Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most elusive subtype of breast cancer that encounters treatment dilemmas owing to the paucity of druggable targets. We found hyperactivation of c-MET and ephrin type-A receptor 2 (EphA2) in patients treated with 5FU driven chemotherapy which correlated with lower disease-free survival. However, silencing of both these genes resulted in a marked decrease in the invasive, migratory, and tumorigenic potential of TNBC cells, indicating that a dual target strategy is actionable. Lupeol is a phytochemical, with potent anticancer efficacy and minimal side effects in preclinical studies. A synergistic strategy with 5FU and Lupeol elicited promising anticancer responses in vitro, in vivo, and in patient-derived ex vivo tumor culture models. This synergistic regimen is effective, even in the presence of HGF, which mechanistically orchestrates the activation of c-MET and EphA2. These data lay the foundation for the clinical validation of this combination therapy for TNBC patients.

Reversing effect of Lupeol on vasculogenic mimicry in murine melanoma progression.

Vasculogenic mimicry, an endothelia-independent tumor microcirculation has been found in various cancers and is thought to be achieved by cancer stem like cells. Dacarbazine resistance is one of the most common features of melanoma and recent studies suggest that the mode of resistance is closely related to the formation of vasculogenic mimicry. In our work, we examined the anticancer effect of Lupeol, a novel phytochemical with Dacarbazine in vivo and in vitro. Results demonstrated adequate cytotoxicity followed by down regulation of CD 133 expression in Lupeol treated B16-F10 cell line. In solid tumor model the drug also inhibited vasculogenic mimicry along with angiogenesis by altering both the cancer stem cell as well as the endothelial progenitor cell population. Lupeol hindered the maturation of bone marrow derived endothelial progenitors and thus, retarded the formation of rudimentary tumor microvessels. Notably, Dacarbazine treatment demonstrated unresponsiveness to B16-F10 cells in both in vivo and in vitro model via upregulation of CD 133 expression and increased formation of vasculogenic mimicry tubes. Together, these data indicate that Lupeol alone can become a proficient agent in treating melanoma, inhibiting vasculogenic mimicry and might play a significant role in subduing Dacarbazine induced drug resistance.

Madhuca indica Inhibits Breast Cancer Cell Proliferation by Modulating COX-2 Expression.

BACKGROUND: Madhuca indica belongs to the family sapotaceae, commonly known as Mahua. It is primarily known for alcoholic beverage production and is reported to have anti-inflammatory, analgesic and antipyretic properties. Madhuca indica has also been reported to be effective in several diseases. OBJECTIVE: This study was undertaken to check the anticancer efficacy and chemopreventive effect of methanolic extract of Mahua flower (ME) on human breast cancer cell lines MCF-7 and MDA-MB-468. METHOD: The cytotoxic and anti-proliferative effects on MCF-7 and MDA-MB-468 cells were studied by MTT, hexosaminidase and colony formation assay. Expression of caspase 3/7 was assessed by flow cytometry and western blot analysis. Expression of COX-2 was evaluated by western blot analysis, luciferase assay and mRNA analysis. RESULTS: ME inhibited the proliferation of breast cancer cells by inducing apoptosis through up-regulating the expression of Caspase 3/7 (P < 0.0001). Our results showed a decrease in the expression of COX-2 mRNA and COX-2 protein in both MCF-7 and MDA-MB-468 cells with an increase in ME concentration. Furthermore synergistic effect of ME and chemotherapeutic drug paclitaxel was also studied in MCF-7 and MDA-MB- 468 cells which were found to be more effective (P < 0.0001) than treatment of either ME or paclitaxel alone. Results were analyzed by ANOVA and Pearson correlation analysis. CONCLUSION: All these experiments suggest that ME inhibits breast cancer cell proliferation and apoptosis by inhibiting the expression of COX-2 in MCF-7 and MDAMB- 468 cells. This work further highlighted that ME may enhance the potentiality of paclitaxel in breast cancer treatment.

