Kirit Pindolia

Pristimerin, a quinonemethide triterpenoid, induces apoptosis in pancreatic cancer cells through the inhibition of pro-survival Akt/NF-κB/mTOR signaling proteins and anti-apoptotic Bcl-2

Lack of effective therapeutics for pancreatic cancer at the present time underscores the dire need for safe and effective agents for the treatment of this malignancy. In the present study, we have evaluated the anticancer activity and the mechanism of action of pristimerin (PM), a quinonemethide triterpenoid, against MiaPaCa-2 and Panc-1 pancreatic ductal adenocarcinoma (PDA) cell lines. Treatment with PM inhibited the proliferation and induced apoptosis in both cell lines as characterized by the increased Annexin V-binding and cleavage of PARP-1 and procaspases -3, -8 and -9. PM also induced mitochondrial depolarization and the release of cytochrome c from the mitochondria. The induction of apoptosis by PM was associated with the inhibition of the pro-survival Akt, NF-κB and mTOR signaling proteins and their downstream intermediaries such as Foxo-3α and cyclin D1 (Akt); Cox-2 and VEGF (NF-κB); p-S6K1 and p-4E-BP1 (mTOR) as well as PKCɛ. Treatment with PM also inhibited the expression of anti-apoptotic Bcl-2 and survivin but not Bcl-xL. The downregulation of Bcl-2 by PM was not due to proteasomal or lysosomal proteolytic degradation of Bcl-2, since treatment with PM in the presence of proteasomal inhibitors MG132 or lactacystin (LAC) or calpain inhibitor MG101 failed to block the downregulation of Bcl-2 by PM. On the other hand, RT-PCR analysis showed the inhibition of Bcl-2 mRNA by PM in a dose-related manner, indicating that inhibition of Bcl-2 by PM is mediated through the suppression of Bcl-2 gene expression. Thus, the mechanistic understanding of the antitumor activity of pristimerin could facilitate in vivo efficacy studies of pristimerin for pancreatic cancer.

Analysis of mutations causing biotinidase deficiencya

Biotinidase deficiency is an inherited disorder in which the vitamin, biotin, is not recycled. Individuals with biotinidase deficiency can develop neurological and cutaneous symptoms if they are not treated with biotin. Biotinidase deficiency screening has been incorporated into essentially all newborn screening programs in the United States and in many countries. We now report 140 known mutations in the biotinidase gene (BTD) that cause biotinidase deficiency. All types of mutations have been found to cause biotinidase deficiency. Variants have been identified throughout the coding sequence. Essentially all the variants result in enzymatic activities with less than 10% of mean normal enzyme activity (profound biotinidase deficiency) with the exception of the c.1330G>C (p.D444H) mutation, which results in an enzyme having 50% of mean normal serum activity. The putative three‐dimensional structure of biotinidase has been predicted by homology to that of nitrilases/amidases. The effect of the various missense mutations can be predicted to affect various important sites within the structure of the enzyme. This compilation of variants causing biotinidase deficiency will be useful to clinical laboratories that are performing mutation analysis for confirmational testing when the enzymatic results are equivocal for children identified through newborn screening.

Inhibition of cell proliferation and induction of apoptosis by oleanane triterpenoid (CDDO-Me) in pancreatic cancer cells is associated with the suppression of hTERT gene expression and its telomerase activity

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a multifunctional oleanane synthetic triterpenoid with potent anti-inflammatory and antitumorigenic properties. The mechanisms of the antisurvival and apoptosis-inducing activities of CDDO-Me and related derivatives of oleanolic acid have been defined; however, to date, no study has been carried out on the effect of CDDOs on human telomerase reverse transcriptase (hTERT) gene or telomerase activity. Here we report for the first time that inhibition of cell proliferation and induction of apoptosis by CDDO-Me in pancreatic cancer cell lines is associated with the inhibition of hTERT gene expression, hTERT telomerase activity and a number of proteins that regulate hTERT expression and activity. Furthermore, abrogation or overexpression of hTERT protein altered the susceptibility of tumor cells to CDDO-Me. These findings suggest that telomerase (hTERT) is a relevant target of CDDO-Me in pancreatic cancer cells.

