Cinnamon and its bioactive compounds inhibit prostate cancer cell proliferation in vitro. The aim of the current study was to assess the chemopreventive efficacy of cinnamon (CN) and its bioactive compounds in vivo using N-methyl-N-nitrosourea (MNU) and testosterone (T) to induce prostate carcinogenesis in male Wistar/National Institute of Nutrition rats. Cancer-induced (CI) rats (n = 10) developed prostatic hyperplasia and prostatic intraepithelial neoplasia. These histopathologic changes were diminished in CI rats fed for 4 months with diets supplemented with either CN (n = 20) or its bioactive compounds (cinnamaldehyde, n = 10 and procyanidin B2, n = 10). Androgen receptor (AR) expression was lower in the prostates of CI rats than in control, but the AR target gene, probasin, was robustly upregulated. Treatment of CI rats with CN or its bioactive compounds upregulated AR expression but inhibited the expression of the 5-alpha reductase genes (Srd5a1 and Srd5a2) and did not further increase probasin expression, suggesting blunted transcriptional activity of AR due to the limited availability of dihydrotestosterone. MNU+T induced an altered oxidant status in rat prostate, which was reflected by an increase in lipid peroxidation and DNA oxidation. These changes were completely or partially corrected by treatment with CN or the bioactive compounds. CN and its active components increased the activity of the apoptotic enzymes caspase-8 and caspase-3 in the prostates of CI rats. In conclusion, our data demonstrate that CN and its bioactive compounds have inhibitory effects on premalignant prostate lesions induced by MNU + T and, therefore, may be considered for the chemoprevention of prostate cancer.

Prevention Relevance:

The research work presented in this article demonstrates the chemopreventive efficacy of CN and its bioactive compounds in a rat model of premalignant prostate cancer.

Prostate cancer is the second most frequently diagnosed cancer in men worldwide and the fifth most common cancer overall (1). Chemotherapy, radiotherapy, and hormone ablation therapy are the most common treatment modalities for prostate cancer. The major drawbacks of hormone ablation therapy are the emergence of hormone-resistant cancer and metastases to sites not available for resection, such as the bone. Treating metastatic disease with radiotherapy and cocktails of chemotherapeutic agents is associated with severe adverse side effects. However, prostate cancer progresses slowly from premalignant lesions or prostatic intraepithelial neoplasia (PIN) to adenocarcinoma and metastatic disease (2). Because malignant tumors are preceded by PIN, which can be easily diagnosed and treated, prostate cancer offers promising prospects for developing chemoprevention strategies (3, 4). A valuable method for screening prostate cancer prevention drugs is the induction of prostate cancer in rodents by administering a combination of the carcinogen N-methyl-N-nitrosourea (MNU) and testosterone. This model has several advantages: first, it mimics the early stages of prostate cancer development in humans; second, the combination of carcinogen and testosterone reproducibly induces a high incidence of prostate cancer (5, 6); and third, the MNU-testosterone treatment induces tumors in the dorsolateral and anterior lobes of the rodent prostate. These lobes of the rat prostate are generally considered homologous to the areas of the human prostate most susceptible to cancer (6). Therefore, the MNU-testosterone model has become a preferred choice for preclinical studies of chemopreventive agents of prostate cancer (7, 8).

Medicinal plants and phytochemicals have emerged in recent years as alternative options for the treatment or prevention of cancer because of the lack of side effects and the lower likelihood of dependence (9). Cinnamon, a popular spice used worldwide since the ancient era in culinary practice, is also known to possess several health benefits (10–12). It is a powerhouse of bioactive compounds such as cinnamaldehyde (CNMD), cinnamic acid (CNA), cinnamtannin B1, procyanidin B2 (PCB2), and eugenol (EUG), which have growth-inhibitory effects on malignant cells (13, 14). Recently, our studies reported the anticancer potential of aqueous cinnamon extract and its PCB2-enriched fraction in human prostate cancer cells (15). We also demonstrated the cause-and-effect relationships between proteasome inhibition and induction of apoptosis by aromatic compounds from cinnamon bark (CNMD, CNA, EUG) in human prostate cancer cells (16). Studies by others demonstrated the growth-inhibitory effect of cinnamon extract on benign prostatic hyperplasia via androgen receptor (AR) signaling (17). Polyphenols in cinnamon are also known for their antioxidant activity, potentially protective against genotoxic damage induced by oxidative stress (18, 19).

