Daun keledek anti kanser Prostat

Polar biophenolics in sweet potato greens extract synergize to inhibit prostate cancer cell proliferation and in vivo tumor growth

Sushma R. Gundala, Chunhua Yang, N. Lakshminarayana, Ghazia Asif, Meenakshi V. Gupta, Shahab Shamsi, Ritu Aneja

https://academic.oup.com/carcin/article/34/9/2039/2463112

Carcinogenesis, Volume 34, Issue 9, September 2013, Pages 2039–2049, https://doi.org/10.1093/carcin/bgt141

Published:

29 May 2013

Abstract

Polyphenolic phytochemicals present in fruits and vegetables indisputably confer anticancer benefits upon regular consumption. Recently, we demonstrated the growth-inhibitory and apoptosis-inducing properties of polyphenol-rich sweet potato greens extract (SPGE) in cell culture and in vivo prostate cancer xenograft models. However, the bioactive constituents remain elusive. Here, we report a bioactivity-guided fractionation of SPGE based upon differential solvent polarity using chromatographic techniques that led to the identification of a remarkably active polyphenol-enriched fraction, F5, which was ~100-fold more potent than the parent extract as shown by IC ₅₀ measurements in human prostate cancer cells. High-performance liquid chromatography–ultraviolet and mass spectrometric analyses of the seven SPGE fractions suggested varying abundance of the major phenols, quinic acid (QA), caffeic acid, its ester chlorogenic acid, and isochlorogenic acids, 4,5-di-CQA, 3,5-di-CQA and 3,4-di-CQA, with a distinct composition of the most active fraction, F5. Subfractionation of F5 resulted in loss of bioactivity, suggesting synergistic interactions among the constituent phytochemicals. Quantitative analyses revealed a ~2.6- and ~3.6-fold enrichment of QA and chlorogenic acid, respectively, in F5 and a definitive ratiometric relationship between the isochlorogenic acids. Daily oral administration of 400mg/kg body wt of F5 inhibited growth and progression of prostate tumor xenografts by ~75% in nude mice, as evidenced by tumor volume measurements and non-invasive real-time bioluminescence imaging. These data generate compelling grounds to further examine the chemopreventive efficacy of the most active fraction of SPGE and suggest its potential usefulness as a dietary supplement for prostate cancer management.

Topic:

• apoptosis
• cell culture techniques
• cell proliferation
• caffeic acids
• chlorogenic acid
• high pressure liquid chromatography procedure
• liquid chromatography
• dietary supplements
• dose fractionation
• fruit
• ipomoea batatas
• mice, nude
• phenols
• prostatic neoplasms
• quinic acid
• solvents
• transplantation, heterologous
• vegetables
• diagnostic imaging
• phytochemicals
• prostate cancer
• tumor growth
• chemopreventive agents
• polyphenols
• tumor volume
• polarity
• bioluminescence

Issue Section:

ORIGINAL MANUSCRIPT

Introduction

Introduction

Nutrition research has long favored a reductionist approach emphasizing single phytochemical-based health benefits. However, the idea of synergy among constituent phytochemicals present in whole foods is gaining momentum. Several reports underscore the benefits of a multitargeted approach offered by a synergistic mixture of phytochemicals present in whole foods ( 1–6 ). It is becoming recognizable that a whole or partially purified extract of a plant offers significant advantages over a single-isolated ingredient. This can be most appropriately described as the ‘herbal shotgun’ approach, as opposed to the ‘silver bullet’ method of conventional medicine ( 7 ) and may partially explain the limited success of clinical trials involving individual phytochemicals such as vitamin E and beta-carotene for cancer chemoprevention ( 8–10 ). Nonetheless, these data solidify the notion that health benefits from fruits and vegetables may not be solely because of the isolated single compounds but are mainly due to additive and/or synergistic interactions among components that ‘partner’ together in the concoction at their relative concentrations. For example, studies with skin-bearing apples have demonstrated strong antiproliferative activity in human colon and hepatic cancer cells compared with apples without skin or its most studied constituent, vitamin C ( 11 ).