Biotinidase deficiency mimicking neuromyelitis optica: Initially exhibiting symptoms in adulthood

Background: Children with untreated biotinidase deficiency can experience variable symptoms depending on their age of presentation. Older children and adolescents can exhibit predominant neurological deficits including para- or tetraparesis and vision loss. Methods: We report the first case of delayed-onset biotinidase deficiency in a young adult. Results: A 22-year-old man presented with a disabling extensive myelopathy and bilateral optic neuropathy which mimicked the findings of a (seronegative) neuromyelitis optica. Imaging investigations were characterized by an MRI T2 hyper-intensity involving the spinal cord, the optic nerves, the fornix and the mammillar bodies, together with an increased 18F-FDG uptake on positron emission tomography. He was ultimately shown to have profound biotinidase deficiency due to a novel missense mutation and was partly improved by oral biotin therapy. Conclusion: This individual exemplifies the need to include biotinidase deficiency in the differential diagnosis of patients with extensive myelopathy and/or bilateral optic neuropathy and argues for newborn screening for the disorder.

DEVELOPMENT AND CHARACTERIZATION OF A MOUSE WITH PROFOUND BIOTINIDASE DEFICIENCY: A BIOTIN-RESPONSIVE NEUROCUTANEOUS DISORDER

Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states.

Ubiquitin-proteasomal degradation of antiapoptotic survivin facilitates induction of apoptosis in prostate cancer cells by pristimerin

Pristimerin (PM), a quinonemethide triterpenoid, is a promising anticancer agent with potent antiproliferative and apoptosis-inducing activities against cancer cell lines. However, the anticancer activity and mechanisms of PM in prostate cancer cells have not been adequately investigated. Here we report that the degradation of survivin plays an important role in the antiproliferative and proapoptotic effects of PM in carcinoma of the prostate (CaP) cell lines. Treatment with PM inhibited proliferation and induced apoptosis in LNCaP and PC-3 cells as characterized by the loss of cell viability and an increase in Annexin V-binding and cleavage of PARP-1, respectively. The antiproliferative and apoptosis-inducing effects of PM were associated with the inhibition of cell cycle regulatory proteins, antiapoptotic survivin and members of the Bcl-2 family. Data showed that response to PM is regulated by survivin since overexpression of survivin rendered CaP cells resistant to PM. Furthermore, downregulation of survivin by PM was mediated through the ubiquitin-proteasomal degradation. Together, these data demonstrate that pristimerin inhibits proliferation and induces apoptosis in CaP cells by abolishing survivin through the ubiquitin-proteasome pathway.

Induction of Apoptosis in Pancreatic Cancer Cells by CDDO-Me Involves Repression of Telomerase through Epigenetic Pathways.

Reactivation of telomerase in cancers provides an attractive target for developing novel agents to selectively destroy tumor cells. Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a synthetic oleanane triterpenoid, inhibited cell proliferation and induced apoptosis in pancreatic cancer cells at very low concentrations. The antiproliferative and apoptosis-inducing effects of CDDO-Me were associated with the inhibition of human telomerase reverse transcriptase (hTERT) mRNA, hTERT protein and reduction in hTERT telomerase activity. CDDO-Me inhibited multiple transcription factors that regulate hTERT expression positively (Sp1, c-Myc and NF-κB) and negatively (CTCF, E2F-1 and MAD1). CDDO-Me inhibited protein levels of DNA methyl transferases DNMT1 and DNMT3a, which also resulted in hypomethylation of hTERT promoter. In addition, transcriptionally active chromatin markers, such as acetylated histone H3 (Lys 9), acetylated histone H4, di-methyl H3 (Lys 4) and tri-methyl H3 (Lys 9) were all reduced in pancreatic cancer cells treated with CDDO-Me. Chromatin immunoprecipitation analysis showed decreased histone deacetylation and histone demethylation at hTERT promoter. Collectively, these results indicate that down-regulation of telomerase through epigenetic mechanisms plays a critical role in induction of apoptosis in pancreatic cancer cells by CDDO-Me.

