Jayalakshmi Pailoor

King cobra (Ophiophagus hannah) venom L-amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumor growth in a tumor xenograft mouse model.

King cobra (Ophiophagus hannah) venom L-amino acid oxidase (OH-LAAO), a heat stable enzyme, has been shown to exhibit very potent anti-proliferative activity against human breast and lung tumorigenic cells but not in their non-tumorigenic counterparts. We further examine its in vitro and in vivo anti-tumor activity in a human prostate adenocarcinoma (PC-3) model. OH-LAAO demonstrated potent cytotoxicity against PC-3 cells with IC50 of 0.05 µg/mL after 72 h incubation in vitro. It induced apoptosis as evidenced with an increase in caspase-3/7 cleavages and an increase in annexin V-stained cells. To examine its in vivo anti-tumor activity, we treated PC-3 tumor xenograft implanted subcutaneously in immunodeficient NU/NU (nude) mice with 1 µg/g OH-LAAO given intraperitoneally (i.p.). After 8 weeks of treatment, OH-LAAO treated PC-3 tumors were markedly inhibited, when compared to the control group (P <0.05). TUNEL staining analysis on the tumor sections showed a significantly increase of apoptotic cells in the LAAO-treated animals. Histological examinations of the vital organs in these two groups showed no significant differences with normal tissues, indicating no obvious tissue damage. The treatment also did not cause any significant changes on the body weight of the mice during the duration of the study. These observations suggest that OH-LAAO cytotoxic effects may be specific to tumor xenografts and less to normal organs. Given its potent anti-tumor activities shown in vitro as well as in vivo, the king cobra venom LAAO can potentially be developed to treat prostate cancer and other solid tumors.

Enzymatic and toxinological activities of Hypnale hypnale (hump-nosed pit viper) venom and its fractionation by ion exchange high performance liquid chromatography

Hypnale hypnale (hump-nosed pit viper) has been recently identified as one of the medically important venomous snakes in Sri Lanka and on the southwestern coast of India. The characterization of its venom is essential for understanding the pathophysiology of envenomation and for optimizing its management. In the present study, the biological properties of Hypnale hypnale venom and venom fractions obtained using Resource Q ion exchange chromatography were determined. The venom exhibited toxic activities typical of pit viper venom, comparable to that of its sister taxon, the Malayan pit viper (Calloselasma rhodostoma). Particularly noteworthy were its high activities of thrombin-like enzyme, proteases, phospholipase A2, L-amino acid oxidase and hyaluronidase. The thrombin-like enzyme was mainly acidic and distributed over several chromatography fractions, indicating its existence in multiple isoforms. The hemorrhagic and necrotic activities of the venom were likely associated with the proteolytic enzyme found mainly in the basic fraction. Phospholipase A2 and phosphomonoesterase exist in both acidic and basic isoforms, while L-amino acid oxidase and hyaluronidase are highly acidic. The venom clotting activity on fibrinogens showed distinct species specificity in the following increasing order for clotting time: bovine < rabbit < goat < human < horse < < dog, and was comparable to that of C. rhodostoma venom. Its clot formation on human fibrinogen is gradual and prolonged, a phenomenon suggestive of consumptive coagulopathy as a complication observed clinically. At an intramuscular sublethal dose, the venom did not cause acute kidney injury in a rodent model, contrary to the positive control group treated with Daboia russelii venom. Nephrotoxicity may result from higher venom doses in the context of coagulopathy, as a complication provoked by venom hematoxicity.

Effect of palm oil carotene on breat cancer tumorigenicity in nude mice

Biological therapies are new additions to breast cancer treatment. Among biological compounds, β-carotene has been reported to have immune modulatory effects, in particular, enhancement of natural killer cell activity and tumor necrosis factor-alpha production by macrophages. The objective of this study was to investigate the effect of palm carotene supplementation on the tumorigenicity of MCF-7 human breast cancer cells injected into athymic nude mice and to explore the mechanism by which palm carotenes suppress tumorigenesis. Forty-eight 4-wk-old mice were injected with 1×106 MCF-7 cells into their mammary fat pad. The experimental group was supplemented with palm carotene whereas the control group was not. Significant differences were observed in tumor incidence (P<0.001) and tumor surface area and metastasis to lung (P<0.005) between the two groups. Natural killer (NK) cells and B-lymphocytes in the peripheral blood of carotene-supplemented mice were significantly increased (P<0.05 and P<0.001, respectively) compared with controls. These results suggest that palm oil carotene is able to modulate the immune system by increasing peripheral blood NK cells and B-lymphocytes and suppress the growth of MCF-7 human breast cancer cells.

