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ORIGINAL ARTICLE
Year : 2014  |  Volume : 3  |  Issue : 3  |  Page : 171-174

Relationship of angiogenic and apoptotic activities in soft-tissue sarcoma


1 Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
2 Department of Pathology, Hospital Umum Sarawak, Jalan Hospital, 93586 Kuching, Sarawak, Malaysia

Date of Web Publication15-Jul-2014

Correspondence Address:
Thin Thin Win
Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan
Malaysia
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Source of Support: This study was funded under Universiti Sains Malaysia Short Term Grant (No: 304/PPSP/6139055)., Conflict of Interest: None


DOI: 10.4103/2278-330X.136799

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  Abstract 

Introduction: Angiogenesis and apoptosis play an essential role in tumor development and progression. Previous studies on apoptosis and angiogenesis of soft-tissue sarcoma (STS) were done separately. This is the first study of the relationship between apoptotic and angiogenic activity. Correlation of expression of anti-apoptotic protein (Bcl-2) and pro-apoptotic protein (Bax) in the tumor cells (TCs) with their expression in endothelial cell (EC) of the tumor blood vessels in STS were also carried out. Materials and Methods: 101 cases of STS; consisting liposarcoma, malignant fibrous histiocytoma, synovial sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma and malignant peripheral nerve sheath tumor; were collected and immunohistochemical reaction of vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined. Results: Higher Bax expression in TCs (54.5%) was seen compared to Bcl-2 expression (44.6%). There was a significant association between Bcl-2 and Bax in TCs with ECs. Significant association was also seen between histological types of STS with Bcl-2 expression; however not with Bax expression. There was an association between VEGF and Bax with high VEGF expression and weak Bax expression. However, VEGF expression was not associated with Bcl-2 expression and histological types. Conclusion: This study supports the role of ECs of tumor blood vessels and apoptosis of TCs in tumor management. Increased angiogenesis may inhibit apoptosis of TCs and lead to tumor growth. Therefore, inhibition of ECs survival or activation of ECs death is promising prospect for tumor therapy. Immunohistochemical antibodies in this study might be potential useful marker for the prognosis of STS.

Keywords: Angiogenesis, apoptosis, Bax, Bcl-2, soft-tissue sarcoma, vascular endothelial growth factor


How to cite this article:
Win TT, Jaafar H, Yusuf Y. Relationship of angiogenic and apoptotic activities in soft-tissue sarcoma. South Asian J Cancer 2014;3:171-4

How to cite this URL:
Win TT, Jaafar H, Yusuf Y. Relationship of angiogenic and apoptotic activities in soft-tissue sarcoma. South Asian J Cancer [serial online] 2014 [cited 2019 Aug 18];3:171-4. Available from: http://journal.sajc.org/text.asp?2014/3/3/171/136799


  Introduction Top


Soft-tissue sarcomas (STS) are a heterogenous group of tumors arising from mesenchymal tissue. Despite the relative rarity (less than 1% of all malignant tumors), incidence and mortality are increasing with a significant diagnostic and therapeutic challenges.

Angiogenesis and apoptosis play a critical role in tumor growth and progression. Neovascularization is an important process in tumor cell (TC) proliferation and also influences TC dissemination. [1] Apoptosis of the TCs and endothelial cells (ECs) of tumor blood vessels also play a role in TC survival and TC death. Studies on apoptotic and angiogenic activities of TCs in STS have been done separately, [2],[3],[4] however no study has been done on the expression of apoptotic markers in ECs. This study aimed to analyze the apoptotic and angiogenic activities in TCs and ECs of tumor blood vessels; and study their association in STS. Apoptotic and angiogenic activities were studied by using immunohistochemical markers. Meanwhile, the correlation of apoptotic activities in TCs and ECs of blood vessels that supply the tumour were also carried out. Background hypothesis of this study was that increased apoptosis of TCs and ECs of tumor blood vessels lead to TC death and regression of the tumor.


  Materials and Methods Top


101 cases of STS which include liposarcoma, malignant fibrous histiocytoma (MFH), synovial sarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma (LMS) and malignant peripheral nerve sheath tumor (MPNST) were included in this study. Formalin-fixed paraffin-embedded tissue blocks of the resected specimens of STS without prior chemotherapy were collected from Pathology Department, Hospital Universiti Sains Malaysia. The study was funded under USM Short Term Grant and it was approved by board of research and ethical committee of School of Medical Sciences, Universiti Sains Malaysia.

