In vitro cytotoxicity assay of D-limonene niosomes: an efficient nano-carrier for enhancing solubility of plant-extracted agents.

The low solubility of the plant-extracted agent like D-limonene in cancer therapy is a critical problem. In this study, we prepared D-limonene-loaded niosomes (D-limonene/Nio) for cancer therapy through in vitro cytotoxicity assay of HepG2, MCF-7, and A549 cell lines. The niosomal formulation was prepared by film hydration technique with Span® 40: Tween® 40: cholesterol (35:35:30 molar ratio) and characterized for vesicle distribution size, morphology, entrapment efficiency (EE%), and in vitro release behaviour. The obtained niosomes showed a nanometric size and spherical morphology with EE% about 87 ± 1.8%. Remarkably prolonged release of D-limonene from niosomes compared to free D-limonene observed. The loaded formulation showed significantly enhanced cytotoxic activity with all three cancer cell lines (HepG2, Macf-7 and A549) at the concentration of 20 µM. These results indicated that niosome loaded with phytochemicals can be a promising nano-carrier for cancer therapy applications.

Diosgenin-loaded niosome as an effective phytochemical nanocarrier: physicochemical characterization, loading efficiency, and cytotoxicity assay.

BACKGROUND: The use of phytochemicals to prevent or suppress tumours is known as chemoprevention. Numerous plant-derived agents have been reported to have anticancer potentials. As one such anticancer phytochemical, diosgenin has several applications which are nevertheless limited due to its low solubility in water. METHODS: We loaded diosgenin into niosome to increase its solubility and hence efficiency. Diosgenin-niosome (diosgenin loaded into niosome) was prepared by thin-film hydration method and characterised by optical microscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), and UV-visible spectrophotometry. Also, loading efficiency, in vitro drug release, and cytotoxicity assay were performed on HepG2 cell line. RESULTS AND DISCUSSION: Diosgenin-niosome has a nanometric size with a normal size distribution and spherical morphology. The loading efficiency of diosgenin was about 89% with a sustainable and controllable release rate. Finally, the viability of free diosgenin was 61.25%, and after loading into niosomes, it was improved to 28.32%. CONCLUSION: The results demonstrated that niosomes increase the solubility of naturally derived hydrophobic chemicals and thus enhance their anticancer effect. Graphical abstract.

Neuroprotective and antihyperalgesic effects of orexin-A in rats with painful diabetic neuropathy.

AIM OF STUDY: Diabetes mellitus is related to the development of neuronal tissue injury in different peripheral and central nervous system regions. A common complication of diabetes is painful diabetic peripheral neuropathy (PDN). We have studied the neuroprotective and anti-nociceptive properties of neuropeptide orexin-A in an animal experimental model of diabetic neuropathy. METHODS: All experiments were carried out on male Wistar rats (220-250 g). Diabetes was induced by a single intraperitoneal injection of 55 mg/kg (i.p.) streptozotocin (STZ). Orexin-A was chronically administrated into the implanted intrathecal catheter (0.6, 2.5 and 5 nM/L, daily, 4 weeks). The tail-flick and rotarod treadmill tests were used to evaluate the nociceptive threshold and motor coordination of these diabetic rats, respectively. Cleaved caspase-3, Bax, Bcl2 and the Bax/Bcl-2 ratio, as the biochemical indicators of apoptosis, were investigated in the dorsal half of the lumbar spinal cord tissue by western blotting method. RESULTS: Treatment of the diabetic rats with orexin-A (5 nM/L) significantly attenuated the hyperalgesia and motor deficit in diabetic animals. Furthermore, orexin-A (5 nM/L) administration suppressed pro-apoptotic cleaved caspase-3 and Bax proteins. Also, orexin-A (5 nM/L) reduced the expression of Bax/Bcl-2 ratio in spinal cord dorsal half of rats with PDN. CONCLUSIONS: Altogether our data suggest that the orexin-A has anti-hyperalgesic and neuroprotective effects in rats with PDN. Cellular mechanisms underlying the observed effects may, at least partially, be related to reducing the neuronal apoptosis.

Effect of a Copper (II) Complex on The Induction of Apoptosis in Human Hepatocellular Carcinoma Cells

Objectives: In the present study, we aimed to identify the anti-proliferative potential of [Cu(L)(2imi)] complex [L = 2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato) and 2imi = 2-methyl imidazole] against HepG2 cells as an in vitro model of human hepatocellular carcinoma and normal mouse fibroblast L929 cells. Methods: The cytotoxic and apoptotic effects of [Cu(L)(2imi)] complex on HepG2 cells and normal fibroblasts (L929) were examined by MTT assay and flow cytometry, respectively. Results: Cytotoxicity induced by [Cu(L)(2imi)] complex was time dependent. Also, there was a positive correlation between cytotoxicity and an increase in Cu complex concentration. For HepG2 cells, the cell viability percentage was 50% at 58 µg/mL after 24 h treatment, whereas in the same concentration and conditions, the viability percentage was surprisingly higher (about 100%) for L929 cells. Also, after 48 h treatment, the viability percentage of HepG2 cells at 55 µg/mL concentration was 50% in contrast with 89.3% for L929 cells in the same conditions. Flow cytometry findings suggest that [Cu(L)(2imi)] complex is capable of decreasing cancer cell viability through apoptosis and did not efficiently activate the necrosis process. Conclusions: Finally, we found that [Cu(L)(2imi)] complex possess the potential for development as an anti-cancer drug for human hepatocellular carcinoma.