Plexiform angiomyxoid myofibroblastic tumour of the stomach

Sir, We report a single case of plexiform angiomyxoid myofibroblastic (PAM) tumour of stomach, a rare entity. This is only the third case reported in the literature. This condition was first described by Takahashi et al. in 2007. A 23-year-old female presented in early August 2008 with a five day history of passing dark coloured stool. There was no history of abdominal pain. Investigations showed normal peripheral blood film, but a low haemoglobin level of 9.4 gm/dL. Her chest X-ray was normal. Computed tomography (CT) scan of the abdomen showed a large mass in the gastric antrum measuring about 4.86 7.2 cm. Gastroscopy revealed a tumour mainly in the wall of the stomach with focal ulceration of the mucosa. She underwent partial gastrectomy. Post-operative recovery was uneventful. She remained well 2 months following surgery. Gross examination of the stomach showed a polypoidal growth in the lesser curvature measuring 8.06 4.0 cm (Fig. 1). Cut section showed a solid tumour mainly in the submucosa, exhibiting mucoid and haemorrhagic areas. A few enlarged lymph nodes were noted along the greater curvature. Histological examination showed tumour tissue extending from the submucosa up to the serosa of the stomach. The tumour exhibited a plexiform pattern (Fig. 2). The tumour cells were spindle-shaped, with no significant nuclear atypia or mitosis. The stroma comprised of myxoid matrix and was rich in small calibre blood vessels (Fig. 3). The lymph nodes showed reactive changes. Immunohistochemistry showed that the tumour cells were positive for vimentin and smooth muscle actin (SMA) (Fig. 4). A few tumour cells were positive for desmin. They were negative for CD34, S-100 protein and c-kit. Based on the histological features, and supported by the immunostain findings, a diagnosis of PAM was made. Takahashi et al. first reported two cases of gastric tumour which were characterised by plexiform growth pattern, bland spindle cells and myxoid stroma rich in blood vessels. These two unusual tumours were named plexiform angiomyxoid myofibroblastic tumour. In their study, the patients were 50 and 68 years old and the tumours were 4.0–4.5 cm in largest dimension. Our patient was younger (only 23 years old) and the tumour was larger, measuring 8 cm in largest dimension. The histogenesis of PAM is debatable, but it is thought to be of myofibroblastic origin based on the immunohistochemical findings such as positivity for SMA and negativity for desmin, as well as ultrastructural findings. In our case, the tumour was diffusely positive for actin and only focally positive for desmin, indicating focal differentiation towards smooth muscle. Although the clinical follow-up data are minimal in our case, the histological features indicate a benign lesion. The differential diagnoses for spindle cell tumour of the stomach include gastrointestinal stromal tumour (GIST), leiomyoma/sarcoma, inflammatory fibroid polyp and inflammatory myofibroblastic tumour and fibromyxoma. GIST is composed of spindle or epithelioid cells. More than 85% of GISTs are immunoreactive for c-kit. The FIG. 1 The stomach is opened to show a polypoidal growth, measuring 8.06 4.0 cm, situated in the lesser curvature. The tumour has a glistening mucoid and haemorrhagic appearance.

Low prevalence of ‘classical’ microscopic colitis but evidence of microscopic inflammation in Asian Irritable Bowel Syndrome patients with diarrhoea

BackgroundThere is increasing evidence for the role of microscopic inflammation in patients with IBS. We aimed to examine the prevalence of microscopic colitis and inflammation in Malaysian IBS patients with diarrhoea (IBS-D).MethodsConsecutive patients who met the Rome III criteria for IBS-D and asymptomatic controls were prospectively recruited. Colonoscopy was performed in all study subjects and systematic biopsies taken from all segments of the colon. The diagnosis of lymphocytic colitis and collagenous colitis was made using previously defined criteria. Patients with post infectious IBS were excluded.Results120 subjects (74 IBS-D, 46 controls) were recruited during the study period. In the IBS-D group, the colonoscopic (macroscopic) findings were as follows; normal findings n = 58 (78.4%), diverticula disease n = 5 (6.8%), diminutive polyps n = 9 (12.2%) and haemorrhoids n = 2(2.7%). No subject under the age of 40 had any significant findings. Microscopically, there was only one case (1.3%) with histology consistent with collagenous colitis. However, the IBS-D patients had a higher prevalence of moderate microscopic inflammation (n = 11, 14.9%) compared to controls (n = 1, 2.2%) (p = 0.005).Conclusions‘Classical’ microscopic colitis is uncommon in Malaysian patients with IBS-D but a significant number of adults showed evidence of microscopic inflammation.

