Valencene post-treatment exhibits cardioprotection via inhibiting cardiac hypertrophy, oxidative stress, nuclear factor- κB inflammatory pathway, and myocardial infarct size in isoproterenol-induced myocardial infarcted rats; A molecular study.

The growing burden of myocardial infarction (MI) becomes a major global health issue that is accountable for considerable mortality worldwide. Hence, it is obligatory to develop a new treatment for MI having lesser side effects. Cardiac hypertrophy, oxidative stress, and inflammatory pathways play crucial roles in the pathogenesis of MI. This investigation established the anti-cardiac hypertrophic, antioxidant, anti-inflammatory, and myocardial infarct size limiting effects of valencene. Rats were induced MI by isoproterenol (100 mg/kg body weight) and then treated with valencene and cardiac sensitive markers, cardiac hypertrophy, oxidative stress, markers of inflammation, nuclear factor- κB inflammatory pathway, and myocardial infarct size was estimated/determined. The serum cardiac diagnostic markers, cardiac hypertrophy, conjugated dienes, markers of inflammation, pro-inflammatory cytokines, and myocardial infarct size were significantly (P < 0.05) increased by isoproterenol. Further, antioxidant enzymes and anti-inflammatory cytokine gene were significantly (P < 0.05) decreased in the heart. The 2, 3, 5-triphenyl tetrazolium chloride dye staining revealed a larger infarct size. Moreover, histological results of myocardial infarcted rat's cardiac tissue revealed separation of cardiac muscle fibers, necrosis, and inflammatory cells. Post-treatment with valencene (12 mg/kg body weight) orally, daily, for two weeks to isoproterenol-induced myocardial infarcted rats reversed all above said structural, biochemical, molecular, and histological parameters investigated, by its anti-cardiac hypertrophic, antioxidant, anti-inflammatory, and myocardial infarct size limiting effects. Thus, valencene is a potential candidate for inhibiting cardiac hypertrophy, oxidative stress, nuclear factor- κB inflammatory pathway, and myocardial infarct size and exhibited cardioprotection in MI.

Significant action of Tridax procumbens L. leaf extract on reducing the TNF-α and COX-2 gene expressions in induced inflammation site in Swiss albino mice.

The leaves of traditionally used herbal plant Tridax procumbens L. contain lots of phytochemicals having potency to reduce inflammation. In this study, the ethanol extract of the leaves of Tridax procumbens L. was analysed for the phytochemicals by GC-MS. The anti-inflammatory activity was then studied with the extract of 10, 50, and 100 mg/kg b.wt in carrageenan-induced mice model by measuring the inflammatory oedema and by analysing the histopathology. The mRNA expression levels of TNF-α and COX2 genes were studied in the inflammatory site to explore the molecular action by reverse transcription PCR and qPCR analyses. A significant (P ≤ 0.01) reduction in mice paw inflammation and a recovered histology were observed in treated groups when compared to control group in 24 h. The RT-PCR results showed a significant (P ≤ 0.01) decrease in the expression levels of TNF-α and COX2 in terms of band density in treated mice compared to control group. The qPCR RQ values also were decreased in treated groups with respect to increasing doses (RQ values of 18.985 ± 0.230, 12.140 ± 1.121, 6.718 ± 0.807 for TNF-α and 15.583 ± 1.043, 7.725 ± 1.013, 5.075 ± 0.615 for COX2, respectively for the three doses) in comparison with the control group (TNF-α 27.107 ± 2.254, COX2 20.626 ± 1.477). Tridax procumbens L. can be, thus, used for the development of a safe, natural, anti-inflammatory drug as it showed a strong inhibitory action on inflammation by acting at molecular level.

Ameliorating effect of lipo-ATRA treatment on the expression of TIG3 and its suppressing effect on PPARγ gene expression in lung cancer animal model.

