PHY906(KD018), an adjuvant based on a 1800-year-old Chinese medicine, enhanced the anti-tumor activity of Sorafenib by changing the tumor microenvironment

PHY906 (KD018) is a four-herb Chinese Medicine Formula. It has been shown to potentially enhance the therapeutic indices of different class anticancer agents in vivo. Here, PHY906 is reported to enhance the anti-tumor activity of Sorafenib in nude mice bearing HepG2 xenografts. Among the four herbal ingredients of PHY906, Scutellaria baicalensis Georgi (S) and Paeonia lactiflora Pall (P) are required; however, S plays a more important role than P in increasing tumor apoptosis induced by Sorafenib with an increase of mouse(m)FasL and human(h)FasR expression. PHY906 may potentiate Sorafenib action by increasing hMCP1 expression and enhancing infiltration of macrophages into tumors with a higher M1/M2 (tumor rejection) signature expression pattern, as well as affect autophagy by increasing AMPKα-P and ULK1-S555-P of tumors. Depletion of macrophage could counteract PHY906 to potentiate the anti-tumor activity of Sorafenib. It was reported that tumor cells with higher levels of ERK1/2-P are more susceptible to Sorafenib, and the S component of PHY906 may increase ERK1/2-P via inhibition of ERK1/2 phosphatase in HepG2 tumors. PHY906 may potentiate the anti-hepatoma activity of Sorafenib by multiple mechanisms targeting on the inflammatory state of microenvironment of tumor tissue through two major ingredients (P and S) of PHY906.

PHY906 (KD018) is a four-herb Chinese Medicine Formula. It has been shown to potentially enhance the therapeutic indices of different class anticancer agents in vivo. Here, PHY906 is reported to enhance the anti-tumor activity of Sorafenib in nude mice bearing HepG2 xenografts. Among the four herbal ingredients of PHY906, Scutellaria baicalensis Georgi (S) and Paeonia lactiflora Pall (P) are required; however, S plays a more important role than P in increasing tumor apoptosis induced by Sorafenib with an increase of mouse(m)FasL and human(h)FasR expression. PHY906 may potentiate Sorafenib action by increasing hMCP1 expression and enhancing infiltration of macrophages into tumors with a higher M1/M2 (tumor rejection) signature expression pattern, as well as affect autophagy by increasing AMPKa-P and ULK1-S555-P of tumors. Depletion of macrophage could counteract PHY906 to potentiate the anti-tumor activity of Sorafenib. It was reported that tumor cells with higher levels of ERK1/2-P are more susceptible to Sorafenib, and the S component of PHY906 may increase ERK1/2-P via inhibition of ERK1/2 phosphatase in HepG2 tumors. PHY906 may potentiate the anti-hepatoma activity of Sorafenib by multiple mechanisms targeting on the inflammatory state of microenvironment of tumor tissue through two major ingredients (P and S) of PHY906.
T he most common type of liver cancer, hepatocellular carcinoma (HCC) has a 5-year survival rate of only ,14%. HCC patients usually present in advanced stages, at which point surgical resection and/or chemical embolism are no longer feasible 1 . The median prognosis of patients with unresectable and recurrent HCC ranges from 3 to 7 months 2 . Sorafenib, an inhibitor of the RAF/MEK/ERK pathways as well as tyrosine kinase receptors such as VEGF, PDGF, and Kit, is the only FDA-approved drug for the treatment of HCC 3 . The most common side effects of Sorafenib are diarrhea, hand-foot skin reactions, nausea, fatigue, and pain. Unfortunately, the treatment only increases median survival time by 3-4 months 4 ; hence, more effective treatments for advanced HCC are needed.
