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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2012 May 3;138(9):1579–1596. doi: 10.1007/s00432-012-1224-0

Schizophyllan inhibits the development of mammary and hepatic carcinomas induced by 7,12 dimethylbenz(α)anthracene and decreases cell proliferation: comparison with tamoxifen

Ahmed Mansour 1, Ayman Daba 4,5, Nahed Baddour 3, Muhammed El-Saadani 2, Eiman Aleem 1,6,✉
PMCID: PMC11824329  PMID: 22552717

Abstract

Background

Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compounds have been isolated from mushrooms. The aim of the present work was to study the anticancer effects of schizophyllan (SCH), a β-d-glucan extracted from the mushroom Schizophyllum commune alone or in combination with tamoxifen (TAM) on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinomas in mice.

Methods

We isolated SCH from S. commune. Female mice received DMBA, SCH, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH or vehicles. We studied mice survival, tumour incidence, histopathology, oestrogen receptor (ER) expression, cell proliferation by immunohistochemical detection of proliferating cell nuclear antigen (PCNA), apoptosis by TUNEL assay, as well as caspase-3 expression.

Results

DMBA treatment resulted in mammary and hepatocellular carcinomas (HCC). Both SCH and TAM reduced the incidence of DMBA-induced mammary tumours by 85 and 75 %, respectively, and equally decreased the PCNA labelling index relative to DMBA. TAM treatment increased the incidence of- and PCNA index in HCCs relative to DMBA, while SCH suppressed these effects. TAM was more effective than SCH in the induction of apoptosis in both mammary and hepatic carcinomas. Caspase-3 levels correlated with the apoptotic index in most experimental groups.

Conclusions

Only one dose of SCH had similar therapeutic effects against DMBA-induced mammary carcinomas as 4 weeks of TAM treatment. This coupled with the ability of SCH to suppress hepatic lesions associated with TAM treatment provides the rationale for further investigating the combined therapeutic effects of TAM+SCH in preclinical models of ER-positive breast cancer, as well as in liver cancer.

Electronic supplementary material

The online version of this article (doi:10.1007/s00432-012-1224-0) contains supplementary material, which is available to authorized users.

Keywords: Mushroom, Schizophyllum commune, Breast cancer, Hepatocellular carcinoma, Tamoxifen, Proliferating cell nuclear antigen, Apoptosis, Caspase-3

Introduction

Breast cancer is the most common malignancy among women and is still today one of the leading causes of cancer mortality despite the development of improved diagnostic tools and novel therapeutic modalities. Treatment options for breast cancer patients include surgery, radiation therapy, chemotherapy and targeting therapies, such as drugs targeting the oestrogen receptor (ER), epidermal growth factor signalling pathway and a number of other kinases (Di Cosimo and Baselga 2008). Tamoxifen (TAM) is widely used as a first-line endocrine therapy for breast cancer patients with positive ER and as a chemoprophylactic agent for women at high risk of developing this disease (Fisher et al. 1998). Besides the significant benefit, long-term administration of TAM has serious adverse effects; including endometrial cancer in women (Killackey et al. 1985; Bernstein et al. 1999). Therefore, alternative therapies with minimal side effects are required. Several anticancer compounds have been isolated from herbs or fungi such as mushrooms. In general, medicinal mushrooms have been shown to improve cardiovascular health, stimulate immune function and contribute to glucose homoeostasis and to modulate detoxification, as well as exert antiallergic, antiviral, antibacterial, antifungal and anti-inflammatory activities (Martin and Brophy 2010). In addition, polysaccharides from mushrooms have exhibited anticancer activities. The bioactive polysaccharides isolated from mushroom fruit bodies, submerged cultured fungal biomass, or liquid culture fermentation broths are either β-d-glucans or β-d-glucan–protein complexes (proteoglycans) (Bohn and BeMiller 1995). A small number have progressed to clinical trials mainly in Japan and China such as lentinan (Lentinus edodes), schizophyllan (SCH) (Schizophyllum commune), PSK and PSP (Trametes versicolor) and Grifron-D (Grifora frondosa) (Sullivan et al. 2006). In almost all cases, the polysaccharides are used as adjuvant treatments with conventional chemotherapy/radiotherapy with many forms of cancer. Their incorporation into treatment regimens significantly reduced the side effects so often encountered by patients. Although the exact molecular mechanism of their antitumour action is still not fully known, these polysaccharides are suggested to enhance the immune responses in vivo and act as biological response modifiers (Ooi and Liu 2000).

SCH from the inedible mushroom S. commune (Mwt ~450 kD), known as split-gill mushroom, is a highly potent antitumour polysaccharide that inhibits solid Sarcoma 180 tumour when injected by intraperitoneal or intravenous route, but has low antitumour activity by subcutaneous route (Komatsu et al. 1969). In humans, it was found ineffective against gastric cancer, but extended survival in patients with head and neck cancer (Kimura et al. 1994; Borchers et al. 1999).

To our knowledge, the efficacy of SCH in the treatment for breast cancer has not been studied before; hence, the present work was conducted to study the potential anticancer effect of SCH alone or in combination with TAM on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinogenesis in mice. DMBA-treated mice developed both mammary- and hepatocellular carcinomas (HCC). An imbalance between proliferation and apoptosis may lead to tumourigenesis. Therefore, in the present study, we investigated the effect of SCH on the incidence of both mammary and hepatic tumours, on the levels of proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation, on apoptosis, as well as on the expression of caspase-3, a central protein in the execution of apoptosis. In the current study, both SCH and TAM alone or in combination decreased mammary tumour incidence through reducing cell proliferation; however, TAM was more effective than SCH in the induction of apoptosis, which was coupled with an increase in caspase-3 levels. Interestingly, TAM increased the HCC incidence in DMBA+TAM-treated mice in comparison with DMBA-treated mice and this was associated with an increase in PCNA labelling index in HCCs. In contrast, SCH reduced the incidence of HCCs in DMBA+SCH- and DMBA+TAM+SCH-treated mice, which was accompanied by a reduction in cellular proliferation. In addition, SCH increased the percentage of apoptotic cells and the levels of caspase-3 expression in HCCs from DMBA- or DMBA+TAM-treated mice but not in normal liver.

