Picrorhiza kurroa, Royle ex Benth:Traditional uses, phytopharmacology, and translational potential in therapy of fatty liver disease.

Picrorhiza kurroa Royle ex Benth, Kutki (P.kurroa) is an important medicinal plant, traditionally recommended and used in Ayurveda for millennia, with certain cautions. There has been a significant revival of keen interest in its pharmacology, pharmacognosy, and phytochemistry for the last few decades. The evidence of its hepatoprotective activity, in experimental and clinical studies, accelerated the correlation of the specific phytochemical constituents of P.kurroa with precise pharmacological activities. Iridoid glycosides, particularly picrosides, emerged as the active molecules. For effective translation of traditional remedies into modern therapy, value addition by mechanistic understanding of molecular actions, drug targets, the degrees of efficacy and safety as well as convenient dosage forms is needed. Reverse pharmacology approach and phytopharmaceutical drug category facilitate such a translation. The present review illustrates how a potential translation of traditional practices of using P.kurroa into a phytochemically standardized, clinically targeted natural product for global unmet medical needs viz. Fatty liver disease can be attained.

Cytologic follow up of Low-grade Squamous Intraepithelial Lesions in Pap smears after integrated treatment with antimicrobials followed by oral turmeric oil extract.

Cervical cancer is preventable because the carcinogenesis is slow and there are opportunities to detect precancerous lesions by Papanicolaou (Pap) smears, colposcopy, or HPV DNA tests and to treat them by antimicrobials, surgery or cold CO2 vapourization. We have earlier reported on the chemopreventive potential of integrated treatment with antimicrobials therapy followed by a standardized oral Turmeric Oil (TO) extract upto 12 weeks in women who had persistent Low-grade Squamous Intra-epithelial Lesion (LSIL) in their Pap smears. In this communication we report their post-therapy follow up for 36 months (N = 18) with Pap smears. We were also able to follow up for 36 months control cases (N = 10) who had only standard therapy with antimicrobials. During 36 months of follow up none of the cases with integrated treatment, progressed to HSIL or cancer. Out of 15/18 cases which had a regression of Pap smear, all 15 remained free of LSIL from 6 to 36 months post-therapy showing persistent therapeutic effect of integrated therapy. In one case there was recurrence of LSIL in Pap smear, ten months post-therapy, which regressed to mild atypia after a second course of oral TO for 8 weeks. In the control group, persistence of LSIL after antimicrobials was observed in Pap smears in 5/10 cases when followed up by Pap smears up to 36 months. This preliminary report indicates some post-therapeutic benefit with integrative treatment as compared to the use of antimicrobials alone. A large scale controlled study is warranted.

Role of cytokines in genesis, progression and prognosis of cervical cancer.

Cytokine research is currently at the forefront in cancer research. Deciphering the functions of these multiple small molecules, discovered within the cell and in intercellular spaces, with their abundance and pleotrophism, was initially a great challenge. Advances in analytical chemistry and molecular biology have made it possible to unravel the pathophysiological functions of these polypeptides/proteins which are called interleukins, chemokines, monokines, lymphokines and growth factors. With more than 5 million women contracting cervical cancer every year this cancer is a major cause of mortality and morbidity the world over, particularly in the developing countries. In more than 95% of cases it is associated with human papilloma virus (HPV) infection which is persistent, particularly in those with a defective immune system. Although preventable, the mere magnitude of prevalence of HPV in the world population makes it a dominating current health hazard. The discovery of cytokine dysregulation in cervical cancer has spurted investigation into the possibility of using them as biomarkers in the early diagnosis of cases at high risk of developing cancer. Their critical role in carcinogenesis and progression of cervical cancer is now being revealed to a great extent. From diagnostics to prognosis, and now with a possible role in therapeutics and prevention of cervical cancer, the cytokines are being evaluated in all anticancer approaches. This review endeavours to capture the essence of the astonishing journey of cytokine research in cervical neoplasia.

Mechanisms of Adipocytokine-Mediated Trastuzumab Resistance in HER2-Positive Breast Cancer Cell Lines.

