Hepatocellular Carcinoma (HCC) is a leading cause of death in Iran and many Asian countries and because of early metastasis and progression its treatment is difficult. How-ever, chemotherapy showed limited effect until now; therefore, other antitumor drugs are needed to be investigated.

Telomeres are tandem repetitive guanine rich sequences of TTAGGG at the end of chromosomes that are seen in non coding regions of DNA ⁽¹⁾. Since DNA polymerase is not able to fully replicate 3′ end of DNA strand, the telomeres of somatic cells were progressively shortened by 50-200 bp with each mitotic division. Telomeres protect chromosomes from degradation by capping the ends of chromosomes. Thus progressive shortening of telomeres interferes with telomeric caps formation which ultimately leads to chromosomal instability and can increase tumor formation by an increase in rate of mutation of tumor suppressor genes and oncogenes ^(2–5). A ribonucleoprotein enzyme i.e. telomerase can build 3′end of chromosomes ⁽⁶⁾. Telomerase is a RNA-dependent DNA polymerase that consists of several subunits including template RNA (TERC) and a catalytic reverse transcriptase (TERT) that adds de novo repetitive sequences of telomeric DNA after each cell division, thus maintains function and length of telomere despite the telomere scrubbing that normally occurs during replication of chromosome ^{(7, 8)}. Cancer cells acquired indefinitely growth capacity and maintenance of telomeres by telomerase activity ^(9–14).

The telomerase activity is not detected in most somatic cells including normal hepatocytes but is detected in many cancer cells including HCC ^(15–17). For this reason, telomerase inhibition by some drugs is a novel approach to cancer therapy ⁽¹⁸⁾.

Bcl-2(B cell lymphoma protein-2) is an anti-apoptotic protein which is located in outer membrane of mitochondria and with another anti-apoptotic protein i.e. BclXL inhibits the release of cytochrome C from the mitochondria ⁽¹⁹⁾; as a result, Bcl-2 inhibits apoptosis in cancer cells and therefore has important role in development of cancer and resistance of cancer to some anticancer drugs. High expression of Bcl-2 is found in many cancer cells and mediates the resistance of cancers to chemotherapeutic drugs ⁽²⁰⁾. Many anticancer drugs act in tumor via apoptosis; so Bcl-2 can interfere with them by blocking the cell death signals. Therefore, inhibition of Bcl-2 by new synthetic drugs will either restore the apoptotic process in tumor cell or sensitize them to drug treatment as it was seen in treatment of non Hodgkin‘s lymphoma in human through inhibition of Bcl-2 by antisense oligonucleotide ⁽²¹⁾. In addition to antisense oligonucleotide ⁽²²⁾, single chain antibodies can target Bcl-2 proteins family in tumor cells and increasingly sensitize tumor cells to chemotherapy ⁽²³⁾.

Azidothymidine is an anticancer drug that not only competitively decreases telomerase activity in tumor cells via active anabolite azidothymidine triphosphate (AZTTP) and ar-rests the tumor cells by favoring apoptosis or inducing senescence ^{(24, 25)}, but also through decreasing Bcl-2 expression and concentration in tumor cells lowers the resistance of the cells to apoptosis and increases their sensitivity to this drug ⁽²⁶⁾.

In the present study, an attempt was made to study telomerase activity and Bcl-2 concentration in HCC induced rats after treatment with platinum azidothymidine (Pt-AZT), a new synthetic platinum compound and to compare these effects with those of azidothymidine (AZT).

Pathogen-free male Wistar rats (n = 100) were purchased from Razi Institute (Karaj, Iran) and were maintained under standard conditions for acclimatization for two weeks. All animals had free access to industrialized food and water.

In this study, one hundred pathogen-free rats were divided randomly to 4 groups (each group contained 25 rats). Group A was considered as the control group (healthy rats). Liver preneoplastic lesions were induced in remaining animals (n = 75) using Solt-Farber resistant hepatocyte protocol and the induction was approved by a pathological laboratory. These animals were randomly allocated in groups B, C and D. Group B was negative control (untreated).