Materials and methods

Preparation of mushroom extract

Schizophyllum commune was collected from a local forest in La Crosse, Wisconsin, USA, and maintained in culture. Slants were incubated at 28 °C for 4 days and stored at 5 °C. Petri dishes were inoculated with 1 cm² of mycelia from slants at room temperature (RT). The culture was grown on potato dextrose agar slants at 28 ± 2 °C for 7 days. A 1 cm2 of mycelia along with agar from such slants were used to inoculate 50 ml of sterile seed culture medium, which was incubated at 28 ± 2 °C for 3 days on a rotary shaker. Polysaccharides were extracted by fermentation. S. commune was screened for SCH production using the media reported by (Rau et al. 1992). Based on the initial screening, S. commune was selected for further studies. The extraction and purification of polysaccharides from mushroom fruit bodies and mycelia were carried out according to the method of (Chihara et al. 1970). Briefly, fresh mushroom fruit bodies or mycelia (1 kg) were washed with water and boiled in 5 L of distilled water for 8–15 h. The suspension was then centrifuged to remove the insoluble matter. The aqueous extract was concentrated under reduced pressure using a rotary evaporator to the point where a slight turbidity was observed. The polysaccharide was obtained by precipitation with an equal volume of absolute ethanol. The fibrous like precipitate was left overnight at 4 °C, collected by centrifugation, washed with absolute ethanol, freeze-dried and then weighed.

Spectroscopy and nuclear magnetic resonance

Ultraviolet and infrared absorptions were determined according to the methods described by (Fujii et al. 1978) (data not shown). The ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy was performed according to the method described by (Abraham and Loftus 1978) in which the polysaccharide sample (50 mg) was dissolved in one millilitre of concentrated dimethylsulfoxide (DMSO) by ultrasonication for 10–15 min. The solution was then introduced into a precision ground tube (5 mm diameter, depth 2–3 cm) and measured. NMR spectra were obtained using a 500 MHz JEOL spectrometer (Japan).

Animals and drug treatment

Female adult swiss albino mice (Mus musculus) with a mean weight of 25 g at the beginning of the experiment were obtained from the animal house at the Faculty of Medicine (Alexandria University, Egypt). Principles of laboratory animal care “NIH publication Np. 85-23, revised 1985” were followed, as well as the guidelines of animal experiments of Alexandria University’s research ethics committee. Five animals were housed per cage and kept on standard diet (mouse chow) and water ad libitum, 22 °C RT, 50 ± 10 % humidity and 12-h light/dark cycle. The animals were divided into six groups, each containing 20 mice. Neoplastic mammary lesions were induced in mice by administering DMBA (Sigma-Aldrich, St. Louis, USA) (40 mg/kg) once/week for 4 weeks in 0.1 mL of sesame oil using oral gavage. Mice were divided into the following groups as shown in Fig. 1: Group 1 received only DMBA and group 2 received only SCH (4 mg/kg, intraperitoneal [i.p.]) in saline once at the beginning of the experiment. Group 3 was divided into two subgroups: group 3a received both DMBA and SCH at the beginning of the experiment and group 3b was treated first with DMBA as in group 1; then, after the appearance of palpable tumours, they received one dose of SCH (4 mg/kg, i.p.). Group 4 received TAM (Sigma-Aldrich, St. Louis, USA) (8 mg/kg/day) in 0.1 mL sesame oil per oral (p.o.) for 4 weeks after the appearance of tumours (DMBA+TAM). Group 5 received both TAM+SCH after the appearance of tumours (DMBA+TAM+SCH). Group 6 was divided into two subgroups, which received vehicles (saline and sesame oil) to serve as negative controls for SCH (saline) and DMBA or TAM (oil), respectively (Fig. 1).

Fig. 1.

Fig. 1

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Experimental groups mice were divided into six groups: group 1 was treated with DMBA at a dose of 40 mg/kg once/week for 4 weeks in 0.1 mL of sesame oil using oral gavage, group 2 was injected intraperitoneal with SCH at a dose of 4 mg/kg only once at the beginning of the experiment. Group 3 was divided into two subgroups: group 3a received both DMBA and SCH at the beginning of the experiment and group 3b was treated first with DMBA as in group 1 then after the appearance of palpable tumours mice received one dose of SCH (4 mg/kg). Group 4 received both DMBA as in group 1 and TAM at a dose of 8 mg/kg/day in 0.1 mL sesame oil per oral for 4 weeks, group 5 received DMBA+TAM+SCH, while group 6 was divided into two subgroups, which received saline (6a) and sesame oil (6b). All the animals were killed after 20 weeks from the first DMBA dose

Histology

Upon necroscopy, mammary and liver tissues and tumours were removed and immediately sliced and fixed in 10 % neutral buffered formalin. The tissues were processed, embedded in paraffin and sectioned at 5 μm thickness. The sections were mounted on glass slides and stained with haematoxylin and eosin (H&E) for histopathological evaluation.

Evaluation of apoptosis in situ

Detection of apoptotic cells in tissue sections was performed using the Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP Nick End Labelling (TUNEL) assay (Apoptosis TUNEL assay IHC kit, AbD Serotec, MorphoSys, Oxford, UK, cat.no. APO002) according to the manufacturer’s protocol. The number of apoptotic cells was measured according to the number of positive cells as well as by morphologic assessment (Garrity et al. 2003). The apoptotic index was calculated as the number of positive cells/total number of positive and negative cells 100× in five different fields per section.

Immunohistochemical detection of ERα, PCNA and caspase-3

For immunohistochemical analysis, formalin-fixed tissue sections were mounted onto coated slides and rehydrated. Antigen retrieval was performed by boiling the slides in 2 mg/L citrate buffer. The slides were then left to cool to RT, and sections were washed in phosphate-buffered saline (PBS) and left to equilibrate. Endogenous peroxidase activity was blocked by incubating the sections in 3 % solution of H2O2 in PBS for 10 min. After washing 3 times in PBS, nonspecific reactivity was blocked by incubation with protein blocker for 5 min. The sections were then incubated with the following monoclonal antibodies: against PCNA (PC10, AM252-5ME, Biogenex Life Sciences, USA), against caspase-3 (CPP32) Ab-3 (clone 3CSP 03) (Thermo scientific, Fremont, CA, USA) and against ERα (RM-9101-S0) (clone SP1) overnight at 4 °C. Sections were then left at RT for 1 h after which they were incubated with the secondary antibody for 30 min at RT. The UltraVision ONE detection system with horseradish peroxidase (HRP) Polymer and DAB Plus Chromogen (Thermo Scientific, Fremont, CA, USA) was used, and sections were counterstained with haematoxylin, mounted and examined. For the verification of the specificity of the immunostaining, the primary antibodies were substituted with their diluting solution on some sections to serve as negative controls. Scoring was performed according to (Allred et al. 1998). For quantitative analysis of the PCNA or caspase-3 positivity, we measured PCNA or caspase-3/Labelling index (LI) as follows: at least 6 fields per section were photographed (400×). Positive and negative cells were counted in each field. We calculated PCNA/LI or caspase-3/LI as the number of positive cells/total number of positive and negative cells.