Acquired resistance to trastuzumab is a clinical problem in the treatment of HER2-over-expressing metastatic breast cancer. Importantly, an earlier report suggested that high body mass index was associated with reduced overall survival and reduced time to progression in patients with early stage or metastatic HER2-positive breast cancer treated with trastuzumab. Adipocyte-secreted factors may stimulate growth of HER2-positive cancers, blocking the growth inhibitory action of trastuzumab. Leptin and growth differentiation factor 15 (GDF15) are two adipocytokines that have been reported to stimulate HER2-PI3K signaling. We previously showed that cells with acquired trastuzumab resistance express increased levels of GDF15, and that GDF15 knockdown restores sensitivity to trastuzumab. The objective of the current study was to identify potential molecular mechanisms by which adipocytes stimulate resistance to trastuzumab in HER2-over-expressing breast cancer cell lines. Cells were cultured in complete media or conditioned media from differentiated adipocytes (CM). Cell viability of trastuzumab-treated cells was examined under anchorage-dependent and -independent conditions. Phosphorylation of Akt was assessed by Western blotting, and response to trastuzumab was reassessed upon treatment with the PI3K inhibitor LY294002 or after transfection with kinase-dead Akt. We report that CM significantly reduced trastuzumab-mediated growth inhibition of HER2-positive cells, and stimulated rapid phosphorylation of Akt. Pharmacologic or genetic inhibition of PI3K overcame CM-mediated trastuzumab resistance. Leptin and GDF15 were both measured in CM, but only GDF15 conferred resistance to trastuzumab. Leptin, on the other hand, abrogated sensitivity to lapatinib but not trastuzumab. Our observations suggest that adipocyte-secreted factors such as GDF15 stimulate PI3K signaling, resulting in reduced response to trastuzumab. The utility of adipocytokines as predictors of drug resistance and approaches to mitigate the cancer-promoting effects of adipocyte-secreted factors should be further examined. Our work supports additional investigation into GDF15 as a potential biomarker of trastuzumab resistance, and development of approaches to therapeutically target GDF15 in HER2-positive breast cancers that have progressed on trastuzumab.

Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion.

Identification of novel molecular markers and therapeutic targets may improve survival rates for patients with ovarian cancer. In the current study, immunohistochemical (IHC) analysis of two human ovarian tumor tissue arrays showed high staining for GDF15 in a majority of tissues. Exogenous stimulation of ovarian cancer cell lines with recombinant human GDF15 (rhGDF15) or stable over-expression of a GDF15 expression plasmid promoted anchorage-independent growth, increased invasion, and up-regulation of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). MMP inhibition suppressed GDF15-mediated invasion. In addition, IHC analysis of human ovarian tumor tissue arrays indicated that GDF15 expression correlated significantly with high MMP2 and MMP9 expression. Exogenous and endogenous GDF15 over-expression stimulated phosphorylation of p38, Erk1/2, and Akt. Pharmacologic inhibition of p38, MEK, or PI3K suppressed GDF15-stimulated growth. Further, proliferation, growth, and invasion of GDF15 stable clones were blocked by rapamycin. IHC analysis demonstrated significant correlation between GDF15 expression and phosphorylation of mTOR. Finally, knockdown of endogenous GDF15 or neutralization of secreted GDF15 suppressed invasion and growth of a GDF15-over-expressing ovarian cancer cell line. These data indicate that GDF15 over-expression, which occurred in a majority of human ovarian cancers, promoted rapamycin-sensitive invasion and growth of ovarian cancer cells. Inhibition of mTOR may be an effective therapeutic strategy for ovarian cancers that over-express GDF15. Future studies should examine GDF15 as a novel molecular target for blocking ovarian cancer progression.

Catalytic deoxyribozyme-modified nanoparticles for RNAi-independent gene regulation.

DNAzymes are catalytic oligonucleotides with important applications in gene regulation, DNA computing, responsive soft materials, and ultrasensitive metal-ion sensing. The most significant challenge for using DNAzymes in vivo pertains to nontoxic delivery and maintaining function inside cells. We synthesized multivalent deoxyribozyme "10-23" gold nanoparticle (DzNP) conjugates, varying DNA density, linker length, enzyme orientation, and linker composition in order to study the role of the steric environment and gold surface chemistry on catalysis. DNAzyme catalytic efficiency was modulated by steric packing and proximity of the active loop to the gold surface. Importantly, the 10-23 DNAzyme was asymmetrically sensitive to the gold surface and when anchored through the 5' terminus was inhibited 32-fold. This property was used to generate DNAzymes whose catalytic activity is triggered by thiol displacement reactions or by photoexcitation at λ = 532 nm. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease degradation, readily enter mammalian cells, and catalytically down-regulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for in vivo work.

p62 is a key regulator of nutrient sensing in the mTORC1 pathway.

The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.

Growth differentiation factor 15 (GDF15)-mediated HER2 phosphorylation reduces trastuzumab sensitivity of HER2-overexpressing breast cancer cells.