Neurological deficits in mice with profound biotinidase deficiency are associated with demylination and axonal degeneration

Biotinidase deficiency is an autosomal recessively inherited disorder characterized by neurological and cutaneous abnormalities. We have developed a transgenic knock-out mouse with biotinidase deficiency to better understand aspects of pathophysiology and natural history of the disorder in humans. Neurological deficits observed in symptomatic mice with biotinidase deficiency are similar to those seen in symptomatic children with the disorder. Using a battery of functional neurological assessment tests, the symptomatic mice performed poorly compared to wild-type mice. Demyelination, axonal degeneration, ventriculomegaly, and corpus callosum compression were found in the brains of untreated, symptomatic enzyme-deficient mice. With biotin treatment, the symptomatic mice improved neurologically and the white matter abnormalities resolved. These functional and anatomical findings and their reversal with biotin therapy are similar to those observed in untreated, symptomatic and treated individuals with biotinidase deficiency. The mouse with biotinidase deficiency appears to be an appropriate animal model in which to study the neurological abnormalities and the effects of treatment of the disorder.

Characterization and functional analysis of cellular immunity in mice with biotinidase deficiency.

Biotinidase deficiency is an autosomal recessively inherited metabolic disorder that can be easily and effectively treated with pharmacological doses of the vitamin, biotin. Untreated children with profound biotinidase deficiency may exhibit neurological, cutaneous and cellular immunological abnormalities, specifically candida infections. To better understand the immunological dysfunction in some symptomatic individuals with biotinidase deficiency, we studied various aspects of immunological function in a genetically engineered knock-out mouse with biotinidase deficiency. The mouse has no detectable biotinidase activity and develops neurological and cutaneous symptoms similar to those seen in symptomatic children with the disorder. Mice with profound biotinidase deficiency on a biotin-restricted diet had smaller thymuses and spleens than identical mice fed a biotin-replete diet or wildtype mice on either diet; however, the organ to body weight ratios were not significantly different. Thymus histology was normal. Splenocyte subpopulation study showed a significant increase in CD4 positive cells. In addition, in vitro lymphocyte proliferation assays consistently showed diminished proliferation in response to various immunological stimuli. Not all symptomatic individuals with profound biotinidase deficiency develop immunological dysfunction; however, our results do show significant alterations in cellular immunological function that may contribute and/or provide a mechanism(s) for the cellular immunity abnormalities in individuals with biotinidase deficiency.

TNF-alpha Gene Therapy with Myeloid Progenitor Cells Lacks the Toxicities of Systemic TNF-alpha Therapy

We examined the antileukemic activity and the toxicity of HPC transduced with human tumor necrosis factor (TNF) cDNA. Both clonal (32Dcl3) and BM-derived primary hematopoietic progenitors (BM-Prog) expressing hTNF-alpha gene (32DTNF-alpha and BMTNF-alpha cells, respectively) inhibited the development of leukemia in mice with a small dose of 32Dp210 cells, a myeloid leukemia cell line. Whether the trans-gene expressing 32DTNF-alpha cells produce toxicities commonly associated with systemic TNF-alpha therapy was determined by examining the effect of TNF-alpha-secreting progenitor cells on body weight, tissue histology, growth of HPC, and engraftment of BMT. Administration of a low or high dose of TNF-alpha-secreting 32DTNF-alpha cells to mice failed to produce loss in body weight, a measure of TNF-alpha-related cachexia. There was also no evidence of tissue necrosis or mononuclear cell (MNC) infiltration in lung, liver, kidney, or intestine of mice injected with transduced progenitor cells. Furthermore, 32DTNF-alpha cells showed no effect on the clonal growth of HPC in colony-forming assays or loss of cellularity in BM, spleen, or blood. Finally, TNF-alpha-secreting cells were found not to interfere with the engraftment of BM transplant and hematopoietic reconstitution thereafter. We conclude from these findings that unlike systemic administration of TNF-alpha, TNF-alpha gene therapy with transduced HPC is nontoxic and may have a role in eradicating residual leukemia after BMT.