This study aimed to find out the molecular therapeutic effect of lipo-ATRA on tumour suppressor TIG3 and cell proliferative biomarker PPARγ in B (a) P-induced lung cancer model. In RT-PCR study, ATRA- and lipo-ATRA-treated mice samples showed relatively higher TIG3 expression and decreased PPARγ expression (Band density) than cancer control. Among treatments, lipo-ATRA showed vital effect than free ATRA by enhancing TIG3 and decreasing PPARγ. The qPCR results also showed significant (p ≤ 0.05) difference in both TIG3 and PPAR (RQ values of TIG3, lipo-ATRA 23.85 ± 1.29; free ATRA 10.43 ± 1.81 and for PPARγ, lipo-ATRA 4.707 ± 1.21; free ATRA 15.78 ± 2.34). From this, we conclude that liposomal ATRA formulation is most preferable for prolonged delivery of ATRA at targeted site to favour molecular action. It implies that the therapeutic effect of lipo-ATRA in lung cancer was exhibited by ameliorating the TIG3 expression and by suppressing the expression of PPARγ.

Enhancement of tumor suppressor RAR-ß protein expression by cationic liposomal-ATRA treatment in benzo(a)pyrene-induced lung cancer mice model.

Targeting the specific molecular proteins or genes which are responsible for the suppression of cancer growth is currently an emerging molecular method to treat cancer. ATRA treatment is now considered as a molecular targeted therapy for many cancers. As ATRA exhibits its therapeutic effect through its receptors, this study was focused to investigate the effect and action of liposomal-ATRA treatment on the expression of RAR-ß protein which is also a tumor suppressor. The liposomal-ATRA was developed with cationic DOTAP and cholesterol by thin-film formation method. The benzo(a)pyrene(50 mg/kg b.wt)-induced mice were treated with free and liposomal-ATRA(0.60 mg/kg b.wt). The RAR-ß protein expression in lung and liver tissue samples were analyzed by immunohistochemistry (IHC) and western blotting (WB) on the 30th and 120th days. Almost nil expression of RAR-ß protein was observed in B(a)P cancer control and liposome alone-treated groups. A comparatively elevated expression was seen in the free ATRA-treated group (IHC score-2+ in lung on the 120th day with band density of 14.46 ± 1.24% in WB). Interestingly, the liposomal-ATRA treatment demonstrated a significantly (p ≤ 0.01) higher RAR-ß expression in lung (35.20 ± 3.398% band intensity and score 4+ in the 120th day) than that of in ATRA alone treatment. This study results indicate that the RAR-ß protein expression was suppressed by B(a)P during cancer induction even on the 30th day itself. The treatment could reactivate the suppression and the lipo-ATRA treatment could show significantly higher RAR-ß expression on the 120th day, which implies that it accumulated more ATRA in target site and sustained it for enhanced action.

Reduced RAR-ß gene expression in Benzo(a)Pyrene induced lung cancer mice is upregulated by DOTAP lipo-ATRA treatment.