On the basis of the aforementioned studies, we hypothesized that the prevention of prostate carcinogenesis by cinnamon compounds might occur by blunting oxidative stress, inhibiting angiogenesis, altering AR signaling, or inhibiting proteasome activity. In the current work, we used the MNU-testosterone model to assess the chemopreventive efficacy of cinnamon and its bioactive compounds (CNMD, PCB2-enriched fraction) on prostate carcinogenesis in male Wistar/NIN (National Institute of Nutrition) adult rats. Furthermore, proteasome inhibitors have been recommended as anticancer and chemopreventive drugs for cancer treatment (20). Because our recent work has provided compelling evidence that cinnamon and its bioactive compounds act as proteasome inhibitors in cultured prostate cancer cells (15, 16), we included a group treated with a known synthetic proteasome inhibitor (MG132) as a reference to determine its potential chemopreventive activity in vivo. We predicted that by comparing the effects of MG132 and the cinnamon compounds on prostate carcinogenesis, we might be able to distinguish the growth-promoting molecular targets that are susceptible to proteasome inhibition and those that are independent of the proteasome and specific to the cinnamon compounds.

In the current study, we demonstrate that cinnamon or its bioactive compounds acted similar to MG132 as proteasome inhibitors, antioxidants, antiangiogenic, antiproliferative, and proapoptotic agents. However, preventing hyperplasia and premalignant lesions, namely PIN, was more efficacious by cinnamon and its compounds than by MG132. A potential mechanism for this difference might be an increase in AR activity by MG132, whereas there was no change in this activity in the cinnamon-treated groups. Our results confirmed the therapeutic potential of cinnamon-derived compounds in prostate cancer prevention.

Chemicals

MNU was purchased from BOC Sciences. Testosterone propionate, trans-cinnamaldehyde, and all other fine chemicals were procured from Sigma-Aldrich. MG132 was purchased from UBP Bio. Proteasome catalytic enzymes, caspase-3, and caspase-8 specific fluorogenic substrates were procured from Enzo Life Sci, Inc. PCNA (sc-56) and AR (sc-815) antibodies were purchased from Santa Cruz Biotechnology; Bax (2772S) was purchased from Cell Signaling Technology Inc., while β-actin (MA1-91399) antibody was purchased from Thermo Fisher Scientific. DNA isolation kit was purchased from Sigma-Aldrich, and the 8(OH)dG kit was procured from EpiGentek. Cinnamon bark (Cinnamomum zeylanicum) was purchased from a local market and powdered using a mixer grinder. C. zeylanicum bark is reported to have a very minimum amount of coumarin (21).

Animal experimentation and induction of prostatic preneoplasia

Seventy Wistar/NIN (RRID: RGD_8655977) male rats, ages around 3 months and weighing 200–250 g were procured from the Animal Facility at the ICMR-NIN, Hyderabad, India. The animals were housed in polypropylene wire-bottomed cages and maintained in an air-conditioned animal facility with a constant 12-hour light/dark cycle. Rats were allowed free access to a standard rat chow diet and drinking water throughout the experimental period. The Institutional Animal Ethics Committee (IAEC) approved the experiment at ICMR-NIN, Hyderabad, India (IAEC No.P3F/IAEC/NIN/5/2018/AYI). Rats were randomly divided into seven groups of 10 rats each. Group I or negative control (CONT) was administered only the vehicle (sesame oil). Rats in groups II–VII received first, daily intraperitoneal injections of testosterone propionate [50 mg/kg body weight (BW)] for 21 consecutive days. On day 23, these rats received daily intraperitoneal injections of testosterone propionate (100 mg/kg BW) in 0.3 mL sesame oil for 3 days. On day 27, all the rats in groups II–VII received a single intravenous dose (50 mg/kg BW) of MNU dissolved in saline at 10 mg/mL through the tail vein. Beginning 1 week after MNU administration, rats in groups II–VII received intraperitoneal injections of testosterone propionate (4 mg/kg BW) on alternate days for 16 weeks. Rats in group II were named CI (cancer-induced). Beginning on day 27, along with the administration of MNU, groups III–VII received treatments as follows: group III received 1.2 g/kg BW of cinnamon powder and was named CI + LCN (low cinnamon). Group IV was given 2.4 g/kg BW cinnamon powder and named CI + HCN (high cinnamon). Group V was given a PCB2-enriched fraction (0.75 mg/kg BW) and named CI + PCB2. Group VI was given 150 mg/kg BW) CNMD and named CI + CNMD. Group VII was injected intraperitoneally with the proteasome inhibitor MG132 (0.5 mg/kg BW) every other day for 16 weeks and named CI + MG132. Cinnamon powder, CNMD, and powdered PCB2-enriched fraction were added directly to the powdered chow diet, mixed thoroughly, and fed to the rats for 16 weeks. Doses of cinnamon and its active compounds administered to rats were chosen based on the following criteria: (i) A recent study demonstrated the beneficial effect of cinnamon (3 g/100 g diet) in a diabetic rat model (22). Assuming that adult rats consume 20 g chow per day, a diet containing 3 g cinnamon/100 g chow is equivalent to daily consumption of 2.4 g/kg BW (HCN). Half that amount (1.2 g/kg BW) was used for the LCN group to determine the safety and efficacy range of cinnamon; (ii) For PCB2-enriched fraction, we chose 0.75 mg/kg BW because 1.2 g cinnamon yields 0.7–0.8 mg of PCB2-enriched fraction prepared as described in ref. 22; (iii) The CNMD content in cinnamon bark is 50%–62% (11). We conducted a pilot study in which rats were fed CNMD at dose levels of 600 mg/kg BW (equivalent to its content in the LCN diet), 300 mg/kg BW, or 150 mg/kg BW for 6 days. Only the rats given 150 mg/kg BW consumed the diet, while the other two doses were not consumed, probably due to the pungent aroma of CNMD. An oral dose of 3,000 mg CNMD/kg BW has been reported to be toxic to rats (23).Toxicity studies with the test compounds may be needed before using them for chemoprevention in humans; (iv) 0.5 mg/kg BW was chosen for MG132, as this is similar to the dose of the PCB2-enriched fraction and a published study used an even higher dose of 2 mg/kg BW (24).