There was not any tumor in liver of healthy rats (group A) at any point of the study; while neoplastic lesions were gradually developed in all lobes of the rats’ liver in groups B, C and D following the neoplasm induction by Solt-Farber resistant hepatocyte model after 8 weeks. In microscopic study of untreated rats’ liver, there were several enlarged nucleuses that showed intensive synthetic phase of cell cycle in these hepatocytes indicating presence of malignant cells (Figures 2A and B).

In treated rats with platinum azidothymidine, there were many regions with karyolysis and pyknosis that were signs of apoptosis and also some regions showed necrosis. Furthermore, in some slides hemorrhage was observed that was not seen in group B (Figures 2C and D). In treated rats with azidothymi-dine, there were several karyolysis and pyk-nosis regions but apoptosis extension was less comparing to the treated rats with Pt-AZT (Figures 2E and F).

At the end of preneoplastic lesions induction protocol, there were a few small size lesions and with passing time there was an increase in both size and number of lesions. After drug treatments, size of some lesions was decreased and some small lesions disappeared; but the efficacy of Pt-AZT was higher as compared to those of AZT. There was no abnormal finding in hepatocyte of normal rat after treatment with AZT or Pt-AZT.

The Bcl-2 concentration and telomerase activity in the studied groups are shown in table 1. The lowest telomerase activity was found in group C (Pt-AZT treated rats). Comparing telomerase activity between groups C and B indicated a significant decrease in group C (p < 0.001); also Bcl-2 concentration was significantly lower in group C as compared with group B (p < 0.001). Similar results were observed comparing these factors between groups D and B indicating significant decrease in both factors in group D (p < 0.001). Also comparison between C and D groups showed lower telomerase activity (p < 0.001) and Bcl-2 concentration (p < 0.001) in group C (Table 1). These results indicated lower telomerase activity and Bcl-2 concentration in Pt-AZT treat-ed group comparing to AZT treated group. Therefore, Pt-AZT was a more potent inhibitor in hepatocellular carcinoma as compared with AZT.

The difference in telomerase activity between group A (healthy group) and group B was significant (0.018±0.011 vs. 0.577±0.116 IU/L, p < 0.001) and similar result was found for Bcl-2 concentration in these two groups (0.19±0.074 vs. 3.94±0.74 ng/ml, p < 0.001). Our result in comparing telomerase activity between groups A and group D showed a significant difference (0.018±0.011 vs. 0.331± 0.06 IU/L p < 0.001), also similar results for Bcl-2 concentration was observed (0.19± 0.074 vs. 2.94±0.594 p < 0.001, Table 1).

Our results indicated a significant negative correlation between telomerase activity and Bcl-2 concentration in untreated rats (group B) (r = -0.43, p = 0.034). However, there were no significant correlation between telomerase activity and Bcl-2 concentration in other studied groups (Table 2).

In this study, it was found that Pt-AZT was more effective than AZT in inhibition of HCC that was induced in rats using resistant hepatocyte Solt-Farber protocol. Our findings showed that telomerase activity and Bcl-2 concentration in Pt-AZT-treated rats was lower than those of AZT-treated rat (Table 1). Also similarly these factors were reduced in AZT-treated rats comparing to the untreated HCC group (Table 1).

A study reported that AZT blocks telomerase activity and effectively inhibits tumor growth and liver metastasis induced by the carcinogen diethyl-nitrosamine (DEN) in rats ⁽²⁶⁾. Our finding indicted that telomerase activity increased after HCC development in untreated group but in the C and D groups that were treated with Pt-AZT and AZT respectively it was significantly decreased. These changes in telomerase activity can be a novel tumor biomarker to detect HCC either at primary or progressive stages. Some authors reported that HCC progression is due to oxidative stress by telomerase activity ⁽²⁹⁾.