Well known for their abundance in fruits and vegetables, polyphenols are versatile molecules containing several hydroxyl groups with multiple aromatic rings. The amphiphilic phenolic moiety of polyphenols blends the hydrophobic character of its planar aromatic core with the hydrophilic nature of its polar hydroxy substituent ( 12 ). The inherent biophysicochemical properties of the phenolic group display a wide repertoire of functional roles, including plant resistance against microbial pathogens and protection against solar radiation. Epidemiological studies suggest an inverse relationship between consumption of polyphenol-rich foods, such as cocoa, red wine, tea, fruits, and vegetables, and the incidence of chronic diseases including cancer ( 13–15 ). Although it is easy to evaluate the protective effect of a single phytochemical, for example, a single polyphenolic compound, the health benefits of dietary polyphenols are difficult to discern when numerous phytochemicals including polyphenolics, flavonoids, lignans, and tannins are active and work synergistically. The complexity of polyphenols in foods limits the identification of definitive compositions of partially purified extracts that display superior efficacy compared with single agents or whole foods. Nevertheless, it is likely that a reductionist approach involving fractionation of a whole extract may result in the increased concentration of bioactive constituents in a particular subfraction, thus enhancing efficacy.

Sweet potato greens (SPG), Ipomoea batatas , a significant source of dietary polyphenols, are widely consumed as a fresh vegetable in Asia, in particular, Taiwan and China ( 16 ). Caffeic, monocaffeoylquinic (chlorogenic acid), dicaffeoylquinic and tricaffeoylquinic acids are reported as the major phenolic constituents of these greens ( 16 , 17 ). Particularly, anthocyanins in SPG have been described to be cyanidin type rather than peonidin type ( 18 ). SPG have been shown radical scavenging, antimutagenic, antidiabetic, antibacterial, anti-inflammatory and anticancer activities ( 19 , 20 ). We recently reported the significant anticancer property of sweet potato greens extract (SPGE) in both in vitro and in vivo prostate cancer models ( 21 ). Although several analytical studies have identified major phenolic compounds in SPG, this study is the first report to detail bioactivity-guided fractionation of SPGE, emphasizing the importance of synergistic interactions among various bioactive components to confer remarkable in vitro and in vivo effects in prostate cancer models.

Materials and methods

Cell culture and materials

Human prostate cancer cell lines, PC-3 cells, were cultured in RPMI-1640 media (Mediatech, Manassas, VA) combined with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin/streptomycin solution. Cells were cultured in a humidified atmosphere at 37°C and 5% CO ₂ . Thiazolyl blue tetrazolium bromide (MTT dye, 98% thin-layer chromatography [TLC]) and dimethyl sulfoxide (DMSO) were from Sigma–Aldrich (St Louis, MO). Quinic acid (QA), chlorogenic acid (ChA), caffeic acid (CA) and Folin–Ciocalteau (FC) reagent, ACS grade methanol, ethyl acetate, hexanes and high-performance liquid chromatography (HPLC) grade solvents were from Sigma–Aldrich. Stably transfected luciferase-expressing PC-3 (PC-3-luc) cells and luciferin were from Caliper Life Sciences (Alameda, CA).

SPGE preparation

SPGE was prepared as described previously ( 21 ). Briefly, young Whatley/Loretan (TU-155) variety of sweet potato ( I. batatas ) greens harvested on day 30 were obtained as part of collaboration with the Nutrition department at Tuskegee University. SPGE was prepared by soaking air-dried leaves in methanol overnight for 3 consecutive days. The supernatant was collected daily and was finally concentrated in vacuo (Buchi Rotavap) followed by freeze-drying using a lyophilizer to a solid-powder form, which was stored at −80°C until tested. SPGE stock solution was prepared by dissolving 10mg in 1ml of DMSO and various concentrations were obtained by appropriate dilutions. Batch-to-batch variation was evaluated by analysis of polyphenolic content in SPGE by FC method.

Determination of total phenol content

Total phenolic content was determined by FC method using ChA as the standard. ChA (0.5g) was dissolved in 10ml ethanol and then diluted to 100ml with water to make a final concentration of 5g/l. A total of 50, 100, 250 and 500mg/l concentrations of standards and 0.5, 1, 2, 3, 4 and 5mg/ml concentrations of test extracts were prepared in distilled water. A total of 20 µl sample of standard or test extract was dissolved in 1.58ml water, followed by 100 µl FC reagent. This mixture was mixed thoroughly and incubated no longer than 8min. Sodium carbonate solution of 300 µl was added to the above mixture and was incubated for 2h at room temperature. A final volume of 2ml was measured for absorbance at 765nm and the results were expressed as milligrams of chlorogenic acid equivalents per gram dry material. The linear range of the calibration curve was 0.02–0.2mg/ml. All samples were analyzed in triplicates.