Food intake and BW were monitored on a daily and weekly basis, respectively. At the end of 16 weeks treatment regimen, the rats were sacrificed. Blood, prostate gland, bone (tibia), and other vital organs were collected. Serum was separated and stored at −80°C for further analysis. A small portion of the prostate tissue was kept in Bouin solution for histopathology analysis, while the rest was frozen in liquid N2 and stored at −80°C for downstream applications.

Blood parameters

Hematologic parameters, such as hemoglobin, hematocrit, total white blood cell (WBC), and red blood cell (RBC) count, were measured using an autoanalyzer (model no. COBAS C311; Roche, Inc.). Serum protein was measured using Lowry method, whereas serum albumin was monitored using a commercial kit (Invitro Biosystems Inc.). Serum calcium was analyzed by atomic absorption spectrophotometry, and phosphorous and alkaline phosphatase levels were estimated using standard methods described earlier (25).

Bone analysis

Tibia bones were taken from the control, CI, and all the treatment groups. The bones were cleaned of any attached muscle and/or cartilage and then dried in an oven at 100°C for 2 hours, followed by overnight drying in an autoclave. The bone ash formed was weighed, dissolved in 1N HCl, and used for calcium and phosphorus estimation by standard methods, as described earlier (25). Bones preserved in formalin were decalcified, and cross-sections were prepared and stained with hematoxylin and eosin (H&E) to assess morphologic differences and osteoclast number.

Prostatic and serum acid phosphatase assay

Acid phosphatase (AcPh) assay was performed in serum and prostate tissue lysates using the method described by Tenniswood and colleagues (26). It is reported that the prostate is the major organ that secretes AcPh and the serum levels of AcPh of prostatic origin increase with the development and/or progression of prostate carcinoma. L-Tartarate significantly inhibited prostate-specific AcPh activity (26). The serum AcPh activity was measured both in the presence and absence of L-tartarate, and the difference between the total activity and that in the presence of tartarate indicated the AcPh activity of prostatic origin in the serum. The activity is expressed as units/L.

Oxidative stress and antioxidant defense markers

All oxidative stress parameters and antioxidant enzymes were tested in prostate tissue. Thiobarbituric acid reactive substances (TBARS) were determined as a measure of lipid peroxidation based on the method of Balasubramanian and colleagues (27). DNA oxidation was assessed by measuring the amount of 8(OH)dG using a commercial kit and was expressed as 8OHdG%. The activity of catalase (CAT) was measured using the method of Sinha and colleagues (28). This method is based on the principle that dichromate in acetic acid is reduced to chromic acetate when heated in the presence of H2O2, forming perchromic acid as an unstable intermediate. The chromic acetate produced was measured calorimetrically at 570–610 nm. CAT activity is expressed as units/mg of protein. The activity of superoxide dismutase (SOD) was measured based on the method of Marklund and Marklund (29). SOD activity was determined based on its ability to inhibit superoxide-mediated reduction. Activity is expressed as units/mg protein, where one unit represents the amount of enzyme that inhibits the oxidation of pyrogallol by 50%.

