Research ArticleResearch Article

Experimental Research of Tankyrase1 Antisense Oligodeoxynucleotides on the Proliferation of Lung Cancer Cell Nodules

Chong LI, Tan LI and Zhong-li ZHAN
Clinical Oncology and Cancer Research June 2010, 7 (3) 181-186; DOI: https://doi.org/10.1007/s11805-010-0515-y

Abstract

OBJECTIVE To observe the effects of sense and antisense oligodeoxynucleotides of tankyrase 1 (TANK1-SODN and TANK1-ASODN) on murine tumor growth following intratumoral injection, investigate the actual suppressing result and mechanism of TANK1-ASODN on cancer cell proliferation, and discuss the possibility of using it in gene therapy on human lung cancer cells.

METHODS After BALB/c nude mice had been subcutaneously inoculated with human lung cancer cell line CALU and it had grown into tumor nodules, we distributed these mice randomly into 3 groups: 4 in saline treatment group, and 5 each in TANK1-SODN group, and TANK1-ASODN groups. Then multiple direct intratumoral injections of synthesized TANK1-ASODN given continuously into tumor nodules for 16 days, this was compared with TANK1-SODN and saline control groups. During the experiment we measured the tumor volume every 5 days with vernier calipers; observed the histopathological characteristics of tumor tissues under microscope; went further to detect the minute changing of ultrastructure of cancer cells by electron microscope; tested the expression levels of ki67 and hTERT protein by means of SABC immunohistochemical method; and detected the lung cancer cells’ hTERT mRNA expression level by hybridization in situ (ISH) in each group.

RESULTS After 16 days of continuous injection, the tumor volume in TANK1-ASODN group was significantly smaller than the other 2 groups (both P < 0.01); quite a lot of tumor cell degeneration and necrosis were observed in mice given TANK1-ASODN. The results of electron microscope also showed that TANK1-ASODN has the power to kill cancer cells in various ways. Moreover, statistically significant decreases in the positive expression ratio of Ki67 Labeling Index (P < 0.01), hTERT protein (P < 0.01), and hTERT mRNA (P < 0.01) were consistently observed in the TANK1-ASODN group.

CONCLUSION Human lung cancer cell line CALU expressed high telomerase activity. TANK1-ASODN had the ability to decline the high expression level of hTERT; inhibit the activity of telomerase, accelerate tumor cell degeneration and necrosis; and then suppress the proliferation of cancer cells.

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Introduction

Telomeres protect chromosome ends from being recognized as DNA double-strand breaks. Telomere shortening, which occurs due to incomplete replication of DNA termini, limits the proliferative capacity of human somatic cells and contributes as a barrier to carcinogenesis. In most human cancer cells, telomerase, a reverse transcriptase that adds telomeric repeats to chromosome ends maintains telomere length whereas TRF1, a telomeric protein, represses telomere access to telomerase. As a negative regulator of telomere length by controlling the action of the XPF nuclease at telomeres[1], the nucleolar accumulation and telomeric association of TRF1 are regulated in vivo by Human PinX1[2]. So in tumor cells and immortalizing cells, with the balance between the positive regulation of telomerase and the negative regulation of TRF1, the length of telomere is maintained at a constant length, indicating that telomerase-mediated telomere elongation is tightly regulated. Recently, scientists discovered a novel telomerase regulating enzyme, TANK1, a protein with homology to ankyrins and to the catalytic domain of poly (adenosine diphosphate-ribose) polymerase (PARP). TANK1 is a multifunctional poly (ADP-ribose) polymerase with a functional role in telomere maintenance, which is found at the centrosome and is associated with vesicular secretion[3]. In metaphase TANK1 localizes to telomeres through its interaction with the shelterin DNA binding subunit TRF1[4,5]. TANK1 binds TRF1 via its ankyrin domain which is classified into 5 conserved subdomains, ARCs (ankyrin repeat clusters) I to V. Each ARC works as an independent binding site for TRF1, ARCs II to V recognize the N-terminal acidic domain of TRF1, whereas ARC I binds to a discrete site between the homodimerization and the Myb-like domains of TRF1[6]. Among them, the most C-terminal ARC V is required for TRF1 PAR sylation and its release from telomeres[7]. TANK1 polymerization is controlled by its sterile alpha motif and poly(ADP-ribose) polymerase domains[8]. Although the catalytic efficiency of TANK1, as expressed by the k(cat)/K(m) value, is approximately 150-fold lower compared to the basal activity of the poly(ADP-ribose) polymerase, PARP1, it regulates telomerase-mediated telomere elongation and plays an important role in cellular senescence and immortalization, elongating telomeres in a telomerase-dependent manner. There is in vitro overexpression of TANK1 in telomerase-positive cells poly(ADP-ribosyl)ates (PARsylates) TRF1, which obviously decreases TRF1’s adhesion ability and in turn dissociates TRF1 from telomeres degraded by ubiquitin-mediated proteolysis. The resulting telomeres become better substrates for telomerase-mediated DNA extension. So TANK1 is considered a positive regulator of telomere length, especially in humans[9], and its RNA interference results in telomere shortening proportional to the level of disintegration[10]. Since the function of TANK1 has already been defined in the process of telomere replication, it has become the topic of great interest in the field of canceration and gene therapy. As related research continues, more and more human cancers in which TANK1 expression level with observed upregulation have been discovered, such as malignant hematopoietic cells[11], B-cell non-Hodgkin lymphomas[12], colon cancer[13,14], breast cancer[15,16], and so on[17,18]. Since the abnormally high expression level of TANK1 leads to telomerase activation and telomere elongation in malignant tumors, our experiment was aimed at investigating the TANK1-ASODN’s potential effect on the proliferation of human lung cancer cell line CALU.

