Med Oncol (2010) 27:20–28 DOI 10.1007/s12032-008-9164-x
ORIGINAL PAPER
Long-term effect of Spirulina platensis extract on DMBA-induced hamster buccal pouch carcinogenesis (immunohistochemical study) Mohammed E. Grawish Æ Ahmed Ragheb Zaher Æ Ashraf I. Gaafar Æ Wesam A. Nasif
Received: 3 December 2008 / Accepted: 30 December 2008 / Published online: 21 January 2009 Ó Humana Press Inc. 2009
Abstract In cancer research, the use of complementary and alternative medicine has increased over the past decade. In this study, 80 male golden Syrian hamsters were divided into four equal groups; the right buccal pouches of the hamster rats in group 1 were painted with 0.5% solution of 7, 12-dimethylbenz[a]anthracene (DMBA), three times a week for 32 weeks. The same pouches of group 2 were subjected to the same DMBA painting; but at the same time, the animals received 10 mg/daily Spirulina platensis extract for the same period. In group 3, the same regimen of DMBA painting was done but for 24 weeks only and the daily systemically S. platensis was received for the 32 weeks. In group 4, neither DMBA painting nor S. platensis administration was done but pouches were painted with saline and served as a control one. Five rats from each group were sacrificed at 12, 24, 28, and 32 weeks, respectively. The required pouches were excised, fixed, and embedded in paraffin to be immunostained with proliferating cell nuclear antigen (PCNA). The results showed that increased PCNA expression was directly related to the severity of pathological alterations from normal epithelium to dysplasia and from dysplasia to squamous cell carcinoma (SCC) in the study groups at the
M. E. Grawish (&) A. R. Zaher Oral Biology Department, Faculty of Dentistry, Mansoura University, Mansoura, Egypt e-mail:
[email protected] A. I. Gaafar Oral Pathology Department, Faculty of Dentistry, Assiut Branch, Al-Azhar University, Cairo, Egypt W. A. Nasif Genetic Engineering Institute, Menofyia University, Menofyia, Egypt
different extended periods of DMBA application and S. platensis extract administration. Analysis of variance and Duncan’s multiple-range test for PCNA labeling index were proved a high significant difference (P \ 0.01) between the different groups. From the pervious results, it can be concluded that S. platensis extract has a beneficial role in regression of cancer progression. Keywords Spirulina platensis 7, 12-Dimethylbenz[a] anthracene Proliferating cell nuclear antigen
Introduction Thirty-five percent of all human cancer deaths appear to be associated with diet and nutrition. Since then numerous experimental, epidemiological, and clinical studies have proved this connection. These studies have demonstrated conclusively that numerous nutrient and non-nutrient constituents in foods in our diet have the potential to confer chemo-preventive properties or enhance conventional therapy [1–3]. There is evidence that vegetables and fruits are rich sources of antioxidants like vitamin C, E, and b-carotene and might protect against different forms of cancer. There is also an evidence to suggest that physiological aging of the immune system may affect cell-mediated immunity that in turn results in cancer development and autoimmune diseases [4]. Radiation, chemotherapy, and surgery therapies cause side effects often worse than the cancer itself. Preventive methods’ utilizing natural products directly or as adjuvant to conventional cancer treatment are therefore the focus of certain research [5]. Spirulina, now named Arthrospira, is a microscopic filamentous blue-green alga that has a long
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history of use as traditional food of some Mexican and African peoples. Its name was derived from the spiral or helical nature of its filaments and it was found in water alkaline volcanic lakes [6]. Early interest in Spirulina focused mainly on its rich content of protein, vitamins, essential amino acids, minerals, and essential fatty acids. Spirulina is 60–70% protein by weight and contains a rich source of vitamins, especially vitamin B12 and provitamin A (b-carotene), and minerals, especially iron, one of the few sources of dietary c-linolenic acid [7]. The therapeutic properties of Spirulina of some prospective studies were to treat certain pathological conditions including iron deficiency anemia [8, 9], pernicious anemia [10], vitamin A deficiency [11–13], inhibition of mother–child transmission of HIV [14], inhibition of infection of T4 helper cells by HIV [15], protein energy disorders [16], cancer prevention through provision of carotenoids [17–19], radiological protection[20], and strengthening immune defenses [21–23]. The studies summarized above suggest a role for Spirulina. Spirulina may offer some degree of protection against certain forms of cancer through its effect on the immune system, through a direct effect in the repair of DNA, and antioxidant protection from reactive oxygen species generated during normal or abnormal metabolism and from toxic substances in the environment. Further researches along these lines are recommended to validate these assumptions. Thus, the purpose of this study was to investigate the long-term effect of Spirulina platensis on 7, 12-dimethylbenz[a]anthracene (DMBA) induced oral carcinogenesis on hamster cheek pouch (HCP) mucosa using proliferating cell nuclear antigen (PCNA) as a diagnostic marker.
