Comprehensive intervention strategies in N-Methyl N-Nitrosourea-testosterone-induced prostate cancer: Exploring the multifaceted impact of dutasteride and docetaxel nanostructured lipid carriers on hormonal, oxidative, immunological and histological parameters

Materials

Samples of Docetaxel (DXT) and dutasteride (DUT) were generously provided as a gift by Mac-Chem Labs Pvt. Ltd, based in Mumbai. The procurement of stearic acid was facilitated through Sigma-Aldrich Pvt. Ltd. (India). Oleic acid was sourced from Sun Pharmaceuticals Ltd, headquartered in Mumbai; The glyceryl monostearate utilized in this study was acquired directly from Croda Chemicals Pvt. Ltd (India); Poloxamer 407, an essential ingredient in the formulation procedure, was generously supplied as a complimentary sample by BASF India Ltd., with its headquarters situated in Mumbai, India. Tween 20, a fundamental element in the formulation, was procured from Central Drug House (Pvt) Ltd., based in New Delhi, India.

Method of NLC preparation

In this study, NLCs loaded with Dxt-NLC and Dut-NLC were developed to enhance therapeutic outcomes for PCa treatment. By selecting biocompatible lipids, and appropriate surfactants and using a hot homogenisation technique followed by ultrasonication, stable NLCs with high drug-loading capacity and nanoscale particle size (average diameter of 103.1 nm) were achieved. Comparative analysis of therapeutic dosages showed that NLCs allow significantly lower doses – 120 mg/kg body weight for Dxt-NLC and 0.5 mg/kg for Dut-NLC – while maintaining or enhancing efficacy, attributed to their improved bioavailability. These reduced dosages minimise systemic toxicity and side effects, potentially improving patient compliance. This study underscores the clinical advantage of NLCs in PCa treatment by providing an effective and safer alternative to traditional formulations.

PCa induction

The experimental protocol involved administering cyproterone acetate to rats at a dosage of 50 mg/kg for 21 consecutive days, dissolved in corn oil. On the 23^rd day, the rats were given intraperitoneal injections of testosterone propionate at 100 mg/kg in corn oil for the subsequent 3 days. On the 27^th day, a single intraperitoneal injection of N-methyl-N-nitrosourea (NMU) was administered at a 50 mg/kg concentration. Following a 1-week interval from NMU administration, rats were given daily intraperitoneal injections of testosterone propionate at 2 mg/kg in corn oil for the next 125 days.

The rats were divided into five groups, each consisting of 10 animals. Group I acted as the control, while PCa was induced in Group II using a modified version of the method described by Liao et al. (2002). The treatment groups (III: Dxt-NLC at 120 mg/kg body weight, IV: Dut-NLC at 0.5 mg/kg body weight and V: Dxt-NLC at 120 mg/kg body weight + Dut-NLC at 0.5 mg/kg body weight) received their respective treatments alongside a promoting dose of testosterone propionate (2 mg/kg body weight intraperitoneally in corn oil) from the 35^th day to the 160^th day. Animals were sacrificed with ketamine 50 mg/kg of the prostate gland and blood was removed for further estimation.^[18-22] The study was conducted following the guidelines provided by the Institutional Ethical Committee Protocol No: IAEC/KIMS/2018/2.

Tissue processing

The processing of prostate tissue began with the removal of the prostate, which was then washed with ice-cold saline. Ventral prostate tissues were prepared at a 10% w/v concentration by homogenising them in chilled phosphate buffer (0.1 M, pH 7.4) using a homogeniser. The homogenate was filtered through muslin cloth and centrifuged at 3000 × g for 10 min at 4°C using a Remi Cooling Centrifuge (C-24 DL) to eliminate nuclear debris. The resulting supernatant was then subjected to a second centrifugation at 12000 × g for 20 min at 4°C to obtain the post-mitochondrial supernatant (PMS) used for various enzyme assays.

Preparation and estimation of parameters from PMS

The PMS obtained from the above centrifugation steps was utilised for the estimation of various biochemical parameters, including lipid peroxidation (LPO), reduced glutathione (GSH), catalase (CAT) activity, glutathione peroxidase (GPx) activity, glutathione reductase (GR) activity and superoxide dismutase (SOD) activity. Each assay was performed according to established protocols as detailed below.^[23-29]

Estimation of hormone levels in serum

Serum levels of dihydrotestosterone (DHT) and testosterone were assessed using specialised enzyme-linked immunosorbent assay (ELISA) kits, following the guidelines provided by the manufacturer. To ensure the precision and consistency of the results, the assays were conducted in triplicate.^[30]

Estimation of PSA levels in serum

Serum PSA levels were measured using a quantitative sandwich enzyme immunoassay technique. The PSA levels were determined according to the kit manufacturer’s protocol, and the results were expressed as ng/mL of serum.^[31]

Measurement of LPO

LPO in the prostate tissues was assessed by measuring the concentration of malondialdehyde, a by-product of LPO. The tissues were homogenised in phosphate buffer (0.1 M, pH 7.4) at 10% w/v, followed by centrifugation as described earlier. The PMS was mixed with thiobarbituric acid and heated at 95°C for 1 h. The absorbance of the resulting pink colour was measured at 532 nm.^[32]

