Preventive effect of Metformin against N-nitrosodiethylamine-initiated hepatocellular carcinoma in rats

1. Introduction

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer worldwide and its incidence is constantly increasing day by day (Parkin et al., 2001). The largest concentration of patients is in Asia and sub-Saharan countries (Hollstein et al., 1993). Treatments for HCC include liver transplantation, resection, local ablative therapy, hepatic artery transcatheter treatment, and systemic treatment. Surgical therapy is the only potentially curative treatment for HCC, but nonsurgical treatment can prolong the survival period and palliate symptoms (Llovet et al., 2003). The median survival of HCC is less than 12 months from diagnosis, reflecting both late presentation and lack of effective therapy (Groupe d’Etude et al., 1995). Given the time course of the disease and the burden of treatment, there is an increase in concerns about the liver diseases and HCC. Metformin is an oral antihyperglycemic agent commonly used in non-insulin dependent (type 2) diabetes mellitus (Klepser and Kelly, 1997). It works by decreasing Gluconeogenesis and activating AMP-activated protein kinase pathway (Lee, 1996). Metformin has been proved by numerous authors as an anticancer agent in lung cancer, breast cancer and ovarian cancer (Esfahanian et al., 2012; Del Barco et al., 2011; Goodwin and Stambolic, 2011; Monteagudo et al., 2011; Schott et al., 2011; Guppy et al., 2011). Several potential mechanisms have been suggested and established for the ability of Metformin to suppress cancer growth in vitro and in vivo: (1) activation of LKB1/AMPK pathway, (2) induction of cell cycle arrest and/or apoptosis, (3) inhibition of protein synthesis, (4) reduction in circulating insulin levels, (5) inhibition of the unfolded protein response (UPR), (6) activation of the immune system, and (7) eradication of cancer stem cells to name a few (Kourelis and Siegel, 2011). Evaluation of Metformin in the treatment of hepatocarcinogenesis, is a novel approach in the field of chemotherapy. The present study will help and assist to design proper medication for patients suffering from diabetes and hepatic cancer.

2. Materials and methods

3. Results

4. Discussion

It is a well known fact that liver cancer is the most important epidemic disease in the world which requires immediate attention. Limited treatment options and poor treatment make HCC one of the leading causes of death (Ei-Serag and Mason, 1999). DENA is reported to be a hepatotoxin and hepatocarcinogenic agent. In the present study, DENA induced hepatocellular damage is clearly evidenced by the marked elevation in serum SGPT, SGOT, GGTP and ALP and a decreased level of glucose in the liver tissue, These biochemical marker enzymes are indicators of tumor response (Thirunavukkarasu and Sakthisekaran, 2003). SGPT, SGOT, GGTP, ALP and decreased level of glucose serve as markers of liver damage and mechanisms of neoplastic process. It has been studied that serum GGTP and ALP levels increase linearly with tumor mass (Carr et al., 2011). Serum GGTP levels increased linearly with increases in small tumor mass, ALP levels are elevated in association with small tumors and further increases with increasing tumor mass (Pancoska et al., 2011). ALP is used as a specific tumor marker during diagnosis in the early detection of cancer. It is well established that (ALT) level signifies the presence of active disease and increases risk, particularly if the ALT is persistently or intermittently elevated over the years (Sherman, 2009).

Metformin does not scavenge ${\text{O}}_2^{-}$ radicals, but is able to react with OH radical. Their results obtained with an in vitro model allow assuming that Metformin, at a molecular level, is not a very good scavenger of ROS. Consequently, it seems that Metformin would certainly exert its in vivo antioxidant activity by different pathways other than the simple free radical scavenging action, such as increasing the antioxidant enzyme activities, decreasing the markers of lipid peroxidation and inhibiting the formation of advanced glycation end products (Khouri et al., 2004). Metformin therapy can enhance the response of patients to DNA damaging agents (chemotherapy and radiation therapy) because of their extended arrest in the S phase. Metformin also enhances RTK inhibitor and anti-EGFR treatment response, because of its action on EGFR and P-MAPK effects (Wang et al., 2008).