Molecular targeted therapy for specific genes is an emerging research. Retinoic Acid Receptor (RAR-ß) is a key tumor suppressor which is found to be lost drastically during much cancer progression. We hence, analyzed the expression level of RAR-ß gene during B(a)P induced lung cancer development in mice and studied the lung cancer targeted action of All Trans Retinoic Acid (ATRA) in DOTAP liposomal formulation. The effect of its treatment on lung cancer was determined by histopathological analysis. RAR-ß gene expression was assessed by RT-PCR and qPCR. A distinct band for RAR-ß gene (density - 0.5123 for lung and 0.5160 for liver) was observed in normal mice, whereas no visible band was observed in cancer induced group, indicating loss of RAR-ß gene expression. Both ATRA and lipo-ATRA treated groups showed detectable RAR-ß expression with relatively lesser density than the normal group. The expression was more intense in lipo-ATRA treatment (density-0.2973) compared with free ATRA treatment (density-0.1549) in lung tissues. The qPCR results also have highlighted a highly significant (p ≤ 0.01) variation RQ values between lipo-ATRA group (15.46 ±â€¯1.54) and free ATRA group (7.58 ±â€¯1.30) in lung tissue sample on 30th day. The mean RQ value for normal lung on 30th day was 20.86 ±â€¯2.58 against the cancer control. The 120th day mice also showed the similar RAR-ß expression pattern with further declined expression levels as there was no treatment given after 30 days. Interestingly, the lipo-ATRA treatment could show a highly significant (p ≤ 0.001) expression (12.00 ±â€¯2.31) when compared with free ATRA treatment (3.31 ±â€¯0.58) which implies that the lipo-ATRA formulation could result in sustained delivery of ATRA in target site. Histopathology of lung and liver on 120th day also revealed an effective therapeutic indication in lipo-ATRA treatment compared to free ATRA treatment due to lipo-ATRA's stealth property and it efficiently inhibited the metastasis to liver. These results revealed that the lipo-ATRA treatment has efficiently delivered ATRA into target site than free ATRA and in-turn it might have induced the expression of RAR-ß gene or prevented loss of RAR-ß gene in cancer animals.

Enhanced expression of tumour suppressor RAR-ß by DSPC nano-formulated lipo-ATRA in the lung of B16F10 cell-implanted C57BL6 mice and in A549 cells.

AIM: All Trans Retinoic acid (ATRA) is an efficient drug for leukemia, but is not efficient therapy for solid cancers. Hence we have used 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol lipo-ATRA to investigate its molecular therapeutic effect on lung cancer. The objective was to find whether it could enhance ATRA receptor, RAR-ß expression in lung cells as it was lost in majority of cancers including lung cancer. MATERIALS AND METHODS: The study was made in an experimental C57BL/6 mice model developed by tail vein injection of B16F10 cells and in A549 human lung cancer cells. The RAR-ß protein expression was studied by Immunohistochemistry/Immunocytochemistry and the mRNA expression was studied by RT-PCR and qPCR methods. KEY FINDINGS: Both free and lipo-ATRA treatments showed an enhancement of RAR-ß protein and gene expressions, indicating its induction on RAR ß. However, lipo-ATRA treatment has shown significant induction when compared with free ATRA treatment. SIGNIFICANCE: Our results implies that the DSPC lipo-ATRA treatment might have accumulated more ATRA in to the target cells which might have resulted in the induction of its receptor RAR-ß expression in a hypothesis of ligand induced receptor expression.

Phytochemical profile and free radical nitric oxide (NO) scavenging activity of Averrhoa bilimbi L. fruit extract.

Averrhoa bilimbi L. belongs to family Oxalidaceae. Traditionally, people use this plant (root, bark, leaves and fruits) for treating several illnesses include itches, boils, syphilis, whooping cough, hypertension, fever and inflammation. The aim of the study was to evaluate the nitric oxide (NO) scavenging activity and GC-MS analysis of A. bilimbi L. fruit extract. Averrhoa bilimbi L. fruits were collected for the preliminary phytochemical analysis, antioxidant scavenging activity and biologically important compounds were identified by GC-MS analysis. The preliminary phytochemicals, GC-MS, total phenolic content and NO scavenging activity of the plant were analysed. In the present investigation, the A. bilimbi L. fruit extract has major phytochemicals. Among the 151 compounds identified in GC-MS, 15 compounds are found to have diverse biological activity. We also observed that the A. bilimbi L. fruit extract has high level of total phenolic compounds at a concentration of 209.25 GAE mg/g. Presence of phenolic compound apparently explains the antioxidant activity of the plant. Antioxidant activity of A. bilimbi L. fruit extract is proven from its high level of NO scavenging activity of potent IC50 value of 108.10. From the above study, it is apparent that the A. bilimbi L. fruit extract is a rich source of phytochemicals (natural products) with biological activity. The GC-MS report on this fruit proves that natural products have pharmacologically and biologically active compounds. A high phenolic content is observed in our study. A. bilimbi L. fruit extract is also found to have NO scavenging activity in our study.