Statistics

Data were analysed and expressed as means ± SD. Statistical analysis was performed using the SPSS statistical package version 16.00 (SPSS Inc, Chicago, Illinois, USA). The differences between groups were analysed using one-way analysis of variance (ANOVA) followed by Fisher Least Significant Difference (LSD) test. The Kruskal–Wallis test was used for nonparametric data, and the Mann–Whitney test was then used for intergroup comparisons. A p value ≤ 0.05 was considered significant.

Results

Identification of polysaccharides extracted from S. commune

In the present study, polysaccharides (Schizophyllan) were extracted and purified from S. commune. The primary structure of the extracted substance identified using different spectroscopic techniques was found to be β-glucan. The 1H NMR spectra exhibited signals at different resonance, which represented the β-anomeric proton and protons of the different hydroxyl groups (Supplementary Fig. 1a, c). The 13C NMR spectrum revealed signals that are characteristic to glucan; the presence of 6 carbon atoms (Supplementary Fig. 1b).

Effect of schizophyllan on mice survival

In the present study, while SCH was found to significantly increase mice’s survival in comparison with control mice receiving vehicle (oil or saline) (p < 0.002, 0.003; respectively), it did not significantly alter the survival of mice, which received DMBA or DMBA and TAM, that is, those harbouring tumours. On the other hand, DMBA alone or DMBA+TAM or DMBA+TAM+SCH decreased the survival of mice significantly in comparison with control mice receiving vehicle (saline or oil) at p < 0.000 (Fig. 2).

Fig. 2.

Fig. 2

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Effect of schizophyllan on mice survival SCH significantly increased mice’s survival in comparison with control mice receiving vehicle (oil or saline) (p < 0.002, 0.003; respectively); however, it did not significantly alter the survival of mice receiving DMBA or DMBA and TAM. Treatment of mice either with DMBA alone, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH decreased animals’ survival significantly in comparison with control and SCH-treated animals. SCHa: mice treated with SCH at the same time as DMBA, SCHb: mice treated with SCH after the appearance of palpable tumours

Effect of schizophyllan and tamoxifen on mammary gland

Schizophyllan reduces the development of DMBA-induced mammary tumours in mice in comparison with tamoxifen

To evaluate the anticancer potential of SCH in vivo, we first induced mammary tumours in female mice using DMBA. Beginning 4 weeks after the last DMBA dose, mice began to develop palpable tumours. We euthanized mice whenever they showed signs of morbidity or when found dead. After 20 weeks from the beginning of DMBA treatment, we euthanized the remaining mice for analysis. While 75 % of DMBA-treated mice developed mammary tumours (group 1), administration of SCH alone before (group 3a) or after (group 3b) the induction of mammary tumours by DMBA resulted in substantial reduction in tumour incidence (from 75 to 15 %). Since SCH had the same effect on tumour incidence whether administered before or after the appearance of tumours, we, herewith, refer to subgroups 3a and 3b collectively as (DMBA+SCH). Treatment with TAM alone was found to reduce the tumour incidence to 25 % (group 4). Combining SCH with TAM was found to further decrease the mammary tumour incidence to 15 % (group 5) in comparison with TAM alone (25 %). SCH-treated mice (group 2) did not develop any tumours similar to the control mice, which received vehicle only (group 6a, b) (Fig. 3a).

Fig. 3.

Fig. 3

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Mammary tumour incidence and histopathology (a) neither the control nor the SCH-treated mice developed any mammary tumours, whereas 75 % of DMBA-treated mice developed mammary tumours. TAM alone reduced the mammary tumour incidence in DMBA-treated mice to 25 %, while TAM in combination with SCH or SCH alone further decreased the mammary tumour incidence to 15 %. b Section of control mouse mammary gland showing normal tissue architecture with ducts and acini (arrowheads), ×200. c SCH-treated mouse mammary tissue showing no pathological changes, arrowheads point to ducts ×200. d DMBA-treated mouse mammary tissue showing ductal carcinoma in situ; the epithelial cells fill and expand the ducts forming glandular spaces (s), a adipocytes, ×200. e DMBA-treated mouse mammary tissue showing lobular carcinoma in situ; the lobules (l) are expanded and filled with epithelial cells separated by stroma (st), ×200. f DMBA+SCH-treated mouse mammary tissue showing atypical ductal hyperplasia; the ducts (arrowheads) are distended by marked proliferation of epithelial cells, ×200. g DMBA+TAM-treated mouse mammary tissue showing invasive ductal carcinoma; malignant epithelial cells (arrows) invading the stroma (st) forming small ductal structures (d), ×400. h DMBA-treated mouse mammary tissue showing positive oestrogen receptor-α immunoreaction, ×100. i The specificity of the ERα immunoreaction (Peroxidase antiperoxidase method) was verified by substitution on a DMBA-treated mouse mammary tissue, ×100. Sections (b–g) are stained with H&E and h is stained with an antibody against ERα

Histopathology and oestrogen receptor status of DMBA-induced mammary lesions

In the present study, no pathological changes were detected either in mammary tissue of control or in SCH-treated mice (Fig. 3b, c). Treatment with DMBA resulted in varying degrees of epithelial hyperplastic changes ranging from mild to atypical changes including ductal carcinoma in situ (DCIS) (Fig. 3d), lobular carcinoma in situ (Fig. 3e) and infiltrating ductal carcinoma (IDC). Mammary glands from mice treated with DMBA+SCH or with DMBA+TAM+SCH still showed epithelial hyperplastic changes such as atypical ductal hyperplasia (Fig. 3f) and DCIS. In the DMBA+TAM-treated group, similar pathological changes to those induced by DMBA including IDC (Fig. 3g) were detected. All mammary tumours induced by DMBA were ERα-positive. A representative section of DMBA-treated mouse mammary gland immunostained with ERα and its negative control are shown in (Fig. 3h, i), respectively.