PHY906(KD018) is currently being developed as an adjuvant for chemotherapy. PHY906 (KD018) is based on the Huang Qin Tang herbal mixture, which was first described in Chinese texts 1800 years ago for treatment of numerous gastrointestinal symptoms, including diarrhea, nausea, and vomiting. The mixture consists of four herbs: Glycyrrhiza uralensis Fisch (G), Paeonia lactiflora Pall (P), Scutellaria baicalensis Georgi (S), and Ziziphus jujuba Mill (Z). PHY906(KD018) was prepared using high-quality herbs picked by experienced herbalists and manufactured according to cGMP (current Good Manufacturing Practice). Consistent preparations of PHY906 have been made over a period of 10 years as demonstrated by Phytomics QC using standardized chemical and biological fingerprints 5 .
In preclinical studies, PHY906 was demonstrated to reduce gastrointestinal toxicity caused by irinotecan (CPT-11) treatment, while enhancing the anti-tumor activity of CPT-11 6 . All four herbs of PHY906 were required to maximally enhance the therapeutic activity of CPT-11 in vivo 6 . PHY906 was shown to reduce CPT-11-induced intestinal inflammation by inhibiting the NFkB, COX2, and iNOS pathways 6 . It was also found to promote intestinal progenitor cell repopulation in the damaged intestine by both increasing expression of several components of the Wnt signaling pathway and potentiating wnt3a action 6 . Recently, PHY906 was demonstrated to decrease toxicity from fractionated abdominal irradiation by promoting faster recovery of the intestine 7 . In tumors, PHY906 was shown to increase the anti-tumor activity of CPT11, resulting in an increase in apoptosis and macrophage infiltration. mRNA array study suggests that PHY906 plus CPT-11 alters the gene expression profiles of tumor to become highly inflamed and pro-apoptotic, favoring tumor rejection 8 .
Currently, three phase I/II and one phase II clinical trials for PHY906 have been finished in U.S. In these clinical studies, PHY906 was demonstrated to reduce chemotherapy-associated side effects for patients with metastatic colorectal cancer (mCRC) (PHY9061 CPT-11/5FU/LV) 9,10 and advanced pancreatic cancers (PHY9061 capecitabine) 11,12 . In a phase I/II clinical trial for advanced hepatocellular carcinoma (HCC), PHY906 appeared to decrease capecitabine-associated toxicity 13 . The median patient survival time was 10.9 months, which is comparable to that of patients treated only with Sorafenib in U.S./European trials and much longer than the 7-month median survival seen in Asian Sorafenib trials 13 . Since Sorafenib is the only approved drug for the treatment of HCC, and since diarrhea is a serious side effect of Sorafenib treatment, we used HepG2-bearing nude mice as a model to study the combination effect of PHY906 and Sorafenib on tumor growth. Our results support the possibility that PHY906 might improve the therapeutic index of Sorafenib in patients with advanced liver cancer, a combination currently entering phase I clinical trials in the United States (National Clinical Trial Identifier: NCT01666756).

Results
PHY906 enhances the antitumor activity of Sorafenib against HepG2 tumors without affecting the body weight of animals. For one cycle (7 day) of drug treatment, PHY906 (500 mg/kg, p.o) was fed twice per day from day 0 to day 3 either with or without Sorafenib (30 mg/kg, b.i.d, p.o, daily) to animals harboring HepG2 tumors, and HepG2 tumor growth and nude mice weight loss were studied. Animal body weights were not affected by any treatments (Fig S1A S2B), and HepG2 tumor growth was not affected by PHY906 treatment (P . 0.05) (Fig. 1A). Whereas Sorafenib reduced HepG2 tumor growth by 35% at day 8 as compared to the control group (Fig. 1A), Sorafenib with PHY906 (So1PHY906) completely inhibited HepG2 tumor growth (P , 0.001) and even enhanced the shrinkage of HepG2 tumors during treatment (Fig. 1A). Increasing Sorafenib treatment to 60 mg/kg or 120 mg/ kg b.i.d did not enhance its antitumor activity, with or without PHY906 co-treatment ( Fig S2A). So1PHY906 could continue inhibit HepG2 tumor growth within two-cycle treatment or three cycle treatment. (Fig S2A S2C). In our study, Sorafenib did not cause histological changes in the small intestine ( Fig S3). In the following preclinical studies, we used treatment concentrations of 500 mg/kg (b.i.d) for PHY906 and 30 mg/kg (b.i.d) for Sorafenib.