Metformin at a dose of 125 mg/kg restored the levels of ALP, SGPT, SGOT and GGTP. A well documented fact is that the levels of SGPT and SGOT increase in HCC (Ramanathan et al., 2011). An increased GGTP activity is found in the preneoplastic foci that enhances cell proliferation and increases tumor promotion (Lucier et al., 1991). The GGTP level elevated significantly in the disease control group as compared to normal control. These results firmly established the role of Metformin as a chemopreventive agent in DENA induced HCC.

Cancer cells require more glucose as energy for survival. In the present research glucose levels were decreased in disease control when compared with the normal control group. It indicates that the liver of the disease control group is necrotic and cell proliferation initiated. Administration of Metformin to therapeutic group decreased the glucose levels as compared to disease controls, making the liver cell deprived of glucose for energy production and utilization, indirectly suggesting its chemopreventive role in the treatment of HCC.

The development of HCC has also been associated with disorders in plasma lipid and lipoprotein metabolism (Jiang et al., 2006). Cancer development is associated with alterations in lipid metabolism, affecting cellular function and growth (Das et al., 1998). The development of hepatocyte nodules in rat liver is associated with changes in lipid parameters and oxidative status (Abel et al., 2009). Alterations in lipid profiles in malignant tissue are of importance due to their effect on membrane integrity, fluidity and regulation of cellular processes related to growth and cell survival (Bartsch et al., 1999; Tapiero et al., 2002).

The increase in cholesterol level increases the membrane fluidity, regulates membrane permeability and alters internal viscosity and also the internal chemical composition. In the present research it was observed that Metformin maintained the lipid profile, hence it can be suggested that Metformin may play the role in inhibition of carcinoma progression.

AFP is a serum protein that is detected in elevated concentration in conditions like hepatocellular carcinoma. It is similar in size, structure and amino acid composition to serum albumin, but it is detectable only in minute amounts in the serum of normal adults. Elevated serum concentrations of this protein can be achieved in the adult by exposure to hepatocarcinogenic agents. AFP has the high specificity for hepatocarcinoma. Its serum concentration confirms hepatocarcinoma and the diagnosis of tumor response to therapy.

In the present study, serum AFP level of DENA treated rats showed a significant increase compared to that of control group, proving the occurrence of premalignant liver changes in DENA treated rats. The elevation of serum AFP in HCC is well documented by many researchers (Borges et al., 2005; Yeo et al., 2006). Co-treatment with Metformin significantly reduced serum AFP.

Histopathological examination of the disease control group showed central veins surrounded by extensive necrosis and inflammation. The tumor cells resembling hepatocytes showed pleomorphism and were seen as 2–8 cell wide trabeculae that were separated by endothelial lined sinusoidal spaces. But in therapeutic group mild bile duct proliferation with no central veins, observation in the photograph suggests the tumor suppressing nature of Metformin at a dose of 125 mg/kg.

Further, as proved by Donadon et al. (2010), Nkontchou et al. (2011) and Chen et al. (2012) on humans a chemopreventive role of Metformin in cirrhosis, and thyroid cancer with existing anticancer drugs. Nowhere the biochemical mechanism of this drug was discussed. In the present study it is an effort to correlate the biochemical mechanism of Metformin as a chemopreventive agent.

The above result indicates that Metformin might be used as chemopreventive by decreasing the level of glucose in the cancer cell. Metformin is an antidiabetic drug which decreases gluconeogenesis process through increase in glucose utilization in the body and decrease in hepatic glucose production. In cancer cells the gluconeogenesis process is increased. Cancer is a process of non recognized cells in the body with some markers, where a decrease in the level of glucose may activate the immune response to retard/inhibit the growth of tumor cells.

5. Conclusion

The above observations suggest that Metformin an antidiabetic drug can possess chemopreventive action. Metformin at reduced dose (125 mg/kg) suppresses the tumors and decreases the biochemical marker which is elevated in HCC. This will open new perspectives that Metformin is a chemopreventive compound to prevent, slow or treat the occurrence of liver cancer.