Mitochondrial ribosomal small subunit (MRPS) MRPS23 protein-protein interaction reveals phosphorylation by CDK11-p58 affecting cell proliferation and knockdown of MRPS23 sensitizes breast cancer cells to CDK1 inhibitors.

BACKGROUND: Post-translational modification of some mitoribosomal proteins has been found to regulate their functions. MRPS23 has been reported to be overexpressed in various cancers and has been predicted to be involved in increased cell proliferation. Furthermore, MRPS23 is a driver of luminal subtype breast cancer. However, its exact role and function in cancer remains unknown. METHODS AND RESULTS: Our previous study identified protein-protein interactions involving MRPS23 and CDK11A. In this study, we confirmed the interaction of MRPS23 with the p110 and p58 isoforms of CDK11A. Phosphoprotein enrichment studies and in vitro kinase assay using CDK11A/cyclin D3 followed by MALDI-ToF/ToF analysis confirmed the phosphorylation of MRPS23 at N-terminal serine 11 residue. Breast cancer cells expressing the MRPS23 (S11G) mutant showed increased cell proliferation, increased expression of PI3-AKT pathway proteins [p-AKT (Ser47), p-AKT (Thr308), p-PDK (Ser241) and p-GSK-3ß (Ser9)] and increased antiapoptotic pathway protein expression [Bcl-2, Bcl-xL, p-Bcl2 (Ser70) and MCL-1] when compared with the MRPS23 (S11A) mutant-overexpressing cells. This finding indicated the role of MRPS23 phosphorylation in the proliferation and survival of breast cancer cells. The correlation of inconsistent MRPS23 phosphoserine 11 protein expression with CDK11A in the breast cancer cells suggested phosphorylation by other kinases. In vitro kinase assay showed that CDK1 kinase also phosphorylated MRPS23 and that inhibition using CDK1 inhibitors lowered phospho-MRPS23 (Ser11) levels. Additionally, modulating the expression of MRPS23 altered the sensitivity of the cells to CDK1 inhibitors. CONCLUSION: In conclusion, phosphorylation of MRPS23 by mitotic kinases might potentially be involved in the proliferation of breast cancer cells. Furthermore, MRPS23 can be targeted for sensitizing the breast cancer cells to CDK1 inhibitors.

Nimbolide, a Neem Limonoid, Inhibits Angiogenesis in Breast Cancer by Abrogating Aldose Reductase Mediated IGF-1/PI3K/Akt Signalling.

BACKGROUND & OBJECTIVE: The insulin/IGF-1R/PI3K/Akt signalling cascade is increasingly being linked to breast cancer development, with aldose reductase (AR) playing a key role in mediating the crosstalk between this pathway and angiogenesis. The current study was designed to investigate whether nimbolide, a neem limonoid, targets the oncogenic signaling network to prevent angiogenesis in breast cancer. METHODS: Breast cancer cells (MCF-7, MDA-MB-231), EAhy926 endothelial cells, MDA-MB-231 xenografted nude mice, and tumour tissues from breast cancer patients were used for the study. The expression of AR and key players in IGF-1/PI3K/Akt signaling and angiogenesis was evaluated by qRT-PCR, immunoblotting, and immunohistochemistry. Molecular docking and simulation, overexpression, and knockdown experiments were performed to determine whether nimbolide targets AR and IGF-1R. RESULTS: Nimbolide inhibited AR with consequent blockade of the IGF-1/PI3K/Akt and /HIF-1alpha/VEGF signalling circuit by influencing the phosphorylation and intracellular localisation of key signaling molecules. The downregulation of DNMT-1, HDAC-6, miR-21, HOTAIR, and H19 with the upregulation of miR-148a/miR-152 indicated that nimbolide regulates AR and IGF-1/PI3K/Akt signaling via epigenetic modifications. Coadministration of nimbolide with metformin and the chemotherapeutic drugs tamoxifen/cisplatin displayed higher efficacy than single agents in inhibiting IGF-1/PI3K/Akt/AR signaling. Grade-wise increases in IGF-1R and AR expression in breast cancer tissues underscore their value as biomarkers of progression. CONCLUSION: This study provides evidence for the anticancer effects of nimbolide in cellular and mouse models of breast cancer besides providing leads for new drug combinations. It has also opened up avenues for investigating potential molecules such as AR for therapeutic targeting of cancer.