Comparing the antitumor activity of Sorafenib to that of So1PHY906 minus one herb component (PHY906-G, PHY906-P, PHY906-S, PHY906-Z) revealed that deletion of either P or S eliminated PHY906's synergistic activity with Sorafenib (Fig. 1B). Thus, P and S are essential to PHY906's enhancement of Sorafenib's antitumor activity.
PHY906 also enhanced the antitumor activity of Sorafenib against Hepa1-6 tumors in both nude and BDF1 mice (immunocompetent mouse) (Fig S1B-D). Therefore, T-cell deficiency is not required for PHY906 to enhance the antitumor activity of Sorafenib.
Deletion of P(P 5 0.002) or S(P 5 0.006) from PHY906 diminished Sorafenib's enhancement of antitumor activity at 96 h posttreatment ( Fig S5). Removal of either component was accompanied by significant decreases in cleaved caspase-3 ( Fig. 2M, S6), cleaved caspase-8 ( Fig. 2N, S6), and cleaved caspase-9 ( Fig. 2O, S6) compared to So1PHY906 treatment (All P values , 0.05). Meanwhile, the number of the cleaved caspases of So1(-S) were lower than those of So1(-P)(All P values , 0.05). These results are consistent with the hypothesis that S plays a more important role than P in increasing apoptosis of HepG2 tumors when combined with Sorafenib.

So1PHY906 increases both macrophage infiltration and the M1/ M2 (tumor rejection) signature expression pattern in tumor cells.
Since macrophage cells could secrete mFasL, the macrophage infiltration of tumor grafts was examined in mice treated with either So1PHY906 or Sorafenib alone. The number of macrophages in HepG2 tumors was increased by So1PHY906 but not Sorafenib as early as 48 h post-treatment (P 5 0.021) (Fig. 4A 4B). At 96 h, Sorafenib induced macrophage infiltration but to a lesser extent than So1PHY906(P 5 0.0008) (Fig. 4A 4C). So1PHY906 treatment could also induced macrophage infiltration in Hepa 1-6 tumors (murine tumor) in both nude mice and BDF1 mice after 9day treatment (Fig S8A-C). In contrast to So1PHY906, Sorafenib did not induce macrophage infiltration of Hepa 1-6 tumors in nude www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9384 | DOI: 10.1038/srep09384 mice but reduced macrophage number of Hepa 1-6 tumor in BDF mice (Fig S8A-C). These results suggest that So1PHY906 induced macrophage infiltration of tumors is not restricted to T cell deficit nude mice. However, Sorafenib might reduce macrophage number of tumors when T cells are present.
In order to determine if infiltration of macrophage is required for PHY906 to enhance the antitumor activity of Sorafenib, clodronate liposomes were used to deplete macrophages during the treatments. Results indicated that clodronate liposomes could significantly deplete macrophages in HepG2 tumors in comparing to control liposome (P 5 0.005) (Fig. 4H, S11A). Under clodronate liposome conditions, PHY906 still enhanced the macrophage infiltration in HepG2 tumors following So1PHY906 treatment (P 5 0.04) but the number of macrophage was significantly lower than that of control liposome group (P 5 0.048) (Fig. 4H, S11A). Although PHY906 still enhanced the anti-tumor activity of sorafenib under macrophage depleted conditions (P 5 0.001) (Fig. 4I), tumors of clodronate liposome/So1PHY906 group are significantly larger than those of control liposome/So1PHY906 group (P 5 0.02) (Fig. 4I). In addition, under macrophage depleted conditions, PHY906 no longer significantly enhanced cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 of HepG2 tumors when co-treated with Sorafenib (P 5 0.45, P 5 0.13, P 5 0.55, respectively) ( Fig. 4J, S11B, S11C). Therefore, macrophage infiltration induced by PHY906 plays a key role in enhancing the antitumor activity of Sorafenib against HepG2 tumor growth.
