MPEG-PCL Nanomicelles Platform for Synergistic Metformin and Chrysin Delivery to Breast Cancer in Mice

Background: Metformin (MET) is a well-known anti-diabetic drug that also has anti-cancer effects. However, high therapeutic doses of MET on cancer cells and the low efficacy of combinatory therapeutic approaches limit its clinical application. Recent studies have shown that chrysin (CHR) can improve the pharmaceutical efficacy of MET by suppressing human telomerase reverse transcriptase (hTERT) and cyclin D1 gene expression. Objective: This study aimed to develop different ratios of methoxy poly(ethylene glycol)-b-poly(e-caprolactone) (MPEG-PCL) micelles for breast cancer to co-deliver a synergistic CHR/MET combination. Methods: CHR/MET drug-loaded micelles were prepared by modified thin-film hydration. Fourier infrared spectrum, gel permeation chromatography, transmission electron microscopy, and high-performance liquid chromatography were used to evaluate the physicochemical properties of nanostructures. Cell proliferation and cell apoptosis were assessed by MTT and Annexin V-FITC/PI double staining method. The gene expression of hTERT and cyclin D1 was measured by real-time PCR assay. A subcutaneous mouse T47D xenograft model was established to evaluate the in vivo efficiency. Results: When the ratio of MPEG-PCL was 1:1.7, the highest drug loading rate and encapsulation efficiency of CHR (11.31±0.37) and MET (12.22±0.44) were observed. Uniform MPEG-PCL micelles of 51.70±1.91 nm allowed MET to incorporate with CHR, which were co-delivered to breast cancer cells. We demonstrated that CHR/MET co-delivery micelles showed a good synergistic effect on inhibiting proliferation in T47D cells (combination index=0.87) by suppressing hTERT and cyclin D1 gene expression. Compared with the free CHR/MET group, the apoptosis rate on T47D cells by CHR/MET nano-micelles significantly improved from 71.33% to 79.25%. The tumour volume and tumour weight of the CHR/MET group increased more slowly than that of the single-drug treatment group (P<0.05). Compared with the CHR/MET group, the tumour volume and tumour weight of the CHR/MET nano-micelle group decreased by 42% and 59%, respectively. Conclusions: We demonstrated that ratiometric CHR/MET micelles could provide an effective technique for the treatment of breast cancer.

Assessment of Physical Performance and Early Treatment Outcomes after Implantation of Modular Prostheses of Femoral and Tibial Shaft

Background. Tumours of bone diaphyses often require resection followed by bone reconstruction. The use of modular pro­sthe­ses permits early limb loading and rapid improvement in physical performance. The aim of this study was to evaluate the func­tioning of patients and early treatment outcomes after the implantation of modular prostheses. The analysis covered the correlation between the extent of resection, physical performance and the number of perioperative complications. Material and methods. 10 patients (5 women and 5 men) with diaphyseal tumours who had modular prostheses implanted were treated at the Orthopaedic Oncology Department in Brzozów between 2014 and 2018. The mean age of the patients was 51.1 years (range: 26-63 years). Functional outcomes were assessed using the MSTS and the Karnofsky scoring system. A VAS was used to evaluate pain intensity. the extent of resection was also analysed, considering bone length and tumour weight. Results. The mean tumour weight was 374g (150-700g). The length of the implants varied from 10 to 25 cm. The mean dura­tion of hospitalisation was 16 days (14-19 days). At 3 months following the surgery, the intensity of pain had decreased from a mean of 6.8 points to 4.2 points (a decrease of 26% from the pre-operative baseline). The MSTS showed improvement of functional performance from a mean of 10.8 points (36%) to 22.9 points (76%). The Karnofsky scores demonstrated an increase in physical performance from 47 to 67 points (20 points on average). Superficial infection of the wound developed in 1 patient. Conclusions. 1. The treatment of diaphyseal tumours with modular prostheses produces good functional outcomes. 2. The extent of the resection and the size of the implant have an effect on the post-operative physical performance of the patients. 3. Pre­operative evaluation of the weight of the tumour may be helpful in predicting the patient’s post-operative functional status. 4. Phy­sical perfor­mance is better after the resection of femoral vs tibial tumours.

Development of an Image-Guided Orthotopic Xenograft Mouse Model of Endometrial Cancer with Controllable Estrogen Exposure