Estimation of reduced GSH level

GSH levels in the prostate tissues were determined using the method of Jollow et al., with modifications. 1 mL of PMS and 1 mL of 4% sulphosalicylic acid were incubated at 4°C for 1 h. After centrifugation at 1200 × g for 15 min at 4°C, the supernatant was collected, and 0.4 mL was added to 2.2 mL of phosphate buffer (0.1 M, pH 7.4) and 0.4 mL of 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB). The yellow colour developed was measured at 412 nm, and GSH content was calculated as nmol DTNB conjugate formed per gram of tissue.^[33]

Estimation of CAT

CAT activity in prostate tissues was measured according to Claiborne’s (1985) method. The reaction mixture consisted of 1.95 mL of 0.1 M phosphate buffer (pH 7.4), 1.0 mL of 0.019 M hydrogen peroxide and 0.05 mL of PMS. The decrease in absorbance at 240 nm due to the breakdown of hydrogen peroxide was monitored, and CAT activity was calculated as nmol H₂O₂ consumed per minute per milligram of protein.^[34]

Estimation of GPx

GPx activity was assessed using the method described by Mohandas et al. (1984). The reaction mixture contained 1.44 ml of phosphate buffer (0.1 M, pH 7.4), 0.1 mL of 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mL of 1.0 mM sodium azide, 0.05 mL of 1 eu/mL GR, 0.05 mL of 1.0 mM reduced GSH, 0.1 mL of 0.2 mM nicotinamide adenine dinucleotide phosphate (NADPH), 0.01 mL of 0.25 mM hydrogen peroxide (H₂O₂) and 0.1 mL of PMS. The reduction of NADPH was monitored at 340 nm, and GPx activity was expressed as nmol NADPH.^[35]

Estimation of GR activity

GR activity was measured using Carlberg and Mannervik’s method (1975). The reaction mixture included 1.65 mL of phosphate buffer (0.1 M, pH 7.6), 0.1 mL of 0.5 mM EDTA, 0.05 mL of 1.0 mM oxidised GSH, 0.1 mL of 0.1 mM NADPH and 0.1 mL of PMS. The decrease in absorbance due to NADPH oxidation was monitored at 340 nm, and GR activity was calculated as nmol NADPH oxidised per minute per milligram of protein using an extinction coefficient of 6.22 × 10³ M^-1 cm^-1.^[36]

Estimation of SOD activity

SOD activity was determined following the method of Marklund and Marklund (1974). The reaction mixture comprised 2.875 mL of Tris- Hydrochloric acid (HCl) buffer (50 mM, pH 8.5), 100 μL of PMS and pyrogallol (24 mM in 10 mM HCl) to a total volume of 3 mL. The auto-oxidation of pyrogallol was measured at 420 nm, and one unit of SOD activity was defined as the amount that inhibits the auto-oxidation by 50%.^[37]

Estimation of cytokines by ELISA

Cytokine levels in the prostate tissues were quantified using ELISA kits. Tumour necrosis factor alpha (TNF-α) levels were measured with an e-Bioscience sandwich ELISA kit, while interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-gamma (IFN-γ) and interleukin-10 (IL-10) levels were determined using ELISA kits from RAY Biotech, according to the manufacturer’s instructions.^[38]

Histological investigation

For histological analysis, prostate tissues were fixed in freshly prepared 10% neutral buffered formalin at 4°C, followed by embedding in paraffin wax. Vertical sections (5 μm thick) were prepared and rehydrated through a graded series of ethyl alcohol, cleared in xylene and re-embedded in paraffin wax. The sections were then deparaffinised, stained with haematoxylin and eosin (H&E) and mounted in DPX. The histological sections were examined under a microscope (Olympus BX 51) for the assessment of morphological changes, including prostatic intraepithelial neoplasia (PIN), secretory cell proliferation and stromal hyperplasia.^[39,40]

Impact of Dut and Dxt nanostructured lipid carriers on serum hormones

In the PCa-induced group (Group II), there was a statistically significant increase in testosterone levels compared to the control group (Group I) (P < 0.01). Treatment with Dxt NLC in Group III, Dut NLC in Group IV and a combination of Dxt and Dut in Group V resulted in a statistically significant, dose-dependent decrease in testosterone levels (P < 0.05, P < 0.01 and P < 0.001, respectively). Similarly, for DHT serum levels, the PCa-induced group (Group II) showed elevated levels compared to the control group (P < 0.01). Administration of Dxt NLC (Group III), Dut NLC (Group IV) and combination therapy (Group V) resulted in a dose-dependent reduction in DHT levels, with the combination therapy showing the most significant effect (P < 0.001). Elevated serum PSA levels were also observed in the PCa-induced group (Group II) compared to the control (P < 0.01). Treatment with Dxt NLC (Group III), Dut NLC (Group IV) and their combination (Group V) led to substantial and dose-dependent reductions in serum PSA levels, with statistical significance (P < 0.001 for the combination therapy). Confidence intervals for the reductions in testosterone, DHT and PSA levels were calculated, demonstrating robust effects across the treatment groups [Graph 1].