So1PHY906 increases autophagy associated with AMP-activated protein kinase a (AMPKa) and ULK1 phosphorylation. In addition to the increase in apoptosis, So1PHY906 also increased autophagy markers in HepG2 tumors at 96 h as indicated by LC3A staining (Fig. 5A-C). There are several pathways leading to autophagy; So1PHY906 treatment had no effect on phosphorylation of either AKT or S6K, which are respectively upstream and downstream of mTOR (Fig S12). ULK S758, which was reported to be dephosphorylated in the case of mTOR inhibition 15 , was not affected by any treatments (Fig S12). Meanwhile, AMPKa-T172-P and ULK1-S555-P, which were reported to be phosphorylated during autophagy 16 , were more highly induced in the So1PHY906 group than in the Sorafenib-only group(P 5 0.002 and P 5 0.00002, respectively) ( Fig. 5D, 5E, 5F). The increase of LC3A and AMPKa-T172-P but not ULK1-S555-P by So1PHY906 could be counteracted by clodronate liposome treatment (Fig. 5G, 5H, 5I). This implies that macrophage infiltration could be an importance factor in promoting autophagy by increasing AMPKa-T172-P of HepG2 tumors under So1PHY906 treatment conditions. P and S were found to be equally important in preserving So1PHY906's induction of autophagy marker LC3A since tumor sections from So1PHY906 minus either P or S exhibited significantly lower levels of LC3A than those treated with complete So1PHY906 at 96 h post-treatment (P values , 0.05) (Fig S13A,  S13B). Deletion of S or P reduced AMPKa-T172-P (P 5 0.03 or P 5 www.nature.com/scientificreports 0.06, respectively) as compared to AMPKa-T172-P of So1PHY906 (Fig S13A, S13C). P and S were also both required for So1PHY906 to increase expression of ULK1-S55P (Fig S13A, S13D).
We further examined if PHY906 could have impact on ERK1/2 phosphorylation in cell culture. Since b-glucuronidase treatment could affect PHY906's activity on different signal pathways 6 , we also compared PHY906 with or without b-glucuronidase treatment on ERK1/2 phosphorylation in HepG2 cell. Results indicated that bglucuronidase-treated PHY906 reduced the dephosphorylation rate of ERK1/2-P but not p38-P, SAPK/JNK-P, or ERK5-P following EGF or H 2 O 2 treatment (Fig. 6C, 6D, S16A and S18A-C). b-glucuronidase itself did not affect the dephosphorylation rate of ERK1/2-P ( Fig  S17A S17B).This suggests that b-glucuronidase-treated PHY906 is quite selective in inhibiting ERK1/2 phosphatase(s). b-glucuronidase-treated S had a significant inhibitory effect on ERK1/2 phosphatase(s) (P , 0.001: S vs Con at 1.5 h) (Fig. 6E, 6F and S16B) but not as strong as b-glucuronidase-treated PHY906 (P 5 0.055: S vs PHY906 at 1.5 h). Deletion of S but no other herbs from PHY906 could abolish the ERK1/2 phosphatase(s) inhibitory effect (P . 0.05: -S vs Con at 1.5 h) (Fig. 6E, 6F and S16B). This result suggests that S plays the most importance role of PHY906 in inhibiting and ERK1/2 phosphatase(s). Other herbs may also contribute some extent to ERK1/2 phosphatase(s) inhibition. Immunohistochemical staining data corroborated these results (Fig. 6A, 6B), demonstrating that deletion of S from So1PHY906 did not increase Erk1/2 phosphorylation in HepG2 tumors. The chemicals and ERK1/2 phosphatase(s) involved this process is currently being investigated.

Discussion
PHY906 enhances the antitumor activity of Sorafenib against hepatoma growth in vivo. Combining PHY906 with either Sorafenib or CPT-11 resulted in increased tumor apoptosis 8 . Moreover, the S and P components of PHY906 are crucial to PHY906's enhancement of both Sorafenib and CPT-11's antitumor activity 19 . S was found to be more important than P for So1PHY906's induction of apoptosis, though both components are crucial for the apoptosis phenotype. This agree with our previous study that found S had stronger activity than P in enhancing CPT-11's antitumor activity 19 . Single herb S or P had much lower activity than full formulation PHY906 in enhancing the action of CPT-11 against tumor growth 19 .
