Suppression of v-Src Transformation by Andrographolide via Degradation of the v-Src Protein and Attenuation of the Erk Signaling Pathway*

Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.

c-Src, at its resting state, is phosphorylated at Tyr-527 and forms an inactive conformation. Upon activation by extracellu-lar signals, c-Src is dephosphorylated at Tyr-527 and phosphorylated at Tyr-416, and then functions as a tyrosine kinase and a signaling molecule to trigger downstream signal transduction. In contrast, v-Src protein, with a deletion of the C-terminal negative-regulatory region, has lost an autoinhibitory phosphorylation site (Tyr-527) and is constitutively active (1). v-Src can promote normal cells to acquire a variety of transformation characteristics, including alteration of morphology, loss of contact inhibition, anchorage dependence and growth factor dependence, and elevation of invasion ability. Oncogenic Src proteins in epithelial cells have been reported to induce a switch between the epithelial and mesenchymal phenotype, the socalled epithelial-mesenchymal transition (2). Oncogenesis induced by v-Src results in aberrant expression or activation of downstream target proteins that are associated with mitogenesis, cell adhesion, motility, and angiogenesis (1,2). v-Src has been shown to suppress expression of the cell cycle inhibitors (p21 waf1 and p27 kip1 ) and induce activation of the cyclin/cyclindependent kinase complexes (3,4). Elevated expression of the transcription factor c-Myc is required for v-Src transformation of fibroblasts (5). Active Src also increases the production of matrix metalloproteinase-2 and -9 (2,6). Furthermore, downregulation of the adhesion molecule E-cadherin by v-Src has been observed in several cell types (6 -8). Expression of v-Src is also required for production of the angiogenesis inducer vascular endothelial growth factor (9). v-Src protein perturbs multiple intracellular signaling pathways. To date, the PI3K, 2 MAPK, and STAT3 signaling pathways have been shown to be essential for v-Src-induced neoplastic transformation (10). In addition, the signaling pathways required for v-Src-mediated transformation are significantly different between fibroblasts and epithelial cells (11)(12)(13)(14)).
An elevated Src protein level and activity have been found in many human tumors, such as colon cancers and breast cancers (15). Furthermore, a truncated mutant of c-Src that is structurally similar to v-Src has been identified in advanced human colon cancer (16). Therefore, the Src protein has been consid-ered to be a promising chemotherapeutic target, and many Src inhibitors have been identified. Most known Src inhibitors restrain the activity of Src by reducing its protein stability, reducing kinase activity or inhibiting protein-protein interactions (17). The benzoquinone ansamycins (geldanamycin and herbimycin A) and the macrocyclic antifungal antibiotic (radicicol) have been shown to bind HSP90 and thus reduce Src protein stability (18). The pyrazolo-pyrimidines (PP1 and PP2) were found to directly inhibit Src tyrosine kinase activity (19). In addition, PP2 increases E-cadherin/catenin levels and reduces cancer metastasis in vivo (20). Another type of Src inhibitor, such as UCS15A, has been shown to disrupt proteinprotein interaction, and thus abolish the downstream signal transduction of Src (21). Nevertheless, only limited Src-targeted drugs have been proven to be efficient in vivo. Currently, there are no Src-specific inhibitors that have clinical application.
In the present study, we established and applied the temperature-inducible v-Src-transformed cell line to identify compounds that inhibit v-Src-mediated cellular transformation. We found that a natural product, andrographolide, significantly suppressed v-Src-induced transformation. Andrographolide is a lactone diterpenoid isolated from the Chinese herb Andrographis paniculata and has been previously shown to exhibit a wide spectrum of pharmacological activities, such as anti-apoptosis, anti-inflammation, anti-angiogenesis, and anti-cancer activity (22)(23)(24)(25)(26). The anti-cancer effect of andrographolide was demonstrated by showing its growthinhibitory activity on various cancer cell lines in vitro and its tumor-suppressive activity in animal models (27). Andrographolide can induce cell cycle arrest through increasing expression of p27 and decreasing expression of cyclin-dependent kinase 4 (27). Andrographolide also triggers apoptosis via the caspase-8 dependent pathway in human cancer cells (28). Through enhancing the cytotoxic T lymphocytes to secret interleukin-2 and interferon-␥, andrographolide inhibits the tumor growth in BALB/c mice (29). Although these studies reveal the potential of andrographolide in cancer therapy, no reports have described the inhibitory effect of andrographolide on v-Src oncogenicity. In this study, we therefore intended to investigate the molecular mechanism(s) by which andrographolide antagonizes the oncogenic properties of v-Src.
