Mechanism of rutin mediated inhibition of insulin amyloid formation and protection of Neuro-2a cells from fibril-induced apoptosis.

Many metabolic and neurodegenerative diseases are associated with protein misfolding and aggregation. Insulin a key hormone, under certain conditions aggregates and forms pathological amyloid fibrils. Several polyphenols have been studied extensively to elucidate their inhibitory effect on amyloid formation. In the present study, we used insulin as an amyloid model to test the mechanism and efficacy of rutin as an anti-amyloidogenic molecule. By using electron microscopy, dynamic light scattering and circular dichroism spectroscopy, we show that rutin inhibits the insulin aggregate and fibril formation. Further, rutin interacts with insulin directly and inhibits fibril formation in a dose-dependent manner as demonstrated by micro scale thermophoresis experiments. The molecular docking study predicted the potential binding pocket of rutin at the interface of chain A and chain B of insulin thereby preventing it from forming the aggregates. Since, rutin is a natural anti-oxidant, we studied its role in diminishing amyloid fibril induced cytotoxicity and apoptosis. Rutin, decreases the insulin amyloid fibrils-induced Neuro-2a cytotoxicity by reducing reactive oxygen species (ROS) levels which in turn downregulates Bax and upregulates Bcl-2 and pBad proteins. These findings suggest the potential action of rutin in preventing protein misfolding, cell death, and serves as a lead structure to design novel anti-amyloidosis compounds.

Evaluation of a polymorphism in MYBPC3 in patients with anthracycline induced cardiotoxicity.

Cardiotoxicity is the most serious side effect of anthracyclines (doxorubicin, daunorubicin or epirubicin). The incidence of anthracycline induced late cardiac toxicity (AIC) that is overt clinically is 3-5% in the Indian population. Polymorphism in intron 32 (deletion of 25bp) of MYBPC3 has been shown to be present exclusively in Asians and more so in South India (3-8%). The frequency of the polymorphism is significantly higher (13%) in patients with cardiomyopathy in India. Fifteen patients were identified to have cardiac dysfunction following treatment for malignant lymphoma with doxorubicin containing regimens. Peripheral blood DNA from control, amplified by polymerase chain reaction yielded a 467bp fragment while in the presence of the 25bp deletion only a 442bp fragment was detected. To confirm the presence or absence of the polymorphism, amplified DNA was restricted using Bgl1 in all samples. Bgl1 restricted amplified DNA only if the 25bp deletion was absent. A 467 base pair band was observed in all the 15 samples, which suggested the absence of polymorphism in MYBPC3. In a sample of DNA from a patient with a deletion in exon 33 (confirmed by sequencing) a 442bp fragment was detected. Amplified DNA from this patient was not restricted with Bgl1. Wild type MYBPC3 when amplified gave a distinct restriction banding pattern consisting of two bands of 401bp and 66bp. Amplified DNA from all peripheral blood samples restricted with Bgl1 suggesting the absence of the polymorphism. In this preliminary report, MYBPC3 does not seem to play a role in anthracycline induced cardiotoxicity.

ß-carotene at physiologically attainable concentration induces apoptosis and down-regulates cell survival and antioxidant markers in human breast cancer (MCF-7) cells.