Schizophyllan and tamoxifen reduce cellular proliferation in DMBA-induced mammary tumours

We next studied the effect of SCH and TAM on cellular proliferation by immunostaining of PCNA in the mammary glands of control and treated mice. In control and SCH-treated mice, PCNA was localized in the ductal epithelial cell nuclei (Fig. 4a, b). The localization and intensity of the signal were similar in both control and SCH-treated mice. The mammary tumours from all the DMBA-treated groups showed a significant increase in the number of PCNA-positive cells. Representative sections from the mammary tumours induced in DMBA+TAM and DMBA+TAM+SCH-treated mice are shown in (Fig. 4c, d). The PCNA signal was localized in the tumour cells in addition to the ductal epithelial cells as shown in a section of the high-grade ductal carcinoma from a DMBA+TAM+SCH-treated mouse shown in (Fig. 4d). The number of PCNA-positive cells was counted in the mammary glands of all groups in six different fields, and the PCNA labelling index (LI) was calculated as described in the “Materials and methods”. The results are presented in (Table 1 and Fig. 4e). Treatment with DMBA significantly increased the PCNA/L1 score to about 8.4-fold of that of control animals (Fig. 4e). In contrast, SCH did not have a significant effect on the PCNA signal in comparison with control mice. However, treatment with either SCH alone or TAM alone or in combination significantly reduced the PCNA/L1 values by approximately 60 % in all treated groups (DMBA+SCH, DMBA+TAM, DMBA+TAM+SCH) in comparison with the DMBA-treated group (Table 1; Fig. 4e).

Fig. 4.

Fig. 4

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Schizophyllan and tamoxifen reduce cell proliferation in DMBA-induced mammary tumours immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) in sections of mouse mammary gland. Control (a) and SCH-treated mouse (b) show normal levels of PCNA expression. PCNA-positive cells appear with brown nuclei in ductal epithelial cells (arrowhead), ×200. Representative sections from DMBA+TAM-treated-(c) and DMBA+TAM+SCH-treated mouse mammary glands (d), ×400 and ×200, respectively. e Histogram showing the effect of different drug treatments (x-axis) on the PCNA labelling index (LI) in the mammary gland (y-axis). Data are expressed as mean ± standard deviation. DMBA treatment resulted in increasing the PCNA LI by 8.4-fold in comparison with control and to SCH. Treatment of mice bearing DMBA-initiated tumours with either SCH or TAM individually or in combination resulted in reduction in the PCNA LI by an average of 60–70 %

Table 1.

PCNA levels in mammary glands from different experimental groups

Control DMBA SCH DMBA+TAM DMBA+SCH DMBA+SCH+TAM
Range 3.0–7.0 38.0–45.0 3.0–6.0 15.0–20.0 14.0–16.0 16.0–19.0
Mean ± SD 4.88 ± 1.25 40.88 ± 2.70 4.63 ± 1.06 16.63 ± 1.69 15.0 ± 0.93 17.38 ± 0.92
F (p) 601.310* (< 0.001)
p1 <0.001* 0.743 <0.001* <0.001* <0.001*
p2 <0.001* <0.001* <0.001* <0.001*
p3 <0.001* <0.001* <0.001*
p4 <0.001* <0.001*
p5 <0.001*
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PCNA levels are expressed as PCNA/LI representing the number of positive cells/number of positive and negative cells in six fields and expressed as mean ± SD

F: F test (ANOVA)

p1: p value of LSD test between control and other groups

p2: p value of LSD test between DMBA and SCH, DMBA+TAM, DMBA+SCH, DMBA+SCH+TAM

p3: p value of LSD test between SCH and DMBA+TAM, DMBA+SCH, DMBA+SCH+TAM

p4: p value of LSD test between DMBA+TAM and DMBA+SCH, DMBA+SCH+TAM

p5: p value of LSD test between DMBA+SCH with DMBA+SCH+TAM

* Statistically significant at p ≤ 0.05

DMBA induces apoptosis in mammary tumours and treatment with TAM or TAM+SCH increases this effect

In the present work, we studied apoptosis in the mammary glands in situ using TUNEL assay (Table 2 and Fig. 5). The percentage of apoptotic cells was equal in the mammary glands from both normal and SCH-treated mice (0.5 %) (Table 2; Fig. 5a, b, g). Treatment of mice with DMBA alone increased the apoptotic index significantly by 11.5-fold in comparison with control (Table 2; Fig. 5c, g). Treatment with either DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH also significantly increased the apoptotic index by 7.5-, 31-, and 40-fold that of control mice (Table 2; Fig. 5d, e, f, g). This indicated that in DMBA+TAM-treated mice, TAM increased the apoptotic index significantly by 2.7-fold that induced by DMBA. Interestingly, combined treatment of SCH and TAM further increased the apoptotic index by 3.5-fold that induced by DMBA. Furthermore, the increase in apoptosis was associated with an increase in caspase-3 expression levels in most experimental groups in comparison with control (Supplementary Fig. 2a).

Table 2.

Apoptotic index determined by TUNEL assay in mammary glands from different experimental groups

Control DMBA SCH DMBA+SCH DMBA+TAM DMBA+SCH+TAM KWp
Range 0.0–1.0 5.0–7.0 0.0–1.0 3.0–5.0 12.0–19.0 19.0–22.0 <0.001*
Mean ± SD 0.50 ± 0.53 5.75 ± 0.89 0.50 ± 0.44 3.75 ± 0.89 15.75 ± 3.06 20.00 ± 1.20
Median 0.50 5.50 0.50 3.50 16.0 20.50
p1 0.001* 1.000 0.001* 0.001* 0.001*
p2 0.001* 0.002* 0.001* 0.001*
p3 0.001* 0.001* 0.001*
p4 0.001* 0.001*
p5 0.001*
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The apoptotic index represents the number of TUNEL-positive cells/number of total positive and negative cells/100 in five fields and expressed as mean ± SD

KWp: p value for Kruskal–Wallis test

p1: p value for Mann–Whitney test control and each other group

p2: p value for Mann–Whitney test between DMBA and each other group

p3: p value for Mann–Whitney test SCH and each other group

p4: p value for Mann–Whitney test DMBA + SCH and each other group

p5: p value for Mann–Whitney test DMBA+TAM and group DMBA+TAM+SCH

* Statistically significant at p ≤ 0.05

Fig. 5.

Fig. 5

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DMBA and tamoxifen but not schizophyllan induce apoptosis in mammary tumours Representative sections of mammary glands from the following treatment groups were processed for TUNEL assay and the positive cells are stained brown. a Control mouse, ×200, b SCH-treated mouse, ×200, c DMBA-treated mouse, ×200, d DMBA+SCH-treated mouse, ×200, e DMBA+TAM-treated mouse, ×200, f DMBA+SCH+TAM-treated mouse, ×200, g Histogram showing the effect of different drug treatments (x-axis) on the percentage of apoptotic cells determined by TUNEL assay (%) in the mammary gland (y-axis). Data are expressed as mean ± standard deviation. DMBA treatment increased the apoptotic index by 11.5-fold in comparison with control. Treatment of mice bearing DMBA-initiated tumours with SCH decreased the apoptotic index by 0.35-fold, while TAM and TAM+SCH increased apoptosis by 2.7- and 3.5-fold, respectively, in comparison with DMBA

