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

Wing Lam, Zaoli Jiang, Fulan Guan, Xiu Huang, Rong Hu, Jing Wang, Scott Bussom, Shwu-Huey Liu, Hongyu Zhao, Yun Yen, Yung-Chi Cheng, Wing Lam, Zaoli Jiang, Fulan Guan, Xiu Huang, Rong Hu, Jing Wang, Scott Bussom, Shwu-Huey Liu, Hongyu Zhao, Yun Yen, Yung-Chi Cheng

Abstract

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.

Conflict of interest statement

Yung-Chi Cheng and Shwu-Huey Liu are the co-inventors of PHY906 for cancer treatment.

Figures

Figure 1. Antitumor activity of PHY906, one…
Figure 1. Antitumor activity of PHY906, one herb deleted PHY906, and/or Sorafenib on HepG2 tumor growth in NCR nude mice.
(A) Effect of PHY906 and/or Sorafenib on HepG2 (human) tumor growth in NCR nude mice. Error bars indicate standard deviations and N = 14. (B) Effect of one herb deleted of PHY906 and Sorafenib on HepG2 tumor growth in NCR nude mice. Error bars indicate standard deviations and N = 5. Sorafenib (30 mg/kg, b.i.d., daily) and PHY906 (500 mg/kg, b.i.d.) was administered orally from day 0 to day 3. Details of experimental procedures are given in Materials and Methods. T-test was used to compare the difference of Sorafenib vs. PHY906 or one herb deleted of PHY906 at day 7. P values are indicated in the graphs.
Figure 2. Effect of PHY906, Sorafenib (So),…
Figure 2. Effect of PHY906, Sorafenib (So), Sorafenib+PHY906 (So+PHY906), Sorafenib+PHY906 deleted P (So+(-P)) and Sorafenib+PHY906 deleted S (So+(-S)) on the induction of apoptosis and growth of HepG2 tumors in NCR nude mice.
Immunohistochemistry staining for cleaved caspase-3 (A, G), cleaved caspase-8(C, I), cleaved caspase-9 (E, K) of HepG2 tumor section after treatment with Sorafenib and So+PHY906 for 48 h and 96 h. Percentage of cleaved caspase-3(B, H, M), cleaved caspase-8(D, J, N), cleaved caspase-9 (F, L, O) stained cell per each view of HepG2 tumor section after the drug treatment for 48 h and 96 h. Each spot represents a mean of the number of heavily stained cells from 4 to 5 views of each tumor section against total live cells (PCNA-stained) in each treatment group. Number of animals are 5 to 9. Sorafenib (30 mg/kg, b.i.d.) and/or PHY906, (-P), (-S) (500 mg/kg, b.i.d.) were administered orally twice a day. Details of experimental procedures are given in Materials and Methods.
Figure 3. Effect of PHY906 and/or Sorafenib…
Figure 3. Effect of PHY906 and/or Sorafenib (So) on the expression of death receptors and their ligands level HepG2 tumor in NCR nude mice.
(A) Heat map for the mRNA expression levels of death receptors and their ligands in HepG2 tumors after 96 h drug treatment (Each bar represents a mean of two to three different experiments (triplicate samples of each; number of animals for 96 h treatment group is 14). The mRNA expression of human FasR (B) and mouse FasL (C) of HepG2 tumor after the drug treatment for 96 h (Each spot represents a mean of two to three different qRT-PCR experiments (triplicate samples of each; number of animals for 48 h treatment group is 5 and number of animals for 96 h treatment group is 14). (D) Immunohistochemistry staining for FasL protein of HepG2 tumor section after the drug treatments (PHY906, Sorafenib (So), Sorafenib+PHY906 (So+PHY906), Sorafenib+PHY906 deleted P (So+(-P)) and Sorafenib+PHY906 deleted S (So+(-S))) for 96 h. (E) Quantification of immunohistochemistry staining of FasL using imaging software. (Each spot represents a mean of the intensity of FasL staining from 5 views of a tumor section; number of animals for 96 h treatment group is 5). Sorafenib (30 mg/kg b.i.d.) and/or PHY906, (-P), (-S) (500 mg/kg b.i.d.) was administered orally twice daily. Details of experimental procedures are given in Materials and Methods.
Figure 4. Effect of PHY906 and/or Sorafenib…
Figure 4. Effect of PHY906 and/or Sorafenib (So) on the infiltration of macrophages in HepG2 tumors in NCR nude mice.
