# Patent

## Title
Hepatopoietin and use thereof

## Abstract

Provided are a hepatopoietin PCn (HPPCn) and its homologous proteins, which can promote hepatocyte proliferation in vitro, promote liver regeneration in vivo, inhibit the growth of tumor cells and promote the apoptosis of tumor cells. The hepatopoietin PCn (HPPCn) and its homologous proteins are useful for the treatment of acute and chronic liver injury, or the treatment of liver fibrosis.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage under 35 USC 371 of International Application Number PCT/CN2007/003722, filed on Dec. 21, 2007, the entire contents of which are hereby incorporated by reference.
TECHNICAL FIELD
The present invention relates to the field of biological products and protein drugs, in particular, to an exogenous hepatic regeneration factor, Hepatopoietin PCn (HPPCn). This protein can promote hepatocyte proliferation and liver regeneration, and can inhibit the growth of tumor cells and promote the apoptosis of tumor cells when present in cells, so that it has a potential value of clinical application.
BACKGROUND OF INVENTION
Liver is a viscus having a powerful ability of regeneration in a body. The mechanism of regulation of liver has been searched for more than 100 years. However, the action of the growth factors associated with liver regeneration as known presently, e.g. hepatocyte growth factors (HGF), transforming growth factor-α (TGF-α), and the like, lacks the specificity for liver, and thus it is difficult to explain the organ-specific regulation mechanism of liver regeneration. Therefore, the studies in this field have been focused on the search of novel regulation factors of liver regeneration. In the 1950s, it was discovered that, in mammalian liver, there are some substances capable of regulating its growth[1]. In 1975, LaBrecque et al.[2] for the first time reported that there is a thermostable mixture, which can specifically promote hepatocyte proliferation, in the regenerated liver tissue of rat, and this mixture is referred to as hepatic stimulator substance (HSS). In the mid-1980s, the same kind of factors were discovered in human fetal liver tissue by the inventors, and the corresponding bioactivity, physicochemical properties, purification of proteins and clinical application were systemically studied. Since this kind of factors exhibited an excellent therapeutic effect in the clinical treatment of sever liver injury, and thus they were of great interest and a relevant US patent was obtained in 1995[3]. However, the components therein were not further identified due to the limitation of the techniques for purifying and identifying proteins, which limits the further development and application of such substances. Meanwhile, the molecular cloning of this kind of factors has been studied in many laboratories worldwide. In 1995, Hagiya et al.[4] isolated an augmenter of liver regeneration from the regenerated liver tissue of rat and performed the cloning and expression thereof, and it was discovered that the recombinant augmenter of liver regeneration could promote the liver regeneration of the partially hepatectomized rat, but did not have the activity of stimulating the primary-cultured hepatocytes and the liver cell lines in vitro[5].
In recent years, the inventors isolated a new hepatocyte growth factor from the liver of newborn calf by utilizing several isolation processes, and it has been discovered that this hepatocyte growth factor can promote the DNA synthesis of hepatocyte and has a protective effect against the acute or chronic liver injury. This hepatocyte growth factor is designated as HPPCn. The sequence analysis thereof showed that it belongs to the family of leucine-rich acidic nuclear protein (LANP). LANP is a multifunctional acidic nuclear protein, which is involved in a variety of biological processes including signal transduction, protein degradation, cytoskeletal dynamics, and morphogenesis[6-13]. However, it is not reported that LANP as a growth factor can stimulate hepatocyte proliferation or liver regeneration.
There are more than 120 millions of patients suffering from viral hepatitis, cirrhosis and/or liver cancer in China Therefore, developing an active factor capable of specifically promoting hepatocyte proliferation and liver regeneration, is of significance for the treatment of liver injury due to various causes.
In addition, the malignant tumors, such as liver cancer, and the like, become one of main killers for human health, and thus it is very socially important to inhibit the growth of tumor cells or tumorigenesis.
CONTENT OF THE INVENTION
It has been discovered in the present invention that Hepatopoietin PCn (HPPCn) is a novel hepatocyte growth factor, which is isolated from the liver of newborn calf The corresponding human nucleotide sequence was obtained from a human cDNA library, and expressed in E. coli to obtain a recombinant human HPPCn protein. The essential characters of HPPCn are as follows: (1) it is a member of the leucine-rich acidic nuclear protein (LANP) family, has a molecular weight of about 30 KD, and is sensitive for proteases and thermostable; (2) it can promote the DNA synthesis of the primary-cultured hepatocytes of rat and the liver cell lines in vitro; (3) it can promote the DNA synthesis in the livers of partially hepatectomized mice in vivo; (4) it can protect liver from acute liver injury and liver fibrosis; (5) its overexpression in tumor cells can inhibit the growth of tumor cells.
Therefore, the first aspect of the present invention relates to HPPCn comprising the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the sequence of HPPCn of the present invention is set forth in SEQ ID NO: 1. The present invention also relates to a homolog of HPPCn, which has at least 80%, preferably at least 85%, 90% or 95% of homology with HPPCn of the present invention, and retains the activity of HPPCn of the present invention.
A further aspect of the present invention relates to a nucleic acid molecule encoding HPPCn or homolog thereof.
A further aspect of the present invention relates to a pharmaceutical composition comprising HPPCn protein of the present invention or homolog thereof or a nucleic acid molecule encoding the same. The pharmaceutical composition may optionally further comprise a pharmaceutically acceptable carrier or other conventional auxiliary agent. These carriers and auxiliary agents are within the skill of a person skilled in the art.
A further aspect of the present invention relates to the use of HPPCn protein or homolog thereof or a nucleic acid molecule encoding the same, for the preparation of a medicament used to promote hepatocyte proliferation and liver regeneration, to treat the liver injury due to various causes, to treat liver fibrosis, to inhibit the growth of tumor cells when present in cells, or to treat various tumors.
Preferably, the medicament of the invention is used to treat liver fibrosis or acute or chronic liver injury.
According to the present invention, the term “hepatocyte proliferation” refers to the enhancement of cell division capability of the primary-cultured parenchyma cells derived from liver, normal liver cell lines and hepatoma cell lines, wherein the proliferation capability of cells is measured mainly by detecting the DNA synthesis.
According to the present invention, the term “liver regeneration” refers to the capability of mammalian liver to spontaneously recuperate after injury.
According to the present invention, HPPCn of the present invention can be expressed in E. coli, Pichia, Saccharomyces cerevisiae or animal cells.