Histopathology analysis and incidence of pathologic lesions in the prostate

The Bouin solution fixed prostate lobes of 10 animals in each group were embedded in paraffin. The paraffin-embedded tissue blocks were sectioned at 3 μm thickness using a rotary microtome and then fixed on microscopic slides. The sections were then covered with glass coverslips, stained with H&E, and permanently mounted with dibutyl phthalate xylene mount and the sections were viewed and photographed under a Nikon H600 L inverted microscope (20× and 40× magnifications). Incidence of hyperplasia and premalignant lesions was expressed as percentage of animals in each group which had histologic evidence of the specified pathology (hyperplasia or PIN) in the H&E-stained prostate tissue samples.

Proteasome, caspase-3, caspase-8 activity assays

Prostate tissue (30 mg) was homogenized in lysis buffer [20 mmol/L Tris HCl (pH 7.2)/0.1 mmol/L EDTA [Ethylenediamine tetraacetic acid]/1 mmol/L 2-mercaptoethanol/5 mmol/L ATP/20% [volume for volume (v/v)] glycerol, 0.04%(v/v) Nonidet P-40], centrifuged at 13,000 × g at 4°C for 15 minutes, and the supernatant was collected. Total protein in the tissue lysates was estimated using Lowry method. The assay was performed using specific fluorogenic substrates, as described previously (15). The results were expressed as nmol amino methyl coumarin released/mg protein/hour.

RNA isolation and qPCR

Total RNA was isolated from 50 mg of prostate tissue using TRIzol reagent according to the manufacturer's instructions (Invitrogen Life Technologies), and cDNA was synthesized from 1 μg of total RNA using the iScript Bio-Rad cDNA synthesis kit. Real-time qPCR was performed using different sets of rat-specific primers for different genes with SYBR Green Supermix (Bio-Rad). All Ct values of the test genes were normalized to the housekeeping gene (Rplp1), and the fold change in gene expression was calculated using the delta-delta Ct method. Data are expressed as fold change taking the CI/group II as “1.” The primer sequences for the different genes used are shown in Supplementary Table S1.

Western blot analysis

Total protein was extracted from 50 mg of frozen prostate tissue by homogenization in ice-cold Tris-buffered HCl 50 mmol/L, pH 8.0, containing 150 mmol/L NaCl, 0.5% IGEPAL, 0.5 mmol/L phenylmethyl sulphonyl fluoride, 0.5 mmol/L dithiothreitol, 5 mmol/L of Na fluoride, and 1X protease inhibitor cocktail. After 30 minutes of rocking at 4°C, the homogenates were centrifuged at 12,000 × g for 20 minutes at 4°C, and the supernatant was retained. The total protein content of the cell extract was estimated using Lowry method, and proteins equivalent to 50 μg were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were processed as described earlier (16). For equal loading of proteins, the membrane was stripped and reprobed with an anti-β-actin antibody.

Statistical analysis

All data are presented as the mean ± SEM of six samples from each group. Data were subjected to statistical analysis using the IBM SPSS (RRID: SCR_002865) Statistics software (version 20). To identify statistically significant differences between groups, one-way ANOVA followed by a post hoc test of the least significant difference was performed. Differences with a “P” value of at least P ≤ 0.05 were considered statistically significant. GraphPad Prism 8 software (RRID: SCR_002798) was used to prepare figures.

Data availability statement

Data generated in this study are available upon request from the corresponding author.

Effect of cinnamon and its bioactive compounds on the hematologic and biochemical parameters in the blood

No differences were observed in blood parameters, such as hemoglobin, hematocrit, and total RBC, between the groups. The total WBC count was observed to be significantly (P ≤ 0.01) lower in the CI group compared with the negative control, and the count remained low in all the treatment groups (Supplementary Table S2). Total protein and albumin levels in the serum did not differ between the groups. The serum calcium levels did not differ between the groups. Interestingly, the serum phosphorus levels were significantly (P ≤ 0.05) lower in the CI group than in the control group, while the serum alkaline phosphatase levels were significantly (P ≤ 0.05) higher in the CI group than in the controls. All treatment groups normalized the phosphorus and alkaline phosphatase levels to those of the control group (Supplementary Table S2).