Effect of schizophyllan and tamoxifen on liver

Schizophyllan reduces and tamoxifen increases hepatic lesions induced by DMBA

In the present study, liver sections from control or SCH-treated mice showed no pathological changes (Fig. 6a, b). Mice treated with DMBA developed HCC in addition to the mammary tumours. Forty-five per cent of the analysed mice developed HCC (Fig. 6i) that were well (Fig. 6c) or poorly differentiated. The remaining mice showed varying degrees of pathological changes such as foci of small cell dysplastic lesions that were more evident in the periportal acinar zone 1. Ductular reaction was also observed. (Fig. 6d). The administration of TAM to DMBA-treated mice increased the severity of hepatic lesions. Fifty-five per cent of DMBA+TAM-treated mice developed HCC (Fig. 6i), mostly were well developed with a trabecular pattern and evidence of bile production. Other hepatic lesions induced by TAM included centrilobular canalicular cholestasis (Fig. 6e), steatohepatitis, which was observed in about 30 % of mice (Fig. 6f), as well as increasing fibrosis. The mice receiving DMBA+TAM+SCH displayed similar histopathological changes as in the DMBA+TAM group such as steatohepatitis, small cell dysplastic lesions, cirrhosis (Fig. 6g) and HCCs; however, these lesions appeared less severe and less numerous. HCC was detected in 35 % of the mice in this group (Fig. 6i). Group 3 that received SCH either before or after the appearance of mammary tumours still developed HCC and dysplastic foci, but were less numerous than those in the DMBA, DMBA+TAM and DMBA+TAM+SCH groups. Dysplastic foci detected in the liver of these mice are shown in (Fig. 6h). HCC was detected in about 30 % of the DMBA+SCH mice. The percentage of mice harbouring HCC in the different experimental groups is shown in (Fig. 6i).

Fig. 6.

Fig. 6

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Histopathology of the liver (a) Section of control liver showing the normal arrangement of hepatocytes within the liver parenchyma, ×200. b SCH-treated liver showing no pathological changes, (cv central vein), ×200. c DMBA-treated mouse liver showing well-differentiated hepatocellular carcinoma, ×200. d DMBA-treated mouse liver showing ductular reaction evident as proliferation of ductular structures at the edges of the portal tracts and encroaching onto the limiting plate, ×200. e DMBA+TAM-treated mouse liver showing cholestasis with accumulation of bile (b) within the hepatic parenchyma, ×200. f DMBA+TAM-treated mouse liver showing steatosis manifest by the accumulation of lipid (arrows), ×200. g DMBA+TAM+SCH-treated mouse liver showing cirrhosis. Nodules (n) are separated by fibrous bands (f), ×200. h DMBA+SCH-treated mouse liver showing dysplastic foci (marked by arrowheads). They were more evident in the periportal acinar zone 1 in the form of increased nuclear/cytoplasmic ratio. Sections are stained with H&E. i Histogram showing the incidence of HCC in experimental groups: neither the control nor the SCH-treated mice developed liver tumours, whereas 45 % of DMBA-treated mice developed HCC. TAM alone increased the HCC incidence in DMBA-treated mice to 55 %, while SCH alone or in combination with TAM decreased the HCC incidence to 30, and 35 %, respectively

Schizophyllan reduces while TAM increases cell proliferation in DMBA-induced HCC

In the present study, PCNA-positive cells were detected in the liver from all control and treated mice (Fig. 7). PCNA was localized in the nuclei of hepatocytes in normal and SCH-treated mice (Fig. 7a, b) and in malignant cells in DMBA-induced HCC (Fig. 7c, d). The PCNA-positive cells were counted, and the PCNA/LI was determined as described in materials and methods and the results are presented in (Table 3, Fig. 7e). As in the mammary glands, SCH did not induce any changes in the PCNA/LI in comparison with control (Table 3, Fig. 7b, e). In contrast, the DMBA-induced HCC displayed a 3.1-fold increase in the number of PCNA/Ll in comparison with control liver. Interestingly, TAM had a synergistic effect with DMBA, and tumour cells from DMBA+TAM-treated mice had a 5.2-fold increase in PCNA/LI relative to the normal cells (170 % increase in comparison with the PCNA/LI value in HCCs from DMBA-treated mice) (Table 3, Fig. 7e). HCCs from DMBA+TAM+SCH-treated mice showed a significant reduction in the PCNA/LI by 52 % in comparison with the value in DMBA+TAM-treated mice (Table 3, Fig. 7e). Similarly, when SCH was administered to DMBA-treated mice either before or after the appearance of tumours the PCNA/LI decreased significantly by 42 % (Table 3, Fig. 7e). The timing of SCH administration did not have a statistically significant effect on the PCNA/LI. These results clearly indicate that while DMBA and TAM increased cell proliferation, administration of SCH significantly reduced this effect.

Fig. 7.

Fig. 7

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Schizophyllan reduces and tamoxifen increases cell proliferation in DMBA-induced liver tumours immunohistochemical analysis of cell proliferation using an antibody against proliferating cell nuclear antigen (PCNA). Control (a) and SCH-treated mouse (b) show normal levels of PCNA expression in liver sections. PCNA-positive cells appear with brown nuclei (arrows), ×200, Representative sections from DMBA+TAM-treated (c) and DMBA+TAM+SCH-treated mouse liver (d) showing increased levels of PCNA-positive nuclei (×100 and ×400, respectively). e Histogram showing the effect of different drug treatments (x-axis) on the PCNA labelling index (LI) in the liver (y-axis). Data are expressed as mean ± standard deviation. While DMBA treatment resulted in increasing the PCNA LI by threefold, treatment of mice with DMBA+TAM increased the PCNA LI by 5.2-fold in comparison with control and to SCH. DMBA+SCH-treated mice showed HCC with 40 % lower PCNA LI in comparison with DMBA-treated mice

Table 3.