CDKN2A-p53 mediated antitumor effect of Lupeol in head and neck cancer.

PURPOSE: The tumor suppressor protein p53 is known to control cell cycle arrest and apoptosis. Lupeol is a phytochemical that has been found to induce apoptosis in different cancer types through the extrinsic pathway. As yet, however, its role in the induction of cell cycle arrest and apoptosis through the intrinsic pathway in head and neck cancer has not been investigated. Here, we aimed at understanding the mechanism underlying the antitumor effect of Lupeol in head and neck cancer. METHODS: The antitumor effect of Lupeol on oral and laryngeal carcinomas was assessed using two in vitro 2D cell line models (HEp-2, UPCI:SCC-131) and, subsequently, an ex vivo 3D tumor explant culture platform that maintains key features of the native tumor microenvironment. The mechanism underlying Lupeol-mediated antitumor responses was delineated using MTT, colony formation, flow cytometry, immunofluorescence, Western blotting and immunohistochemistry assays. RESULTS: We found that Lupeol induced an enhanced expression of p53 in both cell line models tested and, subsequently, cell cycle arrest at the G1 phase. In addition we found that, following Lupeol treatment, p53 induced Bax expression and activated the intrinsic apoptotic pathway (as measured by Caspase-3 cleavage). Interestingly, Lupeol was also found to trigger G1 cell cycle arrest through up-regulation of the expression of CDKN2A, but not p21, resulting in inhibition of CyclinD1. In an ex vivo platform Lupeol was found to impart a potent antitumor response as defined by inhibition of Ki67 expression, decreased cell viability and concomitant activation (cleavage) of Caspase-3. Finally, we found that Lupeol can re-sensitize primary head and neck squamous cell carcinoma (HNSCC) tumor samples that had clinically progressed under a Cisplatin treatment regimen. CONCLUSION: Together, our data indicate that Lupeol may orchestrate a bifurcated regulation of neoplastic growth and apoptosis in head and neck cancers and may serve as a promising agent for the management of tumors that have progressed on a platinum-based treatment regimen.

Resveratrol Alleviates Cadmium-Induced Damage and Overexpression of Epidermal Growth Factor Receptor and its Downstream Signaling Proteins in the Reproductive System of Male Swiss Albino Mice.

Nowadays, exposure to heavy metals and their detrimental effects in humans are grave health concerns. In this study, we investigated the protective effect of resveratrol (RES) against CdCl2 (cadmium chloride)-induced impairment of spermatogenesis, histopathological alterations, and the up-regulation of epidermal growth factor receptor (EGFR) signaling cascade in Swiss albino mice. Two different doses of CdCl2 were injected intraperitoneally into two groups of mice, and in the third group RES was administered orally before injecting CdCl2 (3 times/wk) for 14 days. Sperm motility, count, vitality, and morphology were analyzed. Hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and western blot analyses were performed on testis tissue. In CdCl2-administered animals, significant perturbations of spermatogenesis and histoarchitecture of seminiferous tubules were observed. p-EGFR, p-AKT, AKT1/2/3, NF-κß (p50), and COX-2 of the EGFR cascade were up-regulated. Although there was significant negative correlation between percentage of motile cells and protein expression, we found positive correlation between morphologically abnormal cells and overexpression of proteins in CdCl2-only treated groups. Marked improvement of sperm parameters and histopathological damages as well as down-regulation of the EGFR signaling cascade were observed in the RES-pretreated mice. Hence, the present study elucidates that RES protects against CdCl2-induced perturbation of spermatogenesis and overexpression of EGFR and its downstream signaling proteins.

Cannabis smoke can be a major risk factor for early-age laryngeal cancer--a molecular signaling-based approach.