Fractionation of SPGE

Classical column chromatographic separation was performed on SPGE (3g) that was loaded on to a silica gel column, which was run down using hexane: ethyl acetate solvent system starting with 500ml of 100% hexane. The fraction was collected in a conical flask and stored at 4°C. This was followed by elution using 500ml of hexane:ethyl acetate solution (90:10). Subsequently, a gradient increase in the percentage of ethyl acetate (10% each time) was incurred in the mobile phase to elute various components of SPGE into different fractions ( Supplementary Table 1 , available at Carcinogenesis Online). After the elution of 50:50 hexane:ethyl acetate fraction, hexane was replaced with 50% methanol to elute the highly polar components. With an increment of 10% methanol each time (starting from 50:50 methanol:ethyl acetate), the column was finally eluted with 100% methanol to ensure complete elution of all components. A total of 17 fractions, thus, obtained were concentrated in vacuo (Buchi Rotavap, New Castle, DE) followed by separation on TLC. Based on the observed bands, fractions with similar TLC profiles (Rf values) were pooled to finally obtain seven fractions (F1–F7). All seven fractions were freeze-dried using a lyophilizer and were stored at −80°C until tested

In vivo tumor growth and bioluminescent imaging

A total of 1 × 10 ⁶ PC-3-luc cells were subcutaneously injected in the right flank of 6-week old male BALB/c nude mice (Harlan Laboratories, Indianapolis, IN). When mice developed palpable tumors, they were randomly divided into three groups of eight mice each. Control group received vehicle (phosphate-buffered saline with 0.05% Tween-80, pH = 7.4) and the treatment group received 400mg/kg body wt F5 by oral gavage daily. Real-time bioluminescent imaging of luciferase activity in live mice was employed to monitor tumor growth using the IVIS in vivo imaging system (Caliper Life Sciences) using the Live Imaging software. Briefly, mice anesthetized with isoflurane were intraperitoneally injected 25mg/ml luciferin and imaged with a charge-coupled device camera. An integration of 20 s with four binnings of 100 pixels was used for image acquisition. The relative photon count at the tumor site of the mice from vehicle or F5-treated groups was quantitated as the number of photons leaving a square centimeter of tissue and radiating into a solid angle of 1 steradian (photons/s/cm ² /sr). All animal experiments were performed in compliance with Institutional Animal Care and Use Committee guidelines.

Immunoblot analysis and immunofluorescent microscopy

Tumor lysates treated with vehicle and 400mg/kg body wt F5 were subjected to western blot analysis. Membranes were probed for cleaved caspase-3 and cleaved poly (ADP ribose) polymerase (PARP) along with β-actin, which was used as a loading control. Paraffin-embedded tumor sections from control and F5-treated groups were processed and immunostained with apoptotic markers, cleaved caspase-3, cleaved PARP and the proliferation marker Ki67. Fluorescent images were captured using confocal microscopy.

Human prostate cancer cell lines PC-3 were treated with 10 μg/ml F5 and cell lysates were collected at 0, 6, 12, 24 and 48h. Immunoblot analysis was performed on the F5-treated and control samples by probing for cleaved PARP and β-actin to confirm the induction of apoptosis.

Histopathological analysis

Mice were euthanized after 6 weeks of F5 or vehicle feeding by exposing to CO ₂ for 2min. Blood was collected by cardiocentesis in accordance with our standard Institutional Animal Care and Use Committee protocol. The organs were immediately collected, formalin fixed and paraffin embedded. A total of 5 μm sections were stained with hematoxylin and eosin. Microscopic evaluation was performed by a pathologist in a blinded manner.

Statistical analysis

The mean and standard deviations were calculated for all quantitative experiments using Microsoft-Excel software. The Student’s t -test was used to determine the differences among various treatments, with P -values of ≤ 0.05 considered statistically significant. Furthermore, a two-way analysis of variance was performed to evaluate the differences between vehicle- and F5-fed groups in vivo and P -values were obtained from two-sided tests for statistical significance.