Resistance to the anti-HER2 monoclonal antibody trastuzumab is a major problem in the treatment of HER2-overexpressing metastatic breast cancer. Growth differentiation factor 15 (GDF15), which is structurally similar to TGF beta, has been reported to stimulate phosphorylation of HER2. We tested the hypothesis that GDF15-mediated phosphorylation of HER2 reduces the sensitivity of HER2-overexpressing breast cancer cell lines to trastuzumab. Gene microarray analysis, real-time PCR, and ELISA were used to assess GDF15 expression. Growth inhibition and proliferation assays in response to pharmacologic inhibitors of HER2, TGF beta receptor, or Src were performed on cells stimulated with recombinant human GDF15 or stable GDF15 transfectants. Western blotting was performed to determine effects of GDF15 on HER2 signaling. Cells were infected with lentiviral GDF15 shRNA plasmid to determine effects of GDF15 knockdown on cell survival in response to trastuzumab. Cells with acquired or primary trastuzumab resistance showed increased GDF15 expression. Exposure of trastuzumab-sensitive cells to recombinant human GDF15 or stable transfection of a GDF15 expression plasmid inhibited trastuzumab-mediated growth inhibition. HER2 tyrosine kinase inhibition abrogated GDF15-mediated Akt and Erk1/2 phosphorylation and blocked GDF15-mediated trastuzumab resistance. Pharmacologic inhibition of TGF beta receptor blocked GDF15-mediated phosphorylation of Src. Further, TGF beta receptor inhibition or Src inhibition blocked GDF15-mediated trastuzumab resistance. Finally, lentiviral GDF15 shRNA increased trastuzumab sensitivity in cells with acquired or primary trastuzumab resistance. These results support GDF15-mediated activation of TGF beta receptor-Src-HER2 signaling crosstalk as a novel mechanism of trastuzumab resistance.

Akt-phosphorylated PIKE-A inhibits UNC5B-induced apoptosis in cancer cell lines in a p53-dependent manner.

UNC5B acts as a tumor suppressor, and it induces apoptosis in the absence of its cognate ligand netrins. UNC5B is a direct transcriptional target of p53 upon UV stimulation. Here we show that Akt phosphorylates PIKE-A and regulates its association with UNC5B and inhibits UNC5B-provoked apoptosis in a p53-dependent manner. PIKE-A GTPase binds active Akt and stimulates its kinase activity in a guanine-nucleotide-dependent way. Akt feeds back and phosphorylates PIKE-A on Ser-472 and subsequently enhances its stimulatory effect on Akt kinase activity. Akt activity is significantly reduced in PIKE -/- Mouse Embryonic Fibroblast (MEF) cells as compared to wild-type cells. PIKE-A directly interacts with UNC5B, which is regulated by netrin-1-activated Akt. Overexpression of PIKE-A diminishes UNC5B expression through down-regulation of p53. Knocking down PIKE-A stabilizes p53, increases UNC5B, and escalates UV-triggered apoptosis. Depletion of Akt abrogates PIKE-A's inhibitory effect on both p53 and UNC5B. Hence our findings support the notion that Akt--phosphorylated PIKE-A inhibits UNC5B-elicited apoptosis and reduces its expression level through inactivation of p53.

Menopause and metabolic syndrome: A study of 498 urban women from western India.

INTRODUCTION: Metabolic syndrome (MS) is a cluster of risk factors for future development of type 2 diabetes mellitus and cardiovascular diseases. Menopausal transition with its incidental hormonal changes is considered to contribute to the development of MS. However, age is known to influence MS risk factors. OBJECTIVE: The present study explores the prevalence of MS in pre- and postmenopausal women from western India. METHODS: Four hundred and ninety eight women above 35 years of age, participating in women's health care program were assessed for the prevalence of MS using two criteria- International Diabetes Federation criteria (IDF) and Harmonization (H_MS) criteria. RESULTS: Prevalence of MS amongst postmenopausal women was significantly higher (P < 0.001) than that in premenopausal women by both, IDF (premenopausal 45% and postmenopausal 55%) and H_MS criteria (premenopausal 44% and postmenopausal 56%). However, this significance disappeared when data was adjusted for the confounding variable of age.

Simultaneous inactivation of Par-4 and PTEN in vivo leads to synergistic NF-kappaB activation and invasive prostate carcinoma.