Identification and validation of plasma biomarkers for diagnosis of breast cancer in South Asian women.

Breast cancer is the most common malignancy among women globally. Development of a reliable plasma biomarker panel might serve as a non-invasive and cost-effective means for population-based screening of the disease. Transcriptomic profiling of breast tumour, paired normal and apparently normal tissues, followed by validation of the shortlisted genes using TaqMan® Low density arrays and Quantitative real-time PCR was performed in South Asian women. Fifteen candidate protein markers and 3 candidate epigenetic markers were validated first in primary breast tumours and then in plasma samples of cases [N = 202 invasive, 16 DCIS] and controls [N = 203 healthy, 37 benign] using antibody array and methylation specific PCR. Diagnostic efficiency of single and combined markers was assessed. Combination of 6 protein markers (Adipsin, Leptin, Syndecan-1, Basic fibroblast growth factor, Interleukin 17B and Dickopff-3) resulted in 65% sensitivity and 80% specificity in detecting breast cancer. Multivariate diagnostic analysis of methylation status of SOSTDC1, DACT2, WIF1 showed 100% sensitivity and up to 91% specificity in discriminating BC from benign and controls. Hence, combination of SOSTDC1, DACT2 and WIF1 was effective in differentiating breast cancer [non-invasive and invasive] from benign diseases of the breast and healthy individuals and could help as a complementary diagnostic tool for breast cancer.

Mitochondrial ribosomal small subunit proteins (MRPS) MRPS6 and MRPS23 show dysregulation in breast cancer affecting tumorigenic cellular processes.

Human Mitoribosomal Small Subunit unit (MRPS) family of genes appears to have role in cancer. Gene expression analysis of select MRPS genes (n = 9) in 15 cancer cell lines showed altered expression in cancer cells. Protein levels of MRPS6, MRPS23 showed significant overexpression in breast cancer cells and tissues. Interestingly, their overexpression did not correlate with mitochondrial ribosome translated COX2 protein levels in breast cancer. Subcellular fractionation analysis showed a distinct presence of MRPS23 in the nuclear fraction. GST/MRP6 and GST/MRPS23 pulldown assays identified 32 novel protein-protein interactions (PPIs) and MRPS23-RIPK3 interaction was validated. Co-expression module identification tool (CEMi) analysis of breast cancer gene expression and MRPS6 and MRPS23 interactions revealed hub interactions in gene expression modules having functional roles in cancer-associated cellular processes. Based on PPI network analysis a novel interaction MRPS23-p53 was validated. Knockdown of MRPS6 and MRPS23 decreased proliferation, expression of select mesenchymal markers, oncogenes, and increased expression of tumor suppressor genes. Taken together present study has revealed that MRPS6 and MRPS23 genes have pro-tumorigenic functions in breast cancer.

TGFß C-509T, TGFß T869C, XRCC1 Arg194Trp, IKBα C642T, IL4 C-590T Genetic polymorphisms combined with socio-economic, lifestyle, diet factors and gastric cancer risk: A case control study in South Indian population.

BACKGROUND: Gastric cancer is worldwide the third major cause of cancer related death. Risk factors for gastric cancer includes Helicobacter pylori infection, gastric ulcer, less hygienic condition, use of tobacco, alcohol consumption, use of salted, smoked food, genetic alterations etc. In order to identify the risk factors associated with gastric cancer in South Indian population a case-control study involving 200 proven gastric cancer cases and 400 controls was conducted. METHODS: A structured questionnaire was used to interview all the subjects who participated in our study. Genotyping assay was performed using Taqman allelic discrimination assay for 5 Single Nucleotide Polymorphisms (SNPs)-TGFß C-509T, TGFß T869C, XRCC1 Arg194Trp, IkBα C642T and IL4C-590T. RESULTS: Odds Ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression. Statistical analysis on socio-economic factors, lifestyle factors had showed that subjects from low socio economic status, use of tobacco and consumption of non-vegetarian food had increased risk of developing gastric cancer. Multi-factorial analysis for the SNPs adjusting for the risk factors obtained in this study showed that TGFΒ C-509T TT genotypes had four fold increased risk of gastric cancer (OR = 4.11, CI = 1.02-16.56) and TGFß T869C CC genotype had a decreased risk of gastric cancer (OR = 0.21, CI = 0.05-0.85). CONCLUSION: Economic status, tobacco use and food habits play a significant role in gastric cancer development. TT genotype for TGFß C-509T had an increased risk and CC genotype for TGFß T869C had a decreased risk of gastric cancer in south Indian population after adjusting for socio-economic factors and lifestyle factors.