Materials and methods Eighty pathogen-free male golden Syrian hamsters were used in this study. They were 60–90-days-old and weighed 100–120 g each. The protocol of this study was approved by the Ethics Committee of Mansoura University of Medical Sciences, Mansoura, Egypt, in 2008. The animals were divided into four equal groups; the right buccal pouches of the hamster rats in group 1 were painted with 0.5% solution of DMBA (Sigma Chemical Company, St. Louis, MO) in a heavy mineral oil (Fisher-Scientific Company, Pittsburgh, PA) using no. 4 sable-hair brush three times a week [24–26] for 32 weeks. The same pouches of group 2 were subjected to the same DMBA painting but at the same time, animals received 10 mg/daily [27] S. platensis extract for the same period, which was added to the soft diet supplements. In group 3, the same regimen of DMBA painting was done but
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for 24 weeks only and the daily systemically S. platensis was received for 32 weeks. The hamster rats in group 4 received neither DMBA nor S. platensis extract, painted with mineral oil, and served as a control one. Five rats from each group were sacrificed by ether inhalation at 12, 24, 28, and 32 weeks, respectively. The required pouch of each animal was examined by its exploration for the detection of any clinical changes, and then the pouches were excised, fixed in 10% buffered formalin and embedded in paraffin wax. The specimens were sectioned at 4 lm and prepared for PCNA immunohistochemical staining. PCNA immunohistochemical staining PCNA was used to determine the proliferating cells. For this, representative slices were deparaffinized, rehydrated, and immunostained by the peroxidase anti-peroxidase method. High temperature antigen unmasking technique was employed in 0.01 M citrate buffer pH 6.0 in microwave oven, twice for 5 min each. Blocking of non-specific reaction was performed with 1% normal goat serum and 3% nonfat milk, and PCNA mouse monoclonal antibody (Novo Castra NCL-PCNA) (1:100). After rinsing in phosphate buffered saline (0.01 mol/l PBS, pH 7.4), the sections were incubated in secondary antiserum. They were then washed in PBS and incubated in ABC (avidine–biotine complex) reagents (ABC-kit-Vector) and incubated in peroxidase reaction (3,30 -diaminobenzidine tetra hydrochloride, Sigma) containing 0.01% H2O2 in PBS buffer [28]. Assessment of PCNA labeling index and statistical analysis The slides were examined by light microscopy and the images were captured with a color video camera (MA87 color digital/video microscope eyepiece; Miles Co Scientifics’, Princeton). Counting of immunostained positive cells was done by two investigators simultaneously. They had no knowledge about any clinicopathological data. All identifiable PCNA staining diffuse or granular was considered positive. For the determination of PCNA labeling index (LI); the percentage of cells that were positively stained was obtained from counting 200 cells in five different fields of each slide at a magnification 940, the average of five counts was used as PCNA LI for each analysis of each group at the different periods. Any nuclear staining regardless the intensity was considered immunopositive [29]. The PCNA LI was expressed as a percentage of immunoreactive positive proliferative cells to the total counted resting cells [30]. The data were subjected to the analysis of variance followed by least significant difference
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Table 1 Clinical findings Observation periods Group
12 weeks (5 animals)
24 weeks (5 animals)
28 weeks (5 animals)
Group 1 (20 animals)
Three animals showed thickened mucosa with a rough granular surface and whitish appearance
All animals showed immovable marked tumor growth fixed to the surrounding and underlying tissues
Two animals showed Two animals showed marked marked tumor growth tumor growth, fixed to the manifested with an extra underlying tissues, enlarged oral swelling extra orally, foul odor with bleeding on touch
Group 2 (20 animals)
Group 3 (20 animals)
Group 4 (20 animals)
Two animals showed a small whitish patch Three animals showed thickened mucosa with a rough granular surface and whitish appearance
32 weeks (5 animals)
Three animals were died
Three animals were died
Two animals showed a marked raised area of thickened buccal pouch mucosa
Three animals showed a noticeable raised buccal pouch
Four animals showed a well-developed mass fixed to the underlying tissues and enlarged extra orally
Two animals showed a slightly raised buccal pouch
Three animals showed marked tumor growth fixed to the underlying tissues
Two animals were died
One animal was died
The same clinical findings as their counterpart of group 2
The same clinical findings Three animals showed Four animals showed a as their counterpart of a marked swelling of the marked swelling of the group 2 buccal pouch buccal pouch
No identifiable changes
No identifiable changes
and results were compared by Duncan’s multiple-range test to determine if there is any statistical difference using an IBM-compatible PC and SAS (2004) for windows computer packages.1 A histogram was drawn for the mean value of all groups.