Plasmids-The retroviral constructs, RCAN (Replication-Competent, ALV-LTR, No splice acceptor) and RCAN-ts-vsrc, were kindly provided by Dr. Neil Carragher from the Beatson Institute for Cancer Research, Glasgow, Scotland, UK. RCAN-v-ts-src encodes a temperature-sensitive v-Src mutant (12). Both RCAN and RCAN-ts-v-src can produce avian retroviruses of subgroup A in avian cells. The MKK1-CA (MAPKK⌬ N3/S218E/S222D) plasmid, kindly provided by Dr. Natalie Ahn of the University of Colorado, has been described previously (30). A 1.5-kb XbaI to HindIII fragment from the MKK1-CA plasmid (encoding a HA-tagged-mutated mkk1 sequence) was first cloned into corresponding sites of PBSFI to yield PBSFI-MKK1-CA. RCAS(A)-MKK1-CA was further created by cloning the SfiI fragment of PBSFI-MKK1-CA into RCASFI, a modified version of the replication-competent retroviral vector RCAS(A) (31). The pCDNA3-Src531 plasmid (designated as pcSrc531RI in Ref. 16) carrying a transforming c-Src truncation mutant (Src531) was kindly provided by Dr. Timothy Yeatman at University of South Florida.
Creation of a Temperature-inducible v-Src-transformed Cell Line (ts-v-Src Cells)-The preparation of RCAN-ts-v-src viruses and infection of tv-a-expressing RK3E/tv-a cells have been described previously (6). The infected cells were maintained for 3 weeks and tested in focus formation assays and soft agar assays (at 35°C and 39.5°C, respectively) using protocols as described previously (6). Five of the colonies, formed on soft agar at 35°C, were picked, expanded, and mixed for subsequent experiments.
Creation of RK3E Cell Clones Stably Expressing Src531 (RK3E/Src531 Cell Clones)-RK3E cells were regularly maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin in a 5% CO 2 humidified incubator at 37°C. For transfection, RK3E cells (8 ϫ 10 5 ) were seeded onto 60-mm plates, and 2 g of plasmid (either pcDNA3 or pcDNA3-Src531) was introduced into the cells using FuGENE 6 reagent (Roche Applied Science). Twenty-four hours after transfection, half of the cells were transferred into 100-mm plates and maintained in regular growth medium with G418 (200 g/ml) for 2 weeks. Three pcDNA3 clones (negative control) and six pcDNA3-Src531 clones were picked and expanded for subsequent Western blot analysis to determine protein level of Src531.
Determination of Morphological Change-The ts-v-src cells (10 5 ) were seeded into 6-well plates and grown at 39.5°C. After overnight incubation, the cells were treated with vehicle or drugs, and then immediately shifted to an incubator set at 35°C. Twenty-four hours later cell morphology was observed and photographed under a phase-contrast microscope at 200ϫ magnification.
Soft Agar Assay-Six-well plates were overlaid with 0.6% nutrient agar (Sea Plaque agar in Dulbecco's modified Eagle's medium with 3% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 3 g/ml puromycin) before use. The ts-v-Src cells (10 4 ) grown at 39.5°C were mixed well with 0.3% nutrient agar (Sea Plaque agar in Dulbecco's modified Eagle's medium with 10% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 3 g/ml puromycin) consisting of vehicle or different concentrations of andrographolide, seeded into prepared 6-well plates and transferred to 35°C. Every 3-4 days, the cells were supplied with 0.3% nutrient agar consisting of vehicle or andrographolide. After 2 weeks, colonies were stained with 1% (w/v) p-iodonitrotetrazolium violet solution, photographed, and counted.
Immunofluorescence and Actin Cable Staining-The ts-v-src cells were seeded onto coverslips at 39.5°C. Next day, the cells were incubated with vehicle or andrographolide for 24 h at 35°C, fixed with 4% paraformaldehyde for 10 min, and subsequently permeabilized with 0.5% Triton X-100 for 5 min at room temperature. After blocking with 1% bovine serum albumin/phosphate-buffered saline, the cells were incubated with a primary antibody for 1 h and then treated with a fluorescein isothiocyanate-conjugated secondary antibody for additional hour. Then, the cells were stained with 1 g/ml 4Ј,6-diamidino-2-phenylindole (DAPI) for 10 min. The images were detected using a confocal microscope (FV-300, Olympus Corp., Tokyo, Japan).
To stain actin filaments, the process of fixation and permeabilization were conducted as mentioned above. After blocking, the cells were stained with 2.5 units/ml Texas Red-X phalloidin and 1 g/ml DAPI (4Ј,6-Diamidino-2-phenylindole) for 20 min and 10 min, respectively. The images were detected using a confocal microscope.
