Abstract
Objective: To investigate the correlation between the expressions of human telomerase reverse transcriptase (hTERT) and vascular endothelial growth factor (VEGF) in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to determine if hTERT was correlated with VEGF-promoted tumor cell proliferation in prostate cancer. Materials and Methods: Immunohistochemistry was used to analyze the expressions of hTERT and VEGF in 60 cases of PCa and 60 cases of benign prostatic hyperplasia (BPH). Then their correlation in PCa was analyzed by Spearman correlative analysis. Results: The expressions of hTERT were detected in 38 cases of PCa and 10 cases of BPH. The expressions of VEGF were detected in 46 cases of PCa and 28 cases of BPH. The expressions of hTERT and VEGF in PCa were significantly higher than those in BPH (P < 0.05). As a result of correlation analysis, it was found that with an increase of the expression of VEGF, the expression of hTERT also increased in PCa. Significant correlation was observed between the expressions of hTERT and VEGF in PCa (r = 0.8333, P < 0.05). But there was no significant correlation between the expressions of hTERT and VEGF in BPH (r = 0.3156, P > 0.05). Conclusions: All experiences above indicate that hTERT was one of the important proteins in the proliferation-promoting effect of VEGF on tumor cells in PCa.
INTRODUCTION
In many actively proliferating cells, maintenance of replicative capacity and chromosome stability requires the activity of telomerase, a specialized reverse-transcriptase that adds hexanucleotide repeats of the sequence TTAGGG to the 3′ ends of nuclear DNA, thus counteracting telomeric erosion Citation[1], Citation[2], Citation[3], Citation[4]. Human telomerase consists of at least 3 subunits .Citation[5], Citation[6]. Of them, human telomerase reverse transcriptase (hTERT), which contains the catalytic activity of telomerase, is tightly regulated in its expression Citation[7] and, thus, in most cases is responsible for the variations in telomerase activity levels.
hTERT is expressed in cells that exhibit telomerase activity Citation[8]. Multiple studies have focused on evaluating hTERT or telomerase activity as a marker for prostate cancer Citation[9], Citation[10], Citation[11]. Publications demonstrated that hTERT activity levels exhibit a prevalence range of 63–94% for PCa, and activity has been detected in some cases of high-grade prostatic intraepithelial neoplasia Citation[9], Citation[10], Citation[11]. All these suggested that hTERT might play an important role in the development of prostate cancer.
Angiogenesis in cancers, including prostate cancer, is an important process in maintaining tumor growth and promoting metastasis Citation[12]. Many growth factors are involved in angiogenesis, one of which, the vascular endothelial growth factor (VEGF), is the most important one. It is a cytokine that stimulates the proliferation and migration of vascular endothelial cells, which is a key step in angiogenesis Citation[13]. It was found that expression of VEGF was higher in malignant human prostatic tissues than in non-malignant human prostatic tissues, and high level of VEGF was associated with increased microvascular density and recurrence of PCa Citation[14].
It has been demonstrated that besides stimulating the growth of vascular endothelial cells, VEGF could also promote the proliferation of tumor cells Citation[15]. Experiments demonstrated that telomerase might be a downstream effector on the VEGF signal transduction passway in a rat model of limb ischemia Citation[16]. This study was performed to examine the correlation between the expressions of hTERT and VEGF in PCa and BPH, and then indicate if hTERT was correlated with VEGF-promoted tumor cell proliferation in PCa.
MATERIALS AND METHODS
Patients and Biopsy
Based on the pathology reports, paraffin-embedded sections of 60 prostate cancers were selected randomly from the department of pathology in our hospital. Sixty benign BPH were selected randomly as contrast. All specimens stained by HE were assessed by two pathologists in our hospital. All samples came from 220 patients who were suspected of having prostate cancer under TRUS guidance in our department from November 2001 to July 2003. The patients were 43–85 years of age (average 72 ± 10 years) with PSA levels 0.7–33.3 ng/ml (9.4 ± 6.2 ng/ml). Informed consent was obtained from all patients. This retrospective study was approved by the institutional ethics committee.
Specimens
The biopsy specimens were labeled according to the sites of origin. The sections were sliced 4 μ m in thickness for immunostaining.