Materials and Methods

Materials

RPMI 1640; 10% fetal bovine serum (FBS); female BALB/c nude mice (Ncr nu/nu), 5 to 6 weeks old on arrival, were obtained from Beijing Xiehe Animal hatchery; human lung adenocarcinoma cell line CALU provided by the central laboratory of the Cancer Hospital, Tianjin Medical University; TANK1-ASODN and TANK1-SODN synthesized by Dalian Takara Biological Engineering Co., Ltd; Ki67, hTERT protein immunohistochemistry kits and hTERT mRNA hybridization in situ kit were bought from Beijing Zhongshan Biotechnology Co., Ltd.

Cell culture

Human lung adenocarcinoma cell line CALU was maintained in RPMI 1640 supplemented with 10% FBS, under a humidified atmosphere of 5% carbon dioxide and 95% air at 37°C. This was done every three days for liquid, and every five days for cancer cells’ passage.

The construction of nude mice models with transplant tumor of lung cancer cell CALU

Xenografts were initiated by subcutaneous injection of 1.95 × 107 CALU cells into the right flanks of 2 nude mice. Tumors resulting after 2 weeks were aseptically dissected and mechanically minced; 1-mm3 pieces of tumor tissue were transplanted subcutaneously by trocar needle into 14 nude mice. 2 weeks after tumor transplantation, the tumors’ volume reached (0.547 ± 0.077) cm3, and tumor formation rate was 100%.

The synthesis of TANK1-SODN and TANK1-ASODN sequence

According to TANK1 cDNA sequence, translation initiation site was aimed for synthesizing TANK1-ASODN and TANK1-SODN. The sequences were as follows: 5′ -CGA AGA TGG CGG CGT CGC GT-3′ (sense) and 5′ -ACG CGA CGC CGC CAT CTT CG-3′ (antisense).

In vivo studies

The mice with CALU tumors were randomly divided into 3 groups: 4 in control group in which each mouse was given multiple direct intratumoral injections with 200 μL saline into tumor nodules each day, and 5 in TANK1-SODN group given with 200 μL saline containing 100 μg synthesized TANK1-SODN, the other 5 in TANK1-ASODN group were given with 200 μL saline containing 100 μg synthesized TANK1-ASODN in the same way. During the experiment, tumors were measured every 5 days with microcalipers and tumor volume was calculated according to the following formula: length × width × height × 0.5236. After 16 continuous days, the mice were killed by decapitation while they were under light methoxyflurane anesthesia. The tumors were carefully removed, cleaned, and weighed. In each group, 2 samples were randomly selected from which 2 small pieces of tissue were cut for electron microscope detection. The remaining samples of tumor tissue were fixed in 10% buffered formalin and embedded in Paraffin for making sections and HE dyeing. With the expression levels of ki67 and hTERT protein tested by SABC immunohistochemical method, hTERT mRNA expression level was also observed by ISH in all groups. The positive ratio mentioned above of cancer cells was calculated as follows: cancer cells were counted in 10 standard high-power microscopic fields for 1000 cells, so the positive cancer cell expressing ratio = the sum of positive cancer cells/1000 × 100%. The suppression ratio of transplant tumor growth was calculated as follows: (1-ΔV treatment group/ΔV control group)× 100%, > 60% was considered effective treatment.

Statistical analysis

The statistical analysis was performed with SAS for Windows, version 8.0. Tumor volumes were the quantitative data on which Two-way Repeated-Measures ANOVA was performed. Whereas the positive expression ratios of Ki67 LI, hTERT protein and hTERT mRNA were data on which χ2 test was performed. A difference was considered statistically significant at P < 0.05. All P values were based on 2-sided hypothesis testing.

