thawed for three cycles to lyse the cells. The cell lysates are clarified by centrifugation at 12 000 × g for 20 min at 4°C to remove the whole cells, nuclei, and large mitochondria. After quantification of proteins in cell lysates by using the Bradford reagent, the same amount of proteins for different time is mixed with the same volume of 2 × Laemmli buffer before SDS-PAGE. After finishing the gel electrophoresis and transferring the proteins on the gels to the PVDF membranes for overnight, the membranes are incubated with primary antibodies (1:1000, all antibodies were obtained from Cell Signaling Technology, Danvers, MA, USA) for overnight. The membranes are then incubated with secondary antibodies (1:2000) for 1 h, followed by the reaction with ECL solutions for 10 min and the final detection of proteins. 51 Western blot of L-Phe or ALP co-incubated cells. Similar to the protocol of time dependent western blot, 2 × 10 6 of HeLa cells are seeded onto each 10 cm petri dish initially. After overnight incubation, three random dishes would be incubated with medium containing 300 μM of pDTP and 5 mM of L-phenylalanine (L-Phe). Another three random dishes would be incubated with medium containing 300 μM of pDTP and 10 U/ml of alkaline phosphatase (ALP, Biomatik Corporation, Wilmington, DE, USA). Similarly, we collect the cell lysates at 0, 6, 12, and 24 h, respectively. After quantification of proteins in cell lysates by using the Bradford reagent, the same amount of proteins for different time is used for SDS-PAGE and western blotting.
In vivo tumor model with MES-SA/Dx5. The animal care facilities and programs of the Foster Animal Research Facility in Brandeis University meet the requirements of the law and NIH regulations. The animals are subjected to regular veterinary care on a routine basis. The animal experiments described in this study are performed according to the Guide for the Care and Use of Laboratory Animals published by the NIH. The nu/nu mice are purchased from Charles River Laboratories (Wilmington, MA). MES-SA/Dx5 cells are collected in exponential growth phase. In all, 100 μl of MES-SA/Dx5 cells suspension in complete culture medium at 5 × 10 7 cell/ml is subcutaneously injected into the right flank of nu/nu mice. 52 After 14 days of inoculation, mice bearing MES-SA/Dx5 tumor at average volume of 30 mm 3 are randomly divided into three groups. Subcutaneous peritumoral injections are performed every 3 days for five doses. One group is treated with 100 μl of pDTP at 3.6 mg/ml in PBS buffer; the other group is treated with 100 μl of pDTP at 0.36 mg/ ml in PBS buffer; control group is treated with 100 μl PBS buffer. Based on the average body weight of the mice (22 g) and the molar mass of pDTP (723.23 g/mol), the dosages in gram per kilogram of body weight of pDTP on the mice were calculated as follow: 100 μl × 3.6 mg/ml × 723.23 g/mol/22 g = 12 mg/kg; 100 μl × 0.36 mg/ml × 723.23 g/mol/22 g = 1.2 mg/kg. The number of mice in each group is six. The statistical significance in relative tumor size between the control group and the group treated by 12 mg/kg of pDTP is examined by Studentʹs t-test and Anova statistical analysis.
In vivo assessment of pDTP by intravenous injection. Similar to the protocol mentioned in nu/nu mice, we randomly divide mice into four groups: 10, 20, 50 mg/kg pDTP, and control group (PBS buffer). For each mouse, we would inject pDTP or PBS buffer every 3 days intravenously, and then measure the body weight of mice, organ index, or parameters of enzymes or proteins. In addition, we take H and E staining images of heart, spleen, kidney, liver, and lung from the nu/nu mice. The number of mice in each group is six.