Two animals showed marked tumor growth fixed to the underlying tissues
One animal showed marked tumor growth fixed to the underlying tissues
Faint whitish color of the buccal pouch
Faint whitish color of the buccal pouch
squamous cell carcinoma (SCC) differentiation (well, moderately, and poorly) were observed in the other three experimental groups as shown in Table 2. Samples from died animals were not taken. Immunohistochemical results
Results Clinical findings Variants of clinical findings were seen after scarification of animals, ranging from thickened mucosa with a rough granular surface and whitish appearance to marked tumor growth as shown in Table 1. Died animals were not examined.
In the control group, PCNA antigen was detected exclusively in the nuclei of epithelial cells as a few PCNA positive cells, which were found and distributed in a single or focal pattern mostly in the basal cell layer. No reaction was observed in the cytoplasm (Fig. 1). Meanwhile, the results showed that increased PCNA expression was directly related to the severity of pathological alterations from normal epithelium to dysplasia and from dysplasia to SCC in the study groups at the different extended periods of DMBA application and S. platensis extract administration (Figs. 2, 3, 4, 5, 6, 7).
Histopathological findings The hamster buccal pouch mucosa of the control group consists of thin keratinized stratified squamous epithelium with no rete pegs, sub-epithelial connective tissue, aereolar, and muscular layer. Variants of histological grading of epithelial dysplasia (ED) (mild, moderate, and sever) and 1
SAS (2004). Statistical Analysis System. SAS User Guide: Statistics. SAS Institute Inc. Editors, Cary, NCS.
Statistical results Data represented in Table 3 showed there is a relationship between PCNA LI and tumor pathological features as PCNA LI was closely related to primary tumor status and histological grade; the PCNA LI became higher as the histological grade increased. Moreover, analysis of variance and Duncan’s multiple-range test were clarified a high
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Table 2 Histological findings Observation periods Group
12 weeks (5 animals)
24 weeks (5 animals)
28 weeks (5 animals) 32 weeks (5 animals)
Group 1 (20 animals)
Three animals showed SCC
All animals showed moderately to poorly differentiated SCC
Two animals showed Two animals showed poorly poorly differentiated differentiated SCC SCC
Two animals showed epithelial dysplasia ranging from moderate to severe Group 2 (20 animals)
Group 3 (20 animals)
Group 4 (20 animals)
Three animals were died
Three animals were died
Three animals showed mild to moderate epithelial dysplasia
Two animals showed well differentiated SCC
Three animals Four animals showed showed well well to moderately differentiated SCC differentiated SCC
Two animals showed early invasive SCC
Three animals showed SCC ranging from moderately to poorly differentiated
Two animals were died
The same results as in group 2 The same results as in group 2 regarding number of animals regarding number of animals and and histo-pathological histo-pathological grades grades
Normal epithelium
Normal epithelium
One animal was died
Three animals Four animals showed showed well well differentiated differentiated SCC SCC Two animals showed One animal showed moderately moderately differentiated SCC differentiated SCC Hyperkeratotic normal epithelium
Hyperkeratotic normal epithelium
Fig. 1 Expression of PCNA immunoreactivity in HCP mucosa of the control group in the basal cell layer (original magnification 1609)
Fig. 2 Expression of PCNA immunoreactivity in HCP, 12 weeks after DMBA painting in the all epithelial layers and underlying pathologically altered tissues (original magnification 1609)
significant difference (P \ 0.01) between the different groups at 12, 24, 28, and 32 weeks in response to the effect of S. platensis extract on cell proliferation. Their graphical presentation is shown in Fig. 8.