Endometrial cancer (EC) is the most common gynaecological malignancy in Western society and the majority of cases are estrogen dependent. While endocrine drugs proved to be of insufficient therapeutic value in the past, recent clinical research shows promising results by using combinational regimens and pre-clinical studies and identified potential novel endocrine targets. Relevant pre-clinical models can accelerate research in this area. In the present study we describe an orthotopic and estrogen dependent xenograft mouse model of EC. Tumours were induced in one uterine horn of female athymic nude mice using the well-differentiated human endometrial adenocarcinoma Ishikawa cell line—modified to express the luciferase gene for bioluminescence imaging (BLI). BLI and contrast-enhanced computed-tomograph (CE-CT) were used to measure non-invasive tumour growth. Controlled estrogen exposure was achieved by the use of MedRod implants releasing 1.5 μg/d of 17β-estradiol (E2) in ovariectomized mice. Stable E2 serum concentration was demonstrated by LC-MS/MS. Induced tumours were E2 responsive as increased tumour growth was observed in the presence of E2 but not placebo, assessed by BLI, CE-CT, and tumour weight at sacrifice. Metastatic spread was assessed macroscopically by BLI and histology and was seen in the peritoneal cavity, in the lymphovascular space, and in the thoracic cavity. In conclusion, we developed an orthotopic xenograft mouse model of EC that exhibits the most relevant features of human disease, regarding metastatic spread and estrogen dependency. This model offers an easy to manipulate estrogen dosage (by simply adjusting the MedRod implant length), image-guided monitoring of tumour growth, and objectively measurable endpoints (including tumour weight). This is an excellent in vivo tool to further explore endocrine drug regimens and novel endocrine drug targets for EC.

Chronic Glibenclamide Treatment Attenuates Walker-256 Tumour Growth in Prediabetic Obese Rats

Background/Aims: The sulphonylurea glibenclamide (Gli) is widely used in the treatment of type 2 diabetes. In addition to its antidiabetic effects, low incidences of certain types of cancer have been observed in Gli-treated diabetic patients. However, the mechanisms underlying this observation remain unclear. The aim of the present work was to evaluate whether obese adult rats that were chronically treated with an antidiabetic drug, glibenclamide, exhibit resistance to rodent breast carcinoma growth. Methods: Neonatal rats were treated with monosodium L-glutamate (MSG) to induce prediabetes. Control and MSG groups were treated with Gli (2 mg/kg body weight/day) from weaning to 100 days old. After Gli treatment, the control and MSG rats were grafted with Walker-256 tumour cells. After 14 days, grafted rats were euthanized, and tumour weight as well as glucose homeostasis were evaluated. Results: Treatment with Gli normalized tissue insulin sensitivity and glucose tolerance, suppressed fasting hyperinsulinaemia, reduced fat tissue accretion in MSG rats, and attenuated tumour growth by 27% in control and MSG rats. Conclusions: Gli treatment also resulted in a large reduction in the number of PCNA-positive tumour cells. Although treatment did improve the metabolism of pre-diabetic MSG-rats, tumour growth inhibition may be a more direct effect of glibenclamide.

Reproductive Hormones and Their Receptors May Affect Lung Cancer

Background/Aims: In contrast to men, women have experienced a rapid increase in lung cancer mortality. Numerous studies have found that the sex differences in lung cancer are due to reproductive hormones. Experiments in female mice with and without ovariectomy were performed to explore the possible mechanism by which sex hormones (and their receptors) influence lung cancer. Methods: Twenty-four female C57BL/6 mice aged 56-62 days were randomly divided into the ovariectomized group and the control group. In the ovariectomized group, the bilateral ovaries were removed via the dorsal approach, while the control group underwent a sham operation with bilateral ovarian fat resection at the same sites. After 3 weeks of recovery, Lewis lung cancer cells were transplanted into these mice by subcutaneous inoculation of a tumour cell suspension to establish the ovariectomized lung cancer model. Beginning on the 6th day after subcutaneous inoculation, mouse weight and transplanted tumour volume were measured every 3 days. After 3 weeks, all the mice were killed by cervical dislocation, and we measured the tumour weight. Mouse serum and tumour tissues were removed. Then, the serum levels of E2 (oestradiol) and T (testosterone) were detected by ELISA; the protein expression levels of AR (androgen receptor), ERα (oestrogen receptor α) and ERβ (oestrogen receptor β) were detected by Western Blot and IHC (immunohistochemistry); and the mRNA expression levels of AR, ERα and ERβ were detected by qRT-PCR (quantitative real-time polymerase chain reaction) in the ovariectomized and control groups. Results: Compared with the control group, both mouse weight and transplanted tumour volume increased rapidly in the ovariectomized group, and the transplanted tumour weight was significantly heavier in the ovariectomized group (1.83±0.40 and 3.13±0.43, P<0.05). E2 and T serum levels decreased exponentially in the ovariectomized group, while the E2/T ratio increased compared with the control group (E2: 55.88±11.45 and 78.21±9.37; T: 0.82±0.14 and 1.46±0.16; ratio: 69.62±14.43±29.81 and 52.22±5.42; all P<0.05). The Western blot and IHC results indicated that AR, ERα and ERβ protein expression levels were obviously higher in transplanted tumour and lung tissues from the ovariectomized group, with particular increases in ERβ in transplanted tumour tissue and in ERα in lung tissue. The PCR results also showed markedly higher mRNA expression levels of AR, ERα and ERβ in the ovariectomized group, and in particular, ERβ in transplanted tumour tissue and ERα in lung tissue were significantly increased in the ovariectomized group. Conclusion: Ovariectomy decreased E2 and T serum levels and increased the E2/T ratio in mice, and this imbalance in the internal environment promoted the growth of transplanted tumours. Sex hormone disorder not only promoted transplanted tumour growth but also significantly reduced the protein and mRNA expression levels of sex hormone receptors. The metabolism of E2 and T may affect the growth, proliferation and metabolism of lung cancer cells, and the mechanism by which sex hormones and their receptors influence lung cancer is worthy of further research.