Immunohistochemical expression and localization of cytokines/chemokines/growth factors in gastric cancer.

Our previous studies on gastric cancer tissue and patient plasma samples identified several cytokines/chemokines/growth factors to be differentially expressed, compared to normal samples. In this study our aim was to understand the localization patterns of the markers in gastric tissues. We investigated the expression of PDGFRB, CCL3, MMP3, CXCL8, CXCL10, CCL20, IGFBP3, CXCL9, SPP1, CCL18, TIMP1, CCL15, CXCL5 and CCL4 in gastric tissues using Immunohistochemistry (IHC) on Tissue Microarrays (TMA). The TMA comprised of 25 apparently normal (AN), 87 paired normal (PN) and 134 gastric cancer (T) tissues. The epithelial and stromal expression of markers and their correlation with patient characteristics and outcome were analyzed. Several of the markers [PDGFRB (p<0.001), CCL3 (p<0.001), MMP3 (p<0.001), CXCL8 (p<0.001), CXCL10 (p<0.001), CCL20 (p<0.001), CXCL9 (p<0.001), CCL18 (p<0.001), TIMP1 (p=0.025), CCL15 (p<0.001)] were elevated in the stromal compartment of gastric cancers compared to AN tissues, with some having intermediate levels of expression in PN tissues. Epithelial and stromal PDGFRB (p=0.030, p=0.018) expression was associated with diffuse type gastric cancer. Stromal IGFBP3 (p=0.039), CXCL8 (p=0.008), TIMP1 (p<0.001), CCL4 (p=0.003) and SPP1 (p=0.048) expression was associated with intestinal type gastric cancer. Kaplan-Meier analysis showed higher epithelial PDGFRB (p=0.005 and p=0.004), CXCL8 (p=0.009 and p=0.007) were associated with poor disease free and overall survival. In multivariate analysis, high epithelial PDGFRB (p=0.036 and p=0.02) and SPP1 (p=0.003 and p<0.001) were independent prognostic factors for DFS and OS in patients with gastric cancer. The expression of cytokine/chemokine/growth factor markers is higher in the gastric tumor stroma compared to the normal gastric stroma and PDGFRB and SPP1 may serve as potential prognostic factors in gastric cancer.

Inhibition of ubiquitin conjugating enzyme UBE2C reduces proliferation and sensitizes breast cancer cells to radiation, doxorubicin, tamoxifen and letrozole.

PURPOSE: The objective of this study was to determine radiation, doxorubicin, tamoxifen and letrozole sensitivity of breast cancer cells in response to functional inhibition of the ubiquitin conjugating enzyme UBE2C. METHODS: Taqman Real time PCR was performed to measure UBE2C levels in breast cancer cell lines and control HBL100 and HEK293T cells. A dominant negative form of UBE2C (DN-UBE2C) was used to functionally inhibit wild type UBE2C. Cell proliferation and anchorage independent growth were measured by colorimetric and soft agar assays, respectively. Radiation, doxorubicin, tamoxifen and letrozole responses of the cell lines were assessed by colorimetric and clonogenic assays. RESULTS: Overexpression of UBE2C was observed in all breast cancer cell lines tested using quantitative real time PCR. UBE2C expression was found to be highest in MDAMB231 and relatively lowest in MCF7 cells, compared to control cells. Both the growth rate and the anchorage independent growth of MCF7 and MDAMB231 cells transfected with DN-UBE2C were significantly reduced compared to cells transfected with vector alone. MCF7 and MDAMB231 cells expressing DN-UBE2C were significantly more sensitive to different doses of radiation and doxorubicin compared to both wild type and vector alone transfected cells. In addition, DN-UBE2C transfected MCF7 cells were more sensitive to inhibition by tamoxifen and letrozole compared to wild type and vector alone transfected cells. CONCLUSIONS: Our results show that inhibition of UBE2C sensitizes breast cancer cells to radiation, doxorubicin and hormone blocking agents. UBE2C may, therefore, serve as a potential therapeutic target aimed at inducing radiation and chemo sensitization.