DESCRIPTION OF THE FIGURES
The following figures are only used to illustrate this invention, but not intended to limit the invention.
FIG. 1 shows the amino acid sequence of HPPCn (SEQ ID NO: 1).
FIG. 2 shows the SDS-PAGE results of the expression of HPPCn in E. coli BL21 and of its purification.



The upper panel shows the induced expression of HPPCn in E. coli BL21. 1: negative control; 2: inclusion body protein; 3: soluble protein.
The lower panel shows the purification of HPPCn. 1: inclusion body protein; 2: penetration liquid; 3: product eluted by using eluent; 4: low molecular weight marker.
EXAMPLES
The following examples are used to illustrate this invention, but they are not intended to limit the invention.
Example 1
Production of HPPCn
A protein factor, Hepatopoieton PCn (HPPCn), was purified from the liver of newborn calf and was identified, by chromatography such as DEAE cellulose, Source 15Q, and the like; SDS-PAGE recovery; and MALDY-TOF and Q-TOF mass spectrometry. A human HPPCn cDNA sequence was obtained by screening a human fetal liver cDNA library. The BamH I and Xhol I restriction sites were added at both termini of the gene sequence, and then the sequence was constructed into a prokaryotic expression vector PET-24a to obtain the plasmid PET-24a-HPPCn. The plasmid was transformed into E. coli and expressed by the IPTG induction. A recombinant protein having a purity of more than 95% was obtained by ion exchange and gel filtration. The amino acid sequence of the protein is shown in FIG. 1, and the electrophoretogram of the protein is shown in FIG. 2.
Example 2
Identification of the Activity of the Recombinant Protein HPPCn to Promote Hepatocyte Proliferation
The activity of HPPCn to promote hepatocyte proliferation was detected by 3H-TdR DNA incorporation assay. The primary-cultured hepatocytes of rat were used as the target cells in the detection of bioactivity in vitro. 100 μl of cell suspension (5×104 cells/ml) were seeded onto 96-well plates and incubated for 6 h. Various concentrations of HPPCn were then added and further incubated for 24 h. 1.85×104 Bq 3H-TdR was added to each well, and 3 h later, a liquid scintillation counting was performed. The results showed that HPPCn can promote the DNA synthesis of hepatocytes in a markedly dose-dependent manner (Table 1). Other augmenters of liver regeneration (ALR) do not possess this character of HPPCn.