Western Blot Analysis and Immunoprecipitation-The ts-vsrc cells (3 ϫ 10 5 ) were seeded into a 100-mm plate and grown overnight at 39.5°C. The cells were subsequently treated with vehicle and drugs for various times at 35°C. Afterward, the treated cells were collected by scraping and centrifugation. Cell pellets were then lysed in radioimmune precipitation assay buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 5 mM EDTA (pH 8.0), and 1 mM EGTA (pH 8.0)) in the presence of general protease inhibitors (Sigma-Aldrich). In the experiments for detecting tyrosinephosphorylated proteins, the cells were lysed in radioimmune precipitation assay buffer in the presence of general protease inhibitors and phosphatase inhibitors (sodium fluoride, 1 mM, sodium pyrophosphate, 10 mM). Total protein (50 g) was analyzed by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with 5% nonfat milk/TBS-Tween, the membranes were incubated with primary antibodies overnight at 4°C, and then reacted with horseradish peroxidase-conjugated secondary antibodies for 1 h. After treating the membranes with ECL Western blotting Detection Reagent (Millipore), the signals were detected by exposing the membrane to x-ray film.
For immunoprecipitation, treated cells were collected and lysed in radioimmune precipitation assay buffer containing general protease inhibitors. Total protein (300 -400 g) was incubated with anti-v-Src antibody overnight at 4°C. Protein G-Sepharose slurry (50 l) was then added to and reacted with protein/antibody for further 2 h at 4°C. Afterward the immunoprecipitates were collected by centrifugation for subsequent study.
In Vitro Kinase Assay-To determine kinase activity of v-Src, we used a modified procedure based on the instructions of the Universal Tyrosine Kinase Assay Kit (Takara Bio Inc., Shiga, Japan). For each reaction, 10 6 cells were disrupted in extraction buffer and centrifuged for 10 min at 10,000 ϫ g. The immunoprecipitation was then carried out by incubating cell supernatant with anti-v-Src antibody and protein G-Sepharose. After centrifugation, the immunoprecipitates were washed with 1ϫ phosphate-buffered saline containing 0.05% (v/v) Tween 20, resuspended in ATP-supplemented kinase reaction buffer, and incubated with vehicle or drugs for 30 min. The samples were then transferred to wells precoated with peptide substrate for the kinase reaction (30 min, 37°C), and anti-phosphotyrosinehorseradish peroxidase and horseradish peroxidase substrate were added to each well and incubated for 30 min. After stopping the reaction with 1 N H 2 SO 4 , the absorbance at 450 nm was measured by using a microplate photometer (Multiskan RC, Model 351, Lab Systems, Stockholm, Sweden).
Radiolabeling of Newly Synthesized v-Src Protein-After seeding, the ts-v-src cells were then treated with vehicle and drugs for indicated times at 35°C. Consequently, the cells were incubated with methionine-free medium with 10% dialyzed FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 3 g/ml puromycin, in the presence of vehicle or drug, for 30 min at 35°C. The [ 35 S]methionine-containing medium (methionine-free medium with 10% dialyzed FBS, 100 units/ml penicillin, 100 g/ml streptomycin, 3 g/ml puromycin, and [ 35 S]methionine (100 Ci per dish)) with vehicle or drug was then supplied to cells for an additional 30-min incubation at 35°C. After treatment, cells were harvested, lysed, and thus immunoprecipitated with anti-v-Src antibody. Prepared samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The signals were detected by exposing the membranes directly to x-ray films.
Pulse-chase Assay-After seeding, the ts-v-src cells were first incubated with methionine-free medium with 10% dialyzed FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 3 g/ml puromycin for 30 min at 39.5°C. The cells were then subsequently incubated with [ 35 S]methionine-containing medium for 1 h at 39.5°C. Then, the [ 35 S]methionine-containing medium was removed, the cells were treated with vehicle and drugs for indicated times at 35°C. Finally, the amount of 35 S-labeled v-Src protein was determined using immunoprecipitation and autoradiography.