RESULTS

Fractionation of SPGE

SPGE is non-toxic and inhibits prostate cancer growth both in vitro and in vivo ( 21 ). To gain insights into the nature of compounds present in the whole extract, we employed a ‘top-down logic’ wherein we fractionated the whole extract using classical column chromatography. This led to the sequential separation of subfractions from the complex whole extract based upon their physicochemical characteristics such as polarity and solubility. To achieve optimal fractionation of SPGE, we employed a mobile-phase system that ranged from the non-polar hexanes to highly polar methanol ( Supplementary Figure 1 and Supplementary Data , available at Carcinogenesis Online). The methanolic extract of SPGE was loaded onto the column and binary solvent combinations were used as the mobile phase. Finally, passing 100% methanol through the column ensured complete elution of all compounds. This strategy yielded 17 fractions of varying polarity as shown schematically in Supplementary Figure 1 , available at Carcinogenesis Online. The 17 fractions, thus, obtained were subjected to TLC and fractions with comparable R _f values were pooled together to finally yield seven fractions ( Figure 1A ). Our next step was to perform a comparative quantitation of total polyphenolic content of all seven SPGE fractions. Using FC method, different fractions showed varying total polyphenolic content ( Figure 1B ). The quantitative comparison revealed that F5 contains ~2-fold higher phenolic content compared with SPGE ( Figure 1B ). Given that polyphenolic content has been correlated with bioactivity, these data prompted us to examine the in vitro efficacy of the various SPGE fractions.

A moderately polar fraction, F5, exhibits remarkable antiproliferative activity in prostate cancer cells

Hence, we next determined the half-maximal concentration of growth inhibition (IC ₅₀ ) for the seven SPGE fractions in PC-3 cells using the MTT assay. The IC ₅₀ values of F1–F7 were in the range of ~1–200 μg/ml ( Figure 1C ). Indeed the differential total phenolic content and polarity of various components that define a fraction might underlie the range of antiproliferative activity displayed by these fractions. Intriguingly, among the seven fractions, F5 was the most active and its IC ₅₀ value was initially calculated to be approximately 1 μg/ml ( Figure 1C ). To precisely determine the IC ₅₀ value of F5, we then tested still lower concentrations (0.075, 0.1, 0.5, 1, 5 and 10 μg/ml) of F5 subfraction obtained from four different batches (F5 ₁ , F5 ₂ , F5 ₃ and F5 ₄ ) in PC-3 cells ( Figure 1D ). The IC ₅₀ of F5 was found to be within a range of 0.794–1.5 μg/ml ( Figure 1D ), which was ~100-fold more potent compared with the whole SPGE extract (IC ₅₀ = 100 μg/ml). In addition, F5 exhibited better efficacy in other prostate cancer cell lines (LNCaP, 22Rv1, DU145 and C4-2; Figure 1E ) compared with SPGE, suggesting the generality of the effect of F5 on a variety of prostate cancer cells.

We next performed a clonogenic or colony formation assay to evaluate the capacity of a cell to proliferate to form a colony upon removal of the drug. Antiproliferative activity of F5 was demonstrated when several PC-3 colonies were observed in case of control, and the F5-treated cells were found to only partially retain their colony forming ability ( Figure 1F ). The relative clonogenicity of control versus F5-treated PC-3 cells can be visually observed in the representative micrographs shown above the bar graphical quantitation of the colonies in Figure 1F .

F5 shows enrichment of major phenolic components of SPGE

Having identified the differential bioactivity of SPGE fractions, our next step was to perform a comparative quantitation of the phenolics present in all the seven SPGE fractions by LC-UV/MS analysis. Three major phenolics, QA (m/z = 191), CA (m/z = 179) and ChA (m/z = 353), were identified to be present in SPGE ( 21 ), along with other isochlorogenic acids such as 4,5-di-caffeoylquinic acid (4,5-di-CQA, m/z = 515), 3.5-di-caffeoylquinic acid (3,5-di-CQA, m/z = 515) and 3,4-di-caffeoylquinic acid (3,4-di-CQA, m/z = 515) ( Figure 2Aii , B and Bi ). Further analysis of F1 through F7 compared with SPGE demonstrated the differential relative abundance of the major phenols, QA, CA and ChA, and the three isochlorogenic acids ( Figure 2A , Ai and Aii and Supplementary Tables 2 and 3 , available at Carcinogenesis Online). For example, F1 and F2, the fractions of lower polarity, showed an absence of QA, CA and 4,5-di-CQA ( Figure 2A , Ai and Aii ). The CA content was found to be high in F3 and F4 as opposed to the ChA amounts ( Figure 2Ai , Supplementary Table 2 , available at Carcinogenesis Online). However, the most active fraction F5 exhibited the highest amounts of QA and ChA ( Figure 2A , Ai and Aii and Supplementary Table 2 , available at Carcinogenesis Online). The enrichment of 4,5-di-CQA, 3,4-di-CQA and 3,4-di-CQA was observed from F3 onwards ( Figure 2A and Aii ). However, there was a decrease in their quantities in F6 and F7 compared with F4 and F5 ( Figure 2Aii and Supplementary Table 3 , available at Carcinogenesis Online). F4 was found to be enriched in all the three isochlorogenic acids with 3,5-di-CQA being the most abundant, whereas the content of 3,4-di-CQA is enhanced in F5 ( Figure 2A and Aii and Supplementary Table 3 , available at Carcinogenesis Online). Fractions 6 and 7 exhibited a decrease in the composition of isochlorogenic acids ( Figure 2Aii and Supplementary Table 3 , available at Carcinogenesis Online).

