Analysis of ROCK2 activation in transgenic mouse skin carcinogenesis

The purpose of this study was to investigate ROCK2 activation in squamous cell carcinogenesis and assess its co-operation with rasHa and fos oncogene activation together with loss of PTEN mediated AKT regulation. The analysis of ROCK deregulation with these genes in the MAP Kinase and PI3K pathways, two of the most commonly deregulated signalling systems, employed a well-characterised, transgenic mouse skin model of multi-stage carcinogenesis. A major goal was to study co-operation between these genes in the conversion of benign tumours to malignancy and investigate subsequent progression to aggressive carcinomas, given these are the most significant clinical stages of carcinogenesis from a patient’s viewpoint; and also investigated effects of ROCK2 deregulation on the processes of normal epidermal differentiation. ROCK2 is an effector protein of RhoA, which is a member of the ras superfamily and ROCK2 activation has been associated with tumour progression via an increase in tissue stiffness mediated by changes in actomyosin cytoskeleton leading to increased cellular motility. Thus, ROCK2 expression is commonly associated with the later events in cancer. Furthermore, there are many studies investigating ROCK2 in cancer given that it may be a useful therapeutic target in being downstream of oncogenic ras signalling. Yet, relatively few studies have explored the possibilities of a definite link that confirms the co-operation status of ROCK2 activation with ras/MAPK/fos and /or PTEN/PI3K/AKT pathways in SCC aetiology. Thus, questions exist as to exactly when does ROCK2 activation become causal; and what are the collaborating genes involved in the mechanism that drive the early or late stage events in carcinogenesis. To begin to answer these questions, inducible ROCK2 activation has been introduced into a well-characterised transgenic mouse skin carcinogenesis model that expressed a combination of ras and fos activation, driven by a modified human keratin 1 vector (HK1). Thus, exclusive epidermal expression of activated rasHa and fos oncogenes, in proliferative basal layers, gave hyperplasia and papillomatogenesis; but with no evidence of spontaneous malignant conversion. This stability of phenotype is thus ideal to assess roles for multiple transgene co-operations in the development of benign tumours and their conversion to malignancy. Hence, ROCK transgenic mice that expressed a conditionally active, 4-hydroxytamoxifen (4-HT)-regulated of human ROCK2 transgene were crossed with mice expressing activated rasHa and /or fos exclusively in epidermal transit amplifying keratinocytes (HK1.ras, HK1.fos). Inducible PTEN tumour suppressor gene mutation via exon 5 ablation (K14.Cre/D5PTENflx) and thus loss of AKT regulation was also incorporated into this model. This was achieved via deletion of exon 5 employing the RU486-mediated cre/loxP system, driven by keratin K14 promoter expression in basal layer keratinocytes. Therefore, to facilitate this investigation, a new and unpublished inducible ROCK2 system was employed in order to target the identical keratinocytes as PTEN loss. This new transgenic line of lsl.ROCKer transgenic mice employed the same 4-HT inducible ROCKer transgene but now driven by a generic CAG promoter following cre mediated ablation of the stop cassette once treated with RU486. In bi-genic K14.ROCKer/HK1.ras1205 mice, synergism between ROCK2 activation and (wound-promotion) sensitive HK1.ras1205 line showed direct co-operation and achieved malignant conversion of benign papillomas to well-differentiated squamous cell carcinoma (wdSCCs) histotypes (12 weeks of 4-HT treatment). This placed ROCK2 activity as the causal event driving malignant conversion, but in the absence of a wound promotion stimulus (loss of ear tag), papillomas did not convert. The correct papilloma context was required for ROCK to become causal proved to be the case on employing the wound insensitive HK1.ras1276 line. Here, K14.ROCKer/HK1.ras1276 mice failed to exhibit any papillomas and required the constitutive promotion stimulus from additional fos activation. Interestingly, following cessation of 4-HT, two intriguing observations were recorded. Firstly, that once bi-genic ROCK/ras1205 achieved malignancy, exogenous ROCKer expression appeared to show no involvement once squamous cell carcinomas (SCCs) progressed to poorly differentiated