Immunohistochemistry

Paraffin-embedded tissue blocks were sectioned into 3-4 μm thickness, placed on poly-L-Lysine slides. After deparaffinization, antigen retrieval was carried out. Monoclonal mouse anti-human Bcl-2 (DAKO, Denmark), monoclonal rabbit antiserum of Bax (DAKO, Denmark) and rabbit polyclonal anti-human VEGF (Santa Cruz biotechnology) were used as primary antibodies. For secondary antibody and detection system, horseradish peroxidase polymer solution, buffered solution containing hydrogen peroxide and detector agent of di-amino benzidine were used. All slides were analyzed under microscope (Olympus C × 31) with × 400 magnification.

The scoring for expression of Bcl-2 and Bax was carried out based on combined score of qualitative and quantitative analysis. The intensity (qualitative) of the Bcl-2 and Bax immunostaining was evaluated by dividing the cytoplasmic staining reactions in four score groups: 1 = Weak cytoplasmic staining intensity; 2 = Moderate cytoplasmic staining intensity; 3 = Strong cytoplasmic staining intensity; and 4 = Very strong cytoplasmic staining intensity. The immunostaining was quantified from a total of 100 cells as follows: 0 = No positive staining; 1= <25% of TCs or vessels show cytoplasmic staining positivity; 2 = 25-50% of TCs or vessels show cytoplasmic reactivity; 3 = 50-75% of TCs or vessels showing cytoplasmic reactivity; 4= >75% of TCs or vessels showing cytoplasmic reactivity. A combined score for Bcl-2 and Bax immunostaining was obtained by adding the qualitative and quantitative score; these sums were then divided in three main groups: score = 0, no immunoreactivity; score = 1-4, weak immunoreactivity; and score = 5-8, strong immunoreactivity. [5] To simplify, the results were condensed into two categories only. Negative indicates no immunoreactivity and positive indicates weak and strong immunoreactivity.

The expression of VEGF was assessed based on the percentage of immunoreactive cells in total 1000 cells. Immunoreactivity was graded as follows; more than 10% of the cells staining graded as positive or unequivocal red staining in the TC cytoplasm; and less than 10% of cells staining or no detectable staining graded as negative. [6] Qualitative intensity of staining for VEGF was assessed using a scale between 0 and 3+, with 0 representing no detectable stain, 1 + as weak, 2 + intermediate or moderate and 3 + representing strongest stain. For analyses, VEGF expression was assigned "negative" with a score of 0 and "positive" with a score of 1+ to 3+.

Statistical analysis

All the results were analyzed by using SPSS Inc. PASW Statistics for Windows, Version 18.0. Chicago, Released 2009. Association between expression of Bcl-2 and Bax in TCs with their expression in ECs were analyzed by using Mc-Nemar test. Association between expression of Bcl-2 and Bax in TCs with VEGF in TCs were analyzed by using Pearson's Chi-square test. Association between expression of Bcl-2, Bax and VEGF in TC with histological types was analyzed by using Fisher exact test. P < 0.001 was considered to be statistically significant.


  Results Top


Bcl-2, Bax, VEGF expression in TCs and ECs

Bcl-2 expression in TCs was more than Bcl-2 in ECs. Among the positive Bcl-2 cases in TCs, 27.8% showed strong expression and 16.8% showed weak expression; whereas only 7.9% showed strong expression with 26% weak expression in ECs. As in expression of Bcl-2, the expression of Bax in TCs was stronger than in ECs. The positive staining were 54.4% and 48.5% in tumor and ECs respectively. Majority of the positive cases in both groups showed weak immunoreactivity. Both Bcl-2 and Bax in TCs were directly associated with both expression in ECs (P < 0.001) as shown in [Table 1].
Table 1: Association of Bcl-2 and Bax in tumour cells (TC) with Bcl-2 and Bax in endothelial cells

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VEGF expression was done only in TCs as ECs served as internal positive control and comparison was not done between TCs and ECs. In TCs, 68.3% showed positivity with 54.5% of strong immunoreactivity. Positive immunoreactivities of Bcl-2, Bax and VEGF appear as cytoplasmic staining in tumour cells and in endothelial cells [Figure 1]a and b.
Figure 1: Positive immunoreactivities of Bcl - 2, Bax and VEGF appear as cytoplasmic staining in tumour cells (a) and in endothelial cells (b)

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Association between VEGF expression with Bcl-2 and Bax expression in TCs

There was no significant association between expression of VEGF and expression of Bcl-2 in TCs. However, significant association between VEGF and Bax expression in TCs was seen. More cases (30.7%) revealed weak or no Bax expression with high VEGF expression compared to only 23.8% of strong Bax expression with high VEGF expression. Results are summarized in [Table 2].
Table 2: Association of Bcl-2 and Bax in tumour cells (TC) with VEGF in tumour cells (TC)

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Association between expression of Bcl-2, Bax and VEGF in TCs with histological types