TABLE 1







Detection of the activity of the recombinant HPPCn protein


to promote the primary-cultured hepatocytes proliferation










Dose (ng/ml)
Incorporation amount of 3H (cpm)













0
   560 ± 127.10



10
689.33 ± 92.34



20
 944.33 ± 271.48



50
  1183 ± 241.15



100
1226.33 ± 129.01



200
1190.33 ± 347.74









Example 3
Protective Effect of the Recombinant Protein HPPCn against Acute Liver Injury
The protective effect of the recombinant protein HPPCn on partially hepatectomized mice was determined by detecting the influence of the recombinant protein HPPCn on the DNA synthesis of liver of the 34% hepatectomized mice. Male C57 mice in well health were subjected to the surgical excision of the middle lobe of liver. 2.5 mg HPPCn/kg body weight or a physiological saline in an equivalent volume was injected into the tail vein of the hepatectomized mice at different time points. After treatment for 18 h, 20 μCi 3H-TdR was injected intraperitoneally. After incorporation for 2 h, the animals were sacrificed, and the genomic DNA of liver was extracted. The incorporation amount of 3H-TdR was determined with a liquid scintillation counter. The results showed that the incorporation amounts of 3H-TdR in the treatment group are higher than those in the physiological saline control group at various time points (Table 2), suggesting that HPPCn can enhance the regeneration ability of the hepatectomized liver.







TABLE 2







Effect of the recombinant HPPCn protein on the DNA synthesis


of liver of the 34% hepatectomized mice










Incorporation amount of 3H (cpm/mg DNA)










Time (h)
Recombinant human HPPCn
Physiological saline





24
3244 ± 339.4
1641.3 ± 944.4


36
 4216 ± 1512.7
1582.6 ± 179.8


48
6128 ± 736.1
  4876 ± 867.04


60
4749.66 ± 359.7  
2775 ± 0 


72
3825.333 ± 294.   
2294.66 ± 716.5 


84
2962.667 ± 938.2   
2197.3 ± 823.7









After injection with 1 ml CCl4/kg body weight, 30 Balbc mice in well health were randomly divided into 3 groups: Group I, intravenous injection with 1 mg HPPCn/kg body weight; Group II, intravenous injection with 2.5 mg HPPCn/kg body weight; Group III, intravenous injection with 5 mg HPPCn/kg body weight; Group IV, physiological saline control group. The mice were injected once every 12 h. After 48 h, the alteration of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in blood were detected, and the mice were subjected to a pathological examination. The results showed that the serum levels of AST and ALT in the mice of control group were significantly increased, and a majority of histiocytes of liver necrotized, exhibited the disappearance of cell nucleus and presented vacuolation; while the levels of AST and ALT were recovered to some extent in the mice of HPPCn treatment groups (Table 3), and the number of necrotic hepatocytes and the degree of liver injury were significantly decreased. This suggests that the recombinant protein has a protective effect against the acute liver injury caused by CCl4.







TABLE 3







Effect of the recombinant HPPCn protein on ALT/AST


in the serum of mice with acute liver injury induced by CCl4











Groups
AST (U/L)
ALT (U/L)