Effect of cinnamon and its bioactive compounds on BW, prostate weight, serum, and prostatic AcPh activity

Food intake was not different between the groups throughout the experimental period. However, BWs were significantly lower in CI rats than in control rats (Fig. 1A). None of the treatment groups showed an increase in BW, except in the LCN group, wherein the weights were slightly increased compared with the other groups (Fig. 1A). In contrast, prostate tissue weights were significantly (P ≤ 0.001) higher in the CI group than in the control group (Fig. 1B). Treatment with cinnamon, its bioactive compounds, and MG132 did not alter prostate tissue weights (Fig. 1B). As expected, serum AcPh activity was significantly (P ≤ 0.05) higher in the CI group than in the control group, while prostatic AcPh activity was significantly (P ≤ 0.001) lower in the CI group than in the control group. Treatment with cinnamon, its bioactive compounds, and MG132 appeared to significantly decrease serum AcPh activity (Fig. 1C) and increase prostatic AcPh (Fig. 1D).

Effect of cinnamon and its bioactive compounds on hyperplasia and premalignant lesions in the prostate tissue

In the control group, all 10 animals (100%) exhibited normal prostate tissue as judged by histologic analysis. The prostate glands had large lumina, with some filled with prostatic epithelial secretions, and were lined by a single layer of cuboidal to columnar epithelial cells with basally oriented round to oval nuclei. No mitosis or necrosis was observed (Fig. 2A). As expected in the CI group, the majority of the animals (8/10 = 80%) showed PIN changes with the epithelium lining these glands showing mild dysplasia and stratification ranging from 2–4 layers, with crowding and disorientation of the cells (Fig. 2B). Focal small papillary projections into the lumen were also observed, leading to mild narrowing of the lumen. The cells showed mildly enlarged nuclei with an increased N: C ratio. A loose stroma of connective tissue was observed. One animal (1/10 = 10%) in this group showed hyperplasia with stratification seen in the cells lining the glands and arranged focally in 2–3 layers. In the LCN group, 6/10 animals (60%) displayed predominantly hyperplastic changes in the epithelium, whereas 4/10 animals had normal prostates (Fig. 2C). In the HCN group, 3/10 animals (30%) showed hyperplastic changes in the epithelium, and 7/10 animals (70%) showed histologically normal prostatic epithelium (Fig. 2D). In the PCB2 group, 4/10 animals (40%) showed hyperplastic changes, while the remaining 6/10 animals (60%) showed histologically normal prostatic epithelium with the absence of dysplastic epithelium (Fig. 2E). In the CNMD group, 3/10 animals (30%) showed hyperplastic changes in the epithelium, and 7/10 animals (70%) showed histologically normal prostatic epithelium (Fig. 2F). In the MG132 group, 1/10 (10%) showed features of PIN, 5/10 animals (50%) showed hyperplastic changes in the epithelium with the lining showing focal stratification of 2–3 layers with no dysplastic changes in the epithelial cells (Fig. 2G), and 4/10 animals (40%) were normal. Representative pictures from the control and CI group showing normal epithelium, hyperplastic, and PIN changes at 40× magnification are shown in Fig. 2HJ. Incidence (%) of normal, hyperplasia, and PIN in the different groups is depicted in Fig. 2K.

Effect of cinnamon and its bioactive compounds on the expression levels of AR, its target gene probasin, and 5-alpha reductases in the prostate tissue

Because AR and androgens are the primary contributors to prostate tissue growth (30, 31), we assessed the expression levels of AR, probasin (Pbsn), and 5-alpha reductases (enzymes needed for the conversion of testosterone to dihydrotestosterone). There was a significant (P ≤ 0.05) decrease in the mRNA and protein levels of AR in the CI group compared with those in the control group (Fig. 3AC). Treatment with cinnamon, its bioactive compounds, and MG132 significantly (P ≤ 0.01) increased the levels of AR. Furthermore, there was a significant (P ≤ 0.001) increase in the level of the AR target gene, Pbsn, in the CI group compared with that in the control group (Fig. 3D). However, Pbsn levels did not change after treatment with either cinnamon or its compounds compared with those in the CI group. Interestingly, there was a significant (P ≤ 0.05) increase in PB mRNA levels in the MG132-treated group compared with the CI group. The mRNA expression of both the 5-alpha reductases (Srd5a1 and Srd5a2) was not altered in the CI group compared with that in the control group (Fig. 3E and F). Treatment with either cinnamon or its bioactive compounds decreases the expression of both 5-alpha reductases. However, MG132 significantly (P ≤ 0.01) increased the expression of Srd5a1, whereas it decreased the expression of Srd5a2 in the prostate (Fig. 3E and F).