Immunohistochemistry
The sections were stained with telomerase reverse transcriptase (hTERT) antibody at a dilution of 1:200 and VEGF165 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:300, respectively. The antigen retrieval technique described previously was used Citation[17]. A negative control was performed to ensure the specificity of peroxidase immunostaining by using PBS as the first antibody. Sections were examined on an Olympus microscope, and representative areas were photographed using an Olympus digital camera.
Evaluation of Score
Both the extent and intensity of staining were included when scoring hTERT and VEGF165 expression Citation[18]. The extent of positive staining was scored as follows: Grade 0, ≤10% cells; Grade 1, 10–25% cells; Grade 2, 25–50% cells; Grade 3, 50–75% cells; and Grade 4, > 75% cells. The intensity was scored as follows: Grade 0, negative; Grade +, weak; Grade ++, moderate; and Grade +++, as strong as normal mucosa. The final score was obtained by multiplying the extent scores and intensity scores, producing a range from 0 to 12. Scores 9–12 were defined as preserved or strong staining pattern (++) because there was little difference compared with normal mucosa, scores 0–4 were defined as markedly reduced or lost expression (−), and scores 5–8 were defined as intermediate staining pattern (+). 0
Statistical Analysis
All analyses were performed with STATA 7.0 (Stata Corporation, College Station, TX). Rank sum test and Spearman correlative analysis were used to assess the expressions of hTERT and VEGF, their correlation, and their correlation with serum PSA level. P < 0.05 was considered to be statistically significant.
RESULTS
Expressions of hTERT in PCa and BPH
HTERT was detected in the cytoplasm with brownish pigmentation (see , ). The expression of hTERT was detected in 38 cases of PCa with 12 cases of moderate expression and 26 cases of strong expression. The expression of hTERT was detected in 10 cases of BPH, which were all in moderate expressions. The expression of hTERT in PCa was significantly higher than that in BPH (P < 0.05, see ).
TABLE 1 Difference of hTERT expression between PCa and BPH
Figure 1 Expression of hTERT in PCa (SP, × 400) Staining intensity was detected in the cytoplasm of tumor cells.
Figure 2 Expression of hTERT in BPH (SP, × 400) Staining intensity was detected in the cytoplasm.
Expressions of VEGF in PCa and BPH
VEGF was detected in the cytoplasm with brownish pigmentation (see , ). The expression of VEGF was detected in 46 cases of PCa with 28 cases of moderate expression and 18 cases of strong expression. The expression of VEGF was detected in 28 cases of BPH with 22 cases of moderate expression and 6 cases of strong expression. The expression of VEGF in PCa was significantly higher than that in BPH (P < 0.05, see ).
TABLE 2 Difference of VEGF expressions between PCa and BPH
Figure 3 Expression of VEGF in PCa (SP, × 400) Staining intensity was detected in the cytoplasm of tumor cells.
Figure 4 Expression of VEGF in BPH (SP, × 400) Staining intensity was detected in the cytoplasm.
The Correlation Between the Expressions of hTERT and VEGF in Pca
With the increase of the expression of VEGF, the expression of hTERT also increased in PCa. Significant correlation was observed between the expressions of hTERT and VEGF in PCa (r = 0.8333, P < 0.05, see ).
TABLE 3 Correlation between hTERT and VEGF in Pca
The Correlation Between the Expressions of hTERT and VEGF in BPH
There was no significant correlation between the expressions of hTERT and VEGF in BPH (r = 0.3156, P > 0.05 see ).
TABLE 4 Correlation between hTERT and VEGF in BPH
DISCUSSION
A study by Botchkina et al. Citation[19] showed that all PCa patients revealed high levels of telomerase activity, while the majority of patients with BPH did not express any telomerase activity, or expressed low levels of activity. Re-expression of telomerase is believed to play an important role in immortalization and carcinogenesis. In the present study, we demonstrated that the expression of hTERT significantly increased in PCa (38/60) compared to that in BPH (10/60). Thus, telomerase activity is considered to be a potentially useful diagnostic marker.
Although in situ hybridization (ISH) or the telomeric repeat amplification protocol assay (TRAP) have been used for hTERT assay, these methods are complicated and expensive to use in clinical works, and general and simple in situ detection of hTERT activity for clinical application is needed. The study showed that expression of hTERT protein correlated with that of hTERT mRNA by in situ hybridization (p = 0.044) Citation[20], and immunohistochemistry is a convenient way to detect protein expression. Therefore, we tried immunohistochemistry in our study, and the results indicated that detection of hTERT at the cellular level by immunohistochemistry is achievable and hTERT could serve as a reliable molecular marker of prostate malignancy.