So1PHY906 differentially increased human FasR in tumor cells and murine FasL in host cells. Both P and S were found to be important for inducing FasL protein expression. As the Fas receptor and ligand were both up-regulated, Fas-mediated cell death pathways could be synergistically activated. This hypothesis is supported by previous studies in which Vorinostat (histone deacetylase inhibitor) was demonstrated to promote sorafenib in killing multiple colon cancer cell lines and HepG2 cells by increasing FasR (CD95) activation and/or expression of FAS ligand in an NFkB-dependent fashion 20,21 . Indeed, more cleaved caspase-8 (downstream of death receptors) was detected in tumors following So1PHY906 treatment. It is possible that So1PHY906 indirectly induces cleavage of caspase-9 in HepG2 tumors by increasing caspase-8 expression. Tumor macrophage infiltration was another commonly observed result of both So1PHY906 and CPT-111PHY906 treatment 8 . This macrophage infiltration phenomenon was also observed when So1 PHY906 was administered to nude mice or BDF mice bearing Hepa 1-6 tumors, suggesting that the macrophage infiltration caused by So1PHY906 does not require T-cells. MCP1 is the cytokine responsible for the tumor macrophage infiltration phenotype in mice treated with So1PHY906 and CPT111PHY906. We further dem-onstrate that macrophage infiltration plays a key role for PHY906 to enhance the antitumor activity of Sorafenib against HepG2 tumor growth. We also demonstrated that P and S were essential for PHY906's induction of hMCP1, which, in the presence of Sorafenib, may help attract macrophages to tumors. A biostatistical model suggests that So1PHY906 treatment induces a tumor environment with an increased M1 signature, supporting the presence of M1 macrophages that are believed to have tumor rejection activity 14 . It should be notice that PHY906 could inhibit intestine inflammation triggered by CPT11 by inhibiting many inflammation pathways including NF-kB, COX2 and iNOS 6 while PHY906 could converse tumor inflammation from chronic status to acute status associated with strong macrophage infiltration following CPT-11 8 and Sorafenib treatment. Indeed, PHY906 could have different impacts on gene expression on different tissues 8 . According to our clinical studies, only few compounds of PHY906 can be passed thought the intestine and get into plasma in which many new metabolites of PHY906 were detected 22 . This could partly explain why intestine and tumor tissues had different inflammation responses to PHY906.
Autophagy helps recycle unused cellular material to prolong survival under starvation conditions 23 . Recently, autophagy has also been found to promote cell death in extremely harsh conditions and is resultantly being considered as a novel chemotherapeutic target 24 . Sorafenib has been reported to induce autophagy in different HCC cell lines, including HepG2 25 . Here, PHY906 was demonstrated to enhance Sorafenib-induced autophagy, possibly by increasing the phosphorylation of AMPKa-T172-P and ULK1-S555-P. In addition, macrophage infiltration could be involved for autophagy via AMPKa-T172-P. P and S were found to play critical roles in this process.
It has previously been found that HCC tumors expressing higher levels of ERK1/2 phosphorylation are more susceptible to Sorafenib treatment both in vitro and in vivo 17,18 . Here, we demonstrate that PHY906 treated with recombinant b-glucuronidase increases ERK1/ 2 phosphorylation in HepG2 tumors by inhibiting ERK1/2 phosphatase(s). Potential targets of PHY906 include DUSP5, DUSP6, DUSP7, and DUSP9, which are reported to selectively dephosphorylate ERK1/2 26 . Prolonged ERK activation is believed to promote the antitumor activity of many chemotherapeutic agents 27 through Fas/ caspase-8-mediated apoptosis [28][29][30] . The strong Erk1/2 phosphatase inhibitory activity of S may help to explain why S plays a more important role than P in So1PHY906-triggered apoptosis. In addi-tion, ERK activation is reported to be required for LC3-II formation 31 , induction of beclin 1 31 and BNIP-3 32 , and activation of the RGS19-Gai3-AGS3 signaling cascade 33 during autophagy. Hence, prolonged ERK1/2 phosphorylation by PHY906 may facilitate Sorafenib-induced autophagy.