Results

The suppression effect of TANK1-ASODN on murine tumor growth

After the treatment, the suppression ratio of tumor growth in TANK1-ASODN treatment group was 69.32%, significantly higher than that of the saline treatment group (the tumors’ volume was getting progressively bigger), and that of the TANK1-SODN treatment group (the suppression ratio of tumor growth was less than 40%). The tumor volumes in the 3 groups are shown in Table 1. There was no significant difference in tumor volume among the groups on the first and the fifth treatment day (F = 0.08 and 0.75 respectively, P > 0.05). Whereas on the tenth treatment day, the tumor volume of TANK1-ASODN treatment group was significantly smaller than the other 2 groups (F = 30.49 and 7.68 respectively, P < 0.01). After 16 days of injection treatment, a significant difference in tumor volume was found between TANK1-ASODN and the control group (F = 55.62, P < 0.01), the same between TANK1-ASODN and TANK1-SODN group (F = 52.74, P < 0.01). No significant difference in tumor volume was found between TANK1-SODN and the control group (F = 0.37, P > 0.05).

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Table 1.

Tumor volume of three transplant tumor nodules.

The changing of histopathological characteristics and ultrastructure of tumor tissue in 3 different groups

Under microscope quite a lot of tumor cell degeneration and necrosis was observed in tumor nodules given TANK1-ASODN. The results of the electron microscope showed that the TANK1-ASODN is clearly able to kill cancer cells through various paths, such as necrosis, apoptosis, and oncosis and so on. Whereas the TANK1-SODN had a certain tendency to promote the cancer cells to proliferate and invade the normal surrounding tissue, but this occurrence was not extreme. Necrosis and apoptosis was not observed in TANK1-SODN treated cancer cells. The matched control caner cells treated with saline were observed to grow metastatically.

The expression levels of Ki67 and hTERT protein in 3 different tissue groups

As shown in Fig. 1, all cancer tissue in the 3 groups showed positive expression of Ki67 and hTERT protein, but the coloration intensity in the control group and TANK1-SODN treatment group was stronger than that in TANK1-ASODN treatment group. The labeling index (LI) of Ki67 protein in cancer cells was (81.90 ± 5.13)% in the matched control group, (77.72 ± 9.62)% in TANK1-SODN group, and (47.52 ± 4.62)% in TANK1-ASODN group (Table 2). There was a significant difference in Ki67 LI and hTERT protein expression among the groups (χ2 = 752.91 and 1246.50 respectively, P < 0.01). Moreover, the Ki67 LI and hTERT protein expression of TANK1-ASODN group were significantly lower than either of the other 2 groups (both P < 0.01). Whereas there was no significant difference in Ki67 LI and hTERT protein expression between the control and TANK1-SODN group (χ2 = 3.51 and 0.13 respectively, P > 0.05). The correlation of Ki67 protein and hTERT protein expression levels was highly positive, equaling 0.9561, P < 0.01.

Fig. 1.
Fig. 1.

A, In the control group, cancer tissue showed positive expression of Ki67 protein. More cancer cells which scattered unequally showed positive expression of Ki67 protein localized at cell nucleus clearly with strong coloration intensity of brownish yellow by SABC, 400 ×; B, In the TANK1-ASODN treatment group cancer tissue still showed positive expression of Ki67 protein, but less cancer cells showed positive expression of Ki67 protein, cell nucleus looked unclear with weak coloration intensity of tinted yellow by SABC 400 ×; C, In the control group, cancer tissue showed positive expression of hTERT protein. More cancer cells which scattered unequally showed positive expression of hTERT protein localized at cell nucleus clearly with strong coloration intensity of brownish yellow by 400 ×; D, In the TANK1-ASODN treatment group cancer tissue still showed positive expression of hTERT protein, but less cancer cells showed positive expression of hTERT protein, cell nucleus looked unclear with weak coloration intensity of tinted yellow by SABC 400 ×.

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Table 2.

The positive expression ratio of Ki67 LI and hTERT protein in three transplant tumor nodules (%).

Detecting the expression of hTERT mRNA by ISH in 3 different groups

As shown in Fig. 2, all cancer tissue in the 3 groups showed positive expression of hTERT mRNA, but the coloration intensity in the control group and TANK1-SODN treatment group was stronger than that in TANK1-ASODN treatment group. The positive cells in TANK1-ASODN were less than the other 2 groups. There was a significant difference in hTERT mRNA expression among the groups (χ2 = 839.14, P < 0.01). Moreover, the hTERT mRNA expression level of TANK1-ASODN group was significantly lower than either of the other 2 groups (χ2 = 488.13 and 552.92 respectively, P < 0.01). Whereas, there was no significant difference in hTERT mRNA expression between the control and TANK1-SODN group (χ2 = 0.32, P > 0.05) (Table 3).

Fig. 2.
Fig. 2.