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Graph 1:

Dutasteride and docetaxel nanostructured on impact serum hormone levels. Values are expressed in mean ± standard error of the mean. statistical analysis is done by one-way analysis of variance followed by Bonferroni’s multiple comparison test. ^#P<0.001 when compare with Group I, *P<0.01,**P<0.001 when compared with Group II. DHT: Dihydrotestosterone level, PSA: Prostate-specific antigen level

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Impact of Dut and Dxt nanostructured lipid carriers on antioxidants

The PCa-induced group (Group II) exhibited a significant decline in antioxidant enzyme activities, including GR, GPx and CAT, as well as reduced GSH levels, alongside an increase in LPO (P < 0.01 for all parameters compared to the control group). Administration of Dut and Dxt NLCs in Groups III, IV and V resulted in a statistically significant, dose-dependent restoration of these antioxidant levels to near-normal values (P < 0.05 for GR, GPx and CAT; P < 0.001 for GSH levels and reduction in LPO). Bonferroni’s multiple comparison analysis using confirmed the significance of differences between treatment groups and the PCa-induced group [Table 1].

Impact of Dut and Dxt nanostructured lipid carriers on cytokines and anti-inflammatory cytokine (IL-10) levels

In the PCa-induced group (Group II), there was a significant elevation in pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IFN-γ, accompanied by a decrease in the anti-inflammatory cytokine IL-10 (P < 0.01 for all compared to the control group). Treatment with Dut and Dxt NLCs in Groups III and IV significantly and dose-dependently restored cytokine levels to near-normal values, with a notable increase in IL-10 levels (P < 0.05 for cytokines; P < 0.001 for IL-10). Co-administration of Dut and Dxt NLCs in Group V also exhibited significant restoration of cytokine levels to normal (P < 0.001 for IL-10 restoration) [Graph 2].

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Graph 2:

Dutasteride and docetaxel nanostructured lipid carriers impact on serum cytokine levels. Values are expressed in mean ± standard error of the mean. Statistical analysis is done by one-way analysis of variance followed by Bonferroni’s multiple comparison test. ^#P<0.001 when compared with Group I, *P<0.01,**P<0.001 when compared with Group II. TNF: Tumor necrosis factor, IL: Interleukin

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Impact Dut and Dxt nanostructured lipid carriers on histopathological changes

Histological examination of prostate tissue using H&E staining revealed high-grade PIN with significant secretory cell proliferation and stromal hyperplasia in the PCa-induced group (Group II), compared to the control group. Administration of Dut NLC in Group IV showed low-grade PIN with stromal cell hyperplasia. In contrast, Dxt NLC in Group III demonstrated low-grade PIN with prostate atrophy and acinar degenerative changes. The combination of Dut NLC and Dxt NLC in Group V resulted in the most significant histological improvements, including low-grade PIN, reduced secretory cell proliferation and notable atrophy with degenerative changes in the prostate acini (P < 0.001 for all histopathological changes compared to the PCa-induced group) [Figure 1].

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Figure 1:

Effect of dutasteride and docetaxel nanostructured lipid carriers on histopathology changes. Group I (Control): Normal histoarchitecture of the prostate gland. Well-organized glandular structures with intact epithelial lining. No evidence of neoplasia, cellular proliferation, or inflammatory infiltration. Normal stromal composition without hyperplasia or fibrotic changes. Group II (PCa-Induced Group): High-grade Prostatic Intraepithelial Neoplasia (PIN) observed. Increased secretory cell proliferation with loss of normal glandular architecture. Stromal hyperplasia with an increase in fibroblasts and extracellular matrix deposition. Presence of nuclear pleomorphism, increased mitotic activity, and hyperchromasia. Evidence of inflammatory infiltration and disrupted basement membrane. Group III (DxtNLC Treatment): Low-grade PIN with reduced glandular proliferation. Moderate reduction in stromal hyperplasia, indicating partial recovery. Atrophy of prostate acini, with degenerative changes in epithelial cells. Fewer mitotic figures and reduced nuclear pleomorphism compared to Group II. Group IV (Dut-NLC Treatment): Presence of low-grade PIN, but with less stromal hyperplasia than Group III. Significant reduction in secretory cell proliferation, showing therapeutic impact. Stromal components show moderate fibrosis, but no severe abnormalities. Improved cellular morphology with less nuclear pleomorphism and mitotic activity. Group V (Dxt-NLC + Dut-NLC Co-administration): Marked improvement in histoarchitecture with near-normal glandular structures. Minimal PIN and reduced secretory cell proliferation. Lower stromal hyperplasia compared to other treatment groups. Atrophic and degenerative changes in acinar cells significantly reduced. Enhanced restoration of normal prostate tissue, indicating a synergistic therapeutic effect. Hematoxylin and Eosin (H&E) 400X.

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