Although ß-carotene is known for its anti-carcinogenic and antioxidant properties, a few recent epidemiological and experimental evidence show that at higher concentration it acts as pro-oxidant and induces cancer. Since the global burden of breast cancer exceeds all other types of cancer, and its incidence rates is also in increasing trend, the present study attempted to evaluate the anti-cancer molecular mechanism of ß-carotene (at 1 µM concentration) isolated from Spinacia oleracea in human breast cancer (MCF-7) cells. The carotenoid was purified by open column chromatography and identified by LC-MS. The anti-proliferative effect of ß-carotene at different concentrations was evaluated by WST-1 assay and the changes in cell morphology were examined by microscopic observation. The induction of apoptosis by ß-carotene was observed by DAPI staining and colorimetric caspase-3 assay. The expression of cell survival, apoptotic, and antioxidant marker proteins was measured by western blot analysis. Purified ß-carotene inhibited the viability of MCF-7 cells in a dose-dependent manner, which was well correlated with changes in cell morphology. Increased apoptotic cells were observed in ß-carotene (1 µM)-treated cells. This apoptosis induction was associated with increased caspase-3 activity. The protein expression studies showed that ß-carotene at 1 µM concentration effectively decreases the expression of the anti-apoptotic protein, Bcl-2 and PARP, and survival protein, NF-kB. It also inhibited the activation of intracellular growth signaling proteins, Akt and ERK1/2. The inhibition of Akt activation by ß-carotene results in decreased phosphorylation of Bad. Further, it down-regulated antioxidant enzyme, SOD-2, and its transactivation factor (Nrf-2), and endoplasmic reticulum (ER) stress marker, XBP-1, at protein levels. These findings exhibit the key role of ß-carotene even at a low physiological concentration in MCF-7 cells which further explains its predominant anti-cancer activity.

Long-term outcome of diffuse large B-cell lymphoma: Impact of biosimilar rituximab and radiation.

INTRODUCTION: Rituximab (R)-CHOP improves survival over CHOP in diffuse large B-cell lymphoma (DLBCL). The availability of biosimilar rituximab in India has increased access of this drug. We report on the impact of treatment on outcomes with special emphasis on the impact of biosimilar rituximab and radiation. METHODS: Outcomes of adults (age 15-60 years) treated with CHOP+/- Rituximab radiation were analyzed retrospectively to look at baseline features, treatment, and event-free and overall survival (EFS and OS). RESULTS: In the period 2000-2013, 444 patients (median age 47 years: 15-60; males: 288 [65%]; Stage III/IV: 224 [50%]; age-adjusted international prognostic index [aaIPI] Score 2 or 3 in 50%) received either CHOP (n = 325 [73%]) or RCHOP (n = 119 [27%]) therapy. Biosimilar rituximab and the original were used in 95 (80%) and 24 (20%) patients, respectively. Radiation was given in 134 (30%) patients (Stages I and II, 100/220 [45%] and Stages III and IV, 34/224 [15%]). After a median follow-up of 46 (0.2-126) months, the 5-year EFS and OS were 59% and 68%, respectively. The factors predicting inferior EFS and OS were age> 40 years, performance status 2-4, Stage III/IV, hemoglobin <12 g/dL, the aaIPI Score 2 or 3, and nonuse of rituximab and radiation. Radiation used in early stage disease benefitted all subgroups regardless of bulky disease, use of rituximab, or the number of cycles of chemotherapy. Addition of rituximab improved survival across all categories of aaIPI. CONCLUSION: Availability of biosimilar rituximab has increased access and survival of patients with DLBCL in India. Radiotherapy improved outcomes in early stages.

Pediatric nasopharyngeal carcinoma: Experience from a tertiary cancer center in India.

BACKGROUND: Pediatric nasopharyngeal carcinomas (NPCs) are rare tumors. There is paucity of data on outcomes in pediatric NPC from developing countries. AIM: The present study was conducted to ascertain the outcomes of children with NPC at our center. SETTINGS AND DESIGN: A retrospective analysis of case records of pediatric NPC patients treated at our hospital was performed. PATIENTS AND METHODS: We analyzed the outcomes of 37 consecutive patients <18 years of age with pediatric NPC treated between 2000 and 2015. Patients were treated with concurrent chemoradiotherapy (CTRT) with cisplatin and 5-fluorouracil (5-FU) or CTRT with cisplatin, followed by adjuvant chemotherapy with cisplatin and 5-FU. STATISTICAL ANALYSIS: Survival was analyzed using Kaplan-Meier method, and log rank test was used to compare variables. RESULTS: The median duration of follow-up was 36.6 months. The median age of the patients was 15 years, and 22/37 (59%) patients were male. The most common presenting complaint was neck mass (70%), followed by nasal bleeding (16%). The distribution of Stage I, II, III, and IV patients was 1/37 (3%), 2/37 (6%), 13/37 (35%), and 21/37 (57%), respectively. Distant metastasis at presentation was seen in 3/37 patients. Complete response was seen in 32/37 (86%) patients. The 3-year event-free survival (EFS) for the entire cohort of patients was 60.1%. No significant difference in EFS was observed with age, gender, stage, use of 5-FU with CTRT, and nutritional status. CONCLUSION: Majority of patients with pediatric NPC present with advanced stage disease at our center. No difference in outcomes was seen with the two schedules of CTRT. Local control could be achieved in majority of patients; however, distant metastasis was the most common reason for relapse.