A, In the control group, cancer tissue showed positive expression of hTERT mRNA. More cancer cells which scattered unequally showed positive expression of hTERT mRNA localized at cell nucleus clearly with strong coloration intensity of brownish yellow by ISH 400 ×; B, In the TANK1-ASODN treatment group cancer tissue still showed positive expression of hTERT mRNA, but less cancer cells showed positive expression of hTERT mRNA at cell nucleus unclearly with weak coloration intensity of tinted yellow by ISH 400 ×.

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Table 3.

The hTERT mRNA in 3 transplant tumor nodules (%).

Discussion

TANK1 is a positive regulator of telomere length that elongates telomeres in a telomerase-dependent manner. This function of TANK1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Recent reports implicated TANK1 as tumor antigens and potential targets of anticancer treatment[19-23]. Our experiment was designed to investigate the actual suppressing effect of TANK1-ASODN in human lung adenocarcinoma cell line CALU.

Firstly, our experimental outcome showed that the transplant tumor in TANK1-ASODN group grew slowly. After the injection treatment, the suppression ratio of tumor growth in TANK1-ASODN treatment group was significantly higher than in the other 2 groups. Also the volume of tumor nodules in TANK1-ASODN treatment group was significantly smaller than the other groups. There was no significant difference in tumor volume among the 3 groups on the first and the fifth treatment day, which might result in TANK1-ASODN’s indirectly inhibiting effect on tumor growth by restraining TRF1’ s poly(ADP-ribosyl)ation first, thus in turn increasing the number of associated TRF1 on telomere, and finally suppressing telomerase activation and blocking the access of telomerase to telomeres. So it’s suppressing effect to tumor growth onset is relatively slow. Whereas on the tenth treatment day, the tumor volume of TANK1-ASODN group was significantly smaller than that of the control and TANK1-SODN treatment group, this showed that TANK1-ASODN’s inhibiting effect on tumor growth had already been very clear. After injecting treatment had been all completed, a significant difference in tumor volume was found between TANK1-ASODN and the control group. No difference in tumor volume was found between TANK1-SODN and the control group. Furthermore, the transplant tumors showed a faster growth 10 days later, this prompted that the braking effect of TANK1-ASODN to CALU proliferation was more effective at the early stages of tumor growth when the tumor is smaller.

Secondly, quite a lot of tumor cell degeneration and necrosis were observed in tumor nodules given TANK1-ASODN under microscope. The results of electron microscope also showed that the TANK1-ASODN can clearly kill CALU through various paths, such as necrosis, apoptosis, oncosis and so on. All these results indicated TANK1-ASODN had the obvious ability to suppress the proliferation of human lung cancer cell CALU in BALB/c nude mice. But in the control and TANK1-SODN groups, tumor nodules showed normal growth without negative interference. This finding confirmed it was the specific TANK1-ASODN that hindered the growth of lung cancer cells.

Thirdly, in our experiment the expression levels of Ki67 protein, hTERT protein and hTERT mRNA were extraordinarily high in human lung cancer cell line CALU. Abnormal upregulation of TANK1 and hTERT expression might be an important reason for cancer cells’ active proliferation. The overexpression of TANK1 and hTERT genes might play an important role during lung cancer occurrence and its malignant progression. But in the TANK1-ASODN treated cancer cells, except for the weaker coloration intensity, the Ki67, hTERT protein and hTERT mRNA expression levels were significantly lower than either of the other 2 groups. All these results above indicated TANK1-ASODN could effectively lower the expression levels of Ki67, hTERT protein and hTERT mRNA, clearly down-regulate the activity of telomerase, and decrease the proliferating ability of cancer cells. Inducing a great deal of cancer cells to stop growing and die. Moreover, the experiment also showed that in human lung cancer cell line CALU, there might be a close correlation among the expression levels of TANK1, telomerase and Ki67, so all these 3 proteins could be applied simultaneously to evaluate the propagation activity and differentiation degree of lung cancer cells, predict the lung cancer cell prognosis, and supply a new target for lung caner gene therapy.

Seimiya H’s experiment showed[24] that TANK1 inhibition in human cancer cells enhances telomere shortening by a telomerase inhibitor and accelerates cell death; Shay etc.[25] considers the combination of tankyrase and telomerase inhibitors might offer new opportunities for telomerase inhibition therapy. In antisense oligodeoxynucleotides of TANK1 application field, infecting tongue cancer TCCA-8113 cells could lead to telomere shortening and hasten early tumor cellular crisis[26]. In our experiment, multiple direct intratumoral injections of synthesized TANK1-ASODN into human lung cancer cell nodules also showed a satisfactory suppressing effect on tumor growth. To conclude, with the further study on TANK1 going on, we might find effective means to suppress cancer cell proliferation, and TANK1 supplies a new clue in tumor gene therapy.

Conflict of interest statement

No potential conflicts of interest were disclosed.

  • Received April 8, 2010.
  • Accepted June 10, 2010.

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