F5 phytochemicals exhibit synergism

To corroborate this observation, we next tested commercially available QA, ChA and CA in combination at varying concentrations against PC-3 cells. Quantitative data point out that 1mg of F5 contains 115 µg of QA, 16 μg of ChA and 0.1 μg of CA. Given the IC ₅₀ value of F5 is approximately 1 μg/ml (based on the range of 0.794–1.5 μg/ml, Figure 1D ), F5 (1 μg) actually consists of 115ng QA, 16ng ChA and 0.1ng CA. Assuming that these three compounds are the major players that contribute to F5-A’s activity, we tested the bioactivity of the mixture of the three pure standards by measuring the percentage of cell proliferation using the MTT assay. PC-3 cells were treated with this mixture in an increasing gradient concentration (0.075, 0.1, 0.5, 1, 5 and 10 μg/ml), ensuring that the relative quantities of the three compounds (QA + ChA + CA, the major constituents of F5-A), at each test concentration bore the same ratiometric relationship as was observed between them in F5. This mixture formulation, thus, mimicked the composition of F5-A (as it exists in F5). Evaluation of the in vitro efficacy of this subfraction might also enable exclusion of the possible antagonism of other yet unknown phytochemicals in F5-A. Our data suggested that even at the highest concentration tested (10 μg/ml), the formulated mixture of pure standards did not show 50% inhibition in cell growth. As the pure standard mixture of three compounds could not reproduce equivalent efficacy as of F5, we speculate that the other unknown components in F5-A perhaps did not exert an antagonistic influence. Thus, our results from in vitro experiments testing various combinations of pure standards (QA, ChA and CA) suggested that higher efficacy of F5 could not only be ascribed to enhanced total polyphenolic content but also to possible synergistic interactions associated with definitive ratiometric composition of these phenolics.

The other subfraction F5-B also tested to be non-active. Hence, the loss of bioactivity in both subfractions F5-A and F5-B individually suggested existence of synergism among the characterized and the yet unknown F5 components. It is perhaps likely that other identified compounds such as Qn, nChA, cChA, QnG and astragalin contribute to uphold the superior activity of F5. These data also emphasize the importance of the occurrence of QA, ChA, CA, 4,5-di-CQA, 3,5-di-CQA and 3,4-di-CQA in a distinct ratio, as found in F5 to display remarkable activity. To further substantiate our in vitro data, we tested the efficacy of F5 in an in vivo prostate xenograft model as discussed in the next section.

Oral feeding of F5 inhibits prostate tumor growth in vivo

Given the significant difference in the in vitro antiproliferative activity of F5 compared with SPGE, we next evaluated its in vivo efficacy to inhibit human prostate tumor xenografts subcutaneously implanted in athymic nude mice. We employed a PC-3 cell-line stably expressing luciferase (PC-3-luc), which enables real-time visualization and longitudinal monitoring of prostate cancer growth non-invasively in mice. We have shown previously that SPGE inhibits the in vivo tumor growth by 69% ( 21 ). We found that the treatment group fed with 400mg/kg body wt F5 daily by oral gavage for 6 weeks ( Figure 4Ai and Supplementary Figure 3 , available at Carcinogenesis Online) showed a time-dependent inhibition of tumor growth ( Figure 4Ai , Aii and B ) compared with the vehicle-treated control animals. A relative total flux quantitation revealed a ~75% inhibition in tumor volume with a confidence level of P < 0.05 ( n = 8, Figure 4Aii ) as measured at week 6 for the F5-fed group, compared with vehicle-treated controls. Body weights were recorded twice a week to evaluate the general health and well-being of animals during treatment. Mice in the F5 treatment group exhibited normal weight gain with no signs of discomfort during the treatment regimen. All animals in the control group were euthanized due to tumor overburden, in compliance with Institutional Animal Care and Use Committee guidelines. At the end of week 6, the excised tumors ( Figure 4Di and Dii ) were weighed posteuthanasia and a ~74% reduction in tumor weight was observed in F5-treated groups, compared with controls.