Effect of cinnamon and its bioactive compounds on the oxidative stress parameters, phase I and II metabolizing enzymes, and proteasomal activity in the prostate tissue

Cinnamon is rich in polyphenols (32), and both cinnamon and its bioactive compounds are known to be potent free radical scavengers (33). Hence, we assessed the antioxidant effects of cinnamon and its constituent compounds. The TBARS content, an index of lipid peroxidation, was significantly (P ≤ 0.05) higher in the CI group than in the control group. Treatment with cinnamon, its bioactive compounds PCB2, and CNMD, and MG132 significantly (P ≤ 0.001) decreased TBARS levels (Fig. 4A). SOD levels were lower in the CI group than in the control group (Fig. 4B). Although there appeared to be an increase in SOD activity after treatment with cinnamon and its bioactive compounds, the difference was not statistically significant. However, CAT activity was significantly (P ≤ 0.001) higher in the CI group than in the control group (Fig. 4C). Treatment with cinnamon, its bioactive compounds, and MG132 significantly (P ≤ 0.01, 0.001) decreased CAT activity. 8OHdG levels were significantly (P ≤ 0.01) higher in the CI group than in the control group (Fig. 4D). Treatment with cinnamon, its bioactive compounds, and MG132 significantly (P ≤ 0.05 or 0.01) decreased the 8OHdG levels, except in the PCB2 group, which was not different from the CI group. GSTP1 is a phase II enzyme and a member of the GST enzyme superfamily, which is known to be inactivated in prostate cancer tissue. There was a significant (P ≤ 0.01) decrease in mRNA levels of Gstp1 in the CI group compared with the controls (Fig. 4E). Treatment with either cinnamon or its bioactive compounds significantly (P ≤ 0.001) increased Gstp1 mRNA levels (Fig. 4E). CYP1B1 belongs to the cytochrome P450 (CYP) superfamily and is a potential biomarker for prostate cancer. There was a significant (P ≤ 0.05) increase in mRNA levels of Cyp1b1 in the CI group compared with the controls (Fig. 4F). Treatment with either cinnamon, its bioactive compounds, or MG132 significantly (P ≤ 0.05) decreased the levels of Cyp1b1 mRNA and were similar to those of the control group (Fig. 4F). Cancer initiation and oxidative stress are associated with increased proteasome activity (34). The catalytic activity (Ch-L) of the 26S proteasome was significantly (P < 0.001) higher in the CI group than in the control group (Fig. 4G). Treatment with cinnamon powder and its bioactive compounds significantly (P ≤ 0.01 or P ≤ 0.001, respectively) decreased proteasomal activity.

Effect of cinnamon and its bioactive compounds on the proliferative, metastatic, and apoptotic markers in the prostate tissue

The protein expression of the proliferative marker PCNA was significantly (P ≤ 0.01) higher in the CI group than in the control group. Treatment with cinnamon, its bioactive compounds, or MG132 significantly (P ≤ 0.05 or 0.01) decreased PCNA levels (Fig. 5A). The quantification of PCNA protein levels is shown in Fig. 5B. The proapoptotic protein BAX was significantly (P ≤ 0.05) decreased in the CI group compared with the control, and there appeared to be an increase in the expression of BAX in the treated groups compared with the control group (Fig. 5A). The quantification of BAX protein levels is shown in Fig. 5C. The levels of the angiogenic marker VEGFR were significantly (P ≤ 0.001) increased in the CI group, and this was reversed upon treatment with either cinnamon, its bioactive compounds, or MG132 (Fig. 5D). MMP9 is a member of the matrix metalloproteinase family and is well known to have a role in cancer invasion and metastasis. There was a significant (P ≤ 0.01) increase in mRNA levels of Mmp9 in the CI group compared with the control group (Fig. 5E). Treatment with cinnamon, its bioactive compounds, and MG132 significantly (P ≤ 0.01) decreased the mRNA levels of Mmp9. The caspase-8 and caspase-3 activities, which are markers of apoptosis, were significantly (P ≤ 0.01) higher in the groups treated with either cinnamon or its compounds or MG132 than in the control group (Fig. 5F and G).