VEGF plays a fundamental role in tumor vessel formation, which is required for tumor growth and metastasis. Overexpression of VEGF causes vascular endothelial cells proliferation and increased vascular permeability. Our study demonstrated that the level of VEGF expression was significantly higher in PCa than in BPH, indicating an important role VEGF plays in genesis and development of PCa.
Investigations have been conducted on the correlation between the expression of VEGF and that of hTERT. Up to now there have been a few reports on the possible correlation between hTERT and VEGF and the results are not coincident. Zaccagnini et al. Citation[16] demonstrated that VEGF delivery induced angiogenesis and increased expression of hTERT and telomerase activity in skeletal muscles and endothelial cells. But Kurz et al. Citation[21] got the reverse result that VEGF could not increase telomerase activity in human endothelial cells. Our results showed a strong positive correlation between hTERT and VEGF expressions in PCa with the coefficient of 0.83. This suggests that high level of hTERT expression correlates the likelihood of tumor metastasis. The possible background for this includes that telomerase activity is required for maintenance of ends of chromosomes (telomeres) during cell division Citation[22]. But at the same time, too much expression of telomerase activity will lead to the overgrowth of tumor cells, which needs a lot of oxygen. This leads to a hypoxic micro-environment, which can induce the expression of VEGF Citation[23]. Therefore, telomerase cooperates with VEGF in promoting the growth and proliferation of carcinoma Citation[24].
On the other hand, VEGF may exert its function through hTERT. The study of Zaccagnini et al. Citation[16] has shown that VEGF could upregulate the expression of hTERT through the nitric oxide pathway, and hTERT behaves as an angiogenic factor and a downstream effector of VEGF signaling. In addition, He et al. Citation[25] studied the telomerase activity during in vitro committed differentiation of rabbit bone marrow mesenchymal stem cells (MSCs). In their research, telomerase activity was at a lower level in freshly isolated bone marrowmononuclear cells and MSCs pre-proliferation. Once induced by VEGF, bone marrow MSCs were expanded potentially ex vivo and their telomerase activity was soon up-regulated and kept at a high level in the subsequent five passages.
In the present study, it was demonstrated that with the increase of the expression of VEGF, the expression of hTERT also increased in PCa. Studies have demonstrated that FLT-1 and FLK-1, the receptors of VEGF, could express on tumor cells in PCa, which suggests a potential autocrine function for VEGF (such as regulating tumor cell proliferation) Citation[15]. Then, hTERT might be one of the important effectors in the proliferation-promoting effect of VEGF on tumor cells in PCa.
There was no significant correlation between the expressions of hTERT and VEGF in BPH in this study. BPH is a kind of benign disease, and hTERT might not have been simulated enough. Or this might be caused by the few positive expression cases of hTERT and VEGF, which need further study.
This was a retrospective study. Further investigations should be directed to the exact mechanisms of the correlation between the expressions of VEGF and hTERT in PCa with a prospective study.
CONCLUSION
All experiences above indicate that hTERT was one of the important proteins in the proliferation-promoting effect of VEGF on tumor cells in PCa.
The financial support of National Natural Science Foundation (30440070 and 30670549) is gratefully acknowledged.