In conclusion, PHY906 is able to potentiate the antitumor activity of Sorafenib through multiple mechanisms. First, So1PHY906 could enhance both apoptosis via up-regulation of the Fas/Fas ligand pathway and tumor autophagy via activation of AMPK pathway. Second, PHY906 could increase Erk1/2 phosphorylation which may facilitate Fas/caspase-8-mediated apoptosis [28][29][30] and tumor autophagy [31][32][33] . Third, PHY906 could also potentiate Sorafenib action by up-regulation of the expression of MCP1 which could attract M1 type macrophages to help rejecting tumors. Both P and S are essential components of PHY906, but S plays a more important role in enhancing Sorafenib's induction of apoptosis and Erk1/2 phosphorylation. We propose that dead tumor cells produce local inflammatory substances that interact with PHY906, stimulating the inflammatory process and ultimately triggering infiltration of macrophages with the M1 type phenotype. Thus, PHY906 may potentiate various antitumor agents that work synergistically in the tumor microenvironment. Based on this preclinical study, a phase I/II clinical trial has been initiated for the treatment of HCC in patients using Sorafenib with a PHY906 adjuvant in US.

Methods
Animal Studies. HepG2 cells (1-2 3 10 6 cells in 0.1 ml phosphate-buffered saline) were transplanted subcutaneously into six-week-old female NCR nude mice (Charles River Laboratories, Wilmington, MA). After 10-14 days, mice with tumor sizes of 150-300 mm 3 were selected. Tumor volume was estimated by using the formula length 3 width 2 3 p/6. Unless otherwise indicated, treatment groups each consisted of five mice. Tumor size, body weight, and mortality of the mice were monitored daily. For one cycle of drug treatment, PHY906 was administered orally (p.o.) twice a day for four days (b.i.d., 500 mg/kg) at approximately 10:00am and 3:00pm each day, while Sorafenib was administered orally (p.o.) twice a day for seven days (b.i.d., 30 mg/kg) at approximately 10:00am and 3:00pm each day. On Day 0, PHY906 was administered 30 minutes prior to Sorafenib administration. In the control groups, mice were administered water for oral administration. PHY906 lot number 10 was used for all animal experiments and cell culture experiments. PHY906 lot number 10 has been used in our previous clinical trial 22 and its chemical profile has been reported 22 . Chemical profile of PHY906 lot number 10 is very similar to other lots of PHY906 as indicated by Phytomics Similarity Index (PSI) . 0.9 (1, perfectly identical; 0, no similarities) (Please see supplementary table 1). Clodronate or control liposomes (5 mg/ml) given using i.p. injection at day -2(0.4 ml/mouse), day 0 and day 2 (0.2 ml/mouse). Immunohistochemistry was used to detect protein expression in tumor tissues. qRT-PCR was used to quantify mRNA expression. HepG2, was obtained from American Type Culture Collection (American Type Culture Collection, Manassas, VA) on 2003 without further authenticated. Animal experimental protocols were approved by Yale University Institutional Animal Care and Use Committee (IACUC). All animal experiments were carried out in Immunohistochemistry. Mice (NCR nude bearing HepG2 tumors) were terminated by cervical dislocation at 48 h or at 96 h after initiation of drug treatment (see above). Tumor and intestinal tissues were removed, fixed in formalin, embedded in paraffin, and sectioned into 10mm. The sections were mounted on Superfrost slides, deparaffinized with xylene, and gradually rehydrated. Antigen retrieval was achieved by 10 mM sodium citrate pH6.0 with 0.02% Tween-20 under steam for 30 minutes. The primary antibodies were diluted using Tris-HCl buffer containing 1% BSA and 0.5% Tween-20 and treated slides were incubated at room temperature for one hour. As a negative control, a set of slides was processed without primary antibody. Superpicture immunohistochemistry detection kit (Life Technologies, Inc. Norwalk, CT) was used for detection. The slides were counterstained with hematoxylin and mounted. These antibodies were used: E.coli b-glucuronidase treatment. PHY906 or other herbal extracts (50 mg/ml,Tris-HCl 100 mM,pH 6.8) were incubated with recombinant E.coli b-glucuronidase with 6xHIS at C-terminal (40 mg/ml) at 37uC in a water bath for one hour. After incubation, herbal extracts was sterilized by boiling at 100uC for 5 min.