Establishment of a Temperature-inducible v-Src-transformed
Cell Line-To identify compounds that interfere with the process of v-Src-induced transformation, we first established a temperature-inducible v-Src-transformed cell line (ts-v-Src cells) by infecting RK3E/tv-a cells with RCAN-v-src-derived avian retrovirus. The RK3E/tv-a is a tv-a-expressing rat kidney epithelial cell line that allows efficient gene delivery via avian retroviral infection (6). The RCAN-v-src construct contains a temperaturesensitive v-src mutant carrying a point mutation (Ala-to-Pro substitution at residue 507). This mutant has been previously shown to exhibit temperature-dependent kinase activity and transforming ability (32). We first demonstrated that cellular transformation of ts-v-Src RK3E cells could be regulated by the temperature switch. At the permissive temperature (35°C), tsv-Src cells showed a transformed morphology, were able to induce focus formation, and had acquired anchorage-independent growth ability. In contrast, ts-v-Src cells grown at the nonpermissive temperature (39.5°C) displayed a typical epithelial morphology and did not induce focus formation or soft-agar colony formation (supplemental Fig. S1).
Andrographolide Suppresses v-Src-induced Morphological Transformation and Colony Formation-We thus applied ts-v-Src cells for the screening of anti-transformation compounds (all tested compounds are listed in supplemental Table S1). The ts-v-Src cells grown at 39.5°C were treated with vehicle or natural products, immediately switched to the 35°C incubator, and cultured for 24 h to identify the natural products that were able to impede the v-Src-induced transformation process. For each tested compound, multiple concentrations were initially tested, and sublethal dosages were determined for extended studies. Among the natural products examined, only andrographolide exhibited the ability to block the v-Src-induced morphological transformation. As shown in Fig. 1, ts-v-Src-transformed cells grown at 35°C were more refractile in appearance, had more membrane extensions, showed reduced adhesion, and lost cell-cell contact (Fig. 1B). When different concentrations of andrographolide (1, 3, 5, and 10 M) were added prior to temperature switch, we found that morphological alteration was inhibited by andrographolide at 5 M after incubation at 35°C for 24 h (Fig. 1F). The morphology of the andrographolide-treated cells is flat polygonal with clear cell-cell contacts, which is similar to that of non-transformed ts-v-Src cells grown at 39.5°C (Fig. 1A). Andrographolide at 3 M only exhibited a slightly inhibitory effect on the morphological transformation (Fig. 1E), whereas andrographolide at 1 M did not affect the morphological transformation (data not shown). Radicicol, a known v-Src inhibitor, also blocked the v-Src-mediated morphological transformation (Fig. 1D). In contrast, several well known natural compounds such as naringenin (a natural citrus flavanone), baicalin (the active ingredient form Scutellaria baicalensis), and camptothecin (a plant-derived anti-tumor agent) did not affect the v-Src-mediated morphological transformation ( Fig. 1, G-I).
We also examined whether andrographolide could affect anchorage-independent growth ability, another feature of v-Src-transformed cells. The ts-v-Src cells treated with vehicle and andrographolide at 1 M exhibited anchorage-independent growth ability on soft agar. However, andrographolide at 3 M and 5 M showed slightly and strongly inhibitory effects on colony formation, respectively. These results indicate that andrographolide can restrain the v-Src-induced transformation in a concentration-dependent manner.

Andrographolide Treatment Causes the Disorganization of Actin Filaments and Delays v-Src-mediated E-cadherin Downregulation in v-Src-transformed Cells-
The v-Src activation is known to cause the loss of organized actin filaments (33). To determine the effect of andrographolide on actin organization, we stained the intracellular actin cables of andrographolidetreated cells with Texas Red-phalloidin. The ts-v-Src cells at 39.5°C, with inactive v-Src, showed bundled actin filaments. In contrast, ts-v-Src cells, when shifted to 35°C, lost the integrity of actin fibers due to v-Src activation. However, cells treated with andrographolide or radicicol (the positive control) at 35°C still maintained organized actin filaments ( Fig. 2A). The maintenance of normal actin cable distribution in the andrographolide-treated v-Src cells correlates with the resulting non-transformed morphology.
Compared with untreated v-Src-transformed cells, cell-cell contact was retained among andrographolide-treated cells. v-Src is known to down-regulate E-cadherin expression, which contributes to the loss of cell-cell contact (6 -8). Therefore, we further examined whether the expression of E-cadherin was affected by andrographolide. In the control group, the amount of E-cadherin protein declined with increased dosage or incubation time, but the amount of E-cadherin remained unchanged after 12-h of andrographolide treatment in the test group (Fig. 2, B and C). However, the expression of E-cadherin in andrographolide-treated cells decreases after 24 h. ␤-Catenin, another protein involved in cell-cell adhesion, was also examined but andrographolide did not affect the cellular level of ␤-catenin (Fig. 2C).