Phase II study of interim PET-CT-guided response-adapted therapy in advanced Hodgkin's lymphoma.

BACKGROUND: Combination chemotherapy ABVD (doxorubicin, bleomycin, vinblastine and dacarabazine) cures ∼70% of patients with advanced Hodgkin's lymphoma (aHL, stages IIB, III and IV) while more toxic escalated BEACOPP (EB, combination of bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisolone) increases cure rates to 85%. Patients with a positive interim positron emission tomography-computerized tomography (PET-CT) scan after two cycles (PET-2) of ABVD have very poor outcomes with continued ABVD. Intensifying therapy with EB in PET-2-positive patients ('response-adapted therapy') may improve cure rates, whereas the negative patients can continue ABVD alone. PATIENTS AND METHODS: Eligible patients with newly diagnosed aHL received two cycles of ABVD and underwent PET-2 (scored with semi-quantitative 5-point visual criteria, 'Deauville score'). PET-2-negative patients continued four additional cycles of ABVD, whereas PET-2-positive patients received four cycles of EB. A phase II sample size of 50 was estimated keeping the lower and higher proportion of rejection of the event-free survival (EFS) as 70% and 85%, respectively. RESULTS: Fifty patients [median age 28 (12-60) years; male : female: 39 : 11; stages: IIB-3 (6%), III-29 (58%) and IV-18 (36%); International Prognostic Score (IPS): 0-3: 34 (68%); 4-7: 16 (32%)] were enrolled; 49 underwent PET-2. Eight (16%) were PET-2-positive, whereas 41 (84%) were negative. Forty-seven were evaluable for EFS and all 50 for overall survival (OS). The 2-year EFS was 76% (95% CI: 68-83) and OS was 88% (95% CI: 82-94). PET-2 was strongly prognostic-2-year EFS, negative versus positive: 82% versus 50%; P = 0.013. CONCLUSION: PET-2 response-adapted strategy could not achieve EFS of 85% in aHL. However, escalated therapy improved outcomes in PET-2-positive patients compared with historical data. TRIAL REGISTRATION: CTRI/2012/06/002741 (http://www.ctri.nic.in) and NCT01304849 (http://www.clinicaltrials.gov).

Nutritional profile of pediatric cancer patients at Cancer Institute, Chennai.

BACKGROUND: Malnutrition is widely prevalent in the pediatric population in India. There is paucity of data on the prevalence of malnutrition in pediatric cancer patients and the impact of cancer treatment on nutritional status of Indian children. AIMS: The study was conducted to look at the prevalence of malnutrition and assess the impact of treatment on nutritional status of pediatric cancer patients. SETTINGS AND DESIGN: This was a retrospective study. MATERIALS AND METHODS: Data on the weight of pediatric cancer patients <16 years of age treated at Cancer Institute, Chennai, from January 2013 to May 2014 were analyzed at systematic time points in therapy. Patients' weight were plotted on the Centre for Disease Control (CDC) growth charts. Patients were defined to be undernourished if their weight for age was ≤3rd centile in CDC growth charts and obese if their weight for age was ≥97th centile on CDC growth charts. RESULTS: A total of 295 patient case records were analyzed. Acute lymphoblastic leukemia was the most common malignancy. At diagnosis, under-nutrition was seen in 44% patients, this increased to 46% midway during treatment (end of induction in acute leukemia and completion of 50% of planned treatment in solid tumors) and decreased to 27% at the end of treatment (beginning of maintenance in acute leukemia and completion of planned treatment in solid tumors) (P = 0.0005). There was no significant difference in nutritional status between patients with hematological malignancies and solid tumors (P = 0.8). CONCLUSION: Under-nutrition is present in close to half of the pediatric cancer patients presenting to our institute. Active nutritional intervention and education were able to significantly reduce the prevalence of under-nutrition in patients at the end of treatment.