Protective effect of Averrhoa bilimbi L. fruit extract on ulcerative colitis in wistar rats via regulation of inflammatory mediators and cytokines.

Ulcerative Colitis (UC) is a lingering type of Inflammatory Bowel Disease (IBD) which affects the colon mucosa. Ulcerative colitis is majorly associated with oxidative stress and inflammation in colon tissue leading to damage. Averrhoa bilimbi L. fruit is rich in antioxidant phytochemicals including Vitamin C. In the current research, we have evaluated the defence mechanism of Averrhoa bilimbi L. on Ulcerative Colitis (UC). Male wistar rats were treated with Averrhoa bilimbi L. fruit extract (50mg/kg/bwt and 100mg/kg/bwt) and a standard drug Sulfasalazine (100mg/kg/bwt) for 6 consecutive days via intra peritoneally. After one day fasting, rats were given single dose of 3% 2ml of acetic acid through anal (intra-anal) region to induce Ulcerative Colitis. The protective and therapeutic effect of fruit extract on UC was assessed by comparing the relevant changes observed in the normal and treated group. In treated group the level of mucosal injury was decreased (ulcer score - 2) when compared to the control group (ulcer score - 9). The abnormal increase observed in the inflammation mediator cytokines in control rats, i.e IL-1ß, IL-6, TNF-α levels were decreased significantly (**p<0.01) in the Averrhoa bilimbi L. fruit extract treated groups. The increase in weights of the colon tissue and spleen of the control rats were found to be reduced in treated groups. The levels of inflammatory markers iNOS and COX-2 were also decreased in treated group significantly (**p<0.01) when compared with the control. Furthermore, the treatment with Averrhoa bilimbi L. fruit extract has shown a significant antioxidant activity in the UC condition by reducing the levels of NO and enhancing the levels of SOD and GSH in the colon tissue. These results demonstrate the effective anti-ulcerative colitis activity of the Averrhoa bilimbi L. fruit extract in experimental wistar rats.

Evaluation of immunomodulatory and antitumor activity of all trans retinoic acid (ATRA) in solid tumor bearing mice.

Natural or synthetic agents can modify the immune system and, in some cases, impart a therapeutic benefit. Cancer, a disease of uncontrolled growth and spread of abnormal cells, is a major cause of death. The Vitamin A metabolite all-trans retinoic acid (ATRA) and its other active derivatives are potent modulators of cell growth and differentiation, and because it has an influence on cancer, it can be used as a chemotherapeutic and -preventive agent. To evaluate the immunomodulatory activity of ATRA, the impact of treatment on various parameters, e.g. delayed-type hypersensitivity (DTH), bone marrow cellularity, hematology, and levels of esterase-positive cells, was assessed in Balb/c mice. To evaluate antitumor effects of ATRA, tumor volume and host survival rate were monitored in B16F10 melanoma cell-injected mice. The results showed that administration of ATRA (0.60 mg/kg/dose, IP) caused a decrease in DTH (footpad thickness) in response to challenge with sheep red blood cells (SRBC) in SRBC-sensitized hosts. ATRA also caused increases in WBC counts and bone marrow cell numbers. In tumor-inoculated mice, ATRA caused tumor growth suppression and gave rise to a heightened survival rate. It was also found that ATRA had differential effects on serum levels of reduced glutathione (GSH) and nitric oxide (NO) was reduced in serum. Based on these results, we conclude that ATRA has a potent immunomodulatory potential but also could significantly impact upon solid tumor growth and prolong host survival.

Antioxidant activity of essential oils from Wedelia chinensis (Osbeck) in vitro and in vivo lung cancer bearing C57BL/6 mice.