Andrographolide Reduces the Expression Level of v-Src Protein-We next investigated whether andrographolide blocked v-Src-induced cellular transformation by interfering with the functions of v-Src protein. At first, we examined the protein expression of v-Src after andrographolide treatment. For this purpose, we used an anti-v-Src antibody that specifically recognized exogenous avian Src but not endogenous c-Src. The effects of different concentrations of andrographolide on v-Src protein expression are shown in Fig. 2B. Andrographolide significantly reduced the expression level of v-Src protein in a concentration-dependent manner. In the time-course experiments, we found that v-Src protein expression was substantially reduced after 6 h at 35°C in the presence of andrographolide (Fig. 2C). Furthermore, accompanying the v-Src down-regulation, the phosphorylated Src protein (pY416) also decreased after andrographolide treatment (Fig. 2C).

Andrographolide Changes the Tyrosine Phosphorylation Pattern of v-Src-transformed Cells but Does Not Affect v-Src
Kinase Activity in Vitro-v-Src is an essential tyrosine kinase that regulates various signaling pathways and we therefore next investigated whether andrographolide affects the cellular tyrosine phosphorylation patterns of v-Src-transformed cells. In the presence of andrographolide, global phosphotyrosine patterns were altered (Fig. 3A). Particularly, the tyrosine phosphorylation levels of several proteins were dramatically reduced. Several known downstream targets of v-Src, such as FAK (120 kDa), CAS (94 kDa), paxillin (68 kDa), Shc (52 kDa), annexin II (36kDa), and caveolin-1 (25 kDa) appear to match the sizes of the altered phosphoproteins. However, the identity of these phosphoproteins remains to be verified. Alteration of tyrosine phosphorylation patterns in v-Src-transformed cells by andrographolide treatment may result from the decreased expression of the v-Src protein, or an inhibition of Src kinase activity. We therefore used a universal tyrosine kinase assay to examine whether andrographolide can directly inhibit v-Src kinase activity. As shown in Fig. 3B, andrographolide did not affect v-Src kinase activity. Another possible way to affect functions of v-Src is through interference of proteinprotein interactions. Thus, we also examined whether the binding of v-Src and its-associated proteins is affected by andrographolide. Our results showed that v-Src-mediated protein-protein interactions were considerably reduced by andrographolide (supplemental Fig. S2).

Andrographolide Does Not Affect Expression Level of c-Src Protein-
Several known v-Src inhibitors (geldanamycin, herbimycin A, and radicicol) were previously reported to exhibit distinct effects on c-Src expression. Geldanamycin reduces the level of c-Src protein in cancer cells, while herbimycin A and radicicol show no effects on c-Src expression (34 -38). We thus examined the effect of andrographolide on c-Src expression using vector-infected RK3E/tv-a cells (RCAN). Interestingly, similar to radicicol and herbimycin, andrographolide did not affect protein expression of c-Src (Fig. 4A).
We next investigated whether andrographolide specifically caused degradation of activated v-Src. When ts-v-Src cells grown constantly either at 35°C or 39.5°C were treated with andrographolide, both showed a significant reduction in v-Src protein (Fig. 4B), indicating that the andrographolide caused v-Src reduction regardless of its activation status. Moreover, andrographolide-mediated v-Src protein down-regulation is independent of the cellular transformation status.
Andrographolide Does Not Affect the Transcription and Translation  of v-src-Our data suggested that the reduction in the v-Src protein level by andrographolide was likely the key event causing inhibition of transformation. We thus investigated the molecular mechanisms underlying the andrographolide-mediated v-Src protein down-regulation. We first determined whether andrographolide interfered with viral LTR-driving v-Src transcription. Reverse transcription-PCR experiments with v-Src-specific primers were performed to detect v-src transcripts in andrographolide-treated ts-v-Src cells grown at 35°C for various time periods. As shown in Fig. 4C, andrographolide did not affect mRNA expression of v-src. We further examined whether andrographolide affected the protein synthesis of v-Src. Upon treatment of andrographolide, [ 35 S]methionine-containing medium was used to label newly synthesized v-Src protein. As shown in Fig. 4D, the protein synthesis of v-Src was not affected. Therefore, andrographolide did not affect the transcription and translation of v-src, suggesting that this compound may act on v-Src through the post-translational process.