bioresources, particularly seaweeds, as sources of bioactive substances. Several preparations of seaweeds such as polysaccharide, peptide, and phycobiliproteins were shown to affect the multiplication of tumor cells [18, 31–33]. From a pervious study, in a short-term evaluation, there was an inhibitory effect of S. platensis extract on the induced oral cancer [19]. The purpose of this study was to evaluate the long-term effect of S. platensis extract during cancer induction and progression. In addition, to follow up its impact on cancer progression after cancer inducer stoppage, using PCNA as a diagnostic marker.
Discussion Chemoprevention, the use of drugs or natural substances to inhibit carcinogenesis, is an important evolving subject of cancer research. There has been an interest in marine
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Fig. 3 Expression of PCNA immunoreactivity in HCP, 24 weeks after DMBA painting in the moderately to poorly differentiated SCC (original magnification 1609)
Fig. 4 Expression of PCNA immunoreactivity in HCP, 12 weeks after DMBA painting with the daily administration of 10 mg S. platensis extract, throughout all the pathologically altered dysplastic epithelial layers (original magnification 1609)
With the DMBA painting, the results of this study revealed a progressive change with time in the normal epithelial cells as they transformed into tumor cells as the development of carcinomas presumably may be attributed to the interaction of genetic factors and the locally applied carcinogens. These results were in compliance with Miyata et al. [34] and Rastogi et al. [35] who reported that DMBA is a potent organ-specific carcinogen, can act either as complete carcinogen or as an initiator of skin carcinogenesis. Dihydrodiolepoxide, the ultimate carcinogen of DMBA, mediates skin carcinogenesis by inducing an over production of reactive oxygen species and oxidative DNA damage. In group 2 at 12 weeks, with the administration of S. platensis, there is an inhibitory effect on tumor
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Fig. 5 Expression of PCNA immunoreactivity in HCP, 24 weeks after DMBA painting and daily administration of 10 mg S. platensis extract, in the moderately to poorly differentiated SCC (original magnification 1609)
Fig. 6 Expression of PCNA immunoreactivity in HCP, after 24 weeks DMBA painting and 28 weeks daily administration of 10 mg S. platensis extract, in the moderately differentiated SCC (original magnification 1609)
progression as the number of animals that showed early invasive SCC became less while the remaining animals showed mild to moderate ED. The animals at 24, 28, and 32 weeks showed well-differentiated SCC in contrast to their counterparts of group 1 and this could be attributed to the efficacy of S. platensis extracts as an antioxidant [36], antimutagenic [37], anticancer [18, 38], and immune enhancing [39]. The biological and pharmacological properties of Spirulina were attributed mainly to calciumSpirulina, C-phycocyanin, and b-carotene [33, 40]. In group 3, with the stoppage of DMBA painting and the continuation of Spirulina administration, there is a valuable inhibitory effect on tumor progression regarding the grade of the tumor and this can be attributed to Spirulina ability to enhance cell nucleus enzyme activity and DNA repair
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Fig. 7 Expression of PCNA immunoreactivity in HCP, after 24 weeks DMBA painting and 32 weeks daily administration of 10 mg S. platensis extract, in the well-differentiated SCC (original magnification 1609)
synthesis and it might also prevents oxidative damage to DNA. Pang et al. [41] reported that some common forms of cancer are the results of DNA damage run amok, the polysaccharides from Spirulina support DNA repair through enhancement of endonuclease activity. Hirahashi et al. [42] attributed the inhibitory effect to the eating of Spirulina as it increases the number and effectiveness of immune cells called NK cells. PCNA was originally described as a protein synthesized during the S-phase and named as cyclin. Subsequently, this 29 kDa protein was renamed as PCNA to distinguish it from the cyclin family of proteins [43–45]. PCNA is a useful marker of proliferating cells because its expression and distribution correlate with cellular proliferation rate. Previous studies have shown that PCNA LIs escalate significantly in the order of normal oral mucosa, oral epithelial hyperkeratosis, oral ED, and oral SCC; therefore, it has been used as an indicator of the malignant potential of oral lesions [46–49]. PCNA analysis has the potential to identify cell cycle subpopulations (G1, S, G2, M), dark-staining nuclei represent S-phase cells, light-staining nuclei represent G1/S