AIM: The present investigation was to evaluate the effects of essential oils of Wedelia chinensis (Osbeck) on free radicals and in vivo antioxidant properties. METHODS: Essential oils were extracted using hydro-distillation and compound analysis was performed by GC-MS analysis. Screening for inhibitory activity was conducted by DPPH and OH-scavenging assays. In addition an in vivo study was carried out in cell line implanted cancer bearing mice with assessment of levels of catalase, superoxide dismutase, glutathione peroxidase, lipid peroxidation, nitric oxide and reduced glutathione. Finally, lungs were dissected out for histopathology study of metastasis. RESULTS: GC-MS analysis revealed the presence of carvocrol and trans-caryophyllene as the major compounds with 96% comparison with the Wilily and NBS libraries. The essential oil exhibited significant inhibition in DPPH free radical formation. Whereas reducing power and hydroxyl radical scavenging activity are dose dependent. When compared with the standard, it was found that the essential oil has more or less equal activity in scavenging free radicals produced. In the animal studies, the level of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase, as well as glutathione, were found to be increased in treated groups whereas lipid peroxidation and nitric oxide were reduced. Histopathology report also shows that the essential oil has a significant combating effect against cancer development. CONCLUSION: In all the in vitro assays, a significant correlation existed between the concentrations of the essential oil and percentage inhibition of free radicals. The in vivo studies also has shown a very good antioxidant property for the essential oil during cancer development. From, these results the essential oil can be recommended for treating disease related to free radicals and to prevent cancer development.

Chemoprotective effect of all-trans retinoic acid (ATRA) on oxidative stress and lung metastasis induced by benzo(a)pyrene.

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of cancer. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A under the family retinoids, derived by irreversible oxidation of retinol (vitamin A), the parent compound for all natural retinoids. The aim of the present study is to divulge the chemopreventive and chemoprotective nature of ATRA during benzo(a)pyrene (B(a)P) induced lung cancer development in BALB/c mice. Administration of B(a)P (50 mg/kg body weight) to mice resulted in increased lipid peroxides (LPO), lipid hydroperoxides (LOOH) and nitric oxide (NO) with concomitant decrease in the levels of tissue anti-oxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and vitamin C. ATRA supplementation (0.585 mg/kg body weight) attenuated all these alterations, which indicates the anti-cancer effect that was further confirmed by histopathological analysis. Overall, the above data show that the anti-cancer effect of ATRA is more pronounced when used as an chemopreventive agent against B(a)P-induced lung carcinogenesis.

All trans retinoic acid and cancer.

All-trans retinoic acid (ATRA) is an active metabolite of vitamin A under the family retinoid. Retinoids, through their cognate nuclear receptors, exert potent effects on cell growth, differentiation and apoptosis, and have significant promise for cancer therapy and chemoprevention. Differentiation therapy with ATRA has marked a major advance and become the first choice drug in the treatment of acute promyelocytic leukemia (APL). Conversions of 13-cis-retinoic acid and 9-cis-retinoic acid to all-trans-retinoic acid is very rapid. Currently, two distinct families of retinoid responsive nuclear receptors have been identified and characterized: retinoic acid receptors (RARs) and retinoid receptors (RXRs), each of which include three isoforms, α,ß,and γ. ATRA is being increasingly included in anti-tumour therapeutical schemes for the treatment of various tumoral diseases such as Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer, neuroblastoma and has shown antiangiogenic effects in several systems, inhibiting proliferation in vascular smooth muscle cells (VSMCs) and anti-inflammatory in rheumatoid arthritis. This review helps to understand in details about the ATRA and its role on cancer and it is predicted that modulating the activity of ATRA will soon provide novel prevention and treatment approaches for the cancer patients.

HPV type 18 is more oncopotent than HPV16 in uterine cervical carcinogenesis although HPV16 is the prevalent type in Chennai, India.