Bloodstream infections in pediatric patients at Cancer Institute, Chennai.

BACKGROUND: There is paucity of data on the epidemiology of bloodstream infections in pediatric cancer patients from India. Rationale use of antibiotics in febrile neutropenia is important for reducing morbidity and preventing the emergence of drug resistant bacteria. AIMS: The study was conducted to look at the prevalence of bloodstream bacterial infection and the antibiotic resistance profile at Cancer Institute, Chennai. SETTINGS AND DESIGN: This was a retrospective study. MATERIALS AND METHODS: Data on all blood cultures taken from pediatric cancer patients treated at Cancer Institute, Chennai, during the year 2013 were analyzed. The microbiological profile and sensitivity pattern were analyzed. RESULTS: A total of 1045 blood culture samples were taken, and there were 82/1045 (7.5%) positive blood cultures. Gram-negative organisms accounted for 50/82 (61%) of all positive cultures. Klebsiella pneumoniae (32%) was the most common Gram-negative isolate, and Staphylococcus aureus (93.5%) was the most common Gram-positive. There was high resistance to aminoglycosides and beta-lactam/beta-lactamase inhibitor antibodies. CONCLUSION: Gram-negative organisms are the predominant bacteria isolated. There is high resistance to first-line combination antibiotics used as empiric therapy for treatment of febrile neutropenia.

Factors predicting outcome in high risk febrile neutropenia in patients receiving intensive chemotherapy for acute sleukemia: A prospective, observational study from South India.

BACKGROUND: Outcome of febrile neutropenia (FN) in acute leukemia patients undergoing intensive chemotherapy from India is scanty. MATERIALS AND METHODS: A prospective, observational, single institutional study was conducted to evaluate the clinical features, microbiological aspects, risk factors influencing the outcome of high risk FN during intensive therapy in acute leukemia. RESULTS: Among 115 febrile episodes, though 94 (81.7%) had indwelling central venous catheter (CVC) at the time of diagnosis of FN, infective foci clinically were identified in 70.4% of episodes, with lung as the major site (25.2%) followed by CVC (17.4%). Microbiological documentation was possible in 33% (n = 40) episodes. Gram-negative bacteria isolates were 58.3% and Gram-positive isolates were 41.7% of which Pseudomonas was the predominant Gram-negative and Staphylococcus aureus was the most common Gram-positive isolate. Piperacillin-tazobactam + amikacin were used as first line antibiotic in 93% episodes and second line antibiotics were necessary in 73% episodes. Granulocyte colony stimulating factor was used in 60.9% episodes of high risk FN mostly in acute myeloid leukemia consolidation patients. Eighteen episodes (15.7%) were assigned to have invasive fungal disease. Eleven (9.6%) out of 115 high risk FN had a fatal outcome. Presence of pulmonary infection predicted for fatal outcome (P = 0.02). CONCLUSION: This study reports the outcome of high risk FN in patients with acute leukemia undergoing intensive chemotherapy. Gram-negative isolates are highly sensitive to piperacillin-tazobactum and hence in a cost restraint scenario, carbapenems needs to be judiciously used. Focus of Infection in lungs during FN predicted higher fatal outcomes.

Preliminary experience with the use of bendamustine: a peculiar skin rash as the commonest side effect.