& G2 cells, and non-staining nuclei represent quiescent (G0) cells [50, 51]. In this study, the scores of labeled dark and light-staining nuclei of the sections were counted using light microscopy. PCNA LI was calculated by dividing the number of respective labeled cells by the total number of cells, and the result was expressed as a percentage. In this study, the mean PCNA LI of normal oral epithelium ranged from 9% to 9.5% and was comparable to that reported by Tsuji et al. [48, 52]. In the first group, the mean PCNA LI ranged between 65.5% and 76.0% and this quantification was relatively in accordance with Kobayashi et al. [47] who reported that the mean PCNA LI was 34.5% in ED and 64.7% in SCC and concluded; there is a significant difference in PCNA LI between ED and SCC. On contrary, Tsuji et al. [52] reported that the mean PCNA LI was 16.7% in ED and 22.8% in oral SCC and also concluded; there is a significant difference in PCNA LI between malignant and non-malignant lesions. The reasons for high expression of PCNA in SCC are still not clear. PCNA expression may be due to cell proliferation, unscheduled DNA synthesis, or induction by the presence of tumor cells or growth factors, such as transforming growth factor-a and epidermal growth factor [53–55]. The number of PCNA-positive cells in S. platensis feeding second group was less as compared to their counterparts of the first group. Quantification of these results showed that S. platensis decrease the proliferative index from 65.5% ± 006.3 to 52.6% ± 003.5, 70.4% ± 004.2 to 59.4% ± 004.1, 75.2% ± 007.2 to 58.3% ± 004.0, 76.0% ± 003.2 to 58.2% ± 002.7 at 12, 24, 28, and 32 weeks, respectively. Also when DMBA painting was stopped and administration of S. platensis was extended, the index was decreased from 75.2% ± 007.2 to 46.9% ± 005.4 at 28 weeks and from 76.0% ± 003.2 to 40.0% ± 003.9 at 32 weeks. Interestingly, Shin et al. [49] showed that PCNA expression had increased as tissues progressed from normal epithelium to dysplasia and from dysplasia to SCC in head and neck carcinomas. In the present study, significant differences in PCNA LI were found between the different groups at the extended periods. These results indicate that
Table 3 Mean PCNA LI in each group and results of statistical comparison of the mean PCNA LI Group
N
Observation period Mean PCNA LI ± SD (P value) 12 weeks
24 weeks
28 weeks
32 weeks
Control
10
9.3% ± 003.6 a
9.1% ± 005.4 a
9.0% ± 008.2 a
9.5% ± 002.2 a
Group 1
10
65.5% ± 006.3 b
70.4% ± 004.2 b
75.2% ± 007.2 b
76.0% b ± 003.2 b
Group 2 Group 3
10 10
52.6% ± 003.5 c 52.6% ± 003.5 d
59.4% ± 004.1 c 59.4% ± 004.1 d
58.3% ± 004.0 c 46.9% ± 005.4 d
58.2% ± 002.7 c 40.0% ± 003.9 d
Data represented as mean ± standard deviation. Values followed by different letter within each column indicate results differ significantly
26 Fig. 8 Mean PCNA LI and results of statistical comparison
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32 Week 28 Week 24 Week 12 Week
300 76 250 75.2
200
40 46.9
58.3 150 70.4
59.4
59.4
52.6
52.6
Group 3
Group 2
PCNA LI
58.2
100 65.5 9.5 9 9.1 9.3 Group 1
50
0
Control
Groups
PCNA expression correlates with the degree of pathological changes in these oral lesions. The mean PCNA LI decreased significantly with the daily administration of S. platensis extract.
Conclusion On the basis of this research, the use of S. platensis extracts had regressed cancer progression grading and improved its clinical and histopathological features. Further molecular biology researches are in need to verify these immunohistochemical findings. Conflict of interest statement Potential conflicts do not exist, also no financial relationships: it is a personal one; we do the study design, collecting the data, analyzing, and interpretating it. We do writing without any assistance and we had taken the decision to submit the paper for publication. We had full access to all of the data in this study and had taken complete responsibility for the integrity of the data and the accuracy of the data analysis.
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