(A) Immunohistochemistry staining for F4/80 of HepG2 tumor section after treatment with Sorafenib (So) or So+PHY906 for 48 h and 96 h. Percentage of F4/80 stained cells per view of HepG2 tumor section after the treatments for 48 h (B) and 96 h(C). Each spot represents a mean of the number of F4/80 stained cells from 4 to 5 views of each tumor section against total live cells (PCNA-stained) in each treatment group. (D) Immunohistochemistry of hMCP1 of HepG2 tumor section tumor after PHY906 and/or Sorafenib (So) treatment for 96 h. (E) Quantification of immunohistochemistry staining of hMCP1 using imaging software. (Each spot represents a mean of the intensity of hMCP1 staining from 5 views of a tumor section; N = 14). (F) Heat map for mRNA expression of M1 and M2 macrophage markers in HepG2 tumor at 96 h following the drug treatment (Each bar represents a mean of two to three different qRT- PCR experiments (triplicate samples of each; N = 14). (G) Bayesian analysis for the probability of M1 phenotype. (H) Effect of clodronate liposomes treatment on macrophage infiltration with Sorafenib (So) or So+PHY906 for 96 h. Liposomes were given using i.p. injection at day -2), day 0 and day 2. (I) Effect of clodronate liposomes treatment on HepG2 tumor growth following treatment with Sorafenib (So) or So+PHY906 for 96 h. (J) Effect of clodronate liposomes treatment on apoptosis of HepG2 tumor following treatment with Sorafenib (So) or So+PHY906 for 96 h, N = 5. Sorafenib (30 mg/kg, b.i.d.) and PHY906 (500 mg/kg b.i.d.) were administered orally twice a day. Details of experimental procedures are given in Materials and Methods.
Figure 5. Effect of PHY906 and/or Sorafenib…
Figure 5. Effect of PHY906 and/or Sorafenib (So) on the autophagy of HepG2 tumors in NCR nude mice.
(A) Immunohistochemistry staining for LC3A of HepG2 tumor section after the treatment of Sorafenib and So+PHY906 for 48 h and 96 h. Percentage of LC3A stained cell per each view of HepG2 tumor section after the treatment of PHY906 and/or Sorafenib (So) for 48 h (B) and 96 h(C). Each spot represents a mean of the number of LC3A stained cells from 4 to 5 views of each tumor section against total live cells (PCNA-stained) in each treatment group. The number of animals in the 48 h treatment group is 5 and the number of animals for the 96 h treatment group is 14. (D) Immunohistochemistry staining for AMPKα T172P and ULK1 S555P of HepG2 tumor sections after drug treatments for 96 h. Quantification of immunohistochemistry staining of AMPKα T172P (E) and ULK1 S555P (F) using imaging software. (Each spot represents a mean of the intensity of brown color from 5 views of a tumor section; number of animals for 96 h treatment group is 14). Quantitation of immunohistochemistry staining for LC3A (G), AMPKα T172P (H) and ULK1 S555P (I) of HepG2 tumor sections following treatment of Sorafenib (So) or So+PHY906 with control liposome or clodronate liposome for 96 h. Liposomes were given using i.p. injection at day -2, day 0 and day 2. Each spot represents a mean of the number LC3A stained cells or the intensity of AMPKα T172P and ULK1 S555P staining from 4 to 5 views of each tumor section in each treatment group. Details of experimental procedures are given in Materials and Methods.
Figure 6. Effect of PHY906, Sorafenib (So),…
Figure 6. Effect of PHY906, Sorafenib (So), Sorafenib+PHY906 (So+PHY906), Sorafenib+PHY906 deleted P (So+(-P)), and Sorafenib+PHY906 deleted S (So+(-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 β-glucuronidase treated PHY906 (500 μg/ml) on dephosphorylation rate of Erk1/2 in HepG2 cells following stimulation with EGF (50 ng/ml). β-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 β-glucuronidase treated PHY906 (500 μg/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) (F) Quantification of the Western blot results for the phosphorylated Erk1/2 (Thr202/Tyr204). Details of experimental procedures are given in Materials and Methods.

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