Epidermal growth factor receptor (EGFR) and its downstream elements are overexpressed in most cases of the head and neck squamous cell carcinoma. This study investigated the expression pattern of key proteins linked to the EGFR pathway in laryngeal carcinoma patients with a history of cannabis smoking. We selected 83 male glottic cancer patients, aged between 45 to 75 years with three distinct populations-nonsmoker, cigarette smoker, and cannabis smoker. Immunohistochemical staining was performed for EGFR, protein kinase B (PKB or Akt), nuclear factor kappa B p50 (NF-КB), and cyclooxygenase-2 (COX-2) followed by boolean scoring for statistical analysis. Experimental data showed upregulation of the selected EGFR cascade in tumor cells, stromal expression of EGFR, and nuclear localization of COX-2 in metaplastic gland cells of laryngeal cancer tissue sample. Statistical analyses indicated that overexpression of the EGFR cascade is significantly correlated to cannabis smoking. Cannabis smokers had higher expression (p < 0.01) of these onco-proteins with respect to both nonsmokers as well as cigarette smokers. Risk factor analysis showed high risk of these proteins expression in age <60 years (odds ratio (OR) > 1.5) as the lower age group had relatively higher number of cannabis smokers. This study provides evidence for a direct association between cannabis smoking and increased risk of laryngeal cancer. Higher expression of the EGFR cascade in cannabis smokers revealed that cannabis smoking may be a major cause for the early onset of aggressive laryngeal cancer.

Inhibition of Hep G2 hepatic cancer cell growth and CCl4 induced liver cytotoxicity in Swiss albino mice by Mahua extract.

Mahua flower extract may provide protective effects against hepatotoxicity. The effect of Mahua flower extract (ME) was investigated on Hep G2 cell line and carbon tetrachloride (CCl4)-induced liver damages in Swiss albino mice. To investigate its cytotoxic effect in liver cancer, Hep G2 cells were treated with different doses of ME, and cell proliferation as well as colony formation assays demonstrated dose-dependent cytotoxicity of ME towards Hep G2 cells in tissue culture. Further gene expression studies showed significant down-regulation of AKT1/2/3, p-AKT, and COX-2 proteins including up-regulation of active caspase-3 in ME treated Hep G2 cells. In in vivo experiments, the mice were pretreated with ME for 15 days. On the 16th day CCl4 was injected intraperitoneally and after 24 h all mice were sacrificed. The antioxidant enzyme activities were measured in liver homogenates. CCl4-induced hepatotoxicity was evidenced by significant increase in lipid peroxidation and decrease in activities of antioxidant enzymes such as GST, GSH, SOD, CAT, and GPx. Histological studies showed CCl4-induced centrilobular necrosis and formation of fatty vacuoles in cirrhotic mice liver. Treatment with ME at a dose of 2 mg and 4 mg/kg exhibited the potential to prevent significant liver toxicity. The expression of active caspase-3 protein was down-regulated in ME treated groups compared to CCl4 exposed animals. This study demonstrated ME mediated antioxidant activity and hepatoprotective effects; therefore it could be used in the future for treating hepatic disorders including liver cancer, especially in combination with chemotherapeutics.

Effect of smoking on semen quality, FSH, testosterone level, and CAG repeat length in androgen receptor gene of infertile men in an Indian city.

This study was conducted as part of an epidemiological survey of 126 nonsmokers and 178 smokers, showing primary infertility residing around Kolkata region of Eastern India. Their lifestyle history including smoking habits along with semen and blood were collected. The study examined the association of cigarette smoking with the risk of infertility, by determining the semen quality, follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone levels, and androgen receptor (AR)-CAG repeat length in a group of smokers compared with a control group (non smokers). Based on conventional WHO criteria, lower sperm motility (P < 0.001) and increased sperm morphological defects (P < 0.0001) were associated with smoking habits. Binary logistic regression analysis for the effect of smoking status on sperm DNA integrity demonstrated significant positive correlation (p = 0.006). Serum FSH and LH levels were higher for smokers compared with non-smokers while the testosterone level decreased significantly with the increasing smoking habit. The mean length of CAG repeats in AR gene was significantly higher for smokers with low testosterone compared to non-smokers. The study suggested that smoking is associated with altered semen quality, endocrine hormonal status, and number of CAG repeats in the AR gene.