SOSTDC1 down-regulation of expression involves CpG methylation and is a potential prognostic marker in gastric cancer.

Sclerostin domain containing 1 (SOSTDC1) is reportedly down-regulated in various cancers. Our purpose was to study whether epigenetic mechanisms were involved in the down-regulation of expression in gastric cancer. Expression analysis of SOSTDC1 in gastric cancer cell lines indicated mRNA down-regulation. Our reporter assays and gene reactivation studies using 5-aza-2'-deoxycytidine, a DNA demethylating agent, and trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, demonstrated that epigenetic mechanisms are involved in the down-regulation of SOSTC1 expression. Methylation analysis of the SOSTDC1 promoter CpGs using methylation-specific polymerase chain reaction analysis revealed methylation in gastric cancer cell lines and tissue samples. A majority of tumors (17 of 18) with observed methylation exhibited down-regulation of mRNA expression relative to apparently normal gastric tissues. Immunoreactivity for SOSTDC1 in gastric tumors (24 of 46, 52.1%) was down-regulated relative to normal tissues (36 of 38, 94.7%) (P = 0.00001). The difference in expression between gastric tumor subtypes, intestinal and diffuse, was significant (P = 0.040). Expression of SOSTDC1 in gastric tumors increased the probability of both overall and disease-free survival. When overexpressed in AGS cells, cell proliferation, cell cycle progression, and anchorage-independent growth was repressed. The present findings indicate SOSTDC1 down-regulation involves methylation; SOSTDC1 expression is a potential prognostic factor and tumor suppressor in gastric cancer.

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing's sarcoma in vitro.

BACKGROUND: Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. METHODS: In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. RESULTS: Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. CONCLUSIONS: Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.

Endo-sulfatase Sulf-1 protein expression is down-regulated in gastric cancer.

In our recent report on gene expression in gastric cancer we identified the endo-sulfatase Sulf-1 gene to be up-regulated in gastric tumors relative to apparently normal (AN), and paired normal (PN) gastric tissue samples. In the present report we investigate the protein expression levels of Sulf-1 gene in gastric tumors, AN and PN samples using tissue microarray (TMA) and immunohistochemistry. Expression data was collected from two sets of TMA's containing replicate sections of tissue samples. Scoring data from TMA set-1 revealed a significant difference in Sulf-1 immunoreactivity between tumors and "normals" (PN and AN) (p-value = 0.001928). Also, Sulf-1 expression in tumors was also significantly different from either PN (p-value = 0.019) or AN (p-value = 0.006) samples. Similar results were obtained from analysis of scoring data from the second set of arrays. Comparison of mRNA expression and protein expression in gastric tumor tissues revealed that in 6/20 (30%) tumor samples showed up-regulated protein expression concordant with over-expression of mRNA. However, a discord with mRNA being over-expressed relative to down regulated protein expression was observed in majority 14/20 (70%) of tumor samples. Our study indicates down regulation of Sulf-1 protein expression in gastric tumors relative to PN and AN samples which is discordant with mRNA over-expression seen in tumors.

Intragenic DNA methylation concomitant with repression of ATP4B and ATP4A gene expression in gastric cancer is a potential serum biomarker.

Based on our previous report on gastric cancer which documented ATP4A and ATP4B mRNA down- regulation in gastric tumors relative to normal gastric tissues, we hypothesized that epigenetic mechanisms could be responsible. ATP4A and ATP4B mRNA expression in gastric cancer cell lines AGS, SNU638 and NUGC-3 was examined using reverse transcriptase PCR (RT-PCR). AGS cells were treated with TSA or 5'-AzaDC and methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis were performed. MSP analysis was on DNA from paraffin embedded tissues sections and plasma. Expression analysis revealed downregulation of ATP4A and ATP4B genes in gastric cancer cell lines relative to normal gastric tissue, while treatment with 5'-AzaDC re-activated expression of both. Search for CpG islands in their putative promoter regions did not indicate CpG islands (CGI) but only further downstream in the bodies of the genes. Methylation specific PCR (MSP) in the exon1 of the ATP4B gene and exon7 in ATP4A indicated methylation in all the gastric cancer cell lines tested. MSP analysis in tumor tissue samples revealed methylation in the majority of tumor samples, 15/19, for ATP4B and 8/8 for ATP4A. There was concordance between ATP4B and ATP4A down-regulation and methylation status in the tumour samples tested. ATP4B methylation was detectable in cell free DNA from gastric cancer patient's plasma samples. Thus ATP4A and ATP4B down-regulation involves DNA methylation and methylated ATP4B DNA in plasma is a potential biomarker for gastric cancer.