PCNA levels in liver from different experimental groups

Control DMBA SCH DMBA+TAM DMBA+SCH DMBA+SCH+TAM
Range 54.0–63.0 165.0–188.0 54.0–69.0 587.0–307.0 87.0–112.0 145.0–178.0
Mean ± SD 56.88 ± 3.14 175.1 ± 7.3 60.25 ± 4.95 297.0 ± 7.52 101.38 ± 7.73 154.75 ± 11.03
F (p) 809.783* (< 0.001)
p1 <0.001* 0.864 <0.001* <0.001* <0.001*
p2 <0.001* <0.001* <0.001* <0.001*
p3 <0.001* <0.001* <0.001*
p4 <0.001* <0.001*
p5 <0.001*
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PCNA levels expressed as PCNA/LI representing the number of positive cells/number of positive and negative cells in six fields and expressed as mean ± SD

F: F test (ANOVA)

p1 : p value of LSD test between control and other groups

p2 : p value of LSD test between DMBA with SCH, DMBA+TAM, DMBA+SCH, DMBA+SCH+TAM

p3 : p value of LSD test between SCH with DMBA+TAM, DMBA+SCH, DMBA+SCH+TAM

p4 : p value of LSD test between DMBA+TAM with DMBA+SCH, DMBA+SCH+TAM

p5 : p value of LSD test between DMBA+SCH with DMBA+SCH+TAM

* Statistically significant at p ≤ 0.05

Treatment with schizophyllan or tamoxifen alone or in combination increases apoptosis in HCCs from DMBA-treated mice

In the liver, treatment of mice with SCH did not have a significant effect on the percentage of apoptotic cells detected by TUNEL assay in comparison with normal cells (Table 4; Fig. 8a, b, g). However, DMBA increased the apoptotic index by 1.2-fold the value in normal liver (Table 4; Fig. 8c, g). Administration of SCH to DMBA-treated mice increased the apoptotic index by 1.2-fold in comparison with that induced by DMBA alone (Table 4; Fig. 8d, g). The apoptotic index was further significantly increased in HCCs from DMBA+TAM- or DMBA+TAM+SCH-treated mice almost equally (about twofold), in comparison with that induced by DMBA (Table 4; Fig. 8 e, f, g). This indicates that the administration of SCH alone or TAM alone or in combination into DMBA-treated mice significantly increased the percentage of apoptotic cells in HCCs. Caspase-3 levels were also increased in HCCs from DMBA- and DMBA+TAM-treated mice by 1.3-fold the value of control and in HCCs from DMBA+SCH or DMBA+TAM+SCH by 2.5-fold the value of control (Supplementary Fig. 2b).

Table 4.

Apoptotic index determined by TUNEL assay in liver from different experimental groups

Control DMBA SCH DMBA+SCH DMBA+TAM DMBA+TAM+SCH KWp
Range 15.0–20.0 20.0–25.0 17.0–19.0 22.0–31.0 36.0–44.0 40.0–45.0 <0.001*
Mean ± SD 17.75 ± 2.05 22.0 ± 2.27 18.0 ± 0.76 26.25 ± 3.73 40.75 ± 3.33 43.25 ± 2.19
Median 18.0 21.50 18.0 26.0 41.50 44.0
p1 0.002* 1.000 0.001* 0.001* 0.001*
p2 0.001* 0.034* 0.001* 0.001*
p3 0.001* 0.001* 0.001*
p4 0.001* 0.001*
p5 0.085
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The apoptotic index represents the number of TUNEL-positive cells/number of total positive and negative cells/100 in five fields and expressed as mean ± SD

KWp: p value for Kruskal–Wallis test

p1: p value for Mann–Whitney test control and each other group

p2: p value for Mann–Whitney test between DMBA and each other group

p3: p value for Mann–Whitney test SCH and each other group

p4: p value for Mann–Whitney test DMBA+SCH and each other group

p5: p value for Mann–Whitney test DMBA+TAM and group DMBA+TAM+SCH

* Statistically significant at p ≤ 0.05

Fig. 8.

Fig. 8

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Schizophyllan and tamoxifen increase apoptosis in liver tumours Representative sections of liver from the following treatment groups were processed for TUNEL assay, and the positive cells are stained brown. a Control mouse, ×200, b SCH-treated mouse, ×200, c DMBA-treated mouse, ×200, d DMBA+SCH-treated mouse, ×200, e DMBA+TAM-treated mouse, ×200, f DMBA+SCH+TAM-treated mouse, ×200, g Histogram showing the effect of different drug treatments (x-axis) on the percentage of apoptotic cells determined by TUNEL assay (%) in the liver (y-axis). Data are expressed as mean ± standard deviation. DMBA treatment increased the apoptotic index by 1.2-fold in comparison with control. Treatment of mice bearing DMBA-initiated tumours with SCH increased the apoptotic index by 1.2-fold, while TAM and TAM+SCH increased apoptosis by 1.85- and 1.96-fold, respectively, in comparison with DMBA. The difference between all the groups was statistically significant at p ≤ 0.05 except the groups receiving DMBA+TAM and DMBA+TAM+SCH

Discussion

Dietary mushroom and extracts from medicinal mushrooms have been shown to possess antitumour activities in vitro and in vivo (Yu et al. 1993; Wu et al. 2007; Zhang et al. 2009; Shin et al. 2010). The effect of the inedible mushroom S. commune on mammary cancer in experimental animals has not been published before but a study by (Zhang et al. 2009) reported that dietary intake of fresh mushrooms or dried mushroom powder significantly decreased breast cancer in pre- and postmenopausal women. In another study, 69 % of breast cancer patients consuming whole maitake mushroom powder showed significant cancer regression (Kodama et al. 2002). In the present study, we demonstrated that schizophyllan (SCH), a β-d-glucan extracted from S. commune, markedly reduced the incidence of mammary and hepatic carcinomas and decreased cell proliferation in a mouse model of DMBA-induced carcinogenesis. We also compared the antitumour effects of SCH to those of tamoxifen (TAM).

In the present study, DMBA induced ER-positive mammary tumours. This is in agreement with previous studies showing that DMBA-induced mammary tumours in the rat are essentially similar in morphology, pathogenesis and ER status to human breast cancer and show oestrogen-dependent growth (Russo et al. 1990). In the current work, although mice received only one dose of SCH, it was sufficient to reduce the mammary tumour incidence from 75 % in DMBA-treated mice to 15 % in the groups that received SCH (DMBA+SCH and DMBA+TAM+SCH). The timing of SHC administration whether before or after the appearance of tumours did not differ significantly regarding its effect on tumour incidence or any other biological effect. In comparison, administration of TAM alone (8 mg/kg/day) for 4 weeks into DMBA-treated mice reduced the mammary tumour incidence to 25 %. This result indicates that one dose of SCH was more effective in reducing the mammary tumour incidence in this experimental model than daily administration of TAM for 4 weeks. In the present study, DMBA induced varying degrees of epithelial hyperplastic changes in mammary tissue including both infiltrating ductal and lobular carcinoma and ductal carcinoma in situ (DCIS). Similar mammary lesions were detected in mice, which received DMBA+TAM, albeit with lower incidence. In contrast, DMBA-treated mice receiving SCH showed no infiltrating carcinomas; however, DCIS was detected in these mice. This may indicate that SCH slowed the progression of mammary carcinoma as it is known that in more than 50 % of cases, DCIS may progress to infiltrating carcinoma (Axelrod et al. 2006). Consistent with these findings, the polysaccharide fraction from maitake mushroom was found to hinder metastatic progression, reduce the expression of tumour markers and to increase natural killer cell activity in all breast cancer patients as reported by (Kodama et al. 2003). The powerful anticancer effect of one dose of SCH may be attributed to a long biological half-life of the compound. Consistent with this explanation, (Tokuyasu et al. 2010) reported high plasma concentration of β-d-glucan in a 69-year-old woman 18 years after she was treated with SCH (also known as SPG) for cervical cancer. SPG has been used in combination with radiotherapy to improve the local response to radiation treatment for cervical cancer (Noda et al. 1992). In rats, SPG in the liver is degraded over a period of 6 months to SPG-like substances and it is metabolized in the spleen and mesenteric lymph nodes at a much slower rate, before it is finally excreted in the urine (Tanji et al. 1990). In humans, SPG could be detected in the body after almost 4 years from the last intramuscular injection (a total dose of 280 mg) (Ishizuka et al. 2004). Although the biological half-life of β-d-glucan remains unknown in humans, these reports suggest a long half-life for SCH.