Habitat heterogeneity and prey selection of Aplocheilus panchax: an indigenous larvivorous fish.

OBJECTIVE: The ability of the native larvivorous fish Aplocheilus panchax (Hamilton, 1822) (Cyprinodontiformes: Aplocheilidae) as predator of mosquito larvae was assessed under laboratory conditions using multiple prey and habitat conditions. METHODS: The consumption of larvae of the mosquito Culex quinquefasciatus Say 1823 (Diptera: Culicidae) by A. panchax was evaluated in the presence of tubificid worms and chironomid larvae under complex and simple habitat conditions. The number of mosquito larvae consumed in comparison to other prey types was noted and an index of prey selectivity was used to evaluate the preference for mosquito larvae. RESULTS: Aphlocheilus panchax consumed 53 to 65 mosquito larvae in a three hour feeding bout contrast to 29- 38 tubificid worms and 43-62 chironomid larvae depending on the habitat conditions. The prey consumption differed significantly between the habitats and the prey type. The index of prey selectivity was positive for Cx. quinquefasciatus larvae over other alternative prey in all the habitat conditions. CONCLUSION: It is apparent from the study that the larvivorous fish A. panchax can be employed for biological regulation of mosquitoes in rice -fields and similar wetlands where the multiple prey choices are available under complex habitat conditions. However, field studies including other prey species will be required to substantiate this finding.

The in vitro effect of benzo[a]pyrene on human sperm hyperactivation and acrosome reaction.

OBJECTIVE: To evaluate the in vitro effect of benzo[a]pyrene on sperm hyperactivation and acrosome status in normozoospermic semen samples of nonsmokers analyzed by computer-assisted semen analysis (CASA). DESIGN: Experimental in vitro study. SETTING: Andrology laboratory. PATIENT(S): Thirteen proven fertile, normozoospermic, and nonsmoking men. INTERVENTION(S): Spermatozoa were washed free of seminal plasma and were treated with different concentrations of benzo[a]pyrene and compared with controls treated with medium alone. The benzo[a]pyrene concentrations were: 100, 50, 25, and 12.5 microg/mL. MAIN OUTCOME MEASURE(S): Effect of varying concentrations of benzo[a]pyrene on sperm hyperactivation and acrosomal reaction. RESULT(S): A statistically significant increase in sperm hyperactivation was observed in presence of benzo[a]pyrene at concentrations of >or=50 microg/mL. The result of the acrosome halo test showed that concentrations of benzo[a]pyrene >or=25 microg/mL statistically significantly decreased the percentage of halo formation, indicating an inappropriate (false) acrosome reaction. CONCLUSION(S): Benzo[a]pyrene statistically significantly affected sperm functional competence as evidenced by increased hyperactivation as well as premature acrosomal reaction.

Antidiarrhoeal activity of carbazole alkaloids from Murraya koenigii Spreng (Rutaceae) seeds.

The bioassay guided fractionation of the n-hexane extract of the seeds of Murraya koenigii Spreng (Rutaceae) resulted in the isolation of three bioactive carbazole alkaloids, kurryam (I), koenimbine (II) and koenine (III). The structures of the compounds were confirmed from their (1)H-, (13)C-, and 2D-NMR spectral data. Of the three compounds (I) and (II) exhibited significant inhibitory activity against castor oil-induced diarrhoea and PGE(2)-induced enteropooling in rats. The compounds also produced a significant reduction in gastrointestinal motility in the charcoal meal test in Wistar rats.

Vulnerability of the mosquito larvae to the guppies (Poecilia reticulata) in the presence of alternative preys.