Ubiquitination of v-Src Is Involved in Andrographolide-induced v-Src Degradation-To further investigate how andrographolide reduces the v-Src protein level, we next determined whether andrographolide treatment facilitated v-Src protein degradation. The ts-v-Src cells grown at 39.5°C were pre-treated with cycloheximide for 1 h before andrographolide treatment at 35°C for various time periods. v-Src protein at indicated time points was measured by Western blotting with a v-Src specific antibody. As shown in Fig. 5A, andrographolide treatment in the presence of cycloheximide substantially reduced v-Src protein level as compared with cycloheximide treatment alone. The pulse-chase assay was conducted to further verify the effect of andrographolide on v-Src degradation. [ 35 S]Methionine-labeled cells were treated with vehicle or andrographolide at 35°C for various time periods. The level of labeled v-Src protein was measured at indicated time by auto-radiography. As shown in Fig. 5B, the labeled v-Src protein was decreased in the presence of andrographolide, whereas v-Src protein remains stable after vehicle treatment. These results suggest that andrographolide facilitates v-Src protein degradation.
According to a previous report (39), the active Src protein can be degraded in an ubiquitin-dependent manner. Therefore, we examined whether andrographolide attenuated v-Src level via ubiquitinmediated degradation. Although andrographolide treatment did slightly increase ubiquitination of v-Src versus vehicle treatment, the signals was hardly detectable (lanes 2 and 3 in Fig. 5C). Alternatively, a proteasome inhibitor (lactacystin) was used to block the rapid degradation of ubiquitinated proteins for enhancing the signals. As shown in lanes 4 and 5 in Fig. 5C, andrographolide treatment in the presence of lactacystin increased the ubiquitination of v-Src as compared with lactacystin treatment alone, suggesting that andrographolide facilitated the v-Src protein degradation via an ubiquitin-dependent manner.
Andrographolide-mediated Inhibition of v-Src Transformation Is Associated with the Down-regulation of Erk1/2 Signaling Pathway-The Erk kinase, PI3K, and STAT3 signaling pathways have previously been shown to be crucial for v-Src-induced transformation in different mammalian cell types (13,14,40). However, the significance of the involvement of these pathways in v-Src-transformation of RK3E cells has not yet been reported. To further determine whether these signaling pathways are associated with the inhibitory effect of andrographolide, we compared the effects of several signaling inhibitors on v-Src transformation with that of andrographolide. For each inhibitor, multiple concentrations were examined for toxicity and only sublethal dosages were chosen for subsequent experiments. For those inhibitors blocking v-Src-mediated morphological transformation, cell morphology from the treatment at the lowest effective concentration is shown in Fig. 6A. Conversely, for those drugs not blocking morphological transformation, cell morphology from treatment at the highest concentrations of doses examined is shown (Fig. 6A). Our results indicated that PI3K inhibitor (LY294002) and STAT3 inhibitor apparently did not block v-Src-mediated morphological transformation but MKK inhibitors (PD98059 and U0126) showed an inhibitory effect similar to that of andrographolide. We next examined the effects of andrographolide treatment on the activation of these signaling pathways. As shown in Fig. 6 (B and C), both active Erk1/2 and Akt level were elevated in v-Src-transformed cells but strongly diminished after andrographolide treatment. On the other hand, STAT3 was not significantly activated in v-Src-transformed RK3E cells and levels of active STAT3 were not affected by andrographolide treatment (data not shown). The calpain-calpastatin proteolytic system has been previously demonstrated to participate in the v-Src-induced morphological transformation of chicken embryo fibroblasts (12). However, treatment with ALLN (an inhibitor of calpain-calpastatin proteolytic pathway) did not block the morphological changes of v-Src-transformed RK3E cells (Fig. 6A). Taken together, our results suggest that down-regulation of the Erk signaling pathway plays a crucial role in the inhibition of v-Src-induced transformation by andrographolide.
Constitutive Active Erk Signaling Interferes with Andrographolide-mediated Inhibition of v-Src Transformation-To further determine whether andrographolide inhibits v-Src-induced transformation via down-regulation of the Erk pathway, we next examined the effect of andrographolide on morphological transformation induced by ts-v-Src cells constitutively expressing active MKK1 protein (the upstream activator of Erk1/2). The ts-v-Src RK3E cells grown at 39.5°C were infected with a retrovirus carrying a gene encoding the constitutively active MKK1 (MKK1-CA) or control virus GFP. After infection, these cells were continuously cultured at 39.5°C for 4 days, then treated with vehicle or andrographolide, and grown at 35°C for 24 h. Upon andrographolide treatment, about half of the v-Src cells expressing MKK1-CA (LA29-MKK1-CA) still retained the transformed phenotype. On the other hand, the majority of enhanced GFP-expressing v-Src cells (LA29-GFP) were switched to a non-transformed phenotype after andrographolide treatment (Fig. 7A, left  panel). The percentages of transformed cells among total cells for each treatment were calculated (Fig.  7A, right panel). Furthermore, andrographolide-treated LA29-GFP cells maintained organized actin filaments (Fig. 7B, left panel). However, LA29-MKK1-CA cells lost organized actin filaments after andrographolide treatment, which is consistent with their transformed phenotype (Fig. 7B, right panel). Expression of HA-tagged MKK1, phospho-ErK1/2, GFP, and actin in LA29-GFP or LA29-MKK1-CA cells under the different treatment are shown in Fig. 7C. Andrographolide apparently did not affect the expression of HA-tagged MKK1, phospho-ErK1/2 and GFP. Taken together, our results demonstrated that andrographolide inhibits v-Src-induced transformation via attenuating of the Erk1/2 signaling pathway.