Identification and validation of genes involved in cervical tumourigenesis.

BACKGROUND: Cervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer. MATERIALS AND METHODS: A total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the significant genes by relative quantitation using Taqman Low Density Array Real Time PCR. Immunohistochemistry was used to study the protein expression of MMP3, UBE2C and p16 in normal, dysplasia and cancers of the cervix. The effect of a dominant negative UBE2C on the growth of the SiHa cells was assessed using a MTT assay. RESULTS: Our study, for the first time, has identified 20 genes to be up-regulated and 14 down-regulated in cervical cancers and 5 up-regulated in CIN3. In addition, 26 genes identified by other studies, as to playing a role in cervical cancer, were also confirmed in our study. UBE2C, CCNB1, CCNB2, PLOD2, NUP210, MELK, CDC20 genes were overexpressed in tumours and in CIN3/CIS relative to both Normal and CIN1/CIN2, suggesting that they could have a role to play in the early phase of tumorigenesis. IL8, INDO, ISG15, ISG20, AGRN, DTXL, MMP1, MMP3, CCL18, TOP2A AND STAT1 were found to be upregulated in tumours. Using Immunohistochemistry, we showed over-expression of MMP3, UBE2C and p16 in cancers compared to normal cervical epithelium and varying grades of dysplasia. A dominant negative UBE2C was found to produce growth inhibition in SiHa cells, which over-expresses UBE2C 4 fold more than HEK293 cells. CONCLUSIONS: Several novel genes were found to be differentially expressed in cervical cancer. MMP3, UBE2C and p16 protein overexpression in cervical cancers was confirmed by immunohistochemistry. These will need to be validated further in a larger series of samples. UBE2C could be evaluated further to assess its potential as a therapeutic target in cervical cancer.

Identification and validation of genes involved in gastric tumorigenesis.

BACKGROUND: Gastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets. MATERIALS AND METHODS: We used 24 gastric cancers, 20 Paired normal (PN) and 5 apparently normal gastric tissues obtained from patients with non-gastric cancers (Apparently normal - AN) for the microarray study followed by validation of the significant genes (n = 63) by relative quantitation using Taqman Low Density Array Real Time PCR. We then used a custom made Quantibody protein array to validate the expression of 15 proteins in gastric tissues (4 AN, 9 PN and 9 gastric cancers). The same array format was used to study the plasma levels of these proteins in 58 patients with gastric cancers and 18 from patients with normal/non-malignant gastric conditions. RESULTS: Seventeen genes (ASPN, CCL15/MIP-1δ, MMP3, SPON2, PRSS2, CCL3, TMEPAI/PMEPAI, SIX3, MFNG, SOSTDC1, SGNE1, SST, IGHA1, AKR1B10, FCGBP, ATP4B, NCAPH2) were shown to be differentially expressed between the tumours and the paired normal, for the first time. EpCAM (p = 0.0001), IL8 (p = 0.0003), CCL4/MIP-1ß (p = 0.0026), CCL20/MIP-3α (p = 0.039) and TIMP1 (p = 0.0017) tissue protein levels were significantly different (Mann Whitney U test) between tumours versus AN & PN. In addition, median plasma levels of IL8, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, PDGFR-B and TIMP1 proteins were significantly different between the non-malignant group and the gastric cancer group. The post-surgical levels of EpCAM, IGFBP3, IL8, CXCL10/IP10, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, SPP1/OPN and PDGFR-B showed a uniform drop in all the samples studied. CONCLUSIONS: Our study has identified several genes differentially expressed in gastric cancers, some for the first time. Some of these have been confirmed at the protein level, as well. Some of these proteins will need to be evaluated further for their potential as diagnostic biomarkers in gastric cancers and some could be useful as follow-up markers in gastric cancer.

The influence of sulindac on diabetic cardiomyopathy: a non-invasive evaluation by Doppler echocardiography in streptozotocin-induced diabetic rats.