BACKGROUND & OBJECTIVES: The predatory potential of the larvivorous fishes can be affected by the presence of alternative preys. In the present study the predation pattern of the sewage dwelling Poecilia reticulata (Peters 1872) on the larvae of Culex quinquefasciatus Say 1823 (Diptera: Culicidae) was evaluated in the presence of alternative preys. METHODS: The predation of Cx. quinquefasciatus larvae by different size groups of R. reticulata fishes was evaluated. In addition to this, the niche breadth (N) and diet breadth (B) were measured following Manly's selectivity index (Si) as an indicator of variation of such predation pattern in the presence of alternative prey types, like chironomid larvae and tubificid worms. RESULTS: The consumption of IV instar Cx. quinquefasciatus larvae by individual P. reticulata ranged between 65 and 84 in a 3 h feeding period and varied with the size of fish (F2,33 = 34.91; p < 0.001). The selectivity coefficient revealed a significantly low preference for the Cx. quinquefasciatus larvae (0.16, CL: 0.05-0.27; p < 0.05) compared to the chironomid larvae and tubificid worms, when all the three prey types were present. The niche breadth (N) and diet breadth (B) ranged from 0.77 to 0.92 and 0.69 to 0.93, respectively. The total consumption of all the prey types varied with the predator density, but the selectivity index for the mosquito larvae was significantly low in all the instances. INTERPRETATION & CONCLUSION: P. reticulata can consume a good number of mosquito larvae, with the consumption rate varying with the body size. P. reticulata fishes exhibit low preference for mosquito larvae as prey in the presence of alternative controphic preys like chironomid larvae and tubificid worms. Though establishment and sustenance of P. reticulata in new habitats will be favoured by the presence of alternative preys, but vulnerability of mosquito larvae may be reduced with availability of multiple preys in natural conditions.

Ultrastructural and hormonal changes in the pineal-testicular axis following arecoline administration in rats.

Arecoline is an alkaloid of betel nut of Areca catechu. Betel nut is chewed by millions of people in the world and it causes oral and hepatic cancers in human. It has therapeutic value for the treatment of Alzheimer and schizophrenia. Arecoline has immunosuppressive, mutagenic and genotoxic effects in laboratory animals. It also affects endocrine functions. The objective of this study was to investigate the effects of arecoline on pineal-testicular axis in rats. Since pineal activity is different between day and night, the current study is undertaken in both the photophase and scotophase. The findings were evaluated by ultrastructural and hormonal studies of pineal and testicular Leydig cells, with quantitations of fructose and sialic acid of sex accessories. Arecoline treatment (10 mg/kg body weight daily for 10 days) caused suppression of pineal activity at ultrastructural level by showing dilatation of the cisternae of the rough endoplasmic reticulum (RER), large autophagosome-like bodies with swollen mitochondrial cristae, numerous lysosomes, degenerated synaptic ribbons and reduced number of synaptic-like microvesicles. Moreover, pineal and serum N-acetylserotonin and melatonin levels were decreased with increased serotonin levels in both the gland and serum. In contrast, testicular Leydig cell activity was stimulated with abundance of smooth endoplasmic reticulum (SER), electron-dense core vesicles and vacuolated secretory vesicles, and increased testosterone level in the arecoline recipients. Consequently, the testosterone target, like prostate, was ultrastructurally stimulated with abundance of RER and accumulation of secretory vesicles. Fructose and sialic acid concentrations were also significantly increased respectively in the coagulating gland and seminal vesicle. These results were more significant in the scotophase than the photophase. The findings suggest that arecoline inhibits pineal activity, but stimulates testicular function (testosterone level) and its target organs presumably via muscarinic cholinergic receptor in rats.

Dopamine in vivo inhibits VEGF-induced phosphorylation of VEGFR-2, MAPK, and focal adhesion kinase in endothelial cells.