squamous cell carcinomas (pdSCCs), given the elevated expression of endogenous ROCK upon malignant progression. Secondly, the rapid growth of papilloma appeared upon cessation of 4-HT with highly intense p21 expression indicated the requirement of exogenous ROCK2 for malignant conversion and the possibility of a papilloma inhibition by 4-HT anti-cancer activity. Another major novel finding demonstrated ROCK2 activation could act as an initiator in co-operation with fos activation. Direct co-operation between ROCK2 and fos activation produced benign squamous papillomas yet, whereas ROCK2 activation alone induced hyperplasia as did fos activation alone at this time point, given papilloma formation in HK1.fos mice occur over 4-5 months. However, unlike deregulation of MAP Kinase signalling in bi-genic ROCK/ras1205 mice, in bi-genic ROCK/fos mice, no malignant conversion was observed due to high levels of compensatory p53/p21 expression. Thus, this bi-genic K14.cre/lsl.ROCKer/HK1.fos model suggests the requirement of additional mutation event for malignant conversion. An unexpected result appeared in bi-genic ROCK/Δ5PTENflx co-operation experiments where K14.cre/lsl.ROCKer/Δ5PTENflx cohorts exhibited epidermal hyperplasia with folded papillomatous appearance to the epidermis, but without papillomatogenesis even after seven month of period. This either indicates a weak co-operation between ROCK2 and Δ5PTENflx which may be due to the unexpected low levels of p-AKT from a compensatory increased p21 expression; and additional events needed to fill in the oncogenic gap in this bi-genic ROCK/Δ5PTENflx model, or may possibly highlight redundancy in the oncogenic hits provided by ROCK and PTEN. This latter suggest similar links exist between their normal roles in the epidermis which may be accountable for the alterations observed in keratinocyte differentiation. In both in vitro and in vivo experiments, K14.cre/lsl.ROCKer/Δ5PTENflx cohorts showed alterations in epidermal differentiation via anomalous K1 and low levels of K6 expression. Interestingly, activated ROCK2 appeared to induce or accelerate differentiation activity in K14.cre/lsl.ROCKer keratinocytes via increased K1 (early differentiation marker) and reduced K6 (proliferation marker) expression profiles. These results were consistent with in vivo data where K6 was expressed in low levels in K14.cre/lsl.ROCKer hyperplasia histotypes. In contrast, in K14.cre/lsl.ROCKer/Δ5PTENflx keratinocytes, inactivation of PTEN-mediated AKT activity may be accountable for restored keratin K6 and anomalous keratin K1 expression; as keratin K1 was expressed in a similar fashion of normal keratinocytes in K14.cre/lsl.ROCKer/fos keratinocytes. Interestingly, all tri-genic cohorts: K14.cre/lsl.ROCKer/ras1276/fos, K14.cre/lsl.ROCKer/fos/Δ5PTENflx and K14.cre/lsl.ROCKer/ras1276/Δ5PTENflx synergisms exhibited malignant conversion and /or malignant progression in all animals highlighting a novel role of ROCK2 activation. Further, the stage-specific consequences in each model in this study were shown to be influenced by p53/p21 status, where typically p53 expression disappeared in late stage papillomas yet, p21 expression persisted. This demonstrated the importance of compensatory p53/p21 expression in modulating tumour pathogenesis in all these models. Given that this study incorporated PTEN mutation, the influence of AKT activity was investigated in the SCC progression of tri-genic ROCK/ras1276/PTENflx and ROCK/fos/PTENflx cohorts; revealing a crucial antagonism between p21 and AKT. However, this study revealed that the malignancy in tri-genic ROCK/ras1276/fos cohorts was not influenced by p-AKT expression, and as this tri-genic model achieved wdSCCs only. This suggests that as the roles of ROCK in altering cytoskeletal organisation leading to increase in tissue stiffness are overlaid onto both MAP Kinase and AKT deregulation, the outcome is a very aggressive pdSCC. Thus, suggesting ROCK signalling to be a potential therapeutic target for ras/MAPK/fos pathway in carcinogenesis. Overall, this study showed the involvement of ROCK2 activation in the initiation stage for papillomatogenesis with fos oncogene, and demonstrated ROCK2 as a converter again and also in malignant progression with ras/fos/Δ5PTENflx mutations. This indicates the link of ROCK2 signalling with both MAPK and PI3K pathways, thus targeting ROCK2 would aid in development of cancer therapy