Prostate cancer is one of the most common neoplasias in men. The tumor suppressor Par-4 is an important negative regulator of the canonical NF-kappaB pathway and is highly expressed in prostate. Here we show that Par-4 expression is lost in a high percentage of human prostate carcinomas, and this occurs in association with phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss. Par-4 null mice, similar to PTEN-heterozygous mice, only develop benign prostate lesions, but, importantly, concomitant Par-4 ablation and PTEN-heterozygosity lead to invasive prostate carcinoma in mice. This strong tumorigenic cooperation is anticipated in the preneoplastic prostate epithelium by an additive increase in Akt activation and a synergistic stimulation of NF-kappaB. These results establish the cooperation between Par-4 and PTEN as relevant for the development of prostate cancer and implicate the NF-kappaB pathway as a critical event in prostate tumorigenesis.

Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis.

The atypical PKC-interacting protein, Par-4, inhibits cell survival and tumorigenesis in vitro, and its genetic inactivation in mice leads to reduced lifespan, enhanced benign tumour development and low-frequency carcinogenesis. Here, we demonstrate that Par-4 is highly expressed in normal lung but reduced in human lung cancer samples. We show, in a mouse model of lung tumours, that the lack of Par-4 dramatically enhances Ras-induced lung carcinoma formation in vivo, acting as a negative regulator of Akt activation. We also demonstrate in cell culture, in vivo, and in biochemical experiments that Akt regulation by Par-4 is mediated by PKCzeta, establishing a new paradigm for Akt regulation and, likely, for Ras-induced lung carcinogenesis, wherein Par-4 is a novel tumour suppressor.

Radiosensitization by diospyrin diethylether in MCF-7 breast carcinoma cell line.

The development of radio-resistant tumor cells might be overcome by the use of tumor selective cytotoxic agents in combination with radiation treatment of cancer. Thus, we are exploring the radiomodifying potential of D7, a tumor-inhibitory compound derived from a plant product, diospyrin, in breast carcinoma cells, MCF-7. The present study indicated that D7 could enhance the radiation-induced cytotoxicity and apoptosis through down-regulation of the anti-apoptotic Bcl-2 and COX-2 gene expression, and up-regulation of pro-apoptotic genes, like p53 and p21. The higher expression of PUMA, a pro-apoptotic protein was also observed in the combination treatment. Effect of D7 on up-regulation of p21 expression in irradiated MCF-7 cells was concomitant with the cell cycle arrest in the G1 phase. Thus, it was concluded that D7 could sensitize the effect of radiation in breast carcinoma by regulating the gene expression involved in cell cycle and apoptosis.

Spontaneous or induced regression of cancer a novel research strategy for ayurvidya.

Regression of cancer has been an interiguiny factor for medicinal science. This article is bringing out some interesting data on this issue with a view to generate in the Ayurvedic researchers to see the possibilities of Ayurveda in induced regression of cancer.

Early human safety study of turmeric oil (Curcuma longa oil) administered orally in healthy volunteers.

OBJECTIVE: Turmeric extract and turmeric oil have shown chemoprotective effect against chemically-induced malignancies in experimental animals. They can reverse precancerous changes in oral submucous fibrosis in humans. The use of turmeric or Curcuma longa Linn as a spice and household remedy has been known to be safe for centuries. In view of the long term administration required for cancer prevention a Phase I clinical trial of turmeric oil (TO) was designed to study the safety and tolerance of TO in volunteers for a period of 3 months. MATERIAL AND METHODS: Nine healthy volunteers between 20 and 33 years of age were tested for haemoglobin, blood counts, liver and kidney functions, bleeding and clotting time and serum electrolytes initially and at 1 and 3 months of treatment. They were administered 0.6 ml of TO three times a day for 1 month and 1 ml in 3 divided doses for 2 months. The acute tolerability study on Day 1 was conducted in a Clinical Pharmacology daycare Unit. Blood pressure and pulse were recorded frequently on Day 1 and at 24, 48, 72 and 96 hours and fortnightly till 12 weeks. Volunteers were daily supervised for TO intake as well as for any side effects throughout the study period. RESULTS: Nine volunteers were enrolled for the study. One discontinued on 3rd day for allergic skin rashes which, on discontinuation of TO, gradually disappeared by two weeks. Another discontinued on 7th day for intercurrent fever requiring antibiotic treatment. Seven volunteers completed the study. There was no effect of TO, in two doses, on pulse and blood pressure and no side effects in acute tolerability study on Day 1. There was no effect of TO intake on weight, blood pressure, symptoms and signs upto 12 weeks. There was no clinical, haematological, renal or hepatic-toxicity of TO at 1 month and 3 months. Serum lipids did not show significant change except in one volunteer (reversible). CONCLUSIONS: In view of the potential for reversing oral submucous fibrosis, a precancerous condition for oral cancer, TO, can be recommended directly for a Phase II trial in patients.