Group IV
3450.6 ± 239.5
  3710 ± 177.7



Group I
2861.6 ± 229.9
2915.4 ± 460.7



Group II
  2847 ± 610.4
2904.2 ± 636.0



Group III
2465.8 ± 429.9
  2177 ± 520.5









Example 4
Protective Effect of the Recombinant Protein HPPCn Against Liver Fibrosis
In 40 Wistar rats, a liver fibrosis model was produced by a compound method using ethanol and CCl4. After 4 weeks, the rats were randomly divided into 4 groups: control group, high dose group, medium dose group, and low dose group. The intraperitoneal administration was carried out every day. At week 8, the animals were sacrificed. Blood samples were taken, and the levels of biochemical indicators such as ALT, AST and the like in blood were determined. The liver was removed, and the changes in appearance of liver were observed, the contents of hydroxyproline and malonaldehyde were determined, and histopathological examination was carried out. The results showed that, when compared to the control group, the treatment groups exhibited a decreased level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and a significantly reduced content of hydroxyproline (Hyp), while the content of malonaldehyde (MDA) had no marked change (Table 4). The livers of rats in control group presented a gray yellow and had a non-smooth surface with granules, while the livers of rats in treatment groups were relatively ruddy. The results of HE staining and Masson's trichrome staining of liver tissues showed that cell necrosis and lipid deposition occurred, fibrous tissues proliferated, and pseudolobule formed in the livers of rats of the control group; while the proliferation of fibrous tissues was significantly decreased and the hepatocyte necrosis was reduced in the treatment groups, particularly in the medium dose group. This suggests that the administration of the recombinant protein HPPCn can alleviate the liver fibrosis caused by chronic liver injury.







TABLE 4







Protective effect of the recombinant HPPCn protein


on rats with liver fibrosis












Hydroxy-
Malonalde-




Groups
proline
hyde
AST (U/L)
ALT (U/L)





Normal
0.15 ± 0.072
 0.89 ± 1.29
17.28 ± 1.49 
39.71 ± 5.12


Control
1.02 ± 0.418
11.45 ± 3.15
13.6 ± 3.84
 85.66 ± 15.90


Group






low dose
0.59 ± 0.223
10.15 ± 3.75
10.28 ± 2.13 
59.83 ± 6.55


group






medium
0.50 ± 0.063
11.87 ± 2.23
8.57 ± 2.22
56.83 ± 8.42


dose group






high dose
0.49 ± 0.160
10.99 ± 2.44
9.14 ± 2.47
  58 ± 7.56


group









Example 5
Inhibition of the Growth of Tumor Cells by HPPCn when Present in Cells
HPPCn was constructed into an eukaryotic expression vector, plasmid pEGFP-N1, which was used to transfect human liver cancer SMMC7721 cell line. After 36 h of culture, the cells were immobilized with 4% paraformaldehyde and 70% ethanol, respectively. After PI staining, the changes of cell cycle were detected by FACS. The results showed that the transfected liver cancer cells had an evident G0/G1 phase arrest, and the proportion of cells in G2/M phase among the transfected liver cancer cells was significantly lower than that of the group transfected with empty plasmid (Group transfected with pEGFP-N1) (Table 5). This suggests that the overexpression of HPPCn in cells can markedly inhibit the growth of hepatoma cells.







TABLE 5







Effect of HPPCn overexpression in SMMC7721 cells


on the cell cycle












Groups
G0-G1
G2-M
S















Group transfected with
92.59%
0.12%
7.28%



pEGFP-N1-HPPCn






Group transfected with
45.67%
14.42%
39.91%



pEGFP-N1









Reference:

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## Claims

1. An isolated polypeptide comprising the amino acid sequence SEQ ID NO:1.

2. An isolated nucleic acid, wherein said isolated nucleic acid comprises a nucleic acid sequence encoding the polypeptide of claim 1.

3. A pharmaceutical composition comprising the nucleic acid of claim 2 and a pharmaceutically acceptable carrier.

4. A method of inhibiting growth of one or more liver tumor cells in culture comprising contacting the cells with an effective amount of the pharmaceutical composition of claim 3.

5. A method of promoting proliferation of a hepatocyte in culture, the method comprising contacting the hepatocyte with an effective amount of the pharmaceutical composition of claim 3.

6. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

7. A method of inhibiting growth of one or more liver tumor cells in culture comprising contacting the cells with an effective amount of the pharmaceutical composition of claim 6.

8. A method of promoting proliferation of a hepatocyte in culture, the method comprising contacting the hepatocyte with an effective amount of the pharmaceutical composition of claim 6.