Total Sulfated Glycosaminoglycan (GAG) From Malaysian Sea Cucumbers Stichopus Hermanni And Stichopus Vastus And Its Effects On Wound Healing In Rats

Sea cucumbers have long been exploited as a source of medicinal compounds due to the presence of sulfated glycosaminoglycans (GAGs). The aim of this study was to investigate the occurrence of total sulfated GAG from the integument body wall, the visceral internal organs and the coelomic fluid of Malaysian sea cucumbers Stichopus hermanni and Stichopus vastus and evaluate the effect of total sulfated GAG on wound healing in rats using macroscopic and microscopic evaluations

Biochemical and histological effects of low dose of monosodium glutamate on the liver of adult male Sprague-Dawley rats

Monosodium glutamate (MSG) is widely used as an additive in food. Excess consumption of MSG was reported to cause oxidative stress on brain, liver and renal resulted in increased production of reactive oxygen species (ROS). This study aims to determine the biochemical and histological effects of low dose MSG on the liver of adult male Sprague-Dawley rats. Animals (n = 6 per group) were randomly divided into three groups with two treatment groups: 60 mg/kg (MSG60) and 120 mg/kg (MSG120), and one control group (distilled water). The substances were administered to the rats via force feeding for 28 consecutive days. On day 29, all rats were killed, and liver tissues were biopsied for the biochemical (total protein, liver enzymes, and the status of oxidative stress) and histological analysis. The total protein appeared significantly decreased (p < 0.05) while alanine aminotransferase (ALT) and aspartate aminotransferase (AST) demonstrated a significant increased (p < 0.05) in the MSG120 treatment group as compared to the control group. Malondialdehyde (MDA) levels and the antioxidant levels of superoxide dismutase (SOD) were significantly increase (p < 0.05) in the MSG120 group as compared to the MSG60 and control groups. The histological findings revealed changes to normal liver architecture and accumulation of red blood cells in the central veins in both MSG groups. This study indicates that the MSG consumption at a dose of 120 mg/kg may ALTer the biochemical and histological parameters of the liver

Evaluating physical and biological characteristics of glutaraldehyde (GA) cross-linked nano-biocomposite bone scaffold

In vivo stability of biomaterial-based bone scaffolds often present a significant drawback in the development of materials for tissue engineering purpose. Previously developed nanobiocomposite bone scaffold using alginate and nano cockle shell powder has shown ideal characteristics. However, it showed high degradation rate and reduced stability in an in vivo setting. In this study, we aim to observe the effect of cross-linking glutaraldehyde (GA) in three different concentrations of 0.5%, 1% and 2% during the fabrication process as a potential factor in increasing scaffold stability. Microstructure observations of scaffolds using scanning electron microscope (SEM) showed all scaffolds crossed linked with GA and control had an ideal pore size ranging from 166.8-203.5 μm. Increase in porosity compared to the control scaffolds was observed in scaffolds cross-linked with 2% GA which also presented better structural integrity as scored through semi-quantitative methods. Tested pH values during the degradation period showed that scaffolds from all groups remained within the range of 7.73-8.76. In vitro studies using osteoblast showed no significant changes in cell viability but a significant increase in ALP enzyme levels in scaffold cross-linked with 2% GA. The calcium content released from all scaffold showed significant differences within and between the groups. It can be concluded that the use of GA in the preparation stage of the scaffold did not affect the growth and proliferation of osteoblast and use of 2% GA showed improved scaffold structural integrity and porosity

Combination effect of argan oil and low frequency electromagnetic field on open wound in mice