Inhibitory effect of STAT3 gene combined with CDDP on growth of human Wilms tumour SK-NEP-1 cells

To investigate the effects of signal transducer and activator of transcription 3 (STAT3) combined with cisplatin (CDDP) on the growth of human Wilms tumour (WT) SK-NEP-1 cell subcutaneous xenografts in nude mice and the possible mechanisms. Human WT SK-NEP-1 cells were subcutaneously transplanted to establish the BALB/c nude mice xenograft model. Mice were randomly divided into five groups: blank control group, adenovirus control group (NC group), STAT3 group, CDDP group and STAT3 plus CDDP group (combination group). Tumour volume and tumour weight were observed during the therapeutic process. The expression levels of STAT3, glucose regulatory protein 78 (GRP78) and BCL2-associated X protein (BAX) were evaluated by immunohistochemical analysis. Compared with the STAT3 group or CDDP group, the tumour weight and volume was significantly reduced in the combination group (P<0.05). No statistical significance was found in NC group compared with the blank control group (P > 0.05). Immunohistochemical analysis showed that STAT3, GRP78 and BAX protein levels in the combination group were significantly higher than those in STAT3 group and CDDP group (P<0.05). Exogenous STAT3 and CDDP may synergistically inhibit the xenograft tumour growth through up-regulation of BAX protein via GRP78.

Tumour nuclear oestrogen receptor beta 1 correlates inversely with parathyroid tumour weight

Primary hyperparathyroidism (PHPT) is a common endocrinopathy, frequently caused by a parathyroid adenoma, rarely by a parathyroid carcinoma that lacks effective oncological treatment. As the majority of cases are present in postmenopausal women, oestrogen signalling has been implicated in the tumourigenesis. Oestrogen receptor beta 1 (ERB1) and ERB2 have been recently identified in parathyroid adenomas, the former inducing genes coupled to tumour apoptosis. We applied immunohistochemistry and slide digitalisation to quantify nuclear ERB1 and ERB2 in 172 parathyroid adenomas, atypical adenomas and carcinomas, and ten normal parathyroid glands. All the normal parathyroid glands expressed ERB1 and ERB2. The majority of tumours expressed ERB1 (70.6%) at varying intensities, and ERB2 (96.5%) at strong intensities. Parathyroid carcinomas expressed ERB1 in three out of six cases and ERB2 in five out of six cases. The intensity of tumour nuclear ERB1 staining significantly correlated inversely with tumour weight (P=0.011), and patients whose tumours were classified as ERB1-negative had significantly greater tumour weight as well as higher serum calcium (P=0.002) and parathyroid hormone levels (P=0.003). Additionally, tumour nuclear ERB1 was not expressed differentially with respect to sex or age of the patient. Levels of tumour nuclear ERB2 did not correlate with clinical characteristics. In conclusion, decreased ERB1 immunoreactivity is associated with increased tumour weight in parathyroid adenomas. Given the previously reported correlation with tumour-suppressive signalling, selective oestrogen receptor modulation (SERMs) may play a role in the treatment of parathyroid carcinomas. Future studies of SERMs and oestrogen treatment in PHPT should consider tumour weight as a potential factor in pharmacological responsiveness.

Growth Inhibition of Kirkman-Robbins Hepatoma by l-(l,3-Dihydroxy-2-propoxymethyl)-5,6-tetramethyIeneuraciI and Possible Mechanism of its Biological Activity

Hepatoma, Acyclonucleoside Metabolism , dN and dTMP Kinases. dNTP Pool l-(l,3-D ihydroxy-2-propoxym ethyl)-5,6-tetram ethylene-uracil (DHPTU) is a newly synthe­ sized acyclonucleoside which shows cytostatic properties. It was tested in Syrian hamster 6 days after heterotransplantation of Kirkm an-Robbins hepatoma. A reduction of tumour weight by 61% was found 48 h after its intraperitoneal (i.p .) administration in doses of 20 mg per kg of body weight. Inhibition of tumour growth is accompanied by a reduction of dThd, dGuo and dTMP kinase activities in tumourcytosol (by 91% and 74% and 55% , respectively) and decrease in contents of dTTP, dGTP and dA TP (by 92% , 77% and 67% , respectively) in dNTP pool. DHPTU is not phosphorylated by any tumour dN kinases, but undergoes cleavage with TU release in reaction catalyzed by the tumour cell enzyme, com petitively inhibited by FA . After [14C]DHPTU or [14C]TU had been given i.p. to the animals with the tumour, 90% of the subcellu­lar fraction labelling fell into the nuclear fraction. How ever, if [l4C ]DHPTU was administered with FA and DCF, 27% of radioisotope was found in the nuclear fractions and 68% in cytosol. Since D C F which prevented FA deamination to FB (which is not an inhibitor of the mentioned enzyme) reduces DHPTU-induced changes in activity of dN kinases and dTMP kinase in hepato­ ma cells, the cytostatic activity of DHPTU seems to be connected to an enzyme which releases TU from DHPTU .