6-Methoxyethylamino-numonafide inhibits hepatocellular carcinoma xenograft growth as a single agent and in combination with sorafenib

Abstract

Hepatocellular carcinoma (HCC) is the third leading form of cancer worldwide, and its incidence is increasing rapidly in the United States, tripling over the past 3 decades. The current chemotherapeutic strategies against localized and metastatic HCC are ineffective. Here we report that 6-methoxyethylamino-numonafide (MEAN) is a potent growth inhibitor of murine xenografts of 2 human HCC cell lines. At the same dose and with the same treatment strategies, MEAN was more efficacious in inhibiting tumor growth in mice than sorafenib, the only approved drug for HCC. Treatment by MEAN at an effective dose for 6 wk was well tolerated by animals. Combined therapy using both sorafenib and MEAN enhanced tumor growth inhibition over monotherapy with either agent. Additional experiments revealed that MEAN inhibited tumor growth through mechanisms distinct from those of either its parent compound, amonafide, or sorafenib. MEAN suppressed C-MYC expression and increased expression of several tumor suppressor genes, including Src homology region 2 domain-containing phosphatase-1 (SHP-1) and TXNIP (thioredoxin-interacting protein). As an encouraging feature for envisioned clinical application, the IC₅₀ of MEAN was not significantly changed in several drug-resistant cell lines with activated P-glycoprotein drug efflux pumps compared to drug-sensitive parent cells, demonstrating the ability of MEAN to be effective in cells resistant to existing chemotherapy regimens. MEAN is a promising candidate for clinical development as a single-agent therapy or in combination with sorafenib for the management of HCC.-Liu, Y., Lou, G., Norton, J. T., Wang, C., Kandela, I., Tang, S., Shank, N. I., Gupta, P., Huang, M., Avram, M. J., Green, R., Mazar, A., Appella, D., Chen, Z., Huang, S. 6-Methoxyethylamino-numonafide inhibits hepatocellular carcinoma xenograft growth as a single agent and in combination with sorafenib.

Keywords: C-MYC inhibition; HCC therapeutic; MEAN; combination treatment.

Figure 1.

MEAN inhibited Huh7-luc xenograft tumor growth more effectively than sorafenib under same treatment conditions in mice. Starting at d 11 after inoculation of tumor cells subcutaneously, mice were treated with MEAN (15 mg/kg), sorafenib (15 mg/kg), or MEAN and sorafenib in combination at 15 mg/kg each, or vehicle via i.p. administration at schedule of 5 d on and 2 d off for 42 d. Tumor growth was measured by whole-mount imaging of luciferin florescence (representative images) (A), photon counts twice a week (B), or photon count at end of experiment, d 42 (C), by volume (D), or by tumor weight at d 42 (E). *P < 0.5, **P < 0.01 (Student’s t test). Lack of black bar indicates P > 0.5 (n = 5).

Figure 2.

MEAN inhibited HepG2-luc xenograft tumor growth more effectively than sorafenib under same treatment conditions in mice. Starting at d 11 after inoculation of tumor cells s.c., mice were treated with MEAN (15 mg/kg), sorafenib (15 mg/kg), or MEAN and sorafenib in combination at 15 mg/kg each, or vehicle through i.p. administration at schedule of 5 d on and 2 d off for 42 d. Tumor growth was measured by whole-mount imaging of luciferin florescence (representative images) (A), photon counts twice a week (B), or photon count at end of experiment, d 42 (C), by volume (D), and by tumor weight at d 42 (E). *P < 0.5, **P < 0.01 (Student’s t test). Lack of black bar indicates P > 0.5 (n = 5).

Figure 3.

Combination treatment of sorafenib (15 mg/kg) and reduced MEAN (7.5 mg/kg) enhances tumor growth inhibition in Huh7-luc xenograft model. Starting at d 11 after inoculation of tumor cells subcutaneously, mice were treated with MEAN (7.5 mg/kg), sorafenib (15 mg/kg), or MEAN and sorafenib in combination at corresponding concentrations, or vehicle through i.p. administration at schedule of 5 d on and 2 d off for 42 d. Tumor growth was measured by whole-mount imaging of luciferin florescence (representative images) (A), photon counts twice a week (B), or photon count at end of experiment, d 42 (C), by volume (D), or by tumor weight at d 42 (E). *P < 0.5, **P < 0.01 (Student’s t test). Lack of black bar indicates P > 0.5 (n = 5).

Figure 4.

Toxicity induced by treatment of MEAN, sorafenib, or combination of both was measured against vehicle-treated group through monitoring body weight changes throughout treatment course (A) and detecting serum levels of liver enzymes at experimental end point (B). Liver enzymes were recovered 2 wk after experimental end point (d 42) (C). *P < 0.5, **P < 0.01 (Student’s t test). Lack of black bar indicates P > 0.5 (n = 5).

Figure 5.

MEAN has distinct mechanisms of action compared to parent compound AMN. RNA array analyses show that 2 of 3 gene expression changes in treated HepG2 cells are not shared between MEAN and AMN (A). MEAN and AMN differentially altered gene expression in treated HepG2 and Huh7 cell lines in culture. MEAN significantly reduced C-MYC protein levels when using <10 μM, while AMN did not significantly reduce C-MYC expression at same concentration (B, C). MEAN also reduced SIRT1, and increased SHP-1 and TXNIP protein levels, while AMN did not significantly affect levels of these proteins. Intensity of vehicle treatment is set at 1, and ratios of treatment to vehicle are plotted in each quantitative graph (C). Expression levels of several downstream target genes of C-MYC were reduced in MEAN- and AMN-treated cells, while 2 genes are differentially regulated (D). Y axis represents log fold differences in RNA levels of each indicated gene between agents and DMSO (D). *P < 0.5, **P < 0.01 (Student’s t test). Lack of black bar indicates P > 0.5.

Figure 6.

Sorafenib and MEAN differentially affected gene expression in xenograft tumors. Western blot analysis of tumor tissue treated by MEAN, sorafenib, combination of both, or vehicle demonstrated that MEAN suppressed proteins levels of C-MYC and SIRT1, and increased protein levels of SHP-1, similar to findings in culture cells (A, B). Sorafenib did not significantly affect expression of these genes (A, B). Intensity of vehicle is set at 1, and ratios of treatment to vehicle are plotted in each quantitative graph (B). *P < 0.5, **P < 0.01 (Student’s t test). Lack of black bar indicates P > 0.5.