Vascular permeability factor (VPF)/VEGF is a potent multifunctional cytokine and growth factor that has critical roles in vasculogenesis and in both physiological and pathological angiogenesis. Because it has been recently shown that the neurotransmitter dopamine at pharmacological dose can inhibit VEGF/VPF-mediated microvascular permeability, proliferation, and migration of endothelial cells in vitro, we therefore hypothesized that endogenous dopamine may regulate the actions of VPF/VEGF in vivo. We report that VPF/VEGF-induced phosphorylation of VEGF receptor 2, focal adhesion kinase, and MAPK in the endothelial cells is strikingly increased in both dopamine-depleted and dopamine D(2) receptor knockout mice compared with normal controls, thereby indicating that endogenous dopamine regulate these critical signaling cascades required for the in vivo endothelial functions of VPF/VEGF. Together, these observations provide new mechanistic insight into the dopamine-mediated inhibition of the activities of VPF/VEGF and suggest that endogenous neurotransmitter dopamine might be an important physiological regulator of VPF/VEGF activities in vivo.

Depleted dopamine in gastric cancer tissues: dopamine treatment retards growth of gastric cancer by inhibiting angiogenesis.

PURPOSE: It has been recently shown that the catecholamine neurotransmitter dopamine (DA) strongly and selectively inhibits vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-induced angiogenesis. Gastric cancer is highly angiogenic and is dependent on VEGF for its growth and progression. Because substantial amounts of DA present in normal stomach tissues has been implicated in several gastric functions, we therefore investigated the role, if any, of this neurotransmitter in the growth and progression of gastric cancer. EXPERIMENTAL DESIGN: Initially, the status of DA and tyrosine hydroxylase, the rate-limiting enzyme required for DA synthesis, were determined in human gastric cancer tissues and in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer tissues of rats. On the basis of our observation of inverse correlation between stomach DA and gastric cancer growth, we determined the effect of pharmacological dose of DA on the angiogenesis and growth of MNNG induced gastric cancer in rats and Hs746T human gastric cancer in nude mice. RESULTS: DA and tyrosine hydroxylase were absent in both human and rat gastric cancer tissues. On the contrary, a low nontoxic pharmacological dose of DA significantly retarded tumor angiogenesis by inhibiting VEGFR-2 phosphorylation in tumor endothelial cells, which expressed DA D(2) receptors. This action of DA was associated with the growth inhibition of both MNNG-induced rat malignant gastric tumors and xenotransplanted human gastric cancer in nude mice. CONCLUSIONS: Our study demonstrates that there is an inverse correlation between endogenous stomach DA and gastric cancer and indicates that DA already in clinical use for other purposes might have a role as an antiangiogenic agent in the treatment of gastric cancer.

Reconstruction of facial deformities in leprosy patients.

Almost all the parts of the face may be involved in deformities caused by leprosy. Reconstructive surgery can be undertaken after the disease is arrested by medical treatment. Reconstruction of nose can be done by augmentation with autogenous bone graft, retronasal inlay graft, etc. Loss of eyebrows can be reconstructed with hair bearing skin with hair follicles. Sagging face can be corrected by nasolabial face-lift with correction of glabellar folds. Blepharoplasty is done for correction of some conditions. The temporal muscle sling is a dynamic procedure to reconstruct facial nerve palsy.

Dopamine inhibits cytokine release and expression of tyrosine kinases, Lck and Fyn in activated T cells.

The effect of dopamine (DA) on the release of cytokines from activated human T cells has been evaluated to analyze the mechanism by which physiological concentration of dopamine inhibits T cell proliferation. Dopamine inhibited anti-CD3 mAb-induced release of both Th1 and Th2 cytokines, IL2, IFN-gamma and IL4 from T cells by specific class of dopamine receptors. This action of dopamine was mediated by a new mechanism. Dopamine suppressed non-receptor tyrosine kinases, Lck and Fyn expression which are the initial and pivotal signaling steps in T cell receptor (TCR) mediated different down stream signaling cascades, leading to cytokine release and subsequent clonal expansion of these immune effector cells.