The histological types of the STS showed a significant correlation with expressions of Bcl-2 in TCs [Table 3]. The Bcl-2 expression was very high in synovial sarcoma (100%) and LMS (71.4%); whereas liposarcoma, MFH and MPNST showed <40% of positive cases. There was also no association between expression of both Bax and VEGF with histological types of STS.
Table 3: Association between expression of Bcl-2, Bax, and VEGF in tumour cells (TC) with histological types

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  Discussion Top


Expression of Bcl-2 and Bax

In the development and progression of malignant tumor, two most important apoptosis regulating proteins are Bcl-2 and Bax. Both are functionally antagonistic proteins, which control apoptosis. To the best of our knowledge, this is the first attempt to study the expression of Bcl-2 and Bax in TCs of STS comparing with expression of both in ECs of the tumor blood vessels.

In this study, apoptotic activity was higher in the TCs compared to anti-apoptotic activity, which is consistent with the findings reported by Sabah et al. [2] Unlike Bcl-2 expression; many studies have not been done to evaluate the expression of Bax in STS by immunohistochemistry. Only few studies revealed Bax expression was a common finding in STS. [2],[7] Bcl-2 and Bax are expressed differently in various tissue and cells populations and may respond differently to apoptotic stimuli. [8] Bcl-2 is expressed mostly in the peripheral proliferating tumor areas and decreased in the areas of cell death. However, Bax is highly expressed in the periphery (close to stroma) and remain unchanged or even increased in the areas of cell death. [9] This study showed slight higher expression of Bax compared to Bcl-2, as most of the cases were high-grade tumor with necrosis. It is supported by some research findings which showed a strong association between Bax and Bcl-2 expression; and a trend for high-grade tumors being more frequently Bax-positive. [10]

Association of the expression of Bcl-2 and Bax in TCs and in ECs

This study showed both expression of Bcl-2 and Bax were directly associated between TCs and ECs (P < 0.001). No report was seen in literatures on association between the expression of Bcl-2 and Bax in TCs with the ECs.

Apoptosis in ECs may play an important role in the biology of tumor blood vessels, as the survival of the ECs within the tumor stroma influences the survival of the TCs themselves. ECs isolated from tumor vessels are known to be more resistant to apoptosis than ECs isolated from vessels of corresponding normal tissues. [11] It supported our findings, which is a direct association of Bcl-2 expressions in TCs with Bcl-2 expression in ECs. Based on these results, the inhibition of endothelial survival factors or the activation of ECs death is an attractive anti-tumor strategy, especially when used to supplement strategies directed at the TCs themselves. [12] Several anti-angiogenic agents in clinical trials for cancer treatment appear to exert anti-tumor action by promoting ECs apoptosis.

The apoptosis in ECs may be the triggering factor that selectively causes an increased apoptosis in TCs. This might be the reason for our findings that the higher the expression of Bax, the higher apoptotic activity.

Association between the expression of Bcl-2, Bax and VEGF in TCs with histological types

The significant association was seen between the expressions of Bcl-2 in TCs with histologic types. In this study, the expression of Bcl-2 and Bax were 100% in synovial sarcoma. Bcl-2 expression was also commonly seen in LMS (71.4%) and fibrosarcoma (55.6%) whereas Bax expression was seen in MFH (60%), fibrosarcoma (55.6%), liposarcoma (52.8%) and LMS (50.0%). The other histologic subtypes were expressed less than 50% of Bcl-2 and Bax protein. These findings were consistent with previous report by Sabah et al. [2] No association was seen between Bax and VEGF expression with histological types in this study.

Expression of VEGF and its association with the expression of Bcl-2 and Bax in TCs

Angiogenesis has been shown to play a key role in the maintenance, growth and metastasis of many solid tumors. [13] Most solid tumors secrete and overexpress VEGF, which is one of the potent activators of angiogenesis. In this study, VEGF expression was seen in 69 cases (68.3%) with strong positivity which was almost consistent with findings by Chao et al.[3] Interestingly, diversity was seen in VEGF expression among the various histologic types of STS. In this study, all histological types showed >60% of VEGF expression with 100% for synovial sarcoma and 85.7% for LMS.

Association between angiogenesis and apoptosis is expected in human solid tumors. Because angiogenesis inhibitors can induce and sustain the dormancy of experimental primary tumors and micro-metastases by elevating the incidence of apoptosis in TCs, while the proliferation rate remains unchanged. [14] Incidence of spontaneous apoptosis in TCs is also inversely related to the extent of neovascularization. [15] In a study on colorectal carcinoma, colorectal carcinogenesis is related to the inhibition of apoptosis and proliferative activity. [16] VEGF is also able to induce Bcl-2 expression in ECs of human umbilical vein; which provide an important role of Bcl-2 in VEGF induced cell survival. [17]

This study hypothesized that VEGF expression was higher in association with strong Bcl-2 expression and weak Bax expression. There were no previous published literatures which indicate the association of Bcl-2 and Bax expression with VEGF expression particularly in STS. It showed significant association between VEGF expressions with Bax expression; but no significant association with Bcl-2 expression. It was also able to find that high VEGF expression was correlated with weak Bax expression in TCs. It was evidenced by more cases (30.7%) showing no or weak Bcl-2 expression compared to only 23.8% of strong Bcl-2 expression with high VEGF expression.


