**5.4 Therapy combining NK4 with other treatments**

Anti-cancer chemotherapy is widely used for the suppression of malignant tumors with or without surgical treatment. Therapy regimens that combine anti-cancer chemo drugs and NK4 enhance their anti-tumor effect (Matsumoto *et al*., 2011). Irradiation therapy often enhances cancer metastasis, especially in cases of pancreatic carcinoma, and this is associated with the irradiation-induced up-regulation of HGF in fibroblasts (Qian *et al*., 2003; Ohuchida *et al*., 2004). Thus, NK4 may overcome these irradiation-associated side effects.

Epidermal growth factor receptor (EGFR) kinase inhibitors, such as Gefitinib, are used to treat non-small cell lung cancers that have activating mutations in the EGFR gene, but most of these tumors become resistant to EGFR-kinase inhibitors due to enhancement of HGF-MET signals (Engelman *et al*., 2007; Yano *et al*., 2008; Okamoto *et al*., 2010). Thus, NK4 treatment may reverse HGF-induced resistance to Gefitinib.

Fig. 4. Anti-tumor effects of NK4 on advanced pancreas cancer in mice. (A) Schedules for NK4 treatment of mice with pancreatic cancer. NK4 was injected into mice between 3 and 28

days after the inoculation of human pancreatic cancer cells (SUIT-2). (B) Inhibition of primary tumor growth by NK4. Photographs show appearance of the primary pancreatic cancer. (C) Histological analysis of the effect of NK4-treatment on tumor angiogenesis (left)

and apoptosis (right). NK4-treatment reduced the number of vessel numbers, while apoptotic death of cancers was enhanced by NK4. (D) Inhibitory effects of NK4 on

bearing mice treated with NK4.

**5.4 Therapy combining NK4 with other treatments** 

treatment may reverse HGF-induced resistance to Gefitinib.

with pancreatic cancer.

effects.

peritoneal metastasis. Left: Typical macroscopic findings. Middle: Changes in the number of metstatic nodules. Right: Changes in the ascite volumes. (E) Prolonged survival of tumor-

long-term survival and a recurrence-free status. Targeting tumor angiogenesis and blockade of HGF-mediated invasion of cancer cells may prove to be potential therapy for patients

Anti-cancer chemotherapy is widely used for the suppression of malignant tumors with or without surgical treatment. Therapy regimens that combine anti-cancer chemo drugs and NK4 enhance their anti-tumor effect (Matsumoto *et al*., 2011). Irradiation therapy often enhances cancer metastasis, especially in cases of pancreatic carcinoma, and this is associated with the irradiation-induced up-regulation of HGF in fibroblasts (Qian *et al*., 2003; Ohuchida *et al*., 2004). Thus, NK4 may overcome these irradiation-associated side

Epidermal growth factor receptor (EGFR) kinase inhibitors, such as Gefitinib, are used to treat non-small cell lung cancers that have activating mutations in the EGFR gene, but most of these tumors become resistant to EGFR-kinase inhibitors due to enhancement of HGF-MET signals (Engelman *et al*., 2007; Yano *et al*., 2008; Okamoto *et al*., 2010). Thus, NK4 Recently, it was demonstrated that NK4-mediated tumor regression depends on the infiltration of cytotoxic T lymphocytes (Kubota *et al*., 2009b). Importantly, depletion of CD8+ cells markedly abrogated the anti-tumor activity of NK4 in a mouse model of colon cancer. NK4 enhances immune responses in dendritic cells *in vitro*. Thus, NK4 may also have utility for anti-tumor immunotherapy.

There is now ample evidence that NK4 is useful for the inhibition of growth, invasion and metastasis in various types of tumors, such as gastric carcinoma (Hirao *et al*., 2002), pancreas cancer (Tomioka *et al*., 2001), prostate cancer (Davies *et al*., 2003), multiple myeloma (Du *et al*., 2007) and melanoma (Kishi *et al*., 2009) (**Table-1**). These results support our hypothesis that HGF is a key determinant of tumor malignancy (Matsumoto *et al.*, 1996b).


Endocrine Delivery System of NK4, an HGF-Antagonist and

Anti-Angiogenic Regulator, for Inhibitions of Tumor Growth, Invasion and Metastasis 131

Numerous clinical studies have indicated the apparent increases in serum HGF levels in patients during the progression of cancers (Wu *et al*., 1998; Osada *et al*., 2008). It is likely that HGF in blood protects cancer cell suspension from anoikis-like cell death (Zeng *et al*., 2002). Thus, we predict that over-production of NK4 in blood would overcome the HGF-mediated metastatic events seen in blood flow (and possibly in local sites). Hydrodynamic-based gene delivery is known to achieve an efficient expression of exogenous genes predominantly in the liver but much lesser in the kidney and spleen (Suda *et al*., 2007). Based on this background, we established a method for the induction and maintenance of higher levels of

For hydrodynamic-based gene delivery, 5 microgram of plasmid DNA (pCAGGS-NK4), or pCAGGS-empty (as a control), in saline was injected within 5 seconds into tail veins of mice at 2.4 ml per 30g body weight (Wen *et al*., 2004; 2007). As a result, exogenous NK4 was detected, and plasma NK4 reached a mean value of 49.5 ng/ml 24 hours post-bolus injection and decreased to 15.4 ng/ml on day 3. Following the second and third injections, the plasma NK4 level again reached approximately 70 and 130 ng/ml on days 8 and 15, respectively. Thus, plasma NK4 levels increased following additional administration of the expression plasmid, and were maintained at levels of > 8 ng/ml during 3 weeks post-treatment (**Fig. 5**).

Fig. 5. Hydrodynamics-mediated NK4 delivery system in mice. (A) An experimental protocol of NK4 gene administration. Five microgram of pCAGGS-NK4 was administered intravenously into mice on day 0, 7 and 14. (B) Changes in plasma NK4 levels following repetitive administration of expression plasmid for NK4. Arrows mean the time of plasmid

Colon cancer is one of the most common cancers in the world, with a high propensity to metastasize: 30-40% of patients have metastatic disease at the initial diagnosis. The liver is the most frequent site of metastasis, and hepatic failure is a lethal event during colon cancer. Thus, direct inhibition of the dissociation, spreading and invasion of cancer cells is expected to become efficient treatment. With regard to this, HGF stimulates the invasion of MC-38 mouse colon cancer cells across MatriGel (Parr *et al*., 2000), which is composed of laminin and other matrices and mimics the basement membrane *in vivo*. In this model, NK4 has

administration. See reference (Wen *et al*., 2007) for further information.

**6.2 Inhibition of colon cancer metastasis by NK4 gene delivery** 

**6.1 NK4 supplementation system via hydrodynamic gene delivery in mice** 

NK4 in blood through repeated injections of NK4 cDNA-containing plasmid.


Adeno-NK4, adenoviral vector carrying NK4 cDNA; r-NK4, recombinant NK4 protein; sc, subcutaneous; iv, intravenous; ip, intraperitoneal; im, intramuscular; DC, dendritic cells; and CTL, cytotoxic T lymphocytes.

Table 1. Representative studies to show therapeutic effects of NK4 on distinct types of tumors in animal models