In the present study, SCH did not affect the survival of DMBA-treated mice but it significantly improved the survival of mice, which received SCH alone in comparison with control mice receiving vehicle, and it did not induce any pathological alterations in the mammary gland or liver. This is in agreement with a phase I study for the assessment of safety and tolerability of a soluble form of oral β-glucans (Lehne et al. 2006). β-glucans were given in different doses for 4 consecutive days, and no drug-related adverse effects were observed. In agreement with our finding, (Fujimoto et al. 1991) reported that the median survival time of 130 patients with resectable gastric cancer after 5 years of surgical removal of the whole tumour tissue in addition to the application of SCH, mitomycin and futraful was 72.2 % in comparison with 61.9 % for 134 patients who received chemotherapy alone without SCH. However, SCH had no effect on the survival time when the tumour tissue could not be totally removed. In cervical cancer, SCH prolonged survival and time to recurrence for stage II but not stage III cases (Okamura et al. 1989; Miyazaki et al. 1995) and showed added effectiveness when injected directly into the tumour mass (Nakano et al. 1996). In a randomized trial, SPG combined with conventional chemotherapy improved the long-term survival rate of patients with ovarian cancer (Inoue et al. 1993).

In the present study, although TAM reduced the incidence of DMBA-induced mammary tumours, mice receiving DMBA+TAM developed a higher percentage of HCC in comparison with those which received DMBA only. Our results are in agreement with previous findings that presented TAM as a potent hepatocarcinogen in rats (Greaves et al. 1993; Hard et al. 1993) with both tumour-initiating (Williams et al. 1997) and promoting properties (Dragan et al. 1996). Furthermore, TAM was found to promote mammary cancer development in a mouse model of Brca 1-mutation-related breast cancer (Jones et al. 2005). In humans, several side effects of TAM have been reported. In a Swedish trial using adjuvant TAM 40 mg/day for 2–5 years, 3 cases of liver cancer have been reported and one case was reported in the Breast Cancer Prevention Trial (BCPT, NSABP P-1). The NSABP P-1 was a double-blind, randomized, placebo-controlled 5 years of Tamoxifen citrate therapy (20 mg/day) (Tamoxifen official FDA information, side effects and uses, http://www.drug.com/pro/tamoxifen.html). In the present study, TAM induced additional liver lesions such as steatohepatitis, cholestasis and cirrhosis. These results are consistent with previous studies in humans reporting the incidence of nonalcoholic steatohepatitis in female patients who received TAM (20 mg/day) (Akhondi-Meybodi et al. 2011) or 40 mg/day as postoperative endocrine treatment (Nemoto et al. 2002). In the current study, SCH could reduce the incidence of HCC in DMBA-treated mice from 45 to 30 % and in DMBA+TAM-treated mice from 55 to 35 %. As discussed below, the reduction in HCC incidence was associated with a significant reduction in PCNA labelling index and a slight but statistically significant increase in the apoptotic index coupled with an increase in the levels of caspase-3 protein. Therefore, the protective effect of SCH against “TAM-induced or promoted” liver lesions warrants further investigation.

Our results demonstrated that both TAM and SCH equally decreased cell proliferation in mammary tumours. In agreement with our results, (Martin and Brophy 2010) reported that maitake, crimini, portabella, oyster and white button mushrooms significantly suppressed cellular proliferation with only maitake further inducing apoptosis in MCF-7 breast cancer cells. The inhibition of proliferation of ER-positive cells by TAM is already known and is attributed to the antagonism of ER by TAM and the subsequent inhibition of oestrogen-dependent proliferative events leading to growth arrest of ER-positive cells (Mandlekar et al. 2000). Our findings are, therefore, consistent with several previous reports. For example, de Sousa et al. (2006) demonstrated that TAM reduced the proliferation marker Ki-67 positivity in the breast epithelium of carcinoma patients treated with 10 mg TAM for 14 days (de Sousa et al. 2006). Earlier studies on cell lines revealed that TAM inhibits proliferation of MCF-7 human breast cancer cells through a delay in G1 phase (Osborne et al. 1983).

Furthermore, in the present study, while SCH decreased cell proliferation in HCCs, TAM increased it significantly in DMBA+TAM-treated mice in comparison with DMBA and combined treatment with SCH and TAM reduced the PCNA/LI by 50 % in comparison with that in mice treated with only TAM. Similar to our results, polysaccharides from Lentinus edodes suppressed liver tumour growth in mice (Fu et al. 2011). The increase in cell proliferation by TAM may be attributed to the potential increase in gene expression of cell cycle genes such as Ccnd1 as was previously reported to be induced by TAM in early stages of liver carcinogenesis (Pogribny et al. 2007). Another study reported that the increase in cell proliferation in liver cells by TAM could be attributed to epigenetic reprogramming and prominent increase in the expression of the c-myc proto-oncogene in female rats exposed to TAM (Tryndyak et al. 2007). In the same study, the authors also reported that TAM induced apoptosis in hepatocytes as indicated by a doubling of the number of TUNEL-positive cells (Tryndyak et al. 2007). This is consistent with our results.