BACKGROUND AND OBJECTIVES: Bendamustine has been recently approved for the treatment of low-grade lymphoproliferative disorders. There is little data on the effectiveness or toxicity of this drug outside the trial setting. This is the first report on the use of bendamustine from the Indian subcontinent. SETTINGS AND DESIGN: Retrospective descriptive analysis of response and side effects of bendamustine in eight patients with chronic lymphocytic leukemia and eight patients with follicular lymphoma. METHODS: Data was collated from a review of case records. We examined any association between side effects and clinical parameters. RESULTS: The median age of patients was 52 years and three-quarters had received prior treatment with alkylators or fludarabine. Three different protocols of bendamustine were used (single agent, in combination with rituximab or in combination with vincristine and prednisolone). The overall response rate was 80% (47% complete response, 33% partial response, and 20% progressive disease). The drug was well tolerated with very few grade 3/4 toxicities. More than half the patients (9/16) developed a characteristic erythematous, papular skin rash that resolved after completion of chemotherapy. CONCLUSION: Bendamustine is a safe and useful addition to the drug arsenal against lymphoproliferative disorders. A peculiar skin rash was the commonest side effect noted in Indian patients treated with this drug.

p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors.

We propose that the apoptotic function of p53 has an important role in B-cell homeostasis, which is important for the prevention of B-cell lymphomas. We created a mouse model (mDeltapro) that lacked residues 58-88 of the proline-rich domain of p53. mDeltapro is defective for apoptosis, but is able to arrest cell-cycle progression in hematopoietic tissues. mDeltapro develops late-onset B-cell lymphoma, but not the thymic T-cell tumors found in p53-null mice. Interestingly, mDeltapro lymphomas comprised incorrectly differentiated B cells. B-cell irregularities were also detected in mDeltapro before tumor onset, in which aged mice showed an increased population of inappropriately differentiated B cells in the bone marrow and spleen. We predict that by keeping B-cell populations in check, p53-dependent apoptosis prevents irregular B cells from eventuating in lymphomas.

Initial characterization of hydrogen sulfide effects in female sexual function.

INTRODUCTION: In our male animal models, hydrogen sulfide (H(2)S) displayed significant vasodilatory and smooth muscle relaxant effects suggestive of an endogenous physiological role in erectile process. AIM: In this first exploratory study, we aimed to identify the existence and mechanism of H(2)S pathway in female sexual physiology. METHODS: Vaginal and clitoral cavernosal smooth muscle strips from New Zealand white rabbits (N = 12) were exposed to stable H(2)S donor, sodium hydrosulfide hydrate (NaHS.xH(2)O, 100 microM-1.6 mM), in isometric tension studies. The NaHS responses were repeated after incubations with (i) N(omega)-nitro-L-arginine (50 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 microM) or cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine (MDL 12,330A) (10 microM); and (ii) potassium chloride medium (high K(+) 60 mM/low K(+) 10 mM), tetraethylammonium (10 mM) or glibenclamide (100 microM). Relaxant effect of NaHS was also compared with those of nitroglycerine (0.18-78.2 microM) and sildenafil (0.084-25.3 microM). Additionally, samples (N = 16) were collected for estimations of plasma and tissue H(2)S and expression levels of cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) proteins. MAIN OUTCOME MEASURES: In vitro evidences for H(2)S formation and its physiopharmacological effects. RESULTS: NaHS produced significant concentration-dependent relaxation of vaginal and cavernosal smooth muscles with inhibitions by combination of ODQ and MDL 12,330A (26.4%), N(omega)-nitro-L-arginine (22.2%), high K(+) (15.1%) or glibenclamide (10.1%). Based on molar potency, NaHS was 18.3 and 6.3 times weaker than nitroglycerine and sildenafil, respectively. Quantitative assays indicated that plasma H(2)S level was 16.5 +/- 2.58 microM, and H(2)S was synthesized in the clitoral and vaginal smooth muscles (1.8 and 3.9 nmol/mg soluble protein compared with 26.5 nmol/mg in the liver: positive control). Similarly, western blotting identified the protein expression bands of CSE (44.5 kDa) and CBS (63 kDa) in these genital tissue samples. CONCLUSION: These pilot studies clearly indicate the smooth muscle relaxant effect of H(2)S in female genital tract, mediating through cyclic adenosine 3':5'-monophosphate, nitric oxide-cyclic guanosine monophosphate and K(+)(ATP) channels. Taken together with biochemical and molecular evidences for endogenous formation, H(2)S pathway could be a contributing factor in female sexual responses.