  Conclusion Top


Angiogenic and apoptotic activity plays a pivotal role in the development and progression of STS. Findings in this study support the role of ECs of tumor blood vessels and apoptosis of TCs in tumor management. Increased angiogenesis may inhibit apoptosis of TCs and lead to tumor growth. Therefore, inhibition of ECs survival or activation of ECs death is promising prospect for tumor therapy. Immunohistochemical antibodies in this study might be potential useful marker for the prognosis of STS.


  Acknowledgments Top


This study was funded under Universiti Sains Malaysia Short Term Grant (No: 304/PPSP/6139055).

 
  References Top

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2.Sabah M, Cummins R, Leader M, Kay E. Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: Correlation with histologic grade. Appl Immunohistochem Mol Morphol 2007;15:64-9.  Back to cited text no. 2
    
3.Chao C, Al-Saleem T, Brooks JJ, Rogatko A, Kraybill WG, Eisenberg B. Vascular endothelial growth factor and soft tissue sarcomas: Tumor expression correlates with grade. Ann Surg Oncol 2001;8:260-7.  Back to cited text no. 3
    
4.Potti A, Ganti AK, Tendulkar K, Sholes K, Chitajallu S, Koch M, et al. Determination of vascular endothelial growth factor (VEGF) overexpression in soft tissue sarcomas and the role of overexpression in leiomyosarcoma. J Cancer Res Clin Oncol 2004;130:52-6.  Back to cited text no. 4
    
5.Vakkala M, Lähteenmäki K, Raunio H, Pääkkö P, Soini Y. Apoptosis during breast carcinoma progression. Clin Cancer Res 1999;5:319-24.  Back to cited text no. 5
    
6.Borre M, Nerstrøm B, Overgaard J. Association between immunohistochemical expression of vascular endothelial growth factor (VEGF), VEGF-expressing neuroendocrine-differentiated tumor cells, and outcome in prostate cancer patients subjected to watchful waiting. Clin Cancer Res 2000;6:1882-90.  Back to cited text no. 6
    
7.Dan′ura T, Kawai A, Morimoto Y, Naito N, Yoshida A, Inoue H. Apoptosis and expression of its regulatory proteins in soft tissue sarcomas. Cancer Lett 2002;178:167-74.  Back to cited text no. 7
    
8.Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995;80:285-91.  Back to cited text no. 8
    
9.Shilkaitis A, Graves J, Mehta RR, Hu L, You M, Lubet R, et al. Bcl-2 and Bax are differentially expressed in hyperplastic, premalignant, and malignant lesions of mammary carcinogenesis. Cell Growth Differ 2000;11:437-45.  Back to cited text no. 9
    
10.Veronese S, Mauri FA, Caffo O, Scaioli M, Aldovini D, Perrone G, et al. Bax immunohistochemical expression in breast carcinoma: A study with long term follow-up. Int J Cancer 1998;79:13-8.  Back to cited text no. 10
    
11.Benjamin LE, Golijanin D, Itin A, Pode D, Keshet E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 1999;103:159-65.  Back to cited text no. 11
    
12.Nair S, Boczkowski D, Moeller B, Dewhirst M, Vieweg J, Gilboa E. Synergy between tumor immunotherapy and antiangiogenic therapy. Blood 2003;102:964-71.  Back to cited text no. 12
    
13.Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, Moore DH, et al. Tumor angiogenesis: A new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 1992;84:1875-87.  Back to cited text no. 13
    
14.Ueda M, Ueki K, Kumagai K, Terai Y, Okamoto Y, Ueki M, et al. Apoptosis and tumor angiogenesis in cervical cancer after preoperative chemotherapy. Cancer Res 1998;58:2343-6.  Back to cited text no. 14
    
15.Lu C, Tanigawa N. Spontaneous apoptosis is inversely related to intratumoral microvessel density in gastric carcinoma. Cancer Res 1997;57:221-4.  Back to cited text no. 15
    
16.Tsujitani S, Shirai H, Tatebe S, Sugamura K, Ohfuji S, Gomyo Y, et al. Apoptotic cell death and its relationship to carcinogenesis in colorectal carcinoma. Cancer 1996;77:1711-6.  Back to cited text no. 16
    
17.Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998;273:13313-6.  Back to cited text no. 17
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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