In the present study, DMBA induced apoptosis in both mammary and liver tumours, but more significantly in mammary tumours. DMBA-induced apoptosis was associated with an increase in caspase-3 positivity. Similar to our findings, Tsai-turton et al. (2007) showed that DMBA induced apoptosis in preovulatory follicles, which was mediated by reactive oxygen species and involved Bax and caspase-3 (Tsai-Turton et al. 2007). In further agreement with our results, earlier studies showed that DMBA induced apoptosis in the A20.1 murine B cell lymphoma (Burchiel et al. 1993), and in the adrenal cortex of female Sprague–Dawley rats, which was also associated with activation of caspase-3 (Fu et al. 2005). In the present study, TAM was generally more effective than SCH in the induction of apoptosis in both mammary and liver carcinomas, but combined treatment with DMBA+TAM+SCH increased the percentage of apoptotic cells in mammary carcinomas in comparison with that induced by DMBA+TAM alone. This may explain the reduced mammary tumour incidence in DMBA+TAM+SCH-treated mice (15 %) versus that in DMBA+TAM-treated mice (25 %). In the present study, the caspase-3 labelling index correlated with the apoptosis index in both mammary and liver tissues in the control, DMBA-, SCH- and DMBA+SCH-treated mice. Previous studies demonstrated that the water extract of Chaga mushroom (Inonotus obliquus) induced apoptosis associated with an upregulation of caspase-3 in HT-29 colon cancer cells (Lee et al. 2009), and in melanoma cells in vitro and in vivo (Youn et al. 2009). In the mammary but not the liver tumours, caspase-3 positivity also correlated with the increase in apoptosis by DMBA+TAM. In partial agreement with our results, TAM has been shown before to induce apoptosis in breast cancer cells (Frankfurt et al. 1995), which was through the downregulation of bcl-2 without alterations in p53 levels (Zhang et al. 1999). TAM has also been reported to induce apoptosis in HepG2, Hep1B, Hepa1-6 and MH1C1 hepatoma cells, which was accompanied with an upregulation of caspase 3 and 8 activity, and increased p27, bax, caspase 3 expression (Herold et al. 2002). In the present study, TAM did not increase caspase-3 expression in HCCs but it did in mammary tumours. This may be attributed to the fact that there are two pathways that are responsible for TAM-induced apoptosis, one is ER-dependent, caspase-independent and resembles necrosis-like programmed cell death and the other is ER-independent and caspase-dependent (Obrero et al. 2002). In our experimental setting, the exact mechanism of TAM-induced apoptosis in liver versus mammary tumours requires further investigation.

In spite of the use of mushrooms in clinical trials for cancer patients, particularly in the Far East, the molecular mechanism of their anticancer activity is still not fully understood. Two mechanisms have been proposed to be responsible for the anticancer action of herbs including mushroom; one via direct cytotoxic effect and the other is indirectly through immunomodulatory action (Borchers et al. 2004). It has been reported that polysaccharides from mushrooms are able to stimulate the nonspecific immune system and to exert antitumour activity through the stimulation of the host’s defence mechanism (Chihara et al. 1969). The drugs activate effector cells like macrophages, T lymphocytes and NK cells to secrete cytokines like TNF-α, IFN-γ, IL-1β, which are antiproliferative and induce apoptosis and differentiation in tumour cells (Lindequist et al. 2005). It remains to be elucidated whether the inhibition of mammary tumour development by SCH is associated with a hormone effect.

Conclusions

In conclusion, given the safety level of schizophyllan and its relative low cost, as well as its ability to inhibit mammary carcinomas similar to tamoxifen, and to suppress liver lesions associated with tamoxifen treatment, the present study provides the rationale for the use of schizophyllan in combination with tamoxifen in preclinical models and probably in clinical trials for oestrogen receptor-positive breast cancer therapy. The potential therapeutic value and mechanism of action of SCH deserve further investigation into other types of cancer including liver cancer.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (JPG 294 kb) (293.5KB, jpg)
432_2012_1224_MOESM2_ESM.tif (236KB, tif)

Supplementary material 2. Caspase-3 labelling index in comparison with the apoptotic index in mammary gland and liver. In general increase in the percentage of apoptotic cells was associated with an increase in caspase-3 expression in comparison with control (a) Histogram showing the effect of different drug treatments (x-axis) on the caspase-3 labelling index (LI) in the mammary gland (y-axis) in comparison with the apoptotic index by TUNEL assay. Data are expressed as mean ± standard deviation. DMBA treatment increased the caspase-3 LI by 128-fold, while SCH resulted in twofold increase in comparison with control. Treatment of mice bearing DMBA-initiated tumours with TAM upregulated the caspase-3 LI by twofold in comparison with DMBA. Black columns indicate the percentage of apoptotic cells by TUNEL assay, grey columns indicate the caspase-3 labelling index, (b) Histogram showing the effect of different drug treatments (x-axis) on the caspase-3 labelling index (LI) in liver (y-axis). Data are expressed as mean ± standard deviation. SCH resulted in twofold increase in caspase-3 labelling in comparison with DMBA. Black columns indicate the percentage of apoptotic cells by TUNEL assay and grey columns indicate the caspase-3 labelling index. (TIFF 235 kb)

Acknowledgments

The present study was supported by the Swedish Research Links (VR) to E. Aleem and by an institutional grant from the city for scientific research and technology applications to A. Daba.

Conflict of interest

The authors declare that they have no conflicts of interests.

Abbreviations

DMBA

7,12 Dimethylbenz(α)anthracene

DCIS

Ductal carcinoma in situ

ER

Oestrogen receptor

H&E

Haematoxylin and eosin

HCC

Hepatocellular carcinoma

IDC

Infiltrative ductal carcinoma

ILC

Infiltrative lobular carcinoma

LI

Labelling index

PCNA

Proliferating cell nuclear antigen

SCH

Schizophyllan

TAM

Tamoxifen

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (JPG 294 kb) (293.5KB, jpg)
432_2012_1224_MOESM2_ESM.tif (236KB, tif)

Supplementary material 2. Caspase-3 labelling index in comparison with the apoptotic index in mammary gland and liver. In general increase in the percentage of apoptotic cells was associated with an increase in caspase-3 expression in comparison with control (a) Histogram showing the effect of different drug treatments (x-axis) on the caspase-3 labelling index (LI) in the mammary gland (y-axis) in comparison with the apoptotic index by TUNEL assay. Data are expressed as mean ± standard deviation. DMBA treatment increased the caspase-3 LI by 128-fold, while SCH resulted in twofold increase in comparison with control. Treatment of mice bearing DMBA-initiated tumours with TAM upregulated the caspase-3 LI by twofold in comparison with DMBA. Black columns indicate the percentage of apoptotic cells by TUNEL assay, grey columns indicate the caspase-3 labelling index, (b) Histogram showing the effect of different drug treatments (x-axis) on the caspase-3 labelling index (LI) in liver (y-axis). Data are expressed as mean ± standard deviation. SCH resulted in twofold increase in caspase-3 labelling in comparison with DMBA. Black columns indicate the percentage of apoptotic cells by TUNEL assay and grey columns indicate the caspase-3 labelling index. (TIFF 235 kb)

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