In our study, it was found that telomerase activity in Pt-AZT treated group was significantly lower as compared to AZT treated rats, which is due to presence of platinum that designates manifold anticancer effects of this compound.

There is a report showing that low concentration of platinum compound 2,3-dibromo-succinato [2-(methylaminomethyl) pyridine] platinum (II) (compound E) in treatment of a human hepatoma cell line (HepG2) had the strongest inhibition in telomerase activity and gradually reduced the telomere length, and finally caused apoptosis ⁽³⁰⁾. Our findings confirmed some previously reported results, that platinum compounds and derivatives are more effective in inhibition of telomerase activity than the original compounds. Yamamoto et al prepared the combination of epirubicin-in-corporating micelle NC-6300 and 1,2-di-aminocyclohexane platinum (II) (oxaliplatin parent complex) in 44As3Luc cells and evaluated its antitumor activity in mice bearing 44As3Luc xenografts and showed the higher efficacy of it which was due to the presence of platinum and epirubicin-incorporating micelle NC-6300; as the later complex is a carrier to target cells ⁽³¹⁾. Shimada et al reported that increasing telomerase activity in HCC is accompanied by progression of malignancy ⁽³²⁾.

Some reports show that in vitro treatment of tumor cells with azidothymidine decreased telomerase activity and expression and consequently decreased tumorigenicity and metastatic potential of tumor cells with a substantial increase in apoptotic nuclei and decrease in cell viability ^{(33, 34)}. Liu et al investigated the effect of AZT on human glioblastoma cells in vitro and reported that telomerase activity of these cells that were measured by TRAP assay was significantly reduced after treatment of these cells with 50-100 µmole AZT ⁽³⁵⁾. They concluded that AZT inhibits telomerase and cyclin A that can inhibit passing of cells from G2 to M and S phases and suppresses proliferation of cancer cells ⁽³⁵⁾. Our results indicate that telomerase may be an important target for therapy by controlling cell proliferation and growth. All these studies focused primarily on inhibiting telomerase activity which led to reduced cancer progression and in some cases suppression of it. Secondly, platinum derivatives of these compounds have an effective role in the repression of telomerase activity in comparison to original anticancer compounds. From these studies, it can be concluded that AZT effectively inhibits HCC in vivo. The relationship between Bcl-2 concentration and resistance to drug treatment remains controversial.

According to our results showing the lower Bcl-2 concentrations in Pt-AZT- treated rats compared to AZT-treated group, there was less resistance to the drug in the former group. Comparing the extend of apoptosis which was wider in Pt-AZT-treated group than AZT, it was shown that greater inhibition of Bcl-2 genes expression and concentration decline by Pt-AZT in comparison to AZT. Also according to our findings, it can be concluded that there is a relationship between Bcl-2 concentration and resistance to these drugs. Some studies indicated close inverse relationship between Bcl-2 concentrations and resistance to anticancer drugs ^(36–39). Beale et al showed a statistically significant inverse correlation between inhibition of cell line growth and Bcl-2 levels in human ovarian carcinoma cells treated with cisplatin; over-expression and therefore increased concentration of Bcl-2 in these cells led to resistance to cisplatin as compared to the control ⁽⁴⁰⁾. However, some studies reported no significant correlation between Bcl-2 concentration and anticancer drugs resist-ance ^(41–44). In our study, it was concluded that the relationship depended on some factors such as nature of anticancer drug and the treated concentration.