Clinical Prognostic Factors and Survival Outcome in Renal Cell Carcinoma Patients - A Malaysian Single Centre Perspective

BACKGROUND This study concerns clinical characteristics and survival of renal cell carcinoma (RCC) patients in University Malaya Medical Centre (UMMC), as well as the prognostic significance of presenting symptoms. MATERIALS AND METHODS The clinical characteristics, presenting symptoms and survival of RCC patients (n=151) treated at UMMC from 2003-2012 were analysed. Symptoms evaluated were macrohaematuria, flank pain, palpable abdominal mass, fever, lethargy, loss of weight, anaemia, elevated ALP, hypoalbuminemia and thrombocytosis. Univariate and multivariate Cox regression analyses were performed to determine the prognostic significance of these presenting symptoms. Kaplan Meier and log rank tests were employed for survival analysis. RESULTS The 2002 TNM staging was a prognostic factor (p<0.001) but Fuhrman grading was not significantly correlated with survival (p=0.088). At presentation, 76.8% of the patients were symptomatic. Generally, symptomatic tumours had a worse survival prognosis compared to asymptomatic cases (p=0.009; HR 4.74). All symptoms significantly affect disease specific survival except frank haematuria and loin pain on univariate Cox regression analysis. On multivariate analysis adjusted for stage, only clinically palpable abdominal mass remained statistically significant (p=0.027). The mean tumour size of palpable abdominal masses, 9.5±4.3cm, was larger than non palpable masses, 5.3±2.7cm (p<0.001). CONCLUSIONS This is the first report which includes survival information of RCC patients from Malaysia. Here the TNM stage and a palpable abdominal mass were independent predictors for survival. Further investigations using a multicentre cohort to analyse mortality and survival rates may aid in improving management of these patients.

Epidermal growth factor receptor mutations in non- small cell lung cancers in a multiethnic malaysian patient population.

BACKGROUND Mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in non- small cell lung cancer (NSCLC) are predictive of response to EGFR-targeted therapy in advanced stages of disease. This study aimed to determine the frequency of EGFR mutations in NSCLCs and to correlate their presence with clinical characteristics in multiethnic Malaysian patients. MATERIALS AND METHODS In this prospective study, EGFR mutations in exons 18, 19, 20 and 21 in formalin-fixed paraffin-embedded biopsy specimens of consecutive NSCLC patients were asessed by real-time polymerase chain reaction. RESULTS EGFR mutations were detected in NSCLCs from 55 (36.4%) of a total of 151 patients, being significantly more common in females (62.5%) than in males (17.2%) [odds ratio (OR), 8.00; 95% confidence interval (CI), 3.77-16.98; p<0.001] and in never smokers (62.5%) than in ever smokers (12.7%) (OR, 11.50; 95%CI, 5.08-26.03; p<0.001). Mutations were more common in adenocarcinoma (39.4%) compared to non-adenocarcinoma NSCLCs (15.8%) (p=0.072). The mutation rates in patients of different ethnicities were not significantly different (p=0.08). Never smoking status was the only clinical feature that independently predicted the presence of EGFR mutations (adjusted OR, 5.94; 95%CI, 1.94- 18.17; p=0.002). CONCLUSIONS In Malaysian patients with NSCLC, the EGFR mutation rate was similar to that in other Asian populations. EGFR mutations were significantly more common in female patients and in never smokers. Never smoking status was the only independent predictor for the presence of EGFR mutations.

Granular cell tumour: malignant or benign?