The aim of the present study was to investigate the cardioprotective activity of sulindac as an aldose reductase inhibitor in the development of cardiomyopathy by non-invasive techniques; M-mode and Doppler echocardiography. Diabetes was induced by streptozotocin (45 mg/kg, iv) in the Sprague-Dawley rats. Echocardiography, biochemical and histological studies were carried out in normal control, diabetic untreated, diabetic vehicle (sodium carboxy methyl cellulose, 1%, po) and sulindac (6 mg/kg and 20 mg/kg, po) treated animals at varying time intervals. In the diabetic untreated and vehicle treated rats at 12 weeks after induction of diabetes, there was a significant decrease in the E-wave, an increase in the A-wave and corresponding decrease in the E/A ratio was observed. Significant decrease in the Eat was found after 12 weeks (P < 0.05). Whereas systolic function variables; ejection fraction and fractional shortening were significantly decreased (P < 0.05) after 12 weeks compared to their baseline data. In the sulindac treated animals, there were no significant alterations in the systolic and diastolic parameters were found throughout the study period. Myocardial fructose levels were significantly increased in the diabetic untreated animals compared to normal control rats (P < 0.05), whereas these were significantly decreased in the sulindac (6 mg/kg and 20 mg/kg) treated animals (301.11+/-37.98, 214.11+/-25.31, vs. 914.88+/-56.01 nmol/g) compared to diabetic vehicle treated group (P < 0.05). Extensive focal ischemic myocyte degeneration was observed in the diabetic untreated and vehicle treated rats, whereas in the sulindac (6 mg/kg) treated rats, minimal necrosis was found, with no evidence of necrosis in sulindac (20 mg/kg) group. Our results show for the first time that sulindac has a cardioprotective activity as this agent prevented the development of left ventricular dysfunction in STZ-induced diabetic rats in the 12-week chronic study.

Partial reversal by rutin and quercetin of impaired cardiac function in streptozotocin-induced diabetic rats.

The present investigation was carried out to evaluate the effects of the cyclodextrin complexes quercetin and rutin on left ventricle dysfunction in streptozotocin-induced diabetic rats. Diabetes was induced by streptozotocin (45 mg/kg body mass, i.v.) in Sprague-Dawley rats. Echocardiography and biochemical and histological studies were carried out under normal control, diabetic untreated, normal and diabetic vehicle (beta-cyclodextrin, p.o.), quercetin- (100 and 300 mg/kg, p.o.), and rutin- (100 and 300 mg/kg, p.o.) treated normal and diabetic animals at varying time intervals (1 and 12 weeks). The increase in the serum triglycerides and cholesterol levels was attenuated in the cyclo dextrin complexes of rutin-treated animals significantly more than in the quercetin-treated and diabetic vehicle-treated animals. Left ventricular diastolic dysfunction was observed in diabetic vehicle-treated animals after 12 weeks of the study as determined by a significant decrease in E-wave (45.91%), an increase in the A-wave (75.55%), and a decrease in the E/A ratio (70.14%). However, the percent decrease (after 12 weeks) in the E-wave, increase in the A-wave, and decrease in the E/A ratio were less in the cyclodextrin complexes of rutin-treated animals (100 and 300 mg/kg), which had the following values: E-wave, 12.22% and 13.80%; A-wave, 25.90% and 10.40%; and E/A ratio, 31.01% and 20.52%. In the quercetin-treated animals (100 and 300 mg/kg), which had the following values: E-wave, 40.44% and 36.44%; A-wave, 52.98% and 29.28%; and E/A ratio, 61.70% and 51.11%. Histopathological studies revealed that the degree of myocardial necrosis was less in rutin-treated animals compared with quercetin and diabetic vehicle-treated animals: rutin < quercetin < beta-cyclodextrin. Myocardial fructose levels were significantly increased in the diabetic vehicle-treated animals after 12 weeks of the study, suggesting an increment in the myocardial polyol pathway activity. However, myocardial fructose levels were significantly decreased in the rutin- and quercetin-treated animals compared with the vehicle-treated animals, possibly owing to their aldose reductase inhibitory activity. Quercetin and rutin treatment did not influence the echocardiographical and histo logical parameters in normal animals. Results from the present investigation demonstrated that rutin has a cardioprotective activity, and we conclude that the observed cardioprotection with rutin may be due to its aldose reductase inhibitory activity, as the enhanced aldose reductase pathway is implicated in the development of left ventricle dysfunction by several studies.