CONTEXT: The highest incidence of uterine cervical cancer in India is reported in Chennai. The prevalence and oncopotency are to be considered for the development of vaccines and therapeutic agents. AIMS: The aim of the present study is to analyze the prevalence and oncopotency of high risk type HPV16 and 18 in cervical lesions. SETTINGS AND DESIGN: This study is designed with 130 study subjects for analysis of selected types of HPV 6/11 and 16/18, in four groups, in a course of three years. The Bethesda system of classification is followed for grouping the samples, using histopathologic examination in biopsies. MATERIALS AND METHODS: The biopsy samples were collected in 10% buffered formalin and were embedded in paraffin within 24 hours, for long-term preservation. The presence of HPV types were tested by PCR using type-specific primers for HPV16 and HPV18 in the DNA isolated from the subject's biopsies. The stages of cervical lesions were identified by histopathology using the Hematoxylin Eosin stain. STATISTICAL ANALYSIS USED: The data were subjected to statistical analysis, using the SPSS and INSTAT software packages for their associations and risk estimation, respectively. The Graph Pad Prism 2 x 2 contingency table was used for risk estimation and the Kruskel Wallis test was used for analysis of the associations. RESULTS: In the study population, the data indicated a high prevalence of HPV 16. However, during the course of study (1999 - 2003), four (66.6%) dysplasia cases with HPV 18, three (21.4%) dysplasia cases with HPV 16, and none with low-risk HPV6/11, turned into invasive cancer, within one year. CONCLUSIONS: The observation of the study implied that HPV16 had a high prevalence in uterine cervical cancer compared with HPV18 cases. However, the development of invasive cancer from precancerous lesions was more for HPV18 infected cases than for HPV16 during the study period, which indicated the higher oncopotency of HPV type 18.

HPV-induced carcinogenesis of the uterine cervix is associated with reduced serum ATRA level.

OBJECTIVE: In uterine cervical cancer, certain oncogenic HPV types are considered as key etiologic factor. But the progression of HPV associated cervical precancerous lesions depends on many other factors such as oncogenes, immune system, anti-viral factors etc. This study is therefore focused on the effect of an important dietary anti-viral factor called All Trans Retinoic Acid (ATRA) on the development of HPV associated cervical cancer as it is found higher in poor socioeconomic people. METHOD: We analyzed a total population of 130 including control subjects who have no complaints of uterine cervical lesions and the HPV-6/11, 16/18 infected cases of low grade squamous intraepithelial lesions [SIL], high grade squamous intraepithelial lesions [HSIL], and invasive cancers, for serum ATRA level. This study also focused to find out the association of serum ATRA level with the proliferation status in terms of proliferating cell nuclear antigen (PCNA) expression as it is an anti-proliferation agent and with the grades of cervical lesions, using SPSS statistical package. RESULTS: The results showed a highly significant negative association for serum ATRA level with different stages of cervical lesions (F = 3.305; P = 0.000) by one-way ANOVA and with intensity of PCNA expression (r = -0.825; P < 0.01) by Pearson's correlation test. A highly significant association was observed for the PCNA expression with the grades of cervical lesions too (F = 37.89; P = 0.000). Further, we found from our data that all the invasive cancer cases were infected with HPV-16/18 and none with HPV-6/11. Hence, we analyzed the association of serum ATRA level with HPV-16/18 infected preinvasive cases in developing invasiveness, by Fisher's Exact Test, using Graph Pad Prism as shown in Table 1. The results show an odds ratio (OR) of 36.93 and a relative risk (RR) of 4.99 with an 95% interval being 2.896 to 8.603, which is significant at the level of P = 0.0001 for the reduced [<0.6 mug/ml] serum ATRA level in developing invasive cancer in HPV-16/18 infected preinvasive cases. CONCLUSION: All these results suggest that the serum ATRA level highly influences the progression of cervical lesions to invasive cancer and can be therefore aimed as a marker for progression in combination with HPV-16/18, which helps to enhance the modalities of therapy towards cost effectiveness.