Western Blotting. HepG2 Cells were lysed in 23 SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT, and 0.05% bromophenol blue) and sonicated for 10 s to shear DNA. The whole-cell extracts were then electrophoresed through 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc) with a Mini PROTEANII transfer apparatus (Bio-Rad). The membranes were blocked and probed in TBS-T buffer (13 Tris-HCl 50 mM pH7.6, NaCl 150 mM, 0.2% Tween-20) containing 5% non-fat milk. Primary antibodies (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XPH Rabbit mAb #4370, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb #4668, Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XPH Rabbit mAb #4511, Cell Signaling Technology, Inc.) at 151000 in TBS-T were incubated with the membrane overnight at 4uC. b-actin was used as an internal control for normalization and Figure 6 | Effect of PHY906, Sorafenib (So), Sorafenib1PHY906 (So1PHY906), Sorafenib1PHY906 deleted P (So1(-P)), and Sorafenib1PHY906 deleted S (So1(-S)) on Erk1/2 phosphorylation of HepG2 tumors in NCR nude mice. (A) Immunohistochemistry staining for phosphorylated Erk1/2 (Thr202/Tyr204) in HepG2 tumor sections after the drug treatment for 96 h. (B) Quantification of immunohistochemistry staining of Erk1/2 phosphorylation using imaging software. (Each spot represents a mean of the intensity of brown color from 5 views of a tumor section; number of animals is 5). (C) Western blotting analysis for the effect of PHY906 or E.coli b-glucuronidase treated PHY906 (500 mg/ml) on dephosphorylation rate of Erk1/2 in HepG2 cells following stimulation with EGF (50 ng/ml). b-actin was used as the loading control for normalization. Cropped blots are used in this figure and they have been run under the same experimental conditions (please see the full-length bolts in Fig S16A) (D) Quantification of the Western blot results for the phosphorylated Erk1/2 (Thr202/Tyr204). (E) Western blotting analysis for the effect of E.coli b-glucuronidase treated PHY906 (500 mg/ ml), equivalent concentration of single herbs (G, P, S, Z), or equivalent concentration of a one herb deleted formula (-G, -P, -S, -Z) on dephosphorylation rate of Erk1/2 in HepG2 cells following stimulation with EGF (50 ng/ml). Cropped blots are used in this figure and they have been run under the same experimental conditions (please see the full-lenght bolts in Fig S16B)  After washing with TBS-T, the membranes were then further incubated with horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (155000; Sigma), The immunoreactive bands were visualized by enhanced chemiluminescence reagents (Perkin-Elmer Life Science Products, Boston, MA), and densitometry scanning was performed with the densitometer (Molecular Dynamics).
Quantitative Real-Time PCR. Total RNA was isolated using a modified TRIzolH protocol. Following the manufacturer's instructions, we collected the aqueous phase and then added ethanol. Before centrifuging, this slurry was added to a column (miRNeasy, Qiagen, Venlo, Limburg) for further extraction and simultaneous DNA digestion (RNase-Free DNAse set, Qiagen). cDNA was synthesized using random primers and reverse transcriptase MMLV (New England Biolabs, Ipswich, MA). qPCR assays were performed using iTaq TM SYBRH Green Supermix and the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Primer sets were listed in the supplementary method.
Statistical analysis. Data were analyzed by one-way or two-way ANOVA (GraphPad Prism 4, San Diego, CA), Student's T-test (Microsoft Office Excel). Differences were considered statistically significant when P , 0.05.