Andrographolide Treatment Reduces Protein Level of the c-Src Truncation Mutant (Src531)-An activating truncated mutant of c-Src (Src531), structurally similar to v-Src, was previously identified in advanced human colon cancer cells (16). To examine whether andrographolide exhibits inhibitory effects on Src531 protein, we established a few RK3E stable clones expressing Src531 (RK3E/Src531). The cell clones with clear transformed morphology and elevated Src531 protein expression were further studied for their responses to andrographolide treatment. As shown in Fig. 8, andrographolide treatment reduced protein level of Src531 in these cells. Furthermore, andrographolide treatment caused noticeable cell death and the survived cells mostly remain transformed morphology (supplemental Fig. S3).

DISCUSSION
Andrographolide is a lactone diterpenoid originally isolated from the Chinese herb A. paniculata. In recent years, novel pharmacological effects of andrographolide have been identified, including anti-inflammation, anti-apoptosis, anti-angiogenesis, and anti-cancer (22)(23)(24)(25)(26). Nevertheless, it has never been reported that andrographolide inhibits the activity of the v-Src oncoprotein. In this study, we demonstrated that andrographolide blocks v-Src-induced morphological transformation and anchorage-independent cell growth (Fig. 1). Andrographolide treatment leads to the degradation of v-Src protein via ubiquitination (Figs. 2B, 2C, and 5), the attenuation of the phospho-Erk1/2 level (Fig. 6B), the maintenance of actin organization ( Fig. 2A), and the delay of E-cadherin down-regulation (Fig. 2, B and  C). Furthermore, v-Src-mediated protein-protein interactions are also affected by andrographolide (supplemental Fig. S2). Although andrographolide alters the intracellular tyrosine phosphorylation pattern, it does not directly repress the v-Src kinase activity in vitro (Fig. 3).
We speculate that the reduction in v-Src protein expression is the major event of the andrographolide-mediated inhibition of cellular transformation. Our data showed that andrographolide does not interfere with the transcription and translation of v-Src and we further demonstrated that andrographolide facilitates the degradation of v-Src protein through an ubiquitin-mediated pathway (Fig. 5). Noticeably, it has been described that andrographolide suppressed the expression of inducible nitric-oxide synthase in macrophages (23). However, andrographolide treatment does not cause general protein downregulation, because the levels of several other proteins (including actin, ␤-catenin, E-cadherin, Erk, and Akt) were not affected by andrographolide (Figs. 2B, 2C, and 6). Many small molecules are known to target multiple proteins (19), and we therefore cannot, at present, exclude the possibility that andrographolide may also act on other molecular targets in ts-v-Src cells.
According to our results, andrographolide treatment promotes the ubiquitination and degradation of v-Src protein. The ubiquitin-proteasome system is responsible for most protein degradation in mammalian cells. Ubiquitination is mainly mediated by three major enzymes: ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2) and ubiquitin-protein ligase (E3) (41). Because Cbl protein, an E3 ligase, is known to cause ubiquitination and degradation of Src (42), it is possible that andrographolide facilitates the interaction of v-Src protein with Cbl. Alternatively, andrographolide may attenuate functions of the deubiquitylating enzyme, such as AMSH (associated molecule with the Src homology-3 domain of STAM), to promote protein ubiquitination by the E3 ligases (43). Furthermore, accumulating reports suggest that Hsp90 and its co-chaperones play essential roles in the modulation of Src stability. The strong physical association between v-Src and Hsp90 was previously shown to be essential for the transformation activity of v-Src (44 -46). Two v-Src inhibitors, herbimycin A and radicicol, were known to directly perturb Hsp90 and preferentially destabilize v-Src over c-Src protein (37,38). Herein, we have observed that andrographolide specifically reduces v-Src protein level but not c-Src (Figs. 2C and 4A), similar to the two above-mentioned inhibitors. We thus speculate that andrographolide might regulate v-Src protein via interfering with Hsp90-related functions.