REFERENCES
- Blackburn E. H. Switching and signalling at the telomere. Cell 2001; 106: 661–673
- Blasco M. A. Telomerase beyond telomeres. Nat Rev Cancer 2002; 2: 627–632
- Holt S. E., Wright W. E., Shay J. W. Regulation of telomerase activity in immortal cell lines. Mol Cell Biol. 1996; 16: 2932–2939
- Forsyth N. R., Wright W. E., Shay J. W. Telomerase and differentiation in multicellular organisms: turn it off, turn it on, and turn it off again. Differentiation 2002; 69: 188–197
- Fujimoto R., Kamata N., Taki M., Yokoyama K., Tomonari M., Nagayama M., et al. Gene expression of telomerase related proteins in human normal oral and ectocervical epithelial cells. Oral Oncology 2003; 39: 445–452
- Mitchell J. R., Collins K. Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase. Molecular Cell 2000; 6: 361–371
- Franco J. R., Wasinwong Y. Expression of human telomerase reverse transcriptase in laryngeal tissues. Otolaryngology–Head Neck Surg 2004; 131: 124–125
- Iczkowski K. A., Pantazis C. G., McGregor D. H., Wu Y., Tawfik O. W. Telomerase reverse transcriptase subunit immunoreactivity: a marker for high-grade prostate carcinoma. Cancer 2002; 95: 2487–93
- Crocitto L. E., Korns D., Kretzner L., Shevchuk T., Blair S. L., Wilson T. G., et al. Prostate cancer molecular markers GSTP1 and hTERT in expressed prostatic secretions as predictors of biopsy results. Urology 2004; 64: 821–825
- Orlando C., Gelmini S., Selli C., Pazzagli M. Telomerase in urological malignancy. J Urol 2001; 166: 666–673
- Natarajian S., Chen Z., Wancewicz E. V., Monia B. P., Corey D. R. Telomerase reverse transcriptase (hTERT) mRNA and telomerase RNA (hTR) as targets for downregulation of telomerase activity. Oligonucleotides 2004; 14: 263–273
- Byzova T. V., Goldman C. K., Jankau J., Chen J., Cabrera G., Achen M. G., et al. Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo. Blood 2002; 99: 4434–4442
- Greb R., Wallweiner D., Kiesel L. Vascular endothelial growth factor A (VEGF–A) MRNA expression levels decrease after menopause in normal breast tissue but not in breast cancer lesions. British J Cancer 1999; 81: 225–231
- Ruohola J., Valve E., Karkkainen M., Joukov V., Alitalo K., Harkonen P. Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells. Mol Cell Endocrinology 1999; 49: 29–40
- Ferrer F. A., Miller L. J., Lindquist R., Kowalczyk P., Laudone V. P., Albertsen P. C., et al. Expression of vascular endothelial growth factor receptors in human prostate cancer. Urology 1999; 54(3)567–572
- Zaccagnini G., Gaetano C., Della P. L., Nanni S., Grasselli A., Mangoni R., et al. Telomerase mediates vascular endothelial growth factor-dependent responsiveness in a rat model of hind limb ischemia. J Biol Chem 2005; 280: 14790–14798
- Byzova T. V., Goldman C. K., Jankau J., Chen J., Cabrera G., Achen M. G., et al. Adenovirus encoding vascular endothelial growth factor–D induces tissue-specific vascular patterns in vivo. Blood 2002; 99(12)4434–42
- Hao X. P., Willis J. E., Pretlow T. G., Rao J. S., MacLennan G. T., Talbot I. C., et al. Loss of fragile histidinc traid expression in colorectal carcinomas and premalignant lesions. Cancer Res 2000; 60: 18–21
- Botchkina G. I., Kim R. H., Botchkina I. L., Kirshenbaum A., Frischer Z., Adler H. L. Noninvasive detection of prostate cancer by quantitative analysis of telomerase activity. Clin Cancer Res 2005; 11(9)3243–3249
- Ikeda S., Shibata T., Funakoshi N. Immunohistochemical detection of telomerase (TERT) protein in benign and malignant tumor cells of the breast. Rinsho Byori 2005; 53(6)487–493
- Kurz D. J., Hong Y., Trivier E., Huang H. L., Decary S., Guo H. Z., et al. Fibroblast growth factor-2, but not vascular endothelial growth factor, upregulates telomerase activity in human endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology 2003; 23: 748
- Orlando C., Gelmini S. Telomerase in endocrine and endocrine-dependent tumors. J Steroid Biochem Mol Biol 2001; 78: 201–214
- Li R., Younes M., Wheeler T. M., Scardino P., Ohori M., Frolov A., et al. Expression of vascular endothelial growth factor receptor-3 (VEGFR-3) in human prostate. Prostate 2004; 58: 193–199
- Kirkpatrick K. L., Newbold R. F., Mokbel K. The mRNA expression of hTERT in human breast carcinomas correlates with VEGF expression. J Carcinog 2004; 3: 1–4
- He C. R., Liu X. J., Yan S. R. The telomerase activity during in vitro committed differentiation of rabbit bone marrow mesenchymal stem cells. Basic Med Sci and Clini. 2003; 23: 431–435