As described in the results, our study confirmed a statistically significant negative correlation between Bcl-2 concentration and telomerase activity only in untreated HCC rats (group B, r = -0.43, p = 0.034). But in other studied groups, there were no significant negative correlation between these two factors (Table 2); however, the relationship between telomerase activity and Bcl-2 expression and concentration was reported in some studies ^(45–47). Lida et al reported a possible relationship between telomerase activity and Bcl-2 expression in colorectal carcinoma ⁽⁴⁵⁾. Elkak et al reported no relationship between telomerase activity and Bcl-2 expression in human breast cancer ⁽⁴⁶⁾. Also Ohmura et al suggested that the Bcl-2 protein concentration was conversely correlated with telomerase activity (similar to our findings) and the biological role of Bcl-2 protein differs by degree of tumor aggressiveness in low grade tumor ⁽⁴⁷⁾. Similar to the above mentioned study, induction of HCC in our studied animals was in primary level (pre-neoplastic lesions); therefore, the degree of tumor aggressiveness was in low grade and the inverse correlation between telomerase ac-tivity and Bcl-2 protein was statistically significant.

As it has been indicated in several studies, our results showed that telomerase activity increased after the development of HCC; it ispossibly due to the effect of hTERT protein component of telomerase that regulates telomerase activity in chemically induced heaptocellular carcinoma ⁽⁴⁸⁾. Also, increase in Bcl-2 concentration was reported after the development of HCC ^(49–53).

AZT inhibits synthesis of cancer genome by its active anabolite AZTTP (azidothymidine triphosphate) through chain termination mechanism which may inhibit telomerase activity competitively ⁽⁵⁴⁾. Also, according to the pathological findings, treatment with AZT leads to a decrease in the expression of some genes such as telomerase and Bcl-2. It acts to arrest the cells with inducing senescence and apoptosis in tumor cells ^{(55, 56)}. Another report confirming these facts demonstrates that immortalization has a key role in cell concentration; high telomerase expression is present in 85-90.9% of tumor cells, and telomerase activity increases during the period of normal cells transition towards tumor cells ⁽⁵⁷⁾. AZT, interrupting the reverse transcriptase of cells, blocks the cell cycle and inhibits replication of cells and cell growth ^{(58, 59)}. Also, AZT inhibits several kinds of enzymes in tumor cells among which some contribute to cell cycle regulation such as Mad1. Consequently, reduction of these cell cycle factors inhibits cell growth in S phase and the cell enters apoptosis phase which indicates that AZT is an effective anticancer drug ^(60–62).

Recently, Shah Abadi et al synthesized some antiviral drugs such as platinum complexes e.g. Pt-AZT ⁽⁶³⁾. They studied interaction of Pt-AZT with DNA in vitro and showed that Pt-AZT breaks the backbone of DNA by intercalated mechanism and creation of non-covalent binding between adjacent bases in DNA; subsequently the cells undergo apoptosis ^{(64, 65)}. This effect along with synergistic effects of AZT ^(54–62) destroys cancer cells by Pt-AZT.

Platin - 3-azido-2,3-dideoxythymidine ( Pt-AZT), is a synthetic compound that is made from cisplatin and AZT ^(63–65). Pt-AZT may inhibit telomerase and Bcl-2 in cancer cells and therefore induces apoptosis process. There is no published study showing in vivo effects of Pt-AZT upon HCC. To our knowledge, this is the first investigation to examine the in vivo effects of Pt-AZT in telomerase and Bcl-2 expression in HCC rats that can show their potential as a new drug for HCC treatment.

Our animal model provided an environment for the study of inhibitory effects of Pt-AZT on the growth, progression and metastasis of HCC rat. Since this study was carried out in vivo, it can be considered as an advantage of the study which differs from most of the studies in this field. As a novel finding in this study, for the first time, it was demonstrated that Pt-AZT can reduce the Bcl-2 concentration and telomerase activity more effectively than AZT (the highest levels of Bcl-2 concentration and telomerase activities found in untreated HCC group). These data suggest that Pt-AZT effectively inhibits the growth of liver tumor in rats by extending apoptosis as compared to AZT. Furthermore, our study illustrated that Pt-AZT as a new anticancer drug in vivo can be more efficient than AZT. However, further studies are needed to shed light on the contribution of inhibitory effect of Pt-AZT on the growth of liver tumor in rats by extending apoptosis.