Different signaling pathways have been reported to be crucial for v-Src-induced malignant transformation of different cell types. However, v-Src appears to induce malignant transformation via distinct signaling pathways in different cell types. For example, both the MAPK and PI3K signaling pathways are required for transformation of chicken embryo fibroblasts by v-Src (14), but transformation of rat intestinal epithelial RIE-1 cells by v-Src is independent of MAPK activation (13). Furthermore, activation of the STAT3 (signal transducers and activators of transcription 3) signaling pathway and calpain-calpastatin proteolytic system are essential for v-Src-mediated transformation of NIH3T3 cells and chicken embryo fibroblast cells, respectively (11,12). With respect to the v-Src-mediated transformation of RK3E cells in this study, our data demonstrated that both the Erk and PI3K signaling pathways are activated during v-Src transformation process (Fig. 6, B and C), but only the Erk signaling pathway is required for v-Src-induced morphological transformation (Fig. 6A). Furthermore, calpain and STAT3 are not activated in v-Src-transformed RK3E cells (data not shown) and neither ALLN nor STAT3 inhibitor was able to block morphological changes of v-Src-transformed RK3E cells (Fig. 6A). Taken together, these results support the hypothesis that activation of the Erk signaling pathway is mainly responsible for v-Src-induced morphological transformation of RK3E cells.
According to our data, andrographolide treatment remarkably diminished the activation of Erk1/2 and Akt (Fig. 6, B and C). Similar observations have been previously described in other cell types. Andrographolide treatment is known to abolish complement 5a (C5a)-induced macrophage recruitment through inhibition of Erk1/2 and Akt phosphorylation (47). In the present study, although andrographolide inhibited both the Erk and PI3K signaling pathways, only inhibition of the Erk pathway caused a non-transformed morphology (Fig. 6). Furthermore, expression of constitutively active MKK1 (the upstream activator of Erk signaling) abolished the andrographolide-suppressed morphological inhibition (Fig. 7). These results suggest that andrographolide blocks v-Src-mediated transformation mainly via inhibiting the Erk signaling pathway. Such inhibition may result from direct interference with Erk signaling or down-regulation of v-Src by andrographolide. We suspect that down-regulation of Erk signaling pathway is a consequence of andrographolide-induced v-Src degradation based on the following observations. Firstly, down-regulation of Erk activation correlates well with the reduction of v-Src expression/activation in time-course experiments (Figs. 2C and 6). Secondly, Src-expressing cells with constitutive Erk activation are resistant to andrographolide (Fig. 7), suggesting that this compound does not directly interfere with the Erk signaling pathway.
Andrographolide treatment reduced protein level of the c-Src truncation mutant Src531 (Fig. 8), a similar inhibitory effect as observed with v-Src (Fig. 2, B and C). In addition, andrographolide treatment of Src531-transformed cells caused noticeable cell death and the survived cells mostly remained transformed morphology (supplemental Fig. S3). This phenomenon is consistent to our previous observation that andrographolide treatment of well-transformed ts-v-Src cells, constantly grown at permissive temperature, caused noticeable cell death and the survived cells remained transformed morphology (supplemental Fig. S4). We therefore speculate that the cells with constitutive Src activation (in both Src531 stable clones and ts-v-Src cells constantly grown at permissive temperature) become oncogene-addictive, and treatments of oncogene-targeting agents (such as andrographolide in the present study) drive these cells to undergo cell death, a process called "oncogenic shock" (48,49). Interestingly, pretreatment of andrographolide before induction of v-Src activity could suppress cellular transformation initiated by v-Src ( Fig. 1A and  supplemental Fig. S4). The molecular basis for the discrepancy between treatment responses remained to be further examined. Nevertheless, our observation that andrographolide treatment of Src531-transformed cells can cause cell death and reduction of Src531 protein suggests andrographolide may be therapeutically beneficial in treatment of Src-relevant human cancers.
In summary, the results from the present study clearly demonstrate that andrographolide can inhibit v-Src-induced transformation via ubiquitin-dependent v-Src protein degradation and attenuation of the Erk signaling pathway. Our findings pro- vide evidence that andrographolide can exhibit anti-cancer activity by a novel mechanism involving the degradation of Src oncoprotein. To our knowledge, this is the first report to show possible antagonistic effect of andrographolide on v-Src. Whether andrographolide affects the activity or expression of other oncoproteins remains to be determined. Further studies to elucidate the effects of andrographolide on other cancerrelated proteins should certainly provide crucial information about the anti-cancer mechanism(s) of andrographolide.