Bioengineered human arginase I with enhanced activity and stability controls hepatocellular and pancreatic carcinoma xenografts

Abstract

Hepatocellular carcinoma (HCC) and pancreatic carcinoma (PC) cells often have inherent urea cycle defects rendering them auxotrophic for the amino acid l-arginine (l-arg). Most HCC and PC require extracellular sources of l-arg and undergo cell cycle arrest and apoptosis when l-arg is restricted. Systemic, enzyme-mediated depletion of l-arg has been investigated in mouse models and human trials. Non-human enzymes elicit neutralizing antibodies, whereas human arginases display poor pharmacological properties in serum. Co(2+) substitution of the Mn(2+) metal cofactor in human arginase I (Co-hArgI) was shown to confer more than 10-fold higher catalytic activity (k(cat)/K(m)) and 5-fold greater stability. We hypothesized that the Co-hArgI enzyme would decrease tumor burden by systemic elimination of l-arg in a murine model. Co-hArgI was conjugated to 5-kDa PEG (Co-hArgI-PEG) to enhance circulation persistence. It was used as monotherapy for HCC and PC in vitro and in vivo murine xenografts. The mechanism of cell death was also investigated. Weekly treatment of 8 mg/kg Co-hArgI-PEG effectively controlled human HepG2 (HCC) and Panc-1 (PC) tumor xenografts (P = .001 and P = .03, respectively). Both cell lines underwent apoptosis in vitro with significant increased expression of activated caspase-3 (P < .001). Furthermore, there was evidence of autophagy in vitro and in vivo. We have demonstrated that Co-hArgI-PEG is effective at controlling two types of l-arg-dependent carcinomas. Being a nonessential amino acid, arginine deprivation therapy through Co-hArgI-PEG holds promise as a new therapy in the treatment of HCC and PC.

Figures

Figure 1

After day 1, the IC50 was 0.75 ± 0.36 and 0.44 ± 0.04 nM for HepG2 (A) and Panc-1 (B) cells, respectively, based on mitochondrial reductase activity (MTT). Flow cytometry demonstrated that HepG2 cells treated with 1 nM arginase (C) increased the apoptotic fraction (Annexin V-positive only, P = .001) and dead cell fraction (double positive for Annexin V and 7-AAD, P = .0003) with an appropriate decrease in viability (double negative, P = .0001). A similar decrease in the viability of Panc-1 cells was found (D), albeit less so when compared with the HepG2 cells (P = .004). Necrotic cells were identified as being Annexin V-negative, 7-AAD-positive cellular events.

Figure 2

Protein immunoblot demonstrated increased levels of LC3B-II after a single day of treatment with 1 nM Co-hArgI or placement in l-arg-free medium in HepG2 cells (A). This continued but decreased by day 4 after treatment without medium replacement. Panc-1 cells initially had decreased levels of LC3B-II 1 day after treatment but increased expression by day 4. Activated (cleaved large fragment) caspase-3 increased slightly after 1 day of treatment in HepG2 cells, whereas this was more clearly seen for Panc-1 cells 4 days after treatment. Investigation with flow cytometry demonstrated that more individual Panc-1 cells (B) contained similar levels of ASS-1 (P = .13), increased levels of activated caspase-3 (P = .0009), and similar Ki-67 expression (P = .66) 4 days after l-arg treatment compared with control cells. Likewise, more HepG2 cells (C) expressed ASS-1 (P = .0001), activated caspase-3 (P < .0001), and Ki-67 (P = .0002). All media contained 10% FBS which may contain some l-arg.

Figure 3

(A) Panc-1 subcutaneous xenografts in nude BALB/c mice demonstrated decreased tumor volume due to treatment with 8 mg/kg Co-hArgI-PEG (P = .03). (B) Likewise, HepG2 subcutaneous xenografts also demonstrated significantly smaller tumors, but they did so within 1 week of treatment (P = .001). (C) At the end of the experiment, PEG-Co-hArgI-treated tumors were much smaller as exemplified in the HepG2 tumors.

Figure 4

Although there is some necrosis (*) in HepG2 tumors from mice treated with PBS controls (A; hematoxylin and eosin stain, bright red areas are intravascular blood), there was significantly higher grades of necrosis in the tumors from arginase-treated mice (B). Likewise, a few cells in control tumors expressed activated caspase-3 (C; immunohistochemistry [IHC]), although significantly more cells expressed activated caspase-3 after Co-hArgI treatment (D). Finally, tumors from control-treated mice demonstrated far fewer autophagosomes (E; immunofluorescence [IF]; green label is LC3B-II, whereas blue label is DAPI) than tumors from arginase-treated mice (F). Larger nuclei (blue) are seen with Co-hArgI treatment (D, F).

Figure 5

Arginase-related toxicity was found in mice treated at doses of 15 mg/kg or greater. On necropsy, the only evidence of normal tissue toxicity was massive bone marrow devastation (A), whereas control bone marrow remained normal (B). In addition, weight loss was measured as a function of dose-dependent toxicity (0–32 mg/kg, n = 5 each) after IP injection of l-arg. The greatest observed weight loss occurred 4 days after treatment (C). The 16- and 32-mg/kg groups had a BCS of 1 and were killed for humane reasons. The mice in the 8-mg/kg group had a BCS score of 2 to 3 and regained their original body mass in an additional 2 to 3 days.