INTRODUCTION Granular cell tumours (GrCTs) are uncommon soft tissue tumours that are usually benign (approximately 0.5%-2.0% have been reported as malignant). They are very rarely found at the extremities. Differentiating a malignant GrCT from a benign one is important as the former is aggressive and has a poor prognosis, whereas the latter, after surgical resection, has excellent outcomes. A malignant lesion can be suspected on clinical presentation and confirmed via histopathological examination using the Fanburg-Smith criteria. METHODS This was a retrospective review of all cases of GrCT of the extremities that presented to the Orthopaedic Oncology Unit of University Malaya Medical Centre, Malaysia, from September 2006 to March 2013. RESULTS There were a total of five cases, all of which involved female patients aged 13-40 (mean age 24) years. Three cases involved the upper limbs and two involved the lower limbs. Using the Fanburg-Smith criteria, three cases were classified as benign, one as atypical and one as malignant. Wide local excision was performed in all five cases and the outcomes were excellent except for the patient with a malignant tumour. That patient presented with lung metastasis about three months after surgery. CONCLUSION Malignant and benign GrCTs can be differentiated on clinical presentation and by using the Fanburg-Smith criteria. We believe that wide local excision is the best treatment for both benign and malignant tumours. The role of adjuvant chemotherapy and radiotherapy in malignant GrCTs should be studied. All patients with GrCTs should receive follow-up to check for recurrence and metastasis.

Nutritional composition, antioxidant properties, and toxicology evaluation of the sclerotium of Tiger Milk Mushroom Lignosus tigris cultivar E

The Tiger Milk Mushroom (Lignosus spp.) is an important medicinal mushroom in Southeast Asia and has been consumed frequently by the natives as a cure for a variety of illnesses. In this study, we hypothesized that Lignosus tigris (cultivar E) sclerotium may contain high nutritional value and antioxidant properties, is nontoxic and a potential candidate as a dietary supplement. The chemical and amino acid compositions of the sclerotium were evaluated and antioxidant activities of the sclerotial extracts were assessed using ferric reducing antioxidant power; 1,1-diphenyl-2-picrylhydrazyl; and superoxide anion radical scavenging assays. Acute toxicity of the L. tigris E sclerotium was assessed using a rat model study. The sclerotium was found to be rich in carbohydrate, protein, and dietary fibers with small amounts of fat, calories, and sugar. The amino acid composition of the protein contains all essential amino acids, with a protein score of 47. The sclerotial extracts contain phenolics, terpenoids, and glucan. The ferric reducing antioxidant power values of the various sclerotial extracts (hot water, cold water, and methanol) ranged from 0.008 to 0.015 mmol min(-1) g(-1) extract, while the 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radical scavenging activities ranged from 0.11 to 0.13, and -2.81 to 9.613 mmol Trolox equivalents g(-1) extract, respectively. Acute toxicity assessment indicated that L. tigris E sclerotial powder was not toxic at the dose of 2000 mg kg(-1). In conclusion, L. tigris E sclerotium has the potential to be developed into a functional food and nutraceutical.

Sub-Acute Toxicity Study of Tiger Milk Mushroom Lignosus tigris Chon S. Tan Cultivar E Sclerotium in Sprague Dawley Rats.

Lignosus also known as “Tiger Milk Mushroom,” is classified in the family Polyporaceae and mainly consumed for its medicinal properties in Southeast Asia and China. The sclerotium is known as the part with medicinal value and often used by the natives to treat a variety of ailments. Lignosus tigris Chon S. Tan, one of the species of the Malaysia Tiger Milk mushroom, has recently been successfully cultivated in laboratory. Earlier studies have demonstrated the L. tigris cultivar E sclerotia exhibited beneficial biomedicinal properties. This study evaluated the potential toxicity of L. tigris E sclerotia in a 28-day sub-acute oral administration in Sprague Dawley (SD) rats. L. tigris E sclerotial powder was administered orally at three different doses of 250, 500, and 1000 mg/kg to the SD rats once daily, consecutively for 28-days. Body weight of the rats was recorded and general behavior, adverse effects, and mortality were observed daily throughout the experimental period. At the end of the experiment, blood hematology and biochemistry, relative organ weights, and histopathological analysis were performed. Results showed that there were no mortality nor signs of toxicity throughout the 28-day sub-acute toxicity study. Oral administration of the L. tigris E sclerotial powder at daily dose up to 1000 mg/kg had no significant effects in body weight, relative organ weight, blood hematological and biochemistry, gross pathology, and histopathology of the organs. L. tigris E sclerotial powder did not cause any treatment-related adverse effect in the rats at different treatment dosages up to 1000 mg/kg. As the lethal dose for the rats is above 1000 mg/kg, the no-observed-adverse-effect level (NOAEL) dose is more than 1000 mg/kg.