This study was conducted to evaluate the effect of argan oil with the exposure of low frequency electromagnetic field (EMF) on open wound healing in mice. Eighteen male mice (20-40 g) were divided into three groups: phosphate buffer saline (PBS) as negative control, solcoseryl gel as positive control, and argan oil with the exposure of low frequency EMF, 1.2 mT (treatment group). Full thickness wounds (4 mm diameter) were induced on the shaved dorsal of the mouse. All mice were sacrificed on day 12 after the final treatment. Macroscopic observation, wound contraction rate, histopathology analysis and total protein content were examined in this study. Results showed that wounds treated with argan oil and exposed to low frequency EMF has a significant increase in wound contraction rate (p < 0.05) and total protein content (p < 0.05). Moreover, histopathological analysis on the wound tissues displayed complete re-epithelization with thick and dense collagen fibers in the argan oil with low frequency EMF exposure treated group. In conclusion, topical treatment of argan oil with low frequency EMF exposure yield a better healing progress and showed the ability to accelerate wound healing

Low dose monosodium glutamate induced oxidative damage and histopathological changes on the renal of male rats

Monosodium glutamate (MSG) is a flavour enhancer commonly used in processed food to increase palatability. Several studies have reported that chronic exposure of MSG causes renal fibrosis via oxidative stress mechanism. However, till date, the effects of low dose of MSG on the oxidative stress status and its histopathological observation of renal are still unclear. A total of 18 male Sprague Dawley rats (170 – 200 g) were divided randomly into three groups consisted of the control (received distilled water = 1 ml/kg), MSG 60 (received 60 mg/kg MSG) and MSG 120 (received 120 mg/kg MSG) groups. All of the substances were given via force-feed oral for 28 consecutive days. At the end of the study, all rats were sacrificed and the renal were isolated for biochemical and histological evaluation. The superoxide dismutase (SOD) activity and protein carbonyl (PC) level showed significantly increased (p < 0.05) in MSG 60 and MSG 120 group compared to the control group. However, no significant difference was found in glutathione (GSH) and malondialdehyde (MDA) level in all treated groups. The histology observation showed glomerulus shrinkage in MSG 60 and MSG 120 groups. In conclusion, these findings confirmed low dose of MSG-induced oxidative stress and histopathological changes on the renal of male Sprague-dawley rats. Accordingly, care must be taken on the intake of MSG in our daily basis

The effects of oleuropein on apoptotic rate and oxidative stress profiles during tumour promotion stage in the mouse skin carcinogenesis model

Oleuropein is a phenolic compound that can be abundantly found in the olive plant and it possesses pharmacological properties including anticancer, antioxidant, and anti-inflammatory. This present study was designed to determine the effects of oleuropein on tumour promotion stage, particularly on the histopathological changes, apoptotic rates and oxidative stress profiles by using the mouse skin carcinogenesis model. Female ICR mice were randomly divided into 3 groups (n= 8 mice per group) as follows: Control induced with DMBA/TPA, negative control (acetone) and oleuropein-treated groups. For the treatment group, the mice were initiated with DMBA (200 nmol) followed by pre-treatment with oleuropein (10 mg/kg) and subsequent promotion with TPA (20 nmol). The treatments were topically applied on the shaved dorsal up to 10 weeks. Histopathology analysis showed that oleuropein-pretreated group appeared lack of thickness in epidermal hyperplasia, as compared to thick hyperplasia and epidermal disorganisation in the DMBA/TPA control group. Data also showed that oleuropein pre-treatment resulted in a significant increase of the apoptotic rates (p<0.05) as indicated by the activated caspase-3 labelling compared to DMBA/TPA group. Interestingly, the level of MDA is significantly reduced (p<0.05) in the oleuropein pre-treated group compared to DMBA/TPA group. Next, pre-treatment of oleuropein caused a significant decrease in the GSH levels (p<0.05) along with a significant increase in the SOD levels (p<0.05) compared to the DMBA/TPA group. Overall, this study indicates that oleuropein may act as a potential chemopreventive agent through its apoptotic and antioxidant defence activities on tumour promotion stage in skin carcinogenesis event

Potential Photochemopreventive Effect of Fatty Acids and Terpenoid Rich Leaf Extract of Canarium odontophyllum Miq. on UVB-induced Immortalized Human Keratinocytes (HaCaT) Skin Cancer Model.