Effect of cinnamon and its bioactive compounds on bone mineral content and bone histology

Prostate cancer is known to metastasize to bone, and the early stages of the disease are also reported to affect bone (35). The amount of bone calcium and phosphorous in the tibia was observed to be significantly (P ≤ 0.01) lower in the CI group than in the control group (Fig. 6A and B). Treatment with cinnamon, its bioactive compounds, and MG132 significantly (P ≤ 0.01) increased the levels of both minerals in the bone. H&E sections of the bone indicated an increase in the number of osteoclasts in the CI group compared with that in the control group, indicating increased bone resorption (Fig. 6C). Treatment with either cinnamon, its bioactive compounds, or MG132 significantly (P ≤ 0.01, P ≤ 0.001) decreased the number of osteoclasts in the bone (Fig. 6D).

In the current study, we assessed the efficacy of cinnamon and its bioactive compounds as chemopreventive agents at the early stages of malignancy in the MNU+T model of prostate cancer in rats. In this model, we observed hyperplasia and premalignant lesions, i.e., PIN, feeding them a high dose of cinnamon or its bioactive compounds for duration of 4 months, prevented these lesions by 100% (PIN) and 60%–70% (hyperplasia). It is important to note that while prostate hyperplasia is not considered a premalignant condition in humans, PIN lesions are premalignant. Therefore, the prevention of premalignant lesions by these compounds was highly efficacious.

Chemopreventive drugs exert protective effects by targeting various biological pathways including, cellular damage by inflammation or oxidative stress, and growth-promoting signaling (36). Therefore, we sought to decipher the mechanism(s) for the chemopreventive effect of cinnamon and its bioactive compounds in the MNU+T-induced prostate carcinogenesis by probing these processes.

Uncontrolled oxidative stress can result in mutations of tumor suppressor genes due to DNA damage, which promotes the initiation of neoplastic changes in prostate cancer (37). Simultaneous administration of cinnamon and its bioactive compounds led to the normalization of antioxidant enzyme activities and reduction in DNA oxidation and lipid peroxidation in the prostate, which confirm the function of these compounds as antioxidants during prostate cancer initiation.

GSTP1 is a detoxifying and phase II drug-metabolizing enzyme, and downregulation of its expression by DNA methylation is a common early event in prostate carcinogenesis and serves as an early diagnostic marker (38, 39). Modest suppression of Gstp1 expression in prostates of the CI group and a significant upregulation of its expression by cinnamon or its bioactive compounds, underscores their chemopreventive potential against prostate cancer. Because Gstp1 is a target gene for the antioxidant transcription factor, Nrf2, it is possible that upregulation of its expression by the cinnamon compounds occurs via transcription activation of Nrf2 (40). This possibility is supported by Wondrak and colleagues (41), who demonstrated that the antioxidant effect of CNMD in colon cancer cells is mediated by Nrf2 activation.

Another gene product that contributes to oxidative stress and susceptibility to carcinogens is CYP1B1, a phase I drug-metabolizing enzyme expressed in various cancer tissues (42). Elevated expression of Cyp1b1 is reported in prostate carcinomas and is associated with hyperplasia and tumor progression (43). Robust upregulation of its expression in the CI group confirms its contribution to prostate carcinogenesis in the MNU+T model. Furthermore, suppression of its expression by all cinnamon compounds underscores their efficacy in targeting Cyp1b1 for anticancer therapy (42).

The attenuation of premalignant prostatic lesions by cinnamon and its active compounds was associated with the inhibition of cell proliferation, as reflected by a decrease of PCNA protein and upregulation of apoptosis, driven by an increase of proapoptotic Bax protein and upregulated enzymatic activities of caspase-8 and caspase-3. Androgens, particularly testosterone and its metabolite DHT, are essential for the maintenance of normal prostate tissue, but they also promote prostate tumor growth and progression in both humans and rodents (44, 45). Therefore, we considered disruption in androgen production or action by the cinnamon compounds as a potential mechanism for their growth-inhibitory effects. The results suggest that transcriptional activity of AR in the CI group is robust because PB expression was 20-fold higher than in control, despite a modest decrease in AR expression. However, based on PB expression levels, it appeared that transcriptional activity of AR in the cinnamon compounds-treated groups remained the same as in the CI group, despite their suppression of both Srd5a1 and Srd5a2 possibly because these compounds also upregulated the expression of AR. Therefore, prostate growth inhibition by the cinnamon compounds is probably not due to diminished transcriptional activities of the AR. However, we cannot exclude the possibility that ligand-dependent nongenomic growth-promoting activities of AR are disrupted by the cinnamon compounds due to suppression of Srd5a1 and Srd5a2 expression and DHT production (46).