Dietary feeding of F5 showed enhanced inhibition of human prostate tumor xenograft growth in nude mice compared with SPGE. Male nude mice were subcutaneously injected with 10 ⁶ PC-3-luc cells. ( Ai ) Representative bioluminescent images of one animal per group, indicating progression of tumor growth over 6 weeks. Mice images showing bioluminescent tumors of all animals in vehicle and F5-fed animal groups at week 6 are shown in Supplementary Figure 3 , available at Carcinogenesis Online. ( Aii ) Graphical representation of quantitative radiance measured as the number of photons leaving a square centimeter of tissue and radiating into a solid angle of 1 steradian (photons/s/cm ² /sr) from vehicle- and F5-treated mice for 6 weeks. ( B ) Tumor growth monitored (by vernier calipers) and presented as tumor volume in cubic millimeter over a period of 6 weeks. ( Ci ) Graphical representation of tumor weight. ( Cii ) Photographic images of excised tumors. ( D ) Graphical representation of body weight of vehicle- and F5-treated mice [*, P < 0.05 (two-way analysis of variance), Aii, B and C].

F5 mediates apoptosis and reduction of tumor growth in vivo

To evaluate in vivo inhibition of tumor growth upon oral feeding of F5, we immunostained for Ki67 (MIB-1), a well-known marker of cell proliferation. Essentially, the Ki67 antigen is a non-histone protein expressed in all phases of the cell cycle except G ₀ . We found that Ki67-stained tumor sections from F5-fed animals showed decreased immunoreactivity ( Figure 5A ) compared with vehicle-fed animals. Tumor sections from F5-treated groups also showed an increase in cleaved caspase-3 and PARP staining ( Figure 5Ci and Cii ) compared with vehicle-fed controls, suggesting induction of robust apoptosis in tumors from SPGE-treated mice.

F5 induces apoptosis. Histochemical micrographs of tumor tissue sections from control and 400mg/kg F5-fed groups stained for ( Ai ) hematoxylin and eosin and ( Aii ) Ki67. ( B ) Western blot analysis of tumor tissue lysates probed for cleaved caspase-3, cleaved PARP and cyclin D1. β-Actin was used as a loading control. Immunofluorescence micrographs of tumor sections from control and 400mg/kg body wt F5-treated mice stained for ( Ci ) cleaved caspase-3 and ( Cii ) cleaved PARP. All images and blots shown are representative of three independent experiments.

Furthermore, the tumor tissue lysates were immunoblotted for cyclin D1 and the apoptotic markers, cleaved caspase-3 and cleaved PARP ( Figure 5B ). Cyclin D1 plays a central role in the regulation of proliferation, linking extracellular signaling environment to cell-cycle progression. There was a decrease in cyclin D1 expression in F5-fed tumor lysates suggesting a cessation of cell-cycle progression. Further, as expected, the cleaved caspase-3 and PARP expression ( Figure 5B ) were higher in F5-treated tumors compared with controls. Similar trend was observed in PC-3 cell lysates, where F5 treatment showed increased cleaved PARP expression compared with controls ( Supplementary Figure 4 , available at Carcinogenesis Online).

Non-toxicity of F5 in vivo

Toxicity is overly concerning and is often observed in prostate cancer patients undergoing either radio or chemotherapy. The histopathological evaluation of the tissues of intestine, spleen, liver, lung, brain, heart, adrenal gland, and testes from both vehicle- and F5-fed mice ( Figure 6A ) revealed no detectable differences in architecture. Furthermore, analysis of biochemical markers in the sera (alanine transaminase, aspartate transaminase, alkaline phosphate, lactic acid dehydrogenase, creatinine kinase and urea nitrogen) collected from both vehicle- and F5-fed mice was observed to be within the normal range ( Figure 6Bi–Biii ).

https://academic.oup.com/carcin/article/34/9/2039/2463112

..