Keratinocyte carcinoma is found in skin areas which are often exposed to the sun and a variety of natural products has been developed as a chemoprevention agent. One example is the Canarium odontophyllum Miq, or “Dabai”, which is an indigenous plant to Borneo, Sarawak. Fatty acids & terpenoid-rich extract from the leaf were obtained via extraction using hexane. FRAP assay showed antioxidant capacity for both 500 & 1000 µg/ml extract but not significantly different between doses. Untreated and treated immortalized human keratinocytes (HaCaT) were irradiated with UVB for 6 passages to a cumulative of 180 mJ/cm2 UVB. Findings showed 1000 ug/ml of TRCO significantly reduced p53 expression compared to the untreated group. Both 500 & 1000 µg/ml of TRCO significantly reduced the expression of Ki67 compared to the untreated group. Antioxidant and oxidative stress markers measurement revealed 500 µg/ml of TRCO significantly increased superoxide dismutase activity compared to the untreated group, both 500 & 1000 µg/ml TRCO significantly reduced catalase, glutathione peroxidase, glutathione S-transferase, and protein carbonyls compared to the untreated group. Reduced glutathione peroxidase activity is potentially due to depletion in glutathione by the UVB and extract. In vitro evaluations of TRCO on UVB-induced HaCaT skin cancer model revealed photochemopreventive properties. These promising findings validate further evaluation of C. odontophyllum Miq leaf extract as a potential therapeutic agent

GC-MS ANALYSIS OF TERPENOIDS FROM LEAVES OF Canarium odontophyllum Miq.(DABAI)

Terpenoids are defined as secondary metabolites with carbon backbone molecular structures consisting of isoprene (2-methylbuta-1, 3-diene) units. They demonstrate important biological activities, such as antibacterial, antiviral, antimalarial, antiinflammatory, anticancer and cholesterol synthesis inhibition activities. Canarium odontophyllum Miq. or locally known as “dabai” is an endemic plant in Sarawak, Malaysia. Its leaf compositions were examined by using the GC-MS analysis in order to compare and contrast their volatile terpenoids constituents. The terpenoids content were 36.67% and 14% for hexane and ethanol extracts, respectively. nHexadecanoic acid, phytol and octadecanoic acid were the major terpenoids constituents from the leaves of C. odontophyllum Miq. n-Hexadecanoic acid (20.22%), phytol (8.74%) and octadecanoic acid (7.54%) were found to be predominant in the hexane extract, while phytol(21.02%) and n-hexadecanoic acid (14.52%) were major constituents in the ethanol extract. The C.odontophyllum Miq. leaf constituents are also related to their biological activities and would offer promising therapeutic effects. Further investigation should be conducted to develop it as apotential therapeutic drug

Fatty acids and terpenoids from Canarium odontophyllum MIQ. Leaf and their antioxidant and cytotoxic effects on UVB-induced immortalized human keratinocytes cells (HACAT)

The study evaluated the antioxidant capacity of hexane extract of Canarium odontophyllum Miq. leaf; its fatty acids and terpenoids content; and cytotoxic effects on UVB-induced human keratinocytes (HaCaT). FRAP assay was used to determine antioxidant capacity. GC-MS analysis to identify the fatty acids and terpenoids’ in the hexane extract of Canarium odontophyllum Miq. leaf. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out to measure the cytotoxic effects of the extract on UVB-induced human keratinocytes. Serial doses of up to 1000 µg/mL extract were administered before UVB irradiation of the cells. FRAP assay showed the extract was found to exhibit antioxidant activity but no significant difference in ascorbic acid equivalent antioxidant capacity (AEAC) between dose 500 µg/mL (5.00 ± 0.35 AEAC) and 1000 µg/ mL (5.70 ± 0.29 AEAC) extract. GC-MS analysis showed the extract contained 88.93% of fatty acids and terpenoids, especially n-hexadecanoic acid, spathulenol, and phytol. MTT assay showed no IC50 value for the tested extract dose on UVB-induced HaCaT. Thus, the results suggest the potential application of hexane extract of C. odontophyllum Miq. leaf in terpenoids’ studies. In-depth research and isolation of compounds of interest should be done to develop it as a viable medical phytotherapeutic agent