A decrease in androgen receptor expression in the prostates of the CI group relative to the normal controls is corroborated by another study reporting downregulation of AR in the MNU+T rodent model, especially in prostate adenocarcinoma (47). However, studies in human subjects do not support this trend; wherein AR decreases in the non-epithelial stroma adjacent to the tumor. In contrast, there is either no change or an increase in AR expression in tumor epithelial cells (48). The development of androgen-independent prostate cancer is an inherent feature of progressive prostate carcinogenesis. Loss of AR expression may be the dominant mechanism in rodent models, while hormone-independent activation of AR is more typical in the human prostate.

It is well established that cancer cells possess high proteasome activity that is needed for their uncontrolled growth and proliferation, protection from apoptosis (49), and the initiation of cancer (50).The 26S proteasome is recognized as a molecular target for cancer therapy. Our current study demonstrated significant upregulation of proteasome activity in the CI group and inhibition of this activity by cinnamon and its bioactive compounds. Our in vitro studies demonstrated cause-and-effect relationships between growth inhibition and proteasome inhibition by cinnamon and its compounds in both AR-positive and AR-negative prostate cancer cell lines (16). Therefore, proteasome inhibition in prostate carcinogenesis induced by MNU+T offers a pathway for growth inhibition, independent of AR signaling.

Our experimental design included CI rats treated with the synthetic proteasome inhibitor MG132 as a reference group to distinguish proteasome-dependent and -independent effects of the cinnamon compounds on prostate carcinogenesis. Interestingly, MG132 was somewhat less efficacious than the cinnamon compounds at inhibiting premalignant lesions, suppressing lipid peroxidation and upregulating expression of the antioxidant enzyme Gstp1. Coincidentally, unlike the cinnamon compounds, it also upregulated Srd5a1 and Pbsn expression, suggesting greater transcriptional activity of AR. However, it was as effective as the cinnamon compounds in the upregulation of apoptotic markers and suppressing the expression of Cyp1b1. That MG132 inhibits DNA oxidation effectively can be explained by its very effective suppression of Cyp1b1 expression, which is a likely contributor to DNA adduct formation.

In humans, prostate cancer metastasizes primarily to bone leading to its destruction (51). Despite the absence of bone metastasis in rodent models of prostate cancer, including the MNU+T, there is damage to the bone (35). Our data demonstrate a significant decrease in mineral content and an increase in osteoclast numbers in the bones of rats in the CI group which was prevented by cinnamon and its bioactive compounds. These results indicate that even in the absence of metastasis, prostate cancer progression may affect the bone microenvironment possibly by producing osteolytic factors.

In conclusion, the long latency period and slow progression of prostate cancer render it an ideal disease for chemopreventive interventions (5). Our current study demonstrates that cinnamon and its bioactive compounds can affect several targets and/or pathways involved in prostate cancer initiation and promotion, including susceptibility to carcinogens, and oxidative stress, thereby inhibiting the development of premalignant lesions. Hence, our work warrants further in-depth investigation and clinical trials for assessing the cancer chemopreventive efficacy of cinnamon and its bioactive compounds.

No disclosures were reported.

S. Gopalakrishnan: Formal analysis, investigation, methodology, writing–original draft. M. Dhaware: Data curation, validation, investigation, methodology, writing–review and editing. A.A. Sudharma: Formal analysis, investigation, methodology. S.V. Mullapudi: Formal analysis, investigation, histological analysis. S.R. Siginam: Formal analysis, investigation, methodology. R. Gogulothu: Investigation, methodology. I.A. Mir: Investigation, methodology. A. Ismail: Conceptualization, resources, supervision, funding acquisition, writing–original draft, project administration, writing–review and editing.

This work was supported by grants from the Indian Council of Medical Research (#18-BS-02), GOI (to A. Ismail). We thank the University Grants Commission for providing fellowships (to S. Gopalakrishnan) and the Council for Scientific and Industrial Research, Government of India, for providing fellowships (to A.A. Sudharma and R. Gogulothu). We sincerely thank Dr. Sara Peleg for her critique and feedback on this article. We thank Mr. N. Shivakrishna for maintaining the experimental rats throughout the study.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/).

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