The cytotoxicity effects of a novel Cu complex on MCF-7 human breast cancerous cells.

A variety of biological activities, such as anti-microbial and anti-tumor properties was reported for 1,10-phenanthroline and its copper complexes. In this study, the anti-proliferative activity of a novel  [Cu(L)(phen)] complex was investigated on MCF-7 breast cancer cells using MTT assay. Since chemotherapy is lake of ability to distinguish between normal cells from cancerous cells, therefore we also investigated the effect of  [Cu(L)(phen)] complex on normal L929 cells. The results showed that following 24 and 48 h exposure of cells with  [Cu(L)(phen)] complex, the IC50 values for MCF-7 were significantly lower than that recorded for L929 and normal cells were less sensitive than cancerous cells to the complex. Additionally, the  [Cu(L)(phen)] complex displayed a time- and concentration-dependent cytotoxic response, with MCF-7 and L929 cells. Also flow cytometry findings suggest that  [Cu(L)(phen)] complex is capable of decreasing cancer cell viability through apoptosis and did not efficiently activate the necrosis process.

Induction of cell proliferation, clonogenicity and cell accumulation in S phase as a consequence of human UBE2Q1 overexpression.

Ubiquitination is an important cellular mechanism with a pivotal role in the degradation of abnormal or short-lived proteins and the regulation of cell cycle and cell growth. The ubiquitin-proteasome pathway is altered in multiple types of human malignancies, including colorectal cancer (CRC). The alteration in the expression of the novel human gene ubiquitin-conjugating enzyme E2 Q1 (UBE2Q1), as a putative member of the E2 ubiquitin-conjugating enzyme family, has been reported in several malignancies, including carcinoma of the breast, hepatocellular and colorectal cancer, and pediatric acute lymphoblastic leukemia. In the present study, the effect of UBE2Q1 overexpression on cell growth, clonogenicity, motility and cell cycle was investigated in a CRC cell line. The UBE2Q1 gene was cloned in the pCMV6-AN-GFP expression vector. A series of stable transfectants of SW1116 cells overexpressing UBE2Q1 protein were established and confirmed by fluorescence microscopy and western blotting. Using these cells, MTT assay was performed to evaluate cell growth and proliferation, while crystal violet staining was used for clonogenicity assay. Cell cycle analysis was also performed to survey the ratio of cells accumulated in different phases of the cell cycle upon transfection. The motility of these cells was also studied using wound healing assay. UBE2Q1 transfectants exhibited a faster growth in cell culture, increased colony formation capacity and enhanced motility compared with control non-transfected cells and cells transfected with empty vector (mock-transfected cells). UBE2Q1 overexpression also resulted in a significant decrease in the number of cells accumulated in the G0/G1 phase of the cell cycle. The present findings suggest that UBE2Q1 may function as an oncogene that induces proliferation of cancer cells, and could be a novel diagnostic tool and a potential therapeutic target for CRC.

MGMT-B gene promoter hypermethylation in patients with inflammatory bowel disease - a novel finding.

Inflammatory bowel disease (IBD) is a disease strongly associated with colorectal cancer (CRC) as a well-known precancerous condition. Alterations in DNA methylation and mutation in K-ras are believed to play an early etiopathogenic role in CRC and may also an initiating event through deregulation of molecular signaling. Epigenetic silencing of APC and SFRP2 in the WNT signaling pathway may also be involved in IBD-CRC. The role of aberrant DNA methylation in precancerous state of colorectal cancer (CRC) is under intensive investigation worldwide. The aim of this study was to investigate the status of promoter methylation of MGMT-B, APC1A and SFRP2 genes, in inflamed and normal colon tissues of patients with IBD compared with control normal tissues. A total of 52 IBD tissues as well as corresponding normal tissues and 30 samples from healthy participants were obtained. We determined promoter methylation status of MGMT-B, SFRP2 and APC1A genes by chemical treatment with sodium bisulfite and subsequent MSP. The most frequently methylated locus was MGMT-B (71%; 34 of 48), followed by SFRP2 (66.6 %; 32 of 48), and APC1A (43.7%; 21 of 48). Our study demonstrated for the first time that hypermethylation of the MGMT-B and the SFRP2 gene promoter regions might be involved in IBD development. Methylation of MGMT-B and SFRP2 in IBD patients may provide a method for early detection of IBD-associated neoplasia.

Inhibitory effects of Cyrtopodion scabrum extract on growth of human breast and colorectal cancer cells.

Breast and colorectal cancers rank high in Iran as causes of mortality. Most of the current treatments are expensive and non-specific. The potential anticancer properties of common home gecko, Cyrtopodion scabrum, were investigated in this study. The effects of C. scabrum extract on proliferation, viability and migration of the colorectal cancer (SW-742), breast cancer (MCF-7) and normal (MSC) cell lines were investigated using MTT and in vitro wound healing assay. IC50 values calculated for the extract were 559±28.9 µg/ml for MCF-7 and 339±11.3 µg/ml for SW-742. No toxic effects on the normal control cells were observed. MCF-7 and SW-742 cell growth was inhibited by 32.6% and 62%, under optimum conditions, compared to the untreated control cells. The extract also decreased the motility and migration ability of both cancer cell lines, with no significant effects on the normal control cells. Data suggest C. scabrum extract as a useful natural resource for targeting cancer cells specifically.